Galactose modified liver targeting mesoporous silica material, preparation method and application

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a galactose modified liver targeting mesoporous silica material, a preparation method and application. Mesoporous silica is prepared through a hydrothermal synthesis method, modification is carried out through amino, disulfide bonds, graphene quantum dots and amino-terminated PEG chain galactose, a multifunctional drug carrier (PEG-Gal-MSNs-SS-GQDs) with the redox response release function is prepared, meanwhile, leech active peptide GFAGDDAPRA (GA-10) is loaded into a mesoporous channel, liver targeting release of drugs is achieved, and the liver targeting effect is achieved. The material loaded with the GA-10 has good redox response release characteristics and blood fat reducing activity. The PEG-Gal-MSN-SS-GQDs disclosed by the invention have a wide application prospect, and a new thought is provided for research on application of mesoporous silica in peptide drug loading and blood fat reduction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a galactose-modified liver-targeted mesoporous silica material, a preparation method and an application thereof, and specifically relates to the preparation of a liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal), and a drug-carrying system thereof having a redox-responsive release under a certain concentration of glutathione (GSH) enriched condition in vivo; and an active liver-targeted delivery function thereof after galactose modification. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized by diffuse macrovesicular fatty degeneration of hepatocytes, which is caused by factors other than alcohol and other clear liver damage. Timely intervention and treatment of NAFLD is of practical significance for improving the health level of Chinese residents and alleviating medical pressure. In recent years, many researchers have used leech raw materials or their compound prescriptions to intervene in liver diseases, and the results show that they have good therapeutic effects. In the early stage, our research group established HL animal models such as SD rats and KM mice based on high-fat diet induction, and used leech active peptide GA-10 or enzymatic hydrolysate for intervention. The results showed that leeches have a good lipid-lowering effect. However, pharmacokinetic results showed that GA-10 has a very short retention time in the body, and in vivo imaging results suggest that GA-10 accumulates more in the liver, but the retention time is short. Therefore, the development of a new intelligent drug delivery system for the targeted delivery of GA-10 becomes particularly important. For protein and peptide drugs, sustained-release preparations are considered to be ideal dosage forms, which can effectively solve the problems of fast release of active drugs and short effective action time, and can also reduce the number of dosing and improve the stability of drugs. As a new type of fluorescent material, graphene quantum dots (GQDs) are widely used in the fields of drug delivery, cell imaging and DNA detection in the biomedical field due to their excellent biocompatibility, low cytotoxicity, strong near-infrared absorption, high photothermal conversion efficiency, excellent thermal conductivity and small lateral size. Due to the significant difference in glutathione (GSH) concentrations in the extracellular (2-10μM) and intracellular (10mM) environments, redox potential has attracted much attention in various drug delivery stimuli.

[0003] At present, the current stage of intelligent nano drug delivery systems is widely used in targeted tumor drug delivery with faster effects, but there is a lack of sufficient research on chronic diseases such as hyperlipidemia. Galactose, as a common liver-targeting ligand, has excellent targeting and modification capabilities. It can be modified on the surface of mesoporous silica containing carboxyl groups to form an intelligent drug delivery system with liver-targeting capabilities. However, the current research on liver-targeted mesoporous silica materials covered with graphene quantum dots and modified with galactose is still incomplete.

[0004] Therefore, how to provide a graphene quantum dot-covered, galactose-modified liver-targeted mesoporous silica material and a preparation method thereof is a technical problem that needs to be urgently solved by those skilled in the art. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose. Another technical problem to be solved by the present invention is to provide a method for preparing the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose. Another technical problem to be solved by the present invention is to provide an application of the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose and an evaluation of its blood lipid lowering activity for non-alcoholic fatty liver.

[0006] It should be noted that previous studies have found that the glutathione index of liver cells in high-fat SD rats was measured by GSH kit, and the results showed that the enrichment of glutathione in liver cells was significantly higher than that in other organs. GSH can destroy disulfide bonds, so the redox potential based on disulfide bonds is expected to become an ideal stimulus for controlling drug release.

[0007] Leech active peptide GA-10 is a single peptide prepared and separated by in vitro biomimetic enzymatic hydrolysis technology. It has a variety of other biological activities such as anti-thrombosis, anti-coagulation, antioxidant and lipid-lowering. So far, it has been widely used in many fields such as biotechnology, food and medicine. Leech active peptide GA-10 has good hydrophilicity, and each amino acid is connected by an amide bond, which makes it have good application prospects in the development of functional composite materials.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first technical purpose of the present invention is to provide a liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal). The molecular structure of the material is PEG-Gal-MSNs-SS-GQDs. It is a mesoporous silica prepared by a hydrothermal synthesis method, and modified by amino groups, disulfide bonds, graphene quantum dots and amino-terminated PEG chains with galactose to obtain a multifunctional drug carrier with redox-responsive release.

[0010] Specifically, the structure of the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) is as follows: Figure 1As shown. The yellow ball refers to the mesoporous silica material (MSNs) prepared by hydrothermal synthesis, and the pore size of the mesoporous silica (MSN) is 3-5nm; the blue hexagonal column refers to the mesoporous channel after the template is removed; the chemical structure on the right is the structure after modification of amino groups, disulfide bonds, graphene quantum dots, and PEG-Gal, in which the dark blue hexagon represents graphene quantum dots.

[0011] It should be noted that after synthesis, the surface and mesoporous channels of mesoporous silica contain a large number of silanol groups, which have great adhesion. The silanol groups can bond with the amino groups in triethoxysilane, so that the surface of the mesoporous silica carries a large number of amino groups and the adhesion disappears; L-cystine can be grafted with the amino group through an amidation reaction under the condition of carboxyl activation, and the disulfide bonds are modified on the mesoporous surface; the surface of graphene quantum dots carries a large number of carboxyl groups, which can bond with the amino end of L-cystine through an amidation reaction under the condition of carboxyl activation, thereby achieving the effect of blocking the pore surface; the PEG-modified galactose end carries an amino group, which can react with the carboxyl group of L-cystine, thereby bonding to the surface of the material, so that the material has the ability to actively release liver-targeted drugs.

[0012] The second technical purpose of the present invention is to provide a method for preparing the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) as described above, the method specifically comprising the following steps:

[0013] a. Add NaOH solution (1-2 mL, 2 m NaOH) to hexadecyltrimethylammonium bromide (CTAB) solution (0.1-1 g CTAB dissolved in 200-300 mL deionized water), and stir vigorously at 60-90 ° C; then add tetraethoxysilane (TEOS) (1.5-3 mL) dropwise therein for 5-15 min, stir vigorously for 1-3 h, and obtain a white precipitate. The obtained nanoparticles are collected by repeated centrifugation, and washed with deionized water and ethanol for 3 times. The obtained solid is vacuum dried at 120 ° C for 10-15 h;

[0014] b. The mesoporous silica prepared in step a with a template is subjected to a temperature program and calcined at 500 to 600 ° C for 5 to 8 h using a muffle furnace to remove the template surfactant used to make the pores;

[0015] c. Take 150-300 mg of mesoporous nano-silica with a particle size of 150±30 nm, ultrasonically disperse it in 10-30 mL of anhydrous toluene, add 100-300 μL of 3-aminopropyltriethoxysilane (APTES), and reflux it at 100-120°C for 15-20 hours under stirring; then centrifuge the reaction solution, ultrasonically disperse it, and wash it thoroughly with toluene and methanol, and repeat the operation three times, and then dry it in a vacuum drying oven at 50-80°C overnight to obtain white powder MSNs-NH2;

[0016] d. 5 to 20 mg of L-cystine (L-Cys) was dispersed in 10 to 30 mL of PBS (pH = 5.0) containing 40 to 60 mg of EDC and 20 to 30 mg of NHS. After 1 h, the MSNs-NH2 (30 to 50 mg) synthesized in step c was added thereto and stirred at room temperature for 24 h. The obtained MSNs-SS-NH2 particles were separated by centrifugation and washed with PBS (pH = 5.0) and water to remove excess L-Cys, and then freeze-dried to obtain MSNs-SS-NH2;

[0017] e. 2 to 10 mg of the MSNs-SS-NH2 particles prepared in step d were dispersed in 2 mL of deionized water, and three samples were prepared in parallel for each group; then 5, 10, and 25 mg of GA-10 were added to the above solution and stirred for 20 to 30 h, and then the encapsulation efficiency was determined by centrifugation;

[0018] f. Disperse the purchased graphene quantum dots GQDs (0.5-3 mg), EDC (5-20 mg) and NHS (2-10 mg) in 5-20 mL of deionized water, and stir the solution for 1 h to make the carboxyl groups on the GQDs active;

[0019] g. The MSNs-SS-NH2 particles (10-30 mg) prepared in step d were added to the solution containing GQDs, and the amino groups on the MSNs-SS-NH2 particles were connected to GQDs through an amidation reaction. The mixture was stirred overnight to obtain MSNs-SS-GQDs. The obtained MSNs-SS-GQDs were collected by centrifugation, washed with deionized water, freeze-dried in a vacuum, and stored at 4°C.

[0020] h. Take 5-20 mg of MSN-SS-GQDs and 5-20 mg of solid PEG-Gal, add 1-5 mL of deionized water respectively, and adjust the pH to 7-12 with borate buffer; stir the solution for 10-20 min, then add 30-60 mg of solid sodium triborohydride (Na(CN)BH3), and store the solution under magnetic stirring overnight; collect the galactose functionalized product by centrifugation, wash repeatedly to remove excess reagents, and obtain the final product Gal-PEG-MSN-GQDs, which is stored at 4°C.

[0021] It should be noted that mesoporous silica was synthesized by hydrothermal synthesis, the amino groups in 3-aminopropyltriethoxysilane (APTES) were modified on the mesoporous surface (MSNs-NH2), and then L-cystine was grafted on the mesoporous surface (MSNs-SS-NH2), the leech active peptide GA-10 was loaded into the mesoporous channel, and then the graphene quantum dots were bonded to L-cystine to prepare a redox-responsive drug release system (MSNs-SS-GQDs) affected by glutathione (GSH) concentration, and finally, PEG-modified galactose (PEG-Gal) carrying an amino group at the end was bonded to the carboxyl group on the surface of the mesoporous material through an amidation reaction to prepare a redox drug release system with active liver targeting function.

[0022] Furthermore, the present invention selects mesoporous silica as a matrix, synthesizes mesoporous silica by hydrothermal synthesis, bonds the amino group in 3-aminopropyltriethoxysilane (APTES) to the silanol group, and the amino group is modified on the mesoporous surface (MSNs-NH2), and L-cystine is grafted with disulfide bonds on the mesoporous surface through an amidation reaction between the carboxyl group and the amino group under light-proof conditions (MSNs-SS-NH2), and the leech active peptide GA-10 is loaded into the mesoporous channel in an aqueous solution through electrostatic adsorption and the like, and then the carboxyl group on the graphene quantum dots is bonded to the amino group on the L-cystine under light-proof conditions to prepare a redox-responsive drug release system (MSNs-SS-GQDs) affected by glutathione (GSH) concentration, and finally, PEG-modified galactose (PEG-Gal) carrying an amino group at the end is bonded to the carboxyl group on the surface of the mesoporous material through an amidation reaction to prepare a redox drug release system with active liver targeting function.

[0023] Furthermore, in step c, the sufficient washing is performed three times with toluene and methanol alternately, and the last washing liquid is methanol.

[0024] Furthermore, in step d, after adding L-cystine, the entire reaction process is carried out under light-proof conditions, and the MSNs-NH2 powder is added slowly to allow the reaction to proceed fully and evenly.

[0025] The third technical purpose of the present invention is to provide a liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) as described above for use in pharmaceutical preparations.

[0026] Furthermore, the graphene quantum dot-covered, galactose (Gal)-modified liver-targeted mesoporous silica material is used in the preparation of drugs for preventing or treating non-alcoholic fatty liver disease.

[0027] It should be noted that the post-modified galactose has an active liver-targeted delivery function, and the post-modified galactose has a PEG chain added, which will not cause red blood cells to break and has good biocompatibility.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The synthesis method disclosed in the present invention is simple, and the raw materials are cheap and easily available. By selecting mesoporous silica with a large specific surface area as the basis, more modification sites are provided, and mesoporous silica covered with graphene quantum dots is used, which has a certain redox response release function; and the galactose-modified mesoporous silica material has excellent active liver-targeted delivery ability, and the leech active peptide GA-10 is loaded into the mesoporous channel of silica, and the outermost layer of the channel is covered with graphene quantum dots, which has good application prospects in the loading of polypeptides in new intelligent drug delivery systems.

[0030] 2) The content of the leech active peptide GA-10 loaded in the present invention can be determined by high performance liquid chromatography (HPLC), thereby improving the detection sensitivity of the drug.

[0031] 3) Compared with administering pure GA-10 to SD rats, the present invention has better liver-targeted drug release ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 Schematic diagram of the structure of liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose.

[0034] Figure 2 Diagram of the preparation process of liver-targeted mesoporous silica materials covered with graphene quantum dots and modified with galactose.

[0035] Figure 3 This is a transmission electron microscope image of the mesoporous silica material in Example 1.

[0036] Figure 4 This is the transmission electron microscopy image of MSNs-SS-GQDs in Example 3.

[0037] Figure 5 The Darwin hydrodynamic particle size and Zeta potential diagram of MSN, MSNs-NH2, MSNs-SS-NH2 and MSNs-SS-GQDs modified in Example 3.

[0038] Figure 6 FTIR spectra of MSNs (a), MSNs-containing CTAB (b), MSNs-NH2 (c), MSNs-SS-NH2 (d) and MSNs-SS-GQDs (e).

[0039] Figure 7 The graph shows the 24h cytotoxicity assay of GA-10 and MSNs-SS-GQDs on Hep G2 cells.

[0040] Figure 8 The figure shows the changing trend of rat body weight.

[0041] Fig. 9 This is the appearance of rat liver.

[0042] Fig.10 HE staining of rat liver.

[0043] Fig.11 This is an Oil Red O staining image of rat liver.

[0044] Fig.12 The biochemical indexes of rats in each group were tested. , n=6). DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0047] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0048] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0049] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0050] The invention discloses a galactose-modified liver-targeted mesoporous silica material, a preparation method and an application thereof.

[0051] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0052] Example 1

[0053] First, NaOH solution (1-2 ml, 2 m [with 25 mL, 2 g NaOH]) was added to CTAB solution (0.2-1 g CTAB dissolved in 200-300 ml deionized water) and stirred vigorously at 50-100 ° C; then, TEOS (2.0-3.0 ml) was added dropwise to the above solution for 10 min and stirred vigorously for 1-3 h to obtain a white precipitate. The obtained nanoparticles were collected by repeated centrifugation (9000 rpm, 20 min), washed with deionized water and ethanol for 3 times, and the obtained solid was vacuum dried at 120 ° C for 6-24 h to remove the template. Method 1: Program heating in a muffle furnace: 1 00℃ for 0.5-2h, then raise the temperature to 550℃ within 3h, and finally calcine at 550℃ for 5-8h to remove the template surfactant used to make the pores; Method 2 for removing the template agent: ultrasonically disperse the white powder in a methanol solution of concentrated hydrochloric acid (wherein 50-200mL of methanol and 5-8mL of concentrated hydrochloric acid are added to every 1g of white powder), reflux at 70℃ with strong stirring for 8-15 hours, filter, wash with appropriate amount of methanol 3 times, and then place in a vacuum drying oven overnight to obtain mesoporous silica.

[0054] More preferably, firstly, NaOH solution (1.75 ml, 2 mL [matching 25 mL, 2 g NaOH]) is added to CTAB solution (0.5 g CTAB was dissolved in 240 ml of deionized water) and stirred vigorously at 80°C; then, TEOS (2.5 ml) was added dropwise to the above solution for 10 min and stirred vigorously for 2 h to obtain a white precipitate. The obtained nanoparticles were collected by repeated centrifugation (9000 rpm, 20 min), washed 3 times with deionized water and ethanol, and the obtained solid was vacuum dried at 120°C for 12 h. Method 1 for removing the template agent: programmed temperature was used in a muffle furnace: 100°C for 1 h, then heated to 550°C within 3 h, and finally calcined at 550°C for 6 h to remove the template surfactant used to make the pores; Method 2 for removing the template agent: the white powder was ultrasonically dispersed in a methanol solution of concentrated hydrochloric acid (wherein 100 mL of methanol and 6 mL of concentrated hydrochloric acid were added to every 1 g of white powder), refluxed at 70°C with strong stirring for 12 h, filtered, washed 3 times with an appropriate amount of methanol, and then placed in a vacuum drying oven overnight to obtain mesoporous silica.

[0055] Material Characterization: Figure 3 This is a transmission electron micrograph of MSN.

[0056] Table 1 Characterization of properties of MSNs, MSNs-NH2, MSNs-SS-NH2 and MSNs-SS-GQDs

[0057]

[0058] As shown in Table 1, the specific surface area and average pore size of mesoporous silica (MSN) are 1224.3800±32.2800 m 2 / g and 3.63nm; after modification with graphene quantum dots (GQDs), the specific surface area and average pore size are 49.4348±0.4920m 2 / g and 3.5nm.

[0059] Example 2

[0060] Take 100-300 mg of mesoporous nano-silica with a particle size of 150±30 nm, ultrasonically disperse it in 10-30 mL of anhydrous toluene, add 100-300 μL of 3-aminopropyltriethoxysilane (APTES), and reflux it at 115°C for 15-24 hours under strong stirring. Finally, centrifuge the reaction solution (7800 rpm, 10 min), ultrasonically disperse it, and wash it with toluene and methanol, and repeat the operation three times, and then dry it in a vacuum drying oven at 50-80°C overnight to obtain a white powder.

[0061] First, L-Cys (5-20 mg) was dispersed in 10-30 ml PBS (pH = 5.0) containing EDC (30-80 mg) and NHS (10-50 mg). After 1 hour, the synthesized MSN-NH2 (20-50 mg) was added to the above solution and stirred at room temperature for 18-36 hours. The obtained MSNs-SS-NH2 particles were separated by centrifugation, washed with PBS (pH = 5.0) and water to remove excess L-Cys, and finally the obtained solution was freeze-dried using a freeze dryer.

[0062] Typically, MSNs-SS-NH2 particles (2-10 mg) were dispersed in 1-5 ml of deionized water, and then 5, 10, and 25 mg of GA-10 were added to the solution, stirred for 24 h, and the encapsulation efficiency was determined by centrifugation.

[0063] Further preferably, 200 mg of mesoporous nano-silica with a particle size of 150 ± 30 nm is ultrasonically dispersed in 20 mL of anhydrous toluene, 200 μL of 3-aminopropyltriethoxysilane (APTES) is added, and refluxed for 18 h under strong stirring at 115 ° C. Finally, the reaction solution is centrifuged (7800 rpm, 10 min), ultrasonically dispersed, and fully washed with toluene and methanol, and the operation is repeated three times, and then dried in a vacuum drying oven at 60 ° C overnight to obtain a white powder.

[0064] First, L-Cys (10 mg) was dispersed in 20 ml PBS (pH = 5.0) containing EDC (50 mg) and NHS (25 mg). After 1 h, the synthesized MSN-NH2 (40 mg) was added to the above solution and stirred at room temperature for 24 h. The obtained MSNs-SS-NH2 particles were separated by centrifugation and washed with PBS (pH = 5.0) and water to remove excess L-Cys. Finally, the obtained solution was freeze-dried using a freeze dryer.

[0065] Typically, MSNs-SS-NH2 particles (5 mg) were dispersed in 2 ml of deionized water, and then 5, 10, and 25 mg of GA-10 were added to the above solution, stirred for 24 h, and the encapsulation efficiency was determined by centrifugation.

[0066] Material characterization: Table 2 shows the encapsulation efficiency and drug loading of MSNs-SS-NH2.

[0067] Table 2 Results of encapsulation efficiency and drug loading of GA-10 and materials in different proportions

[0068]

[0069] Example 3

[0070] GQDs were covalently grafted to the outer surface of MSNs through the amidation reaction between the amino groups of MSNs-SS-NH2 and the carboxyl groups of GQDs. Briefly, the obtained GQDs (0.5-2 mg), EDC (5-20 mg) and NHS (2-10 mg) were dispersed in 5-20 ml of deionized water, and the solution was stirred for 0.5-2 h to achieve carboxyl activity and further connect with MSNs-SS-NH2 particles. Then MSNs-SS-NH2 particles (10-30 mg) were added to the above solution and stirred overnight. The obtained MSNs-SS-GQDs were collected by centrifugation, washed with deionized water, freeze-dried in vacuum, and stored at 4 °C. MSN-SS-GQDs, GA-10-MSN-SS-GQDs 0.5-3 ml and solid PEG-Gal 5-20 mg at a concentration of 5 mg / mL were taken in a beaker and the pH was adjusted to 9 with borate buffer. The solution was stirred for 15 min, and then 20-80 mg of solid sodium triborohydride (Na(CN)BH3) was added. The solution was stored under magnetic stirring overnight, and the galactose functionalized product was collected by centrifugation. The excess reagent was repeatedly washed to remove the final product, PEG-Gal-MSN-GQDs, was stored at 4°C.

[0071] Further preferably, GQDs are covalently grafted to the outer surface of MSNs through an amidation reaction between the amino groups of MSNs-SS-NH2 and the carboxyl groups of GQDs. Briefly, the obtained GQDs (1 mg), EDC (10 mg) and NHS (5 mg) were dispersed in 10 ml of deionized water, and the solution was stirred for 1 h to achieve carboxyl activity and further connected with MSNs-SS-NH2 particles. Then MSNs-SS-NH2 particles (20 mg) were added to the above solution and stirred overnight. The obtained MSNs-SS-GQDs were collected by centrifugation, washed with deionized water, freeze-dried in a vacuum, and stored at 4 ° C. MSN-SS-GQDs, GA-10-MSN-SS-GQDs 2 ml and solid PEG-Gal 10 mg at a concentration of 5 mg / mL were taken in a beaker, and the pH was adjusted to 9 with borate buffer. The solution was stirred for 15 min, then 50 mg of solid sodium triborohydride (Na(CN)BH3) was added, the solution was stored under magnetic stirring overnight, the galactose functionalized product was collected by centrifugation, and the excess reagent was removed by repeated washing. The final product PEG-Gal-MSN-GQDs was stored at 4°C.

[0072] Material Characterization: Figure 4 Transmission electron microscopy image of MSNs-SS-GQDs. Figure 5 The particle size distribution and Zeta potential measurement diagram of MSN, MSNs-NH2, MSNs-SS-NH2 and MSNs-SS-GQDs. Figure 6FTIR spectra of MSNs (a), MSNs-containing CTAB (b), MSNs-NH2 (c), MSNs-SS-NH2 (d) and MSNs-SS-GQDs (e).

[0073] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following experiments are conducted to further illustrate the technical features disclosed in the present invention, but they should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above invention without inventive work are also deemed to fall within the scope of protection of the present invention.

[0074] 1) Using the Hep G2 cell line, the MTT method was used to determine the cytotoxicity of the MSN-SS-GQDs mesoporous silica nanodrug carrier and GA-10 peptide drug prepared in the above example. At the same time, the materials were grouped as follows: MSNs-SS-GQDs group, GA-10 group, and all cell experiments were performed three times in parallel. When the Hep G2 cells were in good condition and in the exponential growth phase, they were digested with trypsin, counted, and added to 96-well plates, 100 μL of cell suspension was added to each well, and the cell concentration was 1.5×107L -1 , and then cultured in a sterile incubator for 24 h. Then, different mass concentrations (10, 50, 100, 250, 500, 1000, 2000 μg / mL) of MSN-SS-GQDs and GA-10 solutions were added (5 replicate wells at each concentration), and cultured for 6 h, 12 h, and 24 h. Then, 60 μL of 1.0 g / L MTT solution was added to each well. After 3 h, the plate was shaken, 150 μL of dimethyl sulfoxide was added to each well, and the plate was shaken for 5 min. The absorbance value was measured with an enzyme reader.

[0075] Figure 7 The in vitro cytotoxicity graph of MSN-SS-GQDs and leech active peptide GA-10.

[0076] 2) The GA-10-loaded smart drug delivery system was administered to rats with non-alcoholic fatty liver disease to verify its lipid-lowering activity. The solid phase peptide synthesis company is Shanghai Jier Biotechnology Co., Ltd. 12 SD rats, weighing 180-200g, were randomly divided into a blank group (Con, n=6) and a model group (NAFLD, n=6) after 7 days of adaptive feeding. Con rats were given normal feed, and NAFLD rats were given a high-fat feed (basic feed 65%, lard 15%, cholesterol 5%, egg yolk powder 10%, sodium bile acid 5%), and both were free to eat and drink water. During the period of feeding high-fat feed, the NAFLD group was appropriately supplemented with oral administration of high-fat emulsion (200mL water, 15g lard, 2.5g egg yolk powder, 2.5g cholesterol, 0.5g 6-n-propyl-2-thiouracil) for joint modeling. The weight of the rats was recorded regularly and the status of the rats was observed. At the 12th week of modeling, blood was collected from the orbital venous plexus and placed in an anticoagulant tube containing sodium heparin. The collected rat venous blood samples were centrifuged at 3500r / min for 10min to obtain plasma. The levels of TC, TG, HDL-C and LDL-C in the plasma of each rat were determined using a kit. When the levels of TC, TG, HDL-C and LDL-C in the plasma of NAFLD rats showed significant differences compared with those of Con rats (p<0.05), the modeling was successful.

[0077] After the model was successfully established, the SD rats were grouped: 54 rats were randomly divided into 5 groups, with 10 rats in each group: blank group (Con), NAFLD model group (Mod), GA-10-Gal-PEG-MSN-GQDs high-dose group (HF), GA-10-Gal-PEG-MSN-GQDs low-dose group (LF), and simvastatin positive drug group (F1).

[0078] HF group: GA-10-Gal-PEG-MSN-GQDs were soaked in deionized water at a concentration of 10 mg / kg GA-10 and administered by oral gavage (2 mL) once a day.

[0079] LF group: GA-10-Gal-PEG-MSN-GQDs were soaked in deionized water at a concentration of 5 mg / kg GA-10 and administered by gavage (2 mL) once a day.

[0080] Group F1: 2 mL of 10 mg / kg simvastatin pure powder was soaked in deionized water and administered by gavage once a day.

[0081] The rats in the Con and Mod groups were gavaged with an equal volume of normal saline. Except for the Con group, the rats in all groups were continuously fed with a high-fat diet during the administration period.

[0082] In order to clarify the lipid accumulation in rat liver tissue caused by high-fat diet, the weight data of rats during modeling were recorded in time. Finally, the livers of rats were photographed and recorded during the dissection process to visually observe the changes in the livers of rats in each group. HE staining and Oil Red O staining were performed at the same time to observe the pathological changes and lipid accumulation in liver tissue. The levels of TC, TG, LDL-C, HDL-C, ALT and AST in plasma were detected by microplate reader. The changes in lipid factors in the blood of rats in each group and the degree of liver cell damage were clarified.

[0083] like Figure 8 The results of rat body weight showed that the weight of rats in each group changed significantly over time. Starting from the 6th week of the experiment, compared with the weight growth trend of Con, the weight growth rate of Mod rats increased significantly (p<0.01); at the 19th week of the experiment, compared with Mod, the weight growth rate of rats in HF, LF, and F1 groups slowed down significantly, and there was a significant difference in the weight growth rate between the above-mentioned drug-treated groups and the Mod group (p<0.05). In addition, this study compared the effects of HF, LF, and F1 on the body weight of NAFLD rats. The results showed that starting from the 15th week, the weight growth rate of rats in the LF group was significantly slower than that in the F1 group (p<0.05).

[0084] like Fig. 9 The results of rat liver showed that the liver of rats in the Con group was bright red, smooth and elastic. The liver of rats in the Mod group was dark yellow, rough, with lipid particles and brittle texture, indicating that long-term high-fat diet can cause fat accumulation in the liver of SD rats. Compared with Mod rats, the liver of rats in each treatment group had a darker color, smoother surface, fewer lipid particles, and a soft texture, tending to the liver of rats in the Con group, indicating that GA-10 has a certain repair effect on fat accumulation and liver damage caused by high-fat diet.

[0085] like Fig.10 and Fig.11 The results of rat liver pathological staining showed that the hepatocytes of Con rats were round, plump, and tightly arranged, with the nucleus located in the center of the cell, the hepatic lobule was clearly visible, and there was no obvious fatty degeneration; the hepatocytes of Mod rats were disorderly arranged, of different sizes, the nucleus was squeezed to one side, the lipid vacuolation was severe, there were lipid droplets in the cytoplasm, and there was severe fatty degeneration. The liver pathological sections of the HF group and the LF group showed different degrees of improvement, with clear liver sinusoids, fewer fat droplets in the liver, improved fatty degeneration, and normal hepatocyte morphology. The nucleus was located in the center of the cell, and vacuolation was reduced. In addition, the F1 group was also able to improve liver fatty degeneration and inflammatory infiltration to varying degrees, and had a certain protective effect on the liver.

[0086] like Fig.12The results of rat blood biochemical indicators showed that compared with Con, the TC, TG and LDL-C levels of Mod rats increased significantly (p<0.001), and the HDL-C content decreased significantly (p<0.0001), indicating that the model was successfully established. Compared with Mod, HF, LF, and F1 could significantly reduce the contents of TC, TG and LDL-C (p<0.05), and significantly increase the content of HDL-C (p<0.05). At the same time, the comparison of the four blood lipid values ​​of the HF and LF groups showed that GA-10 showed a certain dose-dependency in lowering blood lipids. In addition, this study compared the differences in the lipid-lowering efficacy of the HF, LF and F1 groups. The results showed that the effects of the LF group in reducing the contents of TC and TG were significantly better than those of the F1 group (p<0.05). The results of rat liver ALT and AST activity assay showed that compared with Con rats, the liver ALT and AST levels of Mod rats were significantly increased (p<0.0001), the ALT level was about 3 times the normal level, and the AST level was about 2 times the normal level, indicating that the Mod group had obvious liver damage. Compared with Mod, the ALT and AST activity levels of each administration group were significantly reduced (p<0.01), and the HF group had a better effect on improving liver damage than the LF group, indicating that Gal-PEG-MSN-GQDs loaded with leech active peptide GA-10 can significantly improve liver damage caused by a high-fat diet, and its effect is dose-dependent.

[0087] Figure 8 This is a graph showing the trend of rat body weight changes. Fig. 9 This is the appearance of rat liver. Fig.10 HE staining of rat liver. Fig.11 This is an Oil Red O staining image of rat liver. Fig.12 Table 3 shows the liver coefficient of rats in each group. Table 4 shows the biochemical index levels of rats in each group.

[0088] Table 3 Liver coefficients of rats in each group ( n=6)

[0089]

[0090] Note: Compared with Con, **p<0.01; Compared with Mod, ### p<0.001, #### p<0.0001

[0091] Table 4 Biochemical index levels of rats in each group ( n=6)

[0092]

[0093] Note: The units of TC, TG, LDL-C, and HDL-C are mmol / L, and the units of ALT and AST are U / L. ## p<0.01, # p<0.05, compared with Mod, ** p<0.01, * p<0.05, compared with HF, a p<0.05; compared with LF, b p<0.05, bb p<0.01

[0094] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal), characterized in that: The molecular structure of the material is PEG-Gal-MSNs-SS-GQDs, which is a mesoporous silica prepared by a hydrothermal synthesis method, and modified by amino groups, disulfide bonds, graphene quantum dots and amino-terminated PEG chain galactose to prepare a multifunctional drug carrier with redox responsive release.

2. A method for preparing the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) as claimed in claim 1, characterized in that: The method specifically comprises the following steps: a. Add NaOH solution (1-2 mL, 2 m NaOH) to hexadecyltrimethylammonium bromide (CTAB) solution (0.1-1 g CTAB dissolved in 200-300 mL deionized water), and stir vigorously at 60-90 ° C; then add tetraethoxysilane (TEOS) (1.5-3 mL) dropwise therein for 5-15 min, stir vigorously for 1-3 h, and obtain a white precipitate. The obtained nanoparticles are collected by repeated centrifugation, and washed with deionized water and ethanol for 3 times. The obtained solid is vacuum dried at 120 ° C for 10-15 h; b. The mesoporous silica prepared in step a with a template is subjected to a temperature-programmed heating and calcined at 500 to 600 ° C for 5 to 8 h using a muffle furnace to remove the template surfactant used to make the pores; c. Take 150-300 mg of mesoporous nano-silica with a particle size of 150±30 nm, ultrasonically disperse it in 10-30 mL of anhydrous toluene, add 100-300 μL of 3-aminopropyltriethoxysilane (APTES), and reflux it at 100-120°C for 15-20 hours under stirring; then centrifuge the reaction solution, ultrasonically disperse it, and wash it thoroughly with toluene and methanol, and repeat the operation three times, and then dry it in a vacuum drying oven at 50-80°C overnight to obtain white powder MSNs-NH2; d. 5 to 20 mg of L-cystine (L-Cys) was dispersed in 10 to 30 mL of PBS (pH = 5.0) containing 40 to 60 mg of EDC and 20 to 30 mg of NHS. After 1 h, the MSNs-NH2 (30 to 50 mg) synthesized in step c was added thereto and stirred at room temperature for 24 h. The obtained MSNs-SS-NH2 particles were separated by centrifugation and washed with PBS (pH = 5.0) and water to remove excess L-Cys, and then freeze-dried to obtain MSNs-SS-NH2; e. 2 to 10 mg of the MSNs-SS-NH2 particles prepared in step d were dispersed in 2 mL of deionized water, and three samples were prepared in parallel for each group; then 5, 10, and 25 mg of GA-10 were added to the above solution and stirred for 20 to 30 h, and then the encapsulation efficiency was determined by centrifugation; f. Disperse the purchased graphene quantum dots GQDs (0.5-3 mg), EDC (5-20 mg) and NHS (2-10 mg) in 5-20 mL of deionized water, and stir the solution for 1 h to make the carboxyl groups on the GQDs active; g. The MSNs-SS-NH2 particles (10-30 mg) prepared in step d were added to the solution containing GQDs, and the amino groups on the MSNs-SS-NH2 particles were connected to GQDs through an amidation reaction. The mixture was stirred overnight to obtain MSNs-SS-GQDs, which were collected by centrifugation, washed with deionized water, freeze-dried in a vacuum, and stored at 4°C. h. Take 5-20 mg of MSN-SS-GQDs and 5-20 mg of solid PEG-Gal, add 1-5 mL of deionized water respectively, and adjust the pH to 7-12 with borate buffer; stir the solution for 10-20 min, then add 30-60 mg of solid sodium triborohydride (Na(CN)BH3), and store the solution under magnetic stirring overnight; collect the galactose functionalized product by centrifugation, wash repeatedly to remove excess reagents, and obtain the final product Gal-PEG-MSN-GQDs, which is stored at 4°C.

3. The method for preparing the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) according to claim 2, characterized in that: In step b, the programmed heating time is to heat to 100° C. for the first hour, maintain for 1 hour, then increase the temperature to 550° C. within 3 hours, and maintain at 550° C. for 6 hours.

4. The method for preparing the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) according to claim 2, characterized in that: In step c, the thorough washing is performed three times with toluene and methanol alternately, and the last washing liquid is methanol.

5. The method for preparing the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) according to claim 2, characterized in that: In step d, after adding L-cystine, the entire reaction process is carried out under light-proof conditions, and the MSNs-NH2 powder is added slowly to ensure that the reaction proceeds fully and evenly.

6. Use of the graphene quantum dot-covered, galactose (Gal)-modified liver-targeted mesoporous silica material as claimed in claim 1 or the graphene quantum dot-covered, galactose (Gal)-modified liver-targeted mesoporous silica material prepared by the method as claimed in claim 2 in a pharmaceutical preparation.

7. The use according to claim 6, characterized in that: The application of the liver-targeted mesoporous silica material covered with graphene quantum dots and modified with galactose (Gal) in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease.

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