Bioinformatics Prediction And Cell Experiment Validation Of Membrane Penetrating Properties Of A Novel Cell-penetrating Peptide P1

Background Biomacromolecule drugs such as peptide,protein,nucleic acid and so on have good therapeutic effect in a variety of diseases.However,due to the existence of cell membrane,it is difficult for these drugs to enter into tissues and cells to exert their effects,which greatly limits their practical clinical application.Therefore,how to safely and efficiently deliver biomacromolecule drugs into cell is an urgent problem to be solved in the current drug delivery system.Cell Penetrating Peptides(CPPs)are a type of short peptides that can penetrate cell membranes,which is a new drug delivery tool that has received wide attention.Objective This study uses bioinformatics methods to analyze and design a new type of CPP-P1,and verify its cell membrane penetration ability and the ability as a vector to mediate the transfection of plasmid DNA into cells,to provide theoretical guidance for P1 as a potential drug delivery tool.Methods(1)Based on the latest research progress and the reported sequence-structure characteristics of CPPs,our research group used bioinformatics technology to screen peptides that might have the ability to penetrate membrane;used bioinformatics server to predict its physicochemical property,secondary structure,3-D structure,immunogenicity,toxicity,hemolysis,sensitization,half-life,membrane penetration ability,etc.(2)FITC-P1 labeled with Fluorescein Isothiocyanate(FITC)and nonsense peptide NCO-FITC acting as a control group were synthesized.FITC-P1 or FITC-NCO was co-cultured with cells,the fluorescence distribution in cells was detected by fluorescence microscopy,intracellular fluorescence of the changes in the fluorescence of P1 penetrating membrane into the cells under different conditions was quantitatively analyzed by multimode spectrophotometry.(3)Fluorescence microscope observation and multimode spectrophotometry reader quantitatively detected the influence of temperature and different endocytosis inhibitors on P1 internalization,so as to preliminarily explore its transmembrane mechanism and predict the interaction between peptides and membranes and cell location through bioinformatics methods to analyze its possible mechanism.(4)MTT assay was used to evaluate the effect of P1 on cell proliferation ability and used hemolysis assay to conduct the effect of P1 on cell membrane integrity.(5)According to the different nitrogen to phosphorus ratio(N/P)of the peptide and the plasmid,a certain amount of P1 and pds Red plasmid were mixed to form an electrostatic complex to transfect different cells,and the expression of the plasmid in the cell was detected by fluorescence microscope.Results(1)A new type of CPP-P1 was screened using bioinformatics methods,and its basic properties were accurately predicted.(2)FITC-P1 of different concentrations could penetrate the cytomembrane and enter the cell interior,and the intracellular fluorescence increased to different degrees with the increase of concentration;in a certain time range,the intracellular fluorescence decreased to different degrees with the extension of time;compared with other CPPs,P1 had higher membrane penetration efficiency;the penetrating effect of P1 had no specificity for cell selection;the membrane penetration efficiency of P1 was significantly improved by DMSO,while sucrose could not significantly improve the membrane penetration efficiency.(3)Low temperature could affect the penetration efficiency of P1;different endocytic inhibitors such as Na N3,NH4 CL,EIPA,chlorpromazine,heparin,MβCD,wortmannin,and serum inhibited the membrane penetration efficiency of P1 to different degrees.(4)The penetrating effect of P1 had no obvious effect on cell viability and cell membrane integrity.(5)Under certain N/P conditions,P1 could mediate the transfection of macromolecular substances such as pds Red plasmid into cells.Conclusion(1)A novel type of CPP-P1 with high membrane penetration efficiency is screened by bioinformatics methods combined with in vitro cell experiments,and DMSO can significantly improve the membrane penetration efficiency of P1.(2)The endocytic pathway may be involved in the uptake of P1.(3)P1 has no obvious cytotoxicity.(4)P1 can mediate the transfection of pds Red plasmid into cells.