Construction And Evaluation Of Toxin Peptide Modified Doxorubicin Liposomes

Nanostructured lipid carrier?NLC?is a nanoscale drug carrier based on lipid.It is a highly developed and promising drug delivery system with low toxicity,low immunogenicity,long metabolic cycle,and easily being modified.It has pretty biological properties,including biocompatibility,biodegradability,and the ability to load hydrophilic and hydrophobic drugs.Lipid carriers include some carrier forms such as liposomes,polymersomes,and micelles.Liposomes?LPs?refer to microvesicles formed by encapsulating a drug in a lipid-like bilayer.As a drug carrier,liposome can improve the solubility of drugs,increase stability,reduce toxic effects,and improve therapeutic effects.Therefore,liposome has great advantages in the field of tumor therapy,particularly in the delivery and targeting release of chemotherapeutic drugs.As a linear cationic peptide,toxin peptide can induce lipid membrane penetration and self-assembly with lipid membrane to form a pore-like structure.Small molecule drugs can rapidly release from the liposome through the pores,thereby exerting therapeutic effects.The hydrophilic layer of hydrophilic polymer polyethylene glycol?PEG?can hinder the recognition and scavenging effect of the reticuloendothelial system on the nanocarrier.Pegylation can offer nanocarrier a longer circulation.PEG has a low effect on the biological properties of the carrier.Therefore PEG-modified nanocarriers are widely used.Matrix metalloproteinases?MMPs?are proteolytic enzymes.MMP-2 and MMP-9 are highly expressed in many malignant tumors,which can promote the secretion of tumor growth factors and accelerate tumor growth,invasion and metastasis.In this study,peptides and PEG were used as raw materials to synthesize functional peptides by Michael addition reaction.The liposomes were modified with synthesized functional peptides.The blank liposome was prepared by thin-film hydration method,and doxorubicin?DOX?was used as a model drug.The DOX entrapment of liposomes was carried out by ammonium sulfate gradient method.The liposomes were characterized by particle size,potential and encapsulation efficiency.The prepared liposomes were evaluated for enzyme sensitivity by dialysis method.The toxic effect of liposomes on human breast MCF-7 cancer cells was investigated by methyl thiazolyl terazolium?MTT?assay.Part one Preparation and characterization of liposomesObjective:To synthesize a series of functional polymers,prepare DOX liposomes and pore-forming peptide-modified DOX liposomes.Characterize these two types of lipid carriers.Methods:The thiol-containing toxin peptide was conjugated to a PEG containing a maleimide group by a Michael addition reaction,and the progress was monitored by a TLC method during the reaction.The blank liposome was prepared by thin-film hydration method.The chemotherapeutic drug doxorubicin was encapsulated by ammonium sulfate gradient method.The functional polymers were modified into liposomes to prepare a pore-forming peptide-modified DOX liposomes.Results:Five kinds of functional polymers were obtained by the synthesis.As presented by thin layer chromatography,the conjugations of peptide to PEG were completed.The two types of DOX liposomes have uniform color.The particle size is normal monomodal distribution,and the particle size is small?100²00 nm?.The polydispersity index?PDI?is low,and the encapsulation efficiency is higher than 70%.Conclusions:In this study,five functional polymers were successfully synthesized,and two kinds of DOX liposomes were constructed.The preparation method was simple with good reproducibility.The encapsulation efficiency was high.Part two Evaluation of enzyme sensitivity of liposomesObjective:The enzyme-sensitivity of the toxin peptide-modified lipsomes was evaluated by measuring the release level of DOX.Methods:The permeability of the toxin peptide DOX liposome was firstly examined by dialysis method.The enzyme sensitivity was evaluated by dialysis in the presence of collagenase.Results:The enzyme sensitivity test of liposomes showed that the liposomes had low permeability to the drug in the absence of collagenase.However,with the addition of collagenase of a certain concentration,the loaded DOX could get different degrees of enzyme-sensitive release.Conclusions:On the basis of the evaluation methods of liposome permeability and enzyme sensitivity,we screened the liposome group with high enzyme-sensitivity.Part three Preliminary evaluation of in vitro activity of liposomesObjective:With the investigation of the pharmacological effect of the peptide-modified DOX liposomes on MCF-7 cells,an evaluation will be conducted for the in vitro activity of the prepared lipid carrier.Methods:The cytotoxicity of the peptide-modified DOX liposomes on MCF-7 cells was evaluated by MTT assay.The effects of incubation time,DOX concentration and collagenase concentration on cytotoxicity were investigated.The optimal liposome group was screened out by comparing the cell viability and IC₅₀ values of each group.Results:The cytotoxicity of the toxin peptide-modified DOX liposomes was strengthened with different level under the action of collagenase.Among them,T-PEG1-DOX-LP showed the most enzyme-sensitivity with a sharp reduction?49.81%?in cell survival rate.Conclusions:The effect of different functional polymers on the enzyme-sensitivity of DOX liposomes,was investigated via MTT assay.The T-PEG1-DOX-LP group exhibited the highest enzyme-sensitivity.