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Method for realizing surface modification of tumor targeted nonviral vector and application thereof

A non-viral vector and surface modification technology, applied in the field of non-viral vectors, can solve the problems of limited clearance speed and application, and achieve the effects of no immunogenicity, high transduction efficiency, and no cytotoxicity.

Inactive Publication Date: 2011-11-16
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Chinese patent CN200810071151.0 discloses a method for preparing gelatin-siloxane nano-hybrid materials by two-step sol-gel reaction. The prepared gelatin-siloxane nanoparticles have good biocompatibility and are non-toxic Side effects, high stability, and can be combined with exogenous genes to realize the transportation and expression of DNA, but the pure gelatin-siloxane nanoparticles have a fast blood clearance in animals, which limits their application

Method used

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  • Method for realizing surface modification of tumor targeted nonviral vector and application thereof
  • Method for realizing surface modification of tumor targeted nonviral vector and application thereof
  • Method for realizing surface modification of tumor targeted nonviral vector and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1) Preparation of gelatin-siloxane nanoparticles (GS)

[0035] 0.2 g of gelatin was dissolved in 20 mL of acetic acid solution with pH 3 and stirred at 50° C. for 30 min. Then, 0.2 g of 3-(2,3-glycidoxy)propyltrimethoxysilane was added to the above 1% gelatin solution. After the reaction solution was stirred and reacted at 30°C for 6 hours, ammonia water was added to adjust the pH of the solution to 9, and the reaction was continued for 12 hours to obtain a milky white suspension. The milky white suspension is subjected to high-speed centrifugation (20,000 rpm, 15 min, 30° C.) to obtain a white precipitate. The white precipitate is washed twice with distilled water to obtain gelatin-siloxane nanoparticles (GS).

[0036] 2) Polyethylene glycol modified gelatin-siloxane nanoparticles (GS-PEG)

[0037] Add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and α- Amino-ω-propionic acid-polyethylene glycol (NH 2 -PEG-COOH). EDC: NHS: NH 2 The mol...

Embodiment 2

[0042] In the pH 8.0 sodium carbonate buffer, add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and α -Amino-ω-propionic acid-polyethylene glycol (NH 2 -PEG-COOH). EDC: NHS: NH 2 -The molar ratio of PEG-COOH is 4.5:4.5:1, shake the reaction for 4h, and then mix it with GS dispersed in pH8.0 sodium carbonate buffer to make NH 2 -PEG-COOH and the amino group on GS (-NH 2 The molar ratio of) is 1:1. After continuing the shaking reaction for 4 hours, the milky white suspension was subjected to high-speed centrifugation (14,000 rpm, 20 min, 20° C.), and a white precipitate was obtained. The white precipitate was centrifuged and washed with distilled water three times to obtain polyethylene glycol-modified gelatin-siloxane nanoparticles (GS-PEG).

[0043] Disperse GS-PEG in a sodium carbonate buffer of pH 8.0, and add 3-(2-pyridinedimercapto) propionate n-hydroxysuccinimide (SPDP) to make SPDP and the amino group on GS-PEG ( -NH 2 The molar ratio of...

Embodiment 3

[0046] Preparation of fluorescently labeled luciferase plasmid pGL3 and GS complex: mix the fluorescent marker propidium iodide (PI) with the pGL3 plasmid at a mass ratio of 0.7:1 evenly in distilled water, and let it stand for 20 minutes at room temperature in the dark. A fluorescently labeled plasmid (pGL3-PI) was obtained. GS and plasmid pGL3-PI were mixed in distilled water at a mass ratio of 50:1 at 25°C, vortexed for 30 seconds, and then left to stand for 1 hour to obtain a gelatin-siloxane nanocomposite suspension (GS / pGL3-PI).

[0047] In the pH 9.0 sodium carbonate buffer, add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and α -Amino-ω-propionic acid-polyethylene glycol (NH 2 -PEG-COOH). EDC: NHS: NH 2 -The molar ratio of PEG-COOH is 3:3:1, the reaction is shaken for 4h, and then it is mixed with the GS / pGL3-PI nanocomposite dispersed in the pH9.0 sodium carbonate buffer to make NH 2 -PEG-COOH and the amino group on GS (-NH 2 The mo...

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Abstract

The invention provides a method for realizing the surface modification of a tumor targeted nonviral vector and application thereof, relating to a surface modification method of a nonviral gene vector. The invention provides a method capable of having longer blood circulating time in vivo and realizing the surface modification of the tumor targeted nonviral gene vector and realizes the transportation of DNA (Deoxyribose Nucleic Acid) by using the modified tumor targeted nonviral gene vector. The method comprises the following steps of: activating the carboxyl (-COOH) of a carboxyl-containing polyethylene glycol molecule by using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxy-succinamide; then reacting with the nonviral vector, i.e. a gelatin-siloxane nanoparticle, to obtain the gelatin-siloxane nanoparticle modified by polyethylene glycol; and modifying the gelatin-siloxane nanoparticle by using a DNA aptamer so as to obtain the gelatin-siloxane nanoparticle corporately modified by the polyethylene glycol and the DNA aptamer. The GS-PEG-Apt (Gelatin-Siloxane-Polytheylene glycol-Aptamer) nanoparticle can be used for effectively loading a foreign gene into a host cell and has high transduction efficiency without immunogenicity or cytotoxicity.

Description

Technical field [0001] The invention relates to a non-viral vector, in particular to a method for surface modification of a non-viral vector that realizes tumor targeting and its application. Background technique [0002] Gene therapy refers to a biomedical technology that introduces normal human genes or therapeutic genes into human target cells in a certain way to correct gene defects or have a therapeutic effect, so as to achieve the purpose of curing diseases. Gene therapy has good application prospects in replacing dysfunctional genes and tumor therapy. Gene therapy has three basic elements: target gene, expression vector and delivery vector. The construction and improvement of delivery vector has always been the focus of gene therapy research. There are two types of delivery vector: viral vector and non-viral vector. Compared with viral vectors, non-viral vectors have the advantages of low toxicity, low immune response, simple use, convenient preparation, and convenient st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/42A61K35/00
Inventor 任磊王天晓王军
Owner XIAMEN UNIV
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