CN107459583A - A kind of height hyperbranched cationic polysaccharide derivative of the group of daiamid containing dendroid and preparation method thereof - Google Patents

Abstract

The invention discloses a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups, a preparation method and application thereof. The structural formula of the highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamide-amine groups is shown in formula (I); the present invention selectively converts dendritic macromolecules through an efficient azide-alkyne click reaction method Molecular polyamide-amine (PAMAM D3 or D4) is coupled to hyperbranched polysaccharide chains to synthesize highly hyperbranched cationic polysaccharide derivatives. The invention has mild reaction conditions, high reaction efficiency and selectivity. The cationic polysaccharide derivatives containing dendritic polyamide-amine groups prepared by the present invention have the characteristics of a highly hyperbranched structure, and can form positively charged nanocomposites with siRNA, which is conducive to carrying siRNA into cells and improving its resistance to siRNA. The transfection efficiency of the gene is expected to be used as a gene carrier and has great application prospects.

Description

Derivatization of a highly hyperbranched cationic polysaccharide containing dendritic polyamide-amine groups substances and their preparation methods

technical field

The invention belongs to the technical field of biomedical engineering. More specifically, it relates to a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups and a preparation method thereof.

Background technique

Gene therapy has been clinically used to treat and prevent a series of diseases (Ginn, S.L., et al. Journal of Gene Medicine, 2013, 15, 65-77), but the key technology of gene therapy is how to design an efficient Safe gene delivery vectors to compact and protect oligonucleotides from degradation by serum nucleases (Niven, R., et al. Journal of Pharmaceutical Sciences, 1998, 87, 1292-1299). Viral vectors have shown high efficiency in gene transfection of many cell lines, but their potential carcinogenicity, immunogenicity, limited DNA compression ability and difficulties in large-scale production limit their clinical application (Anderson, W.F. Nature, 1998, 392, 25-30).

In recent years, non-viral vectors have attracted much attention because of their low immunogenicity, low toxicity and large-scale production (Meredith A., et al. Chemical Reviews, 2009, 109, 259-302). As an important class of non-viral vectors, cationic polymers are currently reported to be polylysine (PLL), polyethyleneimine (PEI), polymethacrylate, cationic polysaccharide derivatives and dendrimers such as Polyamidoamine (PAMAM), polypropyleneimine (PPI) and polylysine (PLL), etc. (Meredith A., et al. Chemical Reviews, 2009, 109, 259-302). Recent studies have found that the structure of cationic polymers is closely related to their biological properties, such as the stability of cationic polymer/gene complexes, cytotoxicity, and gene transfection efficiency (Gao, Y., et al. Biomacromolecules, 2016, 17 ,3640-3647; Wang, R., et al. Biomacromolecules, 2010, 11, 489-495) Compared with linear cationic polymers, cationic polymers with higher degree of branching have lower cytotoxicity, higher Composite gene ability, cell phagocytosis effect and gene transfection efficiency. We have reported that cationic hyperbranched polysaccharide derivatives (such as amylopectin and glycogen) containing small molecular amine groups have low cytotoxicity and can transfect plasmid DNA (pDNA) and small interfering RNA ( siRNA) (Zhou Y.F., et al. Biomaterials, 2012, 33, 4731-4740; Liu Z.Z., et al. International Journal of Nanomedicine, 2015, 10, 2735-2749). However, due to the steric hindrance effect, it is still difficult to synthesize highly hyperbranched cationic polysaccharide derivatives.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of cationic polymers in the prior art, and provide a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups. Synthesis of highly hyperbranched cationic polysaccharide derivatives by selectively coupling dendrimer polyamidoamines (PAMAM D3 or D4) to hyperbranched polysaccharide chains using an efficient azide-alkyne click reaction method; The polysaccharide derivative has a highly hyperbranched structure, which is conducive to improving its gene transfection efficiency, and is expected to be used as a gene carrier.

The object of the present invention is to provide a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups.

Another object of the present invention is to provide a preparation method of the highly hyperbranched cationic polysaccharide derivative.

Another object of the present invention is to provide the application of the highly hyperbranched cationic polysaccharide derivative.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

A highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups, its structural formula is as shown in formula (I):

;

where R is H or , the It is a 3rd or 4th generation polyamide-amine.

The preparation method of the highly hyperbranched cationic polysaccharide derivative containing dendrimer polyamide-amine group is to selectively convert dendrimer polyamide-amine (PAMAM D3 or D4) through an efficient azide-alkyne click reaction method ) coupled to hyperbranched polysaccharide chains to synthesize highly hyperbranched cationic polysaccharide derivatives.

Preferably, the polysaccharide is glycogen or pullulan.

Specifically, its preparation method includes the following steps:

S1. Ice-water bath with inert gas, drop the alcohol solution containing propargylamine into the alcohol solution containing methyl acrylate, react in the ice-water bath and then react at room temperature, evaporate and dry after the reaction to obtain 0.5 generation polyamide-amine; Under certain conditions, the alcohol solution containing 0.5 generation polyamidoamine is added dropwise to the alcohol solution containing ethylenediamine, reacted in an ice-water bath and then reacted at room temperature, evaporated and dried after the reaction to obtain 1 generation polyamidoamine; The first-generation polyamide-amine is reacted with methyl acrylate to prepare the 1.5-generation polyamido-amine; repeat the above steps to prepare alkyne-containing polyamide-amine with different alkyne groups; the reaction process is shown in the reaction formula (II):

S2. Under the protection of an inert gas, dissolve the polysaccharide in an organic solvent, add N,N'-carbonyldiimidazole to activate at room temperature, and then add azidopropylamine to react. After the reaction, the product is dialyzed and dried to obtain azidopropylamine-modified polysaccharide ; Its reaction process is shown in reaction formula (III):

S3. Under the protection of an inert gas, dissolve the azidopropylamine-modified polysaccharide, alkyne-containing polyamide-amine, copper sulfate pentahydrate and sodium ascorbate in a mixed solvent of organic solvent and water, react at 20-40°C, and the reaction ends Afterwards, the product is dialyzed and dried to obtain polyamide-amine modified highly hyperbranched cationic polysaccharide derivatives; the reaction process is shown in the 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 0.5-generation polyamidoamine to ethylenediamine is 1-30:4-200; The mass ratio of the first-generation polyamidoamine to methyl acrylate is 1-30:5-100.

Preferably, the reaction time in the ice-water bath in step S1 is 0.5-6 hours, and the reaction time at room temperature is 24-120 hours.

Preferably, the solution dropping rate in step S1 is 0.1-3 ml/min, and the purpose of dropping is to make methyl acrylate or ethylenediamine excessive, thereby reducing side reactions and increasing reaction yield.

Preferably, the evaporation in S1 is rotary evaporation, the temperature of rotary evaporation is 30-60°C, and the drying is vacuum drying at 30-60°C, the purpose is to remove 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 easy to decompose by absorbing water, the reaction of CDI must be controlled under anhydrous conditions, and the raw materials and solvents used must be fully dehydrated and dried. As a preferred option, the preparation method of the anhydrous dimethyl sulfoxide is: add 0.1 to 20 grams of calcium hydride to 50 to 500 milliliters of dimethyl sulfoxide, stir at room temperature for 1 to 7 days, and let stand for 1 to 7 days. After 7 days, filter, add molecular sieves to the filtrate, and soak for 1 to 7 days.

Preferably, the mass ratio of polysaccharide to N,N'-carbonyldiimidazole in step S2 is 0.1-10:0.01-5.

Preferably, the activation time at room temperature in step S2 is 0.5-5 hours, and the reaction time after adding azidopropylamine is 12-72 hours.

Preferably, the mass ratio of the azidopropylamine-modified polysaccharide, alkyne-containing polyamide-amine, copper sulfate pentahydrate and sodium ascorbate in step S3 is 0.1-10:0.1-100:0.01-10:0.01-10.

Preferably, the reaction at 20-40°C in step S3 is 24-96 hours.

Preferably, the mixed solvent of the organic solvent and water in step S3 is a mixed solvent of 0.5-100 ml of dimethyl sulfoxide and 0.5-10 ml of water.

Preferably, the preparation method of azidopropylamine described in step S2 is as follows: under the protection of an inert gas, add sodium azide to 3-chloropropylamine hydrochloride and potassium chloride aqueous solution, and react at 50-90° C. for 12-72 hours, Cool to room temperature, adjust the pH of the solution to 8-11, then extract the reaction solution with an organic solvent, collect the organic phase, remove water, filter and distill under reduced pressure to obtain azidopropylamine; the reaction process is shown in the reaction formula (V):

More preferably, the adjustment of pH is performed by adding 0.01-10 ml of sodium hydroxide solution.

More preferably, the extraction solvent may be dichloromethane, diethyl ether or chloroform, an organic solvent with high solubility of azidopropylamine, and anhydrous sodium sulfate, anhydrous magnesium sulfate or anhydrous calcium chloride may be used for removing water from the organic phase.

The purpose of dialysis of the product of the present invention is to remove the solvent and unreacted raw materials, and conventional pure water dialysis can be used; as a preferred solution, the dialysis bag with a cut-off molecular weight of 8000-50,000 is selected for dialysis in step S2 or S3 to ensure the removal of small molecules The polysaccharide derivatives will not be dialyzed out while impurities are removed, and the dialysis time is 1 to 5 days.

Preferably, the drying in step S2 or S3 is freeze-drying, so that the polysaccharide derivatives are dried at a low temperature to ensure that their structural properties will not change.

Simultaneously, the present invention feeds inert gas, in step S1 is in order to avoid side reaction of ethylenediamine and carbon dioxide in the air, in step S2 is in order to ensure that reaction is in anhydrous, anaerobic state, avoids reducing CDI activity, in step S3 is In order to ensure that the reaction is in an oxygen-free state and avoid the oxidation of cuprous ions produced, as a preferred solution, the gas is nitrogen, helium or argon.

The highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamide-amine groups prepared by the invention have a highly hyperbranched structure, which is beneficial to improving the transfection efficiency of genes, and is expected to be used as a gene carrier. Therefore, the application of the highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamide-amine groups in the preparation of gene transfection vectors is also within the protection scope of the present invention.

As a preferred embodiment, the preparation method of the highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamide-amine groups of the present invention specifically includes the following steps:

S1. Synthesis of alkyne-containing polyamide-amine dendrimers of different generations:

S11. Synthesis of 1st generation polyamide-amine (D1): Ice-water bath with protective gas, drop 1-100 ml methanol solution containing 0.1-10 g propargyl amine into 10-100 ml methanol solution containing 1-100 g methyl acrylate In ml of methanol solution, stir and react for 0.5-6 hours, warm up to room temperature and stir for 24-72 hours, rotary evaporate, and dry to obtain 0.5 generation polyamide-amine (D0.5); Add 30 grams of 0.5-generation polyamidoamine in 10-200 ml of methanol solution dropwise into 20-500 ml of methanol solution containing 4-200 g of ethylenediamine, stir for 0.5-6 hours, heat up to room temperature and stir for 24-120 hour, rotary evaporation, and drying to obtain the first generation of polyamidoamine (D1);

S12. Synthesis of 2nd-generation polyamidoamine (D2): Ice-water bath with gas protection, add 20-300 ml of methanol solution containing 1-30 grams of 1st-generation polyamido-amine dropwise to 5-100 grams of methyl acrylate 30-500 ml of methanol solution, stirred for 1-10 hours, heated to room temperature and stirred for 24-120 hours, rotary evaporated and dried to obtain 1.5-generation polyamide-amine (D1.5); Add 30 to 500 ml of methanol solution containing 3 to 50 grams of 1.5-generation polyamidoamine dropwise into 50 to 500 ml of methanol solution containing 20 to 300 grams of ethylenediamine, stir and react for 1 to 10 hours, then warm up to room temperature and stir Reaction for 24-120 hours, rotary evaporation, and drying to obtain the 2nd generation polyamidoamine (D2);

S13. Synthesis of 3rd generation polyamidoamine (D3): Ice-water bath with gas protection, 50-500ml methanol solution containing 5-50g 2nd-generation polyamido-amine was added dropwise to 50-300g methyl acrylate 40-600 ml of methanol solution, stirring for 1-10 hours, warming up to room temperature and stirring for 24-120 hours, rotary evaporation, and drying to obtain 2.5-generation polyamide-amine (D2.5); Add 15-100 grams of 2.5-generation polyamidoamine in 50-800 ml of methanol solution dropwise into 50-800 ml of methanol solution containing 40-500 g of ethylenediamine, stir for 1-10 hours, then heat up to room temperature and stir for reaction 36-168 hours, rotary evaporation, drying, to obtain the 3rd generation polyamide-amine (D3);

S14. Synthesis of 4th generation polyamidoamine (D4): Ice-water bath with gas protection, add dropwise 60-700 ml of methanol solution containing 10-80 g of 3rd-generation polyamido-amine to 60-500 g of methyl acrylate 60-800 ml of methanol solution, stirring for 1-10 hours, warming up to room temperature, stirring for 24-120 hours, rotary evaporation, and drying to obtain 3.5-generation polyamide-amine (D3.5); Add 30-100 grams of 3.5-generation polyamidoamine in 60-900 ml of methanol solution dropwise into 60-900 ml of methanol solution containing 50-600 g of ethylenediamine, stir for 1-10 hours, then warm up to room temperature and stir for reaction 36-168 hours, rotary evaporation, drying, to obtain the 4th generation polyamide-amine (D4);

S2. Synthesis of azidopropylamine-modified polysaccharides:

S21. Azidopropylamine synthesis: under the protection of an inert gas, dissolve 10-50 grams of 3-chloropropylamine hydrochloride and 0.01-10 grams of potassium iodide in 50-500 milliliters of deionized water, add 10-200 grams of sodium azide, and heat up Reaction at 50-90 degrees Celsius for 12-72 hours. Cool to room temperature, add 0.01-10 ml of sodium hydroxide solution to adjust the pH of the solution to 8-11. Extract the reaction solution with an organic solvent for 3 to 10 times, separate the solution to collect the organic phase, remove water from the organic phase, filter and distill under reduced pressure to obtain azidopropylamine.

S22. Synthesis of azidopropylamine-modified polysaccharide: under the protection of inert gas, dissolve 0.1-10 grams of polysaccharide in 1-300 ml of dimethyl sulfoxide, and add 0.01-5 grams of N,N'-carbonyldiimidazole (CDI) Activate at room temperature for 0.5-5 hours, add azidopropylamine to react for 12-72 hours. After the reaction, the product was dialyzed in deionized water and dried to obtain the azidopropylamine-modified polysaccharide.

S3. Synthesis of highly hyperbranched cationic polysaccharide derivatives: under the protection of inert gas, 0.01-10 grams of azidopropylamine-modified polysaccharides, 0.1-100 grams of alkyne-containing polyamide-amine dendrimers of different alkyne groups, 0.01-10 grams Copper sulfate pentahydrate and 0.01-10 grams of sodium ascorbate are dissolved in a mixed solvent of 0.5-100 milliliters of dimethyl sulfoxide and 0.5-10 milliliters of water, and the mixture is stirred and reacted at 20-60 degrees Celsius for 24-96 hours. The product is dialyzed in deionized water and dried to obtain polyamide-amine modified highly hyperbranched cationic polysaccharide derivatives of different generations.

The schematic diagram of the product structure and the synthesis reaction mechanism of the preparation method of the present invention are shown in Figure 1 and Figure 2 respectively. Compared with glycogen or pullulan modified by small molecular amines, the synthesis method of highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamidoamines has the following characteristics:

(1) The raw materials of dendritic polyamidoamine and hyperbranched polysaccharide derivatives (pullulan or glycogen) have highly branched structures, which have large reaction steric hindrance and high difficulty in reaction.

(2) The reaction of the present invention adopts azide-alkyne click reaction to synthesize structure-controllable dendritic polyamide-amine modified highly hyperbranched cationic polysaccharide derivatives, and the reaction conditions are mild, efficient and selective.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention adopts azide-alkyne click chemistry reaction to synthesize highly hyperbranched cationic polysaccharide derivatives with controllable structure, and the reaction conditions are mild, efficient and selective.

(2) The dendritic polyamide-amine modified highly hyperbranched cationic polysaccharide derivative prepared by the present invention has good water solubility and can form positively charged nanocomplexes with siRNA, which is beneficial to carry siRNA into cells.

(3) The dendritic polyamide-amine modified highly hyperbranched cationic polysaccharide derivative prepared by the present invention has a highly hyperbranched structure, which is conducive to improving its gene transfection efficiency, and is expected to be used as a gene carrier.

Description of drawings

Fig. 1 is a schematic diagram of the product structure of the present invention, A is a schematic diagram of a hyperbranched polysaccharide structure, and B is a chemical structure of a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine groups.

Fig. 2 is a reaction route diagram of the preparation method of the present invention.

Figure 3 shows the glycogen raw material (Glycogen), azidopropylamine modified glycogen (Gly-N ₃ ), 4th generation polyamide-amine (PAMAM D4) and 4th generation polyamide-amine modified highly hyperbranched cationic sugar of the present invention Infrared spectrum (FTIR) of the original (Gly-D4) product.

Figure 4 shows the glycogen raw material (Glycogen) and azidopropylamine modified glycogen (Gly-N ₃ ), the 4th generation polyamide-amine (PAMAM D4) and the 4th generation polyamide-amine modified highly hyperbranched cationic sugar of the present invention Nuclear Magnetic Spectrum ( ¹ HNMR) of the original (Gly-D4) product.

Figure 5 is an agarose gel electrophoresis image of the 4th generation polyamide-amine modified highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex of the present invention (w/w represents the mass ratio of Gly-D4 to siRNA) .

Figure 6 is the zeta potential diagram of the highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex modified by the 4th generation polyamide-amine product of the present invention (w/w represents the mass ratio of Gly-D4 to siRNA).

Figure 7 is a particle size analysis diagram of the 4th generation polyamide-amine modified highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex of the present invention (w/w represents the mass ratio of Gly-D4 to siRNA).

Fig. 8 is a scanning electron micrograph of the highly hyperbranched cationic glycogen (Gly-D4)/siRNA complex (mass ratio: 10) modified by the 4th generation polyamide-amine product of the present invention.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1

1. Preparation of highly hyperbranched cationic glycogen derivatives (Gly-D4) modified by 4th generation polyamide-amine

(1) Nitrogen protection in an ice-water bath, add 10 ml of methanol solution containing 4 g of propargylamine dropwise to 30 ml of methanol solution containing 13.6 g of methyl acrylate, stir for 3 hours, heat up to room temperature and stir for 24 hours, rotate Evaporate and dry in vacuo to obtain 0.5 generation polyamidoamine (D0.5); ice-water bath with nitrogen protection, 100 ml of methanol solution containing 16.2 g of 0.5 generation polyamidoamine was added dropwise to 100 ml of ethylenediamine containing 50 g In ml of methanol solution, stirred for 3 hours, heated to room temperature and stirred for 24 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain the first-generation polyamide-amine (D1);

(2) Nitrogen protection in an ice-water bath, 50 ml of methanol solution containing 18 g of 1st generation polyamide-amine was added dropwise to 50 ml of methanol solution containing 32 g of methyl acrylate, stirred for 4 hours, warmed to room temperature and stirred for 36 Hours, 30 ° C rotary evaporation, 30 ° C vacuum drying, to obtain 1.5 generation polyamido-amine (D1.5); ice water bath with nitrogen protection, 70 ml of methanol solution containing 28 grams of 1.5 generation polyamido-amine was added dropwise to the containing 80 grams of ethylenediamine in 100 ml of methanol solution, stirred for 3 hours, then heated to room temperature and stirred for 36 hours, 30 ° C rotary evaporation, 30 ° C vacuum drying to obtain the second generation polyamidoamine (D2);

(3) Under nitrogen protection in an ice-water bath, 100 ml of methanol solution containing 35 g of 2nd generation polyamide-amine was added dropwise to 100 ml of methanol solution containing 50 g of methyl acrylate, stirred for 3 hours, heated to room temperature and stirred for 36 hours, Rotary evaporation at 30°C, vacuum drying at 30°C to obtain 2.5-generation polyamide-amine (D2.5); nitrogen protection in an ice-water bath, 100 ml methanol solution containing 45 grams of 2.5-generation polyamide-amine was added dropwise to 105 g Ethylenediamine in 150 ml of methanol solution, stirred for 3 hours, then heated to room temperature and stirred for 36 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain 3rd generation polyamide-amine (D3);

(4) Ice-water bath with nitrogen protection, add 150 ml of methanol solution containing 50 g of 3rd generation polyamide-amine dropwise into 100 ml of methanol solution containing 80 g of methyl acrylate, stir for 3 hours, heat up to room temperature and stir for 36 hours , rotary evaporation at 30°C, and vacuum drying at 30°C to obtain 3.5-generation polyamide-amine (D3.5); nitrogen protection in an ice-water bath, 200 ml methanol solution containing 60 grams of 3.5-generation polyamide-amine was added dropwise to 170 1 g of ethylenediamine in 300 ml of methanol solution, stirred for 3 hours, then heated to room temperature, stirred and reacted for 48 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain 4th generation polyamide-amine (D4);

(5) Under the protection of nitrogen, dissolve 10 g of 3-chloropropylamine hydrochloride and 1 g of potassium iodide in 50 ml of deionized water, add 50 g of sodium azide, and raise the temperature to 80 degrees Celsius for 48 hours. After cooling to room temperature, 1.5 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. The reaction solution was extracted with ether for 6 times, the solution was separated to collect the organic phase, and the organic phase was dehydrated with anhydrous magnesium sulfate, filtered and distilled under reduced pressure to obtain azidopropylamine.

(6) Under nitrogen protection, dissolve 0.1 g of glycogen in 50 ml of dimethyl sulfoxide, add 0.1 g of N,N'-carbonyldiimidazole (CDI) to activate at room temperature for 3 hours, add azidopropylamine to react for 48 hours . After the reaction, the product was put into a dialysis bag with a cut-off molecular weight of 8000, dialyzed against deionized water for 3 days, and freeze-dried to obtain azidopropylamine-modified glycogen (Gly-N ₃ ).

(7) Under nitrogen protection, dissolve 0.08 g of azidopropylamine-modified glycogen, 8.9 g of 4th generation polyamidoamine, 0.05 g of copper sulfate pentahydrate and 0.20 g of sodium ascorbate in 20 ml of dimethyl sulfoxide and 10 ml of In a mixed solvent of water, the mixture was stirred and reacted at 40°C for 48 hours. The product was loaded into a dialysis bag with a cut-off molecular weight of 10,000, dialyzed against deionized water for 3 days, and freeze-dried to obtain the 4th generation polyamide-amine modified highly hyperbranched cationic glycogen derivative (Gly-D4).

2. Results

(1) The obtained glycogen raw material and each product were characterized by infrared spectroscopy, and the obtained FTIR spectrum is shown in Figure 3. It can be seen from Figure 3 that compared with the spectrum of glycogen, the FTIR spectrum of Gly-N ₃ shows the stretching vibration absorption peak of the azido group (-N=N=N) around 2104 cm ^-1 , and the absorption peak of 1709 The C=O stretching vibration absorption peak of carbamate appeared in cm ^-1 , which proved that azidopropylamine had been coupled to glycogen. In addition, compared with the FTIR spectra of Gly-N ₃ and PAMAM D4, the FTIR spectrum of Gly-D4 disappears at 2104 cm ^-1 , and the azido stretching vibration peak appears around 1658, 1552, 1331 cm ^-1 . The amide I, amide II and amide III peaks of amides are the COC stretching vibration absorption peaks on the glycogen sugar ring around 1152 cm ^-1 , and the OH variable angle vibration absorption peaks on the glycogen sugar ring around 1029 cm ^-1 , proving that Four generations of polyamidoamines have been coupled to glycogen.

(2) The obtained glycogen raw material and various products were characterized by nuclear magnetic resonance spectroscopy, and the obtained ¹ H NMR spectrum is shown in FIG. 4 . The proton peaks of the sugar units of glycogen are assigned as follows: the proton peaks of anomeric hydrogen (H1) appear at 5.5-5.1 ppm, and the proton peaks of H2-H6 appear at 4.1-3.2 ppm. Compared with the ¹ H NMR spectrum of glycogen, the following new peaks appeared in the ¹ H NMR spectrum of Gly-N ₃ : 1.7 ppm (b), 2.8 ppm (a) and 3.1 ppm (c) (assignment see Figure 4E) , proving that Gly-N ₃ has been synthesized. In addition, compared with the ¹ H NMR spectra of glycogen and 4th generation polyamidoamine, the following new peaks appeared in the ¹ H NMR spectra of Gly-D4: 2.3 ppm (b), 2.4-3.4 ppm (e, f, g , h, i, j, k) and 8.0 ppm (d), demonstrating the synthesis of Gly-D4 derivatives.

(3) In order to confirm the ability of the 4th generation polyamide-amine modified highly hyperbranched glycogen derivative (Gly-D4) to compound the gene, use Gly-D4 to compound with a small interfering RNA (siRNA) containing 21 base pairs Afterwards, an agarose gel electrophoresis experiment was carried out, and the obtained agarose gel electrophoresis results are shown in FIG. 5 . The mass ratio (w/w) is the mass ratio of Gly-D4 to the mass of siRNA. It can be seen that when the mass ratio is greater than or equal to 10, Gly-D4 and siRNA are completely compounded.

(4) After the obtained 4th generation polyamide-amine modified highly hyperbranched glycogen derivative (Gly-D4) and siRNA were formulated into nanocomposite solutions with different mass ratios, the Zeta potential of the nanocomposite was tested with a Zeta potentiometer. potential, and the results are shown in Figure 6. When the mass ratio of Gly-D4 to siRNA is greater than 1, the zeta potential of the nanocomplex is positive.

(5) After the obtained 4th generation polyamide-amine modified highly hyperbranched glycogen derivative (Gly-D4) and siRNA were formulated into nanocomposite solutions with different mass ratios, the dynamic light scattering instrument was used to test the nanocomposites. The hydrodynamic diameter, the obtained results are shown in Fig. 7. When the mass ratio of Gly-D4 to siRNA is greater than 5, the dynamic mechanical diameter of the nanocomplex is in the range of 100-200nm.

(6) After the obtained 4th generation polyamide-amine modified highly hyperbranched glycogen derivative (Gly-D4) and siRNA were formulated into a nanocomposite solution with a mass ratio of 10, a small amount of the solution was dropped onto an aluminum foil to naturally After drying, its morphology was observed with a scanning electron microscope, and the results are shown in Figure 8. The nanocomposites are irregular spherical with a diameter of about 200 nm.

Example 2

1. Preparation of highly hyperbranched cationic glycogen derivatives (Gly-D3) modified by 3rd generation polyamide-amine

(1) Nitrogen protection in an ice-water bath, drop 5 ml of methanol solution containing 0.1 g of propargylamine into 10 ml of methanol solution containing 1 g of methyl acrylate, stir for 2 hours, warm to room temperature and stir for 24 hours, 40 Rotary evaporation at ℃, vacuum drying at 40℃ to obtain 0.5-generation polyamidoamine (D0.5); nitrogen protection in ice-water bath, 15 ml methanol solution containing 1.2 g 0.5-generation polyamido-amine was added dropwise to 6 g of B Diamine in 20 ml of methanol solution, stirred for 2 hours, heated to room temperature, stirred for 24 hours, 40°C rotary evaporation, 40°C vacuum-dried to obtain 1st generation polyamide-amine (D1);

(2) Nitrogen protection in an ice-water bath, add 20 ml of methanol solution containing 2 grams of 1st generation polyamide-amine dropwise to 25 ml of methanol solution containing 15 grams of methyl acrylate, stir for 2 hours, warm up to room temperature and stir for 24 Hours, 40 ° C rotary evaporation, 40 ° C vacuum drying, to obtain 1.5-generation polyamido-amine (D1.5); ice-water bath with gas protection, 30 ml of methanol solution containing 3.6 grams of 1.5-generation polyamido-amine was added dropwise to the containing 20 g of ethylenediamine in 50 ml of methanol solution, stirred for 2 hours, then heated to room temperature and stirred for 24 hours, 40 ° C rotary evaporation, 40 ° C vacuum drying to obtain the second generation polyamide-amine (D2);

(3) Ice-water bath with nitrogen protection, drop 50 ml of methanol solution containing 5 grams of 2-generation polyamide-amine into 60 ml of methanol solution containing 50 grams of methyl acrylate, stir for 2 hours, warm to room temperature and stir for 24 hours , rotary evaporation at 40°C, and vacuum drying at 40°C to obtain 2.5-generation polyamide-amine (D2.5); the ice-water bath was protected with gas, and 60 ml of methanol solution containing 12 grams of 2.5-generation polyamido-amine was added dropwise to 40 1 g of ethylenediamine in 60 ml of methanol solution, stirred and reacted for 2 hours, then heated to room temperature and stirred for 48 hours, 40 ° C rotary evaporation, 40 ° C vacuum drying, to obtain the 3rd generation polyamide-amine (D3);

(4) Under the protection of nitrogen, dissolve 10 g of 3-chloropropylamine hydrochloride and 1 g of potassium iodide in 50 ml of deionized water, add 50 g of sodium azide, and raise the temperature to 80 degrees Celsius for 48 hours. After cooling to room temperature, 2 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. The reaction solution was extracted 8 times with ether, the solution was separated to collect the organic phase, and the organic phase was dehydrated with anhydrous calcium chloride, filtered and distilled under reduced pressure to obtain azidopropylamine.

(5) Under nitrogen protection, dissolve 0.1 g of glycogen in 20 ml of dimethyl sulfoxide, add 1 g of N,N'-carbonyldiimidazole (CDI) to activate at room temperature for 3 hours, add azidopropylamine to react for 48 hours . After the reaction, the product was put into a dialysis bag with a cut-off molecular weight of 8000, dialyzed against deionized water for 3 days, and freeze-dried to obtain azidopropylamine-modified glycogen (Gly-N ₃ )

(6) Under nitrogen protection, dissolve 0.05 g of azidopropylamine-modified glycogen, 5.3 g of 3rd generation polyamidoamine, 0.5 g of copper sulfate pentahydrate and 2 g of sodium ascorbate in 15 ml of dimethyl sulfoxide and 5 ml of In a mixed solvent of water, the mixture was stirred and reacted at 40°C for 48 hours. The product was loaded into a dialysis bag with a cut-off molecular weight of 10,000, dialyzed against deionized water for 3 days, and freeze-dried to obtain the third-generation polyamide-amine modified highly hyperbranched cationic glycogen derivative (Gly-D3).

Example 3

1. Preparation of highly hyperbranched cationic glycogen derivatives (Gly-D4) modified by 4th generation polyamide-amine

(1) Nitrogen protection in an ice-water bath, add 10 ml of methanol solution containing 1.5 g of propargylamine dropwise to 30 ml of methanol solution containing 12 g of methyl acrylate, stir for 1 hour, heat up to room temperature and stir for 24 hours, rotate Evaporate and dry in vacuo to obtain 0.5-generation polyamidoamine (D0.5); under nitrogen protection in an ice-water bath, 30 ml of methanol solution containing 5 g of 0.5-generation polyamido-amine was added dropwise to 50 ml of 15 g of ethylenediamine In methanol solution, stirred and reacted for 1 hour, heated to room temperature and stirred for 24 hours, 60°C rotary evaporation, 60°C vacuum-dried to obtain 1st generation polyamide-amine (D1);

(2) Nitrogen protection in an ice-water bath, dropwise add 30 ml of methanol solution containing 6 g of 1st generation polyamide-amine to 30 ml of methanol solution containing 6 g of methyl acrylate, stir for 1 hour, warm to room temperature and stir for 24 hour, rotary evaporation, and vacuum drying to obtain 1.5 generation polyamidoamine (D1.5); ice-water bath with gas protection, 30 ml of methanol solution containing 8 g of 1.5 generation polyamidoamine was added dropwise to 25 g of ethylene glycol amine in 50 ml of methanol solution, stirred for 1 hour, then warmed up to room temperature and stirred for 24 hours, 60 ° C rotary evaporation, 60 ° C vacuum drying to obtain the 2nd generation polyamide-amine (D2);

(3) Nitrogen protection in an ice-water bath, add 60 ml of methanol solution containing 10 g of 2nd generation polyamide-amine dropwise into 40 ml of methanol solution containing 50 g of methyl acrylate, stir for 1 hour, heat up to room temperature and stir for 48 hours , rotary evaporation at 60°C, and vacuum drying at 60°C to obtain 2.5-generation polyamide-amine (D2.5); the ice-water bath was protected with gas, and 100 ml of methanol solution containing 15 grams of 2.5-generation polyamido-amine was added dropwise to 60 gram of ethylenediamine in 100 ml of methanol solution, stirred and reacted for 1 hour, then heated to room temperature and stirred for 48 hours, 60°C rotary evaporation, 60°C vacuum-dried to obtain 3rd generation polyamide-amine (D3);

(4) Nitrogen protection in an ice-water bath, add 150 ml of methanol solution containing 18 g of 3rd generation polyamide-amine dropwise to 200 ml of methanol solution containing 70 g of methyl acrylate, stir for 1 hour, heat up to room temperature and stir for 72 hours , rotary evaporation at 60°C, and vacuum drying at 60°C to obtain 3.5-generation polyamide-amine (D3.5); the ice-water bath was protected with gas, and 300 ml of methanol solution containing 31 grams of 3.5-generation polyamido-amine was added dropwise to 200 gram of ethylenediamine in 400 ml of methanol solution, stirred for 1 hour, then heated to room temperature and stirred for 72 hours, 60°C rotary evaporation, 60°C vacuum-dried to obtain 4th generation polyamide-amine (D4);

(5) Under nitrogen protection, dissolve 50 g of 3-chloropropylamine hydrochloride and 5 g of potassium iodide in 500 ml of deionized water, add 100 g of sodium azide, and heat up to 90 degrees Celsius for 72 hours. After cooling to room temperature, 8 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. Extract the reaction solution with dichloromethane for 10 times, separate the solution to collect the organic phase, remove water from the organic phase with anhydrous magnesium sulfate, filter and distill under reduced pressure to obtain azidopropylamine.

(6) Under nitrogen protection, dissolve 1 g of glycogen in 100 ml of dimethyl sulfoxide, add 5 g of N,N'-carbonyldiimidazole (CDI) to activate at room temperature for 3 hours, add azidopropylamine to react for 48 hours . After the reaction, the product was put into a dialysis bag with a cut-off molecular weight of 8000, dialyzed against deionized water for 3 days, and freeze-dried to obtain azidopropylamine-modified glycogen (Gly-N ₃ )

(7) Under nitrogen protection, dissolve 0.8 g of azidopropylamine-modified glycogen, 20 g of 4th generation polyamidoamine, 1 g of copper sulfate pentahydrate and 10 g of sodium ascorbate in 50 ml of dimethyl sulfoxide and 10 ml of In a mixed solvent of water, the mixture was stirred and reacted at 30°C for 96 hours. The product was loaded into a dialysis bag with a cut-off molecular weight of 10,000, dialyzed against deionized water for 3 days, and freeze-dried to obtain the 4th generation polyamide-amine modified highly hyperbranched cationic glycogen derivative (Gly-D4).

Example 4

1. Preparation of highly hyperbranched cationic pullulan derivatives (Amyp-D4) modified by 4th generation polyamide-amine

(1) Nitrogen protection in an ice-water bath, 15 ml of methanol solution containing 4 g of propargylamine was added dropwise to 40 ml of methanol solution containing 16 g of methyl acrylate, stirred for 6 hours, heated to room temperature and stirred for 36 hours, 35 Rotary evaporation at ℃ and vacuum drying at 35°C to obtain 0.5-generation polyamidoamine (D0.5); nitrogen protection in an ice-water bath, 80 ml of methanol solution containing 18 grams of 0.5-generation polyamido-amine was added dropwise to 60 g of B Diamine in 80 ml of methanol solution, stirred for 6 hours, heated to room temperature, stirred and reacted for 36 hours, 35°C rotary evaporation, 35°C vacuum-dried to obtain 1st generation polyamide-amine (D1);

(2) Nitrogen protection in an ice-water bath, dropwise add 80 ml of methanol solution containing 20 g of 1st generation polyamide-amine to 100 ml of methanol solution containing 50 g of methyl acrylate, stir for 6 hours, warm up to room temperature and stir for 48 Hours, 35 ° C rotary evaporation, 35 ° C vacuum drying, to obtain 1.5 generation polyamidoamine (D1.5); ice water bath with nitrogen protection, 100 ml methanol solution containing 28 grams of 1.5 generation polyamidoamine was added dropwise to the containing 70 grams of ethylenediamine in 100 ml of methanol solution, stirred for 6 hours, then heated to room temperature and stirred for 48 hours, 35 ° C rotary evaporation, 35 ° C vacuum drying to obtain the second generation polyamide-amine (D2);

(3) Under nitrogen protection in an ice-water bath, 200 ml of methanol solution containing 30 g of 2nd generation polyamide-amine was added dropwise to 100 ml of methanol solution containing 100 g of methyl acrylate, stirred for 6 hours, heated to room temperature and stirred for 48 hours, Rotary evaporation at 35°C, vacuum drying at 35°C to obtain 2.5-generation polyamide-amine (D2.5); nitrogen protection in an ice-water bath, 150 ml methanol solution containing 35 grams of 2.5-generation polyamido-amine was added dropwise to 105 g In 150 ml of methanol solution of ethylenediamine, stirred and reacted for 6 hours, then warmed up to room temperature and stirred for 72 hours, 35°C rotary evaporation, 35°C vacuum-dried to obtain 3rd generation polyamide-amine (D3);

(4) Ice-water bath with nitrogen protection, 350 ml of methanol solution containing 40 g of 3rd generation polyamide-amine was added dropwise to 150 ml of methanol solution containing 160 g of methyl acrylate, stirred for 6 hours, heated to room temperature and stirred for 48 hours , rotary evaporation at 35°C, and vacuum drying at 35°C to obtain 3.5-generation polyamide-amine (D3.5); nitrogen protection in an ice-water bath, 500 ml of methanol solution containing 50 g of 3.5-generation polyamide-amine was added dropwise to 300 gram of ethylenediamine in 300 ml of methanol solution, stirred for 6 hours, then heated to room temperature and stirred for 120 hours, 35°C rotary evaporation, 35°C vacuum-dried to obtain 4th generation polyamide-amine (D4);

(5) Under the protection of nitrogen, dissolve 5 g of 3-chloropropylamine hydrochloride and 5 g of potassium iodide in 100 ml of deionized water, add 50 g of sodium azide, and heat up to 80 degrees Celsius for 72 hours. After cooling to room temperature, 3 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. The reaction solution was extracted with ether for 10 times, the solution was separated to collect the organic phase, and the organic phase was dehydrated with anhydrous magnesium sulfate, filtered and distilled under reduced pressure to obtain azidopropylamine.

(6) Under nitrogen protection, dissolve 0.5 g of pullulan in 100 ml of dimethyl sulfoxide, add 0.3 g of N,N'-carbonyldiimidazole (CDI) to activate at room temperature for 2 hours, add azidopropylamine to react for 48 Hour. After the reaction, the product was put into a dialysis bag with a cut-off molecular weight of 8000, dialyzed against deionized water for 3 days, and freeze-dried to obtain azidopropylamine-modified pullulan (Amyp-N ₃ ).

(7) Under nitrogen protection, dissolve 0.08 g of azidopropylamine-modified pullulan, 8.9 g of 4th generation polyamidoamine, 0.15 g of copper sulfate pentahydrate and 0.15 g of sodium ascorbate in 20 ml of dimethyl sulfoxide and 5 In the mixed solvent of milliliter water, stir reaction at 40 ℃ for 48 hours. The product was put into a dialysis bag with a cut-off molecular weight of 10,000, dialyzed against deionized water for 3 days, and freeze-dried to obtain the 4th generation polyamide-amine modified highly hyperbranched cationic pullulan derivative (Amyp-D4).

Example 5

1. 4th generation polyamide-amine modified highly hyperbranched cationic pullulan derivatives (Amyp-D4)

(1) Nitrogen protection in an ice-water bath, add 10 ml of methanol solution containing 2 g of propargylamine dropwise to 30 ml of methanol solution containing 15 g of methyl acrylate, stir for 2 hours, warm to room temperature and stir for 24 hours, 30 Rotary evaporation at ℃, vacuum drying at 30°C to obtain 0.5-generation polyamidoamine (D0.5); under nitrogen protection in an ice-water bath, 40 ml of methanol solution containing 5 grams of 0.5-generation polyamido-amine was added dropwise to 30 g of ethylene glycol amine in 50 ml of methanol solution, stirred for 1 hour, heated to room temperature and stirred for 24 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain 1st generation polyamide-amine (D1);

(2) Ice-water bath with nitrogen protection, drop 50 ml of methanol solution containing 5 grams of 1st generation polyamide-amine into 50 ml of methanol solution containing 20 grams of methyl acrylate, stir for 1 hour, warm to room temperature and stir for 24 Hours, 30 ° C rotary evaporation, 30 ° C vacuum drying, to obtain 1.5 generation polyamidoamine (D1.5); ice water bath with gas protection, 50 ml of methanol solution containing 8 grams of 1.5 generation polyamidoamine was added dropwise to the containing 30 g of ethylenediamine in 50 ml of methanol solution, stirred for 1 hour, then raised to room temperature and stirred for 24 hours, 30 ° C rotary evaporation, 30 ° C vacuum drying to obtain the 2nd generation polyamide-amine (D2);

(3) Nitrogen protection in an ice-water bath, dropwise add 100 ml of methanol solution containing 13 g of 2-generation polyamide-amine to 50 ml of methanol solution containing 50 g of methyl acrylate, stir for 1 hour, heat up to room temperature and stir for 48 hours , rotary evaporation at 30°C, and vacuum drying at 30°C to obtain 2.5-generation polyamide-amine (D2.5); the ice-water bath was protected with gas, and 100 ml of methanol solution containing 20 grams of 2.5-generation polyamide-amine was added dropwise to 60 gram of ethylenediamine in 100 ml of methanol solution, stirred and reacted for 1 hour, then heated to room temperature and stirred for 48 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain 3rd generation polyamide-amine (D3);

(4) Nitrogen protection in an ice-water bath, dropwise add 150 ml of methanol solution containing 23 g of 3-generation polyamide-amine to 200 ml of methanol solution containing 70 g of methyl acrylate, stir for 1 hour, heat up to room temperature and stir for 72 hours , rotary evaporation at 30°C, and vacuum drying at 30°C to obtain 3.5-generation polyamide-amine (D3.5); the ice-water bath was protected with gas, and 300 ml of methanol solution containing 31 grams of 3.5-generation polyamido-amine was added dropwise to 100 gram of ethylenediamine in 100 ml of methanol solution, stirred and reacted for 1 hour, then heated to room temperature and stirred for 72 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain 4th generation polyamide-amine (D4);

(5) Under nitrogen protection, dissolve 10 g of 3-chloropropylamine hydrochloride and 5 g of potassium iodide in 300 ml of deionized water, add 100 g of sodium azide, and heat up to 90 degrees Celsius for 72 hours. After cooling to room temperature, 3 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. Extract the reaction solution with diethyl ether for 10 times, separate the solution and collect the organic phase, remove water from the organic phase with anhydrous magnesium sulfate, filter and distill under reduced pressure to obtain azidopropylamine.

(6) Under nitrogen protection, dissolve 1 g of pullulan in 150 ml of dimethyl sulfoxide, add 5 g of N,N'-carbonyldiimidazole (CDI) to activate at room temperature for 3 hours, add azidopropylamine to react for 48 Hour. After the reaction, the product was put into a dialysis bag with a cut-off molecular weight of 8000, dialyzed against deionized water for 3 days, and freeze-dried to obtain azidopropylamine-modified pullulan (Amyp-N ₃ )

(7) Under nitrogen protection, dissolve 1 g of azidopropylamine-modified pullulan, 10 g of 4th generation polyamidoamine, 1 g of copper sulfate pentahydrate and 10 g of sodium ascorbate in 200 ml of dimethyl sulfoxide and 10 In a mixed solvent of milliliters of water, the mixture was stirred and reacted at 30 degrees Celsius for 96 hours. The product was put into a dialysis bag with a cut-off molecular weight of 10,000, dialyzed against deionized water for 3 days, and freeze-dried to obtain the 4th generation polyamide-amine modified highly hyperbranched cationic pullulan derivative (Amyp-D4).

Example 6

1. Preparation of highly hyperbranched cationic pullulan derivatives (Amyp-D3) modified by 3rd generation polyamide-amine

(1) Nitrogen protection in an ice-water bath, dropwise add 10 ml of methanol solution containing 0.5 g of propargylamine to 10 ml of methanol solution containing 2 g of methyl acrylate, stir for 2 hours, warm to room temperature and stir for 24 hours, 30 Rotary evaporation at ℃, vacuum drying at 30℃ to obtain 0.5-generation polyamidoamine (D0.5); nitrogen protection in ice-water bath, 20 ml of methanol solution containing 1 g of 0.5-generation polyamido-amine was added dropwise to 10 g of B Diamine in 20 ml of methanol solution, stirred for 2 hours, heated to room temperature, stirred for 24 hours, 30°C rotary evaporation, 30°C vacuum-dried to obtain 1st generation polyamide-amine (D1);

(2) Nitrogen protection in an ice-water bath, dropwise add 20 ml of methanol solution containing 2 grams of 1st generation polyamide-amine to 30 ml of methanol solution containing 20 grams of methyl acrylate, stir for 2 hours, warm to room temperature and stir for 24 Hours, 30 ° C rotary evaporation, 30 ° C vacuum drying to obtain 1.5 generation polyamido-amine (D1.5); ice water bath with gas protection, 30 ml methanol solution containing 3.5 g 1.5 generation polyamido-amine was added dropwise to the containing 20 g of ethylenediamine in 50 ml of methanol solution, stirred for 2 hours, then heated to room temperature and stirred for 24 hours, 30 ° C rotary evaporation, 30 ° C vacuum drying to obtain the second generation polyamidoamine (D2);

(3) Ice-water bath with nitrogen protection, drop 50 ml of methanol solution containing 5 grams of 2-generation polyamide-amine into 60 ml of methanol solution containing 50 grams of methyl acrylate, stir for 2 hours, warm to room temperature and stir for 24 hours , rotary evaporation at 30°C, and vacuum drying at 30°C to obtain 2.5-generation polyamide-amine (D2.5); the ice-water bath was protected with gas, and 60 ml of methanol solution containing 10.4 g of 2.5-generation polyamido-amine was added dropwise to 60 1 g of ethylenediamine in 60 ml of methanol solution, stirred and reacted for 2 hours, then heated to room temperature and stirred for 48 hours, 30 ° C rotary evaporation, 30 ° C vacuum drying, to obtain the 3rd generation polyamide-amine (D3);

(4) Under nitrogen protection, dissolve 10 g of 3-chloropropylamine hydrochloride and 0.5 g of potassium iodide in 50 ml of deionized water, add 20 g of sodium azide, and heat up to 80 degrees Celsius for 48 hours. After cooling to room temperature, 2 ml of sodium hydroxide solution was added to adjust the pH of the solution to 11. The reaction solution was extracted with chloroform for 6 times, the solution was separated to collect the organic phase, and the organic phase was dehydrated with anhydrous magnesium sulfate, filtered and distilled under reduced pressure to obtain azidopropylamine.

(5) Under nitrogen protection, dissolve 0.1 g of pullulan in 20 ml of dimethyl sulfoxide, add 1 g of N,N'-carbonyldiimidazole (CDI) to activate at room temperature for 1 hour, add azidopropylamine to react for 48 Hour. After the reaction, the product was put into a dialysis bag with a cut-off molecular weight of 8000, dialyzed against deionized water for 3 days, and freeze-dried to obtain azidopropylamine-modified pullulan (Amyp-N ₃ )

(6) Under nitrogen protection, dissolve 0.1 g of azidopropylamine-modified pullulan, 10 g of 3rd generation polyamidoamine, 0.5 g of copper sulfate pentahydrate and 3 g of sodium ascorbate in 15 ml of dimethyl sulfoxide and 5 In the mixed solvent of milliliter water, stir reaction at 40 ℃ for 48 hours. The product was put into a dialysis bag with a cut-off molecular weight of 10,000, dialyzed against deionized water for 3 days, and freeze-dried to obtain the third-generation polyamide-amine modified highly hyperbranched cationic pullulan derivative (Amyp-D3).

Claims (10)

1. a highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine group, is characterized in that, its structural formula is as shown in formula (I): ; where R is H or, the 4.jpg It is a 3rd or 4th generation polyamide-amine. 2. the preparation method of polysaccharide derivative described in claim 1 is characterized in that, comprises the steps: S1. Ice-water bath with inert gas, drop the alcohol solution containing propargylamine into the alcohol solution containing methyl acrylate, react in the ice-water bath and then react at room temperature, evaporate and dry after the reaction to obtain 0.5 generation polyamide-amine; Under certain conditions, the alcohol solution containing 0.5 generation polyamidoamine is added dropwise to the alcohol solution containing ethylenediamine, reacted in an ice-water bath and then reacted at room temperature, evaporated and dried after the reaction to obtain 1 generation polyamidoamine; The 1st generation polyamide-amine is reacted with methyl acrylate to prepare the 1.5th generation polyamide-amine; the above steps are repeated to prepare alkyne-containing polyamide-amines of different generations; S2. Under the protection of an inert gas, dissolve the polysaccharide in an organic solvent, add N,N'-carbonyldiimidazole to activate at room temperature, and then add azidopropylamine to react. After the reaction, the product is dialyzed and dried to obtain azidopropylamine-modified polysaccharide ; S3. Under the protection of an inert gas, dissolve the azidopropylamine-modified polysaccharide, alkyne-containing polyamide-amine, copper sulfate pentahydrate and sodium ascorbate in a mixed solvent of organic solvent and water, react at 20-40°C, and the reaction ends Afterwards, the product is dialyzed and dried to obtain polyamide-amine modified highly hyperbranched cationic polysaccharide derivatives. 3. The preparation method according to claim 2, characterized in that the mass ratio of propargylamine to methyl acrylate in step S1 is 0.1-10:1-100; the 0.5 generation polyamide-amine and ethylenediamine The mass ratio of the 1-generation polyamide-amine to methyl acrylate is 1-30:4-200; the mass ratio of the first-generation polyamide-amine to methyl acrylate is 1-30:5-100. 4. The preparation method according to claim 2, characterized in that the mass ratio of polysaccharide to N,N'-carbonyldiimidazole in step S2 is 0.1-10:0.01-5. 5. The preparation method according to claim 2, wherein the mass ratio of the azidopropylamine-modified polysaccharide described in step S3, polyamide-amine containing alkyne groups, copper sulfate pentahydrate and sodium ascorbate is 0.1 to 10: 0.1～100: 0.01～10: 0.01～10. 6. The preparation method according to claim 2, characterized in that the reaction time in the ice-water bath in step S1 is 0.5-6 hours, and the reaction time at room temperature is 24-120 hours. 7. The preparation method according to claim 2, characterized in that the activation time at room temperature in step S2 is 0.5-5 hours, and the reaction time after adding azidopropylamine is 12-72 hours. 8. The preparation method according to claim 2, characterized in that the reaction at 20-40°C in step S3 is 24-96 hours. 9. according to the preparation method described in any one of claim 2～8, it is characterized in that, the preparation method of azidopropylamine described in step S2 is as follows: under inert gas protection, to 3-chloropropylamine hydrochloride and chlorination Add sodium azide to potassium aqueous solution, react at 50-90°C for 12-72 hours, cool to room temperature, adjust the pH of the solution to 8-11, then extract the reaction solution with an organic solvent, collect the organic phase, remove water, filter and distill under reduced pressure Azidopropylamine is obtained. 10. The application of the highly hyperbranched cationic polysaccharide derivatives containing dendritic polyamide-amine groups according to claim 1 in the preparation of gene transfection vectors.