Granzyme B-mimetic nanozyme for nanovesicle targeted anticancer applications

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: accept

Abstract
Cytotoxic T lymphocytes play a crucial role in anti-tumour immunity, with granzyme B (GrB) being a decisive factor in this process. Developing a GrB-based delivery system to mimic T cell-based immunotherapy holds promise but remains challenging. Here, we present an artificial metalloenzyme (nanozyme) with GrB-like protease activity capable of inducing caspase-dependent cell apoptosis. The nanozyme is based on the site-specific binding of Pd(II) ions to recombinant human heavy chain apo-ferritin nanocages to generate a binuclear catalytic centre consisting of two Pd atoms with bridging cysteine ligands, monodentate methionine and histidine residues, and two water molecules. We show that encapsulation of the Pd-ferritin complex within membrane-fused lipid nanoparticles comprising surface-displayed single-chain antibodies affords GrB-mimicking nanozyme-based nanovesicles capable of receptor-mediated delivery of the nanozyme into the cytoplasm of tumour cells and induction of caspase-dependent apoptosis. This study provides valuable insights into the construction of nano-delivery systems with artificial GrB activity and presents a promising therapeutic option for solid tumours.

Introduction
The primary objective of tumour immunotherapy is to generate and deliver tumour-specific cytotoxic T lymphocytes (CTLs) to tumour sites1,2. The induction of cell apoptosis by granzyme B (GrB), which is the predominant serine protease stored within secretory granules of CTLs, is widely regarded as the key mechanism employed by CTLs to eliminate malignant tumour cells3,4. Upon recognition of the target tumour cells, the activated CTLs deliver GrB through the pore-forming protein perforin into the cytoplasm of target cells, thereby initiating cell apoptosis by mediating the cleavage of intracellular pro-caspases. One representative example that utilises this mechanism is chimeric antigen receptor (CAR)-T therapy, where T cells are genetically engineered to express a single-chain variable fragment (ScFv). This innovative approach has demonstrated promising outcomes in the treatment of specific cancer types, such as acute lymphoblastic leukaemia and lymphomas5–7. However, in solid tumours, CAR-T cell therapy continues to encounter several challenges, including but not limited to off-target effects, T cell exhaustion and ineffective T cell infiltration8–12. These issues largely stem from the insufficient recognition of tumour cells by CAR-T cells and the physical barriers that hinder effective CAR-T cell delivery. Thus, it is crucial to explore potential alternative strategies for overcoming the obstacles of T cell-based immunotherapy.

In recent years, the development of cell-mimicking delivery systems has emerged as a promising approach in the field of nanomedicine. Leveraging their nanoscale size, these systems are engineered to enhance targeting capabilities and overcome biological barriers, improving the delivery of therapeutic agents to solid tumours by replicating the natural characteristics and functions of cells13–18. For example, a smart strategy for mimicking CTL killing was developed by arming platelets with GrB and perforin19. These biomimetic nanocomplexes exhibit the ability to capture circulating tumour cells and actively target nascent metastatic lesions, resulting in effectively suppressing lung metastasis in tumour models. However, the high cost and intrinsic instability of GrB-based delivery systems represent major barriers to their practical application. These challenges highlight the potential of synthetic delivery vehicles embedding artificial GrB-like enzymes as an alternative strategy for tumour immunotherapy, an area in which GrB mimics have rarely been reported.

In this work, we describe an alternative approach using recombinant human heavy chain apo-ferritin nanocages (iron-free form of ferritin nanocages, FTn) to construct a palladium (Pd)-containing metalloenzyme (nanozyme, FTn-Pd) that specifically cleaves the same substrates as natural GrB and when encapsulated within synthetic vesicles induces caspase-dependent cell apoptosis (Fig. 1a). We use protein crystallography analysis to determine the location of the Pd(II) active sites on the surface of the 24-mer FTn and to reveal the potential catalytic mechanism associated with nanozyme-mediated substrate cleavage. Having established the GrB-like activity of the nanozymes, we develop a conceptual design for GrB-mimicking nanozyme-containing nanovesicles (GMNVs) that recapitulate the membrane properties of CAR-T cells and selectively interact with tumour cells (Fig. 1b). To achieve this, we encapsulate the FTn-Pd nanozyme within engineered lipid nanovesicles that display anti-human epidermal growth factor receptor 2 (HER2) ScFv on their membrane surface and explore the potential of the T cell mimics for in vivo targeting and anti-cancer efficacy in tumours with a high expression of HER2. Our results demonstrate that, unlike perforin-dependent mechanisms employed by natural CTLs, the FTn-Pd-containing vesicles induce caspase-dependent tumour cell apoptosis by direct delivery of the nanozymes into the cytoplasm. Taken together, our study provides valuable insights into the construction of targeted delivery systems with artificial GrB activity and presents a promising therapeutic option for solid tumours.

Results
Ferritin-based nanozymes with granzyme B-like activity
One of the critical factors for the de novo design of desired artificial enzymes is the choice of an appropriate protein scaffold. In this regard, we recently employed recombinant human heavy chain FTn as a highly symmetric protein scaffold to synthesise various metal-based nanoparticles/clusters for mimicking peroxidase activity20,21, scavenging superoxide radicals22 or inducing NO generation23. In this study, we sought to utilise FTn as a nanoscale scaffold for the development of nanozymes with distinct metal-ligand active sites engineered specifically for replicating the protease activity of GrB. Initially, we prepared a range of metal-protein complexes by direct binding of different metal ions to FTn and tested their nanozyme activity in the presence of the GrB-specific substrate Ac-IETD-pNA (Ac-Ile-Glu-Thr-Asp-para-nitroaniline) (Fig. 2a, b). Of the 10 metal-ferritin complexes studied, most showed no (Ru, Rh, Ir, Cu, Au, Co, Fe) or minimal activity (Mn, Pt). However, binding of Pd(II) to FTn (FTn-Pd) resulted in high protease-like activity with amide bond cleavage of the derivatized tetrapeptide substrate being completed, with 60 min in the enzymatic reaction buffer at 37 °C (Fig. 2b). Notably, FTn-Pd did not exhibit peroxidase (POD)-mimicking activity (Supplementary Fig. 1a), a primary inducer of free radicals (such as ·OH). However, upon reduction of the bound Pd(II) to Pd(0) using sodium borohydride (NaBH4), the resulting FTn-Pd(0) exhibited POD-mimicking activity (Supplementary Fig. 1b).

Given these observations, we investigated whether FTn-Pd could directly induce the cleavage of pro-caspase-3 and therefore mimic the specific functional operation of native GrB. Western blot analysis of the reaction mixture indicated that the concentration of the cleaved active form of caspase-3 increased progressively at higher concentrations of FTn-Pd (Fig. 2c). To further validate the GrB-like activity of FTn-Pd while excluding POD-like activity, inhibitors for GrB and POD were introduced in the pro-caspase-3 cleavage assay. Western blot analysis demonstrated that all three GrB inhibitors significantly impeded the production of cleaved caspase-3, whereas POD inhibitors did not (Fig. 2d), confirming the GrB-like activity in the developed nanozyme. To confirm the cleavage site of FTn-Pd on caspase-3, we mutated the IETD sequence to IETA. Western blot analysis of the reaction mixture before and after mutation indicated that the mutated caspase-3 was significantly less susceptible to recognition and cleavage by FTn-Pd (Fig. 2e). To further compare the enzymatic activities of FTn-Pd with natural GrB, we measured apparent steady-state kinetic parameters using Ac-IETD-pNA as substrate (Supplementary Fig. 2a, b). The data were fitted to the Michaelis-Menten model, yielding the kinetic parameters presented in Supplementary Fig. 2c. Although the catalytic efficiency (kcat/Km) of FTn-Pd is approximately 1.5-fold lower than that of natural GrB, FTn is known for its high temperature stability24, suggesting that FTn-Pd may offer a stability advantage over natural GrB. To evaluate this, we incubated both FTn-Pd and GrB at various temperatures (10, 37, 60, 80 °C) and pH levels (3, 5, 7, 9, 11) for 2 h, followed by activity measurement under standard conditions. FTn-Pd demonstrated significantly higher stability than GrB across a broad range of temperatures and pH levels (Supplementary Fig. 2d, e).

To further understand the GrB-like activity, we first investigated the structural properties of FTn-Pd. Transmission electron microscopy (TEM) imaging of the recombinant apo-ferritin prior to exposure to Pd(II) ions showed a hollow structure after negative staining, with an outer shell diameter of 12 nm and inner cavity of 8 nm (Fig. 2f). In contrast, intact FTn-Pd nanocages were directly observed under TEM without negative staining, presumably due to the increased electron density associated with the incorporation of Pd atoms. Inductively coupled plasma mass spectrometry (ICP-MS) indicated the presence of a total of 105 Pd atoms per FTn, corresponding to ca. 4 Pd atoms per protein subunit. The Pd atoms were strongly bound and did not readily detach from the protein when incubated in dialysis buffer over time, regardless of the presence of caspase-3 (Supplementary Fig. 3a). Protein crystallography was employed to determine the Pd binding regions within FTn. Protein crystals were prepared using the hanging drop vapour diffusion method, and the structure of the FTn-Pd crystals was refined to 1.85 Å resolution (Supplementary Table 1). Two prominent Pd binding regions, site A (M37/C90/C102/H105) and site B (H173), were revealed within the structurally intact polypeptide shell (Fig. 2g). Site A was located within a single subunit and contained two Pd atoms (Pd1 and Pd2) with occupancies of 0.8 and 0.8, respectively (Supplementary Table 2), resulting in a total of 24 site A Pd(II) atoms exposed on the exterior surface of each protein nanocage. The binuclear centre consisted of two bridging cysteine residues along with monodentate histidine (Pd1) and methionine (Pd2) side chains, and two water molecules coordinated with either Pd1 or Pd2. In contrast, site B consisted of a single Pd atom (Pd3), which was coordinated to four histidine residues of four different FTn subunits located along the six four-fold axes associated with molecular channels in the polypeptide shell25,26. To elucidate whether the sites played a pivotal role in determining GrB-like activity, two FTn mutants were constructed by modifying all four residues of site A (mutant H4-FTn; M37A/C90R/C102A/H105Q) and the single histidine of site B (mutant H1-FTn; H173A). In both cases, an approximately 50% reduction in the level of Pd binding was observed (Supplementary Fig. 3b, c). However, mutation at site B (H1-FTn-Pd) did not affect the GrB-like activity. In contrast, cleavage of Ac-IETD-pNA by H4-FTn-Pd was reduced to the background levels observed for FTn or Pd(II) alone (Fig. 2h), indicating that site A was functionally important for GrB-mimicking activity. This was confirmed by protein crystallography analysis at 1.53 Å resolution, which revealed that the mutation at site A was associated with the absence of Pd1 and Pd2 atoms (Fig. 2i).

Specificity and proposed catalytic mechanism of FTn-Pd nanozyme
To evaluate the catalytic specificity of the FTn-Pd nanozyme, several tetrapeptide substrates were selected, including IEAD (Bid cleavage site), IQAD (caspase-7 cleavage site), VETD (caspase-8 cleavage site), IETD (caspase-3 cleavage site), and IETA (mutant caspase-3 cleavage site). The activities of these substrates in response to FTn-Pd were examined. Molecular docking analysis showed that IETD had the most stable docking position, placing it just 3.1 Å from the catalytic centre of FTn-Pd (site A). In contrast, the distances for IEAD, IQAD, and VETD were significantly greater, at 13.6 Å, 10.2 Å and 10.0 Å, respectively (Fig. 3a). When IETD was mutated to IETA, the distance between the docking site and site A increased to 19.5 Å. The specific cleavage of IETD by FTn-Pd was further confirmed using HPLC analysis (Fig. 3b). Additionally, western blot analysis demonstrated that FTn-Pd exhibited no cleavage activity against recombinant pro-caspase-7, pro-caspase-8 or Bid (Supplementary Fig. 3d–f). These results indicate that the FTn-Pd specifically cleaves caspase-3, without affecting other similar substrates such as caspase-7, caspase-8 or Bid.

Having demonstrated that the GrB-like activity of the FTn-Pd nanozyme was determined by the coordination of two Pd(II) atoms at site A, we sought to elucidate the catalytic mechanism underlying GrB-like activity. Comparing the protein structures of FTn and FTn-Pd showed a local conformational change in the protein structure at site A due to binding of the two Pd(II) ions that gave rise to changes in the arrangement of amino acid residues and the overall architecture of the binding pocket (Fig. 3c). Notably, the histidine H105 side chain was rotated through an angle of 90 degrees. Consequently, the binding pocket of FTn-Pd exhibited enhanced stability by forming bonds with Pd1 and Pd2, along with two coordinated water molecules. Molecular docking analysis of FTn/Ac-IETD-pNA complexes showed two possible regions for substrate binding in regions delineated by K87/D91/R43/S36 and R22/N21/E83 (Fig. 3d, Supplementary Fig. 3g). Significant alterations were observed in these potential substrate binding regions for FTn-Pd/Ac-IETD-pNA complexes, primarily due to the structural changes associated with site A. Specifically, residues Asn25/Asp89/Asn109 were identified as the sole binding sites for Ac-IETD-pNA. Significantly, these residues were near site A, suggesting that preferential access to the catalytic active centre (site A) due to proximity binding was mechanistically important for the catalytic amide bond cleavage of the GrB substrate.

Based on the structural and molecular docking analysis, we propose that the Pd-induced 90-degree flip of H105 enables the histidine side chain to function as a reactive intermediate or proton donor/acceptor for scissile bond cleavage of the GrB substrate bound close to site A (Fig. 3e). This is consistent with the role of histidine in enzyme mechanisms involving proton transfer and reactive intermediate stabilisation27,28. Notably, this catalytic site architecture and mechanism are fundamentally distinct from those of native GrB29 (Supplementary Fig. 3h). This distinction provides a mechanistic explanation for why FTn–Pd selectively cleaves pro-caspase-3 but does not act on caspase-7, caspase-8, or Bid, all of which are substrates of native GrB.

Biomimetic design of FTn-Pd-loaded camouflaged nanovesicles
Having established the GrB-like activity of FTn-Pd, we sought to stimulate the apoptosis of tumour cells by targeted receptor-mediated delivery of the nanozyme using GMNVs. Given that FTn-based particles primarily accumulate in lysosomes after uptake30, we encapsulated FTn-Pd into camouflaged nanovesicles to facilitate direct delivery of the nanozyme into the cytoplasm of tumour cells through lipid-cell membrane fusion (Fig. 4a). To achieve this, we employed FTn-Pd-containing lipid nanoparticles (FNPs) with surface-displayed single-chain antibodies. The latter were obtained using antibody-anchored membrane technology and a genetic engineering strategy31 to display anti-HER2 ScFv on the membrane of Jurkat T cells (Supplementary Fig. 4a, b), and then integrated into the FNPs (FNP-antiHER2) by membrane extrusion (Fig. 4b). TEM images of the FNP-antiHER2 construct showed discrete vesicles that were structurally intact and larger than the precursor LNP and FNPs (Fig. 4c). Dynamic light scattering indicated that incorporation of FTn-Pd and/or the anti-HER2 ScFv-anchored membrane into the lipid nanoparticles (LNPs) resulted in an increase in mean size from 81 ± 3.4 nm (LNPs) to 101 ± 3.8 nm (FNPs) and 135 ± 2.1 nm (FNP-antiHER2), respectively (Fig. 4d), with the latter remaining stable for at least 7 days (Fig. 4e).

To elucidate the role of the anti-HER2 ScFv within a nanomedical context, we generated an MB49 cell line with high expression of the HER2 receptor (MB49-HER2+) using a genetically engineered approach involving a recombinant lentivirus-HER2 expression plasmid. Inclusion of anti-HER2 ScFv in the FNP membrane did not alter the cellular uptake of FNPs in MB49 tumour cells with low HER2 expression but significantly improved intracellular uptake in MB49-HER2+ tumour cells (Supplementary Fig. 4c). The intracellular fate of the FNP-antiHER2 constructs after incubation with MB49-HER2+ cells was determined using real-time imaging under confocal microscopy. During the first hour, the DiO-labelled LNPs and Cy5-labelled FTn-Pd primarily colocalized with the cell membrane, after which a progressive increase in the intracellular distribution of the FTn-Pd nanozyme was observed while most of the LNPs remained on the cell membrane (Fig. 4f, Supplementary Movie 1 and Supplementary Fig. 4d). Further studies using confocal imaging and image-based Pearson’s correlation analysis indicated that the majority of the intracellular FTn-Pd was not distributed within lysosomes (Fig. 4g, Supplementary Fig. 4e). Taken together, these results indicate that the FTn-Pd nanozyme payload of the FNP-antiHER2 construct was gradually released into the cytoplasm of the tumour cells after lipid-membrane fusion.

Given the above observations, we investigated whether the FNP-antiHER2 constructs could serve as a strategy for killing tumour cells by inducing caspase-dependent cell apoptosis after intracellular delivery to SKBR3 tumour cells with naturally high HER2 expression or genetically engineered MB49-HER2+. Compared to vesicles prepared without FTn-Pd (LNPs and LNP-antiHER2), equivalent concentrations of the FNP or FNP-antiHER2 nanoconstructs substantially inhibited cell viability (Fig. 4h). A dose-dependent anti-tumour effect of FNP and FNP-antiHER2 was observed in both types of tumour cells. Furthermore, FNP-antiHER2 exhibited superior anti-tumour ability compared to FNP alone, with cell viability decreasing to approximately 20% for high doses, indicating that delivery of the nanozyme in lipid vesicles displaying antiHER2 enhanced tumour cell apoptosis.

Molecular mechanisms underlying FNP-antiHER2-induced apoptosis
Given that FTn-Pd, as a GrB-mimicking nanozyme, selectively induces pro-caspase-3 cleavage, we next sought to determine whether FNP-antiHER2-induced apoptosis is caspase-3 dependent. To do this, we first assessed caspase-3 activation in SKBR3 cells following different treatments. Western blot analysis showed that treatment with FNP-antiHER2 led to caspase-3 cleavage, comparable to the effect of staurosporine (STS, a known caspase-3 inducer), and this cleavage was effectively blocked by Z-VAD-FMK, an irreversible pan-caspase inhibitor (Fig. 5a). To confirm the functional activity of cleaved caspase-3, we further analyzed the activation of its primary substrate, poly(ADP-ribose) polymerase (PARP). Western blot results revealed significant PARP activation following FNP-antiHER2 treatment, indicating that cleaved caspase-3 successfully propagated downstream apoptotic signalling. Furthermore, cells were immunostained with a cleaved caspase-3 antibody after treatment with FNP-antiHER2, allowing us to visualise intracellular caspase-3 activation by FNP-antiHER2 nanovesicles (Fig. 5b). To further validate that FNP-antiHER2 induces caspase-3-dependent apoptosis, we designed three small interfering RNAs (siRNAs) targeting full-length caspase-3 mRNA (i.e., cas3 siRNA-1, cas3 siRNA-2, cas3 siRNA-3) to downregulate intracellular caspase-3 expression. Western blot analysis revealed a significant reduction in the activation of caspase-3 and its substrate PARP in cells treated with cas3 siRNAs, compared to those treated with control siRNA (ctrl siRNA) (Fig. 5c). Additionally, flow cytometry showed a marked decrease in both early and late apoptosis in cas3 siRNA-3-treated cells following FNP-antiHER2 induction, compared to control siRNA-treated cells (Fig. 5d).

Next, we sought to employ RNA sequencing to compare gene expression in the FNP-antiHER2-treated group versus the control group. The data analysis identified 578 down-regulated and 1035 up-regulated genes in the FNP-antiHER2-treated cells compared to the control (Fig. 5e). Gene Ontology (GO) enrichment analysis of these differentially expressed genes highlighted several key functional impacts of FTn-Pd treatment. As shown in Fig. 5f, enriched GO terms included the regulation of tumour necrosis factor, the regulation of apoptotic signalling pathway, and the regulation of extrinsic apoptotic signalling. Further analysis revealed that these differentially expressed genes were closely linked to apoptotic pathways (Fig. 5g), suggesting that the anti-tumour activity of FNP-antiHER2 is medicated through apoptosis.

In vivo tumour targeting and anti-tumour efficacy
We next sought to evaluate the in vivo behaviour of intravenously administered FNP-antiHER2 in mice bearing SKBR3 tumours with naturally high HER2 expression. First, we examined the plasma pharmacokinetics and observed that FNPs prepared only with cell membranes (FNP-M, control) or with membrane-attached single-chain antibodies (FNP-antiHER2) had significantly prolonged circulation times compared to LNP alone (Fig. 6a). Typically, the particle concentrations in blood plasma at 12 h were determined as 53.5 ± 5.3, 181.5 ± 3.6, 185.1 ± 8.4 ng/mL for the LNP, FNP-M and FNP-antiHER2 constructs, respectively, indicating that cell membrane camouflage of the FNPs improved blood circulation time for LNP. To investigate in vivo accumulation in SKBR3 subcutaneous tumours, we performed in vivo imaging of the mice at the indicated time points after administration of DiR-labelled LNP, FNP-M or FNP-antiHER2. Over time, there was a gradual increase in tumour accumulation, with maximal accumulation attained at 8 h for both LNP and FNP-M (Fig. 6b). In contrast, the highest tumour uptake of FNP-antiHER2 was delayed to approximately 20 h. The enhanced uptake of FNP-antiHER2 could be attributed to both the prolonged blood circulation times and improved retention of particles in the tumour due to receptor-mediated binding of anti-HER2. Furthermore, the tumour tissue retention ability of the LNPs was lower than that of the FNP-M and FNP-antiHER2 nanovesicles, particularly in the case of FNP-antiHER2. Quantification analysis of the isolated tissues showed that the injected particles were primarily distributed in the liver, spleen, lung and tumours 48 h post-administration. Importantly, tumour tissue accumulation of FNP-antiHER2 was increased compared to both LNP and FNP-M (Supplementary Fig. 5).

Given the potential of FNP-antiHER2 to target HER2-positive tumours, we subsequently studied in vivo anti-tumour efficacy of the FNP-antiHER2 nanovesicles using HER2+ tumour models. We established two types of HER2+ tumour models, including mice xenografted with genetically engineered HER2+ tumour cells (MB49-HER2+) and mice xenografted with naturally occurring HER2+ tumour cells (SKBR3). Our initial focus was to evaluate whether the incorporation of the antiHER2-membrane camouflage could enhance therapeutic outcomes of the nanoconstructs in MB49-HER2+ tumours. To achieve this, we inoculated MB49-HER2+ and MB49 (low expression of HER2) tumour cells into the left and right shoulders of the same mice, respectively (Supplementary Fig. 6). Notably, compared to the rapid growth observed in the tumours treated with PBS, the systemically administered FNP-antiHER2 significantly delayed the growth of MB49 tumours (p < 0.05). Next, we further investigated the in vivo anti-tumour efficacy of different treatments in mice with MB49-HER2+ tumours, including PBS, FTn-Pd, LNP-M, LNP-antiHER2, FNP and FNP-antiHER2. Compared to the PBS group, treatment with either the FTn-Pd nanozyme alone or FNP exhibited a moderate inhibition of tumour growth in the MB49-HER2+ tumours (Fig. 6c–e). However, when systemically administered, FNP-antiHER2 displayed a significant suppressive effect on tumour progression. Significantly, Western blot analysis revealed that the levels of cleaved caspase-3 in tumour tissue appeared to increase to a greater extent when mice were treated with FNP-antiHER2 than with other treatments (Fig. 6f), consistent with the GrB-like activity of the FTn-Pd nanozyme payload. We also investigated the in vivo anti-tumour efficacy of the different nanovesicles in SKBR3 tumours, a naturally occurring HER2+ tumour model. Consistent with the results in the MB49-HER2+ tumour model, FNP-antiHER2 demonstrated the most potent anti-tumour efficacy compared to other nanoconstructs when applied to SKBR3 tumours (Fig. 6g–i). The inhibitory effect of the various treatments on tumour growth showed a positive correlation with tumour cell apoptosis, as evidenced by the presence of cleaved caspase-3 (Fig. 6j). Additionally, the MB49-HER2+-bearing mice, SKBR3-bearing mice, and healthy mice receiving FNP-antiHER2 experienced no significant change in body weight (Supplementary Fig. 7a–c). Complete blood count (CBC) parameters (including white blood cells, neutrophils, lymphocytes, monocytes, red blood cells and blood platelets), and serum biochemical parameters (including aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (UREA), and creatinine (CREA)) implied no apparent systemic toxicity associated with FNP-antiHER2 administration (Supplementary Fig. 7d–f). Representative H&E-stained images of the major organs and selected tissues (including heart, liver, spleen, lung, kidney, mammary gland, stomach, small intestine and skin) also showing that FNP-antiHER2 was well tolerated, with no systemic toxicity observed compared with the control group (Supplementary Fig. 8). Taken together, the above results demonstrate that the incorporation of antiHER2-membrane camouflage into FNPs facilitates the targeted delivery of the FTn-Pd nanozyme to MB49-HER2+ and SKBR3 tumours, leading to enhanced stimulation of caspase-dependent apoptosis via a T cell-mimicking pathway involving GrB-mimicking activity.

Methods
Preparation of FTn and mutant FTn
Recombinant human heavy chain FTn was prepared and purified according to our previously reported procedures20,48. The DNA sequence of mutant H4-FTn (M37A/C90R/C102A/H105Q) was synthesised by Genewiz (Suzhou). For mutant H1-FTn (H173A), the DNA sequence was prepared in our laboratory. Then, these genes were cloned into the pET-21a vector. The obtained plasmids were transfected into BL21 Escherichia coli. The expression and purification of H4-FTn and H1-FTn followed the same procedures as FTn. Briefly, overexpression of FTn, H4-FTn and H1-FTn was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. The cells were harvested and resuspended in phosphate buffer solution (PBS) buffer, followed by cell lysis and solution centrifugation. Next, the soluble fraction was heated at 65 °C for 10 min, and centrifugated at 10,000 g for 10 min. The supernatant was concentrated with an Amicon-Ultra (30 kDa, Millipore) and purified with AKTApure protein purification system (GE Healthcare) equipped with Superose 6 increase (GE Healthcare). The protein concentration was determined by the standard BCA method. The obtained FTn was observed following negative staining with 1% uranyl acetate using TEM (JEOL JEM-2800).

Protein crystallisation
FTn and H4-FTn were concentrated to 10–15 mg/mL by ultrafiltration. Crystals were grown by the hanging-drop vapour diffusion method at 10 °C. The drops were prepared by mixing an equal volume (1.2 μL) of concentrated protein solution (10 mg/mL) and the mother liquid (2 M MgCl2, 0.1 M Bicine, pH 9.0). When FTn and H4-FTn were crystallised, 0.2 μL of K2PdCl4 was added to the droplet to the final concentration of 1 mM to afford Pd(II) bound FTn crystals (FTn-Pd). After 24 h incubation, crystals were transferred into the solution with 2 M MgCl2, 100 mM Bicine (pH 9.0) and 10% saturated sucrose (v/v) and subsequently frozen in liquid nitrogen.

X-ray data collection and structure determination
The X-ray diffraction datasets of the crystals were collected either in-house on a Rigaku MIcroMax-007HF X-ray generator with R-AXIS IV++ detector (for FTn-Pd) or at beamline BL02U1 of the Shanghai Synchrotron Radiation Facility (for H4-FTn). The crystals diffracted up to 1.85 Å and 1.53 Å, respectively (Supplementary Table 1). The initial phase of the FTn-Pd structure was obtained using the SAD method by Pd anomalous diffraction signal using the AutoSol pipeline in the PHENIX suite49,50. The model was then manually adjusted and optimised using Phenix. refine. The H4-FTn structure was solved via molecular replacement using Phaser and then refined in the PHENIX suite (using FTn-Pd as the model). Molecular models were visualised using PyMol2 (http://www.pymol.org). The structures of FTn-Pd and H4-FTn-Pd were deposited in the RCSB PDB (Protein Data Bank) with accession codes 9JGO and 9JGP, respectively.

Molecular docking
Molecular docking of the substrate Ac-IETD-pNA (CAS: 219138-21-3), Ac-IETA-pNA, Ac-IQAD-pNA, Ac-VETD-pNA, and Ac-IEAD-pNA to FTn and FTn-Pd was performed using AutoDock 4.251. The structure of FTn was obtained from the Protein Data Bank (PDB ID: 7a6b), while the structure of FTn-Pd was determined by X-ray crystallography. AutoDockTools 1.5.7 was utilised to generate the pdbqt file following the standard AutoDock protocol. All water molecules were removed, and then hydrogens were added to both FTn and FTn-Pd. The substrates Ac-IETD-pNA, Ac-IETA-pNA, Ac-IQAD-pNA, Ac-VETD-pNA and Ac-IEAD-pNA were allowed to rotate during the docking process. A large grid box size of 126/126/126 was employed to cover the entire FTn (centred at 128.207/78.915/88.845) or FTn-Pd (centred at 39.186/27.845/11.436). The genetic algorithm parameters were set with a maximum of 25,000,000 evaluations and 30,000 generations. From the 600 generated poses, the results were selected based on the lowest binding energy. The binding site was analyzed using Discovery Studio 2019 and displayed using PyMol.

Preparation of FTn nanozymes
For preparation of FTn-Fe, FTn-Mn, FTn-Co, FTn-Au, FTn-Cu, FTn-Ir, FTn-Pd, FTn-Pt, FTn-Rh and FTn-Ru, FTn was dissolved in PBS buffer, and adjusted pH was adjusted to 8.0. Then, (NH4)2Fe(SO4)2·6H2O, MnCl2, CoCl2·6H2O, K[AuCl4]·2H2O, CuCl2, IrCl3, K2PdCl4, K2PtCl4, RhCl3·3H2O and RuCl3 were dissolved into deionized water, respectively, followed by adding dropwise into FTn solution separately (equivalent molar ratio, FTn: metal = 1:1000). After stirring for 1 h, free metal ions were removed using a PD-10 desalting column (GE Healthcare), and further purified via dialysis with a molecular weight cutoff of 10 kDa. To obtain reduced FTn-Pd(0), sodium borohydride (NaBH4), a reducing agent (0.089 mM, 500 μL), was slowly added to the Pd(II)-bound FTn (2 mg/mL) solution on an ice bath for 2 h. Finally, the resulting solution was further purified using a PD-10 desalting column. Of note, FTn-Pd nanozymes represent Pd(II)-bound FTn in this study, unless otherwise indicated. The concentration of FTn nanozymes was determined using standard FTn samples as a reference to quantify protein concentration according to SDS-PAGE gel electrophoresis analysis. The metal content of FTn nanozymes was quantified by ICP-MS. The obtained FTn-Pd was directly observed under TEM (JEOL JEM-2800).

Cleavage of GrB substrates
The GrB-like activity of FTn nanozymes was assessed by the hydrolysis of substrate Ac-IETD-pNA (Beyotime) at 37 °C in a 96-well microplate. The released chromogenic groups (paranitroanilide, pNA) by nanozymes were measured by monitoring absorbance at 405 nm. Briefly, 99 μL PBS solution containing various FTn nanozymes with a final concentration of 0.11 μM was added to each well. FTn (0.11 μM), BSA-Pd (0.75 μM), Pd(en)(H2O)2 and aqueous Pd(II) ions (equivalent Pd molar concentration with FTn-Pd) were also added as control groups. Subsequently, 1 μL of different substrates at various concentrations (1.56–200 μM) were added. The absorbance of pNA at 405 nm was recorded every 30 s using a microplate reader (Multiskan FC, Thermo Fisher Scientific). The enzyme activity curve and kinetic parameters were determined based on the Michaelis–Menten equation by GraphPad Prism v9.0 (GraphPad Software). The cleavage activity of various FTn nanozymes against different substrates was also measured using High-performance liquid chromatography (HPLC). After the hydrolysis reaction in the 96-well plate, the solution was transferred to an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The filtrate collected from below the tube was then subjected to HPLC analysis. Separations were conducted on a ChromCore C18 column (5 μM, 4.6 × 250 mm) of an HPLC system, and the column was eluted with methanol:H2O (1:1, v/v) over a period of 15 min, with a flow rate of 0.8 mL/min. The specificity of FTn-Pd towards Ac-IETD-pNA was assessed based on amide bond cleavage of different tetrapeptide substances, including Ac-IETD-pNA (wild-type caspase-3 cleaved site), Ac-IETA-pNA (mutant caspase-3 cleaved site), Ac-IQAD-pNA (caspase-7 cleaved site), Ac-VETD-pNA (caspase-8 cleaved site) and Ac-IEAD-pNA (Bid cleaved site), was also measured using HPLC following the method described above.

Recombinant expression of caspase-3, caspase-7, caspase-8, Bid, and mutant caspase-3
To verify the cleavage effect of FTn-Pd on protein substrates in vitro, we recombinantly expressed full-length proteins, including caspase-3, caspase-7, caspase-8, and Bid, using a prokaryotic expression system. All proteins were prepared by following a previous report52. Briefly, human caspase-3, caspase-7, caspase-8, and Bid cDNAs were extracted from human umbilical vein endothelial cells (HUVEC), and amplified by PCR using following primers: caspase-3 forward: 5’-CGATCGCGCATATGGAGAACACTGAAAACTC-3’; caspase-3 reverse: 5’-CGTCGATCCT CGAGTCAGTGGTGGTGGTGGTGGTGGTGATAAAAATAGAGTTCTTTTGTGAGC-3’. caspase-7 forward: 5’-GGGAATTCCATATGGCAGATGATCAGGGCTG-3’; caspase-7 reverse: 5’-GCCCCGCTCGAGTTGACTGAAGTAGAGTTCCTTG-3’. caspase-8 forward: 5’-CGATCGCGCATATGGACTTCAGCAGAAATC-3’; caspase-8 reverse: 5’-GCCCCGCTCGAGATCAGAAGGGAAGACAAGTT-3’. Bid forward: 5’-GGGAATTCCATATGGACTGTGAGGTCAACAAC-3’; Bid reverse: 5’-CATCGGCTCGAGGTCCATCCCATTTCTGGCTAAG-3’. The PCR products were cloned into the pET-21a plasmid using NdeI and XhoI as restriction sites. The mutant caspase-3 (D175A) was produced by site-directed mutagenesis using pET-21a-caspase-3 (wild-type) as a template. The primers used were: caspase-3 (D175A) forward: GACTGTGGCATTGAGACAGCAAGTGGTG TTGATGATGACATG, and caspase-3 (D175A) reverse: CATGTCATCATCAACACCACTTGCTGTCTC AATGCCACAGTC. All primers were synthesised by Sangon Biotech.

The resulting vectors were transformed into BL21 Escherichia coli, respectively. For protein expression, the following induction conditions were used: cells containing the caspase-3 or caspase-3 (D175A) expression plasmid were induced with 0.2 mM IPTG at 30 °C for 45 min; cells containing the caspase-7 plasmid were induced with 0.2 mM IPTG at 30 °C for 20 min; and cells containing the Bid plasmid were induced with 1 mM IPTG at 30 °C for 2 h. Following induction, the cells were lysed, and the expressed proteins were purified using HisTrap HP columns (GE Healthcare Life Sciences). For the expression and purification of caspase-8, cells containing the caspase-8 plasmid were induced with 0.02 mM IPTG at 30 °C for 3 h, and purified using an inclusion body isolation and refolding method, according to the procedure described in reference 53.

In vitro cleavage of recombinant caspase-3, caspase-7, caspase-8, Bid and mutant caspase-3
To evaluate the effect of FTn-Pd on recombinant protein substrates with a final concentration of 781 nM, varying concentrations of FTn-Pd (1.25, 5 and 20 nM) were co-incubated with the substrates at 37 °C for 1 h. After incubation, the samples were lysed and subjected to Western blot following the standard procedure to detect cleavage products. To further investigate the mechanism of FTn-Pd in promoting caspase-3 cleavage, GrB served as a positive control. Additionally, GrB inhibitors (Z-IETD-FMK, Human SerpinB9, and Z-AAD-CMK) and a POD inhibitor (ascorbic acid) were employed to investigate the involvement of specific pathways in the cleavage process. Briefly, Z-IETD-FMK (100 μM), SerpinB9 (1 μM), Z-AAD-CMK (100 μM) or ascorbic acid (100 μM) was incubated with FTn-Pd (20 nM) in 100 μL PBS buffer at 37 °C for 0.5 h, followed by adding 100 μL of caspase-3 to a final concentration of 781 nM and incubation at 37 °C for an additional 0.5 h. The reaction products were then analyzed via Western blot to assess the impact of the inhibitors on FTn-Pd-mediated cleavage of caspase-3. Uncropped and unprocessed scans of the blots are provided in the Source Data file.

Comparison of enzymatic stability of Granzyme B and FTn-Pd
To evaluate the enzymatic stability, the activities of GrB and FTn-Pd were compared under different temperatures and pH conditions. Briefly, GrB and FTn-Pd were incubated at temperatures of 10, 37, 60, or 80 °C for 2 h. For the pH stability study, GrB and FTn-Pd were incubated at pH levels of 3, 5, 7, 9, and 11 for 2 h. Following incubation, 99 μL of PBS solution (pH 7) containing 0.031 μM GrB or 0.11 μM FTn-Pd was added to each well. Subsequently, 1 μL of 20 mM Ac-IETD-pNA solution was added. Enzymatic activities were monitored by measuring absorbances at 405 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific) every 5 s at 37 °C. Kinetic parameters were calculated using the Michaelis–Menten equation by GraphPad Prism v9.0 software, and enzymatic activities were determined by kcat/Km.

Evaluation of POD-like activity
The POD-like activity of FTn nanozymes was studied by a colour reaction using TMB and H2O2 as substrates. Briefly, 10 μL of FTn nanozymes (100 nM), 10 μL of 2.2 M H2O2 and 10 μL of a series of TMB concentrations (0–41.61 mM) were added in 70 μL hydrogen acetate-sodium acetate (HAc-NaAc) buffer (pH 4.5) in a 96-well microplate. The resulting oxidised TMB were measured as absorbances at 652 nm on a microplate reader (Multiskan FC, Thermo Fisher Scientific) every 5 s. The kinetic parameters were determined based on the Michaelis–Menten equation by GraphPad Prism v9.0 software.

Anti-HER2 ScFv-overexpressing cell membrane
A recombinant lentivirus (LV)-anti-HER2 expression plasmid was first constructed to express anti-HER2 ScFv on the cell membrane of Jurkat T cells. A mCherry tag fused to the platelet-derived growth factor receptor (PDGFR) transmembrane domain was used to analyse the positive antiHER2 ScFv-overexpressing cells using flow cytometry and confocal microscopy. To obtain the packaged LV, LV-anti-HER2 plasmid was transfected into HEK293T cells along with vesicular stomatitis virus G glycoprotein (VSVG) and cytomegalovirus (CMV) packaging plasmids using Fugene@6 (E2691, Promega) transfection reagent following the manufacturer’s instructions. The medium was changed after 16 h incubation, followed by collecting the medium containing LV after 24 h and 48 h incubation. The collected medium was combined and centrifuged at 300 g for 10 min to remove the cell debris. To generate the anti-HER2-overexpressing cell line, the infecting medium containing LV-anti-HER2 and fresh medium (4:1, v/v) was added to Jurkat T cells. The medium was replaced with fresh medium after 24 h of incubation. The cells were collected and sorted based on the mCherry channel by fluorescence-activated cell sorting (BD). The sorted Jurkat T cells were cultured for a period of time and then used for the extraction of cell membranes. The cell membrane extracted from the wide-type Jurkat T cells was used as a control membrane.

Cell membrane extraction and purification
Cell membrane extraction was conducted following the previously published protocol with minor modifications54,55. Briefly, 1 × 10^9 cells were collected and washed with PBS buffer, then resuspended in 5 mL of 4 °C-precooled PBS buffer containing protease inhibitor. The cell suspension was sonicated at a power of 60 W for 20 min on ice to obtain the cell membrane fraction. The supernatant containing the crude cell membrane fraction was collected and pooled, followed by centrifugation at 3000 g for 15 min at 4 °C. The pellet containing cell debris and large cell organelles was discarded, and the supernatant was further centrifuged at 16,000 g for 15 min at 4 °C to collect the pellet. The pellet containing the cell membrane was suspended in PBS buffer containing protease inhibitor and dispensed into volumes of 100 µL. Membrane proteins were quantified using the PierceTM BCA Protein Assay Kit. The extracted cell membranes were stored at −80 °C for further use.

Preparation of Cy5-labelled FTn/FTn-Pd
For fluorescent labelling, Cy5-NHS ester was reacted with FTn/FTn-Pd (molar ratio, 50:1) at room temperature for 2 h in PBS solution (pH 8.0). The resulting mixture was purified with a PD-10 desalting column (GE Healthcare). The number of Cy5 per FTn/FTn-Pd was determined by measuring the concentration of FTn/FTn-Pd and Cy5, respectively. The Cy5 concentration was determined using the absorbance value at 630 nm and the established standard curve of Cy5 samples. In addition, for the preparation of Cy5-labelled FTn-Pd, Cy5-labelled FTn was firstly obtained and purified, and then dissolved in PBS buffer, and the pH was adjusted to 8.0. Next, 1.45 mg of K2PdCl4 was dissolved in deionized water, followed by adding dropwise into 2 mg of FTn in PBS buffer. Cy5-labelled FNP-Pd was also prepared using purification and quantification following the method described above and stored at 4 °C.

Preparation of LNPs
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were first dissolved in chloroform at the molar ratio of 76.2:3.8:20, respectively. The lipid films were then prepared using a mixture of DMPC, DOTAP and DSPE-PEG2000 following established methods46,47. The dried lipid films were formed by evaporating the organic solvent, followed by hydration and extrusion of the films. Briefly, to prepare LNP or FNP, PBS buffer-hydrated or FTn-Pd-hydrated lipid was heated to 40 °C and gently shaken for 10 min. The mixture containing FTn-Pd was then centrifuged at 5000 g for 15 min at 4 °C to remove the free FTn-Pd. Subsequently, the mixture was extruded sequentially through 800, 400, 200 and 100 nm polycarbonate membranes 20 times.

Preparation of cell membrane camouflaged LNP or FNP
To prepare cell membrane camouflaged LNP or FNP, the lipid mixture was firstly hydrated with PBS buffer or PBS buffer containing FTn-Pd. Cell membranes were then added at a specific mass ratio, followed by incubation and purification through centrifugation. The mixture was subsequently extruded through polycarbonate membranes with progressively smaller pore sizes (800, 400, 200 and 100 nm). Specifically, to prepare FNP-antiHER2 (i.e., GMNVs), the lipid films were firstly hydrated with FTn-Pd in PBS buffer, then heated to 40 °C and gently shaken for 10 min. The mixture was centrifugated at 5000 g for 15 min at 4 °C to remove the free FTn-Pd. AntiHER2-anchored cell membranes were added at a mass ratio of lipid: cell membranes of 100:1 (w/w). The mixture was incubated at 37 °C for 1 h, followed by centrifugation at 5000 g for 15 min at 4 °C to remove the free cell membranes. The resulting solution was finally extruded through polycarbonate membranes with progressively smaller pore sizes. FNP-M was also prepared using extrusion following the method described above and stored at 4 °C. The resulting particles needed to be used within one week to ensure stability.

Real-time imaging by confocal microscopy
Tumour cells were plated on glass-bottomed cell culture dishes (Nest Scientific) at a density of 1 × 10^4 and were cultured for 24 h prior to use. LNP and FTn were labelled with fluorescence dye DiO and Cy5, respectively. Then, DiO-labelled LNP was hydrated with Cy5-labelled FTn, followed by centrifugation at 5000 g for 15 min at 4 °C to remove the free FTn. FNP-antiHER2 was generated by the extrusion method, as described above. Then, the cells were incubated with FNP-antiHER2 and imaged in a time series using the living cells workstation (Leica SP8) at 37 °C with 5% CO2. The excitation/emission wavelengths of 484/501 nm were used for DiO-labelled FNP, and 633/670 nm for Cy5-labelled FTn.

Cellular uptake
Tumour cells were plated on glass-bottomed cell culture dishes (Nest Scientific) at a density of 1 × 10^4 and were cultured for 24 h prior to use. DiO-labelled FNP-antiHER2 containing Cy5-labelled FTn-Pd was added to the cells, and then co-incubated for the indicated time points (0, 1, 4, 8, 12, 24, 48, 72 h). Cells were fixed in PFA solution (4%, w/v) for 30 min, followed by staining the nucleus with DAPI and observing using confocal microscopy. FNP-antiHER2 containing Cy5-labelled FTn-Pd was added to the cells, and then co-incubated for the indicated time points. The cells were processed by immunostaining with a lysosome-specific antibody, LAMP-1 (H4A3, Santa Cruz Biotechnology, sc-20011). Briefly, cells were fixed in 4% PFA solution for 30 min, followed by washing with PBS containing Tween-20 (0.5%, v/v). Next, cells were permeabilized with PBS containing Triton X-100 (0.5%, v/v) for 15 min and then washed. Cells were blocked with goat serum (10%, v/v) for 1 h, followed by dilution of LAMP-1 at a volume ratio of 1:100 and adding to cell culture dishes for overnight incubation of cells at 4 °C. After washing the cells with PBS containing 0.5% Tween-20, fluorescent secondary antibody solution (Alexa Fluor® 488 (Thermo, A11001), diluted with 5% goat serum at 1:500 (v/v)) was added dropwise and incubated with cells at room temperature for 1 h, followed by washing again. The co-localisation of FTn and lysosome was observed using confocal microscopy, and the quantification analysis was performed using ImageJ software. The excitation/emission wavelengths of 405/460 nm were used for DAPI-labelled nucleus, 484/501 nm for DiO-labelled FNP, 484/501 nm for Alexa Fluor® 488-labelled LAMP-1 and 633/670 nm for Cy5-labelled FTn, respectively.

Cell viability assay
LNP, LNP-antiHER2, FNP and FNP-antiHER2 were firstly prepared using hydration, extrusion and purification following the method described above. Cytotoxicity was measured by a standard Cell Counting Kit-8 (CCK-8) assay following the manufacturer’s instructions (Beyotime). Briefly, the cells were seeded into 96-well plates (5 × 10^3 cells per well). After 24 h, LNP, LNP-antiHER2, FNP and FNP-antiHER2 were added to the cells. After an additional 36 h incubation, cells were washed with PBS buffer and then subjected to the CCK-8 assay. 96-well plates were measured as absorbances at 450 nm on a microplate reader (Multiskan FC, Thermo Fisher Scientific). Statistical analysis was then performed with GraphPad Prism v9.0 software.

Apoptosis analysis
We assessed apoptosis progression through Western blot and confocal microscopy following treatment with PBS, FTn-Pd, LNP-antiHER2, and FNP-antiHER2 for 48 h. As a positive control, 20 μM STS was used, while cells pretreated with Z-VAD-FMK (50 μM) served as a negative control. For Western blot, the samples were lysed and processed according to standard procedures, using primary antibodies against caspase-3 (Cell signalling technology (CST), 9662S), PARP (CST, 9542S), and actin (ABclonal, AC026). Uncropped and unprocessed scans of the blots are provided in the Source Data file. For microscopic morphological observation, tumour cells were seeded on Glass Bottom Dishes and incubated with PBS, STS, FTn-Pd, LNP-antiHER2, and FNP-antiHER2 for 36 h. After treatments, the cells were fixed with 4% paraformaldehyde and subjected to immunofluorescence staining. Cleaved-caspase3 was detected using a primary antibody (CST, 9664S) and an AF647-labelled goat anti-rabbit secondary antibody (Thermo, A32733). TRITC-labelled phalloidin (Maokangbio, MX4405) was used to stain the cytoskeleton.

Caspase-3 silencing
For silencing caspase-3 gene expression, we used chemically synthesised small interfering RNAs (siRNAs) with the following sense and antisense sequences: caspase-3 siRNA #156, 5′-GCUUCUUCAGAGGCGACUATT-3′ (sense) and 5′-UAGUCGCCUCUGAAGAAGCTT-3′ (antisense); caspase-3 siRNA #256, 5′-GGAUAGUGUUUCUAAGGAATT-3′ (sense) and 5′-UUCCUUAGAAACACUAUCTT-3′ (antisense); caspase-3 siRNA #3, 5′-CGCACAAGCUAGAAUUUAUTT′ (sense) and 5′-AUAAAUUCUAGCUUGUGCGTT-3′ (antisense) (GENCEFE Biotech, China). The Silencer Negative Control siRNA (GENCEFE Biotech, China) was used as a negative control. The siRNAs were transfected into tumour cells using Lipofectamine 2000 and Opti-MEM media (Invitrogen, USA) according to manufacturer recommendations. After 24 h transfection, the cells underwent various treatments for 48 h, followed by apoptosis analysis, including Western blot for PARP (CST, 9542S) and caspase-3 (CST, 9662S), and flow cytometry for Annexin V/PI double staining (Meilun Bio, MA0220-2). The gating strategy was presented in Supplementary Fig. 9. Uncropped and unprocessed scans of the blots are provided in the Source Data file.

RNA-sequencing analysis
To investigate the mechanisms underlying tumour cell apoptosis induced by FTn-Pd, we conducted RNA-sequencing analysis. Tumour cells were treated with FNP-antiHER2 for 24 h. RNA was extracted and sequenced using the Illumina platform. The raw RNA-seq reads were aligned to the mouse genome (GRCm38) by HISAT2 (v2.1.0). For analysis, the read counts for each gene were obtained and normalised as fragments per kilobase of exon model per million mapped fragments (FPKM) mapped fragments. Subsequently, differentially expressed genes (DEGs) between FNP-antiHER2- vs. PBS-treated groups were identified using a threshold of |log2 FC (fold change)| ≥ 1 and an adjusted false discovery rate (FDR) ≤ 0.05. Visualisations, including Volcano plot, Bubble Chart, and bar graph were generated using GraphPad Prism v9 (GraphPad).

Tumour model
All animal experiments were conducted in accordance with the policies and guidelines of the Animal Ethics Committee of Nankai University (2022-SYDWLL-000189). Mice had ad libitum access to food and water and were maintained in a specific pathogen-free (SPF) environment under a 14-h light/10-h dark cycle, at 18–25 °C with 40–60% humidity. Subcutaneous tumour models were successfully constructed by grafting various tumour cells into different mouse species. Mouse-derived tumour cells, namely MB49 cells and MB49-HER2+ cells, were allografted into C57BL/6 mice. Human-derived tumour cells, specifically SKBR3 cells, were xenografted into BALB/c-nude mice. The tumour growth and body weight of mice were monitored daily, and tumour volume was calculated using the formula: (tumour length) × (tumour width)2/2.

Plasma pharmacokinetics
To study the distribution of LNP, FNP-M and FNP-antiHER2 in blood, DiD-labelled LNP were firstly prepared as follows. DMPC, DOTAP and DSPE-PEG2000 were dissolved in chloroform respectively, followed by adding ethanol-dissolved DiD solution dropwise to a final concentration of 1 mM. DiD-labelled LNP, FNP-M and FNP-antiHER2 were then prepared using hydration, extrusion and purification following the method described above. Next, equivalent DiD-labelled LNP, FNP-M and FNP-antiHER2 were intravenously injected into mice via the tail vein at a dose of approximately 0.2 mg DiD per kg of body weight (n = 3/group). Blood samples were collected at specific time points (0.08, 0.17, 0.5, 1, 4, 8, 12, 24, 36, 48 h), and then centrifuged at 3000 g for 10 min to separate the plasma. The concentrations of LNP, FNP-M and FNP-antiHER2 in the plasma were determined by measuring the fluorescence intensity using a microplate reader with excitation/emission wavelengths of 644/665 nm.

Biodistribution studies
To study the distribution of LNP, FNP-M and FNP-antiHER2 in major organs and tumours, DiR-labelled LNP were firstly prepared as follows. DMPC, DOTAP and DSPE-PEG2000 were dissolved in chloroform respectively, followed by adding ethanol-dissolved DiR solution dropwise to a final concentration of 1 mM. DiR-labelled LNP, FNP-M and FNP-antiHER2 were then prepared using hydration, extrusion and purification following the method described above. Next, BALB/c nude mice bearing SKBR3 subcutaneous tumour (n = 5/group) were intravenously injected with equivalent DiR-labelled LNP, FNP-M and FNP-antiHER2 at a dose of approximately 0.2 mg DiR per kg of body weight. The mice were imaged using IVIS Spectrum imaging system (Xenogen) at designated time points (0.5, 1, 4, 8, 24, 36, 48 h) with excitation/emission wavelengths of 754/778 nm. After 48 h, the mice were euthanized, and the tumours and major organs were collected for imaging using IVIS Spectrum imaging system. The mean signal intensity was determined by measuring region of interest (ROI).

In vivo anti-tumour activity and toxicity evaluation
Three experiments were designed to evaluate the in vivo anti-tumour activity. To evaluate HER2-dependent effect of FNP-antiHER2 on different tumours, mouse-derived subcutaneous tumour model was established. Female C57BL/6 mice were inoculated with MB49 and MB49-HER2+ cells into the left and right flank at the same mouse (5 × 10^6 cells per side), respectively. After 8 days, the mice were randomly divided into two groups (n = 6 per group) and intravenously administered with PBS and FNP-antiHER2 at 10 mg per kg (body weight) FTn-equivalent doses every 2 days. For the mouse-derived subcutaneous tumour model, female C57BL/6 mice were inoculated with MB49-HER2+ cells (8 × 10^5 cells per mouse) into the right flank. After 16 days, the mice were randomly divided into six groups (n = 10 per group) and intravenously administered with PBS, FTn-Pd (FTn-equivalent dose of 10 mg/kg), LNP-M (equivalent lipid dose), LNP-antiHER2 (equivalent lipid dose), FNP (FTn-equivalent dose of 10 mg/kg) and FNP-antiHER2 (FTn-equivalent dose of 10 mg/kg) every 2 days. For the human-derived subcutaneous tumour model, female BALB/c-nude mice were inoculated with SKBR3 cells (3 × 10^6 cells per mouse) into the right flank. After 21 days, the mice were randomly divided into five groups (n = 10 per group). Subsequently, the mice were intravenously administered with various treatments every 2 days. The treatments included PBS, FTn-Pd, LNP-antiHER2, FNP and FNP-antiHER2 at 10 mg per kg (body weight) FTn-equivalent doses. For the lipid particles without FTn-Pd, the equivalent lipids used were the same as those in FNP-antiHER2. The tumour size and body weight were measured every day. In parallel, the survival of animals was monitored on a daily basis.

For toxicity evaluation, female C57BL/6 mice (6–8 weeks old) were randomly assigned to six groups (n = 3 per group) with identical treatment strategies. Mice were monitored daily for clinical signs of toxicity. After 15 days of treatment, mice were anaesthetised with isoflurane, and blood was collected via cardiac puncture for haematological and biochemical analyses, including complete blood count (CBC) and serum biochemical parameters (SBP). Subsequently, mice were euthanized by cervical dislocation, and major organs and selected tissues—including heart, liver, spleen, lung, kidney, mammary gland, stomach, small intestine and skin—were harvested and fixed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining.

Statistics and reproducibility
Statistical differences between two groups and among multiple groups were analyzed using two-sided Student’s t-test and one- or two-way ANOVA with Tukey’s multiple comparison test without adjustments, respectively. Analyses were performed using GraphPad Prism v9.0 software. Statistical parameters and sample sizes for each experiment are provided in the figure legends. Each experiment was independently repeated at least three times to ensure reproducibility of the results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In my first review, I pointed out that “an assessment of the real relevance of this new characteristic (as well as the system as a whole) for devising new therapies with respected to existing alternatives is beyond my expertise.” Thus, I focused my second review on the claims related to the nanozyme reactivity and the originality of its design. Specifically, I had expressed two key concerns related to the novelty related to the nanozyme activity of the FTn-Pd system. First, whether the reactivity was in fact due to the Pd-FTn complex, or Pd(II) cations released in water were responsible for the activity reported. Second, if any additional rationale was used in the design of FTn-Pd’s hydrolytic activity introduced. The revision presented by Hu and co-workers did addressed my previous concerns partially.\n\nRegarding the first concern, the authors provided consistent evidence that the Pd is bound to FTn during the hydrolytic reaction, supporting their claims and conclusion regarding the GrB-mimicking nanozymatic activity. I would just encourage the authors to include the “data for reviewer 1” in the paper (f. ex. Fig 2h).\n\nThe second concern was also clarified, but the arguments presented in the point-by-point response revealed that the authors did not apply any specific method to rationally design the catalytic sites responsible for the hydrolytic activity. In terms of catalytic activity of the FTn-Pd nanozyme, the innovation presented is a clever combination of previously known components in the literature. The assessment that the hydrolytic activity is unexpected and unique is, in my opinion, not convincing. Further, the current draft of the manuscript presents still only vague explanations on the choice of the ferritin-Pd combination.\n\nThe remainder of my concerns, which were in line with the ones from other reviewers, were in my opinion sufficiently addressed with revisions and the point-by-point response. Particularly, I enjoyed the authors arguments and new results related to the structure-dependent cleavage selectivity, which given the biochemical tools available nowadays, poses an interesting perspective for the nanozyme field. Likewise, the incorporation of a nanozyme in a delivery system and the demonstration that the reactivity once established at the molecular level can be engineered to be delivered in vivo is indeed an interesting demonstration of the potential of nanozymes. While the molecular innovation from the nanozyme reactivity perspective is not impressive, this demonstration may still have a positive impact in the field, pushing groups to explore more principles governing this design – for example, the SAR discussed may enlighten future nanozyme designs, based on FTn or other protein scaffolds. In this sense, a publication in Nature Nanotechnology can be envisioned, even though the intrinsic chemical reactivity of the nanozymatic system is not new.\n\nFinally, I would encourage the authors to include in their main text the two fragments of their point-by-point response reproduced below:\n1. Include in the manuscript the details about the design given in the passage: “Human heavy chain apo-ferritin (FTn) was chosen as the scaffold for several reasons. FTn has well-documented advantages as a protein scaffold, including ease of production, high thermal stability, and its ability to bind to the transferrin receptor 1. This makes FTn an ideal platform for developing nanozymes with targeted therapeutic applications. Additionally, while the ligation chemistry of Pd(II) ions is well understood, what distinguishes this work is the specific integration of Pd(II) ions into the FTn scaffold, creating a nanozyme with unique protease activity.”\n2. Highlight the distinctive features of the system described in: “Our GrB mimic is based on ferritin (FTn), which can be produced at a large scale and stored under non-harsh conditions. By using FTn as a structural framework, we synthesized FTn-Pd in a simple and rapid manner. FTn-Pd demonstrates superior stability across a range of pH and temperatures compared to natural GrB (new Supplementary Figure 2d and 2e). Additionally, instead of relying on perforin for cellular entry, fusogenic nanoparticles (LNPs) were used to fuse with the cell membrane and deliver FTn-Pd directly to the cytoplasm. This approach bypasses endosomal processing, enhancing delivery efficiency while maintaining the enzymatic activity of FTn-Pd.”", "reviewer_2": "The authors have addressed my concerns and I believe its publication in its present form would be a good contribution to the field of nanozymes. In comparison to previous rounds of review, it is clear the work matured into a balanced and interesting contribution. I agree with the authors that the expectation of mimicking natural systems in their entirety is unrealistic, neither I see it as an objective of the area. In this regard, I consider the publication of this work in Nature Communications in its current form appropriate, as it would be a meaningful contribution for the future rational design of nanozymes with potential biological applications.", "reviewer_4": "This work attempted to test a biomimetic design of GrB-mimicking nanozyme-containing nanovesicles. Although the abstract and introduction gave an exciting impression, a more careful digestion of this work has revealed several flaws at the conceptual, technical and data quality levels, as specified below:\n\n1. Even though their initial design was not aimed at generating a GrB-mimicking nanozyme, it is reasonable to expand the investigation based on their unexpected discovery that FTn-Pd exhibits caspase-cleaving activity when screening the Ac-IETD-pNA substrate. However, the ensuing data sets are insufficient to support FTN-PD as a GrB-mimicking nanozyme. Natural GrB is known to cleave pro caspase-3, -7, -8 and Bid. Yet with recombinant protein substrates, FTN-BD is shown to cleave only pro caspase-3, but has minimal or no cleavage activity towards caspase-7, caspase-8 and Bid, and why the lack of effect on other known substrates is not discussed. On the other hand, pro caspase-3 is also cleaved and activated by the initiator caspases caspase-8 and caspase-9. Figure 2d using recombinant proteins attempted to prove FTN-PD’s GrB-like activity by using a GrB inhibitor, Z-IETD-FMK, which is not specific to GrB and known to inhibit caspase-8. Even worse, Figure 5a used tumor cells to show that a pan-caspase inhibitor, Z-VAD-FMK, completely blocked FNP-antiHER2 induced cleavage of caspase-3 and PARP, strongly suggesting that what FNP possesses is caspase-like activity, not GrB-like activity. Unfortunately, in the point-by-point response, the pan-caspase inhibitor, Z-VAD-FMK, is wrongfully cited as a GrB inhibitor, raising concern about the expertise of the authors on granzyme and caspase biology, as well as whether the title of this work can accurately summarize the reported data.\n\n2. Figure 5c showed only partial knockdown of Caspase 3 by siRNA that is associated with partial inhibition of cell death in Figure 5d induced by FNP-antiHER2. While this result seems reasonable, it is hard to understand the complete loss of caspase-3 cleavage. Why could FNP-antiHER2 not induce cleavage of the remaining pro caspase-3 (>50%) that is not completely knocked out?\n\n3. While the authors provided an extremely lengthy point-by-point response to address most of the concerns raised during the initial review and emphasize the innovation and significance of this work, some key technical rigor and data quality remain unclear. For example, no replicate experiments are described especially for cell-based and in vivo mouse experiments. Even though the data in Figures 5, 6 and 6S look appealing, were the respective experiments performed only once without repeating? Furthermore, in Figure 6s, individual tumor growth curves (b) and mean tumor growth curves (c) look somewhat different: all individual tumor sizes reached 1000 mm3 by day 28 after inoculation, while the mean values were stopped by day 13 after treatment (day 8+13 = day 21 after inoculation). Why were the full time courses not shown for these 3 in vivo experiments? Did the difference in tumor growth have any significant impact on host survival?\n\n4. Figure S7 in vivo toxicity assessments were inadequate without systemic disease scoring to assess overall conditions. In a,b,c, body weights alone in tumor-bearing mice cannot fully reflect potential toxicity. Why are there only two groups in a? Histopathological examinations should not be limited to heart, liver, spleen, lung and kidney, but expand to other organs especially known to express higher levels of HER2 such as mammary gland, skin, and gastrointestinal tracts."}, "author_response": "Response to Reviewers\n\nReviewer #1 (Comments for the Author):\nIn my first review, I pointed out that “an assessment of the real relevance of this new characteristic (as well as the system as a whole) for devising new therapies with respected to existing alternatives is beyond my expertise.” Thus, I focused my second review on the claims related to the nanozyme reactivity and the originality of its design. Specifically, I had expressed two key concerns related to the novelty related to the nanozyme activity of the FTn-Pd system. First, whether the reactivity was in fact due to the Pd-FTn complex, or Pd(II) cations released in water were responsible for the activity reported. Second, if any additional rationale was used in the design of FTn-Pd’s hydrolytic activity introduced. The revision presented by Hu and co-workers did addressed my previous concerns partially.\nResponse: We sincerely thank the reviewer for carefully considering our revised manuscript and for focusing on the mechanistic and design aspects of the FTn-Pd system. We fully recognize the importance of clarifying these issues. Below, we respond to each of the reviewer’s concerns in detail.\nRegarding the first concern, the authors provided consistent evidence that the Pd is bound to FTn during the hydrolytic reaction, supporting their claims and conclusion regarding the GrB-mimicking nanozymatic activity. I would just encourage the authors to include the “data for reviewer 1” in the paper (f. ex. Fig 2h).\nResponse: We thank you for acknowledging that the new data (now included as Fig. 2h) supports the conclusion that Pd remains coordinated to the FTn scaffold during the hydrolytic reaction. We have now integrated this dataset into the main manuscript as suggested.\nThe second concern was also clarified, but the arguments presented in the point-by-point response revealed that the authors did not apply any specific method to rationally design the catalytic sites responsible for the hydrolytic activity. In terms of catalytic activity of the FTn-Pd nanozyme, the innovation presented is a clever combination of previously known components in the literature. The assessment that the hydrolytic activity is unexpected and unique is, in my opinion, not convincing. Further, the current draft of the manuscript presents still only vague explanations on the choice of the ferritin-Pd combination.\nResponse: We acknowledge that the hydrolytic activity arises from a combination of known components, rather than from a de novo design of active sites. While we did not follow a structure-based design strategy for catalysis, our focus was on establishing a modular and biocompatible nanozyme system with in vivo applicability. We have clarified this point in the revised Discussion and reframed our description of novelty to emphasize system-level integration rather than intrinsic catalytic innovation.\nThe remainder of my concerns, which were in line with the ones from other reviewers, were in my opinion sufficiently addressed with revisions and the point-by-point response. Particularly, I enjoyed the authors arguments and new results related to the structure-dependent cleavage selectivity, which given the biochemical tools available nowadays, poses an interesting perspective for the nanozyme field. Likewise, the incorporation of a nanozyme in a delivery system and the demonstration that the reactivity once established at the molecular level can be engineered to be delivered in vivo is indeed an interesting demonstration of the potential of nanozymes. While the molecular innovation from the nanozyme reactivity perspective is not impressive, this demonstration may still have a positive impact in the field, pushing groups to explore more principles governing this design – for example, the SAR discussed may enlighten future nanozyme designs, based on FTn or other protein scaffolds. In this sense, a publication in Nature Nanotechnology can be envisioned, even though the intrinsic chemical reactivity of the nanozymatic system is not new.\nResponse: We sincerely thank the reviewer for the thoughtful and constructive assessment. We are encouraged that the structure-dependent cleavage selectivity and the demonstration of in vivo delivery were recognized as valuable contributions. While we fully acknowledge that the intrinsic reactivity of Pd-based nanozymes is not unprecedented, as the reviewer noted, our study advances the field by introducing a structurally resolved, computation-guided framework for engineering protein-metal hybrid nanozymes in biomedical applications. By integrating protein crystallography, molecular modeling, and nanoscale design, we provide a strategy to systematically decode and harness the complexity of biomolecular-nanomaterial interactions—an enduring challenge in materials-based medicine. We believe this approach establishes a new paradigm for constructing bioactive nanomaterials with atomic-level precision, paving the way for rational nanozyme design and broadening their translational potential in targeted therapeutic delivery.\nFinally, I would encourage the authors to include in their main text the two fragments of their point-by-point response reproduced below:\n1. Include in the manuscript the details about the design given in the passage: “Human heavy chain apo-ferritin (FTn) was chosen as the scaffold for several reasons. FTn has well-documented advantages as a protein scaffold, including ease of production, high thermal stability, and its ability to bind to the transferrin receptor 1. This makes FTn an ideal platform for developing nanozymes with targeted therapeutic applications. Additionally, while the ligation chemistry of Pd(II) ions is well understood, what distinguishes this work is the specific integration of Pd(II) ions into the FTn scaffold, creating a nanozyme with unique protease activity.”\nResponse: We highly appreciate your suggestion. As you suggested, we have incorporated into the main text the rationale for choosing FTn as a scaffold (formerly in our response letter), including its scalability, stability, and relevance to therapeutic delivery. We have also added a concise summary of the delivery mechanism via LNPs and the performance advantages over native GrB (now in the revised Discussion).\n2. Highlight the distinctive features of the system described in: “Our GrB mimic is based on ferritin (FTn), which can be produced at a large scale and stored under non-harsh conditions. By using FTn as a structural framework, we synthesized FTn-Pd in a simple and rapid manner. FTn-Pd demonstrates superior stability across a range of pH and temperatures compared to natural GrB (new Supplementary Figure 2d and 2e). Additionally, instead of relying on perforin for cellular entry, fusogenic nanoparticles (LNPs) were used to fuse with the cell membrane and deliver FTn-Pd directly to the cytoplasm. This approach bypasses endosomal processing, enhancing delivery efficiency while maintaining the enzymatic activity of FTn-Pd.”\nResponse: We thank you again for your detailed feedback and balanced assessment. We have incorporated your suggestions into the revised manuscript.\n\nReviewer #4 (Comments for the Author):\nThis work attempted to test a biomimetic design of GrB-mimicking nanozyme-containing nanovesicles. Although the abstract and introduction gave an exciting impression, a more careful digestion of this work has revealed several flaws at the conceptual, technical and data quality levels, as specified below:\nResponse: We sincerely appreciate the reviewer’s constructive comments and careful evaluation of our work. In response, we have incorporated additional results and clarifications into the revised manuscript to address the concerns raised. We would be pleased to further refine or expand our claims should the reviewer have additional suggestions or specific points that warrant further attention.\n1. Even though their initial design was not aimed at generating a GrB-mimicking nanozyme, it is reasonable to expand the investigation based on their unexpected discovery that FTn-Pd exhibits caspase-cleaving activity when screening the Ac-IETD-pNA substrate. However, the ensuing data sets are insufficient to support FTN-PD as a GrB-mimicking nanozyme. Natural GrB is known to cleave pro caspase-3, -7, -8 and Bid. Yet with recombinant protein substrates, FTN-BD is shown to cleave only pro caspase-3, but has minimal or no cleavage activity towards caspase-7, caspase-8 and Bid, and why the lack of effect on other known substrates is not discussed. On the other hand, pro caspase-3 is also cleaved and activated by the initiator caspases caspase-8 and caspase-9. Figure 2d using recombinant proteins attempted to prove FTN-PD’s GrB-like activity by using a GrB inhibitor, Z-IETD-FMK, which is not specific to GrB and known to inhibit caspase-8. Even worse, Figure 5a used tumor cells to show that a pan-caspase inhibitor, Z-VAD-FMK, completely blocked FNP-antiHER2 induced cleavage of caspase-3 and PARP, strongly suggesting that what FNP possesses is caspase-like activity, not GrB-like activity. Unfortunately, in the point-by-point response, the pan-caspase inhibitor, Z-VAD-FMK, is wrongfully cited as a GrB inhibitor, raising concern about the expertise of the authors on granzyme and caspase biology, as well as whether the title of this work can accurately summarize the reported data.\nResponse: We sincerely thank the reviewer for the thoughtful and professional assessment. In our study, FTn-Pd is designated as a “GrB-mimicking nanozyme” based on three lines of evidence: (i) it specifically cleaves the same synthetic substrates as natural GrB; (ii) it directly cleaves pro-caspase-3, a well-established GrB substrate; and (iii) its cleavage activity toward pro-caspase-3 is substantially blocked by GrB-specific inhibitors (now included in the revised Fig. 2d). We fully agree with the reviewer that natural GrB cleaves multiple substrates, including pro-caspase-3, -7, -8, and Bid. In contrast, FTn-Pd displayed selective activity toward pro-caspase-3 but did not cleave caspase-7, caspase-8, or Bid. This restricted substrate specificity indicates that FTn-Pd does not replicate the complete enzymatic repertoire of natural GrB. In other words, while both FTn-Pd and native GrB catalyze pro-caspase-3 cleavage, their catalytic profiles are not entirely identical. Importantly, FTn-Pd induces tumor cell apoptosis primarily through pro-caspase-3 cleavage, representing a functionally tailored property with direct therapeutic relevance for tumor treatment. Accordingly, and as clarified in our previous revision, we revised the terminology from “artificial GrB” to “GrB-mimicking nanozyme,” which we believe more accurately describes its functional profile while acknowledging the differences from both natural and artificial enzymes. We also consider it reasonable that FTn-Pd cannot fully reproduce the enzymatic spectrum of GrB, given the distinct catalytic active sites involved (active-site analysis and discussion have been added to the revised manuscript). We sincerely welcome any further guidance from the reviewer on terminology that would most precisely reflect the functional characteristics of this system.\nAs the Reviewer #4 mentioned, we would also like to highlight four unique aspects of this work in the context of nanozyme development: i) Nanozyme-specific properties: Unlike conventional artificial enzymes, FTn-Pd nanozymes exhibit superior stability across a wide range of pH and temperatures, can be readily produced at scale using recombinant FTn, and allow facile multifunctionalization through genetic engineering. This offers practical advantages compared to natural GrB. ii) Mechanistic insight: By integrating protein crystallography, molecular modeling, and nanoscale design, we established—for the first time—a structurally resolved framework to understand the catalytic mechanism of protein-metal hybrid nanozymes. Specifically, we identified a binuclear Pd catalytic center coordinated by cysteine, methionine, and histidine residues, along with water molecules. This structural insight provides a paradigm for decoding biomolecular-nanomaterial interactions at atomic resolution, going beyond the material-focused approaches typical of previous nanozyme studies. iii) Catalytic active site composition: Distinct from the active sites of native or artificial GrB (new supplementary Figure 3h), the catalytic center of FTn-Pd comprises two Pd atoms coordinated by residues M37, C90, C102, and H105 of the FTn scaffold. We found that both the Pd atoms and the protein scaffold play essential roles in catalyzing GrB substrates, with the protein framework facilitating substrate accessibility and the Pd atoms providing the hydrolytic activity. This architecture closely resembles metalloprotease active sites. Accordingly, while FTn-Pd mimics GrB by selectively cleaving caspase-3 to induce apoptosis, it is reasonable that its activity does not extend to the full substrate spectrum of natural GrB (i.e., pro-caspase-3, -7, -8, and Bid). iv) Previous studies have shown that Pd(II) complexes, such as Pd(en)(H O ), possess hydrolase activity; however, their specific incorporation into the FTn framework in this study confers a unique protease-like activity not observed in earlier systems. Notably, compared with the catalytic activity of FTn-Pd toward GrB substrates, Pd(en)(H O ) displays negligible activity. Together with structural analyses and the proposed catalytic mechanism, these findings demonstrate that both the Pd centers and the protein scaffold are indispensable for enzymatic function.\nRegarding inhibitor specificity, we agree with the reviewer that Z-IETD-FMK is not GrB-specific and can inhibit caspase-8. To strengthen our claim, we have now validated FTn-Pd’s activity using two GrB-specific inhibitors—human Serpin B9 recombinant protein1 and Z-AAD-CMK2—both of which significantly reduced FTn-Pd-mediated cleavage of recombinant caspase-3 (new Figure 2d). This provides more direct evidence of its GrB-mimicking activity. Importantly, we emphasize that the in vitro assays with recombinant proteins are designed to probe GrB-mimicking activity, whereas the cellular assays demonstrate intracellular delivery and caspase-3 cleavage, without attributing specificity to GrB, given the complex interplay with initiator caspases such as caspase-8 and caspase-9.\nWe sincerely apologize for the misstatement in our original response letter, where Z-VAD-FMK was inadvertently described as a GrB inhibitor. As correctly stated in the manuscript, Z-VAD-FMK is a pan-caspase inhibitor; this error in the response text was unintentional and we regret the confusion it caused.\nFinally, we would like to stress that the central goal of this study was to emulate the functional property of GrB delivery into tumor cells for inducing apoptosis, not to fully replicate its natural substrate spectrum. FTn-Pd reproduces the critical feature of GrB—entry into tumor cells followed by direct cleavage of caspase-3, thereby triggering a caspase-3-dependent apoptotic cascade. This functionally distinguishes our system from endogenous initiator caspase activation pathways, while supporting the designation of FTn-Pd as a “GrB-mimicking nanozyme”. We believe this work provides a meaningful step toward expanding the design principles of bioactive nanozymes for therapeutic applications.\n2. Figure 5c showed only partial knockdown of Caspase 3 by siRNA that is associated with partial inhibition of cell death in Figure 5d induced by FNP-antiHER2. While this result seems reasonable, it is hard to understand the complete loss of caspase-3 cleavage. Why could FNP-antiHER2 not induce cleavage of the remaining pro caspase-3 (>50%) that is not completely knocked out?\nResponse: We thank the reviewer for raising this point. In Figure 5c, the Western blot (WB) image was captured with a long exposure to clearly visualize cleaved caspase-3, which unfortunately led to overexposure of pro-caspase-3 and gave the impression of incomplete knockdown. As shown in the additional short-exposure WB data provided (data for Reviewer), the knockdown efficiency of caspase-3 is in fact high. Furthermore, in the control siRNA-treated group, a small amount of pro-caspase-3 cleavage was already present (indicated by the arrow), making residual pro-caspase-3 after siRNA treatment effectively undetectable. We are happy to provide further clarification or additional experiments if the reviewer considers it necessary.\nData for reviewer. Western blot analysis of pro-caspase-3 (with different exposure time) in three distinct cas3 siRNA-treated cells following FNP-antiHER2 treatment, comparing short- and long-exposure times to demonstrate knockdown efficiency and cleaved caspase-3 detection.\n3. While the authors provided an extremely lengthy point-by-point response to address most of the concerns raised during the initial review and emphasize the innovation and significance of this work, some key technical rigor and data quality remain unclear. For example, no replicate experiments are described especially for cell-based and in vivo mouse experiments. Even though the data in Figures 5, 6 and 6S look appealing, were the respective experiments performed only once without repeating? Furthermore, in Figure 6s, individual tumor growth curves (b) and mean tumor growth curves (c) look somewhat different: all individual tumor sizes reached 1000 mm3 by day 28 after inoculation, while the mean values were stopped by day 13 after treatment (day 8+13 = day 21 after inoculation). Why were the full time courses not shown for these 3 in vivo experiments? Did the difference in tumor growth have any significant impact on host survival?\nResponse: We thank the reviewer for raising these critical points regarding technical rigor and data presentation. All key cell-based assays were performed in at least three independent biological replicates within each experimental run. Statistical analyses of all quantitative data are based on these independent biological replicates. We acknowledge that the description of replicate strategies was insufficiently detailed in the original manuscript; this has now been explicitly clarified in the revised Figure legends and Methods section.\nFor the in vivo mouse studies, the total therapeutic experiments can be considered as three independent batches, with group sizes exceeding n ≥ 6 mice per cohort, to ensure statistical robustness. We employed both nude mice and C57BL/6 mice to establish SKBR3 and MB49-HER2⁺ tumor models, respectively, and evaluated the therapeutic efficacy of the FNP-antiHER2 nanovesicles in both models. In each case, FNP-antiHER2 nanovesicles consistently demonstrated the most pronounced therapeutic benefit (Figure 6). Furthermore, we directly compared their efficacy in MB49-HER2⁺ versus parental MB49 tumors, and the results confirmed superior therapeutic performance in the HER2⁺ model (Supplementary Figure 6).\nIn Supplementary Figure 6, we appreciate the reviewer's careful observation. In Figure S6c (mean tumor growth), data collection stopped at Day 13 post-treatment (i.e., Day 21 post-inoculation) because >50% of animals in the control group had reached the humane endpoint (tumor volume ≥1000 mm³). Continuing to calculate mean tumor volumes beyond this point would have introduced survivorship bias, as only animals with smaller tumors would have remained. In contrast, Figure S6b (individual tumor growth curves) shows the complete course until each tumor reached its endpoint. In the revised manuscript, we have adjusted the presentation so that the x-axis ranges of Figures S6b and S6c are consistent, thereby minimizing potential confusion.\n4. Figure S7 in vivo toxicity assessments were inadequate without systemic disease scoring to assess overall conditions. In a,b,c, body weights alone in tumor-bearing mice cannot fully reflect potential toxicity. Why are there only two groups in a? Histopathological examinations should not be limited to heart, liver, spleen, lung and kidney, but expand to other organs especially known to express higher levels of HER2 such as mammary gland, skin, and gastrointestinal tracts.\nResponse: We sincerely thank the reviewer for these constructive comments on our in vivo toxicity assessment.\nWe agree that tumor burden may confound interpretation of body weight changes. To address this, we repeated the treatment protocol in healthy mice, and the results confirmed that nanomaterial administration had minimal impact on body weight in the absence of tumors (new Supplementary Figure S7c). In addition, to provide a more comprehensive safety evaluation, we expanded our analysis to include complete blood count and serum biochemical parameters (AST, ALT, UREA, CREA), now presented in new Supplementary Figures S7e–f. These data further support the biocompatibility of the nanoplatform.\nWe also agree with the reviewer that HER2-enriched tissues warrant careful examination for potential off-target effects. In response, we have extended histopathological evaluation beyond the initially examined organs (heart, liver, spleen, lung, and kidney) to include stomach, small intestine, skin, and mammary gland. Representative H&E staining images for all examined organs are now provided in new Supplementary Figure S8, and no overt pathological abnormalities were observed.\nRegarding Figure S7a, these data were originally collected as part of the therapeutic study shown in Figure S6, where body weight monitoring was used as an indirect indicator of potential toxicity. We included them under Figure S7a to complement the safety assessment. However, we acknowledge that this placement may have caused confusion. In the revised manuscript, we have relocated these data to Supplementary Figure S6d to maintain consistency in figure organization.\nWe believe these additional experiments and clarifications substantially strengthen the rigor of our toxicity assessment and directly address the reviewer’s concerns.\n\nReviewer #2 (Comments for the Author):\nThe authors have addressed my concerns and I believe its publication in its present form would be a good contribution to the field of nanozymes. In comparison to previous rounds of review, it is clear the work matured into a balanced and interesting contribution. I agree with the authors that the expectation of mimicking natural systems in their entirety is unrealistic, neither I see it as an objective of the area. In this regard, I consider the publication of this work in Nature Communications in its current form appropriate, as it would be a meaningful contribution for the future rational design of nanozymes with potential biological applications.\nResponse: We sincerely thank the reviewer for this thoughtful and encouraging assessment. We appreciate the recognition of the maturation of the work and agree that fully mimicking natural systems is not the primary objective of the field. We are grateful for the reviewer’s support and positive evaluation of the manuscript’s contribution to the rational design of biologically relevant nanozymes."}]

Decision letter