A Nanocomposite Hydrogel with Potent and Broad-Spectrum

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A nanocomposite hydrogel with potent and broad-spectrum antibacterial activity Tianjiao Dai, Changping Wang, Yuqing Wang, Wei Xu, Jingjing Hu, and Yiyun Cheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02527 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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ACS Applied Materials & Interfaces

A nanocomposite hydrogel with potent and broadspectrum antibacterial activity Tianjiao Dai1,†, Changping Wang1,†, Yuqing Wang1, Wei Xu2,*, Jingjing Hu1,*, Yiyun Cheng1,* 1

Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal

University, Shanghai, 200241, P.R. China. 2

Department of Orthopedic Oncology, Changzheng Hospital, the Second Military Medical

University, Shanghai, 200003, P.R. China. †

These authors contributed equally to this manuscript.

KEYWORDS: antibacterial hydrogels, dendrimer, silver nanoparticles, on-demand release, antibacterial coating

ABSTRACT: Local bacterial infection is a challenging task and still remains a serious threat to human health in clinics. Systemic administration of antibiotics has only short-term antibacterial activity, and usually causes adverse effects and bacterial resistance. A bioadhesive hydrogel with broad-spectrum and on-demand antibiotic activity is highly desirable. Here, we designed a pHresponsive nanocomposite hydrogel via a Schiff base linkage between oxidized polysaccharides and cationic dendrimers encapsulated with silver NPs. The antibacterial components, both the cationic dendrimers and silver species, could be released in response to the acidity generated by growing bacteria. The released cationic polymer and silver exhibited a synergistic effect in

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antibacterial activity, and thus the nanocomposite hydrogel showed potent antibacterial activity against both Gram-negative (E. coli and P. aeruginosa) and Gram-positive bacteria (S. epidermidis and S. aureus). The gel showed superior in vivo antibacterial efficacy against S. aureus infection compared with a commercial silver hydrogel at the same silver concentration. In addition, no obvious hemolytic toxicity, cytotoxicity, tissue and biochemical toxicity were observed for the antibacterial hydrogel after incubation with cells or implantation. This study provides a facile and promising strategy to develop smart hydrogels to treat local bacterial infections.


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1. INTRODUCTION Bacterial infections associated with surgical procedures and medical implants are a serious problem in clinics.1 The incision sites are susceptible to bacterial contamination, especially those in non-vascular and high adipose tissues that are fertile infection regions.2 The surgical site infections may lead to complications including retarded wound healing, abscess, sepsis, and even death. For medical implants, pathogenic bacteria may attach themselves to implant surfaces by biofilm formation, which may lead to implant failure.3 In this case, the patient must suffer from a second surgery to replace the implant, which is accompanied with medical risks and further complications. It was reported that bacterial colonization of implants occurs within hours of implant exposure to bacteria, and thus a standard intravenous prophylactic antibiotic regimen combined with local antibiotic treatment of the surgical site is usually adopted immediately after the surgery.4 However, systemic administration of antibiotics has only short-term antibacterial activity, and may cause adverse effects, bacterial resistance and patient noncompliance to frequent dosing schedules. As a result, the use of antibiotics should be prudent, and it is highly desirable to develop on-demand antibiotic delivery systems in which we could conservatively control the release rate of antibiotics for the treatment of bacterial infections. Hydrogels encapsulated with antibiotics are promising formulations for surgical site infections. The hydrogels have exhibited remarkable benefits such as local delivery, bioadhesive property, and sustained payload release which improves bioavailability and minimizes cytotoxic effects of antibiotics.5,6 Antibiotics were physically loaded within cross-linked or supramolecular hydrogels composed of natural polysaccharides,7,8 proteins,9,10 peptides,11 and synthetic polymers.12-14 In antibiotic-loaded hydrogels, the drugs were generally released from the


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hydrogel matrix via passive diffusion or hydrogel degradation. Antibiotics in these gels usually show a burst release behavior at the early stage and followed by a much slower release at later stages, which cannot satisfy the requirements for on-demand drug delivery. To avoid the rapid release of antibiotics from the gels, drugs were loaded in polymers,15 nanoparticles (NPs)16 and liposomes17 to enable a two-stage delivery, or covalently conjugated to scaffolds in the gel via specific linkages.18,19 A recent study reported a smart antibiotic hydrogel via direct cross-linking between aminoglycosides and oxidized polysaccharides, and the gel displayed an on-demand drug release behavior depending on the number of bacteria colonies and the dose of aminoglycosides in the gel.5 The antibiotic hydrogel allows the drug release to synchronize with hydrogel degradation, avoiding burst antibiotic release and risks of foreign-body reactions for non-degradable hydrogels. Though these antibiotic hydrogels have shown great promise in the control of drug release and minimization of adverse effect, the use of conventional antibiotics to treat bacterial infections might be less effective due to rising microbial drug resistance. Antibiotic resistance was estimated to kill 700,000 people per year worldwide, and this number was predicted to increase to 10 million by 2050 if effects are not made to develop new antibiotics.20,21 Exacerbating this problem is the fact that few new families of antibiotics have been introduced to clinics since the 1960s.22 An alternative strategy to relieve the pressure of drug resistance is using unconventional antibiotics with distinct mechanism of action, such as cationic polymers,23,24 dendrimers,25 silver (Ag) NPs,26-28 lysozymes,29 amphiphilic molecules,30,31 host defense peptides32 and biomimetic materials.33 These bioactive components have a broad spectrum of antibacterial activity and low development of microbial drug resistance. Among the bactericidal agents, cationic polymers and Ag NPs were widely incorporated into hydrogels via physical encapsulation, chemical cross-


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linking or in situ synthesis.34-36 For example, a syringe-injectable and bioadhesive hydrogel was prepared for surgical site infections by direct crosslinking oxidized dextran using a cationic polymer polyethylenimine (PEI).2 Generally, cationic polymers kill bacteria through perturbation and disruption of the prokaryotic membrane1, however, the bactericidal polymers are also toxic against eukaryotic cell lines via a similar mechanism.29 Therefore, the dose of cationic polymers in the antibacterial gel should be prudent. Ag has been known for its antibacterial activity since the times of ancient Greece. Ag ions or NPs inhibit bacterial replication by binding to the ATP synthetic enzymes in cell wall or blocking the respiratory chain of microorganisms inside the bacteria.37 The bactericidal effect of colloid Ag NPs is influenced by the particle size: the smaller the Ag NPs, the greater antibacterial activity.38,39 However, small-sized Ag NPs are not stable, which are easily oxidized to Ag ions or aggregate into large particles, and usually require frequent application.40 As a result, Ag NPs were usually used in combination with cationic polymers such as dendrimers and chitosan to improve its stability and antibacterial activity.41-45 In this study, we proposed a nanocomposite hydrogel via a Schiff base linkage between oxidized polysaccharides and cationic dendrimers encapsulated with Ag NPs. The Schiff base is acid-sensitive, which allows the development of pH-responsive antibacterial materials, coatings or surfaces.46-49 The cationic dendrimer was used as a template to synthesize 3-4 nm Ag particles with good stability and bactericidal activity. The acid-labile property of the Schiff base linkage between cationic dendrimers and oxidized polysaccharides enable the bioactive components to release from the hydrogel matrix in response to acidity generated by growing bacteria. The nanocomposite gel exhibited good injectability, limited systemic toxicity, and potent antibacterial activity against both Gram-negative and Gram-positive bacteria. Combination of cationic dendrimer and Ag NPs showed a synergistic effect in the treatment of bacterial


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infections. In vitro and in vivo infection studies showed that bactericidal activity of the hydrogel is superior to gels containing cationic polymers only or commercial Ag hydrogels at the same Ag concentration.

2. EXPERIMENTAL SECTION 2.1. Materials. Amine-terminated generation 5 (G5) polyamidoamine (PAMAM) dendrimer with an ethylenediamine core (G5-NH2) was purchased from Dendritech, Inc. (Midland, USA). Dextran (450-650 kDa) and branched PEI (bPEI, 25 kDa) were purchased from Sigma-Aldrich (St. Louis, USA). Isopropyl-β-D-thiogalactopyranoside (IPTG) and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was purchased from Sangon Biotech (Shanghai, China). Tryptone, yeast extract, tryptic soy broth (TSB), Mueller–Hinton broth (MHB) media and agar used for bacteria culture were purchased from Oxoid (Basingstoke, UK). Commercial silver gel was bought from Hande Biotech (Kunming, China). Crystal violet, ammonium oxalate, aniline, dimethylbenzene used for Gram staining, sodium periodate (NaIO4), ethylene glycol, silver nitrate (AgNO3), sodium borohydride (NaBH4), disodium phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Aladdin Reagent (Shanghai, China). L13152 LIVE/DEAD BacLight™ Bacterial Viability Kits was purchased from Invitrogen (Carlsbad, USA). Escherichia coli (E. coli, DH5α), Staphylococcus epidermidis (S. epidermidis, 1457), Staphylococcus aureus (S. aureus, USA300), and Pseudomonas aeruginosa (P. aeruginosa, PAO1) were obtained from ATCC. 2.2. Synthesis and characterization of dendrimer-encapsulated silver NPs. Typically, G5NH2 PAMAM dendrimer was dissolved in deionized water (DI water) at a concentration of 1 mg/mL. The pH of dendrimer solution was adjusted to 2.5 by the addition of nitric acid. 10 mL


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of PAMAM dendrimer solution was used for the preparation of dendrimer-encapsulated silver NPs. AgNO3 solution dissolved in DI water at a concentration of 10 mg/mL was then added dropwise into the dendrimer solution. The molar ratios of Ag+ to G5-NH2 PAMAM dendrimer are 20:1, 30:1 and 40:1, respectively. The mixture of dendrimer and AgNO3 solutions was stirred in the dark for 1 h. After that, the Ag+ complexed G5-NH2 dendrimer solution was added with 10 mg/mL NaBH4 aqueous solution. The molar ratio of NaBH4 to Ag+ was fixed at 5:1, and the solution was further stirred for 2 h. The reaction solution was then transferred into a dialysis bag (Biosharp, molecular weight cut off = 3500 Da) and then intensively dialyzed against 500 mL DI water for more than 10 times. The G5-Ag nanocomposite solutions were then lyophilized, redissolved at a concentration of 20 mg/mL and used as the stock solutions. The content of Ag in the nanocomposite was quantitatively measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500 CE, Agilent Technologies, USA). The size and morphology of G5-Ag nanocomposite were characterized using a high-resolution transmission electronic microscopy (HRTEM, Hitachi, Japan). 2.3. Synthesis and characterization of oxidized dextran. Oxidized dextran (Dex-CHO) was synthesized by dropwise addition of 2.4 mL NaIO4 solution (107 mg/mL, dissolved in DI water) into 10 mL dextran solution (100 mg/mL, dissolved in DI water), and the solution was magnetically stirred in the dark for 4 h at room temperature. The reaction was stopped by addition of 1 mL ethylene glycol, followed by intensive dialysis against 500 mL DI water for more than 10 times. The solution was concentrated by Millipore filter (15 mL, 3 kDa) to 100 mg/mL and kept under nitrogen atmosphere before use. The oxidization degree of dextran was measured to be 15% by a colorimetric hydroxylamine titration analysis. Generally, 20 mg of the lyophilized Dex-CHO was dissolved in 4 mL hydroxylamine hydrochloride solution (0.25 M),


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and 0.05% methyl orange was added as the pH indicator. The solution was then titrated with 0.1 M NaOH solution, and the pH value of the solution was recorded. Finally, the volume of NaOH was set as the x axis and plotted against the pH value, and then the curve was differentiated. The peak value of the differential curve is the volume of NaOH (∆V, mL) consumed at the end point of titration. Thus, the oxidation degree can be calculated as (∆V0.001nNaOH/0.02)162/2, where 162 is the molar mass of dextran monomer unit. 2.4. Gel formation and characterization. The nanocomposite hydrogels (Dex-G5-Ag) were prepared by simply mixing the G5-Ag solution (20 mg/mL) with Dex-CHO solution (100 mg/mL) at a volume ratio of 1:1 in the vials. 100 µL of the hydrogel was characterized by Cryoscanning electronic microscope (Cryo-SEM, S-4800, Hitachi, Japan). The rheology properties of the gel were evaluated by a rheometer at 37 oC (TA Instrument, USA) using a parallel plate with a diameter of 20 mm. The time-dependent rheology measurement was conducted at 1% strain and 10 rad/s angular frequency. For angular frequency-dependent rheology, the angular frequency swept from 0.1 rad/s to 10 rad/s at 1% strain, while for strain-dependent rheology, the strain swept from 0.01% to 10% at an angular frequency of 10 rad/s. The thixotropic property was studied by continuous step strain measurements of the gels. This was carried out by breaking the gel upon the application of a strain of 200% for the Dex-G5 hydrogel and 1000% for DexG5-Ag (30) hydrogel. After complete rupture of the gel, it can recover at a constant strain of 1%. This breakage and recovery process was repeated for several times to ensure the ability of gel to restore. The entire study was carried out at a constant angular frequency of 10 rad/s. 2.5. In vitro acidity-triggered release and gel degradation. 100 µL Dex-G5-Ag (30) hydrogel was prepared in a vial and 1 mL phosphate buffer (pH 7.4 or pH 5.0) was added into the vial, respectively. The vials were incubated at 37 °C and 100 µL samples were collected after


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24 h. The concentration of released G5 PAMAM dendrimer was evaluated by a well-established ninhydrin assay. Generally, 85 mg ninhydrin and 15 mg hydrindantin were dissolved in 10 mL ethylene glycol-monomethyl ether. 100 µL of the solution was mixed with 100 µL sodium acetate buffer (0.2 M, pH 5.4), and the mixture was added into the 100 µL collected sample. The solution was then incubated in boiling water for 10 min, cooling to room temperature, and added with 300 µL ethanol/water solution (v/v=3/2). Absorbance of the final mixture at 570 nm was recorded using a microplate reader (Thermo, USA). The calibration curve for G5 PAMAM dendrimer is y = 4.7237x + 0.0452 (R2 = 0.9903), where x is the dendrimer concentration (mM), and y is the absorbance of the sample at 570 nm. The absorbance of Ag NPs was subtracted from the sample absorbance to counterbalance the effect of Ag on the ninhydrin assay. The concentration of released Ag from the hydrogel was measured by ICP-MS. Three repeats were conducted for each sample. Rheometer was used to investigate the mechanical moduli of Dex-G5-Ag (30) hydrogel (400 µL) before and after soaking in PB buffers (pH =7.4, 6.5, 6.0 and 5.0). Generally, 400 µL DexG5-Ag (30) hydrogel was prepared in a vial before use, and 1 mL phosphate buffers (pH 7.4, pH 6.5, pH 6.0 or pH 5.0) were added into the vials, respectively. The vials were incubated at room temperature for 1 h, and then the gels’ mechanical modulus was investigated with a rheometer. A 20 mm parallel plate was used for the rheological measurement, which was conducted at 1% strain and 10 rad/s angular frequency. 2.6. Hemolysis assay. Red blood cells (RBC) were separated from the serum of KM mice (Center for Experiment Animals, East China Normal University) and washed three times with sterile PBS by centrifugation at 2000 rpm for 5 min. Then, 200 µL of RBC were dispersed in 9.8 mL PBS to obtain a RBC suspension. 20 µL Dex-G5-Ag (30) hydrogel was immersed into 1 mL


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RBC suspension, and the mixture was incubated at 37 oC for 1 h. After centrifugation at 2000 rpm for 5 min, the supernatant was collected and its absorbance at 540 nm was detected using a microplate reader. Tritox X-100 and PBS were used as positive and negative controls, respectively. To determine the concentration necessary for 10% lysis of RBC (HC10), 300 µL G5 PAMAM dendrimer or G5-Ag (30) nanocomplex solutions at concentrations ranging from 250 µg/mL to 8000 µg/mL were prepared in 1.5 mL EP tubes. The percentage of hemolysis in each well was calculated by hemolysis%=(Asample-APBS)/(ATriton X-100-APBS)×100. 2.7. Cell culture and cytotoxicity assay. NIH 3T3 cells (a mouse embryonic fibroblast cell line, ATCC) was used to evaluate the cytotoxicity of Dex-G5-Ag (30) hydrogel. The cells were plated in a 96-well plate at a density of 104 cells per well, and cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) containing penicillin sulfate (100 units/mL), streptomycin (100 µg/mL) and 10% heat-inactivated fetal bovine serum (FBS, Gemini) at 37 oC under a humidified 5% CO2. Dex-G5-Ag (30) hydrogel (200 µL) was immersed in DMEM (2 mL, without FBS) for 24 h at 37 °C, and then 100 µL of the hydrogel extract was collected to culture the cells for 24 h (10% FBS). The cell viability was measured by a standard MTT assay. Three repeats were conducted for each sample. 2.8. Blood biochemical and histological analysis. Blood biochemical and histological analysis were used to evaluate the in vivo toxicity of Dex-G5-Ag hydrogel. The animal experiments were carried out according to the National Institutes of Health guidelines for care and use of laboratory animals and approved by the ethics committee of East China Normal University. 8-10 week old female KM mice with an average weight of 40 g were divided into two groups (five mice in each group) and subcutaneously injected with 150 µL Dex-G5-Ag hydrogel or PBS (outer and inner diameters of the needle in this study are 0.7 mm and 0.33 mm,


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respectively, and the needle length is 13 mm). All mice were sacrificed after two weeks, and the fresh blood was collected and tested (Adicon Clinical Laboratories, Shanghai, China). The fresh skin tissues around the injection site were collected and fixed in 4% paraformaldehyde. After gradient ethanol dehydration, the tissues were embedded in paraffin, sectioned into slices with a thickness of 4 µm, and stained by haematoxylin and eosin (H&E staining). The sections were observed by a conventional optical microscopy (DM4000B, Leica, Germany). 2.9. Bacterial culture. Fresh culture of E. coli and P. aeruginosa was prepared by isolating a single colony from a Luria-Bertani (LB) plate and suspended in 5 mL sterile LB medium at 37 o

C. S. epidermidis and S. aureus were prepared by inoculating a single bacteria colony from a

tryptic soy agar (TSA) plate in 5 mL sterile TSB medium. The counts of bacteria were quantified by measuring the optical density of medium at 600 nm (OD600) using a microplate plate (Thermo Fisher). For all the assays, the bacteria were allowed to grown to logarithmic phase (OD600 = 0.6 ~ 0.8) before use. 2.10. In vitro antibacterial assay. For the in vitro antibacterial assay, 60 µL Dex-G5-Ag hydrogels were directly formed in a 96-well plate by mixing 30 µL G5-Ag (20 mg/mL) with 30 µL Dex-CHO (100 mg/mL) aqueous solutions. Hydrogels consisted of Dex-CHO and G5 dendrimer (equal Dex-CHO and G5 concentration with the Dex-G5-Ag gel, 60 µL), or Dex-CHO and bPEI (equal Dex-CHO and primary amine concentration, 60 µL), and a commercial Ag hydrogel with the same Ag concentration as G5-Ag (30) (determined by ICP-MS) were tested as control antibiotic hydrogels. Bacteria culture was diluted to 104 CFU/mL with sterile LB or TSB media, and 100 µL of the bacteria solutions were added to the wells containing 60 µL gels. After 24 h incubation at 37 oC, bacteria from the wells were plated on LB or TSA plates. The in vitro


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bactericidal activity was determined by counting the bacterial colonies. The bacteria without any treatment and bacteria-free medium were tested as the controls. The minimum inhibition concentrations (MIC) of G5 and G5-Ag (30) towards E. coli and S. aureus were determined by the following method. A 2-fold serial dilution method was used to determine the MIC value. Briefly, G5 PAMAM dendrimer or G5-Ag (30) nanocomplex (2048 µg/mL) was added into the first column of wells in a 96-well plate, and then the material solutions were 2-fold serial diluted. The bacteria suspension (E. coli and S. aureus) was diluted to 2×106 CFU/mL with sterilized MHB media, and 50 µL of the bacteria suspension was seeded into each well. The bacteria solutions were incubated at 37 °C for 18 h. The wells containing MHB medium only and the wells containing bacteria without any antibacterial agent was set as negative and positive controls, respectively. After that, absorbance of the solutions at 590 nm was recorded, and the lowest concentration in which well the bacteria growth was significantly inhibited was defined as MIC. Three repeats were performed for each material. The inhibition zone measurement was conducted using a poured-plate method. Generally, 18 mL sterilized TSA was mixed with 2 mL S. aureus (107 CFU/mL), and then the mixture was carefully poured into a plate. After its solidification, the S. aureus-TSA mixture was punched with 4 holes, and then each hole was filled with 50 µL Dex-G5 hydrogel, Dex-G5-Ag (20) hydrogel, Dex-G5-Ag (30) hydrogel and Dex-G5-Ag (40) hydrogel, respectively. The diameter of the inhibition zone was recorded after incubation for 16 h at 37 oC. For the Live-dead assay, 60 µL Dex-G5-Ag (30) hydrogels were directly formed in a 96-well plate by mixing 30 µL G5-Ag (30) (20 mg/mL) with 30 µL Dex-CHO (100 mg/mL) aqueous solutions. Hydrogels consisted of Dex-CHO and bPEI (equal Dex-CHO and primary amine concentration, 60 µL), and a commercial Ag hydrogel containing the same Ag concentration with


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the Dex-G5-Ag (30) hydrogel (determined by ICP-MS) were tested as control antibiotic hydrogels. S. aureus was used as the model bacteria. The bacteria medium was diluted to 104 CFU/mL with sterile TSB media, and 100 µL of the bacteria solutions were added to the wells containing 60 µL gels. After 24 h incubation at 37 oC, viability of S. aureus was assessed using LIVE/DEAD BacLight™ Bacterial Viability Kits (Invitrogen, UK) according to the manufacturer’s protocols. The bacteria in the wells were stained with the dyes (6 µM SYTO 9 and 30 µM propidium iodide) for 15 min. Then, the bacteria were mounted onto glass slides and visualized by a fluorescent microscopy (Olympus, Japan). For the detection of SYTO 9, a 488 nm excitation and a 520 nm emission filter was used. For propidium iodide detection, a 543 nm excitation and a 572 nm emission filter was used. The bacteria without any treatment were tested as a control. The morphology of E. coli after contacting with the Dex-G5-Ag hydrogel was observed by SEM. Generally, 60 µL Dex-G5-Ag hydrogel was spread on a piece of glass slide (8×8 mm2). A droplet of the bacteria suspension (concentration of 108 CFU/mL) was added on the hydrogel and incubated for 8 h at 37 oC, and then fixed with glutaraldehyde (2.5%) overnight at 4 oC, and carefully rinsed with PBS three times for 10 min. The bacteria were further fixed with osmic acid for 1 h, and the fixed bacteria were then dehydrated by sequential treatment with 30%, 50%, 75%, 90% and 100% ethanol (10 min for each gradient), and dried with supercritical CO2. Finally, the samples were sputter-coated with platinum and imaged using a Hitachi scanning electron microscope (S-4800, Hitachi, Japan). The bacteria incubated on agar-coated glass slide were observed as a negative control. 2.11. Preparation and characterization of Dex-G5-Ag coating. The 200 µL of the mixed solution of G5-Ag (30) and Dex-CHO (at 1:1 volume ration) was dropped on the glass slide (8×8


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mm2) or 48-well polystyrene plate, and was air-dried for seven days. Thus, we got the Dex-G5Ag (30) hydrogel coated surface (glass slide or polystyrene plate). The uncoated counterpart was used without any treatment. The scanning electronic microscope coupled with energy-dispersive X-ray spectroscopy (SEM-EDS, S-4800, Hitachi, Japan) was used to determine the elements on the glass surface. 2.12. Long-term antibacterial activity of Dex-G5-Ag coated on polystyrene substrate. For the antibacterial assay, red fluorescent protein (RFP) transformed E. coli suspension (300 µL, 104 CFU/mL) was added on the Dex-G5-Ag (30) coated 48-well polystyrene plate. After incubation at 37 oC for 24 h, 100 µL of the bacteria suspension was extracted from the well and cultured on a LB agar plate (bacteria suspension from the coated well was directly cultured on LB agar, and bacteria suspension from the uncoated well was cultured on LB agar 104 times dilution), and then incubated at 37 oC for 12 h, the colonies in the plate were counted to investigate the number of survived bacteria. Then, IPTG was added to the remaining bacteria solution at a final concentration of 1 mM and incubated for another 12 h at 37 oC. The RFP expressed bacteria were directly observed by a fluorescent microscopy (Olympus, Japan). After the first antibacterial assay, the Dex-G5-Ag coating was washed by sterile DI water for ten times, and followed by another two repeated bacteria culture experiments as described above. An uncoated polystyrene plate without any treatment was tested as the control material. 2.13. In vivo antibacterial activity against S. aureus. 8-10 week old female KM mice with an average weight of 40 g were divided into five groups (seven mice in each group). Three groups were injected with 150 µL Dex-G5-Ag (30), Dex-G5 and commercial Ag gels on the dorsal thoracic midline, respectively. Then, 108 CFU/mL S. aureus (70 µL) were directly injected to the hydrogel injection regions. The forth group injected subcutaneously with the same count of S.


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aureus without gel treatment was set as a positive control for S. aureus infection. The fifth group without any treatment was set as a negative control for S. aureus infection. All the mice were euthanized after three days when infection was apparent in the bacteria only group. The skin around the infection area was collected, homogenized in a PBS buffer and diluted by different times (10 to 108), and then 10 µL of the homogenized suspension was added into a TSA plate and incubated at 37 oC for 12 h. After that, bacterial colonies of S. aureus were counted. Fresh tissues collected from the injection site were fixed in 4% paraformaldehyde, and after gradient ethanol dehydration, the tissues were embedded in paraffin and sectioned into slices with thickness of 4 µm. H&E staining and Gram staining were used for histological analysis and S. aureus detection, respectively. The slides were observed by an optical microscopy. 2.15. Statistical analysis. All the data were present as mean ± SD. Two-tailed t-tests were used to determine the significances between two groups. For all the statistical tests, P values < 0.05 were considered to be statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001), while P > 0.05 was considered to be statistically non-significant (N.S.).

3. RESULTS AND DISCUSSION 3.1. Gel formation and characterization. Cationic dendrimers are a family of hyperbranched synthetic polymers with symmetrical topological architecture and abundant surface amine groups.50-52 Compared to linear and branched polymers such as PEIs, dendrimers have a higher density of positive charges on the surface, which is beneficial for antibacterial activity. In addition, the globular shape and interior pockets of dendrimers enable them to be ideal templates from the synthesis of dendrimer-encapsulated metal nanoparticles.53,54 In this study, PAMAM dendrimer was employed as a template to synthesize monodisperse Ag nanoparticle according to


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a well-established method.55 The dendrimer encapsulated Ag (G5-Ag) was further reacted with oxidized dextran through the Schiff base linkage between surface primary amine groups of PAMAM dendrimer and aldehyde groups on Dex-CHO, yielding the Dex-G5-Ag hydrogel (Figure 1 and Figure S1). It is reported that bacterial growth generates acidic products such as carbonic acid and lactic acid during respiration and fermentation, yielding a mild acidic microenvironment.5,16 The acidification of hydrogel environment will lead to degradation of the acid-labile Schiff base linkage and the release of G5-Ag nanocomposite from the gel. In addition, increased acidity will cause the swelling of dendrimer scaffold due to the repulsion of protonated amines in the interior, and the release of Ag NPs from G5 dendrimer.56 These properties allow the Dex-G5-Ag hydrogel to release bactericidal components such as cationic dendrimer and Ag NPs or Ag+ ions in an on-demand manner (Figure 1). The released cationic dendrimer and Ag can kill the bacteria through a synergistic effect of different mechanisms of action.


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Figure 1. Concept of the Dex-G5-Ag hydrogel with potent and broad-spectrum antibacterial activity. The gel was formed via Schiff base linkage between Dex-CHO and G5-Ag nanocomposites. Bacterial growth causes the acidification of the medium, which promotes the degradation of Schiff base linkage within the gel and the release of bactericidal active G5 dendrimer and Ag species. The cationic dendrimer and Ag killed the surrounding bacteria via a synergistic effect. Generally, G5-Ag NPs were synthesized at different Ag/G5 feeding molar ratios (20:1, 30:1 and 40:1, respectively), and the products were termed G5-Ag (20), G5-Ag (30) and G5-Ag (40), respectively. The morphology and size of Ag NPs in the nanocomposites were characterized by HRTEM (Figure 2a, Figure S2-S4). It is observed that the sizes of the Ag NPs were ultrasmall


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and generally monodisperse. The average size of Ag NPs in G5-Ag (20), G5-Ag (30) and G5-Ag (40) is 3.15, 3.33, and 3.87 nm, respectively. The Ag nanoparticle size increased as a function of Ag/G5 feeding ratio. The results suggest that G5 PAMAM dendrimer is an ideal template to synthesize and stabilize small-sized Ag NPs. Dextran was oxidized by sodium periodate to synthesize Dex-CHO. A high oxidation degree on dextran provides more reaction sites to form hydrogel networks, but may cause nonhomogeneous cross-linking in the gel due to rapid gelation, yielding opaque gels, and in severe cases, precipitates. On the contrary, a low oxidation degree on the polymer may lead to failed gelation due to limited cross-linking sites. Therefore, Dex-CHO with a moderate oxidation degree of 15% was chosen to gelate with G5-Ag nanocomposites. As shown in Figure 2b, the Ag NPs were homogeneously dispersed in the forming Dex-G5-Ag hydrogel, and the gel displayed porous nanostructure as revealed by Cryo-SEM (Figure S2a). The dynamic rheological behaviors of Dex-G5-Ag gels were further investigated. Both storage modulus (G’) and loss modulus (G’’) of the Dex-CHO and G5-Ag mixture increased along with the mixing time, and the values equal to each other at a time point around 20 min, indicating a transition from liquid phase to gel-like viscoelastic phase (Figure 2c, Figure S2-S4). We further investigated the thixotropic behavior of Dex-G5 and Dex-G5-Ag (30) hydrogels by dynamic rheometer. It can be observed that the Dex-G5 hydrogel collapsed at the strain of 100% (Figure S5), while the Dex-G5-Ag (30) gel completely collapsed at 1000% (Figure 2d). The higher maximum affordable strain of Dex-G5-Ag (30) gel suggested that incorporation of Ag NPs increases the mechanical strength of gel matrix. The thixotropic property of gels was then investigated by a continuous step strain measurement. The Dex-G5 gel was subjected to alternative 1% and 200% strains, while the Dex-G5-Ag (30) gel was subjected to alternative 1%


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and 1000% strains, respectively (Figure 2e and Figure S5). The results showed that both gels can recover to their original state at a constant strain of 1% after complete rupture. Besides, the gels were also injected from a syringe to support the shear-thinning property. As shown in Figure 2f and Figure S5, the gels became less viscous under shear stress and can pass through the needle tip as fluids, while it could return to the gel again and even stand straight on the flat. These results suggested that the Dex-G5-Ag (30) nanocomposite gel has a good thixotropic property.


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Figure 2. Characterization of the Dex-G5-Ag (30) hydrogel. (a) HRTEM image of G5-Ag (30) NPs, the size distribution of the Ag NPs was shown in the top right corner. (b) TEM image of Dex-G5-Ag (30) gel. The insertion in (b) is the enlarged HRTEM image of G5-Ag (30). (c) Time-dependent rheology measurement of the Dex-G5-Ag (30) gel. (d) Strain-dependent rheology measurement of Dex-G5-Ag (30) hydrogel with the strain sweeping from 0.1% to 1000% at an angular frequency of 10 rad/s. (e) Thixotropic experiment by continuous step strain measurement of Dex-G5-Ag (30) hydrogel. Breaking and recovery of the gel by applying an alternative strain of 1% and 1000%, respectively with progress of time at a constant angular frequency of 10 rad/s. (f) Photograph of the Dex-G5-Ag (30) hydrogel injected from a syringe. (g) Stability of Dex-G5-Ag (30) hydrogel in PBS buffer, LB medium and E. coli suspension. The interface of the gel was labeled on the vial to observe the change in gel thickness. (h) In vitro release of Ag NPs and G5 PAMAM dendrimer from the Dex-G5-Ag (30) gel at pH 5.0 and pH 7.4 for 24 h. (i) Moduli of Dex-G5-Ag (30) hydrogel after immersion in PB buffers of pH 7.4, pH 6.5, pH 6.0 and pH 5.0 for 1 h, respectively. 3.2 Release and degradation of the Dex-G5-Ag hydrogel. It is known that bacterial growth will cause the acidification of agarose medium.5,16 The stability of Dex-G5-Ag hydrogels in PBS buffer, LB medium and bacteria suspension was further investigated. The Dex-G5-Ag (30) gels were prepared in vials, and the gel volume was labeled to observe the thickness, then 1 mL PBS buffer, LB medium or bacteria suspension were added into each vial. The vials were inverted at 0 h, 6 h, 12 h, 24 h, and 48 h to evaluate the gel stability. As shown in Figure 2g, the gel thickness was not obviously changed after incubation in PBS buffer. The Dex-G5-Ag (30) gel showed excellent stability in PBS, gradual degradation in LB medium, and the fast degradation in E. coli bacterial suspension. This is due to the increased acidity during bacteria proliferation and the


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acid-labile property of the Dex-G5-Ag (30) gel. We then investigated the release of G5 dendrimer and Ag from the hydrogel under pH 7.4 and pH 5.0, which mimics the physiological condition and bacteria media, respectively. As shown in Figure 2h, about 17.6% of the Ag in Dex-G5-Ag (30) gel was released within 24 h when the gel was immersed in a pH 5.0 buffer, while the value was only 6% when immersed in a pH 7.4 buffer. Similarly, G5 dendrimer also showed an acidity-triggered release profile from the gel (13% at pH 5.0 versus 6.2% at pH 7.4). The triggered release of G5 dendrimer and Ag species from the gel was attributed to the acidlabile property of the Schiff base linkage in the gel. For PAMAM dendrimers, there is a high density of tertiary amines in the interior. At pH 5.0, the protonation of tertiary amines with a pKa value around 6.5 would result in the change of dendrimer conformation from a congested structure to a swelling one. The protonation weakens the coordination capability of tertiary amine groups to stabilize Ag NPs, and causes the exposure and release of Ag NPs encapsulated within the dendrimer structure.56,57 Besides, Ag NPs in the dendrimer may be oxidized to release Ag+ ions at acidic conditions (Figure S6). Therefore, the released Ag species from Dex-G5-Ag (30) gel is slightly higher than G5 dendrimer at pH 5.0. The immersion of Dex-G5-Ag (30) hydrogel in acidic solutions (pH 6.5, pH 6.0, and pH 5.0) also promoted gel degradation. As shown in Figure 2i, the gel modulus was significantly decreased with the decrease of pH values, suggesting the acidity-triggered degradation behavior of the Dex-G5-Ag hydrogels. 3.3. Biocompatibility assay. We further evaluated the in vitro and in vivo biocompatibility of the Dex-G5-Ag hydrogel. The Dex-G5-Ag (30) gel was selected as the representative material in the biocompatibility assay. The hemolytic toxicity of the gel was first investigated by measuring the hemolytic potential towards RBC. As shown in Figure 3a, less than 1% RBC was lysed when incubated with the hydrogel, and the hemolytic activity is similar to PBS buffer, suggesting


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limited hemolytic toxicity of the Dex-G5-Ag (30) gel. The extremely high HC10 value of G5-Ag (30) (the concentration of a material causes hemolysis of 10% RBC) indicates the good selectivity of nanocomposite towards bacteria and RBC (Table S1). In addition, the extracts from the gel showed minimal toxicity on eukaryotic cells such as NIH 3T3 for 24 h (Figure 3b). After subcutaneous injection of 150 µL Dex-G5-Ag hydrogels to the mice for two weeks, bloods from the animals were collected and the hematological parameters including RBC count, hemoglobin (Hb) concentration, white blood cell (WBC) count, and absolute lymphocyte (LYMPH#) were analyzed. All these parameters of mice treated with the hydrogels showed no significant difference compared with those of the mice treated with PBS (Figure 3c). The fresh skin tissues from the injection sites were collected to observe the tissue toxicity. As shown in Figure 3d and 3e, there was no obvious inflammation around the injection site of mice treated with Dex-G5-Ag (30) gel. All these results suggested that the Dex-G5-Ag (30) hydrogel has limited in vitro and in vivo toxicity.


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Figure 3. In vitro and in vivo toxicity of the Dex-G5-Ag (30) hydrogel. (a) Hemolytic activity of Dex-G5-Ag (30) hydrogel. PBS and Triton X-100 (0.5%) were used as the negative and positive control, respectively. (b) Cytotoxicity of the Dex-G5-Ag (30) gel against NIH 3T3 cells tested by MTT assay. (c) The hematological parameters of mice injected with the Dex-G5-Ag (30) gel or PBS for two weeks. (d and e) H&E staining of skin tissue of mice injected with the Dex-G5-Ag (30) gel (d) and PBS (e) respectively. Scale bar, 100 µm. 3.4. In vitro antibacterial activities. We further evaluated the in vitro antibacterial activities of Dex-G5-Ag hydrogels against both Gram-negative bacteria (E. coli, P. aeruginosa) and Gram-positive bacteria (S. epidermidis, S. aureus). The hydrogels were challenged with these four pathogens at a concentration of 104 CFU/mL and the bacterial quantities were counted after 24 h incubation. Firstly, we compared the antibacterial activities of Dex-G5-Ag (20), Dex-G5Ag (30) and Dex-G5-Ag (40) gels with dextran, G5 dendrimer, Dex-G5 gel without Ag NPs, Dex-bPEI gel, and a commercial Ag gel against E. coli. Free E. coli without treatment was tested as the negative control. The results showed that the Dex-G5-Ag (30) hydrogel has significantly higher bactericidal activity compared with Dex-G5, Dex-G5-Ag (20), and Dex-G5-Ag (40) gels (Figure 4a), and the result is further confirmed by an inhibition zone study (Figure S7). This phenomenon can be explained by the effect of Ag concentration and nanoparticle size on antibacterial activity. Higher Ag concentration in the nanocomposite is expected for a higher bactericidal activity, but the larger Ag nanoparticle size in Dex-G5-Ag (40) may consume more amine groups on cationic dendrimers and thus decrease the antimicrobial effect of Ag nanoparticles. In addition, the antimicrobial activity of Ag NPs decreases with increasing nanoparticle size.38,39 As a result, Dex-G5-Ag (30) showed the highest antibacterial activity against E. coli among the prepared hydrogels. The efficacy of Dex-G5-Ag (30) gel against E.


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coli is higher than a previously reported Dex-bPEI gel at the same concentration of active amine content2. Though commercial Ag hydrogel showed promising antibacterial efficacy against Gram-negative bacteria such as E. coli and P. aeruginosa, but the same formulation failed to efficiently inhibit the growth of Gram-positive bacteria such as S. epidermidis and S. aureus due to the presence of thick and multi-layered peptidoglycans on these bacteria.58 The presence of cationic polymers may help the disruption of peptidoglycan layers and the penetration of Ag ions or NPs into the bacteria. As a result, the efficacy of Dex-G5-Ag (30) gel showed much superior to commercial Ag gels against S. epidermidis and S. aureus, which is attributed to the synergistic effect of cationic polymers and Ag nanoparticles in killing the bacteria (Figure 4b-4d, Figure S8). The results suggested the potent and broad-spectrum antibacterial activity of the Dex-G5-Ag (30) gel. The morphology of E. coli contacted with the Dex-G5-Ag (30) gel was further observed by SEM, and the agar gel was used as a negative control. The substrate was first coated with a thin layer of Dex-G5-Ag (30) hydrogel, and then the bacteria was added on the surface and treated for 8 h. As shown in Figure 4e, lesions and damaged membranes were observed on E. coli contacted with the Dex-G5-Ag (30) gel, in comparison, smooth and intact membranes were observed on the bacteria incubated with the agar substrates (Figure 4f). This result also confirmed the in vitro antibacterial activity of Dex-G5-Ag hydrogel.


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Figure 4. In vitro antibacterial activities of Dex-G5-Ag hydrogels. Antibacterial activity of DexG5-Ag hydrogels against (a) E. coli, (b) P. aeruginosa, (c) S. aureus and (d) S. epidermidis. Free G5 dendrimer, dextran, Dex-G5 gel, Dex-bPEI gel, and a commercial Ag gel were tested as controls. SEM images of E. coli treated with the Dex-G5-Ag (30) gel (e) and agar (f) for 8 h. Scale bar, 1 µm. 3.5. Antibacterial activity of Dex-G5-Ag coating. The Dex-G5-Ag (30) hydrogel was further coated the surface of glass slides or well plates to evaluate its antibacterial activity as coatings (Figure 5a). The gel coating on a glass was demonstrated using SEM-EDS. As shown in Figure 5b, the elements of uncoated glass were analyzed to be silicon (Si), sodium (Na) and calcium (Ca), which are the major elements of glass. After gel coating, the new major elements observed on the surface were carbon (C) and Ag, indicating the successful coating of Dex-G5-Ag (30) gel on the glass (Table S2-S3). The antibacterial ability of the Dex-G5-Ag (30) coating was evaluated using E. coli with RFP expression. It is observed that the E. coli were effectively killed when incubated on the gel


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coated polystyrene substrate. As shown in Figure 5c, nearly no E. coli colony was proliferated for the bacteria suspension collected from the gel coated substrate, while bacteria colonies were observed for the uncoated one. In addition, the uncoated surface incubated with E. coli expressing RFP was observed with bright red fluorescence, while the Dex-G5-Ag (30) coated surface showed no fluorescence, suggesting efficient killing of bacteria (Figure 5d). To investigate the persistence of hydrogel coating, the gel coated surface was washed with DI water and the same antibacterial procedures were repeated for three times. It is observed that the DexG5-Ag (30) coating still show high antibacterial effect after three recycles (Figure 5d). These results confirmed that the Dex-G5-Ag (30) gel has an excellent antibacterial effect and could be used as coatings for local antibacterial purpose.


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Figure 5. Characterization and antibacterial activity of the Dex-G5-Ag (30) coating. (a) Concept of the Dex-G5-Ag (30) gel coating on a glass substrate. (b) The SEM-EDS spectrum and element mapping of uncoated glass (upper panel) and gel coated glass (lower panel). (c) Digital graphs of LB agar inoculated with bacterial suspensions after incubation with uncoated or Dex-G5-Ag (30) coated substrates. The white dots in the plates are live bacteria colonies. (d) Fluorescence images of uncoated and coated glasses. The surfaces were inoculated with E. coli overexpressing RFP. Only live bacteria can be observed with red fluorescence due to the expression of RFP. The antibacterial assay on the coated and uncoated substrates was performed for three recycles. 3.6. In vivo antibacterial activity of Dex-G5-Ag hydrogels. Finally, we investigated the in vivo antibacterial activity of the Dex-G5-Ag (30) hydrogel against S. aureus, the most commonly observed pathogen associated with surgical site infections. The gels were subcutaneously injected to the back of mice and then 108 CFU/mL S. aureus were directly injected the hydrogel site to ensure efficient contact of bacteria and hydrogel. The same concentration of S. aureus was injected subcutaneously into mice without the injection of hydrogels and the group was tested as the positive control for S. aureus infection. Dex-G5 hydrogel and the commercial silver gel containing an equal dose of G5 and silver, respectively were tested as control hydrogels. As shown in Figure 6a and Figure S9, the skins of mice injected with S. aureus only, Dex-G5 gel or commercial silver gel were all observed with a significant skin ulcer, but normal skin morphology was observed for the mice injected with Dex-G5-Ag (30) hydrogel. Gram-positive staining was further performed to detect the S. aureus in the infected lesions (Figure 6b). No obvious difference of the Dex-G5 group and commercial Ag gel group can be observed in comparison with the S. aureus only group, while the mice treated with Dex-G5-Ag (30) gel exhibited much lower bacterial burden in the skin lesion. Moreover, the S. aureus survival at the


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local site of infection was also significantly decreased when treated with the Dex-G5-Ag (30) gel (Figure 6c). It can be observed that the bacteria colonies in the mice pre-treated with the DexG5-Ag (30) gel were decreased by nearly three orders of magnitude compared to the S. aureus only group. The in vivo studies suggested that Dex-G5-Ag (30) gel can effectively prevent local S. aureus infections, and its in vivo bactericidal efficacy is much superior to the commercial Ag hydrogel.

Figure 6. In vivo antibacterial activity of Dex-G5-Ag (30) hydrogel against S. aureus infection. (a) Images of mice treated with Dex-G5-Ag (30) gel and S. aureus, Dex-G5 gel and S. aureus, a commercial Ag gel and S. aureus, S. aureus only, and without any treatment, respectively. (b) Histological examination of S. aureus at the infection site. The tissues were stained by H&E staining and the S. aureus by Gram staining. The blue color indicates S. aureus infection. (c) S. aureus survival (CFU/mL) at the infected site. Scale bar, 200 µm.

4. CONCLUSIONS In summary, we reported a Dex-G5-Ag hydrogel with potent and broad-spectrum antibacterial activities. The gel showed pH-responsive release and degradation behaviors, limited hemolytic


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activity, cytotoxicity, tissue toxicity, and biochemical toxicity. It could be coated on substrates such as glass and polystyrene to act as bactericidal coatings, and efficiently prevented S. aureus infection in a murine infection model. The antibacterial hydrogel should be applicable for combating local infections associated with surgical wound and implanted medical devices.

ASSOCIATED CONTENT The Supplementary data is available free of charge on the ACS Publications via the Internet at http://pubs.acs.org. The scheme of G5-Ag and Dex-CHO synthesis, the scheme of Schiff base linkage, characterization of Dex-G5-Ag (30) hydrogel, Dex-G5-Ag (20) hydrogel and Dex-G5Ag (40) hydrogel, thixotropic property of Dex-G5 and Dex-G5-Ag hydrogel, pH-dependent release of Ag, inhibition zone study of gels, and live-dead staining assay of gels, images of treated mice, MIC and HC10 of G5 and G5-Ag, and elements of the surface of the uncoated and Dex-G5-Ag (30) gel coated glass slides. AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected] *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS


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The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 21404020) and the Shanghai Municipal Science and Technology Commission (17XD1401600) on this work.

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Table of Contents


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A Nanocomposite Hydrogel with Potent and Broad-Spectrum

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