Drug delivery bionic carrier as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, and discloses a drug delivery bionic carrier as well as a preparation method and application thereof. Most of anti-tumor drugs are low in delivery efficiency through intravenous injection, the anti-tumor drugs are'inactivated 'by lysosomes after entering cells and fail, and the drug failure approach is a main reason for reduction of the effectiveness of most of the anti-tumor drugs delivered through intravenous injection. In order to improve the efficiency of targeted delivery of anti-tumor drugs through intravenous injection, a bionic carrier formed by wrapping chalk with a cell membrane is provided to load the anti-tumor drugs, and specifically, chalk releases carbon dioxide in lysosome to break through the lysosome and promote the drugs to escape from the lysosome, so that targeted delivery and intracellular positioning release of the drugs are realized; the anti-tumor effect of the medicine is remarkably improved while the biocompatibility of the carrier is not influenced, and a scientific basis is provided for targeted delivery of the medicine and tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a bionic carrier for drug delivery and a preparation method and application thereof. Background Art

[0002] Malignant tumors are a major public health problem worldwide. Currently, surgical resection, drug therapy, and radiotherapy are commonly used clinical treatment options. Drug therapy is one of the most commonly used treatments for patients with advanced tumors. However, many tumors are not sensitive to drugs, and some anti-tumor drugs are only used as second-line treatments. In addition, systemic drug therapy is often accompanied by defects such as poor treatment effects and severe toxic side effects that greatly reduce patient tolerance.

[0003] Anti-tumor drugs have multiple routes of administration, including oral, intravenous, subcutaneous and arterial infusion, among which intravenous injection is a method of administration in which the drug is directly injected into the vein. This method of administration has the advantages of rapid onset, less trauma and simple operation. Compared with oral administration, intravenous injection can avoid the first-pass effect of drugs through the liver and significantly improve the bioavailability of drugs.

[0004] However, many anti-tumor drugs cannot reach effective blood drug concentration at the target site through intravenous injection, and the therapeutic effect is poor. For example, the patent with application publication number CN105693544A discloses a small molecule material, preparation method and application for anti-tumor drug delivery. The patent connects two molecules of carboxylated D-glucono-δ-lactone with diamine through peptide bonds to form a small molecule material to load paclitaxel, doxorubicin, camptothecin and other anti-tumor drugs, thereby improving the water solubility and biocompatibility of these anti-tumor drugs and targeting effects, and ultimately improving the bioavailability of the drug. However, the patent's method of improving bioavailability still has the following disadvantages: first, the preparation method is complicated, and it is only for anti-tumor drugs of specific structures, and the delivery of most anti-tumor drugs cannot be transferred for application; second, its targeting is passive targeting, and the targeting effect is limited; third, the carrier will have the problem of poor drug release.

[0005] Therefore, the loading and delivery of anti-tumor drugs remains a major challenge. Summary of the invention

[0006] In order to solve the above-mentioned delivery problem of anti-tumor drugs, especially the delivery problem of drugs through intravenous injection, the present invention provides a drug delivery bionic carrier and its preparation method and application. The present invention provides a bionic carrier formed by wrapping chalk with a cell membrane to load the anti-tumor drug, specifically by the chalk releasing carbon dioxide in the lysosome to break through the lysosomal membrane and promote the drug to escape from the lysosome, so as to reduce the ineffectiveness of the anti-tumor drug in the lysosome and increase the amount of the anti-tumor drug that acts on the target tumor cells.

[0007] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a bionic carrier for drug delivery. The bionic carrier provided by the present invention is formed by chalk wrapped by cell membrane, and the drug delivery is delivered by intravenous injection.

[0008] Intravenous injection of drugs is a method of administration in which drugs are directly injected into the vein. Intravenous injection allows drugs to quickly enter the systemic blood circulation and reach the site of action. The present invention has found in experiments that most anti-tumor drugs delivered via intravenous injection will fail in the lysosomes of cells, resulting in reduced accumulation of drugs at the target tumor cell site and reduced amount of drugs that work effectively. This drug failure pathway is the main reason why most anti-tumor drugs delivered via intravenous injection are not very active. In order to reduce the failure of anti-tumor drugs delivered via intravenous injection in lysosomes, the present invention provides a bionic carrier formed by wrapping chalk with a cell membrane to load the anti-tumor drug, specifically by chalk releasing carbon dioxide in the lysosome to break through the lysosome and promote the escape of the drug from the lysosome, so as to reduce the failure of the anti-tumor drug in the lysosome and increase the amount of the anti-tumor drug that works in the target tumor cells.

[0009] Preferably, the cell membrane is a homologous cell membrane of a target cell to which the drug is to be delivered.

[0010] Homologous tumor cell membranes are wrapped around chalk. Since the tumor cell membrane has immune escape ability, the bionic carrier can achieve primary targeting of tumor tissues. Since the tumor cell membrane has homologous targeting characteristics, it deeply penetrates into tumor cells through clathrin-mediated endocytosis, and secondary deep targeting of tumor cells can be achieved. Therefore, the cell membrane used to wrap chalk to form a bionic carrier in the present invention is preferably a homologous cell membrane of the target cell to be delivered.

[0011] Preferably, the average particle size of the bionic carrier is 80-500 nm.

[0012] The nano-bionic carrier prepared by the method of the present invention has a high encapsulation rate under the drug particle size within the acceptable range for intravenous injection in vivo. At the same time, the particle size of the chalk will have a certain influence on the encapsulation rate. A higher encapsulation rate can be obtained by optimizing the particle size of the carrier chalk to increase the effective concentration of the intravenous drug at the target and reduce the amount of the loaded drug used.

[0013] In a second aspect, the present invention provides a method for preparing a bionic carrier for drug delivery, characterized in that: chalk and cell membrane are mixed and ultrasonicated to obtain a nano bionic carrier formed by the cell membrane wrapping the chalk; The particle size of the chalk is 80-500 nm.

[0014] Preferably, the ultrasound is carried out at 10-80 W / m 2 The ultrasound is carried out in the following manner: working for 1 to 10 seconds, with an interval of 1 to 10 seconds, and the total ultrasound time is 2 to 15 minutes.

[0015] Preferably, the ultrasonication is performed under ice bath conditions.

[0016] Preferably, the mixing mass ratio of the chalk to the cell membrane is 0.2-0.9:1.

[0017] In a third aspect, the present invention provides the use of the above-mentioned bionic carrier in the preparation of anti-tumor drugs.

[0018] In a fourth aspect, the present invention provides an injection for intravenous administration, wherein the active drug of the injection is loaded on the above-mentioned bionic carrier.

[0019] Preferably, the active drug is an anti-tumor drug, and the tumor includes one or more of the following tumors: liver cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, bile duct cancer, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, lymphoma, glioma, leukemia and sarcoma.

[0020] Compared with the prior art, the present invention has the following technical effects: (1) In the past, the effective concentration of anti-tumor drugs was often increased by targeted drug administration. The present invention provides a bionic carrier formed by wrapping chalk with a cell membrane to load the anti-tumor drug. Specifically, the chalk releases carbon dioxide in the lysosome to break through the lysosome and promote the escape of the drug from the lysosome, thereby reducing the ineffectiveness of the anti-tumor drug in the lysosome and increasing the amount of the anti-tumor drug that works in the target tumor cells. This provides a new idea for increasing the effective concentration of the anti-tumor drug.

[0021] (2) The nano-bionic carrier prepared by the method of the present invention has a high encapsulation rate at different drug particle sizes within the acceptable range for intravenous injection in vivo. At the same time, the particle size of chalk will have a certain effect on the encapsulation rate. A higher encapsulation rate can be obtained by optimizing the particle size of the carrier chalk, thereby increasing the effective concentration of intravenous drugs at the target site. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The physicochemical properties of the nano-bionic drug carrier prepared in Example 1 of the present invention are characterized in Figure 1, wherein: (A) is the average particle size of H22 CM and C-DOX@H22 CM; (B) is the zeta potential of H22 CM and C-DOX@H22 CM; (C) is the fluorescence spectra (λ Ex 490 nm, λ Em 590 nm); (D) TEM images of Chalk, H22 CM and C-DOX@H22 CM, scale bar: 200 nm; (E) Fluorescence image of C-DOX@H22 CM, scale bar: 100 μm; (F) Protein expression of H22 tumor cell membrane and C-DOX@H22 CM detected by SDS-PAGE (a: Marker, b: C-DOX, c: H22 tumor cell membrane, d: C-DOX@H22 CM).

[0023] Figure 2 This is the pH responsive drug release curve of the nano-bionic drug carrier prepared in Example 1 of the present invention.

[0024] Figure 3 The targeting and immune escape effects of the nano-bionic drug carrier prepared in Example 1 of the present invention on H22 cells, wherein: (A) is the fluorescence intensity of C-DOX@H22 CM in RAW264.7 cells detected by flow cytometry; (B) is the fluorescence intensity of C-DOX@H22 CM in H22 cells detected by flow cytometry; (C) is the cell uptake of C-DOX@H22 CM in different cell types detected by flow cytometry ((a): H22, (b): NIH-3T3, (c): CT26); (D) is the CLSM fluorescence image of C-DOX@H22 CM-DIO prepared by uptake of DIO-labeled H22 cell membrane by different cells, scale bar: 50 μm.

[0025] Figure 4The deep penetration effect of the nano-bionic drug carrier prepared in Example 1 of the present invention on the tumor sphere model, wherein: (A) is the CLSM fluorescence image of the H22 tumor sphere after uptake of C-DOX@H22 CM; (B) is the fluorescence intensity of C-DOX@H22 CM on the 120 μm cross-section of the tumor sphere, scale bar: 50 μm.

[0026] Figure 5 The biocompatibility evaluation of the nano-bionic drug carrier prepared in Example 1 of the present invention, wherein: (A) is the cytotoxicity of the blank bionic carrier C@H22 CM to NIH-3T3 or H22 cells after incubation for 24 hours (n=6); (B) is the hemolysis percentage and hemolysis graph of red blood cells treated with C-DOX@H22 CM at DOX concentrations of 0.1, 1.0, 2.5, 5.0 and 10 μg / mL, and physiological saline and pure water were used as negative (-) and positive (+) controls, respectively.

[0027] Figure 6 This is a graph showing the cytotoxic effect of the nano-bionic drug carrier prepared in Example 1 of the present invention on H22 cells, showing the H22 cell viability measured after incubation of C-DOX@H22 CM with DOX concentrations of 0.1, 1, 2.5, 5, and 10 μg / mL with H22 cells for 24 h, where: *P<0.05, **P<0.01, ***P<0.001 (n=5). DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0029] Example 1 This embodiment provides a homologous tumor cell membrane bionic drug delivery system, and the preparation method thereof comprises the following steps: (1) Extraction of tumor cell membrane Mouse hepatoma cells (H22) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% double antibody. After in vitro culture to a density of 85%~90%, the cells were centrifuged at 1200rpm for 3 min, the supernatant was discarded, and the cells were collected and resuspended with PBS. 10 μL was taken and injected intraperitoneally into the peritoneal cavity of BALB / c mice for ascites culture. The ascites was collected, diluted with PBS, centrifuged at 1200rpm for 3 min, the supernatant was removed, and the washing was repeated twice, and PBS was added to resuspend to prepare a cell suspension.

[0030] The cell suspension was collected, washed with PBS, centrifuged at 4 °C and 600 g for 5 min, and the supernatant was removed. 1 mL-1.5 mL of lysis solution was added and ice-bathed for 15 min. PMSF was added to the lysis solution in advance and pre-cooled. The cells were repeatedly frozen and thawed 5 times at room temperature and liquid nitrogen, centrifuged at 4 °C and 700 g for 10 min, the unlysed cells at the bottom were discarded, the supernatant was taken out, centrifuged at 4 °C and 14000 g for 30 min, the supernatant was removed, and the cell membrane was collected. The Avanti liposome extruder was used to pass through a 400 nm polycarbonate (PCTE) membrane 21 times to obtain tumor cell membrane vesicles H22 CM.

[0031] (2) Preparation of blank nano-bionic carriers Take an appropriate amount of chalk in a mortar, grind it, pour the powder into a sieve, sieve it, and remove the large particles; repeat the above steps 3 times. Weigh the sieved chalk and add it to deionized water. Use a cell ultrasonic disruptor, ice bath, power 390w, ultrasonic for 60 min, work 10 s, interval 10 s, to obtain a small-sized chalk suspension. Take 1mL of the prepared chalk suspension and use a Malvern particle size analyzer to measure the particle size of the chalk. The results show that the average particle size of the chalk is 176±9.1nm.

[0032] Take 500 μL of 500 μg / mL cell membrane H22 CM obtained in step (1) (prepared by adding H22 CM to a PBS solution with a pH of 7.4 and mixing evenly), add 20 μL of 10 mg / mL Chalk (prepared by adding Chalk to deionized water and mixing evenly, freshly prepared and used), and mix evenly. Use a cell ultrasonic disruptor to sonicate the mixture at an intensity of 60 W / m 2 The blank nano-bionic carrier C@H22 CM was obtained by ultrasonication for 15 min with a working time of 3 s and a time interval of 3 s. The mass ratio of chalk to cell membrane was 0.8:1.

[0033] (3) Preparation of nano-bionic drug carriers Take an appropriate amount of chalk in a mortar, grind it, pour the powder into a sieve, sieve it, and remove the large particles; repeat the above steps 3 times. Weigh the sieved chalk and add it to deionized water. Use a cell ultrasonic disruptor, ice bath, power 390w, ultrasonic for 60 min, work 10 s, interval 10 s, to obtain a small-sized chalk suspension. Take 1mL of the prepared chalk suspension and use a Malvern particle size analyzer to measure the particle size of the chalk. The results show that the average particle size of the chalk is 176±9.1nm.

[0034] Accurately weigh an appropriate amount of doxorubicin (DOX) and dissolve it in deionized water. Mix the doxorubicin aqueous solution with the Chalk suspension (mixed at a mass ratio of doxorubicin: Chalk = 1:10), stir on a magnetic stirrer in the dark for 12 h, take out the mixed solution and centrifuge at 8000 rpm for 3 min, collect the precipitate, and obtain C-DOX. The encapsulation efficiency is calculated to be 96.12%. The encapsulation efficiency is calculated as follows: (total amount of drug added - amount of unloaded drug) / total amount of drug added × 100%; the amount of unloaded drug is obtained by measuring the amount of drug in the centrifugal supernatant by fluorescence spectrophotometry.

[0035] Take 500 μg / mL of the cell membrane H22 CM obtained in step (1) (prepared by adding H22 CM to a PBS solution with a pH of 7.4 and mixing evenly), add 20 μL of the 10 mg / mL C-DOX prepared in advance (prepared by adding C-DOX to a PBS solution with a pH of 7.4 and mixing evenly, freshly prepared and used), mix well, and use a cell ultrasonic disruptor to sonicate the mixture at an ultrasonic intensity of 60 W / m 2 The nano-bionic drug carrier C-DOX@H22CM was obtained by ultrasonication for 15 min with a working interval of 3 s and a working interval of 3 s. The mass ratio of chalk to cell membrane was 0.8:1.

[0036] Example 2 This example is used to study the physicochemical properties of nano-bionic drug carriers.

[0037] The nano-bionic drug carrier C-DOX@H22 CM prepared in Example 1 was taken, and the hydrated particle size and Zeta potential of H22 CM and C-DOX@H22 CM after extrusion were measured using a Malvern particle size analyzer; and the emission curves of DOX, C-DOX and C-DOX@H22 CM at an excitation wavelength of 490 nm were measured using a fluorescence spectrometer FS5; and the TEM images of Chalk, H22 CM and C-DOX@H22CM, the fluorescence image of C-DOX@H22 CM, and the protein expression results of H22 tumor cell membrane and C-DOX@H22CM were tested by SDS-PAGE. Figure 1 shown.

[0038] Depend on Figure 1 AB shows that after extrusion by the liposome extruder, the particle size of H22 CM vesicles is about 146 nm, the potential is -23 mV, and the particle size of C-DOX@H22 CM prepared by ultrasound is 250 nm, the potential is -28 mV. The particle size of C-DOX@H22 CM is larger than that of H22 CM, and the potentials are both negative and not much different, which verifies that H22 CM is successfully encapsulated on the surface of C-DOX.

[0039] DOX has the highest fluorescence characteristic peak at 590 nm under the excitation wavelength of 490 nm by FS5 fluorescence spectrometer, such as Figure 1 As shown in Figure C, both C-DOX and C-DOX@H22 CM have the highest peak at 590 nm. At the same DOX concentration, the fluorescence values ​​of C-DOX and C-DOX@H22 CM are lower than those of DOX, which means that Chalk encapsulates DOX and the fluorescence intensity decreases. At the same time, since H22 CM encapsulates C-DOX, the fluorescence intensity of DOX is further reduced. Figure 1 The TEM of D can clearly observe that H22 CM is round and has an average particle size of about 50nm. Since the main component of Chalk is CaCO3, which is an inorganic substance, it cannot be directly photographed by TEM after negative staining, resulting in cavities. However, the cavities in H22 CM can be clearly observed in C-DOX@H22 CM, which also indirectly confirms the successful encapsulation of H22CM. In order to further verify whether H22 CM successfully encapsulates C-DOX, H22CM is loaded with DIO fluorescent probe for labeling. The preparation method is consistent with that of C-DOX@H22 CM to prepare C-DOX@H22 CM-DIO. Take 10 μL of the sample suspension to be tested and drop it on a glass slide. Cover it with a coverslip and use CLSM to shoot the bright field, as shown in the figure. Figure 1 As shown in E, the green fluorescent DIO and the red fluorescent DOX have overlapping parts (yellow), which verifies that H22 CM encapsulates C-DOX successfully. The protein bands of C-DOX, H22 CM and C-DOX@H22 CM were determined by SDS-PAGE gel electrophoresis. Figure 1 As shown in F, C-DOX has no protein bands, and the surface protein bands of C-DOX@H22 CM are consistent with those of H22 CM, indicating that C-DOX@H22 CM retains the surface proteins of tumor cell membranes.

[0040] Example 3 This example is used to study the pH-sensitive drug release characteristics of nano-bionic drug carriers.

[0041] C-DOX@H22 CM was added to a PBS solution with a pH of 7.4 to prepare a 10 mg / mL C-DOX@H22 CM solution. 1 mL of the 10 mg / mL C-DOX@H22 CM solution (pH 7.4) was loaded into a dialysis bag (MWCO: 3500), and the dialysis bag was placed in 15 mL of a PBS solution with a pH of 7.4. C-DOX@H22 CM was added to a PBS solution with a pH of 4.5 to prepare a 10 mg / mL C-DOX@H22 CM solution. 1 mL of the 10 mg / mL C-DOX@H22 CM solution (pH 4.5) was loaded into a dialysis bag (MWCO: 3500), and the dialysis bag was placed in 15 mL of a PBS solution with a pH of 4.5. The two systems were placed in a transdermal diffusion instrument, and the drug release experiment was carried out under the following conditions: 37 °C, 100 rpm. At different time points (0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 56h, 72h and 96h), 3 mL of release solution was taken and supplemented with 3 mL of PBS buffer of the corresponding pH of the two groups of experiments. The fluorescence intensity was measured by fluorescence spectrophotometer after diluting the taken release solution, and the cumulative release rate of the drug was calculated according to the standard curve method. The results are shown in Figure 2 .

[0042] Figure 2 The results showed that C-DOX@H22 CM was released faster in pH 4.5 PBS solution than in pH 7.4 PBS solution. The release rate of C-DOX@H22 CM in pH 4.5 PBS solution was significantly increased, and the cumulative release rate exceeded 60% after 4 h, while the cumulative release rate in pH 7.4 PBS solution was only 19.5% within 96 h.

[0043] Lysosomes are acidic environments with a pH of about 4 to 5. Therefore, it can be seen that C-DOX@H22 CM has better drug release performance in lysosomes in acidic environments. Chalk can release carbon dioxide in lysosomes to break through lysosomes, and drugs can escape lysosomes along the carbon dioxide release site, thereby reducing the ineffectiveness of anti-tumor drugs in lysosomes and ultimately increasing the amount of anti-tumor drugs that work in target tumor cells.

[0044] Example 4 This example is used to evaluate the immune escape characteristics and targeting of the nano-bionic carrier.

[0045] First, the cellular uptake of C-DOX@H22 CM was evaluated in RAW264.7 cells. To investigate the immune escape ability of C-DOX@H22 CM, RAW264.7 cells were incubated with cell-free membrane-encapsulated C-DOX nanoparticles and C-DOX@H22 CM, and then the cells were collected and the fluorescence intensity was measured using flow cytometry. The results are shown in Figure 3 Compared with C-DOX nanoparticles without cell membrane, Figure 3 As shown in A, the fluorescence intensity of C-DOX@H22 CM was lower (P < 0.01).

[0046] Next, this example measured the fluorescence intensity of C-DOX@H22 CM in H22 tumor cells to confirm the homologous targeting ability of C-DOX@H22 CM. The results are shown in Figure 3 B. By Figure 3 B shows that the fluorescence intensity of C-DOX@H22 CM is significantly higher than that of C-DOX without cell membrane encapsulation (P<0.01).

[0047] In order to determine the effect of cell type on cellular uptake and investigate homologous targeting, the biomimetic nanoparticles C-DOX@H22 CM were co-cultured with normal embryonic fibroblasts NIH-3T3, colon cancer cells CT26 and liver cancer cells H22 for 4 h, respectively, with non-homologous cells as controls. The uptake of DOX by cells was analyzed by flow cytometry. The results are shown in Figure 3 C. By Figure 3 The flow cytometry results of C showed that C-DOX@H22 CM had the strongest cellular uptake in H22 cells, while the uptake in normal cells NIH-3T3 and colon cancer cells CT26 was weak, thus confirming the homology specificity of C-DOX@H22 CM.

[0048] In addition, this example also prepared C-DOX@H22CM-DIO by assembling DIO fluorescently labeled H22 cell membrane with C-DOX (the preparation method is the same as that of Example 2). After co-incubation with normal cells NIH-3T3, colon cancer cells CT26, and liver cancer cells H22 for 4 hours, the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 minutes. After rinsing with PBS to remove paraformaldehyde, the cells were stained with DAPI solution for 10 minutes, and the drug uptake was observed using CLSM. The results are shown in Figure 3 D. As Figure 3As shown in Figure D, the CLSM results are consistent with the flow cytometry results. Weak green and red fluorescence signals are shown in the cytoplasm of NIH-3T3 and CT26 cells, while strong yellow fluorescence signals (overlapping green DIO fluorescence and red DOX fluorescence) are observed in the cytoplasm of H22 cells, which is due to the expression of specific proteins on the cell membrane. In addition to the signals in the cytoplasm, purple fluorescence signals (overlapping blue DAPI fluorescence and red DOX fluorescence) are also observed in the cell nucleus, indicating that C-DOX@H22 CM is more easily internalized by homologous cells and quickly releases DOX into the cell nucleus, which is beneficial to the anti-cancer effect in tumor cells, and has superior safety in normal cells, which can reduce side effects.

[0049] In addition, this example also established a 3D tumor sphere model to evaluate the deep targeted penetration ability of C-DOX@H22 CM. H22 and NIH-3T3 cells were seeded on 1.5% agarose in a 96-well plate, and the tumor sphere model was co-cultured with DOX, C-DOX, and C-DOX@H22 CM. After incubation for 24 h, the model was placed on a coverslip and analyzed by CLSM. The results are shown in Figure 4 .like Figure 4 As shown in the figure, the fluorescence signal of free DOX is almost located at the periphery of the tumor sphere, while the penetration depth of the nanoparticles increases after being wrapped by the cell membrane, and the fluorescence signal on the cell membrane is closer to the core of the tumor sphere (reaching 120 μm), verifying the excellent targeted deep penetration ability of C-DOX@H22 CM.

[0050] Therefore, combined with Example 3, it can be verified that C-DOX@H22 CM can effectively increase the amount of anti-tumor drugs acting on target tumor cells.

[0051] Example 5 This example evaluates the biocompatibility of the nano-bionic carrier.

[0052] H22 or NIH-3T3 cells were seeded in 96-well plates and incubated with blank nanobiomimetic carriers C@H22CM (final concentrations of Chalk were 1, 10, 25, 50, and 100 μg / mL) for 24 h. The supernatant was then discarded and replaced with 100 μL of fresh medium containing MTT (5 mg / mL) and incubated for another 4 h. After centrifugation at 1000 rpm for 10 min, the supernatant was removed. 150 μL of DMSO was added to each well and incubated on an oscillator for 10 min. The absorbance (OD) of the samples was measured at 570 nm using a microplate reader, and the results are shown in Table 1. Figure 5 .like Figure 5 As shown in A, the cytotoxicity test showed that the blank nanocarrier had no obvious killing effect on normal cells and tumor cells.

[0053] 1 mL of blood was collected from the mouse orbit and placed in an EDTA anticoagulant tube. It was centrifuged at 4°C and 2000 rpm for 10 min to remove the upper plasma, and washed three times with pre-cooled saline. After dilution, a 20% red blood cell suspension was obtained. The red blood cell suspension was incubated with different concentrations of C-DOX@H22 CM (DOX concentrations were 0.1, 1, 2.5, 5, and 10 μg / mL) at 37°C for 2 h, with saline as a negative control and pure water as a positive control. The red blood cell suspension was centrifuged at 2000 rpm for 10 min, and the supernatant was collected to measure its absorbance OD value at 540 nm. The hemolysis rate calculation formula is: hemolysis rate (%) = (OD sample-OD negative) / (OD positive-OD negative) × 100%. Figure 5 As shown in B, even when red blood cells were treated at a concentration of 10 μg / mL, there was almost no hemolysis, which proved the blood compatibility of the nano-bionic drug carrier and indicated that it can be safely used for intravenous administration.

[0054] Example 6 This example evaluates the anti-tumor effect of the nano-bionic carrier.

[0055] The in vitro antitumor effect of C-DOX@H22 CM on H22 cells was evaluated by the MTT method in Example 5. H22 cells were treated with free DOX or C-DOX@H22 CM at DOX concentrations of 0.1, 1, 2.5, 5, and 10 μg / mL. Figure 6 .like Figure 6 As shown in the figure, the cytotoxicity of C-DOX@H22 CM was concentration-dependent. At a low concentration of 2.5 μg / mL of DOX, the cell survival rate of the C-DOX@H22 CM group was only 55.8±9.8%, showing more obvious cytotoxicity compared with the same amount of DOX (P<0.05). The results showed that C-DOX@H22 CM with tumor cell membranes can enhance the in vitro antitumor effect of free DOX by homologously targeting liver cancer cells, making it a promising antitumor therapeutic option.

[0056] Example 7 This example is used to study the drug loading performance of the nano-bionic carrier.

[0057] First, the nano-bionic carrier C-DOX was prepared by the same preparation method as step (3) of Example 1. When only the average particle size of the screened Chalk was changed and the other preparation conditions were the same as step (3) of Example 1, the encapsulation efficiency of the obtained C-DOX changed significantly. When the average particle sizes of Chalk obtained after grinding, screening and ultrasound were 80±6.1nm, 103±5.7nm, 146±8.2nm, 192±6.4nm, 248±8.5nm and 305±7.5nm, respectively, the encapsulation efficiency of the obtained C-DOX was 80.41%, 87.56%, 96.17%, 97.01%, 93.42% and 92.28%, respectively. Combined with Example 1, the average particle size of Chalk is 176±9.1nm, and its encapsulation efficiency is 96.12%. This shows that the nano-bionic carrier prepared by the method of the present invention has a higher encapsulation efficiency at different drug particle sizes within the acceptable range for intravenous injection in vivo. At the same time, it can be seen that the particle size of Chalk will have a certain effect on the encapsulation efficiency. It can be predicted that a higher encapsulation efficiency can be obtained by optimizing the particle size of the carrier Chalk to increase the effective concentration of intravenous drugs at the target.

[0058] Furthermore, in this example, nano-bionic drug carrier C-DOX@H22 CM was prepared with different mixing mass ratios of chalk to cell membrane. The preparation method was the same as step (3) of Example 1. It was found that when the mixing mass ratio of chalk to cell membrane was 0.2~0.9:1, C-DOX had a better encapsulation condition. When the mixing mass ratio was greater than 1, the encapsulation condition was slightly worse. At the same time, it was found that when the mixing mass ratio was greater than 1, the dispersion effect of the carrier would deteriorate. This indicates that if the amount of C-DOX added was too much, the dispersion effect would deteriorate, thereby leading to a decrease in stability.

[0059] The chalk used in the examples of the present invention is natural chalk purchased from Lingshou County Dehang Mineral Products Co., Ltd. The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0060] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A bionic drug delivery carrier, characterized in that: The bionic carrier is formed by chalk wrapped by cell membrane, and the drug is delivered via intravenous injection.

2. A bionic drug delivery carrier according to claim 1, characterized in that: The cell membrane is a homologous cell membrane of a target cell to which the drug is to be delivered.

3. A bionic drug delivery carrier according to claim 1, characterized in that: The average particle size of the bionic carrier is 80-500 nm.

4. A method for preparing a bionic drug delivery carrier, characterized in that: The chalk is mixed with the cell membrane and subjected to ultrasound to obtain a nano-bionic carrier formed by the cell membrane wrapping the chalk; The particle size of the chalk is 80-500 nm.

5. The preparation method according to claim 4, characterized in that: The ultrasound is at 10-80 W / m 2 The ultrasound is carried out in the following manner: working for 1 to 10 seconds, with an interval of 1 to 10 seconds, and the total ultrasound time is 2 to 15 minutes.

6. The preparation method according to claim 5, characterized in that: The ultrasound was performed in an ice bath.

7. The preparation method according to claim 4, characterized in that: The mixing mass ratio of the chalk to the cell membrane is 0.2-0.9:

1.

8. Use of the bionic carrier according to any one of claims 1 to 3, or the bionic carrier prepared by the preparation method according to any one of claims 4 to 7 in the preparation of anti-tumor drugs.

9. An injection for intravenous administration, characterized in that: The active drug of the injection is loaded on the bionic carrier according to any one of claims 1 to 3, or the bionic carrier prepared by the preparation method according to any one of claims 4 to 7.

10. The injection according to claim 9, characterized in that: The active drug is an anti-tumor drug, and the tumor includes one or more of the following tumors: liver cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, bile duct cancer, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, lymphoma, glioma, leukemia and sarcoma.

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