Temperature-sensitive chitosan-beta-sodium glycerophosphate hydrogel coated IGF-1 sustained-release microsphere preparation, preparation method and application of temperature-sensitive chitosan-beta-sodium glycerophosphate hydrogel coated IGF-1 sustained-release microsphere preparation

Abstract

The invention provides a temperature-sensitive chitosan-beta-sodium glycerophosphate hydrogel coated IGF-1 sustained release microsphere preparation as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. According to the sustained-release microsphere preparation prepared by the method, a hydrogel-coated microsphere system combines precise drug loading of microspheres and intelligent temperature responsiveness of hydrogel, and dual release of drugs in the hydrogel and the microspheres can be realized, so that the sustained-release microsphere preparation has a long-acting sustained-release function, the microspheres can protect the drugs from being degraded, and the sustained-release microsphere preparation has a long-acting sustained-release function; continuous release is realized by adjusting the degradation rate of the polymer, and frequent administration is avoided. In addition, the sustained-release microsphere preparation has the advantage of multi-response regulation, and the hydrogel can respond to pH, temperature or enzyme environment change and cooperate with the microspheres to realize on-demand drug release. Finally, cell-material interaction optimization is achieved, cellular uptake is promoted through the nanoscale structure of the microspheres, the hydrogel provides a bionic microenvironment, the drug effect is synergistically enhanced, and tissue repair is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel wrapped IGF-1 sustained-release microsphere preparation, a preparation method and application. BACKGROUND

[0002] IGF-1 is an important regulatory factor for skeletal muscle growth, which can promote muscle fiber hypertrophy and muscle satellite cell proliferation by activating the PI3K / Akt / mTOR signaling pathway, and significantly enhance muscle contraction. Although IGF-1 has shown potential in animal models, its clinical application in extraocular muscles is still blank, and its clinical translation needs to overcome challenges such as delivery technology and safety. At present, the key difficulties of IGF-1 injection are: 1) Extraocular muscles are rich in fast-twitch fibers, and their metabolic activity is significantly different from slow-twitch fibers. The unique fast-twitch fiber composition of extraocular muscles may lead to differences in drug response; 2) The local half-life of naked IGF-1 is short, and frequent injections are required to maintain an effective concentration, increasing the risk of tissue damage; 3) High-dose IGF-1 may induce local vascular proliferation or systemic metabolic disorders through paracrine effects, and there is a risk of carcinogenesis, and if free IGF-1 enters the blood circulation, it can affect the body's metabolic balance.

[0003] Therefore, the development of a new delivery system with sustained-release properties and the ability to precisely regulate the local bioavailability of IGF-1 is a core challenge to achieve targeted enhancement therapy for extraocular muscles.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] To solve the above technical problems, the present application provides a temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel wrapped IGF-1 sustained-release microsphere preparation, a preparation method and application. The hydrogel wrapped microsphere system designed in the present application combines the precise drug loading of microspheres with the intelligent temperature responsiveness of hydrogels, and the drug can achieve dual release in the hydrogel and the microsphere; at the same time, it realizes long-acting sustained release, protects the drug from degradation, and prolongs the action time of the drug. The prepared preparation can be better applied to eye diseases, realizing the local long-acting dual release of IGF-1.

[0006] In order to achieve the purpose of the present application, the following technical solutions are adopted: The present application provides a preparation method of a temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel wrapped IGF-1 sustained-release microsphere preparation, comprising the following steps: S1, dissolve chitosan in acetic acid solution to prepare a chitosan solution, and store at 4℃ for standby; S2, dissolve sodium beta-glycerophosphate in pre-cooled deionized water to prepare a sodium beta-glycerophosphate solution, and store at 4℃ for standby; S3, slowly drop the beta-glycerophosphate sodium solution into the chitosan solution to obtain a mixed solution; S4, disperse the mesoporous silica microspheres powder in the IGF-1 solution, then add the mixed solution, oscillate and incubate, centrifuge, discard the supernatant, and obtain the drug-loaded particles; S5, wash the drug-loaded particles, disperse them in deionized water, adjust the pH, adjust the concentration, control the concentration of the drug-loaded particle dispersion to be 5-20 mg / mL, put them into a constant temperature water bath, gel, and obtain the product.

[0007] Further, the diameter of the mesoporous silica microspheres is 3-5 nm.

[0008] Further, the mass concentration of the chitosan solution is 1-5%. The mass concentration of the beta-glycerophosphate sodium solution is 25-45%.

[0009] Further, the mass concentration of the chitosan solution is 3.33%. The mass concentration of the beta-glycerophosphate sodium solution is 25%.

[0010] Further, the specific steps of oscillation and incubation in the S4 step are oscillation and incubation at 4°C at a speed of 150 rpm for 12 hours.

[0011] Further, the reagent used for adjusting the pH in the S5 step is a NaOH solution with a mass concentration of ≤0.1 mol / L; the pH is adjusted to 6.8-7.4.

[0012] Further, the way of adjusting the pH is slowly adding the NaOH solution under ice bath and magnetic stirring, and the drop rate of the NaOH solution is 10 µL per 1.5 s.

[0013] The application also provides a temperature-sensitive chitosan-beta-glycerophosphate sodium hydrogel encapsulating IGF-1 sustained-release microsphere preparation prepared by the above preparation method, which is composed of an active ingredient, a carrier skeleton, a temperature-sensitive chitosan-beta-glycerophosphate sodium hydrogel, and a dispersion medium. The active ingredient is composed of IGF-1, which is encapsulated in mesoporous silica microspheres to form drug-loaded microspheres; the temperature-sensitive chitosan-beta-glycerophosphate sodium hydrogel encapsulates the drug-loaded microspheres, and then is dispersed in the dispersion medium.

[0014] Further, the mass ratio of chitosan: beta-glycerophosphate sodium: mesoporous silica microspheres is (1-4): (2-8): (1-4).

[0015] Further, the mass ratio of the chitosan: sodium beta-glycerophosphate: mesoporous silica microspheres is 1:2.5:1.

[0016] The application further provides a use of the above-mentioned IGF-1 sustained-release microsphere preparation wrapped by the temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel in preparation of a medicament for treating ophthalmic diseases.

[0017] Further, the ophthalmic disease is any one of the eye diseases requiring neuroprotection, tissue repair or anti-inflammatory treatment.

[0018] Further, the ophthalmic disease is an extraocular muscle dysfunction.

[0019] Further, the extraocular muscle dysfunction includes any one of comitant strabismus, paralytic strabismus and special types of restrictive strabismus.

[0020] The application has the following technical effects: The sustained-release microsphere preparation prepared by the method has the advantages of long-acting sustained release, multi-response regulation, cell-material interaction optimization, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 is a schematic diagram of the preparation of the temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel and electron microscope scanning; wherein, Figure 1 a is the transformation of the hydrogel from a liquid sol to a gel state; Figure 1 b is a gel structure diagram under a high-magnification scanning electron microscope; Figure 1 c is a gel structure diagram under a low-magnification scanning electron microscope; Figure 2: Schematic diagram for characterization test of temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel; Figure 3 : CCK-8 cell proliferation experiment results of 10 μg / mL in-vitro drug release kinetics test; Figure 4 : CCK-8 cell proliferation experiment results of 1 μg / mL in-vitro drug release kinetics test; Figure 5 : CCK-8 cell proliferation experiment results of 0.1 μg / mL in-vitro drug release kinetics test; Figure 6 : ELISA experiment results of 10 μg / mL in-vitro drug release kinetics test; Figure 7 : ELISA experiment results of 1 μg / mL in-vitro drug release kinetics test; Figure 8 : ELISA experiment results of 0.1 μg / mL in-vitro drug release kinetics test; Figure 9 : Cumulative release concentration of 10 μg / mL in-vitro drug release kinetics test; Figure 10 : Cumulative release concentration of 1 μg / mL in-vitro drug release kinetics test; Figure 11 : Cumulative release concentration of 0.1 μg / mL in-vitro drug release kinetics test. DETAILED DESCRIPTION

[0023] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0024] In a first aspect, the present application provides a preparation method of a temperature-sensitive chitosan-sodium beta-glycerophosphate hydrogel encapsulated IGF-1 sustained-release microsphere preparation, comprising the following steps: S1, dissolving chitosan in acetic acid solution to prepare a chitosan solution, and storing at 4℃ for standby; S2, dissolving sodium beta-glycerophosphate in pre-cooled deionized water to prepare a sodium beta-glycerophosphate solution, and storing at 4℃ for standby; S3, slowly dropping the sodium beta-glycerophosphate solution into the chitosan solution to obtain a mixed solution; S4, disperse mesoporous silica microspheres powder in IGF-1 solution, then add the mixture, oscillation incubation, centrifugation, discard the supernatant, obtain drug-loaded particles; S5, wash the drug-loaded particles, disperse into deionized water, adjust pH, adjust concentration, control the concentration of drug-loaded particle dispersion at 5-20 mg / mL, put into constant temperature water bath, gel, get.

[0025] The method realizes the construction of hydrogel wrapped drug-loaded microspheres through steps S1-S5. Chitosan and β-glycerophosphate sodium form a soluble complex under acidic conditions (S1-S3), which is the precursor of the temperature-sensitive hydrogel. After mesoporous silica microspheres load IGF-1, the surface of the silica is rich in silicon hydroxyl groups (Si-OH), which can graft various functional molecules through mature silanization reaction. This feature is the premise of realizing slow release, and through oscillation incubation, the microspheres are wrapped by the hydrogel precursor, so as to conveniently connect the temperature-sensitive chitosan hydrogel as a slow-release switch, thereby constructing a temperature / pH dual-responsive drug delivery system. The convenience and firmness of this modification are difficult to achieve by other carriers. Finally, adjust the pH to the physiological range (S5), trigger the gelation of the hydrogel at body temperature, and form a stable wrapped structure.

[0026] The structure constructed by this method can make the drug gradually release from the inside of the microspheres and the hydrogel network, realizing the combination of fast initial release and long-term slow release. At the same time, IGF-1 is shielded from enzyme degradation, the hydrogel provides a physical barrier, prolonging the half-life of the drug; in addition, the hydrogel is in sol state at low temperature, which is convenient for injection; at body temperature, it changes to gel, realizing local retention and reducing drug diffusion.

[0027] In some embodiments, the diameter of the mesoporous silica microspheres is 3-5 nm.

[0028] In some embodiments, the mass concentration of the chitosan solution is 1%-5%; The mass concentration of the β-glycerophosphate sodium solution is 25%-45%.

[0029] In some embodiments, the mass concentration of the chitosan solution is 3.33%; The mass concentration of the β-glycerophosphate sodium solution is 25%.

[0030] The concentration of chitosan and β-glycerophosphate sodium is limited, which is a key parameter for the formation of hydrogel. Chitosan provides amino groups for crosslinking with phosphate groups, and β-glycerophosphate sodium acts as a crosslinking agent and pH adjuster. The concentration range ensures moderate crosslinking density. Avoiding too low concentration leading to insufficient gel strength, or too high concentration causing premature gelation, to ensure injectability and gel reliability; at the same time, avoiding concentration affecting the pore size and degradation rate of the hydrogel, so as to adjust the drug diffusion speed.

[0031] In some embodiments, the specific step of oscillation incubation in the S4 step is low-speed oscillation incubation at 4℃ for 12 hours.

[0032] Incubation at 4℃, 150 rpm for 12 hours ensures that IGF-1 is fully dispersed and adsorbed into the pores of the mesoporous silica microspheres, and that the hydrogel precursor uniformly covers the microspheres; low temperature prevents IGF-1 from denaturation, and oscillation promotes mixing, achieving high encapsulation efficiency and drug loading; avoiding microsphere aggregation ensures that each microsphere is completely covered by the hydrogel, ensuring consistent release behavior.

[0033] In some embodiments, the reagent used for adjusting pH in the S5 step is a NaOH solution with a mass concentration of ≤0.1 mol / L; the pH is adjusted to 6.8-7.4.

[0034] Using a low concentration of NaOH to adjust the pH to 6.8-7.4 neutralizes the acetic acid system of chitosan, triggering the phase transition of chitosan-β-glycerophosphate sodium (from sol to gel), and precise pH control avoids local over-alkalization leading to gel rupture or drug burst release, while a physiological pH reduces tissue irritation, making it suitable for sensitive sites such as the eye.

[0035] In some embodiments, the method of pH adjustment is to slowly add the NaOH solution under ice bath and magnetic stirring, and the drop rate of the NaOH solution is 10 µL per 1.5 s.

[0036] Slowly adding NaOH under ice bath and magnetic stirring (10 µL per 1.5 s) ensures uniform pH change and avoids local high concentration; slow addition prevents rapid crosslinking, forming a dense and stable three-dimensional network, optimizing drug encapsulation.

[0037] In a second aspect, the present application also provides an IGF-1 sustained-release microsphere preparation encapsulated by the temperature-sensitive chitosan-β-glycerophosphate sodium hydrogel prepared by the above preparation method; the IGF-1 sustained-release microsphere preparation consists of active ingredients, carrier skeletons, temperature-sensitive chitosan-β-glycerophosphate sodium hydrogel, and dispersion medium. The active ingredients consist of IGF-1, which is encapsulated in mesoporous silica microspheres to form drug-loaded microspheres; the temperature-sensitive chitosan-β-glycerophosphate sodium hydrogel encapsulates the drug-loaded microspheres, which are then dispersed in the dispersion medium.

[0038] The above structure forms a "microsphere-hydrogel" structure, the microsphere can provide primary controlled release based on pore expansion, and the hydrogel can provide secondary controlled release based on degradation and responsiveness, the dual barrier of microspheres and hydrogels reduces drug leakage and degradation to provide multiple protection, and improves bioavailability.

[0039] In some embodiments, the mass ratio of the chitosan: beta-glycerophosphate sodium: mesoporous silica microspheres is (1-4):(2-8):(1-4).

[0040] In some embodiments, the mass ratio of the chitosan: beta-glycerophosphate sodium: mesoporous silica microspheres is 1:2.5:1.

[0041] For chitosan, beta-glycerophosphate sodium, mesoporous silica microspheres balanced hydrogel formation, drug loading and release kinetics, in the above ratio of chitosan provides enough amino, beta-glycerophosphate sodium to achieve efficient crosslinking, microspheres load appropriate amount of IGF-1. Drug release follows first-order kinetics, (Fickian diffusion), the release time is prolonged for several weeks to several months, the size of the silica microspheres can also promote intracellular storage, also facilitate the hydrogel to provide a biomimetic environment, enhance tissue repair.

[0042] In a third aspect, the present application also provides a use of the above-mentioned IGF-1 sustained-release microspheres preparation wrapped by the temperature-sensitive chitosan-beta-glycerophosphate sodium hydrogel in the preparation of a medicament for treating ophthalmic diseases.

[0043] In some embodiments, the ophthalmic disease is any one of the eye diseases requiring neuroprotection, tissue repair or anti-inflammatory treatment.

[0044] In some embodiments, the ophthalmic disease is extraocular muscle dysfunction.

[0045] In some embodiments, the ophthalmic disease includes any one of convergent squint, paralytic squint and special types of restrictive squint.

[0046] The following will be described in conjunction with specific embodiments: Embodiment 1: S1, weigh 1g of chitosan (purchased from Shanghai Ruon Reagent Co., Ltd., item number: RH768220), dissolve in 30mL of 1% acetic acid solution under ice bath condition, prepare a chitosan solution with a mass fraction of 3.33%, stir until completely dissolved, and store at 4℃ for standby.

[0047] S2, weigh 2.5g of beta-glycerophosphate sodium (purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., item number: G806967-25g), dissolve in 7mL of pre-cooled pure water, and finally make up to 10mL to prepare a beta-glycerophosphate sodium solution with a mass fraction of 25%, store at 4℃ for standby.

[0048] S3, slowly drop the prepared beta-glycerophosphate sodium solution into the chitosan solution under ice bath and magnetic stirring, make sure that no white flocculent precipitates are generated during dropping, and finally the mixture is a transparent viscous liquid under ice bath.

[0049] S4, 1.0 g of dry mesoporous silica microspheres powder (purchased from Jiangsu Zhichuan Science and Technology Co., Ltd.) was weighed and dispersed in 1 mL of 10 pg / mL IGF-1 (IGF-I / IGF-1 protein, Mouse, purchased from MedChemExpress Biotechnology Co., Ltd., USA) solution (10 pg / mL), then the mixture was added and placed on a shaker for incubation at 4°C at 150 rpm for 12 hours, then centrifuged at 15000 rpm for 15 minutes.

[0050] S5, the supernatant and drug-loaded particles obtained by centrifugation were collected, and the remaining protein concentration in the supernatant was determined using a BCA protein quantification kit.

[0051] S6, discard the supernatant, gently wash the precipitated drug-loaded particles twice with pre-cooled pure water to wash off the physically adsorbed proteins on the surface. Finally, the drug-loaded particles were re-dispersed in 5 mL of pure water and stored at 4°C for standby.

[0052] S7, adjust the pH: calibrate the pH meter, then prepare a 0.1 mol / L NaOH solution (>0.1 mol / L will make the solution gel instantly and irreversibly), slowly drop under ice bath and magnetic stirring, 10 microliters per 1.5 seconds, measure the pH while adding until the range is 6.8-7.4; the final mixture is diluted to 50 mL with pure water to control the concentration of the drug-loaded particle dispersion at 5 mg / mL-20 mg / mL, and placed in a 37°C constant temperature water bath, observe the gel state every 2 minutes, when the gel state is no longer a flowing liquid, the preparation is complete, as shown in Figure 1 a.

[0053] The prepared gel was characterized and determined, and the experimental results are shown in Figure 1 , wherein the gel under high magnification scanning electron microscope can be seen from Figure 1 b. Figure 1 c is the gel under low magnification scanning electron microscope, which has a dense network cross-linked structure.

[0054] The viscoelastic properties of the prepared gel were determined.

[0055] 1. Test method In order to verify the viscoelastic properties of the hydrogel of the present application, a rotary rheometer was used for rheological characterization. The specific steps are as follows: (1) The obtained hydrogel sample was cut into a circular piece with a diameter of about 25 mm and placed in a parallel plate system of the rheometer (the upper and lower plates have a diameter of 25 mm and a gap of 1 mm).

[0056] (2) The test temperature was kept constant at 25±0.1°C by the temperature control system.

[0057] (3) First, a strain scan was performed at a fixed frequency (1 Hz) to determine the linear viscoelastic region (LVR). The results showed that when the strain was less than 0.5%, the storage modulus (G′) and loss modulus (G″) remained stable. Therefore, 0.5% strain was selected as the subsequent test condition.

[0058] (4) Perform frequency scanning within the LVR, with an angular frequency range of 0.1-100 rad / s.

[0059] (5) Record parameters such as energy storage modulus (G′), loss modulus (G″), complex modulus (|G|), loss factor (tanδ), and complex viscosity (|η|).

[0060] 2. Test Results Test results are as follows Figure 2 As shown in the curves (G′, G″ versus frequency), the test results reveal the characteristics of the hydrogel, i.e., the modulus, as a function of frequency: Storage modulus (G′): In the range of 0.1–100 rad / s, G′ remains consistently at 10. 3 -10 4 The Pa level is significantly higher than G″, indicating a predominantly elastic solid-like characteristic. In the high-frequency region (>80 rad / s), G′ increases slightly, indicating that the hydrogel exhibits higher resistance to deformation under rapid loading conditions. Loss modulus (G″): G″ remains stable at 10 throughout the entire frequency range. 2 The values ​​are around Pa, much lower than G′, indicating limited energy dissipation in the system. A few fluctuations occur at high frequencies, but these do not alter the overall trend. The loss factor (tanδ) is mostly in the 0.1–0.5 range, significantly less than 1, demonstrating the sample's strong elasticity and structural stability. The complex modulus (|G|) gradually increases with frequency, indicating enhanced mechanical rigidity of the hydrogel under high-frequency shear. The complex viscosity (|η|) decreases with increasing frequency, exhibiting typical shear-thinning characteristics, which is beneficial for subsequent injection molding or processing applications.

[0061] 3. Technical Effects The rheological tests described above lead to the conclusion that: (1) the hydrogel of the present invention satisfies G′>>G″ over a wide frequency range, exhibiting excellent elastic dominance and solid-like structural characteristics; (2) the low loss factor (tanδ<0.5) indicates that the system has low energy dissipation and good shape retention capability; (3) the complex viscosity decreases with increasing frequency, indicating good processing fluidity and application stability; (4) in summary, the hydrogel has significant application value in the fields of drug sustained release, tissue engineering, injectable therapy and flexible electronic devices.

[0062] Experimental Example 1: Determination of the effect of thermosensitive chitosan-β-glycerophosphate sodium hydrogel IGF1 sustained-release microsphere formulation on cell proliferation. The specific steps are as follows: 1. Testing Method (1) The hydrogel prepared in this invention was mixed with microspheres / IGF-1 solution to prepare hydrogel experimental groups encapsulating different concentrations of IGF-1 (0.1 μg / ml, 1 μg / ml, 10 μg / ml). At the same time, a blank hydrogel group without IGF-1 was set up as a negative control.

[0063] (2) Select MC3T3-E1 cells, digest and count them, and then divide them into 5×10⁶ cells per well. 3 The cells were evenly seeded at a density in 96-well plates and pre-cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere completely.

[0064] (3) After the cells adhered, the original culture medium was removed. The experimental groups were given complete culture medium containing different concentrations of microspheres / IGF-1 hydrogel; the control group was given an equal volume of blank hydrogel culture medium. Each group was set up with 6 replicates.

[0065] (4) At different time points after drug treatment, add 10 μL of CCK-8 solution to each well. Then put the 96-well plate back into the incubator and continue incubation for 2 hours.

[0066] (5) Use an ELISA reader to measure the absorbance (OD value) of the solution in each well at a wavelength of 450 nm. Record the average OD value of each group at each time point. This value is directly proportional to the number of live cells and can directly reflect the cell proliferation.

[0067] 2. Test Results Experimental results are as follows Figures 3-5 As shown, the CCK-8 assay was used to evaluate the effect of different concentrations of IGF-1 encapsulated in hydrogel on cell proliferation. Figure 3 The results of an in vitro drug release kinetics assay for CCK-8 cell proliferation were obtained using a 10 μg / mL concentration. Figure 4 The results of an in vitro drug release kinetics assay for CCK-8 cell proliferation were obtained using a drug release kinetics assay at a concentration of 1 μg / mL. Figure 5 The results of the CCK-8 cell proliferation assay were obtained using in vitro drug release kinetics at a concentration of 0.1 μg / mL. By comparing the absorbance values ​​of the experimental groups and the control group at different time points using microspheres / IGF-1 hydrogels of different concentrations, the following conclusions can be drawn: (1) Concentration-dependent effect of IGF-1: High concentration group (10 μg / ml IGF-1): The cell proliferation activity (OD value) of this group was significantly higher than that of other groups throughout the observation period. This indicates that the hydrogel can effectively load and release high doses of IGF-1, continuously and strongly promoting cell proliferation. Medium concentration group (1 μg / ml IGF-1): This group also showed a clear proliferation-promoting effect, with an OD value between that of the high concentration group and the low concentration group, indicating moderate biological activity. Low concentration group (0.1 μg / ml IGF-1): At early time points (1 h-24 h), its proliferation-promoting effect was not significantly superior to that of the control group, but a slight promoting effect was still observed at later times (48 h-72 h), indicating that even low doses of IGF-1 can have long-term effects under the sustained release of this hydrogel.

[0068] (2) Time-dependent release and proliferation-promoting kinetics: Short-term effect (1 h-8 h): The OD values of all experimental groups began to show an upward trend, especially the 10 μg / ml group, which showed a strong signal at an early stage, indicating that the hydrogel can achieve initial rapid release of IGF-1, quickly starting the proliferation-promoting process. Long-term effect (24 h-72 h): During the 24-hour to 72-hour period, the OD values of each experimental group and the control group differed most significantly and continued to expand. This strongly demonstrates that the hydrogel and microspheres of the present application can continuously and slowly release IGF-1, rather than a one-time burst release, thereby achieving long-term and stable promotion of cell proliferation. At 72 hours, the OD value of the 10 μg / ml group reached a peak, showing the strongest proliferation-promoting ability.

[0069] 3. Technical effects Through the above cell proliferation experiment, it can be confirmed that: (1) Preservation of biological activity: The preparation process of the hydrogel of the present application does not destroy the growth factor activity of IGF-1, and the encapsulated IGF-1 can still effectively exert its biological function. (2) Sustained release characteristics: The experimental results show a clear time dependence, demonstrating that the hydrogel system can serve as an excellent sustained release carrier for IGF-1, avoiding rapid degradation of the growth factor and extending its action time. (3) Significant proliferation-promoting effect: The microspheres / IGF-1 encapsulated by the hydrogel, especially at concentrations of 10 μg / ml and 1 μg / ml, can significantly promote cell proliferation, far superior to the blank control group.

[0070] Experimental Example 2: In vitro drug release kinetics test ELISA experiment The specific experimental steps are as follows: 1. Test method (1) Accurately weigh a certain amount of the hydrogel of the present application, mix it with a known concentration of microspheres / IGF-1 solution (initial loading concentrations are 0.1 μg / ml, 1 μg / ml, and 10 μg / ml, respectively) uniformly to prepare a drug-loaded hydrogel sample.

[0071] (2) Place the drug-loaded hydrogel sample in a dialysis bag and immerse it in a predetermined release medium (bovine serum albumin solution, BSA). Place the entire system in a constant temperature water bath at 37°C to simulate the internal environment of the human body and maintain uniform concentration.

[0072] (3) At a predetermined time point, quantitatively aspirate a certain volume of sample solution from the release medium.

[0073] (4) Quantitatively analyze the IGF-1 concentration in the collected sample solution by enzyme-linked immunosorbent assay (ELISA). After each sampling, immediately supplement the release system with an equal temperature and volume of fresh release medium.

[0074] (5) According to the measured concentrations at each time point, calculate the cumulative release amount and cumulative release percentage of IGF-1, and draw the release curve.

[0075] 2. Experimental results The release kinetics test results are shown in Figures 6-11 , which are the ELISA experimental results of in vitro drug release kinetics test at 10 μg / mL; Figure 6 , which are the ELISA experimental results of in vitro drug release kinetics test at 1 μg / mL; Figure 7 , which are the ELISA experimental results of in vitro drug release kinetics test at 0.1 μg / mL; Figure 8 , which are the cumulative release concentrations of in vitro drug release kinetics test at 10 μg / mL; Figure 9 , which are the cumulative release concentrations of in vitro drug release kinetics test at 1 μg / mL; Figure 10 , which are the cumulative release concentrations of in vitro drug release kinetics test at 0.1 μg / mL. The release regularity of the hydrogel for microspheres / IGF-1 under different initial loading amounts is shown. Figure 11

[0076] ​(1) Concentration dependence of release behavior: High concentration group (10 pg / ml IGF-1): This group showed the highest cumulative release concentration throughout the 72 hours. The release profile showed a relatively fast release phase in the initial stage (0-8 hours), followed by a slower release rate, entering a steady sustained release period. This indicates that the hydrogel can load and release a large amount of IGF-1. Medium and low concentration groups (1 pg / ml and 0.1 pg / ml IGF-1): The release trends of these two groups are similar to the high concentration group, but the absolute release concentration at each time point is lower, proportional to the initial loading amount. Its release curve also presents the characteristics of fast and slow.

[0077] (2) Typical biphasic release kinetics: The release curves of microspheres / IGF-1 of all concentrations showed a typical biphasic release pattern, which is a key feature of an ideal drug delivery system: Initial burst release phase: Within the first few hours (0-8h), a relatively fast release can be observed. This is mainly due to the rapid diffusion of IGF-1 adsorbed on the surface or near the surface of the hydrogel. Subsequent slow release phase: During 24 hours to 72 hours, the release rate slows down significantly and tends to be stable. The release in this phase is mainly controlled by the swelling of the hydrogel network, the degradation of the microspheres, and the slow diffusion of the IGF-1 wrapped inside. This phase is crucial for achieving the long-acting effect of the drug.

[0078] 3. Technical effects (1) Controllable release ability: The hydrogel of the present application can effectively wrap microspheres / IGF-1 and achieve controllable release, rather than one-time explosive release.

[0079] (2) Excellent sustained release characteristics: Its biphasic release pattern - i.e. moderate initial release to meet early biological needs, and long-term sustained release to maintain long-acting effect - meets the needs of growth factors in tissue regeneration and repair processes.

[0080] (3) Dose dependence: The total amount of release is positively correlated with the initial loading concentration, indicating that the final release dose of growth factors can be accurately controlled by adjusting the drug loading amount, achieving personalized treatment.

[0081] (4) Association with biological function: The release kinetics results are highly consistent with cell proliferation experiments, demonstrating that the sustained release characteristics of the hydrogel and microspheres are the physical basis for their long-acting pro-proliferation effect in biology.

[0082] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation, characterized in that, Includes the following steps: S1. Dissolve chitosan in acetic acid solution to prepare chitosan solution, and store at 4℃ for later use; S2. Dissolve sodium β-glycerophosphate in pre-cooled deionized water to prepare a sodium β-glycerophosphate solution, and store at 4°C for later use. S3. Slowly add the sodium β-glycerophosphate solution dropwise into the chitosan solution to obtain a mixture; S4. Disperse mesoporous silica microsphere powder in IGF-1 solution, then add the mixture, shake and incubate, centrifuge, discard the supernatant, and obtain drug-loaded particles; S5. Wash the drug-loaded particles, disperse them in deionized water, adjust the pH and concentration to control the concentration of the drug-loaded particle dispersion at 5 mg / mL-20 mg / mL, place it in a constant temperature water bath to form a gel, and the product is obtained.

2. The method for preparing the thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 1, characterized in that, The chitosan solution has a mass concentration of 1%-5%; The mass concentration of the β-glycerophosphate sodium solution is 25%-45%.

3. The method for preparing the thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 1, characterized in that, The specific step of the oscillation incubation in step S4 is to oscillate and incubate at 150 rpm at 4°C for 12 hours.

4. The method for preparing the thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 1, characterized in that, In step S5, the reagent used to adjust the pH is a NaOH solution with a mass concentration ≤ 0.1 mol / L; the pH is adjusted to 6.8-7.

4.

5. The method for preparing the thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 4, characterized in that, The pH was adjusted by slowly adding the NaOH solution dropwise in an ice bath with magnetic stirring, at a rate of 10 µL every 1.5 seconds.

6. A thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation prepared by the method described in any one of claims 1-5, characterized in that, The IGF-1 sustained-release microsphere formulation consists of an active ingredient, a carrier backbone, a thermosensitive chitosan-β-glycerophosphate sodium hydrogel, and a dispersion medium. The active ingredient is composed of IGF-1, which is encapsulated in mesoporous silica microspheres to form drug-loaded microspheres; the thermosensitive chitosan-β-glycerophosphate sodium hydrogel encapsulates the drug-loaded microspheres and is then dispersed in the dispersion medium.

7. The thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 6, characterized in that, The mass ratio of chitosan: sodium β-glycerophosphate: mesoporous silica microspheres is (1-4):(2-8):(1-4).

8. The thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 6, characterized in that, The mass ratio of chitosan:sodium β-glycerophosphate:mesoporous silica microspheres is 1:2.5:

1.

9. The use of the thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation as described in claim 8 in the preparation of a medicament for treating ophthalmic diseases.

10. The application of the thermosensitive chitosan-β-glycerophosphate sodium hydrogel-encapsulated IGF-1 sustained-release microsphere formulation according to claim 9 in the preparation of a drug for treating ophthalmic diseases, characterized in that, The ophthalmic disease mentioned is any one of the ophthalmic diseases that requires neuroprotection, tissue repair, or anti-inflammatory treatment.

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