Surface Charge-Modulated Biomimetic Core-Shell Hybrid Nanovesicles for Redox-Triggered Synergistic Cancer Therapy

Surface Charge-Modulated Biomimetic Core-Shell Hybrid Nanovesicles for Redox-Triggered Synergistic Cancer Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Surface Charge-Modulated Biomimetic Core-Shell Hybrid Nanovesicles for Redox-Triggered Synergistic Cancer Therapy Hyeon-Ji Oh, Dong-Yong Hong, Junseob Lee, Jieun Han, Chun Gwon Park, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8862874/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Despite the therapeutic potential of photodynamic therapy (PDT), its clinical efficacy is frequently limited by insufficient photosensitizer (PS) delivery and the strong antioxidant defense mechanisms of tumor cells. In this study, we developed surface charge-modulated biomimetic core-shell nanostructures by encapsulating PS-loaded poly(benzene-1,4-dithiol) (pBDT) nanoparticles within cell-derived nanovesicles (CNVs). To address the electrostatic barriers associated with tumor tissues, the CNV shell was functionalized with a controlled amount of cationic lipids (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP). This modification was designed to modulate surface electrostatic interactions at the tumor interface while maintaining membrane integrity and systemic safety. The resulting hybrid nanovesicles, termed PS/pBDT@cCNVs, integrate the redox-responsive properties of the pBDT core with the intrinsic biological functionalities of the CNV shell. This dual-modal strategy, which combines surface charge modulation with the intrinsic biological properties of CNVs, led to enhanced tumor accumulation and improved intratumoral distribution. Furthermore, the redox-active PS/pBDT core facilitated efficient intracellular ROS generation and glutathione (GSH) depletion, thereby amplifying the phototherapeutic impact. Both in vitro and in vivo evaluations confirmed that PS/pBDT@cCNVs achieved robust tumor ablation with negligible systemic toxicity. Collectively, this work presents a rationally designed, charge-tunable, redox-responsive hybrid nanoplatform that addresses key biological barriers in photodynamic cancer therapy. Photodynamic therapy (PDT) Biomimetic nanovesicles Cell-derived nanovesicles (CNVs) Surface charge modulation Tumor targeting drug delivery Redox-responsive polymer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Cancer remains one of the leading causes of mortality worldwide, and conventional treatments such as surgery, chemotherapy, and radiotherapy often suffer from non-specificity, systemic toxicity, and damage to healthy tissues [ 1 , 2 ]. These limitations have promoted the development of alternative strategies that can achieve accurate tumor targeting while minimizing side effects. Photodynamic therapy (PDT) has emerged as a promising minimally invasive modality, relying on the activation of photosensitizers (PSs) by light to generate reactive oxygen species (ROS) that induce localized tumor cell death [ 3 ]. PDT offers spatiotemporal control and the potential to trigger anti-tumor immune responses [ 4 – 6 ]. However, its clinical translation is hindered by the poor water solubility, instability, and limited tumor accumulation of many hydrophobic PSs [ 6 – 8 ]. Aggregation-induced quenching and rapid systemic clearance further reduce ROS generation efficiency, underscoring the critical need for advanced delivery systems that improve pharmacokinetics and tumor specificity [ 9 – 11 ]. Nanoparticle-based carriers have been widely explored to address these challenges by leveraging the enhanced permeability and retention (EPR) effect or active targeting mechanisms [ 12 – 15 ]. Among these, π-conjugated polymers can stably encapsulate hydrophobic PSs through strong π-π stacking, preventing aggregation and enhancing photodynamic activity [ 16 – 18 ]. To further impart biological targeting capabilities, cell-derived nanovesicles (CNVs) have attracted significant attention. Inheriting the complex membrane protein profile of their source cells, CNVs offer intrinsic biocompatibility, low immunogenicity, and specific homotypic tumor-homing properties [ 19 – 23 ]. Therefore, integrating the high loading capacity of pBDT cores with the biological recognition of CNV shells presents a compelling "core-shell" hybrid strategy. However, a critical barrier remains. The surface charge of natural CNVs is inherently negative. Although CNVs possess targeting ligands, their negative surface charge can induce electrostatic repulsion with the anionic glycocalyx of the tumor endothelium, potentially limiting early interactions at the tumor site [ 24 – 26 ]. Previous studies have reported that electrostatic interactions between cationic nanomaterials and anionic tumor-associated interfaces contribute to vascular association and transendothelial transport [ 29 ]. While cationic nanoparticles are known to interact favorably with anionic tumor tissues, excessive positive charge often leads to opsonization and systemic toxicity [ 27 , 28 ]. Thus, a strategic balance is required. We hypothesized that modulating the CNV surface with a minimal threshold of cationic lipids (e.g. 1,2-dioleoyl-3-trimethylammonium-propane; DOTAP) would enhance electrostatic interaction with the tumor endothelium without compromising the vesicle’s structural integrity or safety. In this study, we present a charge-tunable hybrid nanovesicle platform designed to enhance tumor delivery and photodynamic therapeutic performance (Fig. 1 ). The platform consists of a photosensitizer-loaded poly(benzene-1,4-dithiol) (pBDT) core that enables stable drug retention and a CNV shell minimally modified with cationic lipids to allow controlled modulation of interfacial surface charge. Through this charge engineering strategy, the hybrid nanovesicles promote electrostatic interactions with anionic components of the tumor microenvironment, thereby enhancing tumor accumulation and subsequent cellular uptake, while preserving the structural integrity and biological characteristics of the CNV membrane. As a result, the system demonstrates enhanced photodynamic therapeutic efficacy without inducing overt systemic toxicity. Overall, this work establishes a practical and scalable formulation framework for charge-guided biomimetic nanomedicines, offering a versatile strategy for improving the in vivo performance of photodynamic therapy. 2 Results and Discussion 2.1 Structural Optimization and Preparation of pBDT Nanoparticles In this study, redox-active polymeric nanoparticles were engineered through the oxidative disulfide cross-linking of aromatic dithiol precursors. To achieve structural precision, we evaluated various thiol derivatives based on their ability to form monodisperse nanostructures through the synergy of covalent disulfide bridges and non-covalent π-π stacking interactions ( Fig. S1 ) [ 30 ]. Among the tested candidates, benzene-1,4-dithiol (BDT) was identified as the optimal building block, yielding uniform spherical nanoparticles with an average diameter of ~ 100 nm. In contrast, precursors with ortho- or meta-substitution (e.g., benzene-1,2-dithiol) or bulky substituents produced heterogeneous aggregates. The distinct nanoparticle-forming capability of BDT is likely associated with its linear para-geometry, which favors extended chain propagation and intermolecular stacking, whereas structural isomers may experience steric constraints that hinder long-range ordering [ 31 – 33 ]. Consequently, BDT was selected as the foundational monomer to construct a robust and redox-responsive polymeric matrix. 2.2 Photosensitizer Encapsulation and Redox-Responsive Properties The pBDT core was designed to encapsulate the hydrophobic photosensitizer (PS, pheophorbide A) through strong π-π interactions between the aromatic backbone of the polymer and the planar structure of PS (Fig. 2 a) [ 34 – 37 ]. To optimize the formulation, various PS:BDT feed ratios were evaluated. SEM and DLS analysis confirmed that the 1:200 (PS:BDT) formulation produced uniform spherical nanoparticles (Fig. 2 b, c) with a slightly negative surface charge (Fig. 2 d). The ROS generation capability reached a plateau at the 1:200 ratio (Fig. 2 e). Therefore, the 1:200 formulation was selected as the optimal condition, achieving comparable ROS generation while avoiding unnecessary increases in polymer content. Control experiments confirmed that the polymer matrix itself is photodynamically inactive under irradiation, ensuring that ROS production originates exclusively from the encapsulated PS ( Fig. S2 ). Spectroscopic analysis provided direct evidence of stable supramolecular assembly. In the FT-IR spectrum (Fig. 2 f), the vibrational modes of PS were significantly attenuated, indicating restricted molecular motion within the tightly confined pBDT matrix. Consistently, the fluorescence of PS/pBDT showed a red shift and spectral broadening (Fig. 2 g), suggesting robust π-π stacking between the PS porphyrin ring and the aromatic pBDT framework [ 34 , 35 ]. In addition, PS/pBDT nanoparticles maintained a stable dispersion without noticeable aggregation ( Fig. S3 ), further supporting the formation of a tightly associated and structurally stable supramolecular assembly that preserves photodynamic activity. The structural responsiveness of PS/pBDT to a reductive environment was evaluated via SEM (Fig. 2 h), while the nanoparticles maintained a uniform spherical morphology (~ 84 nm) in PBS, exposure to 10 mM GSH induced significant structural degradation and disassembly, characterized by a loss of spherical integrity and increased polydispersity. This GSH-triggered disassembly is attributed to the cleavage of the disulfide cross-links within the pBDT matrix. To further quantify this interaction, an Ellman’s assay was performed (Fig. 2 i), revealing that pBDT nanoparticles induced a ~ 18–20% reduction in GSH levels through thiol-disulfide exchange. This intrinsic redox reactivity suggests that pBDT does not merely serve as a passive carrier but acts as a redox-active scavenger, potentially sensitizing cancer cells to PDT by perturbing intracellular redox homeostasis. 2.3 Construction of Biomimetic PS/pBDT@CNVs with Homotypic Affinity To enhance the cellular uptake of PS/pBDT nanoparticles, a biomimetic coating strategy using CT26-derived CNVs was adopted. To establish a biologically relevant model for our redox-responsive system, we first performed an in silico analysis of glutathione synthetase (GSS) mRNA expression across multiple cancer types using the GEPIA database ( Fig. S4a ) [ 38 ]. Colorectal cancer exhibited significantly elevated GSS expression compared with normal tissues. To assess whether this transcriptional trend was reflected at the cellular redox level, intracellular glutathione (GSH) concentrations were subsequently measured in representative cancer cell lines ( Fig. S4b ). Among the tested models, the colorectal cancer cell line CT26 displayed the highest basal GSH levels. These characteristics support the suitability of CT26 as a model for evaluating photodynamic and ferroptosis-related therapeutic strategies in which redox regulation plays a critical role. Comparative uptake studies further demonstrated that CT26-derived CNVs exhibited pronounced homotypic affinity toward CT26 cells, whereas CNVs derived from NIH3T3 fibroblasts did not show preferential uptake ( Fig. S5 ). This effect correlated with the expression of adhesion-associated membrane proteins, which have been reported to mediate cell-cell recognition and contribute to homotypic interactions [ 39 , 40 ]. Based on these observations, CT26-derived CNVs were selected as the coating material to impart cell-specific affinity to PS/pBDT nanoparticles. The coating process was subsequently optimized to preserve structural integrity while achieving complete surface coverage (Fig. 3 a). We evaluated various CNV particle ratios to achieve complete surface coverage without inducing aggregation. At a concentration of 1.0 × 10 11 particles/mL, transmission electron microscopy (TEM) revealed a distinct, continuous membrane shell surrounding the pBDT core (Fig. 3 b), accompanied by a monodisperse size distribution (Fig. 3 c) and a zeta potential shift toward the negative charge characteristic of native CNVs (Fig. 3 d). Furthermore, western blot analysis confirmed the preservation of key vesicle-associated proteins, such as TSG101, after the extrusion process (Fig. 3 e). TSG101 expression was also detected in native CNVs ( Fig. S6 ), providing a reference for vesicle-associated protein retention following the coating process. These findings indicate that the coating procedure does not compromise the biological identity of the nanovesicles. 2.4 Interfacial Charge Engineering of cCNVs To address the electrostatic repulsion inherent to native CNVs, we engineered the interfacial surface charge by incorporating the DOTAP. A critical prerequisite was the incorporation of DOTAP without compromising the functional integrity of membrane proteins. We first established the ethanol tolerance of CNVs ( Fig. S7 ). While membrane-associated markers (TSG101 and CD9) remained stable even at 50% ethanol, the luminal marker LC3B began to decrease at ethanol concentrations exceeding 10% (v/v) and showed a pronounced loss above 20%, suggesting increased membrane permeability rather than selective protein degradation. Based on these observations, 10% ethanol was selected as the maximum permissible condition to enable lipid insertion while minimizing structural disruption and unintended loss of vesicle-associated proteins. Under this optimized condition, DOTAP was incorporated into the CNV bilayer at varying concentrations. Zeta potential analysis (Fig. 4 a) revealed a dose-dependent charge reversal, reaching a saturation plateau at 15 mM DOTAP. Crucially, the hydrodynamic diameter remained stable up to 15 mM, but instability and aggregation occurred at higher concentrations (20–25 mM) likely due to excessive surface energy and membrane destabilization (Fig. 4 b). Thus, 15 mM was identified as the optimal engineering concentration, maximizing the positive surface charge while maintaining colloidal stability. The final hybrid nanovesicles, PS/pBDT@cCNVs, were fabricated by coating pBDT cores with these optimized cCNVs (Fig. 4 c). The resulting platforms exhibited a uniform size of ~ 100 nm (Fig. 4 d) and a stable positive surface charge (Fig. 4 e). Importantly, the ROS generation from encapsulated PS was unaffected by DOTAP-mediated post-modulation (Fig. 4 f), confirming that the interfacial engineering did not interfere with the photodynamic properties. Western blot analysis further confirmed the retention of the vesicle marker protein TSG101 in PS/pBDT@cCNVs after charge modulation and coating, indicating preservation of CNV membrane characteristics (Fig. 4 g). In addition, the long-term storage stability of PS/pBDT@cCNVs was validated at 4°C over 105 days, maintaining stable size and charge profiles ( Fig. S8 ), which underscores the structural and electrostatic robustness of our hybrid system. Collectively, these findings establish a quantitative correlation between DOTAP concentration and CNV surface charge, identify a saturation threshold for stable cationic modification, and validate 15 mM DOTAP as the optimal condition for investigating enhanced electrostatic interactions with the tumor microenvironment. 2.5 Charge-Modulated Cellular Uptake and Enhanced Phototoxicity To validate the advantages of the charge-modulated hybrid design, cellular internalization was quantified by flow cytometry (Fig. 5 a). PS/pBDT@cCNVs exhibited a markedly higher internalization rate of 86.3%, representing approximately a 10-fold enhancement compared to free PS (8.9%) and a significant improvement over the unmodulated PS/pBDT@CNVs (66.4%). This hierarchical increase suggests that the cationic surface charge facilitates initial membrane interactions, while the CNV shell contributes to homotypic recognition to promote efficient cellular uptake. The tumor-associated internalization behavior was further examined by fluorescence microscopy using Nile Red-labeled nanovesicles. CT26-derived cCNVs displayed substantially higher uptake in CT26 tumor cells compared with HEK293 non-tumorigenic cells ( Fig. S9 ), supporting preferential internalization associated with the biomimetic coating. The impact of enhanced cellular uptake on multicellular-level delivery was next investigated using 3D tumor spheroids (Fig. 5 b). While free PS was restricted to the periphery, PS/pBDT@cCNVs achieved deep core penetration, distributing uniformly throughout the multicellular architecture. This ability to overcome interstitial diffusion barriers is relevant for treating solid tumors characterized by dense extracellular matrices. Consequently, this superior delivery translated into potent photodynamic toxicity. Live/dead staining assays revealed that PS/pBDT@cCNVs induced the most extensive cell death following irradiation, with a residual viability of 35.1% (Fig. 5 c, d). Dose-response analysis further demonstrated effective phototoxicity even at low PS concentrations of 0.1 µg/mL (Fig. 5 e). Importantly, laser irradiation alone did not induce detectable cytotoxicity ( Fig. S10 ), and all formulations remained non-toxic in the absence of light ( Fig. S11 ), indicating that the system remains biologically inert until photoactivation. 2.6 Oxidative Stress-Driven Cell Death Involving Apoptotic and Ferroptosis-Like Features We investigated the intracellular mechanism underlying this potent cytotoxicity. Cells treated with PS/pBDT@cCNVs exhibited a pronounced reduction in intracellular GSH levels, as evidenced by a fluorescence-based GSH assay using monochlorobimane (MCB) (Fig. 6 a). To determine the appropriate timing for photoactivation, intracellular GSH levels were monitored at multiple time points (2, 4, 8, 12, and 24 h) after PS/pBDT@cCNV treatment, and laser irradiation was subsequently performed at the time point showing maximal GSH depletion ( Fig. S12 ). This preemptive depletion, mediated by the disulfide-rich pBDT core, compromised the cellular antioxidant defense, thereby sensitizing the cells to the subsequent laser-induced ROS burst (Fig. 6 b). This redox imbalance state triggered a cascade of organelle damage. We observed severe lipid peroxidation (Fig. 6 c) and mitochondrial dysfunction (Fig. 6 d), which are hallmarks of extensive oxidative damage. Western blot analysis further supported the involvement of multiple oxidative stress associated death pathways (Fig. 6 e). The increase in Bax and Caspase-9 with reduced Bcl-2 was consistent with activation of the intrinsic apoptotic program. Simultaneously, the depletion of GPX4, a central sentinel of lipid redox homeostasis, strongly suggested the concurrent induction of ferroptosis-like death. Quantitative analyses of fluorescence intensity corresponding to Fig. 6 a-d are summarized in supporting information ( Fig. S13 ). To further examine the contribution of lipid oxidation-sensitive pathways to photodynamic cytotoxicity, cell viability was evaluated in the presence of ferrostatin-1 (Fer-1), a lipophilic radical scavenger that suppresses lipid peroxidation (Fig. 6 f). Fer-1 treatment partially restored cell viability, indicating that lipid peroxidation-associated processes contribute to PS/pBDT@cCNV-mediated cytotoxicity. However, the incomplete rescue suggests that additional oxidative stress-related mechanisms, including apoptotic signaling, also participate in cell death. Together, these results support a multi-pathway mode of action in which redox imbalance, lipid peroxidation, and mitochondrial dysfunction collectively drive photodynamic cell killing. This integrated mechanism is schematically summarized in Fig. S14 and highlights how concurrent engagement of multiple oxidative stress-associated pathways may enhance therapeutic robustness compared with single-pathway interventions. 2.7 Enhanced Tumor Accumulation and Charge-Driven Interstitial Penetration In Vivo The systemic biodistribution and tumor-homing behavior of the engineered nanovesicles were investigated in a CT26 tumor-bearing mouse model (Fig. 7 a). Ex vivo fluorescence imaging at 24 h post-injection revealed that PS/pBDT@cCNVs achieved the highest tumor accumulation (Fig. 7 b-d), accompanied by reduced accumulation in major organs compared with other formulations. This favorable biodistribution profile is attributed to the biomimetic properties of the CNV shell, which may contribute to improved in vivo stability and tumor localization. The PS/pBDT@cCNVs group displayed persistent intratumoral fluorescence, indicating enhanced delivery efficiency and prolonged retention within the tumor site. Quantitative fluorescence analysis (Fig. 7 d) confirmed that PS/pBDT@cCNVs achieved the highest tumor selectivity with minimal accumulation in the liver, spleen, and kidneys. This pattern suggests that cCNV coating improves both homotypic tumor targeting and systemic stability, while reducing nonspecific organ uptake. Confocal imaging of tumor cryosections demonstrated that PS/pBDT@cCNVs were broadly distributed throughout the tumor tissue rather than being confined to the peripheral regions (Fig. 7 e). This enhanced intratumoral distribution is likely associated with the combined effects of biomimetic CNV-mediated tumor interactions and optimized surface charge, which together may facilitate tumor tissue association and interstitial penetration within the tumor microenvironment [ 41 , 42 ]. 2.8 In Vivo Antitumor Efficacy with Systemic Biocompatibility The therapeutic efficacy of the hybrid nanovesicles was evaluated by monitoring tumor growth kinetics following the planned PDT treatment schedule (Fig. 8 a). Free PS and PS/pBDT showed only limited tumor growth suppression, with tumors continuing to enlarge over the observation period, likely due to insufficient tumor accumulation (Fig. 8 b). In contrast, CNV-coated formulations exhibited more pronounced tumor growth inhibition. Notably, while PS/pBDT@CNVs resulted in partial tumor suppression, surface charge-modulated PS/pBDT@cCNVs achieved near-complete tumor ablation with no evident regrowth during the study period (Fig. 8 c, d and Fig. S15 ), underscoring the critical contribution of charge-mediated tumor accumulation. Histological examination of tumor tissues by H&E staining revealed extensive necrosis and structural disruption in the PS/pBDT@cCNV-treated group, whereas tumors from control groups retained largely viable tissue architecture (Fig. 8 e). Importantly, this potent antitumor efficacy was achieved without compromising systemic safety. Throughout the treatment course, mice maintained stable body weights ( Fig. S16 ). In addition, serum biochemical analysis performed at sacrifice showed that liver function markers, including alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as well as renal function markers, including blood urea nitrogen (BUN) and creatinine, exhibited values comparable to those of control groups, with no marked treatment-related deviations observed ( Fig. S17 ). Histological analysis of major organs further confirmed the absence of treatment-related pathological abnormalities ( Fig. S18 ). Collectively, these results demonstrate that PS/pBDT@cCNVs effectively combine strong antitumor activity with favorable systemic biocompatibility. 3 Conclusion In summary, we engineered a biomimetic core-shell hybrid nanoplatform by integrating redox-active pBDT polymer cores with charge-modulated CNVs. This study demonstrates that controlled surface charge modulation, achieved through the incorporation of a minimal amount of cationic lipids, influences tumor accumulation behavior in vivo . By modulating the surface charge of the nanovesicle shell, PS/pBDT@cCNVs exhibited improved tumor accumulation and intratumoral distribution compared with non-modified formulations, addressing key delivery limitations of conventional PDT. Consequently, this optimized design enabled potent tumor ablation and sustained tumor suppression in vivo during the treatment period, without observable systemic toxicity. Beyond PDT, this work demonstrates that surface charge modulation at the CNV interface is a practical and effective strategy for improving the in vivo performance of biomimetic hybrid nanocarriers. This platform may provide a versatile framework for the development of precision nanomedicine strategies targeting solid tumors. Declarations Acknowledgements Illustrations were created with BioRender.com. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (the Ministry of Science & ICT, MSIT) (RS-2024-00350878 and RS-2023-00242443), by the Bio & Medical Technology Development Program of the NRF funded by the MSIT (RS-2024-00440714), by the Korean Fund for Regenerative Medicine (KFRM) funded by MSIT and the Ministry of Health and Welfare, Republic of Korea (RS-2025-02223118), by the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Korean government (MSIT) (RS-2024-00402899), and by the KIST Institutional Program (2E32351-23-130). Data Availability Statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations The authors have no competing interests to declare that are relevant to the content of this article. Statement of Human and Animal Rights All animal experiments were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (SKKUIACUC2024-02-15-1). This article does not contain any studies involving human participants performed by any of the authors. Author contributions H.-J.O. performed the experiments and wrote the manuscript. D.-Y.H., J.S.L., J.H., C.G.P., and H.L. contributed to data analysis and manuscript revision. W.P. and C.G.P. secured funding. W.P. supervised the project. All authors reviewed and approved the final manuscript. Authors and Affiliations Authors: Hyeon-Ji Oh, Dong-Yong Hong, Junseob Lee, Jieun Han, Chun Gwon Park, Hyojin Lee, and Wooram Park* Affiliations Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), Seobu-ro 2066, Jangan-gu, Suwon, Gyeonggi 16419, Republic of Korea Hyeon-Ji Oh, Dong-Yong Hong , Junseob Lee, Jieun Han, and Wooram Park Department of MetaBioHealth, Institute for Cross-disciplinary Studies (ICS), SKKU, Seobu-ro 2066, Suwon, Gyeonggi 16419, Republic of Korea Wooram Park Department of Biomedical Engineering, ICS, SKKU, Seobu-ro 2066, Suwon, Gyeonggi 16419, Republic of Korea Chun Gwon Park Biomaterials Research Center, Korea Institute of Science and Technology (KIST), 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea Chun Gwon Park, Hyojin Lee, and Wooram Park Corresponding authors Correspondence to Wooram Park (email: [email protected] ) Supplementary Information Supplementary data to this article can be found online at the journal website References Siegel RL, Giaquinto AN, Jemal A, Cancer statistics (2024) CA Cancer J Clin. 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J Control Release 328:325–338. 10.1016/j.jconrel.2020.08.062 Zheng N, Zhang ZY, Kuang J, Wang CS, Zheng YB, Lu Q et al (2019) Poly(photosensitizer) Nanoparticles for Enhanced in Vivo Photodynamic Therapy by Interrupting the π-π Stacking and Extending Circulation Time. Acs Appl Mater Inter 11(20):18224–18232. 10.1021/acsami.9b04351 Zhou JJ, Xu M, Jin ZC, Borum RM, Avakyan N, Cheng Y et al (2021) Versatile Polymer Nanocapsules via Redox Competition. Angew Chem Int Edit 60(50):26357–26362. 10.1002/anie.202110829 Tang ZF, Li CW, Kang BX, Gao G, Li C, Zhang ZM (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45(W1):W98–W102. 10.1093/nar/gkx247 Janiszewska M, Primi MC, Izard T (2020) Cell adhesion in cancer: Beyond the migration of single cells. 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Supplementary Files ACHMSupportinginformationOhetal.WRPF.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8862874","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602479460,"identity":"ca9ac100-081b-4bd3-aafd-9a770a9b019c","order_by":0,"name":"Hyeon-Ji Oh","email":"","orcid":"","institution":"Sungkyunkwan University (SKKU)","correspondingAuthor":false,"prefix":"","firstName":"Hyeon-Ji","middleName":"","lastName":"Oh","suffix":""},{"id":602479465,"identity":"a153b965-3fac-4bcf-86b3-6548974ec42a","order_by":1,"name":"Dong-Yong Hong","email":"","orcid":"","institution":"Sungkyunkwan University (SKKU)","correspondingAuthor":false,"prefix":"","firstName":"Dong-Yong","middleName":"","lastName":"Hong","suffix":""},{"id":602479466,"identity":"0582d3c7-809d-4660-b476-2fbfca6200fe","order_by":2,"name":"Junseob Lee","email":"","orcid":"","institution":"Sungkyunkwan University (SKKU)","correspondingAuthor":false,"prefix":"","firstName":"Junseob","middleName":"","lastName":"Lee","suffix":""},{"id":602479467,"identity":"06fbeb03-47ce-46e7-87f4-4b883bbf850e","order_by":3,"name":"Jieun Han","email":"","orcid":"","institution":"Sungkyunkwan University (SKKU)","correspondingAuthor":false,"prefix":"","firstName":"Jieun","middleName":"","lastName":"Han","suffix":""},{"id":602479469,"identity":"54e292d6-fe7d-4b78-ad9a-a8b0e75af478","order_by":4,"name":"Chun Gwon Park","email":"","orcid":"","institution":"Sungkyunkwan University (SKKU)","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"Gwon","lastName":"Park","suffix":""},{"id":602479471,"identity":"a3a4ca19-8cf6-4e76-984d-f1094bf3a794","order_by":5,"name":"Hyojin Lee","email":"","orcid":"","institution":"Korea Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hyojin","middleName":"","lastName":"Lee","suffix":""},{"id":602479473,"identity":"fd31bea4-8a0f-4c8d-88dc-d8cb62795b58","order_by":6,"name":"Wooram Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYHACZiC2YZDgAbHZiNeSRrqWwyRoMWc/+9iYd895e8meMwYMH8oOE9Zi2ZNunMzz7HbibN4eA8YZ54jQYnAgjflwzoHbCXL8PAbMvG3EaDn/DKTlnD1Yy1+itNxIY07OOXCAEeQwZkZitFjOeMZs/OdAcuLMnmMFB3vOpRPWYs6fxiw544CdvcSZ5I0PfpRZE+EwZM4BwurRtYyCUTAKRsEowAoAGIw3AgPNb1kAAAAASUVORK5CYII=","orcid":"","institution":"Sungkyunkwan University (SKKU)","correspondingAuthor":true,"prefix":"","firstName":"Wooram","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2026-02-12 13:56:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8862874/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8862874/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104265077,"identity":"c2f27d96-cf49-4c13-b98c-93ef186b8f16","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":339291,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the design and therapeutic mechanism of PS/pBDT@cCNVs. Benzene-1,4-dithiol (BDT) monomers undergo oxidative polymerization to form poly(benzene-1,4-dithiol) (pBDT) nanoparticles via disulfide bond formation, enabling the encapsulation of photosensitizers (PS) through π-π stacking interactions. The PS/pBDT core is coated with cancer-cell-derived nanovesicles (CNVs) to provide homotypic targeting and prolonged circulation. To address the electrostatic barriers associated with tumor tissues, the CNV shell is further functionalized with a controlled amount of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) to yield cationic-modified CNVs (cCNVs), enhancing tumor accumulation. Upon light irradiation, the combined effects of light-triggered reactive oxygen species (ROS) generation and pBDT-mediated glutathione (GSH) depletion cooperatively induce lipid peroxidation and mitochondrial dysfunction. These dual actions activate multiple oxidative stress associated cell death pathways, including ferroptosis like features and apoptotic signaling, ultimately leading to robust tumor ablation.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/7d5ddb188144b1f894353b33.png"},{"id":104265078,"identity":"8264182b-e9ee-41c5-8767-e27e8abde994","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":962960,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis and characterization of PS/pBDT. (a) Schematic representation of the synthesis of pBDT through oxidative disulfide cross-linking and the concurrent encapsulation of PS via intermolecular π-π stacking. (b) Scanning electron microscopy (SEM) image illustrating the uniform spherical morphology and monodisperse nature of the nanoparticles. (c) Dynamic light scattering (DLS)-based size distributions of PS/pBDT prepared at varied PS-to-BDT molar ratios. (d) Zeta potential profiles of the core particles. (e) ROS generation profiles monitored using Singlet Oxygen Sensor Green (SOSG), showing the optimized 1:200 ratio for maximized photochemical yield. (f) Fourier-transform infrared (FT-IR) spectra showing attenuation of PS specific vibrational modes, suggesting a restricted molecular environment within the pBDT matrix. (g) Fluorescence intensity quantification validating the high loading capacity of PS. (h) SEM images of PS/pBDT nanoparticles before and after exposure to a reductive environment (10 mM GSH), demonstrating GSH-triggered structural disassembly. (i) Quantification of glutathione consumption measured by Ellman’s assay, indicating thiol-disulfide exchange-mediated GSH depletion induced by pBDT nanoparticles.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/2b7b581d605598d39e9da9fc.png"},{"id":104265074,"identity":"c6d3a904-1650-4afc-a67d-2c3ab51c0579","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":461379,"visible":true,"origin":"","legend":"\u003cp\u003eStructural characterization and protein profiling of biomimetic CNV-coated hybrid nanoparticles. (a) Schematic illustration of the co-extrusion process for the interfacial coating of PS/pBDT cores with CNVs. (b) Transmission electron microscopy (TEM) images revealing the formation of a membrane shell surrounding the PS/pBDT core. (c) Hydrodynamic size distribution and (d) zeta potential shift towards negative values, confirming the successful wrapping of the biomimetic membrane. (e) Western blot analysis verifying the retention of key protein markers (e.g., TSG101) within the hybrid architecture, confirming the preservation of the biological identity post-extrusion.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/8fbffa84abe0b033dcb51f0f.png"},{"id":104405034,"identity":"464392ac-5f61-4463-a184-95e5f9ca68f6","added_by":"auto","created_at":"2026-03-11 12:21:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":414218,"visible":true,"origin":"","legend":"\u003cp\u003eDesign optimization of charge-modulated shells and integrated characterization of PS/pBDT@cCNVs. (a) Zeta potential titration of CNVs as a function of DOTAP concentration, showing charge plateauing at 15 mM. (b) Hydrodynamic size monitoring, identifying the 15 mM threshold as the optimal concentration for maintaining colloidal stability without aggregation. (c) Schematic illustration of the multistep coating process for PS/pBDT@cCNVs. (d) DLS-based size distribution and (e) surface charge analysis confirming the successful assembly of the final hybrid nanoplatform. (f) ROS generation profiles, verifying that surface engineering does not impair the photochemical efficiency of the encapsulated PS. (g) Western blot analysis of the marker protein TSG101, confirming the preservation of the biomimetic membrane interface post-integration.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/3f4592c386b59b6e81bfe401.png"},{"id":104265079,"identity":"953724e7-0e87-460b-845c-9ea2a65b1055","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1255291,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced cellular internalization, 3D tumor spheroid penetration, and \u003cem\u003ein vitro\u003c/em\u003e phototherapeutic efficacy. (a) Flow cytometry analysis quantifying the intracellular accumulation of various PS formulations. PS/pBDT@cCNVs exhibited a significant increase in uptake efficiency. (b) Z-stack confocal laser scanning microscopy (CLSM) images of 3D CT26 tumor spheroids, demonstrating the improved deep-core penetration of the charge-modulated vesicles at 2 h and 6 h post-incubation. (c) Representative live/dead staining images and (d) quantitative viability assessment of CT26 cells, demonstrating potent phototoxicity. (e) Dose-dependent cytotoxicity profiles via MTT assay, illustrating the superior phototherapeutic potency of PS/pBDT@cCNVs compared to control groups at equivalent PS concentrations.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/ca457d8240acfc9f2a6a7874.png"},{"id":104265076,"identity":"9d57c80b-20ba-49b0-a5f3-983e2b9ca8ef","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":973719,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic investigation of synergistic oxidative cell death in CT26 cells. (a) Intracellular GSH depletion visualized by monochlorobimane (MCB)-based fluorescence analysis. (b) Amplified intracellular ROS levels visualized by 2,7-dichlorofluorescein (DCF) fluorescence following combined treatment. (c) Lipid peroxidation imaging using C11-BODIPY, indicating oxidative membrane damage. (d) MitoTracker staining illustrates mitochondrial fragmentation and mitochondrial dysfunction. (e) Western blot analysis of key regulatory proteins associated with Stat3 signaling, apoptosis (Bcl-2, Bax, Caspases), and ferroptosis (GPX4). (f) Cell viability assessment with or without the lipid peroxidation inhibitor ferrostatin-1, suggesting the contribution of ferroptosis-like death to the overall cytotoxicity. All scale bars represent 50 µm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/7b48112dfbea4072303db191.png"},{"id":104265082,"identity":"2f981a49-5449-47d4-a16e-ac3e5847e9ca","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":596457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo \u003c/em\u003ebiodistribution and tumor-targeting efficiency of PS/pBDT@cCNVs.\u003cem\u003e \u003c/em\u003e(a) Experimental timeline for the systemic administration and imaging studies. (b) Representative \u003cem\u003eex vivo\u003c/em\u003e fluorescence images of major organs and (c) isolated tumors harvested 24 h post-injection, indicating increased tumor localization of the hybrid vesicles. (d) Quantitative biodistribution analysis supporting the enhanced accumulation of the surface charge-modulated platform compared to free PS. (e) Confocal microscopy of tumor cryosections, revealing extensive intratumoral penetration and improved interstitial distribution of PS/pBDT@cCNVs throughout the tumor parenchyma.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/6c1fa1c09deb53a5a5199b44.png"},{"id":104265080,"identity":"652116c2-2b2e-46ec-8683-a63cf4480bd2","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":591432,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic efficacy of PS/pBDT@cCNVs. (a) Treatment schedule for the \u003cem\u003ein vivo\u003c/em\u003e photodynamic therapy. (b) Individual and (c) average tumor growth kinetics over the treatment period, showing significant tumor growth suppression in the PS/pBDT@cCNV-treated group compared to other treatment groups. (d) Final tumor weights measured at the study endpoint, indicating the enhanced anti-tumor potency of the charge-modulated hybrid platform. (e) Representative histological images of tumor tissues stained with H\u0026amp;E.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/0bffe198e4566d40c588c3d4.png"},{"id":109068118,"identity":"63c9a59d-6ffe-4d1c-a008-87de727418fe","added_by":"auto","created_at":"2026-05-12 10:03:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5875007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/5875b009-499a-4bb2-b314-9c6bd640921b.pdf"},{"id":104265081,"identity":"c4622440-10da-45cc-a224-a9b98af19a76","added_by":"auto","created_at":"2026-03-09 19:46:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3685966,"visible":true,"origin":"","legend":"","description":"","filename":"ACHMSupportinginformationOhetal.WRPF.docx","url":"https://assets-eu.researchsquare.com/files/rs-8862874/v1/b8bcd68ce6d529e2c8fbe61f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Surface Charge-Modulated Biomimetic Core-Shell Hybrid Nanovesicles for Redox-Triggered Synergistic Cancer Therapy","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCancer remains one of the leading causes of mortality worldwide, and conventional treatments such as surgery, chemotherapy, and radiotherapy often suffer from non-specificity, systemic toxicity, and damage to healthy tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These limitations have promoted the development of alternative strategies that can achieve accurate tumor targeting while minimizing side effects. Photodynamic therapy (PDT) has emerged as a promising minimally invasive modality, relying on the activation of photosensitizers (PSs) by light to generate reactive oxygen species (ROS) that induce localized tumor cell death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. PDT offers spatiotemporal control and the potential to trigger anti-tumor immune responses [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, its clinical translation is hindered by the poor water solubility, instability, and limited tumor accumulation of many hydrophobic PSs [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Aggregation-induced quenching and rapid systemic clearance further reduce ROS generation efficiency, underscoring the critical need for advanced delivery systems that improve pharmacokinetics and tumor specificity [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNanoparticle-based carriers have been widely explored to address these challenges by leveraging the enhanced permeability and retention (EPR) effect or active targeting mechanisms [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among these, π-conjugated polymers can stably encapsulate hydrophobic PSs through strong π-π stacking, preventing aggregation and enhancing photodynamic activity [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To further impart biological targeting capabilities, cell-derived nanovesicles (CNVs) have attracted significant attention. Inheriting the complex membrane protein profile of their source cells, CNVs offer intrinsic biocompatibility, low immunogenicity, and specific homotypic tumor-homing properties [\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, integrating the high loading capacity of pBDT cores with the biological recognition of CNV shells presents a compelling \"core-shell\" hybrid strategy.\u003c/p\u003e \u003cp\u003eHowever, a critical barrier remains. The surface charge of natural CNVs is inherently negative. Although CNVs possess targeting ligands, their negative surface charge can induce electrostatic repulsion with the anionic glycocalyx of the tumor endothelium, potentially limiting early interactions at the tumor site [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have reported that electrostatic interactions between cationic nanomaterials and anionic tumor-associated interfaces contribute to vascular association and transendothelial transport [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. While cationic nanoparticles are known to interact favorably with anionic tumor tissues, excessive positive charge often leads to opsonization and systemic toxicity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, a strategic balance is required. We hypothesized that modulating the CNV surface with a minimal threshold of cationic lipids (e.g. 1,2-dioleoyl-3-trimethylammonium-propane; DOTAP) would enhance electrostatic interaction with the tumor endothelium without compromising the vesicle\u0026rsquo;s structural integrity or safety.\u003c/p\u003e \u003cp\u003eIn this study, we present a charge-tunable hybrid nanovesicle platform designed to enhance tumor delivery and photodynamic therapeutic performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The platform consists of a photosensitizer-loaded poly(benzene-1,4-dithiol) (pBDT) core that enables stable drug retention and a CNV shell minimally modified with cationic lipids to allow controlled modulation of interfacial surface charge. Through this charge engineering strategy, the hybrid nanovesicles promote electrostatic interactions with anionic components of the tumor microenvironment, thereby enhancing tumor accumulation and subsequent cellular uptake, while preserving the structural integrity and biological characteristics of the CNV membrane. As a result, the system demonstrates enhanced photodynamic therapeutic efficacy without inducing overt systemic toxicity. Overall, this work establishes a practical and scalable formulation framework for charge-guided biomimetic nanomedicines, offering a versatile strategy for improving the \u003cem\u003ein vivo\u003c/em\u003e performance of photodynamic therapy.\u003c/p\u003e"},{"header":"2 Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Structural Optimization and Preparation of pBDT Nanoparticles\u003c/h2\u003e \u003cp\u003eIn this study, redox-active polymeric nanoparticles were engineered through the oxidative disulfide cross-linking of aromatic dithiol precursors. To achieve structural precision, we evaluated various thiol derivatives based on their ability to form monodisperse nanostructures through the synergy of covalent disulfide bridges and non-covalent π-π stacking interactions (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the tested candidates, benzene-1,4-dithiol (BDT) was identified as the optimal building block, yielding uniform spherical nanoparticles with an average diameter of ~\u0026thinsp;100 nm. In contrast, precursors with ortho- or meta-substitution (e.g., benzene-1,2-dithiol) or bulky substituents produced heterogeneous aggregates. The distinct nanoparticle-forming capability of BDT is likely associated with its linear para-geometry, which favors extended chain propagation and intermolecular stacking, whereas structural isomers may experience steric constraints that hinder long-range ordering [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consequently, BDT was selected as the foundational monomer to construct a robust and redox-responsive polymeric matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Photosensitizer Encapsulation and Redox-Responsive Properties\u003c/h2\u003e \u003cp\u003eThe pBDT core was designed to encapsulate the hydrophobic photosensitizer (PS, pheophorbide A) through strong π-π interactions between the aromatic backbone of the polymer and the planar structure of PS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To optimize the formulation, various PS:BDT feed ratios were evaluated. SEM and DLS analysis confirmed that the 1:200 (PS:BDT) formulation produced uniform spherical nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c) with a slightly negative surface charge (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The ROS generation capability reached a plateau at the 1:200 ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Therefore, the 1:200 formulation was selected as the optimal condition, achieving comparable ROS generation while avoiding unnecessary increases in polymer content. Control experiments confirmed that the polymer matrix itself is photodynamically inactive under irradiation, ensuring that ROS production originates exclusively from the encapsulated PS (\u003cb\u003eFig. S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSpectroscopic analysis provided direct evidence of stable supramolecular assembly. In the FT-IR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), the vibrational modes of PS were significantly attenuated, indicating restricted molecular motion within the tightly confined pBDT matrix. Consistently, the fluorescence of PS/pBDT showed a red shift and spectral broadening (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), suggesting robust π-π stacking between the PS porphyrin ring and the aromatic pBDT framework [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, PS/pBDT nanoparticles maintained a stable dispersion without noticeable aggregation (\u003cb\u003eFig. S3\u003c/b\u003e), further supporting the formation of a tightly associated and structurally stable supramolecular assembly that preserves photodynamic activity.\u003c/p\u003e \u003cp\u003eThe structural responsiveness of PS/pBDT to a reductive environment was evaluated via SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), while the nanoparticles maintained a uniform spherical morphology (~\u0026thinsp;84 nm) in PBS, exposure to 10 mM GSH induced significant structural degradation and disassembly, characterized by a loss of spherical integrity and increased polydispersity. This GSH-triggered disassembly is attributed to the cleavage of the disulfide cross-links within the pBDT matrix. To further quantify this interaction, an Ellman\u0026rsquo;s assay was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), revealing that pBDT nanoparticles induced a\u0026thinsp;~\u0026thinsp;18\u0026ndash;20% reduction in GSH levels through thiol-disulfide exchange. This intrinsic redox reactivity suggests that pBDT does not merely serve as a passive carrier but acts as a redox-active scavenger, potentially sensitizing cancer cells to PDT by perturbing intracellular redox homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Construction of Biomimetic PS/pBDT@CNVs with Homotypic Affinity\u003c/h2\u003e \u003cp\u003eTo enhance the cellular uptake of PS/pBDT nanoparticles, a biomimetic coating strategy using CT26-derived CNVs was adopted. To establish a biologically relevant model for our redox-responsive system, we first performed an \u003cem\u003ein silico\u003c/em\u003e analysis of glutathione synthetase (GSS) mRNA expression across multiple cancer types using the GEPIA database (\u003cb\u003eFig. S4a\u003c/b\u003e) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Colorectal cancer exhibited significantly elevated GSS expression compared with normal tissues. To assess whether this transcriptional trend was reflected at the cellular redox level, intracellular glutathione (GSH) concentrations were subsequently measured in representative cancer cell lines (\u003cb\u003eFig. S4b\u003c/b\u003e). Among the tested models, the colorectal cancer cell line CT26 displayed the highest basal GSH levels. These characteristics support the suitability of CT26 as a model for evaluating photodynamic and ferroptosis-related therapeutic strategies in which redox regulation plays a critical role.\u003c/p\u003e \u003cp\u003eComparative uptake studies further demonstrated that CT26-derived CNVs exhibited pronounced homotypic affinity toward CT26 cells, whereas CNVs derived from NIH3T3 fibroblasts did not show preferential uptake (\u003cb\u003eFig. S5\u003c/b\u003e). This effect correlated with the expression of adhesion-associated membrane proteins, which have been reported to mediate cell-cell recognition and contribute to homotypic interactions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Based on these observations, CT26-derived CNVs were selected as the coating material to impart cell-specific affinity to PS/pBDT nanoparticles.\u003c/p\u003e \u003cp\u003eThe coating process was subsequently optimized to preserve structural integrity while achieving complete surface coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). We evaluated various CNV particle ratios to achieve complete surface coverage without inducing aggregation. At a concentration of 1.0 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e particles/mL, transmission electron microscopy (TEM) revealed a distinct, continuous membrane shell surrounding the pBDT core (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), accompanied by a monodisperse size distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and a zeta potential shift toward the negative charge characteristic of native CNVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Furthermore, western blot analysis confirmed the preservation of key vesicle-associated proteins, such as TSG101, after the extrusion process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). TSG101 expression was also detected in native CNVs (\u003cb\u003eFig. S6\u003c/b\u003e), providing a reference for vesicle-associated protein retention following the coating process. These findings indicate that the coating procedure does not compromise the biological identity of the nanovesicles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Interfacial Charge Engineering of cCNVs\u003c/h2\u003e \u003cp\u003eTo address the electrostatic repulsion inherent to native CNVs, we engineered the interfacial surface charge by incorporating the DOTAP. A critical prerequisite was the incorporation of DOTAP without compromising the functional integrity of membrane proteins. We first established the ethanol tolerance of CNVs (\u003cb\u003eFig. S7\u003c/b\u003e). While membrane-associated markers (TSG101 and CD9) remained stable even at 50% ethanol, the luminal marker LC3B began to decrease at ethanol concentrations exceeding 10% (v/v) and showed a pronounced loss above 20%, suggesting increased membrane permeability rather than selective protein degradation. Based on these observations, 10% ethanol was selected as the maximum permissible condition to enable lipid insertion while minimizing structural disruption and unintended loss of vesicle-associated proteins.\u003c/p\u003e \u003cp\u003eUnder this optimized condition, DOTAP was incorporated into the CNV bilayer at varying concentrations. Zeta potential analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) revealed a dose-dependent charge reversal, reaching a saturation plateau at 15 mM DOTAP. Crucially, the hydrodynamic diameter remained stable up to 15 mM, but instability and aggregation occurred at higher concentrations (20\u0026ndash;25 mM) likely due to excessive surface energy and membrane destabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Thus, 15 mM was identified as the optimal engineering concentration, maximizing the positive surface charge while maintaining colloidal stability.\u003c/p\u003e \u003cp\u003eThe final hybrid nanovesicles, PS/pBDT@cCNVs, were fabricated by coating pBDT cores with these optimized cCNVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The resulting platforms exhibited a uniform size of ~\u0026thinsp;100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and a stable positive surface charge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Importantly, the ROS generation from encapsulated PS was unaffected by DOTAP-mediated post-modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), confirming that the interfacial engineering did not interfere with the photodynamic properties. Western blot analysis further confirmed the retention of the vesicle marker protein TSG101 in PS/pBDT@cCNVs after charge modulation and coating, indicating preservation of CNV membrane characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). In addition, the long-term storage stability of PS/pBDT@cCNVs was validated at 4\u0026deg;C over 105 days, maintaining stable size and charge profiles (\u003cb\u003eFig. S8\u003c/b\u003e), which underscores the structural and electrostatic robustness of our hybrid system.\u003c/p\u003e \u003cp\u003eCollectively, these findings establish a quantitative correlation between DOTAP concentration and CNV surface charge, identify a saturation threshold for stable cationic modification, and validate 15 mM DOTAP as the optimal condition for investigating enhanced electrostatic interactions with the tumor microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Charge-Modulated Cellular Uptake and Enhanced Phototoxicity\u003c/h2\u003e \u003cp\u003eTo validate the advantages of the charge-modulated hybrid design, cellular internalization was quantified by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). PS/pBDT@cCNVs exhibited a markedly higher internalization rate of 86.3%, representing approximately a 10-fold enhancement compared to free PS (8.9%) and a significant improvement over the unmodulated PS/pBDT@CNVs (66.4%). This hierarchical increase suggests that the cationic surface charge facilitates initial membrane interactions, while the CNV shell contributes to homotypic recognition to promote efficient cellular uptake. The tumor-associated internalization behavior was further examined by fluorescence microscopy using Nile Red-labeled nanovesicles. CT26-derived cCNVs displayed substantially higher uptake in CT26 tumor cells compared with HEK293 non-tumorigenic cells (\u003cb\u003eFig. S9\u003c/b\u003e), supporting preferential internalization associated with the biomimetic coating.\u003c/p\u003e \u003cp\u003eThe impact of enhanced cellular uptake on multicellular-level delivery was next investigated using 3D tumor spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). While free PS was restricted to the periphery, PS/pBDT@cCNVs achieved deep core penetration, distributing uniformly throughout the multicellular architecture. This ability to overcome interstitial diffusion barriers is relevant for treating solid tumors characterized by dense extracellular matrices.\u003c/p\u003e \u003cp\u003eConsequently, this superior delivery translated into potent photodynamic toxicity. Live/dead staining assays revealed that PS/pBDT@cCNVs induced the most extensive cell death following irradiation, with a residual viability of 35.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). Dose-response analysis further demonstrated effective phototoxicity even at low PS concentrations of 0.1 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Importantly, laser irradiation alone did not induce detectable cytotoxicity (\u003cb\u003eFig. S10\u003c/b\u003e), and all formulations remained non-toxic in the absence of light (\u003cb\u003eFig. S11\u003c/b\u003e), indicating that the system remains biologically inert until photoactivation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Oxidative Stress-Driven Cell Death Involving Apoptotic and Ferroptosis-Like Features\u003c/h2\u003e \u003cp\u003eWe investigated the intracellular mechanism underlying this potent cytotoxicity. Cells treated with PS/pBDT@cCNVs exhibited a pronounced reduction in intracellular GSH levels, as evidenced by a fluorescence-based GSH assay using monochlorobimane (MCB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). To determine the appropriate timing for photoactivation, intracellular GSH levels were monitored at multiple time points (2, 4, 8, 12, and 24 h) after PS/pBDT@cCNV treatment, and laser irradiation was subsequently performed at the time point showing maximal GSH depletion (\u003cb\u003eFig. S12\u003c/b\u003e). This preemptive depletion, mediated by the disulfide-rich pBDT core, compromised the cellular antioxidant defense, thereby sensitizing the cells to the subsequent laser-induced ROS burst (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThis redox imbalance state triggered a cascade of organelle damage. We observed severe lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) and mitochondrial dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), which are hallmarks of extensive oxidative damage. Western blot analysis further supported the involvement of multiple oxidative stress associated death pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). The increase in Bax and Caspase-9 with reduced Bcl-2 was consistent with activation of the intrinsic apoptotic program. Simultaneously, the depletion of GPX4, a central sentinel of lipid redox homeostasis, strongly suggested the concurrent induction of ferroptosis-like death. Quantitative analyses of fluorescence intensity corresponding to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-d are summarized in supporting information (\u003cb\u003eFig. S13\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo further examine the contribution of lipid oxidation-sensitive pathways to photodynamic cytotoxicity, cell viability was evaluated in the presence of ferrostatin-1 (Fer-1), a lipophilic radical scavenger that suppresses lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Fer-1 treatment partially restored cell viability, indicating that lipid peroxidation-associated processes contribute to PS/pBDT@cCNV-mediated cytotoxicity. However, the incomplete rescue suggests that additional oxidative stress-related mechanisms, including apoptotic signaling, also participate in cell death.\u003c/p\u003e \u003cp\u003eTogether, these results support a multi-pathway mode of action in which redox imbalance, lipid peroxidation, and mitochondrial dysfunction collectively drive photodynamic cell killing. This integrated mechanism is schematically summarized in \u003cb\u003eFig. S14\u003c/b\u003e and highlights how concurrent engagement of multiple oxidative stress-associated pathways may enhance therapeutic robustness compared with single-pathway interventions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Enhanced Tumor Accumulation and Charge-Driven Interstitial Penetration \u003cem\u003eIn Vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe systemic biodistribution and tumor-homing behavior of the engineered nanovesicles were investigated in a CT26 tumor-bearing mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). \u003cem\u003eEx vivo\u003c/em\u003e fluorescence imaging at 24 h post-injection revealed that PS/pBDT@cCNVs achieved the highest tumor accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d), accompanied by reduced accumulation in major organs compared with other formulations. This favorable biodistribution profile is attributed to the biomimetic properties of the CNV shell, which may contribute to improved \u003cem\u003ein vivo\u003c/em\u003e stability and tumor localization.\u003c/p\u003e \u003cp\u003eThe PS/pBDT@cCNVs group displayed persistent intratumoral fluorescence, indicating enhanced delivery efficiency and prolonged retention within the tumor site. Quantitative fluorescence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed) confirmed that PS/pBDT@cCNVs achieved the highest tumor selectivity with minimal accumulation in the liver, spleen, and kidneys. This pattern suggests that cCNV coating improves both homotypic tumor targeting and systemic stability, while reducing nonspecific organ uptake.\u003c/p\u003e \u003cp\u003eConfocal imaging of tumor cryosections demonstrated that PS/pBDT@cCNVs were broadly distributed throughout the tumor tissue rather than being confined to the peripheral regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). This enhanced intratumoral distribution is likely associated with the combined effects of biomimetic CNV-mediated tumor interactions and optimized surface charge, which together may facilitate tumor tissue association and interstitial penetration within the tumor microenvironment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 \u003cem\u003eIn Vivo\u003c/em\u003e Antitumor Efficacy with Systemic Biocompatibility\u003c/h2\u003e \u003cp\u003eThe therapeutic efficacy of the hybrid nanovesicles was evaluated by monitoring tumor growth kinetics following the planned PDT treatment schedule (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Free PS and PS/pBDT showed only limited tumor growth suppression, with tumors continuing to enlarge over the observation period, likely due to insufficient tumor accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). In contrast, CNV-coated formulations exhibited more pronounced tumor growth inhibition. Notably, while PS/pBDT@CNVs resulted in partial tumor suppression, surface charge-modulated PS/pBDT@cCNVs achieved near-complete tumor ablation with no evident regrowth during the study period (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, d \u003cb\u003eand Fig. S15\u003c/b\u003e), underscoring the critical contribution of charge-mediated tumor accumulation. Histological examination of tumor tissues by H\u0026amp;E staining revealed extensive necrosis and structural disruption in the PS/pBDT@cCNV-treated group, whereas tumors from control groups retained largely viable tissue architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). Importantly, this potent antitumor efficacy was achieved without compromising systemic safety. Throughout the treatment course, mice maintained stable body weights (\u003cb\u003eFig. S16\u003c/b\u003e). In addition, serum biochemical analysis performed at sacrifice showed that liver function markers, including alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as well as renal function markers, including blood urea nitrogen (BUN) and creatinine, exhibited values comparable to those of control groups, with no marked treatment-related deviations observed (\u003cb\u003eFig. S17\u003c/b\u003e). Histological analysis of major organs further confirmed the absence of treatment-related pathological abnormalities (\u003cb\u003eFig. S18\u003c/b\u003e). Collectively, these results demonstrate that PS/pBDT@cCNVs effectively combine strong antitumor activity with favorable systemic biocompatibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Conclusion","content":"\u003cp\u003eIn summary, we engineered a biomimetic core-shell hybrid nanoplatform by integrating redox-active pBDT polymer cores with charge-modulated CNVs. This study demonstrates that controlled surface charge modulation, achieved through the incorporation of a minimal amount of cationic lipids, influences tumor accumulation behavior \u003cem\u003ein vivo\u003c/em\u003e. By modulating the surface charge of the nanovesicle shell, PS/pBDT@cCNVs exhibited improved tumor accumulation and intratumoral distribution compared with non-modified formulations, addressing key delivery limitations of conventional PDT.\u003c/p\u003e \u003cp\u003eConsequently, this optimized design enabled potent tumor ablation and sustained tumor suppression \u003cem\u003ein vivo\u003c/em\u003e during the treatment period, without observable systemic toxicity. Beyond PDT, this work demonstrates that surface charge modulation at the CNV interface is a practical and effective strategy for improving the \u003cem\u003ein vivo\u003c/em\u003e performance of biomimetic hybrid nanocarriers. This platform may provide a versatile framework for the development of precision nanomedicine strategies targeting solid tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIllustrations were created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (the Ministry of Science \u0026amp; ICT, MSIT) (RS-2024-00350878 and RS-2023-00242443), by the Bio \u0026amp; Medical Technology Development Program of the NRF funded by the MSIT (RS-2024-00440714), by the Korean Fund for Regenerative Medicine (KFRM) funded by MSIT and the Ministry of Health and Welfare, Republic of Korea (RS-2025-02223118), by the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Korean government (MSIT) (RS-2024-00402899), and by the KIST Institutional Program (2E32351-23-130).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Human and Animal Rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (SKKUIACUC2024-02-15-1). This article does not contain any studies involving human participants performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.-J.O. performed the experiments and wrote the manuscript. D.-Y.H., J.S.L., J.H., C.G.P., and H.L. contributed to data analysis and manuscript revision. W.P. and C.G.P. secured funding. W.P. supervised the project. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors: Hyeon-Ji Oh,\u0026nbsp;Dong-Yong Hong, Junseob Lee,\u0026nbsp;Jieun Han, Chun Gwon Park, Hyojin Lee, and Wooram Park*\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eDepartment of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), Seobu-ro 2066, Jangan-gu, Suwon, Gyeonggi 16419, Republic of Korea\u0026nbsp;\u003cul start=\"12\"\u003e\n \u003cli\u003e\u003cem\u003eHyeon-Ji Oh,\u0026nbsp;\u003c/em\u003e\u003cem\u003eDong-Yong Hong\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Junseob Lee, Jieun Han, and Wooram Park\u003c/em\u003e\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n \u003cli\u003eDepartment of MetaBioHealth, Institute for Cross-disciplinary Studies (ICS), SKKU, Seobu-ro 2066, Suwon, Gyeonggi 16419, Republic of Korea\u0026nbsp;\u003cul start=\"12\"\u003e\n \u003cli\u003e\u003cem\u003eWooram Park\u003c/em\u003e\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n \u003cli\u003eDepartment of Biomedical Engineering, ICS, SKKU, Seobu-ro 2066, Suwon, Gyeonggi 16419, Republic of Korea\u0026nbsp;\u003cul start=\"12\"\u003e\n \u003cli\u003e\u003cem\u003eChun Gwon Park\u003c/em\u003e\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n \u003cli\u003eBiomaterials Research Center, Korea Institute of Science and Technology (KIST), 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea\u0026nbsp;\u003cul start=\"50\"\u003e\n \u003cli\u003e\u003cem\u003eChun Gwon Park, Hyojin Lee, and Wooram Park\u003c/em\u003e\u003c/li\u003e\n \u003c/ul\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Wooram Park (email: [email protected])\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at the journal website\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A, Cancer statistics (2024) CA Cancer J Clin. 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Adv Mater 37(27). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/adma.202503004\u003c/span\u003e\u003cspan address=\"10.1002/adma.202503004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Photodynamic therapy (PDT), Biomimetic nanovesicles, Cell-derived nanovesicles (CNVs), Surface charge modulation, Tumor targeting drug delivery, Redox-responsive polymer","lastPublishedDoi":"10.21203/rs.3.rs-8862874/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8862874/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite the therapeutic potential of photodynamic therapy (PDT), its clinical efficacy is frequently limited by insufficient photosensitizer (PS) delivery and the strong antioxidant defense mechanisms of tumor cells. In this study, we developed surface charge-modulated biomimetic core-shell nanostructures by encapsulating PS-loaded poly(benzene-1,4-dithiol) (pBDT) nanoparticles within cell-derived nanovesicles (CNVs). To address the electrostatic barriers associated with tumor tissues, the CNV shell was functionalized with a controlled amount of cationic lipids (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP). This modification was designed to modulate surface electrostatic interactions at the tumor interface while maintaining membrane integrity and systemic safety. The resulting hybrid nanovesicles, termed PS/pBDT@cCNVs, integrate the redox-responsive properties of the pBDT core with the intrinsic biological functionalities of the CNV shell. This dual-modal strategy, which combines surface charge modulation with the intrinsic biological properties of CNVs, led to enhanced tumor accumulation and improved intratumoral distribution. Furthermore, the redox-active PS/pBDT core facilitated efficient intracellular ROS generation and glutathione (GSH) depletion, thereby amplifying the phototherapeutic impact. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evaluations confirmed that PS/pBDT@cCNVs achieved robust tumor ablation with negligible systemic toxicity. Collectively, this work presents a rationally designed, charge-tunable, redox-responsive hybrid nanoplatform that addresses key biological barriers in photodynamic cancer therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Surface Charge-Modulated Biomimetic Core-Shell Hybrid Nanovesicles for Redox-Triggered Synergistic Cancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-09 19:45:57","doi":"10.21203/rs.3.rs-8862874/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"522241c4-8b58-40a6-8701-dc755a5e16fe","owner":[],"postedDate":"March 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-11T21:46:40+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T21:54:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-09 19:45:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8862874","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8862874","identity":"rs-8862874","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}