Preparation And Selection Of Optimization Of Cationic Gene Loaded MePEG-PLGA Nanoparticles And The Construction Of PEGFP-BDNF

Background: Alzheimer's disease(AD) is a central nervous system(CNS) degenerative diseases which function of cognitive and memory decline in clinical manifestations. Age is the dominant risk factor in Alzheimer's disease. The increase in the number of new cases of AD is the direct consequence of an improvement in life expectancy. The underlying causes of AD are complicated, despite the tremendous corpus of knowledge of genetics, epidemiology, risk factors and neuropathological mechanisms, there is still no cure for AD. Thus, it is urgent to find out the effective therapeutic measures.Brain-derived neurotrophic factor(BDNF) is a member of neurotrophin family. The important role of BDNF has been firmly established in regulating the survival and differentiation of various neural populations including sensory, cerebellar, and spinal motor neurons. Neurons are the major source of BDNF in the nervous system. It has been reported that the neural precursors culturing in vitro seems to be restricted to the neuronal cell population when added BDNF co-culture. BDNF is a kind of natural protein molecule, which can not pass blood brain barrier(BBB). It has so for not been possible to apply BDNF for the treatment of human diseases because of difficulty in delivering sufficient amounts of BDNF to the proper site of lesions in the central nervous systems. And usually injection into brain may lead to new damage or infection of brain. It was discovered in many researchs that outside source recombined BDNF play a safeguard role to different brain harm. Thus, gene transfection is the only therapeutic tool which can provide long term effect at present, which has been extend applied in many nervous system degeneration diseases research. Gene modified cells transplanted into brain is intensive studying。 In the eukaryotic expression vectors, pEGFP-N1 is a general plasmid vector, which has a fluorescence intensity higher 35 times than GFP. It is very strong in duplication that could make them inherit to descendant cell as the division of the target cell. There are tow powfully efficent promotor, SV40 and CMV, which make the target gene steadily expression in proliferous cell. There is a multiple clone site(MCS) in pEGFP-N1, where the target gene could insert. Tow selective lable gene, kanamycin and neomycin, would help to select cells that contain the plasmid EGFP. These special structure could make the target gene steabily express in target cell. Thus, pEGFP-N1 could be selected as the vehicle of recombinated BDNF.Resently, transgene therapy was increasingly attended. But problems about the gene vehicle and immunogenicity, cytotoxicity, safe restrised restricted its development and clinical application. The carrier must be safe, stable, efficient. Compared with viral vector, non-viral vector was non-immunogenicity and non-genetoxic. Nano-carrier has some special characters such as designability of structure, controllability of size, slow-release, smaller particle size, which was increasingly attended and learned. Researches showed that the particles with an average size less than 100 nanometers were 27 times in transfection efficiency higher than particles with the ones larger than 100 nanometers.Poly(D, L-lactide-co-glycolide), PLGA was an bio-degradation and bio- compatible meterial, which was approved by food and drug administration(FDA). PLGA was generally used in histio-engineering and drug vehicle, which plays role both in frame and sllow-relased, which has an potential value as the carrier of pDNA.However, the hydrophobicity of PLGA restrict its advantage. Polyethylene glycol (PEG) was non-ionic hydrophilia fragment that was generally used as modifying molecule, which was great in biocompatibility, hydrophilia, cell penetrability, non-toxicity in vivo and non-immugenicity. It was approved by FDA. It was that PEG was insert in PLGA could improve the hydrophilia and flexibility of polymer, enhance the stability and anti-enzyme activity, improve the interaction with cells. Moreever, hydrophilia micro-environment formed by PEG could increase the efficiency of DNA-loaded, which was benefit to retain drug activity. Cetyl trimethyl ammonium bromide(CTAB) was an cationic surfactant, which was stability and bio-degradation. Modifying MePEG-PLGA-NPs with CTAB could make nanoparticle surface load positive charge then would efficiently associate DNA in negative charge.In this research we prepared cationic gene loaded MePEG-PLGA-NPs by nanoprecipitation method. This method needn't ultra-sonication or high speed homogenization, nor leding-in homo-temperature or toxicity organic solvent. These merits were benefit to retain bio-activity of bio-senstization molecule. Through univariate analysis and orthogonal design, optimization was selected. Nanoparticles with suitable size and higher Zeta-potential ware prepared. More research about particle average size, morphology, drug-carrier rate, capability of protecting pDNA. We will apply RT-PCR to amplify BDNF gene from totle RNA of rat hippocampus tissue, construct recombination BDNF gene vector system, and select and identify the right combine vector.Conclusion:1 Univariate analysis showed that the average size of nanoparticles increased along with increase of the MePEG-PLGA content and the dropwise speed. But it decreased along with increase of magnetically stirring and acetone volume. Average size was smallest when CTAB concentration was 0.25%, increasing or decreasing CTAB concentration would increase the average size.2 Results of orthogonal experimental design and variance analysis showed that MePEG-PLGA content and magnetically stirring significantly affected on the average size, but affection of the dropwise speed was not significant. Associating univariate analysis, the final confirmation of the optimal prescription for the MePEG-PLGA content was 50mg, CTAB concentation was 0.5%, dropwise speed was 2.5mL/min, magnetically stirring speed was 300rpm, acetone volume was 5mL.3 Average size of nanoparticles prepared in optiminal prescription was 89.7nm, Zeta-potential was 28.3mV. Nanoparticles under transmision elecrtron microscope- (TEM) appeared similar spherical in shape and separated from each other. Rate of DNA-carrier was about 80%. These particles could protect gene from DNase degradation. 4 BDNF gene was successfully amplified by RT-PCR method from totle RNA of rat hippocampus tissue. Recombination BDNF gene vector system was constructed, and the right recombined vector was selected and identified.