CN114601801B

CN114601801B - Double-targeting functional LDL-MLN nano-drug and application thereof

Info

Publication number: CN114601801B
Application number: CN202210269447.3A
Authority: CN
Inventors: 杨琼; 何冰虹
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-11-08
Anticipated expiration: 2042-03-18
Also published as: CN114601801A

Abstract

The invention discloses a double-targeting functional LDL-MLN nano-drug and application thereof. The LDL-MLN nano-drug is obtained by embedding a small molecular inhibitor MLN8237 by using LDL particles as drug carriers. The invention is proved by experiments that: the LDL-MLN nano-drug can effectively inhibit the proliferation of HEL cells and MPN primary cells, promote the differentiation and apoptosis of the HEL cells and the MPN primary cells, reduce the disease burden of PMF mice and has good safety. The LDL is a good drug transport carrier aiming at abnormal megakaryocytes, and the LDL-MLN nano-drug prepared by the invention can be used for inhibiting the proliferation of tumor cells and/or promoting the differentiation and apoptosis of the tumor cells, and has good application prospect in preventing and/or treating myeloproliferative tumors, primary myelofibrosis and acute myelogenous leukemia.

Description

Double-targeting functional LDL-MLN nano-drug and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a double-targeting functional LDL-MLN nano-drug and application thereof.

Background

Primary Myelofibrosis (PMF) is a myeloproliferative tumor (MPN) caused by abnormal proliferation of hematopoietic cells, excessive secretion of cytokines leading to proliferation of hematopoietic tissue collagen. The median survival was 5 years and there was a 20% chance of conversion to acute myeloid leukemia.

Research shows that a large number of immature megakaryocytes exist in a patient with myeloproliferative tumors (MPN), the activity of protein kinase Aurora kinase A (AURKA) in the megakaryocytes is increased, and a small molecule inhibitor MLN8237 targeting AURKA can effectively inhibit the proliferation of abnormal megakaryocytes and induce the differentiation and apoptosis of the cells. Subsequently, the small molecule inhibitor MLN8237 enters a clinical first-stage experiment, the MLN8237 alone acts on myeloproliferative tumor patients, but only 1/3 of the patients have better disease burden reduction after undergoing the whole-course treatment, and the treatment effect of the MLN8237 still needs to be further improved.

Low Density Lipoprotein (LDL) is a natural apolipoprotein existing in the body, and plays a role in transporting cholesterol mainly in the blood circulation. The main structure of LDL is an outer phospholipid monolayer, and cholesterol is present in a hydrophobic core. There is also an apolipoprotein ApoB-100 on the surface of LDL, which is a protein that efficiently recognizes LDL receptors on cell membranes. LDL enters cells through ApoB-100 binding to cell surface LDL receptors, and undergoes lysosomal degradation within the cell releasing cholesterol in the hydrophobic core for cellular vital activities.

Disclosure of Invention

The invention aims to provide a double-targeting functional LDL-MLN nano-drug and application thereof.

In order to achieve the above objects, the present invention firstly provides a novel use of a dual targeting functional LDL-MLN nano-drug.

The invention provides an application of LDL-MLN nano-drugs in any one of the following 1) to 8):

1) Preparing a product for preventing and/or treating myeloproliferative tumors;

2) Preventing and/or treating myeloproliferative tumors;

3) Preparing a product for preventing and/or treating primary myelofibrosis;

4) Preventing and/or treating primary myelofibrosis;

5) Preparing a product for preventing and/or treating acute myeloid leukemia;

6) Preventing and/or treating acute myeloid leukemia;

7) Preparing a product for inhibiting the proliferation of the tumor cells and/or promoting the differentiation and apoptosis of the tumor cells;

8) Inhibiting the proliferation of tumor cells and/or promoting the differentiation and apoptosis of tumor cells;

the LDL-MLN nano-drug is obtained by embedding a small molecular inhibitor MLN8237 by using LDL particles as drug carriers.

In the above application, the mass ratio of the LDL particles to the MLN8237 may be 1: (0.02-0.03), specifically 1:0.025.

further, the preparation method of the LDL-MLN nano-drug comprises the following steps:

1) Mixing LDL particles with potato starch, and freeze-drying to obtain LDL-starch mixture;

2) After completing step 1), extracting endogenous lipids from the LDL-starch mixture with heptane to obtain an extracted product, then mixing the MLN solution with the extracted product, and incubating to obtain an LDL-MLN mixture;

the MLN solution consisted of toluene, MLN8237, lauric acid and stearic acid.

Furthermore, in the step 1), the ratio of the LDL particles to the potato starch may be 1 (10-15), specifically 1.

In the step 2), the solvent of the MLN solution is toluene, and the solute and the concentration thereof are MLN 8237.25 mg/mL, lauric acid 8mg/mL and stearic acid 2mg/mL respectively.

The number of times endogenous lipids were extracted from the LDL-starch mixture with heptane was at least 3 times, preferably 3 times.

The incubation was followed by a step of removing toluene under ice bath, argon.

The method further comprises a step of purification. The purification method may comprise the steps of: the LDL-MLN mixture was dispersed in buffer, centrifuged, and the supernatant collected, which was then filtered. The method specifically comprises the following steps: dispersing the LDL-MLN mixture in tricine buffer (10 mM, pH = 8.4), and standing at 4 ℃ for 18 hours; then centrifuging at 2000rpm for 10 minutes, and collecting initial clear liquid; centrifuging the initial clear liquid for 10 minutes at 10000rpm for two rounds, and collecting the supernatant; the supernatant was finally filtered through a 0.22 μm sterile filter.

The LDL-MLN nano-drug also belongs to the protection scope of the invention.

In order to achieve the above object, the present invention also provides a product; the function of the product is any one of the following A-D:

A. preventing and/or treating myeloproliferative tumors;

B. preventing and/or treating primary myelofibrosis;

C. preventing and/or treating acute myeloid leukemia;

D. inhibit the proliferation of tumor cells and/or promote the differentiation and apoptosis of tumor cells.

The active ingredient of the product provided by the invention is the LDL-MLN nano-drug.

In any of the above uses or products, the tumor cell may be a tumor cell caused by megakaryocyte abnormality.

Further, the tumor cell caused by megakaryocyte abnormality may be a myeloproliferative tumor cell or an acute myelogenous leukemia cell.

Further, the myeloproliferative tumor cells may be MPN primary cells, and the acute myeloid leukemia cells may be HEL cells.

The use of LDL particles or LDL proteins as drug delivery vehicles for abnormal megakaryocytes is also within the scope of the present invention.

In order to improve the treatment effect of a small molecular inhibitor MLN8237, LDL protein is used as a drug carrier of the small molecular inhibitor MLN8237, and a novel double-targeting function LDL-embedded MLN8237 nano-drug LDL-MLN is designed so as to further improve the targeting property on abnormal megakaryocytes. Furthermore, the LDL-MLN nano-drug is respectively applied to in vitro and in vivo experiments, so that the proliferation, differentiation and apoptosis level changes of abnormal megakaryocytes after the treatment of the LDL-MLN nano-drug are detected. The results show that: the LDL-MLN nano-drug can effectively inhibit the proliferation of HEL cells and MPN primary cells, promote the differentiation and apoptosis of the HEL cells and the MPN primary cells, reduce the disease burden of PMF mice and has good safety. Experiments prove that the LDL is a good drug transport carrier aiming at abnormal megakaryocytes, and the LDL-MLN nano-drug prepared by the invention has good application prospect.

Drawings

FIG. 1 is a graph showing the determination of IC50 of LDL-MLN/MLN in HEL cells.

FIG. 2 is a HEL cell proliferation curve 72 hours after LDL-MLN/MLN dosing treatment.

FIG. 3 shows HEL cell viability after 72 hours of LDL-MLN/MLN dosing treatment.

FIG. 4 is the HEL apoptosis level 72 hours after LDL-MLN/MLN dosing treatment.

FIG. 5 shows the expression level of HEL cell surface differentiation markers 72 hours after LDL-MLN/MLN administration.

FIG. 6 shows HEL intracellular DNA content determination 72 hours after LDL-MLN/MLN treatment.

FIG. 7 is a determination of IC50 of LDL-MLN/MLN in MPN primary cells.

FIG. 8 shows the viability of MPN primary cells after 48 hours of treatment with LDL-MLN/MLN.

FIG. 9 shows GFP expression levels of MPN primary cells 48 hours after LDL-MLN/MLN administration.

FIG. 10 shows the expression levels of MPN primary cell surface markers 48 hours after LDL-MLN/MLN administration treatment.

FIG. 11 shows the determination of DNA content in MPN primary cells 48 hours after LDL-MLN/MLN administration treatment.

FIG. 12 shows the cell ratio change of each line of bone marrow in LDL-MLN/MLN safety experiment.

FIG. 13 is a pathological section in an LDL-MLN/MLN safety experiment.

FIG. 14 shows changes in the peripheral blood GFP levels of PMF mice after treatment with LDL-MLN/MLN.

FIG. 15 is a pathological section of PMF mice treated with LDL-MLN/MLN.

FIG. 16 is a graph showing the level of spleen cell differentiation in PMF mice treated with LDL-MLN/MLN administration.

FIG. 17 shows the level of differentiation of bone marrow cells of PMF mice treated with LDL-MLN/MLN.

FIG. 18 is a graph showing the detection of GFP expression levels in cells by flow cytometry.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 Synthesis of LDL-Embedded MLN8237 Nanedicharmaceutical LDL-MLN

LDL-embedded MLN8237 nanomedicines (abbreviated as LDL-MLN) were synthesized according to the methods described in the literature (Zhu C, pradhan P, huo D, et al, reconsistition of Low-sensitivity Lipoproteins with Fatty Acids for the Targeted Delivery of Drugs in Cancer cells, angew Chem Int Ed Engl.2017;56 (35): 10399-10402. Doi. The method comprises the following specific steps:

1. preparation of LDL-MLN particles

1) 1mg of native LDL particles (LEE Biosolutions, cat # 360-10) were vortex mixed with 12.5mg of potato starch and then freeze-dried overnight to give an LDL-starch mixture.

2) After the step 1) is finished, extracting endogenous lipid in the LDL-starch mixture obtained in the step 1) with heptane for three times, and removing lipid substances in natural LDL particles to obtain an extracted product; then, 100. Mu.L of premixed payload (solvent of the premixed payload is toluene, solute and concentration thereof are 0.25mg/mL of MLN8237 (Selleck, S1133), 8mg/mL of lauric acid, and 2mg/mL of stearic acid, respectively) was added to the extracted product, and after incubation at-20 ℃ for 20 minutes, toluene was removed under ice bath and argon to obtain LDL-MLN mixture.

While replacing the premixed payload with the control premix, resulting in an LDL-blank mixture. The solvent of the control premix was toluene, and the solute and the concentration thereof were 8mg/mL of lauric acid and 2mg/mL of stearic acid, respectively.

3) After completion of step 2), the LDL-MLN mixture was dispersed in 300. Mu.L of tricine buffer (10 mM, pH = 8.4) (sigma, T0377) and allowed to stand at 4 ℃ for 18 hours. Then centrifuged at 2000rpm for 10 minutes and the initial clear liquid was collected. The initial supernatant was centrifuged at 10000rpm for 10 minutes and the supernatant was collected. Finally, the supernatant was filtered through a 0.22 μm sterile filter (Millipore) to obtain an LDL-MLN particle solution (containing MLN).

While replacing the LDL-MLN mixture with an LDL-blank mixture, an LDL-blank particle solution (without MLN) was obtained.

The LDL-MLN particle solution and LDL-blank particle solution were stored at 4 ℃ for subsequent use.

2. Determination of MLN concentration

To quantify the MLN concentration in the encapsulated LDL-MLN particle solution, the LDL-MLN particle solution was extracted with methanol under sonication. Then, the mixture was centrifuged at 10000rpm for 5 minutes, and the supernatant was collected. The supernatants of both samples were then measured by UV/Vis spectroscopy (Shimadzu, UV-2600). Finally, the MLN concentration was determined with reference to a calibration curve of MLN in methanol. The concentration of MLN in the LDL-MLN particle solution was measured to be 35. Mu. Mol/L.

3. Characterization of LDL-MLN particles

The morphology of all rLDL particles was characterized by transmission electron microscopy.

Example 2 application of LDL-MLN to inhibition of HEL cell proliferation, promotion of HEL cell differentiation and apoptosis

1. IC50 determination of LDL-MLN/MLN in HEL cells

0.8X 10 medium was added to each well (2 mL of culture medium per well) in a 24-well plate ⁶ HEL cells (purchased from a cell bank of Chinese academy of sciences) were then drug-treated by adding LDL-MLN to give final concentrations of 300nM, 100nM, 30nM, 10nM, 5nM, 3nM, 1nM, 0.3nM, 0.1nM and 0nM, respectively, while MLN was used as a control. After 24 hours of drug treatment, the total number of cells per well was calculated by trypan blue staining (cell suspension mixed with 0.4% solution 1:1) and IC50 of the cells was calculated by GraphPad prism 7.0.

The results are shown in FIG. 1 and show that: after 24 hours of in vitro drug treatment, IC50 of LDL-MLN and MLN in HEL cells is measured by trypan blue staining method, and the IC50 of LDL-MLN is relatively low, and the proliferation inhibition effect of LDL-embedded nano-drug on HEL cells is more obvious.

2. LDL-MLN/MLN dosing treatment 72 hour cell proliferation Curve

0.8X 10 aliquots were added to each well (2 mL culture volume per well) of a 24-well plate ⁶ HEL cells (purchased from cell banks of Chinese academy of sciences) were then drug-treated by adding LDL-MLN to a final concentration of 30nM, while MLN, LDL-blank and tricine buffer (Con) were used as controls. After 72 hours of drug treatment, the total number of cells per well was calculated by trypan blue staining (cell suspension mixed with 0.4% solution 1:1).

The results are shown in FIG. 2 and show that: when the change in proliferation of HEL cells was detected by trypan blue staining at a dose of 30nM, it was found that LDL-MLN had a more significant effect on the proliferation of HEL cells.

3. Cell viability assay 72 hours after LDL-MLN/MLN dosing treatment

Cell viability was analyzed using an ACEA flow cytometry analyzer in 1.5mL centrifuge tubes using 50. Mu.L of each HEL cell treated with 30nM final LDL-MLN/MLN/LDL-blank/tricine (Con) for 72 hours.

The results are shown in FIG. 3 and show that: cell survival after 72 hours of cell administration was examined using flow cytometry, and it was found that the LDL-MLN-treated group had higher cell survival and relatively less cytotoxicity to LDL-MLN when the administration dose was 30 nM.

4. Detection of apoptosis level 72 hours after LDL-MLN/MLN administration treatment

Respectively taking 0.5 × 10 ⁶ HEL cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of 30nM for 72 hours were incubated in a 1.5mL centrifuge tube using an apoptosis kit (BD 550474) at room temperature in the absence of light for 15min, and APC signaling was detected using an ACEA flow cytometer after incubation.

The results are shown in FIG. 4 and show that: detection of PS expression levels of phosphatidylserine in HEL cells 72 hours after administration by flow cytometry revealed that MLN at the same concentration was similar to the level of apoptosis induction of LDL-MLN. When the dose is 30nM, HEL cell apoptosis can be induced obviously.

5. Measurement of expression level of cell surface differentiation marker after 72 hours of treatment with LDL-MLN/MLN administration

During the differentiation process of the megakaryocytes, the up-regulation of the expression levels of cell surface differentiation markers CD41 and CD42 is a marker for the differentiation and maturation of the megakaryocytes, and the differentiation condition of the cells can be measured by detecting the expression level change of the cell surface markers.

Respectively taking 0.5 × 10 ⁶ HEL cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of 30nM for 72 hours were plated out in 1.5mL centrifuge tubes using the flow-through antibodies CD41a-APC, CD42a-PE (BD Pharmingen) ^TM APC Mouse Anti-Human CD41a，559777；BD Pharmingen ^TM PE Mouse Anti-Human CD42a, 558819) were incubated at 4 ℃ for 15min in the dark, and APC and PE signal detection was performed with ACEA flow cytometer after incubation.

The results are shown in FIG. 5 and show that: by detecting the expression levels of CD41 and CD42 in HEL cells 72 hours after administration through flow cytometry, LDL-MLN and MLN can effectively promote the expression of CD41 and CD42, wherein the LDL-MLN is more remarkably up-regulated for an early differentiation marker CD41, and the MLN is more remarkably up-regulated for a late differentiation marker CD 42.

6. Intracellular DNA assay 72 hours after LDL-MLN/MLN dosing treatment

The differentiation process of megakaryocytes is accompanied with the replication of DNA in cells, but cytoplasm does not divide, so polyploidy phenomenon can occur, and the detection of the change of DNA content in the cells is a standard for judging the differentiation level of the megakaryocytes.

Respectively taking 0.5 × 10 ⁶ HEL cells treated for 72 hours at a final concentration of 30nM LDL-MLN/MLN/LDL-blank/tricine (Con) were incubated for 40min at 37 ℃ in the absence of light using the DNA dye Hoechst 33342 (Invitrogen H3570) in a 1.5mL centrifuge tube and subjected to Pacific blue signal detection using an ACEA flow cytometer after incubation.

The results are shown in fig. 6 and show that: the DNA content of HEL cells after 72 hours of administration is detected by flow cytometry, and the LDL-MLN and MLN both remarkably improve the intracellular DNA content level, and the multiplylation phenomenon of the LDL-MLN treatment group is more remarkable. In connection with fig. 5, it can be concluded that: LDL-MLN promotes the level of differentiation of whole cells in HEL cells of the heterogeneous cell population.

Example 3 application of LDL-MLN to inhibition of proliferation of MPN primary cells and promotion of differentiation and apoptosis of MPN primary cells

The occurrence of gene mutations is frequently accompanied in myeloproliferative tumors. The MPLW515K/L gene mutation is the main molecular cause for MPN. The MPLW515L mutation was transduced into mouse bone marrow c-kit positive cells by retroviral transduction, and MPN primary cell lines were constructed in vitro to mimic in vivo conditions. And the MPN primary cell line is treated by drug administration, and the proliferation and differentiation level changes of the cells after the drug treatment are detected. The method comprises the following specific steps:

1. construction of MPN Primary cells

1. Mouse bone marrow cells were taken and sorted for c-kit positive bone marrow cells (designated as c-kit + cells) using a c-kit magnetic bead sorting system (Miltenyi Biotec 130-091-224).

2. After completion of step 1, transformation and extraction of MPLW515L plasmid (MPLW 515L plasmid is described in Wen, qiang Jermey et al, "Targeting megakaryocytic-induced fibrosis by myelogenous inflammation by AUA RK inhibition." Nature media vol.21,12 (2015): 1473-80.Doi 10.1038/nm.3995) was performed by Trans10 chemically competent cells.

3. After completion of step 2, 5X 10 cells were transfected one day before transfection ⁶ The individual plat-E cells were seeded in 10 cm dishes using X-treemeGENE ^TM 9 DNA transfection reagent (Roche) the MPLW1515L plasmid was transfected into plat-E cells and the viral supernatant was collected 48 hours after transfection.

4. After completion of step 3, 1mL of viral supernatant was mixed with 2X 10 ⁶ The individual c-kit + cells and 8. Mu.g/ml polybrene (Sigma, TR-1003) were mixed, centrifuged at 2,500rpm at 32 ℃ for 90 minutes, the centrifugation was repeated twice, then the viral supernatant was removed, and cells were added to the pelletThe culture broth was cultured and the level of GFP expression in the cells was detected by flow cytometry (GFP fluorescent protein carried by MPLW515L plasmid) to give c-kit + cells overexpressing MPLW515L and this was designated as MPN primary cells. The GFP flow was 20.2%, and the results are shown in FIG. 18.

2. Determination of IC50 of LDL-MLN/MLN in MPN Primary cells

0.8X 10 aliquots were added to 24-well plates per well (2 mL culture volume per well) ⁶ MPN primary cells were then drug-treated by adding LDL-MLN to final concentrations of 1000nM, 500nM, 300nM, 100nM, 50nM, 30nM, 10nM, 5nM, 1nM and 0nM, respectively, while MLN was used as a control. After 48 hours of drug treatment, the total number of cells per well was calculated by trypan blue staining (cell suspension mixed with 0.4% solution 1:1) and IC50 of the cells was calculated by GraphPad prism 7.0.

The results are shown in FIG. 7 and show that: after 48 hours of in vitro dosing treatment, IC50 of LDL-MLN was relatively low as determined by trypan blue staining for LDL-MLN and MLN in MPN primary cells.

3. Survival rate of MPN primary cells 48 hours after LDL-MLN/MLN administration treatment

Cell viability was analyzed using an ACEA flow cytometry analyzer in 1.5mL centrifuge tubes using 50. Mu.L of each MPN primary cell treated with 300nM final LDL-MLN/MLN/LDL-blank/tricine (Con) for 48 hours.

The results are shown in fig. 8 and show that: the survival rate of MPN primary cells after 48 hours of cell administration was measured by flow cytometry, and it was found that the cell survival rate of the MLN-treated group was higher and the killing effect of LDL-MLN on MPN primary cells was stronger when the administration dose was 300 nM.

4. GFP expression level of MPN primary cells 48 hours after LDL-MLN/MLN administration treatment

The MPN primary cells express GFP, and the survival rate of the MPN cells can be judged by detecting the change of GFP expression level in the cell population.

After 48 hours of LDL-MLN/MLN/LDL-blank/tricine (Con) treatment at a final concentration of 300nM, 50. Mu.L of MPN primary cells were individually placed in a 1.5mL centrifuge tube, the MPLW515L positive cells were autofluorescent with GFP, and the cellular GFP expression levels were analyzed using an ACEA flow cytometry analyzer.

The results are shown in fig. 9 and indicate that: GFP expression levels were examined by flow cytometry and it was found that after 48 hours of treatment with drug, the LDL-MLN and MLN treated MPN primary cell populations had significantly reduced GFP and that LDL-MLN had a stronger killing effect on MPN primary cells.

5. Detection of expression level of MPN primary cell surface differentiation marker 48 hours after LDL-MLN/MLN administration treatment

Respectively taking 0.5 × 10 ⁶ MPN primary cells treated 48 hours with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of 300nM in a 1.5mL centrifuge tube using the flow antibody CD41a-PE-Cy7 (eBioscience) ^TM 25-0411-82) at 4 ℃ and in the dark for 15min, and detecting the PE-Cy7 signal by an ACEA flow cytometer after incubation.

The results are shown in FIG. 10 and show that: the expression level of the MPN primary cell surface differentiation marker CD41 after the drug treatment is detected by flow cytometry, and the CD41 expression of the LDL-MLN treatment group is obviously up-regulated, which indicates that the LDL-MLN has better MPN cell differentiation promoting effect compared with the MLN.

6. Determination of DNA content in MPN Primary cells 48 hours after LDL-MLN/MLN treatment

Respectively taking 0.5 × 10 ⁶ MPN primary cells treated for 48 hours at a final concentration of 300nM LDL-MLN/MLN/LDL-blank/tricine (Con) were incubated for 40min at 37 ℃ in the absence of light using the DNA dye Hoechst 33342 (Invitrogen H3570) in a 1.5mL centrifuge tube and subjected to Pacific blue signal detection using an ACEA flow cytometer after incubation.

The results are shown in FIG. 11 and show that: DNA content in MPN primary cells after 48 hours of administration is detected, and the DNA content in cells of MLN and LDL-MLN treatment groups is obviously increased, and the promotion level of LDL-MLN on cell polyploidy is more obvious. As can be seen from FIG. 10, LDL-MLN had a better differentiation-inducing effect on MPN primary cells than MLN.

Example 4 evaluation of LDL-MLN safety

To evaluate the safety of LDL-MLN at 0.01mg/kg, 8-week-old C57BL/6N mice (purchased from Wittisonia) were selected for tail vein administration at a dose of 0.01mg/kg every 2 days. LDL-blank (administered dose: 0.01 mg/kg), MLN (administered dose: 0.01 mg/kg) and no-administration treatment (WT) were used as controls. Mice were analyzed for changes in peripheral blood cell populations and changes in liver spleen cell populations 2 weeks after treatment with the drug. The specific experimental method is as follows:

1. mouse bone marrow cell flow analysis

1) Cell preparation

Bone marrow cell preparation: after the cells in the bone marrow of the mouse are blown out by PBS, the cells are centrifuged for 10min at 300g, cell precipitates are collected and incubated for 5min at room temperature by erythrocyte lysate, and erythrocytes are removed for standby.

2) Flow analysis

Red series analysis: taking the cell suspension before cracking red blood cells (the cell number is 0.5 multiplied by 10) ⁶ ) Using the flow antibody BD Pharmingen ^TM APC Rat Anti-Mouse TER-119/Erythroid Cells (557909) and BD Pharmingen ^TM And (3) incubating PE Rat Anti-Mouse CD71 (553267) at 4 ℃ in a dark place for 15min, and finally detecting an APC signal and a PE signal by using an ACEA flow cytometry.

Particle system analysis: taking the cell suspension (the cell number is 0.5 multiplied by 10) after the red blood cells are lysed ⁶ ) Using the flow antibody BD Pharmingen ^TM PE Rat Anti-Mouse CD11b (557397) and BD Pharmingen ^TM And (3) incubating the APC Rat Anti-Mouse Ly-6G (560599) under the incubation condition of 4 ℃ in a dark place for 15min, and finally detecting an APC signal and a PE signal by using an ACEA flow cytometry.

Analysis of megakaryocyte series: taking cell suspension (cell number is 0.5 × 10) ⁶ ) Using the flow antibody CD41a-PE-Cy7 (eBioscience) ^TM 25-0411-82), incubating at 4 ℃ in a dark place for 15min, and finally detecting the PE-Cy7 signal by an ACEA flow cytometer.

2. Dyeing

Liver and spleen HE staining: taking a paraffin section of a mouse tissue with the size of 5 mu m, drying, dewaxing to water, staining for 5min by hematoxylin staining solution, staining for 6min by eosin staining solution, fixing, and observing the cell morphology by a microscope.

Blood smear: blood is collected from the tail vein by 10 mu L, the smear is fixed evenly and then stained for 15min by Giemsa staining solution, and the cell morphology is observed by a microscope.

The results are shown in fig. 12 and show that: it was confirmed by flow cytometry that the ratio of cells of each line in the bone marrow of LDL-MLN-administered mice to untreated mice (WT) was not changed, and bone marrow suppression was not caused at the administered dose.

FIG. 13 shows that the cell population of LDL-MLN and MLN-administered treated mice was similar to that of untreated mice (WT) as seen by blood smear. HE staining of liver spleens also showed that the healthy level of mice at this dose was not affected.

Example 5 LDL-MLN was effective in reducing PMF mouse disease burden

1. Construction of PMF mouse model

The PMF mouse model was constructed by injecting MPN primary cells prepared in example 3 (MPLW 515L overexpressing mouse c-kit + cells) into myeloablative mice by bone marrow transplantation. The method comprises the following specific steps: mixing 0.3X 10 ⁶ The MPN primary cells were injected via tail vein into 950cGy gamma-irradiated C57BL/6N mice (myeloablative mice) and two weeks after rearing, to obtain a PMF mouse model.

2. Changes in peripheral blood GFP levels in PMF mice following treatment with LDL-MLN/MLN administration

LDL-MLN administration was started 15 days after modeling, and administered once every 2 days at a dose of 0.01mg/kg, and GFP levels in peripheral blood of PMF mice were measured 7 times after administration at 6, 10, 14, 18, 22, 26, 30, 34, and 38 days after administration, while MLN, LDL-blank and treatment without administration (Con) were used as controls. The GFP level was determined as follows: 20 mu L of tail vein blood is collected, and GFP signals are detected by an ACEA flow cytometry after the tail vein blood is treated by erythrocyte lysate.

The results are shown in fig. 14 and show that: from the change level of GFP in peripheral blood, it was found that both LDL-MLN and MLN were able to reduce the content of MPLW515L cells in peripheral blood after the administration treatment to PMF mice, and that LDL-MLN had a more significant effect of inhibiting the proliferation of these cells, and that tumor cells were almost cleared after 7 administrations.

3. LDL-MLN/MLN treatment of PMF mouse pathological sections

LDL-MLN treatment was started 15 days after modeling, administered once every 2 days at a dose of 0.01mg/kg, and the change in the peripheral blood cell population and the change in the liver spleen cell population after 7 administrations were analyzed while MLN, LDL-blank and treatment without administration (Con) were used as controls. The specific analysis method was the same as in example 4.

Results as shown in fig. 15, PMF mouse peripheral blood smears demonstrated the presence of MPN cells in peripheral blood of each group of PMF mice. In addition to the LDL-MLN-administered group, the peripheral blood of PMF mice in the other administered groups had a large number of tumor cells, and the HE-stained sections of liver spleen also showed tumor cell infiltration. The control group (Con, LDL-blank and MLN) also had a large number of immature megakaryocytes present in the spleen sections. The disease burden of the LDL-MLN group is obviously reduced, a peripheral blood smear shows that the cell group is close to that of a healthy mouse, and meanwhile, liver spleen slices are also not different from that of the healthy mouse.

4. Level of spleen cell differentiation in PMF mice treated with LDL-MLN/MLN administration

Splenomegaly is a typical pathological feature of PMF, and analysis of the level of differentiation of spleen cells in PMF mice can be used as an effective assessment of therapeutic efficacy.

LDL-MLN administration treatment was started 15 days after modeling, administered once every 2 days at a dose of 0.01mg/kg, and after 7 administrations, the tumor cell differentiation level in the spleen of PMF mice after administration treatment was analyzed by flow cytometry, while MLN, LDL-blank and treatment without administration (Con) were used as controls. The specific analysis method is as follows: taking the cell suspension (the cell number is 6 multiplied by 10) after the red blood cells are lysed ⁶ ) Flow-through antibodies ANTI-MO LY-6A/E D PE (eBioscience, 12-5981-81), STREPTAVIDIN PERCP-CYN5.5 (eBioscience, 45-4317-82), ANTI-MO CD117 2B8 APC-EF780 (eBioscience, 47-1171-82), ANTI-MO CD41 MWREG30 PE-CYN7 (eBioscience), respectively, were usednce，25-0411-82)，ANTI-MO CD16/32 93PE-CYN7(eBioscience，25-0161-81)，BD Pharmingen ^TM Alexa

647Rat anti-Mouse CD34 (eBioscience, 560230), incubating at 4 deg.C, protecting from light, 15min, and detecting with ACEA flow cytometry.

The results are shown in fig. 16, and show that: the spleen of mice in the LDL-MLN administration group had significantly reduced tumor cells, and almost cleared. As can be seen from the analysis of LSK cells (Lin-Sca-1-c-Kit +) of GFP + cells, LDL-MLN effectively reduced the content of abnormal stem cells, and the ratio of CMP, GMP and MEP lineages in LSK cells was closer to that of healthy mice.

5. Level of differentiation of bone marrow cells in PMF mice treated with LDL-MLN/MLN administration

LDL-MLN dosing treatment was started 15 days after modeling, and was given every 2 days at a dose of 0.01mg/kg, and the level of tumor cell differentiation in the bone marrow of PMF mice after the dosing treatment was analyzed by flow cytometry 7 times after the dosing treatment, while MLN, LDL-blank and no dosing treatment (Con) were used as controls. The specific analysis method is as follows: taking cell suspension (cell number is 6X 10) after lysing erythrocyte ⁶ ) The flow-through antibodies ANTI-MO LY-6A/E D PE (eBioscience, 12-5981-81), STREPTAVIDIN PERCP-CYN5.5 (eBioscience, 45-4317-82), ANTI-MO CD117 2B8 APC-EF780 (eBioscience, 47-1171-82), ANTI-MO CD41 MWREG30 PE-CYN7 (eBioscience, 25-0411-82), ANTI-MO CD16/32 93PE-CYN7 (eBioscience, 25-0161-81), BD Pharmingen 5363 PE 7 (eBioscience, 25-0161-81) and BD Pharmingen were used ^TM Alexa

647 And incubating Rat anti-Mouse CD34 (eBioscience, 560230) under the incubation condition of 4 ℃ and keeping out of the sun for 15min, and finally detecting by using an ACEA flow cytometry.

The results are shown in fig. 17 and show that: the LDL-MLN administration group mice had a significant reduction in tumor cells in bone marrow with almost zero clearing. The LSK cell content of GFP + cells decreased and the CMP, GMP and MEP lineage cells in LSK cells were more similar to healthy mice. The marked decrease of MEP cells as abnormal megakaryocyte progenitor cells means that the cells overexpressing MPLW515L in bone marrow are decreased and the differentiation level of tumor cells is increased.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

Use of LDL-MLN nano-drugs in any of the following 1) -3):

1) Preparing a product for preventing and/or treating myeloproliferative tumors;

2) Preparing a product for preventing and/or treating primary myelofibrosis;

3) Preparing a product for preventing and/or treating acute myeloid leukemia;

the LDL-MLN nano-drug is obtained by taking LDL particles as drug carriers and embedding a small molecular inhibitor MLN 8237;

the preparation method of the LDL-MLN nano-drug comprises the following steps:

1) Mixing LDL particles with potato starch, and freeze-drying to obtain LDL-starch mixture;

2) After completing step 1), extracting endogenous lipids from the LDL-starch mixture with heptane to obtain an extracted product, then mixing the MLN solution with the extracted product, and incubating to obtain an LDL-MLN mixture;

the MLN solution consisted of toluene, MLN8237, lauric acid and stearic acid.
2. Use according to claim 1, characterized in that: the mass ratio of the LDL particles to the MLN8237 is 1: (0.02-0.03).
3. Use according to claim 1, characterized in that: the mass ratio of the LDL particles to the potato starch is 1 (10-15).
4. Use according to claim 1, characterized in that: the solvent of the MLN solution is toluene, and the solute and the concentration thereof are MLN 8237.25 mg/mL, lauric acid 8mg/mL and stearic acid 2mg/mL respectively.
5. Use according to claim 1, characterized in that: the incubation is followed by a step of removing the toluene.
6. Use according to any one of claims 1 to 5, characterized in that: the method further comprises a step of purification; the purification method comprises the following steps: the LDL-MLN mixture was dispersed in buffer, centrifuged, and the supernatant collected, which was then filtered.
7. The LDL-MLN nanopharmaceutical of any of claims 1-6.
8. A product whose active ingredient is the LDL-MLN nanopharmaceutical of any one of claims 1-6;

the function of the product is any one of the following A-C:

A. preventing and/or treating myeloproliferative tumors;

B. preventing and/or treating primary myelofibrosis;

C. preventing and/or treating acute myelogenous leukemia.