CD71-targeting ROS responsive micelle as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a CD71-targeting ROS responsive micelle as well as a preparation method and application thereof. In order to improve the efficacy of an anti-tumor drug in treating AML patients and reduce toxic and side effects, the invention provides a nano drug delivery system, namely a CD71-targeted ROS responsive micelle, which comprises the following components: amphiphilic molecules with ROS response, DSPE-PEG-DT7 and an anti-tumor drug. The micelle provided by the invention adopts a small molecule polypeptide ligand, so that competition with endogenous Tf is avoided, the micelle has protease digestion resistance, and the targeting property in blood and the in-vivo stability are improved; an amphiphilic block containing ROS responsiveness is introduced as a micelle skeleton, so that specific drug release in AML cells is realized, the curative effect is enhanced, and the side effect is reduced; aiming at AML (acute myelogenous leukemia) cells with ROS (reactive oxygen species) rise caused by FLT3-ITD mutation, the micelle disclosed by the invention can be used for effectively releasing drugs and generating a stronger drug effect reaction.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to ROS-responsive micelles targeting CD71, and a preparation method and application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem cells with significant genetic heterogeneity. Mutations in the FMS-like Tyrosine Kinase 3 (FLT3) gene are among the most common mutations in AML. FLT3 mutations are generally classified as internal tandem duplications (ITDs) and point mutations in the tyrosine kinase domain. Clinically, patients with FLT3-ITD-positive AML often present with high leukemic cell burden, poor response to treatment, high relapse rates, and significantly reduced overall survival, with a 3-year survival rate as low as 24%. This makes FLT3-ITD an independent poor prognostic factor for AML. Therefore, FLT3-ITD is considered a key target in AML treatment.

[0003] Targeted therapies targeting FLT3-ITD mutations, such as the FLT3 inhibitors midostaurin and gilteritinib, can significantly improve patient outcomes, but they are expensive and can lead to drug resistance with long-term use. Studies have shown that homoharringtonine (HHT), isolated from Cephalotaxus truncatum, has a promising therapeutic effect in treating AML patients with FLT3-ITD. Furthermore, the drug is readily available and inexpensive. However, HHT has a short half-life and requires repeated infusions for clinical use. The majority of AML patients are elderly, with high-risk factors, frail constitutions, and multiple comorbidities, who are often unable to tolerate standard doses of chemotherapy drugs. HHT's side effects, primarily myelosuppression and cardiotoxicity, are dose-dependent, and repeated infusions can increase the drug's toxic side effects.

[0004] Therefore, how to enhance the efficacy of HHT while reducing its toxic side effects has become the key and difficulty in the research of AML treatment drugs. It is necessary to develop more new approaches to reduce the toxic side effects of HHT and enhance the therapeutic effect of HHT on AML.

[0005] CD71, or transferrin receptor 1 (TfR1), is a 97 kDa type II transmembrane glycoprotein that binds to transferrin (Tf) and ferritin, mediating cellular iron uptake. Iron is an important metal cofactor for the function of multiple intracellular proteins and is involved in regulating key processes such as cellular respiration, DNA synthesis, and cell proliferation. Tumor cells, including AML cells, have a significantly increased demand for iron to meet the demands of DNA synthesis during active proliferation. Compared to other types of leukemia, AML cells express higher levels of CD71, suggesting that CD71 is a potential drug delivery target. However, while CD71-targeted nano-drug delivery systems have been proposed for the treatment of AML, the targeting ligands used in these systems to mediate cell targeting are Tf, monoclonal antibodies, and ferritin. However, these technical solutions have the following significant disadvantages: (1) Targeting ligands have competitive binding issues: natural ligands such as Tf and ferritin compete with high concentrations of endogenous Tf / ferritin in the blood for binding to CD71, significantly weakening their in vivo targeting and drug delivery efficiency; (2) Lack of responsiveness: most carriers do not have responsive drug release functions, and only two cases have applied pH responsiveness. (3) Complex materials and cumbersome preparation processes make it difficult to scale up production: for example, antibody or Tf modification often involves problems such as protein conformational protection, low coupling efficiency, and difficult quality control. Summary of the Invention

[0006] In order to improve the efficacy of anti-tumor drugs, especially homoharringtonine (HHT) in the treatment of AML patients, and reduce the toxic side effects during use, this application provides a CD71-targeted small molecule peptide-modified, reactive oxygen species (ROS)-responsive micelle, as well as its preparation method and application, based on the delivery of HHT via a nano-based drug delivery system (NDDS).

[0007] To achieve the above application objectives, the technical solutions adopted in this application are as follows: In the first aspect, the present invention provides a ROS-responsive micelle targeting CD71, the components of which include amphiphilic molecules with ROS response in a mass ratio: DSPE-PEG- D T7: Anti-tumor drugs = 80~96:4~20:2.5~10.

[0008] Furthermore, the amphiphilic molecule with ROS response is selected from DSPE-TK-PEG 2000 , DSPE-Se-Se-PEG 2000 DPPE-TK-PEG2000 , DOPE-TK-PEG 2000 Or any one or more combinations of PLGA-TK-PEG.

[0009] Furthermore, the anti-tumor drug is selected from any one or more combinations of HHT, pharmaceutically acceptable salts of HHT or derivatives thereof, and vincristine.

[0010] Preferably, the HHT derivative is selected from any one or more combinations of Omacetaxine, Harringtonine (HT), Deoxyharringtonine (DHT), 11-hydroxyharringtonine or Omacetaxine.

[0011] Furthermore, the DSPE-PEG- D T7 is a combination of DSPE-PEG2000-Mal and peptide D T7 was synthesized via Michael addition reaction.

[0012] Preferably, the polypeptide D The amino acid sequence of T7 is shown in SEQ ID NO: 1: HRPYIAHC.

[0013] Furthermore, the DSPE-PEG- D Peptides in T7 D T7 can be replaced by other peptides with CD71 binding activity.

[0014] In a preferred technical solution of the present invention, the micelle component comprises DSPE-TK-PEG in a mass ratio of 88-92:8-12:2.5-5. 2000 , DSPE-PEG- D T7 and HHT.

[0015] In a more preferred technical solution of the present invention, the micelle component comprises DSPE-TK-PEG in a mass ratio of 92:8:2.5. 2000 , DSPE-PEG- D T7 and HHT.

[0016] In a second aspect, the present invention provides a method for preparing the above-mentioned ROS-responsive micelles targeting CD71, comprising the following steps: (1) Synthesis of DSPE-PEG- D T7: Peptide by Michael addition reaction D T7 and DSPE-PEG 2000 -Mal connection to generate CD71-targeted micellar skeleton material DSPE-PEG- DT7; (2) Dissolve the ROS-responsive amphiphilic molecules, DSPE-PEG- D T7 and anti-tumor drugs are mixed in corresponding mass ratios and stirred evenly; (3) Ultrasound emulsification after adding water; (4) Formation of micelles by solvent evaporation; (5) Ultrafiltration centrifugation is used to remove free antitumor drugs, and the final micelle composition is obtained after the filter membrane is sterilized.

[0017] Furthermore, in step (2), the amphiphilic molecule having ROS response is selected from DSPE-TK-PEG 2000 , DSPE-Se-Se-PEG 2000 DPPE-TK-PEG 2000 , DOPE-TK-PEG 2000 Or any one or more combinations of PLGA-TK-PEG.

[0018] Furthermore, in step (2), the anti-tumor drug is selected from any one or more combinations of HHT, a pharmaceutically acceptable salt of HHT or a derivative thereof, and vincristine.

[0019] Furthermore, according to the mass ratio, the amphiphilic molecules with ROS response: DSPE-PEG- D T7: Anti-tumor drugs = 80~96: 4~20: 2.5~10.

[0020] Furthermore, in step (3), the water may be replaced by other aqueous phases, such as PBS solution.

[0021] In a third aspect, the present invention provides the use of ROS-responsive micelles targeting CD71 in the preparation of treatment and / or prevention of acute myeloid leukemia (AML).

[0022] Furthermore, the AML is FLT3 mutated AML.

[0023] Preferably, the FLT3-mutated AML is selected from FLT3-ITD MOLM-13 or MV4-11 cells.

[0024] Beneficial effect: The present invention provides a ROS-responsive micelle composition targeting CD71, the components of which include amphiphilic molecules with ROS response in a mass ratio: DSPE-PEG- D T7: anti-tumor drugs = 80-96: 4-20: 2.5-10. Among them, the amphiphilic molecules with ROS response give ROS responsiveness and achieve specific release of drugs in AML cells; DSPE-PEG- DT7 provides CD71 targeting; the anti-tumor drug is used as a drug carrier to kill AML cells. Experiments have shown that the micelle composition prepared by the present invention has the following advantages: 1. The micelle of the present invention is a small molecule polypeptide composed of cysteine-modified all-D conformation amino acids. D T7 (amino acid sequence as shown in SEQ ID NO: 1) as a targeting ligand for CD71 avoids the reduced targeting efficiency of traditional antibodies or macromolecular ligands such as transferrin due to competition with endogenous Tf in the body. It also has the ability to resist protease digestion, significantly improving its targeting in the blood environment and in vivo stability. 2. This invention introduces an amphiphilic block containing a ROS-responsive thioketal (TK) bond as a micellar backbone bond, enabling precise drug release specific to AML cells, particularly those with FLT3-ITD, enhancing efficacy and reducing side effects. 3. The micellar composition can effectively deliver anti-tumor drugs, especially HHT, and significantly enhance its efficacy in AML cells; 4. The micelle material of the present invention has a clear source and stable components. The micelle preparation process is simple and reproducible, and has good prospects for large-scale production and transformation applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 (A) CD71 expression levels and ROS levels (C) of AML cell lines compared to HUVEC cells in Example 1; (B) CD71 expression bar graph of AML cell lines HEL (FLT3 wild type), MOLM-13 (FLT3-ITD), and MV4-11 (FLT3-ITD) used in subsequent experiments; Figure 2 Figures show the characterization results of DTP / HHT and TDTP / HHT micelles in Experimental Example 1: (A) Summary of particle size, PDI, potential, and encapsulation efficiency; (BE) micelle size distribution and appearance; Figure 3 Characterization results of DTP / HHT and TDTP / HHT micelles in Experiment 1: (A) Statistics of micelle hemolysis rate; (B) Appearance of micelles after 1 hour of hemolysis test; (C) Statistics of serum stability of micelles; (D) Drug release curve of micelles; Figure 4 (A) Flow cytometry histogram; (B) Statistical graph of cell DiD positive rate; Figure 5 This is the result of confocal microscopy investigation of cell targeting of DTP / DiD and TDTP / DiD micelles in Experimental Example 2.

[0026] Figure 6 This is the result of the MTT experiment of free HHT, DTP / HHT, and TDTP / HHT micelles on HEL cells in Experimental Example 3; Figure 7 This is the result of the MTT experiment of free HHT, DTP / HHT, and TDTP / HHT micelles on MOLM-13 cells in Experimental Example 3; Figure 8 This is the result of the MTT experiment of free HHT, DTP / HHT, and TDTP / HHT micelles on MV4-11 cells in Experimental Example 3; Figure 9 The graph shows the apoptosis results of free HHT, DTP / HHT, and TDTP / HHT micelles on HEL, MOLM-13, and MV4-11 cells in Experiment 3; Figure 10 This is a graph showing the results of the efficacy study of free HHT, DTP / HHT, and TDTP / HHT micelles on MOLM-13 cells in mice in Experimental Example 3. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.

[0028] Explanation of terms: The term "nano-based drug delivery system (NDDS)" refers to a highly efficient system that uses nanotechnology to deliver drugs to specific sites in the body, aiming to improve drug therapeutic effects, reduce side effects and enhance targeting.

[0029] The term "condition-responsive drug release" is a key advantage of NDDS. It refers to an intelligent drug delivery technology that leverages specific environmental stimuli, such as reactive oxygen species (ROS), pH, temperature, enzyme activity, redox potential, and light, to precisely release drugs in vivo. By constructing carrier materials sensitive to specific stimuli, drug release is triggered upon reaching the lesion or encountering an abnormal microenvironment, rapidly increasing intracellular drug concentrations, improving therapeutic efficacy, and reducing systemic toxic side effects.

[0030] In the present invention, homoharringtonine (HHT), chemical formula is C 29 H 39NO9, CAS registration number 26833-87-4, is an alkaloid obtained from Cephalotaxus chinensis or its relatives, members of the Trifoliaceae family. Its remarkable efficacy against FLT3 mutations in AML is closely related to its pharmacological mechanism: FLT3-mutant cells rely on abnormally activated signaling pathways, such as PI3K / Akt, MAPK, and STAT5, to maintain their malignant phenotype. HHT binds to the A site of the ribosome, interfering with protein translation and inhibiting multiple key signaling pathways, such as PI3K / Akt, MAPK / ERK, and Wnt / β-catenin, thereby suppressing cell proliferation and survival. Furthermore, FLT3-mutant cells often exhibit resistance to apoptosis. HHT can degrade the expression of several short-lived proteins, such as MCL-1, Cyclin D1, and c-Myc, which are crucial for cell cycle progression and survival. Their degradation triggers apoptosis, reduces the expression of anti-apoptotic proteins, and activates apoptosis-related proteins, such as Bax, Caspase-3, and Caspase-9, inducing apoptosis. Cells harboring FLT3 mutations often develop resistance to conventional therapeutic agents. HHT, however, overcomes this resistance by directly targeting protein synthesis and survival signaling pathways through non-cross-resistance mechanisms. These mechanisms work together to enable HHT to exhibit significant anti-tumor activity in FLT3-mutant tumor cells. However, the side effects of HHT, primarily myelosuppression and cardiotoxicity, are dose-dependent, and repeated infusions can exacerbate these side effects. Therefore, novel approaches are needed to reduce the toxic side effects of HHT and enhance its therapeutic efficacy in AML.

[0031] Therefore, the applicant discovered in the early stage of research: First, compared to other types of leukemia, AML cells express higher levels of CD71, while other types of blood cells express it at lower levels. Furthermore, it was found that cells carrying FLT3-ITD have higher levels of CD71 than cells with other genetic backgrounds, which means that CD71 is an ideal target for drug delivery.

[0032] On the other hand, due to the enormous potential of NDDS in clinical drug delivery, to give HHT the ability to target AML cells and further improve its efficacy, the applicant discovered that it is possible to design appropriate targeted nanocarriers to deliver HHT, extending its half-life while also altering its in vivo distribution, thereby enhancing its efficacy and reducing its toxic side effects. However, existing CD71-targeted NDDS suffer from strong competitive binding in the blood, low targeting efficiency, and a lack of responsive controlled release.

[0033] The applicant then conducted further research and found that the ROS level of AML cells was significantly higher than that of normal cells, especially cells carrying FLT3-ITD. This means that ROS-responsive drug release can rapidly increase the intracellular drug concentration. Currently, common ROS-responsive structures such as selenium bridges, thioether bonds, and phenylboronic acid esters are widely used in drug controlled release systems, but they have not been combined with blood system-specific targeting methods, especially in the treatment of AML, which is still in the exploratory stage.

[0034] In summary, based on the generally elevated ROS levels in FLT3-ITD AML cells, the applicant has developed a novel CD71-targeted NDDS system with a simple structure, controllable synthesis, strong targeting, and the ability to achieve precise drug release in AML cells with high ROS levels. This system delivers "on-demand" drug release to FLT3-ITD AML cells, improving drug selective toxicity.

[0035] In one embodiment of the present invention, a ROS-responsive micelle composition targeting CD71 is provided, wherein the components thereof include, by mass ratio, amphiphilic molecules having ROS response: DSPE-PEG- D T7: anti-tumor drug = 80-96: 4-20: 2.5-10. Within this ratio range, the composition can achieve the basic functions of targeting CD71 and releasing drugs in response to ROS.

[0036] In some embodiments of the present invention, the amphiphilic molecule having ROS response is selected from DSPE-TK-PEG 2000 , DSPE-Se-Se-PEG 2000 DPPE-TK-PEG 2000 , DOPE-TK-PEG 2000 Or any one or more combinations of PLGA-TK-PEG, which can achieve the basic functions of targeting CD71 and releasing drugs in response to ROS.

[0037] In some embodiments of the present invention, the anti-tumor drug is selected from any one or more combinations of HHT, a pharmaceutically acceptable salt of HHT or its derivatives, and vincristine. In addition to HHT, the nano-drug delivery system prepared using the present invention can also deliver a pharmaceutically acceptable salt of HHT or its derivatives, and vincristine. The HHT derivative is selected from at least one of omacetaxine, harringtonine (HT), deoxyharringtonine (DHT), 11-hydroxyharringtonine, and omega-harringtonine.

[0038] In some embodiments of the present invention, the DSPE-PEG- D Peptides in T7 DT7 can be replaced by other peptides with CD71 binding activity to achieve D T7 has similar effects.

[0039] In some preferred embodiments of the present invention, the polypeptide D The amino acid sequence of T7 is shown in SEQ ID NO: 1: HRPYIAHC.

[0040] Furthermore, the DSPE-PEG- D T7 is DSPE-PEG 2000 -Mal and peptides D T7 was synthesized via Michael addition reaction.

[0041] In a preferred embodiment of the present invention, the micelle component comprises DSPE-TK-PEG in a mass ratio of 88-92:8-12:2.5-5. 2000 , DSPE-PEG- D T7 and HHT. Under this preferred ratio, drug loading efficiency is high, particle size is stable, targeting efficiency is good, and release curve is controllable.

[0042] In a more preferred embodiment of the present invention, the micelle component comprises DSPE-TK-PEG in a mass ratio of 92:8:2.5. 2000 , DSPE-PEG- D The micelles with this ratio had a particle size of approximately 155 nm and a moderate zeta potential, and were more effective in killing AML cells in vitro than free drugs and non-targeted micelles.

[0043] DSPE-TK-PEG 2000 and DSPE-PEG- D T7 builds micelle skeleton to encapsulate drugs. 2000 Endowed with ROS responsiveness, achieving specific release of drugs in AML cells; DSPE-PEG- D T7 provides CD71 targeting; HHT is the drug-carrying component used to kill AML cells.

[0044] In another embodiment of the present invention, a method for preparing ROS-responsive micelles targeting CD71 is provided, comprising the following steps: (1) Synthesis of DSPE-PEG- D T7: Small molecule peptides with terminal cysteine ​​modified by Michael addition reaction D T7 and DSPE-PEG 2000 -Mal was connected to generate the CD71-targeted micelle skeleton material DSPE-PEG- D T7; (2) Dissolve the ROS-responsive amphiphilic molecules, DSPE-PEG- D T7 and anti-tumor drugs are mixed in corresponding mass ratios and stirred evenly; (3) After adding water, emulsification is performed using ultrasonic probe; (4) Formation of micelles by solvent evaporation; (5) Ultrafiltration centrifugation is used to remove free antitumor drugs, and the final micelle composition is obtained after the filter membrane is sterilized.

[0045] The ROS-responsive micelles targeting CD71 prepared by the above method have been experimentally confirmed to have the following effects: 1. Targeted delivery of anti-tumor drugs to tumor cells with high CD71 expression; 2. The targeting ligand used avoids competitive binding with the CD71 ligand Tf in the blood, and the presence of Tf promotes cellular uptake of the vector; 3. It can effectively improve the efficacy of anti-tumor drugs, especially HHT, and reduce the IC of HHT in cell lines with high CD71 expression in vitro. 50 It drops to 0.5 to 0.6 times of free HHT, which can significantly prolong the survival of mice in vivo.

[0046] 4. Studies have found that AML cells carrying FLT3-ITD have higher levels of CD71 expression and ROS levels and are more sensitive to HHT. The present invention is most suitable for the treatment of AML carrying FLT3-ITD.

[0047] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0048] DSPE-TK-PEG used in the examples 2000 , DSPE-PEG 2000 -Mal, HHT, mDSPE-PEG 2000 All are commercially available, cysteine-modified CD71 targeting peptides composed of all D-type amino acids. D T7, amino acid sequence is HRPYIAHC.

[0049] Example 1: Detection of CD71 expression and ROS levels in AML cells Human umbilical vein epithelial cells (HUVECs) were used as normal control.

[0050] (1) CD71 expression: 2×10 cells were stained with isotype control and CD71-APC, respectively. 5 AML cells were incubated at room temperature for 15 min, washed with PBS, and CD71 expression was detected by flow cytometry; (2) ROS level: 5×10 5 AML cells were incubated at 37°C for 30 min and washed with PBS before ROS levels were detected by flow cytometry.

[0051] Detection found ( Figure 1 ) CD71 expression and ROS levels in AML cells were significantly increased, especially in MOLM-13 and MV4-11 cells carrying FLT3-ITD mutation.

[0052] Example 2: Preparation of ROS-responsive micelles targeting CD71 1. DSPE-PEG- D Synthesis of T7: (1) DSPE-PEG 2000 -Mal was dissolved in chloroform at a concentration of 20 mg / mL. D T7 peptide was dissolved in methanol with half the volume of chloroform, DSPE-PEG2000-Mal: D T7 =1:1.5 (molar).

[0053] (2) D The methanol solution of T7 peptide was slowly added dropwise to the chloroform solution of DSPE-PEG2000-Mal, and the reaction was stirred at room temperature under N2 protection for 48 h.

[0054] (3) Remove the organic solvent by rotary evaporation, add DMSO to dissolve, transfer to a dialysis bag with a cutoff of Mw = 2500, and dialyze in deionized water for 72 h.

[0055] (4) DSPE-PEG is obtained after freeze-drying 2000 - D T7.

[0056] (5) Separately mix the raw materials DSPE-PEG 2000 -Mal and the product DSPE-PEG 2000 - D T7 was dissolved in DMSO-d6 and identified by 1H NMR (400 MHz). The 2.5 ppm shift was the DMSO-d6 solvent peak, 6.8 ppm was the Mal group characteristic peak, and 3.64 ppm was the PEG characteristic peak. The PEG characteristic peak remained, while the Mal group characteristic peak disappeared, indicating that DThe sulfhydryl group on T7 reacts with Mal to verify the DSPE-PEG 2000 - D T7 was successfully synthesized.

[0057] 2. Preparation of micelles: (1) Preparation of micelles labeled with fluorescent dye DiD: ① Weigh DSPE-TK-PEG 2000 、mDSPE-PEG 2000 , DSPE-PEG- D T7 and DiD were dissolved in chloroform and used at concentrations of 10 mg / mL, 4 mg / mL, 4 mg / mL, and 0.2 mg / mL, respectively; ② Add the above reagents dissolved in chloroform into the EP tube: add the corresponding volume of DSPE-TK-PEG with a mass ratio of 92:8:2.5 into the control micelle sample DTP / DiD tube 2000 、mDSPE-PEG 2000 , DiD, targeted micelle sample TDTP / DiD tube was added with the corresponding volume of DSPE-TK-PEG with a mass ratio of 92:8:2.5 2000 , DSPE-PEG- D T7, DiD; ③ Mix the EP tube containing the sample thoroughly on a vortex machine; ④ Add 7 times the volume of cold PBS solution; ⑤ Probe ultrasound under ice bath conditions: 100 W, 5 min, 5 s / s; ⑥ Transfer the sample to a 25 mL eggplant-shaped flask and evaporate under reduced pressure at 55°C and 80 rpm to remove chloroform and concentrate the volume to less than 1 mL; ⑦ Filter the sample with a sterile 0.22 μm filter to remove free DiD; ⑧The volume of the sample was supplemented to 1 mL, and 100 μL was taken to measure the fluorescence intensity at Ex = 644 nm, Em = 665 nm. The average value was taken after 5 measurements, and the fluorescence intensity of the two samples was adjusted to the same level to obtain DTP / DiD and TDTP / DiD micelles.

[0058] (2) Preparation of HHT-loaded micelles: ① Weigh DSPE-TK-PEG 2000 、mDSPE-PEG 2000 , DSPE-PEG- D T7 and HHT were dissolved in chloroform and used at concentrations of 10 mg / mL, 4 mg / mL, 4 mg / mL, and 1 mg / mL, respectively; ② Add the above reagents dissolved in chloroform to the EP tube: add the corresponding volume of DSPE-TK-PEG with a mass ratio of 92:8:2.5 to the control micelle sample DTP / HHT tube 2000 、mDSPE-PEG 2000 , HHT, targeted micelle sample TDTP / HHT tube was added with the corresponding volume of DSPE-TK-PEG with a mass ratio of 92:8:2.5 2000 , DSPE-PEG- D T7, HHT; ③ Mix the EP tube containing the sample thoroughly on a vortex machine; ④ Add 7 times the volume of cold PBS solution; ⑤ Probe ultrasound under ice bath conditions: 100 W, 5 min, 5 s / s; ⑥ Transfer the sample to a 25 mL eggplant-shaped flask and evaporate under reduced pressure at 55°C and 80 rpm to remove chloroform and concentrate the volume to less than 5 mL. ⑦Remove free HHT: Fill the sample volume to 5 mL, take 100 μL and mark it as C 总 Set aside, take 4800 μL sample (marked as V 总 ) to the inner tube of the ultrafiltration tube, centrifuge at 3500 rpm for 20 min, and record the volume of the liquid in the outer tube of the ultrafiltration tube and mark it as V 外 , take 100 μL of the outer tube liquid to measure the HHT concentration and mark it as C 外 ; ⑧The inner tube micelle sample was filtered through a sterile 0.22 μm filter head, and 100 μL was taken to measure the HHT concentration. DTP / HHT micelles and TDTP / HHT micelles were obtained and stored at 4°C for subsequent experiments.

[0059] Experimental Example 1: Characterization of DTP / HHT and TDTP / HHT micelles (1) Appearance: Transmission electron microscopy (2) Particle size, potential, PDI: Malvern particle size analyzer (3) Encapsulation efficiency: The HHT concentration in the sample collected during the micelle preparation process was detected by high performance liquid chromatography, and the encapsulation efficiency was calculated according to the formula: Encapsulation efficiency (%) = (1-C 外 *V 外 / C 总 *V 总 )*100%.

[0060] (4) Serum stability: The micelles were mixed with 10% or 50% FBS, with three replicates for each sample; the samples were incubated in a shaker at 37°C and 80 rpm; the micelle particle size was measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h.

[0061] (5) Hemolysis: Peripheral blood samples were collected from two AML patients and prepared into a 7% red blood cell suspension for later use. A negative control group was set up: 500 μL red blood cell suspension + 500 μL PBS. A positive control group was set up: 500 μL red blood cell suspension was centrifuged at 1500 rpm for 5 min, the upper layer of PBS was discarded, and 1 mL of pure water was added to resuspend the red blood cells. Sample group: 3.5% red blood cell suspension was mixed with 500 μg / mL micelles. The samples were placed at 37°C for incubation and mixed every 30 min. The samples were centrifuged at 1500 rpm for 5 min at 1 h, 2 h, 4 h, and 8 h, and 100 μL of the supernatant was taken and the absorbance was measured at 540 nm.

[0062] (6) ROS responsiveness: The micelles were dispersed in PBS or PBS containing 10 mM H2O2 to a final concentration of 1 mg / mL and incubated in a shaker at 37°C. The samples were ultrafiltered at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The volume of the liquid in the outer tube was recorded, and 100 μL of HHT was taken for concentration detection. The cumulative release rate of HHT was calculated.

[0063] from Figure 2 and Figure 3 The DTP / HHT micelles had a particle size of 116 nm, while the TDTP / HHT micelles modified with the targeting ligand increased to 155 nm. Both micelles were uniform spheres with an encapsulation efficiency exceeding 90%. The micelles did not cause hemolysis, exhibited excellent serum stability, and were safe for intravenous administration. The drug release profile demonstrated that the micelles were ROS-responsive.

[0064] Experimental Example 2: Investigation of Micellar Targeting Fluorescent dye-labeled DTP / DiD and TDTP / DiD micelles were co-incubated with AML cells expressing high levels of CD71 for 4 hours. Three replicates were set up: the DTP / DiD group, the TDTP / DiD group, the free peptide pretreatment + TDTP / DiD group, and the 50% human peripheral blood plasma + TDTP / DiD group. Flow cytometry and laser confocal microscopy were used to observe the fluorescence intensity within each cell group to reflect the cellular uptake of the vector.

[0065] The results are as follows Figure 4 and Figure 5As shown, in HUVEC cells with low CD71 expression, the smallest DTP / DiD particle had the highest uptake rate, with no significant difference between the TDTP / DiD group and the free peptide pretreatment group. In AML cell lines with high CD71 expression, however, the TDTP / DiD group had a higher positive rate than the DTP / DiD group. Free peptide pretreatment reduced the TDTP / DiD positive rate, and co-incubation with human peripheral blood plasma significantly increased the TDTP / DiD positive rate. This demonstrates that the vector has CD71 targeting and does not compete with transferrin in human blood.

[0066] Test Example 3: Pharmacodynamics Study (1) MTT assay: Cells in the logarithmic growth phase were collected and cultured at a concentration of 1 × 10 5 90 μL of the culture medium was plated per well in a 96-well plate at a density of 1 mL / mL. The cells were incubated overnight. The next day, 10 μL of the corresponding drug solution was added. The blank group consisted of 90 μL of culture medium plus 10 μL of PBS. The control group consisted of 90 μL of cell suspension plus 10 μL of PBS. The free HHT group consisted of 90 μL of cell suspension plus 10 μL of free HHT solutions containing 50, 100, 200, 400, or 600 nM (for HEL cells); and 90 μL of cell suspension plus 10 μL of free HHT solutions containing 5, 7.5, 10, 15, or 20 nM (for MOLM-13 and MV4-11 cells). DTP / HHT group: 90 μL cell suspension + 10 μL of DTP / HHT solution containing 50, 100, 200, 400, or 600 nM (for HEL cells); 90 μL cell suspension + 10 μL of DTP / HHT solution containing 5, 7.5, 10, 15, or 20 nM (for MOLM-13 and MV4-11 cells). TDTP / HHT group: 90 μL cell suspension + 10 μL of TDTP / HHT solution containing 50, 100, 200, 400, or 600 nM (for HEL cells); 90 μL cell suspension + 10 μL of TDTP / HHT solution containing 5, 7.5, 10, 15, or 20 nM (for MOLM-13 and MV4-11 cells). Three replicate wells were set up for each group. After incubating the 96-well plate in an incubator for 24, 48, and 72 hours, 20 μL of MTT solution (5 mg / mL) was added and incubated for another 4 hours. After 4 hours, 100 μL of MTT solution was added and the plate was incubated overnight at 37°C. Absorbance was measured at 570 nm. Cell viability was calculated for each group using the formula: Cell viability = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0067] The results are as follows Figure 6 、 7As shown in Figure 8, two cell lines carrying FLT3-ITD, MOLM-13 and MV4-11, are sensitive to HHT. TDTP / HHT can convert the IC 50 The level of HHT was reduced to 0.5-0.6 times that of free HHT, and the same was true for HEL cells with wild-type FLT3.

[0068] (2) Cell apoptosis experiment: Cells in the logarithmic growth phase were collected and counted at 1×10 5 Cells were seeded at 400 nM in a 12-well plate and divided into the control group (PBS); the free HHT group: HEL cells were treated with free HHT at a final concentration of 400 nM; MOLM-13 and MV4-11 cells were treated with free HHT at a final concentration of 20 nM. The DTP / HHT group: HEL cells were treated with DTP / HHT at a final concentration of 400 nM; MOLM-13 and MV4-11 cells were treated with DTP / HHT at a final concentration of 20 nM. The TDTP / HHT group: HEL cells were treated with TDTP / HHT at a final concentration of 400 nM; MOLM-13 and MV4-11 cells were treated with TDTP / HHT at a final concentration of 20 nM. After addition of the corresponding solutions, the plates were incubated in an incubator for 48 hours. The cells were harvested, washed with PBS, and stained with PI and Annexin V for 15 minutes. The percentage of apoptotic cells was determined by flow cytometry.

[0069] The results are as follows Figure 9 As shown in the results, compared with the free HHT group and DTP / HHT group, the TDTP / HHT group could significantly accelerate the apoptosis of HEL (FLT3 wild type), MOLM-13 (carrying FLT3-ITD), and MV4-11 (carrying FLT3-ITD) cells, and the apoptosis rate of the FLT3-ITD-carrying cell line MOLM-13 was significantly higher than that of the FLT3 wild type cell HEL.

[0070] (3) In vivo survival experiment: Luciferase was introduced into MOLM-13 (carrying FLT3-ITD) cells using viral vectors to construct a cell line stably expressing luciferase (MOLM-13-Luc). 7-week-old female NCG mice were injected with 5×10 5 MOLM-13-Luc cells were used to establish a CDX mouse AML model. After tumor formation, the mice were randomly divided into four groups: control (PBS), free HHT group, DTP / HHT group, and TDTP / HHT group. The mice were administered at a dose of 1.5 mg / kg every two days, and their survival was observed.

[0071] The results are as follows Figure 10As shown, compared with other groups, the tumor burden of the TDTP / HHT group was significantly reduced as the injection days increased, indicating that the TDTP / HHT micelles prepared in the present invention can significantly prolong the survival of mice.

Claims

1. A ROS-responsive micelle targeting CD71, characterized in that: Its components include amphiphilic molecules with ROS response according to mass ratio: DSPE-PEG- D T7: Anti-tumor drugs = 80~96:4~20:2.5~10.

2. The ROS-responsive micelle targeting CD71 according to claim 1, characterized in that: The amphiphilic molecule with ROS response is selected from DSPE-TK-PEG 2000 , DSPE-Se-Se-PEG 2000 DPPE-TK-PEG 2000 , DOPE-TK-PEG 2000 Or any one or more combinations of PLGA-TK-PEG.

3. The ROS-responsive micelle targeting CD71 according to claim 1 or 2, characterized in that: The anti-tumor drug is selected from any one or more combinations of HHT, pharmaceutically acceptable salts of HHT or derivatives thereof, and vincristine.

4. The ROS-responsive micelle targeting CD71 according to claim 3, characterized in that: The HHT derivative is selected from any one or more combinations of Omacetaxine, Taxol, Deoxyharringtonine, 11-hydroxytaxol or Omacetaxol.

5. The ROS-responsive micelle targeting CD71 according to any one of claims 1 to 4, characterized in that: The DSPE-PEG- D T7 is DSPE-PEG 2000 -Mal and peptides D T7 was synthesized via Michael addition reaction.

6. The ROS-responsive micelle targeting CD71 according to claim 5, characterized in that: The polypeptide D The amino acid sequence of T7 is shown in SEQ ID NO:

1.

7. The ROS-responsive micelle targeting CD71 according to any one of claims 1 to 6, characterized in that: The micelle components include DSPE-TK-PEG in a mass ratio of 88-92:8-12:2.5-5. 2000 , DSPE-PEG- D T7 and HHT; preferably, the micelle component comprises DSPE-TK-PEG in a mass ratio of 92:8:2.5 2000 , DSPE-PEG- D T7 and HHT.

8. The method for preparing the CD71-targeting ROS-responsive micelles according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Synthesis of DSPE-PEG- D T7: Peptide by Michael addition reaction D T7 and DSPE-PEG 2000 -Mal connection to generate DSPE-PEG- D T7; (2) Dissolve the ROS-responsive amphiphilic molecules, DSPE-PEG- D T7 and anti-tumor drugs are mixed in corresponding mass ratios and stirred evenly; (3) Ultrasound emulsification after adding water; (4) Formation of micelles by solvent evaporation; (5) Ultrafiltration centrifugation is used to remove free antitumor drugs, and the final micelle composition is obtained after the filter membrane is sterilized.

9. The preparation method according to claim 8, characterized in that: In step (2), according to the mass ratio, the amphiphilic molecules with ROS response: DSPE-PEG- D T7: anti-tumor drugs = 80-96: 4-20: 2.5-10; The amphiphilic molecule with ROS response is selected from DSPE-TK-PEG 2000 , DSPE-Se-Se-PEG 2000 , DPPE-TK-PEG 2000 , DOPE-TK-PEG 2000 Or any one or more combinations of PLGA-TK-PEG; The anti-tumor drug is selected from any one or more combinations of HHT, pharmaceutically acceptable salts of HHT or derivatives thereof, and vincristine.

10. Use of the CD71-targeting ROS-responsive micelles according to any one of claims 1 to 7 in the preparation of a drug for treating and / or preventing acute myeloid leukemia.

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