A nano-micelle loaded with moclobemide, its preparation method and application

Abstract

The application discloses a nanomicelle loaded with meclofenoxate and a preparation method thereof, which is composed of a carrier and meclofenoxate loaded in the carrier, the carrier is a nanomicelle formed by a PEG-SS-PAE copolymer, and the nanomicelle loaded with meclofenoxate is prepared by adopting a thin film hydration method. The nanomicelle loaded with meclofenoxate prepared by the application has strong anticancer activity, can effectively inhibit tumor growth in a mouse breast cancer model, and has no obvious toxic effect at a cellular level, which is proved by experiments.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a nano-micelle loaded with moxonidine and a preparation method and application thereof. BACKGROUND

[0002] Breast cancer has a high incidence in women worldwide and is a major factor causing female deaths. Cancer cell metastasis in cancer is the cause of death for most cancer patients and is difficult to cure once it occurs. Finding new anticancer drugs and efficient treatment methods is a hot and difficult point in the field of tumor research. In clinical practice, breast cancer is divided into different molecular subtypes by detecting estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 (cell proliferation index). Breast cancer in which ER, PR, and HER2 are all negative is called triple-negative breast cancer (TNBC). Its incidence accounts for about 15% of all breast cancers, and it has characteristics such as high incidence, low age of onset, high invasiveness, early lymph node metastasis, and poor prognosis. Since TNBC is not sensitive to hormone drugs and targeted drugs, the current main treatment methods are surgical resection, drug treatment, and radiotherapy. However, these traditional treatment methods have limitations such as postoperative recurrence, limited tumor suppression, and serious side effects.

[0003] In recent years, in the fields of oncology and immunology, tumor immunity and immunotherapy have received great attention, especially immune checkpoint blockade (ICB) has made breakthrough progress in the field of cancer treatment, and has great clinical significance in inhibiting tumor growth and preventing metastasis and recurrence. Although tumor immunotherapy has developed rapidly, it can strengthen the patient's own immune response, thereby improving the anti-tumor response or eliminating the immunosuppressive effect, bringing new ideas to cancer treatment and achieving outstanding results, but there are still many problems such as low responsiveness, few tumor T lymphocyte infiltrates, and easy to cause serious adverse reactions. Nanoparticle platforms are usually used as carriers and can be passively targeted to tumors by taking advantage of the enhanced permeability and retention (EPR) effect, and can also achieve active targeting by surface modification of targeted molecules, thereby accumulating immune stimulatory drugs (chemotherapeutic drugs, antibodies, immunomodulators, or other functional molecules) at the tumor site, enhancing local immune responses, improving local immunosuppression, and enhancing tumor immunotherapy.

[0004] Monoamine oxidase-A (MAO-A) is a newly reported immune checkpoint that regulates the degradation of serotonin to modulate the anti-tumor activity of T cells, and the use of monoamine oxidase inhibitors (MAOIs) can inhibit the degradation of serotonin to enhance the anti-tumor activity of T cells. MAOIs are closely related to the occurrence and development of some cancers. However, there are few studies on their anti-tumor effects and mechanisms in breast cancer.

[0005] A major side effect of MAOIs is that they can cause tyramine-induced hypertension when patients consume tyramine-rich foods such as aged cheese (this side effect is known as the "cheese effect"), leading to various drug-related cumbersome food restrictions, which is a major obstacle to their clinical application. At the same time, most MAOIs have poor water solubility and poor circulation in the body, and severe damage to the liver and kidneys is also a problem that needs to be solved urgently. Nanocarrier systems have great advantages in drug delivery, can increase the solubility of poorly soluble drugs, and can be functionally modified to achieve precise and controllable drug release and reduce damage to important organs. Nanoparticles are more likely to cross biological barriers due to their small size, penetrate the body efficiently, and greatly improve drug efficacy. Among them, nanomicelles with good biocompatibility, degradability, good membrane penetration, and low cytotoxicity can efficiently deliver drugs. Nanomicelles are self-assembled systems composed of lipids or other amphiphilic molecules. Two or more polymer chains with different hydrophilicities are systematically assembled into a core-shell assembly in an aqueous medium, which is suitable for drug delivery. In recent years, the design of micelles can overcome the difficulty of drug penetration through biological membranes to some extent. However, studies have shown that the anti-biological membrane efficacy of ordinary micelles is greatly limited due to the lack of penetration and retention capacity. pH and GSH dual-responsive micelles are composed of three fragments: hydrophobic fragments, hydrophilic fragments, and pH and GSH sensitive fragments. The hydrophobic fragment can form a solid inner core to load drugs, the hydrophilic fragment can make the micelles escape the rapid clearance of the mononuclear phagocyte system, the pH sensitive fragment is responsible for converting the surface charge when the pH value at the infection site becomes acidic, and the GSH sensitive fragment undergoes redox reaction under the condition of high concentration of GSH to break the disulfide bond between the fragments. The fragments work together to release the drug to the tumor site and exert high anti-tumor effect. SUMMARY

[0006] The application takes the pH and GSH double-response degradable nanomicelle PEG-SS-PAE as a carrier material, encapsulates the hydrophobic drug Moclobemide, increases water solubility, improves bioavailability, and studies the influence of PEG-SS-PAE@Moclobemide on the occurrence and development of breast cancer, and experiments prove that the Moclobemide-loaded nanomicelle prepared by the application has strong anticancer activity, can effectively inhibit tumor growth in a mouse breast cancer model, and experiments prove that it has no obvious toxic effect at the cellular level.

[0007] A Moclobemide-loaded nanomicelle is composed of a carrier and Moclobemide encapsulated in the carrier, the carrier is a nanomicelle formed by a PEG-SS-PAE copolymer, and the Moclobemide-loaded nanomicelle is prepared by adopting a film hydration method, the mass ratio of Moclobemide to PEG-SS-PAE is 1-3:6, preferably the mass ratio of Moclobemide to PEG-SS-PAE is 1:2.5-3.5 or 1:3.

[0008] The PEG-SS-PAE copolymer is PEG5k-SS-PAE10k, the encapsulation efficiency of the Moclobemide-loaded nanomicelle is 97-99.2%, and the drug loading amount is 15-37%.

[0009] The application further provides a preparation method of the above-mentioned Moclobemide-loaded nanomicelle, which comprises the following steps: using a PEG-SS-PAE copolymer as a carrier material, and encapsulating Moclobemide by adopting a film hydration method to obtain the Moclobemide-loaded nanomicelle.

[0010] The preparation method specifically comprises the following steps:

[0011] 1) dissolving the carrier material and Moclobemide in an organic solvent to obtain a mixed solution;

[0012] 2) evaporating the organic solvent in the mixed solution to obtain a compound film;

[0013] 3) dissolving the compound film in sterile water to prepare a suspension, and performing high-speed centrifugation on the suspension, and the supernatant is the Moclobemide-loaded nanomicelle solution.

[0014] Preferably, in step 1), the carrier material and Moclobemide are respectively dissolved in an organic solvent, and after mixing the two solutions, stirring is performed until the liquid is clear and transparent.

[0015] Preferably, the organic solvent is chloroform, and in step 2), the organic solvent in the mixed solution is evaporated by adopting rotary evaporation.

[0016] Preferably, the speed of high-speed centrifugation in step 3) is 5500-7500r, and the supernatant obtained by centrifugation is freeze-dried to obtain the nanomicelle freeze-dried powder loaded with moclomide.

[0017] The application also provides the use of the above-mentioned nanomicelle loaded with moclomide in the preparation of an antitumor drug.

[0018] Preferably, the tumor is breast cancer.

[0019] Preferably, the tumor is triple-negative breast cancer.

[0020] The application adopts a film hydration method to prepare blank micelles and drug-loaded micelle systems. PAE can be combined with polyethylene glycol (PEG) to form PEG-PAE. PEG-PAE has a hydrophilic PEG-shell and a hydrophobic PAE-core. When PAE is in an acidic environment, the protonation of the amine groups in its structure changes, thereby improving its solubility in water. The acidic environment of the tumor is used as a signal, and the characteristic of the twisted charge of PAE under acidic conditions is used to trigger the release of the drug wrapped in PAE in a specific tissue. At the same time, disulfide bonds are used as a connecting agent in the structure of the nanoparticles, and the high concentration of GSH in the tumor site can cause the disulfide bond to break, and the nanomicelles to be disassembled, so that the loaded drug will accumulate in large quantities in the tumor site.

[0021] The co-loaded immune checkpoint inhibitor dual-responsive micelles PEG-SS-PAE@Moclobemide are successfully constructed, and can accumulate in large quantities at the tumor site when injected in vivo. In addition, the safety of PEG-SS-PAE@Moclobemide is evaluated in vitro. The results of the cytotoxicity test of 4T1 cells confirm that PEG-SS-PAE@Moclobemide has no cytotoxicity. The in vivo effect of the drug is also studied through a tumor growth experiment, which shows that PEG-SS-PAE@Moclobemide has good antitumor effect and can significantly slow down the growth of tumors. At the same time, H&E staining experiments and blood biochemical tests confirm that PEG-SS-PAE@Moclobemide has high safety.

[0022] The application constructs a co-loaded immune checkpoint blocker dual-responsive degradable nanomicelle, which has strong anticancer activity, can effectively inhibit tumor growth in a mouse breast cancer model, has no obvious toxicity at the cellular level, has good safety, and also exhibits good therapeutic effect in an animal model, thereby providing a new idea for tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1TEM of PEG-SS-PAE, PEG-SS-PAE@Moclobemide.

[0024] Figure 2 Linear relationship between Moclobemide concentration and UV absorbance.

[0025] Figure 3 Stability of drug-loaded micelles of Moclobemide mixed with PEG-SS-PAE in different ratios in PBS.

[0026] Figure 4 Cytotoxicity of PEG-SS-PAE, Moclobemide, PEG-SS-PAE@Moclobemide on 4T1 cells.

[0027] Figure 5 Body weight change of tumor-bearing mice during treatment.

[0028] Figure 6 H&E staining of major organs of tumor-bearing mice.

[0029] Figure 7 Tumor growth in mice.

[0030] Figure 8 Comparison of tumor weight in each group. DETAILED DESCRIPTION

[0031] The application will be further described in conjunction with the following examples, but the application is not limited by the examples.

[0032] The experimental methods in the following examples are all routine methods unless otherwise specified.

[0033] Example 1, synthesis of mPEG5k-SS-PAE10k

[0034] The copolymer mPEG5k-SS-PAE10k was synthesized by Chongqing Weishanglide Biological Technology Co., Ltd. according to the requirements. The synthesis method is as follows:

[0035] 1. Weigh 2.0 g of mPEG5k-SS-NH2 and dissolve it in 20 ml of chloroform, add acryloyl chloride (10 eq.) and triethylamine (10 eq.) and stir at room temperature for 12 h. Wash the reaction liquid with water 3 times, dry with anhydrous sodium sulfate, concentrate under reduced pressure, pour into a large amount of ice ethanol to precipitate, collect the product by filtration, and dry under vacuum to obtain the product mPEG5K-SS-acryloyl.

[0036] 2. Weigh 1.0 gm of PEG5K-SS-Propylene into 10 ml of chloroform, add 1,6- bis(acryloyloxy)hexane (10.0 eq.) and 1,3-bis(4-piperidinyl)propane (11.0 eq.) and stir to dissolve, then react at 55 °C for 48 h. After the reaction is completed, the reaction solution is concentrated under reduced pressure, then poured into a large amount of ice-ethanol to precipitate, and the product is collected by filtration and dried under vacuum to obtain the product mPEG5K-SS-PAE10k, which is used to load Moclobemide in the present application.

[0037] Wherein the Chinese name of the starting material mPEG-SS-NH2 is methoxy polyethylene glycol-SS-amine or methoxy polyethylene glycol-disulfide bond-amino, and the molecular weight of PEG can be selected: 350, 550, 750, 1k, 2k, 3.4k, 5k. The -NH2 at the end of mPEG-SS-NH2 can be coupled to the surface of peptides, antibodies or proteins containing NHS or COOH groups, and when -ss- encounters reduced material dithiothreitol (DTT), the disulfide bond can be broken to accelerate the release of drugs. mPEG5k-SS-NH2 can be directly purchased from biological and chemical companies.

[0038] Example 2, Construction of PEG-SS-PAE@Moclobemide

[0039] Polymeric micelles are a kind of nanoparticles with a shell-core structure, which can encapsulate drugs in the hydrophobic core, while the hydrophilic shell ensures solubility in the body, and the small particle size of the micelles also enables passive targeting of tumors by enhancing the EPR effect. The pH value of human tumor tissue is generally between 6.5 and 7.0, while the pH value of healthy human subcutaneous tissue remains around 7.4. Therefore, pH-sensitive polymeric micelles can be degraded under acidic conditions in tumors to promote the release of encapsulated drug molecules. GSH-sensitive micelles are used to release drugs into the cytoplasm, which contains 100-1000 times more reduced GSH (about 2-10 mM) than extracellular fluid (about 2-20 μM). Under the action of high concentration of GSH, redox reaction occurs, and the breakage of disulfide bond between them will lead to further complete release of core drugs into the cytoplasm.

[0040] 1 Materials

[0041] Moclobemide (CAS No. 71320-77-9): It is a reversible and selective MAO-A inhibitor. The source is: GlpBio Company (USA).

[0042] PEG5k-SS-PAE10k: It is a copolymer prepared in Example 1, which is a multi-block polymer, polyethylene glycol-SS-poly(β-amino ester).

[0043] 2 Experimental methods

[0044] 2.1 Synthesis of PEG-SS-PAE blank micelles and PEG-SS-PAE@Moclobemide drug-loaded micelles

[0045] (1) 5 mg of PEG5k-SS-PAE10k polymer powder was dissolved in 1 ml of chloroform. After complete dissolution, the solution was transferred to a distillation flask, dried by rotary evaporation for 30 minutes, and the chloroform was completely evaporated to form a thin film on the wall of the flask. The obtained thin film was dissolved in deionized water and placed in an ultrasonic instrument for 30 minutes to form a blank micelle suspension. The collected suspension was pre-cooled in a -80°C refrigerator for 2 hours, placed in the ice trap of a pre-started freeze dryer, and the vacuum pump was started. After freeze-drying the micelle suspension, PEG5k-SS-PAE10k freeze-dried powder was formed and stored at -20°C for future use.

[0046] (2) In this study, thin film hydration method and centrifugation method were used to prepare PEG-SS-PAE@Moclobemide nanomicelles co-loaded with immune checkpoint blockers. 5 mg, 10 mg, and 15 mg of Moclobemide were dissolved in 1 mL, 2 mL, and 3 mL of chloroform, respectively, and vortexed for 30 s to prepare Moclobemide / chloroform solutions. Another 30 mg of PEG-SS-PAE was added to 3 mL of chloroform and shaken to dissolve to prepare a PEG-SS-PAE / chloroform solution. The two solutions were mixed and placed on a magnetic stirrer at 25°C and 150 r / min for 12 h until the liquid became clear and transparent. Then the mixture was transferred to a distillation flask and dried by rotary evaporation for 60 minutes. The chloroform was completely evaporated to form a thin film on the wall of the flask. The obtained thin film was dissolved in deionized water and placed in an ultrasonic instrument for 30 minutes to form a suspension. The collected suspension was placed in a high-speed centrifuge at 6500 r for 15 minutes. The supernatant was pre-cooled in a -80°C refrigerator for 2 hours, placed in the ice trap of a pre-started freeze dryer, and the vacuum pump was started. After freeze-drying the micelle suspension, PEG-SS-PAE@Moclobemide freeze-dried powder (i.e. Moclobemide-loaded nanomicelles) was formed and stored at -20°C for future use.

[0047] 2.2 Morphology, particle size distribution, and zeta potential of blank micelles

[0048] (1) 10 mg of polymer was dissolved in deionized water to obtain a micelle solution. An appropriate amount of micelle solution was dropped onto a copper mesh, and the excess water was absorbed with filter paper. After drying, it was stained with 2% phosphotungstic acid negative stain. After drying, it was observed under a transmission electron microscope.

[0049] (2) The micelle solutions under different conditions were prepared by dissolving the polymers in (1) pH = 7.4; (2) pH = 5.6; (3) pH = 7.4 + 10 mM GSH; (4) pH = 5.6 + 10 mM GSH, respectively. The micelle solutions under different conditions were placed in special cuvettes, and the particle size distribution and zeta potential were measured by a laser particle size analyzer. The measurement temperature was 25°C, and each sample was measured three times.

[0050] 2.3 Drug loading and encapsulation efficiency determination of PEG-SS-PAE@Moclobemide drug-loaded micelles

[0051] A certain amount of Moclobemide was accurately weighed, diluted with dimethyl sulfoxide (DMSO), and then the standard curve was detected by ultraviolet-visible spectrophotometer (UV-vis). 5.0 mg, 10.0 mg, 15.0 mg of Moclobemide were dissolved in 10 mL of DMSO, and then stirred by vortex. Then the polymer (30 mg) was dissolved in 5.0 mL of chloroform. The Moclobemide solution was added to the polymer solution. After that, the mixed solution was transferred to a flask and dried by rotary evaporation for 2 hours. The obtained film was dissolved in an aqueous solution and soaked in a water bath for 30 minutes to form Moclobemide-loaded micelles. After that, PEG-SS-PAE@Moclobemide (10 mg each) was dissolved in distilled water (2 ml), and then the micelles shell was destroyed by ultrasonic method. After 20 minutes of ultrasonic treatment, DMSO (8 mL) was added to dissolve Moclobemide completely, and the obtained liquid was divided into ultrafiltration tubes, and centrifuged at 13500 rpm for 10 minutes at 4°C using a refrigerated high-speed centrifuge, and the solutions inside and outside the ultrafiltration tubes were reserved respectively. The filtrate was analyzed by ultraviolet-visible light at 265 nanometers to determine the concentration of Moclobemide. The encapsulation efficiency (EE%) and drug loading (DL%) were calculated according to the following formula.

[0052] Drug loading (DL%) = (mass of drug in micelles / mass of drug-loaded micelles) * 100%

[0053] Encapsulation efficiency (EE%) = (mass of drug in micelles / mass of input drug) * 100%

[0054] 3 Experimental results

[0055] 3.1 PEG-SS-PAE polymer structure has a dual-responsive characteristic

[0056] The structure of PEF-SS-PAE polymer was identified by nuclear magnetic resonance hydrogen spectrum. The acid-sensitive ester bond and GSH-sensitive disulfide bond can be seen, which indicates that the micelles can theoretically achieve dual response.

[0057] 3.2 Morphology, particle size distribution and zeta potential of blank micelles

[0058] To observe the morphology of the micelles, they were imaged using a transmission electron microscope, and the results are as follows: Figure 1 As shown, PEG-SS-PAE particles are spherical in size and uniform in size. PEG-SS-PAE@Moclobemide particles also exhibit a similar structure, but the center is darker than the surrounding area, possibly due to the drug being loaded in the center. Overall, they maintain a spherical structure.

[0059] To evaluate the acid-sensitive and GSH-sensitive dual-response properties of PEG-SS-PAE, the polymer was dissolved in: (1) pH = 7.4; (2) pH = 5.6; (3) pH = 7.4 + 10 mM GSH; (4) pH = 5.6 + 10 mM GSH to prepare micelle solutions under different conditions, and their particle size and zeta potential were detected. The results (Table 1) show that when the pH is 6.5 and contains a high concentration of GSH, the particle size of PEG-SS-PAE nanomicelles increases significantly, indicating that the nanomicelles rupture under this condition. Since the PAE core encapsulated within is electronegative, the zeta potential undergoes a charge reversal after being released, and becomes extremely unstable.

[0060] Table 1. Particle size and zeta potential of PEG-SS-PAE nanomicelles under different conditions (n=3)

[0061]

[0062] 3.3 Encapsulation efficiency (EE%) and drug loading (LC%) of drug-loaded micelles

[0063] The poor drug loading capacity of nanocarriers is a major challenge in their application, determined by the interaction between the drug and the colloidal core. We first plotted a standard curve of Moclobemide in DMSO using UV spectrophotometry results. The standard curve is shown below. Figure 2 As shown, the standard curve equation is Y = 0.01046X + 0.05562, R0 2 =0.9957, from which the encapsulation efficiency and drug loading of the micelles were calculated. Table 2 shows the EE (%) and LC (%) data for PEG-SS-PAE@Moclobemide. The EE (%) of both micelles reached over 97%. The LC (%) for different mass ratios were approximately 15.99%, 27.72%, and 36.69%. Simultaneously, we tested the stability of PEG-SS-PAE@Moclobemide micelles with three different mass ratios in PBS. Results ( Figure 3) showed that the stability of the micelles was best when the mass ratio of Moclobemide to PEG-SS-PAE was 10:30, so it was chosen as the object of subsequent study.

[0064] Table 2 EE (%) and LC (%) of PEG-SS-PAE@Moclobemide with different ratios

[0065]

[0066] Example 3, in vitro toxicity study of PEG-SS-PAE@Moclobemide

[0067] 1. Experimental materials

[0068]

[0069] 2. Experimental method

[0070] 2.1 Cell recovery, cell passage, cell cryopreservation, and cell counting

[0071] The experimental cells were mouse triple-negative breast cancer cell lines (4T1), and cell recovery, cell passage, cell cryopreservation, and cell counting were performed using conventional methods.

[0072] 2.2 CCK-8 experiment

[0073] In the 4T1 special medium, 5×10 3 cells per well were seeded in a 96-well culture plate at 37°C in the presence of 5% CO2 for 24 hours to allow the cells to adhere to the plate. Then, a portion of the cells was replaced with fresh culture medium containing different concentrations (1, 3, 10, 30, and 100 micrograms / milliliter) of PEG-SS-PAE@Moclobemide micelles. Another portion of the cells was cultured under four conditions: 4T1 special medium, medium + Moclobemide, medium + empty micelles, and medium + PEG-SS-PAE@Moclobemide micelles. After 24, 48, and 72 hours of culture, the original culture medium was discarded, 110 μl of CCK-8 reagent and incomplete medium premix were added to each well, and the cells were incubated at 37°C for 30 minutes before being detected for optical density at 450 nm using a machine to evaluate the cell proliferation ability.

[0074] 2.3 Flow cytometry

[0075] To further detect the effect of drug-loaded micelles on the apoptosis of 4T1 breast cancer cells, flow cytometry was used after Annexin V / PI staining. 1×10 5The cells were seeded in 6-well plates at a density of 1 x 105 cells / well and incubated at 37 °C for 24 h. Then, the culture medium was replaced with fresh medium containing different concentrations (1, 3, 10, 30 and 100 μg / mL) of PEG-SS-PAE@Moclobemide micelles. After 24 h incubation, the cells were washed three times with PBS and apoptosis was observed by flow cytometry using an Annexin V-FITC / PI Apoptosis Detection Kit.

[0076] 3 Experimental results

[0077] The effects of PEG-SS-PAE, Moclobemide and PEG-SS-PAE@Moclobemide at a concentration of 60 μg / mL on cell proliferation were compared, and the results are shown in Figure 4 PEG-SS-PAE@Moclobemide had no cytotoxicity.

[0078] Example 4, Study on the Anti-breast Cancer Effect of PEG-SS-PAE@Moclobemide

[0079] 1 Experimental materials

[0080] 1.1 Experimental cells and experimental animals

[0081] The experimental cells were triple-negative breast cancer cell line (4T1).

[0082] 6-week-old female BALB / C mice weighing 18-19 g (purchased from Hunan Slike Jingda Experimental Animal Co., Ltd.) were used, and the feeding conditions were as follows: SPF level.

[0083] The animal experiment plan of this subject was approved by the Animal Ethics Committee of our school. The feeding and experiments of all animals were in accordance with the relevant regulations.

[0084] 1.2 Experimental materials

[0085]

[0086] 2 Experimental methods

[0087] 2.1 Cell culture

[0088] The 4T1 cells were added with RPMI-1640 medium containing 10% FBS and 1% antibacterial drugs (penicillin-streptomycin double-antibiotic, 10,000 U / mL) and incubated in a 5% CO2 incubator at 37 °C. The cells were passaged every two days, and the experiment was performed when the confluence of the 4T1 cells reached about 80%.

[0089] 2.2 Construction of BALB / C mouse breast cancer tumor 4T1 cell line xenograft model

[0090] 4T1 cells in logarithmic phase were trypsinized, centrifuged, resuspended with pbs, and the cell density of the cell suspension was adjusted to 10 7 After the abdomen of the female mice was depilated, 100 μL of the cell suspension was injected subcutaneously into the fourth pair of mammary fat pads on the right lower abdomen of the mice using a 1 mL syringe. After the inoculation, the mice were continuously fed for 14 days, and 6 mice with tumor volumes greater than 100 mm 3 The above mice were used for experiments.

[0091] 2.3 Safety of PEG-SS-PAE@Moclobemide nanomicelles in vivo

[0092] The safety of the nanomicelles was evaluated by analyzing the weight change curves of the mice in different treatment groups during the administration, the H&E staining images of the main organs of the mice after the administration, and the blood biochemical indicators, including ALT, AST, CREA, and UREA.

[0093] The obtained mouse hearts, livers, spleens, lungs, and kidneys were fixed in 4% paraformaldehyde fixing solution until the fixing solution completely flooded the tissues, and then the tissues were dehydrated and embedded in paraffin to prepare sections. The sections were stained with hematoxylin-eosin and observed by taking photographs.

[0094] The above mice were subjected to eye blood collection, and whole blood samples were collected using ordinary EP tubes, with a certain space reserved for serum separation. After collection, the samples were stored at 4°C, centrifuged at 3500 rpm for 15 min, and the supernatant was detected for the corresponding indicators.

[0095] 2.4 In vivo distribution of PEG-SS-PAE nanomicelles

[0096] DiR-dyed micelles were prepared by the thin film hydration method, and the red fluorescence of DiR under the in vivo imaging instrument was used to indicate the in vivo distribution of the nanomicelles.

[0097] Six mice with tumor volumes greater than 100 mm 3 were selected from the mice inoculated with tumor cells, and randomly divided into two groups, with 3 mice in each group. After the chest and abdomen of the mice were depilated, free-DiR and DiR-micelles (the injection amount of DiR was equivalent to 10 mg / kg of the body weight of the mice) were injected intraperitoneally. At 2 h, 6 h, 12 h, and 24 h after injection, the in vivo distribution of the fluorescent dye DiR in the mice was observed using a small animal in vivo imaging instrument. The detection conditions were: excitation wavelength 748 nm, emission wavelength 780 nm, exposure time 3 s, and the distribution was recorded by taking photographs.

[0098] 24h after the mice were executed by breaking the neck, and their tumors, hearts, livers, kidneys, lungs, and spleens were taken. The fluorescence signal of the fluorescent dye DiR in the main organs was recorded by live imaging technology to show the in vivo distribution of the micelles, and the average fluorescence intensity of each tissue was quantitatively analyzed.

[0099] 2.5 Anti-tumor effect of PEG-SS-PAE@Moclobemide nanomicelles

[0100] From the above mice, 20 BALB / C mice with breast cancer tumor 4T1 cell line transplanted tumor models with tumor volume greater than 100 mm 3 were selected and randomly divided into 4 groups, 5 mice in each group: ① Blank group (simple injection of normal saline); ② PEG-SS-PAE group; ③ Moclobemide group; ④ PEG-SS-PAE@Moclobemide group. The drug was administered by intraperitoneal injection, and the dose was equivalent to 3 mg / kg of drug.

[0101] The day of administration was recorded as 0 day, and the drug was administered every 3 days for a total of 18 days. The tumor diameter was measured every 3 days, and the longest diameter (A) and shortest diameter (B) were measured and substituted into the following formula to calculate the tumor volume (V) of each group. Then, with the tumor volume as the vertical coordinate and the time as the horizontal coordinate, a tumor growth inhibition curve was drawn. On the 18th day, the mice were sacrificed, the tumors were removed, and the weight was recorded. With the tumor weight as the vertical coordinate and the group as the horizontal coordinate, the final weight of the tumors in each group was compared. The volume calculation formula of the tumor is as follows:

[0102]

[0103] 2.6 Effect of PEG-SS-PAE@Moclobemide on cytokine secretion

[0104] The tumor was cut into pieces and placed in a tissue homogenizer. After mixing with RIPA lysis buffer, it was carefully ground on an ice box for 15 minutes. The upper liquid was transferred to a centrifuge tube, and the protein was extracted by centrifuging the sample at 13,000g. The protein concentration was determined using a BCA kit and a standard curve was drawn, and then the expression of IL-2 and IFN-γ was tested according to the instructions of the ELISA kit.

[0105] 3 Experimental results

[0106] 3.1 Successful construction of mouse breast cancer tumor 4T1 cell line transplanted tumor model

[0107] The mouse breast cancer transplanted tumor model was successfully constructed, and the tumor was located in the breast of the mouse.

[0108] 3.2 Safety of PEG-SS-PAE@Moclobemide

[0109] As Figure 5 shown, the body weight of mice was recorded every 3 days from the first administration, and the body weight of mice in each group before administration was 22 g. After 18 days of treatment, the body weight of the NS group was 22.92 ± 0.86 g, the body weight of the PEG-SS-PAE group was 24.34 ± 1.05 g, the body weight of the Moclobemide group was 24.44 ± 0.54 g, and the body weight of the PEG-SS-PAE@Moclobemide group was 23.54 ± 1.22 g. No significant difference in body weight change was observed (p > 0.05), indicating that the drug did not affect the weight gain of mice and had no toxicity.

[0110] From the tissue structure Figure 6 , compared with the control group, the H&E staining pictures of the main organs had no obvious necrotic area. It was shown that PEG-SS-PAE nanomicelles would not cause damage to the main organs, and had the characteristics of safety and low toxicity. At the same time, it could be seen that the tumor necrosis area of the PEG-SS-PAE@Moclobemide group showed an expanding trend compared with other groups, indicating that it had the effect of killing tumor tissue.

[0111] The blood biochemical results showed (Table 3) that the values of ALT, AST, CREA, and UREA in each group were within the normal range, and had no toxic effect on liver and kidney function.

[0112] Table 3 Blood biochemical values of ALT, AST, CREA, and UREA in each group

[0113]

[0114] 3.3 PEG-SS-PAE@Moclobemide inhibits tumor growth

[0115] The in vivo administration scheme was as described above, and the tumor volume of mice was recorded and calculated every 3 days, and the tumor volume change curve was drawn, and the results are shown in Figure 7 and Figure 8 Compared with the PEG-SS-PAE and Moclobemide groups, the PEG-SS-PAE@Moclobemide group had a significant inhibitory effect on tumors.

Claims

1. The application of moclobemide-loaded nanomicelles in the preparation of antitumor drugs, characterized in that: The tumor is breast cancer. The moclobemide-loaded nanomicelles consist of a carrier and moclobemide encapsulated in the carrier. The carrier is a nanomicelle formed from PEG-SS-PAE copolymer PEG5k-SS-PAE10k. The moclobemide-loaded nanomicelles are prepared by thin-film hydration method. The mass ratio of moclobemide to PEG-SS-PAE is 1 to 3:

6. The PEG5k-SS-PAE10k was prepared according to the following steps: 1) Weigh 2.0g of mPEG5k-SS-NH2 and dissolve it in 20ml of chloroform. Add acryloyl chloride and triethylamine and dissolve completely. Stir the reaction at room temperature for 12h. Wash the reaction solution with water 3 times, dry it with anhydrous sodium sulfate, concentrate it under reduced pressure and pour it into a large amount of ice-cold ether to precipitate. Filter to collect the product and dry it under vacuum to obtain the mPEG5K-SS-propylene product. 2) Weigh 1.0g of mPEG5K-SS-propylene and dissolve it in 10ml of chloroform. Add 1,6-bis(acryloyloxy)hexane and 1,3-bis(4-piperidine)propane and dissolve completely. Stir the reaction at 55℃ for 48h. Concentrate the reaction solution under reduced pressure and pour it into a large amount of ice-cold diethyl ether to precipitate. Filter to collect the product and dry it under vacuum to obtain PEG5k-SS-PAE10k.

2. The application according to claim 1, characterized in that: The encapsulation efficiency of the moclobemide-loaded nanomicelles was 97–99.2%, and the drug loading was 15–37%.

3. The application according to claim 1, characterized in that: The method for preparing the moclobemide-loaded nanomicelles includes the following steps: using PEG-SS-PAE copolymer as a carrier material, moclobemide is encapsulated by thin film hydration method to obtain moclobemide-loaded nanomicelles.

4. The application according to claim 3, characterized in that: The preparation method specifically includes the following steps: 1) Dissolve the carrier material and moclobemide in an organic solvent to obtain a mixed solution; 2) Evaporate the organic solvent in the mixed solution to obtain a composite film; 3) The complex was thinly dissolved in sterile water to prepare a suspension. The suspension was centrifuged at high speed, and the supernatant was the nanomicelle solution loaded with moclobemide.

5. The application according to claim 4, characterized in that: In step 1), the carrier material and moclobemide are dissolved in organic solvents respectively, and the two solutions are mixed and stirred until the liquid is clear and transparent.

6. The application according to claim 5, characterized in that: The organic solvent is chloroform, and in step 2), rotary evaporation is used to evaporate the organic solvent in the mixed solution.

7. The application according to claim 4, characterized in that: In step 3), the high-speed centrifugation speed is 5500-7500 r, and the supernatant obtained by centrifugation is freeze-dried to obtain nano micelle freeze-dried powder loaded with moclobemide.

8. The application according to claim 1, characterized in that: The tumor is a triple-negative breast cancer.

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