A Mo-PDA@GOx nanoparticle and its preparation method and application

Abstract

The present invention discloses Mo-PDA@GOx nanoparticles, their preparation method, and applications, relating to the field of drug preparation technology. The nanoparticles are spherical Mo-PDA nanoparticles prepared by mixing Mo-POM phosphomolybdic acid with dopamine, and then loaded with glucose oxidase (GOx). The mixing reaction involves adding Mo-POM phosphomolybdic acid to deionized water, followed by dopamine, with continuous stirring for 1 hour, and then reacting at 150-170°C for 14-18 hours. In this invention, Mo-PDA@GOx nanoparticles for photothermal-chemokinetic-starvation synergistic tumor treatment are synthesized by combining PDA with a Mo-containing polyacid and externally loading glucose oxidase (GOx). The nanoparticles exhibit an excellent photothermal conversion efficiency of 67.2%. These nanoparticles effectively utilize photothermal-chemokinetic-starvation synergistic therapy, enhancing the therapeutic effect on tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to Mo-PDA@GOx nanoparticles and a preparation method and application thereof. Background Art

[0002] Cancer remains a major cause of death worldwide, originating from a multi-stage process in which normal cells transform into tumor cells. Chemotherapy remains the primary treatment for tumors, but the non-targeted delivery of chemotherapy drugs in the body often leads to unnecessary drug accumulation within normal tissues, resulting in suboptimal tumor treatment outcomes, accompanied by adverse reactions and multidrug resistance. The rapid development of nanotechnology and nanomaterials has opened up new avenues for the prevention, diagnosis, and treatment of tumors. The synergistic combination of photothermal therapy, starvation therapy, and chemodynamic therapy can help achieve optimal therapeutic effects from anti-tumor drugs while minimizing toxic side effects.

[0003] Polydopamine (PDA) has attracted increasing attention due to its unique physicochemical properties. Its diverse functional groups, such as catechol and amino groups, can react with metal ions to further modify the material's functionality. PDA exhibits excellent biocompatibility and can be used as a drug carrier for both photothermal and chemotherapeutic applications.

[0004] Chemodynamic therapy (CDT) holds promise for cancer treatment by generating reactive oxygen species (ROS). Mo-containing polysalts, due to their high water solubility, can act as Fenton reagents, reacting with H₂O₂ to generate cytotoxic ROS. However, the effectiveness of CDT is still limited by insufficient endogenous H₂O₂ in the tumor microenvironment (TME). Therefore, it is imperative to enhance the catalytic efficiency of the Fenton reaction by selectively increasing H₂O₂ levels in tumors. Summary of the Invention

[0005] The present invention aims to provide Mo-PDA@GOx nanoparticles.

[0006] Another object of the present invention is to provide a method for preparing the Mo-PDA@GOx nanoparticles.

[0007] The third object of the present invention is to apply the above-mentioned Mo-PDA@GOx nanoparticles.

[0008] In this invention, polydopamine (PDA) and phosphomolybdic acid (Mo-POM) are combined to form a combination of photothermal therapy and chemodynamic therapy for tumor treatment. However, this is still limited by insufficient endogenous H2O2 in the TME, resulting in suboptimal synergistic therapeutic effects. To address this H2O2 limitation, the present invention further incorporates the biocatalyst glucose oxidase (GOx) to effectively convert glucose within the tumor into gluconic acid and H2O2. Furthermore, compared to 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.

[0009] However, in the process of forming a composite carrier with PDA and phosphomolybdic acid Mo-POM and then loading GOx, it was found that the surface functional groups of PDA were excessively wrapped and covered by phosphomolybdic acid Mo-POM, resulting in a decrease in the binding performance between the carrier and GOx. GOx was difficult to load into the composite carrier, resulting in poor drug loading effect and low Gox loading concentration, which led to the inability of GOx to effectively function in the nanoparticles.

[0010] In the present invention, the prepared phosphomolybdic acid Mo-POM and dopamine are reacted at a specific temperature to form a composite carrier with a particle size of about 50 nm. In the formed composite carrier, the polydopamine PDA formed simultaneously exposes more amino groups. The amino groups on the surface of PDA can interact with functional groups (such as carboxyl groups, hydroxyl groups, etc.) in the GOx molecules through covalent bonds (such as amide bonds) or non-covalent bonds (such as hydrogen bonds, electrostatic interactions), thereby achieving GOx fixation. At the same time, the surface charge density of the small-particle carrier is high, which can form a stronger electrostatic interaction with GOx. In addition, the small-particle carrier has a higher specific surface area, which can provide more active sites to interact with GOx through physical adsorption or chemical binding. The carrier prepared by the present invention improves the loading efficiency of GOx through the synergistic effect of the above multiple effects.

[0011] In the Mo-PDA@GOx nanoparticles, which are efficiently loaded with GOx, GOx effectively exerts a specific starvation effect. This starvation treatment results in increased H₂O₂ levels, providing ample substrate for the Fenton reaction. Furthermore, this elevated H₂O₂, combined with CDT, accelerates the generation of •OH. Exogenous stimuli, such as near-infrared (NIR) laser irradiation, can also enhance the CDT effect with the assistance of photothermal agents.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] A Mo-PDA@GOx nanoparticle is characterized in that: the composite nanoparticle Mo-PDA is prepared by a mixed reaction of phosphomolybdic acid Mo-POM and dopamine, and then glucose oxidase (GOx) is loaded to form a spherical Mo-PDA@GOx nanoparticle.

[0014] Furthermore, the mixed reaction is to take phosphomolybdic acid Mo-POM, add deionized water, then add dopamine and stir continuously for 1 hour, and react continuously at 150-170° C. for 14-18 hours.

[0015] Furthermore, the usage ratio of the phosphomolybdic acid Mo-POM, dopamine and deionized water is 45-50 mg: 4-6 mg: 10 mL.

[0016] Furthermore, the phosphomolybdic acid Mo-POM is (NH4)6Mo7O 24 •4H2O and NaH2PO4•2H2O are dissolved in ultrapure water, and ascorbic acid is added while stirring. Subsequently, ethanol is added to precipitate the solution, which is centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, namely phosphomolybdic acid Mo-POM.

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

[0018] A method for preparing Mo-PDA@GOx nanoparticles, characterized by comprising the steps of synthesizing phosphomolybdic acid Mo-POM, synthesizing Mo-PDA nanoparticles and synthesizing Mo-PDA@GOx nanoparticles, specifically using (NH4)6Mo7O 24 •4H2O and NaH2PO4•2H2O were used as raw materials to synthesize phosphomolybdic acid Mo-POM, and 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, which were then compounded with glucose oxidase to synthesize Mo-PDA@GOx nanoparticles.

[0019] Furthermore, the synthesis of phosphomolybdic acid Mo-POM is to (NH4)6Mo7O 24 •4H2O and NaH2PO4•2H2O are dissolved in ultrapure water, and ascorbic acid is added while stirring. Subsequently, ethanol is added to precipitate the solution, which is centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, namely phosphomolybdic acid Mo-POM.

[0020] Furthermore, the (NH4)6Mo7O 24The amounts of •4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid are 2.4-2.6 g: 0.17-0.2 g: 10 mL: 20 mL, and the concentration of ascorbic acid is 0.48-0.51 g / mL.

[0021] Furthermore, the usage ratio of the phosphomolybdic acid Mo-POM, dopamine and deionized water is 45-50 mg: 4-6 mg: 10 mL.

[0022] Furthermore, the mass ratio of the Mo-PDA nanoparticles to GOx is 1:2.

[0023] Most specifically, a method for preparing Mo-PDA@GOx nanoparticles is characterized by comprising the following steps:

[0024] (1) Synthesis of phosphomolybdic acid Mo-POM:

[0025] (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, 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 amounts of 4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid are 2.4-2.6 g: 0.17-0.2 g: 10 mL: 20 mL, and the concentration of ascorbic acid is 0.48-0.51 g / mL;

[0026] (2) Synthesis of Mo-PDA nanoparticles:

[0027] Mo-POM phosphomolybdic acid was weighed, deionized water was added, and then dopamine (DA) was added and stirred continuously for 1 hour. The mixture was placed in a stainless steel reactor and reacted continuously at 150-170°C for 14-18 hours. The product was centrifuged and washed twice with water to obtain Mo-PDA nanoparticles. The dosage ratio of Mo-POM phosphomolybdic acid, dopamine, and deionized water was 45-50 mg: 4-6 mg: 10 mL.

[0028] (3) Synthesis of Mo-PDA@GOx nanoparticles:

[0029] Mo-PDA nanoparticles were mixed with glucose oxidase (GOx), reacted for 24 h in the dark, taken out, centrifuged, and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The mass ratio of the Mo-PDA nanoparticles to GOx was 1:2.

[0030] Application of Mo-PDA@GOx nanoparticles prepared by the above method in the preparation of anti-tumor drugs.

[0031] The present invention has the following technical effects:

[0032] In this study, Mo-PDA@GOx nanoparticles were synthesized by combining PDA with a Mo-containing polysalt and externally loading glucose oxidase (GOx) for synergistic photothermal, chemokinetic, and starvation tumor therapy. The GOx loading in these nanoparticles reached 57.3%. These nanoparticles exhibited an excellent photothermal conversion efficiency of 67.2%. These nanoparticles effectively promoted synergistic photothermal, chemokinetic, and starvation therapy, enhancing 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 nanocomposites in synergistic tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : TEM image of Mo-PDA nanoparticles prepared by the present invention.

[0034] Figure 2 : The hydrated particle size of Mo-PDA nanoparticles and Mo-PDA@Gox nanoparticles prepared in the present invention.

[0035] Figure 3 : Zeta potential diagram of Mo-PDA nanoparticles and Mo-PDA@Gox nanoparticles prepared in the present invention.

[0036] Figure 4 : Particle size distribution diagram of Mo-PDA1 prepared in Comparative Example 2.

[0037] Figure 5 : Scanning electron microscope image of Mo-PDA1 prepared in Comparative Example 2.

[0038] Figure 6 : Mo-PDA nanoparticles aqueous solution with different concentrations was irradiated by 808 nm laser (1.0 W·cm -2 ) warming trend.

[0039] Figure 7 : Mo-PDA nanoparticles under 808 nm laser irradiation (1.0 W·cm -2 )'s photostability.

[0040] Figure 8 : Analysis of the effects of different particle concentrations (a), different H2O2 concentrations (b) and different temperatures (c) on the generation of ROS by Mo-PDA nanoparticles.

[0041] Figure 9:Analysis of the effects of Mo-PDA@Gox and Mo-PDA on catalyzing ROS production from glucose.

[0042] Figure 10 : pH change of Mo-PDA@GOx nanoparticles dispersed in glucose solution.

[0043] Figure 11 : CLSM images of ROS production in HepG2 cells under different treatment conditions (Scale bar: 100 μm).

[0044] 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.

[0045] Figure 13 :The synergistic therapeutic effect of Mo-PDA@Gox nanoparticles was determined by MTT method. DETAILED DESCRIPTION

[0046] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0047] Example 1

[0048] A method for preparing Mo-PDA@GOx nanoparticles comprises the following steps:

[0049] (1) Synthesis of phosphomolybdic acid Mo-POM:

[0050] 2.6 g (NH4)6Mo7O 24 •4H2O and 0.2 g NaH2PO4•2H2O were dissolved in 10 mL ultrapure water, and 20 mL of 0.48 g / mL ascorbic acid was added while stirring. Subsequently, 40 mL of ethanol was added to precipitate the precipitate, which was then centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which was phosphomolybdic acid Mo-POM.

[0051] (2) Synthesis of Mo-PDA nanoparticles:

[0052] 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 hour. Place in a stainless steel reactor and react at 170°C for 14 hours. Centrifuge the product and wash it twice with water to obtain Mo-PDA nanoparticles.

[0053] (3) Synthesis of Mo-PDA@GOx nanoparticles:

[0054] 1 mg of Mo-PDA nanoparticles and 2 mg of Gox were added to 1 mL of water and mixed. After reacting for 24 h in the dark, the mixture was centrifuged and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The Mo-PDA nanoparticles and GOx were mixed.

[0055] Example 2

[0056] A method for preparing Mo-PDA@GOx nanoparticles comprises the following steps:

[0057] (1) Synthesis of phosphomolybdic acid Mo-POM:

[0058] 2.4 g (NH4)6Mo7O 24 •4H2O and 0.17 g NaH2PO4•2H2O were dissolved in 10 mL ultrapure water, and 20 mL of 0.51 g / mL ascorbic acid was added while stirring. Subsequently, 40 mL of ethanol was added to precipitate the precipitate, which was then centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which was phosphomolybdic acid Mo-POM.

[0059] (2) Synthesis of Mo-PDA nanoparticles:

[0060] 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 hour. Place the mixture in a stainless steel reactor and react at 150 °C for 18 hours. The product is centrifuged and washed twice with water to obtain Mo-PDA nanoparticles.

[0061] (3) Synthesis of Mo-PDA@GOx nanoparticles:

[0062] 1 mg of Mo-PDA nanoparticles and 2 mg of Gox were added to 1 mL of water and mixed. After reacting for 24 h in the dark, the mixture was centrifuged and washed twice with water to obtain Mo-PDA@GOx nanoparticles. The Mo-PDA nanoparticles and GOx were mixed.

[0063] Example 3

[0064] A method for preparing Mo-PDA@GOx nanoparticles comprises the following steps:

[0065] (1) Synthesis of phosphomolybdic acid Mo-POM:

[0066] 2.47 g (NH4)6Mo7O 24•4H2O and 0.18 g NaH2PO4•2H2O were dissolved in 10 mL ultrapure water. 20 mL of 0.5 mg / mL ascorbic acid was added while stirring. 40 mL of ethanol was then added to precipitate the product. The product was centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, which was phosphomolybdic acid Mo-POM.

[0067] (2) Synthesis of Mo-PDA nanoparticles:

[0068] 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 hour. Place in a stainless steel reactor and react at 160°C for 16 hours. Centrifuge the product and wash it twice with water to obtain Mo-PDA nanoparticles.

[0069] (3) Synthesis of Mo-PDA@GOx nanoparticles:

[0070] 1 mg of Mo-PDA nanoparticles and 2 mg of glucose oxidase (GOx) were added to 1 mL of water and mixed. After reacting for 24 h in the dark, the mixture was centrifuged and washed twice with water to obtain Mo-PDA@GOx nanoparticles.

[0071] Morphological characterization of nanoparticles:

[0072] The morphology of the synthesized Mo-PDA nanoparticles was characterized, e.g. Figure 1 As shown in the TEM image, it can be seen that the nanoparticles are spherical structures with uniform particle size, the particle size is about 56 nm, and the particles are evenly dispersed.

[0073] Characterization of hydrated particle size and Zeta potential of nanoparticles:

[0074] The hydrated particle size of Mo-PDA nanoparticles and Mo-PDA@Gox nanoparticles was measured to be 76.6 nm and 210.5 nm by nanoparticle size and Zeta potential analyzer, which are slightly larger than the nanoparticle size results characterized by TEM. Figure 2 As shown in Figure 2, this is because the nanoparticles associate with water molecules in the solution to form a hydration film, which increases the particle size. The above results show that the Mo-PDA and Mo-PDA@Gox nanoparticles have uniform particle size and good stability and dispersibility in water. Figure 3 From the Zeta potential diagram shown, we can see that the Zeta potential of Mo-PDA nanoparticles is -68 mV, and the Zeta potential of Mo-PDA@Gox is -26 mV. After Mo-PDA is loaded with Gox, the potential increases, indicating that the Gox loading is successful.

[0075] Comparative Example 1:

[0076] Compared with Example 1, the difference is that the Mo-PDA nanoparticle synthesis step adopts a physical mixing method, and the remaining steps are the same as Example 1, that is, step (2) is specifically as follows:

[0077] Synthesis of polydopamine (PDA): Mix 10 mL of ethanol with 1 mL of 2 mol / L ammonia solution, then add 20 mL of water. Once thoroughly mixed, add 100 mg of dopamine (DA) and continue stirring for 24 hours. Centrifuge at 9000 rpm and wash the product three times with water and then with alcohol.

[0078] 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.

[0079] The Mo-PDA nanoparticles prepared by this method have a low content of Mo-POM phosphomolybdic acid loaded on the PDA surface, which affects the photothermal and chemical kinetic effects of the final Mo-PDA nanoparticles and produces a low content of reactive oxygen species (ROS). Secondly, the final Mo-PDA nanoparticles are synthesized by simple physical mixing of PDA and Mo-POM phosphomolybdic acid. The functional groups on the PDA surface are covered by Mo-POM phosphomolybdic acid, resulting in a low GOx loading in Mo-PDA@GOx.

[0080] Comparative Example 2:

[0081] Compared with Example 1, the difference is that when preparing Mo-PDA, phosphomolybdic acid Mo-POM is replaced with gadolinium phosphomolybdate (K 17 [Gd(P2Mo 17 O 61 )2]·26H2O), to prepare Mo-PDA, and the remaining steps were the same as in Example 1. Among the nanoparticles finally prepared, the nanoparticles corresponding to gadolinium phosphomolybdate were denoted as Mo-PDA1.

[0082] like Figure 4 As shown in Figure 2, the particle size of Mo-PDA1 prepared by this method is about 600 nm and its stability is poor. Figure 5 ) It can be seen from the figure that the particles have undergone coagulation and aggregated into clusters. The particles are poorly dispersed and have irregular morphology.

[0083] The loading efficiency of GOx directly determines the intensity and duration of the starvation therapy (ST) effect. 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 to obtain a supernatant. The supernatant and the original drug solution were assayed for GOx content using a BCA protein assay kit, and the GOx loading efficiency was calculated. The drug loading efficiency was calculated according to the formula:

[0084]

[0085] Calculated GOx drug loading efficiencies for 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. Due to the low GOx drug loading efficiencies of the nanoparticles prepared in Comparative Examples 1 and 2, subsequent efficacy testing was not performed.

[0086] Photothermal conversion effect test:

[0087] In order to investigate the photothermal conversion effect of Mo-PDA nanoparticles prepared in Example 1, a near-infrared (NIR) laser (808 nm, 1.0 W·cm -2 ) were irradiated with five groups of 500 μL of different concentrations (0, 50, 100, 200 and 400 μg·mL -1 ) Mo-PDA nanoparticle solution for 600 s, recording the temperature change every 30 s. After completion, stop irradiation and record the cooling process of the solution. Under the same conditions, deionized water was irradiated with laser as a control group. Calculate the photothermal conversion efficiency ( η ):

[0088]

[0089] h is the heat transfer coefficient, S is the surface area of ​​the container, T max is the equilibrium temperature, T sur is the ambient temperature, Q dis is the heat absorbed by the solvent, I is the power of the irradiating light (1.0 W cm -2 ), A 808 is the UV-vis absorbance value of the Mo-PDA nanoparticle solution at 808 nm, and the value of hS is calculated according to the following formula:

[0090]

[0091] τ s is the sample system time constant, m D and C Dis the mass and specific heat capacity of water as the solvent.

[0092] The photothermal conversion ability of Mo-PDA nanoparticles with different concentrations was analyzed. Under 808 nm laser irradiation, the temperature changes of Mo-PDA nanoparticles with different concentrations were recorded. 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 hardly changes.

[0093] Figure 7 Mo-PDA nanoparticles were irradiated by 808 nm laser (1.0 W·cm -2 The cyclic photostability of Mo-PDA nanoparticles was investigated. The temperature changes of the Mo-PDA nanoparticles were recorded using an IRT instrument. After four repeated cycles of laser irradiation and cooling, the temperature increase of the Mo-PDA nanoparticles remained unchanged. These results demonstrate that Mo-PDA nanoparticles possess excellent photothermal stability and exhibit concentration / irradiation time-dependent photothermal behavior. The photothermal conversion efficiency of the Mo-PDA nanoparticles was calculated to be 67.2%.

[0094] Chemical kinetics performance test:

[0095] In order to test the ROS generation effect of the Mo-PDA nanoparticles prepared in Example 1, o-phenylenediamine (OPD) was selected as a ROS scavenger. This is because ROS can catalyze the oxidation of OPD into 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:

[0096] Mo-PDA nanoparticles were prepared at different concentrations (400, 200, 100 and 50 μg mL -1 ) were dispersed into 1 mL of a mixture of H2O2 and OPD, where the concentration of H2O2 was 50 mM and the concentration of OPD was 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 the ROS production effect.

[0097] The results are as follows Figure 8As shown in the figure, when Mo-PDA nanoparticles react with OPD or H2O2 alone, no characteristic absorption peak at 420 nm is generated. Only when they react with OPD and H2O2 simultaneously can the absorption peak at 420 nm be generated. This shows that Mo-PDA nanoparticles cannot react with OPD or H2O2 alone to produce hydroxyl radicals. Only when they react with OPD and H2O2 simultaneously can hydroxyl radicals be generated. With the increase of Mo-PDA particle concentration, the intensity of the absorption peak at 420 nm increases. Figure 8 As shown in a, it shows that Mo in Mo-PDA nanoparticles 5+ It also tested that the combination of OPD and H2O2 alone could not produce free radicals. After further introduction of Mo-PDA, the intensity of the absorption peak at 420nm increased with the increase of H2O2 concentration under the condition of constant OPD concentration. Figure 8 In addition, in this system, the increase in temperature also promotes the Fenton reaction. When the temperature is increased to 50 o C, the characteristic absorption peak intensity at 420 nm is significantly increased, that is, more hydroxyl radicals are generated, and the hydroxyl radical generation rate is increased, such as Figure 8 As shown in Figure c, this indicates that the effect of Mo-PDA nanoparticles in generating hydroxyl radicals is dependent on particle concentration / H2O2 concentration / temperature.

[0098] In addition, in order to compare the effects of Mo-PDA and Mo-PDA@Gox prepared in Example 1 on the generation of hydroxyl radicals, methylene blue (MB) was selected as a ROS scavenger. Since ·OH can induce the degradation of methylene blue (MB), this can cause a significant decrease in the absorption peak of MB at the maximum wavelength of 664 nm. Therefore, the generation effect of ·OH can be examined by observing the degradation of MB. 100 μg Mo-PDA and Mo-PDA@Gox nanoparticles were added with MB (100 μg·mL -1 ) and glucose (Glu) (1 mg mL -1 ) were added to 500 μL each and reacted at 37°C for 15 min. The mixture was centrifuged and the absorbance of the supernatant at 664 nm was measured using a UV-visible absorption spectrometer.

[0099] The results are as follows Figure 9 As shown in the figure, under the combined action of methylene blue (MB) and glucose, Mo-PDA@Gox nanoparticles produced more hydroxyl radicals and MB was degraded more significantly, indicating that Gox on Mo-PDA@Gox nanoparticles catalyzed glucose to produce more H2O2, thereby generating more hydroxyl radicals.

[0100] Enzyme catalytic properties of nanoparticles:

[0101] like Figure 10 As shown, the autocatalytic process occurs when the Gox on the Mo-PDA@Gox nanoparticles consumes glucose in the tumor site, thereby producing gluconic acid. Monitoring the pH changes when the Mo-PDA@Gox nanoparticles are mixed with glucose confirms the upregulation of acidity in this process. As the reaction progresses, the pH gradually decreases, dropping from 5.96 to 2.91 after 4 hours. This indicates that the Mo-PDA@GOx nanoparticles react effectively with the glucose solution, generating gluconic acid, which lowers the pH and creates an acidic environment.

[0102] Effects of Mo-PDA nanoparticles on ROS production at the cellular level:

[0103] Since the H2O2 content in tumor cells is higher than that in normal cells, in order to better evaluate the highly toxic ROS produced by Mo-PDA nanoparticles in tumor cells, DCFH-DA indicator was selected to detect the presence of ROS in cells. This is because DCFH-DA can react with ROS and show green fluorescence under CLSM. The detection steps are as follows:

[0104] First, select a 6-well plate and culture 1 mL of HepG2 cells in the plate to make the cell concentration 1×10 4 1 mL of nanoparticles (100 μg mL -1 ) and 100 μM H₂O₂ dissolved in PBS-washed cell culture medium and incubated for another 6 h. After incubation, cells were washed three times with cold PBS and incubated for 30 min at room temperature with 10 mM DCFH-DA indicator. Finally, cells were washed three times with PBS and observed under a fluorescence microscope.

[0105] H2O2 in the cell passes through Mo 6+ / Mo 5+ Catalyzes redox reactions to generate highly toxic ROS. Figure 11 As shown in the figure, compared with the control group, the PDA group did not produce green fluorescence signals, while the green fluorescence signal of Mo-PDA nanoparticles was enhanced, and the green fluorescence signal of Mo-PDA@Gox nanoparticles was even stronger, indicating that Mo-PDA nanoparticles induced tumor cells to produce highly toxic ROS, while Mo-PDA@Gox nanoparticles produced H2O2 through the reaction of Gox with glucose in the cells, increasing the H2O2 concentration in the cells, thereby generating more ROS.

[0106] In vitro cytotoxicity test:

[0107] The cytotoxicity of Mo-PDA nanoparticles was detected by MTT method. MCF-7 and HepG2 cells were selected for detection. The cultured cells (MCF-7 cells and HepG2 cells were cultured in a medium (1640) containing 10% fetal bovine serum (FBS), 37°C, 95% air, 5% CO2 in an incubator) were inoculated at a cell density of 1.0×10 4 The cells were plated on a 96-well plate and incubated overnight. Then different concentrations of nanoparticles (3.125, 6.25, 12.5, 25, 50, and 100 μg mL) were added. -1 ) were added to the cells and cultured for 24 h and 48 h. After the culture was completed, the cell activity detected by MTT assay was calculated using the following formula:

[0108]

[0109] After incubating Mo-PDA nanoparticles with two types of tumor cells for 24 h and 48 h, the biosafety of the nanoparticles was tested. Figure 12 As shown in Figure 2, when the concentration of Mo-PDA nanoparticles is 100 μg·mL -1 The activity of the two cell lines (HepG2 and MCF-7) was as high as 90% ( Figure 12 a, b), confirming that Mo-PDA nanoparticles have high biosafety.

[0110] Mo-PDA@GOx nanoparticles synergistic photothermal-chemokinetic-starvation therapy:

[0111] The photothermal-chemodynamic-starvation synergistic therapeutic effect of Mo-PDA@GOx nanoparticles was analyzed by the standard MTT method. HepG2 cells were seeded in 96-well plates, and the control group, 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 -2 , 808 nm). Continue incubation for 24 h, then add 10 μL of MTT detection reagent to each well. Incubate for 2 h, discard the supernatant, add 150 μL of DMSO to each well, and assay cell viability using a microplate reader.

[0112] Regarding the photothermal-chemokinetic-starvation synergistic therapeutic effect of Mo-PDA@Gox nanoparticles, we used the standard MTT method to detect the in vitro antitumor activity of different treatment groups on HepG2 cells ( Figure 13As the concentration increased, cell viability decreased, indicating that cell death in the different treatment groups was concentration-dependent. At the same concentration, the Mo-PDA@Gox nanoparticle + H2O2 + light treatment group exhibited superior antitumor activity. This makes it an ideal agent for photothermal-chemokinetic-starvation synergistic therapy.

Claims

1. A Mo-PDA@GOx nanoparticle, characterized by: Composite nanoparticles Mo-PDA are prepared by mixing phosphomolybdic acid Mo-POM with dopamine, and then Mo-PDA@GOx nanoparticles with spherical structures are loaded with glucose oxidase GOx. The mixing reaction is to take phosphomolybdic acid Mo-POM, add deionized water, then add dopamine and stir continuously for 1 hour, and react continuously at 150-170°C for 14-18 hours. The amount ratio of the phosphomolybdic acid Mo-POM, dopamine and deionized water is 45-50 mg: 4-6 mg: 10 mL. The phosphomolybdic acid Mo-POM is (NH4)6Mo7O 24 •4H2O and NaH2PO4•2H2O are dissolved in ultrapure water, and ascorbic acid is added while stirring. Subsequently, ethanol is added to precipitate the solution, which is centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, namely phosphomolybdic acid Mo-POM.

2. The Mo-PDA@GOx nanoparticle according to claim 1, wherein: The (NH4)6Mo7O 24 The amounts of •4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid are 2.4-2.6 g: 0.17-0.2 g: 10 mL: 20 mL, and the concentration of ascorbic acid is 0.48-0.51 g / mL.

3. A method for preparing Mo-PDA@GOx nanoparticles, characterized by: The invention comprises the synthesis of phosphomolybdic acid Mo-POM, the synthesis of Mo-PDA nanoparticles and the synthesis of Mo-PDA@GOx nanoparticles, wherein the method adopts (NH4)6Mo7O 24 •4H2O and NaH2PO4•2H2O were used as raw materials to synthesize phosphomolybdic acid Mo-POM, and 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, which were then compounded with glucose oxidase GOx to synthesize Mo-PDA@GOx nanoparticles.

4. The method for preparing Mo-PDA@GOx nanoparticles according to claim 3, wherein: The synthesis of phosphomolybdic acid Mo-POM is to (NH4)6Mo7O 24 •4H2O and NaH2PO4•2H2O are dissolved in ultrapure water, and ascorbic acid is added while stirring. Subsequently, ethanol is added to precipitate the solution, which is centrifuged, washed three times with water and ethanol, and freeze-dried to obtain a powder, namely phosphomolybdic acid Mo-POM.

5. The method for preparing Mo-PDA@GOx nanoparticles according to claim 4, wherein: The (NH4)6Mo7O 24 The amounts of •4H2O, NaH2PO4•2H2O, ultrapure water, and ascorbic acid are 2.4-2.6 g: 0.17-0.2 g: 10 mL: 20 mL, and the concentration of ascorbic acid is 0.48-0.51 g / mL.

6. The method for preparing Mo-PDA@GOx nanoparticles according to claim 5, wherein: The usage ratio of the phosphomolybdic acid Mo-POM, dopamine and deionized water is 45-50 mg: 4-6 mg: 10 mL.

7. Use of Mo-PDA@GOx nanoparticles prepared by the method according to claim 6 in the preparation of anti-tumor drugs.

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