Transition metal dichalcogenide (TMD) composite and cancer immunotherapy agent comprising same

Abstract

Provided is a transition metal dichalcogenide (TMD) composite, wherein the transition metal dichalcogenide (TMD) is in the form of a nanosheet, a fatty acid is bonded to the transition metal dichalcogenide (TMD) nanosheet, and the expression type of macrophages is determined according to a combination of the transition metal dichalcogenide and the fatty acid.

Description

Transition metal dichalcogenide (TMD) complex and immuno-oncology agent containing the same

[0001] The present invention relates to a transition metal dichalcogenide (TMD) complex and an immuno-oncology agent comprising the same, and more particularly, to a transition metal dichalcogenide (TMD) complex capable of inducing polarization of macrophages into the M1 expression type and thus being utilized as an immuno-oncology agent, and an immuno-oncology agent comprising the same.

[0002] Macrophages, or phagocytes, are key cells of innate immunity. While most are found sessile throughout the body, some exist in the blood as monocytes. These monocytes can differentiate into dendritic cells or macrophages. Macrophages are a type of white blood cell that performs phagocytic activity, engulfing and digesting non-protein substances present in the healthy body, such as tissue, foreign substances, microorganisms, and cancer cells.

[0003] Macrophages can either induce or suppress inflammation in the body. When inflammation increases in the body or the immune system is stimulated, they can activate to suppress inflammation or release cytokines to reduce the immune response. Macrophages that induce inflammation are called M1 macrophages, while those that reduce inflammation and promote tissue repair are called M2 macrophages, which aid in cancer progression.

[0004] Therefore, the development of novel materials and methods that can selectively reprogram the phenotype of macrophages is very important in the development of immunotherapy-based anticancer or anti-inflammatory treatments.

[0005] Therefore, the problem to be solved by the present invention is to provide a novel material and method capable of reprogramming the phenotype of macrophages, and thereby to provide an anticancer treatment agent that selectively expresses M1 and M2 phenotype macrophages.

[0006] In order to solve the above problem, the present invention provides a transition metal dichalcogenide (TMD) complex, wherein the transition metal dichalcogenide (TMD) is in the form of a nanosheet, a fatty acid is bound to the transition metal dichalcogenide (TMD) nanosheet, and the expression type of a macrophage is determined according to the combination of the transition metal dichalcogenide and the fatty acid.

[0007] In one embodiment of the present invention, the fatty acid is palmitic acid (PA) or linoleic acid (LA).

[0008] In one embodiment of the present invention, the transition metal dichalcogenide (TMD) is any one selected from the group consisting of MoS2, WS2, MoSe2, and WSe2.

[0009] In one embodiment of the present invention, the transition metal dichalcogenide (TMD) complex is a complex in which WSe2 nanosheets are functionalized with palmitic acid (PA) (PA-WSe2) or a complex in which WS2 nanosheets are functionalized with linoleic acid (LA) (LA-WS2).

[0010] In one embodiment of the present invention, the PA-WSe2 promotes polarization of the macrophages to an M1 phenotype, and the LA-WS2 promotes polarization of the macrophages to an M2 phenotype.

[0011] The present invention also provides an immuno-anticancer agent comprising a transition metal dichalcogenide (TMD) complex as an active ingredient, wherein the transition metal dichalcogenide (TMD) is in the form of a nanosheet, a fatty acid is bound to the transition metal dichalcogenide (TMD) nanosheet, and the immuno-anticancer agent is characterized in that it promotes polarization of macrophages into the M1 expression type depending on the combination of the transition metal dichalcogenide and the fatty acid.

[0012] In one embodiment of the present invention, the transition metal dichalcogenide (TMD) complex is PA-WSe2.

[0013] The present invention can selectively determine and manufacture a transition metal dichalcogenide nanosheet immunotherapeutic agent functionalized with a fatty acid through a hydrophobic bond between a fatty acid and a transition metal dichalcogenide, and can bidirectionally control macrophages into M1 or M2 depending on the type of fatty acid and the type of transition metal dichalcogenide used for functionalization. Therefore, such nanosheet immunotherapeutic agent can be utilized as an immuno-oncology agent by inducing polarization of macrophages toward the M1 expression type.

[0014] Figures 1A and 1B are schematic diagrams showing the structural formulas of two types of complexes and fatty acids, respectively, synthesized according to one embodiment of the present invention.

[0015] Figure 2 shows thermodynamic parameters measured by ITC for the interaction between WSe2 (or WS2) nanosheets and PA (or LA), Figure 3 shows TEM and AFM images of the PA-WSe2M1 complex, Figure 4 shows TEM and AFM images of the LA-WS2M2 complex, Figure 5 shows FT-IR spectra, Figure 6 shows W4f XPS spectra, Figure 7 shows C1s XPS spectra of the PA-WSe2 and LA-WS2 nanosheet complexes, and Figure 8 shows the results showing the loading amounts of PA and LA on the PA-WSe2 and LA-WS2 nanosheet complexes.

[0016] Figure 9 shows the results of in vitro macrophage polarization by pure WS2 nanosheets or LA-WS2 complexes, including relative mRNA expression levels of a) CD86, b) CD80, c) CD163, d) CD206, e) iNOS, and f) Arg-1. g) Arg-1 / iNOS expression ratio in LPS-stimulated BM treated with pure WS2 nanosheets or LA-WS2M2 complexes (μM).

[0017] Figure 10 shows the results of in vitro macrophage polarization by pure WSe2 nanosheets or PA-WSe2 complexes, including the relative mRNA expression levels of h) CD86, i) CD80, j) CD163, k) CD206, l) iNOS, and m) Arg-1. n) Arg-1 / iNOS expression ratio in LPS-stimulated BM treated with pure WSe2 nanosheets or PA-WSe2 complexes (μM).

[0018] Figure 11 shows the results of measuring the cell viability of BMDM through CCK-8 analysis after treatment with pure WS2 and LA-WS2 complex functionalized according to the present invention, and Figure 12 shows the results of measuring the cell viability of BMDM through CCK-8 analysis after treatment with pure WSe2 nanosheets or PA-WSe2 complex.

[0019] Figure 13 shows the results of cell viability of RAW 264.7 macrophages measured by CCK-8 assay after treatment with LA-WS2 or PA-WSe2 nanosheet composites at various concentrations for 48 hours.

[0020] Figure 14 shows the expression levels of P-STAT1 and STAT1 in the M1 polarization pathway and P-STAT3 and STAT3 in the M2 polarization pathway from BMDM treated with PA-WSe2 or LA-WS2 nanosheet composites.

[0021] Figure 15 shows a complex (1 mg kg-) of a PA-WSe2 or LA-WS2 nanosheet immune switch according to the present invention. 1 ) shows the photo of the mouse after injection and the size of the tumor.

[0022] Figure 16 shows the results of analyzing tumor size.

[0023] Figure 17 is an H&E stained image of colon tumor sections obtained from xenograft colon cancer mice treated with PA-WSe2 or LA-WS2 complex (1 mg kg-1) or not treated with these complexes.

[0024] Figure 18 shows, from left to right, the relative mRNA expression levels of e) CD86, f) CD80, and g) iNOS in tumor tissues from xenograft colon cancer mice treated or not with PA-WSe2 or LA-WS2 complexes, respectively.

[0025] [Best mode for carrying out the invention]

[0026] Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0027] Before describing the present invention in detail, it should be noted that the terms or words used in this specification should not be interpreted as being unconditionally limited to their usual or dictionary meanings, and the inventor of the present invention may appropriately define and use the concepts of various terms in order to describe his or her invention in the best possible manner.

[0028] Furthermore, it should be noted that these terms and words should be interpreted with meanings and concepts that are consistent with the technical idea of ​​the present invention.

[0029] That is, the terms used in this specification are only used to describe preferred embodiments of the present invention, and are not intended to specifically limit the contents of the present invention.

[0030] It should be noted that these terms are defined taking into account the various possibilities of the present invention.

[0031] Additionally, in this specification, a singular expression may include a plural expression unless the context clearly indicates a different meaning.

[0032] Also, it should be noted that even if similarly expressed in plural, it can contain singular meaning.

[0033] It should be noted that whenever it is stated throughout this specification that a component "includes" another component, it may mean that any other component may be included, rather than excluding any other component, unless specifically stated to the contrary, and that the description of third components or means may be omitted.

[0034] In the drawings attached to this specification, the size, position, connection relationship, etc. of each component constituting the present invention may be described with some exaggeration, reduction, or omission in order to sufficiently clearly convey the idea of ​​the present invention or for convenience of explanation, and therefore the proportions or scales may not be strict.

[0035] In addition, in the following description of the present invention, a detailed description of a configuration that is judged to unnecessarily obscure the gist of the present invention, for example, a known technology including a prior art, may be omitted.

[0036] The present invention is described in more detail below.

[0037] In order to solve the above-described problem, the present invention provides a novel method for determining the expression type of macrophages using a complex composed of transition metal dichalcogenide (hereinafter abbreviated as 'TMD') and fatty acid, and an anticancer agent for immunotherapy capable of killing and suppressing cancer cells therefrom.

[0038] In this specification, TMD is formed by a combination of a transition metal cation (M) and a chalcogen anion (A), and can be represented by the chemical structural formula of MA2. The chalcogen anion (A) is an element corresponding to Group 16 in the periodic table, and may include, for example, sulfur (S), selenium (Se), and tellurium (Te). Such transition metal dichalcogenides (TMD) can be represented by MoS2, WS2, MoSe2, and WSe2.

[0039] Transition metal dichalcogenides (TMDs) have a layered structure similar to graphite, with interlayer spacings of approximately 6–7 angstroms, and van der Waals forces act between the layers. TMD nanosheets are separated by exfoliation from bulk TMDs and consist of a single layer or several layers, with a thickness of only a few nanometers. These TMD nanosheets are distinct from bulk TMDs.

[0040] Meanwhile, fatty acids (fatty acids) are carboxylic acids with long saturated or unsaturated aliphatic chains in chemistry, especially biochemistry, and have unbranched chains consisting of an even number of carbon atoms, from 4 to 28. In particular, the present invention newly discovered that palmitic acid (PA) and linoleic acid (LA) among these fatty acids determine the expression type of macrophages, and can selectively induce anticancer or anti-inflammatory effects therefrom.

[0041] [Form for implementing the invention]

[0042] Figures 1A and 1B are schematic diagrams showing the structural formulas of two types of complexes and fatty acids, respectively, synthesized according to one embodiment of the present invention.

[0043] Referring to Figures 1A and 1B, the two types of complexes synthesized in the present invention are a complex (PA-WSe2) in which WSe2 nanosheets, which are TMDs, are functionalized with palmitic acid (PA), and a complex (LA-WS2) in which WS2 nanosheets, which are TMDs, are functionalized with linoleic acid (LA). In particular, the present invention has confirmed a new fact that PA-WSe2 promotes polarization toward the M1 phenotype, whereas LA-WS2 promotes polarization toward the completely different M2 phenotype, which will be described in more detail through experimental examples below.

[0044] The present invention provides a complex of a transition metal dichalcogenide (TMD) and a fatty acid, wherein the type of polarized macrophage is determined depending on the combination of the transition metal dichalcogenide and the fatty acid, thereby enabling selective production of an M1 immunotherapeutic agent and an M2 immunotherapeutic agent, respectively.

[0045] For example, when M2 is expressed, it can be used as an immune-based anti-inflammatory treatment, and when M1 is expressed, it can be used as an immune-based anti-cancer treatment. The present invention is described in more detail below through preferred examples.

[0046]

[0047] Example

[0048] Exfoliation of WS2 and WSe2 nanosheets in aqueous solution

[0049] A 600 mg portion of bulk WS2 (826.9 mg for WSe2) was added to 20 mL of H2O containing 40 mg of PCL-b-PEG. The mixture was then sonicated in an ice bath for 1 h (pulse-on for 6 s and pulse-off for 2 s), and the resulting solution was centrifuged at 700 xg for 1 h to obtain a supernatant. The obtained supernatant was further centrifuged at 14,500 xg for 1 h to obtain a precipitate, which was then redispersed in 2 mL of water. This centrifugation process at 14,500 xg was repeated twice. After dispersing the precipitate in 2 mL of water, the supernatant was centrifuged at 2,000 xg for 30 min to obtain WS2 or WSe2 nanosheets for further applications.

[0050]

[0051] Synthesis of functionalized nanosheets

[0052] After adjusting the concentration of WS2 or WSe2 nanosheets to 3 mM, 500 μL of LA or PA (750 μM in 95 vol% EtOH / H2O solution) was mixed with 250 μL of the nanosheet solution. The resulting mixture was sonicated in a bath sonar at 25 °C for 90 min and then shaken at 700 rpm for 12 h at 25 °C. The solution was then centrifuged at 14,500 ×g for 45 min to obtain the nanosheet precipitate. A 500 μL portion of 75 vol% EtOH / H2O solution was added to the precipitate, redispersed the nanosheet precipitate, and then centrifuged again at 14,500 ×g for 45 min to remove unbound LA or PA. The precipitate obtained at this time was redispersed in 500 μL of a 50 vol% EtOH / H2O solution and centrifuged under the same conditions to obtain a nanosheet precipitate. Finally, the nanosheet precipitate was redispersed in 500 μL of pure H2O, and the resulting solution was centrifuged at 14,500 ×g for 45 min to obtain a TMD immunoswitch, which is a LA-WS2 or PA-WSe2 nanosheet complex.

[0053]

[0054] RNA extraction and quantitative real-time PCR

[0055] BMDMs were seeded at a density of 1 × 104 cells per well in 6-well plates (SPL, Korea) and cultured in a humidified incubator at 37°C under 5% CO2. After overnight incubation, the old medium was replaced with 2 mL of fresh medium containing 100 μL of PA-WSe2 or LA-WS2 nanosheet immunoswitch (final concentrations: 20, 50, 100, and 200 μM). Then, a 100 μL portion of LPS (final concentration: 100 ng mL-1) was added to the BMDMs 1 h after treatment with the PA-WSe2 or LA-WS2 nanosheet immunoswitch. Total RNA was isolated from BMDMs using TRIzol™ reagent (Invitrogen, USA) 24 h after treatment with the nanosheet immunoswitch. cDNA synthesis was performed using the M-MLV cDNA synthesis kit (Enzynomics, Korea). Quantitative real-time PCR was performed using gene-specific primers and SYBR Green PCR Master Mix (Roche) with QuantStudio™3 (ABI) according to the manufacturer's instructions. Relative expression levels were calculated using the delta-delta Ct method. The following mouse primer sequences were used.

[0056]

[0057] Experimental example

[0058] Figure 2 shows thermodynamic parameters measured by ITC for the interaction between WSe2 (or WS2) nanosheets and PA (or LA), Figure 3 shows TEM and AFM images of the PA-WSe2M1 complex, Figure 4 shows TEM and AFM images of the LA-WS2M2 complex, Figure 5 shows FT-IR spectra, Figure 6 shows W4f XPS spectra, Figure 7 shows C1s XPS spectra of the PA-WSe2 and LA-WS2 nanosheet complexes, and Figure 8 shows the results showing the loading amounts of PA and LA on the PA-WSe2 and LA-WS2 nanosheet complexes.

[0059] Referring to FIGS. 2 to 8, it can be seen that PA and LA fatty acids are effectively bound to and loaded into nanosheets through hydrophobic bonds, and that they still form a sheet shape even after functionalization.

[0060] Figure 9 shows the results of in vitro macrophage polarization by pure WS2 nanosheets or LA-WS2 complexes, including relative mRNA expression levels of a) CD86, b) CD80, c) CD163, d) CD206, e) iNOS, and f) Arg-1. g) Arg-1 / iNOS expression ratio in LPS-stimulated BM treated with pure WS2 nanosheets or LA-WS2M2 complexes (μM).

[0061] Referring to Figure 9, it can be seen that the WS2 complex functionalized with LA decreases CD86 and CD80 expression in a concentration-dependent manner, but conversely increases CD163 and CD206 expression (see Figure 9 a) to d). In addition, referring to Figure 9 e) to g), it can be seen that it decreases iNOS expression, but conversely promotes Arg-1 expression.

[0062] The above results strongly suggest that LA-WS2 of the present invention can be used as an immune-based therapeutic agent by promoting polarization of macrophages to the M2 phenotype.

[0063] In contrast, the PA-functionalized WSe2 complex exhibits an opposite mechanism to WS2, as shown in Fig. 10.

[0064] Figure 10 shows the results of in vitro macrophage polarization by pure WSe2 nanosheets or PA-WSe2 complexes, including the relative mRNA expression levels of h) CD86, i) CD80, j) CD163, k) CD206, l) iNOS, and m) Arg-1. n) Arg-1 / iNOS expression ratio in LPS-stimulated BM treated with pure WSe2 nanosheets or PA-WSe2 complexes (μM).

[0065] The above results strongly experimentally demonstrate that the type of polarized macrophages is determined by the combination of the above transition metal dichalcogenide and fatty acid, and suggest that immuno-oncology agents or immune-based anti-inflammatory agents can be selectively produced depending on the combination.

[0066] Figure 11 shows the results of measuring the cell viability of BMDM through CCK-8 analysis after treatment with pure WS2 and LA-WS2 complex functionalized according to the present invention, and Figure 12 shows the results of measuring the cell viability of BMDM through CCK-8 analysis after treatment with pure WSe2 nanosheets or PA-WSe2 complex.

[0067] Referring to Figures 11 and 12, it can be seen that both the fatty acid-functionalized complex and the pure TMD nanosheets exhibit low cytotoxicity. This indicates that the complex according to the present invention has the potential to be utilized as an in vivo therapeutic agent.

[0068] Figure 13 shows the results of cell viability of RAW 264.7 macrophages measured by CCK-8 assay after treatment with LA-WS2 or PA-WSe2 nanosheet composites at various concentrations for 48 hours.

[0069] Referring to Figure 13, it can be confirmed that the cytotoxicity, especially cytotoxicity against macrophages, is very low, similar to Figures 11 and 12.

[0070] Figure 14 shows the expression levels of P-STAT1 and STAT1 in the M1 polarization pathway and P-STAT3 and STAT3 in the M2 polarization pathway from BMDM treated with PA-WSe2 or LA-WS2 nanosheet composites.

[0071] The results in Figure 14 strongly experimentally demonstrate that PA-WSe2 promotes polarization toward the M1 phenotype, whereas LA-WS2 promotes polarization toward the entirely different M2 phenotype.

[0072] In the present invention, xenograft colon cancer was modeled in mice, and the complex according to the present invention was injected every three days, and the results were analyzed.

[0073] Figure 15 shows a complex (1 mg kg-) of a PA-WSe2 or LA-WS2 nanosheet immune switch according to the present invention. 1 ) shows the photo of the mouse after injection and the size of the tumor.

[0074] Referring to Figure 15, it can be seen that in mice treated with PA-WSe2, the tumor size is significantly reduced.

[0075] Figure 16 shows the results of analyzing tumor size.

[0076] Referring to Figure 16, it can be seen that the tumor size of mice injected with PA-WSe2 decreased in a time-dependent manner. In particular, it can be seen that the tumor size actually increased in mice injected with LA-WS2 compared to mice injected without any substance. This strongly demonstrates the technical concept of the present invention, namely, that the expression type of macrophages is determined by the combination of a transition metal dichalcogenide and a fatty acid, and that anticancer or anti-inflammatory agents can be produced depending on the direction of the crystallization.

[0077] Figure 17 is an H&E stained image of colon tumor sections obtained from xenograft colon cancer mice treated with PA-WSe2 or LA-WS2 complex (1 mg kg-1) or not treated with these complexes.

[0078] Referring to Figure 17, it can be confirmed that tumor tissue is killed when treated with PA-WSe2, an immunotherapy agent according to the present invention.

[0079] Figure 18 shows, from left to right, the relative mRNA expression levels of e) CD86, f) CD80, and g) iNOS in tumor tissues from xenograft colon cancer mice treated or not with PA-WSe2 or LA-WS2 complexes, respectively.

[0080] Referring to Figure 18, it can be seen that when treated with PA-WSe2, CD86, CD80, and iNOS, which exhibit specificity in immunotherapy, are all expressed significantly higher.

[0081] As described above, the present invention can selectively determine and manufacture a transition metal dichalcogenide nanosheet immunotherapeutic agent functionalized with a fatty acid through a hydrophobic bond between a fatty acid and a transition metal dichalcogenide, and can bidirectionally control macrophages into M1 or M2 depending on the type of fatty acid and the type of transition metal dichalcogenide used for functionalization. Therefore, such a nanosheet immunotherapeutic agent can be utilized as an immuno-oncology agent by inducing polarization of macrophages toward the M1 expression type.

[0082] The present invention is recognized as having industrial applicability as an immune-based anticancer treatment.

Claims

1. As a transition metal dichalcogenide (TMD) complex, A transition metal dichalcogenide (TMD) complex characterized in that the above transition metal dichalcogenide (TMD) is in the form of a nanosheet, a fatty acid is bound to the transition metal dichalcogenide (TMD) nanosheet, and the expression type of macrophages is determined according to the combination of the transition metal dichalcogenide and the fatty acid.

2. In paragraph 1, A transition metal dichalcogenide (TMD) complex characterized in that the fatty acid is palmitic acid (PA) or linoleic acid (LA).

3. In paragraph 2, A transition metal dichalcogenide (TMD) complex, characterized in that the above transition metal dichalcogenide (TMD) is any one selected from the group consisting of MoS2, WS2, MoSe2, and WSe2.

4. In paragraph 3, The above transition metal dichalcogenide (TMD) complex is a transition metal dichalcogenide (TMD) complex characterized in that the WSe2 nanosheet is a complex functionalized with palmitic acid (PA) (PA-WSe2) or the WS2 nanosheet is a complex functionalized with linoleic acid (LA) (LA-WS2).

5. In paragraph 4, A transition metal dichalcogenide (TMD) complex characterized in that the above PA-WSe2 promotes polarization of the macrophages to an M1 phenotype, and the above LA-WS2 promotes polarization of the macrophages to an M2 phenotype.

6. An immuno-oncology agent containing a transition metal dichalcogenide (TMD) complex as an active ingredient. The above transition metal dichalcogenide (TMD) is in the form of a nanosheet, and a fatty acid is bound to the above transition metal dichalcogenide (TMD) nanosheet, The above immuno-oncology agent is an immuno-oncology agent characterized in that it promotes polarization of macrophages into the M1 expression type according to the combination of the transition metal dichalcogenide and fatty acid.

7. In paragraph 6, An immuno-oncology agent characterized in that the above transition metal dichalcogenide (TMD) complex is PA-WSe2.

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