A method for preparing polysaccharide-loaded molybdenum nanomaterials and its application

Abstract

This invention discloses a method for preparing polysaccharide-loaded molybdenum nanomaterials and their applications. These polysaccharide-loaded molybdenum nanomaterials exhibit good water dispersibility, stability, photothermal effects, and the ability to consume glutathione, thus reducing the toxic side effects on normal cells and improving anti-tumor efficacy. Under mild in vivo conditions, these polysaccharide-loaded molybdenum nanomaterials can selectively generate sufficient ROS while simultaneously consuming GSH, leading to a severe imbalance of oxidative stress in tumor cells. Combined with photothermal therapy, this achieves the goal of killing tumor cells, providing theoretical and experimental basis for the clinical treatment of tumors and showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a method for preparing polysaccharide-loaded molybdenum nanomaterials and their applications. Background Technology

[0002] Reactive oxygen species (ROS) are cytotoxic byproducts produced during tumor growth and can be used to kill tumor cells. Numerous studies have confirmed that excessive ROS production disrupts the redox homeostasis within tumor cells, leading to oxidative damage to proteins, lipids, and DNA, mitochondrial dysfunction, and ultimately, apoptosis or necrosis. Therefore, stimulating excessive ROS production in tumor cells to disrupt redox balance has the potential to become a novel cancer treatment method. However, how to selectively produce sufficient ROS under mild in vivo conditions remains a significant challenge.

[0003] Nanoparticle-based therapies have been envisioned as effective treatments for cancer, infections, inflammation, and diabetes, as they can disrupt the redox balance in cancer while protecting healthy tissues from the toxicity of conventional chemotherapy. Many reports focus primarily on eliminating reactive oxygen species (ROS), which can damage normal cells. Conversely, disrupting the redox balance by increasing cellular ROS can also be fatal to cancer. Therefore, the rational utilization of oxidative stress plays a crucial role in treating various types of diseases. To date, much research has focused on using nanomaterials to disrupt redox balance. However, most redox-mediated nanomaterials are inorganic (such as SiO2, MgO2, Fe3O4, ZnO2, Au, etc.), and their long-term biosafety and biocompatibility remain unclear. Molybdenum is an essential trace element for the human body and a fundamental component of xanthine oxidase and aldehyde oxidase. Increased molybdenum content will enhance the activity of xanthine oxidase and aldehyde oxidase, thereby causing oxidative stress. To survive and proliferate, cells will utilize more redox substances to combat oxidative stress. Glutathione (GSH), the most abundant thiol-containing substance in cells, can scavenge reactive oxygen species (ROS) and alleviate lipid peroxidation through selenoprotein protease (GPX-4). Thiol and selenool groups in the thioredoxin (TXN) redox system react with ROS, reducing oxidative stress. Therefore, consuming glutathione and increasing ROS may be a potential strategy for cancer treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying polysaccharide-loaded molybdenum nanomaterials. A natural polysaccharide BFP loaded with MoNPs (POM) was designed and synthesized as an anti-tumor nanomedicine, which can significantly induce increased intracellular oxidative stress and GSH consumption, while also exhibiting good photothermal conversion ability.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a method for preparing polysaccharide-loaded molybdenum nanomaterials, comprising the following steps:

[0007] (1) Polysaccharide BFP was extracted from black fungus by hydrothermal extraction. The polysaccharide BFP was dissolved in ultrapure water and stirred to obtain a clear polysaccharide BFP aqueous solution.

[0008] (2) Add phosphomolybdic acid aqueous solution to the polysaccharide BFP aqueous solution obtained in step (1) and stir for 5-10 min. Add tannic acid dropwise and adjust the pH with sodium carbonate. React in the dark for 6-24 h to obtain a clear and transparent solution. After dialyzing through a dialysis bag, freeze dry to obtain POM nanomaterials.

[0009] Further, the specific steps of the hydrothermal extraction method in step (1) are as follows: weigh 250g of dried black fungus, crush it by a wall-breaking machine, remove the oil by 4h of ethyl acetate and acetone circulation distillation and dry it, then soak and stir the dried black fungus in 70% ethanol aqueous solution for 24h, remove the solvent and stir in 0.9% sodium chloride aqueous solution at 85℃ for 4h to break the wall, then continue to stir at room temperature for 24h, centrifuge and take the supernatant, the supernatant is continuously decolorized by 30% hydrogen peroxide and deproteinized by n-butanol / chloroform, centrifuge and take the supernatant to obtain crude black fungus polysaccharide, the crude polysaccharide is subjected to multi-stage acetone recrystallization to obtain polysaccharide BFP.

[0010] Furthermore, the stirring time in step (1) is 2-24 hours.

[0011] Furthermore, the concentration of polysaccharide BFP in step (1) is 1-5 mg / mL.

[0012] Furthermore, in step (2), the volume ratio of polysaccharide BFP aqueous solution, phosphomolybdic acid aqueous solution, and tannic acid is (100-25):1:2.

[0013] Furthermore, the concentration of the phosphomolybdic acid aqueous solution in step (2) is 0.1M.

[0014] Furthermore, in step (2), the concentration of tannic acid is 100 mg / mL, and the pH is adjusted to 10.0-4.0.

[0015] Furthermore, the molecular weight cutoff of the dialysis bag described in step (2) is 3500 Da, and the dialysis time is 1-3 days.

[0016] A polysaccharide-loaded molybdenum nanomaterial obtained by the above preparation method.

[0017] The present invention also provides the application of the above-mentioned polysaccharide-loaded molybdenum nanomaterials in the preparation of antitumor drugs that kill breast cancer cells.

[0018] Furthermore, the polysaccharide-loaded molybdenum nanomaterials obtained in this invention induce cancer cells to produce reactive oxygen species, which consume glutathione within the cancer cells, thereby causing oxidative stress disorder in the cancer cells to kill the cells, while exhibiting low toxicity to normal cells.

[0019] Furthermore, the polysaccharide-loaded molybdenum nanomaterials obtained in this invention can also enhance anticancer activity in conjunction with photothermal therapy.

[0020] Black fungus polysaccharide is a β-glucan isolated from the fruiting body of black fungus. β β-glucan (BFP), with repeating units consisting of a backbone of three β-1,3-glucose residues linked to a side chain of β-1,6-glucose residues, exhibits excellent anti-breast cancer proliferation activity. It induces apoptosis and inhibits tumor angiogenesis by enhancing the immune response, while simultaneously interfering with multiple metabolic pathways within tumor cells, triggering intracellular oxidative stress, and ultimately inhibiting tumor growth to achieve anti-tumor effects. Phosphomolybdic acid is a heteropolyacid with oxidizing properties. Tannic acid is a polyphenol with good reducing and antibacterial properties. BFP is a triple-helix β-glucan that can self-assemble into parallel arrangements in water, self-coiling to form nanotube structures with hydrophobic cavities, making it a good nanocarrier for delivering nanoparticles and anti-tumor drugs. Using BFP to deliver redox-mediated MoNPs to induce oxidative stress in tumors for breast cancer treatment shows great promise; combining it with photothermal therapy can enhance the killing effect of nanomaterials on tumor cells. Furthermore, few nanosystems can simultaneously deplete glutathione and increase ROS for cancer treatment.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) This invention synthesizes a polysaccharide-loaded molybdenum nanomaterial, POM, which has good biocompatibility and low toxicity to normal cells. POM can be used as a GSH scavenger and ROS inducer to inhibit the proliferation of breast cancer cells and induce apoptosis of breast cancer cells. Near-infrared laser thermal imaging analysis revealed that POM also has good photothermal conversion ability and photothermal cycling stability.

[0023] (2) This invention provides a new strategy for treating cancer by consuming glutathione and enhancing redox imbalance, and provides a new perspective for understanding the interaction between anticancer drugs and cancer, which is of great significance to pharmacological research and clinical practice. Attached Figure Description

[0024] Figure 1 : Aqueous solution of BFP obtained by hydrothermal extraction; (a) a photo of black fungus used, (b) solid BFP obtained by extraction, (c) aqueous solution of BFP, and (d) aqueous solution of POM nanomaterials.

[0025] Figure 2 Scanning electron microscope image of POM nanomaterials.

[0026] Figure 3 The photothermal effect of different concentrations of POM under near-infrared laser at 808 nm in this invention is shown in (a) and the photothermal cycling stability of 25 μg / mPOM1 is shown in (b).

[0027] Figure 4 Figure 1: POM induces the generation of reactive oxygen species (a) and consumes glutathione (b).

[0028] Figure 5 : POM activity against breast cancer cells MCF-7 (a) and normal cells L929 (b).

[0029] Figure 6 A graph showing the effects of POM on apoptosis of breast cancer cells with and without near-infrared laser (808nm) irradiation.

[0030] Figure 7 (a) Effects of different concentrations of POM1 and POM2 on oxidative stress in breast cancer cells; (b) Changes in glutaraldehyde content; (c) Changes in superoxide dismutase content. Detailed Implementation

[0031] To facilitate understanding of the present invention, the technical solutions described below are further explained in conjunction with specific embodiments; however, the present invention is not limited thereto. It should be noted that processes not specifically described in detail below are conventional techniques.

[0032] Example 1: Preparation of POM Nanomaterials

[0033] (1) Extraction of polysaccharide BFP: The polysaccharide BFP in this invention was extracted from the fruiting body of black fungus by hydrothermal extraction. Figure 1 (a, originating from Fujian) The specific steps are as follows: Weigh 250g of dried black fungus, crush it using a high-speed blender, remove the oil by 4h of ethyl acetate and acetone circulation distillation and then dry it. Next, soak and stir the dried black fungus in a 70% ethanol aqueous solution for 24h. After removing the solvent, stir in a 0.9% sodium chloride aqueous solution at 85℃ for 4h to break the cell wall. Then continue stirring at room temperature for 24h, centrifuge and collect the supernatant. The supernatant is continuously decolorized with 30% hydrogen peroxide and deproteinized with n-butanol / chloroform, then centrifuged again to obtain crude black fungus polysaccharide. The crude polysaccharide is then subjected to multi-stage acetone reprecipitation to obtain our final neutral black fungus polysaccharide BFP, with a molecular weight of 1*10. 6 The repeating unit is every three β -1,3-D-glucose backbone linked with 2 β The repeating unit structure of the -1,6-D-glucose side chain.

[0034] (2) Preparation of POM1 nanomaterials: 20 mL of BFP (1 mg / mL) was dissolved in ultrapure water and stirred continuously for 24 h to obtain a clear BFP aqueous solution. Figure 1 c). Add 200 μL of phosphomolybdic acid aqueous solution (0.1 M) to BFP aqueous solution and stir for 5-10 minutes. Then, add 400 μL of tannic acid (100 mg / mL) dropwise, and adjust the pH to 9.0 by adding sodium carbonate solution. Continue stirring in the dark for 12 hours until the mixture turns into a clear, transparent orange solution. Dialyze the orange solution in a dialysis bag with a molecular weight cutoff of 3500 Da for 2 days to remove unreacted small molecule raw materials. Finally, freeze-dry to obtain POM1 nanomaterials. Figure 2 This is a scanning electron microscope image of POM1.

[0035] (3) Preparation of POM2 nanomaterials: Other experimental steps are the same as in step (2), except that sodium carbonate is not added to obtain a solution with a pH of 5.0, which yields POM2 nanomaterials.

[0036] Dissolving POM1 nanomaterials in water, such as Figure 1 As shown in d, POM1 nanomaterials have good water solubility.

[0037] Figure 3 The image shows the photothermal imaging of POM1 under 808 nm near-infrared laser irradiation and the photothermal cycling stability diagram with laser on-off switching. As the concentration of POM1 aqueous solution increases from 0-200 μg / mL, its photothermal conversion temperature also increases from 0-50℃. Figure 3 a), and after three laser-switched irradiation cycles, 25 μg / mL POM1 also exhibited excellent photothermal cycling stability ( Figure 3 b).

[0038] Example 2: Detection of reactive oxygen species generation and glutathione consumption

[0039] Glutathione (GSH) consumption and free radical detection were performed using a method reported in the literature [Z. Li, Y. Chen, X. Zeng, X. Zhang, Ultra-small FePt / siRNA loaded mesoporous silica nanoplatform to deplete cysteine ​​for enhanced ferroptosis in breast tumor therapy, NanoToday 38 (2021) 101150.]. PBS (Control group), POM1 (0.2 mg / mL, 1 mL), and POM2 (0.2 mg / mL, 1 mL) were added to GSH (1.25 mM, 50 μL), stirred continuously, and incubated at 37°C for 5 h. 1 mL of PBS was added as a control group. Glutathione levels were detected using a glutathione assay kit.

[0040] The free radical content was detected using 1,3-diphenylisobenzofuran (DPBF). A 10 mM DPBF / DMF solution was prepared. 2 μL of DPBF / DMF was added to 1 mL of ultrapure water to obtain a diluted DPBF aqueous solution. 100 μL of the diluted DPBF aqueous solution was added to each well of a 96-well plate, followed by 50 μL of PBS (Control group), 1 mg / mL POM1, and 1 mg / mL POM2, with three replicates per group. The absorbance at 410 nm for each solution was measured and recorded at the initial and different time intervals using a Spark 10M multi-plate reader (TECAN, Switzerland).

[0041] Figure 4 The POM obtained in Example 1 induces the generation of reactive oxygen species (ROS). Figure 4 a) and a graph showing glutathione consumption ( Figure 4 (b) The results showed that both POM1 and POM2 could induce the generation of reactive oxygen species and consume glutathione.

[0042] Example 3 Cell viability test

[0043] The activity of POM against breast cancer cells MCF-7 and normal cells L929 was determined, respectively. Breast cancer cells MCF-7 showed an activity of 2 × 10⁻⁶ pM. 4 Cells were seeded in 96-well plates and cultured for 24 h. Different concentrations of POM1 and POM2 nanomaterials (0, 1.25, 2.5, 5, 10, and 20 μg / mL) were then added, and the cells were co-cultured for 72 h. The treatment of normal L929 cells was the same as for breast cancer MCF-7 cells, i.e., normal L929 cells were seeded at 2 × 10⁻⁶ cells / well. 4Cells were seeded in 96-well plates and cultured for 24 h. Different concentrations of POM1 nanomaterials (0, 0.061, 0.122, 0.244, 1.95, 3.8, 7.8, and 15.6 μg / mL) were added, and the cells were co-cultured for 72 h. Cell viability was assessed using the MTT assay kit after 72 h of treatment, and absorbance at 490 nm was measured using a Spark 10M microplate reader. Cell viability was calculated as the ratio of absorbance between the treatment group (POM) and the control group (PBS), and results are expressed as percentages.

[0044] Figure 5 Figure a shows the cell viability of POM1 and POM2 on breast cancer cells and normal cells. The results indicate that the killing effect of POM1 and POM2 on breast cancer cells is concentration-dependent. Figure 5 b shows that POM1 has relatively low cytotoxic side effects on normal cells.

[0045] Example 4 Apoptosis Test

[0046] The following treatment groups were set up: Control group (PBS), POM1 group (1.95 μg / mL POM), Control+NIR group (PBS + 1.0 W laser for 5 min), and POM1+NIR group (1.95 μg / mL POM + 1.0 W laser for 5 min). MCF-7 was administered at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates and incubated for 72 h. Cells were then digested, washed, and stained using the Annexin V-FITC / PI kit (BD Biosciences). Apoptotic cells were detected using flow cytometry (BD FACSCalibur, USA). Apoptosis results are shown below. Figure 6 As shown, the results indicate that POM1 chemotherapy can promote apoptosis in breast cancer cells MCF-7, and photothermal therapy can promote early apoptosis and inhibit the production of nutrients required for cell DNA replication.

[0047] Example 5: Oxidative stress in cells

[0048] According to the reactive oxygen species (ROS) detection kit (Beyotime Biotechnology Co., Ltd., Shanghai, China), ROS generation was measured using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The specific steps were as follows: Control group (PBS), low-concentration POM1 group (0.244 μg / mL), high-concentration POM1 group (1.95 μg / mL), low-concentration POM2 group (1.25 μg / mL), and high-concentration POM2 group (2.5 μg / mL) were set up. 100 μL of the ROS detection reagent was added to MCF-7 breast cancer cells, and the cells were incubated for 60 min. Fluorescence images were obtained using a fluorescence microscope, and the fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 385 nm and 535 nm, respectively. For other oxidative stress markers, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were detected using specific detection kits (Solarbio® LIFE SCIENCES, Beijing, China). The tests were conducted according to the corresponding testing protocol, and the levels were normalized to protein content. The protein content was determined using cypermethrin (BCA).

[0049] Figure 7 a indicates that POM (POM1 and POM2) induces a dose-dependent increase in oxidative stress in breast cancer cells, while MDA content increases with increasing POM concentration. Figure 7 b), SOD showed a decreasing trend ( Figure 7 c). MDA serves as a marker of lipid peroxidation in cells, while SOD primarily functions to scavenge reactive free radicals generated within cells. Increased MDA levels and decreased SOD levels both indicate that POM induces oxidative stress in breast cancer cells and promotes lipid peroxidation.

[0050] In summary, POM combined with photothermal therapy has a significant killing effect on breast cancer cells. It induces apoptosis by consuming glutathione in breast cancer cells and promoting oxidative stress to kill tumors, and has low toxicity to normal cells, indicating that POM is a highly promising anti-tumor drug.

[0051] The above description is merely an exemplary embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a polysaccharide-loaded molybdenum POM nanomaterial in the preparation of an antitumor drug that kills breast cancer cells, characterized in that: The polysaccharide-loaded molybdenum POM nanomaterials serve as GSH scavengers and ROS inducers, and also possess photothermal conversion capabilities; the effective concentration of the polysaccharide-loaded molybdenum POM nanomaterials is 10~20 mg / mL; The preparation method of the polysaccharide-loaded molybdenum POM nanomaterial includes the following steps: (1) Polysaccharide BFP was extracted from black fungus by hydrothermal extraction. The polysaccharide BFP was dissolved in ultrapure water and stirred to obtain a clear polysaccharide BFP aqueous solution. (2) Add phosphomolybdic acid aqueous solution to the polysaccharide BFP aqueous solution obtained in step (1) and stir for 5-10 min. Add tannic acid dropwise and adjust the pH. React in the dark for 6-24 h to obtain a clear and transparent solution. After dialysis through a dialysis bag, freeze dry to obtain POM nanomaterials.

2. The application according to claim 1, characterized in that: The specific steps of the hydrothermal extraction method described in step (1) are as follows: Weigh 250g of dried black fungus, crush it using a cell wall breaker, remove the oil by 4h of ethyl acetate and acetone cyclic distillation and dry it, then soak and stir the dried black fungus in 70% ethanol aqueous solution for 24h, remove the solvent and stir in 0.9% sodium chloride aqueous solution at 85℃ for 4h to break the cell wall, then continue stirring at room temperature for 24h, centrifuge to collect the supernatant, continuously decolorize the supernatant with 30% hydrogen peroxide and deproteinize it with n-butanol / chloroform, centrifuge to collect the supernatant to obtain crude black fungus polysaccharide, and obtain polysaccharide BFP by multi-stage acetone recrystallization.

3. The application according to claim 1, characterized in that: The stirring time in step (1) is 2-24 hours.

4. The application according to claim 1, characterized in that: In step (1), the concentration of polysaccharide BFP is 1-5 mg / mL.

5. The application according to claim 1, characterized in that: In step (2), the volume ratio of polysaccharide BFP aqueous solution, phosphomolybdic acid aqueous solution, and tannic acid is (100-25):1:

2.

6. The application according to claim 1, characterized in that: The concentration of the phosphomolybdic acid aqueous solution in step (2) is 0.1M.

7. The application according to claim 1, characterized in that: In step (2), the concentration of tannic acid is 100 mg / mL, and the pH is adjusted to 4.0-10.

0.

8. The application according to claim 1, characterized in that: The molecular weight cutoff of the dialysis bag mentioned in step (2) is 3500 Da, and the dialysis time is 1-3 days.

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