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

Polyamide-amine hyperbranched gene vector and preparation method and application thereof Download PDF

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CN105420277B
CN105420277B CN201510770230.0A CN201510770230A CN105420277B CN 105420277 B CN105420277 B CN 105420277B CN 201510770230 A CN201510770230 A CN 201510770230A CN 105420277 B CN105420277 B CN 105420277B
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邓维
张薇
刘梦艳
洪诗斌
姚子健
张建勇
周龙
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Abstract

The invention discloses a polyamide-amine hyperbranched gene vector, which comprises raw materials of a material A, a material B, a material C and triethylamine; the molecular formula of the material A is as follows: c2(n‑1)H5n‑2NnWherein n is 2,3,4, 5; the structural formula of the material C is as follows:
Figure DDA0000845023780000011
wherein n is 0,1,2,3,4,5,6, R is CmH2m+1M is 1,2,3,4, 5; the structural formula of the material B is as follows:
Figure DDA0000845023780000012
n is 1,2,3,4,5, 6; or

Description

Polyamide-amine hyperbranched gene vector and preparation method and application thereof
Technical Field
The invention relates to the technical field of polyamide-amine gene vectors, in particular to a polyamide-amine hyperbranched gene vector and a preparation method and application thereof.
Background
With the rapid development of synthetic chemistry, many new molecular structures can be designed and discussed on the topological structure of the polymer, and the hyperbranched polymer becomes a current research hotspot due to the unique structure and physicochemical properties; the hyperbranched polymer is widely applied to biosensing, phase separation, battery energy, gene transfer, drug delivery, hydrogel, photocatalyst carriers, biomedicine and the like, and can be synthesized by two divergent methods and two convergent methods due to the special three-dimensional structural characteristics of the hyperbranched polymer. Hyperbranched polymers are gaining increasing 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 has been focused on hyperbranched polymers over the last decades.
At present, hyperbranched polymers are becoming a new generation of gene delivery non-viral vectors, and in the gene delivery non-viral vectors, the hyperbranched polymers have two distinct characteristics: one is structural control and one is chemical properties that can be tailored to various requirements such as drug or gene delivery. The preparation of hyperbranched polymers has received considerable attention over the last years. Non-viral gene vectors, particularly cationic polymers, are also receiving increasing attention. The prior cationic polymer polyamide-amine has poor branching degree, high toxicity when used as a gene vector, poor buffering capacity and higher raw material cost.
Disclosure of Invention
The invention provides a polyamide-amine hyperbranched gene vector, a preparation method and application thereof, the polyamide-amine hyperbranched gene vector has good branching degree, low toxicity and good buffering capacity when used as a gene vector, can be used for anticancer therapy, and has low raw material cost and simple preparation method.
The invention provides a polyamide-amine hyperbranched gene vector, which comprises raw materials of a material A, a material B, a material C and triethylamine;
the molecular formula of the material A is as follows: c2(n-1)H5n-2NnWherein n is 2,3,4, 5;
the structural formula of the material C is as follows:
Figure BDA0000845023760000021
wherein n is 0,1,2,3,4,5,6, R is CmH2m+1，m＝1,2,3,4,5；
The structural formula of the material B is as follows:
Figure BDA0000845023760000022
wherein n is 1,2,3,4,5, 6; or
Figure BDA0000845023760000023
Wherein x-y-1, 2,3,4,5, 6.
Feed A has two effects, one is aminolysis and one is end-capped on the terminal olefin by Michael addition.
Preferably, the material A is composed 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.
Preferably, the molar ratio of the N, N '-methylene-bisacrylamide to the L-cysteine methyl ester hydrochloride is 2-4:1-3, and the molar ratio of the N, N' -methylene-bisacrylamide to the material A is 1: 80-120.
Preferably, the solvent also comprises 1-6 parts of water and 0-5 parts of DMSO in parts by volume.
Preferably, the weight volume ratio g of the material B to the solvent is as follows: the ml is 0.2-0.8: 4-8.
Preferably, the raw materials also comprise cyclodextrin, water and DMSO, wherein the volume ratio of the water to the DMSO is 2-12: 1-10; preferably, the volume weight ratio ml of the mixture of water and DMSO to the material B is as follows: g is 4-8: 0.2-0.8.
The invention also discloses a preparation method of the polyamide-amine hyperbranched gene vector, which comprises the following steps:
and (2) feeding the material B, the material C, triethylamine and a solvent into a reactor for polymerization reaction at the reaction temperature of 55-65 ℃ for 30-40h, adding the material A for ammonolysis at the ammonolysis temperature of 55-65 ℃ for 34-52 h, and obtaining the polyamide-amine hyperbranched gene vector.
The invention also discloses a preparation method of the polyamide-amine hyperbranched gene vector, which 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, carrying out ammonolysis reaction at the ammonolysis temperature of 50-60 ℃ for 36 hours, and dialyzing to obtain a polyamide-amine hyperbranched gene vector; preferably, the molecular weight of the dialysis membrane used for dialysis is 3000-14000.
Preferably, after the ammonolysis reaction, concentration and purification are also included; preferably, the concentration process is carried out 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.
The invention also discloses application of the polyamide-amine hyperbranched gene vector as a gene vector for anticancer therapy.
The polyamide-amine hyperbranched gene vector has the advantages of easily available raw materials and low cost, and the polyamide-amine hyperbranched gene vector containing polyamino is generated by reasonably controlling the proportion of each component in the raw materials, so that the branching degree is good; in addition, products with different branching degrees and the same repeating units and similar molecular weights are prepared by adopting a one-step method, the preparation process is effective, the method is simple, in the specific preparation process, firstly, materials C and B are reacted under the catalysis of triethylamine to obtain materials with better biocompatibility and lower cytotoxicity, secondly, the material A is further added for aminolysis to obtain a polyamide-amine hyperbranched gene vector which has a three-dimensional structure different from a common gene vector, the polyamide-amine hyperbranched gene vector can be better compounded with DNA to obtain more excellent in-vivo and in-vitro transfection efficiency, and the further addition of cyclodextrin can effectively control the branching structure of the polyamide-amine hyperbranched gene vector; when the product is used as a gene vector, the product has the advantages of low toxicity to cells, good buffer capacity, excellent DNA composite compression capacity, easily available raw materials and low cost, can be widely applied to anticancer treatment, greatly reduces the cost of anticancer drugs, and is accepted by the public.
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FIG. 1 is a schematic diagram of the molecular structure of the branched chain of the polyamide-amine hyperbranched gene vector provided by the present invention;
FIG. 2 is a two-dimensional nuclear magnetic spectrum of the polyamide-amine hyperbranched gene vector provided by the invention1H-1H-COSY；
FIG. 3 is a one-dimensional nuclear magnetic spectrum of the polyamide-amine hyperbranched gene vector provided by the invention13C-NMR；
FIG. 4 shows the degree of branching of different hyperbranched molecules of the hyperbranched polyamide-amine gene vector proposed by the present invention;
FIG. 5 is a gel permeation chromatography analysis table of the polyamide-amine hyperbranched gene carrier provided by the invention;
FIG. 6 is the hydromechanical radius of hydration and Zeta potential diagram of the polyamide-amine hyperbranched gene vector provided by the invention;
FIG. 7 is an infrared spectrum of a polyamidoamine hyperbranched gene vector provided by the present invention;
FIG. 8 is a diagram of a gel electrophoresis of a polyamide-amine after ammonolysis according to the invention.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1
A polyamide-amine hyperbranched gene vector comprises raw materials of A material, B material, C material and triethylamine;
the molecular formula of the material A is as follows: c2(n-1)H5n-2NnWherein n is 2,3,4, 5;
the structural formula of the material C is as follows:
Figure BDA0000845023760000051
wherein n is 0,1,2,3,4,5,6, R is CmH2m+1，m＝1,2,3,4,5；
The structural formula of the material B is as follows:
Figure BDA0000845023760000052
n is 1,2,3,4,5, 6; or
Figure BDA0000845023760000053
x＝y＝1,2,3,4,5,6。
Example 2
The polyamide-amine hyperbranched gene vector is prepared with N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, triethylenetetramine and water.
The material A can be ethylenediamine, triethylenetetramine or polyethylene polyamine with the molecular weight of 250-300.
The molar ratio of N, N '-methylenebisacrylamide to L-cysteine methyl ester hydrochloride was 3:2.4, and the molar ratio of N, N' -methylenebisacrylamide to triethylenetetramine was 1: 80. The weight volume ratio g of the N, N' -methylene-bisacrylamide to the solvent water is as follows: ml is 0.8: 4.
Example 3
The material of the polyamide-amine hyperbranched gene carrier includes N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, polyethylene polyamine with molecular weight 275 and solvent.
The molar ratio of N, N '-methylene bisacrylamide to L-cysteine methyl ester hydrochloride was 2.5:2.9, and the molar ratio of N, N' -methylene bisacrylamide to polyethylene polyamine was 1: 120. The weight volume ratio g of the N, N' -methylene-bisacrylamide to the solvent is as follows: ml is 0.2: 8. The solvent comprises 1 part of water and 5 parts of DMSO (dimethyl sulfoxide) according to volume parts.
Example 4
The polyamide-amine hyperbranched gene vector is prepared with N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, ethylenediamine and solvent.
The molar ratio of N, N '-methylene bisacrylamide to L-cysteine methyl ester hydrochloride was 2:3, and the molar ratio of N, N' -methylene bisacrylamide to ethylenediamine was 1: 95. The weight volume ratio g of the N, N' -methylene-bisacrylamide to the solvent is as follows: ml is 0.6: 5. The solvent comprises 5 parts of water and 1 part of DMSO (dimethyl sulfoxide) according to volume parts.
The preparation method of the polyamide-amine hyperbranched gene vector comprises the following steps:
feeding N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine and a solvent into a reactor for polymerization reaction at the reaction temperature of 55 ℃ for 40 hours, adding ethylenediamine for ammonolysis at the ammonolysis temperature of 55 ℃ for 52 hours, and obtaining the polyamide-amine hyperbranched gene vector.
Example 5
The polyamide-amine hyperbranched gene vector is prepared with N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, triethylenetetramine and solvent.
The molar ratio of N, N '-methylenebisacrylamide to L-cysteine methyl ester hydrochloride was 3:2.5, and the molar ratio of N, N' -methylenebisacrylamide to triethylenetetramine was 1: 115. The weight volume ratio g of the N, N' -methylene-bisacrylamide to the solvent is as follows: ml is 0.3: 8. The solvent comprises 4 parts of water and 2 parts of DMSO (dimethyl sulfoxide) according to volume parts.
The preparation method of the polyamide-amine hyperbranched gene vector comprises the following steps:
feeding N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine and a solvent into a reactor for polymerization reaction at the reaction temperature of 65 ℃ for 30 hours, adding triethylenetetramine for ammonolysis at the ammonolysis temperature of 65 ℃ for 34 hours, and obtaining the polyamide-amine hyperbranched gene vector.
Example 6
The material of the polyamide-amine hyperbranched gene carrier includes N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, polyethylene polyamine with molecular weight 275 and solvent.
The molar ratio of N, N '-methylenebisacrylamide to L-cysteine methyl ester hydrochloride was 3:2, and the molar ratio of N, N' -methylenebisacrylamide to 275 molecular weight polyethylenepolyamine was 1: 100. The weight volume ratio g of the N, N' -methylene-bisacrylamide to the solvent is as follows: ml is 0.45: 6. The solvent comprises 3 parts of water and 3 parts of DMSO (dimethyl sulfoxide) according to volume parts.
The preparation method of the polyamide-amine hyperbranched gene vector comprises the following steps:
feeding N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine and a solvent into a reactor for polymerization reaction at the reaction temperature of 60 ℃ for 36 hours, adding polyethylene polyamine with the molecular weight of 275 for aminolysis at the aminolysis temperature of 60 ℃ for 36 hours, and obtaining the polyamide-amine hyperbranched gene vector.
Example 7
The polyamide-amine hyperbranched gene vector comprises raw materials of N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, ethylenediamine, water, DMSO and cyclodextrin.
The molar ratio of the N, N '-methylene bisacrylamide to the L-cysteine methyl ester hydrochloride is 4:1, and the molar ratio of the N, N' -methylene bisacrylamide to the ethylenediamine is 1: 119; volume weight ratio ml of water and DMSO mixture to N, N' -methylene bisacrylamide: g is 7: 0.8. The volume ratio of water to DMSO is 2: 4.
The preparation method of the polyamide-amine hyperbranched gene vector comprises the following steps:
weighing 60wt% of DMSO, adding N, N' -methylene bisacrylamide, dissolving completely to obtain a first solution, uniformly mixing water and the rest DMSO, adding L-cysteine methyl ester hydrochloride, dissolving completely, adding cyclodextrin to saturate to obtain a second solution, dripping the first solution into the second solution, adding triethylamine after the dropwise adding is completed, heating to 62 ℃, reacting for 37h, adding ethylenediamine for aminolysis reaction, wherein the aminolysis temperature is 58 ℃, the aminolysis time is 36h, concentrating in a rotary evaporator, adding cold acetone into the concentrated material for precipitation, filtering, adding distilled water into the obtained precipitate for dissolving, continuously adding cold acetone for precipitation, repeating for 3 times, dialyzing by a dialysis membrane with the molecular weight of 3000, and vacuum freeze-drying for 24 hours to obtain the polyamide-amine hyperbranched gene vector.
Example 8
A polyamide-amine hyperbranched gene vector is prepared from N, N' -methylenebisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, triethylenetetramine, water, DMSO and cyclodextrin.
The molar ratio of N, N '-methylenebisacrylamide to L-cysteine methyl ester hydrochloride was 2.1:1.4, and the molar ratio of N, N' -methylenebisacrylamide to triethylenetetramine was 1: 82. Volume weight ratio ml of water and DMSO mixture to N, N' -methylene bisacrylamide: g is 4: 0.26. The volume ratio of water to DMSO is 2: 4.
the preparation method of the polyamide-amine hyperbranched gene vector comprises the following steps:
weighing 41 wt% of DMSO, adding N, N' -methylene bisacrylamide, dissolving completely to obtain a first solution, uniformly mixing water and the rest DMSO, adding L-cysteine methyl ester hydrochloride, dissolving completely, adding cyclodextrin to saturate to obtain a second solution, dripping the first solution into the second solution, adding triethylamine after the dropwise adding is completed, heating to 53 ℃ for reaction for 33h, adding triethylenetetramine for aminolysis reaction at 52 ℃ for 36h, concentrating in a rotary evaporator, adding ether into the concentrated material for precipitation, filtering, adding distilled water into the obtained precipitate for dissolving, continuously adding ether for precipitation, repeating for 4 times, dialyzing with a dialysis membrane with molecular weight of 14000, and vacuum freeze-drying for 24 hours to obtain the polyamide-amine hyperbranched gene vector.
Example 9
The material of the polyamide-amine hyperbranched gene carrier includes N, N' -methylene bisacrylamide, L-cysteine methyl ester hydrochloride, triethylamine, polyethylene polyamine with molecular weight of 275, water, DMSO and cyclodextrin.
The molar ratio of N, N '-methylene bisacrylamide to L-cysteine methyl ester hydrochloride was 3:2, and the molar ratio of N, N' -methylene bisacrylamide to polyethylene polyamine was 1: 100. Volume weight ratio ml of water and DMSO mixture to N, N' -methylene bisacrylamide: g is 6: 0.45. The volume ratio of water to DMSO is 1: 5.
the preparation method of the polyamide-amine hyperbranched gene vector comprises the following steps:
weighing 50 wt% of DMSO, adding N, N' -methylene bisacrylamide, dissolving completely to obtain a first solution, uniformly mixing water and the rest DMSO, adding L-cysteine methyl ester hydrochloride, dissolving completely, adding cyclodextrin to saturate to obtain a second solution, dripping the first solution into the second solution, adding triethylamine after the dropwise adding is completed, heating to 60 ℃, reacting for 36h, adding polyethylene polyamine for aminolysis reaction, wherein the aminolysis temperature is 60 ℃, the aminolysis time is 36h, concentrating in a rotary evaporator, adding cold acetone and ether into the concentrated material for precipitation, filtering, adding distilled water into the obtained precipitate for dissolving, continuously adding cold acetone and ether for precipitation, repeating for 5 times, dialyzing by a dialysis membrane with the molecular weight of 3500, and freeze-drying in vacuum for 24 hours to obtain the polyamide-amine hyperbranched gene vector.
The polyamide-amine hyperbranched gene vectors described in examples 1-6 can be used as gene vectors for anticancer therapy.
Subjecting the polyamide-amine hyperbranched gene vector to polymer characterization, wherein: one-dimensional and two-dimensional NMR spectra were recorded using a pulse sequence spectrometer of Bruker 500MHz standard (deuterated water D2O-D2 as solvent); by using1H-NMR；13C-NMR；13C,1H-HMQC；1H，1H–COSY；13C，1Spectral characterization of H-HMBC.
The molecular weight was measured by a GPC (gel permeation chromatography) two-detector.
Fourier transform Infrared attenuated Total reflectance Spectroscopy (FTIR-ATR) was performed on an AVATAR370 FT-IR type Infrared spectrometer available from Nikkiso, USA.
Malvern Zeta sizer 3000HS and computer analysis software evaluate the hydrodynamic radius and Zeta potential of the synthesized samples, which were dissolved in deionized water and disrupted in an ultrasonic cell disruptor and uniformly dispersed in deionized water.
As shown in fig. 1, fig. 1 is a schematic view of the molecular structure of the branched chain of the polyamidoamine proposed by the present invention.
FIG. 2 is a two-dimensional NMR spectrum of the polyamidoamine proposed by the present invention, as shown in FIG. 21H-1H-COSY。
As shown in FIG. 3, FIG. 3 is a one-dimensional nuclear magnetic spectrum of the polyamidoamine provided by the invention13C-NMR。
As shown in FIG. 4, FIG. 4 shows the branching degree of different hyperbranched molecules of the polyamide-amine proposed by the present invention.
As shown in FIG. 5, FIG. 5 is a gel permeation chromatography table of the proposed polyamidoamine of the present invention.
As shown in FIG. 6, FIG. 6 is a graph showing the hydrodynamic radius of hydration of the proposed polyamide-amine and its Zeta potential.
As shown in fig. 7, fig. 7 is an infrared spectrum of the polyamide-amine proposed by the present invention.
As shown in FIG. 8, FIG. 8 is a gel electrophoresis diagram of the aminolyzed polyamide-amine proposed by the present invention.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the 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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CN103110954B (en) * 2013-01-31 2015-02-18 北京大学 Cholesterol-modified biodegradable polycation carrier as well as preparation method and application thereof
CN104004196B (en) * 2014-05-04 2016-08-31 健雄职业技术学院 A kind of preparation method and applications of degradable over-branched polyamidoamine

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