Preparation,Characterization And In Vitro Antibacterial Activity Of BLfcin Loaded Choisan Nanoparticle Thermosensitive Hydrogel

Dairy cow mastitis is a disease that causes inflammation of the nipples or mammary gland tissues which caused by multiple factors caused by the combination of pathogenic microorganisms and the environment.Among them,Staphyloccocus aureus(S.aureus),Escherichia coli(E.coli)are the common pathogen in dairy cow mastitis.At present,the treatment of dairy cow mastitis still relies on antibiotics,but the long-term abuse of antibiotics will lead to the production of drug-resistant strains,reduced treatment effect,drug residues in milk and other problems,which will ultimately threaten human health.Bovine lactoferricin(BLfcin)is an antimicrobial peptide with multiple biological functions such as broad-spectrum antibacterial effect,immune regulation,antiviral,antitumor and antioxidation.The aim of this study is to develop a nano-composite preparation that can be used to treat dairy cow mastitis caused by bacteria.Therefore,BLfcin was selected as the raw material to prepare a composite temperature-sensitive hydrogel loaded with BLfcin nanoparticles,which provide a new options for antibiotic replacement and dairy cow mastitis treatment.The research content and results of this article were as follows:(1)Chitosan nanoparticles(LCS-NPs)were prepared by ionic gelation using low molecular weight chitosan(LCS)as carrier and sodium tripolyphosphate(TPP)as crosslinking agent.The optimal preparation scheme was obtained by orthogonal test: under the condition of crosslinking environment temperature of 0-4℃,the LCS solution with the concentration of 1 mg/m L was preheated in water bath at 60℃ for 10 min,and then 1 mg/m L TPP solution(LCS: TPP = 3:1)was dripped and stirred for 30 min.The LCS-NPs have a particle size of 98～122 nm,with a unimodal particle size distribution,and a polydispersity index(PDI)of 0.225 ± 0.015.At the same time,the average surface charge of LCS-NPs is +14.48 ± 1.02 m V.(2)Bovine lactoferricin nanoparticles(BLfcin-NPs)were prepared by loading BLfcin into LCS-NPs.The characterization,cytotoxicity and in vitro antibacterial activity of BLfcin-NPs were studied.The results showed that when the amount of BLfcin was 1 mg,the encapsulation efficiency was 72.18 ± 0.78%,and the drug loading was 2.17 ± 0.03%.The nano system was nearly spherical under transmission electron microscope.The cytotoxicity test showed that BLfcin-NPs had no cytotoxicity on bovine mammary epithelial cells(BMECs)and could promote proliferation.BLfcin-NPs can inhibit bacterial proliferation through sustained drug release.Adding equal volume of5% sucrose aqueous solution to BLfcin-NPs aqueous dispersion system,the freeze-dried nanoparticles were cake like,with good redispersibility and the smallest change rate of particle size and PDI,which was convenient for storage and clinical application.(3)Middle molecular weight chitosan/β-glycerophosphate thermosensitive hydrogel(MCS/β-GP Hydrogel)was prepared,and added BLfcin-NPs in it to make nanoparticle-loaded composite temperature-sensitive hydrogel(BLfcin-NPs Hydrogel).Its characterization was studied,and its biodegradability and antibacterial activity in vitro were studied.Cryo-SEM shows that the nanocomposite hydrogel was a cross-linked network structure.The results of FTIR showed that hydrogen bonds and complexes were formed between MCS and β-GP,but the characteristic absorption bands of BLfcin and BLfcin-NPs were not observed in the infrared absorption spectrum of BLfcin-NPs Hydrogel.XRD results showed that BLfcin-NPs had little effect on the crystallization properties of hydrogels.The rheological test shows that BLfcin-NPs hydrogel has a fast gelation time,and its phase transition point is 24℃.The residual weight of BLfcin-NPs hydrogel was 30.54 ± 3.82%after 21 days,which indicated that the biodegradability of BLfcin-NPs hydrogel was better.Cytotoxicity test showed that the BLfcin-NPs hydrogel has no significant negative effect on BMSCs proliferation.Antibacterial tests prove that the nanocomposite hydrogel has good antibacterial activity in vitro.In summary,this experiment optimizes the preparation conditions of chitosan-based nanoparticles,which can be used to produce chitosan nanoparticles with controllable particle size and good dispersibility.The BLfcin-NPs have good physical and chemical characterization and in vitro bacteriostasis,and can be used as a promising antimicrobial peptide drug release system.The prepared composite hydrogel containing BLfcin-NPs has temperature-sensitive gelation,degradability,and in vitro antibacterial activity,and this composite material can become a new choice for clinical treatment of bacterial dairy cow mastitis.