Simvastatin albumin nanoparticles as well as preparation and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to simvastatin albumin nanoparticles as well as preparation and application thereof, in particular to simvastatin bovine serum albumin nanoparticles as well as a preparation method and application thereof in preparation of antitumor drugs. The simvastatin albumin nanoparticle provided by the invention is composed of simvastatin and serum albumin, and the mass ratio of the simvastatin to the serum albumin is 1: (10-20). And a nano emulsified solvent volatilization method is adopted for preparation. The preparation method comprises preparation of O / W emulsion and preparation of albumin nanoparticles. The average particle size of the prepared nanoparticles is smaller than 500 nm, the polydispersity coefficient is smaller than 0.5, and the encapsulation efficiency is larger than 70%. The surface of the particle is negatively charged, and the zeta potential is-31.25 mV. The nanoparticles have selectivity to tumor cells, have different degrees of inhibition effects on breast cancer, liver cancer, ovarian cancer and colorectal cancer, and have the strongest inhibition effect on colorectal cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to simvastatin albumin nanoparticles as well as preparation and application thereof, in particular to simvastatin bovine serum albumin nanoparticles as well as a preparation method thereof and application of the simvastatin bovine serum albumin nanoparticles in preparation of an antitumor drug. BACKGROUND

[0002] Cancer is a major global public health problem. Chemotherapy, radiotherapy, targeted therapy and immunotherapy are effective tumor treatment options. Due to the toxicity of cancer chemopreventive drugs, people are increasingly concerned about finding adjuvant drugs to alleviate tumors.

[0003] Colorectal cancer (CRC) includes colon cancer (COC) and rectal cancer, which is one of the common malignant tumors of the digestive tract and poses a significant threat to human life and health. For most patients with advanced colorectal cancer, chemotherapy is the main treatment option. Despite some clinical progress, the survival rate of patients with advanced colorectal cancer remains limited, and more treatment options are urgently needed. Cholesterol metabolism plays a key role in carcinogenic signaling pathways and tumor cell development. The function of cholesterol is to maintain cell structure and perform normal biological functions, which is crucial in diseases such as dyslipidemia, obesity, diabetes and cardiovascular disease. Clinical trials have shown that blood cholesterol levels change in cancer patients compared to healthy people.

[0004] Statins are one of the most common lipid-lowering drugs. Statins are divided into hydrophilic and lipophilic. Hydrophilic statins include rosuvastatin and pravastatin, and lipophilic statins include simvastatin (SV), lovastatin, pitavastatin and atorvastatin. It is worth noting that compared with hydrophilic statins, lipophilic statins are more likely to penetrate cell membranes, interact with receptor acyl chains, inhibit cholesterol synthesis in the liver, have stronger bioavailability, and have the ability to affect multiple mevalonate metabolism targets in cancer cells.

[0005] Statins are a class of drugs used to treat hyperlipidemia and atherosclerotic cardiovascular disease, mainly including natural statins, semi-synthetic derivatives of natural statins and synthetic statins. Synthetic statins are different from natural statins in structure, but there is no significant difference in drug efficacy and adverse reactions. The decrease of intracellular cholesterol level can induce the up-regulation of Sterol Response Element Binding Protein (SREBP), leading to the increase of Low-density Lipoprotein (LDL) receptor on the cell surface, promoting the absorption of low-density lipoprotein particles rich in cholesterol in the blood, thereby reducing the plasma LDL-cholesterol level. The pentenylization of Reticular Activating System (RAS) caused by statins is regulated by downstream signaling pathways to control cell development, survival, migration, invasion, metastasis and apoptosis.

[0006] Simvastatin (SV) is a lipophilic statin, a semi-synthetic derivative of lovastatin, and is the most commonly used drug for treating dyslipidemia. It is reported that 42% of users of cholesterol-lowering drugs use SV. SV belongs to BCS class II compounds, with high permeability and low water solubility, and it is difficult to administer SV intravenously. The use of nano delivery system can improve its release characteristics in vivo, and it is a promising drug delivery method.

[0007]

[0008] Albumin as a macromolecular carrier can be degraded into nontoxic, non-immunogenic water-soluble products in vivo. Albumin-based nanoparticle drug delivery systems have received much attention. Albumin has the advantages of high nutritional value, abundant resources and renewable, strong drug binding capacity, easy preservation, stability in vivo, and easy processing in the preparation process. Ovalbumin (OVA), Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) are the three common albumins, which have been widely used in various biomedical research fields. BSA is often used for research due to its low price and easy purification. HSA has a slightly lower yield and is often used in research that needs to avoid the loss caused by animal albumin such as bovine spongiform encephalopathy. OVA has been widely used in the food industry due to its ability to prepare foam and gel network.

[0009] There is no report on the research of SV nano delivery system based on BSA carrier targeting tumor cells, especially COC cells in the prior art. SUMMARY

[0010] The present application provides a simvastatin albumin nanoparticle, which is prepared by encapsulating simvastatin with serum albumin to form simvastatin serum albumin nanoparticles, and has the advantages of water solubility, low toxicity and combination with hydrophobic drugs, so as to improve the solubility of simvastatin in water, enhance the aggregation of simvastatin at tumor sites to enhance the therapeutic effect, and reduce the toxicity to normal tissues.

[0011] The present application is realized by the following technical scheme:

[0012] The simvastatin albumin nanoparticle is composed of simvastatin and serum albumin, wherein the mass ratio of simvastatin to serum albumin is 1:10-20.

[0013] The serum albumin is one of ovalbumin (OVA), human serum albumin (HSA) and bovine serum albumin (BSA).

[0014] The simvastatin albumin nanoparticle is prepared by a nano-emulsification solvent evaporation method.

[0015] Specifically, the simvastatin albumin nanoparticle is prepared by the following method:

[0016] (1) Preparation of O / W emulsion:

[0017] (a) Preparation of water phase: dissolve serum albumin in deionized water to prepare water phase;

[0018] (b) Preparation of drug-containing oil phase: dissolve simvastatin in organic phase to prepare drug-containing oil phase;

[0019] (c) Add the drug-containing oil phase drop by drop into the water phase, and vortex after the addition is completed; immediately after vortexing, ultrasonic at a specific power and working interval probe for a period of time under ice bath to obtain O / W emulsion.

[0020] wherein,

[0021] In step (a), the concentration of serum albumin is 2.5-5 mg / mL, preferably 5 mg / mL;

[0022] In step (b), the organic phase is dichloromethane;

[0023] In step (b), the concentration of simvastatin in dichloromethane is 2.5-5 mg / mL, preferably 2.5 mg / mL;

[0024] The volume ratio of the water phase of step (a) to the drug-containing oil phase of step (b) is 15:1 to 10:1, preferably 10:1;

[0025] In step (c), the ice bath temperature is below 4℃.

[0026] In step (c), the ultrasonic time is 10 to 40 min, preferably 25 to 30 min.

[0027] In step (c), the ultrasonic power is 300 to 400 W, preferably 400 W.

[0028] (2) Preparation of albumin nanoparticles

[0029] The O / W emulsion obtained in step (1) is rotary evaporated under reduced pressure to remove the organic solvent, and then is diluted with deionized water, and centrifuged to obtain simvastatin albumin nanoparticles.

[0030] The centrifugation temperature is -4 to -6℃, and the centrifugation rate is 12000 to 15000 rpm;

[0031] Further, the simvastatin albumin nanoparticles are added with a freeze-drying protective agent to prepare a freeze-dried powder of simvastatin albumin nanoparticles.

[0032] Specifically comprising the following steps:

[0033] (1) The simvastatin albumin nanoparticles are added with deionized water to be reconstituted into a simvastatin albumin nanoparticle solution;

[0034] (2) The solution is transferred to a Schlenk flask, and a freeze-drying protective agent is added to the solution, and if necessary, the freeze-drying protective agent is completely dissolved by gently shaking.

[0035] (3) The solution of step (2) is frozen in a ultra-low temperature refrigerator at -70 to -80℃ for 2 to 3 days, and then is quickly transferred to a freeze dryer at -60℃ for drying for 2 to 3 days to obtain a freeze-dried powder of simvastatin albumin nanoparticles.

[0036] In step (2), the freeze-drying protective agent is one or more of trehalose / glucose, mannitol, and lactose, and the amount of the freeze-drying protective agent is 1 to 5%, preferably 3 to 5%, and preferably, the freeze-drying protective agent is 3 to 5% of mannitol.

[0037] Further, the present application provides the simvastatin albumin nanoparticles or the freeze-dried powder thereof for use in the preparation of an anti-tumor drug.

[0038] The tumor is breast cancer, liver cancer, ovarian cancer, colorectal cancer, and preferably colorectal cancer.

[0039] Compared with the prior art, the present application has the following advantages:

[0040] The present application uses serum albumin as a carrier, and uses nanoparticle albumin binding technology to load common hypolipidemic drug simvastatin in a nanoparticle drug delivery system for treating tumors. Serum albumin has superior biological safety and is biodegradable in vivo, and the preparation technology used does not introduce agents toxic to the human body, which further ensures the safety of the prepared nanoparticles.

[0041] The present application uses nanoemulsion solvent evaporation method to prepare simvastatin albumin nanoparticles, and under transmission electron microscopy, spherical nanoparticles can be observed, with an average particle size of less than 500 nm, preferably less than 250 nm, a polydispersity coefficient of less than 0.5, and an encapsulation rate of more than 70%. The particle surface is negatively charged, with a zeta potential of -31.25 mV.

[0042] Simvastatin albumin nanoparticles release faster than simvastatin in a simulated in vivo environment, with a significantly increased cumulative release rate. It is proved that the nano-dosage form of serum albumin improves the physical properties of simvastatin, improves the in vitro release, and further improves the in vivo absorption.

[0043] Simvastatin albumin nanoparticles have cytotoxicity to tumor cells, with time dependence and concentration dependence. The cytotoxicity of simvastatin albumin nanoparticles is selective in tumor sources. The cytotoxicity of simvastatin albumin nanoparticles is higher than that of simvastatin, and statistical differences can be seen at some concentrations.

[0044] Simvastatin albumin nanoparticles have an inhibitory effect on the migration ability of COC CT26 cells. The higher the concentration of simvastatin albumin nanoparticles, the stronger the inhibitory effect on tumor cell migration, and there is a significant difference between concentrations. Simvastatin albumin nanoparticles can induce COC CT26 cell apoptosis, and the late apoptosis rate induced by simvastatin albumin nanoparticles at some concentrations is significantly different from that of simvastatin. Simvastatin albumin nanoparticles can be successfully taken up by COC CT26 cells. With the extension of incubation time, simvastatin albumin nanoparticles are enriched in CT26 cells. The selectivity of simvastatin albumin nanoparticles for CT26 tumor cells is significantly better than that for other tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Figure 1 is a transmission electron micrograph of SV-BSA-NPs; the scale is 200 nm;

[0046] Figure 2 Figure 3 is the in vitro release curve of SV, SV-BSA-NPs;

[0047] Figure 3 Figure 4 is the inhibitory effect of SV, SV-BSA-NPs on human colon cancer cells (HCT116) and the inhibition curve;

[0048] A, D: 24h B, E: 48h C, F: 72h Mean ± SEM, n = 3

[0049] Figure 4 Inhibition of human colon cancer cells (HT29) by SV, SV-BSA-NP and inhibition curve;

[0050] A, D: 24h B, E: 48h C, F: 72h

[0051] Mean ± SEM, n = 3 *P<0.05; ****P<0.0001

[0052] Figure 5 Inhibition of human colon cancer cells (SW480) by SV, SV-BSA-NP and inhibition curve;

[0053] A, D: 24h B, E: 48h C, F: 72h

[0054] Mean ± SEM, n = 3

[0055] Figure 6 Inhibition of mouse colon cancer cells (CT26) by SV, SV-BSA-NP and inhibition curve;

[0056] A, D: 24h B, E: 48h C, F: 72h

[0057] Mean ± SEM, n = 3 *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001

[0058] Figure 7 Inhibition of human ovarian cancer cells (OVCAR3) by SV, SV-BSA-NP and inhibition curve;

[0059] A, D: 24h B, E: 48h C, F: 72h Mean ± SEM, n = 3

[0060] Figure 8 Inhibition of human liver cancer cells (HuH-7) by SV, SV-BSA-NP and inhibition curve;

[0061] A, D: 24h B, E: 48h C, F: 72h

[0062] Mean ± SEM, n = 3 *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001

[0063] Figure 9 Changes in cell viability of human breast cancer cells (MCF7) by SV, SV-BSA-NP;

[0064] A: 24h B: 48h C: 72h

[0065] Figure 10 Effect of SV, SV-BSA-NP, Blank-BSA-NP on normal cell viability;

[0066] A-C: NIH-3T3 cells A: 24h B: 48h C: 72h

[0067] D-F: HEK293 cells D: 24h E: 48h F: 72h

[0068] Figure 11 Effect of SV, SV-BSA-NP concentration on the scratch healing ability of mouse colon cancer cells (CT26);

[0069] A~H: Photographs of scratches before and after administration of different concentrations of SV for 24h;

[0070] I~P: Photographs of scratches before and after administration of different concentrations of SV-BSA-NP for 24h;

[0071] Figure 12 Relationship between incubation of different concentrations of SV, SV-BSA-NP and the scratch healing rate of mouse colon cancer cells (CT26);

[0072] Mean ± SEM; n = 3, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

[0073] Figure 13 Effect of SV-BSA-NPs on the induction of apoptosis in mouse colon cancer cells (CT26) for 24h;

[0074] A: Comparison of apoptosis rates at different administration concentrations;

[0075] B: Comparison of early apoptosis rates at different administration concentrations;

[0076] C: Comparison of late apoptosis rates at different administration concentrations;

[0077] n = 3, Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

[0078] Figure 14 Effect of SV and SV-BSA-NPs at different concentrations on the induction of late apoptosis in mouse colon cancer cells (CT26);

[0079] A: Apoptosis flow cytometry of negative control, SV, SV-BSA-NP at the concentration of 2.5 μΜ, and the statistical histogram of late apoptosis rate (from left to right).

[0080] B, C: Apoptosis flow cytometry and the statistical histogram of late apoptosis rate at the concentration of 5 μΜ and 10 μΜ, respectively.

[0081] Mean ± SEM n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0082] Figure 15 Qualitative analysis of the uptake of Cou6-BSA-NPs by CT26 cells at different administration times;

[0083] Green fluorescence is the spontaneous fluorescence of Cou6-SV-BSA-NPs, and blue fluorescence is the DAPI staining of cell nuclei; the scale is 100 μm.

[0084] Figure 16 Tumor volume-time curve of mice in each administration group;

[0085] Mean ± SEM n = 8; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.

[0086] Figure 17 Tumor tissue and weight of SV-BSA-NPs in vivo anti-mouse CT26 colon cancer tumor efficacy experiment;

[0087] A: Tumor tissue pictures of mice in each group; B: Tumor weight histogram of each group (n = 8);

[0088] *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001

[0089] Figure 18 Body weight-time curve of mice in each group after drug treatment;

[0090] Figure 19 Blood biochemical liver and kidney indicators of mice in each group after drug treatment

[0091] A: Aspartate aminotransferase (AST); B: Alanine aminotransferase (ALT); C: Urea nitrogen (BUN); D: Creatinine.

[0092] Figure 20 Representative diagram of H&E staining sections of main organs of mice in each administration group after drug treatment, the scale is 50 μm. DETAILED DESCRIPTION

[0093] Example 1:

[0094] Preparation of Simvastatin Serum Albumin Nanoparticles (SV-BSA-NPs) by Nanoemulsion Solvent Evaporation Technique

[0095] The nanoparticle prepared by nanoemulsion solvent evaporation technique has smaller particle size, more uniform distribution and higher encapsulation efficiency. Different preparation conditions affect the particle size, polydispersity index (PDI) and encapsulation efficiency of SV-BSA-NPs to different degrees, and further affect the in vitro release and in vivo efficacy of BA-BSA-NPs. By investigating different preparation conditions, the best simvastatin serum albumin nanoparticles can be obtained.

[0096] Simvastatin albumin nanoparticles (SV-BSA-NPs) were prepared according to the following method:

[0097] BSA was accurately weighed and dissolved in distilled water, and vortexed to disperse uniformly to obtain an aqueous phase. SV was accurately weighed and dissolved in dichloromethane to obtain a drug-containing oil phase. The drug-containing oil phase was slowly added to the aqueous phase, vortexed for 5 min, and then transferred to an ultrasonic cell crusher. The power was 300-400 W, and ice bath ultrasonic was performed for 30 min, with 2 s of ultrasonic and 1 s of stop. An O / W emulsion was obtained. The O / W emulsion was rotary evaporated at 40°C and 30 r / min for 6 min to remove the organic solvent, and then the volume was adjusted with deionized water. Centrifugation was performed at 4°C and 12000 rpm to obtain SV-BSA-NPs.

[0098] Table 1 Effect of different preparation conditions on particle size, PDI and encapsulation efficiency of SV-BSA-NPs

[0099]

[0100] Results of schemes 1-5 show that the nanoparticles prepared by schemes 4 and 5 have too large particle size, which are not suitable for further preparation of qualified nanoparticles. When the volume ratio of the aqueous phase to the oil phase containing the drug is 2:1 and 5:1, the nanoparticles with qualified particle size cannot be obtained, and the encapsulation efficiency is not considered. Therefore, the volume ratio of the aqueous phase to the oil phase containing the drug should be at least 10:1. When the concentration of BSA is 5 mg / mL, the concentration of SV is 2.5-5 mg / mL, the volume ratio of the aqueous phase to the oil phase containing the drug is 10:1, and the power is 300-400 W, the particle size of the prepared SV-BSA-NPs is less than 500 nm, the PDI is less than 0.5, and the encapsulation efficiency is greater than 70%. The smaller the particle size of the nanoparticles, the better the in vitro release and in vivo effect. Therefore, considering the particle size, PDI and encapsulation efficiency, the preferred technical solution of the present application is scheme 3, i.e. the concentration of BSA is 5 mg / mL, the concentration of SV is 2.5 mg / mL, the volume ratio of the aqueous phase to the oil phase containing the drug is 10:1, the power is 400 W, and the mass ratio of SV to BSA is 1:20. Under these conditions, the particle size of the prepared SV-BSA-NPs is less than 250 nm, the PDI is less than 0.3, and the encapsulation efficiency is greater than 70%.

[0101] Example 2: Selection of freeze-drying protectant for SV-BSA-NPs

[0102] SV-BSA-NPs were prepared according to the scheme of scheme 3 of Example 1. The SV-BSA-NPs were freeze-dried without adding any freeze-drying protectant. After complete freeze-drying, the particle size and PDI of the SV-BSA-NPs were measured. The results are shown in Table 2.

[0103] Table 2 Measurement results of particle size of SV-BSA-NPs before and after freeze-drying

[0104]

[0105] The results show that the freeze-drying process changes the particle size of the nanoparticles. It is speculated that part of the nanoparticles are affected by the drastic change of external temperature during pre-freezing or drying, and drug leakage occurs.

[0106] In order to improve the stability of SV-BSA-NPs, 5% freeze-drying protectant was added before freeze-drying. The freeze-drying protectants were mannitol, trehalose or sucrose, respectively.

[0107] The prepared SV-BSA-NPs were collected, and the prescribed amount of deionized water was added to reconstitute the SV-BSA-NPs solution. The solution was transferred to a Schlenk flask. A certain amount of freeze-drying protectant was added to the solution, and the freeze-drying protectant was completely dissolved by gently shaking if necessary. The solution was pre-frozen at -80°C for 24 h, and then quickly transferred to a freeze-drying machine for freeze-drying for 24 h, thereby obtaining SV-BSA-NPs freeze-dried powder. The particle size and appearance after freeze-drying are shown in Table 3.

[0108] Table 3 Particle size measurement results and appearance of samples with added lyophilization protectants (n = 3)

[0109]

[0110] The results in Table 3 show that the sample without added lyophilization protectants is prone to clumping after lyophilization, and the reconstitution process requires assistance from blowing and shaking. The sample with added protectants has a better appearance, with no collapse on the surface and a smoother surface, and the powder is fluffy. The sample with added mannitol has little change in particle size, similar to the sample without added lyophilization protectants, so it is excluded. The samples with added trehalose and sucrose have a smaller particle size after lyophilization than before, and have a good appearance, so the lyophilization protectant is preferably trehalose or sucrose.

[0111] Example 3: Preparation of SV-BSA-NPs

[0112] Precisely weigh 50 mg of BSA and dissolve it in 10 mL of distilled water, and vortex to disperse it uniformly to obtain an aqueous phase. Precisely weigh 2.5 mg of SV and dissolve it in 1 mL of dichloromethane to obtain a drug-containing oil phase. Slowly add the drug-containing oil phase to the aqueous phase, vortex for 5 min, and then quickly transfer it to an ultrasonic cell crusher. Ultrasonic at a power of 400 W for 30 min in an ice bath, with 2 s of ultrasonic and 1 s of pause. Thus, an O / W emulsion is obtained. Remove the organic solvent from the O / W emulsion by rotary evaporation at 40°C and 30 r / min for 6 min, and then use deionized water to make up the volume. Centrifuge at 12000 rpm at 4°C to obtain SV-BSA-NPs.

[0113] The particle size of the prepared SV-BSA-NPs is 213.7 nm, the polydispersity coefficient is 0.226, the zeta potential is -31.25 mV, and the transmission electron microscopy is as shown in Figure 1 .

[0114] Example 4: In vitro release of SV-BSA-NPs

[0115] A certain amount of SV, SV-BSA-NPs (Example 3) of the same drug amount were respectively dissolved with 2 mL release medium, placed in a dialysis bag with a molecular weight cut-off of 3.5 kD, and a mixed solution of 0.5% sodium dodecyl sulfate in 0.01 mol / L sodium dihydrogen phosphate buffer (pH adjusted to 7.0 with 50% sodium hydroxide solution) containing anhydrous ethanol was used as the release medium, wherein the volume ratio of anhydrous ethanol to aqueous solution was 20:80. The in vitro release behavior of SV-BSA-NPs was studied at 37±0.5°C, 100 r / min, and 100 mL of release medium. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h, and an equal amount of medium was added in time. The release curve was plotted with the sampling time as the abscissa X and the cumulative release amount as the ordinate Y, and compared with the SV raw material. The cumulative release rate was calculated as follows: cumulative release amount = cumulative release amount at each time point / total amount of drug in nanoparticles x 100%. The release curve is shown in Figure Figure 2

[0116] The results showed that the cumulative release of SV was 35.11±2.83% within 24 h, 54.24±2.18% at 48 h, and 87.67±4.86% at the last sampling before the end of the experiment. This situation was speculated to be due to the poor water solubility of SV, which released slowly in vivo. SV released less than 50% within 24 h, and its full release required more than 72 h. The SV-BSA-NPs synthesized in this study encapsulated SV in BSA, which improved the water insolubility of SV and greatly increased its hydrophilicity. SV-BSA-NPs showed a rapid release within 1 h after the start of release, with a release rate of 52.11±1.73%, and then the release rate slowed down. The cumulative release rate was 85.48±0.67% at 24 h and 96.61±1.31% at 72 h, which was close to complete release. That is, SV had a faster release rate within 0-8 h, with a cumulative release of 75.57±0.41% at 8 h, and then the release rate slowed down. Therefore, the preparation of SV-BSA-NPs can significantly improve the dissolution and release of SV, and thus improve its bioavailability.

[0117] Example 5 CCK8 method for determining the inhibitory effect of SV-BSA-NPs on COC cell survival rate

[0118] The CCK8 method was used to detect the effect of SV-BSA-NPs (Example 3) on the cell survival rate of common COC cell lines and whether it was time-dependent and concentration-dependent, and compared with SV raw material.

[0119] ​The COC cell line used in the application is mouse COC cell line CT26, human COC cell line HCT116, HT-29, SW480, all of which are from the Cell Bank of Chinese Academy of Sciences.

[0120] Precisely take 10 mg of SV, add 0.4778 mL of cell culture grade DMSO to dissolve, use water bath ultrasonic assisted dissolution, obtain SV stock solution with a concentration of 50 mM, store in a 4°C refrigerator in the dark.

[0121] Collect SV-BSA-NPs containing 3 mg of SV, accurately take 0.1433 mL of PBS for reconstitution, obtain SV-BSA-NPs stock solution with a concentration of 50 mM, store at 4°C in the dark.

[0122] After obtaining the cell suspension in the logarithmic growth phase and in good condition, 10 μL of liquid is taken for counting using a hemocytometer, the cell concentration of the cell suspension is obtained, and then the cell suspension is diluted to a specific concentration using complete medium. The diluted cell suspension is seeded in a 96-well plate at a density of 5×10 3 Each group sets 4-6 replicate wells, and a group of wells with a drug concentration of 0 is reserved as a blank control, and a group of wells with only liquid is reserved as a background zero. Continue to incubate in the carbon dioxide incubator for a period of time. After adding CCK8 detection solution, pay attention to avoid light throughout the operation. Configure CCK8 detection solution, i.e. mix the liquid in a volume ratio of V 完全培养基 : V CCK8 = 9:1, add the above detection solution after reaching the predetermined culture time and discarding the drug-containing culture medium, then incubate at 37°C for 40-60 min, the specific incubation time varies depending on the cell line. Detect on the machine. Set the enzyme marker detection wavelength to 450 nm, record the OD value of each well, and repeat each test not less than three times. Calculate the cell survival rate according to the following algorithm:

[0123] Cell viability (%) = (OD 实验 - OD 背景 ) / (OD 空白 - OD 背景 ) × 100%

[0124] Record the drug concentration and cell survival rate, take the logarithm of the drug concentration as the abscissa X, and take the cell survival rate as the ordinate Y, and statistically analyze the results and calculate the IC 50 value.

[0125] In this experiment, the absorbance of different action times at 2.5-160 μM concentration was detected by CCK8 method, and the IC50 Values.

[0126] The effect of SV-BSA-NPs on the cell viability of human COC cell line HCT116 is shown in Table 1. Figure 3 .

[0127] The results show that SV-BSA-NPs and SV both have inhibitory effect on HCT116 cells at a concentration of 2.5-80 μM, and the inhibitory effect of SV-BSA-NPs is slightly stronger than that of SV. The inhibitory effect on cell viability is enhanced with the increase of concentration, and the result of SV at a concentration of 80 μM and cultured for 48 h is significantly different (P < 0.001). The half inhibitory concentration of SV-BSA-NPs on HCT116 is 178.3 μM, 55.43 μM and 19.71 μM respectively at 24 h, 48 h and 72 h, which is significantly less than 254.4 μM, 114.5 μM and 38.73 μM of free drug respectively, and is 1.43 times, 2.07 times and 1.96 times respectively. It can be seen that the inhibitory effect of SV-BSA-NPs on HCT116 cells is significantly enhanced with the prolongation of action time, and the half inhibitory concentration at 48 h and 72 h is 3.22 times and 9.05 times respectively of that at 24 h.

[0128] The effect of SV-BSA-NPs on the cell viability of human COC cell line HT29 is shown in Table 2. Figure 4 .

[0129] The results show that the inhibitory effect of SV-BSA-NPs on HT29 is slightly enhanced compared with that of SV. The cell survival rate is significantly different at a concentration of 80 μM and cultured for 48 h, and obvious difference is also observed at a concentration of 20 μM and cultured for 72 h. The half inhibitory concentration is 76.25 μM, 62.04 μM and 48.65 μM respectively at 24 h, 48 h and 72 h, and it can be seen that the inhibitory effect on cell viability is time-dependent, and the drug efficacy at 48 h and 72 h is 1.16 times and 1.24 times respectively of that of the original drug.

[0130] The effect of SV-BSA-NPs on the cell viability of human COC cell line SW480 is shown in Table 3. Figure 5 .

[0131] The results show that the inhibitory effect of SV-BSA-NPs on SW480 is also concentration-dependent and time-dependent. In the case of 24h, 48h, 72h, the half inhibitory concentration of SV-BSA-NPs on SW480 is 39.00 μM, 16.91 μM, 9.036 μM, slightly less than 48.41 μM, 23.04 μM, 11.52 μM of free drug, 1.24 times, 1.36 times, 1.27 times, respectively. With the extension of the action time, the half inhibitory concentration of SV-BSA-NPs is reduced to 43.36% and 23.17% of 24h at 48h and 72h, respectively.

[0132] The effect of SV-BSA-NPs on the cell viability of mouse COC cell line CT26 is shown in Figure 6 .

[0133] The results show that with the increase of SV concentration, the cell viability of CT26 is inhibited, and the inhibitory effect is enhanced, which shows that the inhibitory effect is concentration-dependent Figure 6 -A, B, C); with the extension of the culture time, the IC 50 value decreases significantly Figure 6 -D, E), which shows that the inhibitory effect is time-dependent. In the case of 24h, 48h, 72h, the IC 50 of SV-BSA-NPs on CT26 is 5.152 μM, 3.746 μM, 0.613 μM, while the IC 50 of SV is 21.51 μM, 4.817 μM, 1.682 μM, which shows that the inhibitory effect of SV-BSA-NPs on CT26 cells is significantly stronger than that of raw material drug SV, and the difference is significant at multiple concentrations, and there is a significant difference at the extremely low drug concentration of 0.3125 μM. (P<0.0001)

[0134] The method is the same as in Example 5, and the tumor cells are human hepatoma cell line HuH-7, human breast cancer cell line MCF-7, and human ovarian cancer cell line OVCAR3, all from the Chinese Academy of Sciences Typical Culture Collection Committee Cell Bank.

[0135] The effect of SV-BSA-NPs on the cell viability of OVCAR3 cell line is shown in Figure 7 .

[0136] The results show that in the concentration range of 1.25-40 μM, the cell viability is inhibited, and the cell viability gradually decreases, which shows concentration-dependent. From the comparison of IC 50 values, the IC 50was 38.96 μM, much less than 151.5 μM of SV. The inhibitory effect of SV-BSA-NPs was also much stronger than that of free drug at 48 h and 72 h, IC 50 The values were much lower than the results at 24 h, indicating that the toxic effect was time-dependent.

[0137] The effect of SV-BSA-NPs on the cell viability of human hepatoma cell line HuH-7 is shown in Table 4. Figure 8 .

[0138] The results show that the sensitivity of HuH-7 cells to SV-BSA-NPs is lower than that of other cells, and the cell viability is almost not inhibited after 24 h. The slight inhibitory effect is not found to have the concentration-dependent feature. However, the inhibitory effect appears to be concentration-dependent at 48 h and 72 h, and the IC 50 of SV-BSA-NPs at 72 h is 2.69 times that of SV. Significant differences are found at the drug concentrations of 1.25 μM, 2.5 μM and 5 μM, which proves that the therapeutic effect of SV-BSA-NPs is better than that of SV, and the toxic effect is time-dependent.

[0139] The effect of SV-BSA-NPs on the cell viability of human breast cancer cell line MCF7 is shown in Table 5. Figure 9 .

[0140] The results show that the inhibitory effect is slightly enhanced with the increase of the concentration in the range of 1.25-40 μM. The cell viability is more strongly inhibited at the same drug concentration with the extension of the incubation time. For example, the cell survival rate is 99.60±7.16% at 24 h, 91.92±2.46% at 48 h and 83.64±12.98% at 72 h when the drug concentration of SV-BSA-NPs is 20 μM. However, the inhibitory effect is weak, and the IC 50 value cannot be obtained.

[0141] Example 7 Determination of the Toxicity of SV-BSA-NPs to Normal Cells by CCK8 Method

[0142] Blank-BSA-NPs were prepared according to the BSA content of 3 mg SV equivalent of nanoparticles. The nanoparticles were collected by centrifugation, and were re-dissolved with 0.1433 mL PBS to obtain a Blank-BSA-NPs stock solution. The stock solution was stored in a refrigerator at 4°C in the dark, and could be used within one week.

[0143] The CCK8 method was used to detect the toxicity of SV-BSA-NPs and the blank carrier Blank-BSA-NPs without drug to mouse embryonic fibroblast cell line NIH-3T3 and human embryonic kidney cell line HEK293, and the results were compared with those of the raw drug SV. The experimental results are shown in Tables 6 and 7.Figure 10 A-C.

[0144] Results show that: in the concentration range of 0.3125-120 μM, in the range of 24-72 h, the blank nanoparticles have weak inhibitory effect on the viability of NIH-3T3 cells, which proves that they have good safety. The toxicity of SV-BSA-NPs increases with time, but there is no significant difference compared with free drug SV, which shows that the toxicity does not significantly increase after being prepared into nano-dosage form.

[0145] The results of the toxicity test of SV-BSA-NPs and Blank-BSA-NPs on HEK293 cells are shown in Table 2. Figure 10 D-F.

[0146] Results show that: SV-BSA-NPs have strong toxicity on HEK293 cells, and the survival rate of HEK293 cells is obviously inhibited, and the inhibitory effect has no significant difference compared with free drug SV, which shows that the toxicity does not increase in the preparation process. The toxicity of the blank albumin nanoparticles Blank-BSA-NPs prepared according to the optimal prescription is relatively small, and there is obvious inhibitory effect only at the highest concentration of 160 μM under the three action times, which shows that the toxicity is small.

[0147] Example 8 Cell scratch healing test

[0148] The CT26 cells in the logarithmic growth phase were diluted with complete culture medium, and inoculated in a six-well plate at a density of 3x10 5 cells / well, and cultured at 37°C, 5% CO2 for 24 h until the cells covered the bottom of the six-well plate. The complete culture medium in the well was discarded, and washed once with PBS and discarded, and a 200 μL pipette tip was used to draw a line on the bottom of the plate in the vertical direction, and the gun head was perpendicular to the plate, and the hand was gentle and fast. The cells that were washed off were washed again with PBS, and the scratch was photographed, which was recorded as 0 h. Then, 2 mL of incomplete culture medium containing SV and SV-BSA-NPs (Example 3) with concentrations of 0.625, 1.25, 5 μmol / L was added to the well, and placed in a carbon dioxide incubator for 24 h, and then photographed. Image J software was used to analyze and process the pictures, and the scratch healing rate was calculated according to the following algorithm.

[0149] Scratch healing rate = (A 0h -A 24h ) / A 0h x 100%

[0150] Wherein A 0h is the area of the scratch at 0 h, and A 24h is the area of the scratch at 24 h.

[0151] The results of the effect of SV, SV-BSA-NPs on the migration function of CT26 cells are shown in Figure 11 As can be seen from the results shown in Figure 11 that the scratches showed a healing trend within 24h, and the healing degree of the low concentration group was more obvious than that of the high concentration group. The higher the concentration, the worse the healing, and the more inhibited the cell migration ability.

[0152] The pictures were processed by ImageJ software to calculate the cell migration rate, which can be quantitatively evaluated. Figure 12 The healing rate of the control group was 29.86±3.31%, which was significantly higher than that of the SV-BSA-NPs concentration of 0.625μM, 1.25μM, 5μM (P<0.0001), which were 16.13±0.94%, 5.97±1.06%, 2.78±0.56%, respectively. The results showed that the addition of SV-BSA-NPs could effectively inhibit the migration of tumor cells even at very low concentrations.

[0153] Example 9 Flow cytometry detection of the effect of SV-BSA-NPs on the apoptosis rate of COC cells

[0154] CT26 cells in the logarithmic growth phase were diluted with complete culture medium, and inoculated in a six-well plate at a cell density of 2.5×10 5 cells / well, and cultured at 37℃, 5% CO2 for overnight until the cells were completely adherent. The culture medium was replaced with complete culture medium containing a certain concentration of SV and SV-BSA-NPs, and the culture was continued for 24h. The cells at the bottom of the six-well plate were collected by using 200μL trypsin without EDTA, centrifuged at 300g at 4℃, the supernatant was discarded, and pre-cooled PBS was added to resuspend the cells gently, and attention should be paid to not generate bubbles. The washing process was repeated twice, and pre-cooled PBS was used throughout the washing process. After the last washing, binding buffer was added to resuspend the cells, 5μL Annexin V-FITC and 10μL PI dye were added in a dark environment, and the reaction was carried out at room temperature for 15min. Finally, 400μL Binding Buffer was added, mixed well, placed on ice, and detected by flow cytometry within 1h.

[0155] In this study, FITC / PI double staining was used to study the apoptosis of CT26 cells induced by SV-BSA-NPs, and the experiment was processed according to the instructions of the Annexin V-FITC / PI kit. This experiment designed 0, 2.5, 5, 20μM, a total of 4 concentration gradients, and after incubation with SV-BSA-NPs-containing medium at the above concentrations for 24h, the apoptosis of the cells in each group was detected by machine, and the results are shown in Figure 13The results show that the proportion of apoptotic cells in each concentration group is 4.40±0.47%, 15.58±4.55%, 20.43±5.14%, and 18.55±4.99%, respectively; the proportion of early apoptotic cells is 0.78±0.11%, 2.12±1.27%, 3.13±3.07%, and 2.16±1.66%, respectively; and the proportion of late apoptotic cells is 3.61±0.57%, 13.77±4.30%, 17.70±2.19%, and 16.20±4.62%, respectively. With the increase of the concentration of SV-BSA-NPs, the apoptosis rate of CT26 cells gradually increases and is significantly higher than that of the control group, indicating that SV-BSA-NPs have excellent ability to induce apoptosis of CT26 cells and are positively correlated with the concentration.

[0156] Example 10 Comparison of the Effects of SV and SV-BSA-NPs on the Apoptosis of CT26 Cells

[0157] The ability of SV-BSA-NPs and SV to induce apoptosis of CT26 cells was compared at different concentration levels in the same way, and the results and statistical graphs are shown in Figure 14 .

[0158] The results show that when the drug concentration is 2.5 μM, the late apoptosis rate of cells treated with SV-BSA-NPs is significantly higher than that of the negative control group (P<0.01), in which SV-BSA-NP is 13.77±4.30% and the negative control is 3.61±0.57%. When the drug concentration is 20 μM, the late apoptosis rate of cells treated with SV-BSA-NPs is significantly higher than that of the negative control group (P<0.01), in which SV-BSA-NP is 18.55±4.99% and the negative control is 4.40±0.47%. In the three drug concentration groups, the late apoptosis rate of cells treated with SV-BSA-NPs is significantly higher than that of the negative control group; when the drug concentration is 5 μM, the late apoptosis rate of cells treated with SV-BSA-NPs is significantly higher than that of SV (P<0.001), in which SV is 10.57±1.49% and SV-BSA-NPs is 17.70±2.19%. With the gradual increase of the concentration, the difference in apoptosis between SV-BSA-NPs and SV is more and more significant. The above results show that the use of BSA to embed SV can improve its ability to induce apoptosis of tumor cells to a certain extent and is concentration-dependent.

[0159] Example 11 Laser Confocal Microscope Study of the Uptake of SV-BSA-NPs by COC Cells

[0160] Coumarin 6 was weighed and dissolved in 1 mL of methanol to obtain a coumarin 6 stock solution with a concentration of 0.5 mg / mL. A certain volume of the coumarin 6 stock solution was mixed with the SV dichloromethane solution, and then the coumarin 6 SV-BSA-NPs were prepared according to the optimal prescription. Note that the preparation process is in the dark.

[0161] Coumarin 6 was weighed and dissolved in 1 mL of methanol to obtain a coumarin 6 stock solution with a concentration of 0.5 mg / mL. Then, gradient dilution was performed to obtain coumarin 6 solutions with concentrations of 1, 2, 4, 8, 16, and 32 ng / mL.

[0162] 100 μL of each of the above solutions was placed in a 96-well plate, and the fluorescence intensity was measured using a microplate reader with the measurement conditions set as λ ex = 450 nm and λ em = 505 nm. The standard curve was plotted with the concentration as the horizontal coordinate X and the fluorescence intensity OD value as the vertical coordinate Y, and linear regression was performed. The drug concentration was determined according to the standard curve results. Note that the whole operation is in the dark.

[0163] CT26 cells in the logarithmic growth phase were diluted with complete culture medium and inoculated in a cell culture dish at a cell density of 1.5 x 10 5 cells / mL. The dish was placed in a cell incubator and cultured for 24 h until the cells were completely adherent. A certain concentration of Cou6-SV-BSA-NPs was added under light shielding conditions, and the dish was incubated at 37°C and 5% CO2 for a period of time. After incubation, the liquid in the dish was discarded, and the dish was washed with pre-cooled PBS three times. 200 μL of 4% paraformaldehyde solution was added to the dish, and the dish was fixed in the dark for 20 min. After washing with PBS three times, sufficient DAPI was added to the dish, and the dish was incubated in the dark for 5 min. After washing with PBS, the dish was ready for imaging. The uptake of Cou6-SV-BSA-NPs by CT26 cells is shown in Figure 15 .

[0164] The results show that SV-BSA-NPs themselves have no fluorescence, so coumarin 6, which does not react with any component in the prescription and emits green light spontaneously, is added to the preparation process and is embedded in the albumin nanoparticles together with SV. At 2 h, green fluorescence appears in the cells, indicating that Cou6-SV-BSA-NPs have been taken up by CT26 cells 2 h after administration. As the administration time increases, the green fluorescence observed around the cell nuclei stained with DAPI gradually increases, indicating that the intracellular Cou6-SV-BSA-NPs concentration has a tendency to increase with time within 24 h after administration. This experiment shows that SV-BSA-NPs can be successfully taken up by tumor cells and enriched, laying the foundation for the anti-tumor efficacy of SV-BSA-NPs.

[0165] Example 12: In vivo anti-colon cancer efficacy study of SV-BSA-NPs

[0166] Experimental animals: SPF grade BALB / c mice, five weeks old, female, body weight 15±2.5g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. This experiment was approved by the China Medical University Experimental Animal Welfare Ethics Committee, number: CMU20231157.

[0167] Cell line: Mouse COC cell line CT26.WT, China Academy of Sciences Typical Culture Preservation Committee Cell Bank / Cell Resource Center of Shanghai Institutes for Biological Sciences.

[0168] Experimental method:

[0169] Select CT26 cells in the logarithmic growth phase, use a small amount of trypsin containing EDTA for digestion, centrifuge the collection liquid, count, make up the basic medium, and prepare a certain concentration of basic medium cell suspension, which is ready for use at 4°C in the dark environment, avoid shaking, and use a pipette to blow evenly in time, and inoculate in experimental animals within 2h.

[0170] Female BALB / c mice were raised in a barrier environment for a period of time. After the experimental animals fully adapted to the barrier environment and the body weight reached 20±2g, 100-200μL of CT26 cell suspension was injected subcutaneously on the back of the mice, and each individual was injected subcutaneously with 1×10 6 After inoculation, the growth of the mice was observed regularly, and the tumor volume was measured. When the tumor volume reached 100mm 3 , the model was successfully established, and the next experiment could be performed.

[0171] The calculation method of the tumor volume of mice is: tumor volume (mm 3 ) = 0.5×(longest diameter×shortest diameter 2 )

[0172] Tumor-bearing mice were raised in a barrier environment, and the environment was kept at 20-22°C, humidity 55%, and day-night alternating cycle 12 hours, and were observed regularly. When the tumor volume reached 100mm3, the tumor-bearing mice were randomly divided into 4 groups, 8 in each group. The 4 experimental groups were: saline group (Saline), blank albumin nanoparticle group (Blank-BSA-NPs), SV solution group (SV), and SV-BSA-NPs group (Example 3 SV-BSA-NPs).

[0173] When the tumor volume reached the requirement, the mice were randomly divided into groups, and the drug administration was started one day after the grouping. The drug was administered at a dose of 4 mg / kg SV by tail vein injection at a volume of 100 μL. The administration frequency was once every 2 days, and the administration was continued for 10 days. The mice were continuously observed and recorded for the body weight and tumor volume, and the measurement frequency was once every 2 days. The experiment was terminated on the 14th day after the start of the administration. After the mice were anesthetized, the eyeballs were enucleated to obtain 500-1000 μL of whole blood. After standing, the blood was centrifuged, and the serum was stored in a -80°C ultra-low temperature refrigerator for subsequent analysis of blood biochemical indexes of liver and kidney functions. The detection indexes included AST (glutamic-oxaloacetic transaminase), ALT (glutamic-pyruvic transaminase), BUN (urea nitrogen), and CR (creatinine). The mice were euthanized by cervical dislocation, and the tumors, heart, liver, spleen, lung, and kidney were removed and immediately immersed in 4% paraformaldehyde for storage at 4°C for subsequent histological analysis.

[0174] Example 13 Pharmacodynamic evaluation of SV-BSA-NPs

[0175] After the tumor-bearing mice were randomly divided into 4 groups on day 0, the drug administration was started on day 1 according to the planned scheme. After the administration was completed, the mice were further raised in the same environmental conditions for a period of time. The tumor volume was measured and recorded at the same frequency. The mice were euthanized on day 14, and the tumor tissues were removed and weighed. The tumor volume from day 1 of the administration to day 13 before euthanization was recorded, and the tumor volume-time relationship diagram of each group was obtained as shown in FIG. 4. Figure 16

[0176] The results show that the tumor volume growth trends of the normal saline group and the Blank-BSA-NPs group are relatively close. It can be seen that the prepared Blank-BSA-NPs without drug loading has no inhibitory effect on tumor cells. Compared with the other two groups, the tumor grows faster, and the tumor volume is larger. On day 13, the Blank-BSA-NPs group and the SV-BSA-NPs group have a significant difference (P<0.0001), and the normal saline group and the SV solution group and the SV-BSA-NPs group have a significant difference (P<0.0001). The SV group and the SV-BSA-NPs group have a similar trend before the 5th administration, and the tumor volume is similar. No difference in treatment effect is observed. After the administration is completed, the difference gradually increases during the observation period. The tumor volume growth trend of the SV-BSA-NPs group is significantly more gentle than that of the SV group. On day 13, the tumor volume of the SV group and the SV-BSA-NPs group has a significant difference (P<0.01), indicating that the drug efficacy of SV-BSA-NPs for COC is significantly better than that of SV free drug.

[0177] The tumor tissues obtained after dissection on day 14 were washed with normal saline, weighed, and photographed. The tumor tissue pictures of FIG. 4A were obtained, and the weights were statistically analyzed to obtain Figure 17 Figure 17 ​​B-bar chart. The saline group had the largest tumor weight, the Blank-BSA-NPs group had a slightly smaller tumor weight, and the SV-BSA-NPs group had the smallest tumor weight. The tumor weight was statistically different from the SV group, the Blank-BSA-NPs group, and the saline group, indicating that the nanoparticles using BSA to encapsulate SV have superior anti-COC tumor performance.

[0178] Example 14: In vivo toxicity evaluation of SV-BSA-NPs

[0179] Throughout the experiment, the mice's weight was measured and recorded every two days, as shown in the following figures. Figure 18 The graph shows the relationship between mouse body weight and time.

[0180] Mouse body weight is correlated with tumor volume and also reflects the systemic toxicity of the dosing regimen. As shown in the figure, the body weight trends of the four dosing groups were similar throughout the experiment, all increasing slowly with no significant differences. On day 13, the average body weights of the groups were 21.72±0.80g, 21.56±0.94g, 21.32±1.99g, and 22.29±0.45g, respectively. This indicates that tail vein injection of SV at a dose of 4mg / kg for the treatment of colon cancer in mice has low systemic toxicity, and the addition of Blank-BSA-NPs has almost no effect on systemic toxicity. This further demonstrates the superiority of BSA-encapsulated drugs for delivering antitumor drugs.

[0181] After the observation period, blood samples were taken for analysis of liver and kidney function biochemical indicators. The results are as follows: Figure 19 The four detection indicators reflected the liver and kidney function of mice, respectively. While the values ​​of the indicators varied among the groups, no statistically significant differences were observed. This indicates that Blank-BSA-NPs and SV-BSA-NPs did not cause toxic damage to the liver and kidney function of mice, and the prepared SV-BSA-NPs exhibited low in vivo toxicity and good safety.

[0182] After the experiment, sections of major organs from mice were taken, and the tissue sections were stained with hematoxylin and eosin. The results are as follows. Figure 20 As shown, there were no significant differences in the histological characteristics of the major organs of mice in each treatment group. The administration regimen did not produce significant toxicity to the major organs of mice, and the prepared SV-BSA-NPs showed low toxicity to each organ.

Claims

1. Simvastatin albumin nanoparticle characterized in that, consisting of simvastatin and serum albumin, the mass ratio of simvastatin to serum albumin being 1:10-20.

2. The simvastatin albumin nanoparticle of claim 1, wherein the serum albumin is one of ovalbumin, human serum albumin and bovine serum albumin.

3. The process for the preparation of simvastatin albumin nanoparticle as claimed in claim 1 or 2, wherein, comprising the following steps: (1) Preparation of O / W emulsion: (a) Preparation of water phase: dissolve serum albumin in deionized water to prepare water phase; (b) Preparation of drug-containing oil phase: dissolve simvastatin in organic phase to prepare drug-containing oil phase; (c) Drop the drug-containing oil phase into the water phase drop by drop, and vortex after the dropwise addition is completed; immediately after vortexing, sonicate at a specific power and work interval probe for a period of time under ice bath to obtain O / W emulsion; (2) Preparation of albumin nanoparticle The O / W emulsion obtained in step (1) is rotary evaporated under reduced pressure to remove the organic solvent, followed by constant volume with deionized water, and centrifugation to obtain simvastatin albumin nanoparticle.

4. The production method according to claim 3, characterized by In the step (a), the concentration of serum albumin is 2.5-5 mg / mL, preferably 5 mg / mL.

5. The production method according to claim 3, characterized by In the step (b), the organic phase is dichloromethane; The concentration of simvastatin in dichloromethane is 2.5-5 mg / mL, preferably 2.5 mg / mL.

6. The preparation method according to claim 3, characterized in that, The volume ratio of water phase in step (a) to drug-containing oil phase in step (b) is 15:1-10:1, preferably 10:

1.

7. The preparation method according to claim 3, characterized in that, In step (c), the ultrasonic power is 300-400 W, preferably 400 W.

8. A lyophilized powder of simvastatin albumin nanoparticles characterized in that, The simvastatin albumin nanoparticle of claim 1 or 2 is added to a lyophilization protectant to prepare a lyophilized powder.

9. Use of the simvastatin albumin nanoparticle of claim 1 or 2 or the simvastatin albumin nanoparticle lyophilized powder of claim 8 in the preparation of an antitumor drug.

10. Use according to claim 9, characterized in that, The tumor is breast cancer, liver cancer, ovarian cancer, colorectal cancer, preferably colorectal cancer. The tumor is breast cancer, liver cancer, ovarian cancer, colorectal cancer, preferably colorectal cancer.

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