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Beta-cyclodextrin-modified polyamide-dendrimer and preparation method of gold nanoparticle compound of beta-cyclodextrin-modified polyamide-dendrimer

A technology of amine dendrimers and dendrimers, which is applied in the field of preparation of polyamide-dendrimer and its nano-gold particle complex, to avoid aggregation, good gene transfection effect, and improve biocompatibility Effect

Inactive Publication Date: 2015-12-30
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Retrieval of related literature and patents at home and abroad shows that the method of using β-CD modified dendrimers and their gold nanoparticles as carriers for gene transfection of human embryonic kidney cells (293T) has not been reported yet.

Method used

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  • Beta-cyclodextrin-modified polyamide-dendrimer and preparation method of gold nanoparticle compound of beta-cyclodextrin-modified polyamide-dendrimer
  • Beta-cyclodextrin-modified polyamide-dendrimer and preparation method of gold nanoparticle compound of beta-cyclodextrin-modified polyamide-dendrimer
  • Beta-cyclodextrin-modified polyamide-dendrimer and preparation method of gold nanoparticle compound of beta-cyclodextrin-modified polyamide-dendrimer

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Experimental program
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Effect test

Embodiment 1

[0036] Weigh 16.46mg β-CD and 23.51mg CDI, dissolve them in 5mL DMSO respectively, stir and react for 6h. Weigh the fifth generation polyamidoamine dendrimer (G5.NH 2 ) 15.06 mg, dissolved in 5 mL DMSO. Add the activated β-CD solution to G5.NH dropwise 2 solution, magnetically stirred at room temperature, and reacted for 3d to obtain G5.NH 2 -β-CD. G5.NH 2 - β-CD was transferred to a dialysis bag with a molecular weight cut-off of 14000, dialyzed in distilled water for three days (2L×3), and then freeze-dried, and finally the obtained G5.NH 2 - β-CD was dissolved in 5 mL of water, and 322.7 μL of HAuCl was added dropwise 4 Aqueous solution (30mg / mL) was mixed and stirred for 30min, then 445μL of 10mg / mL NaBH was added rapidly 4 solution, reacted for 3h, and obtained {(Au 0 ) 25 -G5.NH 2 -β-CD} solution. After the reaction, the reaction product {(Au 0 ) 25 -G5.NH 2 -β-CD} was transferred to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed in di...

Embodiment 2

[0039] G5.NH 2 , the {(Au 0 ) 25 -G5.NH 2}, {(Au 0 ) 25 -G5.NH 2 -β-CD} was complexed with pDNA and subjected to gel retardation experiments. Prepare 8 wells of 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 pDNA as an example, prepare vector / pDNA complexes according to different N / P (0.25, 0.5, 1, 2, 3, 4, 5), and use DNAmarker as a positive 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. The migration of DNA in the gel was analyzed using a gel imager. The result is as Figure 4 shown. The results showed that {(Au 0 ) 25 -G5.NH 2} and {(Au 0 ) 25 -G5.NH 2 -β-CD} can well complex with DNA at a lower N / P (N / P=1) and block DNA. in, Figure 4 1 is the DNAmarker band, which is the positive control group, and the remaining 2-8 are vector / pDNA complexes prepared with ...

Embodiment 3

[0041] G5.NH 2 , the {(Au 0 ) 25 -G5.NH 2}, {(Au 0 ) 25 -G5.NH 2 -β-CD} under different N / P ratio conditions (1:1, 2.5:1, 5:1, 10:1) and 5 μg pDNA to form vector / pDNA complexes through electrostatic interaction, so that the final volume was fixed at 100 μL , incubate at room temperature for 20 min, and then add 1 mL of deionized water. Characterize its hydrodynamic particle size and surface potential by Malvern laser particle size analyzer (Malvern, MK, 633nm laser), the result is as follows Figure 5 shown. The results show that with the increase of N / P, the sizes and potentials of all the composites first decrease and then slightly increase; under the same N / P, the material potential of the coated gold nanoparticles is higher than that of the uncoated gold nanoparticles. However, the hydrodynamic particle size and surface of all functionalized polyamidoamine dendrimers and their nanocomposites / pDNA The potentials are all within the appropriate transfection range, wh...

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Abstract

The invention relates to a beta-cyclodextrin-modified polyamide-dendrimer and a preparation method of a gold nanoparticle compound of the beta-cyclodextrin-modified polyamide-dendrimer. The preparation method includes: mixing DMSO (dimethyl sulfoxide) solutions in which beta-CD and CD I are dissolved, respectively, adding DMSO solution of G5PAMAM after reacting, performing stirring at room temperature for 3 d, and performing dialysis and freeze-drying to obtain G5.NH2-beta-CD; adding HauCl4.4H2O solution into the obtained G5.NH2-beta-CD solution, adding NaBH4 solution after stirring, performing stirring for 2-4 h, and performing dialysis and freeze-drying to obtain a target compound {(Au0)25-G5.NH2-beta-CD}. The prepared compound is a good gene carrier, capable of successfully carrying and expressing pDNA-transfected human embryonic kidney cells, transfection conditions are mild, operating is easy, transfection efficiency is high, and the compound has a promising application in terms of biomedicine such as gene therapy.

Description

technical field [0001] The invention belongs to the field of preparation methods of polyamide-dendrimer and nano-gold particle composites thereof, and particularly relates to a preparation method of polyamide-dendrimer and nano-gold particle composites modified by β-cyclodextrin . Background technique [0002] Gene therapy is a new technology that combines modern medicine and molecular biology to introduce exogenous normal genes into target cells to correct or compensate diseases caused by gene deletion or abnormalities, so as to achieve the purpose of treating certain diseases. At present, what is most lacking in the application of gene therapy is a safe and effective carrier that can deliver the target gene to various cells, tissues and organs. Currently, the vectors used for gene therapy mainly include viral vectors and non-viral vectors. Viral vectors have high transfection efficiency, but their clinical application is limited by their high immunogenicity, high toxicity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08L87/00C08G81/00C08G83/00C08B37/16C08K3/08C12N15/87C12N15/85
Inventor 史向阳邱洁茹孔令丹曹雪雁
Owner DONGHUA UNIV
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