Prussian blue composite material and preparation method and application thereof

Abstract

The application provides a Prussian blue composite material and a preparation method and application thereof, and belongs to the technical field of biological medicines.The Prussian blue composite material provided by the application comprises hollow mesoporous Prussian blue nanoparticles, liposomes coated on the hollow mesoporous Prussian blue nanoparticles and borane ammine complexes loaded on the hollow mesoporous Prussian blue nanoparticles.The hollow mesoporous Prussian blue nanoparticles of the application can simulate various enzyme-like activities to remove ROS, such as superoxide dismutase, catalase or peroxidase-like activities to relieve inflammatory damage caused by oxidative stress;the borane ammine complexes are loaded on the hollow mesoporous Prussian blue nanoparticles, the borane ammine complexes can continuously supply hydrogen, reach the inflammation site of dry eye syndrome, and thus relieve the symptoms of dry eye syndrome in combination with the hollow mesoporous Prussian blue nanoparticles;the liposomes can specifically deliver the Prussian blue composite material to specific cells or tissues, so as to improve the targeting and curative effect of the drug and relieve the symptoms of dry eye syndrome.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a Prussian blue composite material and a preparation method and application thereof. BACKGROUND

[0002] Dry eye syndrome is a complex ocular surface disease with a high prevalence, which is related to corneal injury, excessive oxidative stress and inflammation. The current treatment strategy is hydrogen therapy, which mainly uses borane ammonia complex (NH3BH3, referred to as AB). The borane ammonia complex is a molecule with low toxicity, strong acid reactivity and strong hydrogen storage capacity. It can release hydrogen very slowly in a pH = 7 ± 0.1 environment at room temperature, and release hydrogen rapidly in an acidic environment, so that there is a large amount of H2 in the cell, thereby effectively blocking the mitochondrial respiratory chain, reducing the production of adenosine triphosphate (ATP), and thereby inhibiting the expression of intracellular ROS, and relieving dry eye syndrome, but the relief effect is effective. Therefore, how to improve the borane ammonia complex to improve the effect of relieving dry eye syndrome has become a technical problem to be solved in the art. SUMMARY

[0003] The purpose of the present application is to provide a Prussian blue composite material and a preparation method and application thereof. The Prussian blue composite material provided by the present application can slowly release hydrogen to the inflammation site of dry eye syndrome, and at the same time can relieve the damage caused by oxidative stress, thereby effectively relieving the symptoms of dry eye syndrome.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0005] The present application provides a Prussian blue composite material, comprising hollow mesoporous Prussian blue nanoparticles, liposomes coated on the hollow mesoporous Prussian blue nanoparticles, and borane ammonia complex loaded on the hollow mesoporous Prussian blue nanoparticles.

[0006] Preferably, the preparation method of the hollow mesoporous Prussian blue nanoparticles comprises:

[0007] The Prussian blue nanoparticles, the first stabilizer and the first acidic solution are mixed to obtain a mixed solution, and then etching is performed to obtain the hollow mesoporous Prussian blue nanoparticles.

[0008] Preferably, the etching temperature is 130-150℃, and the etching time is 3-8h.

[0009] The present application also provides a preparation method of the Prussian blue composite material described in the above technical solutions, comprising the following steps:

[0010] (1) mixing raw materials of liposomes and an organic solvent to obtain a lipid solution;

[0011] (2) performing rotary evaporation on the lipid solution obtained in the step (1) to obtain a lipid film;

[0012] (3) mixing the lipid film, the hollow mesoporous Prussian blue nanoparticles and water obtained in the step (2) to perform coating to obtain the hollow mesoporous Prussian blue nanoparticles coated by the liposome;

[0013] (4) mixing the hollow mesoporous Prussian blue nanoparticles coated by the liposome, the buffer solution and the borane-ammonia complex obtained in the step (3) to perform loading to obtain the Prussian blue composite material.

[0014] Preferably, the raw material of the liposome in the step (1) comprises lecithin, cholesterol and DSPE-mPEG2000.

[0015] Preferably, the mass ratio of the lipid film to the hollow mesoporous Prussian blue nanoparticles in the step (3) is (0.5-4):1.

[0016] Preferably, the temperature for coating in the step (3) is 10-30℃, and the time for coating is 10-30 min.

[0017] Preferably, the mass ratio of the borane-ammonia complex to the hollow mesoporous Prussian blue nanoparticles coated by the liposome in the step (4) is 1:(0.2-17).

[0018] Preferably, the temperature for loading in the step (4) is 4-25℃, and the time for loading is 24-72 h.

[0019] The application further provides application of the Prussian blue composite material in the above technical solution or the Prussian blue composite material prepared by the preparation method in the above technical solution in a pharmaceutical composition for relieving symptoms of dry eye.

[0020] The application provides a Prussian blue composite material, which comprises hollow mesoporous Prussian blue nanoparticles, liposomes coating the hollow mesoporous Prussian blue nanoparticles and borane-ammonia complexes loaded on the hollow mesoporous Prussian blue nanoparticles. The hollow mesoporous Prussian blue nanoparticles used in the application can simulate various enzyme-like activities to scavenge ROS, such as superoxide dismutase (SOD), catalase (CAT) or peroxidase (POD) like activities to relieve inflammatory damage caused by oxidative stress; the borane-ammonia complexes are loaded on the hollow mesoporous Prussian blue nanoparticles, the borane-ammonia complexes can continuously supply hydrogen to reach the inflammation site of dry eye, thereby relieving the symptoms of dry eye in combination with the hollow mesoporous Prussian blue nanoparticles; the liposomes can specifically deliver the Prussian blue composite material to specific cells or tissues, thereby improving the targeting and efficacy of the drug, and further relieving the symptoms of dry eye. Experimental results show that the Prussian blue composite material provided by the application can effectively improve the symptoms and corneal morphological structure of dry eye mice, and also can reduce the ROS expression level of dry eye HCECs cells, and effectively improve the symptoms of dry eye. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Transmission electron microscopy image of HPB in Example 1;

[0022] Figure 2 Transmission electron microscopy image of HPB@Lip in Example 1;

[0023] Figure 3 Transmission electron microscopy image of HPB@Lip@AB prepared in Example 1;

[0024] Figure 4 Anterior segment photograph of corneal fluorescein sodium staining of normal mice;

[0025] Figure 5 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice;

[0026] Figure 6 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice improved by HPB@Lip in Example 1;

[0027] Figure 7 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice improved by AB in Example 1;

[0028] Figure 8 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice improved by HPB@Lip@AB prepared in Example 1;

[0029] Figure 9 Fluorescein sodium staining score of cornea of normal mice, cornea of dry eye mice, respectively after using normal saline, HPB@Lip in Example 1, AB in Example 1 and HPB@Lip@AB prepared in Example 1 to drop eye;

[0030] Figure 10 Tear secretion amount of normal mice, dry eye mice, respectively after using normal saline, HPB@Lip in Example 1, AB in Example 1 and HPB@Lip@AB prepared in Example 1 to drop eye;

[0031] Figure 11 Tear film break-up time of normal mice, dry eye mice, respectively after using normal saline, HPB@Lip in Example 1, AB in Example 1 and HPB@Lip@AB prepared in Example 1 to drop eye;

[0032] Figure 12 H&E staining image of cornea of normal mice;

[0033] Figure 13 H&E staining image of cornea of dry eye mice;

[0034] Figure 14 H&E staining image of mouse cornea after eye-dropping of HPB@Lip prepared in Example 1;

[0035] Figure 15 H&E staining image of mouse cornea after eye-dropping of AB prepared in Example 1;

[0036] Figure 16 H&E staining image of mouse cornea after eye-dropping of HPB@Lip@AB prepared in Example 1;

[0037] Figure 17 DHE staining image of normal HCECs cells in vitro;

[0038] Figure 18 DHE staining image of HCECs cells in vitro with dry eye;

[0039] Figure 19 DHE staining image of HCECs cells in vitro after intervention of HPB@Lip prepared in Example 1;

[0040] Figure 20 DHE staining image of HCECs cells in vitro after intervention of AB prepared in Example 1;

[0041] Figure 21 DHE staining image of HCECs cells in vitro after intervention of HPB@Lip@AB prepared in Example 1;

[0042] Figure 22 DHE staining curves of normal HCECs cells in vitro, HCECs cells in vitro with dry eye, HCECs cells in vitro after intervention of HPB@Lip prepared in Example 1, HCECs cells in vitro after intervention of AB prepared in Example 1 and HCECs cells in vitro after intervention of HPB@Lip@AB prepared in Example 1. DETAILED DESCRIPTION

[0043] The present application provides a Prussian blue composite material, comprising hollow mesoporous Prussian blue nanoparticles, liposomes coated on the hollow mesoporous Prussian blue nanoparticles and borane ammine complex loaded on the hollow mesoporous Prussian blue nanoparticles.

[0044] The present application does not have special limitations on the sources of raw materials, and commercially available products known to those skilled in the art can be used.

[0045] The Prussian blue composite material provided by the present application comprises hollow mesoporous Prussian blue nanoparticles. The hollow mesoporous Prussian blue nanoparticles can simulate various enzyme-like activities to scavenge ROS, such as superoxide dismutase (SOD), catalase (CAT) or peroxidase (POD) like activities to alleviate inflammatory damage caused by oxidative stress, thereby alleviating dry eye symptoms in combination with borane ammine complex.

[0046] In the present application, the preparation method of the hollow mesoporous Prussian blue nanoparticles preferably comprises:

[0047] The Prussian blue nanoparticles, the first stabilizer and the first acidic solution are mixed to obtain a mixed solution, and then etching is performed to obtain the hollow mesoporous Prussian blue nanoparticles.

[0048] In the present application, the particle size of the Prussian blue nanoparticles is preferably 50-300 nm.

[0049] In the present application, the preparation method of the Prussian blue nanoparticles preferably comprises: mixing potassium ferricyanide, a second stabilizer and a second acidic solution to obtain a mixed solution, and then performing hydrothermal reaction to obtain the Prussian blue nanoparticles.

[0050] In the present application, the second stabilizer is preferably polyvinylpyrrolidone; and the polyvinylpyrrolidone is preferably at least one of K15, K30, K60 and K90. In the present application, the polyvinylpyrrolidone can be adsorbed on the surface of the Prussian blue nanoparticles to prevent aggregation and agglomeration, thereby maintaining the dispersibility of the Prussian blue nanoparticles.

[0051] In the present application, the second acidic solution is preferably hydrochloric acid; and the concentration of the hydrochloric acid is preferably 0.005-0.02 M, more preferably 0.01-0.015 M. In the present application, the second acidic solution is used to provide an acidic environment.

[0052] In the present application, the concentration of potassium ferricyanide in the mixed solution is preferably 1-10 mg / mL; and the concentration of the second stabilizer in the mixed solution is preferably 50-200 mg / mL. As an embodiment, the concentration of potassium ferricyanide in the mixed solution can be 2 mg / mL, 3 mg / mL, 3.3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL or 9 mg / mL; and the concentration of the second stabilizer in the mixed solution can be 75 mg / mL, 100 mg / mL, 150 mg / mL or 180 mg / mL.

[0053] The present application does not have special limitations on the operation of mixing the potassium ferricyanide, the second stabilizer and the second acidic solution, and any preparation method known to those skilled in the art can be used.

[0054] In the present application, the temperature of the hydrothermal reaction is preferably 75-85℃, more preferably 80℃; the time of the hydrothermal reaction is preferably 16-24h, more preferably 20h. Limiting the temperature and time of the hydrothermal reaction in the above range can improve the degree of the hydrothermal reaction, thereby improving the yield of the Prussian blue nanoparticles.

[0055] After the hydrothermal reaction, the product obtained from the hydrothermal reaction is preferably subjected to centrifugation, separation, washing and drying in sequence to obtain the Prussian blue nanoparticles.

[0056] The operation of the centrifugation is not particularly limited in the present application, and the operation well known to those skilled in the art can be used.

[0057] The operation of the separation is not particularly limited in the present application, and the operation well known to those skilled in the art can be used to obtain a solid.

[0058] The operation of the washing is not particularly limited in the present application, and the washing to pH 7±0.1 can be used.

[0059] The operation of the drying is not particularly limited in the present application, and the drying to constant weight can be used.

[0060] In the present application, the type of the first stabilizer is preferably the same as that of the second stabilizer, which is not described herein again.

[0061] In the present application, the first acidic substance is preferably hydrochloric acid; the concentration of the hydrochloric acid is preferably 0.8-1.2M, more preferably 0.9-1.0M. In the present application, the first acidic substance is used to etch the Prussian blue nanoparticles to obtain hollow mesoporous Prussian blue nanoparticles.

[0062] In the present application, the concentration of the Prussian blue nanoparticles in the mixed solution is preferably 0.5-2mg / mL; the concentration of the first stabilizer in the mixed solution is preferably 3-10mg / mL. As an embodiment, the concentration of the Prussian blue nanoparticles in the mixed solution can be 0.6mg / mL, 0.8mg / mL, 1mg / mL, 1.2mg / mL, 1.4mg / mL, 1.6mg / mL or 1.8mg / mL; the concentration of the first stabilizer in the mixed solution can be 4mg / mL, 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL or 9mg / mL.

[0063] The operation of mixing the Prussian blue nanoparticles, the first stabilizer and the first acidic solution is not particularly limited in the present application, and the technical solution for preparing a mixture well known to those skilled in the art can be used.

[0064] In the present application, the etching temperature is preferably 130-150 DEG C, more preferably 140 DEG C; the etching time is preferably 3-8 h, more preferably 4-6 h. The present application limits the etching temperature and time within the above range to improve the etching effect.

[0065] After the etching is completed, the product obtained by the etching is preferably subjected to centrifugation, separation, washing and drying in sequence to obtain the hollow mesoporous Prussian blue nanoparticles.

[0066] The present application does not have special limitations on the centrifugation operation, and the operation well known to those skilled in the art can be used.

[0067] The present application does not have special limitations on the separation operation, and the operation well known to those skilled in the art can be used to obtain a solid.

[0068] The present application does not have special limitations on the washing operation, and the washing to pH 7±0.1 can be used.

[0069] The present application does not have special limitations on the drying operation, and the drying to constant weight can be used.

[0070] The hollow mesoporous Prussian blue nanoparticles provided by the present application have good biocompatibility, and have prominent antioxidant and anti-inflammatory properties.

[0071] The Prussian blue composite provided by the present application further comprises a liposome coated on the hollow mesoporous Prussian blue nanoparticles. The liposome used in the present application can specifically deliver the Prussian blue composite to specific cells or tissues, thereby improving the targeting and efficacy of the drug, and further relieving the symptoms of dry eye.

[0072] The Prussian blue composite provided by the present application further comprises a borane ammine complex loaded on the hollow mesoporous Prussian blue nanoparticles. The borane ammine complex is loaded on the hollow mesoporous Prussian blue nanoparticles in the present application, and the borane ammine complex can continuously supply hydrogen to the inflammation site of dry eye, thereby relieving the symptoms of dry eye in combination with the hollow mesoporous Prussian blue nanoparticles.

[0073] The hollow mesoporous Prussian blue nanoparticles used in the present application can simulate various enzyme-like activities to scavenge ROS, such as superoxide dismutase (SOD), catalase (CAT) or peroxidase (POD) like activities to relieve inflammatory damage caused by oxidative stress; the borane ammine complex is loaded on the hollow mesoporous Prussian blue nanoparticles, and the borane ammine complex can continuously supply hydrogen to the inflammation site of dry eye, thereby relieving the symptoms of dry eye in combination with the hollow mesoporous Prussian blue nanoparticles; the liposome can specifically deliver the Prussian blue composite to specific cells or tissues, thereby improving the targeting and efficacy of the drug, and further relieving the symptoms of dry eye.

[0074] The application further provides a preparation method of the Prussian blue composite material.

[0075] (1) mixing raw materials of liposomes and an organic solvent to obtain a lipid solution;

[0076] (2) performing rotary evaporation on the lipid solution obtained in the step (1) to obtain a lipid film;

[0077] (3) mixing the lipid film obtained in the step (2), hollow mesoporous Prussian blue nanoparticles and water to perform coating to obtain liposome-coated hollow mesoporous Prussian blue nanoparticles;

[0078] (4) mixing the liposome-coated hollow mesoporous Prussian blue nanoparticles obtained in the step (3), a buffer solution and a borane-ammonia complex to perform loading to obtain the Prussian blue composite material.

[0079] The application mixes raw materials of liposomes and an organic solvent to obtain a lipid solution.

[0080] In the application, the raw materials of liposomes preferably include lecithin, cholesterol and DSPE-mPEG2000.

[0081] In the application, the mass of the lecithin is preferably 50-70% of the total mass of the lecithin, cholesterol and DSPE-mPEG2000, and more preferably 60%; the mass of the cholesterol is preferably 10-30% of the total mass of the lecithin, cholesterol and DSPE-mPEG2000, and more preferably 20%; and the mass of the DSPE-mPEG2000 is preferably 10-30% of the total mass of the lecithin, cholesterol and DSPE-mPEG2000, and more preferably 20%. Limiting the mass of the lecithin, cholesterol and DSPE-mPEG2000 within the above range can further improve the effect of relieving the symptoms of dry eye; wherein the lecithin is the main structural component of the liposomes; the cholesterol can enhance the stability and rigidity of the liposomes; and the DSPE-mPEG2000 can modify the surface of the liposomes to increase the circulation time and stability thereof in the body.

[0082] In the application, the organic solvent is preferably dichloromethane. The application does not have special limitations on the amount of the organic solvent, as long as each raw material is completely dissolved.

[0083] The application does not have special limitations on the operation of mixing the raw materials of liposomes and the organic solvent, and any technical solution for preparing a mixture known to those skilled in the art can be used.

[0084] After obtaining the lipid solution, the application performs rotary evaporation on the lipid solution to obtain a lipid film.

[0085] The present application does not have special limitation to the operation of the rotary evaporation, as long as the organic solvent is completely evaporated.

[0086] After obtaining the lipid film, the present application mixes the lipid film, the hollow mesoporous Prussian blue nanoparticles and water to perform coating, so as to obtain the hollow mesoporous Prussian blue nanoparticles coated by the liposome.

[0087] In the present application, the mass ratio of the lipid film to the hollow mesoporous Prussian blue nanoparticles is preferably (0.5-4):1. As an embodiment, the mass ratio of the lipid film to the hollow mesoporous Prussian blue nanoparticles can be (1-2):1.

[0088] The present application does not have special limitation to the amount of the inorganic solvent, as long as the raw material is dissolved.

[0089] The present application does not have special limitation to the operation of mixing the lipid film, the hollow mesoporous Prussian blue nanoparticles and water, and the technical solution for preparing the mixture material well known by the person skilled in the art can be adopted.

[0090] In the present application, the temperature of the coating is preferably 10-30℃; the time of the coating is preferably 10-30min; and the coating is preferably performed under the ultrasonic condition. The present application limits the process parameters of the coating within the above range, so as to improve the coating effect of the liposome.

[0091] The present application does not have special limitation to the power of the ultrasonic, and the power well known by the person skilled in the art can be adopted.

[0092] As an embodiment, the temperature of the coating can be 15℃, 20℃ or 25℃; and the time of the coating can be 15min, 20min or 25min.

[0093] After the coating is completed, the present application preferably sequentially performs centrifugation, separation and drying on the product obtained by the coating, so as to obtain the hollow mesoporous Prussian blue nanoparticles coated by the liposome.

[0094] The present application does not have special limitation to the operation of the centrifugation, and the operation well known by the person skilled in the art can be adopted.

[0095] The present application does not have special limitation to the operation of the separation, and the solid can be obtained by the operation well known by the person skilled in the art.

[0096] The present application does not have special limitation to the operation of the drying, and the operation well known by the person skilled in the art can be adopted to dry to constant weight.

[0097] The liposome-encapsulated hollow mesoporous Prussian blue nanoparticles are mixed with a buffer solution and a borane-ammonia complex to obtain the Prussian blue composite material.

[0098] In the present application, the buffer solution is preferably a PBS solution. The present application does not have a special limitation on the concentration of the buffer solution, and a buffer solution well known to those skilled in the art can be used.

[0099] The present application does not have a special limitation on the amount of the buffer solution, as long as the raw materials are completely dissolved.

[0100] In the present application, the mass ratio of the borane-ammonia complex to the liposome-encapsulated hollow mesoporous Prussian blue nanoparticles is preferably 1:(0.2-17), and more preferably 1:(0.5-15). As an embodiment, the mass ratio of the borane-ammonia complex to the liposome-encapsulated hollow mesoporous Prussian blue nanoparticles can be 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, or 1:14.

[0101] The present application does not have a special limitation on the operation of mixing the liposome-encapsulated hollow mesoporous Prussian blue nanoparticles, the buffer solution, and the borane-ammonia complex, and a preparation mixture solution well known to those skilled in the art can be used.

[0102] In the present application, the temperature of the loading is preferably 4-25°C, and the time of the loading is preferably 24-72 h. The loading is preferably carried out under stirring. The present application limits the temperature and time of the loading to the above range to further improve the loading effect.

[0103] The present application does not have a special limitation on the stirring speed, and a well-known operation to those skilled in the art can be used.

[0104] After the loading is completed, the present application preferably sequentially carries out centrifugation, separation, and drying on the product obtained by the loading to obtain the Prussian blue composite material.

[0105] The present application does not have a special limitation on the operation of centrifugation, and a well-known operation to those skilled in the art can be used.

[0106] The present application does not have a special limitation on the operation of separation, and a solid can be obtained by using a well-known operation to those skilled in the art.

[0107] The present application does not have a special limitation on the operation of drying, and a well-known operation to those skilled in the art can be used to dry to a constant weight.

[0108] The preparation method provided by the application has simple process.

[0109] The application further provides application of the Prussian blue composite material or the Prussian blue composite material prepared by the preparation method in a drug composition for relieving symptoms of dry eye.

[0110] The application of the Prussian blue composite material in the drug composition for relieving symptoms of dry eye is not specially limited in operation, and application operation well known to those skilled in the art can be adopted.

[0111] The technical solutions in the application will be clearly and completely described below with reference to the embodiments in the application. Obviously, the described embodiments are only some of the embodiments of the application, but not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0112] Embodiment 1

[0113] A Prussian blue composite material is composed of hollow mesoporous Prussian blue nanoparticles, liposomes coated on the hollow mesoporous Prussian blue nanoparticles and borane ammine complex loaded on the hollow mesoporous Prussian blue nanoparticles;

[0114] The preparation method of the Prussian blue composite material is as follows:

[0115] (1) 660 mg of potassium ferricyanide and 15 g of polyvinylpyrrolidone K30 are added into 200 mL of 0.01M hydrochloric acid, magnetically stirred for 0.5 h to obtain a mixed solution, and then transferred to a high-pressure reaction kettle for hydrothermal reaction at 80℃ for 20 h, and then centrifuged at 12000 rpm for 5 min, followed by separation, and then washed with deionized water until pH = 7, and dried to obtain Prussian blue nanoparticles; wherein the concentration of potassium ferricyanide in the mixed solution is 3.3 mg / mL; and the concentration of polyvinylpyrrolidone K30 in the mixed solution is 75 mg / mL;

[0116] (2) 200 mg of the Prussian blue nanoparticles obtained in step (1) and 1.0 g of polyvinylpyrrolidone K30 are added into 200 mL of 1.0M hydrochloric acid, magnetically stirred for 0.5 h to obtain a mixed solution, and then transferred to a high-pressure reaction kettle for etching at 140℃ for 4 h, and then centrifuged at 12000 rpm for 5 min, followed by separation, and then washed with deionized water until pH = 7, and dried to obtain hollow mesoporous Prussian blue nanoparticles, denoted as HPB; wherein the concentration of Prussian blue nanoparticles in the mixed solution is 1 mg / mL; and the concentration of the first stabilizer in the mixed solution is 5 mg / mL;

[0117] (3) 0.6 g of lecithin, 0.2 g of cholesterol and 0.2 g of DSPE-mPEG2000 were weighed and dissolved in 10 mL of dichloromethane to obtain a lipid solution;

[0118] (4) The lipid solution obtained in step (3) was added to a round-bottom flask, and dichloromethane was removed by rotary evaporation at 40°C to obtain a lipid film;

[0119] (5) 0.5 g of the hollow mesoporous Prussian blue nanoparticles obtained in step (2) and 50 mL of deionized water were added to the round-bottom flask in step (4), and coating was performed by ultrasonicating at 30°C for 25 min, followed by centrifugation at 12000 rpm for 20 min, and the supernatant was discarded. The precipitate was vacuum freeze-dried to obtain hollow mesoporous Prussian blue nanoparticles coated with liposomes, denoted as HPB@Lip; wherein the mass ratio of the lipid film to the hollow mesoporous Prussian blue nanoparticles was 2:1;

[0120] (6) 200 mg of borane-ammonia complex (AB) and 100 mg of the hollow mesoporous Prussian blue nanoparticles coated with liposomes obtained in step (5) were added to 10 mL of a PBS solution (pH 7.2), and loading was performed by stirring at 25°C for 24 h, followed by centrifugation at 12000 rpm for 20 min, and the supernatant was discarded. The precipitate was vacuum freeze-dried to obtain a Prussian blue composite material, denoted as HPB@Lip@AB; wherein the mass ratio of the borane-ammonia complex to the hollow mesoporous Prussian blue nanoparticles coated with liposomes was 1:0.5.

[0121] Figure 1 Figure 1 is a transmission electron microscope image of HPB in Example 1; Figure 2 Figure 2 is a transmission electron microscope image of HPB@Lip in Example 1; Figure 3 Figure 3 is a transmission electron microscope image of HPB@Lip@AB prepared in Example 1.

[0122] From Figures 1 to 3 It can be seen that the Prussian blue composite material prepared in the present application is composed of hollow mesoporous Prussian blue nanoparticles, liposomes coated on the hollow mesoporous Prussian blue nanoparticles, and borane-ammonia complex loaded on the hollow mesoporous Prussian blue nanoparticles.

[0123] A dry eye mouse model was constructed by subcutaneous injection of hydrobromide anisodamine (1.5 mg of hydrobromide anisodamine diluted in physiological saline per time, three times a day, for a total of 7 days), and then the HPB@Lip in Example 1, the HPB@Lip@AB prepared in Example 1, and the AB in Example 1 were respectively used for eye drops, twice a day, for a total of 14 days, and then slit lamp photography was performed, and the photographs are shown in Figures 4 to 8 .

[0124] Figure 4Anterior segment photograph of corneal fluorescein sodium staining of normal mice; Figure 5 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice; Figure 6 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice improved by HPB@Lip in Example 1; Figure 7 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice improved by AB in Example 1; Figure 8 Anterior segment photograph of corneal fluorescein sodium staining of dry eye mice improved by HPB@Lip@AB prepared in Example 1; wherein 1 μL of 1% fluorescein sodium was dropped into the conjunctival sac of the mice, and the corneal staining of the mice in each group was observed after 5 min.

[0125] From Figures 4 to 8 It can be seen that HPB@Lip@AB prepared in Example 1 can significantly improve the dry eye symptoms of mice, and the effect is obviously better than that of HPB@Lip and AB.

[0126] The dry eye mouse model was established by subcutaneous injection of hyoscine hydrobromide (1.5 mg / 0.2 mL / time, three times a day, for 7 days), and then 5 μL of normal saline (NS), 5 μL of 200 ug / mL HPB@Lip in Example 1, 5 μL of 200 ug / mL AB in Example 1 and 5 μL of 200 ug / mL HPB@Lip@AB prepared in Example 1 were used for eye drops, and the corneal fluorescein sodium staining score of the mice was as shown in Figure 9 , the tear secretion amount was as shown in Figure 10 , and the tear film break-up time was as shown in Figure 11 , in the figure, CTRL is the cornea of normal mice, and DED is the cornea of dry eye mice; 1 μL of 1% fluorescein sodium was dropped into the conjunctival sac of the mice, and the corneal staining of the mice in each group was observed after 5 min; the fluorescein sodium staining score method used a 12-point method, i.e., the cornea was divided into 4 quadrants (upper, lower, nasal and temporal sides), each quadrant was 0-3 points, no staining was 0 point, 1-30 punctate coloration was 1 point, > 30 punctate coloration but the staining was not fused was 2 points, and 3 points was the appearance of punctate coloration fusion, filament and ulceration, etc.

[0127] From Figures 9 to 11 It can be seen that, compared with the control group, HPB@Lip@AB has obvious improvement effect on dry eye, and the effect is significantly better than that of HPB@Lip and AB.

[0128] 5 μL of 200 ug / mL HPB@Lip in Example 1, 5 μL of 200 ug / mL AB in Example 1 and 5 μL of 200 ug / mL HPB@Lip@AB prepared in Example 1 were used for eye drops, and then H&E staining was performed, and the results were as shown in Figures 12 to 16 .

[0129] H&E staining image of normal mouse cornea; Figure 12 H&E staining image of dry eye mouse cornea; Figure 13 H&E staining image of dry eye mouse cornea; Figure 14 H&E staining image of mouse cornea after eye point of HPB@Lip in Example 1; Figure 15 H&E staining image of mouse cornea after eye point of AB in Example 1; Figure 16 H&E staining image of mouse cornea after eye point of HPB@Lip@AB prepared in Example 1.

[0130] From the H&E staining image of mouse cornea (x400), it can be seen that the normal cornea structure is complete, the stroma layer is dense, and the endothelial structure is continuous and complete; the corneal epithelial layer of dry eye is thin, the stroma layer is disordered, and the endothelial structure is uneven; after eye point of HPB@Lip@AB, the corneal epithelial thickness is restored, the stroma layer and endothelial layer structure are restored, and the effect is obviously better than HPB@Lip and AB.

[0131] In vitro, HCECs cells (corneal epithelial cells) 550mOsm·L -1 osmotic pressure culture system to construct dry eye model, HPB@Lip in Example 1, HPB@Lip@AB prepared in Example 1 and AB in Example 1 were respectively incubated at 37℃, 5% CO2 incubator for 24h, and the results are shown in Figures 17 to 22

[0132] Figure 17 DHE staining image of normal in vitro HCECs cells; Figure 18 DHE staining image of dry eye in vitro HCECs cells; Figure 19 DHE staining image of in vitro HCECs cells after intervention of HPB@Lip in Example 1; Figure 20 DHE staining image of in vitro HCECs cells after intervention of AB in Example 1; Figure 21 DHE staining image of in vitro HCECs cells after intervention of HPB@Lip@AB prepared in Example 1; Figure 22 DHE staining curve of normal in vitro HCECs cells, dry eye in vitro HCECs cells, in vitro HCECs cells after intervention of HPB@Lip in Example 1, in vitro HCECs cells after intervention of AB in Example 1 and in vitro HCECs cells after intervention of HPB@Lip@AB prepared in Example 1, in the figure, CTRL is normal in vitro HCECs cells, HS is dry eye in vitro HCECs cells.

[0133] From the H&E staining image of mouse cornea (x400), it can be seen that the normal cornea structure is complete, the stroma layer is dense, and the endothelial structure is continuous and complete; the corneal epithelial layer of dry eye is thin, the stroma layer is disordered, and the endothelial structure is uneven; after eye point of HPB@Lip@AB, the corneal epithelial thickness is restored, the stroma layer and endothelial layer structure are restored, and the effect is obviously better than HPB@Lip and AB. Figures 17 to 22It can be seen that the ROS level of normal in-vitro HCECs cells is low; the ROS level of in-vitro HCECs cells of dry eye is obviously increased; after the intervention of HPB@Lip@AB, the ROS level is obviously decreased, and the effect is obviously better than that of HPB@Lip and AB. It is illustrated that HPB@Lip@AB can obviously down-regulate the ROS level of dry eye, and the effect is obviously better than that of HPB@Lip and AB.

[0134] In conclusion, the Prussian blue composite material can effectively improve the symptoms and corneal morphological structure of dry eye mice, and can also reduce the ROS expression level of dry eye HCECs cells, and effectively improve the symptoms of dry eye.

[0135] It can be seen from the above examples that the Prussian blue composite material can slowly release hydrogen to reach the inflammation site of dry eye, and can also relieve the damage caused by oxidative stress, thereby effectively relieving the symptoms of dry eye.

[0136] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A Prussian blue composite material, comprising hollow mesoporous Prussian blue nanoparticles, liposomes coating the hollow mesoporous Prussian blue nanoparticles, and a borane-amine complex loaded on the hollow mesoporous Prussian blue nanoparticles; The method for preparing the hollow mesoporous Prussian blue nanoparticles includes: Prussian blue nanoparticles, a first stabilizer, and a first acidic solution are mixed to obtain a mixture, which is then etched to obtain hollow mesoporous Prussian blue nanoparticles. The first stabilizer is polyvinylpyrrolidone; The first acidic solution is hydrochloric acid; The method for preparing the Prussian blue nanoparticles includes: Potassium ferricyanide, a second stabilizer, and a second acidic solution were mixed to obtain a mixed solution, and then a hydrothermal reaction was carried out to obtain Prussian blue nanoparticles. The second stabilizer is polyvinylpyrrolidone; The second acidic solution is hydrochloric acid; The preparation method of the Prussian blue composite material includes the following steps: (1) The raw materials for liposomes are mixed with an organic solvent to obtain a lipid solution; (2) The lipid solution obtained in step (1) is subjected to rotary evaporation to obtain a lipid film; (3) The lipid film, hollow mesoporous Prussian blue nanoparticles obtained in step (2) are mixed with water and coated to obtain liposome-encapsulated hollow mesoporous Prussian blue nanoparticles. (4) The liposome-encapsulated hollow mesoporous Prussian blue nanoparticles obtained in step (3), buffer solution and borane-ammonia complex are mixed and loaded to obtain Prussian blue composite material. The raw materials for the liposomes in step (1) include lecithin, cholesterol and DSPE-mPEG2000; In step (3), the mass ratio of the lipid film to the hollow mesoporous Prussian blue nanoparticles is (0.5~4):1; In step (4), the mass ratio of the borane-ammonia complex to the liposome-encapsulated hollow mesoporous Prussian blue nanoparticles is 1:(0.2~17).

2. The Prussian blue composite material according to claim 1, characterized in that, The etching temperature is 130~150℃, and the etching time is 3~8h.

3. A method for preparing the Prussian blue composite material according to any one of claims 1 to 2, comprising the following steps: (1) The raw materials for liposomes are mixed with an organic solvent to obtain a lipid solution; (2) The lipid solution obtained in step (1) is subjected to rotary evaporation to obtain a lipid film; (3) The lipid film, hollow mesoporous Prussian blue nanoparticles obtained in step (2) are mixed with water and coated to obtain liposome-encapsulated hollow mesoporous Prussian blue nanoparticles; (4) The liposome-encapsulated hollow mesoporous Prussian blue nanoparticles obtained in step (3), buffer solution and borane-ammonia complex are mixed and loaded to obtain Prussian blue composite material. The raw materials for the liposomes in step (1) include lecithin, cholesterol and DSPE-mPEG2000; In step (3), the mass ratio of the lipid film to the hollow mesoporous Prussian blue nanoparticles is (0.5~4):1; In step (4), the mass ratio of the borane-ammonia complex to the liposome-encapsulated hollow mesoporous Prussian blue nanoparticles is 1:(0.2~17).

4. The preparation method according to claim 3, characterized in that, The coating temperature in step (3) is 10~30℃ and the coating time is 10~30min.

5. The preparation method according to claim 3, characterized in that, In step (4), the temperature of the load is 4~25℃ and the loading time is 24~72h.

6. The use of the Prussian blue composite material according to any one of claims 1 to 2 or the Prussian blue composite material prepared by the preparation method according to any one of claims 3 to 5 in the preparation of a pharmaceutical composition for relieving symptoms of dry eye syndrome.

Images