Cholesterol oxidase-loaded biological material as well as preparation method and anti-tumor application thereof

Abstract

A cholesterol oxidase-loaded biological material is a nanometer material and is characterized by comprising polyacrylic acid, cobalt oxyhydroxide and cholesterol oxidase, cobalt oxyhydroxide is adsorbed on polyacrylic acid to form a compound, and cholesterol oxidase is loaded on the compound. The size of the nano-biological material is about 100-150 nm, the nano-biological material is combined with ultrasound to obtain multiple nano-enzyme activities with enhanced effects and more excellent capabilities of generating active oxygen and relieving hypoxia, and a lipid raft structure on a cell membrane is damaged by the loaded cholesterol oxidase, so that cell migration is inhibited, and the effect of resisting tumor metastasis is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterial technology, and specifically relates to a cobalt oxyhydroxide (CoOOH) nanosheet with multi-enzyme activity, ultrasound enhancement effect and anti-cancer function and a cholesterol oxidase (COD) composite system loaded on its surface. The system achieves tumor treatment and inhibits tumor cell migration by catalyzing the generation of reactive oxygen species (ROS) and consuming cholesterol. Background Art

[0002] Tumor metastasis is the main cause of cancer-related death. Disrupting lipid rafts on cancer cell membranes to inhibit physical movement is a reliable strategy. In addition, a growing number of emerging cancer treatments have attracted widespread attention in recent years, such as photothermal therapy (PTT), enzyme-dynamic therapy (EDT), and sonodynamic therapy (SDT).

[0003] Nanozymes are a novel class of biocatalytic artificial nanomaterials that can catalyze the conversion of enzyme substrates into products under specific conditions according to enzyme kinetics. Their most notable feature is their ability to modulate reactive oxygen species (ROS) levels by accepting or donating electrons (MedComm–Biomaterials and Applications 2023, 2). Although factors such as substrate availability and enzyme activity limit their application, the application of exogenous ultrasound (US) to provide additional energy enhances the catalytic activity of nanozymes, overcoming the limited tissue penetration depth of traditional lasers and improving their clinical applicability. For example, researchers have designed a bionanomaterial called lanthanum ferrite (LaFeO3) that can enhance the production of ROS under ultrasound, thereby strengthening its anti-tumor effect (Advanced Materials 2022, 35).

[0004] In tumor cells, lipid rafts sense signals and regulate migration through surface proteins (Nature 1997, 387, 569). Other studies have found that depleting cholesterol can disrupt the integrity of lipid rafts, impairing integrins and other associated proteins on them, thereby inhibiting cancer cell migration (Journal of Controlled Release 2024, 371, 29). While cholesterol oxidase can be used to lower cholesterol levels (Cell Death & Disease 2014, 5, e1372), the instability of the native enzyme and the hypoxic conditions of the tumor microenvironment prevent this approach from achieving the desired effect. Summary of the Invention

[0005] In view of the above problems, one object of the present invention is to provide a method for preparing and applying a biomaterial that is simple to synthesize, highly effective in treating tumors and capable of inhibiting metastasis.

[0006] Another purpose of the present invention is to solve the problem of distant tumor metastasis by consuming cholesterol in tumor cells through cholesterol oxidase, while at the same time destroying cell membrane lipid rafts to cause further tumor apoptosis, and to jointly treat cancer with the enzyme-dynamic therapy of cobalt hydroxide nanozyme.

[0007] Another object of the present invention is to enhance the therapeutic effect of enzyme kinetics. The use of exogenous ultrasound provides additional energy to enhance the catalytic activity of nanozymes and overcome the limitations of traditional laser tissue penetration depth.

[0008] The biomaterial provided by the present invention (denoted as: CoPC) includes polyacrylic acid, cobalt oxyhydroxide and cholesterol oxidase. Cobalt oxyhydroxide is adsorbed on polyacrylic acid to form a complex (denoted as: CoP). Cholesterol oxidase is loaded on CoP, which has excellent anti-cancer (such as breast cancer) and anti-metastasis properties, and is also resistant to ultrasound (1 MHz; 1-1.5 W / cm 2 ; Duty cycle 50%; 3~5 min) can amplify this effect.

[0009] After being acted upon by ultrasound, the enzyme kinetics of the surface-loaded cholesterol oxidase cobalt oxyhydroxide provided by the present invention is also significantly improved, and can generate a variety of ultrasound-enhanced active oxygen molecules and oxygen.

[0010] In a biomaterial of the present invention, CoP is formed by adsorbing cobalt oxyhydroxide onto polyacrylic acid, and specific adsorption methods include physical adsorption and electrostatic adsorption.

[0011] Another biomaterial of the present invention is prepared by first preparing cobalt oxyhydroxide nanosheets, and then forming CoP with polyacrylic acid.

[0012] Another biomaterial of the present invention is obtained by mixing CoP and cholesterol oxidase in a mass ratio of 2:1 in a buffer solution (such as PBS, pH 7.4).

[0013] In the biomaterial of the present invention, the mass ratio of polyacrylic acid to cobalt oxyhydroxide is 1:10-1:1, and the content of cholesterol oxidase carried is 20.0%-30.2%.

[0014] A method for preparing CoPC, comprising: Cobalt chloride (e.g., 10 mM) is added to a sodium hydroxide solution (1 M) at a molar ratio of 1:3. Ultrasonication is first performed to uniformly disperse the solution. Then, sodium hypochlorite solution (e.g., 0.9 M) is quickly added to the bottle. The solution (pH 10 ± 0.2) turns brown-black. Ultrasonication is repeated (e.g., 5 minutes), and finally, centrifugation is performed to obtain cobalt oxyhydroxide (CoOOH) nanosheets. Next, cobalt oxyhydroxide (CoOOH) nanosheets are dispersed in water, and polyacrylic acid (concentration, e.g., 40 wt%) is added, with the polyacrylic acid and cobalt oxyhydroxide ratio being 1:10 to 1:1 by mass. The mixture is stirred (e.g., for 1 hour) and then centrifuged and washed to obtain a composite CoP. Finally, CoP and cholesterol oxidase COD are reacted in PBS at a mass ratio of 2:1 (eg, 6 hours), and then centrifuged and washed to obtain the cholesterol oxidase-loaded biomaterial CoPC.

[0015] Another method for preparing CoPC comprises: Cobalt chloride is added to a blue-capped bottle containing 10 mL of deionized water to form a solution (concentration, e.g., 10 mM). Stirring is performed to uniformly disperse the solution. 300 μL of 1 mol / L sodium hydroxide solution is added to the 10 mL cobalt chloride solution and ultrasonicated to uniformly disperse the solution. 500 μL of 0.9 mol / L sodium hypochlorite solution is then quickly added to the bottle. The solution quickly turns brown-black. Ultrasonication is performed again (e.g., 5 minutes), and finally centrifugation is performed to wash the solution. Cobalt oxyhydroxide (CoOOH) nanosheets are obtained. 5 mg of cobalt oxyhydroxide nanosheets were dispersed in deionized water, and 1.2 mL of 40% polyacrylic acid was added. The mixture was stirred for 1 hour and then centrifuged to obtain a polyacrylic acid-cobalt oxyhydroxide complex (CoP). The resulting CoP and cholesterol oxidase (COD) were mixed in PBS at a mass ratio of 2:1 and reacted for 6 hours. Finally, the cholesterol oxidase-loaded cobalt oxyhydroxide nanomaterial (CoPC) was obtained by centrifugation.

[0016] Beneficial effects achieved by the technical solution of the present invention: The biomaterial CoPC provided by the present invention is composed of cobalt oxyhydroxide nanosheets and cholesterol oxidase. The cholesterol oxidase is loaded on the cobalt oxyhydroxide nanosheets. The effect of generating reactive oxygen molecules and consuming cholesterol is amplified through ultrasonic intervention, thereby achieving the excellent anti-cancer and anti-metastasis effects of the nanomaterial, solving the problem that traditional nanozymes have poor therapeutic effects and are prone to metastasis.

[0017] The biomaterial CoPC provided by the present invention, when combined with ultrasound, has a stronger ability to exert the activity of various types of enzymes, and consumes cholesterol to produce hydrogen peroxide, thereby enhancing the production of cytotoxic killer molecules and improving the problem of unsatisfactory effects caused by limited hydrogen peroxide in the tumor microenvironment.

[0018] It has been verified that the biomaterial CoPC provided by the present invention does not have adverse effects on normal tissues and cells, and the ultrasonic effect can overcome the limitations of traditional laser tissue penetration depth and reduce tissue burns caused by excessive temperature.

[0019] The method for preparing the biomaterial of the present invention has simple process operation and high repeatability, and can form a biomaterial with good effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A flow chart of the bionanomaterial prepared for the present invention; Figure 2 TEM image of CoPC prepared in the present invention; Figure 3 TEM-EDS image of CoPC; Figure 4 This is the zeta potential diagram before and after cobalt oxyhydroxide is loaded with cholesterol oxidase; Figure 5 This is the curve of the reaction activity of CoPC at a wavelength of 652 nm versus time; Figure 6 This is a fluorescence detection result of CoPC consuming cholesterol to produce hydrogen peroxide; Figure 7 Electron paramagnetic resonance (EPR) spectra of CoPC in the presence and absence of cholesterol; Figure 8 The figure shows the cytotoxicity results of CoPC. DETAILED DESCRIPTION

[0021] The following examples are further given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description herein, and are not limited to the specific numerical values ​​exemplified below.

[0022] The process flow of the bionanomaterial prepared by the present invention is shown in FIG. Figure 1 The biomaterial preparation method of this embodiment is as follows: Add 300 μL of 1 mol / L sodium hydroxide solution to 10 mL of 10 mM cobalt chloride aqueous solution and stir well.

[0023] 500 μL of 0.9 mol / L sodium hypochlorite was quickly injected into the mixture and treated with water bath sonication for 5 minutes without any interference. The precipitate was collected by centrifugation and washed with deionized water.

[0024] 5 mg of the synthesized CoOOH nanosheets were redispersed in water, and 1.2 mL of polyacrylic acid (PAA) was added to the solution, followed by continuous stirring on a magnetic stirrer for 1 hour. Afterwards, the PAA-modified CoOOH (CoP) was collected by centrifugation and washing.

[0025] 40 μL of COD stock solution was dissolved in 5 mL of PBS, and 2 mg of the above-obtained CoP was added to the solution. The mixed solution was stirred at room temperature for 6 hours and finally centrifuged and washed to obtain the target CoPC.

[0026] The TEM image of the biomaterial CoPC synthesized in this example is shown in Figure 2 , intuitively showing that the material is a hexagonal nanosheet with a size of about 100-150 nanometers.

[0027] The results of TEM-EDS detection of CoPC are as follows Figure 3 , the signals of Co, O, P, and S were detected in the same sample area, proving that these elements are co-distributed on the nanomaterials.

[0028] Depend on Figure 4 The Zeta potential presented shows that the potential of the material has changed to varying degrees before and after polyacrylic acid modification and loading of cholesterol oxidase, proving the successful grafting of polyacrylic acid and the successful loading of cholesterol oxidase.

[0029] CoPC catalyzes the hydrogen peroxide reaction, and the generated product oxidizes the colorless TMB (3,3',5,5'-tetramethylbenzidine) to blue. The change in absorbance is used to quantitatively characterize the degree of reaction. Figure 5 Ultrasound significantly enhanced CoPC enzyme activity, demonstrating that the introduction of ultrasound can enhance CoPC's activity in generating hydroxyl radicals. The absorbance of the reaction system under ultrasound was significantly higher than that under non-ultrasound conditions, fully demonstrating the amplification effect of ultrasound on CoPC enzyme activity.

[0030] When cholesterol is added, both CoPC and cholesterol oxidase generate hydrogen peroxide, which is confirmed by the fluorescence generated by the probe after it captures hydrogen peroxide. Figure 6 The performance of CoPC-loaded cholesterol oxidase was still able to function and produce hydrogen peroxide in the presence of cholesterol, which was not greatly affected compared with simple cholesterol oxidase, indicating the stable performance of CoPC in consuming cholesterol.

[0031] Figure 7 It is the electron spin resonance (ESR) test result of the cascade catalytic generation of hydroxyl radicals after the reaction of CoPC with cholesterol, which can prove the ability of CoPC to generate hydroxyl radicals.

[0032] The cytotoxicity test of the samples was evaluated using the classic CCK-8 method. First, the cells were plated at 5×10 3 / Cells were seeded into 96-well plates at the same density (100 μg / ml) and incubated in a humidified CO2 incubator at 37°C with 5% CO2 for 24 hours to allow attachment. The culture medium of the adherent cells was then replaced with fresh culture medium containing the same concentration (the concentration was determined by the mass of cobalt) of CoP (150 μg / ml), CoP+US, CoPC, CoPC+US, and fresh culture medium alone, and incubated for another 4 hours. After incubation, both groups were sonicated for 5 minutes using an ultrasound therapy device at 1.2 MHz, 1.2 W / m2, and a 50% duty cycle. After sonication, the plates were incubated in a humidified CO2 incubator for 3 hours. The culture medium was removed and the plates were washed twice with fresh culture medium. 100 μL of serum-free culture medium containing 10% CCK-8 was added to each well, and the plates were incubated in a humidified CO2 incubator at 37°C with 5% CO2 for 1.5 hours. After gentle shaking, the absorbance was measured on a microplate reader (wavelength = 450 nm). The cytotoxicity index is expressed as the percentage of cell viability after sample treatment relative to the cell viability of the untreated blank control group. It can be seen that the nanomaterial combining natural enzymes and artificial enzymes can achieve a good therapeutic effect of ultrasound-assisted enzyme kinetics. Figure 8 It can be seen that under the conditions of ultrasound and cholesterol consumption, this biomaterial can greatly enhance the therapeutic effect on 4T1 breast cancer cells, and can form a tumor treatment effect that synergistically enhances enzyme dynamics by destroying lipid rafts.

Claims

1. A biomaterial loaded with cholesterol oxidase, which is a nanomaterial, characterized in that The invention comprises polyacrylic acid, cobalt oxyhydroxide and cholesterol oxidase. The cobalt oxyhydroxide is adsorbed on the polyacrylic acid to form a complex, and the cholesterol oxidase is loaded on the complex.

2. The cholesterol oxidase-loaded biomaterial according to claim 1, characterized in that Cobalt oxyhydroxide nanosheets are first prepared and then formed into a complex with polyacrylic acid.

3. The cholesterol oxidase-loaded biomaterial according to claim 1, characterized in that The complex and cholesterol oxidase are mixed and reacted in a buffer solution at a mass ratio of 2:1 to obtain the product.

4. The cholesterol oxidase-loaded biomaterial according to claim 1, characterized in that The mass ratio of the polyacrylic acid to the cobalt oxyhydroxide is 1:10 to 1:

1.

5. The cholesterol oxidase-loaded biomaterial according to claim 1, characterized in that The cholesterol oxidase content is 20.0%~30.2%.

6. A method for preparing the cholesterol oxidase-loaded biomaterial according to any one of claims 1 to 5, characterized in that include: First, cobalt oxyhydroxide nanosheets are prepared by adding cobalt chloride solution to sodium hydroxide solution at a molar ratio of 1:

3. First, ultrasonication is performed to uniformly disperse the solution. Then, sodium hypochlorite solution is quickly added to the bottle, and the solution turns brown-black. Ultrasonication is performed again, and finally, centrifugation is performed to wash the solution to obtain cobalt oxyhydroxide nanosheets. Next, the cobalt oxyhydroxide nanosheets were dispersed in water, and polyacrylic acid was added. The weight ratio of polyacrylic acid to cobalt oxyhydroxide was 1:10 to 1:

1. After stirring, the mixture was centrifuged and washed to obtain CoP. Finally, CoP and cholesterol oxidase COD were reacted in PBS at a mass ratio of 2:1, and then centrifuged and washed to obtain the cholesterol oxidase-loaded biomaterial CoPC.

7. The method according to claim 6, characterized in that The concentration of cobalt chloride is 10mM, and the concentration of sodium hydroxide is 1M.

8. The method according to claim 6, characterized in that The polyacrylic acid concentration is 40wt%.

9. The method according to claim 6, characterized in that The CoP and the cholesterol oxidase were reacted in PBS for 6 hours.

10. Use of the cholesterol oxidase-loaded biomaterial according to claim 1 in the preparation of anti-tumor drugs.

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