Gene transfection method based on polyamide arborescent macromolecule carrier

A technology of polyamidoamine dendrimers and macromolecular carriers, which is applied in gene therapy, the use of carriers to introduce foreign genetic material, and pharmaceutical formulations, etc., to achieve the effects of easy operation, mild transfection conditions, and good application prospects

Inactive Publication Date: 2012-10-03
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Retrieval of related literature and patents at home and abroad shows that there is no report on the method of using lactobionic acid-modified polyamidoamine dendrimer as a targeting carrier for targeted gene transfection of liver cancer

Method used

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  • Gene transfection method based on polyamide arborescent macromolecule carrier
  • Gene transfection method based on polyamide arborescent macromolecule carrier
  • Gene transfection method based on polyamide arborescent macromolecule carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Weigh 15.7 mg of lactobionic acid (La) and dissolve it in 2 mL of phosphate buffer (pH=6.0). Add EDC (0.30M, 0.726mL) and NHS (0.25M, 0.892mL) equivalent to five times the molar number of lactobionic acid carboxyl groups, and react with strong magnetic stirring for 3 hours.

[0049] Weigh the fifth generation polyamidoamine dendrimer (G5.NH 2 ) 41.5mg, dissolved in 4mL of distilled water to prepare a solution with a concentration of 10.4mg / mL. According to La / G5.NH 2 The molar ratio is 11 / 1, add the activated lactobionic acid solution to G5.NH 2 solution, stirred magnetically at room temperature, and reacted for 3 days.

[0050] After the reaction, the reaction product was transferred to a dialysis bag with a molecular weight cut-off of 14,000, and dialyzed in distilled water (2L×6, 3 times a day) for 2 days. Then lyophilized to obtain G5.NH 2 -La reaction product. For the resulting product G5.NH 2 -La carried out NMR characterization, the results are shown in Fi...

Embodiment 2

[0052] G5.NH prepared according to the method of Example 1 2 -La and the fifth-generation polyamidoamine dendrimer G5.NH with amino terminal groups 2 Perform gel retardation experiments with pDNA. Prepare 8-well agarose gel (1.0% w / v) containing ethidium bromide (0.1 μg / mL), and place at room temperature until the agarose gel solidifies. Taking 1 μg of DNA as an example, prepare vector / DNA complexes according to different N / P (0.25, 0.5, 1, 2, 3, 4, 5), and use naked DNA as a control. Get the G5.NH that embodiment 1 obtains 2 -La, dilute to 50 μL with sterile water, then dilute 1 μg DNA to 50 μL with sterile water, then mix well to a final volume of 100 μL, and incubate at 37°C for 30 minutes. Then the corresponding carrier / DNA complexes were added to the wells of the agarose gel respectively, with a voltage of 80V and a time of 35min. The migration of DNA in the gel was analyzed using a gel imager (UVP, USA). The result is as shown in Figure 2. The results showed that G...

Embodiment 3

[0054] G5.NH prepared according to the method of Example 1 2 -La and G5.NH 2 After complexing with 1 μg DNA (N / P=2.5), take 5 μL of the complexed solution and drop it into a mica sheet of appropriate size, and air dry it. The surface morphology of the composite was observed with an atomic force microscope (Veeco Instruments), and the results are shown in Figure 3. The results showed that the complexes were all spherical, indicating that G5.NH 2 -La and G5.NH 2 Both can complex plasmid DNA, making it possible to transfect genes into cells. In addition, G5.NH 2 / DNA complex particle size ratio G5.NH 2 -The particle size of the La / DNA complex is large, indicating that the G5.NH 2 -La compresses DNA better.

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Abstract

The invention relates to a gene transfection method based on a polyamide arborescent macromolecule carrier. The gene transfection method comprises the followings: lactobionic acid decoration to the surface of polyamide arborescent macromolecule to obtain G5. NH2-La; (2), preparation of lactobionic acid decorated polyamide arborescent macromolecule carrier and plasmid compound so as to obtain G5.NH2-La and plasmid compound G5.NH2-La/p DNA; and (3), lactobionic acid decorated polyamide arborescent macromolecule plasmid compound targeting gene transfer and expression to hepatoma carcinoma cells. The functionalized polyamide arborescent macromolecule plays a targeting effect on the hepatoma carcinoma cells; and the gene transfection method provided by the invention has the advantages of temperate transfection conditions, easiness in operation, high transfection efficiency, excellent specificity and the like, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of targeted gene transfection of macromolecule nanocarriers, in particular to a gene transfection method based on polyamidoamine dendrimers. Background technique [0002] At present, the expression regulation of cells has been paid more and more attention, and gene transfection is an important means to realize the regulation of cell expression. The vectors used for gene transfection are divided into viral vectors and non-viral vectors. Although viral vectors have high transfection efficiency, they are limited by their own shortcomings such as immunogenicity, high toxicity, and incapability of large-scale production. Non-viral vectors, especially polyamidoamine dendrimers (PAMAM), have attracted increasing attention due to their low toxicity, high efficiency, and large-scale production. In the expression regulation of specific parts (such as liver tissue), many gene carriers will reduce the efficiency of cell express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/34C12N15/63A61P35/00
Inventor 史向阳姜慧欣刘辉
Owner DONGHUA UNIV
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