ZnO-incorporated chitin hydrogels for infected wound therapy

Chronic wounds caused by pathogenic bacterial infections have been a worldwide medical threat and challenge, ZnO is a promising antibacterial agent to promote infected wound healing. However, ZnO particles need to be with a wound dressing material to improve low-dose antibacterial e�cacy while reducing cytotoxicity. The natural polysaccharide, chitin-based hydrogels can be applied as a preferential supporting matrix for the deposition of ZnO. In this study, we developed chitin/ZnO composite hydrogels (CZG hydrogels), which were applied for the biolm-infected full-thickness wound treatment. The three-dimensional hydrophilic network structure of chitin hydrogels provided a large number of absorption sites for Zn 2+ ions, and CZG hydrogels were prepared by in situ synthesis of ZnO. CZG hydrogels had potent broad-spectrum and long-lasting antibacterial activity, good bacteriostatic ability against high concentration of bacterial �uids. The in vivo studies showed that CZG hydrogels have a signi�cant effect of accelerating biolm-infected wound healing. Collectively, this work con�rmed that chitin hydrogels could be applied as a preferential natural supporting matrix for the deposition of inorganic metal and metal oxide nanoparticles, and provided alternative antibacterial dressing for treating microbial infections and promoting wound healing.


Introduction
Chronic wounds caused by pathogenic bacterial infections have been a worldwide medical threat and challenge (Xu et al., 2023;Du et al., 2022;Falanga et al., 2022).As alternative treatment to antibiotic therapy, inorganic metal and metal oxide nanoparticles have superior antibacterial properties (Wu et al.; Wang et al., 2022).Among the reported metal oxide nanoparticles, zinc oxide (ZnO) nanoparticles have excellent antibacterial effect, and proper concentration of Zn 2+ released from ZnO in wounds can stimulate angiogenesis and promote broblast proliferation and migration, as well as re-epithelialization of the skin (Alavi and Nokhodchi, 2020; Song et al., 2020).Therefore, ZnO is a promising antibacterial agent to promote infected wound healing.However, ZnO particles need to be with a wound dressing material to improve their dispersion and stability, and improve low-dose antibacterial e cacy while reducing cytotoxicity at the bacterial infection site (Yin et al., 2022;Zhang et al., 2021;Raguvaran et al., 2017).
According to the "wet wound healing theory", wounds heal twice as fast in a wet healing environment than in a dry environment (Wang et al., 2023).Due to the unique combination of biological and physical properties, hydrogels with antibacterial property might be ideal dressings to treat infected wound (Rakhshaei and Namazi, 2017;Yadollahi et al., 2015a;Yadollahi et al., 2015b;Yadollahi et al., 2016;Abbasabadi et al., 2022).Thus far, considerable efforts have been made to design and develop naturally occurring polysaccharide-based hydrogels with a combination of antibacterial ZnO (Abbasabadi et al., 2022;Zhou et al., 2022;Yang et al., 2023;Jin et al., 2023).Chitin, the nitrogen-containing polysaccharide in nature and the second most abundant renewable polymer after cellulose, exists mainly in shrimps' and crabs' exoskeletons (Pillai et  hydrogels have multiple advantages such as biocompatibility, biodegradability, and three-dimensional hydrophilic polymer network structure similar to the macromolecular biological tissue, which are extensively used in biomedical application (Huang et al., 2021;Kang et al., 2022;Anitha et al., 2014).To improve its properties and broaden its applications, chitin hydrogel has been modi ed, such as with polyphenol or hydroxyapatite (Cao et al., 2023;Xing et al., 2021;Lin et al., 2021a).In our previous work, Mn 3 O 4 nanoparticles were formed in the network of chitin hydrogels via in situ mineralization process and encapsulated to fabricate novel chitin/Mn 3 O 4 composite hydrogels, with NIR-assisted outstanding photothermal antibacterial and antibio lm activities (Zhang et al., 2023).Thereby, chitin hydrogels can be applied as a preferential natural supporting matrix for the deposition of ZnO.
Hence, in this work, we attempted to fabricate chitin/ZnO composite hydrogels by in situ conversion of Zn 2+ to ZnO throughout chitin hydrogel networks.It is anticipated that chitin/ZnO composite hydrogels combine their unique properties.Therefore, the broad-spectrum antibacterial activity and biocompatibility of chitin/ZnO composite hydrogels were systematically investigated.Furthermore, the wound healing performance of the chitin/ZnO composite hydrogels was evaluated in a mouse full-thickness S. aureus bio lm-infected wound model.

Preparation of chitin/ZnO composite hydrogels
Chitin hydrogels (CG hydrogels) were prepared according to our previous work (Zhang et al., 2023).The CG hydrogels were immersed in Zn(NO 3 ) 2 solution at different concentrations (0.05, 0.1 and 0.25 M) for 24 h.Then NH 3 •H 2 O was dripped into the solution, and Zn 2+ ions combined in chitin hydrogel network transformed into ZnO particles after 24 h.The excess alkali and ZnO particles were washed away with distilled water (DW), and chitin/ZnO composite hydrogels were obtained.Chitin/ZnO composite hydrogels with initial Zn(NO 3 ) 2 concentration of 0.05, 0.1 and 0.25 M, were coded as CZG-1, CZG-2 and CZG-3 hydrogels, respectively.

Antibacterial test
Staphylococcus aureus (S. aureus), Escherichia coli (E.coli), Methicillin-resistant Staphylococcus aureus (MRSA) and Multidrug resistant Escherichia coli (MDR E. coli) were selected to evaluate the antibacterial activity.The CG/CZG hydrogels were placed in 24-well plates, and a bacterial suspension was added dropwise on the surface of the hydrogels and incubated for predetermined time.The antibacterial ability was evaluated using colony forming unit (CFU) counting, the bacterial growth kinetics, crystal violet staining, live/dead uorescence assay, and SEM measurements.The detailed procedures can be found in the supplementary material.

Biocompatibility evaluation
The cytocompatibility of the hydrogels on embryonic mouse broblast cell line (NIH 3T3 cells) was assessed by MTT assay.The blood compatibility of the hydrogels was evaluated using a hemolysis test.
The detailed procedures can be found in the supplementary material.

Animal experiments
Female Kunming mice with the average bodyweight around 20 g (Wuhan Centers for Disease Prevention & Control, China) were used as experimental subjects.
The S. aureus bio lm-infected mice were randomly divided into three groups, and were treated with PBS, CG, and CZG-2 hydrogels, respectively.The wound healing process was monitored for 15 days.On day 15, the wound skin tissue and main organs (heart, liver, spleen, kidney, and lung) of the mice were harvested for hematoxylin-eosin (H&E) and Masson trichrome staining analysis.The detailed procedures can be found in the supplementary material.

Statistical analysis
Data were analyzed by using one-way ANOVA analysis of variance with Tukey's test for multiple comparisons.The values of *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were taken for statistically signi cant, respectively.All data were exhibited as mean ± standard deviations (SD).
3 Results and discussion

Characterization of the chitin/ZnO composite hydrogels
The transparent chitin hydrogels (CG hydrogels) were obtained according to our previous work (Zhang et al., 2023).Then CG hydrogels were immersed into different concentrations of Zn(NO 3 ) 2 solution to make Zn 2+ ions full penetration throughout the nanopores of chitin, Zn 2+ ions anchored onto the stable absorption site on chitin nano bers, followed by alkali treatment, the chitin/ZnO composite hydrogels with different ZnO contents were obtained.It is obvious to observe that the composite hydrogels were opaque because of the formation of ZnO nanostructures (Fig. 1), and the actual ZnO content in the composite hydrogels can be measured by TGA (Figure S1).The actual ZnO content of CZG-1, CZG-2 and CZG-3 was 2.6%, 5.8% and 6.5%, respectively.The micromorphology of both surface and cross-section of the as-prepared CG and CZG composite hydrogels was monitored by scanning electron microscope (SEM), as represented in Fig. 1a and 1b.It can be clearly seen that all the hydrogels simultaneously exhibited the 3D hierarchical porous network structure, but the incorporation of ZnO particles obviously destroyed the original porous structure of CG hydrogels, and the CZG hydrogels exhibited looser structure, which indicated the interaction existed between ZnO particles and chitin matrix.Of note, the content of ZnO particles on and inside the CZG hydrogels both increased signi cantly, with the increasing concentration of Zn(NO 3 ) 2 , and the average size of ZnO particles in CZG hydrogels is basically much smaller than 1 µm (Fig. 1b).The corresponding mapping showed the homogeneous dispersion of ZnO particles in CZG-3 hydrogels (Fig. 1c).
The structure of CZG hydrogels was further investigated.Figure 2a displayed the X-ray diffraction (XRD) patterns of CG and CZG hydrogels.CG hydrogels showed typical peaks at 9.0°, 19.5° and 26.5°, which were corresponded to the 020, 110 and 013 crystal planes of chitin, respectively (Shang et  ).In addition, with the loading of ZnO, the intensity of characteristic peaks of chitin became weak and the degree of crystallization decreased, which indicated that the interaction between ZnO and chitin matrix affected the crystal structure of chitin.The XPS survey spectrum (Fig. 2b) illustrated that the obvious Zn signal peaks appeared after the incorporation of ZnO particles in CZG-3 hydrogels.In the Zn 2p spectrum (Fig. 2c), the binding energies of Zn 2p 3/2 and Zn 2p 1/2 were at 1021. and the absorption band of C-H at 2929 and 2878 cm − 1 for CG hydrogels, respectively, generated bathochromic shift after doping ZnO in CZG hydrogels, indicating that the hydrogen bond interaction between the chitin chains was weakened.Also, the slight change in the shape of the band at 1658 cm − 1 attributed to υ(C = O) of amide groups, indicated the interaction between ZnO and chitin chains (Brugnerotto et al., 2001).The above results prove the successful formation of ZnO in chitin matrix and the tight interaction existing among ZnO and chitin matrix, which might play an important role in the uniform dispersion of ZnO particles.

Antibacterial activity and investigation of bio lm inhibition in vitro
There has been plenty of reports about ZnO with antibacterial activity (Zhou et  As shown in the resultant colony pictures of the agar plate (Fig. 3a), CZG hydrogels exhibited broad-spectrum antibacterial activity against S. aureus, E. coli, MRSA and MDR E. coli, and once we increased the ZnO content in CZG hydrogels, the antibacterial e ciency increased accordingly.The killing rate of CZG-1 hydrogels against S. aureus, E. coli, MRSA, and MDR E. coli was more than 95%, and the killing rate of CZG-2 and CZG-3 hydrogels against the four bacterial strains was up to 99.9%, respectively (Fig. 3b).Then CZG-2 hydrogel was selected for subsequent antibacterial tests, for the reason that the high concentration of ZnO inevitably increased the toxicity of hydrogels (George et al., 2022).We further tested the long-lasting antibacterial activity of CZG-2 hydrogels against MRSA, and MDR E. coli, as shown in Figure S3, CZG-2 hydrogels maintained excellent antibacterial activity for 7 consecutive days.In addition, time-dependent inhibition behavior of CZG-2 hydrogels against S. aureus and E. coli, with the concentration of 10 5 , 10 6 , and 10 7 CFU/mL was monitored by measuring OD 600 of the culture medium (Fig. 3c and 3d).Both of the growth curve of S. aureus and E. coli showed a trend of "S" type within 24 h, and the CZG-2 hydrogels can completely inhibit both S. aureus and E. coli growth when the initial bacterial concentration was 10 5 and 10 6 CFU/mL, which was similar to that in the reported works (Ma et al., 2020;Dutta et al., 2012;Zhang et al., 2022;Reyes et al., 2017).And the inhibitory effect of CZG-2 hydrogels was decreased to some extent after 14 h, when the initial higher bacterial concentration of 10 7 CFU/mL, which was seldom applied.Regardless, CZG-2 hydrogels possessed excellent inhibitory activity for both S. aureus and E. coli.
In order to more detailedly evaluate the antibacterial properties of CZG-2 hydrogels, S. aureus and E. coli after being treated with CZG-2 hydrogels, were stained with Calcein-AM/propidium iodide (PI) double staining kit, and then directly observed by the uorescent microscopy.As shown in Fig. 3e1-f2, both S. aureus and E. coli displayed a strong green uorescence (live bacteria) in the PBS groups, in contrast, all of bacteria (including S. aureus and E. coli) after treatment with CZG-2 hydrogels were stained red, adequately suggesting the signi cant antibacterial properties.Furthermore, the microstructures of S. aureus and E. coli were also observed by SEM (Fig. 3g1-h2).Both S. aureus and E. coli after being respectively treated with PBS retained normal morphology, with clear and smooth cell outline.Importantly, the CZG-2 hydrogels treatment group showed serious damage to both S. aureus and E. coli, the membranes of the two kinds of bacteria are disrupted (the broken membrane structure is marked by green arrows Bio lms form a dense natural barrier that can hinder the penetration of antibacterial drugs, thus prolonging the infection (Mei et al.;Rumbaugh and Sauer, 2020;Sauer et al., 2022).Hence, to prevent the formation of bacterial bio lms is perhaps an effective way to improve treatment e cacy.Therefore, we used crystal violet staining and plate count method to evaluate the inhibitory effects against bio lm formation of CZG hydrogels.As shown in Fig. 4a and 4b, the inhibitory effect of CZG hydrogels on bio lm formation was concentration-dependent, the inhibitory rate of CZG-1 hydrogels group was more than 82%, the inhibitory rate of CZG-2 hydrogels group was over 90%, and there was hardly any S. aureus or E. coli bio lm formation in the CZG-3 hydrogels group.From the CFU enumeration (Fig. 4c and 4d), we obtained similar results, CZG hydrogels could effectively inhibit the bio lm formation.The excellent antibacterial and bio lm-inhibitory performance of CZG hydrogels might be attributed to the release of Zn 2+ from ZnO nanoparticles, which generates ROS through the Fenton reaction, leading to the destruction of intracellular lipids (lipid peroxidation), proteins (e.g., enzymes), and nucleic acids (DNA/RNA) macromolecules (Ning et al., 2015;Rosenberg et al., 2020;Lin et al., 2021b;Sirelkhatim et al., 2015).Speci c antibacterial mechanism study for the work need to be further explored.

Safety evaluation in vitro
Hydrogels dressings act directly at the site of infected wound, it is important to evaluate the biosafety of the dressings (Yang et al., 2022;Zeng et al., 2022).NIH 3T3 cells were used to assess the cellular safety of CZG hydrogels.Figure 5a showed that after incubation for 24, 48, and even 72 h, all components except CZG-3 hydrogels lack apparent cytotoxic effects in whole, and the relatively lower viability of CZG-3 hydrogels was attributed to relatively higher ZnO content.Additionally, after incubation for 72 h, the cells were stained with Calcein-AM/PI dyes to con rm the safety of CZG hydrogels.Figure 5b showed that the cells presented a broblast-like morphology, and no red uorescence was apparent, demonstrating the good cellular safety of CZG hydrogels.For analysis of hemocompatibility, CZG hydrogels were co-incubated with red blood cells, and the rate of hemolysis was measured, with deionized water (DW) as the positive control and PBS as the negative control, respectively.The supernatant of the red blood cell suspension was colorless, except DW group, and no signi cant hemolysis was observed for CZG hydrogels (Fig. 5c).These results indicated that CZG hydrogels had acceptable cellular safety and excellent blood compatibility.

Antibio lm and wound healing performances in vivo
Inspired by the excellent antibacterial effect in vitro, severely infected wound mouse models were established to explore the therapeutic e cacy of CZG hydrogels in vivo (Fig. 6a).After inoculating S. aureus suspension for 3 d to form the initial bio lm, all infected wounds were randomly divided into three groups, and treated with PBS, CG hydrogels, and CZG-2 hydrogels, respectively.After three days of treatment, S. aureus cultured from the infected tissues were counted.Compared to the PBS and CG hydrogels groups, the CZG-2 hydrogels group showed a signi cant elimination effect against bio lms and a signi cant reduction, with antibacterial rate up to 99.9% (Fig. 6b and 6c), owing to the antibacterial role of ZnO.The infected wound area and body weights of all mice were monitored every 3 days.As shown in the Fig. 6e, all groups showed varying degrees of shrinkage in wound area, and the scar size in the CZG-2 hydrogels group was the smallest at each time point.In whole, the wounds treated with CG and CZG-2 hydrogels showed improved recovery, with signi cant differences from day 6 compared to those in the PBS groups (Fig. 6f).Furthermore, there was no signi cant difference between the weights of all groups, and all weights maintained slightly increased (Fig. 6d), showing that the treatments did not affect the growth of mice.
After sacri cing the mice on day 15, the infected tissues were collected and subjected to hematoxylineosin (H&E) and Masson staining.As shown in Fig. 7a, there were still obvious defects in the PBS groups, with large wound areas not covered by epithelial tissue and signi cant in ammatory response (marked with black arrows).Compared with those in PBS groups, the wounds in CG hydrogels group showed abundant mature granulation tissue and complete epithelial structure, indicating that chitin hydrogels can accelerate the epithelialization process by promoting the development of granulation tissue (An et al., 2023;Shi et al., 2023).It is remarkably, however, the wounds in CZG-2 hydrogels group were basically completely healed, and the corneum and basal layers (marked with green dotted line) were intact.The reconstructed dermal tissues at the wound site had more skin appendages, including sweat glands (marked with yellow arrows) and hair follicles (marked with red arrows), which were no difference from normal dermal tissue, and indicated that CZG-2 hydrogels have a potential for scarless repair (Wu et al., 2022).
Collagen is critical for promoting wound healing and dermal reconstruction (Shi et al., 2023;Gaspar-Pintiliescu et al., 2019;Linju and Rekha, 2023).As shown in Fig. 7b, Masson staining results showed that the collagen of the wound of the PBS group was sparse and disordered, and the wounds after CG hydrogels treatment had more collagen deposition.While, the collagen bers in the CZG-2 hydrogels treatment group were more and the organization was highly orderly.The area of collagen deposition percentage of the wound slices of the infected wounds in the three groups was calculated, as shown in Fig. 7c, it was obvious that CZG-2 hydrogels treatment could promote collagen deposition.These results indicate that CZG-2 hydrogels can promote the re-epithelialization of mouse skin wounds, promote the generation and maturation of collagen, and promote the regeneration of skin appendages.The reasons that CZG-2 hydrogels are bene cial to wound healing are speculated as follows.1) The biocompatible CZG-2 hydrogels were used to cover the bacterial infected wounds, allowing them to continuously clear the bacteria and prevent secondary infection.2) The slowly release of Zn 2+ is conducive to the formation of epithelial cells, promote the growth of granulation tissue and accelerate collagen deposition (Liao et al., 2023).In addition, histological analysis of the major organs, including heart, liver, spleen, lung, and kidney, demonstrated no obvious in ammatory lesions or necrosis in the different groups (Figure S4), which supported the biocompatibility of CZG-2 hydrogels.

Conclusion
We successfully fabricated chitin/ZnO composite hydrogels by in situ conversion of Zn 2+ to ZnO throughout chitin hydrogel networks.Strong interactions between ZnO particles and chitin chains promote uniform dispersion of ZnO in chitin hydrogel networks.The in vitro experiments showed that CZG hydrogels had potent broad-spectrum and long-lasting antibacterial activity, which could effectively kill E. coli, MDR E. coli, S. aureus and MRSA.In addition, CZG hydrogels had good bacteriostatic ability against high concentration of bacterial uids and could effectively inhibit the formation of bacterial bio lm.The preliminary biosafety evaluation indicated that CZG hydrogels had acceptable cellular safety and excellent blood compatibility.The in vivo studies showed that CZG hydrogels have a signi cant effect of accelerating S. aureus bio lm-infected wound healing, by promoting the re-epithelialization of mouse skin wounds, the generation and maturation of collagen, and promoting the regeneration of skin appendages.This work con rmed that chitin hydrogels could be applied as a preferential natural supporting matrix for the deposition of inorganic metal and metal oxide nanoparticles, and provided alternative antibacterial dressing for wound treatment.
al., 2009; Duan et al., 2018; Hou et al., 2023).Chitin has been fabricated into hydrogels by physical cross-linking or chemical cross-linking (Huang et al., 2023; Lu et al., 2023).Chitin 3 and 1044.3 eV, respectively, with △E = 23.0 eV, close to the standard reference value of ZnO(Zhang et al., 2010;Wu et al., 2021), while the binding energy value of ZnO in CZG hydrogels is slightly higher than that of pure ZnO.The high-resolution spectra of N 1s, O 1s, and C 1s were further explored in detail.As depicted in Fig.2d, the O 1s spectrum of CG hydrogels exhibited two peaks at 532.4 and 531.6 eV, attributed to C-O and C = O, respectively.The characteristic peak of Zn-O appeared in CZG hydrogels, and the binding energy of O 1s for C = O increased.After the introduction of ZnO, the binding energy of N 1s in the acetamide groups changed slightly (Fig.2e), and the binding energy of C 1s for C = O and C-O/ C-N also changed (FigureS2).The Fourier transform infrared (FTIR) spectra of CZG hydrogels further con rmed the successful formation of ZnO in chitin hydrogels matrix, as shown in Fig.2f.Compared to the CG hydrogels, all spectra of the CZG hydrogels showed the characteristic absorption bands of ZnO in the range of 400-550 cm − 1(Dhanalakshmi et al., 2017).Moreover, the incorporation of ZnO affected the innate hydrogen networks of chitin.The stretching vibration peak of O-H and N-H observed at 3428 and 3270 cm − 1(Xu et al., 2016),

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Figure 4