Injectable methylcellulose hydrogel containing silver oxide nanoparticles for burn wound healing

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Injectable methylcellulose hydrogel containing silver oxide nanoparticles for burn wound healing ⁎⁎

Min Hee Kima, Hanna Parka, Hyung Chan Nama, Se Ra Parkb, Ju-Young Jungb, , Won Ho Parka, a b

⁎

Department of Advanced Organic Materials and Textile Engineering System, Chungnam National University, Daejeon 34134, South Korea Department of Veterinary Medicine, Chungnam National University, Daejeon 43134, South Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Methylcellulose Thermo-sensitive hydrogel Silver oxide nanoparticles In situ formation Wound healing

A thermo-sensitive methylcellulose (MC) hydrogel containing silver oxide nanoparticles (NPs) was prepared via one-pot synthesis in which a silver acetate precursor salt (CH3COOAg) induces a salt-out effect in the MC solution. The silver oxide NPs were synthesized in situ from Ag+ ions during the MC hydrogelation, and the residual CH3COO− ions decreased the gelation temperature of the MC solution through the salt-out effect. The gelation behavior of the MC solution varied according to the CH3COOAg content and was monitored. Also, the formation and structure of the silver oxide NPs in the MC hydrogel was confirmed. From the results, silver oxide NPs was successfully incorporated in MC hydrogels, simultaneously, acetate ion which was counter ion of Ag was affected gelation behavior of Ag. Finally, the antimicrobial activity and wound healing effect was examined using the shaking flask method and burn wound test, respectively. The MC hydrogel with silver oxide NPs showed excellent antimicrobial activity and burn wound healing. Therefore, this thermo-responsive MC hydrogel has great potential as an injectable hydrogel for wound regeneration.

1. Introduction Hydrogels consist of 3-dimensional crosslinked polymer networks that are highly swollen in aqueous environments. Hydrogels can be divided into physical or chemical hydrogels according to the nature of their crosslinking. Chemical hydrogels are produced through covalent crosslinking between the polymer chains, and physical hydrogels form with non-covalent crosslinking, such as through van der Waals interactions, hydrophobic association, or an electrostatic interaction (Wang, Wang, & Teng, 2016). Stimuli-responsive hydrogels are one of physical hydrogels that exhibit a reversible sol-gel transition in response to external stimuli, including temperature, pH, electrical field and light (Hoffman, Schmer, Harris, & Kraft, 1972). Among these, thermo-reversible hydrogels have received a significant amount of attention for use in drug delivery systems due to their sol-gel transition in response to changes in temperature (Hoffman & Ratner, 1975). The activity of thermo-reversible hydrogels can be easily controlled by simply administering them into the body, without the need for chemical or environmental treatment (Kamath & Park, 1993). For this reason, thermoreversible hydrogels are usually applied as injectable hydrogels with a

sol-gel transition upon exposure at body temperature (El-Sherbiny & Yacoub, 2013). Recently, hydrogels with encapsulated drugs have been implanted as locally injectable drug delivery systems to treat injuries, and these are designed to be very sensitive to a given stimulus (Zhu & Marchant, 2011). Methylcellulose (MC) is a water-soluble cellulose derivative that can be used as a binder for pharmaceuticals (drug delivery), water-retention agents, and paint and food thickeners (Woerly, 1997). In particular, the MC with a degree of substitution (DS) of 1.7-2.0 has a unique behavior in that a reversible sol-gel transition could be induced by a hydrophobic interaction in an aqueous solution upon an increase in temperature (Wang, Chen, Xiang, & Ye, 2007; Borkenhagen, Clémence, Sigrist, & Aebischer, 1998; Hoffman, 2012; Li et al., 2001). The sol-gel transition of MC can be controlled by the molecular weight, degree of substitution (DS), concentration, and additive electrolytes. The effect of various salts on the sol-gel transition of MC is particularly consistent with the socalled Hofmeister series, an order of ions that are ranked in terms of how strongly they affect the hydrophobicity of a solute in water (Bawa, Pillay, Choonara, & Lisa, 2009; Jeong, Kim, & Bae, 2002; Hubbell, 1998). In general, the effect of common anions follows the order of ci-

⁎ Corresponding author at: Department of Advanced Organic Materials and Textile System Engineering, College of Engineering, Chungnam National University, Daejeon 305-764, South Korea. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (J.-Y. Jung), [email protected], [email protected] (W.H. Park).

https://doi.org/10.1016/j.carbpol.2017.11.109 Received 6 September 2017; Received in revised form 28 November 2017; Accepted 28 November 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Kim, M.H., Carbohydrate Polymers (2017), https://doi.org/10.1016/j.carbpol.2017.11.109

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Fig. 1. UV–vis absorption spectra of silver oxide NPs synthesized from MC solution containing 1.0 wt% silver acetate for 48 h. Fig. 2. Temperature and time frequency sweep of the MC solution with silver acetate. Gelation temperature measured at a 1 °C/min heating rate and gelation time measured at 37 °C (0.05% strain, 1 rad/s frequency).

trate3− > SO42− = tartrate2− > HPO42− > CrO42− > acetate− > HCO3− > Cl− > NO3− > CIO3− (Gibson & O'Reilly, 2013; Salis & Ninham, 2014). Therefore, the anions are responsible for lowering the gelation temperature of the MC solution by dehydrating the MC molecules (salt-out effect). In contrast, the anions on the right tend to delay the gelation of the MC by hydrating the MC molecules (salt-in effect). Injectable hydrogels are also able to load bioactive NPs in their aqueous phase and can be transformed into a gel when injected into body, resulting in the elimination of surgical procedures, administrated in a minimally invasive manner and can easily fill arbitrary shaped defects (Yu & Ding, 2008). It is also possible to apply as a wound dressing material through the thermosensitive sol-gel transition at wound surface. Silver (Ag) compound such as Ag and AgO NPs has been widely used for wound treatments with respect to antimicrobial activity (Chun et al., 2010). Various types of wound or burn dressing contained Ag compound have been commercialized in market such as Acticoat®. The Ag compound is relatively less toxic compared to other antimicrobial agent, and has a broad spectral antimicrobial activity against a wide range of micro-organism (Wright, Lam, Buret, Olson, & Burrel, 2002). In this study, based on the Hofmeister series, silver acetate salt was from Ag precursor salts for promote burn wound healing bioactive agent with a good salt-out effect. Good salt-out effect Ag precursor salt

was added into the aqueous MC solution. When salt was added into the MC solution, the silver oxide NPs were synthesized in situ from Ag+ and OH−, and the unreacted acetate anions simultaneously decreased the gelation temperature and time for the MC solution. This one-pot synthesis is effective to easily prepare the thermo-sensitive hydrogels containing bioactive NPs, and the resulting MC composite hydrogel was examined as an injectable hydrogel for burn wound regeneration.

2. Experimental 2.1. Materials and preparation of MC hydrogel containing Ag NPs MC with a viscosity of 15 cP (Mn (g/mol) = 44,000; Mw (g/mol) = 110,000; and DS = 1.6–1.7) was obtained from LOTTE Fine Chemical Co., Ltd (Korea), silver acetate (CH3COOAg) (Sigma-Aldrich, U.S.A.) was used as a precursor salt for the Ag NPs. Silver acetate powder was added into the 8 wt% aqueous MC solution and was gently stirred overnight at room temperature. The final concentrations of the CH3COOAg were 0.5 and 1.0 wt%. The MC hydrogels with different CH3COOAg concentrations (0.0–1.0 wt%) were named MC-0.0, MC-0.5, and MC-1.0, respectively.

2

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Fig. 3. SEM images of lyophilized MC hydrogel: (A, D, G) MC-0.0, (B, E, F) MC-0.5, and (C, F, I) MC1.0.

spectroscopy (XPS) measurements were performed on the Thermo Scientific MultiLab 2000 using 400 W monochromated Al Kα radiation A 500 μm X-ray spot was used for the XPS analysis, and the base pressure in the analysis chamber was of about 5 × 10−10 mbar. Binding energies were determined by a fitting process using Gaussian peak shapes. Typically, the C1s line at 286.25 eV from adventitious carbon is used for energy referencing. Both survey scans and individual high-resolution scans for Ag(3d), O(1s) and C(1s) peaks were also recorded.

2.2. Rheological measurements A strain-auto controlled mode by rheometer (MARS 40, Haake, Germany) with a plate geometry (diameter = 60 mm and gap = 1 mm) was used to measure the rheological properties of the various MC samples. The measurement condition was determined by an amplitude sweep that existed in the linear region of the storage modulus (G’) and loss modulus (G”) (0.05% strain and 1 rad/s frequency). The gelation temperature, gelation time, and gel strength were monitored by a frequency sweep at set conditions. A temperature sweep was carried out from 20 to 40 °C at a heating rate of 1 °C/min to measure the gelation temperature. A time sweep was also performed to investigate the gelation time at 37 °C.

2.4. In vitro release profile The in vitro release profile of the silver oxide NPs from MC hydrogel was evaluated using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). CH3COOAg of 0.5 or 1.0 wt% was dissolved in the MC solutions prior to gelation. A release experiment was carried out to observe the release behavior of the Ag ions at a constant shaking rate and body temperature (37 °C). At each time interval, aliquots of 3 ml were collected from the medium following the release of the Ag+ ions. Cumulative release of Ag+ ion was determined by ion spectrum measured by ICP-MS and quantitified.

2.3. Structural characterization of the MC hydrogel containing Ag NPs Diluted MC solution containing CH3COOAg was measured by UV–vis spectrophotometer (UV-2450, Shimadzu, Japan) at certain time interval to confirm the formation of Ag NPs from CH3COOAg. The crystalline structure of the Ag NPs was measured in the range from 2θ = 5–50° (λ = 0.154 nm, 40 kV, 40 mA) using X-ray diffraction (XRD, D8 DISCOVER, Bruker, U.S.A.) at room temperature. The synthesized Ag NPs were analyzed after washing the MC hydrogel obtained at 37 °C, and the MC hydrogel was suspended in distilled water and centrifuged at 3000 rpm for 2 min. This process was repeated 3 times, and the Ag NPs were lyophilized after gelation at 37 °C. The porous structures of the MC hydrogel, morphology of the Ag NPs, and the distribution of the Ag atoms were observed using a field emission scanning electron microscope (FE-SEM, JSM-7000F, JEOL, Japan) in low vacuum mode. The 8 wt% MC hydrogel with or without Ag NPs samples were lyophilized after gelation at 37 °C, and the crosssection was observed. To characterization of synthesized Ag NPs, X-ray photoelectron

2.5. Antimicrobial activity The antimicrobial activity of the MC hydrogel with Ag NPs was evaluated using the shaking flask method (ASTM E2149) (Boonkaew, Suwanpreuksa, Cuttle, Barber, & Supaphol, 2014). Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli were chosen as the bacteria to evaluate the antimicrobial activity. The samples were incubated to induce gelation at 37 °C for 30 min. For the shaking method, 50 ml of phosphate buffer (pH 7.2) was taken, and 1 ml of hydrogel was added to adjust the test sample concentration of 2%. Next, 2.0 × 105 of bacteria were seeded to the test sample and then shaken at 250 rpm at 3

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Fig. 4. XRD patterns and XPS spectra of silver oxide NPs synthesized in MC solution containing silver acetate, (A) XRD patterns, (B) XPS spectra of pure MC hydrogel and MC hydrogel with silver oxide NPs, (C, D) Deconvolution XPS peaks of MC hydrogel with silver oxide NPs(0.5 wt%, 1.0 wt% silver acetate, respectively). Fig. 5. Cumulative release of Ag ions from the MC hydrogel as a function of time.

37 °C for 1 h. The media was finally extracted to evaluate the antimicrobial activity of the MC hydrogel with Ag NPs.

animal care in laboratory research. For all experiments, four-week-old male Sprague-Dawley rats were housed and bred at the experimental animal center of Chungnam National University. The animals were provided with a commercial diet and water ad libitum under temperature-, humidity-, and lighting-controlled conditions (23 ± 2 °C, 55 ± 5%, and a 12:12-h light-dark cycle, respectively). Procedures

2.6. Burn wound healing test The experiment was approved by the institutional committee for 4

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Table 1 Bacterial reduction rate of control, pure MC hydrogel, MC hydrogels with silver oxide NPs. Staphylococcus aureus

Control MC-0.0 MC-0.5 MC-1.0

Klebsiella pneumoniae

Escherichia coli

Number of bacterial after 1 h

Bacterial Reduction rate

Number of bacterial after 1 h

Bacterial Reduction rate

Number of bacterial after 1 h

Bacterial Reduction rate

2.0*105 2.7*105 < 30 < 30

– 0 99.9 99.9

2.0*105 2.7*105 < 30 < 30

– 0 99.9 99.9

2.0*105 2.6*105 < 30 < 30

– 0 99.9 99.9

concentration of CH3COOAg increased to 1.0 wt% (Fig. 2B). Therefore, the CH3COOAg was found to accelerate the gelation of the MC due to a good salt-out effect of the CH3COO− ion. When CH3COOAg was added into the MC solution, Ag+ and CH3COO− ions were promptly generated. According to the Hormeister series, the CH3COO− ion can form a solvated shell via hydrogen bonds with water molecules. Subsequently, the MC molecular chains in the solution were dehydrated, and the MC gelation was accelerated by the increased hydrophobic interaction between the MC molecules.

involving animals and their care were conducted in accordance with institutional guidelines that comply with international laws and policies (Jain, Sandhu, Malvi, & Gupta, 2013). To induce burns with skin damage, a slightly modified soldering iron with a flat contact area of 28.3 mm2 (AD = 6 mm) was made. Before creation of the burn wound, rats were anaesthetized using Zoletil and Rompun, (0.15 ml/kg + 0.15 ml/kg) according to the body weight. Hair on the dorsal side of the rats was removed, and a burn wound was inflicted by placing the circular iron disc (heated to 95 °C) over the dorsal side for 20 s, except for the untreated groups (n = 6 rats/group). Second-degree burns without cellular and tissue structure in the dermis were observed by sections stained with hematoxylin and eosin (H&E). Animals were divided after 3 days of acclimation in a cage and were then equally assigned (n = 24) to one of the following groups: Group 1, untreated control; Group 2, induced control; Group 3, treated with MC-0.0; Group 4, treated with MC-1.0. The sample tissue was enucleated after treatment 1, 3, 7, 14 and 21 days before sacrifice. The skin tissue for a histopathological analysis was fixed in 10% buffered formalin, subsequently dehydrated, and embedded in paraffin. The tissue paraffin was cut into 5 μm sections. The fixed sections were then stained with Hematoxylin and Eosin (H&E) or Masson’s trichrome.

3.3. In situ formation of silver oxide NPs in the MC hydrogel The MC hydrogel containing silver oxide NPs was examined via SEM (Fig. 3). There were no NPs in the pure MC-0.0 gel (Fig. 3A, D), and silver oxide NPs were observed in the MC hydrogel with CH3COOAg (Fig. 3E, F). The MC hydrogel containing silver oxide had uniform pore size and an interconnected porous network structure as shown in Fig. 3B, C. The Ag atom was evenly distributed in the hydrogel sample, as shown in the mapping data (Fig. 3H, I). The intensity of Ag element in MC only is due to the peak resolution of EDX equipment and overlapping x-ray between adjacent atoms (Fig. 3G). This indicates that the silver oxide NPs were synthesized through an in situ reaction and were evenly distributed in the MC hydrogel. Fig. 4A shows the XRD patterns of the silver oxide NPs synthesized in the MC solution. The synthesized NPs showed a strong two intense peaks at 27.9 and 32.3, which corresponds to (110) and (111) peak corresponding to a main peak of silver oxide NPs (Clark, Gebbart, Gonder, Keeling, & Kohn, 1985; Dhoondia & Chakraborty, 2012). Also, the (211) peak corresponded to crystalline peaks of the silver oxide NPs whereas no crystalline peak was observed in the pure MC hydrogel. Fig. 4B shows the XPS spectra of the MC sample containing silver oxide NPs. The characteristic peaks, such as C(1s), O(1s), and Ag(3d) peaks were observed in the MC samples containing silver oxide NPs, while the pure MC sample showed C(1s) and O(1s) peaks. The expanded Ag(3d) peaks for the silver oxide NPs was deconvoluted in Fig. 4C and D. The Ag(3d5/2) and Ag(3d3/2) peaks were observed at binding energies of 368.1 eV and 374.1 eV, respectively. Moreover, the Ag peak slitting of the 3d doublet is 6 eV, which indicating the formation of metallic silver (Breitwieser et al., 2013). To further understand the chemical state of the silver oxide NPs, a detailed deconvolution of the Ag(3d) peak was also performed. The deconvolution analysis revealed the chemical states of Ag0, Ag+ and Ag2+. The binding energy of Ag(3d5/2) for Ag0, Ag2O and AgO is 368.4, 368.1, 367.6 eV, respectively (Jiao et al., 2015). Based on the deconvolution analysisit was found that 41.4% of the silvers were in the Ag0 state, while about 52.5% and 6.4% of them were in Ag+ and Ag2+ chemical state, respectively in the case of 0.5 wt% CH3COOAg (Fig. 4C). While, With the addition of 1.0 wt% CH3COOAg, Ag0 (23.4%) and Ag+ (76.6%) states were exist, however, chemical state of Ag2+ was not found (Fig. 4D). This indicates that the Ag atom in the MC hydrogel exists in various chemical states by a relative amount of Ag and OH−. From the XRD and XPS results, silver oxide NPs were synthesized by a reaction between Ag+ and OH− in the MC solution at pH 7.4. When a

3. Results and discussion 3.1. Reduction of silver acetate precursor in the MC solution In our previous study, gelation behavior of MC solution was primarily associated with anion. Furthermore, we identified salting-out effect of common anions follows the order of Hofmeister series (Park, Kim, Yoon, & Park, 2017). Silver acetate (CH3COOAg) is dissociated into silver (Ag+) and acetate (CH3COO−) ions in aqueous solution. The silver oxide NPs were generated in situ in the MC solution through the reduction and stabilization of MC molecules. Fig. 1A shows the change in the absorbance of silver oxide NPs according to the time for 48 h. The maximum absorbance at 417 nm originated from the formation of silver oxide NPs, and the peak broadening indicates that the NPs were polydisperse. Also, the absorbance intensity of the MC solution increased with time, suggesting that the amount of silver oxide NPs that formed from CH3COOAg precursor increased. Fig. 1B shows the change in the maximum absorbance (417 nm) with time. The absorbance increased sigmoidally with time up to 24 h and reached a plateau after that. From this, the optimum time to synthesize the Ag NPs was found to be 24 h. 3.2. Gelation behavior of MC solution containing silver oxide NPs In this study, CH3COOAg was selected as a precursor salt for silver oxide NPs. Since CH3COO− ion is located on the left side of the Hofmeister series, it was expected to have a good salt-out effect. Fig. 2 shows the gelation temperature and time of the MC solution containing CH3COOAg. The gelation temperature was lowered from 37.0 °C to 33.6 °C as the concentration of CH3COOAg increased from 0 to 1.0 wt% (Fig. 2A). The gelation time also decreased from 120 s to 40 s as the 5

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Fig. 6. (A) Photographic feature of skin of burned rat back, and (B) Evaluation of the histopathology regarding the healing effect of ointments on burn-induced skin damage using Hematoxylin and Eosin (H&E) staining (x40).

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Fig. 6. (continued)

Fig. 7. Evaluation of collagen percentage in the cure effect of MC hydrogel on burn induced skin damage(x100). A. B: The collagen deposition measured in the skin stained with Masson’s trichrome staining at the 7 days.

single Ag+ ion was reacted with OH−, AgO formed. Similarly, Ag2O formed when two Ag+ ions and OH− reacted.

ions was fast. The MC hydrogel containing silver oxide NPs exhibited a burst release behavior, indicating that almost all Ag ions were released in 1 h.

3.4. In vitro release profiles of the silver oxide NPs from the MC hydrogel 3.5. Antibacterial activity of the MC hydrogel containing silver oxide NPs To investigate the release of the Ag ions from the MC hydrogel, CH3COOAg of 0.5 or 1.0 wt% was dissolved in the MC solutions prior to gelation. A release experiment was carried out to observe the release behavior of the Ag ions at a body temperature (37 °C). The cumulative release profile of the Ag ions from the MC hydrogel showed an inverted L-shaped curve (Fig. 5). At MC-1.0, a higher cumulative release (%) of Ag ions was observed in the profiles, and the release of Ag ions was almost completed within 6 h (∼95%). At MC-0.5, approximately, 60% of the Ag was released within 1 h, thereafter, the release increased slightly. These results were due to the osmotic pressure difference between released medium and MC hydrogel. The osmotic pressure of MC1.0 was higher than MC-0.5, accordingly, cumulative release rate of Ag

An antibacterial activity test of the MC hydrogel with silver oxide NPs was carried out at 37 °C for 1 h. The 2 × 105 of initial bacteria were seeded in the test sample. For comparison, the control group and pure MC hydrogel were used. Regardless of the type of bacteria, the MC hydrogel with silver oxide NPs showed an excellent antibacterial activity of 99.9% (Table 1). The Ag compounds are known to have antibacterial activity against a wide range of microorganisms. The bacterial membrane contains sulfur-containing proteins, and the Ag+ from silver oxide NPs interacts with these proteins in the cell as well as with phosphorus containing compounds such as DNA. Since the Ag+ anchors in and penetrates the cell wall of bacteria, it is reasonable to suggest 7

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that the resulting structural change in the cell membrane could cause an increase in the cell permeability, leading to uncontrolled transport through the cytoplasmic membrane, and ultimately cell death (Sondi & Sondi, 2004). For this reason, the MC hydrogel containing silver oxide NPs can be assumed to have an excellent antibacterial activity.

Acknowledgements

3.6. Burn wound healing properties

References

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015R1A2A2A01007954).

The change in the histopathological appearance from burning skin epithelium was significant compared to that of the untreated group. The change from burning epithelium treated with MC-1.0 hydrogel showed a significant restoration effect of skin against burn-induced skin damage (Fig. 6A). The healing pattern of the burn wounds was studied to examine the histology of the untreated, induced, MC-0.0, and MC-1.0 groups on days 1, 3, 7, 14 and 21 using H&E staining (Fig. 6B). The wide necrosis, inflammation induced by burn damage in skin was prevented in the MC-1.0 group. The induced group was shown to have extensive necrosis in the epithelium, and the dermis showed bulla, necrosis and infiltrated inflammation. The MC-0.0 group showed similar characteristics as the induced group. However, on 14 days, the induced, MC-0.0 groups showed little infiltrated inflammation, not bulla. MC-1.0 group showed a little infiltrated inflammation over the 7 days, with a lesion showing infiltrated inflammation in the dermis and the cure process. Otherwise, the untreated group can be shown to be correctly aligned regarding the collagen fiber and fibroblast. The change in the histopathological appearance from burning skin epithelium was significant compare to untreated group. The histopathological appearance showed a change in the burning epithelium treated with the MC-1.0 group, indicating a significant restoration of the skin against the burn-induced skin damage. Masson’s trichrome staining was performed to observe the collagenous components. Heat-denatured collagen in burned skin stains red instead of blue in Masson's trichrome stain (Dhoondia & Chakraborty, 2012). Necrosis was filled in regenerated tissue in the induced and MC-0.0 groups, whereas the MC-1.0 group showed regeneration of the collagen for burn wound healing (Fig. 7A). Furthermore, a significant collagen fiber was found to be restored in the MC1.0 group (Fig. 7B). The change in the histopathological appearance from the burning skin epithelium was significant compared to that of the untreated group, and the change in the burning epithelium treated with MC-1.0 showed a significant restoration of the skin against burninduced skin damage, particularly in the histopathological appearance of the burn-induced damage post-treated with silver.

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4. Conclusions This study implemented the Hofmeister series to select a silver acetate precursor salt with a good salt-out effect. An injectable hydrogel from the thermo-reversible MC solution was prepared using a one-pot reaction for burn wound regeneration. First, the gelation behavior of the pure MC was promoted by a stronger hydrophobic interaction with an increase in the concentration of silver acetate precursor. During hydrogelation, the silver oxide NPs were in situ synthesized and evenly distributed in the gel network. Also, the formation of silver oxide NPs in the hydrogel was confirmed via XRD and XPS spectra. Therefore, the thermo-responsive MC hydrogels containing silver oxide NPs were successfully prepared using a one-pot reaction. The resulting MC hydrogel with silver oxide NPs exhibited an excellent antimicrobial activity and burn wound healing effect. Therefore, this thermo-responsive MC hydrogel has great potential as an injectable hydrogel for wound regeneration.

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