CN116869935A

CN116869935A - Drug-loaded polymer vesicle and preparation method and application thereof

Info

Publication number: CN116869935A
Application number: CN202310554956.5A
Authority: CN
Inventors: 孙欢利; 岳淑静; 钟志远
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2023-05-17
Filing date: 2023-05-17
Publication date: 2023-10-13

Abstract

The invention belongs to the technical field of polymer nano-drugs, and particularly relates to a drug-loaded polymer vesicle and a preparation method and application thereof. According to the invention, the amphiphilic block polymer self-assembles and self-crosslinks, and simultaneously carries the anti-tumor drug, so that the polymer vesicle dual-drug nano-preparation is obtained. The polymer vesicle for co-carrying the medicine has the advantages of simple and controllable preparation, adjustable medicine proportion, small size, easy storage, high stability, excellent biocompatibility, quick and proportional release of the medicine in cells, strong synergistic anti-tumor effect, high in vivo safety, capability of completely eradicating tumor and the like. In general, the polymer vesicle carrying two antitumor drugs has the advantages of simple preparation, safety, high efficiency and synergy, and the like, and is expected to greatly improve the treatment effect of malignant tumors, especially acute myeloid leukemia.

Description

Drug-loaded polymer vesicle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of polymer nano-drugs, and particularly relates to a drug-loaded polymer vesicle and a preparation method and application thereof.

Background

Oncogene Polo-like kinase 1 (PLK 1) is critical for division and proliferation of tumor cells, over-expression exists in leukemia and other various types of tumors, and the expression level is closely related to prognosis defects, so that development of PLK1 molecular targeted drugs is greatly promoted. Of these, volasertib (Vol) is the most advanced PLK1 inhibitor in current clinical research, qualifying as a breakthrough therapy granted by the FDA in 2013 for the treatment of Acute Myeloid Leukemia (AML) patients. However, it has the problems of high in vivo clearance rate, less tumor enrichment, large administration dosage, serious dose-limiting toxicity, easy drug resistance and the like, and has limited clinical efficacy when used alone, and usually needs to be combined with chemotherapy drugs.

Vincristine sulfate (VCR) is a powerful chemotherapeutic agent for inhibiting tubulin polymerization, and is widely used for treating malignant blood tumors such as leukemia, but has low available dosage due to serious neurotoxicity. CN111973556B as the applicant's prior application, the entire disclosure of which is incorporated into the present application as the prior art, discloses a reversible cross-linked degradable polymer vesicle loaded with vincristine sulfate, which is obtained by assembling and cross-linking amphiphilic block polymers, and has an asymmetric membrane structure, wherein the outer shell is polyethylene glycol (PEG), the membrane layer is reversible cross-linked hydrophobic polycarbonate, and the inner shell is polypeptide KD _z Efficient loading of VCRs can be achieved for efficient and specific targeted delivery of vincristine sulfate to multiple myeloma cells. CN110229323a as the applicant's prior application patent, its disclosureThe entire contents of which are incorporated into the present application as prior art.

In contrast to single drug therapy, a dual drug and multi-drug combination regimen may be achieved by acting on the same signaling pathway or multiple related pathways. However, the current multi-drug combination scheme still faces some challenges, on one hand, multi-drug combination is usually accompanied by higher toxic and side effects, and on the other hand, different properties of drugs can have great differences in pharmacokinetics, biodistribution, cell penetration capacity and the like, and are difficult to deliver to a target site in an optimal ratio, resulting in poor synergistic effects. The anti-tumor effect of different drug combinations was unexpected, maria Sol Brassesco et al found that the combination of PLK1 inhibitor and doxorubicin had antagonistic effect (Cancer Biology&Therapeutic 14:7, 648-657, july 2013); marta Halasa et al found that CDDP, CAM did not work synergistically with MCF7, 47D, MDA-MB-231, MDA-MB-468, IC ₅₀ Higher than single drug (int.j.mol.sci.2021, 22, 8573); according to the studies carried out, the polymer-supported CFZ/MID has little synergy in toxicity to Molm-13-Luc, for additive and even antagonistic effects. Therefore, how to achieve efficient and stable entrapment of multiple drugs with different properties in the same nanocarrier and deliver them to the target in a fixed ratio to exert synergistic effects is also very challenging.

Disclosure of Invention

The invention aims to provide a drug-loaded polymer vesicle which is mainly prepared from an amphiphilic block polymer and two or more than two antitumor drugs, and the antitumor effect is synergistically improved.

Another object of the present invention is to provide a method for preparing drug-loaded polymer vesicles and applications thereof.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

a drug-loaded polymer vesicle, which comprises an amphiphilic block polymer and a plurality of anti-tumor drugs; preferably, the plurality of antitumor drugs are two or more different antitumor drugs; further preferably, the drug-loaded polymer vesicles are prepared from amphiphilic block polymers and two or more antitumor drugs.

Wherein the amphiphilic block polymer has a structure of hydrophilic segment-hydrophobic segment-polypeptide segment.

Further, the structure of the amphiphilic block polymer is hydrophilic segment-hydrophobic segment-KD _z Wherein K is lysine and D is aspartic acid; preferably, z is 5 to 15.

Further, at least one of the antitumor drugs is a tubulin inhibitor.

Further, the tubulin inhibitor is any one or more of vincristine sulfate, paclitaxel, docetaxel, cabazitaxel, auristatin, maytansine and Ai Li brin.

Further, the provided drug-loaded polymer vesicles, at least one of which is a targeted oncogene Polo-like kinase 1 inhibitor.

Further, the targeted oncogene Polo-like kinase 1 inhibitor is any one or more of Volasatide, regoraceti, ON01910, BI2536, HMN-214 and GSK 461364.

Further, the provided drug-loaded polymer vesicles, the two or more antitumor drugs are combinations of a tubulin inhibitor and an oncogene Polo-like kinase 1 inhibitor.

Further, two or more antitumor drugs are a combination of vincristine sulfate and volasertib.

Wherein, the feeding mass ratio of the tubulin inhibitor to the targeted oncogene Polo-like kinase 1 inhibitor is 1: (1-48); preferably, the mass ratio of the materials is 1: (8-32); more preferably, the feeding mass ratio is 1: 8. 1:16 or 1:32.

further, the hydrophilic segment in the amphiphilic block polymer is a polyethylene glycol segment, the hydrophobic segment is a random copolymer P (A-DTC), A is an ester monomer or a carbonate monomer, and DTC is a disulfide five-membered ring carbonate unit; p means polymerization, P (A-DTC) is a random copolymer of A-DTC.

Further, the structure of P (A-DTC) is one of the following structures:

further, the ester monomer is one or two of caprolactone and lactide; the carbonate monomer is trimethylene carbonate.

Further, in the amphiphilic block polymer, the molecular weight of the hydrophilic segment is 2000-10000 Da; the molecular weight of the hydrophobic section is 2-10 times of that of the hydrophilic section; the molecular weight of PDTC is 10% -40% of the total molecular weight of the hydrophobic section; z is 5 to 15;

preferably, the molecular weight of the hydrophilic segment is 2000-8000 Da; the molecular weight of the hydrophobic chain segment is 2-6 times of that of the hydrophilic segment; the molecular weight of the PDTC chain segment is 10% -30% of that of the hydrophobic chain segment;

more preferably, the hydrophilic segment has a molecular weight of 5000 to 7500 Da; the molecular weight of the hydrophobic chain segment is 2-4 times of that of the hydrophilic segment; the molecular weight of PDTC is 10% -20% of the total molecular weight of the hydrophobic segment, and z is 5, 10 or 15.

Further, the structure of the amphiphilic block polymer is any one of the following structures:

。

further, the drug-loaded polymer vesicle is mainly prepared from an amphiphilic block polymer, a tubulin inhibitor and a targeted oncogene Polo-like kinase 1 inhibitor, wherein the structure of the amphiphilic block polymer is any one of the following structures:

。

The tubulin inhibitor is vincristine sulfate, and the targeted oncogene Polo-like kinase 1 inhibitor is voratite; preferably, the feeding mass ratio of the tubulin inhibitor to the targeted oncogene Polo-like kinase 1 inhibitor is 1:1 to 48.

Preferably, the feeding mass ratio of the tubulin inhibitor to the targeted oncogene Polo-like kinase 1 inhibitor is 1:8 to 32.

Preferably, the molecular weight of the hydrophilic segment of the amphiphilic block polymer is 2000-8000 Da; the molecular weight of the hydrophobic chain segment is 2-6 times of that of the hydrophilic segment; the molecular weight of the PDTC chain segment is 10% -30% of that of the hydrophobic chain segment.

More preferably, the dosage mass ratio of the tubulin inhibitor to the targeted oncogene Polo-like kinase 1 inhibitor is 1: 8. 1:16 or 1:32.

further preferably, the hydrophilic segment has a molecular weight of 5000 to 7500 Da; the molecular weight of the hydrophobic chain segment is 2-4 times of that of the hydrophilic segment; the molecular weight of PDTC is 10% -20% of the total molecular weight of the hydrophobic segment, and z is 5, 10 or 15.

Further, the invention provides a method for preparing drug-loaded polymer vesicles, comprising the following steps: two or more than two antitumor drugs and amphiphilic block polymers are used as raw materials, and the polymer vesicles carrying the drugs are prepared by a solvent substitution method.

Further, the preparation method comprises the steps of mixing an anti-tumor drug solution and an amphiphilic block polymer solution, standing, and dialyzing to obtain drug-loaded polymer vesicles; the time for the standing is 5 to 50 hours, preferably 8 to 30 hours.

Further, the preparation method of the reversible cross-linked degradable polymer vesicle (Ps-VCR/Vol) for co-loading VCR and Vol comprises the steps of mixing Vol with PEG-P (TMC-DTC) -KD _z Adding the mixed solution of the polymer into the VCR solution under stirring, standing and dialyzing to obtain Ps-VCR/Vol. Specifically, VCR is dissolved in ultrapure water and mixed with a buffer solution, such as N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) buffer (HEPES), to obtain VCR solution; vol was combined with Polymer PEG-P (TMC-DTC) -KD _z Respectively dissolving in DMSO and uniformly mixing, then injecting the mixed solution into a stirred VCR solution, continuously stirring, then placing into a shaking table for standing, and then dialyzing with a buffer solution (such as HEPES) to obtain Ps-VCR/Vol, wherein the feeding mass ratio of VCR to Vol is 1: (8-32).

Furthermore, the invention provides application of the drug-loaded polymer vesicle in preparing an anti-tumor drug.

Furthermore, the invention provides application of the drug-loaded polymer vesicle in preparing a drug for treating acute myeloid leukemia.

Compared with the prior art, the invention has the following advantages:

the invention designs a novel drug-loaded vesicle for co-carrying a tubulin inhibitor and a targeted oncogene Polo-like kinase 1 inhibitor; the vesicle membrane is reversible crosslinked biodegradable poly trimethylene carbonate (PTMC), poly Lactide (PLA) and Polycaprolactone (PCL) with good biocompatibility, and the dithiolane of the side chain can provide reversible crosslinking sensitive to reduction, so that long circulation of the medicine in blood can be ensured, quick crosslinking in cells can be realized, and two medicines can be synchronously released into tumor cells.

2. The VCR and Vol co-carried vesicle disclosed by the invention has remarkable in-vivo and in-vitro synergistic anti-tumor effect, and the polymer has good biocompatibility, can form vesicles with asymmetric membrane structures, and has good drug entrapment and co-delivery effects.

3. The degradable polymer vesicle carrier provided by the invention overcomes the defects of large particle size, poor in-vivo circulation stability, easy leakage of medicine, slow release of intracellular medicine and the like of the existing nano carrier.

4. The vesicle system of the invention has a plurality of unique advantages, including small size, simple and controllable preparation, excellent biocompatibility, high in vivo circulation stability, high release speed of intracellular drugs, remarkable tumor growth inhibition effect and the like. Therefore, the vesicle system is hopeful to become a simple and multifunctional nano platform and is used for efficiently delivering the tubulin inhibitor and the targeted oncogene Polo-like kinase 1 inhibitor to tumor cells, and a strong synergistic anti-tumor effect is exerted.

Drawings

FIG. 1 is a schematic diagram of PEG-P (TMC-DTC) -KD in example one ₁₀ Nuclear magnetic spectrum of (2).

FIG. 2 is a schematic representation of the preparation of Ps-VCR/Vol in example two.

FIG. 3 shows the particle size and particle size distribution of Ps-VCR/Vol in example two.

FIG. 4 shows (A) particle size, particle size distribution variation, and (B) drug leakage during long-term storage at 4℃for Ps-VCR/Vol in example three.

FIG. 5 shows the stability of (A) Ps-VCR/Vol in 50-fold dilution and 10% FBS addition, and (B) the release profile of Ps-VCR/Vol in example three under non-reducing conditions and 10 mM GSH.

FIG. 6 shows anti-AML activity (n=6) of Ps-VCR/Vol, ps-VCR, ps-Vol and free VCR/Vol in cells (A), (D) MV-4-11, (B), (E) Molm-13-Luc and (C), (F) WEHI-3 in example four.

FIG. 7 shows the anti-AML activity of PS-DNR/Vol in WEHI-3 cells.

FIG. 8 shows (A) MV-4-11, (B) Molm-13-Luc and (C) WEHI-3 in example fiveCell cycle analysis after incubation of cells with Ps-VCR/Vol, ps-VCR, ps-Vol, free VCR/Vol and PBS 24 h.

FIG. 9 shows the induction of apoptosis in (A) MV-4-11, (B) Molm-13-Luc, (C) WEHI-3 cells and (D) primary AML cells by Ps-VCR/Vol in example five.

FIG. 10 is a graph showing the effect of Ps-VCR/Vol on the relative protein content of MV-4-11 and Molm-13-Luc cells in example six.

FIG. 11 shows (A) weight change, (B) Kaplan-Meier survival curve, and (C) blood biochemical and blood routine index analysis of mice in each group within 14 days after single administration of Ps-VCR/Vol in example seven.

FIG. 12 is a schematic diagram of (A) in situ Molm-13-Luc AML mouse model establishment, dosing and monitoring, (B) in situ Molm-13-Luc AML mouse in vivo bioluminescence imaging picture, (C-D) semi-quantitative analysis of Luc bioluminescence signal of mice (n=4, dotted line is background signal value of healthy mice), (E) Kaplan-Meier survival curve, (F) change of body weight of mice during treatment.

FIG. 13 shows (A) AML cell infiltration in liver, lung, spleen, peripheral blood and bone marrow of mice and (B) quantitative analysis and (C) weights of spleens of mice in different treatment groups in example eight.

FIG. 14 is an H & E staining photograph of major organs (liver, spleen, kidney, lung and heart) of in situ Molm-13-Luc mice after different treatments in example eight.

FIG. 15 is (A) H & E stained pictures and (B) TRAP stained pictures of in situ Molm-13-Luc mouse leg bones in example eight.

FIG. 16 is a graph of (A) micro-CT and (B) quantitative analysis of each index of femur and tibia of mice in different treatment groups in example eight.

FIG. 17 is a schematic diagram of the establishment, administration and detection of (A) in situ MV-4-11 or WEHI-3 AML mice models in example eight, wherein (B) Kaplan-Meier survival curves and (C) body weight changes of in situ MV-4-11 AML mice were treated differently, and (D) Kaplan-Meier survival curves and (E) body weight changes of in situ WEHI-3 AML mice were treated differently.

Detailed Description

Unless otherwise indicated, the terms used herein have the following meanings:

tubulin inhibitors: and a compound which, by binding to a specific site on tubulin, affects and interferes with the kinetics of polymerization and depolymerization of tubulin, thereby blocking the formation of the spindle in the M phase and causing tumor cell growth to arrest in the G2/M phase, thereby exerting an antitumor effect. The present tubulin inhibitors are vincristine sulfate, paclitaxel, docetaxel, cabazitaxel, auristatin, maytansine, ai Li brin and the like.

PLK1 inhibitors: by inhibiting the activity of PLK1, a compound that causes tumor cell growth inhibition and even apoptosis, but has no significant effect on normal cells. Current PLK1 inhibitors are volasertib, regoracetib, ON01910, BI2536, HMN-214, GSK461364, etc.

Random copolymer: and the main chain is a copolymer with random arrangement of monomer units. If the random copolymer has two monomer units, the monomers M1 and M2 are arranged randomly on the macromolecular chain, and the two monomers are distributed randomly on the main chain. In the invention, the random copolymer is P (A-DTC), A is an ester monomer or a carbonate monomer unit, and DTC is a disulfide five-membered ring carbonate monomer unit.

The invention is further described below with reference to examples and figures, and specific methods of preparation and testing are routine in the art. In the following examples, unless otherwise specified, the amphiphilic block polymer is PEG _5k -P(TMC _15k -DTC _2k )-KD ₁₀ The method comprises the steps of carrying out a first treatment on the surface of the Vincristine sulfate (VCR), volasertib (Vol) and other agents are all commercially available materials. The cell experiment and the animal experiment are routine experiments, and meet the related requirements of the university of Suzhou.

Example Polymer PEG-P (TMC-DTC) -KD ₁₀ Is synthesized by (a)

Polymer PEG-P (TMC-DTC) -KD ₁₀ Activation of PEG-P (TMC-DTC) terminal hydroxyl group by P-NPC, followed by KD ₁₀ Obtained by the reaction, and can be prepared by the method disclosed in the patent CN201910472613.8 or the patent CN 202010845920.9.

Weigh 85 mg KD ₁₀ The polypeptide was placed in a double-necked flask, after evacuation of 1. 1 h, 3.5 mL anhydrous DMSO was added under nitrogen to dissolve the polypeptide, and 153 mg (210. Mu.L) triethylamine was added. Then, an anhydrous DMSO solution (755 mg,7 mL) of PEG-P (TMC-DTC) -NPC was added dropwise thereto, and reacted at 30℃for 48 h. Subsequently, the reaction solution was filled into dialysis bags (MWCO: 3500 Da), 40 h was dialyzed against DMSO containing 10% absolute methanol, the dialysate was changed every 8 h, and 8 h was dialyzed against DCM every 4 h. Finally, precipitating in glacial diethyl ether, and vacuum drying 48 h to obtain white flocculent polymer PEG-P (TMC-DTC) -KD ₁₀ Yield: 87%.

PEG-P(TMC-DTC)-KD ₁₀ A kind of electronic device ¹ As shown in FIG. 1, the H NMR spectra can be obtained for PEG (. Delta.3.23,. Delta.3.49), TMC (. Delta.4.15,. Delta.1.91), DTC (. Delta.4.15,. Delta.3.06) and KD ₁₀ Characteristic peaks of (δ4.47, δ1.15) and by comparing KD at δ4.47 ppm ₁₀ Peak areas of the methylene and TMC methylene at delta 1.91 ppm were calculated to obtain KD ₁₀ The grafting ratio of (2) was 100%.

Example preparation and characterization of two Ps-VCR/Vol

Ps-VCR/VolPrepared by solvent displacement method, wherein VCR and Vol are prepared by combining KD with _z The electrostatic interaction between the two is wrapped in the hydrophilic inner cavity of the vesicle. The mass feed ratios of VCR to Vol were 1:8, 1:16 and 1:32, respectively, with the theoretical Drug Loading (DLC) of Vol constant 16.7 wt%, and the preparation schematic is shown in FIG. 2.

Taking 1:16 as an example, 1.75 mL of Vol in DMSO (20 mg/mL) was combined with 4.3 mL of PEG-P (TMC-DTC) -KD ₁₀ Is uniformly mixed with DMSO solution (40 mg/mL); 0.44 mL of VCR in water (5 mg/mL) was added to 38 mL of HEPES solution (5 mM, pH 6.8), mixed well at 37℃and 300 rpm, then polymer and Vol in DMSO solution were injected into the mixture at a constant speed, stirred for 15 min, transferred to a 37℃shaker, and incubated for 12 h at rest. Then, the resultant was dialyzed (MWCO: 3500 Da) against HEPES (5 mM, pH 7.4) to obtain Ps-VCR/Vol, and concentrated by centrifugal ultrafiltration (vivaspin, MWCO:10 kDa) to give a co-drug-loaded vesicle concentration of 9 mg/mL.

Co-drug-loaded vesicles with feed ratios of 1:8 and 1:32, respectively, were also prepared as above.

The particle size of the obtained Ps-VCR/Vol at different drug ratios was found to be between 27.2 and 29.5 and nm, and the particle size distribution was less than 0.1 (Table 1 and FIG. 3). The VCR content was calculated by HPLC (mobile phase methanol: water=70:30, 0.15% triethylamine was added and the pH was adjusted to 7.0 with phosphoric acid) and the absorbance at wavelength 298 nm (UV) was measured and the Vol content was calculated by ultraviolet spectrophotometer (UV-vis) test.

The results show that the encapsulation rates (DLE) for VCR and Vol are 47.0-49.5% and 71.4-75.4%, respectively.

When the mass feed ratio of VCR to Vol is 1:8, 1:16 and 1:32 respectively, the mass ratio of actual entrapped VCR to Vol is 1:12.2, 1:24.3 and 1:48.7 respectively, abbreviated as Ps-VCR/Vol _1/12 、Ps-VCR/Vol _1/24 And Ps-VCR/Vol _1/48 。

Ps-VCR and Ps-Vol are prepared similarly to Ps-VCR/Vol, and only VCR or Vol is added during the drug encapsulation process, wherein theoretical DLC of VCR and Vol is 5 wt% and 16.7 wt%, respectively, and the particle size and particle size distribution of both are similar to Ps-VCR/Vol, and actual DLC is 4 wt% and 13.24 wt%, respectively.

^a By HPLC; ^b measured by UV-vis; ^c measured by DLS.

With PEG5k-P (TMC 15k-DTC2 k) -KD ₁₀ The Ps-DNR/Vol is prepared by taking a carrier, vincristine sulfate (VCR) and daunorubicin hydrochloride (DNR) as medicines; DNR/Vol feed ratio 1:9 loaded DNR and Vol can achieve an encapsulation efficiency of 28.4% (Vol), DNR/Vol feed ratio 2:5 loaded DNR and Vol can achieve an encapsulation efficiency of 26.4% (Vol), and DNR/Vol actual ratio 1:1.2.

PEG-P (TMC-DTC) -KD according to the method described above _z 、PEG-P(CL-DTC)-KD _z 、PEG-P(LA-DTC)-KD _z Loading VCR and Vol can achieve encapsulation efficiency of 40% or more (VCR), 70% or more (Vol), such as PEG5k-P (TMC 10k-dtc1.5 k) -KD ₁₀ 、PEG5k-P(TMC15k-DTC2k)-KD ₁₅ 、PEG5k-P(TMC15k-DTC2k)-KD ₅ 、PEG5k-P(CL15k-DTC2k)-KD ₁₀ 、PEG5k-P(LA15k-DTC2k)-KD ₁₀ 、PEG7.5k-P(TMC15k-DTC2k)-KD ₁₀ 。

Example stability of three Ps-VCR/Vol and in vitro drug Release

To study the stability of Ps-VCR/Vol, ps-VCR/Vol was used _1/24 For example, the particle size, particle size distribution variation, and VCR and Vol leakage during storage at 4 ℃ were continuously monitored. At days 30, 60, 90 and 300, the particle size and particle size distribution were tested. At 220 and 300 days, 2 mg Ps-VCR/Vol was taken, respectively _1/24 Ultracentrifugation (58000 rpm,1 h) was performed with HEPES diluted to 1 mg/mL, the centrifuged supernatant was taken, and the concentration of Vol and VCR was tested by HPLC and the content of leaked drug was calculated. Drug retention (%) was calculated by comparing the drug content with that at day 0. FIGS. 4A and 4B are Ps-VCR/Vol, respectively _1/24 Particle size, particle size distribution and drug retention profile. The results show that Ps-VCR/Vol _1/24 The particle size and the polydispersity index (PDI) have no obvious change within 300 days when the composition is placed at the temperature of 4 ℃, and the medicine retention amount is higher than 95%. In addition, monitoring by DLSMeasurement of Ps-VCR/Vol _1/24 Particle size distribution after 50-fold dilution with PBS and 10% FBS addition, as shown in FIG. 5A, ps-VCR/Vol maintains good stability when diluted 50-fold or 10% FBS is added.

In Ps-VCR/Vol _1/12 For example, the release of Ps-VCR/Vol was studied by dialysis. Will 0.5 mL Ps-VCR/Vol _1/12 (2 mg/mL) was placed in a drug delivery bag (MWCO: 14000 Da), placed in 20 mL HEPES (5 mM, pH 7.4) or HEPES containing 10 mM GSH, and run in a shaker at 200 rpm at 37 ℃ (n=3). After 0, 1, 2, 4, 6, 8, 10, 12 and 24 h, 5 mL release liquid was removed and 5 mL fresh release medium was replenished, respectively. After the release liquid is freeze-dried, 300 [ mu ] L of mixed liquid (volume ratio is 1:1) of methanol and secondary water is added for dissolution, and HPLC test is carried out after the release liquid is filtered by a 0.22 [ mu ] m filter head. The mobile phase of VCR is methanol: water (0.15% triethylamine) =70: 30, the detection wavelength is 298 nm; the mobile phase of Vol is acetonitrile: water (0.15% triethylamine) =60: 40, the pH was adjusted to 7.0 with phosphoric acid, and the detection wavelength was 330 nm. FIG. 5B shows Ps-VCR/Vol _1/12 In vitro release results for the dual vesicle nanoparticulate formulation are shown. The results show that the release amount of the Ps-VCR/Vol is less than 20% of the release amount of the VCR and the Vol in 24 h under physiological conditions, however, the release amount of the VCR and the Vol reaches more than 90% in the presence of 10 mM GSH, and the release kinetics of the VCR and the Vol are similar, so that the VCR and the Vol can be released according to the set proportion.

Example four Ps-VCR/Vol in vitro synergistic anti-AML experiments

The synergistic antitumor activity of Ps-VCR/Vol in MV-4-11, molm-13-Luc and WEHI-3 AML cells at different drug ratios was determined using the CCK-8 kit. MV-4-11 and Molm-13-Luc cells were cultured in RPMI-1640 medium containing 10% FBS,1% penicillin and streptomycin, and WEHI-3 cells were cultured in DMEM medium (containing 20% FBS,1% penicillin and streptomycin). The 3 cells were plated in 96-well plates (2X 10) ⁴ Each hole) and 20 mu L of Ps-VCR, ps-Vol and Ps-VCR/Vol with different concentrations are respectively added into each hole _1/12 、Ps-VCR/Vol _1/24 、Ps-VCR/Vol _1/48 And free VCR/Vol _1/24 48 and h were incubated in an incubator. Wherein the in-well concentration of the Ps-VCR group VCR is 0.007-6.8 ng/mL, for the Ps-Vol and dual drug combination, the concentration of Vol in wells was 0.1-500 ng/mL when incubated with Molm-13-Luc and WEHI-3 cells, and 0.1-100 ng/mL when incubated with MV-4-11 cells. After 48 h incubation, 10 μl CCK-8 solution was added to each well, and after 3 h incubation was continued, the absorbance at 450 nm (n=6) was measured with a microplate reader, and the cell viability was the ratio of the absorbance of the cells of the sample group to the absorbance of the cells of the PBS group. The synergy of VCR and Vol is evaluated by calculating a synergy index (Combination Index, CI). Namely:

A synergistic effect when CI <1, in particular a strong synergistic effect when CI <0.5, in particular a very strong synergistic effect when CI < 0.3; additive effect when ci=1; antagonism is shown when CI > 1. The evaluation results were as follows:

the research shows that both Ps-VCR and Ps-Vol can effectively inhibit proliferation of AML cells, and IC in three kinds of AML cells ₅₀ 1.3-1.7 ng/mL and 8-21.4 ng/mL, respectively (FIG. 6 and Table 2). The Ps-VCR/Vol dual vesicle nano-formulations exhibit significantly enhanced anti-AML activity at VCR to Vol mass ratios of 1:12, 1:24, and 1:48 compared to Ps-VCR and Ps-Vol single vesicles. In MV-4-11 cells, three VCR and Vol IC ₅₀ Values of 0.032-0.095 ng/mL and 1.3-1.5 ng/mL, respectively, were only 1/13 and 1/5 of Ps-VCR and Ps-Vol, and the synergy index (CI) was as low as 0.20-0.25 (FIG. 6A, D and Table 2), indicating potent synergy anti-AML activity when the VCR and Vol were co-loaded, and a wide range of adjustable drug ratios.

IC of Ps-VCR/Vol in Molm-13-Luc cells and WEHI-3 cells in different VCR and Vol ratios ₅₀ The CI values were also significantly reduced compared to Ps-VCR and Ps-Vol, 0.36-0.41 and 0.18-0.23, respectively (FIG. 6B, C, E, F and Table 2). While free VCR/Vol _1/24 Is weak against AML, its VCR and Vol IC ₅₀ The values are Ps-VCR/Vol, respectively _1/24 3.7-6.9 and 3.0-5.8 times, which shows that the synergistic effect of the vesicle nano-preparation which carries VCR and Vol together is obviously better than that of the free medicine combination.

Referring to the above method, the anti-AML activity of PS-DNR/Vol in WEHI-3 cells is shown in FIG. 7 and Table 3, wherein the drug ratio is the actual ratio, and the CI value of DNR and Vol is 1.1-2.4 when the DNR and Vol are co-carried, and the antagonism is shown when the DNR and Vol are co-carried.

Example five Ps-VCR/Vol synergistically blocks cell cycle and induces apoptosis

The period blocking of AML cells by Ps-VCR/Vol was studied by PI/RNase A staining. 1.8 mL MV-4-11, molm-13-Luc and WEHI-3 cells were plated into 6-well plates (5X 10) ⁵ 0.2 mL PBS, ps-VCR, ps-Vol, ps-VCR/Vol were added to each well) _1/12 、Ps-VCR/Vol _1/24 、Ps-VCR/Vol _1/48 And free VCR/Vol _1/24 . Wherein the concentration of VCR in the wells of the Ps-VCR was 0.4. 0.4 ng/mL and the concentration of Vol in the other groups was fixed at 10. 10 ng/mL. After incubation of 24 h in an incubator, cells were collected by centrifugation and resuspended in 1 mL ice PBS, and slowly added to 4 mL 95% ethanol in vortexing, and fixed at 4 ℃ for 24 h. Then, cells were washed with PBS and resuspended in 0.4 mL staining buffer, 15 μl PI and 4 μl RNase a (2.5 mg/mL) were added to each sample group and directly tested with a flow cytometer after staining for 30 min at 37 ℃. The results show that Ps-VCR/Vol with different VCR and Vol mass ratios can efficiently induce the G2/M phase retardation of MV-4-11 cells at a Vol concentration of 10 ng/mL, the G2/M phase cell ratio is significantly increased from 18.9% to 56.2% in the PBS group (fig. 8A, D), and the vesicle single agent preparation is similar to that in the PBS group (22%). In addition, the free VCR/Vol group induced only 30.8% G2/M phase block, significantly lower than the Ps-VCR/Vol group. Similarly, in Molm-13-Luc cells, three different ratios of Ps-VCR/Vol induced 60-70% of G2/M phase arrest, significantly higher than the free VCR/Vol group (20.6%), however, the Ps-Vol (Vol: 10 ng/mL) vesicle single drug formulation failed Causing cell cycle arrest, the G2/M phase ratio was comparable to that of the PBS group (6%), and the Ps-VCR (VCR: 0.4 ng/mL) increased the G2/M phase arrest by only 16.9%. (FIG. 8B, E). In WEHI-3 cells, the G2/M phase ratio was also significantly higher in the three different proportions of the Ps-VCR/Vol group than in the other control group (FIG. 8C, F).

Both VCR and Vol are capable of inducing cell cycle arrest, thereby causing apoptosis. For this reason, the case of inducing apoptosis of AML cells by Ps-VCR/Vol was further studied using an Annexin V-APC/7-AAD double-dye apoptosis kit. 1.8 mL MV-4-11, molm-13-Luc, WEHI-3 and primary cells extracted from AML patient bone marrow were plated into 6 well plates (5X 10) ⁵ 0.2 mL PBS, ps-VCR, ps-Vol, ps-VCR/Vol were added to each well) _1/12 、Ps-VCR/Vol _1/24 、Ps-VCR/Vol _1/48 And free VCR/Vol _1/24 . The concentration of Ps-VCR in VCR wells was 0.4 ng/mL in the three cell lines, the concentration of Vol in the other group was constant at 10 ng/mL, and the concentrations of VCR and Vol in the primary cells of the patient were 0.2 and 4.8 μg/mL, respectively. After 48. 48 h incubation in an incubator, cells were collected by centrifugation, resuspended in 0.5 mL of 1 Xbinding buffer, 5. Mu.L of Annexin V-APC and 10. Mu.L of 7-AAD were added to each well, and after 5 min of staining at room temperature, tested by flow cytometry. At least 10000 cells were collected per sample. The results show that in MV-4-11 cells, the Ps-VCR/Vol with three different drug ratios can cause significant apoptosis, the apoptosis rate is higher than 90%, wherein the Ps-VCR/Vol _1/24 The highest group, up to 99.7%, is significantly higher than Ps-VCR (49.2%), ps-Vol (36%) and free VCR/Vol _1/24 Group (62.6%) (fig. 9A). In Molm-13-Luc cells, ps-VCR/Vol _1/12 、Ps-VCR/Vol _1/24 And Ps-VCR/Vol _1/48 Results in apoptosis of about 75%, compared with single vesicle formulations of Ps-VCR (34.3%) and Ps-Vol (32.5%) and free VCR/Vol _1/24 (39.7%) was significantly improved (fig. 9B). After incubation of WEHI-3 cells with different Ps-VCR/Vol for 48 h, about 70% of the cells were apoptotic, with Ps-VCR (32.6%), ps-Vol (26.9%) and free VCR/Vol _1/24 (36.3%) was 2.1-2.7 fold higher than the above (FIG. 9C). Furthermore, ps-VCR/Vol _1/24 Also for primary patient specimen cellsHas remarkable pro-apoptotic ability, and is superior to other control groups (Ps-VCR, ps-Vol and free VCR/Vol) in 8 patient samples _1/24 ) All showed the strongest antitumor ability (fig. 9D). Therefore, the Ps-VCR/Vol can efficiently induce the AML cells to be blocked in the G2/M phase under the conditions of three drug ratios (1:12, 1:24 and 1:48), so that the apoptosis is promoted, and the effects are obviously better than that of single-drug preparations of Ps-VCR and Ps-Vol vesicles and free VCR/Vol, and the effects have obvious synergistic effects.

EXAMPLE six Western blotting (Western blot) study of inhibition of Ps-VCR/Vol on AML cellular protein expression

To investigate the synergistic anti-AML mechanism of VCR and Vol, immunoblotting was used to characterize the expression of the relevant proteins. 1.8 mL of Molm-13-Luc and MV-4-11 cell suspension (1X 10) ⁶ Individual/well) was plated into 12-well plates, and 0.2 mL PBS, ps-VCR, ps-Vol, ps-VCR/Vol was added _1/24 Or free VCR/Vol _1/24 The concentration in the wells of VCR and Vol were made 0.2 ng/mL and 4.8 ng/mL, respectively. After incubation in incubator 48 h, washing with ice PBS and adding 100 μl of ice RIPA lysate to lyse on ice for 20 min, then centrifuging (4 ℃ at 12000 rpm) for 15 min to remove the precipitate, and testing the total protein concentration with BCA. 20% of sample buffer solution with volume fraction is added into the protein, and the mixture is boiled for 5 min in a water bath with the temperature of 95 ℃. 20. Mu.g of protein was separated by electrophoresis using 10% SDS-PAGE and transferred to PVDF membrane, blocked with TBST containing 5% skimmed milk at room temperature for 2 h, and MCL-1 antibody, BCL-2 antibody, pHH3 antibody and. Beta.3-tubulin antibody were added separately by cutting a band of appropriate molecular weight and incubated overnight at 4 ℃. The strips were washed 3 times with Tris-HCl buffer/0.1% Tween-20 wash (TBST) and incubated with HRP-labeled secondary antibody at room temperature for 1.5. 1.5 h. After the strip has been treated with the developer, an image is taken with an electrochemiluminescence detection system (Pierce). FIG. 10 is a graph of immunoblotting results. The results show that Ps-VCR/Vol _1/24 The treatment can obviously down regulate two anti-apoptosis proteins of MCL-1 and BCL-2, simultaneously up regulate the expression of the effect Caspase-3 protein, activate the pro-apoptosis pathway, and the effect is obviously better than that of a vesicle single drug preparation and free VCR/Vol _1/24 Control group. Taken together, it is known that Ps-VCR/Vol blocks cells in G2/M phase by synergy,down-regulating anti-apoptosis proteins BCL-2 and MCL-1, regulating balance between BCL-2 family pro-apoptosis and anti-apoptosis proteins, activating Caspase dependent effector pathways, and initiating mitochondrial apoptosis to exert synergistic anti-AML activity.

Example seven in vivo acute toxicity test of Ps-VCR/Vol

In vivo acute toxicity of Ps-VCR/Vol was studied using Kunming mice (Vetolihua, 6 weeks old, male and female halves). The mice were randomly divided into 3 groups of 10 mice each, each half of which was injected with Ps-VCR/Vol through the tail vein in a single injection _1/12 （1 mg VCR&12 mg Vol equiv./kg）、Ps-VCR/Vol _1/24 （1 mg VCR&24 mg Vol equiv./kg）、Ps-VCR/Vol _1/48 （0.5 mg VCR&24 mg Vol equiv./kg). Mice were monitored for body weight, status and survival continuously for 14 days after dosing. On day 5 post-dose, 4 mice (2 females and 2 males) were randomized for each group and tested for blood normative and biochemical by abdominal aortic blood sampling. 4 healthy mice without drug administration were used as controls. The results showed that Ps-VCR/Vol _1/48 Group mice did not lose weight and remained continuously growing at VCR and Vol doses of 0.5 and 24 mg/kg, respectively, and were not abnormal in posture and behavior. Ps-VCR/Vol _1/12 （1 mg VCR&12 The body weight of the group of mg Vol equiv./kg mice decreased slightly within 2 days after the administration<5%) followed by a rapid return to normal without significant toxicity and death. For Ps-VCR/Vol _1/24 （1 mg VCR&24 mg Vol equiv./kg) mice had a decrease in body weight (8%) within 3 days after dosing, and one of the mice (1/10) had retarded activity, but recovered rapidly from day 4, without any death due to toxicity (fig. 11A, B). In addition, the blood routine and blood biochemical index of each group of mice are in the normal range, and there is no obvious difference compared with healthy mice (fig. 11C), indicating that Ps-VCR/Vol of three different drug ratios have higher safety even at high doses and are obviously better than free drugs.

Examples eight Ps-VCR/Vol in vivo synergistic anti-AML activity

The in vivo synergistic anti-AML activity of Ps-VCR/Vol was studied using three AML mouse models, molm-13-Luc, MV-4-11 and WEHI-3 in situ.

To build the in situ Molm-13-Luc model, 200. Mu.L of a Molm-13-Luc cell suspension (5X 10 ⁵ And then injected into NOD.CB17-Prkdc ^scid /IL2rg ^tm1 Bcgen (B-NDG) female mice were vaccinated on day 0. On day 3 post-inoculation, in situ Molm-13-Luc AML mouse models were randomized into 7 groups, each by tail vein injection with PBS, ps-VCR (0.25 mg VCR equiv./kg), ps-Vol (6 mg Vol equiv./kg), ps-VCR/Vol, respectively _1/12 （0.25 mg VCR&3 mg Vol equiv./kg）、Ps-VCR/Vol _1/24 （0.25 mg VCR&6 mg Vol equiv./kg）、Ps-VCR/Vol _1/48 （0.125 mg VCR&6 mg Vol equiv./kg) and free VCR/Vol _1/24 （0.25 mg VCR&6 mg Vol/kg) was injected 4 times per 3 days (FIG. 12A). Wherein each group of 13 mice, 6 mice were used for weight and life cycle monitoring, 4 mice were used for imaging monitoring tumor growth, and 3 mice were used for assays such as tumor infiltration and micro-CT. The PBS group mice were found to develop rapidly, developed symptoms of hind limb paralysis, poor spirit, etc. at about day 15 post-inoculation, and caused death (fig. 12B-E). The proliferation of Molm-13-Luc cells in mice can be effectively inhibited by Ps-VCR/Vol with three different drug ratios, no tumor-related bioluminescence signal was observed within 20 days (at day 32) after the end of administration, wherein the proliferation of Molm-13-Luc cells was inhibited by Ps-VCR/Vol _1/24 At the end of imaging on day 44 no significant signal was seen, luc signal intensity was comparable to healthy mice (fig. 12B-D). However, the Ps-Vol and Ps-VCR vesicle single agent groups proliferated rapidly in vivo AML cells after the end of administration, and tumor Luc bioluminescence signal at day 22 increased 235 and 5.6 fold over that at day 7, respectively. Free VCR/Vol _1/24 Although the group inhibited proliferation of Molm-13-Luc cells in the early stage, recurrence occurred at day 32, tumor Luc signal increased rapidly, and at day 38 compared with Ps-VCR/Vol _1/24 Group mice were 114-fold higher (/ x:)p<0.001). Accordingly, free VCR/Vol _1/24 Group mice died all within 46 days with a Median Survival (MST) of 44 days, and Ps-VCR/Vol _1/24 Group mice were all cured and no morbidity occurred during the 180 day observation periodSymptoms and death were prolonged by more than 6.7 times compared to the Ps-VCR (MST: 27 days) and Ps-Vol (MST: 24 days) vesicle single drug formulation group, by more than 11 times compared to the PBS group (MST: 16 days) (fig. 12E). Furthermore, ps-VCR/Vol _1/12 And Ps-VCR/Vo _1/48 At lower Vol and VCR doses, survival of mice was also significantly prolonged, with MST 55 and 52 days respectively, 3.2 fold more than PBS group. Free VCR/Vol during treatment _1/24 Group mice reduced in weight by 8%, showed slight toxicity, and other groups had no obvious abnormalities in weight (fig. 12F). The results comprehensively show that the Ps-VCR/Vol has better safety and excellent synergistic anti-tumor activity in an in-situ Molm-13-Luc AML model, wherein the Ps-VCR/Vol _1/24 Has the strongest anti-AML activity and realizes complete cure.

At day 43 post inoculation, ps-VCR/Vol was sacrificed _1/12 、Ps-VCR/Vol _1/24 、Ps-VCR/Vol _1/48 Treatment groups were used to examine 3 mice infiltrated, and liver, spleen, lung, bone Marrow (BM) and Peripheral Blood (PB) were collected to test tumor cell infiltration (n=3) therein. Collecting heart, liver, spleen, lung, kidney, etc. main organs and rear leg bones, fixing with 4% paraformaldehyde, embedding in paraffin, and treating with H &E and TRAP staining for histological and osteoclast analysis. On day 15 post-inoculation, PBS group mice were sacrificed as controls. The Ps-VCR and Ps-Vol groups were analyzed by collecting major organs/tissues at the end of the experiment in the sacrificial mice. Consistent with imaging results, ps-VCR/Vol _1/24 Leukemia cells infiltration was not seen in each organ of group mice, and spleen weights were similar to healthy mice (fig. 13), further indicating that they effectively cleared leukemia cells in mice. Ps-VCR/Vol _1/12 And Ps-VCR/Vol _1/48 Although the leukemia cell infiltration rate of each organ of mice in the group was significantly lower than that of PBS group, there was significant leukemia cell infiltration, wherein Ps-VCR/Vol _1/12 The infiltration rates in the liver, spleen, lung and bone marrow were 47%, 9%, 22.8% and 17%, respectively, in group mice (fig. 13A, B). In addition, spleen weights of two groups of mice compare to Ps-VCR/Vol _1/24 There was also a significant increase in the group and healthy mice (fig. 13C).

H&E-stained picture display, PBS groupThe liver, spleen, kidney and lung of mice are almost completely invaded by AML cells, while the AML cells in the liver, spleen, kidney and lung of mice in the groups of Ps-VCR and Ps-Vol are somewhat reduced compared with the PBS group, a large number of AML cells are also present in the liver, normal cells in the spleen and lung are almost completely replaced by AML cells, and a small part of tumor infiltration is also present in the kidney, so that organ injury is serious. Ps-VCR/Vol _1/12 And Ps-VCR/Vol _1/48 The infiltration of leukemia cells in the liver and spleen is obviously inhibited after treatment, and a small amount of leukemia cells still exist in the liver and spleen of mice, while Ps-VCR/Vol _1/24 No significant leukemic cells were found in the group organs. No toxicity-related lesions were seen in the major organs of each group (FIG. 14). Leukemia cells in the leg bones of mice in PBS, ps-Vol and Ps-VCR groups filled the entire bone marrow cavity, almost completely replaced normal bone marrow cells, and had a large number of hematopoietic cells missing. Ps-VCR/Vol _1/12 And Ps-VCR/Vol _1/48 Leukemia cells in the leg bones of group mice were significantly reduced compared to the vesicular single agent group, but were still visible, while Ps-VCR/Vol _1/24 Hematopoietic cells were normal in the leg bones and leukemia cells were not seen (fig. 15A), all of which were consistent with the infiltration results of the flow assay. The proliferation of tumor cells in the bone marrow cavity can cause bone pain due to excessive bone marrow cavity pressure. In addition, NF- κB receptor activating ligand (RANKL) may activate osteoclasts, enhance bone resorption and cause bone destruction. AML cells inhibit osteoprotegerin, which can block RANKL, leading to abnormal activation of osteoclasts and causing reduced bone mass and osteoporosis. The change in bone microenvironment of each group of mice was assessed using tartrate-resistant acid phosphatase (TRAP) staining and micro-CT. From TRAP staining patterns, it was found that PBS and vesicle single drug treatment groups had abnormally increased osteoclast numbers, and that the number of osteoclasts was significantly decreased in the leg bones of mice in each group of Ps-VCR/Vol, especially Ps-VCR/Vol _1/24 The group remained identical to healthy mice (fig. 15B). From the Micro-CT image, the bone trabeculae in the bone marrow cavities of the femur and tibia of the PBS group are largely disappeared, and the cortical bone has the phenomenon of dissolving bone. The single drug group of the Ps-VCR and the Ps-Vol vesicles, although improved over the PBS group, remained at a lower level, ps-VCR/Vol _1/24 Bone damage can be significantly ameliorated (fig. 16A). Bone mineral density of mouse leg bone after Ps-VCR/Vol treatmentThe bone volume fraction, the bone surface area, the bone trabecular thickness and the bone trabecular number are obviously improved, the bone trabecular separation degree is reduced, and the Ps-VCR/Vol is reduced _1/24 The group was not different from healthy mice, whereas the vesicular single drug group had only a slight improvement in PBS (fig. 16B).

In order to further verify the in vivo synergistic anti-AML effects of Ps-VCR/Vol, in situ MV-4-11 and WEHI-3 AML mouse models were established. In situ MV-4-11The AML mouse model was established as that of Molm-13-Luc. To establish in situ WEHI-3AML mouse model, 200 [ mu ] L WEHI-3AML cell suspension (5×10) ⁶ And personal) was injected into 6-week-old BALB/c female mice by tail vein, and the inoculation was on day 0. In situ MV-4-11 and WEHI-3AML mice were dosed on day 3 post-inoculation with 6 mice each, each with PBS, ps-VCR, ps-Vol, ps-VCR/Vol _1/24 And Ps-VCR/Vol _1/48 The dose and frequency of administration were consistent with those in the in situ Molm-13-Luc mouse model (FIG. 17A). Mice were continuously monitored for body weight and survival period during and after dosing. The study shows that in situ MV-4-11 AML mice have higher malignancy and rapid onset, the PBS group MST has a limited therapeutic effect for 20 days, and the single medicine groups of Ps-VCR and Ps-Vol vesicles have only slightly prolonged median survival time, and MST has a single medicine group of 26.5 days and 25 days (figure 17B). However, ps-VCR/Vol _1/24 And Ps-VCR/Vol _1/48 The treatment effectively eliminates leukemia cells in mice, has no disease symptoms in 180 days, realizes complete cure, and prolongs the survival period by more than 7 times and more than 9 times compared with vesicle single drug and PBS respectivelyp<0.001). In situ WEHI-3 AML mouse model, ps-VCR/Vol _1/24 And Ps-VCR/Vol _1/48 The survival time of the mice is effectively prolonged, and the Ps-VCR/Vol is 180 days _1/24 Group mice survived all, ps-VCR/Vol _1/48 Half of the mice survived, and the median survival time was not reached, and there was a significant increase in the survival time compared to the Ps-VCR (MST: 57 days), ps-Vol (MST: 53 days) and PBS (40 days) groupsp<0.001 (fig. 17D). Importantly, no significant decrease in body weight occurred in each group of mice during the treatment period (FIG. 17C, E), indicating good in vivo safety of Ps-VCR/Vol. The above results together show that the Ps-VCR/Vol has powerful body Internally synergistic anti-AML activity, complete cure was achieved in all 3 different in situ AML models.

The Ps-VCR/Vol provided by the invention is obtained by assembling and crosslinking amphiphilic block polymers, and has an asymmetric membrane structure, wherein the outer shell is polyethylene glycol, the membrane layer is reversibly crosslinked hydrophobic polycarbonate, and the inner shell is KD _z Efficient loading of VCR and Vol can be achieved by electrostatic action. The preparation method of the drug-loaded vesicle can be as follows: vincristine sulfate, volasertib, PEG-P (TMC-DTC) -KD _z The Ps-VCR/Vol is prepared by a solvent replacement method. In vivo and in vitro experiments prove that the Ps-VCR/Vol provided by the invention has obvious synergistic effect, obviously reduces the semi-lethal concentration of cells, and particularly greatly improves the median survival time of mice, and has high safety even under high dosage.

It will be apparent to those skilled in the art from this disclosure that various other changes and modifications can be made which are within the scope of the invention as defined in the appended claims.

Claims

1. The polymer vesicle for co-carrying the medicine is characterized by being mainly prepared from amphiphilic block polymers and a plurality of antitumor medicines; the plurality of antitumor drugs are two or more different antitumor drugs.

2. The drug-loaded polymer vesicle of claim 1, wherein the amphiphilic block polymer has a structure of hydrophilic segment-hydrophobic segment-polypeptide segment.

3. The drug co-loaded polymer vesicle of claim 2, wherein the amphiphilic block polymer has a structure of hydrophilic segment-hydrophobic segment-KD _z Wherein K is lysine and D is aspartic acid.

4. The drug-co-loaded polymeric vesicle of claim 1, wherein at least one of the antineoplastic agents is a tubulin inhibitor.

5. The drug-loaded polymer vesicle according to claim 4, wherein the tubulin inhibitor is any one or more of vincristine sulfate, paclitaxel, docetaxel, cabazitaxel, auristatin, maytansine, ai Li brin.

6. The drug-co-loaded polymeric vesicle of claim 1, wherein at least one of the antineoplastic agents is a targeted oncogene Polo-like kinase 1 inhibitor.

7. The drug-loaded polymer vesicle according to claim 6, wherein the targeted oncogene Polo-like kinase 1 inhibitor is any one or more of volasertib, regoracet, ON01910, BI2536, HMN-214, GSK 461364.

8. The drug-co-loaded polymeric vesicle of claim 1, wherein the plurality of anti-tumor drugs is a combination of a tubulin inhibitor and a targeted oncogene Polo-like kinase 1 inhibitor.

9. The drug-loaded polymer vesicle of claim 8, wherein the tubulin inhibitor is vincristine sulfate and the targeted oncogene Polo-like kinase 1 inhibitor is volasertib.

10. The drug-loaded polymer vesicle according to claim 8, wherein the dosage mass ratio of the tubulin inhibitor to the targeted oncogene Polo-like kinase 1 inhibitor is 1:1-48.

11. The drug co-loaded polymer vesicle according to any one of claims 1-9, wherein the hydrophilic segment in the amphiphilic block polymer is a polyethylene glycol segment, the hydrophobic segment is a random copolymer P (a-DTC), a is an ester monomer or a carbonate monomer, and DTC is a disulfide five-membered ring carbonate unit; the ester monomer comprises one or two of caprolactone and lactide; the carbonate monomer comprises trimethylene carbonate.

12. The drug-loaded polymer vesicle according to any one of claims 1-9, wherein the amphiphilic block polymer has a hydrophilic segment with a molecular weight of 2000-10000 Da; the molecular weight of the hydrophobic section is 2-10 times of that of the hydrophilic section; the molecular weight of PDTC is 10% -40% of the total molecular weight of the hydrophobic section; z is 5 to 15.

13. The drug co-loaded polymer vesicle according to any one of claims 1-12, wherein the amphiphilic block polymer has a structure of any one of the following structures:

。

14. the drug-loaded polymer vesicle is characterized by being prepared from an amphiphilic block polymer, a tubulin inhibitor and a targeted oncogene Polo-like kinase 1 inhibitor, wherein the structure of the amphiphilic block polymer is any one of the following structures:

；

the tubulin inhibitor is vincristine sulfate, and the targeted oncogene Polo-like kinase 1 inhibitor is voratite; preferably, the feeding mass ratio of the tubulin inhibitor to the targeted oncogene Polo-like kinase 1 inhibitor is 1: (1-48).

15. The method for preparing the drug-loaded polymer vesicles according to any one of claims 1 to 14, which is characterized in that the drug-loaded polymer vesicles are prepared by a solvent substitution method by taking two or more than two antitumor drugs and amphiphilic block polymers as raw materials; or preparing the polymer vesicle carrying the drug by taking the tubulin inhibitor, the targeted oncogene Polo-like kinase 1 inhibitor and the amphiphilic block polymer as raw materials through a solvent substitution method.

16. Use of a drug co-loaded polymer vesicle according to any one of claims 1-14 for the preparation of an anti-tumour drug.

17. The use according to claim 16, wherein the antineoplastic agent is a medicament for the treatment of acute myeloid leukemia.