PEGylated Poly(2-(Dimethylamino) Ethyl Methacrylate)/DNA Nanoparticles Decorated With TGN Peptide For Brain-targeted Gene Delivery

Gene therapy is becoming an increasing attractive possibility for the treatment of several neurodegenerative diseases. However, therapeutic genes themselves, viral vectors and non-viral vectors can not reach the brain following an intravenous administration due to the presence of blood-brain barrier (BBB). Most genes or current vectors must be given via parenchymal injections which are considered to be highly invasive and unable to deliver genes products to global areas of the brain. Non-viral vectors modified with specific proteins might enable widespread expression of exogenous genes throughout the brain following a non-invasive transvascular pathway. The man problem is the low transfection and expression efficiency. Therefore, developing a brain-targeting and efficient non-viral gene vector to enable the global gene expression in the brain is currently very important for the treatment of neurodegenerative diseases.In this study, a novel efficient brain-targeted non-viral nano-scaled genedelivery system was finally constructed with means of pharmaceutics, macromolecular chemistry and biology. Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) was applied as the main macromolecular gene vector. And the new brain-targeting ligand TGN peptide, obtained by four rounds of in vivo phage display screening from a 12-mer peptide library, was investigated as a brain-targeting ligand in the design of PDMAEMA-based nanoparticles, using polyethyleneglycol (PEG) as a spacer. The characteristics of the vector are showed as follows:(1) employing the cationic macromolecular material, PDMAEMA, as the main gene vector, with high gene encapsulation ability and high transfection efficiency, (2) polyethyleneglycol modified on the surface of the nanoparticles could extend the in vivo residence time, decrease the toxicity of cationic polymers, (3) TGN modified on the surface of the nanoparticles could enhance brain-targeting efficiency.The three sections of this dissertation are as follows:(1) Construction of PEGylated poly (2-(dimethylamino) ethyl methacrylate)/DNA nanoparticles decorated with TGN and its brain delivery characteristics evaluation. (2) The study of TGN-PEG-PDMAEMA used as the gene vector of NGF. (3) The preliminary mechanism study of TGN-PEG-PDMAEMA used as a brain-targeting gene delivery vector.In the first part of this study, MPEG-PDMAEMA and Mal-PEG-PDMAEMA were synthesized by the atom transfer radical polymerization (ATRP) method; the structure of both copolypmers was verified by 1H NMR and GPC. A reaction was performed with Mal-PEG-PDMAEMA and the TGN peptide; the MAL groups of Mal-PEG-PDMAEMA were specifically reacted with the thiol groups of TGN, producing TGN-PEG-PDMAEMA. Appropriate amount of the copolymers was added to pDNA solutions to obtain different N/P ratios nanoparticles. The encapsulation efficiency of different nanoparticles was evaluated by agarose gel electrophoresis and PicoGreen assay. The complete complex formation of copolymers with pDNA was achieved when N/P> 1.25. Comprehensively consideration of particle size, zeta potential, cytotoxicity and in vitro transfection efficiency, N/P= 10 was chose to prepare nanoparticles. Nanoparticles prepared according to the optimal prescription has high encapsulation efficiency (>99.9%), with particle size at around 78.6 nm and zeta potential about 4.82 mV. The cytotoxicity was obviously less than PEI 25 Kd (N/P= 10). The TGN-PEG-PDMAEMA/DNA nanoparticles could resist to external anionic substance and serum while maintain the integrity of the DNA.In order to evaluate the brain-targeting gene delivery characteristics of this TGN modified nanoparticle, two kinds of reporter gene encoding green fluorescent protein and luciferase enzyme were employed for the qualitative and quantitative evaluation. In qualitative study, the GFP expression of TGN-PEG-PDMAEMA/pEGFP was much higher than that of MPEG-PDMAEMA/pEGFP. In quantitative study, the luciferase activity in bEnd.3 cells treated with TGN-PEG-PDMAEMA/pGL polyplexes was approximately 2.96-fold higher than that of MPEG-PDMAEMA/pGL polyplexes. Cellular uptake study also showed similar results. Plasmid DNA was labeled with EMA for the in vivo evaluation of brain targeting. EMA-labeled DNA obviously accumulated in the brains of the mice treated with the TGN-PEG-PDMAEMA/DNA poleplexes, while the fluorescence in the brains of the mice treated with MPEG-PDMAEMA/DNA polyplexes was not as significant. Furthermore, the polyplexes were found mainly distributed in the liver and kidney. Importantly, the conjugation of TGN decreased the accumulation of the conjugates in both the liver and kidney. GFP expression was observed in the section s of the third ventricle, lateral ventricle, hippocampus, substantia nigra, triangular nucleus, hypothalamus and cortical layer and was much higher than that of the MPEG-PDMAEMA/pEGFP polyplexes at 48 h after i.v. injection of 50 ?g/mouse of TGN-PEG-PDMAEMA/pEGFP polyplexes. In quantitative in vivo gene expression assay, the brain gene expression levels of the TGN-PEG-PDMAEMA/pGL polyplexes were approximately 3-fold higher than that of the MPEG-PDMAEMA/pGL polyplexes.In the second part of this study, nerve growth factor gene was chose as the model drug to evaluate the therapeutic effect of TGN-PEG-PDMAEAM/NGF polyplexes in mice model of AD. The TGN-PEG-PDMAEAM/NGF polyplexes has an average particle size of 80 nm, zeta potential of 5 mV. After three times intravenous administration of TGN-PEG-PDMAEAM/NGF polyplexes (1 injection/2 days), the gene product was 1.76-times higher than a single dose. Thereby, the mode of administration was chose for the pharmacodynamic study. The AD model was established by bilateral injection of A?1-40 into mice hippocampus. The Morris water maze experiment was subsequently conducted to evaluate the effect of different NGF loaded nanoparticles on the mice spatial memory deficits. According to the behavioral research and biochemical indicators results, intravenously administration of TGN-PEG-PDMAEMA/pEGFP polyplexes (50 ?g) and MPEG-PDMAEMA/NGF polyplexes (20 ?g) had no improvement on A?1-40 induced AD model mice. The improvement of memory and biochemical index was still limited even the dose was up to 50?g/mouse/time via intravenously administration of MPEG-PDMAEMA/NGF polyplexes. Administration of TGN-PEG-PDMAEMA/NGF polyplexes (10?g) had shown obviously improvement. When the dose of TGN-PEG-PDMAEMA/NGF polyplexes was increased to 20?g/mouse/time, the results demonstrated a protective effect on the hippocampal cells in AD models by decreasing the apoptosis of the nerve cells and lowering the deposition of A? plaque. The trophic action of NGF on the central cholinergic system, including elevating the ChAT activity and maintaining cholinergic nerve function, could weaken the cholinergic system dusfunction and cognitive function impairment caused by AD. Primary safe study revealed that TGN-PEG-PDMAEMA/NGF nanoparticles did not display any side effects on brain, heart, liver, spleen, lung and kidney.In the third part of this study, the preliminary mechanism study of TGN-PEG-PDMAEMA used as a brain-targeting gene delivery vector was conducted. The cellular uptake of TGN-PEG-PDMAEMA/DNA polyplexes was a time, concentration and temperature dependent process. The uptake inhibition test showed that TGN-PEG-PDMAEMA/DNA polyplexes was uptake by a TGN mediated energy-dependent process involving clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis, and had nothing to do with Tf. The confocal laser scanning microscopy study indicated that the TGN-PEG-PDMAEMA/DNA polyplexes were internalized into bEnd.3 cells through a lysosome-mediated pathway and were able to quickly escape from the lysosome, thereby releasing YOYO-1-labeled DNA into the nucleus. Mouse brain tissue sections revealed an obvious accumulation of EMA-labeled TGN-PEG-PDMAEMA/DNA polyplexes, which could be completitively inhibited by TGN equilibrium in advanced. It indicated that TGN played an important role in the gene delivery to brain by TGN-PEG-PDMAEMA/DNA polyplexes. The distribution of GFP in mouse brain sections was observed under a fluorescent microscope. Part of gene product did not co-localized with brain capillaries, indicating the part of TGN-PEG-PDMAEMA/DNA polyplexes crossed the BBB and GFP could be found in neural tissue. Futher invitro cellular uptake assay conducted on PC 12 cells demonstrated that TGN-PEG-PDMAEMA/DNA polyplexes crossed the BBB and scattered in the brain parenchyma but did not target to the nerve cells.