Mitochondria-targeted metal immune drug as well as preparation method and application thereof

Abstract

The invention constructs a mitochondrial targeting metal immune drug (Mito-chaos), and the Mito-chaos is used for tumor treatment and wound repair. The core structure of the drug is Ca < 2 + > and Cu < 2 + > co-doped hollow Prussian blue nanoparticles (Ca / Cu-PB). The particle has a remarkable near-infrared (NIR) light response characteristic, can respond to 1064nm near-infrared light, and generates an efficient photothermal conversion effect. And furthermore, the Mito-chaos with the mitochondrial targeting capability is obtained by loading TPP modified Mito Cur in the pores of the Ca / Cu-PB nano particles, so that the Mito-chaos with the mitochondrial targeting capability is obtained. The Mito-chaos can be slowly degraded in a tumor acidic microenvironment (TME), releases Ca < 2 + > and Cu < 2 + > and delivers the Ca < 2 + > and the Cu < 2 + > into tumor cell mitochondria, induces homeostasis destruction of ions in the mitochondria, activates a tumor cell copper death pathway and plays an efficient tumor cell killing role. And meanwhile, the sustained-release Ca < 2 + > and Cu < 2 + > can play roles in promoting angiogenesis, promoting collagen deposition and improving tissue regeneration capacity, the postoperative wound healing speed is accelerated, and good tissue repair performance and clinical application potential of the medicine are embodied.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a mitochondrial-targeted metal immunodrug, a preparation method thereof, and an application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Melanoma is a highly invasive and metastatic skin malignancy, and surgical resection is the main treatment method. However, its local recurrence and distant metastasis rates are high, which seriously affects the patient's prognosis. Traditional treatments such as chemotherapy and radiotherapy have problems such as strong toxic side effects and poor selectivity, and cannot simultaneously take into account postoperative wound repair. They ignore the need for postoperative wound healing and cannot promote the regeneration and repair of damaged tissues.

[0004] Copper apoptosis, a newly emerging form of programmed cell death, is considered a promising new form of cancer therapy. Its mechanism is that intracellular copper ions, through the combined effects of specific molecules (such as DLATs) and the loss of iron-sulfur cluster proteins, induce cell death. However, tumor cells have a series of homeostatic regulatory mechanisms for copper ions (such as copper ion pumping by ATP7B, GSH chelation, and HSP protein stabilization), resulting in inefficient copper apoptosis induction and limiting its therapeutic efficacy.

[0005] Prussian blue (PB) is an inorganic material that has been approved by the FDA for the treatment of metal poisoning and has good biocompatibility. Hollow structured Prussian blue nanoparticles have the characteristics of large specific surface area and rich internal channels, which are very suitable for loading and delivering drugs and ions. By element doping, PB nanoparticles can be given a variety of functions, such as using Cu 2+ 、Zn 2+ 、Ga 3+ 、Mn 2+ Replacing some of the iron with other metals can impart properties such as chemical catalysis, metabolic regulation, or magnetic resonance imaging. Furthermore, Prussian blue nanoparticles undergo structural decomposition in the weakly acidic microenvironment of tumors, selectively releasing the metal ions and drugs they carry, thereby improving release efficiency at the tumor site and reducing nonspecific leakage. In terms of optical properties, PB nanoparticles exhibit excellent photothermal conversion efficiency for near-infrared light and possess various enzyme-mimicking activities, such as peroxidase. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the purpose of the present invention is to develop a mitochondrial-targeted dual-ion interference metal immunodrug that combines postoperative anti-tumor recurrence and accelerated tissue healing, providing a new solution that is efficient, safe and has transformational potential for the comprehensive postoperative treatment of melanoma.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a mitochondrial-targeted metal immunotherapy drug, comprising Ca 2+ and Cu 2+ Co-doped hollow Prussian blue nanoparticles, and a mitochondrial targeting ligand loaded on the nanoparticles.

[0009] Preferably, the mitochondrial targeting ligand is a triphenylphosphine (TPP)-modified curcumin derivative MitoCur.

[0010] In a second aspect, the present invention further provides a method for preparing the mitochondrial-targeted metal immunodrug as described above, comprising the following steps:

[0011] S1. Mix CaCl2·2H2O, K3[Fe(CN)6]·3H2O, and PVP in 0.01M hydrochloric acid, stir to form a uniform solution, and then perform solvent thermal reaction for 24 hours. Collect the product and etch it with 1M hydrochloric acid to form CaCl2·2H2O. 2+ Doped hollow structured Prussian blue nanoparticles Ca-PB;

[0012] S2. Disperse Ca-PB in deionized water, add copper source, chelating agent Na3C6H5O7·2H2O and PVP, stir and react for 3 hours, then add K3[Fe(CN)6]·3H2O and continue stirring for 3 hours. The resulting mixture is allowed to stand at room temperature for 24 hours, and then centrifuged and washed to obtain Ca 2+ and Cu 2+ Co-doped hollow Prussian blue nanoparticles Ca / Cu-PB;

[0013] S3. Dissolve the mitochondrial targeting ligand in dimethyl sulfoxide (DMSO) and stir to form a homogeneous solution. Then add the hollow Prussian blue nanoparticles Ca / Cu-PB obtained in step S2 and continue stirring at room temperature for 8-12 hours to achieve physical adsorption loading of the drug molecules. The mixed solution is centrifuged and washed to remove the unloaded free mitochondrial targeting ligand, and finally obtain the mitochondrial-targeted metal immunodrug.

[0014] Preferably, the mitochondrial targeting ligand is MitoCur, a curcumin derivative modified with triphenylphosphine (TPP).

[0015] Preferably, the copper source is any one of Cu(CH3COO)2, CuSO4, Cu(NO3)2 or other water-soluble copper salts. More preferably, the copper source is Cu(CH3COO)2.

[0016] In a third aspect, the present invention further provides a use of the mitochondria-targeted metal immunodrug as described above in the preparation of tumor therapeutic drugs.

[0017] Furthermore, the drug further comprises a pharmaceutically acceptable excipient, wherein the pharmaceutical excipient is a pharmaceutically acceptable salt, excipient or carrier, and the carrier is any one or more of a filler, a wetting agent, a binder, a disintegrant or a lubricant.

[0018] Preferably, the pharmaceutical excipient is a natural hydrogel, and the metal immunodrug is coated in the natural hydrogel to prepare an injectable hydrogel.

[0019] According to the above disclosure, this application has the following beneficial technical effects:

[0020] 1) Precise mitochondrial delivery: Triphenylphosphine-modified curcumin derivatives are used to precisely deliver copper and calcium ions into mitochondria, causing ion imbalance at the subcellular level, effectively weakening the anti-stress defense mechanism of tumor cells and enhancing sensitivity to copper toxicity.

[0021] 2) Dual ion synergy: The synergistic interference of copper ions and calcium ions produces a superimposed effect. Calcium ion overload reduces mitochondrial ATP synthesis, inhibits the function of ATP-dependent copper efflux protein (ATP7B) and heat shock protein (HSP), making it difficult for tumor cells to eliminate excess copper ions; at the same time, excess copper ions induce the aggregation of fatty acylated proteins and the loss of iron-sulfur cluster proteins in a low HSP environment, amplifying the copper death effect from two aspects.

[0022] 3) Photothermal and nanozyme combined therapy: This metal immunotherapy drug exhibits excellent NIR-II photothermal conversion capabilities, allowing it to function even under mild photothermal conditions. The photothermal effect directly damages tumor cells while also increasing the peroxidase- and glutathione peroxidase-like activities of the nanoparticles, accelerating the production of reactive oxygen species (·OH) and depleting glutathione, thereby further increasing oxidative stress within tumor cells and synergistically inducing cell death.

[0023] 4) Inducing Immune Activation: This invention induces immunogenic cell death in tumor cells, prompting them to release DAMPs signals, effectively promoting the immune system to recognize and eliminate residual tumors. Experiments in mice showed that treatment with this invention significantly increased the infiltration of cytotoxic T lymphocytes in tumor tissues, and that tumor-associated macrophages polarized toward the anti-tumor M1 type, demonstrating that this invention can effectively reshape the tumor immune microenvironment and reduce the risk of tumor recurrence and metastasis.

[0024] 5) Promote wound regeneration: The Ca released by the present invention 2+ Helps to stop bleeding quickly and provide a scaffold for healing, Cu 2+ This stimulates angiogenesis and collagen matrix reconstruction, which together accelerate postoperative wound healing. Compared with the control, the wound closure time of the treatment group was significantly shortened, the number of new skin appendages (hair follicles) increased, and the collagen deposition was more mature and dense, indicating a higher quality of healing.

[0025] 6) Excellent biocompatibility and safety: The Prussian blue carrier is FDA-approved for safety and gradually degrades in the biological environment. The components of the nanoparticles undergo controlled metabolism in the body. Furthermore, through localized hydrogel administration and triggered release in the slightly acidic tumor environment, this invention achieves controlled, targeted therapeutic action, minimizing damage to surrounding healthy tissue.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, some of the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Schematic diagram of the synthesis mechanism of Mito-chaos shown in Example 1 of the present invention.

[0029] Figure 2 This is a transmission electron microscope image of Mito-chaos shown in Example 1 of the present invention, showing its morphology and structural characteristics.

[0030] Figure 3 This is the ultraviolet-visible (UV-Vis) absorption spectrum of Mito-chaos shown in Example 1 of the present invention.

[0031] Figure 4 This is a photothermal conversion performance test of Mito-chaos shown in Example 2 of the present invention under 1064nm near-infrared laser irradiation.

[0032] Figure 5 This is an in vitro evaluation of the anti-tumor activity of Mito-chaos on melanoma cells as shown in Example 3 of the present invention.

[0033] Figure 6 These are the in vitro experimental results of Mito-chaos-induced calcium overload in tumor cell mitochondria as shown in Example 3 of the present invention.

[0034] Figure 7 This is the in vitro oxidative stress inducing effect of Mito-chaos on tumor cells as shown in Example 3 of the present invention.

[0035] Figure 8 This is the in vitro copper-induced cell death promoting effect of Mito-chaos shown in Example 3 of the present invention.

[0036] Figure 9 This is a physical picture of the Mito-chaos loaded hydrogel shown in Example 4 of the present invention.

[0037] Figure 10 This is the in vivo tumor recurrence inhibition effect of Mito-chaos shown in Example 4 of the present invention.

[0038] Figure 11 Mito-chaos induced ICD and stimulated immune response as shown in Example 5 of the present invention.

[0039] Figure 12 This is the skin healing promotion of Mito-chaos shown in Example 6 of the present invention. DETAILED DESCRIPTION

[0040] In order to better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are conventional methods unless otherwise specified.

[0041] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field; the reagents or materials described are all from commercial channels unless otherwise specified.

[0042] The following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. The following examples are intended only to explain the present invention and are not intended to limit its contents. If the specific experimental conditions are not specified in the examples, they are generally in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] The present invention constructs a mitochondrial-targeted metal immunodrug, including Ca 2+ and Cu 2+Co-doped hollow Prussian blue nanoparticles and mitochondrial targeting ligands loaded on the nanoparticles. The metal immunodrug can accurately deliver Cu 2+ With Ca 2+ It enters the mitochondria of tumor cells, destroying their ion homeostasis, thereby amplifying the copper death effect and efficiently inducing programmed cell death of residual melanoma cells. Combining near-infrared photothermal and nanozyme catalytic mechanisms, it enhances the level of oxidative stress in tumor cells, synergizes the copper death pathway, and further improves the killing efficiency of tumor cells. At the same time, it stimulates anti-tumor immune response, promotes dendritic cell maturation and antigen presentation by inducing immunogenic cell death (ICD), and activates T cell-mediated immune clearance, thereby reducing the risk of postoperative tumor recurrence and metastasis. Finally, through the sustained release of Ca 2+ and Cu 2+ Ions promote tissue repair, achieve synergistic activation of hemostasis, angiogenesis, collagen deposition and other processes, accelerate postoperative wound healing and improve regeneration quality.

[0044] Preferably, the mitochondrial targeting ligand is a triphenylphosphine (TPP)-modified curcumin derivative MitoCur.

[0045] The preparation method of the mitochondria-targeted metal immunodrug comprises the following steps:

[0046] S1. Mix CaCl2·2H2O, K3[Fe(CN)6]·3H2O, and PVP in 0.01M hydrochloric acid, stir to form a uniform solution, and then perform solvent thermal reaction for 24 hours. Collect the product and etch it with 1M hydrochloric acid to form CaCl2·2H2O. 2+ Doped hollow structured Prussian blue nanoparticles Ca-PB;

[0047] S2. Disperse Ca-PB in deionized water, add copper source, chelating agent Na3C6H5O7·2H2O and PVP, stir and react for 3 hours, then add K3[Fe(CN)6]·3H2O and continue stirring for 3 hours. The resulting mixture is allowed to stand at room temperature for 24 hours, and then centrifuged and washed to obtain Ca 2+ and Cu 2+ Co-doped hollow Prussian blue nanoparticles Ca / Cu-PB;

[0048] S3. Dissolve the mitochondrial targeting ligand in dimethyl sulfoxide (DMSO) and stir to form a homogeneous solution. Then add the hollow Prussian blue nanoparticles Ca / Cu-PB obtained in step S2 and continue stirring at room temperature for 8-12 hours to achieve physical adsorption loading of the drug molecules. The mixed solution is centrifuged and washed to remove the unloaded free mitochondrial targeting ligand, and finally obtain the mitochondrial-targeted metal immunodrug.

[0049] Preferably, the mitochondrial targeting ligand is MitoCur, a curcumin derivative modified with triphenylphosphine (TPP).

[0050] Preferably, the copper source is any one of Cu(CH3COO)2, CuSO4, Cu(NO3)2 or other water-soluble copper salts. More preferably, the copper source is Cu(CH3COO)2.

[0051] The mitochondrial-targeted metalloimmunotherapy drug can be used to prepare a tumor treatment drug. The drug also includes a pharmaceutically acceptable excipient, which is a pharmaceutically acceptable salt, excipient, or carrier, and the carrier is any one or more of a filler, wetting agent, binder, disintegrant, or lubricant.

[0052] Preferably, the pharmaceutical excipient is a natural hydrogel, and the metal immunodrug is coated in the natural hydrogel to prepare an injectable hydrogel.

[0053] The mechanism of action of the above-mentioned metal immunotherapy drugs and related drugs is as follows:

[0054] 1) Delivery mechanism: Metal immunotherapy drugs can gradually degrade in response to the acidic microenvironment of tumors, releasing Cu 2+ and Ca 2+ At the same time, MitoCur efficiently targets and delivers ions to the mitochondria of tumor cells through the mediation of the TPP group, inducing an imbalance in mitochondrial ion homeostasis.

[0055] 2) Cell killing mechanism:

[0056] Copper ions are converted into Cu under the catalysis of enzymes such as FDX1 + , inducing abnormal aggregation of DLAT proteins and loss of Fe–S proteins in mitochondria, activating the copper death pathway;

[0057] Calcium ion overload in mitochondria leads to ATP production disorders, decreased membrane potential, and impaired function of anti-copper stress pathways (such as ATP7B copper pump and HSP70 / 90), thereby further amplifying copper toxicity.

[0058] Metal immunodrugs have good photothermal conversion capabilities. They generate heat and enhance nanozyme activity (such as POD and GPx-like activity) under near-infrared laser irradiation, promote the generation of reactive oxygen species and glutathione consumption, and further synergistically induce cell death.

[0059] 3) Immune activation function: During the treatment process, tumor cells undergo immunogenic cell death (ICD), which is manifested by calreticulin (CRT) membrane eversion and high mobility group protein B1 (HMGB1) release, promoting dendritic cell maturation, T cell activation, and stimulating systemic anti-tumor immune response.

[0060] 4) Wound repair promotion: drug-sustaining Ca 2+ Promotes coagulation and cell migration, Cu 2+ Induces angiogenesis and collagen maturation, thereby significantly accelerating postoperative wound healing and improving the quality of regenerated tissue.

[0061] 5) Administration form: The above-mentioned metal immunodrugs can be locally administered by being embedded in an injectable natural hydrogel system, achieving sustained release in the surgical area, controlled light triggering, low systemic toxicity and high tissue compatibility.

[0062] The present application will be further described below with reference to specific embodiments:

[0063] Example 1 Construction and Characterization of Mito-chaos Metal Immune Drugs

[0064] The present invention adopts calcium / copper co-doped Prussian blue (Ca / Cu-PB) as nanoparticles and loads a triphenylphosphine-modified curcumin derivative MitoCur to construct a mitochondrial-targeted dual-ion interference metal immunodrug Mito-chaos.

[0065] like Figure 1 As shown, the synthesis of Mito-chaos includes the following steps:

[0066] Step 1: Preparation of calcium-doped hollow Prussian blue nanoparticles (Ca-PB): 3.0 g of PVP and 131.72 mg of K3[Fe(CN)6]·3H2O were dissolved in 30 mL of 0.01 M hydrochloric acid solution and mixed thoroughly under magnetic stirring to form a transparent solution A. Separately, 1.0 g of PVP and 14.7 mg of CaCl2·2H2O were added to 10 mL of 0.01 M hydrochloric acid and stirred to form a solution B. Solution A was then slowly added dropwise to solution B and stirred at room temperature for 1 hour. The resulting mixture was placed in an 80°C oven for 24 hours. After completion of the reaction, the product was collected by centrifugation (10,000 rpm, 10 minutes), washed three times with deionized water, and dried at 25°C for 24 hours. Twenty mg of the resulting product and 100 mg of PVP were added to 20 mL of 1 M HCl and stirred at room temperature for 4 hours. The mixture was then transferred to a stainless steel reactor and reacted at 140°C for 4 hours to form Ca-PB nanoparticles with a hollow mesoporous structure.

[0067] Step 2, copper ion doping: 50 mg of the above-mentioned Ca-PB was dispersed in 20 mL of deionized water. 22 mg of Cu(CH3COO)2·H2O, 67.5 mg of Na3C6H5O7·2H2O, and 250 mg of PVP were added, and magnetic stirring was performed at room temperature for 3 hours. Subsequently, 66 mg of K3[Fe(CN)6]·3H2O was added, and stirring was continued for 3 hours. The reaction solution was aged at 25°C for 24 hours. The resulting product was collected by centrifugation and washed three times with deionized water to obtain Ca / Cu co-doped hollow Prussian blue nanoparticles (Ca / Cu-PB).

[0068] Step 3, MitoCur loading: Dissolve 5 mg of MitoCur in 2 mL of DMSO and stir magnetically for 30 minutes to form a homogeneous solution. Add 10 mg of Ca / Cu-PB and continue stirring for 12 hours to allow the MitoCur molecules to be physically adsorbed onto the surface and pores of the Ca / Cu-PB. After loading, collect the product by centrifugation (7500 rpm, 20 minutes) and wash it three times with alternating ethanol and deionized water to obtain Ca / Cu-PB (also known as Mito-chaos).

[0069] Figure 2 Transmission electron microscopy image shows that Mito-chaos has a regular nanocubic structure and obvious hollow features. Figure 3 Ultraviolet-visible spectrum analysis shows that the material has good absorption capacity in the near-infrared band.

[0070] Example 2 Photothermal performance of Mito-chaos

[0071] like Figure 4 As shown in the figure, Mito-chaos has excellent photothermal response performance. The experimental results show that Mito-chaos has excellent photothermal response performance under different concentrations (12.5, 25, 50, 100 μg·mL-1). -2 After 10 minutes of laser irradiation, the solution temperature rose to 41°C, 46°C, 53°C and 69°C, respectively, showing an obvious concentration-dependent photothermal effect ( Figure 4 A, B). In addition, at a fixed concentration (50 μg·mL-1), as the laser power density gradually increased from 1.0 to 2.5 W·cm -2 , the temperature continues to rise, showing a good power density dependence ( Figure 4 C). Further studies have shown that Mito-chaos maintains stable photothermal conversion capability after five consecutive heating-cooling cycles, demonstrating excellent photothermal stability ( Figure 4 D).

[0072] Example 3 In vitro anti-tumor activity and mechanism verification of Mito-chaos

[0073] like Figure 5 As shown in the in vitro evaluation of the anti-tumor activity of Mito-chaos, first, the cell activity after different treatments was evaluated by Calcein-AM / PI double staining. Among them, Calcein-AM marks living cells (green fluorescence) and PI marks dead cells (red fluorescence). The results showed that under 50μg / mL Mito-chaos and 1.5W / cm 2 After 5 minutes of combined exposure to a high-intensity 1064nm NIR laser, the cell samples displayed extensive red fluorescence and minimal green fluorescence. Annexin V-FITC / PI flow cytometry was further used to quantify the apoptosis rate in each group. The results showed that Mito-chaos combined with NIR treatment induced an apoptosis rate of up to 82.0% in B16 cells, significantly superior to that of the other groups. The apoptosis rate in the control group was 2.78%; in the NIR treatment alone, it was 8.68%; in the Cu-PBC group, it was 19.15%; in the Cu-PBC + NIR group, it was 63.6%; and in the Mito-chaos treatment alone, it was 44.9%. These results demonstrate that Mito-chaos possesses significant anti-tumor activity under near-infrared laser excitation.

[0074] To further elucidate the anti-tumor mechanism of Mito-chaos, the intracellular calcium ion homeostasis imbalance and oxidative stress levels induced by it were analyzed. B16 cells were labeled with intracytoplasmic and intramitochondrial calcium ion fluorescent probes, respectively. The results showed that after treatment with 50 μg / mL Mito-chaos, the calcium ion fluorescence intensity of the entire cell and the mitochondrial region was significantly increased. The calcium ion content was further quantitatively analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the total calcium content and mitochondrial calcium content of cells treated with Mito-chaos increased to about 4 times that of the control group, indicating that Mito-chaos can induce significant calcium overload in tumor cells ( Figure 6 At the same time, the oxidative stress levels of the treated tumor cells were evaluated by DCFH-DA (detection of intracellular ROS) and MitoSOX Red (detection of mitochondrial ROS) probes. The results showed that compared with the treatment with the same concentration of Cu-PBC (50 μg / mL), Mito-chaos treatment could induce a higher intensity of oxidative stress level ( Figure 7 ).

[0075] The effect of Mito-chaos in inducing copper death in B16 tumor cells was further evaluated. First, the treated B16 cells were stained and observed using RBH probes (copper ion probes, red fluorescence) and Mitotracker Green (mitochondrial probes, green fluorescence). The results showed that after Mito-chaos treatment, intracellular copper ion fluorescence and mitochondrial fluorescence were highly co-localized, suggesting that Mito-chaos can significantly promote the aggregation of intracellular copper ions to mitochondria, resulting in a significant increase in the concentration of copper ions in mitochondria. Further Western Blot protein expression analysis showed that under the conditions of Mito-chaos combined with NIR irradiation, the expression level of intracellular iron-sulfur cluster proteins (Fe-S cluster proteins) was significantly reduced or even lost. This confirms that Mito-chaos effectively activates cell copper death ( Figure 8 ).

[0076] Example 4 In vivo anti-tumor effect of Mito-chaos in a postoperative mouse model

[0077] To achieve local controlled release and postoperative treatment, the Mito-chaos of the present invention can be loaded into sodium alginate hydrogel. It can be applied to the wound surface after surgery to achieve the multiple functions of local drug delivery and photothermal therapy ( Figure 9 ).

[0078] When the volume of subcutaneous B16 melanoma in C57BL / 6 mice reaches about 100 mm 3 Afterwards, about 95% of the tumor tissue was surgically removed to establish a postoperative residual tumor model. Subsequently, 30 C57BL / 6 mice were randomly divided into the following 6 groups (5 mice in each group): control group (Control), near-infrared irradiation group (NIR) alone, Cu-PBC group, Cu-PBC combined with near-infrared irradiation group (Cu-PBC+NIR), Mito-chaos group, Mito-chaos combined with near-infrared irradiation group (Ca / Cu-PBC+NIR). For groups 3 to 6, a hydrogel preparation containing Cu-PBC or Mito-chaos nanoparticles was locally applied to the tumor resection site immediately after surgery. For the NIR irradiation group, 1064nm near-infrared laser irradiation (power density 1.5W / cm 2 The mice were continuously monitored for tumor growth during the experiment and observed for 14 consecutive days. The results showed that the growth of residual tumors in mice treated with Mito-chao combined with NIR irradiation was significantly inhibited, and the tumor volume was significantly reduced; in some mice, the tumors completely disappeared. In contrast, the other treatment groups did not show a significant tumor growth inhibition effect ( Figure 10 ).

[0079] Example 5 Mito-chaos induces immunogenic cell death

[0080] To further elucidate the regulatory effects of Mito-chaos combined with NIR therapy on immune cell infiltration and immune status in the tumor microenvironment (TME), flow cytometry was used to analyze the composition and phenotypic changes of immune cells in tumor tissue at the treatment endpoint. The method is as follows:

[0081] Tumor tissues of mice in each group were collected at the end of treatment, mechanically minced, and prepared into single-cell suspensions. Fluorescently labeled antibodies were used to label the M1 and M2 phenotypes of CD8+ T cells and tumor-associated macrophages (TAMs), respectively. The M1 macrophage phenotype marker was F4 / 80+CD86+CD206-; the M2 macrophage phenotype marker was F4 / 80+CD86-CD206+. The experimental results showed that Mito-chaos combined with NIR irradiation treatment significantly increased the infiltration rate of CD8+ T cells in tumor tissues, reaching 51.6%, which was 1.34 times higher than that of the Cu-PBC combined with NIR group ( Figure 11 A). In addition, further analysis of the phenotypic transformation of tumor-associated macrophages revealed that Mito-chaos combined with NIR irradiation can significantly promote the transformation of macrophages in tumor tissue from M2 to M1. Compared with other treatment groups, Mito-chaos+NIR treatment significantly increased the proportion of M1 macrophages and effectively suppressed the proportion of M2 macrophages. The M1 / M2 ratio in tumor tissue in the Mito-chaos combined with NIR group was approximately 2.1 times higher than that in the Cu-PBC combined with NIR group, suggesting that it can effectively regulate the tumor immune microenvironment, achieve efficient photothermal-immunotherapy, and achieve the effect of inhibiting tumor recurrence ( Figure 11 B,C).

[0082] Example 6 Mito-chaos promotes skin tissue repair

[0083] To further evaluate the effect of Mito-chaos of the present invention in promoting wound healing in vivo, a full-thickness circular skin defect wound model was created on the back of C57BL / 6 mice, and the mice were randomly divided into the following treatment groups: control group (Control), simple hydrogel treatment group (Hydrogel), Cu-PBC nanoparticle hydrogel group (Cu-PBC), Cu-PBC nanoparticle hydrogel combined with near-infrared irradiation group (Cu-PBC+NIR), Ca / Cu-PBC nanoparticle hydrogel group (Ca / Cu-PBC), and Ca / Cu-PBC nanoparticle hydrogel combined with near-infrared irradiation group (Ca / Cu-PBC+NIR). Immediately after surgery, the corresponding hydrogel was applied to the wound in each group, and the near-infrared light irradiation conditions were 1064nm, 1.5W / cm2 , irradiated for 10 minutes. The wounds were observed continuously for 12 days, and the size and morphology of the wounds were recorded and photographed at regular intervals. The results showed that the wounds coated with Cu-PBC and Ca / Cu-PBC nanoparticles healed at a significantly higher rate than the control group and the simple hydrogel group. Especially on the 12th day after surgery, the wound areas of the control group and the simple hydrogel group still remained at approximately 10.3% and 12.9%, respectively; while the wounds in the Cu-PBC combined with NIR group and the Ca / Cu-PBC combined with NIR group were almost completely healed. Further histological analysis results showed that on the 12th day, the new epidermis of the wounds in the Ca / Cu-PBC and Ca / Cu-PBC combined with NIR groups were intact, tightly connected, and significantly thicker. It is worth noting that compared with the control group and the simple hydrogel group, the hair follicle structure and collagen deposition in the wounds of the Cu-PBC, Cu-PBC combined with NIR, Ca / Cu-PBC, and Ca / Cu-PBC combined with NIR groups were significantly increased, suggesting that the Ca / Cu-PBC of the present invention has good wound healing and tissue repair capabilities ( Figure 12 ).

[0084] In summary, the present invention has constructed a mitochondrial-targeted metal immunodrug (Mito-chaos) for tumor treatment and wound repair. The core structure of the metal immunodrug is Ca 2+ and Cu 2+ Co-doped hollow Prussian blue (Ca / Cu-PB) nanoparticles. The particles have significant near-infrared (NIR) light response characteristics and can respond to 1064nm near-infrared light to produce efficient photothermal conversion effects. Furthermore, by loading the mitochondrial targeting ligand MitoCur modified with triphenylphosphine (TPP) into the pores of the Ca / Cu-PB nanoparticles, a Mito-chaos metal immunodrug with mitochondrial targeting ability is obtained. Mito-chaos can slowly degrade in the acidic microenvironment (TME) of the tumor, releasing Ca 2+ and Cu 2+ It is delivered to the mitochondria of tumor cells, inducing the destruction of ion homeostasis in mitochondria, significantly activating the copper death (cuproptosis) pathway of tumor cells, and exerting a highly effective tumor cell killing effect. 2+ and Cu 2+ It can significantly promote angiogenesis, promote collagen deposition and improve tissue regeneration ability, accelerate postoperative wound healing, and reflect the drug's good tissue repair performance and clinical application potential.

[0085] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0086] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A mitochondrial-targeted metal immunodrug, characterized in that: Including Ca 2+ and Cu 2+ Co-doped hollow Prussian blue nanoparticles, and a mitochondrial targeting ligand loaded on the nanoparticles.

2. The mitochondrial-targeted metal immunodrug according to claim 1, characterized in that The mitochondrial targeting ligand is a triphenylphosphine-modified curcumin derivative MitoCur.

3. A method for preparing a mitochondrial-targeted metal immunodrug according to any one of claims 1 to 2, characterized in that: The steps include: S1. Mix CaCl2·2H2O, K3[Fe(CN)6]·3H2O, and PVP in 0.01M hydrochloric acid, stir to form a uniform solution, and then perform solvent thermal reaction for 24 hours. Collect the product and etch it with 1M hydrochloric acid to form CaCl2·2H2O. 2+ Doped hollow structured Prussian blue nanoparticles Ca-PB; S2. Disperse Ca-PB in deionized water, add copper source, chelating agent Na3C6H5O7·2H2O and PVP, stir and react for 3 hours, then add K3[Fe(CN)6]·3H2O and continue stirring for 3 hours. The resulting mixture is allowed to stand at room temperature for 24 hours, and then centrifuged and washed to obtain Ca 2+ and Cu 2+ Co-doped hollow Prussian blue nanoparticles Ca / Cu-PB; S3. Dissolve the mitochondrial targeting ligand in dimethyl sulfoxide (DMSO) and stir to form a homogeneous solution. Then add the hollow Prussian blue nanoparticles Ca / Cu-PB obtained in step S2 and continue stirring at room temperature for 8-12 hours to achieve physical adsorption loading of the drug molecules. The mixed solution is centrifuged and washed to remove the unloaded free mitochondrial targeting ligand, and finally obtain the mitochondrial-targeted metal immunodrug.

4. The preparation method according to claim 4, wherein The mitochondrial targeting ligand is a curcumin derivative MitoCur modified with triphenylphosphine (TPP).

5. The preparation method according to claim 4, wherein The copper source is a water-soluble copper salt.

6. Use of the mitochondrial-targeted metal immunodrug according to any one of claims 1 to 3 in the preparation of tumor therapeutic drugs.

7. The use according to claim 6, characterized in that The drug further comprises a pharmaceutically acceptable excipient, wherein the pharmaceutical excipient is a pharmaceutically acceptable salt, excipient or carrier, and the carrier is any one or more of a filler, a wetting agent, a binder, a disintegrant or a lubricant.

8. The use according to claim 7, characterized in that The pharmaceutical excipient is a natural hydrogel, and the metal immune drug is coated in the natural hydrogel to prepare an injectable hydrogel.

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