Ergosterol peroxide composite slow-release agent as well as preparation method and application thereof

Abstract

The invention discloses an ergosterol peroxide composite sustained-release agent as well as a preparation method and application thereof, and belongs to the technical field of medical antitumor drug preparations. The invention discloses a preparation method of an ergosterol peroxide composite sustained-release agent, which comprises the following steps: compounding ergosterol peroxide powder and soybean lecithin (Soybean lecithin, SPC, purity gradegt, 90%) through methanol, and sequentially carrying out the steps of normal hexane washing, reduced pressure rotary evaporation, filtration and ultrasonic treatment, so as to obtain the ergosterol peroxide composite sustained-release agent. Compared with the anti-tumor effect of pure ergosterol peroxide, the ergosterol peroxide has the characteristics of long circulation, low toxicity, strong targeting property, better treatment effect and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical anti-tumor drug preparation, more particularly to an ergosterol peroxide-soybean phospholipid complex based nanodrug (ES NDs) and a preparation method and application thereof. BACKGROUND

[0002] Ergosterol peroxide (EP), CAS No. 2061-64-5, Mr 428.647. is a steroidal derivative, which is an anticancer drug that can be isolated from various fungi, yeast, lichen or sponges. EP is a colorless or slightly yellow crystalline powder with a melting point of 178°C. It is easily soluble in organic solvents such as ethyl acetate, tetrahydrofuran, dichloromethane, and very slightly soluble in water and ethanol.

[0003] Mechanism of action of EP: Natural ergosterol lacks peroxide bond (O-O), which has been proved to have no significant activity on most cancer cells. Therefore, it is generally believed that the peroxide bridge in ergosterol peroxide is an important functional group of biological activity. The hemolytic cleavage of the peroxide bridge in a reducing environment produces reactive oxygen species (ROS), which may have cytotoxic effects on cancer cells. EP can inhibit the in vitro and in vivo proliferation of tumor cells, and it can act on signal pathways such as β-catenin, JAK2 / STAT3, NF-κB and MAPKs, thereby playing an inhibitory role.

[0004] The drug is an important active ingredient obtained by isolation and extraction from various edible fungi, and has obvious anti-tumor effect on breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma and ovarian cancer, etc. However, it has adverse side effects such as insolubility in water, non-specific distribution and lack of specificity in tumor cell recognition, and there is a risk of damaging normal tissues and organs. The drug molecules in the body fluid also reduce the availability due to the excretion of the urinary system. Therefore, how to improve the in vivo bioavailability and tumor targeting of EP is what people expect and concern.

[0005] Nanoparticle drug belongs to the category of ultrafine particles (particle size is 1-300 nm), which is usually prepared from inorganic materials or natural / synthetic polymer materials. The drug is combined on the surface of the nanoparticle by covalent connection or electrostatic adsorption, or the drug molecules are directly wrapped inside. As a drug carrier, the nanoparticle can effectively reduce the phagocytosis of the human reticuloendothelial system (RES) macrophages, can pass through the intercellular space as a nanoparticle, can pass through the smallest capillary vessels in the human body, and can be absorbed by the cell tissue. The nanoparticle drug carrier can control the drug to be delivered to the tumor tissue by the enhanced permeability and retention effect (EPR) dependent on the abnormal physiological structure of the tumor tissue, reduce the drug dosage, enhance the drug efficacy and reduce the drug toxicity. At the same time, the particle system can avoid the loss of drug activity, and is beneficial to the storage and transportation of the drug. Therefore, the nanoparticle is a very promising new drug dosage form.

[0006] At present, hydrophobic drugs are mainly prepared into nanoparticles with core-shell structure by self-assembly technology. The nanoparticles prepared by the self-assembly method have uniform particle size, less impurities, simple preparation method, and are more and more widely used. However, how to prepare EP nanoparticles with longer circulation, lower toxicity and better therapeutic effect has become a technical problem to be solved in the field of pharmaceutical antitumor drug preparations.

[0007] Therefore, it is an urgent problem for those skilled in the art to provide a complex sustained-release agent of ergosterol peroxide and a preparation method and application thereof. SUMMARY

[0008] Therefore, the application provides a complex sustained-release agent of ergosterol peroxide and a preparation method and application thereof.

[0009] In the preparation method of the complex sustained-release agent of ergosterol peroxide, the ergosterol peroxide powder is compounded with soy lecithin by methanol, and then sequentially washed by n-hexane, reduced pressure rotary evaporation, filtration and other steps, so that the complex sustained-release agent of ergosterol peroxide with high drug loading, long circulation, low toxicity and good therapeutic effect is prepared.

[0010] In order to achieve the above purpose, the application adopts the following technical scheme: A preparation method of a complex sustained-release agent of ergosterol peroxide, comprising the following steps: S1: Ergosterol peroxide powder and soybean lecithin are dissolved in 100 ml of methanol, acetone or tetrahydrofuran (THF) solvent, and reacted vigorously in a water bath at 25-55°C for 4-12 hours to obtain an ergosterol peroxide phospholipid composite solution; the mass ratio of ergosterol peroxide powder to soybean lecithin is 1:1-4; the concentration of ergosterol peroxide powder is 0.4-1.2 mg / mL; S2: The crude ergosterol peroxide phospholipid complex is obtained by rotary evaporation to remove methanol, acetone or tetrahydrofuran from the ergosterol peroxide phospholipid complex solution under vacuum conditions of 0.08~0.09 MPa, water bath temperature of 25-50℃ and rotation speed of 50-120 r / min. S3: The crude product of the above ergosterol peroxide phospholipid complex was put into 100 ml of n-hexane solvent, filtered to remove the ergosterol peroxide that was insoluble in n-hexane and not bound to soybean lecithin, and the ergosterol peroxide phospholipid complex suspended in n-hexane solvent was collected. S4: The n-hexane in the ergosterol peroxide phospholipid complex is removed again by rotary evaporation under conditions of vacuum degree 0.08-0.09MPa, water bath temperature 30-50℃, and rotation speed 50-120 r / min. A thin film formed on the inner wall of the rotary evaporation flask is the refined ergosterol peroxide phospholipid complex. S5: Add 15-30 ml of deionized water at 30-40℃ to the rotary evaporator containing the above-mentioned ergosterol peroxide phospholipid complex refined product, and then continuously sonicate in an ultrasonic cleaner of 100-250W to fully and uniformly disperse the reaction film to obtain an aqueous solution of ergosterol peroxide phospholipid complex refined product. S6: After removing larger particles by passing the above-mentioned ergosterol peroxide phospholipid complex aqueous solution through 0.45μm and 0.22μm microporous filter membranes, 2%-5% by mass of DSPE-mPEG1000 (Maclean Biochemical Technology Co., Ltd.) of the complex refined product is added to obtain ergosterol peroxide complex sustained-release agent (ES NDs).

[0011] Furthermore, the lecithin is soybean lecithin.

[0012] Furthermore, the ergosterol peroxide complex sustained-release agent prepared by the method is described.

[0013] Furthermore, the application of the ergosterol peroxide complex sustained-release agent in the sustained-release of ergosterol peroxide.

[0014] Furthermore, the application of the ergosterol peroxide complex sustained-release agent in the preparation of formulations that enhance the killing effect on cancer cells.

[0015] Furthermore, the cancer cells are solid tumor cancer cells.

[0016] Furthermore, the cancer cells mentioned are cervical cancer cells.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses an ergosterol peroxide complex sustained-release agent, its preparation method and application. The carrier material in the ergosterol peroxide complex sustained-release agent is soybean lecithin. It has good stability. Compared with ergosterol peroxide, in the ergosterol peroxide complex sustained-release agent, the ergosterol peroxide and phospholipid interact through weak interaction forces, rather than the drug being simply encapsulated in vesicles by phospholipids. Therefore, it has the characteristics of high drug loading, long circulation, low toxicity and good therapeutic effect. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is a transmission electron microscope image of the ergosterol peroxide complex sustained-release agent in this invention.

[0020] Figure 2 The X-ray diffraction patterns of ergosterol peroxide (EP), soybean phosphatidylcholine (SPC), and ergosterol peroxide-soybean phospholipid complex based nanodrugs (ESNDs) in this invention are shown.

[0021] Figure 3 The infrared spectrum of the composite sustained-release agent of ergosterol peroxide, soybean lecithin, and ergosterol peroxide in this invention.

[0022] Figure 4 The (A) hydration kinetic diameter distribution and (B) zeta potential of the ergosterol peroxide complex sustained-release agent in this invention are shown.

[0023] Figure 5The stability of ES NPs in this invention in H2O, PBS, 1640 cell culture medium and 1640 cell culture medium containing 10% serum; wherein, A: the trend of particle size change at different time periods; B: the trend of dispersion coefficient change at different time periods.

[0024] Figure 6 The EP drug release curves of the ergosterol peroxide complex sustained-release agent in this invention are shown under different pH conditions.

[0025] Figure 7 The effect of the compound sustained-release agent of ergosterol peroxide, soybean lecithin and ergosterol peroxide on the in vitro toxicity of HeLa cells in this invention; wherein, (A) ergosterol peroxide; (B) soybean lecithin; (C) ergosterol peroxide compound sustained-release agent.

[0026] Figure 8 The study used flow cytometry to detect tumor cell apoptosis and necrosis using ES NDs nanoparticles of different concentrations in this invention.

[0027] Figure 9 This is a time-dependent uptake analysis of ES NPs and EP by HeLa cells in this invention; where A: laser confocal analysis results; B: flow cytometry analysis results.

[0028] Figure 10 These are the plasma pharmacokinetic results of ES NDs and EP in this invention.

[0029] Figure 11 This shows the distribution of ES NDs and EP in mouse organs and tissues 12 hours after intravenous drug administration in this invention.

[0030] Figure 12 This invention is used to study the in vivo antitumor effect of intravenously delivered ES NDs; wherein, A: trend graph of weight change in tumor-bearing mice; B: trend graph of tumor volume change in tumor-bearing mice; C: tumor weight of tumor-bearing mice at the end of the experiment; D: photograph of tumor tissue of tumor-bearing mice.

[0031] Figure 13 The effect of intravenous administration of ES NDs in this invention on liver and kidney function indicators in tumor-bearing mice; wherein, A: alanine aminotransferase (ALT) content; B: aspartate aminotransferase (AST) content; C: blood urea nitrogen (BUN) content; D: creatinine (Cr) content. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1 A method for preparing an ergosterol peroxide complex sustained-release agent includes the following steps: (1) Ergosterol peroxide powder and soybean lecithin were dissolved in 100 ml of methanol solvent and stirred vigorously in a water bath at 35 °C for 8 hours to obtain ergosterol peroxide phospholipid composite solution; the mass ratio of ergosterol peroxide powder to soybean lecithin was 1:2; the concentration of ergosterol peroxide powder was 0.4 mg / mL.

[0034] (2) Methanol in the ergosterol peroxide phospholipid complex solution was removed by rotary evaporation under conditions of vacuum degree between 0.08-0.09 MPa, water bath temperature of 40℃, and rotation speed of 90 r / min, to obtain crude ergosterol peroxide phospholipid complex.

[0035] (3) The crude product of the above ergosterol peroxide phospholipid complex was put into 100 ml of n-hexane solvent, and the ergosterol peroxide that was insoluble in n-hexane and not bound to soybean lecithin was removed by filtration. The ergosterol peroxide phospholipid complex dissolved in n-hexane solvent was collected.

[0036] (4) The n-hexane in the ergosterol peroxide phospholipid complex is removed by rotary evaporation under conditions of vacuum degree 0.08-0.09MPa, water bath temperature 50℃, and rotation speed 90 r / min. The thin film formed on the inner wall of the rotary evaporation flask is the refined product of ergosterol peroxide phospholipid complex.

[0037] (5) Add 20 ml of deionized water at 35°C to the rotary evaporator containing the ergosterol peroxide phospholipid complex refined product, and then use a 200W ultrasonic cleaner to continuously sonicate the reactant film to fully and evenly disperse it, thereby obtaining an aqueous solution of the ergosterol peroxide phospholipid complex refined product.

[0038] (6) After filtering the above-mentioned ergosterol peroxide phospholipid complex aqueous solution through 0.45μm and 0.22μm microporous filter membranes to remove larger particles, add 5% by mass of DSPE-mPEG1000 of the complex refined product and stir thoroughly to obtain ergosterol peroxide complex sustained-release agent (ES NDs).

[0039] (7) Using an X-ray diffractometer (XRD), with a speed of 10° / min and a scanning angle (2θ) of 5-60°, the crystal form of EP, SPC and ES NDs was analyzed.

[0040] (8) A Fourier transform infrared spectrometer (FTIR) was used, with a scanning range of 4000-400 cm⁻¹. -1 The resolution is 4 cm. -1 Under the specified conditions, the dried sample powder was thoroughly mixed with potassium bromide and then compressed into tablets. The infrared spectra of EP, SPC and ES NDs were then measured.

[0041] (9) The particle size, polydispersity index (PDI) and zeta potential of the ergosterol peroxide complex sustained-release agent were analyzed using a Zetasizer Nano ZS nanoparticle size potentiometer.

[0042] (10) The morphology of the ergosterol peroxide complex sustained-release agent was observed by transmission electron microscopy (TEM).

[0043] Results: When the θ angle domain was 5° < 2θ < 30°, XRD analysis showed that ergosterol peroxide and soybean lecithin formed a complex that masked the original spiny, polymorphic structure. Figure 2 FTIR results showed that in the infrared spectra of ES NDs, the hydroxyl stretching vibration of ergosterol peroxide shifted from the original wavenumber 3431 to 3357, and the stretching vibration phosphate group in the lipid structure also migrated from the original wavenumber 1241 to 1251, with no new groups appearing. This indicates that the hydroxyl groups of ES NDs are combined with the phosphate groups of phospholipids through hydrophobic interactions. Figure 3 Particle size analysis showed that the diameter of ES NDs was 238.5 ± 2.5 nm, the PDI was 0.217 ± 0.015, and the potential was -30.4 ± 1.5 mV. This indicates that the ES NDs particles have relatively uniform size and high stability. Figure 4 TEM results showed that ES NDs exhibited a spherical, uniform structure with a particle size of approximately 200 nm, similar to liposomes. Figure 1 This spherical structure, characterized by high dispersion, non-agglomeration, non-clustering, and non-adhesion, is similar to... Figure 4 The particle size test results were consistent.

[0044] Example 2: Evaluation of the antitumor effect of ES NDs 2.1 Stability and in vitro release of ES NDs 1) In vitro stability test of ES NDs: The cells were placed in water, PBS, 1640 cell culture medium and 1640 cell culture medium containing 10% serum for five days respectively. The particle size and dispersion coefficient of the drug were analyzed and measured by DLS at different time points to evaluate the stability.

[0045] 2) Place 1 mL of 0.4 mg / mL ergosterol peroxide complex sustained-release solution into a MWCO 2000 Da sealed dialysis bag, and then place it in PBS solution at pH 4.5, 6.5 or pH 7.4 to simulate the tumor and blood system environment. Shake the dialysis bag containing ergosterol peroxide complex sustained-release solution at 37 °C for 72 h.

[0046] 3) Collect 1 ml of dialysate at 0, 0.5, 1, 2, 6, 12, 24, 48 and 72 h respectively, and analyze the ergosterol peroxide content in each sample by HPLC.

[0047] Results: ES NDs showed good stability in water, PBS, 1640 cell culture medium, and 1640 cell culture medium containing 10% serum, with no significant change in particle size over 5 days. Figure 5 The drug release curves at different pH levels show that at 48 h, >80% of the EP was released into the solution in an acidic environment (pH=4.5), approximately 50% of the EP was released in a simulated slightly acidic environment (pH=6.5) at the tumor site, while <30% of the EP was released in a slightly alkaline body fluid environment (pH=7.4). Figure 6 This indicates that ES NDs have good stability in plasma and can effectively release drugs in the slightly acidic tumor microenvironment.

[0048] 2.2 Effects of ES NDs on tumor cell viability in vitro 1) Weigh 0.25 g of MTT (thiazolyldiphenyl-tetrazolium bromide; Solarbio M8180) powder and place it in a 50 mL centrifuge tube. Add 50 mL of sterile PBS and stir well. After the powder has completely dissolved (ultrasonic aid may be used), filter it through a 0.22 μm filter, then aliquot it into equal portions, seal it, and store it at -20℃.

[0049] 2) Inoculate 5×10⁶ cells into a 96-well plate. 3HeLa tumor cells / well were cultured for 24 h and then treated with different concentrations of drugs: a blank control group (Control), a negative control group (SPC), and a positive control group (30 μg / mL cisplatin, DDP). ES NDs were serially diluted to EP concentrations of 1, 2, 5, 6, 7, 9, and 12 μg / mL, with six replicates selected for each treatment group.

[0050] 3) After culturing HeLa cells for another 24 h, centrifuge at 1200 rpm for 7 min and discard the supernatant. Add 0.5 mg / ml MTT solution, incubate at 37℃ for another 4 h, centrifuge at 1200 rpm for 7 min, discard the MTT solution, add 150 μL DMSO, and vortex for 10 min to help dissolve the methyl crystals completely. After the methyl crystals are completely dissolved, measure the OD value at 490 nm using a microplate reader. Cell viability is calculated as follows: Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0051] Results: The results are as follows Figure 7 As shown, HeLa cells were co-incubated with SPC nanocarriers at concentrations ranging from 1 to 800 μg / mL for 24 h, and the survival rate of HeLa cells exceeded 95%, demonstrating that SPC is a relatively safe drug delivery carrier. Both free EP and ESNDs showed dose-dependent inhibitory effects on the proliferation of cervical cancer cells HeLa. At the same EP concentration, the half-lethal concentration (IC50) of tumor cells decreased from 10.02 for free EP to 6.764 μg / mL for encapsulated EP, indicating that ESNDs were more effective than free EP in inhibiting tumor proliferation (p<0.001).

[0052] 2.3 Detection of apoptosis levels induced by ES NDs 1) Seed 1 × 10⁶ HeLa cells into a 12-well plate. 5 Cells per well, incubated at 37°C for 24 hours.

[0053] 2) Discard the cell culture medium, add 2 mL of different concentrations of ES NDs and free EP solution, and incubate at 37°C for 24 h.

[0054] 3) Add 10 μL of Annexin V-FITC / PI staining solution (1:2), incubate at room temperature for 20 min, filter with a copper mesh, and finally detect apoptosis using a flow cytometer.

[0055] Results: The apoptosis rate of HeLa cells treated with ES NDs nanoparticles was significantly increased compared with the control group (p<0.001), and also significantly higher than that of the same dose of free EP (p<0.001). Figure 8 ).

[0056] 2.4 In vitro cell uptake experiment 1) 100 mg EP and 1.2 times the molar amount of 4-chloro-7-nitro-2,1,3-benzoxoxadiazole (NBD Cl) were dissolved in dichloromethane.

[0057] 2) Add a few drops of triethylamine to the reaction solution.

[0058] 3) React at room temperature for 12 hours.

[0059] 4) Dialyze to purify and then freeze-dry to obtain NBD Cl-EP.

[0060] 5) Synthesize NBD Cl-labeled ES NDs by combining NBD Cl-EP with SPC.

[0061] 6) Using 1×10 5 Cells were seeded at a density of cells / plate and cultured at 37°C with 5% CO2 for 24 h. The culture medium was then removed, and the medium for NBD Cl-EP and NBD Cl-ES NDs samples was added. Cells were cultured for 0, 1, 2, 4, and 8 h, respectively. Cells were washed three times with PBS, and the cell pellet was resuspended in 200 μL PBS. A portion of the pellet was filtered through a 200-mesh copper screen into flow cytometry tubes, and data on EP or ES NDs uptake were collected using a BD FACSCalibur flow cytometer. Uptake information was analyzed using FlowJo software. Another portion was treated with 4% paraformaldehyde, stained with Hoechst, mounted, and the differences in EP or ES NDs uptake were observed using a Nikon A1 R HD25 laser confocal microscope.

[0062] Results: Laser confocal analysis showed that SPC-encapsulated ES NDs had better cellular uptake compared to free EP. Figure 9 This may lead to ( Figure 7 and Figure 8 The reason why ES NDs have better antitumor activity than free EP.

[0063] 2.5 Pharmacokinetics and Organ Distribution of Intravenously Injected ES NDs 1) Mice were given a single tail vein injection of ES NDs and EP (1.75 mg / kg), followed by blood collection from the orbital cavity. The blood samples were placed in EDTA-treated EP tubes and centrifuged at 4°C and 12,000 rpm for 10 min.

[0064] 2) Transfer the supernatant from the centrifuged plasma to a new EP tube, dilute it 4 times with PBS, mix well and prepare the plasma supernatant for later use.

[0065] 3) Twelve hours after administration, mice were sacrificed and their hearts, livers, spleens, lungs, kidneys, and tumors were collected. The samples were cleaned with PBS, wiped with filter paper, and then homogenized with 9 times the volume of PBS solution.

[0066] 4) Centrifuge the tissue homogenate at 4°C and 12,000 rpm for 10 min, and collect the supernatant in a new EP tube to obtain the tissue homogenate supernatant for later use.

[0067] 5) Quantitatively analyze drug content using HPLC based on the EP standard curve.

[0068] Results: Following a single tail vein administration, the blood concentrations of both ES NDs and EP gradually decreased over time, with the rate of decrease initially rapid and then slowing down until the drug concentrations became undetectable at 24 hours. Notably, the blood concentrations of ES NDs remained consistently higher than those of EP, indicating a slower elimination rate of ES NDs from the bloodstream. Figure 10 ); 12 h after tail vein injection of ESNDs and EP, ESNDs mainly accumulated in the liver and tumor, and the content of ESNDs in the tumor was significantly higher than that of EP alone (p<0.001), indicating that nanomedicine enables EP to target tumors ( Figure 11 ).

[0069] 2.6 Evaluation of the antitumor effects of ES NDs 1) Select healthy female C57BL / 6 mice and house them in an SPF-grade experimental environment. Keep the room temperature at around 25°C and allow them to adapt for 2 days. Observe the appearance and behavior of the mice and select healthy mice to establish the tumor-bearing model.

[0070] 2) After tumor cell counting, the TC-1 tumor cell count was adjusted to 5 × 10⁻⁶ cells using PBS. 5 0.1 mL of TC-1 cells per 100 μL were subcutaneously injected into the right posterior region of C57BL / 6 mice.

[0071] 3) When a palpable induration is found in the inoculation area, the experimental mice are randomly divided into 7 groups: Control group, SPC group, EP group (2.5 mg / kg), Cisplatin group (5 mg / kg), ES NDs group (0.875 mg / kg), ES NDs group (1.75 mg / kg), and ES NDs group (3.5 mg / kg), with 5 mice in each group.

[0072] 4) After being divided into groups, the tumor-bearing mice were injected with 0.2 mL of the drug via the tail vein, and the tail vein injection was repeated every other day.

[0073] 5) After treatment begins, the long diameter (A) and short diameter (B) of the mouse tumor are measured with calipers on the same day and every 48 hours and recorded. These measurements are used to calculate the tumor volume. The mouse weight is also measured. Treatment is stopped when the long diameter of the tumor is ≥15 mm, and the mouse is euthanized. Tumor growth curves and mouse weight change curves are plotted. The tumor volume calculation formula is as follows: V mm³ = AB 2 / 2 (mm 3 ).

[0074] 6) Two days after the last administration, blood was collected from the mice, and they were euthanized. Tumors and major organs were removed for physiological and biochemical analysis. Blood samples were also collected for biochemical testing.

[0075] Results: Changes in tumor volume and weight showed that, compared with PBS and SPC controls, the Cisplatin group, EP group, and high, medium, and low dose ES NDs groups all significantly slowed tumor growth and had significantly lighter tumor weights than the PBS and SPC groups; compared with the tumor-suppressing effect of 2.5 mg / kg free EP, the nanoparticles formed by SPC encapsulation of EP significantly improved the therapeutic effect of EP. Figure 12 ).

[0076] 2.7 Serum biochemical factor level detection 1) Place the EP tube containing mouse blood in a 37°C constant temperature incubator for 2 hours, then incubate overnight at 4°C. Centrifuge at 3000 rpm for 20 minutes at 4°C, and transfer the supernatant to a new EP tube to obtain the serum of the tumor model mouse.

[0077] 2) Following the manufacturer's instructions, use the creatine kinase (CK) kit (Solarbio: BC1145), blood urea nitrogen (BUN) test kit (Solarbio: BC1535), alanine aminotransferase (ALT / GPT) test kit (Solarbio: BC1555), and aspartate aminotransferase (AST / GOT) test kit (Solarbio: BC1565) to detect various cardiac, hepatic, and renal injury indicators in the supernatant using the microplate method.

[0078] Results: Compared with the control group, the liver function ALT, AST, and kidney function BUN and Cr levels in the treatment group mice were not significantly increased, indicating that the treatment group did not cause damage to the liver and kidneys. However, the positive control drug Cisplatin caused a significant increase in renal creatinine (Cr) and blood urea nitrogen (BUN), indicating that Cisplatin seriously affected the kidney function of the treated mice. Figure 13 ).

[0079] Compared to ergosterol peroxide alone, this formulation has the characteristics of long circulation, low toxicity, strong targeting, and better therapeutic effect.

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an ergosterol peroxide complex sustained-release agent, characterized in that, Includes the following steps: S1: Ergosterol peroxide powder and soybean lecithin are dissolved in 100 ml of methanol, acetone or tetrahydrofuran solvent, and reacted with vigorous stirring in a water bath at 25-55℃ for 4-12 hours to obtain an ergosterol peroxide phospholipid composite solution; the mass ratio of ergosterol peroxide powder to soybean lecithin is 1:1-4; the concentration of ergosterol peroxide powder is 0.4-1.2 mg / mL; S2: The crude ergosterol peroxide phospholipid complex is obtained by rotary evaporation to remove methanol, acetone or tetrahydrofuran from the ergosterol peroxide phospholipid complex solution under vacuum conditions of 0.08~0.09 MPa, water bath temperature of 25-50℃ and rotation speed of 50-120 r / min. S3: The crude product of the above ergosterol peroxide phospholipid complex was put into 100 ml of n-hexane solvent, filtered to remove the ergosterol peroxide that was insoluble in n-hexane and not bound to soybean lecithin, and the ergosterol peroxide phospholipid complex suspended in n-hexane solvent was collected. S4: The n-hexane in the ergosterol peroxide phospholipid complex is removed again by rotary evaporation under conditions of vacuum degree 0.08-0.09MPa, water bath temperature 30-50℃, and rotation speed 50-120 r / min. A thin film formed on the inner wall of the rotary evaporation flask is the refined ergosterol peroxide phospholipid complex. S5: Add 15-30 ml of deionized water at 30-40℃ to the rotary evaporator containing the above-mentioned ergosterol peroxide phospholipid complex refined product, and then continuously sonicate in an ultrasonic cleaner of 100-250W to fully and uniformly disperse the reaction film to obtain an aqueous solution of ergosterol peroxide phospholipid complex refined product. S6: After removing larger particles by passing the above-mentioned ergosterol peroxide phospholipid complex aqueous solution through 0.45μm and 0.22μm microporous membranes, 2%-5% by mass of DSPE-mPEG1000 is added to obtain the ergosterol peroxide complex sustained-release agent.

2. The method for preparing an ergosterol peroxide complex sustained-release agent according to claim 1, characterized in that, The lecithin mentioned is soybean lecithin.

3. The ergosterol peroxide complex sustained-release agent prepared by the method according to any one of claims 1-2.

4. The application of the ergosterol peroxide complex sustained-release agent according to claim 3 in the sustained-release of ergosterol peroxide.

5. The use of the ergosterol peroxide complex sustained-release agent according to claim 3 in the preparation of a formulation that enhances the killing effect on cancer cells.

6. The application according to claim 5, characterized in that, The cancer cells mentioned are solid tumor cancer cells.

7. The application according to claim 5, characterized in that, The cancer cells in question are cervical cancer cells.

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