PREPARATION METHOD AND APPLICATION OF TUMOR MICROPARTICLES ENCAPSULATING METABOLIC INHIBITORS

Abstract

A preparation method of a tumor microparticle encapsulating a metabolic inhibitor includes following steps. A tumor cell is cultivated, and a statin drug is added to a culture medium after the tumor cell grows stably. Then, the tumor cell is irradiated with an ultraviolet, and then the tumor cell is incubated in a cell incubator for 22˜26 hours. After the incubation, cell supernatant is collected and the tumor microparticle encapsulating the metabolic inhibitor is obtained through a gradient centrifugation. The preparation method uses tumor cell-derived microparticles as drug delivery platforms, with a simple preparation process that preserves functions such as biosafety, biocompatibility, targeting, and intercellular communication. The preparation method discovers new uses of a statin drug, which can inhibit tumor growths with the statin drug, regulate tumors metabolism and improve a tumor microenvironment, and have a potential to enhance a chemotherapy efficacy and delay a chemotherapy resistance.

Claims

1. A preparation method of a tumor microparticle encapsulating a metabolic inhibitor comprising the following steps: step 1, cultivating a tumor cell and adding a statin drug to a culture medium after the tumor cell grows stably; step 2, performing ultraviolet (UV) irradiating on the tumor cell, and then incubating the tumor cell in a cell incubator for 22˜26 hours (h); and step 3, after the incubating, collecting cell supernatant and obtaining the tumor microparticle encapsulating the metabolic inhibitor through a gradient centrifugation.

2. The preparation method of the tumor microparticle encapsulating the metabolic inhibitor as claimed in claim 1, wherein in step 1, 0.2 millimoles per liter (mmol/L) Fluvastatin as the statin drug are added to every 10.sup.8 numbers of the tumor cell.

3. The preparation method of the tumor microparticle encapsulating the metabolic inhibitor as claimed in claim 1, wherein a time of the UV irradiating in step 2 is 20 minutes (min).

4. The preparation method of the tumor microparticle encapsulating the metabolic inhibitor as claimed in claim 1, wherein a time of the incubating in step 2 is 24 h.

5. The tumor microparticle encapsulating the metabolic inhibitor, prepared by the method as claimed in claim 1.

6. An application method of the tumor microparticle encapsulating the metabolic inhibitor, comprising: treating a cancer by using the tumor microparticle encapsulating the metabolic inhibitor as claimed in claim 5.

7. The application method of the tumor microparticle encapsulating the metabolic inhibitor as claimed in claim 6, comprising: preparing an injection reagent by using the tumor microparticle encapsulating the metabolic inhibitor as claim in claim 5 for inhibiting tumor growth.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to illustrate embodiments of the disclosure or technical solutions in the related art more clearly, a brief introduction is made to drawings needed in the embodiments below. Apparently, the drawings described below are only some of the embodiments of the disclosure. For those skilled in the art, other drawings are obtained from the drawings needed in the embodiments below without any creative effort.

[0026] FIG. 1 is a schematic diagram of a preparation process of a tumor microparticle encapsulating a metabolic inhibitor such as Fluvastatin (TMP-F).

[0027] FIG. 2 is a schematic diagram of a transmission electron microscope image of the TMP-F.

[0028] FIG. 3A is a schematic diagram of a size distribution of the TMP-F.

[0029] FIG. 3B is a schematic diagram of a particle concentration of the TMP-F.

[0030] FIG. 4 is a schematic diagram of an expression result of tumor cell-derived microparticles (TMPs) related membrane markers in western blotting (WB).

[0031] FIG. 5A is a schematic diagram of a standard curve of the Fluvastatin detected by high performance liquid chromatography (HPLC).

[0032] FIG. 5B is a schematic diagram of a drug content in the TMP-F, F.sub.0.1, F.sub.0.2, F.sub.0.4, and F.sub.0.5 represent the TMP-F induced by Lewis lung carcinoma (LLC) cells stimulated by 0.1 millimoles per liter (mM), 0.2 mM, 0.4 mM, and 0.5 mM Flu, respectively.

[0033] FIG. 6A is a schematic diagram of a flow cytometry result of a cell apoptosis experiment.

[0034] FIGS. 6B and 6C are schematic diagrams showing cell proliferation detected by cell counting kit-8 (CCK8), FIG. 6B shows percentage of apoptotic cells, FIG. 6C shows cell viability. In FIGS. 6B and 6C, Control represents no external stimulation of the LLC cells, Flu represents the LLC stimulated by the Flu (the concentration is equivalent to the Flu content in the TMP-F) for 24 hours (h); TMP-0 represents the LLC stimulated by the empty TMPs (TMP-0) for 24 h; TMP-F represents the LLC stimulated by the TMPs encapsulating the drugs (TMP-F) for 24 h; and ns is not statistically significant, *P<0.05, **P<0.01, ***P<0.001, **P<0.001.

[0035] FIG. 7A is a schematic diagram of a volume change curve of subcutaneous tumors of mice.

[0036] FIG. 7B is a schematic diagram of a weight change of the mice.

[0037] FIG. 8 is a schematic diagram of a display of nude tumors of the mice.

[0038] FIG. 9A is a schematic diagram of a volume change curve of the subcutaneous tumors during an intervention period.

[0039] FIG. 9B is a schematic diagram of a weight change of the mice during the intervention period.

[0040] FIG. 10A is a first schematic diagram showing the nude tumors in the mice.

[0041] FIG. 10B is a second schematic diagram showing the nude tumors in the mice.

[0042] FIG. 11 is a schematic diagram of a statistical map of pulmonary metastatic nodules, and *, #, and & respectively represent a statistical significance after comparison with phosphate buffered saline (PBS), the Flu, and the TMP-0 groups.

[0043] FIG. 12A is a schematic diagram of a distribution of CD4.sup.+ T cells in a tumor immune microenvironment.

[0044] FIG. 12B is a schematic diagram of a distribution of Treg cells in the tumor immune microenvironment.

[0045] FIG. 12C is a schematic diagram of a distribution of Th1 cells in the tumor immune microenvironment.

[0046] FIG. 12D is a schematic diagram of a distribution of CD8.sup.+ T cells in the tumor immune microenvironment.

[0047] FIG. 12E is a schematic diagram of a distribution of an interferon-γ (IFN-γ) secreted by cytotoxic CD8.sup.+ T cells in the tumor immune microenvironment.

[0048] FIG. 12F is a schematic diagram of a distribution of Granzyme B secreted by the cytotoxic CD8.sup.+ T cells in the tumor immune microenvironment.

[0049] FIG. 13A is a schematic diagram of a distribution of NK cells in the tumor immune microenvironment.

[0050] FIG. 13B is a schematic diagram of a distribution of the Granzyme B secreted by activated NK cells in the tumor immune microenvironment.

[0051] FIG. 13C is a schematic diagram of a distribution of an interferon-γ secreted by the activated NK cells in the tumor immune microenvironment.

[0052] FIG. 14A is a schematic diagram of a distribution of M1 macrophages in the tumor immune microenvironment.

[0053] FIG. 14B is a schematic diagram of a distribution of M2 macrophages in the tumor immune microenvironment.

[0054] FIG. 14C is a schematic diagram of a distribution proportion of the M1 and the M2 macrophages in the tumor immune microenvironment.

DETAILED DESCRIPTION OF EMBODIMENTS

[0055] The following is a description of principles and features of the disclosure in conjunction with accompanying drawings. Embodiments provided are only intended to explain the disclosure and are not intended to limit its scope.

[0056] 1. Preparing a Tumor Cell-Derived Microparticle (TMP) Encapsulating a Metabolic Inhibitor Such as Fluvastatin (TMP-F)

[0057] After the Lewis lung carcinoma (LLC) cell (lung adenocarcinoma cell lines) grows stably, 0.2 millimoles per liter (mM) Fluvastatin is added to a culture medium. Subsequently, the LLC cell is irradiated with an ultraviolet (300 joules per square meter (Jm.sup.−2)) for 20 minutes (min), and then incubated in a cell incubator for 24 hours (h). After the 24 h, cell supernatant is collected, and the TMP-F is obtained by a gradient centrifugation. Specifically, the gradient centrifugation is performed on the cell supernatant at 800 revolutions per minute (rpm) for 10 min, and then the cell precipitate is discarded to obtain first cell supernatant, the gradient centrifugation is performed on the first cell supernatant at a relative centrifuge force of 2000×g (the unit for g is gravitational acceleration, i.e., 9.8 m/s.sup.2) for 30 min, and then the cell debris is discarded to obtain second cell supernatant. The gradient centrifugation is performed on the second cell supernatant at a relative centrifuge force of 16000×g for 60 min to obtain the precipitate (i.e., the supernatant is discarded), the obtained precipitate is the TMP-F. Finally, the TMP-F is cleaned twice with phosphate buffered saline (PBS) and stored at −80° C. (FIG. 1).

[0058] 2. Identifying the TMP-F

[0059] 2.1 A Size of the TMP-F

[0060] The TMP-F is identified by a transmission electron microscopy, a nanoparticle tracking analysis (NTA), and a nanoflow cytometry to determine a shape, the size, and extraction concentration of the TMP-F (FIGS. 2, 3A and 3B). The average particle size of the TMP-F is 154.2 nanometers (nm), with the concentration of 2.75×10.sup.10 particles per milliliter (particles/ml).

[0061] 2.2 Membrane Marking of the TMP-F

[0062] Epithelial cellular adhesion molecule (EPCAM), tumor susceptibility gene 101 protein (TSG101) and CD63 are classic tumor-derived microparticle marker proteins, β-Tubulin is an internal reference protein, and the western blotting indicates that the TMP-F expresses the above proteins, successfully extracting the TMPs (FIG. 4).

[0063] 2.3 A Drug Loading Capacity of the TMP-F

[0064] To identify the content of the drug encapsulated by the TMP-F, a high performance liquid chromatography (HPLC) is used to infer that the TMP-F secreted by the LLC cell stimulated by the 0.2 mM Fluvastatin (Flu) contains about 4.25 micrograms per milliliter (μg/ml) drug (FIGS. 5A and 5B).

[0065] 3. The TMP-F Inhibits Cancer Cell Proliferation at a Cellular Level

[0066] To verify the effect of the TMP-F on lung adenocarcinoma, experiments were first conducted at the cellular level. Results of cell apoptosis and the CCK8 experiment suggest that the TMP-F significantly inhibits the cell proliferation and induces cancer cell apoptosis (FIGS. 6A, 6B and 6C).

[0067] 4. The TMP-F Inhibits Tumor Growth at an Animal Level

[0068] At the animal level, lung adenocarcinoma (LLC cell line) subcutaneous tumors are inoculated on right shoulder backs of C57BL/6 mice. After the growth volume of the subcutaneous tumors reaches 50 cubic millimeters (mm.sup.3), interventions are beginning. The mice are randomly divided into 4 groups, with the 5 mice in each group. The results show that the TMP-F group has a significant inhibitory effect on the tumor growth, but compared to a PBS group, administering a same dose of Flu drug in the TMP-F does not achieve the ideal tumor inhibitory effect (FIGS. 7A and 7B). After the 5 times of the interventions, the subcutaneous tumors obtained from dissection are shown in FIG. 8.

[0069] 5. The TMP-F Enhances Chemotherapy Efficacy and Inhibits the Lung Metastasis at the Animal Level

[0070] In order to explore the anti-tumor effect of TMP-F combined with chemotherapy, two doses of cisplatin chemotherapy (Cis) are added during the intervention process. From a growth trend of the subcutaneous tumors in the mice and gross photos of naked tumors, it is known that compared with the chemotherapy alone or the TMP-F, the TMP-F combined with chemotherapy significantly inhibits tumor growth (FIGS. 9A, 9B, 10A and 10B). In addition, the lungs of mice are extracted, hematoxylin-eosin (H&E) staining is performed on the lungs of mice, and the number of lung metastatic nodules under the light microscope are observed and counted. It is concluded that compared to the PBS intervention group, the chemotherapy alone, the TMP-F, or the TMP-F combined with chemotherapy inhibits the lung metastasis (FIG. 11).

[0071] 6. The TMP-F and TMP-F Combined with Chemotherapy Reshape a Tumor Immunosuppressive Microenvironment at the Animal Level

[0072] 6.1 T Cells

[0073] Because the TMPs carry abundant tumor antigens, impacts on the immune response related cells in the tumor microenvironment (TME) has attracted researchers' attention. Therefore, the local immune response of tumors is studied and the therapeutic mechanisms of TMP-F or TMP-F combined with chemotherapy are explored. The T cell subpopulations are tested in cellular immunity and found that in the TMP-F or the TMP-F combined with chemotherapy group, a level of immunosuppressive Treg is decreased, a distribution of helper Th1 cells assisting cellular immunity is increased, and a level of CD8.sup.+ T cells is increased, along with a secretion of cytokines such as interferon γ (IFN-γ) and Granzyme B is increased, but there is no significant change in CD4.sup.+ T cells among groups (FIGS. 12A to 12F). The result suggests that TMP-F and TMP-F combined with chemotherapy can mobilize the cell immune response to assist or directly kill the tumor cells.

[0074] 6.2 NK Cells

[0075] In addition to cell immunity, the natural immune response is detected in the TME. The TMP-F and TMP-F combined with chemotherapy promote infiltration of the NK cells in tumors, and activate the NK cells to release and increase a cytokine IFN-γ with a tumor killing effect.

[0076] In addition, the release of Granzyme B from the NK cells is significantly increased after the TMP-F combined with chemotherapy (FIGS. 13A, 13B and 13C).

[0077] 6.3 Macrophages

[0078] Tumor M1 type macrophages are considered an anti-inflammatory type and have anti-tumor effects; and M2 type tumor macrophages are considered a pro-inflammatory type, have a tumor promoting effect. The TMP-F and the TMP-F combined with chemotherapy effectively increase the infiltration rate of the M1 type macrophages, reduce M2 type macrophages, and increase a proportion of the M1 and the M2 macrophages, indicating that the TMP-F or the TMP-F combined with chemotherapy has a potential to induce macrophage polarization from M2 to M1 type macrophages (FIGS. 14A, 14B and 14C).

[0079] From the above results of cell immunity and natural immunity, it is known that after the intervention of the TMP-F or the TMP-F combined with chemotherapy, the tumor immunosuppressive microenvironment has a big reversal, and has changed into the immune microenvironment that promotes the tumor killing.

[0080] The embodiments described above are all preferred embodiments in the disclosure, and the parts not detailed are common knowledge of those skilled in the art. A scope of a protection of the disclosure is subject to a content of claims, and any equivalent transformation based on a technical inspiration of the disclosure is within the scope of the protection of the disclosure.