Design Of PH/thermo-sensitive Lipopeptide With Zipper Structure And Its Antitumor Properties

Peptides are widely used as functional biomaterials for their good biocompatibility,responsive secondary structures and variable charge adjusted by pH.Peptide with leucine zipper structure is one of thermo-sensitive molecular for the conformational transition of its secondary structure between 'close' and 'open'at hyperthermia.In this paper,based on the design of lipopeptides with leucine zipper,their applications on antitumor agents were studied,and this study provided a theoretical basis for the design and praperation of novel drug carrier in nanoscale and antitumor peptide.This paper is divided into two parts:Part I:Thermo-sensitive drug carrier in nanoscale(1)Lipopeptide with leucine zipper,C6-Pep,was designed and its thereto-sensitivity was analyzed by Circular Dichroism,and results showed the secondary structure of C6-Pep changed from a-helix to random at 48?.Novel thermo-sensitive liposome-lipopeptide(Lipo-LPe)was prepared by mixing C6-Pep with lipids.The Tm of lipopeptide decreased when it was arranged into liposome,and C6-Pep also increased liposome's stability by measuring the anisotropy of DPH in lipid membranes.DOX@Lipo-LPe were prepared through active loading method and their drug release kinetics were measured at 37? and 45?.Drug leakage from Lipo-LPe at 37? decreased,but it achieved the quick and great amout of drug release at 45?.Finally,the cytotoxicity of Huh7 proved Lipo-LPe had the great antitumor activity at 45?.(2)Based on the feature of C6-Pep,another three lipopeptides,C8-Pep/C10-Pep/C8-AS-Pep,were designed and the corresponding Lipo-LPe were prepared.Circular Dichroism were measured to analyze the thermo-sensitivity of lipopeptides in liposomes.Results showed C 10-Pep held the worst thermo-sensitivity for its longer hydrophobic chain,the thermo-sensitivity of C8-AS-Pep was the best for the modification of Asp-Ser.UV spectrophotometer was used to measure the drug release kinetics from Lipo-LPe,and C8-AS-Pep achieved the best drug release at hyperthermia.The effect of lipid membrane' fluidity on drug release was further studied.It was found that excessively fluid or excessively stable membranes prevented the drug release from liposomes,which was controlled by temperature.Because of the reversible conformational transition of lipopeptides,Lipo-LPe achieved good temperature-controlled stepped drug release process.In conclusion,molecular structure of lipopeptides,lipid membrane properties,and even the release mode all have effect on the thermo-sensitive drug release from Lipo-LPe.Part II:pH/thermo-sensitive antitumor peptide(1)Behaviors of C6-Pep and Pep at interface were studied by combining Langmuir-Blodgett(LB)technology with Polarization Modulation Infrared Reflection Absorption Spectroscopy(PM-IRRAS).Three kinds of lipid monolayers with different charges were prepared at air-water interface.Firstly,the adsorption kinetics of C6-Pep and Pep into pure air-water interface and lipid monolayers were compared.Results showed C6-Pep and Pep in water diffused from subphase to air-water interface by hydrophobic force and the diffusion rate of C6-Pep was bigger than that of Pep for its longer hydrophobic chain.Then,the effects of pH,temperature,and initial surface pressure of lipid monolayer were studied,and results showed C6-Pep could selectively adsorpt and insert into lipid monolayers containing DPPG at pH 5.5 by electrostatic interaction.The higher initial surface pressure prevented the adsorption of C6-Pep from subphase to interface.C6-Pep in lipid monolayer was also thermo-sensitive,and the content of a-helix decreased with the increase of temperature.(2)Three kinds of liposomes with different charges to mimic different cell membranes were prepared by thin-film dispersion method.Circular Dichroism were used to measure the secondary structures of lipopeptides in liposomes.It was found that C6-Pep and Pep selectively adsorpted and inserted into liposomes containing DPPG at pH 5.5.C6-Pep was still in a-helix and was also thermo-sensitive.Meanwhile,Pep's content of a-helix structure increased in DPPG liposome than in water.Dye leakage from Rhodamine 6G@liposome induced by peptides proved the destructiveness of C6-Pep to DPPG liposome was strongest at pH 5.5,and C6-Pep induced a large amount of dye leakage at lower concentration at 45 ? because of its good thermo-sensitivity.High cytotoxicity to Hela cell at pH 5.5 and 45? with low dose of C6-Pep further proved C6-Pep' good antitumor activity,presenting it great application potential as antitumor peptide.