Nanoparticles for synergistically treating tumors and preparation method thereof

Abstract

The invention discloses nanoparticles for synergistically treating tumors and a preparation method of the nanoparticles, and relates to the technical field of preparation of drugs containing inorganic active ingredients. The nanoparticles are composite nanoparticles Mo-PDA prepared by mixing and reacting inorganic active ingredients of phosphomolybdic acid Mo-POM and dopamine; the preparation method comprises the following steps: taking Mo-POM (phosphomolybdic acid), then loading Mo-PDA-coated GOx nanoparticles with a spherical structure formed by glucose oxidase (GOx), and carrying out mixing reaction by taking Mo-POM, adding deionized water, then adding dopamine, continuously stirring for 1 hour, and continuously reacting for 14-18 hours at the temperature of 150-170 DEG C. According to the preparation method disclosed by the invention, PDA is combined with Mo-containing polyacid salt and externally loaded with glucose oxidase (GOx), so that the PTT-modified CDT-hunger nanoparticle Mo-PDA-GOx for cooperatively treating tumors is synthesized, and the PTT-modified CDT-hunger nanoparticle Mo-PDA-GOx has excellent photo-thermal conversion efficiency which reaches 67.2%. The nanoparticles effectively exert synergistic treatment, and the treatment effect on tumors is improved.

Description

[0001] This invention is a divisional application of patent 202510812745.6, entitled "A Mo-PDA@GOx Nanoparticle and its Preparation Method and Application". Technical Field

[0002] This invention relates to the field of drug preparation technology containing inorganic active ingredients, and specifically to a nanoparticle for synergistic tumor treatment and its preparation method. Background Technology

[0003] Cancer remains a leading cause of death worldwide, and chemotherapy has been a common and primary treatment for tumors. However, due to the non-targeted delivery of chemotherapy drugs within the body, unnecessary accumulation of drugs in normal tissues often occurs, leading to unsatisfactory treatment outcomes, adverse reactions, and multidrug resistance. With the rapid development of nanotechnology and nanomaterials, new directions have been provided for the prevention, diagnosis, and treatment of tumors. The synergistic treatment of photothermal therapy, starvation therapy, and chemokinetics can help anti-tumor drugs achieve optimal therapeutic effects and reduce toxic side effects on the body.

[0004] Polydopamine (PDA) possesses various functional groups that can coordinate with metal ions, thereby enabling further functional modification of materials. PDA exhibits good biocompatibility and can serve as a drug carrier for both photothermal therapy (PTT) and chemotherapy. Chemokinetic therapy (CDT) offers promise for cancer treatment by generating reactive oxygen species (ROS). Molybdenum polyphosphates, with their good water solubility, can act as Fenton's reagents, reacting with H₂O₂ to generate cytotoxic ROS. However, the efficacy of CDT is still limited by insufficient endogenous H₂O₂ levels in the tumor microenvironment (TME). Therefore, selectively increasing H₂O₂ levels at the tumor site to enhance the catalytic efficiency of the Fenton reaction is imperative. Summary of the Invention

[0005] The purpose of this invention is to provide a nanoparticle for synergistic tumor treatment, specifically Mo-PDA@GOx nanoparticles.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned nanoparticles for synergistic tumor treatment.

[0007] The objective of this invention is achieved through the following technical solution: A nanoparticle for synergistic tumor treatment, characterized in that: a composite nanoparticle Mo-PDA is prepared by mixing and reacting the inorganic active ingredient phosphomolybdic acid Mo-POM with dopamine, and then loading glucose oxidase (GOx) to form spherical Mo-PDA@GOx nanoparticles. The mixing reaction is carried out by taking phosphomolybdic acid Mo-POM, adding deionized water, then adding dopamine and stirring continuously for 1 hour, and reacting continuously at 150~170℃ for 14~18 hours.

[0008] Furthermore, the ratio of the amount of phosphomolybdic acid Mo-POM, dopamine, and deionized water is 45~50mg:4~6mg:10mL.

[0009] The combination of polydopamine (PDA) and phosphomolybdic acid (Mo-POM) forms a synergistic PTT (post-tumor therapy) and CDT (tumor-specific therapy) combination in tumor treatment. However, it still faces the limitation of insufficient endogenous H2O2 in the tumor microenvironment (TME), resulting in unsatisfactory synergistic therapeutic effects. To address the H2O2 limitation problem, this invention selects to further load the biocatalyst glucose oxidase (GOx) to effectively convert glucose within the tumor into gluconic acid and H2O2. Furthermore, compared with normal tissue cells, glucose plays a more important role in maintaining the survival and proliferation of tumor cells; therefore, GOx can achieve non-invasive specific starvation therapy (ST) through glucose deprivation.

[0010] However, during the process of forming a composite carrier of PDA and Mo-POM phosphomolybdic acid and then loading GOx, it was found that the binding performance between the carrier and GOx decreased due to the excessive coating and coverage of the functional groups on the surface of PDA by Mo-POM phosphomolybdic acid. GOx was difficult to load into the composite carrier, resulting in poor drug loading effect and low drug loading concentration of GOx, which prevented GOx in the nanoparticles from playing an effective role.

[0011] In this invention, a composite support with a particle size of approximately 50 nm is generated by reacting prepared phosphomolybdic acid (Mo-POM) and dopamine at a specific temperature. In the resulting composite support, the simultaneously formed polydopamine (PDA) exposes more amino groups. The amino groups on the PDA surface can interact with functional groups (such as carboxyl groups and hydroxyl groups) in GOx molecules through covalent bonds (such as amide bonds) or non-covalent bonds (such as hydrogen bonds and electrostatic interactions), thereby immobilizing GOx. At the same time, the small-particle-size support has a high surface charge density, which can form a stronger electrostatic interaction with GOx. In addition, the small-particle-size support has a higher specific surface area, which can provide more active sites to interact with GOx through physical adsorption or chemical binding. The composite support prepared by this invention improves the loading efficiency of GOx through the synergistic effect of the above multiple actions.

[0012] In Mo-PDA@GOx nanoparticles formed by highly efficient GOx loading, GOx effectively exerts a specific starvation therapy. This starvation treatment leads to an increase in H2O2 levels, providing sufficient substrate for the Fenton reaction. Simultaneously, the increased H2O2 levels and CDT binding also accelerate the generation of •OH. Exogenous stimuli such as near-infrared (NIR) laser irradiation can also enhance the CDT effect with the assistance of photothermal reagents.

[0013] Furthermore, the phosphomolybdic acid Mo-POM is in the form of (NH4)6Mo7O 24 • 4H2O and NaH2PO4•2H2O were dissolved in ultrapure water, and ascorbic acid was added while stirring. Then ethanol was added to precipitate the precipitate. After centrifugation, the precipitate was washed three times with water and ethanol and freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM.

[0014] Furthermore, the (NH4)6Mo7O 24 The dosage of 4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid is 2.4~2.6g: 0.17~0.2g: 10mL: 20mL, and the concentration of ascorbic acid is 0.48~0.51g / mL.

[0015] A method for preparing nanoparticles for synergistic tumor therapy, characterized by including the synthesis of Mo-POM phosphomolybdic acid, the synthesis of Mo-PDA nanoparticles, and the synthesis of Mo-PDA@GOx nanoparticles, specifically using (NH4)6Mo7O 24 • 4H2O and NaH2PO4•2H2O were used as raw materials to synthesize phosphomolybdic acid Mo-POM. Then, phosphomolybdic acid Mo-POM was dissolved in deionized water, and dopamine was added and mixed. The mixture was reacted at 150~170℃ for 14~18 h to generate Mo-PDA nanoparticles. Then, Mo-PDA@GOx nanoparticles were synthesized by combining them with glucose oxidase.

[0016] Furthermore, the synthesis of phosphomolybdic acid Mo-POM involves reacting NaH2PO4•2H2O with (NH4)6Mo7O 24 • Dissolve 4H2O in ultrapure water, add ascorbic acid while stirring, then add ethanol to precipitate the precipitate, centrifuge, wash three times with water and ethanol, freeze dry to obtain powder, which is phosphomolybdic acid Mo-POM.

[0017] Furthermore, the (NH4)6Mo7O 24 The dosage of 4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid is 2.4~2.6g: 0.17~0.2g: 10mL: 20mL, and the concentration of ascorbic acid is 0.48~0.51g / mL.

[0018] Furthermore, the ratio of the amount of phosphomolybdic acid (Mo-POM), dopamine, and deionized water is 45-50 mg: 4-6 mg: 10 mL. The mass ratio of the Mo-PDA nanoparticles to GOx is 1:2.

[0019] Most specifically, a method for preparing nanoparticles for synergistic tumor therapy is characterized by comprising the following steps: (1) Synthesis of Mo-POM phosphomolybdic acid: (NH4)6Mo7O 24 • 4H2O and NaH2PO4•2H2O were dissolved in ultrapure water, and ascorbic acid was added while stirring. Then, 40 mL of ethanol was added to precipitate the precipitate. The precipitate was centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM. The (NH4)6Mo7O 24 The dosage of 4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid is 2.4~2.6g: 0.17~0.2g: 10mL: 20mL, and the concentration of ascorbic acid is 0.48~0.51g / mL. (2) Synthesis of Mo-PDA nanoparticles: Weigh out Mo-POM phosphomolybdic acid, add deionized water, then add dopamine (DA) and stir continuously for 1 hour. Place the mixture into a stainless steel reactor and react continuously at 150-170℃ for 14-18 hours. Centrifuge the product and wash it twice with water to obtain Mo-PDA nanoparticles. The ratio of Mo-POM phosphomolybdic acid, dopamine and deionized water is 45-50 mg: 4-6 mg: 10 mL. (3) Synthesis of Mo-PDA@GOx nanoparticles: Mo-PDA nanoparticles were mixed with glucose oxidase (GOx) and reacted under light-protected conditions for 24 h. The mixture was then removed, centrifuged, and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The mass ratio of Mo-PDA nanoparticles to GOx was 1:2.

[0020] The present invention has the following technical effects: This invention synthesizes Mo-PDA@GOx nanoparticles for PTT-CDT-starvation synergistic tumor therapy by combining PDA with Mo-containing polyacid salts and externally loading glucose oxidase (GOx). The GOx loading in these nanoparticles reaches 57.3%. These nanoparticles exhibit excellent photothermal conversion efficiency, reaching 67.2%. These nanoparticles effectively utilize the synergistic effects of PTT-CDT-starvation therapy, improving the therapeutic efficacy against tumors. This invention provides a green, simple, low-cost, and environmentally friendly preparation method and offers a new strategy for the application of nanocomposite materials in synergistic tumor therapy. Attached Figure Description

[0021] Figure 1 TEM image of the Mo-PDA nanoparticles prepared in this invention.

[0022] Figure 2 The hydration particle size of Mo-PDA nanoparticles and Mo-PDA@Gox nanoparticles prepared in this invention.

[0023] Figure 3 Zeta potential diagrams of Mo-PDA nanoparticles and Mo-PDA@Gox nanoparticles prepared in this invention.

[0024] Figure 4 Particle size distribution of Mo-PDA1 prepared in Comparative Example 2.

[0025] Figure 5 Scanning electron microscope image of Mo-PDA1 prepared in Comparative Example 2.

[0026] Figure 6 Mo-PDA nanoparticle aqueous solutions of different concentrations under 808 nm laser irradiation (1.0 W·cm⁻¹) -2 The warming trend of ).

[0027] Figure 7 Mo-PDA nanoparticles under 808 nm laser irradiation (1.0 W·cm⁻¹) -2 (Photostability)

[0028] Figure 8 Analysis of the effects of different particle concentrations (a), different H2O2 concentrations (b), and different temperatures (c) on the generation of ROS in Mo-PDA nanoparticles.

[0029] Figure 9 Analysis of the effects of Mo-PDA@Gox and Mo-PDA on the catalytic production of ROS from glucose.

[0030] Figure 10 Graph showing pH changes when Mo-PDA@GOx nanoparticles are dispersed in glucose solution.

[0031] Figure 11 CLSM plot of ROS production in HepG2 cells under different treatment conditions (scale bar: 100 μm).

[0032] Figure 12 Cell viability of HepG2(a) and MCF-7(b) tumor cells after co-incubation with Mo-PDA nanoparticles for 24 and 48 h.

[0033] Figure 13The synergistic therapeutic effect of Mo-PDA@Gox nanoparticles was determined using the MTT assay. Detailed Implementation

[0034] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0035] Example 1 A method for preparing Mo-PDA@GOx nanoparticles includes the following steps: (1) Synthesis of Mo-POM phosphomolybdic acid: 2.6 g (NH4)6Mo7O 24 • 4H2O and 0.2 g NaH2PO4•2H2O were dissolved in 10 mL of ultrapure water. While stirring, 20 mL of ascorbic acid with a concentration of 0.48 g / mL was added, followed by the addition of 40 mL of ethanol to precipitate the precipitate. The precipitate was then centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM. (2) Synthesis of Mo-PDA nanoparticles: Weigh 45 mg of phosphomolybdic acid Mo-POM, add 10 mL of deionized water, then add 4 mg of dopamine (DA) and stir continuously for 1 h. The mixture is then placed in a stainless steel reactor and reacted continuously at 170 °C for 14 h. The product is centrifuged and washed twice with water to obtain Mo-PDA nanoparticles. (3) Synthesis of Mo-PDA@GOx nanoparticles: 1 mg of Mo-PDA nanoparticles and 2 mg of Gox were added to 1 mL of water and mixed. The mixture was reacted under light-protected conditions for 24 h, centrifuged, and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The Mo-PDA nanoparticles and GOx were then reacted.

[0036] Example 2 A method for preparing Mo-PDA@GOx nanoparticles includes the following steps: (1) Synthesis of Mo-POM phosphomolybdic acid: 2.4 g (NH4)6Mo7O 24 • 4H2O and 0.17 g NaH2PO4•2H2O were dissolved in 10 mL of ultrapure water. While stirring, 20 mL of ascorbic acid with a concentration of 0.51 g / mL was added, followed by the addition of 40 mL of ethanol to precipitate the precipitate. The precipitate was then centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM. (2) Synthesis of Mo-PDA nanoparticles: Weigh 50 mg of phosphomolybdic acid Mo-POM, add 10 mL of deionized water, then add 6 mg of dopamine (DA) and stir continuously for 1 h. The mixture is then placed in a stainless steel reactor and reacted continuously at 150 °C for 18 h. The product is centrifuged and washed twice with water to obtain Mo-PDA nanoparticles. (3) Synthesis of Mo-PDA@GOx nanoparticles: 1 mg of Mo-PDA nanoparticles and 2 mg of Gox were added to 1 mL of water and mixed. The mixture was reacted under light-protected conditions for 24 h, centrifuged, and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The Mo-PDA nanoparticles and GOx were then reacted.

[0037] Example 3 A method for preparing Mo-PDA@GOx nanoparticles includes the following steps: (1) Synthesis of Mo-POM phosphomolybdic acid: 2.47 g (NH4)6Mo7O 24 • 4H2O and 0.18 g NaH2PO4•2H2O were dissolved in 10 mL of ultrapure water. While stirring, 20 mL of 0.5 mg / mL ascorbic acid was added, followed by 40 mL of ethanol to precipitate the precipitate. After centrifugation, the precipitate was washed three times with water and ethanol and then freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM. (2) Synthesis of Mo-PDA nanoparticles: Weigh 48 mg of phosphomolybdic acid Mo-POM, add 10 mL of deionized water, then add 5 mg of dopamine (DA) and stir continuously for 1 h. The mixture is then placed in a stainless steel reactor and reacted continuously at 160 °C for 16 h. The product is centrifuged and washed twice with water to obtain Mo-PDA nanoparticles. (3) Synthesis of Mo-PDA@GOx nanoparticles: 1 mg of Mo-PDA nanoparticles and 2 mg of glucose oxidase (GOx) were added to 1 mL of water and mixed. The mixture was reacted under light-protected conditions for 24 h, centrifuged, and washed twice with water to obtain Mo-PDA@GOx nanoparticles.

[0038] Morphological characterization of nanoparticles: The morphology of the synthesized Mo-PDA nanoparticles was characterized, such as... Figure 1 As shown in the TEM image, the nanoparticles are spherical structures with uniform particle size, approximately 56 nm in size, and are uniformly dispersed.

[0039] Characterization of hydrated particle size and Zeta potential of nanoparticles: The hydrated particle size of Mo-PDA nanoparticles was measured to be 76.6 nm using a nanoparticle size and Zeta potential analyzer, and the hydrated particle size of Mo-PDA@Gox nanoparticles was measured to be 210.5 nm. These particle size results are slightly larger than the nanoparticle sizes characterized by TEM. Figure 2 As shown, this is because the nanoparticles associate with water molecules in the solution, forming a hydration film, which increases the particle size. The above results demonstrate that Mo-PDA and Mo-PDA@Gox nanoparticles have uniform particle size and exhibit good stability and dispersibility in water. Figure 3 As shown in the Zeta potential diagram, the Zeta potential of Mo-PDA nanoparticles is -68 mV, and the Zeta potential of Mo-PDA@Gox is -26 mV. The potential increases after Mo-PDA is loaded with Gox, indicating that the loading of Gox was successful.

[0040] Comparative Example 1: Compared with Example 1, the difference is that the Mo-PDA nanoparticle synthesis step adopts a physical mixing method, while the remaining steps are the same as in Example 1, that is, step (2) is as follows: Synthesis of polydopamine (PDA): 10 mL of ethanol and 1 mL of 2 mol / L ammonia solution were mixed, then 20 mL of water was added. After thorough mixing, 100 mg of dopamine (DA) was added, and stirring was continued for 24 h. The mixture was centrifuged at 9000 rpm, and the product was washed three times with water and then with ethanol to obtain the final product. Synthesis of Mo-PDA nanoparticles: PDA nanoparticles were dissolved in water, and then phosphomolybdic acid Mo-POM was added. The mixture was stirred in the dark for 24 h, centrifuged at 9000 rpm, and the product was washed with water three times to obtain the product Mo-PDA.

[0041] In the Mo-PDA nanoparticles prepared by this method, the content of phosphomolybdic acid Mo-POM loaded on the PDA surface is relatively low, which affects the photothermal and chemical kinetic effects of the final Mo-PDA nanoparticles and results in a low content of reactive oxygen species (ROS). Furthermore, the final Mo-PDA nanoparticles are synthesized by a simple physical mixing of PDA and phosphomolybdic acid Mo-POM, where the functional groups on the PDA surface are covered by phosphomolybdic acid Mo-POM, leading to a low GOx loading in Mo-PDA@GOx.

[0042] Comparative Example 2: The difference from Example 1 is that, in the preparation of Mo-PDA, Mo-POM phosphomolybdate is replaced with gadolinium phosphomolybdate (K). 17 [Gd(P2Mo 17 O 61Mo-PDA was prepared by smelting gadolinium phosphomolybdate (2)·26H2O. The remaining steps were the same as in Example 1. The nanoparticles obtained in the final preparation were denoted as Mo-PDA1, with the nanoparticles corresponding to gadolinium phosphomolybdate as the nanoparticles.

[0043] like Figure 4 As shown, the Mo-PDA1 prepared by this method has a particle size of approximately 600 nm and exhibits poor stability. (SEM image) Figure 5 As can be seen from the data, the particles have aggregated and clustered together, exhibiting poor dispersion and irregular morphology.

[0044] The loading efficiency of GOx directly determines the intensity and duration of starvation therapy (ST). High-loading-efficiency Gox can significantly increase the glucose consumption rate and H2O2 production in the tumor microenvironment. The Mo-PDA@GOx nanoparticle solutions synthesized in Example 1, Comparative Example 1, and Comparative Example 2 were centrifuged and washed with water to obtain the supernatant. The GOx content in the supernatant and the original drug solution was determined using a BCA protein assay kit, and the GOx drug loading efficiency was calculated. The drug loading efficiency was calculated according to the formula:

[0045] Calculations showed that the GOx drug loading efficiencies of the Mo-PDA@GOx nanoparticles synthesized in Example 1, Comparative Example 1, and Comparative Example 2 were 57.3%, 21.7%, and 28.6%, respectively. Since the GOx drug loading efficiencies of the nanoparticles prepared in Comparative Example 1 and Comparative Example 2 were low, further efficacy tests were not conducted.

[0046] Photothermal conversion effect test: To investigate the photothermal conversion effect of the Mo-PDA nanoparticles prepared in Example 1, a near-infrared (NIR) laser (808 nm, 1.0 W·cm⁻¹) was used. -2 Irradiation with 500 μL of different concentrations (0, 50, 100, 200 and 400 µg·mL) for five groups -1 Irradiation was performed on a Mo-PDA nanoparticle solution for 600 s, and the temperature change was recorded every 30 s. After completion, irradiation was stopped, and the cooling process of the solution was recorded. Under the same conditions, deionized water was used as a control group irradiated with laser. The photothermal conversion efficiency (PCO) was calculated. η ):

[0047] h is the heat transfer coefficient, S is the surface area of ​​the container, and T is the heat transfer coefficient. max It is the equilibrium temperature, T sur It is the ambient temperature, Q dis I is the heat absorbed by the solvent, and I is the power of the irradiated light (1.0 W / cm²). -2 A808 This is the UV-vis absorbance value of the Mo-PDA nanoparticle solution at 808 nm. The hS value is calculated using the following formula:

[0048] τ s It is the time constant of the sample system, m D and C D It refers to the mass and specific heat capacity of water as a solvent.

[0049] The photothermal conversion capacity of Mo-PDA nanoparticles with different concentrations was analyzed. Temperature changes of Mo-PDA nanoparticles with different concentrations were recorded under 808 nm laser irradiation. The results are as follows: Figure 6 As shown, after 10 min of irradiation, the concentration was 400 µg·mL. -1 The Mo-PDA nanoparticle solution can be heated to 72.3°C, while the temperature of deionized water remains almost unchanged.

[0050] Figure 7 It is Mo-PDA nanoparticles irradiated by an 808 nm laser (1.0 W·cm⁻¹) -2 The photothermal stability of Mo-PDA nanoparticles was assessed by recording temperature changes using an IRT instrument. After four repeated laser irradiation-cooling cycles, the temperature increase of the Mo-PDA nanoparticles did not show significant changes. These results indicate that Mo-PDA nanoparticles possess good photothermal stability and exhibit concentration / irradiation time-dependent photothermal behavior. Based on the calculation formula, the photothermal conversion efficiency of the Mo-PDA nanoparticles was determined to be 67.2%.

[0051] Chemical kinetic performance testing: To test the ROS generation effect of the Mo-PDA nanoparticles prepared in Example 1, o-phenylenediamine (OPD) was selected as the ROS scavenger. This is because ROS can catalyze the oxidation of OPD to yellow 2,3-diaminophenazine (DAP), causing the solution to change from colorless to yellow. DAP has a characteristic absorption peak at 420 nm, and the generated ROS can be detected by UV-vis. The test steps are as follows: Mo-PDA nanoparticles were prepared at different concentrations (400, 200, 100, and 50 µg·mL⁻¹). -1 The particles were dispersed in a 1 mL mixture of H2O2 and OPD, with H2O2 concentration of 50 mM and OPD concentration of 1.0 mM. After reacting in the dark for 2 h, the mixture was centrifuged at 9000 rpm for 10 min, and the absorbance of the supernatant was measured to demonstrate the effect of particle concentration on ROS production.

[0052] The results are as follows Figure 8 As shown, Mo-PDA nanoparticles do not produce a characteristic absorption peak at 420 nm when reacting with OPD or H2O2 alone. Only when reacting with both OPD and H2O2 simultaneously do they produce an absorption peak at 420 nm. This indicates that Mo-PDA nanoparticles cannot generate hydroxyl radicals when reacting with OPD or H2O2 alone; they only generate hydroxyl radicals when reacting with both OPD and H2O2 simultaneously. The intensity of the 420 nm absorption peak increases with increasing Mo-PDA particle concentration. Specifically, as shown... Figure 8 As shown in figure a, this illustrates the Mo in Mo-PDA nanoparticles. 5+ A Fenton reaction occurred with H2O2. It was also tested that the combination of OPD and H2O2 alone did not generate free radicals. Further introduction of Mo-PDA, with a constant OPD concentration, showed that the intensity of the absorption peak at 420 nm increased with increasing H2O2 concentration. Figure 8 As shown in b. Furthermore, in this system, increasing temperature also promotes the Fenton reaction; when the temperature rises to 50°C... o At C, the intensity of the characteristic absorption peak at 420 nm is significantly increased, indicating the generation of more hydroxyl radicals and thus increasing the hydroxyl radical generation rate. Figure 8 As shown in c, this indicates that the effect of Mo-PDA nanoparticles in generating hydroxyl radicals is dependent on particle concentration / H2O2 concentration / temperature.

[0053] Furthermore, to compare the effects of Mo-PDA and Mo-PDA@Gox prepared in Example 1 on generating hydroxyl radicals, methylene blue (MB) was selected as the ROS scavenger. Since ·OH can induce the degradation of methylene blue (MB), this leads to a significant decrease in the absorption peak of MB at its maximum wavelength of 664 nm. Therefore, the effect of ·OH generation can be examined by observing the degradation of MB. 100 μg of Mo-PDA and Mo-PDA@Gox nanoparticles were respectively added with MB (100 μg·mL⁻¹). -1 ) and glucose (Glu) (1 mg·mL) -1 500 µL of each solution were reacted at 37 °C for 15 min. After centrifugation, the absorbance of the supernatant at 664 nm was measured using a UV-Vis absorption spectrometer.

[0054] The results are as follows Figure 9 As shown, under the combined action of methylene blue (MB) and glucose, Mo-PDA@Gox nanoparticles generate more hydroxyl radicals, and MB degrades more significantly. This indicates that Gox on Mo-PDA@Gox nanoparticles catalyzes glucose to produce more H2O2, thereby generating more hydroxyl radicals.

[0055] Enzyme catalytic performance of nanoparticles: like Figure 10 As shown, the autocatalytic process is achieved by the Gox on the Mo-PDA@Gox nanoparticles consuming glucose at the tumor site to produce gluconic acid. Monitoring the pH change when Mo-PDA@Gox nanoparticles were mixed with glucose confirmed that acid upregulation was achieved in this process. With the progress of the reaction, the pH gradually decreased, dropping from 5.96 to 2.91 after 4 hours. This indicates that Mo-PDA@Gox nanoparticles can react well with glucose solution to generate gluconic acid, thus lowering the pH and creating an acidic environment.

[0056] The effects of Mo-PDA nanoparticles on ROS production at the cellular level: Because tumor cells have higher H2O2 content than normal cells, DCFH-DA indicator was chosen to detect the presence of intracellular ROS in order to better assess the production of highly toxic ROS in tumor cells induced by Mo-PDA nanoparticles. This is because DCFH-DA can react with ROS and exhibits green fluorescence under CLSM. The detection steps are as follows: First, select a 6-well plate and culture 1 mL of HepG2 cells in the plate to achieve a cell concentration of 1 × 10⁻⁶. 4 Cells / well, cultured overnight. Add 1 mL of nanoparticles (100 μg / mL) to each well. -1 The cells were incubated for 6 hours after washing with PBS and dissolved in 100 μM H2O2. After incubation, the cells were washed three times with cold PBS, and then incubated for 30 minutes with 10 mM DCFH-DA indicator at room temperature. Finally, the cells were washed three times with PBS and observed under a fluorescence microscope.

[0057] Intracellular H2O2 via Mo 6+ / Mo 5+ Catalysis can lead to redox reactions that generate highly toxic ROS. For example... Figure 11 As shown, compared with the control group, the PDA group did not produce a green fluorescence signal, while the green fluorescence signal of Mo-PDA nanoparticles was enhanced, and the green fluorescence signal of Mo-PDA@Gox nanoparticles was even stronger. This indicates that Mo-PDA nanoparticles induce tumor cells to produce highly toxic ROS, while Mo-PDA@Gox nanoparticles generate H2O2 through the interaction of Gox with intracellular glucose, which increases the intracellular H2O2 concentration and thus produces more ROS.

[0058] In vitro cytotoxicity test: The cytotoxicity of Mo-PDA nanoparticles was detected using the MTT assay, with MCF-7 and HepG2 cells selected for the test. Cultured cells (MCF-7 and HepG2 cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS), at 37°C, 95% air, and 5% CO2) were seeded at a cell density of 1.0 × 10⁶ cells / year. 4 Incubate overnight in 96-well plates with nanoparticles per well. Then, add different concentrations of nanoparticles (3.125, 6.25, 12.5, 25, 50, and 100 μg·mL⁻¹). -1 The cells were added and cultured for 24 h and 48 h. After the culture was completed, the cell viability as detected by the MTT assay was calculated using the following formula:

[0059] After incubating Mo-PDA nanoparticles with two types of tumor cells for 24 h and 48 h, respectively, the biosafety of the nanoparticles was tested, and the results are as follows: Figure 12 As shown, when the concentration of Mo-PDA nanoparticles is 100 μg·mL -1 Both cell lines (HepG2 and MCF-7) showed a cell viability of up to 90%. Figure 12 (a) and (b) confirm that Mo-PDA nanoparticles have high biocompatibility.

[0060] Mo-PDA@GOx nanoparticles synergistic photothermal-chemical kinetics-starvation therapy: The photothermal-chemokinetic-starvation synergistic therapeutic effect of Mo-PDA@GOx nanoparticles was analyzed using the standard MTT assay. HepG2 cells were seeded in 96-well plates, and control, Mo-PDA nanoparticle group, Mo-PDA nanoparticle + H2O2 group, Mo-PDA@GOx nanoparticle group, Mo-PDA nanoparticle + Laser group, and Mo-PDA@GOx nanoparticle + H2O2 + Laser group (1.0 W·cm⁻¹) were established. -2 (808 nm). Continue culturing for 24 h, then add 10 μL of MTT assay reagent to each well. Incubate for 2 h, discard the supernatant, add 150 μL of DMSO to each well, and use a microplate reader to test cell viability.

[0061] To assess the photothermal-chemical kinetics-starvation synergistic therapeutic effect of Mo-PDA@Gox nanoparticles, we used the standard MTT assay to detect the in vitro antitumor activity of different treatment groups on HepG2 cells. Figure 13As the concentration increased, cell viability decreased, indicating that different treatment groups exhibited concentration-dependent cell death. Under the same concentration conditions, the Mo-PDA@Gox nanoparticle + H2O2 + light irradiation group showed better antitumor activity. This could serve as an ideal photothermal-chemokinetic-starvation synergistic therapeutic agent.

Claims

1. A nanoparticle for synergistic tumor therapy, characterized in that: The composite nanoparticles Mo-PDA are prepared by mixing and reacting the inorganic active ingredient phosphomolybdic acid Mo-POM with dopamine, and then loading glucose oxidase GOx to form spherical Mo-PDA@GOx nanoparticles. The mixing reaction involves taking phosphomolybdic acid Mo-POM, adding deionized water, then adding dopamine and stirring continuously for 1 hour, and reacting continuously at 150~170℃ for 14~18 hours.

2. The nanoparticles for synergistic tumor treatment as described in claim 1, characterized in that: The ratio of phosphomolybdic acid (Mo-POM), dopamine, and deionized water is 45-50 mg: 4-6 mg: 10 mL.

3. The Mo-PDA@GOx nanoparticle as described in claim 2, characterized in that: The phosphomolybdic acid Mo-POM is (NH4)6Mo7O 24 • 4H2O and NaH2PO4•2H2O were dissolved in ultrapure water, and ascorbic acid was added while stirring. Then ethanol was added to precipitate the precipitate. After centrifugation, the precipitate was washed three times with water and ethanol and freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM.

4. The nanoparticles for synergistic tumor treatment as described in claim 3, characterized in that: The (NH4)6Mo7O 24 The dosage of 4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid is 2.4~2.6g: 0.17~0.2g: 10mL: 20mL, and the concentration of ascorbic acid is 0.48~0.51g / mL.

5. A method for preparing nanoparticles for synergistic tumor treatment, characterized in that, Includes the following steps: (1) Synthesis of Mo-POM phosphomolybdic acid: (NH4)6Mo7O 24 • 4H2O and NaH2PO4•2H2O were dissolved in ultrapure water, and ascorbic acid was added while stirring. Then, 40 mL of ethanol was added to precipitate the precipitate. The precipitate was centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which is phosphomolybdic acid Mo-POM. The (NH4)6Mo7O 24 The dosage of 4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid is 2.4~2.6g: 0.17~0.2g: 10mL: 20mL, and the concentration of ascorbic acid is 0.48~0.51g / mL. (2) Synthesis of Mo-PDA nanoparticles: Weigh out Mo-POM phosphomolybdic acid, add deionized water, then add dopamine (DA) and stir continuously for 1 hour. Place the mixture into a stainless steel reactor and react continuously at 150-170℃ for 14-18 hours. Centrifuge the product and wash it twice with water to obtain Mo-PDA nanoparticles. The ratio of Mo-POM phosphomolybdic acid, dopamine and deionized water is 45-50 mg: 4-6 mg: 10 mL. (3) Synthesis of Mo-PDA@GOx nanoparticles: Mo-PDA nanoparticles were mixed with glucose oxidase (GOx) and reacted under light-protected conditions for 24 h. The mixture was then removed, centrifuged, and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The mass ratio of Mo-PDA nanoparticles to GOx was 1:2.

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