Polytheneimine transgened carrier of targeted fibroblast growth factor receptor

A technology of growth factor receptors and fibroblasts, which is applied in the field of non-viral transgenic vectors and the preparation of non-viral transgenic vectors, achieving good application prospects, inhibiting tumor growth, and overcoming size limitations

Inactive Publication Date: 2005-05-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The complete bFGF molecule or its oligopeptide-coupled PEI has not been reported yet

Method used

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  • Polytheneimine transgened carrier of targeted fibroblast growth factor receptor
  • Polytheneimine transgened carrier of targeted fibroblast growth factor receptor
  • Polytheneimine transgened carrier of targeted fibroblast growth factor receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The peptide is coupled to PEI through a heterobifunctional cross-linker SPDP (Succinimidyl3-(2-pyridyldithio) propionate) or SMCC (Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate). SPDP interacts with the amino groups of PEI (pH 7-8) to form pyridinedithiol-modified PEI (PDP-PEI); the latter can react with proteins containing sulfhydryl groups. The polypeptide reacts with PDP-PEI via the cysteine ​​sulfhydryl group to produce a disulfide-linked oligopeptide-PEI derivative. SMCC and PEI form maleimidated PEI (maleimided PEI), which reacts with a polypeptide containing a sulfhydryl group, and the oligopeptide and PEI are linked by a disulfide bond. The coupling products of YC25 oligopeptide and PEI with different molar ratios are combined with negatively charged exogenous DNA according to a certain N / P ratio through electrostatic interaction to form a YC25 peptide / cationic polymer / exogenous DNA carrier complex.

[0022] 1. Synthesis of YC25:

[0023] The ...

Embodiment 2

[0042] Example 2 Gel electrophoresis identification of YC25 peptide / PEI / exogenous DNA complex carrier

[0043] According to PEI / DNA N / P=0, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, use HBS as the solvent, prepare YC25-PEI / pEGFP DNA mixture respectively, take 30μl for agarose gel electrophoresis observation DNA blockage conditions. Electrophoresis conditions: 1% agarose (containing 0.5 μg / ml ethidium bromide), 1×TAE buffer, voltage 5 V / cm, electrophoresis time 30 min. The results show that when the N / P ratio is greater than 3, the plasmid DNA can be completely blocked in the sample well (see figure 1 ).

Embodiment 3

[0044] Example 3 Size of YC25-PEI / DNA complex particles under transmission electron microscope

[0045] The size and morphology of PEI derivative / DNA complexes were examined by negative-stain transmission electron microscopy. PEI / DNA complexes, YC25-PEI(5:1) / pEGFP DNA complexes and CP9-PEI(5:1) / pEGFP DNA complexes were prepared as N / P=10. 2 μg of pSVβ plasmid was added to 100 μl of 0.9% NaCl solution, the corresponding amount of PEI derivative was added thereto, and vortexed to mix. After incubation for 30 min, 5 μl of the oligopeptide PEI / DNA complex was carefully dropped on the copper / rhodium grid covered with polyvinyl acetal support film. After 1 min, place the copper / rhodium mesh on the uranyl acetate solution for negative staining for 20 s. Excess solution was carefully blotted off with absorbent paper. The samples were observed under a Philips TECNAI 10 transmission electron microscope with a voltage of 80kV. At N / P=10, the particles of PEI derivatives / DNA complexes...

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Abstract

The invention was involved in the polymine modified vector of fibroblast growth factor receptor, containing peptide YC25 which could specially bind to the alkaline fibroblast growth factor receptor, peptide YC25/positive ion PEI/exogenous DNA.The exogenous DNA could be effectively introduced to the cancer clone and system of high expression of FGFRs to inhibit the growth of tumor. But there was low effect to the tumor tissue and cell liens without expression of FGFRs, so the vector system was tumor-targeting. The non-virus vector system could transfer the exogenous DNA and its size ranged from tens bp to thousands kb to overcome the limit of the size of exogenous gene. So, it could be used to produce the medicament to cure tumor by combined transfer of multi-gene and also to produce gene drug for other disease.

Description

technical field [0001] The invention belongs to biotechnology, in particular, relates to a non-viral transgene carrier targeting fibroblast growth factor receptors (FGFRs) on the surface of tumor cells, which is used to transfer expression plasmids encoding therapeutic genes to into FGFR-rich tumor cells. The present invention also relates to the preparation and application of the non-viral transgene carrier. Background technique [0002] Gene therapy refers to transferring exogenous genes into cells, correcting the structural or functional disorders of human genes by restoring or increasing gene expression, preventing the progression of disease, killing diseased cells, or inhibiting the genetic material of exogenous pathogens copy, so as to achieve the purpose of curing diseases. Gene therapy includes three important links, namely the target gene, transgenic carrier and target cells. Gene transfer system is the core technology of gene therapy. Currently, the vectors use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00C12N15/63C12N15/85C12N15/87
Inventor 王青青李经忠余海王建莉曹雪涛
Owner ZHEJIANG UNIV
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