Surface hydrophobicity and rigidity determines protein corona on orally delivered nanoparticles treating colitis

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

Abstract
Disease-specific protein corona adsorbed on nanocarriers determines in vivo delivery efficiency. Macrophages can orchestrate inflammation resolution and mucosa repair, making them a promising therapeutic target in colitis. Here, we show that surface hydrophobicity and rigidity determine colitis-specific intestinal protein corona (C-IPC) on orally delivered nanoparticles for treating colitis, which improves therapeutic efficiency. We show that high surface hydrophobicity boosts overall protein adsorption, leading to improved colon macrophage delivery and therapeutic effect when loaded with budesonide. Moreover, hydrophobicity with high rigidity results in a corona enriched with macrophage-targeting proteins, notably S100A8, generating an optimal C-IPC characterized by both high protein amount and high proportion of targeting proteins. Consequently, high rigidity nanoparticles more effectively attenuates the inflammatory state and restores the immune homeostasis in male rats with colitis. Our work develops a rational strategy of manipulating protein corona formation through physicochemical properties for efficient oral drug delivery, holding broad promise for diverse nanocarriers and pathologies.

Introduction
Protein corona, a dynamic layer of biomolecules inevitably adsorbed onto nanomedicines in biological environments, critically governs the drug delivery and therapeutic effect in vivo1–3. Emerging evidence highlights that protein corona formed under pathological conditions contain varied functional proteins, display distinct bioactivities to determine the transport and targeting behaviors of nanocarriers4–6. For example, neurodegenerative disease-associated protein corona facilitated blood-brain barrier penetration of nanoparticles by adsorbing the apolipoproteins7,8. The tumor microenvironment-derived protein corona altered nanoparticles biodistribution to enhance their tumor accumulation and targeting9,10. Our pre-study also revealed that the colitis-specific intestinal protein corona (C-IPC) possessed varied functional proteins11, which could moderately enhance the macrophage targeting and internalization of orally administrated nanocarriers. Hence, these disease-specific protein corona possessing unique tissue- or cell-targeting related proteins show great potential in improving the delivery efficiency of nanocarriers. Furthermore, seeking suitable strategy to selectively facilitate the functional protein adsorption may further enhance the targeted drug delivery.

Recent studies have shown that altering the physicochemical properties is promising for governing the protein corona formation on nanocarriers12–15. The surface hydrophobicity and charge of nanocarriers might influence the protein adsorption amount. For instance, hydrophilic polyethyleneglycol (PEG) modified nanoparticles exhibited lower protein adsorption due to weakened hydrophobic interaction16,17. In addition, cationic nanoparticles could promote negatively charged serum protein adsorption through electrostatic interaction18. In another aspect beyond overall corona absorption, rigidity and surface chemistry of nanocarriers affect their interaction with specific proteins, resulting in varied protein corona composition13. High rigidity nanoparticles adsorbed more apolipoproteins but fewer complement proteins, thus prolonging the blood circulation19. Besides, through adjusting the amino/hydroxyl ratios of nanovesicles, immunoglobulin adsorption declined and tumor distinctive proteins increased which prolonged circulation and tumor cell internalization10. Thus far, most studies have focused on singular physicochemical property modulation of nanocarriers10,19–21, which cannot sufficiently promote the functional protein adsorption to obtain the desired protein corona. Therefore, developing multiple-properties modulation strategy to synchronously increase protein adsorption amount and selectively upregulate functional protein proportion may effectively enhance the delivery efficiency of nanocarriers.

Macrophages are key effector cells of the innate immune system for intestinal mucosal homeostasis, yet dysregulated macrophages are a central feature of inflammatory bowel disease (IBD), including colitis22. They accumulate in vast numbers in active colitis, being potent producers of pro-inflammatory mediators, such as reactive oxygen (ROS), TNF-α, IL-1β, IL-6. Concurrently, intestinal macrophages prevent excessive inflammation by facilitating the expansion of regulatory T (Treg) cells, mainly via the production of IL-1023. Given that macrophages can orchestrate inflammation resolution and mucosa repair, they are regarded as attractive therapeutic targets in colitis24. Several macrophage targeting drug delivery systems have been developed to improve the colitis therapy via suppressing inflammation and restoring intestinal homeostasis25–28. Therefore, targeted delivery of therapeutics to colon macrophages represents a promising strategy for colitis treatment.

In this study, a sequential hydrophobicity-rigidity co-modulation strategy was performed to optimize the C-IPC formation on orally administrated nanoparticles (NPs) for efficient oral macrophage targeted delivery and colitis treatment (Fig. 1). Increasing surface hydrophobicity of NPs tended to enhance the protein adsorption amount during C-IPC formation, thus high hydrophobic NPs (Phobic) loaded with anti-inflammatory drug budesonide showed improved colon macrophage targeting and better colitis treatment. Next, the rigidity of NPs was modulated by varying the amount of interior water layer. Increasing the rigidity of Phobic was found to further promote the macrophage targeted drug delivery, and high rigidity NPs (Hard) could effectively attenuate the inflammation and restore the immune homeostasis in colitis rats. Interestingly, possessing high rigidity selectively adsorbed more macrophage-targeting related proteins on NPs including S100A8 protein, contributing to the superior macrophage targeting and internalization. Our work proposed and verified the concept of rationally tailoring the disease-specific protein corona on nanocarriers through multiple-physicochemical properties co-modulation for efficient targeted drug delivery. More importantly, this strategy may be applicable for various types of nanocarriers and diseases.

Results
Preparation and characterization of NPs with varied hydrophobicity
In this study, the soyabean lecithin and cholesterol were lipid component to form the lipid-based NPs. To modulate the surface hydrophobicity, different ratios of DSPE-PEG2000 were added to prepare NPs with varied polyethyleneglycol (PEG) modification degree29,30. As the PEGylation increased gradually from 10%, 40%, to 80% (mass ratio of DSPE-PEG2000 : soybean lecithin) (Fig. 2a), we denoted the NPs as Phobic, Medium and Philic, respectively. All NPs were similar in hydrodynamic diameter ~165 nm measured by dynamic light scattering (DLS) (Fig. 2b, and Supplementary Table 1). The cryo-TEM images (Fig. 2b) showed that NPs possessed the morphologies of spherical shape and particle size ~115 nm, which is lower than the hydrodynamic diameter. For characterization of the hydrophobicity, the hydrophobic domains in the NPs were detected with Coomassie Brilliant Blue (CBB) G-250 dye that participated in strong hydrophobic interactions31,32. We found that the wavelength of the maximum absorption of CBB, a parameter dependent on the hydrophobicity of nanoparticles, gradually increased from Philic, Medium to Phobic (Fig. 2c). In addition, the Rose-Bengal (RB) binding constant33,34 of three NPs gradually increased from 0.005, 0.026 to 0.033 mL/μg (Fig. 2d). Further, to directly measure the hydrophobicity, the contact angles of NPs were detected35. As shown in Fig. 2e, the contact angles increased progressively from average 37.1° for Philic to 45.6° for Medium and 60.5° for Phobic. Taken together, these results suggested that NPs with increased hydrophobicity were successfully prepared (Fig. 2f). All NPs could remain stable in the simulated gastrointestinal (GI) condition without significant change of particle size and PDI (Supplementary Fig. 1). Besides, the Förster resonance energy transfer (FRET) assay using DSPE-PEG2000-FITC and DiI as fluorescent molecule pairs36,37, further verified the structural integrity and stable hydrophilic/hydrophobic modification of NPs after gastric processing in vivo (Supplementary Fig. 2).

Drug-loaded NPs with varied hydrophobicity for colitis therapy
The potency of NPs with increased hydrophobicity as carriers for colitis treatment was investigated. Anti-inflammatory drug budesonide (Bud) was used as the therapeutic cargo38, and the drug-loaded NPs (NPs-Bud) with increased hydrophobicity were prepared as Philic-Bud, Medium-Bud and Phobic-Bud. The drug loading process showed no influence on the particle size, PDI and zeta potential of nanoparticles, and three NPs-Bud all owned a high encapsulation efficiency to ~80% (Supplementary Table 2). The Phobic-Bud had the highest drug loading (2.80 μg/mg), mainly because the Philic-Bud and Medium-Bud contained higher amount of DSPE-PEG2000 therefore increasing the total weight of formulation. Besides, all formulations exhibited similar sustained drug release behavior in the simulated GI condition with ~40% of drug released from NPs-Bud within 10 h (Supplementary Fig. 3).

The treatment regimen was shown in Fig. 3a. The colitis rats were orally administrated with saline, free Bud, Philic-Bud, Medium-Bud and Phobic-Bud, and their body weights were recorded daily. Compared with Saline group, recovery of colitis-induced body weight loss occurred after Phobic-Bud treatment, better than that of Philic-Bud and Medium-Bud groups, while no recovery was observed for Free Bud and Saline groups (Fig. 3b). In Day 8, Phobic-Bud treatment relived the shortening of colon (Fig. 3c, d), reduced the spleen index (Fig. 3e) as well as restored the damaged colon mucosa (Supplementary Fig. 4), which were better than those of Philic-Bud and Medium-Bud groups. Enhanced reactive oxygen species (ROS) generated from infiltrated macrophages leads to pathological inflammatory in colitis39,40. By contrast, Phobic-Bud treatment could decrease the dysregulated ROS level in colon of colitis rats and showed lower ROS level than Philic-Bud and Medium-Bud groups (Fig. 3f). Colitis progression would induce the up-regulation of inflammatory cytokines in colon38–41. After treatment, Phobic-Bud group significantly decreased the abnormal up-regulation of colon TNF-α, IL-1β and IL-6 level in colitis rats, exerting better effect than Philic-Bud and Medium-Bud groups (Fig. 3g–i, Supplementary Fig. 5a–c). Notably, oral administration of free Bud almost showed no therapeutic effect for relieving the colitis progression. Therefore, the Phobic-Bud showed better colitis therapy in alleviating colonic impairment and down-regulating the inflammatory cytokines.

The colitis could induce the disordered immune cells in body including the abnormal regulatory T (Treg) cells and macrophages42,43. For rats suffered with colitis, the Treg cells (CD3+ CD4+ FOXP3+) in colon and spleen both declined significantly compared with healthy rats (Fig. 3j, and Supplementary Fig. 5d). After oral treatment, Phobic-Bud group could upregulate the proportion of Treg cells to about 10.0% in colon and 18.4% in spleen, which were both higher than that of Philic-Bud and Medium-Bud group. IL-10 is an important anti-inflammatory cytokine secreted by various immune effector cells including intestine-resident macrophages to promote expansion and mediate the immunosuppressive function of Treg cells44. Notably, Phobic-Bud group could obviously upregulate the IL-10 level as well compared with Saline group (Supplementary Fig. 5e), which was consistent with the increase of Treg cells. Moreover, Phobic-Bud treatment was able to influence the macrophage polarization and markedly decreased the ratio of M1 type (inflammatory) macrophages (F4/80+ CD11b+ CD86+) to M2 type (anti-inflammatory) macrophages (F4/80+ CD11b+ CD206+) in colon and spleen at the same time (Fig. 3k, and Supplementary Fig. 5f). The free Bud also exhibited very limited effect in regulating the disordered immune cells, demonstrating that the nano-formulations offered enhanced therapeutic benefit for budesonide. Overall, these results demonstrated oral treatment of Phobic-Bud could restore the dysregulated immune cells in colitis. Meanwhile, the result also implied the increased surface hydrophobicity of nanocarriers played an important role on oral drug delivery to colon.

Impact of hydrophobicity on colon macrophage targeting and protein adsorption
Macrophages played a crucial role in the progression of colitis, and targeted delivering therapeutic cargos to colon macrophages was beneficial to suppress inflammation and restore intestinal homeostasis23,27,45. Therefore, the colon macrophage targeting of NPs with increased hydrophobicity in colitis or healthy rats were investigated (Fig. 4a). When oral administration to colitis rat, the overlap of NPs (green) with colon macrophages (red) gradually enhanced with the increased hydrophobicity, and the high hydrophobic NPs (Colitis-Phobic) showed the best distribution in macrophages (Fig. 4b). In comparison, the NPs all barely coincided with the macrophages regardless of their hydrophobicity in the healthy rats (Fig. 4c). Besides, increasing hydrophobicity could promote the macrophage uptake of NPs in colitis rats, and the Colitis-Phobic group showed superior in vivo macrophage uptake among all (Fig. 4e, f). However, this phenomenon was not observed in healthy rats, and the internalized NPs with increased hydrophobicity by macrophages (Healthy-Philic, Healthy-Medium, Healthy-Phobic) were all limited. Interestingly, these results indicated that increasing hydrophobicity could promote the macrophage targeting of NPs only for the colitis rats. We deemed that hydrophobicity modulation altered the colitis-intestinal protein corona (C-IPC) formation, leading to this phenomenon instead of the hydrophobicity change itself.

To explore whether the hydrophobicity of NPs could influence the NPs-protein corona bio-interface, the in situ intestine study was conducted (Fig. 4g). Fluorescence-labeled NPs were injected into the intestinal lumen of colitis rat to form the C-IPC adsorbed NPs (NPs@C-IPC) in vivo, then the intestinal biofluids containing NPs@C-IPC were withdrawn from the lumen and the NPs@C-IPC were collected using Sephadex G50 gel size-exclusion column (No pre-removal group), and three different NPs@C-IPC were abbreviated as Philic@C-IPC, Medium@C-IPC and Phobic@C-IPC. By contrast, the intestinal segments were rinsed with acetylcysteine (NAC) solution several times to pre-remove the intestinal biofluids before tied at both sides, setting as control (Intestinal biofluids pre-removal group). When pre-removal of intestinal biofluids, the protein amount declined sharply compared with No pre-removal group (Fig. 4h), indicating the formation of in vivo C-IPC on NPs. Moreover, the protein adsorption enhanced obviously with the increased hydrophobicity of NPs, and the protein content of C-IPC on Phobic was 4.0-fold and 1.9-fold higher than that of Philic and Medium (Fig. 4i). Hence, increasing hydrophobicity could enhance the protein content of C-IPC which might amplify the macrophage targeting effect of C-IPC.

Impact of C-IPC with enhanced protein amount on macrophage recognition and uptake
To obtain the NPs@C-IPC formed in vitro, the fluorescence-labeled NPs were incubated with the intestinal biofluids extracted from colitis rats (colitis-intestinal biofluids)11. NPs@C-IPC were then separated from free proteins in the colitis-intestinal biofluids, and collected using Sephadex G50 gel size-exclusion column (Fig. 5a). Slight changes in particle size (Fig. 5b) and surface charge (Fig. 5c) were observed in NPs@C-IPC after incubation with intestinal biofluids, indicating the formation of intestinal protein corona. This was further confirmed by the TEM imaging showing that the NPs@C-IPC basically remained the spherical structure though covered with proteins (Fig. 5d). Quantification of the adsorbed C-IPC in vitro showed that the protein amount of Phobic@C-IPC was ~2.8-fold and 1.5-fold of Philic@C-IPC and Medium@C-IPC (Fig. 5e), which was consistent with the findings for C-IPC formed in vivo (Fig. 4i). Besides, NPs with (+) or without (-) fluorescent labeling owned similar contact angle and protein adsorption (Supplementary Fig. 6), indicating the fluorescent labeling will not interfere with the hydrophobicity modulation and C-IPC formation. Collectively, the these results demonstrated NPs with increased hydrophobicity tended to adsorb more C-IPC on the surface.

Next, the influence of C-IPC on the macrophage uptake was investigated. The RAW264.7 cells were used as in vitro macrophage model, and all NPs or NPs@C-IPC showed no cytotoxicity (Supplementary Fig. 7). Notably, after C-IPC adsorption, the macrophage uptake of NPs@C-IPC significantly increased as compared with NPs (Fig. 5f). Among them, the Phobic@C-IPC owned the best macrophage uptake that was 1.8-fold and 5.5-fold higher than Medium@C-IPC and Philic@C-IPC. To verify whether the enhanced effect on macrophage uptake was specific for C-IPC, the healthy-intestinal protein corona (H-IPC) adsorbed NPs (NPs@H-IPC) were also prepared. Though increased hydrophobicity could also enhance the protein content of H-IPC (Supplementary Fig. 8), the improvement in macrophage uptake of NPs@H-IPC were not as profound as NPs@C-IPC (Supplementary Fig. 9). Besides, the epithelial cells (Caco-2 cells) and macrophages (RAW264.7 cells) co-cultured model was established to simulate the colon mucosa in vitro, for investigating the specific enhanced effect of C-IPC on macrophages (Fig. 5g). Caco-2 cells and RAW264.7 cells were co-cultured with different ratios and labeled by fluorescent dye DiI and DiD separately, then the internalized nanoparticles by different cells were detected by flow cytometer (Fig. 5h). In comparison with NPs without protein corona absorption, the relative cell uptake of RAW264.7 cells by NPs@C-IPC were all improved even though the ratio of epithelial cells to macrophages increased to 10:1, and the Phobic@C-IPC could always remain the highest internalization by macrophages (Fig. 5i–k). These results showed that NPs with higher hydrophobicity were preferentially taken up by macrophages, due to its ability to readily form macrophage interacting C-IPC on the surface.

Taken together, increasing hydrophobicity of NPs enhanced protein amount of C-IPC on the surface, endowing the drug-loaded NPs with better delivery efficiency to colon macrophages. However, it should be noted that although the colitis therapy was improved by Phobic-Bud, it still could not be restored to the healthy level in terms of the abnormal inflammatory cytokine level and disordered immune cells (Fig. 3).

Preparation and characterization of high hydrophobic NPs with varied rigidity
The PEGylation degree of NPs was 10% (Phobic) to maintain a high protein adsorption amount, then the rigidity was tuned by varying the amount of interfacial water layer between the polystyrene nanoparticles (PS) core and the lipid shell46,47. As illustrated in Fig. 6a, the PS with hydrodynamic diameter of 90.3 nm (PS-90) or 153.4 nm (PS-150) (Supplementary Table 3) were encapsulated into the NPs by extruding through a mini-extruder device. We named the high hydrophobic NPs encapsulated without PS or with PS-90 and PS-150 as Soft, Semi and Hard, respectively.

The hydrodynamic diameters of Soft, Semi and Hard were uniformly distributed, approximately ranging from 150~160 nm, with similar zeta potential (Fig. 6b, and Supplementary Table 3). The cryo-TEM images also demonstrated the NPs all owned spherical shape with size about 115 nm, and the empty space inside NPs full of water layer was obviously observed in Soft (Fig. 6b). With the encapsulation of PS-90 and PS-150 (Semi and Hard), the interfacial water layer gradually decreased until completely occupied (Fig. 6b). For characterization of the rigidity, internal water layer of NPs was labeled with rhodamine B46. In comparison, the fluorescence intensity of the water layer gradually reduced from Soft, Semi to Hard (Fig. 6c), indicating the gradual decrease of the amount of internal water as well. Subsequently, Young’s modulus was measured using the atomic force microscope (AFM), which was the crucial parameter for rigidity characterization of nanoparticles46–48. The result showed that the Young’s modulus of Soft, Semi and Hard increased from 10.1, 20.9 – 64.5 Mpa (Fig. 6d). Therefore, these results suggested the successful preparation of NPs owned similar physicochemical properties but with increased rigidity (Fig. 6e). They also had good stability in the simulated gastrointestinal conditions (Supplementary Fig. 10).

Hydrophobicity-rigidity co-modulation for effective colitis therapy
The Phobic-Bud with increased rigidity (Soft-Bud, Semi-Bud and Hard-Bud) was prepared. Three formulations also possessed similar encapsulation efficiency, drug loading (Supplementary Table 4) and sustained drug release behavior (Supplementary Fig. 11). Following the procedure illustrated in Fig. 3a, the drug-loaded high hydrophobic NPs with increased rigidity (Soft-Bud, Semi-Bud and Hard-Bud) were oral administered to colitis rats for therapeutic treatment. Compared with Saline group, treatments with three drug-loaded NPs could relieve the body weight loss and shortening of colon, but better efficacy was achieved as the rigidity of NPs increased (Fig. 7a–d). The Hard-Bud group could remarkedly decrease the colon ROS level (Fig. 7e), and recovered the damaged colon mucosa (Supplementary Fig. 12), exerting better efficacy than Soft-Bud and Semi-Bud. For inhibiting the inflammation in colon, Hard-Bud groups showed similar TNF-α level as Semi-Bud group (Fig. 7f), but could maximally down-regulate the IL-1β and IL-6 levels among three drug-loaded NPs (Fig. 7g, h). More importantly, the Hard-Bud treatment could normalize all three inflammatory cytokines to the healthy level (Fig. 7f–h, and Supplementary Fig. 13a–c).

The improvement of immune cells in colitis rats was next investigated. Consistent with the anti-inflammation ability, the Hard-Bud treatment could also maximally upregulate the Treg cells in both colon and spleen to the healthy level (Fig. 7i, and Supplementary Fig. 13d), and increased the expression of anti-inflammatory cytokine IL-10 (Supplementary Fig. 13e). Moreover, the ratios of M1/M2 macrophages in colon and spleen largely decreased after oral treatment, and Hard-Bud group still exhibited the best efficacy among all, which could restore the dysregulated macrophages to healthy level (Fig. 7j, and Supplementary Fig. 13f).

Taken together, Hard-Bud had good therapeutic effect in vivo, significantly recovering the impaired colon mucosa, inhibiting the expression of inflammatory cytokines and restoring the abnormal immune cells induced by colitis. It indicated that, on the basis of maintaining high hydrophobicity, increasing the rigidity of NPs could further improve the colitis therapy.

Impact of hydrophobicity-rigidity co-modulation on colon macrophage targeting and C-IPC formation
A similar in vivo macrophage targeting study of high hydrophobic NPs with increased rigidity was conducted in colitis models (Fig. 4a). Upon oral administration of the fluorescence labeled NPs to colitis rats, much higher distribution of NPs (green) within colon macrophages (red) was observed compared to healthy rats, with the Colitis-Hard group displaying a greater proportion of NPs uptake by macrophages than the Colitis-Soft and Colitis-Semi groups (Fig. 8a). Quantitative analysis further revealed that the Colitis-Hard group had the highest proportion and quantity of NPs taken up by colon macrophages (Fig. 8b–d), demonstrating that the colon macrophage targeting of NPs could be promoted by increasing the rigidity. Further, the potential uptake of NPs@C-IPC by epithelium and other immune cells in colon was evaluated. It showed that the fluorescence intensity of internalized Hard@C-IPC by macrophages is significantly higher than that of epithelial cells and also obviously better than that of T cells, DC cells and neutrophils (Supplementary Fig. 14), indicating the C-IPC adsorption specifically promoted NPs target towards macrophages in colon. In addition, the in vivo formation of C-IPC on high hydrophobic NPs with increased rigidity was also confirmed (Supplementary Fig. 15). Therefore, the distinctive C-IPC anchored on high rigidity NPs could induce the improved macrophage targeting in vivo.

The NPs@C-IPC with increased rigidity were prepared as illustrated in Fig. 5a. Following C-IPC adsorption, an increase in particle size was noted, but minimal changes were observed in the zeta potential and morphological characteristics (Supplementary Fig. 16). Quantitative analysis of the C-IPC protein adsorbed onto NPs of varying rigidity revealed that Soft, Semi, and Hard all retained a substantial protein content, with no statistically significant differences detected between them (Fig. 8e). This observation indicated that modulating the rigidity of NPs does not significantly affect the protein content of the adsorbed C-IPC. Subsequently, we explored the impact of rigidity modulation on macrophage uptake. The NPs@C-IPC demonstrated no cytotoxic effects on RAW264.7 cells (Supplementary Fig. 17). Notably, while the cellular uptake of Soft@C-IPC, Semi@C-IPC and Hard@C-IPC was significantly enhanced compared to NPs without C-IPC adsorption, Hard@C-IPC exhibited the most pronounced enhancement (4.1-fold) and the highest macrophage uptake overall (Fig. 8f). This trend was not mirrored in H-IPC, where only a marginal improvement in macrophage uptake was observed across all three NPs@H-IPC variants (Supplementary Fig. 18).

Together, our findings indicate that the rigidity modulation strategy can further enhance the beneficial effects of C-IPC on macrophage targeting and uptake, building upon the foundation of hydrophobicity modulation. We supposed that the increased rigidity of NPs optimized the protein composition of C-IPC that possessed high protein amount, endowing the nanocarriers with superior colon macrophage targeting and colitis treatment.

Mechanism of C-IPC on macrophage targeting and internalization
To elucidate how rigidity modulation altered the protein composition of C-IPC, the proteomics technique via LC-MS/MS was applied for quantitative analysis (Fig. 9a)11,49,50. According to the proteomic analysis (Proteomics data file 1, 2), there were 278, 315 and 330 proteins identified separately, and the C-IPC on Hard owned the largest variety of proteins (Fig. 9b). The top twenty identified proteins within C-IPC were listed in Supplementary Table 5.

Next, the Gene Oncology (GO) analysis was performed to elucidate the biological function of identified proteins11,51. Interestingly, three C-IPC all contained dozens of proteins involved in the “Receptor-mediated endocytosis” (GO: 0006898), and they also all owned several proteins involved in the receptor binding annotations (Fig. 9c). It’s worth noting that the immunoglobulin receptor, G protein coupled receptor, Toll-like receptor, low-density lipoprotein receptor, EGFR receptor and RAGE receptor were all expressed on the macrophages as reported52–58. Therefore, we deemed that the adsorbed C-IPC could improve the macrophage targeting and uptake in a direct pattern through directly binding to the receptors/transporters on macrophage (Fig. 9d). In addition, three C-IPC all possessed proteins involved in the substance (including carbohydrate, monocarboxylic acid and fatty acid) binding annotations as well (Fig. 9e). These substances could be recognized and transported into cells through specific receptors/transporters, and some of them were already reported to work as ligands for improving the cell uptake of nanoparticles. For instance, the fructose (carbohydrate) and butyrate (monocarboxylic acid) modified nanoparticles were able to improve the Caco-2 cell uptake through glucose transporter (GLUT)59 and monocarboxylic acid transporter (MCT-1)60. Linolenic acid (fatty acid) modification was also reported to improve the cell uptake of micelles through fatty acid transporter (FATP4)61. Based on this, we speculated that the exterior C-IPC could also improve the macrophage targeting and uptake in an indirect pattern, by firstly binding with these substances to form specific ligands and then indirectly binding to the expressed receptors/transporters (Fig. 9f). Consequently, the two receptors/transporters-binding patterns by C-IPC worked together to exert its macrophage targeting effect, and these macrophage receptor binding-related functional proteins within C-IPC were called as macrophage-targeting proteins.

Impact of rigidity on the macrophage-targeting protein selective adsorption
The distinction between the protein composition and protein proportion of C-IPC on NPs with increased rigidity was analyzed. Venn plot analysis showed that unique proteins were observed for each C-IPC (Supplementary Figs. 19a, b). By pairwise comparison, the cluster heatmap and volcano plot analysis both showed the protein composition significantly changed after rigidity modulation of NPs, with multiple of proteins obviously upregulated and down-regulated (Supplementary Figs. 19c, d). Consequently, these altered proteins might generate impact on the macrophage targeting effect of different C-IPC.

In particular, we found several annotations about the receptor binding and substance binding were significantly changed between the C-IPC adsorbed by Semi and Hard (Fig. 10a), including the “Toll-like receptor 4 binding” (GO: 0035662), “Toll-like receptor binding” (GO: 0035325), “RAGE receptor binding” (GO: 0050786) as well as “Mannose binding” (GO: 0005537), “Fatty acid derivative binding” (GO: 1901567), “Long-chain fatty acid binding” (GO: 0036041). Our findings revealed that the C-IPC might mediate NPs binding to receptors/transporters in both direct and indirect pattern to improve the macrophage targeting and uptake (Fig. 9). We deemed that the C-IPC adsorbed by Hard could amplify this mechanism to a greater extent, thus making Hard@C-IPC owned better macrophage targeting than Semi@C-IPC. It’s worth noting that α-mannosidase (MAN2B1), β-mannosidase (MANBA) and protein S100-A8 (S100A8) were the identified functional proteins that involved in almost all of the significantly changed GO annotations (Fig. 10a), and the proportion of them were all upregulated in Hard compared with Semi especially for S100A8 protein (Fig. 10b). Notably, the C-IPC on Hard possessed the highest protein proportion of S100A8 protein among all (Fig. 10c). Then the S100A8 antibody was used to specifically block the S100A8 protein in the C-IPC for evaluating its involvement in macrophage uptake of NPs (Fig. 10d). After pre-blocking S100A8, only the macrophage uptake of Hard@C-IPC was significantly inhibited, while Soft@C-IPC and Semi@C-IPC did not change significantly (Fig. 10e). It suggested that S100A8 was a vital protein in C-IPC adsorbed by Hard to mediate the receptor binding and macrophage uptake (Fig. 10f). In consequence, increased rigidity of NPs could optimize the protein composition of C-IPC by up-regulating the proportion of macrophage-targeting proteins (including S100A8 protein), achieving better macrophage targeting and superior colitis treatment.

In addition, the colitis-intestinal biofluids (C-IF) from rats with mild colitis (Mild) or severe colitis (Severe) (Supplementary Fig. 20) were also detected using the label-free proteomic analysis via LC-MS/MS (Proteomics data file 3). There were 600 and 653 kinds of proteins identified in the Mild and Severe group (Supplementary Fig. 21a), and 239 and 76 kinds of proteins significantly upregulated or down-regulated (≥2-fold) in Severe group compared with Mild group (Supplementary Fig. 21b, c), indicating the variable protein composition and level in the intestinal biofluids with the progression of colitis. Notably, the protein content of S100A8 within C-IF also significantly increased in Severe group which was about 3.2-fold higher than that of Mild group (Supplementary Fig. 21d). It was beneficial for C-IPC formation because S100A8 was the desired macrophage-targeting protein. Therefore, we believe the increased severity of colitis might influence the composition of protein corona on nanocarriers.

Methods
Materials
Soybean lecithin was purchased from Tywei Pharmaceutical Corp., Ltd (Shanghai, China). Cholesterol, Rose-bengal, 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) and Coumarin 6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly(lactic-co-glycolic acid) (PLGA, 50/50, viscosity: 0.15~0.25 dL/g) was purchased from Lactel absorbable polymers (UK). DSPE-PEG2000 and mPEG2000-SH were purchased from Posure Biological (Shanghai, China). Rhodamine B was purchased from Huaxia Chemical Reagents Co., Ltd (Chengdu, China). Coomassie brilliant blue G-250 and Hank’s balanced salt solution (HBSS) were purchased from Solarbio Life Sciences (Beijing, China). Sephadex G50 was purchased from MedChemExpress (New Jersey, USA). Protein LoBind tubes were purchased from Eppendorf (Hamburg, Germany). Polystyrene nanoparticles (PS) with theoretical diameter of 90 or 150 nm were purchased from Tianjin BaseLine ChromTech Research Centre (Tianjin, China). Au nanoparticles (Au-NPs) were purchased from Tomicro technology (Shanghai, China). Polycarbonate membrane for extrusion was purchased from GE healthcare Life Sciences (Pittsburgh, USA). Acetylcysteine (NAC) was purchased from Chengdu Huaxia Chemical Reagent Co., LTD. 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI), 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DiD), 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR), Bradford protein assay kit, BCA protein assay kit, Reactive oxygen species (ROS) assay kit and DAPI were purchased from Beyotime Biotechnology (Shanghai, China). Budesonide was purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Alexa Fluor 647 conjugated secondary antibody (ab150083, 1:100), Alexa Fluor 488 conjugated secondary antibody (ab150113, 1:1000) were provided by Abcam. Anti-CD3-FITC (Elabscience, E-AB-F1013C, clone: 17A2; 1:300), Anti-CD4-Percp/Cy5.5 (Elabscience, E-AB-F1097J, clone: GK1.5; 1:300), Anti-CD11b-PE (Elabscience, E-AB-F1081D, clone: M1/7O; 1:300) were provided by Elabscience. Anti-Foxp3-PE (BioLgend, 560408, clone: MF23; 1:300), anti-F4/80-FITC (BioLgend, B379316, clone: BM8; 1:300), anti-CD86-Percp/Cy5.5 (BioLgend, B422684, clone: GL-1; 1:300), anti-CD206-APC (BioLgend, B441147, clone: C068C2; 1:300) were purchased from Biolegend. Anti-S100A8 antibody (HA723135, clone: PSH09-70; 1:100) was purchased from HUABIO (Hangzhou, China). Rat TNF-α, IL-1β and IL-6 Elisa kit were purchased from Quanzhou Ruixin Biological Tcehnology Co., LTD. All reagents were used as received without further purification unless specified otherwise. All other chemical reagents in this study were at least analytic grade.

Cells and animals
The Caco-2 cells and RAW264.7 macrophages were obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). RAW264.7 macrophages were cultured in Dulbecco’s modified eagle medium (DMEM, Basal Media, China) containing 10% (v/v) fetal bovine serum (FBS, Excell, China), 1% (v/v) penicillin and streptomycin solution (100 IU/mL, Solarbio, China) at 37°C in an incubator with 5% CO2. Caco-2 cells were cultured in DMEM containing 10% FBS, 1% penicillin and streptomycin solution and 1% (v/v) non-essential amino acids (Solarbio, China) under the same conditions.

Sprague-Dawley (SD) rats (male, 200–220 g, 7 weeks old) were obtained from Chengdu Dossy experimental animals Co., Ltd (China). The rats were raised in the qualified animal room under pathogen free conditions at constant temperature (25°C). All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee of Sichuan University (accreditation number, SYXK (Chuan) 2018-113).

The colitis rats were established as in our previous report11. The SD rats were fasted overnight but free access to water. TNBS aqueous solution (5%, w/v) was mixed with equal volume of ethanol to form the TNBS-ethanol solution at a final concentration of 2.5%. Rats were anesthetized by isoflurane (Small Animal Anesthesia Machine, RWD Life Science, China), then medical-grade rubber cannula was inserted about 8 cm through the anus. The TNBS-ethanol solution was infused into the lumen of colon at TNBS dose of 80 mg/kg. Then the rats were kept for a supine position for about 5 min after administration to ensure the sufficient infiltration of TNBS-ethanol in colon. After the treatment, the rats were fed for a week to induce the colitis. According to the “Guideline of assessment for humane endpoints in animal experiment (RB/T 173-2018, China)”, the humane endpoints in this study were set as body weight of rats reduced to more than 20% of initial. These rats would be removed from the experiments and euthanized via intraperitoneal injection of pentobarbital.

Preparation and characterization of NPs with varied hydrophobicity
Soybean lecithin and cholesterol as the lipid component of NPs were mixed with DSPE-PEG2000 at different mass ratios (soybean lecithin: cholesterol: DSPE-PEG2000 = 10:2.5:8, 10:2.5:4 or 10:2.5:1, equal to PEGylation of 80%, 40% and 10%). The mixture was dissolved in 4 mL chloroform, and then the chloroform was removed by a rotary evaporator at 37°C in a vacuum to form the thin lipid film. Then the lipid film was hydrated with 4 mL distilled water for 5 min to form the crude lipid suspensions, followed by sonication using the ultrasonic-processor (Ningbo Scientz Biotechnology Co., Ltd, China) on ice bath (120 W, 3 min) to finally obtain the NPs with different PEG modification. The fluorescence Coumarin 6-labeled NPs were prepared by extra adding the Coumarin 6 dye to the initial lipid and DSPE-PEG2000 mixture before the evaporation process.

The hydrodynamic diameter, polydispersity index (PDI) and zeta potential of NPs were measured by Malvern Zetasizer (Malvern Instruments Ltd., UK). The morphological characteristics of the NPs were examined by cryogenic transmission electron microscopy (cryo-TEM) using a Titan Krios G4 instrument (Thermo Fisher Scientific). For sample preparation, a droplet of the NPs suspension was applied to a glow-discharged, carbon-coated copper grid (300 mesh, R2/1, Quantifoil Micro Tools). Blotting was conducted for 5 seconds under conditions of 4°C and 100% humidity employing a Vitrobot Mark IV (Thermo Fisher Scientific). Immediate vitrification was achieved by rapidly plunging the grid into liquid ethane, after which the vitrified sample was stored in liquid nitrogen at 90 K. Subsequently, the grid was transferred into the microscope, which was operated at an accelerating voltage of 300 kV. Image acquisition was performed with a Falcon 4i detector at a nominal magnification of 81,000×.

Hydrophobicity measurement
The NPs were diluted to the concentration from 2.5 to 0.625 mg/mL (lipid dose), and then 100 μL nanoparticle dilutions were mixed with 100 μL CBB solution (Coomassie brilliant blue G-250). The mixture was incubated at 37°C for 20 min under shaking. Finally, the UV absorbance spectrum of the mixture was measured at 550-650 nm using a microplate reader and the CBB solution without NPs were set as control. The shift of the maximum absorption wavelength of CBB from different mixture were calculated.

Rose-bengal solutions (RB) were mixed with NPs suspensions (1 mg/mL) to a final concentration of 480-320 μg/mL. The mixture was incubated at 37°C for 3 h under shaking, then the RB bound to NPs and the free RB were separated using the ultrafiltration centrifugal tube (100 kDa) by centrifugation (3920g, 30 min). The UV absorbance of the free RB at 549 nm from different samples was measured using a microplate reader. The binding constant of RB was calculated according to the Eq. (1):

r = KN / (1 + Ka) (1)

Where r is the amount of RB adsorbed per nanoparticles (μg/mg), a is equilibrium constant of RB (μg/mL), K is binding constant (mL/μg) and N is maximum amount bound (μg/mg).

The NPs suspensions were vacuum freeze-dried and compressed into tablets, then adhered to glass slides. The contact angles of NPs were measured using a contact angle measuring instrument (OCA50, DataPhysics, Germany). Quantitative analysis of contact angle was performed using ImageJ software.

Preparation and characterization of NPs with varied rigidity
Soybean lecithin, cholesterol as lipid component and DSPE-PEG2000 at mass ratio of 10:2.5:1 (maintain PEGylation of 10%) were dissolved in 4 mL chloroform, and then evaporated to form the thin lipid film. The dry lipid film was then hydrated with 4 mL distilled water without polystyrene nanoparticles (PS), or 4 mL distilled water contained 1 mg of PS with theoretical diameter of 90 nm (PS-90), or 4 mL distilled water contained 1 mg of PS with theoretical diameter of 150 nm (PS-150) for 5 min at 37°C to form the crude lipid suspensions. Finally, the crude suspensions were extruded through a polycarbonate membrane (WhatmanTM, 0.4 μm) using a mini-extruder device (Avanti Polar Lipids, USA) to obtain the NPs encapsulated with/without polystyrene nanoparticles. The fluorescence Coumarin 6-labeled NPs were also prepared by extra adding the Coumarin 6 dye to the initial lipid and DSPE-PEG2000 mixture. The hydrodynamic diameter, PDI, zeta potential and cryo-TEM images of NPs with varied rigidity were also measured.

Water content study
To label the water layer inside NPs, 200 μM rhodamine B in distilled water were applied as the hydration solution. After obtained the labeled NPs, the free rhodamine B was removed from the NPs using a Sephadex G50 gel size-exclusion column, and the collected NPs were diluted to the same concentration. The fluorescence emission spectra of the collected NPs were measured at an excitation wavelength of 510 to 700 nm using a microplate reader (Thermo Scientific, USA).

Young’s modulus measurement
The NPs suspensions were dropped onto the surface of freshly cleaved mica and air-dried at room temperature. The NPs samples were measured using the atomic force microscopy (AFM, Cypher VRS, Oxford instruments, UK). The measuring parameters were: scan rate of 0.5 Hz, the tip (AC240) with a spring constant of 2 N/m, scan lines of 256 and scan points of 256. The Young’s modulus of NPs was then analyzed and calculated using the JKR model in the Igor Pro software.

Preparation and characterization of drug-loaded nanoparticles
Budesonide-loaded NPs (NPs-Bud) were prepared by adding budesonide (0.75 mg) to the initial lipid component before the evaporation process, and then the NPs-Bud were centrifuged (3920g, 1 h) using ultrafiltration centrifuge tube (100 kDa) to separate the free drug. The collected NPs-Bud were re-dispersed with PBS solution for further use. The particle size, PDI and zeta potential of NPs-Bud were detected. For the encapsulation efficiency (EE) and drug loading (DL) measurement, freshly prepared NPs-Bud suspensions were centrifuged to separate the free drug. Then the amount of drug encapsulated in the NPs-Bud was quantified using high performance liquid chromatography (HPLC, Agilent 1260 Infinity II, USA). The encapsulation efficiency (EE) was calculated using the following Eq. (2):

EE (%) = (amount of drug encapsulated in the NPs-Bud (μg) / total amount of the added drug (μg)) × 100% (2)

Besides, the dry mass of NPs-Bud was weighed after freeze-drying, and then the drug loading (DL) was calculated using the following Eq. (3):

DL (%) = (weight of drug in NPs-Bud (μg) / total weight of NPs-Bud (mg)) (3)

Colitis therapy in vivo
In Day 0, SD rats (male, 200–220 g, 7 weeks old) were treated with TNBS solution to induce the colitis and the rats without treatment were set as healthy control group (Healthy). In Day 1, the colitis rats were orally administrated with free Bud, Philic-Bud, Medium-Bud and Phobic-Bud at budesonide dose of 0.6 mg/kg/day once a day, and the colitis rats without treatment of budesonide were set as another control group (Saline). The body weight of rats in each group (n=5) was measured and recorded daily. In Day 8, the rats were sacrificed and the colons and spleens were collected. The collected colons and spleens were gently washed with PBS solution and imaged, then the colon length and spleen weight were measured. The colon mucosa was evaluated by the Hematoxylin-eosin staining (H&E) analysis and Masson analysis. To investigate the ROS level, the colons were grinded on an ice bath and the colon cell suspensions were collected. The ROS level of colon cells was measured using Reactive oxygen species (ROS) assay kit (Beyotime Biotechnology, China). Briefly, the colon cells were incubated with DCFH-DA (10 μM, ROS probe) at 37°C for 20 min. After incubation, the cells were washed with PBS solution twice, and the fluorescence intensity of DCF in the cells was detected by flow cytometry.

To investigate the inflammation level, the colons were evaluated by immunohistochemical analysis. Briefly, the colons were fixed with paraformaldehyde (4%, w/v) and embedded in paraffin. Afterwards, the colons were cut into slices, the sections were washed and incubated with goat serum (10%, v/v) for at 37°C 30 min. Then the colon samples were incubated with anti-TNF-α, anti-IL-1β, anti-IL-6 and anti-IL-10 antibody at 4°C overnight, and second antibody at 37°C for 45 min. At last, the cell nucleus was stained with hematoxylin and the samples were recorded by microscope (BX53, Olympus Corporation, Japan). In addition, the inflammatory cytokine levels of different groups were quantitatively measured according to the protocol of the ELISA kit (Beyotime Biotechnology, China). The inflammatory cytokine concentrations of the colon suspensions were calculated according to the standard curves, and then the protein concentrations of colon suspensions were measured using the BCA protein assay kit. Finally, the inflammatory cytokine concentrations measured by the ELISA kit were normalized by calibrating with the protein concentrations of different groups.

To investigate the immune cells, the colon and spleen cells were stained with FITC-conjugated CD3 antibody and PE/Cyanine7-conjugated CD4 antibody at 4°C for 1 h. These cells were punched and stained with Percp/5.5-conjugated Foxp3 antibody at 4°C for 1 h, and then washed with PBS solution thrice, followed by flow cytometry analysis (Treg cells). For quantitative analysis of the macrophages, the colon and spleen cells were incubated with anti-CD16/32 antibody for 30 min. Then the cells were washed with PBS solution twice and stained with anti-F4/80, anti-CD11b, anti-CD86 and anti-CD206 antibody at 4°C for 1 h. Afterwards, cells were washed with PBS solution and the M1 type and M2 type macrophages were detected by flow cytometry.

The colitis therapy experiment by budesonide-loaded NPs with different rigidity (Soft-Bud, Semi-Bud and Hard-Bud) was also proceeded as described above. The power analysis was conducted to verify the sample number was set reasonably.

Colon macrophage targeting in vivo
The fluorescence labeled NPs were orally administrated to colitis SD rats or healthy SD rats (male, 200-220 g, 7 weeks old). After 6 h, the rats were sacrificed and the colon was collected and washed with saline solution. The colon was fixed with paraformaldehyde (4%, w/v), dehydrated in sucrose (30%, w/v) overnight, embedded into optimum cutting temperature compound (OCT), and sectioned at a depth of 10 mm (Lecia, Germany). Then the colon samples were stained with anti-CD68 antibody and DAPI solution. Finally, the colon samples were imaged by confocal laser scanning microscopy (CLSM) (LSM800, Carl Zeiss, Germany).

For quantitative analysis, the collected colon was grinded on an ice bath and the colon cell suspensions were collected. The colon cell suspensions were stained with anti-F4/80 antibody and anti-CD11b antibody at 4°C for 1 h. Afterwards, cells were washed with PBS solution and detected by flow cytometry.

Preparation and characterization of NPs@C-IPC
Colitis-intestinal biofluids (C-IF) extracted from the colitis rats was applied to simulate the intestinal milieu11. The fluorescence labeled NPs suspensions were mixed with the colitis-intestinal biofluids in the Protein LoBind tube (Eppendorf, Germany). Then the mixture was incubated at 37°C for 1 h under shaking. After incubation, the C-IPC adsorbed nanoparticles (NPs@C-IPC) and the free proteins were separated using Sephadex G50 gel size-exclusion column. The NPs@C-IPC fractions were collected, concentrated by ultrafiltration centrifugation (3920g, 30 min) and finally dispersed with PBS or HBSS solution for further use. The particle size, PDI and zeta potential of NPs@C-IPC were measured by Malvern Zetasizer, and their morphology was detected by TEM. Besides, the H-IPC adsorbed nanoparticles (NPs@H-IPC) were prepared as described above and only the colitis-intestinal biofluids were replaced with healthy-intestinal biofluids from healthy rats.

Macrophage uptake study
RAW264.7 cells were cultured in 24-well plates for 1 day before use. The fluorescence labeled NPs and NPs@C-IPC suspensions (100 μg/mL) were incubated with RAW264.7 cells for 3 h. The RAW264.7 cells were rinsed with PBS solution twice, and then the macrophage uptake was detected by a flow cytometer (Beckman Colter, USA).

In addition, the Caco-2 cells and RAW264.7 cells were stained with fluorescent dye DiI and DiD separately. Afterwards, different ratio (1:1, 5:1, 10:1) of the stained Caco-2 cells and RAW264.7 cells were co-cultured in 24-well plates and then incubated with the fluorescence labeled NPs and NPs@C-IPC suspensions for 3 h. Finally, the internalized nanoparticles by Caco-2 cells or RAW264.7 cells were detected by flow cytometer.

Protein amount of C-IPC
The protein samples from NPs@C-IPC were eluted and then the protein content of the C-IPC formed in vitro was measured using Bradford protein assay kit (Beyotime Biotechnology, China). Briefly, the C-IPC samples (5 μL) were added into 96-well plates, followed by addition of 250 μL Coomassie Blue G-250 staining solution. The absorbance at 590 nm of each sample were recorded by microplate reader (Thermo Scientific, USA). The bovine serum albumin (BSA) solution with the same procedure was recorded for the calibration curve and the protein content of C-IPC samples were calculated. The results were expressed as the protein amount (μg) per nanoparticles (μg).

After anesthesia, the intestinal lumen of colitis rat was rinsed with acetylcysteine (NAC) solution several times to pre-remove the intestinal biofluids before tied at both sides, and then the fluorescence labeled NPs solutions were injected into the lumen for 1 h. Then the suspensions were withdrawn from the lumen and the NPs@C-IPC were collected using Sephadex G50 gel size-exclusion column. Finally, protein content of C-IPC formed in vivo was measured using Bradford protein assay kit, and rat without pre-removal of intestinal biofluids was set as control. In addition, the C-IPC was also detected by SDS-PAGE assay.

Proteomic analysis of C-IPC and C-IF
The C-IPC formed on NPs with different rigidity and the C-IF extracted from mild or severe colitis rats were detected using the proteomic analysis via LC-MS/MS. The filter-aided sample preparation (FASP) procedure was performed. Aliquots of 100 ug of C-IPC or C-IF protein samples were diluted in urea buffer UA (8 M urea, 0.1 M Tris-HCl, pH 8.5) to a final concentration of 0.5% sodium dodecyl sulfate (SDS). Protein samples were further processed in Microcon 30 kDa Centrifugal Filter Units (Merck) in a tempered centrifuge at 24°C. Samples were added to the filter unit, washed with UA buffer, centrifuged for 15 min at 14000g, and incubated with 50 mM IAA for 20 min at room temperature (in the dark). SDS was exchanged by four consecutive washes with UA buffer (centrifugation for 15 min at 14000g) and a single wash with 50 mM ammonium bicarbonate (ABC) buffer followed by centrifugation for 5 min at 14000g. Proteins were digested using trypsin solution (25 μg/mL) in a 1:50 protein-to-enzyme ratio and incubated for 18 h at 37°C on a thermoshaker at 600 rpm. The resulting peptides were recovered by centrifugation at 14000g for 5 min, followed by elution with 50 μL of 50 mM ABC buffer and repeated centrifugation. Combined eluates were acidified using TFA at a final concentration of 1%. Finally, the filtrate was collected and the peptide samples were obtained by lyophilization. Then the peptides dissolved in 0.1% formic acid were loaded onto a Thermo Scientific analytical column (75 μm × 25 cm, 5 μm, C18) and separated by an Easy nanoLC system (Thermo Scientific, USA). Mobile phase A was 0.1% formic acid, and mobile phase B was 0.1% formic acid with acetonitrile. Peptides were separated with a linear gradient from 0 to 5% phase B within 5 min, followed by an increase to 30% phase B within 55 min and further to 35% phase B within 5 min, followed by a washing step at 95% phase B and re-equilibration. Then the separated samples were analyzed by mass spectrometric (MS, Obitrap Fusion Lumos, Thermo Scientific, USA). The results (Raw files) were analyzed using the MaxQuant software (version 2.0.1.0). MS/MS spectra were searched against UniProt database.

The amount of protein was calculated using spectral counting (SpC), which represented the total number of the MS/MS spectra for all peptides attributed to a detected protein. The relative quantitative concentration of each protein identified in C-IPC or C-IF was assessed by calculating the total spectral counting (SpC) of all peptides matched to a specific protein, and then normalized for the molecular weight (Mw) of each protein19,71,72. The SpC of each identified protein was normalized to the protein mass and expressed as the normalized protein content (% total protein) using the following Eq. (4):

NpSpCk = (SpC / Mw)k / Σ (SpC / Mw)i × 100 (4)

where NpSpC was the percentage normalized spectral count for protein k, SpC was the spectral count identified, and M was the molecular weight in kDa for protein k. The heatmaps, volcano plots, venn plots and protein summaries in the manuscript were generated using the TBtools73 and GraphPad Prism 8 software.

The effect of S100A8 protein on macrophage uptake
The effect of S100A8 protein in the C-IPC was investigated by pre-blocking NPs@C-IPC with the S100A8 antibody. Briefly, the NPs@C-IPC suspensions were incubated with S100A8 antibody solution (1 mg/mL in PBS (pH 7.4), HUABIO, China) for 1 h, and then separated using Sephadex G50 gel size-exclusion column. Afterwards, the resulting NPs@C-IPC with S100A8 antibody pre-blocking (S100A8 antibody group) were incubated with RAW264.7 cells for 3 h. Finally, the cells were washed and collected for the cell uptake detection by flow cytometer. In comparison, the NPs@C-IPC without S100A8 antibody pre-blocking were set as control (Control group). The cell uptake result was presented as the percentage of macrophage uptake in S100A8 antibody group compared to Control group.

Statistical analysis
All data were presented as mean ± standard deviations (SD) and they were obtained at least three times via independent data. All statistical analyses were performed using Student’s t-test or one-way analysis of variance (ANOVA) using the IBM SPSS® software (version 23.0), and p<0.05 were defined to be statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper aims to engineer nanoparticle designs that favor the formation of a colitis-specific intestinal biomolecular corona (C-IPC) to enhance therapeutic efficacy in a colitis rat model. By co-modulating hydrophobicity and rigidity, the authors seek to optimize oral macrophage-targeted drug delivery. The in vivo efficacy data are impressive and suggest successful therapeutic optimization. However, the claim that improved efficacy is primarily mediated by C-IPC targeting, particularly involving S100A8, requires stronger mechanistic support. Overall, this is a well-executed and impactful study demonstrating that tuning material properties can enhance drug delivery and that the disease-specific protein corona formation plays a role, though several aspects warrant clarification or further validation prior to publication.\n1. The authors should comment on how disease (colitis) severity and dynamics, such as inflammation and mucosal disruption in colitis, influence the formation and composition of the corona. As a general consideration, would colitis severity affect the C-IPC and thus, the effectiveness of their approach?\n2. In Results Section 1, the sentence “As the PEGylation decreased gradually from 80%, 40%, to 10%...” should be corrected to “As the PEGylation increased gradually from 10%, 40%, to 80%...” to accurately reflect the DSPE-PEG2000:lecithin mass ratios described in Figure 2a.\n3. A more detailed description of the nanoparticle composition and preparation method should be included to improve clarity. For example, the “lipid” component shown in Figure 2a is not sufficiently described in the figure or text.\n4. The budesonide loading (µg drug/mg particle) and encapsulation efficiency for each formulation should be reported. In addition, the authors should state whether drug release profiles were measured, as release kinetics could affect efficacy and influence interpretation of the results.\n5. Additional details regarding the colitis model need to be added to the methods section. For example, please provide the concentration of TNBS. This model seems highly lethal, can the authors comment on the humane endpoints employed as it is mentioned that death occurred, which is less common.\n6. Budesonide administered without a nanoparticle carrier should be included as a control group to determine whether the particle formulations offer enhanced therapeutic benefit.\n7. In Figure 5A, the images of healthy and colitis intestines appear to be switched and should be corrected. In Figures 5B and 5C, the word “coumarin” is misspelled.\n8. In Figure 5H, the gel intensity for the “Phobic” and “Medium” groups appears oversaturated. These samples should be diluted to improve visualization of protein fingerprints.\n9. The observed enrichment of corona proteins on hydrophobic particles is consistent with existing literature and strengthens the claim that particle hydrophobicity modulates corona formation.\n10. The authors should provide raw or normalized abundance values from the mass spectrometry analysis. It is unclear how “% total protein” was calculated, or how the heatmaps, volcano plots, and protein summaries were generated. Clarifying the data processing steps would strengthen the analysis and general understanding.\n11. The uptake data were normalized, but the normalization method should be described more clearly in the Methods.\n12. Although Figures 12F and 12G suggest that adding S100A8 antibody to the Hard@C-IPC particle reduces uptake, the observed reduction is modest (~20%). Given the presence of endogenous immunoglobulins in the corona, the specificity of this effect should be interpreted cautiously.\n13. Have the authors evaluated the protein composition of the colitis fluid itself? The authors should report the concentration/abundance of S100A8 in the colitis fluid to contextualize its biological relevance in the targeting mechanism.\n14. Hydrophobic particles are known to be more readily opsonized than hydrophilic ones due to enhanced binding of complement proteins, immunoglobulins, and other serum components. The current mass spectrometry data do not clearly show the abundance of these opsonins or how they compare with what is known in the literature. Additional analysis and interpretation are warranted.\n15. Opsonization may alter particle interactions with many cell types, not just macrophages. Can the authors provide additional data to support the claim that C-IPC particles specifically “target” macrophages in the colon? It would be helpful to assess potential uptake by other immune and epithelial cell populations.", "reviewer_2": "This manuscript by Wu et al., describes the dual modulation of an orally delivered nanoparticle to treat colitis. Specifically, the authors altered two physicochemical properties at the same time (hydrophobicity and rigidity) and investigated their impact on macrophage targeting for the purpose of colitis treatment. Their results suggest that high hydrophobicity alongside with higher rigidity resulted in not only higher amount of protein corona adsorption, but also in a higher proportion of macrophage targeting proteins. Although modulating physicochemical NP properties to manipulate disease-specific protein corona formation is not novel by itself, rationally modulating two physicochemical properties of NPs to tune protein corona formation and disease specific targeting does distinguish this work from others. Once comments below are addressed, this would be an article that would be of broad interest.\nComments are as follows:\n1. Figure 1: “Philic” not Phillc, targeting proteins “upregulated”\n2. Line 57: the authors mention that most studies have focused on singular physicochemical property modulation of nanocarriers which cannot sufficiently promote the functional protein adsorption to obtain the desired protein corona. Could they please provide references from recent studies?\n3. Line 63: macrophages were mentioned for the first time, but no sufficient background was provided regarding why macrophages are promising therapeutic targets for colitis treatment. What is the role of macrophages in colitis pathogenesis? Similarly in the abstract, please provide a brief rationale for targeting macrophages. A brief discussion on the importance of macrophages seems to occur around line 155, but not the introduction\n4. Figure 2: The use of TEM, which images dehydrated samples, to complement DLS, which measures hydrated samples size is appropriate. However, since TEM requires dehydrated samples, the drying process may cause potential particle shrinkage. And this affects their size measurements. Could the authors discuss this limitation and why they did not use cryo-TEM instead?\n5. Please rationalize and discuss the limitations of using of Coomassie blue and Rose-Bengal, which are indirect methods for measuring hydrophobicity\n6. Line 124: appears to be referencing Figure 3 instead? Please review the manuscript carefully for grammatical and correct figure referencing\n7. Did the authors run a power analysis to determine the sample numbers in Figure 3?\n8. Figure 3: Please state how the ELISA results were normalized\n9. Figure 6: Please briefly state the rationale for the differing macrophage ratios and their physiological relevance\n10. Figure 7: what model was used to calculate their NP Young’s moduli?\n11. Please expand on the results and discussion section to better contextualize the results to recent literature. For example, section 9 is a bit confusing to follow and does not connect results to potential endocytic pathways. Additionally, there is a growing body of literature describing how rigidity enhances certain endocytic/mechanobiology pathways to increase NP uptake in macrophages. Connecting their results to recent literature and contextualizing NP uptake mechanisms would add to the impact of this manuscript.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The study by Wu et al. demonstrates that modulating the surface physicochemical properties of nanocarriers allows precise control over the quantity and composition of the colitis-specific intestinal protein corona (C-IPC) formed on oral nanoparticles (NPs), thereby improving their targeting toward colonic macrophages and enhancing therapeutic outcomes in colitis. Given the promising experimental results, it is recommended that the authors undertake major revisions prior to being accepted.:\nQ. 1. At the beginning of the Results and Discussion section, the authors should provide a clear description of the materials used to prepare the nanoparticles, rather than focusing solely on surface modifications. Furthermore, is it feasible for NPs made of different materials (e.g., metal salts, metal–organic frameworks, liposomes, polymers, etc.) to achieve comparable effects following similar modifications?\nQ. 2. Why was there a high mortality rate in mice treated with Philic-Bud, whereas only minimal mortality was observed in the saline-treated negative control group?\nQ. 3. Variations in hydrophobic modification may influence drug loading capacity. Have the authors quantified the drug loading efficiencies of NPs with different hydrophobicities? How can they ensure that the differences in therapeutic efficacy are not attributable to variations in drug loading?\nQ. 4. Do the NPs undergo structural or physicochemical changes after gastric processing in mice? Specifically, do the hydrophilic/hydrophobic modifications remain stable?\nQ. 5. Could fluorescent labeling interfere with the adsorption of C-IPC onto the NPs? It is likely that fluorescent labeling alters the surface hydrophilicity/hydrophobicity—could this in turn affect protein corona formation?"}, "author_response": "We are grateful for reviewers’ positive comments and recognition of the significance on our work. We have revised the manuscript accordingly and addressed all the concerns of the reviewers. The following list describes the changes that have been made in the revised manuscript and the responses to the reviewers’ comments. The changes in the text, tables and figures of the manuscript are shown in red.\nReviewer #1 (Remarks to the Author):\nThis paper aims to engineer nanoparticle designs that favor the formation of a colitis-specific intestinal biomolecular corona (C-IPC) to enhance therapeutic efficacy in a colitis rat model. By co-modulating hydrophobicity and rigidity, the authors seek to optimize oral macrophage-targeted drug delivery. The in vivo efficacy data are impressive and suggest successful therapeutic optimization. However, the claim that improved efficacy is primarily mediated by C-IPC targeting, particularly involving S100A8, requires stronger mechanistic support. Overall, this is a well-executed and impactful study demonstrating that tuning material properties can enhance drug delivery and that the disease-specific protein corona formation plays a role, though several aspects warrant clarification or further validation prior to publication.\nQ. 1. The authors should comment on how disease (colitis) severity and dynamics, such as inflammation and mucosal disruption in colitis, influence the formation and composition of the corona. As a general consideration, would colitis severity affect the C-IPC and thus, the effectiveness of their approach?\nQ. 13. Have the authors evaluated the protein composition of the colitis fluid itself? The authors should report the concentration/abundance of S100A8 in the colitis fluid to contextualize its biological relevance in the targeting mechanism.\nA. 1&13. Thanks for your comments and suggestions. We combine these two questions for reply.\nWe believe that the severity of colitis will influence the formation and composition of the corona on nanocarriers because the colitis-intestinal biofluids (C-IF) may be changed with the progression of colitis. As far as we know, few studies have distinctly elucidated the changes of protein in the intestinal biofluids, including their type and concentration, during the progression of colitis. Therefore, we perform the proteomics analysis on the C-IF from rats with mild or severe colitis. SD rats are treated with TNBS-ethanol with dose of 40 or 80 mg/kg (80 mg/kg is used for colitis rat induction in the in vivo studies) by clyster, and fed for a week to induce rats with mild colitis (Mild) or severe colitis (Severe). The colon H&E analysis confirms that less mucosal disruption observed in Mild compared with Severe group (Supplementary Fig. 18). The C-IF are extracted and then detected using the label-free proteomic analysis via LC-MS/MS (Proteomics data file 3).\nThe results show that there are 600 and 653 kinds of proteins identified in the C-IF from Mild and Severe group, respectively (Supplementary Fig. 19a). Compared with the Mild group, 239 kinds of proteins are significantly upregulated (≥2-fold change) and 76 kinds of proteins are significantly downregulated (≥2-fold change) in the Severe group (Supplementary Fig. 19b–c), indicating alterations in protein composition and abundance level in the intestinal biofluids during colitis progression. Notably, the protein content of S100A8, one of the vital macrophage-targeting proteins in C-IPC (Fig. 12c-h), also significantly increases in Severe group which is about 3.2-fold higher than that of Mild (Supplementary Fig. 19d). The GO enrichment analysis (Severe vs Mild) reveals these altered proteins are mainly associated with immune cells and immune response (Supplementary Fig. 19e), which is consist with the enhanced inflammatory infiltration in colitis. What's more, dozens of GO annotations about the receptor binding and substance binding are significantly altered between the C-IF from Mild or from Severe group, such as “immunoglobulin receptor binding” (GO: 0034987), “integrin binding” (GO: 0005178) and “fatty acid binding” (GO: 0005504) (Supplementary Fig. 19f).\nIn conclusion, we find the severity of colitis will influence the protein composition and level in the intestinal biofluids. S100A8 is a desired macrophage-targeting protein. Notably, the protein level of S100A8 in the biofluids obviously increases with the progression of colitis, which is conducive to the protein corona formation. Therefore, we believe the increased severity of colitis may influence the composition of protein corona on nanocarriers. These significantly altered proteins in C-IPC can exert a stronger biological effect on immune response, receptor binding, and substance binding, which is beneficial for macrophage-targeted drug delivery.\nSupplementary Fig. 18 H&E analysis of the colon from healthy rats (Healthy) and colitis rats induced by TNBS-ethanol with dose of 40 mg/kg (Mild) or 80 mg/kg (Severe).\nSupplementary Fig. 19 Proteomic analysis of colitis-intestinal biofluids. a The number of identified proteins. Three independent samples detected for each group (n=3). b The number of upregulated and downregulated proteins (Severe vs Mild) with ratio ≥2 (p <0.05). c Volcano plot of the altered proteins. d Proportion of protein S100A8 in colitis-intestinal biofluids. e Top 20 of the GO enrichment analysis in biological process (BP) classification (Severe vs Mild). f Comparative analysis of the identified proteins (Severe vs Mild) for GO annotations with significant change (p<0.05) in molecular function (MF) classification.\nThe raw mass spectrometry proteomics data are available in the ProteomeXchange partner repository with the dataset identifier PXD071199 (https://www.proteomexchange.org/). Besides, the related result, discussion and experimental method are added in the Results, Discussions, Methods and Supplementary methods sections as following:\n“9. Impact of rigidity on the macrophage-targeting protein selective adsorption. In addition, the colitis-intestinal biofluids (C-IF) from rats with mild colitis (Mild) or severe colitis (Severe) (Supplementary Fig. 18) were also detected using the label-free proteomic analysis via LC-MS/MS (Proteomics data file 3). There were 600 and 653 kinds of proteins identified in the Mild and Severe group (Supplementary Fig. 19a), and 239 and 76 kinds of proteins significantly upregulated or downregulated (≥2-fold) in Severe group compared with Mild group (Supplementary Fig. 19b-c), indicating the variable protein composition and level in the intestinal biofluids with the progression of colitis. Notably, the protein content of S100A8 within C-IF also significantly increased in Severe group which was about 3.2-fold higher than that of Mild group (Supplementary Fig. 19d). It was beneficial for C-IPC formation because S100A8 was the desired macrophage-targeting protein.”\n“Discussions. Moreover, through comparing the intestinal biofluids from rats with mild and severe colitis, we found the colitis severity could influence the protein composition and level in the intestinal biofluids. These significantly altered proteins could exert a stronger biological effect on immune response, receptor binding and substance binding (Supplementary Fig. 19e, f). Notably, the protein level of S100A8 obviously increased with the progression of colitis, which was conducive to the protein corona formation because S100A8 was the desired macrophage-targeting protein. Therefore, we believe the increased severity of colitis might influence the composition of protein corona on nanocarriers. These significantly altered proteins in C-IPC could exert a stronger biological effect on immune response, receptor binding, and substance binding, which was beneficial for macrophage-targeted drug delivery.”\n“Proteomic analysis of C-IPC and C-IF. The C-IPC formed on NPs with different rigidity and the C-IF extracted from rats with mild or severe colitis were detected using the proteomic analysis via LC-MS/MS. The filter-aided sample preparation (FASP) procedure was performed. Briefly, C-IPC or C-IF samples were mixed with DTT solution (100 mM) and heated for 5 min.”\n“C-IF extraction from rats with mild or severe colitis. Fasted SD rats are treated with TNBS-ethanol with dose of 40 or 80 mg/kg by clyster, and fed for a week to induce mild colitis (Mild) or severe colitis (Severe). The rats were anesthetized and the colitis-intestinal biofluids (C-IF) were extracted from the lumen. Finally, the freshly collected C-IF from Mild and Severe group were centrifugated at 12000 rpm for several times to remove the insoluble substances, and then detected by proteomic analysis.”\nQ. 2. In Results Section 1, the sentence “As the PEGylation decreased gradually from 80%, 40%, to 10%...” should be corrected to “As the PEGylation increased gradually from 10%, 40%, to 80%...” to accurately reflect the DSPE-PEG2000:lecithin mass ratios described in Figure 2a.\nA. 2. We are sorry for the unclear description. The sentence “As the PEGylation decreased gradually from 80%, 40%, to 10%...” is corrected to “As the PEGylation increased gradually from 10%, 40%, to 80%...” to match the schematic diagram of NPs with different PEGylation in Fig 2a in the revised manuscript.\nQ. 3. A more detailed description of the nanoparticle composition and preparation method should be included to improve clarity. For example, the “lipid” component shown in Figure 2a is not sufficiently described in the figure or text.\nA. 3. Thanks for your suggestion.\n(1) The lipid component of nanoparticles is soybean lecithin and cholesterol. We have supplemented the lipid component description in the schematic diagrams of Fig. 2a and Fig. 7a.\nFig. 2a Schematic diagram of NPs with different PEGylation.\nFig. 7a Schematic diagram of NPs with different rigidity.\n(2) More detailed descriptions of the nanoparticle composition and preparation method are provided in the Methods section as following:\n“Preparation and characterization of NPs with varied hydrophobicity. Soybean lecithin and cholesterol as the lipid component of NPs were mixed with DSPE-PEG at different mass ratio (soybean lecithin: 2000 cholesterol: DSPE-PEG =10:2.5:8, 10:2.5:4 or 10:2.5:1, equal to PEGylation of 80%, 40% and 10%). The 2000 mixture was dissolved in 4 mL chloroform, and then the chloroform was removed by a rotary evaporator at 37 ℃ in a vacuum to form the thin lipid film. Then the lipid film was hydrated with 4 mL distilled water for 5 min to form the crude lipid suspensions, followed by sonication using the ultrasonic-processor (Ningbo Scientz Biotechnology Co., Ltd, China) on ice bath (120 W, 3 min) to finally obtain the NPs with different PEG modification. The fluorescence Coumarin 6-labeled NPs were prepared by extra adding the Coumarin 6 dye to the initial lipid and DSPE-PEG mixture before the evaporation process. 2000\nPreparation and characterization of NPs with varied rigidity. Soybean lecithin, cholesterol as lipid component and DSPE-PEG at mass ratio of 10:2.5:1 (maintain PEGylation of 10%) were dissolved in 4 2000 mL chloroform, and then evaporated to form the thin lipid film. The dry lipid film was then hydrated with 4 mL distilled water without polystyrene nanoparticles (PS), or 4 mL distilled water contained 1 mg of PS with theoretical diameter of 90 nm (PS-90), or 4 mL distilled water contained 1 mg of PS with theoretical diameter of 150 nm (PS-150) for 5 min at 37 ℃ to form the crude lipid suspensions. Finally, the crude suspensions were extruded through a polycarbonate membrane (WhatmanTM, 0.4 μm) using a mini-extruder device (Avanti Polar Lipids, USA) to obtain the NPs encapsulated with/without polystyrene nanoparticles. The fluorescence Coumarin 6-labeled NPs were also prepared by extra adding the Coumarin 6 dye to the initial lipid and DSPE-PEG mixture.” 2000\nQ. 4. The budesonide loading (µg drug/mg particle) and encapsulation efficiency for each formulation should be reported. In addition, the authors should state whether drug release profiles were measured, as release kinetics could affect efficacy and influence interpretation of the results.\nA. 4. Thanks for your suggestion.\n(1) The encapsulation efficiency (EE) and drug loading (DL) of budesonide (Bud) in each formulation are provided. The result in Supplementary Tab. 2 shows that Philic-Bud, Medium-Bud and Phobic-Bud all own a high drug encapsulation efficiency to approximately 80%. However, the Phobic-Bud have the highest drug loading, mainly because the Philic-Bud and Medium-Bud contain higher amount of DSPE-PEG therefore 2000 increasing the total weight of formulation. The Soft-Bud, Semi-Bud and Hard-Bud also all have a high encapsulation efficiency to approximately 79% but similar drug loading (Supplementary Tab. 4). In addition, the drug loading process shows no influence on the particle size, PDI and zeta potential of nanoparticles.\nSupplementary Tab. 2 Characterization of NPs-Bud with varied hydrophobicity (n=3). Encapsulation efficiency (EE), drug loading (DL).\nSize (nm) PDI ZP (mV) EE (%) DL (μg/mg)\nPhilic-Bud 1 0 - 80.2 ±0.4 1\n5 . 3 . \nMedium-Bud 1 0 - 7 1\n2 2 7 4\n5 . 3 9 .\n. 9 . 8\nPhobic-Bud 1 0 - 8 2\n1 2 2 . 9\n1 1 1\n4 . 3 0 .\n. 5 . 1 3\n0.03\n8 2 1 . 8\n1 1 1\nSupplementary Tab. 4 Characterization of NPs-Bud with varied rigidity (n=3).\n2.0 0.021 1.0\n. 3 . 6 0\n0.8 0.04\nSize 1(nm) PD1I ZP (1mV) EE ( %) DL (μ g/mg)\n1.5 0.041 0.5\n0.7 0.10\nSoft-Bud 1 0 - 7 2\n1.5 0.031 0.8\n4 . 3 9 .\nSemi-Bud 1 0 - 7 2\n9 2 0 . 9\n5 . 3 9 .\n. 5 . 4 4\nHard-Bud 1 0 - 7 2\n1 2 2 . 6\n1 1 1\n5 . 3 9 .\n. 7 . 1 1\n0.3 0.01\n4 2 0 . 6\n1 1 1\n(2) The drug release behaviors of NPs-Bud in the simulated GI condition are also investigated. All 3.7 0.011 0.8\n. 3 . 7 3\nformulations exhibit similar susta ined drug rele ase behavior i n simulated0 .g5a stric fluid0s. 0(1S GF), simulated\nintestinal fluids (SIF) and simula1ted colonic flu1ids (SCF), with1 approximate ly 40% of dr ugs released from\n1.9 0.011 0.2\n0.9 0.03\n2.6 0.021 0.4\nNPs-Bud within 10 h (Supplementary Fig. 3, 10). This result demonstrates that the majority of drugs can be\n100\nf\no)\n%\nt SGF SIF SCF\nn( 80\nud Philic-Bud\nou\nmB 60\nMedium-Bud\na d\ne v e s 40 Phobic-Bud\nia\nt a l e e l e 20\nRr\n0\n0 1 2 3 4 5 6 7 8 9 10\nTime (h)\nSupplementary Fig. 3 Drug release behavior of NPs-Bud with varied hydrophobicity in simulated GI condition (n=3).\n100\nf\no)\n%\nt SGF SIF SCF\nn( 80\nud Soft-Bud\nou\nmB 60\nSemi-Bud\na d\ne v e s 40 Hard-Bud\nia\nt a l e e l e 20\nRr\n0\n0 1 2 3 4 5 6 7 8 9 10\nTime (h)\nSupplementary Fig. 10 Drug release behavior of NPs-Bud with varied rigidity in simulated GI condition (n=3).\nThe related result, discussion and experimental method are added in the Results, Methods and Supplementary\nmethods sections as following:\n“2. Drug-loaded NPs with varied hydrophobicity for colitis therapy. The drug loading process showed no\ninfluence on the particle size, PDI and zeta potential of nanoparticles, and three NPs-Bud all owned a high\nencapsulation efficiency to approximately 80% (Supplementary Tab. 2). The Phobic-Bud had the highest drug\nloading (2.80 μg/mg), mainly because the Philic-Bud and Medium-Bud contained higher amount of DSPE-\nPEG therefore increasing the total weight of formulation. Besides, all formulations exhibited similar\n2000\nsustained drug release behavior in the simulated GI condition with approximately 40% of drug released from\nNPs-Bud within 10 h (Supplementary Fig. 3).\n6. Hydrophobicity-rigidity co-modulation for effective colitis therapy. The Phobic-Bud with increased\nrigidity (Soft-Bud, Semi-Bud and Hard-Bud) were prepared. Three formulations also possessed similar\nencapsulation efficiency, drug loading (Supplementary Tab. 4) and sustained drug release behavior\n(Supplementary Fig. 10).”\n“Preparation and characterization of drug-loaded nanoparticles. Budesonide-loaded NPs (NPs-Bud)\nwere prepared by adding budesonide (0.75 mg) to the initial lipid component before the evaporation process,\nand then the NPs-Bud were centrifuged (6500 rpm, 1 h) using ultrafiltration centrifuge tube (100 kDa) to\nseparate the free drug. The collected NPs-Bud were re-dispersed with PBS solution for further use.\nThe particle size, PDI and zeta potential of NPs-Bud were detected. For the encapsulation efficiency (EE)\nand drug loading (DL) measurement, freshly prepared NPs-Bud suspensions were centrifuged to separate the\nfree drug. Then the amount of drug encapsulated in the NPs-Bud was quantified using high performance liquid\nchromatography (HPLC, Agilent 1260 Infinity II, USA). The encapsulation efficiency (EE) was calculated\nusing the following equation (2):\namount of drug encapsulated in the NPs-Bud (μg)\nEE (%) = ×100%\ntotal amount of the added drug (μg)\nBesides, the dry mass of NPs-Bud was weighed after freeze-drying, and then the drug loading (DL) was\ncalculated using the following equation (3):\nweight of drug in NPs-Bud (μg)\nDL (%) =\ntotal weight of NPs-Bud (mg)\n“Drug release from nanoparticles. The NPs-Bud were added into the dialysis tubes (100 kDa) and then\nimmersed in the SGF for 2 h, SIF for 4 h and simulated colonic fluids (SCF) for 4 h under stirring. 100 μL of\nNPs-Bud suspensions were withdrawn from the dialysis tubes at the predetermined time points and the amount\nof drug in NPs-Bud (unreleased Bud) was detected by HPLC. Finally, the drug release profile was constructed\nby plotting the amount of drug released from NPs-Bud over time.”\nQ. 5. Additional details regarding the colitis model need to be added to the methods section. For example, please provide the concentration of TNBS. This model seems highly lethal, can the authors comment on the humane endpoints employed as it is mentioned that death occurred, which is less common.\nQ. 6. Budesonide administered without a nanoparticle carrier should be included as a control group to determine whether the particle formulations offer enhanced therapeutic benefit.\nA. 5&6. Thanks for your comments and suggestions. We combine these two questions for reply.\n(1) The concentration of TNBS aqueous solution (P2297, Sigma-Aldrich) is 5% (w/v), and additional details\nregarding the colitis model are added to the Methods section as following:\n“Cells and animals. Briefly, SD rats were fasted overnight but free access to water. TNBS aqueous solution\n(5%, w/v) was mixed with equal volume of ethanol to form the TNBS-ethanol solution at a final concentration\nof 25 mg/mL. Rats were anesthetized by isoflurane (Small Animal Anesthesia Machine, RWD Life Science,\nChina), then medical-grade rubber cannula was inserted about 8 cm through the anus. The TNBS-ethanol\nsolution was infused into the lumen of colon at TNBS dose of 80 mg/kg. Then the rats were kept for a supine\nposition for about 5 min after administration to ensure the sufficient infiltration of TNBS-ethanol in colon.\nAfter the treatment, the rats were fed for a week to induce the colitis.”\n(2) The humane endpoints in this study are set as body weight of rats reduced to more than 20% of initial,\naccording to the “Guideline of assessment for humane endpoints in animal experiment (RB/T 173-2018,\nChina)”. In the animal experiments, rats with body weight loss over 20% are also regarded as dead, then they\nare removed from the experiments and euthanized via intraperitoneal injection of pentobarbital. The\ndescription about the humane endpoints is added to Methods section as following:\n“Cells and animals. According to the “Guideline of assessment for humane endpoints in animal experiment\n(RB/T 173-2018, China)”, the humane endpoints in this study were set as body weight of rats reduced to more\nthan 20% of initial. These rats would be removed from the experiments and euthanized via intraperitoneal\ninjection of pentobarbital.”\n(3) In this study, the colitis models established by TNBS-ethanol solution showed certain risk of death. A\nreview article about the TNBS-induced colitis animal model mentioned that death may happen, resulting\nmainly from an excessive inflammatory reaction [1]. However, the TNBS-induced colitis rats in Fig. 3 seemed\nunexpectedly lethal especially for rats with insufficient therapeutic effect (Saline and Philic-Bud groups).\nTherefore, we repeat the in vivo experiments with free budesonide group as a control group. The results are\nshown in Fig. 3 and Fig. 4.\nAs shown in the revised Fig. 3 and Fig. 4, oral administration of free drug (Free Bud group) almost shows\nno therapeutic effect for relieving the colitis progression. Phobic-Bud group still exhibits superior colitis\ntherapy including colon mucosa recovery, inflammatory cytokine suppression, and disordered immune cell\nrestoring, demonstrating enhanced therapeutic benefit for budesonide by nanocarriers with high\nhydrophobicity (Phobic). In addition, 80 mg/kg TNBS dose, the same as last time, is applied for the\nestablishment of colitis rat models, and there is no death occurrence for all rat groups. We suppose that the\nvaried physical condition of SD rats may be the possible reason. The previous batch of rats may be more\nsensitive to the TNBS-ethanol treatment for damaging colon mucosa and triggering immunoreaction, thus the\nrats without effective therapy are more easily to die due to the excessive inflammatory reaction [1].\nRef: [1] The TNBS-induced colitis animal model: An overview. Ann Med Surg (Lond). 2016 Aug 19;11:9-15.\nFig. 3 Drug-loaded NPs with varied hydrophobicity for colitis treatment. a Schematic diagram of the therapy of colitis\nrats by oral administration. Sprague-Dawley (SD) rats were treated with TNBS-ethanol solution by clyster to establish the\ncolitis rat model (Day 0). After 24 h (Day 1), the colitis rats were treated with free drug or different drug-loaded NPs once a\nday for continuous 7 days by oral administration. Colitis rats treated with saline (Saline) and Healthy rats (Healthy) were set\nas control groups. At Day 8, rats were sacrificed and evaluated. b Body weight of rats was recorded daily from Day 0 to Day\n7 and the body weight change (percentage of the initial body weight in Day 0) was calculated (n=5). c Rats were sacrificed in\nDay 8, then the colons were collected and imaged. d Length of the collected colons was measured (n=5). e Spleen index was\ncalculated (n=5). f The ROS level of colon was measured by flow cytometry using ROS Assay Kit (n=5). g-i\nImmunohistochemical analysis of the TNF-α, IL-1β and IL-6 level in colon. Scale bar= 100 μm. j-l Quantification of the TNF-\nα, IL-1β and IL-6 level in colon using ELISA assay (n=5).\nFig. 4 The immune cells in colitis rats after oral treatment. a, b Measurement of the proportion of Treg cells in colon and\nspleen by flow cytometry (n=5). c Immunohistochemical analysis of the IL-10 level in colon. Scale bar= 100 μm. d, e\nMeasurement of the ratio of M1 macrophages to M2 macrophages in colon and spleen by flow cytometry (n=5).\nThese figures are provided in the revised manuscript as Fig. 3 and Fig. 4, and the related results are also revised\nin the Methods section as following:\n“2. Drug-loaded NPs with varied hydrophobicity for colitis therapy. The treatment regimen was shown in\nFig. 3a. The colitis rats were orally administrated with saline, free Bud, Philic-Bud, Medium-Bud and Phobic-\nBud, and their body weights were recorded daily. Compared with Saline group, recovery of colitis-induced\nbody weight loss occurred after Phobic-Bud treatment, better than that of Philic-Bud and Medium-Bud groups,\nwhile no recovery was observed for Free Bud and Saline groups (Fig. 3b). In Day 8, Phobic-Bud treatment\nrelived the shortening of colon (Fig. 3c, d), reduced the spleen index (Fig. 3e) as well as restored the damaged\ncolon mucosa (Supplementary Fig. 4), which were better than that of Philic-Bud and Medium-Bud groups.\nEnhanced reactive oxygen species (ROS) generated from infiltrated macrophages leads to pathological\ninflammatory in colitis39,40. By contrast, Phobic-Bud treatment could decrease the dysregulated ROS level in\ncolon of colitis rats and showed lower ROS level than Philic-Bud and Medium-Bud groups (Fig. 3f). Colitis\nprogression would induce the up-regulation of inflammatory cytokines in colon38-41. After treatment, Phobic-\nBud group significantly decreased the abnormal up-regulation of colon TNF-α, IL-1β and IL-6 level in colitis\nrats, exerting better effect than Philic-Bud and Medium-Bud groups (Fig. 3g-l). Notably, oral administration\nof free Bud almost showed no therapeutic effect for relieving the colitis progression. Therefore, the Phobic-\nBud exhibited better colitis therapy in alleviating colonic impairment and down-regulating the inflammatory\ncytokines.\nThe colitis could induce the disordered immune cells in body including the abnormal regulatory T (Treg)\ncells and macrophages42, 43. For rats suffered with colitis, the Treg cells (CD3+ CD4+ FOXP3+) in colon and\nspleen both declined significantly compared with healthy rats (Fig. 4a, b). After oral treatment, Phobic-Bud\ngroup could upregulate the proportion of Treg cells to about 10.0% in colon and 18.4% in spleen, which were\nboth higher than that of Philic-Bud and Medium-Bud group (Fig. 4a, b). IL-10 is an important anti-\ninflammatory cytokine secreted by various immune effector cells including intestine-resident macrophages to\npromote expansion and mediate the immunosuppressive function of Treg cells44. Notably, Phobic-Bud group\ncould obviously upregulate the IL-10 level as well compared with Saline group (Fig. 4c), which was consistent\nwith the increase of Treg cells. Moreover, Phobic-Bud treatment was able to influence the macrophage\npolarization and markedly decreased the ratio of M1 type (inflammatory) macrophages (F4/80+ CD11b+\nCD86+) to M2 type (anti-inflammatory) macrophages (F4/80+ CD11b+ CD206+) in colon and spleen at the\nsame time (Fig. 4d, e). The free Bud also exhibited very limited effect in regulating the disordered immune\ncells, demonstrating that the nano-formulations offered enhanced therapeutic benefit for budesonide. Overall,\nthese results demonstrated oral treatment of Phobic-Bud could restore the dysregulated immune cells in colitis.\nMeanwhile, the result also implied the increased surface hydrophobicity of nanocarriers played an important\nrole on oral drug delivery to colon.”\nQ. 7. In Figure 5A, the images of healthy and colitis intestines appear to be switched and should be corrected.\nIn Figures 5B and 5C, the word “coumarin” is misspelled.\nA. 7. We are sorry for the mistakes. The errors in Fig. 5a-c have been corrected in the revised manuscript.\nFig. 5a-c\nQ. 8. In Figure 5H, the gel intensity for the “ Phobic” and “ Medium” groups appears oversaturated. These\nsamples should be diluted to improve visualization of protein fingerprints.\nA. 8. Thanks for the suggestion. The protein samples are diluted with PBS solution and then detected by SDS-\nPAGE assay to improve visualization of protein fingerprints (Fig. 5h), and the result is added in the revised\nmanuscript.\nFig. 5h SDS-page detection of the in vivo protein corona adsorbed on NPs.\nQ. 9. The observed enrichment of corona proteins on hydrophobic particles is consistent with existing\nliterature and strengthens the claim that particle hydrophobicity modulates corona formation.\nA. 9. Thanks for your recognition.\nQ. 10. The authors should provide raw or normalized abundance values from the mass spectrometry analysis.\nIt is unclear how “% total protein” was calculated, or how the heatmaps, volcano plots, and protein\nsummaries were generated. Clarifying the data processing steps would strengthen the analysis and general\nunderstanding.\nA. 10. Thanks for your suggestions.\n(1) The proteomics analysis data including the protein intensity (Proteomics data file 1) and the normalized\nabundance values (Proteomics data file 2) are provided. In addition, the raw mass spectrometry proteomics\ndata are available in the ProteomeXchange partner repository with the dataset identifier PXD071116\n(https://www.proteomexchange.org/). The relative quantitative concentration of each protein identified in C-\nIPC was assessed by calculating the total spectral counting (SpC) of all peptides matched to a specific protein,\nand then normalized for the molecular weight (Mw) of each protein [1-3]. The SpC of each identified protein\nis normalized to the protein mass and expressed as the normalized protein content (% total protein) using the\nfollowing equation:\n(𝑆𝑝𝐶/𝑀 ) \n𝑁𝑝𝑆𝑝𝐶𝑘 = { 𝑤 𝑘 } × 100\n∑𝑛 (𝑆𝑝𝐶/𝑀 ) \n𝑖=1 𝑤 𝑖\nwhere NpSpCk is the percentage of normalized spectral count for protein k, SpC is the spectral count identified,\nand Mw is the molecular weight in kDa for protein k.\nRef: [1] Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality\nand Protein Corona. ACS Nano. 2016 Apr 26;10(4):4421-30.\n[2] Protein Corona of Magnetic Hydroxyapatite Scaffold Improves Cell Proliferation via Activation of\nMitogen-Activated Protein Kinase Signaling Pathway. ACS Nano. 2017 Apr 25;11(4):3690-3704.\n[3] Nanoparticle elasticity affects systemic circulation lifetime by modulating adsorption of apolipoprotein A-\nI in corona formation. Nat Commun. 2022 Jul 16;13(1):4137.\nThe detailed descriptions about the data processing are added in Methods section as following:\n“Proteomic analysis of C-IPC and C-IF. The amount of protein was calculated using spectral counting (SpC),\nwhich represented the total number of the MS/MS spectra for all peptides attributed to a detected protein. The\nrelative quantitative concentration of each protein identified in C-IPC or C-IF was assessed by calculating the\ntotal spectral counting (SpC) of all peptides matched to a specific protein, and then normalized for the\nmolecular weight (Mw) of each protein19, 71, 72. The SpC of each identified protein was normalized to the\nprotein mass and expressed as the normalized protein content (% total protein) using the following equation\n(4):\n(𝑆𝑝𝐶/𝑀 ) \n𝑁𝑝𝑆𝑝𝐶𝑘 = { 𝑤 𝑘 } × 100 (4)\n∑𝑛 (𝑆𝑝𝐶/𝑀 ) \n𝑖=1 𝑤 𝑖\nwhere NpSpCk was the percentage of normalized spectral count for protein k, SpC was the spectral count\nidentified, and Mw was the molecular weight in kDa for protein k.”\n(2) The heatmaps, volcano plots and venn plots are generated using TBtools software [4]. The protein\nsummaries in Fig. 11 are generated using GraphPad Prism 8 software.\nRef: [4] TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant.\nThe related description is also added in Methods section as following:\n“Proteomic analysis of C-IPC and C-IF. The heatmaps, volcano plots, venn plots and protein summaries in\nthe manuscript were generated using the TBtools73 and GraphPad Prism 8 software.”\nQ. 11. The uptake data were normalized, but the normalization method should be described more clearly in\nthe Methods.\nA. 11. Thanks for your suggestion. The normalization method for cell uptake in Fig. 12g is added in Methods\nsection as following:\n“The effect of S100A8 protein on macrophage uptake. Afterwards, the resulting NPs@C-IPC with S100A8\nantibody pre-blocking (S100A8 antibody group) were incubated with RAW264.7 cells for 3 h. Finally, the\ncells were washed and collected for the cell uptake detection by flow cytometer. In comparison, the NPs@C-\nIPC without S100A8 antibody pre-blocking were set as control (Control group). The cell uptake result was\npresented as the percentage of macrophage uptake in S100A8 antibody group compared to Control group.”\nQ. 12. Although Figures 12F and 12G suggest that adding S100A8 antibody to the Hard@C-IPC particle\nreduces uptake, the observed reduction is modest (~20%). Given the presence of endogenous\nimmunoglobulins in the corona, the specificity of this effect should be interpreted cautiously.\nA. 12. Thanks for your comment. After searching the proteomics analysis data (Proteomics data file 2), we\nfind that only six kinds of immunoglobulins were identified in C-IPC from each group (Table a) and their\nrelative protein content are also limited (total proportion is approximately 3%). Hence, the immunoglobulins\nin C-IPC may have little influence on the macrophage uptake of NPs@C-IPC as shown in Fig. 12g. In addition,\nthe adsorbed S100A8 protein on the surface of nanoparticles is selectively pre-blocked using the S100A8\nmonoclonal antibody which has high specificity for protein binding. Therefore, we think that the comparation\nof the cell uptake of NPs@C-IPC before and after protein pre-blocking can specifically investigate the effect\nof S100A8 protein on the macrophage uptake.\nTable a. Immunoglobulins identified in C-IPC\n% total protein % total protein % total protein\nProtein IDs Protein names\n(Soft) (Semi) (Hard)\nG3V6G1 0.8236 1.118 0.7889\nImmunoglobulin\njoining chain\nImmunoglobulin\nB2RZB2 kappa light chain- 0.7733 0.6283 0.6044\nlike\nImmunoglobulin\nF1LPR6 heavy constant 0.3848 0.3828 0.4540\nepsilon\nSimilar to\nM0RCN6 immunoglobulin 0.4048 0.3040 0.2911\nkappa-chain VK-1\nPolymeric\nF1M7H2 immunoglobulin 0.2817 0.3295 0.2722\nreceptor\nImmunoglobulin\nF1LM30 heavy constant 0.1004 0.1162 0.1073\nepsilon\nQ. 14. Hydrophobic particles are known to be more readily opsonized than hydrophilic ones due to enhanced\nbinding of complement proteins, immunoglobulins, and other serum components. The current mass\nspectrometry data do not clearly show the abundance of these opsonins or how they compare with what is\nknown in the literature. Additional analysis and interpretation are warranted.\nA. 14. Thanks for your comment. Protein corona formed in the blood circulation are reported to be mainly\ncomposed of immunoglobulins, lipoproteins, complement proteins and albumin [1-2]. However, the\nproteomics analysis (Proteomics data file 2) in this study shows that the opsonins and other serum proteins\nare quite few in C-IPC. Besides the immunoglobulins (discussions shown in Q12, Table a), there are only\nthree kinds of complement proteins and low-density lipoprotein identified, which both own low protein\ncontent, and the albumin is also limited (Table b). In comparison, as the protein corona generated in the GI\ntract, the C-IPC contained multiple proteins associated to digestion and metabolism of substances in the\nintestine, such as phospholipase A2, carboxypeptidase A1 and alpha-amylase (Supplementary Tab. 5). This\nfinding is also consistent with our previous study [3]. Therefore, we believe that the protein composition of\ncorona on nanoparticles is closely related to the physiological environment where they generate.\nRef: [1] Nanoparticle elasticity affects systemic circulation lifetime by modulating adsorption of\napolipoprotein A-I in corona formation. Nat Commun. 2022 Jul 16;13(1):4137.\n[2] Cholesterol modulates the physiological response to nanoparticles by changing the composition of protein\ncorona. Nat Nanotechnol. 2023 Sep;18(9):1067-1077.\n[3] Disease-specific protein corona formed in pathological intestine enhances the oral absorption of\nnanoparticles. Acta Pharm Sin B. 2023 Sep;13(9):3876-3891.\nTable b. Complement proteins, lipoproteins and albumin identified in C-IPC\n% total protein % total protein % total protein\nProtein IDs Protein names\n(Soft) (Semi) (Hard)\nM0RBF1 Complement C3 0.2439 0.2944 0.3622\nFA\nA0A8I6ANG0 complementation / 0.0273 0.0256\ngroup I\nFA\nA0A0G2K7F0 complementation 0.0220 / /\ngroup M\nLow-density\nlipoprotein\nP98158 0.0436 0.0476 0.0363\nreceptor-related\nprotein 2\nA0A8I5Y4N3 Albumin 0.0718 0.0533 0.0511\nQ. 15. Opsonization may alter particle interactions with many cell types, not just macrophages. Can the authors\nprovide additional data to support the claim that C-IPC particles specifically “target” macrophages in the\ncolon? It would be helpful to assess potential uptake by other immune and epithelial cell populations.\nA. 15. Thanks for your comment. The in vivo macrophage, epithelial cell and other immune cell targeting\nstudy in colon is supplemented as you suggest. As shown in Supplementary Fig. 12, the fluorescence intensity\nof internalized Hard@C-IPC by macrophages is significantly higher than that of epithelial cells and also\nobviously better than that of other immune cells including T cells, DC cells and neutrophils. Therefore, we\nbelieve the C-IPC adsorption specifically promotes NPs targeting towards macrophages in colon.\nSupplementary Fig. 12 Gating strategy to determine different cells in colon and the in vivo cell uptake of Hard@C-IPC\ndetected by flow cytometer (n=5).\nThe related results and experimental methods are added in the Results and Supplementary methods sections\nas following:\n“7. Impact of hydrophobicity-rigidity co-modulation on colon macrophage targeting and C-IPC\nformation. Further, the potential uptake of NPs@C-IPC by epithelium and other immune cells in colon was\nevaluated. It showed that the fluorescence intensity of internalized Hard@C-IPC by macrophages is\nsignificantly higher than that of epithelial cells and also obviously better than that of T cells, DC cells and\nneutrophils (Supplementary Fig. 12), indicating the C-IPC adsorption specifically promoted NPs target\ntowards macrophages in colon.”\n“Colon epithelium and immune cell targeting in vivo. The fluorescence labeled Hard were orally\nadministrated to colitis rats. After 6 h, the rats were sacrificed and the colon was collected and washed with\nsaline solution. The collected colon was grinded on an ice bath and the colon cell suspensions were collected.\nTo label the different cells in colon, the cell suspensions were stained with anti-Alpi antibody, anti-F4/80\nantibody, anti-CD3 antibody, anti-CD11c antibody and anti-Ly6G antibody separately at 4 ℃ for 1 h.\nAfterwards, cells were washed with PBS solution and detected by flow cytometry.”\nReviewer #2 (Remarks to the Author):\nThis manuscript by Wu et al., describes the dual modulation of an orally delivered nanoparticle to treat colitis.\nSpecifically, the authors altered two physicochemical properties at the same time (hydrophobicity and rigidity)\nand investigated their impact on macrophage targeting for the purpose of colitis treatment. Their results\nsuggest that high hydrophobicity alongside with higher rigidity resulted in not only higher amount of protein\ncorona adsorption, but also in a higher proportion of macrophage targeting proteins. Although modulating\nphysicochemical NP properties to manipulate disease-specific protein corona formation is not novel by itself,\nrationally modulating two physicochemical properties of NPs to tune protein corona formation and disease\nspecific targeting does distinguish this work from others. Once comments below are addressed, this would be\nan article that would be of broad interest.\nComments are as follows:\nQ. 1. Figure 1: “Philic” not Phillc, targeting proteins “upregulated”.\nA. 1. We are sorry for the mistakes. The errors in Fig. 1 have been corrected in the revised manuscript.\nFig. 1\nQ. 2. Line 57: the authors mention that most studies have focused on singular physicochemical property\nmodulation of nanocarriers which cannot sufficiently promote the functional protein adsorption to obtain the\ndesired protein corona. Could they please provide references from recent studies?\nA. 2. Thanks for your comment. Regulating the physicochemical properties of nanocarriers to modulate the\nprotein corona formation have been reported to improve delivery efficiency. For example, nanoparticles with\nintermediate elasticity (75-700 kPa) can preferentially adsorb the apolipoprotein A-I in the corona formation,\nexhibiting longer systemic circulation lifetime [1]. Bovine serum albumin (BSA) nanoparticles with higher\nhydrophobicity show better macrophage uptake due to the upregulated opsonins in protein corona [2]. Carbon\ndots with high zeta-potential (>+30 mV) and charge density (>2 μmol/mg) are highly internalized by the\nmacrophages which is strongly correlated with protein corona enriched in vitronectin, fibulin, fetuin,\nadiponectin and alpha-glycoprotein [3]. Nanovesicles with an amino/hydroxyl ratio of approximately 0.4 are\nfound to efficiently suppress immunoglobulin adsorption which results in prolonged circulation, and\nmeanwhile adsorb abundant tumor distinctive proteins to selectively internalized by tumor cells [4]. These\nstudies regulate and optimize the protein corona formation via single modulation of rigidity, hydrophobicity,\nsurface charge or surface chemistry. We believe if combined with other property modulation, the corona\nformation may be governed more subtly for precise drug delivery.\nRef: [1] Nanoparticle elasticity affects systemic circulation lifetime by modulating adsorption of\napolipoprotein A-I in corona formation. Nat Commun. 2022 Jul 16;13(1):4137.\n[2] Protein Nanoparticle Charge and Hydrophobicity Govern Protein Corona and Macrophage Uptake. ACS\nAppl Mater Interfaces. 2020 Oct 28;12(43):48284-48295.\n[3] Surface charge influences protein corona, cell uptake and biological effects of carbon dots. Nanoscale.\n[4] Regulating protein corona on nanovesicles by glycosylated polyhydroxy polymer modification for efficient\ndrug delivery. Nat Commun. 2024 Feb 7;15(1):1159.\nThe related references are added in the revised manuscript as following:\n“Introduction. Thus far, most studies have focused on singular physicochemical property modulation of\nnanocarriers10, 19-21, which cannot sufficiently promote the functional protein adsorption to obtain the desired\nprotein corona.”\nQ. 3. Line 63: macrophages were mentioned for the first time, but no sufficient background was provided\nregarding why macrophages are promising therapeutic targets for colitis treatment. What is the role of\nmacrophages in colitis pathogenesis? Similarly in the abstract, please provide a brief rationale for targeting\nmacrophages. A brief discussion on the importance of macrophages seems to occur around line 155, but not\nthe introduction.\nA. 3. Thanks for your suggestion. Macrophages are one of the most abundant leukocyte cell types found in\nthe luminal gastrointestinal mucosa. They are key effector cells of the innate immune system, vital for\nintestinal mucosal homeostasis, yet dysregulated macrophages are a central feature of inflammatory bowel\ndisease (IBD) including colitis [1]. Macrophages accumulate in vast numbers in active colitis, being potent\nproducers of pro-inflammatory mediators, such as reactive oxygen (ROS), TNF-α, IL-1β, IL-6 and IL-23.\nConcurrently, intestinal macrophages prevent excessive inflammation in response to harmless commensal\nmicroorganisms and promote tolerance by facilitating the expansion of antigen-specific CD4+CD25+ regulatory\nT (Treg) cells, mainly via the production of IL-10 [2]. Given that macrophages can orchestrate inflammation\nresolution and tissue repair, they are seen as attractive therapeutic targets in colitis [3]. Based on this,\nmacrophage targeting drug delivery systems have been developed to improve the colitis therapy via\nsuppressing inflammation and restoring intestinal homeostasis [4-7]. Therefore, targeted delivery of\ntherapeutics to colon macrophages represents a promising strategy for colitis treatment.\nRef: [1] Human Intestinal Macrophages Are Involved in the Pathology of Both Ulcerative Colitis and Crohn\nDisease. Inflamm Bowel Dis. 2021 Oct 18;27(10):1641-1652.\n[2] Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD. Nat Rev\nGastroenterol Hepatol. 2019 Sep;16(9):531-543.\n[3] Macrophages in intestinal homeostasis and inflammatory bowel disease. Nat Rev Gastroenterol Hepatol.\n[4] Orally deliverable sequence-targeted astaxanthin nanoparticles for colitis alleviation. Biomaterials. 2023\nFeb;293:121976.\n[5] Macrophage-Derived Extracellular Vesicles-Coated Palladium Nanoformulations Modulate Inflammatory\nand Immune Homeostasis for Targeting Therapy of Ulcerative Colitis. Adv Sci (Weinh). 2023\nNov;10(33):e2304002.\n[6] Mulberry Leaf Lipid Nanoparticles: a Naturally Targeted CRISPR/Cas9 Oral Delivery Platform for\nAlleviation of Colon Diseases. Small. 2024 Jun;20(25):e2307247.\n[7] Oral microsphere formulation of M2 macrophage-mimetic Janus nanomotor for targeted therapy of\nulcerative colitis. Sci Adv. 2024 Jun 28;10(26):eado6798.\nThe descriptions about the role of macrophages in colitis pathogenesis and macrophage targeted drug delivery\nhave been added in the Abstract and Introduction section as following:\n“Abstract. Given that macrophages can orchestrate inflammation resolution and mucosa repair, they are\nregarded as promising therapeutic targets in colitis.”\n“Introduction. Macrophages are key effector cells of the innate immune system for intestinal mucosal\nhomeostasis, yet dysregulated macrophages are a central feature of inflammatory bowel disease (IBD)\nincluding colitis22. They accumulate in vast numbers in active colitis, being potent producers of pro-\ninflammatory mediators, such as reactive oxygen (ROS), TNF-α, IL-1β, IL-6. Concurrently, intestinal\nmacrophages prevent excessive inflammation by facilitating the expansion of regulatory T (Treg) cells, mainly\nvia the production of IL-1023. Given that macrophages can orchestrate inflammation resolution and mucosa\nrepair, they are regarded as attractive therapeutic targets in colitis24. Several macrophage targeting drug\ndelivery systems have been developed to improve the colitis therapy via suppressing inflammation and\nrestoring intestinal homeostasis25-28. Therefore, targeted delivery of therapeutics to colon macrophages\nrepresents a promising strategy for colitis treatment.”\nQ. 4. Figure 2: The use of TEM, which images dehydrated samples, to complement DLS, which measures\nhydrated samples size is appropriate. However, since TEM requires dehydrated samples, the drying process\nmay cause potential particle shrinkage. And this affects their size measurements. Could the authors discuss\nthis limitation and why they did not use cryo-TEM instead?\nA. 4. Thanks for your comment. The drying process of NPs for TEM detection may cause potential particle\nshrinkage which influences the particle size measurement. To avoid this limitation, the cryo-TEM images of\nNPs are further detected as you suggest. As shown in Fig. 2b, NPs with different hydrophobicity possess the\nmorphologies of spherical shape and particle size approximately 115 nm, which is lower than the\nhydrodynamic diameter (approximately 165 nm) measured by dynamic light scattering (DLS). Similar\nphenomenon observed for NPs with different rigidity, and they all show spherical shape with size about 115\nnm (Fig 7b) which are also decreased compared with the hydrodynamic diameters (150 ~ 160 nm).\nFig. 2b Particle size distribution of NPs measured by Malvern Zetasizer and the representative images detected by cryo-TEM.\nScale bar=50 nm.\nFig. 7b Particle size distribution of NPs and the representative cryo-TEM images. Scale bar=50 nm.\nThe related results and methods are added in the Results and Methods sections as following:\n“1. Preparation and characterization of NPs with varied hydrophobicity. All NPs were similar in\nhydrodynamic diameter approximating 165 nm measured by dynamic light scattering (DLS) (Fig. 2b,\nSupplementary Tab. 1). The cryo-TEM images (Fig. 2b) showed that NPs possessed the morphologies of\nspherical shape and particle size approximately 115 nm, which is lower than the hydrodynamic diameters.\n5. Preparation and characterization of high hydrophobic NPs with varied rigidity. The hydrodynamic\ndiameters of Soft, Semi and Hard were uniformly distributed approximately ranging from 150~160 nm, with\nsimilar zeta potential (Fig. 7b, Supplementary Tab. 3). The cryo-TEM images also demonstrated the NPs all\nowned spherical shape with size about 115 nm, and the empty space inside NPs full of water layer was\nobviously observed in Soft (Fig. 7b).”\n“Preparation and characterization of NPs with varied hydrophobicity. The morphological characteristics\nof the NPs were examined by cryogenic transmission electron microscopy (cryo-TEM) using a Titan Krios\nG4 instrument (Thermo Fisher Scientific). For sample preparation, a droplet of the NPs suspension was\napplied to a glow-discharged, carbon-coated copper grid (300 mesh, R2/1, Quantifoil Micro Tools). Blotting\nwas conducted for 5 seconds under conditions of 4 °C and 100% humidity employing a Vitrobot Mark IV\n(Thermo Fisher Scientific). Immediate vitrification was achieved by rapidly plunging the grid into liquid\nethane, after which the vitrified sample was stored in liquid nitrogen at 90 K. Subsequently, the grid was\ntransferred into the microscope, which was operated at an accelerating voltage of 300 kV. Image acquisition\nwas performed with a Falcon 4i detector at a nominal magnification of 81,000×.\nPreparation and characterization of NPs with varied rigidity. The hydrodynamic diameter, PDI, zeta\npotential and cryo-TEM images of NPs with varied rigidity were also measured.”\nQ. 5. Please rationalize and discuss the limitations of using of Coomassie blue and Rose-Bengal, which are\nindirect methods for measuring hydrophobicity.\nA. 5. Thanks for your comment. Coomassie blue and Rose-Bengal methods are applied to rank the relative\nhydrophobicity of nanoparticles by determining the relative affinity between the tested nanoparticles and\nspecific hydrophobic probe molecule. To directly verify the hydrophobicity of nanoparticles in this study, the\ncontact angle of NPs with different PEGylation is measured [1]. As shown in Fig 2e, the contact angles\nincrease progressively from average 37.1° for Philic to 45.6° for Medium and 60.5° for Phobic. It is consistent\nwith the results of Coomassie blue and Rose-Bengal assays that the hydrophobicity of NPs enhances along\nwith the decreased PEG modification.\nRef: [1] Harnessing Surface Hydrophilicity of Inhalable Nanoparticles for Precision Delivery of Glucagon-\nlike Peptide-1 Receptor Agonists or Anti-Asthmatic Therapeutics. ACS Nano. 2025 Jun 24;19(24):22357-\n22375.\nFig. 2e Contact angle assay of NPs (n=3).\nBesides, the related result and method are added in the Results and Methods sections as following:\n“1. Preparation and characterization of NPs with varied hydrophobicity. Further, to directly measure the\nhydrophobicity, the contact angles of NPs were detected35. As shown in Fig 2e, the contact angles increased\nprogressively from average 37.1° for Philic to 45.6° for Medium and 60.5° for Phobic. Taken together, these\nresults suggested that NPs with increased hydrophobicity were successfully prepared (Fig. 2f).”\n“Hydrophobicity measurement. The NPs suspensions were vacuum freeze-dried and compressed into tablets,\nthen adhered to glass slides. The contact angles of NPs were measured using a contact angle measuring\ninstrument (OCA50, DataPhysics, Germany). Quantitative analysis of contact angle was performed using\nImageJ software.”\nQ. 6. Line 124: appears to be referencing Figure 3 instead? Please review the manuscript carefully for\ngrammatical and correct figure referencing.\nA. 6. We are sorry for the writing error. The sentence in line 124 “..., exerting better effect than Medium-Bud\ngroup (Fig. 4h-m)” has been corrected to “..., exerting better effect than Philic-Bud and Medium-Bud group\n(Fig. 3g-l)”. In addition, we have carefully reviewed and revised the manuscript for grammatical description\nand figure referencing.\nQ. 7. Did the authors run a power analysis to determine the sample numbers in Figure 3?\nA. 7. Thanks for your comment. Power analysis is performed to determine the sample number for all\nexperiments presented in Figure 3. These analyses, conducted with a significance level of α = 0.05, are based\non pilot data or expected effect sizes for each specific metric, including colon length, spleen index, plasma\nlevels of TNF-α, IL-6, and IL-1β, ROS, as well as the frequencies of Treg cells and macrophages in both\nspleen and colon. The results indicate that to meet the requirements of the power value of 0.8 and above [1-\n2], the minimum total sample number required ranging from 3 to 4 across these diverse endpoints. To ensure\nrobust and consistent experimental conditions and guarantee sufficient statistical power for all assays, a\nuniform sample number of n=5 per group is employed for the entire study. This sample number exceeds the\ncalculated minimum requirement for every individual parameter and yielded a post-hoc statistical power\nranging from 0.912 to 0.990, thus confirming the study is adequately powered to detect the effects of interest.\nRef: [1] The experimental design of postmortem studies: the effect size and statistical power. Forensic Sci\nMed Pathol. 2016 Sep;12(3):343-9.\n[2] Power Analysis for Null Hypothesis Significance Testing. Clin Spine Surg. 2021 Mar 1;34(2):63-65.\nThe descriptions are added in the Methods sections as following:\n“Colitis therapy in vivo. The power analysis was conducted to verify that the sample number was set\nreasonably.”\nQ. 8. Figure 3: Please state how the ELISA results were normalized.\nA. 8. Thanks for your comment. The inflammatory cytokine levels of different groups are quantitatively\nmeasured according to the protocol of the ELISA kit. The inflammatory cytokine concentrations of the colon\nsuspensions are calculated according to the standard curves, and then the protein concentrations of colon\nsuspensions are measured using the BCA protein assay kit. Finally, the inflammatory cytokine concentrations\nmeasured by the ELISA kit are normalized by calibrating with the protein concentrations of different groups.\nMore descriptions about the ELISA assay are added in the Method section as following:\n“Colitis therapy in vivo. In addition, the inflammatory cytokine levels of different groups were quantitatively\nmeasured according to the protocol of the ELISA kit (Beyotime Biotechnology, China). The inflammatory\ncytokine concentrations of the colon suspensions were calculated according to the standard curves, and then\nthe protein concentrations of colon suspensions were measured using the BCA protein assay kit. Finally, the\ninflammatory cytokine concentrations measured by the ELISA kit were normalized by calibrating with the\nprotein concentrations of different groups.”\nQ. 9. Figure 6: Please briefly state the rationale for the differing macrophage ratios and their physiological\nrelevance.\nA. 9. Thanks for your comment. After literature searching, we have not found research reporting the\nquantitative ratio of colon epithelial cells to macrophages. It speculates that colon macrophage activity is\nclosely related to the physiological and pathological states such as inflammation, thus the number of\nmacrophages may change dynamically, making it difficult to quantify. The purpose of Fig. 6g-k is to\ninvestigate when macrophages and epithelial cells co-exist, whether C-IPC can preferentially promote the\nuptake of nanoparticles by macrophages. Therefore, two types of cells with the same quantity (1:1) are firstly\nco-cultured for the cell uptake study. The results are expressed as the percentage of RAW264.7 macrophage\nuptake compared to Caco-2 cell uptake, and we find that C-IPC adsorption can increase the macrophage uptake\nof all nanoparticles with varied hydrophobicity (Fig. 6i). Furthermore, when the proportion of epithelial cells\ngradually increases to be five and ten times of macrophages, that is in an environment with large number of\nepithelial cells but small number of macrophages, it is found that C-IPC can still increase the proportion of\nnanoparticles taken up by macrophages (Fig. 6j, k). Based on this, we believe that C-IPC can selectively\ncontribute to the recognition and internalization of nanoparticles by macrophages.\nFig. 6 g Schematic illustration of cell uptake study of NPs and NPs@C-IPC on Caco-2 and RAW264.7 co-cultured cells. h\nGating strategy to determine the Caco-2 cells (DiI) and RAW264.7 cells (DiD) with different ratios. i-k Cell uptake of\nNPs@C-IPC on Caco-2 and RAW264.7 co-cultured cells with different ratios detected by flow cytometer (n=3). Results were\nexpressed as the percentage of RAW264.7 macrophage uptake compared to Caco-2 cell uptake.\nQ. 10. Figure 7: what model was used to calculate their NP Young’s moduli?\nA. 10. Thanks for your comment. In this study, the Young’s modulus of nanoparticles is calculated using the\nJKR model in Igor Pro software. This information is also added in the Method section as following:\n“Young’s modulus measurement. The Young’s modulus of NPs was then analyzed and calculated using the\nJKR model in the Igor Pro software.”\nQ. 11. Please expand on the results and discussion section to better contextualize the results to recent literature.\nFor example, section 9 is a bit confusing to follow and does not connect results to potential endocytic pathways.\nAdditionally, there is a growing body of literature describing how rigidity enhances certain\nendocytic/mechanobiology pathways to increase NP uptake in macrophages. Connecting their results to recent\nliterature and contextualizing NP uptake mechanisms would add to the impact of this manuscript.\nA. 11. Thanks for your suggestion. There are studies reporting that nanoparticle rigidity can influence its\nendocytic pathways in macrophages [1-2]. Therefore, the influence of C-IPC and rigidity on the macrophage\nendocytic pathways of nanoparticles in this study is supplemented. As shown in Supplementary Fig. 21a, the\nNPs with varied rigidity can be internalized by macrophages through the clathrin-mediated endocytosis,\ncaveolin-mediated endocytosis and micropinocytosis. This result is different from the literatures that the\nRAW264.7 uptake of silica nanocapsules (SNCs) with different rigidity (Young's modulus ranging from\n0.56~1184 Mpa) relies on micropinocytosis and caveolin-mediated endocytosis [1], and the phagocytosis may\nbe the main form of BMDMs uptake of lipid-polymer nanoparticles (LPNPs) with varied rigidity (no Young's\nmodulus data) [2]. We speculate the materials and the Young's modulus may both influence the endocytic\npathways of nanoparticles. Besides, after C-IPC adsorption, the macrophage uptake of NPs@C-IPC is also\ninvolved with the clathrin-pathway, caveolin-pathway and micropinocytosis (Supplementary Fig. 21b). It\nindicates that the increased rigidity and C-IPC adsorption will not change the endocytic pathways of NPs in\nthis study, so the macrophage uptake improvement of Hard@C-IPC are due to the involvement of adsorbed\nfunctional proteins.\nRef: [1] Nanoparticle elasticity regulates phagocytosis and cancer cell uptake. Sci Adv. 2020 Apr\n17;6(16):eaaz4316.\n[2] Modulating Lipid-Polymer Nanoparticles' Physicochemical Properties to Alter Macrophage Uptake. ACS\nBiomater Sci Eng. 2024 May 13;10(5):2911-2924.\nSupplementary Fig. 21 a,b Normalized cell uptake of NPs and NPs@C-IPC using different endocytic pathway inhibitors\nin RAW264.7 cells (n=5).\nThe related discussions and experimental methods are added in the Discussions section and Supplementary\nmethods as following:\n“Discussions. There were studies reporting that the rigidity may influence the endocytic pathways of\nnanoparticles to increase their macrophage uptake68, 69. However, in our study NPs with different rigidity could\nbe internalized by macrophages through clathrin-mediated endocytosis, caveolin-mediated endocytosis and\nmicropinocytosis (Supplementary Fig. 21a). Besides, after C-IPC adsorption, the macrophage uptake of the\nNPs@C-IPC was also involved with these three pathways (Supplementary Fig. 21b). It indicated the increased\nrigidity and C-IPC adsorption will not change the endocytic pathways, thus the improved macrophage uptake\nof Hard@C-IPC relied on the involvement of adsorbed functional proteins.”\n“Endocytic pathways of NPs and NPs@C-IPC in macrophages. RAW264.7 cells were cultured for 1 day.\nThe endocytosis inhibitors including 5-(Nethyl-N-isopropyl) amiloride (EIPA, 1 μg/mL), methyl-β-\ncyclodextrin (m-β-CD, 15 μg/mL) and chlorpromazine (CPZ, 0.3 mg/mL) were pre-incubated with cells for\n30 min to block the micropinocytosis, caveolin-mediated endocytosis and clathrin-mediated endocytosis,\nrespectively. Afterwards, the fluorescence labeled NPs and NPs@C-IPC were incubated with cells for 3 h (the\ninhibitors were maintained). Finally, the macrophage uptake was detected by flow cytometer. Cells without\ninhibitor treatment were set as control group (Control) and the normalized cell uptake was calculated\ncompared with Control.”\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe study by Wu et al. demonstrates that modulating the surface physicochemical properties of nanocarriers\nallows precise control over the quantity and composition of the colitis-specific intestinal protein corona (C-\nIPC) formed on oral nanoparticles (NPs), thereby improving their targeting toward colonic macrophages and\nenhancing therapeutic outcomes in colitis. Given the promising experimental results, it is recommended that\nthe authors undertake major revisions prior to being accepted.:\nQ. 1. At the beginning of the Results and Discussion section, the authors should provide a clear description of\nthe materials used to prepare the nanoparticles, rather than focusing solely on surface modifications.\nFurthermore, is it feasible for NPs made of different materials (e.g., metal salts, metal–organic frameworks,\nliposomes, polymers, etc.) to achieve comparable effects following similar modifications?\nA. 1. Thanks for your suggestions.\n(1) Description about the nanoparticle composition is added at the beginning of Results section as following:\n“1. Preparation and characterization of NPs with varied hydrophobicity. In this study, the soyabean\nlecithin and cholesterol were lipid component to form the lipid-based NPs. To modulate the surface\nhydrophobicity, different ratios of DSPE-PEG were added to prepare NPs with varied polyethylene glycol\n2000\n(PEG) modification degree.”\n(2) In this study, the NPs made of soyabean lecithin and cholesterol own lipid bilayer and inner water layer,\nbelonging to liposomes. Additionally, to investigate whether manipulating the protein adsorption via similar\nsurface PEG modification is applicable for nanoparticles made of different materials, the poly (lactic-co-\nglycolic acid) nanoparticles (PLGA-NPs) composed of polymers and gold nanoparticles (Au-NPs) composed\nof metal material are prepared. First, the PLGA-NPs or Au-NPs with decreased PEG modification are\nabbreviated as P-Philic, P-Medium, P-Phobic or Au-Philic, Au-Medium, Au-Phobic, respectively. As shown\nin Supplementary Tab. 6, PLGA-NPs and Au-NPs with different PEGylation all own similar hydrodynamic\ndiameters (110~125 nm), but zeta potential gradually increased with the decreased PEG on the surface. Similar\nphenomenon is also observed in NPs made of lipid component in this study (Supplementary Tab. 1). Second,\nthe CBB assay shows that with decreased PEGylation can both enhance the wavelength of the maximum\nabsorption of CBB from Philic, Medium to Phobic (Supplementary Fig. 20a, c), demonstrating the increased\nsurface hydrophobicity. Finally, the C-IPC quantification results show the increased surface hydrophobicity\ncan also enhance the protein adsorption amount on the PLGA-NPs and Au-NPs (Supplementary Fig. 20b, d).\nTherefore, we believe the hydrophobicity modulation strategy in this study is feasible for different types of\nnanocarriers to manipulate the protein corona adsorption.\nSupplementary Tab. 6 Characterization of PLGA-NPs and Au-NPs with different PEGylation (n=3). P for PLGA-NPs, Au\nfor Au-NPs.\nSize (nm) PDI ZP (mV)\nP-Philic 1 0 -\n2 . 2\nP-Medium 1 0 -\n4 2 7\n2 . 2\n. 6 .\nP-Phobic 1 0 -\n1 2 2\n7 2 4\n2 . 1\n. 2 .\nAu-Philic 1 0 -\n5 1 6\n0 9 8\n11.0 0.0.15 01.6\n. 3 .\nAu-Medium 1 0 -\n0 2 8\n2 5 6\n01.8 0.0.13 01.6\n. 8 .\nAu-Phobic 1 0 -\n7 2 3\n0 9 8\n32.5 0.0.17 11.0\n. 8 .\n2 2 1\n2 4 0\n1.3 0.023 1.4\n. 8 .\n2 1 2\n1.2 0.024 2.3\n2.0 0.041 2.1\nSupplementary Fig. 20 a, c Changes in the wavelength of the maximum absorption of CBB after incubating with PLGA-\nNPs or Au-NPs (n=3). b, d Quantification of protein corona on PLGA-NPs@C-IPC or Au-NPs@C-IPC (n=3). P for PLGA-\nNPs, Au for Au-NPs.\nThe related discussions, experimental methods and results are added in the Discussions and Supplementary\nmethods sections as following:\n“Discussions. In this study, the NPs made of soyabean lecithin and cholesterol own lipid bilayer and inner\nwater layer, belonging to liposomes. Additionally, we prepared NPs made of different materials including poly\n(lactic-co-glycolic acid) nanoparticles (PLGA-NPs)66 composed of polymers and gold nanoparticles (Au-\nNPs)67 composed of metal material (Supplementary Tab. 6). It showed that by altering the PEGylation, the\nincreased surface hydrophobicity can also enhance the protein adsorption amount on the PLGA-NPs and Au-\nNPs (Supplementary Fig. 20). Therefore, we believed the hydrophobicity modulation strategy in this study\nwas feasible for different types of nanocarriers to manipulate the protein corona adsorption.”\n“Preparation and characterization of PLGA-NPs and Au-NPs with different PEGylation. PLGA-NPs\nwere prepared via the self-assembly nanoprecipitation method. PLGA and cholesterol as the lipid component\nof NPs were mixed with DSPE-PEG at different mass ratios (PLGA: cholesterol: DSPE-\nDMSO. The mixture suspension was added to the aqueous phase dropwise under stirring at 1000 rpm. The\nobtained PLGA-NPs were washed thrice using ultrafiltration centrifuge tube (100 kDa, Millipore) to remove\nthe DMSO. Subsequently, PLGA-NPs were re-dispersed in PBS for the following experiments.\nThiolated polyethylene glycol (mPEG -SH) was covalently grafted to the surface of the Au-NPs for\nwith mPEG -SH at different molar ratios (Au: mPEG -SH=10:8, 10:4 or 10:1, equal to PEGylation of\n2000 2000\n80%, 40% and 10%). The mixture was stirred for 24 h at room temperature. After that, the excess mPEG -\nPEGylation were obtained. For characterization, hydrodynamic diameter, PDI and zeta potential of PLGA-\nNPs and Au-NPs with different PEGylation were measured by Zetasizer Nano ZS90 (Malvern Instruments\nLtd., UK).”\nQ. 2. Why was there a high mortality rate in mice treated with Philic-Bud, whereas only minimal mortality\nwas observed in the saline-treated negative control group?\nA. 2. Thanks for your comment. The humane endpoints in this study are set as body weight of rats reduced to\nmore than 20% of the initial weight, according to the “Guideline of assessment for humane endpoints in animal\nexperiment (RB/T 173-2018, China)”. In the animal experiments, colitis rats with body weight loss over 20%\nare also regarded as dead, then they are removed from the experiments and euthanized via intraperitoneal\ninjection of pentobarbital. Hence, the mortality rate of Saline and Philic-Bud groups also contain the colitis\nrats with body weight loss over 20%, and the survival rate curve result may not fully reflect the actual mortality.\nWe indeed observe the colitis rat models established by TNBS-ethanol solution show certain risk of death. A\nreview article about the TNBS-induced colitis animal model mentioned that death may happen, resulting\nmainly from an excessive inflammatory reaction [1]. In our initial study, the TNBS-induced colitis rats in Fig.\n3 seem unexpectedly lethal especially for rats with insufficient therapeutic effect such as Saline and Philic-\nBud groups. Therefore, we repeat the in vivo experiments and the results are shown in the revised Fig. 3 and\nFig. 4. TNBS dose at 80 mg/kg, the same as last time, is applied for the establishment of colitis rat models,\nand there is no death occurrence for all rat groups. We suppose that the varied physical condition of SD rats\nmay be the possible reason. The previous batch of rats may be more sensitive to the TNBS-ethanol treatment\nfor damaging colon mucosa and triggering immunoreaction, thus the rats without effective therapy are more\neasily to die due to the excessive inflammatory reaction [1].\nRef: [1] The TNBS-induced colitis animal model: An overview. Ann Med Surg (Lond). 2016 Aug 19;11:9-15.\nFig. 3 Drug-loaded NPs with varied hydrophobicity for colitis treatment. a Schematic diagram of the therapy of colitis\nrats by oral administration. Sprague-Dawley (SD) rats were treated with TNBS-ethanol solution by clyster to establish the\ncolitis rat model (Day 0). After 24 h (Day 1), the colitis rats were treated with free drug or different drug-loaded NPs once a\nday for continuous 7 days by oral administration. Colitis rats treated with saline (Saline) and Healthy rats (Healthy) were set\nas control groups. At Day 8, rats were sacrificed and evaluated. b Body weight of rats was recorded daily from Day 0 to Day\n7 and the body weight change (percentage of the initial body weight in Day 0) was calculated (n=5). c Rats were sacrificed in\nDay 8, then the colons were collected and imaged. d Length of the collected colons was measured (n=5). e Spleen index was\ncalculated (n=5). f The ROS level of colon was measured by flow cytometry using ROS Assay Kit (n=5). g-i\nImmunohistochemical analysis of the TNF-α, IL-1β and IL-6 level in colon. Scale bar= 100 μm. j-l Quantification of the TNF-\nα, IL-1β and IL-6 level in colon using ELISA assay (n=5).\nFig. 4 The immune cells in colitis rats after oral treatment. a, b Measurement of the proportion of Treg cells in colon and\nspleen by flow cytometry (n=5). c Immunohistochemical analysis of the IL-10 level in colon. Scale bar= 100 μm. d, e\nMeasurement of the ratio of M1 macrophages to M2 macrophages in colon and spleen by flow cytometry (n=5).\nQ. 3. Variations in hydrophobic modification may influence drug loading capacity. Have the authors quantified\nthe drug loading efficiencies of NPs with different hydrophobicities? How can they ensure that the differences\nin therapeutic efficacy are not attributable to variations in drug loading?\nA. 3. Thanks for your comment. The encapsulation efficiency (EE) and drug loading (DL) of drug-loaded NPs\n(NPs-Bud) with different hydrophobicity are provided. As shown in Supplementary Tab. 2, the Philic-Bud,\nMedium-Bud and Phobic-Bud all own a high encapsulation efficiency to approximately 80%. However, the\nPhobic-Bud have the highest drug loading, mainly because the Philic-Bud and Medium-Bud contain higher\namount of DSPE-PEG to increase the total weight of NPs-Bud. In addition, the NPs-Bud with different\n2000\nrigidity are also provided, and they all have similar encapsulation efficiency and drug loading (Supplementary\nTab. 4). In the therapeutic efficacy experiments, the administration doses of NPs-Bud are calculated according\nto their encapsulation efficiency to ensure the therapeutic dose of all treating groups equal to the dose of Bud\nat 0.6 mg/kg/day. Therefore, the differences in the therapeutic efficacy for different NPs-Bud are not\nattributable to the variations in drug loading.\nSupplementary Tab. 2 Characterization of NPs-Bud with varied hydrophobicity (n=3). Encapsulation efficiency (EE), drug\nloading (DL).\nSize (nm) PDI ZP (mV) EE (%) DL (μg/mg)\nPhilic-Bud 1 0 - 80.2 ±0.4 1\n5 . 3 .\nMedium-Bud 1 0 - 7 1\n2 2 7 4\n5 . 3 9 .\n. 9 . 8\nPhobic-Bud 1 0 - 8 2\n1 2 2 . 9\n1 1 1\n4 . 3 0 .\n. 5 . 1 3\n0.03\n8 2 1 . 8\n1 1 1\nSupplementary Tab. 4 Characterization of NPs-Bud with varied rigidity (n=3).\n2.0 0.021 1.0\n. 3 . 6 0\n0.8 0.04\nSize 1(nm) PD1I ZP (1mV) EE ( %) DL (μ g/mg)\n1.5 0.041 0.5\n0.7 0.10\nSoft-Bud 1 0 - 7 2\n1.5 0.031 0.8\n4 . 3 9 .\nSemi-Bud 1 0 - 7 2\n9 2 0 . 9\n5 . 3 9 .\n. 5 . 4 4\nHard-Bud 1 0 - 7 2\n1 2 2 . 6\n1 1 1\n5 . 3 9 .\nThe related result, discussion and e.xperimental m7ethod are adde.d in the Resu1lts, Methods a1nd Supplementary\n0.3 0.01\n4 2 0 . 6\nmethods sections as following:\n1 1 1\n3.7 0.011 0.8\n. 3 . 7 3\n“2. Drug-loaded NPs with varied hydrophobic ity for colitis t herapy. The0 .d5r ug loading0 .p0r1o cess showed no\ninfluence on the particle size, PD1I and zeta pote1ntial of nanop1articles, and th ree NPs-Bud all owned a high\n1.9 0.011 0.2\nencapsulation efficiency to approximately 80% (Supplementary Tab. 2). The Phobic-Bud had the highest drug\n0.9 0.03\nloading (2.80 μg/mg), mainly because the Philic-Bud and Medium-Bud contained higher amount of DSPE-\n2.6 0.021 0.4\nPEG2000 to increase the total weight of formulation.\n6. Hydrophobicity-rigidity co-modulation for effective colitis therapy. The Phobic-Bud with increased\nrigidity (Soft-Bud, Semi-Bud and Hard-Bud) were also prepared, and three formulations also possessed\nsimilar encapsulation efficiency, drug loading (Supplementary Tab. 4).”\n“Preparation and characterization of drug-loaded nanoparticles. Budesonide-loaded NPs (NPs-Bud)\nwere prepared by adding the budesonide (0.75 mg) to the initial lipid component before the evaporation\nprocess, and then the NPs-Bud were centrifuged (6500 rpm, 1 h) using ultrafiltration centrifuge tube (100 kDa)\nto separate the free drug. The collected NPs-Bud were re-dispersed with PBS solution for further use.\nThe particle size, PDI and zeta potential of NPs-Bud were detected. For the encapsulation efficiency (EE)\nand drug loading (DL) measurement, freshly prepared NPs-Bud suspensions were centrifuged to separate the\nfree drug. Then the amount of drug encapsulated in the NPs-Bud was quantified using high performance liquid\nchromatography (HPLC, Agilent 1260 Infinity II, USA). The encapsulation efficiency (EE) was calculated\nusing the following equation (2):\namount of drug encapsulated in the NPs-Bud (μg)\nEE (%) = ×100%\ntotal amount of the added drug (μg)\nBesides, the dry mass of NPs-Bud was weighed after freeze-drying, and then the drug loading (DL) was\ncalculated using the following equation (3):\nweight of drug in NPs-Bud (μg)\nDL (%) =\ntotal weight of NPs-Bud (mg)\nQ. 4. Do the NPs undergo structural or physicochemical changes after gastric processing in mice? Specifically,\ndo the hydrophilic/hydrophobic modifications remain stable?\nA. 4. Thanks for your comment. We believe the NPs are stable in the gastric condition. First, the hydrodynamic\ndiameter and PDI of NPs show no significant change in the simulated gastric fluids (SGF) for 4 h\n(Supplementary Fig. 1a). Second, to further investigate whether the structural integrity and surface PEG\nmodification of NPs remain stable after gastric processing in vivo, the Förster resonance energy transfer (FRET)\nassay is applied [1-2]. Briefly, dual-labeled NPs (DiI@Phobic-FITC) are prepared by substituting the DSPE-\nPEG with fluorescein isothiocyanate labeled DSPE-PEG (DSPE-PEG -FITC). As shown in\n2000 2000 2000\nSupplementary Fig. 2, the FRET phenomenon of fluorescent molecule pairs (FITC as donor and DiI as\nacceptor) is observed for both DiI@Phobic-FITC before or after gastric processing in SD rat, verifying that\nthe structural integrity and stable hydrophilic/hydrophobic modification of NPs in the gastric environment in\nvivo.\nRef: [1] Airway epithelial-targeted nanoparticle reverses asthma in inhalation therapy. J Control Release. 2024\nMar;367:223-234.\n[2] Bioengineered Milk-derived Extracellular Vesicles Implementing High Drug Loading and Membrane\nIntegrity for Efficient Oral Drug Delivery. Asian J Pharm Sci. (2025): 101093.\nSupplementary Fig. 1 a Particle size and polydispersity index (PDI) change of NPs with increased hydrophobicity in the\nsimulated gastric fluids (SGF) for 4 h (n=3).\ny\nt is 10000\nn\ne 8000\nt DSPE-PEG -FITC\nn 2000\nI\ne 6000 DiI\nc\nn\ne 4000 DiI@Phobic-FITC (Before)\nc\ns DiI@Phobic-FITC (After)\ne 2000\nr\no\nu\n0\nl\nF\n500 550 600 650\nWavelength (nm)\nSupplementary Fig. 2 Fluorescence spectra of DSPE-PEG -FITC, DiI and DiI@Phobic-FITC before or after gastric\n2000\nprocessing in vivo at an excitation wavelength of 490 nm and emission wavelength of 490-650 nm.\nThe related results and method are added in the Results and Methods sections as following:\n“1. Preparation and characterization of NPs with varied hydrophobicity. All NPs could remain stable in\nthe simulated gastrointestinal (GI) condition without significant change of particle size and PDI\n(Supplementary Fig. 1). Besides, the Förster resonance energy transfer (FRET) assay using DSPE-PEG -\nhydrophilic/hydrophobic modification of NPs after gastric processing in vivo (Supplementary Fig. 2).”\n“Integrity of NPs after gastric processing in vivo using Förster resonance energy transfer (FRET) assay.\nDual-labeled NPs (DiI@Phobic-FITC) are prepared by substituting the DSPE-PEG with fluorescein\n2000\nisothiocyanate labeled DSPE-PEG (DSPE-PEG -FITC), and the FITC (donor) and DiI (acceptor) are\n2000 2000\nfluorescent molecule pairs. Fasted SD rat was anesthetized by isoflurane and the stomach was ligated at both\nsides, then the DiI@Phobic-FITC were injected into the stomach for 2 h. Afterwards, the gastric content and\nsuspensions were collected and centrifuged at 2000 × g for 10 minutes, and the DiI@Phobic-FITC in the\nsupernatant were collected using Sephadex G50 gel size-exclusion column. Finally, the fluorescence emission\nspectra (490~650 nm) of DSPE-PEG -FITC, DiI and DiI@Phobic-FITC before or after gastric processing\n2000\nin vivo were recorded using a microplate reader at an excitation wavelength of 460 nm.”\nQ. 5. Could fluorescent labeling interfere with the adsorption of C-IPC onto the NPs? It is likely that\nfluorescent labeling alters the surface hydrophilicity/hydrophobicity—could this in turn affect protein corona\nformation?\nA. 5. Thanks for your comment. The hydrophobic Coumarin 6 is encapsulated into the lipid bilayer of NPs for\nthe fluorescent labeling, and theoretically it will not affect the surface hydrophobicity. To verify this, the\ncontact angles of NPs with (+) or without (-) fluorescent labeling are measured for hydrophobicity evaluation.\nAs shown in Supplementary Fig. 5a, the contact angles of fluorescent labeled NPs are consistent with the NPs\nwithout fluorescent labeling, demonstrating that the fluorescent labeling will not alter the surface\nhydrophobicity. Further, the C-IPC adsorbed on NPs with or without fluorescent labeling are compared\n(Supplementary Fig. 5b, c). The SDS-PAGE analysis and protein quantification results both show that the\nfluorescent labeling will not interfere with the protein corona formation.\nSupplementary Fig. 5 a Contact angle assay of NPs with (+) or without (-) fluorescent labeling (n=3). b SDS-PAGE analysis\nof the C-IPC on the NPs with (+) or without (-) fluorescent labeling. c Quantification of the protein corona on the NPs with\n(+) or without (-) fluorescent labeling (n=3).\nThe related results and method are added in the Results and Methods sections as following:\n“4. Impact of C-IPC with enhanced protein amount on macrophage recognition and uptake. Besides,\nNPs with (+) or without (-) fluorescent labeling owned similar contact angle and protein adsorption\n(Supplementary Fig. 5), indicating the fluorescent labeling will not interfere with the hydrophobicity\nmodulation and C-IPC formation.”\n“Hydrophobicity measurement. The NPs suspensions were vacuum freeze-dried and compressed into tablets,\nthen adhered to glass slides. The contact angles of NPs were measured using a contact angle measuring\ninstrument (OCA50, DataPhysics, Germany). Quantitative analysis of contact angle was performed using\nImageJ software.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed each comment by this reviewer.", "reviewer_2": "The authors have adequately addressed reviewers comments in their revised manuscript.", "reviewer_4": "The authors had endeavored to address the relevant issues. The manuscript with its current status can be accepted for publication."}, "author_response": ""}]

Decision letter