A preparation method of an inhibitor nano particle targeting residual liver cancer tissue

Abstract

The present application relates to the field of nanomedicine, and more particularly to a preparation method of a targeted residual liver cancer tissue inhibitor nanoparticle. The residual liver cancer tissue is a micro-focal lesion, a cutting edge positive lesion, a satellite lesion or a high-risk recurrent lesion left after surgical resection, ablation, TACE, radiotherapy and other treatments of primary liver cancer. In the present application, quercetin is dissolved in an alkaline solution, copper salt and calcium salt are then added in sequence, a copper-polyphenol network skeleton is constructed through coordination chelation, and further calcium ions are combined in the network to form a copper-calcium bimetallic polyphenol network. After dialysis purification, TPP-mPEG surface electrostatic modification and secondary dialysis freeze-drying, a mitochondria-targeted bimetallic polyphenol network inhibitor nanoparticle is obtained. The nanoparticle is enriched in residual liver cancer tissue after ultrasound penetration, and cooperatively inhibits the growth and recurrence of residual liver cancer through copper death and calcium overload.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a method for preparing inhibitor nanoparticles that target residual liver cancer tissue. Background Technology

[0002] Metal-polyphenol networks (MPNs) are formed by the self-assembly of metal ions and natural polyphenols through coordination interactions. They possess advantages such as simple preparation, good biocompatibility, and pH-responsive dissociation, attracting widespread attention in drug delivery and cancer therapy. Recent studies have found that copper ions can generate reactive oxygen species and induce copper death through Fenton-like reactions, while calcium ions can induce mitochondrial calcium overload. The synergistic delivery of these two systems holds promise for enhancing anti-tumor effects through different death pathways. However, most existing MPN systems only load a single metal ion, and systematic research on the synergistic assembly and loading ratio regulation of copper-calcium bimetallic compounds and polyphenol ligands is still lacking.

[0003] Chinese invention patent CN119700995A discloses a mitochondrial-targeting copper nanoclusters based on natural polyphenols. The polyphenols, acting as ligands and stabilizers, react with copper ions under alkaline conditions to form copper nanoclusters, and the polyphenols' mitochondrial targeting ability is used to scavenge reactive oxygen species (ROS) for the treatment of acute kidney injury. However, this patent only relates to single-metal copper nanoclusters for antioxidant ROS scavenging in the treatment of acute kidney injury, and does not address copper-calcium bimetallic systems or tumor therapy. The functional directions and application scenarios of such single-metal systems are significantly limited. Their preparation methods also have shortcomings in pH control during coordination and optimization of the lyophilization process, leading to poor batch-to-batch consistency, unstable targeting efficiency, and decreased dispersibility after lyophilization and reconstitution. For key technical issues such as the synergistic assembly of copper-calcium bimetals and polyphenol ligands, precise control of the bimetallic loading ratio, active mitochondrial targeting modification, and responsive release in the tumor microenvironment, no existing technology has simultaneously met the above requirements. Existing Ca / Cu bimetallic systems are mostly heterogeneous composite structures of CaCO3 support and copper coordination polymer, with calcium and copper existing in different structural domains. Their dual-ion release kinetics are difficult to match precisely, and structural uniformity is limited by the heterogeneous interface.

[0004] Following radical surgery, radiofrequency ablation, microwave ablation, TACE, and radiotherapy for liver cancer, residual microlesions, positive resection margins, satellite lesions, and high-risk recurrence microenvironments that are difficult to completely remove through imaging or pathology are often present. These are collectively referred to as residual hepatocellular carcinoma (HCC) tissue. Residual HCC tissue is often accompanied by a fibrotic barrier, heterogeneous blood supply, weakened EPR effect, and an immunosuppressive microenvironment, making it difficult for conventional nanomedicines to effectively accumulate and deeply penetrate, thus being a significant cause of postoperative recurrence and metastasis in liver cancer. Existing metallopolyphenol networks are mostly used for the treatment of primary liver cancer or subcutaneous xenografts, and nanoparticle design targeting the anatomical and pathological characteristics of residual HCC tissue is still lacking. In particular, there is a lack of technical solutions that combine copper-calcium bimetallic polyphenol networks, TPP-mPEG mitochondrial targeted modification, and ultrasound-enhanced penetration for targeted inhibition of residual HCC tissue.

[0005] Therefore, there is an urgent need for a method to prepare inhibitor nanoparticles that can be enriched in residual liver cancer tissue, overcome the fibrosis barrier, and achieve synergistic killing by copper death and calcium overload, in order to solve the technical problems of lack of precise control in the preparation process of metal polyphenol networks, insufficient research on bimetallic synergistic coordination, and difficulty in controlling the degree of purification in the existing technology. Summary of the Invention

[0006] Therefore, this invention provides a method for preparing inhibitor nanoparticles that target residual liver cancer tissue, in order to solve the technical problems of existing metal polyphenol network preparation processes lacking precise control, insufficient research on copper-calcium bimetallic synergistic coordination, difficulty in controlling the degree of purification, and the difficulty of existing nanoparticles targeting and enriching residual liver cancer tissue and effectively inhibiting its growth and recurrence.

[0007] To achieve the above objectives, the present invention provides a method for preparing inhibitor nanoparticles targeting residual liver cancer tissue, comprising the following steps: Step S1: Dissolve quercetin in sodium hydroxide solution and adjust the pH to 9.0-12.0 to obtain quercetin sodium salt solution; Step S2: Add copper chloride solution dropwise to the sodium quercetin salt solution at a molar ratio of quercetin to copper chloride of 1:(5-6) to carry out the reaction, so that copper ions and quercetin form a chelate structure. During the reaction, sodium hydroxide solution is added dropwise to maintain the pH of the system at 9.0-12.0, and a copper-polyphenol network dispersion is obtained. Step S3: Add calcium chloride solution to the copper-polyphenol network dispersion at a molar ratio of quercetin to calcium chloride of 1:(0.5-0.7) to react and allow calcium ions to combine with the copper-polyphenol network to obtain a bimetallic polyphenol network dispersion. Step S4: The bimetallic polyphenol network dispersion is placed in a dialysis bag for dialysis to remove unreacted metal ions and free quercetin, resulting in a core nanoparticle dispersion. Step S5: Add mitochondrial-targeting modification molecule TPP-mPEG to the core nanoparticle dispersion at a molar ratio of TPP-mPEG to quercetin of (0.08-0.15):1 and stir. Modify the nanoparticles through electrostatic interaction to obtain the nanoparticle modification solution. Step S6: After a second dialysis of the nanoparticle-modified solution, the solution is freeze-dried to obtain the inhibitor nanoparticles targeting residual liver cancer tissue. The inhibitor nanoparticles are used to target and inhibit residual liver cancer tissue, which refers to small lesions, positive resection margins, satellite lesions, or high-risk microenvironments for recurrence remaining after surgical resection, ablation, TACE, or radiotherapy of primary liver cancer.

[0008] Furthermore, quercetin was first dissolved in a sodium hydroxide solution with a concentration of 0.01 mol / L to 0.1 mol / L, and then the pH of the system was adjusted to 9.0 to 12.0 with a sodium hydroxide solution with a concentration of 1 mol / L.

[0009] Furthermore, the reaction conditions include a reaction temperature of 20℃-30℃, a stirring speed of 500rpm-800rpm, and a stirring time of 2h-4h.

[0010] Furthermore, the reaction conditions include a reaction temperature of 20℃-30℃, a stirring speed of 500rpm-800rpm, and a stirring time of 0.5h-1h.

[0011] Furthermore, the molecular weight cutoff of the dialysis bag is 8000Da-14000Da, the dialysis fluid is deionized water, the volume of the deionized water is 50-100 times the volume of the bimetallic polyphenol network dispersion, the dialysis temperature is 4℃-25℃, the dialysis time is 20h-28h, and the dialysis fluid is replaced every 6h-8h.

[0012] Furthermore, the TPP-mPEG has a molecular weight of 600 Da-700 Da; the surface modification temperature is 20℃-30℃, the stirring speed is 400 rpm-600 rpm, and the reaction time is 2h-3h.

[0013] Furthermore, the dialysate for the secondary dialysis is PBS buffer, the dialysis temperature is 4℃-10℃, the dialysis time is 12h-18h, and the lyophilization process includes: The nanoparticle modification liquid was dispensed into freeze-drying containers, placed on the shelf of a freeze dryer, and the shelf temperature was lowered to -40°C to -50°C for 4 to 6 hours of pre-freezing. After pre-freezing, start the vacuum pump to raise the shelf temperature to -20℃ to -30℃, control the vacuum degree to 10Pa-20Pa, and carry out a drying cycle of 20-28 hours. After the first drying cycle, raise the shelf temperature to 20℃-30℃, control the vacuum degree to 1Pa-5Pa, and perform a second drying cycle for 4-8 hours.

[0014] On the other hand, there is an inhibitor nanoparticle that targets residual liver cancer tissue, wherein the hydrated particle size of the inhibitor nanoparticle is 90nm-125nm, PDI≤0.15, and Zeta potential is -25mV to -23mV.

[0015] Furthermore, the use of the inhibitor nanoparticles in the preparation of a drug for targeting and inhibiting residual liver cancer tissue, wherein the residual liver cancer tissue is a small lesion, a positive resection margin, a satellite lesion, or a high-risk microenvironment for recurrence remaining after surgical resection, ablation, TACE, or radiotherapy of primary liver cancer.

[0016] Furthermore, the inhibitor nanoparticles are used in combination with ultrasonic permeation technology, wherein the frequency of the ultrasound is 1MHz-3MHz and the intensity is 0.5W / cm² (measured by spatial average and time average sound intensity). 2 -1.5W / cm 2 The action time is 3-10 minutes.

[0017] Compared with existing technologies, this invention systematically solves the technical problems of lack of precise control in the preparation process of existing metal polyphenol network systems, insufficient research on copper-calcium bimetallic synergistic coordination, and difficulty in controlling the degree of purification by stepwise assembly of copper and calcium ions with quercetin polyphenol ligands, active mitochondrial targeting by TPP-mPEG surface electrostatic modification, and physical enhancement by ultrasound-enhanced permeation. This enables efficient, precise, and synergistic treatment of hepatocellular carcinoma.

[0018] Furthermore, this invention utilizes the differentiated coordination ability of quercetin polyphenol ligands for copper and calcium ions, employing a stepwise assembly strategy of first constructing the framework through copper coordination and then binding calcium. Copper ions form strong coordination bonds with the deprotonated phenolic hydroxyl groups of quercetin, constructing a stable three-dimensional network framework; the remaining copper ions reside within the network as nano-hydroxide clusters (Cu(OH)2 / CuO) under alkaline conditions, forming a composite network structure together with chelated copper; calcium ions reside in the network framework in a bound state through multiple weak interactions, including cation-π interactions, electrostatic attraction, and weak coordination. In the slightly acidic environment of tumors, the coordination bonds dissociate, releasing copper ions and triggering copper death, while the simultaneous release of calcium ions induces mitochondrial calcium overload and apoptosis. In Example 1, the apoptosis rate was 35.8%, significantly higher than that of the calcium-deficient and copper-deficient control groups; the tumor inhibition rate was 51.2%, significantly higher than that of QC-TPP and QCa-TPP, confirming the synergistic effect of copper-calcium bimetallic synthesis.

[0019] Furthermore, this invention utilizes the electrostatic attraction between the negative charge on the nanoparticle surface and the TPP-mPEG quaternary cation to achieve the self-assembly of the targeted ligand; the TPP group precisely delivers the nanoparticles to the mitochondria based on the negative transmembrane potential difference of the mitochondria. In Example 1, the mitochondrial colocalization coefficient was 0.83, significantly higher than the non-targeted control ratio; the tumor inhibition rate was 51.2%, higher than QCC, confirming the necessity of active mitochondrial targeting for improving therapeutic efficacy.

[0020] Furthermore, this invention utilizes the acoustic pore effect of ultrasound to enhance the transcellular transport and deep penetration of nanoparticles. Ultrasound-enhanced penetration, mitochondrial targeting, and copper death combined with calcium overload form a spatiotemporally synergistic therapeutic loop. The apoptosis rate of the QCC-TPP combined with ultrasound group was 52.3%, the tumor inhibition rate was 69.8%, and the necrosis area was 65.3%, all higher than the QCC-TPP group, confirming that ultrasound-enhanced penetration can significantly enhance the anti-tumor effect. These three mechanisms work sequentially to achieve highly efficient, precise, and synergistic treatment of hepatocellular carcinoma. Attached Figure Description

[0021] Figure 1 This is a process flow diagram of the method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to the present invention; Figure 2 Morphology and particle size characterization of QCC and QCC-TPP of the present invention; Figure 3 This is a CLSM image of the in vitro cell phagocytosis experiment of QCC-TPP in this invention; Figure 4 CLSM observation of ROS generation in tumor cells of each group in this invention; Figure 5 These are CLSM images of mitochondrial membrane potential of tumor cells in each group of this invention; Figure 6 This invention provides multimodal molecular imaging of nanoparticles. Detailed Implementation

[0022] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0023] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0024] It should be noted that in the description of this invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and is not intended to indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this invention.

[0025] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0026] The inhibitor nanoparticles described in this invention are primarily used to target residual hepatocellular carcinoma (HCC) tissue. These residual HCC tissues include, but are not limited to: positive margin lesions after hepatocellular carcinoma resection, residual lesions after ablation, residual active lesions after TACE, residual lesions after radiotherapy, satellite lesions, and high-risk microenvironments for postoperative recurrence. After administration via tail vein, the inhibitor nanoparticles accumulate in residual HCC tissue through the EPR effect, ultrasound-enhanced penetration, and TPP-mPEG mitochondrial targeting. They release copper and calcium ions in the tumor's microacidic environment, synergistically inhibiting residual HCC growth and recurrence through copper death and calcium overload.

[0027] The inhibitor nanoparticles are used in conjunction with ultrasound-enhanced permeation technology. Ultrasound treatment can be performed 10 minutes after nanoparticle administration, using a frequency of 1MHz-3MHz and an intensity of 0.5W / cm² (space-averaged time-averaged sound intensity). 2 -1.5W / cm 2 Apply continuously for 3-10 minutes.

[0028] Please see Figure 1 As shown, it is a process flow diagram of a method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to the present invention.

[0029] This invention provides a method for preparing inhibitor nanoparticles targeting residual liver cancer tissue, comprising the following steps: The function of step S1 is to provide a quercetin precursor solution with fully dissociated phenolic hydroxyl groups and coordination reactivity.

[0030] Specifically, quercetin is first dissolved in a sodium hydroxide solution with a concentration of 0.01 mol / L to 0.1 mol / L, and then the pH of the system is adjusted to 9.0-12.0 with a sodium hydroxide solution with a concentration of 1 mol / L.

[0031] Understandably, quercetin (3,3',4',5,7-pentahydroxyflavone) has extremely low solubility in water. Its molecule contains three potential metal coordination domains: the 3',4'-o-dihydroxyl group on ring B, the 5-hydroxy-4-keto group at the intersection of rings A and C, and the 3-hydroxy-4-keto group on ring C. In a neutral state where the molecule is associated with hydrogen bonds, the oxygen atom of the phenolic hydroxyl group has insufficient electron cloud density to provide lone pairs of electrons to the empty orbitals of metal ions to form stable coordination bonds. Only under alkaline conditions do these phenolic hydroxyl groups undergo deprotonation to transform into the phenoxy anion Ar-O. - Subsequently, the negative charge density on the oxygen atom increases significantly, and the coordination activity is greatly enhanced, allowing it to chelate with transition metal ions such as copper ions. In this embodiment, a sodium hydroxide solution with a concentration of 0.01 mol / L to 0.1 mol / L is used as an alkaline medium, and the pH of the system is adjusted to 9.0-12.0. This neutralizes the protons of at least the 3- and 7-hydroxyl groups in the quercetin molecule, converting them into sodium quercetin salt. At the same time, the 4' hydroxyl group is partially deprotonated, thereby endowing quercetin with good water solubility and multidentate coordination ability.

[0032] It is understandable that the ortho-dihydroxyl groups in quercetin molecules are highly oxidatively sensitive under alkaline conditions. Dissolved oxygen can oxidize them to ortho-quinone products, leading to irreversible oxidative polymerization or degradation. This results in a decrease in the effective ligand concentration, loss of coordination sites, and ultimately affects the structural integrity and batch consistency of the copper-polyphenol network. To suppress the above-mentioned side reactions, the following measures were taken in this embodiment: (1) Control of dissolution stage: The dissolution process of S1 is carried out under light-protected conditions, and the cumulative time from the contact between quercetin and sodium hydroxide solution to the completion of ultrasonic dissolution does not exceed 30 minutes, so as to shorten the alkaline exposure time during the dissolution stage; (2) Nitrogen protection during the coordination reaction stage: Nitrogen gas is introduced throughout the S2 coordination reaction to reduce the dissolved oxygen concentration in the reaction system and inhibit the oxidation of phenolic hydroxyl groups by dissolved oxygen; (3) Temperature control during coordination reaction: The temperature of coordination reaction is controlled within a mild range of 20℃-30℃ to avoid accelerated oxidation at high temperatures.

[0033] Through the synergistic control of the above measures, the alkaline exposure time of quercetin during the dissolution stage does not exceed 30 minutes. During the coordination reaction stage, the S2 stirring reaction lasts for 2-4 hours and the S3 stirring reaction lasts for 0.5-1 hour. Oxidation is inhibited by nitrogen protection throughout the process and a mild temperature of 20℃-30℃. The oxidative degradation rate of quercetin during the coordination reaction process can be controlled within an acceptable range, ensuring the high reproducibility of the coordination network structure.

[0034] In this embodiment, step S1 includes the following specific steps: Step S11: Weigh 930 mg of anhydrous quercetin (M = 302.24), place it in a 250 mL beaker, and add 60 mL of 0.05 mol / L sodium hydroxide solution. The molar ratio of quercetin to NaOH is approximately 1:1, so that quercetin is partially salted. In step S12, the pH of the system is adjusted to 10.5 with 1 mol / L sodium hydroxide solution, and the solution is dissolved by ultrasonication at 40 kHz for 10 minutes to obtain a sodium quercetin solution for later use. The entire dissolution process is carried out under light-protected conditions, and the total time from the contact between quercetin and sodium hydroxide solution to the completion of ultrasonic dissolution does not exceed 30 minutes.

[0035] In step S11, a 0.05 mol / L sodium hydroxide solution is used to dissolve quercetin; in step S12, a 1 mol / L sodium hydroxide solution is used to precisely adjust the pH of the system to 10.5. The difference in concentration is due to their different functions: the lower concentration alkaline solution has a larger volume, which facilitates the full dispersion of quercetin and slow salt formation; the higher concentration alkaline solution requires a smaller volume, which allows for precise pH adjustment without significantly diluting the system.

[0036] Step S2 functions by using sodium quercetin as a polydentate ligand, and by adding copper chloride solution dropwise while maintaining an alkaline pH, to induce Cu... 2+ A copper-polyphenol network with a three-dimensional network structure was constructed by coordinating and chelating with the deprotonated phenolic hydroxyl groups of quercetin, providing a structural basis for subsequent calcium ion loading.

[0037] Specifically, the reaction conditions are a reaction temperature of 20℃-30℃, a stirring speed of 500rpm-800rpm, and a stirring time of 2h-4h.

[0038] It is understandable that quercetin molecules, after being converted to sodium quercetin under alkaline conditions, possess the activity of polydentate chelation coordination with transition metal ions. When copper chloride solution is added dropwise to sodium quercetin solution, Cu... 2+ As an electron-deficient metal center, it preferentially coordinates with the ortho-bisphenol hydroxyl anion, which has the highest electron cloud density, to form a five- or six-membered chelate ring structure. Because the quercetin molecule contains at least two or more ortho-hydroxyl pairs that can participate in coordination, a single quercetin molecule can simultaneously coordinate with multiple Cu atoms. 2+ Coordination, while single Cu 2+ It can bridge two or more quercetin molecules, thereby forming a cross-linked network structure in three-dimensional space, namely the copper-polyphenol network.

[0039] In this embodiment, the molar ratio of quercetin to copper chloride is 1:(5-6), with copper ions in significant excess relative to quercetin. The effects of the excess copper ions include: (1) Ensure that as many ortho- and tho-hydroxyl groups as possible in the quercetin molecule are fully occupied, so as to maximize the coordination crosslinking density and form a dense and regular three-dimensional network structure. (2) Under alkaline conditions, excess copper ions are generated in situ as nano-hydrogen hydroxide clusters and reside within the network framework and surface pores, forming a composite network structure together with chelated copper, providing more binding sites and structural support for subsequent calcium ion loading. The judgment that excess copper ions exist in the form of nano-hydrogen hydroxide clusters is supported by the following evidence: First, Table 1 shows the trend of Cu content (33.5 ± 0.8 wt%) and particle size / PDI variation in Examples 1-2: When the Cu:quercetin molar ratio was further increased from 1:5.5 to 1:6, the Cu content only increased slightly, from 33.5 wt% to 34.2 wt%, and the particle size tended to plateau, indicating that the network coordination had become saturated. Excess copper ions mainly existed in the network in the form of hydroxide clusters and had not yet destroyed the overall integrity of the network structure.

[0040] Second, XRD characterization: Slow-scan XRD analysis was performed on the products of Examples 1-2. Weak diffraction peaks appeared at 2θ = 18.5°, 34.2°, and 38.6°, which were initially identified as Cu(OH)2 (JCPDS 13-0420); weak diffraction peaks appeared at 2θ = 35.5° and 38.7°, which were initially identified as CuO (JCPDS 45-0937). Due to the close positions of the characteristic peaks of the two phases and the small size and low crystallinity of the nanoclusters, there is some uncertainty in the XRD identification. Combined with the Cu(II) main peak at 934.5 eV and 954.2 eV and the satellite peak at 940-945 eV in the XPS Cu 2p spectrum, the lattice oxygen peak at 529.8 eV and the hydroxyl oxygen peak at 531.5 eV in the O 1s spectrum, and the Cu enrichment region of 2-5 nm in TEM-EDS, the presence of Cu(OH)2 / CuO nanoclusters in the products is supported.

[0041] Third, XPS characterization: The Cu 2p XPS spectrum shows Cu 2p atoms appearing at 934.5 eV and 954.2 eV. 3 / 2 and Cu2p 1 / 2 The main peak, along with a distinct satellite peak at 940eV-945eV, indicates that copper mainly exists in the Cu(II) valence state. Combined with the lattice oxygen peak at 529.8eV and the hydroxyl oxygen peak at 531.5eV in the O1s spectrum, it is further confirmed that Cu(II) exists in the forms of Cu(OH)2 and CuO.

[0042] Fourth, TEM-EDS elemental distribution: High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) combined with EDS surface scanning showed that Cu was uniformly distributed in the nanoparticles, while Cu-enriched regions with a size of about 2-5 nm existed in local areas inside the network framework, consistent with the size of the nano hydroxide clusters; Ca was mainly distributed in the voids of the network framework, and its distribution was complementary to that of Cu-enriched regions.

[0043] In this embodiment, step S2 includes the following specific steps: Step S21: Take the sodium quercetin solution obtained in step S12 and place it in a constant temperature magnetic stirrer. Under the stirring conditions of 25℃ and 700 rpm, add about 56.5 mL of 0.3 mol / L copper chloride solution at a rate of 1 drop / second. The solution contains about 16.95 mmol of CuCl2, and the molar ratio of quercetin to CuCl2 is about 1:5.5. Nitrogen gas is purged throughout the addition process for protection. At the same time, 1 mol / L sodium hydroxide solution is added dropwise to maintain the pH of the reaction system in the range of 10.0-10.5. In step S22, after the addition is complete, the mixture is stirred and reacted for 3 hours at 25°C and 700 rpm. Nitrogen gas is continuously introduced throughout the reaction to inhibit the oxidative degradation of the phenolic hydroxyl groups of quercetin, thus obtaining a copper-polyphenol network dispersion.

[0044] Table 1 shows the particle size, PDI, Zeta potential, and Cu content of copper-polyphenol network dispersions under different quercetin:CuCl2 molar ratios.

[0045] Table 1. Particle size, PDI, Zeta potential, and Cu content of copper-polyphenol network dispersions under different quercetin:CuCl2 molar ratios.

[0046] Table 1 Note: After purification in step S4, each product from step S22 was diluted 15 times with deionized water for determination. The dilution medium was deionized water. Hydrated particle size and PDI were determined by dynamic light scattering (DLS). The hydrated particle size data was the average of three independent determinations, and the ± value was the standard deviation (n=3) between the three determinations, representing the precision of the determination. PDI was provided by the Cumulant analysis instrument, reflecting the width of the particle size distribution in a single determination. There is no simple square relationship between PDI and the standard deviation. Zeta potential was determined by electrophoretic light scattering (ELS), with each sample measured in triplicate. Cu content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), with each sample measured in triplicate. The results are expressed as mean ± standard deviation (n=3).

[0047] Table 1 shows that Comparative Example 1-1 has a particle size of 142.3 nm, a PDI of 0.19, a Zeta potential of -21.5 mV, and a Cu content of 29.1 ± 1.0 wt%. 2+ Insufficient to fully utilize all coordinating sites of quercetin molecules, lack of coordination cross-linking points, loose network, and structural heterogeneity lead to larger particle size, wider distribution, and insufficient absolute value of Zeta potential. Example 1-1-1-3: Particle size 95.2nm-108.5nm, PDI 0.08-0.11, Zeta potential -23.8mV to -25.3mV, Cu content 31.8-34.2wt%, Cu... 2+ The dosage was sufficient to drive the major coordination sites of quercetin molecules to fully participate in coordination, increasing the coordination crosslinking density and making the network structure more compact and regular. In the 1:5.5 ratio, the particle size was 98.7 nm, PDI was 0.08, Zeta potential was -25.3 mV, and Cu content was 33.5 ± 0.8 wt%. At this ratio, coordination was close to saturation, the structure was the most compact and regular, and the surface charge density was the highest. In Examples 1-3, the Cu content was 34.2 ± 0.9 wt%, the particle size was only slightly smaller than in the 1:5.5 ratio, tending towards a plateau, and the PDI and potential slightly decreased, indicating that the network was saturated and some excess Cu was present. 2+ Initially, the particles reside within or on the surface of the network as nano-hydroxide clusters, causing slight disturbances but not disrupting the overall network structure. Although the Cu content in Comparative Examples 1-2 reached 34.8 ± 1.5 wt%, the particle size rebounded to 134.6 nm, the PDI was 0.32, and the Zeta potential dropped to -20.5 mV. The root cause lies in the Cu content. 2+ When in excessive amounts, it hydrolyzes into Cu(OH)2 precipitate under alkaline conditions, which then co-aggregates with the nanoparticles and neutralizes their charge, thus disrupting the network structure.

[0048] The function of step S3 is to add Ca to the already formed Cu-quercetin coordination network. 2+ , making Ca 2+ Through multiple weak interactions such as cation-π interaction, electrostatic attraction, and weak coordination, it is loaded into the Cu-quercetin network in a bound state to form a Cu-Ca bimetallic polyphenol network, laying the material basis for the subsequent synergistic release of copper death and calcium overload in the microacidic environment of tumors.

[0049] Specifically, the reaction conditions are: reaction temperature of 20℃-30℃, stirring speed of 500rpm-800rpm, and stirring time of 0.5h-1h.

[0050] It is understandable that in the copper-polyphenol network dispersion obtained in step S2, Cu 2+ It has formed a cross-linked network with quercetin, and excess Cu 2+The Cu(OH)₂ / CuO nanoclusters reside within the network framework and surface pores as nano-hydroxyl clusters. Numerous uncoordinated aromatic rings and deprotonated phenolic hydroxyl groups remain on the network surface and in the internal pores. XRD, XPS, and TEM-EDS characterization confirmed the presence of Cu(OH)₂ / CuO nanoclusters. When calcium chloride solution is added to this dispersion, Ca… 2+ On the one hand, it electrostatically attracts the electron-rich π system of the quercetin benzene ring in the network through cation-π interactions; on the other hand, it is enriched in the negatively charged region on the network surface where the deprotonated phenolic hydroxyl groups are located through electrostatic attraction; in addition, a small amount of Ca... 2+ It can be used with networks that have not yet been Cu 2+ The occupied phenolic hydroxyl group undergoes weak coordination. The synergistic effect of these multiple interactions allows Ca... 2+ It is stably bound to the structural voids of the Cu-quercetin network without disrupting the existing Cu-coordination framework, because Ca... 2+ The coordination ability of Cu is much weaker. 2+ It cannot competitively replace the already coordinated Cu. 2+ Ca 2+ After loading, the network structure rearranges due to the insertion of additional cations, and the hydrated particle size increases with Ca. 2+ The dosage initially decreased and then increased; when Ca:quercetin = 0.3:1, Ca... 2+ Insufficient Ca content resulted in low network density, with the peak hydrated particle size of DLS reaching approximately 95.0 nm, close to the 98.7 nm of the product from step S2. When the Ca:quercetin ratio was 0.6:1, the peak hydrated particle size of DLS reached approximately 80.0 nm, lower than the 98.7 nm of the product from step S2, indicating that an appropriate amount of Ca... 2+ Inducing network densification; when Ca:quercetin ≥ 0.7:1, Ca 2+ When the network exceeds its binding site capacity, it expands / aggregates, resulting in increased particle size. The zeta potential decreases slightly due to the neutralization effect of positive charges.

[0051] In this embodiment, step S3 includes the following specific steps: Step S31: Take the copper-polyphenol network dispersion obtained in step S22, place it in a constant temperature magnetic stirrer, and add a calcium chloride solution with a concentration of 0.1 mol / L under stirring conditions of 25℃ and 700 rpm, while maintaining a nitrogen atmosphere during the addition process. In step S32, after the addition is complete, the mixture is stirred and reacted for 0.75 h at 25 °C and 700 rpm. Nitrogen gas is continuously introduced for protection throughout the reaction to obtain the bimetallic polyphenol network dispersion.

[0052] Table 2 shows the particle size, PDI, Zeta potential, and Ca content of bimetallic polyphenol network dispersions under different Ca:quercetin molar ratios.

[0053] Table 2. Particle size, PDI, Zeta potential, and Ca content data of bimetallic polyphenol network dispersions under different Ca:quercetin molar ratios.

[0054] Table 2 Note: The Cu:quercetin = 1:5.5 product from step S22 was reacted in step S3 and purified in step S4, then diluted with deionized water to a suitable concentration for determination. Hydrated particle size and PDI were determined by dynamic light scattering (DLS). The hydrated particle size data is the average of three independent determinations, and the ± value is the standard deviation (n=3) between the three determinations, representing the determination precision. PDI is given by the Cumulant instrument and reflects the width of the particle size distribution in a single determination. There is no simple square relationship between PDI and standard deviation. Zeta potential was determined by electrophoretic light scattering (ELS), with each sample measured in triplicate. Ca content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), with each sample measured in triplicate. The results are expressed as mean ± standard deviation (n=3).

[0055] Table 2 shows that Comparative Example 2-1 has a particle size of 95.0 nm, a PDI of 0.09, a Zeta potential of -26.5 mV, and a Ca content of 0.8 ± 0.2 wt%. 2+ Insufficient dosage resulted in unsaturated binding sites, low network density, and particle size close to the product of step S2. Although the dispersion was good, it was insufficient to meet the needs of subsequent calcium overload therapy. Examples 2-1 to 2-3 showed particle sizes of 80.0 nm-99.8 nm, PDI of 0.10-0.11, Zeta potential of -23.8 to -24.8 mV, and Ca content of 1.5 wt%-2.3 wt%, indicating that Ca... 2+ It successfully incorporated into the Cu-quercetin network through multiple weak interactions, including cation-π interactions, electrostatic attraction, and weak coordination, while maintaining the integrity of the network structure and remaining unaffected by Ca. 2+ The loading process disrupted the system. In Example 2-2, the Ca content was 2.0 ± 0.3 wt%, the particle size was 80.0 nm, the PDI was 0.10, and the Zeta potential was -24.3 mV, still remaining above 20 mV. This indicates that the binding sites were approaching saturation, the network structure remained intact, and the system was in a colloidal stable range. In Example 2-3, the Ca content was 2.3 ± 0.3 wt%, and the particle size increased from 80.0 nm to 99.8 nm. The PDI and Zeta potential slightly decreased, indicating that the binding sites were approaching saturation, and the additional Ca content was insufficient. 2+Initially, a slight disturbance was caused to the network, but all indicators still met the target requirements. In Comparative Example 2-2, although the Ca content reached 2.8 ± 0.4 wt%, the particle size rebounded to 115.3 nm, the PDI rose to 0.22, and the Zeta potential was -22.1 mV, all below the lower limit of the 20 mV stability range of this invention; simultaneously, both the PDI and particle size exceeded the stable range. The root cause lies in the Ca content. 2+ Exceeding the capacity of network binding sites, free Ca 2+ Increasing ionic strength compresses the electric double layer, weakening electrostatic repulsion and leading to network aggregation, increased particle size, wider distribution, and decreased system stability.

[0056] The function of step S4 is to remove unreacted metal ions, free quercetin, counterions and small molecule oligomers from the bimetallic polyphenol network dispersion by dialysis, purify the nanoparticle core dispersion, and obtain a uniform particle size distribution, stable surface charge and clear component ratio in the product, so as to provide a clean nanoparticle substrate for subsequent surface-targeted modification.

[0057] Specifically, the dialysis bag used for dialysis has a molecular weight cutoff of 8000 Da-14000 Da, the dialysis fluid is deionized water, the volume of the deionized water is 50-100 times the volume of the bimetallic polyphenol network dispersion, the dialysis temperature is 4℃-25℃, the dialysis time is 20h-28h, and the dialysis fluid is replaced every 6h-8h.

[0058] Understandably, the dialysis bag used has a molecular weight cutoff of 8000 Da-14000 Da, and its membrane pore size is approximately 2 nm-5 nm. The hydrated particle size of the bimetallic polyphenol network nanoparticles obtained in step S3 is approximately 80 nm-100 nm, much larger than the membrane pore size, and is retained within the dialysis bag; while free Cu in the system... 2+ Ca 2+ Na + Cl - Unreacted quercetin and small-molecule oligomers, with molecular weights far below 8000 Da, can freely diffuse through the semi-permeable membrane into the dialysate outside the bag under the drive of the concentration gradient. The dialysate is deionized water, with a volume 50-100 times that of the dispersion, to maintain a large concentration gradient inside and outside the bag, ensuring sufficient diffusion of impurities. The dialysate is changed every 6-8 hours. This restores the concentration gradient between the inside and outside of the bag, allowing diffusion to continue, and also removes small-molecule impurities that have diffused outside the bag, preventing them from re-equilibrating back inside. The dialysis temperature is controlled between 4℃ and 25℃. Low temperatures help inhibit hydrolysis, oxidation, or structural rearrangement of nanoparticles during purification, while also inhibiting microbial growth; room temperature accelerates the diffusion rate and shortens the dialysis time. A dialysis time of 20-28 hours is sufficient to reduce the impurity concentration to an acceptable level.

[0059] In this embodiment, step S4 includes the following specific steps: Step S41: Take the bimetallic polyphenol network dispersion obtained in step S32 and transfer it to a dialysis bag with a molecular weight cutoff of 8000 Da-14000 Da. The liquid volume of each bag is 60%-80% of the volume of the dialysis bag, and the bag opening is sealed with a dialysis clamp. Step S42: Place the dialysis bag in a dialysis container filled with deionized water (80 times the volume of the dispersion). Dialyze at 25°C for 24 hours, replacing the external dialysis solution every 6 hours. After dialysis, measure the conductivity of the external dialysis solution (<5 μS / cm). Acidify the external dialysis solution with dilute nitric acid, then add 0.1 mol / L AgNO3 solution. No white precipitate forms, confirming Cl... - Complete removal has been achieved; simultaneously, the nanoparticle dispersion inside the bag was subjected to ultrafiltration and centrifugation to remove free metal ions (molecular weight cutoff 10000 Da). The filtrate was then analyzed by ICP-AES to confirm the presence of free Cu. 2+ Residual content less than 5 ppm, free Ca 2+ A core nanoparticle dispersion is obtained when the residual amount is below 5 ppm. It should be noted that the above-mentioned free Cu... 2+ Residual and free Ca 2+ Residual amount refers to metal ions that have not participated in coordination and exist in the form of free ions, excluding copper and calcium that have been coordinated and bound in the copper-polyphenol network framework. The total copper content in the nanoparticle product is approximately 31 wt%, and the total calcium content is approximately 2 wt%, which is a different concept from the residual amount of free metal ions.

[0060] The function of step S5 is to modify the mitochondrial targeting ligand TPP-mPEG onto the surface of the core nanoparticle through electrostatic interaction, forming a composite functional layer on the nanoparticle surface that combines the functions of the TPP end for mitochondrial targeting and the mPEG end for long-cycle cloaking. This endows the nanoparticle with the ability to actively target mitochondria, providing a targeted delivery basis for subsequent synergistic therapy of mitochondrial copper death and calcium overload at the tumor cell level.

[0061] Specifically, the TPP-mPEG has a molecular weight of 600 Da-700 Da; the surface modification temperature is 20℃-30℃, the stirring speed is 400 rpm-600 rpm, and the reaction time is 2h-3h.

[0062] It is understandable that the core nanoparticle dispersion obtained in step S4 is a Cu-quercetin-Ca bimetallic polyphenol network, which has a large number of uncoordinated deprotonated phenolic hydroxyl groups on its surface, giving the nanoparticle surface a strong negative charge.

[0063] Specifically, TPP-mPEG is an amphiphilic functional molecule with a TPP group at one end, existing as a quaternary cation in aqueous solution with a permanent positive charge; the other end is an mPEG chain, exhibiting good hydrophilicity and flexibility. When TPP-mPEG is added to the core nanoparticle dispersion, the TPP-mPEG molecules self-assemble onto the nanoparticle surface through the following mechanism: 1. Electrostatic attraction: The positively charged TPP terminal quaternary cations in the TPP-mPEG molecule are electrostatically attracted to the negatively charged nanoparticle surface, causing the TPP-mPEG molecules to be rapidly adsorbed onto the nanoparticle surface. Due to the small molecular weight of TPP-mPEG, its diffusion rate in solution is relatively fast, and adsorption equilibrium can be reached within 2-3 hours.

[0064] 2. Cation-π Interaction: The TPP group contains three benzene rings. The electron-rich π system on the benzene rings can undergo cation-π stacking interactions with the uncoordinated aromatic rings in the quercetin molecule on the nanoparticle surface. Strong π-π stacking and cation-π interactions exist between the benzene rings of TPP and the B-ring (o-diphenol) and A-ring (resorcinol) of quercetin, further enhancing the anchoring strength of TPP-mPEG on the nanoparticle surface. 3. Surface Coverage of mPEG Segments: After the TPP ends are anchored to the nanoparticle surface, the hydrophilic mPEG segments extend outwards, forming a hydrophilic brush layer (PEG brush layer) on the nanoparticle surface. This PEG layer effectively shields the hydrophobic regions and some charges on the nanoparticle surface, reducing non-specific adsorption of serum proteins and prolonging the half-life of the nanoparticles in blood circulation. Simultaneously, the flexibility of the mPEG segments allows them to move freely under physiological conditions without affecting the targeting function of the exposed TPP groups.

[0065] Specifically, the TPP-mPEG is an amphiphilic molecule formed by covalently linking triphenylphosphine (TPP) and methoxy polyethylene glycol (mPEG), wherein the TPP terminal has a quaternary cation (-P). + (C6H5)3), the mPEG end is a hydrophilic polyethylene glycol segment with a molecular weight of 600 Da-700 Da; TPP-mPEG can be synthesized using methods known in the art or purchased directly. The TPP-mPEG used in this invention is a commercially available product, which can be purchased directly from Sigma-Aldrich or domestic reagent suppliers. This product has a molecular weight of approximately 650 Da and a purity ≥95%.

[0066] In this embodiment, step S5 includes the following specific steps: Step S51: Take the core nanoparticle dispersion obtained in step S42, place it in a constant temperature magnetic stirrer, and add TPP-mPEG under stirring conditions of 25℃ and 500rpm, while maintaining a nitrogen atmosphere during the addition process. In step S52, after the addition is complete, continue stirring the reaction at 25℃ and 500rpm for 2.5h to obtain the nanoparticle modified solution.

[0067] Table 3 shows the particle size, PDI, Zeta potential, and mitochondrial colocalization coefficient of nanoparticles under different TPP-mPEG:quercetin feeding ratios.

[0068] Table 3. Data on particle size, PDI, Zeta potential, and mitochondrial colocalization coefficient of nanoparticles under different TPP-mPEG:quercetin feed ratios.

[0069] Table 3 Note: The core nanoparticles (Cu:quercetin = 1:5.5, Ca:quercetin = 0.6:1) prepared in step S4 were modified in step S5 and purified by secondary dialysis in step S6. They were then diluted with deionized water to a suitable concentration for measurement. Hydrated particle size and PDI were determined using dynamic light scattering (DLS). The hydrated particle size data were the average of three independent measurements, with the ± value representing the standard deviation (n=3) between the three measurements, indicating measurement precision. PDI was provided by the Cumulant instrument and reflected the width of the particle size distribution in a single measurement. There was no simple square relationship between PDI and the standard deviation. Zeta potential was determined using electrophoretic light scattering (ELS), with each sample measured in triplicate. Colocalization coefficients were determined using laser confocal microscopy: HepG2 cells were co-incubated with nanoparticles (50 μg / mL) for 4 h. MitoTracker Green was used to label mitochondria, and images were acquired and analyzed using ImageJ software Coloc. 2. The plugin calculates the Pearson correlation coefficient. The colocalization coefficient is quantitatively evaluated using the Pearson correlation coefficient. Generally, a Pearson coefficient >0.5 indicates significant colocalization, and >0.7 indicates high colocalization. No less than 10 cells are randomly selected from each sample for analysis. The results are expressed as mean ± standard deviation (n=10).

[0070] As shown in Table 3, the colocalization coefficient of Comparative Example 3-1 is only 0.58±0.04, which is lower than the high colocalization threshold (0.7). This indicates that the TPP-mPEG feed is insufficient, and the TPP ligand density on the nanoparticle surface is too low, making it difficult to drive the nanoparticles to efficiently accumulate in mitochondria. At this point, the particle size is 112.8 nm, the PDI is 0.11, and the Zeta potential is -26.8 mV. Although the dispersion is good, the targeting efficiency is insufficient.

[0071] Examples 3-1 to 3-4 showed colocalization coefficients of 0.72-0.83, all reaching a high level of colocalization. This indicates that TPP-mPEG was successfully modified onto the nanoparticle surface through electrostatic interactions. Simultaneously, the particle size was 95.2 nm-100.0 nm, PDI was 0.12-0.13, and Zeta potential was -24.5 mV to -23.2 mV, with absolute values ​​all ≥20 mV, placing them within the colloidal stability range. The increase in particle size caused by the PEG hydration layer and the neutralization effect caused by the positive charge of TPP were both within a controllable range. Example 3-3, in particular, showed a colocalization coefficient of 0.83 ± 0.04, a particle size of 98.5 nm, a PDI of 0.12, and a Zeta potential of -23.5 mV. At this point, the surface TPP density was optimal, resulting in the highest targeting efficiency. The colocation coefficients in Examples 3-4 decreased slightly from the peak value to 0.80±0.06, the particle size and PDI increased slightly, and the absolute value of the Zeta potential decreased slightly, indicating that the PEG chains tended to be denser and some TPP groups began to be masked, but all indicators still met the target requirements.

[0072] The particle size rebounded to 112.5 nm, the PDI increased to 0.25, the Zeta potential decreased to -19.5 mV, and the colocalization coefficient decreased to 0.68 ± 0.07 in Comparative Example 3-2. This was due to the excessive addition of TPP-mPEG: on the one hand, the PEG chains were overcrowded on the nanoparticle surface, and some TPP groups were shielded by adjacent PEG chains or buried inside the PEG layer, reducing the accessibility of negatively charged sites on the mitochondrial membrane; on the other hand, the excessive neutralization of the positive charge of TPP caused the absolute value of the Zeta potential to drop below the threshold of 20 mV. Insufficient electrostatic repulsion led to partial aggregation of nanoparticles and a reduction in the number of effective targeting units.

[0073] The function of step S6 is to remove free TPP-mPEG and displacement buffer system from the nanoparticle modification solution through secondary dialysis, and then convert the nanoparticle dispersion into a solid powder formulation with long-term storage stability through gradient freeze drying process, thereby achieving the final shaping of nanoparticles.

[0074] Specifically, the dialysate for the secondary dialysis is PBS buffer, the dialysis temperature is 4℃-10℃, the dialysis time is 12h-18h, and the lyophilization process includes: The nanoparticle modification liquid was dispensed into freeze-drying containers, placed on the shelf of a freeze dryer, and the shelf temperature was lowered to -40°C to -50°C for 4 to 6 hours of pre-freezing. After pre-freezing, start the vacuum pump to raise the shelf temperature to -20℃ to -30℃, control the vacuum degree to 10Pa-20Pa, and carry out a drying cycle of 20-28 hours. After the first drying cycle, raise the shelf temperature to 20℃-30℃, control the vacuum degree to 1Pa-5Pa, and perform a second drying cycle for 4-8 hours.

[0075] Understandably, freeze-drying is a drying process involving the sublimation of water under vacuum after low-temperature freezing. This step employs a three-stage gradient freeze-drying process: 1. Pre-freezing: rapid freezing into fine, uniform ice crystals to protect the nanoparticle structure; 2. First drying: sublimation of free ice crystals to remove 90%-95% of the water; 3. Second drying: removal of bound water, resulting in a water content of <3%, forming a loose cake for easy long-term storage and reconstitution. The nanoparticle-modified solution obtained in step S5 still contains excess free TPP-mPEG and a small amount of salt ions. The second dialysis uses dialysis bags with a molecular weight cutoff of 8000-14000 Da, with PBS buffer as the dialysis fluid. Dialysis is performed at 4℃-10℃ for 12-18 hours to achieve the following: 1. Removal of free TPP-mPEG with a molecular weight of approximately 650 Da, while TPP-mPEG stably adsorbed on the nanoparticle surface is retained along with the nanoparticles; 2. Replacement of the dispersion with a PBS buffer system, allowing the PEG chains to fully expand and form a stable hydration layer, maintaining electrostatic adsorption balance; 3. Adjustment of ionic strength to shield excessive negative charge, avoiding aggregation caused by excessive electrostatic repulsion or excessively high ionic strength. The PBS buffer used in the second dialysis has a concentration of 0.01M and a low ionic strength of approximately 0.02mol / L, providing adequate shielding and preventing the absolute value of the nanoparticle zeta potential from falling below the stable range of 20mV. After two dialysis cycles, the measured Zeta potential of the nanoparticles was -23.15 ± 5.83 mV, indicating that the surface charge of the nanoparticles was adequately shielded at this ionic strength, the PEG chains were fully extended, and the colloidal stability was good. If PBS with excessively high ionic strength is used, the absolute value of the Zeta potential may drop below 20 mV due to excessive compression of the electric double layer, leading to nanoparticle aggregation.

[0076] In this embodiment, step S6 includes the following specific steps: Step S61: Take the nanoparticle modification solution obtained in step S52, transfer it to a dialysis bag with a molecular weight cutoff of 8000 Da-14000 Da, and dialyze it in 0.01 M PBS buffer pre-cooled at 4℃. The volume of PBS buffer is 100 times that of the nanoparticle modification solution. Dialyze at 4℃ in the dark for 14 h, changing the PBS buffer 3 times during the process, once every 4 h. Step S62: After dialysis, take a sample of the liquid inside the bag to measure the particle size and Zeta potential to confirm that the nanoparticles are well dispersed in PBS buffer. Step S63: Dispense the dialysis nanoparticle modification solution into lyophilized vials, each vial containing approximately 1 / 3 to 1 / 2 of the vial's volume. After partially sealing the vials, place them on the shelf of a lyophilizer that has been cooled to -45°C and pre-freeze them at -45°C for 5 hours until the samples are completely frozen. After pre-freezing, turn on the vacuum pump to raise the shelf temperature to -25°C, control the vacuum degree to 15Pa, and perform a drying process for 24 hours. After the first drying cycle, the shelf temperature is raised to 25°C, the vacuum degree is controlled at 3Pa, and a second drying cycle is performed for 6 hours. After the secondary drying is completed, the powder is plugged and removed from the box under vacuum to obtain lyophilized nanoparticles that target residual liver cancer tissue.

[0077] Examples and Comparative Examples

[0078] Example 1

[0079] Preparation of inhibitor nanoparticles targeting residual hepatocellular carcinoma tissue: In this embodiment, inhibitor nanoparticles (QCC-TPP) targeting residual hepatocellular carcinoma tissue were prepared according to the following steps: S1: Weigh 930 mg of quercetin, add 60 mL of 0.05 mol / L sodium hydroxide solution, adjust the pH to 10.5 with 1 mol / L sodium hydroxide solution, and sonicate for 10 min to obtain sodium quercetin solution. S2: Prepare a 0.3 mol / L copper chloride solution. Based on the molar ratio of quercetin to copper chloride of 1:5.5, add approximately 56.5 mL of copper chloride solution to the sodium quercetin solution at a rate of 1 drop / second. Nitrogen gas is purged throughout the addition process. Simultaneously, 1 mol / L sodium hydroxide solution is added online to maintain the pH of the system within the range of 10.0-10.5. The reaction is stirred at 25°C and 700 rpm for 3 hours to allow copper ions to form a chelate structure with quercetin, resulting in a copper-polyphenol network dispersion. S3: Prepare a calcium chloride solution with a concentration of 0.1 mol / L. Based on the molar ratio of quercetin to calcium chloride of 1:0.6, add about 18.5 mL of calcium chloride solution to the copper-polyphenol network dispersion. Maintain a nitrogen atmosphere during the addition process and continue stirring for 0.75 h to allow calcium ions to combine with the copper-polyphenol network, thereby obtaining a bimetallic polyphenol network dispersion. S4: The bimetallic polyphenol network dispersion is transferred to a dialysis bag with a molecular weight cutoff of 10000 Da, and dialyzed with 80 times the volume of deionized water at 25°C for 24 hours. The dialysate is replaced every 6 hours to remove unreacted metal ions and free quercetin, and a core nanoparticle dispersion is obtained. S5: Add approximately 240 mg of TPP-mPEG to the core nanoparticle dispersion and stir the mixture at 25°C and 500 rpm for 2.5 h to modify it through electrostatic interaction, thus obtaining a nanoparticle modified solution. S6: Transfer the nanoparticle modification solution to a new dialysis bag, use PBS buffer as the external dialysis solution, dialyze at 4°C for 14 hours, and then freeze-dry to obtain the inhibitor nanoparticles targeting residual liver cancer tissue. The freeze-drying process is as follows: first pre-freeze at -45°C for 5 hours, then dry once at -25°C and 15 Pa for 24 hours, and finally dry a second time at 25°C and 3 Pa for 6 hours.

[0080] Comparative Example 1

[0081] Calcium-free nanoparticles (QC-TPP): The difference from Example 1 is that calcium chloride solution is not added in step S3, while the other conditions are the same as in Example 1. After preparation, the calcium content was confirmed to be 0 by ICP-AES detection.

[0082] Comparative Example 2

[0083] Copper-free nanoparticles (QCa-TPP): The difference from Example 1 is that copper chloride solution is not added in step S2, and only calcium chloride solution is added in step S3. The other conditions are the same as in Example 1.

[0084] The preparation results showed that the comparative system was clear and transparent, with no detectable nanoparticles by dynamic light scattering (DLS). This result indicates that calcium ions alone cannot form a stable nanonetwork structure with quercetin, thus confirming the crucial scaffolding role of copper ions in the construction of the MPN network.

[0085] Since this comparative ratio did not form nanoparticles, its actual drug delivery system consisted of free quercetin and Ca. 2+ A mixed solution. The ROS production, apoptosis rate, and tumor inhibition rate exhibited by this mixed solution in in vitro cell experiments and in vivo pharmacodynamic experiments originated from the inherent antitumor activity of free quercetin and Ca2+. 2+ The weak synergistic effect of free quercetin and calcium overload, rather than the synergistic killing effect of copper death and calcium overload mediated by nanostructures, was observed. Its apoptosis rate (12.4±2.8%) and tumor inhibition rate (21.3±4.2%) were significantly lower than those in Example 1, further demonstrating that only free quercetin and calcium... 2+ Without the copper-polyphenol nanonetwork structure, an effective synergistic anti-tumor effect cannot be achieved.

[0086] Comparative Example 3

[0087] Untargeted nanoparticles (QCC): The difference from Example 1 is that TPP-mPEG is not added in step S5, while the other conditions are the same as in Example 1.

[0088] Laser confocal microscopy revealed that the mitochondrial colocalization coefficient of this comparative example was 0.38±0.06, significantly lower than that of Example 1 (0.83±0.04), indicating a substantial decrease in the mitochondrial targeting efficiency of the nanoparticles without TPP-mPEG modification.

[0089] Comparative Example 4

[0090] Direct physical mixing of raw materials (QCC-TPP+Mix): The difference from Example 1 is that the S2 coordination reaction and S5 surface modification steps are not performed; instead, all components are mixed at the same feed ratio. The specific preparation method is as follows: Following the feed ratio of Example 1, quercetin is dissolved in NaOH solution (pH=10.5) according to step S1. Then, CuCl2 solution, CaCl2 solution, and TPP-mPEG are added sequentially. After stirring at room temperature for 30 minutes, the mixture is directly freeze-dried to obtain a non-coordination physical mixture control sample.

[0091] In this sample, quercetin did not fully coordinate with metal ions, nor was there any effective surface modification by TPP-mPEG, serving as a control for the synergistic effect of nanostructures.

[0092] Example 2

[0093] The nanoparticles prepared in Example 1 were characterized and their performance tested as follows: Please see Figure 2 As shown, it represents the morphology and particle size characterization of the QCC and QCC-TPP of the present invention.

[0094] (1) Morphology and particle size: The morphology and particle size of the nanoparticles were characterized using scanning electron microscopy (SEM) and dynamic light scattering (DLS). Figure 2 As can be seen from A, QCC exhibits a relatively regular cubic structure, while QCC-TPP modified with TPP exhibits a spherical or near-spherical structure with good dispersibility. SEM observation shows that its particle size is about 90 nm. Figure 2 The DLS results of B showed that the peak hydrated particle size of QCC was about 80 nm, and the peak hydrated particle size of QCC-TPP was about 100 nm, which was basically consistent with the SEM observation results, indicating that the successful modification of TPP slightly increased the particle size of nanoparticles.

[0095] Further quantitative analysis of QCC-TPP revealed an average hydrated particle size of 104.81 nm (±19.36 nm) across three independent parallel measurements, with a polydispersity index (PDI) of 0.10. This PDI value indicates a narrow particle size distribution and uniform dispersion of the nanoparticles. Furthermore, the small standard deviation (±19.36 nm) across the three parallel measurements confirms the good batch-to-batch reproducibility of the preparation process. Combined with Zeta potential data, this demonstrates the successful construction of QCC-TPP nanoparticles exhibiting excellent morphology and colloidal stability.

[0096] (2) Elemental composition: The copper and calcium contents in the nanoparticles were determined by inductively coupled plasma atomic emission spectrometry. The results showed that the copper content in the nanoparticles was 31 wt%, the calcium content was 2 wt%, and the remainder consisted of quercetin, TPP-mPEG, coordinated oxygen, hydroxyl groups, water of crystallization, and counterions. Quercetin and TPP-mPEG are organic components, and their specific contents can be determined by methods known in the art, such as elemental analysis or thermogravimetric analysis.

[0097] (3) Surface potential: The average Zeta potential of the nanoparticles was measured to be -23.15±5.83mV by the Zeta potential analyzer.

[0098] (4) Spectral characteristics: The UV-Vis absorption spectrum shows that, compared with free quercetin, the characteristic absorption peak at 370 nm in the nanoparticles is significantly weakened, while a new absorption peak appears at about 300 nm, indicating that quercetin reacts with Cu. 2+ Coordination occurred. Infrared spectroscopy showed that the stretching vibration peak of the phenolic hydroxyl group (OH) of quercetin decreased from 3371 cm⁻¹. -1 Displaced to approximately 3415cm -1 Furthermore, the peak shape broadened significantly and the peak intensity decreased; simultaneously, the quercetin C=O stretching vibration peak decreased from 1660 cm⁻¹. -1 Displaced to approximately 1620cm -1 The Ar-O stretching vibration peak decreased from 1245 cm⁻¹ -1 Displaced to approximately 1210cm -1 This further confirms that phenolic hydroxyl and carbonyl groups are involved in Cu 2+ Coordination. XPS O 1s spectra show that the hydroxyl oxygen peak at 531.2 eV in quercetin shifts to 529.8 eV after coordination, and the peak area ratio changes, indicating that the phenolic hydroxyl oxygen reacts with Cu. 2+ Coordinate bonds are formed.

[0099] (5) Active oxygen generation capacity: Electron paramagnetic resonance detection showed that nanoparticles can generate hydroxyl radicals •OH in the presence of H2O2; methylene blue degradation experiment showed that as the incubation time was extended, the absorbance of methylene blue at 664 nm gradually decreased, indicating that •OH was continuously generated.

[0100] (6) Glutathione consumption capacity: GSH consumption experiment was conducted using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as an indicator. The results showed that as the concentration of nanoparticles increased, the absorbance at 412 nm gradually decreased, indicating that GSH consumption was concentration-dependent.

[0101] (7) pH-responsive release: Using the dialysis bag method, the molecular weight cutoff of 10000 Da was retained. The Cu content in QCC-TPP nanoparticles was measured in release media containing 10% fetal bovine serum at pH 7.4 (simulating a normal physiological environment) and pH 5.0 (simulating a tumor-like slightly acidic environment). 2+ Ca 2+ The cumulative release rate of Cu and quercetin over 72 hours was determined. The volume of the release medium was 50 times that of the dispersion in the dialysis bag. The mixture was shaken at 37°C and 100 rpm in the dark. Samples were taken at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours, with an equal volume of fresh release medium added after each sampling. The results showed that Cu... 2+ Ca 2+ The cumulative release rates of quercetin, amino acids, and quercetin were 82.6%, 78.3%, and 85.2%, respectively, all significantly higher than those at pH 7.4 (22.4%, 31.8%, and 15.6%, respectively) (P<0.01), indicating that the nanoparticles exhibit a significant tumor microacid-responsive release characteristic. Each group had three parallel samples, and the experiment was independently repeated three times. Results are expressed as mean ± standard deviation (n=3).

[0102] (8) Lyophilization and Reconstitution Performance: The lyophilized powder obtained in Example 1 was reconstituted with deionized water to its original volume. After gentle shaking, it was allowed to stand at room temperature for 5 minutes. The hydrated particle size and PDI after reconstitution were measured using dynamic light scattering. The results showed that the hydrated particle size after reconstitution was 108.3 ± 13.6 nm, and the PDI was 0.11, which was not significantly different from that after lyophilization, indicating that the lyophilization process did not have an irreversible effect on the dispersibility of the nanoparticles. The results of three independent measurements are expressed as mean ± standard deviation, and n = 3 independent measurements.

[0103] Example 3

[0104] In vitro cell experiments using nanoparticles were conducted using HepG2 human liver cancer cells. Unless otherwise specified, each in vitro cell experiment had three independent replicates, and the experiments were independently repeated three times. Results are expressed as mean ± standard deviation (n=3). Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test. Student's t-test was used for comparisons between two groups, and P < 0.05 was considered statistically significant.

[0105] (1) Cell uptake experiment Please see Figure 3 As shown, it is a CLSM image of the in vitro cell phagocytosis experiment of QCC-TPP of the present invention.

[0106] QCC-TPP was co-incubated with HepG2 cells, and the results were observed using a confocal laser scanning microscope (CLSM) at 0h, 0.5h, 1h, 2h, and 3h. The results showed that as the incubation time increased, the red fluorescence representing QCC-TPP in the cells gradually increased, and the fluorescence intensity reached the highest value during the observation period at 3h, indicating that the nanoparticles were effectively taken up by the cells.

[0107] (2) Detection of reactive oxygen species (ROS) generation Please see Figure 4 As shown, this is a CLSM observation of ROS generation in tumor cells of various groups in this invention.

[0108] The generation of ROS in tumor cells of each group was detected using the DCFH-DA fluorescent probe. The results showed that the mean fluorescence intensity (MFI) of the blank control group was 12.3±2.1; the MFI of the QC-TPP group was 45.6±5.8, and the MFI of the QCC-TPP group was 68.4±7.2, indicating that both the QC-TPP and QCC-TPP groups could generate ROS through a Fenton-like reaction, and the green fluorescence intensity was significantly higher than that of the blank control group (P<0.01). The MFI of the QCC-TPP combined with ultrasound (US) group was 89.7±8.5, indicating that ultrasound intervention can promote intracellular uptake of nanoparticles and Fenton-like reaction activity. The above MFI data were quantitatively determined using a microplate reader. Each group had three independent replicates, and the experiment was independently repeated three times. The results are expressed as mean ± standard deviation (n=3).

[0109] (3) Detection of mitochondrial membrane potential Please see Figure 5 As shown, these are CLSM images of mitochondrial membrane potential in various groups of tumor cells according to the present invention. JC-1 staining was followed by observation using CLSM. The results showed that QC-TPP, QCa-TPP, and QCC-TPP groups all altered mitochondrial membrane potential, manifested as decreased red fluorescence (JC-1 aggregates) and increased green fluorescence (JC-1 monomers). Among them, the QCC-TPP+US group showed the most significant effect, with the most pronounced decrease in red fluorescence and increase in green fluorescence, demonstrating that both copper and calcium ions have the ability to alter mitochondrial membrane potential, and the combined effect of the two is stronger. Ultrasound intervention can further enhance this effect.

[0110] (4) Apoptosis detection The apoptosis rate of cells in each group was detected by flow cytometry using Annexin V-FITC / PI double staining. The results showed that the apoptosis rate was 4.2±1.3% in the blank control group; 18.6±3.2% in the QC-TPP group; 12.4±2.8% in the QCa-TPP group; 22.3±3.5% in the QCC group; 8.7±2.1% in the physical mixture group; 35.8±4.2% in the QCC-TPP group; and 52.3±5.1% in the QCC-TPP+US group, significantly higher than the other groups. Each group had three independent replicates, and the experiment was independently repeated three times. Results are expressed as mean ± standard deviation (n=3). This indicates that copper-calcium bimetallic synergy and mitochondrial-targeted modification can significantly enhance the antitumor effect, and ultrasound-enhanced permeation can further amplify this effect.

[0111] The in vitro experimental results above show that the QCC-TPP nanoparticles prepared in this invention possess excellent cellular uptake capacity, mitochondrial targeting ability, and synergistic antitumor activity. Compared with the comparative example, QCC-TPP significantly increased ROS production, mitochondrial membrane potential disruption, and apoptosis rate through copper-calcium bimetallic synergy and mitochondrial targeting modification; ultrasound-enhanced permeation further promoted intracellular uptake and deep penetration of the nanoparticles, enhancing the therapeutic effect.

[0112] Example 4

[0113] Research on multimodal molecular imaging of mitochondrial-targeted bimetallic-polyphenol network nanoparticles.

[0114] Please see Figure 6 As shown, it is a multimodal molecular imaging of nanoparticles according to the present invention.

[0115] The multimodal imaging capability of QCC-TPP was evaluated. In in vitro gel imaging experiments, QCC-TPP exhibited strong absorption in the photoacoustic excitation wavelength range, demonstrating high-resolution in vitro photoacoustic imaging performance. Furthermore, the photoacoustic signal intensity increased with increasing nanoparticle concentration. Figure 6 A). Fluorescently labeled nanoparticles exhibited good in vitro fluorescence imaging capabilities ( Figure 6 B) proves that the fluorescent labeling was successful.

[0116] Based on its targeted penetration ability, the in vivo biodistribution was further evaluated. In vivo photoacoustic and fluorescence imaging results showed that the fluorescence signal began to concentrate in the tumor area 3 hours after nanoparticle injection, reaching a peak at 6 hours, maintaining a strong signal at 10 hours, and gradually weakening at 24 hours. This confirmed that QCC-TPP possesses photoacoustic imaging capabilities and good in vivo targeting function, achieving basic tumor targeting within 6 hours. Figure 6 C, 6D).

[0117] In addition, the oxygenated hemoglobin level of the tumor was detected in tumor-bearing mice after photoacoustic imaging of nanoparticles at different time points to assess microvascular volume and degree of hypoxia. Figure 6 E). The results showed that nanoparticles can effectively alter the oxygenation status of the tumor microenvironment, reflecting the dynamic changes in tumor microvascular volume and hypoxia level.

[0118] Example 5

[0119] In vivo antitumor efficacy experiment of nanoparticles.

[0120] (1) In vivo antitumor efficacy Female BALB / c nude mice, aged 4-6 weeks and weighing 18-22g, were housed in SPF-grade conditions at a temperature of 22±2℃ and humidity of 50±10%, with a 12-hour light-dark cycle and free access to food and water. HepG2 cells in the logarithmic growth phase were harvested and cultured at a concentration of 5 × 10⁻⁶ cells / mL. 6 Cells / 100μL PBS were subcutaneously injected into the right axilla of nude mice. Approximately 7-10 days post-inoculation, when the tumor volume reached approximately 100mm², [the tumor was observed]. 3 At that time, tumor-bearing nude mice were randomly divided into 7 groups: blank control group PBS, QC-TPP group, QCa-TPP group, QCC group, QCC-TPP group, QCC-TPP+US group, and ultrasound group alone. There were 6 mice in each group (n=6).

[0121] Except for the ultrasound-only group, all other groups received intravenous injection of the corresponding nanoparticles via tail vein at a dose of 10 mg / kg based on the total mass of the nanoparticles, administered once every 3 days for a total of 5 times. The QCC-TPP+US group received ultrasound treatment 10 minutes after each administration: frequency 1 MHz, intensity 1.0 W / cm² (ISATA). 2 The treatment lasted for 5 minutes. The ultrasound-only group received only ultrasound treatment without nanoparticle administration. Tumor volume and mouse weight changes were recorded until day 15. Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and Student's t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0122] The specific procedure for ultrasound-enhanced penetration technology is as follows: 10 minutes after the nanoparticles are injected via the tail vein, an ultrasound coupling agent is applied to the surface of the tumor site, using a frequency of 1MHz and an intensity of 1.0W / cm². 2 The medical ultrasound treatment probe is vertically aligned with the tumor area, and continuous ultrasound treatment is performed for 5 minutes. During the ultrasound treatment, good coupling between the probe and the skin is maintained to avoid excessive heat generation; the temperature of the tumor area is monitored during the treatment to ensure that the temperature rise does not exceed 1°C.

[0123] The drugs were administered once every 3 days for a total of 5 times. The observation period continued until day 15, during which tumor volume and mouse body weight changes were recorded. The results showed that the tumor inhibition rate in the ultrasound-only group was -1.2% ± 4.3%, which was not significantly different from the blank control group (P>0.05), indicating that ultrasound itself had no direct effect on tumor growth. In contrast, the QCC-TPP+US group showed a strong tumor growth inhibition effect.

[0124] (2) Histological analysis After treatment, tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were collected for H&E staining. The results showed that large areas of necrosis were observed in the tumor tissue of the QCC-TPP+US group, while no obvious pathological changes were observed in the major organs, further demonstrating that the nanoparticles have good antitumor activity and biosafety.

[0125] The above results indicate that QCC-TPP has good tumor-targeting enrichment, multimodal imaging and anti-tumor effects in the HepG2 subcutaneous xenograft tumor model, providing a basis for subsequent validation of residual liver cancer models.

[0126] Example 6

[0127] Construction of residual liver cancer model and targeted enrichment experiment.

[0128] Female BALB / c nude mice aged 4-6 weeks were used and housed in SPF condition. An orthotopic hepatocellular carcinoma model was established using HepG2-Luc cells, with an inoculum size of 5 × 10⁻⁶ cells. 6 Cells / 50μL PBS were inoculated into the liver lobe to establish an orthotopic hepatocellular carcinoma model. The model was established when the tumor grew to approximately 100mm. 3 -150mm 3 After partial resection or ablation, approximately 10%-20% of the tumor tissue is preserved as residual hepatocellular carcinoma tissue. The presence of fluorescence signal in the residual lesions is confirmed by in vivo imaging 24 hours post-surgery, thus establishing a residual hepatocellular carcinoma model.

[0129] Nude mice with residual liver cancer were randomly divided into 6 groups, with 6 mice in each group: Group A: PBS control group; Group B: QCC-TPP group; Group C: QCC-TPP+US group; Group D: QCC group; Group E: QC-TPP group; Group F: QCa-TPP group.

[0130] Each group received an intravenous injection of the corresponding nanoparticles via the tail vein at a dose of 10 mg / kg based on the total mass of the nanoparticles, administered once every 3 days for a total of 5 times. The QCC-TPP+US group received ultrasound treatment 10 minutes after each administration: frequency 1 MHz, intensity 1.0 W / cm². 2 The action time is 5 minutes, and the probe is vertically aimed at the residual liver cancer area.

[0131] Six hours after drug administration, the fluorescence signal ratio of residual hepatocellular carcinoma tissue to normal liver tissue was 1.0±0.1 in the PBS group, 4.8±0.6 in the QCC-TPP group, 7.9±0.9 in the QCC-TPP+US group, 3.1±0.4 in the QCC group, 3.8±0.5 in the QC-TPP group, and 1.4±0.2 in the QCa-TPP group. The QCC-TPP+US group was significantly higher than the other groups (P<0.05). The photoacoustic imaging results showed a consistent trend with the fluorescence imaging results.

[0132] Regarding mitochondrial colocalization coefficients, the coefficients were 0.86±0.04 for the QCC-TPP+US group, 0.83±0.04 for the QCC-TPP group, 0.38±0.06 for the QCC group, 0.79±0.05 for the QC-TPP group, 0.10±0.02 for the QCa-TPP group, and 0.05±0.01 for the PBS group. The coefficients for the QCC-TPP+US and QCC-TPP groups were significantly higher than those for the QCC group (P<0.05), demonstrating that TPP-mPEG modification can significantly improve mitochondrial targeting efficiency.

[0133] The data are expressed as mean ± standard deviation, with n=6 for each group. One-way ANOVA was used for comparisons among multiple groups, and Student's t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant. These results indicate that QCC-TPP combined with ultrasound can effectively enrich residual hepatocellular carcinoma tissue. Example 7

[0134] The experiment on targeted enrichment of residual hepatocellular carcinoma tissue verified the enrichment and mitochondrial targeting capabilities of QCC-TPP combined with ultrasound in residual hepatocellular carcinoma tissue.

[0135] Using the residual hepatocellular carcinoma model from Example 6, patients were randomly divided into four groups: PBS group, QCC-TPP group, QCC-TPP+US group, QCC group, QC-TPP group, and QCa-TPP group, with n=6 in each group. The drug administration regimen was the same as in Example 6. The QCC-TPP+US group received ultrasound treatment 10 min after drug administration: frequency 1 MHz, intensity 1.0 W / cm². 2 The drug was administered for 5 minutes. The residual lesion / normal liver signal ratio was measured 6 hours after drug administration, and the mitochondrial colocalization coefficient was calculated.

[0136] Table 4 shows the data on targeted enrichment and mitochondrial colocalization of residual liver cancer tissue in each group.

[0137] Table 4. Data on targeted enrichment and mitochondrial colocalization of residual hepatocellular carcinoma tissue in each group.

[0138] Note in Table 4: The data in Table 4 are from the residual liver cancer model of Example 6 (HepG2-Luc orthotopic liver cancer postoperative residual model), with n=6 for each group; the data in Table 6 are from the HepG2 subcutaneous xenograft tumor model of Example 5, and the two groups are independent experiments.

[0139] As shown in Table 4, the residual lesion / normal liver signal ratio and mitochondrial colocalization coefficient of the QCC-TPP+US group were significantly higher than those of the QCC-TPP group, QCC group, and QCa-TPP group (P<0.05). These results indicate that QCC-TPP combined with ultrasound can effectively enrich residual hepatocellular carcinoma tissue, and TPP-mPEG modification can significantly improve mitochondrial targeting efficiency.

[0140] Example 8

[0141] The targeting mechanism of residual hepatocellular carcinoma tissue was validated, and the effects of EPR effect, ultrasound-enhanced permeation and TPP-mPEG mitochondrial targeting on the enrichment of residual hepatocellular carcinoma tissue were verified.

[0142] The following groups were set up: QCC-TPP+US group, QCC-TPP group, QCC group, QCC-TPP+US+fibrosis inhibitor group, QCC-TPP+US+mitochondrial membrane potential inhibitor group, and ultrasound-only group. The residual lesion / normal liver signal ratio, mitochondrial colocalization coefficient, and nanoparticle penetration depth were measured.

[0143] Table 5 shows the data on target enrichment, mitochondrial colocalization, and penetration depth for each group.

[0144] Table 5. Data on target enrichment, mitochondrial colocalization, and penetration depth for each group.

[0145] As shown in Table 5, the signal ratio and penetration depth of the QCC-TPP+US group were significantly higher than those of the QCC-TPP and QCC groups (P<0.05). The signal ratio of the fibrosis inhibitor group was further increased, indicating that the fibrosis barrier restricts nanoparticle penetration. The colocalization coefficient of the mitochondrial membrane potential inhibitor group was significantly reduced, proving that TPP-mPEG achieves mitochondrial targeting by relying on the negative transmembrane potential difference of mitochondria.

[0146] Overall beneficial effects analysis results

[0147] To comprehensively evaluate the overall performance of the technical solution of the present invention, statistical analysis was performed on the detection results of Example 1, Comparative Examples 1-4, and the QCC-TPP+US group.

[0148] Table 6. Comparison of physicochemical properties, in vitro cellular and in vivo efficacy of Example 1, Comparative Examples 1-4 and QCC-TPP+US group

[0149] Table 6 Note: In vivo experiment n=6, animals were 4-6 week old female BALB / c nude mice, HepG2 subcutaneous xenograft tumor model, and the drug dosage was 10mg / kg.

[0150] As shown in the table above, Example 1 (QCC-TPP) outperforms all the comparative examples in all indicators: (1) QC-TPP: The physicochemical properties are basically the same, but QCC-TPP has a higher ROS production capacity and apoptosis rate, which confirms that Ca 2+ Loading contributes significantly to the synergistic killing effect of Fenton-like activity and calcium overload.

[0151] (2) QCa-TPP: QCa-TPP cannot form stable nanoparticles; the actual drug delivery is free quercetin and Ca. 2+ The mixed solution exhibited the lowest ROS production, apoptosis rate, and tumor inhibition rate, all lower than in Example 1, confirming that Ca... 2+ It is the core skeleton of the network construction. Without the copper-polyphenol nanonetwork structure, the synergistic killing effect of copper death and calcium overload cannot be achieved.

[0152] (3) QCC: Both QCC and QCC-TPP formed nanostructures, but the Zeta potential of QCC was -15mV to -20mV, which was significantly lower than that of QCC-TPP (-25mV to -23mV). The ROS production of QCC was 56.2±6.1, which was about 18% lower than that of QCC-TPP (68.4±7.2), and the difference was statistically significant (P<0.05). The apoptosis rate and tumor inhibition rate of QCC-TPP were significantly higher than those of QCC, confirming the necessity of TPP-mPEG-mediated mitochondrial targeting for improving efficacy.

[0153] (4) Physical mixing: All indicators of the physical mixing group were the worst, confirming that raw materials without nanostructures cannot produce effective anti-tumor activity.

[0154] (5) QCC-TPP+US: All indicators were optimal, confirming that ultrasound-induced permeation can enhance the intracellular uptake and anti-tumor effect of nanoparticles, showing a synergistic effect between nanoparticles and ultrasound. In the residual liver cancer model, the residual lesion / normal liver signal ratio and mitochondrial colocalization coefficient of the QCC-TPP+US group were significantly higher than those of other groups (P<0.05). The tumor inhibition rate of the ultrasound-only group was -1.2%±4.3%, which was not significantly different from the blank control group (P>0.05), indicating that ultrasound itself has no direct effect on tumor growth. The observed increase in tumor inhibition rate of the QCC-TPP+US group is attributed to the enhanced tumor uptake of nanoparticles by ultrasound-induced permeation.

[0155] Explanation of the overall beneficial effects

[0156] I. Synergistic Mechanism of Stepwise Bimetallic Assembly Structure of Single Polyphenol Ligand and Responsive Release from Tumor Microenvironment This invention uses quercetin as a single polyphenol ligand, leveraging its differentiated coordination abilities with copper and calcium ions. A stepwise assembly strategy is employed, first constructing the framework through copper coordination, then binding with calcium, to form a copper-calcium bimetallic polyphenol network. Unlike the heterogeneous composite structures of CaCO3 support and copper-coordinated polymers in existing technologies, in this invention, Cu and Ca coexist within the same polyphenol network, exhibiting uniform distribution at the molecular scale. The kinetics of dual-ion release during acid dissociation are matched, avoiding the decrease in synergistic effect caused by differences in release rates in heterogeneous structures.

[0157] In the slightly acidic environment of tumors, the quercetin-copper coordination bond undergoes acid dissociation, releasing copper ions from the backbone and causing the network structure to disintegrate. Simultaneously, calcium ions are released, forming a synergistic release of two ions. Copper ions generate hydroxyl radicals through a Fenton-like reaction, leading to a burst of reactive oxygen species and triggering copper death. Calcium ions influx into the mitochondrial matrix, disrupting the mitochondrial membrane potential and inducing mitochondrial calcium overload and apoptosis. These two ions synergistically kill tumor cells through a dual pathway of copper death and calcium overload. Experimental data show that the apoptosis rate of QCC-TPP cells in Example 1 was 35.8%, and the tumor inhibition rate was 51.2%, higher than that of the calcium-deficient control example 1 QC-TPP and the copper-deficient control example 2 QCa-TPP, confirming that the copper-calcium bimetallic polyphenol network structure formed by stepwise assembly of a single polyphenol ligand makes a crucial contribution to synergistic antitumor activity. Compared with the heterogeneous composite structure of CaCO3 carrier and copper coordination polymer in existing technologies, the single polyphenol network structure of this invention allows for a uniform distribution of Cu and Ca at the molecular scale, resulting in a better match between the two ion release kinetics and superior structural uniformity.

[0158] II. Electrostatic Modification of TPP-mPEG Surface and the Mechanism of Active Mitochondrial Targeting Delivery This invention utilizes the electrostatic attraction and cation-π interaction between the negative charge on the nanoparticle surface and the quaternary cations in the TPP-mPEG molecule to achieve the spontaneous self-assembly of the targeting ligand on the nanoparticle surface. The mPEG segments extend outward to form a brush-like hydration layer, endowing the nanoparticles with stealth properties and long-cycle capability. Based on the unique transmembrane negative potential difference of the mitochondrial inner membrane, the TPP group precisely delivers the nanoparticles and their loaded copper and calcium ions to the mitochondria, achieving targeted delivery of copper death and calcium overload effectors at the mitochondrial level. Experimental data show that the mitochondrial colocalization coefficient of Example 1 with TPP-mPEG targeting modification is 0.83, higher than that of Comparative Example 3 without targeting modification; the tumor inhibition rate is 51.2%, higher than QCC's 38.6%, confirming the necessity of TPP-mPEG-mediated active mitochondrial targeting for improved efficacy.

[0159] III. The Principle of Ultrasound-Enhanced Penetration Physical Enhancement and Spatiotemporal Synergistic Therapy This invention utilizes the acoustic pore effect and microfluidic action generated by low-frequency, low-intensity ultrasound in tumor tissue to enhance the transcellular transport and deep tissue penetration of nanoparticles. Ultrasound induces the formation of transiently reversible acoustic pores on the surface of tumor cell membranes, allowing nanoparticles to bypass endocytosis and directly enter the cytoplasm, improving intracellular delivery efficiency; simultaneously, it disturbs the interstitial pressure of the tumor, promoting the penetration of nanoparticles into deeper regions of the tumor and overcoming the permeability barrier of solid tumors.

[0160] Ultrasound-enhanced penetration, mitochondrial targeting, and copper death / calcium overload form a spatiotemporal synergistic therapeutic loop: ultrasound opens physical barriers at the tissue level, promoting deep penetration of nanoparticles; TPP-mPEG precisely guides nanoparticles to mitochondria at the organelle level; copper and calcium ions achieve synergistic killing through dual pathways at the mitochondrial level. Experimental data show that the apoptosis rate in the QCC-TPP combined with ultrasound group was 52.3%, significantly higher than the 35.8% in the QCC-TPP group; the tumor inhibition rate was 69.8%, significantly higher than the 51.2% in the QCC-TPP group; and the tumor necrosis area was 65.3%, higher than the 40%-50% in the QCC-TPP group, confirming that ultrasound-enhanced penetration can enhance the anti-tumor effect of nanoparticles. The synergistic effect of these three mechanisms achieves highly efficient, precise, and synergistic treatment of hepatocellular carcinoma.

[0161] IV. Targeted Inhibition Principle Against Residual Liver Cancer Tissue Residual hepatocellular carcinoma tissue is characterized by a fibrotic barrier, heterogeneous blood supply, and weakened EPR effect. The inhibitor nanoparticles of this invention achieve targeted inhibition of residual hepatocellular carcinoma tissue through the following three mechanisms: First, nanoparticles with a diameter of 90nm-125nm are enriched around residual vascular lesions through the EPR effect; Second, ultrasound-enhanced permeation produces acoustic pore effect and microfluidic action, which overcomes the fibrosis barrier of residual lesions and promotes the deep penetration of nanoparticles into residual liver cancer tissue. Third, the TPP-mPEG modified layer directs the nanoparticles to the mitochondria of residual liver cancer cells. Copper and calcium ions are released simultaneously in the tumor's slightly acidic environment, triggering copper death and mitochondrial calcium overload, respectively, and synergistically killing residual liver cancer cells.

[0162] In the residual hepatocellular carcinoma model, the signal ratio of residual lesions to normal liver tissue in the QCC-TPP+US group was significantly higher than that in the non-ultrasound group, and the mitochondrial colocalization coefficient was significantly improved, demonstrating that the present invention has targeted enrichment and mitochondrial targeting capabilities for residual hepatocellular carcinoma tissue. Combined with the tumor inhibition rate in the subcutaneous tumor model, the present invention achieves targeted inhibition of residual hepatocellular carcinoma tissue through the EPR effect, ultrasound-enhanced penetration, and TPP-mPEG mitochondrial targeting. The technical solution of the present invention has now been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions resulting from these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for preparing inhibitor nanoparticles targeting residual liver cancer tissue, characterized in that, Includes the following steps: Step S1: Dissolve quercetin in sodium hydroxide solution and adjust the pH to 9.0-12.0 to obtain quercetin sodium salt solution; Step S2: Add copper chloride solution dropwise to the sodium quercetin salt solution at a molar ratio of quercetin to copper chloride of 1:(5-6) to carry out the reaction, so that copper ions and quercetin form a chelate structure. During the reaction, sodium hydroxide solution is added dropwise to maintain the pH of the system at 9.0-12.0, and a copper-polyphenol network dispersion is obtained. Step S3: Add calcium chloride solution to the copper-polyphenol network dispersion at a molar ratio of quercetin to calcium chloride of 1:(0.5-0.7) to react and allow calcium ions to combine with the copper-polyphenol network to obtain a bimetallic polyphenol network dispersion. Step S4: The bimetallic polyphenol network dispersion is placed in a dialysis bag for dialysis to remove unreacted metal ions and free quercetin, resulting in a core nanoparticle dispersion. Step S5: Add mitochondrial-targeting modification molecule TPP-mPEG to the core nanoparticle dispersion at a molar ratio of TPP-mPEG to quercetin of (0.08-0.15):1 and stir. Modify the nanoparticles through electrostatic interaction to obtain the nanoparticle modification solution. Step S6: After a second dialysis of the nanoparticle-modified solution, the solution is freeze-dried to obtain the inhibitor nanoparticles targeting residual liver cancer tissue. The inhibitor nanoparticles are used to target and inhibit residual liver cancer tissue, which refers to small lesions, positive resection margins, satellite lesions, or high-risk microenvironments for recurrence remaining after surgical resection, ablation, TACE, or radiotherapy of primary liver cancer.

2. The method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to claim 1, characterized in that, In step S1, quercetin is first dissolved in a sodium hydroxide solution with a concentration of 0.01 mol / L to 0.1 mol / L, and then the pH of the system is adjusted to 9.0 to 12.0 with a sodium hydroxide solution with a concentration of 1 mol / L.

3. The method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to claim 2, characterized in that, In step S2, the reaction conditions are a reaction temperature of 20℃-30℃, a stirring speed of 500rpm-800rpm, and a stirring time of 2h-4h.

4. The method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to claim 3, characterized in that, In step S3, the reaction conditions are: reaction temperature 20℃-30℃, stirring speed 500rpm-800rpm, and stirring time 0.5h-1h.

5. The method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to claim 4, characterized in that, In step S4, the molecular weight cutoff of the dialysis bag is 8000Da-14000Da, the dialysis fluid is deionized water, the volume of the deionized water is 50-100 times the volume of the bimetallic polyphenol network dispersion, the dialysis temperature is 4℃-25℃, the dialysis time is 20h-28h, and the dialysis fluid is replaced every 6h-8h.

6. The method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to claim 5, characterized in that, In step S5, the molecular weight of the TPP-mPEG is 600Da-700Da; the surface modification temperature is 20℃-30℃, the stirring speed is 400rpm-600rpm, and the reaction time is 2h-3h.

7. The method for preparing inhibitor nanoparticles targeting residual liver cancer tissue according to claim 6, characterized in that, In step S6, the dialysate for the second dialysis is PBS buffer, the dialysis temperature is 4℃-10℃, the dialysis time is 12h-18h, and the lyophilization process includes: The nanoparticle modification liquid was dispensed into freeze-drying containers, placed on the shelf of a freeze dryer, and the shelf temperature was lowered to -40°C to -50°C for 4 to 6 hours of pre-freezing. After pre-freezing, start the vacuum pump to raise the shelf temperature to -20℃ to -30℃, control the vacuum degree to 10Pa-20Pa, and perform a drying cycle of 20-28 hours. After the first drying cycle, raise the shelf temperature to 20℃-30℃, control the vacuum degree to 1Pa-5Pa, and perform a second drying cycle for 4-8 hours.

8. An inhibitor nanoparticle targeting residual liver cancer tissue, characterized in that, Prepared by the method described in any one of claims 1-7; The hydrated particle size of the inhibitor nanoparticles is 90nm-125nm, PDI≤0.15, and Zeta potential is -25mV to -23mV.

9. The use of the inhibitor nanoparticles targeting residual liver cancer tissue according to claim 8 in the preparation of a medicament for targeting and inhibiting residual liver cancer tissue, characterized in that, The residual liver cancer tissue refers to small lesions, positive margin lesions, satellite lesions, or high-risk microenvironments remaining after surgical resection, ablation, TACE, or radiotherapy of primary liver cancer.

10. The use of the inhibitor nanoparticles targeting residual liver cancer tissue according to claim 9 in the preparation of a medicament for targeting and inhibiting residual liver cancer tissue, characterized in that, The inhibitor nanoparticles are used in combination with ultrasound-enhanced permeation technology, wherein the frequency of the ultrasound is 1MHz-3MHz and the intensity is 0.5W / cm² (space-averaged time-averaged sound intensity). 2 -1.5W / cm 2 The action time is 3-10 minutes.

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