CN103224624A

CN103224624A - Triblock copolymer polyethyleneimine (PEI)-polyethylene glycol (PEG)-PEI

Info

Publication number: CN103224624A
Application number: CN2013101737542A
Authority: CN
Inventors: 方剑群
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-05-10
Filing date: 2013-05-10
Publication date: 2013-07-31

Abstract

The invention discloses a triblock copolymer polyethyleneimine (PEI)-polyethylene glycol (PEG)-PEI, belonging to the field of gene drug carriers. The triblock copolymer PEI-PEG-PEI has the structural formula as shown in the specifications. A PEI-PEG copolymer synthesized by the technical scheme of the invention has the characteristics of high transfection efficiency and low cytotoxicity, and has a wide application prospect.

Description

A kind of triblock copolymer PEI-PEG-PEI

Technical field

The invention belongs to the gene drug carriers field, more particularly, relate to a kind of triblock copolymer PEI-PEG-PEI.

Background technology

Genophore is the prerequisite that realizes gene therapy safely and effectively, because cationic polymers is easy to synthesize, non-immunogenicity, can form supermolecule polymer closely with DNA easily, protection DNA avoids the degraded of nuclease, and promote it to enter cell, thereby become an important kind in the non-viral gene vector; But cationic polymer gene vector, pair cell have the relevant toxicity of electric charge, and transfection efficiency is lower than virus vector, and this becomes the bottleneck that limits its application.

In the polymer cation carrier of using at present, polymine (PEI) all shows high transfection efficiency in vivo and in vitro.Per two carbon atoms just connect nitrogenous primary amine and secondary amine in the repeating structure monomer of PEI, the PEI of branched chain type also comprises tertiary amine, can protonated generation positive polarity amino, become the phosphate radical bonded point of application of going up negative charge with DNA, form nano level polymkeric substance/DNA mixture, can be by cellular uptake.In the PEI of numerous different molecular weights, molecular weight is 22000 and 25000 is most widely used.Polymkeric substance is rich in positively charged ion, and stronger DNA binding ability and cell adhesion ability are arranged, and PEI has only part amino protonated under physiological pH unlike polymers such as poly-lysines.Under physiological pH, just have in per 5,6 amino nitrogens of strong PEI 1 can be doubly protonated, but combine after the nucleic acid, just have in per 2,3 amino nitrogens 1 can be doubly protonated, this is higher than poly-lysine far away.When the intravital pH of lyase descended, PEI can catch proton in a large number, and causes Cl ^-In stream, cause the swelling of lysosome perviousness, last lysosome breaks and need not the booster action of lysosome dissolving peptide, thereby the DNA of endocytosis is discharged in the tenuigenin, " proton sponge " effect of Here it is PEI.

Polymine (PEI) is the important function of gene pharmaceutical carrier, in the research of non-virus carrier consequence is arranged.But molecular weight does not have transfection efficiency substantially less than 800 PEI, and molecular weight is remarkable above the cytotoxicity of the PEI of 25K.

But simple PEI uses as genophore many problems are arranged, when entering blood circulation as the PEI-DNA mixture, the positive electric charge of composite surface has just become barrier one.Mixture with positive electric charge not only causes erythrocytic gathering, also can with composition such as albumin, scleroproein and the complement C3 effect of blood plasma, cause polymeric formation, particle increases, caught by the lungs capillary bed at last in circulation, this brings very big difficulty for the disease beyond the treatment lungs; It also is the toxic major cause of PEI that positive electric charge of carrier surface and polymer form; In addition, target is also very poor.

Summary of the invention

1. the technical problem that will solve

Has the relevant toxicity of electric charge at the cationic polymers pair cell as gene drug carriers in the prior art, and transfection efficiency is lower than the problem of virus vector, the invention provides a kind of triblock copolymer PEI-PEG-PEI, this technical scheme reduces its bio-toxicity when improving the polymine molecular weight and starts with, successfully synthesize the PEI-PEG-PEI(polymine-polyethylene glycol-ethyleneimine of different chain lengths) triblock polymer, this polymkeric substance has high transfection efficiency, the characteristics of low cytotoxicity have a extensive future.

2. technical scheme

Purpose of the present invention is achieved through the following technical solutions.

A kind of triblock copolymer PEI-PEG-PEI of the present invention, its structural formula is as follows:

The synthetic method of a kind of above-mentioned triblock copolymer PEI-PEG-PEI the steps include:

(1) with PEG _k, Et ₃N and Me ₃N.HCl is dissolved in 40ml CH according to the mol ratio of 5:20:1 ₂Cl ₂In, wherein, molecular weight k=1000,2000,3000;

(2) with TsCl(tosic acid acyl chlorides) be dissolved in 40ml CH ₂Cl ₂In, splash into dropping funnel in the mixing solutions of described step (1) preparation, wherein, TsCl and PEG _kMol ratio be 2:1,25 ℃ of following reaction 12h, solution becomes sorrel and has precipitation to generate, with the filtrate decompression distillation, product of distillation precipitates in the 100ml ether after with 40ml tetrahydrofuran (THF) (THF) dissolving after the filtration; Remove the upper strata stillness of night, place dry ventilation to make ether volatilization, product 40ml CH ₂Cl ₂200-300 purpose silica gel pillar, the product after obtaining purifying are crossed in the dissolving back;

(3) product that the described step of dissolving (2) makes in the 40ml acetonitrile, add the 2-ethyl 2-azoles quinoline of 40mmol, tube sealing carries out polyreaction, 70 ℃ were reacted 72 hours down, from tube sealing, take out reactant, place the 100ml ether to precipitate, remove the upper strata stillness of night, after product is put into 30 ℃ of vacuum drying ovens and vacuumized 2h, in vacuum drying oven, leave standstill 12h again;

(4) after the product that step (3) is made dissolves fully with THF, the methanol solution that adds the saturated 7N ammonia (purity that is ammonia is 99.99999%) of 3.5mL, stir 12h under the room temperature, reactant precipitates in the 100ml ether, abandon the upper strata stillness of night after leaving standstill, product leaves standstill 12h after placing 30 ℃ of vacuum drying ovens to vacuumize 2h again in vacuum drying oven;

(5) be that 10% HCl solution dissolves the product that described step (4) makes with the 30ml mass concentration, behind 100 ℃ of following backflow 12h, regulate pH value to 12.5, have solid to separate out with the NaOH solution of 10wt%, filter back with ultrapure water washing, centrifugal, repetitive scrubbing, centrifugal 3 postlyophilizations.

3. beneficial effect

Than prior art, the invention has the advantages that:

Technical scheme of the present invention is by the method for cationoid polymerisation, respectively with ethylene glycol, and PEG ₁₀₀₀, PEG ₂₀₀₀, PEG ₃₀₀₀Be substrate, and, successfully synthesize the PEI-PEG-PEI triblock polymer of different chain lengths, and characterize as can be known, determine that the synthetic material is exactly the PEI-PEG-PEI triblock polymer that expection will obtain by nuclear-magnetism by regulating the monomer add-on.Different is with the multipolymer of synthetic PEI in the past, and PEI-PEG multipolymer in the past is with hydroxy-end capped, and the technical program has added the process of aminolysis at the hydrolysis back, makes the PEI-PEG multipolymer that obtains with amino-terminated.Can improve the reactive behavior of PE-PEG multipolymer so greatly, can react, be cross-linked into the higher PEI-PEG multipolymer of molecular weight, thereby can improve its gene transfection efficient with the linking agent of similar DSP.On the other hand because the intersegmental existence that disulfide linkage is arranged of crosslinked later copolymer chain, thereby can after entering cell, be degraded to the less PEI-PEG multipolymer of molecular weight, reduce its cytotoxicity.PEI-PEG multipolymer behind old friend's connection has high transfection efficiency, and the characteristics of low cytotoxicity have broad application prospects.

Description of drawings

Fig. 1 is the building-up process synoptic diagram of the triblock copolymer PEI-PEG-PEI of the embodiment of the invention 1.

Embodiment

Below in conjunction with Figure of description and specific embodiment, the present invention is described in detail.

Embodiment 1

In conjunction with Fig. 1, a kind of triblock copolymer PEI-PEG-PEI of present embodiment 1, its structural formula is as follows:

As the synthesis flow of Fig. 1, the synthetic method of above-mentioned triblock copolymer PEI-PEG-PEI the steps include:

(1) with PEG _k, Et ₃N and Me ₃N.HCl gets 5mol, 20mol, 1mol respectively according to the mol ratio of 5:20:1, is dissolved in 40ml CH ₂Cl ₂In, wherein, molecular weight k=1000;

(2) with TsCl(tosic acid acyl chlorides) be dissolved in 40ml CH ₂Cl ₂In, splash into dropping funnel in the mixing solutions of described step (1) preparation, wherein, TsCl and PEG _nMol ratio be 2:1,25 ℃ of following reaction 12h, solution becomes sorrel and has precipitation to generate, with the filtrate decompression distillation, product of distillation precipitates in the 100ml ether after with 40ml tetrahydrofuran (THF) (THF) dissolving after the filtration; Remove the upper strata stillness of night, place dry ventilation to make ether volatilization, product 40ml CH ₂Cl ₂200-300 purpose silica gel pillar, the product after obtaining purifying are crossed in the dissolving back;

Below schematically the invention and embodiment thereof are described, this description does not have restricted.So, if those of ordinary skill in the art is enlightened by it, under the situation that does not break away from this creation aim, design the technical scheme similar to this technical scheme without creationary, all should belong to the protection domain of this patent.

Claims

1. triblock copolymer PEI-PEG-PEI, its structural formula is as follows: