N-halamine-Mediated Multifunctional Dressings as Quick

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Novel ZnO/N-halamine-Mediated Multi-functional Dressings as Quick Antibacterial Agent for Biomedical Applications Wei Ma, Lin Li, Xinghuan Lin, Yingfeng Wang, Xuehong Ren, and Tung-Shi Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10857 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 3, 2019

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Novel ZnO/N-halamine-Mediated Multi-functional Dressings as Quick Antibacterial Agent for Biomedical Applications Wei Ma,† Lin Li,† Xinghuan Lin,† Yingfeng Wang,† Xuehong Ren*,†, Tung-Shi Huang‡ †

Key Laboratory of Eco-textiles of Ministry of Education, College of Textiles and Clothing,

Jiangnan University, Wuxi, Jiangsu, 214122, China ‡ Department

of Poultry Science, Auburn University, Auburn, AL 36849, USA

ABSTRACT: Cutaneous hemorrhage often occurs in daily life which may cause infection and even amputation. This research aims to develop a novel chitosan dressings impregnated with ZnO/N-halamine hybrid nanoparticles for quick antibacterial performance, outstanding haemostatic potential, high porosity, and favorable swelling property through combining sonication and lyophilization processing. After 30 days of storage, about 90% bacterial cell viability loss could be observed towards both gram positive Staphylococcus aureus (S. aureus) and gram negative Escherichia coli O157:H7 (E. coli O157:H7) within 30 min of contact by colony counting method. The hybrids assembled much more platelet and red blood cell as compared with pure chitosan control. Moreover, the lower blooding clotting index value gave evidence that these composites could control hemorrhaging and reduce the probability of wound infection. No potential skin irritation and toxicity were detected using in vitro cytocompatibility and skin stimulation test. Therefore, this work demonstrated a facile and cost-effective approach for the preparation of N-halamine-based hybrid sponges which show promising application for wound dressings.


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KEYWORDS: ZnO, N-halamine, chitosan dressing, antibacterial, cytocompatibility INTRODUCTION Uncontrolled bleeding arising from medical trauma, traffic accidents, and war injuries poses serious threats to wound healing. A tremendous number of people suffer from wound infection and severe tissue necrosis each year which are considered as life-threatening. Accordingly, dressings functioning as temporary skin are needed to avoid the fluid and protein from seeping out of wound surface and keep away from bacterial contamination. Once contacted with wound, these strains grow quickly and seriously damage skin, causing inflammation or internal infection which is difficult to treat. It should be noted that wound dressings themselves can cause infection sometimes since they have many necessary properties such as gas exchange, wound exudate absorbing, biocompatibility, simultaneously. Thus, it is urgent to develop wound dressings with antimicrobial features. Of the various antimicrobial agents that are effectively applied in healthcare area, quaternary ammonium compounds,1 silver nanoparticles,2-4 biguanides,5-6 and zinc oxide7 are commonly used in wound dressing applications. Researchers had found that cationic quaternary ammonium compounds showed toxicity when they were presented as monomers.8 Another work reported that silver nanoparticles and biguanides were easily released from substrates, leading to bio-accumulation in the environment.9 Also, the limitations of using silver as antibacterial agent are skin staining, toxicity and lower sterilization efficiency.10 Zinc oxide nanoparticles (ZnO NPs) possess antibacterial activity and could be used in a wound healing area.11-13 During the releasing process of ZnO, the Zn ions could boost the migration of keratinocytes to the wound surface, promoting wound healing.14 However, the photo-dependent antibacterial property restricts its further application. Quaternary ammonium salts, CuO, as well as silver were


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designed to modify ZnO for antibacterial hemostatic dressings in order to enhance their antibacterial efficiencies.15-16 However, they only showed significant antibacterial activity when used in large amounts and their toxicity needed to be addressed.17 In addition, previous work has shown that some antimicrobials agents (e.g. Ag, CuO) had bacteria resistant issues.18 Therefore, it is necessary to develop “ideal” antibacterial agents for wound dressings so as to counter crossinfections. N-halamines have shown to be of great interest and been added to cellulose,19-20 paint,21 and applied for water disinfection,22 attributing to their effective antibacterial efficacy and nontoxicity. For N-halamine materials, size plays a crucial part in antimicrobial response, and attempts to prepare N-halamine based nanoparticles including N-halamine/SiO2,23 Nhalamine/polystyrene,24 and N-halamine/poly(methyl methacrylate) nanoparticles25 have been reported. Another approach of preparing nano-sized electrospinning fibers containing Nhalamine/silica,26 polyamide-based N-halamine,27 and N-halamine/poly(vinyl alcohol-coethylene),28 have been also studied. Additionally, it’s worth mentioning that N-halamines have never been reported to possess bacterial resistance,29 which could make N-halamines useful for biomedical applications. For instance, Malik et al.30 prepared a biodegradable antibacterial suture via electrostatic assembly, that showed high performance in regard to biocidal activity and biocompatibility. Buket et al.31 anchored different N-halamine precursors onto primary Aid® brand dressing material and investigated their antibacterial efficiencies, revealing that these Nhalamine-based materials could inactivate around 6-7 logs of S. aureus and P. aeruginosa within 15 to 60 min, respectively. Furthermore, these dressings met the bio-safety requirements in wound treatment when the toxic properties were compared with silver and PHMB based


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dressings. So far, little research has concentrated on the combination of ZnO/N-halamine nanohybrid materials. Natural chitosan has received increasing research interest for practical applications as it can produce N-acetyl glucosamine by biodegradation. It was found that human body has the same bioactive materials which can largely accelerate the re-epithelization.32 Studies have demonstrated sponge-like chitosan dressings could significantly promote surface induced thrombosis and blood coagulation.33-34 However, chitosan shows only little antibacterial properties under neutral conditions, which limits its further application.35 Thus, we introduced a facile strategy for preparing ZnO based N-halamine hybrid nanoparticles, which were applied to chitosan via both sonication and lyophilization processes (Scheme 1). Up to now, there is no report about the chitosan dressings containing ZnO/N-halamine hybrids for antimicrobial purposes. The unique hybrid structures show prominent antibacterial properties towards two bacterial models S. aureus and E. coli O157:H7. As a hemostatic material, the blood coagulation properties and platelets adhesion ability of chitosan treated with nanomaterials were investigated. Cytotoxicity studies were performed to verify the biocompatibility on rat skin fibroblasts, and the skin stimulation of chitosan dressing containing ZnO/N-halamine hybrids was also tested in vitro.


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Scheme 1. Schematic interpretation of the design and preparation procedure for the CS/ZnOPSPH-Cl dressing. EXPERIMENTAL SECTION Materials. 5,5-Dimethylhydantoin and γ-chloropropyltriethoxysilane were supplied by Hebei Yaguang Fine Chemical Co., and J&K Scientific Ltd, Shanghai, China., Ltd, respectively. Chitosan (average molecular weight of 50 kDa, 90% deacetylation) was provided by Zhejiang Aoxing Biochemical, China. ZnO NPs were provided from Aladdin with average particle size of 30 nm. The remaining chemicals in this study were obtained from Sinopharm Chemical Reagent Co., Ltd.


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Characterization. Scanning electron microscope (SEM) (Hitachi SU1510, Japan) was used to illustrate the microstructure of the as-prepared chitosan dressings. X-ray photoelectron spectroscopy (XPS) of the dressings was carried out on an ESCALAB 250 Xi producing from Thermo Scientific (USA). Absorbance of blood related to thrombogenic activity was measured with a UV-2802S (UNICO, USA) ranging from 200-800 nm. The diffraction angle for 2θ was taken in the angular range of 5o-75o at room temperature on a Philips X'Pert diffractometer. Synthesis of chlorinated ZnO/N-halamine hybrid NPs. Poly[5,5-dimethyl-3-(3'triethoxysilylpropyl)-hydantoin] (PSPH) was prepared through an approach reported in literature.36 100 mL of ethanol, 12.81 g of 5,5-dimethylhydantoin, and 4.16 g of NaOH were putted into a flask, refluxed for 10 min and dried in a vacuum environment. After dissolving in DMF, equal molar amount of γ-chloropropyltriethoxysilane was added and reacted at 95°C overnight. The resulted NaCl was filtrated off and DMF solvent was removed by distillation. Then, the crude monomer product was added to a ethanol-water mixture, and diluted hydrochloric acid was selected to adjust the final pH (4.0). The mixture was reacted under reflux for 6 h. Desired polymer PSPH was obtained after solvent evaporation. Typically, a bath containing an appropriate amount of PSPH and ZnO in a mixture of ethanol and water at a 1:1 weight ratio was prepared and treated with ultrasound for 30 min. Subsequently, the reaction was conducted at 95oC overnight, purified with ethanol, and dried. The preparation of ZnO-PSPH-Cl hybrid was produced by dispersing above NPs into a 10% neutral sodium hypochlorite solution with stirring for 2 h. Any free chlorine on the samples was removed by washing with distilled water and dried for further use.


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Fabrication of CS/ZnO-PSPH-Cl dressings. Chitosan powder (1 g) was dissolved in 50 mL acetic acid solution (at a 1:100 volume ratio). The obtained mixture was then slowly poured into casting plate with a diameter of 12 cm and kept overnight at -36oC. The dressings were formed by lyophilization with the assistance of a freeze dryer (Christ ALPHA 1-4/LD plus). In order to neutralize the residual acetic acid, the formed sponges were dipped in 1.0 mol/L ammonia solution for 2 h and washed with distilled water. Then the dressings were exposed to ZnO-PSPH-Cl solution with concentrations of 2, 4, 6, 8, and 10 g/L, respectively, under sonication for 1 h, vacuum freeze-dried for 24 h and stored for further experiments. The above prepared samples are termed CS/ZnO-PSPH-Cl-2, CS/ZnO-PSPH-Cl-4, CS/ZnO-PSPH-Cl-6, CS/ZnO-PSPH-Cl-8, and CS/ZnO-PSPH-Cl-10, respectively. Unmodified chitosan sponge was served as control. Titration method. Briefly, about 0.1 g of CS/ZnO-PSPH-Cl dressings were soaked into a beaker with 10 mL water and 0.2 g KI. When the stirring time reached 10 min, a drop of starch solution was added. Then sodium thiosulfate was used to reduce the formed iodine to iodide ions, and the endpoint can be obviously determined due to the color change. The Cl+(%) in the CS/ZnO-PSPH-Cl dressings was calculated based on Equation (1).

+

Cl (%)=

N  V  35.45 2m

 100

(1)

Where N and V respectively represent the normality (equiv/L) and volume (L) of reductive titrant, and m represents the mass (g) of CS/ZnO-PSPH-Cl. Porosity, swelling ratio, and liquid retention ability measurements. Absolute ethanol was used to determine the porosity of pre-weighted CS (as a signal control) and CS/ZnO-PSPH-


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Cl dressings. The final weight was recorded when it reached saturation. The porosity was calculated using Equation (2).

P=

W2-W1

(2)

ρV1

Where W2 (g) and W1 (g) represent the weight of CS/ZnO-PSPH-Cl before and after immersing in ethanol, respectively. ρ (g/cm3) represents the density of ethanol, and V1 (cm3) represents the volume of as-prepared samples. The test was triplicate in this procedure. Dressing samples with the same specifications were soaked in multiple sealed bottles containing phosphate buffered saline (PBS) with pH 7.4 at 37oC. For predetermined time intervals, the dressings were immediately weighed after removal of the excess water. The swelling degree was detected by Equation (3).37

DS=

Ww-Wd

(3)

Wd

Where DS represents the swelling degree of dressings, and Ww (g) and Wd (g) represent the mass of the dressings before and after soaking in PBS, respectively. Equal size of dry dressings were weighed, and subsequently exposed to a 0.9% saline solution (pH 7.3) and a simulated body fluid (SBF, pH 7.4). The nominal ion concentration of SBF was prepared according to the reference.38 After adsorption at room temperature for 10 min, the dressings were further treated with 40 mmHg for 1 min and the weight of each dressing was measured. The corresponding liquid retention ratio under pressure (LRRP) towards different models was calculated based on Equation (4) and (5).39 LRRP(g/g)=

W2-W1

(4)

W1


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2

LRRP(g/cm )=

W2-W1

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(5)

A

Where W1 and W2 respectively represent the mass (g) of dressings before and after treatment, A represents the surface area (cm2) of dressings. Whole blood clotting experiment. In this test, the whole blood containing fresh rabbit blood and citrate dextrose were kindly supported by the animal center of Soochow University. Briefly, 100 µL of anticoagulant blood was evenly dispensed onto various dry dressings with an area of one square centimeter. Then, 0.2 M CaCl2 solution (10 µL) was further dripped for the purpose of deflocculation. After kept at 37oC for 5 min, 25 mL of water droplets was carefully added to the glass dishes along the edge and gently shaken at 30 rpm for 10 min. Characteristic hemolysis phenomenon could occur due to the existence of loose red blood cells (RBCs) without immobilization. After that, chitosan samples were air-dried and characterized by SEM. The absorbance of the above hemoglobin solution (Abs of sample), or blank whole blood in deionized water (Abs of blank) was monitored according to the absorbance at 540 nm, and the blood clotting index (BCI) was determined by Equation (6).

BCI index 

100  (Abs of sample) (Abs of blank)

(6)

Platelet adhesion ability of dressing. The platelet-rich plasma (PRP) was collected by centrifugation of citrated whole blood at 1500 rpm for 20 min. Then, PRP with a volume of 3 mL was added to the center of dry square dressing pieces and preserved at 37oC. After 1 h of contact, the non-adherent platelets were subsequently washed with PBS and kept in 2.5% glutaraldehyde for fixation. Afterwards, the treated dressings were fully rinsed with PBS and soaked in different graded ethanol-water mixture for dehydration, naturally dried, and sequentially examined via SEM.


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Stability assessment. CS/ZnO-PSPH-Cl dressings were placed in opaque packaging at room temperature and sealed. After specific storage intervals, the residual chlorine loadings in the dressings were measured by the standard iodometric/thiosulfate titration method according to Equation (1). In addition, the CS/ZnO-PSPH-Cl dressings (1 in. × 1 in.) were respectively added into sealed bottles, which contained 20 mL of alcohol solution (75%). Samples were periodically taken out, dried at 45oC, and titrated. In vitro cytotoxicity and antibacterial tests. XTT assay method according to ISO 10993-5 was performed to assess the cytotoxicity of various chitosan dressings towards 3T3 mouse fibroblasts (ATCC CRL-1213). Besides, a modified AATCC Test Method 100-2004 (termed as sandwich test) was chose to evaluate the antibacterial efficiency of CS and CS/ZnO-PSPH-Cl dressings against both E. coli O157:H7 (ATCC 43895) and S. aureus (ATCC 6538) strains, which was reported in literature.31 Before the test, the chitosan dressings (1 in. × 1 in.) were sterilized with 20 mL of 75% alcohol for 30 min. Skin irritation test. Four New Zealand rabbits, half male and female, weighing 2.5-3 kg were chose to record the skin integrity stimulation. Before the test, the hair on the back was gently removed without damaging the skin. Dry CS (2 cm × 2 cm) and CS/ZnO-PSPH-Cl (2 cm × 2 cm) were used to cover one side of the rabbit back as a control, while the other side was exposed to two layers of bandage. Both sides were fixed with non-irritating taps. Then, the erythema and edema of the rabbits were directly determined every day after 4 h of coverage following our previous work.20 After removal of the bandage on day 7, scoring was performed at 30-60 min, 24, 48 and 72 h intervals. An average score for stimulus intensity at each time (It) and the corresponding maximum It (Itmax) were calculated based on the criteria presenting in the supporting information. Damaged skin was further produced by using needle until hemorrhage


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occurred, and the same simulation evaluation method was conducted. The animal experiments were conducted according to the laws and guidelines by the Department of Science & Technology (Jiangsu Province, China). RESULTS AND DISCUSSION Surface and cross-section SEM micrographs of CS and CS/ZnO-PSPH-Cl dressings are presented in Figure 1 (a, b, d, e). It can be seen that both CS and CS/ZnO-PSPH-Cl dressing have pore-like structures on the surface and interior, with average pore sizes ranging from 50 μm to 200 μm. White dots are observed at higher magnifications, which might indicate the existence of ZnO-PSPH-Cl hybrid nanoparticles. Figure 1c displays the XPS spectrum of chitosan control sample. Characteristic signals at binding energies of 280 eV, 400 eV, and 530 eV matched with C 1s, N 1s, and O 1s peaks, respectively. Once modified with ZnO-PSPH-Cl nanoparticles, new peaks located at 1043 eV, 1021 eV, 200 eV, and 102 eV were detected, which could be assigned to Zn 2p1/2, Zn 2p3/2, Cl 2p, Si 2p, respectively, reflecting the successful assembly of the hybrid nanoparticles on the chitosan dressings.25,

40

The crystalline structure of the samples was

identified by XRD measurements. Besides the characteristic peaks of chitosan control, the diffractograms suggested the typical hexagonal structure of crystalline (wurtzite) ZnO at 31.6°, 34.3°, 36.2°, 56.5°, 62.8°, and 67.8°, respectively.41 The diffraction peak intensities of CS (Fig. 1f) decreased due to a ZnO-PSPH-Cl layer formed on the surface.


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Figure 1. SEM micrographs of the surface and cross-sectional morphology of CS (a, b), CS/ZnO-PSPH-Cl (d, e). The XPS survey of the dressings (c), and XRD pattern of CS and CS/ZnO-PSPH-Cl (f). There were no significant differences in the porosity among the A, B, C, D, and E groups of CS/ZnO-PSPH-Cl. As shown in Figure 2a, all samples displayed porosity in the range of 85%92%. The porosity of hybrid dressings decreased quickly when the weight content of nanoparticles increased to 1% since the nanoparticles may cover pores with smaller diameters. The swelling properties of the dressings in Figure 2b also gradually decreased with the incorporation of ZnO-PSPH-Cl nanoparticles. At day 1, chitosan dressings showed an average swelling ratio of 20, while the composite chitosan dressings had ratios ranging from 15 to 18 due to lower water uptake and retention capacities of ZnO-PSPH-Cl nanoparticles. The swelling ratios of the composite dressings containing a large proportion of nanoparticles did not change


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obviously after 3 days of incubation, after which the swelling equilibrium might have been reached due to the dense structure. Figure 2c displays the chlorine loadings of the dressings as a function of concentration ranging from 2 to 6 g/L. It was found that the corresponding values possessed strong concentration dependence. As depicted in Figure 2d, the active chlorine content tended to become stable after 60 min of ultrasound treatment. A chlorine value of about 0.2% was obtained at a concentration of 6 g/L and an ultrasonic time of 60 min. According to literature,28, 42 this value is sufficient to inactivate bacteria.

Figure 2. Porosity (a), swelling ratios (b), chlorine content of CS/ZnO-PSPH-Cl obtained from different concentrations (c) and after increasing sonication time (d).


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Figure 3 illustrates the liquid retention ratio under pressure of each fluid of CS, CS/ZnOPSPH-Cl, pledget, and bandage samples. Clearly, pledget showed greatest LRRP of normal saline with 8.8 ± 1.2 g/g and 0.20 ± 0.01 g/cm2 due to the irregular structure and hydrophilic properties after degreasing process. CS and CS/ZnO-PSPH-Cl retained comparable mass of saline, 8.2 ± 1.5 g/g and 7.7 ± 0.1 g/g, respectively. When normalized to volume, similar conclusion could be reached in regard to their stable porous structure. In addition, the obtained LRRP in simulated body fluid followed the order CS > CS/ZnO-PSPH-Cl > pledget > bandage. Owing to the structure of the bandage, the value of LRRP after treatment with saline and SBF are smaller than that of other 3D materials. Overall, the prepared chitosan hybrid dressings have better retention ability of SBF than normal saline solution.


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Figure 3. Illustrations of LRRP obtained from mass and area. Here CS, CS/ZnO-PSPH-Cl, pledget, and bandage were selected as testing samples and their blood clotting activity are shown. As can be seen in Figure 4, the blood was completely absorbed into the all samples within 1 min. The CS/ZnO-PSPH-Cl dressing delayed the diffusion compared with the other sample, which was probably due to the presence of the nanoparticles within the porous structure. After 10 min, the blood partial adhered to the surface of the sample. The antithrombogenic activity was monitored by determining the BCI. A lower BCI value represents better hemostatic property of the materials. In comparison with pledget and bandage, CS showed enhanced blood clotting ability with a low BCI of 10% due to the hierarchical structure. This is probably due to the fact that porous chitosan can absorb more moisture from human blood, leading to higher concentration of clotting factors on the wound surface, and thus accelerate blood coagulation. Whereas for the CS/ZnO-PSPH-Cl, the BCI decreased to 5%, exhibiting much stronger hemostatic ability since the bioactive ZnO NPs could denature anticoagulant proteins, thus shortening the coagulation time.


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Figure 4. Blood absorption behaviors of CS, CS/ZnO-PSPH-Cl, pledget, and bandage (left side), and blood clotting index (BCI) (right side). Representative SEM images showed that fewer RBCs were observed in CS control than in the CS/ZnO-PSPH-Cl hybrid samples (Figure 5a-b). At the physiological pH of 7.0, ZnO behaves as a positively charged substrate due to its relatively high isoelectric point (~9.5), and played a important role in mobilization of negatively charged cells via strong electrostatic interaction.43 Furthermore, the adhesion property and morphology structure of the platelets on dressings are of vital importance for the response to thrombus formation. As shown in Figure 5d, more platelets adhered to the CS/ZnO-PSPH-Cl dressings compared to CS control (Figure 5c). The platelets became highly irregular with multiple extensions and interlocked with neighboring osterocytes, confirming that CS/ZnO-PSPH-Cl composites exhibited bioactive properties and could significantly promote platelets activation. Simple nanoparticles formed a layer on the


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surface of dressings, which could increase the roughness of the material and the platelets were more easily stimulated to aggregate to the surface of CS/ZnO-PSPH-Cl dressings. This phenomenon is consistent with the BCI results.

Figure 5. SEM images of red blood cells and platelets adhering to CS (a, c) and CS/ZnO-PSPHCl (b, d) dressings (arrowheads: red blood cell; double arrowheads: ZnO-PSPH-Cl; ellipses: platelets). To further confirm their health impacts, we analyzed the NIH3T3 cells viability by a XTT measurement. Untreated cells were selected as the control, and the biocompatibility was tested at various contact times (from 4 h to 24 h) for CS and CS/ZnO-PSPH-Cl hybrid dressings. From


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Figure 6a, after 8 h of incubation, the cell survival rate of all samples was higher than 96% as expected, confirming no toxicity to mammalian cells. After 24 h, it was found that the relative cell viabilities showed a slight decrease, while still maintaining nearly 90% in the presence of CS/ZnO-PSPH-Cl as compared to that of control, revealing the feasibility of the dressings for in vitro applications. The CS/ZnO-PSPH-Cl dressings were further packed in the dark condition for a month to explore their biocidal efficacies of active chlorine after storage. The chlorine contents decreased from 0.19 to 0.17%, retaining 90% on the average of their initial content. By comparing chlorine contents after treatment with 75% alcohol for various intervals, no obvious difference was detected in Figure 6b. The results showed that the functional N-Cl groups in CS/ZnO-PSPH-Cl were very stable during storage and alcohol sterilization process. Furthermore, the inhibition effect of the produced CS and CS/ZnO-PSPH-Cl dressings (after 4 weeks storage) against both bacterial cells were examined. From Figure 6c-d, it can be seen that pure CS dressings as control showed mild activity against strains in 30 min, with sterilization rates of 45.36% and 16.58% towards both S. aureus and E. coli O157:H7, respectively. While the CS/ZnO-PSPH-Cl dressings with active chlorine of 0.17% showed significant bacteria reduction with inactivation rates of 85.05% and 77.84% of S. aureus and E. coli O157:H7 within 5 min, respectively. The antibacterial activity gradually increased up to 30 min, and 99.93% and 88.01% of S. aureus and E. coli O157:H7 were inactivated, respectively. Obviously, the introduction of ZnO and Nhalamine in the chitosan dressings significantly enhanced antibacterial efficacies. It was noted that the dressings showed a lower antibacterial activity against E. coli O157:H7 than S. aureus since the cell membrane of gram negative bacterial have an external protective lipid which is more difficult to damage by contact with substrates.44-45


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Figure 6. Cell viability of 3T3 fibroblasts with CS and CS/ZnO-PSPH-Cl after various incubation time. *, p

N-halamine-Mediated Multifunctional Dressings as Quick

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