DRUG-LOADED POLYMER VESICLE HAVING ASYMMETRIC MEMBRANE STRUCTURE, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF IN PREPARATION OF DRUGS FOR TREATING ACUTE MYELOID LEUKEMIA

Abstract

A drug-loaded polymer vesicle having an asymmetric membrane structure, a preparation method therefor, and an application thereof in preparation of drugs for treating acute myeloid leukemia. An amphiphilic triblock polymer having polyaspartic acid (PAsp), a targeted amphiphilic block polymer, and a small-molecule drug are assembled together to prepare the targeted small-molecule drug-loaded polymer vesicle having the asymmetric membrane structure. The drug-loaded polymer vesicle has many unique advantages, comprising small size, simple and controllable preparation, reversible crosslinking, in-vivo stability, targeted delivery, high enrichment concentration of intracellular drugs, reduction sensitivity, efficient killing of tumor cells, remarkable tumor growth inhibition effect, etc., and has an effective inhibition effect on both acute myelogenous leukemia cell strains and patient cells. Therefore, the polymer vesicle is expected to become a simple and multifunctional nanoplatform for efficient and specific targeted delivery of the drugs to the tumor cells.

Claims

1. A peptide targeting drug-load polymer vesicle having an asymmetric structure, which is obtained by co-loading an amphiphilic triblock polymer and a peptide targeting amphiphilic block polymer with a small molecule drug; or obtained by co-loading the amphiphilic triblock polymer and a functionalized amphiphilic block polymer with the small molecule drugs and then followed by a peptide preparation; wherein the small molecule drug includes vincristine sulfate, daunorubicin or mitoxantrone; the chemical structural formula of the amphiphilic triblock polymer is as follows: ##STR00009## ##STR00010## ##STR00011## wherein, n is 5-20.

2. The peptide targeting drug-load polymer vesicle having an asymmetric structure according to claim 1, wherein: in the amphiphilic triblock polymer, a molecular weight of a hydrophilic chain segment is 3000-8000 Da; a molecular weight of the hydrophobic chain segment is 2.5-6 times that of the a chain segment; a molecular weight of a PDTC chain segment is 8%-30% of that of the hydrophobic chain segment.

3. Use of the peptide targeting drug-load polymer vesicle having an asymmetric structure according to claim 1 in preparing an anti-tumor drug.

4. The use according to claim 3, wherein the peptide is A6, CLL1 or iNGR.

5. Use of a drug-load polymer vesicle having an asymmetric structure in preparing an anti-acute myeloid leukemia drug; the drug-load polymer vesicle having an asymmetric structure is obtained by loading an amphiphilic triblock polymer and an amphiphilic block polymer with a small molecular drug; the small molecule drug includes vincristine sulfate, daunorubicin or mitoxantrone; the chemical structural formula of the amphiphilic triblock polymer is as follows: ##STR00012## ##STR00013## ##STR00014## wherein, n is 5-20.

6. The use according to claim 5, wherein the method for preparing the drug-load polymer vesicle having an asymmetric structure comprises the following steps: preparing the non-targeting loading drug on the vesicle formed by polymer by a solvent displacement method using the small molecule drug and the amphiphilic triblock polymer as starting materials.

7. A method for preparing the peptide targeting drug-load polymer vesicle having an asymmetric structure according to claim 1, wherein the method comprises the following steps: preparing the targeting loading drug on the vesicle formed by polymer by a solvent displacement method using the small molecule drug, the amphiphilic triblock polymer and targeting amphiphilic block polymer as starting materials; or preparing the loading drug on the vesicle formed by polymer by a solvent displacement method using the small molecule drug, the amphiphilic triblock polymer and the functionalized amphiphilic block polymer as raw materials, then preparing the targeting loading drug on the vesicle formed by polymer by peptide reaction.

8. The method according to claim 7, wherein, the an of the targeted amphiphilic block polymer is 5%-35% of that of the molar sum of the amphiphilic triblock polymer and targeting amphiphilic block polymer; the amount of the functionalized amphiphilic block polymer is 5%-35% of that of the molar sum of the amphiphilic triblock polymer and targeting amphiphilic block polymer.

9. The method according to claim 7, wherein the peptide is A6, CLL1 or iNGR.

10. Use of the peptide targeting drug-load polymer vesicle having an asymmetric structure according to claim 1, the active ingredients of the drug are vincristine sulfate, daunorubicin or mitoxantrone.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is the nuclear magnetic spectrum of PEG-P(TMC-DTC)-NPC in Example 1.

[0037] FIG. 2 is the nuclear magnetic spectrum of PEG-P(TMC-DTC)-PAsp in Example 1.

[0038] FIG. 3 is the nuclear magnetic spectrum of Mal-PEG-P(TMC-DTC) in Example 2.

[0039] FIG. 4 is the nuclear magnetic spectrum of A6-PEG-P(TMC-DTC) in Example 2.

[0040] FIG. 5A is the hydrodynamic particle size diagram and cryo-EM diagram of 4 kinds of A6-cPS-VCR in Example 4.sub.∘

[0041] FIG. 5B shows the VCR release behavior of A6-cPS-VCR under reducing and nonreducing conditions in Example 5.

[0042] FIG. 5C shows the inhibition of cell proliferation of A6-cPS-VCR with different A6 surface densities in the acute myeloid leukemia cell line MV4-11 in Example 6.

[0043] FIG. 5D shows the endocytosis of Cy5-A6-cPS in the acute myeloid leukemia cell line MV4-11 in Example 7.

[0044] FIG. 6 shows the proliferation inhibition of A6-cPS-VCR, cPS-VCR and VCR in CD44-positive acute myeloid leukemia cell lines MV4-11, HL-60 and SHI-1 in Example 8.

[0045] FIG. 7 shows the proliferation inhibition of A6-cPS-VCR, cPS-VCR and VCR in CD44-negative acute myeloid leukemia cell lines YNH-1 and OCI-AML-3 in Example 8.

[0046] FIG. 8 shows the proliferation inhibition of MV4-11 by A6-cPS-DNR, cPS-DNR and DNR in Example 8.

[0047] FIG. 9 shows the apoptosis of MV4-11 cells by A6-cPS-VCR, cPS-VCR and VCR in Example 8.

[0048] FIG. 10 shows the effects of A6-cPS-VCR, cPS-VCR and VCR on the cycle of MV4-11 cells in Example 8.

[0049] FIG. 11 shows the construction of the orthotopic MV4-11-GFP-Luc acute myeloid leukemia transplantation model mice in Example 9.

[0050] FIG. 12 shows the distribution of tumors by bioluminescence fluorescence imaging on the 10th day after modeling of the in situ MV4-11-GFP-Luc acute myeloid leukemia mice in Example 9.

[0051] FIG. 13 shows for 8 hours later Cy5 fluorescence imaging of femur, tibia, and ilium by injecting Cy5-A6-cPS and Cy5-cPS the tenth day after the model of the in situ MV4-11-GFP-Luc acute myeloid leukemia mice in Example 10.

[0052] FIG. 14 is the treatment workflow of the orthotopic MV4-11-GFP-Luc acute myeloid leukemia mouse transplantation model in the Netherlands in Example 11.

[0053] FIG. 15 is a graph showing the therapeutic effect of A6-cPS-VCR on in situ MV4-11-GFP-Luc-bearing acute myeloid leukemia mice in vivo bioluminescence fluorescence imaging in Example 11.

[0054] FIG. 16 is a graph showing the body weight change and Kaplan-Meier survival curve of each group of micein Example 11.

[0055] FIG. 17 shows after dissection the infiltration rates of leukemia cells in the bone marrow, liver and spleen of 3 mice randomly selected from each group.

[0056] FIG. 18 is after dissection the HE section diagram of bone marrow, liver and spleen after 3 mice were randomly selected from each group in Example 11.

[0057] FIG. 19 is the Micro-CT analysis of the femur after dissection of 3 mice randomly selected from each group in Example 11.

[0058] FIG. 20 is a graph showing the therapeutic effect of A6-cPS-DNR on in situ MV4-11-GFP-Luc-bearing acute myeloid leukemia mice by in vivo bioluminescence fluorescence imaging of mice in Example 12.

[0059] FIG. 21 shows the proliferation inhibition and apoptosis experiments of A6-cPS-VCR, cPS-VCR and VCR in AML patient leukemia cells in Example 14.

INVENTION EMBODIMENTS

Examples of the Invention

[0060] The reversible loading a small molecule drug on the vesicle formed by cross-linked biodegradable polymer of the present invention is obtained by self-crosslinking while the amphiphilic triblock polymer is self-assembled; the molecular chain of amphiphilic triblock polymer includes sequentially connected hydrophilic chain segment, hydrophobic chain segment, and PAsp chain segment. The hydrophilic chain segment is PEG with the molecular weight is 3000-8000 Da. The hydrophobic chain segment is PTMC, PLA or PCL with the molecular weight is 2.1-5.7 times that of PEG; the molecular weight of Pasp is 15%-50% that of PEG. The molecular chain of amphiphilic block polymer includes sequentially connected the targeting, hydrophilic chain segment, and hydrophobic chain segment. The molecular weight of PEG is 5000-10000 Da. The hydrophobic chain segment is PTMC, PLA or PCL with the molecular weight is 1.4-3.8 times that of PEG.

Example 1 Synthesis of Amphiphilic Triblock Polymers:

[0061] At First, ring-opening polymerization to synthesize amphiphilic block polymers PEG-P(TMC-DTC), PEG-P(CL-DTC), PEG-P(LA-DTC) ). Then, the terminal hydroxyl groups of the above three amphiphilic block polymers were activated by p-nitrophenyl chloroformate (p-NPC), and then reacted with Pasp to prepare amphiphilic triblock polymers.

[0062] Specifically, taking the synthesis of PEG-P(TMC-DTC)-Pasp as an example, the synthetic route is as follows:

##STR00008##

[0063] In step (i), the reaction conditions were anhydrous dichloromethane (DCM), pyridine, 25° C., 24 hours; in step (ii), the reaction conditions were anhydrous dimethyl sulfoxide (DMSO), Pasp, triethylamine, 30° C., 48 hours.

[0064] The specific synthesis steps are as follows: The synthesis of PEG-P(TMC-DTC)-Pasp is divided into two steps, namely using p-NPC to activate the end of PEG-P(TMC-DTC) (5.0-(15.0-2.0) kg/mol) After the hydroxyl group, it can be obtained by reacting with Pasp. Taking the synthesis of PEG-P(TMC-DTC)-Pasp (n=15) as an example, the specific operation is as follows. PEG-P(TMC-DTC) (1.0 g, 45.5 .Math.mol) was dissolved in 10 mL of free nitrogen under nitrogen atmosphere. In aqueous DCM, then transferred to an ice-water bath and added pyridine (18.0 mg, 227.5 .Math.mol), and after stirring for 10 minutes, a solution of p-NPC (48.4 mg, 240.3 .Math.mol) in DCM (1.0 mL) was added dropwise. After 30 minutes of dropwise addition, the reaction was continued at room temperature for 24 hours, and then the pyridine salt was removed by suction filtration, and the polymer solution was collected and concentrated to ~100 mg/mL by rotary evaporation, precipitated with glacial ether, and dried in vacuo to obtain the product PEG-P(TMC -DTC)-NPC, yield: 90.0%. Subsequently, under nitrogen protection, Pasp15 (60.0 mg, 83.4 .Math.mol) was weighed and dissolved in 4 mL of anhydrous DMSO and triethylamine (4.2 mg, 41.7 .Math.mol) was added, and then was added dropwise to it with stirring PEG-P (TMC-DTC)-NPC in anhydrous DMSO (9.0 mL) was added dropwise over 30 minutes. After reacting at 30° C. for 2 days, dialyze against DMSO containing 5% anhydrous methanol for 36 hours (replaced the medium 4-5 times) to remove unreacted Pasp and p-nitrophenol produced by the reaction, and then dialyze against DCM for 6 hours. Then the polymer solution was collected and concentrated to a polymer concentration of 50 mg/mL by rotary evaporation, precipitated in ice ether and dried in vacuo to obtain a white cotton-like polymer PEG-P(TMC-DTC)-Pasp, yield: 91.0%. FIGS. 1 and 2 are hydrogen NMR spectra of PEG-P(TMC-DTC)-NPC and PEG-P(TMC-DTC)-Pasp. The characteristic peaks of p-NPC (δ 7.41 and δ 8.30 ppm) and the characteristic peaks of PEG-P(TMC-DTC) (δ 2.03, 2.99, 3.38, 3.63, 4.18 and 4.22 ppm) can be seen from FIG. 1, according to the ratio of the integral area of p-NPC characteristic peak to the area of PEG methyl hydrogen peak at δ 3.38 ppm, the grafting rate of NPC was about 100%. FIG. 3 shows that the characteristic peaks of NPC at δ 7.41 and δ 8.30 ppm were disappeared, and a new signal peak appeared at δ 4.54 ppm, which was the characteristic peak of methine in Pasp. The degree of functionalization of Pasp was calculated to be ~100% by comparing the ratio of the peak area at δ 4.54 ppm to the area of the TMC methylene hydrogen peak at δ 1.95 ppm. The successful synthesis of PEG-P(TMC-DTC)-Pasp was demonstrated for the following examples. PEG-P(TMC-DTC)-Pasp (n = 5, 10) were prepared according to the same method, except that the n value of Pasp was changed.

Example 2

[0065] Synthesis of targeting amphiphilic block polymer: the preparation of targeting amphiphilic block polymer was performed in two steps. The functionalized amphiphilic block polymer with Mal functional group and NHS functional group was synthesized at first, and then the targeted amphiphilic block polymer was obtained by reacting the targeting polypeptide with the functionalized amphiphilic block polymer. Specifically, take A6-PEG-P (TMC-DTC) as an example. Mal-PEG-P(TMC-DTC) (7.5-(14.9-2.1) kg/mol) was firstly synthesized by ring-opening polymerization, and then A6-PEG-P(TMC-DTC) was obtained by the Michael addition reaction of the thiol group of A6 with Mal-PEG-P(TMC-DTC). In the nitrogen, 1 mL of Mal-PEG-P(TMC-DTC) (100 mg, 4.1 .Math.mol) in anhydrous DMSO was added dropwise via a constant pressure dropping funnel to 2 mL of A6 (7.47 mg, 8.2 .Math.mol), and reacted at room temperature for 48 hours. After the reaction, the reaction solution was first dialyzed against DMSO for 36 hours (replacement of the medium 4-5 times) to remove unreacted A6, and then dialyzed against DCM for 6 hours, then the polymer solution was collected and concentrated by rotary evaporation to a polymer concentration of about 50 mg/mL, precipitated in ice ether and dried in vacuo to obtain a white cotton flocculent polymer A6-PEG-P(TMC-DTC). Yield: ~95%. FIG. 3 is the NMR spectrum of Mal-PEG-P (TMC-DTC), according to the integrated area of TMC (δ 2.03, 4.24 ppm) and DTC (δ 3.02, 4.19 ppm) characteristic peaks and PEG characteristic peaks (δ 3.65 ppm) From the ratio of, the degree of polymerization of TMC and DTC was calculated to be 147 and 10, respectively; from the ratio of the characteristic peak of Mal (δ 6.75 ppm) and the integration of PEG methoxy (δ 3.37 ppm), the content of Mal was calculated to be 100%, It shows that Mal remains stable during the reaction and processing. FIG. 4 is the hydrogen NMR spectrum of A6-PEG-P (TMC-DTC), the characteristic peak of Mal at δ 6.75 ppm disappears. In addition, the degree of functionalization of A6 was measured to be approximately 90% using the TNBSA method, indicating the successful synthesis of A6-PEG-P(TMC-DTC), which was used in the following examples.

[0066] The synthesis of CLL1 and INGR-targeted amphiphilic block polymers was carried out with reference to the above method, only the Mal-PEG-P(TMC-DTC) polymer was replaced by the NHS-PEG-P(TMC-DTC) polymer. The degree of functionalization is 90~96%.

Example 3

[0067] Preparation of non-targeted drug-loaded polymer vesicles: Non-targeting loading drug on vesicle formed by polymer was prepared by a solvent displacement method, using the electrostatic interaction between the drug and the Pasp chain segment in the amphiphilic triblock polymer. Specifically, the amphiphilic triblock polymer was exemplified by PEG-P(TMC-DTC)-Pasp. PEG-P(TMC-DTC)-Pasp was dissolved in DMSO (40 mg/mL), and 100 .Math.L was dispensed into standing 900 .Math.L HEPES (pH 6.8, 10 mM) containing small molecule drug at 300 rpm After stirring for 3 minutes, incubate at 37° C. for 8 hours. The non- targeting loading drug on vesicle formed by polymer cPS-VCR was obtained by dialysis against HEPES (pH 7.4, 10 mM) for 8 hours.

[0068] Among them, the theoretical loading small-molecule drug of VCR was set at 4.8-9.1 wt%, study found that the particle size of the obtained cPS-VCR was about 30 nm, and the particle size distribution was about 0.1 wt% (Table 1). The drug loading of cPS-VCR was calculated to be 4.6-4.9 wt% by measuring its absorbance at 298 nm by UV-Vis spectroscopy. In other methods, the VCR was replaced with cytarabine hydrochloride, and the results showed that the drug loading of cytarabine was 0.1 wt%. The same method was used to load PEG-P(TMC-DTC) vesicles, the results showed that the drug loading of PEG-P(TMC-DTC) vesicle was only 0.7 wt%, that was only about 15% of vesicle cPS-VCR of PEG-P(TMC-DTC)-Pasp; PEG-P(LA-DTC)-Pasp or PEG-P(CL-DTC)-Pasp was used to carry VCR in vesicle by the same method, and the results showed that their drug loading capacity was about 70% of the drug loading of vesicle on PEG-P(TMC-DTC)-Pasp.

TABLE-US-00001 Characterization of cPS-VCR Polymer Particle size (nm).sup.a PDI.sup.a DLC (wt.%).sup.b DLE.sup.b (%) Theoretical Test PEG-P(TMCSTC)-PAsp 36 0.09 9.1 4.9 51.4 28 0.08 7.0 4.8 66.7 28 0.11 4.8 4.6 97.2 PEG-P(TMC-DTC) 75 0.12 4.8 0.7 14.1 .sup.a testing by DLS; .sup.b testing by UV-vis.

[0069] The preparation method of the small molecule drug cPS-DNR is the same as the above method. It was found that the particle size of the prepared cPS-DNR was about 28 nm, and the loading drug was 9.4 wt% (when the theoretical drug loading was 16.7 wt%) (Table 2). In addition, on the basis of the above loading drug method, the loading drug effect was studied by changing the pH value and incubation time of HEPES, and the results showed that the drug loading amount did not change significantly. Compared with PEG-P(TMC-DTC), PEG-P(LA-DTC) or PEG-P(CL-DTC), the loading vesicle with DNR by the same method, and the results showed that the drug loading of this vesicle was only less than 50% that of PEG-P(TMC-DTC)-Pasp vesicles.

[0070] The preparation method of the small molecule drug mitoxantrone polymer vesicle Is the same as the above method. The results showed that the prepared mitoxantrone-loaded polymersomes (cPS-MTO) had a particle size of 50-130 nm due to different dosages. The drug loading of cPS-MTO was 3.3-9.1 wt% (Table 3). PEG-P(TMC-DTC) vesicles were loaded with MTO by the same method, and the results showed that the drug loading of PEG-P(TMC-DTC) vesicles was as low as 0.3-0.7 wt%.

[0071] To sum up, it is concluded that the amphiphilic triblock polymer of the present invention has unexpected technical effects for loading small molecule drugs.

TABLE-US-00002 Characterization of cPS-DNR Polymer Particle size (nm).sup.a PDI.sup.a DLC (wt.%).sup.b DLE.sup.b (%) Theoretical Test PEG-P(TMC-DTC)-PAsp 28 0.19 16.7 9.4 52.1 27 0.14 9.1 5.5 52.6 PEG-P(TMC-DTC) 54 0.18 16.7 4.8 25.4 52 0.15 9.1 2.2 24.2 .sup.a testing by DLS; .sup.b testing by UV-vis.

TABLE-US-00003 characterization of cPS-MTO Polymer Particle (nm).sup.a PDI.sup.a DLC (wt.%).sup.b DLE.sup.b (%) Theoretical Test PEG-P(TMC-DTC)-PAsp 58 0.11 5.0 3.3 69 81 0.17 10.0 6.4 68 126 0.28 15.0 9.1 67 PEG-P(TMC-DTC) 59 0.11 5.0 0.3 2.9 62 0.12 10.0 0.3 3.1 65 0.10 15.0 0.7 3.5 .sup.a testing by DLS; .sup.b testing by UV-vis.

Example 4

[0072] Preparation of targeting loading a drug on the vesicle formed by polymer: The targeting loading a drug on the vesicle formed by polymer was composed of the amphiphilic triblock polymer synthesized in Example 1 and the targeting amphiphilic polymer in Example 2, drug co-assembly prepared by solvent replacement method. Specifically, take the VCR-loaded A6 polypeptide-directed polymersome (A6-cPS-VCR) as an example. The DMSO solution of A6-PEG-P(TMC-DTC) and PEG-P(TMC-DTC)-Pasp polymers (total polymer concentration was 40 mg/mL, in which A6-PEG-P(TMC-DTC) and the molar ratio of PEG-P(TMC-DTC)-Pasp to obtain different ratios of A6-cPS-VCR), put 0.5 mL into 4.5 mL HEPES (10 mM, pH 6.8) buffer containing VCR, and stirred at 300 rpm for 5 min. A6-cPS-VCR with different ratios was obtained by dialysis with HEPES (pH 7.4, 10 mM) for 8 hours after incubation at 37° C. In the targeting loading a drug on the vesicle formed by polymer, the content of A6-PEG-P(TMC-DTC) polymer was 10.0-30.0 mol.%. The results showed that the particle size of A6-cPS-VCR was 36-47 nm (FIG. 5A), the particle size distribution was narrow (0.05-0.11), and the drug loading efficiency of VCR was between 79.8%-84.3% (Table 4).

TABLE-US-00004 characterization of A6-cPS-VCR ratio of A6-PEG-P(TMC-DTC) Particle.sup.a size (nm) PDI.sup.a DLC (wt.%) .sup.b DLE.sup.b (%) Theoretical Test 10% 42 0.05 4.8 4.0 84.3 45 0.07 4.8 3.8 80.8 30% 47 0.09 4.8 3.8 79.8 .sup.a testing by DLS; .sup.b testing by UV-vis.

[0073] The same method can be used to prepare the carrier based on 20% of A6-PEG-P(TMC-DTC) polymer and 80% of PEG-P(TMC-DTC)-Pasp5 or PEG-P(TMC-DTC)-Pasp10, respectively. VCR vesicle, when the theoretical drug loading amount was 4.8 wt%, obtained VCR with loading drug efficiencies was 62.4% and 73.3%, and particle sizes was 58 nm and 52 nm, respectively.

Example 5

[0074] In vitro drug release of A6-cPS-VCR-targeted polymer vesicle nanomedicine: 20% A6-cPS-VCR was used as a representative to study the in vitro drug release behavior of A6-cPS-VCR-targeted vesicle nanomedicine. The in vitro drug release behavior of A6-cPS-VCR was studied by dialysis with two release media, HEPES (pH 7.4, 10 mM) and HEPES solution containing 10 mM GSH (nitrogen atmosphere). 0.5 mL of A6-cPS-VCR (0.5 mg/mL) was first loaded into a release bag (MWCO: 14 kDa) and then placed in 20 mL of the corresponding release medium in a shaker at 37° C., 100 rpm. At the set time points (0, 1, 2, 4, 7 h), 5 mL of dialysate was withdrawn and supplemented with 5 mL of fresh medium. The content of VCR in the dialysate was determined by HPLC (mobile phase was methanol: water (15% triethylamine was added, pH was adjusted to 7.0 with phosphoric acid) = 70:30). FIG. 5B is a graph showing the results of In vitro release of A6-cPS-VCR targeting vesicle nano-drug. The results showed that under 10 mM of GSH, A6-cPS-VCR released more than 60% of VCR within 7 hours, while under non-reducing conditions, the cumulative release of VCR within 24 hours was only about 23%.

Example 6

[0075] A6-cPS-VCR-targeted polymer vesicle nanomedicine inhibits the proliferation of MV4-11 cells: Firstly, prepared A6-cPS-VCR with different surface densities of A6, namely cPS-VCR (refer to preparation method of Example 3 )and 10% of A6-cPS-VCR, 20% of A6-cPS-VCR, 30% of A6-cPS-VCR (refer to preparation method of Example 4 ), and by the cell proliferation inhibition assay (CCK8 method) to study the effects of acute myeloid cells with high CD44 expression. Firstly, MV4-11 cells were plated in a 96-well plate (2×10.sup.4 cells/well), placed in an incubator for 24 hours, and then 20 .Math.L of 10% A6-cPS-VCR and 20% of A6-cPS-VCR were added to the wells. 30% of A6-cPS-VCR or cPS-VCR (10 ng/mL in VCR well), and the control group were supplemented with 20 .Math.L of PBS. After 4 hours of incubation, the supernatant was removed by centrifugation (3000 rpm, 10 minutes), then fresh RPMI-1640 complete medium was added, the cells were blown off and placed in the incubator for a further 44 hours. 10 .Math.L of CCK8 was added and the incubation continued for 2 hours. Finally, the absorbance at 450 nm wavelength was detected by a microplate reader. The cell viability was calculated by the ratio of the absorbance value of the experimental group to the absorbance value of the control group, and the experiment was performed in parallel with three replicate wells (mean ± SD, n = 3). The test results showed that 20% of A6-cPS-VCR had the highest proliferation inhibitory effect (FIG. 5C). In the following implementation, unless otherwise specified, A6-cPS-VCR was 20% that of A6-cPS-VCR.

Example 7

[0076] Endocytosis behavior of A6-cPS-VCR-targeted polymer vesicle nanomedicine: using Cy5-labeled polymer (refer to Example 2 for preparation method, replace A6 with Cy5), and A6-PEG-P (TMC -DTC) and PEG-P(TMC-DTC)-Pasp were mixed at 0.5:20:79.5 to prepare Cy5-A6-cPS and Cy5-cPS (refer to Example 4 for the preparation method), and A6-cPS was studied by flow cytometry. By cPS-VCR in acute myeloid leukemia cell line MV4-11 with high CD44 expression. Firstly, MV4-11 cells were plated in a 6-well plate (2×10.sup.5 cells/well), placed in an incubator for 24 hours, and then 200 .Math.L of Cy5-A6-cPS or Cy5-cPS (concentration of Cy5 was 2.0 .Math.g/mL) was added, the control group was added with 200 .Math.L PBS. After 4 hours of incubation, cells were collected by centrifugation (800 rpm, 5 min), washed twice with PBS, and finally dispersed with 500 .Math.L of PBS and placed in a flow tube for assay. The test results showed (FIG. 5D) that the endocytosis of Cy5-A6-cPS in MV4-11 cells was significantly higher than that of Cy5-cPS, and its fluorescence intensity was twice that of the Cy5-cPS group, indicating that the introduction of A6 polypeptide can significantly increase the amount of Cy5-A6-cPS. Enhanced cellular uptake of Cy5-cPS.

Example 8

[0077] Inhibition of cell proliferation by A6-cPS-VCR-targeted polymer vesicle nanomedicine: Inhibition of cell proliferation by A6-cPS-VCR on CD44-positive acute myeloid leukemia cell lines MV4-11, HL-60 and SHI-1 The CCK8 method was used for determination. The cells were first plated in 96-well plates (2×10.sup.4 cells/well) and cultured for 24 hours, then 20 .Math.L of A6-cPS-VCR, cPS-VCR or free VCR were added (the final concentrations of VCR were 0.001, 0.01, 0.1, and 1, respectively), 10 and 100 ng/mL), and the control group was supplemented with 20 .Math.L of PBS. After 4 hours of incubation, the supernatant was removed by centrifugation (3000 rpm, 10 minutes), then fresh RPMI-1640 complete medium was added, the cells were blown off and placed in the incubator for a further 44 hours. Add 10 .Math.L of CCK8 and continue to incubate for 2 hours. Finally, the microplate reader detects the absorbance at 450 nm wavelength. Cell viability = (absorbance value of experimental group - absorbance of blank)/(absorbance value of control group - absorbance of blank) × 100%, and the experiment was carried out in parallel with three replicate wells (mean ± SD, n = 3). FIG. 6 shows the results of inhibition of proliferation of MV4-11, HL-60 and SHI-1 cells by A6-cPS-VCR, cPS-VCR and VCR. The results showed that the median inhibitory concentration (IC.sub.50) of A6-cPS-VCR was lower than that of cPS-VCR and VCR in all three cell lines. A6-cPS-VCR, cPS-VCR and VCR had no significant difference in the proliferation inhibition of CD44-negative cell lines YNH-1 and OCI-AML-3 (FIG. 7).

[0078] The cell proliferation inhibition of MV4-11 by A6-cPS-DNR refers to the above method. Only added drugs were replaced with A6-cPS-DNR and cPS-DNR. FIG. 8 shows the results of inhibition of proliferation of MV4-11 by A6-cPS-DNR and cPS-VCR. The results showed that 10% A6-cPS-DNR had the lowest IC.sub.50.

Example 9

[0079] A6-cPS-VCR-targeted polymer vesicle nanomedicine induces apoptosis and cell cycle: First, MV4-11 cells were plated in a 24-well plate at a density of 2 × 10.sup.5 cells/well and cultured for 24 hours. 20 .Math.L of A6-cPS-VCR, cPS-VCR or VCR (concentration of VCR was 10 ng/mL) was added, respectively, and 20 .Math.L of PBS was added to the control group. After 4 hours of incubation, the supernatant was removed by centrifugation (3000 rpm, 10 minutes), then fresh RPMI-1640 complete medium was added, the cells were blown off and placed in the incubator for a further 44 hours. Cells were collected in a flow tube, centrifuged (800 rpm, 5 min) and washed twice with 4° C. cold PBS. Finally, 200 .Math.L of binding buffer was added to resuspend the cells. After pipetting evenly, taken 100 .Math.L of it into the flow tube, added 5 .Math.L of AnnexinV-F647 and 10 .Math.L of PI solution in sequence, and stain at room temperature for 15 minutes in the dark, then add 400 .Math.L of PBS and mix evenly to stop the staining, and detection by a flow cytometer within 1 hour. Among them, the cells that were treated in a 50° C. water bath for 5 minutes and fixed with 4% paraformaldehyde for 5 minutes were added with 5 .Math.L of AnnexinV-F647 solution and 10 .Math.L of PI solution for staining for 15 minutes respectively. Cytometer for testing. FIG. 9 shows the results of A6-cPS-VCR-induced apoptosis of MV4-11 cell line. The results showed that A6-cPS-VCR could effectively induce apoptosis. When the concentration of VCR was 10 ng/mL, it could induce 23.3% apoptosis, and the apoptosis rate was significantly higher than that in the non-targeted control cPS-VCR group (12.8%) and free VCR group (16.7%).

[0080] The cell culture and treatment of the cell cycle experiment of A6-cPS-VCR refer to the above method, and only add 1 mL of PBS to resuspend the cells at the end. Then, 4 mL of ice 95% ethanol was added to fix the cells for 12 hours, and 400 .Math.L of PI staining solution was added for 30 minutes in the dark on a shaker at 37° C. Finally, the cells were tested by flow cytometry. FIG. 10 shows the cycle results of A6-cPS-VCR in MV4-11 cell line. The results showed that A6-cPS-VCR and VCR have similar mechanisms, arresting cells in G2/M phase, and eventually leading to apoptosis.

Example 10

[0081] Construction of an in situ MV4-11-GFP-Luc acute myeloid leukemia mouse model: All animal experiments and operations were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University. Establishment of an orthotopic acute myeloid leukemia model: As shown in FIG. 11, the establishment of an orthotopic acute myeloid leukemia model requires the use of NOD/SCID strain mice (6 weeks old, female, body weight greater than 20 g). Mice were first irradiated with X-rays (2.0 Gy), then the antibody CD122 (10 mg/kg mouse) was injected intraperitoneally, and 6 hours later, MV4-11-GFP-Luc cells (1×10.sup.5 cells/mouse) injected into mice by tail vein. After inoculation, the proliferation and proliferation of leukemia cells in mice were observed by in vivo imaging of small animals (FIG. 12).

Example 11

[0082] Enrichment of Cy5-A6-cPS in orthotopic MV4-11-GFP-Luc mice: Targeted enrichment of Cy5-A6-cPS in the bone marrow of orthotopic MV4-11-GFP-Luc mice The collection situation was obtained by in vivo and ex vivo Cy5 fluorescence imaging analysis. On the 10.sup.th day after inoculation, 200 .Math.L of Cy5-A6-cPS and Cy5-cPS (250 .Math.g Cy5equiv./kg) were injected into the mice through the tail vein, respectively, and the mice were anesthetized using an isoflurane gas anesthesia system for 8 hours. In vivo Cy5 fluorescence imaging (FIG. 13A). The mice were then dissected to remove their femurs, tibias and ilium for ex vivo fluorescence imaging (FIG. 13B). The results showed that Cy5-A6-cPS could be efficiently targeted and enriched in the bone marrow, and the fluorescence signal was significantly higher than that of the non-targeted Cy5-cPS group.

Example 12

[0083] Antitumor effect of A6-cPS-VCR in orthotopic MV4-11-GFP-Luc-bearing mice: To study the antitumor effect of A6-cPS-VCR on orthotopic MV4-11-GFP-Luc bearing mice Tumor effect, the following treatment plan was designed (FIG. 14): the first injection was injected on the 6.sup.th day after the inoculation, which was counted as the 0.sup.th day, and the second injection was given on the 2.sup.nd day. The dose of VCR was 0.50 mg/kg, and the control group was injected with 100 .Math.L PBS. There were 10 tumor-bearing mice in each group. Typical images of mouse bioluminescence fluorescence imaging (FIG. 15A) and quantitative results (FIG. 15B) showed that the MV4-11-GFP-Luc cells in the blank control group continued to proliferate rapidly, and began to die on the 4.sup.th day. The MV4-11-GFP-Luc cells of the mice in the VCR group proliferated slowly during drug administration, but proliferated rapidly after drug withdrawal. The MV4-11-GFP-Luc cells of the mice in the cPS-VCR group stopped proliferating during drug administration, but resumed proliferation after drug withdrawal. The MV4-11-GFP-Luc cells of the mice in the A6-cPS-VCR group were killed during the administration period and proliferated slightly after drug withdrawal, indicating that A6-cPS-VCR could effectively inhibit abnormal proliferation of MV4-11-GFP-Luc in mice. There was no significant decrease In body weight In each group of mice during the administration period (FIG. 16A), indicating that the mice tolerated this dose well. Mice lose weight before dying. In addition, the survival time of the mice in the A6-cPS-VCR group was significantly prolonged (FIG. 16B), and the median survival time of the mice was 16 days, compared with the PBS group (6 days) and the VCR group (9 days). And cPS-VCR (11 days), prolonged 1.5-2.7 times.

[0084] In order to further accurately and quantitatively analyze the proliferation of leukemia cells in mice, on the 4.sup.th day, 3 mice in each group were randomly selected for dissection, and the leukocytes in the liver, spleen and bone marrow were extracted, and the content of leukemia cells was detected by flow cytometry. And the liver, spleen and bone marrow slices were analyzed to observe the damage. As shown in FIG. 17, up to about 55% of leukemia cells were detected in the bone marrow of mice in the PBS group, and different degrees of leukemia cells were also detected in the liver and spleen, indicating that the model had a high degree of malignancy. The leukemia levels in the liver, spleen and bone marrow of mice in the VCR and cPS-VCR groups were reduced, but compared with the A6-cPS-VCR group, the A6-cPS-VCR showed better antitumor effect . The HE staining sections (FIG. 18) showed that the bone marrow and spleen tissues of the A6-cPS-VCR group were almost normal, and the liver tissues were almost normal, which also indicated that A6-cPS-VCR had better anti-tumor effect.

[0085] Acute myeloid leukemia cells abnormally proliferate in the bone marrow of mice, resulting in osteolytic lesions in mice. Therefore, micro-CT was used to evaluate the relevant indexes of the femur of each group of mice. The results (FIG. 19) found that there were severe osteoclasts in the hind leg bones of the mice in the PBS group and the cPS-VCR group, and a large number of bone trabeculae were missing. After A6-cPS-VCR treatment, the osteolytic lesions of the mice were significant improvement improved.

Example 13

[0086] Antitumor effect of A6-cPS-DNR in orthotopic MV4-11-GFP-Luc-bearing mice: To study the antitumor effect of A6-cPS-DNR on orthotopic MV4-11-GFP-Luc-bearing mice Tumor effect (refer to Example 11 for the design scheme). The DNR dose was 2 mg/kg, and A6-cPS-DNR was increased by 3 mg/kg in a high-dose group. The blank control group was injected with 100 .Math.L PBS. There were 3 tumor-bearing mice in each group.

[0087] The typical Images of mouse bioluminescence fluorescence Imaging (FIG. 20A) and quantitative results (FIG. 20B) showed that the MV4-11-GFP-Luc cells in the blank control group continued to proliferate rapidly, and the fluorescence quantification in the DNR group decreased slightly, but Treatment is ineffective. The fluorescence quantification of mice in the A6-cPS-DNR and cPS-DNR groups at 2 mg/kg was similar to that in the DNR group. The A6-cPS-DNR high-dose group had obvious therapeutic effect, but the mice in this group died quickly. This experiment shows that A6-cPS-DNR is highly toxic and has a narrow therapeutic window, and the use of A6-targeted polymer vesicles for the treatment of AML is not suitable for all drugs, and specific problems need to be analyzed.

Example 14

[0088] Inhibitory effect of A6-cPS-DNR on proliferation and apoptosis of primary cells of AML patients: Inhibition of proliferation of primary acute myeloid leukemia cells derived from clinical AML patients by A6-cPS-VCR using trypan blue measured by counting method. Firstly, the patient primary cells were plated in a 96-well plate (2×10.sup.4 cells/well), and 20 .Math.L of A6-cPS-VCR, cPS-VCR or free VCR (the final concentration of VCR was 1 mg/mL, respectively) was added to the control. Add 20 .Math.L of PBS to the group. After 4 hours of incubation, the supernatant was removed by centrifugation (3000 rpm, 10 minutes), then fresh RPMI-1640 complete medium was added, the cells were blown off and placed in the incubator for a further 44 hours. The number of viable cells in each well was counted with trypan blue dye. Cell survival rate = (the number of viable cells in the experimental group - the number of blank viable cells)/ (the number of viable cells in the control group - the number of blank viable cells) × 100%. The results (FIG. 21) showed that the toxicity of A6-cPS-VCR to CD44+ patient primary cells was higher than that of CD44- primary cells, and its toxicity to CD44+ cells was higher than that of cPS-VCR, showing a certain targeted therapeutic effect, while There was no significant targeting effect on CD44-patient cells.

[0089] The apoptosis experiment method of A6-cPS-VCR on primary AML cells of patients refers to the apoptosis experiment of MV4-11 cell line in Example 9. Only the concentration of A6-cPS-VCR, cPS-VCR or VCR of VCR was replaced by 1 mg/mL). The results (FIG. 21) show that A6-cPS-VCR has a certain targeted therapeutic effect on primary cells of CD44+ patients, but has no obvious targeting effect on CD44- patients. Apoptosis of patient cells was consistent with the results of proliferation inhibition experiments.

[0090] In conclusion, the reversible cross-linking reduction-sensitive drug-loaded polymer vesicle nano-drug with asymmetric membrane structure of the present invention can efficiently load vincristine sulfate in the cavity, can significantly improve the curative effect of acute myeloid leukemia in situ, and at the same time its biological It has the advantages of degradation, in vivo safety, and simple preparation process, and has the prospect of clinical transformation.