CN111991419A - Use of platinum-containing particles for the preparation of a medicament and/or a medical product for inducing the differentiation of leukemia cells - Google Patents

Abstract

The application of the platinum-containing particles of the present invention in the preparation of medicines and/or medical products for inducing leukemia cell differentiation. The platinum-containing particles provided by the present invention have the effect of promoting macrolineage and granulocyte differentiation on various blood tumors. The invention finds that platinum-containing granules can induce the differentiation of various leukemia cells to giant lineage and myeloid cells, promote the differentiation of bone marrow-derived mononuclear cells of leukemia patients to giant lineage and myeloid cells, and effectively relieve the mature megakaryocytes caused by the blocked differentiation of leukemia cells. , the number of granulocytes decreased. In addition, the present invention highlights for the first time the potential of platinum-containing particles to promote the differentiation of hematopoietic stem/progenitor cells into the giant lineage and the myeloid lineage.

Description

Application of platinum-containing particles in the preparation of drugs and/or medical products for inducing leukemia cell differentiation

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of platinum-containing particles in the preparation of medicines and/or medical products for inducing differentiation of leukemia cells.

Background technique

Leukemia is a malignant clonal disease of hematopoietic stem cells. Leukemia cells proliferate and accumulate in the bone marrow due to uncontrolled proliferation, impaired differentiation, and blocked apoptosis, and infiltrate other tissues and organs, thereby inhibiting normal hematopoiesis. Traditional leukemia treatment mostly uses radiotherapy and chemotherapy. The main mechanism of action is to inhibit and kill malignantly proliferating tumor cells by destroying DNA replication, transcription and modification, but it also kills normal cells while killing leukemia cells. It has the disadvantages of poor selectivity, easy emergence of drug resistance and large side effects.

Differentiation induction therapy is an effective method to overcome the above shortcomings of radiotherapy and chemotherapy. Under normal circumstances, pluripotent hematopoietic stem cells can differentiate into progenitor cells of various lineages, and then each lineage is gradually differentiated until mature cells are differentiated and released into peripheral blood. For leukemia patients, malignant clones of their multipotent hematopoietic stem cells have occurred, and all differentiated are leukemia cells, which involve different lines and make their differentiation stagnant at a certain stage. In the stage of differentiation stagnation, immature cells are directly released into the peripheral blood, thereby making There are many immature cells in the peripheral blood of leukemia patients. Induced differentiation of tumor cells means that in the presence of differentiation inducers, malignant tumor cells are differentiated in the direction of normal cells in terms of morphology and biology, or even completely transformed into normal cells, with the characteristics of strong pertinence and less side effects.

Various types of leukemias, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS), are caused by the blocked differentiation of bone marrow hematopoietic stem/progenitor cells. The differentiation of leukemia cells includes giant lineage differentiation, myeloid differentiation, erythroid differentiation and monocytic lineage differentiation, among which the giant lineage differentiation is closely related to the formation of platelets, and when it is involved or inhibited, it will cause thrombocytopenia and cause bleeding, etc.; Involvement or inhibition of lineage differentiation can cause leukopenia, which can easily lead to infection. The differentiation-inducing agents that have been discovered so far mainly involve retinoids, arsenic agents, traditional Chinese medicines, molecular targeted therapy drugs, demethylation drugs, histone deacetylation drugs, miRNAs, etc. Among them, in clinical treatment The more mature all-trans retinoic acid is used in the treatment of acute myeloid leukemia (APL). There are very limited reports of differentiation-inducing drugs for other leukemias.

Therefore, there is an urgent need to explore and develop other drugs that can induce leukemia cell differentiation.

SUMMARY OF THE INVENTION

Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art, and to provide an application of platinum-containing particles in the preparation of drugs and/or medical products for inducing differentiation of leukemia cells.

In order to achieve the above object, the first aspect of the present invention provides the use of platinum-containing particles in the preparation of drugs and/or medical products for inducing leukemia cell differentiation.

According to the application of the first aspect of the present invention, the platinum-containing particles are pure platinum nanoparticles or platinum-containing hybrid structure particles;

Preferably, the platinum-containing particles can be uniformly and stably dispersed in the aqueous phase.

According to the application of the first aspect of the present invention, in the drug and/or medical product, the concentration of the platinum-containing particles is not less than 1 pM, preferably 5 pM to 60 mM.

According to the application of the first aspect of the present invention, when the platinum-containing particles are platinum-containing hybrid structure particles, the particle size is 50-200 nm, preferably 120-123 nm;

The platinum-containing hybrid structure particles are selected from one or more of the following: gold core/platinum shell nanorods, gold core/platinum shell nanoparticles, silver platinum nanoparticles, platinum palladium nanoparticles, copper platinum nanoparticles, platinum-containing nanoparticles Particle micelles, liposomes containing platinum particles; preferably gold core/platinum shell nanorods;

More preferably, the gold core/platinum shell nanorod is a gold core/platinum shell structure composed of a cylindrical gold nanorod inner core and an island-shaped porous platinum shell layer coated on the outer surface of the cylindrical gold nanorod inner core. ;

When the platinum-containing particles are pure platinum nanoparticles, the particle size is 3-20 nm, preferably 14-17 nm.

According to the application of the first aspect of the present invention, the leukemia is a disease caused by the blocked differentiation of bone marrow hematopoietic stem/progenitor cells;

Preferably, the leukemia is selected from one or more of the following: chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome.

According to the application of the first aspect of the present invention, the platinum-containing particles induce the differentiation of bone marrow hematopoietic stem/progenitor cells into giant lineage and/or myeloid lineage.

According to the application of the first aspect of the present invention, wherein the cells are derived from infiltration of acute/chronic myeloid leukemia cell lines cultured in vitro, spleen and bone marrow cells derived from relapsed and refractory AML1-ETO acute myeloid leukemia mouse model of leukemia cells and bone marrow-derived mononuclear cells from MDS patients.

The second aspect of the present invention provides a method for inducing leukemia cell differentiation in vitro, the method comprising using platinum-containing particles to induce leukemia cell differentiation;

Preferably, the platinum-containing particles are pure platinum nanoparticles or platinum-containing hybrid structure particles;

Preferably, the platinum-containing particles can be uniformly and stably dispersed in the aqueous phase.

The method according to the second aspect of the present invention, wherein the leukemia cell differentiation is induced using the aqueous dispersion of platinum-containing particles.

The platinum-containing particles are particles containing platinum elements, which can be spherical, rod-shaped and other shapes, and can be pure platinum particles or particles with a hybrid structure.

The level of reactive oxygen species (ROS) in leukemia cells is higher than that in normal cells, and tumor cells are also more sensitive to elevated ROS levels than normal cells. Since most chemotherapeutic drugs induce cell death by increasing intracellular reactive oxygen species (ROS), this property has been applied in tumor chemotherapy, however upregulation of ROS may increase the genetic instability of leukemia cells due to oxidative damage to DNA molecules . Studies have shown that the disruption of redox balance and the increase of reactive oxygen species help to block the oncoprotein MUC1-C, thereby inhibiting the abnormal proliferation of leukemia cells; by down-regulating the level of ROS in human CML cells, realgar nanoparticles can effectively induce Erythroid differentiation of CML cells. These studies suggest that exogenous ROS regulation may be a way to break the proliferation cycle of leukemia cells and induce leukemia cells to differentiate into mature cells.

In recent years, platinum-containing particles have received extensive attention due to their unique and flexible regulation of ROS, with the advantages of good stability, low cost, and easy combination with a variety of drugs. For example, platinum (Pt) nanoparticles have oxidase-like, peroxidase, polyphenol oxidase, ferric oxidase, catalase, and superoxide dismutase-like enzymatic activities. These enzymatic activities can be involved in interactions between particles and various ROS (hydroxyl radicals, superoxide, singlet oxygen, and hydrogen peroxide) and are context-dependent. Therefore, platinum-containing particles can be applied to the induction and differentiation of leukemia through the regulation of ROS.

Differentiation-inducing drugs have been found in hematological tumors, mainly including: all-trans retinoic acid, which induces acute promyelocytic leukemia cells to differentiate into myeloid, monocyte and giant lineage; arsenic, which induces acute promyelocytic leukemia cells Myeloid, erythroid, and giant lineage differentiation of leukemia cells; histone deacetylase inhibitors (hydroxamic acids, benzamides, cyclic peptides), induction of T/B cell lymphoma cells, multiple Myeloma cells differentiate into myeloid and giant lineage; Vidaza (azacitidine injection), induces erythroid, giant and myeloid differentiation of MDS, AML, and CML cells; homoharringtonine, induces acute non-lymphocyte differentiation Leukemia cells (eg, acute myeloid leukemia cells, acute promyelocytic leukemia cells, acute monocytic leukemia cells) differentiate into the myeloid lineage. But so far, there is no literature report on the use of platinum-containing particles as a leukemia-inducing differentiation agent.

The present invention aims to provide the application of platinum-containing particles as an inducer of differentiation of bone marrow hematopoietic stem/progenitor cells in giant lineage and myeloid lineage.

The purpose of the present invention is to provide the application of platinum-containing particles in the leukemia differentiation inducer for the gap existing in the existing demand.

The platinum-containing particles include pure platinum particles or hybrid structure particles thereof.

The differentiation-inducing agent is used as a drug for the treatment of diseases caused by the blocked differentiation of bone marrow hematopoietic stem/progenitor cells, including chronic myeloid leukemia (CML), acute myeloid leukemia ( AML), myelodysplastic syndrome (MDS), etc.

Using the platinum-containing particles to induce the differentiation of bone marrow hematopoietic stem/progenitor cells into giant lineage and myeloid lineage, the experimental concentration of cells in vitro is not less than 1 pM, preferably 5-60 mM; the experimental concentration in mice is not less than 1 mg/kg, preferably 4-60 mM 8mg/kg.

The method of the present invention can have but is not limited to the following beneficial effects:

The platinum-containing particles used in the present invention include pure platinum particles containing platinum elements or particles of hybrid structures thereof. It can be uniformly and stably dispersed in the water phase.

The invention discloses that after co-incubating the platinum-containing particle dispersion with leukemia cells from different sources, the levels of cell surface differentiation marker molecules, including giant lineage differentiation marker (CD41a) and myeloid differentiation marker (CD11b), are detected. After treatment with platinum-containing particles, it was found that the differentiation marker molecules on the cell surface were significantly increased in a dose-dependent manner, indicating that platinum-containing particles can effectively induce the differentiation of chronic myeloid leukemia cells into giant lineage and myeloid lineage. Cells, bone marrow mononuclear cells derived from the bone marrow of patients with MDS, leukemia cells infiltrated in the spleen and bone marrow cells derived from the relapsed and refractory AML1-ETO acute myeloid leukemia mouse model, etc. have this effect of inducing differentiation. The induction effect can effectively alleviate the abnormal cell differentiation caused by the blocked differentiation of leukemia cells, especially the abnormal differentiation of giant lineage and myeloid lineage. The differentiation of leukemia cells can avoid systemic side effects caused by large doses of drugs.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1 shows a transmission electron microscope picture of the platinum-containing hybrid structure and pure platinum particles in Test Example 1 of the present invention; Figure 1(a) is a transmission electron microscope picture of the platinum-containing hybrid structure particle, and Figure 1(b) is a transmission electron microscope picture of pure platinum particles.

Figure 2 shows the particle size distribution and potential diagram of the platinum-containing hybrid structure and pure platinum particles in the aqueous phase in Test Example 2 of the present invention; wherein Figure 2(a) is the particle size distribution of the platinum-containing hybrid structure particles and Potential diagram, Figure 2(b) is the particle size distribution and potential diagram of pure platinum particles.

Figure 3 shows the effect of the platinum-containing hybrid structure and pure platinum particles on the giant lineage differentiation of human chronic myeloid leukemia cells K562 in Test Example 3 of the present invention; wherein Figure 3(a) is the result of the platinum-containing hybrid structure , Figure 3(b) is the result of pure platinum particles.

Figure 4 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 4 of the present invention on the myeloid differentiation of human chronic myeloid leukemia cells K562 cells; wherein Figure 4(a) is the result of the platinum-containing hybrid structure, Figure 4(b) shows the results for pure platinum particles.

Figure 5 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 5 of the present invention on the giant lineage differentiation of human acute myeloid leukemia cells Kasumi-1 cells; wherein Figure 5(a) is the platinum-containing hybrid structure Figure 5(b) is the result of pure platinum particles.

Figure 6 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 6 of the present invention on the myeloid differentiation of human acute myeloid leukemia cells Kasumi-1 cells; wherein Figure 6(a) is the platinum-containing hybrid structure Figure 6(b) is the result of pure platinum particles.

Figure 7 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 7 of the present invention on the giant lineage differentiation of leukemia cells infiltrated in the spleen and bone marrow in the AML1-ETO mouse model; 7(b) is the result of the platinum-containing hybrid structure, and FIG. 7(c) and FIG. 7(d) are the results of pure platinum particles.

Figure 8 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 8 of the present invention on the myeloid differentiation of leukemia cells infiltrating in the spleen and bone marrow in the AML1-ETO mouse model; 8(b) is the result of the platinum-containing hybrid structure, and FIG. 8(c) and FIG. 8(d) are the results of pure platinum particles.

Figure 9 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 9 of the present invention on the giant lineage differentiation of bone marrow clinical samples from two MDS leukemia patients; wherein Figures 9(a) and 9(b) are two Figure 9(c) and Figure 9(d) are the results of pure platinum particles in two patients.

Figure 10 shows the effect of the platinum-containing hybrid structure and pure platinum particles in Test Example 10 of the present invention on the myeloid differentiation of the bone marrow clinical samples of two MDS leukemia patients; wherein Figures 10(a) and 10(b) are two Figure 10(c) and Figure 10(d) are the results of pure platinum particles in two patients.

Figure 11 shows the effect of several other types of platinum-containing hybrid structure particles in Example 2 of the present invention on the giant lineage differentiation of human chronic myeloid leukemia cells K562 cells; wherein Figure 11(a) is silver platinum nanoparticles Figure 11(b) is the result of platinum-palladium nanoparticles, Figure 11(c) is the result of copper-platinum nanoparticles, Figure 11(d) is the result of micelles containing platinum particles, and Figure 11(e) is the result of Results of platinum particle liposomes.

Figure 12 shows the effect of several other types of platinum-containing hybrid structure particles in Example 2 of the present invention on the granulocyte differentiation of human chronic myeloid leukemia cells K562; Figure 12(a) is silver-platinum nanoparticles Figure 12(b) is the result of platinum-palladium nanoparticles, Figure 12(c) is the result of copper-platinum nanoparticles, Figure 12(d) is the result of micelles containing platinum particles, and Figure 12(e) is the result of Results of platinum particle liposomes.

Figure 13 shows the effect of several other types of platinum-containing hybrid structure particles in Example 2 of the present invention on the giant lineage differentiation of human acute myeloid leukemia cells Kasumi-1 cells; wherein Figure 13(a) is silver The results of platinum nanoparticles, Fig. 13(b) is the result of platinum-palladium nanoparticles, Fig. 13(c) is the result of copper-platinum nanoparticles, Fig. 13(d) is the result of micelles containing platinum particles, Fig. 13(e) ) are the results for liposomes containing platinum particles.

Figure 14 shows the effect of several other types of platinum-containing hybrid structure particles in Example 2 of the present invention on the myeloid differentiation of human acute myeloid leukemia cells Kasumi-1 cells; wherein Figure 14(a) is silver The results of platinum nanoparticles, Fig. 14(b) is the result of platinum-palladium nanoparticles, Fig. 14(c) is the result of copper-platinum nanoparticles, Fig. 14(d) is the result of micelles containing platinum particles, Fig. 14(e) ) are the results for liposomes containing platinum particles.

Detailed ways

The present invention is further described below through specific examples, but it should be understood that these examples are only used for more detailed and specific description, and should not be construed as being used to limit the present invention in any form.

This section provides a general description of the materials and test methods used in the tests of the present invention. While many of the materials and methods of operation used for the purposes of the present invention are known in the art, the present invention is described in as much detail as possible. It is clear to those skilled in the art that, in the context, if not specifically stated, the materials and methods of operation used in the present invention are well known in the art.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through regular channels.

Unless otherwise specified, the human chronic myeloid leukemia cell K562 cell line and the human acute myeloid leukemia cell Kasumi-1 cell line used in the following examples were purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences.

Unless otherwise specified, the solvents of the aqueous solutions used in the following examples are sterile ultrapure aqueous solutions.

Unless otherwise specified, the reagents used in the following examples are of analytical grade.

Unless otherwise specified, the PBS solutions used in the following examples are all 1×PBS solutions.

The reagents and instruments used in the following examples are as follows:

Reagents:

Platinum nanoparticles (PtNPs), purchased from nanocomposix company; sterile ultrapure aqueous solution, from MerkMillipore company, model Milli-Q Advantage A10;

Cetyltrimethylammonium bromide, sodium borohydride, ascorbic acid, potassium tetrachloroplatinite, sodium polystyrene sulfonate (PSS), tetrachloropalladium(II) acid (H ₂ PdCl ₄ ) were purchased from Alfa Aesar, tetrachloroauric acid, silver nitrate, sulfuric acid, copper sulfate (CuSO ₄ ), citric acid, and sodium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd., regenerated cellulose dialysis tubes were purchased from VWR Company, and hydrochloric acid was purchased from Nacalai Company, THF was purchased from Wako Company, soybean phosphatidyl choline (SPC-3), cholesterol, dipalmitoyl phosphatidyl glycerol (DPPG) and methoxy polyethylene glycol distearoyl phosphatidyl ethanolamine (mPEG 2000- DSPE) was purchased from Lipoid Company in Germany, RPMI 1640 medium was purchased from Hyclone Company, anti-human CD41a antibody was purchased from eBioscience Company, anti-human CD11b antibody was purchased from Biolegend Company, and human lymphocyte separation liquid was purchased from Daktronics Company.

instrument:

Transmission electron microscope, purchased from JEOL, Tokyo, Japan, model TEM-1400plus;

Nano particle size analyzer, purchased from Malvern, UK, model Nano ZS90;

Flow cytometer, purchased from BD Company in the United States, model Accuri C6.

Example 1

This example is used to illustrate the preparation method of the platinum-containing hybrid structure particles of the present invention, and the platinum-containing hybrid structure nanoparticles are named Au@Pt.

The present invention uses the gold core/platinum shell nanorod dispersion disclosed in the patent application with the application number of 200910093031.5 as a typical platinum-containing hybrid structure particle.

A gold core/platinum shell nanorod solution (Au@Pt) was prepared, and the preparation steps were as follows:

(1) Preparation of gold seed solution:

Take 7.5mL of 0.1M cetyltrimethylammonium bromide aqueous solution, add 100.4μL of 24.7mM tetrachloroauric acid aqueous solution to it, mix well and dilute the volume to 9.4mL, under the condition of magnetic stirring Add 0.6mL concentration of 0.01M sodium borohydride aqueous solution (temporarily prepared before use and placed in ice water) to obtain a first mixed solution, the cetyltrimethylammonium bromide in the first mixed solution , tetrachloroauric acid and sodium borohydride material ratio=0.75: 0.0025: 0.006; stop after stirring for three minutes, let stand for 3 hours to obtain a gold seed solution containing gold seeds, the concentration of gold in the gold seed solution is 0.25mM;

(2) Preparation of gold nanorod solution:

Take 100mL of 0.1M aqueous solution of cetyltrimethylammonium bromide, add 2.05mL of 24.7mM aqueous tetrachloroauric acid solution, 1mL of 10mM aqueous silver nitrate solution and 2mL of 0.5M aqueous sulfuric acid solution , after mixing evenly, add 800 μL of ascorbic acid aqueous solution with a concentration of 0.1M, the obtained mixed solution changes from orange to colorless, and then add 240 μL of the gold seed solution prepared in step 1 to obtain a second mixed solution; Put it into a constant temperature water bath at 30°C; the solution began to appear color after 20 minutes, and finally turned purplish red after 14 hours, indicating that a gold nanorod solution was formed; the cetyltrimethyl bromide in the second mixed solution The weight ratio of ammonium chloride, tetrachloroauric acid, silver nitrate, sulfuric acid, ascorbic acid and gold seeds = 5:0.025:0.0025:0.5:0.04:0.000015;

(3) Purification of gold nanorods

The prepared gold nanorod solution was first placed in a constant temperature water bath at 30°C, and then centrifuged twice for 10 minutes at 12,000 rpm to remove unreacted ions and excess hexadecane. trimethylammonium bromide to obtain a purified gold nanorod solution, and then add deionized water to control the concentration of gold nanorods in the purified gold nanorod solution to be 0.5mM;

(4) Preparation of gold core/platinum shell nanorod solution:

Take a portion (1 mL) of the purified gold nanorod solution after the above centrifugation and put it into a test tube, and add 1 mL of deionized water, 40 μL of 2mM potassium tetrachloroplatinite aqueous solution and 8 μL of 0.1 M aqueous solution of ascorbic acid to it and mix it uniformly. The third solution; the weight ratio of ascorbic acid, potassium tetrachloroplatinite and gold nanorods in the third solution is 20:1:3;

Then put it in a constant temperature water bath at 30 °C, and after 2 hours of reaction, the solution changed from purplish red to dark gray (indicating the formation of gold core/platinum shell nanorods), and then hexadecyl trisulfoxide was added to the third solution. Aqueous methyl ammonium bromide solution, and the hexadecyl trimethyl ammonium bromide concentration in the solution after adding hexadecyl trimethyl ammonium bromide is 0.03M; then centrifugal separation is performed to obtain gold core/ Platinum shell nanorod solution;

(5) PSS modification of gold core/platinum shell nanorod solution:

The gold core/platinum shell nanorods obtained in step 4 were centrifuged at 12,000 rpm for 5 minutes, the centrifuged supernatant was discarded, and an equal volume of the modified solution with the supernatant was added to the precipitate, The modified solution is composed of sodium polystyrene sulfonate (PSS) solution and sodium chloride solution (the concentration of sodium polystyrene sulfonate (PSS) solution is 1 mg/mL, and the concentration of sodium chloride solution is 3.0 mM); The obtained mixed solution was then sonicated in an ultrasonic cleaner for 10 minutes, and then allowed to stand for 3 hours to obtain a gold core/platinum shell nanorod solution modified by sodium polystyrene sulfonate (PSS);

(6) Purification of modified gold core/platinum shell nanorod solution

The modified gold core/platinum shell nanorod solution prepared in step 5 was centrifuged at 12,000 rpm for 5 minutes, the supernatant after centrifugation was discarded, and an equal volume of the supernatant was added. The ionized water is uniformly dispersed to obtain a purified modified gold core/platinum shell nanorod solution, and the obtained modified gold core/platinum shell nanorod solution is obtained.

Commercial platinum particles (PtNPs, from nanocomposix company, commercial product LotNumber: DAG2419, named 5nm Citrate BioPure ^™ Platinum) are used as a typical pure platinum particle in the examples and test examples of the present invention. These particles are uniformly and stably dispersed in the aqueous phase.

Example 2

This example is used to illustrate the preparation method of the platinum-containing hybrid structure particles of the present invention.

The inventor has also prepared other types of platinum-containing hybrid structure particles, and the preparation method is as follows:

(1) Silver platinum nanoparticles: 200 μL of 2 mM PtCl ₄ ^2- and 40 μL of 10 mM AgNO ₃ were mixed with 2 mL of 0.25 mM CTAB aqueous solution. Then, 10x 0.1M AA was added. Shake vigorously immediately and then place in a water bath at 30°C for 5h. When the solution turned dark gray, it indicated that silver platinum nanoparticles were synthesized. The obtained AgPt NPs were purified by centrifugation twice at 12 000 rpm and 5 min.

(2) Platinum palladium nanoparticles: 1 mL of purified Au NRs was mixed with 40 μL of PtCl ₄ ^2- (2 mM) and 2 mM of H ₂ PdCl ₄ . 10x more AA (0.1M) was added and shaken vigorously. Adjust the final volume to 3 mL with water. The color changes from brown to gray, indicating the synthesis of platinum-palladium nanoparticles.

(3) Copper platinum nanoparticles: 1.2 mM CuSO ₄ and 0.4 mM K ₂ PtCl ₄ were added to a solution containing 0.05 mg/mL PSS, followed by AA (160 mM). The mixed solution was incubated in a 30° C. water bath for 2 hours, centrifuged once (12,000 rpm, 5 min), and the obtained precipitate was dispersed in 100 μL of H ₂ O to obtain copper-platinum nanoparticles.

(4) Platinum particle-containing micelles: Dissolve 5-10 parts of platinum nanoparticles in 1-10 mg/mL THF, mix with 100-300 parts of DSPE-PEG, slowly pour into 10 mL of water, and stir rapidly. After stirring for 2 hours, the micelles were obtained by overnight dialysis with 10k molecular weight cut-off regenerated cellulose dialysis tubing to obtain platinum particle-containing micelles.

The specific preparation method of the platinum particle-containing micelles used in the following test examples 11 and 12 is as follows: 5 parts of platinum nanoparticles (purchased from nanocomposix company, product number DAG2419, the diameter under TEM is 4.6±0.8nm) are dissolved in 8 mg/mL THF , mixed with 200 parts of DSPE-PEG. 500L of the suspension was slowly poured into 10mL (18M) water with rapid stirring. After stirring for 2 hours, the micelles were obtained by overnight dialysis with 10k molecular weight cut-off regenerated cellulose dialysis tubing to obtain platinum particle-containing micelles.

(5) Liposomes containing platinum particles: lipids include 50-200 parts of soybean phosphatidylcholine (SPC-3), 10-50 parts of cholesterol, 10-30 parts of dipalmitoyl phosphatidylglycerol (DPPG) and 1- 10 parts methoxypolyethylene glycol distearoylphosphatidylethanolamine (mPEG 2000-DSPE). The above lipids were added to 10 mL of anhydrous ethanol solvent to obtain a mixed solution. Dissolve 1-5 parts of platinum-containing particles in citrate buffer with pH 3.8, mix the platinum-containing particle solution and the liposome solution, and incubate at 55°C for 1 h. A white suspension (reverse-phase micelles) was obtained after sonication with a probe under nitrogen protection. The organic phase was removed by rotary evaporation to form a white gelatinous substance, and then rotary evaporation was continued to obtain a liposome solution containing platinum particles.

The platinum particle-containing liposomes used in the following Test Examples 11 and 12 included 200 parts of soybean phosphatidylcholine (SPC-3), 50 parts of cholesterol, 20 parts of dipalmitoyl phosphatidylglycerol (DPPG) and 10 parts of methoxy Polyethylene glycol distearoylphosphatidylethanolamine (mPEG 2000-DSPE). The above lipids were added to 10 mL of anhydrous ethanol solvent to obtain a mixed solution. Take 5 parts of platinum-containing particles (purchased from nanocomposix company, product number DAG2419, the diameter under TEM is 4.6±0.8nm) and dissolve them in citric acid buffer of pH 3.8, and mix the platinum-containing particle solution and liposome solution for 55 minutes. Incubate at ℃ for 1 h. A white suspension (reverse-phase micelles) was obtained after sonication with a probe under nitrogen protection. The organic phase was removed by rotary evaporation to form a white gelatinous substance, and then rotary evaporation was continued to obtain a liposome solution containing platinum particles.

Test Example 1: Transmission Electron Micrograph

The transmission electron microscope images of the platinum-containing hybrid structure particles and pure platinum nanoparticles prepared in Example 1 are shown in FIG. 1 .

It can be seen from Figure 1 that the platinum-containing hybrid structure particles have a rod-like structure, and the platinum nanoparticles are uniformly distributed on the gold rods; the pure platinum nanoparticles are spherical particles.

Test Example 2: DLS Analysis of Particle Size Distribution and Potential

Take 10 μL of platinum-containing nanoparticles prepared in Example 1, add 990 μL of sterile ultra-pure aqueous solution to make a suspended dispersion, draw 1 mL of the above dispersion and place it in a cuvette, and use Nano ZS90 nanometer particle size analyzer to test the particle size and electric potential, the results obtained are shown in Figure 2.

It can be seen from Figure 2 that the average hydrated particle size of the platinum-containing hybrid structure nanoparticles in the dispersion is about 121.8±0.9nm; the average hydrated particle size of the pure platinum nanoparticles is about 15.6±0.8nm. The average potential of platinum-containing hybrid nanoparticles is about -31.5±0.2mV; the average potential of pure platinum nanoparticles is about -17.6±6.3mV, indicating that the particles have better stability.

Test Example 3: The effect of platinum-containing nanoparticles on the giant lineage differentiation of K562 cells

K562 cells were seeded into 24-well cell culture plates at a density of 1×10 ⁵ cells/well, 250 μL per well. Take the platinum-containing nanoparticles prepared in Example 1 and disperse them in the medium for culturing K562 cells, and add 250 μL of the above dispersion to the culture system, so that the final concentrations of the platinum-containing hybrid structure nanoparticles in the medium are 10 pM, 15 pM and 10 pM respectively. 20 pM, and the final concentrations of pure platinum nanoparticles were 6 pM and 60 pM, respectively. After incubating the cells with the nanoparticle suspension for 24 hours at 37°C, the cells were harvested and washed with PBS. Cells were incubated with FITC-labeled giant differentiation signature molecule (CD41a) for 40 minutes at room temperature in the dark. The mean fluorescence intensity of CD41a on the surface of K562 cells was detected by flow cytometry. All groups have three replicate wells. The results are shown in Figure 3.

The results showed that platinum-containing hybrid nanoparticles and pure platinum nanoparticles could significantly increase the mean fluorescence intensity of CD41a, indicating that K562 cells could be induced to differentiate into giant lineage. Within the experimental concentration range, the effect of inducing differentiation is significant.

Test Example 4: The effect of platinum-containing nanoparticles on the differentiation of K562 cells

The sample processing method was the same as that of Test Example 3. The final concentrations of the platinum-containing hybrid structure nanoparticles were 15 pM and 20 pM, respectively, and the concentrations of pure platinum particles were 6 pM and 60 pM, respectively. The mean fluorescence intensity of myeloid differentiation signature molecule (CD11b) on the surface of K562 cells was detected by flow cytometry. The results obtained are shown in Figure 4.

The results showed that platinum-containing hybrid nanoparticles and pure platinum nanoparticles could significantly increase the mean fluorescence intensity of CD11b, indicating that platinum-containing nanoparticles could also induce K562 to differentiate into granulocytes. Within the experimental concentration range, the effect of inducing differentiation is significant.

Test Example 5: The effect of platinum-containing nanoparticles on the giant lineage differentiation of Kasumi-1 cells

Kasumi-1 cells are acute myeloid leukemia cells. In this experiment, Kasumi-1 cells are used as an example to verify the effect of the inducer of the present invention on their differentiation level. The cell seeding density and sample processing method were the same as those in Test Example 3. The final concentrations of platinum-containing hybrid structure nanoparticles were 10 pM and 20 pM, respectively, and the concentrations of pure platinum particles were 6 pM and 60 pM, respectively. The obtained results are shown in Figure 5.

The results showed that platinum-containing hybrid nanoparticles and pure platinum nanoparticles could significantly increase the mean fluorescence intensity of CD41a, indicating that the nanoparticles could also induce AML cell lines to differentiate into giant lineages. Within the experimental concentration range, the effect of inducing differentiation is significant.

Test Example 6: The effect of platinum-containing nanoparticles on the differentiation of Kasumi-1 cells

The sample processing method was the same as that of Test Example 3, except that CD41a was replaced with a granulocyte differentiation characteristic molecule (CD11b). The obtained results are shown in Figure 6.

The results showed that platinum-containing hybrid nanoparticles and pure platinum nanoparticles could significantly increase the mean fluorescence intensity of CD11b, indicating that the nanoparticles could also induce AML cell lines to differentiate into granulocytes. Within the experimental concentration range, the effect of inducing differentiation is significant.

Test Example 7: The effect of platinum-containing nanoparticles on the giant lineage differentiation of infiltrating leukemia cells in the spleen and bone marrow of AML1-ETO mice effect

The mice used in this experiment were 6-week-old female C57BL/6 mice, which were raised in an SPF environment of the Experimental Animal Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences. Animals were acclimated to laboratory conditions one week before the experiment. 1×10 ⁶ AML1-ETO mouse primary spleen cells (GFP labeled) were injected via tail vein after sublethal irradiation (450 cGy). About 7 days after inoculation, compared with healthy mice, the peripheral blood GFP+ cells of the model mice reached about 6%, and the GFP+ cells in the spleen and bone marrow reached about 20%, indicating that the leukemia model mice had been successfully modeled. From the 8th day after transplantation, the experimental group was intraperitoneally injected with 100 μL of platinum-containing nanoparticles and pure platinum nanoparticles prepared in Example 1, while the control group was intraperitoneally injected with the same volume of 5% glucose solution. After continuous administration for 1 week, they were sacrificed by cervical dislocation after blood collection by enucleation of the eyeballs, and spleen samples were collected to collect bone marrow cells. The mean fluorescence intensity of CD41a was analyzed by flow cytometry on the collected spleen and bone marrow cells. The results obtained are shown in Figure 7.

The results showed that the platinum-containing hybrid structure nanoparticles and pure platinum nanoparticles described in this paper can significantly promote the giant lineage differentiation of leukemia cells infiltrated in the spleen and bone marrow cells of AML1-ETO mice, and the effect is significant at higher concentrations in the experiment.

Test Example 8: The effect of platinum-containing nanoparticles on the myeloid differentiation of infiltrating leukemia cells in the spleen and bone marrow of AML1-ETO mice effect

The sample processing method was the same as that of Test Example 7. The mean fluorescence intensity of CD11b was analyzed by flow cytometry on the collected spleen and bone marrow cells. The obtained results are shown in Figure 8.

The results show that the platinum-containing hybrid nanoparticles and pure platinum nanoparticles described in this paper can also significantly promote the differentiation of leukemia cells infiltrated in the spleen and bone marrow cells of AML1-ETO mice into myeloid cells, and the effect is significant at higher concentrations in the experiment. .

Test Example 9: Effect of platinum-containing nanoparticles on giant lineage differentiation of clinical samples from two MDS leukemia patients

Clinical bone marrow samples were first diluted with PBS and then added to an equal volume of human lymphocyte separation solution for centrifugation. Bone marrow mononuclear cells (BM-MNCs) in the middle layer were collected and resuspended in culture medium. The cells were grown on a 24-well cell culture plate at a density of 1×10 ⁵ cells/well, and the operation was performed according to the method in Test Example 3. The final concentrations of the platinum-containing hybrid structure nanoparticles were 5pM, 10pM, 15pM and 20pM, respectively. , the pure platinum particle concentrations were 0.6 pM, 6 pM and 60 pM, respectively. The obtained results are shown in Figure 9.

The results showed that platinum-containing hybrid nanoparticles and pure platinum nanoparticles could significantly increase the mean fluorescence intensity of CD41a in bone marrow clinical samples of two MDS leukemia patients, indicating that the nanoparticles could induce BM-MNC cells to differentiate into giant lineage.

Test Example 10: The effect of platinum-containing nanoparticles on the myeloid differentiation of clinical samples from two MDS leukemia patients

The operations were carried out with reference to the methods in Test Example 3 and Test Example 9, respectively. The results obtained are shown in Figure 10.

The results showed that platinum-containing hybrid nanoparticles and pure platinum nanoparticles could significantly increase the mean fluorescence intensity of CD11b in bone marrow clinical samples of two MDS leukemia patients, indicating that the nanoparticles could induce BM-MNC cells to differentiate into myeloid lineage.

Test Example 11: The effects of several other types of platinum-containing hybrid structure particles in Example 2 on K562 cell giant lineage and granules The role of lineage differentiation

Operation was carried out with reference to the methods in Test Example 3 and Test Example 4, respectively. The results obtained are shown in Figures 11 and 12.

The results showed that several other types of platinum-containing hybrid particles could significantly increase the average fluorescence intensity of CD41a and CD11b on the surface of K562 cells, indicating that these types of platinum-containing hybrid particles could induce K562 cells to become giant and Granular differentiation.

Test Example 12: Effect of other types of platinum-containing hybrid structure particles in Example 2 on the Kasumi-1 cell giant line and myeloid differentiation

The operations were carried out with reference to the methods in Test Example 5 and Test Example 6, respectively. The results obtained are shown in Figures 13 and 14.

The results show that other types of platinum-containing hybrid particles can significantly increase the average fluorescence intensity of CD41a and CD11b on the surface of kasumi-1 cells, indicating that these types of platinum-containing hybrid particles can induce kasumi-1 cells. Differentiation into macrolineage and granuloid lineage.

The results show that both the platinum-containing hybrid structure particles and pure platinum particles used in the present invention can significantly increase the proportion of CD41a and CD11b positive cells in leukemia cell populations from different sources, indicating that the platinum-containing particles have the ability to induce leukemia cells to enter the giant lineage and the myeloid lineage. Differentiation potential, which means that platinum-containing particles can be used to treat diseases related to the blocked induction of differentiation of bone marrow hematopoietic stem/progenitor cells.

Although this invention has been described to a certain extent, it will be apparent that suitable changes in various conditions may be made without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the embodiments described, but is to be included within the scope of the claims, which include equivalents for each of the elements described.

Claims (10)

1. Application of platinum-containing particles in the preparation of drugs and/or medical products for inducing leukemia cell differentiation. 2. The application according to claim 1, wherein the platinum-containing particles are pure platinum nanoparticles or platinum-containing hybrid structure particles; Preferably, the platinum-containing particles can be uniformly and stably dispersed in the aqueous phase. The application according to claim 1 or 2, characterized in that, in the drug and/or medical product, the concentration of the platinum-containing particles is not less than 1 pM, preferably 5 pM to 60 mM. 4. The application according to any one of claims 1 to 3, wherein when the platinum-containing particles are platinum-containing hybrid structure particles, the particle size is 50-200 nm, preferably 120-123 nm; and /or The platinum-containing hybrid structure particles are selected from one or more of the following: gold core/platinum shell nanorods, gold core/platinum shell nanoparticles, silver platinum nanoparticles, platinum palladium nanoparticles, copper platinum nanoparticles, containing Platinum particle micelles, platinum particle-containing liposomes; preferably gold core/platinum shell nanorods; More preferably, the gold core/platinum shell nanorod is a gold core/platinum shell structure composed of a cylindrical gold nanorod inner core and an island-shaped porous platinum shell layer coated on the outer surface of the cylindrical gold nanorod inner core. ; When the platinum-containing particles are pure platinum nanoparticles, the particle size is 3-20 nm, preferably 14-17 nm. 5. The use according to any one of claims 1 to 4, wherein the leukemia is a disease caused by blocked differentiation of bone marrow hematopoietic stem/progenitor cells. 6. The use according to claim 5, wherein the leukemia is selected from one or more of the following: chronic myeloid leukemia, acute myeloid leukemia, and myelodysplastic syndrome. 7. The use according to any one of claims 1 to 6, wherein the platinum-containing particles induce bone marrow hematopoietic stem/progenitor cells to differentiate into giant lineage and/or myeloid lineage. 8. The application according to any one of claims 1 to 7, wherein the hematopoietic stem/progenitor cells comprise acute/chronic myeloid leukemia cell lines derived from in vitro culture, relapsed and refractory AML1-ETO Leukemia cells infiltrated in spleen and bone marrow cells derived from a mouse model of acute myeloid leukemia and bone marrow mononuclear cells derived from bone marrow of MDS patients. 9. A method for inducing differentiation of leukemia cells in vitro, wherein the method comprises using platinum-containing particles to induce differentiation of leukemia cells; Preferably, the platinum-containing particles are pure platinum nanoparticles or platinum-containing hybrid structure particles; The platinum-containing particles can be uniformly and stably dispersed in the aqueous phase. 10. The method of claim 8, wherein the leukemia cell differentiation is induced using the aqueous dispersion of platinum-containing particles.

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