Antitumor Compositions, Preparation Methods and Applications

Abstract

This invention discloses an antitumor composition, its preparation method, and its application. The antitumor composition comprises atmospheric pressure cold plasma and a therapeutically effective amount of an MTH1 inhibitor. This invention uses atmospheric pressure cold plasma and an MTH1 inhibitor as a pharmaceutical composition. Through the synergistic effect of atmospheric pressure cold plasma and the MTH1 inhibitor, it significantly amplifies DNA damage in cancer cells, blocks cancer cell repair pathways, and strongly induces tumor cell apoptosis. This overcomes the limitation of poor penetration of directly using atmospheric pressure cold plasma, enabling deep tumor tissue treatment.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, and in particular to an antitumor composition, its preparation method, and its application. Background Technology

[0002] Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature, rich in various reactive oxygen species (RONS), common RONS including H2O2, O3, and O2. - ·OH, ¹O2, NO, NO2 NO3 ONOO It can induce tumor cell death. However, it has two limitations in the field of tumor treatment. On the one hand, its anti-tumor therapeutic effect when used alone is moderate. On the other hand, the tissue penetration ability of CAP when used directly is poor, which limits its application in deeper organs or tissues and makes it impossible to achieve continuous and precise intratumoral delivery.

[0003] Therefore, there is an urgent need to develop an anti-tumor composition that can strongly induce tumor cell death and can be continuously and precisely delivered to achieve deep tissue therapy. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present invention proposes an anti-tumor composition that, through the synergistic effect of atmospheric pressure cold plasma and MTH1 inhibitor, significantly amplifies DNA damage in cancer cells, blocks cancer cell repair pathways, strongly induces tumor cell apoptosis, overcomes the limitation of poor penetration of CAP, and achieves deep tumor tissue treatment.

[0005] A second aspect of the present invention also provides a method for preparing an antitumor composition.

[0006] A third aspect of the present invention also provides a gel.

[0007] A fourth aspect of the present invention also provides the use of an antitumor composition.

[0008] According to a first aspect of the present invention, an antitumor composition is provided, comprising atmospheric pressure cold plasma and a therapeutically effective amount of an MTH1 inhibitor.

[0009] According to a preferred embodiment of the present invention, the antitumor composition comprises a therapeutically effective amount of plasma-activated liquid formed by atmospheric pressure cold plasma and a therapeutically effective amount of MTH1 inhibitor.

[0010] According to a preferred embodiment of the present invention, the antitumor composition further includes a delivery carrier.

[0011] According to a preferred embodiment of the present invention, the delivery carrier includes at least one of sodium alginate, chitosan, collagen, gelatin, hyaluronic acid, silk fibroin, dextran, polylactic acid, polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer, poly(N-isopropylacrylamide), and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer.

[0012] According to a preferred embodiment of the present invention, the concentration of the therapeutically effective MTH1 inhibitor is 0.01~30 mg / kg.

[0013] According to a preferred embodiment of the present invention, the MTH1 inhibitor includes at least one of the following: MTH1 inhibitor TH588, MTH1 inhibitor TH287, MTH1 inhibitor TH1579, or (S)-clotinib.

[0014] According to a preferred embodiment of the present invention, the plasma activation liquid is prepared by the following steps: The aqueous solution was treated in a plasma device under working gas for 30 s to 1200 s to obtain the plasma activation solution.

[0015] According to a preferred embodiment of the present invention, the working gas includes at least one of helium, argon, nitrogen, oxygen, or air.

[0016] According to a preferred embodiment of the present invention, the operating voltage of the plasma device is 4~30 kV.

[0017] According to a preferred embodiment of the present invention, the frequency of the plasma device is 5~25 kHz.

[0018] The antitumor composition according to embodiments of the present invention has at least the following beneficial effects: This invention uses atmospheric pressure cold plasma and a therapeutically effective dose of an MTH1 inhibitor as an anti-tumor composition. Through the synergistic effect of atmospheric pressure cold plasma and the MTH1 inhibitor, it significantly amplifies DNA damage in cancer cells, blocks cancer cell repair pathways, and strongly induces tumor cell apoptosis. It overcomes the limitation of poor penetration of directly using atmospheric pressure cold plasma, achieving deep tumor tissue treatment.

[0019] Furthermore, the mechanism of action of the present invention is as follows: RONS (H2O2, NO2) generated by atmospheric pressure cold plasma. - NO3 -MTH1 inhibitors induce DNA oxidative damage, and MTH1 inhibitors prevent the clearance of 8-oxo-dGTP, enhancing mutation accumulation. The two work synergistically to lead to a rapid accumulation of DNA damage (including nuclear DNA and mitochondrial DNA). The damage activates the p53 signaling pathway, upregulating Bax and downregulating Bcl-2; it also activates Caspase-9 / 3, inducing tumor cell apoptosis; the mitochondrial membrane potential decreases, cell cycle arrest occurs, and ultimately, cell death occurs.

[0020] According to a second aspect of the present invention, a method for preparing an antitumor composition as described in the first aspect of the present invention is provided, comprising the following steps: The solution is obtained by mixing atmospheric pressure cold plasma activation liquid and MTH1 inhibitor.

[0021] According to a preferred embodiment of the invention, the mixing further includes a delivery carrier.

[0022] A third aspect of the present invention provides a gel comprising the antitumor composition described in the first aspect of the present invention. A fourth aspect of the present invention provides the use of the antitumor composition described in the first aspect of the present invention or the gel described in the third aspect of the present invention in the preparation of a therapeutic antitumor drug.

[0023] According to a preferred embodiment of the present invention, the tumor includes at least one of melanoma, breast cancer, skin cancer, liver cancer, colorectal cancer, oral cancer, pancreatic cancer, lung cancer, or glioma.

[0024] Definitions and general terms The "therapeutic effective amount" as described in this invention includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0025] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram illustrating the principle of the antitumor composition according to an embodiment of the present invention; Figure 2The cytotoxicity of CAP treatment time in (A) B16F10 cells, (D) 4T1 cells, and (G) 3T3 cells; and the cytotoxicity of TH588 concentration in (B) B16F10 cells, (E) 4T1 cells, and (H) 3T3 cells. n=4. (C) Bliss synergistic heatmap of CAP and TH588 in B16F10 cells and (F) 4T1 cells. Color gradients represent Bliss score (δE) values ​​(red: synergistic effect; blue: antagonistic effect; δE>0). n=4. (I) ΔE for fixed ratio combinations (B16F10: 30s, 4T1: 45s, TH588: 86 μM and 59.13 μM). n=5.

[0027] Figure 3 The MTH1 inhibitor TH588 synergistically enhances the in vitro anticancer efficacy of CAP. Intracellular (A) ROS and (B) RNS levels in B16F10 cells after treatment with MTH1 inhibitor TH588 and CAP. n = 3. Cytotoxicity of MTH1 inhibitor TH588 and CAP on (C) B16F10 cells and (D) 4T1 cells. n = 5. Effect of MTH1 inhibitor TH588 and CAP on mitochondrial membrane potential in B16F10 cells and (F) 4T1 cells. n = 5. Live / dead cell staining of (G) B16F10 cells and (H) 4T1 cells using Calcein-AM (live cells, green) and PI (dead cells, red). Scale bar, 100 μm. (I) Flow cytometry analysis of apoptosis in untreated or (J) MitoQ-pretreated B16F10 cells subsequently exposed to control, CAP, TH588, or CAP+TH588 combination. Cells were co-stained with annexin V-FITC (an apoptosis marker) and PI. Data are expressed as mean ± standard deviation. Differences between groups were analyzed using one-way ANOVA combined with Tukey's post-hoc test. Significance levels were set at: *P<0.05; **P<0.01; ***P<0.001.

[0028] Figure 4 This image shows the colocalization of 8-oxo-dG with mitochondria in B16F10 and 4T1 cells after treatment with control, CAP, TH588, or CAP+TH588. Cells were co-stained with MitoTracker Red (mitochondria), avidin-Alexa Fluor 488 (green), and DAPI (nucleus). Scale bar: 10 µm.

[0029] Figure 5This is a Western blot analysis of apoptosis-related proteins (p53, cleaved caspase 3, caspase 3, BAX, Bcl-2, p21) in B16F10 cells and 4T1 cells after treatment with control, CAP, TH588, or CAP+TH588.

[0030] Figure 6 B16F10 cells and 4T1 cells were treated with control, CAP, TH588, or CAP+TH588. p53 , caspase 3 , caspase 8 , caspase 9 , BAX , Bcl-2 and p21 qRT-PCR quantification of mRNA levels.

[0031] Figure 7 This represents the biodistribution of CAP solution or CAP hydrogel in B16F10 tumor-bearing mice (n = 6).

[0032] Figure 8 (A) Timeline of treatment experiments in B16F10 tumor-bearing mice (n = 14). (B) Tumor growth kinetics at which the tumor tissue in mice reached the ethical endpoint (tumor volume > 1500 mm³). (C) Body weight changes, (D) Tumor images (n = 5), (E) Tumor weight (n = 6), (F) Kaplan-Meier survival curves (n = 8) of B16F10 tumor-bearing mice.

[0033] Figure 9 (A) H&E and (B) TUNEL staining of tumor tissue sections excised from mice after treatment. Scale bar, 5 mm, 50 μm. (C) H&E staining histopathology of major organs (heart, kidney, liver, lung, spleen) in B16F10 tumor-bearing mice after treatment. Scale bar, 50 μm.

[0034] Figure 10 This is a Western blot analysis of apoptosis-related proteins (p53, cleaved caspase 3, caspase 3, BAX, Bcl-2, p21) in B16F10 tumors after treatment.

[0035] Figure 11(A) is a volcano plot of differentially expressed genes (DEGs) between the antitumor composition and control tumors of the present invention (|log2FC|>0, p<0.05). (B) Hierarchical clustering heatmap of significant DEGs between different treatment groups. (C) Gene Ontology functional enrichment analysis (GO) of DEGs associated with cell death pathways and (D) KEGG pathway enrichment analysis (by -log2FC). 10 (Top 20 terms ranked by p-value). Tumors were collected at the ethical endpoint. n = 3.

[0036] Figure 12 Apoptosis-related genes in tumor tissue ( p53 , p21 , Bax , Bcl-2 , Caspase 3 , Caspase 8 , Caspase 9 RT-qPCR validation (n = 4).

[0037] Figure 13 This is a diagram illustrating the mechanism by which the antitumor composition of this invention induces apoptosis in cancer cells. Detailed Implementation

[0038] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0039] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0040] In some embodiments of the present invention, an antitumor composition is provided, comprising atmospheric pressure cold plasma and a therapeutically effective amount of an MTH1 inhibitor.

[0041] It is understood that the atmospheric pressure cold plasma (CAP) of this invention is an ionized gas that generates reactive oxygen species (RONS). This invention uses atmospheric pressure cold plasma and a therapeutically effective amount of an MTH1 inhibitor as an antitumor composition. Through the synergistic effect of atmospheric pressure cold plasma and the MTH1 inhibitor, it significantly amplifies DNA damage in cancer cells, blocks cancer cell repair pathways, and strongly induces tumor cell apoptosis. This overcomes the limitation of poor penetration of directly using atmospheric pressure cold plasma, enabling deep tumor tissue treatment.

[0042] Furthermore, the mechanism of action of the present invention is as follows: RONS (egH2O2, NO2) generated by atmospheric pressure cold plasma. - NO3 -MTH1 inhibitors induce DNA oxidative damage, prevent the clearance of 8-oxo-dGTP, and enhance mutation accumulation. The two work together to cause a rapid accumulation of DNA damage (including nuclear DNA and mitochondrial DNA). The damage activates the p53 signaling pathway, upregulates Bax and downregulates Bcl-2; activates Caspase-9 / 3, and enhances the induction of tumor cell apoptosis; decreases mitochondrial membrane potential, arrests the cell cycle, and ultimately leads to death.

[0043] In some embodiments of the present invention, an antitumor composition is provided, the antitumor composition comprising a therapeutically effective amount of plasma activating fluid formed by atmospheric pressure cold plasma and a therapeutically effective amount of MTH1 inhibitor.

[0044] In some embodiments of the present invention, the antitumor composition further includes a delivery carrier.

[0045] In some embodiments of the present invention, sodium alginate, chitosan, collagen, gelatin, hyaluronic acid, silk fibroin, dextran, polylactic acid, polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer, poly(N-isopropylacrylamide), and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer (e.g., Pluronic F-127) are used. Thus, the delivery carrier of the present invention can achieve local retention and sustained-release properties, control release behavior, form local sustained-release capability, and achieve continuous and precise intratumoral delivery.

[0046] Furthermore, when the delivery carrier is sodium alginate, the precursor liquid obtained by mixing sodium alginate, MTH1 inhibitor, and plasma activation solution is injected into the tumor tissue and placed in a physiological ionic environment (Ca²⁺). + / Mg² + It rapidly forms a hydrogel. Therefore, the antitumor composition of the present invention has sustained-release capability, enabling continuous and precise intratumoral delivery.

[0047] In some embodiments of the present invention, the concentration of sodium alginate in the antitumor composition is 5-30 mg / mL. For example, it includes 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 25 mg / mL, 28 mg / mL, 30 mg / mL, or any sub-range of two of the above values. Therefore, when the concentration of sodium alginate is within the above range, the antitumor composition forms a gel more rapidly in vivo.

[0048] In some embodiments of the present invention, the concentration of the therapeutically effective MTH1 inhibitor is 0.01 to 30 mg / kg, for example including 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.08 mg / kg, 0.1 mg / kg, 0.12 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, or a subrange consisting of any two of the above values.

[0049] In some embodiments of the present invention, the MTH1 inhibitor includes at least one of the following: MTH1 inhibitor TH588, MTH1 inhibitor TH287, MTH1 inhibitor TH1579, or (S)-clotinib.

[0050] In some embodiments of the present invention, the plasma activation liquid is prepared by the following steps: The aqueous solution was treated in a plasma device under working gas for 30 s to 1200 s to obtain the plasma activation solution.

[0051] In some embodiments of the present invention, the processing time under the working gas is, for example, 30 s, 40 s, 50 s, 60 s, 70 s, 80 s, 90 s, 100 s, 200 s, 300 s, 400 s, 500 s, 600 s, 700 s, 800 s, 900 s, 1000 s, 1100 s, 1200 s, or a subrange consisting of any two of the above values.

[0052] In some embodiments of the present invention, the working gas includes at least one of helium, argon, nitrogen, oxygen, or air.

[0053] In some embodiments of the present invention, the operating voltage of the plasma device is 4 to 30 kV. For example, 4 kV, 5 kV, 8 kV, 10 kV, 15 kV, 20 kV, 22 kV, 25 kV, 28 kV, 30 kV, or any sub-range of any two of the above values.

[0054] In some embodiments of the present invention, the frequency of the plasma device is 5 to 25 kHz. For example, it includes 5 kHz, 8 kHz, 10 kHz, 12 kHz, 15 kHz, 18 kHz, 20 kHz, 22 kHz, 25 kHz, or any subrange consisting of two of the above values.

[0055] In some embodiments of the present invention, the aqueous solution includes at least one of deionized water, physiological saline, phosphate buffer, Ringer's solution, Hanks' solution, and 5% glucose solution.

[0056] In some embodiments of the present invention, a method for preparing an antitumor composition as described in the first aspect of the present invention is provided, comprising the following steps: The solution is obtained by mixing atmospheric pressure cold plasma with MTH1 inhibitor.

[0057] In some embodiments of the invention, the mixing further includes a delivery carrier.

[0058] In some embodiments of the present invention, the antitumor composition described in the first aspect of the present invention is included. Thus, the gel possesses all the effects of the antitumor composition described in the first aspect of the present invention. Furthermore, the gel of the present invention also has sustained-release capability, enabling continuous and precise intratumoral delivery.

[0059] In some embodiments of the present invention, the use of the antitumor composition described in the first aspect of the present invention in the preparation of a therapeutic antitumor drug is provided.

[0060] In some embodiments of the present invention, the tumor includes at least one of melanoma, breast cancer, skin cancer, liver cancer, colorectal cancer, oral cancer, pancreatic cancer, lung cancer, or glioma.

[0061] The raw materials used in the embodiments of this invention are as follows: Unless otherwise specified, all reagents were purchased from Sigma-Aldrich, Thermo Fisher Scientific, Gibco, or Beyotime. Deionized water was prepared using Milli-Q direct 8 (Millipore) with a resistivity greater than 18.2 MΩ·cm. Mouse melanoma cells (B16F10, RRID: CVCL_0159) and mouse breast cancer cells (4T1, RRID: CVCL_0125) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, respectively. The cell culture temperature was maintained at 37°C in the presence of 5% CO2. Mycoplasma, bacterial, and fungal tests were negative. STR analysis confirmed the cells were of sole mouse origin, with no evidence of cross-contamination with human cells. Healthy male C57BL / 6 mice aged 6–8 weeks were purchased from Guangdong Yaokang Biotechnology Co., Ltd. All mouse-related studies were approved by the Animal Experiment Ethics Committee of the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (SIAT-IACUC-240705-YGS-CZTA2839).

[0062] 1. Configuration and characterization of the CAP device The CAP device (such as a dielectric barrier discharge (DBD) device) was designed and assembled in the laboratory. Its operating principle involves ceramic electrodes connected to a high-voltage transformer, with air as the working gas. The discharge voltage was measured using a Tektronix TDS2024C oscilloscope with a Tronovo TR9340A high-voltage probe. The generated ROS and RNS in the plasma were detected using a fiber-coupled spectrometer (Brolight BIM-6602A) located 10 mm from the center of the ceramic electrode, with an integration time of 10,000 ms. The discharge process was recorded by an ICCD camera (TRC411), directly triggered by a pulse signal from a high-voltage AC power supply. Plasma was generated in ambient air using a 10 kV, 10 kHz AC power supply (period approximately 100 μs).

[0063] 2. Western blot analysis: B16F10 and 4T1 cells were treated with CAP, TH588, or the antitumor composition of the present invention, respectively. Samples were separated by SDS-polyacrylamide gel electrophoresis on a 10% gel at 200V for 50 minutes. Proteins from the SDS-PAGE gel were transferred to a PVDF membrane using Trans-Blot® Turbo™ for 9 minutes. The membrane was blocked with TBST and EveryBlotBlocking Buffer (Bio-Rad) at room temperature for 10 minutes. The membrane was incubated with antibodies against p53, p21, BAX, Bcl-2, cleaved caspase-3, caspase-3, and β-actin (Abcam) at 4 °C for 12 hours, followed by incubation at room temperature with the peroxidase-conjugated secondary antibody goat anti-rabbit IgG H&L (HRP) (Abcam). Band intensities were analyzed and quantified using GelView9000 Lite (BLT) and ImageJ software. The expression levels of other proteins were determined by calculating their relative intensity to β-actin.

[0064] 3. In vitro apoptosis treatment Apoptosis was detected using the annexin V–FITC / PI apoptosis detection kit. The distribution of apoptotic cells was determined according to the manufacturer's protocol. B16F10 and 4T1 cells were cultured at 2.0 × 10⁻⁶ cells / year. 5 Cells / well were seeded in 6-well plates for 24 hours, then the medium was replaced with fresh sample-containing medium (1 mL). Cells were analyzed using CytExpert (Beckman), and data were analyzed and processed using FlowJo. In another group, cells were pretreated with or without the mitochondrial-targeting antioxidant MitoQ (0.2 M). The procedure was the same as described above.

[0065] 4. RT-qPCR analysis B16F10 and 4T1 cells were collected 24 hours after treatment, and tumors were collected when mice reached the treatment endpoint. Collected samples were treated on ice and placed in tubes containing lysis buffer and zirconium oxide beads, then ground at 70 Hz in a cryogenic grinder at -50 °C (Servicebio). Total RNA was extracted using the AxyPrep™ Multisource Total RNA Miniprep Kit (Axygen) and quantified using a DS-11FX (DeNovix). cDNA was reverse transcribed from total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Detection was performed using qTOWER3G (Analytikjena) and WitEnzy 2× SYBR Green qPCR Master Mix (Dakewe). β-actin , p53 , p21 , Bcl-2 , Bax , Caspase 3 , Caspase 9 and Caspase 8 The level.

[0066] Statistical analysis All results are expressed as mean ± standard deviation. One-way ANOVA was used for multiple comparisons. The log-rank test was used to determine survival rates. All statistical analyses were performed using the Prism software package. The threshold for statistical significance was P < 0.05.

[0067] The following are some abbreviations of this invention: CAP (Atmospheric Pressure Cold Plasma), PAM (Plasma Activation Fluid), RONS (Reactive Oxygen Nitrogen Substances), Alg (Sodium Alginate), MTH1 (MutT Homolog 1), TH588 (MTH1 Inhibitor), 8-oxo-dG (8-oxo-2'-deoxyguanosine), 8-oxo-dGTP (8-oxo-dG Triphosphate), nDNA (Nuclear DNA), and mtDNA (Mitochondrial DNA).

[0068] Example 1 This example first prepares an antitumor composition for cell experiments, including a plasma activation solution formed by atmospheric pressure cold plasma, the MTH1 inhibitor TH588, and sodium alginate. The specific steps are as follows: S1. Preparation of plasma activation solution: Using a dielectric barrier discharge (DBD) device, 2 mL of deionized water in a 6-well plate was placed 10 mm below the ceramic, and the output voltage of the DBD was 10 kV and the frequency was 10 kHz, with a processing time of 30 s.

[0069] S2. Dissolve sodium alginate and MTH1 inhibitor TH588 in plasma activation solution to obtain an antitumor composition, wherein the concentration of sodium alginate is 15 mg / mL and the concentration of inhibitor TH588 is 16 µM.

[0070] The levels of ROS and RNS in the plasma activation solution in step S1 were detected using a hydrogen peroxide detection kit (Beyotime S0038) and a nitrite detection kit (Beyotime S0021M), respectively. The results showed that ROS was 100 µM and RNS was 220 µM.

[0071] Example 2 This example describes the preparation of an antitumor composition for animal experiments, comprising a plasma-activated solution formed by atmospheric pressure cold plasma, the MTH1 inhibitor TH588, and sodium alginate. The specific steps are as follows: S1. Preparation of plasma activation solution: Using a plasma jet (APPJ) device, 1.5 mL of deionized water in a 24-well plate was placed 35 mm below the jet tube. The output voltage of the APPJ was 17 kV and the frequency was 8 kHz. The processing time was 10 min. S2. Dissolve sodium alginate and MTH1 inhibitor TH588 in plasma activation solution to obtain an antitumor composition, wherein the concentration of sodium alginate is 15 mg / mL and the concentration of inhibitor TH588 is 2 mg / kg.

[0072] The levels of ROS and RNS in the plasma activation solution in step S1 were detected using a hydrogen peroxide detection kit (Beyotime S0038) and a nitrite detection kit (Beyotime S0021M), respectively. The results showed that ROS was 1000 μM and RNS was 200 μM.

[0073] Example 3 This study investigated the effects of different treatment times and concentrations of the MTH1 inhibitor TH588 on the cytotoxicity of B16F10, 4T1, and 3T3 cells using the CCK-8 assay. The results are as follows: Figure 2 As shown, where, Figure 2 In this context, A, D, and G represent the cytotoxicity of CAP treatment at different times in B16F10, 4T1, and 3T3 cells, respectively. Figure 2 In this context, B, E, and H represent the cytotoxicity of TH588 concentrations in B16F10, 4T1, and 3T3 cells, respectively.

[0074] The results showed that the CAP of the present invention exhibited time-dependent cytotoxicity, while TH588 exhibited a concentration-dependent effect. CAP showed less cytotoxicity to normal cells (such as 3T3 fibroblasts), indicating that the higher oxidative stress levels in tumor cells made them more likely to reach the lethal threshold, and that CAP had a highly selective killing effect on cancer cells.

[0075] Furthermore, the synergistic effect between CAP and TH588 was quantitatively assessed using the Bliss independence model, with the Bliss score (ΔE) calculated as ΔE = E AB -(E A + E B -E A ×E B ), where E AB E A and E B The values ​​represent the inhibition rates of combination therapy and monotherapy, respectively, revealing a time-dependent synergistic effect. The results are as follows: Figure 2 As shown in C, F, and I, when the CAP exposure time exceeds 30 seconds and is associated with IC 50 When TH588 is used in combination at different concentrations, the ΔE value becomes significantly positive (ΔE>0), demonstrating a clear synergistic activity between CAP and TH588.

[0076] Example 4 This study investigated intracellular ROS and RNS levels and cell viability in vitro. Tumor cells (including B16F10 and 4T1 cells) were seeded in 6-well plates and cultured for 24 hours. Cells were then co-incubated for 24 hours with different treatment groups (control group, CAP group, TH588 group, and CAP + TH588 group; for B16F10 cells, the CAP treatment time was 30 s and the TH588 concentration was 64 μM; for 4T1 cells, the CAP treatment time was 45 s and the TH588 concentration was 59.13 μM). The cells were then stained for 1 hour with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (a fluorescent probe for intracellular ROS) (Beyotime S0033M) or 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FMDA, a fluorescent probe for intracellular RNS) (Beyotime S0020M). Finally, the cells were analyzed using flow cytometry (Beckman).

[0077] The results of intracellular ROS and RNS levels in B16F10 cells are as follows: Figure 3 A and Figure 3 As shown in B, the combined use of the plasma activation solution of the present invention and the MTH1 inhibitor TH588 significantly increased the ROS / RNS level.

[0078] Example 5 Cell viability was measured using mouse melanoma cells (B16F10) and mouse breast cancer cells (4T1) via the CCK-8 assay. Each group of cells was sputtered at 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL per well in 96-well cell culture plates containing DMEM. After incubation for 24 hours, the medium was replaced with fresh sample medium. CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 2 hours. The absorbance at 450 nm was measured using an Infinite 200 Pro (Tecan). B16F10 and 4T1 cells were seeded at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells per well in 6-well plates and incubated for 24 hours. The culture medium was then replaced with fresh sample medium (1 mL) and four treatment groups were added (control group, CAP group, TH588 group, and CAP+TH588 group).

[0079] After 24 hours of incubation, the cytotoxicity of the treated cells is shown in the figure. Figure 3 C and Figure 3 As shown in Figure D, compared with CAP alone or TH588 alone, the combined use of the plasma activation solution of the present invention and the MTH1 inhibitor TH588 significantly reduced cell viability. In summary, the accumulation of ROS and RNS drives tumor cell death.

[0080] Furthermore, the mitochondrial membrane potential of cells in vitro was measured, as follows: B16F10 and 4T1 cells were used at 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated for 24 hours. The culture medium was then replaced with fresh medium (1 mL) for four groups (control group, CAP group, TH588 group, and CAP+TH588 group). After 24 hours of incubation, the cells were washed and stained with Rhodamine 123 working solution (C2008S, Beyotime) in the dark at 37°C for 30 minutes. After removing the staining solution and washing, the fluorescence intensity was detected using a microplate reader at an excitation wavelength of 507 nm and an emission wavelength of 529 nm. The results are as follows: Figure 3 As shown in E and F, CAP reduces mitochondrial membrane potential, and CAP+TH588 exhibits a cumulative effect on the reduction of cell viability.

[0081] Furthermore, after 24 hours of incubation, the cells were washed twice with PBS and co-stained with Calcein-AM / PI reagent, then observed using a confocal laser scanning microscope (CLSM). The results are as follows: Figure 3As shown in G and H, Calcein-AM (green) labels live cells and PI (red) labels dead cells, indicating that cells treated with CAP+TH588 have significant cytotoxicity.

[0082] Furthermore, flow cytometry using Annexin V / PI co-staining yielded the following results: Figure 3 As shown in Figure I, cell apoptosis was confirmed to be increased in the CAP, TH588, and combined groups, with over 50% of cells undergoing early or late apoptosis after CAP+TH588 treatment. These results indicate that the MTH1 inhibitor enhances the anticancer efficacy of CAP.

[0083] Furthermore, pretreatment of B16F10 cells with the mitochondrial antioxidant MitoQ yielded the following results: Figure 3 As shown in Figure J, apoptosis was reduced during combination therapy. This indicates that mitochondrial DNA (mtDNA) mutations contributed to CAP+TH588-induced mitochondrial apoptosis. The survival of tumor cells after MitoQ treatment suggests that CAP-induced oxidative damage is necessary for TH588-mediated MTH1 inhibition. CAP also impairs DNA repair, disrupting the ROS defenses of tumor cells through MTH1 inhibitors. Therefore, MTH1 inhibition enhances CAP-induced cancer cell death.

[0084] Example 6 This example further explores the mechanism of oxidative DNA damage amplification, as detailed below: To elucidate the mechanism of oxidative DNA damage amplification, 8-oxo-dG in DNA was imaged using Alexa Fluor 488-conjugated avidin, leveraging the high binding affinity of avidin for 8-oxo-dG. Cells were incubated at 37 °C with MitoTracker Red CMXRos (200 nM) for 30 min, followed by Calcein-AM / PI staining using the same protocol. Cells were fixed in 4% paraformaldehyde at room temperature for 30 min, washed twice with TBST (tris-buffered saline containing 0.1% Triton X-100). Blocking was performed for 2 h at room temperature using 15% FBS solution containing TBST. Cells were then incubated at 37 °C for 1 h in blocking solution containing Alexa Fluor 488-conjugated avidin (10 µg / mL), followed by washing twice with TBST for 5 min each time, blocking with a mounting medium (containing DAPI), and imaging was performed using a super-resolution laser confocal scanning microscope (AXR-NSPARC, Nikon). Mutants in the cell nucleus and mitochondria were estimated by calculating the green fluorescence intensity from the overlapping green / blue and green / red regions, respectively.

[0085] An imaging diagram of B16F10 cells is shown below. Figure 4 As shown in Figure A, the imaging schematic of 4T1 cells is as follows: Figure 4 As shown in D, immunofluorescence reveals that the antitumor composition of the present invention significantly increases the accumulation of the oxidative damage marker 8-oxo-dG in the cell nucleus and mitochondrial DNA.

[0086] Furthermore, Western blot analysis was performed, and the results are as follows: Figure 5 As shown in Figures A and B, Western blot analysis of apoptosis-related proteins revealed the anti-proliferative mechanism of the CAP-based combination therapy. p53, a key apoptosis-inducing factor following DNA damage, was significantly upregulated in all treatment groups, with the highest expression in the combination therapy group. This was accompanied by an increase in the level of the apoptosis-executing protease cleaved caspase 3, while CAP and TH588 significantly reduced p21 expression (10.63-fold). The p21 level in the combination group was lower than that in the single treatment group, indicating that although p53 was strongly activated, the inhibition of this anti-apoptotic factor mediated by CAP promoted cell death. Mitochondrial apoptosis pathway analysis revealed differential regulation of Bcl-2 family proteins, with the combination therapy producing a significant pro-apoptotic shift. The altered BAX / Bcl-2 ratio indicated exacerbated mitochondrial damage, with CAP particularly maximizing the downregulation of Bcl-2 and the upregulation of BAX. These changes confirm that TH588, combined with CAP, synergistically inhibits proliferation by enhancing apoptosis execution.

[0087] Furthermore, PCR analysis was performed on B16F10 and 4T1 cells, and the results are as follows: Figure 6 As shown in A and B, quantitative PCR analysis revealed that... p53 mRNA expression was significantly upregulated (increased 2.82-fold), accompanied by downstream pro-apoptotic effector factors. Bax (Increased by 7.52 times) and p21 Transcriptional activation increased by 9.57 times.

[0088] In summary, the mechanism of the antitumor composition of the present invention is as follows: treatment upregulates p53 and Bax, downregulates Bcl-2, resulting in a sharp increase in the Bax / Bcl-2 ratio, and activates Caspase-9 and Caspase-3, inducing apoptosis in the mitochondrial pathway.

[0089] Example 7 The biodistribution of CAP (the antitumor composition of Example 2) in vivo was investigated. The metabolism of CAP solution and gel in vivo was visualized using an in vivo fluorescence imaging system (IVIS), with the near-infrared fluorescent dye Cy7 covalently labeled into the solution. 100 μL of CAP solution (left back) and CAP@Alg solution (Example) (right back) were injected intratumorally into B16F10 mice. Fluorescence images of the mice were captured spectrally using the imaging system to assess in situ drug retention. Tumors, liver, spleen, kidney, lung, and heart were extracted and imaged using IVIS (Ex=720 nm, Em=790 nm) at 48 hours post-injection (n=6).

[0090] Its image is as follows Figure 7 As shown, the antitumor composition of this invention can remain at the tumor site for more than 48 hours, while the free solution group is diffused within 24 hours.

[0091] Example 8 This study investigated the antitumor effect in an in vivo mouse model, using 1×10⁻⁶ mice. 6 B16F10 cells were seeded into the right side of the animal's back, resulting in a tumor size of approximately 50-100 mm. 3 Mice were divided into 4 groups (n = 14). Every 5 days, the tumor-bearing mice were treated with PBS gels (control group), CAP gels, TH588 gels, and CAP+TH588 gels (the antitumor composition of Example 2), for a total of 4 injections. Every 2 days, the mice's weight and tumor size were recorded, and the tumor volume was measured using calipers. The formula was: width = 2 Calculated as length × 0.5. When the tumor size exceeds ~1500 mm. 3 Mice were euthanized upon reaching the treatment endpoint. After euthanasia, major organs, including the heart, liver, spleen, lungs, and kidneys, were harvested for preparing paraffin sections for H&E staining. Tumors were collected for paraffin sectioning and H&E and TUNEL staining assays.

[0092] in, Figure 8 A is the experimental timeline graph. Figure 8 B is the tumor growth curve. Figure 8 C is a graph showing changes in body weight. Figure 8 D is a photograph of the tumor. Figure 8 E represents the tumor weight graph. Figure 8 F represents the survival curves. These graphs show that in the B16F10 melanoma mouse model, the tumor volume inhibition rate of the CAP+TH588@Alg gels treatment group reached 54%, intratumoral administration reduced tumor weight by 67.44±0.27%, and significantly prolonged the survival of mice.

[0093] Furthermore, after euthanasia, major organs, including the heart, liver, spleen, lungs, and kidneys, were harvested for preparing paraffin sections for H&E staining. The staining results are as follows: Figure 9 As shown in A and 9B, hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining revealed large areas of apoptosis in the tumor tissue of the combination therapy group, with TUNEL staining showing significant apoptosis; this confirmed excellent tumor targeting, significant tumor suppression effect, induction of tumor cell apoptosis, and good biocompatibility.

[0094] Furthermore, Figure 9 The "C" in the figure represents the H&E staining of the major organs. As can be seen from the figure, no obvious toxicity was found in the H&E staining of the major organs.

[0095] Furthermore, Western blot analysis was performed on tumor tissue to evaluate the roles of CAP and TH588 in regulating apoptosis proteins in tumor tissue after combination therapy. The results are as follows: Figure 10 As shown, the combination therapy significantly upregulated p53 protein expression (1.44-fold compared to the control group), which activated the mitochondrial apoptosis pathway. This was demonstrated by increased expression of the pro-apoptotic protein Bax and decreased levels of the anti-apoptotic protein Bcl-2, leading to an increased Bax / Bcl-2 ratio (14.53, compared to 0.25 in the control group). Simultaneously, compared to the control group, the levels of Caspase 3 precursors were decreased (1.64-fold reduction), while Cleaved Caspase 3 was increased (7.16-fold increase), confirming the execution phase of apoptosis.

[0096] Furthermore, tumor tissue from B16F10 mice was collected at the study endpoint for RNA sequencing. RNA library preparation and sequencing were performed by Novogene (https: / / cn.novogene.com / ). Volcano plots of important genes, heatmaps of differentially expressed top genes, and Gene Ontology (GO) functional enrichment analysis were conducted using Novogene's online database. Genes with log2 fold changes > 0 and p < 0.05 were designated as differentially expressed genes (DEG).

[0097] The result is as follows Figure 11 As shown, where Figure 11 In the diagram, A represents the differentially expressed gene volcano plot. Compared with the control group, the combination therapy of CAP+TH588 induced 1436 differentially expressed genes, of which 266 were significantly upregulated and 1170 were significantly downregulated.

[0098] Figure 11The "B" in the diagram represents a differential gene heatmap, revealing the regulation of apoptosis-related genes after treatment. The results showed that in the CAP+TH588 treatment group, pro-apoptotic factors (e.g., PARP1 ) was significantly upregulated, while anti-apoptotic mediators (e.g. NOTCH1 , NFKBIA ) and caspase family regulators (e.g. BCL6 , BCL2L1 Downregulation. DNA repair components (e.g.) PARP1 , PDIA3 ) and markers of oxidative stress (e.g. CAT , GCLM The coordinated regulation of these factors suggests that the dual induction of genomic instability and redox imbalance is a key trigger for apoptosis. Furthermore, cell cycle regulators (e.g., CDC25A , CDKN1C The altered expression of these changes suggests accompanying cell cycle arrest, which may create a permissive state for apoptosis. These transcriptomic changes collectively depict a multimodal mechanism underlying the synergistic pro-apoptotic effect of combined CAP and TH588 therapy.

[0099] Figure 11 The C in the figure represents GO enrichment analysis. The results showed that differentially expressed genes were significantly enriched in biological processes such as oxidative stress response, double-strand break repair and DNA metabolism, cellular components such as the nucleus, endoplasmic reticulum and extracellular matrix, and molecular functions such as enzyme inhibitor activity.

[0100] Figure 11 The D in the figure represents the KEGG pathway enrichment analysis. The results showed that the differentially expressed genes were significantly enriched in the DNA replication (mmu03030), microRNA in cancer (mmu05206), and cell cycle regulation (mmu04110) pathways.

[0101] In summary, as can be seen from these figures, the antitumor compositions of the present invention significantly affect signaling pathways (such as the p53 pathway, cell cycle pathway, etc.) related to DNA damage response, oxidative stress, cell cycle, and apoptosis.

[0102] GO and KEGG enrichment analyses further confirmed that DNA replication, repair processes, and apoptosis pathways were deeply activated.

[0103] Furthermore, qPCR validation was performed on tumor tissue, and the results were as follows: Figure 12 As shown, qPCR analysis of tumor tissue confirmed the coordinated regulation of apoptosis and cell cycle pathways after CAP+TH588 combination therapy. DNA damage triggered by the combination regimen was strongly upregulated. p53 Expression, its transcriptional activation p21 To induce cell cycle arrest and simultaneously increase pro-apoptotic levels Bax And inhibits anti-apoptosis Bcl-2 The resulting Bax / Bcl-2 The ratio increases significantly, driving mitochondrial outer membrane permeability (MOMP) and cytochrome c release, thereby allowing... Caspase 9 and Caspase 3 Cleavage activates the intrinsic apoptotic cascade. Notably, the extrinsic apoptotic pathway synergizes with mitochondrial signaling: Caspase 8 activation mediated by the assembly of the death-inducing signal complex (DISC) or Bid cleavage amplifies the apoptotic cascade. Dynamic regulation of p21 is observed in this process—early cell cycle arrest may provide a window for DNA repair, but sustained apoptotic signaling (via caspase activation) ultimately overrides the p21-mediated survival effect, leading to irreversible cell death (the mechanism is as follows). Figure 13 (As shown).

[0104] In summary, the schematic diagram of the antitumor composition of the present invention is shown below. Figure 1 As shown, where Figure 1 In this context, A represents the preparation of the antitumor composition of the present invention and the intratumoral injection formation process. Figure 1 The "B" in the figure indicates the synergistic mechanism: CAP produces RONS, causing DNA damage, while TH588 inhibits MTH1 repair function, leading to damage accumulation and ultimately triggering apoptosis.

[0105] The present invention has been described in detail above with reference to the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An antitumor composition, characterized in that, This includes atmospheric pressure cold plasma and therapeutically effective doses of MTH1 inhibitors.

2. The antitumor composition according to claim 1, characterized in that, The antitumor composition comprises a therapeutically effective amount of plasma-activated liquid formed by atmospheric pressure cold plasma and a therapeutically effective amount of MTH1 inhibitor.

3. The antitumor composition according to claim 1 or 2, characterized in that, The antitumor composition further includes a delivery carrier; And / or, the delivery carrier includes at least one of sodium alginate, chitosan, collagen, gelatin, hyaluronic acid, silk fibroin, dextran, polylactic acid, polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer, poly(N-isopropylacrylamide), and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer.

4. The antitumor composition according to claim 1 or 2, characterized in that, The MTH1 inhibitor includes at least one of the following: MTH1 inhibitor TH588, MTH1 inhibitor TH287, MTH1 inhibitor TH1579, or (S)-Clotinib.

5. The antitumor composition according to claim 2, characterized in that, The plasma activation solution is prepared through the following steps: The aqueous solution was treated in a plasma device under working gas for 30 s to 1200 s to obtain the plasma activation solution; And / or, the working gas includes at least one of helium, argon, nitrogen, oxygen, or air.

6. A method for preparing the antitumor composition according to any one of claims 1 to 5, characterized in that, Includes the following steps: The solution is obtained by mixing atmospheric pressure cold plasma activation liquid and MTH1 inhibitor.

7. The preparation method according to claim 6, characterized in that, The mixture also includes a delivery carrier.

8. A gel, characterized in that, Includes the antitumor composition according to any one of claims 1 to 5.

9. The use of the antitumor composition according to any one of claims 1 to 5 or the gel according to claim 8 in the preparation of therapeutic and / or preventive antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor includes at least one of melanoma, breast cancer, skin cancer, liver cancer, colorectal cancer, oral cancer, pancreatic cancer, lung cancer, or glioma.

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