Tumor treatment platform based on endogenous and exogenous zinc overload strategy, preparation method and application

Abstract

The present application relates to the field of biological medicine, in particular to a tumor treatment platform based on endogenous and exogenous zinc overload strategy, a preparation method and application. For the treatment platform, it comprises an acid-sensitive carrier, zinc ions and a sound-sensitive organic ligand; the sound-sensitive organic ligand can generate reactive oxygen under ultrasonic irradiation; the sound-sensitive organic ligand and the zinc ions are connected by a coordination bond to form a metal-organic complex; and the metal-organic complex is loaded on the acid-sensitive carrier. The present application proposes a tumor treatment strategy of "endogenous-exogenous synergy", combines "exogenous Zn 2+ " delivery with "endogenous Zn 2+ " mobilization, successfully induces "Zn 2+ overload" in cells through the synergistic effect of the two, realizes the precise, efficient and controllable induction of local Zn 2+ overload in tumors, and improves the effectiveness of pyroptosis. Through the present application, the limitations of traditional methods in controllability, efficacy and safety are overcome.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a tumor therapy platform based on an endogenous and exogenous zinc overload strategy, its preparation method, and its application. Background Technology

[0002] While immune checkpoint inhibitors have revolutionized cancer treatment, their limited efficacy in unresponsive "cold" tumors (clinical response rates below 30%) remains a significant therapeutic challenge. In fact, the immunogenic potential of tumor cell death fundamentally depends on its death pattern, as different death pathways drive differentiated immune responses. Pyroptosis, a highly immunogenic and lysogenic form of programmed cell death, offers a promising alternative to the immune-silenced apoptosis typically induced by traditional antitumor therapies. Due to its unique ability to activate the immune system, pyroptosis has become a powerful strategy for enhancing antitumor immune responses.

[0003] During pyroptosis, plasma membrane disruption leads to cellular permeability swelling and the explosive release of cytoplasmic contents, including pro-inflammatory cytokines (such as IL-1β and IL-18) and damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and ATP. This cell death pattern, accompanied by an inflammatory cascade, can establish a self-amplifying immune activation cycle within the tumor microenvironment (TME), effectively transforming immunologically "cold" tumors into "hot" tumors. Despite its significant potential in stimulating anti-tumor immunity, achieving effective and controllable induction of tumor-specific pyroptosis remains a key obstacle. Existing treatments (such as chemotherapy drugs) not only easily cause irreversible damage to healthy tissues due to their non-specific cytotoxic effects, but also increase off-target effects due to poor spatiotemporal control, making it difficult to achieve a balance between efficacy and safety.

[0004] Ion interference therapy, as an emerging strategy, can efficiently and safely induce pyroptosis in tumor cells. Among numerous metal ions, zinc, as the second most abundant transition metal in organisms, has become an ideal candidate for controllable ion interference therapy due to its good metabolic safety and immunomodulatory capabilities. Recent studies have shown that zinc ions (Zn) within tumor cells... 2 + Excessive accumulation of Zn-based molecules can disrupt plasma membrane integrity and trigger the release of damage-associated molecular patterns (DAMPs), highlighting the importance of Zn-based molecules. 2+ The application prospects of overload treatment strategies in the efficient and safe induction of pyroptosis.

[0005] However, achieving tumor-specific Zn 2+ Overload still presents significant challenges. Existing Zn... 2+ Delivery methods (such as inorganic zinc salts and zinc-based nanomaterials) often lack sufficient tumor selectivity, leading to Zn 2+Precise and controllable accumulation at the tumor site is difficult to achieve. Furthermore, most intracellular Zn... 2+ By binding to metallothioneins (MTs) and existing in an inert form, this significantly reduces the amount of bioavailable free Zn. 2+ The concentration limits its effectiveness as a pyroptosis inducer, and it is only through Zn 2+ Delivery methods enhance intracellular Zn 2+ At high concentrations, the therapeutic effect is very limited. Summary of the Invention

[0006] This invention aims to provide a tumor therapy platform, preparation method, and application based on an endogenous and exogenous zinc overload strategy. This invention proposes an "endogenous-exogenous synergistic" tumor therapy strategy, which utilizes "exogenous Zn..." 2+ "Delivery and "Endogenous Zn" 2+ "By combining the two and working synergistically, we successfully induced the intracellular 'Zn'..." 2+ "Overload", achieving local Zn in tumors 2+ The precise, efficient, and controllable induction of overload improves the effectiveness of pyroptosis. This invention overcomes the limitations of traditional methods in terms of controllability, efficacy, and safety.

[0007] To achieve the above objectives, on the one hand, the present invention adopts the following technical solution: the application of a tumor therapy platform based on endogenous and exogenous zinc overload strategy in the preparation of cell-specific pyroptosis drugs.

[0008] To achieve the above objectives, on the other hand, the present invention adopts the following technical solution: for a tumor treatment platform based on an endogenous and exogenous zinc overload strategy, it includes an acid-sensitive carrier, zinc ions, and a sound-sensitive organic ligand; The acoustically sensitive organic ligand can generate reactive oxygen species under ultrasonic irradiation. The acoustically sensitive organic ligand and zinc ions are linked by coordination bonds to form a metal-organic complex. The metal-organic complex is loaded on an acid-sensitive support.

[0009] The principles and advantages of the above scheme are as follows: 1. A tumor therapy platform based on an endogenous and exogenous zinc overload strategy includes an acid-sensitive carrier, thereby achieving TME-responsiveness in the nanoplatform. It remains stable under physiological pH conditions but rapidly degrades only in acidic TMEs, thus selectively releasing Zn. 2+ And sound-sensitive organic ligands, the released Zn 2+ Achieved tumor-specific "exogenous Zn" 2+ "deliver.

[0010] 2. After the release of the acoustically sensitive organic ligand, during local ultrasound irradiation of the tumor site, the released acoustically sensitive organic ligand generates a large amount of reactive oxygen species in situ. These reactive oxygen species oxidize and attack the zinc-metallothionein complex within tumor cells, releasing zinc ions from the zinc-metallothionein complex. This effectively releases the intracellularly stored "endogenous Zn". 2+ ".

[0011] 3. Achieving self-amplification through a positive feedback loop: "Zn" 2+ "Storm": Exogenous Zn 2+ and endogenous Zn 2+ The two work together to flow into the cell, leading to intracellular Zn 2+ The concentration increased rapidly and significantly. Excessive Zn 2+ Accumulation can further exacerbate intracellular oxidative stress by impairing mitochondrial function, thereby promoting the release of free Zn into the cell. 2+ The maximum release is achieved. This self-reinforcing cycle amplifies "Zn". 2+ The storm ultimately triggers irreversible pyroptosis, a process that causes cell death.

[0012] 4. Experiments show that this treatment platform can effectively inhibit tumor growth and reprogram the immunosuppressive TME, while also significantly enhancing the therapeutic effect of αPD-L1 and effectively inhibiting the growth of primary and distant tumors.

[0013] In summary, conventional pyroptosis inducers in the current technology have relatively strong side effects and off-target toxicity, and cannot achieve endogenous Zn. 2+ The utilization of existing methods has limited effects in inducing pyroptosis. However, the tumor therapy platform based on endogenous and exogenous zinc overload strategy proposed in this application can achieve specific exogenous Zn. 2+ Release and endogenous Zn at tumor sites 2+ Release, without the application of external ultrasound, of endogenous Zn 2+ It will not release, nor will pyrolysis occur. Endogenous Zn will only be released after ultrasound is applied to a specific location (tumor area). 2+ Only then will tumor-specific pyroptosis be induced, achieving local Zn deposition in the tumor. 2+ Precise overload (specific release of endogenous and exogenous Zn at the tumor site) 2+ High efficiency (endogenous and exogenous Zn) 2+ The combined release of the two increases Zn 2+ Concentration), controllable (ultrasound-controlled spatiotemporal endogenous Zn) 2+ This significantly improves the effectiveness of inducing pyroptosis.

[0014] Due to "Zn" 2+ The "storm" mechanism relies on the unique high endogenous Zn content in tumor tissue. 2+At this level, the process cannot be activated in normal tissues, thus ensuring higher safety and tumor specificity. Zinc is the second most abundant metallic element in the human body, and Zn is used. 2+ Inducing pyroptosis through overload also has a certain degree of safety and biocompatibility.

[0015] Preferably, as an improvement, the sonosensitive organic ligand has a macrocyclic conjugated structure; the sonosensitive organic ligand has a nitrogen atom and / or a carboxyl group that can coordinate with zinc ions. The macrocyclic conjugated structure is easily excited by ultrasound, thereby generating a large amount of reactive oxygen species.

[0016] Preferably, as an improvement, the sonicated organic ligands include, but are not limited to, one or more of tetra(4-carboxyphenyl)porphyrin, protoporphyrin IX, dihydroporphyrin e6, tetraphenylporphyrin, and porphyrin derivatives.

[0017] Preferably, as an improvement, the sonosensitive organic ligand is tetrakis(4-carboxyphenyl)porphyrin.

[0018] Preferably, as an improvement, the acid-sensitive carrier includes, but is not limited to, one or more of calcium carbonate, calcium phosphate, manganese carbonate, mesoporous silica, pH-sensitive liposomes, and acid-sensitive polymer carriers.

[0019] Preferably, as an improvement, calcium carbonate is used as the acid-sensitive carrier. Calcium carbonate naturally exists in living organisms, therefore, calcium carbonate has a certain degree of safety as an acid-sensitive carrier.

[0020] To achieve the above objectives, in a third aspect, the present invention adopts the following technical solution: a method for preparing a tumor therapy platform based on an endogenous and exogenous zinc overload strategy (specifically Zn-TCPP@CaCO3), comprising the following steps: S1. Preparation of calcium carbonate nanoparticles: Add ethanol containing CaCl2-2H2O to a container covered with aluminum foil, which has several small holes. Then, ammonium bicarbonate was placed in a vacuum drying chamber along with the container and kept at 40°C for 24 hours to obtain calcium carbonate nanoparticles. S2. Mix the calcium carbonate suspension with the PVP K30 ethanol solution and stir at room temperature; Then add TCPP ethanol solution and continue stirring; Next, add Zn(NO3)2 ethanol solution and stir; Finally, the Zn-TCPP@CaCO3 product was separated by centrifugation and washed multiple times with ethanol.

[0021] Thus, this scheme enables the development of a tumor therapy platform based on an endogenous and exogenous zinc overload strategy.

[0022] Preferably, as an improvement, it also includes, S3. Disperse Zn-TCPP@CaCO3 in anhydrous ethanol, then mix it with DOPA chloroform solution, and sonicate the mixture in a 37°C water bath for 30 minutes. Next, the DOPA-coated nanoparticles obtained by centrifugation purification were resuspended in a solution containing cholesterol, DPPC, and DSPE-PEG. 5000 The synthesized Zn-TCPP@CaCO3 was stirred at room temperature for 12 hours in a chloroform solution, and then the chloroform was removed by a rotary evaporator. Finally, the synthesized Zn-TCPP@CaCO3 was purified by centrifugation.

[0023] Therefore, this approach improves the biocompatibility of Zn-TCPP@CaCO3.

[0024] Fourthly, this invention provides the following technical solution: a drug for treating tumors, including an immune checkpoint inhibitor and the aforementioned tumor treatment platform based on an endogenous and exogenous zinc overload strategy. Thus, this drug provides a promising strategy for addressing the challenge of poor efficacy of immune checkpoint inhibitors in "cold tumors." Attached Figure Description

[0025] Figure 1 The accompanying figures illustrate the synthesis and characterization of Zn-TCPP@CaCO3. (A) A schematic diagram of the Zn-TCPP@CaCO3 synthesis process, including the subsequent PEGylation step; (B) A representative TEM image of Zn-TCPP@CaCO3 (Zn...). 2+ (C) Representative TEM image of Zn-TCPP@CaCO3 (Zn 2+ (D) HAADF-STEM image of Zn-TCPP@CaCO3, showing the elemental distribution of C, O, Ca, and Zn; (E) XPS spectrum of Zn-TCPP@CaCO3; (F) UV-Vis absorption spectra of calcium carbonate, TCPP, and Zn-TCPP@CaCO3; (G) FTIR spectra of calcium carbonate, TCPP, and Zn-TCPP@CaCO3; (H) DLS data of Zn-TCPP@CaCO3; (I) Zn in Zn-TCPP@CaCO3. 2+ (J) Time-dependent release curves; (J) Fluorescence of SOSG in PBS, TCPP and Zn-TCPP@CaCO3 under different ultrasound irradiation times.

[0026] Figure 2 In vitro antitumor effects and Zn-TCPP@CaCO3-induced Zn 2+The accompanying figures are related to the storm. (AB) FCM analysis of intracellular uptake of Zn-TCPP@CaCO3 by 4T1 cells after different incubation times; (C) Viability of 4T1 cells treated with different concentration formulations (n ​​= 5); (D) Viability of 4T1 cells treated with different formulations (n ​​= 5); (E) CLSM images showing intracellular ROS levels detected using the DCFH-DA probe after different treatments; (F) Quantitative analysis of intracellular ROS levels after different treatments; (G) Intracellular Zn after different treatments. 2+ Quantitative analysis of levels; (H) Detection of intracellular Zn after different treatments using an ethyl zinc ester probe. 2+ CLSM images of horizontal expression; (I) CLSM images of MTF expression in cells after different treatments; (J) Zn 2+ Schematic diagram of storm mechanism; (K) CLSM images of MT expression in cells after different treatments.

[0027] Figure 3 Figures are attached to illustrate the in vitro pyroptosis and immunostimulatory effects induced by Zn-TCPP@CaCO3. (A) Transmission electron microscopy (TEM) image of 4T1 cells after PBS treatment; (B) TEM image of 4T1 cells after Zn-TCPP@CaCO3 + sonication treatment; (C) Western blot analysis of pyroptosis-related proteins in 4T1 cells under different treatments; (DE) Relative levels of IL-1β and IL-18 released after different treatments; (FG) Relative levels of HMGB1 and ATP released after different treatments; (H) CLSM images of CRT expression in cells after different treatments; (I) Schematic diagram of the Transwell system used for co-culturing cells; (JK) FCM analysis of dendritic cell maturation after different treatments; (L) In vitro photoacoustic (PA) imaging, with PA signal linearly correlated with nanoparticle concentration; (M) In vivo PA imaging; (NO) In vivo fluorescence imaging and ex vivo organ imaging; (P) Schematic diagram of the pyroptosis-immune activation mechanism.

[0028] Figure 4 Activating Zn with sound waves 2+ The accompanying figures illustrate the in vivo antitumor effect assessment triggered by the "storm". (A) Schematic diagram of the experimental design for assessing the in vivo antitumor effect in the 4T1 tumor mouse model; (B) Changes in tumor volume in mice of different treatment groups; (C) Tumor weight after different treatments; (D) Tumor growth curves after different treatments; (E) Microscopic photograph of the tumor removed at the end of the treatment period; (F) Changes in mouse body weight during treatment.

[0029] Figure 5 This illustrates the dendritic cell (DC) maturation detection.

[0030] Figure 6 The DC maturity rate is shown.

[0031] Figure 7 The T lymphocyte subset analysis is illustrated.

[0032] Figure 8 CD8 is shown + T cell ratio.

[0033] Figures 9-10 This illustrates the detection of pro-inflammatory factor concentrations.

[0034] Figure 11 This indicates Foxp3 + Statistics on the proportion of Tregs (immunosuppressive cells).

[0035] Figure 12 The CD80 was shown. + / F4 / 80 + Statistics on the proportion of M1 macrophages (anti-tumor macrophages).

[0036] Figure 13 For "sound-activated Zn" 2+ Figures related to the in vivo antitumor therapy study of "Storm" combined with αPD-L1; including: (A) bilateral tumor treatment process; (BC) bilateral tumor growth curves; (DE) quantitative statistics of tumor volume; (F) mouse survival curve; (G) visual tumor photographs; (H) immune evaluation process; (I) DC maturation flow cytometry density map; (J) T cell subset flow cytometry density map; (K) DC maturation rate statistics; (LM) CD8 + Statistics on the percentage of T cells.

[0037] Figure 14 The illustration shows the mitochondrial membrane potential levels assessed using the JC-1 staining method in different treatment groups. Detailed Implementation

[0038] The following detailed description illustrates the specific implementation method: This embodiment discloses a tumor therapy platform based on an endogenous and exogenous zinc overload strategy, including an acid-sensitive carrier, zinc ions, and a sound-sensitive organic ligand; The acoustically sensitive organic ligand can generate reactive oxygen species under ultrasonic irradiation. The acoustically sensitive organic ligand and zinc ions are linked by coordination bonds to form a metal-organic complex. The metal-organic complex is loaded on an acid-sensitive support.

[0039] The sonosensitive organic ligands possess a macrocyclic conjugated structure; they also have nitrogen atoms and / or carboxyl groups capable of coordinating with zinc ions. The macrocyclic conjugated structure is readily excited by ultrasound, thereby generating a large amount of reactive oxygen species.

[0040] For sound-sensitive organic ligands, there are one or more of tetra(4-carboxyphenyl)porphyrin, protoporphyrin IX, dihydroporphyrin e6, tetraphenylporphyrin, and porphyrin derivatives. In this embodiment, tetra(4-carboxyphenyl)porphyrin (TCPP) is preferred.

[0041] For acid-sensitive carriers, one or more of the following are included, but are not limited to: calcium carbonate, calcium phosphate, manganese carbonate, mesoporous silica, pH-sensitive liposomes, and acid-sensitive polymer carriers. In this embodiment, calcium carbonate is preferred.

[0042] The aforementioned tumor therapy platform based on endogenous and exogenous zinc overload strategies can be applied to the preparation of drugs that induce cell-specific pyroptosis. This platform responsively releases exogenous zinc ions at the tumor site. Under ultrasound activation, TCPP generates reactive oxygen species (ROS), which oxidize and attack the Zn-MT complex, releasing endogenous zinc ions. The exogenous and endogenous zinc ions synergistically form tumor-specific Zn. 2+ Storms disrupt cellular zinc homeostasis, induce mitochondrial dysfunction and oxidative stress cascades, and induce tumor cell pyroptosis in a spatiotemporally controllable manner, thereby breaking the immunosuppressive microenvironment and enhancing the efficacy of antitumor immunotherapy.

[0043] This embodiment also discloses a method for preparing a tumor therapy platform based on an endogenous and exogenous zinc overload strategy (specifically, Zn-TCPP@CaCO3) (in conjunction with...). Figure 1 As shown in A), the following steps are included: S1. Preparation of calcium carbonate nanoparticles: Add 100 ml of ethanol containing 150 mg of CaCl2-2H2O to a container (specifically a beaker) covered with aluminum foil, the aluminum foil having several small holes. Then, 6 grams of ammonium bicarbonate were placed in a vacuum drying chamber along with the container and kept at 40°C for 24 hours to obtain calcium carbonate nanoparticles.

[0044] S2. Mix 3 mL of calcium carbonate suspension (concentration of 3 mg / mL in ethanol) with 0.5 mL of polyvinylpyrrolidone K30 (PVP K30) ethanol solution (60 mg / mL) and stir at room temperature for 5 minutes. Then add 0.5 mL of TCPP ethanol solution (2 mg / mL) and continue stirring for 5 minutes; Next, add 1 mL of Zn(NO3)2 ethanol solution (30 mg / mL) and stir for 4 h; Finally, the Zn-TCPP@CaCO3 product was separated by centrifugation (11,000 rpm, 10 min) and washed three times with ethanol.

[0045] S3. Disperse 20 mg of Zn-TCPP@CaCO3 in 5 mL of anhydrous ethanol, then mix with 5 mL of DOPA chloroform solution (2 mg / mL), and sonicate the mixture in a 37°C water bath (30 kHz) for 30 minutes. Next, the DOPA-coated nanoparticles obtained by centrifugation (11000 r for 10 min) were purified and resuspended in a solution containing 2 mg cholesterol, 4 mg 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and 8 mg 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DSPE-PEG). 5000 The synthesized Zn-TCPP@CaCO3 was stirred in a chloroform solution (2 mL) at room temperature for 12 hours. The chloroform was then removed using a rotary evaporator, and the final product was purified by centrifugation (11000 rpm, 10 min). Through S3, compared to S2, a stable lipid layer formed on the surface of Zn-TCPP@CaCO3, and the lipid-modified nanoparticles exhibited superior dispersibility in various media compared to the unmodified nanoparticles.

[0046] In this embodiment, DOPA refers to sodium 1,2-dioleoyl-sn-glycerol-3-phosphate.

[0047] experiment The following series of experiments demonstrate the properties and effects of the aforementioned treatment platform.

[0048] I. Characterization Study of Zn-TCPP@CaCO3 Combination Figure 1 As shown in B-1C, transmission electron microscopy (TEM) analysis shows that the nanoparticles prepared in Example S2 exhibit a uniform spherical hollow structure.

[0049] The nanoparticles prepared in this embodiment were systematically characterized using a variety of analytical techniques. First, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) revealed that the elemental signals of C, O, Ca, and Zn were uniformly distributed within the obtained nanoparticles. Figure 1 This result was further confirmed by X-ray photoelectron spectroscopy (XPS). Figure 1 E). By recording the UV-Vis spectrum of Zn-TCPP@CaCO3, characteristic absorption peaks of TCPP were observed at 414 nm and 648 nm, confirming the successful integration of TCPP into the nanoparticles. Figure 1F). Fourier transform infrared spectroscopy analysis showed that the hydroxyl groups in Zn-TCPP@CaCO3 had disappeared. Furthermore, the absorption peak corresponding to the C=O symmetric stretching vibration of the carboxyl group shifted to lower frequencies compared to free TCPP, indicating the formation of Zn-TCPP coordination bonds. Figure 1 G). Dynamic light scattering (DLS) measurements show that the average hydrodynamic diameter of Zn-TCPP@CaCO3 is approximately 141 nm ( Figure 1 H). Furthermore, the zinc content in Zn-TCPP@CaCO3 was determined to be 11.2% ± 2.67% using inductively coupled plasma optical emission spectrometry (ICP-OES). In summary, these results confirm the successful synthesis and structural integrity of the Zn-TCPP@CaCO3 nanoplatform.

[0050] Calcium carbonate has been widely used as a drug carrier, enabling pH-responsive drug release. This study aimed to evaluate the pH sensitivity of Zn-TCPP@CaCO3 and the properties of Zn... 2+ Release capabilities, studied Zn 2+ The time-dependent release curve was obtained. Specifically, 4 mg of Zn-TCPP@CaCO3 nanoparticles were dispersed in 2 mL buffer solutions at different pH levels (pH 7.4, 6.5, and 5.5). Figure 1 As shown in Figure I, under physiological conditions (pH 7.4), Zn 2+ The release of Zn is slow and limited, with a cumulative release of only 6.3% over 24 hours, indicating excellent stability of the nanoparticles in a neutral environment. Under simulated TME acidic conditions (pH 6.5), Zn... 2+ Release was significantly enhanced, reaching 43.3% within 24 hours. Under simulated lysosomal acidic conditions (pH 5.5), Zn 2+ The release was faster and more complete, with a release rate of 60.6% within the same time period. These results indicate that Zn-TCPP@CaCO3 exhibits excellent acid-responsive release characteristics, enabling selective release of Zn in acidic TME. 2+ This confirms the TME responsiveness of "exogenous Zn". 2 + "The feasibility of delivery."

[0051] The reactive oxygen species (ROS) generation capacity of Zn-TCPP@CaCO3 was evaluated using a singlet oxygen sensor green (SOSG) probe, with PBS as a control. Following ultrasonic irradiation (40 kHz, 5 min), the ROS levels of Zn-TCPP@CaCO3 increased in a time-dependent manner, confirming its efficacy as a sonic sensitizer and providing a basis for spatiotemporally controllable acoustic activation of endogenous Zn. 2+ "The mobilization mechanism provided crucial support ( Figure 1 J).

[0052] II. In vitro antitumor effects and Zn-TCPP@CaCO3-induced Zn 2+ Storm Research The antitumor effect of Zn-TCPP@CaCO3 depends on its efficient intracellular uptake. To investigate the cellular uptake behavior of the nanoparticles, Zn-TCPP@CaCO3 was co-incubated with 4T1 cells at different time intervals (0, 3, 6, and 12 hours) (co-incubation procedure: 20 mg / ml of nanoparticles and 4T1 cells were co-incubated at 37°C and 5% CO2 concentration for 24 h). Flow cytometry analysis showed that the fluorescence intensity of TCPP in 4T1 cells increased over time. Figure 2 A- Figure 2 (B) indicates that Zn-TCPP@CaCO3 can be effectively phagocytosed by 4T1 cells. Subsequently, the CCK-8 assay was used to assess the cytotoxicity of Zn-TCPP@CaCO3 on 4T1 cells, with calcium carbonate and TCPP@CaCO3 as controls. The results showed that when Zn... 2+ No significant toxicity was observed in cells at concentrations below 50 μM. However, when Zn... 2+ When the concentration was increased from 50 μM to 70 μM, we observed a significant decrease in cell viability to approximately 30%. Figure 2 C), indicating a high concentration of Zn 2+ It has significant cytotoxic effects.

[0053] Based on these findings, Zn with no significant toxicity was selected. 2+ The concentration (30 μM) was used to further investigate its synergistic effect with sonodynamic therapy (SDT). 4T1 cells were treated with: (1) PBS; (2) TCPP@CaCO3 (TCPP = 5 μM); (3) TCPP@CaCO3 (TCPP = 5 μM) + US; (4) Zn-TCPP@CaCO3 (TCPP = 5 μM, Zn 2+ = 30μM); (5) Zn-TCPP@CaCO3 (TCPP = 5 μM, Zn 2+ = 30μM) + US; each group was incubated for 24 h, with corresponding ultrasonic treatment: 40 kHz for 5 min. The study found that even in non-toxic Zn... 2+ At a concentration (30 μM), compared with SDT alone (Group 3) or Zn 2+ Compared to group 4, simultaneous administration of SDT and Zn 2+ The viability of treated 4T1 cells was significantly reduced. Figure 2 D), explaining the effects of ultrasonic treatment and exogenous Zn. 2+ The processing has a synergistic effect.

[0054] To investigate the mechanism of this synergistic effect, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used to detect the generation of intracellular reactive oxygen species (ROS). When zinc ions (Zn)... 2+ When combined with SDT (group 5), the detected ROS level was significantly higher than that of the group using SDT alone (group 3). Figure 2 E- Figure 2 F), which indicates the addition of Zn 2+ After (SDT and exogenous Zn) 2+ Co-processing (Zn) can significantly enhance ROS generation. However, using Zn alone... 2+ The amount of ROS generated in the treatment group was quite limited. This phenomenon indicates that under these concentration conditions, "exogenous Zn..." 2+ "It is not a potent ROS inducer itself; its significant ROS-promoting effect requires synergy with SDT to be fully realized. This is because the TCPP-mediated SDT effect is the main source of ROS generation in this study system. The initial ROS generated through the SDT effect triggers the storage of 'endogenous Zn' in cells." 2+ "Release, endogenous Zn" 2+ The reason for this release is that zinc-metallothioneins (MTs) are highly expressed in tumor cells and have redox-sensitive regulatory functions, which can regulate endogenous Zn. 2+ The combination and release of SDT-induced ROS attack the antioxidant system, represented by MTs, leading to the degradation of endogenous Zn. 2+ The release of "endogenous Zn". 2+ "Exogenous Zn delivered by nanoparticles" 2+ "The synergistic effect of endogenous and exogenous factors leads to intracellular Zn..." 2+ The level rose sharply in a short period of time, ultimately amplifying the level of reactive oxygen species (ROS) in the cells.

[0055] To verify the "endogenous-exogenous synergistic effect", we used Zn 2+ The specific fluorescent probe Zinquin ethyl ester tracks intracellular Zn 2+ .like Figure 2 G- Figure 2 As shown in Figure H, intracellular Zn was observed after applying SDT alone (Group 3). 2+ The elevated levels further confirm that SDT can indeed induce endogenous Zn 2+ "release.

[0056] In addition, co-processing group (Zn) 2+ The fluorescence signal intensity of + SDT (Group 5) showed a more dramatic increase, and its level was significantly higher than that of Zn alone. 2+Treatment group (Group 4). These results indicate that nanoparticles have effectively incorporated Zn. 2+ Delivered into cells, thereby providing "exogenous Zn" 2+ On the other hand, SDT-induced "endogenous Zn" 2+ "The release effect is achieved by adding exogenous Zn" 2+ "Further enhanced. Experiments show that this amplification effect originates from intracellular Zn." 2+ Mitochondrial damage due to excessive accumulation: Mitochondrial membrane potential measurements using the JC-1 probe, combined with Figure 14 As shown, the results indicated that Zn-TCPP@CaCO3+ US treatment induced the most significant mitochondrial depolarization (manifested as enhanced green monomeric fluorescence) compared to other groups, indicating impaired mitochondrial function, which in turn became a potent source of reactive oxygen species. This further exacerbated intracellular oxidative stress, ultimately triggering complete "endogenous Zn" depolarization. 2+ "Release, forming high-strength 'Zn'" 2+ "Storm", thus establishing a "self-amplifying Zn2+ storm cycle" mechanism (combined with Figure 2 As shown in J).

[0057] Further exploration of "Zn" 2+ The molecular mechanism of "storm" was studied, and it was found that after SDT treatment, the expression levels of metallothionein (MT) and metal-regulated transcription factor 1 (MTF1) were significantly upregulated. Figure 2 I and Figure 2 K). MT and MTF1 are key regulators of intracellular zinc homeostasis, and studies have reported that their expression levels are related to intracellular Zn. 2+ The concentrations are positively correlated, therefore the upregulation of MTF1 and MT not only provides intracellular Zn 2+ The mechanisms and intensity of storms provide molecular-level evidence and directly confirm Zn as a core mechanism for regulating intracellular zinc homeostasis. 2+ -MTF1-MT compensation axis is activated.

[0058] Furthermore, this Zn-TCPP@CaCO3+ ultrasound-induced cytotoxicity exhibited selectivity for tumor cells. Under the same treatment conditions as 4T1 cells (Zn-TCPP@CaCO3+ ultrasound, Zn... 2+ =30 μM), no significant cytotoxic effect was observed in human umbilical vein endothelial cells (HUVECs), which is related to the low MT expression level in HUVECs. In short, compared with normal cells, 4T1 tumor cells have higher endogenous Zn content due to their high MT expression level. 2+ Reserves. Therefore, even when exposed to the same concentration of exogenous Zn... 2+Non-tumor cells also cannot exceed the intracellular zinc toxicity threshold because they cannot release enough endogenous Zn. 2+ This avoids the death of a large number of cells. Therefore, this "endogenous-exogenous synergistic" strategy achieves specific killing of tumor cells by utilizing the difference in MT expression between tumor tissue and normal tissue, while the nanoparticles are safe for normal cells.

[0059] III. Study on Zn-TCPP@CaCO3-induced pyroptosis and its effects on immunostimulation We further investigated whether Zn-TCPP@CaCO3+ US could effectively trigger pyroptosis in tumor cells (pyroptosis is a unique form of programmed cell death characterized by cell swelling). To explore Zn-TCPP@CaCO3+ US-induced pyroptosis, we used optical microscopy to analyze cells treated with PBS and Zn-TCPP@CaCO3 (TCPP = 5 μM, Zn...). 2+ = 30 μM ) + US treated 4T1 cells (co-incubated for 24 h, followed by sonication of the nanoparticle group after 24 h) for morphological observation. The results showed that cells treated with Zn-TCPP@CaCO3+ US exhibited a swollen morphology with large vesicle formation under a light microscope. Further observation using bio-electron microscopy (Bio-TEM) revealed significant morphological changes in cells treated with Zn-TCPP@CaCO3+ US, including plasma membrane rupture, mitochondrial swelling, and vacuolation, characteristics highly consistent with pyroptosis. Figure 3 A- Figure 3 B).

[0060] To further validate pyroptosis at the molecular level, we performed Western blot analysis to detect the expression of cleaved caspase-1 and GSDMD-N, two key proteins in the pyroptosis pathway. Cleaved caspase-1 initiates the pyroptosis pathway by cleaving GSDMD and releasing its pore-forming N-terminal fragment (GSDMD-N), thereby driving the pyroptosis cascade. Western blot analysis showed that Zn-TCPP@CaCO3+ US treatment significantly enhanced the expression of these two proteins. Figure 3 C). Furthermore, caspase-1 activation not only induces cell membrane porosimetry by cleaving GSDMD, but also activates and releases pro-inflammatory factors. ELISA analysis showed that the levels of IL-1β and IL-18 were significantly increased in the Zn-TCPP@CaCO3+ US group ( Figure 3 D- Figure 3 E), thus providing further evidence for pyroptosis at the cytokine level.

[0061] During pyroptosis, the release of damage-associated molecular patterns (DAMPs) is crucial for initiating an antitumor immune response. We investigated changes in representative DAMPs after different treatments, including calreticulin (CRT, exposed on the cell surface), ATP, and HMGB1 (released into the extracellular space). Figure 3 F- Figure 3 As shown in G, compared with other groups, the Zn-TCPP@CaCO3+US group induced significantly higher levels of HMGB1 and ATP release. Immunofluorescence staining further confirmed these findings, indicating that Zn-TCPP@CaCO3+US treatment significantly enhanced CRT exposure on the cell surface compared with monotherapy. Figure 3 H). In summary, these results indicate that Zn 2+ Storm-induced pyroptosis effectively promotes the release of DAMPs, which act as "danger signals" to alert the immune system.

[0062] Dendritic cells (DCs) are the most potent antigen-presenting cells, and mature DCs can effectively promote the activation of anti-tumor T cells. Therefore, we used the Transwell co-culture system to evaluate the ability of Zn-TCPP@CaCO3+US to promote DC maturation. Figure 3 I), the basic operation is as follows: (1) First, immature DCs are seeded into the lower chamber of a 12-well plate. (2) Collect 4T1 cells from each group after different interventions, adjust the cell concentration, and then seed them into the upper chamber of a Transwell with a pore size of 0.4 μm. (3) Carefully place the upper chamber into the lower chamber containing immature DCs and incubate it in a cell culture incubator at 37°C and 5% CO2 for 24 h. (4) After co-culture, collect the BMDCs in the lower chamber and transfer them to a flow cytometry tube, wash with PBS, and centrifuge and resuspend. Flow cytometry (FCM) analysis showed that Zn-TCPP@CaCO3+US can significantly promote DC maturation ( Figure 3 J- Figure 3 K).

[0063] IV. In vivo biocompatibility and biodistribution studies of Zn-TCPP@CaCO3 To assess the potential systemic toxicity of Zn-TCPP@CaCO3, mice were intravenously injected with Zn-TCPP@CaCO3 (TCPP injection dose: 10 mg / kg). Hematological evaluation and H&E-stained histopathological examination of major organs (heart, liver, spleen, lung, and kidney) were performed on healthy mice at 7, 14, and 30 days post-injection. Results showed no significant differences in routine blood parameters and biochemical parameters compared to the untreated control group after Zn-TCPP@CaCO3 treatment. Furthermore, no significant pathological changes were observed in the H&E-stained tissues of major organs. In summary, these results indicate that Zn-TCPP@CaCO3 possesses excellent biocompatibility.

[0064] Effective accumulation at the tumor site is crucial for achieving optimal therapeutic effects. Firstly, in vitro photoacoustic (PA) imaging showed a strong linear correlation between the PA signal intensity and the concentration of Zn-TCPP@CaCO3. Figure 3 L), confirming its excellent PA imaging capability. Further in vivo PA imaging tracking showed that the PA signal in the tumor region of tumor-bearing mice gradually increased after intravenous injection of Zn-TCPP@CaCO3, reaching a peak at 24 hours post-injection. Figure 3 M). To comprehensively assess the in vivo biodistribution and metabolic behavior of Zn-TCPP@CaCO3, fluorescence imaging was also performed, such as... Figure 3 As shown in Figure N, the fluorescence (FL) signal in the tumor region gradually increased within 24 hours after injection, consistent with the PA imaging results. 24 hours after injection, tumor tissue and major organs were collected for ex vivo fluorescence imaging. The results showed that the FL signal intensity in the tumor was significantly higher than that in other major organs (…). Figure 3 These findings collectively confirm that Zn-TCPP@CaCO3 can effectively accumulate at tumor sites.

[0065] V. In vivo antitumor effect evaluation study Considering that Zn-TCPP@CaCO3+ US can effectively induce pyroptosis in 4T1 cells and trigger ICD in vitro, demonstrating significant anti-tumor potential, its in vivo therapeutic effect was subsequently evaluated using a 4T1 tumor-bearing mouse model. The 4T1 tumor-bearing mouse model was established as follows: 4T1 cells in the logarithmic growth phase were collected, digested with trypsin, washed by centrifugation, resuspended in pre-cooled sterile PBS, and the cell concentration was adjusted. The backs of the mice were shaved, and cells containing 1×10⁻⁶ cells were injected using a microsyringe. 650 μL of 4T1 cell suspension was subcutaneously injected into the left back of each mouse to establish a primary 4T1 subcutaneous tumor-bearing mouse model. Specifically, 7 days after 4T1 cell inoculation, mice were randomly divided into five groups: (1) G1: PBS group; (2) G2: TCPP@CaCO3 group; (3) G3: TCPP@CaCO3+ US group; (4) G4: Zn-TCPP@CaCO3 group; (5) G5: Zn-TCPP@CaCO3+ US group; the drug concentration for each group was calculated as TCPP, which was 10 mg / kg, and the corresponding sonication treatment was 40 kHz for 5 min. The treatment regimen and experimental timeline are as follows. Figure 4 As shown in Figure A. Tumor volume and body weight were monitored every two days during treatment. Results showed that the Zn-TCPP@CaCO3+ US group exhibited the most significant tumor growth inhibition compared to all other groups. Figure 4 B- Figure 4 E). No significant weight loss or mortality was observed throughout the treatment period. Figure 4 F) further confirms its good in vivo safety.

[0066] VI. Study on the in vivo immune response triggered by sound-activated "Zn2+ storm" To further elucidate the immune activation induced by ultrasound in Zn-TCPP@CaCO3+, we systematically analyzed the infiltration of immune cells in tumor tissue 72 hours after treatment. The results showed that, combined with… Figure 5 and Figure 6 As shown, Zn-TCPP@CaCO3+ ultrasound significantly promoted the maturation of dendritic cells in tumor tissue, indicating a significantly enhanced antigen-presenting capacity, which is beneficial for promoting the activation of naive T cells. Furthermore, combined with… Figure 7 and Figure 8 As shown, for tumor-infiltrating lymphocytes (TILs), the key effector cell CD8 + The proportion of T cells was significantly increased in the Zn-TCPP@CaCO3+ ultrasound group. Specifically, CD3+ was present in the tumor tissue of this group. + CD8 in T cells + The mean percentage of T cells was 33.7%, more than double that of the control group (15.3%), and significantly higher than that of the SDT group (20.2%) or Zn. 2+ Monotherapy group (18.0%). These results indicate that "sound-activated Zn..." 2+ The "storm" effectively activated and recruited cytotoxic T cells into the tumor mesotherapy area (TME) by enhancing DC-mediated antigen presentation. Furthermore, ELISA analysis of cytokine levels in tumor homogenates showed that... Figure 9 and Figure 10As shown, the concentrations of pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ were significantly increased in the Zn-TCPP@CaCO3+ ultrasound therapy group, while the response in the monotherapy group was negligible. These results further confirm that SDT and Zn 2+ The combined application of these substances can effectively activate the inflammatory immune response within the TME.

[0067] This immunosuppressive tumor microenvironment (TME), characterized by lactate accumulation, regulatory T cell (Treg) infiltration, and enrichment of M2 phenotype tumor-associated macrophages (TAMs), represents a significant obstacle to effective tumor immunotherapy. In this study, Zn-TCPP@CaCO3+ ultrasound (US) significantly reduced the proportion of Tregs within the TME. Figure 11 This effect promoted the phenotypic shift of TAMs from the M2 to the M1 phenotype, thereby reversing the immunosuppressive state. This effect was attributed to the ability of calcium carbonate to neutralize lactate within the TME. The ability of nanoparticles to alleviate tumor acidity was assessed by directly measuring intratumoral pH using a commercially available pH microelectrode. The results showed that, compared with the PBS control group, the tumor pH was significantly increased 24 hours after intravenous injection of Zn-TCPP@CaCO3 ( Figure 12 However, calcium carbonate treatment alone only partially alleviated the acidic conditions but failed to completely reverse immunosuppression, indicating that pH regulation alone is insufficient to fully remodel the immune microenvironment. In the Zn-TCPP@CaCO3+US group, tumor acidity was effectively neutralized, accompanied by a significant pyroptosis response, leading to significant activation of pro-inflammatory signaling pathways. These synergistic effects jointly promoted the polarization of TAMs from the M2 phenotype to the M1 phenotype and reduced the proportion of Tregs within the TME ( Figures 11-12 Overall, these findings demonstrate that Zn-TCPP@CaCO3 can transform immunosuppressive "cold" TMEs into immunoactivating "hot" TMEs.

[0068] 7. "Sound-activated Zn" 2+ In vivo antitumor therapy study of "Storm" combined with αPD-L1 Although immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, their limited efficacy against non-responsive "cold" tumors with clinical response rates below 30% remains a major treatment challenge.

[0069] The above studies demonstrate that Zn-TCPP@CaCO3+ US can effectively remodel the tumor immune microenvironment and stimulate anti-tumor immune responses. To further enhance therapeutic efficacy and evaluate its induced systemic immune activation, we used a bilateral tumor mouse model to investigate the effect of "sound-activated Zn..." 2+The combined potential of "storm" and αPD-L1, and the specific modeling method of bilateral tumor mouse model: First, on day -7, subcutaneously inject 1×10⁻⁶ dpi into the left back of the mouse. 6 A primary tumor was constructed using 4T1 cells, and then, on day -4, an equal number of 4T1 cells were injected subcutaneously into the right back of mice to simulate distant metastatic tumors. αPD-L1 exerts its effect by blocking the PD-1 / PD-L1 inhibitory pathway, thereby alleviating T cell suppression and supplementing Zn-TCPP@CaCO3+ US-induced in situ immune activation. Therefore, this combination therapy synergistically enhances anti-tumor immunity, effectively inhibits primary tumor growth, and simultaneously induces strong distant effects to suppress the progression of untreated distant tumors.

[0070] The entire experimental design is as follows Figure 13 As shown in Figure A, mice were randomly divided into four groups: (1) control group; (2) αPD-L1 group; (3) Zn-TCPP@CaCO3+ US group; (4) Zn-TCPP@CaCO3+ US + αPD-L1 group (10 mg kg) -1 In this study, the drug concentrations and ultrasound parameters for each group were the same as described above, with αPD-L1 at 10 mg / kg. Results showed that αPD-L1 monotherapy inhibited the growth of primary and distant tumors to some extent, but its efficacy was limited, possibly due to insufficient T-cell infiltration. In contrast, the combination therapy group exhibited the most significant anti-tumor effect, characterized by significant inhibition of primary and distant tumor growth and a markedly prolonged survival. Figure 13 B- Figure 13 G). These results confirm that the combination therapy not only inhibits the growth of the primary tumor but also induces significant distant effects, thus demonstrating the activation of the systemic antitumor immune response.

[0071] Further mechanistic research ( Figure 13 H) indicates that the combination therapy of Zn-TCPP@CaCO3 + US + αPD-1 significantly enhanced CD8+ in tumor draining lymph nodes (TDLNs). + T cell infiltration and dendritic cell maturation ( Figure 13 I- Figure 13 J, Figure 13 M). Notably, CD8+ in distal tumor tissues in the combination therapy group + The proportion of T cells was significantly higher than that of the control group (40.1% vs. 14.4%). Figure 13 K- Figure 13Immunofluorescence analysis showed a decreased abundance of regulatory T cells (Tregs) and a significantly increased infiltration of pro-inflammatory M1 macrophages in distal tumor tissue, indicating that the TME was effectively reprogrammed into an anti-tumor state. Furthermore, ELISA results showed that Zn-TCPP@CaCO3+ US treatment significantly increased serum levels of pro-inflammatory cytokines, including IL-6, TNF-α, and IFN-γ. These findings suggest that "sound-activated Zn..." 2+ The "storm" not only directly releases inflammatory mediators through pyroptosis but also broadly activates the immune response. When used in combination with αPD-L1, the levels of these cytokines are further synergistically upregulated, with a particularly significant increase in IFN-γ, reflecting effective activation of T cell function. Overall, these results indicate that Zn-TCPP@CaCO3+ ultrasound (US) can elicit a strong immune response, providing an initial stimulus and inflammatory microenvironment ("ignition") for initiating antitumor immunity by inducing pyroptosis, promoting the release of damage-associated molecular patterns (DAMPs), and upregulating pro-inflammatory cytokines. Simultaneously, αPD-L1 blockade alleviates immunosuppression of the immune system, particularly restoring CD8+. + T cell activity. The synergistic effect of these two mechanisms ultimately drives a significant systemic antitumor immune response, providing a promising strategy for overcoming the limited efficacy of immune checkpoint inhibitors (ICIs) in cold tumors.

[0072] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A tumor therapy platform based on endogenous and exogenous zinc overload strategies, characterized in that: Including acid-sensitive carriers, zinc ions, and acoustically sensitive organic ligands; The acoustically sensitive organic ligand can generate reactive oxygen species under ultrasonic irradiation. The acoustically sensitive organic ligand and zinc ions are connected by coordination bonds to form a metal-organic complex. The metal-organic complex is loaded on an acid-sensitive support.

2. The tumor therapy platform based on endogenous and exogenous zinc overload strategy according to claim 1, characterized in that: Sound-sensitive organic ligands have macrocyclic conjugated structures; Sound-sensitive organic ligands have nitrogen atoms and / or carboxyl groups that can coordinate with zinc ions.

3. The tumor therapy platform based on endogenous and exogenous zinc overload strategy according to claim 2, characterized in that: The sonosensitive organic ligands include, but are not limited to, one or more of tetra(4-carboxyphenyl)porphyrin, protoporphyrin IX, dihydroporphyrin e6, tetraphenylporphyrin, and porphyrin derivatives.

4. The tumor therapy platform based on endogenous and exogenous zinc overload strategy according to claim 1, characterized in that: The acid-sensitive carrier includes, but is not limited to, one or more of the following: calcium carbonate, calcium phosphate, manganese carbonate, mesoporous silica, pH-sensitive liposomes, and acid-sensitive polymer carriers.

5. A method for preparing a tumor therapy platform based on an endogenous and exogenous zinc overload strategy, characterized in that: Includes the following steps: S1. Preparation of calcium carbonate nanoparticles: Add ethanol containing CaCl2-2H2O to a container covered with aluminum foil, which has several small holes. Then, ammonium bicarbonate was placed in a vacuum drying chamber along with the container and kept at 40°C for 24 hours to obtain calcium carbonate nanoparticles. S2. Mix the calcium carbonate suspension with the PVP K30 ethanol solution and stir at room temperature; Then add TCPP ethanol solution and continue stirring; Next, add Zn(NO3)2 ethanol solution and stir; Finally, the Zn-TCPP@CaCO3 product was separated by centrifugation and washed multiple times with ethanol.

6. The method for preparing a tumor therapy platform based on an endogenous and exogenous zinc overload strategy according to claim 5, characterized in that: It also includes, S3. Disperse Zn-TCPP@CaCO3 in anhydrous ethanol, then mix it with DOPA chloroform solution, and sonicate the mixture in a 37°C water bath for 30 minutes. Next, the DOPA-coated nanoparticles obtained by centrifugation purification were resuspended in a solution containing cholesterol, DPPC, and DSPE-PEG. 5000 The synthesized Zn-TCPP@CaCO3 was stirred at room temperature for 12 hours in a chloroform solution, and then the chloroform was removed by a rotary evaporator. Finally, the synthesized Zn-TCPP@CaCO3 was purified by centrifugation.

7. The application of the tumor therapy platform based on the endogenous and exogenous zinc overload strategy as described in any one of claims 1-4 in the preparation of drugs that induce cell-specific pyroptosis.

8. The application according to claim 7, characterized in that: The treatment platform has the functions of inhibiting tumor growth and reprogramming the immunosuppressive TME, enhancing the therapeutic effect of immune checkpoint inhibitors, and inhibiting the growth of primary and distant tumors.

9. A drug for treating tumors, characterized in that: Including immune checkpoint inhibitors and tumor therapy platforms based on endogenous and exogenous zinc overload strategies as described in any of claims 1-4.

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