BDNF gene-PEI derivative nano-particle composition, preparation method and application thereof
A nanoparticle and derivative technology, applied in gene therapy, drug combination, pharmaceutical formulation, etc., to achieve the effects of high transfection efficiency, uniform particle size distribution, and strong DNA binding ability
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Embodiment 1
[0045] Example 1 Preparation of BDNF 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 800Da, X is 2,5-imidazole dicarbaldehyde) and dissolve it in water to make the concentration 2.0 mg / ml, pass through 0.45 μm water After membrane filtration, it is ready for use; draw a certain amount of BDNF gene solution and dilute it with water to form a 20 μg / ml BDNF gene stock solution. When preparing the complex of PEI derivatives and BDNF gene nanoparticles, according to the set mass ratio of a series of PEI derivatives and BDNF genes, the solution of PEI derivatives is diluted to the corresponding concentration, and then quickly added to the same volume and fixed concentration of BDNF In the gene solution, the final concentration of the BDNF gene was 20 μg / ml. Finally, the solution was uniformly mixed at 1000 rpm, and incubated at room temper...
Embodiment 2
[0047] Example 2 Agarose gel electrophoresis of BDNF 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, referring to the preparation method of the nanoparticle composite solution in Example 1, prepare nanoparticle composite solutions with different mass ratios, the mass ratios of PEI derivatives and BDNF genes in the nanoparticle composite solution are 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× loading buffer ...
Embodiment 3
[0049] Example 3 Transmission electron microscope observation of BDNF 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 BDNF gene of 20 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 the BDNF gene molecule, complexed by electrostatic force to form a complex, showing a spherical nano-particle, and its shape is regular and uniform. It is obvious that the BDNF gene molecule is wrapped in the polymer Inside, the nanoparticles have a particle size of 200 - 300 nm.
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