gentamicin nanocarrier for synergistic bacteria disinfection and wound healing application

Chemical Engineering Journal 380 (2020) 122582

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Bacteria-triggered hyaluronan/AgNPs/gentamicin nanocarrier for synergistic bacteria disinfection and wound healing application

T

Ningxiang Yua, Xiaoya Wanga, Liang Qiub, Taimei Caic, Chengjia Jianga, Yong Suna, Yanbin Lid, ⁎ ⁎ Hailong Penga,c, , Hua Xionga, a

State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China Centre for Translational Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330004, China c School of Resources, Environmental, and Chemical Engineering, Nanchang University, No. 999 Xuefu Avenue, Nanchang 330031, China d Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA b

H I GH L IG H T S

G R A P H I C A L A B S T R A C T

new Hyaluronan/AgNPs/ • AGentamicin (HB/Ag/g) nanocarrier has been prepared.

exhibited bacteria con• HB/Ag/g trollable release of silver and gentamicin.

showed synergistic anti• HB/Ag/g bacterial activities. was coated on chitin hy• HB/Ag/g drogel for disinfecting coatings construction (HB/Ag/g@CPH).

showed good bio• HB/Ag/g@CPH compatibility and intelligent application in wound healing.

A R T I C LE I N FO

A B S T R A C T

Keywords: Hyaluronic acid Bacteria-triggered nanocarrier Synergistic antibacterial Disinfecting coatings Wound healing

Here, we report on a bacteria-triggered hyaluronan/AgNPs/gentamicin nanocarrier (HB/Ag/g) that has highly efficient synergistic bacteria disinfection capability, and intelligent application in wound healing when used in combination with mussel-inspired chitin hydrogel. Briefly, HB/Ag/g was prepared using a simple self-assembly process and exhibited a controllable release of Ag and gentamicin that was triggered by either pH or hyaluronidase (HAase) which was released from bacteria. The Ag and gentamicin released from HB/Ag/g showed robust synergistic antibacterial activities because of their different antibacterial mechanisms. Furthermore, HB/ Ag/g was used as a coating block for the construction of disinfectant coatings on polydopamine modified chitin hydrogel (CPH) by a facile dip-drying procedure, and the product was named HB/Ag/g@CPH. HB/Ag/g@CPH exhibited strong inhibition of the growth and adhesion of bacteria, and did not affect cell attachment and proliferation. Most importantly, in vivo results indicate that HB/Ag/g@CPH can be conveniently used for wound disinfection and accelerated wound healing in a wound infected with S. aureus.

⁎ Corresponding authors at: State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China (H. Peng). E-mail addresses: [email protected] (H. Peng), [email protected] (H. Xiong).

https://doi.org/10.1016/j.cej.2019.122582 Received 26 April 2019; Received in revised form 20 August 2019; Accepted 21 August 2019 Available online 22 August 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. (a) Schematic illustration of preparing HB/Ag/g nanocarrier and designing synergetic antibacterial hydrogel for wound disinfection; (b) Overview of fabrication process for HB/Ag/g coatings on dopamine-grafted chitin hydrogel (CPH).

1. Introduction

investigated for the synergistic inhibitory impact on the growth of bacteria, and this has become a promising approach to decrease the dosage and toxic side effects of drugs [12–14]. Many researchers have studied the synergistic mechanism. Some studies found that the combination of AgNPs with antibiotics inhibited the formation of the biofilm that is primarily associated with multidrug-resistant and chronic bacterial infections [15]. Others pointed out that the formation of AgNPs-antibiotics complexes surrounding the AgNPs core enhanced the antibacterial ability [16]. Moreover, it was proposed that some antibiotics can enhance the binding of Ag or the release of Ag+, thus creating a temporary high concentration of Ag+ near the bacterial cell wall that inhibits the bacterial growth [17]. In previous studies, synergistic therapy was achieved through the direct mixture of AgNPs and antibiotics. However, these preparation methods have been deemed unfavorable because of aggregation and high biotoxicity and are unable to exert long-lasting antibacterial activity, which can sharply limit the use of this approach in the antibacterial treatment field [15,17]. To improve these drawbacks, many researchers have used natural polymers as nanocarriers to load AgNPs and antibiotics. Hyaluronic acid (HA) is a natural polysaccharide with a repeat unit composed of D-glucuronic acid and N-acetyl glucosamine linked with alternating β-(1–4) and β-(1–3) glycosidic bonds found in all vertebrates as a key component of the extracellular matrix [18]. HA has been increasingly studied for targeted therapies, tissue engineering, and drug delivery in the last twenty years because of its advantageous biological properties such as hydrophilicity, biodegradability, and virtually no biotoxicity [18–22]. Since HA has a high surface area with abundant

Bacterial resistance to antibiotics has been increasing over the years because of the overuse of antibiotics, and this increased resistance has become a major problem for contemporary medicine [1]. Therefore, exploration of new antimicrobial agents with broader efficiencies that do not engender bacterial resistance is urgently needed. Benefitting from the rapid development of nanotechnology, nanomaterials have opened up a new avenue for eliminating the growing number of drugresistant bacteria [2,3]. Ag nanoparticles (AgNPs) are considered to be excellent antibacterial agents, and have been successfully applied in clinical treatment because of their broader-spectrum antibacterial activity and limited bacterial resistance [4]. AgNPs can penetrate the bacterial membrane, and damage the bacterial proteins and DNA, leading to bacterial death [5]. Unfortunately, the use of traditional AgNPs as antibacterial agents has been limited by the disadvantages of their easy aggregation and uncontrolled release [6,7]. To solve these problems, natural polymers have been utilized to prepare AgNPs-based biomimetic nanomaterials to avoid aggregation and achieve controlled release ability [8–10]. However, the inherent biotoxicity toward mammal cells at high dosages of AgNPs has always been considered in the preparation of AgNPs-based biomimetic nanomaterials, which limited the application of it in biomedical field [11]. Thus, the development of an effective approach to reduce the dosage of AgNPs is crucial for the preparation of AgNPs-based nanomaterials. In the past decade, combinations of AgNPs with conventional antibiotics such as ampicillin, tetracycline, and gentamicin (Gen) were 2

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of Gen. Finally, HB/Ag/g and HB/Ag nanocarriers were dialyzed at 4 °C. The concentration of Ag element in HB/Ag/g and HB/Ag nanocarriers was verified using ICP-MS.

acetylamino groups and carboxyl groups that are available for chemical conjugation, many researchers have focused on the use of functionalized HA for the preparation of nanocarriers and successfully loading and delivering compounds such as curcumin [19], manassantin B [20], metal NPs [21], and tannic acid (TA) [22]. These HA-modified nanocarriers have shown enhanced antibacterial infection, wound restoring, and tumor ablation properties, compared to those of traditional agents. In particular, since several bacteria secrete hyaluronidase (HAase) that is capable of decomposing HA enzymolysis, many HAase-triggered nanocarriers have been successfully designed for the treatment of bacterial infections [23,24]. However, to the best of our knowledge, the synthesis of HA modified drug nanocarriers to load both AgNPs and Gen and the use of these as an antibacterial agents has not been reported. As shown in Fig. 1, a facile pH and HAase-sensitive self-assembly carbohydrate polymer (HB) was successfully synthesized from HA. HB can be used as a stabilizing agent to immobilize AgNPs via an in situ synthesis method because of its abundant acetylamino groups and carboxyl groups. The phenylboronic acid groups in the outer surface of HB can bind to the diols of the AgNPs reduced by TA and form cyclic boronate esters that can be reversibly cleaved under acidic conditions. Therefore, the AgNPs and Gen loaded HB nanocarrier (HB/Ag/g) with bacteria (pH and HAase) triggered release were constructed via the combination of intermolecular forces. It was verified that HB/Ag/g showed a strong synergistic antibacterial effect, that allowed a dramatical decrease in the dosage of drugs (AgNPs and Gen). In addition, HB/Ag/g can also serve as an important and versatile building block for coating biomaterial interfaces because of its biocompatibility and substrate recognition capability. To achieve this, polydopamine-grafted chitin hydrogel (CPH) was obtained from chitin hydrogel (CH) as a model substrate through a mussel-inspired approach. Owing to the abundance of amine groups of polydopamine, a novel HB/Ag/g coating was easily constructed on the CPH through a facile dip-drying process, and is named HB/Ag/g@CPH. HB/Ag/g@CPH exhibits strong antibacterial activity, a favorable anti-adhesion property for bacteria, and acceptable biocompatibility. The constructed HB/Ag/g@CPH was used for wound disinfection in vivo to facilitate biofilm formation and to destroy S. aureus.

2.3. Hyaluronidase (HAase) triggered activity To study the HAase triggered release of AgNPs from an HB/Ag nanocarrier, different concentrations of HAase were added to 5 mL of 30 mg/mL HB/Ag nanocarrier suspension (PBS, pH = 7.4), respectively. After incubation with vibration for 30 min, the obtained suspension was centrifuged at 10,000 rpm for 15 min, and the released AgNPs were obtained from the upper solution. The absorbance at 405 nm was measured to determine the amount of released AgNPs [25]. 2.4. Antibacterial activity of nanocarriers 2.4.1. Minimal inhibition concentration (MIC) assays The microtiter broth dilution method was performed by measuring MIC at OD600 nm. Briefly, E. coli, S. aureus, and MRSA in glycerol were grown separately in a Luria-Bertani (LB) medium under agitation (180 rpm) at 37 °C for 18 h, and then subcultured in LB medium for 4–6 h to the mid-log phase. The bacteria were harvested by centrifugation (4000 rpm) for 10 min and washed three times with a sterile saline. The bacterial suspensions were then diluted to an optical density of 1 at 600 nm (OD600 = 1) using a sterile saline and were diluted 1:100 to obtain a 1 × 106 cfu/mL bacterial suspension. The concentration of Ag element in initial HB/Ag/g and HB/Ag nanocarrier solutions which performed to the MIC experiment were 259.4 ± 1.5 μg/mL and 262.9 ± 1.8 μg/mL, respectively. Firstly, initial nanocarrier solutions (1 mL) with different concentrations were prepared in sterile glass tubes using double dilution methods, and then the bacterial suspension (1 mL) was mixed with each sample dilution. The glass tubes including the 5 × 105 cfu/mL bacterial suspension and different dilutions were incubated at 37 °C for 24 h, and the lowest sample concentration that can inhibit bacterial growth by more than 90% was used as the MIC. The fractional inhibitory concentration (FIC) was used to evaluate the synergistic action of compound A (Ag) and compound B (Gen) and was calculated as follows:

2. Experimental section

FIC 2.1. Synthesis of 3aminophenylboronic acid grafted hyaluronic acid

=

HA (500 mg) was dissolved in a HEPES buffer (100 mL, 50 mM, pH 7.4) and stirred for approximately 3 h at room temperature to obtain a viscous transparent HA solution. Then, a solution (10 mL) containing 3aminophenylboronic acid (3-APBA, 50 mg) and DMTMM (225 mg) was added dropwise into the HA solution. The reaction was allowed to proceed for 24 h under magnetic stirring, and then approximately 1 L of ethanol was added to finish the reaction. After 24 h, the precipitate was collected via centrifugation at 5000 rpm for 10 min. The precipitate was dissolved in deionized water and dialyzed at 4 °C for three days. Spongy-like white solid 3-APBA grafted HA named HB was obtained by freeze-drying [22].

MIC of compound A in combination MIC of compound A alone MIC of compound B in combination + MIC of compound B alone

The FIC ≤ 0.5 was defined as synergistic action [26]. 2.4.2. Bacterial growth monitoring in presence of nanocarriers Bacterial suspensions of E. coli, S. aureus, and MRSA (20 mL) were grown respectively with HB/Ag/g nanocarrier at its MIC, and the bacterial concentrations were measured by detecting the optical density (OD600) at different intervals. The same experiment was performed with the HB/Ag nanocarrier and the HB/g nanocarrier, and their concentrations were equal to the amounts of Ag and Gen in the HB/Ag/g nanocarrier.

2.2. Preparation of HB/Ag/g nanocarrier HB (10 mg) was dissolved in water (5 mL, pH 8.5) under vigorous magnetic stirring at room temperature. This was followed by the addition of fresh AgNO3 (100 μL) and Gen (100 μL) solutions. The final concentrations of Ag+ and Gen in the obtained solution were 1.25–10 mM and 5–100 mg/L, respectively. After stirring for 2 h, a certain amount of TA acting as a reducing agent was added into the reaction system, and the molar ratio of TA:Ag+ was 3:40. The mixtures were then continuously stirred for 10 h, and grey products were obtained that were named HB/Ag/g nanocarriers. The HB/Ag nanocarriers were prepared by the method described above without addition

2.5. Preparation of disinfectant coatings via chitin hydrogel In this study, chitin hydrogel (CH) prepared using the procedure described in the previous report was used as the substrate to study the potential of the HB/Ag/g as a coating material [27]. Briefly, a transparent chitin solution was obtained from a mixture of NaOH, urea, and distilled water (with a weight ratio of 8:4:88) through a freeze-thaw process. After centrifugal degassing, the chitin solution was spread on a glass plate and then was immersed into alcohol. The CH was thoroughly washed with de-ionized water to remove the residual reagent. The 3

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microscopy (Nikon, Japan).

prepared CH was immersed in 2 mg/mL of a dopamine hydrochloride solution (Tris-HCl buffer, pH 8.5) for 24 h to obtain polydopaminegrafted chitin hydrogel, named CPH. The HB/Ag, HB/g, and HB/Ag/g solutions were fixed to the surface of the CPH through a layer-by-layer dip-drying procedure, and the obtained samples were named HB/ Ag@CPH, HB/g@CPH, and HB/Ag/g@CPH, respectively.

2.9. In vivo mice wound model and healing process The in vivo animal study was approved by the experimental animal science and technology center of the Jiangxi university of traditional Chinese medicine. Eight-week old SD female rats with an average weight of 250 g were evenly divided into three groups (eight parallel rats in each group): a gauze group (negative control group), an HB/ Ag@CPH group (positive control group), and an HB/Ag/g@CPH group (experimental group). The rats were anesthetized by isoflurane, and two 15-mm diameter partial wounds were prepared on the right and left sides of the back. The wounds were infected by injecting 200 μL of S. aureus (1 × 106 CFU/mL) and incubating for 30 min. The infected wounds of the three groups were tightly covered with gauze, HB/ Ag@CPH, and HB/Ag/g@CPH, respectively. The rats were individually kept at a standardized temperature on a 12:12 L/D cycle. The dressing materials were changed at every two days, and the wounds were photographed. The wound areas were measured using IPP 6.0 software. The exudate from the wounds were collected using sterile swabs on Days 4, 8, and 12, and dispersed into 1 mL sterilized saline water. The suspensions (100 μL) were spread on LB agar plates and cultured at 37 °C overnight. The skin tissue samples were harvested on Days 4, 8, and 14, and fixed with 10% formalin for histological examination. Hematoxylin and erosin (H&E) staining and immunohistochemistry staining were used for the histological analysis of the wound tissues [28].

2.6. Ag and Gen release from HB/Ag/g@CPH The Ag release behavior from the HB/Ag/g@CPH was tested in PBS. Briefly, the three HB/Ag/g@CPH and HB/Ag@CPH samples (2 × 2 cm) were immersed in 40 mL PBS at 37 °C, respectively. The pH of the PBS were set at 5.5 and 7.4, respectively. 2.0 mL of release medium was removed at different intervals, and the same amount of PBS was added. The concentration of Ag was determined by ICP-MS. To study the Gen release behavior, HB/Ag/g@CPH was incubated into 4 mL of PBS at different intervals. The obtained suspension was then centrifuged, and the supernatant was collected. The concentration of Gen was established by HPLC-ELSD using 0.2 mol/L trifluoroacetic acid and methanol (92:8) as the mobile phase. 2.7. Antibacterial properties of HB/Ag/g@CPH The optical density monitoring, bactericidal assays, and bacterial adhesion assays were performed to evaluate the antibacterial properties of HB/Ag/g@CPH. For the optical density monitoring, bacterial suspensions (2 mL, 5 × 105 CFU/mL) were incubated with different samples for 12 h, and the bacterial concentrations were measured by detecting the optical density at 600 nm (OD600). For the bactericidal assays, S. aureus suspensions (2 mL, approximately 5 × 104 CFU/mL) were incubated with different samples for 2 h. The resulting mixtures (100 μL) were then pipetted out and dispersed onto LB agar plates and incubated for 24 h. The bacterial kill ratios were obtained by counting the bacterial colonies on the culture plates. For the bacterial adhesion assays, after incubation with a bacterial suspension (approximately 107 CFU/mL), the samples were washed with a sterile saline solution and immediately fixed by glutaraldehyde (2.5 wt%) and dehydrated in a series of ethanol solutions. The bacteria attached on the samples were observed by scanning electron microscopy (SEM).

2.10. Statistical analysis All data are expressed as means ± standard deviations. The statistical significance was evaluated using the Student's t-test and rectified by ANOVA for comparisons between multiple groups. Results with P values less than 0.05 were considered statistically significant.

3. Results and discussion 3.1. Characterization of HB In this study, 3aminophenylboronic acid (APBA), a pHdependent agent that binds to TA-reduced AgNPs, was conjugated to HA (Mw = 2.48 × 106 g/mol, Fig. S1A) via amide bond formation to obtain HB (Mw = 2.35 × 106 g/mol, Fig. S1B). The successful preparation of HB was verified by 1H NMR and FT-IR. As shown in Fig. 2A, the peak at 1.89 ppm showed the chemical shifts for the protons of the N-acetyl group of HA, and the peak at approximately 7.36–7.76 ppm is a signature of the aromatic rings of APBA, indicating the conjugation of APBA to HA [22]. The degree of functionalization of HB by APBA was determined quantitatively from the ratio of the integration area (7.36–7.76 ppm × 4H)/(1.89 ppm × 3H), and the grafting yield for APBA was approximately 9.13% according to the 1H NMR spectra [20,22]. The successful synthesis of HB was also confirmed by the FT-IR spectra, and the spectral range of 500–4000 cm−1 for HA and HB is shown in Fig. 2B. The characteristic peak of HA at approximately 3412 cm−1 can be ascribed to OeH stretching [29]. The peaks at 1617 and 1410 cm−1 were attributed to the C]O and CeO vibration of the carboxyl group, respectively, and the strong band at 1321 cm−1 corresponds to the amide band III, demonstrating the presence of the amide groups of HA [29]. However, the peaks at 2895, 1151, and 1044 cm−1 were ascribed to CeH, CeOeC, and CeOH vibrations, respectively, and represent the polysaccharide skeleton of HA [29]. A new peak of HB conjugates appeared at 1554 cm−1 indicating NeH bending, and the peak of the C]O stretching vibration was shifted from 1617 to 1631 cm−1, implying the formation of an amide bond between the amine group of APBA and the carboxylic acid group of HA [20].

2.8. In vitro cytocompatibility study 2.8.1. Cell viability In vitro cell viability of the nanocarrier was estimated by the observation of flow cytometer (Beckman, CytoFLEX) using human dermal fibroblasts (CCC-HSF-1). Briefly, 100 μL of cell suspension (about 5 × 103 cells/well) were seeded into 96-well plates in high-glucose Dulbecco's modified Eagle's medium (H/DMEM), and incubated for 24 h at 37 °C in 5% CO2. The cells were then treated with various concentrations of nanocarrier (0, 15.63, 31.25, and 62.50 μg/mL) or different films and further incubated for 24 h at 37 °C. Followly, the cells were collected and washed with PBS. After having removed the PBS, the cells were digested by pancreatic enzymes and stained with PI (100 μg/mL) for 8 min. The cell viability was detected by flow cytometer with the excitation and emission waves at 488 nm and 585 nm, respectively. 2.8.2. Cell adhesion and spreading study The cells were incubated with different films in a 48-well plate for 1–5 days. The cells adhered on the films were washed with PBS (pH 7.4) and then fixed with a 4% formaldehyde solution for 30 min. After washing with PBS (pH 7.4) to remove the residual formaldehyde, the cells were stained with FITC-phalloidin (1:200, 2 h, Servicebio) to add color to the cytoskeletal actin. The nuclei of the cells were then counterstained with 4, 6-diamidino-2-phenylindole (DAPI, 1:600, 5 min, Servicebio). Finally, the cells were observed by fluorescence 4

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Fig. 2. (A) 1H NMR spectrum, and (B) FT-IR spectrum of HA and HB; (C) TEM images and Transmission electron diffraction patterns of HB/Ag/g nanocarrier; (D) EDX analysis of HB/Ag/g nanocarrier; (E) Light-scattering photo (Tyndall effect) of different solutions; (F) X-ray photoelectron spectroscopy (XPS) wide scan; Inset is elemental analysis of HB, HB/Ag, and HB/Ag/g, which was estimated from XPS wide spectra. (G) Ag 3d spectra, and (H) B1s spectra for HB, HB/Ag, and HB/Ag/g.

(Scheme 1b). Meanwhile, the size distribution obtained from TEM observation presented in the inset of Fig. S3B-F showed much smaller sizes than the sizes measured by DLS, which was attributed to the shrinking of the HB/Ag/g nanocomposites after drying [17]. Interestingly, the negative zeta potential values of HB/Ag/g decreased from −31.2 ± 3.3 to −41.3 ± 5.2 mV with an increase in concentration of Gen, which may be due to the formation of hydrogen bonding between AgNPs and Gen. Light scattering (Tyndall effect) can be observed from different nanocomposites, verifying the formation of a nanoscale structure (Fig. 2E) [17]. As shown in Fig. 2C, the HB/Ag/g nanocarrier exhibited nearly spherical nanostructures with size in the 10–30 nm range. The observed polycrystalline structure of the HB/Ag/g nanocarrier from the selected area electron diffraction (SAED) pattern is presented in Fig. 2C. The result of energy-dispersive spectrometry (EDS) measurements showed that C, O, and Ag elements were distributed uniformly in the HB/Ag/g nanocarrier (Fig. 2D), demonstrating the successful preparation of the HB/Ag/g nanocarrier, which was also verified by the XPS spectrum. As shown in Fig. 2F, four peaks corresponding to the C1s, O1s, B1s, and N1s states were found in the spectrum of HB, and new peaks of Ag3d states were observed in the spectra of HB/Ag and HB/Ag/g, indicating that AgNPs were successfully immobilized in HB. Fig. 2G clearly shows two individual peaks at approximately 374.1 and 368.2 eV that can be attributed to the Ag (0) 3d5/2 and Ag (0) 3d3/2 electrons of metallic Ag, respectively [13]. In addition, the contents (atom %) of different elements in the three nanocarriers were calculated from the XPS wide scan, and are presented in Fig. 2F. The Ag contents of the HB/Ag and HB/Ag/g nanocarrier were 0.67 atom % (approx. 5.02 wt%) and 0.66 atom % (approx. 4.95 wt%), respectively [33].

3.2. Preparation and characterization of HB/Ag/g nanocarrier An efficient and environmentally-friendly method was proposed to prepare the HB/Ag/g nanocarrier, with the plausible synthesis mechanism shown in Scheme S1. In this process, TA, a natural and readily available polyphenol, was chosen as the reductant and stabilizer. As shown in Scheme S1a, the phenolic hydroxyl groups of TA were easily oxidized to form quinones and donated electrons to Ag+, leading to the formation of AgNPs in a mildly alkaline environment [30]. However, only 10 pairs of o-dihydroxyphenyl participated in the redox reactions [31]. The remaining catechol (1, 2diol) of TA bonded with the boronic acid groups of HB to form cyclic boronate esters that reversibly dissociated in the acidic environment (Scheme S1b). Simultaneously, by taking advantage of the abundance of acetyl amino groups of HB and phenolic hydroxyl groups of TA, the AgNPs were stabilized and immobilized in the HB-TA macromolecule matrix via coordination bond and hydrogen bond [31,32]. Here, the ability of stabilization AgNPs by HB-TA macromolecule matrix was studied. As shown in Fig. S2A, the UV/Vis spectra of different concentrations of AgNPs loaded nanocomposites presented a typical localized surface plasmon resonance band of AgNPs, and the absorption peak intensity increased with an increase in the AgNO3 concentration, strongly suggesting that more AgNPs were embedded in the HB-TA matrix with a higher concentration of AgNO3 [32]. As shown in Fig. S2B, compared with the HB spectra, two diffraction peaks at 2θ = 38° (1 1 1) and 44° (2 0 0) appeared in the AgNPs loaded nanocomposite, indicating that the face centered cubic (fcc) lattice of the Ag crystal was retained in the Ag loaded nanocomposite. Clearly, these results were mainly attributed to the fact that the rate of the spontaneous nucleation of the Ag atom increased significantly with an increase in AgNO3 concentration, thereby improving the growth rate of AgNPs [30–32]. As shown in Fig. S3A, the addition of Gen influenced the typical localized surface plasmon resonance band of the AgNPs, which could be due to the formation of Gen-AgNPs complexes connected by the OeAg bond [13]. The DLS results presented in Fig. S3B-F showed that larger size distributions were observed when the Gen concentration increased, possibly because of the electrostatic interactions between HB and Gen

3.3. Bacterial disinfection properties of the HB/Ag/g nanocarrier To investigate the synergistic antibacterial capability of Ag and Gen, the minimum inhibitory concentration (MIC) values of the HB/Ag/g nanocarrier against E. coli, S. aureus, and MRSA were evaluated using the microdilution method. Figs. 3A and S4 show the MIC values of HB/ Gen, HB/Ag, and HB/Ag/g nanocarriers, indicating their ability to 5

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Fig. 3. (A) MIC values of HB/Gen, HB/Ag, and HB/Ag/g required for inhibition of E. coli, S. aureus, and MRSA, respectively (the data were obtained from Fig. S4); Bacterial growth kinetics of (B) E. coli, (C) S. aureus, and (D) MRSA incubated with different nanocarriers (Concentrations of HB/Ag/g are the MIC for inhibition of different bacterias, and concentrations of HB/Gen and HB/Ag are equal to the amount of silver and gentamicin in HB/Ag/g); (E) UV–vis absorbance of released AgNPs in the supernatant controlled by HAaes; (F) Bacterial morphology images observed by TEM and the EDS patterns.

However, the cell membranes of the two types of bacteria treated with nanocarriers underwent melting (red arrow), and the intracellular densities (green rectangles) decreased compared to the cases of the untreated bacteria, indicating the presence of significant intracellular substrate leakage. In particular, the bacteria treated with HB/Ag/g showed smaller intracellular densities than those of the HB/Ag-treated groups. It is postulated that the presence of Gen in HB/Ag/g enhances the penetration of Ag into the cell membrane [15]. Meanwhile, the lack of integrity of the bacterial cell membrane makes it easier for the Gen to enter the bacterial cell and kill the bacteria through the inhibition of protein synthesis by binding of gentamicin to the ribosomal subunits [36]. In addition, EDS detected the presence of the Ag element inside the broken bacteria, demonstrating the penetration of Ag into the cell membrane that resulted in an osmotic collapse and loss of some of the intracellular substrates [37]. To further investigate the antibacterial mechanisms of HB/Ag/g, the morphological changes in the two types of bacteria before and after HB/Ag/g treatment with different concentration were observed by SEM. As shown in Fig. S5, both E. coli and S. aureus presented normal morphologies with smooth and intact cell surfaces with no ruptures. However, after being treated with HB/Ag/g at the concentration of 30.02 μg/mL for 4 h, the bacteria lost their cellular integrity and underwent osmotic collapse. When the HB/Ag/g concentrations increased, the degree of collapse of cell structures increases significantly. In addition, the release of intracellular materials from the cracked membrane of bacteria treated with 60.05 μg/mL of nanocarrier was observed (red arrow).

inhibit different bacterial strains. The obtained data show that the MIC values of the HB/Ag/g nanocarrier were 6.07 μg/mL silver and 0.59 μg/ mL Gen against E. coli, and 3.04 μg/mL silver and 0.29 μg/mL against S. aureus. However, the MIC values of HB/Gen and HB/Ag were 3.13 μg/ mL Gen and 32.86 μg/mL silver towards E. coli, and 0.78 μg/mL Gen and 24.65 μg/mL silver towards S. aureus. A significant enhancement of antibacterial effect was associated with HB/Ag/g compared to those associated with HB/Gen and HB/Ag, resulting in a strong decrease in the amount of Ag and Gen used for bacterial inhibition. To evaluate the synergistic effect between Ag and Gen, the fractional inhibitory concentration (FIC) index was calculated [34]. The FIC index values were 0.373 and 0.495 for E. coli and S. aureus, respectively, both below 0.5, indicating a synergistic effect between Ag and Gen. Moreover, the dosedependent growth kinetics curves of E. coli, S. aureus, and MRSA were examined by measuring the optical density at 600 nm to assess the antibacterial activity of the HB/Gen, HB/Ag, and HB/Ag/g nanocarrier, respectively. As shown in Fig. 3B–D, bacterial growth was observed in the HB/Gen- and HB/Ag-treated groups. However, bacterial proliferation was inhibited by HB/Ag/g, indicating that the therapeutic efficacies of silver and Gen in the HB/Ag/g were enhanced by the presence of the other component [15,17]. In addition, the HB/Ag and HB/Ag/g nanocarriers both showed better inhibition effects toward S. aureus than that toward E. coli. This phenomenon may be attributed to two possible reasons: the first is that the produced hyaluronidase (HAase) from S. aureus leads to the specific cleavage of the HB/Ag nanocarrier that can enhance the release of Ag or Gen [23,24]. Release of AgNPs from the HB/Ag nanocarrier triggered by HAase was explored experimentally. As shown in Fig. 3E, more AgNPs were released from the HB/Ag nanocarrier when HAase was added. On the other hand, the cell wall structure of S. aureus is composed of peptidoglycan and has abundant pores that renders the cell wall more susceptible to reactive species, leading to cell disruption. However, the outer layer cell wall of E. coli consists of lipopolysaccharide, lipoprotein, and phospholipids and is less vulnerable to attack by a reactive species [35]. To understand the interaction between the bacteria and nanocarriers, the cell membrane damage in the presence of nanocarriers was examined by TEM using E. coli and S. aureus as examples. As shown in Fig. 3F, the untreated E. coli and S. aureus bacteria show normal morphology with distinct cell walls and compact intracellular substrates.

3.4. Construction and properties of HB/Ag/g@CPH HB/Ag/g was found to exhibit the HAase-triggered release of AgNPs and versatile antibacterial activity, demonstrating its potential for application in wound disinfection. Meanwhile, the rheological properties of HB/Ag/g were investigated. As shown in Fig. S6, the intrinsic viscosity of HB/Ag/g was significantly increased with an increase in the concentrations of HB and Gen, which was ascribed to the possible electrostatic interactions between hydroxyl, carboxyl, and acetamide groups of HB and Gen. Due to its intrinsic viscosity and overall microstructure, HB/Ag/g was suitable for surface coating preparation. In this study, HB/Ag/g@CPH was fabricated by a facile dip-drying process 6

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colorless and transparent CH. After coating with HB/Ag/g, the HB/Ag/ g@CPH had a black color. The surface morphology of different hydrogels was examined by SEM. As shown in Fig. 4A, the CH displayed reticular nanostructures composed of chitin nanofibrils (with diameters of approximately 30 nm) that were interconnected through electrostatic interactions and hydrogen bonding. After being covered with dopamine, white granule protuberances with diameters in the range from 80 to 300 nm were observed (red arrow) which may be macro/nano particles formed by the uncontrollable self-polymerization of the polydopamine molecules [41]. The AgNPs (white dots indicated by red circle) were evenly distributed on the surface of HB/Ag/g@CPH, and the diameters of the AgNPs were below 50 nm. The successful grafting of the polydopamine layer and the coating of HB/Ag/g on the surface of CH were further confirmed by XPS and FTIR measurements, respectively, the results of which are shown in Fig. S8. In addition, the results of water absorption capacity, surface wettability, tensile strength, and elongation measurements are shown in Fig. S9, indicating that HB/Ag/ g@CPH showed applicable wettability and mechanical strength that are beneficial for biomedical applications. These results are discussed in detail in Supporting Information.

using chitin hydrogel (CH) as the model coating substrate to enhance the application of HB/Ag/g in wound disinfection. The CH was prepared by a green sol–gel transition method by wetting chitin–NaOH–urea aqueous solution in ethanol, and CH was formed via the hydrogen bonding interaction without an external crosslinker [27]. CH has been demonstrated to have versatile applications in biomedical fields, with advantages of biocompatibility and biodegradability, and it was therefore chosen as the model coating substrate in this study [38]. However, it was found that HB/Ag/g coated poorly on the surface of CH because of the hydrophobic interactions between the polymeric chains of CH. In addition, the abundant acylamido groups of CH weaken the electrostatic interactions with glycosaminoglycan of the cells that will prevent cell adhesion and proliferation, thereby limiting the application of CH in the biological field [38,39]. Herein, a polydopamine layer was formed on the surface of CH via oxidative selfpolymerization at the dopamine solution under alkaline conditions as shown in Fig. S7, endowing it with a positively charged surface and introducing numerous eNH2 groups that give rise to electrostatic interactions between eNH2 and the eCOOH or eOH of the negatively charged HB/Ag/g [40]. Subsequently, the HB/Ag/g was coated on the surface of chitin/polydopamine hydrogel (CPH) via a layer-by-layer dip-drying procedure, and the obtained material was named HB/Ag/ g@CPH. The optical photographs of CH, CPH, and HB/Ag/g@CPH are shown in the inset of Fig. 4A. CPH has a brown appearance with a certain degree of transparency after polydopamine covered the surface of the

3.5. Release property of HB/Ag/g@CPH The pH-triggered release of Ag+ and Gen from HB/Ag@CPH and HB/Ag/g@CPH in PBS was monitored on a daily basis, and the obtained results are shown in Fig. 4B. The total Ag element contents of

Fig. 4. (A) SEM images of different sample surfaces (Inset photographs were their appearance); (B) Ag+ and Gen release from HB/Ag@CPH and HB/Ag/g@CPH in PBS at different pH, respectively; (C) Optical density (600 nm) values of bacterial solutions (2 × 105 CFU/mL) after incubation with samples for 12 h; (D) The killing ratios of S. aureus for the different samples calculated from the agar plate count results; (E) SEM images that revealed the bacterial adhesion on the different sample surfaces within 4 h. 7

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HB/Ag/g@CPH and HB/Ag@CPH were 19.51 ± 0.89 μg/cm2 and 20.31 ± 1.21 μg/cm2, respectively. These two groups displayed a similar release trend to that of Ag+; the release of Ag+ was rapid in the first stage (0–5 days) followed by a gradual stabilization as the immersion time increased (5–21 days), indicating long-term release durability. The amount of Ag+ released from HB/Ag/g@CPH on the first day was approximately 0.41 mg/L, which was slightly higher than that of HB/Ag@CPH (0.36 mg/L). This result may be explained by the formation of Gen-AgNPs complexes in HB/Ag/g@CPH that enhanced the Ag+ release [11]. Moreover, it was observed for HB/Ag/g@CPH that the release capacity of Ag+ at pH 5.5 (0.58 mg/L on the first day) was higher than that at pH 7.4, indicating that the release behavior of the Ag+ is pH-dependent. This phenomenon can be attributed to the pHsensitive groups (hydroxyl, carboxyl, and phenylboronic acid groups) of HB/Ag/g that can spontaneously release AgNPs in an acidic environment. The AgNPs can be oxidized to Ag+ by dissolved oxygen and H+ in an aqueous solution as expressed by the formula: 4AgNPs + O2 (aq) + 4H+ = 4Ag+ (aq) + 2H2O [42]. Therefore, a reduction in pH is associated with a higher H+ concentration formed in the medium that produced more Ag+ from AgNPs. The pH-triggered release of Gen was also observed in Fig. 4E. Due to the presence of ionized carboxyl groups on HB in an acidic environment, the interaction between HB and Gen was weakened, increasing the release of Gen. This pH-triggered release property of HB/Ag/g@CPH is favorable for disinfection applications because bacteria generate metabolic products and acidify their local environment, leading to slightly lower pH values than in the neutral environment [43].

membranes and could oxidize the surface proteins on the plasma membrane and consequently led to structural changes in cell membranes [4,5]. (3) Meanwhile, the Ag+ released from AgNPs destroyed the integrity of the cell membrane, causing morphological collapse and a significant increase in membrane permeability [31]. (4) AgNPs and Ag+ have also been well-documented to form reactive oxygen species (ROS) that can damage the cell membranes [31,33]. (5) Membrane leakage may diminish the transmembrane proton electrochemical gradient and thereby inactivate energy-dependent reactions such as ATP synthesis, ion transport and metabolite sequestration [5]. (6) The increased permeability of cell membranes makes it easier for Gen to enter the bacterial cell. (7) Gen can inhibit protein synthesis through biochemically binding to the ribosome at two sites (h44 in the 30S subunit and H69 in the 50S subunit), thereby exerting its antibacterial effect [36]. (8) Additionally, the AgNPs and Ag+ that entered the cell can interact with intracellular enzyme, inflict damage on the DNA, and lead to disordered metabolism of the cell by inducing intracellular ROS [45,46]. (9) Previous studies of the interaction of silver and antibiotics have revealed that the formed antibiotic-AgNPs complexes interact more strongly with the bacteria and cause greater Ag+ release, thus creating a temporary high concentration of Ag+ in the vicinity of the bacteria cell wall that leads to the growth inhibition of bacteria [13]. (10) Thirumurugan et al. confirmed the possible synergistic antibacterial mechanism where the combination of silver and antibiotics gave rise to an increased ROS level, and membrane damage following protein release, K+ leakage and biofilm inhibition [47]. 3.8. In vitro biocompatibility

3.6. Antibacterial activity of HB/Ag/g@CPH 3.8.1. Cytotoxicity test Some studies have reported that AgNPs exhibit cytotoxicity to normal cells by releasing Ag+ ions in a high concentration [6]. Ag+ can permeate through cell membranes and then react with the negatively charged functional groups of protein and DNA, leading to their dysfunction [17]. Therefore, prior to using our AgNPs-based biomaterials in biomedical applications, the cytotoxicity of these biomaterials to normal cells should be carefully evaluated. In this study, 24 h MTT assays were performed to investigate the cytotoxicity of the samples using CCC-HSF-1, and the obtained results are shown in Fig. 6A and 6B. The blank HB exhibited excellent cell viability that was attributed to the nature of its functional groups, because of the nontoxicity of HA, as shown in Fig. 6A [23]. Clearly, HB/Ag and HB/Ag/g had little impact on cell growth when their concentrations were below 31.25 μg/mL. This is because the AgNPs loaded by HB had inhibited toxicity, since HB could slow down the release of Ag+ and prevent the direct interaction between AgNPs and cell membranes [21,23]. In addition, the interaction between the released Ag+ and TA ultimately reduced the cytotoxicity of the HB/Ag and HB/Ag/g [31,44]. HB/Ag/g still showed a good cell survival rate at a dosage of 62.5 μg/mL. This phenomenon can be explained by the possible mechanism of AgNPs first forming a complex with Gen that increased the cell survival rate of HB/Ag/g [15]. However, the specific coordination between AgNP and Gen is still unclear and the details of the mechanisms of the enhancement of the cell survival rate must still be explored. The cytotoxicity of the films was also evaluated, and the results are shown in Figs. 6B and S11. It was found that the cell viability for the cells co-cultured with HB/Ag/ g@CPH was similar to that of the control and CPH, revealing that the HB/Ag/g coating had no significant effect on the cellular compatibility of CPH.

The antibacterial activities of HB/Ag/g@CPH were investigated through OD600 values, bacterial kill ratios, and bacterial adhesion observations. As shown in Fig. 4C, the final OD value was monitored to evaluate the antibacterial property of the coatings. Turbid media were observed for the CH-, CPH-, and HB/Ag@CPH-treated groups that revealed rapid bacterial growth. Conversely, bacterial growth was significantly suppressed in the HB/Ag/g@CPH-treated group because of the excellent synergistic antibacterial activity of Ag and Gen. The bacterial killing ratio was further applied to investigate the bactericidal efficacy of different coatings. As shown in Fig. S10, compared to the CH- and CPH-treated groups, a negligible difference of bacterial colony with turbid plates was observed for the HB/Ag@CPH-treated group. However, after treatment with HB/Ag/g@CPH, the bacteria were almost completely inhibited, and the bacterial killing ratio against S. aureus was approximately 89 ± 5.4%, as shown in Fig. 4D. The bacterial anti-adhesion property of HB/Ag/g@CPH was investigated by SEM observation. Fig. 4E shows a large amount of both E. coli (typical rod shape) and S. aureus (spherical shape) bacteria adhesion, proliferation, and aggregation on the surface of CPH. However, the adhesion of the bacteria was inhibited on the surface of HB/Ag@CPH. Nearly no S. aureus and several E. coli with cracked membranes were observed on the surface of the HB/Ag/g@CPH, indicating its good bacterial anti-adhesion property. 3.7. Mechanism of synergistic antibacterial activity In this study, the synergistic antibacterial mechanism of HB/Ag/ g@CPH was assumed to account for the above-mentioned results. It was revealed that ten-step pathways may be the main origin of synergistic antibacterial activity. As shown in Fig. 5, (1) the HAase secreted by gram-positive bacteria (S. aureus) led to the specific cleavage of the HB/ Ag/g nanocarrier [23,24]; and in addition, an acidic environment (H+) was created in the vicinity of the proliferating bacteria due to the metabolic behavior of the bacteria [17], and both HAase and H+ can trigger the HB/Ag/g to release AgNPs and Gen (Fig. 3E and 4E). (2) The released AgNPs were found to adhere and accumulate on the cell

3.8.2. Cell adhesion The cell–scaffold interaction at the first stage in tissue engineering repair is determined by the initial adhesion and spreading behavior of the cells on the interface that affects the ability of cells to proliferate and differentiate [33,37]. In this study, the capability of CCC-HSF-1 cells for developing the cytoskeleton on film surfaces was visualized by 8

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Fig. 5. Schematic illustration of the possible antibacterial mechanism of HB/Ag/g@CPH.

3.9. In vivo evaluation of wounds healing

confocal microscopy via the staining of F-actin and the nucleus with FITC–phalloidin and DAPI, respectively. As shown in Figs. 6C and S12, many adhering and proliferating cells were found on CPH cultured for 2 days, indicating that the formation of the polydopamine coating layer effectively enhanced the cells’ adhesion on chitin. After culturing for 5 days on CPH, the adhesion of more cells with well-spreading cytoskeleton networks was observed. This result indicates the biocompatibility of the CPH. Many studies have shown that the polydopamine coating layer produced a large amount of positively charged amine groups on the surface of the scaffold, thus allowing for stronger electrostatic interactions with glycosaminoglycan that promoted cell adhesion, spreading, and growth [38,39]. Clearly, the cells’ adhesion and proliferation on HB/Ag/g@CPH after being cultured for 2 days were similar to that of CPH, even though the cells expression of F-actin filaments and filopodia were less pronounced than that on CPH. The spreading out of filopodia with cytoskeletal tension and stress was visualized on the surface of HB/Ag/g@CPH in the following days, and finally, a cytoskeleton network was observed on the entire surface after 5 days. These results demonstrated that the added HB/Ag/g coating had no obvious impact on the cell adhesion and proliferation of CPH except at the initial stage. To elucidate the adhesion and proliferation of the cells on the HB/Ag/g@CPH, the dehydrated and lyophilized sample was observed by SEM. As shown in Fig. S13, the cells that were welladhered on the HB/Ag/g@CPH exhibited elongated morphologies and a large number of filopodia (red arrows). More importantly, the cells were well spread out with considerable extensions and interconnections through filopodia. It is speculated that the multi-triggered AgNPs and Ag+ release approach allowed the AgNPs and Ag+ to be protected by biocompatible HA without affecting the adhesion and proliferation of fibroblast on the HB/Ag/g@CPH [23,24]. Overall, the MTT, fluorescence microscopy, and SEM results suggested that HB/Ag/g@CPH had good biocompatibility and promising potential for biomedical applications.

To evaluate the therapeutic efficacy in antibacterial activity and wound healing of HB/Ag/g@CPH in vivo, the dorsal wound infection model of a rat was built. First, full-thickness round wounds with a diameter of 18 mm were built on the dorsum of female rats and exposed to S. aureus. Then, the S. aureus infected models were divided into three groups and treated with PBS-based gauze, HB/Ag@CPH, and HB/Ag/ g@CPH, respectively. Figs. S14 and 7A showed digital photographs of the wound healing process for different groups and their corresponding histologic analyses results of the wound skin staining with hematoxylin and eosin. Briefly, an inflammatory reaction with ulceration, maturation, and edema on the wound was found for both the gauze group and the HB/Ag@CPH group based on the digital photograph of the wound on Day 4. The wounds of HB/Ag/g@CPH group became smaller than that of the gauze group (p < 0.05, as shown in Fig. 7B), and no ulceration or edema appeared, demonstrating the superior ability of HB/Ag/g@CPH to effectively prevent wound infection and significantly accelerate wound healing. For all of the wounds on Day 6, epidermal necrosis and separation with dermal were observed on the histologic analyses (red circle). Moreover, a large number of inflammatory cells, such as neutrophils and lymphocytes (black arrow), emerged on the connective tissue of the dermis for all of the groups, indicating that the wounds had an inflammatory response [48]. As shown in Fig. 7B on Day 8, wound sizes of the HB/Ag/g@CPH group decreased considerably in contrast to the wounds of the other two groups (p < 0.05 relative to the HB/ Ag@CPH group and p < 0.01 relative to the gauze group), indicating the superior ability of HB/Ag/g for accelerating wound healing. After the 10-day treatment (shown in Fig. 7A), elongated fibroblasts (yellow arrow) in the dermis were observed in all of the wound dressing groups, indicating that a recovery procedure of the wounds occurred [49]. However, compared to the case of the HB/Ag/g@CPH group, more 9

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Fig. 6. Cell viability of Human skin fibroblast cells after treated with (A) different concentration of HB, HB/Ag, and HB/Ag/g nanocarrier (B) control, CPH, HB/ Ag@CPH, and HB/Ag/g@CPH for 24 h; (C) Fluorescent images of CCC-HSF-1 cells cultured on different samples for 2 days and 5 days (scale bar: 50 μm).

revealed the strongest disinfection effect on the wound. However, the CFU count of gauze and HB/Ag@CPH groups on Day 7 were approximately 360 ± 60 and 240 ± 50 CFU/mL, respectively, which were greater than that of the HB/Ag/g@CPH treated group. After 8 days of treatment, approximately 140 ± 50 CFU/mL bacterial colonies were still present in the gauze group, which may be because the gauze not only lacked antibacterial properties, but also provided a breeding ground for bacteria [50].

inflammatory cells (black arrow) remained at the gauze treated wound and HB/Ag@CPH treated wound, validating that these wounds still maintained an inflammatory response. Furthermore, a new and thin epidermis was observed for the HB/Ag/g@CPH group, but the epidermis of the other two groups still exhibited necrosis and dermal separation (red circle). On Day 14, Figs. 7B and S14 showed that the wounds treated with HB/Ag/g@CPH had healed, while small wounds with scabs were observed for the other two groups. While the granulation tissue was organized into fibrous connective tissue in all of the groups, a thicker epidermis (blue arrow) and denser keratinocytes (red arrow) were clearly observed on the HB/Ag/g@CPH treated wound, indicating better wound healing. However, local hemorrhage (green arrow) and necrotic foci (green rectangles) appeared on the gauze treated wound and HB/Ag@CPH treated wound, respectively, suggesting that the wounds had not yet healed. These results clearly indicate that HB/Ag/g@CPH effectively accelerates wound healing. Additionally, the bacterial infection status was further assessed in vivo by the bacterial colonies extracted from the wound exudates on Days 4, 8, and 12 using the standard plate counting method. It was observed from Figs. 7C and S15 that the number of colony forming units (CFU) obviously decreased after treatment with HB/Ag/g@CPH, which

3.10. Assessment of inflammation and cell proliferation on wounds To investigate the underlying mechanism of the observed effects, the dynamic change in inflammation activity was assessed by detecting the level of IL-1β, an important marker for inflammation which influences the granulation tissue synthesis, fibroblast proliferation, and formation of collagen. As shown in Fig. 8A and C, the wounds treated with HB/Ag/g@CPH and HB/Ag@CPH had lower levels of IL-1β than that of the gauze group after a 6-day treatment (p < 0.05), which may be attributed to the ability of alleviating the inflammatory reaction and enhanced wound closure through the inhibited inflammatory enzymes of the AgNPs. However, the HB/Ag/g@CPH treated group showed 10

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Fig. 7. (A) Histological images of skin tissues stained by H&E dissected in the different intervals (Inset were the representative photographs of wounds from different treated groups at different intervals); (B) Wound area closure ratio at indicated time points; (C) Colonies of bacteria in the wound at indicated time points.

efficient synergistic activity of silver and Gen, HB/Ag/g@CPH significantly inhibited bacteria growth and adhesion. Moreover, it exhibited favorable biocompatibilities that suitability for the attachment and proliferation of CCC-HSF-1 cells which benefit from the nontoxicity and controlled release ability of HB. Most importantly, HB/Ag/g@CPH was used as a wound dressing for inhibiting infection, decreasing inflammation, and enhancing cell proliferation in an S. aureus infected wound. The low concentration Ag and Gen used in HB/Ag/g@CPH with synergistic antibacterial activity and favorable biocompatibilities demonstrated its potential for applications in medical implant materials and wound dressings. To advance this technology to bedside, more clinical pathogen will be chosen to detect the antibacterial properties of HB/Ag/g@CPH.

lower inflammation than that of the HB/Ag@CPH treated group after a 14-day treatment (p < 0.05), and it was speculated that the Gen had amplified the anti-inflammatory ability of the AgNPs. Furthermore, it has been reported that the aminoglycoside Gen can reduce the pro-inflammatory cytokines of certain cells including IL-1β and IL-6 in human and mouse proximal tubule cells [51]. To further research the underlying mechanism of HB/Ag/g@CPH on the promotion of cell proliferation, the dynamic changes in PCNA levels in the newly regenerated epithelium and granulation tissues were detected. As shown in Fig. 8B and D, after a 6-day treatment, more PCNApositive fibroblast-like cells were found in the HB/Ag/g@CPH group than that of gauze group (p < 0.01) and HB/Ag@CPH group (p < 0.05), indicating that HB/Ag/g@CPH significantly promoted cell proliferation in the newly regenerated epithelium and granulation tissues [52]. In addition, After a 14-day treatment, the number of PCNA in the HB/Ag/g@CPH group was significantly lower than that of the 6 days treatment, which implies that the wound healing stages transitioned from a cell proliferation to a cell maturation phase [53].

Acknowledgments This work was supported by Planning Subject of “the Twelfth FiveYea-Plan” National Science and Technology for the Rural development of China (2013AA102203-05), the National Natural Science Foundation of China (31660482 and 21667018), and Outstanding Youth Foundation of Jiangxi (20171BCB23010).

4. Conclusion We successfully developed a new kind of multi-responsive antibacterial nanocarrier, hyaluronan/AgNPs/Gen (HB/Ag/g). The HB/Ag/ g nanocarrier exerted synergistic antibacterial effects that enabled it to overcome bacterial resistance through the different antibacterial mechanisms of the effects of silver and Gen. Furthermore, we successfully achieved a HB/Ag/g coating on the CPH, which was named HB/Ag/ g@CPH. HB/Ag/g@CPH can simultaneously deliver Ag+ and Gen to an inflammation site under the triggers of H+ and HAase. Due to the

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2019.122582.

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Fig. 8. Representative images of immunohistochemical staining of (A) IL-1β and (B) PCNA on wounds from different treated groups after the 6-Days treatment and 14-days treatment, respectively; (C) Mean optical density values of the IL-1β which obtained from images with each group after the 6-Days treatment and 14-Days treatment; (D) The number of PCNA-positive keratinocytes of images with each group after the 6-Days treatment and 14-Days treatment (Data of each group were counted and analyzed from three images by two independent researchers).

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