CN107459583B - Highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine group and preparation method thereof - Google Patents
Highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine group and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups, and a preparation method and application thereof. The structural formula of the highly hyperbranched cationic polysaccharide derivative containing the dendritic polyamide-amine group is shown as a formula (I); the invention selectively couples dendritic macromolecular polyamide-amine (PAMAM D3 or D4) to a hyperbranched polysaccharide chain by an efficient azide-alkyne click reaction method to synthesize the highly hyperbranched cationic polysaccharide derivative. The method has the advantages of mild reaction conditions, high reaction efficiency and selectivity. The dendritic polyamide-amine group-containing cationic polysaccharide derivative prepared by the invention has the characteristic of a highly hyperbranched structure, can well form an electropositive nano-composite with siRNA, is beneficial to carrying siRNA into cells, improves the transfection efficiency of the siRNA to genes, is expected to be used as a gene carrier, and has a wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering. More particularly, relates to a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups and a preparation method thereof.
Background
Gene therapy has been used clinically to treat and prevent a range of diseases (Ginn, S.L., et al. Journal of Gene Medicine, 2013, 15, 65-77), but the technical key to the existence of Gene therapy is how to design a highly efficient and safe Gene transfection vector to compress and protect oligonucleotides from degradation by serum nucleases (Niven, R., et al. Journal of Pharmaceutical Sciences, 1998, 87, 1292-1299). Viral vectors exhibit high efficiency in gene transfection of many cell lines, but their clinical applications are limited by the potential carcinogenicity, immunogenicity, limited DNA compaction capacity, and the difficulty of large-scale production (Anderson, w.f. Nature,1998, 392, 25-30).
In recent years, non-viral vectors have attracted considerable attention because of their low immunogenicity, low toxicity and large-scale production (Meredith A., et al. Chemical Reviews, 2009,109, 259-302). Cationic polymers as an important class of non-viral vectors, Polylysine (PLL), Polyethyleneimine (PEI), polymethacrylates, cationic polysaccharide derivatives and dendrimers such as Polyamidoamine (PAMAM), polypropyleneimine (PPI) and Polylysine (PLL) are currently reported (Meredith A., et al, Chemical Reviews, 2009,109, 259-. Recently, the structure of the cationic polymer is closely related to the biological properties thereof, such as stability, cytotoxicity and gene transfection efficiency of the cationic polymer/gene complex (Gao, Y., et al, Biomacromolecules, 2016, 17, 3640-. We have reported that cationic hyperbranched polysaccharide derivatives containing small amine groups (such as pullulan and glycogen) have low cytotoxicity and can efficiently transfect plasmid DNA (pDNA) and small interfering RNA (siRNA) (Zhou Y.F., et al. Biomaterials, 2012, 33, 4731-. However, due to steric hindrance effects, there is still a great difficulty in how to synthesize polysaccharide derivatives containing highly hyperbranched cations.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of cationic polymers in the prior art and provides a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups. Selectively coupling dendritic macromolecular polyamide-amine (PAMAM D3 or D4) to a hyperbranched polysaccharide chain by adopting a high-efficiency azide-alkyne click reaction method to synthesize a highly hyperbranched cationic polysaccharide derivative; the polysaccharide derivative has a highly hyperbranched structure, is beneficial to improving the transfection efficiency of the polysaccharide derivative on genes, and is expected to be used as a gene carrier.
The invention aims to provide a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups.
It is another object of the present invention to provide a process for the preparation of said highly hyperbranched cationic polysaccharide derivatives.
It is a further object of the present invention to provide the use of said highly hyperbranched cationic polysaccharide derivatives.
The above object of the present invention is achieved by the following technical solutions:
a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups has a structural formula shown as a formula (I):
wherein R is H or
Is a 3 or 4 generation polyamidoamine.
The preparation method of the highly hyperbranched cationic polysaccharide derivative containing the dendritic polyamide-amine group comprises the step of selectively coupling dendritic macromolecular polyamide-amine (PAMAM D3 or D4) to a hyperbranched polysaccharide chain by a high-efficiency azide-alkyne click reaction method to synthesize the highly hyperbranched cationic polysaccharide derivative.
Preferably, the polysaccharide is glycogen or amylopectin.
Specifically, the preparation method comprises the following steps:
s1, introducing inert gas into an ice-water bath, dropwise adding alcohol solution containing propargylamine into alcohol solution containing methyl acrylate, reacting at room temperature after the ice-water bath reaction, and evaporating and drying after the reaction is finished to obtain 0.5-generation polyamide-amine; under the same condition, dripping alcohol solution containing 0.5 generation of polyamide-amine into alcohol solution containing ethylenediamine, reacting in ice water bath, reacting at room temperature, evaporating after the reaction is finished, and drying to obtain 1 generation of polyamide-amine; then 1 generation of polyamide-amine reacts with methyl acrylate to prepare 1.5 generation of polyamide-amine; repeating the steps to prepare alkynyl-containing polyamide-amine with different generations; the reaction process is shown as a reaction formula (II):
s2, under the protection of inert gas, dissolving polysaccharide in an organic solvent, adding N, N' -carbonyldiimidazole for activation at room temperature, adding azidopropylamine for reaction, and dialyzing and drying a product after the reaction is finished to obtain azidopropylamine modified polysaccharide; the reaction process is shown as a reaction formula (III):
s3, under the protection of inert gas, dissolving azidopropylamine modified polysaccharide, alkynyl-containing polyamide-amine, copper sulfate pentahydrate and sodium ascorbate in a mixed solvent of an organic solvent and water, reacting at 20-40 ℃, dialyzing a product after the reaction is finished, and drying to obtain a polyamide-amine modified highly hyperbranched cationic polysaccharide derivative; the reaction process is shown as a reaction formula (IV):
preferably, the alcohol solution in step S1 is a methanol solution.
Preferably, the mass ratio of propargylamine to methyl acrylate in step S1 is 0.1-10: 1-100; the mass ratio of the 0.5-generation polyamide-amine to the ethylenediamine is 1-30: 4-200; the mass ratio of the 1-generation polyamide-amine to the methyl acrylate is 1-30: 5 to 100.
Preferably, the reaction time of the ice-water bath in the step S1 is 0.5-6 hours, and the reaction time at room temperature is 24-120 hours.
Preferably, the dropping speed of the solution in the step S1 is 0.1-3 ml/min, and the dropping is performed to make the methyl acrylate or the ethylenediamine excessively large, so as to reduce the occurrence of side reactions and improve the reaction yield.
Preferably, the evaporation of S1 is rotary evaporation, the temperature of the rotary evaporation is 30-60 ℃, and the drying is vacuum drying at 30-60 ℃ for removing unreacted methyl acrylate, ethylenediamine and methanol.
Preferably, the organic solvent in step S2 is anhydrous dimethyl sulfoxide.
Since N, N' -Carbonyldiimidazole (CDI) is sensitive to water and is liable to be decomposed by absorption of water, the reaction of CDI must be carried out under anhydrous conditions, and the raw materials and solvent used must be sufficiently dehydrated and dried. As a preferable scheme, the preparation method of the anhydrous dimethyl sulfoxide comprises the following steps: adding 0.1-20 g of calcium hydride into 50-500 ml of dimethyl sulfoxide, stirring at room temperature for 1-7 days, standing for 1-7 days, filtering, adding a molecular sieve into the filtrate, and soaking for 1-7 days.
Preferably, the mass ratio of the polysaccharide to the N, N' -carbonyldiimidazole in the step S2 is 0.1-10: 0.01 to 5.
Preferably, the room-temperature activation time of step S2 is 0.5 to 5 hours, and the reaction time after the addition of azidopropylamine is 12 to 72 hours.
Preferably, in the step S3, the azidopropylamine-modified polysaccharide, the alkynyl-containing polyamide-amine, the copper sulfate pentahydrate and the sodium ascorbate have a mass ratio of 0.1-10: 0.1 to 100: 0.01-10: 0.01 to 10.
Preferably, the reaction at 20-40 ℃ in the step S3 is carried out for 24-96 hours.
Preferably, the mixed solvent of the organic solvent and water in step S3 is a mixed solvent of 0.5 to 100 ml of dimethyl sulfoxide and 0.5 to 10 ml of water.
Preferably, the preparation method of azidopropylamine in the step S2 is as follows: under the protection of inert gas, adding sodium azide into 3-chloropropylamine hydrochloride and potassium chloride aqueous solution, reacting for 12-72 hours at 50-90 ℃, cooling to room temperature, adjusting the pH of the solution to 8-11, extracting the reaction solution by using an organic solvent, collecting an organic phase, removing water, filtering, and distilling under reduced pressure to obtain propylamine azide; the reaction process is shown as a reaction formula (V):
more preferably, the pH is adjusted by adding 0.01-10 ml of sodium hydroxide solution.
More preferably, the extraction solvent can be selected from dichloromethane, ether or chloroform which are organic solvents with high azidopropylamine solubility, and the organic phase can be selected from anhydrous sodium sulfate, anhydrous magnesium sulfate or anhydrous calcium chloride after removing water.
The product dialysis aims at removing the solvent and unreacted raw materials, and the conventional pure water dialysis is adopted; as a preferable scheme, a dialysis bag with the cut-off molecular weight of 8000-50,000 is selected for dialysis in the step S2 or S3, so that the polysaccharide derivative is not dialyzed while small molecular impurities are removed, and the dialysis time is 1-5 days.
Preferably, the drying in step S2 or S3 is freeze-drying, so that the polysaccharide derivative is dried at low temperature, ensuring that its structural properties are not changed.
Meanwhile, inert gas is introduced, in step S1, a side reaction between ethylenediamine and carbon dioxide in the air is avoided, in step S2, the reaction is in an anhydrous and anaerobic state, and the reduction of CDI activity is avoided, in step S3, the reaction is in an anaerobic state, and the generated cuprous ions are prevented from being oxidized, and as a preferred scheme, the gas is nitrogen, helium or argon.
The highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups prepared by the invention has a highly hyperbranched structure, is beneficial to improving the gene transfection efficiency, and is expected to be used as a gene carrier. Therefore, the application of the highly hyperbranched cationic polysaccharide derivative containing the dendritic polyamide-amine group in the preparation of a gene transfection vector is also within the protection scope of the invention.
As a preferred possible embodiment, the process for the preparation of the highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamidoamine groups according to the invention comprises in particular the following steps:
s1, synthesizing alkynyl-containing polyamide-amine dendrimers of different generations:
S11.Synthesis of the polyamidoamines (D1): introducing protective gas into an ice-water bath, dripping 1-100 ml of methanol solution containing 0.1-10 g of propargylamine into 10-100 ml of methanol solution containing 1-100 g of methyl acrylate, stirring and reacting for 0.5-6 hours, heating to room temperature, stirring and reacting for 24-72 hours, performing rotary evaporation, and drying to obtain 0.5-generation polyamide-amine (D0.5); introducing protective gas into an ice-water bath, dripping 10-200 ml of methanol solution containing 1-30 g of 0.5-generation polyamide-amine into 20-500 ml of methanol solution containing 4-200 g of ethylenediamine, stirring and reacting for 0.5-6 hours, heating to room temperature, stirring and reacting for 24-120 hours, performing rotary evaporation, and drying to obtain 1-generation polyamide-amine (D1);
S12.Synthesis of polyamidoamine generation 2 (D2): introducing gas protection into an ice-water bath, dropwise adding 20-300 ml of methanol solution containing 1-30 g of 1-generation polyamidoamine into 30-500 ml of methanol solution containing 5-100 g of methyl acrylate, stirring for 1-10 hours, heating to room temperature, stirring for reaction for 24-120 hours, performing rotary evaporation, and drying to obtain 1.5-generation polyamidoamine (D1.5); carrying out ice-water bath aeration body protection, namely dripping 30-500 ml of methanol solution containing 3-50 g of 1.5-generation polyamidoamine into 50-500 ml of methanol solution containing 20-300 g of ethylenediamine, stirring and reacting for 1-10 hours, then heating to room temperature, stirring and reacting for 24-120 hours, carrying out rotary evaporation, and drying to obtain 2-generation polyamidoamine (D2);
S13.Synthesis of polyamidoamine generation 3 (D3): carrying out ice-water bath aeration body protection, dripping 50-500 ml of methanol solution containing 5-50 g of 2-generation polyamidoamine into 40-600 ml of methanol solution containing 50-300 g of methyl acrylate, stirring for 1-10 hours, heating to room temperature, stirring for reaction for 24-120 hours, carrying out rotary evaporation, and drying to obtain 2.5-generation polyamidoamine (D2.5); carrying out ice-water bath aeration body protection, namely dripping 50-800 ml of methanol solution containing 15-100 g of 2.5-generation polyamidoamine into 50-800 ml of methanol solution containing 40-500 g of ethylenediamine, stirring and reacting for 1-10 hours, then heating to room temperature, stirring and reacting for 36-168 hours, carrying out rotary evaporation, and drying to obtain 3-generation polyamidoamine (D3);
S14.Synthesis of the 4-generation polyamidoamines (D4): carrying out ice-water bath aeration body protection, namely dripping 60-700 ml of methanol solution containing 10-80 g of 3-generation polyamidoamine into 60-800 ml of methanol solution containing 60-500 g of methyl acrylate, stirring for 1-10 hours, heating to room temperature, stirring for reaction for 24-120 hours, carrying out rotary evaporation, and drying to obtain 3.5-generation polyamidoamine (D3.5); carrying out ice-water bath ventilation body protection, namely dripping 60-900 ml of methanol containing 30-100 g of 3.5-generation polyamide-amine into 60-900 ml of methanol solution containing 50-600 g of ethylenediamine, stirring and reacting for 1-10 hours, then heating to room temperature, stirring and reacting for 36-168 hours, carrying out rotary evaporation, and drying to obtain 4-generation polyamide-amine (D4);
s2, synthesis of azidopropylamine modified polysaccharide:
s21, synthesis of azidopropylamine: under the protection of inert gas, 10-50 g of 3-chloropropylamine hydrochloride and 0.01-10 g of potassium iodide are dissolved in 50-500 ml of deionized water, 10-200 g of sodium azide is added, and the temperature is raised to 50-90 ℃ for reaction for 12-72 hours. Cooling to room temperature, and adding 0.01-10 ml of sodium hydroxide solution to adjust the pH of the solution to 8-11. And extracting the reaction solution for 3-10 times by using an organic solvent, separating the solution, collecting an organic phase, removing water from the organic phase, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
S22, synthesis of azidopropylamine modified polysaccharide: under the protection of inert gas, 0.1-10 g of polysaccharide is dissolved in 1-300 ml of dimethyl sulfoxide, 0.01-5 g of N, N' -Carbonyldiimidazole (CDI) is added to activate at room temperature for 0.5-5 hours, and azidopropylamine is added to react for 12-72 hours. And after the reaction is finished, dialyzing the product in deionized water, and drying to obtain the azidopropylamine modified polysaccharide.
S3, synthesis of highly hyperbranched cationic polysaccharide derivatives: under the protection of inert gas, 0.01-10 g of propylamine azide modified polysaccharide, 0.1-100 g of dendritic macromolecule containing alkynyl polyamide-amine in different generations, 0.01-10 g of copper sulfate pentahydrate and 0.01-10 g of sodium ascorbate are dissolved in a mixed solvent of 0.5-100 ml of dimethyl sulfoxide and 0.5-10 ml of water, and the mixture is stirred and reacted for 24-96 hours at the temperature of 20-60 ℃. And dialyzing the product in deionized water, and drying to obtain the polyamide-amine modified highly hyperbranched cationic polysaccharide derivatives with different generations.
The product structure schematic diagram and the synthesis reaction mechanism of the preparation method are respectively shown in figure 1 and figure 2. Compared with glycogen or amylopectin modified by small molecular amine, the synthesis method of the highly hyperbranched cationic polysaccharide derivative containing the dendritic polyamidoamine has the following characteristics:
(1) the raw materials of the dendritic polyamide-amine and the hyperbranched polysaccharide derivative (amylopectin or glycogen) have a highly branched structure, and have high reaction steric hindrance and high reaction difficulty.
(2) The reaction of the invention adopts azide-alkyne click reaction to synthesize the dendritic polyamide-amine modified highly hyperbranched cationic polysaccharide derivative with controllable structure, and the reaction condition is mild, high-efficiency and selective.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts azide-alkyne click chemistry reaction to synthesize the highly hyperbranched cationic polysaccharide derivative with controllable structure, and has mild reaction condition, high efficiency and selectivity.
(2) The dendritic polyamide-amine modified highly hyperbranched cationic polysaccharide derivative prepared by the invention has good water solubility, can well form an electropositive nano compound with siRNA, and is beneficial to carrying siRNA to enter cells.
(3) The dendritic polyamide-amine modified highly hyperbranched cationic polysaccharide derivative prepared by the invention has a highly hyperbranched structure, is beneficial to improving the transfection efficiency of the derivative on genes, and can be expected to be used as a gene carrier.
Drawings
Fig. 1 is a schematic diagram of the product structure of the present invention, wherein a is a schematic diagram of the hyperbranched polysaccharide structure, and B is a chemical structure of a highly hyperbranched cationic polysaccharide derivative containing a dendritic polyamidoamine group.
FIG. 2 is a reaction scheme of the preparation process of the present invention.
FIG. 3 shows Glycogen raw material (Glycogen) and azidopropylamine modified Glycogen (Gly-N) according to the present invention3) An infrared spectrum (FTIR) of the products of polyamidoamine 4 generation (PAMAM D4) and polyamidoamine 4 generation modified highly hyperbranched cationic glycogen (Gly-D4).
FIG. 4 shows Glycogen raw material (Glycogen) and azidopropylamine modified Glycogen (Gly-N) according to the present invention3) 4-generation polyamidoamines (PAMAM D4) and 4-generation polyamidoamines modified with high levels ofNuclear magnetic spectrum of highly hyperbranched cationic glycogen (Gly-D4) product (1HNMR）。
FIG. 5 is the agarose gel electrophoresis of the 4 generation polyamidoamine modified highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex of the invention (w/w represents the mass ratio of Gly-D4 to siRNA).
FIG. 6 is zeta potential diagram (w/w represents the mass ratio of Gly-D4 to siRNA) of 4-generation polyamidoamine modified highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex of the invention.
FIG. 7 is a graph of particle size analysis of 4 generation polyamidoamine modified highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex of the invention (w/w represents the mass ratio of Gly-D4 to siRNA).
FIG. 8 is a scanning electron microscope image of the 4-generation polyamidoamine modified highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex (mass ratio of 10) of the invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1
1. Preparation of 4-generation polyamidoamine modified highly hyperbranched cationic glycogen derivative (Gly-D4)
(1) Introducing nitrogen for protection in ice-water bath, dropwise adding 10 ml of methanol solution containing 4 g of propargylamine into 30 ml of methanol solution containing 13.6 g of methyl acrylate, stirring for reaction for 3 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation, and performing vacuum drying to obtain 0.5-generation polyamide-amine (D0.5); introducing nitrogen for protection in ice-water bath, dripping 100 ml methanol solution containing 16.2 g of 0.5-generation polyamide-amine into 100 ml methanol solution containing 50 g of ethylenediamine, stirring for reaction for 3 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 1-generation polyamide-amine (D1);
(2) introducing nitrogen for protection in an ice-water bath, dripping 50 ml of methanol solution containing 18 g of 1-generation polyamidoamine into 50 ml of methanol solution containing 32 g of methyl acrylate, stirring for 4 hours, heating to room temperature, stirring for reaction for 36 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 1.5-generation polyamidoamine (D1.5); introducing nitrogen for protection in ice-water bath, dropwise adding 70 ml of methanol solution containing 28 g of 1.5-generation polyamide-amine into 100 ml of methanol solution containing 80 g of ethylenediamine, stirring for reaction for 3 hours, then heating to room temperature, stirring for reaction for 36 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 2-generation polyamide-amine (D2);
(3) ice water bath nitrogen protection, dripping 100 ml methanol solution containing 35 g 2 generation polyamidoamine into 100 ml methanol solution containing 50 g methyl acrylate, stirring for 3 hours, heating to room temperature, stirring for reaction for 36 hours, rotary evaporating at 30 ℃, and vacuum drying at 30 ℃ to obtain 2.5 generation polyamidoamine (D2.5); introducing nitrogen for protection in ice-water bath, dripping 100 ml methanol solution containing 45 g 2.5 generation polyamidoamine into 150 ml methanol solution containing 105 g ethylene diamine, stirring and reacting for 3 hours, then heating to room temperature, stirring and reacting for 36 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 3 generation polyamidoamine (D3);
(4) introducing nitrogen for protection in ice-water bath, dropwise adding 150 ml of methanol solution containing 50 g of 3-generation polyamidoamine into 100 ml of methanol solution containing 80 g of methyl acrylate, stirring for 3 hours, heating to room temperature, stirring for reaction for 36 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 3.5-generation polyamidoamine (D3.5); introducing nitrogen for protection in ice-water bath, dropwise adding 200 ml of methanol solution containing 60 g of 3.5-generation polyamidoamine into 300 ml of methanol solution containing 170 g of ethylenediamine, stirring for reaction for 3 hours, then heating to room temperature, stirring for reaction for 48 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 4-generation polyamidoamine (D4);
(5) under the protection of nitrogen, 10 g of 3-chloropropylamine hydrochloride and 1 g of potassium iodide are dissolved in 50 ml of deionized water, 50 g of sodium azide is added, and the temperature is raised to 80 ℃ for reaction for 48 hours. Cooled to room temperature and 1.5 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. And extracting the reaction solution with diethyl ether for 6 times, separating the solution, collecting an organic phase, removing water from the organic phase with anhydrous magnesium sulfate, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
(6) Under the protection of nitrogen, 0.1 g of glycogen is dissolved in 50 ml of dimethyl sulfoxide, 0.1 g of N, N' -Carbonyldiimidazole (CDI) is added to activate at room temperature for 3 hours, and azidopropylamine is added to react for 48 hours. After the reaction is finished, the product is put into a dialysis bag with the cut-off molecular weight of 8000 to be dialyzed for 3 days against deionized water, and is frozen and dried to obtain the azidopropylamine modified glycogen (Gly-N)3）。
(7) Under the protection of nitrogen, 0.08 g of propylamine azide-modified glycogen, 8.9 g of 4-generation polyamidoamine, 0.05 g of copper sulfate pentahydrate and 0.20 g of sodium ascorbate were dissolved in a mixed solvent of 20 ml of dimethyl sulfoxide and 10 ml of water, and the reaction was stirred at 40 ℃ for 48 hours. And putting the product into a dialysis bag with cut-off molecular weight of 10000, dialyzing the product for 3 days against deionized water, and freeze-drying the dialyzed product to obtain the 4-generation polyamidoamine modified highly hyperbranched cationic glycogen derivative (Gly-D4).
2. Results
(1) The glycogen raw material and the products are subjected to infrared spectrum characterization, and the obtained FTIR spectrum is shown in figure 3. As can be seen from fig. 3: Gly-N compared to glycogen profile3Has an FTIR spectrum of 2104cm-1An extensional vibration absorption peak of azido group (-N = N = N) was observed in the vicinity of 1709 cm-1C = O stretching shock absorption peaks of the carbamate appeared, demonstrating that azidopropylamine has been coupled to glycogen. Furthermore, with Gly-N3And comparing the FTIR spectrum of PAMAM D4, the FTIR spectrum of Gly-D4 is 2104cm-1Disappearance of the azido stretching vibration peak of (2) at 1658,1552,1331 cm-1The amide I peak, the amide II peak and the amide III peak of the secondary amide appear nearby, and are 1152 cm-1Near the absorption peak of C-O-C stretching vibration on glycogen sugar ring, at 1029 cm-1The vicinity is an O-H variable angle vibration absorption peak on glycogen, and the fact that the 4 generation polyamidoamine is coupled to the glycogen is proved.
(2) Subjecting the glycogen raw material and each product to nuclear magnetic resonance spectroscopy characterization to obtain1The H NMR spectrum is shown in FIG. 4. The proton peaks of the sugar units of glycogen are assigned as follows: the proton peak of the anomeric hydrogen (H1) appears at 5.5-5.1 ppm, and the proton peak of H2-H6 appears at 4.1-3.2 ppm. With glycogen1Comparison of H NMR spectra, Gly-N3Is/are as follows1The H NMR spectrum showed the following new peaks: 1.7 ppm (b), 2.8 ppm (a) and 3.1 ppm (c) (see FIG. 4E), demonstrating that Gly-N was synthesized3. In addition to glycogen and 4-generation polyamidoamines1Comparison of H NMR spectra, of Gly-D41The H NMR spectrum showed the following new peaks: 2.3 ppm (b), 2.4-3.4 ppm (e, f, g, h, i, j, k) and 8.0 ppm (D) prove that Gly-D4 derivatives are synthesized.
(3) To confirm the ability of 4-generation polyamidoamine-modified highly hyperbranched glycogen derivative (Gly-D4) to complex genes, Gly-D4 was complexed with 21 base pair-containing small interfering RNA (siRNA) and then subjected to agarose gel electrophoresis, the results of which are shown in FIG. 5. The mass ratio (w/w) was Gly-D4, and it was found that Gly-D4 was completely complexed with siRNA when the mass ratio was 10 or more.
(4) After the obtained 4-generation polyamidoamine modified highly hyperbranched glycogen derivative (Gly-D4) and siRNA were prepared into nanocomposite solutions with different mass ratios, the Zeta potential of the nanocomposite was tested with a Zeta potential meter, and the obtained results are shown in FIG. 6. When the mass ratio of Gly-D4 to siRNA is more than 1, the zeta potential of the nano-composite is positive.
(5) After the obtained 4-generation polyamidoamine modified highly hyperbranched glycogen derivative (Gly-D4) and siRNA were formulated into nanocomposite solutions with different mass ratios, the hydrodynamic diameter of the nanocomposite was tested with a dynamic light scattering instrument, and the obtained results are shown in fig. 7. When the mass ratio of Gly-D4 to siRNA is more than 5, the dynamic mechanical diameter of the nano-composite is within the range of 100-200 nm.
(6) After the obtained 4-generation polyamidoamine modified highly hyperbranched glycogen derivative (Gly-D4) and siRNA are prepared into a nano compound solution with the mass ratio of 10, a trace amount of solution is dropwise added onto an aluminum foil, the aluminum foil is naturally dried, the appearance of the aluminum foil is observed by using a scanning electron microscope, and the obtained result is shown in figure 8. The nano-composite is irregular and spherical and has a diameter of about 200 nm.
Example 2
1. Preparation of 3-generation polyamide-amine modified highly hyperbranched cationic glycogen derivative (Gly-D3)
(1) Introducing nitrogen for protection in ice-water bath, dropwise adding 5 ml of methanol solution containing 0.1 g of propargylamine into 10 ml of methanol solution containing 1 g of methyl acrylate, stirring for reaction for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 40 ℃, and performing vacuum drying at 40 ℃ to obtain 0.5-substituted polyamidoamine (D0.5); introducing nitrogen for protection in ice-water bath, dropwise adding 15 ml of methanol solution containing 1.2 g of 0.5-generation polyamide-amine into 20 ml of methanol solution containing 6 g of ethylenediamine, stirring for reaction for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 40 ℃, and performing vacuum drying at 40 ℃ to obtain 1-generation polyamide-amine (D1);
(2) introducing nitrogen for protection in an ice-water bath, dripping 20 ml of methanol solution containing 2 g of 1-generation polyamidoamine into 25 ml of methanol solution containing 15 g of methyl acrylate, stirring for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 40 ℃, and performing vacuum drying at 40 ℃ to obtain 1.5-generation polyamidoamine (D1.5); introducing gas for protection in an ice-water bath, dropwise adding 30 ml of methanol solution containing 3.6 g of 1.5-generation polyamide-amine into 50 ml of methanol solution containing 20 g of ethylenediamine, stirring for reaction for 2 hours, then heating to room temperature, stirring for reaction for 24 hours, carrying out rotary evaporation at 40 ℃, and carrying out vacuum drying at 40 ℃ to obtain 2-generation polyamide-amine (D2);
(3) introducing nitrogen for protection in ice-water bath, dripping 50 ml of methanol solution containing 5 g of 2-generation polyamidoamine into 60 ml of methanol solution containing 50 g of methyl acrylate, stirring for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 40 ℃, and performing vacuum drying at 40 ℃ to obtain 2.5-generation polyamidoamine (D2.5); introducing gas for protection in an ice-water bath, dropwise adding 60 ml of methanol solution containing 12 g of 2.5-generation polyamidoamine into 60 ml of methanol solution containing 40 g of ethylenediamine, stirring and reacting for 2 hours, then heating to room temperature, stirring and reacting for 48 hours, carrying out rotary evaporation at 40 ℃, and carrying out vacuum drying at 40 ℃ to obtain 3-generation polyamidoamine (D3);
(4) under the protection of nitrogen, 10 g of 3-chloropropylamine hydrochloride and 1 g of potassium iodide are dissolved in 50 ml of deionized water, 50 g of sodium azide is added, and the temperature is raised to 80 ℃ for reaction for 48 hours. Cooled to room temperature and 2 ml of sodium hydroxide solution are added to adjust the pH of the solution to 11. Extracting the reaction solution with diethyl ether for 8 times, separating the solution, collecting an organic phase, removing water from the organic phase with anhydrous calcium chloride, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
(5) Under the protection of nitrogen, 0.1 g of glycogen is dissolved in 20 ml of dimethyl sulfoxide, 1 g of N, N' -Carbonyldiimidazole (CDI) is added to activate at room temperature for 3 hours, and azidopropylamine is added to react for 48 hours. After the reaction is finished, the product is put into a dialysis bag with the cut-off molecular weight of 8000 to be dialyzed for 3 days against deionized water, and is frozen and dried to obtain the azidopropylamine modified glycogen (Gly-N)3）
(6) 0.05 g of propylamine azide-modified glycogen, 5.3 g of 3-generation polyamidoamine, 0.5 g of copper sulfate pentahydrate and 2 g of sodium ascorbate were dissolved in a mixed solvent of 15 ml of dimethyl sulfoxide and 5 ml of water under the protection of nitrogen, and the reaction was stirred at 40 ℃ for 48 hours. And putting the product into a dialysis bag with cut-off molecular weight of 10000, dialyzing the product for 3 days against deionized water, and freeze-drying the dialyzed product to obtain the 3-generation polyamidoamine modified highly hyperbranched cationic glycogen derivative (Gly-D3).
Example 3
1. Preparation of 4-generation polyamidoamine modified highly hyperbranched cationic glycogen derivative (Gly-D4)
(1) Introducing nitrogen for protection in ice-water bath, dropwise adding 10 ml of methanol solution containing 1.5 g of propargylamine into 30 ml of methanol solution containing 12 g of methyl acrylate, stirring for reaction for 1 hour, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation, and performing vacuum drying to obtain 0.5-generation polyamide-amine (D0.5); under the protection of ice water bath nitrogen, dripping 30 ml of methanol solution containing 5 g of 0.5-generation polyamidoamine into 50 ml of methanol solution containing 15 g of ethylenediamine, stirring and reacting for 1 hour, heating to room temperature, stirring and reacting for 24 hours, performing rotary evaporation at 60 ℃, and performing vacuum drying at 60 ℃ to obtain 1-generation polyamidoamine (D1);
(2) introducing nitrogen for protection in ice-water bath, dropwise adding 30 ml of methanol solution containing 6 g of 1-generation polyamidoamine into 30 ml of methanol solution containing 6 g of methyl acrylate, stirring for 1 hour, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation, and performing vacuum drying to obtain 1.5-generation polyamidoamine (D1.5); introducing gas for protection in an ice-water bath, dropwise adding 30 ml of methanol solution containing 8 g of 1.5-generation polyamidoamine into 50 ml of methanol solution containing 25 g of ethylenediamine, stirring and reacting for 1 hour, then heating to room temperature, stirring and reacting for 24 hours, carrying out rotary evaporation at 60 ℃, and carrying out vacuum drying at 60 ℃ to obtain 2-generation polyamidoamine (D2);
(3) introducing nitrogen for protection in ice-water bath, dripping 60 ml of methanol solution containing 10 g of 2-generation polyamidoamine into 40 ml of methanol solution containing 50 g of methyl acrylate, stirring for 1 hour, heating to room temperature, stirring for reaction for 48 hours, performing rotary evaporation at 60 ℃, and performing vacuum drying at 60 ℃ to obtain 2.5-generation polyamidoamine (D2.5); introducing gas for protection in an ice-water bath, dropwise adding 100 ml of methanol solution containing 15 g of 2.5-generation polyamidoamine into 100 ml of methanol solution containing 60 g of ethylenediamine, stirring and reacting for 1 hour, then heating to room temperature, stirring and reacting for 48 hours, carrying out rotary evaporation at 60 ℃, and carrying out vacuum drying at 60 ℃ to obtain 3-generation polyamidoamine (D3);
(4) introducing nitrogen for protection in ice-water bath, dripping 150 ml methanol solution containing 18 g of 3-generation polyamidoamine into 200 ml methanol solution containing 70 g of methyl acrylate, stirring for 1 hour, heating to room temperature, stirring for reaction for 72 hours, performing rotary evaporation at 60 ℃, and performing vacuum drying at 60 ℃ to obtain 3.5-generation polyamidoamine (D3.5); introducing gas for protection in an ice-water bath, dropwise adding 300 ml of methanol solution containing 31 g of 3.5-generation polyamidoamine into 400 ml of methanol solution containing 200 g of ethylenediamine, stirring and reacting for 1 hour, then heating to room temperature, stirring and reacting for 72 hours, carrying out rotary evaporation at 60 ℃, and carrying out vacuum drying at 60 ℃ to obtain 4-generation polyamidoamine (D4);
(5) under the protection of nitrogen, 50 g of 3-chloropropylamine hydrochloride and 5 g of potassium iodide are dissolved in 500 ml of deionized water, 100 g of sodium azide is added, and the temperature is raised to 90 ℃ for reaction for 72 hours. Cooled to room temperature and 8 ml of sodium hydroxide solution are added to adjust the pH of the solution to 11. And extracting the reaction solution for 10 times by using dichloromethane, separating the solution, collecting an organic phase, removing water by using anhydrous magnesium sulfate of the organic phase, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
(6) Under the protection of nitrogen, 1 g of glycogen is dissolved in 100 ml of dimethyl sulfoxide, 5 g of N, N' -Carbonyldiimidazole (CDI) is added to activate at room temperature for 3 hours, and azidopropylamine is added to react for 48 hours. After the reaction is finished, the product is put into a dialysis bag with the cut-off molecular weight of 8000 to be dialyzed for 3 days against deionized water, and is frozen and dried to obtain the azidopropylamine modified glycogen (Gly-N)3）
(7) Under the protection of nitrogen, 0.8 g of azidopropylamine-modified glycogen, 20 g of 4-generation polyamidoamine, 1 g of copper sulfate pentahydrate and 10 g of sodium ascorbate were dissolved in a mixed solvent of 50 ml of dimethyl sulfoxide and 10 ml of water, and the reaction was stirred at 30 ℃ for 96 hours. And putting the product into a dialysis bag with cut-off molecular weight of 10000, dialyzing the product for 3 days against deionized water, and freeze-drying the dialyzed product to obtain the 4-generation polyamidoamine modified highly hyperbranched cationic glycogen derivative (Gly-D4).
Example 4
1. Preparation of 4-generation polyamide-amine modified highly hyperbranched cationic amylopectin derivative (Amyp-D4)
(1) Introducing nitrogen for protection in ice-water bath, dropwise adding 15 ml of methanol solution containing 4 g of propargylamine into 40 ml of methanol solution containing 16 g of methyl acrylate, stirring and reacting for 6 hours, heating to room temperature, stirring and reacting for 36 hours, performing rotary evaporation at 35 ℃, and performing vacuum drying at 35 ℃ to obtain 0.5-substituted polyamide-amine (D0.5); introducing nitrogen for protection in ice-water bath, dropwise adding 80 ml of methanol solution containing 18 g of 0.5-generation polyamidoamine into 80 ml of methanol solution containing 60 g of ethylenediamine, stirring for reaction for 6 hours, heating to room temperature, stirring for reaction for 36 hours, performing rotary evaporation at 35 ℃, and performing vacuum drying at 35 ℃ to obtain 1-generation polyamidoamine (D1);
(2) introducing nitrogen for protection in ice-water bath, dripping 80 ml of methanol solution containing 20 g of 1-generation polyamidoamine into 100 ml of methanol solution containing 50 g of methyl acrylate, stirring for 6 hours, heating to room temperature, stirring for reaction for 48 hours, performing rotary evaporation at 35 ℃, and performing vacuum drying at 35 ℃ to obtain 1.5-generation polyamidoamine (D1.5); introducing nitrogen for protection in ice-water bath, dripping 100 ml methanol solution containing 28 g 1.5 generation polyamidoamine into 100 ml methanol solution containing 70 g ethylene diamine, stirring and reacting for 6 hours, then heating to room temperature, stirring and reacting for 48 hours, carrying out rotary evaporation at 35 ℃, and carrying out vacuum drying at 35 ℃ to obtain 2 generation polyamidoamine (D2);
(3) under the protection of ice water bath and nitrogen, dropwise adding 200 ml of methanol solution containing 30 g of 2-generation polyamidoamine into 100 ml of methanol solution containing 100 g of methyl acrylate, stirring for 6 hours, heating to room temperature, stirring for reaction for 48 hours, performing rotary evaporation at 35 ℃, and performing vacuum drying at 35 ℃ to obtain 2.5-generation polyamidoamine (D2.5); introducing nitrogen gas for protection in ice water bath, dripping 150 ml methanol solution containing 35 g 2.5 generation polyamidoamine into 150 ml methanol solution containing 105 g ethylene diamine, stirring and reacting for 6 hours, then heating to room temperature, stirring and reacting for 72 hours, carrying out rotary evaporation at 35 ℃, and carrying out vacuum drying at 35 ℃ to obtain 3 generation polyamidoamine (D3);
(4) introducing nitrogen for protection in ice-water bath, dropwise adding 350 ml of methanol solution containing 40 g of 3-generation polyamidoamine into 150 ml of methanol solution containing 160 g of methyl acrylate, stirring for 6 hours, heating to room temperature, stirring for reaction for 48 hours, performing rotary evaporation at 35 ℃, and performing vacuum drying at 35 ℃ to obtain 3.5-generation polyamidoamine (D3.5); introducing nitrogen for protection in ice-water bath, dropwise adding 500 ml of methanol solution containing 50 g of 3.5-generation polyamidoamine into 300 ml of methanol solution containing 300 g of ethylenediamine, stirring for reaction for 6 hours, then heating to room temperature, stirring for reaction for 120 hours, carrying out rotary evaporation at 35 ℃, and carrying out vacuum drying at 35 ℃ to obtain 4-generation polyamidoamine (D4);
(5) under the protection of nitrogen, 5 g of 3-chloropropylamine hydrochloride and 5 g of potassium iodide are dissolved in 100 ml of deionized water, 50 g of sodium azide is added, and the temperature is raised to 80 ℃ for reaction for 72 hours. After cooling to room temperature, the solution was adjusted to pH 11 by adding 3 ml of sodium hydroxide solution. And extracting the reaction solution with diethyl ether for 10 times, separating the solution, collecting an organic phase, removing water from the organic phase with anhydrous magnesium sulfate, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
(6) Under the protection of nitrogen, 0.5 g of amylopectin is dissolved in 100 ml of dimethyl sulfoxide, 0.3 g of N, N' -Carbonyldiimidazole (CDI) is added for activation for 2 hours at room temperature, and azide is addedPropylamine was reacted for 48 hours. After the reaction is finished, the product is put into a dialysis bag with the cut-off molecular weight of 8000 to be dialyzed for 3 days against deionized water, and is frozen and dried to obtain the azidopropylamine modified amylopectin (Amyp-N)3）。
(7) Under the protection of nitrogen, 0.08 g of propylamine azide modified amylopectin, 8.9 g of 4-generation polyamide-amine, 0.15 g of copper sulfate pentahydrate and 0.15 g of sodium ascorbate are dissolved in a mixed solvent of 20 ml of dimethyl sulfoxide and 5 ml of water, and the mixture is stirred and reacted for 48 hours at the temperature of 40 ℃. And putting the product into a dialysis bag with cut-off molecular weight of 10000, dialyzing the product for 3 days against deionized water, and freeze-drying the dialyzed product to obtain the 4-generation polyamidoamine modified highly hyperbranched cationic amylopectin derivative (Amyp-D4).
Example 5
1. 4-generation polyamide-amine modified highly hyperbranched cationic amylopectin derivative (Amyp-D4)
(1) Introducing nitrogen for protection in ice-water bath, dropwise adding 10 ml of methanol solution containing 2 g of propargylamine into 30 ml of methanol solution containing 15 g of methyl acrylate, stirring and reacting for 2 hours, heating to room temperature, stirring and reacting for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 0.5-substituted polyamide-amine (D0.5); ice water bath nitrogen protection, dripping 40 ml methanol solution containing 5 g 0.5 generation polyamidoamine into 50 ml methanol solution containing 30 g ethylene diamine, stirring and reacting for 1 hour, heating to room temperature, stirring and reacting for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 1 generation polyamidoamine (D1);
(2) introducing nitrogen for protection in ice-water bath, dropwise adding 50 ml of methanol solution containing 5 g of 1-generation polyamidoamine into 50 ml of methanol solution containing 20 g of methyl acrylate, stirring for 1 hour, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 1.5-generation polyamidoamine (D1.5); carrying out ice-water bath aeration body protection, dripping 50 ml of methanol solution containing 8 g of 1.5-generation polyamide-amine into 50 ml of methanol solution containing 30 g of ethylenediamine, stirring and reacting for 1 hour, then heating to room temperature, stirring and reacting for 24 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 2-generation polyamide-amine (D2);
(3) introducing nitrogen for protection in ice-water bath, dripping 100 ml of methanol solution containing 13 g of 2-generation polyamidoamine into 50 ml of methanol solution containing 50 g of methyl acrylate, stirring for 1 hour, heating to room temperature, stirring for reaction for 48 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 2.5-generation polyamidoamine (D2.5); introducing gas for protection in ice-water bath, dropwise adding 100 ml of methanol solution containing 20 g of 2.5-generation polyamidoamine into 100 ml of methanol solution containing 60 g of ethylenediamine, stirring and reacting for 1 hour, then heating to room temperature, stirring and reacting for 48 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 3-generation polyamidoamine (D3);
(4) introducing nitrogen for protection in ice-water bath, dripping 150 ml methanol solution containing 23 g of 3-generation polyamidoamine into 200 ml methanol solution containing 70 g of methyl acrylate, stirring for 1 hour, heating to room temperature, stirring for reaction for 72 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 3.5-generation polyamidoamine (D3.5); introducing gas for protection in an ice-water bath, dropwise adding 300 ml of methanol solution containing 31 g of 3.5-generation polyamidoamine into 100 ml of methanol solution containing 100 g of ethylenediamine, stirring and reacting for 1 hour, then heating to room temperature, stirring and reacting for 72 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 4-generation polyamidoamine (D4);
(5) under the protection of nitrogen, 10 g of 3-chloropropylamine hydrochloride and 5 g of potassium iodide are dissolved in 300 ml of deionized water, 100 g of sodium azide is added, and the temperature is raised to 90 ℃ for reaction for 72 hours. After cooling to room temperature, the solution was adjusted to pH 11 by adding 3 ml of sodium hydroxide solution. And extracting the reaction solution for 10 times by using ether, separating the solution, collecting an organic phase, removing water by using anhydrous magnesium sulfate of the organic phase, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
(6) Under the protection of nitrogen, 1 g of amylopectin is dissolved in 150 ml of dimethyl sulfoxide, 5 g of N, N' -Carbonyldiimidazole (CDI) is added to activate at room temperature for 3 hours, and azidopropylamine is added to react for 48 hours. After the reaction is finished, the product is put into a dialysis bag with the cut-off molecular weight of 8000 to be dialyzed for 3 days against deionized water, and is frozen and dried to obtain the azidopropylamine modified amylopectin (Amyp-N)3）
(7) Under the protection of nitrogen, 1 g of azidopropylamine modified amylopectin, 10 g of 4-generation polyamide-amine, 1 g of copper sulfate pentahydrate and 10 g of sodium ascorbate are dissolved in a mixed solvent of 200 ml of dimethyl sulfoxide and 10 ml of water, and the mixture is stirred and reacted for 96 hours at the temperature of 30 ℃. And putting the product into a dialysis bag with cut-off molecular weight of 10000, dialyzing the product for 3 days against deionized water, and freeze-drying the dialyzed product to obtain the 4-generation polyamidoamine modified highly hyperbranched cationic amylopectin derivative (Amyp-D4).
Example 6
1. Preparation of 3-generation polyamide-amine modified highly hyperbranched cationic amylopectin derivative (Amyp-D3)
(1) Introducing nitrogen for protection in ice-water bath, dropwise adding 10 ml of methanol solution containing 0.5 g of propargylamine into 10 ml of methanol solution containing 2 g of methyl acrylate, stirring for reaction for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 0.5-substituted polyamidoamine (D0.5); introducing nitrogen for protection in ice-water bath, dripping 20 ml methanol solution containing 1 g of 0.5 generation polyamidoamine into 20 ml methanol solution containing 10 g of ethylenediamine, stirring for reaction for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 1 generation polyamidoamine (D1);
(2) introducing nitrogen for protection in ice-water bath, dripping 20 ml of methanol solution containing 2 g of 1-generation polyamidoamine into 30 ml of methanol solution containing 20 g of methyl acrylate, stirring for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 1.5-generation polyamidoamine (D1.5); introducing gas for protection in ice-water bath, dropwise adding 30 ml of methanol solution containing 3.5 g of 1.5-generation polyamide-amine into 50 ml of methanol solution containing 20 g of ethylenediamine, stirring for reaction for 2 hours, then heating to room temperature, stirring for reaction for 24 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 2-generation polyamide-amine (D2);
(3) introducing nitrogen for protection in ice-water bath, dripping 50 ml of methanol solution containing 5 g of 2-generation polyamidoamine into 60 ml of methanol solution containing 50 g of methyl acrylate, stirring for 2 hours, heating to room temperature, stirring for reaction for 24 hours, performing rotary evaporation at 30 ℃, and performing vacuum drying at 30 ℃ to obtain 2.5-generation polyamidoamine (D2.5); introducing gas for protection in an ice-water bath, dropwise adding 60 ml of methanol solution containing 10.4 g of 2.5-generation polyamidoamine into 60 ml of methanol solution containing 60 g of ethylenediamine, stirring for reaction for 2 hours, then heating to room temperature, stirring for reaction for 48 hours, carrying out rotary evaporation at 30 ℃, and carrying out vacuum drying at 30 ℃ to obtain 3-generation polyamidoamine (D3);
(4) under the protection of nitrogen, 10 g of 3-chloropropylamine hydrochloride and 0.5 g of potassium iodide are dissolved in 50 ml of deionized water, 20 g of sodium azide is added, and the temperature is raised to 80 ℃ for reaction for 48 hours. Cooled to room temperature and 2 ml of sodium hydroxide solution are added to adjust the pH of the solution to 11. And extracting the reaction solution with chloroform for 6 times, separating the solution, collecting an organic phase, removing water from the organic phase with anhydrous magnesium sulfate, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
(5) Under the protection of nitrogen, 0.1 g of amylopectin is dissolved in 20 ml of dimethyl sulfoxide, 1 g of N, N' -Carbonyldiimidazole (CDI) is added to activate at room temperature for 1 hour, and azidopropylamine is added to react for 48 hours. After the reaction is finished, the product is put into a dialysis bag with the cut-off molecular weight of 8000 to be dialyzed for 3 days against deionized water, and is frozen and dried to obtain the azidopropylamine modified amylopectin (Amyp-N)3）
(6) Under the protection of nitrogen, 0.1 g of propylamine azide modified amylopectin, 10 g of 3-generation polyamide-amine, 0.5 g of copper sulfate pentahydrate and 3 g of sodium ascorbate are dissolved in a mixed solvent of 15 ml of dimethyl sulfoxide and 5 ml of water, and the mixture is stirred and reacted for 48 hours at the temperature of 40 ℃. And putting the product into a dialysis bag with cut-off molecular weight of 10000, dialyzing the product for 3 days against deionized water, and freeze-drying the dialyzed product to obtain the 3-generation polyamidoamine modified highly hyperbranched cationic amylopectin derivative (Amyp-D3).
Claims (9)
1. A highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups is characterized in that the structural formula is as follows:
the above-mentioned
Is a 3 or 4 generation polyamidoamine;
the preparation method of the highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups comprises the following steps:
s1, introducing inert gas into an ice-water bath, dropwise adding alcohol solution containing propargylamine into alcohol solution containing methyl acrylate, reacting at room temperature after the ice-water bath reaction, and evaporating and drying after the reaction is finished to obtain 0.5-generation polyamide-amine; under the same condition, dripping alcohol solution containing 0.5 generation of polyamide-amine into alcohol solution containing ethylenediamine, reacting in ice water bath, reacting at room temperature, evaporating after the reaction is finished, and drying to obtain 1 generation of polyamide-amine; then 1 generation of polyamide-amine reacts with methyl acrylate to prepare 1.5 generation of polyamide-amine; repeating the steps to prepare 3 or 4 substituted alkynyl-containing polyamide-amine;
s2, under the protection of inert gas, dissolving polysaccharide in an organic solvent, adding N, N' -carbonyldiimidazole for activation at room temperature, adding azidopropylamine for reaction, and dialyzing and drying a product after the reaction is finished to obtain azidopropylamine modified polysaccharide;
s3, under the protection of inert gas, dissolving propylamine azide modified polysaccharide, 3-or 4-substituted alkynyl-containing polyamide-amine, copper sulfate pentahydrate and sodium ascorbate in a mixed solvent of an organic solvent and water, reacting at 20-40 ℃, dialyzing a product after the reaction is finished, and drying to obtain the polyamide-amine modified highly hyperbranched cationic polysaccharide derivative; the polysaccharide is glycogen and amylopectin;
the structural formula of the 0.5-substituted polyamide-amine is shown in the specification
The structural formula of the 1-generation polyamide-amine is shown as
The structural formula of the 3-generation alkynyl-containing polyamide-amine is shown in the specification
4-substituted alkynyl-containing polyamidoaminesHas the structural formula
2. The polysaccharide derivative according to claim 1, wherein the mass ratio of propargylamine to methyl acrylate in step S1 is 0.1-10: 1-100; the mass ratio of the 0.5-generation polyamide-amine to the ethylenediamine is 1-30: 4-200; the mass ratio of the 1-generation polyamide-amine to the methyl acrylate is 1-30: 5 to 100.
3. The polysaccharide derivative according to claim 1, wherein the mass ratio of the polysaccharide to N, N' -carbonyldiimidazole in step S2 is 0.1-10: 0.01 to 5.
4. The polysaccharide derivative according to claim 1, wherein the azidopropylamine-modified polysaccharide obtained in step S3, the 3-or 4-substituted alkynyl-containing polyamide-amine, the copper sulfate pentahydrate and the sodium ascorbate are present in a mass ratio of 0.1 to 10: 0.1 to 100: 0.01-10: 0.01 to 10.
5. The polysaccharide derivative according to claim 1, wherein the reaction time of the ice-water bath in step S1 is 0.5-6 hours, and the reaction time at room temperature is 24-120 hours.
6. The method of claim 1, wherein the room temperature activation time of step S2 is 0.5-5 hours, and the reaction time after the addition of azidopropylamine is 12-72 hours.
7. The polysaccharide derivative according to claim 1, wherein the reaction at 20-40 ℃ in step S3 is carried out for 24-96 hours.
8. The polysaccharide derivative according to any one of claims 1 to 7, wherein the azidopropylamine of step S2 is prepared by the following method: under the protection of inert gas, adding sodium azide into 3-chloropropylamine hydrochloride and potassium chloride aqueous solution, reacting for 12-72 hours at 50-90 ℃, cooling to room temperature, adjusting the pH of the solution to 8-11, extracting the reaction solution by using an organic solvent, collecting an organic phase, removing water, filtering, and distilling under reduced pressure to obtain the azidopropylamine.
9. Use of the highly hyperbranched cationic polysaccharide derivatives containing dendrimer-amine groups according to claim 1 for the preparation of gene transfection vectors.
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