Ultrabroad-spectrum Antimicrobial Quaternized Chitin Derivative For Infected Wound Healing

Objective Infections of wounds can cause delayed healing and have consistently been recognized as serious threats to human.Thus,developing novel antimicrobial wound dressings with both efficient antimicrobial properties and accelerated wound-healing effects is quite valuable for clinical application.Chitin is biodegradable,non-toxic,and biocompatible natural polymer material,and recognized as promising for various biomedical applications,particularly in wound dressings.Thus,this research aims to fabricate quaternized chitin derivatives(QC-s)from a high-efficiency and sustainable route using aqueous KOH/urea solution,and to verify its antimicrobials activities,especially for multidrug resistant pathogens,biocompatibility and underlying antimicrobial mechanism.Besides,a porcine infected full-thickness wounds model will be established to preclinically evaluate QC-4 on in vivo antimicrobial activities and infected wound healing improvement.Methods(1)A series of QC-s with varying degrees of deacetylation(DD)and degrees of quaternization(DQ)were fabricated from aqueous KOH/urea solution and their antimicrobial effect against Escherichia coli(E.coli)was examined to elucidate the relationship between the structure of QC-s and its antimicrobial properties and select the optimal antimicrobial candidate(QC-4).(2)Eight microbial pathogens were used to determine the antimicrobial properties of QC-4,including clinically prevalent multidrug-resistant Escherichia coli(MDR-E.coli),methicillin resistant Staphylococcus aureus(MRSA),multidrug-resistant Pseudomonas aeruginosa(MRPA),and multidrug-resistant Acinetobacter baumannii(MDR-A.baumannii).The antimicrobial mechanism was examined by an imaging-based approach and quantitative flow cytometry.The morphological changes of microbes after incubation with QC-4 were observed by scanning electron microscope(SEM),and the microbial membrane permeability and potential were examined by Calcein-AM/PI live/dead bacterial assay and DiOC2(3)membrane potential flow cytometry assay.The antimicrobial activity assays of QC-4 in the presence of lipoteichoic acid(LTA)and lipopolysaccharide(LPS)were also performed.(3)The in vitro biocompatibility was examined through CCK-8 cell viability assays and Calcein-AM/PI live/dead staining of primary normal human dermal fibroblasts(NHDF)and hemolytic test of erythrocytes.The effects of subcutaneous implantation of QC-4 for 4 weeks on the architecture and function of large organs in mice were detected to evaluate the in vivo systemic toxicity of QC-4.(4)QC-4 was applied in a porcine S.aureus infected skin full-thickness wound to test its abilities to disrupt bacterial aggregates and promote wound healing by immunofluorescence staining and pathological Masson staining assay,in comparison with the widely used commercial silver nanoparticle-based Alginate-Ag dressing and chitosan-based Chitoclot Bandage dressing.And a mice subcutaneously implanted model was established to examine the multidrug resistant bacteria-killing performance of QC-4.Results(1)We synthesized 9 QC-s with varying DD and DQ in aqueous KOH/urea solution and found that the antimicrobial properties of QC-s became stronger along with the increasing DD and DQ.Among them,QC-4(DD=78.9%,DQ=0.46)showed the best antimicrobial performance and was hence selected as the optimal antimicrobial candidate.(2)QC-4 exerted ultrabroad-spectrum and high-efficiency antimicrobial activity against Gram-negative E.coli,Gram-positive S.aureus,fungi Candida albicans(C.albicans),anaerobic Bacteroides fragilis(B.fragilis),MDR-E.coli,MRSA,MRPA,and MDR-A.baumannii even at a very low concentration range of 500 ng/mL to 5 ?g/mL.QC-4 could bind to the microbial surface through electrostatic interaction between the cationic sites carried by QC-4 and the negatively charged LTA and LPS on the bacterial cell wall,thus disrupting microbial membrane integrity and enhancing microbial membrane permeability and depolarization,and ultimately killing the microbial pathogens.(3)QC-4 demonstrated excellent cytocompatibility and hemocompatibility in vitro even at a very high concentration of 500 ?g/mL.No obvious damages of architecture and function on major organs were observed after subcutaneous implantation of QC-4 for 4 weeks.Conversely,silver accumulation was indeed observed in liver from mice subcutaneously implanted with commercial Alginate Ag dressing for 4 weeks.(4)Both QC-4 dressing and commercial Alginate Ag dressing demonstrated potent efficiency of microbial aggregate damage and promoted re-epithelialization and collagen formation for wound healing on the porcine infected skin full-thickness wound.But the commercial Chitoclot Bandage dressing and gauze were not effective in reducing the microbial aggregate and promoting wound healing.The infected wounds treated with QC-4 presented roughly equivalent antimicrobial effects and healing profiles with respect to the commercial silver nanoparticle-based Alginate Ag dressing and completely superior treatment effect to that of commercial chitosan-based Chitoclot Bandage dressing and gauze.Conclusions This study demonstrated that a novel quaternized chi tin derivative QC-4 synthesized from aqueous KOH/urea solution had ultrabroad-spectrum antimicrobial activity,especially against multidrug resistant pathogens,excellent biocompatibility,and accelerated wound healing performance,which might substantiate its use as antimicrobial agents in clinical practice.