阅读说明：本技术 一种pH和温度双响应UV交联壳聚糖可注射水凝胶的制备方法 (Preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel ) 是由 李保强 丁海昌 冯玉杰 贾德昌 周玉 于 2020-06-04 设计创作，主要内容包括：本发明涉及一种pH和温度双响应的UV交联壳聚糖可注射水凝胶的制备方法,属于生物医用水凝胶领域。本发明首先分别合成pH响应的烯丙基壳聚糖(OAL-CS)和温度响应性的巯基改性聚异丙基丙烯酰胺(HS-PNIPAM),然后基于巯基-烯点击化学能够在UV辐照下可将含有光引发剂的OAL-CS/HS-PNIPAM溶液快速转化为水凝胶；通过调整OAL-CS/HS-PNIPAM质量比,可实现控制水凝胶的pH和温度响应性溶胀大小。本发明不但实现了OAL-CS/HS-PNIPAM溶液在UV辐照下快速形成凝胶,而且赋予UV交联壳聚糖可注射水凝胶的溶胀行为具有pH和温度的智能响应性；由于该水凝胶体系可快速UV固化,可以通过UV固化或UV光刻技术制备用于组织工程、药物释放和创面修复等领域的快速成型的水凝胶。(The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, belonging to the field of biomedical hydrogel. Firstly, respectively synthesizing pH-responsive allyl chitosan (OAL-CS) and temperature-responsive sulfydryl-modified polyisopropylacrylamide (HS-PNIPAM), and then quickly converting an OAL-CS/HS-PNIPAM solution containing a photoinitiator into hydrogel under UV irradiation based on sulfydryl-alkene click chemistry; by adjusting the mass ratio of OAL-CS/HS-PNIPAM, the control of the pH and temperature response swelling size of the hydrogel can be realized. The invention not only realizes that the OAL-CS/HS-PNIPAM solution quickly forms gel under UV irradiation, but also endows the swelling behavior of the UV cross-linked chitosan injectable hydrogel with intelligent responsiveness of pH and temperature; because the hydrogel system can be rapidly UV cured, the rapidly formed hydrogel used in the fields of tissue engineering, drug release, wound repair and the like can be prepared by UV curing or UV photoetching technology.)

1. A pH and temperature double-response UV cross-linked chitosan injectable hydrogel is characterized in that the pH and temperature double-response UV cross-linked chitosan injectable hydrogel is O-allyl chitosan (OAL-CS) and sulfydryl PNIPAM (HS-PNIPAM), and the O-allyl chitosan and the sulfydryl PNIPAM have the following structural formula (I).

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2. A method for preparing the pH and temperature dual-responsive UV cross-linked chitosan injectable hydrogel according to claim 1, which comprises the following specific steps:

the method comprises the following steps: synthesizing O-allyl chitosan (OAL-CS) with pH response by using an amino protection strategy for chitosan; synthesizing PNIPAM with temperature response by utilizing a reversible addition-fragmentation chain transfer polymerization technology, and then synthesizing sulfydryl PNIPAM (HS-PNIPAM) with temperature response through an esterification reaction;

step two: dissolving the two responsive substances in deionized water, normal saline or a cell culture medium to prepare an OAL-CS/HS-PNIPAM solution, wherein the mass ratio of the OAL-CS/HS-PNIPAM is 2-6; UV is adopted to irradiate OAL-CS/HS-PNIPAM solution containing photoinitiator Irgacure2959 for a certain time to obtain the UV-cured double-response hydrogel.

3. The preparation method of the pH and temperature dual-response UV cross-linked chitosan injectable hydrogel according to claim 2, wherein the pH and temperature dual-response UV cross-linked chitosan injectable hydrogel OAL-CS/HS-PNIPAM can be rapidly cured to form hydrogel in 10-50 seconds under UV irradiation, and the swelling behavior of the hydrogel has pH and temperature responsiveness.

4. The preparation method of the pH and temperature dual-responsive UV cross-linked chitosan injectable hydrogel according to claim 2, wherein the concentration of the photoinitiator Irgacure2959 is 0.05% (w/v); the wavelength of the UV is 360-480 nm, and the UV irradiance is 4mW/cm²And the irradiation time is 5-10 seconds.

5. The preparation method of the pH and temperature dual-response UV cross-linked chitosan injectable hydrogel according to claim 2, wherein the concentration of the prepared O-allyl chitosan aqueous solution is 20-25 mg/mL, and the concentration of the sulfhydryl group PNIPAM is 4-10 mg/mL.

6. The method for preparing the pH and temperature dual-responsive UV cross-linked chitosan injectable hydrogel according to claim 2, wherein the O-allyl chitosan is synthesized with NaOH as a catalyst at a concentration of 0.5%.

7. The method for preparing the pH and temperature dual-responsive UV cross-linked chitosan injectable hydrogel according to claim 2, wherein the sulfhydryl PNIPAM uses DMF and H in a solvent₂The volume ratio of O is 1: 1, the reaction time is 3 days at normal temperature.

Technical Field

The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, belonging to the field of biomedical hydrogel.

Background

A smart responsive hydrogel refers to a hydrogel that undergoes a significant change in some properties, including volume, osmotic pressure, phase, etc., upon exposure to an external stimulus, such as temperature, pH, light, electric field, magnetic field, oxidizing/reducing agents, etc. These property changes can be controlled, giving intelligent hydrogels a wide range of demanding applications. Among these external stimuli, pH and temperature are the most easily regulated changes of the human environment and are closely related to the physiological activities of the human body. It is reported that almost all pH and temperature responsive hydrogels are synthetic macromolecules and are not biodegradable, which cannot meet the requirement of in vivo biodegradability. Therefore, there is a need to develop a biodegradable pH and temperature dual-responsive hydrogel based on natural products.

The chitosan is the only alkalescent natural polysaccharide with amino in nature, and can be applied to the fields of drug release, tissue engineering, wound dressing and the like due to good biocompatibility and biodegradability. However, the chitosan is not dissolved in water, physiological saline or aqueous medium with neutral pH such as simulated body fluid and the like due to the strong hydrogen bond action in molecules and among molecules. Amino groups in chitosan molecules endow the chitosan with pH responsiveness and biological activity, but the chitosan itself lacks temperature responsiveness, and temperature responsive groups need to be introduced into the chitosan molecules. At present, temperature-responsive polyisopropylacrylamide is subjected to free radical polymerization to introduce chitosan molecules, and the temperature-sensitive chitosan hydrogel is successfully prepared.

However, the above method for synthesizing the UV curable chitosan derivative still has the following problems: (1) the grafting rate of the temperature-responsive polyisopropylacrylamide in the chitosan molecule is difficult to control; (2) the pH responsiveness of the chitosan hydrogel is lost. The invention synthesizes a hydrogel system which can be rapidly cured by UV and can be dissolved in water and has pH and temperature double responses, wherein O-allyl chitosan provides pH responsiveness, and sulfhydryl PNIPAM provides temperature responsiveness. After the two substances are mixed, the hydrogel can be rapidly cured under UV irradiation, and the volume of the gel can be reversibly changed. The system can be rapidly UV cured, so that hydrogel (in the size range of micron to centimeter) with any shape and size can be prepared by UV photoetching or UV curing technology, and the system has potential application in the fields of tissue engineering scaffolds, drug release, wound repair and the like.

Disclosure of Invention

The present invention is directed to the problem of prior art UV curable chitosan hydrogels that do not have intelligence, such as pH and temperature responsiveness. Synthesizing chitosan having pH responsiveness based on pH responsiveness possessed by chitosan when the chitosan contains a large number of amino groups; synthesizing a sulfydryl PNIPAM with temperature responsiveness based on the temperature responsiveness of the PNIPAM; the two substance solutions can be used for rapidly preparing the dual-response hydrogel (20-60 seconds) during UV curing through sulfydryl-alkene click chemistry.

The pH and temperature double-response UV cross-linked chitosan injectable hydrogel provided by the invention is O-allyl chitosan (OAL-CS) and sulfydryl PNIPAM (HS-PNIPAM), wherein the O-allyl chitosan and the sulfydryl PNIPAM have the following structural formula (I).

The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, which comprises the following specific steps:

the method comprises the following steps: synthesizing O-allyl chitosan (OAL-CS) with pH response by using an amino protection strategy for chitosan; synthesizing PNIPAM with temperature response by utilizing a reversible addition-fragmentation chain transfer polymerization (RAFT) technology, and then synthesizing sulfydryl PNIPAM (HS-PNIPAM) with temperature response through an esterification reaction;

step two: dissolving the two responsive substances in deionized water, normal saline or a cell culture medium to prepare an OAL-CS/HS-PNIPAM solution, wherein the mass ratio of the OAL-CS/HS-PNIPAM is 2-6; UV is adopted to irradiate OAL-CS/HS-PNIPAM mixed solution containing photoinitiator Irgacure2959 for a certain time to obtain UV cured double-responsiveness hydrogel;

the invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, which is OAL-CS/HS-PNIPAM capable of being irradiated under UV (the wavelength is 360-480 nm, and the UV irradiance is 4 mW/cm)²) The hydrogel is rapidly cured in 10-50 seconds to form the hydrogel, and the swelling behavior of the hydrogel has pH and temperature responsiveness.

The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, wherein the concentration of a photoinitiator Irgacure2959 is 0.05% (w/v); the wavelength of the UV is 360-480 nm, and the UV irradiance is 4mW/cm²And the irradiation time is 5-10 seconds.

The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, wherein the concentration of the prepared O-allyl chitosan aqueous solution is 20-25 mg/mL, and the concentration of sulfydryl PNIPAM is 4-10 mg/mL.

The invention relates to a preparation method of UV cross-linked chitosan injectable hydrogel with pH and temperature dual responses, wherein during the synthesis of O-allyl chitosan, the concentration of NaOH as a catalyst is 0.5%.

The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, wherein the solvent used by sulfydryl PNIPAM is DMF: h₂O is 1: 1, and the reaction time is 3 days at normal temperature.

The invention relates to a preparation method of pH and temperature dual-response UV cross-linked chitosan injectable hydrogel, which synthesizes pH-response chitosan and temperature-response PNIPAM, can maintain the pH responsiveness of chitosan by reserving a large amount of amino groups in the chitosan, and simultaneously, allyl groups on molecules provide cross-linkable performance; the sulfydryl PNIPAM not only provides temperature responsiveness, but also provides crosslinkable performance through the sulfydryl groups on molecules; irradiating the mixed aqueous solution of the two substances containing the photoinitiator by using UV (ultraviolet), and obtaining the chitosan hydrogel within a short irradiation time (5-10 seconds); the system has controllable structure, simple and convenient gel operation, and adjustable swelling ratio of gel.

Drawings

FIG. 1 shows a structural formula of a method for preparing a pH and temperature dual-responsive UV cross-linked chitosan injectable hydrogel.

FIG. 2 shows the reaction of chitosan with O-allyl chitosan in example¹H NMR spectrum.

FIG. 3 is an FTIR spectrum of chitosan and O-allyl chitosan in example

FIG. 4 shows PNIPAM and mercapto PNIPAM in examples¹H NMR spectrum.

Fig. 5 shows swelling control of the pH and temperature dual-responsive hydrogels of the examples, (a) swelling ratio at different temperatures at pH 7.2, and (B) swelling ratio at different pH conditions at 37 ℃.

Detailed Description