Mitochondria-targeted triple-response nano-platform as well as preparation method and application thereof

Abstract

The invention relates to a mitochondrial targeting triple response nano platform and a preparation method thereof. The preparation method comprises the following steps: preparing a nano core; the nano core is loaded with a photosensitizer; preparing the core-shell nanoparticles; and preparing the core-shell nanoparticles. The mitochondria-targeted triple-response nano-platform disclosed by the invention has the advantages of accurate liver cancer mitochondria targeting capability, multi-stimulation response and multi-mechanism synergistic treatment effect, real-time imaging and treatment monitoring, high safety and excellent biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of nanozyme technology, and in particular to a mitochondrial-targeted triple-response nanoplatform, its preparation method, and its applications. Background Technology

[0002] Liver cancer, one of the deadliest malignant tumors globally, has seen a continuous rise in incidence and mortality rates in recent years, seriously threatening human health and life. Especially in my country, the number of patients with primary liver cancer is enormous. Due to the insidious nature of early symptoms, rapid tumor progression, and the tendency for multicentric occurrence, clinical diagnosis is often delayed, and treatment options are limited. Furthermore, liver cancer is highly invasive and prone to recurrence, and traditional methods such as surgical resection, chemotherapy, and radiotherapy often fail to significantly improve long-term survival rates. Chemotherapy drugs, in particular, often have limited efficacy due to drug tolerance, systemic toxicity, and the tumor microenvironment barrier; while radiotherapy or local ablation therapy easily causes damage and dysfunction to normal liver tissue, making it difficult to achieve radical cure of tumor cells. Therefore, current liver cancer treatment urgently needs a new technological system that can overcome the tumor microenvironment barrier and improve drug targeting and therapeutic synergy.

[0003] In recent years, nanomedicine and tumor microenvironment regulation have become emerging research hotspots in the comprehensive treatment of liver cancer. Multifunctional nanosystems based on nanozymes, photosensitizers, and peptide-targeting molecules, in particular, have attracted significant attention due to their high responsiveness to the acidity, oxidative stress, and biological barriers of the tumor microenvironment, enabling precise local treatment of tumor cells. For example, nanozymes, as artificial enzyme systems combining the activity of natural enzymes with the unique tunable properties of nanomaterials, can efficiently catalyze the production of reactive oxygen species (ROS) from hydrogen peroxide (H2O2) within tumor cells, inducing cellular oxidative stress and death. Photosensitizers such as indocyanine green (ICG) can generate additional ROS and release photothermal energy upon near-infrared (NIR) irradiation, further amplifying the cell damage effect. Meanwhile, the mitochondrial-targeting peptide SS31, due to its high affinity for mitochondrial membrane potential, can guide nanomaterials to actively locate to mitochondria, precisely disrupting energy metabolism hubs and inducing ferroptosis and apoptosis. All of these single functional components have shown potential applications in the comprehensive treatment of liver cancer.

[0004] However, existing nanoprobe or multifunctional nanomaterial systems still face the following key technical bottlenecks. First, most nanozymes or photosensitizers are prone to rapid in vivo clearance and lack targeted delivery mechanisms, leading to drug distribution in non-tumor tissues and increasing systemic toxicity. Second, single-response modes (such as pH-only or light-only responses) are insufficient to address the complex and variable characteristics of the liver cancer microenvironment (such as acidity, elevated ROS, and local hypoxia), limiting therapeutic efficacy and increasing the risk of recurrence due to residual tumor cells. Simultaneously, the targeting efficiency of some mitochondrial-targeting nanomaterials is affected by the tumor's multilayered biomembrane barriers and intracellular transport obstacles, making it difficult to achieve high-density accumulation in mitochondria, resulting in weak ferroptosis and energy destruction cascade effects. Traditional composite nanomaterial systems also face problems such as synergistic failure and uncontrolled component release when integrating multi-response and multifunctional components. For example, the activity of nanozymes and photosensitizers is often reduced or interferes with each other due to the influence of the microenvironment, leading to decreased stability of the therapeutic system. Multifunctional nanoformulations, primarily based on physical encapsulation or simple surface modification, are susceptible to protein coating and enzymatic degradation during in vivo delivery. They exhibit insufficient responsiveness to acidic environments and limited photosensitizer activation efficiency, making precise spatiotemporal control difficult. Furthermore, excessive ROS bursts induced by some metal-based nanozymes may damage normal cellular mitochondria and the surrounding microenvironment, causing uncontrollable side effects. These technical limitations restrict the application value of multi-response, mitochondrial-targeted comprehensive therapeutic systems in the clinical translation of liver cancer.

[0005] To address the aforementioned issues, there is an urgent need to develop a novel nanoplatform system that combines multiple response mechanisms (such as pH, light, and ROS), highly efficient mitochondrial targeting capabilities, and synergistic release of multiple components. An ideal nanozyme system should possess the ability to dynamically sense the tumor microenvironment of liver cancer, intelligently disintegrate in the acidic regions of liver cancer cells, precisely release functional components, catalyze a ROS cascade eruption and synergistically photoactivate, efficiently induce ferroptosis and energy collapse within tumor mitochondria, and achieve complete elimination of liver cancer cells. Simultaneously, it should possess sufficient system stability, biocompatibility, and component controllability to minimize side effects and systemic toxicity to normal tissues. Current technologies lack integrated innovation for these key indicators, necessitating a deep cross-disciplinary approach involving nanomedicine, materials chemistry, and bioengineering to overcome the technical bottlenecks of conventional single-functional materials and traditional combined therapies, and construct a liver cancer treatment nanozyme platform system with multi-level response, precise targeting, and strong synergy. Summary of the Invention

[0006] To address the shortcomings of existing liver cancer treatment technologies, such as insufficient targeting of photosensitizing drugs, poor responsiveness to the tumor microenvironment, and low efficiency in regulating reactive oxygen species (ROS), this invention aims to solve the technical challenges of limited efficacy, poor system stability, and insufficient targeting of mitochondria in single photodynamic or enzyme-catalyzed therapies. In existing treatment systems, photosensitizers are easily photobleached or rapidly eliminated in vivo, leading to a decline in photothermal and photodynamic effects. Furthermore, traditional nanozyme catalytic systems exhibit unstable catalytic activity in the complex acidic and reducing tumor environment, making it difficult to maintain sustained ROS generation. In addition, the lack of an active targeting mechanism makes it difficult for drugs to accumulate within the mitochondria of tumor cells, thus hindering the efficient induction of apoptosis or ferroptosis and significantly reducing treatment efficiency.

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing a mitochondrial-targeted triple-response nanoplatform, characterized by comprising the following steps:

[0008] Preparation of nanonuclei (MnFe2O4): Iron and manganese salts were dissolved, sodium acetate (NaAc) was added, the mixture was stirred, the pH was adjusted, a hydrothermal reaction was carried out, the mixture was cooled, the precipitate was separated, washed, and vacuum dried to obtain the nanonuclei. Photosensitizer loaded on nanonuclei: The photosensitizer was dissolved, sonicated, and the photosensitizer solution was added to the nanonuclei dispersion. Buffer solution was added, and the mixture was incubated in the dark. The precipitate was collected by centrifugation, washed, and resuspended to obtain photosensitizer-loaded nanonuclei (MnFe2O4-ICG). The photosensitizer included indocyanine green (ICG). Preparation of core-shell nanoparticles (MnFe2O4-ICG@ZIF-8): The photosensitizer-loaded nanoparticle dispersion was mixed with methanol, sonicated, stirred and injected with zinc nitrate solution, stirred, 2-methylimidazolium methanol solution was added, stirring was continued, the precipitate was collected by centrifugation, washed and vacuum dried to obtain the product; Mitochondrial-targeting peptide modification: A core-shell nanoparticle dispersion was taken, buffer solution was added, and the mixture was sonicated. Dopamine hydrochloride was added, and the mixture was stirred, centrifuged to collect the precipitate, washed, and resuspended to obtain dopamine-coated core-shell nanoparticles. The resuspended dopamine-coated core-shell nanoparticles were then added to a mixed solvent of dimethyl sulfoxide and water, followed by the addition of succinic anhydride. The mixture was stirred at room temperature, centrifuged, washed, and resuspended to obtain carboxyl-rich dopamine-coated core-shell nanoparticles. The resuspended carboxyl-rich dopamine-coated core-shell nanoparticles were then added to a buffer solution, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-succinimide ester. The mixture was shaken, pH adjusted, and mitochondrial-targeting peptide solution was added dropwise. The mixture was reacted in the dark, centrifuged to collect the precipitate, washed, and resuspended to obtain a mitochondrial-targeting triple-response nanoplatform (MnFe2O4-ICG@ZIF-8-SS31). The mitochondrial-targeting peptide includes SS31.

[0009] In one embodiment, the iron salt comprises ferric chloride (FeCl3), and the manganese salt comprises manganese chloride (MnCl2).

[0010] In one embodiment, the dimethyl sulfoxide and water mixture is in a volume ratio of (3~5):1.

[0011] In the specific preparation process of the mitochondrial-targeted triple-response nanoplatform of this invention, there are obvious conflicts among the components in terms of solvent system, pH conditions, nucleation and growth behavior, and surface chemistry. This invention solves the following key technical difficulties through step-by-step design and condition reconstruction: 1. Stability of MnFe2O4-ICG composite cores under ZIF-8 in-situ growth conditions The MnFe2O4-ICG composite core used in this invention operates in methanol / water and Zn... 2+ It is highly likely to occur in a 2-methylimidazole mixture: (1) Mn 2+ Fe 3+ It dissolves from the surface, affecting core magnetism and enzyme-like activity; (2) ICG rapidly desorbs, aggregates, and even photodegrades under polar organic solvent + metal salt conditions, resulting in a severe decrease in photothermal / photosensitive properties.

[0012] To resolve the aforementioned conflict, this invention does not directly introduce MnFe2O4 and ICG into the ZIF-8 growth system, but instead uses: (1) First, control the mass ratio of MnFe2O4 to ICG (about 8:1) in the aqueous phase, and complete the adsorption under mild pH (7.4) and HEPES conditions to obtain the MnFe2O4-ICG composite core, avoiding the organic solvent exposure time of ICG; (2) MnFe2O4-ICG was then transferred into the methanol system, and Zn was optimized. 2+ Concentration (0.1 mol L) -1 ), 2-methylimidazole ratio (0.4 mol L) -1 The reaction time (2h) allows ZIF-8 to rapidly nucleate in situ on the outer layer, playing a role in "synergistic immobilization and shielding" of MnFe2O4 and ICG.

[0013] 2. The balance between the acid-sensitive disintegration and ICG stability of ZIF-8 It is known that ZIF-8 will gradually degrade under conditions of pH < 6.0, thus achieving drug release. However, ICG is extremely prone to aggregation and photobleaching in acidic and metal ion-rich environments. The conventional "ZIF-8 encapsulation" method often results in slow leakage under neutral conditions and sudden release and rapid inactivation under acidic conditions.

[0014] This invention achieves a controllable "neutral-stable - acidic-rapid release" mode by controlling the mass ratio of MnFe2O4-ICG to ZIF-8, the shell thickness, and the pore structure, resulting in an extremely low leakage rate of ICG at pH 7.4, while significantly increasing cumulative release at a tumor-simulated pH of 5.5. This pH responsiveness relies on the formation of a dense core by MnFe2O4-ICG, followed by the induction of orderly growth of the ZIF-8 shell.

[0015] 3. Conditional Conflicts and Synergistic Designs of PDA Coating, Succinic Anhydride Carboxylation, and SS31 Coupling The difficulty of this invention lies in: (1) PDA self-polymerization is extremely sensitive to pH. At low pH, polymerization is slow and the shell does not form a film; at high pH, ​​a large number of free PDA aggregates form, blocking the ZIF-8 channels and severely affecting the enzyme-like activity of MnFe2O4 and the release of ICG. Through screening, we found that PDA self-polymerization only occurs at pH = 8.5 and [DA] ≈ 1 mg / mL. -1 Only under stirring conditions of 3 hours can a continuous, thin PDA coating rich in -NH2 / -OH be formed without significantly damaging the ZIF-8 structure; (2) Succinic anhydride carboxylation requires an organic solvent / DMSO environment, while ZIF-8 is prone to partial dissociation in highly polar organic solvents. This invention uses a 4:1 DMSO / water mixture and precisely controls the amount of succinic anhydride (50 mg / 10 mL) and reaction time (4 h) to balance the full ring-opening of PDA-NH2 with the integrity of the ZIF-8 framework. Comparative experiments show that if carboxylation is carried out directly in a pure DMSO system, the ZIF-8 framework collapses significantly, and the pH-triggered release performance disappears; (3) SS31 coupling requires EDC / NHS activation without damaging the nanozyme and ICG activity. In this invention, carboxyl activation is first performed in MES (pH=6.0), and then the pH is quickly adjusted to 7.0~7.4 to introduce SS31. The entire process is kept mild and light-protected to reduce the degradation of SS31 and ICG, while ensuring that the structure of MnFe2O4-ICG@ZIF-8 is not impacted by acid / alkali. The final SS31 loading efficiency is approximately 70.3%, while the nanozyme activity and photothermal properties are still preserved and synergistically amplified.

[0016] In one embodiment, the preparation of the nanonucleus includes the following steps: Iron and manganese salts are dissolved in water, followed by the addition of sodium acetate. The mixture is stirred and the pH is adjusted to 12-14. A hydrothermal reaction is then carried out, followed by cooling, separation of the precipitate, washing, and vacuum drying to obtain the final product. According to the molar ratio, the iron salt: the manganese salt is (1~3):1.

[0017] In one embodiment, the hydrothermal reaction conditions are 190~210℃ for 8~12 hours.

[0018] In one embodiment, the nanonucleus-loaded photosensitizer includes the following steps: Dissolve the photosensitizer in water, sonicate, add the photosensitizer solution to the nanonucleus dispersion, add 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), incubate in the dark, centrifuge to collect the precipitate, wash, and resuspend to obtain the photosensitizer-loaded nanonucleus; According to the mass ratio, the nanonucleus:the photosensitizer is (7~9):1.

[0019] In one embodiment, the light-protected incubation time is 5-8 hours.

[0020] In one embodiment, the preparation of the core-shell nanoparticles includes the following steps: Take the photosensitizer-loaded nanonucleus dispersion and mix it with methanol, sonicate, inject the methanol solution of zinc nitrate (Zn(NO3)2) under stirring, stir, then add 2-methylimidazolium methanol solution, continue stirring for 1~3h, centrifuge to collect the precipitate, wash, and vacuum dry to obtain the product; According to the dosage ratio, the photosensitizer-loaded nanonucleus: zinc nitrate: 2-methylimidazole is (3~5) mg: (0.4~0.6) mmol: (7~9) mmol.

[0021] In one embodiment, the concentration of the zinc nitrate methanol solution is 0.1 mol / L; and the concentration of the 2-methylimidazolium methanol solution is 0.4 mol / L.

[0022] In one embodiment, the stirring time followed by the addition of the 2-methylimidazolium methanol solution is 3 to 8 minutes.

[0023] In one embodiment, the mitochondrial-targeting peptide modification includes the following steps: Take the core-shell nanoparticle dispersion, add tris-(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl), sonicate, add dopamine hydrochloride, stir for 2-4 h, centrifuge to collect the precipitate, wash, and resuspend to obtain dopamine-coated (PDA) core-shell nanoparticles (MnFe2O4-ICG@ZIF-8@PDA); take the dopamine-coated core-shell nanoparticle resuspension, add a mixed solvent of dimethyl sulfoxide (DMSO) and water, then add succinic anhydride, stir at room temperature for 3-5 h, centrifuge, wash, and resuspend to obtain carboxyl-rich dopamine-coated core-shell nanoparticles. Shell nanoparticles (MnFe2O4-ICG@ZIF-8@PDA-COOH); 2-morpholine ethanesulfonic acid buffer (MES) was added to the resuspension of carboxyl-rich dopamine-coated core-shell nanoparticles, followed by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-succinimide ester (NHS). The mixture was shaken, and the pH was adjusted to 7.0-7.4. Mitochondrial-targeting peptide solution was added dropwise, and the mixture was reacted in the dark for 10-14 h. The product was collected by centrifugation, washed, and resuspended to obtain the mitochondrial-targeting triple-response nanoplatform. According to the mass ratio, the core-shell nanoparticles: the dopamine hydrochloride is 1:(2~3); According to the mass ratio, the dopamine-coated core-shell nanoparticles: succinic anhydride is 1: (12~13). According to the mass ratio, the core-shell nanoparticles with carboxyl-rich dopamine coating: the mitochondrial-targeting peptide is (1~3):1.

[0024] In one embodiment, the final concentration of the dopamine hydrochloride after dissolution is 1 mg / mL, and the final concentration of the succinic anhydride after dissolution is 5 mg / mL.

[0025] In one embodiment, the oscillation time is 25-35 minutes.

[0026] In a second aspect, the present invention also provides a mitochondrial-targeted triple-response nanoplatform obtained by the above preparation method.

[0027] A third aspect of the present invention also provides the application of the mitochondrial-targeted triple-response nanoplatform obtained by the above preparation method or the above mitochondrial-targeted triple-response nanoplatform in the preparation of drugs for treating liver cancer.

[0028] In addition, the present invention also provides a drug for treating liver cancer, comprising the mitochondrial-targeted triple-response nanoplatform obtained by the above preparation method or the above mitochondrial-targeted triple-response nanoplatform.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. Precise targeting capability of liver cancer mitochondria: The nanozyme platform of this invention introduces SS31 functional peptide to achieve efficient targeting and enrichment of liver cancer cell mitochondria, effectively breaking through the tumor microenvironment barrier, improving the positioning accuracy of therapeutic factors, and thus significantly enhancing the killing efficiency of liver cancer cells.

[0030] 2. Synergistic Therapeutic Effects of Multiple Stimulus Responses and Mechanisms: This invention integrates a triple stimulus response mechanism of pH, light, and ROS, enabling intelligent activation and targeted drug release based on changes in the microenvironment of liver cancer cells. The nanoplatform disintegrates in the acidic environment of the tumor area, and near-infrared light irradiation promotes the production of ROS by photosensitizers. Simultaneously, nanoenzymes synergistically activate oxidative stress reactions, achieving a synergistic effect of multiple mechanisms such as ferroptosis and energy deprivation, significantly improving the therapeutic effect of liver cancer.

[0031] 3. Real-time imaging and treatment monitoring: This platform integrates the photosensitizer ICG to give nanoparticles near-infrared fluorescence imaging capabilities, making the treatment process monitorable and quantifiable, which helps in the development of individualized treatment plans and efficacy evaluation.

[0032] 4. High safety and excellent biocompatibility: Using highly biocompatible materials such as ZIF-8, the platform has good stability in vivo, which can effectively reduce side effects on normal liver tissue and other organs. It also has good metabolic and excretion characteristics, reducing the risk of material accumulation.

[0033] 5. Strong controllability of preparation process: The preparation process of the nanoplatform of this invention is simple, the component ratio is easy to control, and the synthesis efficiency is high. It is conducive to large-scale production and clinical translation, reduces the industrialization threshold and cost, and has good application prospects. Attached Figure Description

[0034] Figure 1 This is a graph showing the cumulative release rate of ICG under different pH conditions. Figure 2 The image shows the hemolysis test results of MnFe2O4-ICG@ZIF-8-SS31. Figure 3 To investigate the toxicity of different concentrations of MnFe2O4-ICG@ZIF-8-SS31 on L929 cells; Figure 4 The effects of different treatment groups on Huh7 cell clone formation. Detailed Implementation

[0035] This invention provides a pH / light / ROS multi-stimulation responsive mitochondrial-targeted nanozyme system (MIZSS) based on MnFe2O4-ICG@ZIF-8-SS31. Designed based on a triple-response mechanism for synergistic treatment of liver cancer, it constructs an integrated multifunctional nanoplatform with a MnFe2O4 nanozyme as the core, loaded with the photosensitizer ICG, encapsulated in a metal-organic framework ZIF-8, and modified with the mitochondrial-targeting peptide SS31. By organically combining the multi-response nanostructure with the mitochondrial-targeting peptide SS31, this invention achieves precise recognition and deep penetration of liver cancer cells. Under local acidic environment and near-infrared light irradiation, it synergistically generates heat, oxygen, and catalyzes free radical generation, significantly enhancing the ROS accumulation effect, thereby achieving efficient, safe, and controllable liver cancer treatment. This invention, with a multi-response nanozyme as its core, achieves a synergistic improvement in targeting, stability, and therapeutic effect, providing a novel comprehensive technical solution for the catalytic-photothermal-mitochondrial targeted synergistic treatment of liver cancer.

[0036] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0038] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0039] Example I. This invention provides a method for preparing a mitochondrial-targeted triple-response nanoplatform, comprising the following steps: 1. Synthesis of MnFe2O4 nanozymes Prepare a metal salt solution by dissolving 1.0 mmol FeCl3·6H2O and 0.5 mmol MnCl2·4H2O in deionized water; then add 5.5 g NaAc and stir at room temperature for 1 h until completely dissolved. Under magnetic stirring at 600 rpm, slowly add 1.5 mol L... - 1NaOH solution was added until the system pH ≈ 13. The obtained precursor was transferred to a 100 mL polytetrafluoroethylene autoclave and hydrothermally reacted at 200 °C for 10 h. After natural cooling to room temperature, the black precipitate was separated using a neodymium iron boron (NdFeB) magnet, washed three times by centrifugation with ethanol / water (v / v = 1:1), and dried under vacuum at 75 °C for 8 h to obtain brownish-black MnFe2O4 nanoparticles.

[0040] 2. Preparation of MnFe2O4-ICG complex Weigh 2.5 mg of indocyanine green (ICG), dissolve it in 1 mL of deionized water, and sonicate for 2 min to obtain 2.5 mg / mL. -1 ICG stock solution. Take 4 mL of MnFe2O4 aqueous dispersion (2 mg / mL). -1 Place the sample in a 10 mL centrifuge tube. Add 0.4 mL of ICG stock solution (2.5 mg / mL). -1 This resulted in a MnFe₂O₄ to ICG mass ratio of approximately 8:1 and a total volume of 4.4 mL. A small amount of 10 mmol L⁻¹ was added. - 1 Adjust the ionic strength with HEPES buffer (pH=7.4) to a final volume of 5 mL. Incubate at 25°C and 150 rpm for 6 h in the dark to promote ICG adsorption onto the MnFe₂O₄ surface via electrostatic and hydrophobic interactions. After incubation, collect the precipitate by centrifugation at 8000 rpm for 10 min, wash three times with PBS to remove unbound free ICG. Finally, resuspend the precipitate in PBS (pH=7.4) and adjust the MnFe₂O₄-ICG concentration to 2 mg / mL. -1 Store at 4°C, protected from light.

[0041] 3. Synthesis of MnFe2O4-ICG@ZIF-8 core-shell nanoparticles Take 2 mL of MnFe2O4-ICG aqueous dispersion (2 mg / mL) -1 Mix with 8 mL of methanol and sonicate at 40 kHz and 100 W for 5 min to obtain a homogeneous suspension; under magnetic stirring at 600 rpm and 25 °C, rapidly inject 5 mL of Zn(NO3)2·6H2O methanol solution (0.1 mol / L). -1 Continue stirring for 5 minutes. Then add 20 mL of 2-methylimidazolium methanol solution (0.4 mol / L) all at once. -1 The mixture was stirred continuously at room temperature for 2 hours (the solution instantly changed from brownish-black to milky, indicating in-situ growth of the ZIF-8 shell). After the reaction was completed, the precipitate was collected by centrifugation at 10000g for 8 minutes, washed three times with deionized water and once with ethanol, and dried under vacuum at 60℃ for 6 hours to obtain brownish-black MnFe2O4-ICG@ZIF-8 powder, which was then stored in a desiccator away from light.

[0042] 4. Covalent grafting of SS31 peptide (1) PDA surface coating: Take 2 mL of MnFe2O4-ICG@ZIF-8 dispersion (2 mg / mL) -1 Add 8 mL of 10 mmol / L -1 Tris-HCl buffer (pH=8.5) and sonicate for 3 min. Weigh 10 mg of dopamine hydrochloride and dissolve it in the above suspension (final concentration approximately 1 mg / mL). -1 The mixture was stirred at room temperature and 500 rpm for 3 hours to allow it to self-polymerize and form a PDA coating. The precipitate was collected by centrifugation at 8000 rpm for 10 minutes, washed three times with deionized water to obtain MnFe2O4-ICG@ZIF-8@PDA, and resuspended in PBS (pH=7.4) at a concentration of 2 mg / mL. -1 .

[0043] (2) Surface carboxylation: Take 2 mL of MnFe2O4-ICG@ZIF-8@PDA dispersion (2 mg / mL) -1 Add 8 mL of DMSO / water mixed solvent (V / V=4:1), add 50 mg of succinic anhydride, and stir at room temperature for 4 h. After the reaction is complete, centrifuge at 8000 rpm for 10 min, wash 3 times with PBS to obtain MnFe2O4-ICG@ZIF-8@PDA-COOH with carboxyl groups on the surface, and resuspend in PBS.

[0044] (3) SS31 coupling: Take 2 mL of MnFe2O4-ICG@ZIF-8@PDA-COOH dispersion and add 8 mL of 0.1 mol / L solution. - 1 MES buffer (pH=6.0) was added, followed by 20 mg EDC·HCl and 12 mg NHS. The mixture was gently shaken at room temperature for 30 min to activate the surface carboxyl groups. The pH was then adjusted to 7.0–7.4 with PBS, and 1 mL of SS31 solution (2 mg / mL) was slowly added dropwise. -1 The product was dissolved in PBS and reacted at room temperature or 4°C in the dark for 12 hours. After the reaction, the product was collected by centrifugation at 8000 rpm for 10 minutes, washed three times with PBS, and finally resuspended in PBS (pH=7.4). The solid content was adjusted to approximately 2 mg / mL. -1 The target product MnFe2O4-ICG@ZIF-8-SS31 was obtained and stored at 4℃ in the dark.

[0045] Example 1 Synthesis of MnFe2O4 nanozymes: Weigh out 1.0 mmol FeCl3·6H2O and 0.5 mmol MnCl2·4H2O, dissolve them in an appropriate amount of deionized water to prepare a mixed metal salt solution; then add 5.5 g sodium acetate (NaAc), and stir magnetically for 1 h at room temperature until completely dissolved to obtain a homogeneous precursor solution. Under stirring at 600 rpm, slowly add 1.5 mol L... -1 The pH of the system was adjusted to approximately 13 using NaOH solution. The resulting mixture was transferred to a 100 mL high-pressure reactor lined with polytetrafluoroethylene (PTFE) and subjected to a hydrothermal reaction at 200 °C for 10 h. After the reaction, the mixture was allowed to cool naturally to room temperature, and magnetic separation was performed on the outside using an NdFeB permanent magnet to collect the black precipitate. The precipitate was washed three times by centrifugation with a 1:1 volume fraction ethanol / water mixture to remove residual ions, and finally dried under vacuum at 75 °C for 8 h to obtain brownish-black MnFe₂O₄ nanoparticle powder.

[0046] Example 2 Preparation of MnFe2O4-ICG complex: Weigh 2.5 mg of indocyanine green (ICG), dissolve it in 1 mL of deionized water, and sonicate for 2 min to obtain 2.5 mg / mL of the solution. -1 ICG stock solution. Take 4 mL of MnFe2O4 aqueous dispersion (2 mg / mL). -1 Place the solution in a 10 mL centrifuge tube for later use. Add 0.4 mL of ICG stock solution (2.5 mg / mL) to the above MnFe2O4 dispersion. -1 The mass ratio of MnFe₂O₄ to ICG in the system was adjusted to approximately 8:1, with a total volume of 4.4 mL. Then, an appropriate amount of 10 mmol L⁻¹ was added. -1 The ionic strength was adjusted using HEPES buffer (pH=7.4), and the total volume was adjusted to 5 mL. Under light-protected conditions, the mixture was placed in a shaker and incubated at 25°C and 150 rpm for 6 h, allowing ICG to spontaneously adsorb onto the MnFe₂O₄ surface through electrostatic and hydrophobic interactions. After incubation, the precipitate was collected by centrifugation at 8000 rpm for 10 min and washed three times with PBS (pH=7.4) to remove unbound free ICG. Finally, the precipitate was resuspended in PBS (pH=7.4) to adjust the MnFe₂O₄-ICG concentration to 2 mg / mL. -1 Stored at 4°C protected from light for subsequent ZIF-8 coating steps. The actual ICG loading and loading efficiency were calculated by measuring the absorbance of ICG in the supernatant and combining it with a standard curve. The results are shown in Table 1.

[0047] Table 1 Drug loading and loading efficiency of ICG and SS31

[0048] (Note: Load efficiency is the ratio of actual load to initial load.) As shown in Table 1, the ICG loading was 7.8 wt%, and the loading efficiency was 75.7%. This indicates that MnFe2O4 nanoparticles can effectively load ICG photosensitizers, laying the foundation for subsequent photothermal therapy.

[0049] Example 3 Synthesis of MnFe2O4-ICG@ZIF-8 core-shell nanoparticles: Take 2 mL of MnFe2O4-ICG aqueous dispersion (2 mg / mL) -1 The solution was mixed with 8 mL of methanol and sonicated at 40 kHz and 100 W for 5 min to obtain a homogeneous suspension. Under magnetic stirring at 25 °C and 600 rpm, 5 mL of a 0.1 mol / L Zn(NO3)2·6H2O methanol solution was rapidly injected. -1 Continue stirring for 5 minutes. Then add 20 mL of 2-methylimidazolium methanol solution (0.4 mol / L) all at once. -1 The mixture was stirred continuously at room temperature for 2 hours. During the reaction, the solution gradually changed from brownish-black to milky, indicating that the ZIF-8 shell grew in situ on the MnFe2O4-ICG surface, forming a core-shell structure. After the reaction, the brownish-black precipitate was collected by centrifugation at 10000g for 8 minutes. It was then washed three times with deionized water and once with ethanol to remove unreacted precursors and small molecules. Finally, the powder was vacuum dried at 60℃ for 6 hours to obtain MnFe2O4-ICG@ZIF-8 powder, which was then stored in a desiccator away from light for later use. The cumulative release rate of ICG in MnFe2O4-ICG@ZIF-8 under different pH conditions was determined (e.g., ...). Figure 1 As shown in the figure, the results indicate that the material exhibits low ICG leakage under neutral conditions, while under acidic conditions (pH=5.5) simulating a tumor microenvironment, the shell gradually disintegrates, and ICG release increases significantly, verifying that the core-shell structure has good pH responsiveness and drug controlled release performance.

[0050] Example 4 Synthesis of MnFe2O4-ICG@ZIF-8 core-shell nanoparticles: (1) Preparation of PDA surface coating Take 2 mL of MnFe2O4-ICG@ZIF-8 dispersion (2 mg / mL) -1 Add 8 mL of 10 mmol / L -1 Tris-HCl buffer (pH=8.5) was used to sonicate for 3 min to obtain a homogeneous suspension. 10 mg of dopamine hydrochloride was weighed and added to the suspension (final concentration approximately 1 mg / mL). -1The mixture was magnetically stirred at 500 rpm for 3 hours at room temperature. During the reaction, dopamine self-polymerized to form a polydopamine (PDA) coating on the surface of MnFe2O4-ICG@ZIF-8. After the reaction, the precipitate was collected by centrifugation at 8000 rpm for 10 min, washed three times with deionized water, and MnFe2O4-ICG@ZIF-8@PDA with PDA coating was obtained. The precipitate was resuspended in PBS (pH=7.4) and the concentration was adjusted to 2 mg / mL. -1 ,spare.

[0051] (2) Surface carboxylation Take 2 mL of MnFe2O4-ICG@ZIF-8@PDA dispersion (2 mg / mL) -1 Add 8 mL of DMSO / water mixed solvent (volume ratio 4:1), weigh 50 mg of succinic anhydride and add it to the system. Stir magnetically at room temperature for 4 h to allow the amino groups on the PDA surface to undergo a ring-opening reaction with the succinic anhydride, introducing carboxyl functional groups. After the reaction is complete, collect the precipitate by centrifugation at 8000 rpm for 10 min, wash three times with PBS to obtain MnFe2O4-ICG@ZIF-8@PDA-COOH with a carboxyl-rich surface, resuspend in PBS (pH=7.4) for later use.

[0052] (3) SS31 peptide coupling Take 2 mL of MnFe2O4-ICG@ZIF-8@PDA-COOH dispersion (2 mg / mL) -1 Add 8 mL of 0.1 mol L -1 MES buffer (pH=6.0) was added, followed by the addition of 20 mg EDC·HCl and 12 mg NHS, and the mixture was gently shaken at room temperature for 30 min to activate the surface carboxyl groups. The pH was then adjusted to 7.0–7.4 using PBS, and 1 mL of SS31 peptide solution (2 mg / mL) was slowly added dropwise. -1 The SS31 was dissolved in PBS and reacted in the dark at room temperature or 4°C for 12 h to covalently couple to the surface of the nanoparticles via amide bonds. After the reaction, the product was collected by centrifugation at 8000 rpm for 10 min, washed three times with PBS to remove uncoupled free SS31, and finally resuspended in PBS (pH=7.4) and adjusted to a solid content of approximately 2 mg / mL. -1The target product, MnFe2O4-ICG@ZIF-8-SS31 (MIZSS), was obtained and stored at 4°C in the dark. The actual loading amount and loading efficiency of SS31 were calculated by measuring the concentration of SS31 in the supernatant. Combined with ICG loading data, the results are summarized in Table 1: the SS31 loading amount was 5.7 wt%, and the loading efficiency was 70.3%. This indicates that the SS31 peptide can be effectively coupled to the surface of nanoparticles, providing a guarantee for subsequent mitochondrial targeted therapy.

[0053] Example 5 Using 808nm near-infrared laser (power density 1Wcm²) -2 The photothermal properties of MnFe2O4, MnFe2O4-ICG, and MnFe2O4-ICG@ZIF-8-SS31 were evaluated. Each sample was dispersed in PBS at a fixed concentration (100 μg / mL). -1 The solution was placed in a quartz cuvette at an initial room temperature of 25.0℃. Under continuous NIR irradiation for 5 min, temperature changes were recorded in real time using an infrared thermal imager or digital thermometer. The solution temperature and temperature rise (ΔT) at 0 min and 5 min for each group are shown in Table 2.

[0054] Table 2. Photothermal temperature rise curves (NIR laser 808nm, 1W / cm²) 2 (5 min)

[0055] As shown in Table 2, under the same ICG dosage and the same NIR irradiation conditions (808 nm, 1 W / cm²), 2 At 5 min: the temperature rise ΔT of the MnFe2O4 solution was only about 3.1℃, while the ΔT of the MnFe2O4-ICG system increased to 18.5℃. The ΔT of the MnFe2O4-ICG@ZIF-8-SS31 system of the present invention was further increased to 23.2℃. This shows that the combination of ZIF-8+PDA+SS31 is not a passive "encapsulation" of ICG, but rather forms a local photothermal enrichment and thermal insulation microenvironment in the microenvironment, which significantly improves the photothermal conversion efficiency per unit ICG.

[0056] Example 6 L929 cytotoxicity evaluation (CCK-8 assay) L929 cells were seeded into 96-well plates, with approximately 7000 cells per well, and incubated at 37°C in a 5% CO2 incubator for 24 hours to ensure adequate cell adhesion. After cell adhesion, the old culture medium was discarded, and 100 μL of DMEM complete medium containing different concentrations (0, 25, 50, 100, 200, 400 μg / mL) of MnFe2O4-ICG@ZIF-8-SS31 nanomaterials was added to each well. Six replicates were made for each group. Incubation continued for another 24 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, and incubation was carried out in the dark for 1 hour. Finally, the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0057] like Figure 3 As shown, the MnFe2O4-ICG@ZIF-8-SS31 nanomaterial maintained a survival rate of over 80% in L929 cells even at a concentration as high as 400 μg / mL. This indicates that the nanomaterial possesses good biocompatibility and low toxicity to normal cells.

[0058] Example 7 Cytotoxicity evaluation of Huh7 hepatocellular carcinoma cells under different treatment groups and NIR conditions (CCK-8 assay) Human hepatocellular carcinoma Huh7 cells were seeded into 96-well plates, with approximately 7000 cells per well, and cultured at 37°C in a 5% CO2 incubator for 24 hours to ensure adequate cell adhesion. Subsequently, the cells were divided into the following treatment groups: blank control group (culture medium only), NIR irradiation group, MnFe2O4 group, MnFe2O4-ICG group, MnFe2O4-ICG@ZIF-8 group, and MnFe2O4-ICG@ZIF-8-SS31 group. Different material concentrations (50 μg / mL) were set for each group. -1 and 100 μg mL -1 Each group was configured with multiple duplicate wells. After incubation with nanomaterials for 6 hours, for the treatment group marked "+NIR", an 808nm laser (1W cm⁻¹) was used. -2 Irradiation was performed for 5 min; the NIR-irradiated group was irradiated under the same conditions. After irradiation, all groups continued to incubate in an incubator for the total treatment time of 24 h. After treatment, the supernatant was discarded, and the cells were gently washed 2-3 times with PBS to remove free nanoparticles. Culture medium containing 10% CCK-8 reagent was added to each well (e.g., 100 μL of culture medium + 10 μL of CCK-8 per well), and incubated at 37°C in the dark for 1 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader, and the cell viability of each group was calculated with the OD value of the control group as 100%. The viability results of Huh7 cells under different treatment groups and NIR conditions are shown in Table 3.

[0059] Table 3. Survival rates of Huh7 cells under different treatment groups and NIR conditions.

[0060] As shown in Table 3, under the same concentration and irradiation conditions: the cell viability of the MnFe2O4 group at 100 μg / mL + NIR was approximately 74.2%, the MnFe2O4-ICG group was approximately 38.9%, the MnFe2O4-ICG@ZIF-8 group further decreased to 34.5%, and the MnFe2O4-ICG@ZIF-8-SS31 group of this invention significantly decreased to 18.7%. If the ROS killing effect of MnFe2O4 nanozyme and the photothermal / photodynamic effect of ICG are simply superimposed, the theoretical trend of its toxicity enhancement should be close to that of the MnFe2O4-ICG group. However, after the introduction of ZIF-8-PDA-SS31, the killing effect was non-linearly amplified, indicating that: (1) ZIF-8 imparts site-specific release and activation of MnFe2O4 activity within acidic tumors; (2) SS31 mediates the specific enrichment of the nanoplatform in mitochondria, causing ROS and thermal effects to concentrate on the mitochondrial membrane, triggering irreversible mitochondrial functional collapse.

[0061] Example 8 Different treatment groups affected mitochondrial ROS, membrane potential, and mitochondrial calcium in Huh7 cells. 2+ The influence of level Huh7 cells were seeded in 24-well plates at a density of 2.5 × 10⁶ cells per well. 4 Cells were cultured for 24 hours and then treated according to the following groups: Control group (culture medium only), NIR group, MnFe2O4 group, MnFe2O4-ICG group, MnFe2O4-ICG@ZIF-8 group, and MnFe2O4-ICG@ZIF-8-SS31 group. All experimental groups were added with the same mass concentration of nanomaterials and incubated at 37℃ for 6 hours. Except for the Control group, the remaining groups were irradiated with 808nm laser for 5 minutes and then incubated for another 6 hours, followed by fluorescent probe staining and detection.

[0062] (1) Detection of mitochondrial ROS levels: After treatment, discard the culture medium and wash the cells 2-3 times with PBS. Add serum-free culture medium containing MitoSOX Red probe (prepared according to the instructions) and incubate at 37°C in the dark for 30 minutes. After incubation, wash the cells 3 times with PBS to remove excess probe, add an appropriate amount of PBS, collect fluorescence signals using a fluorescence microscope, and calculate the relative fluorescence intensity using image analysis software, normalizing to 1 for the Control group.

[0063] (2) Detection of mitochondrial membrane potential: Mitochondrial membrane potential was detected using the JC-1 probe. After treatment, cells were washed 2-3 times with PBS, and then incubated with working solution containing JC-1 at 37°C in the dark for 30 min. After incubation, cells were washed 3 times with buffer, and the changes in the red / green fluorescence ratio were observed using a fluorescence microscope. The red / green ratio was calculated, and the membrane potential level of the Control group was set as 1.

[0064] (3) Mitochondrial Ca 2+ Leveling inspection: After treatment, discard the culture medium and wash the cells 2-3 times with PBS. Add mitochondrial calcium... 2+ The serum-free medium containing the fluorescent probe was incubated at 37°C in the dark for 30 min. After incubation, the probe was washed three times with PBS, resuspended in PBS, and the fluorescence intensity was immediately detected under a fluorescence microscope and normalized to 1 using the Control group.

[0065] (4) Detection of mitochondrial permeability transition pore (mPTP) opening: After treatment, discard the culture medium and wash the cells 2-3 times with PBS. Add Calcein-AM / Co 2+ The staining working solution was incubated at 37°C in the dark for 30 min. After incubation, the sample was washed three times with PBS, resuspended in PBS, and the fluorescence intensity was immediately detected under a fluorescence microscope and normalized to 1 using the Control group.

[0066] Mitochondrial ROS levels, mitochondrial membrane potential levels, and mitochondrial calcium levels in each treatment group 2+ The quantitative results of the level and the degree of opening of the mitochondrial permeability transition pore (mPTP) are shown in Table 4.

[0067] Table 4. Mitochondrial ROS, mitochondrial membrane potential, and mitochondrial Ca2+ induced by different treatment groups 2+ Results of level and mitochondrial permeability transition pore (mPTP) opening

[0068] As shown in Table 4, under the same treatment conditions, the MnFe2O4-ICG@ZIF-8-SS31 group induced the following in Huh7 cells: mitochondrial ROS levels increased to 3.10 times that of the control group, mitochondrial membrane potential decreased to 0.32 times, and mitochondrial Ca2+ levels decreased. 2+The level increased by 3.2-fold, and the Calcein fluorescence intensity indicated by mPTP decreased by 0.29-fold. Compared with the groups containing only MnFe2O4 or MnFe2O4-ICG, all mitochondrial-related indicators showed changes in the same direction and with a higher degree of polarization, indicating that this nanoplatform achieved a "four-hit linkage" of ROS surge, calcium overload, membrane potential collapse, and permeability transition pore opening at the same organelle level. This invention is the first to use SS31 in reverse as a mitochondrial targeting ligand. By covalently linking it with the ROS-producing MnFe2O4-ICG@ZIF-8 system, ROS is highly enriched in the mitochondrial microenvironment targeted by SS31, thereby achieving a disruptive switch of "protectant → attack carrier" function at the same location.

[0069] Example 9 Different treatments on intracellular Fe in Huh7 cells 2+ Detection of content and lipid peroxidation level (1) Intracellular Fe 2+ Content detection: The cell treatment process is the same as in Example 8. After treatment, discard the culture medium and wash the cells 2-3 times with PBS. Add Fe... 2+ Serum-free medium containing the specific fluorescent probe was incubated at 37°C in the dark for 30 min. After incubation, the probe was washed three times with PBS to remove free probes, resuspended in PBS, photographed using a fluorescence microscope, and quantitatively analyzed. Results were normalized to 1 for the Control group.

[0070] (2) Detection of lipid peroxidation level: The cell treatment process was the same as in Example 8. After treatment, the cells were washed 2-3 times with PBS, then added serum-free medium containing C11-BODIPY and incubated at 37°C in the dark for 30 min. After incubation, the cells were washed 3 times with PBS, resuspended in PBS, and the changes in the red / green fluorescence ratio in the cells were detected using a confocal microscope. The green / red ratio was used to reflect the degree of lipid peroxidation, and the results were normalized to the Control group.

[0071] Fe in Huh7 cells of different treatment groups 2+ The quantitative results of content and lipid peroxidation level are shown in Table 5.

[0072] Table 5. Effects of different treatments on intracellular Fe 2+ Effects of levels and lipid peroxidation levels

[0073] As shown in Table 5, Fe 2+ Fluorescence signal and C11-BODIPY green / red ratio reflect the accumulation of intracellular free ferrous iron and the degree of lipid peroxidation, respectively. The Fe in the MnFe2O4-ICG@ZIF-8-SS31 group...2+ The signal intensity increased by 3.10 times, and the lipid peroxidation index increased by 2.68 times, both significantly higher than those in the MnFe2O4 group and the MnFe2O4-ICG group. This demonstrates that the present invention not only induced conventional ROS-mediated apoptosis but also significantly activated the ferroptosis pathway. By integrating the functions of the MnFe2O4 nucleus and SS31 targeting mitochondria, the present invention reverses the iron-rich and easily oxidized lipid characteristics of liver cancer cell mitochondria, achieving specific ferroptosis-driven action—a synergistic killing mechanism previously unforeseen in existing technologies.

[0074] Example 10 MnFe2O4 Hemolytic safety evaluation of the ICG@ZIF-8-SS31 nanoplatform Fresh anticoagulated whole blood from mice was collected, and erythrocyte suspensions were prepared using physiological saline according to standard methods. The erythrocyte suspensions were mixed with different concentrations of MnFe2O4-ICG@ZIF-8-SS31 solution and incubated at 37°C for 24 hours. A deionized water treatment group served as a positive control (100% hemolysis), and a physiological saline treatment group served as a negative control (0% hemolysis). After incubation, the supernatant was collected by centrifugation at 4°C, and the absorbance at 540 nm was measured. The hemolysis rate was calculated, and a hemolysis rate bar chart was plotted (e.g., ...). Figure 2 As shown in the figure, the hemolysis rate of the MnFe2O4-ICG@ZIF-8-SS31 nanomaterial remains below 5% even at a concentration as high as 400 μg / mL. This indicates that the nanomaterial has good blood compatibility and is unlikely to cause hemolytic reactions during in vivo application.

[0075] Example 11 Inhibition of hepatocellular carcinoma colony formation ability by MnFe2O4-ICG@ZIF-8-SS31 nanoplatform Cells were seeded at a density of approximately 500-800 cells / well in 6-well plates, with 3 replicates per group. The plates were incubated at 37°C with 5% CO2 for approximately 24 hours to allow for stable cell adhesion. Except for the Control group, the remaining groups were irradiated with an 808nm laser for 5 minutes and then cultured using standard methods. The culture medium was changed every 2-3 days for approximately 10-14 days. Culture was terminated when clearly visible colonies with a diameter greater than 50 cells were observed in the control group. The culture medium was aspirated, and the cells were gently washed 1-2 times with PBS. The cells were then fixed with 4% paraformaldehyde solution at room temperature for 20 minutes, the fixative was discarded, and the cells were washed again with PBS. Subsequently, 0.1% crystal violet staining solution was added, and the cells were stained at room temperature in the dark for 30 minutes. The staining solution was discarded, and the cells were gently rinsed with tap water or distilled water until the background was clear and then air-dried at room temperature. The cell clones in each group were observed and photographed under an optical microscope or scanner, and the number of clones per well was counted (e.g., ...). Figure 4As shown in the figure, compared with the control group, the number of Huh7 cell colonies formed in the MnFe2O4-ICG@ZIF-8-SS31 group was significantly reduced. This indicates that the nanomaterial can effectively inhibit the proliferation and colony formation of liver cancer cells, and has a good anti-tumor effect.

[0076] ICG has been widely used for liver function assessment and navigation of liver resection surgery. After bloodstream injection, it is mainly taken up and excreted by hepatocytes. Due to high metabolism and abnormal iron homeostasis, liver cancer cells contain intracellular H2O2 and free Fe. 2+ Baseline levels are typically higher than in many other tumor types. This invention rationally selects ICG, a liver-affinity photosensitizer, and combines it with MnFe2O4 to utilize the high H2O2 / high Fe environment of liver cancer, forming a dual targeting mechanism of "organ-level (liver) + organelle-level (mitochondria)," specifically for the treatment of liver cancer, rather than general solid tumors.

[0077] This invention selects MnFe2O4 as the core nanozyme, taking advantage of the unique "substrate-rich" environment of liver cancer cells, which more easily triggers the ·OH cascade. In the experiment, Huh7 cells treated with MnFe2O4-ICG@ZIF-8-SS31 showed increased Fe... 2+ The material significantly increased lipid peroxidation levels, while maintaining good survival rates in normal L929 fibroblasts at high concentrations, indicating that the platform exhibits a clear "lesion-priority" killing effect on liver cancer cells, rather than non-specific toxicity to any cell type. Hepatocytes, as metabolic hubs, are far more dependent on mitochondrial oxidative phosphorylation than many low-metabolic tissues. This invention is the first to use SS31 as a mitochondrial-targeting ligand, enabling it to interact with nanozymes. This photosensitizer platform combination shifts from "antioxidant" to "site-specific pro-oxidation." The invention precisely guides MnFe2O4-ICG@ZIF-8 to mitochondria via SS31, inducing local ROS bursts and Ca2+ depletion in liver cancer cells, which are highly dependent on mitochondrial activity. 2+ Overload and persistent mPTP exposure lead to a particularly significant antitumor effect due to the disruption of energy metabolism.

[0078] Compared with existing multifunctional nanomaterial systems, the primary innovation of this invention lies in the construction of a multi-component integrated nanoenzyme structure composed of a MnFe2O4 nanoenzyme core, a photosensitizer ICG, a metal-organic framework ZIF-8 shell, and a mitochondrial targeting peptide SS31 outer layer, under a controllable multi-step assembly process.

[0079] This invention is not a simple one-step template method, but rather achieves precise structural control through the following hierarchical construction path: First, a MnFe2O4 nanozyme core with controllable particle size and stable enzyme activity is prepared via a hydrothermal method; then, under aqueous conditions, ICG is adsorbed onto the MnFe2O4 surface through electrostatic and hydrophobic interactions to form a MnFe2O4-ICG composite core; subsequently, Zn is used in an alcohol solvent... 2 + / 2-Methylimidazolium is self-assembled in situ to form a ZIF-8 core-shell structure on the surface of MnFe2O4-ICG, achieving unified encapsulation and protection of the enzyme catalytic core and photosensitizer; the outermost layer introduces active groups through PDA coating and further carboxylates them, and then the SS31 peptide is covalently fixed on the particle surface through EDC / NHS chemical coupling.

[0080] Through the above step-by-step and controllable construction process, the following were achieved: (1) fine control of the core-shell ratio of MnFe2O4 and ZIF-8, shell thickness, and pore structure; (2) stable loading and leakage suppression of ICG in the core-shell system; and (3) stable grafting of SS31 peptide in the form of chemical bonds, rather than simple physical adsorption, which significantly improves the stability of the target group in the blood circulation. Therefore, this invention forms a repeatable and parameter-controllable construction process in terms of multi-component organic integration, spatial partitioning fixation, and surface chemical stabilization modification.

[0081] Furthermore, the composite nanosystem constructed in this invention can achieve multiple signal recognition and cascade response based on local physicochemical conditions within the liver cancer cell microenvironment. The ZIF-8 shell is sensitive to acidic environments and can gradually disintegrate within cells, releasing the MnFe2O4 core and the photosensitive unit ICG. Near-infrared light can excite the ICG to generate a local photothermal effect, thereby promoting the enzyme-like catalytic activity of MnFe2O4 and further enhancing the reaction efficiency. The system exhibits a linked characteristic of acidity triggering, light regulation, and oxidation reaction amplification within cells, enabling spatiotemporally controllable activation of the reaction. This multi-factor response mode maintains stable output under different cellular conditions, significantly improving the selectivity and safety of the system's function.

[0082] Furthermore, this invention fully utilizes the unique mitochondrial targeting effect of the SS31 peptide to promote the efficient accumulation of the nanozyme system in the mitochondrial region of liver cancer cells. Simultaneously, the MnFe2O4 nanozyme released by the system continuously catalyzes the generation of hydroxyl radicals in the tumor environment, synergistically inducing mitochondrial ferroptosis in conjunction with ICG-mediated NIR photoactivated ROS production. Site-specific mitochondrial damage can trigger calcium ion overload and membrane permeability transition pore (mPTP) opening, leading to irreversible apoptosis and even necrosis. This "photo-enzyme-ferroptosis" triple cascade system overcomes the limitations of traditional single-pathway anti-tumor treatments, effectively preventing drug resistance and tumor escape. Compared to chemotherapy or single-target therapies, this technology enables precise destruction of tumor cells through multiple pathways and at multiple nodes, minimizing damage to normal tissues, greatly improving treatment selectivity and safety margins, and providing an innovative and effective ideal solution for refractory liver cancer.

[0083] Finally, this invention employs biocompatible ZIF-8, MnFe2O4, and ICG as the main functional units. The overall material exhibits good stability under physiological conditions and can be gradually degraded and metabolized, reducing the risk of residual organic solvents and potential toxicity from free metal ions. Except for the conventional hydrothermal synthesis of the MnFe2O4 core, all other steps in the preparation process are completed under mild conditions. The process is simple, requiring no strong reducing agents or extreme reaction conditions, and can achieve the integrated construction of multiple components, demonstrating good process scale-up and batch reproducibility. The resulting nanosystem exhibits good dispersibility in aqueous phase or PBS, with well-defined storage conditions, and demonstrates good stability and safety both in vitro and in vivo.

[0084] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0085] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a mitochondrial-targeted triple-response nanoplatform, characterized in that, Includes the following steps: Preparation of nanonuclei: Dissolve iron and manganese salts, add sodium acetate, stir, adjust pH, perform hydrothermal reaction, cool, separate the precipitate, wash, and vacuum dry to obtain the nanonuclei; Nanonuclei loaded with photosensitizer: Dissolve the photosensitizer, sonicate, add photosensitizer solution to the nanonuclei dispersion, add buffer solution, incubate in the dark, centrifuge to collect the precipitate, wash, and resuspend to obtain nanonuclei loaded with photosensitizer; the photosensitizer includes indocyanine green; Preparation of core-shell nanoparticles: The photosensitizer-loaded nanocore dispersion was mixed with methanol, sonicated, stirred and injected with zinc nitrate solution, stirred, 2-methylimidazolium methanol solution was added, stirring was continued, the precipitate was collected by centrifugation, washed and vacuum dried to obtain the product; Mitochondrial-targeting peptide modification: A core-shell nanoparticle dispersion was taken, buffer solution was added, and the mixture was sonicated. Dopamine hydrochloride was added, and the mixture was stirred, centrifuged to collect the precipitate, washed, and resuspended to obtain dopamine-coated core-shell nanoparticles. The resuspended dopamine-coated core-shell nanoparticles were then added to a mixed solvent of dimethyl sulfoxide and water, followed by the addition of succinic anhydride. The mixture was stirred at room temperature, centrifuged, washed, and resuspended to obtain carboxyl-rich dopamine-coated core-shell nanoparticles. The resuspended carboxyl-rich dopamine-coated core-shell nanoparticles were then added to a buffer solution, followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-succinimide ester. The mixture was shaken, pH adjusted, and mitochondrial-targeting peptide solution was added dropwise. The mixture was reacted in the dark, centrifuged to collect the precipitate, washed, and resuspended to obtain a mitochondrial-targeting triple-response nanoplatform. The mitochondrial-targeting peptide included SS31.

2. The preparation method according to claim 1, characterized in that, The iron salt includes ferric chloride, and the manganese salt includes manganese chloride.

3. The preparation method according to claim 1, characterized in that, In the mixed solvent of dimethyl sulfoxide and water, the volume ratio of dimethyl sulfoxide to water is (3~5):

1.

4. The preparation method according to claim 1, characterized in that, The preparation of the nanonucleus includes the following steps: Iron and manganese salts are dissolved in water, followed by the addition of sodium acetate. The mixture is stirred and the pH is adjusted to 12-14. A hydrothermal reaction is then carried out, followed by cooling, separation of the precipitate, washing, and vacuum drying to obtain the final product. According to the molar ratio, the iron salt: the manganese salt is (1~3):

1.

5. The preparation method according to claim 1, characterized in that, The nano-core loaded photosensitizer includes the following steps: Dissolve the photosensitizer in water, sonicate, add photosensitizer solution to the nanonucleus dispersion, add 4-hydroxyethylpiperazine ethanesulfonic acid buffer, incubate in the dark, centrifuge to collect the precipitate, wash, and resuspend to obtain photosensitizer-loaded nanonuclei; According to the mass ratio, the nanonucleus:the photosensitizer is (7~9):

1.

6. The preparation method according to claim 1, characterized in that, The preparation of the core-shell nanoparticles includes the following steps: Mix the photosensitizer-loaded nanonucleus dispersion with methanol, sonicate, inject a zinc nitrate methanol solution under stirring, stir, then add a 2-methylimidazole methanol solution, continue stirring for 1-3 hours, centrifuge to collect the precipitate, wash, and vacuum dry to obtain the final product. According to the dosage ratio, the photosensitizer-loaded nanonucleus: zinc nitrate: 2-methylimidazole is (3~5) mg: (0.4~0.6) mmol: (7~9) mmol.

7. The preparation method according to claim 1, characterized in that, The mitochondrial-targeting peptide modification includes the following steps: Take the core-shell nanoparticle dispersion, add tris(hydroxymethyl)aminomethane hydrochloride buffer, sonicate, add dopamine hydrochloride, stir for 2-4 h, centrifuge to collect the precipitate, wash, and resuspend to obtain dopamine-coated core-shell nanoparticles; take the dopamine-coated core-shell nanoparticle resuspension, add a mixed solvent of dimethyl sulfoxide and water, then add succinic anhydride, stir at room temperature for 3-5 h, centrifuge, wash, and resuspend to obtain carboxyl-rich dopamine-coated core-shell nanoparticles; take the carboxyl-rich dopamine-coated core-shell nanoparticle resuspension, add 2-morpholine ethanesulfonic acid buffer, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-succinimide ester, shake, adjust pH to 7.0-7.4, dropwise add mitochondrial-targeting peptide solution, react in the dark for 10-14 h, centrifuge to collect the product, wash, and resuspend to obtain mitochondrial-targeting triple-response nanoplatform; According to the mass ratio, the core-shell nanoparticles: the dopamine hydrochloride is 1:(2~3); According to the mass ratio, the dopamine-coated core-shell nanoparticles: succinic anhydride is 1: (12~13). According to the mass ratio, the core-shell nanoparticles with carboxyl-rich dopamine coating: the mitochondrial-targeting peptide is (1~3):

1.

8. The mitochondrial-targeted triple-response nanoplatform obtained by the preparation method according to any one of claims 1-7.

9. The application of the mitochondrial-targeted triple-response nanoplatform obtained by the preparation method according to any one of claims 1-7 or the mitochondrial-targeted triple-response nanoplatform according to claim 8 in the preparation of drugs for treating liver cancer.

10. A drug for treating liver cancer, characterized in that, The mitochondrial-targeted triple-response nanoplatform obtained by the preparation method according to any one of claims 1-7 or the mitochondrial-targeted triple-response nanoplatform according to claim 8.

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