Nanozyme FePGOGA, preparation method and application

Abstract

The present invention discloses a nanozyme FePGOGA, which is made of glucose oxidase, GAP19 and Fe-PDAP nanozyme, and provides its corresponding preparation method and application. The present invention loads glucose oxidase and GAP19 through Fe-PDAP nanozyme to make it have glucose oxidase, POD, GSHO X The present invention has multiple catalytic activities and excellent cascade enzyme catalytic ability. The present invention loads glucose oxidase (GOx) and GAP19 on the Fe-PDAP nanozyme through charge adsorption and ππ interaction, and then separates and purifies them. The preparation method is simple and easy to operate, which is convenient for industrial promotion and application. The GAP19 of the present invention can block the gap junctions of cells, block the efflux of ROS, enable ROS to be effectively accumulated, eliminate resistance to catalytic treatment, and block the transport of extracellular glucose, achieving efficient starvation and low-temperature photothermal therapy of tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biomedical materials, and specifically relates to a nanoenzyme FePGOGA, a preparation method and an application thereof. Background Art

[0002] Cancer is a major threat to human health, and there is an urgent need to design new drug formulations for cancer diagnosis and treatment. Compared with traditional cancer drugs, the formation of nanoparticles can improve drug biodistribution and enhance drug accumulation at the tumor site, thereby reducing drug side effects and enhancing therapeutic effects.

[0003] As biocatalysts, enzymes possess excellent specificity and high efficiency, mediating diverse reactions in biological processes such as signal transduction, metabolism, and digestion. However, natural enzymes are subject to high production costs, low yields, and are subject to volatilization and inactivation, making their widespread application difficult. Consequently, researchers have gradually developed nanozymes, nanomaterials that follow enzyme kinetics under physiological conditions and can catalyze chemical reactions involving enzyme substrates. Nanozymes possess high catalytic activity (accelerating biochemical reactions), good stability, low production costs, and simple preparation and purification steps. To date, a large number of nanomaterials, such as metal nanoparticles, metal oxide nanoparticles, carbon-based nanomaterials, and metal-organic frameworks, have been demonstrated to function like natural enzymes. These nanozymes offer significant advantages in biomedical applications, particularly in cancer treatment. However, research on nanozymes with multiple catalytic activities is currently lacking. Summary of the Invention

[0004] The object of the present invention is to provide a nanozyme FePGOGA, and providing its corresponding preparation method and application is another object of the present invention.

[0005] Based on the above objectives, the present invention adopts the following technical solutions:

[0006] A nanozyme FePGOGA is made of glucose oxidase, GAP19, and Fe-PDAP nanozyme.

[0007] The Fe-PDAP nanozyme is loaded with glucose oxidase and GAP19.

[0008] The loading amount of glucose oxidase on the Fe-PDAP nanozyme is (0.1-3.5) μg·mg -1 The loading amount of GAP19 on Fe-PDAP nanozyme was (1-150) μg·mg -1 .

[0009] The loading amount of glucose oxidase on Fe-PDAP nanozyme is (0.2-3.5) μg·mg -1 The loading amount of GAP19 on Fe-PDAP nanozyme was (20-150) μg·mg -1 .

[0010] The method for preparing the nanozyme FePGOGA comprises the following steps:

[0011] 1) Dissolve glucose oxidase, GAP19, and Fe-PDAP nanozyme in water and stir to mix in the dark;

[0012] 2) Separation and purification to obtain nanozyme FePGOGA.

[0013] In step 1), the dosage ratio of Fe-PDAP nanozyme, glucose oxidase, GAP19 and water is 1000 μg: (1-100) μg: (10-400) μg: (0.5-3) mL; in step 2), dialysis is used for separation and purification.

[0014] In step 1), the dosage ratio of Fe-PDAP nanozyme, glucose oxidase, GAP19, and water is 1000 μg: (2-100) μg: (50-400) μg: (0.5-3) mL.

[0015] In step 1), the preparation method of Fe-PDAP nanozyme is as follows: FeCl3·6H2O is dissolved in water, 2,6-diaminopyridine is added for polymerization, and then separated, purified, and freeze-dried to obtain Fe-PDAP nanozyme.

[0016] In the preparation method of Fe-PDAP nanozyme, the molar ratio of FeCl3·6H2O and 2,6-diaminopyridine is 4:1; the polymerization reaction conditions are: temperature 35-40°C, polymerization time 22-26h; and dialysis is used for separation and purification.

[0017] Application of nanozyme FePGOGA in the preparation of anti-tumor drugs.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The present invention loads glucose oxidase and GAP19 through Fe-PDAP nanozyme, making it have glucose oxidase, POD (peroxidase), GSHO X (Glutathione oxidase) has multiple catalytic activities and excellent cascade enzyme catalytic ability;

[0020] 2) The present invention loads glucose oxidase (GOx) and GAP19 onto Fe-PDAP nanozymes through charge adsorption and ππ interaction, respectively, and then separates and purifies them. The preparation method is simple and easy to operate, facilitating industrial promotion and application.

[0021] 3) The Fe-PDAP nanozyme, GAP19 of the present invention can block the gap junctions of cells, block the efflux of ROS, enable ROS to be effectively accumulated, eliminate resistance to catalytic therapy, and block the transport of extracellular glucose, thereby achieving efficient starvation and low-temperature photothermal therapy of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an image of Fe-PDAP nanozyme; Figure 1 (A) is the image of Fe-PDAP nanozyme; Figure 1 (B) TEM images of FeP nanozymes at day 1 and day 21, scale bar = 200 nm; Figure 1 (C) Tyndall effect image of Fe-PDAP nanozyme in PBS.

[0023] Figure 2 Preparation and characterization of nanozyme FePGOGA; Figure 2 (A) Schematic diagram of the preparation of FePGOGA;

[0024] Figure 2 (B) FeP size determination by DLS; Figure 2 (C) TEM image of FeP nanozyme, scale bar = 200 nm; Figure 2 (D) AFM image of FeP nanozyme; Figure 2 (E) FT-IR spectrum of FeP nanozyme; Figure 2 (F) is the XPS image of FeP nanozyme.

[0025] Figure 3 The stability and safety of FeP nanozymes; Figure 3 (A) is the stability of FeP; Figure 3 (B) The hemolytic ability results of FeP nanozymes at different concentrations.

[0026] Figure 4 UV-visible spectra of FeP nanozymes with and without 808 nm laser irradiation.

[0027] Figure 5 It is the photothermal effect of FeP nanozyme; Figure 5 (A) FeP nanozymes with different concentrations were generated by 808 nm laser at 1.5 W·cm -2 Image after 10 minutes of irradiation; Figure 5(B) FeP nanozymes with different concentrations were exposed to 808 nm laser at 1.5 W·cm -2 Temperature curve after 10 minutes of irradiation; Figure 5 (C) FeP nanozyme after six laser “on / off” cycles (1.5 W·cm -2 ) Temperature changes under irradiation.

[0028] Figure 6 It is the photothermal effect of FeP nanozyme; Figure 6 (A) FeP nanozyme (0.5 mg mL -1 ) at different powers (0.3, 0.5, 0.8, 1.0 and 1.5 W·cm -2 ) under 808nm laser irradiation for 10min;

[0029] Figure 6 (B) is the light-heat curve distribution.

[0030] Figure 7 Images of FePGO, FePGA, and FePGOGA preparations.

[0031] Figure 8 Characterization of cargo-loaded FeP nanozymes; Figure 8 (A) is the size of different NPs (n=3); Figure 8 (B) UV-visible spectra analysis of different NPs.

[0032] Figure 9 UV-visible spectrum for loaded cargo; Figure 9 (A) Absorption spectra of FITC series; Figure 9 (B) is the standard curve of FITC; Figure 9 (C) Absorption spectra of a series of DIDs; Figure 9 (D) is the standard curve of DID.

[0033] Figure 10 is the release curve of the loaded cargo; FeP FITC Release curves of (A) FITC and (B) DID of GODID with and without light irradiation (n=3).

[0034] Figure 11 Characterization of the nanozyme FePGOGA of the present invention; Figure 11 (A) is the size of different NPs; Figure 11 (B) Potentials of different NPs (n=3); Figure 11 (C) Stability of different nanozymes in different solutions (n=3).

[0035] Figure 12 is the enzymatic activity of nanozyme FePGOGA; Figure 12(A) Fluorescence image of O2 consumption during GOx catalysis using RDPP as a probe; Figure 12 (B)Fe 2+ Release curves from FeP by GSHox simulated activity (0, 0.2, and 2.0 mM GSH) (n=3); Figure 12 (C)FeP (0, 50, 100, 200 and 500 μg mL -1 )(n=3) Concentration of GSH remaining in the solution after the solution was mixed with GSH, and (inset) photograph of the solution; Figure 12 (D) UV-visible spectrum and (inset) photograph of FeP consuming H2O2 using MB as a probe.

[0036] Figure 13 is the GOx activity of FePGO nanozyme; Figure 13 (A) Various FePGO nanozymes (0, 2, 4, 6 μg·mL -1 Quantitative O2 levels in GOx) (n=4) solutions; Figure 13 (B) RDPP in FePGO nanozymes (0, 2, 4, 6 μg mL -1 UV-visible spectra of GOx in solution.

[0037] Figure 14 Glutathione oxidase (GSHOx) mimetic ability of FeP; Figure 14 (A) UV-visible absorption spectra of the GSH series; Figure 14 (B) Standard curve of GSH; Figure 14 (C) UV-visible spectra of different FeP in the presence of GSH.

[0038] Figure 15 FeP FITC Cellular uptake of GODID; Figure 15 (A) and free FITC (2.5 μg mL -1 ),free FITC GOx (2.5 μg mL -1 FITC), free DID (5 μg·mL -1 ) and FeP FITC GODID (2.5 μg mL -1 CLSM images of cal27 cells after 6 h of incubation with FITC (scale bar = 50 μm); Figure 15 Flow cytometric analysis of (B) FITC and (C) DID fluorescence intensities.

[0039] Figure 16 FeP FITC Quantitative analysis of cellular uptake of GODID; Figure 16 (A) Figure 15 FITC fluorescence intensity quantified in B; Figure 16 (B) Figure 15 DID fluorescence intensity quantified in C; Figure 16 (C)FeP FITC Lysosomal escape of GA, scale bar = 50 μm.

[0040] Figure 17 Western blot analysis of intracellular Cx43 expression in cal27 cells after treatment with different FeP contents and different nanoformulations for 24 h; ae represent different treatments: a: PBS, b: FeP, c: FePGO, d: FePGA, e: FePGOGA.

[0041] Figure 18 Immunofluorescence images of Cx43; cal27 cells were treated with different NPs for 24 hours (2 μg mL -1 GOx, 40 μg mL -1 GAP19), and then the nucleus and Cx43 were stained with DAPI (blue) and anti-Cx43 (red), respectively, scale bar = 50 μm.

[0042] Figure 19 is the ROS generated by FePGOGA in cells; Figure 19 (A) CLSM image, scale bar = 50 μm; Figure 19 (B) Flow cytometry analysis.

[0043] Figure 20 The in vitro cascade nanozyme-like catalytic activity of FePGOGA; Figure 20 (A) CLSM images of intracellular O2 levels depleted by GOx, scale bar = 50 μm; Figure 20 (B) Flow cytometric analysis of intracellular O2; Figure 20 (C) IVIS images of intracellular O2; Figure 20 (D) Intracellular Fe 2+ Horizontal CLSM images, scale bar = 50 μm; Figure 20 (E) Intracellular Fe 2+ horizontal flow cytometric analysis; Figure 20 (F) Quantification of Fe 2+ Fluorescence intensity (n=3); Figure 20 (G) Flow cytometric analysis of intracellular LPO levels. Figure 21 cal27 cells were treated with different nanoparticles; Figure 21 (A) Changes in RDPP fluorescence intensity; Figure 21 (B) GPX4, (C) HSP90, and (D) HSP70 protein expression changes.

[0044] Figure 22 For cell viability analysis; Figure 22 (A) cal27 and (B) scc7 cell cytotoxicity; Figure 22 (C) Flow cytometric analysis of cal27 cells treated with different nanosystems and double-stained with Annexin V-FITC / PI; CLSM images of cal27 cells treated with different nanosystems and double-stained with (D) calcein-AM (green, live cells) and propidium iodide (PI) (red, dead cells) and (E) crystal violet, scale bar = 50 μm.

[0045] Figure 23 The apoptosis rate of cal27 cells after treatment with different NPs.

[0046] Figure 24 The in vivo antitumor efficacy of FePGOGA; Figure 24 (A) Timeline of FePGOGA-induced low-temperature PTT initiation; Figure 24 (B) Mice bearing cal27 tumors were injected with FePDID and free DID (5 mg kg -1 Fluorescence imaging at different time intervals after DID; Figure 24 (C) Using 808nm laser (1.5W·cm -2 Thermal images of tumor-bearing mice after irradiation (10 min) and 12 h after injection of FeP, FePGOGA, and PBS. Figure 25 is the distribution of FePGOGA in the body; Figure 25 (A) Ex vivo fluorescence images of tumors and major organs (including heart (H), liver (LI), spleen (SP), lungs (LU), and kidneys (K)) and tumors (T) 12 h after injection of FePDID and free DID (5 mg kg -1 ); Figure 25 (B) Statistical results of the fluorescence intensity of FePDID and free DID in tumors and major organs corresponding to (A).

[0047] Figure 26 FeP, FePGOGA and PBS were irradiated with 808 nm laser at 1.5 W·cm -2 Temperature rise curve of the tumor after 10 minutes of irradiation.

[0048] Figure 27 For the anti-tumor effect in vivo; Figure 27 (A) Schedule of FePGOGA-induced tumor starvation, catalytic therapy, and low-temperature photothermal therapy in tumor-bearing mice; on days 1, 4, and 7, tumor-bearing cal27 mice (150 mm 3 ) injected with PBS, FePGO, FePGA and FePGOGA (2 mg kg -1GOD and 40 mg kg -1 GAP19), and for the FePGOGA-L group, the tumor site was irradiated 12 hours after injection (808 nm, 1.5 W cm -2 , 10 minutes); Figure 27 (B), (C) Tumor volume; Figure 27 (D) Representative tumor images;

[0049] Figure 27 (E) Tumor weight.

[0050] Figure 28 Tumor-bearing mice (initial tumor volume 150 mm 3 )Observation period images.

[0051] Figure 29 The in vivo antitumor efficacy of FePGOGA in the late stage of tumor-bearing mice; Figure 29 (A) Timeline of FePGOGA-initiated tumor starvation, catalytic therapy, and low-temperature PTT; Cal27 tumor-bearing mice (500 mm 3 ) were injected with PBS, FePGO, FePGA, and FePGOGA (2 mg·kg -1 GOx and 40 mg kg -1 For the FePGOGA-L group, 808 nm laser (1.5 W·cm -2 , 10 minutes) irradiate the tumor site; Figure 29 (B), (C) Tumor volume; Figure 29 (D) Representative tumor images; Figure 29 (E) Tumor weight and (F) body weight during the 16-day observation period (n=5); Figure 29 (G) H&E staining and (H) TUNEL staining of tumor tissues harvested on day 16 after injection, and Figure 29 (I) Immunofluorescence images of tumor sections stained with anti-Cx43 antibody; cell nuclei and Cx43 were stained with DAPI (blue) and anti-Cx43 antibody (red), respectively; scale bar = 100 μm.

[0052] Figure 30 Tumor-bearing mice (initial tumor volume 500 mm 3 )Observation period images.

[0053] Figure 31 These are the staining results of tumor tissues harvested 20 days after injection; Figure 31 (A) H&E staining; Figure 31 (B) TUNEL staining, scale bar = 100 μm; Figure 31(C) Immunofluorescence images of tumor sections stained with anti-Cx43 antibody. Cell nuclei and Cx43 were stained with DAPI (blue) and anti-Cx43 antibody (red), respectively.

[0054] Figure 32 Immunofluorescence images of tumor sections stained with anti-HSP70 / 90 / GPX4 antibodies (initial tumor volume 150 mm 3 ).

[0055] Figure 33 Immunofluorescence images of tumor sections stained with anti-HSP70 / 90 / GPX4 antibodies (initial tumor volume 500 mm 3 ).

[0056] Among them, in order to better illustrate the effect, Figure 15 、 16 , 18, 19, 20, 22, 31, 32, 33. Color drawings are also provided. For details, please refer to other supporting documents. DETAILED DESCRIPTION

[0057] In this application, Fe-PDAP nanozyme is abbreviated as FeP nanozyme, and glucose oxidase is abbreviated as GOx.

[0058] GAP19: is a nonapeptide derived from the Cx43 cytoplasmic loop (CL) that acts as a selective connexin 43 (Cx43) hemichannel blocker.

[0059] Example 1

[0060] A nanozyme FePGOGA is made of glucose oxidase, GAP19, and Fe-PDAP nanozyme, and the Fe-PDAP nanozyme is loaded with glucose oxidase and GAP19.

[0061] The method for preparing the nanozyme FePGOGA comprises the following steps:

[0062] 1) a. Dissolve 80 mmol of FeCl3·6H2O in 400 mL of deionized water and stir at room temperature (25°C) for 1 h. Then, add 20 mmol of 2,6-diaminopyridine and stir at 37°C for 24 h. After polymerization, dialyze the reaction solution with deionized water (MWCO = 10 kDa) for 24 h, changing the deionized water every 2 h during the dialysis period. The solution is freeze-dried using a freeze dryer to obtain Fe-PDAP nanozyme. b. Dissolve 10 μg of glucose oxidase, 100 μg of GAP19, and 1000 μg of Fe-PDAP nanozyme in 1 mL of deionized water and stir at 300 r / min for 24 h in the dark.

[0063] 2) Dialysis was performed with deionized water (molecular weight cut-off MWCO = 10 kD) for 24 h, with the deionized water replaced every 2 h during the dialysis period, to remove free GOx and GAP19 and obtain the nanozyme FePGOGA.

[0064] The nanozyme FePGOGA prepared by the above method was calculated by ultraviolet absorption spectroscopy. The loading amount of glucose oxidase on Fe-PDAP nanozyme was 3.5 μg·mg -1 The loading amount of GAP19 on FeP nanozyme was 85 μg·mg -1 The size of the nanozyme FePGOGA was determined to be approximately 118.5 nm and the potential was 9.7 mV by DLS and TEM.

[0065] Application of nanozyme FePGOGA in the preparation of anti-tumor drugs.

[0066] Example 2

[0067] This embodiment is different from Example 1 in that in step b, 2 μg glucose oxidase, 200 μg GAP19, and 1000 μg Fe-PDAP nanozyme are dissolved in 3 mL deionized water, and the rest is the same as Example 1.

[0068] The nanozyme FePGOGA prepared by this method was calculated by ultraviolet absorption spectroscopy, and the loading amount of glucose oxidase on FeP nanozyme was 0.74 μg·mg -1 The loading amount of GAP19 on Fe-PDAP nanozyme was 148 μg·mg -1 The size of the nanozyme FePGOGA was determined to be approximately 126.8 nm and the potential was 13.1 mV by DLS and TEM.

[0069] Example 3

[0070] This embodiment is different from Example 1 in that, in step b, 5 μg glucose oxidase, 200 μg GAP19, and 1000 μg Fe-PDAP nanozyme are dissolved in 2 mL deionized water, and the rest are the same as Example 1.

[0071] The nanozyme FePGOGA prepared by this method was calculated by ultraviolet absorption spectroscopy, and the loading amount of glucose oxidase on Fe-PDAP nanozyme was 2.3 μg·mg -1 The loading amount of GAP19 on FeP nanozyme was 108 μg·mg -1 The size of the nanozyme FePGOGA was determined to be approximately 132.4 nm and the potential was 12.4 mV by DLS and TEM.

[0072] Example 4

[0073] This embodiment is different from Example 1 in that in step b, 10 μg glucose oxidase, 200 μg GAP19, and 1000 μg FeP nanozyme are dissolved in 0.5 mL deionized water, and the rest is the same as Example 1.

[0074] The nanozyme FePGOGA prepared by this method was calculated by ultraviolet absorption spectroscopy, and the loading amount of glucose oxidase on Fe-PDAP nanozyme was 3.2 μg·mg -1 The loading amount of GAP19 on FeP nanozyme was 134 μg·mg -1 The size of the nanozyme FePGOGA was determined to be approximately 125.4 nm and the potential was 10.4 mV by DLS and TEM.

[0075] Example 5

[0076] This embodiment is different from Example 1 in that, in step b, 100 μg glucose oxidase, 100 μg GAP19, and 1000 μg FeP nanozyme are dissolved in 1.5 mL deionized water, and the rest are the same as Example 1.

[0077] The nanozyme FePGOGA prepared by this method was calculated by ultraviolet absorption spectroscopy, and the loading amount of glucose oxidase on FeP nanozyme was 0.2 μg·mg -1 , the loading amount of GAP19 on FeP nanozyme was 25 μg·mg -1 The size of the nanozyme FePGOGA was determined to be approximately 134.5 nm and the potential was 25.6 mV by DLS and TEM.

[0078] Example 6 Test Example

[0079] The samples and parameters of Example 2 of the present invention were used in the test example.

[0080] 1. Characterization, hemolytic ability, stability, and photothermal testing of FeP nanozymes

[0081] 1.1 Characterization of FeP Nanozyme in Example 2

[0082] The morphology and size of FeP nanozymes were characterized by DLS and TEM. Figure 1 and 2 shown.

[0083] Figure 1 (A) is an image of FeP nanozyme, which is dark brown in color. Figure 1 (B) TEM images of FeP nanozymes at day 1 and day 21, scale bar = 200 nm. Figure 1(C) is the Tyndall effect image of FeP nanozyme in PBS. A typical Tyndall effect was detected in the FeP solution.

[0084] Preparation of FeP nanozymes by surfactant-free oxidative polymerization of 2,6-diaminopyridine and FeCl3·6H2O Figure 1 (A), dark brown in color), as Figure 2 (A) As shown. Under dynamic light scattering (DLS), the average size of FeP nanozymes was determined to be approximately 105 nm, and the size distribution showed a polydispersity index (PDI) of 0.25 ( Figure 2 (B)). Transmission electron microscopy (TEM) images show the spindle-shaped morphology of FeP nanozymes ( Figure 2 (C)). Atomic force microscopy (AFM) confirmed that the thickness of FeP is about 8.5nm ( Figure 2 (D)). Significant changes in Fourier transform infrared (FT-IR) spectra, such as the peak at approximately 3000 cm -1 Disappears at about 1640 and 1593 cm -1 The increase at 845 cm indicates the formation of C=N bonds during the polymerization process. -1 The peak at indicates that the FeP nanozyme is successfully polymerized ( Figure 2 (E)). Fe2p in the X-ray photoelectron spectroscopy (XPS) curve of FeP nanozyme 1 / 2 and Fe2p 3 / 2 The typical binding energy peak of Fe 3+ Stability in FeP nanostructures ( Figure 2 (F)).

[0085] 1.2 Stability and hemolytic ability of Fe-PDAP nanozyme (FeP nanozyme)

[0086] 1) Stability of FeP nanozymes

[0087] The concentration of FeP nanozyme solution was adjusted (FeP nanozyme was directly dissolved in deionized water), the size and PDI of FeP nanozyme were measured at different concentrations, and the size of FeP nanozyme under different conditions was characterized by DLS. Figure 1 (B) and 3(A).

[0088] As shown in the figure, FeP nanozyme remains stable for 7 days under physiological conditions. Figure 3 (A)), while gradually self-biodegrading on the 21st day ( Figure 1 (B)).

[0089] 2) Hemolytic ability of FeP nanozymes

[0090] The specific method is as follows:

[0091] (1) Remove serum from blood by centrifugation (3000 rpm, 15 min) to obtain red blood cells;

[0092] (2) The cells were dispersed in PBS and incubated with FeP nanozymes (0, 50, 100, 200, 500, 1000 μg mL -1 )mix;

[0093] (3) Incubate at 37°C for 2 h and then measure at 450 nm using a microplate reader. Figure 3 (B).

[0094] Depend on Figure 3 (B) It was found that FeP nanozyme has excellent biocompatibility (negligible hemolysis (<5%)).

[0095] 1.3 Photothermal evaluation of FeP nanozymes

[0096] Specific test methods:

[0097] (1) Photothermal effect of FeP: FeP nanozyme solutions with different concentrations (FeP nanozyme directly dissolved in deionized water) (0, 0.2, 0.5, 1, 1.5 mg mL -1 ) were placed in a cuvette (2 mL) and irradiated with 808 nm laser (0.3, 0.5, 0.8, 1.0, 1.5 W·cm -2 , 10 min) and the temperature was recorded by a digital thermometer and an infrared thermal imaging camera.

[0098] (2) Light stability of FeP: 808nm (1.5W·cm -2 , 10 min) irradiated with FeP (1.0 mg mL -1 ), then turn off the laser until the heating temperature is reached. Figure 5 and 6 shown.

[0099] UV-visible spectra of FeP nanozymes with or without 808 nm laser irradiation, e.g. Figure 4 shown.

[0100] Depend on Figure 4 It was found that FeP nanozyme exhibited a strong photothermal effect under 808nm laser irradiation.

[0101] At 1.5 W·cm -2 After irradiation for 10 minutes, FeP nanozyme (0.5 mg mL -1 ) increased from 23°C to 45°C, indicating that the temperature depends on the FeP nanozyme concentration, laser power, and laser time ( Figure 5In contrast, under the same irradiation conditions, the temperature of phosphate buffered saline (PBS) only increased to 28°C ( Figure 6 (A) and 6 (B)). In addition, six stable irradiation on-off cycles were performed to confirm the photostability of FeP nanozymes ( Figure 5 (C)), which is consistent with the UV-visible absorption spectrum of FeP nanozyme irradiated with 808 nm laser ( Figure 4 ), which means that FeP nanozymes have good photothermal capabilities.

[0102] 2. Characterization of Nanozyme FePGOGA (Labeled as FePGOGA)

[0103] 2.1 Determination of drug encapsulation capacity of FePGOGA by UV absorption spectroscopy

[0104] Based on the FeP nanozyme, multifunctional nanozymes were further developed. Nanozyme preparations FePGO (preparation method refers to Example 1 of this application), FePGA (preparation method refers to Example 1 of this application) and nanozyme FePGOGA of Example 2 of the present invention, such as Figure 7 shown.

[0105] Depend on Figure 7 It was found that under the set preparation conditions, the prepared nanozyme preparation was well dispersed.

[0106] In order to test the loading capacity of FeP nanozymes, we used (FeP FITC GODID) electrostatic and ππ interactions will FITC GOx (GOx labeled with fluorescein isothiocyanate (FITC)) and DID (a model drug that replaces GAP19) were encapsulated in FeP nanozymes to control drug release under NIR irradiation. Figure 8 As shown in (A), due to the loading of cargo, FeP FITC GO and FeP FITC The size of GODID increases to 120-200nm. FITC The UV-visible spectrum of GODID is used to characterize the cargo loading efficiency, such as Figure 8 (B) Typical peaks at 488 nm (FITC) and 633 nm (DID) ( Figure 8 (B)) confirmed FITC GOx and DID on FeP FITC Successful co-loading in GODID, with a loading capacity calculated to be 0.74 μg mg -1 (GOx / FeP) and 148 μg·mg -1 (DID / FeP)( Figure 9 ).

[0107] 2.2 The morphology and size of FePGOGA were determined by DLS and TEM. FePGOGA was irradiated with 808 nm laser, and the release of loaded drugs GOx and GAP19 was determined by fluorescence spectroscopy. The stability of different nanozymes in different solutions was also determined. Figure 10 and 11 shown.

[0108] GOx and GAP19 were co-loaded onto FeP nanozymes, and the average size of the prepared nanozymes FePGOGA was about 120 nm ( Figure 11 (A)). In addition, after GOx and GAP19 were co-loaded, the potential of FeP nanozyme decreased ( Figure 11 (B)), indicating that GOx and GAP19 were successfully co-loaded. The stability of FeP, FePGA, FePGO and FePGOGA nanozymes in H2O, PBS and DMEM containing 10% FBS (n=3) is shown in Figure 3. Figure 11 (C).

[0109] like Figure 10 As shown in (A), 35.2% and 63.5% of FITC were released without and with laser irradiation, respectively, indicating that 808 nm laser irradiation significantly promoted the release of cargo from nanozymes. The same release curve was also detected by DID ( Figure 10 (B)), reveals the DID or FITC The interaction between GOx and FeP was dissociated due to hyperthermia.

[0110] 3. Catalytic effect of nanozyme FePGOGA

[0111] The ability of GOx to consume O2 was investigated using the O2 probe [Ru(dpp)3]Cl2(RDPP). Figure 12 (A)). Figure 12 As shown in (A) and 13, the fluorescence of RDPP decreases with the concentration of GOx and sufficient glucose. Then, the mimetic ability of GSHox was examined, that is, GSH was catalyzed to GSSH and released Fe 2+ , and the two products were detected using (5,5-dithiobis-(2-nitrobenzoic acid)] and phenanthroline, respectively. The results showed that 80% of Fe 2+ Release within 24 hours ( Figure 12 (B)). In addition, Fe 3+ Reduction to Fe 2 + , reducing GSH to 40% ( Figure 12(C) and 14), indicating the effective GSHox activity of FeP nanozymes. In addition, the POD mimicking ability of FeP nanozymes was evaluated using methylene blue (MB), that is, H2O2 was catalyzed to ·OH with high cytotoxicity. The absorption of MB at 663nm decreased sharply after adding FeP nanozymes, as shown in Figure 2. Figure 14 As shown in Figure 5, the degradation of H2O2 and the generation of ·OH were the main reasons for the degradation of H2O2. These results indicate that FePGOGA nanozymes have excellent cascade enzyme-like catalytic ability.

[0112] Reaction mechanism:

[0113]

[0114] FeP+GSH→Fe 2+ +GSSH

[0115] Fe 2+ +H2O2→Fe 3+ +·OH+OH -

[0116] Fe 3+ +H2O2→Fe 2+ +·OOH+H +

[0117] IV. Evaluation of Multifunctional Nanozyme In Vitro Uptake, Cx43 Blockade, Catalytic Therapy, Mild Photothermal Therapy, and Anti-tumor Therapy

[0118] 4.1 In vitro uptake experiment of multifunctional nanozymes

[0119] Specific test methods:

[0120] (1) Cal27 cells were seeded into glass bottom culture dishes (5.0×10 5 ) overnight;

[0121] (2) Add free FITC, free DID and FeP to the culture dish FITC GODID (FITC 2.5 μg mL -1 , DID 5μg·mL -1 ), culture for 6 h;

[0122] (3) Wash with PBS three times, and DAPI (5 μg mL -1 , 10 min) staining, paraformaldehyde (4%, 10 min) fixation, CLSM observation (FITC:λ ex =488nm,λ em =520nm,DID:λ ex =634nm,λ emIn a parallel experiment, cal27 cells were seeded in 6-well plates and treated as described above, and the fluorescence intensity of FITC and DID in the cells was detected by FCM.

[0123] To observe lysosomal escape, FITC-labeled GAP19 was loaded into FeP nanozymes to form FeP FITC After incubation for 6 h, the cells were illuminated and washed three times with PBS and stained with DAPI (5 μg mL -1 , 10 min), LysoTracker@Red (0.5 μM, 30 min), paraformaldehyde fixation (4%, 10 min) and observation by CLSM (FITC: λ ex =488nm,λ em =520nm, DID:λ ex =577nm,λ em =590nm).

[0124] Evaluation of FeP using CLSM FITC Cellular uptake of GODID. Due to the poor transmembrane permeability of GOx, free FITC GOx-treated cells showed minimal green (FITC) fluorescence. FITC GO and FeP FITC Strong green (FITC) fluorescence was detected in GODID-treated cells, confirming the function of FeP nanozymes as high-performance nanocarriers that can enhance the cellular uptake of GOx, e.g. Figure 15 (A) (Scale bar = 50 μm). FITC Green (FITC) and red (DID) fluorescence were observed in GODID-treated cells, confirming the successful co-loading and internalization of GOx and DID. Flow cytometry analysis detected consistent results ( Figure 15 (B), 15(C) and Figure 16 (A) Figure 16 (B)).

[0125] Lysosomal escape is an important process for GAP19 peptide to block Cx43 channels, e.g. Figure 16 (C) shows that green fluorescence is largely separated from red fluorescence in cal27 cells, demonstrating lysosomal escape of GAP19.

[0126] 4.2 Multifunctional nanozyme blocking Cx43 in vitro experiment

[0127] 1) Changes in Cx43 expression

[0128] The specific method is as follows:

[0129] (1) Cal27 cells were seeded into 6-well plates (5.0×10 5 ) cells overnight;

[0130] (2) FeP (0, 50, 100, 200, 500 μg·mL -1 ), FePGO, FePGA and FePGOGA (2μg·mL -1 GOx and 40 μg mL -1 GAP19) were added to the culture medium and incubated for 24 h;

[0131] (3) Wash with PBS three times, lyse with RIPA, and detect the expression of Cx43 by western blot (protein immunoblotting). Figure 17 shown.

[0132] Cx43 forms intercellular channels and exchanges metabolites and signals to protect cells from damage, making tumors highly resistant to treatment. Figure 17 It was found that Cx43 protein levels increased after 24 hours of treatment with FeP nanozyme due to the gradual increase in oxidative stress. In addition, the Cx43 blocking ability of GAP19 was also studied, and the results showed that Cx43 protein was drastically reduced, and GAP19 exhibited excellent blocking ability. Similar results were detected in cal27 cells treated with FeP, FePGO, FePGA, and FePGOGA. Figure 17 As shown in Figure 3, the expression of Cx43 was slightly increased in cells treated with FeP and FePGO, while the expression of Cx43 in cells treated with FePGA and FePGOGA was greatly suppressed. Immunofluorescence staining of Cx43 revealed consistent results ( Figure 18 ).

[0133] 2) Changes in intracellular ROS content

[0134] The specific method is as follows:

[0135] (1) Cal27 cells were seeded into 6-well plates (5.0×10 5 ) cells overnight;

[0136] (2) FeP (0, 50, 100, 200, 500 μg·mL -1 ), FePGO, FePGA and FePGOGA (2μg·mL -1 GOx and 40 μg mL -1 GAP19) were added to the culture medium and incubated for 24 h;

[0137] (3) The cells were washed three times with PBS. After DCFH-DA (5 μM) staining for 30 min, DAPI (5 μg / mL, 10 min) staining, paraformaldehyde (4%, 10 min) fixation, and CLSM observation (λ ex =488nm,λ em =520nm). In a parallel experiment, the amount of intracellular ROS was detected by flow cytometry. Figure 19 shown.

[0138] Depend on Figure 19 (A) It was found that higher ROS fluorescence intensity was detected in cal27 cells treated with FePGO and FePGA due to the catalytic effect of GOx and the blocking effect of GAP19. Flow cytometry analysis showed that the average fluorescence intensity of DCF in cal27 cells treated with different preparations was 2×10 3 (FeP)<4×10 3 (FePGO)<4.5×10 3 (FePGA)<7.5×10 3 The order of (FePGOGA) increases ( Figure 19 (B)).

[0139] 5. Cascade nanozyme-like catalytic activity of nanozyme FePGOGA

[0140] Figure 20 The in vitro cascade nanozyme catalytic activity of the nanozyme FePGOGA. Figure 20 (A) CLSM images of intracellular O2 levels depleted by GOx. Scale bar = 50 μm. Figure 20 (B) Flow cytometric analysis of intracellular O2. Figure 20 (C) IVIS images of intracellular O2. Figure 20 (D) Intracellular Fe 2+ Horizontal CLSM images. Scale bar = 50 μm. Figure 20 (E) Intracellular Fe 2+ Flow cytometric analysis of the levels. Figure 20 (F) Quantification of Fe 2+ Fluorescence intensity (n=3). cal27 cells were treated with different concentrations of FeP nanozymes for 12 hours and then probed with FerroOrange. Figure 20 (G) CLSM images of intracellular LPO levels. Scale bar = 50 μm.

[0141] The cascade nanozyme-like catalytic activity of FePGOGA was studied in cal27 cells. Figure 20As shown. First, the intracellular O2 level consumed during GOx catalysis was analyzed. Red fluorescence (RDPP, a luminescent oxygen sensor) was significantly reduced only after FePGO treatment. The change in red fluorescence in cells treated with free GOx was negligible, which was attributed to the poor transmembrane permeability of GOx. Flow cytometric analysis of RDPP ( Figure 20 (B) and Figure 21 ) and in vivo imaging system (IVIS) images of living animals ( Figure 20 (C), Cal27 cells treated with and without FeP nanozymes and GOx for 12 h and then probed with RDPP) revealed similar phenomena, confirming that FeP nanozymes are high-performance nanocarriers that can enhance the intracellular uptake of GOx and the nanozyme-like catalytic activity of GOx. The GSHOx-like activity of FeP was investigated using FerroOrange (a Fe 2+ Obviously, the intracellular Fe 2+ The concentration increased with the increase of FeP concentration in cal27 cells, e.g. Figure 20 (D) Flow cytometry ( Figure 20 (E) and 20(F)) were used to quantify this increase, Fe 2+ The concentration almost quadrupled, with the FeP concentration at 0.5 mg mL -1 , thus verifying the GSHOx-like activity of FeP nanozyme and its ability to generate Fe 2+ ability.

[0142] 5. Nanozyme FePGOGA Anti-tumor Therapy in Vitro

[0143] Specific methods:

[0144] (1) FePGO, FePGA, FePGOGA, cal27 and scc7 cells were seeded into 96-well plates (1×10 4 ), and then FeP, FePGO (2 μg·mL -1 GOx), FePGA (40 μg mL -1 GAP19), FePGOGA (2 μg·mL -1 GOx and 40 μg mL - 1 GAP19) were added to the cells and incubated for 6 h;

[0145] (2) With or without 808 nm laser (1.5 W·cm -2 , 10 min) irradiated cells, cultured for another 24 h, and then evaluated by MTT assay;

[0146] (3) In another parallel experiment, cal27 cells were seeded onto 6-well plates and treated as described above. The cells were then washed three times with PBS, stained with calcein-AM (20 nM) and PI (4 μM), and then observed using CLSM (calcein-AM: λ ex =488nm,λ em =520nm, PI:λ ex =633nm,λ em =664nm);

[0147] (4) In another parallel experiment, CAL27 cells were seeded onto 6-well plates and treated as described above. In another parallel experiment, CAL27 cells were seeded onto 6-well plates and treated as described above. The cells were then washed three times with PBS, stained with Annexin V-FITC / PI and crystal violet, and observed using FCM and CLSM, respectively.

[0148] The antitumor effect of FePGOGA was investigated in cal27 and scc7 cells using PBS, FePGO, and FePGA as controls. Cells treated with FePGOGA exhibited higher cytotoxicity than those treated with FePGO and FePGA, even without irradiation, which can certainly be attributed to the combination of starvation and catalytic therapy, as Figure 22 (A) and 22 (B) As shown in Figure 22, the photothermal conversion effect of FeP nanozymes, the cell inhibition efficiency of FeP, FePGO and FePGA was 1.5 W·cm -2 The cytotoxicity of FePGOGA was significantly enhanced after 808 nm laser irradiation for 10 min. Due to the multiple therapeutic effects of starvation, catalytic therapy and low-temperature PTT, cells treated with FePGOGA under the same conditions showed the highest cytotoxicity. Figure 22 Annexin v-FITC / PI assay in the PLGA matrix revealed the most significant antitumor effect of FePGOGA under 808 nm laser irradiation.

[0149] In addition, FeP, FePGO (2 μg·mL -1 GOx), FePGA (40 μg mL -1 GAP19) and FePGOGA (2 μg·mL -1 GOx and 40 μg mL -1 GAP19) treated cal27 cells for 6 h with and without irradiation (808 nm, 1.5 W cm -2 , 10 min) and further incubated for 24 h before staining with crystal violet to confirm the effective combination of these three treatments ( Figure 23 ).

[0150] 6. In vivo circulation distribution, photothermal conversion ability, and anti-tumor effect of the nanozyme FePGOGA 6.1 In vivo circulation distribution and photothermal conversion ability of the nanozyme FePGOGA

[0151] To analyze the in vivo distribution of nanozyme FePGOGA, mice bearing cal27 tumors were intravenously injected with FePDID or free DID (5 mg kg -1 DID). The fluorescence intensity of DID was analyzed at different time intervals using a live animal imaging system ( Figure 24 (A)). Significant DID fluorescence signals appeared in the tumor area, e.g. Figure 24 (B) The fluorescence intensity peaked 12 hours after FePDID injection, indicating that it effectively targeted the tumor due to the enhanced permeability and retention (EPR) effect. In contrast, the fluorescence of free DID decreased rapidly. In addition, the fluorescence intensity of various organs and tumors 12 hours after injection was consistent with these results ( Figure 25 The photothermal activity of FePDID in vivo was also investigated based on its distribution. -2 Tumor temperature was recorded in mice irradiated with 808 nm laser for 10 min, and the temperature increased rapidly from 34°C to 44°C in only 5 min. Figure 24 (C) Figure 26 , which is high enough for low-temperature PTT. However, for mice treated with PBS, the local temperature increase of the tumor was negligible under the same irradiation.

[0152] Here’s how:

[0153] (1) Cal27 cells (10 6 cells in 100 μL PBS);

[0154] (2) To tumor-bearing mice (200 mm 3 ) were injected intravenously with free DID and FePDID, and at the determined time points (0, 2, 4, 6, 12 and 24 h), the live animal imaging system (λ ex =634nm,λ em =670nm) to observe mice;

[0155] (3) Mice were sacrificed 12 h after injection, and tumors and major tissues, including heart, liver, spleen, lung, and kidney, were collected and imaged as described above;

[0156] (4) Twelve hours after injection, 808 nm laser (1.5 W·cm -2 , 10 min) to irradiate the tumor and then record it with a digital thermometer and infrared thermal imager.

[0157] 6.2 Nanozyme FePGOGA Anti-tumor Therapy in Vivo

[0158] The specific method is as follows:

[0159] (1) When the tumor volume of the tumor-bearing mice reached 150 mm 3 and 500mm 3 The mice were randomly divided into 5 groups (n=5) and injected with PBS, FePGO, FePGA, FePGOGA and FePGOGA(L) through the tail vein, respectively. The FePGOGA(L) group was exposed to light 12 hours after the injection.

[0160] (2) Repeat the above treatment every 2 days for a total of 3 times. Measure the weight and tumor volume of the mice every 1 day starting from the injection of the drug to observe the combined anti-tumor effects of the catalytic treatment of the multifunctional nanomedicine, starvation treatment, and low-temperature photothermal therapy.

[0161] (3) After the tumor inhibition experiment, tumor tissues were collected and analyzed by immunohistochemistry (IHC), IF, and WB techniques to analyze the blocking effect of GAP19 on intercellular space proteins and the changes in intracellular ROS and ATP after intercellular space protein blocking. The combined anti-tumor effect of the multifunctional nanozyme was analyzed by H&E and TUNEL.

[0162] The therapeutic effect of FePGOGA was further evaluated in mice bearing cal27 tumors. Figure 27 (A) Mice bearing cal27 tumors were randomly divided into five groups (n=5): PBS, FePGO, FePGA, FePGOGA, and FePGOGA(L) (the suffix -L represents the use of 808 nm laser, 1.5 W·cm -2 Irradiate the tumor site for 10 minutes). The drug dosage is 2 mg kg -1 GOx and 40 mg kg -1 GAP19, once every three days, for a total of three times. Twelve hours after injection, mice in the FePGOGA(L) group were treated with 1.5W·cm -2 The mice were irradiated with 808 nm laser for 10 minutes. The tumor volume and body weight of the mice were measured every other day to evaluate the therapeutic effect and systemic toxicity.

[0163] Compared with the PBS group, the FePGO and FePGA groups showed tumor inhibition due to tumor starvation treatment and catalytic treatment, as shown in Figure 27(BD) and 28. It is worth noting that the tumor inhibition in mice treated with FePGOGA was significantly better than that in the FePGO and FePGA groups. After irradiation of mice with 808nm laser (FePGOGA(L)), the tumor inhibition effect was significantly enhanced, which was due to the synergistic effect of tumor starvation, catalytic therapy and low-temperature PTT, and even some tumors were completely eliminated. In addition, the tumor weight of mice 20 days after treatment also confirmed that the tumor inhibition effect of the FePGOGA(L) group was the highest ( Figure 27 (E)).

[0164] In addition to analyzing the changes in tumor size and weight, tumor tissues were also subjected to immunohistochemical analysis. As revealed by hematoxylin and eosin (H&E) staining, the FePGOGA(L) group showed the most severe anti-tumor effect ( Figure 31 In addition, TUNEL staining showed that the apoptosis signal of this group of cells was the strongest ( Figure 31 ), the cell proliferation signal is the weakest.

[0165] In clinical practice, the late stage of tumor usually leads to the death of patients. In order to further prove the anti-tumor effect of FePGOGA nanozyme, we selected 500mm 3 Cal27 tumor-bearing mice were treated with various Figure 29 (A) ), and tumor volume and body weight were recorded. The results showed that FePGO or FePGA treatment had negligible inhibition on tumor growth, as Figure 29 (BD) and 30. Although FePGOGA treatment (starvation and catalytic therapy) achieved significant regression of tumor growth, the tumor volume remained approximately 500 mm on day 16 after treatment. 3 In contrast, the tumor volume of mice in the FePGOGA(L) group (starvation, catalytic therapy, and low-temperature PTT) decreased significantly to 100 mm on day 16 after treatment. 3 ( Figure 29 (BD) and Figure 30 ), showing a strong synergistic anti-tumor effect. On the 16th day after treatment, the FePGOGA ( Figure 29 (G) and (H) H&E and TUNEL staining of tumors in mice also showed that FePGOGA had the highest rates of cell necrosis and apoptosis under light irradiation. In addition, all treatments had negligible effects on the body weight and major organs (heart, liver, spleen, lung, and kidney) of the mice.

[0166] We investigated the detailed mechanism of the synergistic effects of tumor starvation, catalytic therapy, and low-temperature PTT. Figure 31 and Figure 29As shown in (I), red fluorescence (Cx43 expression) in tumor tissue decreased significantly after injection of FePGA or FePGOGA. Tumors treated with the nanozyme FePGOGA exhibited the weakest red fluorescence (GPX4 expression) compared to those treated with FePGO or FePGA, suggesting enhanced catalytic therapy. Based on the results of cell experiments, FePGOGA treatment significantly induced the degradation of HSPs.

[0167] On day 20 after intravenous injection, mice bearing cal27 xenograft tumors (initial tumor volume 150 mm) were isolated. 3 ) Tumors were collected and injected with different NPs. Cell nuclei and HSP70 / 90 / GPX4 were stained with DAPI (blue) and anti-HSP70 / 90 / GPX4 antibodies (red), respectively. Scar bar = 50 μm. Immunofluorescence images, such as Figure 32 On day 16 after intravenous injection, mice bearing cal27 xenograft tumors (initial tumor volume 500 mm 3 ) Tumors were collected. Cell nuclei and HSP70 / 90 / GPX4 were stained with DAPI (blue) and anti-HSP70 / 90 / GPX4 antibodies (red), respectively. Scale bar = 50 μm, Figure 33 As shown in Figure 2, lower tumor HSP90 / 70 expression levels were detected after FePGOGA treatment and 808 nm laser irradiation, owing to HSP90 / 70 degradation induced by starvation and catalytic therapy. Thus, FePGOGA treatment induces starvation and enhances catalytic therapy efficiency, while simultaneously reducing HSPs and enhancing their sensitivity to low-temperature PTT.

Claims

1. A nanozyme FePGOGA, characterized in that Made of glucose oxidase, GAP19, and Fe-PDAP nanozyme; The preparation method of the Fe-PDAP nanozyme comprises dissolving FeCl3·6H2O in water, adding 2,6-diaminopyridine for polymerization, and obtaining the Fe-PDAP nanozyme.

2. The nanozyme FePGOGA according to claim 1, characterized in that The Fe-PDAP nanozyme is loaded with glucose oxidase and GAP19.

3. The nanozyme FePGOGA according to claim 2, characterized in that The loading amount of glucose oxidase on Fe-PDAP nanozyme is 0.1-3.5 μg·mg -1 The loading amount of GAP19 on Fe-PDAP nanozyme was 1-150 μg·mg -1 .

4. The nanozyme FePGOGA according to claim 3, characterized in that The loading amount of glucose oxidase on Fe-PDAP nanozyme is 0.2-3.5 μg·mg -1 The loading amount of GAP19 on Fe-PDAP nanozyme was 20-150 μg·mg -1 .

5. Use of the nanozyme FePGOGA as described in any one of claims 1 to 4 in the preparation of anti-tumor drugs.

Images