A self-assembling nano drug and its preparation method and pharmaceutical application in cardiovascular and cerebrovascular diseases

Abstract

The present invention relates to the field of medical technology, and in particular to a self-assembled nano drug and a preparation method thereof and pharmaceutical application in cardiovascular and cerebrovascular diseases. The self-assembled nano drug comprises the following components: macitentan, a photosensitizer Ce6 and a divalent manganese ion. The present invention has the advantage of excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a self-assembling nano drug and a preparation method thereof, and pharmaceutical application thereof in cardiovascular and cerebrovascular diseases. Background Art

[0002] The indications of Macitentan include pulmonary hypertension, chronic thromboembolic pulmonary hypertension (CTEPH), pulmonary hypertension-related interstitial lung disease (PH-ILD), etc. Pulmonary hypertension is a rare but serious cardiovascular disease characterized by high blood pressure in the pulmonary artery. Macitentan is an endothelin receptor antagonist that can reduce pulmonary artery blood pressure in patients with pulmonary hypertension by inhibiting the action of endothelin. At the same time, it has the effects of improving the tumor immune microenvironment, activating T cell proliferation, and downregulating Tregs cells, which can promote anti-tumor immunotherapy. However, macitentan drug treatment alone has the problems of low drug utilization, poor biocompatibility, and large side effects.

[0003] Photodynamic therapy (PDT) is a new method of treating tumors, precancerous lesions, proliferative skin diseases, and vascular diseases using photosensitive drugs and laser activation. Irradiating the lesion site with a specific wavelength can activate the photosensitive drugs that selectively accumulate in the lesion tissue, triggering a photochemical reaction to destroy the lesion. The photosensitizers in the new generation of photodynamic therapy (PDT) transfer energy to the surrounding oxygen to generate highly active singlet oxygen. Singlet oxygen can undergo oxidation reactions with nearby biomacromolecules, produce cytotoxicity, and then kill diseased cells. Compared with traditional therapies, the advantage of PDT is that it can accurately and effectively treat, and the side effects of this therapy are also very small.

[0004] The principle of photodynamic therapy is a photochemical reaction, and its basic elements are oxygen, photosensitizer and visible light (usually laser). First, the photosensitizer is selectively taken up and stored inside, and then after local irradiation with light of appropriate wavelength, the photosensitizer is activated and produces a photosensitizing effect.

[0005] Studies have shown that various divalent inorganic metal ions such as Co 2+ 、Zn 2+ , Cu 2+ and Mn 2+ etc., play an important role in the immune regulation process. Compared with other metal ions, Mn 2+ It can directly activate cGAS protein and enhance the production and cytoplasmic enrichment of endogenous STING agonist cGAMP by enhancing cGAS's recognition of double-stranded DNA (dsDNA), thereby enhancing the activation efficiency of the STING signaling pathway. 2+It is difficult to break through the intracellular delivery barrier, and the low content of double-stranded DNA in the tumor limits the application of this strategy.

[0006] However, the above drugs are prone to adverse reactions and have poor efficacy. Therefore, it is particularly important to develop a new dosage form that responds to the tumor-specific environment. With the development of nanotechnology, targeted drug carriers based on nanomaterials provide a new solution for achieving tumor-specific drug delivery. Compared with traditional drug dosage forms, nano drug carriers can improve the bioavailability, permeability and stability of anticancer drugs, which further ensures continuous and controllable drug delivery.

[0007] Chinese patent application CN117138042A discloses a divalent inorganic metal ion / photosensitizer protein nanoparticle and its preparation and application. The nanoparticle adopts a new technical idea to effectively solve the problem that the high-valent inorganic oxides usually delivered need to be reduced to a divalent state in the cell to play an immunotherapy role. The nanoparticles are successfully prepared at room temperature in a certain pH aqueous solution. The divalent inorganic metal ions encapsulated therein form a complex with the photosensitizer. The complex has the advantages of stable valence and small size, and can penetrate deep into the tumor tissue through the blood vessel wall. After rapid dissociation in the slightly acidic environment of the lysosome, the divalent inorganic metal ions are released, and the phototherapy effect is exerted. It can destroy the lysosomal membrane, break through the lysosomal barrier and directly release Mn 2+ It is transported to the cytoplasm, induces the release of double-stranded DNA after illumination, produces cGAMP in situ in the cytoplasm, activates the STING signaling pathway, improves the immune response rate, and enhances the efficacy of tumor immunotherapy. Experimental verification shows that it can completely inhibit tumor growth in a mouse in situ breast cancer model. However, the preparation method of this technology is complex and time-consuming. The synthesis and preparation of nanoparticles takes more than 1-6 hours, and its efficacy needs to be further improved.

[0008] Therefore, it is very necessary to develop a self-assembled nanomedicine and its preparation method and pharmaceutical application in cardiovascular and cerebrovascular diseases that can solve the above technical problems. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a self-assembled nano drug with excellent anti-tumor effect, simple preparation process and short time consumption, as well as a preparation method and pharmaceutical application in cardiovascular and cerebrovascular diseases.

[0010] The present invention is achieved through the following technical solutions:

[0011] The first aspect of the present invention provides a self-assembled nano drug composition, comprising the following components: macitentan, photosensitizer Ce6 and divalent manganese ions.

[0012] Among them, Ce6 is dihydrochlorin e6.

[0013] In some embodiments of the present invention, the divalent manganese ion is selected from manganese chloride.

[0014] In some embodiments of the present invention, the mass ratio of macitentan, photosensitizer Ce6 and divalent manganese ions is 200-300:100-200:5.

[0015] In some preferred embodiments of the present invention, the mass ratio of macitentan, photosensitizer Ce6 and divalent manganese ions is 250:150:5.

[0016] The second aspect of the present invention provides a self-assembled nano drug, comprising the self-assembled nano drug composition and pharmaceutically acceptable excipients.

[0017] In some embodiments of the present invention, the pharmaceutically acceptable excipient includes DSPE-PEG2000.

[0018] DSPE-PEG2000 is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0019] The mass ratio of the DSPE-PEG2000 to the macitentan is 1:1.

[0020] The third aspect of the present invention provides a method for preparing a self-assembled nanomedicine, comprising the following steps:

[0021] (1) using solvent A to prepare a divalent manganese ion solution; using solvent B to prepare macitentan solution, Ce6 solution and DSPE-PEG2000 solution respectively;

[0022] (2) first adding water into a container, and then adding a divalent manganese ion solution to obtain a solution 1;

[0023] (3) mixing the macitentan solution, the Ce6 solution and the DSPE-PEG2000 solution to obtain solution 2, adding the solution to solution 1 and stirring to obtain solution 3;

[0024] (4) After centrifuging solution 3, remove the supernatant and resuspend the precipitate in water.

[0025] In some embodiments of the present invention, the mass ratio of the raw material addition amounts of macitentan, photosensitizer Ce6 and divalent manganese ions is 20-42:20-42:200.

[0026] In some preferred embodiments of the present invention, the mass ratio of the raw material addition amounts of macitentan, photosensitizer Ce6 and divalent manganese ions is 30:30:200.

[0027] In some embodiments of the present invention, the solvent A in step (1) is water.

[0028] In some embodiments of the present invention, the solvent B is DMSO.

[0029] In some embodiments of the present invention, the concentration of the divalent manganese ion solution is 8-12 mg / mL, preferably 10 mg / mL.

[0030] In some embodiments of the present invention, the concentration of the macitentan solution is 8-12 mg / mL, preferably 10 mg / mL.

[0031] In some embodiments of the present invention, the concentration of the Ce6 solution is 8-12 mg / mL, preferably 10 mg / mL.

[0032] In some embodiments of the present invention, the concentration of the DSPE-PEG2000 solution is 8-12 mg / mL, preferably 10 mg / mL.

[0033] In some embodiments of the present invention, the volume ratio of the water to the divalent manganese ion solution in step (2) is 4-6:1, preferably 5:1.

[0034] In some embodiments of the present invention, the volume ratio of the macitentan solution, the Ce6 solution and the DSPE-PEG2000 solution in step (3) is 0.8-1.2:0.8-1.2:0.8-1.2, preferably 1:1:1.

[0035] In some embodiments of the present invention, the volume ratio of the solution 2 to the divalent manganese ion solution is 8-10:20, preferably 9:20.

[0036] The fourth aspect of the present invention provides the use of the self-assembled nano-drug composition or the self-assembled nano-drug or the self-assembled nano-drug prepared by the above-mentioned preparation method in the preparation of drugs for treating cardiovascular and cerebrovascular diseases and / or anti-tumor diseases.

[0037] In some embodiments of the invention, the tumor comprises breast cancer.

[0038] In some embodiments of the present invention, the cardiovascular and cerebrovascular diseases include pulmonary hypertension.

[0039] The beneficial effects of the present invention are:

[0040] The present invention adopts macitentan, photosensitizer Ce6 and divalent manganese ions to compound in the range of mass ratio of 20-42:20-42:200, and can form a nano drug with good stability, and the drug loading mass ratio is 200-300:100-200:5, and through synergistic photodynamic therapy, immunogenic cell death is promoted, and the EPR effect of nano drugs in tumors can be enhanced at the same time, so as to play a good synergistic anti-tumor purpose, especially for breast cancer. The therapeutic effect is good and the synergistic effect is obvious. At the same time, the present invention can improve the effect of macitentan in treating cardiovascular and cerebrovascular diseases, especially pulmonary hypertension, and after being prepared into nano drugs, its cardiovascular and cerebrovascular disease and anti-tumor efficacy are further significantly improved.

[0041] The process for preparing the nano drug of the present invention is simple and takes a short time, and can be completed within 35-45 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a scanning electron micrograph of MCM, scale bar = 1 μm.

[0043] Figure 2A These are the particle size test results of MCM, MnCe6, Ce6Mac and MnMac.

[0044] Figure 2B These are the test results for the uniformity of particle size distribution of MCM, MnCe6, Ce6Mac and MnMac.

[0045] Figure 2C Particle size distribution diagram of MCM, MnCe6, Ce6Mac and MnMac.

[0046] Figure 2D This is the standard curve for determining the content of MCM nanodrugs.

[0047] Figure 3A The pictures were taken by laser microscope after 4T1 cells were treated with different concentrations of Ce6 and MCM. Scale bar = 10 μm.

[0048] Figure 3B The statistical results of laser microscope fluorescence values ​​after 4T1 cells were treated with different concentrations of Ce6 and MCM.

[0049] Figure 4 The statistical results of flow cytometric fluorescence values ​​after 4T1 cells were treated with different concentrations of Ce6 and MCM.

[0050] Figure 5 It is the ratio of the fluorescence value of each nanodrug sample to the fluorescence value of the blank control group SOSG.

[0051] Fig. 6ALaser microscope photos of 4T1 cells after treatment with each nanodrug sample and incubation with the DCFH-DA probe. Scale bar = 20 μm.

[0052] Figure 6B Statistical results of laser microscopy fluorescence values ​​after 4T1 cells were treated with each nanodrug sample and incubated with the DCFH-DA probe.

[0053] Figure 7 The MTT toxicity test results of each nanodrug sample on 4T1 cells.

[0054] Fig. 8A Live-dead staining confocal imaging results of the effects of various nanodrug samples on 4T1 cell viability, scale bar = 100 μm.

[0055] Figure 8B Live-dead staining confocal imaging fluorescence statistics of each nanodrug sample on 4T1 cell viability.

[0056] Fig. 9 The statistical results of protein immunoblotting bands and band grayscale values ​​of each nanodrug sample on 4T1 cells.

[0057] Fig.10 Confocal imaging results of immunofluorescence staining of 4T1 cells by various nanodrug samples, scale bar = 10 μm.

[0058] Fig.11 The results of fluorescence imaging of various tissues after injection of MCM or Ce6 into 4T1 breast cancer tumor-bearing mice.

[0059] Fig. 12A This is the tumor size of T1 tumor-bearing mice during the drug treatment stage 4.

[0060] Fig. 12B This is a picture of the tumor volume in 4T1 tumor-bearing mice on the 21st day of drug treatment.

[0061] Fig. 12C This is the tumor weight of 4T1 tumor-bearing mice on day 21 of drug treatment.

[0062] Fig.13A Tunel apoptosis staining of 4T1 tumor-bearing mice on day 21 after drug treatment. Scale bar = 100 μm.

[0063] Fig. 13B Ki67 staining of tumors in 4T1 tumor-bearing mice on day 21 after drug treatment. Scale bar = 100 μm.

[0064] Fig. 13C H&E staining of tumors in 4T1 tumor-bearing mice on day 21 after drug treatment. Scale bar = 200 μm.

[0065] Fig.14 H&E staining of liver, spleen, kidney, and heart tissues of 4T1 tumor-bearing mice 21 days after drug treatment. Scale bar = 200 μm. DETAILED DESCRIPTION

[0066] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0067] Glossary:

[0068] MAC: Macitentan, an endothelin receptor antagonist;

[0069] Ce6: photosensitizer;

[0070] Mn 2+ : Manganese chloride;

[0071] MCM:Mn 2+ , nanodrugs formed by self-assembly of three drugs Ce6 and MAC;

[0072] MnCe6:Mn 2+ Nanodrugs formed by self-assembly of two drugs, Ce6;

[0073] Ce6Mac: Nanodrug formed by self-assembly of two drugs Ce6 and MAC;

[0074] MnMac:Mn 2+ Nanodrugs formed by self-assembly of two drugs, MAC.

[0075] The experimental groups in the present invention are:

[0076] Control: normal healthy mouse group;

[0077] Blank: blank control group;

[0078] Mac: pure free Macitentan;

[0079] Mn 2+ : Pure free manganese chloride;

[0080] C- or Ce6: photosensitizer dark-treated group;

[0081] M+C+M- or M+C+M: free Mn 2+, Ce6 and MAC three drug dark treatment groups;

[0082] MCM- or MCM: Mn 2+ , the dark-treated nanodrug group formed by self-assembly of three drugs Ce6 and MAC;

[0083] C+ or Ce6(+): photosensitizer illumination group;

[0084] M+C+M+ or M+C+M(+): free Mn 2+ , Ce6 and MAC three drug light groups;

[0085] MCM+ or MCM(+): Mn 2+ , the nanodrug light-irradiated group formed by self-assembly of three drugs Ce6 and MAC;

[0086] Among them, each dark treatment group has no light source irradiation, and each light treatment group uses laser irradiation to activate the photosensitizer to exert its effect.

[0087] The preparation methods of each nano drug are as follows:

[0088] 10mg / mL Mn 2+ : Dissolve manganese chloride in ultrapure water;

[0089] 10 mg / mL MAC: DMSO dissolves MAC;

[0090] 10mg / mL Ce6: DMSO dissolves Ce6;

[0091] 10 mg / mL DSPE-PEG2000: Dissolve DSPE-PEG2000 in DMSO.

[0092] (1)MCM preparation:

[0093] ① Prepare a 1-2mL chromatography bottle, add 1mL of ultrapure water, and then add 200μL of 10mg / mL Mn 2+ .

[0094] ② Take 30 μL of 10 mg / mL MAC, 10 mg / mL Ce6, and 10 mg / mL DSPE-PEG2000, mix them, and then add them dropwise into the chromatographic bottle of ①, then stir at 2000 rpm at room temperature and avoid light for 5 minutes.

[0095] ③ After stirring for 5 minutes, the mixed solution was centrifuged (14680rpm, 30min) to remove the remaining free Ce6 and MAC. After the centrifugation, the supernatant was removed and the precipitate was resuspended in 1mL ultrapure water to obtain the material MCM. The scanning electron microscopy image of MCM is shown in Figure 1 Shown, scale bar = 1 μm.

[0096] (2) Preparation of MnCe6:

[0097] ① Prepare a 1-2mL chromatography bottle, add 1mL of ultrapure water, and then add 200μL of 10mg / mL Mn 2+ .

[0098] ② Take 30 μL of 10 mg / mL Ce6 and 10 mg / mL DSPE-PEG2000 respectively, mix them, and then add them dropwise into the chromatographic bottle of ①, then stir at 2000 rpm at room temperature and avoid light for 5 minutes.

[0099] ③ After stirring for 5 minutes, the mixed solution was centrifuged (14680rpm, 30min) to remove the remaining free Ce6. After the centrifugation, the supernatant was removed and the precipitate was resuspended with 1mL of ultrapure water to obtain the material MnCe6.

[0100] (3) Ce6Mac preparation:

[0101] ① Prepare a 1-2mL chromatography bottle and add 1mL of ultrapure water.

[0102] ② Take 30 μL of 10 mg / mL MAC, 10 mg / mL Ce6, and 10 mg / mL DSPE-PEG2000, mix them, and then add them dropwise into the chromatographic bottle of ①, then stir at 2000 rpm at room temperature and avoid light for 5 minutes.

[0103] ③ After stirring for 5 minutes, the mixed solution was centrifuged (14680rpm, 30min) to remove the remaining free Ce6 and MAC. After the centrifugation, the supernatant was removed and the precipitate was resuspended with 1mL of ultrapure water to obtain the material Ce6Mac.

[0104] (4) MnMac preparation:

[0105] ① Prepare a 1-2mL chromatography bottle, add 1mL of ultrapure water, and then add 200μL of 10mg / mL Mn 2+ .

[0106] ② Take 30 μL of 10 mg / mL MAC and 10 mg / mL DSPE-PEG2000 respectively, mix them, and then add them dropwise into the chromatographic bottle of ①, and then stir at 2000 rpm at room temperature in the dark for 5 minutes.

[0107] ③ After stirring for 5 minutes, the mixed solution was centrifuged (14680 rpm, 30 minutes) to remove the remaining MAC. After the centrifugation, the supernatant was removed and the precipitate was resuspended with 1 mL of ultrapure water to obtain the material MnMac.

[0108] The prepared MCM, MnCe6, Ce6Mac and MnMac were placed at room temperature for 7 days, and the particle size was measured every day using a nanoparticle size analyzer. The average particle size results are as follows: Figure 2A The particle size distribution uniformity results are shown in Figure 2B As shown, Figure 2B The smaller the PDI, the more uniform the particle size distribution. The particle size distribution on the 7th day is shown in the figure below. Figure 2C shown.

[0109] Next, the content of each drug in the MCM nanomedicine was determined using a high performance liquid chromatograph (conditions: the mobile phase consisted of 0.2% trifluoroacetic acid as solvent A and acetonitrile as solvent B) to form a standard calibration curve (e.g. Figure 2D shown).

[0110] The final measured content of macitentan in MCM was 250 mg / L; Ce6 150 mg / L; manganese ion 5 mg / L; the drug loading ratio of macitentan: Ce6: manganese ion was: 250:150:5.

[0111] 1. Confocal Concentration Endocytosis

[0112] 4T1 cells were inoculated into confocal microplates and incubated for 24 h. After the cells adhered to the wall, the original culture medium was discarded. Then Ce6 (5, 10, 15 μg / mL, solvent DMSO) and MCM of equal volume and concentration were added to 4T1 cells (5.0×10 4 The cells were incubated in the dark for 8 h with PBS, and then stained with Hoechst 33342 for 15 min in the dark. The cells were then washed 3 times with PBS, and fresh culture medium was added. The fluorescence characteristics of the cells were observed and analyzed using a laser microscope (CLSM), and the fluorescence changes in the cells were analyzed. Figure 3A The fluorescence value statistics are shown in Figure 3B shown.

[0113] 2. Flow Cytometry Concentration Endocytosis

[0114] 4T1 cells were seeded in six-well plates and incubated for 24 h. After the cells adhered to the wall, the original culture medium was discarded, and then Ce6 (5, 10, 15 μg / mL) and MCM of equal volume and concentration were added to 4T1 cells (5.0×10 5 The cells were incubated in the dark for 8 h, and then gently washed with PBS for 3 times. Finally, the cells were collected and analyzed by flow cytometry for the fluorescence changes in the cells. Figure 4 shown.

[0115] 3. Singlet oxygen reactive oxygen species fluorescence probe SOSG reactive oxygen species determination

[0116] SOSG (5 μM) was mixed with each nanodrug sample in PBS. The concentration of each nanodrug sample was 15 μg / mL Ce6 as the standard. Each group took drugs of the same corresponding concentration, and SOSG (5 μM) without nanodrug sample was set as a blank control group and exposed to laser (635 nm, 80 mW / cm 2 The fluorescence intensity of each group was recorded for 10 seconds / time, with an excitation wavelength of 488 nm and an emission wavelength of 537 nm. The dark treatment group was not irradiated with laser. The ratio of the fluorescence value of each group to the fluorescence value of SOSG in the blank control group was calculated and recorded as F t / F, the result is as follows Figure 5 shown.

[0117] 4. Reactive oxygen species (ROS) determination using the fluorescent probe DCFH-DA

[0118] After 4T1 cells were inoculated in a confocal dish and incubated for 24 h, they were washed with PBS. Then, each group used 2 μg / mL Ce6 as the standard and took the same concentration to treat 4T1 cells for 8 h. After washing with PBS three times, the cells were incubated with DCFH-DA (10 μM) at room temperature for 20 min and then irradiated (1.8 J / cm 2 , 29.8mW / cm 2 ) for 2 min, and the dark treatment group was not irradiated with laser. DCFH-DA (10 μM) without nanodrug sample was set as blank control group (Blank), and the green fluorescence of active oxygen in cells of each group was observed under CLSM microscope. Fig. 6A The fluorescence value statistics are shown in Figure 6B shown.

[0119] 5. MTT assay

[0120] Appropriate amounts of 4T1 cells were inoculated in 96-well plates. After 24 h, each nanomedicine group was set up with a gradient according to the concentration of Ce6 in MCM (1.32 mg / L, 1.98 mg / L, 2.96 mg / L, 4.44 mg / L, 6.67 mg / L and 10.00 mg / L). Six parallel samples were set for each concentration, i.e., n=6. After incubation with 4T1 cells for 8 h, laser irradiation (1.8 J / cm 2 , 29.8mW / cm 2 After 90s), the dark treatment group was not irradiated with laser and incubated for 24h in dark conditions. After adding 20μL MTT (5mg / mL) and incubating for 4h, the culture medium was discarded, 150μL DMSO was added and shaken to dissolve for 20min, and then detected by microplate reader. The toxicity of other groups to 4T1 cells was detected under the same experimental conditions. The results are shown in Figure 7 shown.

[0121] 6. Live-dead staining

[0122] Each nanomedicine group was incubated with 4T1 cells at an equal concentration of Ce6 (2 mg / L) (prepared in serum culture medium) for 8 h, and then laser (1.8 J / cm 2 , 29.8mW / cm 2 ) were irradiated for 6min and 10min respectively. The dark treatment group was not irradiated with laser. The culture medium was discarded under light-proof conditions and washed 3 times with PBS. A 4T1 cell group without nanodrug samples (incubated with serum culture medium) was set as a blank control group (Blank). Then, the cells were stained with a live / dead cell double staining kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) and the cell viability was observed and analyzed using CLSM. The confocal imaging results are shown in Fig. 8A As shown, the confocal imaging fluorescence statistics are shown in Figure 8B shown.

[0123] 7. Western Blot and Immunofluorescence Staining

[0124] 7.1 Western Blot:

[0125] (1) Protein extraction

[0126] 4T1 cells were inoculated in a 6-well plate. After the cells adhered to the wall, the drugs were added and incubated. The incubation concentration of the experimental group was mainly 15 mg / L of Ce6 in MCM. The drug concentration of the control group was calculated according to the equivalent concentration. After 8 hours of drug addition, the cells were illuminated for 10 minutes and then incubated for another 4 hours before treatment. Three replicate wells were set for each group. The lysis buffer was prepared in proportion in advance, with a volume ratio of RIPABuffer lysis buffer: protease inhibitor: phosphatase inhibitor = 100:1:1 to prevent protein degradation and inhibit protein phosphorylation. The original culture medium was discarded, and 2 mL of 4°C precooled PBS was added to each well for washing 3 times. Then, the PBS was discarded and 100 μL of precooled prepared lysis buffer was added to each well. After lysis for 20 minutes, protein was extracted with a cell scraper. Before use, the cell scraper was soaked in anhydrous ethanol, rinsed 1-3 times with double distilled water, and the attached cells were gently scraped with a cell scraper. The cells and lysate were transferred to a centrifuge tube on ice. Use an ultrasonic pulverizer to pulverize cells and break genomic DNA to prevent the sample from being too viscous and affecting the loading. Then centrifuge at 12000r / min for 20min at 4℃ to extract the supernatant protein and record the supernatant protein volume for quantitative protein dilution. All steps are performed on ice.

[0127] (2) BCA method for protein concentration determination

[0128] Take 5 μL of supernatant protein sample and dilute it 4 times with ultrapure water for protein quantification. Prepare BCA working solution in the ratio of A: B = 50: 1, 95 μL per well, and add it to a 96-well plate. Divide the 96-well plate into a standard solution area and a sample area. Prepare 6 gradient concentrations of standard solution, with 2-3 replicates for each gradient; 2-3 replicates for each group of samples, take 5 μL / well of standard solution and add it to the standardization area, take 5 μL / well of the 4-fold diluted sample and add it to the sample area, then add 95 μL / well of the prepared BCA working solution to the standardization area and the sample area, and do not add samples to the outer edges of the 96-well plate. Place in a 37°C incubator and incubate in the dark for 30 minutes. Detect the OD value at a wavelength of 562 nm with an enzyme-labeled detector; calculate the standard curve and calculate the protein concentration. According to the protein concentration, add lysate and 5× protein loading buffer to prepare protein samples of equal concentration and volume. Vortex mix, heat to denature at 100°C for 10 min, cool on ice and store in a -80°C refrigerator until use.

[0129] (3) Glue preparation

[0130] Select a pair of 1.5cm glass plates with glue, make the bottoms flush, put the plates into the gel maker, add ultrapure water to check for leaks, and observe the degree of level drop after 20 minutes. Prepare 10% SDS-PAGE gel according to the instructions of the kit and the molecular weight of the target protein required. First prepare 7-8mL of the lower layer separation gel, use a 1mL pipette to stick to the wall of the glass plate and add it to the middle of the glass plate to 2cm from the upper edge of the short glass plate, add water or isopropanol to seal the upper gap and drive away all bubbles. Let it stand for 20-30 minutes, and after the gel solidifies, discard the sealed isopropanol; use absorbent paper to absorb all residual water, then prepare 2-3mL of the upper layer concentrated gel, and slowly insert a 1.5mm thick 10-hole comb after the gel is poured, let it stand for 15-20 minutes, and wait for the gel to solidify.

[0131] (4) Sample loading and electrophoresis

[0132] Add the prepared gel to the electrophoresis device, add 1x SDS electrophoresis buffer until the glass plate is completely covered, gently pull out the comb in parallel, add the protein sample in the middle of the card slot, the protein loading amount is 20μg, add markers at both ends, 5μL at the left end and 3μL at the right end, when loading, aim the gun tip at the back glass and slowly add it, plug the positive and negative electrodes, red to red, black to black. Perform electrophoresis in two stages, the first stage voltage is 80v, 20-30min, so that the sample runs out of the separation gel and into the concentration gel. The second stage voltage is 120-180mv, until the bromophenol blue at the front runs out of the gel, about 60-90min.

[0133] (5) Transfer

[0134] Cut a PVDF membrane of the same size as the gel and soak it in methanol for 60 seconds for activation. Use the wet transfer method for electrotransfer. Place the sponge gasket, filter paper, gel, PVDF membrane, filter paper, sponge gasket in order from the negative electrode to the positive electrode. Use the "sandwich" clamp method to remove the bubbles between the gel and the PVDF membrane. Place the corresponding positive and negative electrodes in the transfer rack and transfer tank, pour in the pre-cooled electrotransfer solution, and ice bath. The current is adjusted to 120V and the time is set to 90min. The time can be adjusted appropriately according to the molecular weight of the protein.

[0135] (6) Closed

[0136] Take out the transferred membrane and mark the front and back. Add 5% skim milk, that is, weigh 2.5g skim milk powder and add 50mL TBST. Incubate on a shaker at room temperature at 60r / min for 90min. Put the small box on the shaker and incubate at 60r / min for 90min.

[0137] (7) Incubation with primary antibody

[0138] Take out the blocked PVDF membrane and wash the membrane 5 times with 1×TBST, 110-120r / min for 5min each time; determine the target range, compare the colorimetric molecule card, cut the desired target protein band; put it into the marked strip box; prepare the primary antibody, only the target protein specifically binds to the primary antibody. Dilute with a ratio of primary antibody: primary antibody diluent = 1:2000; soak each strip with 3mL of primary antibody, put the strip box in a shaker in a 4℃ refrigerator, and incubate the primary antibody overnight for 12h. The primary antibody selected is the immunogenic cell death marker protein HMGB-1, CRT and the internal reference protein β-Actin.

[0139] (8) Incubation with secondary antibody

[0140] Recover the primary antibody. Wash the strips 5 times with 1×TBST, 110-120r / min, 5min each time. Prepare the secondary antibody, select the corresponding mouse / rabbit secondary antibody according to the primary antibody; prepare the secondary antibody according to the ratio of secondary antibody: 5% skim milk powder = 1:2000; soak each strip with 3mL of secondary antibody, and then place it on a shaker at room temperature, 60r / min, 90min.

[0141] (9) Exposure

[0142] Recover the secondary antibody. Wash the strips 5 times with 1×TBST, 110-120r / min, 5min each time. Prepare the luminescent solution, black and white in a 1:1 ratio, the luminescent solution can bind to the binding site of the secondary antibody. Take out the strips, absorb the TBST buffer with paper, lay the strips flat, remove bubbles, and add luminescent solution. Turn on the multifunctional imaging system, set the exposure time, mode and save path, detect the strips, and analyze the grayscale value with Image J software. The results are as follows Fig. 9 shown.

[0143] 7.2 Immunofluorescence staining

[0144] 4T1 cells were inoculated in 6-well plates. After the cells adhered to the wall, drugs were added for incubation. The incubation concentration of the experimental group was mainly 15 mg / L of Ce6 in MCM. The drug concentration of the control group was calculated according to the equivalent concentration. After 8 hours of drug addition, the cells were illuminated for 10 minutes and then incubated for another 4 hours before treatment. The cells in the Blank group were incubated without any drugs and only with serum culture medium. The incubation time and operation time of this group were consistent with those of other groups. The other groups were incubated with serum culture medium with corresponding drug concentrations. Three replicates were set for each group. The original culture medium was discarded and washed 3 times with 1 mL PBS. Fix with 1 mL 4% paraformaldehyde (15 min). The fixative was discarded and washed 3 times with 1 mL PBS. Block with 1 mL 10% goat serum (9 mL PBS + 1 mL goat serum) for 1 hour, discard the blocking solution, and wash 3 times with 1 mL PBS. The primary antibodies used were HMGB-1, CRT, a marker of immunogenic cell death, and β-Actin, an internal reference protein. Then, the primary antibody dilution solution (PD-L1 antibody: 10% goat serum = 1:500) was prepared, 500 μL for the light-treated group and 150 μL for the dark-treated group, overnight. The primary antibody was recovered, washed 3 times with 1 mL of PBS, and incubated with fluorescent secondary antibody for 1 hour {secondary antibody (1:200 = 5% BSA: goat anti-rabbit IgG H&L)--where 5% BSA is 150 mg powder + 3 ml PBS}. Washed 3 times with PBS, stained the nucleus with DAPI for 15 minutes, washed 3 times with PBS, and observed using a confocal microscope. The results are as follows Fig.10 shown.

[0145] 8. Animal Imaging

[0146] Female BALB / c mice, 20-24 g, 6-8 weeks old, were selected as experimental subjects and purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mice were kept under the following conditions: laboratory rodent feed pellets, supplemented with adequate food and water. The ambient temperature was 22±2°C, with a 12-h dark / light cycle.

[0147] 4T1 breast cancer cells (2×10 5 ) was injected subcutaneously into the right hind leg of BALB / c mice to establish a 4T1 breast cancer tumor-bearing mouse model. When the average tumor volume reached 100 mm 3Around 12 hours after administration, 400 μL of MCM or Ce6 was injected into the tail vein, and the fluorescence changes in the mice were recorded in real time at a certain time interval under an excitation wavelength of 630 nm. At the predetermined time intervals of 2, 6, 8 and 12 hours, the real-time fluorescence intensity of the tumor site of the mice was measured by the in vivo fluorescence imaging system. 12 hours after administration, the mice were killed and the heart, liver, spleen, lung, kidney and tumor were collected by dissection for organ ex vivo fluorescence imaging analysis, and the in vitro fluorescence imaging of each tissue was measured and analyzed. The results are shown in Figure 2. Fig.11 As shown, a high fluorescence intensity indicates a large amount of drug accumulation, and a low fluorescence intensity indicates a small amount of drug accumulation, indicating that the drug has a good accumulation effect in the tumor site within 12 hours.

[0148] 9. Inhibition of animal tumor growth

[0149] The tumor-bearing mice were randomly divided into Control group, Blank group, Mn group 2+ There were 9 groups in total, including group, MAC group, C- group, MCM- group, C+ group, M+C+M+, and MCM+, with 5 mice in each group. Each group was treated at a predetermined time point, that is, after the grouping was completed, 400 μL of MCM was injected into the tail vein of 4T1 tumor-bearing mice on the 1st day, 4th day, and 7th day (3 treatments in total), and the other groups were converted according to equal volume and equal concentration. The dark-treated group was not irradiated with laser, and each light-treated group was irradiated with laser for 10 minutes (65V) 6 hours after administration. The body weight and tumor volume of each mouse were observed and recorded every 2 days, the changes in the longest and shortest diameters of the mouse tumor were measured, and the changes in tumor volume were monitored. The results are shown in the figure. Fig. 12A The tumor volume was calculated as (tumor width) 2 × (tumor length) / 2. On the 21st day after administration, the mice were killed, the tumor tissue was removed, the tumor size was photographed, and the tumor weight was recorded. The results are shown in Fig. 12B and Fig. 12C As shown, after 21 days of drug treatment, the tumor growth rate of mice in the MCM+ group was significantly inhibited compared with other groups. Fig. 12B There were 5 mouse tumors in the MCM+ group. Due to the obvious tumor suppression effect in this group, the tumor at the bottom was extremely small and difficult to see with the naked eye, so it was circled with a dotted line to indicate the suppression result of the tumors in 5 mice.

[0150] At the same time, the tumor tissues were collected for TdT-mediated dUTP nick-end labeling (Tunel), nuclear-associated antigen Ki67 and H&E staining to analyze the apoptosis and proliferation of tumor cells in each group. The results are shown in Fig.13A , Fig. 13BAs shown in the figure, the green fluorescence in the Tunel signal of the MCM+ group is the strongest, and the green fluorescence in the Ki67 signal is the weakest, indicating that in animal experiments, MCM+ can also maximize the apoptosis of tumor cells and inhibit the proliferation of tumor cells. In addition, H&E staining ( Fig. 13C ) analysis also confirmed that after MCM+ treatment, tumor cells showed extensive and obvious necrosis.

[0151] 10. H&E staining

[0152] After taking out the above-mentioned fresh heart, liver, spleen, and kidney important organ tissues for perfusion treatment, they were made into slices. The slice samples were immersed in xylene I and xylene II in turn for 15 minutes, and then dewaxed. After ensuring that there was no wax residue, they were placed in ethanol gradient dehydration in turn. The stained area was completely covered with hematoxylin staining solution. After 3 minutes, it was gently rinsed with running water for 1-3 seconds. Put it in 1% hydrochloric acid ethanol, and after returning to blue for 10-30 seconds, it was rinsed with running water for 10-15 minutes, and then stained with 1% eosin solution for 1-3 minutes. The sample was transferred to a gradient ethanol solution and placed for 3-5 seconds for dehydration, and then transferred to xylene I for 3-5 seconds and xylene II for 3-5 minutes. Finally, it was sealed with neutral gum. The results are as follows Fig.14 As shown, there were no obvious pathological changes in the important organ tissues of the heart, liver, spleen, and kidney of 4T1 tumor-bearing mice during the administration of the nanodrug, indicating that the drug had good biosafety.

[0153] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A self-assembled nanomedicine, characterized in that: The invention comprises the following components: macitentan, photosensitizer Ce6, divalent manganese ions and DSPE-PEG2000, wherein the mass ratio of macitentan, photosensitizer Ce6 and divalent manganese ions is 200-300:100-200:5; The method for preparing the self-assembled nanomedicine comprises the following steps: (1) Solvent A was used to prepare a divalent manganese ion solution; solvent B was used to prepare macitentan solution, Ce6 solution and DSPE-PEG2000 solution respectively; (2) First add water into a container, then add divalent manganese ion solution to obtain solution 1; (3) mixing the macitentan solution, the Ce6 solution and the DSPE-PEG2000 solution to obtain solution 2, adding the solution to solution 1 and stirring to obtain solution 3; (4) After centrifuging solution 3, remove the supernatant and resuspend the precipitate in water.

2. The self-assembled nanomedicine according to claim 1, characterized in that: The divalent manganese ion is selected from manganese chloride.

3. The self-assembled nanomedicine according to claim 1, characterized in that: The mass ratio of macitentan, photosensitizer Ce6 and divalent manganese ions is 250:150:

5.

4. A method for preparing the self-assembled nanomedicine according to any one of claims 1 to 3, characterized in that: The steps include: (1) Solvent A was used to prepare a divalent manganese ion solution; solvent B was used to prepare macitentan solution, Ce6 solution and DSPE-PEG2000 solution respectively; (2) First add water into a container, then add divalent manganese ion solution to obtain solution 1; (3) mixing the macitentan solution, the Ce6 solution and the DSPE-PEG2000 solution to obtain solution 2, adding the solution to solution 1 and stirring to obtain solution 3; (4) After centrifuging solution 3, remove the supernatant and resuspend the precipitate in water.

5. The preparation method according to claim 4, characterized in that: In step (1), the solvent A is water; the solvent B is DMSO; the concentration of the divalent manganese ion solution is 8-12 mg / mL; the concentration of the macitentan solution is 8-12 mg / mL; the concentration of the Ce6 solution is 8-12 mg / mL; the concentration of the DSPE-PEG2000 solution is 8-12 mg / mL; In step (2), the volume ratio of water to the divalent manganese ion solution is 4-6:1; In step (3), the volume ratio of the macitentan solution, the Ce6 solution and the DSPE-PEG2000 solution is 0.8-1.2:0.8-1.2:0.8-1.2; the volume ratio of the solution 2 to the divalent manganese ion solution is 8-10:

20.

6. Use of the self-assembled nano drug according to any one of claims 1 to 3 or the self-assembled nano drug prepared by the preparation method according to any one of claims 4 to 5 in the preparation of drugs for treating cardiovascular and cerebrovascular diseases and / or anti-tumor diseases.

7. The use according to claim 6, characterized in that: The tumor includes breast cancer.

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