A preparation method and application of biofilm-encapsulated drug nanocrystals

Abstract

The present invention belongs to the field of pharmacy, and relates to a method for preparing biofilm-coated drug nanocrystals, and the application of the method in constructing a nano drug delivery system with biofilm-coated nanocrystals. The present invention uses drug nanocrystals as a rigid support skeleton, and directly fills them into biofilm capsules to prepare biofilm-encapsulated biomimetic nano drug preparations with the characteristics of high drug loading, good biocompatibility, long systemic circulation time, and good drug sustained release effect. The results of in vivo and in vitro studies show that the drug can be successfully encapsulated in the biofilm nano capsule after being prepared into nanocrystals, and the biomimetic nano drug preparation retains the advantages of drug nanocrystals and biofilms respectively; it can significantly improve the safety of drug use, prolong the drug circulation time in the blood, and improve the drug treatment and diagnostic effects. The present invention has good clinical application prospects and development value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and relates to a method for preparing biofilm-coated drug nanocrystals, and the application of the method in constructing a nano drug delivery system using biofilm-coated nanocrystals, and in particular to a nano drug delivery system in which the drug nanocrystals are directly used as a rigid support skeleton and coated in a biofilm to prepare a biofilm-coated drug nanocrystal ... Background Art

[0002] The prior art discloses that the biofilm-coated nano drug delivery system is a drug delivery system formed by coating a layer of biofilm on a rigidly supported nanoparticle, and the supporting nanoparticles are usually formed of organic polymer materials or inorganic materials. Compared with traditional drug delivery systems such as liposomes, polymer micelles and nanoparticles, the biofilm-coated nano drug delivery system has the following advantages: 1) good biocompatibility and high safety; 2) long systemic circulation time; 3) compared with traditional long-circulation nano preparations, there is no PEGylation modification and low immunogenicity. In addition, through homologous tropism or modification of surface target molecules, the biofilm-coated nano drug delivery system can also have a targeting function.

[0003] The drug loading of biofilm-coated nano drug delivery system can be achieved by adsorbing the drug on the biofilm or encapsulating it in the support frame. The drug loading method adsorbed on the membrane is generally used for substances that have specific adsorption to the biofilm, such as using the natural adsorption of bacterial toxins on the red blood cell membrane to make the red blood cell membrane-coated nanoparticles adsorb toxins for immune prevention and treatment, but this method of drug loading is not suitable for most drugs. The drug loading method of encapsulating the drug on the support frame is to first encapsulate the drug in nanoparticles formed by organic polymer carriers (commonly PLGA) or inorganic carriers (mesoporous materials), and then encapsulate it in the biofilm by extrusion or ultrasound to form a drug-loaded biofilm-coated nano drug delivery system; however, drug loading with PLGA nanoparticles often leads to low drug loading due to poor compatibility between drug molecules and carriers; although drug loading with inorganic mesoporous materials has a higher drug loading capacity, it is limited in practical applications because the inorganic carriers are non-degradable and have poor biocompatibility.

[0004] Drug nanocrystals refer to drug crystals with nanoscale and rigidity formed during the crystallization process of drug molecules. They serve as a drug intermediate to improve drug solubility and enhance drug bioavailability. In addition, the drug exists in the form of nanocrystals, giving it a sustained-release function. Studies have reported the use of drug nanocrystals in the development of oral preparations.

[0005] Based on the current state of the art, the inventors of this application intend to provide a method for preparing biofilm-coated drug nanocrystals and the application of this method in constructing a nano drug delivery system using biofilm-coated nanocrystals. This method has great potential application value. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing biofilm-coated drug nanocrystals and the application of the method in constructing a nano drug delivery system using biofilm-coated nanocrystals, in view of the shortcomings of the prior art. The method has great potential application value.

[0007] The present invention uses rigid drug nanocrystals as a supporting skeleton to prepare a biofilm-coated nano drug delivery system, wherein the drug nanocrystals replace PLGA or inorganic mesoporous materials commonly used in the prior art to prepare the biofilm-coated nano drug delivery system, thereby providing the supporting structure required for the biofilm-coated nano drug delivery system and realizing drug loading at the same time. The nano drug delivery system of biofilm-coated drug nanocrystals constructed by the present invention has many advantages: 1) a large drug loading amount, which can meet the dosage requirements of clinical medication; 2) avoiding the use of carrier materials, thereby improving the biocompatibility and safety of the drug delivery system; 3) reducing the specific surface energy of the drug nanocrystals and increasing the stability of the drug nanocrystals; 4) combining the sustained-release performance of the drug nanocrystals with the advantages of the long in vivo circulation of the biofilm-coated nano drug delivery system.

[0008] Specifically, in the present invention, a nano drug delivery system of biomembrane-coated drug nanocrystals is prepared by constructing the drug into nanocrystals and then directly coating the biomembrane as a rigid support skeleton; the drug nanocrystals can be prepared by physical or chemical treatment, and the obtained nanocrystal particle size is controlled at 10nm to 1000nm, preferably 10nm to 200nm.

[0009] In the present invention, the drug nanocrystals are composed of therapeutic drugs or diagnostic drugs; the therapeutic drugs are anti-tumor drugs (such as taxane drugs such as paclitaxel, anthracycline drugs such as doxorubicin, camptothecin drugs, vincristine drugs, bortezomib and other zolmi drugs, cisplatin and other platinum drugs, irinotecan, parthenolide drugs, etc.), anti-infective drugs (such as ceftriaxone, cefoxitin, aztreonam, streptomycin, amphotericin B, vancomycin, tigecycline, teicoplanin, morpholino, adenosine, acyclovir, etc.), anti- Drugs for cardiovascular and cerebrovascular diseases (such as gangliosides, ferulic acid, chuanxiongzine, troxerutin, sodium ozagrel, etc.), drugs for lymphatic system diseases (such as panobinostat, etc.), drugs for immune system diseases (such as methylprednisolone, cyclosporine, etc.), analgesics (such as morphine, methadone, etc.); the diagnostic drugs are fluorescent substances (such as Fluorescein, Rhodamine, etc.) that can be prepared into nanocrystals by physical or chemical methods, near-infrared dyes (such as cy5.5, IR820, DiR, etc.), magnetic resonance imaging agents (such as Gd agents, iron oxide, etc.). The selected biological membrane is a membrane structure with a lipid bilayer, which can be a natural cell membrane, such as an erythrocyte membrane, a platelet membrane, a macrophage membrane, a leukocyte membrane, and a tumor cell membrane, etc., or an artificial biological membrane, such as a liposome membrane, etc.

[0010] In the embodiments of the present invention, a nano drug delivery system of irinotecan nanocrystals coated with erythrocyte membranes and platelet membranes, a nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membranes, and a nano drug delivery system of docetaxel nanocrystals coated with RGD-modified erythrocyte membranes were constructed using the provided method; the constructed nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membranes and RGD-modified erythrocyte membranes can further implement tumor-targeted drug delivery in vivo for anti-tumor treatment.

[0011] Furthermore, the preparation method of the biofilm-coated drug nanocrystals of the present invention is used to construct a nano drug delivery system of biofilm-coated nanocrystals. The nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane constructed in the embodiments of the present invention is used as the basis for tumor treatment, and experimental detection and evaluation are carried out.

[0012] 1) Preparation and characterization of nano drug delivery system with irinotecan nanocrystals coated with red blood cell membrane and platelet membrane

[0013] The target cells (red blood cells or platelets) are taken from a living animal, and the cell membranes are extracted after the cells are broken to obtain a cell membrane suspension; an appropriate amount of surfactant is added to irinotecan, and a drug nanocrystal suspension is obtained after film formation and hydration; the cell membrane suspension and the drug nanocrystal suspension are mixed by ultrasound to obtain a nano drug delivery system of irinotecan nanocrystals coated with a biomembrane, and its morphology is characterized by transmission electron microscopy;

[0014] 2) Preparation and characterization of the nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0015] The nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane was prepared and characterized by the same method as above;

[0016] The modification of RGD is carried out by first modifying streptavidin onto the surface of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membranes by lipid insertion method, and then incubating with biotinylated RGD to obtain the nano drug delivery system of docetaxel nanocrystals coated with RGD-modified erythrocyte membranes, and measuring the in vitro release of the drug by the dialysis bag method;

[0017] 3) In vivo pharmacokinetic study of the nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0018] Commercially available docetaxel injection, docetaxel nanocrystals, nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membranes, and nano-drug delivery system of docetaxel nanocrystals coated with RGD-modified erythrocyte membranes were administered to ICR mice via tail vein. Blood was collected at a certain time, and the drug concentration in the blood was determined by HPCL method, and the pharmacokinetic curve was drawn.

[0019] 4) Investigation of in vivo tissue distribution of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane nano-drug delivery system

[0020] Commercially available docetaxel injection, erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system, and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system were administered via the tail vein of nude mice bearing U87 subcutaneous transplanted tumor models. Tissues, organs, and whole blood were collected at certain time points, and drug concentrations in tissues and blood were determined by HPLC.

[0021] 5) In vitro pharmacodynamic evaluation of the nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0022] The MTT method was used to investigate the in vitro growth inhibitory effects of commercially available docetaxel injection, docetaxel nanocrystals, erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system, and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system on brain glioma U87 cells.

[0023] 6) In vivo pharmacodynamic evaluation of the nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0024] Normal saline, commercially available docetaxel injection, docetaxel nanocrystals, erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system, and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system were injected into the tail vein of nude mice bearing U87 subcutaneous transplanted tumor models. The anti-tumor effect in vivo was evaluated using tumor volume and survival time as indicators.

[0025] 7) Safety evaluation of the nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0026] Normal nude mice were injected with normal saline, commercially available docetaxel injection, erythrocyte membrane-coated docetaxel nanocrystals nano-drug delivery system, and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nano-drug delivery system through the tail vein. The in vivo safety was evaluated using the white blood cell level and mouse creatinine clearance as indicators.

[0027] The experimental results show that the constructed nano-drug delivery system of biofilm-coated drug nanocrystals has a clear core-membrane structure as shown by transmission electron microscopy, and the stability of the drug nanocrystals is significantly improved, indicating that the drug nanocrystals are successfully coated by the biofilm; in vitro release shows that the nano-drug delivery system of biofilm-coated drug nanocrystals has obvious drug sustained-release performance; in vivo pharmacokinetics show that the nano-drug delivery system of biofilm-coated drug nanocrystals has the advantage of long circulation, significantly prolonging the circulation time of the drug in the body; the nano-drug delivery system of biofilm-coated and targeted molecule-modified biofilm-coated drug nanocrystals can be enriched in tumor tissues through passive or active targeting, and has a stronger inhibitory effect on tumor growth.

[0028] The nano drug delivery system of the biofilm-coated drug nanocrystals of the present invention is safer than commercially available drug preparations and drug nanocrystals. The preparation method of the biofilm-coated drug nanocrystals provided by the present invention can be used to prepare the nano drug delivery system of the biofilm-coated drug nanocrystals, and has greater advantages than the currently existing biofilm-coated nano drug delivery system and its drug loading method, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Electron microscopic photos of the nano drug delivery system with irinotecan nanocrystals coated with red blood cell membrane and platelet membrane.

[0030] As can be seen from the figure, the irinotecan nanocrystals (A) are rod-shaped; the nano-drug delivery system in which the irinotecan nanocrystals are coated with red blood cell membranes (B) and platelet membranes (C) is spherical, with a particle size of about 40 nm.

[0031] Figure 2 , electron microscope photos of the nano drug delivery system of docetaxel nanocrystals coated with red blood cell membrane,

[0032] As can be seen from the figure, docetaxel nanocrystals (A) are spherical with a particle size of about 30nm; the nano drug delivery system of docetaxel nanocrystals coated with red blood cell membranes (B) is spherical with a distinct core-membrane structure and a particle size of about 70nm.

[0033] Figure 3 In vitro release curves of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane.

[0034] The figure shows the release of docetaxel nanocrystals (DTX NCs), red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RBC / DTX NCs) and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RGD-RBC / DTX NCs) in PBS (pH 7.4) solution. The results show that docetaxel has a good sustained-release effect after being coated with red blood cell membrane, and the modification of targeting molecules has no effect on the release.

[0035] Figure 4 , erythrocyte membrane and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nano-drug delivery system in vivo pharmacokinetic curves,

[0036] As can be seen from the figure, the nano drug delivery system of red blood cell membrane-coated docetaxel nanocrystals (RBC / DTX NCs), whether modified with RGD or not, has a longer circulation time in mice compared with docetaxel nanocrystals (DTX NCs) and commercially available docetaxel injection (DTX), and the targeted molecule modification has no obvious effect on its pharmacokinetic behavior in mice.

[0037] Figure 5 , erythrocyte membrane and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nano-drug delivery system in vivo tissue distribution bar graph,

[0038] As can be seen from the figure, the nano drug delivery system of red blood cell membrane-coated docetaxel nanocrystals (RBC / DTX NCs), regardless of whether it is modified with RGD or not, has reduced distribution in the mouse liver and increased distribution in the blood and tumors compared with the commercially available docetaxel injection (DTX), and targeted molecular modification can significantly increase the accumulation of the drug at the tumor site.

[0039] Figure 6 The in vitro anti-U87 cell activity curve of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane,

[0040] As can be seen from the figure, after U87 cells were incubated with commercially available docetaxel injection (DTX), docetaxel nanocrystals (DTXNCs), red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RBC / DTX NCs), and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RGD-RBC / DTX NCs) for 48 hours, the IC 50 They were 41.6, 49.7, 233.8 and 13.8 nM, respectively. The results showed that RBC / DTX NCs with or without RGD modification could inhibit the in vitro growth of U87 cells, and the in vitro antitumor activity of RGD-modified RBC / DTX NCs was much better than that of the non-target group.

[0041] Figure 7 Evaluation of the nano-drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane against U87 subcutaneous tumor

[0042] Figure A is a curve showing the change of tumor volume over time in nude mice with subcutaneous transplanted tumors of U87; Figure B is a survival curve of nude mice with subcutaneous transplanted tumors of U87; Figure C is a comparison of tumor inhibition rates of each group on the 28th day after administration; Figure D is a comparison of tumor inhibition rates of each group at the median survival time after administration. The results showed that docetaxel nanocrystals (DTX NCs) rapidly caused death of model nude mice after administration, while red blood cell membrane coating (RBC / DTX NCs) overcame its toxicity, and compared with normal saline (PBS) (median survival time 27.5 days) and commercially available docetaxel injection (DTX) (median survival time 38 days), the nano drug delivery system of docetaxel nanocrystals coated with red blood cell membranes (RBC / DTX NCs) significantly prolonged the survival time of model nude mice (median survival time 42 days), and the nano drug delivery system of docetaxel nanocrystals coated with RGD-red blood cell membranes (RGD-RBC / DTX NCs) significantly prolonged the survival time of model nude mice (median survival time 42 days). The survival time of the RBC / DTX NCs group was most significantly prolonged (median survival time 47 days). On the 28th day after administration, the tumor inhibition rates of each group were: 89.18±6.75% in the RBC / DTX NCs group and 97.28±2.46% in the RGD-RBC / DTX NCs group, both significantly higher than the tumor inhibition rate of the DTX group (53.28±19.79%). After the median survival time was reached after administration, the tumor inhibition rates of each group were: 33.76%±6.37% in the RBC / DTX NCs group and 77.24%±6.58% in the RGD-RBC / DTX NCs group, both significantly higher than the tumor inhibition rate of the DTX group (4.93%±2.52%). The results show that RGD-RBC / DTX NCs has the best in vivo tumor inhibition efficacy.

[0043] Figure 8 In vivo safety evaluation of nano-drug delivery system containing docetaxel nanocrystals encapsulated by erythrocyte membrane and RGD-modified erythrocyte membrane

[0044] Figure A shows the changes in the number of white blood cells in the whole blood of normal nude mice within 12 days after administration. The results show that the commercially available docetaxel injection (DTX) significantly reduces the level of white blood cells in mice, reaching the lowest value 5 days after administration and returning to normal levels 10 days after administration. The nano-drug delivery system of docetaxel nanocrystals coated with red blood cell membranes (RBC / DTX NCs) and the nano-drug delivery system of docetaxel nanocrystals coated with RGD-modified red blood cell membranes (RGD-RBC / DTX NCs) can significantly slow down the decline in their white blood cell count; Figure B shows the creatinine clearance rate of normal nude mice after 12 days of administration. The results show that DTX significantly reduces the creatinine clearance rate of mice and has nephrotoxicity. There is no significant difference in the creatinine clearance rate of mice in the RBC / DTX NCs, RGD-RBC / DTX NCs and normal saline groups. DETAILED DESCRIPTION

[0045] The following embodiments will help to further understand the present invention, but the present invention is not limited to the scope of the following description.

[0046] Example 1 Preparation and characterization of nano drug delivery system of irinotecan nanocrystals coated with red blood cell membrane and platelet membrane

[0047] 1) Preparation and characterization of irinotecan nanocrystals coated with erythrocyte membranes

[0048] Whole blood of male ICR mice was collected and centrifuged at 1000 g / min for 5 minutes at 4°C. The upper serum and white blood cell layer were discarded. The lower red blood cells were washed with 1×PBS and resuspended in 0.25×PBS at 4°C for 30 minutes. Hemoglobin was removed by centrifugation at 15000 g / min for 7 minutes at 4°C. The obtained light red red red blood cell membrane was resuspended and stored in double distilled water. The membrane protein concentration was detected by BCA kit. 4 mg of irinotecan and an appropriate amount of surfactant F127 were weighed in a 25 mL eggplant-shaped bottle, and an appropriate amount of methanol was added to dissolve the membrane and hydrate it to prepare irinotecan nanocrystals with good dispersion. The red blood cell membrane suspension was sonicated at 100 W for 3 minutes to obtain red blood cell membrane vesicles. Then, the 4 mg / mL irinotecan nanocrystal solution was mixed with red blood cell membrane vesicles (2:1, w / w) and sonicated to obtain a nano drug delivery system of red blood cell membrane-coated irinotecan nanocrystals. The morphology was observed by uranyl acetate negative staining electron microscopy. The results are as follows: Figure 1 As shown;

[0049] 2) Preparation and characterization of platelet membrane-coated irinotecan nanocrystals nano-drug delivery system

[0050] Whole blood of male ICR mice was collected and centrifuged at 300 g / min and 4°C for 5 minutes. The supernatant was collected and centrifuged at 2000 g / min for 5 minutes. The supernatant was discarded. The lower layer of platelets was washed with double distilled water and then repeatedly frozen and thawed 3 times. The platelet membrane was centrifuged at 20000 g / min for 7 minutes. The obtained white platelet membrane was resuspended and stored in double distilled water, and its membrane protein concentration was detected by BCA kit. The platelet membrane suspension and irinotecan nanocrystal solution were subjected to the same operation as above to prepare a nano drug delivery system of platelet membrane-coated irinotecan nanocrystals. Figure 1 The morphology observed by electron microscopy with uranyl acetate negative staining is shown.

[0051] Example 2 Preparation and characterization of nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0052] 1) Preparation and characterization of the nano-drug delivery system of red blood cell membrane-coated docetaxel nanocrystals

[0053] The preparation and characterization methods were the same as those of the nano drug delivery system of irinotecan nanocrystals coated with red blood cell membranes. Figure 2 As shown;

[0054] 2) Preparation of nano drug delivery system of docetaxel nanocrystals coated with RGD-modified erythrocyte membrane

[0055] The basic preparation process is the same as that of the nano drug delivery system of docetaxel nanocrystals coated with red blood cell membranes. The RGD modification method is as follows: 40 μL of streptavidin-PEG 3400 -DSPE PBS solution (5 mg / mL) was incubated with erythrocyte membrane vesicles obtained from 100 μL whole blood in a 37°C water bath for 30 minutes to prepare streptavidin-erythrocyte membrane vesicles. The obtained streptavidin-erythrocyte membrane vesicles were mixed with docetaxel nanocrystals and then sonicated to obtain a nano drug delivery system of erythrocyte membrane-coated nanocrystals with surface modified streptavidin, and then 100 μL biotin-PEG was added. 3500 -RGD PBS solution (0.1 mg / mL) was incubated in a 37°C water bath for 10 minutes to prepare the RGD-modified erythrocyte membrane-coated docetaxel nanocrystal nano-drug delivery system.

[0056] Example 3 In vitro release test of docetaxel nanocrystal drug delivery system coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0057] The in vitro release was determined by the dialysis bag method. 0.3 mL of docetaxel nanocrystals (DTX NCs), erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system (RBC / DTX NCs) and RGD-modified erythrocyte membrane-coated docetaxel nanocrystals nanodrug delivery system (RGD-RBC / DTX NCs) were placed in dialysis bags (molecular weight cutoff 7 kDa), sealed, placed in 6 mL of pH 7.4 PBS solution (containing 1% sodium dodecyl sulfate), shaken at 37°C, and 0.2 mL of release medium was taken at 15 and 30 min, 1, 1.5, 2, 4, 8, 24, 48 and 72 h, respectively, and the same volume of fresh medium was added. The taken solution was appropriately diluted, and the docetaxel concentration was determined by HPLC. The release curve was plotted. The results are shown in Figure 2. Figure 3 shown.

[0058] Example 4 In vivo pharmacokinetic study of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0059] ICR mice were injected with 150 μL of commercially available docetaxel injection (DTX), docetaxel nanocrystals (DTX NCs), red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RBC / DTX NCs) and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RGD-RBC / DTX NCs) through the tail vein. 50 μL of whole blood was collected at 5, 15 and 30 min, 1, 2, 4, 8, 24, 48 and 72 h, respectively, and extracted twice with ether / tetrahydrofuran (1:4) solution, evaporated to dryness, and then re-dissolved in acetonitrile and analyzed by HPLC. The results are shown in Figure 2. Figure 4 , and as shown in Table 1.

[0060] Table 1 In vivo pharmacokinetic parameters of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane,

[0061]

[0062] Example 5 In vivo tissue distribution test of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0063] U87 subcutaneous tumor animal model was established, and 150 μL of commercially available docetaxel injection (DTX), red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RBC / DTX NCs), and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RGD-RBC / DTX NCs) were injected into the tail vein. Tumor, liver, kidney, and whole blood were collected at 2 h and 24 h, respectively. After tissue homogenization, the samples were extracted twice with ether / tetrahydrofuran (1:4) solution, evaporated to dryness, and reconstituted in acetonitrile for HPLC analysis. The results are shown in Figure 2. Figure 5shown.

[0064] Example 6 In vitro pharmacodynamics test of the nano drug delivery system of erythrocyte membrane and RGD-modified erythrocyte membrane coated with docetaxel nanocrystals

[0065] The MTT method was used to determine the in vitro growth inhibitory effects of commercially available docetaxel injection (DTX), docetaxel nanocrystals (DTX NCs), red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RBC / DTX NCs) and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nanodrug delivery system (RGD-RBC / DTX NCs) on U87 tumor cells; U87 cells in the logarithmic growth phase were taken, digested with 0.25% trypsin and blown into single cells, the cells were suspended in DMEM culture medium containing 10% FBS, and seeded in a 96-well cell culture plate at a density of 3000 cells per well, with a volume of 0.2 mL per well, three wells with culture medium without cells were set aside as blank wells, and the plates were cultured in a carbon dioxide incubator for 24 hours, and each group of drugs was diluted sixfold in sequence with cell culture medium. The cell culture medium in the 96-well plate was aspirated, and 200 μL of a series of drug solutions were added to each well. Three replicate wells were set up for each concentration, and three wells with only culture medium added were reserved as control wells. After 48 hours of culture, 20 μL of MTT reagent (5 mg / mL) was added to the experimental wells, control wells and blank wells and incubated for 4 hours. The culture medium in the wells was discarded, and 150 μL of dimethyl sulfoxide was added to each well. After shaking to fully dissolve the generated blue-purple crystals, the absorbance (A) of each well at 490 nm was measured with an enzyme-labeled instrument, and the cell survival rate was calculated according to the following formula:

[0066] Survival rate = (A 490实验孔 -A 490空白孔 ) / (A 490对照孔 -A 490空白孔 )×100%

[0067] GraphPad Prism software was used to plot the survival rate against the logarithmic value of drug concentration (e.g. Figure 6 The inhibitory concentration (IC 50 ).

[0068] Example 7 In vivo pharmacodynamic evaluation of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0069] Construct a U87 subcutaneous tumor animal model and regularly observe the tumor size. When the tumor size reaches 150 mm 3The mice were divided into groups for the experiment and injected with PBS (pH 7.4), commercial docetaxel injection (DTX), docetaxel nanocrystals (DTXNCs), red blood cell membrane-coated docetaxel nanocrystals nano-drug delivery system (RBC / DTX NCs) and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nano-drug delivery system (RGD-RBC / DTX NCs) through the tail vein. The total dose of docetaxel was 25 mg / kg, and the survival time of nude mice was recorded (eg Figure 7 The long diameter (a) and short diameter (b) of the tumor were measured every other day, and the tumor volume of each group of nude mice was calculated according to the formula, and the change curve of tumor volume over time was drawn (as shown in Figure 7 B) and calculate the tumor inhibition rate (as shown in Figure 7 C), tumor volume calculation formula:

[0070] V 瘤体积 =0.5(a×b 2 )

[0071] Tumor inhibition rate calculation formula:

[0072] Tumor inhibition rate (%) = (1-V 实验组瘤体积 / V 对照组瘤体积 )×100

[0073] Example 8 Safety evaluation of the nano drug delivery system of docetaxel nanocrystals coated with erythrocyte membrane and RGD-modified erythrocyte membrane

[0074] Normal nude mice were injected with normal saline, commercial docetaxel injection (DTX), red blood cell membrane-coated docetaxel nanocrystals nano-drug delivery system (RBC / DTX NCs) and RGD-modified red blood cell membrane-coated docetaxel nanocrystals nano-drug delivery system (RGD-RBC / DTX NCs) through the tail vein. The total dose of docetaxel was 25 mg / kg, and the whole blood was collected on the 1st, 3rd, 5th, 7th, 9th and 11th days after administration to determine the white blood cell count (such as Figure 8 A), serum was collected on the 12th day after administration to determine creatinine clearance (as shown in Figure 8 B).

Claims

1. A nano drug delivery system of biomembrane-coated drug nanocrystals with targeting function, characterized in that The drug is directly filled into the biomembrane as a rigid support skeleton in the physical form of nanocrystals. The drug is docetaxel, and the biomembrane is an erythrocyte membrane. The preparation method of the nano drug delivery system is as follows: 40 μL of PBS solution (5 mg / mL) of streptavidin-PEG3400-DSPE is incubated with erythrocyte membrane vesicles obtained from 100 μL of whole blood in a 37°C water bath for 30 minutes to obtain streptavidin-erythrocyte membrane vesicles; 4 mg of docetaxel and an appropriate amount of surfactant F127 are weighed and added to In a 25 mL eggplant-shaped bottle, an appropriate amount of methanol was added to dissolve the film and hydrate it to prepare docetaxel nanocrystals; streptavidin-erythrocyte membrane vesicles were mixed with docetaxel nanocrystals and then sonicated to obtain a nano-drug delivery system of erythrocyte membrane-coated nanocrystals with surface modified streptavidin, and then 100 μL of biotin-PEG3500-RGD PBS solution (0.1 mg / mL) was added and incubated in a 37°C water bath for 10 minutes to obtain a nano-drug delivery system of RGD-modified erythrocyte membrane-coated docetaxel nanocrystals.

2. The nano drug delivery system of biofilm-coated drug nanocrystals with targeting function as claimed in claim 1, characterized in that Drug nanocrystals as skeleton materials and drug reservoirs.

3. The nano drug delivery system of biofilm-coated drug nanocrystals with targeting function as claimed in claim 1, characterized in that Drug nanocrystals are nanoscale crystals formed by treating drugs through physical or chemical methods.

4. The nano drug delivery system of biofilm-coated drug nanocrystals with targeting function as claimed in claim 1, characterized in that The particle size of drug nanocrystals is 10nm to 1000nm.

5. The nano drug delivery system of biofilm-coated drug nanocrystals with targeting function as claimed in claim 4, characterized in that The particle size of drug nanocrystals is 10nm to 200nm.

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