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

A non-viral carrier and surface modification technology, applied in the field of non-viral carrier, can solve the problem of limited application of clearance speed, achieve the effect of no immunogenicity, prolong circulation time, and no cytotoxicity

Inactive Publication Date: 2012-11-28
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] Dissolve 0.2g of gelatin in 20mL of acetic acid solution with a pH of 3, and stir at 50°C for 30min. 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 value of the solution to 9, and the stirring reaction was continued for 12 hours to obtain a milky white suspension. The milky white suspension was subjected to high-speed centrifugation (20,000 rpm, 15 min, 30° C.) to obtain a white precipitate. The white precipitate was 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-propionyl-polyethylene glycol (NH 2 -PEG-COOH). EDC: NHS: ...

Embodiment 2

[0042] In sodium carbonate buffer at pH 8.0, add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and α -Amino-omega-propionyl-polyethylene glycol (NH 2 -PEG-COOH). EDC: NHS: NH 2 - The molar ratio of PEG-COOH is 4.5:4.5:1, shaken for 4h, and then mixed with GS dispersed in pH 8.0 sodium carbonate buffer to make NH 2 -PEG-COOH and the amino group on GS (-NH 2 ) in a molar ratio of 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.) to obtain a white precipitate. The white precipitate was centrifuged and washed three times with distilled water to obtain polyethylene glycol-modified gelatin-siloxane nanoparticles (GS-PEG).

[0043] Disperse GS-PEG in the sodium carbonate buffer solution of pH 8.0, add 3-(2-pyridine dimercapto) propionic acid n-hydroxysuccinimide ester (SPDP), make SPDP and the amino group on GS-PEG ( -NH 2 ) in a molar rat...

Embodiment 3

[0046] Preparation of fluorescence-labeled luciferase plasmid pGL3 and GS complex: uniformly mix the fluorescent marker propidium iodide (PI) with pGL3 plasmid in distilled water at a mass ratio of 0.7:1, and stand in the dark for 20 minutes at room temperature. 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 s, and then allowed to stand for 1 h to obtain a gelatin-siloxane nanocomposite suspension (GS / pGL3-PI).

[0047] In sodium carbonate buffer at pH 9.0, add 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and α -Amino-omega-propionyl-polyethylene glycol (NH 2 -PEG-COOH). EDC: NHS: NH 2 -The molar ratio of PEG-COOH was 3:3:1, and the reaction was shaken for 4h, and then it was mixed with the GS / pGL3-PI nanocomposite dispersed in pH9.0 sodium carbonate buffer to make the NH 2 -PEG-COOH and the amino group on GS (-NH 2 ) in a 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 carrier, in particular to a surface modification method and application of the non-viral carrier for tumor targeting. Background technique [0002] Gene therapy refers to the biomedical technology that introduces human normal genes or genes with therapeutic effects into human target cells in a certain way to correct gene defects or play a therapeutic role, so as to achieve the purpose of treating diseases. Gene therapy has promising applications in replacing dysfunctional genes and in tumor therapy. Gene therapy has three basic elements: target gene, expression vector, and delivery vector. The construction and improvement of delivery vectors has always been the focus of gene therapy research. There are two types of delivery vectors: viral vectors and non-viral vectors. Compared with viral vectors, non-viral vectors have the advantages of low toxicity, low immune response, simple use, convenient preparation, and con...

Claims

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Application Information

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