Study On The Preparation And Performance Of Polyamidoamine-polyethylenimine Copolymers And Their Folate-modified Derivatives As Gene Vectors

Many diseases, such as cancer, HIV and genetic disorders, cannot be completely cured by traditional treatment and technology. With advances in the completion of the human genome project, molecular biology and biotechnology, it has been realized that many diseases can be understand in genetic aspects and cured by gene therapy. Gene therapy can be defined as the treatment of human disease using the delivery of genetic materials, such pDNA, siRNA, MicroRNA and shRNA, into targeted cells to express therapeutic proteins, repair or replace the defective gene, or silence unwanted gene expression. Because naked genetic materials are easily degraded, leading to the loss of therapeutic effect. Therefore, the development of gene delivery vectors is very important for the successes of gene therapy. It has been shown that treatment outcome or gene delivery efficiency is largely depended on the types of vector, which generally can be divided into viral vectors and nonviral vectors. Viral vectors have high transfection efficiency but with some limitations, including immunogenicity, carcinogenesis, limited DNA packaging capacity and difficulty and high cost of manufacture. However, these limitations, especial safety concerns, can be addressed by using non-viral vectors for gene therapy. Despite great progress in the development of non-viral vectors, their relatively low gene delivery efficiency, poor stability, and lack of specificity to diseased cells are still hindering their clinical applications. Many recent studies mainly focused on improving the transfection activity of non-viral vectors. Among the various nonviral gene carriers, cationic polymers, such as polyamidoamine (PAMAM) and polyethylenimine (PEI), have been shown to be effective vectors for in vitro and in vivo gene delivery. Generally, high molecular weight PEIs and high-generation primary amine-terminated PAMAMs display high transfection efficiency and serious cytotoxicity. In contrast, low molecular weight (LMW) PEIs and low-generation (LG) PAMAMs display low cytotoxicity along with low transfection efficiency. Many studies suggested that the transfection efficiency of LMW PEI and LG PAMAM could be improved by conjugation to a biocompatible backbone or core. These vectors also could keep low cytotoxicty. To improve the stability, PEG has been widely used to modify cationic polymers and as a spacer for linking ligand and vector to improve target ability. The aim of this study was to synthesize a kind of copolymer based on LG PAMAM and LMW PEI and its folate modified derivatives for gene delivery.The first part focused on the synthesis, transfection activity and transfection mechanism of PAMAM-PEI copolymer. PAMAM-PEI copolymers were prepared by conjugating PEI 1800 (PEI-1.8k) onto the surface of PAMAM G1.5 or G2.5 by amidation reaction. Their structures were confirmed by FT-IR,1H NMR and GPC. The copolymer with G1.5 or G2.5 as core was named as PAPE-1 or PAPE-2, respectively. The DNA combination capability and the stability against nuclease degradation were evaluated by gel retardation electrophoresis. The results showed that both PAPE-1 and PAPE-2 could completely condense DNA at N/P=2 like gold-standard PEI 25K and could efficiently protect DNA from nuclease degradation. PAPEs were able to self-assemble with pDNA and form spherical nanoparticles with sizes of 70-200 nm and zeta potentials of+13-+33 mV. These were very benefit for cellular uptake and transport in cells. The transfection activity of PAPEs/DNA complexes were first evaluated on HEK 293T cells. The results showed that both PAPE-1 and PAPE-2 in the absence of serum displayed the highest transfection efficiency at N/P=25, which was significantly higher than that of PEI 25K and PAMAM G5 and was lower than that of Lipofectamine 2000 (P<0.05). However, both PAPE complexes in the presence of serum had higher transfection efficiency than that of all positive controls (p<0.05). Compared to PAPE-1, PAPE-2 was more efficient in gene transfection for HEK 293T with the presence of serum (P<0.05). To further study the potential of PAPEs for in vitro gene delivery, PAPEs/DNA complexes at their two optimal N/P ratios of 25 and 90 were incubated with BEL-7402 and COS-7 cells. The transfection efficiencies of PAPEs in both cells were much higher than those of PEI 25k whether under serum conditions or not(P <0.05). PAPE-2 had stronger serum-resistance than PAPE-1. The cytotoxicity of PAPEs was lower than that of PEI 25K and PAPEs/DNA complexes at their two optimal N/P ratios had over 78% cell viability. The transfection mechanism was evaluated by endocytosis inhibition with specific inhibitors, time-dependent transfection, and intracellular trafficking inspection by CLSM. The high level of transgene expression mediated by PAPEs was attributed to caveolae-mediated cellular uptake, the reduced entry into lysosomes and the entry into the nucleus through mitosis.The second part focused on the relationship between transfection activity/cytotoxicity and the structure of PAMAM-PEI copolymer, the synthsies of differnernt divalent folate modified PAMAM G2.5-PEI 423(PME) copolymers and the evaluation of their targeting ability. Due to lower cytotoxicity, PEI 423 was used to instead of PEI 1800 Da for reacting with PAMAM G2.5. The result conjugate was named PME and was confirmed by FT-IR,1H NMR and GPC. Transfection results from various cell lines showed that PME displayed higher transfection activity but lower cytotoxicity than PAPE-2. To improve the stability and specificity to targeted cells, PME was modified with divalent folate-conjugated PEG (PEG-FA2). PME modified with PEG alone or monovalent folate-conjugated PEG (PEG-FA1) was prepared as controls. Their structures were confirmed by’H NMR and UV spectroscopy. The number of PEG, PEG-FA1, or PEG-FA2 values per PME was determined from the integral ratios of the peak of PEG to the peaks of PME. On average, about 1.69 PEG3.5k,1.66 PEG3.4k-FA1, and 1.72 PEG34k-FA2 molecules were conjugated to one PME molecule, respectively. These copolymers were named PME-(PEG3.5k)1.69, PME-(PEG3.4k-FA1)1.66, and PME-(PEG3.4k-FA2)1.72, respectively. PME-(PEG3.4k-FA2)1.72 exhibited strong DNA condensation capacity like parent polymer PME which was not significantly influenced by PEG. PME-(PEG3.4k-FA2)1.72/DNA complexes at N/P= 10 had a diameter-143 nm and zeta potential ～+13 mV and showed the lowest cytotoxicity and hemolysis and the highest transfection efficiency among all tested polymers. In folate receptor positive (FR-positive) cells, the cellular uptake and transfection efficiency were increased with the increase in the number of folates coupled on PEG; the order was PME-(PEG3.4k-FA2)1.72> PME-CPEG3.4k-FA1)1.66> PME-(PEG3.5k)1.69 (all P< 0.05). Folate competition assays showed that PME-(PEG3.4k-FA2)1.72 complexes had stronger targeting ability than PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66 complexes due to their higher folate density per PEG molecule (p<0.05). Cellular uptake mechanism study showed that the folate density on PEG could change the endocytosis pathway of PME-(PEG3.sk)1.69 from clathrin-mediated endocytosis to caveolae-mediated endocytosis, leading to less lysosomal degradation. Distribution and uptake in 3D multicellular spheroid assays showed that divalent folate could offer PME-(PEG3.4k-FA2)1.72 complexes stronger penetrating ability and higher cellular uptake. With these advantages, PME-(PEG3.4k-FA.2)1.72 may be a promising nonviral vector candidate for efficient gene delivery.