Click‐hydrogel delivered aggregation‐induced emissive nanovesicles for simultaneous remodeling and antibiosis of deep burn wounds

As a high‐risk trauma, deep burns are always hindered in their repair process by decreased tissue regeneration capacity and persistent infections. In this study, we developed a simultaneous strategy for deep burn wounds treatment using functional nanovesicles with antibacterial and tissue remodeling properties, delivered via a click‐chemistry hydrogel. An aggregation‐induced emission photosensitizer of 4‐(2‐(5‐(4‐(diphenylamino)phenyl)thiophen‐2‐yl)vinyl)‐1‐(2‐hydroxyethyl) pyridin‐1‐ium bromide (THB) with excellent photodynamic properties was first prepared, and then combined with readily accessible adipose stem cells‐derived nanovesicles to generate the THB functionalized nanovesicles (THB@ANVs). The THB@ANVs showed strong antibacterial activity against Gram‐positive bacteria (up to 100% killing rate), and also beneficial effects on tissue remodeling, including promoting cell migration, cell proliferation, and regulating immunity. In addition, we prepared a click‐hydrogel of carboxymethyl chitosan for effective delivery of THB@ANVs on wounds. This hydrogel could be injected to conform to the wound morphology while responding to the acidic microenvironment. In vivo evaluations of wound healing revealed that the THB@ANVs hydrogel dressing efficiently accelerated the healing of second‐degree burn wounds by reducing bacterial growth, regulating inflammation, promoting early angiogenesis, and collagen deposition. This study provides a promising candidate of wound dressing with diverse functions for deep burn wound repair.


INTRODUCTION
Burns are one of the most disruptive forms of trauma. [1,2]The recovery of deep burn wounds is hindered by impaired tissue regeneration in the deep dermis and persistent infections, which can potentially lead to sepsis or even mortality. [3,4]chieving burn wound repair with a single pharmaceutical component is difficult because the complex process of tissue regeneration requires the cooperation of multiple tissues and cells. [5,6]Thus, stem cells have emerged as the most promising cells for refractory wound treatment with numerous capabilities, such as repairing damaged tissue, mobilizing wound repair-related cells, and regulating immunity. [7]And adipose-derived stem cells (ADSCs) which can be extracted from adult adipose tissue and expanded in vitro are the optimal choice with abundant supply. [8]17][18][19] The invasion of bacterial infection into dermis necrotic tissues of deep burns, accompanied by the secretion of toxins and other metabolites, can result in persistent infection and may even lead to fatal systemic inflammatory response. [20,21]lthough nanovesicles have excellent wound repair functions, their anti-infectious ability still needs enhancement.Systemic antibiotics for localized infection treatment can cause antibiotic resistance. [22]In contrast, photodynamic therapy (PDT) has attracted widespread attentions owing to its ability to generate reactive oxygen species (ROS), which is less likely to lead to drug resistance in antibiosis. [23,24]Tang et al. have developed a variety of aggregation-induced emission (AIE) photosensitizers with enhanced ROS generation efficiencies and good photostabilities for the PDT of tumors and microbial infections. [25,26]Therefore, the combination of AIEgens with ADSCs-derived nanovesicles has a great potential to synergistically realize both enhanced anti-infectious function and tissue-regenerative abilities.
The continuous delivery of nanovesicles is also crucial for the long-term treatment of wounds. [27]Dropwise or solution injection administration methods commonly exhibit the limitation of rapid degradation or metabolism of nanovesicles on wounds, leading to shortened effective times in their application. [28]Hydrogel wound dressings can protect the nanovesicles and realize long-term effective concentration on wounds. [29]Moreover, they can protect wounds from infections, absorb excess exudate, and promote the dissolution and exfoliation of deep necrotic tissue. [29,30]However, hydrogels for controllable release are usually prepared using UV light, heating, or catalysts, which may potentially harm the essential biological components of nanovesicles. [31]Although temperature-sensitive hydrogel PF-127 has been utilized for in situ gelation in esophagus without affecting the functions of nanovesicles, [32] it is not suitable for skin with unstable temperatures.
In this study, we developed nanovesicles with antibiosis and tissue remodeling functions, delivered with a clickhydrogel for deep burn wound treatment (Scheme 1).Adipose stem cells-derived nanovesicles (ANVs) were easily prepared and combined with the photodynamic AIEgen of 4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)-1-(2-hydroxyethyl) pyridin-1-ium bromide (THB) to obtain THB@ANVs.The THB@ANVs retained the functionality of both stem cells and THB in promoting wound healing and inducing antibacterial effects.To enable the delivery of THB@ANVs without affecting their activity, we prepared an acid-responsive cross-linking hydrogel carrier through a heat-, light-, and catalyst-free click-reaction between the amino groups of carboxymethyl chitosan (CMC) and di-activated alkynes-modified polyethylene glycol (PEG-DA). [33]Furthermore, the mechanical behaviors and injectability of the hydrogels were assessed.The prepared THB@ANVs hydrogel dressing can be injected through a needle according to the shape of wound.In vivo burn wound-healing experiments were finally conducted on rats to evaluate the effectiveness of this hydrogel dressing for treating bacterial infections, promoting angiogenesis, collagen synthesis, and cell proliferation.

Preparation and characterization of ANVs
We used rat ADSCs as the cellular source to prepare ANVs through a simple extrusion method, aiming to mimic the functions of ADSC exosomes. [17]The fragmented cell membranes of ADSCs was reconstructed into nanovesicles by repeated extrusion through a polycarbonate membrane with nanosized pores, allowing precise control over the size of the nanovesicles.Figure 1A (left) displayed the transmission electron microscopy (TEM) image of ANVs, which exhibited a tea cup holder-like structure with a size of 100-200 nm.The structure closely resembled that of natural exosomes, [34] confirming the successful preparation of ANVs.As illustrated in Figure 2I, nanoparticle tracking analysis (NTA) revealed that the size of ANVs was 168.3 ± 4.5 nm, with a considerable yield of 1.99 × 10 10 /10 6 cells.
The proposed extrusion method inherited a higher concentration of active ingredients from stem cells than traditional S C H E M E 1 Schematic illustration of the construction and working mechanism of THB@ANVs hydrogel dressing.The deep burn wound-healing process promoted by THB@ANVs hydrogel is presented from right to left.methods for preparing EVs, evidenced by a high protein concentration (approximately 201 μg/10 10 ). Figure 1B showed that ANVs displayed protein profiles similar to ADSCs, as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that the characteristic proteins of ADSCs were retained in the ANVs obtained from the proposed method.Furthermore, classic EV protein markers CD9 (MIC III) and TSG101 were characterized through Western blotting (Figure 1C).EV exclusion marker calnexin was also assessed, which were not detected in ANVs.These results revealed the capability of the ANVs to preserve essential stem cell components while exhibiting a remarkable resemblance to natural vesicles.

Preparation and characterization of 4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2yl)vinyl)−1-(2-hydroxyethyl) pyridin-1-ium bromide
[37] In this study, we synthesized a hydrophilic THB compound with a hydroxypyridinium moiety, which was expected to show strong antibacterial effects.The THB was synthesized through facile organic reactions with high yield, and the structures were confirmed by 1 H and 13 C nuclear magnetic resonance spectroscopy and highresolution mass spectroscopy (Figures S1-S5).As shown in Figure 1D,E, the THB displayed a typical AIE effect, where the photoluminescent (PL) intensity increased significantly in solution with an increasing fraction of hexane as a poor solvent.The enhanced emissions in aggregates could be attributed to the rotor motions restriction, which activated the radiative decay process.The maximum absorption of THB was observed at 464 nm (Figure S6), while the maximum emission in the aggregated state was located at 647 nm, indicating its red-emission property and a large Stokes shift.

2.1.3
Preparation and characterization of THB@ANVs THB@ANVs were prepared by co-incubating THB nanoparticles (NPs) with 400 nm ANVs and extruding through a 200 nm polycarbonate membrane.The different initial concentrations of THB NPs were evaluated at a fixed ANVs concentration of 100 μg/mL (Figure S7).An optimal initial concentration of 50 μM THB NPs was selected, resulting in a hydrodynamic diameter of 204.1 nm and a polydispersity index (PDI) of 0.194 for THB@ANVs, close to that of ANVs (230.6 nm, PDI = 0.202).The low PDI value indicated consistent quality control across different batches of preparation.Further increasing the concentration of THB NPs did not enhance the amount of loaded THB.Additionally, a high concentration of excess THB NPs resulted in the deposition of large particles on the polycarbonate membrane, affecting the production of THB@ANVs.Calculated from the standard curve of THB (Figure S7B), a highest loading concentration of 35 nmol THB per 100 μg ANVs per milliliter can be obtained, corresponding to a loading content ([weight of THB/total weight of THB@ANVs] × 100%) of approximately 19.6%. [38]s illustrated in Figure 1F, the UV-vis absorption spectra of THB@ANVs exhibited a characteristic absorption band of THB at 464 nm.The TEM morphology of THB@ANVs (Figure 1A, right) confirmed the presence of loaded THB NPs.The particle size and Zeta potentials of the ANVs and THB@ANVs were measured using dynamic light scattering (DLS) and NTA (Figure 1G-J).The hydration effect resulted in larger particle size measurements by DLS compared to NTA.The diameters of the isolated ANVs and THB@ANVs ranged from 30 to 300 nm, consistent with the TEM results.Besides, the coupling of THB with ANVs was further confirmed by the co-localization of the red fluorescence of THB with the membrane labeled with 3,3-dioctadecyloxacarbocyanine perchlorate (DiO) (Figure 1K).

Inflammatory regulation ability
Macrophages are innate immune cells vital in host defense, wound healing, and immunity regulation. [39]In the early stages of wound healing, M1 macrophages can cause inflammation and resist infection.However, after the antibacterial treatment of a wound, the M1 macrophages must be allowed to quickly convert to the M2 type to promote effective wound repair.The ADSCs have been proven to promote wound repair by regulating inflammation. [40,41]Thus, to investigate the inflammatory regulation ability of ANVs and THB@ANVs, we incubated M1 macrophages with ANVs or THB@ANVs at a protein concentration of 4 μg/mL, according to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay results in Figure 2A.Lipopolysaccharide (LPS)-induced M1 macrophages were used as a positive control, while inactivated M 0 macrophages served as a negative control.As illustrated in Figure 2B-D, the ANVs or THB@ANVs group exhibited significant inhibition of proinflammatory tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and promotion of anti-inflammatory factor interleukin-10 (IL-10), proving that ANVs derived from ADSCs would also possess the ability to regulate inflammation.

Cell migration and proliferation promoting effects
ADSCs and their EVs have also been reported to promote tissue remodeling. [42,43]Since the composition of the asprepared ANVs was similar to that of ADSCs, the functions should be similar.The migration-promoting effects of ANVs and THB@ANVs were investigated through cell scratch tests and Transwell assays with immortalized human keratinocytes (HaCaT cells).The in vitro scratch wound model is a straightforward and reliable approach for studying cell migration and wound closure.As shown in Figure 2E,F, ANVs and THB@ANVs significantly enhanced HaCaT cell migration from the scratch edge after 24 h compared to the control group.Moreover, a higher number of HaCaT cells passed through the membrane from the upper Transwell chamber to the lower chamber when ANVs or THB@ANVs were added (Figure 2G,H), consistent with the results of the cell scratch test.As shown in Figure 2I,J, treatment with ANVs and THB@ANVs increased the number of 5-ethynyl-2′deoxyuridine (EdU)-positive cells, indicating enhanced DNA replication levels.The number of cells in MTT also increased considerably in a dose-dependent manner after incubation with ANVs or THB@ANVs (Figure 2A).These indicated that the ability of ADSCs to promote cell proliferation was retained in the as-prepared ANVs and THB@ANVs.No statistical difference was observed between the ANVs and THB@ANVs groups, suggesting that the addition of THB did not adversely affect cell proliferation and migration.Furthermore, ANVs and THB@ANVs exhibited similar effects on other types of wound repair-related cells, such as human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs), as detailed in Figure S8.All the cells exhibited minimal repulsion toward ANVs extracted from different individuals, proving these nanovesicles suitable for broad applications.

Preparation of hydrogel
In this study, we synthesized and characterized a di-activated alkyne-modified PEG-4000 cross-linking agent (PEG-DA) according to the literature (Figure S9). [44,45]Commercially available CMC with good water solubility, biocompatibility, and biodegradability was used to provide amino groups for the click-reaction with alkynes at room temperature to form a hydrogel of carboxymethyl chitosan and di-activated alkynemodified PEG (CMC-DA hydrogel). [46]The successful preparation of the hydrogel was confirmed through Fouriertransform infrared (FT-IR) spectroscopy (Figure 3A).The characteristic peaks of amino and alkyne groups did not appear, and the ester group peak shifted to lower wavenumbers in hydrogel, indicating the formation of β-aminoacrylate bonds in the hydrogel.Furthermore, a 1:1 ratio of activated alkyne and amine groups was used to optimize the hydrogel concentration.Gelation proceeded smoothly when the mass percentage exceeded 3% (Table S1).Increasing the concentration could further enhance gelation speed and mechanical strength.

Mechanical behaviors and injectability of hydrogel
Optimal repair can be achieved by soft hydrogels with mechanical characteristics fit to the wounds.We assessed the mechanical properties of 4% and 5% CMC-DA hydrogels, which showed fast gelation speed and good self-supporting ability.Strain sweep tests were performed to determine the breakage strains of hydrogels.The corresponding values of elastic modulus (G′) and loss modulus (G″) at an oscillation frequency of 1 Hz were shown in Figure 3B,C.Both G′ and G″ increased with the increase in strain, and a cross-over point between G′ and G″ was observed, indicating a transition from an elastic hydrogel to a sol-like behavior due to extreme deformation and ultimate breakage.The higher concentration of hydrogel resulted in lower breaking strain due to increased brittleness.The 4% hydrogel exhibited better elasticity and lower brittleness, making it more suitable for wound repair applications.
Injectability is a desirable feature for the biomedical applications of therapeutic hydrogels.These hydrogels can be transformed into liquid state under shear forces during injection, and rapidly solidify once injected to maintain their desired shape.The viscoelastic properties of the CMC-DA hydrogels were evaluated through a frequency-sweep test to indicate the transition from a solid to a liquid state.(Figure 3D,E).Both hydrogels demonstrated stable elastic properties between a linear response range of 1 to 10 Hz.The significantly higher values of G′ than G′′ suggested a solidlike behavior.The intersection frequencies of 4% hydrogel is lower, and the viscosity decreased rapidly with shear force increase, confirming its good injectability (Figure 3F).It was successfully injected into different patterns using a 1 mL syringe with a 16 G needle (Figure 3G).The injectability of the hydrogel enables it to fill irregular irregular shapes of real wounds, making it highly convenient for clinical applications.As illustrated in Figure 3H,I, cryo-scanning electron microscopy (SEM) revealed a uniform pore size distribution in the 4% CMC-DA hydrogel, with an average diameter of approximately 12.6 μm.The high porosity of the hydrogel provides sufficient space for nanovesicle loading.

pH-responsive swelling and degradation of hydrogel
Hydrogels can absorb exudate from wound tissue and regulate wound humidity.As shown in Figure 3J, the CMC-DA hydrogel could slowly swell and reach a plateau after 6 h.However, the swelling capacity was decreased in acidic environment.Since the β-aminoacrylate bond in the hydrogel has been reported to break in response to pH changes, [47] we investigated the pH-responsive properties of the hydrogel.As shown in Figure 3K,L, the release kinetics were faster in acidic environments than under neutral or alkaline pH conditions, and over 80% of THB were released from the hydrogel after 3 days.This indicated that degradation plays a more significant role over free exchange in the sustained release process under acidic conditions.Figure 3K demonstrated that the hydrogel initially swelled and then gradually degraded during 3 days.Thus, the release of THB@ANVs could be accelerated in infected wounds with lower pH values.Moreover, changes in pH affected the mechanical strength of hydrogels (Figure 3M-O).Under neutral conditions, Young's modulus values of the 4% and 5% hydrogels were 2.88 and 4.00 kPa, respectively, which decreased under alkaline or acidic conditions.These results demonstrated the promising potential of CMC-DA hydrogel for drug delivery and tissue engineering applications.

2.4
In vitro effects of the hydrogel

Biocompatibility
To assess the biocompatibility of the CMC-DA hydrogel with wounds, MTT and live/dead staining assay were performed on HaCaT cells (Figure 4A,B).The results indicated that different hydrogel concentrations had no negative effects on cellular viability.Both the control and hydrogel groups comprised predominant live cells and only a few dead cells, indicating the satisfactory cytocompatibility.Additionally, the hemocompatibility of the hydrogels was evaluated using a hemolysis assay, considering their potential contact with blood (Figure 4C).The results indicated that neither the hydrogel nor THB@ANVs induced significant hemolysis in red blood cells (<5% hemolysis ratio).
The release process of proteins from THB@ANVs encapsulated hydrogel was also evaluated (Figure 4D,E, Figure S11).After storage at 4 • C for 3, 7, and 14 days, the hydrogel was incubated in PBS solution at 37 • C, and the released proteins were quantified using a bicinchoninic acid (BCA) kit.The results indicated that the hydrogel better preserved the components of ANVs than their corresponding solution within the first 3 days of storage at 4 • C.However, over time, the water content in the hydrogel gradually decreased, resulting in a decreased protective effect.Therefore, storing hydrogels at 4 • C after preparation and using them within 3 days is recommended.The good biocompatibility and ability to protect proteins in THB@ANVs make the hydrogel suitable for use as burn wound dressing.

Antibacterial ability assay
The ROS generation capability of THB was assessed using commercially available 2′,7′-dichlorodihydrofluorescein diacetate (DCFH) and 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) (Figure 4F,G).In the presence of a low concentration of THB, the fluorescence of 2′,7′dichlorofluorescein (DCF) increased with white light exposure, indicating efficient generation of total ROS by THB.And the absorbance of ABDA decreased with increased singlet oxygen generation, suggesting the potential photodynamic antibacterial activity of THB.We then analyzed the killing effect of THB NPs, THB@ANVs, blank hydrogel, and THB@ANVs hydrogel on Staphylococcus aureus and Escherichia coli through spread plate method (Figure 4H).All THB-containing groups effectively killed Gram-positive S. aureus (nearly 100%) under white light, but had a limited inhibitory effect on Gram-negative E. coli.
(Figure S12).The negative charge of bacteria facilitates the stain of THB, but the thick outer membranes of Gram-negative bacteria may impede this process.As shown in Figure 4I, the AIE property of THB was used for bacteria imaging, with S. aureus demonstrating higher labeling efficiency compared to E. coli.SEM analysis (Figure 4J) revealed shrinkage and fusion of S. aureus upon treatment with THB@ANVs, providing direct evidence of bacteria-killing effects.However, the morphology of E. coli did not undergo significant damage despite the partial aggregation of particles on the surface.Additionally, the blank hydrogel exhibited antibacterial activity against Gram-negative bacteria, potentially due to its disruption of cell membrane integrity through CMC. [48]Given the remarkable bactericidal activity of THB against Gram-positive bacteria, the minimum inhibitory concentration (MIC) of THB in THB@ANVs was compared with the widely used antibiotic penicillin (Figure 4K).The results indicated that THB@ANVs exhibited a significantly lower MIC against S. aureus when exposed to light.The THB@ANVs demonstrated high antibacterial activity, low cytotoxicity, and reduced bacterial resistance, making them a superior alternative to conventional antibiotics for effective antibacterial treatment.

2.5
In vivo evaluation of deep second-degree burn wound healing

In vivo wound assay
The skin offers a primary defense against external bacterial infections.However, in the case of burn injuries, the skin barrier is damaged, allowing bacteria to easily infiltrate the body during the prolonged wound-healing process.
In vitro studies have demonstrated the significant potential of the THB@ANVs hydrogel in bacterial resistance and wound repair.Therefore, we established the model of deep second-degree burn wounds (16 mm in diameter) in rats using burn equipment with parameters set at 500 g, 95 • C, and 15 s (Figure S13).As illustrated in Figure 5A, hydrogels were applied to the wounds after debridement treatment, and S. aureus was dropped onto the wound surface to investigate the tissue repair promoting and anti-infectious ability of THB@ANVs hydrogel.Unlike in humans, the primary wound closure of rat relies more on the contracture of loose skin, rather than re-epithelialization and deposition of connective tissue. [49]Thus, silicone fixing rings were used to increase the tension of rat skin in the wound-healing assessment.In addition, commercially available 3 M ™ Tegaderm ™ film was applied to protect the wounds from scratches.Figure 5B presented photographs of the wounds at different time points (days 1, 3, 7, and 14) in the deep second-degree burn model of rats treated with PBS, blank hydrogels, ANVs hydrogels, or THB@ANVs hydrogels.To evaluate wound regeneration, we measured and analyzed the wound size reductions (Figure 5C).The THB@ANVs hydrogel group exhibited high antibacterial activity, with only a few bacterial colonies observed after photodynamic treatment (Figure 5D).As a result, the repair speed of the THB@ANVs hydrogel group was evidently accelerated with granulation tissue formation observed on day 3.However, bacteria nourished polysaccharide fragments might be released during the long-term biodegradation of hydrogel and promoted the proliferation of bacteria in hydrogel and ANVs hydrogel group.The wound areas of the ANVs or THB@ANVs hydrogel group on the day 7 and day 14 were smaller than that of the other two groups, confirming the ).(H) Inhibition of Staphylococcus aureus and Escherichia coli growth by THB NPs, THB@ANVs, hydrogel, or THB@ANVs hydrogel under white light irradiation (100 mW cm −2 , corresponding concentration of THB is 5 μM).(I) Fluorescence and merged images of E. coli and S. aureus incubated with THB@ANVs.Scale bar: 50 μm.(J) SEM images of E. coli and S. aureus treated with THB@ANVs before and after white light irradiation (100 mW cm −2 ).Scale bar: 500 nm.(K) MIC tests for penicillin and THB@ANVs before and after white light irradiation (100 mW cm −2 ).Penicillin or THB in THB@ANVs at concentrations of 0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, 5.12, and 10.24 μg/mL were incubated with 10 7 CFU/mL S. aureus for plate culture.wound-healing ability of the ANVs.On day 21, the ANVs and THB@ANVs hydrogel groups exhibited higher vascular densities, with a greater number of vascular master junctions and meshes (Figure 5E-H).This finding demonstrates that ANVs could enhance nutrient supply to wounds by promoting angiogenesis, thereby accelerating wound healing.The above results indicated that THB@ANVs hydrogel is a promising wound dressing for preventing bacterial infections and promoting wound regeneration simultaneously.

Histological analysis
To further investigate the healing effects of the hydrogels on wound tissues, we conducted hematoxylin and eosin (H&E) and Masson's trichrome staining on days 7 and 14 on the dissected wound tissues (Figure 6A).Collagen deposition plays a crucial role during skin remodeling by improving tissue tensile strength and epidermal integrity.Owing to the exceptional inhibitory effect of THB on early bacterial infection, the THB@ANVs hydrogel group exhibited a notable reduction in inflammation and higher collagen expression on day 7, while the ANVs hydrogel group exhibited a slight delay.On day 14, both ANVs and THB@ANVs hydrogel groups demonstrated significantly enhanced collagen deposition at the regenerated tissue sites, surpassing the control group, indicating the pivotal role of ANVs in this process.These histological observations were consistent with the above wound repair promotion experiments, and demonstrated the remarkable efficacy of THB@ANVs hydrogel in enhancing the process of burn wound healing.
In the proliferation phase, neovascularization is critical to ensure the transport of nutrients and oxygen to the wound site for sustaining fibroblast proliferation, collagen synthesis, and re-epithelialization.As shown in Figure 6B, we used immunofluorescence staining of CD31 and α-smooth muscle actin (α-SMA) to assess angiogenesis on day 7.The control group exhibited fewer newly formed blood vessels, possibly attributable to the inhibitory effects of bacterial infection on angiogenesis.In contrast, the THB@ANVs hydrogel group showed the highest levels of -SMA and CD31 positive expression among all groups, demonstrating the effectiveness of the anti-infection treatment.Furthermore, CD86 and CD163 staining verified that the M2/M1 macrophage expression in the THB@ANVs hydrogel group was the highest among all groups, indicating that wound healing had entered the proliferative phase earlier than in other groups.Besides, H&E staining of major organs from rats treated with PBS and THB@ANVs hydrogel revealed no significant signals of organ damage (Figure S14).These results indicated that THB@ANVs hydrogel is a promising burn wound treatment dressing with low biotoxicity.

CONCLUSIONS
In this study, we prepared aggregation-induced emissive nanovesicles delivered by click-hydrogel for simultaneous deep burn wound remodeling and antibiosis.We synthesized the AIE photosensitizer THB and combined it with ADSCs-derived nanovesicles to obtain THB@ANVs through a feasible extrusion method.The THB@ANVs exhibited exceptional antibacterial efficacy while also emulating the tissue repair-promoting capabilities of stem cells.A biocompatible CMC-DA hydrogel was prepared with click-chemistry to protect and deliver THB@ANVs for deep burn wound treatment.This hydrogel could be injected and adapt to the irregular shapes of wounds, absorb exudate from the tissue, and release in response to lower pH in infected wounds.We observed remarkable effects of the THB@ANVs hydrogel in inhibiting bacterial proliferation, regulating inflammation, and promoting neovascularization in a rat model of seconddegree burn wounds.Therefore, the developed THB@ANVs hydrogel dressing provides a promising solution for promoting the repair of deep burn wounds and preventing burn sepsis.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflicts of interest.

E T H I C S S TAT E M E N T
All animal experiments of the present study were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and approved by the Animal Ethics Committee of the Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China (2022, L399-1).All the authors listed have approved the animal study.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F
I G U R E 4 (A) Cell viability of HaCaT cells evaluated by MTT assay after treatment with hydrogels of different concentrations (n = 3).(B) Live/dead staining assay (Calcein-AM/PI) of HaCaT cells before and after incubating with hydrogel.Scale bar: 100 μm.(C) Blood compatibility and hemolysis ratio of 5 mg/mL hydrogel, THB@ANVs hydrogel, or corresponding quantity of THB@ANVs (n = 3).(D) Protein release rate of THB@ANVs encapsulated hydrogel before and after 3 days of storage at 4 • C, assessed using the BCA kit.(E) Release percentage of proteins from THB@ANVs hydrogel after storage at 4 • C for 3, 7, and 14 days.(F) Reactive oxygen species (ROS) generation of 1 μM THB NPs based on dichlorodihydrofluorescein diacetate indicator under white light irradiation (100 mW cm −2 ).(G) Singlet oxygen generation of 1 μM THB NPs based on 9,10-anthracenediylbis(methylene)-dimalonic acid indicator under white light irradiation (100 mW cm −2

F I G U R E 5
In vivo evaluation of the therapeutic efficacy of hydrogels on second-degree burn wounds.(A) Schematic diagram of the burn molding process and the timeline for assessing the therapeutic effects of hydrogels.(B) Representative images of the wounds on days 0, 1, 3, 7, and 14 and diagrams showing the time-evolved wound areas.(C) Statistical analysis of the wound area (n = 3).(D) Photographs of bacteria from the wound tissues on day 3, cultured on Luria-Bertani (LB) medium plate and Staphylococcus aureus selective mannitol salt agar (MSA) medium plate after 1000-fold dilution.(E) Representative images of blood vessels at the wound sites on day 21 after different treatments.Vascular regeneration at the wound sites on day 21 was estimated based on (F) the number of master junctions, (G) the number of meshes, and (H) total length of blood vessels.*p < 0.05; **p < 0.01.F I G U R E 6 Histological analysis of wound tissues.(A) Representative images of H&E staining and Masson's trichrome staining of the wound tissues on day 7 and day 14.Scale bar: 500 μm.(B) Representative immunofluorescence images of wounds on day 7 stained to show the expression of CD31 (green) and α-smooth muscle actin (red).Scale bar: 100 μm.(C) Representative immunofluorescence images of wounds on day 7 stained to show the expression of CD86 (green) and CD163 (red).Scale bar: 100 μm.
National Key Research and Development Program of China (2021YFC2302200), Natural Science Fund of Guangdong Province for Distinguished Young Scholars (2022B1515020089), and Hubei Province Natural Science Foundation for Distinguished Young Scholars (2022CFA089).We thank KetengEdit (www.ketengedit.com)for its linguistic assistance during the preparation of this manuscript.