Lycorine-loaded functionalized methylacryloylated chitosan injection, gel, preparation method and application

Abstract

The invention provides lycorine-loaded functionalized methylacryloylated chitosan injection, gel, a preparation method and application, and relates to the field of medicines. The preparation method comprises the following steps: dropwise adding methacrylic anhydride into the chitosan-acetic acid solution, and reacting for 24 hours; dialyzing the reaction liquid, and performing freeze drying to obtain methacrylated chitosan CSMA; the preparation method comprises the following steps: dissolving CSMA in water, and stirring at room temperature for 12-24 hours until the solution is transparent to obtain a CSMA solution; adding the lycorine stock solution into the CSMA solution, and stirring for 30 minutes in a dark place; adding a photoinitiator, and stirring in a dark place until the photoinitiator is completely dissolved to obtain a CSMA-lycorine injection; and carrying out illumination curing on the injection to obtain the gel. The swelling property of the gel in the early stage can support the damaged centrum, CSMA is gradually degraded along with the recovery of the intervertebral disc in the middle and late stages, and the normal function and recovery of the intervertebral disc in the late stage cannot be influenced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically, to a lycorine-loaded functionalized methacrylated chitosan injection, a gel, a preparation method therefor, and its application. Background Technology

[0002] Currently, the hydrogel materials used in the treatment of intervertebral disc degeneration and regeneration are mainly traditional thermosensitive hydrogels. After implantation, these materials can form an extracellular matrix-like structure through thermosensitive phase transition behavior, which can alleviate local inflammation to some extent and provide initial mechanical support for the degenerated intervertebral disc. However, these hydrogels still have several significant limitations in practical applications, severely restricting their clinical translation and therapeutic efficacy.

[0003] First, traditional thermosensitive hydrogels generally have poor mechanical properties, lacking sufficient toughness and fatigue resistance, making it difficult to withstand the complex cyclic loads generated during spinal movement over long periods. They are prone to cracking or deformation, leading to failure of their support function. Second, their degradation behavior is difficult to match with the tissue regeneration process, often resulting in degradation rates that are too fast or too slow: too fast degradation leads to premature loss of mechanical support, while too slow degradation may hinder the ingrowth of new tissue and trigger chronic inflammatory responses.

[0004] Furthermore, existing hydrogels have relatively limited functions, mostly serving only as fillers and physical barriers, lacking the bioactivity to promote cell migration, proliferation, and differentiation, and failing to integrate multiple therapeutic functions (such as anti-inflammatory, antioxidant, and angiogenesis inhibitory effects). In terms of operability, these hydrogels typically have long curing times, which not only prolong surgical time but also increase uncertainty during implantation. More seriously, due to their weak initial rheological properties, they are easily squeezed out of the implantation area under the high pressure environment within the intervertebral disc before complete curing, leading to treatment failure and even complications in surrounding tissues.

[0005] In conclusion, although traditional thermosensitive hydrogels have some potential in the repair of intervertebral disc degeneration, their significant shortcomings in mechanical properties, degradation characteristics, biological functions, and operational applicability limit the effectiveness and reliability of their further clinical applications. Summary of the Invention

[0006] The purpose of this invention is to provide a lycorine-loaded functionalized methacrylated chitosan injection. The lycorine-loaded CSMA can achieve sustained drug release. In the early stage, the swelling properties of CSMA provide support for the damaged vertebral body. In the middle and late stages, as the intervertebral disc recovers, the CSMA gradually degrades without affecting the normal function and recovery of the intervertebral disc in the late stage.

[0007] Another objective of this invention is to provide a method for preparing a lycorine-loaded functionalized methacrylated chitosan injection, which allows for sustained drug release.

[0008] The third objective of this invention is to provide an application of a lycorine-loaded functionalized methacrylated chitosan injection or gel, which, when applied to the treatment of intervertebral disc degeneration, can provide both support and sustained drug release. Furthermore, the CSMA has suitable support time and degradation rate, and minimal side effects on the intervertebral disc.

[0009] The technical problem solved by this invention is achieved by the following technical solution.

[0010] On one hand, embodiments of the present invention provide a method for preparing a lycorine-loaded functionalized methacryloxylated chitosan injection, comprising the following steps: Synthesis of S1, methacrylamide chitosan (CSMA): Chitosan was dissolved in an aqueous acetic acid solution and magnetically stirred for 2-4 hours until completely dissolved to obtain a chitosan-acetic acid solution. Under continuous stirring, methacrylic anhydride was added dropwise to the chitosan-acetic acid solution, and the reaction was carried out for 24 h. The pH was then adjusted to 7.0 with saturated NaHCO3 solution to terminate the reaction. The reaction solution was dialyzed and freeze-dried to obtain methacrylated chitosan (CSMA), which was dried and stored at 4°C for later use. Preparation of S2, CSMA-Lycorine Injection: Dissolve CSMA in water and stir at room temperature for 12-24 h until clear to obtain a CSMA solution; Add the lycorine stock solution to the CSMA solution and stir in the dark for 30 min; add the photoinitiator and stir in the dark until completely dissolved to obtain CSMA-lycorine injection.

[0011] In some embodiments of the present invention, in step S1, the degree of deacetylation of the chitosan is ≥75% and the viscosity-average molecular weight is 50-190 kDa.

[0012] In some embodiments of the present invention, in step S1, the dialysis includes: transferring the reaction solution into a dialysis bag with a molecular weight cutoff of 3.5-8 kDa, dialyzing with deionized water for 3 days, and changing the water twice a day.

[0013] In some embodiments of the present invention, in step S1, the freeze-drying temperature is -50 °C and the time is 48-72 h.

[0014] In some embodiments of the present invention, in step S1, the molar ratio of methacrylic anhydride to chitosan amino is 1:1-1.2.

[0015] In some embodiments of the present invention, in step S2, the concentration of lycorine in the CSMA-lycorine injection is 20 μM.

[0016] In some embodiments of the present invention, in step S2, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite, and the concentration of the photoinitiator in the CSMA-lycorine injection solution is 0.2-0.3% (w / v).

[0017] On the other hand, embodiments of the present invention provide a lycorine-loaded functionalized methacrylated chitosan injection, which is prepared by the above method.

[0018] Thirdly, embodiments of the present invention also provide a lycorine-loaded functionalized methacrylated chitosan gel, which is obtained by irradiating the injection solution prepared by the above method under 405nm blue light or 365nm ultraviolet light for 10-30 s and then curing it.

[0019] Fourthly, embodiments of the present invention also provide the application of a lycorine-loaded functionalized methacrylated chitosan injection or gel in the preparation of a medicament for treating intervertebral disc degeneration.

[0020] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: The injection and gel provided by this invention use methacrylated chitosan as a carrier and are loaded with lycorine. Lycorine is a natural isoquinoline alkaloid with broad-spectrum anti-inflammatory, antioxidant, and antitumor activities. Methacrylated chitosan (CSMA) can be rapidly cross-linked under blue / ultraviolet light to form a three-dimensional network, achieving sustained drug release. Using lycorine-loaded CSMA in an IVDD rat model allows for localized drug administration and orderly release, improving inflammatory responses within the vertebral bodies of rats with intervertebral disc degeneration, and aiding in the recovery of normal function. Furthermore, the early swelling properties of CSMA provide support to the damaged vertebral bodies, while in the middle and late stages, as the intervertebral disc recovers, CSMA gradually degrades without affecting the late-stage normal function and recovery of the intervertebral disc. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1The above are SEM images of CSMA and Lyc@CSMA in Embodiment 1 of the present invention; Figure 2 This is a statistical diagram of the water contact angle of CSMA and Lyc@CSMA in Embodiment 1 of the present invention; Figure 3 This is a pressure-strain curve diagram of CSMA and Lyc@CSMA in Embodiment 1 of the present invention; Figure 4 The Young's modulus of CSMA and Lyc@CSMA in Embodiment 1 of the present invention; Figure 5 This is a diagram showing the cell viability status in each group during the live / dead staining experiment. Figure 6 This is a statistical chart of cell viability for CSMA and Lyc@CSMA in Example 1 of the present invention; Figure 7 The images show the staining patterns of cells in each group after incubation with Aggrecan in a cell protein fluorescence staining experiment. Figure 8 The images show the staining patterns of cells in each group after Collagen incubation in a cell protein fluorescence staining experiment. Figure 9 The intensity of protein fluorescence in a cell protein fluorescence staining experiment; Figure 10 Results of Western blot (WB) assay for inflammatory protein expression; Figure 11 shows the results of HE staining (first row), safranin staining (second row), and MRI experiments (third row) of rat intervertebral disc cells. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0025] The abbreviations for each substance are shown below: CSMA: Methacrylamide chitosan; LAP: Photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphite DMSO: Dimethyl sulfoxide Lycorine, Lyc: Lycorine Lyc@CSMA: Methacrylated chitosan hydrogel loaded with lycorine The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] Example 1 The injection solution of this embodiment was prepared as follows: Synthesis of S1. Methacrylated chitosan (CSMA) 1.1 Chitosan with a degree of deacetylation ≥75% and a viscosity-average molecular weight of 100 kDa was dissolved in a 1% (v / v) aqueous acetic acid solution at a ratio of 1 g:100 mL and magnetically stirred at 25 °C for 2-4 h until completely dissolved to obtain a chitosan-acetic acid solution. 1.2 Under continuous stirring, methacrylic anhydride was slowly added dropwise at a molar ratio of 1:1 to chitosan amino groups, and the reaction was carried out at 25 °C for 24 h. 1.3 The reaction was terminated by adjusting the pH to 7.0 with a saturated NaHCO3 solution; 1.4 Transfer the reaction solution into a dialysis bag with a molecular weight cutoff of 3.5-8 kDa, and dialyze with deionized water for 3 days, changing the water twice a day; 1.5 Freeze-dry (-50 °C, 48 h) to obtain a white, fluffy solid, namely CSMA, which is then dried and stored at 4 °C.

[0027] Preparation of S2.CSMA-Lycorine Injection 2.1 Dissolve CSMA in sterile ultrapure water at 2% (w / v) and stir at room temperature for 12-24 h until clear to obtain CSMA solution; 2.2 Based on a final concentration of 20 μM for lycorine, slowly add the lycorine stock solution (1 mM) to the above CSMA solution to make the final concentration of DMSO ≤0.1%, and stir for 30 min in the dark. 2.3 Based on the final concentration of the photoinitiator in the injection solution being 0.25% (w / v), add the photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphite), stir in the dark until completely dissolved, and obtain CSMA-lycorine injection solution.

[0028] Preparation of CSMA-lycorine gel: CSMA-lycorine injection solution is injected into the mold; it is then irradiated with 405nm blue light for 30 seconds to solidify, thus forming CSMA-lycorine gel (Lyc@CSMA) in situ.

[0029] Example 2 The difference from Example 1 is that the viscosity-average molecular weight of chitosan is 50 kDa, while the rest of the preparation method and raw material ratio are the same as in Example 1.

[0030] Example 3 The difference from Example 1 is that the viscosity-average molecular weight of chitosan is 150 kDa, while the rest of the preparation method and raw material ratio are the same as in Example 1.

[0031] Example 4 The difference from Example 1 is that the viscosity-average molecular weight of chitosan is 190 kDa, while the rest of the preparation method and raw material ratio are the same as in Example 1.

[0032] Example 5 The difference from Example 1 is that the molar ratio of methacrylic anhydride to chitosan amino group is 1:1.2, while the rest of the preparation method and raw material ratio are the same as in Example 1.

[0033] Example 6 The difference from Example 1 is that the final concentration of the photoinitiator in the injection solution is 0.2% (w / v), while the rest of the preparation method and raw material ratio are the same as in Example 1.

[0034] Example 7 The difference from Example 1 is that the final concentration of the photoinitiator in the injection solution is 0.3% (w / v), while the rest of the preparation method and raw material ratio are the same as in Example 1.

[0035] Example 8 The difference from Example 1 is that, in preparing the gel, it was cured by irradiation with 365 nm ultraviolet light for 10 s, while the rest of the preparation method and raw material ratios are the same as in Example 1.

[0036] Experimental Example SEM Experiment: The CSMA sample from Example 1 was subjected to liquid nitrogen embrittlement to obtain a fresh fracture surface. The fracture surface was fixed upwards on conductive adhesive and sputtered with gold (15mA current, 60 seconds) to eliminate the charging effect. During SEM observation, a low accelerating voltage (5kV) and a relatively small beam current were used to avoid electron beam damage. The sample was placed in the sample chamber and evacuated to 10⁻⁴ Pa. The working distance was adjusted to 8-10 mm. In secondary electron mode, low-magnification positioning was first used, followed by gradual magnification to the desired magnification to observe the phase structure, particle dispersion, and interfacial bonding. The results are as follows: Figure 1 As shown.

[0037] Water contact angle experiment: The static water contact angle was measured using the seated drop method. A 1-2 μL droplet of deionized water was placed on a flat sample surface using a micro-syringe, and images were acquired using a high-speed camera system within (60±10) s. The contact angle was calculated by fitting the droplet profile using the Young-Laplace equation. At least three different sites were randomly selected for repeated measurements on each sample, and ImageJ software was used for quantitative analysis. Results are expressed as mean ± standard deviation, and outliers were removed after calculating the 95% confidence interval. The results are as follows: Figure 2 As shown.

[0038] Compression-strain test: Uniaxial compression testing was performed using a universal testing machine. A standard specimen (5mm x 5mm cylinder) was prepared, and axial pressure was applied at a constant loading rate (1mm / min). Load-displacement curves were recorded until specimen failure. Stress and strain data were collected simultaneously, and stress-strain curves were plotted. Figure 3 and Figure 4 As shown, Figure 3 This is the stress-strain curve. Figure 4 Young's modulus, calculated from the slope of the linear elastic phase of the stress-strain curve (typically strain <0.2%), is given by E = Δσ / Δε, where the least squares method is used to fit the linear relationship between stress and strain. At least three valid specimens were tested in each sample group, and results are expressed as mean ± standard deviation. 95% confidence intervals were calculated. One-way ANOVA was used to assess the significance of differences among multiple groups, with P < 0.05 considered significant. At least three valid specimens were tested in each sample group.

[0039] Live / dead cell staining assay: Cells were co-cultured with material extracts (blank group: no material, normal cells in complete culture medium; CSMA group: pre-swollen CSMA hydrogel at a concentration of 0.1 g / mL immersed in complete culture medium, i.e., CSMA + complete culture medium; Lyc@CSMA group: pre-swollen Lyc@CSMA hydrogel at a concentration of 0.1 g / mL immersed in complete culture medium, i.e., Lyc@CSMA + complete culture medium) for 24-48 hours. After washing 2-3 times with PBS, staining working solution containing 2 μM Calcein AM and 4.5 μM PI was added, and the cells were incubated at room temperature in the dark for 15-20 minutes. Under a fluorescence microscope, live cells showed green fluorescence (494 / 517 nm), and dead cells showed red fluorescence (535 / 617 nm). The cell viability status of each group was as follows: Figure 5 As shown. At least 5 fields of view were randomly selected from each sample, and the proportion of live cells was quantitatively analyzed using ImageJ software, as shown. Figure 6 As shown. Data are expressed as mean ± standard deviation. One-way ANOVA was used to compare differences between groups, and P < 0.05 was considered statistically significant. Figure 6In the graph, the horizontal axis represents the grouping, and the vertical axis represents the cell survival rate.

[0040] Cellular protein fluorescence staining experiment: divided into blank group, Lyc@CSMA group, and LPS group.

[0041] Cells cultured in the Lyc@CSMA group of the above live / dead staining experiment were seeded in six-well plates. 2 mL of 14% paraformaldehyde was added to each well for fixation for 15 minutes, followed by permeabilization with 0.1-0.5% Triton X-100 for 10 minutes, and blocking with 5% BSA for 1 hour. Primary antibody (Aggrecan or Collagen, 1:200) was incubated overnight at 4°C, followed by incubation with fluorescent secondary antibody at room temperature in the dark for 1 hour. Nuclei were stained with DAPI (100 ng / mL) for 5 minutes. After mounting with an anti-quenching agent, the cells were imaged using a confocal microscope. Figure 7 and Figure 8 As shown in the figure. ImageJ software was used to quantitatively analyze the proportion of positive cells and fluorescence intensity. At least three fields of view were randomly selected for each sample. Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze differences between groups, with P < 0.05 considered statistically significant. The results are shown in the figure. Figure 9 As shown, the horizontal axis represents the grouping, and the vertical axis represents the protein fluorescence intensity.

[0042] Establishment of a rat model of intervertebral disc degeneration: Three-month-old male SD rats (weighing 200–250 g) were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg) and fixed in a prone position. The Co7 / 8 intervertebral disc of the caudal vertebra was used as the surgical segment. After shaving and disinfection, a longitudinal incision (approximately 1 cm) was made on the dorsal midline, and the subcutaneous tissue was bluntly dissected to expose the intervertebral space. A 21G sterile puncture needle (0.8 mm in diameter) was inserted perpendicularly to the surface of the intervertebral disc, penetrating the annulus fibrosus and nucleus pulposus to a depth of 5 mm (through the entire disc). After insertion, the needle was slowly rotated 180°–360° and left in place for 30 seconds. When withdrawing the needle, a sterile cotton ball was used to press the needle tract for 30 seconds to prevent the nucleus pulposus from spilling out. The skin was then sutured layer by layer. In the Sham group, only the skin was incised to expose the intervertebral disc; no acupuncture was performed. Rats were randomly divided into three groups (n=3): Control group, CSMA group, and Lyc@CSMA group. A second surgery was performed on day 14 post-surgery: after anesthesia and disinfection as before, a needle was inserted through the original incision, and 2 μL of material was injected into the nucleus pulposus via the original needle tract using a sterile microsyringe (no injection in the control group, CSMA hydrogel in the CSMA group, and Lyc@CSMA in the Lyc@CSMA group). Post-surgery, rats were housed separately with free access to food and water. The following experiments were then conducted.

[0043] Western blot analysis to detect inflammatory protein expression: Protein extraction from rat intervertebral disc tissue: The nucleus pulposus, annulus fibrosus, or endplate tissue was rapidly separated on ice. RIPA lysis buffer containing protease / phosphatase inhibitors was added at a ratio of 50 mg / mL. After homogenization, the mixture was lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was used to determine the concentration using the BCA method. Western blotting: Protein was mixed with loading buffer and boiled for 5 minutes. After separation by SDS-PAGE, the protein was wet-transferred to a PVDF membrane, blocked with 5% BSA for 1 hour, incubated with primary antibody overnight at 4°C, washed three times with TBST, incubated with secondary antibody at room temperature for 1 hour, and then developed by ECL. ImageJ was used to quantify the band gray values, and the target protein was normalized to the internal control. Statistical analysis was performed using GraphPadPrism 9.5.1 software. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant. Results are as follows: Figure 10 As shown in the figure, the horizontal axis represents grouping, and the vertical axis represents the results after normalizing the gray values ​​of different protein bands.

[0044] HE staining method: After sacrificing rats, the L4 / 5 intervertebral disc and adjacent vertebral bodies were collected, fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% EDTA for 4 weeks, routinely dehydrated and embedded in paraffin, and 4μm serial sections were prepared. After dewaxing and hydration, hematoxylin staining was performed for 3 minutes, followed by differentiation in 1% hydrochloric acid alcohol, eosin staining for 1 minute, and then dehydration, clearing, and mounting. The structure of the nucleus pulposus (NP) and annulus fibrosus (AF) was observed under a panoramic scanning microscope. The results are attached. Figure 11 The first row (HE row) is shown.

[0045] Safranin staining (PSG) experiment: After routine dewaxing of paraffin sections to water, the nuclei were stained with Weigert iron hematoxylin for 5 minutes, followed by 0.001% Fast Green staining for 3 minutes, differentiation with 1% acetic acid, and then stained with 0.1% Safranin O for 5 minutes. Proteoglycans appeared red or orange-red, and collagen fibers appeared green. The results are as follows: Figure 11 As shown in the second row (PSG row).

[0046] MRI experiment on intervertebral discs in SD rats: Using a 3.0T MRI scanner, rats were anesthetized with intraperitoneal injection of 10% sodium pentobarbital (40 mg / kg), fixed in a prone position, and subjected to sagittal T2-weighted imaging (slice thickness 1.5 mm). The results are as follows: Figure 11 The third row (MRI row) is shown.

[0047] In summary, the injection and gel provided in this invention use methacrylated chitosan as a carrier and are loaded with lycorine. Lycorine is a natural isoquinoline alkaloid with broad-spectrum anti-inflammatory, antioxidant, and antitumor activities. Methacrylated chitosan (CSMA) can be rapidly cross-linked under blue / ultraviolet light to form a three-dimensional network, achieving sustained drug release. Using lycorine-loaded CSMA in an IVDD rat model allows for localized drug administration and orderly release, improving inflammatory responses within the vertebral bodies of rats with intervertebral disc degeneration, and aiding in the recovery of normal function. Furthermore, the early swelling properties of CSMA provide support to the damaged vertebral bodies, while in the middle and late stages, as the intervertebral disc recovers, CSMA gradually degrades without affecting the late-stage normal function and recovery of the intervertebral disc.

[0048] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a lycorine-loaded functionalized methacryloxylated chitosan injection, characterized in that, Includes the following steps: Synthesis of S1, methacrylamide chitosan (CSMA): Chitosan was dissolved in an aqueous acetic acid solution and magnetically stirred for 2-4 hours until completely dissolved to obtain a chitosan-acetic acid solution. Under continuous stirring, methacrylic anhydride was added dropwise to the chitosan-acetic acid solution, and the reaction was carried out for 24 h. The pH was then adjusted to 7.0 with saturated NaHCO3 solution to terminate the reaction. The reaction solution was dialyzed and freeze-dried to obtain methacrylated chitosan (CSMA), which was dried and stored at 4°C for later use. Preparation of S2, CSMA-Lycorine Injection: Dissolve CSMA in water and stir at room temperature for 12-24 h until clear to obtain a CSMA solution; Add the lycorine stock solution to the CSMA solution and stir in the dark for 30 min; add the photoinitiator and stir in the dark until completely dissolved to obtain CSMA-lycorine injection.

2. The preparation method of the lycorine-loaded functionalized methacryloxylated chitosan injection according to claim 1, characterized in that, In step S1, the chitosan has a degree of deacetylation ≥75% and a viscosity-average molecular weight of 50-190 kDa.

3. The preparation method of the lycorine-loaded functionalized methacryloxylated chitosan injection according to claim 1, characterized in that, In step S1, the dialysis includes: transferring the reaction solution into a dialysis bag with a molecular weight cutoff of 3.5-8 kDa, dialyzing with deionized water for 3 days, and changing the water twice a day.

4. The method for preparing the lycorine-loaded functionalized methacryloxylated chitosan injection according to claim 1, characterized in that, In step S1, the freeze-drying temperature is -50 °C and the time is 48-72 h.

5. The preparation method of the lycorine-loaded functionalized methacryloxylated chitosan injection according to claim 1, characterized in that, In step S1, the molar ratio of methacrylic anhydride to chitosan amino is 1:1-1.

2.

6. The preparation method of the lycorine-loaded functionalized methacryloxylated chitosan injection according to claim 1, characterized in that, In step S2, the concentration of lycorine in the CSMA-lycorine injection solution is 25 μM.

7. The method for preparing the lycorine-loaded functionalized methacryloxylated chitosan injection according to claim 1, characterized in that, In step S2, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite, and the concentration of the photoinitiator in the CSMA-lycorine injection solution is 0.2-0.3% (w / v).

8. A lycorine-loaded functionalized methacryloxylated chitosan injection, characterized in that, It is prepared by the method described in any one of claims 1-7.

9. A lycorine-loaded functionalized methacryloxylated chitosan gel, characterized in that, The injection solution prepared by the method according to any one of claims 1-7, or the injection solution according to claim 8, is irradiated under 405nm blue light or 365nm ultraviolet light for 10-30 s to solidify, thereby obtaining the gel.

10. The use of a lycorine-loaded methacrylated chitosan injection or gel in the preparation of a drug for treating intervertebral disc degeneration.

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