阅读说明：本技术 一种葡聚糖接枝树枝状聚酰胺-胺聚合物及其制备方法和应用 (Glucan grafted dendritic polyamide-amine polymer and preparation method and application thereof ) 是由 马栋 刘璐 胡云峰 薛巍 于 2019-09-19 设计创作，主要内容包括：本发明属于生物医学工程材料领域,公开了一种葡聚糖接枝树枝状聚酰胺-胺聚合物及其制备方法和应用。所述聚合物包括以下操作步骤：(1)将叠氮乙酸溶于DMF中,然后依次加入EDC·HCl、NHS和葡聚糖在室温下反应得到叠氮化葡聚糖；(2)将含炔基的聚酰胺-胺溶于水后,依次加入叠氮化葡聚糖、无水硫酸铜和还原剂进行加热反应得到最终产物。所得到的产物生物相容性好、表面基团丰富、易于进行化学改性；将其用于负载NO时,负载量较高,且负载的NO可以有效抑制细菌和真菌的生长和繁殖,并且在一定程度上可以消除炎症以及促进伤口的愈合,为其在制备生物医药工程材料的应用提供支持。(The invention belongs to the field of biomedical engineering materials, and discloses a glucan grafted dendritic polyamide-amine polymer and a preparation method and application thereof. The polymer comprises the following operation steps: (1) dissolving azidoacetic acid in DMF, and then sequentially adding EDC & HCl, NHS and glucan to react at room temperature to obtain azido glucan; (2) dissolving polyamide-amine containing alkynyl in water, and sequentially adding glucan azide, anhydrous copper sulfate and a reducing agent for heating reaction to obtain a final product. The obtained product has good biocompatibility and rich surface groups, and is easy to carry out chemical modification; when the supported NO is used for loading NO, the supported NO has high loading capacity, can effectively inhibit the growth and reproduction of bacteria and fungi, can eliminate inflammation and promote the healing of wounds to a certain extent, and provides support for the application of the supported NO in the preparation of biomedical engineering materials.)

1. A dextran grafted dendritic polyamidoamine polymer characterized by the following molecular formula:

wherein m is an integer of 80 to 100, and n is an integer of 10 to 20.

2. A process for the preparation of the dextran grafted dendritic polyamidoamine polymer according to claim 1, characterized in that it comprises the following operative steps:

(1) synthesis of azido group-modified dextrans

Dissolving azidoacetic acid in N-N-dimethylformamide, then sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and activating N-hydroxysuccinimide to obtain an azidoacetic acid mixed solution; dissolving glucan in water, then adding the glucan into the azido-acetic acid mixed solution for reaction, and obtaining azido glucan after the reaction is finished;

(2) synthesis of dextran grafted dendritic polyamidoamine polymers

And (2) dissolving polyamide-amine containing alkynyl into water, then sequentially adding the azido glucan obtained in the step (1), anhydrous copper sulfate and a reducing agent to carry out heating reaction, and obtaining the glucan grafted dendritic polyamide-amine polymer after the reaction is finished.

3. A method of preparing the glucan graft dendrimer-polyamidoamine polymer according to claim 2, wherein:

the molecular weight of the glucan in the step (1) is 1000-80000;

the mole ratio of the glucan, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the N-hydroxysuccinimide and the azidoacetic acid in the step (1) is 1: (1-12): (1-12): (1-12);

the dosage of the N, N-dimethylformamide in the step (1) is calculated by adding 1-5 g of azidoacetic acid into every 10mL of the N, N-dimethylformamide; the dosage of the water is calculated by adding 0.1g to 5g of azido acetic acid into every 10 mL.

4. A method of preparing the glucan graft dendrimer-polyamidoamine polymer according to claim 2, wherein:

the mole ratio of the glucan azide to the alkynyl polyamide-amine in the step (2) to the anhydrous copper sulfate to the reducing agent is 1: (1-12): (1-12): (2.5-18).

5. A method of preparing the glucan graft dendrimer-polyamidoamine polymer according to claim 2, wherein:

the alkynyl-containing polyamide-amine in the step (2) is prepared by referring to Chinese patent authorization text CN 106046382B; the reducing agent is sodium ascorbate;

the amount of the water in the step (2) is calculated by adding 5-15 g of alkynyl-containing polyamide-amine to every 100mL of water.

6. A method of preparing the glucan graft dendrimer-polyamidoamine polymer according to any one of claims 2 to 5, wherein:

the activation time of the step (1) is 30 min-3 h; the reaction in the step (1) is carried out at room temperature for 16-32 h;

the heating reaction in the step (2) is carried out for 24-48 h at 40-75 ℃.

7. Use of the dextran grafted dendritic polyamidoamine polymer according to claim 1 for loading nitric oxide.

8. A nitric oxide-loaded cationic polymer prepared from the dextran grafted dendritic polyamidoamine polymer according to claim 1, characterized by being prepared by the following steps:

adding the glucan grafted dendritic polyamide-amine polymer into an anhydrous methanol solution of sodium methoxide, introducing NO gas, stirring for reaction, and obtaining the nitric oxide loaded cationic polymer after the reaction is finished.

9. The nitric oxide-loaded cationic polymer of claim 8, wherein:

the molar ratio of the glucan grafted dendritic polyamide-amine polymer to sodium methoxide is 1: 400-500;

the anhydrous methanol solution of sodium methoxide is calculated by adding 2-3 g of sodium methoxide into every 100mL of methanol.

10. Nitric oxide loaded cationic polymer according to claim 8 or 9, characterized in that:

after NO gas is communicated, 5-6 atmospheric pressures are maintained;

the stirring reaction is carried out for 5-7 days at room temperature.

Technical Field

The invention belongs to the field of biomedical engineering materials, and particularly relates to a glucan grafted dendritic polyamide-amine polymer and a preparation method and application thereof.

Background

In recent years, the possibility of fungal infection has been increasing year by year due to low immune function, severe malnutrition, extensive burns and organ transplantation, or complications caused by malignant tumors, diabetes and blood diseases. Meanwhile, due to the wide use of broad-spectrum antibiotics, hormones and immunosuppressants, a plurality of drug-resistant fungi appear, the number and the types of the drug-resistant fungi are in a growing trend, and the drug-resistant fungi generally have cross drug resistance compared with bacteria. Although fungal resistance caused by antibiotic abuse is not as great as bacterial resistance, it is worth noting that drugs against different fungal infections are still very limited at present (mainly including azole drugs, polyene drugs, nucleoside drugs, echinocandin drugs), and the mortality rate caused by invasive fungal infections is increased year by year, 90% of the deaths are caused by fungi belonging to the classification of candida, aspergillus, cryptococcus, mucor and rhizopus (Trends Microbiol 2010,18:195-204), and the newly discovered invasive pathogen fungal infections have attracted more and more attention at present.

Another infection with a higher incidence than invasive is a superficial infection of the skin or mucosa with fungi, usually caused by trichophyton, epidermophyton or microsporum (International journal of microbiology 2012), which greatly interferes with the normal life of the patient. Fungi are eukaryotic organisms with the same cellular structure and metabolic function as the host, and the toxicity of the fungi to the host cells is considered when selecting a therapeutic target, so that the development of a novel antifungal preparation applied to the field of biomedicine is extremely urgent.

Clinically used antifungal drugs generally cause bacterial death by four ways: (1) inhibiting sterol synthesis in fungal cell membranes (2) acting on fungal cell walls to disable synthesis or to disrupt nucleic acid production of genetic material in fungi (3). However, such antifungal drugs often cause a series of side effects, such as affecting the overall level of leukocytes and impaired liver function, and prolonged use may cause a decrease in androgen in the blood and impaired adrenal function. One important way to change the resistance of fungi is to inhibit the formation of fungal biofilms, and it has been found that PGE 2 is a regulator of fungal cell membrane development. For dermatophytes and other filamentous fungi that infect keratinized host structures, they may emit sulfite as a reducing agent in the degradation of keratin, cleaving cystine in keratin directly into cysteine and thiosulphur. Thus, sulfite transporters may also serve as potential antifungal targets.

NO, as a small gas signaling molecule, has been shown to be involved in a variety of physiological processes such as angiogenesis, apoptosis, immune response, neurotransmission, and cardiovascular homeostasis. In vivo, NO is produced by endogenous arginine under catalysis by Nitric Oxide Synthase (NOs), which has a broad spectrum of activity as an antibacterial agent, and biomedical applications for exogenous NO mainly include treatment of bacterial infections and bacterial adhesion. NO produces many oxidized/nitrosated active species when reacting with oxygen or reactive oxygen intermediates, which may interact with microbial proteins, DNA and enzymes, destroying important cellular functions and structures, thereby manifesting good antimicrobial efficacy.

Related experimental studies have previously confirmed that NO also has antifungal efficacy, and is a gas regulator for controlling biofilms, and endogenous NO can induce the lysis of biofilms, thereby inhibiting fungal infection and adhesion. Gailde et al designed a NO-carrying compound DETA-NO and verified that the compound had bacteriostatic activity against six species of Candida, while the compound showed significant synergy when used in combination with azole antifungals (Antimicrobial Agents and thermology 1998,42: 2342-2346). Weller et al found that nitrite produces an active nitrogen intermediate in acidic environments and that NO molecules may be produced in the presence of nitric oxide synthase, and experiments demonstrated that different concentrations of acidified nitrite have different degrees of inhibition of several fungi that are infected with skin (Journal of applied Microbiology 2001,90: 648-652).

Although NO has a good inhibition effect on bacteria and fungi and cannot generate drug resistance, the NO loading capacity is limited, the half-life period in vivo is short, the storage process is unstable, the biological safety of the material is uncertain and the like, so that the application of the NO in clinical treatment is greatly limited. Therefore, under the condition of not adding other reducing agents, the azido acetic acid is used for azidizing the glucan, the glucan is coupled with the dendritic polyamide-amine containing alkynyl, and then NO is loaded to generate a polymer material with good biocompatibility and stability for treating dermatophyte infection, so far, NO report is available.

Disclosure of Invention

In order to solve the disadvantages and shortcomings of the prior art, the primary object of the present invention is to provide a dextran grafted dendritic polyamidoamine polymer. The polymer has a definite and controllable structure, good biocompatibility, stable NO load and excellent antibacterial performance, and can show an important application prospect in the aspect of treating skin fungal infection.

The invention also aims to provide a preparation method of the glucan grafted dendritic polyamide-amine polymer. The method comprises the steps of firstly synthesizing the glucan modified by the azide group through a substitution reaction, and then carrying out a coupling reaction on the glucan modified by the azide group and alkynyl on polyamide-amine (PAMAM) through a click reaction to synthesize the cationic polymer capable of being used as an NO donor.

The invention also aims to provide the application of the glucan grafted dendritic polyamide-amine polymer in loading nitric oxide.

It is a further object of the present invention to provide a nitric oxide-loaded cationic polymer prepared by grafting the dendritic polyamidoamine polymer with dextran as described above.

The invention further aims to provide a preparation method of the nitric oxide-loaded cationic polymer.

Still another object of the present invention is to provide the use of the above nitric oxide-loaded cationic polymer for antifungal purposes.

The purpose of the invention is realized by the following technical method:

a dextran grafted dendritic polyamidoamine polymer having the formula:

wherein m is an integer of 80 to 100, and n is an integer of 10 to 20.

A preparation method of the glucan grafted dendritic polyamide-amine polymer comprises the following operation steps:

(1) synthesis of azido group-modified dextrans:

reacting azidoacetic acid (N)₃-CH₂-COOH) is dissolved in N-N-Dimethylformamide (DMF), then 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) is added in sequence, and N-hydroxysuccinimide (NHS) is activated to obtain an azidoacetic acid mixed solution; dissolving glucan in water, then adding the glucan into the azido-acetic acid mixed solution for reaction, and obtaining azido glucan after the reaction is finished;

(2) synthesis of dextran grafted dendritic polyamidoamine polymers

And (2) dissolving polyamide-amine containing alkynyl into water, then sequentially adding the azido glucan obtained in the step (1), anhydrous copper sulfate and a reducing agent to carry out heating reaction, and obtaining the glucan grafted dendritic polyamide-amine polymer after the reaction is finished.

The molecular weight of the glucan in the step (1) is 1000-80000;

the mole ratio of the glucan, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the N-hydroxysuccinimide and the azidoacetic acid in the step (1) is 1: (1-12): (1-12): (1-12);

the dosage of the N, N-dimethylformamide in the step (1) is calculated by adding 1-5 g of azidoacetic acid into every 10mL of the N, N-dimethylformamide; the dosage of the water is calculated by adding 0.1g to 5g of azido acetic acid into every 10 mL.

The activation time of the step (1) is 30 min-3 h; the reaction in the step (1) is carried out at room temperature for 16-32 h.

And (2) after the reaction in the step (1) is finished, dialyzing the obtained product dialysis bag in pure water for 2-3 days, and then freeze-drying.

The alkynyl-containing polyamide-amine in the step (2) is prepared by referring to Chinese patent authorization text CN 106046382B.

The reducing agent in the step (2) is preferably sodium ascorbate.

The mole ratio of the glucan azide to the alkynyl polyamide-amine in the step (2) to the anhydrous copper sulfate to the reducing agent is 1: (1-12): (1-12): (2.5-18);

the amount of the water in the step (2) is calculated by adding 5-15 g of alkynyl-containing polyamide-amine to every 100mL of water;

the heating reaction in the step (2) is carried out for 24-48 h at 40-75 ℃.

And (3) after the reaction in the step (2) is finished, dialyzing the obtained product for 2-4 days by using a dialysis bag, and then freeze-drying.

The cut-off molecular weight of the dialysis bag in the steps (1) and (2) is 1000-80000.

Step (2) is preferably carried out under a noble gas or nitrogen.

Use of the dextran grafted dendritic polyamidoamine polymer described above for loading nitric oxide.

A nitric oxide-loaded cationic polymer prepared according to the dextran-grafted dendritic polyamidoamine polymer described above.

The nitric oxide-loaded cationic polymer is prepared by the following steps:

adding the glucan grafted dendritic polyamide-amine polymer into an anhydrous methanol solution of sodium methoxide, introducing NO gas, stirring for reaction, and obtaining the nitric oxide loaded cationic polymer after the reaction is finished.

The molar ratio of the glucan grafted dendritic polyamide-amine polymer to sodium methoxide is 1: 400-500;

the anhydrous methanol solution of sodium methoxide is calculated by adding 2-3 g of sodium methoxide into every 100mL of methanol; the preparation method of the sodium methoxide comprises the steps of adding 10-20 g of metal sodium into 500mL of methanol solution, and refluxing for 2-5 h at 50-65 ℃ to obtain the sodium methoxide;

the preparation method of the anhydrous methanol comprises the following operation steps: adding calcium hydride into methanol, stirring for 12-24 hours, and then distilling at normal pressure to obtain anhydrous methanol, wherein the addition amount of the calcium hydride is 1-2 g per 500mL of methanol;

the preparation process of the nitric oxide-loaded cationic polymer is preferably carried out in a high-pressure reaction kettle; before the NO gas is introduced, N is preferably used₂Removing air in the reaction kettle for 20-120 min; after NO gas is communicated, 5-6 atmospheric pressures are maintained;

the stirring reaction is carried out for 5-7 days at room temperature.

After the reaction is completed, N is used₂Removing unreacted NO gas for 20-120 min; then washing the obtained product with anhydrous methanol and diethyl ether in sequence, and then drying in vacuum at room temperature; the obtained nitric oxide-loaded cationic polymer is stored at the temperature of-10 to-20 ℃.

The application of the nitric oxide loaded cationic polymer in antifungal field.

The room temperature and unspecified reaction temperature are 15-37 ℃;

compared with the prior art, the invention has the following advantages and beneficial effects:

dendritic polyamide-amine is taken as a main chain of a carrier, azide groups are introduced by click chemical coupling of azide dextran, then the azide groups react with NO under certain pressure to prepare a high molecular polymer material capable of releasing nitric oxide gas, and finally the high molecular polymer which does not need electron transfer, auxiliary factors and enzyme participation and spontaneously releases NO gas at fixed points under physiological conditions is obtained.

(1) The natural glucan has good biological activity, contains a large amount of hydroxyl groups on the molecular structure, has good water solubility and biocompatibility, and is easy to carry out chemical modification;

(2) natural glucan is adopted for modification, so that the stability of azide groups can be improved, and the cytotoxicity of cationic polymer polyamide-amine is greatly reduced;

(3) the dendritic polyamide-amine molecular structure is definite and controllable, and the number of nucleophilic sites can be regulated and controlled by using dendritic molecules with different generations, so that the release of NO becomes controllable;

(4) the dendritic polyamide-amine and the azido glucan are coupled through click reaction, so that the reaction efficiency is high, the molecular weight distribution is single, and the stability of the polymer is enhanced;

(5) the surface of the material presents positive electric property, which is beneficial to the affinity with cells and has good biocompatibility;

(6) the preparation method of the polyamide-amine dendrimer is mild, the operation is simple and convenient, the product is relatively single and easy to separate and purify, and the cytotoxicity caused by the by-product is reduced to a certain extent;

(7) the branch chain of the dendritic macromolecule contains a large number of modifiable groups, and the dendritic macromolecule can be further chemically modified to expand the application of the dendritic macromolecule in the field of biological materials or grafted with natural compounds to improve the biocompatibility of the dendritic macromolecule.

Drawings

FIG. 1 is a graph of the cumulative amount of NO released over different times for the nitric oxide loaded dextran/polyamidoamine dendrimer cationic polymer of example 8.

Fig. 2 is a graph showing the bacteriostatic effect of the products obtained in example 6 and example 9 on candida albicans at different concentrations, wherein (a) is the polymeric material of example 6 without loading nitric oxide, and (b) is the polymeric material of example 9 with loading nitric oxide.

FIG. 3 is an infrared spectrum of dextran grafted dendritic polyamidoamine obtained in example 4.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

The preparation of dry methyl acrylate, ethylenediamine, propargylamine, and anhydrous methanol as described in the following examples was carried out according to the following procedure: adding anhydrous sodium sulfate into methyl acrylate, stirring for 12-24 hours, and then distilling at normal pressure to obtain anhydrous methyl acrylate, wherein the addition amount of the anhydrous sodium sulfate is calculated by adding 1-2 g of anhydrous sodium sulfate into every 500mL of methyl acrylate; adding anhydrous potassium hydroxide into ethylenediamine, stirring for 12-24 hours, and then distilling under reduced pressure to obtain anhydrous ethylenediamine, wherein the addition amount of the anhydrous potassium hydroxide is calculated by adding 1-2 g of anhydrous potassium hydroxide into every 500mL of ethylenediamine; the preparation method of the dried propargylamine comprises the following operation steps: adding calcium hydride into propargylamine, stirring for 12-24 hours, and then carrying out reduced pressure distillation to obtain dry propargylamine; the addition amount of the calcium hydride is calculated by adding 0.1-0.2 g of calcium hydride in each 10mL of propargylamine. The preparation method of the anhydrous methanol comprises the following operation steps: adding calcium hydride into methanol, stirring for 12-24 hours, and then distilling at normal pressure to obtain anhydrous methanol, wherein the addition amount of the calcium hydride is calculated by adding 1-2 g of calcium hydride into every 500mL of methanol.