Catechol Cross-linked Antimicrobial Peptide Hydrogels Prevent Multidrug-resistant Acinetobacter Baumannii And MRSA Infection In Burn Wound And Accelerate Wound Healing

Part 1:Catechol cross-linked antimicrobial peptide hydrogels prevent multidrug-resistant Acinetobacter baumannii infection in burn woundBaclground:Hospital-acquired infections are common in burn patients and it is a major cause of morbidity and mortality.Bacterial infections such as Acinetobacter baumannii(A.baumannii)are hard to treat due to its rapid acquiring resistance to antibiotics and biofilm formation.Hydrogel dressings are considered as an important component in wound care and management.It has a variety of applications.Hydrogels can maintain moisture on the wound surface,essential for wound cleaning and debridement.They can prevent bacteria and oxygen reaching the wound surface,creating a barrier for infections.Antimicrobial peptides(AMPs)have gained a lot of attention due to its antimicrobial activity and established safety.epsilon-poly-L-lysine(EPL),an AMP,is known for its broad antimicrobial activity against Gram-positive and Gram-negative bacteria,yeasts and moulds.Also,EPL is water soluble,stable,biodegradable and has low toxicity.Catechol is a colourless naturally occurring organic compound(C6H4(OH)2).It possesses ortho isomer of the three isomeric benzenediols.Researchers promote the use of catechol in the design of functionalized biomaterials.Catechol low molecular weight and good water solubility makes it a good candidate for biomimetic biomedical materials.This work presents a newly developed EPL and catechol hydrogel that has the potential for treating contaminated burn wound infection and biofilm eradication.Methods:The hydrogel formulation is based on EPL and catechol,which was crosslinked via mussel-inspired chemistry between the amine and phenol groups.A series of EPL-catechol hydrogels were prepared by mixing different ratios of EPL and catechol,i.e.,Gel 1:(0.3:0.1),Gel 2:(0.4:0.1)and Gel 3:(0.5:0.1)mol/L.Scanning electron microscopy(SEM)was used to analyze the microporous structures of hydrogels.Multidrug-resistant A.baumannii(MRAB),clinical strain,were used to analyze the antimicrobial and antibiofilm properties of hydrogels in vivo and in vitro.EPL-catechol hydrogel biocompatibility was assessed using clonal mouse myoblast cell line(C2C12).Furthermore,we created a second-degree burn wound on mice dorsal skin surface followed by contamination with MRAB.To evaluate the hydrogel antimicrobial activity,wound bacterial burden was analyzed on day 1 and 2 of infection.SEM was used to observe antimicrobial effect hydrogel against A.baumannii both in vivo and in vitro.Also,EPL-catechol hydrogel was applied topically and implanted suboutaneously in mice to evaluate its good biocompatibility.Results:The SEM observations of hydrogels revealed highly interconnected porous structures of the EPL-catechol hydrogels.The pore size decreased with increasing EPL concentration in various hydrogels.Hydrogels showed high water contents ranging from 87%to 94%.In vitro studies,EPL-catechol hydrogels showed promising antimicrobial activity against MRAB.The zone of inhibition of various EPL-catechol hydrogels increased as the EPL concentration increased.A 100%MRAB kill was noticed when treated EPL-catechol hydrogel.SEM analysis showed,remarkable structural alterations i.e.,disrupted,withered and wrinkled surfaces morphology of MRAB upon treatment with EPL-catechol hydrogel.EPL-catechol hydrogel inhibited MRAB biofilm formation.The antibiofilm assay showed that only a few live/dead MRAB cells were observed on EPL-catechol hydrogel treatment compared with TCPS(control),where most of the bacteria were alive.In addition,the EPL-catechol hydrogel showed good biocompatibility with the clonal mouse myoblast cell line(C2C12).Cells showed periodic growth in the presence in of EPL-catechol hydrogel.Burn wound inoculation with of MRAB,followed by hydrogel treatment showed a logio reduction of 4.76 compared with the control group.After 48 hours of hydrogel application,5.70 logio reductions were observed compared with the control group.SEM observations revealed few bacterial growths on the MRAB infected burn wound treated with EPL-catechol hydrogel compared with the control group,where MRAB proliferate in high number.On histological studies,no inflammatory cells infiltration,oedema or congestion were seen on day 1 and day 2 of topical application of EPL-catechol hydrogel on mice,when compared with the untreated control group.Furthermore,the sub-cutaneous hydrogel implantation in mice following day 2 and 5 days,displayed normal architecture of epidermis,dermis,and subcutaneous tissues and no signs of inflammation and when compared with the untreated control group.These results indicate EPL-catechol hydrogel good biocompatibility.Conclusion:In this study,for the first time,we established a novel interlaced microporous EPL cross-linked with catechol hydrogel.Through this study,we demonstrated that this hydrogel not only exhibits antimicrobial but also antibiofilm activity towards MRAB.We noticed that the EPL-catechol hydrogel is biocompatible and non-cytotoxic with mammalian cells.Both,in vitro and in vivo studies indicated the significant reduction of bacteria.The histological examination of mice skin upon hydrogel application did not exhibit any signs of inflammation and tissue alteration.Based on these results,we concluded that EPL-catechol hydrogel is an interesting and promising future biomaterial to fight against multidrug-resistant bacterial infections.Part 2:Mussel-inspired hydrogel with potent in vivo contact-active antimicrobial and wound healing-promoting activitiesBackground:Open wounds(e.g.,burns and trauma)are always challenged by various opportunistic bacteria.There is an urgent need for developing novel wound dressing that is able to prevent bacterial infection and promote the healing simultaneously.Herein,we developed a new type of antimicrobial hydrogels for the open wound healing through imitating a facile mussel-inspired catechol/polyamine chemistry.Antimicrobial peptides(AMP),which disrupts the cell membrane of microbes,are believed to have less tendency in developing drug-resistance.Epsilon-poly-L-lysine(EPL),a natural antimicrobial peptide produced by Streptomyces albulus,has potent broad-spectrum antimicrobial property.Unlike other AMPs,EPL could be produced at a relatively low cost by fermentation.EPL-based derivatives have been used to prepare hydrogels with in vitro antimicrobial activity.However,the preparation of these hydrogels usually involves multiple steps of chemical synthesis such as the modification of EPL molecules.Mussel-inspired chemistantimicrobial ry which takes place in mild conditions is a facile method for preparation of materials.Recently studies reported the reaction between catechol and polyamines.The phenol groups of catechol reacted with the amines thus generated mussel foot proteins like thin films.EPL which contains 25-30 lysine is a typical polyamine with plenty of side amine groups,thus could react with catechol to generate cross-linked networks.This work presents a newly developed catechol and EPL hydrogels that have the potential for treating MRSA infection and accelerate wound healing.Methods:Three types of EPL-catechol based hydrogels were synthesized.i.e.,EPL(10 mmol/L,20 mmol/L,and 30 mmol/L)and catechol(30 mmol),named as Gel 1:(10:30 mmol/L)EPL-catechol hydrogel,Gel 2:(20:30 mmol/L)EPL-catechol hydrogel and Gel 3:(30:30 mmol/L)EPL-catechol hydrogel respectively.The microstructures of hydrogels were observed by SEM.The antimicrobial activities of the hydrogels were determined against E.coli and MRSA.SEM was used to examine bacterial morphologies.LIVE/DEAD Baclight was used to access the EPL-catechol hydrogel antibiofilm activity.C2C12 cells line was used to determine in vitro biocompatibility of the EPL-catechol hydrogel.Cell migration assay was performed using mouse vascular endothelial cells(bEnd.3)on EPL-catechol(Gel 3)hydrogel was carried out according to reported protocol.For in vivo studies,mice burn wound infected with MRSA was developed to observe EPL-catechol(Gel 3)hydrogel antimicrobial activity.Hematoxylin and eosin(H&E)staining,immunohistochemical staining of IL-6 and TNF-?,and immunofluorescence labelling of VEGF were used to analyze anti-inflammatory and wound healing properties of EPL-catechol hydrogel.Results:The EPL-catechol hydrogels were synthesized by the crosslinking of the EPL with catechol.the EPL-catechol hydrogel turned to brownish to the oxidation of catechol to a quinone.EPL-catechol hydrogels swelled rapidly in water.The hydrogel EPL-catechol contained more EPL and exhibited the highest swelling ratio.SEM images of hydrogels examination revealed porous structures.The EPL-catechol hydrogels showed potential antimicrobial activity against both E.coli and MRSA.EPL-catechol(Gel 1)reduced 79.2%of E.coli and 59.4%for MRSA(0.88 ± 0.32 and 0.66 ± 0.02 in log reduction).EPL-catechol(Gel 2)hydrogel against E.coli and MRSA were increased to 2.8±0.31(99.8%Kill)and 1.5 ± 0.24(97.5%Kill)respectively.The EPL-catechol(Gel 3)hydrogel showed the most potent antimicrobial efficacy with log reductions at 5.5 ±0.28(>99.999%Kill)and 2.95±0.35(99.9%Kill)for E.coli and MRSA respectively.Biofilm formation was noticed on the quartz plate(control group).However,EPL-catechol inhibited biofilm formation,resulting in effective biofilm prevention.The in vitro cytotoxicity of the EPL-catechol hydrogel on day 1 and 3 showed stable growth rate compared with control(TCPS),indicating the non-cytotoxicity of EPL-catechol(Gel 3)hydrogel.Also,the EPL-catechol hydrogel increased the cell migration of mouse microvascular endothelial cells when compared with the TCPS control,suggesting that EPL-catechol hydrogel facilitated the formation of new blood vessels.In vivo antimicrobial studies,we observed the EPL-catechol hydrogel application on MRSA infected burn wound for 3 days,significantly reduced 99.9%and 99.8%(3.13 and 2.73 in the log)bacterial load compared with the control saline and PEGDA groups(P<0.01).Hematoxylin and eosin(H&E)staining revealed a large number of inflammatory cells(stained blue)infiltrated in the tissue of control(saline)and PEGDA groups on day 1 and 3.On the other hand,the EPL-catechol hydrogel reduced the inflammatory effects at the MRSA infected burn wound site.Furthermore,SEM observations show,EPL-catechol(Gel 3)hydrogel reduced MRSA burden in burn wound.The EPL-catechol hydrogel exhibited remarkable wound repair and skin regeneration properties compared with the control(saline)and PEGDA hydrogel groups.EPL-catechol hydrogel treated wound was nearly completely closed at day 10 compared with the saline group and PEGDA hydrogel group.Histological analysis on day 3 showed reduced inflammatory cells in the wound treated with EPL-catechol hydrogel.On day 6,hair follicles and blood vessels appeared with EPL-catechol hydrogel treatment,while no hair follicles and epithelium observed in saline and PEGDA treated groups.At day 10,in EPL-catechol hydrogel treated group,a large number of hair follicles,blood vessels and complete epidermis appeared compared with saline and PGDA groups showed mild acute inflammatory responses.These results indicate outstanding wound healing of EPL-catechol hydrogel.In addition,immunohistochemical staining showed reduced expression of pro-inflammatory cytokines indicating anti-inflammatory activity and that helps wound healing.The EPL-catechol hydrogel showed high vascular endothelial growth factor(VEGF)expression on day 3,6,and 10 compared with the other two control groups(saline and PGDA).Conclusion:In this study,we demonstrated a hydrogel prepared by a very simple mussel-inspired polyamine/catechol reaction.EPL was directly crosslinked with catechol by single step mixing at room temperature.The EPL-catechol hydrogels exhibited significant contact-active antimicrobial efficacy.In vitro experiments indicated that the EPL-catechol hydrogel not only exhibited striking bactericidal performance against E.coli and MRSA but also showed the efficacy of preventing biofilm formation.Also,EPL-catechol hydrogel also possessed good biocompatibility with murine myoblast cells(C2C12)and promoted the migration of murine vascular endothelial cells in vitro.More importantly animal model of burn wound infection and a full-thickness cutaneous wound showed that the EPL-catechol hydrogel not only prohibited a burn wound MRSA infection but also accelerated wound healing through reducing the expression of pro-inflammatory cytokines IL-6 and TNF-a and increasing the expression of vascular endothelial growth factor(VEGF).Those findings demonstrated that EPL-catechol hydrogel is a promising biomaterial for applications of wound dressings which can prevent wound infection and accelerate the wound healing.