阅读说明：本技术 一种白蛋白水凝胶、其制备方法及应用 (Albumin hydrogel, and preparation method and application thereof ) 是由 贺超良 王天然 张震 李杲 陈学思 于 2020-06-08 设计创作，主要内容包括：本发明提供了一种白蛋白水凝胶,由白蛋白、交联剂和溶剂混合制备得到；所述交联剂包含具有式(I)结构的重复单元和具有式(II)结构的端基。本发明开发了一种快速交联、成胶浓度低、无生物毒性的白蛋白水凝胶,以应用于细胞培养、组织工程和药物递送领域。另外,作为良好的紫杉醇局部治疗药物载体,本发明解决了紫杉醇溶解性差的缺点,且不引入具有生物毒性的聚氧乙烯蓖麻油,与全身制剂相比,本发明有效减少了给药次数、增加了肿瘤处药物浓度以及降低全身毒性。以包裹紫杉醇的纳米粒子为凝胶的成胶组分,可以实现紫杉醇在凝胶中的均匀分布,以实现稳定且持续的释放。(The invention provides an albumin hydrogel which is prepared by mixing albumin, a cross-linking agent and a solvent; the cross-linking agent comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II). The invention develops an albumin hydrogel which is fast in crosslinking, low in gelling concentration and free of biotoxicity, and is applied to the fields of cell culture, tissue engineering and drug delivery. In addition, as a good paclitaxel local treatment drug carrier, the invention solves the defect of poor paclitaxel solubility, does not introduce polyoxyethylene castor oil with biotoxicity, and effectively reduces the administration times, increases the drug concentration at the tumor and reduces the systemic toxicity compared with a systemic preparation. The nanometer particle coating the taxol is used as the gel forming component of the gel, so that the taxol can be uniformly distributed in the gel, and the stable and continuous release can be realized.)

1. An albumin hydrogel is characterized by being prepared by mixing albumin, a cross-linking agent and a solvent;

the cross-linking agent comprises a repeating unit with a structure shown in a formula (I) and an end group with a structure shown in a formula (II);

2. the albumin hydrogel of claim 1, wherein the cross-linking agent is selected from one or more compounds having the structures of formula (III) to formula (v);

wherein n is the degree of polymerization, and n is more than or equal to 50 and less than or equal to 100; m is polymerization degree, and m is more than or equal to 25 and less than or equal to 50; p is polymerization degree, and p is more than or equal to 67 and less than or equal to 133.

3. The albumin hydrogel of claim 1, wherein the albumin is selected from the group consisting of bovine serum albumin, human serum albumin, bovine serum albumin nanoparticles, human serum albumin nanoparticles, bovine serum albumin/paclitaxel nanoparticles, and human serum albumin/paclitaxel nanoparticles.

4. The albumin hydrogel of claim 3, wherein the bovine serum albumin nanoparticles, human serum albumin nanoparticles, bovine serum albumin/paclitaxel nanoparticles, or human serum albumin/paclitaxel nanoparticles are prepared by a desolvation method.

5. The albumin hydrogel of claim 1, wherein the solvent is selected from the group consisting of water, physiological saline, a buffer solution, a tissue culture solution, and a body fluid.

6. A method of preparing an albumin hydrogel according to any one of claims 1 to 5, comprising the steps of:

and mixing the albumin solution with the cross-linking agent solution to obtain the albumin hydrogel.

7. The method according to claim 6, wherein the albumin solution is a mixture of albumin and a solvent, and the albumin solution has a mass volume concentration of 4% to 20%.

8. The method according to claim 6, wherein the crosslinking agent solution is a mixture of a crosslinking agent and a solvent, and the mass volume concentration of the crosslinking agent in the crosslinking agent solution is 4 to 20%.

9. The method according to claim 6, wherein the mixing temperature is 25 to 37 ℃.

10. Use of the albumin hydrogel of any one of claims 1 to 5 in tissue engineering and drug delivery.

Technical Field

The invention belongs to the technical field of high polymer materials, and particularly relates to an albumin hydrogel, and a preparation method and application thereof.

Background

Chemically crosslinked hydrogels are materials that are crosslinked by covalent bonds, have a three-dimensional (3D) network structure, and contain a large amount of water or biological fluids, and generally have good stability, durability, and mechanical properties. The hydrogel can be divided into two categories, namely natural polymer and synthetic polymer according to different sources of the components. The natural polymer mainly comprises polysaccharides such as chitosan and hyaluronic acid, and proteins such as collagen, gelatin and albumin; the synthetic polymer mainly comprises polymethacrylic acid derivatives, polyester, polyether, polyamino acid materials and the like. Among such a wide variety of hydrogels, protein-based hydrogels constructed using natural proteins as basic structural units have a wide application prospect in the aspects of sensing, drug delivery, tissue engineering, artificial organs, and the like due to good biocompatibility, viscoelasticity, and in-vivo degradability, and among them, albumin is often used in the biomedical field as a protein-based gel material due to its characteristics of good solubility, good stability, in-tumor accumulation, easy availability, biodegradability, nontoxicity, and the like.

International cancer research machine according to 2018Recent data from the Institute of America (IARC) survey showed that breast cancer has an incidence of 24.2% in female cancers worldwide, and is the first place for female cancers. Paclitaxel, one of the most excellent natural anticancer drugs found at present, has been widely used for treating breast cancer clinically. However, paclitaxel has very poor water solubility, which greatly affects its therapeutic efficiency. Therefore, it is commonly used in clinicA preparation with polyoxyethylated castor oil and ethanol as solvent for improving solubility of paclitaxel is provided. However, the introduction of polyoxyethylated castor oil may cause allergic reactions in the body. In order to reduce the toxicity and increase the therapeutic efficacy of paclitaxel, researchers have developed a number of carriers for the delivery of drugs, such as paclitaxel lipid physically pamoate, micellar paclitaxel, which have been used clinically

And albumin-bound paclitaxelHowever, the paclitaxel preparations are systemic therapeutic preparations and have the disadvantages of more administration times and large systemic toxicity. Therefore, it is important to develop new topical therapeutic formulations that can reduce the number of administrations, increase the drug concentration at the tumor, and reduce systemic toxicity. Injectable hydrogels are a commonly used local treatment carrier, and have received extensive attention in the local drug release and tumor treatment fields with the advantages of being minimally invasive, site-specific, prolonging drug release time, low systemic toxicity, and being capable of delivering hydrophilic and hydrophobic agents.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a fast-crosslinking, low-gel-forming concentration, non-biotoxic albumin hydrogel, which is applied in the fields of cell culture, tissue engineering and drug delivery.

The invention provides an albumin hydrogel which is prepared by mixing albumin, a cross-linking agent and a solvent;

the cross-linking agent comprises a repeating unit with a structure shown in a formula (I) and an end group with a structure shown in a formula (II);

preferably, the crosslinking agent is selected from one or more compounds with structures from formula (III) to formula (V);

wherein n is the degree of polymerization, and n is more than or equal to 50 and less than or equal to 100; m is polymerization degree, and m is more than or equal to 25 and less than or equal to 50; p is polymerization degree, and p is more than or equal to 67 and less than or equal to 133.

Preferably, the albumin is selected from bovine serum albumin, human serum albumin, bovine serum albumin nanoparticles, human serum albumin nanoparticles, bovine serum albumin/paclitaxel nanoparticles or human serum albumin/paclitaxel nanoparticles.

Preferably, the bovine serum albumin nanoparticles, the human serum albumin nanoparticles, the bovine serum albumin/paclitaxel nanoparticles or the human serum albumin/paclitaxel nanoparticles are prepared by a desolvation method.

Preferably, the solvent is selected from water, physiological saline, a buffer solution, a tissue culture solution or a body fluid.

The invention also provides a preparation method of the albumin hydrogel, which comprises the following steps:

and mixing the albumin solution with the cross-linking agent solution to obtain the albumin hydrogel.

Preferably, the albumin solution is a mixture of albumin and a solvent, and the mass volume concentration of albumin in the albumin solution is 4-20%.

Preferably, the cross-linking agent solution is a mixture of a cross-linking agent and a solvent, and the mass volume concentration of the cross-linking agent in the cross-linking agent solution is 4-20%.

Preferably, the mixing temperature is 25-37 ℃.

The invention also provides application of the albumin hydrogel in tissue engineering and drug delivery.

Compared with the prior art, the invention provides the albumin hydrogel which is prepared by mixing albumin, a cross-linking agent and a solvent; the cross-linking agent comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II). The invention develops an albumin hydrogel which is fast in crosslinking, low in gelling concentration and free of biotoxicity, and is applied to the fields of cell culture, tissue engineering and drug delivery. In addition, as a good paclitaxel local treatment drug carrier, the invention solves the defect of poor paclitaxel solubility, does not introduce polyoxyethylene castor oil with biotoxicity, and effectively reduces the administration times, increases the drug concentration at the tumor and reduces the systemic toxicity compared with a systemic preparation. The nanometer particle coating the taxol is used as the gel forming component of the gel, so that the taxol can be uniformly distributed in the gel, and the stable and continuous release can be realized.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of an o-phthalaldehyde derivative prepared in example 9 of the present invention;

FIG. 2 is a NMR spectrum of 4aPEG-OPA prepared in example 10 of the present invention;

FIG. 3 shows the results of mechanical strength tests of albumin hydrogel with a mass concentration of 4%;

FIG. 4 shows the results of mechanical strength tests of albumin hydrogel with a mass concentration of 8%;

FIG. 5 shows the results of the measurement of the mechanical strength of the hydrogel composed of albumin-paclitaxel nanoparticles with mass concentrations of 8%, 10%, and 12%;

FIG. 6 shows the results of testing the mechanical strength of hydrogels composed of albumin-paclitaxel nanoparticles at 8%, 10% and 12% by mass concentration;

FIG. 7 is a scanning electron microscope photograph of the gel prepared in example 17;

FIG. 8 is a scanning electron microscope photograph of the gel prepared in example 18;

FIG. 9 shows the results of an in vitro degradation experiment for the gel prepared in example 19;

FIG. 10 shows the cytotoxicity results of gel materials at different concentrations on L929 cells.

Detailed Description

The invention provides an albumin hydrogel which is prepared by mixing albumin, a cross-linking agent and a solvent;

the cross-linking agent comprises a repeating unit with a structure shown in a formula (I) and an end group with a structure shown in a formula (II);

in some embodiments of the invention, the crosslinking agent is selected from one or more compounds having the structures of formula (III) to formula (v);

wherein n is the degree of polymerization, and n is more than or equal to 50 and less than or equal to 100; m is polymerization degree, and m is more than or equal to 25 and less than or equal to 50; p is polymerization degree, and p is more than or equal to 67 and less than or equal to 133.

In some embodiments of the invention, the crosslinking agent is selected from compounds having the structure of formula (III).

In the present invention, the albumin is selected from bovine serum albumin, human serum albumin, bovine serum albumin nanoparticles, human serum albumin nanoparticles, bovine serum albumin/paclitaxel nanoparticles or human serum albumin/paclitaxel nanoparticles.

Wherein the bovine serum albumin nanoparticles, the human serum albumin nanoparticles, the bovine serum albumin/paclitaxel nanoparticles or the human serum albumin/paclitaxel nanoparticles are prepared by a desolvation method.

Specifically, a certain amount of bovine serum albumin or human serum albumin is dissolved in deionized water, and after stirring and dissolving, the pH is adjusted to 7.0-9.0, preferably 7.7, so as to obtain a protein solution.

Dissolving a certain amount of paclitaxel in anhydrous ethanol to obtain paclitaxel ethanol solution.

Wherein the mass ratio of albumin to paclitaxel is 20: 1-10: 1, and the volume ratio of deionized water to absolute ethyl alcohol is 1: 2-1: 4

Then, setting the rotating speed at 400-600 rpm, dropwise adding the absolute ethyl alcohol or paclitaxel ethanol solution into the protein solution, and stirring for 24 hours at room temperature. Dialyzing for 3 days after the reaction, and freeze-drying to obtain bovine serum albumin nanoparticles or human serum albumin nanoparticles or bovine serum albumin/paclitaxel nanoparticles or human serum albumin/paclitaxel nanoparticles.

In the present invention, the solvent is selected from water, physiological saline, a buffer solution, a tissue culture solution or a body fluid, and preferably a buffer solution.

The invention also provides a preparation method of the albumin hydrogel, which comprises the following steps:

and mixing the albumin solution with the cross-linking agent solution to obtain the albumin hydrogel.

Specifically, albumin is dissolved in a solvent to obtain an albumin solution. The solvent is selected from water, physiological saline, buffer solution, tissue culture solution or body fluid, preferably buffer solution. The mass volume concentration of albumin in the albumin solution is 4-20%, preferably 5-15%.

And dissolving the crosslinking agent in a solvent to obtain a crosslinking agent solution. The solvent is selected from water, physiological saline, buffer solution, tissue culture solution or body fluid, preferably buffer solution. The mass volume concentration of the cross-linking agent in the cross-linking agent solution is 4-20%, preferably 5-15%.

And then mixing the albumin solution with the cross-linking agent solution to obtain the albumin hydrogel. The mixing temperature is 25-37 ℃.

The invention also provides application of the albumin hydrogel in tissue engineering and drug delivery.

The albumin hydrogel provided by the invention has the following beneficial effects:

1. the invention has short gelling time and low gelling concentration, can realize quick crosslinking gelling without other crosslinking agents, and can conveniently regulate and control the gelling time and the mechanical strength of the gel by changing the concentration ratio of the components. When the albumin component is the drug-loaded nano-particles, the drug-loaded amount of the gel can be changed by changing the concentration of the nano-particles, so that the subsequent medical application is facilitated.

2. The invention takes the drug-loaded nano particles as the gel-forming component of the gel, can uniformly distribute the paclitaxel in the gel, and is beneficial to realizing the stable and long-term release of the paclitaxel.

3. The invention is a local therapeutic preparation, which avoids the use of toxic solvent, reduces the administration times, increases the drug concentration at the tumor and reduces the systemic toxicity.

In order to further understand the present invention, the albumin hydrogel, the preparation method and the application thereof provided by the present invention are illustrated below with reference to the following examples, and the scope of the present invention is not limited by the following examples.