Anti-immunological rejection FK506 ZIF-8 / HTCC ophthalmic gel and preparation method thereof

Abstract

The invention relates to an anti-immunological rejection FK506 (at) ZIF-8 / HTCC ophthalmic gel and a preparation method thereof, FK506 and a zeolite imidazole skeleton (ZIF-8) are combined to prepare FK506 loaded nanoparticles (FK506 (at) ZIF-8), and quaternized chitosan is innovatively used as a sol of the FK506 (at) ZIF-8 to prepare the ophthalmic gel for use. The problems that a novel potent immune preparation tacrolimus administration mode is short in drug retention time, fast in metabolism, poor in cornea permeability and insoluble in water, and consequently the bioavailability of the FK506 eye drops is low are solved. Compared with an FK506 direct administration mode, the administration mode of the novel nanoparticle drug-loaded ophthalmic gel is higher in drug utilization rate, has a treatment effect on corneal transplantation immunological rejection, and delays the occurrence and development of transplantation rejection.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of hydrogel based on nano drug-loading platform (ZIF-8), quaternary ammonium chitosan (HTCC) and tacrolimus (FK506), for treating immune rejection reaction occurred after corneal transplantation, belong to medical material technical field. BACKGROUND

[0002] Corneal opacity is the second leading cause of blindness worldwide, and corneal transplantation surgery is the only effective method to treat corneal blindness caused by corneal opacity. There are more than 180,000 cases of corneal transplantation surgery worldwide each year to restore the vision of corneal blind patients. However, about 30% of the transplanted patients will experience postoperative immune rejection, and one-third of the patients eventually lead to graft failure. Therefore, alleviating or even eliminating the immune-mediated immune rejection of the graft is crucial for the prognosis of corneal transplant recipients. In order to prevent and treat immune rejection, hormone drugs and immunosuppressants are commonly used in clinical practice. In recent years, a new type of powerful immunoreagent, tacrolimus (Tacrolimus), also known as FK506, has shown better application effect than traditional immunosuppressant cyclosporine A (CsA) in the prevention and treatment of corneal graft rejection. However, due to the short drug retention time, rapid metabolism, poor corneal permeability and drug insolubility in water of the traditional drug delivery method, the bioavailability of FK506 eye drops is low. Therefore, it is particularly important to develop a new drug delivery system to improve the retention time and permeability of the drug in the cornea.

[0003] At present, the cause of corneal graft opacity caused by immune rejection is not completely clear. Related studies have shown that corneal transplant immune rejection is a kind of immune response mainly based on cellular immunity, and various factors cooperate together. Graft immune rejection is usually caused by the complex interaction between lymphangiogenesis (afferent end of the immune loop), angiogenesis (efferent end of the immune loop) and inflammation. In short, the host is first sensitive to donor antigens, then APC is activated, and then induces T cell proliferation in the draining lymph node. Subsequently, the inflammatory cytokines (such as IL-2, IFN-γ, IL-4, IL-8) secreted by CD4+ and CD8+ T cells are transported to the graft site through blood vessels. Finally, the rejection reaction starts, and the graft will be continuously attacked until it fails. The presence of perfused blood vessels around the corneal bed after corneal transplantation is an important channel for transporting T cells and various immune factors. Penetrating keratoplasty will replace the autologous corneal endothelium with allogeneic corneal endothelial tissue due to full-thickness lesions of the cornea, increasing the risk of rejection. And the repeated entry of intraocular instruments into the anterior chamber during surgery increases the inflammatory response in the anterior chamber, which is an important factor that causes the disturbance of the intraocular immune 'pardon' environment. To prevent the occurrence of immune rejection after corneal transplantation, in addition to avoiding full-thickness corneal transplantation during surgery, minimizing the range of lesions drilled, and removing or blocking new blood vessels around the graft bed, drug application is the main way to prevent immune rejection after corneal transplantation.

[0004] In clinical practice, patients with high-risk corneal transplantation who develop immune rejection within 2 months usually use systemic glucocorticoid application combined with local treatment, and the rejection signs can be observed to subside. Early diagnosis and appropriate glucocorticoid treatment can reverse most immune rejection reactions, but glucocorticoids have long-term side effects and related complications, limiting their more lasting role in preventing immune rejection. Therefore, finding more potent immunosuppressive drugs has become an important research direction for preventing immune rejection.

[0005] The immunosuppressive agents CsA and FK506 are most widely used in clinical applications, and the preventive rejection effects of systemic application of both have been reported at home and abroad. The results of the study show that the in vitro inhibitory effect of FK506 on lymphocyte activity is 10-100 times that of CsA. FK506 is a calcineurin inhibitor, which binds to FK506 binding protein to reduce the activity of calcineurin, reduces the immune response through NFAT and IL-2 signal transduction, and thus destroys the activity of T cells. In ophthalmic clinics, FK506 eye drops can effectively prolong the survival time of corneal grafts in patients, but due to the presence of the blood-ocular barrier, it is difficult for the drug to maintain a therapeutic concentration in the eye for a long time, and long-term systemic use has obvious side effects, such as nephrotoxicity, hypertension, hypersensitivity, and central nervous system changes. Although eye drops have the advantages of easy administration and good compliance, the corneal barrier leads to poor absorption of drug molecules, and the lacrimal and nasolacrimal ducts expel the drug out of the eye and into the systemic circulation through other pathways, so transplant recipients need higher drug doses or administration frequency to maintain an effective therapeutic concentration in the anterior chamber.

[0006] Based on the above, the development of a drug delivery system not only improves the treatment time of the drug in the eye, but also increases the penetration of the drug on the cornea, and the above-mentioned several ways to improve the bioavailability of water-insoluble drugs such as FK506 in the eye are the problems that the skilled in the art are eager to solve. SUMMARY

[0007] Based on the characteristics of ZIF-8 biological safety, corneal permeability, drug release (pH = 7.4), and positive potential, we chose ZIF-8 as the drug delivery platform for FK506, and synthesized FK506@ZIF-8 nanoparticles. Due to the large surface area of FK506@ZIF-8 nanopowder, it is easy to aggregate and precipitate, which limits its penetration ability on the cornea, and FK506@ZIF-8 nanopowder is not easy to use directly. The biological safety of chitosan has been proven in the field of ophthalmology, and experiments have shown that chitosan can replace sodium hyaluronate as a viscoelastic agent for cataract surgery, and there is no statistical significance in the incidence of postoperative high intraocular pressure compared with the use of sodium hyaluronate. The introduction of quaternary ammonium groups on the amino groups of chitosan molecules can generate cationic HTCC. Through the electrostatic interaction between the quaternary ammonium salt groups in HTCC and the negatively charged proteins on the surface of the cornea, the nanoparticles are fixed on the surface of the cornea, prolonging the retention time of the nanoparticles. The present application uniformly disperses FK506@ZIF-8 nanopowder in viscous HTCC to form an ophthalmic sol, and finally prepares FK506@ZIF-8 / HTCC ophthalmic gel for use.

[0008] The application discusses in detail the composition, structure, drug loading and in-vitro drug release behavior of the prepared FK-506@ZIF-8 nanoparticles. In addition, the dispersibility and long-term stability of FK-506@ZIF-8 nanoparticles in HTCC sol are evaluated. New Zealand rabbits are used for drug corneal retention and penetration experiments, a high-risk corneal transplantation model of rats is established, clinical observation scores are established, HE staining is performed, and the number of CD4+ and CD8+ T cells is observed by immunohistochemistry to verify the therapeutic effect of the gel.

[0009] The purpose of the application is to solve the problems of low drug utilization rate, short action time and poor patient dependence of FK506 eye drops in treating immune rejection after corneal transplantation. The application realizes the penetration of FK506 through the cornea by loading FK506 on the ZIF-8 nano-drug delivery platform combined with quaternary ammonium chitosan sol, and adjusting the pH to about 7, which has the functions of pH-responsive release and long-acting immunity.

[0010] To achieve the above purpose, the application provides a preparation method of a novel anti-immune rejection FK506@ZIF-8 / HTCC ophthalmic gel, comprising the following steps: (1) Preparation of ZIF-8 nanoparticles Zn(NO3)2·6H2O is dissolved in deionized water, and C4H6N2 is dissolved in methanol. The two solutions are mixed and reacted on a vortex instrument for 5 minutes to obtain a white suspension. Then the suspension is centrifuged to obtain a centrifuged product (ZIF-8 nanoparticles). The obtained nanoparticles are irradiated with a UV lamp for 30-60 min, and then stored in a 4℃ refrigerator to obtain ZIF-8 nanoparticles. The centrifugation meets the following conditions: rotation speed is 12000 rpm / min, and time is 15 minutes.

[0011] (2) Preparation of FK506@ZIF-8 nanoparticles Zn(NO3)2·6H2O is dissolved in 5ml deionized water, and C4H6N2 and FK506 drug powder are dissolved in methanol. The two solutions are mixed and vortexed on a vortex instrument for 5 minutes to obtain a white suspension.

[0012] The white suspension is dispersed and centrifuged to obtain a centrifuged product (FK506@ZIF-8 nanoparticles). Then it is dispersed and dissolved in anhydrous ethanol solution using a centrifuge, eluted 3 times, and the residual methanol solution is removed. The final product is irradiated with a UV lamp for 40 min, and then stored in a 4℃ refrigerator to obtain FK506@ZIF-8 nanoparticles.

[0013] The centrifugal treatment satisfies the following conditions: a rotation speed of 12000 rpm / min and a time of 15 minutes.

[0014] (3) FK506@ZIF-8 / HTCC eye gel High-pressure sterilized double distilled water is added to the chitosan quaternary ammonium salt powder in a proportion, and a magnetic stirrer is stirred to obtain a quaternized chitosan sol of 0.1-2% mass concentration (preferably a quaternized chitosan sol of 1% mass concentration is configured); FK506@ZIF-8 nanoparticles are uniformly mixed with the chitosan sol, and an acid-base buffer is used to adjust the pH to about 7 to prepare the FK506@ZIF-8 / HTCC eye gel, which is stored in a refrigerator at 4°C. The present application has the following advantages and improvements over the current problems: the present application combines FK506 with ZIF-8 to prepare FK506@ZIF-8 nanoparticles, and uses quaternized chitosan as a sol of FK506@ZIF-8 to prepare an eye gel, which has a higher drug utilization rate through the administration method, has a therapeutic effect on corneal transplantation immune rejection, and delays the occurrence and development of transplantation rejection. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can also be obtained from the provided drawings without creative labor.

[0016] Figure 1 The figure is the action diagram and use schematic diagram of the FK506@ZIF-8 / HTCC eye gel.

[0017] Figure 2 A: ZIF-8 nanoparticles B: FK506@ZIF-8 nanoparticles C: FK506@ZIF-8 / HTCC eye gel D: FK506@ZIF-8 / HTCC eye gel, the pH value of which is about 7.

[0018] Figure 3 A: ZIF-8 nanoparticles B: FK506@ZIF-8 nanoparticles C: FK506@ZIF-8 / HTCC eye gel D: FK506@ZIF-8 / HTCC eye gel, the pH value of which is about 7.

[0019] Figure 4Transmission electron microscopy (TEM) of ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles, A: ZIF-8 nanoparticles (100 nm and 10 nm); B: FK506@ZIF-8 nanoparticles (100 nm and 10 nm).

[0020] Figure 5 DLS results of ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles, A: average particle size of ZIF-8 nanoparticles; B: average particle size of FK506@ZIF-8 nanoparticles; C: Zeta potential of ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles.

[0021] Figure 6 Fourier transform infrared spectroscopy (FTIR) of (A) FK506 powder, ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles; (B) local magnification in high frequency region.

[0022] Figure 7 XRD detection, BET specific surface area detection, UV-Vis detection results, A: XRD test pattern of FK506 powder, ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles; B: BET pattern of ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles; C: UV-Vis absorption spectrum of FK506 powder, ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles.

[0023] Figure 8 (A) ZIF-8 nanoparticles thermogravimetric and differential thermal (DTG) diagram; (B) FK506@ZIF-8 nanoparticles DTG diagram; (C) FK506 powder DTG diagram.

[0024] Figure 9 (A) Standard curve of FK506 (B) 24h in vitro cumulative release curve of FK506@ZIF-8 nanoparticles.

[0025] Figure 10 Release model fitting curve results, A: in vitro release curve; B: Weibull model C: first-order model D: Higuchi model E: Korsmeyer-Peppas model F: Kopcha model.

[0026] Figure 11For CCK8 cell viability test and live / dead cell staining test results, A: CCK8 cell viability test results B: live / dead cell staining test results.

[0027] Figure 12 For (A) simple FK506 fluorescein eye drops and FK506@ZIF-8 / HTCC gel eye drops corneal retention time comparison chart. (B) is the FK506@ZIF-8 / HTCC ophthalmic gel can effectively improve the penetration of the drug in the cornea chart.

[0028] Figure 13 For the general eye photograph of rats after allogeneic in situ corneal transplantation (12 days and 24 days); control group, FK506@ZIF-8 / HTCC ophthalmic gel group.

[0029] Figure 14 For the rat corneal graft survival curve chart of FK506 eye drop group.

[0030] Figure 15 For the effect chart of FK506@ZIF-8 / HTCC ophthalmic gel on the treatment of allogeneic in situ corneal transplantation, A rat corneal HE staining, immunohistochemical results B: CD4+ T cell average optical density statistical results C: CD8+ T cell average optical density statistical results. DETAILED DESCRIPTION

[0031] Example 1 The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0032] The present embodiment provides a kind of nanoparticle drug-loaded ophthalmic gel and its preparation method, including FK506 is loaded in ZIF-8 nanoparticle, and is prepared into FK506@ZIF-8 / HTCC ophthalmic gel in combination with HTCC;Including the following steps: (1) preparation of ZIF-8 nanoparticle 150mg of Zn (NO3) 2·6H2O is dissolved in deionized water, and 330mg of C4H6N2 is dissolved in methanol; The two solutions are mixed and reacted on a vortex instrument at room temperature for 5 minutes to obtain a white suspension, and then the suspension is centrifuged to obtain a centrifuged product (ZIF-8 nanoparticle); The obtained nanoparticles are stored in a refrigerator at 4°C after irradiation with an ultraviolet lamp for 40 min to prepare ZIF-8 nanoparticles.

[0033] The centrifugal treatment meets the following conditions: a rotation speed of 12000 rpm / min and a time of 15 min.

[0034] (2) Preparation: FK506@ZIF-8 nanoparticles 150 mg of Zn(NO3)2·6H2O is dissolved in 5 ml of deionized water, and 330 mg of C4H6N2 and 10 mg of FK506 drug powder are dissolved in methanol; The two solutions are mixed and vortexed on a vortex instrument for 5 min to obtain a white suspension.

[0035] The white suspension is dispersed and centrifuged to obtain a centrifugal product (FK506@ZIF-8 nanoparticles), which is then dispersed in anhydrous ethanol solution and washed with a centrifuge for 3 times to remove residual methanol solution. The final product is stored in a refrigerator at 4°C after irradiation with an ultraviolet lamp for 40 min to prepare FK506@ZIF-8 nanoparticles.

[0036] The centrifugal treatment meets the following conditions: a rotation speed of 12000 rpm / min and a time of 15 min.

[0037] (3) FK506@ZIF-8 / HTCC eye gel High-pressure sterilized double-distilled water is added to the chitosan quaternary ammonium salt powder in a proportion, and a magnetic stirrer is used for stirring to obtain a 1% mass concentration of quaternized chitosan sol; the FK506@ZIF-8 nanoparticles and the chitosan sol are fully mixed, and an acid-base buffer is used to adjust the pH to about 7 to prepare FK506@ZIF-8 / HTCC eye gel, which is stored in a refrigerator at 4°C.

[0038] The prepared ZIF-8 nanoparticles, FK506@ZIF-8 nanoparticles and FK506@ZIF-8 / HTCC eye gel are shown in Figure 2 as shown in Figure 2 B The FK506@ZIF-8 nanoparticles are yellowish compared with the ZIF-8 nanoparticles, and the pH value of the FK506@ZIF-8 / HTCC eye gel is about 7.

[0039] Example 2 In this example, the surface morphology of the ZIF-8 nanoparticles, FK506@ZIF-8 nanoparticles and FK506@ZIF-8 / HTCC eye gel prepared in Example 1 is detected.

[0040] Morphology inspection: (1) such as Figure 3 As shown, SEM was used to observe the morphology of ZIF-8 nanoparticles, FK506@ZIF-8 nanoparticles, and the lyophilized powder of FK506@ZIF-8 / HTCC ophthalmic gel. Compared with the three-dimensional structure of ZIF-8 nanoparticles, FK506@ZIF-8 nanoparticles have an irregular morphology and smaller volume, and the FK506@ZIF-8 nanoparticles are distributed in HTCC.

[0041] (2) For example Figure 4 As shown, high-resolution TEM observations revealed the internal morphology of ZIF-8 and FK506@ZIF-8 nanoparticles. The ZIF-8 nanoparticles exhibited a smooth surface without obvious macropores or irregular structures, consistent with typical ZIF-8 nanoparticle characteristics. The FK506@ZIF-8 nanoparticles showed slight deformation, indicating that drug loading may have affected their surface structure. Compared to the unloaded form, the FK506@ZIF-8 nanoparticles were significantly smaller, a difference more pronounced under high magnification.

[0042] Hydrogel structure detection (1) such as Figure 5 As shown, the FK506@ZIF-8 nanoparticles are positively charged, which is beneficial for interacting with the negatively charged surface of the cornea and facilitating corneal diffusion. The average particle size of the ZIF-8 nanoparticles is 210.5±0.9075 nm; the Zeta potential is 12.2±0.85 mV. Compared with the SEM results, the average particle size of the nanoparticles is relatively large. This may be because the hydration dispersion in methanol solvent may lead to the material detection result being larger than the actual size. The average particle size of the FK506@ZIF-8 nanoparticles is 91.68±0.1258 nm; the Zeta potential is 2.77±0.245 mV. The inclusion of ZIF-8 in FK506 results in a reduction in nanosize and an alteration in charge properties.

[0043] (2) For example Figure 6As shown in A, the FTIR spectrum obtained by scanning in the range of 4000-500 cm⁻¹ shows that the peaks at 1420 cm⁻¹, 1144 cm⁻¹, 933 cm⁻¹, 760 cm⁻¹, 690 cm⁻¹, etc. are related to the C-N, C-H vibration modes of the imidazole ring in ZIF-8 (black). FK506 (red) has obvious O-H or N-H stretching vibration in the region above 3000 cm⁻¹, C-H stretching vibration at 2928 cm⁻¹, and multiple peaks in the region of 1000-1750 cm⁻¹, reflecting the complex molecular structure of FK506. In FK506@ZIF-8 (green), the peak at 3135 cm⁻¹ decreases, indicating that the N-H / O-H vibration absorption is affected by the drug loading. The C-H stretching vibration at 2928 cm⁻¹ remains, but the peak intensity decreases slightly, and the peak positions at 1420 cm⁻¹, 1144 cm⁻¹, etc. remain unchanged, indicating that the structure of ZIF-8 is basically maintained. Figure 6 As shown in B, the local magnification of the high-frequency region shows that the N-H / O-H absorption peak changes significantly, indicating strong molecular interaction.

[0044] (3) As shown in Figure 7 the analysis results confirm that FK506 is successfully loaded onto ZIF-8 nanoparticles, and it is shown that the process has an impact on the structure and properties of ZIF-8. As shown in FIG. 7A, the diffraction peaks at 7.34, 10.41, 12.72, 16.47, 18.11, 19.51 and 22.16 confirm the sodalite structure of ZIF-8, and the crystal structure of ZIF-8 remains stable after loading FK506, indicating that the framework has not changed significantly. As shown in FIG. 7B, ZIF-8 has strong adsorption of nitrogen in the low pressure section, and the nitrogen adsorption capacity is 530 cm³ / g in the middle pressure section, and FK506@ZIF-8 is 380 cm³ / g. Both of them show typical I-type adsorption-desorption curve, with micropore characteristics (pore size <2 nm). The specific surface area of ZIF-8 and FK506@ZIF-8 is 1408 m² / g and 1188 m² / g, respectively, and the specific surface area decreases after loading FK506, proving that the drug is successfully loaded and sealed in the MOFs, corresponding to the FTIR results. As shown in FIG. 6C, FK506 (red) has multiple absorption peaks between 200-300 nm, especially a strong absorption at 290 nm; ZIF-8 (black) mainly has absorption between 230-250 nm, without obvious characteristic peaks. FK506@ZIF-8 has multiple sharp peaks between 200-235 nm, and a characteristic peak appears at 276 nm, which may be related to the change of coordination bond of FK506.

[0045] As shown in FIG. 8A, the DTG graph of ZIF-8 nanoparticles: the residual mass percentage at 800°C: 36.33%. As shown in FIG. 8B, the DTG graph of FK506: the residual mass percentage at 620°C: 0.88%. As shown in FIG. 8C, the DTG graph of FK506@ZIF-8 nanoparticles: the residual mass percentage at 800°C: 34.70%.

[0046] Example 3 This example detects the performance of the ZIF-8 nanoparticles, FK506@ZIF-8 nanoparticles, FK506@ZIF-8 / HTCC ophthalmic gel prepared in Example 1.

[0047] The thermal gravimetric analyzer (TGA) was used to detect FK506 powder, ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles to calculate the drug loading rate of FK506. First, 5 mg of each sample was weighed and evenly distributed in the aluminum alloy sample pan of the TGA. The test conditions were set as follows: nitrogen flow rate 25 ml / min, heating rate 5°C / min, temperature range 25-800°C, and then TGA analysis was performed to obtain the DTG curves of the three samples. The loading amount and encapsulation efficiency were calculated.

[0048] According to the DTG curve, the equation was established to calculate the residual mass percentage of the composite material. Assuming that MFK506mg of FK506 is loaded in M total mg of nanoparticles at a ratio of X%, the loading rate is y%. R composite=(1−y)×RZIF-8+y×RFK506; solve y%, i.e. LE(%) EE(%)=the amount of drug loaded in the carrier / the total amount of drug × 100% The dynamic dialysis method was used to evaluate the drug release period of FK506@ZIF-8 nanomaterials.

[0049] (1) Draw the standard curve: Precisely take 2 mg of FK506 powder, dissolve in acetonitrile, configure into a 2 mg / mL solution, dilute by ratio to configure into a standard solution with concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, respectively transfer to the sample bottle, machine detection, mobile phase is acetonitrile and 0.1% formic acid aqueous solution (70 / 30), high performance liquid chromatography column selects C18 (4.6mmx250mm, 5µm), column temperature 50℃, flow rate 1 mL / min, sample volume 20µL, detection wavelength 220 nm, detection time 15 min. Establish linear relationship curve.

[0050] (2) In vitro drug release determination: 1. Preparation of in vitro samples Precisely take 40 mg of FK506@ZIF-8 nanomaterials, dissolve in 5 mL of PBS, configure 1xPBS (pH 7.4), and add 0.5% Tween 80 to improve the solubility of FK506. Place 40 mg of FK506@ZIF-8 nanomaterial solution in a 3,500 Da dialysis bag, seal both ends with a dialysis clamp, immerse the dialysis bag in a 50 mL centrifuge tube containing 40 mL of 1xPBS + 0.5% Tween 80, and place the centrifuge tube in a constant temperature shaker at 100 rpm and 37℃ to simulate the human microenvironment. 2. Time point sampling and liquid addition Take 1 mL of liquid sample at each time point (1h, 2h, 4h, 8h, 12h, 24h), place it in a 2 mL centrifuge tube, and add 1 mL of PBS to the release system to maintain the total volume. After each sampling, centrifuge the sample at 15000 rpm for 10 min, take the supernatant and filter through a 0.22 µm filter to ensure cleanliness, then transfer it to the sample bottle, and repeat the experiment 3 times.

[0051] 3. Draw the curve The HPLC sample detection method is consistent with the standard curve sample detection method; calculate the cumulative release rate of FK506 from the self-assembled peptide hydrogel.

[0052] The calculation formula is:

[0053] Q represents the cumulative release rate of FK506, Cn represents the concentration of FK506 at the sampling time, Vn represents the volume of each sample, C n-1The FK506 concentration represented by the last sampling point, V represents the volume of the release medium, and D represents the mass of FK506 in PBS. According to the results of 3 experiments, the cumulative release curve of FK506 in 1xPBS + 0.5% Tween 80 within 24h was drawn and fitted with a drug release model using OriginPro2021 software.

[0054] As shown in FIG. 9A, the standard curve of FK506, the regression equation is R 2 = 0.9997, close to 1, which indicates that the fitting degree of the standard curve is very high and can be used to accurately calculate the concentration of FK506. As shown in FIG. 9B, the cumulative release curve of FK506@ZIF-8 nanoparticles in a simulated human microenvironment, within the first 5 hours, FK506 was rapidly released, from 0% to about 20%. This may be due to the rapid diffusion of FK506 adsorbed on the surface into the medium. Between 5 hours and 24 hours, the drug release was relatively smooth and slow, indicating that the main drug release may depend on the degradation and diffusion process of the ZIF-8 carrier, and finally reached about 55%. FK506@ZIF-8 nanoparticles are suitable for sustained drug release anti-rejection therapy.

[0055] As can be seen from the mathematical model in FIG. 10B-F, the in vitro sustained release curve conforms to the Weibull model (R 2 > 0.98), indicating that the release rate of the drug during the entire release process is relatively constant, which is a relatively ideal sustained release model.

[0056] Example 4 This test example detects the biocompatibility of the nanoparticle drug-loaded eye gel prepared in Example 1, and the effect on the state of human corneal epithelial cells (HCEC).

[0057] (1) CCK-8 was used to measure the effect of ZIF-8 nanoparticle and FK506@ZIF-8 nanoparticle leaching liquid on HCEC cell activity.

[0058] 1. HCEC cells were cultured in a 37°C, 5% CO2 environment, using DMEM medium containing 10% FBS and 1% penicillin-streptomycin, until the cells reached 80%-90% confluence.

[0059] 2. In a clean bench, accurately weigh 1 mg of ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticles, and mix them separately into 5 ml of DMEM (-) and soak for 48 hours. Obtain the corresponding concentrations of ZIF-8 nanoparticles and FK506@ZIF-8 nanoparticle soaking solutions by equal dilution method.

[0060] 3. In the experiment, a blank control group (100 μL of DMEM medium containing 10% FBS), a ZIF-8 nanoparticle group, and an FK506@ZIF-8 nanoparticle group were set up. The concentration gradient of the nanoparticle groups was 5 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL of medium soaking solution.

[0061] 4. Spread 100 μL of cell suspension (density 1×10^4 cells / mL) evenly into 96-well plates and incubate at 37℃ and 5% CO2 for 24 hours. When HCEC cells adhere and reach 80%-90% confluence, begin the nanomaterial soaking solution intervention. The experimental groups were treated with the corresponding concentrations of ZIF-8 nanoparticle soaking solution and FK506@ZIF-8 nanoparticle soaking solution, respectively, while the control group was treated with an equal volume of DMEM(-) solution, ensuring a consistent volume (10 μL / well). The total culture medium volume was maintained at 100 μL / well. Each experimental and control group had 6 replicates.

[0062] 5. After 24 and 48 hours of intervention with the two groups of nanomaterials, remove the 96-well plates and add 10 μL of CCK-8 reagent to each well. Continue incubation at 37℃ and 5% CO2 for 1 hour. After incubation, measure the absorbance of each well at 450 nm using a microplate reader. A blank control well (containing only culture medium and CCK-8 reagent, without cells and drugs) should be set up simultaneously for background correction during each measurement to ensure the accuracy of the experimental results. The experiment was repeated three times.

[0063] 6. After background correction of the absorbance data, calculate the relative survival rate of each group of cells relative to the control group. The formula is as follows: Relative survival rate (%) = Absorbance of experimental group - Absorbance of blank control group / Absorbance of control group - Absorbance of blank control group × 100%.

[0064] (2) The cytotoxic effect of nanoparticles on HCEC cells was detected by live / dead cell staining. The effect of nanoparticles on HCEC cell apoptosis was detected by flow cytometry.

[0065] 1. Cells were seeded in 96-well plates for microscopic observation and cultured to 80%-90% confluence in a 37°C, 5% CO2 incubator.

[0066] 2. Control group: Add the same volume of DMEM (-) solution to each well. ZIF-8 group: Add 20 μg / mL of ZIF-8 nanoparticle soaking solution to each well. FK506@ZIF-8 group: Add 20 μg / mL of FK506@ZIF-8 nanoparticle soaking solution to each well. Incubate the cells in each group in a 37°C, 5% CO2 incubator for 24 hours.

[0067] 3. PBS washing: Remove the culture medium and wash the cells 2-3 times with gentle PBS buffer to remove residual soaking solution and culture medium and reduce background interference. Use live / dead cell staining reagent for detection and use a fluorescence microscope to observe the cells. Live cells will show green fluorescence (Calcein-AM labeling) and dead cells will show red fluorescence (PI labeling). Randomly select multiple areas under each field for photography to ensure representativeness. Each group was subjected to 3 repeated experiments.

[0068] As shown in Figure 11 A, CCK8 was used to detect the effect of FK506@ZIF-8 nanoparticles and ZIF-8 nanoparticles on HCEC cell activity. With increasing drug concentration, cell viability decreased significantly. At a concentration of 0-20 μg / ml, FK506@ZIF-8 nanoparticles had less effect on HCEC cell activity. As shown in Figure 11 B, live / dead cell staining was used to detect the effect of 20 μg / ml nanoparticles on HCEC cells co-cultured for 24 hours. The results showed that most cells remained in a viable state, indicating that nanoparticles at a concentration of 20 μg / ml had less effect on HCEC cell activity.

[0069] Example 5 This test evaluates the irritation of FK506@ZIF-8 / HTCC ophthalmic gel to rabbit eyes and verifies its safety from an in vivo perspective.

[0070] 1. Adult healthy New Zealand white rabbits (body weight 2.0-2.5 kg), male and female (18). Animal grouping: the rabbits were randomly divided into 3 groups, 6 rabbits in each group; negative control group: 0.1 ml of 0.9% normal saline was dropped into the right eye of each rabbit. Positive control group: 0.1 ml of 20 μg / mL ZIF-8 / HTCC eye gel was dropped into the right eye of each rabbit. Experimental group: 0.1 ml of 20 μg / mL FK506@ZIF-8 / HTCC eye gel was dropped into the right eye of each rabbit. The left eye of all rabbits was dropped with the same amount of PBS as an excipient, as an internal control, to eliminate the differences between individuals.

[0071] 2. The animals were gently fixed, and the upper and lower eyelids were gently separated with one hand. The corresponding reagent (0.1 mL) of each group was dropped into the conjunctival sac of the right eye of the rabbit with a dropper, and the same amount of PBS was dropped into the left eye of each rabbit.

[0072] 3. The irritation reaction of the rabbit eye was observed at 6 hours, 24 hours, 48 hours, 72 hours, and on the 4th day and the 7th day after administration. At each time point, the rabbit eye was observed with a microscope, and the degree of corneal epithelial damage was observed under cobalt blue light irradiation with 2% fluorescein sodium. The irritation score was evaluated using the Draize scoring system.

[0073] As shown in Table 1: the Draize scores of each group were <10, indicating that the FK506@ZIF-8 / HTCC eye gel had no irritation to the rabbit eye.

[0074] Example 6 This test example detects the corneal retention time of the nanoparticle drug-loaded eye gel prepared in Example 1, the corneal penetration effect of the nanoparticles, and the evaluation of the inhibition effect of the gel on the occurrence of corneal immune rejection.

[0075] The FK506@ZIF-8 nanoparticles labeled with fluorescein sodium were obtained, which facilitated the subsequent evaluation and recording of the example. Add PBS buffer, ultrasonic dispersion to uniform, then add 10 mg / mL fluorescein sodium solution dropwise into the nanoparticle solution, magnetic stirring for 2-4 hours, and finally use centrifugation method to remove the unbound fluorescein sodium.

[0076] Corneal retention time evaluation in rabbit eyes. Rabbits were randomly divided into 2 groups, 6 in each group; control group: FK506 eye drops labeled with fluorescein sodium were used. Experimental group: FK506@ZIF-8 / HTCC eye gel labeled with fluorescein sodium was used. The animals were gently fixed, and the upper and lower eyelids were gently separated with one hand. Control group: About 0.1 mL of fluorescein sodium-labeled FK506 eye drops was dropped into the conjunctival sac of the right eye of the rabbit with a dropper. Experimental group: About 0.1 mL of fluorescein sodium-labeled FK506@ZIF-8 / HTCC eye gel was dropped into the conjunctival sac of the right eye of the rabbit with a dropper (the concentration of fluorescein sodium was consistent with the control group). The left eye of each rabbit in each group was dropped with the same amount of normal saline. At the specified time point after administration, the rabbit eyes were irradiated with a purple light (wavelength 365 nm), and the fluorescence images at each time point were recorded using a photographic device.

[0077] Evaluation of nanoparticle corneal permeability in rabbit eyes. Rabbits were randomly divided into 2 groups, 6 in each group. Control group: The same volume of normal saline was dropped. Experimental group: Fluorescein sodium-labeled FK506@ZIF-8 / HTCC eye gel was dropped. The rabbits were gently fixed, and the right eye of the rabbit was opened with an eye speculum; 0.1 mL of FK506@ZIF-8 / HTCC eye gel was dropped into the conjunctival sac of the rabbit eye in the experimental group; the same volume of normal saline was dropped in the control group, and observed for 1 h. After the rabbit was anesthetized, the right cornea was removed, and the surface of the cornea was gently washed with normal saline to remove the unabsorbed drugs. Frozen section: The corneal tissue was prepared into a 5-10 µm thick frozen section, and immediately observed and recorded the images using a fluorescence microscope.

[0078] Animal corneal transplantation model was established to evaluate the immune effect of FK506@ZIF-8 / HTCC eye gel. Animal selection: female SD rats were selected as corneal graft recipients (60), and male Wistar rats were selected as corneal graft donors (30). Animal anesthesia: the rats were adaptively fed for one week, and were weighed one by one before the model was established. General anesthesia was performed using sodium pentobarbital. The rat's mustache and eyelashes were removed using ophthalmic scissors to ensure that the surgical field was fully exposed. After the conjunctival sac was flushed with normal saline and the hair around the eye was disinfected with iodophor for three times, a sterile drape made of gauze was laid. The operating table was disinfected with 75% alcohol, and the rat was placed on the operating table in a lateral position, and the microscope was adjusted so that the rat's eyeball was in the center of the microscope field of view. During the entire operation process, the rat's heart rate, respiration and other vital signs need to be closely monitored. A completely randomized grouping design scheme was adopted, and the rats that successfully received corneal transplantation were randomly divided into three groups: control group: normal saline was given to the eye (12). FK506@ZIF-8 / HTCC eye gel group: 20 μg / ml FK506@ZIF-8 / HTCC eye gel was given after operation (12). FK506 eye drop group: 0.1% FK506 eye drop treatment (12). 6 rats in each group were taken for survival analysis, and the remaining rats in each group were used for detection experiments. In the donor rat eye, a 3.5mm diameter graft was drilled in the center of the cornea using a corneal trephine, a small amount of viscoelastic agent Avise was injected into the anterior chamber, and a 10-0 suture was left at 12 o'clock. The removed graft was placed in a culture dish containing sodium hyaluronate for protection. In the recipient rat right eye, compound tropicamide eye drops were used to dilate the pupil, and propamocaine hydrochloride was used for surface anesthesia. A 3.25mm diameter corneal trephine was used to drill a hole in the central corneal area of the recipient rat to prepare a graft bed. The donor corneal graft was placed on the recipient's graft bed, and 10-0 suture was used for intermittent suture (8 needles) to ensure that the knot was exposed to the outside and was not embedded. A toothless forceps was used to fix the eyeball, and a Hamilton syringe with a 30g needle was used to puncture the anterior chamber at 3 o'clock of the ring mark. When the aqueous humor flowed out, it was ready. Note: when puncturing, care should be taken not to damage the rat's lens. After the operation, tobramycin eye ointment was applied in the conjunctival sac, and 6-0 suture was used to suture the rat's eyelid. The rat's limbs were appropriately trimmed to prevent the rat from scratching the eye after operation, and the postoperative recovery was closely observed.

[0079] Corneal transplantation in rats postoperative administration and treatment. The first day after surgery, the eye lid suture was removed, and the graft status was observed. To prevent postoperative infection, each group was given levofloxacin eye drops within 5 days after surgery, and the eye was dropped, 2 times / day, 0.01 ml each time, and the corneal suture was removed on the 7th day after surgery. Control group: during the observation period or on the day when the cornea was taken out for further experiment: give normal saline eye drops, 2 times a day, (9:00 in the morning, 17:30 in the afternoon), 0.01 ml each time. (12). FK506@ZIF-8 / HTCC gel group: give FK506@ZIF-8 / HTCC gel, 2 times a day, 0.01 ml each time (9:00 in the morning, 17:30 in the afternoon), (12). FK506 eye drop group: give FK506 eye drops, 2 times a day, 0.01 ml (9:00 in the morning, 17:30 in the afternoon), (12). All surgical operations in the experiment were completed by the same operator, and the eye condition was closely observed. If there is iris adhesion, intraocular hemorrhage, lens opacity, endophthalmitis, it will be removed in time and supplemented with rats meeting the same experimental conditions. Randomly grab 1 rat in each group, and take a general photo of the eye on the 12th day and the 24th day after surgery.

[0080] Postoperative observation of mouse corneal grafts and treatment. According to the scoring standard of corneal transplantation of Larkin et al., the degree of opacity, edema and neovascularization of the graft were scored. Kaplan-Meier method was used for survival analysis of corneal grafts. After successfully modeling, 6 rats in each group were randomly selected and sacrificed by cervical dislocation method 16 days later for histological analysis. The selected individuals do not participate in the statistical analysis of clinical score data. When operating, the rat was placed in left lateral position under the table magnifying glass, and the right upper and lower eyelids were cut off with ophthalmic scissors. Then, the skin around the eye socket was fixed with a toothed forceps, and the connective tissue and muscle between the eyeball and the eye socket were slowly separated until only the optic nerve was connected. At this time, the optic nerve root was clamped with a toothed forceps, and it was cut along the lateral edge. Then, the eyeball was gently taken out and washed with a syringe to remove blood and hair on its surface. Finally, the treated eyeball was placed in a pre-prepared 4% paraformaldehyde fixing solution and stored in a centrifuge tube.

[0081] In order to evaluate the immune rejection of corneal transplantation after the application of hydrogel, the mouse corneal grafts were observed and treated after surgery: the corneal grafts were observed under a microscope every day and quantitatively scored, and the survival curve was made. The rejection of corneal transplantation and the repair of cornea in each group were observed from the appearance and histology, and the eyeball was taken out for HE staining and immunohistochemical observation of CD4+ and CD8+ T cell infiltration on the 16th day after surgery, and the corresponding statistical analysis was made on the above results. For example Figure 12As shown in Figure A, compared with the control group, the FK506@ZIF-8 / HTCC ophthalmic gel exhibited a longer residence time on the cornea, demonstrating a more sustained drug release effect and contributing to improved treatment efficiency. Control group (FK506): Fluorescence began to diffuse and rapidly weakened, essentially disappearing after 15 minutes. Experimental group (FK506@ZIF-8 / HTCC): Fluorescence significantly diffused and persisted, maintaining a high intensity before gradually weakening; fluorescence was still visible after 120 minutes.

[0082] like Figure 12 As shown in Figure B, FK506@ZIF-8 / HTCC ophthalmic gel effectively enhances drug penetration in the cornea. In the rabbit eye control group (FK506 eye drops), fluorescence was mainly concentrated on the corneal surface. Magnified images show that sodium-labeled FK506 failed to penetrate the deeper corneal tissues. In the experimental group (FK506@ZIF-8 / HTCC ophthalmic gel), fluorescence was not only concentrated on the corneal surface but also penetrated into the deeper corneal tissues. Magnified images show that sodium-labeled FK506@ZIF-8 / HTCC was distributed in the deeper layers of the cornea.

[0083] Figure 13 As shown, gross ocular images of normal tissue and control group, FK506@ZIF-8 / HTCC ophthalmic gel group and FK506 eye drops group at 12 and 24 days.

[0084] like Figure 14 As shown, FK506@ZIF-8 / HTCC ophthalmic gel significantly prolongs the drug retention time in the eye. FK506@ZIF-8 nanoparticles possess corneal permeability. The survival times of corneal grafts in each group were (20.3±1.9) days in the FK506@ZIF-8 / HTCC ophthalmic gel group, (11.1±1.2) days in the control group, and (14.5±1.0) days in the FK506 eye drops group. The survival of corneal grafts in each group was analyzed using the Kaplan-Meier method. The survival of corneal grafts in the FK506@ZIF-8 / HTCC ophthalmic gel group was superior to that in the control group and the FK506 eye drops group, and the difference was statistically significant (p<0.001).

[0085] As shown in FIG. 15A, HE staining results show that FK506@ZIF-8 / HTCC eye gel can effectively reduce the inflammatory reaction after corneal transplantation. The normal group of corneal section structure is complete, the level is clear, and the tissue has no obvious inflammatory cell infiltration. The control group has a disordered corneal structure, obvious tissue deformation and a large number of inflammatory cell infiltration, showing a severe immune rejection reaction. The FK506 eye drop group has a certain deformation of corneal tissue structure compared with the normal group. The inflammatory cell infiltration is less than that of the control group, but there is still obvious inflammatory reaction. The corneal tissue structure of the FK506@ZIF-8 / HTCC eye gel group is relatively complete, and the inflammatory cell infiltration is significantly reduced, and the anti-inflammatory effect is significantly better than that of the control group and the eye drop group. The HE staining results show that the blank group has no inflammatory reaction, and the inflammatory reaction of the remaining groups from strong to weak is: control group > FK506 eye drop group > FK506@ZIF-8 / HTCC eye gel group. The deeper the color of CD4+ and CD8+ T cells in the tissue, the higher the cell infiltration. As shown in FIG. 15A, the results of immunohistochemical staining under low power (100X) and high power (400X) show that the number of CD4+ and CD8+ T cells in the blank group is very small under 100X and 400X fields, and there is almost no obvious staining. The number of CD4+ and CD8+ T cells in the control group under 100X and 400X fields is significantly increased, which belongs to high infiltration. The staining of CD4+ and CD8+ T cells in the FK506 eye drop group under 100X and 400X fields is reduced, and most of the cells are observed to be stained, which belongs to medium infiltration. The staining of CD4+ and CD8+ T cells in the FK506@ZIF-8 / HTCC eye gel group under 100X and 400X fields is reduced compared with the control group, which belongs to low infiltration.

[0086] As shown in FIG. 15B, the statistical results of the average optical density of CD4+ T cells in the control group and the FK506 eye drop group and the FK506@ZIF-8 / HTCC eye gel group show that the difference in the average optical density of CD4+ T cells between the two groups has statistical difference (P<0.05, ****P<0.001, ****P<0.0001, respectively). p <0.05,**** p <0.0001)。

[0087] As shown in FIG. 15C, the statistical results of the average optical density of CD8+ T cells in the control group and the FK506 eye drop group and the FK506@ZIF-8 / HTCC eye gel group show that the difference in the average optical density of CD8+ T cells between the two groups has statistical difference (P<0.05, ****P<0.001, ****P<0.0001, respectively). p <0.001,**** p<0.0001). The above results show that FK506 has a significant inhibitory effect on the activity of CD4+ T cells and CD8+ T cells, and FK506@ZIF-8 / HTCC ophthalmic gel has a stronger immunosuppressive effect than FK506 eye drops alone.

[0088] Table 1 Summary of FK506@ZIF-8 / HTCC ophthalmic gel irritation Draize scores (n = 6)

[0089] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing an FK506@ZIF-8 / HTCC ophthalmic gel with anti-immune rejection properties, characterized in that, Includes the following steps: S1. Add Zn(NO3)2·6H2O aqueous solution to an alcohol solution containing a mixture of C4H6N2 and FK506, and react under stirring. After centrifugation and light irradiation, FK506@ZIF-8 nanoparticles are obtained. S2. After thoroughly mixing FK506@ZIF-8 nanoparticles with quaternized chitosan sol, the pH value was adjusted to neutral to obtain FK506@ZIF-8 / HTCC ophthalmic gel.

2. The method for preparing the FK506@ZIF-8 / HTCC ophthalmic gel for resisting immune rejection according to claim 1, characterized in that, In step S1, the ratio of Zn(NO3)2·6H2O, C4H6N2 and FK5O6 is 15:33:

1.

3. The method for preparing the FK506@ZIF-8 / HTCC ophthalmic gel for resisting immune rejection according to claim 1, characterized in that, In step S1, the alcohol solution includes C1-C5 alcohols.

4. The method for preparing the FK506@ZIF-8 / HTCC ophthalmic gel for resisting immune rejection according to claim 1, characterized in that, In step S1, FK506@ZIF-8 nanoparticles were obtained after centrifugation and irradiation with a UV lamp for 30-60 min.

5. The method for preparing the FK506@ZIF-8 / HTCC ophthalmic gel for resisting immune rejection according to claim 1, characterized in that, In step S2, the mass ratio of FK506@ZIF-8 nanoparticles to quaternized chitosan is 1:

50.

6. The method for preparing the FK506@ZIF-8 / HTCC ophthalmic gel against immune rejection according to claim 1, characterized in that, In step S2, quaternized chitosan is mixed with water to form a quaternized chitosan sol with a mass concentration of 0.1-2%.

7. The method for preparing the FK506@ZIF-8 / HTCC ophthalmic gel against immune rejection according to claim 6, characterized in that, In step S2, quaternized chitosan is mixed with water to form a quaternized chitosan sol with a mass concentration of 1%.

8. The FK506@ZIF-8 / HTCC ophthalmic gel for resisting immune rejection prepared according to any one of claims 1-7, characterized in that, FK506 was successfully loaded onto ZIF-8 nanoparticles, and its characteristic absorption peaks were observed near 7.34, 10.41, 12.72, 16.47, 18.11, 19.51, and 22.16 in the XDR diffraction pattern.

9. The FK506@ZIF-8 / HTCC ophthalmic gel for resisting immune rejection prepared according to the method of claim 8, characterized in that, The average particle size of FK506@ZIF-8 / HTCC nanoparticles is 91.68±0.1258 nm; the zeta potential is 2.77±0.245 mV.

10. The use of the FK506@ZIF-8 / HTCC ophthalmic gel according to claim 8 or 9, which resists immune rejection, in the preparation of a medicament for treating keratitis, characterized in that... FK506@ZIF-8 / HTCC ophthalmic gel prolongs the retention time of the drug in the eye to more than 72 hours, more preferably more than 4 days, and even more preferably more than 7 days.

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