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Tumor targeted gene delivery system and application of same

A gene delivery and tumor targeting technology, applied in the field of medicine, can solve the problems of strong cytotoxicity, easy dissociation, poor transfection effect, etc., and achieve the effects of improved therapeutic index, high transfection efficiency, and low toxicity and side effects.

Inactive Publication Date: 2015-08-12
JIAXING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PEI still has two outstanding problems as a gene carrier: first, there is a contradiction between transfection efficiency and cytotoxicity
Although small molecule PEI has low cytotoxicity, it is easy to dissociate with DNA under physiological ion concentration, resulting in poor transfection effect; although PEI with a molecular weight above 20kDa has a relatively ideal transfection efficiency, because the surface of PEI is rich in positive High molecular weight PEI exhibits strong cytotoxicity due to its charge and non-degradability in vivo
Second, polyethyleneimine has poor targeting
[0006] However, the bifunctional peptide-modified polyethyleneimine derivatives composed of DR5 short peptides and TAT cell-penetrating peptides and their applications have not been reported at home and abroad.

Method used

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  • Tumor targeted gene delivery system and application of same
  • Tumor targeted gene delivery system and application of same
  • Tumor targeted gene delivery system and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The calculation of the activation of embodiment 1 Pluronic (Pluronic) and its productive rate

[0047] Pluronic (Pluronic) is an amphiphilic block copolymer, which is a non-ionic triblock composed of two segments of hydrophilic polyoxyethylene (EO) connected to the side of a hydrophobic polyoxypropylene (PO) chain in the middle. Copolymer (polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer). The present invention uses Pluronic as a bridging molecule to connect low-molecular-weight polyethyleneimine (PEI) in series. Firstly, the hydroxyl groups at both ends of the Pluronic must be activated to react with two identical polyethyleneimine molecules. Use triphosgene + N-hydroxysuccinimide to activate Pluronic, and the activation process of Pluronic will be described in detail below by taking the activation of Pluronic 123 (P123) as an example.

[0048] (1) Activation of P123

[0049] The hydroxyl group of Pluronic 123 (P123) is condensed with triphosgene, ...

Embodiment 2

[0061] The synthesis of embodiment 2 Pluronic-PEI

[0062] Pluronic was activated and reacted with low molecular weight PEI (LMW-PEI), and then the structure of the obtained product was determined by infrared scanning and nuclear magnetic resonance.

[0063] In this example, PEI was linked to P123 activated in Example 1 to solve the problem of low transfection efficiency of low-molecular-weight PEI and high cytotoxicity of high-molecular-weight PEI. The reaction equation is as follows:

[0064]

[0065] Dissolve the above-mentioned activated P123 in 10ml of dichloromethane, dissolve PEI 2K in 10ml of dichloromethane (the molar ratio of the two is close to 1:2), slowly add the above two solutions to 10ml of dichloromethane methane, stirred overnight. Discard the precipitate by high-speed centrifugation, and vacuum rotary evaporation. After removing the solvent, the product was dissolved with ultrapure water. The dissolved product was placed in a dialysis bag and dialyzed...

Embodiment 3

[0070] Example 3 Synthesis of bifunctional peptide DR5-TAT (D21)

[0071] The DR5 receptor is highly expressed on the surface of most human tumor cells. The present invention selects a DR5 short peptide that is specifically compatible with the DR5 receptor and links it with the cell-penetrating peptide TAT to synthesize a bifunctional peptide that has tumor cell-targeting and carrier-penetrating effects. Peptide DR5-TAT (D21 for short).

[0072] The sequence of the bifunctional polypeptide D21 is as follows:

[0073] Tyr-Cys-Lys-Val-Ile-Leu-Thr-His-Arg-Cys-Tyr-Cys-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO. 1).

[0074] The D21 polypeptide was synthesized by the Fmoc method, and the MW of the D21 polypeptide was 2823.5.

[0075] The sequence of the synthesized bifunctional peptide D21 is identified by electrospray mass spectrometry, the content is determined by high performance liquid chromatography (HPLC), and the relative percentage content is determined by the normali...

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Abstract

The invention discloses a tumor targeted gene delivery system which includes: a polyethyleneimine derivative connected with a difunctional peptide, wherein the polyethyleneimine derivative is pluronic-modified low-molecular-weight polyethyleneimine and the difunctional peptide contains DR5 oligopeptide having specifically affinity with a DR5 acceptor, and cell-penetrating peptide TAT. The invention also provides a preparation method and an application of the gene delivery system. The tumor targeted gene delivery system is high in transfection efficiency, is high in tumor cell and tissue targeting property and is excellent in safety and in-vivo and in-vitro stability, can achieve oriented killing effect on tumor cells, is low in toxic and side effects, is high in therapeutic index and has a wide application prospect.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a bifunctional peptide-modified tumor-targeted gene delivery system and its application. Background technique [0002] Tumor has become a major disease that threatens human life and health, and there is still a lack of effective treatment methods. Traditional drugs for treating tumors have poor curative effect, large side effects and poor targeting effect. The biological cause of tumor development is gene mutation. Targeted therapy to diseased genes and tumor cells is currently the most powerful treatment method, and it is also the most active field of modern research on tumor treatment. [0003] To successfully implement gene therapy, three key factors must be in place: targeted therapeutic gene; gene delivery system; gene expression regulation system. Among them, the gene delivery system is the core technology of gene therapy. An ideal gene delivery carrier system should hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/42A61K47/34A61K47/32A61K48/00A61P35/00C07K19/00
Inventor 丁宝月高申丁雪鹰武鑫吴兆勇詹淑玉郑永霞傅应华黄越燕黄嬛姚翀范伟王翔刘克海
Owner JIAXING UNIV
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