Permeable nanoreactor eye drop for enzymatic cascade-mediated treatment for acute retinal injury model mimicking geographic atrophy

Domain: biology
Published: 2026-03-17
Editorial outcome: major_revision

Abstract
Geographic atrophy (GA), the most prevalent form of age-related macular degeneration (AMD), remains clinically challenging due to its complex pathogenesis. Using an acute retinal injury model that mimics GA pathology (oxidative stress-induced retinal apoptosis), we identify enzymatic cascade restoration as a promising therapeutic strategy. To overcome ocular delivery barriers, we engineer zwitterionic nanocages for co-delivering catalase (CAT) and superoxide dismutase (SOD) (Z(CAT&SOD)). These optimized nanocages achieve a remarkable 9.32% ocular penetration rate, which significantly outperforms free peroxidase (0.54%), through conjunctival-sclera-retina and conjunctival-blood-retina penetration pathways, and achieve therapeutic efficacy comparable to intravitreal injection. In the chronic management of GA, the non-invasive Z(CAT&SOD) eye drops show superior therapeutic effects to the current gold standard clinical antioxidants and lutein supplement. Therefore, this work reveals the possible association between retinal peroxidase deficiency and GA progression, and proposes a non-invasive, highly effective zwitterionic nanocage for AMD treatment.

Introduction
Age-related macular degeneration (AMD) is a progressive and degenerative disease characterized by dysfunction and death of retinal pigment epithelium (RPE) cells and photoreceptors, leading to irreversible vision loss. It is the leading cause of permanent visual impairment worldwide, affecting more than 180 million people, mainly over the age of 551, 2. AMD manifests in two primary forms: geographic atrophy (GA) and choroidal neovascularization (CNV). Among them, GA patients constitute approximately 85% to 90%, and the rate of blindness approaches nearly 20%3. Since the introduction of intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF), patients with CNV have experienced substantial improvements in retinal function. However, despite the effectiveness of anti-VEGF therapies for CNV, these antibodies have proven ineffective in treating GA4. Therefore, there is currently no approved treatment for GA, primarily due to its complex and poorly understood pathogenesis. Clinically, supplementing antioxidants such as lutein appears to be the only viable approach to slowing down the deterioration of GA5. Numerous studies have revealed that relative pathogenesis of GA involves processes such as complement system activation, disturbances in lipid metabolism, oxidative stress, abnormal autophagy, and the chronic inflammation pathways and etc6-8. In response, various therapeutic strategies have been explored, including the suppression of complement activation, inhibition of angiogenesis, and exertion of anti-inflammatory effects9-11. However, these approaches have not yet demonstrated specific, positive impacts in clinical trials involving GA patients12-14. Therefore, the exploration of alternative therapeutic targets for GA treatment remains crucial.

Oxidative stress disrupts the physiological functions in most biological tissues and can lead to cell death15, 16. In the retina, oxidative stress triggers a cascade of events that result in focal loss of RPE and photoreceptors, leading to structural changes in proteins, lipids and DNA, which is increasingly recognized as a major contributing factor to the development of GA12, 15, 17. Numerous studies have demonstrated that the removal of free radicals from the retina can effectively slow the progression of AMD18. Additionally, in AMD treatment research, strategies targeting the elimination of reactive oxygen species (ROS) have been proposed, including the use of inorganic nanozymes like Pt, Rh, CeO2 to perform antioxidant function19-22. However, these nanozymes face challenges such as biocompatibility, biodegradability, and unclear therapeutic efficacy. Endogenous antioxidant enzyme, such as superoxide dismutase (SOD) and catalase (CAT), neutralize free radicals and maintain cellular homeostasis, with SOD targeting superoxide anion (O2•−) and CAT decomposing hydrogen peroxide (H2O2). These enzymes play essential roles in defending against oxidant stress, particularly in regulating ROS in inflammatory diseases23-26. However, the specific roles of CAT and SOD in the pathogenesis of age-related macular degeneration, especially during the geographic atrophy stage, require further investigation.

In this study, we discovered that the cellular antioxidant defense mechanisms were obviously compromised in sodium iodate (SI)-induced acute retinal injury model (ARI), pathological feature of which mimic that of human GA disease27, as evidenced by a noticeable decrease in the expression of CAT and SOD in the retina. While supplementation with CAT and SOD partially alleviated retinal atrophy and visual impairment, the therapeutic effects were not satisfactory. These limitations arise from poor ocular delivery and enzyme instability. Hence, we explored the use of zwitterions, which can polymerize in situ on protein surfaces to effectively shield charges, enhance permeability and preserve enzyme activity in complex physical environment28-32. By ingeniously employing zwitterionic materials, we coated the CAT and SOD with sulfobetaine methacrylate (SBMA), Z(CAT&SOD), a compound widely recognized for its charge regulation, mucosal and tissue penetration and biocompatibility. Remarkably, the Z(CAT&SOD) exhibited improved stability and activity against protease. Moreover, Z(CAT&SOD) demonstrated the ability to facilitate free diffusion across ocular barriers penetration capabilities by promoting clathrin-dependent and caveolae-mediated endocytosis33. As expected, following intravitreal injection of Z(CAT&SOD) in ARI model, the enzymatic cascade reactions exhibited superior ROS scavenging capacity and suppressed the activation of NF-κB-HMGB1-ILs inflammatory pathway. Ultimately, they protected RPEs and photoreceptors from ROS-induced apoptosis, which is crucial for preserving retinal function and preventing degenerative changes. Given the chronic nature and long-term treatment requirements of this disease, we attempted to develop a non-invasive peroxidase eye drop formulation, compared with the clinical gold-standard treatment strategy, supplementing with lutein. Unexpectedly, optimizing the components of zwitterionic polymers led to a significant enhancement in the ocular barrier permeability of zwitterionic-peroxidase, which achieved an impressive 9.32% ocular penetration (the ocular penetration of free peroxidase was only 0.54%) by increasing the permeability through both conjunctiva-sclera-retinal and blood-retinal pathways. Remarkably, the therapeutic effects of zwitterionic-peroxidase eye drops were comparable to those achieved through intravitreal injections in the ARI model, and both of them demonstrated greater efficacy compared to oral lutein therapy. Our research results emphasize that the progression of GA is associated with clear oxidative stress damage to the retina. The innovative and non-invasive eye drop we developed, which is based on a bimetallic ion nanocage coating of peroxidase, provides a transformative and promising therapeutic approach for dry AMD (Fig. 1).

Results
Oxidative stress and impaired enzyme cascade caused retina damage in acute retinal injury model
SI, an oxidizing agent, is known to induce the ARI model including RPE cell death, geographic atrophy and progressive visual dysfunction. This dosage is well-established to consistently induce oxidative damage in RPE cells and photoreceptors, creating an ARI mouse model that recapitulates key oxidative stress-related cellular events in GA34-36. Previous studies have demonstrated that SI could induce intense oxidative stress, which is concomitant with mitochondrial dysfunction in the retina35, 36. To demonstrate the successful establishment of ARI model, NaIO3 (10 mg/kg) was intravenously injected on day 0. Subsequently, the retinal structure and visual function of the mice were assessed at one or two weeks post-treatment using fundus fluorescein angiography (FFA), optical coherence tomography (OCT), and electroretinography (ERG) (Fig. 2a). As shown in Fig. 2b, in alignment with the pathogenesis of clinical GA, fundus imaging combined with angiographic data revealed that the mice with ARI model exhibited secondary vascular inflammation and significant drusen-like deposits in the retina, and the area of these drusen-like deposits progressively increased over time. OCT imaging provided detailed visualization of the retinal layers, revealing irregular accumulation and atrophy of the RPE layer in both the outer and inner nuclear layers (ONL and INL) (Supplementary Fig. 1a). By two-week time points, the ONL had nearly disappeared, accompanied by significant changes in overall retinal thickness. Further analysis of retinal thickness using Image J demonstrated a reduction of approximately 33% at two weeks (Fig. 2c). ERG analysis revealed a marked reduction in both the a and b waves, reflecting impaired activity of photoreceptors and ON-bipolar cells. These reductions were positively correlated with retinal thickness (Fig. 2d, 2e and 2f). These findings closely mirrored those observed in clinical GA patients, suggesting the relevance of this model for studying GA.

To assess the underlying mechanisms, we quantified the total ROS levels using fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and evaluated retinal apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. As shown in Fig. 2g and 2h, intense DCF fluorescence signals were observed in both the RPE layer and ONL, indicating substantial ROS generation. Apoptotic signals were also detected, indicating that ROS may induce apoptosis in retinal cells. Additionally, we collected retinas from ARI mice at both one week and two weeks, and quantified the expression levels of CAT and SOD by quantitative real-time polymerase chain reaction (qPCR). At two weeks, a significant reduction in the expression of both CAT and SOD was observed (Fig. 2i). Enzyme activity assays further confirmed this downregulation, revealing impaired antioxidant defense mechanism in the retina following SI treatment (Supplementary Fig. 1b and 1c). This impairment of antioxidant enzyme expression and activity coincided with increased oxidative stress and retinal cell apoptosis, thereby exacerbating the progression of retinal injury. Collectively, these results highlight the crucial role of oxidative stress in the pathogenesis of ARI model. The supplementation of natural antioxidant enzymes may potentially counteract the intense oxidative stress, providing a therapeutic strategy to alleviate retinal damage and visual impairment associated with ARI model.

Inspired by the above finding, we sought to assess the protective effects of CAT and SOD in the mouse model of SI-induced ARI. Three hours after intravenous injection of SI, 1×PBS (1 μL, Untreated group as negative control), SOD (2 mg/mL, 50000 U/kg, 1 μL), CAT (2 mg/mL, 80000 U/kg, 1 μL), or a combination of both enzymes (SOD&CAT, CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL) was promptly injected into the vitreous cavity. In addition, healthy mice were used as the positive control. The degree of retinal degeneration in each group of mice with ARI was assessed one week late using fundus imaging, FFA, OCT, and ERG (Fig. 2j). As shown in Fig. 2k and 2l, prominent retinal deposits were observed in both PBS- and SOD-injected groups, accompanied by subretinal debris accumulation (likely originating from degenerated RPE and photoreceptors), inflammatory edema, and oxidative stress-induced vascular abnormalities (Fig. S2a). After different treatments, a blinded evaluation was also performed based on fundus imaging and OCT imaging to assess retinal structural integrity and thickness reduction (Fig. S2b). Notable deformation of the RPE and ONL were also evident in these groups. In contrast, mice treated with CAT or SOD&CAT exhibited milder retinal damage, characterized by fewer drusen-like deposits, less microvascular abnormality, and reduced degeneration of the RPE and ONL. Quantitative analysis of retinal thickness using Image J from OCT images, revealed that the thickness of retinas treated with CAT or SOD&CAT remained approximately 80% of the original thickness, in sharp contrast to the significant thinning observed in the untreated group and SOD injection groups (Fig. 2m and Supplementary Fig. 2c). ERG results further corroborated these findings, showing a significant decline in retinal function following SI induction (Fig. 2n). Analysis of the a- and b- waves, which reflect photoreceptor and ON-bipolar cell activity, respectively, demonstrated partial recovery of electrophysiological responses in mice treated with either CAT or SOD&CAT injections (Supplementary Fig. 2d and 2e). Nevertheless, despite these improvements, the retinal still did not completely recover to the levels seen in healthy retinal, indicating that oxidative stress-induced retinal damage was not completely alleviated, which warrant further investigation.

Zwitterionic nanocages enhanced the permeability of peroxidases
Due to the limited therapeutic effect of intravitreal injection of peroxidase on ARI model, we speculated that drug delivery may be limited by complex ocular structures and physiological barriers. Therefore, we further verified the composition of intravitreal and related structural barriers and drug delivery barriers. The vitreous is primarily composed of collagen fibers and acidic mucopolysaccharides, which can easily form coulombic interactions with enzymes36. Additionally, the dense vitreous body membrane, known as the inner limiting membrane, separates the retina from the vitreous cavity, restricting the diffusion of enzymes and limiting their effectiveness37. Therefore, it is important to increase the diffusion of drugs in the vitreous cavity and the barrier penetration function for the treatment of GA retinopathy with peroxidase. Considering that zwitterionic platform’s proven ability to effectively penetrate mucosal barrier in the intestinal tract29, 31, we aim to develop zwitterionic nanocages to enhance the permeability of peroxidases (Fig. 3a). The encapsulation process involved enriching acrylamide, acrylic acid, sulfobetaine methacrylate (SBMA) and bisacrylamide (BIS; a cross-linker) around CAT through noncovalent interactions, followed by the growth of a thin polymer shell around each CAT by in situ polymerization. To evaluate the necessity of individual components for nanocage formation, we established multiple nanocages with various compositions, ranging from Z1 to Z8. Among them, Z1 and Z6 are cationic, Z2 and Z7 are anionic, Z3 contains only SBMA, and Z4 and Z8 incorporate cationic, anionic and SBMA. Moreover, Z6, Z7, and Z8 include crosslinking agent BIS and the initiator APS. We compared these nanocages based on size, surface charge, protein encapsulation ability, and permeability (Fig. 3b). As shown in Fig. 3c and 3d, different nanocages exhibit different sizes and charges. Notably, attempts to encapsulate peroxidase using Z1 to Z4 were unsuccessful, as nanocages could not be detected. In contrast, formulations Z5 and Z8 maintained stable sizes ranging from 100 nm to 200 nm. To further assess the encapsulation efficiency of Z5 to Z8 nanocages, we conducted gel electrophoresis and transmission electron microscope (TEM). As shown in Supplementary Fig. 3, the results indicated that Z8 nanocages, when formulated with BIS and APS, achieved the most effective enzyme encapsulation. TEM images further confirmed that Z8 nanocages exhibited a uniform particle size distribution approximately 200 nm (Fig. 3e).

To identify which nanocage formulations could effectively penetrate the ILM barrier, we tested the retinal permeability of Z5 to Z8 in mice after intravitreal injecting by tracking the FITC fluorescent signal labeled on the CAT. As shown in Figure 3f, compared to free CAT-FITC, most fluorescence signals were confined to the vitreous cavity (VC), while those in the Z8 group successfully penetrated the inner membrane and distributed across various retina layers (R). We further optimized the ratio of CAT to zwitterionic components, testing molar ratios of 1:2000, 1:4000, 1:8000, and 1:12000, and observed the resulting changes in particle size. As shown in Supplementary Fig. 4, at molar ratios of 1:2000 and 1:4000, the encapsulation of peroxidase was either uneven or incomplete. Conversely, at the molar ratio was 1:12000, the particle size of the peroxidase nanocages increased significantly, suggesting aggregation or gel formation. However, at a molar ratio of 1:8000, the zwitterionic nanocages exhibited a uniform size, indicating the completely encapsulation of CAT. Therefore, we selected the mole ratio of 1:8000 as the optimal ratio and chose Z8 as the polymer formulation for the preparation of zwitterionic nanocages. We further characterized the physical properties and drug-loading performance at this optimal ratio. Dynamic light scattering analysis revealed a nearly neutral surface charge (Supplementary Table 1). Notably, the nanocages exhibited excellent drug-loading capacity of 94.84 ± 1.55% with encapsulation efficiency of 35.32 ± 4.43%. Furthermore, circular-dichroism (CD) spectroscopy confirmed that the zwitterionic nanocage encapsulation preserved the native secondary structure of the peroxidases (Supplementary Fig. 5). To further investigate the interaction between zwitterionic nanocages and the vitreous, we measured the surface charge of Z(CAT) nanocages after co-incubation with the isolated rabbit vitreous (Supplementary Fig. 6). We found that the near-neutral surface of the zwitterionic nanocages remained stable in the vitreous environment, facilitating enhanced diffusion of the encapsulated protein drugs. To explore the mechanism by which Z(CAT) enhances permeability across ocular barriers, we conducted a series of tests on pathways such as paracellular pathway and endocytosis related to ocular barrier permeability. Firstly, we cultured human conjunctival epithelial cells (HConEpiC) to form a tightly connected cell barrier layer. After incubating the cells with Z(CAT) or free CAT for 24 hours, we performed immunofluorescence staining for E-cadherin, a key tight junction protein39. As shown in Fig. 3g, the morphology and fluorescence intensity of E-cadherin remained unchanged, suggesting that Z(CAT) does not enhance permeability via the paracellular pathway. Subsequently, we cultured HConEpiC cells in the upper chamber of a transwell to construct the conjunctival epithelial barrier in vitro. Barrier formation was monitored daily using a cell resistance meter until the resistance value plateaued, signifying that the barrier was fully formed. We then added either Z(CAT) or CAT to the upper chamber and incubated for 6 hours. Subsequently, we measured transepithelial electrical resistance (TEER) and observed nearly no significant changes following treatment with either Z(CAT) or free CAT (Supplementary Fig. 7), further confirming that Z(CAT) does not compromise tight junction integrity or utilize the paracellular route for ocular penetration.

To investigate whether Z(CAT) facilitates permeability through endocytosis, we co-cultured Cy5.5-labeled CAT and Z(CAT-Cy5.5) with HConEpiC cells for different time intervals (1, 3, 6 hours). As shown in Fig. 3h and 3j, Z(CAT) demonstrated significantly higher endocytosis compared to free CAT. After 6 hours of incubation, the average fluorescence intensity per cell for Z(CAT-Cy5.5) was significantly higher than that of free CAT-Cy5.5. Additionally, we quantified the remaining fluorescence quantity of Z(CAT-Cy5.5) and CAT-Cy5.5 in HConEpiC cells after replacing with fresh medium for different durations (3, 6, 12 hours). Three hours later, the fluorescence intensity in cells pre-incubation of Z(CAT-Cy5.5) retained about one-fifth of the original signal, and by 12 hours, the fluorescence had nearly disappeared (Fig. 3i and 3k), indicating that its enhanced barrier permeability of Z(CAT) is primarily due to the enhanced endocytosis. To further elucidate the cellular uptake mechanism of zwitterionic nanocages, we employed a range of endocytosis inhibitors. As shown in Fig. 3l, treatment with amiloride hydrochloride (AM) exhibited negligible inhibitory impact on cellular uptake, indicating that macropinocytosis may not be an important pathway for the assimilation of zwitterionic nanocages. Notably, treatment with chlorpromazine (CPZ, a clathrin inhibitor), resulted in a substantial decline in cellular uptake. Furthermore, the caveolae-mediated pathway was also instrumental in the endocytosis of zwitterionic nanocages, as evidenced by the marked reduction in the endocytosis when treated with inhibitors, genistein (Geni) and β-cyclodextrin (β-CD). Above all, through clathrin-dependent and caveolae-mediated endocytosis, zwitterionic nanocages could facilitate the effective penetration of peroxidase to break through the eye barriers (Fig. 3m).

To investigate potential organelle-specific effects, we incubated RPE cells with Cy5.5 labeled Z(CAT) for 6 hours and performed mitochondrial co-localization analysis using a mitochondrial tracker. Confocal imaging revealed minimal mitochondrial accumulation of the nanocages (Supplementary Fig. 8), indicating that their therapeutic mechanism does not rely on direct mitochondrial targeting. Instead, the nanocage predominantly localized to the cytosol and peri-mitochondrial space, a critical region for ROS diffusion and redox signaling. This spatial distribution is particularly advantageous for catalase activity, as it positions the therapeutic agent near the sites of ROS generation while allowing for effective diffusion throughout the cytosol. To further characterize the cellular response to treatment, we analyzed the activation of endogenous antioxidant pathways in oxidatively stressed RPE cells after Z(CAT&SOD) treatment using qPCR. RPE cells were firstly treated with 200 μg/mL SI for 6 hours to induce oxidative stress, followed by treatment with free CAT&SOD or Z(CAT&SOD) for additional 6 hours. The qPCR analysis revealed that Z(CAT&SOD) treatment significantly downregulated the Nrf2 pathway and its downstream targets (e.g. GPX1, NQO1, GCLC, HMOX-1) (Supplementary Fig. 9a). This oxidative stress induction was effectively attenuated by both free CAT/SOD and the zwitterion-protected formulation, with the latter exhibiting superior protective efficacy, likely due to enhanced enzyme stability and improved cellular delivery. To further evaluate mitochondrial protection, we analyzed mitochondria integrity in stressed RPE cells after Z(CAT&SOD) treatment (Supplementary Fig. 9b). Although the nanocage does not directly localize to mitochondria, its cytosolic ROS-scavenging activity effectively preserved mitochondrial membrane potential and reduced secondary mitochondrial ROS production. These findings highlight the significant organelle-protective mechanism, reinforcing the therapeutic potential of our platform.

Moreover, considering the complex enzymatic metabolic reactions in vivo, we also assessed whether the zwitterionic nanocages could protect peroxidase from enzymatic degradation. To simulate the in vivo degradation environment, we exposed Z(SOD), Z(CAT), free SOD and free CAT to proteinase K at 37 °C. After 3 hours, free SOD retained only 51% enzymatic activity and free CAT only 39%, with both activities nearly vanishing after 12 hours. In contrast, Z(SOD) maintained 83% enzymatic activity, and Z(CAT) retained 74% even after 12 hours (Fig. 3n). We further evaluated the enzymatic stability of Z(SOD), Z(CAT), and their free counterparts under different storage conditions (4 ℃, 25 ℃ and 37 ℃). While free enzymes exhibited rapid activity loss at 25 ℃ and 37 ℃, the zwitterionic nanocages-encapsulated enzymes retained most of their initial activity for four weeks, indicating their superior protective effect of zwitterionic nanocage (Supplementary Fig. 10). These results demonstrated that zwitterionic nanocages effectively protect protein activity, ensuring that SOD and CAT function as peroxidase in scavenging free radicals within complex physiological environment.

Intravitreal injection of Z(CAT&SOD) for treatment of acute retinal injury
Motivated by the permeability and stability of enzyme provided by zwitterionic nanocages, we aimed to explore the therapeutic effect of enzymatic cascade in eradicating ROS within the retina. We firstly induced ARI in mice using SI on Day 0 and randomly divided them into four groups for timely treatments: Untreated (1×PBS, 2 μL), Z(CAT) (2 mg/mL, 80000 U/g, 1 μL), Z(SOD) (2 mg/mL, 50000 U/g, 1 μL), and Z(CAT&SOD) (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL). In addition, healthy mice were used as the positive control. Three hours after SI injection, we intravitreally administrated PBS, Z(CAT), Z(SOD), or Z(CAT&SOD) into each group. The retina was evaluated using retinal imaging at one- and two-weeks post-injection (Fig. 4a). As shown in Fig. 4b and Supplementary Fig. 11, PBS- or Z(SOD)-injected mice developed progressive subretinal drusen-like deposits, secondary vascular inflammation, widespread retinal cell apoptosis, and atrophy over time. In contrast, Z(CAT) treated mice exhibited a significant reduction in subretinal drusen-like deposits, milder vascular inflammation and improved retinal cells survival. Surprisingly, the retina of mice treated with Z(CAT&SOD) displayed nearly no subretinal drusen-like deposits, healthy retinal vasculature without edema, clear retinal layers, and no signs of atrophy. The retinas of mice treated with Z(CAT&SOD) intravitreal injections were even comparable to those of healthy retinas. Furthermore, zwitterionic nanocages without peroxidase demonstrated nearly no therapeutic effect on the SI-induced ARI model.

Two weeks post-treatment, we further performed hematoxylin and eosin (H&E) staining to assess the structure of the retinal layers. In the Z(CAT&SOD) group, the retinal layers were well-preserved, with high cell density in both the INL and ONL, and clearly defined photoreceptor and RPE layers. In contrast, mice treated with PBS or Z(SOD) showed severe degeneration, including almost complete loss of the INL, photoreceptor layer, and RPE layer. The ONL had sparse cells, reduced thickness, and overall retinal thinning. A blinded evaluation based on fundus imaging and OCT were conducted two weeks after treatment (Fig. 4c). Under the influence of SI, both the Untreated and Z(SOD) groups had retinal scores of less than 1, indicating severe damage. In contrast, the Z(CAT&SOD) group showed scores comparable to a healthy retina, with significant improvement over the Z(CAT) group, which exhibited only partial relief. Additionally, we measured retinal thickness using Image J software, with results consistent with the scoring system. The Z(CAT&SOD) nanocages, coated with zwitterionic polymers, effectively inhibited retinal atrophy caused by oxidative stress, with significant differences observed compared to the other treatment groups (Fig. 4d). Furthermore, we assessed the visual function of mice using ERG. Mice injected with PBS and Z(SOD) exhibited severe visual impairment, with their visual function reduced to one-third of normal levels. Mice injected with Z(CAT) also showed compromised visual function, with a reduction to two-thirds of normal levels. However, mice treated with Z(CAT&SOD) displayed nearly no visual impairment (Fig. 4f, 4g and Supplementary Fig. 12). The restoration of ERG amplitudes in Z(CAT&SOD)-treated mice, alongside intact ONL and RPE morphology, provides direct evidence that photoreceptor-targeted ROS neutralization is both a mechanistic requirement and an alternative therapeutic strategy for preserving retinal function.

Recognizing that significant optic nerve degeneration in ARI model is associated with chronic inflammation caused by oxidative stress40,41, we then investigated the regulatory effects of an antioxidant enzyme cascade on inflammation during oxidative stress resistance. One week after intravitreal injection of PBS, Z(CAT), Z(SOD), or Z(CAT&SOD), we assessed retinal apoptosis and inflammatory pathway responses using immunofluorescent staining, flow cytometry and enzyme-linked immunosorbent assay (ELISA). DCFH-DA labeling experiments demonstrated that Z(CAT&SOD) significantly reduces ROS levels in the retina, indicating that Z(CAT&SOD) successfully eliminating ROS (Supplementary Fig. 13a). TUNEL analysis further revealed that Z(CAT&SOD) effectively reduced SI-induced retinal damage by inhibiting photoreceptor apoptosis, particularly in the ONL and INL (Fig. 5a). To explore the molecular mechanisms underlying SI-triggered retinal inflammation and cell death, we examined the expression and distribution of NF-κBp65, IL-1β, and HMGB1 in retinal tissues following different treatments. As illustrated in Fig. 5b-5d, supplementation with Z(CAT&SOD) resulted in a significant decrease in the inflammatory markers of the NF-κB-HMGB1-ILs pathway, indicating that Z(CAT&SOD) effectively mitigated inflammation. Moreover, chronic inflammation often triggers the release of VEGF family proteins, increasing vascular permeability and potentially leading to vascular swelling and secondary vascular abnormalities40. Therefore, we also evaluated VEGF and ZO-1 protein expression in retinal tissue. As anticipated, the untreated group and those receiving only Z(SOD) or Z(CAT) showed decreased ZO-1 and increased VEGF levels in the retina, suggesting that a malignant microenvironment characterized by inflammation and angiogenesis. This increased vascular permeability disrupts the eye’s immune microenvironment, creating a vicious cycle. In contrast, the Z(CAT&SOD) group displayed a more intact retinal structure and lower inflammation levels, comparable to the healthy retinal microenvironment. This dual-enzyme approach not only mitigates inflammation but also helps maintain the integrity of the retinal structure by reducing vascular permeability and preventing secondary vascular abnormalities (Supplementary Fig. 13b and 13c).

Additionally, we evaluated the proportions of M1 and M2 macrophage subpopulations in retinal tissue using flow cytometry, and measured cytokine levels in the retina by ELISA following different treatments. As shown in Fig. 5e-5g, there was a marked increase in CD206+F4/80+ cells in retinas treated with Z(CAT&SOD) and Z(CAT), whereas a decline was observed in those treated with PBS and Z(SOD). Notably, the healthy group, along with the Z(CAT) and Z(CAT&SOD) groups, predominantly exhibited CD206+F4/80+ macrophages, indicative of an anti-inflammatory M2 phenotype. In contrast, the untreated control group and the Z(SOD) group were mainly characterized by CD80+F4/80+ cells, reflecting a pro-inflammatory M1 phenotype. Furthermore, treatment with Z(CAT) and Z(CAT&SOD) resulted in a significant reduction in retinal inflammatory factor levels, bringing the close to those seen in healthy mice. This was in stark contrast to the untreated and Z(SOD) groups, which maintained higher levels of inflammation (Fig. 5h-5k). Importantly, Z(CAT&SOD) demonstrated a more pronounced inhibitory effect on both inflammatory pathways and inflammatory cells and cytokines compared to Z(CAT) alone, indicating that the enzymatic cascade reaction was more effective at eliminating ROS in the retina. Therefore, the enzymatic cascade reaction mediated by Z(CAT&SOD) synergistically reduced key inflammatory markers such as NF-κBp65, IL-1β, and HMGB1 in retinal tissues while promoting macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotypes. These findings highlight Z(CAT&SOD) as a promising therapeutic candidate for retinal disorders involving oxidative stress and inflammation (Fig. 5l).

Z(CAT&SOD)-based eye drop for acute retinal injury treatment
Motivated by the exceptional permeability of the zwitterionic nanocage, we endeavored to treat GA in a non-invasive manner using eye drops. Considering the unique barrier system of the eye, we tested the capacity of nanocages with distinct charge properties to penetrate the conjunctival and scleral barriers. Among them, Z6 was cationic, Z7 was anionic, while Z5 and Z8 were zwitterionic based on SBMA and Z8 crosslinked with BIS and the initiator APS. Six hours post-application of eye drops in different formulation of Z5 to Z8, the permeability of Z8 obviously surpassed that of both free CAT-FITC and the Z5 to Z7 eye drop groups, with the fluorescence signal of CAT-FITC evident in the conjunctiva, sclera (C/S) and especially in various layers of the retina (R) (Fig. 6a and 6b). Consequently, the zwitterionic SBMA-constructed nanocages (Z8) exhibit superior tissue permeability compared to their cationic or anionic counterparts, which make them an ideal choice for formulating peroxidase eye drops and was selected for further investigation for ocular permeability.

We further examined the permeability and localization of the selected Z(CAT-FITC) (Z8) in the eye. Following the administration of Z(CAT-FITC) and free CAT-FITC eye drops to mice, eye samples were collected at intervals of 1, 3, 6, 9 and 12 hours to measure total fluorescence intensity within the eyeballs. As shown in Fig. 6c, Z(CAT-FITC) achieved a peak eyeball penetration of 9.32% at 6 hours post-administration, while in which of free CAT-FITC was 0.54%. Even after 12 hours, Z(CAT-FITC) maintained a penetration rate of 4.09%, outperforming the free CAT-FITC (0.12%). Confocal imaging of whole-eye sections revealed that the zwitterionic nanocages might penetrate the conjunctival-scleral barrier, reaching the choroid and retina (Fig. 6d). Quantitative analysis of CAT-FITC fluorescent signals revealed a conjunctiva/sclera-to-retina ratio of 6.67:1 (Supplementary Fig. 14a), suggesting efficient transscleral delivery. In addition, pharmacokinetic profiling following topical administration of Z(CAT-FITC) eye drops (measured at 1, 3, 6, 9, and 12 h) showed that Z(CAT-FITC) exhibited significantly higher blood accumulation than free CAT-FITC at 6 hours (Supplementary Fig. 14b), indicating the existence of a blood mediated retinal delivery pathway. Additionally, to more directly determine the ocular permeability of zwitterionic peroxidase by measuring the content of peroxidase, we monitored CAT enzyme activity in retinal tissue at different time points (1, 3, 6, 9 and 12 hours) post-administration using a catalase activity assay kit. Results indicated significantly higher CAT enzyme activity in the retina of the Z(CAT) group compared to free CAT group during the period from 3 hours to 12 hours (Supplementary Fig. 14c). This finding prompted us to reflect that the zwitterionic nanocages not only enhanced the permeability of peroxidase in the eye, but also protected it from degradation during delivery, thereby preserving its enzymatic activity. Therefore, it clearly demonstrated that zwitterionic peroxidase nanocages could effectively facilitate peroxidase penetration within complex structural barriers without inactivation.

Leveraging the effective ocular permeation and enzyme activity protection provided by zwitterionic nanocages for peroxidase, we attempted to use this eye drop for treating ARI model. To benchmark against the clinical gold standard treatment2, we established a positive control group that receive oral lutein supplementation. After intravenous injection of SI, subjects were randomly divided into six groups: Untreated (1×PBS eye drop, 2 μL twice daily for a week), CAT&SOD eye drops (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL twice daily for a week), CAT&SOD intravitreal injection (same concentration, single 2 μL injection), Z(CAT&SOD) eye drops (same concentration, 2 μL twice daily for a week), Z(CAT&SOD) intravitreal injection (same concentration, single 2 μL injection), and lutein oral administration (0.5 μg lutein per day for one week). Retinal evaluations were conducted using imaging systems one and two weeks later (Supplementary Fig. 15a). Unlike the PBS group and the uncoated groups (CAT&SOD i.v.t and CAT&SOD e.d.), which exhibited severe subretinal drusen-like deposits, secondary vascular abnormalities, and reduced retinal thickness, the Z(CAT&SOD) eye drops group demonstrated significantly decreased subretinal drusen-like deposits, reduced vascular abnormality, and maintained normal retinal thickness (Figure 6e and Supplementary Fig. 15b). Clinical scoring indicated that Z(CAT&SOD) eye drop effectively relieved symptoms of mice with ARI and maintained the thickness of RPE and retina (Supplementary Fig. 15c and 15d), with therapeutic effect comparable to intravitreal injections over time. ERG tests conducted two weeks post-treatment revealed that Z(CAT&SOD) effectively protected photoreceptors and ON-bipolar cells, showing no significant difference compared to intravitreal injection (Fig. 6f, Supplementary Fig. 15e and 15f). H&E staining results two weeks after treatment confirmed that Z(CAT&SOD) eye drop maintained cell density and thickness across all retinal layers (Fig. 6e). In contrast, the untreated group and the uncoated groups experienced significant reductions in retinal thickness and cell density, particularly in the ONL and photoreceptor layer, correlating with ERG findings of visual impairment. These effects were comparable to those seen with intravitreal injection, indicating that zwitterionic nanocage effectively promoted enzyme penetration across the ocular barrier, facilitating ROS clearance and protecting retina and vascular tissues. Interestingly, the mice with ARI supplemented with daily oral lutein exhibited some improvement in retinal atrophy compared to the untreated group and those supplemented with unmodified peroxidase. This was evidenced by a reduction in subretinal drusen-like deposits, preservation of retinal thickness, and mitigation of retinal cell apoptosis (Fig. 6e, 6f and Supplementary Fig. 16). This indicated that effective lutein supplementation could indeed alleviate the progression of dry macular degeneration. However, when compared to the coated peroxidase supplementation, lutein’s therapeutic effect was less satisfactory. Mice treated with Z(CAT&SOD) via intravitreal injection or eye drops exhibited the least retinal deposition, the mildest secondary vascular abnormalities, optimal maintenance of retinal thickness, and best preservation of retinal optic nerve function, which may be attributed to the limited ROS-scavenging ability of lutein and its absorption.

Given the high sensitivity of ocular tissues, we conducted a thorough biocompatibility assessment of Z(CAT&SOD) to ensure its safety for long-term use. A battery of ophthalmic evaluations including slit-lamp microscope, OCT, ERG and intraocular pressure (IOP), were performed on mice four weeks after treatment with Z-polymer and Z(CAT&SOD) eye drop. As illustrated in Supplementary Fig. 17a and 17b, the corneas of treated mice remained clear and intact, with no observable differences compared to healthy controls. Moreover, prolonged administration of Z-polymer and Z(CAT&SOD) did not obviously alter tear secretion (Supplementary Fig. 17c). Corneal OCT imaging confirmed that neither formation affected corneal thickness (Supplementary Fig. 17d and 17e), while fundus imaging and retina thickness measurements revealed no structural abnormalities (Supplementary Fig. 17f to 17i). In addition, IOP values remained stable after long-term treatment, indicating that the Z(CAT&SOD) eye drop dose not induce ocular hypertension or increase the risk of glaucoma (Supplementary Fig. 17j). To assess systemic toxicity, we performed histological analysis of major organs (heart, liver, spleen, lung, kidney and eye) via H&E staining. Tissues form Z-polymer and Z(CAT&SOD)-treated mice exhibited nearly no pathological changes compared to healthy mice (Supplementary Fig. 17k). Additionally, hematological analysis of blood samples confirmed that all parameters remained in the normal range, indicating negligible systemic toxicity even after prolonged subconjunctival retention (Supplementary Fig. 17l). Therefore, these findings demonstrate that both Z-polymer and Z(CAT&SOD) exhibit excellent biocompatibility after long-term administration.

Methods
Materials, cells and animals
The acrylamide, acrylic acid, sulfobetaine methacrylate (SBMA), bisacrylamide (BIS; cross-linker), TMEDA, sodium iodate (SI), H2O2 and fluorescein sodium were purchased from Aladdin Industrial Co. (Shang hai, China). The Catalase Assay Kit and the Superoxide Dismutase Assay Kit were purchased form Beyotime Biotechnology co. (Shang hai, China). Catalase (CAT) was obtained from Bidepharm co. (Shang hai, China), while Superoxide dismutase (SOD) was obtained from Solarbio co. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin, were purchased from Life Technologies Co. (New York, USA). E-cadherin antibody (ab40772, Abcam, Dilution:1:10000), IL-1β antibody (ab234437, Abcam, Dilution:1:50), NF-κB antibody (ab203568, Abcam, Dilution:1:50), HMGB1 antibody (ab79823, Abcam, Dilution:1:250), VEGFA antibody (ab52917, Abcam, Dilution:1:500), ZO-1 antibody (ab221547, Abcam, Dilution:1:100) were purchased from Abcam. The Elisa kits of mouse IL-1β (88-7013A-86)，mouse IL-6 (88-7064-88), mouse IL-12P70 (88-7121-88), mouse IFN-γ (88-7314-88) and mouse TNFα (88-7324-88) were purchased from Thermo Fisher Scientific. PE/Cyanine7 anti-mouse F4/80 Antibody (Biolenged, Catalog:123113), PE anti-mouse CD80 Antibody (Biolenged, Catalog: 104707), FITC anti-mouse CD11b Antibody (Biolenged, Catalog: 101205), APC anti-mouse CD206 (MMR) Antibody (Biolenged, Catalog: 141707), Purified anti-mouse CD16/32 Antibody (Biolenged, Catalog: 101301) were purchased from BioLegend co. The DCFHDA kit (S0033M), Mito-Tracker Green Probe (C1048), Mitochondrial membrane potential assay kit with JC-1 (C2006) and TUNEL kit (C1091) were purchased from Beyotime Biotechnology co. (Shang hai, China). Human conjunctival epithelial cells (HConEpiC) and adult retinal pigment epithelial cell line-19 (RPE19) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in DMEM containing 1% penicillin/streptomycin and 10% FBS at 37 °C in 5% CO2. Seven-week-old female C57BL/6 mice were purchased from Nanjing Pengsheng Biological Technology Co. The animals were acclimated in SPF environment for one week prior to conducting follow-up experiments. The mice were maintained under a 14-hour light/10-hour dark cycle, with temperatures ranging from 18 to 23 °C and humidity levels maintained between 40 and 60%. The mice were housed in microisolator cages to ensure the containment of pathogens. All animal procedures complied with relevant laws and were approved by the Institutional Animal Care and Use Committee of Soochow University (No. ECSU-2019000198).

Construction and evaluation of acute retinal injury mice model
Sodium iodate (SI) was prepared at a concentration of 2 mg/mL in sterile phosphate-buffered saline (PBS) and stored at 4 °C until use. SI was administered via tail vein injection to induce the model. Control mice received equivalent volumes of PBS in accordance with standard protocols. To visualize the formation of CNV lesions in the eyes, female or male mice were intraperitoneally injected with 0.1 ml of 2% fluorescein sodium. Fundus images and OCT images were captured using a Phoenix Micron Ⅳ multifunctional high-resolution retinal imaging system (Phoenix, USA). Additionally, ERG was performed under scotopic condition to examine the optic function of both mice with acute retinal injury and healthy mice. The A waves and B waves were recorded, quantified and analyzed to evaluate retinal function.

In Vivo Imaging (Fundoscopy, OCT and ERG): All imaging was performed using a Micron IV retinal imaging system (Phoenix Research Labs) coupled with a high-resolution OCT module and ERG module. Prior to imaging, mice were anesthetized via intraperitoneal injection of ketamine (80 mg/kg) and xylazine, followed by pupil dilation with with topical administration of 1% tropicamide.

Fundoscopy: Color fundus images were captured using the integrated color fundus camera under standard white light illumination.

Spectral-Domain OCT (SD-OCT): Retinal cross-sectional scans were acquired using the scanning protocol centered on the optic nerve head. Subsequent quantification of retinal layer thicknesses was performed using image J system.

Electroretinography (ERG): Full-field ERG recording was performed in accordance with the International Society for Clinical Electrophysiology of Vision (ISCEV) standard protocol for rodents. Following overnight dark adaptation (>12 hours), mice were anesthetized as above and their pupils were dilated under dim red illumination. Scotopic (rod-dominated) responses were firstly recorded using a series of flashes with increasing intensities (3 log cd·s/m2). For photopic (cone-mediated) responses, mice were light-adapted for 10 minutes under a 30 cd/m2 background before recording responses to a 1.0 log cd·s/m2 flash stimulus. ERG waveform analysis included: a-wave amplitude (baseline to trough of initial negative deflection), b-wave amplitude (a-wave trough to the peak of subsequent positive wave), implicit times for both components. All recordings and analyses were performed using the integrated ERG module of the Micron IV retinal imaging system.

Measurement of ROS activity and cell apoptosis in the retina of mice with acute retinal injury
ROS production in the retinas of mice with acute retinal injury was quantified using a 2’,7’-dichlorofluorescein diacetate (DCFHDA) assay kit, while cell apoptosis was measured using a TUNEL assay kit, following established protocols. Briefly, frozen eye tissue sections were fixed with eyeball fixation solution for 15 min, washed with PBS three times, permeabilized using freshly prepared 0.1% Triton X-100, and incubated with terminal deoxynucleotidyl transferase solution for 60 min at 37°C. Subsequently, tissues were incubated with DCFH-DA and DAPI at 37°C for another 30 min. The fluorescence intensity was examined by confocal microscope (Zeiss, USA).

Determination of CAT and SOD in mice with acute retinal injury
Retinas were extracted from the eyeballs of SI-mice and lysed using a sonic disruptor in Western and IP sample lysates (500 μL) at 0°C. The supernatant was then centrifuged, and 100 microliters were used for CAT and SOD assay tests. Additionally, RNA expression levels of CAT and SOD were assessed by qPCR analysis. Retinal cells isolated from mice with acute retinal injury were lysed with Trizol, and the RNA was diluted to a final concentration of 200 ng/μL for analysis. Primer and probe sequences were as follows: CAT forward primer: AAATGCTTCAGGGCCGCCTT; CAT reverse primer: GTAGGGACAGTTCACAGGTA; SOD forward primer: AACAATGGTGAAGGCTGTTGCTGT; SOD reverse primer: TTGTGCATGCACATGGAACCCATG; GAPDH forward primer: GTGACGTTGACATCCGTAAAGA; GAPDH reverse primer: GTSAACAGTCCGCCTAGAAGCAC. These primes were synthesized by Servicebio.

Preparation of Z(CAT) nanocages
Under continuous low-speed stirring, a 20 mg/mL CAT solution was added gradually into a round-bottom flask. Acrylamide, acrylic acid, SBMA, bisacrylamide (cross-linker) and TMEDA were sequentially added with different molar ratio, with each addition followed by 10 minutes of stirring to ensure thorough mixing. After all reagents were added, the solution was stirred continuously for an additional two hours. Unreacted small molecules were removed by dialysis to terminate the reaction. Finally, the concentration and enzymatic activity of Z(CAT)s were determined using the BCA protein assay kit and the CAT activity assay kit. The preparation method of Z(CAT&SOD) nanocages is the same as that mentioned above. And enzymatic activity of SOD was determined using the SOD activity assay kit.

Retina penetration of intravitreal injection with Z(CAT)s
To assess the penetration of Z(CAT)s through the inner limiting membrane (ILM) barrier into the retina, CAT was labeled with FITC. Z(CAT-FITC) nanocages (Z5 to Z8, with CAT concentration at 2 mg/mL, 2 μL) were intravitreally injected into the eyes of anesthetized mice. Six hours post-injection, the mice were euthanized, and their eyes were harvested to determine the location of CAT-FITC using a confocal microscope (Zeiss, USA). ImageJ was used for semi-quantitative analysis of FITC fluorescence within the retina.

Retina penetration of Z(CAT)s eyedrops
To identify the optimal Z(CAT) formation for penetrating ocular barriers, we evaluated FITC fluorescence in the retina after administration of Z5(CAT-FITC) to Z8(CAT-FITC) eyedrops in mice. Each eyedrop contained Z-CAT nanocapsules (CAT: 2 mg/ml, 2 μL) or free CAT (CAT: 2 mg/ml, 2 μL). The eyedrops were applied to anesthetized mice, and the animals were euthanized at different time points (1, 3, 6, 9, 12 hours post administration). Retina in different groups were collected f at corresponding time points, homogenized at 4°C to isolate proteins, and the fluorescence intensity of CAT-FITC was measured using a microplate reader (Thermo Fisher, USA). Additionally, the concentration of CAT in mouse eyes was determined using a CAT-specific assay kit.

Immunocytochemistry analysis of tight junction proteins
HConEpiCs were cultured to form a corneal epithelial layer and then co-cultured with either CAT or Z(CAT) at a concentration of 50 μg/mL. Six hours later, the cells were washed with PBS, fixed in 4% paraformaldehyde at 4°C for 20 min, and permeabilized with 0.5% TritonX-100 at room temperature for 20 min. The cells were then blocked with 2% bovine serum albumin (BSA) in PBS at room temperature for 1 hour. Following blocking, they were incubated overnight at 4°C with the primary antibody, anti-E-cadherin (1:500). After washing away unbound primary antibodies, the cells were further incubated with the FITC-conjugated secondary antibody (1:1000) at room temperature for 1 hour. Fluorescence imaging was conducted using the confocal microscope (Zeiss, USA).

Detection of protein expression related to the endogenous antioxidant system
RPE cells were incubated with Sodium iodate (at an incubation concentration of 200 ug/mL) for 6 hours to induce the oxidative stress state of RPE cells. Then, we divided them into three groups: untreated, free CAT&SOD, and Z(CAT&SOD). After another 6-hour incubation, we detected the endogenous antioxidant system status of RPE cells. Additionally, RNA expression levels of HMOX1, GPX1, Nrf, NQO1 and RPLP0 were assessed by qPCR analysis. RPE cells after different treatments were lysed with Trizol, and the RNA was diluted to a final concentration of 200 ng/μL for analysis. Primer and probe sequences were as follows: HMOX1 forward primer: CCAGGCSAGAGAATGCTGAGTTC; HMOX1 reverse primer: AAGACTGGGCTCTCCTTGTTGCE; GPX1 forward primer: GTGCTCGGCTTCCCGTGCAAC; GPX1 reverse primer: CTCGAAGAGCATGAAGTTGGGC; Nrf forward primer: CACATCCAGTCAGAAACCAGTGGP; Nrf reverse primer: GGAATGTCTGCGCCAAAAGCTG; NQO1 forward primer: CCTGCCATTCTGAAAGGCTGGT; NQO1 reverse primer: GTGGTGATGGAAAGCACTGCCT; GCLC forward primer: GGAAGTGGATGTGGACACCAGA; GCLC reverse primer: GCTTGTAGTCAGGATGGTTTGCG β-actin forward primer: GTCTCCETCTGACTTCAACAGCG; β-actin reverse primer: ACCACCCTGTTGCTGTAGCCAA; RPLP0 forward primer: TGGTCATCCAGCAGGTGTTCGA RPLP0 reverse primer: ACAGACACTGGCAACATTGCGG. These primes were synthesized by Servicebio.

Cellular endocytosis and mitochondrial co-localization tests of Z(CAT) nanocages
Cellular uptake of Z(CAT) nanocages by HConEpiCs was assessed using flat-bottom, 24-well plates, with a seeding density of 1×105 cells per well. The cells were exposed to 50 µg/mL of Z(CAT) or free CAT in serum-free medium. After incubation for 3, 6 and 12 hours, the medium was removed, and the cells were washes with PBS. Then, the cells were fixed with 4% polyformaldehyde and stained with DAPI for 30 minutes. Fluorescence intensity was visualized and analyzed by the confocal microscope (Zeiss, USA).

Based on this, we further investigated whether the two-ion-type nanocages could be co-localized with the mitochondrial organelles. The RPE cells were exposed to 50 µg/mL of Z(CAT) or free CAT in serum-free medium. After incubation for 3, 6 and 12 hours, the medium was removed, and the cells were washes with PBS. Then, the cells were incubated with Mito-Tracker Green Probe for 30 min. Next, the Mito-Tracker Green staining solution was removed and the fresh cell culture medium pre-warmed at 37°C was added. Subsequently, the samples were observed using a confocal microscope (Zeiss, USA).

Mitochondrial damage detection
RPE cells were incubated with Sodium iodate (at an incubation concentration of 200 µg/mL) for 6 hours to induce the oxidative stress state of RPE cells. Then, we divided them into three groups: untreated, free CAT&SOD, and Z(CAT&SOD). After another 6-hour incubation, we detected the endogenous antioxidant system status of RPE cells. Then, the culture medium was removed and the cells were washed with PBS, and then incubated with JC-1 (Mitochondrial membrane potential assay kit) at staining working solution for 20 minutes. After the incubation, the supernatant was removed and the cells were washed twice with the JC-1 staining buffer. Then, the cell nuclei were stained with DAPI for 15 minutes, and then observed using a confocal microscope (Zeiss, USA).

TEER measurement assay
HConEpiCs were seeded into Transwell insert chambers with the density of 5×104 cells per well, and the resistivity (R) of the cell layer was measured every two days using an Epithelium R/V Meter (RE1600; Beijing King Technology Co. Ltd.). TEER values were calculated using the standard method. Once the TEER value stabilized, CAT or Z(CAT) with equivalent CAT concentration of 50 μg/mL was added to the insert chambers for 6 hours, and TEER values were recorded at 0, 6 and 24 hours.
The following formula was used to calculate TEER (ohm·cm2).
TEER (ohm·cm2) = (R1 -R0) × 0.33;
R1 is the TEER value of inserts with cells;
R0 is the TEER value of inserts without cells;
The insert membrane area in the plate was 0.33 cm2.

Intravitreal injection and eyedrop application of Z(CAT&SOD) nanocages in mice with acute retinal injury
Initially, a dry macular lesion model was induced in mice via intravenous injection of NaIO3. Three hours post-injection, the mice were anesthetized and randomly assigned to four groups: Untreated (1×PBS, 2 μL), Z(CAT) (2 mg/mL, 80000 U/g, 1 μL), Z(SOD) (2 mg/mL, 50000 U/g, 1 μL), and Z(CAT&SOD) (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL). The respective treatments were administered via intravitreal injections. Live fundus evaluations, including fundus imaging, FFA, OCT and ERG, were conducted one- and two-weeks post-treatment. For the evaluation of topical eyedrop treatment, the mice were randomly divided into six groups: After intravenous injection of SI, subjects were randomly divided into six groups: Untreated (1×PBS eyedrop, 2 μL twice daily for a week), CAT&SOD eye drops (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL twice daily for a week), CAT&SOD intravitreal injection (same concentration, single 2 μL injection), Z(CAT&SOD) eye drops (same concentration, 2 μL twice daily for a week), Z(CAT&SOD) intravitreal injection (same concentration, single 2 μL injection), and lutein oral administration (0.5 μg lutein per day for one week). Live fundus evaluations were conducted again at one- or two-weeks post-treatment. Two weeks after different treatments, eyes from different groups were collected for histological examination and immunofluorescence staining using TUNEL kit and IL-1β, ZO-1, NF-κB, HMGB1 and VEGF antibodies.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents an approach to mitigate oxidative stress–induced retinal degeneration through co-encapsulation of catalase (CAT) and superoxide dismutase (SOD) in polymeric nanocages. While antioxidant supplementation in retinal disease is a well-explored area, the co-delivery of these two enzymes within a single carrier represents a technically interesting strategy that could have therapeutic potential. However, several major concerns must be addressed before the manuscript can be considered for publication.\nFirst, the nanocages are composed of acrylamide and bisacrylamide-based polymers, materials known to have neurotoxic properties. The manuscript does not sufficiently address the safety or biocompatibility of these polymers for intraocular use, which is essential for translational relevance. Additionally, although the title refers to an eye drop–based therapy, the data presented are limited to intravitreal delivery; this discrepancy is misleading and should be clarified.\nA more significant concern lies in the conceptual accuracy of several claims. The manuscript contains multiple errors in ocular definitions and physiological interpretations, which diminish confidence in the scientific rigor. For example, retinal ganglion cells are not the primary cells affected in geographic atrophy (GA); the a-wave of the ERG is primarily associated with photoreceptor (particularly rod) activity, not cones; and the b-wave is typically driven by bipolar cells rather than directly by rods. Furthermore, evaluating choroidal neovascularization (CNV) as an outcome measure is not appropriate for GA, which is a non-neovascular form of age-related macular degeneration. These inaccuracies suggest a need for more careful literature review and refinement of disease-specific understanding.\nLastly, while the sodium iodate model does recapitulate oxidative stress–related RPE damage, it reflects an acute injury rather than the chronic, progressive degeneration seen in human GA. Therefore, while the protective effects observed are encouraging, their relevance to GA pathophysiology should be presented more cautiously.\nOverall, the concept may be of interest if reframed with appropriate disease context, technical clarifications, and stronger emphasis on translational considerations.", "reviewer_2": "The manuscript identifies a critical deficit in retinal peroxidase activity (specifically catalase and superoxide dismutase) during GA progression, which exacerbates oxidative stress, activates the NF-κB-HMGB1-ILs inflammatory pathway, and drives retinal cell apoptosis. To counteract this, the authors developed charge-shielded zwitterionic nanocages (Z(CAT&SOD)) that protect enzyme activity, enhance permeability across ocular barriers, and facilitate delivery via clathrin-dependent and caveolae-mediated endocytosis. The study is well-organized and provides comprehensive insights into the potential of this innovative therapeutic strategy. The authors shoud address the following questions to further enhance the quality of the manuscript:\n1. As a nano-delivery system intended for ocular applications, the biosafety of zwitterionic nanocages (Z (CAT&SOD)) constitutes a fundamental prerequisite for clinical translation; however, existing research lacks systematic evaluation. It is need to conduct supplementary long-term safety assessments, encompassing evaluations of ocular irritation, intraocular pressure variations, corneal integrity, corneal transparency, and retinal pathology.\n2. It is necessary to elucidate the molecular basis of zwitterionic charge shielding: to investigate the dynamic changes in surface charge (as monitored by real-time zeta potential measurements) of the CAT&SOD complex in the vitreous microenvironment.\n3. Supplementary stability data for dosage forms are required: The particle size distribution of the ophthalmic solution, enzyme activity retention rate (for a minimum of 1 month), and the presence of nanocage aggregation (e.g., through long-term DLS monitoring) should be assessed under various storage conditions (4°C, room temperature, 37°C).\n4. It is necessary to include negative controls, such as a control group utilizing blank zwitterionic nanocages (enzyme-free), to eliminate potential confounding effects of the material itself on inflammation or retinal structure.\n5. The study employs a sodium iodate-induced model of dry macular degeneration, which phenotypically approximates the human dry macular degeneration model. Please elucidate the underlying mechanisms involved.\n6. Regarding the concern of the barrier integrity of tight junction proteins, the authors need tested the longer incubation with Z (CAT&SOD) for 6 hours. As the corneal epithelial cells are the protection of the eyes, it still brings concern about the use of Z (CAT&SOD) in vivo, such as infection.\n7. Figure 5l fails to elucidate the mechanistic role of macrophages, which warrants supplementation regarding their functional mechanism in the treatment of dry macular degeneration with Z (CAT&SOD) therapy. Clarify how Z(CAT&SOD) drives M2 polarization (e.g., via reduced ROS levels inhibiting NF-κB, or direct modulation of macrophage metabolism).\n8. The drug loading and drug encapsulation efficiency of Z(CAT&SOD) should be reported.\n9. The rationale for using peroxidases (CAT and SOD) in GA therapy should be strengthened with literature support linking their deficiency to human GA: Cite clinical studies showing reduced CAT/SOD activity in retinas or aqueous humor of GA patients (vs. age-matched controls). Discuss how CAT/SOD deficiencies exacerbate oxidative stress in GA: For example, do they impair ROS clearance in RPE cells, leading to lipofuscin accumulation or mitochondrial DNA damage (as reported in human GA)? Compare with other antioxidant strategies (e.g., nanozymes, small-molecule ROS scavengers) to highlight the advantages of enzymatic cascades (e.g., specificity, biocompatibility).\n10. Minor point: Supplementary figures (e.g., Figure S1–S17) are not cited in the order they appear in the text, which hinders readability. For example, Figure S3 is referenced before Figure S2. The authors should reorder supplementary figure citations to align with their appearance in the main text, ensuring a logical flow.", "reviewer_3": "This study presents an interesting and potentially translationable strategy for the treatment of geographic atrophy by constructing a zwitterionic nanocage-based delivery system that enables non-invasive co-delivery of catalase (CAT) and superoxide dismutase (SOD) to the retina. The work demonstrates strong innovation, solid in vivo data, and a relatively in-depth mechanistic exploration. However, several scientific and mechanistic issues still need to be clarified or reinforced, and some aspects of data presentation require revision. I recommend accept this manuscript after addressing the following specific comments:\n1. The manuscript emphasizes the synergistic effect of CAT and SOD as an enzymatic cascade. However, there is no quantitative analysis confirming whether the combined use is superior to individual enzymes in terms of ROS scavenging efficiency (e.g., rate of radical elimination, half-life of ROS). It is recommended to include in vitro kinetic assays to validate whether this is a true cascade reaction or simply an additive effect.\n2. The authors propose that Z(CAT&SOD) eyedrops can penetrate the retina through both the conjunctiva–sclera–retina and conjunctiva–blood–retina routes. However, there is no quantitative analysis comparing the relative contribution of each route. I suggest performing quantitative tissue fluorescence distribution (e.g., FITC signal intensity in different ocular layers) or using pathway-specific inhibitors to confirm the predominant transport route.\n3. The suppression of inflammation is attributed mainly to ROS reduction, but it remains unclear whether CAT/SOD also exerts ROS-independent immunomodulatory effects, particularly on the NF-κB pathway. The authors are encouraged to explore whether changes in key signaling proteins (e.g., p65 phosphorylation) precede complete ROS clearance.\n4. Although the study shows that encapsulation preserves enzymatic activity under proteolytic stress, it would strengthen the claims to include structural or kinetic data (e.g., circular dichroism spectra, DLS, changes in Michaelis–Menten kinetics) to assess whether zwitterionic polymerization alters the enzyme’s conformation or access to the active site.\n5. The sodium iodate-induced model used in this study mimics acute oxidative damage and may primarily represent late-stage GA. Please discuss the limitations of this model in simulating the chronic progression of GA and whether the proposed therapeutic strategy could be effective in early-stage disease (e.g., before lipofuscin accumulation).\n6. While ERG measurements showed improved visual function after Z(CAT&SOD) treatment, the source of improvement (e.g., photoreceptor protection vs. retinal ganglion cell preservation vs. synaptic conduction) is not clearly discussed. Consider correlating specific ERG wave changes (e.g., a-wave vs. b-wave) with histological data to identify the principal site of action.\n7. The authors demonstrate M2 macrophage polarization and inflammation suppression following treatment, but there is no follow-up data on whether this immune modulation is reversible or potentially immunosuppressive. I recommend including 1–2 weeks of post-treatment follow-up to evaluate the persistence and safety of immune alterations.\n8. The manuscript is generally readable but contains some repetitive phrasing and grammatical issues (e.g., the phrase “has historically challenging to treat” in the Abstract is incorrect). Professional language editing by a native speaker or editing service is strongly advised.\n9. The figure legends are somewhat inconsistent in statistical notation. Please standardize the indication of significance (e.g., p<0.05, *p<0.01, etc.) and clearly state the statistical methods used in each figure legend."}, "author_response": "Responses to reviewer’s comments\nWe sincerely thank the reviewers for the valuable comments and suggestions. Below we have provided point-to-point responses to the comments and have revised the manuscript accordingly.\nReviewer #1:\nThe manuscript presents an approach to mitigate oxidative stress-induced retinal degeneration through co-encapsulation of catalase (CAT) and superoxide dismutase (SOD) in polymeric nanocages. While antioxidant supplementation in retinal disease is a well-explored area, the co-delivery of these two enzymes within a single carrier represents a technically interesting strategy that could have therapeutic potential. However, several major concerns must be addressed before the manuscript can be considered for publication.\n1. First, the nanocages are composed of acrylamide and bisacrylamide-based polymers, materials known to have neurotoxic properties. The manuscript does not sufficiently address the safety or biocompatibility of these polymers for intraocular use, which is essential for translational relevance. Additionally, although the title refers to an eye drop-based therapy, the data presented are limited to intravitreal delivery; this discrepancy is misleading and should be clarified.\nReply: Thanks so much for the reviewer’s suggestion. We fully agree that the safety profile of any intraocular or topical ophthalmic formulation is of utmost importance for its translational potential. While acrylamide and bisacrylamide monomers have been associated with neurotoxicity in their unpolymerized forms, it is important to emphasize that the polymerized forms used in our nanocage system are chemically stable macromolecules that are not expected to exert systemic toxicity under normal physiological conditions. To directly address this concern, we conducted a comprehensive 4- week ocular and systemic safety evaluation of both empty zwitterionic nanocages and Z(CAT&SOD) nanocages following topical ocular administration in healthy mice. The results showed that there were no significant abnormalities observed in any of the tested parameters. Both empty and enzyme-loaded nanocages were well-tolerated, with no signs of ocular irritation, no increase in IOP, no corneal or retinal damage, and no systemic toxicity (Figure R1). These findings were consistent with previous reports demonstrating that zwitterionic polymers exhibit excellent biocompatibility, low immunogenicity, and minimal protein adsorption, making them suitable for biomedical applications, including ocular drug delivery.\nFurthermore, we sincerely apologize for any confusion caused by our title regarding the administration method in this study. In this manuscript, intravitreal injection was used as a proof-of-concept to rigorously evaluate the therapeutic efficacy of enzyme-loaded nanocages for dry AMD. Considering the clinical preference for non-invasive therapies, we finally developed and optimized a topical eye drop formulation using the same zwitterionic nanocage platform. Notably, this formulation demonstrated promising therapeutic efficacy in treating oxidative stress-induced dry macular degeneration in mice.\n“Given the high sensitivity of ocular tissues, we conducted a thorough biocompatibility assessment of Z(CAT&SOD) to ensure its safety for long-term use. A battery of ophthalmic evaluations including slit-lamp microscope, optical coherence tomography (OCT), electroretinogram (ERG) and intraocular pressure (IOP), were performed on mice four weeks after treatment with Z-polymer and Z(CAT&SOD) eyedrops. As illustrated in Figure S14a and S14b, the corneas of treated mice remained clear and intact, with no observable differences compared to healthy controls. Moreover, prolonged administration of Z-polymer and Z(CAT&SOD) did not obviously alter tear secretion (Figure S14c). Corneal OCT imaging confirmed that neither formation affected corneal thickness (Figure S14d and S14e), while fundus imaging and retina thickness measurements revealed no structural abnormalities (Figure S14f to S14i). In addition, IOP values remained stable after long-term treatment, indicating that the Z(CAT&SOD) eyedrop dose not induce ocular hypertension or increase the risk of glaucoma (Figure S14j). To assess systemic toxicity, we performed histological analysis of major organs (heart, liver, spleen, lung, kidney and eye) via H&E staining. Tissues form Z-polymer and Z(CAT&SOD)-treated mice exhibited nearly no pathological changes compared to healthy mice (Figure S14k). Additionally, hematological analysis of blood samples confirmed that all parameters remained in the normal range, indicating negligible systemic toxicity even after prolonged subconjunctival retention (Figure S14l). Therefore, these findings demonstrate that both Z-polymer and Z(CAT&SOD) exhibit excellent biocompatibility after long-term administration.”\nFigure R1. Safety assessment of Z-polymer and Z(CAT&SOD) (n=3). (a) Representative images of cornea’s light fields and (b) fluorescein cornea staining of mouse eyes after different treatments for 2 and 4 weeks by slit-lamp microscopy. (c) The tear secretion amount of mouse eyes after different treatments for 2 and 4 weeks (d) Representative images of OCT images of cornea and (e) retina for 2 and 4 weeks. (f) Statistical analysis of relative retina thickness. (g) Representative images of retina’s light fields and (h) fluorescein staining of retina after different treatments for 2 and 4 weeks. (i) Statistical analysis of IOPs of eyes in mice after different treatments for 2 and 4 weeks. (j) H&E staining images of different organs including liver, spleen, lung, kidney, heart and eye collected from mice in different groups (scale bars: 500 μm). (k) Blood biochemistry and complete blood analysis of mice with different treatments. The following parameters were tested: percentage of white blood cell (WBC); percentage of lymphocytes (Lymph); percentage of monocytes (MON); percentage of neutrophilic granulocyte (Gran); red blood cell (RBC) counts; hemoglobin (HGB); hematocrit (HCT); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); platelets (PLT); alanine aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP) and blood urea nitrogen (BUN). Data were represented as means ± SD.\n2. A more significant concern lies in the conceptual accuracy of several claims. The manuscript contains multiple errors in ocular definitions and physiological interpretations, which diminish confidence in the scientific rigor. For example, retinal ganglion cells are not the primary cells affected in geographic atrophy (GA); the a-wave of the ERG is primarily associated with photoreceptor (particularly rod) activity, not cones; and the b-wave is typically driven by bipolar cells rather than directly by rods. Furthermore, evaluating choroidal neovascularization (CNV) as an outcome measure is not appropriate for GA, which is a non-neovascular form of age-related macular degeneration. These inaccuracies suggest a need for more careful literature review and refinement of disease-specific understanding.\nReply: We apologize for the confusion. We sincerely thank the reviewer for pointing out these critical inaccuracies in ocular terminology and physiological interpretations. We fully acknowledge that precision in disease-specific descriptions is essential for scientific rigor and translational credibility. In response, we have carefully revised the manuscript to address the following key issues. Firstly, we have corrected the misinterpretation of ERG waves. The a-wave is now correctly described as reflecting photoreceptor activity (primarily rods), and the b-wave as originating from ON-bipolar cells, not rods or cones directly. Then, we have corrected all references to “retinal ganglion cells” as primary targets in GA, and accurately identified photoreceptors and RPE cells as the main cell types affected. Moreover, we have explicitly distinguished geographic atrophy (non-neovascular) from choroidal neovascularization (neovascular), and the evaluation of this model was reorganized (Figure R2 and R3). In light of the abnormal retinal vasculature observed by FFA, we have moved these data to the Supporting Information (Figure S2a, Figure S9b, Figure S13b).\n“This treatment effectively mitigated ROS-induced apoptosis and suppressed the NF-κB-HMGB1-ILs inflammatory pathway, providing substantial protection to photoreceptors and retinal pigment epithelial (RPE) cells, which represents a groundbreaking therapeutic strategy for dry AMD treatment and offers a novel approach to addressing the challenges associated with this progressive retinal disorder.”\n“ERG analysis revealed a marked reduction in both the a and b waves, reflecting impaired activity of photoreceptors and ON-bipolar cells.”\n“Analysis of a- and b- waves, which correspond to photoreceptors and ON-bipolar cell activity, demonstrated partial restoration of visual acuity in mice treated with CAT or SOD&CAT injections.”\n“Post-treatment ERG assessments at two weeks confirmed that Z(CAT&SOD) effectively preserved photoreceptors and ON-bipolar cell function, with no statistically significant difference compared to intravitreal injection.”\n“As shown in figures 2k and 2l, prominent retinal deposits were observed in PBS- and SOD-injected groups, accompanied by subretinal debris accumulation (likely derived from damaged RPE and photoreceptors), inflammatory edema, and secondary vascular abnormalities induced by oxidative stress (Figure S2a).”\n“In contrast, untreated and uncoated groups exhibited severe retinal thinning and reduced cell density, particularly in the outer nuclear layer (ONL) and photoreceptor layer, which are critically affected in GA. These structural deficits correlated with ERG-documented visual impairment.”\n“As shown in Figure 4b, PBS- or Z(SOD)-injected mice developed progressive subretinal deposits, retinal vasculitis, widespread retinal cell apoptosis, and atrophy over time.”\nFigure R2 (in previous manuscript’s figure 4). Representative fundus images, OCT images and H&E images of mice with different treatments (Health, Untreated, Z-polymer, Z(CAT), Z(SOD) and Z(CAT&SOD)) at one and two weeks (n=4, scale bar: 100 μm).\nFigure R3 (in previous manuscript’s figure 6). Representative fundus images, OCT images and H&E images of mice with different treatments (Untreated, CAT&SOD eyedrop, CAT&SOD intravitreal injection, Z(CAT&SOD) eyedrop, Z(CAT&SOD) intravitreal injection and Lutein oral administration) at one and two weeks (n=4, scale bar: 100 μm).\n3. Lastly, while the sodium iodate model does recapitulate oxidative stress-related RPE damage, it reflects an acute injury rather than the chronic, progressive degeneration seen in human GA. Therefore, while the protective effects observed are encouraging, their relevance to GA pathophysiology should be presented more cautiously. Overall, the concept may be of interest if reframed with appropriate disease context, technical clarifications, and stronger emphasis on translational considerations.\nReply: We sincerely appreciate the reviewer’s comment regarding the distinction between the sodium- iodate model, which induces acute oxidative damage, and the chronic, low-grade oxidative stress characteristic of human geographic atrophy. We fully acknowledge this critical difference and agree that caution must be exercised when extrapolating our findings to clinical GA. The NaIO₃ rapidly triggers key pathological features of late-stage GA, including RPE/photoreceptor degeneration, complement activation, and microglial recruitment, thereby recapitulating the terminal oxidative microenvironment of GA1. Given that CAT/SOD function by acutely neutralizing ROS, the data suggest that targeted antioxidant intervention may help halt or slow active atrophic expansion2–5. As a proof-of-concept, these findings demonstrate that rapid ROS scavenging can preserve residual photoreceptors in the presence of oxidative damage. For more accurate understanding, we have added related discussion in the revised manuscript.\n“In the SI-induced GA mouse model, we observed pathological features resembling those in clinical GA patients, including subretinal deposits and retinal atrophy. Mechanistic investigations revealed that RPE and photoreceptor cell apoptosis was closely associated with the microenvironment characterized by high oxidative stress and diminished peroxidase activity, particularly reduced levels of CAT and SOD. As a proof-of-concept, our findings suggest that timely antioxidant supplementation may preserve residual photoreceptors when oxidative damage as the primary driver of degeneration.”\nReference\n1. The retinal pigment epithelium in Sorsby Fundus Dystrophy shows increased sensitivity to oxidative stress-induced degeneration. Redox Biol. 37, 101681 (2020).\n2. Hollyfield, J. G. et al. Oxidative damage–induced inflammation initiates age-related macular degeneration. Nat. Med. 14, 194–198 (2008).\n3. Lauvås, A. J. et al. Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure. NeuroToxicology 92, 33–48 (2022).\n4. Comparative mechanistic study of RPE cell death induced by different oxidative stresses. Redox Biol. 65, 102840 (2023).\n5. Kerur, N. et al. cGAS drives noncanonical-inflammasome activation in age-related macular degeneration. Nat. Med. 24, 50–61 (2018).\n----------------------\nReviewer #2:\nThe manuscript identifies a critical deficit in retinal peroxidase activity (specifically catalase and superoxide dismutase) during GA progression, which exacerbates oxidative stress, activates the NF- κB-HMGB1-ILs inflammatory pathway, and drives retinal cell apoptosis. To counteract this, the authors developed charge-shielded zwitterionic nanocages (Z(CAT&SOD)) that protect enzyme activity, enhance permeability across ocular barriers, and facilitate delivery via clathrin-dependent and caveolae-mediated endocytosis. The study is well-organized and provides comprehensive insights into the potential of this innovative therapeutic strategy. The authors should address the following questions to further enhance the quality of the manuscript:\n1. As a nano-delivery system intended for ocular applications, the biosafety of zwitterionic nanocages (Z (CAT&SOD)) constitutes a fundamental prerequisite for clinical translation; however, existing research lacks systematic evaluation. It is need to conduct supplementary long-term safety assessments, encompassing evaluations of ocular irritation, intraocular pressure variations, corneal integrity, corneal transparency, and retinal pathology.\nReply: Thanks so much for the reviewer’s suggestion. We recognize the critical importance of safety for translational ophthalmic formulations. In response to reviewer’s comments, we evaluated both empty and CAT&SOD-loaded zwitterionic nanocages in healthy mice for 4 weeks. Both the nanocages showed excellent tolerability with no observed ocular irritation, IOP elevation, corneal/ retinal damage, or systemic toxicity (Figure R4). These findings confirmed zwitterionic polymers’ excellent biocompatibility, low immunogenicity, and minimal protein adsorption-ideal properties for ocular drug delivery.\n“Given the high sensitivity of ocular tissues, we conducted a thorough biocompatibility assessment of Z(CAT&SOD) to ensure its safety for long-term use. A battery of ophthalmic evaluations including slit-lamp microscope, optical coherence tomography (OCT), electroretinogram (ERG) and intraocular pressure (IOP), were performed on mice four weeks after treatment with Z-polymer and Z(CAT&SOD) eyedrops. As illustrated in Figure S14a and S14b, the corneas of treated mice remained clear and intact, with no observable differences compared to healthy controls. Moreover, prolonged administration of Z-polymer and Z(CAT&SOD) did not obviously alter tear secretion (Figure S14c). Corneal OCT imaging confirmed that neither formation affected corneal thickness (Figure S14d and S14e), while fundus imaging and retina thickness measurements revealed no structural abnormalities (Figure S14f to S14i). In addition, IOP values remained stable after long-term treatment, indicating that the Z(CAT&SOD) eyedrop dose not induce ocular hypertension or increase the risk of glaucoma (Figure S14j). To assess systemic toxicity, we performed histological analysis of major organs (heart, liver, spleen, lung, kidney and eye) via H&E staining. Tissues form Z-polymer and Z(CAT&SOD)-treated mice exhibited nearly no pathological changes compared to healthy mice (Figure S14k). Additionally, hematological analysis of blood samples confirmed that all parameters remained in the normal range, indicating negligible systemic toxicity even after prolonged subconjunctival retention (Figure S14l). Therefore, these findings demonstrate that both Z-polymer and Z(CAT&SOD) exhibit excellent biocompatibility after long-term administration.”\nFigure R4. Safety assessment of Z-polymer and Z(CAT&SOD) (n=3). (a) Representative images of cornea’s light fields and (b) fluorescein cornea staining of mouse eyes after different treatments for 2 and 4 weeks by slit-lamp microscopy. (c) The tear secretion amount of mouse eyes after different treatments for 2 and 4 weeks (d) Representative images of OCT images of cornea and (e) retina for 2 and 4 weeks. (f) Statistical analysis of relative retina thickness. (g) Representative images of retina’s light fields and (h) fluorescein staining of retina after different treatments for 2 and 4 weeks. (i) Statistical analysis of IOPs of eyes in mice after different treatments for 2 and 4 weeks. (j) H&E staining images of different organs including liver, spleen, lung, kidney, heart and eye collected from mice in different groups (scale bars: 500 μm). (k) Blood biochemistry and complete blood analysis of mice with different treatments. The following parameters were tested: percentage of white blood cell (WBC); percentage of lymphocytes (Lymph); percentage of monocytes (MON); percentage of neutrophilic granulocyte (Gran); red blood cell (RBC) counts; hemoglobin (HGB); hematocrit (HCT); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); platelets (PLT); alanine aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP) and blood urea nitrogen (BUN). Data were represented as means ± SD. P values in (c, d, e, g and h) were calculated by using one-way ANOVA.\n2) It is necessary to elucidate the molecular basis of zwitterionic charge shielding: to investigate the dynamic changes in surface charge (as monitored by real-time zeta potential measurements) of the CAT&SOD complex in the vitreous microenvironment.\nReply: Thanks so much for the reviewer’s suggestion. To elucidate the molecular mechanism of zwitterionic charge shielding in the vitreous, we performed real-time zeta potential measurements of Z(CAT&SOD) nanocages incubated in fresh rabbit vitreous humor at physiological temperature (37°C) for different times. As show in Figure R5, the ζ-potential of Z(CAT&SOD) maintained stable throughout the observation period, demonstrating excellent charge homeostasis in the vitreous milieu. This charge stabilization phenomenon can be attributed to the unique zwitterionic architecture of Z(CAT&SOD), where the protonation quilibrium between acidic and basic side chains creates a dynamic charge-shielding effect, which effectively neutralizes electrostatic interactions with anionic vitreous components and preserves the native ζ-potential. We have added these results and discussion to the revised manuscript as following:\n“To further investigate the interaction between zwitterionic nanocages and the vitreous, we measured the surface charge of Z(CAT&SOD) nanocages after co-incubation with the isolated rabbit vitreous (Figure S6). We found that the near-neutral surface of the zwitterionic nanocages remained stable in the vitreous environment, facilitating enhanced diffusion of the encapsulated protein drugs.”\nFigure R5. Zeta potential of Z(CAT&SOD) incubated in rabbit vitreous body for different times.\n3) Supplementary stability data for dosage forms are required: The particle size distribution of the ophthalmic solution, enzyme activity retention rate (for a minimum of 1 month), and the presence of nanocage aggregation (e.g., through long-term DLS monitoring) should be assessed under various storage conditions (4°C, room temperature, 37°C).\nReply: Thanks so much for the reviewer’s suggestion. To evaluate formulation stability, we systematically compared the enzymatic activity retention of zwitterionic nanocage-encapsulated enzymes (Z(CAT&SOD)) with free enzymes under various storage conditions (4°C, 25°C, and 37°C) for 4 weeks. Weekly activity assessments revealed that while both formulations maintained over 90% initial activity at 4°C throughout the study period, marked difference emerged at higher temperatures. Free enzymes exhibited rapid activity decay, losing significant functionality by day 7 at both 25 °C and 37 °C. In contrast, Z(CAT&SOD) nanocages demonstrate the superior stability (Figure R6).\n“We further evaluated the enzymatic stability of Z(SOD), Z(CAT), and their free counterparts under different storage conditions (4°C, 25°C and 37°C). While free enzymes exhibited rapid activity loss at 25°C and 37°C, the zwitterionic nanocages-encapsulated enzymes retained most of their initial activity for four weeks, indicating their superior protective effect of zwitterionic nanocage (Figure S8).”\nFigure R6. The enzyme activity stability of Z(SOD), Z(CAT), free SOD and free CAT at different storage environments (4°C, 25°C and 37°C).\n4) It is necessary to include negative controls, such as a control group utilizing blank zwitterionic nanocages (enzyme-free), to eliminate potential confounding effects of the material itself on inflammation or retinal structure.\nReply: Thanks so much for pointing it out. To further validate the therapeutic mechanism, we performed additional experiments using “blank” zwitterionic nanocages (Z-polymer nanocages without CAT or SOD) in the GA mouse model. Both OCT and fundus imaging demonstrated that these blank nanocages failed to preserve photoreceptor integrity or RPE morphology, with outcomes indistinguishable from untreated control. These data confirmed that the therapeutic effects are exclusively mediated by the activities of CAT and SOD (Figure R7). The related results and discussion have been added in the revised manuscript as following:\n“Furthermore, zwitterionic nanocages without peroxidase demonstrated nearly no therapeutic effect on the SI-induced GA model.”\nFigure R7 (in previous manuscript’s figure 4). Treatment of GA via intravitreal injection of Z(CAT&SOD). (a) Schematic illustration of different treatments on SI-induced GA mouse model and the fundus assessment after one week. (b) Representative fundus images, OCT images and H&E images of mice after different treatments at one and two weeks (n=4, scale bar: 100 μm). (c) Clinical grading of GA damage according to a leakage score system (n=4). (d & e) Statistical analysis of retinal thickness of mice in different groups at one and two weeks (n=4). (f) Representative ERG wave responses in mice after different treatments (n=4). (g & h) Statistical analysis of A waves (g) and B waves (h) in retina of mice in different groups under scotopic condition (n=4). Data were represented as means ± SD. P values in (c, d, e, g and h) were calculated by using one-way ANOVA.\n5) The study employs a sodium iodate-induced model of dry macular degeneration, which phenotypically approximates the human dry macular degeneration model. Please elucidate the underlying mechanisms involved.\nReply: Thanks so much for the reviewer’s comment. Sodium iodate (NaIO₃) serves as a well- characterized oxidative stress inducer that selectively targets the retinal pigment epithelium (RPE), initiating a degenerative cascade that faithfully recapitulates the cardinal features of human geographic atrophy (GA)1. The mechanistic underpinnings of this model involve three interrelated pathological proceess: 1) NaIO₃-mediated ROS overproduction induces RPE mitochondrial dysfunction, membrane depolarization, and subsequent necroptotic cell death6; 2) secondary photoreceptor degeneration occurs through disrupted retinoid cycling and loss of metabolic support, manifesting as outer nuclear layer (ONL) thinning and diminished a-wave amplitudes on ERG; and 3) sustained neuroinflammation develops via microglial activation, Muller cell gliosis, and macrophage infiltration, accompanied by elevated inflammatory mediators (IL-1β, CCL2, IL-33, and TGF-β)7,8. This mark of RPE dysfunction, photoreceptor loss, and chronic inflammation mirrors both the structural and dysfunction, photoreceptor loss, and chronic inflammation indicated both the structural and functional deficits observed in GA patient9. The NaIO₃ model thus provides a robust preclinical platform that captures the complex pathophysiology of dry AMD, particularly for evaluating antioxidant therapies targeting oxidative stress-mediated degeneration. We have added the related discussion in the revised manuscript as following:\n“The SI-induced GA model faithfully recapitulated key clinical manifestations of human GA, including subretinal deposits and retinal atrophy. Through mechanistic studies, we identified two critical pathologies features: oxidative stress-medicated apoptosis in both RPE and photoreceptor cells and severe deficiency of endogenous antioxidant defenses (CAT/SOD). Notably, our therapeutic experiments established that photoreceptor survival could be improved through antioxidant supplementation during the critical window of oxidative stress dominance.”\nReference\n1. The retinal pigment epithelium in Sorsby Fundus Dystrophy shows increased sensitivity to oxidative stress-induced degeneration. Redox Biol. 37, 101681 (2020).\n6. Hanus, J., Anderson, C. & Wang, S. RPE necroptosis in response to oxidative stress and in AMD. Ageing Res. Rev. 24, 286–298 (2015).\n7. Liu, B. et al. Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells. Cell Death Dis. 12, 230 (2021).\n8. Khandhadia, S. & Lotery, A. Oxidation and age-related macular degeneration: insights from molecular biology. Expert Rev. Mol. Med. 12, e34 (2010).\n9. Yaspan, B. L. et al. Targeting factor D of the alternative complement pathway reduces geographic atrophy progression secondary to age-related macular degeneration. Sci. Transl. Med. 9, eaaf1443 (2017).\n6) Regarding the concern of the barrier integrity of tight junction proteins, the authors need tested the longer incubation with Z (CAT&SOD) for 6 hours. As the corneal epithelial cells are the protection of the eyes, it still brings concern about the use of Z (CAT&SOD) in vivo, such as infection.\nReply: Thanks so much for the reviewer’s suggestion. To evaluate the potential impact of Z(CAT&SOD) on corneal epithelial barrier function, we extended the incubation time in our transwell-based transepithelial electrical resistance (TEER) assay from 1 hour to 6 hours of direct exposure, specifically assessing tight junction integrity in human corneal epithelial cells (HCECs). The results suggested that prolonged exposure to Z(CAT&SOD) did not disrupt epithelial barrier integrity, indicating a low risk of compromising ocular surface infection under the tested conditions (Figure R8). The related discussion has been added in the revised manuscript as following:\n“Subsequently, we measured transepithelial electrical resistance (TEER) and observed nearly no significant changes following treatment with either Z(CAT) or free CAT (Figure S7), further confirming that Z(CAT) does not compromise tight junction integrity or utilize the paracellular route for ocular penetration.”\n%\n/)e 120 PBS CAT Z-CAT\nu\nla\nv 100\nla\nitin 80\nI\no 60\nt\ne\nv\nita 40\nle\nR 20\n(\nR\nE 0\nE 0 6 1224 0 6 1224 0 6 1224\nT\nTime (h)\nFigure R8. TEER measurements of HCECs at various time points after different treatments (n=3).\n7) Figure 5l fails to elucidate the mechanistic role of macrophages, which warrants supplementation regarding their functional mechanism in the treatment of dry macular degeneration with Z (CAT&SOD) therapy. Clarify how Z(CAT&SOD) drives M2 polarization (e.g., via reduced ROS levels inhibiting NF-κB, or direct modulation of macrophage metabolism).\nReply: Thanks so much for the reviewer’s comment. Z(CAT&SOD) rapidly scavenges H₂O₂ in the injured retina (Figure R9a), alleviating mitochondrial oxidative stress and suppressing the release of DAMPs such as HMGB1 (Figure R9d). Reduced DAMPs and ROS inhibit phosphorylation and nuclear translocation of NF-κB p65 (Figure R9b), thereby downregulating M1-polarizing cytokines (IL-1β, TNF-α, IL-6, IL-12) at the transcriptional level (Figure R9h-k). Concomitantly, NF-κB suppression restored mitochondrial respiration, creating a metabolic milieu conducive to M2 polarization. Accordingly, Z(CAT&SOD)-treated retinas exhibited an increased CD206⁺F4/80⁺ M2 population and a decrease in CD80⁺F4/80⁺ M1 macrophages (Figure R9e, R9f and R9g). Thus, these data demonstrate that Z(CAT&SOD) eliminates ROS-driven inflammatory signals, inactivates the NF- κB pathway, and promotes endogenous M2 programming in the retinal micro-environment as summarized in Figure R9l. The related discussion has been added in the revised manuscript as following:\n“Therefore, the enzymatic cascade reaction mediated by Z(CAT&SOD) synergistically reduced key inflammatory markers such as NF-κBp65, IL-1β, and HMGB1 in retinal tissues while promoting macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotypes. These findings highlight Z(CAT&SOD) as a promising therapeutic candidate for retinal disorders involving oxidative stress and inflammation.”\nFigure R9 (in previous manuscripts’ figure 5). Mechanism of Z(CAT&SOD) in treating GA. Representative immunofluorescence staining images showing the expression of TUNEL(a), NF-κB (b), IL-1β (c) and HMGB1(d) in the retina of mice in different groups (scale bar: 50 μm). (e) Representative flow cytometry analysis of macrophage polarization in retina of mice in different groups as indicated (n=3). (f) and (g) Percentages of F4/80+CD206+ macrophages and F4/80+CD80+ macrophage cells by flowcytometry (n=3). (h to k) Concentrations of IL-12P70, TNF-α, IL-6 and IFN- γ in the retinas of mice in different groups evaluated by ELISA (n=4). (l) Schematic illustration of NF- κB-HMGB1-ILs pathway-mediated inflammatory effects associated with apoptosis due to decreased peroxidase activity and increased ROS levels. Data were represented as means ± SD. P values in (f, g, h, i, j and k) were calculated by using one-way ANOVA.\n8) The drug loading and drug encapsulation efficiency of Z(CAT&SOD) should be reported.\nReply: Thanks so much for the reviewer’s comment. Using the BCA assay, we quantified the drug loading capacity and encapsulation efficiency of the Z(CAT&SOD) nanocages. The optimized formulation demonstrated a high drug loading capacity of 94.84 ± 1.55% with encapsulation efficiency of 35.32 ± 4.43%.\n“We further characterized the physical properties and drug-loading performance at this optimal ratio. Dynamic light scattering analysis revealed a nearly neutral surface charge (Table S1). Notably, the nanocages exhibited excellent drug-loading capacity of 94.84 ± 1.55% with encapsulation efficiency of 35.32 ± 4.43%).”\nTable R1. The particle size, surface charge, drug loading and drug encapsulation efficiency of Z(CAT&SOD) nanocages.\n9) The rationale for using peroxidases (CAT and SOD) in GA therapy should be strengthened with literature support linking their deficiency to human GA: Cite clinical studies showing reduced CAT/SOD activity in retinas or aqueous humor of GA patients (vs. age-matched controls). Discuss how CAT/SOD deficiencies exacerbate oxidative stress in GA: For example, do they impair ROS clearance in RPE cells, leading to lipofuscin accumulation or mitochondrial DNA damage (as reported in human GA)? Compare with other antioxidant strategies (e.g., nanozymes, small-molecule ROS scavengers) to highlight the advantages of enzymatic cascades (e.g., specificity, biocompatibility).\nReply: Thanks so much for the reviewer’s comment. To strengthen the therapeutic potential of CAT/SOD supplementation in GA, we highlight clinical evidence demonstrating significantly reduced CAT and SOD activities in both retinal tissue and aqueous humor of GA patients compared to age-matched controls9,10. This enzymatic deficiency compromises ROS clearance in RPE cells, thereby promoting key pathological features of GA, including lipofuscin accumulation, mitochondrial DNA oxidation, and drusen formation11. Unlike small-molecule antioxidants or nanozyme, the CAT/SOD enzymatic cascade provides substrate-specific, catalytic efficiency and physiologically compatible elimination of O₂•⁻ and H₂O₂, offering a more sustainable and endogenous strategy to mitigate oxidative damage in GA.\nReference\n9. Yaspan, B. L. et al. Targeting factor D of the alternative complement pathway reduces geographic atrophy progression secondary to age-related macular degeneration. Sci. Transl. Med. 9, eaaf1443 (2017).\n10. Senabouth, A. et al. Transcriptomic and proteomic retinal pigment epithelium signatures of age- related macular degeneration. Nat. Commun. 13, 4233 (2022).\n11. Eyedrop delivery of therapeutic proteins with zwitterionic polymers to treat dry age-related macular degeneration. Biomaterials 305, 122429 (2024).\n10) Minor point: Supplementary figures (e.g., Figure S1–S17) are not cited in the order they appear in the text, which hinders readability. For example, Figure S3 is referenced before Figure S2. The authors should reorder supplementary figure citations to align with their appearance in the main text, ensuring a logical flow.\nReply: Thanks for pointing out this. We have now re-checked the entire manuscript and reordered all Supplementary Figure citations so that they appear strictly sequentially in line with their first mention in the text. This adjustment improves readability and is reflected in the revised file.\n----------------------\nReviewer #3:\nThis study presents an interesting and potentially translationable strategy for the treatment of geographic atrophy by constructing a zwitterionic nanocage-based delivery system that enables non- invasive co-delivery of catalase (CAT) and superoxide dismutase (SOD) to the retina. The work demonstrates strong innovation, solid in vivo data, and a relatively in-depth mechanistic exploration. However, several scientific and mechanistic issues still need to be clarified or reinforced, and some aspects of data presentation require revision. I recommend accept this manuscript after addressing the following specific comments:\n1) The manuscript emphasizes the synergistic effect of CAT and SOD as an enzymatic cascade. However, there is no quantitative analysis confirming whether the combined use is superior to individual enzymes in terms of ROS scavenging efficiency (e.g., rate of radical elimination, half-life of ROS). It is recommended to include in vitro kinetic assays to validate whether this is a true cascade reaction or simply an additive effect.\nReply: We thank the reviewer for this critical point. While we acknowledge that direct kinetic data would further strengthen our mechanistic interpretation, our results demonstrate that the SOD-CAT cascade operates through functional complementarity. Specifically, CAT converts the toxic byproduct of SOD activity (H₂O₂) into harmless H₂O and O₂, thereby protecting SOD from oxidative inactivation and ensuring sustained antioxidant capacity. This cooperative mechanism prevents •OH generation via Fenton reaction, achieving complete ROS detoxification. To validate this synergy, we evaluated the ROS-scavenging capacity of Z(CAT), Z(SOD), and Z(CAT&SOD) nanocages. As shown in Figure R10, both modified enzymes retained their catalytic efficiency: SOD effectively eliminated superoxide anions, while CAT degraded H₂O₂. Notably, the combined system (Z(CAT&SOD)) exhibited enhanced radical scavenging, demonstrating cooperative action for optimal free radical elimination.\nFigure R10. (a) Superoxide scavenging kinetics monitored at 550 nm (A-A ) for xanthine/xanthine 0 oxidase reactions with different nanocages (1 μg/mL). (b) Oxygen evolution kinetics from H₂O₂ decomposition (50 mM) by nanocages (1 μg/mL), measured via dissolved oxygen content.\n2) The authors propose that Z(CAT&SOD) eyedrops can penetrate the retina through both the conjunctiva-sclera-retina and conjunctiva-blood-retina routes. However, there is no quantitative analysis comparing the relative contribution of each route. I suggest performing quantitative tissue fluorescence distribution (e.g., FITC signal intensity in different ocular layers) or using pathway-specific inhibitors to confirm the predominant transport route.\nReply: Thanks so much for the reviewer’s suggestion. To quantify the relative contribution of different pathways, we measured FITC fluorescence in dissected sclera and retina 6h post-instillation and simultaneously determined the blood concentration of CAT-FITC-loaded zwitterionic nanocages. The conjunctiva/sclera-to-retina fluorescence ratio was about 6.67:1, suggesting preferential scleral uptake. In addition, we performed pharmacokinetic analysis by collecting blood samples at 1, 3, 6, 9, and 12 hours after topical administration of Z(CAT-FITC) eye drops. At 6 hours, the signal of Z(CAT-FITC) in the blood was significantly higher than that of free CAT-FITC, indicating the presence of a blood-to-retina pathway (Figure R11). The related results and discussion have been added in the revised manuscript as following:\n“Confocal imaging of whole-eye sections revealed that the zwitterionic nanocages might penetrate the conjunctival-scleral barrier, reaching the choroid and retina (Figure 6d). Quantitative analysis of CAT- FITC fluorescent signals revealed a conjunctiva/sclera-to-retina ratio of 6.67:1 (Figure S11a), suggesting efficient transscleral delivery. In addition, pharmacokinetic profiling following topical administration of Z(CAT-FITC) eye drops (measured at 1, 3, 6, 9, and 12 h) showed that Z(CAT-FITC) exhibited significantly higher blood accumulation than free CAT-FITC at 6 hours (Figure S11b), indicating the existence of a blood mediated retinal delivery pathway.”\nFigure R11. (a) Statistical analysis of total fluorescence intensity of CAT-FITC in the conjunctiva/sclera (C/S), retina (R) and vitreous cavity of mice after different eyedrops treatments (n=3). (b) The CAT-FITC concentration in blood after different treatments for 0, 1, 3, 6, 9, 12 hours.\n3) The suppression of inflammation is attributed mainly to ROS reduction, but it remains unclear whether CAT/SOD also exerts ROS-independent immunomodulatory effects, particularly on the NF- κB pathway. The authors are encouraged to explore whether changes in key signaling proteins (e.g., p65 phosphorylation) precede complete ROS clearance.\nReply: Thanks so much for the reviewer’s comment. CAT and SOD lack kinase-inhibitory domains or scaffold functions, as their primary role is redox regulation. While transient H₂O₂ can activate IKKβ, chronic oxidative stress and DAMP release sustain NF-κB signalling in GA. Z(CAT&SOD) rapidly scavenges H₂O₂ and O₂•⁻, eliminating the oxidative trigger for IKKβ activation and consequently suppressing downstream p65 phosphorylation without requiring a separate, ROS-independent mechanism. Our data show that phospho-p65, IL-1β and HMGB1 levels decrease concomitantly with the DCF signal attenuation (Figure R12), further supporting ROS-dependent NF-κB activation. Therefore, our findings demonstrate that NF-κB suppression occurs predominantly as a secondary consequence of ROS clearance rather than through direct, ROS-independent modulation of the signaling cascade by CAT or SOD.\nFigure R12. Mechanism of Z(CAT&SOD) in treating GA (in previous manuscripts’ figure 5). Representative immunofluorescence staining images showing the expression of TUNEL(a), NF-κB (b), IL-1β (c) and HMGB1(d) in the retina of mice in different groups (scale bar: 50 μm). (e) Representative flow cytometry analysis of macrophage polarization in retina of mice in different groups as indicated (n=3). (f) and (g) Percentages of F4/80+CD206+ macrophages and F4/80+CD80+ macrophage cells by flowcytometry (n=3). (h to k) Concentrations of IL-12P70, TNF-α, IL-6 and IFN- γ in the retinas of mice in different groups evaluated by ELISA (n=4). (l) Schematic illustration of NF- κB-HMGB1-ILs pathway-mediated inflammatory effects associated with apoptosis due to decreased peroxidase activity and increased ROS levels. Data were represented as means ± SD. P values in (f, g, h, i, j and k) were calculated by using one-way ANOVA.\n4) Although the study shows that encapsulation preserves enzymatic activity under proteolytic stress, it would strengthen the claims to include structural or kinetic data (e.g., circular dichroism spectra, DLS, changes in Michaelis–Menten kinetics) to assess whether zwitterionic polymerization alters the enzyme’s conformation or access to the active site.\nReply: Thanks so much for the reviewer’s suggestion. To verify whether zwitterionic encapsulation alters enzyme conformation or active-site integrity, we performed circular dichroism (CD) spectra of both free CAT and Z(CAT) nanocages. The results demonstrated nearly identical α-helical and β-sheet content before and after polymer coating, confirming that the native secondary structure was preserved (Figure R13). The related results and discussion have been added in the revised manuscript as following.\n“Furthermore, circular-dichroism (CD) spectroscopy confirmed that the zwitterionic nanocage encapsulation preserved the native secondary structure of the peroxidases (Figure S5).”\n20\n)\n1\n-lo\n10\nm\n2\nm\n0\nc\ng\ne\nd -10\n3 ( free CAT\n-0\n1 × -20 Z(CAT)\n]\nθ\n[\n-30\n200 220 240 260 280\nFigure R13. Circular dichroism spectrum of Z(CAT) and free CAT.\n5）The sodium iodate-induced model used in this study mimics acute oxidative damage and may primarily represent late-stage GA. Please discuss the limitations of this model in simulating the chronic progression of GA and whether the proposed therapeutic strategy could be effective in early- stage disease (e.g., before lipofuscin accumulation).\nReply: Thanks for pointing out this. While the sodium iodate model rapidly induces RPE and photoreceptor degeneration that mimics late-stage geographic atrophy, its acute oxidative insult fails to recapitulate the chronic, low-grade oxidative stress underlying human disease progression, particularly the gradual lipofuscin accumulation characteristic of early pathogenesis9. We therefore recognize this as a key limitation: while our data demonstrate therapeutic efficacy under acute, advanced-like conditions, they do not address intervention efficacy during the critical pre-lipofuscin. Notably, the CAT/SOD cascade specifically targets the ROS implicates in lipofuscinogenesis10,11. This suggests that prophylactic administration of Z(CAT&SOD) during early-stage dry AMD could effectively mitigate disease progression by neutralizing free radical before irreversible damage occurs.\n“In the SI-induced GA mouse model, we observed pathological features resembling those in clinical GA patients, including subretinal deposits and retinal atrophy. Mechanistic investigations revealed that RPE and photoreceptor cell apoptosis was closely associated with the microenvironment characterized by high oxidative stress and diminished peroxidase activity, particularly reduced levels of CAT and SOD. As a proof-of-concept, our findings suggest that timely antioxidant supplementation may preserve residual photoreceptors when oxidative damage as the primary driver of degeneration.”\nReference\n9. Yaspan, B. L. et al. Targeting factor D of the alternative complement pathway reduces geographic atrophy progression secondary to age-related macular degeneration. Sci. Transl. Med. 9, eaaf1443 (2017).\n10. Senabouth, A. et al. Transcriptomic and proteomic retinal pigment epithelium signatures of age- related macular degeneration. Nat. Commun. 13, 4233 (2022).\n11. Eyedrop delivery of therapeutic proteins with zwitterionic polymers to treat dry age-related macular degeneration. Biomaterials 305, 122429 (2024).\n6) While ERG measurements showed improved visual function after Z(CAT&SOD) treatment, the source of improvement (e.g., photoreceptor protection vs. retinal ganglion cell preservation vs. synaptic conduction) is not clearly discussed. Consider correlating specific ERG wave changes (e.g., a-wave vs. b-wave) with histological data to identify the principal site of action.\nReply: Thanks so much for the reviewer’s suggestion. The 70% reduction in ERG amplitude observed in the SI group correlated with commensurate thinning of the outer nuclear layer (ONL) in H&E-stained sections and intense TUNEL labeling in photoreceptor segments. Z(CAT&SOD) treatment not only restored a-wave amplitude but also preserved ONL thickness and reduced photoreceptor apoptosis, indicating primary neuroprotection at the photoreceptor level. While the b-wave originates from bipolar cells, its recovery temporally mirrored both a-wave restoration and ONL survival. This temporal correspondence supports the established dependence of bipolar cell function on intact photoreceptor input: by mitigating ROS-mediated photoreceptor degeneration, Z(CAT&SOD) preserved synaptic transmission to second-order neurons, thereby facilitating b-wave recovery. Thus, these ERG improvements reflect a primary photoreceptor rescue mechanism with consequent restoration of downstream signaling pathways. The related discussion has been added in the revised manuscript as following:\n“The restoration of ERG amplitudes in Z(CAT&SOD)-treated mice, alongside intact ONL and RPE morphology, provides direct evidence that photoreceptor-targeted ROS neutralization is both a mechanistic requirement and a novel therapeutic strategy for preserving retinal function.”\n7) The authors demonstrate M2 macrophage polarization and inflammation suppression following treatment, but there is no follow-up data on whether this immune modulation is reversible or potentially immunosuppressive. I recommend including 1-2 weeks of post-treatment follow-up to evaluate the persistence and safety of immune alterations.\nReply: Thanks so much for the reviewer’s suggestion. To determine whether the observed retinal immune modulation was sustained, we included a one-week drug-free observation period following treatment. Retinal tissues were collected one week after the final administration, and IL-6, IL-12 P70 levels were quantified via ELISA. The lack of inflammatory rebound suggests that a single 7-day course of Z(CAT&SOD) effectively mitigated oxidative stress, maintained retinal homeostasis, and prevented secondary inflammatory activation (Figure R14). The related results and discussion have been added in the revised manuscript as following.\nFigure R14. Concentrations of IL-12P70 and IL-6 in the retinas of mice one week after the last different treatments evaluated by ELISA (n=4).\n8) The manuscript is generally readable but contains some repetitive phrasing and grammatical issues (e.g., the phrase “has historically challenging to treat” in the Abstract is incorrect). Professional language editing by a native speaker or editing service is strongly advised.\nReply: Thanks so much for the reviewer’s comment. All grammatical errors, awkward phrasing and repetitions have been corrected as detailed above.\n“Age-related macular degeneration (AMD), particularly in its most prevalent form geographic atrophy (GA), has historically been challenging to treat due to its complex pathogenesis.”\n“Our pioneering study revealed the critical association between retinal peroxidase deficiency and GA progression, and proposed an innovative zwitterionic nanocage-based peroxidase, which exhibits great potential as a non-invasive yet highly effective therapeutic strategy for AMD.”\n“To demonstrate the successful establishment of GA mouse model, NaIO (10 mg/kg) was 3 intravenously injected on day 0.”\n“Analysis of a- and b- waves, which correspond to photoreceptors and ON-bipolar cell activity, demonstrated partial restoration of visual acuity in mice treated with CAT or SOD&CAT injections.”\n9) The figure legends are somewhat inconsistent in statistical notation. Please standardize the indication of significance (e.g., p<0.05, *p<0.01, etc.) and clearly state the statistical methods used in each figure legend.\nReply: Thanks for pointing out this. We have now standardized all figure legends to use the same significance notation (*p < 0.01, **p < 0.001, ***p < 0.0001) and explicitly state the statistical test (one-way ANOVA) used for each panel."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript contains significant conceptual, methodological, and technical flaws related to ocular biology, which limit the validity of the study. The authors have addressed some prior concerns and added new experiments, but key issues remain.\n1. Safety of Nanocages\n1. Polymerized materials are generally inert, but incomplete polymerization or residual monomers may pose risks and should be acknowledged.\n2. Conceptual Accuracy and Ocular Terminology\n2. FFA detects vascular abnormalities; in SI-treated mice, increased FFA reflects RPE barrier disruption, not neovascularization.\n3. Neovascularization is not expected in the SI injury model.\n4. Fig. S12 references CNV incorrectly; SI injury does not induce CNV, and GA does not involve CNV.\n5. ERG a- and b-waves measure retinal function, not visual acuity.\n6. Missing statistical summaries of ERG amplitudes is a critical issue; plotting style conventions are minor (see Section 5).\n3. SI-Induced Retinal Damage Model\nModel Understanding and Terminology\n7. The rationale for using 10 mg/kg SI is not provided.\n8. The SI model should not be referred to as GA; it is an acute chemical injury distinct from human geographic atrophy.\n9. Mice lack a macula; human GA scoring is not applicable. GA-like lesions in mice should be described as RPE/photoreceptor atrophy.\nFigure 1 and Related Panels\n10. “Neurodeatrophia” is nonstandard; “neurodegeneration” or “neuronal atrophy” is correct.\n11. “Atrophy of optic nerve cells” is incorrect; the shapes shown do not resemble photoreceptor outer segments.\n12. “Drusen” is misapplied; SI models do not produce true drusen and may represent pseudodrusen.\n13. Antioxidant dysfunction is not illustrated in the RPE.\n14. Panels showing “conjunctival barrier” and “sclerotic barrier” inside cells are anatomically incorrect.\n15. The scheme does not accurately show how CAT and SOD reach the RPE; Z(CAT&SOD) contains both enzymes but this is not clear.\n16. HCECs and HConEpiCs are irrelevant; SI injury affects RPE and photoreceptors.\nMethods and Experimental Design\n17. Methods list ARPE-32, HUVECs, and RAW 264.7 cells, but none appear in the results. HCECs are not listed in Methods and are corneal endothelial cells, not relevant to the study.\n18. The subtitle “Construction of a GA model” is misleading; the model is an acute chemical injury.\n19. Methods for ocular assays (fundoscopy, OCT, ERG), instrumentation, and analyses are not described, indicating lack of ocular biology expertise.\n20. Intravitreal injections of 5 µL are physiologically impossible in mice (vitreous ≈2 µL).\nFundus and FAF\n21. Hyperfluorescent spots from FAF images are not quantified.\n4. Overinterpretation and Therapeutic Claims\n22. Claims of “pioneering a new therapeutic paradigm for dry AMD” overstate the study.\n23. The SI model is not representative of human GA or AMD.\n24. Simply increasing SOD/CAT is not universally protective; overexpression can have unintended effects.\n25. Evidence regarding GPx/TAS in AMD, delivery of CAT/SOD to RPE mitochondria, and SOD mimetics or gene therapy as clinically feasible strategies is not addressed.\n5. Minor Issues\n26. Fig. R1 (now Fig. S14) legend does not specify whether assays were performed in untreated or SI-treated animals.\n27. Figures S9b and S13b lack clear legends, treatment descriptions, and baseline (“before treatment”) images.\n28. Terms such as “thickness length of CNV” are unclear.\n29. Missing “respectively” when defining a- and b-waves (p.4, line 144; p.5, line 180).\n30. Plotting style of some ERG panels (e.g., Fig. 4g) is unconventional.\n31. “Eye drop” should be two words.\n32. Abstract and introduction are excessively long; discussion does not include limitations.\n33. Missing references (p.3, lines 94–96; p.11, lines 452–455).\n34. Many figure legends lack treatment details, orientation, and arrow explanations.\n35. IR fundus images (Fig. 4b, 6e) do not indicate whether they are from the same animals.\n36. OCT images lack optic nerve localization and measurement indicators.\n37. Source tissue for mRNA analyses is unspecified.\n38. TUNEL staining in the RPE is not indicated.\n39. Individual data points are missing from plots, especially Fig. 3l and ERG amplitude analyses.\n40. Some ERG plots are partially obscured by axis labels.\n41. Fig. 5a: retinal orientation unclear; TUNEL higher in “Healthy” vs. “Untreated” is unexplained.\n42. Fig. 5l schematic is not informative.\n43. Fig. 6e: timing of lutein treatment relative to SI is unclear; prophylactic vs. post-injury effects differ.\n44. Fig. 6f: statistical analysis of a- and b-wave amplitudes for all animals is missing.\n45. CAT-FITC enzymatic activity is not reported.\nSummary: The manuscript contains fundamental flaws in experimental design, methodology, and biological interpretation. Major issues include misrepresentation of the SI injury model as GA, inappropriate in vitro cell models, physiologically impossible intravitreal injections, missing descriptions of ocular assays and analyses, and multiple inaccuracies in figures and data interpretation. These flaws significantly compromise the validity of the study.", "reviewer_2": "The authors have satisfactorily addressed my concerns; I recommend acceptance.", "reviewer_3": "The authors did a nice job to address my concerns and I would like to reccomend accept this manuscript without scientific change."}, "author_response": "Responses to reviewer’s comments\nWe sincerely thank all the reviewers for their time and valuable comments. We are particularly pleased to see the recognition of our work from reviewer 2# and 3#. However, we are very concerned about some of the comments from reviewer 1#. We believe that some of these comments are based on misunderstandings of the work. And we will provide detailed responses point by point below in an attempt to clarify the misunderstandings.\nReviewer #1:\nThe manuscript contains significant conceptual, methodological, and technical flaws related to ocular biology, which limit the validity of the study. The authors have addressed some prior concerns and added new experiments, but key issues remain.\n1. Polymerized materials are generally inert, but incomplete polymerization or residual monomers may pose risks and should be acknowledged.\nReply: Thanks so much for the reviewer’s suggestion. The synthesis protocol for zwitterionic polymer is well-established and widely recognized for producing biomedical-grade polymers, as reported in previous literatures1-5. Furthermore, our comprehensive in vivo safety data, presented in the initial response (Figure R1 / S14), provide the most biologically relevant confirmation. Critically, no local or systemic toxicity was observed, even after long-term topical administration. Therefore, these findings demonstrate the safety of our nanocage system for its intended ophthalmic application.\nReference\n1. Fang, H. et al. In Situ Polymerization of Zwitterions on Therapeutic Proteins to Enable Their Effective Oral Delivery. ACS Nano 17, 1128-1143 (2023).\n2. Fan, W. et al. Mucus Penetrating and Cell-Binding Polyzwitterionic Micelles as Potent Oral Nanomedicine for Cancer Drug Delivery. Advanced Materials 34, 2109189 (2022).\n3. Zhou, Y. et al. A pH-Triggered Self-Unpacking Capsule Containing Zwitterionic Hydrogel-Coated MOF Nanoparticles for Efficient Oral Exendin-4 Delivery. Advanced Materials 33, 2102044 (2021).\n4. Zhou, Q. et al. Enzyme-activatable polymer-drug conjugate augments tumour penetration and treatment efficacy. Nature Nanotechnology 14, 799-809 (2019).\n5. Han, X. et al. Zwitterionic micelles efficiently deliver oral insulin without opening tight junctions. Nature Nanotechnology 15, 605-614 (2020).\nFigure R1. Safety assessment of Z-polymer and Z(CAT&SOD) in normal mice (n=3). (a) Representative images of cornea’s light fields and (b) fluorescein cornea staining of mouse eyes after different treatments for 2 and 4 weeks by slit-lamp microscopy. (c) The tear secretion amount of mouse eyes after different treatments for 2 and 4 weeks (d) Representative images of OCT images of cornea and (e) retina for 2 and 4 weeks. (f) Statistical analysis of relative retina thickness. (g) Representative images of retina’s light fields and (h) fluorescein staining of retina after different treatments for 2 and 4 weeks. (i) Statistical analysis of IOPs of eyes in mice after different treatments for 2 and 4 weeks. (j) H&E staining images of different organs including liver, spleen, lung, kidney, heart and eye collected from mice in different groups (scale bars: 500 μm). (k) Blood biochemistry and complete blood analysis of mice with different treatments. The following parameters were tested: percentage of white blood cell (WBC); percentage of lymphocytes (Lymph); percentage of monocytes (MON); percentage of neutrophilic granulocyte (Gran); red blood cell (RBC) counts; hemoglobin (HGB); hematocrit (HCT); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); platelets (PLT); alanine aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP) and blood urea nitrogen (BUN). Data were represented as means ± SD.\n2. FFA detects vascular abnormalities; in SI-treated mice, increased FFA reflects RPE barrier disruption, not neovascularization.\nReply: We sincerely appreciate the reviewer’s insightful comment, which allows us to better clarify a critical aspect of our experimental model. We fully agree that in typical human geographic atrophy (GA), increased FFA signal is primarily attributed to RPE barrier disruption rather than neovascularization. However, it’s important to note that the sodium iodate (SI)-induced model in our study, while effectively mimicking oxidative stress-driven photoreceptor/RPE degeneration characteristic GA, often elicits a more extensive secondary inflammatory response involving retinal vasculature. The vascular abnormalities we observed via FFA (has been changed as “vascular inflammation” or “secondary vascular abnormalities” in the revised main text) do not represent neovascularization, but rather reflect inflammation-associated vascular leakage specific in this model. The observed fluorescence enhancement indeed stems from blood-retinal barrier disruption, encompassing both RPE and endothelial cell damage under inflammatory conditions. Here, FFA serves as a valuable in vivo method to evaluate the extent of secondary vascular inflammation and overall blood-retinal barrier integrity, both important indicators of retinal health in our model. We have moved this data to Supporting Information to avoid overstating its role in the core narrative of GA-like atrophy, while still presenting it as a supporting metric of disease severity and treatment efficacy against the multifaceted insult induced by SI.\nWe have carefully revised the manuscript to clarify that the FFA findings reflect model-specific inflammatory responses rather than neovascularization. This distinction is now explicitly addressed in the discussion to avoid any potential misunderstanding regarding neovascular and atrophic pathways.\n“FFA images showing therapeutic effects on secondary vascular inflammation in SI-induced retinal degeneration”\n“As shown in Figure 2k and 2l, prominent retinal deposits were observed in both PBS- and SOD-injected groups, accompanied by subretinal debris accumulation (likely originating from degenerated RPE and photoreceptors), inflammatory edema, and oxidative stress-induced vascular abnormalities (Figure S2a).”\n3. Neovascularization is not expected in the SI injury model.\nReply: We appreciate the reviewer’s insightful comment regarding the sodium iodate (SI) model’s pathophysiology. While SI primarily targets the RPE and photoreceptors, and is not a classic model for choroidal neovascularization (CNV), extensive literatures demonstrates that its severe oxidative injury triggers robust secondary inflammatory response. This inflammation response extends beyond the RPE/photoreceptor layer, affecting retinal vasculature and leading to blood-retinal barrier (BRB) breakdown, vascular leakage, and in some reports, even aberrant vascular changes6-9. Importantly, these effects are inflammation-mediated and distinct from VEGF-driven neovascularization in wet AMD. In our revised manuscript, we have consistently termed these observation as “secondary vascular abnormalities”, “vascular inflammation” or “vascular leakage” to accurately reflect this inflammatory vasculopathy.\n“In contrast, the Z(CAT&SOD) group displayed a more intact retinal structure and lower inflammation levels, comparable to the healthy retinal microenvironment. This dual-enzyme approach not only mitigates inflammation but also helps maintain the integrity of the retinal structure by reducing vascular permeability and preventing secondary vascular abnormalities.”\nReference\n6. Hollyfield, J. G. et al. Oxidative damage-induced inflammation initiates age-related macular degeneration. Nat. Med. 14, 194-198 (2008).\n7. Yao, T. et al. Comparative mechanistic study of RPE cell death induced by different oxidative stresses. Redox Biol. 65, 102840 (2023).\n8. Kerur, N. et al. cGAS drives noncanonical-inflammasome activation in age-related macular degeneration. Nat. Med. 24, 50-61 (2018).\n9. Zhang, R. et al. A pH/ROS dual-responsive and targeting nanotherapy for vascular inflammatory diseases. Biomaterials 230, 119605 (2020).\n4. Fig. S12 references CNV incorrectly; SI injury does not induce CNV, and GA does not involve CNV.\nReply: We thank the reviewer for this valuable feedback. Upon careful consideration, we recognized that the original phrase “lesions in GA mice” could be misleading, as classic GA is pathologically characterized by atrophic lesions (RPE/photoreceptor loss) rather than vascular lesions detectable by FFA. To ensure conceptual clarity, we have revised the figure legend to describe these observations more accurately in the revised manuscript as following. In addition, we would like to clarify the scientific rationale for including this assay. While the SI model primarily recapitulates the oxidative stress and photoreceptor/RPE degeneration characteristic of GA, it also elicits a significant secondary inflammatory response. Importantly, this inflammation can disrupt the blood-retinal barrier (BRB), resulting in quantifiable vascular abnormalities detectable by FFA.\n“FFA images showing therapeutic effects on secondary vascular inflammation in SI-induced retinal degeneration”\n“As shown in Figure 2k and 2l, prominent retinal deposits were observed in both PBS- and SOD-injected groups, accompanied by subretinal debris accumulation (likely originating from degenerated RPE and photoreceptors), inflammatory edema, and oxidative stress-induced vascular abnormalities (Figure S2a).”\n5. ERG a- and b-waves measure retinal function, not visual acuity.\nReply: We sincerely appreciate the reviewer’s insightful comment and fully agree with this important clarification. We acknowledge that certain terms in our original description required greater precision. Specifically, we have revised phrases such as “restoration of visual acuity” in the context of ERG data with more precise descriptions as following in the revised manuscript.\n“Analysis of the a- and b- waves, which reflect photoreceptor and ON-bipolar cell activity, respectively, demonstrated partial recovery of electrophysiological responses in mice treated with either CAT or SOD&CAT injections.”\n6. Missing statistical summaries of ERG amplitudes is a critical issue; plotting style conventions are minor (see Section 5).\nReply: Thanks so much for pointing it out. We would like to clarify that the complete statistical analyses for the ERG amplitude data are provided in the Supporting Information as Figure S12 in our original manuscript.\nFigure R2. Representative ERG wave responses of acute retinal injury mice under scotopic conditions following different treatments, along with statistical analysis of A and B waves measured by ERG (n=4).\n7. The rationale for using 10 mg/kg SI is not provided.\nReply: We appreciate the reviewer’s question regarding the SI dosage. The 10 mg/kg sodium iodate (administered intravenously) was selected based on well-established protocols to ensure a robust and reproducible model of oxidative retinal injury10-12. The related discussion has been added in the revised manuscript as following:\n“This dosage is well-established to consistently induce oxidative damage in RPE cells and photoreceptors, creating an acute retinal injury mouse model that recapitulates key oxidative stress-related cellular events in GA33-35.”\nReference\n10. Liu, B. et al. Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells. Cell Death & Disease 12, 230 (2021).\n11. Chen, Y. et al. Oxidative stress induces mitochondrial iron overload and ferroptotic cell death. Scientific Reports 13, 15515 (2023).\n12. Zhou, Y. et al. Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation. Cell Death & Disease 13, 689 (2022).\n8. The SI model should not be referred to as GA; it is an acute chemical injury distinct from human geographic atrophy.\nReply: Thanks so much for the reviewer’s important comment regarding disease modeling terminology. We fully agree that the sodium iodate (SI) model represents an acute chemical injury model and does not fully replicate the chronic, multifactorial pathogenesis of human geographic atrophy (GA). We selected the SI model because it is a well-established and widely used preclinical model that reliably induces oxidative stress-mediated RPE and photoreceptor degeneration, the key cellular events in GA pathogenesis13-14. This model allows us to specifically test whether our antioxidant nanocage platform can protect these critical cell types from the oxidative damage, a central mechanism in GA progression. We have revised the text to clarify the model’s limitations while maintaining its scientific rational: to evaluate whether targeted antioxidant delivery can rescue retinal cells from oxidative stress, a therapeutic strategy directly relevant to dry AMD/GA. We thank the reviewer for this valuable opportunity to improve our manuscript’s clarity.\nTitle: “Permeable nanoreactor eyedrop for enzymatic cascade-mediated treatment for acute retinal injury model mimicking geographic atrophy”\n“Collectively, our work demonstrates that the zwitterionic nanocage platform enables effective topical delivery of antioxidant enzymes to the retina, presenting a promising therapeutic strategy for combating oxidative stress in conditions such as dry AMD.”\nReference\n13. Fleckenstein, M. et al. Age-Related Macular Degeneration: A Review. JAMA 331, 147-157 (2024).\n14. Ambati, J. et al. An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr- 2-deficient mice. Nature Medicine 9, 1390-1397 (2003).\n9. Mice lack a macula; human GA scoring is not applicable. GA-like lesions in mice should be described as RPE/photoreceptor atrophy.\nReply: We sincerely appreciate the reviewer’s insightful comment regarding species-specific anatomical considerations. As correctly noted, mice lack a macula, which precludes direct application of human GA clinical scoring criteria that rely on macular lesion characteristics. In response to this important observation, we would like to clarify that the scoring system presented in Table R1 represents a customized, semi-quantitative histopathological grading scale specifically developed for this study. This tailored approach enables systematic assessment of two key pathological features in our mouse model: the severity of RPE layer degeneration and the degree of overall retinal thinning. By establishing this species-appropriate evaluation method, we aimed to maintain rigorous pathological assessment while respecting fundamental anatomical differences between murine and human ocular structures.\n“After different treatments, a blinded evaluation was performed based on fundus imaging and OCT imaging to assess retinal structural integrity and thickness reduction(Figure S2b).”\nTable R1. The scoring rubric of acute retinal injury mice model\n10. “Neurodeatrophia” is nonstandard; “neurodegeneration” or “neuronal atrophy” is correct.\nReply: We appreciate the reviewer’s careful attention to terminology. As correctly noted, “Neurodeatrophia” is a nonstandard term. We have replaced all instances with the standard term, “neurodegeneration” throughout the manuscript and figure in this revised manuscript (Figure R3).\nFigure R3. Schematic illustration of antioxidant mechanism disruption and therapeutic intervention in acute retinal injury mice model. (a) In the progression of acute retinal injury model, neurodegeneration is associated with the accumulation of oxidative stress and reduction in peroxidase levels. This imbalance triggers retinal inflammatory pathways, leading to the atrophy and antioxidant dysfunction of RPEs. (b) A groundbreaking and noninvasive eye drop formulation utilizing zwitterionic polymer-modified peroxidase (Z(CAT&SOD)) was developed for GA treatment. The Z(CAT&SOD) eye drops could effectively penetrate the ocular barriers through both conjunctiva-sclera-retina and conjunctiva-blood-retina pathways to reduce oxidative stress and mitigate inflammation by harnessing the via enzymatic cascade, offering a promising therapeutic approach for GA management.\n11. “Atrophy of optic nerve cells” is incorrect; the shapes shown do not resemble photoreceptor outer segments.\nReply: We sincerely appreciate the reviewer’s valuable feedback on our schematic diagram. In response to above comment, we have revised “Atrophy of optic nerve cells” to the accurate term “neurodegeneration”. The diagram was designed to conceptually illustrate the pathological progression from an organized, healthy structure to a damaged, degenerative state. The abstract shapes were intended to represent dystrophic and fragmented photoreceptor remnants within the atrophic zone, consistent with our histological observations.\n12. “Drusen” is misapplied; SI models do not produce true drusen and may represent pseudodrusen.\nReply: Thanks so much for the reviewer’s suggestion. Our schematic aimed to conceptually represent subretinal deposit accumulation in our model’s retinal degenerative phenotype, which shares a conceptual similarity with the deposit-driven pathology in human AMD. While we acknowledge that “Drusen” specifically refers to extracellular deposits located between the RPE and Bruch’s membrane in human eyes, a feature not fully recapitulated in our model, we have revised the terminology to “Drusen-like deposits” in both the schematic and manuscript to reflect these distinctions more accurately while maintaining the conceptual parallel.\nFigure R4. Schematic illustration of the retina of acute retinal injury mice model.\n13. Antioxidant dysfunction is not illustrated in the RPE.\nReply: We sincerely appreciate the reviewer’s constructive suggestion to enhance the conceptual clarity of our schematic. We fully agree that explicitly illustrating antioxidant dysfunction within the RPE cell would better represent this critical pathological mechanism. Accordingly, we have modified the schematic to incorporate this important element, which will significantly enhance its scientific accuracy and educational value.\n14. Panels showing “conjunctival barrier” and “sclerotic barrier” inside cells are anatomically incorrect.\nReply: Thanks so much for the reviewer’s suggestion. Our schematic aimed to illustrate the key ocular barriers that a topically applied formulation must overcome. In response to the comment, we have adjusted the illustration to better highlight the cell-cell interfaces and junctional complexes that form these functional barriers. Additionally, we will update the figure legend to explicitly state: “Schematic representation of the cellular barriers formed by conjunctival and scleral cells.”\n15. The scheme does not accurately show how CAT and SOD reach the RPE; Z(CAT&SOD) contains both enzymes but this is not clear.\nReply: We sincerely appreciate the reviewer’s comment regarding the schematic. We would like to clarify that this illustration is intended as a simplified, conceptual overview highlighting the key steps of our therapeutic strategy. The detailed mechanism of cellular uptake and intracellular delivery of the enzymes (CAT and SOD) by our zwitterionic nanocage, Z(CAT&SOD), is rigorously supported by experimental evidence presented in the manuscript. In the part where we studied the penetration section of Z(CAT&SOD), we provided a detailed description of its penetration mechanism again. See Figure R5 (Figure 3m). What’s more, we also demonstrate the co-loading and quantitative encapsulation efficiency of both CAT and SOD in Table S1. What’s more, we also tested the enzyme activity of CAT and SOD in the zwitterionic polymer nanocages.\nFigure R5. Schematic illustration of penetration mechanism of zwitterion peroxidase.\n16. HCECs and HConEpiCs are irrelevant; SI injury affects RPE and photoreceptors.\nReply: We sincerely appreciate the reviewer’s comment. This provides us with a valuable opportunity to clarify the dual purpose of our schematic. This illustration is not limited to depicting the SI injury model; rather, it presents a comprehensive overview of our drug delivery strategy: from topical application and traversal of anterior segment barriers to therapeutic action at the retinal disease site. Importantly, removing the anterior segment cells (HCECs, HConEpiCs) would mispresent our study, as these elements are essential for demonstrating the delivery challenge that our zwitterionic nanocage specifically addresses.\n17. Methods list ARPE-32, HUVECs, and RAW 264.7 cells, but none appear in the results. HCECs are not listed in Methods and are corneal endothelial cells, not relevant to the study.\nReply: We sincerely appreciate the reviewer’s careful evaluation of our Methods and Results sections. We acknowledge and apologize for the identified discrepancies, which resulted from an oversight during manuscript preparation. We are grateful for the opportunity to address these issues and have conducted a comprehensive, cross-referential review of the entire manuscript to ensure full consistency between the methodologies described and the results presented.\n“Human conjunctival epithelial cells (HConEpiC) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in DMEM containing 1% penicillin/streptomycin and 10% FBS at 37 °C in 5% CO . Seven-week-old female C57BL/6 mice 2 were purchased from Nanjing Pengsheng Biological Technology Co. The animals were acclimated in SPF environment for one week prior to conducting follow-up experiments. All animal procedures complied with relevant laws and were approved by the Institutional Animal Care and Use Committee of Soochow University (No. ECSU-2019000198).”\n18. The subtitle “Construction of a GA model” is misleading; the model is an acute chemical injury.\nReply: We appreciate the reviewer’s valuable suggestion and fully agree with the recommendation. Accordingly, we have modified the subtitle from “Construction of a GA model” to the more accurate description “Construction and evaluation of acute retinal injury mice model” in this revised manuscript as following:\n“Construction and evaluation of acute retinal injury mice model”\n19. Methods for ocular assays (fundoscopy, OCT, ERG), instrumentation, and analyses are not described, indicating lack of ocular biology expertise.\nReply: We sincerely appreciate the reviewer’s comment regarding the methodological aspect. We apologize for this oversight, which stemmed from our assumption that these protocols were standard practice rather than from any technical limitations. We have provided the detailed methodological specifications that were inadvertently omitted in this revised manuscript as following:\n“In Vivo Imaging (Fundoscopy, OCT and ERG): All imaging was performed using a Micron IV retinal imaging system (Phoenix Research Labs) coupled with a high-resolution OCT module and ERG module. Prior to imaging, mice were anesthetized via intraperitoneal injection of ketamine (80 mg/kg) and xylazine, followed by pupil dilation with with topical administration of 1% tropicamide.\nFundoscopy: Color fundus images were captured using the integrated color fundus camera under standard white light illumination.\nSpectral-Domain OCT (SD-OCT): Retinal cross-sectional scans were acquired using the scanning protocol centered on the optic nerve head. Subsequent quantification of retinal layer thicknesses was performed using image J system.\nElectroretinography (ERG): Full-field ERG recording was performed in accordance with the International Society for Clinical Electrophysiology of Vision (ISCEV) standard protocol for rodents. Following overnight dark adaptation (>12 hours), mice were anesthetized as above and their pupils were dilated under dim red illumination. Scotopic (rod-dominated) responses were first recorded using a series of flashes with increasing intensities (3 log cd·s/m2). For photopic (cone-mediated) responses, mice were light-adapted for 10 minutes under a 30 cd/m2 background before recording responses to a 1.0 log cd·s/m2 flash stimulus. ERG waveform analysis included: a-wave amplitude (baseline to trough of initial negative deflection), b-wave amplitude (a-wave trough to the peak of subsequent positive wave), implicit times for both components. All recordings and analyses were performed using the integrated ERG module of the Micron IV retinal imaging system.”\n20. Intravitreal injections of 5 µL are physiologically impossible in mice (vitreous≈2 µL).\nReply: We sincerely appreciate the reviewer’s comment regarding the critical technical aspect. The reviewer is correct in noting that a 5 µL intravitreal injection would exceed physiological limits in the mouse eye (with a vitreous volume of approximately 2 µL) and could potentially cause ocular damage. We would like to clarify that in all experimental procedures, we strictly adhered to the standard injection volumes of either 1 µL or 2 µL, as explicitly detailed in Section 4 of the methods. These volumes are well-established and well-tolerated in murine models. The reference to “5 µL” appears to be an inadvertent typographical error that may have appeared in supplementary materials or figure legends. We have immediately conducted a thorough, line-by-line review of the entire manuscript to ensure the intravitreal injection volume is correct.\n“We first induced acute retinal injury model in mice using SI on Day 0 and randomly divided them into four groups: Untreated (1×PBS, 2 μL), Z(CAT) (2 mg/mL, 80000 U/g, 1 μL), Z(SOD) (2 mg/mL, 50000 U/g, 1 μL), and Z(CAT&SOD) (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL).”\n“After intravenous injection of SI, subjects were randomly divided into six groups: Untreated (1×PBS eyedrop, 2 μL twice daily for a week), CAT&SOD eye drops (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL twice daily for a week), CAT&SOD intravitreal injection (same concentration, single 2 μL injection), Z(CAT&SOD) eye drops (same concentration, 2 μL twice daily for a week), Z(CAT&SOD) intravitreal injection (same concentration, single 2 μL injection), and lutein oral administration (0.5 μg lutein per day for one week).”\n21. Hyperfluorescent spots from FAF images are not quantified.?\nReply: Thanks so much for the reviewer’s attention of the imaging data. We would like to clarify that the images were obtained using fluorescein angiography (FFA) or color fundus photography (CFP) rather than fundus autofluorescence (FAF). We apologize for any confusion and have verified that all figures and descriptions in the manuscript accurately reflect the imaging techniques employed in our study.\n22. Claims of “pioneering a new therapeutic paradigm for dry AMD” overstate the study.\nReply: We sincerely appreciate the reviewer’s the reviewer’s valuable feedback regarding the tone of our conclusion. We fully acknowledge that the phrase “pioneering a new therapeutic paradigm” may be considered overly ambitious, and we grateful for the opportunity to revise the concluding statements to reflect the scope and significance of our findings while maintaining scientific rigor more precisely.\n“This work develops a novel therapeutic platform that tackles two critical challenges in dry AMD treatment including non-invasive delivery and combined antioxidant therapy.”\n23. The SI model is not representative of human GA or AMD.\nReply: We appreciate the reviewer’s insightful comment. We fully acknowledge that nearly no animal model perfectly recapitulates the complex, chronic progression of human GA/AMD. However, our study is primarily a mechanism-driven proof-of-concept investigation, designed to test whether non-invasive delivery of antioxidant enzyme can protect the retina from oxidative injury-a key pathogenic driver in dry AMD. For this specific purpose, the SI model serves as is a validated experimental system that reliably induces oxidative stress-mediated RPE and photoreceptor degeneration, which are hallmark cellular events in GA. Given the current lack of effective GA therapies, exploring novel strategies in reproducible models remains critically important. Additionally, we have carefully revised the manuscript wording better reflect these considerations as following.\nTitle: “Permeable nanoreactor eyedrop for enzymatic cascade-mediated treatment for acute retinal injury model mimicking geographic atrophy”\n24. Simply increasing SOD/CAT is not universally protective; overexpression can have unintended effects.\nReply: We appreciate the reviewer’s insightful comment regarding the potential risks of antioxidant enzyme overexpression. We completely agree that constitutive overexpression can disrupt redox homeostasis, a well-known challenge in antioxidant therapy. However, our approach differs fundamentally: rather than genetically inducing chronic enzyme production, we employ localized delivery of precisely dosed SOD and CAT co-encapsulated in a protective nanocage. This pharmacologic strategy ensures stoichiometric enzyme delivery to injury sites without genetic alteration. Importantly, our in vivo safety data (Figure R1/S14) demonstrate no detectable toxicity, after single or repeated administration, directly addressing the reviewer’s concern. While we acknowledge the biological complexities of antioxidant interventions, our study provides the first evidence that transient, dual-enzyme delivery can achieve efficacy without the off-target effects associated with traditional antioxidant strategies. In summary, while we share the reviewer’s caution, our data show that controlled, localized enzyme delivery circumvents the historical pitfalls of nonspecific antioxidant approaches.\n“In summary, this work demonstrated that the pathogenesis of acute retinal injury involves excessive retinal ROS production and dimished peroxidase activity. Replenishment of CAT and SOD effectively attenuated retinal oxidative stress and inflammation, thereby reducing cellular apoptosis. Moreover, we developed a localized delivery system featuring precisely dosed SOD and CAT co-encapsulated in a protective nanocage, enabling stoichiometric enzyme delivery to injury sites while avoiding genetic alteration.”\n25. Evidence regarding GPx/TAS in AMD, delivery of CAT/SOD to RPE mitochondria, and SOD mimetics or gene therapy as clinically feasible strategies is not addressed.\nReply: We appreciate the reviewer’s valuable perspective on this issue. While we fully acknowledge the important roles of glutathione GPx and TAS in AMD pathophysiology, our decision to focus on the CAT/SOD cascade was driven by our experiment findings showing their specific downregulation in our GA model (Figure 2i, S1b, c) and their direct causal link to ROS-mediated apoptosis. This formed the basis for our targeted therapeutic strategy of replenishing these compromised endogenous enzymes. We have incorporated discussion of GPx/TAS and their complementary antioxidant roles in the revised manuscript.\nRegarding subcellular targeting, we agree that mitochondrial delivery would be ideal given its role as a major ROS source. While organelle-specific targeting remains challenge for protein therapies, our work establishes a crucial foundation by achieving efficient delivery of active enzymes to retinal cells (RPE/photoreceptors), as demonstrated by the confocal imaging (Figure 3f) and functional rescue data (Figs. 4-6). Future platform iterations incorporating mitochondrial-targeting motifs represent an exciting direction, which have highlighted in the revised manuscript.\nCollectively, our system represents a non-genetic protein replacement strategy that leverages the natural catalytic efficiency of evolutionarily optimized enzymes. Our nanocage system effectively preserves their full native activity while enabling non-invasive delivery.\n“Our findings highlight the therapeutic potential of enzymatic antioxidant therapy while revealing important avenues for further research. First, comprehensive investigation of endogenous antioxidant systems, particularly the glutathione peroxidase (GPx)/total antioxidant status (TAS) and related peroxidases, could uncover synergistic mechanisms. Developing organelle-specific delivery platforms may further optimize therapeutic efficacy in oxidative retinal injury models. Second, while the SI-induced acute model has provided valuable mechanistic insights, its limitations in modeling the chronic, multifactorial nature of human dry AMD should be acknowledged. Complementary model systems that better recapitulate disease progression will be crucial for translating these antioxidant strategies into clinically viable therapies.”\n26. Fig. R1 (now Fig. S14) legend does not specify whether assays were performed in untreated or SI-treated animals.\nReply: Thanks so much f for pointing it out. The long-term safety test was conducted in healthy mice, and we have included this detail in the SI section.\n“Figure S14. Safety assessment of Z-polymer and Z(CAT&SOD) in healthy mice (n=3).”\n27. Figures S9b and S13b lack clear legends, treatment descriptions, and baseline “( before treatment”) images.\nReply: We sincerely appreciate the reviewer’s f suggestion and apologize for any confusion caused. Regarding the referenced figures (Figure S9b and S13b), they comprehensively document the complete treatment protocols and dosage information corresponding to Chapters 3 and 4, respectively. We would like to clarify that the absence of pre-treatment assessment stems from our experimental design: therapeutic intervention was intentionally initiated immediately after establishing the SI-induced acute retinal injury model. This approach was specifically to prevent irreversible pathological progression in this of the acute chemical-induced retinal damage model. Therefore, mice in each group were in a healthy state prior to treatment, and this baseline condition was included as a control group in the study.\n28. Terms such as “thickness length of CNV” are unclear.\nReply: Thanks so much for pointing it out. We have made corresponding revisions in the revised manuscript.\n“Figure S12. (a) Schematic illustration of the treatments in SI-induced acute retinal injury model mouse model, along with fundus assessment conducted after one and two weeks. (b) Statistical analysis of the thickness length of retina by Image J (n=4).”\n29. Missing “respectively” when defining a- and b-waves (p.4, line 144; p.5, line 180).\nReply: Thanks so much for pointing it out. We have made corresponding revisions in the revised manuscript.\n“Analysis of a- and b- waves, which correspond to photoreceptors and ON-bipolar cell activity respectively, demonstrated partial restoration of visual acuity in mice treated with CAT or SOD&CAT.”\n30. Plotting style of some ERG panels (e.g., Fig. 4g) is unconventional.\nReply: We sincerely appreciate the reviewer’s comment. The figure presents the statistical analysis of a-wave and b-wave amplitudes, which have been refined for improved clarity and visual presentation.\nFigure R6. Statistical analysis of A waves and B waves in retina of mice in different groups under scotopic condition (n=4).\n31. “Eye drop” should be two words.\nReply: Thanks so much for pointing it out. We have changed \"eyedrop\" to the standard form \"eye drop\" throughout the manuscript.\n32. Abstract and introduction are excessively long; discussion does not include limitations.\nReply: Thanks so much for pointing it out. We have revised abstract and introduction.\n“Abstract:\nAge-related macular degeneration (AMD), particularly in its most prevalent form geographic atrophy (GA), has historically been challenging to treat due to its complex pathogenesis. A key driver of GA progression is the oxidative stress-induced apoptosis of retinal cells, ultimately leading to blindness. In our study using a retinal acute injury mouse model to mimic the reactive oxygen species (ROS)-induced retinal degeneration pathogenesis of GA, we consistently observed effective recovery of enzymatic cascade may be an effective therapeutic strategy for this model. To overcome ocular delivery barriers, we engineered zwitterionic nanocages for co-delivering CAT and SOD. This system significantly alleviated oxidative damage and inflammation in an SI-induced retinal injury model. Given the chronic and progressive nature of GA, zwitterionic-peroxidase was optimized to develop a non-invasive eye drop formulation for GA management. Surprisingly, the zwitterionic-peroxidase achieved a remarkable 9.32% penetration rate into the eyeball, significantly outperforming the free peroxidase (0.54%), through conjunctival-sclera-retina and conjunctival-blood-retina penetration pathways, and achieved therapeutic efficacy comparable to intravitreal injections. Additionally, extra supplementation of Z(CAT&SOD) in acute retinal injury model showed superior therapeutic effects compared to the current gold standard clinical treatment of supplementing antioxidants and lutein. Our pioneering study revealed the possible association between retinal peroxidase deficiency and GA progression, and proposed an innovative zwitterionic nanocage-based peroxidase, which exhibits great potential as a non-invasive yet highly effective therapeutic strategy for AMD.”\n“Introduction:\nAge-related macular degeneration (AMD) is a progressive and degenerative disease characterized by dysfunction and death of retinal pigment epithelium (RPE) cells and photoreceptors, leading to irreversible vision loss1, 2. It is the leading cause of permanent visual impairment worldwide, affecting more than 180 million people, mainly over the age of 55. AMD manifests in two primary forms: geographic atrophy (GA) and choroidal neovascularization (CNV). Among them, GA patients constitute approximately 85% to 90%, and the rate of blindness approaches nearly 20%3. Since the introduction of intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF), patients with CNV have experienced substantial improvements in retinal function. However, despite the effectiveness of anti-VEGF therapies for CNV, these antibodies have proven ineffective in treating GA4. Therefore, there is currently no approved treatment for GA, primarily due to its complex and poorly understood pathogenesis. Clinically, supplementing antioxidants such as lutein appears to be the only viable approach to slowing down the deterioration of GA5. Numerous studies have revealed that relative pathogenesis of GA involves processes such as complement system activation, disturbances in lipid metabolism, oxidative stress, abnormal autophagy, and the chronic inflammation pathways and etc6-8. In response, various therapeutic strategies have been explored, including the suppression of complement activation, inhibition of angiogenesis, and exertion of anti-inflammatory effects9-11. However, these approaches have not yet demonstrated specific, positive impacts in clinical trials involving GA patients12-14. Therefore, the exploration of alternative therapeutic targets for GA treatment remains crucial.\nOxidative stress disrupts the physiological functions in most biological tissues and can lead to cell death15, 16. In the retina, oxidative stress triggers a cascade of events that result in focal loss of RPE and photoreceptors, leading to structural changes in proteins, lipids and DNA, which is increasingly recognized as a major contributing factor to the development of GA12, 15, 17. Numerous studies have demonstrated that the removal of free radicals from the retina can effectively slow the progression of AMD18. Additionally, in AMD treatment research, strategies targeting the elimination of reactive oxygen species (ROS) have been proposed, including the use of inorganic nanozymes like Pt, RhZ, CeO to perform antioxidant function19-22. However, these nanozymes face challenges such as 2 biocompatibility, biodegradability, and unclear therapeutic efficacy. Endogenous antioxidant enzyme, such as superoxide dismutase (SOD) and catalase (CAT), neutralize free radicals and maintain cellular homeostasis, with SOD targeting superoxide anion (O •−) and CAT decomposing hydrogen peroxide 2 (H O ). These enzymes play essential roles in defending against oxidant stress, particularly in 2 2 regulating ROS in inflammatory diseases23-26. However, the specific roles of CAT and SOD in the pathogenesis of age-related macular degeneration, especially during the geographic atrophy stage, require further investigation.\nIn this study, we discovered that the cellular antioxidant defense mechanisms were obviously compromised in SI-induced acute retinal injury model, pathological feature of which mimic that of human GA disease27, as evidenced by a noticeable decrease in the expression of CAT and SOD in the retina. While supplementation with CAT and SOD partially alleviated retinal atrophy and visual impairment, the therapeutic effects were not satisfactory. These limitations arise from poor ocular delivery and enzyme instability. Hence, we explored the use of zwitterions, which can polymerize in situ on protein surfaces to effectively shield charges, enhance permeability and preserve enzyme activity in complex physical environment28-32. By ingeniously employing zwitterionic materials, we coated the CAT and SOD with sulfobetaine methacrylate (SBMA), Z(CAT&SOD), a compound widely recognized for its charge regulation, mucosal and tissue penetration and biocompatibility. Remarkably, the Z(CAT&SOD) exhibited improved stability and activity against protease. Moreover, Z(CAT&SOD) demonstrated the ability to facilitate free diffusion across ocular barriers penetration capabilities by promoting clathrin-dependent and caveolae-mediated endocytosis. As expected, following intravitreal injection of Z(CAT&SOD) in acute retinal injury model, the enzymatic cascade reactions exhibited superior ROS scavenging capacity and suppressed the activation of NF-κB-HMGB1-ILs inflammatory pathway. Ultimately, they protected RPEs and photoreceptors from ROS-induced apoptosis, which is crucial for preserving retinal function and preventing degenerative changes. Given the chronic nature and long-term treatment requirements of this disease, we attempted to develop a non-invasive peroxidase eye drop formulation, compared with the clinical gold-standard treatment strategy, supplementing with lutein. Unexpectedly, optimizing the components of zwitterionic polymers led to a significant enhancement in the ocular barrier permeability of zwitterionic-peroxidase, which achieved an impressive 9.32% ocular penetration (the ocular penetration of free peroxidase was only 0.54%) by increasing the permeability through both conjunctiva-sclera-retinal and blood-retinal pathways. Remarkably, the therapeutic effects of zwitterionic-peroxidase eye drops were comparable to those achieved through intravitreal injections in the acute retinal injury model, and both of them demonstrated greater efficacy compared to oral lutein therapy. Our research results emphasize that the progression of GA is associated with clear oxidative stress damage to the retina. The innovative and non-invasive eye drop we developed, which is based on a bimetallic ion nanocage coating of peroxidase, provides a transformative and promising therapeutic approach for dry AMD (Figure 1).”\n“Discussion:\nIn the SI-induced acute retinal injury model, we observed pathological features resembling those in clinical GA patients, including subretinal drusen-like deposits and neurodegeneration. Mechanistic investigations revealed that RPE and photoreceptor cell apoptosis was closely associated with the microenvironment characterized by high oxidative stress and diminished peroxidase activity, particularly reduced levels of CAT and SOD. As a proof-of-concept, our findings suggest that timely antioxidant supplementation may preserve residual photoreceptors when oxidative damage as the primary driver of degeneration. However, the coulombic interactions within the vitreous body and the ILM barrier in the vitreous cavity posed significant challenges to the diffusion and penetration of peroxidases into the RPE and optic nerve cells, even when employing intravitreal injection as a benchmark ophthalmic drug delivery modality. To overcome these challenges, we developed zwitterionic nanocages for CAT and SOD to enhance diffusion and barrier penetration by shielding surface charges and promoting endocytosis pathways. Following the intravitreal injection of Z(CAT&SOD), we observed a significant mitigation of retinal damage in SI-induced acute retinal injury model. This was evidenced by reduced ROS-induced damage in RPE and optic nerve cells, suppression of the NF-κB-HMGB1-ILs inflammation pathways. Therefore, the strategy of using zwitterionic peroxidase with intravitreal injection shows promise for slowing the progression of dry AMD.\nHowever, early-stage dry AMD patients often delay therapy due to concerns about traumatic scarring and postoperative infection, thereby missing the optimal treatment window. Moreover, patients with advanced atrophic dry AMD require sustained therapeutic drug concentrations over an extended period. Developing an effective, non-invasive ophthalmic treatment strategy based on peroxidase is of great importance. To address this, we optimized the components of zwitterionic polymers to select the nanocages with the strongest barrier penetration as carriers for a peroxidase eye drop formulation. Unexpectedly, eyeball penetration of Z(CAT&SOD) eye drop was 9.32%, much higher than that of free peroxidase (the eyeball penetration was only 0.54%). Furthermore, after applying the Z(CAT&SOD) eye drop for two weeks, we observed a significant reduction in retinal cell apoptosis induced by SI, alongside stabilization of retinal thickness and visual function, achieving comparable therapeutic effects to those observed in the intravitreal injection of Z(CAT&SOD). More interestingly, compared with the therapeutic effects of both intravitreal injection and eye drops of Z(CAT&SOD), lutein exhibited inferior therapeutic efficacy. This may be attributed to the retina was affected by the blood-eye barrier in the systemic circulatory system, resulting in limited absorption and the limitation of ROS-scavenging ability of lutein. In summary, this work reported that the pathogenesis of acute retinal injury model is linked to the overproduction of retinal ROS and reduced peroxidase levels. Replenishing CAT and SOD could reduce retinal oxidative stress and inflammation, decreasing cell apoptosis. And we employed localized delivery of precisely dosed SOD and CAT co-encapsulated in a protective nanocage, which ensured stoichiometric enzyme delivery to injury sites without genetic alteration. Collectively, our work demonstrates that the zwitterionic nanocage platform enables effective topical delivery of antioxidant enzymes to the retina, presenting a promising therapeutic strategy for combating oxidative stress in conditions such as dry AMD.\nOur findings highlight the therapeutic potential of enzymatic antioxidant therapy while revealing important avenues for further research. First, comprehensive investigation of endogenous antioxidant systems, particularly the glutathione peroxidase (GPx)/total antioxidant status (TAS) and related peroxidases, could uncover synergistic mechanisms. Developing organelle-specific delivery platforms may further optimize therapeutic efficacy in oxidative retinal injury models. Second, while the SI-induced acute model has provided valuable mechanistic insights, its limitations in modeling the chronic, multifactorial nature of human dry AMD should be acknowledged. Complementary model systems that better recapitulate disease progression will be crucial for translating these antioxidant strategies into clinically viable therapies.”\n33. Missing references (p.3, lines 94–96; p.11, lines 452–455).\nReply: We have added the references according to the reviewer’s suggestion.\n34. Many figure legends lack treatment details, orientation, and arrow explanations.\nReply: Thanks so much for pointing it out. We have added orientation and arrow explanations in some figures.\n35. IR fundus images (Fig. 4b, 6e) do not indicate whether they are from the same animals.\nReply: We sincerely appreciate the reviewer’s valuable observation. The IR fundus images in Figure 4b and 6e represent longitudinal tracking of the same animal cohort. This can be confirmed through careful examination of each mouse’s unique retinal vascular signature, including specific vessel branching patterns, trajectories and bifurcation points, which remains consistent across different time points, which serves as a reliable intrinsic identifier.\n36. OCT images lack optic nerve localization and measurement indicators.\nReply: Thanks so much for pointing it out. We have marked the location of the optic nerve on the corresponding OCT images, and described the measurement method in the methods section.\nFigure R7. Representative fundus images and OCT images of lesions in healthy versus acute retinal injury model mice at different timepoints. Internal Limiting Membrane (ILM); Nerve Fiber Layer (NFL); Ganglion Cell Layer (GCL); Inner Plexiform Layer (IPL); Inner Nuclear Layer (INL); Outer Plexiform Layer (OPL); Outer Nuclear Layer (ONL); External Limiting Membrane (ELM); Photoreceptor Layer (Inner Segment/IS and Outer Segment/OS); Retinal Pigment Epithelium (RPE).\n37. Source tissue for mRNA analyses is unspecified.\nReply: Thanks so much for the reviewer highlighting this methodological detail, and we have specified this clearly in the revised Methods section.\n“Additionally, we collected retinas from acute retinal injury model mice at both one week and two weeks, and quantified the expression levels of CAT and SOD by quantitative real-time polymerase chain reaction (qPCR). At two weeks, a significant reduction in the expression of both CAT and SOD was observed (Figure 2i).”\n“Retinas were extracted from the eyeballs of SI-mice and lysed using a sonic disruptor in Western and IP sample lysates (500 μL) at 0 oC. The supernatant was then centrifuged, and 100 microliters were used for CAT and SOD assay tests. Additionally, RNA expression levels of CAT and SOD were assessed by qPCR analysis. Retinal cells isolated from GA mice were lysed with Trizol, and the RNA was diluted to a final concentration of 200 ng/μL for analysis. Primer and probe sequences were as follows: CAT forward primer: AAATGCTTCAGGGCCGCCTT; CAT reverse primer: GTAGGGACAGTTCACAGGTA; SOD forward primer: AACAATGGTGAAGGCTGTTGCTGT; SOD reverse primer: TTGTGCATGCACATGGAACCCATG; GAPDH forward primer: GTGACGTTGACATCCGTAAAGA; GAPDH reverse primer: GTAACAGTCCGCCTAGAAGCAC. These primes were synthesized by Servicebio.”\n38. TUNEL staining in the RPE is not indicated.\nReply: We sincerely appreciate the reviewer’s comment regarding the dynamic pathological progression in our acute injury model. The observation can be explained by both technical considerations and the temporal progression of SI-induced damage. The focal signal detected in Healthy RPE layers represents tissue autofluorescence, a technical artifact rather than genuine apoptosis. Regarding the absence of TUNEL signal in the RPE layer of SI-injured mice, this is finding accurately reflects the model’s pathophysiology: SI induces rapid, severe oxidative damage that primarily targets RPE cells, leading to early apoptosis/necrosis and subsequent clearance of these vulnerable cells by the 1-week endpoint (our tissue harvest time). Consequently, the remaining RPE layer appears depopulated or severely damaged, explaining the lack of late-stage TUNEL signal. Conversely, the prominent TUNEL signal observed in the outer nuclear layer and inner nuclear layer (INL) at this later time point represents secondary, progressive apoptosis in neuronal cells, driven by the ongoing oxidative stress and inflammatory microenvironment following the initial RPE injury.\n“TUNEL analysis further demonstrated that Z(CAT&SOD) significantly attenuated SI-induced acute retinal injury by suppressing retinal cells apoptosis, particularly in the RPE layer. Notably, without timely ROS-scavenging intervention, RPE cell apoptosis progressed to near-total depletion, highlighting the critical window for therapeutic efficacy (Figure 5a).”\nFigure R8. Representative immunofluorescence staining images showing the expression of TUNEL in the retina of mice in different groups (scale bar: 50 μm; the arrow indicates the direction of the retina).\n39. Individual data points are missing from plots, especially Fig. 3l and ERG amplitude analyses.\nReply: We have revised all relevant bar graphs (including Figure 3l and all ERG amplitude analyses) to display scatter plots (individual data points) overlaid with the mean ± SEM (or SD) according to the reviewer’s suggestion.\n✱✱✱✱\n%100 ns\ne\nk\na 80\ntp\nu 60\nlle\nc 40\ne\nv\nita 20\nle\nR 0\nntreate\nd\n+ A\nM\n+ Ge\nni\n+ β- C\nD\n+ C P\nZ\nU\nFigure R9. Relative uptake of Z(CAT-Cy5.5) by cells after incubation with different pathway inhibitors (n=6).\n40. Some ERG plots are partially obscured by axis labels.\nReply: Thanks so much for pointing it out. We have adjusted the layout of the affected ERG figures to ensure all plot elements are fully visible and not obscured by axis labels in the revised manuscript.\n41. Fig. 5a: retinal orientation unclear; TUNEL higher in “Healthy” vs. “Untreated” is unexplained.\nReply: Thanks so much for pointing this out. We acknowledge that the current version lacks explicit anatomical labeling and we have revised the figure accordingly to ensure clarity in the manuscript. The focal, bright signal observed in the RPE layer of the “Health” sample represents a known technical artifact. As the RPE contains abundant melanin and other pigments, these can cause autofluorescence and/or non-specific background signals in certain TUNEL assay conditions, leading to localized false-positive signals. In contrast, the diffuse and intense TUNEL staining observed throughout all nuclear layers in the “Untreated” (SI-injured) group represents genuine, widespread apoptosis induced by oxidative stress.\nFigure R10. Representative immunofluorescence staining images showing the expression of TUNEL in the retina of mice in different groups (scale bar: 50 μm; the arrow indicates the direction of the retina).\n42. Fig. 5l schematic is not informative.\nReply: We sincerely appreciate the reviewer’s comment regarding Figure 5l. This schematic serves as an integrative summary, consolidating key molecular pathway findings from the preceding panels (Figure 5a to 5k) and related data. Specifically, it illustrates how the oxidative insult triggers activation of the NF-κB-HMGB1-ILs inflammatory axis (as demonstrated by antibody-based localization in Figure 5b, 5c and 5d) and drives macrophage polarization toward a pro-inflammatory state, ultimately driving retinal cell apoptosis.\nFigure R11. Schematic illustration of the oxidative stress triggers activation of the NF-κB-HMGB1- ILs inflammatory axis in relative retinal cells.\n43. Fig. 6e: timing of lutein treatment relative to SI is unclear; prophylactic vs. post-injury effects differ.\nReply: We appreciate the reviewer’s insightful question regarding the timing of lutein administration. In in this study, including oral lutein supplementation (0.5 µg/day), were initiated therapeutically after SI-induced injury. Specifically, lutein administration began on the same day following SI injection and continued daily for one week, paralleling the treatment window for the enzyme-based interventions. This design was chosen to mimic a clinically relevant therapeutic approach, as patients typically start antioxidant supplementation after disease onset or diagnosis.\n“After intravenous injection of SI, subjects were randomly divided into six groups (Figure S12a): Untreated (1×PBS eyedrop, 2 μL twice daily for a week), CAT&SOD eye drops (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL twice daily for a week), CAT&SOD intravitreal injection (same concentration, single 2 μL injection), Z(CAT&SOD) eye drops (same concentration, 2 μL twice daily for a week), Z(CAT&SOD) intravitreal injection (same concentration, single 2 μL injection), and lutein oral administration (0.5 μg lutein per day for one week).”\nFigure R12. Schematic illustration of the treatments in SI-induced acute retinal injury mouse model, along with fundus assessment conducted after one and two weeks.\n44. Fig. 6f: statistical analysis of a- and b-wave amplitudes for all animals is missing.\nReply: Thanks so much for pointing it out. We would like to clarify that the full statistical analyses for the ERG amplitude data are included in the Supporting Information as Figure S12 in the original manuscript.\n45. CAT-FITC enzymatic activity is not reported.\nReply: We sincerely appreciate the reviewer’s comment regarding the use of CAT-FITC conjugate. This fluorescent tracer was used exclusively as a subset of in vitro and ex vivo experiments (e.g., cellular uptake studies) to qualitatively visualize the intracellular localization and delivery efficiency of our nanocage platform. While full retention of enzymatic activity would be ideal, it was not a primary requirement for these proof-of-concept imaging studies. Moreover, we have provided direct experimental evidence that our core therapeutic formulation successfully maintains activity of enzymes.\nFigure R13. The enzyme activity stability of Z(SOD), Z(CAT), free SOD and free CAT at different storage environments (4 °C, 25 °C and 37 °C).\nSummary: The manuscript contains fundamental flaws in experimental design, methodology, and biological interpretation. Major issues include misrepresentation of the SI injury model as GA, inappropriate in vitro cell models, physiologically impossible intravitreal injections, missing descriptions of ocular assays and analyses, and multiple inaccuracies in figures and data interpretation. These flaws significantly compromise the validity of the study.\n1. Reply: We sincerely appreciate the reviewer’s thoughtful evaluation of our work. In response to the comprehensive feedback, we have implemented the following substantive revisions to the manuscript: Model clarification: the experimental model has been explicitly defined and consistently referenced throughout the manuscript to eliminate any ambiguity.\n2. Technical correction: we have verified and confirmed that intravitreal injection volume were correctly reported as 1 µL or 2 µL in our original manuscript. The reviewer’s mention of of 5 µL appears to stem from a misinterpretation, which we have now clarified in the revised text. .\n3. Methodological rigor: all ocular assay protocols have been expanded with granular detail, including instrumentation parameters, quality control measures, and validation steps to ensure full reproducibility.\n4. Comprehensive revisions: every textual statement and graphical element has undergone dual verification for numerical accuracy, terminological precision, and logical consistency with the dataset.\nThrough these revisions, we have strengthened the manuscript’s scientific rigor and transparency. We are confident the current version provides robust evidentiary support for its conclusions and respectfully submit it for reevaluation.\nReviewer #2:\nThe authors have satisfactorily addressed my concerns; I recommend acceptance.\nReply: Thanks so much for the reviewer’s positive comment.\nReviewer #3:\nThe authors did a nice job to address my concerns and I would like to reccomend accept this manuscript without scientific change.\nReply: Thanks so much for the reviewer’s positive comment."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The study reports that dual-enzyme (CAT/SOD) delivery via nanocages can reduce oxidative injury in a sodium iodate-induced acute retinal injury model. However, due to fundamental flaws in experimental design, model choice, and data interpretation, these results cannot be considered reliable or generalizable to human GA/AMD. The study overinterprets acute toxin-induced injury as a model for GA/AMD, and its findings do not provide a clear advance over established antioxidant delivery studies.\nThe conclusions regarding therapeutic potential for dry AMD are unsupported. Key claims rely on misapplied animal and cellular models, schematic figures that may mislead readers, and incomplete or misinterpreted imaging and functional data. While the authors corrected several points to accommodate the concerns, overall credibility is diminished by rewriting and over-simplifications in response to prior criticisms. Multiple conceptual, methodological, and interpretive flaws remain, including inappropriate use of the SI model to mimic GA, overemphasis on vascular outcomes, missing statistical analyses in main figures, and schematics that do not clearly depict experimental or anatomical reality.\nMethodological concerns include use of irrelevant in vitro cell types, incomplete descriptions of imaging and electrophysiology protocols, and misrepresentation of animal pathology. Although some methodological details have been clarified, critical information remains inconsistent or inaccurate, particularly regarding relevant cell types, imaging quantification, and schematic representation of enzyme delivery. Consequently, reproducibility is not ensured, and the manuscript does not meet field standards.\nThe authors’ responses and revisions do not adequately address key conceptual, methodological, and technical concerns raised in prior rounds. Overall, the manuscript's conclusions regarding therapeutic efficacy for dry AMD remain unsupported.", "reviewer_4": "This manuscript offers compelling evidence that a zwitterionic nanocage-based peroxidase eye drop therapy could be a game-changer for treating oxidative stress-related retinal diseases. These diseases may include geographic atrophy (GA) and dry age-related macular degeneration (AMD). The work addresses a pressing gap in ophthalmology—therapeutic options for dry AMD remain scarce. By pairing an innovative delivery platform with a deeper understanding of oxidative injury, this study could really move the needle in the field.\nThe authors thoughtfully build on earlier antioxidant strategies. These include inorganic nanozymes such as Pt-, RhZ-, and CeO₂-based systems, which have struggled with poor biocompatibility and questionable efficacy. The zwitterionic nanocage platform sidesteps many of these issues. It boosts molecular stability, tissue permeability, and catalytic activity, making it a more practical therapeutic option. The non-invasive eye drop formulation improves patient compliance and accessibility, especially for people in the early stages of disease who hesitate to pursue more invasive treatments. After incorporating and addressing the reviewers' comments, the work is substantially more coherent.\nThat said, there are some limitations worth addressing to strengthen the translational relevance of these findings. The authors lean heavily on the sodium iodate (NaIO₃) model. While this model is widely used and practical for inducing RPE degeneration, it doesn’t fully mirror human AMD. NaIO₃ causes acute, chemically driven oxidative injury, leading to rapid RPE death. This occurs through apoptosis, necroptosis, and ferroptosis. Human AMD, on the other hand, unfolds slowly and involves a complex mix of lipid and protein buildup, chronic inflammation, complement dysregulation, and genetic factors. Also, NaIO₃ results are highly dose-dependent and can vary significantly from animal to animal. So the model captures only one piece of the AMD puzzle and might make the therapy appear more robust than it would be in chronic, real-world disease. These limitations don’t undermine the study, but they do suggest the authors should be cautious about claims of disease modification. The response to the review does not fully cover this.\nThe modified experimental design, data analysis, and interpretation are rigorous and well supported by the evidence presented. The reported therapeutic efficacy, tissue penetration, and non-invasive nature of the nanocage eye drops are convincingly demonstrated. However, the study evaluates treatment effects over only two weeks. This interval is insufficient to model the long-term progression of GA or dry AMD. Extending the treatment and follow-up durations would substantially strengthen the conclusions and better reflect clinical conditions. To clarify mechanisms, further studies should analyze the roles of endogenous antioxidant pathways and organelle-specific effects, and examine how the nanocage formulation interfaces with oxidative and inflammatory cascades.\nFor transparency and reproducibility, the authors should include details of how they calculated sample sizes and explain their rationale for selecting specific statistical methods. Clearly justifying these decisions and ensuring adequate statistical power and rigor will help build confidence in the reported therapeutic effects.\nOverall, this manuscript presents a really innovative therapeutic strategy with strong potential to shape AMD research and clinical practice. While there’s more work to be done, especially in chronic disease models, mechanistic validation, and longer-term studies, the approach is scientifically sound. The findings are compelling. With revisions that address the concerns I’ve outlined, this study would be a valuable addition to Nature Communications and the broader vision science community."}, "author_response": "Responses to reviewer’s comments\nWe sincerely thank all the reviewers for their time and valuable comments. We are particularly pleased to see the recognition of our work from reviewer 4#. Below we have provided point-to-point responses to the comments and have revised the manuscript accordingly.\nReviewer #4:\nThis manuscript offers compelling evidence that a zwitterionic nanocage-based peroxidase eye drop therapy could be a game-changer for treating oxidative stress-related retinal diseases. These diseases may include geographic atrophy (GA) and dry age-related macular degeneration (AMD). The work addresses a pressing gap in ophthalmology-therapeutic options for dry AMD remain scarce. By pairing an innovative delivery platform with a deeper understanding of oxidative injury, this study could really move the needle in the field.\nThe authors thoughtfully build on earlier antioxidant strategies. These include inorganic nanozymes such as Pt-, RhZ-, and CeO₂-based systems, which have struggled with poor biocompatibility and questionable efficacy. The zwitterionic nanocage platform sidesteps many of these issues. It boosts molecular stability, tissue permeability, and catalytic activity, making it a more practical therapeutic option. The non-invasive eye drop formulation improves patient compliance and accessibility, especially for people in the early stages of disease who hesitate to pursue more invasive treatments. After incorporating and addressing the reviewers' comments, the work is substantially more coherent.\nThat said, there are some limitations worth addressing to strengthen the translational relevance of these findings. The authors lean heavily on the sodium iodate (NaIO₃) model. While this model is widely used and practical for inducing RPE degeneration, it doesn’t fully mirror human AMD. NaIO₃ causes acute, chemically driven oxidative injury, leading to rapid RPE death. This occurs through apoptosis, necroptosis, and ferroptosis. Human AMD, on the other hand, unfolds slowly and involves a complex mix of lipid and protein buildup, chronic inflammation, complement dysregulation, and genetic factors. Also, NaIO₃ results are highly dose-dependent and can vary significantly from animal to animal. So the model captures only one piece of the AMD puzzle and might make the therapy appear more robust than it would be in chronic, real-world disease. These limitations don’t undermine the study, but they do suggest the authors should be cautious about claims of disease modification. The response to the review does not fully cover this.\nThe modified experimental design, data analysis, and interpretation are rigorous and well supported by the evidence presented. The reported therapeutic efficacy, tissue penetration, and non-invasive nature of the nanocage eye drops are convincingly demonstrated. However, the study evaluates treatment effects over only two weeks. This interval is insufficient to model the long-term progression of GA or dry AMD. Extending the treatment and follow-up durations would substantially strengthen the conclusions and better reflect clinical conditions. To clarify mechanisms, further studies should analyze the roles of endogenous antioxidant pathways and organelle-specific effects, and examine how the nanocage formulation interfaces with oxidative and inflammatory cascades.\nFor transparency and reproducibility, the authors should include details of how they calculated sample sizes and explain their rationale for selecting specific statistical methods. Clearly justifying these decisions and ensuring adequate statistical power and rigor will help build confidence in the reported therapeutic effects.\nOverall, this manuscript presents a really innovative therapeutic strategy with strong potential to shape AMD research and clinical practice. While there’s more work to be done, especially in chronic disease models, mechanistic validation, and longer-term studies, the approach is scientifically sound. The findings are compelling. With revisions that address the concerns I’ve outlined, this study would be a valuable addition to Nature Communications and the broader vision science community.\nReply: We are deeply appreciative of the reviewer’s thoughtful and constructive evaluation, as well as the encouraging recognition of our work’s potential impact. The insightful suggestions have been invaluable in strengthening the translational relevance and mechanistic depth of our study. Below, we provide a point-by-point response to the specific concerns raised, detailing the revisions and additional experiments we have conducted.\n1. Model terminology, limitations, and claims of disease modification.\nWe fully agree with the reviewer that the sodium iodate (NaIO₃) model represents an acute chemical injury and does not fully recapitulate the slow, multifactorial pathogenesis of human geographic atrophy (GA) or dry age-related macular degeneration (AMD). In the revised manuscript, we have consistently replaced “GA mouse model” with “NaIO₃-induced acute retinal injury model (ARI)” in the text, titles, and figures. More importantly, we have added a dedicated subsection in the Discussion as following, where we explicitly acknowledge that this model primarily mirrors the oxidative stress component of retinal degeneration, and avoided over-interpreting its direct equivalence to chronic human disease. We clarify that our study is a mechanistic proof-of-concept designed to validate whether targeted enzyme delivery can ameliorate oxidative stress in the retina—but a key driver in GA pathogenesis.\n“In the acute retinal injury model designed to recapitulate the reactive oxygen species (ROS)-induced degeneration pathology of GA, we consistently observed that restoration of the enzymatic cascade represents a promising therapeutic strategy for this condition.”\n“Collectively, our findings demonstrate that the zwitterionic nanocage platform enables effective topical delivery of antioxidant enzymes to the retina, presenting a promising therapeutic strategy for mitigating oxidative stress-related retinal damage. While the SI-induced acute oxidative injury model serves as a valuable tool for investigating oxidative stress, a key driver of RPE and photoreceptor degeneration, it represents a simplified, accelerated system that lacks the chronic, multifactorial complexity of human dry AMD. Future studies will need to validate this proof-of-concept in more physiologically relevant chronic models that better mimic the slow human disease progression. Nonetheless, by addressing the critical challenge of ocular drug delivery, our platform provides a versatile and patient-friendly foundation for developing disease-modifying therapies for dry AMD.”\n2. Treatment duration and long-term therapeutic evaluation\nWe really appreciate the reviewer’s insightful comment regarding the limited observation window for modeling chronic disease progression. In response, we would like to highlight that during the preliminary phase of this study, we conducted an exploratory extended-duration experiment in which mice were treated with Z(CAT&SOD) for one month following NaIO₃-induced injury (Figure R1). Intriguingly, while this longer-term assessment confirmed sustained preservation of retinal structure and function compared to controls, the therapeutic benefits plateaued after the initial two-week period. We ultimately selected the two-week endpoint for the main study based on following reasons: (i) this duration aligns with the acute progression of NaIO₃-induced retinal injury, (ii) the two-week data robustly demonstrates the fundamental efficacy of our delivery platform, and (iii) this approach adheres to the 3R principles (Replacement, Reduction, Refinement) by minimizing animal use while maintaining scientific validity.\nFigure R1. Representative fundus images and OCT images of mice after different treatments at (a) two weeks and (c) one month. Statistical analysis of retinal thickness of mice in different groups at (b) two weeks and (d) one month (n=6). Data were represented as means ± SD. P values in (c, d, e and g) were calculated by using one-way ANOVA.\n3. Elucidation of endogenous antioxidant pathways and organelle-specific effects\nIn response to the reviewer’s valuable suggestion regarding mechanistic insights, we examined the effect of the zwitterionic nanocage’s internalization into cells on the mitochondria. We evaluated the subcellular localization of Z(CAT) labeled with Cy5.5 in RPE cells using confocal microscopy with a mitochondrial tracker (Figure R2). The imaging showed minimal mitochondrial accumulation of the nanocage, indicating that its therapeutic mechanism does not rely on direct organelle targeting. Instead, the nanocage predominantly localized to the cytosol and peri-mitochondrial space, a critical region for ROS diffusion and redox signaling. This spatial distribution is particularly beneficial for catalase activity, as ROS generated in mitochondria readily into the cytosol, where excessive accumulation amplifies oxidative stress and inflammatory signaling. By scavenging cytosolic ROS, our strategy indirectly mitigates oxidantive damage while preserving mitochondrial homeostasis.\nTo further characterize the therapeutic mechanism of Z(CAT&SOD), we conducted systematic analyses of endogenous antioxidant responses and mitochondrial protection in oxidatively stressed RPE cells. Quantitative PCR revealed that SI-induced oxidative stress triggered a robust activation of the Nrf2/ARE pathway, as evidenced by the significant upregulation of Nrf2 and its downstream targests (HMOX-1, NQO1, GCLC, and GPX1). Notably, this oxidative stress response was effectively attenuated by both free enzymes and Z(CAT&SOD), with the zwitterion-protected formulation exhibiting superior efficacy (Figure R3a). We next evaluated mitochondrial protection by measuring membrane potential and ROS generation. While confocal imaging confirmed minimal mitochondrial accumulation of Z(CAT&SOD), functional assays revealed its potent ability to preserve mitochondrial membrane integrity and attenuate secondary ROS production (Figure R3b). These findings demonstrate the therapeutic advantage of Z(CAT&SOD) in preventing mitochondrial oxidative damage by effectively scavenging cytosolic ROS without requiring direct organelle targeting..\nWe have integrated these findings into the Results and Discussion, expanding our mechanistic narrative from one of mere “replacement” to “activation and synergy” with endogenous systems.\n“To investigate potential organelle-specific effects, we incubated RPE19 cells with Cy5.5 labeled Z(CAT) for 6 hours and performed mitochondrial co-localization analysis using a mitochondrial tracker. Confocal imaging revealed minimal mitochondrial accumulation of the nanocages (Supplementary Fig. 8), indicating that their therapeutic mechanism does not rely on direct mitochondrial targeting. Instead, the nanocage predominantly localized to the cytosol and peri-mitochondrial space, a critical region for ROS diffusion and redox signaling. This spatial distribution is particularly advantageous for catalase activity, as it positions the therapeutic agent near the sites of ROS generation while allowing for effective diffusion throughout the cytosol. To further characterize the cellular response to treatment, we analyzed the activation of endogenous antioxidant pathways in oxidatively stressed RPE cells after Z(CAT&SOD) treatment using qPCR. RPE cells were first treated with 200 μg/mL SI for 6 hours to induce oxidative stress, followed by treatment with free CAT&SOD or Z(CAT&SOD) for additional 6 hours. The qPCR analysis revealed that Z(CAT&SOD) treatment significantly downregulated the Nrf2 pathway and its downstream targets (e.g. GPX1, NQO1, GCLC, HMOX-1) (Supplementary Fig. 9a). This oxidative stress induction was effectively attenuated by both free CAT/SOD and the zwitterion-protected formulation, with the latter exhibiting superior protective efficacy, likely due to enhanced enzyme stability and improved cellular delivery. To further evaluate mitochondrial protection, we analyzed mitochondria integrity in stressed RPE cells after Z(CAT&SOD) treatment (Supplementary Fig. 9b). Although the nanocage does not directly localize to mitochondria, its cytosolic ROS-scavenging activity effectively preserved mitochondrial membrane potential and reduced secondary mitochondrial ROS production. These findings highlight the significant organelle-protective mechanism, reinforcing the therapeutic potential of our platform.”\n“In summary, this study demonstrated that the pathogenesis of ARI is characterized by excessive retinal ROS production and reduced peroxidase activity. Our therapeutic intervention with CAT and SOD exhibits multi-model protection effects by effectively mitigating oxidative stress and inflammation responses while reducing cellular apoptosis and preserving mitochondrial protection. These findings establish a comprehensive antioxidant therapeutic approach for ARI management.”\nFigure R2. Tracing of zwitterionic nanocage and mitochondria within RPE cells.\nFigure R3. (a) Relative GPX1, GCLC, Nrf, NQO1 and HMOX-1 mRNA abundance (2-ΔΔCt) in RPE cells with different treatments. (b) Mitochondrial damage changes in RPE cells with different treatments.\n4. Statistical transparency and reproducibility\nIn response to the reviewer’s request, we have expanded the Methods section to provide a complete description of our statistical analysis framework. This include the rationale for sample size determination and the selection of specific statistical tests (including one-way, two-way and t-test). To ensure full compliance with journal policy and maximal transparency, we have prepared a comprehensive Source Data file containing all individual quantitative measurements underlying the graphical representations. This dataset is accompanied by detailed annotations specifying the exact statistical test applied to each comparison.\nOnce again, we extend our sincere gratitude to Reviewer #4 for the exceptionally constructive and forward-looking critique, which has substantially elevated the quality of our manuscript. We believe the revised version, with its clarified scope, deeper mechanistic insight, and enhanced transparency, now offers a compelling and robust case for the therapeutic potential of our zwitterionic nanocage platform."}, {"round": 4, "reviewer_comments": {"reviewer_4": "Thank you for adressing the question."}, "author_response": ""}]

Decision letter
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ATTACHED REVIEWER REPORT — IMPORTANT
The text below is a complete reviewer report submitted as a PDF
attachment. It appears after the Creative Commons license block.
Assign it to whichever reviewer wrote 'see pdf' or had no inline text.
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Responses to reviewer’s comments
We sincerely thank the reviewers for the valuable comments and suggestions. Below we have provided
point-to-point responses to the comments and have revised the manuscript accordingly.
Reviewer #1:
The manuscript presents an approach to mitigate oxidative stress-induced retinal degeneration
through co-encapsulation of catalase (CAT) and superoxide dismutase (SOD) in polymeric nanocages.
While antioxidant supplementation in retinal disease is a well-explored area, the co-delivery of these
these
two enzymes within a single carrier represents a technically interesting strategy that could have
therapeutic potential. However, several major concerns must be addressed before the manuscript can
be considered for publication.
1. First, the nanocages are composed of acrylamide and bisacrylamide-based polymers, materials
known to have neurotoxic properties. The manuscript does not sufficiently address the safety or
biocompatibility of these polymers for intraocular use, which is essential for translational relevance.
Additionally, although the title refers to an eye drop-based therapy, the data presented are limited to
intravitreal delivery; this discrepancy is misleading and should be clarified.
Reply: Thanks so much for the reviewer’s suggestion. We fully agree that the safety profile of any
intraocular or topical ophthalmic formulation is of utmost importance for its translational potential.
While acrylamide and bisacrylamide monomers have been associated with neurotoxicity in their
unpolymerized forms, it is important to emphasize that the polymerized forms used in our nanocage
system are chemically stable macromolecules that are not expected to exert systemic toxicity under
normal physiological conditions. To directly address this concern, we conducted a comprehensive 4-
week ocular and systemic safety evaluation of both empty zwitterionic nanocages and Z(CAT&SOD)
nanocages following topical ocular administration in healthy mice. The results showed that there were
no significant abnormalities observed in any of the tested parameters. Both empty and enzyme-loaded
nanocages were well-tolerated, with no signs of ocular irritation, no increase in IOP, no corneal or
retinal damage, and no systemic toxicity (Figure R1). These findings were consistent with previous
reports demonstrating that zwitterionic polymers exhibit excellent biocompatibility, low
immunogenicity, and minimal protein adsorption, making them suitable for biomedical applications,
including ocular drug delivery.
Furthermore, we sincerely apologize for any confusion caused by our title regarding the administration
method in this study. In this manuscript, intravitreal injection was used as a proof-of-concept to
rigorously evaluate the therapeutic efficacy of enzyme-loaded nanocages for dry AMD. Considering
the clinical preference for non-invasive therapies, we finally developed and optimized a topical eye
drop formulation using the same zwitterionic nanocage platform. Notably, this formulation
demonstrated promising therapeutic efficacy in treating oxidative stress-induced dry macular
degeneration in mice.
“Given the high sensitivity of ocular tissues, we conducted a thorough biocompatibility assessment of
Z(CAT&SOD) to ensure its safety for long-term use. A battery of ophthalmic evaluations including
slit-lamp microscope, optical coherence tomography (OCT), electroretinogram (ERG) and intraocular
pressure (IOP), were performed on mice four weeks after treatment with Z-polymer and Z(CAT&SOD)
eyedrops. As illustrated in Figure S14a and S14b, the corneas of treated mice remained clear and intact,
with no observable differences compared to healthy controls. Moreover, prolonged administration of
Z-polymer and Z(CAT&SOD) did not obviously alter tear secretion (Figure S14c). Corneal OCT
imaging confirmed that neither formation affected corneal thickness (Figure S14d and S14e), while
fundus imaging and retina thickness measurements revealed no structural abnormalities (Figure S14f
to S14i). In addition, IOP values remained stable after long-term treatment, indicating that the
Z(CAT&SOD) eyedrop dose not induce ocular hypertension or increase the risk of glaucoma (Figure
S14j). To assess systemic toxicity, we performed histological analysis of major organs (heart, liver,
spleen, lung, kidney and eye) via H&E staining. Tissues form Z-polymer and Z(CAT&SOD)-treated
mice exhibited nearly no pathological changes compared to healthy mice (Figure S14k). Additionally,
hematological analysis of blood samples confirmed that all parameters remained in the normal range,
indicating negligible systemic toxicity even after prolonged subconjunctival retention (Figure S14l).
Therefore, these findings demonstrate that both Z-polymer and Z(CAT&SOD) exhibit excellent
biocompatibility after long-term administration.”
Figure R1. Safety assessment of Z-polymer and Z(CAT&SOD) (n=3). (a) Representative images of
cornea’s light fields and (b) fluorescein cornea staining of mouse eyes after different treatments for 2
and 4 weeks by slit-lamp microscopy. (c) The tear secretion amount of mouse eyes after different
treatments for 2 and 4 weeks (d) Representative images of OCT images of cornea and (e) retina for 2
and 4 weeks. (f) Statistical analysis of relative retina thickness. (g) Representative images of retina’s
light fields and (h) fluorescein staining of retina after different treatments for 2 and 4 weeks. (i)
Statistical analysis of IOPs of eyes in mice after different treatments for 2 and 4 weeks. (j) H&E
staining images of different organs including liver, spleen, lung, kidney, heart and eye collected from
mice in different groups (scale bars: 500 μm). (k) Blood biochemistry and complete blood analysis of
mice with different treatments. The following parameters were tested: percentage of white blood cell
(WBC); percentage of lymphocytes (Lymph); percentage of monocytes (MON); percentage of
neutrophilic granulocyte (Gran); red blood cell (RBC) counts; hemoglobin (HGB); hematocrit (HCT);
mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); platelets (PLT); alanine
aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP) and blood
urea nitrogen (BUN). Data were represented as means ± SD.
2. A more significant concern lies in the conceptual accuracy of several claims. The manuscript
contains multiple errors in ocular definitions and physiological interpretations, which diminish
confidence in the scientific rigor. For example, retinal ganglion cells are not the primary cells affected
in geographic atrophy (GA); the a-wave of the ERG is primarily associated with photoreceptor
(particularly rod) activity, not cones; and the b-wave is typically driven by bipolar cells rather than
directly by rods. Furthermore, evaluating choroidal neovascularization (CNV) as an outcome measure
is not appropriate for GA, which is a non-neovascular form of age-related macular degeneration. These
inaccuracies suggest a need for more careful literature review and refinement of disease-
specific understanding.
Reply: We apologize for the confusion. We sincerely thank the reviewer for pointing out these critical
inaccuracies in ocular terminology and physiological interpretations. We fully acknowledge that
precision in disease-specific descriptions is essential for scientific rigor and translational credibility.
In response, we have carefully revised the manuscript to address the following key issues. Firstly, we
have corrected the misinterpretation of ERG waves. The a-wave is now correctly described as
reflecting photoreceptor activity (primarily rods), and the b-wave as originating from ON-bipolar cells,
not rods or cones directly. Then, we have corrected all references to “retinal ganglion cells” as primary
targets in GA, and accurately identified photoreceptors and RPE cells as the main cell types affected.
Moreover, we have explicitly distinguished geographic atrophy (non-neovascular) from choroidal
neovascularization (neovascular), and the evaluation of this model was reorganized (Figure R2 and
R3). In light of the abnormal retinal vasculature observed by FFA, we have moved these data to the
Supporting Information (Figure S2a, Figure S9b, Figure S13b).
“This treatment effectively mitigated ROS-induced apoptosis and suppressed the NF-κB-HMGB1-ILs
inflammatory pathway, providing substantial protection to photoreceptors and retinal pigment
epithelial (RPE) cells, which represents a groundbreaking therapeutic strategy for dry AMD treatment
and offers a novel approach to addressing the challenges associated with this progressive retinal
disorder.”
“ERG analysis revealed a marked reduction in both the a and b waves, reflecting impaired activity of
photoreceptors and ON-bipolar cells.”
“Analysis of a- and b- waves, which correspond to photoreceptors and ON-bipolar cell activity,
demonstrated partial restoration of visual acuity in mice treated with CAT or SOD&CAT injections.”
“Post-treatment ERG assessments at two weeks confirmed that Z(CAT&SOD) effectively preserved
photoreceptors and ON-bipolar cell function, with no statistically significant difference compared to
intravitreal injection.”
“As shown in figures 2k and 2l, prominent retinal deposits were observed in PBS- and SOD-injected
groups, accompanied by subretinal debris accumulation (likely derived from damaged RPE and
photoreceptors), inflammatory edema, and secondary vascular abnormalities induced by oxidative
stress (Figure S2a).”
“In contrast, untreated and uncoated groups exhibited severe retinal thinning and reduced cell density,
particularly in the outer nuclear layer (ONL) and photoreceptor layer, which are critically affected in
GA. These structural deficits correlated with ERG-documented visual impairment.”
“As shown in Figure 4b, PBS- or Z(SOD)-injected mice developed progressive subretinal deposits,
retinal vasculitis, widespread retinal cell apoptosis, and atrophy over time.”
Figure R2 (in previous manuscript’s figure 4). Representative fundus images, OCT images and
H&E images of mice with different treatments (Health, Untreated, Z-polymer, Z(CAT), Z(SOD) and
Z(CAT&SOD)) at one and two weeks (n=4, scale bar: 100 μm).
Figure R3 (in previous manuscript’s figure 6). Representative fundus images, OCT images and
H&E images of mice with different treatments (Untreated, CAT&SOD eyedrop, CAT&SOD
intravitreal injection, Z(CAT&SOD) eyedrop, Z(CAT&SOD) intravitreal injection and Lutein oral
administration) at one and two weeks (n=4, scale bar: 100 μm).
3. Lastly, while the sodium iodate model does recapitulate oxidative stress-related RPE damage, it
reflects an acute injury rather than the chronic, progressive degeneration seen in human GA.
Therefore, while the protective effects observed are encouraging, their relevance to GA
pathophysiology should be presented more cautiously. Overall, the concept may be of interest if
reframed with appropriate disease context, technical clarifications, and stronger emphasis on
translational considerations.
Reply: We sincerely appreciate the reviewer’s comment regarding the distinction between the sodium-
iodate model, which induces acute oxidative damage, and the chronic, low-grade oxidative stress
characteristic of human geographic atrophy. We fully acknowledge this critical difference and agree
that caution must be exercised when extrapolating our findings to clinical GA. The NaIO₃ rapidly
triggers key pathological features of late-stage GA, including RPE/photoreceptor degeneration,
complement activation, and microglial recruitment, thereby recapitulating the terminal oxidative
microenvironment of GA1. Given that CAT/SOD function by acutely neutralizing ROS, the data
suggest that targeted antioxidant intervention may help halt or slow active atrophic expansion2–5. As a
proof-of-concept, these findings demonstrate that rapid ROS scavenging can preserve residual
photoreceptors in the presence of oxidative damage. For more accurate understanding, we have added
related discussion in the revised manuscript.
“In the SI-induced GA mouse model, we observed pathological features resembling those in clinical
GA patients, including subretinal deposits and retinal atrophy. Mechanistic investigations revealed that
RPE and photoreceptor cell apoptosis was closely associated with the microenvironment characterized
by high oxidative stress and diminished peroxidase activity, particularly reduced levels of CAT and
SOD. As a proof-of-concept, our findings suggest that timely antioxidant supplementation may
preserve residual photoreceptors when oxidative damage as the primary driver of degeneration.”
Reference
1. The retinal pigment epithelium in Sorsby Fundus Dystrophy shows increased sensitivity to
oxidative stress-induced degeneration. Redox Biol. 37, 101681 (2020).
2. Hollyfield, J. G. et al. Oxidative damage–induced inflammation initiates age-related macular
degeneration. Nat. Med. 14, 194–198 (2008).
3. Lauvås, A. J. et al. Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a
human mixed culture of neurons and astrocytes undergoing differentiation in concentrations
relevant for human exposure. NeuroToxicology 92, 33–48 (2022).
4. Comparative mechanistic study of RPE cell death induced by different oxidative stresses. Redox
Biol. 65, 102840 (2023).
5. Kerur, N. et al. cGAS drives noncanonical-inflammasome activation in age-related macular
degeneration. Nat. Med. 24, 50–61 (2018).
----------------------
Reviewer #2:
The manuscript identifies a critical deficit in retinal peroxidase activity (specifically catalase and
superoxide dismutase) during GA progression, which exacerbates oxidative stress, activates the NF-
κB-HMGB1-ILs inflammatory pathway, and drives retinal cell apoptosis. To counteract this, the
authors developed charge-shielded zwitterionic nanocages (Z(CAT&SOD)) that protect enzyme
activity, enhance permeability across ocular barriers, and facilitate delivery via clathrin-dependent
and caveolae-mediated endocytosis. The study is well-organized and provides comprehensive insights
into the potential of this innovative therapeutic strategy. The authors should address the following
questions to further enhance the quality of the manuscript:
1. As a nano-delivery system intended for ocular applications, the biosafety of zwitterionic nanocages
(Z (CAT&SOD)) constitutes a fundamental prerequisite for clinical translation; however, existing
research lacks systematic evaluation. It is need to conduct supplementary long-term safety assessments,
encompassing evaluations of ocular irritation, intraocular pressure variations, corneal integrity,
corneal transparency, and retinal pathology.
Reply: Thanks so much for the reviewer’s suggestion. We recognize the critical importance of safety
for translational ophthalmic formulations. In response to reviewer’s comments, we evaluated both
empty and CAT&SOD-loaded zwitterionic nanocages in healthy mice for 4 weeks. Both the nanocages
showed excellent tolerability with no observed ocular irritation, IOP elevation, corneal/ retinal damage,
or systemic toxicity (Figure R4). These findings confirmed zwitterionic polymers’ excellent
biocompatibility, low immunogenicity, and minimal protein adsorption-ideal properties for ocular
drug delivery.
“Given the high sensitivity of ocular tissues, we conducted a thorough biocompatibility assessment of
Z(CAT&SOD) to ensure its safety for long-term use. A battery of ophthalmic evaluations including
slit-lamp microscope, optical coherence tomography (OCT), electroretinogram (ERG) and intraocular
pressure (IOP), were performed on mice four weeks after treatment with Z-polymer and Z(CAT&SOD)
eyedrops. As illustrated in Figure S14a and S14b, the corneas of treated mice remained clear and intact,
with no observable differences compared to healthy controls. Moreover, prolonged administration of
Z-polymer and Z(CAT&SOD) did not obviously alter tear secretion (Figure S14c). Corneal OCT
imaging confirmed that neither formation affected corneal thickness (Figure S14d and S14e), while
fundus imaging and retina thickness measurements revealed no structural abnormalities (Figure S14f
to S14i). In addition, IOP values remained stable after long-term treatment, indicating that the
Z(CAT&SOD) eyedrop dose not induce ocular hypertension or increase the risk of glaucoma (Figure
S14j). To assess systemic toxicity, we performed histological analysis of major organs (heart, liver,
spleen, lung, kidney and eye) via H&E staining. Tissues form Z-polymer and Z(CAT&SOD)-treated
mice exhibited nearly no pathological changes compared to healthy mice (Figure S14k). Additionally,
hematological analysis of blood samples confirmed that all parameters remained in the normal range,
indicating negligible systemic toxicity even after prolonged subconjunctival retention (Figure S14l).
Therefore, these findings demonstrate that both Z-polymer and Z(CAT&SOD) exhibit excellent
biocompatibility after long-term administration.”
Figure R4. Safety assessment of Z-polymer and Z(CAT&SOD) (n=3). (a) Representative images of
cornea’s light fields and (b) fluorescein cornea staining of mouse eyes after different treatments for 2
and 4 weeks by slit-lamp microscopy. (c) The tear secretion amount of mouse eyes after different
treatments for 2 and 4 weeks (d) Representative images of OCT images of cornea and (e) retina for 2
and 4 weeks. (f) Statistical analysis of relative retina thickness. (g) Representative images of retina’s
light fields and (h) fluorescein staining of retina after different treatments for 2 and 4 weeks. (i)
Statistical analysis of IOPs of eyes in mice after different treatments for 2 and 4 weeks. (j) H&E
staining images of different organs including liver, spleen, lung, kidney, heart and eye collected from
mice in different groups (scale bars: 500 μm). (k) Blood biochemistry and complete blood analysis of
mice with different treatments. The following parameters were tested: percentage of white blood cell
(WBC); percentage of lymphocytes (Lymph); percentage of monocytes (MON); percentage of
neutrophilic granulocyte (Gran); red blood cell (RBC) counts; hemoglobin (HGB); hematocrit (HCT);
mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); platelets (PLT); alanine
aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP) and blood
urea nitrogen (BUN). Data were represented as means ± SD.
2) It is necessary to elucidate the molecular basis of zwitterionic charge shielding: to investigate the
dynamic changes in surface charge (as monitored by real-time zeta potential measurements) of the
CAT&SOD complex in the vitreous microenvironment.
Reply: Thanks so much for the reviewer’s suggestion. To elucidate the molecular mechanism of
zwitterionic charge shielding in the vitreous, we performed real-time zeta potential measurements of
Z(CAT&SOD) nanocages incubated in fresh rabbit vitreous humor at physiological temperature (37°C)
for different times. As show in Figure R5, the ζ-potential of Z(CAT&SOD) maintained stable
throughout the observation period, demonstrating excellent charge homeostasis in the vitreous milieu.
This charge stabilization phenomenon can be attributed to the unique zwitterionic architecture of
Z(CAT&SOD), where the protonation quilibrium between acidic and basic side chains creates a
dynamic charge-shielding effect, which effectively neutralizes electrostatic interactions with anionic
vitreous components and preserves the native ζ-potential. We have added these results and discussion
to the revised manuscript as following:
“To further investigate the interaction between zwitterionic nanocages and the vitreous, we measured
the surface charge of Z(CAT&SOD) nanocages after co-incubation with the isolated rabbit vitreous
(Figure S6). We found that the near-neutral surface of the zwitterionic nanocages remained stable in
the vitreous environment, facilitating enhanced diffusion of the encapsulated protein drugs.”
Figure R5. Zeta potential of Z(CAT&SOD) incubated in rabbit vitreous body for different times.
3) Supplementary stability data for dosage forms are required: The particle size distribution of the
ophthalmic solution, enzyme activity retention rate (for a minimum of 1 month), and the presence of
nanocage aggregation (e.g., through long-term DLS monitoring) should be assessed under various
storage conditions (4°C, room temperature, 37°C).
Reply: Thanks so much for the reviewer’s suggestion. To evaluate formulation stability, we
systematically compared the enzymatic activity retention of zwitterionic nanocage-encapsulated
enzymes (Z(CAT&SOD)) with free enzymes under various storage conditions (4°C, 25°C, and 37°C)
for 4 weeks. Weekly activity assessments revealed that while both formulations maintained over 90%
initial activity at 4°C throughout the study period, marked difference emerged at higher temperatures.
Free enzymes exhibited rapid activity decay, losing significant functionality by day 7 at both 25 °C
and 37 °C. In contrast, Z(CAT&SOD) nanocages demonstrate the superior stability (Figure R6).
“We further evaluated the enzymatic stability of Z(SOD), Z(CAT), and their free counterparts under
different storage conditions (4°C, 25°C and 37°C). While free enzymes exhibited rapid activity loss at
25°C and 37°C, the zwitterionic nanocages-encapsulated enzymes retained most of their initial activity
for four weeks, indicating their superior protective effect of zwitterionic nanocage (Figure S8).”
Figure R6. The enzyme activity stability of Z(SOD), Z(CAT), free SOD and free CAT at different
storage environments (4°C, 25°C and 37°C).
4) It is necessary to include negative controls, such as a control group utilizing blank zwitterionic
nanocages (enzyme-free), to eliminate potential confounding effects of the material itself on
inflammation or retinal structure.
Reply: Thanks so much for pointing it out. To further validate the therapeutic mechanism, we
performed additional experiments using “blank” zwitterionic nanocages (Z-polymer nanocages
without CAT or SOD) in the GA mouse model. Both OCT and fundus imaging demonstrated that these
blank nanocages failed to preserve photoreceptor integrity or RPE morphology, with outcomes
indistinguishable from untreated control. These data confirmed that the therapeutic effects are
exclusively mediated by the activities of CAT and SOD (Figure R7). The related results and discussion
have been added in the revised manuscript as following:
“Furthermore, zwitterionic nanocages without peroxidase demonstrated nearly no therapeutic effect
on the SI-induced GA model.”
Figure R7 (in previous manuscript’s figure 4). Treatment of GA via intravitreal injection of
Z(CAT&SOD). (a) Schematic illustration of different treatments on SI-induced GA mouse model and
the fundus assessment after one week. (b) Representative fundus images, OCT images and
H&E images of mice after different treatments at one and two weeks (n=4, scale bar: 100 μm). (c)
Clinical grading of GA damage according to a leakage score system (n=4). (d & e) Statistical analysis
of retinal thickness of mice in different groups at one and two weeks (n=4). (f) Representative ERG
wave responses in mice after different treatments (n=4). (g & h) Statistical analysis of A waves (g) and
B waves (h) in retina of mice in different groups under scotopic condition (n=4). Data were represented
as
represented as means ± SD. P values in (c, d, e, g and h) were calculated by using one-way ANOVA.
5) The study employs a sodium iodate-induced model of dry macular degeneration, which
phenotypically approximates the human dry macular degeneration model. Please elucidate the
underlying mechanisms involved.
Reply: Thanks so much for the reviewer’s comment. Sodium iodate (NaIO₃) serves as a well-
characterized oxidative stress inducer that selectively targets the retinal pigment epithelium (RPE),
initiating a degenerative cascade that faithfully recapitulates the cardinal features of human geographic
atrophy (GA)1. The mechanistic underpinnings of this model involve three interrelated pathological
proceess: 1) NaIO₃-mediated ROS overproduction induces RPE mitochondrial dysfunction, membrane
depolarization, and subsequent necroptotic cell death6; 2) secondary photoreceptor degeneration
occurs through disrupted retinoid cycling and loss of metabolic support, manifesting as outer nuclear
layer (ONL) thinning and diminished a-wave amplitudes on ERG; and 3) sustained neuroinflammation
develops via microglial activation, Muller cell gliosis, and macrophage infiltration, accompanied by
elevated inflammatory mediators (IL-1β, CCL2, IL-33, and TGF-β)7,8. This mark of RPE dysfunction,
photoreceptor loss, and chronic inflammation mirrors both the structural and dysfunction,
photoreceptor loss, and chronic inflammation indicated both the structural and functional deficits
observed in GA patient9. The NaIO₃ model thus provides a robust preclinical platform that captures
the complex pathophysiology of dry AMD, particularly for evaluating antioxidant therapies targeting
oxidative stress-mediated degeneration. We have added the related discussion in the revised
manuscript as following:
“The SI-induced GA model faithfully recapitulated key clinical manifestations of human GA,
including subretinal deposits and retinal atrophy. Through mechanistic studies, we identified two
critical pathologies features: oxidative stress-medicated apoptosis in both RPE and photoreceptor cells
and severe deficiency of endogenous antioxidant defenses (CAT/SOD). Notably, our therapeutic
experiments established that photoreceptor survival could be improved through antioxidant
supplementation during the critical window of oxidative stress dominance.”
Reference
1. The retinal pigment epithelium in Sorsby Fundus Dystrophy shows increased sensitivity to
oxidative stress-induced degeneration. Redox Biol. 37, 101681 (2020).
6. Hanus, J., Anderson, C. & Wang, S. RPE necroptosis in response to oxidative stress and in AMD.
Ageing Res. Rev. 24, 286–298 (2015).
7. Liu, B. et al. Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells.
Cell Death Dis. 12, 230 (2021).
8. Khandhadia, S. & Lotery, A. Oxidation and age-related macular degeneration: insights from
molecular biology. Expert Rev. Mol. Med. 12, e34 (2010).
9. Yaspan, B. L. et al. Targeting factor D of the alternative complement pathway reduces geographic
atrophy progression secondary to age-related macular degeneration. Sci. Transl. Med. 9, eaaf1443
(2017).
6) Regarding the concern of the barrier integrity of tight junction proteins, the authors need tested the
longer incubation with Z (CAT&SOD) for 6 hours. As the corneal epithelial cells are the protection of
the eyes, it still brings concern about the use of Z (CAT&SOD) in vivo, such as infection.
Reply: Thanks so much for the reviewer’s suggestion. To evaluate the potential impact of
Z(CAT&SOD) on corneal epithelial barrier function, we extended the incubation time in our transwell-
based transepithelial electrical resistance (TEER) assay from 1 hour to 6 hours of direct exposure,
specifically assessing tight junction integrity in human corneal epithelial cells (HCECs). The results
suggested that prolonged exposure to Z(CAT&SOD) did not disrupt epithelial barrier integrity,
indicating a low risk of compromising ocular surface infection under the tested conditions (Figure R8).
The related discussion has been added in the revised manuscript as following:
“Subsequently, we measured transepithelial electrical resistance (TEER) and observed nearly no
significant changes following treatment with either Z(CAT) or free CAT (Figure S7), further
confirming that Z(CAT) does not compromise tight junction integrity or utilize the paracellular route
for ocular penetration.”
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Figure R8. TEER measurements of HCECs at various time points after different treatments (n=3).
7) Figure 5l fails to elucidate the mechanistic role of macrophages, which warrants supplementation
regarding their functional mechanism in the treatment of dry macular degeneration with Z (CAT&SOD)
therapy. Clarify how Z(CAT&SOD) drives M2 polarization (e.g., via reduced ROS levels inhibiting
NF-κB, or direct modulation of macrophage metabolism).
Reply: Thanks so much for the reviewer’s comment. Z(CAT&SOD) rapidly scavenges H₂O₂ in the
injured retina (Figure R9a), alleviating mitochondrial oxidative stress and suppressing the release of
DAMPs such as HMGB1 (Figure R9d). Reduced DAMPs and ROS inhibit phosphorylation and
nuclear translocation of NF-κB p65 (Figure R9b), thereby downregulating M1-polarizing cytokines
(IL-1β, TNF-α, IL-6, IL-12) at the transcriptional level (Figure R9h-k). Concomitantly, NF-κB
suppression restored mitochondrial respiration, creating a metabolic milieu conducive to M2
polarization. Accordingly, Z(CAT&SOD)-treated retinas exhibited an increased CD206⁺F4/80⁺ M2
population and a decrease in CD80⁺F4/80⁺ M1 macrophages (Figure R9e, R9f and R9g). Thus, these
data demonstrate that Z(CAT&SOD) eliminates ROS-driven inflammatory signals, inactivates the NF-
κB pathway, and promotes endogenous M2 programming in the retinal micro-environment as
summarized in Figure R9l. The related discussion has been added in the revised manuscript as
following:
“Therefore, the enzymatic cascade reaction mediated by Z(CAT&SOD) synergistically reduced key
inflammatory markers such as NF-κBp65, IL-1β, and HMGB1 in retinal tissues while promoting
macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotypes. These
findings highlight Z(CAT&SOD) as a promising therapeutic candidate for retinal disorders involving
oxidative stress and inflammation.”
Figure R9 (in previous manuscripts’ figure 5). Mechanism of Z(CAT&SOD) in treating GA.
Representative immunofluorescence staining images showing the expression of TUNEL(a), NF-κB
(b), IL-1β (c) and HMGB1(d) in the retina of mice in different groups (scale bar: 50 μm). (e)
Representative flow cytometry analysis of macrophage polarization in retina of mice in different
groups as indicated (n=3). (f) and (g) Percentages of F4/80+CD206+ macrophages and F4/80+CD80+
macrophage cells by flowcytometry (n=3). (h to k) Concentrations of IL-12P70, TNF-α, IL-6 and IFN-
γ in the retinas of mice in different groups evaluated by ELISA (n=4). (l) Schematic illustration of NF-
κB-HMGB1-ILs pathway-mediated inflammatory effects associated with apoptosis due to decreased
peroxidase activity and increased ROS levels. Data were represented as means ± SD. P values in (f, g,
h, i, j and k) were calculated by using one-way ANOVA.
8) The drug loading and drug encapsulation efficiency of Z(CAT&SOD) should be reported.
Reply: Thanks so much for the reviewer’s comment. Using the BCA assay, we quantified the drug
loading capacity and encapsulation efficiency of the Z(CAT&SOD) nanocages. The optimized
formulation demonstrated a high drug loading capacity of 94.84 ± 1.55% with encapsulation efficiency
of 35.32 ± 4.43%.
“We further characterized the physical properties and drug-loading performance at this optimal ratio.
Dynamic light scattering analysis revealed a nearly neutral surface charge (Table S1). Notably, the
nanocages exhibited excellent drug-loading capacity of 94.84 ± 1.55% with encapsulation efficiency
of 35.32 ± 4.43%).”
Table R1. The particle size, surface charge, drug loading and drug encapsulation efficiency of
Z(CAT&SOD) nanocages.
9) The rationale for using peroxidases (CAT and SOD) in GA therapy should be strengthened with
literature support linking their deficiency to human GA: Cite clinical studies showing reduced
CAT/SOD activity in retinas or aqueous humor of GA patients (vs. age-matched controls). Discuss
how CAT/SOD deficiencies exacerbate oxidative stress in GA: For example, do they impair ROS
clearance in RPE cells, leading to lipofuscin accumulation or mitochondrial DNA damage (as
reported in human GA)? Compare with other antioxidant strategies (e.g., nanozymes, small-molecule
ROS scavengers) to highlight the advantages of enzymatic cascades (e.g., specificity, biocompatibility).
Reply: Thanks so much for the reviewer’s comment. To strengthen the therapeutic potential of
CAT/SOD supplementation in GA, we highlight clinical evidence demonstrating significantly reduced
CAT and SOD activities in both retinal tissue and aqueous humor of GA patients compared to age-
matched controls9,10. This enzymatic deficiency compromises ROS clearance in RPE cells, thereby
promoting key pathological features of GA, including lipofuscin accumulation, mitochondrial DNA
oxidation, and drusen formation11. Unlike small-molecule antioxidants or nanozyme, the CAT/SOD
enzymatic cascade provides substrate-specific, catalytic efficiency and physiologically compatible
elimination of O₂•⁻ and H₂O₂, offering a more sustainable and endogenous strategy to mitigate
oxidative damage in GA.
Reference
9. Yaspan, B. L. et al. Targeting factor D of the alternative complement pathway reduces geographic
atrophy progression secondary to age-related macular degeneration. Sci. Transl. Med. 9, eaaf1443
(2017).
10. Senabouth, A. et al. Transcriptomic and proteomic retinal pigment epithelium signatures of age-
related macular degeneration. Nat. Commun. 13, 4233 (2022).
11. Eyedrop delivery of therapeutic proteins with zwitterionic polymers to treat dry age-related
macular degeneration. Biomaterials 305, 122429 (2024).
10) Minor point: Supplementary figures (e.g., Figure S1–S17) are not cited in the order they appear
in the text, which hinders readability. For example, Figure S3 is referenced before Figure S2. The
authors should reorder supplementary figure citations to align with their appearance in the main text,
ensuring a logical flow.
Reply: Thanks for pointing out this. We have now re-checked the entire manuscript and reordered all
Supplementary Figure citations so that they appear strictly sequentially in line with their first mention
in the text. This adjustment improves readability and is reflected in the revised file.
----------------------
Reviewer #3:
This study presents an interesting and potentially translationable strategy for the treatment of
geographic atrophy by constructing a zwitterionic nanocage-based delivery system that enables non-
invasive co-delivery of catalase (CAT) and superoxide dismutase (SOD) to the retina. The work
demonstrates strong innovation, solid in vivo data, and a relatively in-depth mechanistic exploration.
However, several scientific and mechanistic issues still need to be clarified or reinforced, and some
aspects of data presentation require revision. I recommend accept this manuscript after addressing
the following specific comments:
1) The manuscript emphasizes the synergistic effect of CAT and SOD as an enzymatic cascade.
However, there is no quantitative analysis confirming whether the combined use is superior to
individual enzymes in terms of ROS scavenging efficiency (e.g., rate of radical elimination, half-life of
ROS). It is recommended to include in vitro kinetic assays to validate whether this is a true cascade
reaction or simply an additive effect.
Reply: We thank the reviewer for this critical point. While we acknowledge that direct kinetic data
would further strengthen our mechanistic interpretation, our results demonstrate that the SOD-CAT
cascade operates through functional complementarity. Specifically, CAT converts the toxic byproduct
of SOD activity (H₂O₂) into harmless H₂O and O₂, thereby protecting SOD from oxidative inactivation
and ensuring sustained antioxidant capacity. This cooperative mechanism prevents •OH generation via
Fenton reaction, achieving complete ROS detoxification. To validate this synergy, we evaluated the
ROS-scavenging capacity of Z(CAT), Z(SOD), and Z(CAT&SOD) nanocages. As shown in Figure
R10, both modified enzymes retained their catalytic efficiency: SOD effectively eliminated superoxide
anions, while CAT degraded H₂O₂. Notably, the combined system (Z(CAT&SOD)) exhibited enhanced
radical scavenging, demonstrating cooperative action for optimal free radical elimination.
Figure R10. (a) Superoxide scavenging kinetics monitored at 550 nm (A-A ) for xanthine/xanthine
0
oxidase reactions with different nanocages (1 μg/mL). (b) Oxygen evolution kinetics from H₂O₂
decomposition (50 mM) by nanocages (1 μg/mL), measured via dissolved oxygen content.
2) The authors propose that Z(CAT&SOD) eyedrops can penetrate the retina through both the
conjunctiva-sclera-retina and conjunctiva-blood-retina routes. However, there is no quantitative
analysis comparing the relative contribution of each route. I suggest performing quantitative tissue
fluorescence distribution (e.g., FITC signal intensity in different ocular layers) or using pathway-specific
inhibitors to confirm the predominant transport route.
Reply: Thanks so much for the reviewer’s suggestion. To quantify the relative contribution of different
pathways, we measured FITC fluorescence in dissected sclera and retina 6h post-instillation and
simultaneously determined the blood concentration of CAT-FITC-loaded zwitterionic nanocages. The
conjunctiva/sclera-to-retina fluorescence ratio was about 6.67:1, suggesting preferential scleral uptake.
In addition, we performed pharmacokinetic analysis by collecting blood samples at 1, 3, 6, 9, and 12
hours after topical administration of Z(CAT-FITC) eye drops. At 6 hours, the signal of Z(CAT-FITC)
in the blood was significantly higher than that of free CAT-FITC, indicating the presence of a blood-
to-retina pathway (Figure R11). The related results and discussion have been added in the revised
manuscript as following:
“Confocal imaging of whole-eye sections revealed that the zwitterionic nanocages might penetrate the
conjunctival-scleral barrier, reaching the choroid and retina (Figure 6d). Quantitative analysis of CAT-
FITC fluorescent signals revealed a conjunctiva/sclera-to-retina ratio of 6.67:1 (Figure S11a),
suggesting efficient transscleral delivery. In addition, pharmacokinetic profiling following topical
administration of Z(CAT-FITC) eye drops (measured at 1, 3, 6, 9, and 12 h) showed that Z(CAT-FITC)
exhibited significantly higher blood accumulation than free CAT-FITC at 6 hours (Figure S11b),
indicating the existence of a blood mediated retinal delivery pathway.”
Figure R11. (a) Statistical analysis of total fluorescence intensity of CAT-FITC in the
conjunctiva/sclera (C/S), retina (R) and vitreous cavity of mice after different eyedrops treatments
(n=3). (b) The CAT-FITC concentration in blood after different treatments for 0, 1, 3, 6, 9, 12 hours.
3) The suppression of inflammation is attributed mainly to ROS reduction, but it remains unclear
whether CAT/SOD also exerts ROS-independent immunomodulatory effects, particularly on the NF-
κB pathway. The authors are encouraged to explore whether changes in key signaling proteins (e.g.,
p65 phosphorylation) precede complete ROS clearance.
Reply: Thanks so much for the reviewer’s comment. CAT and SOD lack kinase-inhibitory domains
or scaffold functions, as their primary role is redox regulation. While transient H₂O₂ can activate IKKβ,
chronic oxidative stress and DAMP release sustain NF-κB signalling in GA. Z(CAT&SOD) rapidly
scavenges H₂O₂ and O₂•⁻, eliminating the oxidative trigger for IKKβ activation and consequently
suppressing downstream p65 phosphorylation without requiring a separate, ROS-independent
mechanism. Our data show that phospho-p65, IL-1β and HMGB1 levels decrease concomitantly with
the DCF signal attenuation (Figure R12), further supporting ROS-dependent NF-κB activation.
Therefore, our findings demonstrate that NF-κB suppression occurs predominantly as a secondary
consequence of ROS clearance rather than through direct, ROS-independent modulation of the
signaling cascade by CAT or SOD.
Figure R12. Mechanism of Z(CAT&SOD) in treating GA (in previous manuscripts’ figure 5).
Representative immunofluorescence staining images showing the expression of TUNEL(a), NF-κB
(b), IL-1β (c) and HMGB1(d) in the retina of mice in different groups (scale bar: 50 μm). (e)
Representative flow cytometry analysis of macrophage polarization in retina of mice in different
groups as indicated (n=3). (f) and (g) Percentages of F4/80+CD206+ macrophages and F4/80+CD80+
macrophage cells by flowcytometry (n=3). (h to k) Concentrations of IL-12P70, TNF-α, IL-6 and IFN-
γ in the retinas of mice in different groups evaluated by ELISA (n=4). (l) Schematic illustration of NF-
κB-HMGB1-ILs pathway-mediated inflammatory effects associated with apoptosis due to decreased
peroxidase activity and increased ROS levels. Data were represented as means ± SD. P values in (f, g,
h, i, j and k) were calculated by using one-way ANOVA.
4) Although the study shows that encapsulation preserves enzymatic activity under proteolytic stress,
it would strengthen the claims to include structural or kinetic data (e.g., circular dichroism spectra,
DLS, changes in Michaelis–Menten kinetics) to assess whether zwitterionic polymerization alters the
enzyme’s conformation or access to the active site.
Reply: Thanks so much for the reviewer’s suggestion. To verify whether zwitterionic encapsulation
alters enzyme conformation or active-site integrity, we performed circular dichroism (CD) spectra of
both free CAT and Z(CAT) nanocages. The results demonstrated nearly identical α-helical and β-sheet
content before and after polymer coating, confirming that the native secondary structure was preserved
(Figure R13). The related results and discussion have been added in the revised manuscript as
following.
“Furthermore, circular-dichroism (CD) spectroscopy confirmed that the zwitterionic nanocage
encapsulation preserved the native secondary structure of the peroxidases (Figure S5).”
20
)
1
-lo
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m
2
m
0
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]
θ
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-30
200 220 240 260 280
Figure R13. Circular dichroism spectrum of Z(CAT) and free CAT.
5）The sodium iodate-induced model used in this study mimics acute oxidative damage and may
primarily represent late-stage GA. Please discuss the limitations of this model in simulating the
chronic progression of GA and whether the proposed therapeutic strategy could be effective in early-
stage disease (e.g., before lipofuscin accumulation).
Reply: Thanks for pointing out this. While the sodium iodate model rapidly induces RPE and
photoreceptor degeneration that mimics late-stage geographic atrophy, its acute oxidative insult fails
to recapitulate the chronic, low-grade oxidative stress underlying human disease progression,
particularly the gradual lipofuscin accumulation characteristic of early pathogenesis9. We therefore
recognize this as a key limitation: while our data demonstrate therapeutic efficacy under acute,
advanced-like conditions, they do not address intervention efficacy during the critical pre-lipofuscin.
Notably, the CAT/SOD cascade specifically targets the ROS implicates in lipofuscinogenesis10,11. This
suggests that prophylactic administration of Z(CAT&SOD) during early-stage dry AMD could
effectively mitigate disease progression by neutralizing free radical before irreversible damage occurs.
“In the SI-induced GA mouse model, we observed pathological features resembling those in clinical
GA patients, including subretinal deposits and retinal atrophy. Mechanistic investigations revealed that
RPE and photoreceptor cell apoptosis was closely associated with the microenvironment characterized
by high oxidative stress and diminished peroxidase activity, particularly reduced levels of CAT and
SOD. As a proof-of-concept, our findings suggest that timely antioxidant supplementation may
preserve residual photoreceptors when oxidative damage as the primary driver of degeneration.”
Reference
9. Yaspan, B. L. et al. Targeting factor D of the alternative complement pathway reduces geographic
atrophy progression secondary to age-related macular degeneration. Sci. Transl. Med. 9, eaaf1443
(2017).
10. Senabouth, A. et al. Transcriptomic and proteomic retinal pigment epithelium signatures of age-
related macular degeneration. Nat. Commun. 13, 4233 (2022).
11. Eyedrop delivery of therapeutic proteins with zwitterionic polymers to treat dry age-related
macular degeneration. Biomaterials 305, 122429 (2024).
6) While ERG measurements showed improved visual function after Z(CAT&SOD) treatment, the
source of improvement (e.g., photoreceptor protection vs. retinal ganglion cell preservation vs.
synaptic conduction) is not clearly discussed. Consider correlating specific ERG wave changes (e.g.,
a-wave vs. b-wave) with histological data to identify the principal site of action.
Reply: Thanks so much for the reviewer’s suggestion. The 70% reduction in ERG amplitude observed
in the SI group correlated with commensurate thinning of the outer nuclear layer (ONL) in H&E-
stained sections and intense TUNEL labeling in photoreceptor segments. Z(CAT&SOD) treatment not
only restored a-wave amplitude but also preserved ONL thickness and reduced photoreceptor apoptosis,
indicating primary neuroprotection at the photoreceptor level. While the b-wave originates from
bipolar cells, its recovery temporally mirrored both a-wave restoration and ONL survival. This
temporal correspondence supports the established dependence of bipolar cell function on intact
photoreceptor input: by mitigating ROS-mediated photoreceptor degeneration, Z(CAT&SOD)
preserved synaptic transmission to second-order neurons, thereby facilitating b-wave recovery. Thus,
these ERG improvements reflect a primary photoreceptor rescue mechanism with consequent
restoration of downstream signaling pathways. The related discussion has been added in the revised
manuscript as following:
“The restoration of ERG amplitudes in Z(CAT&SOD)-treated mice, alongside intact ONL and RPE
morphology, provides direct evidence that photoreceptor-targeted ROS neutralization is both a
mechanistic requirement and a novel therapeutic strategy for preserving retinal function.”
7) The authors demonstrate M2 macrophage polarization and inflammation suppression following
treatment, but there is no follow-up data on whether this immune modulation is reversible or
potentially immunosuppressive. I recommend including 1-2 weeks of post-treatment follow-up to
evaluate the persistence and safety of immune alterations.
Reply: Thanks so much for the reviewer’s suggestion. To determine whether the observed retinal
immune modulation was sustained, we included a one-week drug-free observation period following
treatment. Retinal tissues were collected one week after the final administration, and IL-6, IL-12 P70
levels were quantified via ELISA. The lack of inflammatory rebound suggests that a single 7-day
course of Z(CAT&SOD) effectively mitigated oxidative stress, maintained retinal homeostasis, and
prevented secondary inflammatory activation (Figure R14). The related results and discussion have
been added in the revised manuscript as following.
Figure R14. Concentrations of IL-12P70 and IL-6 in the retinas of mice one week after the last
different treatments evaluated by ELISA (n=4).
8) The manuscript is generally readable but contains some repetitive phrasing and grammatical issues
(e.g., the phrase “has historically challenging to treat” in the Abstract is incorrect). Professional
language editing by a native speaker or editing service is strongly advised.
Reply: Thanks so much for the reviewer’s comment. All grammatical errors, awkward phrasing and
repetitions have been corrected as detailed above.
“Age-related macular degeneration (AMD), particularly in its most prevalent form geographic atrophy
(GA), has historically been challenging to treat due to its complex pathogenesis.”
“Our pioneering study revealed the critical association between retinal peroxidase deficiency and GA
progression, and proposed an innovative zwitterionic nanocage-based peroxidase, which exhibits great
potential as a non-invasive yet highly effective therapeutic strategy for AMD.”
“To demonstrate the successful establishment of GA mouse model, NaIO (10 mg/kg) was
3
intravenously injected on day 0.”
“Analysis of a- and b- waves, which correspond to photoreceptors and ON-bipolar cell activity,
demonstrated partial restoration of visual acuity in mice treated with CAT or SOD&CAT injections.”
9) The figure legends are somewhat inconsistent in statistical notation. Please standardize the
indication of significance (e.g., p<0.05, *p<0.01, etc.) and clearly state the statistical methods used
in each figure legend.
Reply: Thanks for pointing out this. We have now standardized all figure legends to use the same
significance notation (*p < 0.01, **p < 0.001, ***p < 0.0001) and explicitly state the statistical test
(one-way ANOVA) used for each panel.
Responses to reviewer’s comments
We sincerely thank all the reviewers for their time and valuable comments. We are particularly pleased
to see the recognition of our work from reviewer 2# and 3#. However, we are very concerned about
some of the comments from reviewer 1#. We believe that some of these comments are based on
misunderstandings of the work. And we will provide detailed responses point by point below in an
attempt to clarify the misunderstandings.
Reviewer #1:
The manuscript contains significant conceptual, methodological, and technical flaws related to ocular
biology, which limit the validity of the study. The authors have addressed some prior concerns and
added new experiments, but key issues remain.
1. Polymerized materials are generally inert, but incomplete polymerization or residual monomers
may pose risks and should be acknowledged.
Reply: Thanks so much for the reviewer’s suggestion. The synthesis protocol for zwitterionic polymer
is well-established and widely recognized for producing biomedical-grade polymers, as reported in
previous literatures1-5. Furthermore, our comprehensive in vivo safety data, presented in the initial
response (Figure R1 / S14), provide the most biologically relevant confirmation. Critically, no local
or systemic toxicity was observed, even after long-term topical administration. Therefore, these
findings demonstrate the safety of our nanocage system for its intended ophthalmic application.
Reference
1. Fang, H. et al. In Situ Polymerization of Zwitterions on Therapeutic Proteins to Enable Their
Effective Oral Delivery. ACS Nano 17, 1128-1143 (2023).
2. Fan, W. et al. Mucus Penetrating and Cell-Binding Polyzwitterionic Micelles as Potent Oral
Nanomedicine for Cancer Drug Delivery. Advanced Materials 34, 2109189 (2022).
3. Zhou, Y. et al. A pH-Triggered Self-Unpacking Capsule Containing Zwitterionic Hydrogel-Coated
MOF Nanoparticles for Efficient Oral Exendin-4 Delivery. Advanced Materials 33, 2102044
(2021).
4. Zhou, Q. et al. Enzyme-activatable polymer-drug conjugate augments tumour penetration and
treatment efficacy. Nature Nanotechnology 14, 799-809 (2019).
5. Han, X. et al. Zwitterionic micelles efficiently deliver oral insulin without opening tight junctions.
Nature Nanotechnology 15, 605-614 (2020).
Figure R1. Safety assessment of Z-polymer and Z(CAT&SOD) in normal mice (n=3). (a)
Representative images of cornea’s light fields and (b) fluorescein cornea staining of mouse eyes after
different treatments for 2 and 4 weeks by slit-lamp microscopy. (c) The tear secretion amount of mouse
eyes after different treatments for 2 and 4 weeks (d) Representative images of OCT images of cornea
and (e) retina for 2 and 4 weeks. (f) Statistical analysis of relative retina thickness. (g) Representative
images of retina’s light fields and (h) fluorescein staining of retina after different treatments for 2 and
4 weeks. (i) Statistical analysis of IOPs of eyes in mice after different treatments for 2 and 4 weeks. (j)
H&E staining images of different organs including liver, spleen, lung, kidney, heart and eye collected
from mice in different groups (scale bars: 500 μm). (k) Blood biochemistry and complete blood analysis of
mice with different treatments. The following parameters were tested: percentage of white blood cell
(WBC); percentage of lymphocytes (Lymph); percentage of monocytes (MON); percentage
of neutrophilic granulocyte (Gran); red blood cell (RBC) counts; hemoglobin (HGB); hematocrit
(HCT); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); platelets (PLT);
alanine aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase (ALP) and
blood urea nitrogen (BUN). Data were represented as means ± SD.
2. FFA detects vascular abnormalities; in SI-treated mice, increased FFA reflects RPE barrier
disruption, not neovascularization.
Reply: We sincerely appreciate the reviewer’s insightful comment, which allows us to better clarify a
critical aspect of our experimental model. We fully agree that in typical human geographic atrophy
(GA), increased FFA signal is primarily attributed to RPE barrier disruption rather than
neovascularization. However, it’s important to note that the sodium iodate (SI)-induced model in our
study, while effectively mimicking oxidative stress-driven photoreceptor/RPE degeneration
characteristic GA, often elicits a more extensive secondary inflammatory response involving retinal
vasculature. The vascular abnormalities we observed via FFA (has been changed as “vascular
inflammation” or “secondary vascular abnormalities” in the revised main text) do not represent
neovascularization, but rather reflect inflammation-associated vascular leakage specific in this model.
The observed fluorescence enhancement indeed stems from blood-retinal barrier disruption,
encompassing both RPE and endothelial cell damage under inflammatory conditions. Here, FFA
serves as a valuable in vivo method to evaluate the extent of secondary vascular inflammation and
overall blood-retinal barrier integrity, both important indicators of retinal health in our model. We
have moved this data to Supporting Information to avoid overstating its role in the core narrative of
GA-like atrophy, while still presenting it as a supporting metric of disease severity and treatment
efficacy against the multifaceted insult induced by SI.
We have carefully revised the manuscript to clarify that the FFA findings reflect model-specific
inflammatory responses rather than neovascularization. This distinction is now explicitly addressed in
the discussion to avoid any potential misunderstanding regarding neovascular and atrophic pathways.
“FFA images showing therapeutic effects on secondary vascular inflammation in SI-induced retinal
degeneration”
“As shown in Figure 2k and 2l, prominent retinal deposits were observed in both PBS- and SOD-
injected groups, accompanied by subretinal debris accumulation (likely originating from degenerated
RPE and photoreceptors), inflammatory edema, and oxidative stress-induced vascular abnormalities
(Figure S2a).”
3. Neovascularization is not expected in the SI injury model.
Reply: We appreciate the reviewer’s insightful comment regarding the sodium iodate (SI) model’s
pathophysiology. While SI primarily targets the RPE and photoreceptors, and is not a classic model
for choroidal neovascularization (CNV), extensive literatures demonstrates that its severe oxidative
injury triggers robust secondary inflammatory response. This inflammation response extends beyond
the RPE/photoreceptor layer, affecting retinal vasculature and leading to blood-retinal barrier (BRB)
breakdown, vascular leakage, and in some reports, even aberrant vascular changes6-9. Importantly,
these effects are inflammation-mediated and distinct from VEGF-driven neovascularization in wet
AMD. In our revised manuscript, we have consistently termed these observation as “secondary
vascular abnormalities”, “vascular inflammation” or “vascular leakage” to accurately reflect this
inflammatory vasculopathy.
“In contrast, the Z(CAT&SOD) group displayed a more intact retinal structure and lower inflammation
levels, comparable to the healthy retinal microenvironment. This dual-enzyme approach not only
mitigates inflammation but also helps maintain the integrity of the retinal structure by reducing
vascular permeability and preventing secondary vascular abnormalities.”
Reference
6. Hollyfield, J. G. et al. Oxidative damage-induced inflammation initiates age-related macular
degeneration. Nat. Med. 14, 194-198 (2008).
7. Yao, T. et al. Comparative mechanistic study of RPE cell death induced by different oxidative
stresses. Redox Biol. 65, 102840 (2023).
8. Kerur, N. et al. cGAS drives noncanonical-inflammasome activation in age-related macular
degeneration. Nat. Med. 24, 50-61 (2018).
9. Zhang, R. et al. A pH/ROS dual-responsive and targeting nanotherapy for vascular inflammatory
diseases. Biomaterials 230, 119605 (2020).
4. Fig. S12 references CNV incorrectly; SI injury does not induce CNV, and GA does not involve CNV.
Reply: We thank the reviewer for this valuable feedback. Upon careful consideration, we recognized
that the original phrase “lesions in GA mice” could be misleading, as classic GA is pathologically
characterized by atrophic lesions (RPE/photoreceptor loss) rather than vascular lesions detectable by
FFA. To ensure conceptual clarity, we have revised the figure legend to describe these observations
more accurately in the revised manuscript as following. In addition, we would like to clarify the
scientific rationale for including this assay. While the SI model primarily recapitulates the oxidative
stress and photoreceptor/RPE degeneration characteristic of GA, it also elicits a significant secondary
inflammatory response. Importantly, this inflammation can disrupt the blood-retinal barrier (BRB),
resulting in quantifiable vascular abnormalities detectable by FFA.
“FFA images showing therapeutic effects on secondary vascular inflammation in SI-induced retinal
degeneration”
“As shown in Figure 2k and 2l, prominent retinal deposits were observed in both PBS- and SOD-
injected groups, accompanied by subretinal debris accumulation (likely originating from degenerated
RPE and photoreceptors), inflammatory edema, and oxidative stress-induced vascular abnormalities
(Figure S2a).”
5. ERG a- and b-waves measure retinal function, not visual acuity.
Reply: We sincerely appreciate the reviewer’s insightful comment and fully agree with this important
clarification. We acknowledge that certain terms in our original description required greater precision.
Specifically, we have revised phrases such as “restoration of visual acuity” in the context of ERG data
with more precise descriptions as following in the revised manuscript.
“Analysis of the a- and b- waves, which reflect photoreceptor and ON-bipolar cell activity, respectively,
demonstrated partial recovery of electrophysiological responses in mice treated with either CAT or
SOD&CAT injections.”
6. Missing statistical summaries of ERG amplitudes is a critical issue; plotting style conventions are
minor (see Section 5).
Reply: Thanks so much for pointing it out. We would like to clarify that the complete statistical
analyses for the ERG amplitude data are provided in the Supporting Information as Figure S12 in our
original manuscript.
Figure R2. Representative ERG wave responses of acute retinal injury mice under scotopic conditions
following different treatments, along with statistical analysis of A and B waves measured by ERG
(n=4).
7. The rationale for using 10 mg/kg SI is not provided.
Reply: We appreciate the reviewer’s question regarding the SI dosage. The 10 mg/kg sodium iodate
(administered intravenously) was selected based on well-established protocols to ensure a robust and
reproducible model of oxidative retinal injury10-12. The related discussion has been added in the revised
manuscript as following:
“This dosage is well-established to consistently induce oxidative damage in RPE cells and
photoreceptors, creating an acute retinal injury mouse model that recapitulates key oxidative stress-
related cellular events in GA33-35.”
Reference
10. Liu, B. et al. Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19
cells. Cell Death & Disease 12, 230 (2021).
11. Chen, Y. et al. Oxidative stress induces mitochondrial iron overload and ferroptotic cell death.
Scientific Reports 13, 15515 (2023).
12. Zhou, Y. et al. Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell
activation via stimulating oxidative phosphorylation. Cell Death & Disease 13, 689 (2022).
8. The SI model should not be referred to as GA; it is an acute chemical injury distinct from human
geographic atrophy.
Reply: Thanks so much for the reviewer’s important comment regarding disease modeling
terminology. We fully agree that the sodium iodate (SI) model represents an acute chemical injury
model and does not fully replicate the chronic, multifactorial pathogenesis of human geographic
atrophy (GA). We selected the SI model because it is a well-established and widely used preclinical
model that reliably induces oxidative stress-mediated RPE and photoreceptor degeneration, the key
cellular events in GA pathogenesis13-14. This model allows us to specifically test whether our
antioxidant nanocage platform can protect these critical cell types from the oxidative damage, a central
mechanism in GA progression. We have revised the text to clarify the model’s limitations while
maintaining its scientific rational: to evaluate whether targeted antioxidant delivery can rescue retinal
cells from oxidative stress, a therapeutic strategy directly relevant to dry AMD/GA. We thank the
reviewer for this valuable opportunity to improve our manuscript’s clarity.
Title: “Permeable nanoreactor eyedrop for enzymatic cascade-mediated treatment for acute retinal
injury model mimicking geographic atrophy”
“Collectively, our work demonstrates that the zwitterionic nanocage platform enables effective topical
delivery of antioxidant enzymes to the retina, presenting a promising therapeutic strategy for
combating oxidative stress in conditions such as dry AMD.”
Reference
13. Fleckenstein, M. et al. Age-Related Macular Degeneration: A Review. JAMA 331, 147-157 (2024).
14. Ambati, J. et al. An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-
2-deficient mice. Nature Medicine 9, 1390-1397 (2003).
9. Mice lack a macula; human GA scoring is not applicable. GA-like lesions in mice should be
described as RPE/photoreceptor atrophy.
Reply: We sincerely appreciate the reviewer’s insightful comment regarding species-specific
anatomical considerations. As correctly noted, mice lack a macula, which precludes direct application
of human GA clinical scoring criteria that rely on macular lesion characteristics. In response to above
important observation, we would like to clarify that the scoring system presented in Table R1
represents a customized, semi-quantitative histopathological grading scale specifically developed for
this study. This tailored approach enables systematic assessment of two key pathological features in
our mouse model: the severity of RPE layer degeneration and the degree of overall retinal thinning.
By establishing this species-appropriate evaluation method, we aimed to maintain rigorous
pathological assessment while respecting fundamental anatomical differences between murine and
human ocular structures.
“After different treatments, a blinded evaluation was performed based on fundus imaging and OCT
imaging to assess retinal structural integrity and thickness reduction(Figure S2b).”
Table R1. The scoring rubric of acute retinal injury mice model
10. “Neurodeatrophia” is nonstandard; “neurodegeneration” or “neuronal atrophy” is correct.
Reply: We appreciate the reviewer’s careful attention to terminology. As correctly noted,
“Neurodeatrophia” is a nonstandard term. We have replaced all instances with the standard term,
“neurodegeneration” throughout the manuscript and figure in this revised manuscript (Figure R3).
Figure R3. Schematic illustration of antioxidant mechanism disruption and therapeutic intervention in
acute retinal injury mice model. (a) In the progression of acute retinal injury model, neurodegeneration
is associated with the accumulation of oxidative stress and reduction in peroxidase levels. This
imbalance triggers retinal inflammatory pathways, leading to the atrophy and antioxidant dysfunction
of RPEs. (b) A groundbreaking and noninvasive eye drop formulation utilizing zwitterionic polymer-
modified peroxidase (Z(CAT&SOD)) was developed for GA treatment. The Z(CAT&SOD) eye drops
could effectively penetrate the ocular barriers through both conjunctiva-sclera-retina and conjunctiva-
blood-retina pathways to reduce oxidative stress and mitigate inflammation by harnessing the via
enzymatic cascade, offering a promising therapeutic approach for GA management.
11. “Atrophy of optic nerve cells” is incorrect; the shapes shown do not resemble photoreceptor outer
segments.
Reply: We sincerely appreciate the reviewer’s valuable feedback on our schematic diagram. In
response to above comment, we have revised “Atrophy of optic nerve cells” to the accurate term
“neurodegeneration”. The diagram was designed to conceptually illustrate the pathological
progression from an organized, healthy structure to a damaged, degenerative state. The abstract shapes
were intended to represent dystrophic and fragmented photoreceptor remnants within the atrophic zone,
consistent with our histological observations.
12. “Drusen” is misapplied; SI models do not produce true drusen and may represent pseudodrusen.
Reply: Thanks so much for the reviewer’s suggestion. Our schematic aimed to conceptually represent
subretinal deposit accumulation in our model’s retinal degenerative phenotype, which shares a
conceptual similarity with the deposit-driven pathology in human AMD. While we acknowledge that
“Drusen” specifically refers to extracellular deposits located between the RPE and Bruch’s membrane
in human eyes, a feature not fully recapitulated in our model, we have revised the terminology to
“Drusen-like deposits” in both the schematic and manuscript to reflect these distinctions more
accurately while maintaining the conceptual parallel.
Figure R4. Schematic illustration of the retina of acute retinal injury mice model.
13. Antioxidant dysfunction is not illustrated in the RPE.
Reply: We sincerely appreciate the reviewer’s constructive suggestion to enhance the conceptual
clarity of our schematic. We fully agree that explicitly illustrating antioxidant dysfunction within the
RPE cell would better represent this critical pathological mechanism. Accordingly, we have modified
the schematic to incorporate this important element, which will significantly enhance its scientific
accuracy and educational value.
14. Panels showing “conjunctival barrier” and “sclerotic barrier” inside cells are anatomically
incorrect.
Reply: Thanks so much for the reviewer’s suggestion. Our schematic aimed to illustrate the key ocular
barriers that a topically applied formulation must overcome. In response to the comment, we have
adjusted the illustration to better highlight the cell-cell interfaces and junctional complexes that form
these functional barriers. Additionally, we will update the figure legend to explicitly state: “Schematic
representation of the cellular barriers formed by conjunctival and scleral cells.”
15. The scheme does not accurately show how CAT and SOD reach the RPE; Z(CAT&SOD) contains
both enzymes but this is not clear.
Reply: We sincerely appreciate the reviewer’s comment regarding the schematic. We would like to
clarify that this illustration is intended as a simplified, conceptual overview highlighting the key steps
of our therapeutic strategy. The detailed mechanism of cellular uptake and intracellular delivery of the
enzymes (CAT and SOD) by our zwitterionic nanocage, Z(CAT&SOD), is rigorously supported by
experimental evidence presented in the manuscript. In the part where we studied the penetration section
of Z(CAT&SOD), we provided a detailed description of its penetration mechanism again. See Figure
R5 (Figure 3m). What’s more, we also demonstrate the co-loading and quantitative encapsulation
efficiency of both CAT and SOD in Table S1. What’s more, we also tested the enzyme activity of CAT
and SOD in the zwitterionic polymer nanocages.
Figure R5. Schematic illustration of penetration mechanism of zwitterion peroxidase.
16. HCECs and HConEpiCs are irrelevant; SI injury affects RPE and photoreceptors.
Reply: We sincerely appreciate the reviewer’s comment. This provides us with a valuable opportunity
to clarify the dual purpose of our schematic. This illustration is not limited to depicting the SI injury
model; rather, it presents a comprehensive overview of our drug delivery strategy: from topical
application and traversal of anterior segment barriers to therapeutic action at the retinal disease site.
Importantly, removing the anterior segment cells (HCECs, HConEpiCs) would mispresent our study,
as these elements are essential for demonstrating the delivery challenge that our zwitterionic nanocage
specifically addresses.
17. Methods list ARPE-32, HUVECs, and RAW 264.7 cells, but none appear in the results. HCECs
are not listed in Methods and are corneal endothelial cells, not relevant to the study.
Reply: We sincerely appreciate the reviewer’s careful evaluation of our Methods and Results sections.
We acknowledge and apologize for the identified discrepancies, which resulted from an oversight
during manuscript preparation. We are grateful for the opportunity to address these issues and have
conducted a comprehensive, cross-referential review of the entire manuscript to ensure full consistency
between the methodologies described and the results presented.
“Human conjunctival epithelial cells (HConEpiC) were obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA), and cultured in DMEM containing 1%
penicillin/streptomycin and 10% FBS at 37 °C in 5% CO . Seven-week-old female C57BL/6 mice
2
were purchased from Nanjing Pengsheng Biological Technology Co. The animals were acclimated in
SPF environment for one week prior to conducting follow-up experiments. All animal procedures
complied with relevant laws and were approved by the Institutional Animal Care and Use Committee
of Soochow University (No. ECSU-2019000198).”
18. The subtitle “Construction of a GA model” is misleading; the model is an acute chemical injury.
Reply: We appreciate the reviewer’s valuable suggestion and fully agree with the recommendation.
Accordingly, we have modified the subtitle from “Construction of a GA model” to the more accurate
description “Construction and evaluation of acute retinal injury mice model” in this revised manuscript
as following:
“Construction and evaluation of acute retinal injury mice model”
19. Methods for ocular assays (fundoscopy, OCT, ERG), instrumentation, and analyses are not
described, indicating lack of ocular biology expertise.
Reply: We sincerely appreciate the reviewer’s comment regarding the methodological aspect. We
apologize for this oversight, which stemmed from our assumption that these protocols were standard
practice rather than from any technical limitations. We have provided the detailed methodological
specifications that were inadvertently omitted in this revised manuscript as following:
“In Vivo Imaging (Fundoscopy, OCT and ERG): All imaging was performed using a Micron IV retinal
imaging system (Phoenix Research Labs) coupled with a high-resolution OCT module and ERG
module. Prior to imaging, mice were anesthetized via intraperitoneal injection of ketamine (80 mg/kg)
and xylazine, followed by pupil dilation with with topical administration of 1% tropicamide.
Fundoscopy: Color fundus images were captured using the integrated color fundus camera under
standard white light illumination.
Spectral-Domain OCT (SD-OCT): Retinal cross-sectional scans were acquired using the scanning
protocol centered on the optic nerve head. Subsequent quantification of retinal layer thicknesses was
performed using image J system.
Electroretinography (ERG): Full-field ERG recording was performed in accordance with the
International Society for Clinical Electrophysiology of Vision (ISCEV) standard protocol for rodents.
Following overnight dark adaptation (>12 hours), mice were anesthetized as above and their pupils
were dilated under dim red illumination. Scotopic (rod-dominated) responses were first recorded using
a series of flashes with increasing intensities (3 log cd·s/m2). For photopic (cone-mediated)
responses, mice were light-adapted for 10 minutes under a 30 cd/m2 background before recording
responses to a 1.0 log cd·s/m2 flash stimulus. ERG waveform analysis included: a-wave amplitude
(baseline to trough of initial negative deflection), b-wave amplitude (a-wave trough to the peak of
subsequent positive wave), implicit times for both components. All recordings and analyses were
performed using the integrated ERG module of the Micron IV retinal imaging system.”
20. Intravitreal injections of 5 µL are physiologically impossible in mice (vitreous≈2 µL).
Reply: We sincerely appreciate the reviewer’s comment regarding the critical technical aspect. The
reviewer is correct in noting that a 5 µL intravitreal injection would exceed physiological limits in the
mouse eye (with a vitreous volume of approximately 2 µL) and could potentially cause ocular damage.
We would like to clarify that in all experimental procedures, we strictly adhered to the standard
injection volumes of either 1 µL or 2 µL, as explicitly detailed in Section 4 of the methods. These
volumes are well-established and well-tolerated in murine models. The reference to “5 µL” appears
to be an inadvertent typographical error that may have appeared in supplementary materials or figure
legends. We have immediately conducted a thorough, line-by-line review of the entire manuscript to
ensure the intravitreal injection volume is correct.
“We first induced acute retinal injury model in mice using SI on Day 0 and randomly divided them
into four groups: Untreated (1×PBS, 2 μL), Z(CAT) (2 mg/mL, 80000 U/g, 1 μL), Z(SOD) (2 mg/mL,
50000 U/g, 1 μL), and Z(CAT&SOD) (CAT: 1 mg/mL, 40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2
μL).”
“After intravenous injection of SI, subjects were randomly divided into six groups: Untreated (1×PBS
eyedrop, 2 μL twice daily for a week), CAT&SOD eye drops (CAT: 1 mg/mL, 40000 U/g, SOD: 1
mg/mL, 25000 U/g, 2 μL twice daily for a week), CAT&SOD intravitreal injection (same
concentration, single 2 μL injection), Z(CAT&SOD) eye drops (same concentration, 2 μL twice
daily for a week), Z(CAT&SOD) intravitreal injection (same concentration, single 2 μL injection),
and lutein oral administration (0.5 μg lutein per day for one week).”
21. Hyperfluorescent spots from FAF images are not quantified.?
Reply: Thanks so much for the reviewer’s attention of the imaging data. We would like to clarify that
the images were obtained using fluorescein angiography (FFA) or color fundus photography (CFP)
rather than fundus autofluorescence (FAF). We apologize for any confusion and have verified that all
figures and descriptions in the manuscript accurately reflect the imaging techniques employed in our
study.
22. Claims of “pioneering a new therapeutic paradigm for dry AMD” overstate the study.
Reply: We sincerely appreciate the reviewer’s the reviewer’s valuable feedback regarding the tone of
our conclusion. We fully acknowledge that the phrase “pioneering a new therapeutic paradigm” may
be considered overly ambitious, and we grateful for the opportunity to revise the concluding statements
to reflect the scope and significance of our findings while maintaining scientific rigor more precisely.
“This work develops a novel therapeutic platform that tackles two critical challenges in dry AMD
treatment including non-invasive delivery and combined antioxidant therapy.”
23. The SI model is not representative of human GA or AMD.
Reply: We appreciate the reviewer’s insightful comment. We fully acknowledge that nearly no animal
model perfectly recapitulates the complex, chronic progression of human GA/AMD. However, our
study is primarily a mechanism-driven proof-of-concept investigation, designed to test whether non-
invasive delivery of antioxidant enzyme can protect the retina from oxidative injury-a key pathogenic
driver in dry AMD. For this specific purpose, the SI model serves as is a validated experimental system
that reliably induces oxidative stress-mediated RPE and photoreceptor degeneration, which are
hallmark cellular events in GA. Given the current lack of effective GA therapies, exploring novel
strategies in reproducible models remains critically important. Additionally, we have carefully revised
the manuscript wording better reflect these considerations as following.
Title: “Permeable nanoreactor eyedrop for enzymatic cascade-mediated treatment for acute retinal
injury model mimicking geographic atrophy”
24. Simply increasing SOD/CAT is not universally protective; overexpression can have unintended
effects.
Reply: We appreciate the reviewer’s insightful comment regarding the potential risks of antioxidant
enzyme overexpression. We completely agree that constitutive overexpression can disrupt redox
homeostasis, a well-known challenge in antioxidant therapy. However, our approach differs
fundamentally: rather than genetically inducing chronic enzyme production, we employ localized
delivery of precisely dosed SOD and CAT co-encapsulated in a protective nanocage. This
pharmacologic strategy ensures stoichiometric enzyme delivery to injury sites without genetic
alteration. Importantly, our in vivo safety data (Figure R1/S14) demonstrate no detectable toxicity,
after single or repeated administration, directly addressing the reviewer’s concern. While we
acknowledge the biological complexities of antioxidant interventions, our study provides the first
evidence that transient, dual-enzyme delivery can achieve efficacy without the off-target effects
associated with traditional antioxidant strategies. In summary, while we share the reviewer’s caution,
our data show that controlled, localized enzyme delivery circumvents the historical pitfalls of
nonspecific antioxidant approaches.
“In summary, this work demonstrated that the pathogenesis of acute retinal injury involves excessive
retinal ROS production and dimished peroxidase activity. Replenishment of CAT and SOD effectively
attenuated retinal oxidative stress and inflammation, thereby reducing cellular apoptosis. Moreover,
we developed a localized delivery system featuring precisely dosed SOD and CAT co-encapsulated in
a protective nanocage, enabling stoichiometric enzyme delivery to injury sites while avoiding genetic
alteration.”
25. Evidence regarding GPx/TAS in AMD, delivery of CAT/SOD to RPE mitochondria, and SOD
mimetics or gene therapy as clinically feasible strategies is not addressed.
Reply: We appreciate the reviewer’s valuable perspective on this issue. While we fully acknowledge
the important roles of glutathione GPx and TAS in AMD pathophysiology, our decision to focus on
the CAT/SOD cascade was driven by our experiment findings showing their specific downregulation
in our GA model (Figure 2i, S1b, c) and their direct causal link to ROS-mediated apoptosis. This
formed the basis for our targeted therapeutic strategy of replenishing these compromised endogenous
enzymes. We have incorporated discussion of GPx/TAS and their complementary antioxidant roles in
the revised manuscript.
Regarding subcellular targeting, we agree that mitochondrial delivery would be ideal given its role as
a major ROS source. While organelle-specific targeting remains challenge for protein therapies, our
work establishes a crucial foundation by achieving efficient delivery of active enzymes to retinal cells
(RPE/photoreceptors), as demonstrated by the confocal imaging (Figure 3f) and functional rescue data
(Figs. 4-6). Future platform iterations incorporating mitochondrial-targeting motifs represent an
exciting direction, which have highlighted in the revised manuscript.
Collectively, our system represents a non-genetic protein replacement strategy that leverages the
natural catalytic efficiency of evolutionarily optimized enzymes. Our nanocage system effectively
preserves their full native activity while enabling non-invasive delivery.
“Our findings highlight the therapeutic potential of enzymatic antioxidant therapy while revealing
important avenues for further research. First, comprehensive investigation of endogenous antioxidant
systems, particularly the glutathione peroxidase (GPx)/total antioxidant status (TAS) and related
peroxidases, could uncover synergistic mechanisms. Developing organelle-specific delivery platforms
may further optimize therapeutic efficacy in oxidative retinal injury models. Second, while the SI-
induced acute model has provided valuable mechanistic insights, its limitations in modeling the
chronic, multifactorial nature of human dry AMD should be acknowledged. Complementary model
systems that better recapitulate disease progression will be crucial for translating these antioxidant
strategies into clinically viable therapies.”
26. Fig. R1 (now Fig. S14) legend does not specify whether assays were performed in untreated or SI-
treated animals.
Reply: Thanks so much f for pointing it out. The long-term safety test was conducted in healthy mice,
and we have included this detail in the SI section.
“Figure S14. Safety assessment of Z-polymer and Z(CAT&SOD) in healthy mice (n=3).”
27. Figures S9b and S13b lack clear legends, treatment descriptions, and baseline “( before treatment”)
images.
Reply: We sincerely appreciate the reviewer’s f suggestion and apologize for any confusion caused.
Regarding the referenced figures (Figure S9b and S13b), they comprehensively document the complete
treatment protocols and dosage information corresponding to Chapters 3 and 4, respectively. We would
like to clarify that the absence of pre-treatment assessment stems from our experimental design:
therapeutic intervention was intentionally initiated immediately after establishing the SI-induced acute
retinal injury model. This approach was specifically to prevent irreversible pathological progression
in this of the acute chemical-induced retinal damage model. Therefore, mice in each group were in a
healthy state prior to treatment, and this baseline condition was included as a control group in the study.
28. Terms such as “thickness length of CNV” are unclear.
Reply: Thanks so much for pointing it out. We have made corresponding revisions in the revised
manuscript.
“Figure S12. (a) Schematic illustration of the treatments in SI-induced acute retinal injury model
mouse model, along with fundus assessment conducted after one and two weeks. (b) Statistical analysis
of the thickness length of retina by Image J (n=4).”
29. Missing “respectively” when defining a- and b-waves (p.4, line 144; p.5, line 180).
Reply: Thanks so much for pointing it out. We have made corresponding revisions in the revised
manuscript.
“Analysis of a- and b- waves, which correspond to photoreceptors and ON-bipolar cell activity
respectively, demonstrated partial restoration of visual acuity in mice treated with CAT or SOD&CAT.”
30. Plotting style of some ERG panels (e.g., Fig. 4g) is unconventional.
Reply: We sincerely appreciate the reviewer’s comment. The figure presents the statistical analysis of
a-wave and b-wave amplitudes, which have been refined for improved clarity and visual presentation.
Figure R6. Statistical analysis of A waves and B waves in retina of mice in different groups under
scotopic condition (n=4).
31. “Eye drop” should be two words.
Reply: Thanks so much for pointing it out. We have changed "eyedrop" to the standard form "eye
drop" throughout the manuscript.
32. Abstract and introduction are excessively long; discussion does not include limitations.
Reply: Thanks so much for pointing it out. We have revised abstract and introduction.
“Abstract:
Age-related macular degeneration (AMD), particularly in its most prevalent form geographic
atrophy (GA), has historically been challenging to treat due to its complex pathogenesis. A key driver
of GA progression is the oxidative stress-induced apoptosis of retinal cells, ultimately leading to
blindness. In our study using a retinal acute injury mouse model to mimic the reactive oxygen species
(ROS)-induced retinal degeneration pathogenesis of GA, we consistently observed effective recovery
of enzymatic cascade may be an effective therapeutic strategy for this model. To overcome ocular
delivery barriers, we engineered zwitterionic nanocages for co-delivering CAT and SOD. This system
significantly alleviated oxidative damage and inflammation in an SI-induced retinal injury model.
Given the chronic and progressive nature of GA, zwitterionic-peroxidase was optimized to develop a
non-invasive eye drop formulation for GA management. Surprisingly, the zwitterionic-peroxidase
achieved a remarkable 9.32% penetration rate into the eyeball, significantly outperforming the free
peroxidase (0.54%), through conjunctival-sclera-retina and conjunctival-blood-retina penetration
pathways, and achieved therapeutic efficacy comparable to intravitreal injections. Additionally, extra
supplementation of Z(CAT&SOD) in acute retinal injury model showed superior therapeutic effects
compared to the current gold standard clinical treatment of supplementing antioxidants and lutein.
Our pioneering study revealed the possible association between retinal peroxidase deficiency and GA
progression, and proposed an innovative zwitterionic nanocage-based peroxidase, which exhibits great
potential as a non-invasive yet highly effective therapeutic strategy for AMD.”
“Introduction:
Age-related macular degeneration (AMD) is a progressive and degenerative disease characterized
by dysfunction and death of retinal pigment epithelium (RPE) cells and photoreceptors, leading to
irreversible vision loss1, 2. It is the leading cause of permanent visual impairment worldwide, affecting
more than 180 million people, mainly over the age of 55. AMD manifests in two primary forms:
geographic atrophy (GA) and choroidal neovascularization (CNV). Among them, GA patients
constitute approximately 85% to 90%, and the rate of blindness approaches nearly 20%3. Since the
introduction of intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF), patients
with CNV have experienced substantial improvements in retinal function. However, despite the
effectiveness of anti-VEGF therapies for CNV, these antibodies have proven ineffective in treating
GA4. Therefore, there is currently no approved treatment for GA, primarily due to its complex and
poorly understood pathogenesis. Clinically, supplementing antioxidants such as lutein appears to be
the only viable approach to slowing down the deterioration of GA5. Numerous studies have revealed
that relative pathogenesis of GA involves processes such as complement system activation,
disturbances in lipid metabolism, oxidative stress, abnormal autophagy, and the chronic inflammation
pathways and etc6-8. In response, various therapeutic strategies have been explored, including the
suppression of complement activation, inhibition of angiogenesis, and exertion of anti-inflammatory
effects9-11. However, these approaches have not yet demonstrated specific, positive impacts in clinical
trials involving GA patients12-14. Therefore, the exploration of alternative therapeutic targets for GA
treatment remains crucial.
Oxidative stress disrupts the physiological functions in most biological tissues and can lead to
cell death15, 16. In the retina, oxidative stress triggers a cascade of events that result in focal loss of RPE
and photoreceptors, leading to structural changes in proteins, lipids and DNA, which is increasingly
recognized as a major contributing factor to the development of GA12, 15, 17. Numerous studies have
demonstrated that the removal of free radicals from the retina can effectively slow the progression of
AMD18. Additionally, in AMD treatment research, strategies targeting the elimination of reactive
oxygen species (ROS) have been proposed, including the use of inorganic nanozymes like Pt, RhZ,
CeO to perform antioxidant function19-22. However, these nanozymes face challenges such as
2
biocompatibility, biodegradability, and unclear therapeutic efficacy. Endogenous antioxidant enzyme,
such as superoxide dismutase (SOD) and catalase (CAT), neutralize free radicals and maintain cellular
homeostasis, with SOD targeting superoxide anion (O •−) and CAT decomposing hydrogen peroxide
2
(H O ). These enzymes play essential roles in defending against oxidant stress, particularly in
2 2
regulating ROS in inflammatory diseases23-26. However, the specific roles of CAT and SOD in the
pathogenesis of age-related macular degeneration, especially during the geographic atrophy stage,
require further investigation.
In this study, we discovered that the cellular antioxidant defense mechanisms were obviously
compromised in SI-induced acute retinal injury model, pathological feature of which mimic that of
human GA disease27, as evidenced by a noticeable decrease in the expression of CAT and SOD in the
retina. While supplementation with CAT and SOD partially alleviated retinal atrophy and visual
impairment, the therapeutic effects were not satisfactory. These limitations arise from poor ocular
delivery and enzyme instability. Hence, we explored the use of zwitterions, which can polymerize in
situ on protein surfaces to effectively shield charges, enhance permeability and preserve enzyme
activity in complex physical environment28-32. By ingeniously employing zwitterionic materials, we
coated the CAT and SOD with sulfobetaine methacrylate (SBMA), Z(CAT&SOD), a compound
widely recognized for its charge regulation, mucosal and tissue penetration and biocompatibility.
Remarkably, the Z(CAT&SOD) exhibited improved stability and activity against protease. Moreover,
Z(CAT&SOD) demonstrated the ability to facilitate free diffusion across ocular barriers penetration
capabilities by promoting clathrin-dependent and caveolae-mediated endocytosis. As expected,
following intravitreal injection of Z(CAT&SOD) in acute retinal injury model, the enzymatic cascade
reactions exhibited superior ROS scavenging capacity and suppressed the activation of NF-κB-
HMGB1-ILs inflammatory pathway. Ultimately, they protected RPEs and photoreceptors from ROS-
induced apoptosis, which is crucial for preserving retinal function and preventing degenerative changes.
Given the chronic nature and long-term treatment requirements of this disease, we attempted to
develop a non-invasive peroxidase eye drop formulation, compared with the clinical gold-standard
treatment strategy, supplementing with lutein. Unexpectedly, optimizing the components of
zwitterionic polymers led to a significant enhancement in the ocular barrier permeability of
zwitterionic-peroxidase, which achieved an impressive 9.32% ocular penetration (the ocular
penetration of free peroxidase was only 0.54%) by increasing the permeability through both
conjunctiva-sclera-retinal and blood-retinal pathways. Remarkably, the therapeutic effects of
zwitterionic-peroxidase eye drops were comparable to those achieved through intravitreal injections
in the acute retinal injury model, and both of them demonstrated greater efficacy compared to oral
lutein therapy. Our research results emphasize that the progression of GA is associated with clear
oxidative stress damage to the retina. The innovative and non-invasive eye drop we developed, which
is based on a bimetallic ion nanocage coating of peroxidase, provides a transformative and promising
therapeutic approach for dry AMD (Figure 1).”
“Discussion:
In the SI-induced acute retinal injury model, we observed pathological features resembling those
in clinical GA patients, including subretinal deposits and retinal atrophy. Mechanistic investigations revealed that
RPE and photoreceptor cell apoptosis was closely associated with the microenvironment characterized
by high oxidative stress and diminished peroxidase activity, particularly reduced levels of CAT and
SOD. As a proof-of-concept, our findings suggest that timely antioxidant supplementation may
preserve residual photoreceptors when oxidative damage as the primary driver of degeneration. However,
the coulombic interactions within the vitreous body and the ILM barrier in the vitreous cavity posed
significant challenges to the diffusion and penetration of peroxidases into the RPE and optic nerve cells,
even when employing intravitreal injection as a benchmark ophthalmic drug delivery modality. To
overcome these challenges, we developed zwitterionic nanocages for CAT and SOD to enhance
diffusion and barrier penetration by shielding surface charges and promoting endocytosis pathways.
Following the intravitreal injection of Z(CAT&SOD), we observed a significant mitigation of retinal
damage in SI-induced acute retinal injury model. This was evidenced by reduced ROS-induced damage
in RPE and optic nerve cells, suppression of the NF-κB-HMGB1-ILs inflammation pathways. Therefore,
the strategy of using zwitterionic peroxidase with intravitreal injection shows promise for slowing the
progression of dry AMD.
However, early-stage dry AMD patients often delay therapy due to concerns about traumatic
scarring and postoperative infection, thereby missing the optimal treatment window. Moreover,
patients with advanced atrophic dry AMD require sustained therapeutic drug concentrations over an
extended period. Developing an effective, non-invasive ophthalmic treatment strategy based on
peroxidase is of great importance. To address this, we optimized the components of zwitterionic
polymers to select the nanocages with the strongest barrier penetration as carriers for a peroxidase eye
drop formulation. Unexpectedly, eyeball penetration of Z(CAT&SOD) eye drop was 9.32%, much
higher than that of free peroxidase (the eyeball penetration was only 0.54%). Furthermore, after
applying the Z(CAT&SOD) eye drop for two weeks, we observed a significant reduction in retinal cell
apoptosis induced by SI, alongside stabilization of retinal thickness and visual function, achieving
comparable therapeutic effects to those observed in the intravitreal injection of Z(CAT&SOD). More
interestingly, compared with the therapeutic effects of both intravitreal injection and eye drops of
Z(CAT&SOD), lutein exhibited inferior therapeutic efficacy. This may be attributed to the retina was
affected by the blood-eye barrier in the systemic circulatory system, resulting in limited absorption
and the limitation of ROS-scavenging ability of lutein. In summary, this work reported that the
pathogenesis of acute retinal injury model is linked to the overproduction of retinal ROS and reduced
peroxidase levels. Replenishing CAT and SOD could reduce retinal oxidative stress and inflammation,
decreasing cell apoptosis. And we employed localized delivery of precisely dosed SOD and CAT co-
encapsulated in a protective nanocage, which ensured stoichiometric enzyme delivery to injury sites
without genetic alteration. Collectively, our work demonstrates that the zwitterionic nanocage
platform enables effective topical delivery of antioxidant enzymes to the retina, presenting a promising
therapeutic strategy for combating oxidative stress in conditions such as dry AMD.
Our findings highlight the therapeutic potential of enzymatic antioxidant therapy while revealing
important avenues for further research. First, comprehensive investigation of endogenous antioxidant
systems, particularly the glutathione peroxidase (GPx)/total antioxidant status (TAS) and related
peroxidases, could uncover synergistic mechanisms. Developing organelle-specific delivery platforms
may further optimize therapeutic efficacy in oxidative retinal injury models. Second, while the SI-
induced acute model has provided valuable mechanistic insights, its limitations in modeling the
chronic, multifactorial nature of human dry AMD should be acknowledged. Complementary model
systems that better recapitulate disease progression will be crucial for translating these antioxidant
strategies into clinically viable therapies.”
33. Missing references (p.3, lines 94–96; p.11, lines 452–455).
Reply: We have added the references according to the reviewer’s suggestion.
34. Many figure legends lack treatment details, orientation, and arrow explanations.
Reply: Thanks so much for pointing it out. We have added orientation and arrow explanations in some
figures.
35. IR fundus images (Fig. 4b, 6e) do not indicate whether they are from the same animals.
Reply: We sincerely appreciate the reviewer’s valuable observation. The IR fundus images in Figure
4b and 6e represent longitudinal tracking of the same animal cohort. This can be confirmed through
careful examination of each mouse’s unique retinal vascular signature, including specific vessel
branching patterns, trajectories and bifurcation points, which remains consistent across different time
points, which serves as a reliable intrinsic identifier.
36. OCT images lack optic nerve localization and measurement indicators.
Reply: Thanks so much for pointing it out. We have marked the location of the optic nerve on the
corresponding OCT images, and described the measurement method in the methods section.
Figure R7. Representative fundus images and OCT images of lesions in healthy versus acute retinal
injury model mice at different timepoints. Internal Limiting Membrane (ILM); Nerve Fiber Layer
(NFL); Ganglion Cell Layer (GCL); Inner Plexiform Layer (IPL); Inner Nuclear Layer (INL);
Outer Plexiform Layer (OPL); Outer Nuclear Layer (ONL); External Limiting Membrane (ELM);
Photoreceptor Layer (Inner Segment/IS and Outer Segment/OS); Retinal Pigment Epithelium (RPE).
37. Source tissue for mRNA analyses is unspecified.
Reply: Thanks so much for the reviewer highlighting this methodological detail, and we have specified
this clearly in the revised Methods section.
“Additionally, we collected retinas from acute retinal injury model mice at both one week and two
weeks, and quantified the expression levels of CAT and SOD by quantitative real-time polymerase
chain reaction (qPCR). At two weeks, a significant reduction in the expression of both CAT and SOD
was observed (Figure 2i).”
“Retinas were extracted from the eyeballs of SI-mice and lysed using a sonic disruptor in Western and
IP sample lysates (500 μL) at 0 oC. The supernatant was then centrifuged, and 100 microliters were
used for CAT and SOD assay tests. Additionally, RNA expression levels of CAT and SOD were
assessed by qPCR analysis. Retinal cells isolated from GA mice were lysed with Trizol, and the RNA
was diluted to a final concentration of 200 ng/μL for analysis. Primer and probe sequences were as
follows: CAT forward primer: AAATGCTTCAGGGCCGCCTT; CAT reverse primer:
GTAGGGACAGTTCACAGGTA; SOD forward primer: AACAATGGTGAAGGCTGTTGCTGT;
SOD reverse primer: TTGTGCATGCACATGGAACCCATG; GAPDH forward primer:
GTGACGTTGACATCCGTAAAGA; GAPDH reverse primer: GTAACAGTCCGCCTAGAAGCAC.
These primes were synthesized by Servicebio.”
38. TUNEL staining in the RPE is not indicated.
Reply: We sincerely appreciate the reviewer’s comment regarding the dynamic pathological
progression in our acute injury model. The observation can be explained by both technical
considerations and the temporal progression of SI-induced damage. The focal signal detected in
Healthy RPE layers represents tissue autofluorescence, a technical artifact rather than genuine
apoptosis. Regarding the absence of TUNEL signal in the RPE layer of SI-injured mice, this is finding
accurately reflects the model’s pathophysiology: SI induces rapid, severe oxidative damage that
primarily targets RPE cells, leading to early apoptosis/necrosis and subsequent clearance of these
vulnerable cells by the 1-week endpoint (our tissue harvest time). Consequently, the remaining RPE
layer appears depopulated or severely damaged, explaining the lack of late-stage TUNEL signal.
Conversely, the prominent TUNEL signal observed in the outer nuclear layer and inner nuclear layer
(INL) at this later time point represents secondary, progressive apoptosis in neuronal cells, driven by
the ongoing oxidative stress and inflammatory microenvironment following the initial RPE injury.
“TUNEL analysis further demonstrated that Z(CAT&SOD) significantly attenuated SI-induced acute
retinal injury by suppressing retinal cells apoptosis, particularly in the RPE layer. Notably, without
timely ROS-scavenging intervention, RPE cell apoptosis progressed to near-total depletion,
highlighting the critical window for therapeutic efficacy (Figure 5a).”
Figure R8. Representative immunofluorescence staining images showing the expression of TUNEL
in the retina of mice in different groups (scale bar: 50 μm; the arrow indicates the direction of the
retina).
39. Individual data points are missing from plots, especially Fig. 3l and ERG amplitude analyses.
Reply: We have revised all relevant bar graphs (including Figure 3l and all ERG amplitude analyses)
to display scatter plots (individual data points) overlaid with the mean ± SEM (or SD) according to the
reviewer’s suggestion.
✱✱✱✱
%100 ns
e
k
a 80
tp
u 60
lle
c 40
e
v
ita 20
le
R 0
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Figure R9. Relative uptake of Z(CAT-Cy5.5) by cells after incubation with different pathway inhibitors
(n=6).
40. Some ERG plots are partially obscured by axis labels.
Reply: Thanks so much for pointing it out. We have adjusted the layout of the affected ERG figures
to ensure all plot elements are fully visible and not obscured by axis labels in the revised manuscript.
41. Fig. 5a: retinal orientation unclear; TUNEL higher in “Healthy” vs. “Untreated” is
unexplained.
Reply: Thanks so much for pointing this out. We acknowledge that the current version lacks explicit
anatomical labeling and we have revised the figure accordingly to ensure clarity in the manuscript.
The focal, bright signal observed in the RPE layer of the “Health” sample represents a known technical
artifact. As the RPE contains abundant melanin and other pigments, these can cause autofluorescence
and/or non-specific background signals in certain TUNEL assay conditions, leading to localized false-
positive signals. In contrast, the diffuse and intense TUNEL staining observed throughout all nuclear
layers in the “Untreated” (SI-injured) group represents genuine, widespread apoptosis induced by
oxidative stress.
Figure R10. Representative immunofluorescence staining images showing the expression of TUNEL
in the retina of mice in different groups (scale bar: 50 μm; the arrow indicates the direction of the
retina).
42. Fig. 5l schematic is not informative.
Reply: We sincerely appreciate the reviewer’s comment regarding Figure 5l. This schematic serves as an
integrative summary, consolidating key molecular pathway findings from the preceding panels (Figure
5a to 5k) and related data. Specifically, it illustrates how the oxidative insult triggers activation of the
NF-κB-HMGB1-ILs inflammatory axis (as demonstrated by antibody-based localization in Figure 5b,
5c and 5d) and drives macrophage polarization toward a pro-inflammatory state, ultimately driving
retinal cell apoptosis.
Figure R11. Schematic illustration of the oxidative stress triggers activation of the NF-κB-HMGB1-
ILs inflammatory axis in relative retinal cells.
43. Fig. 6e: timing of lutein treatment relative to SI is unclear; prophylactic vs. post-injury effects
differ.
Reply: We appreciate the reviewer’s insightful question regarding the timing of lutein administration.
In in this study, including oral lutein supplementation (0.5 µg/day), were initiated therapeutically after
SI-induced injury. Specifically, lutein administration began on the same day following SI injection and
continued daily for one week, paralleling the treatment window for the enzyme-based interventions.
This design was chosen to mimic a clinically relevant therapeutic approach, as patients typically start
antioxidant supplementation after disease onset or diagnosis.
“After intravenous injection of SI, subjects were randomly divided into six groups (Figure S12a):
Untreated (1×PBS eyedrop, 2 μL twice daily for a week), CAT&SOD eye drops (CAT: 1 mg/mL,
40000 U/g, SOD: 1 mg/mL, 25000 U/g, 2 μL twice daily for a week), CAT&SOD intravitreal injection
(same concentration, single 2 μL injection), Z(CAT&SOD) eye drops (same concentration, 2 μL twice
daily for a week), Z(CAT&SOD) intravitreal injection (same concentration, single 2 μL injection),
and lutein oral administration (0.5 μg lutein per day for one week).”
Figure R12. Schematic illustration of the treatments in SI-induced acute retinal injury mouse model,
along with fundus assessment conducted after one and two weeks.
44. Fig. 6f: statistical analysis of a- and b-wave amplitudes for all animals is missing.
Reply: Thanks so much for pointing it out. We would like to clarify that the full statistical analyses
for the ERG amplitude data are included in the Supporting Information as Figure S12 in the original
manuscript.
45. CAT-FITC enzymatic activity is not reported.
Reply: We sincerely appreciate the reviewer’s comment regarding the use of CAT-FITC conjugate.
This fluorescent tracer was used exclusively as a subset of in vitro and ex vivo experiments (e.g.,
cellular uptake studies) to qualitatively visualize the intracellular localization and delivery
efficiency of our nanocage platform. While full retention of enzymatic activity would be ideal, it was
not a primary requirement for these proof-of-concept imaging studies. Moreover, we have provided
direct experimental evidence that our core therapeutic formulation successfully maintains activity of
enzymes.
Figure R13. The enzyme activity stability of Z(SOD), Z(CAT), free SOD and free CAT at different
storage environments (4 °C, 25 °C and 37 °C).
Summary: The manuscript contains fundamental flaws in experimental design, methodology, and
biological interpretation. Major issues include misrepresentation of the SI injury model as GA,
inappropriate in vitro cell models, physiologically impossible intravitreal injections, missing
descriptions of ocular assays and analyses, and multiple inaccuracies in figures and data
interpretation. These flaws significantly compromise the validity of the study.
1. Reply: We sincerely appreciate the reviewer’s thoughtful evaluation of our work. In response to
the comprehensive feedback, we have implemented the following substantive revisions to the
manuscript: Model clarification: the experimental model has been explicitly defined and
consistently referenced throughout the manuscript to eliminate any ambiguity.
2. Technical correction: we have verified and confirmed that intravitreal injection volume were
correctly reported as 1 µL or 2 µL in our original manuscript. The reviewer’s mention of of 5 µL
appears to stem from a misinterpretation, which we have now clarified in the revised text. .
3. Methodological rigor: all ocular assay protocols have been expanded with granular detail,
including instrumentation parameters, quality control measures, and validation steps to ensure full
reproducibility.
4. Comprehensive revisions: every textual statement and graphical element has undergone dual
verification for numerical accuracy, terminological precision, and logical consistency with the
dataset.
Through these revisions, we have strengthened the manuscript’s scientific rigor and transparency. We
are confident the current version provides robust evidentiary support for its conclusions and
respectfully submit it for reevaluation.
Reviewer #2:
The authors have satisfactorily addressed my concerns; I recommend acceptance.
Reply: Thanks so much for the reviewer’s positive comment.
Reviewer #3:
The authors did a nice job to address my concerns and I would like to reccomend accept this
manuscript without scientific change.
Reply: Thanks so much for the reviewer’s positive comment.
Responses to reviewer’s comments
We sincerely thank all the reviewers for their time and valuable comments. We are particularly pleased
to see the recognition of our work from reviewer 4#. Below we have provided point-to-point responses
to the comments and have revised the manuscript accordingly.
Reviewer #4:
This manuscript offers compelling evidence that a zwitterionic nanocage-based peroxidase eye drop
therapy could be a game-changer for treating oxidative stress-related retinal diseases. These diseases
may include geographic atrophy (GA) and dry age-related macular degeneration (AMD). The work
addresses a pressing gap in ophthalmology-therapeutic options for dry AMD remain scarce. By
pairing an innovative delivery platform with a deeper understanding of oxidative injury, this study
could really move the needle in the field.
The authors thoughtfully build on earlier antioxidant strategies. These include inorganic nanozymes
such as Pt-, RhZ-, and CeO₂-based systems, which have struggled with poor biocompatibility and
questionable efficacy. The zwitterionic nanocage platform sidesteps many of these issues. It boosts
molecular stability, tissue permeability, and catalytic activity, making it a more practical therapeutic
option. The non-invasive eye drop formulation improves patient compliance and accessibility,
especially for people in the early stages of disease who hesitate to pursue more invasive treatments.
After incorporating and addressing the reviewers' comments, the work is substantially more coherent.
That said, there are some limitations worth addressing to strengthen the translational relevance of
these findings. The authors lean heavily on the sodium iodate (NaIO₃) model. While this model is
widely used and practical for inducing RPE degeneration, it doesn’t fully mirror human AMD. NaIO₃
causes acute, chemically driven oxidative injury, leading to rapid RPE death. This occurs through
apoptosis, necroptosis, and ferroptosis. Human AMD, on the other hand, unfolds slowly and involves
a complex mix of lipid and protein buildup, chronic inflammation, complement dysregulation, and
genetic factors. Also, NaIO₃ results are highly dose-dependent and can vary significantly from animal
to animal. So the model captures only one piece of the AMD puzzle and might make the therapy appear
more robust than it would be in chronic, real-world disease. These limitations don’t undermine the
study, but they do suggest the authors should be cautious about claims of disease modification. The
response to the review does not fully cover this.
The modified experimental design, data analysis, and interpretation are rigorous and well supported
by the evidence presented. The reported therapeutic efficacy, tissue penetration, and non-invasive
nature of the nanocage eye drops are convincingly demonstrated. However, the study evaluates
treatment effects over only two weeks. This interval is insufficient to model the long-term progression
of GA or dry AMD. Extending the treatment and follow-up durations would substantially strengthen
the conclusions and better reflect clinical conditions. To clarify mechanisms, further studies should
analyze the roles of endogenous antioxidant pathways and organelle-specific effects, and examine how
the nanocage formulation interfaces with oxidative and inflammatory cascades.
For transparency and reproducibility, the authors should include details of how they calculated
sample sizes and explain their rationale for selecting specific statistical methods. Clearly justifying
these decisions and ensuring adequate statistical power and rigor will help build confidence in the
reported therapeutic effects.
Overall, this manuscript presents a really innovative therapeutic strategy with strong potential to
shape AMD research and clinical practice. While there’s more work to be done, especially in chronic
disease models, mechanistic validation, and longer-term studies, the approach is scientifically sound.
The findings are compelling. With revisions that address the concerns I’ve outlined, this study would
be a valuable addition to Nature Communications and the broader vision science community.
Reply: We are deeply appreciative of the reviewer’s thoughtful and constructive evaluation, as well as
the encouraging recognition of our work’s potential impact. The insightful suggestions have been
invaluable in strengthening the translational relevance and mechanistic depth of our study. Below,
we provide a point-by-point response to the specific concerns raised, detailing the revisions and
additional experiments we have conducted.
1. Model terminology, limitations, and claims of disease modification.
We fully agree with the reviewer that the sodium iodate (NaIO₃) model represents an acute
chemical injury and does not fully recapitulate the slow, multifactorial pathogenesis of human
geographic atrophy (GA) or dry age-related macular degeneration (AMD). In the revised manuscript,
we have consistently replaced “GA mouse model” with “NaIO₃-induced acute retinal injury model
(ARI)” in the text, titles, and figures. More importantly, we have added a dedicated subsection in the
Discussion as following, where we explicitly acknowledge that this model primarily mirrors the
oxidative stress component of retinal degeneration, and avoided over-interpreting its direct
equivalence to chronic human disease. We clarify that our study is a mechanistic proof-of-concept
designed to validate whether targeted enzyme delivery can ameliorate oxidative stress in the retina—
but a key driver in GA pathogenesis.
“In the acute retinal injury model designed to recapitulate the reactive oxygen species (ROS)-induced
degeneration pathology of GA, we consistently observed that restoration of the enzymatic cascade
represents a promising therapeutic strategy for this condition.”
“Collectively, our findings demonstrate that the zwitterionic nanocage platform enables effective
topical delivery of antioxidant enzymes to the retina, presenting a promising therapeutic strategy for
mitigating oxidative stress-related retinal damage. While the SI-induced acute oxidative injury model
serves as a valuable tool for investigating oxidative stress, a key driver of RPE and photoreceptor
degeneration, it represents a simplified, accelerated system that lacks the chronic, multifactorial
complexity of human dry AMD. Future studies will need to validate this proof-of-concept in more
physiologically relevant chronic models that better mimic the slow human disease progression.
Nonetheless, by addressing the critical challenge of ocular drug delivery, our platform provides a
versatile and patient-friendly foundation for developing disease-modifying therapies for dry AMD.”
2. Treatment duration and long-term therapeutic evaluation
We really appreciate the reviewer’s insightful comment regarding the limited observation window
for modeling chronic disease progression. In response, we would like to highlight that during the
preliminary phase of this study, we conducted an exploratory extended-duration experiment in which
mice were treated with Z(CAT&SOD) for one month following NaIO₃-induced injury (Figure R1).
Intriguingly, while this longer-term assessment confirmed sustained preservation of retinal structure
and function compared to controls, the therapeutic benefits plateaued after the initial two-week period.
We ultimately selected the two-week endpoint for the main study based on following reasons: (i) this
duration aligns with the acute progression of NaIO₃-induced retinal injury, (ii) the two-week data
robustly demonstrates the fundamental efficacy of our delivery platform, and (iii) this approach
adheres to the 3R principles (Replacement, Reduction, Refinement) by minimizing animal use while
maintaining scientific validity.
Figure R1. Representative fundus images and OCT images of mice after different treatments at (a) two
weeks and (c) one month. Statistical analysis of retinal thickness of mice in different groups at (b) two
weeks and (d) one month (n=6). Data were represented as means ± SD. P values in (c, d, e and g) were
calculated by using one-way ANOVA.
3. Elucidation of endogenous antioxidant pathways and organelle-specific effects
In response to the reviewer’s valuable suggestion regarding mechanistic insights, we examined
the effect of the zwitterionic nanocage’s internalization into cells on the mitochondria. We evaluated
the subcellular localization of Z(CAT) labeled with Cy5.5 in RPE cells using confocal microscopy
with a mitochondrial tracker (Figure R2). The imaging showed minimal mitochondrial accumulation
of the nanocage, indicating that its therapeutic mechanism does not rely on direct organelle targeting.
Instead, the nanocage predominantly localized to the cytosol and peri-mitochondrial space, a critical
region for ROS diffusion and redox signaling. This spatial distribution is particularly beneficial for
catalase activity, as ROS generated in mitochondria readily into the cytosol, where excessive
accumulation amplifies oxidative stress and inflammatory signaling. By scavenging cytosolic ROS,
our strategy indirectly mitigates oxidantive damage while preserving mitochondrial homeostasis.
To further characterize the therapeutic mechanism of Z(CAT&SOD), we conducted systematic
analyses of endogenous antioxidant responses and mitochondrial protection in oxidatively stressed
RPE cells. Quantitative PCR revealed that SI-induced oxidative stress triggered a robust activation of
the Nrf2/ARE pathway, as evidenced by the significant upregulation of Nrf2 and its downstream
targests (HMOX-1, NQO1, GCLC, and GPX1). Notably, this oxidative stress response was effectively
attenuated by both free enzymes and Z(CAT&SOD), with the zwitterion-protected formulation
exhibiting superior efficacy (Figure R3a). We next evaluated mitochondrial protection by measuring
membrane potential and ROS generation. While confocal imaging confirmed minimal mitochondrial
accumulation of Z(CAT&SOD), functional assays revealed its potent ability to preserve mitochondrial
membrane integrity and attenuate secondary ROS production (Figure R3b). These findings
demonstrate the therapeutic advantage of Z(CAT&SOD) in preventing mitochondrial oxidative
damage by effectively scavenging cytosolic ROS without requiring direct organelle targeting..
We have integrated these findings into the Results and Discussion, expanding our mechanistic
narrative from one of mere “replacement” to “activation and synergy” with endogenous systems.
“To investigate potential organelle-specific effects, we incubated RPE19 cells with Cy5.5 labeled
Z(CAT) for 6 hours and performed mitochondrial co-localization analysis using a mitochondrial
tracker. Confocal imaging revealed minimal mitochondrial accumulation of the nanocages
(Supplementary Fig. 8), indicating that their therapeutic mechanism does not rely on direct
mitochondrial targeting. Instead, the nanocage predominantly localized to the cytosol and peri-
mitochondrial space, a critical region for ROS diffusion and redox signaling. This spatial distribution
is particularly advantageous for catalase activity, as it positions the therapeutic agent near the sites of
ROS generation while allowing for effective diffusion throughout the cytosol. To further characterize
the cellular response to treatment, we analyzed the activation of endogenous antioxidant pathways in
oxidatively stressed RPE cells after Z(CAT&SOD) treatment using qPCR. RPE cells were first treated
with 200 μg/mL SI for 6 hours to induce oxidative stress, followed by treatment with free CAT&SOD
or Z(CAT&SOD) for additional 6 hours. The qPCR analysis revealed that Z(CAT&SOD) treatment
significantly downregulated the Nrf2 pathway and its downstream targets (e.g. GPX1, NQO1, GCLC,
HMOX-1) (Supplementary Fig. 9a). This oxidative stress induction was effectively attenuated by both
free CAT/SOD and the zwitterion-protected formulation, with the latter exhibiting superior protective
efficacy, likely due to enhanced enzyme stability and improved cellular delivery. To further evaluate
mitochondrial protection, we analyzed mitochondria integrity in stressed RPE cells after Z(CAT&SOD)
treatment (Supplementary Fig. 9b). Although the nanocage does not directly localize to mitochondria,
its cytosolic ROS-scavenging activity effectively preserved mitochondrial membrane potential and
reduced secondary mitochondrial ROS production. These findings highlight the significant organelle-
protective mechanism, reinforcing the therapeutic potential of our platform.”
“In summary, this study demonstrated that the pathogenesis of ARI is characterized by excessive retinal
ROS production and reduced peroxidase activity. Our therapeutic intervention with CAT and SOD
exhibits multi-model protection effects by effectively mitigating oxidative stress and inflammation
responses while reducing cellular apoptosis and preserving mitochondrial protection. These findings
establish a comprehensive antioxidant therapeutic approach for ARI management.”
Figure R2. Tracing of zwitterionic nanocage and mitochondria within RPE cells.
Figure R3. (a) Relative GPX1, GCLC, Nrf, NQO1 and HMOX-1 mRNA abundance (2-ΔΔCt) in
RPE cells with different treatments. (b) Mitochondrial damage changes in RPE cells with different
treatments.
4. Statistical transparency and reproducibility
In response to the reviewer’s request, we have expanded the Methods section to provide a
complete description of our statistical analysis framework. This include the rationale for sample size
determination and the selection of specific statistical tests (including one-way, two-way and t-test).
To ensure full compliance with journal policy and maximal transparency, we have prepared a
comprehensive Source Data file containing all individual quantitative measurements underlying the
graphical representations. This dataset is accompanied by detailed annotations specifying the exact
statistical test applied to each comparison.
Once again, we extend our sincere gratitude to Reviewer #4 for the exceptionally constructive and
forward-looking critique, which has substantially elevated the quality of our manuscript. We believe
the revised version, with its clarified scope, deeper mechanistic insight, and enhanced transparency,
now offers a compelling and robust case for the therapeutic potential of our zwitterionic nanocage
platform.