Multifunctional liposome of cisplatin, preparation method and application thereof

Abstract

The application provides a multifunctional liposome of cisplatin and a preparation method and application thereof. The liposome is prepared from epigallocatechin gallate, cisplatin, zinc peroxide nanoparticles, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 long-acting fragments, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-estrogen targeting fragments, lecithin and cholesterol, and has a particle size of 80-130 nm. The multifunctional liposome can realize the slow release of oxygen in the tumor microenvironment by introducing zinc peroxide nanoparticles, thereby reversing hypoxia; through the modification of the targeting fragments, the multifunctional liposome can realize the active targeting effect by combining with the estrogen receptors highly expressed on the surface of breast cancer cells; through the co-loading of epigallocatechin gallate and cisplatin, the sensitivity of the chemotherapeutic drugs can be improved, the toxic and side effects thereof can be reduced, and the multifunctional liposome has a good anti-breast cancer effect and can significantly reduce the tumor volume.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an active targeted liposome based on zinc peroxide nanoparticles self-generating oxygen and co-loading epigallocatechin gallate and cisplatin, as well as a preparation method and application thereof, for anti-tumor treatment targeting breast cancer. Background Art

[0002] Breast cancer is a malignant tumor that develops in the epithelial tissue of the mammary gland. Due to its high mortality and morbidity rates, it remains a major health threat to women. The 2022 American Cancer Society's Annual Cancer Statistics report shows that breast cancer remains the leading cancer among women in the United States, accounting for approximately 31% of all cancers in women, and breast cancer deaths account for 15% of all cancer deaths in women. Epidemiological trends over the past few decades have shown that breast cancer mortality rates in some high-income countries have continued to decline significantly, with annual declines exceeding 2%. However, mortality rates in many middle-income and low-income countries remain stagnant or even increasing. The latest cancer report published by the National Cancer Center of my country in the Journal of the National Cancer Center shows that breast cancer also leads the incidence of cancer among women in China, accounting for 16.72% of all new cancers, and the mortality rate continues to rise. Breast cancer is a major cancer that poses a serious threat to the health of women in my country, causing severe physical and mental trauma. Early prevention, early detection, and early, standardized treatment are key to breast cancer prevention and treatment.

[0003] Human breast cancer has been classified using a multidimensional framework based on histopathological classification, clinical features, and molecular typing. At diagnosis, tumors are broadly classified histologically as either in situ or invasive carcinoma, depending on the spread of malignant cells from the breast lobules or ducts into the surrounding stroma. In addition to morphological classification, breast tumors are clinically divided into three major categories based on the expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2): ER-positive, HER2-positive, and triple-negative breast cancer. ER-positive tumors account for approximately 70% of all breast cancers.

[0004] At present, the exact cause of breast cancer is still not very clear. A large number of studies have shown that the occurrence and development of breast cancer are affected by many risk factors, including hormones, diet, lifestyle, radiation and genetic factors. The breast is the most important target organ of female hormones, and its development process is affected by a variety of hormones, including endogenous hormones such as estrogen, androgen, progesterone, oxytocin and prolactin, as well as some metabolism-related hormones involved in breast development such as thyroxine, insulin, growth hormone and corticosteroids. Many studies have confirmed that these hormones are closely related to the occurrence and development of breast cancer. Estrogen receptors and progesterone receptors have been widely used in clinical practice as important indicators reflecting the biological characteristics of breast cancer. The role of estrogen in breast cancer has been confirmed, and its role as a breast carcinogen has long been a focus of attention. It is speculated that the following three mechanisms may be involved in the carcinogenic effect of estrogen: (1) stimulating cell proliferation through its receptor-mediated hormone activity; (2) increasing the mutation rate through cytochrome P450-mediated metabolic activation to produce genotoxic effects; (3) inducing the production of aneuploidy. Estrogen receptor (ER) is highly expressed in breast cancer. ERα is expressed in less than 10% of healthy breast cells, but its expression increases to 80% in breast cancer tissue. Approximately 83% of breast cancer patients over 50 years old are hormone receptor-positive (ER+ / PR+).

[0005] Tumor cells and their surrounding components dynamically interact through signaling molecules, forming a unique tumor microenvironment (TME), considered the "soil" for cancer development. Stromal cells within this microenvironment differ from cells in normal tissues, altering certain inherent characteristics through functional suppression or hyperactivity primarily induced by tumor cells. Furthermore, various stromal cells and their secreted factors coordinate important homeostatic pathways, providing favorable conditions for tumor proliferation, invasion, and metastasis. Rapid tumor growth leads to an inability to keep up with blood and oxygen supply, resulting in a state of hypoxia at the tumor site. Hypoxia further induces tumor angiogenesis. Unlike normal vascular tissue, newly formed tumor vessels are disorganized and highly permeable, reducing drug delivery while increasing the potential for distant invasion and migration of tumor cells. Currently, the clinical treatment of breast cancer remains primarily surgical resection, supplemented by radiotherapy, chemotherapy, and immunotherapy. Chemotherapy is the most commonly used and effective adjuvant treatment. Combination chemotherapy for breast cancer generally includes alkylating agents, anthracyclines, and antimetabolites, which can significantly reduce the risk of breast cancer recurrence. The use of combination chemotherapy can not only improve the efficacy of breast cancer chemotherapy, but also reduce the dose of drugs, reduce drug toxicity, and reduce or delay the development of drug resistance. Platinum drugs have long been used in clinical anti-tumor chemotherapy. Cisplatin, as the first-generation platinum anti-tumor drug, has a broad anti-cancer spectrum, strong effects, high activity, and is easily compatible with other anti-tumor drugs. The low cross-resistance is conducive to combination therapy. However, the use of cisplatin often causes various significant side effects, such as nephrotoxicity, bone marrow suppression, peripheral neuropathy, ototoxicity, and allergic reactions, which limit its high-dose and long-term use in clinical practice. In addition, the hypoxic tumor microenvironment reduces the sensitivity of breast cancer cells to cisplatin.

[0006] Catechins are the primary secondary metabolites of the tea plant and the main bioactive components of green tea, accounting for approximately 12% to 24% of the dry weight of tea leaves. Epigallocatechin gallate (EGCG) is a common catechin compound, accounting for approximately 50% to 80% of the catechins in tea leaves. Its phenolic hydroxyl-rich structure gives it strong antioxidant and free radical scavenging abilities. EGCG also has anti-oxidative and anti-inflammatory effects, lowers blood lipids and blood sugar, and has anti-tumor effects. EGCG's anti-cancer mechanisms include angiogenesis inhibition, tumor cell death induction, and tumor growth inhibition. EGCG inhibits tumor angiogenesis by suppressing the expression of hypoxia-inducible factor 1α (HIF-1α) and its downstream protein, vascular endothelial growth factor (VEGF), in tumor cells.

[0007] Nanoplatforms based on metal peroxides (such as magnesium peroxide, calcium peroxide, and zinc peroxide) can be degraded in normal physiological environments or acidic tumor microenvironments to generate metal ions and hydrogen peroxide, where hydrogen peroxide is further degraded to release oxygen, thereby directly supplying oxygen to tumor cells. Zinc peroxide is different from magnesium peroxide and calcium peroxide. It is stable under normal physiological conditions, but it degrades rapidly in acidic environments. At the same time, the zinc ions generated after its degradation enter the mitochondria, disrupt their respiratory chain, promote the production of reactive oxygen species, and thus exert chemical kinetics to induce tumor cell apoptosis.

[0008] By using liposomes as drug carriers, the aforementioned components can be co-encapsulated and delivered to the tumor site. Furthermore, due to the good biocompatibility of liposomes, the toxic side effects of free drugs can be reduced. However, upon entering the body, traditional liposomes are rapidly adsorbed by plasma opsonin proteins, stimulating the mononuclear macrophage system and thus being rapidly cleared. Therefore, the introduction of the long-acting DSPE-mPEG2000 fragment can prolong the in vivo circulation time of the liposomes through the spatial barrier effect of the PEG molecule. However, due to the enhanced permeability and retention effect (EPR) of tumor tissue, simply PEG-modified liposomes still do not achieve satisfactory therapeutic effects. Therefore, ligand-modified liposomes are used for active targeted drug delivery. Estrone, as a ligand for the estrogen receptor, can actively target and bind to the estrogen receptor highly expressed on the surface of breast cancer cells, thereby achieving the effect of active targeted drug delivery. Summary of the Invention

[0009] The purpose of the present invention is to provide an active targeting liposome based on zinc peroxide nanoparticles self-generated oxygen and co-loaded with epigallocatechin gallate and cisplatin, as well as a preparation method and application. The liposome can target the estrogen receptor on the surface of MCF-7 cells, increase the targeting and uptake of the liposome, and at the same time prolong the circulation time in the body, enhance the killing effect on breast cancer cells, have a good anti-ovarian cancer effect, and significantly reduce tumor volume.

[0010] In order to achieve the above object, the present invention provides an active targeting liposome based on zinc peroxide nanoparticles self-generated oxygen co-loaded with epigallocatechin gallate and cisplatin, the liposome is composed of lecithin, cholesterol, epigallocatechin gallate, cisplatin, zinc peroxide nanoparticles, DSPE-mPEG 2000 Long-acting fragments and DSPE-PEG 2000-ES targeting fragment reaction, its particle size is 80-130nm; the zinc peroxide nanoparticles in the liposome can be degraded to generate zinc ions and hydrogen peroxide in the acidic microenvironment of the tumor, destroying the mitochondrial membrane potential, promoting the production of reactive oxygen species and directly supplying oxygen to tumor cells, reversing the tumor hypoxic microenvironment; the epigallocatechin gallate and cisplatin in the liposome have a synergistic anti-tumor effect. This synergistic strategy can increase the sensitivity of breast cancer cells to the chemotherapy drug cisplatin and reduce the toxic side effects of cisplatin; the DSPE-PEG in the liposome 2000 -ES targeting fragment is a fragment that specifically recognizes estrogen receptors, specifically recognizes and binds to estrogen receptors on the surface of MCF-7 cells, enters cells through estrogen receptor-mediated endocytosis, and increases the uptake of the liposomes; DSPE-mPEG in the liposomes 2000 The long-acting fragment reduces the interference between liposomes and albumin and the reticuloendothelial system in the body, and prolongs the circulation time of liposomes in the body; the IC 50 The values ​​were 10.78-16.21 μM, and the tumor inhibition rate was 65-83%.

[0011] Preferably, the zinc peroxide nanoparticles are obtained by reacting zinc peroxide with hydrogen peroxide and sodium hydroxide in a methanol solution with vigorous stirring. Preferably, the liposomes are obtained by mixing lecithin, cholesterol, epigallocatechin gallate, zinc peroxide nanoparticles and DSPE-mPEG. 2000 The long-acting fragment alcohol was dissolved in a chloroform / methanol (v / v=1:1) mixed solution to form a translucent film, and then cisplatin / normal saline solution was added. After hydration, it was mixed with DSPE-PEG 2000 -ES targeting fragment co-incubation obtained.

[0012] Preferably, the lecithin is egg yolk lecithin, the molar ratio of egg yolk lecithin to cholesterol is 2-5:1, the molar ratio of cisplatin to phospholipid is 1:10-30, and the molar ratio of cisplatin to epigallocatechin gallate is 1:4-4:1.

[0013] Preferably, the dosage of the zinc peroxide nanoparticles is 1-5 mg.

[0014] Preferably, the zeta potential of the liposome is -21.24±0.84 mV, the encapsulation efficiency of cisplatin is 43.21±2.13%, and the encapsulation efficiency of epigallocatechin gallate is 96.61±2.97%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the solutions in this application, a brief introduction will be given below to the drawings required for use in the description of the embodiments of this application. Obviously, the drawings described below are some embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] Figure 1 Transmission electron microscopy image of zinc peroxide nanoparticles in Example 1 of the present invention.

[0017] Figure 2 Example 1 of the present invention is an in vitro oxygen release experiment of zinc peroxide nanoparticles.

[0018] Figure 3 Transmission electron microscopy images of liposomes of ZnO2@EPL-CDDP / EGCG in Example 2 of the present invention and ZnO2@L-CDDP / EGCG, ZnO2@PL-CDDP / EGCG and ZnO2@EL-CDDP / EGCG in Comparative Examples 2-4.

[0019] Figure 4 Storage stability and dispersion stability of Example 2 of the present invention.

[0020] Figure 5 In vitro drug release curves of Example 2 of the present invention and Comparative Examples 1-4.

[0021] Figure 6 In vitro cellular uptake study of Example 2 of the present invention and Comparative Examples 1-4.

[0022] Figure 7 Cytotoxicity and IC of Example 2 and Comparative Examples 1-4 on MCF-7 cells 50 value.

[0023] Figure 8 In vivo anti-tumor effect study of Example 2 of the present invention and Comparative Examples 1-4. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0025] Example 1

[0026] A method for preparing zinc peroxide nanoparticles, comprising:

[0027] Weigh 0.5 g of zinc chloride and dissolve it in 5 mL of methanol solution to prepare solution A. Weigh 0.3 g of sodium hydroxide and dissolve it in 5 mL of methanol solution to prepare solution B. Slowly add 1 mL of 30% hydrogen peroxide solution and solution B to solution A at 150 rpm and 25°C in a constant temperature oscillator. After reacting for 4 hours, the mixed solution is centrifuged at 11000 rpm for 10 minutes, the supernatant is discarded, the precipitate is washed 3 times with methanol solution, and dried at 60°C to obtain ZnO2 nanoparticles.

[0028] After testing, the particle size of the zinc peroxide nanoparticles was 27.78±2.16 nm.

[0029] The transmission electron microscopy images of the zinc peroxide nanoparticles obtained in this example are shown in the attached Figure 1 .

[0030] The in vitro oxygen release of the zinc peroxide nanoparticles obtained in this example is shown in the attached Figure 2 .

[0031] Example 2

[0032] A method for preparing active targeted liposomes based on zinc peroxide nanoparticles self-generating oxygen and co-loading epigallocatechin gallate and cisplatin, comprising:

[0033] 40 mg of lecithin, 7 mg of cholesterol, 2 mg of epigallocatechin gallate, 13 mg of the long-acting DSPE-mPEG2000 fragment, and 2 mg of ZnO2 nanoparticles were dissolved in 10 mL of a chloroform / methanol (v / v = 1:1) mixture. The mixture was mixed at atmospheric pressure for 3-5 minutes to allow the drug and phospholipid to mix thoroughly. After that, the mixture was slowly evacuated to form a translucent film. 1.5 mL of saline solution containing cisplatin (0.4 mg / mL) was added to the film, where the molar ratio of cisplatin to epigallocatechin gallate was 3:1. The film was rotated and hydrated at 37°C for 2 hours. Then, 1.5 mg of the targeting DSPE-PEG2000-ES fragment was added to the solution and incubated at 37°C for 30 minutes. The resulting liquid was ultrasonicated for 3 seconds and cooled for 3 seconds in an ice-water bath for a total of 10 minutes. It was then extruded three times through an 80 nm polycarbonate membrane using a liposome extruder to obtain active targeted liposomes based on zinc peroxide nanoparticles self-generated oxygen and co-loaded with epigallocatechin gallate and cisplatin, namely ZnO2@EPL-CDDP / EGCG. The preparation method of DSPE-PEG2000-ES is as follows (the same as in the following examples and comparative examples):

[0034] Estrone (ES, 500.0 mg, 1.85 mmol) was dissolved in dry tetrahydrofuran (THF), and succinic anhydride (SAA, 370.0 mg, 3.7 mmol), dried triethylamine (TEA, 561.0 mg, 5.54 mmol), and 4-dimethylaminopyridine (DMAP, 23.0 mg, 0.185 mmol) were added. The mixture was reacted under magnetic stirring for 24 hours. The resulting mixture was concentrated and dissolved in 10 mL of water. The pH of the solution was adjusted to 7-8 with potassium carbonate (K2CO3) and to pH 3 with 12N HCl. The solution was allowed to precipitate, and the contents were filtered to obtain 674 mg of ES-COOH.

[0035] ES-COOH (50.0 mg, 0.135 mmol) was dissolved in 1 mL of dichloromethane in a 10 mL round-bottom flask. N,N-carbonyldiimidazole (CDI, 2.19 mg, 0.0135 mmol) was added. DSPE-PEG2000-NH2 (54.0 mg, 0.027 mmol) was added under magnetic stirring at room temperature and allowed to react overnight. The solution was concentrated under reduced pressure, and ether was slowly added dropwise with stirring. The resulting precipitate was evenly dispersed and allowed to stand, filtered, and dried to obtain DSPE-PEG2000-ES.

[0036] The transmission electron micrograph of the multifunctional active targeting liposome obtained in this example is shown in the attached Figure 3 D.

[0037] The stability of the multifunctional active targeting liposomes obtained in this example is shown in the attached Figure 4 .

[0038] The in vitro drug release curve of the multifunctional active targeting liposomes obtained in this example is shown in the attached Figure 5 .

[0039] The in vitro cellular uptake of the multifunctional active targeting liposomes obtained in this example is shown in the attached Figure 6 .

[0040] The in vitro cytotoxicity of the multifunctional active targeting liposomes obtained in this example is shown in the attached Figure 7 .

[0041] The in vivo anti-tumor effect of the multifunctional active targeting liposomes obtained in this example is shown in the attached Figure 8 .

[0042] Comparative Examples 1-4

[0043] Comparative Examples 1-4 are free drug ZnO2@CDDP / EGCG, common liposome ZnO2@L-CDDP / EGCG ( Figure 3 A), long-acting liposome ZnO2@PL-CDDP / EGCG ( Figure 3B) and targeted liposome ZnO2@EL-CDDP / EGCG ( Figure 3 C), the preparation method is basically the same as that of Example 2, except that: the ordinary liposomes are not added with DSPE-mPEG 2000 and DSPE-PEG 2000 -ES; targeted liposomes without DSPE-mPEG 2000 ; Long-acting liposomes without DSPE-PEG 2000 -ES.

[0044] Characterization of Example 1

[0045] 1. Observation of ZnO2 nanoparticle morphology

[0046] Example 1 was observed using a JEM-2100F transmission electron microscope, and the results were as follows:

[0047] As attached Figure 1 As shown, ZnO2 nanoparticles are spherical and uniform in size, about 20nm.

[0048] 2. Determination of ZnO2 nanoparticle size, PDI value and Zeta potential

[0049] Example 1 was dispersed in an ultrapure aqueous solution, and its particle size, PDI value, and zeta potential were measured using a Malvern particle size analyzer. The results are shown in Table 1. The zinc peroxide nanoparticles of Example 1 had a particle size of less than 30 nm and a PDI value of less than 0.3, indicating uniform size. The zeta potential was -3.19 mV.

[0050] Table 1 Particle size, PDI value and zeta potential of Example 1 of the present invention (n=3)

[0051]

[0052] 3. In vitro oxygen release assay of ZnO2 nanoparticles

[0053] Example 1 was dispersed in PBS (pH 7.4) buffer solution and acetate (pH 5.5) buffer solution, and the oxygen content in the solution was monitored in real time using a DOG 3082 dissolved oxygen meter. The results are shown in the attached figure. Figure 2 As shown, the oxygen production rate of zinc peroxide nanoparticles in Example 1 of the present invention in an acidic environment is faster than that in a physiological environment, indicating that ZnO2 nanoparticles can directly supply oxygen to tumor tissues.

[0054] Determination of encapsulation efficiency and drug loading in Example 2 and Comparative Examples 2-4

[0055] Example 2 and Comparative Examples 2-4 were demulsified, and the encapsulation efficiency (EE) and drug loading (DL) of cisplatin and epigallocatechin gallate were determined by HPLC. The results are shown in Table 2. The introduction of the long-acting fragment and the targeting fragment did not affect the encapsulation efficiency of the two drugs, nor did the two drugs affect each other. On the contrary, the introduction of the long-acting fragment and the targeting fragment could enhance the stability of the plasma membrane and slightly improve the encapsulation efficiency.

[0056] Table 2 Encapsulation efficiency and drug loading of liposomes of Example 2 of the present invention and Comparative Examples 2-4 (n=3)

[0057]

[0058] Characterization of Example 2 and Comparative Examples 1-4

[0059] 1. Morphological observation

[0060] JEM-2100F transmission electron microscope was used to observe Example 2 and Comparative Examples 2-4. The results are shown in the attached figure. Figure 3 As shown in Figures 2-4, AC are electron micrographs of ZnO2@L-CDDP / EGCG, ZnO2@PL-CDDP / EGCG, and ZnO2@EL-CDDP / EGCG liposomes in Comparative Examples 2-4 of the present invention, and D is an electron micrograph of ZnO2@EPL-CDDP / EGCG liposomes in Example 2 of the present invention. All four liposomes are spherical with uniform size distribution.

[0061] 2. Determination of particle size, PDI value and Zeta potential

[0062] The diluted solutions of Example 2 and Comparative Examples 2-4 were taken and the particle size, PDI, and zeta potential of the liposomes were measured using a Malvern particle size analyzer. The results, shown in Table 3, show that the liposome particle size increased with the introduction of the long-acting segment and the targeting segment. The PDI values ​​of all liposomes were less than 0.3, indicating a uniform liposome size distribution. The zeta potential values ​​of the liposomes were all negative, and the absolute value of the zeta potential decreased with the addition of the long-acting segment.

[0063] Table 3 Particle size, PDI value and Zeta potential of liposomes of Example 2 of the present invention and Comparative Examples 2-4 (n=3)

[0064]

[0065] 3. Stability testing

[0066] Storage stability: Example 2 was stored at 4°C and 25°C for 14 days, and samples were taken at specific time intervals to determine the changes in its encapsulation efficiency. In addition, Example 2 was stored at 4°C for 14 days, and a Malvern particle size analyzer was used to detect changes in its particle size, PDI value, and zeta potential. Dispersion stability: Example 2 was placed in three release media to determine the changes in particle size and PDI value within 48 hours. The three media were PBS solution, serum, and complete cell culture medium (10% FBS + 90% culture medium). PBS solution and serum are the most commonly used release media in in vitro release experiments, and the complete cell culture medium is to simulate the growth environment of human cells. The results are shown in the attached figure. Figure 4 As shown in the figure, A and B show the changes in particle size, PDI and zeta potential of ZnO2@EPL-CDDP / EGCG in Example 2 after storage at 4°C for 14 days. There is no significant change in the three, indicating that the ZnO2@EPL-CDDP / EGCG in Example 2 has good storage stability at 4°C. C and D show the changes in the encapsulation efficiency of ZnO2@EPL-CDDP / EGCG in Example 2 after storage at 4°C and 25°C for 14 days, respectively. The two encapsulated drugs in Example 2 have higher storage stability at 4°C. E and F show the dispersion stability of ZnO2@EPL-CDDP / EGCG in Example 2 in three release media. There is no significant difference in the particle size and PDI values, indicating that the ZnO2@EPL-CDDP / EGCG in Example 2 has good stability in the simulated human cell growth environment.

[0067] 4. Investigation of in vitro release efficiency

[0068] The samples of Example 2 and Comparative Examples 1-4 were placed in dialysis bags, which were immersed in a release medium (PBS) and shaken on a thermostatic shaker at 37°C and 100 rpm for 48 hours. At predetermined time intervals, samples were taken to detect the drug concentration and a cumulative release curve was plotted. The results are shown in the attached figure. Figure 5 As shown, A is the cumulative release curve of CDDP from Example 2 of the present invention and Comparative Examples 1-4, and B is the cumulative release curve of EGCG from Example 2 of the present invention and Comparative Examples 1-4. The free drug ZnO2@CDDP / EGCG is released rapidly, and the cumulative release of both drugs exceeds 80% within 8 hours, while the liposome formulation shows a slow release kinetic behavior, and the cumulative release of both drugs within 8 hours is less than 60%. CE is the ZnO2@CDDP / EGCG release curve under different pH conditions. 2+ , CDDP and EGCG cumulative release curves from Example 2 of the present invention. The results show that Example 2 of the present invention has pH-dependent drug release kinetics, which can effectively increase the drug concentration in the tumor site and reduce the toxic side effects on normal tissues.

[0069] In vitro cellular uptake of Example 2

[0070] To observe the uptake of Example 2 and Comparative Examples 1-4 by MCF-7 cells, in vitro cellular uptake experiments were conducted using the fluorescent dye rhodamine B (Rh B) as a missing indicator. Example 2 and Comparative Examples 1-4 were co-incubated with MCF-7 cells, followed by Hoechst 33342 staining of the cell nuclei. Changes in cellular uptake of the different formulations were observed under a fluorescence microscope. Hoechst 33342 fluoresces blue under a fluorescence microscope, while rhodamine B fluoresces red under a fluorescence microscope.

[0071] As attached Figure 6 As shown, the cell fluorescence images of free Rh B, L-Rh B, PL-Rh B, EL-Rh B and EPL-Rh B in Example 2 of the present invention and Comparative Examples 1-4 show that the fluorescence intensity of free Rh B in the cell is the weakest, while the fluorescence intensity of the preparation group in the cell is stronger than that of free Rh B, among which EL-Rh B has the strongest fluorescence intensity in the cell. This is because the estrogen modification plays an active targeting role by targeting the estrogen receptor highly expressed on the surface of breast cancer cells. The fluorescence intensity of the EPL-Rh B group is slightly weaker than that of the EL-Rh B group due to the steric hindrance effect of PEG. The above results show that the targeting fragment can specifically recognize and bind to the estrogen receptor on the surface of MCF-7 cells, enter the cell through receptor-mediated endocytosis, and increase the uptake of liposomes.

[0072] In vitro cytotoxicity of Example 2 and Comparative Examples 1-4

[0073] MCF-7 cells in the logarithmic growth phase (cultured in MEM medium containing 10% fetal bovine serum and 1% streptomycin and penicillin mixed solution) were placed in a 25 cm 2 After digestion, the cells were centrifuged at 1000 rpm for 5 min, resuspended in the culture medium, and diluted to an appropriate multiple to make the cell concentration 5 × 10 4 Take 100 μL of diluted cell suspension (5×10 3 Cells were inoculated into 96-well cell culture plates (cells / well). Control wells, blank wells, and drug-addition wells were set up simultaneously. When the cells in the wells grew to 80%, drugs were added in triplicate and different concentrations of drugs were added. The cells were incubated at 37°C with 5% CO2 for 24 hours. 10 μL of CCK-8 working solution was added, and the cells were incubated for another 2 hours before detection at a wavelength of 450 nm using a microplate reader. The relative cell viability was calculated, and the IC 50 The results are as follows Figure 7 As shown, A is the MCF-7 cell viability, B is the IC of each preparation 50All preparations showed dose-dependent cytotoxic effects on MCF-7 cells, among which ZnO2@EPL-CDDP / EGCG showed the strongest tumor cell killing ability and had the lowest IC 50 The value is 14.20μM.

[0074] In vivo antitumor effects of Example 2 and Comparative Examples 1-4

[0075] Healthy female BALB / c nude mice weighing 18 ± 2 g were selected and housed in a clean environment at room temperature of 25°C with a 12-h day and night cycle. MCF-7 cells in the logarithmic growth phase were taken and 0.1 mL of cell suspension (5 × 10 6 When the tumor volume grows to about 70 mm 3 At 14 days, the mice were intravenously injected with drugs (CDDP: 6.0 mg / kg, EGCG: 9.0 mg / kg) through the tail vein, once every four days, for a total of five doses. The mice were observed for signs, and the weight and tumor volume of each group were measured. After 28 days, the mice were killed, the tumor tissues were removed and weighed, and the tumor inhibition rate of each group was calculated. The results are shown in the attached figure. Figure 8 As shown, A is the tumor volume change curve, B is the tumor weight change curve, C is the tumor inhibition rate, and D is the mouse body weight change curve. The ZnO2@EPL-CDDP / EGCG in Example 2 of this study showed the strongest tumor growth inhibition, with the highest tumor inhibition rate of 85.36%. There were no significant differences in mouse body weights among the formulation groups, indicating that at this therapeutic dose, each formulation group was highly safe and did not produce systemic toxicity.

[0076] In summary, the long-acting targeted co-loaded liposomes based on ZnO2 of the present invention have the ability to release oxygen in an acidic microenvironment and can reverse the hypoxic microenvironment of the tumor. In addition, by introducing long-acting fragments and targeting fragments, the liposomes have a good killing effect on breast cancer cells and tumor tissues with less toxic side effects. This new self-generated oxygen co-loaded nano drug delivery system will make an important contribution to the development of the field of pharmaceutical preparations, provide a new and more effective treatment method for a variety of clinical malignant tumors, and provide a more important experimental basis for improving the targeted treatment effect of clinical combined chemotherapy drugs, reducing their toxic side effects, and improving the quality of life of clinical tumor patients. It has good application prospects.

[0077] Although the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as limiting the present invention.

Claims

1. A multifunctional liposome of cisplatin, characterized in that: The liposome is made of lecithin, cholesterol, epigallocatechin gallate, cisplatin, zinc peroxide nanoparticles, distearoylphosphatidylethanolamine-polyethylene glycol 2000 long-acting fragment and distearoylphosphatidylethanolamine-polyethylene glycol 2000-estrone targeting fragment.

2. The multifunctional liposomes of cisplatin according to claim 1, which are self-oxygenating, actively targeted, long-circulating co-loading liposomes, have a particle size of 80-130 nm, a zeta potential of -21.24 ± 0.84 mV, an encapsulation efficiency of cisplatin of 43.21 ± 2.13%, and an encapsulation efficiency of epigallocatechin gallate of 96.61 ± 2.97%.

3. A method for preparing a multifunctional cisplatin liposome, characterized in that: The liposomes are prepared by dissolving lecithin, cholesterol, epigallocatechin gallate, zinc peroxide nanoparticles and distearoylphosphatidylethanolamine-polyethylene glycol 2000 long-acting fragment in a v / v = 1:1 chloroform / methanol mixture to form a translucent film, which is then added with cisplatin / normal saline solution and co-incubated with distearoylphosphatidylethanolamine-polyethylene glycol 2000-estrone targeting fragment after hydration.

4. The method for preparing a multifunctional cisplatin liposome according to claim 3, wherein: The preparation method of the zinc peroxide nanoparticles comprises the following steps: Zinc chloride is dissolved in methanol to prepare solution A, and sodium hydroxide is dissolved in methanol to prepare solution B. While vigorously stirring at room temperature, 30% hydrogen peroxide solution and solution B are slowly added dropwise to solution A, wherein the molar ratio of zinc chloride to sodium hydroxide is 1:2-4, and the molar ratio of zinc chloride to hydrogen peroxide is 1:2-4. The particle size of the resulting zinc peroxide nanoparticles is 20-30 nm.

5. The method for preparing a multifunctional cisplatin liposome according to claim 3, wherein: The lecithin is egg yolk lecithin, the molar ratio of egg yolk lecithin to cholesterol is 2-5:1, the molar ratio of cisplatin to phospholipid is 1:10-30, the molar ratio of cisplatin to epigallocatechin gallate is 1:4-4:1, and the dosage of the zinc peroxide nanoparticles is 1-5 mg.

6. Use of the multifunctional liposome of cisplatin according to claim 1 in the preparation of a drug for treating breast cancer.

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