Design Of Functional Peptides And Applications In Gene Transfection

Gene transfection,a common method in gene therapy,is the frontier and hot topic in biology and science.During the research of biomaterials,many non-viral gene vectors including liposomes,inorganic nanoparticles,cationic polymers and peptides have been produced to solve the problems in gene transfection.Among the numerous gene vectors materials,peptides have attracted more and more attention and have great potential for being used as excellent gene carriers due to their easy production,high biocompatibility and functional diversity.In this paper,we designed and synthesized several functional peptides,studied their self-assembly behavior in buffer solutions,cytotoxicity towards different cell lines,their interactions with DNA and applications in gene transfection.The main results are concluded as follows:（1）We designed and synthesized a series of functional peptide molecules,which had three functional fragments including a transmembrane peptide sequence,a matrix metalloproteinase 7（MMP7）response site and a tumor-targeting site.Their molecular structure are Ac-RGDGPLGLAGI₃GR₈-NH₂（RR-22）,Ac-RGDGPLGLAGI₃GR₆-NH₂（RR-20）and Ac-RGDGPLGLAGI₃GR₄-NH₂（RR-18）.Through cytotoxicity experiments,a molecule RR-22,which can kill the cancer cells selectively,was picked out from these three molecules and further studied.In the concentration range of 50-100μM,the survival rate of normal cells is above 90%,and the cancer cells is only about 20%.The further research results showed that RR-22 itself could not form ordered structures in buffer solution.In addition,MMP7 could act on the molecular enzymolysis site,PLGLAG,to cleave the molecule,and the fragment LAGI₃GR₈-NH₂ had the cell-killing function.RGD sequence could target to the surface of cancer cells,effectively increase the cell selectivity.When the+/-charge ratio of peptide to DNA is 5,DNA can be condensed into dense aggregates which diameter are about200nm.These dense aggregates can prevent the degradation of DNA by nuclease.The transfection efficiency of DNA can be as high as 70%,indicating the great potential as gene vectors.Therefore,the multifunctional peptide,RR-22,is expected to achieve both selective killing of cancer cells and efficient gene transfection.（2）A series of amphiphilic peptides were designed and synthesized.Their molecular structure are Ac-IGPLGLAGGR₈-NH₂（IR-17）,Ac-I₃GPLGLAGGR₈-NH₂（IR-19）,Ac-I₅GPLGLAGGR₈-NH₂（IR-21）and Ac-GPLGLAGI₃GR₈-NH₂（GR-19）The hydrophilic end of the peptide consisted of eight arginines and the difference was the number of isoleucine at the hydrophobic end.Studies showed that the self-assembly ability,the DNA binding ability,the ability to load hydrophobic drugs,and the cytotoxicity were all enhanced with increasing the hydrophobicity of peptide molecule.IR-21 is the ideal drug carrier among the three molecules,which can form a long fibrous structure with a diameter of about 9nm and a length of more than 1μm in the buffer solution and can efficiently encapsulate the hydrophobic dye of Nile red with a loading rate of 0.8%.What’s more,the transfection efficiency is above 20%when+/-charge ratio equals 5,because IR-21 can induce DNA to form aggregation with diameter about 300nm efficiently to protect DNA from nuclease hydrolysis and transfer it into cells successfully.During the gene transfection,the IR-21 has few side effect on cells.Therefore,it may be used as a carrier to transport both hydrophobic drugs and gene simultaneously into cells to realize synergistic effect of chemotherapy and gene transfection.