Method for gene transfection by utilizing PEG (polyethylene glycol) functionalized PAMAM (poly(amidoamine)) dendrimer carrier encapsulating gold nanoparticles

A technology of nano-gold particles and dendrimers, applied in the field of gene transfection of PAMAM dendrimers

Inactive Publication Date: 2014-04-09
DONGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Retrieval of relevant literature and patents at home and abroad shows that the method of using PEGylated

Method used

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  • Method for gene transfection by utilizing PEG (polyethylene glycol) functionalized PAMAM (poly(amidoamine)) dendrimer carrier encapsulating gold nanoparticles
  • Method for gene transfection by utilizing PEG (polyethylene glycol) functionalized PAMAM (poly(amidoamine)) dendrimer carrier encapsulating gold nanoparticles
  • Method for gene transfection by utilizing PEG (polyethylene glycol) functionalized PAMAM (poly(amidoamine)) dendrimer carrier encapsulating gold nanoparticles

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Experimental program
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Embodiment 1

[0064]Taking the combination of carrier H1 as an example, weigh 10 mg of (mPEG-COOH) with a molecular weight of 2000 and dissolve it in 3 ml of DMSO. Weigh 1.7mg EDC and dissolve it in 2ml DMSO, add the EDC solution to mPEG-COOH, and stir for 3 hours. Weigh the fifth generation PAMAM dendrimers (G5.NH 2 ) 13mg was dissolved in 4mL DMSO to prepare a solution with a concentration of 10.0mg / mL. According to mPEG-COOH / G5.NH 2 The molar ratio is 10 / 1, add the activated mPEG-COOH solution to G5.NH 2 solution, stirred magnetically at room temperature, and reacted for 3 days. Get sample G5.NH 2 -mPEG2k 10 solution. Add 171.49 μL HAuCl dropwise to the above solution 4 Aqueous solution (30mg / mL), mix and stir for 30min, then quickly add 23.6μL of 10mg / mL NaBH 4 Solution, reduction reaction 3h. 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 for 3 days (3 times a day). Freeze-d...

Embodiment 2

[0066] Gel electrophoresis experiments were used to test the ability of the vector to complex pDNA or siRNA and to determine the N / P ratio at which the material was able to fully compress pDNA or siRNA. Firstly, the carrier material and 1 μg pDNA or siRNA complexes were configured to form complexes at different N / P (0.125, 0.25, 0.5, 1, 2, 5) ratios, and reacted at room temperature for 20-30 minutes. Prepare ethidium bromide (0.1 μg / mL) agarose gel (1.0% w / v), and place at room temperature until the agarose gel solidifies. Using naked pDNA or siRNA as a control, add the corresponding vector / pDNA or vector / siRNA complexes to the wells of the agarose gel respectively, with a voltage of 80V and a time of 30min. Use a gel imager to analyze the migration of pDNA or siRNA in the gel. The results are shown in Figures 3(a) and 3(b). It shows that all four vectors can completely compress pDNA and siRNA when N / P>0.5, and this result also provides a reference for the selection of N / P i...

Embodiment 3

[0068] 5μg pDNA or siRNA (N / P=1, 2.5, 5, 10) were mixed with four vectors and G5.NH 2 After complex formation, the volume was made up to 1 mL with deionized water. The particle size and surface potential of the composite were measured using a Malvern laser particle size analyzer (Malvern, UK, 633nm laser), and the results are shown in Figure 4(a) and 4(b). It shows that the surface potentials of the complexes are all positive, indicating that the complexes can be easily combined with cells through electrostatic. The particle size of the complexes is about 200nm, which is conducive to the phagocytosis of cells.

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Abstract

The invention relates to a method for gene transfection by utilizing a PEG (polyethylene glycol) functionalized PAMAM (poly(amidoamine)) dendrimer carrier encapsulating gold nanoparticles. The method comprises the following steps: (1) preparing an activated mPEG (methoxypolyethylene glycol) solution, weighing a fifth-generation PAMAM dendrimer and dissolving the fifth-generation PAMAM dendrimer in DMSO (dimethyl sulfoxide), then adding the activated mPEG solution and carrying out magnetic stirring under room temperature to react to obtain a G5.NH2-mPEG10 solution; (2) dropwise adding an HAuCl4 aqueous solution to the G5.NH2-mPEG10 solution and quickly adding a NaBH4 solution after stirring to obtain a reaction product; (3) preparing a carrier/gene compound solution with the reaction product and pDNA (plasmid deoxyribonucleic acid) or siRNA (small interfering ribonucleic acid); (4) carrying out cell transfection by adopting the carrier/gene compound solution. The prepared carrier has the advantages of mild transfection conditions, easiness in operation, high transfection efficiency, good specificity and the like when being used for gene transfection and has good application prospect in cancer therapy and the like.

Description

technical field [0001] The invention belongs to the field of gene transfection using functionalized polymer nanocarriers, and in particular relates to a gene transfection method of a PEG-functionalized PAMAM dendritic macromolecule carrier encapsulating nano-gold particles. Background technique [0002] At present, with the development of science and technology, gene therapy has brought good news to the treatment of cancer and some genetic diseases, and is considered to be the most promising treatment technology in future cancer treatment. Compared with traditional gene therapy methods, the advantages of gene therapy for cancer treatment are: high targeting, remarkable effect, less pain for patients during treatment, and less damage to normal cells during treatment. [0003] The key to gene therapy is to find safe and efficient gene carriers. Usually, the carriers used for gene delivery include viral vectors and non-viral vectors. Although the research on viral vectors star...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N15/85
Inventor 史向阳侯文秀郭睿孔令丹
Owner DONGHUA UNIV
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