CN114601801B - A dual-targeted LDL-MLN nanomedicine and its application - Google Patents

Abstract

The invention discloses a LDL-MLN nanomedicine with dual-targeting function and its application. The LDL-MLN nanomedicine is obtained by embedding the small molecule inhibitor MLN8237 with LDL particles as a drug carrier. The present invention proves through experiments that LDL-MLN nanomedicine can effectively inhibit the proliferation of HEL cells and MPN primary cells, promote the differentiation and apoptosis of HEL cells and MPN primary cells, reduce the disease burden of PMF mice, and has good safety sex. It shows that LDL is a good drug transport carrier for abnormal megakaryocytes, and the LDL-MLN nanomedicine prepared by the present invention can be used to inhibit tumor cell proliferation and/or promote tumor cell differentiation and apoptosis, and is useful in preventing and/or treating myeloproliferative tumors. , primary myelofibrosis, acute myeloid leukemia has a good application prospect.

Description

A kind of LDL-MLN nano drug with dual targeting function and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an LDL-MLN nano-medicine with dual targeting functions and an application thereof.

Background technique

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) caused by abnormal hematopoietic cell proliferation and excessive secretion of cytokines leading to hematopoietic tissue collagen hyperplasia. The median survival is 5 years, and there is a 20% chance of transformation to acute myeloid leukemia.

Studies have shown that there are a large number of immature megakaryocytes in patients with myeloproliferative neoplasms (MPN), and the activity of protein kinase Aurora kinase A (AURKA) in this type of megakaryocytes is increased, and the small molecule inhibitor MLN8237 targeting AURKA can effectively inhibit Proliferation of abnormal megakaryocytes, induction of cell differentiation and apoptosis. Subsequently, the small molecule inhibitor MLN8237 entered the first phase of clinical trials. MLN8237 alone acts on patients with myeloproliferative neoplasms, but only 1/3 of the patients have a better disease burden reduction phenomenon after undergoing a full course of treatment, and its therapeutic effect still needs to be further studied. improve.

Low-density lipoprotein (LDL) is a kind of natural apolipoprotein existing in the body, which mainly undertakes the role of transporting cholesterol in the blood circulation. The main structure of LDL is the outer phospholipid monolayer, and cholesterol exists in the hydrophobic core. There is also an apolipoprotein ApoB-100 on the surface of LDL, which can effectively recognize the LDL receptor on the cell membrane. LDL enters the cell through the combination of ApoB-100 and the LDL receptor on the cell surface, and releases the cholesterol in the hydrophobic core through the degradation of the lysosome in the cell for the life activities of the cell.

Contents of the invention

The object of the present invention is to provide a kind of LDL-MLN nano-medicine with double targeting function and its application.

In order to achieve the above purpose, the present invention firstly provides a new application of LDL-MLN nanomedicine with dual targeting function.

The present invention provides the application of LDL-MLN nanomedicine in any one of the following 1)-8):

1) Preparation of products for the prevention and/or treatment of myeloproliferative neoplasms;

2) Prevention and/or treatment of myeloproliferative neoplasms;

3) Preparation of products for the prevention and/or treatment of primary myelofibrosis;

4) Prevention and/or treatment of primary myelofibrosis;

5) Preparation of products for the prevention and/or treatment of acute myeloid leukemia;

6) Prevention and/or treatment of acute myeloid leukemia;

7) Prepare products that inhibit tumor cell proliferation and/or promote tumor cell differentiation and apoptosis;

8) Inhibit tumor cell proliferation and/or promote tumor cell differentiation and apoptosis;

The LDL-MLN nano-medicine is obtained by embedding the small molecule inhibitor MLN8237 by using LDL particles as a drug carrier.

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 nanomedicine comprises the following steps:

1) LDL granules are mixed with potato starch, then freeze-dried to obtain the LDL-starch mixture;

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

The MLN solution consists of toluene, MLN8237, lauric acid and stearic acid.

Further, in the above 1), the ratio of the LDL granules to the potato starch can be 1:(10-15), specifically 1:12.5.

In said 2), the solvent of the MLN solution is toluene, the solute and its concentration are respectively 0.25 mg/mL of MLN8237, 8 mg/mL of lauric acid, and 2 mg/mL of stearic acid.

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

After the incubation, the step of removing toluene in ice bath and argon is also included.

The method also includes a purification step. The purification method may include the following steps: dispersing the LDL-MLN mixture in a buffer, centrifuging, collecting the supernatant, and then filtering the supernatant. Specifically, the following steps may be included: disperse the LDL-MLN mixture in tricine buffer solution (10mM, pH=8.4), and place it at 4°C for 18 hours; then centrifuge at 2000rpm for 10 minutes, and collect the initial clear liquid; The initial supernatant was subjected to two rounds of centrifugation at 10,000 rpm for 10 minutes, and the supernatant was collected; finally, the supernatant was filtered through a 0.22 μm sterile filter.

The above-mentioned LDL-MLN nano-medicine also belongs to the protection scope of the present 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. Prevention and/or treatment of myeloproliferative neoplasms;

B. Prevention and/or treatment of primary myelofibrosis;

C. Prevention and/or treatment of acute myeloid leukemia;

D. Inhibit tumor cell proliferation and/or promote tumor cell differentiation and apoptosis.

The active ingredient of the product provided by the invention is the above-mentioned LDL-MLN nano-medicine.

In any of the above applications or products, the tumor cells may be tumor cells caused by abnormal megakaryocytes.

Further, the tumor cells caused by abnormal megakaryocytes may be myeloproliferative tumor cells or acute myeloid leukemia cells.

Furthermore, the myeloproliferative tumor cells can specifically be MPN primary cells, and the acute myeloid leukemia cells can specifically be HEL cells.

The application of LDL particles or LDL protein as a drug delivery carrier for abnormal megakaryocytes also belongs to the protection scope of the present invention.

In order to improve the therapeutic effect of the small molecule inhibitor MLN8237, the present invention uses LDL protein as the drug carrier of the small molecule inhibitor MLN8237, and designs a new type of nano drug LDL-MLN with dual targeting functions LDL-embedded MLN8237 to further improve Targeting of abnormal megakaryocytes. Further, by applying LDL-MLN nanomedicine to in vitro and in vivo experiments, the changes in the proliferation, differentiation and apoptosis levels of abnormal megakaryocytes after LDL-MLN nanomedicine treatment were detected. The results showed that LDL-MLN nanomedicine can effectively inhibit the proliferation of HEL cells and MPN primary cells, promote the differentiation and apoptosis of HEL cells and MPN primary cells, reduce the disease burden of PMF mice, and has good safety. The present invention proves through experiments that LDL is a good drug transport carrier for abnormal megakaryocytes, and the LDL-MLN nano-medicine prepared by the present invention has good application prospects.

Description of drawings

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

Figure 2 is the HEL cell proliferation curve after LDL-MLN/MLN administration for 72 hours.

Figure 3 shows the survival rate of HEL cells after LDL-MLN/MLN administration for 72 hours.

Figure 4 shows the apoptosis level of HEL cells after LDL-MLN/MLN administration for 72 hours.

Figure 5 shows the expression levels of differentiation markers on the surface of HEL cells after treatment with LDL-MLN/MLN for 72 hours.

Fig. 6 is the measurement of DNA content in HEL cells after LDL-MLN/MLN administration and treatment for 72 hours.

Figure 7 is the determination of IC50 of LDL-MLN/MLN in MPN primary cells.

Figure 8 shows the survival rate of MPN primary cells after LDL-MLN/MLN administration for 48 hours.

Figure 9 shows the expression level of GFP in MPN primary cells after LDL-MLN/MLN administration for 48 hours.

Figure 10 shows the expression levels of surface markers of MPN primary cells after treatment with LDL-MLN/MLN for 48 hours.

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

Figure 12 shows the changes in the proportions of various bone marrow cell lines in the LDL-MLN/MLN safety experiment.

Figure 13 is a pathological section in the LDL-MLN/MLN safety experiment.

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

Fig. 15 is a pathological section of PMF mice after treatment with LDL-MLN/MLN.

Figure 16 shows the differentiation level of spleen cells in PMF mice after treatment with LDL-MLN/MLN.

Figure 17 shows the differentiation level of bone marrow cells in PMF mice after treatment with LDL-MLN/MLN.

Figure 18 is the detection of GFP expression level in cells by flow cytometry.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1, Synthesis of LDL-embedded MLN8237 nano drug LDL-MLN

According to the literature (Zhu C, Pradhan P, Huo D, et al. Reconstitution of Low-Density Lipoproteins with Fatty Acids for the Targeted Delivery of Drugs into Cancer Cells. Angew Chem Int Ed Engl.2017; 56(35):10399-10402.doi : 10.1002/anie.201704674) to synthesize LDL-embedded MLN8237 nanomedicine (abbreviation: LDL-MLN). Specific steps are as follows:

1. Preparation of LDL-MLN particles

1) Vortex-mix 1 mg of native LDL granules (LEE Biosolutions, product number 360-10) with 12.5 mg of potato starch, and freeze-dry overnight to obtain a LDL-starch mixture.

2) After completing step 1), use heptane to extract the endogenous lipids in the LDL-starch mixture obtained in step 1) three times to remove the lipid substances in the natural LDL granules to obtain the extracted product; then 100 μL of the premixed The payload (the solvent of the premixed payload is toluene, the solute and its concentration are respectively 0.25 mg/mL of MLN8237 (Selleck, S1133), 8 mg/mL of lauric acid, and 2 mg/mL of stearic acid) were added to the post-extraction product. After incubating at -20°C for 20 minutes, the toluene was removed in an ice bath under argon to obtain a LDL-MLN mixture.

At the same time, the premix payload was replaced with the control premix to obtain the LDL-blank mixture. The solvent of the contrast premix is toluene, the solute and its concentration are respectively 8 mg/mL of lauric acid and 2 mg/mL of stearic acid.

3) After step 2), the LDL-MLN mixture was first dispersed in 300 μL tricine buffer solution (10 mM, pH=8.4) (sigma, T0377), and placed at 4° C. for 18 hours. Then centrifuge at 2000rpm for 10 minutes to collect the initial supernatant. Then the initial supernatant was centrifuged at 10000rpm for 10 minutes for two rounds, 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).

At the same time, the LDL-MLN mixture was replaced by the LDL-blank mixture to obtain the LDL-blank particle solution (without MLN).

The LDL-MLN particle solution and LDL-blank particle solution were stored at 4°C 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 ultrasonic conditions. Then centrifuge at 10000rpm for 5 minutes, and collect the supernatant. The supernatants of the two samples were then measured by a UV/Vis spectrometer (Shimadzu, UV-2600). Finally, the MLN concentration was determined with reference to the calibration curve of MLN in methanol. After testing, the concentration of MLN in the LDL-MLN particle solution was 35 μmol/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 in Inhibiting HEL Cell Proliferation and Promoting HEL Cell Differentiation and Apoptosis

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

Add 0.8×106 HEL cells (purchased from the ^Cell Bank of Chinese Academy of Sciences) to each well of a 24-well plate (the volume of culture medium in each well is 2 mL), and then add LDL-MLN for drug treatment, so that the final concentrations are 300 nM, 100nM, 30nM, 10nM, 5nM, 3nM, 1nM, 0.3nM, 0.1nM and OnM, and 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 (1:1 mixing of cell suspension and 0.4% solution), and the IC50 of cells was calculated by GraphPad prism 7.0.

The results are shown in Figure 1. The results show that: after 24 hours of drug treatment in vitro, the IC50 of LDL-MLN and MLN in HEL cells can be determined by trypan blue staining. It can be found that the IC50 of LDL-MLN is relatively low, and that of LDL After embedding, the nano-medicine has a more significant inhibitory effect on the proliferation of HEL cells.

2. Cell proliferation curve of LDL-MLN/MLN administration treatment for 72 hours

Add 0.8×106 HEL cells (purchased from the ^Cell Bank of the Chinese Academy of Sciences) to each well of a 24-well plate (the volume of culture medium in each well is 2 mL), and then add LDL-MLN for drug treatment so that the final concentration is 30 nM. 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 (1:1 mixing of cell suspension and 0.4% solution).

The results are shown in Figure 2, and the results show that: by detecting the proliferation of HEL cells at a concentration of 30 nM by trypan blue staining, it can be found that LDL-MLN has a more significant effect on the proliferation of HEL cells.

3. Detection of cell viability after LDL-MLN/MLN administration for 72 hours

Take 50 μL of HEL cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of 30 nM for 72 hours and place them in a 1.5 mL centrifuge tube, and analyze the cell viability using ACEA flow cytometry analyzer.

The results are shown in Figure 3, and the results show that: using flow cytometry to detect the cell survival rate after 72 hours of cell administration, it can be found that when the dosage is 30nM, the cell survival rate of the LDL-MLN treatment group is higher, and the LDL - MLN is relatively less cytotoxic.

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

Take 0.5×106 HEL cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of ^30nM for 72 hours in a 1.5mL centrifuge tube, use the apoptosis kit (BD 550474) in Incubate for 15 min at room temperature and in the dark, and detect APC signals with ACEA flow cytometer after incubation.

The results are shown in Figure 4, and the results showed that the expression level of phosphatidylserine PS in HEL cells was detected by flow cytometry after 72 hours of administration, and the apoptosis induction level of MLN and LDL-MLN at the same concentration was similar. When the dose is 30nM, it can significantly induce the apoptosis of HEL cells.

5. Detection of expression levels of cell surface differentiation markers after LDL-MLN/MLN administration for 72 hours

During the differentiation process of megakaryocytes, the up-regulation of the expression of cell surface differentiation markers CD41 and CD42 is a sign of the differentiation and maturity of megakaryocytes, and the differentiation of cells can be measured by detecting the changes in the expression levels of cell surface markers.

Take 0.5×106 HEL cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of ^30nM for 72 hours in a 1.5mL centrifuge tube, use flow cytometry antibodies CD41a-APC, CD42a- PE (BD Pharmingen ^TM APC Mouse Anti-Human CD41a, 559777; BD Pharmingen ^TM PE Mouse Anti-Human CD42a, 558819) was incubated at 4°C for 15 minutes in the dark, and after incubation, the APC signal and PE Signal Detection.

The results are shown in Figure 5, and the results show that the expression levels of CD41 and CD42 in HEL cells after 72 hours of administration can be detected by flow cytometry, and it can be found that both LDL-MLN and MLN can effectively promote the expression of CD41 and CD42, wherein, The upregulation of early differentiation marker CD41 was more significant in LDL-MLN, and the upregulation of late differentiation marker CD42 was more significant in MLN.

6. Determination of intracellular DNA content after LDL-MLN/MLN administration for 72 hours

The differentiation process of megakaryocytes is accompanied by the replication of intracellular DNA without cytoplasmic division, so polyploidy will occur. Detection of changes in the DNA content of cells is the standard for judging the level of megakaryocyte differentiation.

Take 0.5×106 HEL cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of ^30nM for 72 hours in a 1.5mL centrifuge tube, use DNA dye Hoechst 33342 (Invitrogen H3570) in Incubate for 40 min at 37°C in the dark, and then detect the Pacific blue signal with an ACEA flow cytometer.

The results are shown in Figure 6, and the results show that: the DNA content of HEL cells was detected by flow cytometry after 72 hours of administration, and it was found that LDL-MLN and MLN all significantly increased the level of intracellular DNA content, and the LDL-MLN treatment group's The phenomenon of polyploidy is more obvious. Combining with Figure 5, it can be concluded that LDL-MLN promotes the differentiation level of the overall cells in the heterogeneous cell population HEL cells.

Example 3. Application of LDL-MLN in Inhibiting MPN Primary Cell Proliferation and Promoting MPN Primary Cell Differentiation and Apoptosis

Gene mutations are often present in myeloproliferative neoplasms. MPLW515K/L gene mutation is the main molecular cause of MPN. The MPLW515L mutation was transduced into mouse bone marrow c-kit positive cells by means of retroviral transduction, and the MPN primary cell line was constructed in vitro to simulate the in vivo conditions. And the MPN primary cell line was given drug treatment, and the changes of cell proliferation and differentiation level after drug treatment were detected. Specific steps are as follows:

1. Construction of primary MPN cells

1. Take mouse bone marrow cells, and use c-kit magnetic bead sorting system (Miltenyi Biotec 130-091-224) to sort out c-kit positive bone marrow cells (denoted as c-kit+ cells).

2. After completing step 1, carry out the MPLW515L plasmid through Trans10 chemically competent cells (MPLW515L plasmid is recorded in the following literature: Wen, Qiang Jeremy et al. "Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition." Nature medicine vol.21, 12(2015):1473-80.doi:10.1038/nm.3995) transformation and extraction.

3. After completing step 2, inoculate 5×10 ⁶ plat-E cells in a 10 cm culture dish one day before transfection, and use X-tremeGENE ^TM 9 DNA transfection reagent (Roche) to transfect the MPLW1515L plasmid into For plat-E cells, virus supernatants were collected 48 hours after transfection.

4. After completing step 3, mix 1 mL of virus supernatant with 2×10 ⁶ c-kit+ cells and 8 μg/ml polybrene (Sigma, TR-1003), and then centrifuge at 32°C and 2,500 rpm for 90 Minutes, repeat the centrifugation twice, then remove the virus supernatant, add cell culture medium to the pellet for culture, detect the expression level of GFP in the cells by flow cytometry (MPLW515L plasmid carries GFP fluorescent protein), and obtain the overexpressed MPLW515L c -kit+ cells, which were designated as MPN primary cells. The GFP detected by flow cytometry was 20.2%, and the result is shown in FIG. 18 .

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

Add 0.8×106 MPN primary cells to each well of a ²⁴ -well plate (the volume of culture medium in each well is 2mL), and then add LDL-MLN for drug treatment, so that the final concentrations are 1000nM, 500nM, 300nM, 100nM, 50nM , 30nM, 10nM, 5nM, 1nM and OnM, 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 (1:1 mixing of cell suspension and 0.4% solution), and the IC50 of cells was calculated by GraphPad prism 7.0.

The results are shown in Figure 7. The results showed that: after 48 hours of administration in vitro, the IC50 of LDL-MLN and MLN in MPN primary cells was measured by trypan blue staining. It can be found that the IC50 of LDL-MLN is relatively Low.

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

Take 50μL of LDL-MLN/MLN/LDL-blank/tricine(Con) with a final concentration of 300nM and treat the primary MPN cells for 48 hours in a 1.5mL centrifuge tube, and use the ACEA flow cytometry analyzer to analyze the cell survival rate .

The results are shown in Figure 8, and the results show that: the survival rate of MPN primary cells detected by flow cytometry after 48 hours of cell administration can be found that when the administration dose is 300nM, the cell survival rate of the MLN treatment group is higher, LDL-MLN had a stronger killing effect on MPN primary cells.

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

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

Take 50 μL of LDL-MLN/MLN/LDL-blank/tricine (Con) with a final concentration of 300nM and treat MPN primary cells for 48 hours in a 1.5mL centrifuge tube. MPLW515L positive cells can autofluoresce GFP, and use ACEA flow cytometry The cytometry analyzer analyzes the expression level of GFP in the cells.

The results are shown in Figure 9. The results showed that: GFP expression levels detected by flow cytometry found that after 48 hours of drug treatment, GFP in the MPN primary cell population treated with LDL-MLN and MLN was significantly reduced, and LDL-MLN It has a stronger killing effect on MPN primary cells.

5. Detection of the expression of differentiation markers on the surface of MPN primary cells after LDL-MLN/MLN administration for 48 hours

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

The results are shown in Figure 10, and the results show that: the expression level of CD41, the surface differentiation marker CD41 on the surface of MPN primary cells after drug treatment, can be found by flow cytometry, and the expression of CD41 in the LDL-MLN treatment group is significantly up-regulated, indicating that LDL-MLN Compared with MLN, it has a better role in promoting the differentiation of MPN cells.

6. Determination of DNA content in MPN primary cells after LDL-MLN/MLN administration for 48 hours

Take 0.5×10 ⁶ MPN primary cells treated with LDL-MLN/MLN/LDL-blank/tricine (Con) at a final concentration of 300nM for 48 hours in a 1.5mL centrifuge tube, use DNA dye Hoechst 33342 (InvitrogenH3570) Incubate for 40 min at 37°C in the dark, and then detect the Pacific blue signal with an ACEA flow cytometer.

The results are shown in Figure 11, the results show that: detection of DNA content in MPN primary cells after 48 hours of administration can be found, the intracellular DNA content of MLN and LDL-MLN treatment groups were all significantly increased, and LDL-MLN has a significant effect on the cells The level of promotion for polyploidy was even more pronounced. Combining with Figure 10, it can be seen that LDL-MLN has a better effect of inducing the differentiation of primary MPN cells than MLN.

Embodiment 4, LDL-MLN safety evaluation

In order to evaluate the safety of LDL-MLN at a dose of 0.01 mg/kg, 8-week-old C57BL/6N mice (purchased from Weitong Lihua) were selected for tail vein administration, and the frequency was once every 2 days. Each administration dose is 0.01mg/kg. At the same time, LDL-blank (dosage: 0.01 mg/kg), MLN (dosage: 0.01 mg/kg) and no treatment (WT) were used as controls. After 2 weeks of administration, the changes of peripheral blood cell populations and liver spleen cell populations were analyzed. The specific experimental method is as follows:

1. Flow cytometric analysis of mouse bone marrow cells

1) Cell preparation

Bone marrow cell preparation: Blow out the cells in the mouse bone marrow with PBS, centrifuge at 300g for 10 minutes, collect the cell pellet, incubate with red blood cell lysate for 5 minutes at room temperature, remove red blood cells, and set aside.

2) Stream analysis

Erythroid analysis: take the cell suspension before lysing red blood cells (the number of cells is 0.5×10 ⁶ ), use flow cytometry antibodies BDPharmingen ^TM APC Rat Anti-Mouse TER-119/Erythroid Cells (557909) and BDPharmingen ^TM PE Rat Anti-Mouse CD71 (553267) was incubated under the condition of 4°C, protected from light, for 15 minutes, and finally the APC signal and PE signal were detected by ACEA flow cytometer.

Granuloid analysis: take the cell suspension after lysing erythrocytes (the number of cells is 0.5×10 ⁶ ), and use flow cytometry antibodies BDPharmingen ^TM PE Rat Anti-Mouse CD11b (557397) and BD Pharmingen ^TM APC Rat Anti-MouseLy-6G (560599 ) for incubation under the conditions of 4° C., protected from light, for 15 min, and finally the APC signal and PE signal were detected with ACEA flow cytometer.

Megakaryotic lineage analysis: take the cell suspension after lysing erythrocytes (the number of cells is 0.5×10 ⁶ ), and incubate with the flow cytometry antibody CD41a-PE-Cy7 (eBioscience ^TM 25-0411-82) at 4°C and avoid Light, 15min, and finally use ACEA flow cytometer to detect PE-Cy7 signal.

2. Dyeing

Liver and spleen HE staining: 5 μm tissue paraffin sections of mice were taken, dried, dewaxed to water, stained with hematoxylin staining solution for 5 minutes, stained with eosin staining solution for 6 minutes, fixed, and the cell morphology was observed under a microscope.

Blood smear: 10 μL of blood was collected from the tail vein, and the uniform smear was fixed and stained with Giemsa staining solution for 15 minutes, and the cell morphology was observed under a microscope.

The results are shown in Figure 12, and the results show that: through flow cytometry analysis, it can be seen that the proportion of cells of each line in the bone marrow of LDL-MLN administration treated mice and untreated mice (WT) has not changed. No myelosuppression occurs at the administered dose.

The results in Figure 13 show that, as can be seen from blood smears, the cell populations of mice treated with LDL-MLN and MLN administration are similar to those of untreated mice (WT). The HE staining results of the liver and spleen also showed that the health level of the mice was not affected by this dose.

Embodiment 5, LDL-MLN effectively reduces the disease burden of PMF mice

1. Construction of PMF mouse model

The MPN primary cells prepared in Example 3 (overexpressing MPLW515L mouse c-kit+ cells) were injected into myeloablated mice by means of bone marrow transplantation to construct a PMF mouse model. The specific steps are as follows: 0.3×10 ⁶ MPN primary cells were injected into C57BL/6N mice (myeloablative mice) irradiated with 950cGy γ-rays through the tail vein, and after two weeks of feeding, the PMF mouse model was obtained.

2. Changes of GFP level in peripheral blood of PMF mice after LDL-MLN/MLN administration

After 15 days of modeling, LDL-MLN administration treatment was started, given once every 2 days, and each administration dose was 0.01mg/kg. , 22, 26, 30, 34, and 38 days, the GFP levels in the peripheral blood of PMF mice, while MLN, LDL-blank and no administration treatment (Con) were used as controls. The detection method of GFP level is as follows: 20 μL of blood is collected from the tail vein, and the GFP signal is detected by ACEA flow cytometer after being treated with erythrocyte lysate.

The results are shown in Figure 14, and the results show that: from the change level of GFP in peripheral blood, it can be seen that after administration of PMF mice, both LDL-MLN and MLN can reduce the content of overexpressed MPLW515L cells in peripheral blood, and LDL- MLN has a more significant inhibitory effect on the proliferation of the cells, and the tumor cells are almost cleared after 7 administrations.

3. Pathological sections of PMF mice treated with LDL-MLN/MLN

After 15 days of modeling, LDL-MLN administration treatment was started, once every 2 days, each administration dose was 0.01mg/kg, and the changes of peripheral blood cell groups and liver spleen cells were analyzed after administration for 7 times Group changes, while MLN, LDL-blank and no drug treatment (Con) were used as controls. Concrete analysis method is with embodiment 4.

The results are shown in Figure 15, the peripheral blood smears of PMF mice showed the presence of MPN cells in the peripheral blood of PMF mice in each group. Except for the LDL-MLN administration group, there were a large number of tumor cells in the peripheral blood of the PMF mice in the other administration groups, and at the same time, the HE-stained sections of the liver and spleen also showed the infiltration of tumor cells. In the control group (Con, LDL-blank and MLN), there were also a large number of immature megakaryocytes in the spleen sections. However, the disease burden in the LDL-MLN group was significantly reduced, and the peripheral blood smears showed that the cell populations were close to those of healthy mice, and liver and spleen sections were no different from those of healthy mice.

4. Differentiation level of splenocytes in PMF mice after LDL-MLN/MLN administration

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

647Rat anti-Mouse CD34(eBioscience，560230)进行孵育，孵育条件为4℃、避光、15min，最后用ACEA流式细胞分析仪进行检测。After 15 days of modeling, LDL-MLN administration treatment was started, once every 2 days, each administration dose was 0.01mg/kg, and the PMF mice after administration were analyzed by flow cytometry after administration for 7 times The differentiation level of tumor cells in the spleen, while MLN, LDL-blank and no drug treatment (Con) were used as controls. The specific analysis method is as follows: take the cell suspension after lysing red blood cells (the number of cells is 6×10 ⁶ ), and use flow cytometry antibodies ANTI-MO LY-6A/E D7 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 ^™ Alexa

647Rat anti-Mouse CD34 (eBioscience, 560230) was incubated at 4°C, protected from light, for 15 min, and finally detected by ACEA flow cytometer.

The results are shown in Figure 16, and the results showed that the tumor cells in the spleen of mice administered with LDL-MLN were significantly reduced and almost cleared. The analysis of LSK cells (Lin-Sca-1-c-Kit+) of GFP+ cells shows that LDL-MLN effectively reduces the content of abnormal stem cells, and the proportion of CMP, GMP and MEP lineage cells in LSK cells tends to increase. similar to that of healthy mice.

5. Differentiation level of bone marrow cells in PMF mice after LDL-MLN/MLN administration

647 Rat anti-Mouse CD34(eBioscience，560230)进行孵育，孵育条件为4℃、避光、15min，最后用ACEA流式细胞分析仪进行检测。After 15 days of modeling, LDL-MLN administration treatment was started, once every 2 days, each administration dose was 0.01mg/kg, and the PMF mice after administration were analyzed by flow cytometry after administration for 7 times Differentiation level of tumor cells in bone marrow, while MLN, LDL-blank and no drug treatment (Con) were used as controls. The specific analysis method is as follows: take the cell suspension after lysing red blood cells (the number of cells is 6×10 ⁶ ), use the flow cytometry antibody ANTI-MO LY-6A/E D7 PE (eBioscience, 12-5981-81), STREPTAVIDIN PERCP- CYN5.5 (eBioscience, 45-4317-82), ANTI-MO CD1172B8 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), BDPharmingen ^™ Alexa

647 Rat anti-Mouse CD34 (eBioscience, 560230) was incubated at 4°C, protected from light, for 15 minutes, and finally detected by ACEA flow cytometer.

The results are shown in Figure 17, and the results showed that the tumor cells in the bone marrow of mice administered with LDL-MLN were significantly reduced and almost cleared. The content of LSK cells in GFP+ cells decreased, and the proportion of CMP, GMP and MEP lineage cells in LSK cells was closer to that of healthy mice. MEP cells, which are abnormal megakaryocyte progenitors, were significantly reduced, implying fewer cells overexpressing MPLW515L in the bone marrow and increased levels of tumor cell differentiation.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (8)

1. Application of LDL-MLN nanomedicine in any of the following 1)-3): 1) Preparation of products for the prevention and/or treatment of myeloproliferative neoplasms; 2) Preparation of products for the prevention and/or treatment of primary myelofibrosis; 3) Preparation of products for the prevention and/or treatment of acute myeloid leukemia; The LDL-MLN nanomedicine is obtained by embedding the small molecule inhibitor MLN8237 with LDL particles as a drug carrier; The preparation method of described LDL-MLN nano medicine comprises the steps: 1) Mix LDL granules with potato starch, then freeze-dry to obtain LDL-starch mixture; 2) After completing step 1), extract endogenous lipids from the LDL-starch mixture with heptane to obtain an extracted product, then mix the MLN solution with the extracted product and incubate to obtain an LDL-MLN mixture ; The MLN solution consists of toluene, MLN8237, lauric acid and stearic acid. 2. The application according to claim 1, characterized in that the mass ratio of the LDL particles to the MLN8237 is 1: (0.02-0.03). 3. The application according to claim 1, characterized in that: the mass ratio of the LDL particles to the potato starch is 1:(10-15). 4. The application according to claim 1, characterized in that: the solvent of the MLN solution is toluene, the solute and its concentration are respectively 0.25 mg/mL of MLN8237, 8 mg/mL of lauric acid, and 2 mg/mL of stearic acid . 5. The application according to claim 1, characterized in that: the step of removing toluene is also included after the incubation. 6. The application according to any one of claims 1-5, characterized in that: the method further comprises a purification step; the purification method comprises the steps of: dispersing the LDL-MLN mixture in a buffer, Centrifuge to collect the supernatant, which is then filtered. 7. The LDL-MLN nanomedicine described in any one of claims 1-6. 8. A product whose active ingredient is the LDL-MLN nano-medicine described in any one of claims 1-6; The function of the product is any one of the following A-C: A. Prevention and/or treatment of myeloproliferative neoplasms; B. Prevention and/or treatment of primary myelofibrosis; C. Prevention and/or treatment of acute myeloid leukemia.

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