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Process for preparig dentritic poly lysine derivative carrier for gene therapy

A technology of polylysine and fluorene methoxycarbonyl lysine is applied in the field of preparation of non-viral gene therapy vectors, and can solve problems such as differences in metabolic pathways and toxicity, limited application, and pharmacological uncertainty.

Inactive Publication Date: 2005-10-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the main reasons for this situation is that it has a certain molecular weight distribution, which causes differences in metabolic pathways and toxicity and pharmacological uncertainty, thus limiting its clinical application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Take 0.5 g of Wang resin of 100-200 mesh and Fmocylglycine modification density of 0.5 mmol / g, swell in dichloromethane, add 50% piperidine / dichloromethane solution to react for 40 min; use ethanol twice, Dichloromethane washed the resin 4 times to neutrality. Add 1mmol of N to the deprotected resin α , N ε-In the mixed solution of bisfluorenylmethoxycarbonyl lysine, 1ml DMF and 34mg HOBt, add 0.15ml DIC and 1.85ml dichloromethane, react for 24 hours; the resin after the reaction is drained and washed with dichloromethane to obtain Resins containing first generation products (Fmoc) 2 -Lys-Gly-Wang resin; the resin containing the second-generation polylysine product can be obtained by repeating the above synthesis process, and N α , N ε - The addition of bisfluorenylmethoxycarbonyl lysine is 2mmol, the addition of DMF and HOBt is 2ml and 68mg respectively, the addition volume of DIC and dichloromethane is 0.3ml and 3.7ml, and the reaction is 48 hours; it also contain...

Embodiment 2

[0019] The resin containing the third-generation polylysine product obtained in Example 1 was added to a 50% piperidine / dichloromethane solution to react for 90 minutes; the resin was washed with ethanol twice and dichloromethane four times until neutral. The deprotected resin was added to a mixed solution of 4 mmol of fluorenylmethoxycarbonyl phenylalanine, 4 ml of DMF and 136 mg of HOBt, and then 0.6 ml of DIC and 7.4 ml of dichloromethane were added to react for 72 hours. After the reaction, the liquid phase in the system was added to 50% piperidine / dichloromethane solution to react for 90 minutes; the resin was washed with ethanol 2 times and dichloromethane 4 times to neutrality; the deprotected resin was added with a volume ratio of 1 Trifluoroacetic acid / dichloromethane solution reacted with the deprotected resin at 15°C for 60 minutes, and finally rinsed the resin with dichloromethane solution for 3 times, combined the filtrate, and rotary evaporated until the volume di...

Embodiment 3

[0021] The resin containing the third-generation polylysine product obtained in Example 1 was added to a 50% piperidine / dichloromethane solution to react for 90 minutes; the resin was washed with ethanol twice and dichloromethane four times until neutral. The deprotected resin was added to a mixed solution of 4 mmol of acetic acid, 4 ml of DMF and 136 mg of HOBt, and then 0.6 ml of DIC and 7.4 ml of dichloromethane were added to react for 72 hours. After the reaction, the liquid phase in the system was discarded, and then a trifluoroacetic acid / dichloromethane solution with a volume ratio of 1 was added to react with the deprotected resin at 15°C for 60 minutes, and finally the resin was rinsed 3 times with a dichloromethane solution. Merge the filtrates, rotovap until the volume no longer changes, add a large amount of anhydrous ether and collect by centrifugation, the resulting precipitate is the product (((Ac) 2 -Lys) 2 -Lys) 2 -Lys-Gly, the single-step yield was 97%. Th...

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Abstract

The preparation process of dentritic polylysine derivative includes the stepped repeated condensation reaction of fluorylmethoxylcarbonyl amino acid modified poly(4-hydroxymethylphenoxymethyl styrene) resin as solid phase synthesized medium and N, N-difluorylmethoxylcarbonyl lysine to produce 3G dentritic polylysine with protecting fluorylmethoxylcarbonyl radical; further reaction with carboxy radical containing compound to produce polylysine derivative resin with protecting radical; deprotection with piperidine / methane dichloride; cracking with trifluoroacetic acid to cut down the polylysine derivative from the resin; and precipitation in anhydrous ethyl ether to obtain the one-side branched polylysine derivative. The polylysine derivative thus prepared has high uniqueness and high biocompatibility, and may be used as nucleic acid carrying carrier for gene treating process and may be also used as carrier for antigen and diagnosis contrast.

Description

technical field [0001] The invention relates to a biodegradable dendritic polymer method for gene therapy constructed from amino acids, which belongs to the preparation technology of non-viral gene therapy vectors. Background technique [0002] The gene therapy technology developed since the 1990s has fundamentally changed the concept of traditional drug therapy, and has brought hope for the cure of many single-gene diseases, tumors and genetic diseases. Efficient and safe gene therapy vectors are the most important link in gene therapy programs. For quite a long time in the past, viral vectors represented by retroviruses and adenoviruses have been playing a pivotal role in gene therapy. In 1999, a major incident occurred in the United States in which volunteers died during gene therapy. The vector used at that time was an adenovirus vector; a similar situation occurred in France after that. The occurrence of this series of events has caused p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/34A61K48/00C08G69/10
Inventor 史清洪郝挥红董晓燕白姝孙彦
Owner TIANJIN UNIV
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