Poly-L-Lysine In-Situ Forming Hydrogels For Treatment Of Infected Wounds

Bacterial infections easily occur in skin wounds,heavily influencing wound healing,leading to tissue necrosis and even amputation.Systemic and topical antibiotics are the usual treatments of infections,although the excessive application of them commonly produces antibiotic-resistant strains.Variously shaped wounds need the wound wholly covered drugs;otherwise,wound healing and infection therapy would be hindered.Therefore,it is of great scientific significance and potential clinical value to search for new anti-traumatic infection drugs and their preparations.As a new antibacterial peptide,ε-Poly-L-lysine(EPL)is produced by fermentation of Streptomyces albicans.It is a natural cationic antibacterial polypeptide and exerts antibacterial activity by destroying the bacterial plasma membrane.EPL possesses the advantages of wide antibacterial spectrum and good biocompatibility,which has great potential in the treatment of skin wound infections.Hydrogels are considered to be an important means of wound treatment and care,and have been widely used in the field of wound repair.Moreover,the network structure of hydrogels is similar to that of natural extracellular matrix(ECM)and absorbs excess exudate around the wound and allows oxygen permeation,which provides a suitable environment for wound healing.However,traditional hydrogels usually lack the functions such as shape adaptability and infection control,limiting their clinical application.Therefore,the development of an EPL-based multifunctional hydrogel with wound shape adaptability,excellent antibacterial properties,safety and promote wound healing is of great significance for the treatment of skin wound infection.The specific research contents are as follows.(1)Preparation of EPL hydrogelsSodium periodate was used to oxidize hyaluronic acid(HA)to obtain the oxidized hyaluronic acid(OHA),dopamine(DA)was grafted on the OHA to obtain oxidized hyaluronic acid-dopamine(OHA-DA)to improve the adhesion of the hydrogels,and EPL was combined with OHA-DA by Schiff base reaction to obtain dopamine-hyaluronic acid-ε-poly-L-lysine(DA-HA-EPL).A pro-hydrogel,basically composed of acrylamide(AM)and DA-HA-EPL,was prepared.It was filled into the V.vulnificus-infected wound.A 405-nm blue light was exerted on the wound for rapid photopolymerization of AM to its polymer polyacrylamide(PAM).A physical hydrogel network of PAM/DA-HA-EPL immediately formed on site within several seconds.(2)Characteristics of EPL hydrogelsThe 72-h swelling rate turn of hydrogels at p H 5 and p H 8 was PAM/DA-HA >PAM/DA-HA-EPL-60 > PAM/DA-HA-EPL-20 > PAM/DA-HA-EPL-40,indicating that PAM/DA-HA-EPL-60 absorbed more wound exudates than PAM/DA-HA-EPL-20 and PAM/DA-HA-EPL-40.The adhesion strengths of PAM/DA-HA and PAM/DA-HA-EPL-60 were about 5 k Pa,which were the same as that of the fibrin glue(about 5 k Pa).Therefore,PAM/DA-HA and PAM/DA-HA-EPL-60 hydrogels had suitable adhesion strength.Moreover,after the hydrogels underwent stretching or compression,PAM/DA-HA-EPL-60 hydrogels returned to its original state,which demonstrated that PAM/DA-HA-EPL-60 displayed shape adaptability.(3)Antibacterial activity and biocompatibility of EPL hydrogelsEPL hydrogels achieved nearly 100% surface antibacterial ability for S.aureus,E.coli and V.vulnificus by surface antibacterial test.In the inhibition zone test,antibacterial ability of PAM/DA-HA-EPL was as the following turn:PAM/DA-HA-EPL-60 > PAM/DA-HA-EPL-40 > PAM/DA-HA-EPL-20,where the antibacterial ability of PAM/DA-HA-EPL-60 was strongest.Moreover,the SEM images of the bacteria clearly displayed the effect of PAM/DA-HA-EPL-60 on the bacterial morphologies that were wrinkled or ruptured,indicating the disruption of bacterial membranes.Compared with PBS group,PAM/DA-HA-EPL-60 group showed few live bacteria in the live/dead bacterial assay,indicating PAM/DA-HA-EPL-60 exhibited outstanding antibacterial activities.PAM/DA-HA and PAM/DA-HA-EPL-60 showed good biocompatibility.Bright green fluorescence and almost no red fluorescence were observed in the live/dead assay.The green fluorescence intensity gradually increased in three days,indicating the normal proliferation of L929 cells.The CCK-8 assay showed no cytotoxicity of the extracts of the hydrogels within three days.The cell migration experiment demonstrated that PAM/DA-HA-EPL hydrogels improved the migration of L929 cells much more than DMEM alone,although less than the DMEM containing FBS.Moreover,PAM/DA-HA and PAM/DA-HA-EPL-60 showed good hemocompatibility,the absorbance value was lower than 0.1 and erythrocyte hemolysis did not occur.(4)The efficacy of EPL hydrogels in the treatment of wound infectionIn the mouse skin infection wound model,compared with the model and Ag dressing group,the hydrogel PAM/DA-HA-EPL significantly accelerated the wound repair rate.The wound healing rates were 75.5%,85.1%,and 92.2%,respectively,the collagen volume fractions were 90.7%,85.9%,and 96.5%,respectively,and the granulation tissue thicknesses were 704.0 μm,795.5 μm,and 1056.9 μm,respectively.The mean fluorescence intensity of α-SMA(α-smooth actin)was 11.4,12.1,and 30.0,respectively.All results suggested that PAM/DA-DA-EPL had a good healing effect for wound closure,tissue regeneration,collagen deposition and vascular remodeling.Therefore,PAM/DA-HA-EPL exhibited strong antibacterial property,good biocompatibility,proper adhesion and shape adaptability,which has great potential in the treatment of skin wound infection.