A targeting dual anti-apoptotic protein polymer micelle and a preparation method and application thereof

Abstract

The application discloses a kind of targeted dual anti-apoptotic protein polymer micelles and its preparation method and application, to overcome single drug resistance with two kinds of small molecule drugs for inhibiting the expression of anti-apoptotic protein BCL-2 and MCL-1 simultaneously loaded by polymer carrier.VEN and SOR in clinical treatment are obviously hematological toxicity due to the need to use high dose, reduce the overall treatment effect, the application discloses new targeted dual anti-apoptotic protein polymer micelles, effectively solve the problems of large dose, toxicity in the prior art.The experimental results show that TPMs-V / S effectively reduces the dose, toxic side effects of VEN and SOR, effectively improves the curative effect of double drug.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a targeted dual anti-apoptotic protein polymer micelle, its preparation method and application, for active targeted therapy in an orthotopic acute myeloid leukemia mouse model. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant hematologic disease caused by abnormal clones of myeloid hematopoietic stem cells in the bone marrow. It is highly susceptible to drug resistance, relapse, and metastasis. The disease exhibits high tumor heterogeneity and can activate multiple anti-apoptotic drug resistance signaling pathways. Monotherapy often leads to relapse and drug resistance, making it difficult to achieve sustained and in-depth remission. Therefore, combined targeted therapy targeting multiple pathways may be a more effective approach to improve the survival rate of the AML population. Theoretically, the FLT3 inhibitor SOR can be used in combination with VEN to simultaneously inhibit both BCL-2 and MCL-1 targets. However, both VEN and SOR have significant hematological toxicity in clinical treatment due to the need for high doses, reducing overall therapeutic efficacy [DiNardo, CD; Pratz, KW; Letai, A.; Jonas, BA; Wei, AH, et al., Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-randomised, open-label, phase 1b study. The Lancet Oncology 2018, 19 (2), 216-228]. Currently, the key challenge facing this combination therapy is to address the additive toxicity of the combination therapy, enhance the targeting selectivity for tumor cells, reduce toxicity to normal tissues and organs, and broaden the therapeutic window.

[0003] Currently, several nanoparticle formulations have been approved or entered different stages of clinical trials. For example, liposomal cytarabine (Depocyte) has been used in clinical research for AML, but its accumulation at tumor sites is low. This class of drugs still faces challenges such as low tumor accumulation, poor target selection, and weak endocytosis, resulting in limited improvement in efficacy. To improve tumor accumulation and tumor cell uptake, peptides are bonded to the surface of nanosystems; to further improve treatment efficacy and overcome monotherapy resistance, AML treatment often uses combinations of two or more anticancer drugs to enhance therapeutic effects. However, it is worth noting that these dual-drug nanoparticle formulations still face problems such as poor in vivo stability, low tumor accumulation, and slow drug release. Summary of the Invention

[0004] Existing VEN and SOR treatments require high doses in clinical practice, resulting in significant hematologic toxicity and reduced overall therapeutic efficacy. This invention discloses a novel targeted dual anti-apoptotic protein polymer micelle, which effectively solves the problem of high toxicity in existing technologies.

[0005] The present invention adopts the following technical solution:

[0006] A polymeric micelle targeting dual anti-apoptotic proteins includes a polymeric carrier and a small molecule drug that simultaneously inhibits the expression of anti-apoptotic proteins BCL-2 and MCL-1; preferably, the small molecule drug that simultaneously inhibits the expression of anti-apoptotic proteins BCL-2 and MCL-1 is two drugs, preferably veteclans (VEN) and sorafenib (SOR).

[0007] In this invention, polymer micelles are assembled from non-targeted polymers or from a combination of non-targeted and targeted polymers. The non-targeted polymers consist of hydrophilic and hydrophobic segments; the targeted polymers consist of a target molecule, hydrophilic segments, and hydrophobic segments. The hydrophilic segments have a molecular weight of 2–10 kDa, and the hydrophobic segments have a molecular weight 0.8–3 times, preferably 1–2 times, that of the hydrophilic segments; the hydrophilic segments are preferably polyethylene glycol segments. The hydrophobic segments are obtained by copolymerizing 1,2-dithiopentanetrimethylene carbonate with other monomers, such as ester monomers or carbonate monomers, like CL, TMC, and LA; the molecular weight of the segments composed of 1,2-dithiopentanetrimethylene carbonate units is 20–45%, preferably 25–35%, of the hydrophobic segment molecular weight. The target molecule is preferably a polypeptide.

[0008] In this invention, non-targeted polymers and small molecule drugs that simultaneously inhibit the expression of anti-apoptotic proteins BCL-2 and MCL-1 are used as raw materials, or non-targeted polymers and targeted polymers and small molecule drugs that simultaneously inhibit the expression of anti-apoptotic proteins BCL-2 and MCL-1 are used as raw materials; targeted dual anti-apoptotic protein polymer micelles are prepared by solvent displacement method.

[0009] Preferably, in the non-targeted polymer and the targeted polymer, the molar fraction of the targeted polymer is 0-30% and does not include 0, more preferably 10-30%, and even more preferably 10-20%.

[0010] This invention discloses the application of the above-mentioned targeted dual anti-apoptotic protein polymer micelles in the preparation of a drug for treating hematologic malignancies. Preferably, the hematologic malignancy is leukemia, and more preferably, the hematologic malignancy is acute myeloid leukemia.

[0011] This invention, PMs-V / S, can significantly kill AML cells at a fixed drug synergistic ratio. Furthermore, by modifying the T22 peptide, the cytotoxicity of PMs-V / S against AML cells is significantly enhanced, efficiently inducing apoptosis, especially with low toxicity to normal cells. Free veteclam (VEN) and sorafenib (SOR) can effectively kill AML cells, but they suffer from high dosage, low bioavailability, and significant additive toxicity. Nanocarriers can stably encapsulate drugs and improve drug circulation; however, these nanocarriers are limited by a lack of tumor targeting, weak tumor cell uptake, and / or slow intracellular drug release. This invention designs and prepares targeted T22 peptide-functionalized, highly efficient disulfide cross-linked polymer micelle nanomedicines (TPMs-V / S) loaded with VEN and SOR. These nanomedicines efficiently target and rapidly release VEN and SOR into leukemia cells, while simultaneously inhibiting the expression of anti-apoptotic proteins BCL-2 and MCL-1, initiating the apoptosis process in AML cells, thereby inhibiting AML cell proliferation and metastasis, especially with low dosage, high bioavailability, and low additive toxicity. Attached Figure Description

[0012] Figure 1 T22 peptide-modified disulfide cross-linked micelles co-deliver veteccralfate and sorafenib (TPMs-V / S) for highly effective targeted therapy in a mouse model of orthotopic MV4-11 acute myeloid leukemia.

[0013] Figure 2 Synthetic routes for (A) PEG-bP(CL-co-DTC) and (B) T22-PEG-bP(CL-co-DTC).

[0014] Figure 3The 1H NMR spectra (400 MHz, DMSO-d6) of (A) PEG-bP(CL-co-DTC), (B) NHS-PEG-bP(CL-co-DTC), (C) DBCO-PEG-bP(CL-co-DTC) and (D) T22-PEG-bP(CL-co-DTC).

[0015] Figure 4 GPC plots for (A) PEG-bP(CL-co-DTC) and (B) NHS-PEG-bP(CL-co-DTC).

[0016] Figure 5 Characterization of TPMs-V / S. (A) Particle size distribution of a series of drug-loaded micelles determined by DLS. (B) TEM image of T20PMs-V / S 1:4, scale bar at 50 nm. (C) UV-Vis spectra of PEG-bP (CL-co-DTC) solution and its micelles. Particle size changes of T20PMs-V / S 1:4 after storage at 4°C for 40 days, storage in 10% FBS for 24 h, 100-fold dilution in PBS (D), and 0 h, 12 h, and 30 h under 10 mM GSH (E). (F) HPLC spectra of free SOR and VEN standards at different concentrations. (G) Drug release of T20PMs-V / S 1:1 determined by HPLC after 12 h of storage with / without 10 mM GSH. (H) In vitro release behavior of TPMs-V / S in PB with and without 10 mM GSH.

[0017] Figure 6 To investigate the effect of T22 peptide modification on the endocytosis of TPMs-Cy5 by (A) MV4-11, (B) MOLM-13, (C) HL-60, and (D) OCI-AML3 cells using flow cytometry, the incubation time was 4 h. MV4-11 and MOLM-13 cells pretreated with free T22 peptide were used as controls for incubation with Cy5-labeled T20PMs-Cy5.

[0018] Figure 7 CLSM image of MV4-11 cells after incubation with T20PMs-Cy5 for 4 h (scale bar: 25 μm).

[0019] Figure 8 CLSM image of MOLM-13 cells after incubation with T20PMs-Cy5 for 4 h (scale bar: 25 μm).

[0020] Figure 9To detect the cytotoxicity of PMs-V, PMs-S, and PMs-V / S with different dual-drug ratios in (A) MV4-11 and (B) MOLM-13 cells (n = 6), CCK-8 assay was used, and the synergistic effect of VEN and SOR in (C) MV4-11 and (D) MOLM-13 cells was evaluated using CompuSyn software. The cytotoxicity of free V / S, PMs-V / S, and T20PMs-V / S in (E) MV4-11 and (F) MOLM-13 cells after co-incubation for 48 h was also assessed (n = 4). The cytotoxicity of empty micelle PMs and T20PMs in (G) MV4-11 and (H) MOLM-13 cells was also assessed (n = 4).

[0021] Figure 10 The 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) Hemolysis rate of samples (different concentrations of free V / S1:4, PMs-V / S1:4, T20PMs-V / S1:4) in red blood cell suspension at 37°C, 200 rpm for 3 h, with PBS and Triton-X100 treated samples as negative and positive controls, respectively (n = 4, ***p<0.001, ****p<0.0001).

[0022] Figure 11 To determine by flow cytometry the apoptosis induced by T20PMs-V / S, PMs-V / S, free V / S, PMs-S, and PMs-V in (A) MV4-11 cells (VEN / SOR concentration of 2 / 8 ng / mL) and (B) MOLM-13 cells (VEN / SOR concentration of 1 / 2 ng / mL) at the optimal drug synergy ratio (for a total incubation of 48 h).

[0023] Figure 12 Acute toxicity study of T20PMs-V / S. (A) Dosing regimen and monitoring flowchart of the model. (B) Weight change of mice in each group within 8 days of administration (n = 4), (C) Blood routine index analysis (n = 3) and (D) Blood biochemical index analysis (*p<0.05, **p<0.01, ***p<0.001, n = 4).

[0024] Figure 13 Fluorescence imaging images of leg bones in different treatment groups (A) and quantitative fluorescence analysis of major organs (n ​​= 3) (B). Samples were taken 8 h after intravenous injection of T20PMs-Cy5 and PMs-Cy5, respectively, with a Cy5 concentration of 2.5 µg / mL.

[0025] Figure 14(A) The workflow for constructing and treating the orthotopic MV4-11 AML mouse transplantation model (grey arrows indicate dosing time in the treatment groups). (B) Quantitative peripheral blood leukemia cell infiltration to monitor disease progression in the orthotopic mouse model. (C) Body weight changes in mice during dosing in some treatment groups. (D) Kaplan-Meier survival curves (n = 5, TPMs-V / S1:4 (H) versus free V / S group (po), TPMs-V / S1:1 (H), TPMs-V / S1:4 (L), PMs-V / S1:4 (L), PMs-V (L), PMs-S (L), Free-V / S (L, iv), and PBS group, **p<0.01). (E) Spleen weight of surviving mice dissected on day 32.

[0026] Figure 15 Representative flow cytometry scatter plots (with CD45-positive AML cell populations within rectangular gates) of MV4-11 cells in bone marrow (BM), liver (Liver), lung (Lung), peripheral blood (PB), and spleen (Spleen) of mice in different treatment groups are presented for (A) and statistical analysis (n = 3).

[0027] Figure 16 Blood routine (A) and blood biochemistry (B) parameters (n = 3) were analyzed in mice from different treatment groups. Abbreviations: Red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), red blood cell distribution width (RDW), and hemoglobin-related parameters: hemoglobin concentration (HGB), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), hemoglobin distribution width (HDW), platelet count (PLT), plateletcrit (PCT), mean platelet concentration (PCV), white blood cell count (WBC), eosinophils (EOS), neutrophils (Neut), and lymphocytes (Lymph).

[0028] Figure 17 H&E staining images of the heart, kidney, lung, liver, and spleen of mice in different treatment groups. Scale bar: 100 μm.

[0029] Figure 18 H&E staining images of the femur and tibia of mice in different treatment groups (the black circle indicates the area infiltrated by leukemia cells). Figure A: 500 μm; Figure B: 100 μm.

[0030] Figure 19 TRAP staining of osteoclasts (white arrows) in the hind leg bone of different treatment groups (green bar: 500 μm, black bar: 100 μm).

[0031] Figure 20Micro-CT images of the tibia and femur in mice from different treatment groups (A) and statistical analysis of the tibia (B) and femur (C) (n = 3). Detailed Implementation

[0032] This invention utilizes PEG and polycarbonate (PTMC) to design and prepare a targeted peptide-modified disulfide crosslinked, biodegradable polymer micelle (TPMs-V / S) co-loaded with VEN and SOR for active targeted therapy in an orthotopic mouse MV4-11 AML model, achieving optimal synergistic delivery and efficient, controllable release of VEN and SOR (Figure 1). TPMs-V / S possesses advantages such as high efficiency, stability, controllable drug loading, tunable surface peptide density, rapid drug release under reduction response, and good targeting. Results show that TPMs-V / S can efficiently target MV4-11 cells in an orthotopic FLT3-ITD subtype AML mouse model, strongly inhibiting tumor growth and significantly prolonging mouse survival.

[0033] Succinimidyl ester-functionalized polyethylene glycol hydroxyl groups (NHS-PEG-OH, M n = 5.0 kDa (Xi'an Ruixi Biotechnology) can be used directly after purchase. Monomethoxy polyethylene glycol (MeO-PEG-OH) M n = 5.0 kDa (Beijing Jiankai Technology Co., Ltd.) is used after azeotropic distillation with toluene. ε-caprolactone (ε-CL, 99%, Alfa Aesar) is dried with calcium hydride and distilled under reduced pressure before use. 1,2-Dithiopentane trimethylene carbonate (DTC) is used after purification. Diphenyl phosphate (DPP, >99%, TCI) is dried under vacuum for 2 h before use. 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 bridge: Cys5-Cys18, Cys9-Cys14), >85%, Jier Biochemical Co., Ltd.) and amino-functionalized dibenzocyclooctyne (DBCO-NH2, >95%, Xi'an Ruixi Biotechnology) can be used directly after purchase. PEG- b -P(CL- co-DTC)-Cy5 (molecular weight 5.0-4.0-2.0 kDa) is synthesized directly using conventional methods. Dichloromethane (DCM, Sinopharm Group) and N,N-dimethylformamide (DMF, Sinopharm Group) are used after being purified by a solvent purification system to remove water and oxygen. Anhydrous diethyl ether (Sinopharm Group), anhydrous methanol (Sinopharm Group), Tween 80 (TW80, Maclean Biochemical Technology Co., Ltd.), and HPLC-grade acetonitrile (ACN, >99%, Sigma Alrich, USA) are purchased and used directly. Sorafenib (SOR, >99%, MCE), Viteclax (VEN, >99%, MCE), glutathione (GSH, >99%, KOSYN Biotech), 4,6-diamidino-2-phenylindole (DAPI, Beyotime), paraformaldehyde-glutaraldehyde fixative (Solepro), polylysine (PDL, Beyotime), PE anti-human CD184 antibody (PE-anti-human-CXCR4, Biolegend), CCK-8 assay kit (Suzhou Meilun Biotechnology Co., Ltd.), apoptosis assay kit (Annexin V-APC / 7-AAD, Linko Biotechnology), and dialysis bags with different molecular weight cutoffs (MWCO) (Xi'an Youbo Biotechnology Co., Ltd.) were all purchased and used directly. The cell culture medium was RPMI 1640 (Roswell Park Memorial Institute 1640, HyClone), supplemented with 1% penicillin, streptomycin (Gino Biotechnology), and 15% fetal bovine serum (Gibco) before use. Human peripheral blood mononuclear cells (PBMCs) were obtained from Soochow University.

[0034] The NMR solvent for the polymer is deuterated dimethyl sulfoxide (DMSO-). d6) Chemical shifts were compared with solvent peaks. Polymer molecular weight and molecular weight distribution were determined using a Waters 1515 gel permeation chromatography (GPC, Waters 1515), with a series of monodisperse linear polymethyl methacrylates used as standard samples. The particle size and size distribution of polymer micelles were determined at room temperature using a dynamic light scattering (DLS, Zetasizer Nano-ZS, Malvern Instruments). The morphology of drug-loaded micelles was determined using a Tecnai G220 transmission electron microscope (TEM, USA) at 120 kV accelerating voltage. Receptor expression levels, apoptosis, micelle endocytosis behavior, and tumor cell progression in animal models were determined by flow cytometry (FACS Calibur, BDBiosciences, USA) and analyzed using Flowjo software (Tree Star). Laser confocal microscopy (CLSM) images were taken using a Leica TCS SP5 (Wetzlar, Germany). A Thermo Multiskan FC microplate reader was used to measure the absorbance of the products of the cell-CCK-8 reaction at 450 nm, with PBS used as a control sample. In vitro mouse imaging was performed using a near-infrared imaging system (Caliper IVIS Lumina II, Ex 643 nm, Em 668 nm) and analyzed using Living Image software. Hematoxylin-eosin (H&E) staining and trap staining images were captured using an inverted fluorescence microscope (Nikon Eclipse Ti). Complete blood counts were performed using a blood analyzer (advia2120i, Siemens, Germany), and blood biochemistry was performed using an automated biochemical analyzer (Mindray BS-420) at 3000 rpm / min and 4 °C. Bone tissue structure was analyzed using micro-computed tomography (Micro CT, SkyScan 1176, Aartselaar, Belgium) (65 kV, 385 mA, and a 1 mm Al filter).

[0035] All data in this paper are presented as mean ± standard deviation (SD). Statistical differences among three or more groups were assessed using one-way ANOVA, and a result was considered statistically significant when the result was statistically significant. p (<0.05), Tukey's post hoc test was used for comparisons between groups. p <0.05 indicates a significant difference. p <0.01 and *** p <0.001 indicates a highly significant difference.

[0036] The specific preparation operations and testing methods used in the examples are conventional techniques.

[0037] PEG- with a theoretical molecular weight of 5.0-4.0-2.0 kDa b -P(CL- co The synthesis of -DTC is carried out in the presence of the catalyst diphenyl phosphate (DPP) and the macromolecular initiator MeO-PEG-OH (-DTC). M n It is obtained by ring-opening polymerization of DTC and ε-CL initiated by (e.g., 5.0 kg / mol), and the specific synthesis reaction is based on existing technology. It is a pale yellow blocky solid.

[0038] T22-PEG- b -P(CL- co The synthesis of -DTC) polymers consists of three steps: The first step involves using the macromolecular initiator NHS-PEG-OH (…). M n = 5.0 kg / mol) under the catalysis of DPP initiated the ring-opening polymerization of DTC and ε-CL to obtain NHS-PEG- b -P(CL- co -DTC) polymer. Preparation method, feed molar ratio, and purification method are similar to PEG- b -P(CL- co The synthesis of -DTC is the same, the difference being that the macromolecular initiator used is NHS-PEG-OH. The second step, NHS-PEG- b -P(CL- co -DTC) reacts with DBCO-NH2 via an amidation reaction to yield DBCO-PEG- b -P(CL- co -DTC). Specifically, NHS-PEG- was thoroughly dissolved in anhydrous DMF solution. b -P(CL- co -DTC (1110 mg, 0.1 mmol) and DBCO-NH2 (40.5 mg, 0.15 mmol) were reacted in a 25 mL sealed reactor under nitrogen atmosphere. The former was added dropwise over 15 min to a DMF solution of DBCO-NH2, followed by reaction in an oil bath at 37 °C and 300 rpm for 48 h. The solution was then dialyzed overnight in DMF (MWCO: 3.5 kDa), then dialyzed in DCM for 12 h, with four solvent changes. The solution was precipitated three times with ice-cold diethyl ether to obtain DBCO-PEG- b -P(CL- co -DTC). Third step, T22-N3 and DBCO-PEG- b -P(CL- coT22-PEG- (-DTC) was obtained through a click chemistry reaction. b -P(CL- co -DTC). Specifically, under nitrogen atmosphere, DBCO-PEG- is added to a 25 mL sealed reactor. b -P(CL- co -DTC (387.5 mg, 0.034 mmol), DBCO-NH2 (40.5 mg, 0.0378 mmol), and anhydrous DMF (3 mL) were mixed and thoroughly dissolved, then transferred to an oil bath at 37°C and 300 rpm for 48 h. The purification process was the same as that for DBCO-PEG- b -P(CL- co -DTC). Product yield: 89.9%.

[0039] TPMs-V / S is made from T22-PEG- b -P(CL- co -DTC) and PEG- b -P(CL- co T22-PEG- is assembled in an aqueous solution after being mixed with VEN and SOR in a certain proportion (-DTC). b -P(CL- co The synthesis of -DTC is first performed by NHS-PEG- b -P(CL- co -DTC) and DBCO-NH2 are amidated to yield the intermediate DBCO-PEG- b -P(CL- co -DTC), and then obtained by click chemistry using the azide group of T22 and the macrocyclic alkyne group of DBCO. See the synthetic route diagram. Figure 2 PEG- b -P(CL- co The theoretical molecular weight of -DTC is 5.0-4.0-2.0 kg / mol. Figure 3 for 1 1H NMR spectrum. GPC test results show that the polymer has a narrow molecular weight distribution ( M w / M n = 1.1 (Table 1), indicating that the ring-opening copolymerization reaction was well controllable and the polymer was successfully synthesized. The product was a pale yellow blocky solid. T22-PEG- b -P(CL- co -DTC), NHS-PEG- b -P(CL- co -DTC), DBCO-PEG- b-P(CL- co See the NMR spectrum of -DTC. Figure 3 The ratio of the characteristic peak areas of NHS and PEG matches the theoretical value, with an NHS grafting rate of 99.9%. Furthermore, the molecular weight of each polymer segment can be calculated based on the ratio of the integrated areas of the corresponding characteristic peaks of DTC, ε-CL, and PEG (Table 1), which is close to the theoretical molecular weight. Additionally, GPC (… Figure 4 The measured polymer molecular weight distribution is relatively narrow. M w / M n =1.2). The above indicates that T22-PEG- was successfully synthesized via click chemistry. b -P(CL- co -DTC); the grafting rate of T22 peptide was 88.2% as determined by the BCA protein kit.

[0040]

[0041] a right 1 The peak area of ​​H NMR was obtained by integrating; b Measured by GPC.

[0042] Example 1: Preparation and Characterization of Drug-Loaded Micelles

[0043] All polymer micelles were prepared by solvent displacement method. First, PEG- b -P(CL- co -DTC) and T22-PEG- b -P(CL- co The T22-PEG-DTC polymer was prepared as a 10 mg / mL DMF solution. The two polymer solutions were mixed thoroughly at a specific volume ratio, for example, when T22-PEG-DTC was mixed with DMF at a specific volume ratio. b -P(CL- co When the molar percentage of -DTC is 20%, T is obtained. 20 PMs are obtained when their molar percentage is 0. The specific preparation process is as follows: 0.1 mL of the polymer solution obtained by mixing according to the ratio is injected into 0.9 mL of PB (pH 7.4, 10 mM) at a uniform rate. The mixture is blown with a pipette to make it uniform. Then the sample is placed in an environment of 37 ℃ and left to stand overnight. Then it is dialyzed with PB buffer (MWCO: 7.5 kDa) for 6 h, and the buffer is changed 5 times to remove organic solvents. Finally, PMs or TPMs are obtained. Their particle size and particle size distribution are determined by DLS.

[0044] The preparation methods for SOR-loaded single-drug micelles (PMs-S), VEN-loaded single-drug micelles (PMs-V), co-loaded micelles (PMs-V / S), and targeted co-loaded micelles (TPMs-V / S) are similar to those for empty micelles. The difference lies in the following: first, powdered VEN or SOR is dissolved separately in DMF to prepare a 10 mg / mL drug solution. Then, the solution is thoroughly mixed with the polymer solution according to a predetermined drug mass ratio and injected into 9 times the volume of PB buffer. The particle size and distribution of the polymer-loaded drug micelles were determined by DLS. The drug concentrations of VEN and SOR were determined by HPLC.

[0045] T22-PMs-Cy5 is based on T22-PEG- b -P(CL- co -DTC) solution mixed with 2% Cy5-labeled PEG- b -P(CL- co A polymer solution (10 mg / mL) of T22-PMs-Cy5 was prepared to study its cellular uptake capacity.

[0046] The nanomicelles used in the in vivo antitumor experiment were prepared in the same way as the drug-loaded micelles mentioned above, except that the concentrations of the polymer solution and the free drug solution were increased by 10 times.

[0047] To investigate the degree of self-crosslinking of micelles, PEG-P(CL-DTC) was dissolved in DMF to a concentration of 10 mg / mL, and then micelle TPMs (1 mg / mL) were prepared in PB (10 mM, pH 7.4). PB was then used as a blank control, and the changes in UV absorption of the micelles at around 330 nm were monitored by UV-Vis spectrophotometry.

[0048] To test the stability of TPMs-V / S in 10% fetal bovine serum (FBS), 100 μL of FBS was added to 900 μL of TPMs-V / S micelles, and the mixture was placed in a shaker at 37 °C and 200 rpm. Changes in particle size and size distribution were monitored using DLS. To test the stability of TPMs-V / S at dilution, the TPMs-V / S micelle concentration was diluted 100-fold with PB buffer, and changes in particle size and size distribution were monitored using DLS. To test the long-term stability of TPMs-V / S, the micelles were stored at 4 °C, and changes in particle size and size distribution were monitored on day 0 and day 40. To test the reduction responsiveness of TPMs-V / S, 10 mM GSH was added to TPMs-V / S, and changes in particle size and size distribution were monitored at h 0, h 6, and h 30.

[0049] To test the drug release performance of TPMs-V / S, two aliquots of TPMs-V / S with a concentration of 5 mg / mL were prepared. One aliquot was added with 10 mM GSH and incubated for 12 h, while the other served as a control. The drug release of TPMs-V / S under reducing conditions was monitored using HPLC. Furthermore, the dual-drug release behavior of TPMs-V / S was studied using dialysis with two different release media: PB (pH 7.4, 10 mM) containing 0.1% Tween 80 and PB solution containing 0.1% Tween 80 and 10 mM GSH (under nitrogen atmosphere). First, 1 mL of TPMs-V / S (1 mg / mL) was placed in a release bag (MWCO: 100 kDa) and then placed in 25 mL of the corresponding release buffer, and stored at 37 ºC and 100 rpm on a shaker. At each pre-set time point, 5 mL of dialysate was aspirated, and then the same volume of fresh buffer was added. The resulting sample was freeze-dried using a lyophilizer. 300 μL of ACN was used to dissolve VEN and SOR, and the drug concentration in each sample was determined by HPLC. Three parallel samples were set up for each group.

[0050] Both VEN and SOR are highly hydrophobic drugs with significant hematologic toxicity at clinically used doses. Therefore, stable loading, synergistic delivery, and controlled release to prevent leakage and ensure efficacy are crucial. To achieve efficient and stable loading and synergistic targeted delivery of VEN and SOR, disulfide crosslinked micelles modified with T22 peptides based on PEG and polycarbonate (PTMC) were designed and prepared. These micelles consist of two amphiphilic block copolymers containing disulfide pentane groups: PEG- b -P(CL- co -DTC) and T22-PEG- b -P(CL- co -DTC) is assembled in aqueous solution. Uniform cross-linked micelles were prepared under the conditions of DMF as the polymer and drug solvent, an organic phase to aqueous phase ratio of 1:9, and no stirring during dropwise addition. DLS analysis showed that the polymer could assemble into micelles with a particle size of approximately 39±2 nm in PB, and could also stably encapsulate VEN and / or SOR through hydrophobic interactions. Figure 5 (A). Among them, the single-drug micelles PMs-S or PMs-V have a particle size of 37-41 nm and a narrow particle size distribution (PDI: 0.06-0.2) (Table 2). When the theoretical drug loading of VEN is less than 20 nm... wt At a drug loading rate of .%, PMs-V encapsulation efficiency can reach over 92%, even with a theoretical drug loading of 30. wt At a concentration of 0.05%, the encapsulation efficiency remains above 70%, indicating that PMs have highly efficient encapsulation capabilities for VENs. Furthermore, PMs encapsulate SORs with a theoretical drug loading of (5~8%). wtIt maintained a high drug loading efficiency (69.3%~77.4%) when the .% was low.

[0051]

[0052] a The drug concentration was determined by HPLC and then calculated. b Measured by DLS.

[0053] By adjusting the drug feed ratio, a series of PMs-V / S with actual drug mass ratios (V / S) of 1:1, 1:2, 1:4, and 1:6 were obtained (Table 3). All PMs-V / S had particle sizes of approximately 37-41 nm, a narrow particle size distribution (PDI: 0.04-0.07), and high encapsulation efficiency (DLE) of VEN and SOR (>70%). The T22 peptide-functionalized co-loaded micelles TPMs-V / S were obtained by using PEG- b -P(CL- co -DTC) and T22-PEG- comprising 10%, 20% or 30% (molar content) of the total polymer. b -P(CL- co After mixing with DTC, it is obtained using a solvent exchange method. For example, when T22-PEG- b -P(CL- co When the molar percentage of -DTC is 20%, T is obtained. 20 PMs are obtained when their molar percentage is 0. Their particle size (39-40 nm) and particle size distribution (PDI) (0.04-0.06) are not significantly different from those of PMs-V / S. It is worth noting that, compared with PMs-S, the addition of VEN in the dual-load micelles significantly improves the drug loading capacity and drug loading efficiency of SOR.

[0054]

[0055] a The drug concentration was determined by HPLC and then calculated. b Measured by DLS.

[0056] With T 20 PMs-V / S 1:4 The morphology, stability, reduction response, and drug release behavior of targeted drug-loaded micelles were studied using this example. TEM images show T... 20 PMs-V / S 1:4 It has a spherical micelle structure (B in Figure 5) and uses PEG- b -P(CL- coThe self-crosslinking of the DTC structure was studied using empty micelles prepared with DTC. The results showed that crosslinking occurred immediately after the micelles were prepared: the absorbance of the micelles at 330 nm was significantly lower than that of the DMF solution of the polymer. Figure 5 The cross-linking degree was further enhanced after storage at 4 °C for 12 h. After being placed at 4 °C for 40 days, stored in 10% FBS solution for 24 h, or diluted 100 times with PBS, the particle size and particle size distribution of the micelles remained essentially unchanged. Figure 5 Figure 5(D) provides evidence for the stable circulation of micelles in the blood, preventing leakage of drugs loaded in the nucleus. Under simulated intracellular reducing conditions (10 mM GSH, pH 7.4), the polymer micelles (5 mg / mL) decrosslinked, increasing in size within 12 h, and exhibiting obvious impurity peaks after 30 h (Figure 5(E)). Using a liquid chromatography method capable of separating and quantifying the characteristic peaks of VEN and SOR (Figure 5(F)), the standard curve obtained by this method showed good correlation (correlation coefficient > 0.999). The characteristic peaks of SOR and VEN, at a ratio of acetonitrile:water (containing 0.05% phosphoric acid) = 72:28, had retention times of approximately 5.4 min and 6.9 min, respectively. Based on this liquid chromatography method, the release behavior of TPMs-V / S under reducing conditions with and without 10 mM GSH was explored. The red line indicates that the release of VEN and SOR from drug-loaded micelles without added GSH was extremely low. Figure 5 The release amounts of VEN and SOR from the disulfide cross-linked micelles (GSH-loaded micelles) were only 1.8% of those released by micelles loaded with GSH, indicating that the disulfide cross-linked micelles released the drug under reducing conditions and exhibited excellent reduction responsiveness. Under simulated intracellular reducing conditions (10 mM GSH, pH 7.4), the release amounts of SOR and VEN from TPMs-V / S reached 84% and 81%, respectively, within 30 h, while under conditions without GSH, the release amounts of SOR and VEN were only 17% and 16%, respectively (H in Figure 5).

[0057] Example 2: Cell Endocytosis Experiment

[0058] Flow cytometry was used to investigate the endocytosis of polymeric micelle TPMs with different T22 targeting densities in MV4-11, MOLM-13, OCI-AML3, and HL-60 cells. Since VEN and SOR are not fluorescent, Cy5-labeled PMs and TPMs with different targeting densities (10%, 20%, and 30%) were used. First, 800 μL of cell suspension (2 × 10⁻⁶ cells / mL) was used. 5Cells were evenly seeded in 6-well plates (number of cells / well) and incubated for 24 h. Then, 200 μL of PBS and Cy5-labeled TPMs or PMs (2.0 μg Cy5 / mL) were added. After co-incubation for 4 h, cells were collected and centrifuged (1000 rpm, 3 min). The supernatant was removed, and the cells were washed twice with PBS and then dispersed in 200 μL of ice-cold PBS for flow cytometry analysis using FlowJo-10 software. The receptor blocking assay was performed similarly to the endocytosis assay, except that 100 μL of low, medium, and high concentrations of free T22 were used to block the CXCR4 receptor on the surface of tumor cells 2 h before sample addition.

[0059] The endocytosis of Cy5-labeled TPMs in MV4-11 and MOLM-13 cells was investigated using CLSM. In the CLSM experiment, small discs were first placed in 24-well plates, and 500 μL of 0.1 mg / mL PDL solution was added for 8 h to help MV4-11 cells adhere. The PDL was then removed, and the cells were washed twice with ice-cold PBS. MV4-11 cells were then cultured at 2 × 10⁻⁶ cells / well. 5 Cells were seeded at a density equal to the density of the wells in 24-well plates and incubated for 24 h. Then, 200 μL of Cy5-labeled T22-PMs or PMs (40 μg Cy5 / mL) were added, and the cells were incubated for another 4 h. After washing twice with ice-cold PBS, 5 μL of PE-anti-human-CXCR4 was added and incubated for 20 min, followed by 4 washes with PBS. Cells were then fixed with 4% paraformaldehyde for 15 min, followed by 4 washes with PBS. The nuclei were stained with DAPI for 5 min, followed by 4 washes with ice-cold PBS. Finally, 10 μL of glycerol was added for mounting, and the plates were protected from light with aluminum foil. The resulting samples were photographed using a CLSM (Leica, TCS SP5) and analyzed using LAS X software. The treatment method for TPMs-Cy5 in MOLM-13 cells was the same as for MV4-11.

[0060] Flow cytometry results showed that TPMs-Cy5 was present in MV4-11 ( Figure 6 (A) and MOLM-13 ( Figure 6 The amount of endocytosis in cells B was significantly higher than that in PMs-Cy5, among which T 10 The fluorescence intensity of PMs-Cy5 was 7.6 times and 8.4 times that of the PMs-Cy5 control group, respectively, and was comparable to that of T. 20 Cells co-incubated with PMs-Cy5 showed the highest uptake, with fluorescence intensities 14.8 times and 11.8 times higher than the PMs-Cy5 control group, respectively (Table 4). Meanwhile, T... 30Cellular uptake of PMs-Cy5 no longer increased, and the fluorescence intensity was 13.8 times and 10.9 times that of the PMs-Cy5 control group, respectively, indicating that the T22 targeting density reached saturation at 20%. Pretreatment of MV4-11 and MOLM-13 cells with low (L), medium (M), and high (H) concentrations of free T22 peptides was used to retest TPMs-Cy5 uptake, and the results are shown in (…). Figure 6 (A, B). For two types of AML cells HL-60 ( Figure 6 (C), OCI-AML3 ( Figure 6 The intake of TPMs-Cy5 is much lower than that of PMs-Cy5, with the highest intake levels being only 2.7 times and 1.8 times, respectively (Table 4).

[0061]

[0062] a Abbreviation for average fluorescence intensity; b Multiples relative to the PBS group.

[0063] T was further studied using CLSM. 20 PMs-Cy5 and PMs-Cy5 in MV4-11 cells ( Figure 7 ) and MOLM-13 cells ( Figure 8 The internalization situation in T. 20 After incubation of PMs-Cy5 with MV4-11 or MOLM-13 cells for 4 h, significant red fluorescence was observed around the cell nucleus, while the fluorescence in cells incubated with PMs-Cy5 was weaker. Importantly, T... 20 Compared to the PMs-Cy5 group, the yellow fluorescence of CXCR4 in the PMs-Cy5 group was significantly weakened, indicating that T 20 PMs-Cy5 preemptively bound to and blocked the CXCR4 receptor before monoclonal antibody labeling. These results indicate that T22-modified nanomicelles possess excellent CXCR4 targeting and efficient, rapid cellular uptake capabilities, enabling them to quickly endocytose cancer cells. This is significant for rapidly and efficiently promoting the combined intracellular entry of VEN and SOR to inhibit BCL-2 and MCL-1 expression, thereby inducing cytotoxicity. Furthermore, the aforementioned cells also express the CXCR4 receptor intracellularly.

[0064] Subsequent studies all used T with a target density of 20%. 20 The PMs-V / S study was conducted, with the drug mass ratio (S / V) being the measured ratio.

[0065] Example 3 Cytotoxicity Experiment

[0066] Cytotoxicity assays were performed using human acute myeloid leukemia cell lines MV-4-11 and MOLM-13, healthy mouse fibroblast cell line L929, mouse myeloid dendritic cells (DC 2.4), and human peripheral blood mononuclear cells (PBMCs). The effects of different drug synergistic ratios and peptide densities on the cytotoxicity of TPMs-V / S were investigated, and the cytotoxicity of empty micelles and the cytotoxicity of TPMs-V / S to healthy cells were tested.

[0067] The in vitro synergistic antitumor activity of VEN and SOR was determined in MV4-11 and MOLM-13 cell lines. The combination index (CI) was calculated using CCK-8 staining kit, microplate reader measurement of complex absorbance, and Graphpad Prism 8 software to determine the median lethal concentration (IC50) of PMs-V / S at different mass ratios. 50 The MV4-11 cells (2 × 10⁻⁶) were obtained first. 4 Cells were seeded in 96-well plates (80 μL / well) and incubated at 37 ºC, 5% CO2 for 4 h. Then, 20 μL of PMs-V / S (SOR concentration range 0.1-100 ng / mL), PMs-V (VEN concentration range 0.1-1000 ng / mL), and 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 co-incubation with the samples for 48 h, 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 3 h. The absorbance of the complexes at 450 nm was measured using a microplate reader. Cells with added PBS served as the control group. Cell viability was the ratio of absorbance of each well to the absorbance of the control group (n = 6). The final results are presented as mean ± SD. The half-maximal inhibitory concentration (IC50) for cell growth was also measured. 50 The synergistic ratio of PMs-V / S in MOLM-13 was calculated using nonlinear regression on the cell viability curve. The method for testing the synergistic ratio of PMs-V / S in MOLM-13 was the same as that for MV4-11, except that the concentrations of each sample group were different: PMs-V / S group (SOR concentration range 0.001-10 ng / mL), PMs-S group (SOR concentration range 0.01-50 ng / mL), and PMs-V group (VEN concentration range 0.01-50 ng / mL).

[0068] The formula for calculating cell viability is:

[0069] Whether SOR and VEN have a synergistic effect is assessed by calculating the Combination Index (CI). The CI-Fa (fraction affected) curve is obtained by fitting using Compusyn software. When CI > 1, it shows an antagonistic effect; when CI = 1, it shows an additive effect; when CI < 1, it shows a synergistic effect; and when CI < 0.5, it shows a strong synergistic effect. The CI value is defined as the IC of SOR and VEN used together versus when they are used individually. 50 The sum of ratios, that is

[0070] The targeting and cytotoxicity of TPMs-V / S in MV4-11 and MOLM-13 cells were investigated. MV4-11 cells were seeded in 96-well plates (80 μL, 2 × 10⁻⁶ cells / well). 4 / hole). Add T22 with a surface density of 20% T. 20 PMs-V / S 1:4 PMs-V / S 1:4 and free V / S 1:4 (20 μL, SOR concentration range 0.01-30 ng / mL), after co-incubation for 48 h, absorbance was measured using the CCK-8 assay with a microplate reader. This ensured the survival of MOLM-13 cells. The treatment method for TPMs-V / S in MOLM-13 cells was the same as for MV4-11, except that the SOR to VEN mass ratio was 2:1, and the SOR concentration range was 0.001-2.5 ng / mL.

[0071] When verifying the toxicity of empty micelle TPMs and PMs to MV4-11 and MOLM-13 cells, the cell plate number was 2×10⁻⁶. 4 / well, co-incubation time is 48 h, micelle concentration in well is 1-400 μg / mL, the remaining steps are the same as above.

[0072] The CCK-8 assay results showed that the IC50 values ​​of single-drug micelles PMs-V and PMs-S in MV4-11 cells were [not specified]. 50 The values ​​were 38.58 ng / mL and 9.61 ng / mL, respectively. Figure 9 (See Table 5). This indicates that SOR micelles are significantly more toxic than VEN micelles. The drug-to-drug ratios (VEN:SOR) were 1:1, 1:2, 1:4, and 1:6, with CI values ​​all less than 1 (0.27-0.71), showing a good synergistic effect. The lowest CI value, PMs-V / S, was observed when the mass ratio of loaded VEN to SOR was 1:4. 1:4They exhibit the strongest synergistic effect. Tests in MOLM-13 cells showed that PMs-V and PMs-S were more toxic to MOLM-13 cells than to MV4-11 cells. Figure 9 (China B, Table 5), IC 50 The values ​​were 12.00 ng / mL and 5.28 ng / mL, respectively. Surprisingly, the CI values ​​were all less than 0.1 (0.07-0.09) for the four dual-drug ratios mentioned above, indicating a very strong synergistic effect. The CI value was the lowest when the mass ratio of VEN to SOR was 1:2.

[0073] Based on the above cytotoxicity results, the effects of VEN and SOR on MV4-11 cells were further confirmed using CompuSyn software. Figure 9 (C) and MOLM-13 cells ( Figure 9 The synergistic effect of VEN and SOR in the two cell types was observed. CompuSyn-fitted CI-Fa simulation curves showed that VEN and SOR exhibited strong synergistic effects on both cell types over a wide concentration range.

[0074] Unless otherwise specified, the mass ratio of VEN to SOR in the dual-drug micelles was 1:4 and 1:2 in MV4-11 and MOLM-13 cells, respectively, for further investigation in subsequent experiments.

[0075] T was studied 20 The in vitro antitumor activity of PMs-V / S in MV4-11 (V:S = 1:4) and MOLM-13 (V:S = 1:2) cells was evaluated, with free V / S and PMs-V / S as controls. Results showed that after co-incubation with MV4-11 cells for 48 h (… Figure 9 (E), T 20 PMs-V / S 1:4 IC of VEN and SOR 50 The values ​​were 0.24 and 0.94 ng / mL, respectively, compared to PMs-V / S 1:4 The decrease was significant (0.55 and 2.21 ng / mL) compared to the free V / S 1:4 (0.80 and 3.21 ng / mL) decreased by approximately 3.2-fold. After co-incubation with MOLM-13 cells for 48 h ( Figure 9 (F), T 20 PMs-V / S 1:2 IC of VEN and SOR 50 The values ​​were 0.096 and 0.192 ng / mL, respectively, compared to PMs-V / S 1:2 (0.211 and 0.423 ng / mL) decreased by approximately 2.2-fold compared to free V / S 1:2The levels (0.251 and 0.502 ng / mL) decreased by approximately 2.6 times.

[0076] Empty micelles PMs and T 20 PMs concentrations of 1-400 μg / mL were effective against MV4-11 ( Figure 9 (G) and MOLM-13 ( Figure 9 No significant cytotoxicity was observed in H1N1 cells, indicating that the vector has good biocompatibility.

[0077]

[0078] The toxicity of TPMs-V / S, PMs-V / S, and free V / S on healthy cells was investigated. Mouse fibroblasts (L929), mouse myeloid dendritic cells (DC 2.4), and human peripheral blood mononuclear cells (PBMCs) were used. The cell seeding numbers were: L929, DC 2.4 (5000 cells / well), and PBMCs (5 × 10⁶ cells / well). 5 Samples were incubated with cells for 48 h at a VEN to SOR mass ratio of 1:4, with SOR concentrations ranging from 0.1 to 1000 ng / mL. The remaining steps were the same as described above. The effects of VEN and SOR-loaded micelle formulations on human peripheral blood mononuclear cells (PBMCs) were investigated. Figure 10 (A) Mouse myeloid dendritic cells DC 2.4 ( Figure 10 (B) and mouse fibroblast L929 ( Figure 10 Biocompatibility in normal cells such as C) was studied. Results showed that even at SOR / VEN concentrations of 1000 / 250 ng / mL, T... 20 PMs-V / S 1:4 and PMs-V / S 1:4 It also did not show obvious toxicity, and the cell survival rate was close to 100%.

[0079] Clinical trials have reported severe hematologic toxicity of VEN and SOR. This invention prepares disulfide crosslinked polycarbonate micelles to encapsulate VEN and SOR, and preliminarily evaluates the hematologic toxicity of TPMs-V / S, PMs-V / S, and free V / S through hemolysis experiments. First, fresh blood was collected from the eyeballs of healthy mice. The blood was resuspended in ice-cold PBS and centrifuged (3600 rpm, 10 min). This process was repeated five times until the supernatant became clear. The bottom red blood cells were then treated with ice-cold PBS to obtain a 2% red blood cell suspension. 400 μL of the red blood cell suspension and 400 μL of different concentrations of Free-V / S, PMs-V / S, or TPMs-V / S (SOR concentration range 1-100 μg / mL, V / S = 1:4) were added to EP tubes. PBS was used as a negative control, and Triton X-100 as a positive control. The EP tubes were then incubated at 37 ℃ and 200 rpm for 3 h on a shaker. After centrifugation (1500 rpm, 5 min), 100 μL of the supernatant was added to a 96-well plate, and the absorbance at 545 nm was measured using a multi-mode microplate reader. The hemolysis rate formula is as follows:

[0080]

[0081] The results of the hemolysis test showed that ( Figure 10 D), T 20 PMs-V / S 1:4 Compared to free V / S 1:4 and PMs-V / S 1:4 It has better blood compatibility. Specifically, within the SOR concentration range of 5-100 μg / mL, T... 20 PMs-V / S 1:4 With PMs-V / S 1:4 Compared to free V / S 1:4 The hemolysis rate was reduced by 35-47 times and 25-30 times, respectively. These results collectively indicate that TPMs-V / S have low cytotoxicity to normal cells, good biocompatibility, and may reduce systemic toxicity during in vivo circulation.

[0082] Example 4: Apoptosis Experiment

[0083] First press 2.5×10 5MV4-11 or MOLM-13 cells were evenly seeded into 12-well plates at a density of cells / well and incubated for 4 h. Then, 200 μL of TPMs-V / S, PMs-V / S, free V / S, PMs-S, and PMs-V were added to each well (the SOR / VEN concentrations for MV4-11 or MOLM-13 were 8 / 2 or 2 / 1 ng / mL, respectively). The control group was the PBS group. After incubation for 48 h, samples were collected in flow cytometry tubes and centrifuged (1000 rpm, 3 min). The supernatant was discarded, and the cells were resuspended in ice-cold PBS. The accumulated MV4-11 cells at the bottom of the flow cytometry tube were then repeated twice. Two PBS groups were prepared by resuspending the cells in 200 μL of binding buffer. One PBS group was then mixed with 500 μL of positive control solution and incubated on ice for 0.5 h. The cells were then washed with ice-cold PBS to remove the supernatant. Resuspend the cells in binding buffer, then mix with the untreated PBS group. Divide the mixture into three equal portions: two for positive monostaining controls and one for a blank control. In the early-apoptosis monostaining group, add 5 μL of Annexin V-APC to one group of cell suspensions; in the late-apoptosis monostaining group, add 10 μL of 7-AAD to the cell suspension of another group for staining. In the sample group, resuspend the cells in 200 μL of binding buffer (approximately 1 × 10⁻⁶ cells per group). 6 (The text appears to be incomplete and contains several errors. A more accurate translation would require the full context.) 20 Apoptosis of PMs-V / S, PMs-V / S, free V / S, PMs-S, and PMs-V after 48 h of incubation with MV4-11 and MOLM-13 cells at the optimal drug synergistic ratio. In the formulation co-incubated with MV4-11 cells, the concentration of VEN was 2 ng / mL and the concentration of SOR was 8 ng / mL; in the formulation co-incubated with MOLM-13 cells, the concentration of VEN was 1 ng / mL and the concentration of SOR was 2 ng / mL. Figure 11 It can be seen that T 20 PMs-V / S effectively induced apoptosis in both cell types. After co-incubation with MV4-11 for 48 h, T... 20 PMs-V / S induced 36.2% apoptosis, significantly higher than the control group (27.1%), free V / S group (26.5%), PMs-S group (19.1%), and PMs-V group (9.9%). Similarly, after co-incubation with MOLM-13 for 48 h, T... 20PMs-V / S 1:2 It can induce 26.5% of cells to undergo apoptosis, which is significantly higher than that of the control PMs-V / S group (18.0%), the free V / S group (13.5%), the PMs-S group (8.7%), and the PMs-V group (5.7%). Moreover, the PMs-V / S group showed a stronger synergistic effect compared with both the PMs-S group and the PMs-V group.

[0084] Example 5: Establishment of Animal and Tumor Models

[0085] 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. Acute toxicity experiments used 8-week-old female Balbc mice (Vitalivan) weighing approximately 19-21 g, while biodistribution and antitumor therapy experiments used 9-week-old female NOD.CB17-Prkdcscid / IL2rgtm1 / Bcgen (B-NDG, Biocytogen).

[0086] Establishment of an in situ MV4-11 AML tumor model: 200 μL of MV4-11 cells (5 × 10⁻⁶ cells) were injected into the tumor using a 1 mL syringe. 6 (Number of mice per mouse) was injected into B-NDG mice via the tail vein, with the day of injection designated as day 0. Treatment began on day 3 post-injection, and mouse weight was monitored every three days.

[0087] To assess the disease progression of MV4-11 cells in mice, orbital blood was collected every three days after inoculation, and the development of AML cell infiltration in peripheral blood of mice was measured by flow cytometry.

[0088] Example 6 Acute Toxicity and Biodistribution Experiment

[0089] To further evaluate the hematologic toxicity of TPMs-V / S, healthy Balbc female mice (19–21 g, 8 weeks old) were randomly assigned to three groups of four mice each, including TPMs-V / S. 20 PMs-V / S, healthy group, and oral administration group. 20 The PMs-V / S group (2.5 / 10 mg VEN / SOR equiv. / kg) was administered via tail vein injection, once every two days for a total of 4 injections. The free drug group (150 / 30 mg VEN / SOR equiv. / kg) was administered orally via gavage for 7 consecutive days. Mice were continuously monitored for body weight and health status for 8 days after administration. On day 9, mice in each group were dissected, and blood samples were collected from the peritoneum for complete blood count and blood biochemistry.

[0090] Both VEN and SOR have shown significant hematologic toxicity in the clinical treatment of AML due to their high dosage, limiting the dosage and therapeutic effect. In particular, the combined use of VEN and SOR produces even more pronounced additive toxicity, making hematologic toxicity a major challenge for this combination therapy. This invention evaluated the acute toxicity and mouse tolerance of TPMs-V / S in healthy female Balbc mice. The dosing regimen is described below. Figure 12 In the control group, free V / S was administered orally for 7 consecutive days according to the reported dosage and ratio. The results showed that when the dosage of free V / S was 150 / 30 mg VEN / S OR equivalent / kg, the mice experienced a continuous decrease in body weight during the administration period. Figure 12 (B) By day 8, the animal had reached nearly 85% of its initial body weight, accompanied by symptoms such as hair loss, loss of appetite, dull coat color, and weakness in the hind limbs, while (T) 20 PMs-V / S (2.5 / 10 mg VEN / SOR equiv. / kg, iv It was well tolerated, and no abnormal toxic side effects were observed in the control group. Blood routine data ( Figure 12 The results (C) showed that the levels of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelets (PLT), neutrophils, and lymphocytes in the oral V / S group were significantly abnormal compared to the healthy group, while T... 20 In the PMs-V / S group, except for decreased lymphocyte count, all indicators were close to those of the healthy group, indicating that the TPMs-V / S group significantly improved the toxicity of combination therapy compared to the free V / S group. Blood biochemistry data ( Figure 12 The results from the study (D) showed that the liver function indicators AST (aspartate aminotransferase), ALT (alanine aminotransferase), and serum alkaline phosphatase (ALP) and the kidney function indicators creatinine (CREA) and urea (UREA) in the oral V / S group were significantly lower than those in the healthy group, indicating severe liver and kidney damage. Meanwhile, the T... 20 The PMs-V / S group significantly improved damage to AST, CREA, and UREA. These results collectively indicate that TPMs-V / S effectively reduced the toxic side effects of VEN and SOR and broadened their dosing window, demonstrating good in vivo biocompatibility. A 15-fold reduction in VEN dosage and a 2-fold reduction in SOR dosage resulted in longer survival.

[0091] The tumor-targeting ability of T22-PMs labeled with Cy5 in an orthotopic AML mouse model was investigated using in vitro fluorescence imaging and Cy5-labeled T22-PMs. On day 20 post-inoculation, mice were randomly divided into two groups of three mice each. Two groups of three mice were injected intravenously with 200 μL of T22-PMs-Cy5 and PMs-Cy5 (0.5 µg Cy5 / mouse). Eight hours after injection, leg bones were harvested for in vitro fluorescence imaging, and the images were analyzed using Lumia II software. On day 20 of orthotopic MV4-11 AML modeling, PMs-Cy5 and TPMs-Cy5 were injected intravenously via the tail vein. Eight hours later, mice were sacrificed, and major organs, foreleg bones, and hindleg bones were harvested for fluorescence imaging. In vitro imaging results showed that the fluorescence intensity of the TPMs-Cy5 group in the foreleg and hindleg bones was significantly higher than that of the PMs-Cy5 group. Figure 13 The presence of TPMs-Cy5 indicates its strong ability to specifically target the bone marrow, where AML cells are highly concentrated. The TPMs-Cy5 group also showed significantly increased enrichment in the lungs. Figure 13 (Middle B), this is because in the late stage of the MV4-11 model, AML cells have invaded the lungs and produced obvious tumor infiltration.

[0092] Example 7: In vivo antitumor activity experiment and histological analysis

[0093] To investigate the antitumor effects of TPMs-V / S, PMs-V / S, PMs-V, PMs-S, and free V / S on orthotopic AML mice, an orthotopic xenograft model was established in mice using MV4-11 cells. Before administration, mice were randomly divided into 10 groups of 9 mice each. Four mice were used for autopsy to measure infiltration, complete blood count, and blood biochemistry, while five mice were used to monitor body weight and survival. The low-dose group (L) received VEN = 1.25 mg / kg or SOR = 5 mg / kg, while the high-dose group (H) received VEN = 2.5 mg / kg, 10 mg / kg, or SOR = 10 mg / kg. TPMs-V / S were administered via tail vein injection. 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, iv ) and a control group (PBS group). In addition, a separate control group was set up, which received free V / S (150 / 30 mg VEN / SOR equiv. / kg) orally by gavage, denoted as Free-V / S ( poIn each tail vein administration group, administration began on day 3 post-inoculation, with 200 μL administered 10 times. The oral administration group received two cycles (10 times total) of the maximum tolerated dose reported in the literature, with 5 oral administrations per cycle, followed by a 9-day rest period. Survival was continuously monitored during treatment, and mouse weight was recorded every 3 days. The PBS group was dissected at near death, and organs, leg bones, and peritoneal blood were collected as controls for the experimental groups. Survival was continued after treatment. On day 32 after inoculation, the mice in the dual-drug non-targeted micelle group showed a decrease in body weight. Four mice from each remaining surviving group were dissected, and blood was collected from the celiac artery to monitor complete blood count and blood biochemistry. The spleen weight was measured. Blood samples from the liver, spleen, lungs, hind leg bones, and peripheral blood were collected and the degree of leukemia infiltration was monitored by flow cytometry. Heart, spleen, liver, kidneys, lungs, and hind leg bones were collected and fixed with 4% paraformaldehyde. Histopathological analysis was performed by H&E and TRAP staining. The femur and tibia were scanned by Micro CT (SkyScan 1176, Belguim), and the bone tissue damage was analyzed and quantified using NRecon, DataViewer, and CTAn software.

[0094] Different formulation groups were designed to investigate the effects of modified T22 peptide, different drug ratios, and different doses on the antitumor properties of TPMs-V / S. Simultaneously, the results were compared with those reported in the literature for oral high-dose drugs and tail vein injection of free drug to explore the clinical application potential of TPMs-V / S. Firstly, B-NDG mice were injected via tail vein with 5×10... 5 A mouse orthotopic model was constructed using MV4-11 cells. On day 3 post-inoculation, mice were randomly assigned to groups and began treatment. Each tail vein injection group received one injection every 3 days for a total of 10 injections. The treatment groups were free V / S, PMs-V, PMs-S, PMs-V / S, and TPMs-V / S groups. Three TPMs-V / S groups were established to investigate the effect of dose and mass ratio on the treatment effect. The free V / S group included an oral administration group and a tail vein injection group as controls. The workflow for constructing and treating the orthotopic MV4-11 AML mouse transplantation model is as follows (…). Figure 14 (Middle A). The oral free V / S control group was administered the drug via gavage at the reported free V / S dosage (150 / 30 mg VEN / SORequiv. / kg). PBS was used as a control. Nine tumor-bearing mice were used in each treatment group; four were dissected for AML-related marker analysis, and five were used for weight monitoring and survival observation.

[0095] The degree of peripheral blood infiltration in the PBS group was monitored by orbital blood sampling and flow cytometry. Figure 14 (B) Monitoring showed that tumor cell infiltration increased continuously over time until the peripheral blood infiltration reached about 5%-10%, at which point the mice died. Figure 15Dissection of dying PBS mice in the study revealed AML cell infiltration in the bone marrow, liver, and spleen at approximately 20%, 70%, and 40%, respectively. These findings collectively indicate the successful establishment of the in situ MV4-11 model. Experimental results ( Figure 14 The results (C) show that mice in all tail vein injection groups did not experience significant changes in body weight during administration, indicating low toxicity. In contrast, the oral administration of free V / S groups resulted in continuous weight loss during administration, followed by gradual recovery after treatment cessation, suggesting that the V / S dose (150 / 30 mg VEN / SOR equiv. / kg) had strong toxicity in mice. As a control, existing technology suggests that combinations of drugs inhibiting BCL-2 and MCL-1 proteins have significant systemic toxicity in mouse orthotopic models. Furthermore, oral administration of clinical doses of VEN or SOR to AML patients typically produces high levels of toxicity and may lead to treatment discontinuation.

[0096] Survival results show ( Figure 14 (D): First, the disease progressed rapidly in the PBS group mice. On day 18 post-inoculation, they began exhibiting symptoms such as weight loss, lethargy, hind limb paralysis, darkened fur, hair loss, and hard, black feces. Mice began dying on day 19, and all mice died by day 21, with a median survival of 20 days. Second, the formulation group showed changes in PMs-V (L), PMs-S (L), and free V / S (L). iv ) and PMs-V / S 1:4 (L) demonstrated some tumor-suppressive ability, with median survival of 21 days, 26 days, 25 days, and 34 days, respectively. PMs-V / S 1:4 (L) Compared to the first two formulations, the survival time was extended by 1.6 times (**, p = 0.0026<0.01), 1.3 times (**, p = (0.0018 < 0.01), demonstrating good synergistic ability and showing superior efficacy compared to free drug combinations (**, p =0.0021<0.01). Third, TPMs-V / S 1:4 (L) The median survival reached 39 days, which is PMs-V / S 1:4 (L) 1.2 times (**, p =0.0021<0.01). Fourth, the high-dose targeted group TPMs-V / S 1:1 (H) and TPMs-V / S 1:4 (H) The median survival was 41 and 46 days (**, p =0.0019<0.01). Fifth, TPMs-V / S 1:4 (H) The therapeutic effect was stronger than that of the free V / S group ( po 38 days (**, p=0.0018<0.01), and the spleen weight of the two groups was similar to that of the healthy mice and significantly lower than that of the oral administration group and other treatment groups, and no splenomegaly was observed. Figure 14 (E).

[0097] The above results demonstrate that the TPMs-V / S formulation obtained by limiting the proportion, dosage, and targeting ability of the micelle formulation in this invention not only outperforms the intravenously administered free drug group in terms of efficacy, but also exhibits better safety and anti-tumor properties compared to the oral drug route used in clinical practice. It effectively inhibits the proliferation of AML cells and prolongs the survival of mice. Importantly, the oral treatment group has already reached the maximum tolerated dose, while the TPMs-V / S formulation, due to its good biocompatibility and active selection ability, still has more room for dose escalation, showing great potential to broaden its therapeutic efficacy. For highly malignant tumors like AML, early administration of high doses of drugs close to the tolerated dose is extremely important for rapidly reducing the number of tumor cells, preventing drug resistance, and prolonging survival. Therefore, subsequent experiments with TPMs-V / S could increase the dose to achieve a longer survival in AML mice.

[0098] Tumor cell infiltration is an important indicator for evaluating treatment efficacy. Flow cytometry was used to monitor tumor cell infiltration in the major organs, bone marrow, and peripheral blood of model mice to compare the therapeutic effects of various agents. On day 32 post-vaccination, when mice in the PMs-V / S group were near death, four mice from each group were dissected, and peripheral blood, liver, spleen, lung, and hind leg bones were collected. After cell collection, MV4-11 cells labeled with APC-anti-human-CD45 antibody were added, and AML cell infiltration in each organ was detected by flow cytometry. The PBS group served as a control, and mice in this group were dissected near death (days 19-20). The results showed that significant leukemia cell infiltration was observed in the liver (LI), spleen (SP), bone marrow (BM), and peripheral blood (PB) of mice in both the PBS and PMs-V / S groups. Figure 15 (A) indicates that the tumor cells in this model metastasize to multiple organs throughout the body, consistent with previous reports. Notably, treatment with high-dose TPMs-V / S(H) significantly reduced the leukemia burden in mice, with no obvious leukemia infiltration in the liver, spleen, bone marrow, and peripheral blood, and infiltration rates all below 0.5%. Figure 15 (B), where TPMs-V / S 1:4(H) The lowest tumor infiltration rates across all sites indicate that a VEN / SOR ratio of 1:4 provides the optimal synergistic effect. Secondly, TPMs-V / S(L) significantly reduced tumor cell infiltration compared to PMs-V / S(L). Finally, although the oral V / S group significantly alleviated tumor burden compared to the PBS group, and even showed stronger efficacy than the non-targeted PMs-V / S(L) group, it only partially reduced leukemia infiltration in the liver, spleen, bone marrow, and peripheral blood, and its therapeutic effect was inferior to that of the TPMs-V / S(H) group. These results collectively suggest that TPMs-V / S increases the anti-cancer effects of VEN and SOR in the orthotopic MV4-11 AML mouse model, demonstrating greater application potential compared to high-dose oral medications used in clinical practice. TPMs-V / S, in particular, effectively inhibited the proliferation and metastasis of AML cells in mice, and almost completely suppressed leukemia cells in the bone marrow (99.8%). Because most anti-AML therapies fail due to low utilization of BM, TPMs-V / S are of particular importance in effectively alleviating leukemia cells in the BM niche.

[0099] In clinical treatment, routine blood tests and blood biochemistry tests are often used to assess the tolerance of AML patients to drugs. Routine blood tests and blood biochemistry indicators in mice were monitored by collecting peritoneal blood to help observe treatment efficacy and hematological toxicity. The PBS group was sacrificed and monitored at the point of near death (days 19-20), and the healthy group served as the control group in this experiment. The other groups were monitored on day 32 (at which time the oral group ended treatment on day 11, and the formulation group ended treatment on day 2).

[0100] The blood routine test results showed that ( Figure 16 In the PBS group (as in group A), compared to the healthy group, the red blood cell, platelet, and hemoglobin levels were significantly reduced. This is because a large number of leukemia cells filled the bone marrow, limiting the capacity of the bone marrow cavity and suppressing normal hematopoietic function. However, in both TPMs-V / S(H) groups, there were no significant differences in red blood cell, hemoglobin, platelet, and white blood cell counts compared to the healthy group. Other blood routine indicators also showed no significant hematological burden during treatment, indicating that its therapeutic effect and hematological toxicity were superior to TPMs-V / S(L), PMs-V / S(L), and the oral group. It is worth noting that although the oral group showed a lower V / S ratio in survival experiments and tumor invasion detection... po The oral V / S (L) formulation showed stronger efficacy than the PMs-V / S (L) group, but its various blood routine indicators were significantly lower than those of the healthy group, resulting in significant hematological toxicity. This is extremely detrimental to the treatment of hematological malignancies. The formulation significantly improved this deficiency, further confirming the conclusion of the acute toxicity test that the micelle formulation has good biocompatibility. In addition, the oral V / S (L) group... poThe erythrocyte-related parameters (red blood cell count, hematocrit (HCT), mean corpuscular volume (MCV), red blood cell distribution width (RDW)) and hemoglobin-related parameters (hemoglobin concentration (HGB), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), and hemoglobin distribution width (HDW)) all showed significant toxicity compared to the healthy group, indicating that the mice exhibited severe anemia. Secondly, the oral administration group showed V / S (… po The platelet parameters of the oral group, including platelet count (PLT), plateletcrit (PCT), and mean platelet concentration (PCV), were significantly worse than those of the healthy group and the various formulation groups, suggesting that the free drug has strong toxicity to platelets. Furthermore, the V / S ratio in the oral group was significantly lower than that in the healthy group and the formulation groups. po Various immune cell indicators, including white blood cell count (WBC), eosinophil count (EOS), neutrophil count (Neut), and lymphocyte count (Lymph), also showed significant toxic side effects, while TPMs-V / S(H) was similar to the healthy group and did not exhibit this toxicity. The micellar formulation group did not show effective relief for lymphocyte toxicity. In summary, the hematological toxicity observed in the oral group is consistent with clinically reported hematological toxicity of high-dose VEN and SOR. Unexpectedly, the micellar formulation TPMs-V / S of this invention can effectively alleviate this hematological toxicity.

[0101] Blood biochemistry data indicate that ( Figure 16 In the oral administration group (B), the levels of aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly higher than in other treatment groups and the healthy group, only better than in the PBS group. This indicates that the oral administration group caused greater liver damage, while the micelle formulations effectively alleviated the drug's hepatotoxicity. Furthermore, the oral administration group showed a severe decrease in alanine aminotransferase (ALT), suggesting acute hepatitis symptoms, which were alleviated in the micelle formulations. The oral administration group also showed an abnormally high level of creatinine (CREA), indicating that the mice in this treatment group also experienced some kidney damage, while no significant differences were observed in other treatment groups. These hematological data suggest that the TPMs-V / S group has better therapeutic efficacy and lower toxicity compared to clinically used oral therapies.

[0102] Histological analysis of each treatment group was performed using paraffin sections, H&E staining, and Trap staining. The PBS group and healthy groups served as controls. Samples were collected from PBS mice at the point of death (days 19-20), and from one mouse in each of the other groups on day 32 after treatment. Results showed that the PBS group and PMs-V / S... 1:4In group (L) mice, scattered infiltration and growth of AML cells were observed in the liver, lungs, and spleen. Hepatocyte morphology was altered, with increased intercellular spaces, blurred edges, and irregular arrangement. The morphology of the structures surrounding the bronchi and pulmonary arteries in the lungs was significantly altered compared to the healthy group. Red blood cells were significantly reduced. Clusters of AML cells were observed infiltrating the spleen, and red pulp was reduced. It is noteworthy that free V / S ( po In group 1, severe splenic hemorrhage was observed (white dashed line area), indicating strong additive toxicity of the free drug. In contrast, no obvious AML cells were observed in the liver and spleen of the other groups. Hepatocytes were normal in morphology, homogeneous, and orderly arranged, with no congestion or edema in the hepatic sinusoids. The spleen had abundant and evenly distributed red and white pulp, with no obvious primary or secondary lymphoid follicles. The lung structure was clear, and there was no widening of the pulmonary interstitium. Figure 17 In addition, no obvious abnormalities were observed in the hearts and kidneys of mice in each group. The myocardial fibers were continuous and the structure was clear, without congestion or edema. The glomerular structure was clear, the renal tubules were evenly distributed, and there was no obvious tumor infiltration.

[0103] H&E staining results of the tibia and femur showed ( Figure 18 ), PBS group and PMs-V / S 1:4 In group (L) mice, a large number of AML cells were infiltrated in the bone marrow (black dashed line area), and hematopoietic stem cells were reduced, while free V / S ( po Group and TPMs-V / S 1:4 (L) group mice showed a small amount of infiltration in the bone marrow and some cell morphology changes, TPMs-V / S 1:1 (L) group and TPMs-V / S 1:4 In group (H), no AML cells were found in the bone marrow, just like in healthy mice, and no obvious abnormalities were observed in the morphology and structure of bone marrow hematopoietic stem cells.

[0104] The progression of AML is often accompanied by bone damage. To assess the effect of TPMs-V / S treatment on bone damage in AML mice, tartrate-resistant acid phosphatase (TRAP) staining and micro-computed tomography (MicroCT) analysis were performed to evaluate osteoclast content and bone resorption. AML cells in the bone marrow stimulate osteoclast proliferation, enhanced bone resorption, leading to osteolysis and osteoporosis. High expression of tartrate-resistant acid phosphatase (TRAP) is one of the main markers of osteoclast activity. TPMs-V / S... 1:4 (H) group and TPMs-V / S 1:1 The osteoclast content (white arrow) in group (H) was significantly lower than that in the untreated PBS group and PMs-V / S 1:4 (L) group, free V / S ( po Group and TPMs-V / S 1:1 (L) group ( Figure 19 ), where TPMs-V / S 1:4 Group (H) was similar to the healthy group, indicating that TPMs-V / S effectively inhibited osteoclast activity and significantly alleviated bone injury in mice. Micro CT images showed ( Figure 20 (A), PMs-V / S 1:4 (L) group and free V / S ( po Significant loss of trabeculae in group 1, TPMs-V / S 1:4 (L) group showed significant improvement in bone structure, while TPMs-V / S 1:4 Group (H) had similar results to the healthy group. Quantitative analysis using MicroCT showed that ( Figure 20 In the BC group, bone mineral density (BMD), trabecular bone thickness (Tb.Th), and bone volume fraction (BV / TV) were significantly lower in mice compared to the healthy group. Meanwhile, TPMs-V / S 1:4 In group (H), the trabecular bone tissue was intact and the bone density was similar to that of healthy mice, which is consistent with our observation that there was very little leukemia cell infiltration and osteoclast production in the bone marrow of this group. All of the above results confirm that the micelle TPMs-V / S of this invention exhibits remarkable targeting, therapeutic efficacy, and safety against AML cells in vivo, and has a good preventive and protective effect against bone damage caused by AML.

[0105] For FLT3-ITD subtype AML, this invention designs and prepares T22 peptide-modified disulfide crosslinked polymer micelles (TPMs-V / S) co-loaded with VEN and SOR for highly effective targeted therapy in situ MV411 (FLT3-ITD subtype) AML mice. TPMs-V / S possesses excellent properties such as small size (~40 nm), good reduction responsiveness, controllable drug ratio, adjustable surface peptide density, specific receptor targeting, and low toxicity. It can stably co-load VEN and SOR at an optimal synergistic ratio, effectively inhibiting AML cell proliferation. The small size of TPMs-V / S facilitates accumulation at AML infiltration sites, and its reduction-responsive drug release increases the effective drug concentration at the tumor site, enhancing anti-tumor effects and reducing toxicity. Cell experiments show that TPMs-V / S can be efficiently taken up by AML cells MV4-11 and MOLM-13, inducing cytotoxicity and apoptosis, while exhibiting minimal cytotoxicity to normal cells, demonstrating excellent anti-tumor effects. Acute toxicity and biodistribution experiments showed that TPMs-V / S significantly reduced the hematological toxicity of free drug and, compared to untargeted micelle PMs-V / S, specifically accumulated in the liver, spleen, and bone marrow infiltrated by AML cells. Treatment experiments in an orthotopic MV4-11 (FLT3-ITD subtype) AML mouse model demonstrated that TPMs-V / S significantly reduced drug dosage and systemic toxicity, greatly alleviated hematological damage, inhibited AML cell proliferation and metastasis in mice, almost completely inhibited AML cells in the bone marrow, effectively suppressed osteoclast activity, improved bone damage in mice, protected bone tissue, and significantly prolonged median survival. This targeted TPMs-V / S micelle nanomedicine represents an effective strategy for the clinical treatment of FLT3-ITD subtype acute myeloid leukemia.

Claims

1. A polymeric micelle targeting dual anti-apoptotic proteins, comprising a polymeric carrier and a small molecule drug that simultaneously inhibits the expression of anti-apoptotic proteins BCL-2 and MCL-1, characterized in that, The small molecule drugs that simultaneously inhibit the expression of anti-apoptotic proteins BCL-2 and MCL-1 are veteclare and sorafenib; the polymer carrier is assembled from a non-targeting polymer and a targeting polymer. The non-targeting polymer is composed of hydrophilic and hydrophobic segments; the targeting polymer is composed of a targeting molecule, hydrophilic segments, and hydrophobic segments; the hydrophobic segments are obtained by copolymerizing 1,2-dithiopentanetrimethylene carbonate with other monomers, with the molecular weight of the hydrophilic segments being 2–10 kDa and the molecular weight of the hydrophobic segments being 0.8–3 times that of the hydrophilic segments; in the hydrophobic segments, the molecular weight of the segments composed of 1,2-dithiopentanetrimethylene carbonate units is 20–45% of the molecular weight of the hydrophobic segments; the targeting molecule is peptide T22; the non-targeting polymer is PEG-bP(CL-co-DTC), and the targeting polymer is T22-PEG-bP(CL-co-DTC); the mass ratio of veteclare to sorafenib is 1:4, and the concentration of SOR is 5–100 μg / mL.

2. The method for preparing the targeted dual anti-apoptotic protein polymer micelles according to claim 1, characterized in that, Using small molecule drugs that simultaneously inhibit the expression of anti-apoptotic proteins BCL-2 and MCL-1 as raw materials, non-targeted polymers and targeted polymers were used to prepare polymer micelles targeting dual anti-apoptotic proteins via solvent displacement method.

3. The method for preparing targeted dual anti-apoptotic protein polymer micelles according to claim 2, characterized in that, In the non-targeted polymer and the targeted polymer, the molar fraction of the targeted polymer is 0 to 30% and does not include 0.

4. The application of the targeted dual anti-apoptotic protein polymer micelles according to claim 1 in the preparation of hematologic malignancy treatment drugs, characterized in that, The hematologic tumor is leukemia.

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