Method for preparing biofilm-coated drug nanocrystal and application thereof

Abstract

A nano drug delivery system includes a biofilm-coated drug nanocrystal. A drug in a physical form of nanocrystal is directly used as a rigid supporting skeleton, and is filled in a biofilm. The nano drug delivery system has the advantages of high drug loading capacity, good biocompatibility, long systemic circulation time and drug sustained release.

Claims

1. A nano drug delivery system, comprising a biofilm-coated drug nanocrystal, wherein the drug is in the physical form of a nanocrystal, and the drug nanocrystal is coated with a biofilm, wherein, a surface of the nano drug delivery system is further modified by a targeting molecule to construct the nano drug delivery system as a nano drug delivery system of a biofilm-coated nanocrystal with an active targeting capability; the targeting molecule is a polypeptide targeting molecule; and the polypeptide targeting molecule is one or more selected the group consisting of RGD, VAP, WVAP, A7R, and CDX; wherein, the drug is at least one selected from the group consisting of a therapeutic drug and a diagnostic agent; wherein, the therapeutic drug is one or more selected from the group consisting of: an anti-tumor drug, an anti-infective drug, an anti-cardiovascular disease drug, an anti-lymphatic system disorder drug, an anti-immune system disorder drug, and an analgesic drug, and the therapeutic drug is in the physical form of the nanocrystal; wherein, the anti-tumor drug is one selected from the group consisting of: a taxane drug, an anthracycline drug, a camptothecin drug, a vincristine drug, and a zomib drug, irinotecan and a parthenolide drug; the anti-infective drug is one selected from the group consisting of ceftriaxone, cefoxitin, aztreonam, streptomycin, amphotericin B, vancomycin, tigecycline, teicoplanin, moroxydine, vidarabine and acyclovir; the anti-cardiovascular disease drug is one selected from the group consisting of ganglioside, ferulic acid, ligustrazine, troxerutin, and sodium ozagrel the anti-lymphatic system disorder drug is panobinostat; the anti-immune system disorder drug is one selected from the group consisting of methylprednisolone and cyclosporine; and the analgesic drug is one selected from the group consisting of morphine and methadone; wherein, the diagnostic agent is a fluorescent substance and is in the physical form of the nanocrystal; and wherein, the biofilm has a membrane structure with a lipid bilayer, and the biofilm is a natural cell membrane; wherein the natural cell membrane is one or more selected from the group consisting off an erythrocyte membrane, a platelet membrane, a macrophage membrane, a leukocyte membrane, and a tumor cell membrane.

2. The nano drug delivery system of claim 1, wherein, the drug nanocrystal has a particle size of 10-1000 nm.

3. A method comprising administering the nano drug delivery system of claim 1 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-IC show electron micrographs of a nano drug delivery system of an erythrocyte membrane-coated irinotecan nanocrystal and a nano drug delivery system of a platelet membrane-coated irinotecan nanocrystal:

(2) as can be seen from the figure, a irinotecan nanocrystal in FIG. 1A is rod-shaped; the nano drug delivery system of the erythrocyte membrane-coated irinotecan nanocrystal in FIG. 1B and the nano drug delivery system of the platelet membrane-coated irinotecan nanocrystal in FIG. 1C are spherical with a particle size of about 40 nm.

(3) FIGS. 2A-2B show electron micrographs of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal:

(4) as can be seen from the figure, a docetaxel nanocrystal in FIG. 2A is spherical with a particle size of about 30 nm; the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal in FIG. 2B is spherical with a distinct nuclear-membrane structure and a particle size of about 70 nm.

(5) FIG. 3 shows in vitro release curves of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal:

(6) the figure shows releases of a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) in PBS solution with a pH of 7.4. The results show that docetaxel have a good sustained release capability after being coated with erythrocyte membrane, and the modification of the targeting molecule has no effect on the release.

(7) FIG. 4 shows in vivo pharmacokinetic curves of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal:

(8) as can be seen from the figure and table, whether being modified with RGD or not, the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs) has a longer circulation time in mice, and the modification of the targeting molecule has no significant effect on the pharmacokinetic behavior in the mice, compared with a docetaxel nanocrystal (DTX NCs) and a commercially available docetaxel injection (DTX).

(9) FIG. 5 is a bar graph showing tissue distributions of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal in U87 subcutaneous xenograft model mice:

(10) as can be seen from the figure, whether being modified with RGD or not, the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs) has a reduced distribution in livers of mice, and an increased distribution in bloods and tumors, and targeting molecular modification can significantly increase an accumulation of drugs at a tumor site, compared with a commercially available docetaxel injection (DTX).

(11) FIG. 6 is a bar graph showing tissue distributions of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal in U87 intracerebral orthotopic tumor model nude mice:

(12) as can be seen from the figure, whether being modified with RGD or not, the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs) has a reduced distribution in livers of mice, and an increased distribution in bloods, and targeting molecular modification can carry the nano drug delivery system across a blood-brain tumor barrier and significantly increase an accumulation of drugs at a tumor site, compared with a commercially available docetaxel injection (DTX).

(13) FIG. 7 shows in vitro anti-U87 cell activity curves of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal:

(14) as can be seen from the figure, U87 cells are incubated with a commercially available docetaxel injection (DTX), a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC-DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) for 48 h, respectively, and their IC.sub.50 are 41.6 nM, 49.7 nM, 233.8 nM, and 13.8 nM, respectively. The results show that, whether being modified with RGD or not, RBC/DTX NCs can inhibit the growth of the U87 cells in vitro.

(15) An in vitro anti-tumor activity of the RGD modified RBC/DTX NCs is much better than that of the group without target.

(16) FIGS. 8A-8D show anti-U87 subcutaneous tumor evaluations of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal:

(17) FIG. 8A shows changing curves of tumor volumes of U87 subcutaneous transplanted nude mice over time; FIG. 8B shows survival curves of U87 subcutaneous transplanted nude mice over time; FIG. 8C is a comparison of tumor inhibition rates of various groups on 28.sup.th day after administration; and FIG. 8D is a comparison of tumor inhibition rates of various groups at a median survival time after administration. The results show that administrating with a docetaxel nanocrystal (DTX NCs) immediately causes model nude mice to die. While the docetaxel nanocrystals after being coated with erythrocyte membrane (RBC/DTX NCs) losses its toxicity, moreover, compared with normal saline (phosphate buffer saline (PBS)) (a median survival time is 27.5 days) and a commercially available docetaxel injection (DTX) (a median survival time is 38 days), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC,DTX NCs) significantly prolongs a survival time of model nude mice (a median survival time is 42 days). The nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) prolongs the survival time of the model nude mice most significantly (a median survival time is 47 days). Tumor inhibition rates of the various groups on the 28.sup.th day after administration are respectively shown as follows: the tumor inhibition rate of the RBC/DTX NCs group is 89.18±6.75%, and the tumor inhibition rate of the RGD-RBC/DTX NCs group is 97.28±2.46%, which are significantly higher than that of the DTX group (53.28±19.79%). Tumor inhibition rates the various groups at the median survival time after administration are respectively shown as follows: the tumor inhibition rate of the RBC/DTX NCs group is 33.76%±6.37%, and the tumor inhibition rate of the RGD-RBC/DTX NCs group is 77.24%±6.58%, which are also significantly higher than that of the DTX group (4.93%±2.52%). The results show that the RGD-RBC/DTX NCs has the best anti-tumor effect in vivo.

(18) FIG. 9 shows anti-U87 intracerebral orthotopic tumor evaluations of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal: Survival curves of U87 intracerebral orthotopic tumor model nude mice are shown in the figure. The results show that, compared with normal saline (PBS) (a median survival time is 32 days), a commercially available docetaxel injection (DTX) (a median survival time is 32.5 days) and the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs) (a median survival time is 34.5 days), the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) significantly prolongs mouse survival time (a median survival time is 62 days).

(19) FIGS. 10A-10B show in vivo safety evaluations of a nano drug delivery system of an erythrocyte membrane-coated docetaxel nanocrystal and a nano drug delivery system of an RGD modified erythrocyte membrane-coated docetaxel nanocrystal:

(20) FIG. 10A shows a change in the number of white blood cells in whole blood within 12 days after administration in normal nude mice. The results show that a commercially available docetaxel injection (DTX) significantly reduces a level of white blood cells in mice, reaching a minimum value 5 days after administration, and returning to a normal level 10 days after the administration; and the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs) and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) can significantly slow down the decrease of the number of the white blood cells. FIG. 10B shows a creatinine clearance rate 12 days after administration to normal nude mice. The results show that the DTX significantly reduces the creatinine clearance rate in mice and has nephrotoxicity, and the creatinine clearance rates of the RBC,DTX NCs, RGD-RBC/DTX NCs and normal saline groups are not distinctly different.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(21) The following embodiments are provided to help a further understanding of the present invention, but the present invention is not limited to the scope of the following description.

Embodiment 1

Preparation and Characterization of Nano Drug Delivery System of Erythrocyte Membrane-Coated/Platelet Membrane-Coated/Tumor Cell Membrane-Coated Irinotecan/Cabazitaxel Nanocrystal

(22) 1. Preparation and Characterization of Nano Drug Delivery System of Erythrocyte Membrane-Coated Irinotecan Nanocrystal

(23) Whole blood of a male ICR mouse was taken, centrifuged at 1000 g/min for 5 min at 4° C. The serum and leukocytes at an upper layer were discarded. The erythrocytes at a lower layer were washed with 1×PBS, and then resuspended in 0.25×PBS at 4° C. for 30 min. The hemoglobin was removed by centrifugation at 15000 g/min for 7 min at 4° C. The obtained light red erythrocyte membrane was resuspended and stored in double distilled water, and the membrane protein concentration thereof was detected by a bicinchoninic acid (BCA) kit. 4 mg of irinotecan and an appropriate amount of surfactant F127 were weighed and placed in a 25 mL eggplant-shaped flask, followed by adding an appropriate amount of methanol to dissolve for film forming and hydration to obtain an irinotecan nanocrystal with a good dispersibility. The erythrocyte membrane suspension was ultrasonicated at 100 W for 3 min to obtain erythrocyte membrane vesicles. Then, 4 mg/mL of an irinotecan nanocrystal solution was mixed with the erythrocyte membrane vesicles (2:1, w/w) and then ultrasonicated to obtain the nano drug delivery system of the erythrocyte membrane-coated irinotecan nanocrystal. The morphology was observed by a uranyl acetate negative stain electron microscopy method. The results are shown in FIGS. 1A-1C.

(24) 2. Preparation and Characterization of Nano Drug Delivery System of Platelet Membrane-Coated Irinotecan Nanocrystal

(25) Whole blood of a male ICR mouse was taken, centrifuged at 300 g/min for 5 min at 4° C. to obtain a first supernatant, followed by centrifuging at 2000 g/min for 5 min, and a second supernatant was discarded. The platelets at a lower layer were washed with double distilled water, and then repeatedly frozen and thawed three times, followed by centrifuging at 20000 g/min for 7 min. The obtained white platelet membrane was resuspended and stored in double distilled water, and the membrane protein concentration thereof was detected by a BCA kit. The platelet membrane suspension was treated with an irinotecan nanocrystal solution as above to obtain the nano drug delivery system of the platelet membrane-coated irinotecan nanocrystal. The morphology was observed by a uranyl acetate negative stain electron microscopy method. The results are shown in FIGS. 1A-1C.

(26) 3. Preparation of Nano Drug Delivery System of Tumor Cell Membrane-Coated Irinotecan Nanocrystal

(27) Tumor cells (U87) were transferred to a buffer solution (obtained by dissolving 20.5 g of mannitol and 13 g of sucrose in 500 mL of Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, pH 7.5), and centrifuged at 800 g/min for 5 min. A first supernatant was discarded, and the residue was added with the above buffer, followed by adding ethylenediaminetetraacetic acid and a protease inhibitor. Ultrasonic homogenization was performed, and the homogenized solution was centrifuged for 5 min at 800 g/min to obtain a second supernatant, followed by centrifuging at 8000 g/min at −20° C. for 25 min to obtain a third supernatant. Then, the third supernatant was centrifuged at 30000 g/min at −20° C. for 35 min to obtain tumor cell membranes by discarding a fourth supernatant. The tumor cell membranes were resuspended and stored in a 0.2 mM ethylenediaminetetraacetic acid solution, and the membrane protein concentration thereof was detected by a BCA kit. The tumor cell membrane suspension was treated with an irinotecan nanocrystal solution as above to obtain the nano drug delivery of the tumor cell membrane-coated irinotecan nanocrystal.

(28) 4. Preparation of Nano Drug Delivery System of Erythrocyte Membrane-Coated/Platelet Membrane-Coated/Tumor Cell Membrane-Coated Cabazitaxel Nanocrystal

(29) The preparation method was the same as the nano drug delivery system of the erythrocyte membrane-coated/platelet membrane-coated/tumor cell membrane-coated irinotecan nanocrystal.

Embodiment 2

Preparation and Characterization of Nano Drug Delivery System of Erythrocyte Membrane-Coated/RGD, VAP, WVAP, A7R, or CDX Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(30) 1. Preparation and Characterization of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(31) The methods of the preparation and characterization were the same as the nano drug delivery system of the erythrocyte membrane-coated irinotecan nanocrystal. The results are shown in FIGS. 2A-2B.

(32) 2. Preparation of Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(33) The preparation process was basically the same as the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal. The RGD modification method was as follows. 40 μL of streptavidin-PEG.sub.3400-DSPE PBS solution (5 mg/mL) was incubated with erythrocyte membrane vesicles obtained from 100 μL of whole blood in a water bath at 37° C. for 30 min to obtain streptavidin-erythrocyte membrane vesicles. The obtained streptavidin-erythrocyte membrane vesicles were mixed with docetaxel nanocrystals and then ultrasonicated to obtain a nano drug delivery system of a streptavidin-modified erythrocyte membrane-coated nanocrystal. Then, 100 μL of biotin-PEG.sub.3500-RGD PBS solution (0.1 mg/mL) was added and incubated in the water bath at 37° C. for 10 min to obtain the nano drug delivery system of the RGD-modified erythrocyte membrane-coated docetaxel nanocrystal.

(34) 3. Preparation of Nano Drug Delivery System of VAP, WVAP, A7R, or CDX Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(35) The preparation method was the same as the nano drug delivery system of the RGD-modified erythrocyte membrane-coated docetaxel nanocrystal.

Embodiment 3

In Vitro Release Tests of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal and Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(36) The in vitro release was determined by a dialysis bag method. 0.3 mL of a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) were respectively placed in a dialysis bag (molecular weight cut-off of 7 kDa) and sealed, and then placed in 6 mL of PBS solution having a pH of 7.4 (containing 1% sodium dodecyl sulfate) and shaken at 37° C. 0.2 mL of release medium was respectively taken at 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, 24 h, 48 h and 72 h, and a same volume of fresh medium was added. The taken solution was properly diluted, the docetaxel concentration was determined by an HPLC method, and the release curve was plotted. The results are shown in FIG. 3.

Embodiment 4

In Vivo Pharmacokinetic Tests of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal and Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(37) ICR mice were administered with 150 μL of a commercially available docetaxel injection (DTX), a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC,DTX NCs) by tail vein injections, respectively. 50 μL of whole blood was respectively taken at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h and 72 h, extracted twice with a solution composed of diethyl ether and tetrahydrofuran at a volume ratio of 1:4, evaporated and dried, and redissolved with acetonitrile for HPLC analysis. The results are shown in FIG. 4 and Table.

(38) TABLE-US-00001 TABLE Parameters of nano drug delivery system of erythrocyte membrane-coated docetaxel nanocrystal and nano drug delivery system of RGD modified erythrocyte membrane-coated docetaxel nanocrystal Pharmacokinetic RGD-RBC/DTX parameter DTX DTX NCs RBC/DTX NCs NCs T.sub.1/2 β (h)  3.7 ± 1.5  2.5 ± 0.6 20.4 ± 2.2 19.7 ± 1.1 AUC.sub.0-∞ (mg/L × h) 163.2 ± 48.1 143.1 ± 47.7 3714.5 ± 132.2 3852.3 ± 139.3

Embodiment 5

In Vivo Tissue Distribution Tests of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal and Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(39) U87 subcutaneous xenograft/orthotopic tumor animal models were constructed, and were administered with 150 μL of a commercially available docetaxel injection (DTX), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) by tail vein injections, respectively. Tissue and whole blood were taken at 2 h and 24 h, respectively. The tissue was homogenized, extracted twice with a solution composed of diethyl ether and tetrahydrofuran at a volume ratio of 1:4, evaporated and dried, and redissolved with acetonitrile for HPLC analysis. The results are shown in FIGS. 5 and 6.

Embodiment 6

In Vitro Pharmacodynamic Tests of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal and Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(40) In vitro growth inhibition effects of a commercially available docetaxel injection (DTX), a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC,DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) on U87 tumor cells are determined by MTT assay. The U87 cells in logarithmic growth phase were digested with 0.25% trypsin and blown into single cells. The cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, and inoculated in a 96-well cell culture plate at a density of 3000 cells/well and a volume of 0.2 mL/well. Three wells were remained to add with the medium without the cells as blank wells. A culture was performed in a carbon dioxide incubator for 24 h. Each group of the drugs was serially diluted six times with the cell culture medium. The cell culture medium in the 96-well cell culture plate was removed, and 200 μL of each of the the serial concentrations of the drug solutions was added to each well. Three repeated wells were set for each concentration, and three wells were remained to merely add with the culture medium as control wells. After culturing for 48 h, 20 μL of MTT reagent (5 mg/mL) was added to the test wells, control wells and blank wells for incubation for 4 h, and then the culture medium was discarded. 150 μL of dimethyl sulfoxide was added to each well, and shaken to sufficiently dissolving the generated blue-violet crystals. The absorbance (A) of each well was measured by a microplate reader at 490 nm. The cell survival rate was calculated according to the following formula:
Survival rate=(A.sub.490 test well−A.sub.490 blank well)/(A.sub.490 control well−A.sub.490 blank well)×100%

(41) The survival rate over the logarithm of drug concentration were plotted by GraphPad Prism software (FIG. 7), and the half maximal inhibitory concentration (IC.sub.50) was calculated.

Embodiment 7

In Vivo Pharmacodynamic Evaluations of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal and Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(42) The U87 subcutaneous tumor animal models were constructed, and the tumor size was observed regularly. When the tumor size reached 150 mm.sup.3, the test was performed in groups. The groups were administered with PBS (pH 7.4), a commercially available docetaxel injection (DTX), a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC,DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) by tail vein injections, respectively. The docetaxel was administered at a total dose of 25 mg/kg in a single dose. The survival time of the nude mice was recorded (FIG. 8A), and the large diameter (a) and small diameter (b) of the tumor were measured every other day. The tumor volume of each group of nude mice was calculated according to a formula, and the curve of tumor volume over time was plotted (FIG. 8B) and the tumor inhibition rate was calculated (FIGS. 8C and 8D). The tumor volume was calculated according to the following formula:
V.sub.tumor volume=0.5(a×b.sup.2)

(43) The tumor inhibition rate was calculated to the following formula:
Tumor inhibition rate (%)=(1−V.sub.test group tumor volume/V.sub.control group tumor volume)×100

(44) The U87 orthotopic tumor animal models were constructed, and were administered with PBS (pH 7.4), a commercially available docetaxel injection (DTX), a docetaxel nanocrystal (DTX NCs), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC/DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) by tail vein injections 10 days after tumor implantation, respectively. The docetaxel was administered at a total dose of 25 mg/kg in a single dose. The survival time of the nude mice was recorded (FIG. 9).

Embodiment 8

Safety Evaluations of Nano Drug Delivery System of Erythrocyte Membrane-Coated Docetaxel Nanocrystal and Nano Drug Delivery System of RGD Modified Erythrocyte Membrane-Coated Docetaxel Nanocrystal

(45) Normal nude mice were administered with normal saline, a commercially available docetaxel injection (DTX), the nano drug delivery system of the erythrocyte membrane-coated docetaxel nanocrystal (RBC,DTX NCs), and the nano drug delivery system of the RGD modified erythrocyte membrane-coated docetaxel nanocrystal (RGD-RBC/DTX NCs) by tail vein injections, respectively. The docetaxel was administered at a total dose of 25 mg/kg in a single dose. Whole blood was taken on days 1, 3, 5, 7, 9, and 11 after administration for measuring the number of leukocytes (FIG. 10A). The serum was taken on day 12 after administration to determine the creatinine clearance rate (FIG. 10B).