阅读说明：本技术 一种乏氧响应性壳聚糖药物载体及其制备方法与应用 (Hypoxia responsive chitosan drug carrier and preparation method and application thereof ) 是由 郭东波 石松庆 缑泽明 凌世长 黄林旋 张洋 何佳鹏 游柏浩 于 2019-11-13 设计创作，主要内容包括：本发明公开了一种乏氧响应性壳聚糖药物载体及其制备方法与应用。所述乏氧响应性壳聚糖药物载体的结构如式Ⅰ所示,其侧链带有硝基咪唑基团,R为带有苯基、对苯基和甲基中至少一种的烷烃链,a为0-100,b为0-100,c为0-100,d为1-100,n为0-100。本发明中的乏氧响应性壳聚糖药物载体可用于装载抗癌药物、抗生物、蛋白等药物,在肿瘤微微环境下,疏水性的硝基咪唑基团降解成亲水的氨基咪唑基团,可控释放药物,起到治疗效果。<Image he="242" wi="700" file="DDA0002270675300000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention disclosesDiscloses a hypoxia responsive chitosan drug carrier and a preparation method and application thereof. The structure of the hypoxia-responsive chitosan drug carrier is shown as a formula I, a side chain of the drug carrier is provided with a nitroimidazole group, R is an alkane chain provided with at least one of phenyl, p-phenyl and methyl, a is 0-100, b is 0-100, c is 0-100, d is 1-100, and n is 0-100. The hypoxia-responsive chitosan drug carrier can be used for loading anticancer drugs, antibiotics, protein and other drugs, and under the slight environment of tumors, hydrophobic nitroimidazole groups are degraded into hydrophilic aminoimidazole groups, so that the drugs can be controllably released, and the therapeutic effect is achieved.)

1. A hypoxia-responsive chitosan drug carrier is characterized in that the structural formula is shown as formula I:

wherein a is 0-100, b is 0-100, c is 0-100, d is 1-100, n is 0-100, R is C1-25 alkane chain or H, and the alkane chain has at least one of phenyl, p-phenyl and methyl.

2. The hypoxia-responsive chitosan drug carrier according to claim 1, wherein a is 1-100, n is 1-20; the structural formula of the hypoxia-responsive chitosan drug carrier is shown as a formula II:

3. the preparation method of the hypoxia-responsive chitosan drug carrier of any one of claims 1-2, characterized by comprising the following steps:

(1) dissolving alkyl acid with nitroimidazole as an end group, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a solvent, reacting at 0-60 ℃ for 0.5-4 h, then adding an amino chitosan acid solution, controlling the pH of the system to be 2-7, continuing to react for 0.5-48 h, finishing the reaction, adjusting the pH to 7-10, precipitating, and dialyzing to obtain hypoxia-responsive chitosan A;

(2) dissolving hypoxic responsive chitosan A in an acid solution, then adding an ethanol water solution of sodium cyanoborohydride and RCHO, or adding RCOOH, or adding RC-CH, stirring and reacting at 0-80 ℃ for 1-48 h, finishing the reaction, adjusting the pH value of the system to 2-10, centrifuging, and dialyzing to obtain hypoxic responsive chitosan B;

the hypoxia-responsive chitosan A and the hypoxia-responsive chitosan B are both hypoxia-responsive chitosan drug carriers.

4. The method for preparing the hypoxia-responsive chitosan drug carrier according to claim 3, wherein the molar ratio of the alkyl acid with the nitroimidazole as the terminal group, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide in the step (1) is 1: (0.8-1.5): (0.8 to 1.5); the molar ratio of amino in the amino chitosan acid solution to alkyl acid with the end group of nitroimidazole is (0.01-1000): 1;

the molar ratio of the hypoxia-responsive chitosan A to the RCHO or RCOOH or RC-CH in the step (2) is (0.01-1000): 1; the molar ratio of the sodium cyanoborohydride in the RCHO and sodium cyanoborohydride ethanol aqueous solution is (0.01-1000): 1.

5. the preparation method of the hypoxia-responsive chitosan drug carrier according to claim 4, wherein the carbon number of the alkyl acid with the end group of nitroimidazole in the step (1) is 3-103; and (2) R in the RCHO, RCOOH and RC ═ CH is an alkane chain with 1-25 carbon atoms or H, and the alkane chain carries at least one group of phenyl, p-phenyl and methyl.

6. The preparation method of the hypoxia-responsive chitosan drug carrier according to claim 5, wherein the number of carbon atoms in the alkyl group in the alkyl acid with the end group of nitroimidazole in the step (1) is 4-23; the alkyl acid with the end group of nitroimidazole is at least one of nitroimidazole propionic acid, 6- (2-nitroimidazole) hexanoic acid, 7- (2-nitroimidazole) heptanoic acid, 8- (2-nitroimidazole) octanoic acid and 12- (2-nitroimidazole) dodecanoic acid;

the concentration of the alkyl acid with the end group of nitroimidazole in the solvent in the step (1) is 0.5-5 mg/ml; the mass concentration of the amino chitosan acid solution is 0.1-5%; and (3) the mass concentration of the hypoxic responsive chitosan A in the acid solution in the step (2) is 0.1-5%.

7. The preparation method of the hypoxia-responsive chitosan drug carrier according to claim 6, wherein the solvent in the step (1) is dimethylformamide and water according to a volume ratio (0.1-10): 1, mixing to obtain; the acid solution in the amino chitosan acid solution in the step (1) is at least one of an acetic acid solution, a hydrochloric acid solution, a nitric acid solution and a sulfuric acid solution, wherein the mass concentration of the acid solution is 0.1-1%; stirring rotation speed of the reaction in the step (1) is 100-1000 rpm;

the acid solution in the step (2) is at least one of an acetic acid solution, a hydrochloric acid solution, a nitric acid solution and a sulfuric acid solution with the mass concentration of 0.1-1%; the RCHO in the step (2) is p-octadecyloxybenzaldehyde; the stirring speed of the reaction in the step (2) is 150-1000 rpm; the mass ratio of ethanol to water in the ethanol aqueous solution of sodium cyanoborohydride in the step (2) is (0.1-100): 100.

8. the application of the hypoxia-responsive chitosan drug carrier of any one of claims 1-2 in the fields of drug loading and drug preparation.

9. A hydrogel hypoxia-responsive chitosan carrier drug is characterized by being prepared by the following method:

dispersing 1-10 mg of drug in 1-10 mL of hypoxic responsive chitosan drug carrier solution with the concentration of 1-10 mg/mL, as described in any one of claims 1-2, and stirring or ultrasonic dispersing for 1-24 hours to obtain the hydrogel-like hypoxic responsive chitosan carrier drug.

10. A hypoxia-responsive chitosan drug stent is characterized by being prepared by the following method:

the hydrogel hypoxia-responsive chitosan drug carrier of claim 9 is freeze-dried at-40 to 0 ℃ for 1 to 48 hours to obtain a hypoxia-responsive chitosan drug scaffold.

Technical Field

The invention belongs to the technical field of tumor treatment materials, and particularly relates to a hypoxic responsive chitosan drug carrier, and a preparation method and application thereof.

Background

Chitosan materials are naturally degradable materials and are often used in biomedical and clinical fields. Among them, since the side chain has a modifiable amino group, it is often used for designing various drug carriers. However, when the existing chitosan material is used for treating tumors, due to the microenvironment of the tumors, such as high osmotic pressure, few blood vessels, hypoxic oxygen and the like, the requirements of treatment cannot be met.

On one hand, in clinical practice, the treatment of tumors on the surface of a human body is mainly performed by an operation, but tumor cells remain at a tumor focal site after the operation, so that the phenomena of tumor metastasis, recurrence and the like are easily caused. The existing anticancer drugs are mostly small molecules, so that the problems of low drug efficiency, large side effect, drug resistance and the like easily occur. Therefore, there is a need to design a drug delivery device to overcome the above problems. On the other hand, tumor resection is often accompanied by focal site loss, such as: bone defects, muscle defects and the like, and the drug carrier is often required to have a tissue regeneration function during treatment. The degradation products of the chitosan material are monosaccharide and other substances which are easily absorbed by human bodies, but the linear structure of the chitosan material hardly supports the three-dimensional space environment for tissue regeneration.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a hypoxic responsive chitosan drug carrier. According to the hypoxic-responsive chitosan drug carrier, the hypoxic responsiveness is realized by modifying a nitroimidazole group on a side chain of a chitosan material, the chitosan is endowed with the controllable drug release capacity through the chemical structure change of a nitro group in a hypoxic microenvironment, and the introduction of a rigid group (such as the nitroimidazole group) provides a growth space for normal cells, so that the hypoxic-responsive chitosan drug carrier can be applied to tumor treatment and tissue regeneration, and the introduction of an alkane chain is beneficial to adsorbing bacteria and red blood cells and is beneficial to antibiosis and hemostasis.

The invention also aims to provide a preparation method of the hypoxia-responsive chitosan drug carrier.

The invention also aims to provide application of the hypoxia-responsive chitosan drug carrier.

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

a hypoxia-responsive chitosan drug carrier has a structural formula shown in formula I:

wherein a is 0-100, b is 0-100, c is 0-100, d is 1-100, n is 0-100, R is C1-25 alkane chain or H, and the alkane chain has at least one of phenyl, p-phenyl and methyl.

Preferably, a is 1-100, and n is 1-20.

Preferably, b is 1-100, and c is 1-100.

Preferably, the structural formula of the hypoxia-responsive chitosan drug carrier is shown as formula II:

the hypoxia-responsive chitosan drug carrier with the structure of formula II has the functions of antibiosis and hemostasis.

The preparation method of the hypoxia responsive chitosan drug carrier comprises the following steps:

(1) dissolving alkyl acid with nitroimidazole as an end group, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a solvent, reacting at 0-60 ℃ for 0.5-4 h, adding an amino chitosan acid solution, controlling the pH of the system to be 2-7, continuing to react for 0.5-48 h, finishing the reaction, adjusting the pH to 7-10, precipitating, and dialyzing to obtain hypoxic responsive chitosan A;

(2) dissolving hypoxic responsive chitosan A in an acid solution, then adding an ethanol water solution of sodium cyanoborohydride and RCHO, or adding RCOOH, or adding RC-CH, stirring and reacting at 0-80 ℃ for 1-48 h, finishing the reaction, adjusting the pH value of the system to 2-10, centrifuging, and dialyzing to obtain hypoxic responsive chitosan B;

the hypoxia-responsive chitosan A and the hypoxia-responsive chitosan B are both hypoxia-responsive chitosan drug carriers.

Preferably, the number of carbon atoms of an alkyl group in the alkyl acid with the end group of nitroimidazole in the step (1) is 3-103, and preferably 4-23; the alkyl acid with the end group of the nitroimidazole is preferably at least one of nitroimidazole propionic acid, 6- (2-nitroimidazole) hexanoic acid, 7- (2-nitroimidazole) heptanoic acid, 8- (2-nitroimidazole) octanoic acid and 12- (2-nitroimidazole) dodecanoic acid.

When the alkyl acid with the end group of nitroimidazole in the step (1) is 6- (2-nitroimidazole) hexanoic acid, the structural formula of the hypoxia-responsive chitosan A is shown as a formula III:

preferably, the molar ratio of the alkyl acid with the end group of nitroimidazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the step (1) is 1: (0.8-1.5): (0.8 to 1.5); the molar ratio of amino in the amino chitosan acid solution to alkyl acid with the end group of nitroimidazole is (0.01-1000): 1.

preferably, the solvent in the step (1) is Dimethylformamide (DMF) and water according to a volume ratio of (0.1-10): 1 are mixed to obtain the product.

Preferably, the concentration of the alkyl acid with the end group of nitroimidazole in the step (1) in the solvent is 0.5-5 mg/ml.

Preferably, the mass concentration of the amino chitosan acid solution in the step (1) is 0.1-5% (referring to that 100g of the amino chitosan acid solution contains 0.1-5 g of amino chitosan); the acid solution in the amino chitosan acid solution is at least one of an acetic acid solution, a hydrochloric acid solution, a nitric acid solution and a sulfuric acid solution, wherein the mass concentration of the acid solution is 0.1-1%.

Preferably, the stirring speed of the reaction in the step (1) is 100-1000 rpm.

Preferably, the dialysis in steps (1) and (2) refers to the purification of the reaction product mixture by dialysis in water.

Preferably, the mass concentration of the hypoxic responsive chitosan A in the acid solution in the step (2) is 0.1-5% (referring to that 100ml of the acid solution contains 0.1-5 g of the hypoxic responsive chitosan A); the acid solution is at least one of an acetic acid solution, a hydrochloric acid solution, a nitric acid solution and a sulfuric acid solution with the mass concentration of 0.1-1%.

Preferably, the molar ratio of the hypoxia-responsive chitosan a and RCHO or RCOOH or RC ═ CH of step (2) is (0.01 to 1000): 1; the molar ratio of the sodium cyanoborohydride in the RCHO and sodium cyanoborohydride ethanol aqueous solution is (0.01-1000): 1.

preferably, in step (2), R in RCHO, RCOOH and RC ═ CH is as defined for R in formula i, i.e. R is an alkane chain with 1 to 25 carbon atoms or H, said alkane chain carrying at least one of phenyl, p-phenyl and methyl.

Preferably, the RCHO of step (2) is para-octadecyloxybenzaldehyde.

When the alkyl acid with the end group of nitroimidazole in the step (1) is 6- (2-nitroimidazole) hexanoic acid and the RCHO in the step (2) is p-octadecyloxybenzaldehyde, the structural formula of the obtained hypoxia-responsive chitosan B is shown as a formula IV:

preferably, the stirring speed of the reaction in the step (2) is 150-1000 rpm.

Preferably, the concentration of the sodium cyanoborohydride in the ethanol aqueous solution of the sodium cyanoborohydride in the step (2) is 0.5-10 mg/mL.

Preferably, the mass ratio of ethanol to water in the ethanol aqueous solution of sodium cyanoborohydride in the step (2) is (0.1-100): 100.

the hypoxic responsive chitosan drug carrier is applied to the fields of drug loading and drug preparation.

A hydrogel hypoxia-responsive chitosan carrier drug is prepared by the following method:

dispersing 1-10 mg of drug in 1-10 mL of the hypoxic responsive chitosan drug carrier solution with the concentration of 1-10 mg/mL, and stirring or ultrasonically dispersing for 1-24 hours to obtain the hydrogel-like hypoxic responsive chitosan carrier drug.

Preferably, the solvent of the hypoxic responsive chitosan drug carrier solution is at least one of an acetic acid solution, a hydrochloric acid solution, a nitric acid solution and a sulfuric acid solution with a mass concentration of 0.2-5%.

Preferably, the drug loading rate of the hypoxia-responsive chitosan drug carrier in the hypoxia-responsive chitosan drug carrier solution is 1-100%.

Preferably, the medicament is at least one of anticancer drugs, antibiotics and proteins, and the medicament is preferably at least one of cisplatin, adriamycin, paclitaxel, camptothecin, cephalosporins, β -lactam antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, gemcitabine, growth factors and inhibitors.

A hypoxia-responsive chitosan drug stent is prepared by the following method:

and (3) freeze-drying the hydrogel hypoxia-responsive chitosan carrier drug at-40-0 ℃ for 1-48 hours to obtain the hypoxia-responsive chitosan drug scaffold.

The hypoxia-responsive chitosan drug carrier prepared by the invention has a stable three-dimensional structure and can be used for repairing tissue defects; under a tumor or tissue hypoxia microenvironment, the hydrophobic nitroimidazole group is reduced into a hydrophilic aminoimidazole group, so that the hydrophilicity and the hydrophobicity of the chitosan material are changed, and the drug can be controllably released.

The deacetylation degree of the hypoxic responsive chitosan material drug carrier prepared by the method is 50-90%, and the method can be used for controlling the degradation rate of the chitosan material and controlling the procedural progress of drug release and tissue regeneration.

In order to realize the bacteriostatic and hemostatic functions of the chitosan material in the operation process, the side chain of the chitosan material is modified with alkane chains with different lengths, so that the hypoxia-responsive chitosan drug carrier is obtained. The hypoxia responsive chitosan drug carrier is mixed with broad-spectrum anticancer drugs, antibiotics or proteins to form gel, and then the drug carriers can be loaded.

The hypoxic responsive chitosan drug carrier prepared by the invention is injected into an operation focal part in a hydrogel form or implanted into the focal part in a drug stent form, and forms a certain three-dimensional network for tissue regeneration while treating tumors. The hypoxic responsive chitosan drug carrier can also control the drug release rate and the degradation rate of materials by changing the grafting rate of nitroimidazole and the deacetylation degree of chitosan.

According to the hypoxic responsive chitosan material, under a hypoxic environment, a hydrophobic nitroimidazolidine hydrocarbon chain is reduced into a hydrophilic aminoimidazolidine hydrocarbon chain, so that the hydrophilicity and the hydrophobicity of the chitosan material are changed, and the chitosan material releases a drug and is used for treating tumors, resisting bacterial infection, promoting tissue growth and the like.

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

1. the invention has simple reaction conditions, and is prepared from commercial raw materials; meanwhile, the post-treatment is simple, and the method is suitable for industrial production.

2. The nitroimidazole group on the hypoxic responsive chitosan drug carrier prepared by the invention is reduced into the aminoimidazole group under a tumor hypoxic microenvironment, so that the hydrophilicity and the hydrophobicity of the chitosan are changed, the drug can be controllably released, and the problem of poor drug controlled release of the existing chitosan material is solved.

3. The deacetylation degree of the hypoxic responsive chitosan drug carrier is controlled by the grafting ratio of the nitroimidazole alkane chain and the R alkane chain, so that the degradation rate of the material can be adjusted, the drug release is facilitated, the three-dimensional structure of the material can be maintained, and the tissue defect repair and the programmed process of metabolic absorption of the material are facilitated.

Drawings

FIG. 1 is a synthesis scheme of hypoxic responsive chitosan prepared in example 1.

FIG. 2 is a synthesis scheme of hypoxic responsive chitosan prepared in example 2.

FIG. 3 is a graph showing the preparation of hypoxic responsive chitosan in example 1¹H NMR spectrum.

FIG. 4 is a graph showing the preparation of hypoxic responsive chitosan in example 2¹H NMR spectrum.

FIG. 5 is the in vitro release profile of the hydrogel-type doxorubicin-loaded hypoxia-responsive chitosan prepared in example 3.

Fig. 6 is a graph showing the swelling ratio of the hypoxic responsive chitosan material prepared in example 2.

Fig. 7 is a graph showing the biocompatibility of the hypoxic responsive chitosan material prepared in example 2.

FIG. 8 is a zone diagram of the doxorubicin hypoxia-responsive chitosan drug-loaded stent material prepared in example 4.

FIG. 9 is a graph showing the toxicity of the doxorubicin loaded hypoxic-responsive chitosan prepared in example 3 on brain glioma tumor cells.

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.

Those who do not specify specific conditions in the examples of the present invention follow conventional conditions or conditions recommended by the manufacturer. The raw materials, reagents and the like which are not indicated for manufacturers are all conventional products which can be obtained by commercial purchase.

The amino chitosan in the embodiment of the application is purchased from Zhejiang gold Chitosan pharmaceutical industry Co., Ltd (product name: chitosan special for pharmaceutical excipients).