Nanoparticles, nanofomulations, methods of making and using the same

Abstract

The present application relates to the field of biological medicine preparation, in particular to a kind of nanoparticle, nano-preparation and its preparation method and application.The nanoparticle contains drug molecules and glucose oxidase for wrapping the drug molecules, the drug molecules are rapamycin and / or rapamycin derivative, the nano-preparation contains above-mentioned nanoparticle and biological membrane coated on the surface of the nanoparticle.The nano-preparation prepared by the nanoparticle has better therapeutic effect on tumor, and preparation process is simple, and can be industrialized large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine preparation, in particular to a kind of nanoparticle, nano-preparation and its preparation method and application. BACKGROUND

[0002] Tumor is an important disease affecting human health, and has become the second largest cause of death in the world. Compared with normal tissues, tumor tissues need more nutrient uptake, transfer and transformation. Therefore, nutrient deprivation, also known as starvation therapy, is a targeted treatment method with high specificity and strong effectiveness. However, the metabolic plasticity of tumor cells is extremely strong, and has the ability to utilize different nutrient substrates and the ability to process nutrient substrates in different ways. When a certain metabolic pathway of breast cancer cells is blocked, the cancer cells themselves will rely on other substances to maintain life activities through some compensatory mechanisms. Current treatment methods still cannot meet the needs of tumor treatment, and there is an urgent need to develop safer and more effective treatment methods to improve treatment effectiveness and reduce recurrence and metastasis after treatment. SUMMARY

[0003] The purpose of the present application is to solve the problem of low drug efficacy or toxic side effects in the prior art for treating tumors. A kind of nanoparticle, nano-preparation and its preparation method and application are provided. The nano-preparation has good therapeutic effect on tumors, high delivery efficiency and small toxic side effects.

[0004] To achieve the above purpose, the first aspect of the present application provides a kind of nanoparticle, which contains glucose oxidase and drug molecules loaded in the glucose oxidase, and the drug molecules are rapamycin and / or rapamycin derivatives.

[0005] Further preferably, in the nanoparticle, the content of the glucose oxidase is ≥70wt%.

[0006] The second aspect of the present application provides a kind of nano-preparation, which contains the aforementioned nanoparticle and biological membrane coated on the surface of the nanoparticle.

[0007] Preferably, the biological membrane is selected from at least one of tumor cell membrane, red blood cell membrane, phospholipid bilayer membrane and neutrophil membrane.

[0008] The third aspect of the present application provides a kind of preparation method of nanoparticle, which comprises: mixing glucose oxidase, drug molecules and solvent for reaction, and obtaining the nanoparticle after purification, and the drug molecules are rapamycin and / or rapamycin derivatives.

[0009] Further preferably, the addition amount of the drug molecules is 0.1-0.4g relative to 1g of the glucose oxidase.

[0010] Preferably, the solvent comprises an organic solvent and an inorganic solvent, and the method of the mixed reaction comprises the following steps:

[0011] (1) mixing the glucose oxidase and the inorganic solvent to obtain a glucose oxidase solution, and mixing the drug molecules and the organic solvent to obtain a solution containing drug molecules;

[0012] (2) mixing the glucose oxidase solution and the solution containing drug molecules to perform a reaction.

[0013] Preferably, the organic solvent is at least one selected from ethanol, acetone, dichloromethane and trichloromethane; and the inorganic solvent is water or a buffer solution.

[0014] Preferably, in step (2), the reaction conditions at least include: a time of 1-10 min and a temperature of 40-70℃.

[0015] The fourth aspect of the present application provides a preparation method of a nano preparation, comprising: mixing nano particles with a biological membrane and then performing homogenization treatment to obtain the nano preparation.

[0016] The nano particles are the nano particles prepared by the preparation method of the nano particles.

[0017] Preferably, the biological membrane is at least one selected from tumor cell membranes, red blood cell membranes, phospholipid bilayer membranes and neutrophil membranes.

[0018] Preferably, the mass ratio of the biological membrane to the nano particles is 1:0.2-2.

[0019] The fifth aspect of the present application provides an application of the aforementioned nano particles, the aforementioned nano preparation, the nano particles prepared by the preparation method of the aforementioned nano particles or the nano preparation prepared by the preparation method of the aforementioned nano preparation in the preparation of a tumor treatment drug.

[0020] Preferably, the tumor treatment drug is at least one selected from a breast cancer treatment drug, a colon cancer treatment drug and a melanoma treatment drug.

[0021] Through the above technical solutions, the present application has the following beneficial effects:

[0022] The nano particles provided by the present application can effectively interfere with the energy metabolism of tumors, thereby inducing excessive autophagy and apoptosis of tumor tissues and improving the treatment effect of tumors. Moreover, the nano particles provided by the present application have high delivery efficiency in vivo, can quickly and accurately reach tumor cells, thereby further improving the treatment effect of tumors, and can effectively reduce the toxic and side effects of drug molecules themselves.

[0023] The nano-preparation provided by the present application can significantly improve the accumulation of drugs in tumor tissues, reduce the distribution of the preparation in normal tissues, and thus improve the treatment effect of tumors, through the interaction of the nanoparticles containing the aforementioned structure and the biological membrane coated on the surface of the aforementioned nanoparticles. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The particle size distribution graph of the nano-preparation prepared in Example 1 is shown in Figure 1.

[0025] Figure 2 The killing effect of the nano-preparation prepared in Example 1 on breast cancer cells is shown in Figure 2.

[0026] Figure 3 The application effect of the nano-preparation prepared in Example 1 in the treatment of breast cancer is shown in Figure 3.

[0027] Figure 4 The transmission electron microscope graph of the nano-preparation prepared in Example 2 is shown in Figure 4.

[0028] Figure 5 The application effect of the nano-preparation prepared in Example 2 in the treatment of colon cancer is shown in Figure 5.

[0029] Figure 6 The content change graph of the nano-preparation prepared in Example 4 in breast cancer tissue is shown in Figure 6. DETAILED DESCRIPTION

[0030] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the present application. The endpoints of the ranges and any values are provided as approximations only and are understood to encompass a range of values around the endpoints. Any numerical value, however, can be expressed as a range by either discarding a single digit of the numerical value or by adding or subtracting a lower limit and / or an upper limit from the numerical value. For example, a numerical value expressed as 100 can be expressed as a range from 90 to 110, or from 91 to 109, or from 92 to 108, etc.

[0031] The first aspect of the present application provides a nanoparticle containing a drug molecule and glucose oxidase for wrapping the drug molecule, wherein the drug molecule is rapamycin and / or a rapamycin derivative.

[0032] According to the present application, the drug molecule is loaded inside the glucose oxidase, which better combines the drug molecule with the glucose oxidase, and the morphology of the nanoparticle is observed by transmission electron microscopy, which is nearly spherical and uniformly distributed, as shown in Figure 1. Figure 4 .

[0033] The inventors have found that the nanoparticles provided by the present application can interfere with the energy metabolism of tumors in a therapeutic manner, and can overcome drug resistance of tumors, so that the obtained nanoparticles have better effects on treating tumors. Moreover, the nanoparticles provided by the present application have high delivery efficiency in vivo, can quickly and accurately reach tumor cells, and can further improve the therapeutic effect on tumors, and can effectively reduce the toxic and side effects of antibiotics.

[0034] According to the present application, the rapamycin derivative can be temsirolimus and everolimus, and more preferably, the drug molecule is rapamycin. The nanoparticles obtained by combining glucose oxidase and rapamycin can further induce excessive autophagy and apoptosis of tumor tissues, thereby further improving the therapeutic effect on tumors.

[0035] According to the present application, it is further preferred that the content of the glucose oxidase in the nanoparticles is ≥70 wt%, that is, the weight of the glucose oxidase accounts for ≥70% of the total weight of the nanoparticles. Under the above content, the glucose oxidase and the drug molecule have better coordination effect, and the nanoparticles have better therapeutic effect on tumors.

[0036] The second aspect of the present application provides a nano-preparation containing the aforementioned nanoparticles and a biological membrane coated on the surface of the nanoparticles. The morphology of the nano-preparation is observed by transmission electron microscopy, and the outer layer of the near-spherical nanoparticles has a uniform distribution of a biological membrane, as shown in Figure 4 .

[0037] The nano-preparation not only has the effect of the nanoparticles itself, but also the inventors have found that the half-life of the nano-preparation obtained by coating the nanoparticles with a biological membrane is significantly prolonged in blood circulation, and the accumulation and distribution of the nano-preparation in tumor tissue regions are significantly increased, which has high delivery efficiency and does not affect the effect of the nanoparticles itself, so that the therapeutic effect of the nanoparticles on tumor tissues can be better exerted.

[0038] Preferably, the biological membrane is selected from at least one of tumor cell membranes, red blood cell membranes, phospholipid bilayer membranes and neutrophil membranes. Studies have found that the nano-preparation obtained by coating the nanoparticles with the above biological membranes has higher delivery efficiency and better therapeutic effect. More preferably, the biological membrane is a tumor cell membrane, a red blood cell membrane and a phospholipid bilayer membrane.

[0039] The third aspect of the present application provides a preparation method of nanoparticles, which comprises: mixing glucose oxidase, a drug molecule and a solvent for reaction, and purifying to obtain the nanoparticles, wherein the drug molecule is rapamycin and / or a rapamycin derivative.

[0040] The nanoparticles prepared by the method have better effect on treating tumors and can overcome drug resistance of tumors.

[0041] According to the application, the rapamycin derivative can be temsirolimus and everolimus, and more preferably, the drug molecule is rapamycin. The nanion obtained by combining glucose oxidase and rapamycin can further induce excessive autophagy and apoptosis of tumor tissues, thereby further improving the treatment effect on tumors.

[0042] According to the application, preferably, the drug molecule is added in an amount of 0.1-0.4 g relative to 1 g of the glucose oxidase. Specifically, the drug molecule can be added in an amount of 0.1, 0.2, 0.3, 0.4 g or any value within the foregoing range relative to 1 g of the glucose oxidase. Under the foregoing conditions, the drug molecule can achieve a higher loading amount on the glucose oxidase, which can reduce waste of raw materials, and the nanoparticles prepared have better effect on treating tumors. In order to further improve the treatment effect on tumors, preferably, the drug molecule is added in an amount of 0.2-0.3 g relative to 1 g of the glucose oxidase.

[0043] Preferably, the solvent includes an organic solvent and an inorganic solvent, and the method of the mixing reaction includes the following steps:

[0044] (1) mixing the glucose oxidase and the inorganic solvent to obtain a glucose oxidase solution, and mixing the drug molecule and the organic solvent to obtain a solution containing the drug molecule;

[0045] (2) mixing the glucose oxidase solution and the solution containing the drug molecule to perform reaction.

[0046] According to the application, the organic solvent is at least one selected from ethanol, acetone, dichloromethane and trichloromethane, the inorganic solvent is water or buffer, the water is ultrapure water, and the addition amount of the organic solvent or the inorganic solvent is at least an amount capable of completely dissolving the drug molecule or the glucose oxidase. Under the foregoing conditions, the solvent can achieve better dispersion effect of the drug molecule or the glucose oxidase, thereby improving the reaction effect between the reactants and further improving the effect of the prepared nanoparticles on treating tumors. More preferably, the organic solvent is acetone, the inorganic solvent is phosphate buffer, and the pH of the phosphate buffer is 6-8.

[0047] Preferably, in step (2), the reaction is carried out under stirring, and the stirring rate is not particularly limited, as long as the glucose oxidase and the drug molecules can be sufficiently contacted. The reaction conditions further include a time of 1-10 min and a temperature of 40-70℃. Under the above conditions, the solution containing the glucose oxidase and the solution containing the drug molecules can be better reacted, so that the glucose oxidase and the drug molecules can be better combined, and the effect of the prepared nanoparticles on treating tumors is improved. Further preferably, the reaction time is 4-6 min, and the reaction temperature is 50-65℃.

[0048] According to the present application, the purification process can be carried out in any feasible way, and preferably, the purification is carried out by ultrafiltration, and the molecular weight cut-off after the ultrafiltration membrane treatment is 20-40KD. Under the above conditions, the purification can better remove free drugs, reduce the side effects of free drugs, and thus improve the effect of treating tumors.

[0049] The present application provides a preparation method of the nano-preparation in a fourth aspect, comprising: mixing the nanoparticles with the biological membrane and then homogenizing to obtain the nano-preparation.

[0050] The nano-preparation prepared by the above method has a better effect on treating tumors. The nano-preparation obtained by coating the nanoparticles with the biological membrane has a significantly prolonged half-life in blood circulation, can increase the accumulation and distribution of the nano-preparation in the tumor tissue region, and thus improve the drug delivery efficiency and the treatment effect on tumors. Moreover, the biological membrane has good biocompatibility, and thus can further reduce the toxic side effects of the nano-preparation.

[0051] The nanoparticles are the nanoparticles prepared by the above nanoparticle preparation method.

[0052] According to the present application, preferably, the biological membrane is selected from at least one of tumor cell membranes, red blood cell membranes, phospholipid bilayer membranes, and neutrophil membranes. Research has found that the nano-preparation obtained by coating the nanoparticles with the above biological membrane has a higher delivery efficiency and can better exert the therapeutic effect. More preferably, the biological membrane is a tumor cell membrane, a red blood cell membrane, or a phospholipid bilayer membrane.

[0053] Preferably, the mass ratio of the biological membrane to the nanoparticles is 1:0.2-2. Under the above conditions, the biological membrane can be sufficiently combined with the nanoparticles, the loss of raw materials is reduced, and the yield of the nano-preparation is improved. Further preferably, the mass ratio of the biological membrane to the nanoparticles is 1:0.5-1.

[0054] Preferably, the homogenization treatment can be any method capable of achieving homogenization treatment, exemplarily, can be extrusion treatment and / or homogenizer homogenization, the extrusion treatment can be carried out by an extruder. Specifically, the number of cycles of the extrusion treatment is 8-12 times, and the working conditions of the homogenizer are: working pressure 200-400 Bar, cycle number 4-6 cycles. Under the above conditions, the homogenization effect is good, and the yield of the prepared nano-preparation is high.

[0055] The fifth aspect of the present application provides the use of the above-mentioned nanoparticles, nano-preparation, nanoparticles prepared by the preparation method of nanoparticles, or nano-preparation prepared by the preparation method of nano-preparation in the preparation of a tumor treatment drug.

[0056] The tumor treatment drug prepared by the above-mentioned nanoparticles or nano-preparation has a good delivery efficiency and a good therapeutic effect on tumors. Moreover, the tumor treatment drug prepared by the above-mentioned nanoparticles or nano-preparation has less toxic and side effects.

[0057] Preferably, the tumor treatment drug is at least one of a breast cancer treatment drug, a colon cancer treatment drug, and a melanoma treatment drug. The nanoparticles or nano-preparation provided by the present application has a better therapeutic effect on the above-mentioned tumors. Further preferably, the tumor treatment drug is a breast cancer treatment drug.

[0058] As a relatively preferred embodiment of the present application, a preparation method of a nano-preparation is provided, comprising:

[0059] (1) mixing glucose oxidase with a phosphate buffer solution with a pH of 6-8 at a temperature of 55-65℃ for 1-2 min to obtain a glucose oxidase solution;

[0060] mixing rapamycin with acetone to obtain a rapamycin solution;

[0061] (2) mixing the glucose oxidase solution with the rapamycin solution under constant stirring at a temperature of 50-65℃ for 4-6 min, and then ultrafiltrating with an ultrafiltration membrane with a molecular weight cut-off of 30-40 KD to remove free drugs, to obtain nanoparticles;

[0062] The addition amount of the rapamycin is 0.2-0.3 g relative to 1 g of the glucose oxidase;

[0063] (3) mixing tumor cell membranes with the nanoparticles at a mass ratio of 1:0.5-1, and then extruding through an extruder for 8-12 cycles, or through a homogenizer at 200-400 Bar for 4-6 cycles, to prepare an enzyme nano-preparation;

[0064] The biological membrane is selected from at least one of tumor cell membrane, red blood cell membrane and phospholipid bilayer membrane.

[0065] The nanometer preparation prepared by the preparation method provided above can effectively improve the effect of treating tumors.

[0066] The application will be described in detail below through examples.

[0067] In the following examples, the specific conditions of the experiments are not specified, and the experiments are generally carried out according to the conventional conditions or the conditions recommended by the reagent companies; the reagents, consumables and the like used in the following examples can be obtained through commercial channels unless otherwise specified; the experimental water is pure water; and the method is a conventional process method in the art unless otherwise specified.

[0068] In the following examples, the biological membrane is obtained by the following method:

[0069] (1) Preparation method of tumor cell membrane: tumor cells are purchased from ATCC cell bank, and after the tumor cells are cultured under the corresponding recommended conditions, the tumor cells are collected, centrifuged at 600 rpm for 4 min by a centrifuge, tumor cell membrane extraction solution (containing 75 mM sucrose, 25 mM mannose, 0.5 mM benzylsulfonyl fluoride, 5 mg / mL bovine serum albumin) is added, repeated freeze-thawing is performed for 3 times (-80℃ to room temperature), protease inhibitors (containing 100 μg / mL benzylsulfonyl fluoride, 2 μg / mL serine protease inhibitor, 2 μg / mL serine / cysteine protease inhibitor, 1 μg / mL pepsin inhibitor) are added, ultrasonic crushing is performed for 5 min under ice bath condition, centrifugation is performed at 3000 rpm for 5 min by a centrifuge, the supernatant is discarded, and the precipitate is reserved. Phosphate buffer solution (pH = 7.4) is added, resuspended, centrifuged at 15000 rpm for 1 h by a centrifuge, and the precipitate obtained by centrifugation is stored at -80℃ for later use.

[0070] (2) Preparation method of red blood cell membrane: animal or human blood is collected into an anticoagulant tube, centrifuged at 3000 rpm for 5 min by a centrifuge, and the supernatant is discarded. Three times the volume of the precipitate is added to the phosphate buffer solution, resuspended, and the precipitate is collected by centrifugation. 60 times the volume of the precipitate is added to the pre-cooled 0.25× phosphate buffer solution, and stirring is continuously performed for 4 h. After centrifugation at 8000 rpm for 15 min by a centrifuge, the supernatant is discarded, the precipitate is resuspended by adding 0.25× phosphate buffer solution, and centrifugation is repeated again, which is repeated for three times until the supernatant solution is colorless. Finally, the red blood cell membrane precipitate is resuspended with an appropriate volume of 1× phosphate buffer solution, and the large red blood cell membrane is changed into a suitable size by using low-power ultrasonic (32W, 1 min).

[0071] (3) The preparation method of the phospholipid bilayer film: dissolve lecithin and cholesterol in dichloromethane at a ratio of 1:0.1-0.5, form a phospholipid film at the bottom of the container by rotary evaporation for 5 min, then add phosphate buffer solution, shake well, and obtain the phospholipid bilayer film by low-frequency ultrasonic for 5 min.

[0072] (4) The preparation method of the neutrophil membrane: collect neutrophils from animal or human blood or bone marrow, separate and purify the neutrophils by neutrophil extraction kit (Solarbio, P9201), add neutrophil lysis solution (containing 75 mM sucrose, 25 mM mannose, 0.5 mM benzylsulfonyl fluoride, 5 mg / mL bovine serum albumin, 30 mM Tris-HCl) and protease inhibitors (containing 2 μg / mL serine protease inhibitor, 2 μg / mL serine / cysteine protease inhibitor, 1 μg / mL pepsin inhibitor), ultrasonic breakage for 5 min under ice bath condition, then centrifuge at 3000 rpm for 5 min, discard the supernatant, and keep the precipitate. Add phosphate buffer solution (pH = 7.4), resuspend, centrifuge at 15000 rpm for 1 h, and store at -80°C for use.

[0073] In the following examples, the high-performance liquid chromatograph is purchased from Shimadzu Corporation of Japan, the model number is LC-40, the dynamic light scattering instrument is purchased from Brookhaven Corporation of the United States, the model number is 90Plus Zeta, the glucose oxidase is purchased from Shanghai Baoman Biological Technology Co., Ltd., the number is E0016, the extruder is purchased from Avanti Company of Germany, the multifunctional enzyme label instrument is purchased from BioTek Corporation of the United States, the model number is Synergy H1, the homogenizer is purchased from PilotGenizer Company of the United States, the rapamycin is purchased from Widely Technology Co., Ltd., and the CCK-8 reagent is purchased from Hunan Butterfly Biological Technology Co., Ltd.

[0074] Example 1

[0075] Dissolve 40 mg of glucose oxidase in 40 mL of phosphate buffer solution with pH of 7.4, heat to 60°C, keep for 1 min, add 2 mL of 5 mg / mL rapamycin acetone solution under the condition of stirring at 500 rpm, continue to react for 5 min, cool to 4°C quickly after the end, and then ultrafilter the solution by using an ultrafiltration membrane with a molecular weight cut-off of 30 KD to remove free drugs, to obtain nanoparticles.

[0076] Mix the tumor cell membrane with the nanoparticles according to the mass ratio of 2:1, extrude by the extruder for 10 cycles to prepare the nano-preparation.

[0077] The obtained nano-preparation is uniform and has no precipitate, and is light blue.

[0078] The particle size of the nano-preparation is 100-200 nm, as detected by a dynamic light scattering instrument. Figure 1 The content of rapamycin was determined by high performance liquid chromatography, the mobile phase was acetonitrile: water (v:v=75:25), the detection wavelength was 278 nm, and the column temperature was 60°C. The content of glucose oxidase was calculated to be 85% through the content of rapamycin. The nano-preparation was incubated with breast cancer cells, and the ultraviolet absorption at 450 nm was detected by using CCK-8 reagent and a multifunctional enzyme marker. The absorption value of the physiological saline group was taken as 100%, and the cell survival rate of the nano-preparation group was calculated. After treatment with the nano-preparation, the cell survival rate of breast cancer was 40%, as shown in Figure 2 The nano-preparation was injected into the tail vein of a breast cancer tumor-bearing mouse, the size of the subcutaneous tumor was measured by using a vernier caliper, the tumor volume was calculated according to the length×width 2 / 2, and the tumor volume of the physiological saline group was taken as 100%. The tumor inhibition rate of the nano-preparation was calculated to be 81%, as shown in Figure 3 , and the treatment effect of the tumor is shown in Table 1.

[0079] Example 2

[0080] The nano-preparation was prepared by mixing the red blood cell membrane with the nanoparticles at a mass ratio of 1:1, and then passing through a homogenizer at 300 Bar for 5 cycles.

[0081] The obtained nano-preparation is uniform and has no precipitate, and is light blue.

[0082] The particle size of the nano-preparation is 120-200 nm, as detected by a dynamic light scattering instrument. The particle morphology of the drug-loaded nano-preparation was observed by using a transmission electron microscope. The particles were uniformly distributed and had a near-spherical shape, and the size was similar to the measurement result of the dynamic light scattering instrument, as shown in Figure 4 The content of rapamycin was determined by high performance liquid chromatography, the mobile phase was acetonitrile: water (75:25), the detection wavelength was 278 nm, and the column temperature was 60°C. The content of glucose oxidase was calculated to be 85% through the content of rapamycin. The nano-preparation was injected into the tail vein of a colon cancer tumor-bearing mouse, the size of the subcutaneous tumor was measured by using a vernier caliper, the tumor volume was calculated according to the length×width 2 / 2, and the tumor volume of the physiological saline group was taken as 100%. The tumor inhibition rate of the nano-preparation was calculated to be 60%, as shown in Figure 5 , and the treatment effect of the tumor is shown in Table 1.

[0083] Example 3

[0084] Nanoparticles were prepared according to the method in Example 1. Then, the phospholipid bilayer film and the nanoparticles were thoroughly mixed at a mass ratio of 1:1 and extruded through an extruder for a total of 10 cycles to prepare a nano-formulation.

[0085] Observation shows that the obtained nano-preparation is uniform, free of precipitate, and light blue.

[0086] Dynamic light scattering analysis revealed that the particle size of the nanoparticles was 100-180 nm. The content of rapamycin was determined using high-performance liquid chromatography (HPLC) with acetonitrile:water (75:25) as the mobile phase, a detection wavelength of 278 nm, and a column temperature of 60 °C. Based on the rapamycin content, the glucose oxidase content was calculated to be 80%. The nanoparticles were injected intravenously into melanoma-bearing mice, and the size of the subcutaneous tumors was measured using calipers, according to the length × short diameter. 2 / 2 The tumor volume was calculated, with the tumor volume of the saline group as 100%. The tumor inhibition rate of the nano-formulation was calculated to be 65%. The therapeutic effect of the tumor is shown in Table 1.

[0087] Example 4

[0088] Nanoparticles were prepared according to the method in Example 1. Then, neutrophil membranes and nanoparticles were thoroughly mixed at a mass ratio of 3:1 and homogenized in a homogenizer for 5 cycles at 300 Bar to prepare a nano-formulation.

[0089] Observation revealed that the obtained nano-formulation was uniform, free of precipitation, and pale blue. Dynamic light scattering analysis showed that the average particle size of the nano-formulation was 110-200 nm. Equal volumes of the nano-formulation and nanoparticles were injected into breast cancer-bearing mice via the tail vein. The distribution of the nano-formulation and nanoparticles within the tumor over time was observed using an in vivo imaging system. The nano-formulation coated with neutrophil membranes accumulated more extensively in the tumor tissue. (See [link to relevant documentation]). Figure 6 The treatment effects on the tumor are shown in Table 1.

[0090] Example 5

[0091] The nanoparticles were prepared according to the method in Example 1, except that 0.8 mL of 5 mg / mL rapamycin acetone solution was added to thoroughly mix the tumor cell membranes and nanoparticles at a mass ratio of 5:1. The therapeutic effects of the above nanoparticles on breast cancer are shown in Table 1.

[0092] Example 6

[0093] The nanoscale preparation was prepared according to the method of Example 1, except that 3.2 mL of 5 mg / mL rapamycin acetone solution was added, and the tumor cell membrane and the nanoparticles were mixed at a mass ratio of 1:2. After treatment with the nanoscale preparation, the treatment effect of breast cancer was as shown in Table 1.

[0094] Example 7

[0095] The nanoscale preparation was prepared according to the method of Example 1, except that 1.6 mL of 5 mg / mL rapamycin acetone solution was added, and the tumor cell membrane and the nanoparticles were mixed at a mass ratio of 4:5. After treatment with the nanoscale preparation, the treatment effect of breast cancer was as shown in Table 1.

[0096] Example 8

[0097] The nanoscale preparation was prepared according to the method of Example 1, except that 2.4 mL of 5 mg / mL rapamycin acetone solution was added, and the tumor cell membrane and the nanoparticles were mixed at a mass ratio of 1:1. After treatment with the nanoscale preparation, the treatment effect of breast cancer was as shown in Table 1.

[0098] Comparative Example 1

[0099] The nanoscale preparation was prepared according to the method of Example 1, except that the glucose oxidase was replaced by bovine serum albumin without biological catalytic activity. After treatment with the nanoscale preparation, the treatment effect of breast cancer was as shown in Table 1.

[0100] Comparative Example 2

[0101] The nanoscale preparation was prepared according to the method of Example 1, except that 2 mL of 5 mg / mL rapamycin acetone solution was replaced by 2 mL of acetone solution. After treatment with the nanoscale preparation, the treatment effect of breast cancer was as shown in Table 1.

[0102] Comparative Example 3

[0103] 10 mg of rapamycin was dissolved in a small amount of acetone solution, then dispersed with PEG200, and then mixed directly with 40 mg of glucose oxidase to obtain a mixture. After treatment with the mixture, the treatment effect of breast cancer was as shown in Table 1.

[0104] Table 1

[0105] Tumor cell survival rate Tumor tissue inhibition rate Example 1 40％ 81％ Example 2 62％ 60％ Example 3 60％ 65％ Example 4 43％ 72％ Example 5 64％ 57％ Example 6 58％ / Example 7 53％ / Example 8 52％ / Comparative Example 1 71％ 40％ Comparative Example 2 83％ 20％ Comparative Example 3 74％ 25％

[0106] As can be seen from the results of the examples, the tumor cell survival rate of the example group is significantly lower than that of the comparative example group, and the tumor tissue inhibition rate of the example group is significantly higher than that of the comparative example group, indicating that the nanoscale preparation provided by the present application can better inhibit the growth of tumor cells and achieve better tumor treatment effect.

[0107] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A nano-formulation, characterized in that, The nano-formulation contains nanoparticles and a biofilm coating the surface of the nanoparticles. The nanoparticles contain drug molecules and glucose oxidase for encapsulating the drug molecules. The drug molecules are rapamycin. The content of glucose oxidase in the nanoparticles is ≥70% by weight. The method for preparing the nanoparticles includes: mixing and reacting the glucose oxidase and the drug molecules at a temperature of 40-70°C in the presence of a solvent, and then purifying the mixture to obtain the nanoparticles. The amount of the drug molecule added is 0.1-0.4g relative to 1g of the glucose oxidase; the biomembrane is selected from at least one of tumor cell membrane, erythrocyte membrane, phospholipid bilayer membrane and neutrophil membrane.

2. The nano-formulation according to claim 1, characterized in that, The solvent includes organic solvents and inorganic solvents, and the mixed reaction method includes the following steps: (1) The glucose oxidase and the inorganic solvent are mixed to obtain a glucose oxidase solution, and the drug molecules and the organic solvent are mixed to obtain a solution containing drug molecules; (2) The glucose oxidase solution and the solution containing drug molecules are mixed and reacted.

3. The nano-formulation according to claim 2, characterized in that, The organic solvent is selected from at least one of ethanol, acetone, dichloromethane, and chloroform; the inorganic solvent is water or a buffer solution.

4. The nano-formulation according to claim 3, characterized in that... In step (2), the reaction conditions include at least the following: a time of 1-10 min.

5. The method for preparing the nano-formulation according to any one of claims 1-4, characterized in that, include: The nanoparticles were mixed with a biomembrane and then homogenized to obtain the nano-formulation.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the biofilm to the nanoparticles is 1:0.2-2.

7. The application of the nanoformulation according to any one of claims 1-4 or the nanoformulation prepared by the preparation method according to claim 5 or 6 in the preparation of drugs for treating tumors; The tumor treatment drug is selected from at least one of the following: drugs for treating breast cancer, drugs for treating colon cancer, and drugs for treating melanoma.

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