Tumor-targeted nano generator for chemically generating peroxynitrite through light control as well as preparation method and application of nano generator

Abstract

The invention provides a tumor-targeting nanometer generator for optically-controlled chemical generation of peroxynitrite and a preparation method and application thereof.The tumor-targeting nanometer generator for optically-controlled chemical generation of peroxynitrite comprises an inner core and a coating layer coating the inner core, the inner core comprises BNN-6-loaded Prussian blue, and the coating layer comprises a BNN-6-loaded Prussian blue and a BNN-6-loaded Prussian blue. The coating layer comprises a tumor cell membrane. According to the present invention, the specific generation of ONOO <-> on tumor cells under the irradiation of the near-infrared I region light can be achieved, the activity of pyruvate kinase (PK) and glutaminase (GLS) can be inhibited, and the glycolysis and the glutamine metabolism of the tumor cells can be destroyed;

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a photo-controlled chemical generator for the targeted generation of peroxynitrite ions in tumors, its preparation method, and its application. Background Technology

[0002] peroxynitrite (ONOO) - Nitric oxide (NO) is a highly reactive nitrogenous substance that can alter the structure and function of proteins by nitrifying them, thereby regulating signaling pathways. The formation of peroxynitrite depends on nitric oxide (NO) and superoxide anion (O2). - The interaction between these two molecules (• and •) is limited by their short intracellular lifespan and limited diffusion distance, resulting in a low probability of them meeting and reacting, thus restricting the formation of peroxynitrite. Therefore, there is an urgent need to develop a new strategy for the efficient generation of peroxynitrite. Literature reports that in test tubes, peroxynitrite (ONOOH, i.e., protonated ONOO) can be generated. - ) can be overcome by nitrite (NO2) - NO is chemically synthesized from acidic hydrogen peroxide (H2O2), while NO can react with oxygen (O2) to produce NO2. - Therefore, generating NO and relatively stable, highly diffuse oxygen (O2) in the unique weakly acidic environment of tumor cells and the overexpression of hydrogen peroxide (H2O2) is a potential strategy for the generation of peroxynitrite. Summary of the Invention

[0003] In view of this, the present invention provides a photo-controlled chemical generation nanogenerator for targeting tumors to produce peroxynitrite, its preparation method and application, which can photochemically generate ONOO under near-infrared I region light irradiation. - It can achieve the specific generation of ONOO on tumor cells under near-infrared I region light irradiation. - It inhibits the activity of pyruvate kinase (PK) and glutaminase (GLS), thereby disrupting glycolysis and glutamine metabolism in tumor cells.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a photocontrolled chemically generated peroxynitrite nanogenerator targeting tumors, comprising a core and a coating layer covering the core, the core comprising Prussian blue loaded with BNN-6, and the coating layer comprising tumor cell membranes.

[0005] Preferably, the tumor cell membrane comprises a 4T1 cell membrane.

[0006] Secondly, the present invention provides a method for preparing the aforementioned photocontrolled chemical generation nanogenerator for targeting tumors to generate peroxynitrite, comprising the following steps: S1, Obtain Prussian Blue; S2. Disperse the Prussian blue in anhydrous ethanol to obtain a Prussian blue dispersion; S3. Add BNN-6 to the Prussian blue dispersion, ultrasonically disperse, place in an ice bath and stir, centrifuge, take the precipitate and disperse it in water to obtain a Prussian blue dispersion loaded with BNN-6. S4. Disperse the tumor cell membrane in PBS buffer to obtain a tumor cell membrane dispersion; S5. The Prussian blue dispersion loaded with BNN-6 is mixed with the tumor cell membrane dispersion and sonicated to obtain a nanogenerator that generates peroxynitrite ions by photocontrolled chemical generation targeting tumors.

[0007] Preferably, step S1 includes: Potassium ferricyanide, polyvinylpyrrolidone, and water were mixed, then concentrated hydrochloric acid was added, the mixture was heated to react, and centrifuged to obtain Prussian blue.

[0008] Preferably, in step S3, the mass ratio of BNN-6 to Prussian blue is greater than or equal to 2:1.

[0009] Preferably, in step S3: The mixture was placed in an ice bath and stirred for 12 hours; and / or, The centrifugation temperature was 4℃, the centrifugation speed was 10000 rpm, and the centrifugation time was 30 min.

[0010] Preferably, in step S4, the concentration of the tumor cell membrane dispersion is greater than or equal to 1 mg / mL.

[0011] Preferably, in step S5, the ultrasound time is 15 min to 30 min.

[0012] Thirdly, the present invention provides a photo-controlled chemical peroxynitrite nanogenerator for targeting tumors, or a photo-controlled chemical peroxynitrite nanogenerator for targeting tumors prepared by the aforementioned method, for the application in inhibiting pyruvate kinase activity and / or glutaminase activity.

[0013] Fourthly, the present invention provides a photo-controlled chemical generator for generating peroxynitrite ions targeting tumors, or a photo-controlled chemical generator for generating peroxynitrite ions targeting tumors prepared by the aforementioned method, and its application in the preparation of drugs for treating tumors.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The photo-controlled chemical generation nanogenerator for targeting tumors of the present invention generates heat under near-infrared I region laser stimulation, which causes BNN-6 to release NO, and NO reacts with O2 to generate NO2. - Subsequently, it reacts with H2O2 under acidic conditions to generate ONOO. - This enables the specific generation of ONOO on tumor cells under near-infrared I region light irradiation. - .

[0015] (2) The photocontrolled chemical generation nanogenerator for targeting tumors in this invention generates highly diffuse ONOO - It can nitrate tyrosine residues, inhibit the activity of pyruvate kinase (PK) and glutaminase (GLS), disrupt the glycolysis and glutamine metabolism of tumor cells, and completely cut off the energy supply of tumor cells, providing a new and effective treatment strategy for tumor treatment. Attached Figure Description

[0016] Figure 1 A scanning electron microscope image of BPM and a mapping elemental analysis diagram obtained by transmission electron microscopy are provided for an embodiment of the present invention. Figure 2 The absorption spectra of PB, Mem, BNN-6, and BPM are provided in an embodiment of the present invention. Figure 3 This is an embodiment of the BPM in vitro photothermal heating curve provided by the present invention; Figure 4 The dissolved oxygen content of BNN-6, PB, and BPM in water under conditions containing H2O2 and without H2O2, as provided in an embodiment of the present invention; Figure 5 The fluorescence intensity of NO produced by Mem, PB, BNN-6, and BPM under both illuminated and dark conditions, according to an embodiment of the present invention; Figure 6 NO2 generated by Mem, PB, BNN-6, and BPM under different dissolved oxygen conditions, as provided in an embodiment of the present invention. - The concentration; Figure 7 According to an embodiment of the present invention, PB, BNN-6, and BPM are used to generate ONOO under conditions of light and no light, and with and without H2O2. - The concentration; Figure 8 Semi-quantitative MFIs of 4T1 cells incubated with BPM for different times and cell viability of 4T1 cells incubated with light for different times are provided in an embodiment of the present invention. Figure 9This invention provides a confocal imaging of BPM after incubation with a DAX-J2 TMPON Green probe under conditions of illumination and darkness, hypoxia and normoxic conditions; Figure 10 The changes in the activities of pyruvate kinase and glutaminase in BPM-treated cells provided in an embodiment of the present invention; Figure 11 This invention provides in vitro imaging of tumor tissue at different BPM injection times according to an embodiment of the invention. Figure 12 The images show the tumor volume changes of PB and BPM mice under light and no light conditions, as well as the tumor tissue after 14 days of treatment, provided as an embodiment of the present invention. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0018] In a first aspect, the present invention provides a photocontrolled chemically generated peroxynitrite nanogenerator targeting tumors, comprising a core and a coating layer covering the core, the core comprising Prussian blue loaded with BNN-6, and the coating layer comprising tumor cell membranes.

[0019] The present invention relates to a photocontrolled chemical peroxynitrite nanogenerator (BNN-6@PB@Mem, BPM) for targeting tumors. Utilizing Prussian blue (PB) as a nanocarrier, which possesses excellent photothermal conversion properties and peroxidase-like characteristics, BNN-6, which generates NO upon thermal stimulation, is loaded onto the nanocarrier and then coated with tumor cell membranes exhibiting homologous targeting effects. Leveraging the homologous targeting ability of the tumor cell membrane, the device specifically targets tumor cells. BPM decomposes H2O2 to generate O2, increasing the O2 concentration within the tumor cells. Under near-infrared I region laser stimulation, the heat generated by BPM causes BNN-6 to release NO, which then reacts with O2 to generate NO2. - Subsequently, it reacts with H2O2 under acidic conditions to generate ONOO. - This enables the specific generation of ONOO on tumor cells under near-infrared I region light irradiation. - Highly diffused ONOO - It can nitrate tyrosine residues in tumor cells, inhibit the activity of pyruvate kinase (PK) and glutaminase (GLS), disrupt glycolysis and glutamine metabolism in tumor cells, and completely cut off the energy supply to tumor cells, providing a new and effective treatment strategy for tumor therapy.

[0020] Furthermore, the tumor cell membrane includes a 4T1 cell membrane.

[0021] Secondly, the present invention provides a method for preparing the aforementioned photocontrolled chemical generation nanogenerator for targeting tumors to generate peroxynitrite, comprising the following steps: S1, Obtain Prussian Blue; S2. Disperse the Prussian blue in anhydrous ethanol to obtain a Prussian blue dispersion; S3. Add BNN-6 to the Prussian blue dispersion, ultrasonically disperse, place in an ice bath and stir, centrifuge, take the precipitate and disperse it in water to obtain a Prussian blue dispersion loaded with BNN-6. S4. Disperse the tumor cell membrane in PBS buffer to obtain a tumor cell membrane dispersion; S5. The Prussian blue dispersion loaded with BNN-6 is mixed with the tumor cell membrane dispersion and sonicated to obtain a nanogenerator that generates peroxynitrite ions by photocontrolled chemical generation targeting tumors.

[0022] It is understandable that in step S2, ultrasonic dispersion can be used for dispersion; in step S3, after centrifugation to remove the supernatant and obtain the precipitate, ultrapure water can be added to the precipitate and ultrasonic dispersion can be used to disperse the precipitate in the water.

[0023] Further, step S1 includes: Potassium ferricyanide, polyvinylpyrrolidone, and water were mixed, then concentrated hydrochloric acid was added, the mixture was heated to react, and centrifuged to obtain Prussian blue.

[0024] It is understandable that in step S1, the mass concentration of concentrated hydrochloric acid is 36%~38%, and the water can be ultrapure water.

[0025] Furthermore, in step S3, the mass ratio of BNN-6 to Prussian blue is greater than or equal to 2:1.

[0026] Furthermore, in step S3, the stirring time in an ice bath is 12 h.

[0027] Furthermore, the centrifugation temperature was 4℃, the centrifugation speed was 10000 rpm, and the centrifugation time was 30 min.

[0028] Furthermore, in step S4, the concentration of the tumor cell membrane dispersion is greater than or equal to 1 mg / mL.

[0029] Furthermore, in step S5, the ultrasound time is 15 min to 30 min.

[0030] Thirdly, the present invention provides a photo-controlled chemical peroxynitrite nanogenerator for targeting tumors, or a photo-controlled chemical peroxynitrite nanogenerator for targeting tumors prepared by the aforementioned method, for the application in inhibiting pyruvate kinase activity and / or glutaminase activity.

[0031] Fourthly, the present invention provides a photo-controlled chemical generator for generating peroxynitrite ions targeting tumors, or a photo-controlled chemical generator for generating peroxynitrite ions targeting tumors prepared by the aforementioned method, and its application in the preparation of drugs for treating tumors.

[0032] Example 1 The synthesis of a nanogenerator for the photocontrolled chemical generation of peroxynitrite ions targeting tumors includes the following steps: (1) Add K3[Fe(CN)6] (potassium ferricyanide, 226.7 mg) and PVP (polyvinylpyrrolidone, 3 g) to 40 mL of ultrapure water and stir vigorously; then add 35 μL of concentrated hydrochloric acid to the aqueous solution; then heat the mixture at 80 °C for 20 h. (2) Centrifuge the above reaction solution at 10,000 rpm for 30 min at room temperature, remove the supernatant, and obtain the precipitate, which is Prussian blue. (3) Take 1 mg of Prussian blue and disperse it in anhydrous ethanol by ultrasonication to obtain a Prussian blue (PB) dispersion; (4) Add 2 mg BNN-6 to the Prussian blue dispersion according to the ratio of 1 mg PB: 2 mg BNN-6, disperse by ultrasonication, stir in an ice bath for 12 hours, centrifuge at 10000 rpm for 30 min at 4℃, remove the supernatant, obtain the precipitate, add ultrapure water, disperse by ultrasonication, and obtain the Prussian blue (BNN-6@PB) dispersion loaded with BNN-6; (5) Take 1 mg of 4T1 cell membrane, add 1 mL of PBS, and sonicate for 30 s to obtain a 1 mg / mL cell membrane (Mem) dispersion; (6) Mix the Prussian blue dispersion loaded with BNN-6 with 1 mg / mL cell membrane dispersion and sonicate for 15 min to obtain a photo-controlled chemically generated peroxynitrite nanogenerator (BPM) that targets tumors.

[0033] The synthesized BPM nanoparticles were characterized as follows: (1) Morphology and elemental composition of BPM The synthesized nanoparticles (PB and BPM) were diluted in solution at appropriate concentrations, dropped onto copper grids, and dried. Transmission electron microscopy (TEM) images were then acquired using a JEM-2100 TEM, and mapping elemental distribution images were acquired using a Talos F200 TEM. The synthesized BPM nanoparticles were diluted in solution at appropriate concentrations, dropped onto silicon wafers, and dried. Scanning electron microscopy (SEM) images were then acquired using a Zeiss Sigma FESEM. The results are shown in the table below. Figure 1 .

[0034] like Figure 1 As shown in figure a, the BPM nanoparticles are uniformly square in shape, well-dispersed, and have a particle size of approximately 256.9 nm; Figure 1 As shown in b, a thin film with a thickness of approximately 13 nm was successfully modified onto the surface of the BPM nanoparticles, indicating successful modification of the 4T1 cell membrane. Due to the modification of the outer cell membrane, the particle size of BPM is slightly increased compared to PB, approximately 13 nm. Further characterization results are shown in... Figure 1 The elemental analysis of the mapping by transmission electron microscopy of b shows that Fe is uniformly distributed within the nanoparticles, and P is uniformly distributed on the surface of the square particles, further indicating the successful modification of the 4T1 cell membrane on the surface of the nanoparticles.

[0035] (2) Ultraviolet absorption spectroscopy detection Different samples (PB, Mem, BNN-6, BPM) provided in this embodiment were dissolved in ultrapure water, and their ultraviolet absorption spectra were detected using a UV-Vis spectrophotometer. The results are shown in the figure. Figure 2 .

[0036] like Figure 2 As shown, the characteristic absorption peak of PB is around 780 nm, and the characteristic absorption peak of BNN-6 is around 264 nm.

[0037] (3) Photothermal performance study of BPM The in vitro photothermal properties of BPM were detected using a thermal imager, including: different materials (H2O, PB, PM, BPM) under 808nm laser light at 0.6 W / cm². 2 The photothermal heating curve at power is shown below. Figure 3 a. PM was obtained by directly coating 4T1 cell membranes with PB, without loading BNN-6; different concentrations of BPM were subjected to 808 nm laser at 0.6 W / cm². 2 The photothermal heating curve at power is shown below. Figure 3 b; The photothermal heating curves of 25 μg / mL BPM under different powers of 808 nm laser are shown in [reference needed]. Figure 3 c.

[0038] like Figure 3 As shown in a, at an 808 nm laser with a strength of 0.6 W / cm² 2 Under high power irradiation, the temperature of nanomaterials containing PB gradually increases with increasing irradiation time; for example... Figure 3 As shown in b, with a fixed laser power (808 nm laser, 0.6 W / cm²), 2 At power levels, the temperature of the BPM increases with increasing material concentration; for example... Figure 3 As shown in Figure c, at a fixed BPM concentration (25 μg / mL), the temperature of BPM increases with increasing 808 nm laser power.

[0039] (4) Study on the oxygen production performance of BPM by decomposing H2O2 The dissolved oxygen content in water was measured using a dissolved oxygen meter. The dissolved oxygen content in water for different materials (Blank, BNN-6, PB, BPM) was compared under conditions with and without 3% H2O2 aqueous solution. Figure 4 Blank is ultrapure water (blank control).

[0040] like Figure 4 As shown, when nanomaterials containing PB are added to an aqueous solution containing H2O2, the dissolved oxygen content in the water gradually increases over time.

[0041] (5) NO performance study of BPM The NO emission performance of BPM was detected by DAF-FM. The fluorescence intensity of NO produced by different materials (PBS, Mem, PB, BNN-6, BPM) under both illuminated and dark conditions is shown in the figure. Figure 5 The illumination conditions were 808 nm laser at 0.6 W / cm². 2 Irradiate with high power for 5 minutes.

[0042] like Figure 5 As shown, at an 808 nm laser with a strength of 0.6 W / cm² 2 Under high power irradiation, the fluorescence of BPM increased significantly, indicating that BPM can produce NO under illumination.

[0043] (6) NO2 in BPM - Performance Research NO2 in BPM was detected using a genomic reagent assay kit. - Performance, NO2 production of different materials (PBS, Mem, PB, BNN-6, BPM) under light irradiation, hypoxic, normoxic, oxygen-enriched, and 3% H2O2 conditions. - Concentration see Figure 6 The illumination conditions were 808 nm laser at 0.6 W / cm². 2Irradiate with high power for 5 minutes.

[0044] like Figure 6 As shown, under illumination and in oxygen-deficient conditions, only BPM produces trace amounts of NO2. - As oxygen content increases, the NO2 produced by BPM... - Increase NO2 in oxygen-rich environments - The concentrations were 19.2 times and 2.3 times higher in hypoxic and normoxic environments, respectively.

[0045] (7) BPM's ONOO - Performance Research The generation of ONOO by BPM in an aqueous solution containing H2O2 was detected using BboxiProbe® O56. - The performance of different materials (H2O, PB, BNN-6, and BPM) under the following conditions: illumination + no H2O2 (-H2O2+light), no illumination + 3% H2O2 (+H2O2-light), and illumination + 3% H2O2 (+H2O2+light). - Concentration see Figure 7 The illumination conditions were: 808 nm laser at 0.6 W / cm². 2 Irradiate with high power for 5 minutes.

[0046] like Figure 7 As shown, under illumination, BPM exhibits significant fluorescence compared to the condition without H2O2, indicating that ONOO - Effective generation.

[0047] Example 2 Cell uptake and cell killing assay In the cell uptake assay, 4T1 cells were evenly seeded at an appropriate density in confocal dishes and incubated for 12 h to allow cell adhesion. Then, BPM (prepared in Example 1) was specifically labeled with DiD (a commercially available fluorescent cell membrane probe). The cells were then incubated with fresh culture medium containing DiD-labeled BPM for 0 h, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively. Finally, the culture medium was discarded, the cells were washed three times with PBS, and images were captured using a confocal fluorescence microscope. The semi-quantitative MFIs of 4T1 cells incubated with BPM at different times are shown below. Figure 8 a. In the apoptosis detection experiment, the MTT assay was used to detect apoptosis. The illumination conditions were 808 nm laser at 0.6 W / cm². 2 Power, PB concentration of PM and BPM was 50 μg / mL, cell viability of 4T1 cells incubated with PM and BPM at different light exposure times is shown in the figure. Figure 8 b.

[0048] like Figure 8 As shown in a, the uptake of BPM nanoparticles within cells gradually increases over time; as Figure 8 As shown in b, BPM nanoparticles exhibit significant cell-killing effects under light irradiation.

[0049] Example 3 Intracellular ONOO - Production and its effects on glycolysis and glutamine metabolism (1) Intracellular ONOO - Performance research Intracellular ONOO - Generation is fully explored using the DAX-J2™ PON Green probe, which is compatible with ONOO. - Following a specific reaction, bright green fluorescence is emitted. 4T1 cells, after incubation with different materials under light and dark, hypoxic and normoxic conditions, were imaged using confocal imaging after incubation with the DAX-J2™ PON Green probe. Results are shown in [Figure number missing]. Figure 9 The BPM was prepared according to Example 1, wherein the illumination conditions were an 808 nm laser with a wavelength of 0.6 W / cm². 2 power.

[0050] like Figure 9 As shown, in the absence of light, almost no green fluorescence was observed in cells treated with either PM or BPM. After illumination, significant fluorescence was observed in cells treated with BPM, indicating that ONOO in the cells... - The efficient generation of ONOO; furthermore, under hypoxic conditions, cells treated with BPM also showed significant fluorescence under light, indicating that because PB's catalase-like activity can decompose excess intracellular hydrogen peroxide, BPM can overcome the limitations of tumor hypoxia and efficiently produce ONOO. - .

[0051] (2) Effects of BPM on glycolysis and glutamine metabolism The activities of intracellular pyruvate kinase (PK) and glutaminase (GLS) were detected using a pyruvate kinase and glutaminase assay kit. Specifically, 4T1 cells were evenly seeded at an appropriate density in 6-well plates and incubated for 12 h to allow cell adhesion. The culture medium was then discarded and replaced with fresh culture medium containing different samples for co-incubation. Cells requiring illumination were then irradiated with 808 nm light for 4 h. After incubation, the culture medium was discarded, and the cells were washed three times with PBS before lysing. The activities of pyruvate kinase and glutaminase were measured using the pyruvate kinase and glutaminase assay kit. Cells treated with blank culture medium served as a control. The experiment was performed three times. The relative content refers to the index in cells treated with blank culture medium as 100%, and the index in other treated cells divided by the index in cells treated with blank culture medium, multiplied by 100. Results are shown below. Figure 10 The BPM was prepared according to Example 1, under the illumination conditions of 808 nm laser at 0.6 W / cm². 2 power.

[0052] like Figure 10 As shown, the activities of pyruvate kinase and glutaminase in cells treated with BPM were significantly reduced, indicating that ONOO - Nitrifying tyrosine residues in tumor cells inhibits the activity of pyruvate kinase (PK) and glutaminase (GLS).

[0053] Example 4 Study on tumor-targeting enrichment ability of BPM in mice 4T1 cells (1×10⁴ cells per mouse) 7 (Number of samples) were inoculated into the right hind leg of mice to construct a tumor model. When the tumor reached a certain size, different samples were injected into the mice via the tail vein and in vivo fluorescence imaging was performed using a 460 / 530 NIR-II fluorescence imaging system. The results are shown in [Figure number missing]. Figure 11 .

[0054] like Figure 11 As shown, after being injected into mice via the tail vein, PyTPE@PB@Mem can achieve efficient enrichment at the tumor site, thereby enabling accurate tumor imaging and efficient drug delivery, which is also key to achieving precision diagnosis and treatment.

[0055] Example 5 Tumor suppression test 4T1 cells (1×10⁴ cells per mouse) 7 (One tumor sample) was injected into the right hind leg of mice to establish a tumor model; once the tumor grew to approximately 80-100 mm... 3 Different samples were injected into mice via the tail vein. Twenty-four hours later, the tumor sites of mice requiring light exposure were irradiated with laser. The size of the mouse tumors (volume = length × width) was measured daily.2 ×0.5) and body weight were measured once, and the results were taken once for 14 days. On day 14, a photograph of the tumor removed from the mouse was taken. The results are shown in the table below. Figure 12 In PM+L and BPM+L, L refers to illumination, specifically 0.6 W / cm² for an 808 nm laser. 2 Irradiate with high power for 5 minutes.

[0056] like Figure 12 As shown in Figure a, under illumination, BPM exhibits a more significant tumor growth inhibition effect compared to PM; for example... Figure 12 As shown in b, the tumor images of mice on day 14 clearly demonstrate that BPM exhibits excellent tumor growth inhibition under light irradiation; indicating that the photo-controlled chemical generation of ONOO based on BPM... - Advantages of simultaneous inhibition of nitroxypyruvate kinase and glutaminase in metabolic therapy for cancer treatment.

[0057] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0058] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanogenerator for the photocontrolled chemical generation of peroxynitrite ions targeting tumors, characterized in that, It includes a core and a coating layer covering the core, the core including Prussian blue loaded with BNN-6, and the coating layer including tumor cell membranes.

2. The photo-controlled chemical generation nanogenerator for targeting tumors to produce peroxynitrite ions according to claim 1, characterized in that, The tumor cell membrane includes the 4T1 cell membrane.

3. The method for preparing the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite ions according to claim 1 or 2, characterized in that, Includes the following steps: S1, Obtain Prussian Blue; S2. Disperse the Prussian blue in anhydrous ethanol to obtain a Prussian blue dispersion; S3. Add BNN-6 to the Prussian blue dispersion, ultrasonically disperse, place in an ice bath and stir, centrifuge, take the precipitate and disperse it in water to obtain a Prussian blue dispersion loaded with BNN-6. S4. Disperse the tumor cell membrane in PBS buffer to obtain a tumor cell membrane dispersion; S5. The Prussian blue dispersion loaded with BNN-6 is mixed with the tumor cell membrane dispersion and sonicated to obtain a nanogenerator that generates peroxynitrite ions by photocontrolled chemical generation targeting tumors.

4. The method for preparing the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite ions according to claim 3, characterized in that, Step S1 includes: Potassium ferricyanide, polyvinylpyrrolidone, and water were mixed, then concentrated hydrochloric acid was added, the mixture was heated to react, and centrifuged to obtain Prussian blue.

5. The method for preparing the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite ions according to claim 3, characterized in that, In step S3, the mass ratio of BNN-6 to Prussian blue is greater than or equal to 2:

1.

6. The method for preparing the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite ions according to claim 3, characterized in that, In step S3: The mixture was placed in an ice bath and stirred for 12 hours; and / or, The centrifugation temperature was 4℃, the centrifugation speed was 10000 rpm, and the centrifugation time was 30 min.

7. The method for preparing the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite ions according to claim 3, characterized in that, In step S4, the concentration of the tumor cell membrane dispersion is greater than or equal to 1 mg / mL.

8. The method for preparing the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite ions according to claim 3, characterized in that, In step S5, the ultrasound time is 15 min to 30 min.

9. The photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite as described in claim 1 or 2, or the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite prepared by the preparation method described in any one of claims 3-8, for the application in inhibiting pyruvate kinase activity and / or glutaminase activity.

10. The photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite as described in claim 1 or 2, or the photo-controlled chemical generation nanogenerator for targeting tumors to generate peroxynitrite prepared by the preparation method described in any one of claims 3-8, in the preparation of drugs for treating tumors.

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