NGF (Nerve Growth Factor) gene and PEI (Polyether Imide) derivative nano-particle compound and preparing method and application thereof
A nanoparticle and derivative technology, applied in other methods of inserting foreign genetic materials, gene therapy, drug combination, etc., to achieve uniform particle size distribution, strong DNA binding ability, and high transfection efficiency.
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Embodiment 1
[0045] Example 1 Preparation of NGF gene-PEI derivative nanoparticle complex
[0046] Weigh a certain amount of PEI derivatives (PEI and imidazole dicarbaldehyde to form a copolymer, the weight average molecular weight of PEI is 1500Da, X is 2,5-imidazole dicarbaldehyde) and dissolve it in water so that the concentration is 2.0 mg / ml, passing through 0.45 μm water After membrane filtration, it is ready for use; draw a certain amount of NGF gene solution and dilute it with water to form a 20 μg / ml NGF gene stock solution. When preparing the complex of PEI derivatives and NGF gene nanoparticles, according to the set mass ratio of a series of PEI derivatives and NGF genes, the PEI derivative solution is diluted to the corresponding concentration, and then quickly added to the same volume of NGF with a fixed concentration In the gene solution, make the final concentration of NGF gene 20 μg / ml, and finally mix the solution evenly at 1000 rpm, and incubate at room temperature for 2...
Embodiment 2
[0047] Example 2 Agarose gel electrophoresis of NGF gene-PEI derivative nanoparticle complex
[0048] Weigh 1.0 g of agarose, add 100 ml of 1×TAE buffer, heat and dissolve in a microwave oven, wait until the temperature drops to 65°C, add ethidium bromide (EB) to prepare a 1.0% agarose solution (containing 0.5 μg / ml ethidium bromide), pour it into the gel-making tank, insert the sample comb, and let it stand at room temperature for 0.5-1 hour until the gel solidifies. Then, pull out the sample comb, add TAE buffer to the electrophoresis tank to cover the gel, and wait for the sample to be loaded. Next, with reference to the preparation method of the nanoparticle composite solution in Example 1, nanoparticle composite solutions with different mass ratios were prepared, and the mass ratios of PEI derivatives and NGF genes in the nanoparticle composite solution were 0, 1, 2, 3, 5, 7, 10, 15, 20, 30, 50, 70, 80, 100. Marker chooses DS TM 5000 (100 – 5000 bp), load 2 μl; 6× loa...
Embodiment 3
[0049] Example 3 Transmission electron microscope observation of NGF gene-PEI derivative nanoparticle complex
[0050] According to the preparation method of the nanoparticle complex solution in Example 1, a nanoparticle complex solution with a mass ratio of PEI derivative to NGF gene of 30 was prepared, and the sample volume was 100 μl. First, 10 μl of the nanoparticle complex solution was slowly dropped on a 400-mesh copper grid, and dried naturally at room temperature. Finally, a transmission electron microscope was used to observe the shape of the sample, and the transmission electron microscope picture was recorded. Transmission electron microscopy shows that: PEI derivatives are added to NGF gene molecules, complexed by electrostatic force to form a complex, showing spherical nanoparticles, and its shape is regular and uniform. It is obvious that NGF gene molecules are wrapped in polymers. Inside, the nanoparticles have a particle size of 150 - 300 nm.
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