CN105420277B - Polyamide-amine hyperbranched gene vector and preparation method and application thereof - Google Patents

Abstract

其中n＝0,1,2,3,4,5,6，R＝C_mH_2m+1，m＝1,2,3,4,5；B物料结构式为：

n＝1,2,3,4,5,6；或

x＝y＝1,2,3,4,5,6。本发明还公开了一种聚酰胺‑胺超支化基因载体的制备方法和应用。本发明支化程度好，用作基因载体时毒性小，缓冲能力好，可用作抗癌治疗，原料成本低，制备方法简单。The invention discloses a polyamide-amine hyperbranched gene carrier, the raw materials of which include material A, material B, material C and triethylamine; the molecular formula of material A is: C _2(n-1) H _5n-2 N _n , Among them, n=2,3,4,5; the structural formula of C material is:

where n=0,1,2,3,4,5,6, R=C _m H _2m+1 , m=1, 2, 3, 4, 5; the structural formula of B material is:

n=1,2,3,4,5,6; or

x=y=1,2,3,4,5,6. The invention also discloses a preparation method and application of a polyamide-amine hyperbranched gene carrier. The invention has good branching degree, low toxicity when used as a gene carrier, good buffering capacity, can be used for anticancer treatment, low cost of raw materials and simple preparation method.

Description

A kind of polyamide-amine hyperbranched gene vector and its preparation method and application

technical field

The invention relates to the technical field of polyamide-amine gene carriers, in particular to a polyamide-amine hyperbranched gene carrier and a preparation method and application thereof.

Background technique

With the rapid development of synthetic chemistry, many new molecular structures can be designed to explore the polymer topology. Hyperbranched polymers have become a current research focus due to their unique structure and physicochemical properties; hyperbranched polymers are widely used in biological Sensing, phase separation, battery energy, gene delivery, drug delivery, hydrogels, photocatalyst carriers and biomedicine, etc., due to the special three-dimensional structural characteristics of hyperbranched polymers, they can be synthesized by two divergent and convergent methods. Hyperbranched polymers have attracted more and more attention from scientists because of their unique physical and chemical properties, such as low viscosity, high solubility, high compatibility, and high reactivity. A great deal of research in the past decades has focused on hyperbranched polymers.

At present, hyperbranched polymers are becoming a new generation of non-viral vectors for gene delivery. Among non-viral vectors for gene delivery, hyperbranched polymers have two distinct characteristics: one is that the structure is controllable, and the other is that the chemical properties can be adapted to various requirements such as drug or gene delivery. The preparation of hyperbranched polymers has received considerable attention in the past few years. Non-viral gene carriers, especially cationic polymers, have also received increasing attention. The current cationic polymer polyamide-amine has a poor degree of branching, is highly toxic when used as a gene carrier, has poor buffering capacity, and has high raw material costs.

SUMMARY OF THE INVENTION

The invention provides a polyamide-amine hyperbranched gene carrier and a preparation method and application thereof, which have good branching degree, low toxicity when used as a gene carrier, good buffering ability, can be used as anti-cancer treatment, low cost of raw materials, and can be prepared The method is simple.

The polyamide-amine hyperbranched gene carrier proposed by the present invention comprises the raw materials of A material, B material, C material and triethylamine;

The molecular formula of material A is: C _2(n-1) H _5n-2 N _n , where n=2,3,4,5;

其中n＝0,1,2,3,4,5,6，R＝C_mH_2m+1，m＝1,2,3,4,5；The structural formula of C material is:

where n=0,1,2,3,4,5,6, R=C _m H _2m+1 , m=1, 2, 3, 4, 5;

其中n＝1,2,3,4,5,6；或The structural formula of B material is:

where n = 1, 2, 3, 4, 5, 6; or

其中x＝y＝1,2,3,4,5,6。

where x=y=1,2,3,4,5,6.

Material A has two effects, one is aminolysis, and the other is capped by Michael addition to the terminal olefin.

Preferably, A material is composed of one or more of ethylenediamine, triethylenetetramine and polyethylene polyamine with a molecular weight of 250-300, B material is N,N'-methylenebisacrylamide, C The material is L-cysteine methyl ester hydrochloride.

Preferably, the molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 2-4:1-3, N,N'-methylenebisacrylamide and The molar ratio of A material is 1:80-120.

Preferably, a solvent is also included, and the solvent includes 1-6 parts of water and 0-5 parts of DMSO by volume.

Preferably, the weight-to-volume ratio g:ml of the B material to the solvent is 0.2-0.8:4-8.

Preferably, the raw materials also include cyclodextrin, water and DMSO, and the volume ratio of water to DMSO is 2-12:1-10; the volume-to-weight ratio ml:g of the mixture of water and DMSO to material B is preferably 4-8 : 0.2-0.8.

The invention also discloses a preparation method of a polyamide-amine hyperbranched gene carrier, comprising the following steps:

Feed B material, C material, triethylamine and solvent into the reactor for polymerization reaction, the reaction temperature is 55-65 ℃, the reaction time is 30-40h, add A material to carry out ammonolysis, the ammonolysis temperature is 55-65 ℃, the aminolysis time is 34-52 hours, and the polyamidoamine hyperbranched gene vector is obtained.

The invention also discloses a preparation method of a polyamide-amine hyperbranched gene carrier, comprising the following steps:

Weigh 40-60wt% DMSO, add material B to it to dissolve completely to obtain the first solution, mix water with the remaining DMSO evenly, add material C to it to dissolve completely, add cyclodextrin to saturation to obtain the second solution, and mix the first solution. Add the first solution dropwise to the second solution, add triethylamine after the dropwise addition is complete, heat up to 52-62°C for 32-38h, add material A to carry out ammonolysis reaction, the ammonolysis temperature is 50-60°C, and the aminolysis time is 50-60°C. For 36 hours, the polyamide-amine hyperbranched gene vector is obtained by dialysis; preferably, the molecular weight of the dialysis membrane used for dialysis is 3000-14000.

Preferably, after the ammonolysis reaction, it also includes concentration and purification; preferably, the concentration process is carried out in a rotary evaporator; in the purification process, cold acetone and/or ether are added to the material obtained by concentration for precipitation, filtration, and Distilled water is added to the obtained precipitate to dissolve, and cold acetone and/or ether are continued to be added for precipitation, which is repeated 3-5 times in turn.

The invention also discloses the application of a polyamidoamine hyperbranched gene carrier as a gene carrier for anticancer therapy.

The raw materials of the polyamide-amine hyperbranched gene carrier of the present invention are easy to obtain and the cost is low. Better; in addition, by adopting a one-step method to prepare products with different branching degrees and the same repeating unit and similar molecular weight, the preparation process is effective and the method is simple. The material obtained by the reaction under catalysis has good biocompatibility and low cytotoxicity. Secondly, the polyamide-amine hyperbranched gene carrier obtained after further adding material A after ammonolysis has a three-dimensional structure that is different from that of ordinary gene carriers. It can be better compounded with DNA to obtain more excellent transfection efficiency in vivo and in vitro, and the further added cyclodextrin can effectively control the branched structure of the polyamidoamine hyperbranched gene carrier; when the product is used as a gene carrier, it is suitable for The cell has low toxicity, good buffering ability, excellent DNA composite compression ability, easy availability of raw materials, and low cost. It can be widely used in anti-cancer treatment, greatly reducing the cost of anti-cancer drugs, and is accepted by the public.

Description of drawings

Fig. 1 is the molecular structure schematic diagram of the branched chain of the polyamidoamine hyperbranched gene carrier proposed by the present invention;

Fig. 2 is the two-dimensional nuclear magnetic spectrum of the polyamidoamine hyperbranched gene carrier proposed by the present invention. Fig. ¹ H- ¹ H-COSY;

Fig. 3 is the one-dimensional nuclear magnetic spectrum diagram ¹³ C-NMR of the polyamidoamine hyperbranched gene carrier proposed by the present invention;

Fig. 4 is the branching degree of different hyperbranched molecules of the polyamidoamine hyperbranched gene carrier proposed by the present invention;

Fig. 5 is the gel permeation chromatography analysis table of the polyamidoamine hyperbranched gene carrier proposed by the present invention;

Fig. 6 is the hydration hydrodynamic radius and Zeta potential diagram of the polyamidoamine hyperbranched gene carrier proposed by the present invention;

Fig. 7 is the infrared spectrogram of the polyamidoamine hyperbranched gene carrier proposed by the present invention;

Fig. 8 is the polyamide-amine gel electrophoresis diagram after the aminolysis proposed by the present invention.

Detailed ways

Hereinafter, the technical solutions of the present invention will be described in detail through specific embodiments.

Example 1

A polyamide-amine hyperbranched gene carrier, the raw materials of which include A material, B material, C material and triethylamine;

The molecular formula of material A is: C _2(n-1) H _5n-2 N _n , where n=2,3,4,5;

其中n＝0,1,2,3,4,5,6，R＝C_mH_2m+1，m＝1,2,3,4,5；The structural formula of C material is:

where n=0,1,2,3,4,5,6, R=C _m H _2m+1 , m=1, 2, 3, 4, 5;

n＝1,2,3,4,5,6；或The structural formula of B material is:

n=1,2,3,4,5,6; or

x＝y＝1,2,3,4,5,6。

x=y=1,2,3,4,5,6.

Example 2

A polyamide-amine hyperbranched gene carrier whose raw materials include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, triethylenetetramine and water.

A material can be ethylenediamine, triethylenetetramine or polyethylene polyamine with a molecular weight of 250-300.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 3:2.4, and the molar ratio of N,N'-methylenebisacrylamide to triethylenetetramine 1:80. The weight-to-volume ratio g:ml of N,N'-methylenebisacrylamide to solvent water was 0.8:4.

Example 3

A polyamide-amine hyperbranched gene carrier, its raw materials include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, polyvinylpolyamine with a molecular weight of 275 and solvent.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 2.5:2.9, and the molar ratio of N,N'-methylenebisacrylamide to polyvinylpolyamine 1:120. The weight-to-volume ratio g:ml of N,N'-methylenebisacrylamide to the solvent was 0.2:8. Solvents include 1 part water and 5 parts DMSO by volume.

Example 4

A polyamide-amine hyperbranched gene carrier, the raw materials of which include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, ethylenediamine, and a solvent.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 2:3, and the molar ratio of N,N'-methylenebisacrylamide to ethylenediamine is 1 :95. The weight-to-volume ratio g:ml of N,N'-methylenebisacrylamide to the solvent was 0.6:5. Solvents include 5 parts water and 1 part DMSO by volume.

The preparation method of described polyamidoamine hyperbranched gene carrier, comprises the steps:

The N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine and solvent were sent into the reactor for polymerization reaction, the reaction temperature was 55°C, and the reaction time was 40h, Ethylenediamine was added for ammonolysis, the temperature of ammonolysis was 55°C, and the time of ammonolysis was 52 hours to obtain a polyamide-amine hyperbranched gene vector.

Example 5

A polyamide-amine hyperbranched gene carrier whose raw materials include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, triethylenetetramine, and a solvent.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 3:2.5, and the molar ratio of N,N'-methylenebisacrylamide to triethylenetetramine 1:115. The weight-to-volume ratio g:ml of N,N'-methylenebisacrylamide to the solvent was 0.3:8. Solvents included 4 parts water and 2 parts DMSO by volume.

The preparation method of described polyamidoamine hyperbranched gene carrier, comprises the steps:

The N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine and solvent were sent into the reactor to carry out the polymerization reaction, the reaction temperature was 65°C, and the reaction time was 30h, Triethylenetetramine was added for ammonolysis, the temperature of ammonolysis was 65°C, and the time of ammonolysis was 34 hours to obtain a polyamide-amine hyperbranched gene carrier.

Example 6

A polyamide-amine hyperbranched gene carrier, the raw materials of which include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, polyvinylpolyamine with a molecular weight of 275, and solvent.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 3:2, and the molar ratio of N,N'-methylenebisacrylamide to polyethylene polyamine with a molecular weight of 275 The molar ratio is 1:100. The weight-to-volume ratio g:ml of N,N'-methylenebisacrylamide to the solvent was 0.45:6. Solvents included 3 parts water and 3 parts DMSO by volume.

The preparation method of described polyamidoamine hyperbranched gene carrier, comprises the steps:

The N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine and solvent were sent into the reactor to carry out the polymerization reaction, the reaction temperature was 60°C, and the reaction time was 36h, Polyethylene polyamine with molecular weight of 275 was added for aminolysis, the temperature of aminolysis was 60°C, and the time of aminolysis was 36 hours to obtain a polyamide-amine hyperbranched gene carrier.

Example 7

A polyamide-amine hyperbranched gene carrier, the raw materials of which include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, ethylenediamine, water, DMSO, Cyclodextrin.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 4:1, and the molar ratio of N,N'-methylenebisacrylamide to ethylenediamine is 1 : 119; the volume weight ratio of water and DMSO mixture to N,N'-methylenebisacrylamide ml:g is 7:0.8. The volume ratio of water to DMSO was 2:4.

The preparation method of described polyamidoamine hyperbranched gene carrier, comprises the steps:

Weigh 60wt% DMSO, add N,N'-methylenebisacrylamide to it, dissolve completely to obtain the first solution, mix water and remaining DMSO evenly, add L-cysteine methyl ester hydrochloride to it The salt is completely dissolved, add cyclodextrin to saturation to obtain the second solution, add the first solution dropwise to the second solution, add triethylamine after the dropwise addition is complete, heat up to 62 ° C for 37 hours, add ethylenediamine for ammonolysis Reaction, the aminolysis temperature is 58 ° C, the aminolysis time is 36 hours, the concentration is carried out in a rotary evaporator, cold acetone is added to the concentrated material for precipitation, filtered, and distilled water is added to the obtained precipitate to dissolve, and continue to add Precipitation with cold acetone was repeated three times in turn, followed by dialysis with a dialysis membrane with a molecular weight of 3000, and vacuum freeze-drying for 24 hours to obtain a polyamidoamine hyperbranched gene carrier.

Example 8

A polyamide-amine hyperbranched gene carrier comprising N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, triethylenetetramine, water, DMSO , and cyclodextrins.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 2.1:1.4, and the molar ratio of N,N'-methylenebisacrylamide to triethylenetetramine 1:82. The volume weight ratio of water and DMSO mixture to N,N'-methylenebisacrylamide ml:g is 4:0.26. The volume ratio of water to DMSO was 2:4.

The preparation method of described polyamidoamine hyperbranched gene carrier, comprises the steps:

Weigh 41wt% DMSO, add N,N'-methylenebisacrylamide to it, dissolve it completely to obtain the first solution, mix water and remaining DMSO evenly, add L-cysteine methyl ester hydrochloride to it The salt is completely dissolved, add cyclodextrin to saturation to obtain the second solution, add the first solution dropwise to the second solution, add triethylamine after the dropwise addition is complete, heat up to 53 ° C and react for 33h, add triethylenetetramine for ammonia The hydrolysis reaction, the aminolysis temperature is 52 ° C, the aminolysis time is 36 hours, the concentration is carried out in a rotary evaporator, ether is added to the concentrated material for precipitation, filtered, and distilled water is added to the obtained precipitate to dissolve, and continue to add The ether precipitation was repeated 4 times in turn, then a dialysis membrane with a molecular weight of 14,000 was used for dialysis, and the polyamide-amine hyperbranched gene carrier was obtained by vacuum freeze-drying for 24 hours.

Example 9

A polyamide-amine hyperbranched gene carrier, the raw materials of which include N,N'-methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, polyvinylpolyamine with a molecular weight of 275, Water, DMSO, and Cyclodextrin.

The molar ratio of N,N'-methylenebisacrylamide to L-cysteine methyl ester hydrochloride is 3:2, and the molar ratio of N,N'-methylenebisacrylamide to polyvinylpolyamine 1:100. The volume weight ratio of water and DMSO mixture to N,N'-methylenebisacrylamide ml:g is 6:0.45. The volume ratio of water to DMSO was 1:5.

The preparation method of described polyamidoamine hyperbranched gene carrier, comprises the steps:

Weigh 50wt% DMSO, add N,N'-methylenebisacrylamide to it, dissolve it completely to obtain the first solution, mix water and remaining DMSO evenly, add L-cysteine methyl ester hydrochloride to it The salt is completely dissolved, add cyclodextrin to saturation to obtain the second solution, add the first solution dropwise to the second solution, add triethylamine after the dropwise addition is complete, heat up to 60 ° C for 36h reaction, add polyethylene polyamine for ammonia Hydrolysis reaction, the aminolysis temperature is 60 ° C, the aminolysis time is 36 hours, concentrated in a rotary evaporator, cold acetone and ether are added to the concentrated material for precipitation, filtered, and distilled water is added to the obtained precipitate to dissolve , continue to add cold acetone and ether to carry out precipitation, repeat 5 times in turn, then use a dialysis membrane with a molecular weight of 3500 to carry out dialysis, and vacuum freeze-drying for 24 hours to obtain a polyamide-amine hyperbranched gene carrier.

The polyamidoamine hyperbranched gene vectors described in Examples 1-6 can be used as gene vectors for anticancer therapy.

The polyamide-amine hyperbranched gene carrier is characterized by polymer, wherein: one-dimensional and two-dimensional NMR spectra are recorded using a Bruker 500MHz standard pulse sequence spectrometer (deuterated water D2O-d2 is a solvent); ¹ H-NMR is used; ¹³ C-NMR; ¹³ C, ¹ H-HMQC; ¹ H, ¹ H-COSY; ¹³ C, ¹ H-Spectral characterization of HMBC.

Molecular weights were measured by GPC (gel permeation chromatography) two detectors.

Fourier Transform Infrared Attenuated Total Reflectance Spectroscopy (FTIR-ATR) was performed on an AVATAR370 FT-IR infrared spectrometer spectrometer produced by Nicholas Corporation of the United States.

A Malvern Zeta sizer 3000HS and computer analysis software were used to evaluate the hydrodynamic radius and Zeta potential of the synthesized samples. The synthesized samples were dissolved in deionized water and disrupted in an ultrasonic cell disruptor and dispersed uniformly in deionized water.

As shown in FIG. 1 , FIG. 1 is a schematic diagram of the molecular structure of the branched chain of the polyamide-amine proposed by the present invention.

As shown in FIG. 2 , FIG. 2 is a two-dimensional nuclear magnetic spectrum diagram ¹ H- ¹ H-COSY of the polyamide-amine proposed by the present invention.

As shown in FIG. 3 , FIG. 3 is the one-dimensional nuclear magnetic spectrum diagram ¹³ C-NMR of the polyamide-amine proposed by the present invention.

As shown in FIG. 4 , FIG. 4 is the branching degree of different hyperbranched molecules of polyamide-amine proposed by the present invention.

As shown in Fig. 5, Fig. 5 is a gel permeation chromatography analysis table of the polyamide-amine proposed by the present invention.

As shown in FIG. 6 , FIG. 6 is the hydrodynamic radius of hydration and its Zeta potential diagram of the polyamide-amine proposed by the present invention.

As shown in FIG. 7 , FIG. 7 is the infrared spectrum of the polyamide-amine proposed by the present invention.

As shown in FIG. 8 , FIG. 8 is a polyamide-amine gel electrophoresis image after aminolysis proposed by the present invention.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

Claims (6)

1. A polyamide-amine hyperbranched gene vector is characterized in that raw materials comprise a material A, a material B, a material C and triethylamine;

the material A consists of one or more than two of ethylenediamine, triethylenetetramine and polyethylene polyamine with the molecular weight of 250-300, the material B is N, N' -methylene bisacrylamide, and the material C is L-cysteine methyl ester hydrochloride;

the mol ratio of the N, N '-methylene bisacrylamide to the L-cysteine methyl ester hydrochloride is 2-4:1-3, and the mol ratio of the N, N' -methylene bisacrylamide to the material A is 1: 80-120;

the method for preparing the polyamide-amine hyperbranched gene vector specifically comprises the following steps: feeding the material B, the material C, triethylamine and a solvent into a reactor for polymerization, wherein the polymerization specifically comprises the following steps: weighing 40-60wt% of DMSO (dimethyl sulfoxide), adding a material B into the DMSO, completely dissolving the material B to obtain a first solution, uniformly mixing water with the rest of DMSO, adding a material C into the DMSO, completely dissolving the material C, adding cyclodextrin into the mixture until the mixture is saturated to obtain a second solution, dropwise adding the first solution into the second solution, adding triethylamine into the solution after the dropwise adding is completely added, heating the solution to 52-62 ℃, reacting for 32-38 hours, adding a material A into the solution, performing ammonolysis at the ammonolysis temperature of 50-60 ℃ for 36 hours to obtain the polyamide-amine hyperbranched gene vector, wherein the solvent comprises 1-6 parts by volume of water, 0-5 parts by volume of DMSO and g of the weight-volume ratio of the material B to the solvent: the ml is 0.2-0.8: 4-8.

2. The polyamide-amine hyperbranched gene vector of claim 1, wherein the raw materials further comprise cyclodextrin, water and DMSO, and the volume ratio of water to DMSO is 2-12: 1-10.

3. The polyamide-amine hyperbranched gene vector of claim 2, wherein the volume-to-weight ratio ml of the mixture of water and DMSO to the material B is: g is 4-8: 0.2-0.8.

4. The polyamidoamine hyperbranched gene vector as claimed in claim 1, wherein the molecular weight of the dialysis membrane used for dialysis is 3000-14000.

5. The method for preparing a polyamide-amine hyperbranched gene vector according to claim 1 or 4, further comprising concentration and purification after the ammonolysis reaction.

6. The method for preparing a polyamide-amine hyperbranched gene vector according to claim 5, wherein the concentration process is performed in a rotary evaporator; in the purification process, adding cold acetone and/or ether into the concentrated material for precipitation, filtering, adding distilled water into the obtained precipitate for dissolving, continuously adding cold acetone and/or ether for precipitation, and repeating for 3-5 times.

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