Preparation and Application of Glucose Oxidase-Metal-Curcumin Self-Assembled Nanoparticles

Abstract

The present invention discloses a glucose oxidase-metal-curcumin self-assembled nanoparticle and its preparation and application, belonging to the field of nanomaterial preparation and biomedical application. 2+ , curcumin as self-assembly units, and the coordinated hydrophobic force is used to drive the three to self-assemble at the nanoscale to obtain self-assembled particles, thereby achieving a high loading of GOx (54%). 2+ The competitive inhibitory effect on GOx enzyme activity can achieve precise pH regulation of enzyme activity, allowing starvation therapy to selectively "turn on" and "turn off". By modifying the target molecule RGD on the surface of the self-assembled particles, the differentiated inhibitory effect of the self-assembled particles on normal cells and cancer cells is further enhanced, so that the obtained self-assembled nanoparticles can achieve high inhibition (75%) of tumor cells at a very low GOx dose (1.43μg / ml) relying only on a single starvation therapy.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial preparation and biomedical application, and specifically relates to glucose oxidase-metal-curcumin self-assembled nanoparticles and a preparation method and application thereof. Background Art

[0002] Starvation therapy (ST) is an effective method for inhibiting tumor growth and proliferation by blocking blood flow or depleting essential nutrients and energy. Among these strategies, glucose oxidase (GOx)-mediated blocking of the intracellular glucose supply has garnered widespread attention in tumor suppression. Glucose is the primary energy provider for tumor growth and proliferation. GOx can specifically catalyze the conversion of glucose into gluconic acid and H2O2. By directly consuming glucose, ST deprives cancer cells of energy, thereby inducing cancer cell death. Simultaneously, the generated H2O2 promotes the Fenton reaction, significantly increasing the production of intracellular hydroxyl radicals (•OH), causing further severe oxidative damage to cancer cells. Therefore, in recent years, integrating GOx-mediated tumor starvation therapy with other cancer treatments—for example, combining ST with hypoxia-activated therapy, oxidation therapy, photodynamic therapy, and phototherapy—has become a significant research hotspot.

[0003] However, GOx-based cancer therapy is still in its infancy, and many urgent challenges remain before its clinical application. First, because glucose and oxygen substrates are ubiquitous in the body, nonspecific GOx catalysis inevitably causes other adverse side effects, such as sugar fever, tissue hypoxia, and reactive oxygen species (ROS) generation, causing significant damage to normal tissues. Second, GOx is easily degraded and inactivated by proteases during transport in the body, and also suffers from drawbacks such as poor drug absorption, poor bioavailability, and rapid in vivo metabolism. Third, GOx is currently primarily delivered into the body via various designed carriers, which generally suffer from low loading rates (<25%) and high drug dosages. These issues have seriously hindered the clinical application of GOx-mediated starvation therapy. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a glucose oxidase-metal-curcumin self-assembled nanoparticle and its preparation method and application. The self-assembled nanoparticle has the performance characteristics of high GOx loading rate (54%), precise pH regulation of GOx enzyme activity and active targeting of tumor tissue, and can achieve the efficient tumor inhibition effect of starvation therapy that mainly relies on GOx.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing glucose oxidase-metal-curcumin self-assembled nanoparticles, wherein glucose oxidase (GOx), copper ions (Cu 2+ ) and curcumin (Cur) through self-assembly, and then Arg-Gly-Asp peptide (RGD) is modified on its surface; the specific steps include:

[0007] 1) Fully dissolve GOx in ultrapure water to obtain a GOx solution;

[0008] 2) Cu 2+ Dissolve in deionized water to obtain Cu 2+ solution;

[0009] 3) Fully dissolving Cur in an organic solvent to obtain a Cur solution;

[0010] 4) Under stirring conditions, add an appropriate amount of Tris-HCl buffer to the GOx solution obtained in step 1) to adjust the pH, stir at room temperature for 10 minutes, and then add the Cu obtained in step 2) 2+ The solution and the Cur solution obtained in step 3) were reacted for 5 to 30 minutes, and then centrifuged at 10,000 rpm for 20 minutes. The supernatant was placed in a dialysis bag (WMCO 300 kDa) and dialyzed with phosphate buffer solution for 24 hours (the dialysate was changed every 8 hours). The dialyzed supernatant was then purified by ultrafiltration to obtain self-assembled particles GOx-Cu. 2+ -Cur NPs (GCC NPs);

[0011] 5) The GCC NPs obtained in step 4) were activated by adding EDC and NHS, and then RGD was added to react overnight. The reaction solution was ultrafiltered to obtain the self-assembled nanoparticles RGD-GOx-Cu 2+ -Cur NPs.

[0012] Furthermore, the organic solvent in step 3) includes any one of dimethyl sulfoxide, anhydrous ethanol, and anhydrous acetone.

[0013] Furthermore, the stirring speed in step 4) is 900 rpm.

[0014] Furthermore, in step 4), the pH is adjusted to a range of 8 to 9.

[0015] Furthermore, in step 4), GOx solution, Cu 2+ The amount of solution and Cur solution is based on the GOx, Cu 2+ The mass ratio of Cur is 1.5:(0.15~2):(1~4) for conversion.

[0016] Furthermore, the self-assembled particles obtained in step 4) are spherical monodisperse particles with a size of 40-45 nm.

[0017] Furthermore, in step 5), the mass ratio of RGD to GOx is 0.2:1.5.

[0018] Furthermore, the glucose oxidase-metal-curcumin self-assembled nanoparticles obtained in step 5) have a particle size of 50-55 nm, and have the performance characteristics of high GOx loading rate, precise pH regulation of GOx enzyme activity, and active targeting of tumor tissue.

[0019] The glucose oxidase-metal-curcumin self-assembled nanoparticles can precisely regulate GOx enzyme activity through pH, ​​allowing starvation therapy to be selectively "turned on" and "turned off," thereby achieving controllable starvation therapy to inhibit tumor cell growth, and can therefore be used to prepare anti-tumor drugs.

[0020] The present invention combines glucose oxidase (GOx), copper ions (Cu 2+ ), curcumin (Cur) as self-assembly units, and the coordinated hydrophobic force is used to drive the three to self-assemble at the nanoscale to obtain self-assembled particles, thereby achieving a high loading of GOx (54%). 2+ It has a strong inhibitory effect on the enzyme activity of GOx. Under normal physiological conditions, Cu 2+ It can occupy the active site of the enzyme and effectively inhibit the activity of GOx; in the weak acid environment of the tumor site, Cu 2+ After being stripped, the activity of GOx is restored, so it can be 2+ The inhibitory competition with GOx enables precise pH regulation of enzyme activity, allowing the starvation therapy to selectively "turn on" and "off." Surface modification with the target molecule RGD further enhances the differentiated inhibitory effect of the self-assembled particles on normal and cancer cells. As a result, the resulting self-assembled nanoparticles can achieve a high inhibition rate (75%) against tumor cells at a very low GOx dose (1.43 μg / ml) using only starvation therapy.

[0021] The significant advantages of the present invention are:

[0022] (1) The present invention effectively achieved a high GOx loading (54.43%) by using GOx as the basic unit for self-assembly (rather than adsorption loading).

[0023] (2) The present invention uses Cu 2+ Coordination complexation with GOx, using Cu 2+ The inhibitory competitive effect on GOx enzyme activity can achieve precise pH regulation of enzyme activity, allowing starvation therapy to be selectively "turned on" and "turned off".

[0024] (3) The self-assembled nanoparticles obtained in the present invention can achieve high efficiency of tumor inhibition through starvation therapy at a low GOx dose (1.43 μg / ml), and can effectively avoid the side effects caused by high drug doses. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 GOx-Cu prepared in Example 2+ -Cur NPs and RGD-GOx-Cu 2+ -Cur NPs particle size diagram (A) and potential diagram (B).

[0026] Figure 2 GOx-Cu prepared in Example 2+ -Cur NPs (A) and RGD-GOx-Cu 2+ -TEM image and particle size distribution of Cur NPs (B).

[0027] Figure 3 RGD-GOx-Cu prepared in Example 2+ -Particle size changes of Cur NPs in PBS (A) and DMEM (B) over 14 days.

[0028] Figure 4 RGD-GOx-Cu prepared in Example 2+ -Comparison of H2O2 production by Cur NPs and GOx incubated with 10 mM glucose solution at different pH conditions for different times (6, 12, 18, and 24 h).

[0029] Figure 5 RGD-GOx-Cu prepared in Example 2+ -Comparison of H2O2 production after Cur NPs were incubated with different concentrations of glucose solutions (1, 2, 4, 6, 8, 10 mM) under different pH conditions for 24 h.

[0030] Figure 6 RGD-GOx-Cu prepared in Example 2+ -Cur NPs respond to pH stimulation of Cu 2+ (A) and Cur (B) release curves.

[0031] Figure 7 RGD-GOx-Cu prepared in Example 2+ -Characterization diagram of the biocompatibility of Cur NPs to LO2 normal cells.

[0032] Figure 8 RGD-GOx-Cu prepared in Example 2+-Graph showing the inhibitory effect of Cur NPs on the growth of HepG2 cancer cells.

[0033] Figure 9 The results of the experiments on LO2 cells (6 h) and HepG2 cells (2, 4, 6 h) for RGD-GOx-Cu 2+ -Confocal images of Cur NPs uptake.

[0034] Figure 10 RGD-GOx-Cu prepared in Example 2+ -Confocal fluorescence images of ROS generated by Cur NPs in HepG2 cells.

[0035] Figure 11 RGD-GOx-Cu prepared in Example 2+ Confocal fluorescence images of HepG2 cells stained with AM-PI after treatment with -Cur NPs. DETAILED DESCRIPTION

[0036] 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 implementation methods, but the present invention is not limited thereto. Example

[0037] 1) Weigh 10.0 mg of GOx and dissolve it in 1.0 mL of ultrapure water to prepare a 10.0 mg / mL GOx solution.

[0038] 2) Weigh 1.705 mg of CuCl2·2H2O and dissolve it in 1.0 mL of ultrapure water to prepare 0.01 M CuCl2·2H2O. 2+ solution;

[0039] 3) Weigh 5 mg of Cur and dissolve it in 1.0 mL of dimethyl sulfoxide (DMSO) to prepare a 5.0 mg / mL Cur solution;

[0040] 4) Weigh 25.0 mg of RGD and dissolve it in 1.0 mL of ultrapure water to prepare a 25.0 mg / mL RGD solution.

[0041] 5) Add 150 μL of GOx solution to the reaction flask, then add 55 μL of 0.1M Tris-HCl buffer to adjust the pH to 8, stir at 900 rpm for 10 minutes at room temperature, and then add 100 μL of Cu 2+The solution and 200 μL Cur solution were added and stirred for 15 min. The reaction solution was taken out and centrifuged at 10,000 rpm for 20 min. The supernatant was placed in a dialysis bag (WMCO300 kDa) and dialyzed with phosphate buffer solution for 24 h (the dialysate was changed every 8 h). The dialyzed supernatant was then purified by ultrafiltration to obtain GOx-Cu. 2+ -Cur NPs (GCC NPs);

[0042] 6) Add 20 μL of 5 mg / mL EDC solution and 150 μL of 20 mg / mL NHS solution to 2 mg / mL GCC NPs, and finally add 8 μL of RGD solution, react overnight, and wash twice with ultrafiltration water to obtain RGD-GOx-Cu 2+ -Cur NPs.

[0043] Application Examples

[0044] 1. RGD-GOx-Cu obtained in Example 2+ -Cur NPs and GOx-Cu 2+ -Cur NPs were added into pure water and their particle size and potential were measured. The results are as follows Figure 1 shown.

[0045] Depend on Figure 1 It can be seen that RGD-GOx-Cu 2+ -Cur NPs and GOx-Cu 2+ The particle sizes of -Cur NPs are 58.77 nm and 43.76 nm (a), and the potentials are -8.75 mV and -19.4 mV (b), respectively.

[0046] 2. Prepared GOx-Cu 2+ -Cur NPs and RGD-GOx-Cu 2+ -Cur NPs were fully diluted, and 10 μl of each solution was pipetted onto a clean ultra-thin carbon film. After the sample dried, the ultra-thin carbon film was placed into the sample chamber. The morphology of the nanoparticles was characterized at an accelerating voltage of 200 kV, and the particle sizes of the two nanoparticles were calculated using software. The results are shown in Figure 2. Figure 2 shown.

[0047] Depend on Figure 2 It can be seen that GOx-Cu 2+ -Cur NPs have a particle size of about 39 to 46 nm, and RGD-GOx-Cu 2+ The particle size of -Cur NPs is approximately 46 to 56 nm, and the sizes of both nanoparticles are uniform and well dispersed.

[0048] 3. The RGD-GOx-Cu obtained in Example 2+ -Cur NPs were added to PBS and DMEM culture medium, sealed and stored, and samples were taken to measure the changes in particle size. The results are as follows Figure 3 shown.

[0049] Depend on Figure 3 It can be seen that the particle size of the nanoparticles in PBS and DMEM culture medium did not change significantly within two weeks, indicating that the RGD-GOx-Cu 2+ -Cur NPs have good dispersibility and stability.

[0050] 4. To evaluate the RGD-GOx-Cu 2+ -Cur NPs pH-responsive catalytic activity, the prepared RGD-GOx-Cu 2 + -CurRGCC NPs or glucose oxidase were mixed with glucose aqueous solution and immersed in PBS buffer solution with pH 7.4 or 5.5 respectively, so that the concentration of glucose was 10 mM. After 6 h, 12 h, 18 h, and 24 h of reaction, the mixture was shaken and 100 μL of the reaction solution was aspirated with a pipette. The hydrogen peroxide content after the reaction was detected using a hydrogen peroxide detection kit. The results are shown in Figure 2. Figure 4 shown.

[0051] Depend on Figure 4 It can be seen that RGD-GOx-Cu 2+ -Cur NPs were incubated in PBS buffer at pH 5.5, and the amount of hydrogen peroxide produced increased greatly with time, while the amount of hydrogen peroxide produced in PBS buffer at pH 7.4 increased slowly, which confirmed that under the physiological environment of pH 7.4, RGD-GOx-Cu 2+ -Cur NPs have only low catalytic activity, but their catalytic activity is reactivated in a slightly acidic environment.

[0052] To study the effect of different concentrations of glucose on RGD-GOx-Cu 2+ -Cur NPs catalytic activity release, equal amounts of RGD-GOx-Cu 2+ -Cur NPs were mixed with glucose aqueous solutions of different concentrations (1, 2, 4, 6, 8, 10 mM) and immersed in PBS (10 mM) buffer solution with a pH of 7.4 or 5.5. After 24 h of reaction, the mixture was shaken and 100 μL of the reaction solution was pipetted. The hydrogen peroxide content was detected according to the standard procedure of the hydrogen peroxide content detection kit. The results are shown in Figure 2. Figure 5 shown.

[0053] Depend on Figure 5 It can be seen that RGD-GOx-Cu2+ -Cur NPs in PBS buffer at pH 5.5, the amount of hydrogen peroxide produced increased significantly with the increase of glucose concentration. 2+ -Cur NPs only weakly produce hydrogen peroxide in PBS buffer at pH 7.4. This is mainly because under alkaline conditions, copper ions occupy the active site of glucose oxidase, making it impossible for glucose oxidase to effectively catalyze glucose even in the presence of a large amount of glucose.

[0054] 5. To further study RGD-GOx-Cu 2+ -Cur NPs release catalytic activity in response to pH. 2+ -Cur NPs were transferred to a dialysis bag (MWCO 8000 Da) and immersed in PBS (10 mM) with pH 7.4 or 5.5, and slowly stirred for 48 h in the dark at room temperature. At 0 h, 1 h, 3 h, 5 h, 7 h, 9 h, 12 h, 16 h, 20 h, 24 h, 30 h, 36 h, and 48 h of dialysis, the buffer was removed and replenished with fresh PBS buffer. The Cu was determined by ICP-AES. 2+ At the same time, RGD-GOx-Cu 2+ -Cur NPs were transferred to a dialysis bag (MWCO 8000 Da) and immersed in a PBS (10 mM, pH 7.4 or 5.5) buffer solution containing 5% Tween 80. The solution was slowly stirred in the dark at room temperature for 48 h. At the time of dialysis 0 h, 1 h, 3 h, 5 h, 7 h, 9 h, 12 h, 16 h, 20 h, 24 h, 30 h, 36 h, and 48 h, the buffer solution was removed and replenished with fresh PBS buffer. The Cur content was determined by UV-visible spectrophotometry. The results were given by Figure 6 shown.

[0055] Depend on Figure 6 It can be seen that at pH 5.5, Cu 2+ The release amount of Cur was much greater than that at pH 7.4, which indicated that Cu 2+ It can be released responsively with Cur under acidic conditions.

[0056] 6. To investigate the RGD-GOx-Cu 2+ -Cur NPs biocompatibility, RGD-GOx-Cu 2+ -Cur NPs were co-incubated with normal human liver cells (LO2 cells) to determine the 2+-Toxicity of Cur NPs. When LO2 cells grow to about 90%, cell passage operation is performed. The operation steps are as follows: remove the old culture medium in the culture flask, then add 2.0 mL PBS solution to wash 3 times, then add 1.0 mL trypsin to digest the cells for about 1 minute, remove the trypsin, add 2.0 mL DMEM culture medium to stop digestion and gently blow the cells to suspend the cells to obtain cell stock solution, take 1 / 4 of the cell stock solution and put it into a new culture flask, place it in a 37°C, 5% CO2 incubator for further culture. Take out a part of the LO2 cell stock solution and dilute it with DMEM culture medium to a density of 10 5 / mL, and 10 4 Cells were seeded into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 hours. The old culture medium in the well plate was removed and RGD-GOx-Cu was added with GOx concentrations of 0, 0.55, 0.77, 0.99, 1.21, and 1.43 μg / mL. 2+ -Cur NPs culture medium, set up 4 replicate wells for each concentration. After 6 hours of culture, the culture medium was aspirated and washed twice with PBS, 100 μL of fresh culture medium was added and cultured for another 18 hours, 10 μL of 5.0 mg / mL MTT was added to each well, incubated for 4-6 hours, the culture medium was carefully removed, 150 μL of DMSO was added, and the cells were placed in a constant temperature shaker at 37°C and 150 rpm for 15 minutes, and then the absorbance of each well solution at 490 nm was measured with a microplate reader to calculate the survival rate to evaluate the survival rate of RGD-GOx-Cu. 2+ -Cur NPs biocompatibility, the results are as follows Figure 7 shown.

[0057] Depend on Figure 7 It can be seen that RGD-GOx-Cu 2+ -Cur NPs, the cell survival rate was above 80% when the GOx concentration was 1.43 μg / mL, indicating that RGD-GOx-Cu 2+ -Cur NPs have good biocompatibility.

[0058] At the same time, in order to investigate the 2+ -Cur NPs in vitro therapeutic effect, RGD-GOx-Cu 2+ -Cur NPs were co-incubated with liver cancer cells (HepG2 cells) to determine the 2+ The toxicity of -Cur NPs was investigated. The operation steps were as follows: First, the HepG2 cell suspension in the passage process was redispersed with culture medium and the cell density was adjusted to 5×10 4The redispersed cell suspension was inoculated into a 96-well plate, with four parallel wells per group and 100 μL per well. The plate was then placed in a cell culture incubator at 37°C and 5% CO2 for 24 h to allow the cells to fully adhere to the wall. The old culture medium was removed, and RGD-GOx-Cu was added to each well at concentrations of 0 μg / mL, 0.55 μg / mL, 0.77 μg / mL, 0.99 μg / mL, 1.21 μg / mL, and 1.43 μg / mL, respectively. 2+ -Cur NPs solution (DMEM medium as solvent, glucose concentration is 10 mM) was cultured for 4 h to remove RGD-GOx-Cu 2+ -Cur NPs solution, wash the cells three times with PBS, add new culture medium and culture for 20 h, add 10 μL of 5 mg / mL MTT solution to each well, move to the incubator and continue to culture for 4 h, carefully remove the culture medium to prevent the blue-purple formazan crystals from being sucked out, add 150 μL of DMSO to each well, and finally shake in a 37°C constant temperature shaker in the dark for 15 min. After waiting for the blue-purple formazan crystals to dissolve completely, use a microplate reader to measure the absorbance of each well at 490 nm and calculate the cell survival rate. The results are as follows Figure 8 shown.

[0059] Depend on Figure 8 It can be seen that RGD-GOx-Cu 2+ -Cur NPs only when the GOx concentration was 1.43 μg / mL, the cell death rate reached more than 70%, indicating that RGD-GOx-Cu 2+ -Cur NPs showed excellent therapeutic effects in vitro.

[0060] 7. Comparison of LO2 and HepG2 cell responses to RGD-GOx-Cu by laser scanning confocal microscopy 2+ -CurNPs to explore the uptake of RGD-GOx-Cu 2+ -Cur NPs targeting ability to cancer cells. The operation steps are as follows: the cell density is 1×10 4 LO2 cells and HepG2 cells were seeded in confocal culture dishes at a concentration of 400 μL per well, and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h to allow the cells to adhere completely. The culture medium was discarded and RGD-GOx-Cu labeled with rhodamine B was added. 2+ -Cur NPs serum-free culture medium, placed in a cell culture incubator to culture LO2 cells for 6 h, HepG2 cells were cultured for 2, 4, and 6 h, the culture medium was discarded, the cells were washed twice with PBS, culture medium was added, and the confocal culture dish was placed on a laser confocal microscope to capture the fluorescence spectrum with an excitation wavelength of 488 nm. The results are shown in Figure 9 shown.

[0061] Depend on Figure 9 It can be seen that at 2h, HepG2 cells 2+ The uptake of RGD-GOx-Cu NPs by HepG2 cells was similar to that of LO2 cells at 6 h. 2+ -Cur NPs were much more uptaken by cells than those by LO2 cells, indicating that RGD-GOx-Cu 2+ -Cur NPs have stronger targeting ability to HepG2 cancer cells.

[0062] 8. Detection of RGD-GOx-Cu by DCFH-DA probe 2+ -Cur NPs produced ROS in HepG2 cells. The operation steps are as follows: the cell density is 1×10 4 HepG2 cells at a concentration of 100 μg / mL were seeded in a confocal culture dish and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h to allow the cells to fully adhere to the wall. The culture medium was discarded, and the negative control group was set up with cells cultured in serum-free medium, and the positive control group was set up with cells cultured in serum-free medium containing 0.1 mM H2O2. The experimental groups were added with RGD-GOx-Cu containing 2.2 μg / mL of GOx. 2+ -Cur NPs and GOx-Cu 2+ -Cur NPs serum-free medium, after culturing for 6 h, the old culture medium was removed, the cells were washed with PBS once, and then the DCFH-DA probe was added to each group of cells and incubated for 30 min. Finally, the cells were gently washed twice with PBS, serum-free medium was added, and the confocal culture dish was placed on a laser confocal microscope to capture the fluorescence spectrum with an excitation wavelength of 488 nm. The results are shown in Figure 2. Figure 10 shown.

[0063] Depend on Figure 10 It can be seen that RGD-GOx-Cu 2+ -Cur NPs produced a fluorescence similar to that of the positive control group. 2+ -Cur NPs lack the targeting function, and the green fluorescence generated by ROS is significantly lower than that of RGD-GOx-Cu 2+ -Cur NPs.

[0064] 9. In order to explore the 2+The specific killing effect of -Cur NPs on HepG2 cells was investigated by laser scanning confocal microscopy to capture confocal images of live / dead cells stained with Calcein-AM / PI fluorescent probes. The operation steps were as follows: the cells were plated at a density of 5×10 4 HepG2 cells at a concentration of 100 μg / mL were seeded in a confocal culture dish and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h to allow the cells to fully adhere to the wall. The culture medium was discarded, and the negative control group was cultured in serum-free medium. The experimental groups were treated with RGD-GOx-Cu containing 2.2 μg / mL of GOx. 2+ -Cur NPs and GOx-Cu 2+ -CurNPs serum-free medium, after 4 h of culture, the old culture medium was removed, the cells were washed twice with PBS, and then each group was incubated with fresh serum-free culture medium for 20 h. The old culture medium was removed, Calcein-AM / PI fluorescent dye was added and incubated for 40 min, the cells were gently washed twice with PBS, serum-free medium was added, and imaging was performed by laser scanning confocal microscopy. The results are shown in Figure 2. Figure 11 shown.

[0065] Depend on Figure 11 It can be seen that there is almost no red fluorescence produced by dead cells in the control group; 2+ -Cur NPs group has both red and green fluorescence, indicating that GOx-Cu 2+ -Cur NPs have a certain killing effect on HepG2 cells; while RGD-GOx-Cu 2+ -Cur NPs group has a lot of red fluorescence and trace green fluorescence, indicating that RGD-GOx-Cu 2+ -Cur NPs showed significant therapeutic effects in in vivo experiments.

[0066] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A glucose oxidase-metal-curcumin self-assembled nanoparticle, characterized in that: The self-assembled nanoparticles are made by self-assembling glucose oxidase, copper ions, and curcumin, and then surface-modifying them with RGD peptide. These nanoparticles can precisely regulate glucose oxidase activity through pH, ​​allowing starvation therapy to selectively "turn on" and "off," thereby inhibiting tumor cell growth. The preparation method of the self-assembled nanoparticles specifically comprises the following steps: 1) Dissolving glucose oxidase, copper ions, and curcumin separately to obtain corresponding solutions; 2) The solutions obtained in step 1) are mixed in proportion, the pH is adjusted, and the mixture is reacted for a period of time, followed by centrifugation, and the supernatant is dialyzed to obtain self-assembled particles; 3) Adding EDC and NHS to the self-assembled particles obtained in step 2) for activation, then adding RGD peptide to react overnight, and ultrafiltration to obtain the self-assembled nanoparticles.

2. The glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1, characterized in that In step 2), the corresponding solutions are mixed according to a mass ratio of glucose oxidase, copper ions and curcumin of 1.5:(0.15-2):(1-4).

3. The glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1, characterized in that In step 2), the pH is adjusted to 8-9.

4. The glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1, characterized in that The reaction time in step 2) is 5 to 30 minutes.

5. The glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1, characterized in that Step 2) The self-assembled particles obtained are spherical monodisperse particles with a size of 40-45 nm.

6. The glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1, characterized in that In step 3), the mass ratio of RGD peptide to glucose oxidase is 0.2:1.

5.

7. The glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1, characterized in that Step 3) The particle size of the self-assembled nanoparticles obtained is 50-55 nm.

8. Use of the glucose oxidase-metal-curcumin self-assembled nanoparticles according to claim 1 in the preparation of anti-tumor drugs.

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