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In Situ Forming and H2O2-Releasing Hydrogels for Treatment of Drug-Resistant Bacterial Infections Yunki Lee, Kyong-Hoon Choi, Kyung Min Park, Jong-Min Lee, Bong Joo Park, and Ki Dong Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 6, 2017

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ACS Applied Materials & Interfaces

In Situ Forming and H2O2-Releasing Hydrogels for Treatment of DrugResistant Bacterial Infections

Yunki Lee1,†, Kyong-Hoon Choi2,†, Kyung Min Park3, Jong-Min Lee4, Bong Joo Park2,*, and Ki Dong Park1,*

1

Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea

2

Department of Electrical and Biological Physics, Kwangwoon University, Seoul 138-701, Republic of

Korea 3

Division of Bioengineering, College of Life Sciences and Bioengineering, Incheon National University,

Incheon 22012, Republic of Korea 4

†

College of Medicine, Dongguk University, Goyang 10326, Republic of Korea

These authors contributed equally to this work.

*Correspondence to: K.D. Park, Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea, Tel.: +82 31-219-1846, E-mail: [email protected] B.J. Park, Department of Electrical and Biological Physics, Kwangwoon University, Seoul 138-701, Republic of Korea, Tel.: +82 2-940-8629, E-mail: [email protected]

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Abstract Various types of commercialized wound dressings (e.g., films, foams, gels, and nanofiber meshes) have been clinically used as a physical barrier against bacterial invasion and as wound-healing materials. Although these dressings can protect the wounded tissue from the external environment, they cannot treat the wounds that are already infected with bacteria. Herein, we report in situ H2O2-releasing hydrogels as an active wound dressing with antibacterial properties for treatment of drug-resistant bacterial infection. In this study, H2O2 was used for two major purposes: (1) in situ gel formation via a horseradish peroxidase (HRP)/H2O2-triggered cross-linking reaction, and (2) antibacterial activity of the hydrogel via its oxidative effects. We found that there were residual H2O2 in the matrix after in situ HRP-catalyzed gelling, and varying the feed amount of H2O2 (1−10 mM; used to make hydrogels) enabled control of H2O2 release kinetics within a range of 2−509 µM. In addition, although the gelatin-hydroxyphenyl propionic acid (GH) gel called “GH 10” (showing the greatest H2O2 release, 509 µM) slightly decreased cell viability (to 82−84%) of keratinocyte (HaCaT) and fibroblast (L-929) cells in in vitro assays, none of the hydrogels showed significant cytotoxicity toward tissues in in vivo skin irritation tests. When the H2O2-releasing hydrogels that promote in vivo wound healing, were applied to various bacterial strains in vitro and ex vivo, they showed strong killing efficiency toward gram-positive bacteria including Staphylococcus aureus, S. epidermidis, and clinical isolate of methicillin-resistant S. aureus (MRSA, drug-resistant bacteria), where the antimicrobial effect was dependent on the concentration of the H2O2 released. The present study suggests that our hydrogels have great potential as an injectable/sprayable antimicrobial dressing with biocompatibility and antibacterial activity against drug-resistant bacteria including MRSA for wound and infection treatment.

Keywords: In situ forming hydrogels; Hydrogen peroxide; Drug-resistant bacteria; HRP-catalyzed crosslinking; Wound dressing material


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1. Introduction Polymeric hydrogels have been proposed as a promising wound dressing material to facilitate wound healing because they provide a moist environment, absorb wound exudates, and protect wounds against external infection.1,2 These hydrogel dressings can be applied to an injured tissue either as a preformed type or as a liquid type that is cross-linked after injection. Particularly, in situ forming hydrogels have been widely utilized due to their unique advantages [over traditional wound dressings (e.g., bandages and gauze) and preformed semisolid gels], such as good tissue integration via accommodation of the substrate interface with irregular defect size, ease of application, high patient compliance, and comfort.3-5 Moreover, hydrogel materials can serve as an implantable platform for the delivery of bioactive agents with a controlled and continuous release, which provides an advanced functionality to current passive dressings for wound treatment.6 Thus, in situ forming hydrogels can be designed as an active antimicrobial dressing by incorporating antibacterial drugs, polymer/peptides, or metal nanoparticles. Drug-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA) are pathogens that predominantly cause a variety of serious healthcare-related infections.7,8 Such infections are readily acquired during a stay in a hospital or at other healthcare facilities. If the bacteria invade a wound during the wound-healing process, they can cause severe wound infection. Eventually, these wound infections may prolong or impair wound healing; this problem leads to significant morbidity, mortality, and increased hospital costs.9,10 So far, various types of commercialized wound dressings (e.g., films, foams, gels, and nanofiber meshes) have been clinically used as a physical barrier against bacterial invasion as well as wound-healing materials.11 Although these dressings can protect the wounds from the external environment, they cannot treat the wounds that are already infected with bacteria. Traditionally, as the most common approach to prevent and suppress microbial contamination, silver (Ag) particles have been incorporated into dressing materials, but there are still some hurdles that need to be overcome for successful clinical application.6 These problems include potential detrimental effect of Ag particles on mammalian cells and tissues, and difficulty with ensuring the desired Ag release kinetics because of their selfaggregation.5,12 Therefore, the antibacterial wound dressing with biocompatibility and controllable release kinetics of the loaded therapeutic antibiotics is required in the clinic. 3 Environment ACS Paragon Plus


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Recently, many researchers have studied reactive oxygen species [ROS; e.g., superoxide (O2-), 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

hydrogen peroxide (H2O2), hydroxyl radical (OH), and peroxynitrite (ONOO−)] for their versatile therapeutic applications such as angiogenesis13-15, wound healing16,17, anticancer effects18,19, and bacterialinfection treatment20,21. It has been demonstrated that adequate ROS concentration determines the signaling activity in a relevant metabolic pathway.22 Although ROS function as a part of normal cellular metabolism at low levels, an excessive amount of ROS induce oxidative stress, and damage critical components of cells. Indeed, it is well known that ROS act as a signaling molecule exerting antimicrobial activity in host defense mechanism against pathogens.23,24 Among various ROS, H2O2 has been recognized as a biocide for sterilization, disinfection, and antisepsis in the medical field. In addition, the US Food and Drug Administration (FDA) approved its use at concentrations of up to 3% (980 mM).25,26 Therefore, H2O2releasing biomaterials or wound dressings have been developed to provide antibacterial properties by simply loading H2O2 or by exploiting H2O2-generating reactions.27-29 As an antimicrobial mechanism, previous studies demonstrated that the released or generated H2O2 from biomaterials destroyed the outer membrane of bacterial cells, thereby causing cell death. We have previously reported a horseradish peroxidase (HRP)-catalyzed cross-linking strategy to fabricate in situ forming hydrogels for tissue engineering and regenerative medicine applications: phenolrich polymers undergo in situ cross-linking in the presence of HRP and H2O2.30,31 This gelling system enables easy control of the desired hydrogel properties such as cross-linking density and rate.32,33 In this system, H2O2 plays a critical role as a cross-linking agent where H2O2 is decomposed by HRP for in situ gel formation, and then is converted into water molecules. In the present study, we sought to extend this strategy with the goal of imparting antibacterial properties to hydrogels. We hypothesized that 1) the H2O2 would remain after HRP-catalyzed cross-linking reaction for in situ gelling, and 2) this residual H2O2 in a hydrogel matrix would act as an antimicrobial reagent via its oxidative stress. Herein, we demonstrate injectable/sprayable hydrogels that have intrinsic antibacterial activity via the residual H2O2 release (without additional drugs or antibiotics) for wound infection treatment. We analyzed the H2O2 release kinetics from gelatin-hydroxyphenyl propionic acid (GH) hydrogels formed at various concentrations of H2O2, and then evaluated in vitro and ex vivo antibacterial activities against various bacterial strains including clinical 4 Environment ACS Paragon Plus

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isolates of drug-resistant strains. Furthermore, we also confirmed in vivo wound healing efficacy of these 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

GH hydrogels.

2. Materials and Methods 2.1. Materials Gelatin (>300 Bloom, type A from porcine skin), 3-(4-hydroxyphenyl)propionic acid (HPA), 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), peroxidase from horseradish (HRP, type VI, 250–330 units per milligram of solid substance) and hydrogen peroxide (H2O2, 30 wt% in H2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethylformamide (DMF) was obtained from Junsei (Tokyo, Japan). Dulbecco’s phosphate-buffered saline (DPBS) was purchased from Gibco BRL (Grand Island, NY, USA). All reagents used in this study were of analytical grade and acquired from commercial sources without further purification. All solutions were prepared in DPBS. To confirm the antibacterial activity of the GH hydrogels, we used seven strains of bacteria, including drug-resistant bacteria: Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 9027, and S. aureus ATCC 14458 were acquired from the American Type Culture Collection (ATCC, Rockville, MD, USA), and methicillin-resistant S. aureus (MRSA KCCM 40510) and S. epidermidis KCCM 35494 were purchased from the Korean Culture Center of Microorganisms (KCCM, Sedaemun-Gu, Seoul, Korea). MRSA-1 isolated from patients at the Yonsei Medical Center in Seoul and MRSA-2 isolated at Korea University Anam Hospital in Seoul, were kindly provided by each hospital. All the strains were stored frozen at −70 °C until the experiments. Gram-negative and gram-positive bacteria, E. coli (ATCC 11775) and S. aureus (ATCC 14458), respectively, were grown on plate count agar (PCA, Becton, Dickinson and Company, Sparks, MD, USA) at 35 °C for 24 h. Drug-resistant bacteria (MRSA) were grown on Brain Heart Infusion agar (BHIA, Becton, Dickinson and Company) at 35 °C for 24 h. All the bacterial strains were passaged twice at 48-h intervals before use.



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2.2. Synthesis of GH conjugates 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The GH polymer was synthesized as previously described.34 Briefly, HPA was activated with EDC and NHS as the coupling reagents in water as a cosolvent of DMF (volume ratio = 3:2), and the solution was then applied to a gelatin solution preheated at 40 °C. After a 24-h reaction, the resulting solution was subjected to sequential dialysis against deionized water. The purified solution was filtered and lyophilized to obtain a GH polymer (phenol content = 140 µM per gram of GH polymer). The degree of substitution (DS) of HPA was measured using a UV/VIS spectrophotometer (V-750, Jasco, Japan).

2.3. Preparation of H2O2-releasing GH hydrogels These hydrogels were prepared by mixing two GH solutions (A and B) with either HRP or H2O2. Briefly, HRP (0.02 mg/mL) was dissolved in DPBS, followed by mixing with 5.56 wt% GH (volume ratio GH:HRP = 9:1) to prepare solution A. Solution B was prepared by mixing 5.56 wt% GH and H2O2 (20−1000 mM; volume ratio GH:H2O2 = 9:1). To prepare 5 wt% GH hydrogels, solutions (A and B) were mixed in a microtube at room temperature and gently shaken. The detailed final conditions for the preparation of GH hydrogels are given in Table S1.

2.4. In vitro H2O2 release kinetics from GH hydrogels To analyze the release kinetics of H2O2 remaining in GH hydrogels, the amounts of H2O2 released from the GH hydrogels were quantified by a spectrophotometric method using copper (II) ions and 2,9dimethyl-1,10-phenanthroline (DMP). This protocol is based on a method reported previously.35 The principle of this method is the reduction of Cu(II) ions to Cu(I) by H2O2, yielding a Cu(I)-DMP complex. Under the conditions used, the stoichiometry is given by 2Cu2+ + 4DMP + H2O2 → 2Cu(DMP)2+ + O2 + 2H+ The product, Cu(DMP)2+, is a light-yellow complex that shows maximum absorbance at 454 nm. Furthermore, this product is stable in air- or oxygen-saturated solutions and possesses a molar extinction 6 Environment ACS Paragon Plus

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coefficient (ε) of 15 × 103 L⋅mol-1⋅cm-1.36 Therefore, it is conveniently measured spectrophotometrically by means of its visible absorption band. First, stock solutions, i.e., 10 mL each of 1% DMP in methanol, 0.01 M copper (II) sulfate, or phosphate buffer (pH 7.4), were prepared. To explore the kinetics of the release of H2O2 from the GH hydrogel matrix, 100 µL of a GH hydrogel was prepared in a 48-well plate and incubated with 900 µL of deionized water. At predetermined time points, 50 µL of each medium was collected into a 96-well plate. An aliquot of 50 µL of each stock solution was added to the collected media. After mixing, the absorbance of the sample at 454 nm was measured on a multimode microplate reader (Synergy HT, BioTek Instruments, Inc., VT, USA). Using the difference in absorbance between the sample and blank solutions, a calibration curve was generated.

2.5. In vitro and in vivo biocompatibility assays We performed two kinds of assays such as an in vitro cytotoxicity test and an in vivo skin irritation test to evaluate the biocompatibility of the H2O2-releasing GH hydrogels. To assess in vitro biocompatibility, cytotoxicity tests were performed on mouse fibroblasts (L-929 cells) and human skin keratinocytes (HaCaT cells), as previously described.37,38 To visualize the cellular morphology, the cells were transfected with green fluorescent protein (GFP) as follows; the coding region of copGFP was amplified by PCR from pCDH-EF1-copGFP (SBI: CD511B-1). A 759 bp coding sequence region of copGFP was amplified by using the sense primer 5’-TCTAGAGCCACCATGGA GAGCGACGAGAGCG-3’ and the antisense primer 5’-GAATTCTTAGCGAGATCCGGTGGAG-3’. The amplified copGFP sequence was then inserted into the pGEM-T-Easy vector (Promega) to yield pGEM-TcopGFP and the sequence was verified by DNA sequencing. Then, XbaI-EcoRI fragment containing copGFP ORF was ligated into multi-cloning sites (MCS) of the pCDH-EF1-Puro (SBI: CD510B-1) to yield pCDH-CMV-copGFP-EF1-Puro (Figure S1). For lentivirus production, 293FT cells were co-transfected with the pCDH-CMV-copGFP-EF1-Puro, and lentiviral packaging mixture (pLP1, pLP2 and pLP/VSVG, Invitrogen). After 48 h, viral supernatants were collected and stored at -80 °C for further use. L-929 and HaCaT cells were seeded onto a 6-well plate was transduced with lenti-copGFP supernatant for 12 h in the 7 Environment ACS Paragon Plus


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presence of 8 µg/mL of polybrene at a multiplicity of infection (MOI) of 40. When confluence of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

lentiviral transduced L-929 and HaCaT cells was attained around 90%, the cells were treated with 4 µg/mL puromycin for 24 h. The puromycin resistant cells were maintained in complete medium containing 1 µg/ml puromycin until confluence of the cells was attained again around 90%. Briefly, GFP-transfected HaCaT and L-929 cells were cultured with high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of FBS and 1% of the antibiotic/antimycotic solution. All cells were maintained at 37 °C in a humidified atmosphere (5% of CO2 and 95% of air). The two types of pre-cultured cells were seeded in 24-well plates (Coster Corp., MA, USA) at 2.0 × 105 cells/well, and the plates were incubated at 37 °C in an atmosphere containing 5% of CO2 for 24 h in order to obtain confluent monolayers of the cells prior to use. All the cells were treated with four kinds of GH hydrogels (GH 1, GH 2.5, GH 5, or GH 10), and incubated at 37 °C in an atmosphere containing 5% of CO2 for 24 h. The GH hydrogels were removed from each well of the plate, and the cells were incubated with a Cell Counting Kit-8 (CCK-8) solution (Dojindo Laboratories, Kumamoto, Japan) for 30 min after threetimes washing with DPBS. Cell viability on each GH hydrogel was calculated by measuring the absorbance of each well at 450 nm on a microplate reader (Synergy HT; BioTek Instruments, Inc., VT, USA). The cell viability was expressed as the percentage of surviving cells in relation to the number of control cells. The morphology of cells treated with GH gels for 24 h was also imaged using an imaging reader (Cytation 3; BioTek Instruments, Inc., VT, USA). To confirm biocompatibility in vivo, a skin irritation test was performed on hairless mice using a modified version of the method described in ISO 10993-10.39 We used female hairless mice, SKH-1 (Charles River Corp. Inc., Barcelona, Spain) weighing 20–25 g. Twenty healthy hairless mice were obtained from an approved supplier, traceable using the records available at the Kwangwoon University. The mice were acclimated to the animal laboratory for 1 week and were kept in individual cages. All the experiments were conducted according to the Kwangwoon University Institutional Animal Care and Use Committee directives. SKH-1 mice were randomly subdivided into five groups. Group 1 served as a control and groups 2 to 5 were treated with various GH hydrogels (GH 1, GH 2.5, GH 5, or GH 10). There were four mice in each group. Mice in group 1 were patched with wet filter paper soaked in DPBS as a control, and each 8 Environment ACS Paragon Plus

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mouse in the other groups was topically patched with GH hydrogels (100 µL) on the dorsal skin, as shown in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2c. Signs of edema (ED) or erythema (ER) were evaluated at 24, 48, and 72 h after the hydrogel application, the primary dermal irritation index (PDII) was recorded based upon dermal scores, and any responses at the test sites were also noted according to the criteria of ISO 10993-10. Moreover, histological changes in the skin caused by the GH hydrogels were examined after staining with hematoxylin and eosin (H&E).

2.6. Assessment of in vitro antibacterial activity of the GH hydrogels To confirm the antibacterial activity of our GH hydrogels, we used two methods: a quantitative method (bacterial cell counting) and a qualitative method, in which zones of inhibition were measured. In the quantitative method, bacterial cells from each bacterial colony were resuspended at ~106–107 colony forming units (CFU)/mL in nutrient broth for E. coli and S. aureus or BHI broth for drug-resistant bacteria. Bacterial cells were diluted 10-fold to ~105–106 CFU/mL and inoculated into 24-well plates, then incubated with various GH hydrogels (GH 1, GH 2.5, GH 5, or GH 10) at 35 °C for 24 h. After that, the bacterial cells in each well were incubated for additional 24 h after serial 10-fold dilutions (10 to 107) and were inoculated into PCA or BHIA. Cell viability was determined by counting CFU, and the antibacterial activity was determined by plotting the total number of viable bacterial cells as CFU/mL versus the types of hydrogel. Antibacterial activity was defined as a >3 log decrease in CFU/mL. As for the qualitative method, the antibacterial activity of the GH hydrogels was evaluated using a inhibition zone assay based on a modified disk diffusion method from the National Committee for Clinical Laboratory Standards (NCCLS 2003a), protocol M2-A8. Each bacterial suspension was prepared by direct resuspension of each bacterial colony in sterilized 0.85% saline solution, and the optical density was adjusted to 0.5 McFarland turbidity standards (~107 CFU/mL). Each bacterial suspension was applied to the agar plates along with each GH hydrogel. After incubation at 35 °C for 24 or 48 h, the zone of inhibition was measured by subtracting the diameter of each hydrogel from the diameter of the total inhibition zone.



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To confirm the antibacterial activity of the GH hydrogels by microscopic examination of bacterial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

morphological features, each bacterial strain was resuspended in normal saline, bacterial cells at ~106–107 CFU/mL were inoculated onto sterilized cover glasses coated with poly-L-lysine in 24-well plates, and incubated for 1 h to allow the cells to adhere to the cover glasses. Bacterial cells still in suspension were discarded after this incubation, and the cover glasses in each well were gently rinsed three times with sterilized saline to remove the unattached bacterial cells. Bacterial cells on the cover glasses were incubated with GH 10 hydrogel for 1, 2, and 4 h. Live and dead bacterial cells on the cover glass were counted by means of the Live/Dead BacLight Bacterial Viability Kit (Molecular Probes, Oregon, USA). The live and dead bacterial cells were also analyzed under a laser scanning confocal fluorescence microscope (FV-1200, Olympus, Tokyo, Japan) with 10× and 20× objective lenses and fluorescence optics (excitation at 485 nm for SYTO 9 and propidium iodide [PI] and emission at 530 nm for SYTO 9 and 630 nm for PI). Images of the live and dead bacterial cells were analyzed using imaging software (Imaris, Bitplane, Concord, MA, USA).

2.7. Assessment of in vivo wound healing and ex vivo antibacterial activity of the GH hydrogels The wound-healing efficacy of H2O2-releasing GH hydrogels was assessed with mice (male, SKH-1 hairless weighing 20−25 g). Sixteen healthy mice were obtained from an approved supplier. The mice were acclimated to the animal laboratory for one week, and then were randomly divided into four groups. Group I and II were served as normal and negative control (PBS), and Group III and IV were treated with GH 5 and 10 hydrogels. Four mice were used for each group, and they were kept in individual cages. Wounds of Group II, III, and IV were created on the dorsal surface as previously described.40-42 Briefly, the skin of each mouse was disinfected with povidone-iodine followed by a rinse with 70% ethanol after anesthesia with isoflurane, and the skin was punched with a biopsy punch (8 mm in diameter) to create full-thickness excisional wounds besides the midline. After creating the wounds, each surrounding wound was splinted by a silicon ring (12/20 mm in internal/external diameter) to prevent self-contraction of the skin. Each hydrogel (100 µL of GH 5 and GH 10) was placed into wound region, and the wounds were covered with a transparent oxygen permeable wound dressing (Tegaderm film, 3M, USA). The dressing in each wound site 10 Environment ACS Paragon Plus

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was changed every other day. The mice of negative control (Group II) were treated with PBS in the same 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

way. Wound closures in each mouse were observed using a stereo microscope (SZX16, Olympus, Japan) every other day, and histological changes were examined after 12 days by staining with hematoxylin and eosin (H&E) to evaluate the wound-healing effect of hydrogels. For ex vivo antibacterial activity of the hydrogels, skin infection experiment was conducted using a modified skin infection method.43 The skin of SKH-1 hairless mouse was disinfected with povidone-iodine and 70% ethanol after anesthesia, and the full-thickness skin was taken out from the mouse dorsal skin by punched with a biopsy punch (12 mm in diameter). The surface of each skin taken was slightly scratched out with a knife, and was placed onto DMEM agar media to keep the cell viability in each skin tissue. Bacterial cells at ~105–106 CFU/mL were inoculated onto each skin surface placed on DMEM agar media in 12-well plates, and incubated for 10 min to allow the bacterial cells to adhere to the skin surface. After that, the hydrogels were treated on the skin surface, and incubated at 35 °C for 24 h. After that, the bacterial cells in each skin sample were incubated for additional 24 h after serial 10-fold dilutions, and were inoculated into PCA or BHIA. Bacterial cell viability in skin samples was determined by counting CFU, and the antibacterial activity was determined as described above.

2.8. H2O2-scavenging activity of bacterial lipopolysaccharide (LPS) To explain the selective antibacterial activity of GH hydrogels against gram-positive bacteria, we evaluated the H2O2-scavenging activity of LPS isolated from gram-negative cell walls at different H2O2 concentrations (100 and 200 µM) using the spectrophotometric method described above. 500 µL of H2O2 solutions were mixed with 500 µL of an LPS solution (8, 12, 16, 20, or 24 µg/mL). The mixture was then incubated with vigorous stirring at room temperature for 2 h. Next, the H2O2-scavenging activity of each solution was estimated using the H2O2 detection method. The LPS treatments at concentrations above 30 µg/mL were excluded as they affect the H2O2 quantification assay (data not shown).

2.9. Statistical analysis



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Quantitative data are expressed as mean ± standard deviation (SD). Statistical comparisons involved 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Student’s t-test, and a difference with p < 0.05 was considered statistically significant.

3. Results and Discussion 3.1. Characterization of the H2O2 release from HRP/H2O2-based in situ forming hydrogels We previously reported that in situ cross-linkable GH hydrogels formed via HRP-catalyzed crosslinking show a great potential for wound healing applications, particularly for the development of wound dressing materials with suitable tissue-adhesive properties.4,34 It was demonstrated that these hydrogels could serve as an implantable platform to deliver protein drugs to the wound during a desired time period by controlling the cross-linking density of the hydrogel network.44,45 In addition, based on HRP/H2O2 gelling system, Wang et al. reported the epsilon-poly-L-lysine hydrogels with inherent antibacterial activity for wound infection treatment.46 Unlike their strategy using this specific polymer with intrinsic antibacterial property, we sought to verify that the residual H2O2 within injectable/sprayable hydrogel dressings after the HRP-mediated cross-linking reaction could act as an antibacterial agent for prevention or treatment of bacterial infections (Figure 1a and b). To characterize the in vitro release kinetics of the residual H2O2 after gel formation from GH hydrogels formed at different concentrations of H2O2, the released amounts were quantified as a function of time using a spectrophotometric method. As shown in Table S1, the feed amount of H2O2 was varied from 1 to 50 mM, while maintaining both GH polymer (5 wt%) and HRP (0.001 mg/mL) concentrations consistent. Figure 1c shows the H2O2 concentrations released from GH hydrogels into the medium. At the initial time point (< 1 h), the residual H2O2 was rapidly released from the gel matrix, and the H2O2 amount was strongly dependent on the feed concentration of H2O2 used to fabricate the hydrogel. Specifically, GH 1 and GH 2.5 hydrogels showed a small H2O2 release (< 100 µM) during 20 h, whereas 300−509 µM H2O2 was released from the GH 5 and GH 10 hydrogels. In addition, the release patterns were different between GH 1 and GH 2.5 hydrogels after 3 h. As shown in Figure S2 and Table S1, we characterized the time-course hydrogel formation by varying the H2O2 concentrations (1−50 mM). In this gelling system, the interaction between 12 Environment ACS Paragon Plus

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HRP and H2O2 turns phenol molecules into phenoxy radicals, and results in di-phenol coupling for in situ gel formation, where H2O2 is primarily used to manipulate the cross-linking density of the hydrogel network.34,47,48 Thus, an increase in the H2O2 concentration led to higher mechanical stiffness in a certain range of H2O2 (1−2.5 mM). However, further increase in the H2O2 concentration caused a decrease in both the elastic modulus and cross-linking efficiency due to HRP inactivation by excess H2O2.49,50 On the basis of this result, we infer that the difference of released H2O2 concentration between GH 1 and GH 2.5 is due to greater H2O2 consumption by the HRP activity during hydrogel formation with greater mechanical strength. Taken together, our results suggest that there are residual H2O2 in the hydrogel matrix after in situ gel formation via the HRP-catalyzed cross-linking reaction, and the H2O2 release kinetics can be controlled by means of the feed amounts of H2O2.



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Figure 1. Schematic illustration of injectable/sprayable antimicrobial hydrogels formed via HRP/H2O2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cross-linking reaction and in vitro H2O2 release behavior: (a) H2O2 was used both for in situ hydrogel formation and for creation of the antibacterial effects through the residual H2O2 release after the crosslinking reaction. (b) Digital images of injectable/sprayable GH hydrogel dressings, and (c) release kinetics of residual H2O2 from the hydrogel matrix formed at different H2O2 concentrations (1–10 mM; n = 3).

3.2. Biocompatibility of GH hydrogels releasing residual H2O2 It is necessary to assess the safety and the potential of GH hydrogels for clinical applications. To confirm the biocompatibility of our GH hydrogels, we performed a cytotoxicity test on HaCaT and L-929 cells as previously reported.30-34 As shown in Figure 2a and b, it was found that there was no significant cytotoxicity of the hydrogels toward L-929 and HaCaT cells, and no changes in cellular morphology after treating GH gels, compared to control cells. Under most gel conditions, viability of HaCaT and L-929 cells was >80%; the GH 1 gel slightly increased viability of HaCaT cells by approximately 4%. However, GH 10 hydrogels decreased the viability of HaCaT (to 84%) and L-929 (to 82%) because of relatively high level of oxidative stress caused by the released H2O2 (509 µM). We next carried out the skin irritation assay on the skin of hairless mice. As shown in Table S2, none of the GH hydrogels triggered skin irritation at any patched site, nor did they induce any intracutaneous irritation. No symptoms of cytotoxicity toward skin tissues or abnormal behaviors, such as convulsions or prostration, were observed even after treatment with GH 10 hydrogels (Figure 2c and d). We believe that the relatively milder cytotoxic effect of the GH 10 gel in in vivo environment than in vitro is may be due to enzyme activities in surrounding tissues that catalyze decomposition of ROS including H2O2. As a result, we demonstrated that the H2O2 released from GH hydrogels are barely toxic to mammalian cells and skin, and these hydrogels are biocompatible for use as a wound dressing material.


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Figure 2. (a) photographs of copGFP-transfected HaCaT and L-929 cells incubated with GH hydrogels. The pictures are magnified from the inserted images in each picture, which is a cell image taken by 4 x lens. Scale bars represent 100 µm. (b) Cytotoxicity of H2O2-releasing GH hydrogels toward mouse fibroblasts (L929 cells) and human keratinocytes (HaCaT cells) (n = 4). (c) Photographs of the skin irritation test results using GH 10, and (d) images of representative histological slices of mouse skin after staining with hematoxylin and eosin (H&E). Scale bars are 100 µm.

3.3. In vitro antibacterial activities of the hydrogel dressings Hydrogels are attractive biomaterials for use as wound dressing materials because they provide a moist and occlusive environment at a wound site, facilitating cellular activities essential to the wound healing process. However, it was also reported that the hydrated conditions can induce bacterial infection.51 Accordingly, in addition to the primary functional role of hydrogels, their antibacterial effects are desirable. In this study, we sought to verify that the released H2O2 from hydrogels after in situ gelling have enough inhibitory effect on bacterial growth by measuring the diameter of the total inhibition zone and the total number of viable bacterial cells in various bacterial strains (gram-negative and gram-positive bacteria 15 Environment ACS Paragon Plus


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including clinical MRSA isolates). Figure 3a and Table 1 show the growth inhibition zones of bacteria after 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

24 and 48 h of incubation with GH hydrogels releasing different H2O2 concentrations. If the concentration of H2O2 is enough to kill bacteria (above minimum inhibitory concentration; MIC), growth inhibition zone will be formed and maintained around gel disk. In addition, this zone size depends on how effective the H2O2 is for stopping the growth of bacteria where a stronger antibacterial property will create a lager zone. It was found that GH hydrogels with a greater released amount of H2O2 had a stronger antibacterial effect. Although GH 1 and GH 2.5 hydrogels releasing H2O2 at < 100 µM had no inhibitory effect on bacterial growth (n/d; not determined), GH 5 (< 1.80 ± 0.11 mm) and GH 10 (< 8.87 ± 0.55 mm) hydrogels releasing H2O2 at 300−509 µM had antibacterial activity against gram-positive bacteria (S. aureus, S. epidermidis, and MRSA) (Table 1). Particularly, the GH 10 hydrogel with the largest H2O2 release amount showed wellpronounced growth inhibition zones (Figure 3a). In addition, when we further assessed their antibacterial properties by a quantitative method that involves counting the number of CFU, GH 10 hydrogel showed significant killing efficiency toward S. aureus as well as clinical isolates of MRSA, which is a similar trend with the one seen for the H2O2 concentration-dependent antibacterial effect in the qualitative analysis (Figure 3b). The strong antibacterial activity of GH 10 gels was also confirmed in Live/Dead staining images. There were the greatest number of dead cells (red color) of gram-positive bacteria (S. aureus and MRSA) within 4 h (Figure 3c). Nonetheless, the inhibitory effect on gram-negative bacteria such as E. coli was not observed even when GH 10 hydrogel was used.


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Figure 3. (a) Photographs of the bacterial growth inhibition zone after incubation of H2O2-releasing GH hydrogels for 24 and 48 h, and (b) the total number of viable bacterial cells (gram-negative and grampositive bacteria including clinical isolates of drug-resistant strains; n = 4) (*P < 0.05, **P < 0.005 compared to the control). (c) Live/Dead staining images of bacteria (E. coli, S. aureus, methicillin-resistant S. aureus [MRSA]) after incubation of the GH 10 hydrogel. Scale bars are 50 µm.

Table 1. Zone-of-inhibition diameters for interpretation of the susceptibility of bacteria to H2O2-releasing GH hydrogels. Zone-of-inhibition diameter (mm) Microorganism

E. coli (ATCC11775)

GH 1

GH 2.5

GH 5

GH 10

n/d*

n/d

n/d

n/d



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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P. aeruginosa (ATCC9027)

n/d

n/d

n/d

1.02 ± 0.20

S. aureus (ATCC14458)

n/d

n/d

n/d

5.87 ± 0.52

S. epidermidis (KCCM35494)

n/d

n/d

1.20 ± 0.15

6.27 ± 0.29

n/d

n/d

1.65 ± 0.21

8.87 ± 0.55

n/d

n/d

1.80 ± 0.11

8.13 ± 0.36

MRSA-1 (Clinical isolate) MRSA-2 (Clinical isolate) * n/d = not determined

3.4. In vivo wound healing and ex vivo antibacterial activities of the hydrogels To assess in vivo wound-healing efficacy of the hydrogels, full-thickness excisional wounds were created on the back of mice, and samples (PBS, GH 5 and GH 10) were then applied on the top of the wound site. Figure 4a shows images of skin wounds taken at different time intervals after treating with PBS or GH hydrogels. The wound beds in GH hydrogel-treated groups were fully recovered without skin contraction over 12 days of implantation, whereas the mouse skin in negative control group had a scab in the wound site. In addition, GH 10 gels exhibited the significantly accelerated wound closure at day 6 and 9 compared to the PBS control (Figure 4b). In comparison with control group (72% and 84%), the wound closure rate of GH 10 hydrogel reached about 83% and 95%, respectively (*P < 0.05). This result might be due to the enhanced re-epithelization; 1) by the moist environment of the hydrogel dressing, and 2) by the released H2O2 from hydrogels. It is well known that the moisture-retentive wound dressings effectively promote cell proliferation and migration, thereby facilitating wound healing.52,53 Additionally, we believe that the released H2O2 provided beneficial effect on the cellular activities for wound healing. It can be supported by several previous studies reporting that H2O2 at such concentrations (250−500 µM) resulted in enhanced cellular growth and migration in a keratinocyte scratch wound model.17,54 The skin slides were also stained with H&E for the histological analysis (Figure 4c). In histological images of the skin treated with GH 5 or GH 10 hydrogels, the re-epithelialization with thick epidermis and fibroblast proliferation with skin adnexa and dense dermal fiber (blue area) were observed. However, the negative control showed slightly thin epidermis, and relatively less dermal fibroblast proliferation and 18 Environment ACS Paragon Plus

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collagen deposition, compared to GH hydrogel-treated groups. Thus, the obtained results indicate that the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

moist environment and the released H2O2 from GH hydrogel dressing can accelerate the wound healing with rapid re-epithelialization and increased skin repair. In ex vivo antibacterial experiment, GH 10 hydrogel showed significant killing efficiency on grampositive bacteria (S. aureus as well as clinical isolates of MRSA; Figure 4d), which is comparable to the results of in vitro antibacterial activity assessment. However, the ex vivo antibacterial efficiency of GH 5 and 10 hydrogels on gram-positive bacteria was less than that in in vitro environment. This result can be explained by the antioxidant activity of enzymes (e.g., catalase (CAT) and glutathione peroxidase) released from the damaged mouse skin cells. It is known that mammalian skin has a network of antioxidant enzymes.55 Thus, these enzymes can decompose H2O2 released from GH hydrogels, which was also observed in the in vivo skin irritation test. In case of gram-negative bacteria (E. coli), we could not observe any inhibitory effect of GH hydrogels.



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Figure 4. In vivo wound-healing effect in the full-thickness mouse wound model and ex vivo antibacterial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

activity of GH hydrogels. (a) Stereo microscopic images and (b) wound closure rate treated with PBS (negative control), GH 5 and GH 10 hydrogels over 12 days after post-wounding. Scale bars represent 2 mm. (n = 4, *P < 0.05 compared to the control) (c) Histological images of hematoxylin and eosin (H&E) staining of normal, negative control, and GH 5 and GH 10 hydrogel-treated skin. The representative images of H&E stained histological sections at day 12 after post-wounding. Blue area and red arrows mean adnexa in dermis of skin and repaired skin area, respectively. Scale bars represent 500 µm. (d) Ex vivo antibacterial activity of GH hydrogels. The total number of viable bacterial cells after 24 h post treated GH hydrogels on the surface of scratched skin. (n = 4, *P < 0.05 compared to the control).

In in vitro and ex vivo experiments, we found that the antibacterial activity of the GH hydrogels was more effective against gram-positive bacteria than against gram-negative bacteria. These results may be explained by the antioxidant activities of LPS present on the surface of gram-negative bacteria. Grampositive and gram-negative bacteria have a different cell wall structure.56 The gram-negative bacteria have both inner and outer lipid membranes, while gram-positive bacteria have inner membrane only. The outer membrane of gram-negative bacteria invariably contains a unique component, LPS as well as proteins and phospholipids.57 According to recent researches, chemical structures such as polysaccharides and α-keto carboxylic acid that are similar with LPS, were found to have an antioxidant property against ROS. Their mechanisms include prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging.58-61 Therefore, it is expected that LPS in gram-negative bacteria act as a scavenger to oxidative stress of H2O2. To confirm the scavenging effect of LPS on H2O2, and to explain the selective activity of GH hydrogels against gram-positive bacteria, we confirmed the changes in H2O2 concentrations after incubation with various concentrations of LPS (0−12 µg/mL). As shown in Figure 5, whereas H2O2 levels were stable in a condition without LPS, the LPS treatment resulted in a gradual decrease in H2O2 levels with respect to the initial concentrations. The H2O2 (100 and 200 µM) were decomposed by 77.8−78.6% at 12 µg/mL of LPS.


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Therefore, this result supports that gram-negative bacteria are less susceptible to the H2O2 oxidative stress than gram-positive bacteria because of the antioxidant activities of LPS in the outer membrane.

Figure 5. Scavenging activity of lipopolysaccharide (LPS) isolated from gram-negative cell walls toward H2O2 (n = 4).

4. Conclusions Herein, we report in situ H2O2-releasing hydrogels as an active wound dressing with antibacterial properties for prevention or treatment of bacterial infections. In this study, H2O2 was used for two major objectives: (1) in situ gel formation through HRP/H2O2-triggered cross-linking reaction and (2) antibacterial activity of the hydrogel. As an injectable/sprayable wound dressing material, GH hydrogels were fabricated in situ through rapid cross-linking of GH polymers in the presence of HRP and H2O2. We found that there were residual H2O2 in the gel matrix after in situ gel formation, and varying the H2O2 feed amount (1−10 mM) enabled control of the later H2O2 release kinetics within a range of 2−509 µM. Although the GH 10 gel (the largest H2O2 release, 509 µM) showed slightly decreased cell viability (to 82−84% of the control) of L929 and HaCaT cells in in vitro assays, they did not cause significant cytotoxicity toward tissues in in vivo skin irritation tests. When the H2O2-releasing hydrogels were applied to various bacterial strains in vitro and ex vivo, they exhibited strong killing efficiency towards gram-positive bacteria including S. aureus, S. epidermidis, and MRSA (drug-resistant bacteria). Their antimicrobial effects depended on the concentration 21 Environment ACS Paragon Plus


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of H2O2 released from the hydrogels. Nevertheless, the antibacterial activity of H2O2-releasing hydrogels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was insufficient against gram-negative bacteria (e.g., E. coli) because of the antioxidant effected by LPS. In conclusion, these results suggest that our hydrogels, which show biocompatibility, enhanced wound-healing and antibacterial activities against drug-resistant bacteria such as MRSA, hold great promise as an injectable/sprayable antimicrobial dressing for wound and infection treatment.

Associated Content This document file contains Supporting Information.

Figure S1. Schematic illustration of lentiviral vector for expression of copGFP in keratinocyte (HaCaT cells) and fibroblast (L-929 cells). Figure S2. Gelation kinetics and elastic modulus of GH hydrogels formed by HRP-catalyzed cross-linking at various H2O2 concentrations (1−50 mM). Table S1. Preparation of GH hydrogels formed via an HRP/H2O2 cross-linking reaction. Table S2. The primary dermal irritation index (PDII) in the skin irritation test.

Author information Corresponding Author * Tel.: +82 31-219-1846, E-mail: [email protected] * Tel.: +82 2-940-8629, E-mail: [email protected] Author Contributions The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. †These authors contributed equally to this work. Notes The authors declare no competing financial interest. 22 Environment ACS Paragon Plus

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Acknowledgements This work was supported financially by a grant from the National Research Foundation of Korea (NRF-2015M3A9E2066855, NRF-2010-0027963, and NRF-2015R1A2A1A14027221) funded by the Korean government (MSIP), by the Ministry of Science, ICT & Future Planning, and by the Research Grant from Kwangwoon University in 2016.

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