The Development Of Nonviral Gene Delivery Vectors Based On Polyethylenimine As Backbone

The basic concept underlying gene therapy is the treatment of diseases by the delivery of normal genes or therapeutic genes into specific cells of patients in order to correct or supplement defective genes responsible for disease development. The carriers that deliver the genes are called vectors. Four critical elements are involved in the manipulation of gene therapy: the functional genes, gene delivery vectors, targeting cells (tissues) and suitable delivery routes. Now the vectors are divided into two categories including viral and non-viral vectors, with viral vectors more commonly employed. Though viruses are quite effective gene delivery vectors, it should also be noted that there are still considerable problems such as immunogenicity of viruses, the limited carrying capacity and carcinogenicity. During the past two decades, more scientists have shifted their interest to develop gene delivery systems based on non-viral vectors. The non-viral vectors include liposomes, nanoparticles and colloidal particles with the advantages of low cost, simple synthesis, easily manipulation, large carrying capacity and high safety. However, there are some disadvantages with non-viral vectors such as relatively low gene delivery efficiency and no specific cells or tissues targeting abilities.Polyethylenimine (PEI) is a kind of polymer commonly used in the paper manufacture, formed with the polymerization of aziridine monomers with the catalysis of acids. Due to the difference of the synthesis, there are 2 forms of PEI: linear and branch ones. Under the physiologic pH conditions, only 1 nitrogen of 5-6 amines of PEI is protonated and with the combination of nucleic acid, 1 nitrogen of 2-3 amines of PEI can be protonated. When PEI enters into endosomes, the nitrogenof the amines can be further protonated with the decreased pH. The more protons are captured by PEI and cause the Cl^- inflow over the membranes of endosomes, resulting that the endosomes are disrupted by the osmotic pressure and the plasmids DNA are released into cytoplasm. PEI is one of the non-viral vectors with high efficiency and acts as a benchmark in the research field of non-viral vectors. However, the disadvantages such as lower transgene efficiency compared with viral vectors, high cytotoxicity with high molecular weight PEI, non-degradable characters and having no specific cells or tissues targeting ability limit the usage of PEI especially in vivo. In order to improve the characters of PEI for better behaviors in gene delivery and get ideal nonviral vectors, it is necessary to modify PEI. Some strategies like ligand-conjugation and PEGylation have being studied, but no commercial reagents of modified PEI are available yet.The purpose of the study was to modify PEI to acquire ideal vectors with advantages like high gene delivery efficiency low cytotoxicity specific cells or tissues targeting ability and suitability for in vivo employment. The whole study was divided into three parts and three strategies were used to modify PEI: ligand peptides conjugation, PEGylation and juncture of low molecular weight PEI using 8armPEG as a core structure to get novel vectors. On the basis of successful synthesis of vectors, the physicochemical characters and cytotoxicities of the vector, the interaction with plasmids DNA, the in vitro gene delivery efficiencies, specific cells targeting ability and in vivo gene delivery efficiency were investigated; at the same time the transgene mechanism was also studied.In the first part of the study, considering most cancer cells have high expression level of FGFR and integrin, two peptides, targeting to the FGFR and integrin on cells surface respectively, were co-conjugated onto PEI25k with certain ratio to make up dual receptors targeting vectors. The aim of this part of study was to observe whether the dual targeting vectors would enhance transgenic efficiency, have lower cytotoxicity and higher cells targeting ability. In this part of work, immunhistochemistry methods and peptides YC25 labeled with FITC were firstly employed to screen the FGFR positively expressed cells (COS-7 and HeLa cells) andFGFR negatively expressed cells (PC-3 cells) to act as model cells for gene delivery experiments. In the results of FTIR and UV absorption spectrum, the appearance of peaks at 1630cm^-1 and 270 nm validated the successful synthesis of peptides conjugated PEL The conjugation ratios of YC25 and CP9 were 3.6 and 6.4 respectively by the detection and the calculation of the peaks integral in the ¹H NMR spectrum, with the identification of the characteristic peaks of -CH3, benzene ring derived from peptides and H proton derived from PEL The synthesized vector YC25-PEI-CP9 could efficiently condense pDNA at N/P ratio 4 and visualized by gel retardation assays. Through electrical microscope observation, YC25-PEI-CP9 was found to condense plasmids DNA into nanoparticles with about 200 nm in diameter when the N/P ratio was 10 which were further proved by particle size determination. The particle zeta potential tests proved the zeta potential of the nanopartilces were about 30.9 ± 3.6 mV at N/P ratio 10. The MTT assay in COS-7 cells displayed the lower cytotoxicity of the YC25-PEI-CP9. At N/P ratio 10, YC25-PEI-CP9 displayed the highest gene delivery efficiency whether compared with PEI25k or single peptide conjugated PEI25k. The FGFR and integrin targeting ability of the synthesized vectors was proved by free peptides inhibition tests. In the in vivo experiments, YC25-PEI-CP9 also displayed the highest efficiency in the nude mice with HeLa cells inoculated, however, in that with PC-3 cells inoculated, the synthesized CP9-PEI showed the highest efficiency. YC25-PEI-CP9 showed about half of the toxicity compared with PEI25k in the acute toxicity experiments underwent in SD rats and ICR mice. Through the first part of the study, a novel kind of nonviral vector with dual receptors targeting ability was constructed. It showed advantages like high gene delivery efficiency and specific cells or tissues targeting ability, however, high cytotoxicy and the positive zeta potential of the polyplexes made it unsuitable for employment in vivo through systemic route.In the second part of the study, in order to shield the positive zeta potential of the polyplexes and decrease the cytotoxicity of the vectors for constructing novel kind of vectors which is suitable for in vivo application, polyethylene glycol (PEG) with molecular weight 3400 Da was employed to modify PEI25k and furthermore, thepeptides GT10 targeting FGFR were adopted to conjugate onto the PEGylated PEI25k with different linkage ratio (1 2 and 4). Firstly, NIH3T3 and HepG2 cells, but not PC-3 cells were proved the positive combination of the peptides GT10 by free peptides combination experiments. The disappearance of the absorption peaks of 1732 cm^-1 and the appearance of the peaks of 1658 cm^-1 on FTIR spectrum suggested the successful synthesis of the vectors PEI-PEG-GT10. The conjugation ratio of 1 2 and 4 of the PEGylation and the peptides GT10 was proved by ¹H NMR. The vectors PEI-PEG-GT10 could efficiently condense plasmids DNA at N/P ratio 4-5 by gel retardation assay. The particle size assays showed that when N/P ratio was above 30, the vectors could condense the plasmids DNA into nanoparticles with about 200 nm in diameter. The nanoparticles could counteract the aggregation of the particles with the time lasting. The spherical loose structure of the peri-nanoparticles was observed by electrical microscope. The zeta potential determination assays showed that at N/P ratio 30, PEI-PEG-GT10 could condense plasmids DNA into particles with nearly neutral zeta potential. MTT assay in vitro showed the much decreased cytotoxicity of PEI-PEG-GT10. PEI-PEG-GT10(1) showed the highest gene delivery efficiency at N/P ratio 30 in NIH3T3 cells and HepG2 cells, but in PC-3 cells, the conjugation of peptides GT10 did not bring the benefit of the gene delivery efficiency to PEI-PEG The FGFR targeting ability of PEI-PEG-GT10 was proved by free peptides GT10 inhibition tests. The gene delivery experiments in transfer system with 10% serum showed PEI-PEG-GT10 could efficiently counteract the inhibition of serum. In the acute toxicity experiments in SD rat or ICR mice, PEI-PEG-GT10(1) showed much decreased toxicity compared with PEI25k. In the nude mice inoculated with HepG2 cells, PEI-PEG-GT10(1) showed magnificent gene delivery efficiency in the tumor tissues by tail vein injection and anti-tumor growth effect when carrying plasmids pCSK-α-IFN as therapeutic genes. Through the second part of the study, PEGylated PEI conjugated with peptides targeting FGFR of different ratio was successfully constructed. It showed advantages like high transgene efficiency, capability of counteracting serum inhibition effect and specific cells or tissues targeting ability. When employed in vivo with therapeutic genes, it displayed most suitable charactersand could efficiently inhibit the growth of tumor. However, it still showed some cytotoxicity.In the third part of the study, referring to the low cytotoxicity of low molecular weight PEI (PEI600) and the potential shielding capability of PEG, the author explored the possibility of developing a novel kind of vector. In this part of study, 8armPEG was employed to link PEI600 into high molecular weight polymers and based on the synthesized polymers, FGFR targeting peptides MC11 were sequentially conjugated to endow them the specific receptors targeting ability. The appearance and the disappearance of the absorption peaks of 1730 cm^-1 and 1670 cm^-1 on FTIR spectrum proved the successful synthesis of the 8armPEG-PEI600-MC11. The changed thermo-decomposition character of 8armPEG-PEI600-MC11 was proved by TGA. The molecular weight of 8armPEG-PEI600-MC11 was proved through GPC methods using dextran as molecular weight markers. The conjugation ratio of PEI600 and MC11 was testified by ¹H NMR. The results of gel retardation assay showed that at N/P ratio 3, 8armPEG-PEI600-MC11 could efficiently condense plasmids DNA and retard the DNA mobility and to PEI600, the corresponding N/P ratio was 4. Through particle size and zeta potential assay, 8armPEG-PEI600-MC11 was found to efficiently condense plasmids DNA into nanoparticles with about 200 nm in diameter and some less than 20 mV in zeta potential at N/P ratio 30. The MTT tests in vitro showed that 8armPEG-PEI600-MC11 had very low cytotoxicity like PEI600. HepG2 and PC-3 cells were then employed to testify the gene delivery efficiency of 8armPEG-PEI600-MC11 in vitro and check the role of the conjugated peptides MC11. The results showed that 8armPEG-PEI600 could reach its highest efficiency at N/P ratio 30 and the conjugation of MC11 could efficiently improve the efficiency in HepG2 cells, showing 11 times of efficiency than PEI25k with N/P ratio 10. The free peptides MC11 inhibition tests in vitro proved the FGFR targeting ability of 8armPEG-PEI600-MC11. In vivo tests proved that a large part of 8armPEG-PEI600-MC11 vectors could reach the tumor tissues by tail vein injections and bring about 18 times of gene deliver efficiency than PEI25k. If the plasmids pCSK-α-IFN acted as therapeutic genes, 8armPEG-PEI600-MC11 group showedefficient tumor growth effect by tail vein injections. Through the third part of study, the novel vector 8armPEG-PEI600-MC11 was successfully constructed and found with the advantages such as high gene delivery efficiency, very low cytotoxicity and outstanding FGFR targeting ability. But at the same time, the positive of the polyplexes, though less than PEI25k, brought hindrance for better applications in vivo. In summary, three strategies were employed to modify PEI in the study: 1) dual targeting peptides conjugation, 2) PEGylation and 3) linkage of low molecular weight PEI600. The all synthesized vectors could efficiently condense plasmids DNA into nanoparticles suitable for gene delivery with optimal conditions. With the conjugation of peptides, the vectors were endowed with specific receptors targeting ability. All the vectors showed high gene delivery efficiency compared with PEI25k and prospect in usage of in vivo. However, the synthesized vectors in this study still bear some disadvantages along with the advantages of high gene delivery efficiency, targeting ability, low zeta potential and low cytotoxicity, need more study for further improvement. As to YC25-PEI-CP9 and PEI-PEG-GT10, they still displayed disadvantages like certain cytotoxicity, positive zeta potential of the formed nanoparticles and the non-degradable characters of the main backbone. As to 8armPEG-PEI-MC11, though with very low cytotoxicity, the comparatively positive zeta potential of the polyplexes made it difficulty to use in vivo. The wide coverage of multiple disciplines like organic chemistry analytical chemistry macromolecule chemistry pharmacology molecular biology and oncology in the study brings novel research ideas for constructing novel kind of nonviral gene delivery vectors. The target selected in the study has great prospect in the cancer gene therapy. Kinds of physico-chemical analysis methods the design and the modification strategies of the targeting peptides have great innovative. The definite synthesis route of the vectors with favorable repeatablity and feasibility of practice lays a good foundation for batch industrial preparation. Though the novel vectors synthesized in the study still has some disvantages, we convince that ideal vectors can be developed with further modification strategies like reconstruction of the backbone PEI25k with integration of PEG chain, acylation of the amine groups or conjugation with hydrophobic groupsand construction of co-delivery vectors carrying genes and drugs in future.