CN103804686A

CN103804686A - Preparation method of modified polyethyleneimine (PEI)

Info

Publication number: CN103804686A
Application number: CN201410060569.7A
Authority: CN
Inventors: 朱利民; 巫寒冰; 权静
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2014-02-21
Filing date: 2014-02-21
Publication date: 2014-05-21
Anticipated expiration: 2034-02-21
Also published as: CN103804686B

Abstract

The invention relates to a preparation method of modified polyethyleneimine (PEI), which comprises the following steps: (1) adding PEI into tri-distilled water, and heating and stirring in a water bath till dissolution to obtain a solution A, wherein the dosage ratio of PEI to tri-distilled water is 3g:(6-15mL); (2) introducing carbon dioxide (CO2) into the solution A, continuously bubbling for 3-8 hours at room temperature, and stirring to obtain a solution B; (3) freeze-drying the solution B and grinding to obtain modified PEI. The preparation method provided by the invention is quick, simple, efficient, cheap and convenient to operate, can be applied to gene transfection and biomedicine, and has a good application prospect.

Description

A kind of preparation method of modified polyethyleneimine

Technical field

The invention belongs to the preparation field of polyethylenimine derivates, particularly a kind of preparation method of modified polyethyleneimine.

Background technology

Gene therapy can be with the treatment of ten ancestor genetic diseases (hemophilia, muscular dystrophy, cyst cystic fibrosis etc.) and the serious acquired disease day after tomorrow (cardiovascular disorder, wound, infectious diseases and cancer etc.).But transporting carrier, the gene that lacks high-efficiency low-toxicity becomes the major obstacle of gene therapy.In recent years, non-virus carrier receives publicity, and because non-virus carrier is the important supplement approach of virus vector, transfection efficiency is lower, but generally than virus vector safety, in body, can repeatedly apply.Based on the transfection of expression plasmid, people make great efforts to improve non-virus carrier at present.Non-virus carrier utilizes polyvalent cation polymkeric substance or liposome to wrap up plasmid DNA or antisense oligonucleotide conventionally, utilizes the unnecessary cationic charge in surface to stick to cell surface, enters cell.Nearly decades, non-virus carrier is greatly improved, cationic polymer gene vector used by ten have without gene limitation of size, can produce in a large number and quality product is reproducible, price is low, easy to use, be easy to the advantages such as modification, be subject to investigator's extensive concern.Wherein, polyethylene industry amine (Polyethylenimine, PEI) being paid close attention to widely owing to having compared with high electric density and good proton surge capability, is one of cationic polymer gene vector of current most study: the linear PEI of 25kDa branching PEI (25kDa bPEI) and 22kDa (22kDa1PEI) is known as " golden standard " in current high polymer gene carrier field.But the molecular weight of these two kinds of PEI is higher, its composite surface electric density is higher, have higher cytotoxicity, its main reason is that surface has a large amount of amino, causes cytotoxicity very large, therefore PEI is very restricted aspect gene transfection, because PEI is to CO ₂there is very strong adsorption, and relatively stable after absorption, and decomposition temperature is at 100 ℃～200 ℃.In view of above reason, the PEI derivative after this modification seems significant, and current Research Literature and patent, do not utilize CO ₂pEI is carried out to the article of modification.I utilize CO ₂gas passes into the method for PEI solution, obtained a kind of more simply, more save time, the method for modified PE I is prepared PEI derivative faster method.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of preparation method of modified polyethyleneimine, and the method is quick, simple and direct, efficient, cheap and easy to operate; The starting material that this invention is used are easy to get, and have good biocompatibility, and it does the potentiality that rear related experiment is analyzed to have application.

The preparation method of a kind of modified polyethyleneimine of the present invention, comprising:

(1) polymine PEI is added in tri-distilled water, heating in water bath is stirred to and is dissolved into transparent liquid completely, obtains solution A; Wherein the amount ratio of polymine PEI and tri-distilled water is 3g:6-15mL;

(2) by carbonic acid gas CO ₂pass in solution A, under room temperature condition, continue bubbling 3-8h, stirring reacts fully, and obtains solution B;

(3), by above-mentioned solution B lyophilize, grinding, obtain modified polyethyleneimine (the yellow solid powder after modification).

In described step (1), the weight-average molecular weight of polymine PEI is 25000.

In described step (1), heating in water bath stirs as using digital display magnetic force thermostatic mixer, and bath temperature is controlled at 35-40 ℃, heated and stirred 10-20min.

In described step (2), stir as stirring with digital display magnetic force thermostatic mixer.

Lyophilize be first freezing under-80 ℃ of conditions in described step (3), is dried being transferred in Freeze Drying Equipment.

In described step (3), sublimation drying is 2-3d.

The EP pipe that in described step (3), lyophilize container used is 4mL.

In described step (3), be ground to yellow solid lyophilize being obtained with mortar and grind to form yellow powder.

In described step (3) in the modified polyethyleneimine of gained, in each PEI molecule, there is 56.3% amino to be amidated.

The present invention is for application CO ₂to the preliminary research of PEI modification, reduce PEI surface amino groups quantity, for the PEI derivative of understanding after modification, Cytotoxic impact and application are had great importance.

beneficial effect

(1) the inventive method is quick, simple and direct, efficient, cheap and easy to operate;

(2) starting material used in the present invention are cheap and easy to get, have good biocompatibility, and it does the potentiality that rear related experiment is analyzed to have application.

Accompanying drawing explanation

Fig. 1 is that the branching PEI aqueous solution passes into CO ₂the design sketch of front and back;

Fig. 2 is respectively by branching PEI and prepared yellow powder PEI-CO ₂be dissolved in deuterated water, shown carbon-13 nmr spectra figure;

Fig. 3 is branching PEI and prepared yellow powder PEI-CO ₂infrared spectrogram;

Fig. 4 is branching PEI and prepared yellow powder PEI-CO ₂thermogravimetric analysis (TG) figure.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.

Embodiment 1

(1) add heated and stirred in 15mL tri-distilled water that it is dissolved rapidly 3g branching PEI, obtain solution A;

(2) by carbonic acid gas (CO ₂) pass in A solution, under room temperature, continue bubbling 5h, and stir react fully, obtain solution B.The A obtaining according to above step and B solution photo are as shown in Figure 1.

Solution B is got in 2mL and EP pipe, and lyophilize, grinds, and obtains the yellow solid powder after modification.The D2O that gets respectively 80mg and be dissolved in 1.5mL according to yellow powder that above step obtains and branching PEI, does carbon-13 nmr spectra, and institute's result that obtains as shown in Figure 2.

Embodiment 1 is obtained to yellow powder and branching PEI does ultimate analysis, obtain analyzing and testing result according to above step as shown in table 1.Table 1 is branching PEI and prepared yellow powder PEI-CO ₂ultimate analysis detect table;

Table 1

Embodiment 1 is obtained to yellow powder and branching PEI does infrared spectra, according to result that above step obtains as shown in Figure 3.Embodiment 1 is obtained to yellow powder and branching PEI and under the protection of nitrogen, carry out thermogravimetric analysis (TG) and characterize, result as shown in Figure 4.

Claims

1. a preparation method for modified polyethyleneimine, comprising:

(1) polymine PEI is added in tri-distilled water, heating in water bath is stirred to dissolving, obtains solution A; Wherein the amount ratio of polymine PEI and tri-distilled water is 3g:6-15mL;

(2) by carbonic acid gas CO ₂pass in solution A, under room temperature condition, continue bubbling 3-8h, stir, obtain solution B;

(3) by above-mentioned solution B lyophilize, grinding, obtain modified polyethyleneimine.

2. the preparation method of a kind of modified polyethyleneimine according to claim 1, is characterized in that: in described step (1), the weight-average molecular weight of polymine PEI is 25000.

3. the preparation method of a kind of modified polyethyleneimine according to claim 1, is characterized in that: in described step (1), heating in water bath stirs and is: with digital display magnetic force thermostatic mixer, bath temperature is controlled at 35-40 ℃, heated and stirred 10-20min.

4. the preparation method of a kind of modified polyethyleneimine according to claim 1, is characterized in that: in described step (3), lyophilize is for first freezing, then dry in Freeze Drying Equipment at-80 ℃.

5. the preparation method of a kind of modified polyethyleneimine according to claim 1, is characterized in that: in described step (3), sublimation drying is 2-3d.

6. the preparation method of a kind of modified polyethyleneimine according to claim 1, is characterized in that: in described step (3), in each polymine PEI molecule, have 56.3% amino to be amidated.