A FLT3 and BCL-2 dual inhibitor micelle and its preparation method and application

Abstract

The present invention belongs to biomedical technology, and specifically relates to a FLT3 and BCL-2 dual inhibitor micelle and its preparation method and application. b ‑P(A‑ What ‑DTC), peptide amphiphilic polymers T‑PEG‑ b ‑P(A‑ What ‑DTC), FLT3 inhibitor and BCL‑2 inhibitor are mixed in a solution to obtain the FLT3 and BCL‑2 dual inhibitor micelles. The present invention designs and prepares two multifunctional biodegradable micelles to efficiently load and deliver two small molecule targeted drug combinations to AML cells at an optimal synergistic ratio. The nanocarriers with excellent performance have the advantages of stable drug loading, long circulation, and targeting tumor cells. It is expected that the drug combination at the optimal synergistic ratio and dosage can be specifically targeted to tumor cells, greatly reducing the toxic and side effects of the drugs, and efficiently achieving drug efficacy, so as to improve the therapeutic effect on AML.

Description

Technical Field

[0001] The present invention belongs to biomedical technology, and specifically relates to a FLT3 and BCL-2 dual inhibitor micelle and a preparation method and application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is the most common blood tumor, characterized by abnormal proliferation and blocked differentiation of myeloid progenitor cells. In order to improve the prognosis of AML patients, various small molecule targeted drugs have been developed and applied to the clinical treatment of AML, targeting the widespread genetic abnormalities and AML-promoting survival signaling pathways in AML patients, and have achieved certain therapeutic effects. The clinical application of a single small molecule inhibitor often shows significant efficacy in the early stage of medication, but after a period of time, it falls into the dilemma of poor efficacy, and then drug resistance and relapse. Studies have shown that multiple inhibitors are often required to be used in combination in the treatment of AML to block multiple pathways at the same time. The prior art discloses VEN and sorafenib (SOR) targeted dual anti-apoptotic protein polymer micelles for active targeted therapy of in situ acute myeloid leukemia mouse models, but the advantage of sorafenib drugs is not in AML, and AML indications have not been approved, which limits the practical application significance of the system. The FLT3 inhibitor midostaurin (MID) and the BCL-2 inhibitor Venetoclax (VEN) are two AML targeted drugs that have been approved for marketing. However, clinical trials have also shown that drug combinations based on simple superposition of doses bring about common and varying degrees of toxic side effects, especially severe suppression of the bone marrow, which leads to interruptions in continuous dosing and reduced efficacy. In addition, due to the independent and different physical and chemical properties of different drugs, traditional preparations cannot allow the active ingredients of the drugs to reach AML cells at the same time, or cannot reach effective concentrations in the tumor site, so that the possible pathways for tumor escape cannot be effectively blocked at the same time, reducing or losing efficacy. Summary of the invention

[0003] Since AML has extremely strong heterogeneity, single-drug treatment is usually ineffective, and drug resistance and relapse are common. Therefore, two or more drugs are often needed in the treatment of AML. However, when multiple drugs are used in combination, due to the different pharmacokinetics of the drugs, the drug concentration and ratio reaching the target are uncontrollable, and the effect of synergistic treatment cannot be achieved. In addition, multi-drug combination is also faced with the problem of dose superposition, excessive toxic and side effects, and the need to interrupt administration in clinical treatment. Therefore, the present invention designs and prepares two multifunctional biodegradable micelles, and two small molecule targeted drug combinations are efficiently loaded and delivered to AML cells according to the optimal synergistic ratio. The nanocarrier with excellent performance has the advantages of stable drug loading, long circulation, and targeting tumor cells. It is expected that the drug combination at the optimal synergistic ratio and dosage will be specifically targeted to tumor cells, greatly reducing the toxic and side effects of the drugs, and efficiently achieving drug efficacy, in order to improve the therapeutic effect on AML.

[0004] The present invention adopts the following technical solution:

[0005] A FLT3 and BCL-2 dual inhibitor micelle comprises a polymer micelle and a FLT3 inhibitor and a BCL-2 inhibitor loaded thereon; the mass ratio of the FLT3 inhibitor to the BCL-2 inhibitor is 1: (0.1-1). Preferably, the mass ratio of the FLT3 inhibitor to the BCL-2 inhibitor is 1: (0.2-0.75).

[0006] Preferably, the present invention uses polymer micelles and FLT3 inhibitors and BCL-2 inhibitors loaded therein to form FLT3 and BCL-2 dual inhibitor micelles.

[0007] In the present invention, the polymer micelles are composed of amphiphilic polymer PEG- b -P(A- co -DTC) and polypeptide amphiphilic polymer T-PEG- b -P(A- co -DTC) are co-assembled to form, A is an ester or carbonate monomer unit, preferably a cyclic ester or cyclic carbonate monomer unit, such as one of a trimethylene carbonate monomer, a lactide monomer, and a caprolactone monomer, as common sense, the cyclic ester or cyclic carbonate monomer is ring-opened to form a cyclic ester or cyclic carbonate monomer unit; T is a polypeptide, including T22 polypeptide; FLT3 inhibitors include midostaurin, gilteritinib, quizartinib, lestaurine, and crellalanib, and BCL-2 inhibitors include veteclaz; DTC is dithiolane trimethylene carbonate; all are existing products.

[0008] In the present invention, T-PEG- b -P(A- co -DTC) is PEG- b -P(A- co -DTC) and T-PEG-b -P(A- co -DTC) of the total molar amount, preferably 10-30%.

[0009] In the present invention, PEG- b -P(A- co -DTC) and T-PEG- b -P(A- co -DTC), the number average molecular weight of the PEG segment is independently selected from 2000 to 12000 Da, preferably 3000 to 8000 Da; P(A- co The number average molecular weight of the PDTC segment is 2 to 10 times, preferably 2 to 6 times, that of the PEG segment; the number average molecular weight of the PDTC segment is 2 to 10 times, preferably 2 to 6 times that of the PEG segment; co -DTC) segment total number average molecular weight of 5% to 40%, preferably 10 to 30%, PDTC segment refers to the segment composed of DTC units, P(A- co -DTC) segment is a hydrophobic segment, which means a segment formed by random copolymerization of A and DTC.

[0010] The present invention discloses a method for preparing the above-mentioned FLT3 and BCL-2 dual inhibitor micelles, comprising the following steps: b -P(A- co -DTC), polypeptide amphiphilic polymer T-PEG- b -P(A- co -DTC), FLT3 inhibitor and BCL-2 inhibitor are mixed in a solution to obtain the FLT3 and BCL-2 dual inhibitor micelles. Specifically, the amphiphilic polymer PEG- b -P(A- co -DTC), polypeptide amphiphilic polymer T-PEG- b -P(A- co -DTC) solution is added to the mixture of FLT3 inhibitor, BCL-2 inhibitor and buffer to obtain the above-mentioned FLT3 and BCL-2 dual inhibitor micelles; preferably, after mixing, conventional stirring and dialysis are performed to obtain the above-mentioned FLT3 and BCL-2 dual inhibitor micelles. The amphiphilic block polymer forms a reduction-sensitive disulfide cross-linked micelle for loading the FLT3 and BCL-2 dual inhibitor.

[0011] The present invention discloses a combined medicine, comprising the above-mentioned FLT3 and BCL-2 dual inhibitor micelles and other anti-tumor drugs.

[0012] The present invention discloses the use of the above-mentioned FLT3 and BCL-2 dual inhibitor micelles or combined drugs in the preparation of anti-tumor drugs. Preferably, the anti-tumor drug is a drug for preventing and treating leukemia. Further preferably, the drug for preventing and treating leukemia is a drug for preventing and treating acute myeloid leukemia.

[0013] The present invention is based on the amphiphilic copolymer polyethylene glycol- b -Poly(dithiolane trimethylene carbonate)- co -ε-caprolactone)(PEG- b -P(DTC- co -CL)) and T22 polypeptide modified T22-PEG- b -P(DTC- co -CL) polymer was designed and prepared to prepare an active targeted disulfide cross-linked micelle T22-Ms-M / V that can be loaded with FLT3 inhibitor MID and BCL-2 inhibitor VEN at the same time for the treatment of MV4-11 AML mouse xenograft orthotopic model. T22-Ms-M / V has the advantages of high efficiency, stability, controllable drug loading, adjustable surface peptide density, rapid drug release under reduction response, and targeting CXCR4 receptor. The optimal synergistic ratio of drugs was determined by CCK-8 cytotoxicity experiment, apoptosis experiment and animal pre-experiment. T22-Ms was loaded with drugs in this ratio to achieve targeted delivery and controlled release. T22-Ms-M / V entered the mouse blood circulation through tail vein injection, enriched on the surface of AML cells with high expression of CXCR4 in mice, and entered the cells through CXCR4-mediated endocytosis, releasing the proportionally loaded dual drugs in a reducing environment, activating downstream apoptosis pathways, and promoting AML cell apoptosis. Studies have shown that T22-Ms-M / V can target MV4-11 cells in AML mouse xenograft models, inhibit tumor growth and significantly prolong mouse survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For (A) PEG- b -P(CL- co -DTC) and (B) T22-PEG- b -P(CL- co -DTC) synthesis route.

[0015] Figure 2 For (A) PEG- b -P(CL- co -DTC), (B)NHS-PEG- b -P(CL- co -DTC) and (C)T22-PEG- b -P(CL- co-DTC) H NMR spectrum (600 MHz, DMSO- d 6 ). Illustration: H NMR spectrum of T22 peptide (600 MHz, DMSO- d 6 ).

[0016] Figure 3 For PEG- b -P(CL- co -DTC), NHS-PEG- b -P(CL- co -DTC) and T22-PEG- b -P(CL- co -DTC) FTIR analysis spectrum.

[0017] Figure 4 For DTC and PEG- b -P(CL- co -DTC) in DMF.

[0018] Figure 5 Characterization of drug-loaded micelles, including: (A) Particle size and size distribution of single-drug and dual-drug micelles determined by DLS (Inset: TEM image of 20%T22-Ms-M / V, scale bar is 50 nm). Particle size changes of 20%T22-Ms-M / V (B) stored at 4 °C for 0 and 15 days, (C) diluted 10 and 100 times in PBS (0.01 M), (D) stored in 10% FBS for 0, 4 and 24 h, and (E) in 10 mM GSH for 0 h, 24 h and 48 h. (F) UV spectra of Ms before and after dialysis.

[0019] Figure 6 (A) Flow cytometry analysis of the effect of T22 peptide density on the internalization of T22-PMs-Cy5 by MV4-11 cells. The sample incubation time was 4 h. (B) CLSM images of MV4-11 cells after incubation with Ms-Cy5 and T22-Ms-Cy5 for 4 h. Scale bar: 25 μm.

[0020] Figure 7 (A) Cytotoxicity of Ms-V and Ms-M on MV4-11 cells (n = 5). (B) The synergistic effect of MID and VEN in MV4-11 cells was evaluated by fitting the CI value calculated by the multidrug combination effect calculation software (CompuSyn) (Inset: Ms-M / V 1 / 0.3 and Ms-M / V 1 / 12 The CI value is between 0 and 1). (C) Free M / V 1 / 0.3 , Ms-M / V 1 / 0.3and T22-Ms-M / V 1 / 0.3 Cytotoxicity against MV4-11 cells (n = 5). (D) T22-Ms-M / V 1:0.3 Toxicity in HUVSMC and L-929 cells (n = 5). Co-incubation time was 48 h.

[0021] Figure 8 The cytotoxicity of Ms (A) and T22-Ms (B) on MV4-11 cells and MOLM-13 cells was determined by CCK-8 (n = 5). The cytotoxicity of Ms (C) and T22-Ms (D) on L-929 cells and HUVSMC cells was determined by CCK-8 (n = 5). Cells were incubated with the preparations for 48 h.

[0022] Fig. 9 Determination of the pro-apoptotic activity and CI values ​​of Ms-M, Ms-V and Ms-M / V with different drug contents in MV4-11 cells, including: (A) Early apoptosis, late apoptosis and total apoptosis rates after 24 h of co-incubation with different preparations. (B) Representative scatter plot of apoptosis of MV4-11 cells after 24 h of co-incubation with different preparations. (C) The synergistic effect of MID and VEN was evaluated by fitting and calculating the CI value using the multi-drug combination effect calculation software (CompuSyn). The affected fraction (Fraction affected) refers to the total apoptosis rate.

[0023] Fig.10 Preliminary experiment of Ms-M / V treatment in AML xenograft mouse model, including: (A) Schematic diagram of the construction, treatment and monitoring of MV4-11 AML xenograft mouse model. (B) Kaplan-Meier survival curves of PBS, Ms-M / V 10 / 3 mg / kg, Ms-M / V 10 / 7.5 mg / kg, Ms-M / V 10 / 15 mg / kg and Ms-M / V 15 / 11.25 mg / kg (* p < 0.05, ** p < 0.01, n = 3). (C) Changes in mouse body weight during administration (n = 3). (D) Peripheral blood AML cell infiltration ratios of different treatment groups on day 19.

[0024] Fig.11The treatment of AML xenograft mouse model, including: (A) Schematic diagram of the construction, treatment and monitoring of MV4-11 AML xenograft mouse model. (B) Changes in mouse body weight during administration (n = 6). (C) Kaplan-Meier survival curves of mice in the PBS, Ms-V (6 mg / kg), Ms-M (20 mg / kg), free-M / V (20 / 6 mg / kg), Ms-M / V (20 / 6 mg / kg) and T22-Ms-M / V (20 / 6mg / kg) groups (* p < 0.05, ** p < 0.01,*** p < 0.001, **** p <0.0001, n = 6).

[0025] Fig.12 Representative flow cytometry dot plots of MV4-11 cell infiltration in the bone marrow (BM), liver (Liver), lung (Lung), peripheral blood (PB) and spleen (Spleen) of mice in different treatment groups (the rectangular gate indicates the CD45-positive AML cell population) (A) and statistical analysis (n = 3) (B).

[0026] Fig.13 H&E staining images of the main organs of different treatment groups and healthy mice. Scale bar: 100 μm.

[0027] Fig.14 Femur and tibia of AML mice in different treatment groups were analyzed, including: (A) H&E staining images for histopathological analysis. Scale bar: 100 μm. (B) TRAP staining for osteoclast analysis. Scale bar: 100 μm. (C) Micro-CT images. (D) Quantitative analysis of Tb.N, Tb.Th and Tb.Sp (n = 3, Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular separation).

[0028] Fig.15 MID and VEN dual-loaded polymer micelles (T22-Ms-M / V) modified with T22 peptide on the surface were used for efficient treatment of MV4-11 AML mouse xenograft model. DETAILED DESCRIPTION

[0029] Polyethylene glycol monomethyl ether (MeO-PEG-OH, M n = 5.0 kg / mol, M w / M n= 1.03), α-N-hydroxysuccinimide-ω-hydroxypolyethylene glycol (NHS-PEG-OH, M n = 7.5 kDa, M w / M n = 1.06) was purchased and used directly, and T22 cyclic peptide (sequence: Lys(Azido)-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Arg-NH2 (disulfide bonds: Cys5-Cys18, Cys9-Cys14), >85%, Gill Biochemical Co., Ltd.) was purchased and used directly. b -P(CL- co -DTC)-Cy5 (molecular weight 5.0-(4.0-2.0) kDa) and 1,2-dithiolane trimethylene carbonate (DTC) are existing products, which can be found in the inventor's previous literature. The material weighing of the polymerization reaction was carried out in a glove box equipped with a gas purification system, and the working gas was 99.99% high-purity nitrogen.

[0030] Midostaurin (MID, >99%), venetoclax (VEN, >99%), glutathione (GSH, >99%), 1,4-dithio-D,L-threitol (DTT, >99%), 4,6-diamidino-2-phenylindole (DAPI), paraformaldehyde-glutaraldehyde fixative, poly-lysine (PDL), APC anti-human CD45 antibody, APC anti-human CXCR4 antibody, CCK-8 kit, apoptosis kit (annexin V-APC / 7-AAD), BCA protein detection kit, dialysis bag (MWCO 7 kDa), etc. were all used directly after purchase.

[0031] Nuclear magnetic resonance (NMR) testing uses CDCl3 or DMSO- d 6 as solvent, DD2-600 liquid superconducting NMR spectrometer measured 1 H NMR spectrum, chemical shift (ppm) of sample peaks referenced to residual solvent peak (DMSO: 1 H NMR: δ 2.50) or internal standard tetramethylsilane (TMS: 1H NMR: δ 0.00). The molecular weight distribution of the copolymer was tested by gel permeation chromatography (GPC) with DMF as the eluent, a flow rate of 1.0 mL / min, and a column temperature of 30°C. A series of narrow molecular weight distribution polymethyl methacrylate (PMMA) was used as the standard sample to calibrate the chromatographic column. The infrared absorption spectrum of the polymer was measured by Fourier transform infrared spectrometer. The micelle size was measured by dynamic laser light scattering at 25°C, using a He / Ne laser light source with a wavelength of 633 nm and a backscattering detector. The Zeta potential of the micelle was measured by a dynamic laser light scattering instrument equipped with a standard capillary electrophoresis instrument. The absorbance change of dithiolane in the micelle was tested by an ultraviolet spectrophotometer (UV-2600). The morphological characteristics of the drug-loaded micelles were measured by HT7700 transmission electron microscopy (TEM) at an accelerating voltage of 120 KV. The samples were stained with 1% phosphotungstic acid solution for 0.5 min before testing. The CCK-8 experiment used a multifunctional microplate reader to test the absorbance of the samples at a wavelength of 450 nm. The receptor expression, cell apoptosis, micelle endocytosis behavior, and tumor cell infiltration in animal models were measured by flow cytometry and analyzed by Flowjo software. The quantification of MID and VEN was tested by high-performance liquid chromatography, with the mobile phase being H3PO4 (pH2.4, 9.5 mM) / ACN (v / v = 35 / 65), using a BR-C18 column (4.6×250 mm, 5 μm, 120 Å), the column temperature being 30 ℃, the flow rate being 1.0 mL / min, the detection wavelength being 289 nm, and the injection volume being 20 μL. Confocal laser scanning microscopy (CLSM) images were taken using a TCS SP5 confocal system. Hematoxylin-eosin (H&E) staining images were taken using an inverted fluorescence microscope. The bone structure and tissue morphology images of the femur and tibia of the mouse hind legs were obtained by scanning with a micro-computed tomography scanner.

[0032] All data in this article are mean ± standard deviation (SD). The statistical differences between the data of more than three groups were evaluated by one-way analysis of variance (ANOVA). When the results were significant (p<0.05), multiple comparison tests were used for comparison between groups. *p<0.05 indicates a significant difference, **p<0.01 and ***p<0.001 indicate a highly significant difference.

[0033] The following is an explanation of the technical progress of the present invention through specific experiments, wherein the specific experimental operations and performance tests are conventional techniques.

[0034] Example 1 Synthesis and Characterization of Polymers

[0035] Polymer PEG- b -P(CL- co-DTC) was synthesized using DPP as catalyst and MeO-PEG-OH ( M n = 5.0 kDa) as a macromolecular initiator to initiate the ring-opening polymerization of DTC and ε-CL, and the theoretical molecular weight of the obtained polymer is 5.0-(4.0-2.0) kDa. First, in the nitrogen environment of the glove box, MeO-PEG-OH (500 mg, 0.1 mmol), ε-caprolactone (ε-CL) (400 mg, 3.5 mmol) and DTC (200 mg, 1.0 mmol) were weighed and placed in a reaction bottle, dissolved with anhydrous DCM (4.56 mL), and then DPP (125 mg, 0.5 mmol) was added and mixed evenly. After that, the closed reactor was sealed and removed from the glove box, and reacted at a constant temperature of 37°C and stirring at 400 rpm for 72 h; after the reaction, 30 volumes of ice ether were used for precipitation twice, and the product was filtered and vacuum dried to constant weight to obtain a light yellow solid product with a mass of 941.6 mg and a yield of 85.6%.

[0036] Polymer T22-PEG- b -P(CL- co The synthesis of NHS-PEG- b -P(CL- co -DTC). Specifically, DPP is used as the catalyst, NHS-PEG-OH ( M n = 7.5 kDa) as a macromolecular initiator to initiate the ring-opening polymerization of DTC and ε-CL, and the post-reaction treatment was similar to that of PEG- b -P(CL- co -DTC) with a yield of 92.2% and a theoretical molecular weight of 7.5-(4.0-2.0) kDa. The second step is to combine the NH2 on the T22 polypeptide with the polymer NHS-PEG- b -P(CL- co -DTC) was subjected to amidation reaction to obtain polymer T22-PEG- b -P(CL- co -DTC). The specific operation steps are as follows: T22 peptide (396 mg, 0.15 mmol) and NHS-PEG- b -P(CL- co-DTC) (1000 mg, 0.09 mmol) was dissolved in 3 mL of anhydrous DMF, and then reacted at 40°C and 300 rpm for 48 h. After the reaction, it was dialyzed in 30 volumes of DMSO for 12 h (MWCO: 7.0 kDa), and then dialyzed in 30 volumes of DCM for 12 h (MWCO: 7.0 kDa), with the dialyzate replaced every 3 h. Finally, it was precipitated with 30 volumes of ice ether for 3 times, filtered and dried in vacuum to constant weight.

[0037] Polymer PEG- b -P(CL- co -DTC) synthesis route such as Figure 1 Medium A, theoretical molecular weight is 5.0-(4.0-2.0) kDa. 1 There are characteristic peaks of each part in the H NMR spectrum: PEG ( δ 3.51, 3.24), ε-CL ( δ 1.29, 1.52, 2.26, 4.00-4.14) and DTC ( δ 3.04, 4.04-4.14)( Figure 2 By comparing CL ( δ 1.29) and DTC ( δ 3.04) and the methylene group in PEG ( δ 3.51) integrated area, the degree of polymerization (DP) of CL and DTC was determined to be 31.6 and 9.3 respectively, and the number average molecular weight of PEG-P(CL-DTC) was obtained to be 5.0-(3.6-1.8) kg / mol. The GPC test results showed that the molecular weight distribution of the polymer was narrow ( M w / M n = 1.1), indicating that the ring-opening copolymerization reaction has good controllability and the polymer was successfully synthesized with a yield of 85.6%.

[0038] T22-PEG- b -P(CL- co -DTC) synthesis route such as Figure 1 Middle B). NHS-PEG- b -P(CL- co -DTC) 1 There are characteristic peaks of each part in the HNMR spectrum: NHS ( δ 2.83), PEG ( δ 3.51, 3.24), ε-CL ( δ 1.29, 1.52, 2.26, 4.00-4.14) and DTC ( δ3.04, 4.04-4.14)( Figure 2 By comparing CL ( δ 1.29) and DTC ( δ 3.04) and the methylene group in PEG ( δ 3.51), the degree of polymerization (DP) of CL and DTC were determined to be 31.6 and 11.8, respectively, and NHS-PEG- b -P(CL- co -DTC) is 7.5-(4.0-2.3) kg / mol, which is close to the theoretical molecular weight. In addition, the molecular weight distribution of the polymer measured by GPC is relatively narrow ( M w / M n = 1.1). The above shows that NHS-PEG- b -P(CL- co -DTC), yield 92.2%. T22-PEG- b -P(CL- co -DTC) NMR ( Figure 2 Middle C) shows the T22 characteristic peak (δ 6.61-7.70) and the NHS characteristic peak ( δ 2.83) disappears, Fourier infrared spectrum ( Figure 3 ) showed that T22-PEG- b -P(CL- co -DTC) at 1735 cm -1 The absorption peak of peptide bond (-CO-NH-) appeared at , indicating that T22 peptide was attached to the polymer through amidation reaction. BCA test showed that the functionality of T22 peptide was 60%. DTC and PEG- b -P(CL- co -DTC) solution and tested its absorbance at 330 nm. The results showed that the absorbance of the two at 330 nm was similar ( Figure 4 ), indicating that the polymer did not undergo cross-linking during the dialysis process.

[0039] Example 2 Preparation and Characterization of Blank and Drug-Loaded Micelle

[0040] Blank micelles were prepared using the solvent replacement method. First, PEG- b -P(DTC- co -CL) and T22-PEG- b -P(DTC- co -CL) was prepared into a 10 mg / mL solution and mixed according to the calculated ratio. 100 μL PEG- b-P(DTC- co -CL) and T22-PEG- b -P(DTC- co -CL) was added to 900 μL of room temperature phosphate buffer (PB, 10 mM, pH 7.4) under stirring at 200 rpm. After the addition, stirring was continued at 200 rpm for three minutes to form evenly dispersed micelles. Then, the micelles were dialyzed for 6 h (MWCO: 7.0 kDa) with PB (10 mM, pH 7.4) as the dialysate, and the dialysate was changed every hour to obtain blank micelles Ms (using only PEG- b -P(DTC- co -CL)), blank micelles T22-Ms modified with T22 polypeptide.

[0041] The preparation methods of single-drug micelles (Ms-M and Ms-V), co-loaded micelles (Ms-M / V) and targeted co-loaded micelles (T22-Ms-M / V) are similar to those of empty micelles, except that in the first step, 10 mg / mL of MID solution, VEN solution or MID and VEN mixed solution is mixed with the above polymer solution according to the calculated ratio and then added to the buffer solution. The rest of the operations are the same as those for the preparation of blank micelles. The particle size of the nanoparticles was tested by DLS, the drug concentration of VEN and MID was determined by HPLC, and the drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated from the data measured by HPLC.

[0042] DLC ( wt .%) = × 100

[0043] DLE(%) = × 100

[0044] The preparation of Cy5-labeled micelles Ms-Cy5 and T22-Ms-Cy5 was similar to that of empty micelles, except that 98 μL T22-PEG- b -P(CL- co -DTC) and PEG- b -P(CL- co -DTC) mixed solution (10 mg / mL) and 2 μL PEG- b -P(CL- co -DTC)-Cy5 (10 mg / mL) solution and mix well.

[0045] Empty micelles (Ms), empty targeted micelles (T22-Ms), single-drug micelles loaded with MID (Ms-M), single-drug micelles loaded with VEN (Ms-V), dual-drug micelles loaded with MID and VEN (Ms-M / V), and dual-drug targeted micelles (T22-Ms-M / V) were all prepared by solvent replacement. b -P(CL- co -DTC) was dissolved in an organic solvent and added dropwise to PB for self-assembly. The preparation of targeted drug-loaded micelles also includes T22-PEG- b -P(CL- co -DTC) polymer. Empty micelles are micelles that do not contain any drug. b -P(CL- co -DTC) and T22-PEG- b -P(CL- co -DTC) molar ratio, a series of micelles T22-Ms with different peptide targeting densities were prepared (Table 1). b -P(CL- co -DTC) in the polymer mixture was named 10%T22-Ms, 20%T22-Ms, 30%T22-Ms and 40%T22-Ms. DLS results showed that the particle sizes of Ms and T22-Ms were small and the particle size distribution was narrow. With the increase of T22 polypeptide density on the micelle surface, the surface potential gradually increased.

[0046] The concentration of a single drug that can be loaded into micelles was tested by HPLC. According to the theoretical amount of drug added, it can be calculated that when the theoretical drug loading is 5, 10 and 20 wt .%, the drug loading efficiency (DLE) of MID was between 73.3 and 78.3%, the particle size was between 39.1 and 41.2 nm, and the PDI was between 0.05 and 0.07 (Table 2, Figure 5 At the same theoretical drug loading, the DLE of VEN was significantly higher than that of MID, ranging from 91.4 to 97.1%, the particle size was between 40.3 and 46.6 nm, and the PDI was 0.04 to 0.09 (Table 2, Figure 5 Middle A).

[0047] There is no literature report on the co-loading of FLT3 inhibitors and BCL-2 inhibitors into carriers. In order to achieve the fixed ratio delivery of MID and VEN, the present invention uses polymer micelles to load MID and VEN at the same time, and prepares dual-drug micelles Ms-M / V containing different mass ratios of MID and VEN. During the preparation, the sum of the masses of the two drugs is fixed to 15% of the polymer mass. At the same time, in order to explore the effect of micelle surface modification of T22 polypeptide on dual-drug loading, a series of 20% T22-Ms-M / V (Table 3) with the sum of the masses of the two drugs being 15% of the polymer mass was prepared. The results show that when the ratio of the two drugs is changed during the preparation of dual-drug micelles, whether it is Ms-M / V or 20% T22-Ms-M / V, the particle size is between 44.0 and 48.6 nm, and the particle size distribution is narrow ( Figure 5 In the untargeted dual-drug micelle Ms-M / V, the DLE of MID was 69.1%-84.5%, and the DLE of VEN was 69.5%-76.7% (Table 3). wt .%, the DLE of MID can be increased to 91.1%, and the high DLE when two drugs are co-loaded makes the preparation of dual-drug micelles with different ratios controllable. By changing the ratio of the two drugs during micelle preparation, dual-drug micelles with any desired ratio can be obtained. In summary, T22-Ms and Ms can simultaneously load MID and VEN with adjustable ratios, and the DLE of the two drugs is about 70% to 90%.

[0048] To test the stability of T22-Ms-M / V, 100 μL of fetal bovine serum (FBS) was added to 900 μL of T22-Ms-M / V micelles to prepare a mixture containing 10% fetal bovine serum, which was placed in a shaker at 37°C and 200 rpm. Samples were taken at 0, 4, and 24 h to test the changes in particle size and particle size distribution using DLS. To test the changes of T22-Ms-M / V under dilution conditions, the concentration of T22-Ms-M / V micelles was diluted 10 times and 100 times with PBS (0.01 M), and the changes in particle size and particle size distribution were tested using DLS. To test the changes of T22-Ms-M / V after long-term storage, the micelles were stored in a refrigerator at 4°C, and samples were taken on days 0 and 15 to test the changes in particle size and particle size distribution using DLS. To test the reduction responsiveness of T22-Ms-M / V, 10 mM GSH was added to T22-Ms-M / V, and samples were taken at 0, 24, and 48 h to monitor the changes in particle size and particle size distribution.

[0049] Taking 20% ​​T22-Ms-M / V (the mass ratio of MID to VEN is 1 / 0.3) as an example, the TEM image shows that it has a spherical micelle structure ( Figure 5 Middle A). Place at 4°C for 15 days ( Figure 5B), diluted 10-fold or 100-fold with PBS ( Figure 5 C), stored in 10% FBS solution for 4 h or 24 h ( Figure 5 (D) The particle size and particle size distribution of the micelles remained basically unchanged, and the absorbance of the micelles at 330 nm decreased significantly after dialysis, indicating that the micelles had completed cross-linking when the dialysis was completed ( Figure 5 F). This cross-linking can be decross-linked under reducing conditions, i.e., reduction response. Under conditions simulating the intracellular environment (10 mM GSH, pH 7.4), the polymer micelles (1 mg / mL) increased in size and the particle size distribution became wider after 24 h. At 48 h, the micelles swelled significantly and two peaks appeared ( Figure 5 In summary, based on PEG- b -P(CL- co -DTC) and T22-PEG- b -P(CL- co -DTC) disulfide cross-linked micelles 20% T22-Ms-M / V have the characteristics of stable blood circulation and responsive release in the reducing environment of tumor cells, which is very important for the stable proportional delivery of drug combinations and rapid drug release in tumor cells, and provides a basis for later applications.

[0050] When the prior art uses gelatin membrane-protected gold nanoparticles to load MID, when the drug concentration is 30.72 μg / ml, the DLE is 80% and the particle size is 147.5 nm. In comparison, the drug-loaded micelle solution prepared by the present invention has a higher drug concentration, a smaller particle size, and a narrower particle size distribution. The prior art uses fucoidan sulfate and indocyanine green dye IR-783 as carriers to load VEN, with a DLE of 43.84% and a particle size of 68±7 nm; the prior art uses liposomes to load VEN, with a DLE of only 6.3% and a particle size of 145±20 nm; the prior art uses PEG-modified poly (lipoic acid) as a carrier to load VEN, with a DLE of 54.2% and a particle size of 102 nm; compared with these literature reports, the present invention has a higher DLE, a smaller particle size, and a narrower particle size distribution.

[0051] Table 1 Particle size, particle size distribution and surface potential of Ms and T22-Ms characterized by DLS

[0052]

[0053] Table 2 Drug loading performance, particle size and particle size distribution of Ms-M and Ms-V

[0054]

[0055] Table 3 Characterization of Ms-M / V and 20%T22-Ms-M / V

[0056]

[0057] a The drug concentration was calculated after being obtained by HPLC test (mobile phase: H3PO4 (pH 2.4, 9.5 mM) / ACN (v / v = 35 / 65), flow rate = 1 mL / min, detection wavelength: 289 nm, 30°C);

[0058] b Tested in PB (pH 7.4, 10 mM) at 25 °C.

[0059] Example 3 Cell endocytosis experiment

[0060] The uptake capacity of MV4-11 cells for T22-Ms was tested by flow cytometry. In order to make the targeted micelles detectable and quantified by the machine, PEG- b -P(CL- co -DTC)-Cy5 was used to label micelles with different T22 targeting densities (0%, 10%, 20%, 30% and 40%). MV4-11 cell suspension (2.0×10 per well) was added to a 6-well plate. 5 cells), and then 100 μL T22-Ms-Cy5 (2.0 μg Cy5 / mL) or PBS with different targeting densities was added to each well. After 4 h of co-incubation, all cells were collected and washed 3 times with PBS. The washed cells were resuspended in 200 μL PBS, tested by flow cytometry, and the flow data were analyzed using conventional software. The results showed that the amount of T22-Ms-Cy5 endocytosis in MV4-11 cells increased significantly with the increase of T22 polypeptide density. The fluorescence intensity of 10% T22-PMs-Cy5 and 20% T22-Ms-Cy5 in MV4-11 cells was 3.0 times and 10.6 times that of the Ms-Cy5 control group, respectively. After the surface polypeptide density exceeded 20%, the fluorescence intensity continued to increase, but the increase in multiples slowed down significantly ( Figure 6 A in the middle). Considering that as the density of T22 peptides on the micelle surface increases, the positive charge carried by the micelles also increases, making it easier to be taken up by macrophages in the lungs, liver and spleen, reducing the circulation time of the micelles in vivo. 20%T22-Ms-M / V was selected for subsequent experiments to extend the circulation time of the targeted dual-drug micelles in vivo and increase their uptake by CXCR4 high-expressing cells. In the following text, where the T22 density is not specifically marked, T22-Ms-M / V refers to 20%T22-Ms-M / V.

[0061] CLSM was used to study the endocytosis of Cy5-labeled Ms and T22-Ms in MV4-11 cells. In the CLSM experiment, the small disc was first placed in a 24-well plate and 500 μL of 0.1 mg / mL PDL solution was added and soaked for 8 h to help MV4-11 cells adhere to the wall. Then PDL was removed and washed twice with 4°C PBS. MV4-11 cell suspension (2.0×10 cells per well) was added to the 24-well plate. 5 Cells were placed in an incubator and incubated for 24 hours to allow the cells to adhere to the wall. 200 μL Cy5-labeled T22-Ms or Ms (40 μgCy5 / mL) was added. After incubation for 4 hours, all cells were collected and washed 3 times with 4°C PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed 4 times with PBS, and then the cell nuclei were stained with DAPI for 5 minutes. The cells were washed 4 times with 4°C PBS. Finally, 10 μL glycerol was added to seal the slides and the samples were stored in the dark. The obtained samples were photographed using CLSM (TCS SP5) and analyzed using LAS X software. The internalization of T22-Ms-Cy5 and Ms-Cy5 in MV4-11 cells can be found in Figure 6 In the figure, the blue fluorescence shows the cell nucleus stained with DAPI dye, and the red fluorescence shows the micelles labeled with Cy5. When MV4-11 cells were incubated with T22-Ms-Cy5 for 4 h, obvious red fluorescence appeared around the cell nucleus, while the cells incubated with Ms-Cy5 had weak intracellular fluorescence, indicating that the uptake of T22-Ms-Cy5 by CXCR4+ MV4-11 cells was significantly higher than that of Ms-Cy5 in the same period of time, which was consistent with the flow cytometry results. The above endocytosis experimental results show that T22-Ms has excellent CXCR4 targeting and the ability to efficiently and quickly internalize CXCR4+ cells, which is of great significance for quickly and efficiently promoting the combination of MID and VEN drugs to enter the cell to inhibit FLT3 and BCL-2 pathways and induce cell apoptosis.

[0062] Example 4 Cytotoxicity and cell apoptosis experiments

[0063] In order to study the toxicity of T22-Ms-M / V preparation on AML cells and normal cells, AML cells MV4-11 and MOLM-13, normal cells mouse fibroblasts L929 and human umbilical vein smooth muscle cells HUVSMC were selected for toxicity study. The specific operation was to add the cell suspension into a 96-well plate (2.0×10 4Cells), then 20 μL of empty vector (Ms or T22-Ms), single-drug micelles or micelles loaded with different mass ratios of dual drugs (MID / VEN=1 / 0.3, 1 / 0.75, 1 / 1.5, 1 / 3 or 1 / 12) were added to each well. After the samples and cells were co-incubated in an incubator at 37°C and 5% CO2 for 48 h, 10 μL of CCK-8 solution was added to each well and incubated in the incubator for 4 h. The absorbance of each well at 450 nm was measured with an enzyme-labeled instrument, and the cell survival rate was calculated.

[0064] Cell survival rate (%) = × 100

[0065] In order to study the ability of drug-loaded micelles to induce apoptosis of AML cells, MV4-11 cells were selected for apoptosis study. The specific operation was to add the cell suspension to a 6-well plate (2.0×10 5 Cells) were added to each well, and then 100 μL of single-drug micelles or micelles loaded with different mass ratios of dual drugs (MID / VEN=1 / 0.3, 1 / 0.75 or 1 / 1.5) were added to each well. After the samples and cells were co-incubated in an incubator at 37°C and 5% CO2 for 48 h, the cells in each well of the six-well plate were collected, washed twice with PBS, and resuspended in 200 μL binding buffer. According to the instructions, the dyes membrane-linked protein V-APC and 7-AAD were used for light staining, and detected by flow cytometry after 5 minutes.

[0066] In multi-drug combination therapy, drug dosage and ratio play an important role in drug efficacy. One of the advantages of nanomedicine is that it can deliver drugs at the best synergistic ratio to target cells. The present invention takes human FLT3 mutation-positive AML cells MV4-11 as an example, and reflects the best synergistic ratio of two drugs through CCK-8 cell experiments and cell apoptosis experiments.

[0067] IC values ​​of single-drug micelles and micelles loaded with two drugs at different mass ratios were obtained by conventional fitting of CCK-8 cell experimental results. 50 The relationship curve between drug concentration and cell survival rate. The results showed that both single-drug micelles and dual-drug micelles showed obvious cytotoxicity. The IC 50 The IC of Ms-M is 26.85 nM. 50 was 44.0 nM, and the cytotoxicity of Ms-V was stronger than that of Ms-M (Table 4, Figure 7 Middle A).

[0068] Table 4 Half lethal concentration and synergistic index of drug-loaded micelles on MV4-11 cells

[0069]

[0070] a It was obtained by fitting and calculation using multi-drug combination effect calculation software (CompuSyn).

[0071] The cytotoxicity of Ms-M / V was investigated in a wide range of drug mass ratios (M / V = 1 / 0.3, 1 / 0.75, 1 / 1.5, 1 / 3 or 1 / 12), and the IC 50 (half lethal concentration, Table 4). The concentration of drugs in different parts of the body is uneven. For AML, a blood tumor with tumors distributed throughout the body, tumor cells in different parts may be exposed to low or high concentrations of drugs. The synergistic effects of drugs with the same ratio are different when their concentrations are different. The ideal optimal synergistic ratio is that the two drugs show synergy in all affected ratios (Fa). In order to evaluate the relationship between the synergistic index (CI, conventional technology) and the drug ratio and Fa when the two drugs are used in combination, the software was used to fit the experimental results of CCK-8 cells, and the corresponding drug synergistic index (CI) values ​​for different Fa were calculated (CI>1 represents antagonism, CI = 1 represents superposition, and CI < 1 represents synergy). All selected ratios have CI values ​​less than 1 at most concentrations, but M / V mass ratios of 1 / 0.75 and 1 / 1.5 show antagonism (CI value>1) when the Fa value is low. It is worth noting that when the M / V mass ratios are 1 / 0.3 and 1 / 12, the CI values ​​corresponding to all Fa values ​​are less than 1 ( Figure 7 (B) When the CI value is 1 / 0.3, it is even lower, showing the best synergistic effect.

[0072] After determining the optimal synergistic ratio of drugs in micelles, the bare drug (M / V 1 / 0.3 ), dual-drug non-targeted micelles (Ms-M / V 1 / 0.3 ) and dual-drug targeting micelles (T22-Ms-M / V 1 / 0.3 ) cytotoxicity, and found that M / V 1:0.3 、Ms-M / V 1:0.3 and T22-Ms-M / V 1:0.3 IC for MID 50 The values ​​were 46.47, 41.05, and 42.57 nM, respectively, indicating that the toxicity of targeted and non-targeted dual-drug micelles is very similar at the cellular level ( Figure 7 Middle C). It is worth mentioning that T22-Ms-M / V 1:0.3 When the concentration of MID reached 1000 nM, it still showed low toxicity to normal cells such as HUVSMC and L-929 cells ( Figure 7 Middle D).

[0073] Empty micelles Ms and T22-Ms were co-incubated with AML cells MV4-11, MOLM-13 and normal cells HUVSMC and L-929 for 48 h to test their cytotoxicity. The results showed that no obvious cytotoxicity was observed in these cells treated with Ms and T22-Ms at a concentration of 25-500 μg / mL ( Figure 8 ), indicating that Ms and T22-Ms had no obvious toxicity to cells, that is, the carrier had good biocompatibility.

[0074] Four concentrations of single-drug micelles and micelles loaded with dual drugs at different mass ratios (MID / VEN = 1 / 0.3, 1 / 0.75, or 1 / 1.5) were selected and incubated with MV4-11 cells for 24 h, and the proportion of induced cell apoptosis was determined by annexin V-APC / 7-AAD double staining and flow cytometry ( Fig. 9 A and B in the figure, and use conventional software to fit and calculate the CI value to obtain the corresponding CI value when Fa is different ( Fig. 9 The results show that the CI values ​​under the three ratios are less than 1 in the entire range of Fa changes, but when M / V is 1 / 0.3, the CI value decreases with the increase of Fa, that is, the larger the apoptosis ratio, the more obvious the synergistic effect of the drug (when the Fa value is 0.9, CI = 0.25, strong synergy), while the CI change trends of the other two ratios are opposite ( Fig. 9 C). Combined with the cytotoxicity and apoptosis data, M / V of 1 / 0.3 is the best synergistic ratio for MV4-11 cells. b -Poly (2-aminohexadecanoic acid) (mPEG- b -PAPA)) micelles loaded with MID and CFZ (Ms-M / C), the best Ms-M / C 1 / 1 When the Fa value is in the range of 0.1 to 0.9, the CI is greater than 0.6, and the synergistic effect is significantly lower than that of the present invention.

[0075] Example 5 Establishment and treatment of AML xenograft model

[0076] All animal experiments and procedures were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University. Female mice aged 9 weeks and weighing 20-22 g (B-NDG, Biocytogen) were used in the experiment.

[0077] The specific steps of using MV4-11 cells to establish an AML xenograft model are as follows: the cultured MV4-11 cells were collected in a centrifuge tube, washed three times with PBS, and then resuspended in PBS to a density of 2.5×10 6200 μL MV4-11 cells (5×10 5 The day of inoculation was recorded as day 0.

[0078] In the in vivo experiment, in addition to the 1 / 0.3 (w / w) ratio, Ms-M / V also added two ratios, 1 / 0.75 and 1 / 1.5. In order to more intuitively observe the effect of drug ratio on the therapeutic effect, except for the high-dose group, the dose of MID in each group was fixed at 10 mg / kg, and the specific groups were PBS group, Ms-M / V (10 / 3 mg / kg, n = 3) group, Ms-M / V (10 / 7.5 mg / kg, n = 3) group and Ms-MI / V (10 / 15 mg / kg, n = 3) group. At the same time, in order to study the effect of dose on the final efficacy and the tolerance of mice to dose, the Ms-M / V (15 / 11.25 mg / kg, n = 3) group was added to determine the dosage for the next experiment. This group has the same drug ratio as the Ms-M / V (10 / 7.5 mg / kg) group, and the dose is increased by 1.5 times, which is called the high-dose group. The administration method was to administer the drug through the tail vein on the third day after inoculation, once every three days, for a total of 10 times ( Fig.10 (A, B).

[0079] During the administration period, the mice had slight decreases and increases, and the relative body weight of each group was always above 85%, indicating that the mice tolerated the dose well ( Fig.10 C). Taking the day of inoculation as day 0, the median survival of mice in the PBS group was 20 days. Among the three groups with the same MID dose, the Ms-M / V 10 / 3 mg / kg group had the longest median survival of 29 days ( Fig.10 Figure B shows that the drug ratio of 1 / 0.3 is optimal. On the 19th day, the mice in the PBS group showed obvious symptoms of AML, and the mice were on the verge of death. Peripheral blood was collected from all groups of mice by orbital sampling, and labeled with APC-anti-human-CD45 antibody. Flow cytometry was used to detect the infiltration of AML cells in the peripheral blood ( Fig.10 D), the infiltration of Ms-M / V 10 / 3 mg / kg was the lowest among the three groups with the same MID dose. Therefore, in the in vivo experiment, the M / V mass ratio of 1 / 0.3 was the optimal ratio for the combination of MID and VEN to treat the MV4-11 AML xenograft model, and this ratio was used in subsequent animal experiments. In the experiment, increasing the dose can significantly prolong the survival of mice. The mouse survival curve showed that compared with the median survival of 26 days in the Ms-M / V 10 / 7.5 mg / kg group, the Ms-M / V 15 / 11.25 mg / kg group extended the survival to 32 days (** p , Fig.10 In D), when peripheral blood was collected on day 19 to measure tumor infiltration, the tumor infiltration in the Ms-M / V15 / 11.25 mg / kg group was also significantly lower than that in the Ms-M / V10 / 7.5 mg / kg group (* p ), indicating that appropriately increasing the dosage can improve the efficacy of drug combination and prolong the survival of mice.

[0080] In the following animal experiments, the M / V ratio was maintained at 1 / 0.3 (w / w), the drug dose was appropriately increased within the safety range, and the targeted carrier T22-Ms was used to further improve the anti-tumor effect. The same modeling and administration method was used for formal animal experiments, and the number of mice observed for survival in each group increased to 6, and 3 mice in each group were used for dissection and analysis of relevant indicators of AML treatment. B-NDG mice with MV4-11 AML xenograft model were randomly divided into 6 groups on day 3 after inoculation and injected with 200 μL of PBS (n = 9), Ms-M (20 mg / kg, n = 9), Ms-V (6 mg / kg, n = 9), Ms-M / V (20 / 6 mg / kg, n = 9), T22-Ms-M / V (20 / 6 mg / kg, n = 9) or free drug solution free-M / V (20 / 6 mg / kg, n = 9) through the tail vein every 3 days for a total of 10 times (days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30). The body weight of mice was recorded every 3 days. Six mice in each group were used to observe the survival period. When the PBS group was moribund, three mice were taken from each group for dissection. The liver, spleen, lung, hind leg bone and celiac artery blood cells were collected and labeled with antibodies. The degree of leukemia infiltration was monitored by flow cytometry. The heart, spleen, liver, kidney and lung were collected and fixed with 4% paraformaldehyde fixative. Histopathological analysis was performed by H&E staining ( Fig.11 Except for the PBS group, all mice died on day 21. The weight of mice in other groups fluctuated slightly in the early stage of drug administration, and gradually increased after the dose was tolerated. Only the free-M / V group lost weight due to the rapid progression of AML in the late stage of drug administration ( Fig.11 The mouse survival curve showed that the median survival time of the PBS group was 20 days, and the single-drug micelle treatment groups Ms-V and Ms-M only extended the median survival time by 2.5 days and 5 days, respectively. Compared with the single-drug MID micelle Ms-M, the dual-drug micelles Ms-M / V and T22-Ms-M / V significantly prolonged the survival time of mice (25 vs 38.5, *** p ; 25 vs 43, **** p , Fig.11In the middle C), the effect of single-drug treatment of AML is not good, and a drug combination is needed to improve the efficacy. When MID and VEN are used to treat AML simultaneously, in order to study the effect of fixed-ratio delivery on the efficacy, the same dose of drugs are administered in the form of free drugs and micelle loading. The survival of mice in the free drug free-M / V group is 32 days, while the micelle group Ms-M / V group significantly extends the survival of mice to 38.5 days (** p , Fig.11 In the middle C, micelle delivery can ensure that the drug exerts a higher therapeutic effect in the target cells at the best synergistic ratio. The survival time of the T22-Ms-M / V group modified with T22 peptide was the longest (43 days), which was significantly higher than that of the non-targeted group Ms-M / V (38.5 vs 43, * p ) and free-M / V (32 vs 43, *** p ), indicating that T22 peptide modification can further enhance the anti-tumor effect of micellar drug combination.

[0081] The infiltration of AML cells in peripheral blood, major organs and bone marrow is an important indicator for evaluating the therapeutic effect. On the 19th day when the mice in the PBS group were dying, 3 mice in each group were dissected and cells in peripheral blood, liver, spleen, lung and hind leg bones were collected. APC anti-human CD45 antibody was added to label AML cells and the infiltration ratio of AML cells was determined by flow cytometry. The results showed that there was obvious leukemia cell infiltration in the liver, spleen, bone marrow, lung and abdominal aorta blood of mice in the PBS group ( Fig.12 A and B in the figure indicate that the tumor cells in this model will metastasize to multiple organs throughout the body. The two single-drug micelle groups Ms-M and Ms-V had very high infiltration in various organs on the 19th day, second only to the PBS group, indicating that the single-drug treatment did not inhibit the proliferation of AML cells very well and the therapeutic effect was very poor. Compared with the single-drug micelle treatment, the Ms-M / V and T22-Ms-M / V treatment groups significantly reduced the leukemia burden of mice, and there was no obvious leukemia infiltration in the liver, bone marrow, lungs and peripheral blood, proving that the introduction of a synergistic micelle drug combination can significantly improve the therapeutic effect of AML. Compared with the free drug group at the same dose, the Ms-M / V group and the T22-Ms-M / V group also had lower infiltration in various organs, indicating that after the drug is encapsulated in the micelle, its ability to deliver the drug to the target cells in proportion improves the therapeutic effect of the drug.

[0082] On the 19th day of moribundity in the PBS group, one mouse was dissected from each group, and the heart, kidney, liver, spleen, and lung were collected and preserved in 4% paraformaldehyde. Histological analysis was then performed by paraffin sectioning and H&E staining. The PBS group and the healthy group were used as controls. The observation surface was the largest section of the organ ( Fig.13). The results showed that obvious AML cell infiltration was observed at the edge of the lungs of mice in the PBS group, Ms-V group, and Ms-M group, while no AML cells were observed in the lungs of the free-M / V group. AML cells were observed to grow in clusters and dispersed infiltration in the livers of mice in the PBS group, Ms-V group, and Ms-M group (black dotted area), while a small number of AML cells were observed to be dispersed infiltration in the free-M / V group (black arrows). AML cell infiltration was observed at the edge of the spleens of mice in the PBS group, Ms-V group, and Ms-M group, and a small number of AML cells were observed inside the spleens. No AML cells were observed at the edge of the spleens of the free-M / V group, but a small number of AML cells were observed inside the spleens. No AML cells were observed in the lungs, livers, and spleens of the Ms-M / V group and the T22-Ms-M / V group. The hepatocytes were normal, uniform, and orderly arranged. The red pulp of the spleen was abundant, and the lung structure was intact. In addition, no obvious abnormalities were observed in the hearts and kidneys of mice in each group.

[0083] HE analysis of mouse femur and tibia showed that ( Fig.14 (A) A large number of leukemia cells and only a small number of hematopoietic cells were distributed in the bone marrow cavity of mice in the PBS, Ms-V and Ms-M groups, while the infiltration of leukemia cells in the bone marrow cavity was significantly reduced in the free-M / V group. It is worth noting that basically no leukemia cells were seen in the bone marrow cavity of mice in the Ms-M / V and T22-Ms-M / V groups after treatment. Osteoclasts and osteoblasts in the bones of healthy mice are in a dynamic balance. The increase of osteoclasts often causes osteolytic lesions, eventually leading to osteoporosis and osteolysis. Tartrate-resistant acid phosphatase (TRAP) is the main marker of osteoclasts, which can be shown by TRAP staining ( Fig.14 Middle B) There are a large number of osteoclasts in the leg bones of mice in the PBS, Ms-V, and Ms-M groups, while there are fewer osteoclasts in the free-M / V group. The number of osteoclasts in the Ms-M / V and T22-Ms-M / V groups is significantly reduced after treatment. During the progression of AML disease, the infiltration of AML cells into the bone marrow is often accompanied by bone dissolution, which can also be observed in leukemia patients and mouse models. The tissue structure of the femur and tibia of each group of mice was photographed. Fig.14 As shown in C, the femur and tibia of mice in the PBS, Ms-V and Ms-M groups showed obvious osteolytic destruction and severe loss of trabeculae. The free-M / V group showed slight relief, while the Ms-M / V and T22-Ms-M / V groups could completely inhibit bone destruction and maintain intact bone structure. Quantitative results further showed that the number and thickness of trabeculae in the tibia and femur of mice in the Ms-M / V and T22-Ms-M / V groups were significantly higher than those in the other control groups, and the trabecular separation was significantly lower ( Fig.14 Middle D).

[0084] In order to improve the drug combination regimen for FLT3 mutant AML, the present invention designed and prepared T22 polypeptide-modified disulfide cross-linked polymer micelles co-loaded with MID and VEN (T22-Ms-M / V) for efficient targeted therapy of in situ MV4-11 AML mice, see Fig.15 . T22-Ms-M / V has the advantages of small size, good reduction responsiveness, controllable loading drug combination ratio, adjustable surface peptide density, and targeting CXCR4+ AML cells. It can stably co-load and target release MID and VEN in different ratios, and effectively inhibit the proliferation of AML cells. Cell experiments showed that T22-Ms-M / V can be efficiently taken up by MV4-11 cells, and the uptake is positively correlated with the density of T22 peptides modified on the micelle surface; when the dual drug ratio M / V is 1 / 0.3 (w / w), T22-Ms-M / V has the strongest synergistic effect, the strongest killing and apoptosis effect on MV4-11 cells, and no obvious toxicity to normal cells. The results of the treatment experiment on the in situ MV4-11 AML mouse model showed that compared with single-drug or dual-drug non-target micelles, T22-Ms-M / V significantly inhibited the proliferation and metastasis of AML cells in mice, prolonged the median survival of mice, and improved the bone damage of mice, while the toxic side effects were not obvious.

[0085] Specifically, the present invention prepares T22 polypeptide-modified disulfide cross-linked polymer micelles (T22-Ms-M / V) for stable loading and efficient delivery of two clinical small molecule inhibitors MID and VEN into AML cells, thereby achieving efficient treatment of AML. T22-Ms-M / V micelles are composed of amphiphilic polymers PEG- b -P(CL- co -DTC) and an amphiphilic polymer T22-PEG- b -P(CL- co -DTC) co-assembled to form micelles, while encapsulating MID and VEN through water transport. Among them, T22 polypeptide can target AML cells with high expression of CXCR4 receptor (about 1 / 3), increase the uptake of AML cells, and thus increase the effective concentration of drugs in AML cells. MID and VEN will be loaded and delivered at the optimal synergistic ratio in order to obtain the best synergistic efficacy. The disulfide cross-linked structure of T22-Ms-M / V also has the performance of long-term circulation stability and rapid release of drugs in the reducing environment of tumor cells. This CXCR4 receptor-targeted T22-Ms-M / V micellar nanodrug may be an effective strategy for the clinical treatment of FLT3 mutant AML.

Claims

1. A FLT3 and BCL-2 dual inhibitor micelle, characterized in that: The invention comprises a polymer micelle and a FLT3 inhibitor and a BCL-2 inhibitor loaded thereon; the mass ratio of the FLT3 inhibitor to the BCL-2 inhibitor is 1:0.1-1; the FLT3 inhibitor is midostaurin, and the BCL-2 inhibitor is vetraclon; the polymer micelle is co-assembled by an amphiphilic polymer PEG-bP (A-co-DTC) and a polypeptide amphiphilic polymer T-PEG-bP (A-co-DTC), wherein A is an ester or carbonate monomer unit, and T is a T22 polypeptide; The molar amount of T-PEG-bP(A-co-DTC) is 5 to 20% of the sum of the molar amounts of PEG-bP(A-co-DTC) and T-PEG-bP(A-co-DTC); in PEG-bP(A-co-DTC) and T-PEG-bP(A-co-DTC), the number average molecular weight of the PEG segment is independently selected from 2000 to 12000 Da, the number average molecular weight of the P(A-co-DTC) segment is 2 to 10 times the number average molecular weight of the PEG segment, and the number average molecular weight of the PDTC segment is 5% to 40% of the total number average molecular weight of the P(A-co-DTC) segment.

2. The FLT3 and BCL-2 dual inhibitor micelle according to claim 1, characterized in that: The mass ratio of the FLT3 inhibitor to the BCL-2 inhibitor is 1:0.

3.

3. The method for preparing the FLT3 and BCL-2 dual inhibitor micelles according to claim 1, characterized in that: The method comprises the following steps: mixing an amphiphilic polymer PEG-bP (A-co-DTC), a polypeptide amphiphilic polymer T-PEG-bP (A-co-DTC), a FLT3 inhibitor and a BCL-2 inhibitor in a solution to obtain the FLT3 and BCL-2 dual inhibitor micelles.

4. A combined drug, characterized in that: It comprises the FLT3 and BCL-2 dual inhibitor micelles as described in claim 1 and other anti-tumor drugs.

5. Use of the FLT3 and BCL-2 dual inhibitor micelles according to claim 1 or the combined drug according to claim 4 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that: Anti-tumor drugs are drugs used to prevent and treat leukemia.

7. The use according to claim 6, characterized in that: Drugs for preventing and treating leukemia are drugs for preventing and treating acute myeloid leukemia.

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