Preparation method and application of novel polysaccharide molybdenum-loaded nano material

Abstract

The invention discloses a preparation method and application of a novel polysaccharide molybdenum-loaded nano material. The polysaccharide molybdenum-loaded nano material has good water dispersibility, stability, photo-thermal effect and glutathione consumption capability, and can relieve toxic and side effects of normal cells and improve the anti-tumor curative effect. The polysaccharide molybdenum-loaded nano material can selectively generate enough ROS and consume GSH at the same time under the mild in-vivo condition, so that oxidative stress in a tumor is seriously unbalanced, meanwhile, photothermal therapy is combined, the aim of killing tumor cells is achieved, theoretical and experimental bases are provided for clinical treatment of tumors, and the polysaccharide molybdenum-loaded nano material has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and specifically relates to a preparation method and application of a novel polysaccharide-supported molybdenum nanomaterial. Background Art

[0002] Reactive oxygen species (ROS) are cytotoxic byproducts produced during tumor growth and can be used to kill tumor cells. Many studies have confirmed that excessive production of ROS can disrupt redox homeostasis in tumor cells, leading to oxidative damage to proteins, lipids, and DNA, mitochondrial dysfunction, and ultimately cell apoptosis or necrosis. Therefore, stimulating excessive production of ROS in tumor cells to disrupt redox balance may become a new cancer treatment method. However, how to selectively produce enough ROS under mild conditions in vivo remains a big problem.

[0003] Nanoparticle (NPs)-based therapies have been envisioned as effective treatments for cancer, infection, inflammation, and diabetes, which can disrupt the redox balance in cancer while protecting healthy tissues from the toxicity of traditional chemotherapy. Many reports focus on the elimination of ROS, which can cause damage to normal cells. Conversely, disrupting the redox balance by increasing cellular ROS may also be fatal to cancer. Therefore, the rational use of oxidative stress plays an important role in the treatment of different types of diseases. So far, many studies have focused on using nanomaterials to disrupt the redox balance. However, redox-mediated nanomaterials mostly use inorganic materials (such as nanomaterials SiO2, MgO2, Fe3O4, ZnO2, Au, etc.), and their long-term biosafety and biocompatibility are still unclear. Molybdenum is an important trace element essential to the human body and is one of the basic components of xanthine oxidase and aldehyde oxidase. The increase in molybdenum content will increase the activity of xanthine oxidase and aldehyde oxidase, thereby causing oxidative stress. In order to survive and proliferate, cells will use more redox substances to fight oxidative stress. Glutathione (GSH), as the most abundant thiol-containing substance in cells, can scavenge ROS and alleviate lipid peroxidation through selenoproteinase (GPX-4). The thiol and selenol groups in the thioredoxin (TXN) redox system react with ROS to reduce oxidative stress. Therefore, consuming glutathione and increasing ROS may become a potential strategy for treating cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a new type of polysaccharide-loaded molybdenum nanomaterial, and to design and synthesize an anti-tumor nanodrug of natural polysaccharide BFP loaded with MoNPs (POM), which can significantly induce increased intracellular oxidative stress and increased GSH consumption, and also has good photothermal conversion ability.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a method for preparing a novel polysaccharide-supported molybdenum nanomaterial, comprising the following steps: (1) extracting polysaccharide BFP from black fungus by hydrothermal extraction, dissolving polysaccharide BFP in ultrapure water and stirring 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 minutes, add tannic acid dropwise and adjust the pH with sodium carbonate, and react in the dark for 6-24 hours to obtain a clear and transparent solution, which is dialyzed through a dialysis bag and freeze-dried to obtain POM nanomaterials.

[0007] Furthermore, the hydrothermal extraction method in step (1) specifically comprises the following steps: weighing 250 g of dried black fungus, crushing it with a wall-breaking machine, cyclically distilling ethyl acetate and acetone for 4 hours to remove oil and dry it, then soaking and stirring the dried black fungus in a 70% ethanol aqueous solution for 24 hours, removing the solvent and stirring it in a 0.9% sodium chloride aqueous solution at 85° C. for 4 hours to break the wall, then continuing to stir at room temperature for 24 hours and centrifuging to obtain the supernatant, decolorizing the supernatant with 30% hydrogen peroxide and deproteinizing it with n-butanol / chloroform, and then centrifuging to obtain the supernatant to obtain crude black fungus polysaccharide, and the crude polysaccharide is subjected to multi-stage acetone reprecipitation to obtain polysaccharide BFP.

[0008] Furthermore, the stirring time in step (1) is 2-24 hours.

[0009] Furthermore, in step (1), the concentration of the polysaccharide BFP is 1-5 mg / mL.

[0010] Furthermore, in step (2), the volume ratio of the polysaccharide BFP aqueous solution, the phosphomolybdic acid aqueous solution, and the tannic acid is (100-25):1:2.

[0011] Furthermore, the concentration of the phosphomolybdic acid aqueous solution in step (2) is 0.1M.

[0012] Furthermore, in step (2), the concentration of tannic acid is 100 mg / mL, and the pH is adjusted to 10.0-4.0.

[0013] Furthermore, the molecular weight cutoff of the dialysis bag in step (2) is 3500Da, and the dialysis time is 1-3 days.

[0014] A novel polysaccharide-loaded molybdenum nanomaterial obtained by the above preparation method.

[0015] The present invention also provides the use of the novel polysaccharide-loaded molybdenum nanomaterial in the preparation of anti-tumor drugs that kill breast cancer cells.

[0016] Furthermore, the polysaccharide-loaded molybdenum nanomaterial obtained by the present invention induces cancer cells to produce reactive oxygen, consumes glutathione in cancer cells, causes oxidative stress disorder in cancer cells, thereby achieving the purpose of killing cells, and has less toxicity to normal cells.

[0017] Furthermore, the polysaccharide-loaded molybdenum nanomaterial obtained by the present invention can also cooperate with photothermal therapy to enhance anti-cancer activity.

[0018] Black fungus polysaccharide is a β-glucan isolated from black fungus fruiting body. β -glucan, BFP), with a repeating unit of three β-1,3-glucose residues connected to the main chain of β-1,6-glucose residues, has good anti-breast cancer proliferation activity, induces cell apoptosis and inhibits tumor angiogenesis by enhancing immune response, interferes with multiple metabolic pathways in tumor cells, induces intracellular oxidative stress, and finally inhibits tumor growth to achieve the purpose of anti-tumor. Phosphomolybdic acid is a heteropolyacid with oxidizing properties. Tannic acid is a polyphenolic substance with good reducing and antibacterial properties. BFP is a β-glucan with a triple helical conformation. It can self-assemble in parallel in water and curl itself to form a nanotube structure with a hydrophobic cavity. It can be used as a good nanocarrier to carry nanoparticles and anti-tumor drugs. Using BFP to carry redox-mediated MoNPs to induce oxidative stress in tumors to treat breast cancer has broad prospects. At the same time, combined with photothermal therapy, it can enhance the killing effect of nanomaterials on tumor cells. In addition, few nanosystems can simultaneously achieve the purpose of consuming glutathione and increasing ROS to treat cancer.

[0019] The beneficial effects of the present invention are: The present invention synthesized a new type of polysaccharide-supported molybdenum nanomaterial POM, which has good biocompatibility and low toxicity to normal cells. The POM can be used as a GSH scavenger and ROS inducer to inhibit breast cancer cell proliferation and induce breast cancer cell apoptosis. Near-infrared laser thermal imaging analysis found that POM also has good photothermal conversion ability and photothermal cycle stability.

[0020] (2) This invention provides a new strategy for treating cancer by depleting glutathione and enhancing redox imbalance, and provides a new perspective for in-depth understanding of the interaction between anticancer drugs and cancer, which is of great significance to pharmacological research and clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : A BFP aqueous solution obtained by hydrothermal extraction; (a) a photo of the black fungus used, (b) the BFP solid obtained by extraction, (c) an aqueous solution of BFP, and (d) an aqueous solution of POM nanomaterials.

[0022] Figure 2 : Scanning electron microscope image of POM nanomaterials.

[0023] Figure 3 : The photothermal effect diagram of different concentrations of POM in the present invention under near-infrared laser 808nm (a) and the photothermal cycle stability diagram of 25μg / mLPOM1 (b).

[0024] Figure 4 : Diagram showing the production of reactive oxygen free radicals (a) and consumption of glutathione (b) induced by POM.

[0025] Figure 5 : Activity of POM on breast cancer cells MCF-7 (a) and normal cells L929 (b).

[0026] Figure 6 :The effect of POM on apoptosis of breast cancer cells with or without near-infrared laser (808nm) irradiation.

[0027] Figure 7 :(a)The 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 DESCRIPTION

[0028] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementations, but the present invention is not limited thereto. It should be noted that the processes not specifically described below are all conventional techniques.

[0029] Example 1 Preparation of POM nanomaterials (1) Extraction of polysaccharide BFP: The polysaccharide BFP in the present invention is extracted from black fungus fruiting bodies ( Figure 1 a, origin Fujian), the specific steps are as follows: weigh 250g of dry black fungus, break it with a wall breaking machine, cyclically distill ethyl acetate and acetone for 4h to remove oil and dry it, then soak and stir the dried black fungus in 70% ethanol aqueous solution for 24h, remove the solvent and stir it in 0.9% sodium chloride aqueous solution at 85℃ for 4h to break the wall, then continue to stir at room temperature for 24h and centrifuge to obtain the supernatant, the supernatant is continuously decolorized by 30% hydrogen peroxide and deproteinized by n-butanol / chloroform, and then centrifuged to obtain the supernatant to obtain crude black fungus polysaccharide, and the crude polysaccharide is 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 three β -1,3-D-glucose main chain connects 2 β -1,6-D-glucose side chain repeating unit structure.

[0030] (2) Preparation of POM1 nanomaterials: 20 mL of BFP (1 mg / mL) was dissolved in ultrapure water and stirred 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 the BFP aqueous solution and stir for 5-10 minutes. Then add 400 μL of tannic acid (100 mg / mL) dropwise thereto, and adjust the pH to 9.0 by adding sodium carbonate solution dropwise. Stir for 12 hours in a dark environment until the mixture is converted into a clear, transparent orange solution. The orange solution is dialyzed in a dialysis bag with a molecular weight cutoff of 3500 Da for 2 days to remove the unreacted small molecule raw materials, and finally freeze-dried to obtain POM1 nanomaterials. Figure 2 This is a scanning electron micrograph of POM1.

[0031] (3) Preparation of POM2 nanomaterials: The other experimental steps are the same as step (2), except that no sodium carbonate is added to obtain a solution with a pH of 5.0, thereby obtaining POM2 nanomaterials.

[0032] Dissolve POM1 nanomaterials in water, such as Figure 1 As shown in (d), POM1 nanomaterials have good water solubility.

[0033] Figure 3 The photothermal imaging of POM1 under 808nm near-infrared laser irradiation and the photothermal cycle stability diagram with the 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 cycles of laser on-off irradiation, 25 μg / mL POM1 also exhibited excellent photothermal cycling stability ( Figure 3 b).

[0034] Example 2 Detection of reactive oxygen free radical generation and glutathione consumption Glutathione (GSH) consumption and free radical detection were performed using the 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. The group with 1 mL of PBS was used as the control group, and the glutathione content was detected using a glutathione detection kit.

[0035] 1,3-Diphenylisobenzofuran (DPBF) was used to detect the free radical content. A DPBF / DMF solution with a concentration of 10 mM 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 a 96-well plate, and then 50 μL of PBS (Control group), 1 mg / mL POM1 and 1 mg / mL POM2 were added to each well, with three replicates for each group. The initial and different time period absorbance values ​​of each well solution at 410 nm were measured and recorded using a SPARK10M multifunctional microplate reader (TECAN, Switzerland).

[0036] Figure 4 The POM obtained in Example 1 induces the generation of active oxygen free radicals ( Figure 4 a) and the consumption of glutathione ( Figure 4 b) The results showed that both POM1 and POM2 could induce the production of reactive oxygen free radicals and consume glutathione.

[0037] Example 3 Cell activity test The activity of POM on breast cancer cells MCF-7 and normal cells L929 was measured. 4 The cells / well were inoculated in a 96-well plate and cultured for 24 h. Different concentrations of POM1 and POM2 nanomaterials (0, 1.25, 2.5, 5, 10, and 20 μg / mL) were added and cultured for 72 h. The treatment steps of normal cell L929 were the same as those of breast cancer cell MCF-7 cells, that is, normal cell L929 was treated with 2×10 4Cells / well were inoculated 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 cultured for 72 h. The cell viability after 72 h of treatment was detected by MTT kit, and the absorbance at 490 nm after cell treatment was measured by SPARK 10M microplate reader. The cell viability was calculated by the absorbance ratio of the treatment group (POM) and the control group (PBS), and the results were expressed as percentages.

[0038] Figure 5 a is the cell viability diagram of POM1 and POM2 on breast cancer cells and normal cells. The results show that POM1 and POM2 kill breast cancer cells in a concentration-dependent manner. Figure 5 b shows that POM1 has less toxic side effects on normal cells.

[0039] Example 4 Cell apoptosis test The 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) were set up. MCF-7 was added at 1×10 6 Each well was inoculated in a 6-well plate and incubated for 72 h, then digested, washed and stained with Annexin V-FITC / PI kit (BD Biosciences). Apoptotic cells were detected by flow cytometry (BD FACSCalibur, USA). Figure 6 As shown, the results showed that POM1 chemotherapy can promote apoptosis of breast cancer cells MCF-7, photothermal therapy can promote early cell apoptosis and inhibit the production of nutrients required for cell DNA replication.

[0040] Example 5 Oxidative stress in cells ROS generation was determined by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) according to the Reactive Oxygen Species Detection Kit (Biyuntian Biotechnology Co., Ltd., Shanghai, China). The specific steps were as follows: Control group (PBS), low-concentration POM1 group (0.244μg / mL), high-concentration POM1 group (1.95μg / mL), low-concentration POM2 group (1.25μg / mL), and high-concentration POM2 group (2.5μg / mL) were set up. 100μL of ROS detection reagent was added to breast cancer MCF-7 cells and incubated for 60 min. Fluorescence images were obtained by fluorescence microscopy, and fluorescence intensity was measured by microplate reader at excitation and emission wavelengths of 385nm and 535nm, respectively. For other oxidative stress markers, malondialdehyde (MDA) and superoxide dismutase (SOD) levels (Solarbio®LIFE SCIENCES, Beijing, China) were detected using specific detection kits. The assays were performed according to the corresponding assay protocols, and the levels were normalized to protein content, which was determined using bicyandiamide (BCA) assay.

[0041] Figure 7 a shows that POM (POM1 and POM2) induces the increase of oxidative stress in breast cancer cells in a dose-dependent manner, and the MDA content increases with the increase of POM concentration ( Figure 7 b), SOD showed a decreasing trend ( Figure 7 c). MDA is a marker of lipid peroxidation in cells, and the main function of SOD is to remove active free radicals produced in cells. The increase in MDA content and the decrease in SOD content both indicate that POM induces oxidative stress in breast cancer cells and promotes lipid peroxidation.

[0042] In summary, POM combined with photothermal therapy has a significant killing effect on breast cancer cells. It consumes glutathione in breast cancer cells and promotes oxidative stress to induce cell apoptosis to achieve the purpose of killing tumors, and has less toxicity to normal cells, indicating that POM is a highly potential anti-tumor drug.

[0043] The above description is only 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 in the protection scope of the present invention.

Claims

1. A method for preparing a novel polysaccharide-supported molybdenum nanomaterial, characterized in that: The following steps are involved: (1) extracting polysaccharide BFP from black fungus by hydrothermal extraction, dissolving polysaccharide BFP in ultrapure water and stirring to obtain a clear polysaccharide BFP aqueous solution; (2) Add phosphomolybdic acid aqueous solution to the polysaccharide BFP aqueous solution obtained in step (1), stir for 5-10 minutes, add tannic acid dropwise and adjust the pH, react in the dark for 6-24 hours to obtain a clear and transparent solution, dialyze through a dialysis bag and then freeze-dry to obtain POM nanomaterials.

2. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The specific steps of the hydrothermal extraction method in step (1) are as follows: weighing 250g of dry black fungus, breaking it with a wall breaking machine, cyclically distilling ethyl acetate and acetone for 4 hours to remove oil and dry it, then soaking and stirring the dried black fungus in a 70% ethanol aqueous solution for 24 hours, removing the solvent and stirring it at 85°C for 4 hours in a 0.9% sodium chloride aqueous solution to break the wall, then continuing to stir at room temperature for 24 hours and centrifuging to obtain the supernatant, and continuously decolorizing the supernatant with 30% hydrogen peroxide and deproteinizing it with n-butanol / chloroform, and then centrifuging to obtain the supernatant to obtain crude black fungus polysaccharide, and the crude polysaccharide is subjected to multi-stage acetone reprecipitation to obtain polysaccharide BFP.

3. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The stirring time in step (1) is 2-24 hours.

4. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The concentration of the polysaccharide BFP in step (1) is 1-5 mg / mL.

5. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The volume ratio of the polysaccharide BFP aqueous solution, the phosphomolybdic acid aqueous solution and the tannic acid in step (2) is (100-25):1:

2.

6. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The concentration of the phosphomolybdic acid aqueous solution in step (2) is 0.1M.

7. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The tannic acid concentration in step (2) is 100 mg / mL, and the pH is adjusted to 10.0-4.

0.

8. The method for preparing the novel polysaccharide-supported molybdenum nanomaterial according to claim 1, characterized in that: The molecular weight cutoff of the dialysis bag in step (2) is 3500Da, and the dialysis time is 1-3 days.

9. A novel polysaccharide-supported molybdenum nanomaterial obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the novel polysaccharide-loaded molybdenum nanomaterial as claimed in claim 9 in the preparation of anti-tumor drugs for killing breast cancer cells.

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