阅读说明：本技术 可注射型抗菌止血水凝胶粘合剂及其制备方法和应用 (Injectable antibacterial hemostatic hydrogel adhesive and preparation method and application thereof ) 是由 周祺惠 郝源萍 卢云 尹祥意 于 2020-07-23 设计创作，主要内容包括：本发明公开了一种可注射型抗菌止血水凝胶粘合剂及其制备方法和应用,该可注射型抗菌止血水凝胶粘合剂由醛基封端的聚乙二醇的可交联衍生物与带有氨基的高分子化合物交联得到,制备得到的可注射型抗菌止血水凝胶粘合剂综合了各原料众多优良功能和生物活性,不仅能为细胞提供生长、增殖以及分化的理想微环境,而且具有良好的抗菌活性,此外,该可注射型水凝胶粘合剂还具有较强的粘附性、自修复性能及较强的机械性能,在改善伤者伤口流血及感染和创面修复领域有非常好的应用前景。(The invention discloses an injectable antibacterial hemostatic hydrogel adhesive and a preparation method and application thereof, the injectable antibacterial hemostatic hydrogel adhesive is obtained by crosslinking a crosslinkable derivative of aldehyde-group-terminated polyethylene glycol and a macromolecular compound with amino, the prepared injectable antibacterial hemostatic hydrogel adhesive integrates a plurality of excellent functions and biological activities of raw materials, not only can provide an ideal microenvironment for growth, proliferation and differentiation for cells, but also has good antibacterial activity, and in addition, the injectable hydrogel adhesive also has stronger adhesiveness, self-repairing performance and stronger mechanical performance, and has very good application prospects in the fields of improving wound bleeding and infection of a wound and wound repair of a wound.)

1. The injectable antibacterial hemostatic hydrogel adhesive is characterized by being obtained by crosslinking a crosslinkable derivative of aldehyde-terminated polyethylene glycol and a macromolecular compound with amino.

2. The injectable antibacterial hemostatic hydrogel adhesive according to claim 1, wherein the crosslinkable derivative of polyethylene glycol is one or a mixture of two of star-shaped multi-arm polyethylene glycol and PF 127; the macromolecular compound with amino is one or a mixture of more than two of water-soluble carboxymethyl chitosan derivative, Polyethyleneimine (PEI), gelatin and amino derivative of star-shaped multi-arm polyethylene glycol; the number of arms of the star-shaped multi-arm polyethylene glycol is 2-8.

3. An injectable antibacterial haemostatic hydrogel adhesive according to claim 2, wherein said crosslinkable derivative of aldehyde-terminated polyethylene glycol is a four-armed polyethylene glycol aldehyde (4-arm-PEG-CHO); the macromolecular compound with amino is carboxymethyl chitosan.

4. An injectable antibacterial haemostatic hydrogel adhesive according to claim 2 or 3, wherein the star-shaped multi-arm polyethylene glycol has a molecular weight of not less than 2000.

5. A method for preparing an injectable antibacterial haemostatic hydrogel adhesive according to any of claims 1-4, comprising the steps of:

s1, dissolving carboxymethyl chitosan in PBS buffer solution to obtain carboxymethyl chitosan solution for later use;

s2, synthesizing a four-arm polyethylene glycol aldehyde group, and dissolving the four-arm polyethylene glycol aldehyde group in a PBS buffer solution to obtain a four-arm polyethylene glycol aldehyde group solution;

s3, quickly mixing the carboxymethyl chitosan solution obtained in the step S1 and the four-arm polyethylene glycol aldehyde solution obtained in the step S2 in equal volume to obtain the injectable antibacterial hemostatic hydrogel adhesive.

6. The method according to claim 5, wherein the carboxymethyl chitosan solution contains W, which is the weight percentage of carboxymethyl chitosan₁1% -15%; the weight percentage content W of the four-arm polyethylene glycol aldehyde group in the four-arm polyethylene glycol aldehyde group solution₂1% -20%; w₁/W₂Is 14:1-1: 14.

7. The method according to claim 5, wherein the pH of the PBS buffer is 7.4.

8. The preparation method according to claim 5, wherein the method for synthesizing the four-arm polyethylene glycol aldehyde group specifically comprises:

(1) sequentially adding 4-carboxybenzaldehyde, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 4-dimethylaminopyridine and four-arm polyethylene glycol (4-arm-PEG-OH) into a dry solvent under an inert gas atmosphere, and stirring at 20-40 ℃ for 12-36 h; the molar ratio of the four-arm polyethylene glycol (4-arm-PEG-OH) to the 4-carboxybenzaldehyde is 1 (1-10);

(2) filtering, washing the solid, and vacuum drying to obtain white solid powder.

9. The process according to claim 8, wherein the molar ratio of 4-carboxybenzaldehyde, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and 4-dimethylaminopyridine in step (1) is 2:2: 1; the reaction temperature is 37 ℃, and the reaction time is 18-24 h; when washing, the mixture is sequentially washed with 1M HCl solution and saturated NaHCO₃The solution was washed with brine.

10. Use of the injectable antibacterial hemostatic hydrogel adhesive of any one of claims 1 to 4 in wound tissue sealing and leakage prevention, tissue filling, tissue repair and regeneration, and preparation of bacteriostatic skin dressings, bacteriostatic drug formulations, drug delivery, cell encapsulation and delivery, tissue engineering scaffolds, 3D printing inks.

Technical Field

The invention relates to the technical field of polymer composite materials, in particular to an injectable antibacterial hemostatic hydrogel adhesive and a preparation method and application thereof.

Background

Under critical conditions such as battlefields, traffic accidents, natural disasters and the like, a large number of wounded persons can be generated in a short time. Uncontrolled bleeding and infection are the most serious risk factors for death in these critical situations, with more than half of the victims dying from these factors. In many extreme cases, it is not possible to apply immediate professional treatment to a wound, and therefore simple and effective bleeding control and emergency measures to prevent infection are of great importance to save the lives of the injured before the patient receives professional treatment.

Aiming at the problems of bleeding and susceptibility to infection of wounds, how to prepare a novel biomaterial which can quickly stop bleeding, prevent infection and can be repaired, and the novel biomaterial integrates multiple functions of stopping bleeding, inhibiting bacteria, repairing and the like, and high adhesion, high mechanical property and the like, thereby meeting the requirements of the domestic market on quick hemostasis and the persistence of medical materials for preventing wound infection, realizing organic combination of research and production, and being an actual problem to be solved urgently in the market of medical instruments in China.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention provides an injectable antibacterial hemostatic hydrogel adhesive with high adhesiveness and high mechanical strength.

In order to achieve the purpose, the invention provides the following technical scheme:

the injectable antibacterial hemostatic hydrogel adhesive is obtained by crosslinking a crosslinkable derivative of aldehyde-terminated polyethylene glycol and a macromolecular compound with amino.

Preferably, the crosslinkable derivative of polyethylene glycol is one or a mixture of two of star-shaped multi-arm polyethylene glycol and PF 127.

Preferably, the macromolecular compound with amino is one or a mixture of more than two of water-soluble carboxymethyl chitosan derivative, Polyethyleneimine (PEI), gelatin and amino derivatives of star-shaped multi-arm polyethylene glycol.

Preferably, the number of arms of the star-shaped multi-arm polyethylene glycol is 2 to 8. Particularly preferably, the star-shaped multi-arm polyethylene glycol has an arm number of 3 or 4.

Further preferably, the crosslinkable derivative of aldehyde-terminated polyethylene glycol is a four-arm polyethylene glycol aldehyde (4-arm-PEG-CHO).

Further preferably, the molecular weight of the star-shaped multi-arm polyethylene glycol is not less than 2000.

Further preferably, the polymer compound having an amino group is carboxymethyl chitosan.

The invention also aims to provide a preparation method of the injectable antibacterial hemostatic hydrogel adhesive, which comprises the following steps:

s1, dissolving carboxymethyl chitosan in PBS buffer solution to obtain carboxymethyl chitosan solution for later use;

s2, synthesizing a four-arm polyethylene glycol aldehyde group, and dissolving the four-arm polyethylene glycol aldehyde group in a PBS buffer solution to obtain a four-arm polyethylene glycol aldehyde group solution;

s3, quickly mixing the carboxymethyl chitosan solution obtained in the step S1 and the four-arm polyethylene glycol aldehyde solution obtained in the step S2 in equal volume to obtain the injectable antibacterial hemostatic hydrogel adhesive.

Preferably, the weight percentage content W of the carboxymethyl chitosan in the carboxymethyl chitosan solution ₁1 to 15 percent. Further preferably, the weight percentage of the carboxymethyl chitosan in the carboxymethyl chitosan solution is 2% -15%. Particularly preferably, the carboxymethyl chitosan solution has a weight percentage of 5% -10%.

Preferably, the weight percentage content W of the four-arm polyethylene glycol aldehyde group in the four-arm polyethylene glycol aldehyde group solution₂Is 1 to 20 percent. Further preferably, the weight percentage of the four-arm polyethylene glycol aldehyde group in the four-arm polyethylene glycol aldehyde group solution is 5% -20%. Particularly preferably, the weight percentage of the four-arm polyethylene glycol aldehyde group in the four-arm polyethylene glycol aldehyde group solution is 12-15%.

Preferably, the weight percentage of the four-arm polyethylene glycol aldehyde group solution to the weight percentage of the carboxymethyl chitosan solution (W)₁/W₂) Is 14:1-1: 14. Further preferably, the weight percentage of the four-arm polyethylene glycol aldehyde group solution to the weight percentage of the carboxymethyl chitosan solution (W)₁/W₂) Is 10: 5.

Preferably, the PBS buffer has a pH of 7.4.

Preferably, the method for synthesizing the four-arm polyethylene glycol aldehyde group specifically comprises the following steps:

(1) sequentially adding 4-carboxybenzaldehyde, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 4-dimethylaminopyridine and four-arm polyethylene glycol (4-arm-PEG-OH) into a dry solvent under an inert gas atmosphere, and stirring at 20-40 ℃ for 12-36 h; the molar ratio of the four-arm polyethylene glycol (4-arm-PEG-OH) to the 4-carboxybenzaldehyde is 1 (1-10);

(2) filtering, washing the solid, and vacuum drying to obtain white solid powder.

Further preferably, the molar ratio of 4-carboxybenzaldehyde, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and 4-dimethylaminopyridine in step (1) is 2:2: 1.

Further preferably, the reaction temperature is 37 ℃, and the reaction time is 18-24 hours, more preferably 24 hours; when washing, the mixture is sequentially washed with 1M HCl solution and saturated NaHCO₃The solution was washed with brine.

The invention also aims to provide the application of the injectable antibacterial hemostatic hydrogel adhesive in wound tissue sealing and leakage prevention, tissue adhesion prevention, tissue filling, tissue repair and regeneration, preparation of antibacterial skin dressing, antibacterial medicinal preparation, medicament delivery, cell encapsulation and delivery, tissue engineering scaffold and 3D printing ink.

The invention has the beneficial effects that:

1. the injectable antibacterial hemostatic hydrogel adhesive provided by the invention integrates a plurality of excellent functions and biological activities of various raw materials, not only can provide an ideal microenvironment for growth, proliferation and differentiation of cells, but also has good antibacterial activity.

2. The injectable hydrogel adhesive also has strong adhesion, self-repairing performance and strong mechanical performance. Therefore, the injectable antibacterial hemostatic hydrogel adhesive provided by the invention can be applied to wound tissue sealing and leakage prevention, tissue filling, tissue repair and regeneration, preparation of antibacterial skin dressing, antibacterial drug preparation, drug delivery, cell encapsulation and delivery, tissue engineering scaffold and 3D printing ink.

3. In the preparation method of the injectable antibacterial hemostatic hydrogel adhesive, the 4-arm-PEG-CHO solution and the CMCS solution are injected into a mold through a double-syringe to obtain the injectable antibacterial hemostatic hydrogel adhesive.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a schematic diagram of an injectable hydrogel adhesive gel formulation provided by an embodiment of the present invention;

FIG. 2 is a rheological test of an injectable hydrogel adhesive provided by an embodiment of the present invention;

FIG. 3 is an electron microscope image of an injectable hydrogel adhesive provided in an embodiment of the present invention;

FIG. 4 is an infrared spectrum of an injectable hydrogel adhesive and components provided in accordance with an embodiment of the present invention;

FIG. 5A is a graph illustrating the self-healing effect of injectable hydrogel adhesives provided by embodiments of the present invention;

FIG. 5B is a rheological characterization of injectable hydrogel adhesive self-healing provided by embodiments of the present invention;

FIG. 6 is a swelling ratio measurement of injectable hydrogel adhesives provided in accordance with an embodiment of the present invention;

FIG. 7 is a shear adhesion measurement of an injectable hydrogel adhesive provided in accordance with an embodiment of the present invention on pig skin;

FIG. 8 is a burst pressure measurement of an injectable hydrogel adhesive for pigskin incisions provided in accordance with an embodiment of the present invention;

FIG. 9A is a graph showing the growth of stained cells upon evaluation of cytotoxicity of an injectable hydrogel adhesive provided in an embodiment of the invention;

FIG. 9B is a graph showing the proliferation of cells during cytotoxicity assessment of an injectable hydrogel adhesive according to an embodiment of the present invention;

FIG. 10 shows the surface bacteriostatic properties of an injectable hydrogel adhesive material according to an embodiment of the present invention;

fig. 11 shows the hemostatic properties of an injectable hydrogel adhesive material according to an embodiment of the present invention.

Detailed Description

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

The invention provides an injectable antibacterial hemostatic hydrogel adhesive, which can be obtained by crosslinking a crosslinkable derivative of aldehyde-terminated polyethylene glycol and a macromolecular compound with amino.

Specifically, the crosslinkable derivative of polyethylene glycol is one or a mixture of two of star-shaped multi-arm polyethylene glycol and PF 127.

Specifically, the macromolecular compound with amino is one or a mixture of more than two of water-soluble carboxymethyl chitosan derivative, Polyethyleneimine (PEI) and amino derivative of star-shaped multi-arm polyethylene glycol.

As a preferred embodiment, the star-shaped multi-arm polyethylene glycol has from 2 to 8 arms; the molecular weight of the star-shaped multi-arm polyethylene glycol is not less than 2000.

As a particularly preferred embodiment, the crosslinkable derivative of aldehyde-terminated polyethylene glycol is a four-armed polyethylene glycol aldehyde (4-arm-PEG-CHO).

In a preferred embodiment, the polymer compound having an amino group is carboxymethyl chitosan.

The aldehyde-terminated star-shaped multi-arm polyethylene glycol used in the present invention can be purchased commercially or synthesized synthetically. During synthesis, the method for synthesizing the four-arm polyethylene glycol aldehyde group specifically comprises the following steps:

(1) sequentially adding 4-carboxybenzaldehyde, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and 4-dimethylaminopyridine into a dry solvent under an inert gas atmosphere, adding 0.5-5g of four-arm polyethylene glycol (4-arm-PEG-OH), and stirring at 20-40 ℃ for 12-36 h;

(2) filtering, washing the solid, and vacuum drying to obtain white solid powder.

For an example of the synthesis of 4-arm-PEG-CHO, the synthesis method is described in the literature (Biomacromolecules,2011,12, 2894-2901). The specific synthesis method comprises the following steps: 4-carboxybenzaldehyde 1-ethyl-3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) were dissolved in dry dichloromethane under nitrogen and 4-arm-PEG-OH was added. After the system reacts for 24 hours at 37 ℃, the reaction mixed liquid is sequentially added with 1M HCl and saturated NaHCO₃And washing with saturated brine, standing, and vacuum drying to obtain white solid powder.

4-arm-PEG-CHO is a high quality multi-arm PEG derivative, modified at each end of the four arms with an aldehyde functional group, which can form with amino and protein imines containing a C ═ N double bond. The material has good biocompatibility and tissue adhesion. CMCS contains a large number of amino functional groups in its structure, and thus has an antibacterial property. The two are combined according to the optimal proportion, so that the material has the characteristics of injectability, self-repairing and the like, has the functions of quickly stopping bleeding and preventing infection, and has higher mechanical strength and adhesiveness.

The polyethylene glycol (PEG) mentioned in the present invention is represented by the general formula H- (O-CH)₂-CH₂)_n-OH (CAS No: 25322-68-3).

The PBS buffer was phosphate buffered saline.

The present invention will be further described with reference to specific examples and comparative examples.