Targeted nano-drug delivery system based on bionic composite membrane modification and application thereof

Abstract

The invention belongs to the technical field of nano medicine, and particularly relates to a targeted nano drug delivery system based on bionic composite membrane modification and application of the targeted nano drug delivery system. The preparation method comprises the following steps: firstly, loading hydrophobic drugs astragaloside and atorvastatin in a phospholipid bilayer of a liposome to obtain a drug-loaded liposome, then mixing an erythrocyte membrane with a macrophage membrane to obtain a hybrid composite membrane, coating the drug-loaded liposome with the hybrid composite membrane to obtain a liposome-composite membrane, and preparing the drug-loaded liposome-composite membrane. Finally, hyaluronic acid is inserted into the outer layer of the liposome-composite membrane, and the targeted nano drug delivery system is obtained. Compared with treatment by singly using drugs, the targeted nano drug delivery system provided by the invention has a remarkable combined synergistic effect, and a feasible treatment strategy is provided for effectively treating atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a targeted nano drug delivery system based on bionic composite membrane modification and application thereof. Background Art

[0002] Atherosclerosis is a chronic disease that seriously threatens human health and is the main cause of cardiovascular morbidity and mortality. Its pathological characteristics are characterized by intravascular plaque formation, and its advanced lesions can cause serious downstream effects, such as acute myocardial infarction and stroke. Currently, intravascular stent implantation is an invasive strategy widely used in clinical practice, but there are problems with late thrombosis and postoperative restenosis. In the management of atherosclerosis, the use of therapeutic drug intervention is more acceptable due to its low fatal risk and low medical cost.

[0003] Currently, there are many drugs available for the prevention and treatment of atherosclerosis, such as atorvastatin, simvastatin, clopidogrel, and astragaloside IV. Astragaloside IV (AS-IV) is the main component of the traditional Chinese medicine Astragalus. It can treat atherosclerosis by anti-oxidative stress, anti-inflammation, regulating calcium homeostasis, improving myocardial energy metabolism, anti-apoptosis, regulating myocardial autophagy, and improving myocardial microcirculation. However, astragaloside IV has poor solubility in water and low bioavailability. Atorvastatin (AT), as a lipid-lowering statin used on a large scale in clinical practice, is characterized by rapid onset and strong lipid-lowering effect. However, due to the obvious first-pass effect of atorvastatin in the liver, its oral bioavailability is low, and it is difficult to obtain satisfactory efficacy and safety after long-term administration. Therefore, it is urgent to adopt advanced strategies to integrate enhanced targeted drug delivery, reduce systemic side effects caused by drugs, and improve drug bioavailability.

[0004] With the rapid development of nanotechnology, nanomedicine has also made great progress. Nanomedicine refers to the use of nanoscale materials, such as nanoparticles (NPs) with good biocompatibility. NPs are a general term for materials with a size of less than a few hundred nanometers. Liposomes (LP), as a common type of nanomaterial, have the characteristics of nanoscale size, similar biological membrane structure, and good biocompatibility. As a delivery system, they have good application prospects in drug development. After ordinary nano drug delivery systems enter the human blood circulation system, the phagocytic system in the body easily identifies nanoparticles as foreign substances and quickly removes them. In addition, most nanomaterials with short circulation times cannot effectively accumulate at the site of lesions, resulting in limited therapeutic effects on diseases. Summary of the invention

[0005] In view of the above-mentioned problems, the purpose of the present invention is to provide a targeted nano-drug delivery system based on bionic composite membrane modification and its application, which can prolong the blood circulation time of nano-materials in the body, enhance immune escape and precise targeting capabilities, so that the drug can achieve optimal accumulation in the atherosclerotic plaque area, and provide a feasible therapeutic strategy for the effective treatment of atherosclerosis.

[0006] The first aspect of the present invention provides a targeted nano drug delivery system, which has a core-shell structure, with a liposome loaded with a hydrophobic drug as the core material and a hybrid cell membrane-hyaluronic acid as the shell material; the hydrophobic drug is astragaloside IV and / or atorvastatin; the targeted nano drug delivery system is prepared by the following steps:

[0007] After mixing the mixed solution of lecithin and cholesterol with the hydrophobic drug, removing the solvent, adding water for ultrasonication, centrifuging and collecting the precipitate to obtain the drug-loaded liposome;

[0008] The erythrocyte membrane and the macrophage membrane are mixed to obtain a hybrid composite membrane;

[0009] The hybrid composite membrane is mixed with the drug-loaded liposome, so that the hybrid composite membrane is attached to the surface of the drug-loaded liposome to obtain a composite membrane-liposome;

[0010] The hyaluronic acid is mixed with the composite membrane-liposome, and the hyaluronic acid is inserted into the hybrid composite membrane to obtain a targeted nano drug delivery system; the particle size of the targeted nano drug delivery system is 100-200nm.

[0011] Furthermore, the mixing mass ratio of the erythrocyte membrane to the macrophage membrane is 1:1-3.

[0012] Furthermore, the mass ratio of the lecithin, cholesterol, astragaloside IV and atorvastatin is 4-6:1:1:1.

[0013] Furthermore, the mass ratio of the lecithin to the hybrid composite membrane is 18-22:1; the mass ratio of the lecithin to the hyaluronic acid is 8-12:1.

[0014] Furthermore, the reaction conditions for mixing the red blood cell membrane and the macrophage membrane are 36-37° C. for 55-65 min.

[0015] Furthermore, the reaction conditions for mixing the hybrid composite membrane and the drug-loaded liposomes are 36-37° C. for 55-65 min.

[0016] Furthermore, the reaction conditions for mixing the hyaluronic acid with the composite membrane-liposome are 36-37° C. for 25-35 min.

[0017] The second aspect of the present invention provides a use of the above-mentioned targeted nano drug delivery system in the preparation of drugs for preventing / treating atherosclerosis.

[0018] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0019] The present invention designs a safe and effective targeted nano drug delivery system, firstly, the hydrophobic drug is loaded into the phospholipid molecular layer of the nano material liposome, then the erythrocyte membrane and the macrophage membrane are mixed to obtain a hybrid composite membrane, and finally hyaluronic acid is inserted into the hybrid composite membrane to modify the drug-loaded liposome together, thereby obtaining a targeted nano drug delivery system HA-M@LP@ST. The present invention utilizes the long half-life of the erythrocyte membrane, prolongs the blood circulation time of the modified liposome, utilizes the immune homology of the macrophage membrane to give it an immune escape effect, and utilizes hyaluronic acid for final modification, further increases the circulation time of the drug in the body, and achieves optimal accumulation when the drug targets the atherosclerotic plaque area, improves the efficacy of the drug, and reduces the liver and kidney toxicity caused by non-targeting, thereby realizing the targeted delivery of the bionic nano drug delivery system. The present invention provides new theoretical support for the development of new drugs for the prevention and treatment of atherosclerosis and related clinical prevention and treatment, and has important scientific significance, application value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure A is a transmission electron micrograph of liposomes, and Figure B is a transmission electron micrograph of the targeted nano-drug delivery system HA-M@LP@ST prepared in Example 1.

[0021] Figure 2 Figure A shows the particle size measurement results of Liposome, LP@ST, M@LP@ST, and HA-M@LP@ST, and Figure B shows the potential measurement results of Liposome, LP@ST, M@LP@ST, and HA-M@LP@ST.

[0022] Figure 3 The figures are the results of membrane fusion experiment and stability experiment; Figure A is the macrophage membrane labeled with Dio dye, Figure B is the red blood cell membrane labeled with DiI dye, and Figure C is the fusion image of Figure A and Figure B; Figure D is the liposome labeled with Dio dye, Figure E is the composite membrane labeled with Dil dye, and Figure F is the fusion image of Figure D and Figure E; Figure G is the particle size result of the prepared HA-M@LP@ST after storage in sodium chloride, high glucose medium (DEME) and medium containing 10% fetal bovine serum (10% FBS) for 7 days; Figure H, from top to bottom, each row represents the image of HA-M@LP@ST after culture in sodium chloride, high glucose medium, and medium containing 10% fetal bovine serum for 7 days.

[0023] Figure 4 The results of the cell uptake experiment; A to C are confocal fluorescence images of the uptake of LP@Ce6, M@LP@Ce6 and HA-M@LP@Ce6 labeled with Ce6 in normal macrophages; D to F are confocal fluorescence images of the uptake of M@LP@Ce6, HA-M@LP@Ce6 and HA-M@LP@Ce6 pretreated with hyaluronic acid in inflammatory macrophages stimulated with homocysteine ​​(Hcy); G is the statistical result of the uptake experiment.

[0024] Figure 5 The figures are the results of blood compatibility experiment; Figures A to E are the inverted fluorescence microscope images of red blood cells in PBS, Water, AS-IV+AT, LP@ST, and HA-M@LP@ST solutions of the hemolysis experiment; Figure F is the statistical result of the hemolysis experiment; Figure G is the ep tube image of red blood cells in PBS, Water, and different concentrations of AS-IV+AT, LP@ST, and HA-M@LP@ST; Figure H is the statistical result of platelets in thrombin, PBS, AS-IV+AT, LP@ST, and HA-M@LP@ST solutions of the coagulation experiment.

[0025] Figure 6 The results of the reactive oxygen species scavenging experiment; Figures A to D are the confocal fluorescence images of the negative control group, model group, AS-IV+AT group, and HA-M@LP@ST group, respectively; Figure E is the statistical results of the reactive oxygen species scavenging experiment.

[0026] Figure 7 This is the Oil Red O (ORO) experiment of macrophage-derived foam cells; Figures A to D are the inverted microscope images of the negative control group, model group, AS-IV+AT group, and HA-M@LP@ST group, respectively; Figure E is the statistical results of the ORO experiment.

[0027] Figure 8 For ApoE - / - Results of targeted experiments on atherosclerotic plaques in mice; Figure A shows the targeting of Ce6, M@LP@Ce6, and HA-M@LP@Ce6 labeled with Ce6 at ApoE - / - Fluorescence localization map of mouse aorta. Three mouse aortas in each group were used for repeated experiments. Figure B shows the statistical results of fluorescence intensity.

[0028] Fig. 9 ApoE - / - Results of mouse aorta ORO experiment; Figures A to E are ApoE in the model group, AS-IV group, AT group, AS-IV+AT group, and HA-M@LP@ST group, respectively. - / -ORO staining images of mouse aortas. Four mouse aortas in each group were used for repeated experiments. Figure B shows the statistical results of ORO staining of aortas.

[0029] Fig.10 ApoE - / - Results of in vivo safety experiments in mice; Figures A to E are ApoE in the model group, AS-IV group, AT group, AS-IV+AT group, and HA-M@LP@ST group, respectively. - / - HE staining images of mouse heart, liver, spleen, lung and kidney. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0031] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0032] Current drugs used to prevent and treat different forms of atherosclerosis have problems with low bioavailability and high systemic toxicity, and it is difficult to obtain satisfactory therapeutic effects after long-term administration. After ordinary nano-drug delivery systems enter the human blood circulation system, the body's phagocytic system easily recognizes nanoparticles as foreign substances and quickly removes them. In addition, most nanomaterials with short circulation times cannot effectively accumulate at the lesion site, resulting in limited therapeutic effects on the disease.

[0033] The present invention provides a targeted nano drug delivery system based on biomimetic composite membrane modification and its application. The present invention first loads hydrophobic drugs astragaloside IV and atorvastatin in the phospholipid bilayer of liposomes to prepare drug-loaded liposomes, then mixes erythrocyte membranes with macrophage membranes to obtain hybrid composite membranes, and coats drug-loaded liposomes with hybrid composite membranes to obtain liposome-composite membranes, and finally inserts hyaluronic acid into the outer layer of the liposome-composite membrane to obtain a targeted nano drug delivery system. The targeted nano drug delivery system provided by the present invention can increase the circulation time of drugs in the body and obtain the ability of immune escape, realize the precise targeting effect of drugs, and introduces the two fat-soluble drugs astragaloside IV and atorvastatin into activated macrophages in the atherosclerotic plaque site in a targeted manner to improve the efficacy of drugs and reduce liver and kidney toxicity caused by non-targeting, thereby realizing the targeted delivery of the biomimetic nano drug delivery system.

[0034] Example 1: Preparation of targeted nano-drug delivery system HA-M@LP@ST

[0035] S1. Preparation of drug-loaded liposomes LP@ST

[0036] Use chloroform to fully dissolve lecithin and cholesterol, use methanol to fully dissolve astragaloside IV (AS-IV) and atorvastatin (AT), and add them together into a round-bottom flask, evaporate under reduced pressure at 45°C until a uniform transparent film is formed on the inner wall of the flask, and then add distilled water for hydration. After hydration, use water bath ultrasound to mix the liquid in the bottle, and then continue to use 80w probe ultrasound for 5 minutes. The precipitate obtained by centrifuging the above solution is LP@ST, which is stored at 4°C. The specific preparation steps are as follows:

[0037] Step ①: Dissolve 4 mg of lecithin in 0.1 mL of chloroform to form a solution with a concentration of 40 mg / mL, dissolve 1 mg of cholesterol in 0.02 mL of chloroform to form a liquid with a concentration of 50 mg / mL, dissolve 1 mg of AS-IV in 0.2 mL of methanol to form a liquid with a concentration of 5 mg / mL, and dissolve 1 mg of AT in 0.025 mL of methanol to form a liquid with a concentration of 40 mg / mL. Use chloroform to make up the remaining liquid to a total volume of 5 mL, mix well, and add to a round-bottom flask.

[0038] Step ②: The solution prepared in step ① was ultrasonicated for 5 min using an 80W probe.

[0039] Step ②: Centrifuge the solution obtained in step ② at 13600 rpm and 4°C for 30 min. The resulting precipitate is LP@ST, which is stored at 4°C.

[0040] S2. Preparation of hybrid composite membrane

[0041] Obtaining red blood cell membrane (RBCm): Collect whole blood from C57 BL / 6 mice, remove plasma and wash with 1×PBS and centrifuge until the supernatant is clear. Resuspend the obtained red blood cell precipitate with 0.25×PBS and lyse on ice at 4°C overnight; use 80w probe ultrasound for 5min to completely break the red blood cells to obtain membrane fragments; centrifuge to obtain the membrane fragments in the middle layer of the precipitate and store at -80°C.

[0042] Obtaining macrophage membranes RAW264.7 cells in the cell culture dish were collected, centrifuged at 1000 rpm and 4°C for 10 min to obtain cell pellets, and macrophage membranes were extracted according to the instructions of the cell membrane protein and cytoplasm protein extraction kit (purchased from Biyuntian Company) at 4°C. After centrifugation, the cell pellets were stored at -80°C.

[0043] Preparation of composite membrane: Take the RBCm collected above, Precipitation, RBCm, The mixture was mixed in a mass ratio of 1:1 and then stirred in a water bath at 37°C for 1 h to obtain a hybrid composite membrane.

[0044]

[0045] S3. Preparation of liposome-composite membrane M@LP@ST

[0046] The composite membrane prepared in step S2 was mixed with the LP@ST prepared in step S1, wherein the mixing mass ratio of lecithin in LP@ST to the composite membrane was 20:1. After stirring in a 37°C water bath for 1 h, a liposome-composite membrane M@LP@ST was obtained and stored at 4°C.

[0047] S4. Preparation of targeted nano drug delivery system HA-M@LP@ST

[0048] Preparation of hyaluronic acid (HA-PEG2000-DSPE, HA): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and hyaluronic acid were added to PBS in sequence and stirred at room temperature for 30 minutes; then phospholipid polyethylene glycol amino MW: 2000 (DSPE-PEG2000-NH2) was added and stirred at 37°C for 24 hours; finally, the resulting solution was dialyzed for 12 hours using a dialysis bag with a molecular weight cutoff of 2.5 kDa to remove free NHS and ED to obtain hyaluronic acid liquid, which was then freeze-dried and the freeze-dried powder was stored at -20°C for future use.

[0049] The obtained HA was mixed with M@LP@ST prepared in step S3, wherein the mixing mass ratio of lecithin to HA was 10:1. After stirring in a 37°C water bath for 30 min, the obtained targeted nano-drug delivery system HA-M@LP@ST was collected and stored at 4°C.

[0050] Example 2: Preparation of targeted nano-drug delivery system HA-M@LP@ST

[0051] S1. Preparation of drug-loaded liposomes LP@ST

[0052] Use chloroform to fully dissolve lecithin and cholesterol, use methanol to fully dissolve astragaloside IV (AS-IV) and atorvastatin (AT), and add them together into a round-bottom flask, evaporate under reduced pressure at 45°C until a uniform transparent film is formed on the inner wall of the flask, and then add distilled water for hydration. After hydration, use water bath ultrasound to mix the liquid in the bottle, and then continue to use 80w probe ultrasound for 5 minutes. The precipitate obtained by centrifuging the above solution is LP@ST, which is stored at 4°C. The specific preparation steps are as follows:

[0053] Step ①: Dissolve 5 mg of lecithin in 0.1 mL of chloroform to form a solution with a concentration of 40 mg / mL, dissolve 1 mg of cholesterol in 0.02 mL of chloroform to form a liquid with a concentration of 50 mg / mL, dissolve 1 mg of AS-IV in 0.2 mL of methanol to form a liquid with a concentration of 5 mg / mL, and dissolve 1 mg of AT in 0.025 mL of methanol to form a liquid with a concentration of 40 mg / mL. Use chloroform to make up the remaining liquid to a total volume of 5 mL, mix well, and add to a round-bottom flask.

[0054] Step ②: The solution prepared in step ① was ultrasonicated for 5 min using an 80W probe.

[0055] Step ②: Centrifuge the solution obtained in step ② at 13600 rpm and 4°C for 30 min. The resulting precipitate is LP@ST, which is stored at 4°C.

[0056] S2. Preparation of hybrid composite membrane

[0057] Obtaining red blood cell membrane (RBCm): Collect whole blood from C57 BL / 6 mice, remove plasma and wash with 1×PBS and centrifuge until the supernatant is clear. Resuspend the obtained red blood cell precipitate with 0.25×PBS and lyse on ice at 4°C overnight; use 80w probe ultrasound for 5min to completely break the red blood cells to obtain membrane fragments; centrifuge to obtain the membrane fragments in the middle layer of the precipitate and store at -80°C.

[0058] Obtaining macrophage membranes RAW264.7 cells in the cell culture dish were collected, centrifuged at 1000 rpm and 4°C for 10 min to obtain cell pellets, and macrophage membranes were extracted according to the instructions of the cell membrane protein and cytoplasm protein extraction kit (purchased from Biyuntian Company) at 4°C. After centrifugation, the cell pellets were stored at -80°C.

[0059] Preparation of composite membrane: Take the RBCm collected above, Precipitation, RBCm, The mixture was mixed in a mass ratio of 1:2 and then stirred in a water bath at 36°C for 55 min to obtain a hybrid composite membrane.

[0060]

[0061] S3. Preparation of liposome-composite membrane M@LP@ST

[0062] The composite membrane prepared in step S2 was mixed with the LP@ST prepared in step S1, wherein the mixing mass ratio of lecithin in LP@ST to the composite membrane was 18:1. After stirring in a 36°C water bath for 55 min, M@LP@ST was obtained and stored at 4°C.

[0063] S4. Preparation of targeted nano drug delivery system HA-M@LP@ST

[0064] Preparation of hyaluronic acid (HA-PEG2000-DSPE, HA): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and hyaluronic acid were added to PBS in sequence and stirred at room temperature for 30 minutes; then phospholipid polyethylene glycol amino MW: 2000 (DSPE-PEG2000-NH2) was added and stirred at 37°C for 24 hours; finally, the resulting solution was dialyzed for 12 hours using a dialysis bag with a molecular weight cutoff of 2.5 kDa to remove free NHS and ED to obtain hyaluronic acid liquid, which was then freeze-dried and the freeze-dried powder was stored at -20°C for future use.

[0065] The obtained HA was mixed with the M@LP@ST prepared in step S3, wherein the mixing mass ratio of lecithin to HA was 8:1. After stirring in a 36°C water bath for 25 minutes, the HA-M@LP@ST was collected and stored at 4°C.

[0066] Example 3: Preparation of targeted nano-drug delivery system HA-M@LP@ST

[0067] S1. Preparation of drug-loaded liposomes LP@ST

[0068] Use chloroform to fully dissolve lecithin and cholesterol, use methanol to fully dissolve astragaloside IV (AS-IV) and atorvastatin (AT), and add them together into a round-bottom flask, evaporate under reduced pressure at 45°C until a uniform transparent film is formed on the inner wall of the flask, and then add distilled water for hydration. After hydration, use water bath ultrasound to mix the liquid in the bottle, and then continue to use 80w probe ultrasound for 5 minutes. The precipitate obtained by centrifuging the above solution is LP@ST, which is stored at 4°C. The specific preparation steps are as follows:

[0069] Step ①: Dissolve 6 mg of lecithin in 0.1 mL of chloroform to form a solution with a concentration of 40 mg / mL, dissolve 1 mg of cholesterol in 0.02 mL of chloroform to form a liquid with a concentration of 50 mg / mL, dissolve 1 mg of AS-IV in 0.2 mL of methanol to form a liquid with a concentration of 5 mg / mL, and dissolve 1 mg of AT in 0.025 mL of methanol to form a liquid with a concentration of 40 mg / mL. Use chloroform to make up the remaining volume to a total volume of 5 mL, mix well, and add to a round-bottom flask.

[0070] Step ②: The solution prepared in step ① was ultrasonicated for 5 min using an 80W probe.

[0071] Step ②: Centrifuge the solution obtained in step ② at 13600 rpm and 4°C for 30 min. The resulting precipitate is LP@ST, which is stored at 4°C.

[0072] S2. Preparation of hybrid composite membrane

[0073] Obtaining red blood cell membrane (RBCm): Collect whole blood from C57 BL / 6 mice, remove plasma and wash with 1×PBS and centrifuge until the supernatant is clear. Resuspend the obtained red blood cell precipitate with 0.25×PBS and lyse on ice at 4°C overnight; use 80w probe ultrasound for 5min to completely break the red blood cells to obtain membrane fragments; centrifuge to obtain the membrane fragments in the middle layer of the precipitate and store at -80°C.

[0074] Obtaining macrophage membranes RAW264.7 cells in the cell culture dish were collected, centrifuged at 1000 rpm and 4°C for 10 min to obtain cell pellets, and macrophage membranes were extracted according to the instructions of the cell membrane protein and cytoplasm protein extraction kit (purchased from Biyuntian Company) at 4°C. After centrifugation, the cell pellets were stored at -80°C.

[0075] Preparation of composite membrane: Take the RBCm collected above, Precipitation, RBCm, The mixture was mixed in a mass ratio of 1:3 and then stirred in a 37°C water bath for 65 min to obtain a hybrid composite membrane.

[0076]

[0077] S3. Preparation of liposome-composite membrane M@LP@ST

[0078] The composite membrane prepared in step S2 was mixed with the LP@ST prepared in step S1, wherein the mixing mass ratio of lecithin in LP@ST to the composite membrane was 22:1. After stirring in a 37°C water bath for 65 min, M@LP@ST was obtained and stored at 4°C.

[0079] S4. Preparation of targeted nano drug delivery system HA-M@LP@ST

[0080] Preparation of hyaluronic acid (HA-PEG2000-DSPE, HA): 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and hyaluronic acid were added to PBS in sequence and stirred at room temperature for 30 minutes; then phospholipid polyethylene glycol amino MW: 2000 (DSPE-PEG2000-NH2) was added and stirred at 37°C for 24 hours; finally, the resulting solution was dialyzed for 12 hours using a dialysis bag with a molecular weight cutoff of 2.5 kDa to remove free NHS and ED to obtain hyaluronic acid liquid, which was then freeze-dried and the freeze-dried powder was stored at -20°C for future use.

[0081] The obtained HA was mixed with the M@LP@ST prepared in step S3, wherein the mixing mass ratio of lecithin to HA was 12:1. After stirring in a 37°C water bath for 35 minutes, the HA-M@LP@ST was collected and stored at 4°C.

[0082] In order to verify the application effect of the targeted nano drug delivery system HA-M@LP@ST prepared by the present invention, the inventors conducted the following experiments:

[0083] Since Examples 1 to 3 have similar effects, for the convenience of subsequent discussion and reference, the test results of Example 1 shall prevail. The following are the relevant test results of Example 1.

[0084] Experimental Example 1: Characterization of HA-M@LP@ST

[0085] 1. Transmission electron microscopy observation

[0086] The HA-M@LP@ST prepared in Example 1 was observed by transmission electron microscopy, and liposomes were used as a control.

[0087] The results are as follows Figure 1 As shown, it can be seen that the HA-M@LP@ST prepared in Example 1 is evenly dispersed and spherical, with a particle size of 100 to 200 nm, and the preparation is successful.

[0088] 2. Particle size and potential analysis

[0089] The present invention characterizes the particle size and charge properties of LP@ST, M@LP@ST, and HA-M@LP@ST prepared in Example 1, and Liposome is used as a control.

[0090] The results are as follows Figure 2 As shown, the particle size of HA-M@LP@ST is between 150 and 200 nm, and the potential is between -20 and -60 mV.

[0091] 3. Fluorescence confocal microscopy observation

[0092] The present invention is for RBCm, The specific steps for conducting membrane fusion confocal fluorescence experiments with Liposome are as follows:

[0093] ① Add fluorescent probes DiO and DiI to and RBCm, obtain and Dil-RBCm, and then the two solutions are mixed and stirred to obtain The solution was finally photographed under a fluorescence confocal microscope.

[0094] The results are as follows Figure 3As shown in Figures A, B and C, the Merge results indicate that the composite membrane fusion was successful.

[0095] ②Add DiO and DiI to Liposome and In the solution, DiO-Liposome and The two solutions were stirred and mixed to obtain DiO / DiI-M@LP solution; finally, the solution was photographed under a fluorescence confocal microscope.

[0096] The results are as follows Figure 3 Figures D, E, and F show that the Merge results suggest that the composite membrane successfully modified M@LP.

[0097] Finally, the present invention conducted a stability test on the prepared HA-M@LP@ST.

[0098] The results are as follows Figure 3 As shown in Figure G, the particle diameter of HA-M@LP@ST after storage in sodium chloride, high glucose medium, and medium containing 10% fetal bovine serum for 7 days is approximately 100-200 nm, indicating that HA-M@LP@ST has good stability.

[0099] Experimental Example 2: HA-M@LP@ST uptake experiment on macrophages

[0100] Using the HA-M@LP@ST prepared in Example 1, a cellular uptake experiment of HA-M@LP@ST was conducted in vitro using a quantitative method of detecting fluorescence intensity.

[0101] First, LP@Ce6, M@LP@Ce6, and HA-M@LP@Ce6 labeled with the photosensitizer dihydrochlorin e6 (Ce6) were co-cultured with normal macrophages and inflammatory macrophages stimulated with homocysteine ​​(Hcy) for 4 hours, respectively; one group was pretreated with hyaluronic acid for 2 hours and then HA-M@LP@Ce6 was added and co-cultured with inflammatory macrophages for 4 hours; then the cells were stained with DAPI and observed under a confocal microscope.

[0102] The results are as follows Figure 4 As shown in the figure, compared with the LP@Ce6 group, the HA-M@LP@Ce6 group aggregated more in inflammatory macrophages, indicating that the HA-M@LP@Ce6 prepared in Example 1 can effectively target the inflammatory site. At the same time, the HA-M@LP@Ce6 group pretreated with hyaluronic acid had a reduced uptake by macrophages, indicating that hyaluronic acid played a major role in targeting. In the normal macrophage group, HA-M@LP@Ce6 was less taken up by normal macrophages than LP@Ce6, indicating that HA-M@LP@Ce6 had acquired the ability to escape.

[0103] Experimental Example 3: In vitro safety evaluation of HA-M@LP@ST

[0104] The safety of HA-M@LP@ST was evaluated using blood compatibility experiments.

[0105] Using the HA-M@LP@ST prepared in Example 1, PBS, different concentrations of AS-IV+AT, LP@ST and HA-M@LP@ST were co-cultured with red blood cells and platelets for 6 hours, respectively. Water and thrombin were set as positive control groups. The experimental results were then reflected by photographing under an inverted microscope and detecting the absorbance using a microplate.

[0106] The results are as follows Figure 5 As shown, HA-M@LP@ST achieved excellent safety in vitro.

[0107] Experimental Example 4: Evaluation of the efficacy of HA-M@LP@ST on atherosclerosis in vitro

[0108] 1. ROS scavenging experiment

[0109] Fluorescence imaging was used to determine the ability of HA-M@LP@ST prepared in Example 1 to clear ROS produced by inflammatory macrophages.

[0110] The experimental results are as follows Figure 6 As shown, the HA-M@LP@ST group was more effective in clearing ROS produced by inflammatory macrophages compared with the AS-IV+AT group alone.

[0111] 2. ORO experiment of macrophage-derived foam cells

[0112] An inverted microscope was used to measure the effect of HA-M@LP@ST prepared in Example 1 on the internalization of macrophage-derived foam cells.

[0113] The experimental results are as follows Figure 7 As shown, the HA-M@LP@ST group had a stronger internalization effect on macrophage-derived foam cells compared with the AS-IV+AT group alone.

[0114] Experimental Example 5: Evaluation of in vivo targeting ability of HA-M@LP@ST

[0115] The ability of HA-M@LP@ST prepared in Example 1 to target atherosclerotic plaques in vivo was determined by quantitatively detecting fluorescence intensity.

[0116] ApoE - / -After mice were fed a high-fat, high-protein diet for 12 weeks, 5 mg / kg of Ce6, M@LP@Ce6 and HA-M@LP@Ce6 were injected into the tail vein. Twelve hours later, the mouse aortas were taken for fluorescence imaging, with 3 mice in each group.

[0117] The results are as follows Figure 8 As shown, compared with the Ce6 and M@LP@Ce6 groups, the HA-M@LP@Ce6 group significantly aggregated in the aortic arch and abdominal aorta, indicating that HA-M@LP@ST can effectively target the site of atherosclerotic plaques.

[0118] Experimental Example 6: In vivo efficacy evaluation of HA-M@LP@ST

[0119] The HA-M@LP@ST prepared in Example 1 is used for the treatment of atherosclerosis.

[0120] ApoE - / - The mice were fed a high-fat, high-methionine diet for 12 weeks, and treatment began at the 5th week. They were divided into the following groups: model group; drug-treated group; the drug-treated groups were divided into AS-IV (8 mg / kg), AT (2 mg / kg), AS-IV+AT (8 mg / kg+2 mg / kg) and HA-M@LP@ST (8 mg / kg+2 mg / kg) groups. The mice were fed a high-fat, high-methionine diet at the same time. After the treatment, the aorta of the mice was stripped and stained with Oil Red O to examine the therapeutic effects of the different treatment groups, with 4 mice in each group.

[0121] The results are as follows Fig. 9 As shown, compared with other groups, the HA-M@LP@ST treatment group most significantly reduced the formation of atherosclerotic plaques, indicating that the nanoformulation constructed by the present invention can effectively treat atherosclerosis.

[0122] Experimental Example 7: In vivo safety evaluation of HA-M@LP@ST

[0123] The treatment regimen of Example 6 was used to treat ApoE - / - After the treatment of mice, the main organs (heart, liver, spleen, lung, and kidney) of mice with different treatment components were collected for H&E staining to investigate the biological safety of different treatment groups.

[0124] The results are as follows Fig.10 As shown, no obvious organic lesions were found in the organs of mice in different treatment groups, indicating that the nanoformulation constructed by the present invention has good biosafety.

[0125] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0126] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A targeted nano drug delivery system, characterized in that: The targeted nano drug delivery system has a core-shell structure, with liposomes loaded with hydrophobic drugs as core materials and hybrid cell membrane-hyaluronic acid as shell materials; the hydrophobic drugs are astragaloside IV and / or atorvastatin; the targeted nano drug delivery system is prepared by the following steps: After mixing the mixed solution of lecithin and cholesterol with the hydrophobic drug, removing the solvent, adding water for ultrasonication, centrifuging and collecting the precipitate to obtain the drug-loaded liposome; The erythrocyte membrane and the macrophage membrane are mixed to obtain a hybrid composite membrane; The hybrid composite membrane is mixed with the drug-loaded liposome, so that the hybrid composite membrane is attached to the surface of the drug-loaded liposome to obtain a composite membrane-liposome; The hyaluronic acid is mixed with the composite membrane-liposome, and the hyaluronic acid is inserted into the hybrid composite membrane to obtain a targeted nano drug delivery system.

2. The targeted nano drug delivery system according to claim 1, characterized in that: The mixing mass ratio of the erythrocyte membrane to the macrophage membrane is 1:1-3.

3. The targeted nano drug delivery system according to claim 1, characterized in that: The mass ratio of the lecithin, cholesterol, astragaloside IV and atorvastatin is 4-6:1:1:

1.

4. The targeted nano drug delivery system according to claim 1, characterized in that: The mass ratio of the lecithin to the hybrid composite membrane is 18-22:1; the mass ratio of the lecithin to the hyaluronic acid is 8-12:

1.

5. The targeted nano drug delivery system according to claim 1, characterized in that: The reaction conditions for mixing the red blood cell membrane and the macrophage membrane are 36-37° C. for 55-65 minutes.

6. The targeted nano drug delivery system according to claim 1, characterized in that: The reaction conditions for mixing the hybrid composite membrane and the drug-loaded liposomes are 36-37° C. for 55-65 minutes.

7. The targeted nano drug delivery system according to claim 1, characterized in that: The reaction conditions for mixing the hyaluronic acid and the composite membrane-liposome are 36-37° C. for 25-35 minutes.

8. Use of the targeted nano drug delivery system according to claim 1 in the preparation of drugs for preventing / treating atherosclerosis.

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