Bone marrow stromal cell membrane bionic drug-loading CX4945 nanometer delivery system as well as preparation method and application thereof

Abstract

The invention discloses a bone marrow stromal cell membrane bionic drug-loading CX4945 nano delivery system as well as a preparation method and application thereof. The bone marrow stromal cell membrane bionic drug-loading CX4945 nano delivery system takes a CX4945 drug coated by a high-molecular polymer as a core and a stromal cell membrane as a shell layer, so that the CX4945 can be slowly released, the half-life period of the drug is prolonged, and the medication frequency of the CX4945 is reduced; through specific binding of CXCL12 on a matrix cell membrane and CXCR4 over-expressed on the surface of a B-ALL cell, the uptake of the B-ALL cell to a CX4945 drug is improved, the killing of the drug to tumor cells is enhanced, and accurate delivery of the CX4945 drug is realized; the homing of B-ALL cells can be prevented by blocking a CXCR4 / CXCL12 biological axis, and the problems of transfer, relapse, drug resistance and the like of the B-ALL cells are solved.

Description

Technical Field

[0001] The present invention relates to a bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system, a preparation method thereof and an application thereof, and belongs to the field of drug-loaded nano-delivery systems and their preparation and applications. Background Art

[0002] Acute leukemia (AL) is a type of hematological malignancy that seriously endangers human health. According to the types of affected cells and different treatment methods, it can be divided into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Secondly, according to the differences in cellular immunophenotypes, acute lymphoblastic leukemia can be specifically classified into B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL). In recent years, with the continuous progress of targeted therapy technology, the treatment of acute leukemia has also developed rapidly, but the recurrence, intractability and side effects of acute B-lymphoblastic leukemia are still problems to be solved urgently. Therefore, it is urgent to explore new, efficient and low-toxic treatment methods.

[0003] CX4945 is a selective CK2 inhibitor that can competitively bind to the CK2α subunit with ATP, preventing their binding and playing a role in inhibiting CK2 activity. The expression and activity of CK2 in various tumors including acute leukemia are higher than those in normal tissues. CK2 phosphorylates IKAROS, resulting in the dysregulation of the transcriptional regulatory function of IKAROS, and then abnormalities in cell cycle, cellular sublocalization, DNA binding ability, chromatin remodeling, etc., ultimately leading to the occurrence of leukemia. IKAROS is a protein with important regulatory functions and plays a key role in the occurrence and development of acute leukemia. Existing studies have found that the transcription factor IKAROS encoded by the IKZF1 gene is an important leukemia inhibitory factor, and CK2 blocks its binding to DNA by phosphorylating IKAROS, resulting in the dysregulation of the regulation of downstream target genes. CX4945 can reshape the function of IKAROS and exert its anti-leukemia effect. In addition, in previous work, it was confirmed that CK2 is a very promising new target for the treatment of B-ALL, and CX4945 has a significant effect on inhibiting the proliferation of B-ALL cells.

[0004] In recent years, biomimetic targeted therapy of bone marrow stromal cell membranes has provided new ideas for tumor targeted therapy, enhanced the stability of nanostructures, and reduced drug leakage. In addition, the biomimetic cell membranes help the nano-drugs escape the clearance of the immune system, improve the biocompatibility and specific targeting of the drugs in the body, deliver the drugs accurately and targetedly to the target location, achieve precise treatment, and reduce the side effects of the drugs. The bone marrow stromal cell membrane is a thin film extracted from the surface of bone marrow stromal cells, mainly composed of a lipid bilayer and proteins, and retains the biological activity of its membrane proteins. The chemokine CXCL12 secreted by bone marrow stromal cells can regulate the homing and colonization of hematopoietic stem cells. The CXCR4 receptor highly expressed on the surface of acute leukemia cell membranes specifically binds to the CXCL12 ligand secreted by bone marrow stromal cells to form a CXCR4 / CXCL12 biological axis, causing leukemia cells to produce chemotactic and migratory responses. Through the CXCR4 / CXCL12 biological axis, leukemia cells home to the bone marrow microenvironment, forming minimal residual disease and leading to leukemia recurrence. Therefore, the bone marrow stromal cell membrane biomimetic nano-drug can specifically bind to acute B lymphocytes highly expressing CXCR4, precisely target and treat B-ALL cells, and block the homing of B-ALL cells to the bone marrow.

[0005] The CX4945 drug has shown good prospects for the treatment of acute leukemia. It can restore its normal function by reducing IKAROS phosphorylation. However, as a small molecule drug, CX4945 has a low bioavailability and a fast degradation rate, thus limiting its wide application in the treatment of hematological malignancies. In addition, the homing of B-ALL cells to the bone marrow depends on the regulation of the CXCR4 receptor. CXCR4 specifically binds to the CXCL12 ligand secreted by bone marrow stromal cells to form a CXCR4 / CXCL12 biological axis, causing B-ALL cells to produce chemotactic and migratory responses and home to the bone marrow microenvironment. Summary of the Invention

[0006] Object of the Invention: The technical problem to be solved by the present invention is to provide a bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system capable of achieving precise delivery of the CX4945 drug, and its preparation method and application.

[0007] Technical solution: To solve the above technical problems, the present invention provides a biomimetic drug-loaded CX4945 nanodelivery system based on bone marrow stromal cell membrane, which uses CX4945 drug coated with a polymer as the core, and the core particle size is distributed in 48.364 ± 2.357 nm. The stromal cell membrane is used as the shell layer, and the particle size after coating is distributed in 114.233 ± 2.375 nm. The appearance of CX4945@PLGA-PEG nanoparticles presents a spherical shape under a transmission electron microscope. In addition, it also presents a spherical shape under a transmission electron microscope after coating the stromal cell membrane, and there is a clear cell membrane shell layer on the surface of CX4945@PLGA-PEG@CM.

[0008] Among them, the particle size of the biomimetic drug-loaded CX4945 nanodelivery system based on bone marrow stromal cell membrane is 48 - 120 nm.

[0009] Among them, the bone marrow stromal cell membrane can be replaced by bone marrow mesenchymal stem cells or bone marrow hematopoietic stem cells, and the biological activity of the cell membrane can be retained.

[0010] The present invention also provides a method for preparing the biomimetic drug-loaded CX4945 nanodelivery system based on bone marrow stromal cell membrane, including the following steps: dissolving poly(lactic-co-glycolic acid)-polyethylene glycol and CX4945 drug together in tetrahydrofuran, then adding it to an aqueous solution of polyvinylpyrrolidone, sonicating and disrupting in an ice bath, stirring, centrifuging, and then mixing with the bone marrow stromal cell membrane, sonicating in an ice bath, and centrifuging to remove the excessive cell membrane.

[0011] Among them, the bone marrow stromal cell membrane has CXCL12 chemokine activity.

[0012] Among them, the bone marrow stromal cell membrane is prepared by a method combining hypotonic solution lysis and repeated freezing and thawing with liquid nitrogen.

[0013] Among them, the method combining hypotonic solution lysis and repeated freezing and thawing with liquid nitrogen is as follows: resuspending the bone marrow stromal cells with a hypotonic solution to swell and break the cells, then repeatedly freezing and thawing, and centrifuging to obtain the bone marrow stromal cell membrane.

[0014] Among them, the liquid nitrogen freezing time is 8 - 15 s, and the number of operations of repeated freezing and thawing is 7 - 12 times.

[0015] Among them, centrifugation includes low-speed centrifugation and high-speed centrifugation; the rate of the low-speed centrifugation is 1500 - 2500 rpm, and the time is 8 - 12 min; the rate of the high-speed centrifugation is 11000 - 15000 rpm, and the time is 8 - 12 min.

[0016] Among them, the power of ultrasonic disruption is 120 - 250 W, the working time is 2 - 5 min, the on-time of ultrasound is controlled at 5 - 10 s, and the off-time of ultrasound is controlled at 2 - 5 s. The speed of stirring and volatilization is 300 - 500 rpm, and the stirring time is 2 - 5 h. The molecular weight cut-off of the ultrafiltration centrifugal tube is 30 - 100 KDa, the centrifugation time is 8 - 10 min, the centrifugation speed is 4000 - 5000 rpm, and the number of centrifugation washes is 3 - 5 times.

[0017] Among them, the molecular weight of poly (lactic-co-glycolic acid)-polyethylene glycol is 10K - 12K.

[0018] Among them, the matrix cell membrane biomimetic drug-loaded CX4945 nanodelivery system is spherical in morphology under transmission electron microscopy and has a shell structure. The hydrodynamic size is about 120 nm, and the Zeta potential is between -20 and -30 mV.

[0019] The present invention also provides the application of the matrix cell membrane biomimetic drug-loaded CX4945 nanodelivery system in the preparation of drugs for the treatment of acute leukemia.

[0020] The present invention also provides a drug for the treatment of acute leukemia, which contains the matrix cell membrane biomimetic drug-loaded CX4945 nanodelivery system.

[0021] The present invention will elaborate on the method for preparing the matrix cell membrane biomimetic drug-loaded CX4945 nanodelivery system and its application in B-cell acute lymphoblastic leukemia. The specific binding of CXCL12 expressed on the surface of the matrix cell membrane to CXCR4 overexpressed on the surface of B-ALL cells realizes precise drug targeting delivery, delays the half-life of the CX4945 drug, enhances the killing of B-ALL cells by CX4945, and at the same time blocks the homing of B-ALL cells to the bone marrow microenvironment, providing a basic theoretical basis for the future application of the matrix cell membrane biomimetic drug-loaded CX4945 nanodelivery system in the clinical treatment of acute leukemia.

[0022] The mechanism of the present invention: As Figure 1 shown, first, poly (lactic-co-glycolic acid)-polyethylene glycol and CX4945 drug are dissolved in tetrahydrofuran, and after stirring and volatilization, ultrafiltration and washing are carried out to prepare CX4945@PLGA-PEG. Secondly, the extracted matrix cell membrane is mixed with CX4945@PLGA-PEG and coated by ultrasound, and the excessive cell membrane is removed by centrifugation and washing to obtain the CX4945@PLGA-PEG@CM drug delivery system. Since CXCL12 expressed on the surface of the coated matrix cell membrane can specifically bind to CXCR4 highly expressed on the surface of B-ALL cells, precise drug targeting delivery is realized.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0024] (1) The biomimetic drug-loaded CX4945 nanodelivery system based on bone marrow stromal cell membrane can enable the slow release of CX4945, prolong the half-life of the drug, and reduce the dosing frequency of CX4945;

[0025] (2) The biomimetic drug-loaded CX4945 nanodelivery system based on bone marrow stromal cell membrane can specifically bind to CXCR4 overexpressed on the surface of B-ALL cells through CXCL12 on the stromal cell membrane, improve the uptake of CX4945 drug by B-ALL cells, enhance the killing of tumor cells by the drug, and achieve the precise delivery of CX4945 drug;

[0026] (3) The biomimetic drug-loaded CX4945 nanodelivery system based on bone marrow stromal cell membrane can also block the CXCR4 / CXCL12 biological axis, prevent the homing of B-ALL cells, and solve problems such as their metastasis, recurrence, and drug resistance;

[0027] (4) The biomimetic of bone marrow stromal cell membrane retains the biological activity of stromal cell membrane proteins, improves the escape of nanoparticles from immune clearance, and at the same time improves the compatibility of nanodrugs;

[0028] (5) Poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) as the coating polymer material has good properties. The combination of its hydrophilicity and hydrophobicity enables it to bind well with a variety of drugs, delay the release of drugs, and at the same time it has good biocompatibility, reducing the irritation and immune response to organisms. Brief Description of the Drawings

[0029] Figure 1 Schematic diagram of the preparation mechanism of the biomimetic drug-loaded CX4945 nanodelivery system CX4945@PLGA-PEG@CM NPs based on bone marrow stromal cell membrane;

[0030] Figure 2 Morphologies of the biomimetic drug-loaded CX4945 nanodrug at each stage under transmission electron microscope: (A) PLGA-PEG NPs; (B) CX4945@PLGA-PEG NPs; (C) CX4945@PLGA-PEG@CM NPs; (D) Energy spectrum analysis of CX4945@PLGA-PEG@CM NPs;

[0031] Figure 3 Expression levels of CXCL12 on CX4945@PLGA-PEG@CM NPs, CM, and MS-5;

[0032] Figure 4 Hydrodynamic size (Figure A) and PDI (Figure B) changes of CX4945@PLGA-PEG@CM NPs stored at 4°C in distilled water for 7 days;

[0033] Figure 5 The hydrodynamic size (Figure A) and PDI (Figure B) changes of CX4945@PLGA-PEG@CM NPs after incubation in RPMI1640 medium containing 10% FBS at 37 °C for 0, 6, 12, 24, 48, and 72 h;

[0034] Figure 6 The in vitro release curve of CX4945 from CX4954@PLGA-PEG@CM NPs;

[0035] Figure 7 The hemolysis assay of red blood cells with different concentrations of CX4945@PLGA-PEG@CM and CX4945@PLGA-PEG NPs;

[0036] Figure 8 The determination of CXCR4 expression level in NALM6 cells;

[0037] Figure 9 The viability of NALM6 cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG, and CX4945@PLGA-PEG@CM for 24 h (A) or 48 h (B);

[0038] Figure 10 The spleen size and morphology of mice (n = 7) and (B) spleen weight of mice (*P < 0.05, **P < 0.01, ***P < 0.001, n = 7) after treatment with different drugs;

[0039] Figure 11 The proportion of human CD19-positive cells in the spleen (A) and bone marrow (B) of mice after treatment with different drugs (n = 7);

[0040] Figure 12 The determination of CXCR4 expression level in THP-1 cells;

[0041] Figure 13 The determination of CXCR4 expression level in JURKAT cells;

[0042] Figure 14 The viability assay of THP-1 cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG, and CX4945@PLGA-PEG@CM for 48 h;

[0043] Figure 15 The viability assay of JURKAT cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG, and CX4945@PLGA-PEG@CM for 48 h.

[0044] Figure 16 Determination of NALM6 cell viability after incubation with cell membranes of different concentrations for 48 h. Detailed implementation mode

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0046] Example 1 Preparation of biomimetic drug-loaded CX4945 nano-delivery system based on bone marrow stromal cell membrane

[0047] (1) Preparation of CX4945@PLGA-PEG NPs

[0048] Dissolve 20 mg of poly (lactic-co-glycolic acid)-polyethylene glycol PLGA(10K)-PEG(2K) (Xi'an Ruixi Biotechnology Co., Ltd.; R-PL-012) and 1 mg of CX4945 drug in 500 μL of tetrahydrofuran organic solvent (purity 99.9%), assist dissolution by ultrasonic wave, and then add it to 10 mL of aqueous solution containing 2% polyvinylpyrrolidone (PVP K30). Ice bath and ultrasonic fragmentation (parameters of ultrasonic crusher: working time is 3 min, ultrasonic on time is 10 s, ultrasonic off time is 2 s, power is 195 W). After the reaction, transfer the above solution to a 50 mL three-necked flask and mechanically stir at 400 rpm in a fume hood for 4 h. Centrifuge at 4000 rpm for 10 min (ordinary centrifugation, removing nano-drugs with larger particle size), centrifuge at 4000 rpm for 10 min (centrifugation with a 100KD ultrafiltration tube, removing free CX4945 or unexcessive PLGA-PEG and PVPK30), wash 3 times with ddH2O to obtain yellow CX4945@PLGA-PEG NPs.

[0049] (2) Preparation of bone marrow stromal cell membrane

[0050] The bone marrow stromal cell membrane was prepared by a method combining hypotonic solution lysis and repeated freezing and thawing with liquid nitrogen. Collect bone marrow stromal cells in good growth state, resuspend them with 2 mM PBS solution (pH = 7.4) of hypotonic solution, aliquot them into sterile EP tubes, freeze them in liquid nitrogen for 10 s after cell swelling and fragmentation, and dissolve them at room temperature. Repeat this operation 8 times. Then centrifuge at low speed of 2000 rpm for 5 min, carefully aspirate the supernatant. Collect the supernatant, and then centrifuge at high speed of 12000 rpm for 10 min to remove the supernatant. Finally, resuspend the collected cell membrane and store it in a -80 °C refrigerator.

[0051] (3) Preparation of CX4945@PLGA-PEG@CM NPs

[0052] Mix the prepared CX4945@PLGA-PEG NPs drug with an equal amount of bone marrow stromal cell membrane and sonicate it in an ice bath for 30 min. Subsequently, centrifuge it three times at 6500 rpm to remove the excess cell membrane, and CX4945@PLGA-PEG@CM NPs can be obtained and stored in a -80 °C refrigerator.

[0053] Example 2 Observation of Bone Marrow Stromal Cell Membrane Biomimetic Drug-Loaded CX4945 Nanodelivery System

[0054] The newly prepared bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nanodelivery system solution all showed yellow color, without precipitation and was relatively uniformly dispersed.

[0055] Table 1 Particle size, Zeta potential and PDI distribution of different types of nanoparticles

[0056]

[0057] As shown in Table 1, the particle size of empty nanoparticles (PLGA-PEG) was distributed around 40 nm, the particle size of CX4945@PLGA-PEG nanodrug was distributed around 48 nm; the particle size of CX4945@PLGA-PEG@CM nanodrug was distributed around 114 nm. The Zeta potential all showed negative potential, which helped to inhibit the aggregation of nanoparticles and enhanced the stability of the solution to a certain extent. The charge range was -24.6 to -28.4 mV, indicating that the coating of the biomimetic membrane could not reverse the electrostatic potential of the nanoparticles. In addition, the PDI of the three kinds of nanoparticles was less than 0.2, indicating a uniform particle size distribution, which was very beneficial for the preparation of intravenous administration formulations.

[0058] Take empty nanoparticles (PLGA-PEG), CX4945@PLGA-PEG and CX4945@PLGA-PEG@CM nanodrugs, drop them on copper grids respectively, and observe the morphology through transmission electron microscopy. The results are as Figure 2 shown that all nanoparticles were spherical. There was a clear cell membrane shell layer on the surface of CX4945@PLGA-PEG@CM, which proved the successful synthesis of the bone marrow stromal cell membrane biomimetic CX4945@PLGA-PEG@CM nanodrug with a core-shell structure. Scanning electron microscopy elemental analysis containing Cl, P and S elements also confirmed the successful preparation of CX4945@PLGA-PEG@CM NPs.

[0059] The expression level of CXCL12 in CX4945@PLGA-PEG@CM, CM and MS-5 cells was analyzed by Western blot method. As Figure 3As shown, CXCL12 was expressed on both CX4945@PLGA-PEG@CM NPs, CM, and MS-5 cells, indicating that CXCL12 was retained during the extraction and preparation of CX4945@PLGA-PEG@CM NPs from the bone marrow stromal cell membrane.

[0060] Example 3 Determination of the Storage Stability of the Biomimetic Nanodrug CX4945@PLGA-PEG@CM

[0061] The stability of a drug delivery system plays an important role in the safety and effectiveness of the administration process. CX4945@PLGA-PEG@CM NPs were dispersed in H2O, and sampling time points were set for storage at 4°C. After sampling, the hydrodynamic size and PDI dispersion index were detected by DLS.

[0062] As Figure 4 shown, the hydrodynamic size diameter of CX4945@PLGA-PEG@CM NPs in H2O for 7 days was 115.967 ± 7.333 to 119.800 ± 3.407 nm, and the PDI was 0.111 ± 0.008 to 0.156 ± 0.014, indicating that CX4945@PLGA-PEG@Pt NPs could remain stable under the condition of storage at 4°C for 7 days.

[0063] Example 4 Determination of the Stability of the Biomimetic Nanodrug CX4945@PLGA-PEG@CM in a Physiological Environment

[0064] CX4945@PLGA-PEG@CM NPs were dispersed in RPMI1640 medium (containing 10% FBS). After incubation at 37°C for 0, 6, 12, 24, 48, and 72 h, the hydrodynamic size and PDI dispersion index were detected by DLS.

[0065] As Figure 5 shown, the hydrodynamic size diameter of CX4945@PLGA-PEG@CM NPs in a physiological environment for 72 h was 115.067 ± 9.712 to 122.300 ± 5.212 nm, and the PDI was 0.141 ± 0.015 to 0.169 ± 0.034, indicating that CX4945@PLGA-PEG@CM NPs could remain stable under physiological conditions.

[0066] Example 5 In Vitro Release Curve of CX4945 in the CX4954@PLGA-PEG@CM Nanodrug

[0067] The dialysis method was used to determine the in vitro release behavior of the nano-drug. A total of 1.5 mg of the CX4945 biomimetic nano-drug delivery system CX4954@PLGA-PEG@CM was placed in a 3500 Da dialysis bag, and the dialysis bag was sealed with a clip and immersed in 50 mL of PBS with a pH of 7.4 or 4.5, respectively, and magnetically stirred at 37 °C (200 rpm / min). 1 mL of the dialysis fluid was collected at the specified times (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, 120 h) and replenished with an equal volume of PBS. The content of CX4945 was measured using a UV spectrophotometer and the release curve was plotted.

[0068] As Figure 6 shown, CX4945 was slowly released from the CX4945@PLGA-PEG@CM nano-drug, and the total release amounts of CX4945 reached 80.606% ± 1.569 (pH = 4.5) and 78.10% ± 1.61 (pH = 7.4) after 120 h, respectively. Compared with the neutral pH condition, the drug release rate was slightly increased under acidic conditions due to the degradation of the membrane under weak acidic conditions. In summary, the chemotherapeutic drug can be slowly released from the biomimetic nanoparticles at the extracellular level, enabling effective drug delivery for leukemia treatment.

[0069] Example 6 Blood compatibility of the biomimetic nano-drug CX4945@PLGA-PEG@CM

[0070] Mouse whole blood was centrifuged at 400 g for 5 min and washed five times with PBS solution to obtain pure red blood cells. Sample addition and grouping in 1 mL centrifuge tubes: (1) Positive control: 800 μL of H2O + 200 μL of red blood cells; (2) Negative control: 800 μL of PBS + 200 μL of red blood cells; (3) Experimental group: 800 μL of materials with different concentrations + 200 μL of red blood cells; Incubate at 37 °C for 3 h, after completion, centrifuge at 800 g for 5 min, take 100 μL of the supernatant to a 96-well plate, and measure the absorbance at 540 nm.

[0071] The safety of the nano-drug determines its wide clinical application. As Figure 7 shown, no obvious hemolysis of red blood cells was observed after the nano-drug (CX4945 concentrations of 5, 25, 50, 100 μg / mL) acted for 3 h, indicating that the nano-drug has good biocompatibility.

[0072] Example 7 Determination of the killing effect of the biomimetic nano-drug CX4945@PLGA-PEG@CM NPs on NALM6 cells

[0073] The bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system is effective against tumor cells expressing CXCR4 on the cell surface, and has a poor killing effect on tumor cells that do not express it. The main reason is that the CXCL12 expressed on the stromal cell membrane can specifically bind to CXCR4 on the tumor cell surface, which helps the biomimetic membrane target leukemia cells and enhances the uptake of biomimetic nano-drugs. In addition, it can also prevent tumor cell homing in vivo. In this example, the expression of CXCR4 on the surface of NALM6 tumor cells was detected, and the killing effect of the CX4945@PLGA-PEG@CM nano-drug is directly related to the expression level of CXCR4 on the tumor cell surface.

[0074] 1. Determination of CXCR4 expression level in NALM6 cells

[0075] Collect NALM6 cells in the exponential growth phase by centrifugation, wash them with Staining Buffer, centrifuge and resuspend for counting. Divide the cells into two tubes and dilute them to 1×10 6 cells in 100 μL. Then add the CD107 (BD Biosciences, 555802) antibody to both tubes and incubate for 10 min to block non-specific binding. Next, add the CD184 antibody (BD Biosciences, 557145) to one of the tubes and incubate for 30 min. Finally, centrifuge, wash, resuspend and detect with a flow cytometer.

[0076] As Figure 8 shown, after staining with the CXCR4 antibody, the CXCR4 expression in NALM6 cells was 90% higher than that in the unstained control group. The data indicate that the overexpression of CXCR4 on the surface of NALM6 cells is beneficial to the specific targeting binding of biomimetic nano-drugs to cells and enhances the uptake of drugs by cells.

[0077] 2. Detect the effect of nano-drugs on the activity of acute leukemia cells using the CCK8 kit. After culturing NALM6 cells at a density of 3×10 4 cells / well with different concentrations of drugs (the final concentrations of CX4945 in different types of nano-drugs: 0, 2, 4, 6, 8, 10 μM) for 24 h or 48 h, add 10 μL of CCK8 detection solution and continue to incubate for 4 h. After incubation, measure the absorbance at 450 nm (the experiment was repeated 3 times, with 3 replicates each time).

[0078] After the drugs act for 24 h or 48 h ( Figure 9 ), the killing ability of CX4945 coated with nano-particles on NALM6 cells was significantly enhanced, and the killing intensity of cells showed a trend of CX4945@PLGA-PEG@CM > CX4945@PLGA-PEG > free CX4945 drug.

[0079] Therapeutic Effect of CX4945@PLGA-PEG@CM NPs Nanodrug on B-ALL Mouse Model

[0080] Each mouse requires 1×10 5 NALM6 cells, and the volume of the injected cell suspension is 200 μL, that is, the concentration of the cell suspension to be prepared is 5×10 5 cells / mL. After successful mouse modeling, they were randomly divided into four groups (n = 7). Intravenous injection of Na2HPO4, CX4945, CX4945@PLGA-PEG, and CX4945@PLGA-PEG@CM were performed via the tail vein, and the equivalent concentration of CX4945 in the injection dose was 20 mg / kg. Drug treatment was carried out every two days. After 27 days of treatment, waiting for the mice to develop the disease, the spleens and bone marrows of the mice were collected, and the peripheral blood of the mice was collected regularly. Finally, the therapeutic effect of the nanodrug was evaluated by flow cytometry to determine the proportion of leukemia cells.

[0081] After treatment with the drug (the equivalent concentration of CX4945 was 20 mg / kg), the size and weight of the spleen were as Figure 10 shown, showing a trend of CX4945@PLGA-PEG@CM < CX4945@PLGA-PEG < free CX4945 drug < control group. The size (0.093 ± 0.026 g, P<0.001) and weight of the spleens of the mice in the CX4945@PLGA-PEG@CM treatment group were significantly smaller than those of other drug treatment groups (CX4945@PLGA-PEG group: 0.246 ± 0.042, free CX4945 group: 0.360 ± 0.019, control group: 0.426 ± 0.034 g, P<0.001). After treatment, the proportion of leukemia cells in the bone marrow and spleen was detected by flow cytometry ( Figure 11)Showed a trend of CX4945@PLGA-PEG@CM < CX4945@PLGA-PEG < free CX4945 drug < control group. Compared with mice in other groups, the proportion of human CD19 positive cells in the spleen (CX4945@PLGA-PEG@CM group: 14.400 ± 2.322%, CX4945@PLGA-PEG group: 33.686 ± 2.961%, free CX4945 group: 53.257 ± 1.740%, control group: 70.414 ± 3.484%, P<0.001) and bone marrow (CX4945@PLGA-PEG@CM group: 23.300 ± 2.821%, CX4945@PLGA-PEG group: 47.414 ± 2.719%, free CX4945 group: 61.600 ± 2.251%, control group: 76.057 ± 2.599%) of the CX4945@PLGA-PEG@CM treatment group was significantly reduced. In addition, no significant changes were found in the detection of mouse blood biochemical indexes, and their fluctuations were also within the normal range, without obvious toxic and side effects (Table 2).

[0082] Table 2 Toxicity study after treating leukemia mouse models with different drugs (n = 3)

[0083]

[0084] Example 9

[0085] In this example, the expression of CXCR4 on the surface of THP-1 and JURKAT tumor cells was detected, and the killing effect of the CX4945@PLGA-PEG@CM nanodrug was directly related to the expression level of CXCR4 on the surface of tumor cells.

[0086] According to the method of Example 7, the CXCR4 expression levels of THP-1 and JURKAT cells were measured, and the viability of THP-1 and JURKAT cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG and CX4945@PLGA-PEG@CM for 48 h was detected.

[0087] 1. Measurement of CXCR4 expression levels of THP-1 and JURKAT cells:

[0088] The results were as Figure 12 and Figure 13 shown. After staining with CXCR4 antibody, THP-1 cells had 97.9% higher CXCR4 expression than the unstained control group. JURKAT cells had 90.0% higher CXCR4 expression than the unstained control group. Both cell lines were determined to have high CXCR4 expression by experiments.

[0089] 2. Detection of the viability of THP-1 and JURKAT cells after incubation with different concentrations of CX4945, CX4945@PLGA-PEG, and CX4945@PLGA-PEG@CM for 48 h:

[0090] The results are as Figure 14 and Figure 15 shown. After 48 h of drug treatment, CX4945 coated with biomimetic nanoparticles significantly enhanced the killing of THP-1 and JURKAT cells. In addition, the killing intensity of the biomimetic drug-loaded CX4945 delivery system against leukemia cells also showed the same trend as that of NALM6 cells, with the trend of CX4945@PLGA-PEG@CM > CX4945@PLGA-PEG > free CX4945 drug.

[0091] Example 10: Determination of the killing effect of stromal cell membrane on NALM6 cells

[0092] In this example, the cell viability of NALM6 cells after 48 h of treatment with stromal cell membrane was detected using the same method as in Example 7. The results are as Figure 16 shown. The stromal cell membrane did not show obvious killing effect on leukemia cells, indicating that the stromal cell membrane has good biosafety and is suitable for drug delivery systems.

Claims

1. A bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system, characterized in that: It is composed of CX4945 drug coated with polylactic acid-co-glycolic acid-polyethylene glycol as the core and bone marrow stromal cell membrane as the shell.

2. According to claim 1, the bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system is characterized in that: The particle size of the bone marrow stromal cell membrane bionic drug-loaded CX4945 nano-delivery system is 48-120 nm.

3. A method for preparing the bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system according to claim 1 or 2, characterized in that: The following steps are involved: Polylactic acid-co-glycolic acid-polyethylene glycol and CX4945 drug are dissolved in tetrahydrofuran, added into polyvinyl pyrrolidone aqueous solution, ice-bathed and ultrasonically broken, stirred and centrifuged, mixed with bone marrow stromal cell membrane, ultrasonically treated in ice-bath, and centrifuged to remove excess cell membrane.

4. The method according to claim 3, characterized in that: The bone marrow stromal cell membrane has CXCL12 chemokine activity.

5. The method according to claim 3, characterized in that: The bone marrow stromal cell membrane is prepared by a hypotonic solution lysis method combined with liquid nitrogen repeated freezing and thawing.

6. The method according to claim 5, characterized in that: The method of combining the hypotonic solution lysis method with liquid nitrogen repeated freezing and thawing is as follows: resuspending the bone marrow stromal cells in a hypotonic solution to swell and break the cells, and then repeatedly freezing, thawing, and centrifuging to obtain the bone marrow stromal cell membrane.

7. The method according to claim 6, characterized in that: The liquid nitrogen freezing time is 8 to 15 seconds, and the number of repeated freezing and thawing operations is 7 to 12 times.

8. The method according to claim 6, characterized in that: Centrifugation includes low-speed centrifugation and high-speed centrifugation; the rate of the low-speed centrifugation is 1500-2500 rpm, and the time is 8-12 min; the rate of the high-speed centrifugation is 11000-15000 rpm, and the time is 8-12 min.

9. Use of the bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano-delivery system according to claim 1 or 2 in the preparation of drugs for treating acute leukemia.

10. A drug for treating acute leukemia, characterized in that: It contains the bone marrow stromal cell membrane biomimetic drug-loaded CX4945 nano delivery system as described in claim 1 or 2.

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