A Novel Lectin-like Bioadhesive Peptide: Human α-defensin HNP1

Defensins are a family of small, cationic, cys-rich antimicrobial proteins of molecule weight in range from 3000 to 5000, which play important roles in broad spectrum of antimicarobial activity and innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Previous researches were performed to confirm that defensins highly bound with glycosylated protein which aboundant on mucin surface due to the specific conservative properties of defensins. The targeting sites of bioadhesive delivery system exist in surfaces of oral, nasal cavacity, ocular, vigina and so on. The aim of this study is to design and evaluate a novel bioadhesive liposome delivery system covalently modified with defensins.Linear peptide HNP1 was synthesized via Boc solid-phase synthesis method, bioactive peptides were achieved after being folded by using Cysteine and Cystine. The product was determined and purified by RP-HPLC, and the structure of product was identified by 1H-NMR. Through utilizing fluoresceins Kodak 670 and IR 783, the eliminations of peptides from rabbits’ cornea in vitro were traced by fluorescence-labeled peptides. Furthermore, biotoxicity of peptide was examined through haemagglutination test. The results showed that the product was HNP1 and the purity of it was more than 95%. The elimination characteristics of HNP1 on excised cornea of rabbits showed that HNP1 was not only a potential bioadhesive material but also safe to use.Bioadhesive drug delivery system was developed with formulation of HNP1. EPC/cholesterol/DSPE-PEG3400/DSPE-PEG-HNP1 (4:1:0.5:0.1, w/w/w/w) were used to prepare HNP1 modified liposome by reverse evaporation technique. Liposome size, size distribution and in vitro fluoresceins FAM release were evaluated immediately after preparation. The morphological evaluations of modified liposomes were performed by transmission electron microscopy (TEM) following negative staining with sodium phosphotungstate solution. Mucin was added into HNP1-liposomes to evaluate the change of particle size. Results showed that the mean diameter of the HNP1-liposomes was around 200 nm, no difference in the pattern of in vitro drug release was observed between HNP1-liposomes and unmodified liposomes. After incubation with mucin, the change tendency of particle size of HNP1-liposomes was dissimilar with those unformulated liposomes.To prepare fluorouracil liposomes by reverse evaporation technique and evaluate its quality. The method of entrapment efficiency was built, particle size and entrapment eficiency were investigated. Consequently, using HPLC to test entrapment efficiency of 5-Fu liposomes was efficient, the obtained liposomes were with average size of 200 nm, and entrapment eficiency is 17% which was as the same as reports.All in all, this study aimed at developing a novel bioadhesive delivery system via ocular administration. Possibility of HNP1 as a bioadhesive material was tested by confirming the interaction between HNP1 and mucin, evaluating the elimination of HNP1 modified liposomes by labeling peptides with fluoresceins. HNP1-liposomes and 5-Fu liposomes were prepared for further study the utilization of HNP1 in drug delivery system.