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Carbon nano tube complex gene vector system and preparation method thereof

A carbon nanotube composite, carbon nanotube technology, applied in gene therapy, drug combination, pharmaceutical formulations, etc., can solve problems such as destroying carbon nanotube structure, achieve good biocompatibility, excellent anti-tumor effect, and simple operation easy-to-control effects

Inactive Publication Date: 2014-06-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Covalent modification connects hydrophilic groups through the chemical reaction of the head end and side wall of carbon nanotubes; although covalent modification can greatly increase the solubility of carbon nanotubes, it will destroy the structure of carbon nanotubes

Method used

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  • Carbon nano tube complex gene vector system and preparation method thereof
  • Carbon nano tube complex gene vector system and preparation method thereof
  • Carbon nano tube complex gene vector system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Synthesis of polyethyleneimine-cholesterol

[0023] Weigh 1 g of branched polyethylenimine (0.6kDa, 1.8kDa, 10kDa, 25kDa) with different molecular weights, put them in a 50mL round bottom flask, add 10mL of anhydrous dichloromethane (dehydrated with molecular sieve) and 100uL The mixed solution of triethylamine was stirred in an ice bath for 30 minutes to obtain a PEI solution; 1000, 330, 60, and 24 mg of acid chloride cholesterol were weighed, dissolved in 5 mL of ice anhydrous dichloromethane, and the solution was slowly added dropwise to the above PEI solution The dropwise addition was completed within 30 min; the mixture was stirred and reacted for 12 h under ice-bath conditions. The reacted solution was transferred to an eggplant flask, and the solvent was removed by rotary evaporation. The obtained product was dried in vacuum, dissolved by adding 50mL HCl (0.1M), and extracted 3 times with 100mL dichloromethane to remove unreacted acyl chloride cholest...

Embodiment 2

[0025] Embodiment 2: Preparation of carbon nanotube composite system

[0026] Weigh 1 mg of single-walled carbon nanotubes (timesnano) or 1 mg of multi-walled carbon nanotubes (Nanjing Xianfeng Nano Material Technology Co., Ltd.), 10-50 mg of PEI-Chol prepared in Example 1 (respectively: PEI600-Chol50 mg , PEI1800-Chol30mg, PEI10000-Chol20mg, PEI25000-Chol10mg) and 2mg DSPE-PEG 2000(Xi'an Ruixi Biotechnology Co., Ltd.) Add 5 mL of deionized distilled water to a 50 mL round-bottomed flask, and clean it with an ultrasonic wave with a power of 300-600 w for more than 30 min. Take the carbon nanotube dispersion, centrifuge at 10,000-13,000rpm for 30 minutes, then take the supernatant, discard the carbon nanotube aggregates, centrifuge to get the supernatant, repeat the centrifugation for 3 times, collect the supernatant in a 100kDa centrifugal filter (Millipore), and centrifuge Wash, centrifuge at 4000rpm for more than 30min, discard free polymer molecules, transfer the concentra...

Embodiment 3

[0027] Example 3: Characterization of polyethyleneimine-cholesterol

[0028] Take the PEI-Chol obtained in Example 1, and use IR and 1H-NMR to characterize its functional group and chemical structure; use hydrochloric acid to perform potentiometric titration to measure its buffering capacity. IR results such as figure 2 As shown, 3300~3500cm -1 The nearby -NH absorption peak is -NH of PEI-Chol 2 stretching vibration peak; 2900cm -1 The peak represents the methylene C-H stretching vibration, indicating the presence of -NH- and CH in the polymer PEI 2 -; figure 2 1776cm in A -1 It is the absorption peak of the C=O bond, because the electron-withdrawing induction effect of the chlorine atom in cholesterol formyl chloride moves the position of the carbonyl absorption peak to the high frequency direction; figure 2 The carbonyl absorption peak in B-E is 1650cm -1 , which indicates that -Cl in C=O in cholesterol formyl chloride is replaced by amino group in PEI to form an a...

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Abstract

The invention discloses a carbon nano tube complex gene vector system, and researches the effectiveness, biocompatibility, cytotoxicity, transfection efficiency and the like of the carbon nano tube complex gene vector system. Polyethyleneimine-cholesterol (PEI-Chol) and polyethylene glycol-distearoyl phosphatidyl ethanolamine (DSPE-PEG) are combined on the wall of the carbon nano tube through hydrophobic interaction, so as to obtain the carbon nano tube complex gene vector system. The preparation method comprises specific steps of (1) synthesizing polyethyleneimine-cholesterol by using polyethyleneimine and cholesteryl chloroformate through a dehydrating agent triethylamine under anhydrous low temperature condition; (2) obtaining the water-soluble positive ion carbon nano tube complex system by using polyethyleneimine-cholesterol and polyethylene glycol-distearoyl phosphatidyl ethanolamine through ultrasonic dispersion. The carbon nano tube complex system can be steadily and uniformly dispersed in phosphate solution (pH=7.4), cell medium and serum in 24 hours, has low toxicity, effectively combines and concentrates DNA molecules, and has high transfection efficiency.

Description

(1) Technical field [0001] The invention relates to a carbon nanotube composite gene carrier system and a preparation method thereof. (2) Background technology [0002] Gene therapy is a promising therapeutic approach in the treatment or prevention of congenital and acquired diseases. Currently, most genes are delivered in vivo by viral vectors or non-viral vectors. Although the transfection efficiency of viral vectors is high, there are some defects, including causing inflammation and immune response in the human body. Nonviral vectors include polycationic liposomes, cationic polymers, nanoparticles. Although nonviral vectors are less immunogenic, they are also less able to reach and traverse the nuclear membrane. Therefore, it is imperative to find a gene carrier with low toxicity, low immunogenicity and high membrane-penetrating ability. [0003] Carbon nanotubes are divided into single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). Since ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/32A61K47/34A61K47/02A61K48/00A61P35/00
Inventor 金一孔芬芬沈松王成润
Owner ZHEJIANG UNIV
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