Composite nano-drug loaded with mir-873 simulant and mir-20a inhibitor as well as preparation method and application of composite nano-drug

Abstract

The invention provides a composite nano-drug loaded with a mir-873 simulant and a mir-20a inhibitor as well as a preparation method and application of the composite nano-drug, and belongs to the technical field of drug preparation. According to the application disclosed by the invention, the fact that mir-873 and mir-20a can be used as composite targets of prostatic cancer is found on the basis of earlier-stage research, and a composite nano-drug loaded with a mir-873 simulant and a mir-20a inhibitor is prepared on the basis of the two targets of the mir-873 and the mir-20a; the composite nano-drug takes a mir-873 simulant and a mir-20a inhibitor as active substances, the active substances are coated with a composite nano-carrier, and the composite nano-carrier is obtained by compounding a cationic liposome, an endoplasmic reticulum membrane and bombesin polypeptide; the composite nano-drug is used for efficiently targeting prostatic cancer and inhibiting proliferation and metastasis of prostatic cancer, and the composite drug can be compounded with docetaxel to further efficiently inhibit prostatic cancer, so that the composite nano-drug has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and specifically relates to a composite nano drug loaded with mir-873 mimics and mir-20a inhibitors, and a preparation method and application thereof. Background Art

[0002] Prostate cancer is one of the leading causes of cancer-related death in men worldwide. Although castration therapy has a certain effect on controlling prostate cancer, some patients develop castration-resistant or highly invasive prostate cancer that has metastasized. At this time, even if chemotherapy drugs such as docetaxel are used, the prognosis of patients is poor in most cases. Therefore, it is urgent to develop new treatment strategies to improve the problem of poor chemotherapy effects in prostate cancer patients.

[0003] The prior art discloses drugs that can specifically target prostate cancer cells and effectively reduce protein expression, but the drug itself is made of a cationic polymer. Although it is more conducive to binding to cancer cells, its cationic properties may cause it to have a shorter circulation time in the body; its material is not derived from a biological material, and its long-term biological toxicity, especially its biological safety in the body, is unknown; it has not been tested in animals in vivo. The prior art also inhibits the proliferation of prostate cancer through miRNA targets, but it has not established a drug delivery system based on this, and its inhibitory effect cannot be expected. In addition, the main problem of miRNA (microRNA) delivery is the digestion of miRNA by nucleases that are ubiquitous in the human body, and the endosomal / lysosomal degradation pathway of miRNA in cells. Therefore, a reliable drug carrier is developed to deliver miRNA to the tumor site and prevent degradation by the endosomal / lysosomal pathway. Summary of the invention

[0004] In view of some deficiencies in the prior art, the present invention provides a composite nano drug loaded with a mir-873 mimetic and a mir-20a inhibitor, and a preparation method and application thereof; based on previous studies, the present invention found that mir-873 and mir-20a can be used as composite targets for prostate cancer, and based on the two targets of mir-873 and mir-20a, a composite nano drug loaded with a mir-873 mimetic and a mir-20a inhibitor is prepared; the composite nano drug uses the mir-873 mimetic and the mir-20a inhibitor as active substances, and the active substances are externally coated with a composite nano carrier, and the composite nano carrier is obtained by compounding a cationic liposome, an endoplasmic reticulum membrane and a bombesin polypeptide; the composite nano drug can efficiently target prostate cancer, inhibit the proliferation and metastasis of prostate cancer, and the composite drug can be compounded with docetaxel to further efficiently inhibit prostate cancer, and has good practicality.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention first provides a composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitors, wherein the composite nanomedicine uses mir-873 mimics and mir-20a inhibitors as active substances, and the active substances are externally coated with a composite nanocarrier, which is obtained by compounding cationic liposomes, endoplasmic reticulum membranes and bombesin polypeptides.

[0007] The present invention also provides a method for preparing the composite nanomedicine containing the mir-873 mimetic and the mir-20a inhibitor, the preparation method comprising:

[0008] (1) dissolving DSPE-PEG2k-mal (phospholipid-polyethylene glycol-maleimide) in DMF, and mixing it with Cys-PEG-BBN[7-14] (cysteine-polyethylene glycol-maleimide), reacting at room temperature, and obtaining a bombesin polypeptide-lipid conjugate after the reaction is completed, which is set aside;

[0009] Dioleoyl-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol and bombesin polypeptide-lipid conjugate are mixed to obtain a mixed solution, the mixed solution is dissolved in chloroform to obtain a premixed solution, and the premixed solution is rotary evaporated to dry to form a film-like substance;

[0010] (2) adding mir-873 mimics and mir-20a inhibitors to a glucose aqueous solution to obtain solution a;

[0011] Solution a was added to the obtained film-like substance, and stirred until the film-like substance was completely dissolved, and then the reaction solution was repeatedly extruded on a liposome extruder to obtain a bombesin polypeptide and cationic liposome solution loaded with mir-873 mimics and mir-20a inhibitors, which was recorded as solution b;

[0012] (3) Using PBS to resuspend the endoplasmic reticulum membrane (ERM) isolated from prostate cancer cells PC-3, and then adding solution b thereto, and mixing evenly by ultrasonication under stirring conditions to obtain the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor.

[0013] Preferably, in step (1), the molar ratio of DSPE-PEG2k-mal to Cys-PEG-BBN[7-14] is 1:1 to 1.1;

[0014] The reaction time at room temperature is 12 hours.

[0015] Preferably, in step (1), the molar ratio of dioleoyl-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol and bombesin polypeptide-lipid conjugate is 1:0.5:0.75:0.15.

[0016] Preferably, in step (2), the concentration of the mir-873 mimetic and mir-20a inhibitor in the glucose aqueous solution is 0.75 mg / mL;

[0017] The concentration of the glucose aqueous solution is 5 wt %.

[0018] Preferably, in step (2), the concentration of DOTAP in the film-like substance in solution a is 7.84 mg / mL; preferably, in step (3), the volume ratio of the endoplasmic reticulum membrane to solution b is 1:2 (volume ratio).

[0019] The present invention also provides the use of the composite nano drug loaded with mir-873 mimics and mir-20a inhibitor in the preparation of drugs targeting prostate cancer.

[0020] The present invention also provides a drug targeting prostate cancer, wherein the drug uses the composite nano drug loaded with the mir-873 mimetic and the mir-20a inhibitor as an active ingredient.

[0021] Preferably, the drug further comprises docetaxel; the docetaxel is loaded on the cationic liposome in the composite nanocarrier in the composite nanomedicine loaded with the mir-873 mimetic and the mir-20a inhibitor.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention combines cationic liposomes with miRNA therapeutic drugs and endoplasmic reticulum (ER) membrane-encapsulated nanoparticles isolated from prostate cancer cells, so that the internal therapeutic drugs can be delivered through the Golgi apparatus-endoplasmic reticulum pathway in prostate cancer cells, greatly avoiding degradation by lysosomes. At the same time, the liposome and biomimetic membrane structure can also protect the packaged miRNA (mimics and inhibitors) from degradation by external nucleases.

[0024] (2) The present invention uses bio-coupling technology to couple the bombesin polypeptide on the surface (the bombesin polypeptide has a strong affinity with the gastrin-releasing polypeptide receptor highly expressed on the surface of prostate cancer cells), so that the nanoparticles can be specifically carried into prostate cancer. In addition to characterizing the properties of the composite nano-drug itself, the present invention also evaluates the biomedical functions and in vitro and in vivo therapeutic effects of the composite nano-drug. Two different castration-resistant prostate cancer cell lines are used in the experiment to simulate castration-resistant prostate cancer. The composite drug of the present invention has been shown in in vitro and in vivo experiments to be able to efficiently target prostate cancer and inhibit the proliferation and metastasis of prostate cancer. In addition, the present invention can efficiently inhibit the growth of prostate cancer in experimental mice by loading composite docetaxel with liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of the reaction between DSPE-PEG2k-Mal and Cys-PEG-BBN[7-14] (where 7-14 means the seventh to fourteenth amino acids in the classic 14 amino acid sequence of bombesin polypeptide, and all are directly abbreviated as BBN thereafter).

[0026] Figure 2 The mass spectrometry results of synthesized Cys-PEG-BBN.

[0027] Figure 3 For DSPE-PEG2K-Cys-PEG-BBN 1 H-NMR spectrum.

[0028] Figure 4 The synthesis and characterization of miRNA@CL@ERM composite nanodrug particles, where miRNA refers to a mixture of mir-873m and mir-20ai; in the figure, a is the morphology of miRNA@CL and miRNA@CL@ERM composite nanodrug particles observed by transmission electron microscopy, scale: 100nm; b is the hydrodynamic diameter and zeta potential of miRNA@CL and miRNA@CL@ERM nanoparticles; c is Coomassie blue staining of miRNA@CL@ERM nanoparticles and ERM alone; d is Western blotting of ERM-specific protein.

[0029] Figure 5 This is the gel electrophoresis diagram of miRNA@CL@ERM nanoparticles.

[0030] Figure 6Confocal fluorescence microscopy images of PC3 cells incubated with Cy5-labeled miRNA@CL or miRNA@CL@ERM nanoparticles for different treatment times; in the figure, Lamp-2, KDEL and GM130 mark lysosomes, endoplasmic reticulum and Golgi apparatus, respectively, scale bar: 10 μm.

[0031] Figure 7 The graphs show the changes in the expression levels of mir-20a and mir-873 in prostate cancer cells PC3 (a, c) and DU145 (b, d).

[0032] Figure 8 The results of CCK8 detection in PC3 (a) or DU145 (b) cells after different treatments.

[0033] Fig. 9 The results of Transwell migration experiments of PC3 and DU145 cells in different treatment groups, scale bar: 100 μm; in the figure, a is the result of PC3 cell migration experiment, c is its data statistics; b is the result of DU145 cell migration experiment, d is its data statistics.

[0034] Fig.10 Near-infrared fluorescence imaging (a) and signal intensity measurement (b) of major organs of mice treated with Cy5.5-labeled CL@ERM or CL@ERM-BBN nanoparticles.

[0035] Fig.11 Prostate tumor images (a) and size change statistics (b) of different mouse groups.

[0036] Fig.12 Photos of mouse prostate tumors in the DOC alone and DOC-loaded nanoparticles treatment groups (a), statistics of tumor size changes (b), and H&E staining and Ki67 staining of prostate tumor sections of mice in each group (c). Scale bar: 200 μm.

[0037] Fig.13 Photos of liver metastases in mice in different treatment groups (a) and H&E stained sections (b).

[0038] Fig.14 Statistics of liver metastases in mice in different treatment groups.

[0039] Fig.15 This is the in vivo toxicity evaluation of nanoparticles; in the figure, a is the weight change of mice in each group; b is the change of blood indicators such as ALP, ALT, AST, etc. of mice in each group.

[0040] Fig.16 H&E staining of the main organs of mice in each group (scale bar: 50 μm). DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, the technical means used are conventional means well known to those skilled in the art. Various processes and methods not described in detail are conventional methods known in the art. The source, trade name and the components of the reagents used are indicated when they first appear, and the same reagents used thereafter are not specifically stated, and are the same as the first stated content; the reagents, materials, etc. involved are obtained from commercial channels unless otherwise specified.

[0042] Example 1: Preparation of composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor

[0043] (1) DSPE-PEG2K-Mal and Cys-PEG-BBN were dissolved in DMF respectively to obtain DSPE-PEG2K-Mal with a concentration of 33.3 mg / mL and Cys-PEG-BBN with a concentration of 30 mg / mL; then DSPE-PEG2K-Mal and Cys-PEG-BBN were mixed at a molar ratio of 1:1.1 at room temperature for reaction. After the reaction, a bombesin polypeptide-lipid conjugate was obtained for later use.

[0044] Dioleoyl-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol and bombesin polypeptide-lipid conjugate were mixed evenly at a molar ratio of 1:0.5:0.75:0.15 to obtain a mixed solution, and then the mixed solution was dissolved in chloroform to obtain a premixed solution, in which the DOTAP concentration in the premixed solution was 4 mg / mL. The premixed solution was rotary evaporated to dry to form a film-like substance.

[0045] Combination Figures 1 to 3 It can be seen that DSPE-PEG2k-Mal undergoes a condensation reaction with the cysteine ​​amide group of Cys-PEG-BBN[7-14] through maleimide; the peak diagram of DSPE-PEG2k-Cys-PEG-BBN retains the characteristic peak of BBN of Cys-PEG-BBN, and has the characteristic peaks of PEG and DSPE, which proves the success of the coupling reaction between the two, that is, the bombesin polypeptide-lipid conjugate is successfully prepared.

[0046] (2) Add mir-873 mimics and mir-20a inhibitors to a 5 wt% aqueous glucose solution at an N:P ratio of 4:1 (N:P is the mass ratio of nitrogen atoms in DOTAP to phosphorus atoms in nucleic acids) relative to DOTAP to obtain solution a. At this time, the concentrations of mir-873 mimics and mir-20a inhibitors in solution a are both 0.75 mg / mL.

[0047] Solution a was added to the obtained film-like substance so that the concentration of DOTAP in solution a reached 7.84 mg / mL; the film-like substance was stirred until it was completely dissolved to obtain a reaction solution, and the reaction solution was repeatedly extruded 20 times on a liposome extruder to obtain a bombesin polypeptide and cationic liposome solution loaded with mir-873 mimics and mir-20a inhibitors, which was recorded as solution b.

[0048] (3) Use PBS to resuspend the endoplasmic reticulum membrane (ERM) isolated from prostate cancer cells PC3, and then add solution b thereto so that the volume ratio of the endoplasmic reticulum membrane solution to solution b is 1:2, stir and perform ultrasonic treatment, the ultrasonic condition is continuous for 2 minutes, wherein the ultrasonication is stopped for 2 seconds every 5 seconds, and the power is 10 watts, to obtain the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor.

[0049] from Figure 4 It can be seen that the composite nanocarrier composed of cationic liposomes, endoplasmic reticulum membrane and bombesin polypeptide was successfully synthesized, and its morphology was mainly vesicle-like ( Figure 4 a); the hydrodynamic radii of miRNA@CL and miRNA@CL@ERM were 145.6±16.4 and 156.5±19.5 nm, and the zeta potentials were 39.7±3.6 and 26.6±2.2 mV, respectively ( Figure 4 b). Since the coupling of only a small amount of bombesin polypeptide will not significantly affect the morphology and surface potential of the nanosystem, the composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitor of the present invention is vesicle-shaped; the hydrodynamic radius and zeta potential are similar to those of miRNA@CL@ERM.

[0050] In addition, from Figure 4 As can be seen from the figure, the ERM-loaded nanoparticles have similar Coomassie brilliant blue bands compared to the extracted ERM ( Figure 4 c), the characteristic proteins of ERM can be detected on the nanoparticles ( Figure 4 d), which indicates that the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor of the present invention retains the components of ERM.

[0051] In order to obtain the optimal N:P ratio, different ratios of mir-873m to nanocarrier were tried, followed by gel electrophoresis. Figure 5 As shown in the figure, when N:P is less than 4:1, nucleic acids cannot be packaged, which is shown as bright bands in the figure, while when N:P=4:1, the best packaging ratio can be guaranteed.

[0052] Example 2: Preparation of composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitor

[0053] (1) DSPE-PEG2K-Mal and Cys-PEG-BBN were dissolved in DMF to obtain DSPE-PEG2K-Mal with a concentration of 20 mg / mL and Cys-PEG-BBN with a concentration of 20 mg / mL; DSPE-PEG2K-Mal and Cys-PEG-BBN were then mixed at a molar ratio of 1:1.2 at room temperature for reaction. After the reaction, a bombesin polypeptide-lipid conjugate was obtained for later use.

[0054] Dioleoyl-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol and bombesin polypeptide-lipid conjugate were mixed evenly at a molar ratio of 1:0.5:0.75:0.15 to obtain a mixed solution, and then the mixed solution was dissolved in chloroform to obtain a premixed solution, in which the DOTAP concentration in the premixed solution was 2 mg / mL. The premixed solution was rotary evaporated to dry to form a film-like substance.

[0055] (2) Add mir-873 mimics and mir-20a inhibitors to a 5% aqueous glucose solution at an N:P ratio of 4:1 (N:P is the mass ratio of nitrogen atoms in DOTAP to phosphorus atoms in nucleic acids) relative to DOTAP to obtain solution a. At this time, the concentrations of mir-873 mimics and mir-20a inhibitors in solution a are both 0.75 mg / mL.

[0056] Solution a was added to the obtained film-like substance so that the concentration of DOTAP in solution a reached 7.84 mg / mL; the film-like substance was stirred until it was completely dissolved to obtain a reaction solution, and the reaction solution was repeatedly extruded on a liposome extruder for 5 times to obtain a bombesin polypeptide and cationic liposome solution loaded with mir-873 mimics and mir-20a inhibitors, which was recorded as solution b.

[0057] (3) Use PBS to resuspend the endoplasmic reticulum membrane (ERM) isolated from prostate cancer cells PC3, then add solution b thereto, stir and perform ultrasonic treatment, the ultrasonic condition is continuous for 2 minutes, wherein the ultrasonication is stopped for 2 seconds after every 5 seconds, and the power is 5 watts, to obtain the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor.

[0058] In this example, PC3 cells were seeded on glass slides, incubated with Cy5-miRNA@CL@ERM nanoparticles (where Cy5-miRNA was synthesized by GenePharma Biotech (Shanghai, China) and was a mir-873 mimic labeled with the fluorescent dye Cy5) at different time points, and then fixed with 4% formaldehyde solution. The endoplasmic reticulum, lysosomes, and Golgi bodies were labeled with KDEL, LAMP-2, and GM130 antibodies (all purchased from CST (Danvers, Massachusetts, USA)), respectively, and stained with AlexaFluor@488-labeled secondary antibodies. The sections were observed using a Zeiss LSM 800 confocal microscope and analyzed using ImageJ software. The analysis results are shown in Figure 2. Figure 6 shown.

[0059] As can be seen from the figure, after being loaded with ERM, Cy5-miRNA@CL@ERM nanoparticles rarely enter the lysosome, and most of them enter the endoplasmic reticulum-Golgi pathway, proving that the presence of ERM can assist the composite nanodrug to enter the cell through the endoplasmic reticulum-Golgi pathway, which is beneficial for helping miRNA enter the cell without being degraded in large quantities by the lysosome.

[0060] Example 3: Preparation of composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor

[0061] (1) DSPE-PEG2K-Mal and Cys-PEG-BBN were dissolved in DMF respectively to obtain DSPE-PEG2K-Mal with a concentration of 40 mg / mL and Cys-PEG-BBN with a concentration of 40 mg / mL; then DSPE-PEG2K-Mal and Cys-PEG-BBN were mixed at a molar ratio of 1:1 at room temperature for reaction. After the reaction, a bombesin polypeptide-lipid conjugate was obtained for later use.

[0062] Dioleoyl-trimethylammonium chloride (DOTAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol and bombesin polypeptide-lipid conjugate were mixed evenly at a molar ratio of 1:0.5:0.75:0.15 to obtain a mixed solution, and then the mixed solution was dissolved in chloroform to obtain a premixed solution, in which the DOTAP concentration in the premixed solution was 6 mg / mL. The premixed solution was rotary evaporated to dry to form a film-like substance.

[0063] (2) Add mir-873 mimics and mir-20a inhibitors to a 5% aqueous glucose solution at an N:P ratio of 4:1 (N:P is the mass ratio of nitrogen atoms in DOTAP to phosphorus atoms in nucleic acids) relative to DOTAP to obtain solution a. At this time, the concentrations of mir-873 mimics and mir-20a inhibitors in solution a are both 0.75 mg / mL.

[0064] Solution a was added to the obtained film-like substance so that the concentration of DOTAP in solution a reached 7.84 mg / mL; the film-like substance was stirred until it was completely dissolved to obtain a reaction solution, and the reaction solution was repeatedly extruded 20 times on a liposome extruder to obtain a bombesin polypeptide and cationic liposome solution loaded with mir-873 mimics and mir-20a inhibitors, which was recorded as solution b.

[0065] (3) Use PBS to resuspend the endoplasmic reticulum membrane (ERM) isolated from prostate cancer cells PC3, and then add solution b thereto so that the volume ratio of the endoplasmic reticulum membrane solution to solution b is 1:2, stir and perform ultrasonic treatment, the ultrasonic condition is continuous for 1 minute, wherein the ultrasonication is stopped for 2 seconds every 5 seconds, and the power is 15 watts, to obtain the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor.

[0066] Embodiment 4:

[0067] In order to investigate the intervention effect of composite nanomedicines simultaneously encapsulating mir-20ai and mir-873m on the proliferation and migration of prostate cancer, the present invention used mir-20ai@CL@ERM, mir-873m@CL@ERM, DOC and DOC+mir-20ai+873m@CL@ERM as controls, and used CCK8, Transwell chamber migration assay and other experiments to characterize the in vitro efficacy of mir-20ai+873m@CL@ERM, and tested the effects of the main components of the final product, except bombesin, on inhibiting the proliferation and migration of prostate cancer cells.

[0068] The control products mir-20ai@CL@ERM and mir-873m@CL@ERM were prepared by maintaining the total amount of miRNA unchanged and adding only one miRNA regulating drug to the 5wt% glucose solution. The preparation method of DOC+mir-20ai+873m@CL@ERM was to dissolve DOC (with a mass ratio of 1:3 to DOTAP) in the lipid premix by chloroform, and the remaining steps were consistent with the preparation method of the final product.

[0069] (1) miRNA regulation experiment:

[0070] The experimental group was mir-20ai+873m@CL@ERM, and the control group was NC (control in the figure), mir-20ai@CL@ERM, and mir-873m@CL@ERM.

[0071] Prostate cancer cells PC3 and DU145 (both from the American Type Culture Collection (ATCC), PC3 ATCC number is CRL-1435, DU145 ATCC number is HTB-81) were incubated with the experimental and control groups of nanoparticles for 24 hours, and then the cells were counted and their RNA was extracted for real-time quantitative PCR (Real-time qPCR) determination. The results were as follows: Figure 7 As shown in the figure, it can be seen that nanoparticles loaded with mir-20ai, mir-873m or both can significantly inhibit mir-20a or increase the level of mir-873 in PC3 cells and DU145 cells.

[0072] (2) CCK8 cell viability assay:

[0073] In order to verify whether the composite nanomedicine loaded with two miRNA regulatory drugs has a stronger regulatory ability on the proliferation and migration of prostate cancer cells than a single regulatory method, this example uses mir-20ai+873m@CL@ERM as the experimental group, mir-20ai@CL@ERM and mir-873m@CL@ERM as the control group. First, prostate cancer cells PC3 and DU145 are inoculated in a 96-well plate. After applying different drugs, CCK8 assays are performed at different experimental points (Yisheng's CCK8 kit, 2000 cells per well of a 96-well plate). The assay results are shown in Figure 2. Figure 8 As shown in the figure, it can be seen that nanoparticles loaded with both miR-20ai and miR-873m have better tumor cell growth inhibition ability than single loading.

[0074] In this step, DOC+mir-20ai+873m@CL@ERM was used as the experimental group and DOC was used as the control group to verify whether docetaxel (DOC) loaded in mir-20ai+873m@CL@ERM composite nanomedicine has better efficacy than DOC, a first-line chemotherapy drug, administered alone. The results are shown in Figure 8 As shown in the figure, it can be seen that compared with the single administration of DOC, the efficacy of docetaxel (DOC) loaded on mir-20ai+873m@CL@ERM composite nanomedicine is significantly improved. Therefore, DOC can be combined with the composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitors described in the present invention to effectively inhibit prostate cancer.

[0075] (3) Transwell cell migration assay:

[0076] In this step, mir-20ai+873m@CL@ERM was used as the experimental group, and NC@CL@ERM, mir-20ai@CL@ERM, and mir-873m@CL@ERM were used as the control group. 20,000 prostate cancer cells PC3 or DU145 were planted in each well of the transwell chamber. The migration ability of each group was evaluated by counting the cells in the transwell chamber 24 hours after cell inoculation. The results are shown in Figure 2. Fig. 9 shown.

[0077] As can be seen from the figure, nanoparticles loaded with both mir-20ai and mir-873m have better inhibitory ability on cell migration than single loading ( Fig. 9 a); The composite nanomedicine carrying mir-20ai and mir-873m has a significant ability to inhibit the proliferation and migration of prostate cancer, and the composite nanomedicine carrying both has a more significant efficacy than that carrying a single miRNA ( Fig. 9 b).

[0078] In summary, the composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitors described in the present invention can significantly inhibit mir-20a or increase the level of mir-873 in PC3 cells and DU145 cells, has a more significant efficacy when loaded with a single miRNA, and can be used in combination with DOC to effectively inhibit prostate cancer.

[0079] Embodiment 5:

[0080] In this example, in vivo experiments were conducted on a subcutaneous tumor model of prostate cancer mice and a liver metastasis model to examine the biological distribution of nanomedicines in vivo, verify that the composite nanomedicine containing mir-873 mimics and mir-20a inhibitors of the present invention inhibits the proliferation and metastasis of prostate cancer in the mouse model, and at the same time, detect whether the composite nanomedicine has biological toxicity in vivo. The specific steps are as follows:

[0081] (1) Prostate cancer subcutaneous tumor modeling experiment:

[0082] The mice used in this step were 3-4 week old Balb / c male nude mice (purchased from Shanghai Slack Company). The mice were cared for according to the guidelines of the National Institutes of Health (NIH) of the United States, and the specific process was supervised by the Animal Ethics Committee of Shanghai Cancer Institute.

[0083] In a temperature- and humidity-controlled room, mice were housed in sterile microisolator cages, 5 per cage, with a 12-h light and dark period per day, fed with autoclaved rodent chow, and provided with water. 6 The epithelial tumor-bearing model was established by using PC3 tumor cells, which could grow into relatively uniform and obvious subcutaneous prostate cancer tumors in 3 to 4 weeks.

[0084] In the liver metastasis model, 10 6 PC3 cells were suspended in PBS and injected into the spleen. The liver tissue could be dissected and the metastatic foci of prostate cancer could be visually observed after 4 weeks. The mice were intravenously injected with different groups of therapeutic agents or imaging agents 3 to 4 weeks after tumor cell inoculation.

[0085] (2) In vivo biodistribution study:

[0086] In order to test whether the composite nanomedicine can be efficiently accumulated in tumor cells in vivo, and to see whether the coupling of BBN can give the nanoparticles the ability to actively target prostate cancer in vivo, the in vivo biodistribution test of the composite nanomedicine was performed on the Indigo optical imaging system (Indigo, Jena, Thuringia, Germany). The specific steps are as follows:

[0087] Preparation of Cy5.5@CL@ERM: Cy5.5 was added to the lipid premix, wherein the molar ratio of Cy5.5 to DOPE was 0.005:1, and the other preparation methods were consistent with the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor described in the present invention.

[0088] Mice with a subcutaneous prostate cancer model were injected with 1 mg / kg dose of Cy5.5-labeled CL@ERM or CL@ERM-BBN (peg-modified), three mice in each group, and were killed 24 hours after injection. The tumors and major organs were collected, and the target areas of the tumors and major organs were mapped. The intensity percentage was calculated by dividing the fluorescence intensity of each organ by the total fluorescence intensity of all organs, and the biodistribution of the drug in each organ was quantitatively evaluated. The results are shown in Fig.10 shown.

[0089] As can be seen from the figure, nanoparticles are mainly enriched in tumors, except for the main excretory organs and metabolic organs of liver and kidney. The enrichment level of Cy5.5@CL@ERM-BBN in tumors is significantly higher than that of Cy5.5@CL@ERM, indicating that the coupling of BBN can indeed significantly enhance the ability of nanoparticles to actively target prostate cancer.

[0090] (3) In vivo therapeutic effect:

[0091] S1. In this step, mir-20ai+873m@CL@ERM-BBN is used as the experimental group, and PBS, mir-NC@CL@ERM, mir-20ai+873m@CL, mir-20ai@CL@ERM, mir-873m@CL@ERM, and mir-20ai+873m@CL@ERM are used as the control group to investigate whether the combined administration of two miRNA therapeutic drugs has better in vivo efficacy than a single miRNA, and the ability of BBN and ERM to improve the in vivo efficacy of the composite nanomedicine. The specific steps are as follows:

[0092] Different groups of nanoparticles were intravenously injected into mice at a dose of 1 mg / kg (the amount of miRNA), and the weight changes of the mice were recorded during the process. The results are as follows: Fig.11 As shown in the figure, it can be seen that the composite nucleic acid drug loaded with mir-20ai and mir-873m has a better inhibitory effect on tumor growth than a single drug.

[0093] S2. This step also uses DOC+mir-20ai+873m@CL@ERM-BBN as the experimental group, and PBS, DOC, and DOC@CL@ERM-BBN as the control group, to examine whether this carrier and the combined regulation of mir-20ai+873m after DOC is loaded on mir-20ai+873m@CL@ERM-BBN can give the clinically commonly used first-line drug DOC a more significant prostate cancer treatment effect.

[0094] In the experiment, the docetaxel dose for each mouse was 5 mg / kg. The other steps were as shown in S1. The weight changes of the mice were recorded during the experiment. The results are shown in Fig.12 As shown in the figure, it can be seen that the nanomedicine encapsulated with docetaxel has a significantly improved inhibitory effect on tumor growth compared with single docetaxel or single nucleic acid nanomedicine ( Fig.12 a and 12b); DOC+mir-20ai+873m@CL@ERM-BBN showed a significant decrease in the total number of cells and the proportion of Ki67-positive cells compared with other control groups ( Fig.12 c).

[0095] S3. In this step, DOC+mir-20ai+873m@CL@ERM-BBN was used as the experimental group, and PBS, DOC, and DOC@CL@ERM-BBN were used as the control group to investigate whether the carrier loaded with mir-20ai+873m@CL@ERM-BBN and the combined regulation of mir-20ai+873m can give the clinically commonly used first-line drug DOC a more significant inhibitory effect on the metastasis of prostate cancer in vivo. The results of the investigation are as follows: Fig.13 and14 shown.

[0096] As can be seen from the figure, the DOC treatment group compounded with nucleic acid nanodrugs has a better inhibitory effect on liver metastasis than single DOC, and can significantly reduce the number of liver metastases.

[0097] (4) In vivo toxicity test:

[0098] In this step, mir-20ai+873m@CL@ERM-BBN, DOC+mir-20ai+873m@CL@ERM-BBN, mir-NC@CL@ERM, mir-20ai+873m@CL, mir-20ai+873m@CL@ERM, and DOC were used as experimental groups, and PBS was used as the control group. The weight changes of the mice, immunohistochemical staining of the main tissues, and liver pathological markers were used to investigate whether each composite nano-drug had obvious in vivo toxicity. The results are shown in the figure. Fig.15 and 16 shown.

[0099] As can be seen from the figure, the different treatment groups did not significantly affect the body weight of mice ( Fig.15 a); ALP (serum alkaline phosphatase), ALT (alanine aminotransferase), AST (aspartate aminotransferase) did not change significantly ( Fig.15 b); 21 days later, the mice were killed, and the heart, liver, kidney, spleen, lung and other major organs were taken for paraffin sectioning and H&E to examine their histological characteristics. It was found that there were no pathological changes in these major organs of mice under different treatment groups ( Fig.16 ).

[0100] It can be seen that the miRNA@CL@ERM composite nanodrug loaded with two miRNA regulators has a more obvious inhibitory effect on the proliferation of prostate cancer in vivo than the composite nanodrug loaded with only a single component miRNA. In addition, the miRNA@CL@ERM composite nanodrug loaded with the first-line drug DOC has significantly improved in vivo therapeutic efficacy and the ability to inhibit liver metastases compared to the administration of DOC alone. Finally, from various aspects such as changes in mouse weight, liver toxicity markers, and pathological sections of major organs, it was proved that the composite nanodrug had no obvious toxicity in vivo.

[0101] In summary, the present invention is based on previous studies and found that mir-873 and mir-20a can be used as composite targets for prostate cancer, and based on the two targets of mir-873 and mir-20a, a composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitors is prepared; the composite nanomedicine uses mir-873 mimics and mir-20a inhibitors as active substances, and the active substances are wrapped with composite nanocarriers on the outside, and the composite nanocarriers are composited by cationic liposomes, endoplasmic reticulum membranes and bombesin polypeptides; the composite nanomedicine efficiently targets prostate cancer, inhibits the proliferation and metastasis of prostate cancer, and the composite drug can be compounded with docetaxel to further efficiently inhibit prostate cancer, and has good practicality.

[0102] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitor, characterized in that: The composite nanomedicine uses mir-873 mimics and mir-20a inhibitors as active substances, and the active substances are coated with composite nanocarriers, which are obtained by compounding cationic liposomes, endoplasmic reticulum membranes and bombesin polypeptides; The size range of the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor is 159.2±19.9 nm.

2. The method for preparing the composite nanomedicine containing mir-873 mimics and mir-20a inhibitor according to claim 1, characterized in that: The preparation method comprises: (1) dissolving DSPE-PEG2k-mal in DMF, and mixing it with Cys-PEG-BBN[7-14], reacting at room temperature, and obtaining a bombesin polypeptide-lipid conjugate after the reaction, which is set aside; DOTAP, DOPE, cholesterol and bombesin polypeptide-lipid conjugate are mixed to obtain a mixed solution, the mixed solution is dissolved in chloroform to obtain a premixed solution, and the premixed solution is rotary evaporated to dry to form a film-like substance; (2) adding the mir-873 mimetic and the mir-20a inhibitor to the glucose aqueous solution to obtain solution a; Solution a was added to the obtained film-like substance, and stirred until the film-like substance was completely dissolved, and then the reaction solution was repeatedly extruded on a liposome extruder to obtain a bombesin polypeptide and cationic liposome solution loaded with mir-873 mimics and mir-20a inhibitors, which was recorded as solution b; (3) Using PBS to resuspend the endoplasmic reticulum membrane (ERM) isolated from prostate cancer cells PC-3, and then adding solution b thereto, and mixing evenly by ultrasonication under stirring conditions to obtain the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor.

3. The method for preparing the composite nanomedicine containing mir-873 mimics and mir-20a inhibitor according to claim 3, characterized in that: In step (1), the molar ratio of DSPE-PEG2k-mal to Cys-PEG-BBN[7-14] is 1:1 to 1.1; The reaction time at room temperature is 12 hours.

4. The method for preparing the composite nanomedicine containing mir-873 mimics and mir-20a inhibitor according to claim 3, characterized in that: In step (1), the molar ratio of DOTAP, DOPE, cholesterol and bombesin polypeptide-lipid conjugate is 1:0.5:0.75:0.

15.

5. The method for preparing the composite nanomedicine containing mir-873 mimics and mir-20a inhibitor according to claim 3, characterized in that: In step (2), the concentration of the mir-873 mimetic and mir-20a inhibitor in the glucose aqueous solution is 0.75 mg / mL; The concentration of the glucose aqueous solution is 5 wt %.

6. The method for preparing the composite nanomedicine containing mir-873 mimics and mir-20a inhibitor according to claim 3, characterized in that: In step (2), the concentration of DOTAP in the film-like substance in solution a is 7.84 mg / mL.

7. The method for preparing the composite nanomedicine loaded with mir-873 mimics and mir-20a inhibitor according to claim 3, characterized in that: In step (3), the volume ratio of the endoplasmic reticulum membrane to solution b is 1:

2.

8. Use of the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor as claimed in claim 1 in the preparation of drugs targeting prostate cancer.

9. A drug targeting prostate cancer, characterized in that: The drug uses the composite nano drug loaded with mir-873 mimetic and mir-20a inhibitor as claimed in claim 1 as an active ingredient.

10. The drug according to claim 8, characterized in that The drug also contains docetaxel, which is loaded on the cationic liposome in the composite nanocarrier of the composite nanomedicine loaded with mir-873 mimetic and mir-20a inhibitor.

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