Design And Synthesis Of Functional Peptides Based On Different Cellular Uptake Mechanisms And Their Applications In Gene Delivery And Anti-tumor

Cell penetrating peptides（CPPs）are a class of short peptides with cell penetrating function,usually composed of 5～30 amino acid residues and positively charged,which can bind to negatively charged cell membranes through electrostatic attraction and then penetrate cell membranes in a non-specific manner.CPPs can be used for membrane penetration or membrane disruption,and the specific use of CPPs mainly depends on their cellular uptake mechanisms:those CPPs that penetrate membranes non-destructively are generally used as drug delivery vehicles,while those penetrating membranes destructively are generally used for antimicrobial and antitumor applications.The cellular uptake pathways of CPPs include endocytic pathways and direct translocation pathways.Among them,all endocytic pathways and some direct translocation pathways are non-destructive,while some translocation pathways are destructive.Whether the cellular uptake pathway of CPPs is destructive or non-destructive depends mainly on the following factors:CPPs’concentration,cell type and cell membrane properties,CPPs’structural properties and CPPs’self-assembly.These factors are important references in this thesis when selecting different types of CPPs for different applications.Given that CPPs and their cargoes uptaken by cells through endocytosis will first stay in endosomes before entering the cytoplasm,those CPPs and their cargoes that need to function in the cytoplasm have to achieve endosomal escape after cellular uptake.In this thesis,based on the application characteristics of CPPs in gene delivery and anti-tumor research,functional peptides that enter cells in non-destructive and destructive manners are designed and used as gene delivery vehicles and antitumor peptides,respectively.Additionally,such peptides can achieve endosomal escape when entering cells through endocytosis.The thesis is mainly divided into the following two parts:1.In order to overcome the difficulty of gene delivery into glioma cells,peptide segments with different functions were combined together to obtain a series of L-type and D-type peptide-based gene delivery vectors（P-01～P-13）.The functional segments in these vectors mainly include:transmembrane functional segment TAT,endosomal escape functional segment（LLHH）₃ and targeting functional segment RVG29.Among them,the cationic CPP TAT is inclined to deliver DNA into cells through a non-destructive endocytic pathway,（LLHH）₃ can help the peptide vector to escape from endosomes through the"proton sponge"mechanism,and RVG29 can help the peptide vector to cross the BBB and specifically transfect glioma cells.Besides,cysteine and/or glycine residues were introduced at both ends of the（LLHH）₃ segment to improve the stability of peptide/DNA complexs.This part of the work is mainly divided into two stages.In the first stage,the peptide vectors P-01～P-07 without the targeting functional segment were synthesized.We explored the effect of peptide chirality and cysteine residues on the gene transfection efficiency of peptide vectors,and obtained the L-type peptide vector P-03,which could efficiently and specifically transfect glioma cells.In the second stage,the targeting functional segment RVG29 was introduced into P-03 and P-04 to enhance their specific transfection ability in glioma cells（P-10 and P-11）.However,the introduction of RVG29 did not improve the gene transfection efficiency of P-03 and P-04,which could be resulted from the undesired interaction between RVG29 and the rest part of P-10 and P-11,respectively.Subsequent studies confirmed that P-03/DNA complexes could be taken up by glioma cells through a non-destructive endocytic pathway,followed by endosomal escape and nuclear entry,indicating that different functional segments in P-03 could play a synergistic effect.In addition,P-03 could carry p GL3 plasmids across the in vitro BBB model and transfect C6 glioma cells.In vivo,the EGFP gene delivered by P-03 successfully entered the zebrafish brain and achieved gene expression.2.In order to develop a nonspecific anti-tumor peptide with synergistic anti-tumor effect,the anti-tumor peptide pep5-TAT（PT-05）was modified from the perspectives of improving the delivery efficiency and introducing the membrane-lytic mechanism,subsequently a series of nonspecific anti-tumor peptides（PT-06～PT-09）were obtained.In terms of improving the delivery efficiency,the cell-penetrating functional segment R₉and the endosomal escape functional segment（LLHH）₃ were introduced into pep5-TAT to improve the efficiency of cell penetration and endosomal escape respectively,thereby delivering the apoptosis-inducing peptide segment pep5 into the cytoplasm as much as possible;in terms of introducing the membrane-lytic mechanism,the membrane-lytic functional segment s C18₂ was introduced into pep5-TAT.SC18₂ could selectively enter tumor cells and cause tumor cell necrosis through membrane-disruption.Among PT-06～PT-09,PT-09 containing peptide segments pep5,（LLHH）₃ and s C18₂ showed the strongest antitumor activity.PT-09 could enter tumor cells through both non-destructive endocytosis and destructive direct translocation,and the PT-09 entering tumor cells through endocytosis could achieve endosomal escape.Subsequent studies showed that the s C18₂ and pep5 in PT-09 could play a synergistic anti-tumor effect,which acted on tumor cell membranes and the proteasome in tumor cells respectively,thus leading to the necrosis and apoptosis of tumor cells.These processes were accompanied by the production of excess ROS.In vivo,PT-09 possessed stronger ability to inhibit tumor growth than PT-05.In this thesis,a series of functional peptides for gene delivery and anti-tumor applications were designed and synthesized based on different cellular uptake pathways.A peptide-based gene delivery vector with specific transfection ability of glioma cells and a nonspecific anti-tumor peptide with a synergistic anti-tumor mechanism were obtained.This study provided valuable insights into the design of functional peptides for different applications,and had important scientific significance.