Lovastatin liposome, preparation method therefor, and use thereof

Abstract

A lovastatin liposome, a preparation method therefor, and use thereof in the preparation of an anti-tumor drug. The liposome comprises: lovastatin, phospholipid, cholesterol, and sodium dodecyl sulfate, wherein the mass ratio of lovastatin to phospholipid is 1:15 to 1:30, the mass ratio of cholesterol to phospholipid is 1:5 to 1:20, and the mass ratio of lovastatin to sodium dodecyl sulfate is 1:0.5-1. The lovastatin liposome has a small particle size, high encapsulation efficiency, good stability, and a certain sustained-release effect. The liposome not only can improve the bioavailability of the drug, but also has a significant inhibitory effect on tumor cells, especially in the colorectum, where the inhibitory effect is the best.

Description

Liposome of lovastatin, preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a liposome of lovastatin, a preparation method and application thereof, in particular to the liposome of lovastatin, a preparation method and application thereof in preparation of an antitumor drug. BACKGROUND

[0002] Worldwide, cancer is a major obstacle to human health, and its mortality rate is only second to that of cardiovascular disease. Colorectal cancer (CRC) includes colon cancer (COC) and rectal cancer (RC), accounting for about 11% of all cancers, ranking third in the world in terms of incidence (6.1%), but ranking second in terms of mortality (9.2%), and is a great challenge in the field of cancer today. In countries with rapid economic development, the incidence of CRC has risen sharply, and it is estimated that the number of global CRC deaths will increase by about 60% by 2035.

[0003] Lovastatin (LV) is a natural product isolated from the fermentation broth of Penicillium citrinum by Japanese microbiologist Endo in 1970, which can inhibit the conversion of hydroxy-methylglutaryl coenzyme A (HMG-CoA) to mevalonic acid (MVA) by competitively blocking the active site of HMG-CoA, thereby reducing cholesterol synthesis. By reducing the source of cholesterol biosynthesis and inducing changes in low-density lipoprotein receptor expression, LV can reduce plasma cholesterol levels, and is the first statin approved by FDA for the treatment of hypercholesterolemia and cardiovascular disease.

[0004] The chemical name of lovastatin is (S)-2-methylbutanoic acid-(1S,3S,7S,8S,8aR) 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-{2-[(2R,4R)-4-hydroxy-6-oxo-2-tetrahydropyranyl]-ethyl}-1-naphthalene ester, the molecular formula is C 24 H 36 O5, and the molecular weight is 404.55. Lovastatin is a white powder, easily soluble in acetone, acetonitrile and ethanol, and almost insoluble in water.

[0005] At present, the dosage forms of lovastatin on the market include tablets, capsules, sustained-release tablets, etc. Due to poor water solubility, liver first-pass effect, oral absorption rate of only 31%, in-vivo elimination half-life of only 3h, short half-life drug, and low bioavailability, the clinical application of lovastatin is limited. Therefore, a suitable drug delivery system is needed, which can not only prolong the half-life of the drug, but also achieve the effect of increasing efficacy and reducing toxicity.

[0006] Liposomes are colloidal spherical structures formed by self-assembly of phospholipid molecules, which have both hydrophilic groups and two non-polar hydrophobic chains. Due to the multifunctionality, high biocompatibility, biodegradability, non-toxicity and non-immunogenicity of the structure, liposomes have become an excellent drug delivery system. Phospholipids exhibit amphiphilicity in solution, and their structure is similar to that of biological membranes, so liposomes can interact with cell membranes in the human body, effectively promoting the uptake of liposomes by cells.

[0007] There is no report in the prior art on the preparation of lovastatin into liposomes and its use in anti-tumor, especially anti-colorectal cancer. SUMMARY

[0008] In order to overcome the defects of the prior art, the present application provides a lovastatin liposome (LV-Lips), which has small particle size, high encapsulation efficiency and good stability, and has a certain sustained-release effect, and can increase the uptake ability, cytotoxicity, inhibit cell migration and induce apoptosis of tumor cells.

[0009] The present application is realized by the following technical solutions:

[0010] The present application provides a lovastatin liposome, which comprises: lovastatin, phospholipid, cholesterol, and sodium dodecyl sulfate.

[0011] The mass ratio of lovastatin to phospholipid is 1:15-1:30, preferably 1:18-1:1:25.

[0012] The mass ratio of cholesterol to phospholipid is 1:5-1:20, preferably 1:10-1:20.

[0013] The mass ratio of lovastatin to sodium dodecyl sulfate is 1:0.5-1.

[0014] The phospholipid is selected from the group consisting of egg yolk lecithin, hydrogenated soybean phospholipid, egg yolk phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, soybean lecithin, egg yolk sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol.

[0015] Further, the lovastatin liposome contains 4mg of lovastatin, 60-120mg of phospholipid, 3-24mg of cholesterol and 2-4mg of sodium dodecyl sulfate per 5mL of the liposome.

[0016] Preferably, the lovastatin liposome contains 4mg of lovastatin, 72-100mg of phospholipid, 3.6-10mg of cholesterol and 2-4mg of sodium dodecyl sulfate per 5mL of the liposome.

[0017] More preferably, the lovastatin liposome contains 4mg of lovastatin, 72-80mg of phospholipid, 4-8mg of cholesterol and 2mg of sodium dodecyl sulfate per 5mL of the liposome.

[0018] Further, the present application provides a preparation method of the lovastatin liposome, which comprises the following steps:

[0019] (1) weigh the prescribed amount of phospholipid, cholesterol, lovastatin and sodium dodecyl sulfate in a tomato-shaped flask, add anhydrous ethanol, vortex, and dissolve by ultrasonic for 10min.

[0020] (2) form a thin film of the lipids in the flask by vacuum rotary evaporation, and continue vacuum rotary evaporation for 1h to obtain a uniform thin film.

[0021] (3) add deionized water, vortex, and hydrate after ultrasonic, and finally obtain a lovastatin liposome solution by probe ultrasonic at 300-400W.

[0022] In the step (1), the concentration of the phospholipid in the anhydrous ethanol is 12-16mg / mL.

[0023] In the step (3), the hydrating temperature is 50-60℃.

[0024] In the step (3), the hydrating time is 30-60min.

[0025] In the step (3), the probe ultrasonic is 10-20min, working for 2s and stopping for 1s.

[0026] The lovastatin liposome of the present application can be prepared into a lovastatin liposome lyophilized powder with a lyophilization protective agent selected from one or more of mannitol, sucrose and trehalose. The lyophilization protective agent is preferably 3-7% of trehalose or sucrose.

[0027] The present application also provides the use of the lovastatin liposome or the lovastatin liposome lyophilized powder in improving the bioavailability of drugs.

[0028] The present application also provides the use of the lovastatin liposome or the lovastatin liposome lyophilized powder in preparing anti-tumor drugs.

[0029] The tumor is colorectal cancer, liver cancer, ovarian cancer, breast cancer, preferably colorectal cancer.

[0030] The beneficial technical effects of the present application are:

[0031] The present application provides a kind of lovastatin liposome, the preparation method of the liposome is simple, and the prepared liposome can improve the bioavailability of lovastatin. In this study, lovastatin liposome is successfully prepared by thin film dispersion method, which is simple in operation and good in repeatability. The prepared lovastatin liposome has small particle size, high encapsulation efficiency, good stability, and also has certain sustained-release effect. It has the effects of increasing tumor cell uptake capacity, cytotoxicity, inhibiting cell migration and inducing apoptosis on COC and other tumor cells, which provides a new research idea for lovastatin liposome as an anti-tumor therapeutic drug. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a transmission electron microscope picture of lovastatin liposome with a magnification of 60000, and the scale is 50nm;

[0033] Figure 2 is the in vitro release curve of lovastatin and lovastatin liposome n=3, ****P<0.0001;

[0034] Figure 3 is the inhibition of lovastatin and lovastatin liposome on CT26 cell viability and IC 50 value;

[0035] A, C, E are the cell survival rates at 24h, 48h and 72h respectively; B, D, F are the IC 50 values at 24h, 48h and 72h respectively; n=3, *P<0.05, **P<0.01, ***P<0.001;

[0036] Figure 4 is the inhibition of lovastatin and lovastatin liposome on HCT116 cell viability and IC 50 value;

[0037] A, C, E are the cell survival rates at 24h, 48h and 72h respectively; B, D, F are the IC 50 values at 24h, 48h and 72h respectively; n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0038] Figure 5 is the inhibition of lovastatin and lovastatin liposome on HT29 cell viability and IC 50 value;

[0039] A, C, E are cell viability of 24h, 48h, 72h, respectively, B, D, F are IC50 value of 24h, 48h, 72h, respectively 50 n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

[0040] Figure 6 is the inhibition of HuH7 cell viability by lovastatin and lovastatin liposomes and IC50 values 50

[0041] A, B, C are cell viability of 24h, 48h, 72h, respectively; D, E, F are IC50 value of 24h, 48h, 72h, respectively 50 n = 3, **P < 0.01

[0042] Figure 7 is the inhibition of MCF-7 cell viability by lovastatin and lovastatin liposomes and IC50 values 50

[0043] A, C are cell viability of 48h, 72h, respectively; B, D are IC50 value of 48h, 72h, respectively 50

[0044] n = 3, *P < 0.05, **P < 0.01

[0045] Figure 8 is the inhibition of OVCAR3 cell viability by lovastatin and lovastatin liposomes and IC50 values 50

[0046] A, C are cell viability of 48h, 72h, respectively; B, D are IC50 value of 48h, 72h, respectively

[0047] n = 3, *P < 0.05, **P < 0.01, ***P < 0.001

[0048] Figure 9 is the effect of blank liposomes Blank-Lips on HEK-293 cell viability

[0049] A, B, C are cell viability of 24h, 48h, 72h, respectively (n = 3)

[0050] Figure 10 is the uptake of COU-6-containing lovastatin liposomes in CT26 cells at different times (n = 3)

[0051] Green fluorescence represents COU6-labeled LV-Lips, blue fluorescence represents DAPI-stained nuclei, and the scale bar is 50 μm

[0052] ​​​​Figure 11 is the effect of lovastatin and lovastatin liposomes on CT26 cell scratch healing at different times (n = 3);

[0053] Figure 12 is the effect of lovastatin and lovastatin liposomes on CT26 cell scratch healing rate at different times in CT26 cells;

[0054] n = 3, **P < 0.01, ****P < 0.0001;

[0055] Figure 13 is the effect of lovastatin and lovastatin liposomes on HCT116 cell scratch healing at different times;

[0056] Figure 14 is the effect of lovastatin and lovastatin liposomes on HCT116 cell scratch healing rate at different times;

[0057] n = 3, **P < 0.01, ****P < 0.0001;

[0058] Figure 15 is the effect of lovastatin and lovastatin liposomes on CT26 cell apoptosis rate detected by flow cytometry after 48h treatment (n = 3);

[0059] Q1 represents cells with incomplete cell membranes, Q2 represents late apoptotic cells, Q3 represents live cells, and Q4 represents early apoptotic cells;

[0060] Figure 16 is the effect of lovastatin and lovastatin liposomes of different concentrations on the total apoptosis rate of CT26 cells (n = 3);

[0061] Figure 17 is the effect of lovastatin and lovastatin liposomes on the morphology of CT26 cells;

[0062] A, B, C are blank control group, lovastatin at a dosage of 30μM, and lovastatin liposomes at a dosage of 30μM, respectively. DETAILED DESCRIPTION

[0063] Example 1: Formulation screening of lovastatin liposomes

[0064] Take lovastatin 4mg, cholesterol 4mg, egg yolk lecithin, and SDS 2mg, and accurately weigh them in a 50mL tomato flask. Add 5mL anhydrous ethanol, vortex, and dissolve in a water bath ultrasonic for 10min to make it fully dissolved. Place it in a 50℃ water bath rotary evaporator to remove the organic solvent and form a uniform lipid film. Add 5mL deionized water and shake to make the lipid film on the inner wall of the bottle fall off. Ultrasonic in water bath for 5min, hydrate at 50℃ for 60min, ultrasonic power 300W, work 2s, intermittent 1s, ice bath ultrasonic 10min, and pass through 0.22μm filter membrane to obtain lovastatin liposomes.

[0065] Particle size and Zeta potential determination of lovastatin liposome:

[0066] Take 200 μL lovastatin liposome sample, add 3 mL deionized water, mix well and transfer to a cuvette, place in the sample cell and measure the particle size and PDI at room temperature.

[0067] Take 200 μL prepared lovastatin liposome sample, add 3 mL deionized water, mix well and transfer to an electrode cuvette, be careful not to generate bubbles during the process, insert the electrode tip, place it in the sample cell and adjust the particle size meter to Zeta potential mode, and measure the Zeta potential at room temperature.

[0068] Transmission electron microscopy detection of lovastatin liposome:

[0069] Take the prepared lovastatin liposome and dilute it with deionized water to the appropriate concentration, take 10 μL sample and drop it on a copper grid for 1 min, and use filter paper to remove the floating liquid. Dry for a few minutes at room temperature, and perform electron microscopy detection imaging at 80-120 kV. Observe the morphology of the liposome under a transmission microscope and collect images.

[0070] Encapsulation efficiency determination of lovastatin liposome:

[0071] Use the dextran gel microcolumn centrifugation method, fill the swelled dextran gel inside the syringe with two layers of filter paper, centrifuge at 4000 rpm for 5 min, discard the aqueous solution, and repeat several times to ensure that the dextran gel in the syringe is free of water. Add 100 μL lovastatin liposome to the dextran, centrifuge for 5 min, add 100 μL deionized water, centrifuge for 5 min, and repeat 3 times.

[0072] Collect the filtrate, detect, and calculate the encapsulation efficiency:

[0073] Encapsulation efficiency% = W LV-Lips / W 总 x 100%

[0074] W LV-Lips is the drug amount loaded by the liposome, W 总 is the total drug amount of the liposome

[0075] (1) The effect of the ratio of drug to phospholipid (drug-lipid ratio) on the particle size and encapsulation efficiency of lovastatin liposome:

[0076] The drug and phospholipid ratio is adjusted to be 1:15, 1:18, 1:20, 1:25 and 1:30, and the lovastatin liposomes are prepared according to the above method, and the particle size, PDI and encapsulation rate are taken as the evaluation indexes, and the results are shown in Table 1. With the increase of the drug-lipid ratio, the average particle size shows a trend of first decreasing and then increasing in a small range. The small drug-lipid ratio may cause the liposome to be overloaded, and the particle size is large, so the particle size gradually decreases with the increase of the drug-lipid ratio. However, too much lipid may cause drug leakage, and the particle size increases instead, but the change is not obvious. When the drug-lipid ratio is 1:15-1:30, the particle size of the liposome is less than 250 nm, and the encapsulation rate is more than 80%. However, when the drug-lipid ratio is 1:15, the PDI is large, when the drug-lipid ratio is 1:20, the particle size is the smallest, and the encapsulation rate is the largest. Therefore, the drug-lipid ratio of the application is selected to be 1:18-1:30, preferably 1:18-1:25.

[0077] Table 1 Influence of different drug-lipid ratios on particle size, PDI and encapsulation rate (n=3)

[0078] (2) Influence of the mass ratio of cholesterol to phospholipid on the particle size and encapsulation rate of lovastatin liposomes

[0079] The mass ratio of drug to phospholipid is 1:18, the amount of cholesterol is changed, the mass ratio of cholesterol to phospholipid is adjusted to be 1:5, 1:10, 1:15 and 1:20, and the lovastatin liposomes are prepared according to the above method, and the particle size, PDI and encapsulation rate are taken as the evaluation indexes, and the results are shown in Table 2. With the decrease of the amount of cholesterol, the particle size does not change obviously, but the encapsulation rate gradually increases. Cholesterol can adjust the fluidity of the liposome membrane, resist phospholipid oxidation, and appropriate amount of cholesterol is integrated in the lipid membrane, which can make the lipids tightly packed, improve the acyl directional arrangement order, increase the thickness of the lipid bilayer, reduce the membrane permeability, and maintain the stability of the liposome. However, too high content of cholesterol may cause the liposome to rupture, resulting in poor stability and increased leakage of the liposome. Therefore, the mass ratio of cholesterol to phospholipid is selected to be 1:5-1:20, preferably 1:10-1:20, and more preferably 1:20.

[0080] Table 2 Influence of the mass ratio of cholesterol to phospholipid on particle size, PDI and encapsulation rate (n=3)

[0081] (3) Influence of different organic solvents on the particle size, encapsulation rate and stability of lovastatin liposomes

[0082] The lovastatin liposomes were prepared by the above method with the mass ratio of drug and phospholipid being 1:18, the mass ratio of cholesterol and phospholipid being 1:20, and anhydrous ethanol, dichloromethane or trichloromethane as the solvent, and the particle size, PDI and encapsulation rate were used as the evaluation indexes, and the results are shown in Table 3.

[0083] Table 3 Influence of different organic solvents on particle size, PDI and stability (n=3)

[0084] (4) Selection of surfactant type

[0085] The lovastatin liposomes were prepared by the above method with the mass ratio of drug and phospholipid being 1:18, the mass ratio of cholesterol and phospholipid being 1:20, and anhydrous ethanol as the solvent, and the type and adding time of surfactant were changed to investigate the influence of the type and adding time of surfactant on particle size, PDI and stability. The results are shown in Table 4, and the stability of SDS as the surfactant is better, and the particle size of the lovastatin liposomes prepared by adding the surfactant before hydration is smaller. The stability of the liposomes can be increased by using poloxamer 407 and SDS as the surfactant, and the particle size can be reduced by selecting a suitable surfactant. Therefore, the present application preferably adds SDS before hydration.

[0086] Table 4 Influence of different surfactants on particle size, PDI and stability (n=3)

[0087] (5) Selection of preparation temperature

[0088] The composition and preparation conditions are the same as those in (4), SDS is added before hydration, and the water bath temperature is changed to 40℃, 50℃ or 60℃ when the organic solvent is removed by rotary evaporation, and the particle size, PDI and stability of the liposomes are investigated. The results are shown in Table 5, and the particle size cannot be measured at 40℃, which may be due to the fact that the organic solvent is not completely volatilized at too low a temperature, so that the liposome film cannot be formed, and the particle size of the lovastatin liposomes prepared at 50℃ and 60℃ is not much different, therefore, the temperature is selected to be 50-60℃. However, considering that high temperature can cause oxidation of phospholipids, leading to instability of the liposomes and drug leakage, 50℃ is selected as the preparation temperature.

[0089] Table 5 Influence of preparation temperature on particle size, PDI and stability (n=3)

[0090] (6) Selection of hydration medium type

[0091] The composition and preparation conditions are the same as (5), SDS is added before hydration, the preparation temperature is selected as 50°C, and the hydration medium is changed to investigate the influence of different hydration media on the particle size, PDI and stability of the liposomes. The results are shown in Table 6. When the solution is hydrated with PBS buffer at pH 7.0, the solution is white and turbid, the particle size is large and unstable, so deionized water is selected as the hydration medium.

[0092] Table 6 Influence of hydration medium on particle size, PDI and stability (n = 3)

[0093] (7) Selection of hydration time

[0094] Hydration refers to the process of hydrating and shedding the lipid film at the phase transition temperature of phospholipids, self-assembling to form a closed spherical structure, i.e. liposomes. The composition and preparation conditions are the same as (6), deionized water is used as the hydration medium, and the hydration time is changed to investigate the influence of different hydration times on the particle size, PDI and stability of the liposomes. The results are shown in Table 7. It can be seen that the hydration time has little effect on the particle size, but the liposomes with longer hydration time are more stable, so the hydration time is determined to be 30-60 min, preferably 60 min.

[0095] Table 7 Influence of hydration time on particle size, PDI and stability (n = 3)

[0096] Example 2 Preparation of lovastatin liposomes

[0097] Take lovastatin 4 mg, cholesterol 4 mg, egg yolk lecithin 80 mg, SDS 2 mg, accurately weigh in a 50 mL tomato bottle, add 5 mL of anhydrous ethanol, vortex, dissolve in a water bath ultrasonic for 10 min to make it fully dissolved, place it in a 50°C water bath rotary evaporator to remove the organic solvent, form a uniform lipid film, add 5 mL of deionized water, shake, make the lipid film on the inner wall of the bottle fall off, water bath ultrasonic for 5 min, hydrate at 50°C for 60 min, ultrasonic power 300 W, work for 2 s and intermittent 1 s, ice bath ultrasonic for 10 min, pass through 0.22 μm filter membrane, get lovastatin liposomes.

[0098] Prepare three batches of lovastatin liposomes according to the above method, the particle size, PDI and encapsulation efficiency of the three batches of samples are shown in Table 8.

[0099] Table 8 Reproducibility of three batches of samples

[0100] Example 3 Preparation of lovastatin liposome lyophilized powder

[0101] The sample of lovastatin liposome prepared by the method of Example 2 was mixed uniformly after adding the freeze-drying protective agent, put into a vial, sealed with a sealing film leaving several small air holes, pre-frozen for 2 h in a refrigerator at -20 °C, frozen for 12 h in a refrigerator at -80 °C, and dried for 24 h in a vacuum freeze-drying apparatus to obtain lovastatin lipid freeze-dried powder.

[0102] Example 4: Selection of freeze-drying protective agent and amount

[0103] The lovastatin liposome was prepared according to the method of Example 2, and the prepared liposome was divided into three groups, each group of four, each 2 mL, and different kinds of freeze-drying protective agents trehalose, mannitol, sucrose were added, with a concentration of 1%, 3%, 5%, 7% (w / v), and then mixed uniformly after complete dissolution in a vial. The sample without adding freeze-drying protective agent and the sample with different protective agents were freeze-dried. After obtaining the freeze-dried powder, the appearance characteristics were observed and recorded, and the results are shown in Table 9. After reconstituting with 2 mL of deionized water, the solution state was observed, and the changes in particle size, PDI and encapsulation rate before and after freeze-drying were measured. The results are shown in Table 10.

[0104] Table 9: Effect of different freeze-drying protective agents on appearance

[0105] Table 10: Effect of different freeze-drying protective agents on particle size

[0106] The results of adding mannitol, trehalose and sucrose with a mass concentration of 1%, 3%, 5% and 7% are shown in Table 10. The particle size before freeze-drying was 121.60 ± 1.45 nm, and the particle size of lovastatin freeze-dried powder without freeze-drying protective agent was 699.50 ± 46.47 nm. Freeze-drying had a greater impact on the particle size of liposomes without protective agent. From the effect on particle size, the addition of 5% and 7% trehalose and sucrose as freeze-drying protective agent was the best. The change in encapsulation rate before and after freeze-drying was used to evaluate the best freeze-drying protective agent. The encapsulation rates before freeze-drying, 5% trehalose, 7% trehalose, 5% sucrose and 7% sucrose were 84.28%, 75.21%, 73.30%, 71.77% and 72.3%, respectively. Considering comprehensively, 5% trehalose was selected as the freeze-drying protective agent to prepare lovastatin liposome freeze-dried powder.

[0107] Example 5: In vitro release of lovastatin liposome of Example 2

[0108] About 8 mg lovastatin was accurately weighed and dissolved in 10 mL 2% SDS / ethanol solution (90:10). Three 2 mL samples of the lovastatin solution were taken and put into dialysis bags, which were then clamped into bottles containing 100 mL release medium. Three 2 mL samples of lovastatin liposome solution with a concentration of 0.8 mg / mL were taken and put into dialysis bags, which were then clamped into bottles containing 100 mL release medium.

[0109] At 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 and 72 h, 1 mL of dialysate was taken and 1 mL of release medium at the same temperature was added. The dialysate was filtered and then analyzed by HPLC. The cumulative release percentage at each time point was calculated, and the release time was taken as the abscissa and the cumulative release percentage as the ordinate to draw the in vitro release curve. The release of lovastatin and lovastatin liposome was compared, and the release model was fitted.

[0110] As shown in Figure 2, the release of lovastatin was relatively rapid within 24 h, and the cumulative release amount reached 44.78 ± 3.35%. At 72 h, the cumulative release amount reached 72.80 ± 3.85%. The release of lovastatin liposome was relatively rapid within 6 h, and the release amount increased slowly after 8 h. The cumulative release amounts at 24 h, 48 h and 72 h were 39.00 ± 1.78%, 42.76 ± 1.79% and 43.72 ± 1.95%, respectively. Compared with lovastatin, lovastatin liposome had a certain sustained-release effect (P < 0.0001).

[0111] After processing the in vitro release data of lovastatin and lovastatin liposome, the data was subjected to regression analysis by zero-order kinetic equation, first-order kinetic equation, Higuchi equation and Ritger-Peppas equation to investigate the in vitro release model of the drug. The results showed that the in vitro release behavior of lovastatin and lovastatin liposome was best fitted to the first-order kinetic model, and the cumulative release amount increased with time. 2 The R values were 0.9976 and 0.9921, respectively (Tables 11 and 12). In the Ritger-Peppas equation, the release index of lovastatin liposome was less than 0.45, which was a Fickian diffusion mechanism, and the release index of lovastatin was greater than 0.45 and less than 0.89, which was a non-Fickian diffusion mechanism.

[0112] Table 11 Mathematical model fitting of in vitro release of lovastatin

[0113] Table 12 Mathematical model fitting of in vitro release of lovastatin liposome

[0114] Example 6 CCK-8 method for detecting the inhibition rate of lovastatin and lovastatin liposomes on different tumor cells

[0115] The COC cells in the logarithmic growth phase were taken out from the incubator, and the cell morphology was observed under a microscope. When the cell density reached more than 80%, a single cell suspension was prepared according to the cell subculture procedure. The cell concentration was 5000 cells / mL by counting with a cell counting plate, and the cell density was 100 μL / well by diluting with the culture medium. PBS solution was added to the last row to reduce the edge effect. After 24 h, the cells were observed to have adhered and grown under a microscope, the original culture medium was discarded by aspiration, and 10, 20, 30, 60, 90, and 120 μM of LV and LV-Lips (Example 2) prepared with complete culture medium were added, respectively. Six replicate wells were set for each concentration, and each group of experiments was performed in triplicate. After 24 h, 48 h, and 72 h, the 96-well plates were taken out, the drug-containing culture medium was carefully aspirated and discarded along the well wall, CCK-8 reagent was added in the dark, and the plates were incubated in the incubator for 30 min. The absorbance value (A) was measured at 450 nm wavelength on a microplate reader. The cell survival rate was calculated according to the following formula: Cell viability = (A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 ) × 100%

[0116] A 实验组 is the absorbance value of different concentrations of drugs, A 空白组 is the absorbance value without inoculation of cells, and A 对照组 is the absorbance value without addition of drugs.

[0117] Complete culture medium = 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin + 89% incomplete culture medium

[0118] CT26 and OVCAR3 cells were cultured with RPMI-1640 complete medium, and HCT116, HT29, HuH7, HEK293, and MCF-7 cells were cultured with DMEM complete medium.

[0119] The effects of lovastatin and lovastatin liposomes on the inhibition rates of CT26, HCT116, HT29, MCF-7, HuH7, and OVCAR3 cells were determined according to the above method, and the half maximal inhibitory concentration (IC 50 ) values were calculated for statistical analysis.

[0120] Example 7 Inhibition rate of lovastatin and lovastatin liposomes on COC cells

[0121] CCK-8 method and IC 50 values of lovastatin and lovastatin liposome (Example 2) on COC cell lines CT26, HCT116, HT29 cells were evaluated. CCK8 kit can quickly and sensitively detect cell proliferation and cytotoxicity. The principle is that in the presence of electronic coupling reagent, WST-8 can be reduced by dehydrogenase in the mitochondria of living cells to form a highly water-soluble orange yellow formazan product, the color depth is proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at 450 nm wavelength by using a microplate reader, which can indirectly reflect the number of living cells.

[0122] The results of lovastatin and lovastatin liposome on CT26 inhibition are shown in Figure 3. Lovastatin and lovastatin liposome at different concentrations have obvious inhibition on the survival rate of CT26 cells, and the inhibition effect is enhanced with the increase of drug concentration and the extension of action time. There is no significant difference in the inhibition effect of the drug on the cells at 24h and 72h, which shows that the preparation into liposome preparation does not affect the function of the drug. The inhibition rate of the low concentration preparation group on CT26 cells is lower than that of the raw material drug group, which may be due to the slow release of the drug wrapped in the liposome.

[0123] For CT26 cells, the IC 50 of lovastatin at 24h, 48h, 72h is 48.35μM, 12.76μM, 13.27μM respectively, and the IC 50 of lovastatin liposome is 47.55μM, 47.76μM, 18.94μM respectively.

[0124] The results of lovastatin and lovastatin liposome on HCT116 inhibition are shown in Figure 4. The inhibition rate of lovastatin liposome on HCT116 is significantly higher than that of lovastatin (P<0.0001). The IC 50 of lovastatin liposome group at 24h and 48h after administration is lower than that of lovastatin, which may be because the liposome membrane is similar in structure to the cell membrane, so it has good affinity, making the liposome preparation better absorbed by cells, so it shows better inhibition effect. After 72h of administration, lovastatin at low concentration has no inhibitory effect on cell activity, and the cells begin to proliferate, but lovastatin liposome has a slow release effect. Therefore, at low concentration, it also has an inhibitory effect on HCT116 cells.

[0125] For HCT116 cells, the IC 50 of lovastatin liposome at 24h, 48h, 72h is 40.47μM, 20.22μM, 52.52μM respectively, and the IC 50 of lovastatin is 132.5μM, 45.73μM, 64.01μM respectively.

[0126] The results of lovastatin and lovastatin liposome inhibition on HT29 are shown in Figure 5. Lovastatin and lovastatin liposome showed similar trends in HT29 cells as in HCT116 cells. Lovastatin liposome also had a significant inhibitory effect on HT29 cells and had a significant difference compared with lovastatin (P < 0.001) and was time- and concentration-dependent.

[0127] For HT29 cells, the IC 50 of lovastatin liposome was 53.67 μM, 33.20 μM, and 36.59 μM at 24 h, 48 h, and 72 h, respectively, and the IC 50 of lovastatin was 91.69 μM, 73.62 μM, and 61.25 μM, respectively, indicating that lovastatin liposome was more effective in HT29 cells.

[0128] The above results show that lovastatin liposome has a better effect on human COC cells HCT116 and HT29 than on mouse COC cells CT26, and has better cytotoxicity than free lovastatin.

[0129] Example 8 Inhibition rate of lovastatin and lovastatin liposome on liver cancer, breast cancer, and ovarian cancer cells

[0130] The cytotoxicity of lovastatin and lovastatin liposome (Example 2) on liver cancer cells HuH7, breast cancer cells MCF-7, and ovarian cancer cells OVCAR3 was detected by CCK-8 method (Figures 6-8). The IC 50 of lovastatin liposome on HuH7 cells was 138.75 μM, 118.85 μM, and 72.39 μM at 24 h, 48 h, and 72 h, respectively, and the IC 50 decreased with the extension of the action time. After lovastatin and lovastatin liposome acted on MCF-7 cells for 48 h, the IC 50 of lovastatin was 23.73 μM, lower than the IC 50 of lovastatin liposome (47.33 μM), but there was no significant difference between the two groups with the increase of the concentration. Lovastatin and lovastatin liposome had similar inhibition rates on the survival of OVCAR3 cells, but there was no statistical difference.

[0131] Example 9 CCK-8 method for detecting the cytotoxicity of Blank-Lips on HEK-293 cells

[0132] The cytotoxicity of blank liposome material on human embryonic kidney cells HEK-293 was investigated by CCK-8 reagent according to the method of Reference Example 6 to preliminarily investigate the safety of liposome preparation raw materials on human embryonic kidney cells.

[0133] Blank-Lips without lovastatin were prepared according to the method of Example 2, and the effect of Blank-Lips on the viability of HEK-293 cells was detected using CCK-8 reagent at 24 h, 48 h, and 72 h. The survival rate of HEK-293 cells treated with high concentrations of Blank-Lips at 72 h was more than 80% (Figure 9), indicating that the carrier material is safe.

[0134] Example 10 Uptake of lovastatin liposomes in CT26 cells

[0135] Coumarin (COU-6) labeled LV-Lips fluorescent liposomes of Example 2 were prepared by dissolving COU-6 in methanol: ethanol 1:1 (v / v) and diluting to 1, 3, 5, 10, and 20 ng / mL. The fluorescence intensity was measured on a microplate reader, and a fluorescence curve was plotted to ensure that the fluorescence intensity was within an appropriate range and not overexposed.

[0136] Fluorescently labeled lovastatin liposomes were prepared according to the method of Example 2 by adding 30 μL COU-6, and a free lovastatin solution was also prepared.

[0137] CT26 cells in the logarithmic growth phase were digested with 0.5% trypsin containing EDTA for 2 min. After observing cell shrinkage and rounding under a microscope, the digestion was stopped by adding RPMI-1640 medium, and the cells were centrifuged at 1000 rpm for 5 min in a low-speed centrifuge. After centrifugation, the supernatant was discarded, 1 mL of complete culture medium was added to resuspend the cells, and the cell concentration was adjusted to 1.5 x 10 5 The confocal dish was removed, the original culture medium was discarded, 200 μL of drug-containing RPMI-1640 medium was added, and the dish was placed in a cell incubator for continued culture for 0.5 h, 1 h, and 4 h.

[0138] After the culture ended, the original culture medium was discarded, and the cells were washed with PBS buffer three times. 200 μL of 4% paraformaldehyde was added for fixation for 20 min. After fixation, the cells were washed with PBS buffer three times, and an appropriate amount of propidium iodide (PI) was added for incubation for 5 min. Subsequently, the liquid was discarded, and the cells were washed with PBS buffer three times. 100 μL of PBS was added, and the uptake of drugs by CT26 cells was detected using a confocal microscope. The results are shown in Figure 10.

[0139] It can be seen from Figure 10 that the CT26 cells at 0.5, 1, 4h all have blue fluorescence, indicating that the cell state is good. The blue fluorescence in the figure represents the cell nucleus dyed by DAPI, the green fluorescence represents the lovastatin liposome containing COU-6, and the MERGE represents the image after superimposing the two channels of DAPI and COU-6, reflecting the distribution of the liposome in the cell. It can be seen from the figure that the liposome is mainly distributed in the cytoplasm of the cell, and the green fluorescence intensity shows a gradually increasing trend with the increase of the administration time. The cell uptake experiment shows that the lovastatin liposome can be taken up by the CT26 cell within 4h.

[0140] Example 11 Cell scratch experiment of lovastatin liposome

[0141] The CT26 and HCT116 cells in the logarithmic growth phase were digested by 0.5% trypsin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended with culture medium. After dilution, the cells were counted by cell counting plate, and the cell concentration was adjusted to 2.5x10 5 When the cell density reached more than 90% and the entire bottom of the hole plate was covered under the microscope, a vertical scratch was drawn on the bottom of the hole plate with a 200μL pipette tip, the culture medium was discarded, and the floating cells were washed away with PBS. The serum-free culture medium was used to prepare free lovastatin and lovastatin liposome (Example 2) with a concentration of 30μM. The control group was set without adding drugs, and 3 groups each with 3 replicates. At 0h, 24h and 48h, the floating cells were washed away, and the morphology of CT26 and HCT116 cells was observed under a fluorescence inverted microscope and photographed. Three scratch fields were randomly selected, the scratch area was calculated by Image J software, and the scratch healing rate was calculated according to the following formula:

[0142] Scratch healing rate (%) = (0h scratch area-24h or 48h scratch area) / 0h scratch area x 100%

[0143] The results of the scratch test of lovastatin and lovastatin liposome on CT26 cells are shown in Figures 11 and 12. As shown in Figures 11 and 12, the scratch healing rate of the blank control group was 50.01 ± 5.40% at 24 h and 73.20 ± 6.69% at 48 h. After treatment with 30 μM lovastatin, the scratch healing rate was 45.21 ± 3.96% at 24 h and 50.56 ± 5.30% at 48 h. After treatment with 30 μM lovastatin liposome, the scratch healing rate was 27.32 ± 3.69% at 24 h and 33.19 ± 2.86% at 48 h. Compared with the control group, the scratch healing rates of CT26 cells were reduced by 22.64% and 40.01% at 48 h after treatment with lovastatin and lovastatin liposome, respectively. In combination with the above results, lovastatin liposome has a relatively strong anti-CT26 cell migration ability, and the liposome preparation may have a stronger anti-tumor migration activity than the free drug because it is more easily taken up by tumor cells.

[0144] The results of the scratch test of lovastatin and lovastatin liposome on HCT116 cells are shown in Figures 13 and 14. As shown in Figures 13 and 14, the scratch healing rates of lovastatin and lovastatin liposome in HCT116 cells are similar to those in CT26 cells. The scratch healing rate of the blank control group was 11.09 ± 0.41% at 24 h and 21.43 ± 1.06% at 48 h. After treatment with 30 μM lovastatin, the scratch healing rate was 6.65 ± 0.76% at 24 h and 14.86 ± 1.13% at 48 h. After treatment with 30 μM lovastatin liposome, the scratch healing rate was 4.29 ± 0.3% at 24 h and 6.83 ± 0.62% at 48 h. Compared with the control group, the scratch healing rates of HCT116 cells were reduced by 6.57% and 14.6% at 24 h and 48 h after treatment with lovastatin and lovastatin liposome, respectively. The lovastatin liposome has a better inhibitory effect on the migration rate of HCT116 cells than the free lovastatin (P < 0.0001).

[0145] Example 12 Flow cytometry for detecting apoptosis experiment

[0146] CT26 cells in the logarithmic growth phase were digested with 0.5% trypsin, resuspended into a single cell suspension with RMPI-1640 complete culture medium, centrifuged at 1000 rpm for 5 min in a low-speed centrifuge, counted with a cell counting chamber, and adjusted to a cell density of 5 × 10 5The cells were inoculated in 6-well plates at 5 x 105cells / well and cultured in an incubator for 24 h. After observing the cell adhesion under a microscope, the original culture medium was discarded, and the control group was added with new complete culture medium, and the free lovastatin and lovastatin liposomes (Example 2) were replaced with complete culture medium at a concentration of 20, 30 and 60 μM, and then the cells were continuously cultured in an incubator for 48 h. The cells were digested with trypsin without EDTA, and after observing the cell shrinkage and rounding under a microscope, the digestion was stopped by adding culture medium, and the cells were blown down gently, and the operation was gentle to avoid mechanical damage to the cells. After collecting the cells, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed with pre-cooled PBS and centrifuged again for 3 times. 100 μL of 1x Binding Buffer was added, and the cells were blown down gently to a single cell suspension. One group of cells was not stained, one group of cells was stained with 5 μL of PI Staining, one group of cells was stained with 5 μL of Annexin V-FITC, and the other groups of cells were double-stained with 5 μL of PI Staining and 5 μL of Annexin V-FITC, and then the cells were blown down gently. After incubation at room temperature for 10 min in the dark, 400 μL of 1x Binding Buffer was added, and the cells were mixed uniformly. The apoptosis was measured by flow cytometry.

[0147] The apoptosis of CT26 cells treated with 20, 30 and 60 μM of lovastatin and lovastatin liposomes for 48 h was tested by flow cytometry (Figure 15). The total apoptosis rates of CT26 cells induced by lovastatin at low, medium and high concentrations were 27.01%, 15.81% and 13.70%, respectively, and the total apoptosis rates of CT26 cells induced by lovastatin liposomes were 27.71%, 14.85% and 17.35%, respectively. The results showed that lovastatin and lovastatin liposomes at low concentrations could induce apoptosis of CT26 cells.

[0148] Example 13: Observation of the effect of lovastatin and lovastatin liposomes on the morphology of CT26 cells by inverted microscope

[0149] The CT26 cells in the logarithmic growth phase were trypsinized, centrifuged in a low-speed centrifuge, resuspended with complete culture medium, counted by a cell counting plate, and the cell density was adjusted to 5 x 105cells / well. 5 The cells were inoculated in 6-well plates at 5 x 105cells / well and cultured in an incubator for 24 h. After observing the cell adhesion under a microscope, the original culture medium was discarded, and the control group was added with new complete culture medium, and the free lovastatin and lovastatin liposomes (Example 2) were replaced with complete culture medium at a concentration of 20, 30 and 60 μM, and then the cells were continuously cultured in an incubator for 48 h. The cells were digested with trypsin without EDTA, and after observing the cell shrinkage and rounding under a microscope, the digestion was stopped by adding culture medium, and the cells were blown down gently, and the operation was gentle to avoid mechanical damage to the cells. After collecting the cells, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed with pre-cooled PBS and centrifuged again for 3 times. 100 μL of 1x Binding Buffer was added, and the cells were blown down gently to a single cell suspension. One group of cells was not stained, one group of cells was stained with 5 μL of PI Staining, one group of cells was stained with 5 μL of Annexin V-FITC, and the other groups of cells were double-stained with 5 μL of PI Staining and 5 μL of Annexin V-FITC, and then the cells were blown down gently. After incubation at room temperature for 10 min in the dark, 400 μL of 1x Binding Buffer was added, and the cells were mixed uniformly. The apoptosis was measured by flow cytometry.

[0150] From Figure 17 we can see that the control group cell density is large, cell morphology is clear, and the growth state is good. After drug treatment, the number of cells is reduced, the intercellular space is large, and the cell morphology is round.

[0151] The above experimental results show that:

[0152] In CT26 cells, free lovastatin and lovastatin liposomes have a killing effect on CT26 cells. At 24h, the inhibition effect is similar. At 48h, lovastatin liposomes may release drugs more slowly, so the lovastatin inhibition effect is better. At 72h, because lovastatin liposomes have a slow-release effect, the inhibition effect at high concentration is better than that of free lovastatin.

[0153] In HCT116 cells, the IC 50 of the lovastatin liposome group is less than that of the lovastatin group (P<0.05). After 24h and 48h of lovastatin and lovastatin liposome administration, the IC 50 of lovastatin is 3.2 and 1.99 times higher than that of lovastatin liposomes. With the increase of drug concentration, the inhibition of cell growth is enhanced. Nanometer-sized liposomes with a cutoff limit of 100-800nm for tumor vasculature can be passively accumulated in tumors, so the cytotoxicity of lovastatin liposomes to HCT116 cells is stronger than that of free lovastatin.

[0154] In HT29 cells, after 24h, 48h, and 72h of lovastatin and lovastatin liposome administration, the IC 50 of lovastatin is 1.7, 2.21, and 1.67 times higher than that of lovastatin liposomes.

[0155] Lovastatin and lovastatin liposomes also have cytotoxicity to HuH7, MCF-7, and OVCAR3 cells, but the cytotoxicity to COC cells is stronger.

Claims

1. Liposomes of lovastatin, characterized in that, The liposome comprises: lovastatin, phospholipid, cholesterol, sodium dodecyl sulfate, wherein the mass ratio of lovastatin to phospholipid is 1:15-1:30, preferably 1:18-1:25; the mass ratio of cholesterol to phospholipid is 1:5-1:20, preferably 1:10-1:20; and the mass ratio of lovastatin to sodium dodecyl sulfate is 1:0.5-1.

2. The lovastatin liposome of claim 1, wherein, In the lovastatin liposome, 4 mg of lovastatin, 60-120 mg of phospholipid, 3-24 mg of cholesterol, and 2-4 mg of sodium dodecyl sulfate are contained in 5 mL of the liposome.

3. The lovastatin liposome of claim 1, wherein, In the lovastatin liposome, 4 mg of lovastatin, 72-100 mg of phospholipid, 3.6-10 mg of cholesterol, and 2-4 mg of sodium dodecyl sulfate are contained in 5 mL of the liposome.

4. The lovastatin liposome of claim 1, wherein, In the lovastatin liposome, 4 mg of lovastatin, 72-80 mg of phospholipid, 4-8 mg of cholesterol, and 2 mg of sodium dodecyl sulfate are contained in 5 mL of the liposome.

5. The method of producing the liposome of lovastatin according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) A prescribed amount of phospholipid, cholesterol, lovastatin, and sodium dodecyl sulfate are weighed in an eggplant-shaped flask, anhydrous ethanol is added, vortexed, and dissolved by ultrasonic; (2) The lipids are formed into a thin film in the flask by reduced pressure rotary evaporation, and uniform thin film is obtained by continuing vacuum rotary evaporation; (3) Deionized water is added, vortexed, and hydrated after ultrasonic, and the probe is ultrasonically treated at 300-400 W to obtain the lovastatin liposome. In the step (1), the concentration of the phospholipid in the anhydrous ethanol is 12-16 mg / mL. The lovastatin liposome of any one of claims 1-4 is prepared by adding a lyoprotectant.

6. The production method according to claim 5, characterized by 8. Use of the lovastatin liposome of any one of claims 1-4 or the lovastatin liposome lyophilized powder of claim 7 in the preparation of a drug for improving bioavailability.

7. A lyophilized powder of lovastatin liposome characterized in that, 9. Use of the lovastatin liposome of any one of claims 1-4 or the lovastatin liposome lyophilized powder of claim 7 in the preparation of an antitumor drug. The tumor is colorectal cancer, liver cancer, ovarian cancer, breast cancer, and preferably colorectal cancer. ​ 10. Use according to claim 9, characterized in that, ​

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