Nano material loaded with tacrolimus as well as preparation method and application of nano material

Abstract

The invention relates to a tacrolimus-loaded nano material as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a preparation method of a tacrolimus-loaded nano material, which comprises the following steps: dispersing MnO2 nanosheets in water, carrying out ultrasonic treatment, and centrifuging to obtain a supernatant; mixing the supernate with dopamine hydrochloride, adjusting the pH value, stirring, centrifuging to obtain supernate, and dialyzing to obtain MnO2 / PDA NPs; and mixing a tacrolimus chloroform solution with the obtained MnO2 / PDA NPs, centrifugally collecting the precipitate, and washing to obtain the nano material loaded with tacrolimus. According to the method, MnO2 is subjected to ultrasonic treatment and then centrifuged, MnO2 / PDA NPs containing polydopamine is prepared from MnO2 and dopamine hydrochloride, polydopamine serves as a functional medium, tacrolimus is effectively loaded on a MnO2 substrate through coordination self-assembly, and the nano material loaded with tacrolimus is successfully synthesized.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a tacrolimus-loaded nanomaterial, its preparation method, and its application. Background Technology

[0002] Organ transplantation is the ultimate treatment for patients with end-stage organ failure. However, acute rejection remains a major cause of graft failure due to the activation of the recipient's immune system mediated by T cells, B cells, macrophages, and natural killer (NK) cells. Redox signaling and inflammatory cell-driven reactive oxygen species (ROS) play crucial roles in ischemia-reperfusion injury (IRI) and graft rejection. Therefore, inhibiting inflammatory cells and reducing ROS production can protect grafts from immune-mediated damage.

[0003] Clinical immunosuppressive regimens (including antimetabolites, calcineurin inhibitors (CNIs), and steroids) have significantly improved short-term graft survival. However, these therapies have significant limitations: all drugs are administered systemically and cannot specifically target the graft. CNIs (such as tacrolimus (FK506) and cyclosporine A) can cause graft toxicity, and long-term high-dose use can lead to interstitial fibrosis and graft dysfunction. More importantly, these drugs primarily act on T cells, with limited inhibitory effects on B cells, macrophages, and NK cells.

[0004] Therefore, there is an urgent need to develop a nanomaterial that can effectively improve transplant prognosis and increase the long-term survival rate of inhibitors. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a tacrolimus-loaded nanomaterial, its preparation method and application, wherein the tacrolimus-loaded nanomaterial has the effect of improving transplant prognosis and increasing the long-term survival rate of inhibitors.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing tacrolimus-loaded nanomaterials, comprising the following steps: (1) Disperse MnO2 nanosheets in water, sonicate and centrifuge to obtain supernatant; (2) Mix the supernatant obtained in step (1) with dopamine hydrochloride, adjust the pH to 10-11, stir, centrifuge to obtain the supernatant, dialyze to obtain MnO2 / PDA NPs; (3) Mix the tacrolimus chloroform solution with the MnO2 / PDA NPs obtained in step (2), centrifuge to collect the precipitate, wash, and obtain FK506@MnO2 / PDA NPs, which are nanomaterials loaded with tacrolimus.

[0007] This invention involves ultrasonically treating MnO2 followed by centrifugation to disperse monolayer MnO2 nanosheets into the supernatant. These nanosheets are then mixed with dopamine hydrochloride to prepare MnO2 / PDA NPs containing polydopamine. Polydopamine acts as a functional mediator, effectively loading tacrolimus (FK506) onto the MnO2 substrate through coordination self-assembly. This overcomes the chemical incompatibility between MnO2 and FK506 molecules, successfully synthesizing tacrolimus-loaded nanomaterials FK506@MnO2 / PDA NPs. FK506@MnO2 / PDA NPs are nanomaterials constructed by encapsulating FK506 with MnO2 NPs as a carrier, enabling the sustained release of FK506 and ROS scavenging at target sites.

[0008] In a preferred embodiment of the preparation method described in this invention, the ultrasonic treatment time in step (1) is 5-7 hours.

[0009] In a preferred embodiment of the preparation method of the present invention, the ultrasonic treatment time in step (1) is 6 hours.

[0010] As a preferred embodiment of the preparation method of the present invention, in step (1), the centrifugation conditions are 1800-2500 rpm and 25-35 min.

[0011] As a preferred embodiment of the preparation method of the present invention, in step (1), the centrifugation conditions are 2000 rpm and 30 min.

[0012] As a preferred embodiment of the application described in this invention, in step (2), the mass ratio of the MnO2 nanosheets to dopamine hydrochloride is MnO2 nanosheets: dopamine hydrochloride = (20-40): (40-60).

[0013] As a preferred embodiment of the application described in this invention, in step (2), the mass ratio of the MnO2 nanosheets to dopamine hydrochloride is MnO2 nanosheets: dopamine hydrochloride = 30:50.

[0014] In a preferred embodiment of the application described in this invention, the stirring time in step (2) is 1.5-3 hours.

[0015] In a preferred embodiment of the application described in this invention, the stirring time in step (2) is 2 hours.

[0016] As a preferred embodiment of the application described in this invention, in step (2), the centrifugation conditions are 11000-13000 rpm and 8-12 min.

[0017] As a preferred embodiment of the application described in this invention, in step (2), the centrifugation adjustment is 12000 rpm for 10 min.

[0018] As a preferred embodiment of the application described in this invention, in step (2), the dialysis is performed by placing the obtained supernatant in a dialysis bag and dialyzing for 12-16 hours.

[0019] As a preferred embodiment of the application described in this invention, in step (3), the mass ratio of tacrolimus to MnO2 / PDA NPs in the tacrolimus chloroform solution is tacrolimus:MnO2 / PDA NPs=1:(8-12).

[0020] As a preferred embodiment of the application described in this invention, in step (3), the mass ratio of tacrolimus to MnO2 / PDA NPs in the tacrolimus chloroform solution is tacrolimus:MnO2 / PDA NPs = 1:10.

[0021] As a preferred embodiment of the application described in this invention, in step (3), the mass ratio of tacrolimus to MnO2 / PDA NPs in the tacrolimus chloroform solution is tacrolimus:MnO2 / PDA NPs = 1:1.

[0022] As a preferred embodiment of the application described in this invention, in step (3), the mixing is performed by stirring at 23-27°C for 12-16 hours.

[0023] As a preferred embodiment of the application described in this invention, in step (3), the centrifugation conditions are 1800-2200 rpm and 35-39°C.

[0024] As a preferred embodiment of the application described in this invention, in step (3), the centrifugation conditions are 2000 rpm and 37°C.

[0025] Secondly, this invention provides a tacrolimus-loaded nanomaterial, mainly prepared by the above-described method. Experiments revealed that the tacrolimus-loaded nanomaterial can effectively catalyze the generation of O2 from H2O2 and promote reactive oxygen species scavenging. Due to its dual mechanism of FK506 targeted delivery and reactive oxygen species scavenging, it reduces the infiltration of macrophages, B cells, and NK cells in the graft while inhibiting T cells, thereby exerting a broader immunosuppressive effect.

[0026] Thirdly, this invention provides the application of the aforementioned tacrolimus-loaded nanomaterials in the preparation of anti-rejection drugs. The tacrolimus-loaded nanomaterials of this invention overcome the limitations of traditional immunosuppressants, achieving graft-targeted delivery and sustained release of FK506, significantly reducing drug dosage, minimizing adverse reactions, and improving treatment efficiency. Furthermore, its mechanism of action transcends T-cell suppression, further inhibiting B cells, macrophages, and NK cells by scavenging reactive oxygen species. It can be used as a novel anti-rejection drug to improve organ transplant survival rates and reduce systemic side effects in transplant prognosis.

[0027] As a preferred embodiment of the application described in this invention, the dosage form of the anti-rejection drug includes, but is not limited to, at least one of solid dosage form, liquid dosage form, semi-solid dosage form and gaseous dosage form.

[0028] Fourthly, the present invention provides an anti-rejection drug comprising the above-mentioned tacrolimus-loaded nanomaterial and pharmaceutically acceptable excipients.

[0029] As a preferred embodiment of the application described in this invention, the pharmaceutically acceptable excipients include, but are not limited to, at least one of fillers, binders, wetting agents, disintegrants, lubricants, flow aids, preservatives, colorants, solvents, and cosolvents.

[0030] As a preferred embodiment of the application described in this invention, the filler includes, but is not limited to, at least one of starch, lactose, dextrin, sucrose, dicalcium phosphate, and microcrystalline cellulose.

[0031] As a preferred embodiment of the application described in this invention, the adhesive and wetting agent include, but are not limited to, at least one of water, gelatin, and polyethylene glycol.

[0032] As a preferred embodiment of the application described in this invention, the disintegrant includes, but is not limited to, at least one of crospovidone, sodium carboxymethyl starch, and crospovidone carboxymethyl cellulose.

[0033] As a preferred embodiment of the application described in this invention, the lubricant and flow aid include, but are not limited to, at least one of silica, polyethylene glycol, magnesium stearate, micronized silica gel, and talc.

[0034] As a preferred embodiment of the application described in this invention, the preservative includes, but is not limited to, at least one of benzoic acid and its salts, propylene glycol, ethyl acetate, and sorbic acid.

[0035] As a preferred embodiment of the application described in this invention, the solvent and co-solvent include, but are not limited to, at least one of water, ethanol, glycerol and polysorbate 80.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention disperses monolayer MnO2 nanosheets into the supernatant by ultrasonic treatment followed by centrifugation. The supernatant is then mixed with dopamine hydrochloride to prepare MnO2 / PDA NPs containing polydopamine. Polydopamine acts as a functional mediator, effectively loading tacrolimus (FK506) onto the MnO2 substrate through coordination self-assembly, thus overcoming the chemical incompatibility between MnO2 and FK506 molecules and successfully synthesizing tacrolimus-loaded nanomaterials FK506@MnO2 / PDA NPs. FK506@MnO2 / PDA NPs are nanomaterials constructed by encapsulating FK506 with MnO2 NPs as a carrier, enabling the sustained release of FK506 and ROS scavenging at the target site.

[0037] (2) Through experiments, this invention found that nanomaterials loaded with tacrolimus can effectively catalyze the generation of O2 from H2O2 and promote the clearance of active oxygen. Due to the dual mechanism of FK506 targeted delivery and reactive oxygen scavenging, it can reduce the infiltration of macrophages, B cells and NK cells in the graft while inhibiting T cells, thereby exerting a wider immunosuppressive effect.

[0038] (3) The tacrolimus-loaded nanomaterials described in this invention overcome the limitations of traditional immunosuppressants. By achieving graft-targeted delivery and sustained release of FK506, they significantly reduce drug dosage, decrease adverse reactions, and improve treatment efficiency. Furthermore, their mechanism of action transcends T-cell suppression, further inhibiting B cells, macrophages, and NK cells by scavenging reactive oxygen species. They can be used as a novel anti-rejection drug to improve organ transplant survival and reduce systemic side effects in transplant prognosis. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating the preparation process of tacrolimus-loaded nanomaterials according to the present invention. Figure 2 This is a scanning electron microscope image of the nanomaterial obtained in Example 1 of the present invention; Figure 3 The particle size distribution (A) and potential measurement results (B) of the nanomaterial obtained in Example 1 of the present invention are shown in Example 1. Figure 4 The ultraviolet-visible absorption spectrum of the nanomaterial obtained in Example 1 of the present invention is shown in Example 1. Figure 5 The in vitro drug release curve (A) and the UV-Vis absorption spectrum of serum stability over 72 hours of FK506@MnO2 / PDA NPs obtained in Example 1 of the present invention are shown in Example 1. Figure 6 The effect of different concentrations of the nanomaterials obtained in Example 1 on the viability of AC16 cells (A) and Bend.3 cells (B) is shown in Example 2 of the present invention. Figure 7 The results of the catalytic oxygen production capacity determination of the nanomaterials obtained in Example 1 at different concentrations in Example 3 of the present invention; Figure 8 The results of ROS scavenging ability determination of nanomaterials obtained in Example 1 at different concentrations in Example 3 of the present invention; Figure 9 The effects of different nanomaterials obtained in Example 1 of the present invention on T cells are shown in Example 4 of the present invention. A is a representative flow cytometry graph and B is the statistical result of flow cytometry. Figure 10 The effects of different nanomaterials obtained in Example 1 of the present invention on T cell pro-inflammatory factors are shown in Example 4 of the present invention, where A is IL-17 and B is IFN-γ; Figure 11 The results of in vitro and in vivo uptake of nanomaterials obtained in different Examples 1 of the present invention are shown in Example 5. Figure 12 The statistical results of the survival rates of mice in different treatment groups in Example 6 of the present invention; Figure 13 This is a representative HE staining image of the heart transplanted mice in different treatment groups on day 6 after heart transplantation in Example 6 of the present invention. Figure 14 Immunohistochemical representative images and quantitative results of the grafts from mice in different treatment groups on day 6 after heart transplantation in Example 6 of the present invention; Figure 15 This is a representative scatter plot of the infiltrating immune cell detection results of the sixth graft in mice after heart transplantation in different treatment groups in Example 6 of the present invention, where A represents leukocytes, B represents T cells, C represents macrophages, D represents NK cells, and E represents B cells. Figure 16 The cell frequency and absolute quantitative results of the infiltrating immune cells of the graft in mice in different treatment groups after heart transplantation in Example 6 of the present invention are shown. A represents white blood cells, B and C represent T cells, D represents macrophages, E represents NK cells, and F represents B cells. Figure 17 The results of the safety assessment of mice in different treatment groups on day 6 after heart transplantation in Example 6 of the present invention; Figure 18 The figures show in vivo imaging results of mice in different treatment groups at different times after drug injection in Example 6 of the present invention. The circles in the figures indicate the heart transplant area. Figure 19 This is the quantitative result of fluorescence intensity in in vivo imaging of transplanted mice after drug injection in different treatment groups in Example 6 of the present invention; Figure 20 These are the in vitro imaging results of mice in different treatment groups at different times after drug injection in Example 6 of the present invention; Figure 21 This is the quantitative result of fluorescence intensity in in vitro imaging of transplanted mice after drug injection in different treatment groups in Example 6 of the present invention; Figure 22 The results of comparing the fluorescence intensity of the graft and the mouse's own heart after injection of the drug in different treatment groups in Example 6 of the present invention are shown. A is the result of injection of FK506-Cy7, and B is the result of injection of FK506@MnO2 / PDA NPs. Figure 23 This invention relates to a treatment strategy for acute rejection following transplantation using tacrolimus-loaded nanomaterials. Detailed Implementation

[0040] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0041] Unless otherwise specified, all other materials and reagents used in the examples, comparative examples, and effect examples are commercially available.

[0042] The MnO2 nanosheets described in this invention can be obtained from various commercial channels or prepared in-house. The MnO2 nanosheets in the following examples and comparative examples were prepared by the following methods: Add 40 mL of 0.6 mol / L tetramethylammonium hydroxide pentahydrate and 10 mL of 3 wt.% H2O2 to 20 mL of 0.3 mol / L MnCl2 solution and stir overnight at room temperature (23-27℃). Collect the obtained MnO2 nanosheets by centrifugation at 2000 rpm for 30 min, wash successively with methanol and deionized water, and dry at 60℃ for later use.

[0043] Tacrolimus (FK506) was purchased from APExBIO Inc. in the United States, with product number B2143.

[0044] The reactive oxygen species (ROS) level assay kit was purchased from ThermoFisher Scientific, USA, catalog number I36007.

[0045] PE-cy7-CD69 antibody was purchased from BioLegend, Inc., USA, catalog number 104511.

[0046] Enzyme-linked immunosorbent assay (ELISA) kits were purchased from ThermoFisher Scientific, USA, catalog numbers BMS6001TEN and KMC4021C.

[0047] Adult male BALB / c and C57BL / 6 wild-type mice (weighing 20-22g) were purchased from Spiefer (Suzhou) Biotechnology Co., Ltd., and housed in a specific pathogen-free environment at Cyagen Biosciences Co., Ltd. (Guangzhou, China). All animal experiments were conducted in accordance with the guidelines of the Ethics Committee of Cyagen Biosciences Co., Ltd., and were approved by the committee (Approval No.: JENNIO-IACUC-2024-A058).

[0048] Both Cy7-labeled FK506-Cy7 and FK506@MnO2 / PDA-Cy7 were prepared according to the Cy7 labeling kit instructions. The Cy7 labeling kit was purchased from Wuhan E-LK-C005C.

[0049] Example 1 This embodiment provides a tacrolimus-loaded nanomaterial and its preparation method. The preparation process of the tacrolimus-loaded nanomaterial is as follows: Figure 1 As shown, the preparation method includes the following steps: S1. Disperse 30 mg of MnO2 nanosheets in 300 mL of deionized water, sonicate for 6 h, and centrifuge at 2000 rpm for 30 min to obtain the supernatant; (2) Mix the supernatant obtained in step (1) with 50 mg of dopamine hydrochloride, adjust the pH to 10-11 with ammonia, stir for 2 h, centrifuge at 12000 rpm for 10 min to obtain the supernatant, transfer it into a dialysis bag and dialyze for 12 h to obtain MnO2 / PDA NPs; (3) Mix 1 mg / mL tacrolimus chloroform solution with 1 mg / mL MnO2 / PDA NPs obtained in step (2) at a mass ratio of tacrolimus:MnO2 / PDA NPs = 1:10 in the tacrolimus chloroform solution, stir at room temperature for 12 h, centrifuge to collect the precipitate, wash with deionized water to obtain FK506@MnO2 / PDA NPs, which is the tacrolimus-loaded nanomaterial.

[0050] Example 2 This embodiment provides a tacrolimus-loaded nanomaterial and its preparation method. The preparation method includes the following steps: S1, 30 mg of MnO2 nanosheets are dispersed in 300 mL of deionized water, ultrasonically treated for 5 h, and centrifuged at 1800 rpm for 35 min to obtain the supernatant; (2) The supernatant obtained in step (1) was mixed with 40 mg of dopamine hydrochloride, the pH was adjusted to 10-11 with ammonia water, stirred for 1.5 h, and centrifuged at 11000 rpm for 12 min to obtain the supernatant. The supernatant was then transferred to a dialysis bag and dialyzed for 16 h to obtain MnO2 / PDA NPs. (3) Mix 1 mg / mL tacrolimus chloroform solution with 1 mg / mL MnO2 / PDA NPs obtained in step (2) at a mass ratio of tacrolimus:MnO2 / PDA NPs = 1:10 in the tacrolimus chloroform solution, stir at room temperature for 12 h, centrifuge to collect the precipitate, wash with deionized water to obtain FK506@MnO2 / PDA NPs, which is the tacrolimus-loaded nanomaterial.

[0051] Example 3 This embodiment provides a tacrolimus-loaded nanomaterial and its preparation method. The preparation method includes the following steps: S1, 30 mg of MnO2 nanosheets are dispersed in 300 mL of deionized water, ultrasonically treated for 7 h, and centrifuged at 2500 rpm for 25 min to obtain the supernatant; (2) The supernatant obtained in step (1) was mixed with 60 mg of dopamine hydrochloride, the pH was adjusted to 10-11 with ammonia water, stirred for 1.5 h, and centrifuged at 13000 rpm for 8 min to obtain the supernatant. The supernatant was then transferred to a dialysis bag and dialyzed for 16 h to obtain MnO2 / PDA NPs. (3) Mix 1 mg / mL tacrolimus chloroform solution with 1 mg / mL MnO2 / PDA NPs obtained in step (2) at a mass ratio of tacrolimus:MnO2 / PDA NPs = 1:10 in the tacrolimus chloroform solution, stir at room temperature for 16 h, centrifuge to collect the precipitate, wash with deionized water to obtain FK506@MnO2 / PDA NPs, which is the tacrolimus-loaded nanomaterial.

[0052] Example 1 The MnO2 / PDA NPs, FK506@MnO2 / PDA NPs, and MnO2 NPs obtained in Example 1 were characterized using the following specific methods: 1. Observation was performed using a high-resolution transmission electron microscope, and the results are as follows: Figure 2 As shown, the presence of polydopamine promotes the self-assembly process, enabling FK506@MnO2 / PDA NPs to form aggregated nanospheres with a nanoflower-like structure.

[0053] 2. The particle size and zeta potential of the nanoparticles were measured using a dynamic light scattering instrument under the following conditions: ±10 μcm / Vs, conductivity range 0-200 mS / cm, and temperature range 2-90℃. The results are as follows: Figure 3 As shown, FK506@MnO2 / PDA NPs exhibit a uniform particle size distribution with an average size of 142 nm, significantly larger than the initial particle size of MnO / PDA NPs (50 nm). This is attributed to controlled crystallite growth and surface drug modification. Figure 3 A); Zeta potential testing showed that all NPs were negatively charged, with the FK506@MnO / PDA NPs having an average potential of -14.3mV. Figure 3 B) This negative charge can reduce non-specific protein adsorption and opsonization, prolong blood circulation time, and reduce the tendency to accumulate in the liver and spleen.

[0054] 3. The maximum absorption peak of FK506@MnO2 / PDA NPs was determined using a UV-Vis spectrophotometer in the wavelength range of 200-900 nm. The operating temperature was 15-35℃ and the humidity was 30-80%.

[0055] Serum UV-vis stability of MnO2@PDA@FK506 (250nm) was tested over 72 hours. Two samples with a concentration of 0.1 mg / mL were detected using a Shimadzu UV-2600 UV spectrophotometer. The operating temperature was 15-35℃ and the operating humidity was 30%-80%. The fluorescence wavelength range of 200-900 nm was selected for detection.

[0056] The FK506 content in the filtrate was quantitatively analyzed by measuring the absorbance of the filtrate at 200 nm and comparing it with the FK506 concentration standard curve. The encapsulation efficiency and drug loading of FK506 were calculated using the following formulas: Encapsulation efficiency (%) = (FK506 - FK506) / FK506 × 100% Drug loading (%) = (FK506 - FK506) / NP × 100% Where FK506 is the total amount of FK506 fed, FK506 is the amount of free unloaded FK506, and NP is the total amount of nanoparticles.

[0057] The above results are shown in Figure 4-5 .

[0058] like Figure 4As shown, MnO2 has a strong absorption peak in the visible light region (consistent with its black appearance). The overlapping of the infrared absorption spectrum after PDA loading confirms the successful recombination of MnO2 / PDA. The characteristic peak shift in the 250-400nm band verifies the successful loading of FK506 in MnO2 / PDA and the formation of FK506@MnO2 / PDA NPs.

[0059] like Figure 5 As shown, FK506 exhibits rapid initial release under physiological conditions (30% release within 6 hours), followed by a sustained release phase, with a cumulative release rate of 92% over 60 hours. The UV absorption peak of NPs remains stable over 72 hours, indicating its suitability for subsequent cell and animal experiments.

[0060] Example 2 The in vitro and in vivo toxicity of the MnO2 / PDA NPs and FK506@MnO2 / PDA NPs obtained in Example 1 was evaluated using the following method: 1. In vitro experiments.

[0061] Human cardiomyocytes AC16 and mouse brain endothelial cells Bend.3 were used as experimental subjects. AC16 and Bend.3 cells were stored at 1×10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and allowed to adhere overnight. Virulence was assessed by treating cells with MnO2 / PDA NPs or FK506@MnO2 / PDA NPs at concentrations of 0, 5, 25, 50, 100, 200, 400, and 800 μg / mL for 24 hours. After incubation, 10 μL of LCK-8 reagent was added to each well, and incubation continued for 1 hour. Cell viability was then calculated by measuring absorbance at 450 nm. Results are shown below. Figure 6 .

[0062] like Figure 6 As shown, concentrations of MnO2 / PDA NPs of 5, 25, 50, and 100 μg / mL had no effect on the viability of AC16 and Bend.3 cells, while concentrations of 200, 400, and 800 μg / mL significantly reduced cell viability. Similarly, concentrations of FK506@MnO2 / PDA NPs of 5, 25, and 50 μg / mL had no significant effect on cells, but concentrations of 100, 200, 400, and 800 μg / mL significantly reduced cell viability. These results indicate that MnO2 / PDA NPs exhibit cytotoxicity only at concentrations exceeding 200 μg / mL, while FK506@MnO2 / PDA NPs exhibit cytotoxicity at concentrations exceeding 100 μg / mL. Therefore, a concentration of 50 μg / mL was selected as the working concentration for MnO2 / PDA and FK506@MnO2 / PDA NPs in subsequent in vitro experiments.

[0063] 2. In vivo experiments.

[0064] Male C57BL / 6 mice (weighing 20-22 g) were used as experimental subjects and administered MnO2 / PDA NPs or FK506@MnO2 / PDA NPs via intraperitoneal injection at doses of 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg / kg. Injections were given every two days for a total of four times. Following administration of the nanoparticles, the mice's survival rate, activity level, appetite, and body weight were closely monitored to assess potential toxic effects.

[0065] A clear dose-dependent toxicity trend was observed after administration: high-dose groups (80 and 160 mg / kg) showed 100% lethality within one week of injection; medium-dose groups (20 and 40 mg / kg) exhibited significant behavioral changes, including lethargy, anorexia, and progressive weight loss; while low-dose groups (1.25–10 mg / kg) showed no statistically significant toxic side effects during the 14-day observation period. These results confirm that 10 mg / kg is the maximum tolerated dose, and significant toxicological manifestations occur at doses exceeding 20 mg / kg. Based on these safety data, the 10 mg / kg dosage regimen was selected for subsequent in vivo pharmacodynamic studies.

[0066] Example 3 The catalytic oxygen production capacity and ROS scavenging capacity of the FK506@MnO2 / PDA NPs obtained in Example 1 were evaluated using the following method: 1. The catalytic oxygen production capacity was measured.

[0067] Quantitative assessment was performed using a portable dissolved oxygen meter. Specifically, 50 μg / mL of FK506@MnO2 / PDANPs was co-incubated with different concentrations (25, 50, and 100 nM) of H2O2 for 10 minutes, and the dissolved oxygen content in the solution was measured at specific time points. As a control experiment, the dissolved oxygen level in a 25 nM H2O2 solution without FK506@MnO2 / PDA NPs was simultaneously measured. The results are shown in [Figure number missing]. Figure 7 .

[0068] like Figure 7 As shown, by adding different concentrations of H2O2 to the cell culture medium to simulate the pathological oxidative microenvironment, rapid oxygen generation can be observed within 100s after adding FK506@MnO / PDA NPs, and higher concentrations of H2O2 can maintain continuous oxygen production for more than 600s.

[0069] 2. Test the ROS removal capability.

[0070] AC16 cells were seeded at a density of 1 × 10⁶ cells / well in 96-well plates and cultured overnight. Cells were then treated with MnO₂ / PDA NPs (obtained in Example 1) or FK506@MnO₂ / PDA NPs at concentrations of 5, 25, and 50 μg / mL, respectively, and incubated for 24 h. Untreated cells were used as a control group to determine baseline ROS levels. After incubation, 10 μL of the intracellular reactive oxygen species (ROS) fluorescent probe was added to each well according to the instructions of the intracellular ROS level assay kit, and incubation was continued for 1 h. Finally, intracellular ROS levels were quantitatively analyzed by measuring absorbance at 480 nm. The results are shown in the table below. Figure 8 .

[0071] like Figure 8 As shown, compared with the control group, the intracellular reactive oxygen species (ROS) levels in the culture systems treated with MnO2 / PDA NPs and FK506@MnO2 / PDA NPs were significantly reduced. This inhibitory effect showed a clear concentration-dependent trend. Among them, FK506@MnO2 / PDA NPs had a stronger ROS scavenging efficiency than MnO2 / PDA NPs.

[0072] Example of effect 4 To investigate the immunomodulatory effects of MnO2 / PDA NPs and FK506@MnO2 / PDA NPs obtained in Example 1, MnO2 / PDA NPs or FK506@MnO2 / PDA NPs were added to an in vitro cell culture and activation system, and the expression of related proteins was measured. Specifically: Lymph node cells from wild-type C57BL / 6 mice were purified using nylon hair columns to obtain a T cell population with a purity >90%; the purified cells were resuspended at 2×10⁻⁶. 6 Cells were cultured at a concentration of 4 μg / mL in complete medium containing lipopolysaccharide. Cells were then treated with 50 μg / mL MnO2 / PDA NPs or FK506@MnO2 / PDA NPs in 48-well plates for 72 h. After incubation, T cell activation was assessed by flow cytometry using anti-mouse PE-cy7-CD69 antibody. Simultaneously, the culture supernatant was collected, and the levels of pro-inflammatory cytokines IL-17 and IFN-γ were quantified using an enzyme-linked immunosorbent assay (ELISA) kit. Results are shown below. Figure 9-10 .

[0073] like Figure 9 As shown, treatment with MnO2 / PDA NPs or FK506@MnO2 / PDA NPs significantly inhibited T cell activation, and the inhibitory effect of FK506@MnO / PDA NPs on T cell activation was significantly stronger than that of MnO / PDA NPs; Figure 10As shown, the levels of pro-inflammatory cytokines (including IL-17 and IFN-γ) were significantly reduced, and the cytokine levels in the FK506@MnO2 / PDA NPs group were further reduced compared to the MnO2 / PDA NPs group, indicating a stronger immunosuppressive effect. These results demonstrate that FK506@MnO / PDA NPs can effectively inhibit T cell activation and reduce the production of pro-inflammatory cytokines, highlighting its potential application as an immunosuppressant.

[0074] Example 5 To evaluate the cell compatibility of the MnO2 / PDA NPs or FK506@MnO2 / PDA NPs obtained in Example 1, in vitro and in vivo experiments were conducted, with the specific protocols as follows: 1. In vitro experiments.

[0075] Mouse brain endothelial cells Bend.3 were injected at a rate of 1 × 10⁻⁶ per well. 5 Cells were seeded at a density of 1000 cells / well in 12-well plates and cultured overnight. Cells were then treated with 50 μg / mL Rhodamine 6G (R6G)-labeled FK506@MnO2 / PDA NPs (yellow fluorescence) and incubated for 6 h. After incubation, cells were stained with 5 μg / mL DAPI (blue fluorescence) for 15 min, followed by washing three times with PBS. Cells were then incubated with 5 μg / mL DIO (green fluorescence) under the same conditions. The co-localization of R6G and DIO was observed using a fluorescence microscope, confirming the endocytosis effect of FK506@MnO2 / PDA NPs. Results are shown in [Figure number missing]. Figure 10 .

[0076] like Figure 11 As shown, extremely high cell uptake efficiency was observed after 12 hours of co-incubation of R6G-labeled FK506@MnO2 / PDA NPs with Bend.3 cells, indicating that the material has good biosafety and cell compatibility.

[0077] 2. Male C57BL / 6 mice (20-22g) were intraperitoneally injected with 10 mg / kg of R6G-labeled FK506@MnO2 / PDA NPs. Twenty-four hours later, the spleens were removed and frozen for tissue section preparation. The sections were stained with PE-labeled anti-CD11b and anti-CD11c antibodies (green fluorescence) to label macrophages and dendritic cells, respectively, to assess the uptake of NPs by immune cells. Results are shown in [Figure number missing]. Figure 11 .

[0078] like Figure 11As shown, 24 hours after injection of FK506@MnO2 / PDA NPs, fluorescence microscopy revealed the co-localization of CD11b and R6G, confirming macrophage endocytosis of FK506@MnO2 / PDA NPs. Similarly, the co-localization of CD11c and R6G demonstrated dendritic cell uptake of FK506@MnO2 / PDA NPs, indicating that splenic macrophages and dendritic cells are capable of uptake of FK506@MnO2 / PDA NPs.

[0079] The above results indicate that FK506@MnO2 / PDA NPs have good cell compatibility and can be taken up by cells.

[0080] Example 6 To evaluate the efficacy of FK506@MnO2 / PDA NPs obtained in Example 1 as an immunosuppressant for transplant prognosis, a heart transplant model was constructed and FK506@MnO2 / PDA NPs were administered. The specific protocol is as follows: 1. Experiment content.

[0081] 1.1 A mouse model of heterotopic heart transplantation was established by obtaining heart grafts from BALB / c mice and transplanting them into the peritoneal cavity of C57BL / 6 recipient mice. The aorta and pulmonary artery of the grafts were anastomosed to the abdominal aorta and inferior vena cava of the recipient mice, respectively. All surgical procedures were performed under isoflurane inhalation anesthesia. Mice were euthanized at specific time points by inhaling an overdose of isoflurane. Graft survival was monitored daily by palpation, and cardiac arrest was defined as loss of graft function.

[0082] The experiment was conducted in four groups: a control group, an FK506 group, a MnO2 / PDA NPs group, and an FK506@MnO2 / PDA NPs group. Recipient mice in the MnO2 / PDA NPs group and the FK506@MnO2 / PDA NPs group were intraperitoneally injected with 10 mg / kg of MnO2 / PDA NPs and FK506@MnO2 / PDA NPs, respectively; the FK506 group was injected with 0.263 mg / kg of FK506 (this dose was calculated to match the FK506 loading in the FK506@MnO2 / PDA NPs group); and the control group was injected with an equal volume of saline. All groups were administered the drugs every two days for a total of four times. Survival rate statistics are shown below. Figure 12 .

[0083] 1.2 On day 6 post-transplantation, heart grafts, autologous hearts, livers, spleens, lungs, and kidneys were harvested from mice in each group. After formalin fixation, they were paraffin-embedded and preserved. Tissue samples were prepared into 4 μm thick sections, dewaxed, rehydrated, and then hematoxylin-eosin (HE) staining was used to assess pathological changes in the grafts and other organs. HE staining results are shown below. Figure 13 .

[0084] Sequential sections of paraffin-embedded tissue, 2 μm thick, were subjected to immunohistochemical staining. Except for CD3 staining, which required 0.01 M citrate buffer (pH 6.0), all other antigen retrievals were performed using EDTA buffer (pH 9.0) via pressure heating for 30 minutes. Sections were incubated with 3 v / v% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Subsequently, the sections were incubated overnight at 4°C with the following monoclonal primary antibodies: CD3 (ab16669, Abcam; 1:200), CD4 (25229T, CST; 1:100), CD8 (ab217344, Abcam; 1:400), CD68 (GB113109, Servicebio; 1:400), CD19 (ab245235, Abcam; 1:1000), and CD138 (ab128936, Abcam; 1:500). The film was developed using DAB+ chromogenic substrate at room temperature for 30 seconds. Finally, the entire film was scanned using the Aperio Scan Scope system, and positive cell quantification was performed using the positive pixel counting algorithm in ImageScope software. The qualitative and quantitative analysis results of positive cells are shown below. Figure 14 See Table 1.

[0085] 1.3 Fresh heart grafts were obtained on day 6 post-transplantation, ground, and filtered through a 200-mesh nylon sieve. Collected cells were stained with the following fluorescently labeled antibodies: CD45 (APC-Cy7), CD4 (APC), CD8 (PE), CD11b (PE), F4 / 80 (APC), CD3 (AF700), NK1.1 (FITC), and CD19 (PE-Cy5). The stained cells were then analyzed using a CytExpert flow cytometer (Beckman Coulter, USA). Results are shown below. Figure 15-16 And Table 2.

[0086] 1.4 To evaluate the safety of FK506@MnO2 / PDA NPs in a heart transplantation model, safety was assessed in mice of each group on day 6 post-transplantation. Abnormal behaviors (such as lethargy, decreased appetite, or weight loss) were recorded, and tissues from major organs (including the patient's own heart, liver, spleen, lungs, and kidneys) were collected for HE staining. The results are shown in [Figure number missing]. Figure 17 .

[0087] 1.5 To investigate the biodistribution and retention characteristics of FK506@MnO2 / PDA NPs after in vivo injection, recipient mice were intraperitoneally injected with 10 mg / kg of FK506@MnO2 / PDA-Cy7 NPs on day 3 post-transplantation. As a control, FK506-Cy7, with equivalent fluorescence intensity, was injected based on previous in vitro fluorescence detection results. Following injection of FK506-Cy7 or FK506@MnO2 / PDA-Cy7, mice were monitored for 7 days using a small animal in vivo imaging system (IVIS Spectrum, China), and images were acquired at specific time points. The fluorescence intensity of the entire abdominal region and the cardiac graft site (determined by palpation) was quantitatively analyzed using the AniViewPro system; the results are shown in 18-19.

[0088] In another independent experimental group, autologous heart and heart graft samples were obtained from recipient mice at predetermined time points following injection of FK506-Cy7 or FK506@MnO2 / PDA-Cy7. These samples were finely scanned and images were acquired using a small animal in vivo imaging system. Finally, the fluorescence intensity of the autologous heart and heart graft at specific time points was calculated using the AniViewPro system. The results are shown below. Figure 20-22 .

[0089] 2. Experimental results.

[0090] 2.1 As Figure 12 As shown, in a mouse model of acute cardiac graft rejection, the graft survival times in the control group, FK506 group, MnO2 / PDANPs group, and FK506@MnO2 / PDA NPs group were 7.00±0.58 days, 7.50±0.50 days, 8.83±0.69 days, and 12.50±0.96 days, respectively. Low-dose FK506 treatment (0.263 mg / kg) did not significantly prolong graft survival time, while both MnO2 / PDA NPs and FK506@MnO2 / PDA NPs significantly prolonged survival, with the FK506@MnO2 / PDA NPs group achieving the longest survival time.

[0091] 2.2 such as Figure 13 As shown, on the sixth day after transplantation, both MnO2 / PDA NPs and FK506@MnO2 / PDA NPs significantly reduced cardiac graft injury and inflammatory cell infiltration, with FK506@MnO2 / PDA NPs exhibiting superior efficacy.

[0092] 2.3 such as Figure 14As shown in Table 1, on day 6 post-transplantation, immunohistochemical staining revealed that FK506@MnO2 / PDA NPs were more effective than MnO2 / PDA in reducing T cell (CD3, CD4, CD8), macrophage (CD68), B cell (CD19), and plasma cell (CD138) infiltration. In contrast, low-dose FK506 failed to significantly reduce inflammatory cell infiltration.

[0093] Table 1. Statistical results of cell infiltration in immunohistochemistry of different treatment groups. 2.4 such as Figure 15-16 As shown in Table 2, on the sixth day post-transplantation, the infiltration of inflammatory cells in the graft was analyzed by flow cytometry. The results showed that low-dose FK506 treatment had no significant effect on the proportion and number of infiltrating immune cells, including leukocytes (CD45), T cells (CD4 and CD8), macrophages (CD11bF4 / 80), NK cells (CD3NK1.1), and B cells (CD19). In contrast, treatment with MnO2 / PDA NPs and FK506@MnO2 / PDA NPs significantly reduced the proportion and number of infiltrating leukocytes, with FK506@MnO2 / PDA NPs exhibiting superior therapeutic efficacy. Figure 15 A, Figure 16 A). In this acute rejection model, T cells accounted for approximately 80% of the total infiltrating inflammatory cells. Although the proportions of CD4 and CD8 cells remained unchanged, their absolute numbers were significantly reduced after NP treatment, and the decrease was more significant in the FK506@MnO / PDA NPs group than in the MnO2 / PDA NPs group. Figure 15 B Figure 16 BC). Furthermore, both MnO2 / PDA NPs and FK506@MnO2 / PDA NPs significantly inhibited macrophage and NK cell infiltration, with the FK506@MnO2 / PDA group showing a more pronounced effect. Figure 15 CD Figure 16 DE). B cells also showed a similar trend: FK506@MnO2 / PDA NPs decreased in absolute numbers, although the overall proportion remained unchanged ( Figure 15 E, Figure 16 F). These findings fully demonstrate that FK506@MnO2 / PDA NPs possess potent immunosuppressive properties, effectively reducing inflammatory cell infiltration and improving graft survival.

[0094] Table 2. Absolute quantitative results of immune cells in flow cytometry for different treatment groups. 2.5 Based on the previously established safety profile of MnO2 / PDA and FK506@MnO2 / PDA NPs at a dose of 10 mg / kg in healthy mice, the safety was further evaluated in a heart transplantation model, such as... Figure 17 As shown, compared with the control group and the low-dose FK506 group, mice treated with MnO2 / PDA or FK506@MnO2 / PDA NPs did not exhibit significant behavioral abnormalities such as lethargy, decreased appetite, or weight loss. Furthermore, histopathological analysis of major organs (including the heart, liver, spleen, lungs, and kidneys) showed no signs of cellular or tissue damage after NP administration.

[0095] 2.6 To evaluate the ability of FK506@MnO2 / PDA NPs to sustain FK506 release in acutely rejected cardiac grafts, Cy7-labeled FK506@MnO2 / PDA (FK506@MnO2 / PDA-Cy7) NPs were used for in vivo fluorescence imaging, with Cy7-labeled FK506 (FK506-Cy7) serving as a control. Figure 18 As shown, the fluorescence signal in the peritoneal cavity and graft area significantly decreased within one day after FK506-Cy7 injection; while the signal remained relatively strong throughout the one-week observation period after FK506@MnO2 / PDA-Cy7 NPs injection. Figure 19 As shown, at all observation time points starting from 5 minutes post-injection, the intraperitoneal fluorescence intensity of the FK506@MnO2 / PDA-Cy7 NPs group was significantly higher than that of the FK506-Cy7 group. Furthermore, starting from 30 minutes post-injection, the fluorescence intensity of the cardiac graft region in the FK506@MnO2 / PDA-Cy7 NPs group was significantly superior to that in the FK506-Cy7 group at all time points. These results demonstrate that the MnO carrier enhances the retention capacity of FK506 in the recipient's peritoneum and the graft.

[0096] In another independent experimental group, in vitro fluorescence imaging analysis revealed that at different time points after injection of FK506-Cy7 or FK506@MnO2 / PDA-Cy7 NPs, the fluorescence intensity of both the autologous heart and the heart graft in the FK506@MnO2 / PDA-Cy7 group was significantly higher than that in the FK506-Cy7 group. Figure 21 This further confirms the long-lasting retention characteristics of FK506@MnO2 / PDA in recipients and grafts. Notably, the fluorescence intensity of the heart graft at each time point after FK506-Cy7 injection was only slightly higher than that of the autologous heart. Figure 22 A); while after injection of FK506@MnO2 / PDA-Cy7 NPs, the fluorescence intensity of the heart graft at each time point was significantly higher than that of the autologous heart (A); Figure 22B). These findings demonstrate that FK506@MnO2 / PDA NPs can effectively target grafts that have experienced acute rejection.

[0097] In summary, this invention designs a tacrolimus-loaded nanomaterial, FK506@MnO2 / PDA NPs, which can be used to achieve sustained release of FK506 in grafts to alleviate acute rejection. This nanomaterial can also reduce graft inflammation by catalyzing the conversion of H2O to O2 and scavenging reactive oxygen species. A schematic diagram of the nanomaterial's operation is shown below. Figure 23 .

[0098] This invention successfully synthesized FK506@MnO2 / PDA NPs via in-situ polymerization, using polydopamine (PDA) as an intermediate carrier to load FK506 onto MnO2 nanosheets. Physicochemical characterization of the nanoparticles showed an average diameter of 142 nm and a negative zeta potential, which helps prolong blood circulation time and enhance graft targeting efficiency. Drug release analysis showed that FK506 was rapidly released within the first 6 hours, followed by a sustained-release phase, with a cumulative release rate of 92% after 60 hours. Stability testing confirmed that the nanosystem maintained stability for 72 hours. Safety assessment determined its in vitro safe dose to be less than 50 μg / mL and its in vivo safe dose to be less than 10 mg / kg (healthy mice). At these doses, FK506@MnO2 / PDA NPs exhibited excellent cellular uptake efficiency and biocompatibility, and effectively inhibited T cell activation and pro-inflammatory cytokine secretion.

[0099] Simultaneously, it was confirmed that FK506@MnO2 / PDA NPs can effectively catalyze the generation of O from HO and promote the scavenging of reactive oxygen species (ROS). ROS play a crucial role in post-transplant ischemia-reperfusion injury (IRI) and graft rejection. IRI is a major pathological challenge in organ transplantation, significantly leading to postoperative complications such as graft rejection and functional impairment. During IRI, hypoxia and oxidative stress drive cell death, mainly due to cellular energy depletion and pro-oxidative imbalance caused by excessive ROS production. These effects cause microcirculatory disturbances, inflammation, and apoptosis. During transplantation, hypoxia-induced cell death activates the immune system, further exacerbating graft inflammation and rejection. Damaged and dead cells release potent pro-inflammatory substances extracellularly, amplifying the immune response. In addition, graft-infiltrating cells such as monocytes and macrophages participate in the inflammatory cascade triggered by danger signals during IRI. Therefore, targeting and alleviating the hypoxic microenvironment and oxidative stress induced by excessive ROS can significantly improve transplant prognosis.

[0100] In a mouse model of acute rejection following heart transplantation, low-dose FK506 (0.263 mg / kg) monotherapy failed to effectively alleviate acute rejection, while nanoparticles loaded with the same dose of FK506 showed significant therapeutic effects. Although MnO2 / PDANPs also showed some efficacy, their effect was significantly lower than that of FK506@MnO2 / PDA NPs. These findings indicate that this nanosystem can achieve graft-targeted delivery of FK506, promoting sustained drug release and improving utilization, and remains effective even at non-therapeutic doses. Unlike traditional FK506, which primarily inhibits T cells, FK506@MnO2 / PDA NPs exert a broader immunosuppressive effect by combining FK506 targeted delivery with reactive oxygen species scavenging through a dual mechanism. This not only inhibits T cells but also reduces the infiltration of macrophages, B cells, and NK cells in the graft.

[0101] In vitro and in vivo fluorescence imaging confirmed that, compared to free FK506, FK506@MnO2 / PDA NPs significantly prolonged retention time and preferentially accumulated in cardiac grafts. Following a single dose, the nanoparticles remained detectable in the peritoneal cavity and grafts of recipient mice for over a week. The fluorescence intensity in the grafts was 2-4 times higher than in the autologous heart, demonstrating superior targeting efficiency.

[0102] The design of FK506@MnO2 / PDA NPs successfully overcomes the limitations of traditional immunosuppressants. By achieving graft-targeted delivery and sustained release of FK506, it significantly reduces drug dosage, minimizes adverse reactions, and improves treatment efficacy. Furthermore, its mechanism of action transcends T-cell suppression, further inhibiting B cells, macrophages, and NK cells by scavenging reactive oxygen species. This targeted immunotherapy strategy will open up innovative research directions for improving organ transplant survival rates and reducing systemic side effects.

[0103] In summary, the FK506@MnO2 / PDA NPs developed in this invention significantly enhance FK506 accumulation in grafts, alleviating acute rejection and prolonging graft survival. Mechanistic studies confirm that this system inhibits T cell activation through FK506 release and regulates the inflammatory microenvironment by scavenging reactive oxygen species and reducing oxidative stress. This study provides a novel, targeted, efficient, and low-toxicity immunosuppressive strategy for organ transplantation, which is expected to improve transplant prognosis and increase long-term graft survival.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing tacrolimus-loaded nanomaterials, characterized in that, Includes the following steps: (1) Disperse MnO2 nanosheets in water, sonicate and centrifuge to obtain supernatant; (2) Mix the supernatant obtained in step (1) with dopamine hydrochloride, adjust the pH to 10-11, stir, centrifuge to obtain the supernatant, dialyze to obtain MnO2 / PDA NPs; (3) Mix the tacrolimus chloroform solution with the MnO2 / PDA NPs obtained in step (2), centrifuge to collect the precipitate, wash, and obtain FK506@MnO2 / PDA NPs, which are nanomaterials loaded with tacrolimus.

2. The preparation method according to claim 1, characterized in that, In step (1), the ultrasonic treatment time is 5-7 hours.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of MnO2 nanosheets to dopamine hydrochloride is MnO2 nanosheets: dopamine hydrochloride = (20-40): (40-60).

4. The preparation method according to claim 1, characterized in that, In step (2), the stirring time is 1.5-3 hours.

5. The preparation method according to claim 1, characterized in that, In step (2), the dialysis involves placing the obtained supernatant in a dialysis bag and dialyzing for 12-16 hours.

6. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of tacrolimus to MnO2 / PDA NPs in the tacrolimus chloroform solution is tacrolimus:MnO2 / PDA NPs=1:(8-12).

7. The preparation method according to claim 1, characterized in that, In step (3), the mixing is performed by stirring at 23-27°C for 12-16 hours.

8. A nanomaterial loaded with tacrolimus, characterized in that, It is mainly prepared by the preparation method described in any one of claims 1-7.

9. The use of the tacrolimus-loaded nanomaterial as described in claim 8 in the preparation of anti-rejection drugs.

10. An anti-rejection drug, characterized in that, It includes the tacrolimus-loaded nanomaterials of claim 8 and pharmaceutically acceptable excipients.

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