In situ reduction of silver nanoparticles by sodium alginate to obtain silver-loaded composite wound dressing with enhanced mechanical and antimicrobial property

Journal Pre-proof In situ reduction of silver nanoparticles by sodium alginate to obtain silver-loaded composite wound dressing with enhanced mechanical and antimicrobial property

Kai Chen, Fengyan Wang, Siyu Liu, Xiaofang Wu, Linmin Xu, Dekun Zhang PII:

S0141-8130(19)39374-2

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.156

Reference:

BIOMAC 14460

To appear in:

International Journal of Biological Macromolecules

Received date:

18 November 2019

Revised date:

9 January 2020

Accepted date:

16 January 2020

Please cite this article as: K. Chen, F. Wang, S. Liu, et al., In situ reduction of silver nanoparticles by sodium alginate to obtain silver-loaded composite wound dressing with enhanced mechanical and antimicrobial property, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.01.156

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© 2020 Published by Elsevier.

Journal Pre-proof

In situ reduction of silver nanoparticles by sodium alginate to obtain silver-loaded composite wound dressing with enhanced mechanical and antimicrobial property Kai Chen1, Fengyan Wang1, Siyu Liu1, Xiaofang Wu2, Linmin Xu1, Dekun Zhang1 (1. School of Materials and Physics, China University of Mining and Technology, Xuzhou 221116, China; 2. School of Mechatronic Engineering, China University of Mining and Technology, Xuzhou 221116, China)

Abstract: Wound dressings provide barrier protection during wound treatment while providing an

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environment suitable for wound healing. However, traditional wound dressings have disadvantages, such as easily adhering to wounds, poor barrier effects, and poor haemostasis.

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Therefore, it is of great significance to design a new wound dressing that does not cause further

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injury, has good antibacterial effect and promotes wound healing in view of the disadvantages of traditional wound dressings. In this paper, silver nanoparticles (AgNPs) were reduced by in situ

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reduction with sodium alginate (SA), and construct a silver-loaded PVA/SA/CMCS hydrogel antibacterial wound dressing. The properties of antibacterial hydrogel were evaluated. The results

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show that SA can successfully reduce AgNPs, and the particle size is small and uniform, which meets the requirements of antibacterial material. The AgNPs are evenly distributed inside the

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hydrogel and have stable performance. The silver-loaded hydrogel was formed uniform pores inside the material, and had excellent water absorption and water retention, which can absorb a

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large amount of wound exudate while maintaining a moist wound environment. The antibacterial hydrogel exhibited good mechanical properties, antibacterial activity and biocompatibility. In summary, the silver-loaded hydrogel is an ideal wound dressing. Keywords: Wound dressings; PVA/SA/CMCS hydrogel; Sodium alginate; Silver nanoparticle; Antibacterial

1. Introduction As the largest organ of the body, skin plays an important role in physical, chemical and biological barrier protection[1]. It can protect the internal environment of the body, resist the invasion of external bacteria and avoid excessive fluid loss. However, as the first line of defence of the body, the skin is in direct contact with the outside environment. Therefore, it is often damaged. When the skin is injured and cannot heal in time, the injury will cause many local or even systemic problems, such as bacterial infection, increased metabolism, excessive loss of water

*Corresponding author: Kai Chen and Linmin Xu E-mail address: [email protected]; [email protected]

Journal Pre-proof and protein, and immune system disorders, which may be life-threatening. Therefore, after the skin is damaged, it is necessary to restore the integrity of the skin and re-establish homeostasis in time[2]. Wound dressings can provide temporary protection to replace damaged skin during wound healing and treatment. It can avoid or control wound infections and provide a suitable healing environment. The most common wound dressings are gauze and cotton balls, but these traditional wound dressings have inherent defects[3], such as easy adhesion to wounds and poor barrier function. Through a large number of experimental studies, many scholars found that moderately

of wet wound healing[4-5] was proposed and widely accepted.

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humid environments were more suitable for cell proliferation than dry environments. The theory

The hydrogel is a polymer material with a network structure that has a hydrophilic group

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inside. Therefore, it can absorb a large amount of water and also firmly combine with water.

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Hydrogel medical dressing[6] is a new type of wound dressing. Compared with traditional dressings, it can promote wound healing, relieve pain, inhibit bacterial growth, and improve the

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micro-environment. Liang[7] et al prepared a compound hydrogel for burn wounds using water-soluble sodium carboxymethyl cellulose, sodium alginate and chitosan. The hydrogel has

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good water vapor permeability, self-adjustment and anti-adhesion properties, which can promote the healing of deep secondary burn wounds. Honglei[8-9] designed and prepared an injectable

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self-healing hydrogel of chitosan and konjac glucomannan by a Schiff base reaction. The self-healing property can prolong the service life and better protect the wound surface. In addition,

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it also has adhesion, antibacterial properties and biocompatibility. Qian[10] et al studied the effects of different divalent ions (copper, zinc, bismuth, and calcium) cross-linked with sodium alginate/polyacrylamide hydrogels on various properties. Park[11] et al used a freeze-thaw method to synthesize PVA/PVP copolymer hydrogels for dressing. In addition, aloe vera can accelerate wound healing. Zhang[12] designed and prepared a three-dimensional layered nanofiber sponge, which increased the interaction with blood cells and promoted haemostasis. At the same time, it had the ability to promote wound healing. To avoid wound infection, wound dressings usually need to be antibacterial[13]. Generally, antibacterial hydrogels are divided into self-antibacterial and loaded antibacterial agents. Wang[14] designed a composite material composed of natural bacterial cellulose (BG), polyethylene glycol (PEG) and polyhexamethylene biguanide (PHMB), which had good physicochemical properties, biocompatibility, and continuously strong antibacterial effects. Chitosan (CS) is considered to be one of the most promising wound dressing materials due to its excellent antibacterial, adhesive, oxygen permeability and connective regeneration properties[15]. However, chitosan has poor water

Journal Pre-proof solubility and weak antibacterial properties. The antibacterial effect of self-antibacterial hydrogels is very limited. Therefore, antibacterial agents are usually added to wound dressings. Antibiotics are the most common antibacterial agents. Antibiotics can effectively inhibit the growth and reproduction of bacteria and even kill bacteria. In addition, it has no toxic side effects on the human body. However, in recent years, the abuse of antibiotics has led to the emergence of drug-resistant microorganisms, which has reduced the efficacy of anti-infection[15]. Silver nanoparticles (AgNPs) are the most promising inorganic antibacterial agent due to their excellent antibacterial properties and no cytotoxicity[16-21]. At present, there are two main methods for preparing silver-loaded hydrogels: direct addition and in

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situ synthesis. The direct addition method is simple to operate, and the AgNPs concentration is easy to control, but the AgNPs are easily agglomerated and affect the antibacterial effect. The

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particle size distribution and crystal shape of AgNPs can be controlled by in situ synthesis. In

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addition, AgNPs are more stable in hydrogels. Song[22] et al used sodium borohydride as a reducing agent in situ to reduce AgNPs in sodium alginate solution and prepared sodium alginate

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composite sponges that contained AgNPs. The composite sponge has synergistic antibacterial and anti-inflammatory effects. However, the reducing agent used in the preparation of AgNPs by the

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traditional method has some toxicity and safety hazards. Therefore, it is necessary to find a green and safe method to prepare AgNPs. Juby[23] et al prepared a polyethylene glycol/gum (PVA/GA)

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hydrogel, which was cross-linked and synthesized with AgNPs by gamma-ray induction. Anisha[24] et al used glucose to synthesize AgNPs and prepared chitosan/hyaluronic acid/nano-silver

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antibacterial sponges. This sponge has the potential to become a DFU wound dressing for drug-resistant bacteria. Varaprasad[25] et al prepared polyacrylamide/polyvinyl alcohol/silver nanoparticle composite hydrogels. The polyvinyl alcohol is a stabilizer for stabilizing AgNPs, and the diameter of silver particles can be controlled at 2-3 nm. Gan[26] et al prepared a plant-based hydrogel with long-term and repeatable adhesion by using silver lignin nanoparticles as initiators. It also has good cell affinity and antibacterial activity. Although there are many studies by scholars on hydrogel wound dressings, there are still many problems. For example, chitosan can only dissolve under acidic conditions and exert antibacterial effects. The single chitosan material cannot meet the clinical antibacterial requirements. The potential cytotoxicity of the AgNPs and the potential safety of the preparation method restrict its clinical application. Wound dressings generally have problems such as poor mechanical properties and unsafe preparation methods. In this paper, carboxymethyl chitosan (CMCS), sodium alginate (SA) and polyvinyl alcohol (PVA) were selected as raw materials to construct antibacterial hydrogel dressings. The AgNPs

Journal Pre-proof were reduced in situ by SA, and a silver-loaded PVA/SA/CS hydrogel dressing was prepared by the freeze-thaw method and calcium ion cross-linking method. The AgNPs can be uniformly distributed in the hydrogel and are synergistically antibacterial with CMCS. The microstructure, internal morphology, mechanical properties and biological properties of the hydrogel were characterized.

2. Experimental section 2.1. Materials Polyvinyl alcohol (PVA, degree of polymerization 1750±50) and silver nitrate (AgNO3) were

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purchased from sinopharm chemical reagent Co.,Ltd. Sodium alginate (SA, viscosity 200±20mpa. S) was purchased from Shanghai Aladdin biochemical technology Co., Ltd. Carboxymethyl

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chitosan (CMCS, ≥80%) and anhydrous calcium chloride (CaCl2) were purchased from Shanghai

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McLean biochemical technology Co., Ltd. Deionized water is produced in the laboratory.

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2.2. Preparation of the antibacterial dressing Preparation of SA-Ag solution: AgNPs were prepared by using SA as reductant and stabilizer,

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AgNO3 as silver source. The SA was added to deionized water. The mixture was stirred at 50℃ until reaching homogeneous SA solution (0.2%). AgNO3 was dissolved in deionized water to

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obtain a 1% AgNO3 solution. The SA solution and the AgNO3 solution were mixed at a mass ratio of 0:100, 1:100, 3:100, and 5:100. The mixture was heated in water bath at 90℃, stirred violently

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and protected from light for 10 hours.

Preparation of silver-loaded PVA/SA/CMCS hydrogel dressing: The PVA was added to deionized water and stirred at 95℃ to obtain PVA solution. Then, the CMCS and the SA were added to PVA solution. In 50℃ water bath, the mixture was uniformly stirred to obtain a PVA/SA/CMCS solution. SA-Ag solution and PVA/SA/CMCS solution were mixed at a volume ratio of 1:1. In the finally obtained mixed solution, the PVA concentration was 5%, the CMCS was 1%, and the SA was 0.8%. The mixed solution was ultrasonicated for 30 min to remove air bubbles, and then poured into the mold. The samples were frozen for 6 hours at -20℃ and thawed for 2 hours at room temperature, with 4 cycles. After the last thawing, the samples were immersed in a 2% CaCl2 solution for crosslinking. After 6 hours, the samples were taken out and soak in deionized water (change the water every 6 hours and 6 times). Silver-loaded PVA/SA/CMCS hydrogels

were

obtained,

which

were

respectively

called

PVA/SA/CMCS/Ag1, PVA/SA/CMCS/Ag3 and PVA/SA/CMCS/Ag5.

PVA/SA/CMCS/Ag0,

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2.3. Characterization 2.3.1. Microstructure The formation of the AgNPs was qualitatively analyzed by measuring the spectra of the SA-Ag solution at 300-500 using an ultraviolet-visible spectrophotometer (UV-VIS, Evolution 300, Thermo Scientific, USA). Multiangle particle size and high sensitivity Zeta potential analyzer (NanoBbrook, Brookhaven, USA) was used to measure the particle size distribution of AgNPs reduced by SA. Fourier transform infrared spectroscopy (FT-IR, VTRTEX 80v, Bruker, Germany) was used to evaluate the chemical bonds and functional groups of SA-Ag and hydrogels. The

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composition and crystallization of the SA-Ag and hydrogels were characterized by X-ray diffraction analysis (XRD, D8 ADVANCE, Bruker, Germany). The samples were dried before

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testing.

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2.3.2. Micromorphological

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The pore structure of the hydrogel was observed using a scanning electron microscope (SEM, Quanta 250, FEI, USA). The hydrogel was treated with liquid nitrogen to obtain a cross section,

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and then freeze-dried. Prior to observation, the samples were sprayed with gold. Contact Angle measurement instrument (JC2000D2A, zhongchen digital equipment Co., Ltd., China) was used to

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measure the wetting contact Angle of different hydrogels.

2.3.3. Mechanical properties

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The tensile test was carried out by microcomputer controlled electronic universal testing machine (WDW-2, China ). The diameter and length of the tensile sample were 3mm and 20mm respectively. Before the test, the tensile sample was fully immersed in deionized water. The tensile speed was 10 mm/min until the sample broke. The dynamic mechanical properties were characterized by analyzing the storage modulus, loss modulus of the sample using an Aaton Paar Physica rotary rheometer (MCR301/302, Aaton Paar, Austria). The diameter and thickness of the sample were 25mm and 2mm.

2.3.4. Water absorption and water retention The water absorption and water retention of the material were characterized by gravimetric method. The sample was freeze-dried and the quality was recorded as W0. The sample was placed in deionized water, removed at various time points, and the excess moisture on the surface was erased. The quality was recorded as Wi. Calculate the water absorption rate and plot the swelling

Journal Pre-proof curve. The water absorption rate was calculated as follows:

Water absorption (% )  (

Wi - W0 )  100 % W0

The freeze-dried sample (mass denoted as M0) was immersed in deionized water and full swelling, mass denoted as Me. The fully inflated samples were put into a 37℃ constant temperature drying box, removed at various time points. The quality was recorded as Wi. The

water retention was calculated as follows:

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Mi  M0 )  100 % Me  M0

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Water retention( %)  (

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2.3.5. Antibacterial evaluation

The antibacterial properties were characterized by inhibition zone method and liquid medium

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inhibition test. Escherichia coli and Staphylococcus aureus spore suspension were applied to the autoclaved agar medium. Then, the sample was placed on an agar medium and incubated at 37℃.

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The diameter of the inhibition zone was measured to compare the antibacterial effects of different hydrogels.

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S. aureus and E. coli were inoculated into the LB liquid medium under 220 rpm and 37℃, until the OD value at around 600 nm was about 0.6. The bacterial solution was diluted at a volume

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ratio of 1:1000. The sterilized sample was added to 3 mL of the bacterial solution, and incubated at 220 rpm, 37℃ for 24 hours. The OD value at 600 nm was measured.

2.3.6. Analysis of cytotoxicity in vitro NIH-3T3 cells were used to evaluate the cytotoxicity of the sample. Before the experiment, the samples were subjected to UV-disinfection for 1h. They were placed in 96-well plate. Subsequently, NIH-3T3 cells (1×104 cells/well) at logarithmic phase were collected and inoculated into a 96-well plate cultured 24h. 10 μL of CCK-8 solution was added to each well, followed by incubation for 1h under same conditions. The absorbance at 450nm was measured. The relative survival rate is calculated as:

Relative Viability( %) 

ODexperimental groups ODcontrastive groups

 100 %

Journal Pre-proof In order to observe cell growth directly, live and dead cells were stained using the Live/dead staining kit (BB-4126; Bebo Bio; China) and observed using a Laser confocal fluorescence microscope (TCS SP8; Leica; Germany). The living cells are stained green and the dead cells are red.

3. Results and discussion 3.1. Characterization of silver nanoparticles AgNPs were reduced using SA as a reducing agent and stabilizer. The SA solution and AgNO3 solution were both transparent solutions. After the two were mixed, the solution turned

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bright yellow, and the colour of the solution became darker as the concentration of AgNO3 increased. This indicated that sodium alginate successfully reduced the AgNPs (Fig. 1C).

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Analyzing the X-ray diffraction patterns, compared with that of the SA, the SA-Ag showed diffraction peaks at 38.21°, 44.28°, 64.41°, and 77.72°, which coincided with the characteristic

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peaks of AgNPs, indicating that SA reduced the AgNPs (Fig. 1A). Moreover, the peaks were sharp, indicating that the prepared AgNPs have good crystallinity.

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The formation of AgNPs can be characterized by UV-visible spectroscopy, which exhibits an

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absorption peak at approximately 415 nm. The SA-Ag solution peaked at approximately 415 nm, further demonstrating that the AgNPs were reduced (Fig. 1C). In addition, as the concentration of AgNO3 increases, the peak value of the absorption peak increases; the position of the peak also

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shows a slight redshift, and the half-height width becomes wider. This indicates that the concentration of the AgNPs increases, the particle size increases, and the particle size range

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becomes wider. This is because SA not only acts as a reducing agent to reduce AgNPs but also as a stabilizer to avoid agglomeration. With increasing concentrations of AgNO3, more AgNPs were prepared, and the stabilizing effect was limited. Therefore, the AgNPs were prone to agglomeration. The size of the AgNPs is concentrated at approximately 12 nm, and the particle size range is concentrated and evenly distributed (Fig. 1D). The particle size formed meets the requirements for effective antibacterial materials. Comparing the infrared spectra of SA and SA-Ag solutions, it can be seen that the position and value of peaks changed significantly. The absorption peak corresponding to the stretching vibration of -OH was moved from 3579 cm-1 to 3742 cm-1, and the absorption band became narrower and weaker. For SA, asymmetric and symmetrical vibration absorption of carboxyl groups showed a wide peak at 1607 cm-1 and a narrow peak at 1417 cm-1. After reduction of AgNPs by sodium alginate, the peak moved to 1595 cm-1 and 1403 cm-1, and the peak strength became weaker. The above shift indicates that the hydroxyl and carboxyl groups in SA participated in the reduction process of AgNPs. A new characteristic peak appears at 1730 cm-1,

Journal Pre-proof which belongs to the vibrational absorption peak of the carbonyl (C=O) group. This indicates that

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the reducing functional group (O-H) on SA was oxidized to a carbonyl group (Fig. 1B).

Fig. 1. Characterization of AgNPs. (A) X-Ray diffraction patterns for SA and SA-Ag. (B) Infrared spectra for SA and SA-Ag. (C) UV-Vis Spectra of SA-Ag. (D) Particle size distribution of AgNPs. (E) Reduction mechanism of AgNPs.

The mechanism of reducing AgNPs by SA is as follows: SA plays a dual role as both a stabilizer and reducing agent. The adjacent hydroxyl group and carboxyl group of SA chelate Ag+. The chelate produces Ag0 on heating. The hydroxyl group is oxidized to form a carbonyl group.

Journal Pre-proof SA is easily soluble in water, and the free carbonyl group in water can effectively control the nucleation and growth of AgNPs (Fig. 1E).

3.2. Characterization of silver-loaded PVA/SA/CMCS hydrogel 3.2.1. Characterization of basic properties The internal network structure of the hydrogel affects the water absorbing and moisturizing properties. The SEM images of different hydrogels show that the addition of AgNPs affects the internal structure of the hydrogels. When the content of AgNPs is 0.01%, the hydrogel is relatively dense, and the pore size decreases. When 0.03% AgNPs are added, the pore size significantly increases, and the morphology is similar to that of a silver-free hydrogel. The pore distribution

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was uniform and interconnected. As the AgNPs concentration continues to increase, the size and quantity of pores decrease (Fig. 2A). This is because when a small amount of AgNO3 solution was

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added , part of the sodium alginate, which is not involved in the reduction, participates in the

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cross-linking of the hydrogel. Therefore, the cross-linking points increased. As the concentration of AgNPs increases, the AgNPs are uniformly distributed in the hydrogel, which is beneficial to

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the formation of the mesh structure. When the concentration of the AgNPs is too high, a large number of silver particles tend to agglomerate. Therefore, inhibiting the formation of a hydrogel

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network structure.

The surface contact angle reflects the water absorption capacity of the material. The contact

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angles of all the samples were approximately 20°. This indicates that they are all hydrophilic materials (Fig. 2B). Moreover, the contact angle of hydrogels was not significantly different with

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different AgNPs concentrations.

To promote wound healing and avoid wound infection, wound dressings should have the characteristics of absorbing wound exudate and providing a moist environment. From the swelling curve, it can be seen that hydrogels with different AgNPs concentrations have good water absorption capacity, and the swelling balance is reached at approximately 250 min (Fig. 2D). The water absorption rates of hydrogels containing 0%, 0.01%, 0.03%, and 0.05% AgNPs were 655%, 393%, 966%, and 526%, respectively. In general, an increased number of pores and increased uniformity in the hydrogel results in better water absorption performance. According to the moisture retention rate curve, the addition of AgNPs slightly improves the moisture retention of hydrogels. The hydrogels with the four different AgNPs concentrations all reached equilibrium at approximately 300 min (Fig. 2E). In general, the internal structure of the hydrogel is relatively compact, with better moisture retention performance. In the X-ray diffraction pattern of the silver-loaded hydrogel, diffraction peaks appeared at 38.1°, 44.3°, 64.4°, and 77.4° (Fig. 2C) compared with the silver-free hydrogel. These results

Journal Pre-proof indicate that the AgNPs are uniformly distributed in the hydrogel and have good crystallinity. The characteristic diffraction peaks of PVA appear near 19.64° and 41.38°. The addition of AgNPs promotes the crystallization of PVA, so it has better mechanical properties. The formation of the hydrogel network is the result of the physical blending and cross-linking of the components. Compared with PVA, CS and SA, the absorption peak at approximately 3400 cm-1 widens and moves to a lower wave number in the infrared spectrum of silver-loaded hydrogel. The results indicate that PVA, SA and CS interact with each other to form hydrogen bonds. Compared with the silver-free hydrogel, the silver-loaded hydrogel showed a new absorption peak at 1653 cm-1, which is a characteristic absorption peak of C=N in the Schiff base structure. This

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shows that oxidized sodium alginate is formed during the process of reducing AgNPs in the Schiff base reaction with carboxymethyl chitosan (Fig. 2F). Therefore, the main mechanism of hydrogel

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cross-linking is the egg-box structure by SA and Ca2+, the electrostatic interaction between each

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molecular chain, the crystallisation of PVA, and a small amount of Schiff base structure (Fig. 2G).

Journal Pre-proof Fig. 2. (A) Micrographs of (a) PVA/SA/CS, (b) PVA/SA/CS/0.01%Ag, (c) PVA/SA/CS/0.05%Ag, (d)PVA/SA/CS/0.01%Ag. (B) Contact angles of (a) PVA/SA/CS, (b) PVA/SA/CS/0.01%Ag, (c) PVA/SA/CS/0.05%Ag, (d)PVA/SA/CS/0.01%Ag. (C) X-Ray diffraction patterns for PVA/SA/CS and PVA/SA/CS/Ag. (D) Water absorption of different hydrogels. (E) Water retention of different hydrogels. (F) Infrared spectra for PVA, SA, CS, PVA/SA/CS and PVA/SA/CS/Ag. (G) Mechanism of hydrogel crosslinking.

3.2.2. Characterization of mechanical strength It is required to have excellent elasticity as a wound dressing hydrogel. The hydrogel can be bent into any shape (Fig. 3A). It is soft and conforms to the skin (Fig. S1). It has good mechanical properties and can withstand at least 100 g of force (Fig. 3B). As the content of AgNPs increases,

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the tensile strength first increases and then decreases (Fig. 3C). This may be attributed to the fact that the synthesized AgNPs are uniformly dispersed in the hydrogel and play a strengthening role.

the formation of the grid structure of the hydrogel.

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However, with an increase in the concentration of AgNPs, the easy aggregation of AgNPs affects

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When applied to the wound surface, hydrogel dressing not only needs to bear the force of surrounding skin and tissues but also needs to cope with the sudden changes in the external

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environment. Therefore, it is necessary to study the dynamic rheological properties of dressings.

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Studies on the effect of strain changes on the rheological properties of hydrogels show that the linear viscoelastic zones of different hydrogels are 0.01-1%. After the strain is greater than 1%, the storage modulus (G’) decreases, the loss modulus (G’’) increases, and the G’’ is gradually greater

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than the G’ (Fig. 3D-a). It is indicated that with increasing strain, the internal network structure of the hydrogel is destroyed, and inelastic deformation begins to appear. The energy storage

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performance of the hydrogel increases as the AgNPs concentration increases. This is because the AgNPs uniformly dispersed in hydrogel to enhance the effect. The AgNPs increase the density of cross-linking, which makes the hydrogel network structure more complete and enhances the anti-deformation ability. The addition of AgNPs has little effect on the G’’ of hydrogel. As the concentration of AgNPs increases, the loss factor first decreases and then increases. However, the overall is much lower than that of the silver-free hydrogel (Fig. 3D-a’). The results show that the silver-loaded hydrogels have good elasticity under large strain. Frequency variation has little effect on the rheological properties of hydrogels. The G’, G’’ and loss factor of hydrogels with four components increased slightly with frequency change. In addition, G’ is always much larger than G’’. The hydrogel mainly has elastic deformation. The G’ of silver-loaded hydrogels is higher than that of the silver-free hydrogel. The G’’ of the silver-loaded hydrogel is slightly lower than that of the silver-free hydrogel (Fig. 3D-b). Therefore, the loss factor of silver-loaded hydrogels is also low (Fig. 3D-b’). This indicates that the addition of AgNPs improves the elastic properties of hydrogels and can better cope with the complex

Journal Pre-proof external environment associated with frequency change. The temperature change has little effect on the G’ and G’’ of different hydrogels, and the G’ is much higher than the G’’. As the concentration of AgNPs increases, G’ first increases and then decreases, and G’’ first decreases and then increases (Fig. 3D-c). Therefore, the loss factor decreases first and then increases (Fig. 3D-c’). The above results indicate that the addition of an appropriate amount of AgNPs can better cope with the influence of temperature change and improve the thermal stability. In general, adding AgNPs can improve the rheological properties of hydrogels to different

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Fig. 3. (A) Macroscopic elastic characterization of hydrogels. (B) Macroscopic mechanical characterization of hydrogels. (C) Tensile strength of different hydrogels. (D) Rheological properties of different hydrogels.

3.2.3. Characterization of antibacterial properties Injured skin is vulnerable to bacterial infections, especially S. aureus, because of its lack of barrier protection. Antibacterial properties were evaluated by inhibition zone (Fig. 4A, Fig. 4B) and OD value (Fig. 4C). The results showed that the antibacterial effect of the silver-free hydrogel is weak, and the addition of AgNPs significantly improves the antibacterial ability. This indicates that the CMCS has limited antibacterial activity and cannot meet the antibacterial requirements. The inhibition zone appeared around the silver-loaded hydrogel with obvious boundaries. Moreover, the inhibition zone did not shrink for a long time and even expanded slightly, indicating that the antibacterial effect could be maintained for a long time (Fig. S2). In addition, the

Journal Pre-proof antibacterial activity of silver-loaded hydrogel against E. coli was greater than that against S. aureus. The excellent antibacterial properties of silver-loaded PVA/SA/CMCS hydrogels are achieved by the synergy between the CMCS and AgNPs. The protonated ammonium on the CMCS chain can interact with the negatively charged cell membrane of bacteria allowing entrance into the bacteria and then disturbing its metabolism. Therefore, the CMCS has an antibacterial effect[29]. The hypothesis of the antibacterial mechanism of AgNPs[30] mainly includes contact

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reactions and reactive oxygen-catalysed reactions (Fig. 4D).

Fig. 4. Antibacterial analysis of different hydrogels using (A) E. coli and (B) S. aureus. (C) Optical density at 600 nm measured 24 h after incubating with different hydrogels. (D) Antibacterial mechanism of AgNPs.

3.2.4. Characterization of cytotoxicity in vitro Cell activity in vitro is an important index to evaluate biocompatibility. The silver-loaded

Journal Pre-proof PVA/SA/CMCS hydrogels exhibited excellent biocompatibility (Fig. 5A). Compared with that of the silver-free hydrogel, the relative cell survival rate of silver-loaded hydrogel is slightly lower, but the cell survival rate is still very high. The results of fluorescence staining showed that the cells could still grow and proliferate normally after co-culture with the samples, and a large number of cells proliferated after 72 hours (Fig. 5B). CMCS and SA have excellent biocompatibility, and compounds with AgNPs can effectively control the release of nanoparticles and reduce the damage to cells. The mechanism of silver-loaded PVA/SA/CMCS hydrogel-induced wound healing[14] is mainly as follows: First, the hydrogel dressing covers the wound on the skin and acts as a

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temporary barrier to prevent water loss and bacterial infection. At the same time, the hydrogel has good antibacterial properties. It can play an effective bacteriostatic effect. Second, the hydrogel

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dressing exhibits excellent water absorption and moisturizing properties. Therefore, the hydrogel

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can absorb excess wound exudate while continuously providing a moist environment. In addition,

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CMCS and SA in hydrogels have haemostatic and healing-promoting effects (Fig. 5C).

Fig. 5. (A) Fluorescent staining of the bacteria on the surface of materials after culturing for 24h and 72h. (B) Relative Viability 24 h after incubating with different hydroge lof the cells after 24 h culture with different hydrogels. (C) The main mechanism of PVA/SA/CS/Ag promoting wound healing.

4. Conclusion SA is used as a reducing agent and a stabilizer to reduce AgNPs. The prepared AgNPs have small and uniform particle sizes, and the preparation process does not use reagents with potential

Journal Pre-proof toxicity. The preparation method is greener and safer. Hydrogel dressings were prepared with CMCS, PVA and SA as raw materials. CMCS and SA have excellent biocompatibility, and composites with PVA can improve the performance of hydrogels. The AgNPs are uniformly distributed in the hydrogel to make their performance more stable. The silver-loaded hydrogel dressings had better mechanical properties than the silver-free hydrogel. In addition, it had good water absorption properties and water retention, continuously providing a moist environment for wounds. More importantly, the antibacterial performance of the silver-loaded hydrogel is significantly improved, and the antibacterial effect can be sustained for a long time, which enables the wound to remain sterile and avoid bacterial infection. In vitro cell activity experiments

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indicated that silver-loaded PVA/SA/CMCS hydrogel dressings have good biocompatibility and promote cell proliferation. On the whole, it basically meets the requirements of an ideal wound

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dressing.

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Acknowledgements

This research is supported by National Natural Science Foundation of China (Grant No.

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51705517, 51875564) and China Postdoctoral Science Found (Grant No. 2018M630622).

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[1] Rahimi M, Ahmadi R, Kafil H S, et al. A novel bioactive quaternized chitosan and its silver-containing nanocomposites as a potent antimicrobial wound dressing: Structural and

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Author statement Kai Chen: Conceptualization, Methodology, Writing - Review & Editing, Funding acquisition. Fengyan Wang: Investigation, Formal analysis, Writing- Original draft preparation. Siyu Liu: Visualization, Software. Xiaofang Wu: Visualization, Data Curation. Linmin Xu: Project administration, Resources.

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Dekun Zhang: Supervision.

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Highlights

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A safer method for in-situ reduction of nanometer silver was adopted. The mechanism of reduction are analyzed. The proportion of PVA, CMCS, SA and AgNPs was introduced. The antibacterial and biocompatibility were excellent.

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