CN113350265A

CN113350265A - Celecoxib-loaded injectable temperature-sensitive chitosan hydrogel, preparation method and application thereof in treatment of intervertebral disc degeneration

Info

Publication number: CN113350265A
Application number: CN202110479390.5A
Authority: CN
Inventors: 西永明; 杜钰堃; 李建毅; 隋坤艳
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-09-07

Abstract

The invention discloses an injectable temperature-sensitive chitosan hydrogel loaded with celecoxib, a preparation method and application thereof in treatment of intervertebral disc degeneration, wherein the hydrogel comprises celecoxib, chitosan and beta-sodium glycerophosphate, wherein the chitosan and the beta-sodium glycerophosphate are crosslinked into an interpenetrating network, and the celecoxib is uniformly loaded in the interpenetrating network; the celecoxib-loaded temperature-sensitive injectable chitosan hydrogel has good biocompatibility, can be implanted into a body in an injection mode, has a temperature-sensitive characteristic, can be converted into gel in situ in injection in a short time, can effectively block local defect of a fibrous ring, relieve local inflammation, maintain mechanical stability of an intervertebral disc and further delay degeneration of the intervertebral disc; the composite hydrogel material provides a new treatment prospect for further developing intervertebral disc regeneration and repair materials in the future, preventing the re-protrusion of intervertebral discs and delaying the degeneration of intervertebral discs.

Description

Celecoxib-loaded injectable temperature-sensitive chitosan hydrogel, preparation method and application thereof in treatment of intervertebral disc degeneration

Technical Field

The invention relates to the field of medicines, in particular to an injectable temperature-sensitive chitosan hydrogel loaded with celecoxib, a preparation method and application thereof in treatment of intervertebral disc degeneration.

Background

Intervertebral Disc Degeneration (IDD) is the pathological basis for a variety of spinal diseases, such as lumbar disc herniation, cervical spondylosis and lumbar spinal stenosis, and is also the main cause of lumbago and leg pain. The causes of IDD are numerous, such as trauma, inflammation, long-term shock and immunity. Statistically, the population suffering from lumbago in the world has reached 6.5 hundred million, and lumbago has become one of the main causes of disability of patients. It is widely believed that disc degeneration is mainly caused by the decrease of Proteoglycan (PG) and type II collagen in nucleus pulposus, which causes the change of nucleus pulposus water decrease, disc nutrition supply disorder, etc., and further causes rupture of the annulus fibrosus, so that nucleus pulposus tissue flows out from the rupture port on the annulus fibrosus, which causes the compression of spinal cord and nerve root, and the protruded nucleus pulposus tissue as foreign body in vivo can cause various immune inflammatory reactions, causing different degrees and parts of lumbocrural pain symptoms of patients. With the recent intensive research at home and abroad, many factors are related to IDD, such as inflammatory factors, oxidative stress, apoptosis, intervertebral disc nutrition pathways, tissue inhibitors of metalloproteases and the like. The inflammatory reaction induced by the tissue of the herniated nucleus pulposus is an important factor of IDD, and related research mainly focuses on inflammatory factors such as Interleukin (IL), tumor necrosis factor alpha (TNF- α), prostaglandin E2(PGE2), Nitric Oxide Synthase (NOS), and the like, because various inflammatory factors are always throughout the generation and development process of IDD. The current treatment for IDD mainly comprises drug treatment, minimally invasive treatment, open surgery treatment and the like. However, conservative treatment and surgical treatment can only improve the symptoms of patients, and both can not effectively prevent and reverse the development of intervertebral disc degeneration, so that at present, domestic and foreign research is widely and deeply conducted around how to fundamentally inhibit the development of IDD.

At present, after the lumbar intervertebral disc protrusion is removed in the operation treatment, the intervertebral disc fibrosis is destroyed, the intervertebral disc of a postoperative patient is not blocked by effective materials, the inner nucleus pulposus has the risk of re-protrusion, the local inflammation is aggravated, the nerve root is stimulated to cause the nerve root pain again, and in addition, after the intervertebral disc is destroyed, the intervertebral disc is accelerated to degenerate to enable the intervertebral disc to lose the original functions.

Chinese patent CN108606969A discloses application of Aspirin in preparation of a medicament for treating intervertebral disc degeneration, wherein an intervertebral disc degeneration model is constructed by applying LPS to induce oxidative stress, the treatment effect of Aspirin on intervertebral disc degeneration is observed, and the treatment effect of Aspirin on intervertebral disc degeneration is evaluated by observing various indexes of oxidative stress and intervertebral disc degeneration. Western blot proves that the Aspirin reduces oxidative stress and relieves the brief mechanism of intervertebral disc degeneration. Chinese patent CN110279654A discloses a slow-release aspirin liposome hydrogel, a preparation method thereof and application thereof in medicines for treating recurrent lumbar disc herniation, wherein the hydrogel is adopted to realize the slow-release effect of aspirin, reduce inflammatory reaction, repair defective fibrous rings and delay intervertebral disc degeneration. However, this patent only teaches that aspirin can be coated onto the hydrogel.

Celecoxib is commonly used for relieving symptoms and signs of osteoarthritis, relieving symptoms and signs of adult rheumatoid arthritis and treating acute adult pain. The inventor tries to coat celecoxib by adopting the method of the patent CN110279654A, however, the liposome hydrogel formed after the aspirin is replaced by the celecoxib is not suitable for treating the intervertebral disc degeneration no matter in the aspects of slow release performance, biocompatibility or other characteristics.

Disclosure of Invention

The invention aims to provide an injectable temperature-sensitive chitosan hydrogel loaded with celecoxib, a preparation method and application thereof in treatment of intervertebral disc degeneration, so as to solve the problems in the prior art.

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

the invention provides an injectable temperature-sensitive chitosan hydrogel loaded with celecoxib, which comprises celecoxib, chitosan and beta-sodium glycerophosphate, wherein the chitosan and the beta-sodium glycerophosphate are crosslinked into an interpenetrating network, and the celecoxib is uniformly loaded in the interpenetrating network.

Further, the degree of deacetylation of the chitosan was 95%.

The invention also provides a preparation method of the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel, which comprises the following steps:

(1) dissolving celecoxib in water to obtain a celecoxib solution, dissolving chitosan in dilute acid to obtain a chitosan solution, and dissolving beta-sodium glycerophosphate in water to obtain a beta-sodium glycerophosphate solution;

(2) under the condition of continuous stirring, dropwise adding the celecoxib solution into the chitosan solution, and continuously stirring to obtain a celecoxib-chitosan mixed solution;

(3) and (3) dropwise adding the beta-sodium glycerophosphate solution into the celecoxib-chitosan mixed solution to obtain the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel.

Further, the dilute acid is dilute hydrochloric acid or dilute acetic acid, and the concentration is 0.01 mol/L.

Further, the concentration of the celecoxib solution is 56 g: 100mL, the concentration of the chitosan solution is (2-5) g: 100mL, wherein the concentration of the beta-sodium glycerophosphate solution is 2 g: 100 mL.

Further, in the step (2), the volume ratio of the celecoxib solution to the chitosan solution is 1-1.5: 9; in the step (3), the volume ratio of the beta-sodium glycerophosphate solution to the celecoxib-chitosan mixed solution is 1: 10-10.5.

The invention also provides application of the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel in preparation of a medicament for treating intervertebral disc degeneration.

The invention also provides a pharmaceutical preparation for treating intervertebral disc degeneration, which comprises an effective treatment amount of the injectable temperature-sensitive chitosan hydrogel loaded with celecoxib and pharmaceutically acceptable auxiliary materials.

Further, the mass content of the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel is 1-99%.

Furthermore, the weight content of the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel is 10-90%.

The invention discloses the following technical effects:

the anti-inflammatory drug celecoxib is added into the chitosan hydrogel, so that the hydrogel has a certain local anti-inflammatory capacity aiming at solving the problem of inflammatory reaction generated locally on intervertebral discs after spinal surgery, and meanwhile, the hydrogel can provide a proper microenvironment for cell proliferation and differentiation. The research of the invention finds that the chitosan hydrogel for repairing the intervertebral disc needs to have the following characteristics: appropriate pH: the pH value of the chitosan hydrogel can be changed along with the addition of the proportion of beta-sodium glycerophosphate (beta-GP), and the pH value of the hydrogel can be close to neutrality by adjusting the proportion of the beta-GP, which is beneficial to the growth and survival of nucleus pulposus cells in a hydrogel compound; stable gel form and proper mechanical properties; (iii) lower toxicity: the beta-GP has certain toxicity, and the higher concentration of the beta-GP can change the normal physiological osmotic pressure to cause cell dehydration, thereby inhibiting the cell growth and even causing cell death. The beta-GP concentration applied by the invention is slightly higher, but the use content is lower, so the cytotoxicity is better controlled, and the later cell compatibility experiment also proves the point. Fourthly, the ideal gelation time: to achieve good impregnation of the hydrogel into the injection site, the hydrogel should be in a slightly viscous liquid state at room temperature, and when injected into the body, gelation should be achieved within a relatively short time when the external temperature is normal temperature in the body (i.e., about 37 ℃), so that local defects are blocked, and the gelation time cannot be too long. The hydrogel prepared by the invention has the gelation time of about 10 minutes, and can better meet the local plugging effect required in clinical operation.

According to the invention, the CCK-8 and Hoechst staining experiments are used for detecting the cytotoxicity and biocompatibility of the prepared celecoxib-loaded temperature-sensitive chitosan hydrogel, and compared with a control group, the composite hydrogel does not have behavior of obviously inhibiting cell proliferation and growth. In addition, the prepared composite hydrogel does not cause obvious inflammatory reaction of surrounding tissues after being implanted under the skin of New Zealand white rabbits for histocompatibility detection, the stepwise metabolism disappears in vivo along with the prolonging of time, and the metabolite does not cause obvious inflammatory reaction. Meanwhile, the observation result of the animal histocompatibility experiment shows that the celecoxib-loaded chitosan hydrogel can exist in vivo for a long time, so that the local blocking and filling of the fibrous ring incision of the intervertebral disc are facilitated, and the local inflammation is controlled for a long time.

The invention successfully prepares a new Zealand white rabbit intervertebral disc degeneration model by utilizing a method of sucking nucleus pulposus through acupuncture. After 2 weeks, 1 month and 2 months of operation, the imaging examination of the discs of the new zealand white rabbits by X-ray and MRI shows that the intervertebral space height and the disc degeneration grade of the needle-prick degeneration group (L3/4) are obviously reduced compared with the normal control group (L2/3), and the difference has statistical significance (P is less than 0.05); the hydrogel group and the celecoxib-loaded hydrogel group (L4/5 and L5/6) have the reduction of the intervertebral space height and the degenerative disc grade, but the reduction range is smaller than that of the acupuncture degenerative group (L3/4), and the statistical significance (P <0.05) exists, so the hydrogel group and the celecoxib-loaded hydrogel group also have the important significance for treating the degenerative disc disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a photograph showing chitosan hydrogel appearance with different molecular mass and different concentration;

FIG. 2 shows the general structure (a) of the temperature-sensitive chitosan hydrogel and the general structure (b) of the temperature-sensitive chitosan hydrogel loaded with celecoxib;

FIG. 3 is an electron microscope image (a) of chitosan hydrogel with the same molecular mass and different concentrations and an electron microscope image (b) of celecoxib-loaded chitosan hydrogel (the arrow is celecoxib);

FIG. 4 is EDS (EDS Spectroscopy) data analysis of celecoxib-loaded chitosan hydrogel;

fig. 5 is the liquid-solid gel transition time of the hydrogel (# ═ P <0.05, n ═ 5);

FIG. 6 shows the cell proliferation measured on

days

1, 3 and 5 using CCK-8;

FIG. 7 shows the cell proliferation measured by the Hoechst staining test for 1, 3 and 5 days, in which panel a is a control group, panel b is a hydrogel group, and panel c is a celecoxib-loaded hydrogel group;

fig. 8 is X-ray results 2 weeks, 1 month and 2 months after surgery (#, # ═ P <0.05, n ═ 4);

fig. 9 is MRI T2 weighted image (a) and disc Thompson score (b) at 2 weeks, 1 month and 2 months post-surgery (P <0.05, n-4);

FIG. 10 is a postoperative HE staining of intervertebral discs, wherein A is a normal control group; b is a degeneration group; c is a chitosan hydrogel group; d is a celecoxib-loaded hydrogel group;

figure 11 is postoperative disc HE staining score.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the present invention, which should not be construed as limiting the invention but rather as providing a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 preparation and characterization of celecoxib loaded injectable temperature sensitive chitosan hydrogel

1. Preparation of celecoxib-loaded injectable temperature-sensitive chitosan hydrogel

1.1 preparation of related raw materials

(1) Preparation of 2%, 3% and 5% (W/V) Chitosan solutions

Respectively weighing 2g, 3g and 5g of chitosan (degree of deacetylation is 95%), respectively dissolving in 0.1mol of dilute hydrochloric acid, fully stirring at room temperature for about 6-10 hours (along with the increase of concentration, the required stirring time is prolonged) to respectively prepare chitosan solutions with the total amount of 2%, 3% and 5% of 100ml, subpackaging, sterilizing at 121 ℃ for about 15-20 minutes, and storing in a refrigerator at 4 ℃ for later use.

(2) Preparation of 56% (W/V) beta-sodium glycerophosphate solution

Weighing 5.6g of beta-sodium glycerophosphate powder, taking 10ml of ultrapure water, fully stirring at room temperature for about 2 hours to completely dissolve the powder to obtain 56% of beta-sodium glycerophosphate solution, filtering and sterilizing by using a filter membrane (22 mu m), and placing the solution in a refrigerator at 4 ℃ for later use.

(3) Preparation of 2% celecoxib solution

Weighing 1g of celecoxib powder, taking 50ml of ultrapure water, fully stirring for about 2 hours at room temperature to completely dissolve the celecoxib powder to obtain a 2% celecoxib solution, sterilizing for about 2 hours by using ultraviolet rays, and then placing the celecoxib solution in a refrigerator at 4 ℃ for later use.

1.2 preparation Process of injectable temperature-sensitive Chitosan hydrogel

1ml of 56% sodium beta-glycerophosphate solution is respectively dropwise added into 9ml of 2%, 3% and 5% chitosan solutions, the whole process is preferably carried out under slow stirring, the duration is about 10-15min, the two solutions are completely mixed and dissolved to obtain temperature-sensitive liquid chitosan hydrogel (pH is 6.9-7.4) with different concentrations, then the temperature-sensitive liquid chitosan hydrogel is respectively injected into 15ml sample retention bottles and placed in a constant temperature box at 37 ℃ for about 8-10min to form the solid chitosan hydrogel.

1.3 preparation Process of celecoxib-loaded injectable temperature-sensitive Chitosan hydrogel

1-1.5ml of 2% celecoxib solution is dropwise added into 9ml of 2% chitosan solution, the whole process is carried out under continuous stirring, the celecoxib is completely dissolved in the 2% chitosan solution after stirring for 2h to obtain milk-white celecoxib-chitosan mixed solution, 1ml of 56% beta-sodium glycerophosphate solution is dropwise added into the celecoxib-chitosan mixed solution to obtain the temperature-sensitive celecoxib-chitosan hydrogel, the hydrogel is in a liquid state at room temperature and room temperature, the pH value is about 7.3, the hydrogel is injected into a 15ml sample retention bottle, and the sample retention bottle is placed in a constant temperature box at 37 ℃ for about 8-10min to form the milk-white solid chitosan hydrogel.

2. Characteristics of celecoxib-loaded injectable temperature-sensitive chitosan hydrogel

2.1 scanning Electron microscopy

(1) Rinsing chitosan hydrogel with different concentrations for 2-3 times with 4 deg.C pre-cooled 0.01M PBS solution, each time for 5 min;

(2) fixing chitosan with 3% glutaraldehyde for 2h, sucking off the fixing solution, and soaking and rinsing the fixing solution with 0.01M PBS for 10-15min for 2 times;

(3) alcohol dehydration: after rinsing, the sample is dehydrated by alcohol or acetone with gradually increasing concentration, and then enters an intermediate solution, generally isoamyl acetate is used as the intermediate solution.

(4) Drying and spraying gold: placing the hydrogel specimen in a freeze dryer, freeze-drying, and spraying gold for coating;

(5) and (3) observing by an electron microscope: placing different samples on a scanning electron microscope for observation and shooting;

2.2 Transmission Electron microscopy EDS energy Spectroscopy data analysis

The element types and contents of the material domain components can be analyzed by Energy Dispersive Spectrometer (EDS). The component of the celecoxib is C₁₇H₁₄F₃N₃O₂S, the element of the chitosan is formed into C₆H₁₂NO₄And the elemental composition of sodium beta-glycerophosphate is C₃H₁₅Na₂O₁₀P, therefore, the supported celecoxib chitosan hydrogel is subjected to EDS (electronic Desorption) energy spectrum data analysis to detect F and S elements, and the distribution of the celecoxib in the chitosan hydrogel can be qualitatively and quantitatively observed.

2.3 injectable Studies of Chitosan hydrogel and Selenib-loaded Chitosan hydrogel

About 5ml of liquid 2% temperature-sensitive chitosan hydrogel and celecoxib-loaded temperature-sensitive chitosan hydrogel are respectively extracted by a 10ml syringe, and are ejected by a proper force through a 10ml syringe needle to simulate the in-vivo injection process of the hydrogel.

2.4 study of temperature sensitivity of Chitosan hydrogel and Selenib-loaded Chitosan hydrogel

Respectively extracting 2% of temperature-sensitive chitosan hydrogel and about 3ml of celecoxib-loaded chitosan hydrogel, respectively placing the two samples in 10ml round-cover sample test tubes, respectively taking 5 parts of the samples, placing the samples in an incubator at 37 ℃, approximately observing the gelation conversion condition through glass on the incubator, taking the samples out at intervals of about 1min, measuring the liquid-solid conversion time of the hydrogel by using a test tube inversion method, and exploring the temperature-sensitive characteristic of the hydrogel.

3. Statistical analysis

The experimental result data in the experiment are analyzed by using SPSS 23.0 statistical software, single-factor variance analysis is adopted for multi-group comparison, independent sample t test is adopted for inter-group comparison, and the difference is considered to have statistical significance if p is less than 0.05.

4. Results

4.1 appearance of temperature-sensitive Chitosan hydrogel

As shown in fig. 1, the chitosan is white powder in appearance, the deacetylation degree is about 95%, the viscosity is about 25mpa.s, and the liquid chitosan hydrogel obtained after the chitosan is dissolved in dilute hydrochloric acid gradually becomes milky white with the increase of the concentration; the 56% aqueous solution of sodium beta-glycerophosphate is a colorless transparent liquid. After the beta-sodium glycerophosphate solution is gradually added into the chitosan solution, the obtained chitosan-beta-sodium glycerophosphate hydrogel compound is gradually changed into white liquid hydrogel, the pH value is about 7.0 and is close to the normal pH value of a human body, and a relatively ideal internal growth environment is provided for nucleus pulposus cells and the like. After being injected into a 15ml sample retention bottle, the sample retention bottle is placed in a thermostat with the temperature of 37 ℃ for about 8-15min, and can be coagulated into a solid state, has certain adhesiveness and is easy to adhere to surrounding tissues, but the viscosity is increased and the shearing stress is increased along with the increase of the concentration of chitosan, so that the higher the force required by injection is, the larger the size of the injector is, the less the force can be applied to facilitate the injection.

4.2 appearance morphology of celecoxib-loaded temperature-sensitive chitosan hydrogel

As shown in fig. 2, celecoxib is a white granular substance, is fully dissolved in ultrapure water to obtain a celecoxib solution with the concentration of 2%, a chitosan solution with the concentration of 2% is dropwise added, celecoxib is fully dissolved in chitosan to obtain a celecoxib-chitosan mixed hydrogel solution with uniform distribution, the celecoxib-chitosan mixed hydrogel solution is milk white in appearance, a temperature-sensitive chitosan hydrogel loaded with celecoxib in a liquid state is obtained after a 56% beta-sodium glycerophosphate solution is added, a 15ml sample retention bottle is filled, and the sample retention bottle is placed in a 37 ℃ thermostat for about 8-15min to be condensed into a solid state.

4.3 Transmission Electron microscopy of hydrogels

As shown in FIG. 3, when the chitosan hydrogel is observed by a transmission electron microscope, the chitosan hydrogel has a porous loose structure inside, and has vacuoles and different sizes. In addition, after the chitosan temperature-sensitive hydrogel with different concentrations forms solid gel, the observation of a scanning electron microscope shows that the pore size and the compactness of the gel are different, and the higher the concentration of the chitosan solution is, the smaller the pore size is, the thicker the pore wall is, and the higher the compactness is. For the temperature-sensitive chitosan hydrogel loaded with the celecoxib, as the celecoxib is fully mixed with the chitosan solution in advance, the celecoxib can be uniformly distributed in the interpenetrating network of the chitosan-beta-sodium glycerophosphate when severe dehydration reaction occurs when the temperature rises.

4.4 EDS (EDS) energy spectrum data analysis of celecoxib-loaded temperature-sensitive chitosan hydrogel

According to the experiment, after the elements F and S in the celecoxib-loaded temperature-sensitive chitosan hydrogel are analyzed and detected through EDS (enhanced dispersive Spectroscopy) energy spectrum data, as shown in figure 4, the elements F and S are uniformly distributed in the chitosan hydrogel, and the uniform distribution of celecoxib in the chitosan hydrogel can be indirectly proved, so that the celecoxib can be successfully loaded in the chitosan hydrogel prepared by the experiment.

4.5 Observation of injectability of hydrogels

In order to test the injectability of the hydrogel, after liquid hydrogels with different concentrations are extracted, the in vivo injection process is simulated through the syringe needle, the hydrogels with different concentrations can be successfully injected, but the shear stress of the chitosan hydrogel is increased along with the increase of the concentration of the chitosan hydrogel, the thrust required by injection is also continuously increased, and the injection pressure can be reduced by increasing the size of the syringe, so that the injection is convenient.

4.6 liquid-solid gel transformation analysis of temperature sensitive hydrogel

In order to test the temperature sensitivity of the hydrogel and evaluate the liquid-solid gel conversion capability of the hydrogel, the liquid-solid gel conversion capability of the temperature-sensitive chitosan hydrogel is tested by adopting a test tube inversion method. Through a multi-time point measurement method, the time required for temperature-sensitive chitosan hydrogel with different concentrations to be converted from a liquid state to a solid state is different. As shown in fig. 5: the liquid-solid gel conversion time of the 5 percent chitosan hydrogel is 8.40 +/-1.14 min, and the gel time is shortest; the liquid-solid gel transformation time of the 2% chitosan hydrogel and the 2% celecoxib-loaded chitosan hydrogel is 12.80 +/-1.48 min and 12.20 +/-0.84 min respectively, and the required time is longer than that of the 5% chitosan hydrogel and that of the 3% chitosan hydrogel. It can be seen that the higher the hydrogel concentration, the longer the time required for the liquid-solid gel to transform, but that the addition of celecoxib did not significantly affect the time to transform the liquid to solid state of the chitosan hydrogel.

Example 2 biocompatibility of celecoxib-loaded temperature-sensitive injectable chitosan hydrogel

CCK-8 cytotoxicity assays

Preparation of hydrogel samples

In order to detect the cell compatibility of the hydrogel sample, the cytotoxicity of the hydrogel sample is detected by using a CCK-8 method, firstly, the solid temperature-sensitive chitosan hydrogel and the temperature-sensitive chitosan hydrogel sample loaded with celecoxib are respectively prepared by the method in the embodiment 1, and the sample is sterilized for later use.

Hydrogel cytotoxicity assay grouping

Preparing the disinfected solid temperature-sensitive chitosan hydrogel and the temperature-sensitive chitosan hydrogel sample loaded with celecoxib into leaching solutions with different concentrations, and grouping the samples according to different leaching solution concentrations of different samples to obtain a cell control group, a leaching solution stock solution, a 50% leaching solution group and a 25% leaching solution group.

Experimental procedure

(1) Taking normally cultured L929 cells, absorbing original culture solution, adding PBS (phosphate buffer solution), washing, adding pancreatin for digesting for 3-5min, stopping digestion, blowing and beating into single cells by using a pipette gun, taking 20ul of cell suspension, adding 20ul of trypan blue for counting, centrifuging the rest cell suspension at 1000rpm for 5min, re-suspending by using a culture medium, plating by using 4000 cells/hole (96-hole plate), adding leaching liquor after the cells adhere to the wall, repeating every group for three times, and culturing by 5% CO2 at 37 ℃ overnight;

(2) add 10. mu.L of CCK-8 solution to each well. Incubating for 4 hours in a cell incubator, and adding a stop solution;

(3) and detecting the absorbance at 450nm by using a full-function microplate detector.

(4) Samples were tested for cytotoxicity on

days

1, 3 and 5 following the above procedure.

Hoechst staining experiment

2.1 Experimental groups

Referring to the CCK-8 toxicity result, 50% of temperature-sensitive chitosan hydrogel and the temperature-sensitive chitosan hydrogel sample leaching solution loaded with celecoxib are respectively selected and divided into a 50% hydrogel leaching solution group and a 50% drug-loaded hydrogel leaching solution combined blank control group.

2.2 Experimental procedures

(1) Taking out the cover glass from 75% ethanol by using cover slip forceps, and wiping the cover glass clean by using sterile silk cloth;

(2) gently place coverslips into 24-well plates (one per well);

(3) irradiating for 2-3 hours at a position 20-30 cm away from the direct irradiation range of the ultraviolet lamp;

(4) transfer the counted cell suspension to a culture plate (4 × 104/well) to completely immerse the coverslip in the culture medium;

(5) adding medicine after the cells adhere to the wall overnight;

(6) after adding the medicine for 5 days, dyeing treatment is carried out. Adding 10 mu g/ml Hoechst 33342 staining solution into each well, and incubating for 15min at 37 ℃;

(7) washing with PBS for three times, fixing the slide with 4% paraformaldehyde for 15min, and washing with PBS for 3 times;

(8) and (3) absorbing the liquid on the slide by using absorbent paper, sealing the slide by using sealing liquid containing an anti-fluorescence quenching agent, and observing and acquiring an image under a fluorescence microscope.

3. Hydrogel in vivo biocompatibility and degradability observations

3.1 preparation of hydrogels

In order to detect the histocompatibility and degradability of the hydrogel sample, the hydrogel sample is implanted under the skin in front of the big leg of a New Zealand white rabbit, the in-vivo histocompatibility and in-vivo degradability of the hydrogel are observed, firstly, the temperature-sensitive chitosan hydrogel and the temperature-sensitive chitosan hydrogel sample loaded with celecoxib are respectively prepared according to the preparation process of the hydrogel in example 1, and the sample is sterilized for later use.

3.2 animal groups

Dividing 18 New Zealand white rabbits into 3 groups, wherein each group comprises 6 control groups, hydrogel groups and drug-loaded hydrogel groups, injecting physiological saline, chitosan hydrogel and 3ml of drug-loaded hydrogel under the front of the thigh of each New Zealand white rabbit, killing 2 rabbits in each group 2 weeks, 1 month and 2 months after injection, cutting the front skin of the right thigh, and observing the tissue compatibility and degradability of the hydrogel sample in vivo.

3.3 subcutaneous injection of hydrogel samples

Each rabbit was weighed and numbered before anesthesia, and was fasted for 12h and forbidden for 6h before surgery. The rabbit is placed in a anaesthesia fixing box, the rabbit is pacified to gradually adapt to the environment of the fixing box, the edge of the ear of the rabbit is shaved, the ear vein with moderate thickness and obvious thickness is found to be used as a puncture blood vessel, a disposable scalp needle is connected to a 20ml injector full of urethane, the rabbit is punctured to the ear vein, the puncture angle is about 20-25 degrees, if the puncture is successful, a small amount of blood backflow can be seen, at the moment, an assistant can slowly inject 20% urethane solution about 7-8ml, the rabbit can enter an anaesthesia state in about 5-10min, and 60 ten thousand units of penicillin is injected to the muscle of the rabbit before an operation. The rabbit is placed on an animal operating table, the left lateral decubitus position is taken, the right thigh of the rabbit is fixed on the animal table through a thin rope, the front side of the right thigh is shaved, the diameter of the right thigh is about 3cm, 3ml of physiological saline, hydrogel and drug-loaded hydrogel are injected to the front side of the right thigh of the rabbit subcutaneously through an injector, the operation time is about 10min, the rabbit is placed on a constant-temperature heating operating table after the operation is finished, the rabbit is convenient to keep the body temperature to be anesthetized and revived, and 80 ten thousand units of penicillin is injected to the muscle of the rabbit for three consecutive days after the operation.

4. Statistical analysis

As a result:

1. analysis of hydrogel cytotoxicity

As chitosan and beta-sodium glycerophosphate are chemical materials, celecoxib is a clinically common anti-inflammatory drug, CCK-8 is used for detecting cells on

days

1, 3 and 5, and the proliferation conditions of L929 fibroblasts in different groups are detected. As shown in fig. 6, the chitosan hydrogel groups had no significant effect on L929 fibroblast proliferation compared to the blank control group at

days

1, 3 and 5, but the high concentration of chitosan hydrogel leaching solution had slight inhibition on L929 fibroblast proliferation as the cell culture time was extended. In addition, the celecoxib-loaded chitosan hydrogel slightly inhibits the proliferation of L929 fibroblasts by the leach liquor stock solution, and has no obvious inhibition on the proliferation of the L929 fibroblasts by other concentrations.

Results of Hoechst staining experiments

Hoechst 33342 is a blue fluorescent dye that can penetrate the cell membrane, has low toxicity to cells, and can stain living cells blue. As shown in FIG. 7, compared with the control group, the temperature-sensitive chitosan hydrogel group and the celecoxib-loaded temperature-sensitive chitosan hydrogel group both have no obvious inhibition on cell growth and proliferation, have good cell proliferation and have no obvious cytotoxicity through fluorescent microscope observation.

3. Observation result of subcutaneous hydrogel injection on thigh of New Zealand white rabbit

The 18 new zealand white rabbits in the experiment are anaesthetized and do not die in the operation process, the new zealand white rabbits are successfully revived after anaesthetization and operation, and all the rabbits have no sign of infection. All rabbits were in good condition at 2 weeks, 1 month, 2 months post-surgery, and no evidence of infection was found.

When the control group is observed for 2 weeks, after the primary injection area is shaved, the subcutaneous skin dome in front of the right thigh of the rabbit disappears, after the skin is cut, no physiological saline residue is seen in the subcutaneous part, no obvious infection sign appears, and the subcutaneous fascia and the muscle are ruddy and smooth, so the rabbit in the remaining control group is selected and kept and is not observed any more.

The hydrogel group was sacrificed 2 rabbits at 2 weeks, 1 month, and 2 months after the operation, respectively. At 2 weeks after operation, the hydrogel is found to be obviously remained under the rabbit skin, is wrapped between the subcutaneous mucous membrane and the muscle and has obvious boundary with the surrounding tissues, but the surrounding tissues have no obvious infection signs; the hydrogel still obviously remains under the rabbit skin at 1 month after operation, but compared with 2 weeks, the hydrogel residue is obviously reduced and is in a gradually decomposed state, the boundary with surrounding tissues is still obvious, and no obvious infection sign exists; at 2 months after the operation, compared with 1 month after the operation, the subcutaneous hydrogel of the rabbit basically completely disappears, only a little milky white residue is visible, and the surrounding tissues have no obvious infection signs.

After the drug-loaded hydrogel is implanted under the skin of a rabbit, the drug-loaded hydrogel is found to be obviously remained between a subcutaneous mucosa and muscle and has clear boundary with surrounding tissues and no obvious infection sign in the surrounding tissues in 2 weeks after operation; the liquid medicine carrying gel still obviously remains under the rabbit skin after 1 month of operation, but compared with 2 weeks, the hydrogel residue is reduced and is in a gradually decomposed state, the size of the residue is equivalent to that of the hydrogel group in the same period, the boundary with surrounding tissues is obvious, the liquid medicine carrying gel is attached to the surface layer of muscle fascia, and no obvious infection sign exists; compared with the postoperative hydrogel for 1 month, the drug-loaded hydrogel basically and completely disappears under the skin of a rabbit after 2 months of operation, a little milky residue is visible, the residue amount is equivalent to that of the hydrogel group in the same period, and the surrounding tissues have no obvious infection signs.

Example 3 study of celecoxib-loaded temperature-sensitive injectable hydrogel on degeneration of intervertebral disc

The experimental method comprises the following steps:

1. grouping of laboratory animals

The animal experiment is divided into 4 groups, as shown in table 1, namely a normal control group, a needle-prick degeneration group, a temperature-sensitive chitosan hydrogel group and a temperature-sensitive chitosan hydrogel group loaded with celecoxib. Different lumbar vertebra segments in the same rabbit body are selected for research, and the individual difference of experimental animals is mainly avoided. 4 rabbits were sacrificed at 2 weeks, 1 month and 2 months post-surgery for further observation.

TABLE 1 groups of experimental animals

2. Establishment of rabbit intervertebral disc degeneration model

Each rabbit was weighed and numbered before anesthesia, and was fasted for 12h and forbidden for 6h before surgery. The rabbit is placed in an anaesthesia fixing box, the rabbit is pacified to gradually adapt to the environment of the anaesthesia fixing box, the edge of the ear of the rabbit is shaved, the ear vein with moderate thickness and obvious thickness is found to be used as a puncture blood vessel, the disposable scalp needle is connected to a 20ml injector full of urethane, the ear vein of the rabbit is punctured, the puncture angle is about 20-25 degrees, if the puncture is successful, a small amount of blood backflow can be seen, at the moment, an assistant can slowly inject 20% urethane solution about 7-8ml, the rabbit can enter an anaesthesia state within about 5-10min, and 60 ten thousand units of penicillin is injected into the muscle of the rabbit before an operation. Placing the rabbit on an animal operating table, taking the left lateral lying position, performing an operation by adopting a lateral extraperitoneal approach, and selecting the end of the left side 12 costal margin to the iliac spine as an operation area. The reason is mainly that the new Zealand white rabbit has 7 lumbar vertebrae, 4 lumbar vertebrae are supposed to be exposed in the operation, and the 6 th lumbar vertebra of the iliac spine is taken as a reference, and L2/3, L3/4, L4/5 and L5/6 intervertebral discs are respectively exposed upwards by taking the reference as a positioning point.

After shaving and skin preparation in an operation area, applying iodine for disinfection, paving a disposable hole towel, making a longitudinal incision with the length of about 10cm from the tail end of the left side 12 costal margin to the iliac spine, locally injecting ropivacaine to the subcutaneous part, additionally adding 3-5ml of urethane along the ear vein in the operation (the anesthesia death risk can be reduced by small quantity intravenous injection in times), sequentially cutting open the skin and subcutaneous tissues, stopping bleeding at a bleeding point by using an electrocoagulation hemostatic knife, separating and cutting the extraabdominal oblique muscle, the intra-abdominal oblique muscle and the transverse abdominal muscle, touching the transverse process with the finger abdomen for positioning, and then separating the psoas major muscle by using a scalpel handle, wherein an assistant can support the spinal column of the rabbit at the back side during the period, so that the rabbit slightly rotates towards the left side, and the white intervertebral disc fibrous ring is convenient to be exposed. L2/3, L3/4, L4/5 and L5/6 intervertebral discs are positioned in sequence, a 20G needle penetrates into the intervertebral discs for about 5mm from the front of the side of the L3/4, L4/5 and L5/6 intervertebral discs in sequence, certain breakthrough feeling can be felt when the intervertebral discs are penetrated, the needle is rotated for three circles after the penetration, the needle is pulled out after the suction and the stop for 30 seconds, obvious needle holes are left on the fibrous ring in the front of the side of the intervertebral discs, and a little transparent jelly-like substance can flow out along the needle holes (mainly nucleus pulposus tissues).

3. Rabbit intervertebral disc hydrogel injection

After the intervertebral disc defect model is established, about 1ml of temperature-sensitive chitosan hydrogel is injected into the L4/5 intervertebral disc by an injector along the original puncture needle hole, and about 1ml of temperature-sensitive chitosan hydrogel loaded with celecoxib is injected into the L5/6 intervertebral disc along the original puncture needle hole after a new injector is replaced. After the incision is flushed by normal saline, the incision is sequentially sutured, and sterile dressing is covered and fixed after the iodine is sterilized. The operation time is about 30-50min, and after the operation is finished, the rabbit is placed on a constant-temperature heating operation table, so that the rabbit can be conveniently kept in a body temperature for anesthesia and recovery. The rabbits were given an intramuscular injection of 80 million units of penicillin three consecutive days after surgery.

4. Imaging examination of the intervertebral discs of New Zealand white rabbits

4.1X-ray examination

X-ray examination is carried out on the rabbit 2 weeks, 1 month and 2 months after the operation, ear vein anesthesia is carried out by referring to a 2.2 method before the examination, the rabbit is placed on an X-ray machine in a lateral position, and an X-ray picture is taken. In the experiment, the height of the vertebral body and the height of the intervertebral space are measured through imaging measurement software, and the Bradner Disc Index (BDI) is further calculated to estimate the height change of the intervertebral space.

4.2 MRI examination

Before MRI examination is carried out on rabbits, New Zealand white rabbits are anesthetized firstly, the rabbits are placed on an MRI machine in a prone position, knee joint coils are used, MRI parameters are adjusted to enable images to clearly show intervertebral discs of the New Zealand white rabbits, sagittal position scanning of the intervertebral discs is carried out, T2 weighted image sagittal position MRI images of the white rabbits are collected, regression grading analysis is carried out on T2 weighted image sagittal position MRI images of a collection according to improved Thompson grading, and statistical analysis is carried out according to I grade, II grade, III grade and IV grade (the standards are shown in Table 2).

TABLE 2 improved Thompson ranking

5. Histopathological observation of intervertebral discs

After 2 weeks of surgery, New Zealand white rabbits were sacrificed by means of air embolization of the ear vein, and all experimental segments were removed and fixed with paraformaldehyde. After fixing with paraformaldehyde for several hours, muscle tissues around the spine are easier to strip, the intervertebral disc of the experimental segment is obtained after being washed clean with normal saline, the marking and recording are carried out, calcium is removed by using 20% EDTA for about 1 month, and the intervertebral disc is sliced conventionally, dyed, observed and photographed.

5.1 making pathological sections

Firstly, fixing and decalcifying: the clean intervertebral disc tissues are placed into 4% paraformaldehyde for fixation for 2-3 days, decalcification is carried out by using 20% EDTA decalcification solution for about 1-2 months (the decalcification solution is replaced every 2-3 days) until upper and lower sclerotin of the intervertebral disc become soft, redundant sclerotin is cut off by a knife, the intervertebral disc is kept and washed by parallel flow water, and the decalcification solution on the surface is removed.

And (2) dehydrating: the washed disc tissue was placed in 50%, 65%, 75%, 85%, 95%, 100% ethanol solutions in sequence, each concentration for about 3 hours.

③ transparent: the dehydrated disc tissue was placed in turn in 1/2 xylene +1/2 ethanol solution, 100% xylene solutions I and II, each for about 30 minutes.

Soaking wax and embedding: placing the transparent intervertebral disc tissue into 1/2 dimethylbenzene +1/2 paraffin for 1h, paraffin solution I and paraffin solution II for 2h in sequence. Then the intervertebral disc tissues soaked in paraffin are placed into an embedding mould for embedding.

Slicing and baking: the embedded disc tissue was fixed in a paraffin microtome, the paraffin block was trimmed to size and sectioned with a microtome (5 um thick per section). And putting the cut slices into a warm water tank at about 42 ℃ to unfold the slices, fishing out the unfolded slices by using a glass slide, marking the slices, and putting the slices on a baking sheet machine to bake water. And collecting the dried paraffin sections, and storing at room temperature for later use.

5.2HE staining

Baking slices: and (3) placing the processed paraffin sections on a baking sheet machine for baking sheets at 56 ℃ for 2 h.

Dewaxing and rehydration: the paraffin sections are sequentially placed in 100% dimethylbenzene solutions I and II for 10min respectively, 1/2 dimethylbenzene +1/2 ethanol solutions for 3min, 100% absolute ethyl alcohols I and II for 3min, 95%, 85%, 75%, 65% and 50% ethyl alcohols for 3min respectively and distilled water for 5min respectively according to the following sequence.

③ dyeing with hematoxylin: putting the rehydrated slices into hematoxylin solution for 15min, taking out, and putting into running water to turn blue for 15 min.

Fourthly, first dehydration: the slices were sequentially placed in 50%, 65%, 75%, 85% ethanol for 3min each.

Fifthly, red dyeing: sections dehydrated into 85% ethanol were stained in eosin solution for 5 s.

Sixthly, second dehydration: placing the slices in 95% ethanol solution to remove residual eosin, and sequentially placing the slices in 100% anhydrous ethanol I and II for 3min each.

And c, transparent and sealing: and (3) sequentially placing the slices into 100% dimethylbenzene solutions I and II for 5min respectively, taking out, drying at room temperature, and sealing with neutral resin.

Observing with a microscope: and (4) observing the prepared tissue section under a microscope, and selecting a proper multiple to take a photograph. Disc tissue sections were scored and statistically analyzed according to histological grading. (Table 3)

TABLE 3 grading Scale of intervertebral disc histology

Statistical analysis:

statistical analysis was performed using SPSS19.0 statistical software and the experimental data are expressed as mean. + -. standard error (X. + -. SD). Analysis of variance between groups was performed at different time points for different lumbar segments, and statistical differences were found when P < 0.05.

As a result:

1. intervertebral disc degeneration model modeling

In the experiment, 2 deaths occurred in 12 New Zealand white rabbits during anesthesia and surgery, 1 death occurred too fast due to anesthetic injection, and 1 death occurred on the 2 nd day after surgery. 2 cases of New Zealand white rabbits were supplemented in time after discovery. The rest of the new zealand white rabbits were anesthetized and successfully revived after surgery, and all rabbits showed no signs of infection. After 2 weeks, 1 month and 2 months of operation, all rabbits were in good condition and showed no obvious signs of infection, and the specimen was successfully obtained.

X-ray examination result

As shown in fig. 8, the change of the intervertebral disc height can be visually displayed by the X-ray examination after the operation, and the change of the intervertebral disc height of 2 weeks, 1 month and 2 months after the operation can be reasonably and accurately judged by the BDI. After the X-ray images are collected and measured, the intervertebral space height of the normal control group does not change obviously in the whole observation period, and the intervertebral space height of the degenerative group is narrowed obviously in 2 weeks, 1 month and 2 months after operation. The intervertebral space heights of the chitosan hydrogel group and the temperature-sensitive chitosan hydrogel group loaded with celecoxib are reduced compared with those of a normal control group, the intervertebral space heights between the two groups are equivalent in change, but the reduction range is smaller than that of a needle-prick degeneration group.

Results of MRI examination

Normal nucleus pulposus tissue contains more water, and the water content gradually decreases as the nucleus pulposus degenerates. Water appears as a high signal on the T2 weighted image of the MRI, and thus the signal appearance of the nucleus pulposus tissue of the intervertebral disc can be indirectly reflected by looking at the sagittal MRIT2 weighted image, and the intensity of the disc signal can be quantitatively compared using the modified Thompson score. As shown in FIG. 9a, the intervertebral space of the postoperative needle-pricked degenerative group was significantly narrowed, the margin was blurred, and the signal of the nucleus pulposus tissue was significantly attenuated compared to the control group. Nucleus pulposus signals of the temperature-sensitive chitosan hydrogel group and the temperature-sensitive chitosan hydrogel group loaded with celecoxib are reduced in week 2, and are not obviously reduced in

months

1 and 2. In addition, as shown in fig. 9b, the temperature-sensitive chitosan hydrogel group and the celecoxib-loaded temperature-sensitive chitosan hydrogel group were significantly different from the needle punch degeneration group 2 weeks after the operation (P <0.05), but no significant difference was observed between the 1 st month and the 2 nd month after the operation (P > 0.05).

4. Disc HE staining results and pathology scores

HE staining stains chromatin in the nucleus and ribosomes in the cytoplasm bluish purple and the cytoplasm and extracellular matrix red. The normal nucleus pulposus tissue can be clearly seen after HE staining, and the nucleus pulposus with an oval shape at the inner part and the fibrous ring with an onion shape at the periphery can be seen. (FIG. 10A) the nucleus pulposus cells, the nucleus pulposus matrix and the collagen are all distributed within the inner nucleus pulposus tissue. In addition, the nucleus pulposus is surrounded by the intact cartilage end plates at the upper and lower parts, and the chondrocytes are arranged in order. For the degenerated group of intervertebral discs, disappearance of inner nucleus pulposus cells can be seen after HE staining, and an unorganized area exists, so that the nucleus pulposus and the annulus fibrosus in the intervertebral disc are difficult to distinguish. (FIG. 10B) however, after HE staining, nucleus pulposus tissues can still be seen in the center of the inner part of the intervertebral disc compared with the degeneration group of the chitosan hydrogel group and the celecoxib-loaded hydrogel group, and the arrangement of nucleus pulposus cells is disordered compared with the normal intervertebral disc, but no obvious difference is seen between the two groups. (FIG. 10C, D) histological grading of the intervertebral discs showed significant degeneration of the interior of the discs in the needle-pricked degenerative group. The hydrogel group and the celecoxib-loaded hydrogel group also have degeneration compared with the normal group, but the degeneration grade is better than that of the intervertebral disc of the degeneration group. (FIG. 11, P <0.05)

The above-mentioned embodiments are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention made by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The celecoxib-loaded injectable temperature-sensitive chitosan hydrogel is characterized by comprising celecoxib, chitosan and beta-sodium glycerophosphate, wherein the chitosan and the beta-sodium glycerophosphate are crosslinked into an interpenetrating network, and the celecoxib is uniformly loaded in the interpenetrating network.

2. The celecoxib-loaded injectable thermo-sensitive chitosan hydrogel of claim 1, wherein the chitosan has a degree of deacetylation of 95%.

3. A method for preparing the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel according to claim 1 or 2, which comprises the following steps:

4. The method according to claim 3, wherein the dilute acid is dilute hydrochloric acid or dilute acetic acid, and the concentration is 0.01 mol/L.

5. The process according to claim 3, wherein the celecoxib solution has a concentration of 56 g: 100mL, the concentration of the chitosan solution is (2-5) g: 100mL, wherein the concentration of the beta-sodium glycerophosphate solution is 2 g: 100 mL.

6. The method according to claim 3, wherein in the step (2), the ratio of the celecoxib solution to the chitosan solution is 1-1.5:9 by volume; in the step (3), the volume ratio of the beta-sodium glycerophosphate solution to the celecoxib-chitosan mixed solution is 1: 10-10.5.

7. Use of the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel of claim 1 or 2 in the preparation of a medicament for treating intervertebral disc degeneration.

8. A pharmaceutical preparation for treating intervertebral disc degeneration, which comprises a therapeutically effective amount of celecoxib-loaded injectable temperature-sensitive chitosan hydrogel of claim 1 or 2 and pharmaceutically acceptable excipients.

9. The pharmaceutical preparation according to claim 8, wherein the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel has a mass content of 1-99%.

10. The pharmaceutical preparation according to claim 9, wherein the celecoxib-loaded injectable temperature-sensitive chitosan hydrogel comprises 10-90% by mass.