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Method for applying functionalized poly(amidoamine) dendrimer and nanometer compound thereof in gene transfection

A technology of amine dendritic macromolecules and nanocomposites, which is applied in the field of targeted gene transfection of polymer nanocarriers to achieve the effect of improving transfection ability

Inactive Publication Date: 2013-12-11
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Retrieval of related literature and patents at home and abroad shows that: the method of using PEGylated RGD-modified polyamide-amine dendrimers wrapped with gold nanoparticles as a carrier for targeted gene transfection has not been reported yet.

Method used

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  • Method for applying functionalized poly(amidoamine) dendrimer and nanometer compound thereof in gene transfection
  • Method for applying functionalized poly(amidoamine) dendrimer and nanometer compound thereof in gene transfection
  • Method for applying functionalized poly(amidoamine) dendrimer and nanometer compound thereof in gene transfection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Weigh mPEG-COOH23.16mg, dissolved in 5mL DMSO. Weigh 4.0mg EDC and 2.4mg NHS, dissolve them in 1mL DMSO respectively, add the EDC solution and NHS solution dropwise to the mPEG-COOH solution, and stir for 3 hours. Weigh the fifth generation polyamidoamine dendrimer (G5.NH 2 ) 15.06mg, dissolved in 5mL DMSO. Add the activated mPEG-COOH solution dropwise to G5.NH 2 Solution, magnetic stirring at room temperature, reaction 3d. Get sample K1:G5.NH 2 -mPEG 20 solution.

[0069] Add 198.67 μL HAuCl dropwise to the above solution 4 The aqueous solution (30mg / mL) was mixed and stirred for 30min, and then 27.4μL of 10mg / mL NaBH was added rapidly 4 Solution, reaction 3h. Get sample K2:

[0070] {(Au 0 ) 25 -G5.NH 2 -mPEG 20} DENPs.

[0071] Weigh NH 2 - PEG-COOH 11.58 mg, dissolved in 5 mL DMSO. While stirring, 6-MAL with a dry weight of 1.785 mg dissolved in 1 mL of DMSO solution was added dropwise, and reacted for 8 hours to obtain a MAL-PEG-COOH solution. Add ...

Embodiment 2

[0079] G5.NH prepared according to the method of Example 1 2 -mPEG 20 (K1), {(Au 0 ) 25 -G5.NH 2 -mPEG 20}DENPs(K2), G5.NH 2 -(PEG-RGD) 10 -mPEG 10 (K3), {(Au 0 ) 25 -G5.NH 2 -(PEG-RGD) 10 -mPEG 10}DENPs(K4) formed complexes with pDNA, and performed gel retardation experiments. Prepare 8 wells of agarose gel (1.0% w / v) containing ethidium bromide (1mg / mL), and place at room temperature until the agarose gel solidifies. According to different N / P ratios of 0.125, 0.25, 0.5, 1, 2, and 5, the amount of pDNA was 1 μg / well, and the vector / pDNA complex was prepared and incubated for 30 minutes, and naked pDNA was used as a control. Then the corresponding vector / pDNA complexes were respectively added to the wells of the agarose gel with a voltage of 80V and a time of 30min. Migration of pDNA in the gel was analyzed using a gel imager. The result is attached to the manual Figure 4 shown. The results showed that G5.NH 2 -mPEG 20 (K1), {(Au 0 ) 25 -G5.NH 2 -mPEG ...

Embodiment 3

[0081] G5.NH prepared according to the method of Example 1 2 -mPEG 20 (K1), {(Au 0 ) 25 -G5.NH 2 -mPEG 20}DENPs(K2), G5.NH 2 -(PEG-RGD) 10 -mPEG 10 (K3), {(Au 0 ) 25 -G5.NH 2 -(PEG-RGD) 10 -mPEG 10}DENPs(K4) were combined with 5 μg pDNA (N / P=1, 2.5, 5) respectively, the final volume was fixed at 100 μL, incubated at room temperature for 20 min, and then 1 mL of PBS was added. Its particle size and surface potential were characterized by Malvern laser particle size analyzer (Malvern, MK, 633nm laser), and the results are as attached to the description. Figure 5 shown. The results show that the size of the complexes tends to decrease with the increase of N / P. Under the same N / P (1:1, 2.5:1, 5:1), G5.NH 2 -(PEG-RGD) 10 -mPEG 10 (K3) and {(Au 0 ) 25 -G5.NH 2 -(PEG-RGD) 10 -mPEG 10}DENPs(K4) / pDNA complexes were larger than those of G5.NH 2 -mPEG 20 (K1) and {(Au 0 ) 25 -G5.NH 2 -mPEG 20} The size of the DENPs(K2) / pDNA complex is smaller. Show G5.NH 2 ...

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Abstract

The invention relates to a method for applying a functionalized poly(amidoamine) dendrimer and a nanometer compound thereof in gene transfection. The method comprises the following steps: preparation of the functionalized poly(amidoamine) dendrimer and the nanometer compound thereof, surface functional modification and characterization; preparation of functionalized poly(amidoamine) dendrimer-nanometer compound / pDNA; and research on gene transfection efficiency of the functionalized poly(amidoamine) dendrimer-nanometer compound / pDNA. The advantages of easy operation, simple transfection conditions, high transfection efficiency, strong specificity and the like are obtained in application of the functionalized poly(amidoamine) dendrimer and the nanometer compound thereof in gene transfection, and the functionalized poly(amidoamine) dendrimer and the nanometer compound thereof have good application prospects in aspects like gene therapy of cancers.

Description

technical field [0001] The invention belongs to the field of targeted gene transfection of macromolecular nanocarriers, in particular to a method for functionalized polyamide-amine dendrimers and their nanocomposites for gene transfection. Background technique [0002] Gene therapy has great application prospects in the treatment of various genetic diseases and acquired diseases. Gene therapy is now being considered as a novel medicinal strategy in many ongoing clinical trials for cancer, genetic disorders, and other conditions. However, one of the major hurdles currently in gene therapy is the lack of safe and effective transfer and expression vectors to deliver genetic material to the desired location in an organism. [0003] External genes can be delivered into organisms with the aid of viruses and viral vectors. Due to its high gene transfer efficiency, the inactivated virus shell or infection system has been applied to clinical tests of various gene therapies, but it ...

Claims

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Application Information

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IPC IPC(8): C08G83/00A61K48/00A61K47/34A61P35/00C12N15/63
Inventor 史向阳孔令丹
Owner DONGHUA UNIV
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