Simvastatin albumin nanoparticle and preparation and use thereof

Abstract

A simvastatin albumin nanoparticle and preparation and use thereof. The simvastatin albumin nanoparticle is composed of simvastatin and serum albumin, wherein the mass ratio of simvastatin to serum albumin is 1:10-20. The simvastatin albumin nanoparticles are prepared by means of a nano-emulsification solvent volatilization method. The preparation method specifically comprises the preparation of an O / W emulsion and the preparation of albumin nanoparticles. The prepared nanoparticles have an average particle size of less than 500 nm, a polydispersity coefficient of less than 0.5, and an encapsulation efficiency of greater than 70%. The surface of the particles is negatively charged, and the zeta potential thereof is -31.25 mV. The nanoparticles can selectively target tumor cells, and exhibit different degrees of inhibitory effects on breast cancer, liver cancer, ovarian cancer, and colorectal cancer, with the strongest inhibitory effect observed on colorectal cancer.

Description

Simvastatin albumin nanoparticles and preparation and application thereof TECHNICAL FIELD

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

[0002] Cancer is a major global public health problem. According to the national statistical data in 2023, since 2010, cancer has been the leading cause of death in China, and its incidence, mortality and burden have been increasing. In other countries, cancer is also a health problem that cannot be ignored. Cancer is the leading cause of death in people under the age of 85 in the United States. In 2024, it is estimated that there will be about 2 million new cancer cases and about 610,000 cancer deaths in the United States. 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 major 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, perform normal biological functions, and 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. Notably, compared with hydrophilic statins, lipophilic statins are more easily transported across 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] 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 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 is widely used in the food industry due to its ability to prepare foam and gel network.

[0008] There is no report on SV nano delivery system based on BSA carrier targeting tumor cells, especially COC cells in the prior art. SUMMARY

[0009] The present application solves the technical problems of the prior art by providing 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 the tumor site to enhance the therapeutic effect, and reduce the toxicity to normal tissues. The simvastatin albumin nanoparticle has the characteristics of active and passive targeting of tumors, and enhances the anti-tumor activity of simvastatin.

[0010] The present application is realized by the following technical solutions:

[0011] A simvastatin albumin nanoparticle is composed of simvastatin and serum albumin, wherein the mass ratio of simvastatin to serum albumin is 1:10-20.

[0012] The serum albumin is one of ovalbumin (OVA), human serum albumin (HSA) and bovine serum albumin (BSA).

[0013] The simvastatin albumin nanoparticle is prepared by a nano-emulsification solvent evaporation method.

[0014] Specifically, the simvastatin albumin nanoparticle is prepared by the following method:

[0015] (1) Preparation of O / W emulsion:

[0016] (a) Preparation of aqueous phase: dissolve serum albumin in deionized water to prepare an aqueous phase;

[0017] (b) Preparation of drug-containing oil phase: dissolve simvastatin in an organic phase to prepare a drug-containing oil phase;

[0018] (c) Add the drug-containing oil phase drop by drop to the aqueous phase, and vortex after the addition is completed; immediately after vortexing, sonicate at a specific power and work interval probe for a period of time to obtain an O / W emulsion.

[0019] wherein,

[0020] In step (a), the concentration of serum albumin is 2.5-5 mg / mL, preferably 5 mg / mL;

[0021] In step (b), the organic phase is dichloromethane;

[0022] In step (b), the concentration of simvastatin in dichloromethane is 2.5-5 mg / mL, preferably 2.5 mg / mL;

[0023] The volume ratio of the water phase of step (a) to the drug-containing oil phase of step (b) is 15:1-10:1, preferably 10:1;

[0024] In step (c), the ice-bath temperature is below 4℃.

[0025] In step (c), the ultrasonic time is 10-40 min, preferably 25-30 min.

[0026] In step (c), the ultrasonic power is 300-400 W, preferably 400 W.

[0027] (2) Preparation of albumin nanoparticles

[0028] 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 nanoparticles.

[0029] The centrifugation temperature is -4 to -6℃, and the centrifugation rate is 12000-15000 rpm;

[0030] Further, the simvastatin albumin nanoparticles are added with a freeze-drying protective agent to prepare a freeze-dried powder of simvastatin albumin nanoparticles.

[0031] Specifically comprising the following steps:

[0032] (1) The simvastatin albumin nanoparticles are reconstituted with deionized water to obtain a simvastatin albumin nanoparticle solution;

[0033] (2) The solution is transferred to a Schlenk flask, and a freeze-drying protective agent is added to the solution, and the freeze-drying protective agent is completely dissolved by gently shaking if necessary.

[0034] (3) The solution of step (2) is frozen in a ultra-low temperature refrigerator at -70 to -80℃ for 2-3 days, and then quickly transferred to a freeze dryer at -60℃ for drying for 2-3 days to obtain a freeze-dried powder of simvastatin albumin nanoparticles.

[0035] 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 1-5%, preferably 3-5%, preferably the freeze-drying protective agent is 3-5% mannitol.

[0036] 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.

[0037] The tumor is breast cancer, liver cancer, ovarian cancer, colorectal cancer, preferably colorectal cancer.

[0038] Compared with the prior art, the present application has the following advantages:

[0039] 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.

[0040] 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.

[0041] Simvastatin albumin nanoparticles release faster than simvastatin in a simulated in vivo environment, with a significant increase in cumulative release rate. It is proved that the nanoparticle dosage form wrapped by serum albumin improves the physical properties of simvastatin, improves the in vitro release, and further improves the in vivo absorption.

[0042] 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.

[0043] 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

[0044] Figure 1 is a transmission electron microscopy image of SV-BSA-NPs; the scale is 200 nm;

[0045] Figure 2 is the in vitro release curve of SV, SV-BSA-NPs;

[0046] Figure 3 is the inhibitory effect of SV, SV-BSA-NP on human colon cancer cells (HCT116) and the inhibition curve;

[0047] A, D: 24h B, E: 48h C, F: 72h Mean±SEM, n=3

[0048] Figure 4 is the inhibition effect and inhibition curve of SV, SV-BSA-NP on human colon cancer cells (HT29);

[0049] A, D: 24h B, E: 48h C, F: 72h

[0050] Mean ± SEM, n = 3 *P<0.05; ****P<0.0001

[0051] Figure 5 is the inhibition effect and inhibition curve of SV, SV-BSA-NP on human colon cancer cells (SW480);

[0052] A, D: 24h B, E: 48h C, F: 72h

[0053] Mean ± SEM, n = 3

[0054] Figure 6 is the inhibition effect and inhibition curve of SV, SV-BSA-NP on mouse colon cancer cells (CT26);

[0055] A, D: 24h B, E: 48h C, F: 72h

[0056] Mean ± SEM, n = 3 *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001

[0057] Figure 7 is the inhibition effect and inhibition curve of SV, SV-BSA-NP on human ovarian cancer cells (OVCAR3);

[0058] A, D: 24h B, E: 48h C, F: 72h Mean ± SEM, n = 3

[0059] Figure 8 is the inhibition effect and inhibition curve of SV, SV-BSA-NP on human hepatoma cells (HuH-7);

[0060] A, D: 24h B, E: 48h C, F: 72h

[0061] Mean ± SEM, n = 3 *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001

[0062] Figure 9 is the change of SV, SV-BSA-NP on human breast cancer cell (MCF7) cell viability;

[0063] A: 24h B: 48h C: 72h

[0064] Figure 10 is the effect of SV, SV-BSA-NP, Blank-BSA-NP on normal cell viability;

[0065] A-C: NIH-3T3 cells A: 24h B: 48h C: 72h

[0066] D-F: HEK293 cells D: 24h E: 48h F: 72h

[0067] Figure 11 is the effect of SV, SV-BSA-NP concentration on mouse colon cancer cell (CT26) cell scratch healing ability;

[0068] A~H: Scratch photos before and after administration of different concentrations of SV for 24h;

[0069] I~P: Scratch photos before and after administration of different concentrations of SV-BSA-NP for 24h;

[0070] Figure 12 is the relationship between incubation of different concentrations of SV, SV-BSA-NP and mouse colon cancer cell (CT26) cell scratch healing rate;

[0071] Mean ± SEM; n = 3, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

[0072] Figure 13 is the effect of SV-BSA-NPs on mouse colon cancer cell (CT26) cell apoptosis induction for 24h;

[0073] A: Comparison chart of apoptosis rates at different administration concentrations;

[0074] B: Comparison chart of early apoptosis rates at different administration concentrations;

[0075] C: Comparison chart of late apoptosis rates at different administration concentrations;

[0076] n = 3, Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

[0077] Figure 14 is the effect of SV and SV-BSA-NPs at different concentrations on mouse colon cancer cell (CT26) cell late apoptosis induction;

[0078] A: Apoptosis flow cytometry graphs of negative control, SV, SV-BSA-NP, and statistical bar chart of late apoptosis rates (from left to right) at administration concentration of 2.5 μΜ.

[0079] B, C: Apoptosis flow cytometry graphs and statistical bar chart of late apoptosis rates at administration concentrations of 5 μΜ and 10 μΜ, respectively;

[0080] Mean ± SEM n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001;

[0081] Figure 15 is the qualitative analysis results of the uptake of Cou6-BSA-NPs by CT26 cells at different administration times;

[0082] Green fluorescence is the spontaneous fluorescence of Cou6-SV-BSA-NPs, and blue fluorescence is DAPI staining of cell nuclei; the scale is 100 μm.

[0083] Figure 16 is a graph of the tumor volume of mice in each administration group versus time;

[0084] Mean ± SEM n = 8; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001;

[0085] Figure 17 is a graph of the tumor tissue and weight of the in vivo anti-mouse CT26 colon cancer tumor efficacy experiment of SV-BSA-NPs;

[0086] A: is the tumor tissue picture of each group of mice, and B is the tumor weight column chart (n = 8);

[0087] *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001

[0088] Figure 18 is a graph of the body weight of mice in each group versus time after drug treatment;

[0089] Figure 19 is the blood biochemical liver and kidney index situation of mice in each group after drug treatment

[0090] A: glutamic-oxaloacetic transaminase (AST); B: glutamic-pyruvic transaminase (ALT); C: urea nitrogen (BUN); D: creatinine.

[0091] Figure 20 is a representative graph of H&E staining sections of the main organs of mice in each administration group after drug treatment, with a scale of 50 μm. DETAILED DESCRIPTION

[0092] Example 1:

[0093] Preparation of simvastatin serum albumin nanoparticles (SV-BSA-NPs) by nanoemulsion solvent evaporation technology

[0094] The nano-particles prepared by nano-emulsification solvent evaporation technology have smaller particle size, more uniform distribution and higher encapsulation rate. In the preparation process, different preparation conditions affect the particle size, polydispersity index (PDI) and encapsulation rate of SV-BSA-NPs, and further affect the in-vitro release and in-vivo efficacy of BA-BSA-NPs. The optimal simvastatin serum albumin nano-particles are obtained by investigating different preparation conditions.

[0095] Simvastatin albumin nano-particles (SV-BSA-NPs) are prepared according to the following method:

[0096] BSA is precisely weighed and dissolved in distilled water to prepare an aqueous phase. SV is precisely weighed and dissolved in dichloromethane to prepare a drug-containing oil phase. The drug-containing oil phase is slowly added to the aqueous phase, vortexed for 5 min, and then transferred to an ultrasonic cell crusher. The power is 300-400 W, and ice bath ultrasonic treatment is performed for 30 min. The ultrasonic treatment is 2 s and the pause is 1 s. An O / W emulsion is obtained. The O / W emulsion is subjected to rotary evaporation under reduced pressure at 40 ℃ and 30 r / min for 6 min to remove the organic solvent. Then, the volume is adjusted with deionized water. Centrifugation is performed at 4 ℃ and 12000 rpm to obtain SV-BSA-NPs.

[0097] Table 1 Effect of different preparation conditions on the particle size, PDI and encapsulation rate of SV-BSA-NPs

[0098] The results of schemes 1-5 show that the particle size of the nano-particles prepared by schemes 4 and 5 is too large, which is not suitable for further preparation of qualified nano-particles. When the volume ratio of the aqueous phase to the drug-containing oil phase is 2:1 and 5:1, the particle size of the obtained nano-particles is not qualified, and the encapsulation rate is not considered. Therefore, the volume ratio of the aqueous phase to the drug-containing oil phase should be at least 10:1. When the BSA concentration is 5 mg / mL, the SV concentration is 2.5-5 mg / mL, the volume ratio of the aqueous phase to the drug-containing oil phase 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 rate is greater than 70%. The smaller the particle size of the nano-particles, the better the in-vitro release and in-vivo effect. Therefore, considering the particle size, PDI and encapsulation rate, the technical solution of scheme 3 is preferred in the present application, i.e. the BSA concentration is 5 mg / mL, the SV concentration is 2.5 mg / mL, the volume ratio of the aqueous phase to the drug-containing oil phase 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 rate is greater than 70%.

[0099] Example 2: Selection of freeze-drying protectant for SV-BSA-NPs

[0100] SV-BSA-NPs were prepared according to the procedure of Example 1, Scheme 3. SV-BSA-NPs were lyophilized without any cryoprotectant. After lyophilization, the particle size and PDI of the reconstituted SV-BSA-NPs were measured. The results are shown in Table 2.

[0101] Table 2. Particle size of SV-BSA-NPs before and after lyophilization

[0102] The results show that the particle size of the nanoparticles changed during the lyophilization process. It is speculated that some of the nanoparticles were affected by the drastic change in temperature during the pre-freezing or drying process, resulting in drug leakage.

[0103] To improve the stability of SV-BSA-NPs, 5% of a cryoprotectant was added before lyophilization. The cryoprotectants were mannitol, trehalose, or sucrose, respectively.

[0104] The prepared SV-BSA-NPs were collected and reconstituted with the prescribed amount of deionized water to obtain a SV-BSA-NPs solution. The solution was transferred to a Schlenk flask. A certain amount of cryoprotectant was added to the solution, and if necessary, the cryoprotectant was completely dissolved by gently shaking. The solution was pre-frozen at -80°C for 24 h, then quickly transferred to a freeze dryer, and lyophilized for 24 h to obtain SV-BSA-NPs lyophilized powder. The particle size and appearance after lyophilization are shown in Table 3.

[0105] Table 3. Particle size and appearance of samples with added cryoprotectant (n = 3)

[0106] The results in Table 3 show that the sample without added cryoprotectant was prone to clumping after lyophilization, and reconstitution required the assistance of blowing and shaking. The samples with added cryoprotectant had a better appearance, with no collapse on the surface and a smooth surface, and the powder was fluffy. The sample with added mannitol had little change in particle size, similar to the sample without added cryoprotectant, so it was excluded. The samples with added trehalose and sucrose had a smaller particle size after lyophilization than before, and had a better appearance. Therefore, the preferred cryoprotectant is trehalose or sucrose.

[0107] Example 3: Preparation of SV-BSA-NPs

[0108] Precisely take 50 mg BSA and dissolve in 10 mL distilled water, vortex to disperse evenly to get the water phase. Precisely take 2.5 mg SV and dissolve in 1 mL dichloromethane to get the drug-containing oil phase. Slowly drop the drug-containing oil phase into the water phase, vortex for 5 min, then quickly transfer to the ultrasonic cell crusher, ice bath ultrasonic at 400 W for 30 min, 2 s on and 1 s off, to get the O / W emulsion. Remove the organic solvent from the O / W emulsion at 40 °C, 30 r / min under reduced pressure for 6 min, then use deionized water to constant volume, centrifuge at 4 °C at a speed of 12000 rpm to get the SV-BSA-NPs.

[0109] The prepared SV-BSA-NPs have a particle size of 213.7 nm, a polydispersity coefficient of 0.226, and a zeta potential of -31.25 mV. The transmission electron microscope image is shown in Figure 1.

[0110] Example 4: In vitro release of SV-BSA-NPs

[0111] Dissolve a certain amount of SV and an equal amount of SV-BSA-NPs (Example 3) in 2 mL release medium, respectively, and place in a dialysis bag with a molecular weight cut-off of 3.5 kD. Use a mixture of 0.01 mol / L sodium dihydrogen phosphate buffer containing 0.5% sodium dodecyl sulfate (pH adjusted to 7.0 with 50% sodium hydroxide solution) and anhydrous ethanol, with a volume ratio of 20:80, as the release medium. At 37±0.5 °C, 100 r / min, and a release medium volume of 100 mL, study the in vitro release behavior of SV-BSA-NPs, and take samples at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h, and promptly make up the same amount of medium at the same temperature. Plot the sampling time points as the abscissa X and the cumulative release amount as the ordinate Y to get the release curve, and compare with the SV raw material. The calculation method of the above cumulative release rate is: 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 2.

[0112] The results show that the cumulative release of SV is 35.11±2.83% within 24h, 54.24±2.18% at 48h, and 87.67±4.86% at the last sampling before the end of the experiment. It is speculated that this situation is due to the poor water solubility of SV, which is released slowly in vivo. SV is less than 50% released within 24h, and its full release of drug efficacy requires more than 72h. The SV-BSA-NPs synthesized in this study encapsulate SV in BSA, which is hydrophilic, and its water insolubility is improved by BSA, and its hydrophilic performance is greatly increased. The release rate of SV-BSA-NPs is fast at the beginning, and the release rate can reach 52.11±1.73% at 1h, and then the release rate slows down, and the cumulative release rate is 85.48±0.67% at 24h, and the release is nearly complete at 96.61±1.31% at 72h. That is, the release rate of SV is faster within 0-8h, and the cumulative release is 75.57±0.41% at 8h, and then the release rate is slow. It can be seen that the preparation of SV-BSA-NPs can significantly improve the solubility and release of SV, and thus can improve its bioavailability.

[0113] Example 5 CCK8 method for determining the inhibitory effect of SV-BSA-NPs on COC cell survival rate

[0114] The CCK8 method was used to detect the influence of SV-BSA-NPs (Example 3) on the cell survival rate of common COC cell lines and whether it has time dependence and concentration dependence, and compared with SV raw materials.

[0115] The COC cell lines used in the present application are mouse COC cell line CT26, human COC cell lines HCT116, HT-29, and SW480, which are all from the China Academy of Sciences Typical Culture Preservation Committee Cell Library.

[0116] Precisely take 10mg of SV, add 0.4778mL of cell culture grade DMSO for dissolution, use water bath ultrasonic assisted dissolution to obtain a SV stock solution with a concentration of 50mM, and store it in a 4°C refrigerator in the dark.

[0117] Collect SV-BSA-NPs containing 3mg of SV, accurately take 0.1433mL of PBS for reconstitution to obtain a SV-BSA-NPs stock solution with a concentration of 50mM, and store it at 4°C in the dark.

[0118] After obtaining a cell suspension in the logarithmic growth phase and in good condition, 10μL of liquid is counted using a hemocytometer to obtain the cell concentration of the cell suspension, and then the cell suspension is diluted to a specific concentration using complete culture medium. The diluted cell suspension is seeded in a 96-well plate with a density of 5×10 3The cells were cultured overnight at 37°C in 5% CO2. After the cells were completely adherent, complete medium containing a certain concentration of SV and SV-BSA-NPs was added, respectively, 4-6 replicate wells were set in each group, one group of drug-free wells was reserved as a blank control, and one group of wells with only liquid was reserved as a background zero. Incubation was continued in the carbon dioxide incubator for a period of time. After the CCK8 detection solution was added, the operation was noted to avoid light at all times. The CCK8 detection solution was prepared by mixing the liquid in a ratio of V 完全培养基 :V CCK8 = 9:1. After the predetermined incubation time, the drug-containing medium was discarded and the detection solution was added, followed by incubation at 37°C for 40-60 min. The specific incubation time varied depending on the cell line. The instrument was detected. The wavelength of the enzyme marker was set to 450 nm, and the OD value of each well was recorded. Each test was repeated not less than three times. The cell survival rate was calculated according to the following algorithm: Cell viability (%) = (OD 实验 -OD 背景 ) / (OD 空白 -OD 背景 ) x 100%

[0119] The drug concentration and cell survival rate were recorded, and the log of the drug concentration was used as the abscissa X and the cell survival rate as the ordinate Y. The results were statistically analyzed, and the IC 50 value was calculated.

[0120] In this experiment, the absorbance of SV-BSA-NPs at concentrations of 2.5-160 μM and different action times was detected by CCK8 method, and the IC 50 value of the drug was calculated.

[0121] The effect of SV-BSA-NPs on the cell viability of human COC cell line HCT116 is shown in Figure 3.

[0122] The results show that SV-BSA-NPs and SV have an inhibitory effect on HCT116 cells at a drug concentration of 2.5-80 μM, and the inhibitory effect of SV-BSA-NPs is slightly stronger than that of SV. As the concentration increases, the inhibition of cell viability also increases, and the results of SV concentration of 80 μM cultured for 48 h are significantly different (P < 0.001). In the case of 24 h, 48 h, and 72 h of culture, the half-inhibitory concentration of SV-BSA-NPs on HCT116 is 178.3 μM, 55.43 μM, and 19.71 μM, respectively, which is significantly less than the free drug of 254.4 μM, 114.5 μM, and 38.73 μM, respectively, which is 1.43 times, 2.07 times, and 1.96 times, respectively. It can be seen that as the action time increases, the inhibitory effect of SV-BSA-NPs on HCT116 cells is significantly enhanced, and the half-inhibitory concentration of 48 h and 72 h is 3.22 times and 9.05 times that of 24 h, respectively.

[0123] The effect of SV-BSA-NPs on the cell viability of human COC cell line HT29 is shown in Figure 4.

[0124] The results show that the inhibitory effect of SV-BSA-NPs on HT29 is slightly enhanced compared with the original drug SV. There is a significant difference in cell survival rate at a drug concentration of 80 μM for 48 h, and a significant difference at a drug concentration of 20 μM for 72 h. The half-inhibitory concentration at 24, 48, and 72 h is 76.25 μM, 62.04 μM, and 48.65 μM, respectively. It can be seen that the effect of inhibiting cell viability has a time-dependent effect, and the drug efficacy at 48 h and 72 h is 1.16 times and 1.24 times that of the original drug, respectively.

[0125] The effect of SV-BSA-NPs on the cell viability of human COC cell line SW480 is shown in Figure 5.

[0126] The results show that the inhibitory effect of SV-BSA-NPs on SW480 also has concentration dependence and time dependence. The half-inhibitory concentration of SV-BSA-NPs on SW480 is 39.00 μM, 16.91 μM, and 9.036 μM at 24 h, 48 h, and 72 h, respectively, which is slightly less than that of the free drug 48.41 μM, 23.04 μM, and 11.52 μM, respectively, which is 1.24 times, 1.36 times, and 1.27 times, respectively. With the extension of the action time, the half-inhibitory concentration of SV-BSA-NPs at 48 h and 72 h is reduced to 43.36% and 23.17% of that at 24 h, respectively.

[0127] The effect of SV-BSA-NPs on the cell viability of mouse COC cell line CT26 is shown in Figure 6.

[0128] The results show that as the SV concentration increases, the cell viability of CT26 is inhibited, and the inhibitory effect has a tendency to increase, which indicates that the inhibitory effect has a concentration dependence (Figures 6-A, B, and C); as the culture time is extended, the IC 50 value decreases significantly (Figures 6-D and E), indicating that the inhibitory effect has a time dependence. The IC 50 of SV-BSA-NP on CT26 is 5.152 μM, 3.746 μM, and 0.613 μM at 24, 48, and 72 h, respectively, while the IC 50 of SV is 21.51 μM, 4.817 μM, and 1.682 μM, respectively, which indicates that the inhibitory effect of SV-BSA-NPs on CT26 cells is significantly stronger than that of the original drug SV, and the difference is statistically significant at multiple concentrations, and there is a significant difference at an extremely low drug concentration of 0.3125 μM. (P<0.0001)

[0129] Example 6 CCK8 method for determining the inhibitory effect of SV-BSA-NPs on other tumor cells

[0130] 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 China Academy of Sciences Typical Culture Preservation Committee Cell Library.

[0131] The effect of SV-BSA-NPs on the cell viability of OVCAR3 cell line is shown in Figure 7.

[0132] The results show that in the concentration range of 1.25-40 μM, the cell viability is inhibited, and the cell viability gradually decreases, with concentration dependence. From the IC 50 values, the IC 50 of SV-BSA-NPs at 24 h is 38.96 μM, which is much smaller than that of SV (151.5 μM). The inhibitory effect of SV-BSA-NPs is also much stronger than that of free drug at 48 and 72 h, with IC 50 values much lower than the 24 h results, indicating that the toxic effect is time-dependent.

[0133] The effect of SV-BSA-NPs on the cell viability of human hepatoma cell line HuH-7 is shown in Figure 8.

[0134] 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, and the slight inhibitory effect is not found to have concentration dependence. In the results at 48 and 72 h, the inhibitory effect appears to be concentration-dependent, and the IC 50 of SV-BSA-NPs at 72 h is 2.69 times that of SV, and there is a significant difference 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 also proves that the toxic effect is time-dependent.

[0135] The effect of SV-BSA-NPs on the cell viability of human breast cancer cell line MCF7 is shown in Figure 9.

[0136] The results show that in the concentration range of 1.25-40 μM, the inhibitory effect is slightly enhanced with increasing concentration, and at the same drug concentration, the cell viability is more strongly inhibited with the extension of incubation time, such as when the SV-BSA-NPs drug concentration is 20 μM, 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. However, due to the weak inhibitory effect, the results cannot be fitted to obtain the IC 50Values.

[0137] Example 7 CCK8 method for determining the toxicity of SV-BSA-NPs on normal cells

[0138] 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 reconstituted with 0.1433 mL of 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.

[0139] The CCK8 method was used to detect the toxicity of SV-BSA-NPs and the unloaded blank carrier Blank-BSA-NPs on mouse embryonic fibroblast cell line NIH-3T3 and human embryonic kidney cell line HEK293, and the results were compared with the raw material drug SV. The experimental results are shown in FIGS. 10A-C.

[0140] The results show that in the concentration range of 0.3125-120 μM, within the action time range of 24-72 h, the inhibition effect of the blank nanoparticles on the viability of NIH-3T3 cells is weak, proving 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, indicating that the toxicity does not significantly increase after being prepared into a nano dosage form.

[0141] The results of the toxicity test of SV-BSA-NPs and Blank-BSA-NPs on HEK293 cells are shown in FIGS. 10D-F.

[0142] The results show that SV-BSA-NPs have strong toxicity on HEK293 cells, and the survival rate of HEK293 cells is significantly inhibited, and the inhibition effect has no significant difference compared with free drug SV, indicating that the toxicity does not increase during 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 only a significant inhibition at the highest concentration of 160 μM under the three action times, indicating that the toxicity is small.

[0143] Example 8 Cell scratch healing test

[0144] CT26 cells in the logarithmic growth phase were diluted with complete culture medium to 3 x 10 5Cells were seeded at a density of 1 cell / well in six-well plates and cultured at 37°C and 5% CO2 for 24 hours until the cells covered the bottom of the plate. The complete culture medium in the wells was discarded, and the cells were rinsed once with PBS and discarded. Using a 200 μL pipette tip, a shallow vertical line was gently drawn on the bottom of the plate, ensuring the pipette tip was perpendicular to the plate and the drawing motion was gentle and quick. The cells were rinsed again with PBS to remove any detached cells, and the scratch was photographed; this was recorded as 0 h. Then, 2 mL of incomplete culture medium containing SV and SV-BSA-NPs (Example 3) at concentrations of 0.625, 1.25, and 5 μmol / L were added to the wells, respectively. The plates were incubated in CO2 for another 24 hours and photographed. The images were analyzed and processed using ImageJ software, and the scratch healing rate was calculated using the following algorithm: Scratch healing rate = (A... 0h -A 24h ) / A 0h ×100%

[0145] Where A 0h A is the area of ​​the scratch at time 0h. 24h The area of ​​the scratch after 24 hours.

[0146] The effects of SV and SV-BSA-NPs on the migration function of CT26 cells are shown in Figure 11. Figure 11 shows that the scratches tended to heal within 24 hours, with the low-concentration group showing more significant healing than the high-concentration group. Higher drug concentrations resulted in poorer healing and greater inhibition of cell migration.

[0147] ImageJ software was used to process the images and calculate the quantitatively evaluable cell migration rate (Figure 12). The scratch healing rate in the control group was 29.86±3.31%, significantly higher than that at SV-BSA-NP concentrations of 0.625μM, 1.25μM, and 5μM (P<0.0001), which were 16.13±0.94%, 5.97±1.06%, and 2.78±0.56%, respectively. These results indicate that the addition of SV-BSA-NPs can effectively inhibit tumor cell migration even at extremely low concentrations.

[0148] Example 9: Flow cytometry detection of the effect of SV-BSA-NPs on the apoptosis rate of COC cells

[0149] CT26 cells in the logarithmic growth phase were diluted with complete culture medium at 2.5 × 10⁻⁶. 5The cells were seeded in six-well plates at a density of 1 x 105 cells per well and incubated overnight at 37°C in 5% CO2 until the cells were completely adherent. The medium was replaced with complete medium containing a certain concentration of SV and SV-BSA-NPs, and the cells were incubated for 24 h. The cells at the bottom of the six-well plate were collected by using 200 μL of trypsin without EDTA, centrifuged at 300 g at 4°C, the supernatant was discarded, and the cells were resuspended by adding pre-cooled PBS and gently blowing. The washing process was repeated twice, and pre-cooled PBS was used throughout the washing process. After the last washing, the cells were resuspended by adding binding buffer, and 5 μL of Annexin V-FITC and 10 μL of PI dye were added in the dark. The reaction was performed at room temperature for 15 min. Finally, 400 μL of Binding Buffer was added, mixed, placed on ice, and analyzed by flow cytometry within 1 h.

[0150] In this study, FITC / PI double staining was used to study the apoptosis of CT26 cells induced by SV-BSA-NPs. The experiment was processed according to the instructions of the Annexin V-FITC / PI kit. Four concentration gradients of 0, 2.5, 5, and 20 μM were designed. After incubation with the above concentrations of SV-BSA-NPs-containing medium for 24 h, the apoptosis of the cells in each group was detected by flow cytometry. The results are shown in Figure 13. The results show that the proportion of apoptotic cells in each concentration group was 4.40 ± 0.47%, 15.58 ± 4.55%, 20.43 ± 5.14%, and 18.55 ± 4.99%, respectively. The proportion of early apoptotic cells was 0.78 ± 0.11%, 2.12 ± 1.27%, 3.13 ± 3.07%, and 2.16 ± 1.66%, respectively. The proportion of late apoptotic cells was 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 increased and was significantly higher than that of the control group, indicating that SV-BSA-NPs had excellent ability to induce apoptosis of CT26 cells and was positively correlated with the concentration.

[0151] Example 10 Comparison of the Effects of SV and SV-BSA-NPs on the Apoptosis of CT26 Cells

[0152] The ability of SV-BSA-NPs and SV to induce apoptosis of CT26 cells was compared at different concentration levels by the same method. The results and statistical chart are shown in Figure 14.

[0153] The results show that: at the concentration of 2.5 μM, the late apoptosis rate of the cells treated by SV-BSA-NPs is significantly greater than that of the negative control group (P<0.01), wherein the SV-BSA-NP is 13.77±4.30%, and the negative control is 3.61±0.57%. At the concentration of 20 μM, the late apoptosis rate of the cells treated by SV-BSA-NPs is significantly greater than that of the negative control group (P<0.01), wherein the SV-BSA-NP is 18.55±4.99%, and the negative control is 4.40±0.47%. In the three groups of drug concentrations, the late apoptosis rate of the cells treated by SV-BSA-NPs is significantly greater than that of the negative control group; at the concentration of 5 μM, the late apoptosis rate of the cells treated by SV-BSA-NPs is significantly greater than that of SV (P<0.001), wherein the SV is 10.57±1.49%, and the SV-BSA-NPs is 17.70±2.19%. With the gradual increase of the concentration, the difference between SV-BSA-NPs and SV in apoptosis is more and more significant. The above results show that the use of BSA to embed SV can improve the ability of inducing tumor cell apoptosis to a certain extent, and has a concentration-dependent property.

[0154] Example 11 Laser confocal microscope study on the uptake of SV-BSA-NPs by COC cells

[0155] 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 coumarin 6 stock solution was mixed with SV dichloromethane solution, and then coumarin 6 SV-BSA-NPs were prepared according to the optimal prescription. Note that the preparation process is in the dark.

[0156] 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.

[0157] 100 μL of the above solutions were respectively placed in a 96-well plate, and the fluorescence intensity was measured using an enzyme marker. The measurement conditions were set as λ ex = 450 nm and λ em = 505 nm. The standard curve graph 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.

[0158] CT26 cells in the logarithmic growth phase were diluted with complete culture medium to 1.5×10 5The cells were inoculated at a density of 1 x 105 / mL in a glass bottom confocal dish and placed in a cell incubator for 24 h to allow the cells to adhere completely. A certain concentration of Cou6-SV-BSA-NPs was added under light shielding conditions, and the incubation was continued under light shielding at 37°C and 5% CO2 for a period of time. After the incubation, the liquid in the dish was discarded, and the dish was washed with pre-cooled PBS three times. Then, 200 μL of 4% paraformaldehyde solution was added to the dish, and the dish was fixed under light shielding for 20 min. After washing with PBS three times, sufficient DAPI was added to the dish, and the dish was incubated under light shielding 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 FIG. 15.

[0159] 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 during preparation 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 over time within 24 h after administration. This experiment shows that SV-BSA-NPs can be successfully taken up and enriched by tumor cells, laying the foundation for the anti-tumor efficacy of SV-BSA-NPs.

[0160] Example 12 In vivo Anti-colon Cancer Efficacy of SV-BSA-NPs

[0161] Experimental animals: SPF grade BALB / c mice, five weeks old, female, weighing 15 ± 2.5 g, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. This experiment was approved by the China Medical University Experimental Animal Welfare Ethics Committee, number: CMU20231157.

[0162] 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, Chinese Academy of Sciences.

[0163] Experimental method:

[0164] CT26 cells in the logarithmic growth phase were selected, and a small amount of trypsin containing EDTA was used for digestion. The liquid was centrifuged, counted, and the basic medium was supplemented to prepare a certain concentration of basic medium cell suspension. The suspension was stored at 4°C in the dark and was not shaken. The suspension was blown evenly with a pipette gun in time and was inoculated into experimental animals within 2 h.

[0165] Female BALB / c mice were bred in a barrier environment for a period of time. When the experimental animals were fully adapted to the barrier environment and weighed 20±2 g, 1×10 6 CT26 cells were injected subcutaneously into the back of each mouse. After inoculation, the mice were observed regularly for growth and tumor volume was measured. When the tumor volume reached 100 mm 3 , the model was successfully established and the next experiment could be performed.

[0166] The tumor volume of the mice was calculated as follows: Tumor volume (mm 3 ) = 0.5 × (longest diameter × shortest diameter 2

[0167] The tumor-bearing mice were bred in a barrier environment, and the environment was kept at 20-22°C, 55% humidity, and a 12-hour alternating cycle of day and night, and was observed regularly. When the tumor volume reached 100 mm3, the tumor-bearing mice were randomly divided into 4 groups, 8 in each group. The 4 experimental groups were: saline group (Saline), blank BSA nanoparticle group (Blank-BSA-NPs), SV solution group (SV), and SV-BSA-NPs group (Example 3 SV-BSA-NPs).

[0168] When the tumor volume reached the required value, the mice were randomly divided into groups, and 1 day after the grouping, the drug administration was started. The SV was administered at a dose of 4 mg / kg by tail vein injection of the corresponding drug, and the injection volume was 100 μL. The administration frequency was once every 2 days, and the administration was continued for 10 days. The mice were continuously observed and the tumor volume was recorded, and the measurement frequency was once every 2 days. The experiment was terminated on the 14th day after the start of administration, and 500-1000 μL of whole blood was taken by enucleation under anesthesia, and after standing, the serum was centrifuged and stored in a -80°C ultra-low temperature refrigerator for subsequent analysis of blood biochemical indicators of liver and kidney function. 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 tumor, heart, liver, spleen, lung, and kidney were removed and immediately immersed in 4% paraformaldehyde and stored at 4°C for subsequent histological analysis.

[0169] Example 13 Pharmacodynamic evaluation of SV-BSA-NPs

[0170] 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 schedule, and after the administration was completed, the mice were bred for a period of time under the same environmental conditions, and the tumor volume was measured and recorded at the same frequency. The mice were euthanized on day 14, and the tumor tissue was removed and weighed. The tumor volume from day 1 of administration to day 13 before euthanasia was recorded, and the tumor volume-time relationship diagram of each group was obtained as shown in FIG. 16.

[0171] ​The results show that the saline group and the Blank-BSA-NPs group have similar tumor volume growth trends, and the prepared Blank-BSA-NPs have no inhibitory effect on tumor cells. Compared with the other two groups, the tumor grows faster and the tumor volume is larger. On the 13th day, the Blank-BSA-NPs group and the SV-BSA-NPs group have a significant difference (P<0.0001), and the 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 similar trends before the 5th administration, and the tumor volume is similar. The treatment effect has no difference, and the difference gradually increases during the observation period after the administration ends. The tumor volume growth trend of the SV-BSA-NPs group is significantly slower than that of the SV group. On the 13th day, the tumor volume of the SV group and the SV-BSA-NPs group has a significant difference (P<0.01), indicating that the SV-BSA-NPs have a significantly better effect on COC than the SV free drug.

[0172] On the 14th day, the tumor tissue was dissected, washed with saline, weighed, and photographed. The tumor tissue picture of FIG. 17A was obtained, and the histogram of FIG. 17B was obtained by counting the weight. The saline group has the largest tumor weight, the Blank-BSA-NPs administration group is slightly smaller, and the SV-BSA-NPs administration group has the smallest tumor weight, which is statistically different from the SV group, the Blank-BSA-NPs group, and the saline group, indicating that the BSA-loaded SV nanoparticles have superior anti-COC tumor performance.

[0173] Example 14 In vivo toxicity evaluation of SV-BSA-NPs

[0174] The body weight of the mice was measured and recorded at a frequency of once every 2 days throughout the experiment, and the mouse body weight-time relationship graph of FIG. 18 was obtained.

[0175] The body weight of the mice is related to the tumor volume on the one hand, and also reflects the systemic toxicity of the administration scheme on the other hand. As can be seen from the figure, the body weight trends of the four administration groups are similar throughout the experiment, and there is no significant difference. On the 13th day, the average body weight of each group was 21.72±0.80g, 21.56±0.94g, 21.32±1.99g, and 22.29±0.45g, respectively. It can be seen that the systemic toxicity of the mice treated with SV at a dose of 4mg / kg for colon cancer is small, and the addition of Blank-BSA-NPs has little effect on the systemic toxicity, which again proves the superiority of BSA-wrapped drugs for delivering anti-tumor drugs.

[0176] After the observation, blood samples were taken for liver and kidney function biochemical index analysis, and the results are shown in Figure 19. The four detection indexes respectively reflect the liver and kidney function of the mice, and the index values of each group are not completely the same, but there is no statistical difference. It shows that Blank-BSA-NPs and SV-BSA-NPs have no toxic damage to the liver and kidney function of mice, and the prepared SV-BSA-NPs have less toxicity in vivo and better safety.

[0177] After the experiment, the main organs of the mice were taken for hematoxylin-eosin staining of the tissue sections, and the results are shown in Figure 20. It can be seen that there is no obvious difference in the histology of the main organs of the mice in each group, and the administration scheme does not produce significant toxicity to the main organs of the mice, and the prepared SV-BSA-NPs have less toxicity to each organ.

Claims

1. Simvastatin albumin nanoparticles, characterized in that, It consists of simvastatin and serum albumin, with a mass ratio of simvastatin to serum albumin of 1:10-20.

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

3. The method for preparing simvastatin albumin nanoparticles according to claim 1 or 2, characterized in that, Includes the following steps: (1) Preparation of O / W emulsion: (a) Preparation of aqueous phase: Dissolve serum albumin in deionized water to prepare aqueous phase; (b) Preparation of drug-containing oil phase: Simvastatin was dissolved in the organic phase to prepare the drug-containing oil phase; (c) The drug-containing oil phase is added dropwise to the aqueous phase, and the addition is vortexed after the addition is completed; after the vortexing is completed, the mixture is immediately sonicated for a period of time under an ice bath with a probe of specific power and working interval to obtain an O / W emulsion; (2) Preparation of albumin nanoparticles The organic solvent in the O / W emulsion obtained in step (1) was removed by rotary evaporation under reduced pressure, and then diluted with deionized water and centrifuged to obtain simvastatin albumin nanoparticles.

4. The preparation method according to claim 3, characterized in that, In step (a), the concentration of serum albumin is 2.5–5 mg / mL, preferably 5 mg / mL.

5. The preparation method according to claim 3, characterized in that, In step (b), the organic phase is dichloromethane; the concentration of simvastatin 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 the aqueous phase in step (a) to the drug-containing oil phase in step (b) is 15:1 to 10:1, preferably 10:

1.

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

8. Simvastatin albumin nanoparticle lyophilized powder, characterized in that, The simvastatin albumin nanoparticles according to claim 1 or 2 are added to a lyophilization protectant to prepare a lyophilized powder.

9. The use of simvastatin albumin nanoparticles according to claim 1 or 2 or the lyophilized powder of simvastatin albumin nanoparticles according to claim 8 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumors mentioned are breast cancer, liver cancer, ovarian cancer, and colorectal cancer, with colorectal cancer being the preferred type.

Images