Multifunctional DNA hydrogels with light-triggered gas-therapy and controlled G-Exos release for infected wound healing

Abstract

Infectious wound healing remains a significant medical challenge due to chronic inflammation and bacterial colonization. Effective antimicrobial and anti-inflammatory therapies are essential to facilitate wound recovery. Herein, we introduce a highly biocompatible, ROS-responsive DNA hydrogel (LGAH), modified with aggregationinduced emission luminogens (AIEgen) and incorporating ginseng-derived exosomes (G-Exos) and nitric oxide (NO) donor-L-arginine (L-Arg) to promote healing of infected wounds. The hydrogel degrades in response to elevated ROS levels, releasing therapeutic agents. Upon laser irradiation, AIEgen generates 1O2, which activates L-Arg to produce NO, leading to a synergistic antimicrobial effect. NO is particularly effective at inhibiting bacterial growth and promoting angiogenesis, supporting wound healing. G-Exos modulate immune responses, reduce inflammation, and promote the transition from the inflammatory to the proliferative phase. They also enhance cell proliferation, migration, and collagen production, which are key to tissue regeneration. In vivo experiments demonstrated that LGAH significantly accelerates S. aureus-infected wound healing by modulating the wound microenvironment and promoting tissue regeneration. Transcriptomic analysis revealed that LGAH down-regulates gene expression in inflammation and immune response signaling pathways while up-regulating genes related to energy metabolism. Biosafety evaluations at cellular and animal levels have demonstrated that LGAH possesses excellent biocompatibility and biodegradability, making it ideal for tissue repair and regeneration. This multifunctional DNA hydrogel system offers a safe and promising strategy for the clinical treatment of infected wounds.

Headnote

ABSTRACT

Infectious wound healing remains a significant medical challenge due to chronic inflammation and bacterial colonization. Effective antimicrobial and anti-inflammatory therapies are essential to facilitate wound recovery. Herein, we introduce a highly biocompatible, ROS-responsive DNA hydrogel (LGAH), modified with aggregationinduced emission luminogens (AIEgen) and incorporating ginseng-derived exosomes (G-Exos) and nitric oxide (NO) donor-L-arginine (L-Arg) to promote healing of infected wounds. The hydrogel degrades in response to elevated ROS levels, releasing therapeutic agents. Upon laser irradiation, AIEgen generates 1O2, which activates L-Arg to produce NO, leading to a synergistic antimicrobial effect. NO is particularly effective at inhibiting bacterial growth and promoting angiogenesis, supporting wound healing. G-Exos modulate immune responses, reduce inflammation, and promote the transition from the inflammatory to the proliferative phase. They also enhance cell proliferation, migration, and collagen production, which are key to tissue regeneration. In vivo experiments demonstrated that LGAH significantly accelerates S. aureus-infected wound healing by modulating the wound microenvironment and promoting tissue regeneration. Transcriptomic analysis revealed that LGAH down-regulates gene expression in inflammation and immune response signaling pathways while up-regulating genes related to energy metabolism. Biosafety evaluations at cellular and animal levels have demonstrated that LGAH possesses excellent biocompatibility and biodegradability, making it ideal for tissue repair and regeneration. This multifunctional DNA hydrogel system offers a safe and promising strategy for the clinical treatment of infected wounds.

Keywords: DNA hydrogel Gas therapy Ginseng-derived exosomes Infected wound healing Aggregating-induced emission luminogen Tissue regeneration

1. Introduction

The healing of infected wounds caused by different reasons presents a complex and significant challenge in the medical field [1-3], such as burn infection, post-traumatic infection, pressure ulcer infection [4], diabetic foot infection [5]. Current treatments often struggle to achieve optimal therapeutic outcomes due to severe obstacles posed by the wound microenvironment, chronic inflammation, and bacterial colonization [6]. Traditional antibiotic therapy faces limitations with rising bacterial resistance and inadequate wound microenvironment for tissue repair [7-9]. Therefore, there is an urgent need for non-antibiotic strategies that can both inhibit bacterial growth and modulate the wound microenvironment, thereby accelerating the healing process of infected wounds.

Gas-based antibacterial therapy is an innovative approach that utilizes physiologically significant gases, such as nitric oxide (NO) [10], carbon monoxide (CO) [11,12], and sulfur dioxide (SO2), to diffuse into the tissues of infected wounds and exert antibacterial effects within bacterial cells or biofilms [13-15]. At present, there are both single gas delivery and composite gas delivery for antibacterial purposes, such as NO/H2S gases [16]. L-arginine (L-Arg), a common NO precursor [17], generates NO through the oxidation of its guanidino group, enabling NO to covalently bind to biological macromolecules like DNA and proteins of pathogens, thereby exerting antibacterial effects without fostering resistance [18]. Additionally, NO supports angiogenesis and accelerates wound healing by enhancing vascular regeneration at the injury site [19,20]. However, the controlled, on-demand release of these gases to optimize wound healing remains a significant technical challenge.

The invasion of pathogenic bacteria, such as S. aureus, into open wounds triggers the release of inflammatory cytokines that impact sur rounding skin cells and recruited immune cells, leading to an abnormal wound microenvironment that impairs healing [7,21]. One of the pri mary reasons for persistent redness, swelling, and pain in infected wounds that are slow to heal is the excessive inflammatory response driven by the large infiltration of macrophages. During the tory phase, the accumulation of M1 phenotype macrophages axacerbates the inflammatory response, hindering the healing process. Moreover, an abundance of M1 macrophages at the wound site has been shown to inhibit the transition from the inflammatory phase to the proliferative phase, further delaying recovery [22]. Therefore, a multi functional system that both reduce bacterial load and modulates the inflammatory response is crucial for promoting efficient wound healing.

Exosomes are nano-sized extracellular vesicles [23], approximately 30~200 nm, secreted by cells and play a significant role in intercellular communication [24], immune response [25], tissue homeostasis [26-28], cancer progression, and neurodegenerative diseases [29]. They have shown potential in regenerative medicine [30,31], particularly in wound healing [32]. In recent years, plant-derived exosomes, with their high biocompatibility, immune regulation effects, promotion of angiogenesis, promotion of cell proliferation and migration, ease of large-scale production, and high safety, have attracted increasing attention from researchers [33-35]. Among them, ginseng-derived exosomes (G-Exos) with high biocompatibility, have demonstrated good effect in promoting cell proliferation, increasing collagen production, and reducing inflammatory [36], making them promising candidates for therapeutic strategies in wound healing.

Hydrogels are recognized as a kind of wound friendly dressing and widely used in wound repair [37,53], which hydrogels would have different shapes to adapt to different types of wounds, such as light-sensitive hydrogel patches suitable for oral ulcers [38]. DNA hydrogels, 3D polymeric scaffolds with DNA as the primary constituent, have attracted considerable interest due to their biofunctional attributes and mechanical robustness [39]. Utilized in biomedical research [40,41], molecular diagnostics [42,43], and environmental applications [44], these hydrogels offer enhanced biocompatibility and degradability over traditional hydrogels, minimizing immunological responses [45, 54]. Their 3D architecture emulates the extracellular matrix, promoting cellular adhesion, proliferation, and migration, which is crucial for tissue engineering and regenerative medicine [46]. Notably, DNA hydrogels are programmable and can be tailored to achieve multiple functions through sequence design, making them an ideal material for tissue repair dressings.

In summary, we envision the construction of a ROS-responsive aggregation-induced emission luminogen (AIEgen)-modified DNA hydro gel loaded with G-Exos and controlled releasing of NO for the healing of infectious wounds. Initially, linear DNA strand (named as Linker) with disulfide bonds are modified with AIE molecule through amide reactions to form AIE-Linkers. In addition, the linker is designed to contain a self- complementary palindromic sequence, which can decrease the amount of linker used and reduce the cost of DNA hydrogels. Y-shaped building units (Yabc) with three "sticky ends" are designed, and an AIE-Linker with "sticky ends" at both ends is introduced. Through the hybridization of the "sticky ends" of both the Y-shaped building units and the AIE- Linkers, and loaded with G-Exos and L-Arg, a multifunctional drug-

loaded DNA hydrogel is prepared. During the inflammatory phase, after applying the DNA hydrogel to the infected wound site, the DNA hydrogel responds to the high ROS in the wound microenvironment, and the disulfide bonds in the three-dimensional network structure are oxidized, leading to the degradation of the DNA hydrogel and further release of the cargos. Under laser irradiation, A1E molecule generates a large amount of Oj, which oxidizes L-Arg, producing NO with antimicrobial activity; O2 and NO work together on S. aureus, exerting a combined bactericidal effect. In addition, C-Exos reprogram macrophages, regulate the release of inflammatory factors, alleviate inflammatory responses at the wound site, and accelerate the transition of the wound from the inflammatory phase to the proliferative phase. During the proliferative phase, C-Exos promote the proliferation and migration of cells, promote collagen production and deposition, and additionally, the generated NO enhances neovascularization. Therefore, the multifunctional DNA hydrogel system with high biosafety can promote rapid healing of infected wounds through antibacterial, anti-inflammatory, and enhanced angiogenesis, providing a comprehensive treatment strategy with clinical translation potential for the repair of infected wounds (see Scheme 1).

2. Experiment and methods

2. 1. Maccnab and chemicals

Ya, Yb, Yc and Linker was obtained from Sangon Biotech (Shanghai, China) Co., Ltd. L-Arg, EDS and NHS were purchased from Aladdin Biochemical Technology Co., LTD (Shanghai, China).

SYBR Green 1 (Cat No. 10222ES60) was purchased from Yeasen Biotechnology (Shanghai, China). LPS, Gel red and Griess reagent were purchased from Beyotime Biotechnology (Shanghai, China). Edu Cell Proliferation Kit with Alexa Fluor 555 was purchased from Meilun Biotechnology Co. Ltd (Dalian, China). CDG6-FITC, anti-mouse CDG6 and anti-rabbit CD206 antibodies were purchased from Proteintech Group, Inc (Wuhan, China). Anti-rabbit iNOs (Cat No. ER1706-G9) were acquired from HUABIO. Cryogenic Vial was purchased from NEST Biotechnology Co. Ltd (Wuxi, China). BCA Protein Concentration Determination Kit and Hydrogen peroxide (H2O2) content detection kit were purchased from Suolaibao Technology Co. Ltd (Beijing, China). Mouse IL-6, Mouse IL-IO and Mouse TNF-a EUSA Kit were purchased from Bioswamp Life science Lab (Wuhan, China). The solvents were all analytical pure and used directly without treatment.

2.2. Characterization

The morphology of C-Exos was observed using a Hitachi HT7G00 transmission electron microscope (TEM). The particle size and concentration of G-Exos were studied using a Nanosight NS300 nanoparticle tracking analyzer (NTA). The morphological observation of the freeze-dried D.N A hydrogel was conducted using a TESCAN M1RA LMS scanning electron microscope (SEM) from the Czech Republic. The mechanical properties of the hydrogel were assessed using an Anton Paar MCR 302 rheometer (Austria) which an angular frequency sweep was performed, ranging from 0.1 to 100 rad/s. The distribution of G-Exos within the D.NA hydrogel was observed using a Confocal microscope.

2.3. Extraction and purification of C-exosGinseng is washed and cut into small pieces, mixed with an appropriate amount of PBS, homogenized using a homogenizer, and filtered to obtain the crude ginseng extract 1. The crude ginseng extract I is centrifuged at 3000xg for 30 min and 10,000xg for 1 h, respectively. The supernatant is taken to obtain the crude ginseng extract II. The crude ginseng extract II is centrifuged using an ultracentrifuge at 100,000 xg for 2 h, the precipitate is resuspended in PBS, and vortexed thoroughly to obtain the crude G-Exos. Different concentrations of sucrose solution are sequentially added to the centrifuge tube containing the crude ginseng exosome, and centrifuged again at 100,000×g for 2 h. The sample layer is aspirated, PBS is added to fill the tube, and centrifuged at 100,000×g for 1 h for removing excess sucrose. The precipitate is resuspended in PBS, and the purified G-Exos are obtained.

2.4. Preparation and characterization of LGAH

TPA-COOH is synthesized from compound 1 and compound 2, as shown in Fig. S7. The synthesis process is as follows: Compound 1 (26.0 mg, 0.1 mmol) and Compound 2 (41.6 mg, 0.1 mmol) were mixed in EtOH (20 mL) in 100 mL round flask, then the mixture was refluxed for 4 h. Excess solvent was removed in vacuo and the crude product was purified by column chromatography (CH2Cl2: CH3OH = 5:1) to afford TPA-COOH (30 mg, 46 %).

TP-COOH was characterized by high resolution mass spectrometer (HRMS) and nuclear magnetic resonance(NMR), as shown in Fig. S8 and S9: 1H NMR (600 MHz, CDCl3) δ 8.41 (s, 1H), 8.22 (s, 1H), 7.72 (s, 1H), 7.47-7.33 (m, 6H), 7.21 (s, 1H), 7.03-7.02 (d, J = 6.0 Hz, 4H), 6.83-6.77 (m, 6H), 4.69 (s, 2H), 3.78 (s, 6H), 3.03-3.00 (m, 2H), 2.62 (s, 2H), 2.51 (s, 2H), 1.71 (s, 6H). HRMS (ESI, positive ion mode) m/z: calcd. for [C41H41N2O4S]+, 657.2782; found 657.2722. The UV-visible absorption spectrum (and fluorescence emission spectrum of TPA-COOH are shown in Fig. S10 (A) and (B).

TPA-COOH is mixed with EDC and NHS at a molar ratio of 1:10:10, stirred at room temperature for 2 h to activate the carboxyl groups of TPA-COOH. Linker solution is added dropwise and the reaction continues for 24 h. After the reaction is complete, the reaction mixture is dialyzed for 2 d and then lyophilized, to obtain AIE-Linker.

Ya, Yb, Yc, and AIE-Linker at molar ratios of 1:1:1:3 are maintained at 95 °C for 3 min. They are mixed in an appropriate amount of Tri- HCl&Mg2+ buffer solution to obtain AIE-hydrogel (A-hydrogel). G-Exos and L-Arg are mixed into the A-hydrogel to prepare L-Arg@G-Exos@Ahydrogel (LGAH). FT-IR spectroscopy was used to determine the successful synthesis of AIE-Linker. The internal morphologies of DNA hydrogels were characterized by SEM.

2.5. In vitro NO release

The in vitro release of NO from LCAH hydrogel was studied with different groups: (i) PBS, (ii) LCAH, (ii) LCAH I laser. PBS was used as blank control group, LCAH was used as negative control group. Briefly, LCAH hydrogels were prepared as described above. LCAH was placed into the EP tube, the same amount of releasing medium was added, and the laser was irradiated for 15 min with power of 300 mW/cm". Collect different groups of release media, and that were examined by Criess reagent at room temperature for NO.

2.6, Scratch test

HaCat cells and HUVECs were cultured in 6-wel! plates for 12 h. A Scratch was made in the middle of the well and cleaned with PBS, followed by the addition a fresh medium containing PBS, LPS, or LPS · G-Exos, respectively. The scratches at the same position were photographed at 0 h, 10 h, and 24 h after treatment in the well, and the scratch width was analyzed by image J.

2.7. In vitro anti-inflammatory of C-Exos

RAW264.7 cells were cultured in Confocal dishes for 12 h. RAW264.7 cells were then incubated with C-Exos and LPS (500 ng/mL) for 12 h. Fresh medium was used as blank control group, LPS was used as positive control group. After the incubation, RAW264.7 cells were treated with anti-iNOs/CD206 antibody, corresponding fluorescent labelled secondary antibody and DAP1, and observed by Confocal microscope. The concentrations of 1L-6, TNF-a and 1L-10 within the cell supernatant of each group were determined by enzyme-linked immunosorbent assay (ELISA).

2.8. In vitro antibacterial activity of hydrogch

The in vitro inhibited effect of the LC AH on S. aureus was determined by inhibition zone method, spread plate method, Live/Dead staining and scanning electron microscope (SEM). Firsdy, the antibacterial activity was assayed by inhibition zone method. S. aureus (10 uL, 1 x 108 CFU/ mL) were added to pre-formulated LB solid medium in petri dishes (90 mm). Thereafter, a hole was dug out on the LB solid medium and followed by the addition of PBS, L-Arg, C-Exos, A-hydrogel, LCAH. The laser group requires laser irradiation for 15 min with power of 300 mW/ cm". Finally, the petri dishes were placed in a constant temperature incubator at 37 °C for 12 h. The inhibition activity of each formulation was evaluated by measuring the diameter of inhibition circle. The experiments of spread plate method were separated into seven groups: PBS, L-Arg, C-Exos, A-hydrogel, A-hydrogel , laser, LGAH, LCAH + laser. S. aureus was cultivated in LB liquid medium until logarithmic phase (1 x 10s CFU/mL). Dilute the bacterial solution to 1 x 106 CFU/ mL and then the diluted bacterial solution (100 uL) was added into EP tube followed by different treatments. After co-incubation in a constant temperature incubator at 37 "C for 16 h, the co-cultured bacterial solution was removed and diluted to an appropriate multiple, then lOuLof the dilution was taken and spread to agar plates. The bacterial colony was calculated using Image J and the inhibition efficiency was calculated. The in vitro Live/Dead experiment was conducted by DMAO/P1 double stain kit. Bacteria treated with PBS, A-hydrogel + laser and LCAH t laser were collected and washed by centrifugation. Then they were fixed in glutaraldehyde (2.5 %) at 4 "C for 3 h. The fixed samples were dehydrated using ethanol, followed by lyophilization and gold coating before SEM observation. The SEM observation were provided by Scientific Compass (www.shiyanjia.com).

2.9. In vitro biosafry of DNA hydrogel

HaCat and RAW264.7 Cells were inoculated in 96-well plates. The cells were incubated in a cell culture incubator for 12 h and replaced with 100 µL of medium containing hydrogel extract (50 µM 1-200 µM). The cells cultured in fresh medium were used as a control. After 24 h of incubation, the cytotoxicity was determined by CCK-8 assay. HaCat and RAW264.7 cells were inoculated in 6-well plates. After 12 h incubation, the medium containing 100 µM of hydrogel extract was added to continue the culture for 24 h and 48 h, respectively, to observe cell proliferation. The cell proliferation was evaluated by Live/Dead cell staining assay.

2.10.Proliferative property of LGAH on cells

HaCat cells and HUVECs were cultured in 6-well plates for 12 h. Then the hydrogel leaching solutions (A-hydrogel and LGAH), L-Arg, G-Exos and PBS were added to the cell medium, respectively. HaCat cells and HUVECs were then incubated with above mediums for 12 h. After the incubation, HaCat cells and HUVECs were stained with EdU Cell Proliferation Kit with Alexa Fluor 555 and Hoechst, and observed by inverted fluorescence microscope.

2.11.In vitro anti-inflammatory of LGAH

RAW264.7 cells were cultured in Confocal disj hydrogel leaching solutions (A-hydrogel and LGAH), L-Arg, G-Exos and PBS were added to the DMEM medium, respectively. RAW264.7 cells were then incubated with above mediums and LPS (500 ng/niL) for 12 h. After the incubation, RAW264.7 cells were treated with anti-CD86/ CD206 antibody, corresponding fluorescent labelled secondary antibody and DAPI, and observed by Confocal microscope. Some RAW264.7 cells were collected and stained by CD86-FITC antibody and detected by flow cytometry.

2.12. In vivo healing of fall-thickness wounds on S. Aureus-infected mice

The effect of LGAH on infected wound healing was evaluated on mice with S. aureus-infected full-thickness wound model. C57 male mice were selected to establish a wound model. After anesthetizing the mice with isoflurane, the back hair was shaved, and the back skin was disinfected with 75 % ethanol solution. A circular full-thickness cutaneous wound was made on the back of the anesthetized mice using a skin sampler with a diameter of 6 mm, followed by the introduction of S. aureus suspension (10 µL, 1 ×108 CFU/mL). All model mice were separated into 7 groups (n =5): PBS, L-Arg, G-Exos, A-hydrogel, A-hydrogel +laser, LGAH, and LGAH +laser. Therapy was performed on Day 1, 3 and 5 after molding. The group requiring laser treatment was irradiated for 10 min with a power of 300 mW/cm2. During the treatments, the wounds were photographed every two days, and trauma size quantified using Image J, the percentage of wound healing in each group was calculated. Apply the following formula:

Relative wound area ...

(Ao: wound area on the 0 d; An: wound area on n d)

To determine the remaining bacteria on the wounds, using a swab to sample the wound at 4th day, soak the swab in an equal amount of PBS, then the above solution was used for plate coating and cultured in a bacterial incubator for 12 h to form bacterial colonies. The in vivo biocompatibility of LGAH hydrogels was evaluated. Briefly, after subcutaneous injection of PBS, A-hydrogel, and LGAH into the back of mice for three days, the organ tissues were excised for hematoxylin-eosin (H&E) staining analysis. The blood was collected for blood routine and serum biochemical detection.

Histological analysis of the wound was performed on 8th day. wound tissues were collected, cleaned with PBS, fixed with 4 % paraformaldehyde, and embedded with paraffin wax. Then, sections were made with a tissue microtome and H&E staining and Masson staining were performed according to the instructions, analyzed by Image J.

2.13. Microenvironment healing mechanism of infected wound

For immunofluorescence detection, traumatic tissue sections were first stained with anti-CD31 and anti-collagen I antibodies, respectively, followed by the corresponding fluorescently labelled secondary antibodies and DAPI. To detect the types of macrophages in wound tissue, tissue sections were first stained with anti-iNOs and anti-CD206 antibodies, respectively, followed by the corresponding fluorescently labelled secondary antibodies and DAPI. The fluorescence was examined by Confocal microscope. The expression of IL-6, TNF-a and IL-10 within the wound tissues were determined by immunofluorescence staining.

2.14. Statistical analysis

The results of the calculated data are expressed as mean ± standard deviation. Statistical differences were assessed by Origin for univariate ANOVA (including Tukey correction). Significance levels were set at a regular pattern o f ·p < 0.05, ··p < 0.01 and ···p < 0.001.

3. Results and discussion

3.1. Characterization and promoting wound-healing function of G-exos G-Exos exhibit multiple functions, including promoting cell proliferation and modulating macrophage activity and inflammation (Fig. 1A). They are isolated through a combination of ultracentrifugation and density gradient centrifugation, ensuring high purity and functionality [47]. As shown in Fig. 1B and S1, the transmission electron microscope (TEM) image of G-Exos had a classic "exosome" morphology with a near-circular shape and a diameter of about 100 nm, which was smaller than its hydrated diameter (145.36 nm), possibly because G-Exos contracted after drying. The NTA results (Fig. 1C) demonstrated that the extracted G-Exos has a relatively uniform size distribution. Furthermore, their zeta potential was - 24 mV, suggesting good stability in solution (Fig. S2). To confirm the positive functions of G-Exos on wound healing, HaCat cells and HUVECs were used for the CCK-8 assay. As shown in Fig. 1D, as the concentration of G-Exos increased to 2.5 µg/mL, the cell viability increased, but the cellviability decreased when the concentration exceeded to 5 µg/mL, indicating the effective concentration threshold of G-exos has been reached. This biological effect, which promotes proliferation or has a protective effect at low concentrations while causing toxicity at high concentrations, is commonly found in exosomes [48]. Further, the scratch assay was used to test the effect of G-Exos on cell proliferation and migration. As shown in Fig. 1E and 1F and S3, compared to Control group, G-Exos groups demonstrated a gradual gap shortening in the scratch as the concentration increased, which was in agreement with the results of CCK-8 assay, indicating that G-Exos promote cell proliferation and migration beneficial for wound healing.

Co-incubating G-Exos with DiO, Confocal microscope was applied for confirming whether DiO-labellecl G-Exos could be effectively taken up by cells (Fig- 1G). Compared to Control group, the green fluorescence of DiO was clearly visible in the interior of cell after adding G-Exos, indicating that G-Exos can be effectively taken up by cells. LPS can induce macrophages to highly express iNOs, activated Ml phenotype. Compared to PBS group, the cells in LPS group had strong green fluorescence and weak red fluorescence, indicating that they highly expressed iNOs and lowly expressed CD206. After adding G-Exos, the green fluorescence of the cells decreased while the red fluorescence increased, indicating that the expression of iNOs in the cells decreased and the expression of CD206 increased. The results of the semiquantitative fluorescence chart were in agreement with the fluorescence images (Fig. 1H). These results demonstrated that G-Exos can convert LPS-induced M1 macrophages with high expression of iNOs to M2 macrophages with high expression of CD206.

Since Ml macrophages secrete pro-inflammatory factors, exacerbating inflammatory responses, to verify whether G-Exos can alleviate the inflammatory response caused by a large number of Ml macrophages, ELISA assays were used to detect the pro-inflammatory factors (IL-6 and TNF-a) and anti-inflammatory factor (IL-10) secreted by macrophages in different groups. As shown in Fig. ]J, IK, and 1L, compared with Control group, the cells treated with LPS increased their secretion of IL-6 and TNF-a and decreased that of IL-10, indicating that LPS promotes inflammatory responses. Compared to LPS group, TNF-a and IL-6 were significantly reduced, and IL-10 was increased in the presence of G-Exos. These results demonstrated that G-Exos have the ability to reduce the release of pro-inflammatory factors and increase that of anti-inflammatory factors. The above experiments strongly proved that the extracted G-Exos has the ability to promote the proliferation and migration of various cells. In addition, G-Exos also host antiinflammatory advantages, regulating pro-inflammatory Ml macrophages, promoting their transformation into anti-inflammatory M2 macrophages, and reducing the release of pro-inflammatory factors.

3.2, Preparation and characterization of multifunctional LGAH

After mixing the linker with the other three DNA strands (Yabc), a DNA hydrogel is formed through the complementary base pairing between the DNA strands (Fig. 2A). The Linker and Yabc were stained with nucleic acid dyes of different colors, and the resulting hydrogel has a distinct bevel after mixing, indicating the forming of hydrogel (Fig. 2B). Fluorescence (FL) results and atomic force microscopy (AFM) images confirmed the crosslinking between the linker and Ya (Fig. 2C and 2D). Under the AFM, it could be observed that there are significant DNA structural changes after cross-linking of linker and Ya. Other DNA strands also can be cross-linked successfully shown in Fig. 2D. Among them, data points with larger particle sizes appeared after the mixing of Linker, Ya and cYa, indicating a higher degree of cross-linking. SEM observation of the hydrogel morphology demonstrated that the blank DNA hydrogel has a three-dimensional porous structure (Fig. 2E). Agarose gel electrophoresis results confirmed the crosslinking between the DNA strands (Fig. 2F and S4).

The modulus of the DNA hydrogel was measured by a rheometer, amplitude sweep test determined the linear viscoelastic region and yield strain of the hydrogel (Fig. S5A), the yield strain is greater than 50 %, indicating that the hydrogel maintains viscoelasticity and avoids rupture under extreme deformation. Frequency sweep test showed that energy storage modulus G' is always higher than loss modulus G" with the increase of angular frequency, indicating that a hydrogel with certain elasticity (Fig. 2G). Furthermore, the viscosity of the hydrogel was measured at a shear rate of 50 s . The viscosity is approximately 700 mPa s and suitable for wounds (Fig. S5B). In addition, the swelling rate of the hydrogel reached 463 % within 24 h, indicating that it has excellent water retention capacity (Fig. S5C). The durability of the hydrogel under low strain (1 %) and high strain (10 %) conditions was evaluated at a frequency of 1 Hz, as shown in Fig. S6. The modulus of the hydrogel slightly increased after 100 cycles, possibly due to reversible bond breakage and recombination under cyclic loading, eventually forming more uniform cross-linked structure, thereby increasing the modulus. Furthermore, G 'is always greater than G', indicating that the hydrogel maintains good viscoelasticity under cyclic loading.

To endow the DNA hydrogel with multifunctionality, AIE molecules were comiected to the Linker through an amide reaction to prepare AIE-linker. The successful synthesis of AIE was verified by H NMR and HRMS, and its maximum absorption peak and excitation light were determined by UV-Vis absorption and fluorescence spectra. AIE exhibits a strong absorption peak of at about 648 nm and maximum emission wavelength is 808 nm. Furthermore, the singlet oxygen generation of AIE was verified through DPBF (Fig. S8-SI1). The formation of the amide structure was characterized by infrared spectroscopy (Fig. S12), with the appearance of amide II band and III band at 1536 cm and 1217 cm , indicating that AIE-linker is successfully prepared. Mixing AIE-linker, Yabc, G-Exos and L-Arg resulted in the immediate formation of LGAH (Fig. 2A). The microstructure of the hydrogel was observed by SEM, and it was found that the porous structure of the hydrogel is not affected by loaded cargos (Fig. 2H). Meanwhile, loaded G-Exos were observed on the surface of LGAH. To further determine the loading of G-Exos in the hydrogel, Gel Red and DiO were used to stain the hydrogel and G-Exos, respectively. The distribution of G-Exos in the DNA hydrogel was measured by Z-Stack Confocal imaging. Confocal fluorescence images demonstrated that G-Exos is successfully loaded and distributed in the hydrogel (Fig. 21 and SI3).

To achieve controlled release of loaded cargos from the DNA hydrogel in infected wounds, the Linker was modified with disulfide bonds. As shown in Fig. 2J, compared to Control group, the fluorescence in the H0O2 groups decreased with the addition of H2O2, indicating that the hydrogel dissolved and released AIE. As the concentration of H2O2 increased, the fluorescence gradually decreased, indicating that more AIE molecule was released. The results demonstrated that LGAH is ROS-responsive, capable of responding to the high ROS levels in infected wounds and releasing the loaded cargos. In addition, the responsive release of G-Exos was detected. The results proved that DNA hydrogels with ROS responsiveness could respond to ROS and achieve the rapid release of cargos (Fig. SI4). It was further verified that compared with normal wounds, infectious wounds have a higher concentration of H2O2 to ensure the realization of hydrogel responsiveness at the animal model (Fig. S15).

To detect the generation of NO in LGAH, Griess reagent was used to detect the concentration of generated NO. In Fig. 2K, compared to NO concentration in PBS group, that elevated in LGAH group as the irradiation time increased, indicating that laser-induced NO release. Compared to LGAH, LGAH - laser has significant NO generation in Fig. 2L, indicating that LGAH requires '02 generated by PDT of AIE molecule to activate L-Arg, thereby generating the required NO for infected wound healing.

3.3. In vitro antibacterial effects and biocoinpatibility of LGAH

The antibacterial effects of LGAH were identified on S. aureus, which inhibition zone method and spread plate method were applied for measuring inhibition zone diameter and counting bacterial colonies, respectively. As shown in Fig. 3A and S16, compared with G1 treated with PBS, a bacteriostatic region appeared in G5, showing that PDT of AIE molecule has certain antibacterial effect. However, the treatment of LAGH +laser in G7 showed a remarkable antibacterial effect with the largest inhibition zone area and diameter, indicating that 1O2 produced by PDT activates L-Arg to generate NO, enhancing the overall antibacterial effect of LGAH. From the plates of each group (Fig. 3B), it was observed that the number of colonies was lower in G5 compared to G1, G2, G3, G4 and G6, while G7 had the least colonies distributed. In the statistical analysis of colony counting (Fig. 3C), the survival rate of bacteria in G5 was 31.18 ±11.82 %, while the survival rate in G7 was 3.66 ± 1.71 %. The Live/Dead staining experiment and SEM observation were utilized to further confirm the antibacterial performance of LGAH. From Fig. 3D and 3E, G5 and G7 displayed red fluorescence and reduced proportion of live bacteria, while G7 exhibited remarkable red fluorescence with the least live bacteria, indicating that the combination of 1O2 and NO has a better antibacterial effect. As shown in Fig. 3F, the morphology of S. aureus in G1 was intact, indicating that the bacteria have not been damaged. Compared with G1, a small number of bacteria in G5 exhibited cell wall depression and cell membrane rupture, indicating that the bacteria were damaged by PDT of AIE molecule. However, a large number of damaged bacteria appeared in G7, indicating that the combination of 1O2 and NO produces ideal killing effect to bacteria.

The biocompatibility of DNA hydrogel was evaluated to determine the cytotoxicity, which CCK-8 assay was used to assess the cytotoxicity of DNA hydrogel to HaCat and RAW264.7 cells. As shown in Fig. 3G, different concentrations of hydrogels were coincubated with HaCat and RAW264.7 cells for 24 h, the survival rate of cells was maintained above 95 %, and there was no significant difference compared to Control group (0 uM). From Fig. 3H, almost all cells in Control group and DNA hydrogel group exhibited green fluorescence. The results showed that the cells could survive after incubated with the hydrogel for 2 days. Z-Stack Confocal imaging were used to observe the distribution of cells on DNA hydrogel to investigate the cytocompatibility between cells and DNA hydrogel. These cells were incubated with DNA hydrogel, and it was found that the cells could grow on the stereoscopic DNA hydrogel (Fig. 31), indicating that DNA hydrogel did not inhibit the cell activity, which further proved that the DNA hydrogel has good cytocompatibility. As shown in Fig. 3J, a significant color difference between the DNA hydrogel groups and positive control (Triton X-l 00) group could be observed, and all DNA hydrogels exhibited only a slight hemolysis (<4 %). In addition, the degradation property of hydrogels is one of the testing elements for the safety of hydrogels. As shown in Fig. SI 7, approximately 74 % of the hydrogel was degraded in PBS containing H2O2 for 24 h, while the degradation rate in PBS was only about 33 %. This ROS responsiveness was also confirmed in model animals (Fig. SI 8), which indicated that the ROS responsiveness of the hydrogel accelerated the degradation of the hydrogel both in vivo and in vitro experiments, which was conducive to the release of the cargos and increase safety. All these findings demonstrated that the LGAH has an excellent antibacterial effects and biocompatibility, which could be applied for infected wound healing treatment.

3.4. In vitro functional verification of LGAH

Edu cell proliferation assay was used to examine the effect of LGAH in promoting cell proliferation on HaCat cells and HUVECs. From Fig. 4A and 4B, G-Exos and LGAH group exhibited more red fluorescence and Edu positive regions than Control, L-Arg and A-hydrogel group. This result indicated that these two groups have a superior ability to promote cell proliferation than the others, which is derived from G-Exos. The ability of LGAH to promote proliferation and migration of HaCat cells was further confirmed by scratch test. As shown in Fig. SI 9, compared with other groups, the cell scratch width in LGAH group was always the smallest over time, and the cell scratch completely disappeared after 24 h, indicating that LGAH can accelerate cell proliferation and promote cell migration.

The in vitro anti-inflammation activity of LGAH was evaluated to determine the polarization of Ml macrophages and M2 macrophages (Fig. 4C). Compared with Control group, Control group treated with LPS had significant red fluorescence (CD86) and weak green fluorescence (CD206), indicating that the cells in this group were pro-inflammatory Ml phenotype macrophages. After treatment with G-Exos and LGAH, the red fluorescence intensity of the cells decreased and the green fluorescence intensity increased, indicating a decrease in Ml proinflammatory macrophages and an increase in M2 anti-inflammatory macrophages. In addition, these also demonstrated that the encapsulation of DNA hydrogel does not affect the anti-inflammatory activity of G-Exos. These results demonstrated that LGAH exhibited inhibited activity on the Ml polarization, more promoted activity on M2 polarization. Flow cytometry results were also consistent with Confocal images (Fig. 4D), indicating that LGAH can achieve the regulation of macrophage phenotype.

In order to test in vitro angiogenesis, HUVECs tube formation experiments were carried out and made the quantitative analysis using Image J. The results demonstrated that compared with Gl, the number of nodes, segments and meshes and the total segment length were significantly increased, indicating that G7 has superior angiogenic promoting ability. In addition, the number of nodes, segments and meshes, and total segment length in G7 were significantly higher than those in G6 and G5, indicating that the angiogenic ability of G7 originated from NO generated by the oxidation of L-Arg by PDT (Fig. S20). To assess collagen production and matrix remodeling, hydroxyproline detection kit was applied to quantify the collagen production in different groups and matrix remodeling of L929 cells under different treatments was analyzed by the activity of matrix metalloproteinase-9 (MMP9) using immunofluorescence. Compared with Gl, the hydroxyproline levels of G3 and G7 containing G-Exos were significantly increased, indicating more collagen production. In addition, the levels in G5 also increased, possibly due to the cellular protective mechanism that resists PDT-generated ROS (Fig. S21). Meanwhile, the activity of MMP9 in the cells was detected. The results demonstrated that compared to G'l, the activity level of MMP9 in the group containing G-Exos was decreased (Fig. S22). The above results indicated that LGAH can regulate extracellular matrix homeostasis by promoting collagen synthesis and inhibiting MMP9-mediated matrix degradation.

3.5. In vivo therapeutic effect of LGAH on infected wounds

Healthy mice were randomly divided into 7 groups and the hair on the back was removed. When the mice were under anesthesia, full-cut skin wounds with a diameter of 6 mm were made on the back of mice. In addition, bacteria solution of S. aureus was added to the surface of the wound to construct the S. aareus-infected wound. The diameter alteration of the wounds was recorded and photographed every two days. As illustrated in Fig. 5B-5D, there was no significant difference in Gl and G2, and the wound closure rate was about 65.57 % and 63.99 %, respectively. Compared with Gl and G2, other groups had better wound healing effects, among which G7 had the best wound closure effect. The S. aureus -infected wound in G7 almost completely healed on the 8th day, which the wound closure rate was as high as 92.16 %. To further investigate the in vivo antibacterial activity of LGAH on S. aureus-infected wounds, the relative amounts of residual bacteria on infected wounds were detected by the spread plate method on the 4th day. As shown in Fig. 5E, the bacterial colonies were reduced in G5 compared with other groups, which was due to the antibacterial activity of generated 'c^. G7 exhibited better anti-bacterial effect than G5 since an amount of NO generated by ' 02-activated L-Arg could further inhibit the bacterial proliferation. Expectedly, the fewest bacterial colonies were observed in G7, which is mainly contributed from the combined antibacterial activity of 'C^ and NO from LAGH.

The effect of LGAH on S. aureus-infected wound repair was further assessed by H&E and Masson staining on 8th day (Fig. 5F). From the results of H&E staining, all groups had different degrees of wound healing. Among them, G7 had the best wound healing with the smallest length and depth of the wound, and the least area of inflammatory cell infiltration (Fig. 5G and 5H). The result was consistent with the optical photographs in Fig. 5B. Masson staining is a classical method of dyeing collagen fibers, which is mainly used to distinguish collagen fibers from muscle fibers. After staining, the muscle fibers in the tissue appear red and the collagen fibers appear blue. Obviously, the wound tissues in G7 had the deepest and widest range of blue stains, which meant that there were a lot of collagen fibers in the wound tissues of G7 (Fig. 5F). The proportion of collagen deposition was approximately 49.48 % (Fig. 51). These results preliminarily confirmed that LGAH accelerates the healing of infected wounds by inhibiting bacterial proliferation, reducing inflammatory cell infiltration and accelerating collagen deposition.

3.6. The underlying mechanisms of infected wound repair enhanced by LGAH

Angiogenesis plays a crucial role in the wound healing process. It not only provides the wound with essential oxygen and nutrients but also helps to remove waste and regulate immune responses, which are vital for wound healing [49]. It has proven that NO not only plays an important role in antibacterial, but also promotes angiogenesis during the process of wound healing. CD31, also known as platelet endothelial cell adhesion molecule-1(PECAM-1), is a glycoprotein expressed on the surface of platelets and endothelial cells. CD31 plays an important role in angiogenesis [50]. Therefore, detecting the expression of CD31 in wound tissue can help assess the effects of LGAH on angiogenesis. As shown in Fig. 6A, G7 exhibited significant green fluorescence, indicating the highest expression of CD31 in the wound tissue. Compared to G6, the stronger green fluorescence in G7 suggested that the 1O2 generated by AIE molecule in LAGH under laser irradiation activates L-Arg to produce NO, thereby promoting angiogenesis, while the angiogenic effect is diminished lacking laser activation. Additionally, to further examine collagen deposition, the collagen of the tissue was detected by immunofluorescence and hydroxyproline method. The staining results from Fig. 6A and the collagen quantification result (Fig. S23) were in agreement with the that of Masson staining.

Since LGAH has been proven to have an immunomodulatory effect on macrophages in vitro, the markers of different macrophages were stained in the wound tissue of mice after treatments. Yellow fluorescence (iNOS) indicates M1 macrophages, and green fluorescence (CD206) indicates M2 macrophages. As shown in Fig. 6B, compared to the significant yellow fluorescence in G1, G2, and G4, other groups exhibit obvious green fluorescence, suggesting the presence of a large number of M2 macrophages and fewer M1 macrophages in the tissues of these groups. This results of G3, G6, and G7 could be attributed to the

immunomodulatory effect of G-Exos and that of C5 may be due to the killing of Ml macrophages by PDT. In addition, the levels of proinflammatory and anti-inflammatory factors in the wound tissue were examined by immune-histochemical staining. As shown in Pig. 6C and 6D, compared to other groups., the positive area of IL-6 and TNF-ci were higher and that of 1L-! 0 was lower in C1, G2, and C4. Among them, the anti-inflammatory effect of C-Exos reduced the inflammatory response in G3, C6, and C7. C5 may have observed a decrease in inflammatory response due to the bactericidal effect of PDT. The wound tissue of mice treated with LCAH | laser demonstrated the fewest Nil macrophage distribution, the weakest inflammatory response, and the best vascular regeneration effect, indicating that LCAH t laser promoted the healing of infected wounds through the mechanism of enhancing the neovascularization of wound tissue, accelerating collagen deposition, reducing the proportion of M1 macrophages, reducing the release of proinflammatory factors and increasing the release of anti-inflammatory factors.

The biosafety of LCAH was assessed through H&E staining on major organs, routine blood test and serum biochemical assays. The H&E staining of major organs exhibited no histological damage following the treatment with the DNA hydrogel, and there was no significant infiltration of immune cells (Fig. 524). The results of routine blood test indicated no abnormalities in white blood cells, red blood cells, or other parameters (Pig. S25). Furthermore, the serum of mice in each group was tested for biochemical indices (AST, ALT, UA and CREA) related to liver and kidney function. As shown in Fig. S26, the four indices of both Control group and DNA hydrogel groups were all within the reference range for normal mice, indicating that LCAH does not exhibit hepatorenal toxicity in mice. These above results collectively demonstrated that the good biosafety profile of LCAH.

3.7. The gene regulation of infected wound repair by LCAH

To further explore the mechanism of LC AH in the repair of S. aureus-infected wounds, wound tissue of mice treated by PBS and LC AH t laser was collected and transcriptomic analysis was performed (Pig 7A). Volcano map analysis revealed significant differences in gene expression between Control and Treatment group, with 333 down-regulated genes and 314 up-regulated genes (Fig 7B). Fig. 7C demonstrated the differentially expressed genes (DECs) between Control and Treatment group. Compared with Control group, skin tissue in the Treatment group showed significant differences in the expression of genes related to immune response, skin repair, and barrier function. Examples included IL19, Trem 1, Ly6g, Trem3 (involved in immune regulation), Defb3 (with antimicrobial properties), Sprr2i, Sprr2d, Sprr2e (involved in skin keratinization), and Lce3e (involved in skin barrier function). Through quantitative PCR analysis, it was found that the expressions of IL-19, Treml, Trem3, sprr2d, sprr2e, sprr2i, and Defb3 in the wound tissues of Treatment group were significandy down-regulated compared with those of Control group (Fig. S27). This result suggested that the treatment significantly suppressed the local excessive immune response, alleviated the inflammatory state, promoted the regeneration of normal epidermis and reduced demand for antibacterial agents, indirectly indicating the effectiveness of infection control. The three joindy have pointed to the fact that the wound microenvironment tends to be stable, confirming that this treatment accelerates the healing process through multi-pathway synergy.

These DECs were functionally classified by gene ontology (GO) analysis (Fig. 7D and 7E), and the bubble map demonstrated that up-regulated genes were mainly enriched in biological processes such as cell energy metabolism and cell development Down-regulated genes are mainly involved in inflammatory response and multiple aspects of immune response, including activation, migration and differentiation of immune cells, as well as cytokine production and immune response regulation. In addition, Kyoto encyclopedia of genes and genomes (KECC) analysis was performed for the first 10 significantly different signaling pathways to determine the effect of LCAH on these pathways (Fig. 7F). These signaling pathways ate mainly involved in inflammatory and immune responses. The bubble map showed that in Treatment group, these KECC signaling pathways are suppressed, indicating that LCAH I laser can reduce the excessive inflammatory response in the infected wound and reduce the possibility of excessive immune activation in severe infection trauma by regulating the local immune cells in the wound. The results of gene set enrichment analysis (GSEA) were generally consistent with the above findings, showing that gene sets related to inflammation and immune response were suppressed in Treatment group, while that related to energy metabolism were promoted, indicating that after LCAH < Laser treatment, the cells in the infected wound effectively used nutrients to produce energy, maintained the stability of the intracellular environment, promoted cell proliferation and migration, and thus realized rapid wound healing (Pig. 7G).

4.Conclusion

In this study, we introduced a ROS-responsive AIEgen-modified DNA hydrogel (LCAH) incorporated C-Exos and L-Arg, which realizes the controlled release of G-Exos and NO, responds to and regulates the wound microenvironment for infectious wounds. Confocal and SEM images substantiated the successful encapsulation of C-Exos and confirmed that DNA hydrogel did not compromise the functionality of G-Exos. Laser-induced NO release experiments revealed that the "02 generated by the PDT effect of A1E molecule can effectively stimulate L-Arg within LCAH to generate the requisite NO for infected wound healing. In vitro studies have demonstrated that LCAH significantly suppresses the growth of 5. aureus, and promotes the proliferation and migration of norma! cells. Furthermore, LCAH exhibits a certain modulatory effect on macrophages, effectively curbing inflammatory responses. In vivo experiments confirmed that LCAH treatment markedly expedites S. ourcus-infected wound healing by regulating the wound microenvironment through the combined bactericidal action of NO and "02, the pro-angiogenic function of NO, as well as the immunomodulatory effects of C-Exos. Additionally, both in vivo and in vitro experiments have demonstrated the high biosafety of LCAH. In addition, gene transcriptomic analysis also proved that after LCAH treatment, the signal pathways related to inflammation and immune response of infected wounds were effectively inhibited, and the gene expression related to energy metabolism were activated. LCAH innovatively integrates ROS-responsive DNA hydrogels, AIE photodynamic molecules, gas therapy, and exosome therapy, demonstrating significant clinical potential while addressing key challenges in infected wound treatment. Its core innovation lies in three aspects: 1) ROS-triggered hydrogel degradation enables dynamic release of exo-somes to regulate immunity, combined with photoactivated NO and '02 forming a dual antibacterial mechanism that avoids antibiotic resistance risks. 2) The system responds to microenvironmental signals and utilizes external lasers to achieve spatiotemporally controlled NO release, overcoming the uncontrolled-release NO limitations of traditional materials. 3) DNA hydrogel endows this system with high biological safety. Experimental results validate its triple therapeutic mechanism of antibacterial-anti-inflammatory-regenerative effects, accelerating wound healing through synergistic interactions between multiple therapeutic agents. Compared with conventional dressings, LCAH exhibits unique advantages in precise drug delivery and dynamic microenvironment adaptation, along with excellent biocompatibility and no observed biological toxicity. However, despite providing an integrated solution for intelligent wound repair, its clinical translation still requires systematic exploration to address remaining practical challenges.

Although the hydrogel preparation process is controllable on a laboratory scale and has good stability (Fig. 52G). when it comes to large-scale production, the problems of DNA strand stability and the dispersion uniformity of the substrates need to be solved. Microfluidic technology can be drawn upon to achieve standardized preparation [51,52].

Secondly, the metabolic pathways and immunogenicity of long-term used hydrogels need to be further verified through experiments on primates. Finally, as a combined product containing exosomes and chemical donors, it is necessary to follow the regulatory standards for biological products and medical devices in a coordinated manner and gradually advance through phased clinical trials.

In conclusion, we have successfully engineered an AIEgen-modified DNA hydrogel integrated with L-Arg and G-Exos to achieve the controlled release G-Exos and NO, combined antibacterial effect of NO and 1O2, the pro-angiogenic effect of NO, as well as immunomodulatory effects of G-Exos to address the challenges associated with the prolonged healing of infected wounds. We anticipate that this multifunctional DNA hydrogel may serve as a potent therapeutic agent for effective management of infected wounds.

5.Ethics approval and consent to participate

The procedure of animal experiments was approved by the Institutional Animal Care and Use Committee (IACUC), Kunming Medical University (Kmmu20241912).

CRediT authorship contribution statement

Yuyun Ye: Writing - original draft, Visualization, Methodology, Investigation, Conceptualization. Yan Liu: Writing - original draft, Methodology, Investigation, Conceptualization. Shengchao Ma: Writing - original draft, Methodology, Investigation. Xipeng Li: Writing - original draft, Methodology, Investigation. Wei Wang: Writing - re view & editing, Visualization, Methodology. Xu Chen: Writing - review & editing, Methodology, Conceptualization. Judun Zheng: Writing - review & editing, Visualization, Validation, Supervision, conceotualization. Zhijin Fan: Writing - review & editing, Supervision, Project administration, Formal analysis, Data curation, Conceptualization. Yideng Jiang: Writing - review & editing, Supervision, Resources. Yuhui Liao: Writing - review & editing, Supervision, Resources, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

Yuyun Ye, Yan Liu, Shengchao Ma and Xipeng Li contributed equally to this work. This work was supported by the National Key R&D Program of China (2024YFC231103), National Natural Science Foundation of China (82322042, and 82272248), and Natural Science Fund of Guangdong Province for Distinguished Young Scholars (2022B1515020089).

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ARTICLE INFO

Peer review under the responsibility of editorial board of Bioactive Materials.

Received 13 February 2025; Received in revised form 30 April 2025; Accepted 2 June 2025

Available online 14 June 2025

References

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