Targeted bionic nano-drug loaded with tanshinone IIA and rosuvastatin as well as preparation method and application of targeted bionic nano-drug

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a targeted bionic nano-drug loaded with tanshinone IIA and rosuvastatin as well as a preparation method and application of the targeted bionic nano-drug loaded with the tanshinone IIA and the rosuvastatin. And wrapping the outer layer of the loaded polylactic acid-glycolic acid copolymer with a macrophage membrane, and modifying the surface of the macrophage membrane by using the phosphatidylated hyaluronic acid. According to the targeted bionic nano-drug system, the polylactic acid-glycolic acid copolymer is used for loading two drugs at the same time, inhibition of endothelial cell mesenchymal transition by tanshinone IIA and regulation of macrophage lipid metabolism by rosuvastatin are organically combined, and the effect of combined treatment of atherosclerosis is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a targeted biomimetic nanomedicine loaded with tanshinone IIA and rosuvastatin, its preparation method and application. Background Technology

[0002] Atherosclerosis, abbreviated as AS, includes aortic atherosclerosis, carotid atherosclerosis, and cerebral atherosclerosis. Atherosclerosis is a typical chronic inflammatory vascular disease and a major pathological basis for common cardiovascular diseases. It has a high incidence and mortality rate, and is the leading cause of death worldwide. Studies have shown that homocysteine ​​(Hcy) is a new independent risk factor for AS, closely related to its occurrence and development. It has been confirmed that Hcy can activate macrophages and cause macrophage inflammation. In the early stages of AS, damage to some endothelial cells leads to the diffusion of low-density lipoprotein (LDL) and Hcy from the blood to the subendothelial layer. Stimulated endothelial cells secrete chemokines, continuously recruiting monocytes to migrate to the subendothelial layer and differentiate into macrophages. Macrophages, under the influence of oxidized LDL and Hcy, differentiate into pro-inflammatory M1 macrophages and secrete inflammatory factors such as IL-1β, inducing mesenchymal transition in endothelial cells, further promoting endothelial damage, forming a vicious cycle. On the other hand, inflammatory macrophages take up oxidized low-density lipoprotein, forming foam cells, which eventually die and rupture, releasing deposited lipids. As asthenospermia (As) progresses, lipids accumulate beneath endothelial cells, eventually forming plaques. Inhibiting endothelial damage and lipid deposition are considered two fundamental strategies for the prevention and treatment of Asthenospermia.

[0003] Currently, rosuvastatin, a first-line clinical treatment for atherosclerosis (AS), has a significant lipid-lowering effect, but its overall therapeutic efficacy is poor. This is because the drug has a short residence time in the body, lacks specific targeting, has a single therapeutic effect, and its therapeutic efficacy is limited. Long-term high-dose use can cause significant toxic side effects. Tanshinone IIA, or TIIA, has the effect of repairing vascular endothelium and is mainly used for the treatment of atherosclerotic cardiovascular disease. Studies have shown that TIIA inhibits glycolysis and lactate production, which may slow down end-mode endothelial damage, thereby protecting against endothelial injury. However, this drug has poor water solubility and low bioavailability, limiting its clinical application. In conclusion, there is an urgent need to develop novel drug formulations for atherosclerosis. Summary of the Invention

[0004] This invention provides a targeted biomimetic nanomedicine loaded with tanshinone IIA and rosuvastatin, which achieves the simultaneous delivery and enrichment of TIIA and RSV to the plaque site, exerting the combined effect of the two drugs to effectively treat As.

[0005] The first object of the present application is to provide a targeted biomimetic nanodrug loaded with TIIA and RSV. The targeted biomimetic nanodrug is co-loaded with tanshinone IIA and rosuvastatin by polylactic acid-glycolic acid copolymer, and the outer layer of the polylactic acid-glycolic acid copolymer after loading is wrapped with a macrophage membrane, and the surface of the macrophage membrane is modified by phosphatidylated hyaluronic acid.

[0006] The second object of the present application is to provide a preparation method of the above-mentioned targeted biomimetic nanodrug, comprising the following steps: 1. Preparation of TIIA-loaded PLGA dispersion: dissolve TIIA and PLGA in dimethyl sulfoxide solution, mix uniformly, drop the mixture into v / v 1% PVA aqueous solution drop by drop under water bath ultrasonic, dialysis, freeze-drying, and PBS reconstitution to obtain TIIA-loaded PLGA dispersion.

[0007] 2. Preparation of RSV-loaded PLGA dispersion: dissolve RSV and PLGA in dimethyl sulfoxide solution, mix uniformly, stir on a magnetic stirrer, drop the mixture into v / v 1% PVA aqueous solution drop by drop, dialysis, freeze-drying, and PBS reconstitution to obtain RSV-loaded PLGA dispersion.

[0008] 3. Preparation of biomimetic macrophage membrane-wrapped TIIA and RSV-loaded PLGA dispersion: mix the TIIA-loaded PLGA dispersion and the RSV-loaded PLGA dispersion, and repeatedly extrude to prepare the biomimetic macrophage membrane-wrapped TIIA and RSV-loaded PLGA dispersion.

[0009] 4. Preparation of targeted biomimetic nanodrug loaded with TIIA and RSV: phosphatidylate hyaluronic acid and mix it with the macrophage membrane-coated TIIA and RSV-loaded PLGA dispersion uniformly, water bath stirring, so that the phosphatidylated hyaluronic acid is combined with the biomimetic macrophage membrane, and the inner layer is the TIIA and RSV-loaded PLGA nanoparticles.

[0010] Preferably, the mass ratio of TIIA to PLGA is 1:5-10, mixed in a 250mL round-bottom flask, the volume ratio of dimethyl sulfoxide solution to v / v 1% PVA solution is 1:10-15, the water bath ultrasonic power is 150W, and the dropwise addition speed of the mixture is 1 drop per 5s-8s. The dialysis adopts a 1kD dialysis bag, and the dialysis time is 10h-12h.

[0011] Preferably, the mass ratio of the RSV to the PLGA is 1:3-5, and the mixture is in a 50-60 mL glass sample bottle. The volume ratio of the dimethyl sulfoxide solution to the v / v 1% PVA solution is 1:5-10, and the magnetic stirring speed is 300-500 rpm. The dropping speed of the mixture is 3-5 s per drop. The dialysis uses a 3.5 KD dialysis bag, and the dialysis time is 10-12 h.

[0012] The molar concentration ratio of the TIIA-loaded PLGA dispersion and the RSV-loaded PLGA dispersion is 1:2, the mass ratio of the macrophage membrane to the co-loaded TIIA and RSV PLGA is 1:2-5, and the repeated extrusion refers to repeated extrusion 8-12 times by a micro-extruder.

[0013] Preferably, the method for preparing the macrophage membrane comprises the following steps: The RAW264.7 cells are cultured, and when the cell density reaches 70-80%, the cells are collected, washed with PBS three times, suspended in a lysis solution, repeatedly frozen and thawed to fully break the cells, centrifuged to remove the cell nuclei and unbroken cells, and then centrifuged to collect the cell membrane precipitate.

[0014] Further, the repeated freezing and thawing condition is repeated freezing and thawing 3-5 times at-80°C and 37°C to fully break the cells, the centrifugation parameter for removing the cell nuclei and unbroken cells is 4°C, 500-700 g, and the centrifugation time is 5-10 min, and the centrifugation parameter for collecting the cell membrane precipitate is 4°C, 12000-14000 g, and the centrifugation time is 20-30 min.

[0015] Further, it further comprises resuspending the collected cell membrane precipitate with PBS, activating the cell membrane by probe ultrasonic, and the time is 5-10 min and the power is 100-150 W.

[0016] The mass ratio of the PLGA to the phosphatidylated hyaluronic acid is 2-10:1, and the water bath stirring parameter is 37°C and the stirring speed is 100-300 rpm.

[0017] Preferably, the phosphatidylated hyaluronic acid comprises the following steps: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and hyaluronic acid are dissolved in PBS, mixed by stirring at room temperature, and then di-stearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino is added to the mixture, and the phosphatidylated hyaluronic acid is obtained by stirring, dialysis, and freeze-drying.

[0018] Further, the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid mass ratio is 5:10:1; the stirring mixing parameter is a rotation speed of 500 rpm~800 rpm, and a time of 30 min~60 min, the hyaluronic acid and distearyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino mass ratio is 1:5; the stirring parameter is a rotation speed of 500 rpm~800 rpm, and a time of 16 h~24 h; and the dialysis is using a 3.5kDa dialysis bag, and the dialysis time is 16 h~24 h.

[0019] A third object of the present application is to provide the application of the TIIA and RSV loaded targeted biomimetic nanodrug in the preparation of the drug for treating atherosclerosis.

[0020] Compared with the prior art, the present application has the following beneficial effects: The present application provides a kind of TIIA and rosuvastatin loaded targeted biomimetic nanodrug, the targeted biomimetic nanodrug is co-loaded by polylactic acid-glycolic acid copolymer to TIIA and rosuvastatin, the outer layer of polylactic acid-glycolic acid copolymer after loading is wrapped macrophage membrane, and the surface of macrophage membrane is modified using phosphatidylated hyaluronic acid.The targeted biomimetic nanodrug of the present application simultaneously loads two kinds of atherosclerosis treatment drugs, reduces the dosage of single drug, and simultaneously solves the clinical drug problems of low solubility and low availability of TIIA, high toxicity and strong side effects of RSV.Two drugs target endothelial damage and lipid deposition, the key pathogenic factors of atherosclerosis, and play a synergistic effect, with obvious effect, providing a new scheme for clinical treatment of atherosclerosis, and having potential application prospect.

[0021] The targeted biomimetic nanodrug can effectively target the lesion site, improve the bioavailability of target cells to drugs at the lesion site, prolong the half-life of the drugs, has the function of site-specific release, has good effect on the treatment of atherosclerosis, and effectively avoids the shortcomings of traditional Chinese and western medicines, such as easy clearance, non-specific distribution, and single treatment effect.

[0022] The drug of the present application loads TIIA and rosuvastatin using PLGA, and is wrapped by macrophage membrane, effectively solving the problems of easy clearance, poor biocompatibility and non-specific distribution.The outer layer of the nanodrug of the present application is modified with hyaluronic acid, which interacts with CD44 molecules on the surface of cells in the plaque site, so that the two drugs are efficiently delivered to the lesion site and released at the site, TIIA inhibits endothelial mesenchymal transition at the plaque site, endothelial repair is promoted, and RSV inhibits lipid deposition in macrophages, achieving targeted treatment of As.The nanodrug system provided by the present application has good biological safety.

[0023] The nano drug has good biological safety. The preparation material PLGA is non-toxic, degradable and has good biocompatibility, and has been officially included in the US Pharmacopoeia as a pharmaceutical excipient; macrophages and hyaluronic acid widely exist in human and animal tissues. The nano drug has no any toxic effect on the heart, liver, spleen, lung, kidney and other organs, and is safe and reliable while improving the treatment effect.

[0024] The nano drug preparation scheme has obvious advantages and certain clinical medical value. The PLGA nano material has acidified lysosomes and low pH response characteristics at the lesion site, and is an excellent drug delivery material. The macrophage membrane has immune escape ability and homing characteristics, realizes effective enrichment and penetration at the lesion site. The phosphatidyl hyaluronic acid modification further prolongs the in-vivo circulation time and improves the drug targeting ability. The drug not only has the effects of repairing endothelium and inhibiting lipid deposition, but also has anti-inflammatory properties. The above preparation scheme and the nano material can also be applied to the treatment of other diseases, and is not limited to atherosclerosis, such as liver disease, kidney disease, tumor, arthritis and the like. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The appearance pictures of HAMP@TR NPs prepared by the application and the produced Tyndall phenomenon, A is the solution of TIIA and the solution of RSV; B is the appearance picture of P@TIIA NPs, P@RSV NPs and HAMP@TR NPs; C is the produced Tyndall phenomenon of P@TIIA NPs, P@RSV NPs and HAMP@TR NPs under light source irradiation.

[0026] Figure 2 The transmission electron microscope pictures of HAMP@TR NPs and empty carrier PLGA NPs prepared by the application, A is PLGA NPs, and B is HAMP@TR NPs.

[0027] Figure 3 The outer macrophage membrane marker immunoblotting result of HAMP@TR NPs prepared by the application.

[0028] Figure 4 The outer macrophage membrane and inner PLGA fusion experiment result of HAMP@TR NPs prepared by the application.

[0029] Figure 5 The in-vitro targeting evaluation result of macrophages and endothelial cells, A: representative images of macrophages and endothelial cells; B: statistical result of macrophages; C: statistical result of endothelial cells.

[0030] Figure 6For aortic enrichment results of HAMP@ce6 in mice, A: representative images; B: statistical results of A figure.

[0031] Figure 7 For half-life determination of HAMP@ce6 NPs in mice, A: representative images; B: statistical results of A figure.

[0032] Figure 8 For results of HAMP@TR NPs inhibiting lipid deposition of macrophages, A: results of DiI-oxLDL uptake experiment; B: results of oil red O staining; C: statistical results of A figure; D: statistical results of B figure.

[0033] Figure 9 For results of HAMP@TR NPs prepared by the application inhibiting inflammatory response, A-D are ELISA detection results of IL-1β, TNF-α, IL-6 and IL-10, respectively.

[0034] Figure 10 For HAMP@TR NPs prepared by the application inhibiting endothelial cell mesenchymal transformation, A: WB analysis results; B: statistical results of VE-Cadherin and CD31; C: statistical results of ICAM-1 and Vimentin.

[0035] Figure 11 For vascular oil red O staining pictures of different treatment groups treating atherosclerosis, A: vascular gross staining picture; B: representative images of 5 repetitions of mouse aortic roots of different treatment groups; C: ApoE - / - Frozen section ORO staining of mouse aortic roots.

[0036] Figure 12 For serum lipid levels and Hcy levels after different drug treatments, A-E are TG, CHO, LDL-C, HDL-C and Hcy, respectively.

[0037] Figure 13 For H&E staining pictures of hearts, livers, spleens, lungs and kidneys of atherosclerotic mice treated by different treatment groups of the application. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions of the present application and implement the same, the present application will be further described below in conjunction with specific embodiments, but the embodiments are not intended to limit the present application. In the description of the present application, if not specifically stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0039] The inventive concept of the present application is as follows: The clinical first-line drug for As, rosuvastatin, has obvious lipid-lowering effect, but the overall treatment effect is poor. The reason is that the drug has short residence time in the body, lacks targeting, has single treatment effect, the treatment effect is limited, and long-term high-dose use will cause obvious toxic side effects; Danshensu II A has the effect of repairing vascular endothelium, and is mainly used for the treatment of atherosclerotic cardiovascular diseases. Research shows that TIIA inhibits glycolysis and lactic acid production, and may have the effect of slowing down End MT, thereby protecting endothelial damage. However, the drug has poor water solubility, low bioavailability, and limited clinical application. In summary, the two drugs are used in combination, which reduces the dosage of single drug, targets the treatment of endothelial damage and vascular wall lipid deposition, and is expected to produce good results.

[0040] In recent years, with the development of nanotechnology, nanomedicine system has shown obvious advantages in disease treatment. PLGA nanoparticles have biodegradability and controllable release characteristics, and can improve the stability and biocompatibility of drugs in drug delivery. Nanoparticles disguised as biomimetic macrophages have immune escape function and chemotactic properties, further prolonging the half-life of the drug and effectively improving the targeting of the drug. In the process of As, activated macrophages and endothelial cells in the plaque site highly express CD44 molecules. Using the property that hyaluronic acid specifically binds to CD44 molecules, hyaluronic acid is modified on the outermost layer of the nanoparticle, which can improve the targeting of the nanoparticle and enhance the bioavailability of the drug to target cells, providing a new scheme with potential application prospect for the prevention and treatment of atherosclerosis. The PLGA used in the application has a CAS number of 34346-01-5.

[0041] The abbreviation table of the application is shown in Table 1.

[0042] Table 1 Abbreviation table of the application Example 1 A preparation method of a targeted biomimetic nanoparticle loaded with TIIA and RSV, comprising the following steps: S1, wrapping danshensu II A in polylactic acid-glycolic acid copolymer to prepare a TIIA-loaded PLGA dispersion liquid, and the specific operation process is as follows: 15mg PLGA and 1.5mg TIIA were dissolved in 1mL DMSO, mixed well; 10mL 1% PVA v / v aqueous solution was added to a 250mL round-bottom flask; the mixed solution of PLGA and TIIA was added dropwise into the PVA aqueous solution in the round-bottom flask under water bath ultrasonic condition, the water bath ultrasonic power was 150W, the dropwise adding speed was 5s per drop, after dropwise adding was completed, it was placed in a 1kDa dialysis bag, dialysis for 12h, after freeze-drying, it was dissolved with 1mL PBS, to prepare the TIIA-loaded PLGA dispersion, recorded as P@TIIA NPs, the encapsulation efficiency was about 85%.

[0043] S2, risedronate was wrapped in polylactic acid-glycolic acid copolymer to prepare RSV-loaded PLGA dispersion, the specific operation process was as follows: 10mg PLGA and 2mg RSV were dissolved in 1mL DMSO solution; 5mL 1% PVA v / v aqueous solution was added to a 50mL glass sample bottle; the glass sample bottle was placed on a magnetic stirrer and stirred at a speed of 400rpm, under stirring, the mixed solution of PLGA and RSV was added dropwise into the PVA solution, 3s per drop, after dropwise adding was completed, it was placed in a 3.5KD dialysis bag, dialysis for 12h, after freeze-drying, it was re-dissolved with 1mL PBS, to prepare the RSV-loaded PLGA dispersion, recorded as P@RSV NPs, the encapsulation efficiency was about 70%.

[0044] S3, macrophage cells were cultured, macrophage membrane was extracted, and a biomimetic membrane solution was prepared, the specific operation process was as follows: RAW264.7 cells were cultured in a 15cm culture dish, when the cell density reached 80%, the cells were collected, washed with PBS for three times, suspended in lysis solution, and repeated freeze-thawing for 5 times under the conditions of-80℃ and 37℃, so that the cells were fully broken. The cell nuclei and unbroken cells were removed by centrifugation at 4℃, 700g for 10min, and the cell membrane precipitate was collected by centrifugation at 4℃, 12000g for 10min. The collected cell membrane precipitate was resuspended with PBS, and the cell membrane was activated by probe ultrasonic for 5min at a power of 100W, to obtain a biomimetic membrane solution, recorded as Møm or M.

[0045] S4, the TIIA-loaded PLGA dispersion and the RSV-loaded PLGA dispersion were mixed with the biomimetic membrane solution, and extruded, to prepare a macrophage membrane-coated TIIA and RSV polylactic acid-glycolic acid copolymer dispersion, the specific operation process was as follows: The TIIA-loaded PLGA dispersion liquid and the RSV-loaded PLGA dispersion liquid are mixed according to a molar concentration ratio of 2:1, a biomimetic membrane solution is taken, and the mass ratio of PLGA to macrophage membrane is 5:1. The biomimetic macrophage membrane-coated TIIA and RSV-loaded PLGA dispersion liquid is obtained by repeatedly extruding 10 times by using an extruder, and is denoted as MP@TR.

[0046] S5, the phosphatidylated hyaluronic acid is mixed with the polylactic acid-glycolic acid copolymer dispersion liquid of tanshinone IIA and rosuvastatin coated by macrophage membranes to obtain the targeted biomimetic nanodrug, and the specific operation process is as follows: 25mg EDC, 50mg NHS, and 5mg hyaluronic acid are dissolved in 1mL PBS, stirred at room temperature for 30min at a speed of 800rpm, 25mg DSPE-PEG 2000 -NH2 is added to the mixture, and stirred at 37℃ for 24h at a speed of 800rpm. The phosphatidylated hyaluronic acid is dialyzed by using a 3.5KD dialysis bag for 24h, and freeze-dried to obtain the phosphatidylated hyaluronic acid, denoted as HA.

[0047] The phosphatidylated hyaluronic acid is redissolved in 1mL PBS solution, 10mg / mL phosphatidylated hyaluronic acid solution is taken and added to the MP@TR NPs, so that the mass ratio of phosphatidylated hyaluronic acid to PLGA reaches 1:10. The TIIA and RSV-loaded targeted biomimetic nanodrug, namely HAMP@TR NPs, is obtained by stirring at 37℃ in a water bath for 30min.

[0048] Figure 1 The appearance picture of the HAMP@TR NPs prepared in the application and the produced Tyndall phenomenon are shown in FIG. 1. Figure 1 As shown in FIG. 1, the appearance of the HAMP@TR NPs prepared in Example 1 presents a slight turbidity and opalescence, which is obviously different from a pure clear drug solution. The HAMP@TR NPs dispersion liquid presents the Tyndall phenomenon, which meets the characteristics of a nanomaterial dispersion liquid.

[0049] Figure 2 The transmission electron microscope (TEM) pictures of the HAMP@TR NPs and the empty carrier PLGA NPs prepared in the application are shown in FIG. 2, wherein A is the PLGA NPs and B is the HAMP@TR NPs. Figure 2 As shown in FIG. 2, the carrier PLGA NPs and the HAMP@TR NPs are spherical, regular in shape, and uniformly colored. The outer layer of the HAMP@TR NPs can be coated by macrophage membranes, and the particle size is 50nm-150nm. The above results show that the HAMP@TR NPs have the morphological characteristics of a nanoparticle, and are successfully coated by macrophage membranes.

[0050] Figure 3HAMP@TR NPs outer macrophage membrane marker immunoblotting results prepared for the present application. As shown in Figure 3

[0051] Figure 4 HAMP@TR NPs outer macrophage membrane and inner PLGA fusion experiment results prepared for the present application. Co-localization analysis shows that the macrophage membrane successfully wraps the PLGA NPs.

[0052] Example 2 The application of the targeted biomimetic nanodrug loaded with TIIA and RSV is as follows: 1. The targeted biomimetic nanodrug loaded with TIIA and RSV inhibits macrophage lipid deposition and inhibits endothelial mesenchymal transformation.

[0053] P@TR NPs, MP@TR NPs and HAMP@TR NPs were labeled with chlorin e6 to prepare P@ce6 NPs, MP@ce6 NPs and HAMP@ce6 NPs, respectively.

[0054] The preparation method of P@ce6 NPs is as follows: 2 mg of ce6 and 10 mg of PLGA are dissolved in 1 mL of DMSO. After thorough mixing, 10 mL of 1% v / v PVA aqueous solution is added dropwise on a magnetic stirrer, with stirring at a stirring speed of 400 rpm and a dropwise addition speed of 5 s per drop. The above obtained mixture is dialyzed in a 2.5 kD dialysis bag for 12 h. Centrifugation at 14000 rpm for 10 min, the precipitate is redissolved with 1 mL of PBS to prepare P@ce6 NPs dispersion, which is recorded as P@ce6.

[0055] The preparation method of MP@ce6 NPs is as follows: the biomimetic membrane solution is prepared by the method described in Example 1. 200 μL of biomimetic membrane solution is added to 1 mL of P@ce6 dispersion, so that the mass ratio of membrane to PLGA is 1:5. The extruder is repeatedly extruded for 10 times to obtain MP@ce6 NPs.

[0056] The preparation method of HAMP@ce6 NPs is as follows: phosphatidylated hyaluronic acid HA is prepared by the method described in Example 1. 100 μL of 10 mg / mL HA is added to 1.2 mL of MP@ce6 NPs dispersion prepared above, so that the mass ratio of HA to PLGA is 1:10. Stirring at 37°C for 30 min to prepare HAMP@ce6 NPs.

[0057] 1.1, Evaluation of the targeting and half-life of HAMP@TR NPs.​

[0058] 1.1.1, In vitro evaluation of the targeting property of HAMP@TR NPs.

[0059] RAW 264.7 cells were seeded at 1 x 10 5 cells / well, and HUVECs cells were seeded at 1 x 10 4 cells / well in 24-well plates. After 24 h incubation at 37 °C, 5% CO2v / v, 500 μM Hcy was added to RAW 264.7 culture dish for 24 h; 10 ng / mL IL-1β was added to HUVECs culture dish for 24 h. Then different NPs were added for 3 h, washed with PBS for 3 times, fixed with 4% paraformaldehyde for 10 min, stained with DAPI for 5 min, and imaged by laser confocal microscope.

[0060] Figure 5 For the results of macrophage and endothelial cell uptake of HAMP@ce6 NPs, the uptake rate of HAMP@ce6 NPs was significantly higher than that of other groups, followed by MP@ce6 group. Macrophage membrane wraps PLGA, giving nanoparticles chemotactic properties. After modification with HA, the prepared nanoparticles actively target cell surface receptor molecules CD44, promoting macrophages and endothelial cells to further uptake HAMP@ce6 NPs.

[0061] 1.1.2, In vivo evaluation of the targeting property of HAMP@TR NPs.

[0062] 8-week-old ApoE - / - Mice were fed with high-fat and high-protein diet for three months, and then injected with 200 μL of ce6, P@Ce6 NPs and HAMP@Ce6 NPs dispersion solution at a dose concentration of 5 mg / kg through the tail vein. After 12 h, the aortas of the mice were dissected for fluorescence imaging. High-fat and high-protein diet formula: NRV 2% methionine + 1.5% cholesterol + 15% fat + basal feed.

[0063] Figure 6 For the results of HAMP@ce6 accumulation in the aortas of mice. Compared with other groups, HAMP@ce6 NPs were significantly enriched in the aortas, indicating that membrane-wrapped PLGA, followed by HA modification, can escape the clearance of the immune system and efficiently target the lesion site.

[0064] C57BL / 6 mice were injected with 200 μL of Ce6 and HAMP@Ce6 NPs dispersion solution at a dose concentration of 5 mg / kg through the tail vein, and blood samples were collected at different time points, 0 h, 1 h, 2 h, 4 h, 4 h, 8 h, 12 h and 24 h, for fluorescence intensity determination.

[0065] Figure 7 The half-life of HAMP@Ce6 NPs in mice was determined. The results showed that the blood circulation half-life of Ce6 and HAMP@Ce6 was 1.313 h and 2.457 h, respectively. It is shown that the biomimetic membrane camouflage of nanomedicine is conducive to prolonging its blood circulation time.

[0066] 1.2, HAMP@TR NPs inhibit lipid deposition in macrophages.

[0067] 1.2.1, HAMP@TR NPs inhibit the uptake of Dil-oxLDL by macrophages.

[0068] RAW264.7 cells were inoculated in 24-well plates at 1×10 5 cells / well, except for the Control group, other groups were added with 500 μM Hcy for co-incubation for 24 h; the drug administration group was treated with different components of drugs for 24 h, and then 40 ng / mL Dil-oxLDL was added for 4 h; the cells were taken out, washed with PBS for 3 times, fixed with 4% paraformaldehyde for 10 min, and stained with DAPI for 30 min, and then photographed by confocal microscope. The TR of the drug administration group was 5 μM TIIA+2.5 μM RSV; the HAMP@TR group was added with 20 μL HAMP@TR NPs, which was equivalent to the content of TIIA and RSV of 5 μM+2.5 μM.

[0069] Figure 8 A of FIG. 1 is the Dil-oxLDL uptake inhibition experiment, and the results show that compared with the model group, HAMP@TR NPs have a significant inhibitory effect on oxLDL uptake.

[0070] 1.2.2, HAMP@TR NPS removes lipid droplets in macrophages.

[0071] RAW264.7 cells were inoculated in 24-well plates at 1×10 5 cells / well, except for the Control group, other groups were added with 500 μM Hcy for co-incubation for 24 h, and then different drugs were added for treatment for 24 h, and then 80 μg / mL ox-LDL was added for 24 h; the culture medium was removed, washed with PBS for 3 times, and then 3% ORO staining solution was added for staining for 30 min; and then photographed by microscope.

[0072] Figure 8 B of FIG. 1 is the macrophage oil red O staining result, and the HAMP@TR NPs group is obviously lighter, which shows that HAMP@TR NPs have the effect of removing lipid droplets in macrophages and inhibiting the formation of foam cells.

[0073] 1.3, HAMP@TR NPs inhibit the inflammatory response of macrophages.

[0074] RAW264.7 cells were divided into groups of 1×10⁻⁶. 5 Cells were seeded in 24-well plates. After incubation with 500 μM Hcy for 24 h, different drugs were added for 24 h each. Cell supernatant was collected, and inflammatory factors secreted by cells in each group were detected using a double-antibody sandwich ELISA.

[0075] Figure 9 The results of ELISA analysis of macrophage pro-inflammatory factors IL-1β, TNF-α, IL-6, and anti-inflammatory factor IL-10 are presented. The results indicate that HAMP@TR NPs loaded with TIIA and RSV exhibit significant anti-inflammatory effects.

[0076] 1.4 HAMP@TR NPs inhibit endothelial cell mesenchymal transition.

[0077] HUVECs were seeded in 6 cm culture dishes. When the cell density reached 70%, 10 ng / mL IL-1β was added for stimulation for 24 h, followed by co-incubation with different drugs for another 24 h. The cells were washed three times with PBS, digested with 0.25% trypsin v / v, centrifuged at 1200 rpm for 5 min, and the cell pellet was collected. The cells were lysed with RIPA lysis buffer containing 1% PMSF for 60 min, and the supernatant protein sample was collected by centrifugation at 14000 rpm for 30 min for Western blotting.

[0078] Figure 10 Western blot analysis results of markers related to endothelial cell mesenchymal transition under different drug interventions. During the development of atherosclerosis, when macrophages are stimulated by substances such as homocysteine ​​(Hcy), they secrete large amounts of the inflammatory factor IL-1β, which stimulates endothelial-mesenchymal transition (EMT) in lesion sites, causing endothelial cell damage. This promotes lipid entry into plaque sites, exacerbating lipid deposition in macrophages and creating a vicious cycle. VE-Cadherin and CD31 are markers of endothelial cells, while ICAM-1 and Vimentin are markers of mesenchymal cells. When mesenchymal transition is inhibited, the expression of endothelial markers increases, while the expression of mesenchymal markers decreases. Figure 10 The results showed that HAMP@TR NPs inhibited endothelial-mesenchymal transition.

[0079] 2. HAMP@TR NPs were synthesized using the method described in Example 1 for subsequent experiments.

[0080] 2.1 HAMP@TR NPs biomimetic nanoparticle formulations for the treatment of atherosclerosis.

[0081] 8-week-old male ApoE - / -The mice were randomly divided into a Model group, a TIIA group, an RSV group, a TR group and a HAMP@TR group, and C57BL / 6 mice of the same age were used as a Control group. The Control group was given a regular diet, and the rest of the groups were given a high-fat and high-protein HMD+HFD diet. After one month of the high-fat and high-protein diet, the mice were injected with drugs through the tail vein, once every three days, for two months of continuous administration. After the treatment, the aortas of the mice were stripped for sectioning and oil red O staining, and the serum of the mice was collected to detect the changes in homocysteine and low-density lipoprotein, so as to investigate the treatment effects of different treatment groups. The specific grouping is shown in Table 2.

[0082] Table 2 Grouping In Table 2, "-" represents that this item is not available.

[0083] Figure 11 Fig. 1 is a general staining picture of atherosclerosis of the aorta after treatment of different drug groups, wherein A is the general staining picture of atherosclerosis of the aorta after treatment of different drug groups. As shown in the figure, the white in the upper circle represents a plaque, and the red below represents lipid droplets on the plaque. It was observed that, in the Model group, atherosclerotic plaques were distributed throughout the aorta, especially enriched at the root of the aorta, the thoracic aorta and the abdominal aorta branch. Compared with the Model group, the aortic plaque and lipid droplets of the mice after HAMP@TR treatment were significantly reduced.

[0084] Figure 11 Fig. 1 is a general staining picture of atherosclerosis of the aorta after treatment of different drug groups, wherein B and C are oil red O staining pictures of the root of the aorta after treatment of different drug groups. As shown in the figure, the plaque lipid droplets of the HAMP@TR group were significantly reduced, indicating that the treatment effect was significant.

[0085] Figure 12 Fig. 1 is a general staining picture of atherosclerosis of the aorta after treatment of different drug groups, wherein Fig. 1 is the change in the levels of triglycerides, low-density lipoprotein, high-density lipoprotein, homocysteine and the like in the serum after treatment of different drug groups. After HAMP@TR treatment, the levels of triglycerides, low-density lipoprotein and homocysteine in the serum were significantly reduced, and the level of high-density lipoprotein was not changed, indicating that the application can improve the blood lipid level after a high-fat and high-protein diet, and has a significant therapeutic effect on atherosclerosis.

[0086] 2.2, in vivo biological safety evaluation of HAMP@TR NPs biomimetic nanodrugs.

[0087] Using the above-mentioned treatment scheme, ApoE - / - After the treatment of the mice, the main organs of the mice, including the heart, liver, spleen, lung and kidney, were collected for H&E staining to investigate the biological safety of different treatment groups.

[0088] Figure 13 Fig. 2 is the HE staining results. The results show that there are no obvious organic lesions in the organs of the mice in different treatment groups, indicating that the biomimetic nanodrugs constructed by the application have good biological safety.

[0089] Each technical feature of the above-described embodiments can be combined with any other technical feature, and for the sake of brevity, not all possible combinations are described, but it is understood that the scope of the specification includes all such possible combinations.

[0090] The above-described embodiments are merely representative of several embodiments of the present application, and the description is relatively specific and detailed, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application.

Claims

1. A targeted biomimetic nanodrug loaded with tanshinone IIA and rosuvastatin, characterized in that, The targeting biomimetic nanodrug is prepared by co-loading tanshinone IIA and rosuvastatin on polylactic acid-glycolic acid copolymer, coating the macrophage membrane on the outer layer of the polylactic acid-glycolic acid copolymer after loading, and modifying the surface of the macrophage membrane by phosphatidylated hyaluronic acid.

2. The method for preparing targeted biomimetic nanomedicine as described in claim 1, characterized in that, The method comprises the following steps: The tanshinone IIA is wrapped in the polylactic acid-glycolic acid copolymer to prepare a tanshinone IIA-loaded polylactic acid-glycolic acid copolymer dispersion liquid; The rosuvastatin is wrapped in the polylactic acid-glycolic acid copolymer to prepare a rosuvastatin-loaded polylactic acid-glycolic acid copolymer dispersion liquid; The macrophage membrane solution is prepared by culturing the macrophages and extracting the macrophage membrane; The tanshinone IIA-loaded polylactic acid-glycolic acid copolymer dispersion liquid and the rosuvastatin-loaded polylactic acid-glycolic acid copolymer dispersion liquid are mixed with the biomimetic membrane solution to obtain a macrophage membrane-coated tanshinone IIA and rosuvastatin polylactic acid-glycolic acid copolymer dispersion liquid by extrusion; The phosphatidylated hyaluronic acid is synthesized, and the phosphatidylated hyaluronic acid is uniformly mixed with the macrophage membrane-coated tanshinone IIA and rosuvastatin polylactic acid-glycolic acid copolymer dispersion liquid to obtain the targeting biomimetic nanodrug.

3. The production method according to claim 2, wherein When the tanshinone IIA-loaded polylactic acid-glycolic acid copolymer dispersion liquid is prepared, the mass ratio of tanshinone IIA to polylactic acid-glycolic acid copolymer is 1:5-10.

4. The production method according to claim 2, wherein When the rosuvastatin-loaded polylactic acid-glycolic acid copolymer dispersion liquid is prepared, the mass ratio of rosuvastatin to polylactic acid-glycolic acid copolymer is 1:3-5.

5. The production method according to claim 2, wherein When the macrophage membrane-coated tanshinone IIA and rosuvastatin polylactic acid-glycolic acid copolymer dispersion liquid is prepared, the molar ratio of the tanshinone IIA-loaded polylactic acid-glycolic acid copolymer dispersion liquid to the rosuvastatin-loaded polylactic acid-glycolic acid copolymer dispersion liquid is 1-2:

1.

6. The production method according to claim 2, wherein When the macrophage membrane-coated tanshinone IIA and rosuvastatin polylactic acid-glycolic acid copolymer dispersion liquid is prepared, the mass ratio of the macrophage membrane to the polylactic acid-glycolic acid copolymer is 1:2-5.

7. The production method according to claim 2, wherein When the phosphatidylated hyaluronic acid is uniformly mixed with the macrophage membrane-coated tanshinone IIA and rosuvastatin polylactic acid-glycolic acid copolymer dispersion liquid, the mass ratio of the phosphatidylated hyaluronic acid to the polylactic acid-glycolic acid copolymer is 1:2-10.

8. The production method according to claim 2, wherein The method for synthesizing the phosphatidylated hyaluronic acid is as follows: The 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino are dissolved in PBS, and after stirring at 37℃ for 22-26h, dialysis and freeze-drying are performed to obtain the phosphatidylated hyaluronic acid; The mass ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, hyaluronic acid and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-amino is 5-6:8-10:1:5-6.

9. The use of claim 1, wherein the targeted biomimetic nanomedicine is used for the treatment of cancer. The targeting biomimetic nanodrug is used for preparing a preparation for treating atherosclerosis.

10. The use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The atherosclerosis includes at least one of aortic atherosclerosis, carotid atherosclerosis, and cerebral atherosclerosis.

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