Conductive coordination nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a conductive coordination nano-enzyme and a preparation method and application thereof, the conductive coordination nano-enzyme has a core-shell structure, metal ions and 1, 5-naphthalenediol are coordinated to form a core crystal structure, and the metal ions and a particle size regulator are coordinated to form a shell amorphous structure; the metal ions comprise any one or a combination of at least two of Cu < 2 + >, Fe < 3 + >, Mn < 2 + >, Co < 2 + >, Fe < 2 + >, Ni < 2 + >, Pt < 2 + >, Ag < + >, Au < 3 + > or Ga < 3 + >; the particle size regulator comprises any one or a combination of at least two of cysteine, lipoic acid, glutathione, mercaptoethanol, mercaptoacetic acid, mercaptopropionic acid, formic acid, acetic acid, benzoic acid, trifluoroacetic acid, citric acid, oxalic acid, maleic acid or salicylic acid. The conductive coordination nano-enzyme provided by the invention can stimulate a tumor site to continuously generate hydroxyl radicals to kill tumors, oxidize DHN to be converted into juglone, and specifically induce pyroptosis of tumors.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nanomaterials and biomedicine, and particularly relates to a conductive coordination nanozyme, a preparation method thereof and an application, and more particularly to the application of the conductive coordination nanozyme in tumor-specific pyroptosis, cuproptosis or ferroptosis. Background Art

[0002] Tumor immunotherapy is currently considered to be one of the most promising treatment means for combating tumors. However, most tumors are immunogenic "cold" tumors, and the response rate of tumor immunotherapy is low (about 20%), resulting in unsatisfactory clinical effects of tumor immunotherapy. Pyroptosis is a programmed cell death mediated by the specific cleavage of the Gasdermin protein family by Caspases, which is characterized by cell swelling and enlargement, with many bubble-like protrusions (pyroptotic bodies), and the formation of pores on the cell membrane. Compared with other cell death methods, pyroptosis can rapidly release a large amount of cell contents and induce a strong inflammatory response. Inducing tumor pyroptosis can activate a stronger anti-tumor immune response in the body and maximize the response rate of tumor immunotherapy.

[0003] The existing nano-drug formulations disclosed in the prior art often induce tumor pyroptosis by activating Caspases. However, in fact, the Gasdermin protein family is regulated by DNA methyltransferases, is highly expressed in normal tissues, and is in a silent state in most tumors. The therapy of non-tumor-specific activation of Caspases often has poor tumor pyroptosis effects and is more likely to cause damage to normal tissues.

[0004] In order to increase the content of Gasdermin protein in the tumor site, the prior art delivers DNA methyltransferase inhibitors (such as gemcitabine, etc.) to the tumor site or directly delivers Gasdermin protein or its expression plasmid. However, these strategies usually need to rely on additional drugs to activate Caspases in order to cleave Gasdermin protein and induce pyroptosis of tumor cells.

[0005] However, the in vivo pharmacokinetics of different drugs vary significantly, and they cannot exert a synergistic therapeutic effect synchronously in vivo. There is an urgent need for a more intelligent co-delivery nanocarrier to synchronize the pharmacokinetics of different drugs, achieve an ideal tumor pyroptosis treatment effect, and reduce the toxicity risk of normal tissues. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a conductive coordination nanozyme, a preparation method thereof and an application. The conductive coordination nanozyme provided by the present invention can continuously stimulate the tumor site to generate hydroxyl radicals for a long time to kill tumors, and continuously oxidize DHN to convert it into juglone, and further specifically induce tumor pyroptosis to improve the response of tumor immunotherapy.

[0007] To achieve the object of the present invention, the following technical solutions are adopted in the present invention:

[0008] In a first aspect, the present invention provides a conductive coordination nanozyme, characterized in that the conductive coordination nanozyme has a core-shell structure, metal ions and 1,5-naphthalenediol coordinate to form an inner core crystalline structure, and metal ions and a particle size regulator coordinate to form an outer shell amorphous structure;

[0009] The metal ions include Cu 2+ , Fe 3+ , Mn 2+ , Co 2+ , Fe 2+ , Ni 2+ , Pt 2+ , Ag + , Au 3+ or Ga 3+ or any combination of at least two of them;

[0010] The particle size regulator includes any one or a combination of at least two of cysteine, lipoic acid, glutathione, mercaptoethanol, mercaptoacetic acid, mercaptopropionic acid, formic acid, acetic acid, benzoic acid, trifluoroacetic acid, citric acid, oxalic acid, maleic acid or salicylic acid.

[0011] The present invention applies chemodynamic therapy (CDT), in which metal ions catalyze H2O2 highly expressed in tumor sites to generate highly toxic hydroxyl radicals (·OH) to kill tumor cells; 1,5-naphthalenediol (DHN) contains a naphthalene ring electron conjugation structure and has excellent electron transfer performance, which can mediate the efficient electron transfer of metal ions between the inner core and the outer shell of the conductive coordination nanozyme, improve the utilization rate of metal ions, greatly expand the active sites of metal ions, consume a large amount of glutathione (GSH), and exert an excellent tumor killing effect; the particle size regulator regulates the size of the conductive coordination nanozyme and optimizes its specific surface area, thereby exposing more active sites and significantly improving the tumor killing effect.

[0012] While a large number of hydroxyl radicals are induced by metal ions, DHN is oxidized to juglone, which further activates the Caspase 1-initiated Gasdermin D cleavage pathway to specifically induce pyroptosis of tumor cells and improve the response of tumor immunotherapy. DHN can not only mediate the electron transfer of metal ions, but also act as a prodrug of juglone to mediate tumor-specific pyroptosis, which is non-toxic to normal tissues and overcomes the problems of poor solubility and strong off-target toxicity of juglone.

[0013] The conductive coordination nanozyme provided by the present invention can continuously stimulate the tumor site to generate hydroxyl radicals for a long time to kill tumors, and continuously oxidize DHN to convert it into juglone, and further specifically induce tumor pyroptosis to improve the response of tumor immunotherapy. The metal ions and DHN synergistically induce the specific death of tumor cells, showing excellent tumor killing effect.

[0014] Preferably, the metal ions include Cu 2+ , Fe 3+ , Mn 2+ or Co 2+ or a combination of any one or at least two of them.

[0015] In the present invention, the metal ions are preferably metal ions with peroxidase (POD)-like activity, which can catalyze the highly expressed H2O2 at the tumor site to generate highly toxic hydroxyl radicals, inducing cuproptosis and / or ferroptosis of tumors.

[0016] More preferably, the metal ions are Cu 2+ .

[0017] In the present invention, the metal ions are further preferably Cu with the ability to activate both cuproptosis and ferroptosis 2+ .

[0018] Preferably, the particle size regulator includes any one or a combination of at least two of cysteine, lipoic acid, glutathione, mercaptoethanol, mercaptoacetic acid or mercaptopropionic acid.

[0019] In the present invention, the particle size regulator is preferably a small molecule particle size regulator containing a single mercapto group, which has good biocompatibility, can better regulate the size of the conductive coordination nanozyme, expose more metal ion catalytic sites, improve the peroxidase (POD)-like activity, and enhance the effect of tumor cuproptosis and / or ferroptosis.

[0020] More preferably, the particle size regulator is cysteine.

[0021] In the present invention, the particle size regulator is further preferably cysteine, and the conductive coordination nanozyme can expose more metal ion catalytic sites.

[0022] Preferably, the molar ratio of the metal ions, 1,5-naphthalenediol and the particle size regulator is (0.25 - 2):1:(0.01 - 2).

[0023] The specific point values in (0.25 - 2) can be 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0024] Specific point values within (0.01 - 2) can be 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.

[0025] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0026] Preferably, the molar ratio of the metal ion, 1,5 - naphthalenediol, and the particle size regulator is (0.5 - 1):1:(0.01 - 1).

[0027] Specific point values within (0.5 - 1) can be 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, etc.

[0028] Specific point values within (0.01 - 1) can be 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0029] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0030] Preferably, the particle size of the conductive coordination nanozyme is 40 - 150 nm, and can be, for example, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, etc.

[0031] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0032] In a second aspect, the present invention provides a method for preparing the conductive coordination nanozyme as described in the first aspect, and the preparation method includes:

[0033] (1) Dissolve 1,5 - naphthalenediol in an organic solvent, then add deionized water and mix. Adjust the pH value to 7.5 - 8.5 (which can be, for example, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, etc.) with a pH regulator to obtain a weakly alkaline solution;

[0034] (2) Mix the weakly alkaline solution obtained in step (1) with an aqueous metal salt solution and carry out a reaction;

[0035] (3) Charge an inert gas and add a particle size regulator, carry out a light - shielding reaction, and centrifuge to obtain the conductive coordination nanozyme.

[0036] The preparation method of the conductive coordination nanozyme provided by the present invention is simple, has a short synthesis route, simple process, mild conditions, good repeatability, and is easy for batch production and application; the product prepared by this method has a high yield and strong tumor killing effect.

[0037] Preferably, in step (1), the organic solvent includes any one or a combination of at least two of dimethyl sulfoxide, acetone, methanol, or N,N-dimethylformamide.

[0038] Preferably, the pH regulator includes any one or a combination of at least two of triethylamine, aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous sodium carbonate solution, or aqueous sodium bicarbonate solution.

[0039] Preferably, in step (2), the specific process of the mixing is to drop the metal salt aqueous solution into the weakly alkaline solution under stirring.

[0040] Preferably, the reaction time is 2 - 4 h, for example, it can be 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, 3 h, 3.2 h, 3.4 h, 3.6 h, 3.8 h, or 4 h, etc.

[0041] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0042] Preferably, in step (3), the inert gas includes argon and / or nitrogen.

[0043] Preferably, the temperature of the light-shielded reaction is 20 - 40 °C, for example, it can be 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, or 40 °C, etc.; the time of the light-shielded reaction is 10 - 30 h, for example, it can be 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, or 30 h, etc.

[0044] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0045] Preferably, the rotation speed of the centrifugation is 20000 - 30000 rpm, for example, it can be 20000 rpm, 21000 rpm, 22000 rpm, 23000 rpm, 24000 rpm, 25000 rpm, 26000 rpm, 27000 rpm, 28000 rpm, 29000 rpm, or 30000 rpm, etc.; the centrifugation time is 60 - 120 min, for example, it can be 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min, etc.

[0046] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0047] Preferably, in step (3), a step of ultrasonic treatment is further included before centrifugation.

[0048] Preferably, the power of the ultrasonic wave is 200 - 400 W, for example, it can be 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, 320 W, 340 W, 360 W, 380 W or 400 W, etc.; the time of the ultrasonic wave is 10 - 40 min, for example, it can be 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min, etc.

[0049] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0050] In a third aspect, the present invention provides an application of the conductive coordination nanozyme as described in the first aspect in the preparation of nano - drugs for tumor - specific pyroptosis, cuproptosis or ferroptosis.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The conductive coordination nanozyme provided by the present invention can continuously stimulate the tumor site to generate hydroxyl radicals for a long time to kill tumors, and continuously oxidize DHN to convert it into juglone, activate the Caspase 1 - initiated Gasdermin D cleavage pathway, and further specifically induce tumor pyroptosis to improve the response of tumor immunotherapy. DHN can not only mediate the efficient electron transfer of metal ions, improve the utilization rate of metal ions, expand the active sites, but also act as a prodrug of juglone to mediate tumor - specific pyroptosis, which is non - toxic to normal tissues, overcoming the problems of poor solubility and strong off - target toxicity of juglone. Metal ions and DHN synergistically induce the specific death of tumor cells, showing excellent tumor - killing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a schematic diagram of the synthesis route of the conductive coordination nanozyme;

[0054] Figure 2 It is a dynamic light scattering diagram of the conductive coordination nanozyme Cu - DHN prepared in Example 1;

[0055] Figure 3 It is a transmission electron microscope image of the conductive coordination nanozyme Cu - DHN prepared in Example 1;

[0056] Figure 4 It is an elemental mapping diagram of the conductive coordination nanozyme Cu - DHN prepared in Example 1;

[0057] Figure 5 Dynamic light scattering diagrams of the conductive coordination nanozyme Fe-DHN prepared in Example 2, the conductive coordination nanozyme Mn-DHN prepared in Example 3, and the conductive coordination nanozyme Co-DHN prepared in Example 4;

[0058] Figure 6 Transmission electron microscopy and dynamic light scattering diagrams of the nanoparticles Mg-DHN prepared in Comparative Example 1, the nanoparticles Cu-Juglone prepared in Comparative Example 2, and the nanoparticles Cu-Cys prepared in Comparative Example 3;

[0059] Figure 7 Hydroxyl radical generation rate diagram (a), ion transfer binding energy change diagram (b), ion transfer schematic diagram (d) of the conductive coordination nanozyme Cu-DHN prepared in Example 1, and ion transfer binding energy change diagram (c) of the nanoparticles Cu-Cys prepared in Comparative Example 3;

[0060] Figure 8 Hydroxyl radical production (a) and juglone conversion yield diagram (b) of the conductive coordination nanozyme Cu-DHN prepared in Example 1;

[0061] Figure 9 Cell survival rate diagrams of the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles prepared in Comparative Examples 1-3 after treating 4T1 cells for 24 h;

[0062] Figure 10 Cell survival rate diagrams of the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles Cu-Juglone prepared in Comparative Example 2 after treating HUVEC, NIH 3T3, and HSF cells for 24 h;

[0063] Figure 11 Schematic diagram of the experimental process (a), tumor volume change diagram (b), tumor weight change diagram (c), tumor inhibition rate diagram (d), and survival rate diagram (e) of the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles prepared in Comparative Examples 1-3 for treating 4T1 breast cancer orthotopic tumor mouse models;

[0064] Figure 12 Immunohistochemistry and HE staining diagrams of the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles prepared in Comparative Examples 1-3 for treating 4T1 breast cancer orthotopic tumor mouse models;

[0065] Figure 13Schematic diagram of the experimental process of using the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticle Cu-Juglone prepared in Comparative Example 2 to treat the 4T1 breast cancer orthotopic tumor mouse model (a), liver and kidney function evaluation diagrams (b-g), platelet count diagram (h), and platelet volume diagram (i);

[0066] Figure 14 HE staining diagrams of major organs of the 4T1 breast cancer orthotopic tumor mouse model treated with the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticle Cu-Juglone prepared in Comparative Example 2;

[0067] Figure 15 Graph of relative body weight changes in the 4T1 breast cancer orthotopic tumor mouse model treated with the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticle Cu-Juglone prepared in Comparative Example 2;

[0068] Figure 16 After treating 4T1 cells with the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles prepared in Comparative Examples 1-3, the K + efflux diagram (a), NLRP3 concentration diagram (b), Caspase-1 activity diagram (c), Gasdermin D concentration diagram (d), Gasdermin D-N concentration diagram (e), and IL-1β concentration diagram (f);

[0069] Figure 17 After treating 4T1 cells with the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles prepared in Comparative Examples 1-3, hydroxyl radical fluorescence detection diagram (a), lipid peroxide detection diagram (b), intracellular GSH content diagram (c), and GPX-4 activity diagram (d);

[0070] Figure 18 After treating 4T1 cells with the conductive coordination nanozyme Cu-DHN prepared in Example 1 and the nanoparticles prepared in Comparative Examples 1-3, intracellular copper content diagram (a) and ferredoxin 1 content diagram (b). Detailed implementation manners

[0071] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solutions of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0072] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be purchased through regular channels.

[0073] Example 1

[0074] This example provides a conductive coordination nanozyme (Cu-DHN), and its preparation method is as follows:

[0075] (1) Dissolve 1,5-naphthalenediol (DHN) in dimethyl sulfoxide to prepare a 10 mg / mL DHN mother liquor, then dissolve it in deionized water, and adjust the pH value to 8 with 0.1 M triethylamine to obtain a weakly alkaline solution;

[0076] (2) Under the condition of stirring at 750 rpm, dropwise add a 5 mg / mL CuCl2 aqueous solution to the weakly alkaline solution obtained in step (1), and react for 3 h;

[0077] (3) Dropwise add 5 mg / mL cysteine (Cys) for particle size adjustment, and set different molar ratios of metal ions, DHN, and particle size regulators (Cu:DHN:Cys = 0.5:1:0; Cu:DHN:Cys = 0.6:1:0.2; Cu:DHN:Cys = 0.75:1:0.5; Cu:DHN:Cys = 1:1:1); Fill with argon to discharge oxygen to ensure that the metal ions can fully coordinate with DHN and the particle size regulator, and react for 24 h in the dark at 30 °C; Ultrasonic treatment (power 300 W) for 30 min to form a stable colloidal solution by homogenization treatment; Centrifuge at 25000 rpm for 30 min and repeat three times to obtain the conductive coordination nanozyme (abbreviated as Cu-DHN); Redisperse in PBS buffer (pH = 7.4) to maintain the stability of the nanoparticles, and store at 4 °C for use.

[0078] Example 2

[0079] This example provides a conductive coordination nanozyme (Fe-DHN), and its preparation method is as follows:

[0080] (1) Dissolve 1,5-naphthalenediol (DHN) in acetone to prepare a 10 mg / mL DHN mother liquor, then dissolve it in deionized water, and adjust the pH value to 7.5 with 0.1 M sodium hydroxide aqueous solution to obtain a weakly alkaline solution;

[0081] (2) Under the condition of stirring at 800 rpm, dropwise add a 5 mg / mL Fe(NO3)3 aqueous solution to the weakly alkaline solution obtained in step (1), and react for 2 h;

[0082] (3) Add 5 mg / mL cysteine for particle size adjustment, and set different molar ratios of metal ions, DHN, and particle size regulator (Fe:DHN:Cys = 0.5:1:0; Fe:DHN:Cys = 0.6:1:0.2; Fe:DHN:Cys = 0.75:1:0.5; Fe:DHN:Cys = 1:1:1); fill with argon to expel oxygen to ensure that metal ions can fully coordinate with DHN and particle size regulator, and react at 20 °C in the dark for 30 h; perform ultrasonic treatment (power 200 W) for 40 min to form a stable colloidal solution by homogenization; centrifuge at 20000 rpm for 40 min and repeat three times to obtain the conductive coordination nanozyme (abbreviated as Fe-DHN); redisperse in PBS buffer (pH = 7.4) to maintain the stability of nanoparticles and store in an environment at 4 °C for use.

[0083] Example 3

[0084] This example provides a conductive coordination nanozyme (Mn-DHN), and its preparation method is as follows:

[0085] (1) Dissolve 1,5-naphthalenediol (DHN) in methanol to prepare a 10 mg / mL DHN mother liquor, and then dissolve it in deionized water. Adjust the pH value to 8.5 with 0.1 M potassium hydroxide aqueous solution to obtain a weakly alkaline solution.

[0086] (2) Under the condition of stirring at 700 rpm, dropwise add 5 mg / mL MnSO4 aqueous solution to the weakly alkaline solution obtained in step (1) and react for 4 h.

[0087] (3) Add 5 mg / mL cysteine for particle size adjustment, and set different molar ratios of metal ions, DHN, and particle size regulator (Mn:DHN:Cys = 0.5:1:0; Mn:DHN:Cys = 0.6:1:0.2; Mn:DHN:Cys = 0.75:1:0.5; Mn:DHN:Cys = 1:1:1); fill with argon to expel oxygen to ensure that metal ions can fully coordinate with DHN and particle size regulator, and react at 40 °C in the dark for 10 h; perform ultrasonic treatment (power 400 W) for 10 min to form a stable colloidal solution by homogenization; centrifuge at 30000 rpm for 20 min and repeat three times to obtain the conductive coordination nanozyme (abbreviated as Mn-DHN); redisperse in PBS buffer (pH = 7.4) to maintain the stability of nanoparticles and store in an environment at 4 °C for use.

[0088] Example 4

[0089] This example provides a conductive coordination nanozyme (Co-DHN), and its preparation method is as follows:

[0090] (1) Dissolve 1,5-naphthalenediol (DHN) in N,N-dimethylformamide to prepare a 10 mg / mL DHN stock solution, then dissolve it in deionized water and adjust the pH value to 8.2 with 0.1 M sodium carbonate aqueous solution to obtain a weakly alkaline solution;

[0091] (2) While stirring at 720 rpm, dropwise add a 5 mg / mL CoCl2 aqueous solution to the weakly alkaline solution obtained in step (1) and react for 3.5 h;

[0092] (3) Dropwise add 5 mg / mL cysteine for particle size adjustment, and set different molar ratios of metal ions, DHN and particle size regulator (Co:DHN:Cys = 0.5:1:0; Co:DHN:Cys = 0.6:1:0.2; Co:DHN:Cys = 0.75:1:0.5; Co:DHN:Cys = 1:1:1); Charge argon to remove oxygen to ensure that metal ions can fully coordinate with DHN and particle size regulator, react for 15 h in the dark at 35 °C; Ultrasonic treatment (power 350 W) for 20 min to form a stable colloidal solution by homogenization treatment; Centrifuge at 28000 rpm for 35 min and repeat three times to obtain the conductive coordination nanozyme (abbreviated as Co-DHN); Redisperse in PBS buffer (pH = 7.4) to maintain the stability of nanoparticles and store at 4 °C for use.

[0093] Comparative Example 1

[0094] This comparative example provides a kind of nanoparticle (Mg-DHN), and the difference from Example 1 is only that: replace the CuCl2 aqueous solution with MgCl2 aqueous solution, and the molar ratio of metal ions, DHN and particle size regulator is Mg:DHN:Cys = 0.6:1:0.2, and other steps remain unchanged.

[0095] Comparative Example 2

[0096] This comparative example provides a kind of nanoparticle (Cu-Juglone), and the difference from Example 1 is only that: replace 1,5-naphthalenediol (DHN) with juglone, and the molar ratio of metal ions, juglone and particle size regulator is Cu:Juglone:Cys = 0.6:1:0.2, and other steps remain unchanged.

[0097] Comparative Example 3

[0098] This comparative example provides a kind of nanoparticle (Cu-Cys), and the difference from Example 1 is only that: do not add 1,5-naphthalenediol (DHN), and the molar ratio of metal ions and particle size regulator is Cu:Cys = 0.6:0.2, and other steps remain unchanged.

[0099] Particle Size and Morphology Characterization of Conductive Coordination Nanozymes in Test Example 1

[0100] In the present invention, metal ions and DHN drive co-assembly to form a nano-network through coordination interactions, and a particle size regulator is used to control the particle size, obtaining conductive coordination nanozymes with different particle sizes ( Figure 1 ). The conductive coordination nanozyme (Cu-DHN) prepared in Example 1 has a particle size distribution in the range of 40 - 150 nm, good dispersibility and uniform size under different molar ratios of metal ions, DHN, and the particle size regulator ( Figure 2-3 ). The elemental mapping images ( Figure 4 ) show that Cu and O elements are evenly distributed in Cu-DHN, while S element is mainly distributed on the surface of Cu-DHN, indicating that Cys binds to the surface of Cu-DHN as a particle size regulator to terminate the continuous growth of particles. By regulating the molar ratio of Cys, the particle size of Cu-DHN can be precisely controlled.

[0101] The conductive coordination nanozymes (abbreviated as M-DHN (M = Fe 3+ , Mn 2+ , Co 2+ )) prepared in Examples 2 - 4, although the particle sizes of the nanozymes corresponding to different metal ions are not exactly the same, their changing trends of particle size with the DHN:Cys ratio are consistent ( Figure 5 ). When the feeding ratio of DHN:Cys is 1:0.2, the prepared M-DHN (M = Fe 3+ , Mn 2+ , Co 2+ ) has the smallest particle size, indicating that the strategy of Cys regulating the particle size of Cu-DHN is universal for different metal ions with POD activity. As Figure 6 shows, the average particle size and dispersibility of the nanoparticles prepared in Comparative Examples 1 - 3 meet the requirements of the control materials.

[0102] Test Example 2 Activity Evaluation of Conductive Coordination Nanozymes

[0103] (1) The conductive coordination nanozyme (Cu-DHN) prepared in Example 1 and the nanoparticles (Cu-Cys) prepared in Comparative Example 3 (both with a metal ion content of 0.2 mM) are respectively mixed with glutathione (GSH) (1.0 mM) in a PBS (pH = 6.4) solution to explore the time dependence of the GSH-depleting ability of Cu-DHN. At different incubation times, 150 μL of the above-mentioned mixed solution is taken, 1350 μL of PBS solution is added, and then 6 μL of DTNB stock solution is added, and the absorbance at 412 nm is detected using a UV-visible spectrophotometer.

[0104] Test results: Compared with Cu-Cys, the GSH depletion ability of Cu-DHN showed obvious time-dependence. The naphthalene ring electron conjugation structure of DHN could mediate the rapid electron exchange between the surface and the core copper ions of Cu-DHN, enabling both to react with GSH. Without this structure, only the surface copper ions of Cu-Cys reacted with GSH, resulting in slow electron transfer, a single oxidation state of copper element, and high Cu + content, and thus poor ability to deplete GSH. Therefore, the high efficiency of Cu-DHN in depleting GSH is directly related to the electron exchange and transport mediated by the DHN ligand.

[0105] (2) The conductive coordination nanozyme (Cu-DHN) prepared in Example 1 and the nanoparticles (Cu-Cys) prepared in Comparative Example 3 (both with a metal ion content of 1.0 mM) were respectively mixed with GSH (1.0 mM) at room temperature in PBS (pH = 6.4) solution. After incubation for 10 min, H2O2 (10 mM) was added. The above mixture was quickly added to a quartz cuvette containing 1 mM TMB, and the absorption spectrum at 650 nm was measured with a UV-visible spectrophotometer to reflect the generation amount of hydroxyl radicals (·OH). The ·OH production rate after the reaction of Cu-DHN with different concentrations of H2O2 was measured with an enzyme-labeling instrument, and V max and K 0.5 were calculated.

[0106] Test results: The spectrum of the co-incubation of TMB and H2O2 showed no obvious change compared with the control group, and no ·OH was produced without the catalysis of Cu-DHN; the pre-incubation of Cu-DHN with GSH could catalyze the production of ·OH by H2O2, and the smaller the particle size of Cu-DHN, the higher the ·OH yield ([[]] Figure 7 in (a)), proving the advantage of the particle size regulation strategy in improving the POD activity of nanozymes. The ·OH yield of Cu-Cys with a similar particle size in the reaction with H2O2 was significantly lower than that of Cu-DHN ([[]] Figure 7 in (b-c)), because the conjugated structure of DHN mediated the electron transfer between the shell and the core copper ions of Cu-DHN, greatly expanding the available reaction interface ([[]] Figure 7 in (d)).

[0107] (3) The conductive coordination nanozyme (Cu-DHN) prepared in Example 1 (with a metal ion content of 0.2 mM) was respectively mixed with GSH (0.2 mM) at room temperature in PBS (pH = 6.4) solution. After incubation for 10 min, H2O2 was added to make the final concentration 2.0 mM. A 10 μg / mL methylene blue (MB) solution was added, and at different time points, the degree of decrease in absorbance at 660 nm was read through a UV-visible spectrophotometer to reflect the generation amount of ·OH.

[0108] Test results: In the presence of Cu-DHN and H2O2, the absorbance intensity of MB at 650 nm was the highest (Figure 8 In (a), it continuously decreased within 180 min, indicating that Cu-DHN has the ability to continuously generate ·OH for a long time, which can continuously kill tumors and oxidize the conversion of DHN to juglone ( Figure 8 in (b)).

[0109] Test Example 3 Cytotoxicity and Tumor Killing Specificity of Conductive Coordination Nanozymes

[0110] (1) To study the cytotoxicity and tumor killing specificity of Cu-DHN, 4T1 cells were seeded in a 96-well plate at a density of 1×10 4 cells / well. Then, 200 μL of culture media containing different concentrations of DHN, CuCl2, CuCl2 + Juglone, CuCl2 + DHN, nanoparticles prepared in Comparative Examples 1-3, and conductive coordination nanozyme (Cu-DHN) prepared in Example 1 were added to each well (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent). They were co-incubated for 24 h. The CCK-8 method was used to detect the viability of 4T1 cells. The absorbance value of each group was subtracted by the absorbance value of the blank well, and the ratio of the experimental group to the untreated control group was used as the percentage of cell viability.

[0111] Test results: The IC50 values of Cu-DHN for 4T1 tumor cells were 0.026 mM Cu and 0.042 mM DHN, lower than the IC50 values of Cu-Cys for 4T1 cells ( Figure 9 ). This is because Cu-DHN has a significantly stronger ability to generate intracellular ROS than Cu-Cys, and the ROS generated by it can convert non-toxic DHN into highly toxic juglone, further enhancing its cytotoxicity to 4T1 cells.

[0112] (2) To explore the safety of Cu-DHN for normal cells, HUVEC, NIH 3T3, and HSF cells were seeded in a 96-well plate at a density of 1×10 4 cells / well. Then, 200 μL of culture media containing different concentrations of conductive coordination nanozyme (Cu-DHN) prepared in Example 1 and nanoparticles (Cu-Juglone) prepared in Comparative Example 2 were added to each well (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent). They were co-incubated for 24 h. The CCK-8 method was used to detect the cell viability. The absorbance value of each group was subtracted by the absorbance value of the blank well, and the ratio of the experimental group to the untreated control group was used as the percentage of cell viability.

[0113] Test results: Cu-Juglone showed obvious dose-dependent toxicity to the three normal cells, while Cu-DHN still maintained more than 75% cell viability when the DHN concentration reached 0.1 mM ( Figure 10) This indicates that Cu-DHN has good biocompatibility with normal cells and specific tumor-killing ability. The concentrations of GSH and H2O2 in normal cells are relatively low and are not sufficient to react with Cu-DHN to generate ROS, so they cannot oxidize DHN into highly toxic juglone. Therefore, Cu-DHN has the superior ability to selectively kill tumor cells.

[0114] Test Example 4 In Vivo Antitumor Effect and Biosafety Evaluation of Conductive Coordination Nanozyme

[0115] (1) To examine the inhibitory effect of intratumoral injection of Cu-DHN on tumor growth, 6-week-old female BALB / c mice were selected and inoculated with 1.0×10 6 4T1 cells at the left mammary pad of the abdomen to establish a mouse 4T1 breast cancer orthotopic tumor model ( Figure 11 as shown in (a) in [reference]). When the tumor volume grew to approximately 200 mm 3 , Cu-DHN and different control materials were injected into the BALB / c mice by intratumoral injection (the content of metal ions, DHN or juglone, and particle size regulator in each group was kept consistent). Thereafter, the tumor volume and body weight of the mice were measured and recorded every two days, and the survival curve of tumor-bearing mice was plotted. When the mice died naturally or the tumor volume reached 1500 mm 3 , the mice were regarded as dead, and the relevant data were recorded. On the 14th day after administration, the mice were euthanized, and the main organ tissues such as the liver and kidneys were quickly removed, rinsed clean with physiological saline, and fixed in 10% neutral formalin solution. Subsequently, the tumors of the mice were dissected and weighed, and the tumor inhibition rate was calculated. The frozen sections of the tumors were labeled with Cleaved Gasdermin D Rabbit mAb and FDX1 antibody respectively, then fluorescently labeled with Cy3 goat anti-rabbit IgG, and finally the pyroptosis and cuproptosis of the tumor sections were observed and photographed by CLSM.

[0116] Test Results: The tumor weight in the Cu-DHN treatment group was significantly lower than that in the Mg-DHN and free CuCl2+DHN administration groups, and was comparable to the ability of Cu-Juglone to inhibit tumor weight growth; there was no difference in the tumor growth of mice in the Mg-DHN treatment group and the PBS group; due to Mg 2+There is no POD activity, ROS cannot be generated and DHN cannot be activated, so there is almost no tumor inhibition effect; the free CuCl2+DHN administration group only showed a weak tumor growth inhibition effect, and there was no significant difference with the PBS group. This is because the ROS production in the free CuCl2+DHN group is extremely low, which can neither effectively kill tumors nor efficiently oxidize DHN to Juglone; at the same time, the cell internalization amount of the free drug group is significantly lower than that of the Cu-DHN nanoparticle group, which leads to its poor anti-tumor effect ( Figure 11 The tumor inhibition rate of Cu-DHN was about 75% ( Figure 11 (d)), showing good tumor growth inhibition effect and significantly prolonging the survival time of tumor-bearing mice ( Figure 11 (e)). Tumor sections were tested ( Figure 12 ), the results showed that in the tumors treated with Cu-DHN, the gasdermin-N protein content and lipid peroxide (LPO) level increased significantly, and the ferredoxin 1 (FDX-1) content decreased significantly. This indicates that Cu-DHN specifically induced tumor pyroptosis, copper death, and iron death, resulting in a significant decrease in tumor cell density.

[0117] (2) 1.0×10 6 4T1 cells, and wait until the tumor volume grows to 200mm 3 When around, ( Figure 13 (a)) The conductive coordination nanozyme (Cu-DHN) prepared in Example 1 or the nanoparticles (Cu-Juglone) prepared in Comparative Example 2 were injected into BALB / c mice by intratumoral injection (the metal ions, DHN or Juglone, and particle size regulator content in each group were kept consistent, and the injection dose was 3.78 μmol / kg Cu, 6.24 μmol / kg DHN or Juglone). On the third day after treatment, the mice were blooded from the orbits, and the samples without sodium heparin were centrifuged at 3000 rpm for 10 min to collect serum for blood biochemical indexes, and the samples with sodium heparin were directly subjected to routine blood analysis.

[0118] Test results: Compared with the PBS treatment group, the liver and kidney function evaluation indicators of the Cu-Juglone treatment group were significantly abnormal. Cu-Juglone caused off-target pyroptosis in the liver and kidney tissues, which led to liver and kidney dysfunction in the mice. However, there was no significant difference between the liver and kidney function indicators of the Cu-DHN group and the PBS group, which fully demonstrated that Cu-DHN did not cause obvious off-target pyroptosis in the liver and kidneys. Figure 13In (b-g)). The off-target pyroptosis caused by Cu-Juglone reduced the platelet count and hematocrit in tumor-bearing mice, suggesting coagulation disorders in tumor-bearing mice; while Cu-DHN did not cause significant changes in platelets and hematocrit ( Figure 13 In (h-i)). Through conventional paraffin sectioning and HE staining, it was found that ( Figure 14 ), the tumor-bearing mice in the Cu-Juglone treatment group showed characteristic glomerular swelling, confirming its nephrotoxicity; in sharp contrast, all major organs (heart / liver / spleen / lung / kidney) in the Cu-DHN treatment group maintained normal tissue structure, showing excellent systemic biocompatibility. Body weight monitoring data ( Figure 15 ) further corroborated the toxicity differences. The mice in the Cu-Juglone group showed weight loss since the 7th day, suggesting the cumulative effect of multi-organ function damage; while the Cu-DHN group maintained stable body weight throughout the process, verifying that it had no significant interference with the hematopoietic system and digestive system.

[0119] Test Example 5 Anti-tumor Molecular Mechanism of Conductive Coordination Nanozymes - Pyroptosis

[0120] Inoculate 10 million 4T1 cells in a 15-cm cell culture dish. After the cells adhered, add the culture medium containing Cu-DHN and different control materials (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent), and incubate with 4T1 cells. After incubation, remove the nano materials such as Cu-DHN, and wash the 4T1 cells 6 times with pre-cooled PBS. Scrape the cells with a cell scraper, centrifuge at 1500 rpm for 5 min to remove PBS, collect the cells and resuspend them with PBS for cell counting.

[0121] (1) Determination of K + efflux in 4T1 cells: Incubate with 4T1 cells for 6 h. After incubation, take 1 million cells for digestion, and make up the volume to 20 mL after nitric acid digestion. Use ICP-MS to measure the K + content in the cells. Use the cells untreated with Cu-DHN as the blank to measure the inherent K + content in 4T1 cells to analyze the K +Outflow situation. (2) Determination of the content of intracellular activated NLRP3: Co-incubate with 4T1 cells for 18 h. After incubation, add 4 million cells into 1 mL of extraction solution, perform ultrasonic disruption under ice bath conditions, and then centrifuge at 12,000 rpm for 5 min at 4 °C. Take 10 μL of the supernatant sample and add it to 40 μL of dilution solution, and perform the operation test according to the ELISA kit instruction manual to measure the content of intracellular activated NLRP3. (3) Determination of intracellular Caspase-1 activity: Co-incubate with 4T1 cells for 18 h. After incubation, resuspend 4 million cells with 50 μL of working cell lysis buffer, place on ice for lysis for 30 min, and then centrifuge at 12,000 rpm for 5 min at 4 °C. Take 50 μL of the supernatant, add 50 μL of working reaction buffer and 5 μL of Ac-YVAD-pNA (4 mM), mix well, and incubate at 37 °C for 2 h. Detect the absorbance value at 405 nm and calculate the Caspase-1 activity. (4) Determination of the content of intracellular Gasdermin D and Gasdermin D-N proteins: Co-incubate with 4T1 cells for 18 h. After incubation, add 4 million cells into 1 mL of extraction solution, perform ultrasonic disruption under ice bath conditions, and then centrifuge at 12,000 rpm for 5 min at 4 °C. Take 10 μL of the supernatant sample and add it to 40 μL of dilution solution, and perform the operation test according to the ELISA kit instruction manual to measure the content of intracellular Gasdermin D and Gasdermin D-N proteins. (5) Determination of the content of extracellular secreted IL-1β: Co-incubate with 4T1 cells for 18 h. After incubation, aspirate the supernatant into a sterile centrifuge tube, centrifuge at 4000 rpm for 10 min at 4 °C to remove excess materials, and aspirate the supernatant and place it in a 4 °C refrigerator for later use. Aspirate 100 μL of the supernatant into the sample well, perform the operation according to the standard procedure of the IL-1β ELISA kit, detect the absorbance value of the sample well at 450 nm, and substitute it into the standard curve to calculate the corresponding IL-1β concentration.

[0122] Test results: The results of ICP-MS determination of intracellular K + content showed that ( Figure 16 (a) in), Cu-DHN and Cu-Juglone showed similar abilities in promoting K + efflux. K + efflux is an important initial event for the activation of a series of intracellular signal transduction pathways, and it can trigger the activation of the NLRP3 inflammasome ( Figure 16 (b) in). As an important intracellular immune regulatory complex, the activation of the NLRP3 inflammasome can further activate Caspase 1 ( Figure 16In (c)). ELISA results further confirmed that the levels of full-length Gasdermin D and Gasdermin D-N terminal fragments in 4T1 cells treated with Cu-DHN and Cu-Juglone increased significantly ( Figure 16 In (d-e)). Cu-DHN generates ROS in tumor cells, and ROS oxidizes DHN to juglone, inhibiting the activity of DNA methyltransferase. When the activity of DNA methyltransferase is inhibited, the transcription of the originally silenced Gasdermin D gene is reactivated at the epigenetic level, and then the translation level of its corresponding protein Gasdermin D is upregulated. Its sulfur-containing amino acid fragment is attacked by ROS and is unstable, which also facilitates the promotion of pyroptosis, generating Gasdermin D-N fragments with membrane perforation activity. These Gasdermin D-N fragments form pyroptotic pores on the cell membrane, resulting in the release of cell contents, including inflammatory factors such as IL-1β ( Figure 16 In (f)), ultimately triggering cell pyroptosis.

[0123] Test Example 6 Antitumor Molecular Mechanism of Conductive Coordination Nanozyme - Ferroptosis

[0124] (1) Intracellular ·OH production: 4T1 cells were seeded in a confocal dish at a density of 1 million cells per dish. After the cells adhered, media containing Cu-DHN and different control materials were added (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent). The cells were co-incubated with the materials for 6 h, then the nanomaterials such as Cu-DHN were aspirated, and the cells were washed twice with pre-cooled PBS. A DCFH-DA solution with a concentration of 10 μM was added to each dish of cells and incubated for 30 min, then the cells were fixed with 200 μL of 4% paraformaldehyde for 20 min, and the nuclei were stained with 5.0 μg / mL DAPI for 20 min. The DCF fluorescence images were observed and taken using a confocal laser scanning microscope (CLSM) to detect the intracellular ·OH production. (2) Intracellular GSH content: 10 million 4T1 cells were seeded in a 15-cm-diameter cell culture dish. After adhesion, media containing Cu-DHN and different control materials were added (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent). After co-incubation with the cells for 6 h, the nanomaterials were removed, and the cells were washed three times with pre-cooled PBS. The cells were scraped off with a cell scraper, collected, resuspended in PBS, and then counted. 4 million cells were taken, centrifuged at 1500 rpm for 5 min to remove PBS, and the cells were added to 1 mL of extraction solution and sonicated under ice bath conditions. Then, it was centrifuged at 12,000 rpm for 5 min at 4 °C. 20 μL of the supernatant sample was taken and added to 140 μL of reagent II and 40 μL of reagent III, mixed well, and left to stand at room temperature for 2 min. The absorbances of the measurement tube, standard tube, and blank tube were measured at 412 nm, and the intracellular GSH content was calculated according to the instructions of the kit. (3) Intracellular GPX-4 activity: 10 million 4T1 cells were seeded in a 15-cm-diameter cell culture dish. After adhesion, media containing Cu-DHN and different control materials were added (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent). After incubation for 18 h, the nanomaterials were removed, and the cells were washed three times with pre-cooled PBS. The steps of cell collection and lysis were the same as those for the GSH content detection. The supernatant sample after centrifugation was taken to detect the intracellular GPX-4 activity of 4T1 cells treated with Cu-DHN and control materials. (4) Detection of cell lipid peroxidation: 4T1 cells were seeded in a confocal dish at a density of 1 million cells per dish. After the cells adhered, media containing Cu-DHN and different control materials were added (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent). After co-incubation for 12 h, the nanomaterials were aspirated, and a Liperfluo solution with a concentration of 1.0 μM was added to each dish of cells and stained for 30 min, then fixed with 4% paraformaldehyde for 20 min, and then the nuclei were stained with 5.0 μg / mL DAPI for 20 min.Lipid peroxide (LPO) fluorescence photographs were observed and taken using CLSM to detect the degree of lipid peroxidation in 4T1 cells treated with Cu-DHN and control materials.

[0125] Test results: Compared with the free CuCl2 group and its simple mixed administration groups with juglone and DHN, the Cu-DHN and Cu-Juglone groups produced significantly more intracellular ROS, manifested as a significant increase in the green fluorescence intensity of DCF ( Figure 17 (a) in the figure), which is because the nanoparticle preparation improved the cellular uptake of the free drug group. Most importantly, the regulation of particle size and conductive structure significantly improved the POD performance of Cu-DHN, while Cu-Cys with lower conductivity had a significantly lower intracellular ROS production than Cu-DHN. In addition, the ·OH generated by Cu-DHN in tumor cells can also induce lipid peroxidation ( Figure 17 (b) in the figure), and the reaction of Cu-DHN with GSH reduces the GSH content ( Figure 17 (c) in the figure), thereby resulting in a decrease in the content of GPX-4 ( Figure 17 (d) in the figure), weakening the ability of tumors to repair lipid peroxidation and triggering tumor-specific ferroptosis.

[0126] Test Example 7 Antitumor Molecular Mechanism of Conductive Coordination Nanozyme - Cuproptosis

[0127] (1) Intracellular copper content of cells: 4 million 4T1 cells were seeded in a cell culture dish with a diameter of 7.5 cm. After the cells adhered, the culture medium containing Cu-DHN and different control materials was added (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent), and they were co-incubated for 6 h. After the incubation, Cu-DHN was removed, and the 4T1 cells were washed 3 times with pre-cooled PBS. The cells were scraped off with a scraper, centrifuged at 1500 rpm for 5 min to remove PBS, and the cells were collected and resuspended with PBS for cell counting. 1 million cells were taken for nitric acid digestion, and after digestion, the volume was fixed to 20 mL. ICP-MS was used to measure the uptake of Cu-DHN by the cells, and untreated cells were used as a blank control to deduct the inherent Cu content in 4T1 cells. (2) Detection of the content of intracellular ferredoxin 1 (FDX-1) to measure the degree of cuproptosis: 10 million 4T1 cells were seeded in a cell culture dish with a diameter of 15 cm. After adhesion, the culture medium containing Cu-DHN and different control materials was added (the content of metal ions, DHN or Juglone, and particle size regulator in each group was kept consistent), and they were co-incubated with 4T1 cells for 18 h. After the incubation was completed, the nanomaterials were removed, and the cells were washed 3 times with pre-cooled PBS. The cells were scraped off with a scraper, collected and resuspended with PBS for counting, 4 million cells were taken, and centrifuged at 1500 rpm for 5 min to remove PBS. The cells were added to 1 mL of extraction solution, sonicated and broken in an ice bath, and centrifuged at 12000 rpm for 5 min at 4 °C. Take the supernatant, add 50 μL of the sample to each well and mix evenly, incubate at 37 °C for 1 h, and perform subsequent operations and tests according to the instructions of the FDX-1 ELISA kit to detect the FDX-1 content in 4T1 cells after treatment with Cu-DHN and control materials, and measure the degree of cuproptosis.

[0128] Test results: Within 6 h of co-incubation with 4T1 cells, the internalization amount of Cu-DHN increased significantly with the prolongation of the incubation time and reached saturation at 6 h of incubation. When the incubation time of 6 h was selected as the time point for research, the uptake amount of Cu-DHN by 4T1 cells was significantly higher than that of free DHN and copper ions, indicating that the nanonization of Cu-DHN is beneficial to the internalization of tumor cells ( Figure 18 as shown in (a) of the figure). Cu-DHN itself contains a certain amount of Cys-pre-reduced Cu + , which helps to enhance the ability of Cu-DHN to induce cuproptosis in tumors. By detecting the change in the content of FDX-1, it was found that compared with the treatment groups such as free copper ions, after treatment with Cu-DHN, the content of FDX-1 in 4T1 cells decreased significantly ( Figure 18 as shown in (b) of the figure), indicating that Cu-DHN induced strong tumor cuproptosis.

[0129] The present invention illustrates a conductive coordination nanozyme, its preparation method and application through the above embodiments. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the products of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and disclosure scope of the present invention.

[0130] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0131] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A conductive coordination nanozyme, characterized in that, The conductive coordination nanozyme has a core-shell structure, where metal ions and 1,5-naphthalenediol coordinate to form a crystalline core structure, and metal ions and a particle size regulator coordinate to form an amorphous shell structure; The metal ions include Cu 2+ , Fe 3+ , Mn 2+ , Co 2+ , Fe 2+ , Ni 2+ , Pt 2+ , Ag + , Au 3+ , or Ga 3+ or a combination of any one or at least two of them; The particle size regulator includes any one or a combination of at least two of cysteine, lipoic acid, glutathione, mercaptoethanol, mercaptoacetic acid, mercaptopropionic acid, formic acid, acetic acid, benzoic acid, trifluoroacetic acid, citric acid, oxalic acid, maleic acid or salicylic acid.

2. The conductive coordination nanozyme according to claim 1, wherein The metal ions include Cu 2+ , Fe 3+ , Mn 2+ or Co 2+ or a combination of any one or at least two of them; Preferably, the particle size regulator includes any one or a combination of at least two of cysteine, lipoic acid, glutathione, mercaptoethanol, mercaptoacetic acid or mercaptopropionic acid.

3. The conductive coordination nanozyme according to claim 1 or 2, characterized in that, The molar ratio of the metal ions, 1,5-naphthalenediol and the particle size regulator is (0.25 - 2):1:(0.01 - 2); Preferably, the molar ratio of the metal ions, 1,5-naphthalenediol and the particle size regulator is (0.5 - 1):1:(0.01 - 1).

4. The conductive coordination nanozyme according to any one of claims 1-3, characterized in that, The particle size of the conductive coordination nanozyme is 40 - 150 nm.

5. The preparation method of the conductive coordination nanozyme according to any one of claims 1-4, characterized in that, The preparation method includes: (1) Dissolve 1,5-naphthalenediol in an organic solvent, then add deionized water and mix, and adjust the pH value to 7.5 - 8.5 with a pH regulator to obtain a weakly alkaline solution; (2) Mix the weakly alkaline solution obtained in step (1) with an aqueous metal salt solution and carry out a reaction; (3) Charge an inert gas and add a particle size regulator, carry out a light-shielded reaction, and centrifuge to obtain the conductive coordination nanozyme.

6. The preparation method according to claim 5, characterized in that, In step (1), the organic solvent includes any one or a combination of at least two of dimethyl sulfoxide, acetone, methanol or N,N-dimethylformamide; Preferably, the pH regulator includes any one or a combination of at least two of triethylamine, aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous sodium carbonate solution or aqueous sodium bicarbonate solution.

7. The preparation method according to claim 5, characterized in that, In step (2), the mixing process is specifically to drop the aqueous metal salt solution into the weakly alkaline solution under stirring; Preferably, the reaction time is 2 - 4 h.

8. The preparation method according to claim 5, characterized in that, In step (3), the inert gas includes argon and / or nitrogen; Preferably, the temperature of the light-shielded reaction is 20 - 40 °C, and the time of the light-shielded reaction is 10 - 30 h; Preferably, the centrifugation speed is 20000 - 30000 rpm, and the centrifugation time is 60 - 120 min.

9. The preparation method according to claim 5, characterized in that, In step (3), before centrifugation, there is also a step of ultrasonic treatment; Preferably, the ultrasonic power is 200 - 400 W, and the ultrasonic time is 10 - 40 min.

10. Use of the conductive coordination nanozyme according to any one of claims 1 - 4 in the preparation of nano-drugs for tumor-specific pyroptosis, cuproptosis or ferroptosis.

Images