Polyethylenimine Derivate Modified By Different Functional Peptides As Novel Gene Vectors

Based on genetic engineering and molecular genetics, gene therapy is a new method to cure cancers. The nature of gene therapy is to feed therapeutic genes into target cells by gene vectors to cure diseases caused by genetic defects and abnormal. Gene therapy can therapy a variety of diseases such as cancer, cardiovascular disease and genetic diseases. The present study mainly focused on the treatment of tumor. Searching for a reliable and effective gene delivery system is the key of gene therapy. Gene carrier include viral vectors and non-viral gene vectors. Non-viral gene vector transfer gene by receptor mediated method, it has non-infection,easier to preparation and has the capability of carrying large amounts of genetic materials. Compared with viral gene vector, the non-viral gene vector has lower toxicity, lower immunogenicity and the gene it carried would not connected to the host cells. Therefore, the non-viral gene vector had became a hot topic in recent years. Among non-viral vectors, cationic polymers have been widely explored in gene delivery research.Polyethylenemine(PEI) and its derivatives are the most extensivelyinvestigated because of the “proton sponge” effect. There are some problems in the application of PEI as gene vector. It has been documented that the high molecular weight PEI has high gene transfection ef?ciency but high cytotoxicity; Low molecular weight PEI exhibits low cytotoxicity but low transfection ef?ciency. In addition, there is an inconsistency between its stability in cytoplasm and cellular uptake. It has been proved that PEI derivatives obtained by crosslinking low molecular weight PEI with degradable materials display higher transfection efficiency and lower cytotoxicity. And last, PEI had been proved to be lack of targeting effect,because it was connected to the cells by electrostatic interaction.Electrostatic interaction has no cell specificity.Pluronic is a kind of triblock copolymer, which consist of hydrophilic ethylene oxide(EO)- hydrophobic propylene oxide(PO)- hydrophilic ethylene oxide(EO) blocks. These blocks formed a PEO-PPO-PEO structure. Pluronic are amphiphilic molecules that can be used as structural elements of the polycation gene-delivery systems. Pluronic are non-toxic,non-stimulation, non-immunogenicity and soluble in cytoplasm, and become one of the most common materials for the preparation of polymeric micelle. The structure of Pluronic is flexible. The high PEO blocks rate lead to high water solubility. The high PPO blocks rate lead to low water solubility. Pluronic form molecular micelles in water solutionspontaneously, which size is similar to the size of molecular micelles formed by virus. Therefore, Pluronic are suitable for transport in vivo.Integrin is an important kind of cell surface receptor. Integrins can recognize different distinct proteins as receptors. Integrins are made of α(120-185 k D) and β(90-110 k D) subunits. So far over 20 integrins have been found. Among them αvβ3 receptors are highly expressed on tumor cells and tumor blood vessels but rarely detectable on quiescent blood vessels. So αvβ3 has been used as the targets for antitumor drugs.Arginine-glycine-aspartate(RGD) peptide is a key binding moiety that has been shown to bind specifically to αvβ3 integrin receptors. This had provided a new way to improve the targeting effect for cancer therapy.Cell-penetrating peptides(CPPs) are amino acid long peptides, which have the capable of translocate various macromolecules across the plasma membrane and target to the cell nucleus. One of the most typical CPPs is a small stretch of basic amino acids with the sequence RKKRRQRRR and is known as TAT(amino acids 49-57 of the HIV TAT protein). The nuclear localization signal NLS is a necessary signal sequence that mediates the proteins entering the cell nucleus through the nuclear pore complex. NLS has been suggested as a means to promote nuclear delivery of expression constructs. The three functional peptides play different roles in the process of complex entering into cells. Designed three kinds of peptides based onRGD, coupling with PEI derivatives to form three new different PEI derivatives as gene vector. Evaluate the role of three different peptides in the application of gene vector by comparing their physicochemical properties and cell transfection efficiency.Therefore, a PEI derivate was synthesized by cross-linking low molecular weight PEI with P123, which exhibited good degradability and low cytotoxicity. Meanwhile, we synthesized 3 kind of peptides:(1) RGD peptide(named R4);(2) RGD peptide conjunct with NLS to yield a bifunctional peptide RGD-NLS(named R11);(3) RGD peptide conjunct with TAT(49-57) and NLS to yield a new chimeric peptide RGD-TAT-NLS(named R18). Adopting the 3 kinds of functional peptide R4、R11 and R18.were respectively conjugated to P123-PEI to form new polymeric gene vector P123-PEI-R4、P123-PEI-R11 and P123-PEI-R18. Comparing their physicochemical properties and transfection efficiency to evaluate the role of three different peptides in the application of gene vector.Chapter one gave an overview on the application of PEI, Pluronic P123 and functional peptide as gene vector. Characteristics of PEI derivatives and Pluronic were introduced. The research hotspots of functional peptide were also analyzed.Chapter two was synthesis and characterization of three new polymeric gene vector. At first, these functional peptide R4, R11 and R18 was prepared by the solid phase method. The structure and content were performed respectively by MS and HPLC. Then we crosslinked P123 activated by bis-(trichloromethyl)-carbonate and solid N-hydroxysuccinimide to PEI 2 KDa to get a high molecular weight PEI derivate P123-PEI. At last P123-PEI was modified by R4, R11, R18 respectively and the novel gene vector was obtained as P123-PEI-R4 、P123-PEI-R11 and P123-PEI-R18. Every product was characterized by1H-NMR. The results indicated that R4, R11, R18 was synthesized successfully with the correct sequence and its content was as high as 95%.The functional peptide R4, R11, R18 was also binded to P123-PEI by SMCC successfully.Chapter three was the physicochemical property of the new gene vectors P123-PEI-R4, P123-PEI-R11 and P123-PEI-R18. Buffering capacities of gene vectors was measured by acid-base titration. The particle sizes and zeta potentials of these polymer/DNA complexes were also measured. The capacity of DNA condensation and protect DNA were determined by agarose gel electrophoresis. The cytotoxicity of these polymers on the B16 cells were measured by the MTT assay. The results indicated that P123-PEI-R4, P123-PEI-R11 and P123-PEI-R18 had good buffering capacities compared with pure water. The particle sizes and zeta potential of these complexes were proper. P123-PEI-R4, P123-PEI-R11 andP123-PEI-R18 was able to condense DNA effectively and neutralized its charge at w/w ratio of 3.0, 0.4 and 2.0. All these polymers can protect DNA from being digested by DNase I and FBS at high concentrations.Furthermore, P123-PEI-R4, P123-PEI-R11 and P123-PEI-R18 showed significantly lower cytotoxicity in B16 cell line compared with PEI 25 k Da.Chapter four was the measurement of gene transfection efficiency of the vectors. We examined the ability of P123-PEI-R4, P123-PEI-R11 and P123-PEI-R18 to transfect in B16 cells using the plasmid p EGFP-N2 and p GL3-Control as reporter genes.GFP transfection and luciferase activity were respectively determined using inverted microscope and a luminometer so that we can the quantitatively and qualitatively studied the transfection of P123-PEI-R4/DNA, P123-PEI-R11/DNA and P123-PEI-R18/DNA complexes in vitro. The transfection experiments in vitro showed that transfection efficiency of P123-PEI-R4/DNA, P123-PEI-R11/DNA and P123-PEI-R18/DNA complexes increased as the w/w ratio increasing. All synthesized complexes showed much higher gene transfection efficiency compared with P123-PEI. We compared P123-PEI-R4/DNA,P123-PEI-R11/DNA,P123-PEI-R18/DNA and P123-PEI-R13/DNA(research group synthesized previous), showed that transfection efficiency of P123-PEI-R11 is the best, and the NLS can promote nuclear delivery of expression.We developed three kinds of novel gene vector P123-PEI-R4,P123-PEI-R11 and P123-PEI-R18, which had low cytotoxicity and high gene transfection efficiency. According to the result of compare,P123-PEI-R11 is the best as the gene vector, NLS is the key to promote nuclear delivery of expression.This study can form the base of problems solving and practical applications of PEI as a non-viral gene delivery vector.The contents of this work display theoretical as well as practical values.