Multifunctional cisplatin liposome as well as preparation method and application thereof

Abstract

The invention provides a multifunctional cisplatin liposome as well as a preparation method and application thereof. The liposome is prepared from epigallocatechin gallate, cis-platinum, zinc peroxide nanoparticles, a distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 long-acting fragment, a distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-oestrone targeting fragment, lecithin and cholesterol, and the particle size of the liposome is 80 to 130 nm. According to the multifunctional liposome disclosed by the invention, the zinc peroxide nanoparticles are introduced, so that the function of slowly releasing oxygen in a tumor microenvironment can be realized, and oxygen deficit is reversed; through targeting fragment modification, an active targeting effect can be realized by combining with an estrogen receptor highly expressed on the surface of a breast cancer cell; the epigallocatechin gallate and cis-platinum are co-loaded, so that the sensitivity of chemotherapeutic drugs can be improved, the toxic and side effects of the chemotherapeutic drugs are reduced, a good breast cancer resisting effect is achieved, and the tumor volume is remarkably reduced.

Description

Technical Field

[0001] The 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 occurs in the epithelial tissue of the human mammary gland. It has become one of the major diseases that affect women's health due to its high mortality and morbidity. The 2022 Annual Cancer Statistics Report released by the American Cancer Society shows that the incidence of breast cancer in the United States still ranks first among female cancers, accounting for about 31% of all cancers in women, and the number of deaths from female breast cancer accounts for 15% of all women with cancer. From the epidemiological trends of the past few decades, the mortality rate of breast cancer in some high-income countries has continued to decline significantly, with an annual mortality rate of more than 2%, but the mortality rate in many middle-income and low-income countries remains stagnant or even continues to rise. The latest cancer report published by the National Cancer Center of my country in the Journal of the National Cancer Center shows that the incidence of breast cancer in China also ranks first among female cancers, accounting for 16.72% of all new cancers, and the mortality rate of female breast cancer is still on the rise. Breast cancer is a major cancer that seriously endangers the health of women in my country, and its occurrence will cause serious trauma to women both physically and mentally. Early prevention, early detection and early standardized treatment are the key to the prevention and treatment of breast cancer.

[0003] Human breast cancer has been classified into a multidimensional framework based on histopathological classification, clinical features, and molecular typing. At the time of diagnosis, tumors are broadly classified histologically as either carcinoma in situ or invasive carcinoma, depending on the spread of malignant cells from the breast lobules or ducts to the surrounding stroma. In addition to morphological classification, breast tumors are clinically divided into three major categories based on the expression of estrogen receptor (ER), progesterone receptor (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 unclear. 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. 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. The expression of ERα is less than 10% in 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 show dynamic tandem through signaling molecules, forming a unique tumor microenvironment (TME), which is considered the "soil" for cancer development. The stromal cells in this environment are different from cells in normal tissues. They change certain inherent characteristics through functional inhibition or overactivity mainly induced by tumor cells. In addition, various stromal cells and their secreted factors coordinate important homeostatic pathways, providing favorable conditions for tumor proliferation, invasion and metastasis. The rapid growth of tumors leads to the inability of blood supply and oxygen supply to keep up with demand, so the tumor site shows a state of hypoxia. Hypoxia further induces tumor angiogenesis. The newly generated tumor blood vessels are different from normal vascular tissues. They are arranged in a disorderly manner and have high permeability, which reduces drug delivery and increases the possibility of tumor cell invasion and migration to distant sites. At present, the clinical treatment of breast cancer is still mainly surgical resection, supplemented by radiotherapy, chemotherapy and immunotherapy. Chemotherapy is the most commonly used and most effective adjuvant treatment method. Combination chemotherapy for breast cancer generally includes the combination of alkylating agents, anthracyclines and anti-metabolites, which can significantly reduce the risk of breast cancer recurrence. The use of combined chemotherapy can not only improve the efficacy of breast cancer chemotherapy, but also reduce the dose of drugs, reduce the toxicity of drugs, and reduce or delay the occurrence 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. Less cross-resistance is conducive to combined use. However, the use of cisplatin often results in 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 main secondary metabolites of tea plants and the main bioactive ingredients in green tea, accounting for about 12% to 24% of the dry weight of tea leaves. Epigallocatechin gallate (EGCG) is a common catechin compound, accounting for about 50% to 80% of catechins in tea leaves. The structure rich in phenolic hydroxyl groups gives it a strong antioxidant and free radical scavenging ability. In addition, EGCG also has the effects of inhibiting oxidative stress and inflammation, lowering blood lipids and blood sugar, and anti-tumor. The anti-cancer mechanism of EGCG includes angiogenesis inhibition, tumor cell death induction, and tumor growth inhibition. EGCG inhibits tumor angiogenesis by inhibiting 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 to generate metal ions and hydrogen peroxide in normal physiological environments or acidic tumor microenvironments, 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 in normal physiological environments, but it will degrade rapidly in acidic environments. At the same time, the zinc ions generated after its degradation enter the mitochondria, destroy their respiratory chain, promote the production of reactive oxygen, and thus exert chemical kinetics to induce tumor cell apoptosis.

[0008] By using liposomes as drug carriers, the above-mentioned ingredients can be co-encapsulated and delivered to the tumor site at the same time. At the same time, based on the good biocompatibility of liposomes, the toxic and side effects of free drugs can be reduced. However, after entering the body, traditional liposomes will be quickly adsorbed by plasma opsonin proteins, stimulate the mononuclear macrophage system, and thus be quickly cleared. Therefore, the introduction of DSPE-mPEG2000 long-acting fragments can prolong the in vivo circulation time of liposomes through the spatial barrier effect of PEG molecules. However, due to the enhanced permeability and retention effect (EPR) of tumor tissue, liposomes modified with PEG alone still do not achieve satisfactory therapeutic effects. Therefore, ligand-modified liposomes are used for active targeted drug delivery. Estrone, as a ligand of 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 targeted liposome based on zinc peroxide nanoparticles self-generated oxygen co-loaded with epigallocatechin gallate and cisplatin, as well as a preparation method and application, which 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 the tumor volume.

[0010] In order to achieve the above object, the present invention provides an active targeting liposome based on self-generated oxygen from zinc peroxide nanoparticles 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, with a particle size of 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 and directly supplying oxygen to tumor cells, reversing the hypoxic microenvironment of the tumor; the epigallocatechin gallate and cisplatin in the liposome have a synergistic anti-tumor effect, and 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 liposome; DSPE-mPEG in the liposome 2000 The long-acting fragment reduces the interference between liposomes and albumin and the reticuloendothelial system in vivo, prolonging the circulation time of liposomes in vivo; 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 vigorously stirring zinc peroxide, hydrogen peroxide and sodium hydroxide in a methanol solution. Preferably, the liposomes are prepared 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 and hydrated 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 scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying 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 technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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, centrifuge the mixed solution at 11000 rpm for 10 min, discard the supernatant, wash the precipitate three times with methanol solution, and dry it at 60 ° C to obtain ZnO2 nanoparticles.

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

[0029] The transmission electron microscopy image of the zinc peroxide nanoparticles obtained in this example is 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 DSPE-mPEG2000 long-acting fragment and 2 mg of ZnO2 nanoparticles were weighed and dissolved in 10 mL of chloroform / methanol (v / v=1:1) mixed solution, the mixed solution was mixed at normal pressure for 3-5 min, the drug and phospholipid were fully mixed, and then vacuumed slowly to form a translucent film, and then 1.5 mL of physiological saline solution containing cisplatin (cisplatin concentration of 0.4 mg / mL) was added to the film, wherein the molar ratio of cisplatin to epigallocatechin gallate was 3:1, and the film was rotated and hydrated at 37°C for 2 h. Then 1.5 mg of DSPE-PEG2000-ES targeting fragment was added to the above liquid and incubated at 37°C for 30 min. The obtained liquid was subjected to ultrasonic treatment for 3s and cooled for 3s in an ice-water bath for a total of 10 minutes, and extruded three times through a polycarbonate 80nm membrane through a liposome extruder to obtain an active targeted liposome based on self-oxygenation of zinc peroxide nanoparticles 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), succinic anhydride (SAA, 370.0 mg, 3.7 mmol), triethylamine (TEA, 561.0 mg, 5.54 mmol) and 4-dimethylaminopyridine (DMAP, 23.0 mg, 0.185 mmol) after drying were added, and the mixture was reacted for 24 h under magnetic stirring. The mixture was obtained by concentration and dissolved in 10 mL of water. The pH value of the solution was adjusted to 7-8 with potassium carbonate (K2CO3) and adjusted to pH 3 with 12N HCl. The solution was precipitated as it was, and then 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 and placed in a 10 mL round-bottom flask. N, N-carbonyl diimidazole (CDI, 2.19 mg, 0.0135 mmol) was added. DSPE-PEG2000-NH2 (54.0 mg, 0.027 mmol) was added after magnetic stirring at room temperature and the reaction was allowed to proceed overnight. The solution was concentrated under reduced pressure, and ether was slowly added dropwise under stirring. After the precipitate was evenly dispersed, it was 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 antitumor 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 thereof is substantially the same as that of Example 2, except that: the common 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] The JEM-2100F transmission electron microscope was used to observe Example 1, and the results were as follows:

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

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

[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 particle size of the zinc peroxide nanoparticles in Example 1 of the present invention is less than 30 nm, and the PDI is less than 0.3, indicating that the size is uniform, and the zeta potential is -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. Determination of oxygen release from ZnO2 nanoparticles in vitro

[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 will not affect the encapsulation efficiency of the two drugs, and the two drugs will not affect each other. On the contrary, the introduction of the long-acting fragment and the targeting fragment can 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] Example 2 and Comparative Examples 2-4 were observed using a JEM-2100F transmission electron microscope. The results are shown in the attached figure. Figure 3 As shown, AC is the electron micrograph 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 the electron micrograph of ZnO2@EPL-CDDP / EGCG liposomes in Example 2 of the present invention. These four liposomes are all spherical and have 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 value and zeta potential of the liposomes were detected using a Malvern particle size analyzer. The results are shown in Table 3. With the introduction of the long-acting fragment and the targeting fragment, the liposome particle size increased, and the values ​​of the dispersion coefficient PDI of each liposome were less than 0.3, indicating that the liposome particle size distribution was uniform. The zeta potential values ​​of the liposomes were all negative, and with the insertion of the long-acting fragment, the absolute value of the zeta potential value decreased.

[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 test

[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, and the changes in particle size and PDI value within 48 hours were measured. 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, A and B are 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 ZnO2@EPL-CDDP / EGCG in Example 2 has good storage stability at 4°C. C and D are the changes in encapsulation efficiency of ZnO2@EPL-CDDP / EGCG in Example 2 after storage at 4°C and 25°C for 14 days, respectively. The two drugs encapsulated in Example 2 have higher storage stability at 4°C. E and F are the dispersion stability of ZnO2@EPL-CDDP / EGCG in Example 2 in three release media. There is no significant difference in particle size and PDI value, indicating that 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] Example 2 and Comparative Examples 1-4 were placed in a dialysis bag, which was immersed in a release medium (PBS) and shaken on a constant temperature 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 EGEG 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 preparation exhibits a slow release kinetic behavior, and the cumulative release of both drugs is less than 60% within 8 hours. CE is the cumulative release of Zn 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 a pH-dependent drug release kinetic behavior, 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] In order to observe the uptake of Example 2 and Comparative Examples 1-4 by MCF-7 cells, an in vitro cell uptake experiment was performed 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, and then the cell nuclei were stained with Hoechst 33342 dye, and the changes in the cell uptake of different preparations were observed under a fluorescence microscope. Hoechst 33342 showed blue fluorescence under a fluorescence microscope, and rhodamine B showed red fluorescence 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, which is due to the active targeting effect of estrogen modification 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, which is 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, and the culture medium was resuspended and diluted to an appropriate multiple to make the cell concentration 5×10 4 Take 100 μL of the diluted cell suspension (5×10 3 10 μL CCK-8 working solution was added, and the cells were incubated for another 2 hours at 37°C for detection at a wavelength of 450 nm. The relative cell survival rate 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±2g were selected and kept in a clean environment at room temperature of 25℃ with 12h day and night alternation. MCF-7 cells in logarithmic growth phase were taken and 0.1mL cell suspension (5×10 6 When the tumor volume grows to about 70 mm 3 At 14:00 pm, mice were intravenously injected with drugs (6.0 mg / kg CDDP, 9.0 mg / kg EGCG) through the tail vein, once every four days, for a total of five times. The physical signs of the mice were observed, and the weight and tumor volume of each group of mice were measured. The mice were killed after 28 days, and the tumor tissues were removed and weighed to calculate the tumor inhibition rate of each group. The results are shown in the attached figure. Figure 8 As shown, A is the curve of tumor volume change, B is the curve of tumor weight change, C is the tumor inhibition rate, and D is the curve of mouse weight change. ZnO2@EPL-CDDP / EGCG in Example 2 of this study has the strongest inhibitory effect on tumor growth, with the highest tumor inhibition rate of 85.36%. There is no significant difference in the weight of mice in each preparation group, indicating that at this therapeutic dose, each preparation group has high safety and no 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 targeted fragments, the liposomes have a good killing effect on breast cancer cells and tumor tissues and have 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 new and more effective treatment methods for a variety of clinical malignant tumors, and provide 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, and 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. A multifunctional cisplatin liposome according to claim 1, which is a self-oxygenated, actively targeted, long-circulating co-loading liposome with 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 liposome is prepared by dissolving lecithin, cholesterol, epigallocatechin gallate, zinc peroxide nanoparticles and distearoylphosphatidylethanolamine-polyethylene glycol 2000 long-acting fragment in a chloroform / methanol (v / v=1:1) mixed solution to form a translucent film, then adding cisplatin / normal saline solution, and co-incubating with distearoylphosphatidylethanolamine-polyethylene glycol 2000-estrone targeting fragment after hydration to obtain the liposome.

4. The method for preparing a multifunctional cisplatin liposome according to claim 3, characterized in that: Zinc chloride is dissolved in methanol to prepare solution A, sodium hydroxide is dissolved in methanol to prepare solution B, and 30% hydrogen peroxide solution and solution B are slowly added dropwise to solution A while vigorously stirring at room temperature, 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, and the particle size of the obtained zinc peroxide nanoparticles is 20-30 nm.

5. The method for preparing a multifunctional cisplatin liposome according to claim 3, characterized in that: 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. An application of a multifunctional liposome of cisplatin, characterized in that: The liposome is used as a drug for preparing a drug for treating breast cancer. The distearoylphosphatidylethanolamine-polyethylene glycol 2000-estrone targeting fragment in the liposome serves as a ligand for the estrogen receptor highly expressed on the surface of breast cancer cells, specifically recognizes and binds to the estrogen receptor on the surface of breast cancer cells, and increases the intracellular uptake of the liposome through ligand-receptor mediated endocytosis. The zinc peroxide nanoparticles in the liposome can be degraded to generate zinc ions and hydrogen peroxide in the acidic microenvironment of the tumor, promote the production of reactive oxygen species and directly supply oxygen to tumor cells, thereby reversing the hypoxic microenvironment of the tumor.

7. The use of a multifunctional cisplatin liposome according to claim 6, characterized in that: The multifunctional cisplatin liposome has a significant inhibitory effect on breast cancer cells, with a half inhibitory concentration (IC 50 ) was 10.78-16.21 μM, and the tumor inhibition rate was 65-83%.

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