ROS-Responsive Microneedle Patch for Targeted Delivery of Cryptotanshinone in Synergistic Antibacterial and Antioxidant Therapy of Infected Wounds

ROS-Responsive Microneedle Patch for Targeted Delivery of Cryptotanshinone in Synergistic Antibacterial and Antioxidant Therapy of Infected Wounds | 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 Article ROS-Responsive Microneedle Patch for Targeted Delivery of Cryptotanshinone in Synergistic Antibacterial and Antioxidant Therapy of Infected Wounds Wenbo Zeng, Weijie Sun, Yangjuan Tang, Dong Wan, Jixiang Tan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7630804/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 Chronic infected wounds remain a major clinical challenge due to persistent oxidative stress, bacterial colonization, and delayed tissue regeneration. To address these issues, we developed a reactive oxygen species (ROS)-responsive microneedle (MN) patch incorporating cryptotanshinone (CPT), a traditional Chinese medicine monomer with proven antioxidant and antibacterial properties. The microneedles were fabricated using a mold-casting technique based on a hydrogel matrix formed by tannic acid–quaternized chitosan (TA-QCTS), which endowed the patch with ROS-triggered degradation and controlled drug release. The resulting CPT-loaded microneedles exhibited uniform morphology, adequate mechanical strength (> 0.25 N/needle), and reliable skin insertion capacity. In vitro release assays confirmed efficient CPT encapsulation and sustained release under oxidative conditions mimicking the wound microenvironment. Antibacterial tests demonstrated potent inhibition against Staphylococcus aureus, Escherichia coli, and Streptococcus pyogenes. Moreover, the CPT-MN system effectively suppressed intracellular ROS, reduced pro-inflammatory cytokines (TNF-α, IL-6), upregulated anti-inflammatory IL-10, and promoted fibroblast proliferation and migration. Overall, this ROS-responsive microneedle platform combines smart drug delivery with bioactive phytochemicals, offering a promising strategy for localized treatment of chronic infected wounds. Biological sciences/Biotechnology Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Microbiology ROS-responsive hydrogel Cryptotanshinone delivery Infected wound healing Antibacterial microneedles Oxidative stress modulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Chronic infected wounds, such as diabetic foot ulcers, pressure sores, and post-surgical infections, pose a significant clinical burden due to persistent microbial colonization, excessive inflammation, and elevated oxidative stress. These factors disrupt the orderly phases of wound healing, resulting in prolonged tissue damage, delayed re-epithelialization, and a higher risk of complications including amputation or reinfection[ 1 , 2 ]. Standard treatments such as systemic antibiotics and passive wound dressings often fall short in addressing the complex pathophysiology of infected wounds and may even contribute to the emergence of antibiotic-resistant strains[ 3 ]. Thus, there is an urgent need for multifunctional, localized therapies that can simultaneously combat pathogens, regulate the inflammatory milieu, and restore redox balance within the wound microenvironment[ 4 , 5 ]. In recent years, microneedle (MN) patches based on hydrogel matrices have emerged as promising platforms for transdermal drug delivery in wound care[ 6 , 7 ]. These minimally invasive systems can penetrate the stratum corneum painlessly and deliver therapeutics directly to the dermal layer, improving bioavailability and targeting capability. Moreover, incorporating responsive materials into MN matrices allows for stimulus-triggered drug release at sites of injury or infection. Among various stimuli (e.g., pH, temperature, enzymes), reactive oxygen species (ROS) are particularly relevant in the context of infected wounds. Excess ROS not only damage surrounding tissues but also serve as biomarkers of inflammation, making them an ideal internal trigger for smart drug release[ 8 , 9 ]. Tannic acid (TA)-modified quaternized chitosan hydrogels (TA-QCTS) also abbreviate as QCTH have attracted attention as ROS-responsive biomaterials due to their redox-sensitive degradation behavior, excellent biocompatibility, and inherent antimicrobial properties[ 10 , 11 ]. When integrated into microneedle arrays, such hydrogels can serve as both structural support and functional matrices for controlled therapeutic release in oxidative environments. However, most studies to date focus on synthetic antibiotics or inorganic nanomaterials, with limited exploration of bioactive phytochemicals in MN platforms. Cryptotanshinone (CPT), a lipophilic diterpenoid derived from Salvia miltiorrhiza, exhibits broad-spectrum antimicrobial activity, strong antioxidant capacity, and immunomodulatory effects through downregulation of inflammatory mediators like TNF-α and IL-6[ 12 – 14 ]. Despite its pharmacological potential, the clinical translation of CPT has been hindered by poor water solubility and low bioavailability. Encapsulating CPT within a ROS-degradable hydrogel-based MN system offers a feasible strategy to overcome these limitations and achieve targeted, responsive delivery for wound healing. In this study, we designed a ROS-responsive microneedle patch based on TA-QCTS hydrogel incorporating cryptotanshinone for the localized treatment of infected wounds. We systematically evaluated its physicochemical properties, drug release kinetics, antibacterial performance, and in vitro biological effects including ROS scavenging, cytokine regulation (TNF-α, IL-6, IL-10), and fibroblast migration and proliferation. This platform integrates smart material engineering with the therapeutic potential of traditional herbal compounds, providing a synergistic approach for the treatment of infected wounds. 2. Materials and Methods 2.1 Materials Cryptotanshinone (CPT, ≥ 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The ROS-responsive hydrogel (Tannic acid modified quaternized chitosan hydrogels, TA-QCTS/QCTH) was obtained from Xi’an Qiyue Biotechnology Co., Ltd (Xi’an, China). NIH-3T3 fibroblasts were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Streptococcus pyogenes were purchased from the China General Microbiological Culture Collection Center (CGMCC). All cell culture reagents were purchased from Gibco (USA). Commercial ELISA kits for IL-6, IL-10, and TNF-α were obtained from MultiSciences (Hangzhou, China). All other reagents were of analytical grade and used as received. 2.2 Fabrication of CPT-Loaded ROS-Responsive Hydrogel Microneedle Patches To prepare the CPT@QCTH microneedle system, CPT was first dissolved in a small amount of ethanol (final concentration: 100 µg/mL) and homogeneously mixed with the ROS-responsive hydrogel under gentle stirring. The hydrogel-drug mixture was then cast into PDMS microneedle molds and centrifuged at 3000 rpm for 5 minutes to remove air bubbles and ensure complete mold filling. The molds were incubated at 37°C for 12 hours to allow hydrogel curing. The formed microneedle patches were carefully demolded and stored at 4°C until use. 2.3 Physicochemical Characterization The surface morphology and structural uniformity of the microneedles were observed using scanning electron microscopy (SEM, Hitachi SU8010). Mechanical properties were measured with a universal testing machine (TA.XT Plus, Stable Micro Systems). The fracture force per needle was recorded by compressing each tip against a flat stainless-steel plate. The drug loading content and encapsulation efficiency were determined by dissolving a known number of CPT@QCTH patches in ethanol, followed by UV–Vis spectrophotometry at 430 nm. Encapsulation efficiency (%) was calculated as the ratio of encapsulated CPT to the initially added CPT. 2.4 In Vitro Drug Release Behavior To investigate CPT release kinetics, the CPT-loaded microneedle patches were immersed in PBS (pH 7.4) containing either 0 mM or 0.5 mM H₂O₂ at 37°C under shaking. At predetermined time intervals (0–72 h), 1 mL of release medium was withdrawn and replaced with fresh buffer. CPT concentration was quantified using UV–Vis spectroscopy at 430 nm. The cumulative release profile was plotted, and data were expressed as mean ± SD (n = 3). 2.5 In Vitro Antibacterial Activity Agar diffusion assay: Sterile MN patches were placed on LB agar plates pre-seeded with S. aureus, E. coli, or S. pyogenes (10⁸ CFU/mL). After 24 h incubation at 37°C, inhibition zones were measured. 2.6 Scratch Wound Healing Assay To assess the pro-migratory effect of the CPT@QCTH microneedle extract, a scratch wound healing assay was performed using NIH-3T3 fibroblasts. Cells were seeded into 6-well plates at a density of 5 × 10⁵ cells/well and cultured until reaching 90% confluence. A straight scratch was created in the cell monolayer using a sterile 200 µL pipette tip. The wells were gently washed with PBS to remove detached cells, and the medium was replaced with fresh DMEM containing 1% FBS and different treatments: (1) Control, (2) ROS (100 µM H₂O₂), (3) CPT, and (4) CPT@QCTH extract (equivalent CPT concentration). Images of the scratch area were captured at 0 h and 24 h using an inverted microscope (Olympus, Japan). The percentage of wound closure was quantified using ImageJ software by comparing the remaining wound area at 24 h to the initial area. 2.7 Cytocompatibility and ROS Scavenging NIH-3T3 fibroblasts were seeded in 96-well plates (5 × 10³ cells/well) and incubated with microneedle extracts (equivalent CPT concentration) for 24 h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, with absorbance recorded at 450 nm. For ROS detection, fibroblasts were stimulated with LPS (1 µg/mL) for 4 h, followed by treatment with CPT@QCTH extract. Cells were then stained with DCFH-DA (10 µM, 30 min), and intracellular ROS levels were measured using a fluorescence microplate reader (excitation/emission: 485/535 nm). Fluorescence microscopy images were also obtained. 2.8 Inflammatory Cytokine Analysis To assess anti-inflammatory effects, NIH-3T3 cells were stimulated with LPS (1 µg/mL) and then treated with CPT@QCTH extracts or blank hydrogel controls for 24 h. The supernatants were collected, and the levels of IL-6, TNF-α, and IL-10 were determined using commercial ELISA kits according to the manufacturer’s protocol. IL-6 and TNF-α were used as pro-inflammatory markers, while IL-10 served as a protective anti-inflammatory marker. 2.9 Statistical Analysis All experiments were conducted in triplicate. Data are presented as mean ± standard deviation (SD). One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons. Statistical significance was set at p < 0.05. GraphPad Prism 9.0 was used for statistical analysis and graphing. 3. Results 3.1 Characterization of ROS-Responsive Hydrogel and Microneedle Patch The ROS-responsive hydrogel precursor solution exhibited a rapid sol-to-gel transition upon exposure to 0.5 mM H₂O₂ (simulating pathological ROS levels), as visually confirmed by the fluid-to-solid phase change in Figure 1A(a, b). SEM images revealed a porous microstructure, suggesting good internal network formation (Figure 1B). The microneedle patch displayed a regular array when placed on a fingertip (Figure 1C), with intact pyramidal tips observed under optical microscopy (Figure 1D). SEM confirmed the sharp and uniform morphology of the microneedles (Figure 1E). FITC-labeled microneedles demonstrated localized fluorescence in the tip region, indicating successful drug encapsulation (Figure 1F). 3.2 Mechanical Performance, Skin Penetration, and Dissolution Behavior Mechanical testing showed that the microneedles maintained sufficient compressive strength, with an average force above the threshold required for skin insertion (Figure 2A). Trypan blue staining on mouse skin confirmed successful penetration (Figure 2B). The microneedles gradually dissolved within 120 minutes under physiological conditions (Figure 2C), and quantitative data demonstrated over 50% dissolution by the end of the period (Figure 2D). 3.3 Drug Loading, ROS-Responsive Release, and Antibacterial Performance The microneedle patch achieved a drug loading content of ~34.6 µg（about 36.8%）and an encapsulation efficiency of ~79.2% (Figure 3A). Under oxidative conditions (0.5 mM H₂O₂), the cumulative release of CPT reached approximately 88% over 72 h, compared with <30% in non-ROS environments (Figure 3B). Antibacterial tests showed distinct inhibition zones against E. coli, S. aureus, and S. haemolyticus (Figure 3C–D), indicating broad-spectrum antimicrobial activity. 3.4 Cytocompatibility and Cell Migration under Oxidative Stress Phase-contrast images of NIH-3T3 fibroblasts cultured under various conditions showed that cells maintained a normal morphology without signs of cytotoxicity in all groups (Figure 4A). A scratch wound healing assay was employed to assess the effect of the CPT@QCTH microneedle extract on fibroblast migration under oxidative stress (Figure 4B). After 24 h, the CPT@QCTH group exhibited significantly enhanced wound closure compared to both the untreated control and CPT-only groups. CCK-8 assays further demonstrated good cytocompatibility, with relative cell viability exceeding 115% after treatment with CPT@QCTH extracts (Figure 4C). Quantification of scratch closure revealed a markedly higher migration rate in the CPT@QCTH group (Figure 4D), suggesting that the hydrogel microneedles not only maintained biocompatibility but also promoted cell motility under stress conditions. 3.5 ROS Scavenging and Modulation of Inflammatory Cytokines DHE staining revealed that CPT@QCTH microneedles effectively reduced intracellular ROS levels compared to the ROS group (Figure 5A–B). RT-qPCR analysis showed that CPT@QCTH significantly decreased the expression of IL-6 and TNF-α while upregulating IL-10 (Figure 5C). Consistent results were obtained by ELISA, where The CPT@QCTH group showed notable downregulation of IL-6 and TNF-α while increasing IL-10, indicating effective inflammatory modulation (Figure 5D–F), highlighting its antioxidant and anti-inflammatory effects. 4. Discussion The ROS-responsive microneedle platform developed in this study integrates responsive drug release, antimicrobial efficacy, and cytoprotective functionality, offering a promising approach for treating infected wounds. The uniform microneedle architecture and mechanical strength exceeding 0.25 N per needle ensured effective skin penetration, which is critical for localized transdermal delivery without inducing tissue trauma[ 13 , 14 ]. Moreover, the successful encapsulation and retention of cryptotanshinone (CPT) within the microneedle tips, as confirmed by FITC fluorescence and controlled release profiles, validate the structural integrity and responsiveness of the delivery system. By integrating tannic acid-modified quaternary chitosan, the hydrogel gained ROS-sensitive characteristics, allowing context-dependent CPT diffusion. This feature is especially advantageous for chronic and infected wounds, where ROS levels are abnormally elevated and often disrupt healing processes[ 15 , 16 ]. Similar ROS-triggered platforms have demonstrated efficacy in diabetic wound models and inflammatory skin disorders[ 17 ], supporting the rationality of our material selection. CPT retained its antimicrobial activity when delivered via the microneedle platform and produced distinct inhibition zones against E. coli, S. aureus, and S. haemolyticus. This aligns with prior reports describing CPT’s ability to disrupt bacterial membrane integrity and inhibit bacterial respiration[ 18 , 19 ]. Importantly, the use of microneedles enables localized delivery, reducing systemic exposure and potentially mitigating antibiotic resistance—a growing concern in chronic wound care[ 20 ]. In addition to its antibacterial function, CPT@QCTH patches reduced ROS levels and suppressed inflammatory cytokines, including TNF-α and IL-6, while promoting anti-inflammatory IL-10 expression. These effects confirm the dual antioxidant and immunomodulatory role of CPT[ 21 , 22 ]. Our data also demonstrated that the system promotes fibroblast viability and migration in a scratch assay model under oxidative stress, suggesting beneficial effects on cellular regeneration. This is consistent with previous studies showing that moderate ROS scavenging combined with anti-inflammatory signaling facilitates re-epithelialization and tissue repair[ 23 ]. Clinically, microneedles improve patient tolerance, enable painless dosing, and bypass hepatic metabolism, offering benefits over systemic or topical routes.[ 24 , 25 ]. It also enables higher drug localization, bypassing gastrointestinal degradation and hepatic metabolism. The integration of ROS responsiveness with herbal bioactivity addresses key limitations of conventional wound dressings, particularly in managing inflamed, infected, and slow-healing wounds. Nonetheless, this study is limited to in vitro evaluations. Future work will focus on in vivo validation using animal wound models to assess real-time therapeutic efficacy, inflammatory modulation, and biocompatibility. Further optimization will also be directed toward long-term stability, cost-effective fabrication, and clinical scalability. Limitations Although the CPT@QCTH microneedle patch demonstrated promising antibacterial, antioxidant, and anti-inflammatory effects in vitro, several limitations should be noted. First, this study was limited to in vitro assays, and no in vivo wound healing models were employed to validate therapeutic efficacy, biosafety, and pharmacokinetics under physiological conditions. Second, the ROS-responsiveness of the hydrogel system was only evaluated using H₂O₂ stimulation; further studies are needed to confirm responsiveness under dynamic and heterogeneous ROS levels in real infected tissue environments. Third, the antibacterial assessment was conducted primarily using inhibition zone assays, which provide qualitative evidence. More quantitative approaches, such as colony-forming unit (CFU) counts and time-kill kinetics, are warranted to fully characterize the antimicrobial potency. These limitations highlight the need for further preclinical studies using animal models and more comprehensive microbiological evaluations to fully establish the translational potential of this microneedle-based platform. 5. Conclusion In this study, we developed a ROS-responsive hydrogel microneedle patch incorporating cryptotanshinone (CPT), a natural compound with antibacterial and antioxidant properties. The patch exhibited uniform morphology, sufficient mechanical strength, and ROS-triggered drug release suitable for infected microenvironments. In vitro results demonstrated that CPT-loaded microneedles effectively inhibited Staphylococcus aureus and Escherichia coli, reduced intracellular ROS levels, and suppressed pro-inflammatory cytokines (TNF-α, IL-6) in stimulated fibroblasts, while maintaining excellent cytocompatibility. These findings highlight the potential of integrating traditional herbal agents with smart microneedle platforms for localized infected wound therapy. Further in vivo studies are warranted to confirm therapeutic efficacy and guide clinical translation. Declarations Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. Acknowledgements The authors declare no external funding was received for this study. All authors acknowledge the institutional support from Chongqing Medical University. Ethics Statement All animal experiments were conducted under protocols approved by the Animal Ethics Committee of Chongqing Medical University (Protocol NO.IACUC-CQMU-2025-0444). Competing Interests The authors declare no competing interests. Author Contributions Zeng Wenbo designed the study and performed the data analysis. Sun Weijie and Tang Yangjuan were responsible for data collection and bioinformatics analyses. Wan Dong and Tan Jixiang supervised the research and provided guidance throughout the study. Zeng Wenbo and Tan Jixiang wrote the manuscript. All authors reviewed and approved the final version of the manuscript. References Zarei M, Soleimanian-Zad S. Antibacterial activity of bioactive wound dressing materials: a review. J Tissue Viability. 2022;31(4):582–90. Xia G, Wang L, Wang Y, et al. Current advancements of bioactive hydrogels for wound healing. Front Bioeng Biotechnol. 2022;10:915922. Yang J, Chen Z, Pan D, et al. A bioinspired self-powered dressing to accelerate wound healing through ROS scavenging and electrical stimulation. Adv Sci (Weinh). 2023;10(11):e2206755. Chen X, Qin H, Hu Y, et al. ROS-responsive hydrogel for synergistic antibacterial and wound healing via photothermal-enhanced release of antibiotics. Adv Funct Mater. 2023;33(4):2208183. Xu Q, He Y, Wang H, et al. ROS-responsive hydrogels for tissue engineering and regenerative medicine applications. J Mater Chem B. 2022;10(17):3195–216. Zhang Y, Zhang Y, Zhang H, et al. Reactive oxygen species-responsive microneedles with antibacterial activity for accelerated wound healing. Int J Pharm. 2022;612:121345. Ye Y, Yu J, Wen D, et al. Microneedles integrated with pancreatic cells and synthetic glucose-signal amplifiers for smart insulin delivery. Adv Mater. 2020;32(8):e1906759. Zhang Y, Jiang P, Ye M, et al. 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Cryptotanshinone enhances wound healing in type 2 diabetes with modulatory effects on inflammation, angiogenesis and extracellular matrix remodelling. Pharm Biol. 2020;58(1):845-853. Li Z, Wei W, Zhang M, et al. Cryptotanshinone-Doped Photothermal Synergistic MXene@PDA Nanosheets with Antibacterial and Anti-Inflammatory Properties for Wound Healing. Adv Healthc Mater. 2023;12(28):e2301060. Zhao H, Huang J, Li Y, et al. ROS-scavenging hydrogel to promote healing of bacteria infected diabetic wounds. Biomaterials. 2020;258:120286. Liang Y, Zhao X, Hu T, et al. Adhesive hemostatic bioengineered sponge with antibacterial activity for deep wound repair. Bioact Mater. 2021;6(1):154–167. Xie J, Chen J, Li C, et al. Photocrosslinked hydrogel loaded with cryptotanshinone promotes healing of infected wounds. Int J Pharm. 2022;621:121807. Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":8309624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of ROS-responsive hydrogel and microneedle patch.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Visual demonstration of ROS-triggered sol-gel transition. (a) Free-flowing liquid state of QCTH precursor solution; (b) Solid hydrogel formed after oxidative crosslinking induced by 0.5 mM H₂O₂.\u003c/p\u003e\n\u003cp\u003e(B) SEM images showing the porous microstructure of the hydrogel at low (a) and high (b) magnification.\u003c/p\u003e\n\u003cp\u003e(C) Macroscopic image of the microneedle array patch placed on a fingertip.\u003c/p\u003e\n\u003cp\u003e(D) Optical microscopic view of the complete microneedle array.\u003c/p\u003e\n\u003cp\u003e(E) SEM image of microneedle tips indicating sharp and uniform structure.\u003c/p\u003e\n\u003cp\u003e(F) Fluorescence image of FITC-labeled microneedles confirming drug distribution within the microneedle tips.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/13f2c61152bf688bbe2bd7ed.png"},{"id":94020325,"identity":"53405717-2407-4a88-927e-a76fc3450d66","added_by":"auto","created_at":"2025-10-21 12:13:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6687656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanical strength, skin penetration ability, and dissolution behavior of the CPT@QCTH microneedle patch.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Compression force–displacement curve showing the mechanical strength of the microneedles.\u003c/p\u003e\n\u003cp\u003e(B) Skin application of microneedle patch on mice: (a) before and (b) after trypan blue staining, indicating successful skin penetration.\u003c/p\u003e\n\u003cp\u003e(C) Time-dependent dissolution process of microneedles at 0, 30, 60, 90, and 120 min in PBS at 37 °C.\u003c/p\u003e\n\u003cp\u003e(D) Quantitative dissolution profile of the microneedle patch over 120 min (n = 3, mean ± SD).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/e676bb8352dc94ea697f910c.png"},{"id":94020339,"identity":"edbf37e5-f763-4f61-abb9-b5d74cad4d55","added_by":"auto","created_at":"2025-10-21 12:13:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18119347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrug loading, ROS-responsive release, and antibacterial properties of CPT@QCTH microneedle patch.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Drug loading content and encapsulation efficiency of the microneedle patch.\u003c/p\u003e\n\u003cp\u003e(B) In vitro cumulative drug release profiles under ROS and non-ROS conditions.\u003c/p\u003e\n\u003cp\u003e(C) Inhibition zone diameter ratios for E. coli, S. aureus, and S. haemolyticus.\u003c/p\u003e\n\u003cp\u003e(D) Representative images of inhibition zones（red circle）formed by microneedles against E. coli, S. aureus, and S. haemolyticus after 24 h culture.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/e23a77f83ddcf2fa8b5458e0.png"},{"id":94020793,"identity":"fe38d6e1-ea80-44f7-9de4-2ce3157696f4","added_by":"auto","created_at":"2025-10-21 12:21:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6758766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytocompatibility and wound healing ability of CPT@QCTH microneedles under oxidative stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative phase-contrast images showing fibroblast morphology under different treatment conditions.\u003c/p\u003e\n\u003cp\u003e(B) Scratch wound healing assay at 0 h and 24 h in Control, ROS, CPT, and CPT@QCTH groups.\u003c/p\u003e\n\u003cp\u003e(C) Relative cell viability measured by CCK-8 assay after 24 h treatment.\u003c/p\u003e\n\u003cp\u003e(D) Quantified migration rates of fibroblasts based on scratch wound closure (n = 3, mean ± SD; p \u0026lt; 0.05, p \u0026lt; 0.01, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/fd87f01ff14223ccef33e386.png"},{"id":94021652,"identity":"26de62eb-15e0-4da5-93d7-a5cfd63f05cf","added_by":"auto","created_at":"2025-10-21 12:29:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1302262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROS scavenging ability and anti-inflammatory effects of CPT@QCTH microneedles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative DHE staining images (red fluorescence) of intracellular ROS levels.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of DHE fluorescence intensity.\u003c/p\u003e\n\u003cp\u003e(C) Relative mRNA expression levels of IL-6, TNF-α, and IL-10 measured by RT-qPCR.\u003c/p\u003e\n\u003cp\u003e(D–F) Total protein levels of IL-6 (D), TNF-α (E), and IL-10 (F) measured by ELISA (n = 3, mean ± SD; p \u0026lt; 0.05, p \u0026lt; 0.01, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/bcf88f8942592147e5bb7f42.png"},{"id":94525523,"identity":"82e3e46e-5423-4841-b71a-2168f61c4d87","added_by":"auto","created_at":"2025-10-28 16:59:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":39284333,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/f081caa0-3694-4d56-a05e-d7aae42c6309.pdf"},{"id":94020324,"identity":"d62190db-8d95-4561-ab60-514f3c9cb2fb","added_by":"auto","created_at":"2025-10-21 12:13:24","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":577094,"visible":true,"origin":"","legend":"","description":"","filename":"rawdata.zip","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/3c94581a9c7467bfcd6fad30.zip"},{"id":94020811,"identity":"a9495272-7c38-4852-9f1e-ba8b648e0eb9","added_by":"auto","created_at":"2025-10-21 12:21:25","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14745850,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract01.tif","url":"https://assets-eu.researchsquare.com/files/rs-7630804/v1/f2eae4b473b0031f727e2afa.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"ROS-Responsive Microneedle Patch for Targeted Delivery of Cryptotanshinone in Synergistic Antibacterial and Antioxidant Therapy of Infected Wounds","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic infected wounds, such as diabetic foot ulcers, pressure sores, and post-surgical infections, pose a significant clinical burden due to persistent microbial colonization, excessive inflammation, and elevated oxidative stress. These factors disrupt the orderly phases of wound healing, resulting in prolonged tissue damage, delayed re-epithelialization, and a higher risk of complications including amputation or reinfection[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Standard treatments such as systemic antibiotics and passive wound dressings often fall short in addressing the complex pathophysiology of infected wounds and may even contribute to the emergence of antibiotic-resistant strains[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, there is an urgent need for multifunctional, localized therapies that can simultaneously combat pathogens, regulate the inflammatory milieu, and restore redox balance within the wound microenvironment[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, microneedle (MN) patches based on hydrogel matrices have emerged as promising platforms for transdermal drug delivery in wound care[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These minimally invasive systems can penetrate the stratum corneum painlessly and deliver therapeutics directly to the dermal layer, improving bioavailability and targeting capability. Moreover, incorporating responsive materials into MN matrices allows for stimulus-triggered drug release at sites of injury or infection. Among various stimuli (e.g., pH, temperature, enzymes), reactive oxygen species (ROS) are particularly relevant in the context of infected wounds. Excess ROS not only damage surrounding tissues but also serve as biomarkers of inflammation, making them an ideal internal trigger for smart drug release[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTannic acid (TA)-modified quaternized chitosan hydrogels (TA-QCTS) also abbreviate as QCTH have attracted attention as ROS-responsive biomaterials due to their redox-sensitive degradation behavior, excellent biocompatibility, and inherent antimicrobial properties[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. When integrated into microneedle arrays, such hydrogels can serve as both structural support and functional matrices for controlled therapeutic release in oxidative environments. However, most studies to date focus on synthetic antibiotics or inorganic nanomaterials, with limited exploration of bioactive phytochemicals in MN platforms.\u003c/p\u003e\u003cp\u003eCryptotanshinone (CPT), a lipophilic diterpenoid derived from Salvia miltiorrhiza, exhibits broad-spectrum antimicrobial activity, strong antioxidant capacity, and immunomodulatory effects through downregulation of inflammatory mediators like TNF-α and IL-6[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Despite its pharmacological potential, the clinical translation of CPT has been hindered by poor water solubility and low bioavailability. Encapsulating CPT within a ROS-degradable hydrogel-based MN system offers a feasible strategy to overcome these limitations and achieve targeted, responsive delivery for wound healing.\u003c/p\u003e\u003cp\u003eIn this study, we designed a ROS-responsive microneedle patch based on TA-QCTS hydrogel incorporating cryptotanshinone for the localized treatment of infected wounds. We systematically evaluated its physicochemical properties, drug release kinetics, antibacterial performance, and in vitro biological effects including ROS scavenging, cytokine regulation (TNF-α, IL-6, IL-10), and fibroblast migration and proliferation. This platform integrates smart material engineering with the therapeutic potential of traditional herbal compounds, providing a synergistic approach for the treatment of infected wounds.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eCryptotanshinone (CPT, \u0026ge;\u0026thinsp;98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The ROS-responsive hydrogel (Tannic acid modified quaternized chitosan hydrogels, TA-QCTS/QCTH) was obtained from Xi\u0026rsquo;an Qiyue Biotechnology Co., Ltd (Xi\u0026rsquo;an, China). NIH-3T3 fibroblasts were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Streptococcus pyogenes were purchased from the China General Microbiological Culture Collection Center (CGMCC). All cell culture reagents were purchased from Gibco (USA). Commercial ELISA kits for IL-6, IL-10, and TNF-α were obtained from MultiSciences (Hangzhou, China). All other reagents were of analytical grade and used as received.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Fabrication of CPT-Loaded ROS-Responsive Hydrogel Microneedle Patches\u003c/h2\u003e\u003cp\u003eTo prepare the CPT@QCTH microneedle system, CPT was first dissolved in a small amount of ethanol (final concentration: 100 \u0026micro;g/mL) and homogeneously mixed with the ROS-responsive hydrogel under gentle stirring. The hydrogel-drug mixture was then cast into PDMS microneedle molds and centrifuged at 3000 rpm for 5 minutes to remove air bubbles and ensure complete mold filling. The molds were incubated at 37\u0026deg;C for 12 hours to allow hydrogel curing. The formed microneedle patches were carefully demolded and stored at 4\u0026deg;C until use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Physicochemical Characterization\u003c/h2\u003e\u003cp\u003eThe surface morphology and structural uniformity of the microneedles were observed using scanning electron microscopy (SEM, Hitachi SU8010). Mechanical properties were measured with a universal testing machine (TA.XT Plus, Stable Micro Systems). The fracture force per needle was recorded by compressing each tip against a flat stainless-steel plate.\u003c/p\u003e\u003cp\u003eThe drug loading content and encapsulation efficiency were determined by dissolving a known number of CPT@QCTH patches in ethanol, followed by UV\u0026ndash;Vis spectrophotometry at 430 nm. Encapsulation efficiency (%) was calculated as the ratio of encapsulated CPT to the initially added CPT.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 In Vitro Drug Release Behavior\u003c/h2\u003e\u003cp\u003eTo investigate CPT release kinetics, the CPT-loaded microneedle patches were immersed in PBS (pH 7.4) containing either 0 mM or 0.5 mM H₂O₂ at 37\u0026deg;C under shaking. At predetermined time intervals (0\u0026ndash;72 h), 1 mL of release medium was withdrawn and replaced with fresh buffer. CPT concentration was quantified using UV\u0026ndash;Vis spectroscopy at 430 nm. The cumulative release profile was plotted, and data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 In Vitro Antibacterial Activity\u003c/h2\u003e\u003cp\u003eAgar diffusion assay: Sterile MN patches were placed on LB agar plates pre-seeded with S. aureus, E. coli, or S. pyogenes (10⁸ CFU/mL). After 24 h incubation at 37\u0026deg;C, inhibition zones were measured.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Scratch Wound Healing Assay\u003c/h2\u003e\u003cp\u003eTo assess the pro-migratory effect of the CPT@QCTH microneedle extract, a scratch wound healing assay was performed using NIH-3T3 fibroblasts. Cells were seeded into 6-well plates at a density of 5 \u0026times; 10⁵ cells/well and cultured until reaching 90% confluence. A straight scratch was created in the cell monolayer using a sterile 200 \u0026micro;L pipette tip. The wells were gently washed with PBS to remove detached cells, and the medium was replaced with fresh DMEM containing 1% FBS and different treatments: (1) Control, (2) ROS (100 \u0026micro;M H₂O₂), (3) CPT, and (4) CPT@QCTH extract (equivalent CPT concentration).\u003c/p\u003e\u003cp\u003eImages of the scratch area were captured at 0 h and 24 h using an inverted microscope (Olympus, Japan). The percentage of wound closure was quantified using ImageJ software by comparing the remaining wound area at 24 h to the initial area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Cytocompatibility and ROS Scavenging\u003c/h2\u003e\u003cp\u003eNIH-3T3 fibroblasts were seeded in 96-well plates (5 \u0026times; 10\u0026sup3; cells/well) and incubated with microneedle extracts (equivalent CPT concentration) for 24 h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, with absorbance recorded at 450 nm.\u003c/p\u003e\u003cp\u003eFor ROS detection, fibroblasts were stimulated with LPS (1 \u0026micro;g/mL) for 4 h, followed by treatment with CPT@QCTH extract. Cells were then stained with DCFH-DA (10 \u0026micro;M, 30 min), and intracellular ROS levels were measured using a fluorescence microplate reader (excitation/emission: 485/535 nm). Fluorescence microscopy images were also obtained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Inflammatory Cytokine Analysis\u003c/h2\u003e\u003cp\u003eTo assess anti-inflammatory effects, NIH-3T3 cells were stimulated with LPS (1 \u0026micro;g/mL) and then treated with CPT@QCTH extracts or blank hydrogel controls for 24 h. The supernatants were collected, and the levels of IL-6, TNF-α, and IL-10 were determined using commercial ELISA kits according to the manufacturer\u0026rsquo;s protocol. IL-6 and TNF-α were used as pro-inflammatory markers, while IL-10 served as a protective anti-inflammatory marker.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were conducted in triplicate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used for multiple group comparisons. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GraphPad Prism 9.0 was used for statistical analysis and graphing.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Characterization of ROS-Responsive Hydrogel and Microneedle Patch\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ROS-responsive hydrogel precursor solution exhibited a rapid sol-to-gel transition upon exposure to 0.5 mM H₂O₂ (simulating pathological ROS levels), as visually confirmed by the fluid-to-solid phase change in Figure 1A(a, b). SEM images revealed a porous microstructure, suggesting good internal network formation (Figure 1B). The microneedle patch displayed a regular array when placed on a fingertip (Figure 1C), with intact pyramidal tips observed under optical microscopy (Figure 1D). SEM confirmed the sharp and uniform morphology of the microneedles (Figure 1E). FITC-labeled microneedles demonstrated localized fluorescence in the tip region, indicating successful drug encapsulation (Figure 1F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Mechanical Performance, Skin Penetration, and Dissolution Behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMechanical testing showed that the microneedles maintained sufficient compressive strength, with an average force above the threshold required for skin insertion (Figure 2A). Trypan blue staining on mouse skin confirmed successful penetration (Figure 2B). The microneedles gradually dissolved within 120 minutes under physiological conditions (Figure 2C), and quantitative data demonstrated over 50% dissolution by the end of the period (Figure 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Drug Loading, ROS-Responsive Release, and Antibacterial Performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microneedle patch achieved a drug loading content of ~34.6 \u0026micro;g（about \u0026nbsp;36.8%）and an encapsulation efficiency of ~79.2% (Figure 3A). Under oxidative conditions (0.5 mM H₂O₂), the cumulative release of CPT reached approximately 88% over 72 h, compared with \u0026lt;30% in non-ROS environments (Figure 3B). Antibacterial tests showed distinct inhibition zones against E. coli, S. aureus, and S. haemolyticus (Figure 3C\u0026ndash;D), indicating broad-spectrum antimicrobial activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Cytocompatibility and Cell Migration under Oxidative Stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhase-contrast images of NIH-3T3 fibroblasts cultured under various conditions showed that cells maintained a normal morphology without signs of cytotoxicity in all groups (Figure 4A). A scratch wound healing assay was employed to assess the effect of the CPT@QCTH microneedle extract on fibroblast migration under oxidative stress (Figure 4B). After 24 h, the CPT@QCTH group exhibited significantly enhanced wound closure compared to both the untreated control and CPT-only groups.\u003c/p\u003e\n\u003cp\u003eCCK-8 assays further demonstrated good cytocompatibility, with relative cell viability exceeding 115% after treatment with CPT@QCTH extracts (Figure 4C). Quantification of scratch closure revealed a markedly higher migration rate in the CPT@QCTH group (Figure 4D), suggesting that the hydrogel microneedles not only maintained biocompatibility but also promoted cell motility under stress conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 ROS Scavenging and Modulation of Inflammatory Cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDHE staining revealed that CPT@QCTH microneedles effectively reduced intracellular ROS levels compared to the ROS group (Figure 5A\u0026ndash;B). RT-qPCR analysis showed that CPT@QCTH significantly decreased the expression of IL-6 and TNF-\u0026alpha; while upregulating IL-10 (Figure 5C). Consistent results were obtained by ELISA, where The CPT@QCTH group showed notable downregulation of IL-6 and TNF-\u0026alpha; while increasing IL-10, indicating effective inflammatory modulation (Figure 5D\u0026ndash;F), highlighting its antioxidant and anti-inflammatory effects.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe ROS-responsive microneedle platform developed in this study integrates responsive drug release, antimicrobial efficacy, and cytoprotective functionality, offering a promising approach for treating infected wounds. The uniform microneedle architecture and mechanical strength exceeding 0.25 N per needle ensured effective skin penetration, which is critical for localized transdermal delivery without inducing tissue trauma[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, the successful encapsulation and retention of cryptotanshinone (CPT) within the microneedle tips, as confirmed by FITC fluorescence and controlled release profiles, validate the structural integrity and responsiveness of the delivery system.\u003c/p\u003e\u003cp\u003eBy integrating tannic acid-modified quaternary chitosan, the hydrogel gained ROS-sensitive characteristics, allowing context-dependent CPT diffusion. This feature is especially advantageous for chronic and infected wounds, where ROS levels are abnormally elevated and often disrupt healing processes[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similar ROS-triggered platforms have demonstrated efficacy in diabetic wound models and inflammatory skin disorders[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], supporting the rationality of our material selection. CPT retained its antimicrobial activity when delivered via the microneedle platform and produced distinct inhibition zones against E. coli, S. aureus, and S. haemolyticus. This aligns with prior reports describing CPT\u0026rsquo;s ability to disrupt bacterial membrane integrity and inhibit bacterial respiration[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Importantly, the use of microneedles enables localized delivery, reducing systemic exposure and potentially mitigating antibiotic resistance\u0026mdash;a growing concern in chronic wound care[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to its antibacterial function, CPT@QCTH patches reduced ROS levels and suppressed inflammatory cytokines, including TNF-α and IL-6, while promoting anti-inflammatory IL-10 expression. These effects confirm the dual antioxidant and immunomodulatory role of CPT[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our data also demonstrated that the system promotes fibroblast viability and migration in a scratch assay model under oxidative stress, suggesting beneficial effects on cellular regeneration. This is consistent with previous studies showing that moderate ROS scavenging combined with anti-inflammatory signaling facilitates re-epithelialization and tissue repair[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eClinically, microneedles improve patient tolerance, enable painless dosing, and bypass hepatic metabolism, offering benefits over systemic or topical routes.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It also enables higher drug localization, bypassing gastrointestinal degradation and hepatic metabolism. The integration of ROS responsiveness with herbal bioactivity addresses key limitations of conventional wound dressings, particularly in managing inflamed, infected, and slow-healing wounds.\u003c/p\u003e\u003cp\u003eNonetheless, this study is limited to in vitro evaluations. Future work will focus on in vivo validation using animal wound models to assess real-time therapeutic efficacy, inflammatory modulation, and biocompatibility. Further optimization will also be directed toward long-term stability, cost-effective fabrication, and clinical scalability.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough the CPT@QCTH microneedle patch demonstrated promising antibacterial, antioxidant, and anti-inflammatory effects in vitro, several limitations should be noted. First, this study was limited to in vitro assays, and no in vivo wound healing models were employed to validate therapeutic efficacy, biosafety, and pharmacokinetics under physiological conditions. Second, the ROS-responsiveness of the hydrogel system was only evaluated using H₂O₂ stimulation; further studies are needed to confirm responsiveness under dynamic and heterogeneous ROS levels in real infected tissue environments. Third, the antibacterial assessment was conducted primarily using inhibition zone assays, which provide qualitative evidence. More quantitative approaches, such as colony-forming unit (CFU) counts and time-kill kinetics, are warranted to fully characterize the antimicrobial potency. These limitations highlight the need for further preclinical studies using animal models and more comprehensive microbiological evaluations to fully establish the translational potential of this microneedle-based platform.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we developed a ROS-responsive hydrogel microneedle patch incorporating cryptotanshinone (CPT), a natural compound with antibacterial and antioxidant properties. The patch exhibited uniform morphology, sufficient mechanical strength, and ROS-triggered drug release suitable for infected microenvironments. In vitro results demonstrated that CPT-loaded microneedles effectively inhibited Staphylococcus aureus and Escherichia coli, reduced intracellular ROS levels, and suppressed pro-inflammatory cytokines (TNF-α, IL-6) in stimulated fibroblasts, while maintaining excellent cytocompatibility. These findings highlight the potential of integrating traditional herbal agents with smart microneedle platforms for localized infected wound therapy. Further in vivo studies are warranted to confirm therapeutic efficacy and guide clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no external funding was received for this study. All authors acknowledge the institutional support from Chongqing Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted under protocols approved by the Animal Ethics Committee of Chongqing Medical University (Protocol NO.IACUC-CQMU-2025-0444).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZeng Wenbo designed the study and performed the data analysis. Sun Weijie and Tang Yangjuan were responsible for data collection and bioinformatics analyses. Wan Dong and Tan Jixiang supervised the research and provided guidance throughout the study. Zeng Wenbo and Tan Jixiang wrote the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZarei M, Soleimanian-Zad S. Antibacterial activity of bioactive wound dressing materials: a review. J Tissue Viability. 2022;31(4):582\u0026ndash;90.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eXia G, Wang L, Wang Y, et al. Current advancements of bioactive hydrogels for wound healing. Front Bioeng Biotechnol. 2022;10:915922.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYang J, Chen Z, Pan D, et al. A bioinspired self-powered dressing to accelerate wound healing through ROS scavenging and electrical stimulation. Adv Sci (Weinh). 2023;10(11):e2206755.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChen X, Qin H, Hu Y, et al. ROS-responsive hydrogel for synergistic antibacterial and wound healing via photothermal-enhanced release of antibiotics. Adv Funct Mater. 2023;33(4):2208183.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eXu Q, He Y, Wang H, et al. ROS-responsive hydrogels for tissue engineering and regenerative medicine applications. J Mater Chem B. 2022;10(17):3195\u0026ndash;216.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhang Y, Zhang Y, Zhang H, et al. Reactive oxygen species-responsive microneedles with antibacterial activity for accelerated wound healing. Int J Pharm. 2022;612:121345.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eYe Y, Yu J, Wen D, et al. Microneedles integrated with pancreatic cells and synthetic glucose-signal amplifiers for smart insulin delivery. Adv Mater. 2020;32(8):e1906759.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhang Y, Jiang P, Ye M, et al. Cryptotanshinone: a review of its pharmacology and clinical use. Am J Chin Med. 2022;50(4):913\u0026ndash;41.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWang Y, Chen Y, Wang Y, et al. Cryptotanshinone inhibits inflammation and ROS production via activation of Nrf2/HO-1 pathway in macrophages. Int Immunopharmacol. 2021;99:108078.\u003c/li\u003e\n \u003cli\u003eLiu X, Li M, Xia L, et al. Cryptotanshinone alleviates colitis by regulating intestinal flora and ROS/NF-\u0026kappa;B signaling. Biomed Pharmacother. 2023;160:114387.\u003c/li\u003e\n \u003cli\u003eYin W, Yu J, Lv F, et al. ROS-responsive nanomaterials for inflammation treatment. J Nanobiotechnology. 2022;20(1):120.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eQiao Y, Wang X, Yang C, et al. ROS-responsive nanoplatform for synergistic antibacterial and anti-inflammatory therapy. Biomaterials. 2020;261:120280.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhao X, Liu S, Yao M, et al. ROS-responsive hydrogel for antibacterial and wound-healing applications. Chem Eng J. 2021;403:126399.\u003c/li\u003e\n \u003cli\u003eWang C, Wang C, Huang Y, et al. Bioinspired nanocomposite for wound healing and infection control. Adv Funct Mater. 2020;30(3):1904156.\u003c/li\u003e\n \u003cli\u003eLee H, Han J, Shin H, et al. ROS-cleavable polythioketal hydrogel for skin wound healing. Biomaterials. 2020;263:120360.\u003c/li\u003e\n \u003cli\u003eSun X, Liu X, Li J, et al. Antibacterial activity and mechanism of cryptotanshinone against MRSA. Front Microbiol. 2022;13:857543.\u003c/li\u003e\n \u003cli\u003eJiang J, Fan L, Chen J, et al. Cryptotanshinone exerts anti-inflammatory effects via the Nrf2 pathway in keratinocytes. Int Immunopharmacol. 2020;84:106561.\u003c/li\u003e\n \u003cli\u003eLin J, Zhang X, Zhang W, et al. Cryptotanshinone regulates IL-6 and TNF-\u0026alpha; expression via MAPK and NF-\u0026kappa;B pathways in LPS-stimulated RAW264.7 cells. Inflammation. 2022;45(1):98\u0026ndash;110.\u003c/li\u003e\n \u003cli\u003eZhang L, Guo S, Wu Y, et al. Dual antioxidant and anti-inflammatory activities of polyphenol hydrogel accelerate diabetic wound healing. Adv Funct Mater. 2022;32(3):2107510.\u003c/li\u003e\n \u003cli\u003eChen S, Han Y, Li Y, et al. Transdermal delivery of herbal therapeutics via microneedles. Adv Drug Deliv Rev. 2021;178:113957.\u003c/li\u003e\n \u003cli\u003eSong M, Chen L, Zhang L, et al. Cryptotanshinone enhances wound healing in type 2 diabetes with modulatory effects on inflammation, angiogenesis and extracellular matrix remodelling. Pharm Biol. 2020;58(1):845-853.\u003c/li\u003e\n \u003cli\u003eLi Z, Wei W, Zhang M, et al. Cryptotanshinone-Doped Photothermal Synergistic MXene@PDA Nanosheets with Antibacterial and Anti-Inflammatory Properties for Wound Healing. Adv Healthc Mater. 2023;12(28):e2301060.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhao H, Huang J, Li Y, et al. ROS-scavenging hydrogel to promote healing of bacteria infected diabetic wounds. Biomaterials. 2020;258:120286.\u003c/li\u003e\n \u003cli\u003eLiang Y, Zhao X, Hu T, et al. Adhesive hemostatic bioengineered sponge with antibacterial activity for deep wound repair. Bioact Mater. 2021;6(1):154\u0026ndash;167.\u003c/li\u003e\n \u003cli\u003eXie J, Chen J, Li C, et al. Photocrosslinked hydrogel loaded with cryptotanshinone promotes healing of infected wounds. Int J Pharm. 2022;621:121807.\u003c/li\u003e\n\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":"ROS-responsive hydrogel, Cryptotanshinone delivery, Infected wound healing, Antibacterial microneedles, Oxidative stress modulation","lastPublishedDoi":"10.21203/rs.3.rs-7630804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7630804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic infected wounds remain a major clinical challenge due to persistent oxidative stress, bacterial colonization, and delayed tissue regeneration. To address these issues, we developed a reactive oxygen species (ROS)-responsive microneedle (MN) patch incorporating cryptotanshinone (CPT), a traditional Chinese medicine monomer with proven antioxidant and antibacterial properties. The microneedles were fabricated using a mold-casting technique based on a hydrogel matrix formed by tannic acid\u0026ndash;quaternized chitosan (TA-QCTS), which endowed the patch with ROS-triggered degradation and controlled drug release. The resulting CPT-loaded microneedles exhibited uniform morphology, adequate mechanical strength (\u0026gt;\u0026thinsp;0.25 N/needle), and reliable skin insertion capacity. In vitro release assays confirmed efficient CPT encapsulation and sustained release under oxidative conditions mimicking the wound microenvironment. Antibacterial tests demonstrated potent inhibition against Staphylococcus aureus, Escherichia coli, and Streptococcus pyogenes. Moreover, the CPT-MN system effectively suppressed intracellular ROS, reduced pro-inflammatory cytokines (TNF-α, IL-6), upregulated anti-inflammatory IL-10, and promoted fibroblast proliferation and migration. Overall, this ROS-responsive microneedle platform combines smart drug delivery with bioactive phytochemicals, offering a promising strategy for localized treatment of chronic infected wounds.\u003c/p\u003e","manuscriptTitle":"ROS-Responsive Microneedle Patch for Targeted Delivery of Cryptotanshinone in Synergistic Antibacterial and Antioxidant Therapy of Infected Wounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 12:13:19","doi":"10.21203/rs.3.rs-7630804/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":"99954492-049f-4880-9b8c-7d4d5258c2bc","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56532331,"name":"Biological sciences/Biotechnology"},{"id":56532332,"name":"Biological sciences/Drug discovery"},{"id":56532333,"name":"Health sciences/Medical research"},{"id":56532334,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-10-27T14:39:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-21 12:13:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7630804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7630804","identity":"rs-7630804","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}