阅读说明：本技术 一种基于透明质酸的双亲性接枝共聚物及其制备方法和应用 (Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof ) 是由 孙少平 梁娜 李树鹏 于 2020-04-10 设计创作，主要内容包括：本发明提供了一种基于透明质酸的双亲性接枝共聚物及其制备方法和应用。本发明共聚物包括：作为母体骨架的透明质酸(HA)、接枝在骨架上的去氧胆酸(DCA)、甲氧基聚乙二醇(mPEG)单元和N-乙酰-L-半胱氨酸(NAC)基团。本发明还提供了所述共聚物的制备方法,其中采用mPEG、DCA和NAC对HA进行修饰来制得所述共聚物。本发明还提供了由所述共聚物制得的止血海绵、药物载体和药物组合物。本发明共聚物生物相容性好,吸水速度快,主动被动双重靶向和氧化还原转化,因此可以用于制备快速吸水止血海绵和抗癌药物释放载体,实现药物的延长血液循环、高摄取和快速释药,有效提高肿瘤细胞内药物浓度,具有广阔的应用前景。(the invention provides an amphiphilic graft copolymer based on hyaluronic acid, a preparation method and application thereof.)

1. A hyaluronic acid-based amphiphilic graft copolymer, comprising:

(1) Hyaluronic acid as a matrix scaffold;

(2) A deoxycholic acid group grafted onto a first primary alcohol group of hyaluronic acid;

(3) Methoxypolyethylene glycol units grafted onto the carboxyl groups of hyaluronic acid;

(4) an N-acetyl-L-cysteine group grafted onto the second primary alcohol group of hyaluronic acid.

2. The amphiphilic graft copolymer of claim 1, wherein the amphiphilic graft copolymer has a molecular structure as shown below:

Wherein n is 8-453, and m is 7-659;

Preferably, the average molecular weight of the methoxypolyethylene glycol units is 350-20000 Da; and/or the average molecular weight of the hyaluronic acid is 5000-500000 Da.

3. a process for preparing the amphiphilic graft copolymer of claim 1 or 2, wherein the amphiphilic graft copolymer is prepared by reacting hyaluronic acid, deoxycholic acid, N-acetyl-L-cysteine and methoxypolyethylene glycol as raw materials.

4. A method according to claim 3, characterized in that the method comprises the steps of:

(1) Synthesizing HA-mPEG by using hyaluronic acid and methoxypolyethylene glycol;

(2) Synthesizing DCA-HA-mPEG using deoxycholic acid and the HA-mPEG;

(3) synthesizing NAC-HA-mPEG-DCA as the amphiphilic graft copolymer using N-acetyl-L-cysteine and the DCA-HA-mPEG;

wherein HA represents hyaluronic acid as a parent skeleton, DCA represents deoxycholic acid group, mPEG represents methoxypolyethylene glycol unit, and NAC represents N-acetyl-L-cysteine group;

preferably, in the step (1), hyaluronic acid is dissolved in a first reaction solvent, a first catalyst is added, catalysis is carried out for 0.5-24 hours at 0-60 ℃, then methoxypolyethylene glycol is added, the mixture is stirred and reacted for 2-96 hours at 0-60 ℃, and then HA-mPEG is obtained through separation, and/or in the step (2), N-acetyl-L-cysteine is dissolved in a second reaction solvent, a second catalyst is added, catalysis is carried out for 0.5-24 hours at 0-60 ℃, then HA-mPEG is added, the mixture is stirred and reacted for 2-96 hours at 0-60 ℃, and then NAC-HA-mPEG is obtained through separation, and/or in the step (3), cholic acid deoxidized is dissolved in a third reaction solvent, a third catalyst is added, catalysis is carried out for 0.5-24 hours at 0-60 ℃, then NAC-HA-mPEG is added, and NAC-DCA is obtained through separation after stirring for 2-96 hours at 20-80 ℃.

5. The method of claim 3 or 4, wherein:

The average molecular weight of the mPEG is 350-20000 Da; and/or

The average molecular weight of the hyaluronic acid is 5000-500000 Da.

6. The method according to any one of claims 3 to 5, wherein:

The first, second, and third reaction solvents are each independently selected from the group consisting of water, formamide, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, N-dimethylacetamide;

The first catalyst, the second catalyst, and the third catalyst are independently selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide.

7. The method of any of claims 3 to 6, wherein:

In the step (1), the molar ratio of the carboxyl of the hyaluronic acid to the hydroxyl of the methoxypolyethylene glycol is (1:10) to (100: 1); and/or the molar ratio of the dosage of the first catalyst to the carboxyl of the hyaluronic acid is (1:10) - (10: 1);

in the step (2), the molar ratio of the N-acetyl-L-cysteine to the hydroxyl group of the HA-mPEG is (1:100) - (10:1), and/or the molar ratio of the second catalyst to the N-acetyl-L-cysteine is (1:10) - (10:1), and/or

In the step (3), the molar ratio of deoxycholic acid to hydroxyl groups of the NAC-HA-mPEG is (1:100) - (10: 1); and/or the molar ratio of the dosage of the third catalyst to the deoxycholic acid is (1:10) - (10: 1).

8. A hemostatic sponge made from the amphiphilic graft copolymer of claim 1 or 2 or the amphiphilic graft copolymer made by the method of any one of claims 3 to 7.

9. An anticancer drug delivery vehicle, wherein the anticancer drug delivery vehicle is prepared from the amphiphilic graft copolymer of claim 1 or 2 or the amphiphilic graft copolymer prepared by the method of any one of claims 3 to 7; preferably, the anticancer drug is selected from the group consisting of docetaxel, paclitaxel, epirubicin, and camptothecin.

10. Use of the amphiphilic graft copolymer of claim 1 or 2 or the amphiphilic graft copolymer prepared by the method of any one of claims 3 to 7 for the preparation of a hemostatic sponge or an anticancer drug release carrier; preferably, the anticancer drug is selected from the group consisting of docetaxel, paclitaxel, epirubicin, and camptothecin.

Technical Field

The invention belongs to the technical field of high polymer materials and pharmaceutical preparations, and particularly relates to an amphiphilic graft copolymer based on hyaluronic acid, a preparation method thereof and application thereof in preparation of hemostatic sponges and anticancer drug release carriers.

Background

Cancer is a serious disease threatening human health, and its treatment and diagnosis have been the work focus and difficulty of many biologists, chemists and physicians. The existing cancer treatment means mainly comprise surgical treatment, radiotherapy and chemotherapy, wherein the chemotherapy is an effective measure for clinically treating the cancer at present, most of the used anti-cancer drugs are fat-soluble, and the problems of toxic and side effects, drug resistance and the like exist. Therefore, it is highly desirable to provide an anticancer drug delivery vehicle or a delivery composition comprising the same and an anticancer drug, which can solve the above problems.

the chitosan hemostatic material, although having non-toxic, non-antigenic, antibacterial and biocompatible properties and being degradable and absorbable in vivo, HAs great advantages in developing a rapid hemostatic material, however, the existing chitosan hemostatic material HAs problems of insufficient water absorption capacity, slow wound healing and insufficient adhesion to tissues, and the like, and the Hyaluronic Acid (HA) is a natural linear polysaccharide formed by repeated β - (1 → 4) -D-glucuronic acid and beta- (1 → 3) -N-acetyl-D-glucosamine units.

Disclosure of Invention

In order to solve the problems of the prior art, the present invention provides in a first aspect a hyaluronic acid-based amphiphilic graft copolymer comprising:

(1) Hyaluronic acid as a matrix scaffold;

(2) A deoxycholic acid group grafted onto a first primary alcohol group of hyaluronic acid;

(3) Methoxypolyethylene glycol units grafted onto the carboxyl groups of hyaluronic acid;

(4) an N-acetyl-L-cysteine group grafted onto the second primary alcohol group of hyaluronic acid.

in a second aspect, the present invention provides a process for preparing the amphiphilic graft copolymer of the first aspect of the present invention, which is prepared by reacting hyaluronic acid, deoxycholic acid, N-acetyl-L-cysteine, and methoxypolyethylene glycol as raw materials.

In a third aspect, the present invention provides a hemostatic sponge made from the amphiphilic graft copolymer of the first aspect of the present invention or made by the method of the second aspect of the present invention.

In a fourth aspect, the present invention provides an anticancer drug delivery vehicle prepared from the amphiphilic graft copolymer of the first aspect of the present invention or the amphiphilic graft copolymer prepared by the method of the second aspect of the present invention. Preferably, the anticancer drug is selected from the group consisting of docetaxel, paclitaxel, epirubicin, and camptothecin.

In a fifth aspect, the present invention provides the use of the amphiphilic graft copolymer of the first aspect of the present invention or the amphiphilic graft copolymer prepared by the method of the second aspect of the present invention in the preparation of a hemostatic sponge or an anticancer drug release carrier; preferably, the anticancer drug is selected from the group consisting of docetaxel, paclitaxel, epirubicin, and camptothecin.

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

(1) The amphiphilic graft copolymer of the hyaluronic acid derivative is used as a drug carrier, has high drug loading rate, high entrapment rate, long and stable in-vivo circulation, increased drug utilization rate, good biocompatibility, small toxic and side effects and is degradable in vivo.

(2) The invention provides a tumor tissue microenvironment based on high-concentration biological reductive glutathione, selects the amphiphilic hyaluronic acid derivative containing sulfydryl, the material is easy to form disulfide bonds in an oxidation environment, and the disulfide bonds are opened by utilizing an oxidation-reduction mechanism in the tumor tissue microenvironment, so that the rapid targeted release of the anticancer drug in a living body can be realized.

(3) The invention takes polyethylene glycol and hyaluronic acid as main hydrophilic ends of an amphiphilic polymer drug carrier, takes hyaluronic acid as a target head, can actively target to the surface of a tumor cell, is combined with a CD44 receptor on the surface of the tumor cell and enters the tumor cell through the endocytosis of the cell, and overcomes the problem of low cell uptake capacity of a common carrier micelle carrier.

(4) The drug carrier disclosed by the invention is simple and convenient to prepare, has good biocompatibility and wide raw material sources, has multiple functional groups in a repeating unit, is easy to modify the structure, can quickly generate redox action in tumor cells to release the drug, thereby generating high-efficiency treatment effect, and has huge application potential in the controlled release of the drug.

(5) After the hyaluronic acid derivative disclosed by the invention is prepared into the hemostatic sponge, the water absorption capacity is large, and the water absorption speed is high.

Drawings

FIG. 1 is a scheme showing the synthesis scheme of mPEG-hyaluronic acid-N-acetyl-L-cysteine-deoxycholic acid in example 1.

FIG. 2 is an IR spectrum of the final product of example 1.

FIG. 3 is a NMR spectrum of the final product of example 1.

Detailed Description

The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.

As described above, the present invention provides in a first aspect a hyaluronic acid-based amphiphilic graft copolymer comprising:

(1) Hyaluronic acid as a matrix scaffold;

(2) A deoxycholic acid group grafted onto a first primary alcohol group of hyaluronic acid;

(3) Methoxy polyethylene glycol units grafted to the carboxyl groups of hyaluronic acid;

(4) an N-acetyl-L-cysteine group grafted onto the second primary alcohol group of hyaluronic acid.

in the present invention, the position of the first primary alcohol group grafted with a deoxycholic acid group, the carboxyl group grafted with a methoxypolyethylene glycol unit, and the second primary alcohol group grafted with an N-acetyl-L-cysteine group on the backbone is not particularly limited, and the first primary alcohol group and the second primary alcohol group are used only for distinguishing one from another, and there is no limitation on the order and importance thereof on the backbone.

In some preferred embodiments, the amphiphilic graft copolymer has a molecular structure as shown below:

Wherein n is 8 to 453 (e.g., 50, 65, 80), and m is 7 to 659 (e.g., 100, 150, 200).

In further preferred embodiments, the value of n is such that the average molecular weight of the methoxypolyethylene glycol units is from 350 to 20000Da, for example 500, 1000, 2000, 5000, 10000 or 15000 Da.

In further preferred embodiments, the value of m is such that the hyaluronic acid has an average molecular weight of 5000-500000Da, such as 10000, 15000, 20000 or 25000 Da.

the HA used for chemical modification includes carboxyl, hydroxyl and amino groups resulting from N-acetyl deacetylation, and can be used for chemical modification, for example, by esterification, crosslinking, grafting, and like modifying means.

in addition, the inventors have found that modification of HA improves the stability of HA and results in a derivative having superior properties, in which methoxy polyethylene glycol (mPEG) HAs good hydrophilicity, biocompatibility, nontoxicity and nonimmunogenicity, and that it is also a three-dimensional protective body of nanocarriers, can extend the circulation time of nanocarriers in vivo, HAs good compatibility with human tissues, HAs little toxic and side effects, HAs low irritation, and the like.

in a second aspect, the present invention provides a process for preparing the amphiphilic graft copolymer of the first aspect of the present invention, which is prepared by reacting hyaluronic acid, deoxycholic acid, N-acetyl-L-cysteine, and methoxypolyethylene glycol as raw materials.

in some preferred embodiments, the method comprises the steps of (1) synthesizing HA-mPEG using hyaluronic acid and methoxypolyethylene glycol, (2) synthesizing DCA-HA-mPEG using deoxycholic acid and the HA-mPEG, and (3) synthesizing NAC-HA-mPEG-DCA as the amphiphilic graft copolymer using N-acetyl-L-cysteine and the DCA-HA-mPEG, wherein HA represents hyaluronic acid as a parent backbone, DCA represents a cholic acid group, mPEG represents a methoxypolyethylene glycol unit, and NAC represents an N-acetyl-L-cysteine group.

In other preferred embodiments, in step (1), the HA-mPEG is isolated after dissolving hyaluronic acid in a first reaction solvent, adding a first catalyst, catalyzing at 0-60 ℃ (e.g., 10, 20, 30, 40, or 50 ℃) for 0.5-24 hours (e.g., 1, 3, 6, 9, 12, 15, 18, or 21 hours), then adding methoxypolyethylene glycol, reacting at 0-60 ℃ (e.g., 10, 20, 30, 40, or 50 ℃) for 2-96 hours (e.g., 2, 6, 12, 24, 48, 60, 72, or 84 hours) with stirring.

in other preferred embodiments, in step (2), the NAC-HA-mPEG is isolated after dissolving the N-acetyl-L-cysteine in the second reaction solvent, adding the second catalyst, catalyzing at 0-60 ℃ (e.g., 10, 20, 30, 40, or 50 ℃) for 0.5-24 hours (1, 3, 6, 9, 12, 15, 18, or 21 hours), adding the HA-mPEG, and reacting at 0-60 ℃ (e.g., 10, 20, 30, 40, or 50 ℃) for 2-96 hours (e.g., 2, 6, 12, 24, 48, 60, 72, or 84 hours) with stirring.

In other preferred embodiments, in step (3), deoxycholic acid is dissolved in a third reaction solvent, a third catalyst is added, the mixture is catalyzed at 0-60 ℃ (e.g., 10, 20, 30, 40 or 50 ℃) for 0.5-24 hours, NAC-HA-mPEG is added, and after stirring at 20-80 ℃ (e.g., 20, 30, 40, 50, 60 or 70 ℃) for 2-96 hours (e.g., 2, 6, 12, 24, 48, 60, 72 or 84 hours), NAC-HA-mPEG-DCA is isolated.

In further preferred embodiments, the value of n is such that the average molecular weight of the methoxypolyethylene glycol units is from 350 to 20000Da, for example 500, 1000, 2000, 5000, 10000 or 15000 Da.

In further preferred embodiments, the value of m is such that the hyaluronic acid has an average molecular weight of 5000-500000Da, such as 10000, 15000, 20000 or 25000 Da.

In other preferred embodiments, the first reaction solvent is selected from the group consisting of water, formamide, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide, and preferably, the first reaction solvent is selected from the group consisting of formamide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide.

In other preferred embodiments, the second reaction solvent is selected from the group consisting of water, formamide, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide, and preferably, the second reaction solvent is selected from the group consisting of formamide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide.

In other preferred embodiments, the third reaction solvent is selected from the group consisting of water, formamide, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide, and preferably, the third reaction solvent is selected from the group consisting of formamide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide.

The first catalyst, the second catalyst, and the third catalyst are independently selected from the group consisting of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), DMAP (4-dimethylaminopyridine), DCC (dicyclohexylcarbodiimide). The first catalyst, the second catalyst and the third catalyst may be the same or different. For example, the first catalyst may be selected from the group consisting of EDC, NHS, DCC, and DMAP; the second catalyst may be selected from the group consisting of EDC, NHS, DCC, DMAP; the third catalyst may be a group consisting of EDC, NHS and DCC.

In other preferred embodiments, in step (1), the molar ratio of the carboxyl groups of the hyaluronic acid to the hydroxyl groups of the methoxypolyethylene glycol is (1:10) to (100:1), for example 100:1, 50:1, 10:1, 1:5 or 1: 10. Additionally or alternatively, the molar ratio of the first catalyst to the carboxyl groups of the hyaluronic acid is (1:10) to (10:1), for example 1:0.1, 1:0.5, 1:1, 1:5 or 1: 10.

in other preferred embodiments, in step (2), the molar ratio of N-acetyl-L-cysteine to the hydroxyl groups of the HA-mPEG is (1:100) to (10:1), such as 1:1, 10:1, 1:50 or 1:100, additionally or further alternatively the molar ratio of the second catalyst to N-acetyl-L-cysteine is (1:10) to (10:1), such as 1:0.1, 1:0.5, 1:1, 1:5 or 1: 10.

In other preferred embodiments, in step (3), the molar ratio of deoxycholic acid to hydroxyl groups of the NAC-HA-mPEG is (1:100) to (10:1), for example 1:0.1, 1:0.5, 1:1, 1:5 or 1: 10. Additionally or alternatively, the molar ratio of the third catalyst to deoxycholic acid is (1:10) to (10:1), for example 1:0.1, 1:0.5, 1:1, 1:5 or 1: 10.

In some more specific embodiments, the method comprises the steps of:

(1) Synthesis of HA-mPEG: dissolving hyaluronic acid in a first reaction solvent, adding a first catalyst, and catalyzing at 0-60 ℃ for 0.5-24 hours. Then adding mPEG, stirring and reacting for 2-96 hours at the temperature of 0-60 ℃, dialyzing and drying to obtain the HA-mPEG.

(2) and (2) synthesizing NAC-HA-mPEG, namely dissolving N-acetyl-L-cysteine in a second reaction solvent, adding a second catalyst, catalyzing at 0-60 ℃ for 0.5-24 hours, adding the HA-mPEG derivative, stirring and reacting at 0-60 ℃ for 2-96 hours, dialyzing, and drying to obtain the NAC-HA-mPEG.

(3) Synthesis of NAC-HA-mPEG-DCA: and dissolving deoxycholic acid in a third reaction solvent, adding a third catalyst, catalyzing for 0.5-24 hours at 0-60 ℃, then adding the NAC-HA-mPEG, stirring for 12-96 hours at 20-80 ℃, dialyzing, purifying and freeze-drying a reaction product to obtain the NAC-HA-mPEG-DCA serving as the amphiphilic graft copolymer.

The amphiphilic graft copolymer based on hyaluronic acid (sometimes referred to as hyaluronic acid derivative) of the present invention can be used as a material for hemostatic sponges. Accordingly, in a third aspect, the present invention provides a haemostatic sponge made from an amphiphilic graft copolymer according to the first aspect of the invention or made by the process according to the second aspect of the invention. When the hemostatic sponge is prepared, the amphiphilic graft copolymer can be suspended in water to prepare an aqueous suspension, then calcium chloride is added for crosslinking, and finally the hemostatic sponge is obtained by freeze-drying. For example, the amphiphilic graft copolymer can be prepared into a 200mg/ml solution, and after being added with 30mg/ml calcium chloride for crosslinking for 2 hours, the solution is lyophilized to obtain the NAC-HA-mPEG-DCA hemostatic sponge.

In a fourth aspect, the present invention provides an anticancer drug delivery vehicle prepared from the amphiphilic graft copolymer of the first aspect of the present invention or the amphiphilic graft copolymer prepared by the method of the second aspect of the present invention. The amphiphilic graft copolymer can be used for encapsulating anticancer drugs, so that a multistage targeting polymer which releases the anticancer drugs in tumor cells under the triggering of redox is prepared. Accordingly, the present invention may also provide an anticancer drug releasing composition comprising: (a) an amphiphilic graft copolymer according to the first aspect of the present invention or obtainable according to the second aspect of the present invention; and (b) an anticancer drug entrapped in the amphiphilic graft copolymer. The anticancer drug can realize multi-stage targeted redox-triggered drug release. Therefore, the anti-cancer drug release carrier or the anti-cancer drug containing the carrier has the characteristics of multi-stage tumor targeting, enhancement of in vivo long circulation and redox-triggered drug release (sensitive to redox) in tumor cells.

In preparing the anticancer drug preparation, an anticancer drug such as paclitaxel may be dissolved in a solvent such as dichloromethane to prepare a paclitaxel solution. In addition, an aqueous solution of the amphiphilic graft copolymer was prepared. Then, the paclitaxel solution was added dropwise to the amphiphilic graft copolymer aqueous solution while stirring. After the dropwise addition, the stirring is continued so that the anticancer drug is sufficiently loaded in the amphiphilic graft copolymer. Then, the resultant is filtered to remove the aggregates of the free anticancer drug not being entrapped and the amphiphilic graft copolymer as a carrier, thereby preparing the anticancer drug releasing composition.

In a fifth aspect, the present invention provides the use of the amphiphilic graft copolymer of the first aspect of the present invention or the amphiphilic graft copolymer prepared by the method of the second aspect of the present invention in the preparation of a hemostatic sponge or an anticancer drug release carrier. It is preferable that the first and second liquid crystal layers are formed of,

In various embodiments of the present reference to the anticancer drug, the anticancer drug may be selected from the group consisting of docetaxel, paclitaxel, epirubicin, and camptothecin.

the invention provides a hyaluronic acid derivative and a preparation method and application thereof, wherein hyaluronic acid is used as a matrix skeleton, and deoxycholic acid is used for modifying the hyaluronic acid to prepare the amphiphilic hyaluronic acid derivative, on the basis, N-acetyl-L-cysteine and mPEG are used for modifying the hyaluronic acid to form a novel material which has good biocompatibility and can be used for carrying medicine and preparing hemostatic sponges.