CN116650415A - Targeted dual anti-apoptosis protein polymer micelle and preparation method and application thereof - Google Patents

Abstract

The invention discloses a targeted double anti-apoptosis protein polymer micelle, a preparation method and application thereof, wherein a polymer carrier is used for loading two small molecular drugs which simultaneously inhibit the expression of anti-apoptosis proteins BCL-2 and MCL-1, so as to overcome single drug resistance. The invention discloses a novel targeted double anti-apoptosis protein polymer micelle, which effectively solves the problems of large dosage and large toxicity existing in the prior art. Experimental results comprehensively show that the TPMs-V/S of the invention effectively reduce the dosage of VEN and SOR, have toxic and side effects, and effectively improve the curative effect of the double medicines.

Description

Targeted dual anti-apoptosis protein polymer micelle and preparation method and application thereof

Technical Field

The invention belongs to the nano-drug technology, and particularly relates to a targeted dual anti-apoptosis protein polymer micelle, a preparation method and application thereof, which are used for active targeted treatment of an in-situ acute myeloid leukemia mouse model.

Background

Acute myeloid leukemia (Acute Myeloid Leukemia, AML) is a malignant hematological disease of abnormal clones of myeloid hematopoietic stem cells, which is highly resistant, relapsing and metastatic, has a high degree of tumor heterogeneity, can activate multiple anti-apoptotic drug resistance signaling pathways, often results in recurrent drug resistance with difficulty in achieving sustained profound remissions, and thus combined targeted therapy for multiple pathways may be a more effective approach to improve survival in AML populations. In theory, the FLT3 inhibitor SOR could be used in combination with VEN to inhibit both BCL-2 and MCL-1 dual targets, but both VEN and SOR produced significant hematologic toxicity in clinical treatment due to the high doses required, reducing overall therapeutic efficacy [ DiNardo, C.D.; pratz, K.W.; letai, A.; jonas, B.A.; wei, A.H.; et al Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-random, open-label, phase 1b study. The Lancet Oncology 2018, 19 (2), 216-228]. The key challenge faced by the present combination therapy is to solve the superimposed toxicity caused by the combination therapy, enhance the targeting selection capability on tumor cells, weaken the toxicity on normal tissues and organs, and widen the dosage treatment window.

A variety of nanoformulations are currently approved or entered into clinical trials at different stages. For example, liposomal cytarabine Depocyte has been used in clinical studies of AML, but is less enriched at the tumor site. At present, the medicine still faces the challenges of small tumor enrichment, poor targeting selection capability, weak endocytic capability and the like, so that the curative effect is improved only to a limited extent. In order to improve the tumor enrichment and the uptake capacity of tumor cells, people bond polypeptides and the like on the surface of a nano system; to further enhance the therapeutic effect, treatments for AML that overcome single drug resistance typically use a combination of two or more anticancer drugs to enhance the therapeutic effect. However, it is worth noting that the double-drug nano-preparation still faces the problems of poor in vivo stability, low tumor enrichment, slow drug release and the like.

Disclosure of Invention

The invention discloses a novel targeted double anti-apoptosis protein polymer micelle, which effectively solves the problem of high toxicity in the prior art.

The invention adopts the following technical scheme:

a targeted dual anti-apoptosis protein polymer micelle comprises a polymer carrier and a small molecule drug which simultaneously inhibits the expression of anti-apoptosis proteins BCL-2 and MCL-1; preferably, the small molecule drugs that inhibit the expression of both anti-apoptotic proteins BCL-2 and MCL-1 are two, preferably Viterbi (VEN) and Sorafenib (SOR).

In the invention, the polymer micelle is assembled by a non-targeting polymer or is assembled by the non-targeting polymer and the targeting polymer. In the invention, the non-targeting polymer consists of a hydrophilic chain segment and a hydrophobic chain segment; the targeting polymer consists of a targeting molecule, a hydrophilic chain segment and a hydrophobic chain segment. The molecular weight of the hydrophilic chain segment is 2-10 kDa, and the molecular weight of the hydrophobic chain segment is 0.8-3 times, preferably 1-2 times, that of the hydrophilic chain segment; the hydrophilic segment is preferably a polyethylene glycol segment. In the invention, the hydrophobic chain segment is obtained by copolymerizing 1, 2-dithiolane trimethylene carbonate with other monomers, wherein the other monomers are ester monomers or carbonate monomers, such as CL, TMC, LA and the like; among the hydrophobic segments, the molecular weight of the segment constituted by 1, 2-dithiolane trimethylene carbonate units is 20 to 45%, preferably 25 to 35% of the molecular weight of the hydrophobic segment. The targeting molecule is preferably a polypeptide.

In the invention, a non-targeting polymer and a small molecular medicine which simultaneously inhibits the expression of anti-apoptosis proteins BCL-2 and MCL-1 are taken as raw materials, or the non-targeting polymer and the targeting polymer are taken as raw materials, and the small molecular medicine which simultaneously inhibits the expression of the anti-apoptosis proteins BCL-2 and MCL-1 is taken as raw materials; the targeted dual anti-apoptotic protein polymer micelle is prepared by a solvent substitution method.

Preferably, the mole fraction of the non-targeting polymer to the targeting polymer is 0 to 30% and excluding 0, preferably 10 to 30%, more preferably 10 to 20%.

The invention discloses application of the targeted dual anti-apoptosis protein polymer micelle in preparing a hematological tumor therapeutic drug, preferably, hematological tumor is leukemia, and further preferably, hematological tumor is acute myelogenous leukemia.

The PMs-V/S can obviously kill AML cells under a fixed drug synergistic ratio, and meanwhile, the cytotoxicity of the PMs-V/S to the AML cells can be obviously improved by modifying the T22 polypeptide, so that the apoptosis is efficiently induced, and the cytotoxicity is especially smaller to normal cells. Free Viterbi (VEN) and Sorafenib (SOR) are effective in killing AML cells, but have problems of high doses, low bioavailability, and high additive toxicity. The nano-carrier can stably encapsulate the medicine and improve the circularity of the medicine, but the nano-carrier is limited by the problems of lack of tumor targeting, weak uptake of tumor cells, slow release of the medicine in the cells and the like. The invention designs and prepares targeted T22 polypeptide functionalized disulfide cross-linked polymer micelle nano-drug (TPMs-V/S) for efficiently loading VEN and SOR, which is used for efficiently targeting and rapidly releasing VEN and SOR into leukemia cells, simultaneously inhibiting the expression of anti-apoptosis proteins BCL-2 and MCL-1, and starting the apoptosis process of AML cells, thereby inhibiting the proliferation and transfer of AML cells, and especially has low dosage, high bioavailability and low superposition toxicity.

Drawings

FIG. 1 is a diagram of T22 polypeptide modified disulfide cross-linked micelles co-delivering Viterbi and sorafenib (TPMs-V/S) for efficient targeted therapy of in situ MV4-11 acute myeloid leukemia mice transplantation model.

FIG. 2 shows the synthetic routes of (A) PEG-B-P (CL-co-DTC) and (B) T22-PEG-B-P (CL-co-DTC).

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum (400 MHz, DMSO-D6) of (A) PEG-B-P (CL-co-DTC), (B) NHS-PEG-B-P (CL-co-DTC), (C) DBCO-PEG-B-P (CL-co-DTC), and (D) T22-PEG-B-P (CL-co-DTC).

FIG. 4 is a GPC chart of (A) PEG-B-P (CL-co-DTC) and (B) NHS-PEG-B-P (CL-co-DTC).

FIG. 5 is a characterization of TPMs-V/S. (A) particle size distribution of a series of drug-loaded micelles measured by DLS. (B) TEM photograph of T20PMs-V/S1:4, scale 50 nm. (C) PEG-b-P (CL-co-DTC) solution and ultraviolet-visible spectrum of its micelles. T20PMs-V/S1:4 stored at 4℃for 40 days, 24 h in 10% FBS, 100-fold dilution in PBS (D) and particle size change (E) of 0 h, 12 h, 30 h in 10 mM GSH. (F) HPLC spectra of different concentrations of free SOR and VEN standard. (G) The drug release of T20PMs-V/S1:1 was determined by HPLC with 12 h placed with/without 10 mM GSH. (H) In vitro release behavior of TPMs-V/S in PB with or without 10 mM GSH.

FIG. 6 is a graph showing the effect of T22 polypeptide modification on endocytosis of TPMs-Cy5 by (A) MV4-11, (B) MOLM-13, (C) HL-60, and (D) OCI-AML3 cells using flow cytometry. Incubation time was 4 h. MV4-11 and MOLM-13 cells pretreated with free T22 peptide were incubated with Cy 5-labeled T20PMs-Cy5 as controls.

FIG. 7 is a photograph of CLSM (scale: 25 μm) after incubation of MV4-11 cells with T20PMs-Cy5 for 4 h.

FIG. 8 is a photograph of CLSM (scale: 25 μm) after incubation of MOLM-13 cells with T20PMs-Cy5 for 4 h.

FIG. 9 shows the toxicity of PMs-V, PMs-S and PMs-V/S containing different doublet ratios in (A) MV4-11 and (B) MOLM-13 cells using CCK-8 (n=6) and the synergy of VEN and SOR in (C) MV4-11 and (D) MOLM-13 cells was evaluated by CompuSyn software. Free V/S, PMs-V/S, T20PMs-V/S cytotoxicity (n=4) of 48 h co-incubated with (E) MV4-11 and (F) MOLM-13 cells. Toxicity of empty micelle PMs and T20PMs in (G) MV4-11 cells and (H) MOLM-13 cells (n=4).

FIG. 10 shows toxicity of T20PMs-V/S1:4 and PMs-V/S1:4 in (A) PBMC, (B) DC 2.4 and (C) L929 cells (n=5). (D) Samples (free V/s1:4, PMs-V/s1:4, T20 PMs-V/s1:4) were incubated with erythrocyte suspensions at 37 ℃,200 rpm for 3h of hemolysis, PBS and Triton-X100 treated samples served as negative and positive controls, respectively (n=4, ×p <0.001, ×p < 0.0001).

FIG. 11 shows the flow cytometer assay (co-incubation 48 h) of T20PMs-V/S, PMs-V/S, free V/S, PMs-S, PMs-V induced apoptosis of (A) MV4-11 cells (VEN/SOR concentration 2/8 ng/mL) and (B) MOLM-13 cells (VEN/SOR concentration 1/2 ng/mL) at optimal drug co-ratios.

FIG. 12 is an acute toxicity study of T20 PMs-V/S. Dosing regimen and monitoring flow chart for model (a). (B) Body weight change (n=4) over 8 days of dosing of mice in each group, (C) blood routine index analysis (n=3) and (D) blood biochemical index analysis (< p <0.05, < p <0.01, < p <0.001, n=4).

Fig. 13 is a photograph of fluorescence imaging of the leg bones of different treatment groups (a) and a quantitative analysis of fluorescence of the main organs (n=3) (B). The tail vein was injected with T20PMs-Cy5 and PMs-Cy5 8 h, respectively, and then sampled, and the concentration of Cy5 was 2.5. Mu.g/mL.

FIG. 14 is a construction of (A) in situ MV4-11 AML mouse transplantation model and treatment workflow (gray arrows indicate treatment group dosing time). (B) Peripheral blood quantifies leukemia cell infiltration to monitor disease progression in the in situ mouse model. Part (C) mice in the treatment group had varied body weight during the period of administration. (D) Kaplan-Meier survival curves (n=5, TPMs-V/S1:4 (H) versus Free V/S group (p.o.), TPMs-V/S1:1 (H), TPMs-V/S1:4 (L), PMs-V (L), PMs-S (L), free-V/S (L, i.v.) and PBS group, ×p < 0.01). (E) spleen weight of surviving mice dissected on day 32.

Fig. 15 shows (a) representative flow scattergrams of MV4-11 cell infiltration in Bone Marrow (BM), liver (river), lung (Lung), peripheral Blood (PB) and Spleen (Spleen) of mice from different treatment groups (CD 45 positive AML cell population in rectangular gate) and (B) statistical analysis (n=3).

Fig. 16 is a blood routine (a) and blood biochemical (B) (n=3) index analysis for mice of different treatment groups. Abbreviations: red blood cell count (RBC), hematocrit (HCT), mean red blood cell volume (MCV), red blood cell distribution width (RDW), hemoglobin-related index hemoglobin concentration (HGB), mean red blood cell hemoglobin content (MCH), mean hemoglobin concentration (MCHC), hemoglobin Distribution Width (HDW), platelet count (PLT), platelet volume (PCT), mean Platelet Concentration (PCV), white blood cell count (WBC), eosinophil (EOS), neutrophil (Neut), and lymphocyte (Lymph).

FIG. 17 is a graph of H & E staining of heart, kidney, lung, liver, spleen of mice from different treatment groups. Ruler: 100. μm.

Fig. 18 is an H & E staining of the femur and tibia of mice from different treatment groups (black circled portions indicate leukemic cell infiltration areas). A icon ruler: 500. μm, B icon ruler: 100. μm.

FIG. 19 shows TRAP staining (green scale: 500 μm, black scale: 100 μm) of the osteoclasts of the rear leg bones (white arrows) of the different treatment groups.

Fig. 20 is a graph of Micro-CT of Tibia (Tibia) and Femur (Femur) and Femur (C) of (a) different treatment groups of mice, statistical analysis of Tibia (B) and Femur (C) (n=3).

Detailed Description

The invention designs and prepares a targeting peptide modified disulfide cross-linked biodegradable polymer micelle (TPMs-V/S) for co-carrying VEN and SOR based on PEG and Polycarbonate (PTMC) for active targeting treatment of a mouse in-situ MV4-11 AML model, and realizes optimal synergistic ratio delivery and efficient and controllable release of VEN and SOR (figure 1). The TPMs-V/S has the advantages of high efficiency, stability, controllable drug loading, adjustable surface polypeptide density, rapid drug release under reduction response, good targeting property and the like. Research results show that the TPMs-V/S can efficiently target MV4-11 cells in an in-situ FLT3-ITD subtype AML mouse model, strongly inhibit tumor growth and remarkably prolong the survival period of the mouse.

Succinimide ester functionalized polyethylene glycol hydroxyl (NHS-PEG-OH,M _n =5.0 kDa, sienna ruixi organisms) Directly after purchase, monomethoxy polyethylene glycol (MeO-PEG-OH, M _n =5.0 kDa, beijing key kawa technologies co.ltd) was used after azeotropic distillation with toluene. Epsilon-caprolactone (epsilon-CL, 99%) was dried over calcium hydride and distilled under reduced pressure before use. The 1, 2-Dithiolane Trimethylene Carbonate (DTC) was used after purification. Diphenyl phosphate (DPP,>99%, TCI), dried under vacuum 2 h prior to use. T22 cyclopeptides (sequence: lys (Azido) -Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Arg-NH) ₂ (Disulfide bridge: Cys5-Cys18, Cys9-Cys14)，>85%, jil Biochemical Co., ltd.) and amino-functionalized dibenzocyclooctyne (DBCO-NH) ₂ ，>95%, of the sienna-xi organism) are purchased and used directly. PEG-b-P(CL-co-DTC) -Cy5 (molecular weight 5.0-4.0-2.0 kDa) was synthesized according to conventional methods and used as such. Dichloromethane (DCM, national medicine group) and N, N-dimethylformamide (DMF, national medicine group) are used after water and oxygen removal by a solvent purification system. Anhydrous diethyl ether (national drug group), anhydrous methanol (national drug group), tween 80 (TW 80, mikrin biochemical technology limited), and HPLC grade acetonitrile (ACN,>99%, sigma Alrich, USA) was used directly after purchase. Sorafenib (SOR,>99%, MCE), viterbi (VEN,>99%, MCE), glutathione (GSH, >99%, kesaint biosciences), 4, 6-diamidino-2-phenylindole (DAPI, biyun), paraformaldehyde-glutaraldehyde fixative (Solebao), polylysine (PDL, biyun), PE anti-human CD184 antibody (PE-anti-human-CXCR 4, bioleged), CCK-8 kit (Suzhou Mei-ren biosciences Co., ltd.), apoptosis kit (Annexin V-APC/7-AAD, union), dialysis bags of different molecular weight cut-off (MWCO), etc. are all used directly after purchase. The cell culture medium was RPMI 1640 (Roswell Park Memorial Institute 1640, hyClone) and was used after further addition of 1% penicillin, streptomycin (keno organism) and 15% fetal bovine serum (Gibco). Human Peripheral Blood Mononuclear Cells (PBMC) were from university of su.

The nuclear magnetic test solvent of the polymer is deuteriumDimethyl sulfoxide (DMSO)d ₆ ) Chemical shift is referenced to the solvent peak. The polymer molecular weight and molecular weight distribution were determined by a Waters 1515 gel permeation chromatograph (GPC, waters 1515) using a series of monodisperse linear polymethyl methacrylates as standard samples. The particle size and particle size distribution of the polymer micelles were measured using a dynamic light scattering instrument (DLS, zetasizer Nano-ZS, malvern Instruments) at room temperature. The morphology of the drug-loaded micelles was measured by Tecnai G220 transmission electron microscopy (TEM, USA) at an accelerating voltage of 120 kV. Receptor expression, apoptosis, micelle endocytic behavior, tumor cell progression in animal models, and the like were measured by flow cytometry (FACS Calibur, BD Biosciences, USA), and analyzed with Flowjo software (Tree Star). Laser confocal microscopy (CLSM) pictures were taken using Leica TCS SP5 (Wetzlar, germany). The microplate reader (Thermo Multiskan FC) was used to determine the absorbance of the cell-CCK-8 reaction product at 450 nm using PBS as a sample control. Mice were imaged Ex vivo using a near infrared imaging system (Caliper IVIS Lumina II, ex 643 nm, em 668 nm) and analyzed using the live image software. Hematoxylin-eosin (H) &E) Staining, trap staining pictures were taken by an inverted fluorescence microscope (Nikon Eclipse Ti). Blood was routinely monitored by a blood analyzer (advia 2120i, siemens, germany), and blood biochemistry was monitored by a fully automated biochemical analyzer (merebs-420) at 3000 rpm/min,4 ℃. Bone tissue structure was examined by microcomputer tomography (Micro CT, skyScan 1176, aartselaar, belgium) (65 kV, 385 mA, and a 1 mm Al filter).

All data herein are mean ± Standard Deviation (SD). Statistical differences between three or more groups of data were assessed by ANOVA single factor ANOVA, when the results were significantp <0.05 Group-to-group comparisons were made using Tukey's post hoc test. *p <0.05 represents a significant differencep <0.01 and%p <0.001 indicates a highly significant difference.

The examples employ specific preparation procedures and testing methods as conventional techniques.

PEG with theoretical molecular weight of 5.0-4.0-2.0 kDab-P(CL-coDTC) is synthesized by using macromolecular initiator MeO-PEG-OH under the action of catalyst diphenyl phosphate (DPP)M _n =5.0 kg/mol) to initiate ring-opening polymerization of DTC and epsilon-CL, the specific synthesis reaction is a pale yellow block solid.

T22-PEG-b-P(CL-co-DTC) the synthesis of the polymer is divided into three steps: the first step, using macromolecular initiator NHS-PEG-OH [ ]M _n =5.0 kg/mol) initiating DTC and epsilon-CL ring-opening polymerization under the catalysis of DPP to obtain NHS-PEG-b-P(CL-co-DTC) polymer. Preparation method, feeding molar ratio, purification method and PEG-b-P(CL-co-DTC) is synthesized identically, except that a macroinitiator is used NHS-PEG-OH. In the second step, NHS-PEG-b-P(CL-co-DTC) and DBCO-NH ₂ Obtaining DBCO-PEG through amidation reactionb-P(CL-co-DTC). Specifically, NHS-PEG was dissolved well in anhydrous DMF solution, respectivelyb-P(CL-coDTC) (1110 mg,0.1 mmol) with DBCO-NH ₂ (40.5 mg,0.15 mmol) in a nitrogen atmosphere, the former was dropwise added to DBCO-NH in a 25 mL closed reactor over 15 min ₂ Then transferred to an oil bath at 37℃and 300 rpm for reaction 48 h. The solution was then dialyzed (MWCO: 3.5 kDa) in DMF overnight, then dialyzed 12 h in DCM, changed 4 times, and precipitated three times with glacial diethyl ether to give DBCO-PEG-b-P(CL-co-DTC). Third step, T22-N ₃ With DBCO-PEG-b-P(CL-coDTC) to obtain T22-PEG through click chemistry reactionb-P(CL-co-DTC). Specifically, DBCO-PEG was added to a 25 mL closed reactor under nitrogen atmosphereb-P(CL-co-DTC)（387.5 mg，0.034 mmol）、DBCO-NH ₂ (40.5 mg,0.0378 mmol) anhydrous DMF (3 mL), after mixing, was fully dissolved and subsequently transferred to an oil bath at 37℃and 300 rpm for reaction 48 h. The purification process is the same as DBCO-PEG b-P(CL-co-DTC). Product yield: 89.9%.

TPMs-V/S are derived from T22-PEG-b-P(CL-coDTC) and PEG-b-P(CL-co-DTC) is mixed with VEN and SOR in a certain ratio,and assembling in an aqueous solution. T22-PEG-b-P(CL-coDTC) is synthesized by NHS-PEG-b-P(CL-co-DTC) and DBCO-NH ₂ Obtaining an intermediate DBCO-PEG through amidation reactionb-P(CL-coDTC) and then re-using the azide group of T22 with the macrocyclic alkynyl group of DBCO by click chemistry, see fig. 2 for a synthetic scheme. PEG-b-P(CL-co-DTC) theoretical molecular weight of 5.0-4.0-2.0 kg/mol, FIG. 3 ¹ H NMR spectrum. GPC test results show that the molecular weight distribution of the polymer is narrowerM _w /M _n =1.1) (table 1) shows that the controllability of the ring-opening copolymerization reaction is good, and the polymer is successfully synthesized. The product was a pale yellow block solid. T22-PEG-b-P(CL-co-DTC)、NHS-PEG-b-P(CL-co-DTC) 、DBCO-PEG-b-P(CL-co-DTC) see fig. 3. The ratio of the areas of the characteristic peaks of NHS and PEG accords with the theoretical value, the grafting rate of NHS reaches 99.9 percent, and the molecular weight of each section of the polymer can be calculated according to the ratio of the integral areas of the corresponding characteristic peaks of DTC, epsilon-CL and PEG (table 1), which is close to the theoretical molecular weight. In addition, the molecular weight distribution of the polymer measured by GPC (FIG. 4) is narrowM _w /M _n =1.2). The above description describes the successful synthesis of T22-PEG by click chemistryb-P(CL-co-DTC); the grafting rate of the T22 peptide is 88.2% by the test of BCA protein kit.

^a For a pair of ¹ H NMR peak area integration; ^b measured by GPC.

Example one preparation and characterization of drug-loaded micelles

All polymer micelles were prepared by solvent displacement. Firstly, PEG-b-P(CL-coDTC) and T22-PEG-b-P(CL-co-DTC) polymers were formulated as DMF solutions at a concentration of 10 mg/mL, respectively. The two polymer solutions are mixed uniformly in a certain volume ratio, for example, when T22-PEG-b-P(CL-co-DTC) molar ratio of 20%When T is obtained ₂₀ PMs are obtained when the molar ratio is 0. The preparation process comprises the steps of taking 0.1 mL polymer solution obtained by mixing according to the proportion, injecting the polymer solution into 0.9 mL PB (pH 7.4, 10 mM) at a constant speed, blowing by a pipette to make the mixed solution uniform, then placing a sample in a 37 ℃ environment for standing overnight, dialyzing (MWCO: 7.5 kDa) 6 h by using PB buffer solution, changing the solution for 5 times to remove organic solvents, and finally obtaining PMs or TPMs, wherein the particle size and the particle size distribution are determined by using DLS.

SOR-carrying single drug micelle (PMs-S), VEN-carrying single drug micelle (PMs-V), co-carrying micelle (PMs-V/S), targeting co-carrying micelle (TPMs-V/S) and the like are similar to empty micelles, except that DMF is used to dissolve powdery drug VEN or SOR respectively, and is configured into 10 mg/mL of drug solution, and then fully mixed with polymer solution according to a preset drug mass ratio, and injected into PB buffer solution with 9 times of volume. The particle size and distribution of the polymer drug loaded micelles were determined by DLS. Drug concentrations of VEN and SOR were determined by HPLC.

T22-PMs-Cy5 is at T22-PEG-b-P(CL-coDTC) solution was mixed with Cy 5-labeled PEG in an amount of 2% by volumeb-P(CL-co-DTC) was prepared from a polymer solution (10 mg/mL) for studying the cell uptake capacity of T22-PMs-Cy 5.

The nano micelle used in the in vivo anti-tumor experiment is basically the same as the preparation method of the drug-loaded micelle, and the difference is that the concentration of the used polymer solution and the free drug solution is improved by 10 times.

To investigate the degree of self-crosslinking of micelles, PEG-P (CL-DTC) was dissolved to 10 mg/mL with DMF, followed by preparation of micelle TPMs (1 mg/mL) in PB (10 mM, pH 7.4), followed by monitoring the change in UV absorption at around 330 nm with PB as a blank and UV-visible spectrophotometry.

To test the stability of TPMs-V/S in 10% Fetal Bovine Serum (FBS), 100. Mu.L of FBS was added to 900. Mu.L of TPMs-V/S micelles, which were then placed in a shaker at 37℃and 200 rpm, and the change in particle size and particle size distribution was monitored with DLS. To test the stability of TPMs-V/S upon dilution, TPMs-V/S micelle concentration was diluted 100-fold with PB buffer and changes in particle size and particle size distribution were monitored using DLS. To test the long-term stability of TPMs-V/S, micelles were stored in a refrigerator at 4 ℃ and particle size distribution changes were monitored on day 0 and day 40, respectively. To test the reduction responsiveness of TPMs-V/S, 10 mM GSH was added to TPMs-V/S, and the particle size and particle size distribution changes were monitored at Nos. 0 h and 6 and 30 h, respectively.

To test the drug release properties of TPMs-V/S, two additional portions of TPMs-V/S at a concentration of 5 mg/mL were taken, one portion was added with 10 mM GSH and placed for 12 h, and the other portion was used as a control, and the release of the drug from TPMs-V/S under reducing conditions was monitored by HPLC. In addition, the dual drug release profile of TPMs-V/S was studied by dialysis using 2 different release media, PB (pH 7.4, 10 mM) with 0.1% tween 80 and PB solution (nitrogen atmosphere) with 0.1% tween 80 and 10 mM GSH, respectively. First, 1 mL of TPMs-V/S (1 mg/mL) was placed in a release bag (MWCO: 100 kDa), then placed in a 25 mL corresponding release buffer, and stored in a shaking table at 37℃and 100 rpm. The 5 mL dialyzate is sucked at each preset time point, then the fresh buffer solution with the same volume is added, after the obtained samples are freeze-dried by a freeze dryer, 300 mu L of ACN is taken to dissolve VEN and SOR, and then the drug concentration in each sample is measured by HPLC, and 3 parallel samples are arranged in each group.

Both VEN and SOR are very hydrophobic drugs with significant hematologic toxicity at the clinically used doses, so it is critical to stably load, co-deliver and controllably release them to avoid leakage and to ensure efficacy. In order to realize the efficient and stable loading and the synergistic targeting delivery of VEN and SOR, the T22 polypeptide modified disulfide cross-linked micelle based on PEG and Polycarbonate (PTMC) is designed and prepared by two amphiphilic block copolymers PEG containing dithiolane groups b-P(CL-coDTC) and T22-PEG-b-P(CL-co-DTC) is assembled in an aqueous solution. Under the conditions that DMF is taken as a polymer and a drug solvent, the ratio of an organic phase to a water phase is 1:9, and stirring is not carried out during dripping, a crosslinked micelle with uniform particle size is prepared, and DLS test shows that the polymer can be assembled into a micelle with the particle size of about 39+/-2 nm in PB, and VEN and/or SOR can be stably entrapped through hydrophobic effect (A in figure 5). Which is a kind ofThe particle size of the traditional Chinese medicine micelle PMs-S or PMs-V is 37-41 nm, and the particle size distribution is narrow (PDI: 0.06-0.2) (Table 2). When the theoretical drug loading of VEN is lower than 20wtWhen the content of PMs is equal to or higher than 92%, the encapsulation rate of PMs-V can reach even if the theoretical drug loading rate is 30%wt% encapsulation efficiency was still higher than 70%, indicating that PMs have efficient encapsulation ability for VEN. In addition, PMs encapsulate SOR at a theoretical drug loading (5-8wtHigher drug loading efficiency (69.3% -77.4%) is maintained when the concentration is lower.

^a The drug concentration is measured by HPLC and then calculated; ^b measured by DLS.

By regulating the drug feeding ratio, a series of PMs-V/S with the actual drug mass ratio (V/S) of 1:1, 1:2, 1:4 and 1:6 (Table 3) are obtained, the particle size of all PMs-V/S is about 37-41 nm, the particle size distribution is narrower (PDI: 0.04-0.07), and the encapsulation rate (DLE) of VEN and SOR is higher >70%). T22 polypeptide functionalized co-supported micelles TPMs-V/S are prepared by reacting PEG-b-P(CL-coDTC) and 10%, 20% or 30% by mole of T22-PEG based on the total polymerb-P(CL-co-DTC) is obtained by means of solvent exchange after mixing. For example when T22-PEG-b-P(CL-co-DTC) at a molar ratio of 20% gives T ₂₀ PMs are obtained when the molar ratio is 0. Their particle size (39-40 nm) and particle size distribution PDI (0.04-0.06) were not significantly different from those of PMs-V/S. Notably, the addition of VEN in double-loaded micelles significantly improved the drug loading and drug loading efficiency of the SOR compared to PMs-S.

^a The drug concentration is measured by HPLC and then calculated; ^b measured by DLS.

By T ₂₀ PMs-V/S _1:4 For example, the morphology, stability, reduction response and drug release of the targeted drug-loaded micelle are studiedIs the following. TEM image display T ₂₀ PMs-V/S _1:4 Has a spherical micelle structure (B in FIG. 5), and PEG-b-P(CL-co-DTC) prepared empty micelles to study self-crosslinking of DTC structures, which showed that the micelles were crosslinked after preparation: the absorbance of the micelle was significantly reduced at 330 nm compared to the DMF solution of the polymer (C in fig. 5), and the degree of cross-linking was further enhanced upon storage at 4 ℃ at 12 h. After storage for 40 days at 4℃or storage in 10% FBS solution for 24 h and dilution with PBS for 100 times, the particle size and particle size distribution of the micelles are substantially unchanged (D in FIG. 5), providing evidence for stable circulation of the micelles in the blood, preventing leakage of the drug entrapped in the core, decrosslinking the polymer micelles (5 mg/mL) under simulated intracellular reduction conditions (10 mM GSH, pH 7.4), increasing the particle size in 12 h, and appearance of a distinct peak (E in FIG. 5) after 30 h, the correlation of the labeled curves measured by this method is better (correlation coefficient) using a liquid chromatography method (F in FIG. 5) capable of separating and quantifying VEN and SOR characteristic peaks >0.999 The characteristic peaks of SOR and VEN are in pure acetonitrile; retention times were around 5.4 min and 6.9 min when water (0.05% phosphoric acid) =72:28, respectively, based on which the release behavior of TPMs-V/S with or without 10 mM GSH reduction was explored. The red line shows that the release amount of VEN and SOR in the drug-loaded micelle without GSH is extremely low (G in figure 5), and the release amount of VEN and SOR is only 1.8% of the release amount of the drug-loaded micelle without GSH by integrating the peak area, which shows that the disulfide crosslinked micelle releases the drug under the reducing condition, and has excellent reduction responsiveness. TPMs-V/S released SOR and VEN in 30H to 84% and 81% respectively under simulated intracellular reduction conditions (10 mM GSH, pH 7.4), whereas SOR and VEN released only 17% and 16% in the absence of GSH (H in FIG. 5).

Example two endocytic experiments

Endocytosis of polymer micelle TPMs of different T22 targeting densities in MV4-11, MOLM-13, OCI-AML3, HL-60 cells was studied using a flow cytometer. Since VEN and SOR are not fluorescent per se, cy 5-labeled PMs were used to advance TPMs of different targeting densities (10%, 20%, 30%)Study was performed. mu.L of the cell suspension (2X 10) ⁵ Individual/well) were plated in 6-well plates, incubator incubated 24 h, then 200 μl of PBS, cy 5-labeled TPMs, or PMs (2.0 μg Cy 5/mL) were added. After co-incubation of 4 h, the cells were collected and centrifuged (1000 rpm,3 min) with a centrifuge, the supernatant was removed, the cells were collected after washing twice with PBS and re-dispersed into 200. Mu.L of ice PBS, which was measured by flow cytometry and analyzed by FlowJo-10 software. The receptor blocking procedure was identical to the endocytic assay, except that 100 μl of free T22 at low, medium, and high concentrations was used to block CXCR4 receptors on the tumor cell surface prior to addition of sample 2 h.

Endocytosis of Cy 5-labeled TPMs in MV4-11, MOLM-13 cells was studied using CLSM. In the CLSM assay, small discs were first placed in 24-well plates and immersed in 8 h by adding 500. Mu.L of PDL solution at a concentration of 0.1 mg/mL to aid in MV4-11 cell attachment. Subsequently PDL was removed and washed 2 times with ice PBS, and MV4-11 cells were washed 2X 10 ⁵ The density of/well was spread in 24 well plates and placed in incubator for incubation 24 h, 200. Mu.L Cy 5-labeled T22-PMs or PMs (40. Mu.g Cy 5/mL) were added, incubated 4 h, washed 2 times with ice PBS, 5. Mu.L PE-anti-human-CXCR4 were added for 20 min, washed 4 times with PBS, cells were fixed with 4% paraformaldehyde for 15 min, washed 4 times with PBS, nuclei were stained with DAPI for 5 min, washed 4 times with ice PBS, finally 10. Mu.L of glycerol-sealed sheet was added, and light was protected with tinfoil. The resulting samples were photographed using CLSM (Leica, TCS SP 5) and analyzed with LAS X software. The method of treatment of TPMS-Cy5 in MOLM-13 cells was the same as that of MV4-11.

Flow cytometry results showed that the endocytosis of TPMs-Cy5 was significantly higher in MV4-11 (FIG. 6A) and MOLM-13 (FIG. 6B) cells than PMs-Cy5, where T ₁₀ The fluorescence intensities of PMs-Cy5 are 7.6 times and 8.4 times respectively compared with that of the PMs-Cy5 control group, and T ₂₀ The cells incubated with PMs-Cy5 had the highest uptake with fluorescence intensities 14.8 and 11.8 times, respectively, compared to the PMs-Cy5 control (Table 4), while T ₃₀ The cellular uptake of PMs-Cy5 was no longer increased, fluorescence intensities were 13.8-fold and 10.9-fold, respectively, compared to the PMs-Cy5 control, indicating that the T22 targeting density reached 20% saturation. Pre-preparation using three concentrations of free T22 polypeptide, low (L), medium (M), high (H)The MV4-11 and MOLM-13 cells were treated and the uptake of TPMs-Cy5 was again tested, as shown in FIG. 6 (A, B). For both AML cells HL-60 (C in FIG. 6) and OCI-AML3 (D in FIG. 6), uptake of TPMs-Cy5 was very low compared to PMs-Cy5, with the highest uptake being only 2.7 and 1.8 fold, respectively (Table 4).

^a Average fluorescence intensity abbreviation; ^b fold relative to PBS group.

Further study of T Using CLSM ₂₀ Endocytosis of PMs-Cy5 and PMs-Cy5 in MV4-11 cells (FIG. 7) and MOLM-13 cells (FIG. 8). At T ₂₀ After incubation of PMs-Cy5 with MV4-11 or MOLM-13 cells for 4 h, the nuclei exhibited a pronounced red fluorescence, whereas the cells incubated with PMs-Cy5 exhibited a weaker fluorescence. Importantly, T ₂₀ The CXCR4 yellow fluorescence was significantly reduced in the PMs-Cy5 group compared to the PMs-Cy5 group, indicating T ₂₀ PMs-Cy5 has previously bound and blocked the CXCR4 receptor prior to mab labelling. The results show that the T22 modified nano micelle has excellent CXCR4 targeting and high-efficiency and rapid cell uptake capacity, can be rapidly endocytosed into cancer cells, has important significance for rapidly and efficiently promoting the combination of VEN and SOR to enter into cells to inhibit the expression of BCL-2 and MCL-1 and cause cytotoxicity, and the cells express CXCR4 receptor in the cells.

Subsequent studies all employed T with a targeting density of 20% ₂₀ PMs-V/S was studied, and the drug mass ratio (S/V) was the actual measurement ratio.

Example three cytotoxicity experiments

Cytotoxicity assays were selected from human acute myeloid leukemia cell lines MV-4-11 and MOLM-13 and healthy mouse fibroblast line L929, mouse myeloid dendritic cells (DC 2.4) and human Peripheral Blood Mononuclear Cells (PBMC). The effect of different drug synergistic ratios and polypeptide densities on the cytotoxicity of TPMs-V/S was studied, and the cytotoxicity of empty micelles and the toxicity of TPMs-V/S on healthy cells were tested.

In vitro synergic anti-tumor activity of VEN and SORThe assay was performed in MV4-11, MOLM-13 cell lines, the two-drug synergy index (Combination Index, CI) was determined by CCK-8 kit staining, enzyme-labelling assay of complex uptake values, software Graphpad prism 8 calculated median lethal concentrations (IC) of PMs-V/S at different mass ratios ₅₀ ) Obtained. MV4-11 cells (2X 10) ⁴ Well, 80 μl) was plated in 96-well plates at 37 ℃ with 5% CO ₂ After incubation of 4 h in an incubator (a), 20. Mu.L of PMs-V/S (SOR concentration range 0.1-100 ng/mL), PMs-V (VEN concentration range 0.1-1000 ng/mL), PMs-S (SOR concentration range 0.1-80 ng/mL) containing different drug mass ratios (S/V=1:1, 2:1, 4:1 and 6:1) were added. After incubation of the samples with cells 48 h, 10 μl of CCK-8 solution was added to each well, incubated in an incubator protected from light for 3 h, and absorbance values of the complex at 450 nm were determined using an microplate reader. Cells with PBS added as a control group had viability as a ratio of absorbance of each well to absorbance of the control group (n=6), and the final presented result was mean ± SD. Half maximal Inhibitory Concentration (IC) on cells ₅₀ ) Is calculated by nonlinear regression in a cell viability curve. The synergy ratio test method of PMs-V/S in MOLM-13 is the same as that of MV4-11, and the difference is that the concentrations of each sample group are different, namely, the PMs-V/S group (SOR concentration range is 0.001-10 ng/mL), the PMs-S group (SOR concentration range is 0.01-50 ng/mL) and the PMs-V group (VEN concentration range is 0.01-50 ng/mL).

The cell viability was calculated as:

Whether or not the SOR and VEN have a synergistic effect is assessed by calculating a synergy index (Combination Index, CI). CI value-Fa (fraction affected) curve was fitted by Compusyn software. When CI is>1 exhibits antagonism, superposition when ci=1, and CI<1 is synergistic when CI<At 0.5, a strong synergy is achieved. The CI value formula is defined as the IC when SOR and VEN are used in combination with each alone ₅₀ The sum of the ratios, i.e

Targeting and cytotoxicity of TPMS-V/S in MV4-11 and MOLM-13 cells was investigated. MV4-11 cells were seeded in 96-well plates (80. Mu.L, 2X 10) ⁴ /hole). T22 with a surface Density of 20% was added ₂₀ PMs-V/S _1:4 ，PMs-V/S _1:4 And free V/S _1:4 (20. Mu.L, SOR concentration range 0.01-30 ng/mL), incubating for 48 h, and measuring absorbance by CCK-8 method with enzyme labeling instrument. Ensuring MOLM-13. The method of treatment of TPMs-V/S in MOLM-13 cells was similar to MV4-11 except that the SOR to VEN mass ratio was 2:1 and the SOR concentration was in the range of 0.001-2.5 ng/mL.

When the toxicity of empty micelle TPMs and PMs to MV4-11 and MOLM-13 cells is verified, the number of cell plating is 2 multiplied by 10 ⁴ The incubation time of the wells was 48 and h, the micelle concentration in the wells was 1-400. Mu.g/mL, and the rest of the procedure was the same as described above.

As can be seen from the CCK-8 test results, the single drug micelle PMs-V and PMs-S have IC in MV4-11 cells ₅₀ The values were 38.58 ng/mL and 9.61 ng/mL (FIG. 9A, table 5), respectively, i.e., SOR micelles were significantly more toxic than VEN micelles. The ratio of the two medicines (VEN: SOR) is 1:1, 1:2, 1:4 and 1:6 respectively, and CI values are smaller than 1 (0.27-0.71), which show good synergistic effect, and when the mass ratio of the carried VEN to SOR is 1:4, the CI value is the lowest, namely PMs-V/S _1:4 Has the strongest synergistic effect. The results of the test in MOLM-13 cells showed that PMs-V and PMs-S were more toxic to MOLM-13 cells than to MV4-11 cells (FIG. 9B, table 5), IC ₅₀ Values of 12.00 ng/mL and 5.28 ng/mL, respectively, and unexpectedly, the CI values were all less than 0.1 (0.07-0.09) at the four drug ratios described above, i.e., a very strong synergy, with the CI value being the lowest at a VEN and SOR loading mass ratio of 1:2.

Based on the cytotoxicity results described above, the synergy of VEN and SOR in MV4-11 cells (C in FIG. 9) and MOLM-13 cells (D in FIG. 9) was further confirmed by CompuSyn software. The results of the CI-Fa simulation obtained by CompuSyn fitting showed that VEN and SOR showed strong synergy for both cells over a broad concentration range.

Subsequent experiments were carried out, as described, with the mass ratio of VEN to SOR in the double drug micelles in MV4-11 and MOLM-13 cells being 1:4 and 1:2, respectively, for continued study.

Study T ₂₀ In vitro antitumor activity of PMs-V/S in MV4-11 (V: S=1:4) and MOLM-13 (V: S=1:2) cells was controlled with free V/S, PMs-V/S. The results showed that T after 48 h co-incubation with MV4-11 cells (E in FIG. 9) ₂₀ PMs-V/S _1:4 IC of medium VEN and SOR ₅₀ Values of 0.24 and 0.94 ng/mL, respectively, compared to PMs-V/S _1:4 (0.55 and 2.21 ng/mL) decrease significantly compared to free V/S _1:4 (0.80 and 3.21 ng/mL) by a factor of about 3.2. T after incubation with MOLM-13 cells 48 h (F in FIG. 9) ₂₀ PMs-V/S _1:2 IC of medium VEN and SOR ₅₀ Values of 0.096 and 0.192 ng/mL, respectively, compared to PMs-V/S _1:2 (0.211 and 0.423 ng/mL) about 2.2-fold decrease compared to free V/S _1:2 (0.251 and 0.502 ng/mL) by a factor of about 2.6.

Empty micelle PMs and T ₂₀ At concentrations of PMs of 1-400. Mu.g/mL, no significant cytotoxicity was observed on both MV4-11 (G in FIG. 9) and MOLM-13 (H in FIG. 9) cells, indicating good biocompatibility of the vector.

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The toxicity of TPMs-V/S, PMs-V/S and free V/S on healthy cells selected from the group consisting of mouse fibroblasts (L929), mouse myeloid dendritic cells (DC 2.4) and human Peripheral Blood Mononuclear Cells (PBMC) was studied. The number of cell plating was: l929, DC 2.4 (5000/well) and PBMC (5X 10) ⁵ Number/well), samples were incubated with cells for 48 h with a VEN to SOR mass ratio of 1:4 and SOR concentration ranging from 0.1 to 1000 ng/mL. The remaining steps are the same as described above. Biocompatibility of the vector VEN and SOR micelle preparation in normal cells such as human peripheral blood mononuclear cell PBMC (a in fig. 10), mouse myeloid dendritic cell DC 2.4 (B in fig. 10), and mouse fibroblast L929 (C in fig. 10) was studied. The results show that even when the SOR/VEN concentration reaches 1000/250 ng/mL, T ₂₀ PMs-V/S _1:4 And PMs-V/S _1:4 Nor showed significant toxicity, with cell viability approaching 100%.

Clinical experiments report that VEN and SOR have serious hematological toxicity, the disulfide-crosslinked polycarbonate micelle is prepared to encapsulate the VEN and the SOR, and the hematological toxicity of TPMs-V/S, PMs-V/S and free V/S is primarily evaluated through a hemolysis experiment. Fresh blood was first obtained by taking healthy mice eyeballs, re-suspending with ice PBS and centrifuging (3600 rpm,10 min), repeating the operation for 5 times until the supernatant became clear, treating the bottom red blood cells with ice PBS to obtain 2% red blood cell suspension, adding 400. Mu.L of red blood cell suspension and 400. Mu.L of Free-V/S, PMs-V/S or TPMs-V/S (SOR concentration range of 1-100. Mu.g/mL, V/S=1:4) respectively to the EP tube, setting PBS as negative control, triton (Triton X-100) as positive control, then incubating the EP tube at 37℃with 200 rpm for 3 h, centrifuging the supernatant with a centrifuge (1500 rpm,5 min), adding 100. Mu.L of supernatant to a 96-well plate, and measuring absorbance at 545 nm with a multifunctional microplate reader. The formula of the hemolysis rate is as follows:

The hemolysis experiment result shows (D in FIG. 10), T ₂₀ PMs-V/S _1:4 Compared with free V/S _1:4 And PMs-V/S _1:4 Has better blood compatibility. Wherein T is in the concentration range of 5-100 mug/mL of SOR concentration ₂₀ PMs-V/S _1:4 With PMs-V/S _1:4 Compared with free V/S _1:4 The hemolysis rate is reduced by 35-47 times and 25-30 times respectively, and the results comprehensively show that TPMs-V/S has low toxicity to normal cells, good biocompatibility and possibility of reducing systemic toxicity in the in vivo circulation process.

Example four apoptosis experiments

Firstly, according to 2.5 multiplied by 10 ⁵ Density of individual/well MV4-11 or MOLM-13 cells were uniformly seeded in 12-well plates, then placed in an incubator for incubation 4 h, and 200. Mu.L of TPMs-V/S, PMs-V/S, free V/S, PMs-S and PMs-V (MV 4-11 or MOLM-13 were added with SOR/VEN at 8/2 or 2/1 ng/mL in wells, respectively, the control group was PBS group, after incubation in incubator 48 h, samples were collected in flow tubes, centrifuged (1000 rpm,3 min) using a centrifuge, the supernatant was decanted, and then the bottom of the flow tubes was resuspended in ice PBS to accumulate MV4-11 cells, and repeated twice. 200. Mu.L of Binding buffer was resuspended in two PBS groups, then one group was mixed with 500. Mu.L of positive control solution, and the mixture was left to stand in ice for 0.5. 0.5 h, followed by washing with ice PBS, and the supernatant was removed. And re-suspending with Binding buffer, mixing with untreated PBS group, and dividing into three parts, wherein two parts are positive single-dyeing control, and one part is blank control. In the single-dye early-apoptotic group, 5. Mu.L of Annexin V-APC was mixed into a group of cell suspensions; in the single-dye late-apoptotic group, 10. Mu.L of 7-AAD was added to the cell fluid of the other group for staining. In the sample group, 200. Mu.L of Binding buffer resuspended cells (density of about 1X 10 per group) ⁶ mu.L of Annexin V-APC and 10. Mu.L of 7-AAD dye were mixed into each experimental group, stained with tinfoil for 5 min at room temperature in the dark, and measured in 1 h using a flow cytometer. T was studied by the annexin V-APC/7-AAD double-dyeing technique ₂₀ Apoptosis after 48 h incubation of PMs-V/S, PMs-V/S, free V/S, PMs-S and PMs-V with MV4-11 and MOLM-13 cells at optimal drug agreement ratios. In the preparation incubated with MV4-11 cells, the concentration of VEN was 2 ng/mL and the SOR concentration was 8 ng/mL; in the preparation incubated with MOLM-13 cells, the concentration of VEN was 1 ng/mL and the SOR concentration was 2 ng/mL. As can be seen from fig. 11, T ₂₀ PMs-V/S is effective in inducing apoptosis in both cells. After 48-h co-incubation with MV 4-11T ₂₀ PMs-V/S induced 36.2% apoptosis, significantly higher than control (27.1%), free (26.5%), PMs-S (19.1%) and PMs-V (9.9%). Similarly, T after 48-h co-incubation with MOLM-13 ₂₀ PMs-V/S _1:2 Can induce 26.5% apoptosis, the apoptosis rate is obviously higher than that of a control PMs-V/S group (18.0%) and a free V/S group (13.5%), a PMs-S group (8.7%) and a PMs-V group (5.7%), and the PMs-V/S group shows stronger synergistic effect compared with the PMs-S group and the PMs-V group.

Example five animal and tumor model establishment

All animal experiments and procedures were approved by the university of su laboratory animal center and the university of su animal care and use committee. Balbc female mice (Vetolihua) with the age of 8 weeks and the weight of 19-21 g are used in the acute toxicity test, and NOD.CB17-Prkdcscid/IL2rgtm1/Bcgen (B-NDG, baioerskin) female mice with the age of 9 weeks and the weight of 20-22 g are used in the biodistribution test and the anti-tumor treatment test.

In situ MV4-11 AML tumor model establishment: mu.L MV4-11 cells (5X 10) were injected using a 1 mL syringe ⁶ And/or) was injected into the B-NDG mice via the tail vein, and the inoculation was on day 0. Treatment was started on day 3 post inoculation, while mice body weight was monitored every three days.

To assess the disease progression of MV4-11 cells in mice, orbital blood was taken every three days after inoculation and the infiltration development of AML cells in the peripheral blood of mice was measured by flow cytometry.

EXAMPLE six acute toxicity and biodistribution experiments

To further evaluate hematological toxicity of TPMs-V/S, experiments used healthy Balbc female mice (19-21 g,8 weeks old), randomly grouped, 4 in each group, 3 in total, each including T ₂₀ PMs-V/S, healthy and oral. T (T) ₂₀ The PMs-V/S group (2.5/10 mg VEN/SOR equiv./kg) was administered by tail vein injection for two days, one needle for 4 total needles, and the free drug group (150/30 mg VEN/SOR equiv./kg) was administered by oral gavage for 7 consecutive days. Mice were observed for body weight and health continuously for 8 days after dosing, and groups of mice were dissected on day 9, and blood was collected intraperitoneally for routine blood measurement and blood biochemistry.

Both VEN and SOR produce significant hematologic toxicity in the clinical treatment of AML due to large doses, limiting the dosage and therapeutic effect of the drug, especially the combination of VEN and SOR produces more significant additive toxicity, which is a challenge for the combination therapy. The acute toxicity of TPMs-V/S and mouse tolerance of healthy Balbc female mice were evaluated, and the dosing regimen is shown in FIG. 12A. Wherein the control group free V/S was orally administered at the dosages and ratios reported in the literatureThe administration mode was continuously followed by gastric lavage for 7 days, and the results showed that when the administration dose of free V/S was 150/30 mg VEN/SOR equiv/kg, the mice continued to lose weight during the administration period (B in FIG. 12), approaching 85% of the initial weight at day 8, and were accompanied by characteristics of dehairing, loss of appetite, darkening hair, weakness of hind limbs, etc., while T was the same as the above ₂₀ PMs-V/S（2.5/10 mg VEN/SOR equiv./kg，i.v.) Has better tolerance and does not show abnormal toxic and side effects with the healthy group. Blood routine data (C in FIG. 12) shows that oral V/S group White Blood Cells (WBC), red Blood Cells (RBC), hemoglobin (HGB), platelets (PLT), neutrophils (Neut) and lymphocytes (Lymph) were markedly abnormal, whereas T was markedly abnormal in the healthier group ₂₀ The PMs-V/S group indicators, except for lymphocyte lowering, were all close to the healthy group data, indicating a significant improvement in toxicity of the combination treatment compared to the free V/S group TPMs-V/S group. Blood biochemical data (D in FIG. 12) shows that oral administration of V/S showed significantly reduced liver function index glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), serum alkaline phosphatase (ALP) and renal function index Creatinine (CREA), UREA (UREA) compared with healthy group, and showed stronger liver and kidney injury, while T ₂₀ The PMs-V/S group significantly improved the damage to AST, CREA, UREA. The results comprehensively show that the TPMs-V/S effectively reduce the toxic and side effects of VEN and SOR, widen the administration window, and have good in vivo biocompatibility. The VEN dose was reduced by 15 times and the SOR was reduced by two times, resulting in a longer survival.

Tumor targeting ability was examined in MV4-11 in situ AML mouse xenograft model using ex vivo fluorescence imaging and Cy 5-labeled T22-PMs, mice were randomly grouped on day 20 post-inoculation, 200 μ L T-PMs-Cy 5 and PMs-Cy5 (0.5 μg Cy5 /) were injected into mice via tail vein, two groups of three mice each, and the calf bones were removed for ex vivo fluorescence imaging at 8 h post-injection, and then imaged pictures were analyzed using Lumia II software. PMs-Cy5 and TPMs-Cy5 were injected via tail vein respectively on day 20 of modeling of in situ MV4-11 AML mice, and after 8 hours the mice were sacrificed to remove major viscera, anterior calf bone and posterior calf bone for fluorescence imaging. The in vitro imaging results showed that the fluorescence intensity of the TPMs-Cy5 group was significantly higher than that of PMs-Cy5 in both the anterior and posterior leg bones of mice (a in fig. 13), indicating that its ability to specifically target AML cells at highly concentrated bone marrow was very strong. The enrichment of the TPMs-Cy5 group in the lung was also significantly enhanced (B in fig. 13) because AML cells had invaded the lung and produced significant tumor infiltration in the late MV4-11 model.

Example seven in vivo anti-tumor Activity assay and histological analysis

To study the antitumor effect of TPMs-V/S, PMs-V/S, PMs-V, PMs-S and free V/S on in situ AML mice, in situ xenograft models of mice were established with MV4-11 cell vaccination, mice were randomly divided into 10 groups of 9, 4 for anatomical infiltration, blood convention, blood biochemistry, etc., 5 for monitoring body weight and for observing survival prior to dosing. The administration groups were defined as low dose group (L) with ven=1.25 mg/kg or sor=5 mg/kg, and high dose group (H) with ven=2.5, 10 mg/kg or sor=10 mg/kg, respectively by tail vein injection of TPMs-V/S _1:4 （H）、TPMs-V/S _1:1 （H）、TPMs-V/S _1:4 （L）、PMs-V/S _1:4 （L）、PMs-V（L）、PMs-S（L）、Free-V/S（L,i.v.) And a control PBS group, in addition, a control group was provided, and the Free V/S (150/30 mg VEN/SOR equiv./kg) was orally administered by stomach irrigation, denoted as Free-V/S #p.o.). Each tail vein dosing group was dosed at day 3 post-inoculation, 200 μl each, 10 total times, and the oral group was given a total of 10 gastric lavages per cycle, 5 consecutive times per cycle, followed by 9 days of rest, according to the maximum tolerated dose of the literature. The survival period was continuously observed during the treatment period, and the body weight of the mice was recorded every 3 days. PBS group was dissected at the time of dying, and viscera, leg bones, and abdominal blood were taken as controls for each experimental group. Mice survival was observed continuously after treatment. On the 32 th day of inoculation, the weight of the double-drug targeting-free micelle group is reduced, 4 mice in each group are dissected, and abdominal arterial blood is taken for monitoring blood routine and blood biochemistry; weighing spleen weight; taking liver, spleen, lung, rear leg bones and peripheral blood, and monitoring the leukemia infiltration degree by a flow cytometer; heart, spleen, liver, kidney, lung and rear leg bones were collected and fixed with 4% paraformaldehyde by H &E. TRAP stainingHistopathological analysis was performed, femur and tibia were scanned by Micro CT (SkyScan 1176, belguim), and bone tissue damage was analyzed and quantified using NRecon, dataViewer and CTAn software.

Different preparation groups are designed to examine the influence of modified T22 polypeptide, different medicine proportions and different dosages on the anti-tumor performance of the TPMs-V/S, and meanwhile, compared with oral large-dose medicines and tail vein injection free medicines reported in literature, the clinical application potential of the TPMs-V/S is examined. First, B-NDG mice were injected 5X 10 by tail vein ⁵ The individual MV4-11 cells construct a mouse in situ model. The treatment was randomized and started on day 3 post-inoculation, with one needle every 3 days for each tail iv group, for a total of 10 needles, with each of the free V/S, PMs-V, PMs-S, PMs-V/S and TPMs-V/S groups, respectively, with TPMs-V/S set in three groups to explore the effect of dose and mass ratio on the treatment effect, with the free V/S group set with oral and tail iv groups as controls. The construction and treatment workflow for in situ MV4-11 AML mouse transplantation model is as follows (A in FIG. 14). The oral free V/S control group was administered by intragastric injection at the doses reported in the literature for free V/S (150/30 mg VEN/SOR equiv./kg) with PBS as the control. There were 9 tumor-bearing mice per treatment group, of which 4 were anatomically analyzed for AML related indicators and 5 were used to monitor body weight and observe survival.

The degree of peripheral blood infiltration of the PBS group was monitored by orbital blood extraction and flow cytometry (B in FIG. 14), and tumor cell infiltration was monitored to increase over time until about 5% -10% of mice died by peripheral blood infiltration, while about 20%, 70%, 40% of AML cell infiltration in bone marrow, liver, spleen was found by dissected dying PBS mice in A in FIG. 15, and the above combinations indicated successful establishment of the in situ MV4-11 model. The experimental results (C in fig. 14) showed that all mice in the tail intravenous group had no significant change in body weight during the administration period, indicating that they had lower toxic side effects, whereas the oral free V/S group had a continuous decrease in body weight during the administration period, and slowly recovered after discontinuing drug treatment, indicating that this dose of V/S (150/30 mg VEN/SOR equivalent/kg) had stronger toxic side effects on the mice. As a control, the prior art considers that pharmaceutical combinations that jointly inhibit BCL-2 and MCL-1 proteins have significant systemic toxicity to the murine in situ model, and furthermore, oral clinical doses of VEN or SOR in AML patients often produce higher toxic side effects and may lead to discontinuation of treatment.

Survival outcome shows (D in fig. 14): first, the PBS group mice developed rapidly, symptoms such as weight loss, slow action, paralysis of lower limbs, dark hair, hair loss and black and hard feces began to appear at day 18 after inoculation, mice died at day 19, all the mice died at day 21, and median survival time was 20 days. Second, the formulation group PMs-V (L), PMs-S (L), free V/S (L, i.v.) PMs-V/S _1:4 (L) exerts a certain tumor-inhibiting ability, and the median survival time is 21 days, 26 days, 25 days and 34 days, respectively. PMs-V/S _1:4 (L) a 1.6-fold increase in survival compared to the first two formulations,p =0.0026<0.01 1.3 times (x),p =0.0018<0.01 Exhibits good synergy and shows a therapeutic effect superior to that of the free pharmaceutical combination,p =0.0021<0.01). Third, TPMs-V/S _1:4 The median survival of (L) reached 39 days, was PMs-V/S _1:4 1.2 times (x) of (L),p =0.0021<0.01). Fourth, high dose targeting group TPMs-V/S _1:1 (H) And TPMs-V/S _1:4 (H) Median survival was 41 and 46 days (x,p =0.0019<0.01). Fifth, TPMs-V/S _1:4 (H) The curative effect is stronger than that of the free V/S groupp.o.) Is set for the first time period of 38 days,p =0.0018<0.01 The weights of the spleens of the two groups are similar to that of the mice in the healthy group, and are significantly lower than those of the other treatment groups such as the oral group, and the phenomena of splenomegaly and the like do not occur (E in fig. 14).

The results show that the proportion, the dosage and the targeting capability of the micelle preparation are limited, the obtained TPMs-V/S preparation is superior to a free drug group for intravenous administration in curative effect, and compared with a clinically used oral drug mode, the TPMs-V/S preparation also has better safety and anti-tumor performance, effectively inhibits proliferation of AML cells and prolongs the survival period of mice. Importantly, the oral treatment group has reached a large tolerating dose, and TPMs-V/S formulations have a great potential for widening the therapeutic effect due to good biocompatibility and active selectivity, and more dose-enhancing space. For highly malignant tumors such as AML, early administration of high doses of drugs approaching tolerizing doses is extremely important for rapid reduction of tumor cell numbers, prevention of drug resistance, and prolongation of survival, so subsequent experiments with TPMs-V/S can increase doses to achieve longer survival in AML mice.

Tumor cell infiltration is an important index for evaluating treatment effect, and the main organs, bone marrow and peripheral blood of a model mouse are monitored by adopting flow cytometry, so that the treatment effects of various preparations are compared. On day 32 post-inoculation, 4 mice were dissected from each group when PMs-V/S mice were nearly killed, peripheral blood, liver, spleen, lung and rear leg were collected, APC-anti-human-CD45 antibody-labeled MV4-11 cells were added after cell collection, and AML cell infiltration of each organ of the mice was examined by flow cytometry, with PBS as a control, dissected when the group was nearly killed (day 19-20). The results showed that the Liver (LI), spleen (SP), bone Marrow (BM) and Peripheral Blood (PB) of PBS, PMs-V/S mice had significant leukemia cell infiltration (A in FIG. 15), indicating that tumor cells of the model were metastasized throughout multiple organs, consistent with literature reports. Notably, the leukemia burden in mice was significantly reduced following treatment with targeted high dose group TPMs-V/S (H), no significant leukemia infiltrate in liver, spleen, bone marrow and peripheral blood, with infiltration rates below 0.5% (B in fig. 15), where TPMs-V/S _1:4 (H) The tumor infiltration of each part is the lowest, which shows that the VEN/SOR=1:4 has the optimal synergistic effect. Second, TPMs-V/S (L) significantly reduce tumor cell infiltration compared to PMs-V/S (L). Finally, although oral V/S group significantly reduced tumor burden compared to PBS group and even had better efficacy than non-targeted group PMs-V/S (L), it only partially reduced leukemia infiltration in liver, spleen, bone marrow and peripheral blood, with poorer efficacy than TPMs-V/S (H) group. The results comprehensively show that the TPMs-V/S increases the anticancer effect of VEN and SOR in the in-situ MV4-11 AML mouse model, has greater application potential than the clinical oral large-dose medicine, and has the TPMs-V/S is particularly effective in inhibiting proliferation and metastasis of AML cells in mice, and can almost completely inhibit leukemia cells in bone marrow (99.8%). Just as most anti-AML therapies fail due to low BM availability, TPMs-V/S are of particular importance for effective alleviation of leukemia cells in the BM niche.

Clinical treatment is often assisted by routine blood and biochemical blood tests to determine the tolerance of AML patients to drugs. Mice were monitored for blood normative and blood biochemical indicators by taking peritoneal blood to aid in the observation of therapeutic effects and hematological toxicity, with PBS groups sacrificed at the time of dying (19-20 days) and healthy groups as controls for the experiment, with the remaining groups monitored on day 32 (at which time the oral group ended treatment day 11, the formulation group ended treatment day two).

Blood routine results indicated (fig. 16 a) that the PBS group showed a significant decrease in red blood cells, platelets, hemoglobin compared to the healthy group, because the large number of leukemia cells filled the bone marrow, the volume of the bone marrow cavity was limited, and normal hematopoietic function was inhibited. Compared with the healthy group, red blood cells, hemoglobin, platelets, white blood cells and the like of the two groups of TPMs-V/S (H) have no obvious difference, and other blood routine indexes also indicate that no obvious hematological burden is generated in treatment, so that the treatment effect and hematological toxicity of the TPMs-V/S (L) are superior to those of the TPMs-V/S (L), PMs-V/S (L) and the oral group. Notably, although oral group V/S was found in survival experiments and tumor infiltration assaysp.o.) The therapeutic effect is stronger than that of PMs-V/S (L) group, but the conventional indexes of blood are obviously lower than that of healthy group, so that the great hematological toxicity is caused, the treatment of hematopathy tumor is extremely unfavorable, the preparation group obviously improves the defect, and the conclusion of acute toxicity test, namely, the micelle preparation has good biocompatibility, is again proved. In addition, oral administration of V/Sp.o.) Red blood cell related indicators red blood cell number (RBC), hematocrit (HCT), mean red blood cell volume (MCV), red blood cell distribution width (RDW), hemoglobin related indicators hemoglobin concentration (HGB), mean red blood cell hemoglobin content (MCH), mean hemoglobin concentration (MCHC), hemoglobin distribution Width (HDW) and the like all showed significant toxicity compared with healthy groups, and the mice were shown to have severe anemia. Secondly, oral administration group V/Sp.o.) Platelet indicators including platelet count (PLT), platelet Pressure (PCT) and average Platelet Concentration (PCV) were significantly worse than in healthy and formulation groups, suggesting that the free drug has greater toxicity to platelets. In addition, oral administration group V/Sp.o.) Indicators of various immune cells, including white blood cell count (WBC), eosinophil (EOS), neutrophil (Neut) and lymphocyte (Lymph) also showed greater toxic side effects, whereas TPMs-V/S (H), similar to the healthy group, did not show such toxicity. The micelle formulation group did not show effective relief of lymphocyte (Lymph) toxicity. Taken together, the oral group showed hematologic toxicity consistent with clinical reports of high doses of VEN and SOR, and unexpectedly, the micelle formulation TPMs-V/S of the present invention was effective in alleviating such hematologic toxicity.

Blood biochemical data show (fig. 16B) that glutamic oxaloacetic transaminase (AST) and alkaline phosphatase (ALP) of the oral group are significantly higher than those of other treatment groups and healthy groups, and are only superior to those of PBS group, which indicates that the oral group has great damage to liver function, and each micelle preparation group effectively relieves the toxicity of the drug to liver. In addition, the glutamic pyruvic transaminase (ALT) index of the oral group is severely reduced, which suggests that the oral group has acute hepatitis symptoms, and the micelle preparation group has alleviation. Creatinine (CREA) was also abnormally elevated in the oral group, indicating that the renal function of mice in this treatment group was also impaired to some extent, while no significant differences were seen in the other treatment groups. The above hematological data indicate that the TPMs-V/S group has better therapeutic effect and lower toxicity than clinically employed oral therapies.

By tissue paraffin section and H&Histological analysis was performed on each treatment group by E staining and Trap staining, PBS group and healthy group were used as controls, PBS was sampled at the time of dying (19-20 days), and the other groups were sampled after treatment by selecting one mouse at day 32 of inoculation. The results show that: PBS group and PMs-V/S _1:4 AML cells were seen as scattered infiltration in liver, lung, spleen, and hepatocyte morphology in group (L) miceChanges, gap increases, edge blurring, irregular arrangement, obvious change of morphology, great reduction of red blood cells, infiltration of AML cells which can be seen as clusters in spleen and reduction of red marrow compared with healthy groups of peripheral structures of pulmonary bronchus and pulmonary arterial vessels. Notably, free V/Sp.o.) The group spleen shows severe bleeding phenomenon (white dotted line area), which indicates that the free medicine has strong superposition toxicity, but no obvious AML cells are found in the liver and spleen of the other groups, the liver cells are normal and uniform in morphology, ordered in arrangement, and free from congestion and edema in liver sinus; the spleen red and white marrow are abundant and evenly distributed, no obvious primary and secondary lymphatic follicles are seen, the lung structure is clear, and the lung interstitium is not widened (fig. 17). In addition, the heart and the kidney of each group of mice are not obviously abnormal, myocardial fibers are continuous, the structure is clear, congestion and edema are avoided, the glomerular structure is clear, the tubular distribution is uniform, and no obvious tumor infiltration exists.

H of tibia and femur&E-staining results show (FIG. 18), PBS group and PMs-V/S _1:4 The bone marrow of group (L) mice was seen to be infiltrated with a large number of AML cells (black dotted line area) and hematopoietic stem cells were reduced with free V/S [ (]p.o.) Group and TPMs-V/S _1:4 Small infiltration was seen in the bone marrow of group (L) mice and partial cell morphology was changed, TPMs-V/S _1:1 Group (L) and TPMs-V/S _1:4 (H) As with healthy mice, AML cells are not found in bone marrow, and no obvious abnormality is found in the morphology and structure of bone marrow hematopoietic stem cells.

Progression of AML is often accompanied by the occurrence of bone lesions. To evaluate the effect of TPMs-V/S treatment on AML mice bone injury, tartaric acid-resistant phosphatase (TRAP) staining analysis and microcomputer tomography (Micro CT) analysis were performed to evaluate osteoclast content and bone resorption. AML cells in bone marrow stimulate osteoclastogenesis, increase bone resorption, cause osteolysis and osteoporosis, and high expression of tartrate-resistant acid phosphatase (TRAP) is one of the main markers of osteoclasts. TPMs-V/S by TRAP staining _1:4 (H) Group and TPMs-V/S _1:1 (H) The osteoclast (white arrow) content of the group was evidentBelow untreated PBS group, PMs-V/S _1:4 Group (L), free V/Sp.o.) Group and TPMs-V/S _1:1 Group (L) (FIG. 19), wherein TPMs-V/S _1:4 (H) The group is similar to the healthy group, which shows that the TPMs-V/S effectively inhibit the activity of the osteoclast and obviously relieve the bone injury condition of mice. Micro CT image display (FIG. 20A), PMs-V/S _1:4 Group (L) and free V/Sp.o.) Trabecular mass loss of group bones, TPMs-V/S _1:4 The bone structure of group (L) was significantly improved, while TPMs-V/S _1:4 (H) The group is similar to the healthy group. The results of Micro CT quantitative analysis showed that the Bone Mineral Density (BMD), bone trabecular thickness (Tb. Th) and bone volume fraction (BV/TV) were significantly reduced in the PBS group of mice in the healthier group (B-C in FIG. 20). While TPMs-V/S _1:4 (H) The trabecular tissue integrity and bone density of the group were similar to those of healthy mice, which was consistent with the observation that the group of bone marrow had very little leukemia cell infiltration and very little osteoclast generation. The results prove that the micelle TPMs-V/S provided by the invention has remarkable targeting, therapeutic effect and safety on AML cells in vivo, and has good prevention and protection effects on bone injury caused by AML.

Aiming at the AML of the FLT3-ITD subtype, the invention designs and prepares the disulfide cross-linked polymer micelle (TPMs-V/S) of the T22 polypeptide modified co-carried VEN and SOR for the high-efficiency targeted treatment of the in-situ MV411 (FLT 3-ITD subtype) AML mice. The TPMs-V/S has the excellent properties of small size (40-nm), good reduction responsiveness, controllable drug proportion, adjustable surface polypeptide density, specific targeting receptors, low toxic and side effects and the like, can stably co-load in an optimal synergistic ratio, release VEN and SOR in a targeted manner, and effectively inhibit AML cell proliferation. The TPMs-V/S has small size, is easy to enrich in the AML infiltration part, and can increase the effective drug concentration of the tumor part and enhance the anti-tumor effect and reduce the toxic and side effects by reducing the responsive drug release. Cell experiments show that TPMs-V/S can be efficiently ingested by AML cells MV4-11 and MOLM-13 to cause cytotoxicity and apoptosis, and has very little cytotoxicity to normal cells and excellent anti-tumor effect. Acute toxicity and biodistribution experiments show that TPMs-V/S greatly reduce the hematological toxicity of free drugs, and compared with non-targeting micelle PMs-V/S, the TPMs-V/S can be specifically enriched to the liver, spleen and bone marrow infiltrated by AML cells. Treatment experiments aiming at an in-situ MV4-11 (FLT 3-ITD subtype) AML mouse model show that TPMs-V/S remarkably reduce drug dosage and systemic toxicity, greatly relieve hematological injury, inhibit proliferation and transfer of AML cells in a mouse body, almost completely inhibit AML cells in bone marrow, efficiently inhibit osteoclast activity, improve bone injury condition of the mouse, protect bone tissue of the mouse and remarkably prolong median survival time of the mouse. The targeted TPMs-V/S micelle nano-drug is an effective strategy for clinically treating FLT3-ITD subtype acute myeloid leukemia.

Claims (10)

1. A targeted dual anti-apoptosis protein polymer micelle comprises a polymer carrier and a small molecule drug which simultaneously inhibits the expression of anti-apoptosis proteins BCL-2 and MCL-1.

2. The targeted dual anti-apoptotic protein polymer micelle according to claim 1, wherein said small molecule drugs that inhibit expression of anti-apoptotic proteins BCL-2 and MCL-1 simultaneously are two.

3. The targeted dual anti-apoptotic protein polymer micelle according to claim 2, wherein said small molecule drugs that inhibit expression of anti-apoptotic proteins BCL-2 and MCL-1 simultaneously are viterbi and sorafenib.

4. The targeted dual anti-apoptotic protein polymer micelle according to claim 1, wherein said polymer micelle is assembled from a non-targeted polymer or assembled from a non-targeted polymer and a targeted polymer.

5. The targeted dual anti-apoptotic protein polymer micelle according to claim 4, wherein the non-targeted polymer is comprised of a hydrophilic segment and a hydrophobic segment; the targeting polymer consists of a targeting molecule, a hydrophilic chain segment and a hydrophobic chain segment; the hydrophobic chain segment is obtained by copolymerizing 1, 2-dithiolane trimethylene carbonate and other monomers.

6. The targeted dual anti-apoptotic protein polymer micelle according to claim 5, wherein the molecular weight of the hydrophilic segment is 2-10 kDa and the molecular weight of the hydrophobic segment is 0.8-3 times of the molecular weight of the hydrophilic segment; among the hydrophobic chain segments, the molecular weight of the chain segment formed by the 1, 2-dithiolane trimethylene carbonate units is 20-45% of the molecular weight of the hydrophobic chain segment; the targeting molecule is a polypeptide.

7. The method for preparing the targeted dual anti-apoptotic protein polymer micelle according to claim 1, wherein a non-targeted polymer and a small molecule drug which simultaneously inhibits the expression of anti-apoptotic proteins BCL-2 and MCL-1 are used as raw materials, or a non-targeted polymer and a targeted polymer are used as raw materials, and a small molecule drug which simultaneously inhibits the expression of anti-apoptotic proteins BCL-2 and MCL-1 is used as raw materials; the targeted dual anti-apoptotic protein polymer micelle is prepared by a solvent substitution method.

8. The method for preparing the targeted dual anti-apoptotic protein polymer micelle according to claim 7, wherein the mole fraction of the targeting polymer is 0-30% and not including 0 in the non-targeting polymer and the targeting polymer.

9. The use of the targeted dual anti-apoptotic protein polymer micelle of claim 1 in the preparation of a hematological tumor treatment drug.

10. The use according to claim 9, wherein the hematological neoplasm is leukemia.