Preparation methods and applications of multi-charge electrostatic self-assembled nanocomposites

Abstract

The application discloses a preparation method of a multi-charge electrostatic self-assembled nanocomposite and application thereof, and relates to the technical field of nanomaterials. The method comprises the following steps: (1) synthesizing CAT-Ce6; (2) synthesizing negative electric Ag2S-3MPA QDs; (3) synthesizing positive electric Ag2S-NH2 QDs; and (4) preparing Ag2S@CAT-Ce6@Oxa nanocomposite: ultrasonic mixing of Ag2S-NH2 QDs solution and CAT-Ce6 solution to obtain a mixed solution, dropwise adding Oxa dissolved in a mixture of methanol and DMSO into the mixed solution, stirring in a dark environment at room temperature to obtain Ag2S@CAT-Ce6@Oxa crude product, and purifying the Ag2S@CAT-Ce6@Oxa crude product to obtain Ag2S@CAT-Ce6@Oxa nanoparticle pure product. The application is helpful to realize controllable release of drugs, improve curative effect, effectively relieve tumor hypoxia in vivo, provide a better environment for photodynamic therapy and other treatments, realize accurate positioning and observation of tumors, effectively inhibit tumor growth and prolong survival time, improve curative effect and reduce side effects, and has good biocompatibility and wide application prospect in vivo.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a method for preparing multi-charge electrostatic self-assembled nanocomposite materials and their applications. Background Technology

[0002] Colorectal cancer (CRC) ranks third in incidence but second in mortality, making it a major disease burden worldwide. Traditional treatments for CRC, such as surgery and chemotherapy, often cause excessive damage to the body or unavoidable side effects, and the five-year survival rate is only 12% when CRC metastasizes to other organs. Recently, photodynamic therapy (PDT), a process in which a light-activated photosensitizer (PS) generates cytotoxic reactive oxygen species to trigger apoptosis, has become a safe and effective modality in cancer treatment due to its non-invasiveness, high selectivity, and minimal side effects. PDT has also shown significant clinical efficacy in treating superficial tumors. Most PS in PDT primarily follow an oxygen-dependent type II mechanism, which is limited by oxygen supply. Studies have shown that hypoxia is a hallmark of malignant tumors, and the overexpression of hypoxia-inducible factor (HIF-1α) is associated with increased tumor metastasis and mortality. However, the implementation of PDT is often hampered by a hypoxic tumor microenvironment (TME) with pO2 ≤ 2.5 mmHg, which may, in turn, exacerbate tumor hypoxia to some extent. Therefore, alleviating tumor hypoxia is crucial for effectively delaying tumor progression, preventing tumor metastasis, and improving the efficacy of PDT.

[0003] Various strategies have been proposed to overcome tumor tissue hypoxia, such as direct oxygen delivery, improving intratumoral blood flow and in-situ oxygen generation, as well as economical methods to reduce oxygen consumption. Considering the high levels of hydrogen peroxide (H2O2) present in solid tumors, utilizing catalase (CAT), a highly efficient and specialized catalytic enzyme, to decompose H2O2 into oxygen is an attractive strategy and has been successfully used in several types of therapeutic nanosystems to alleviate tumor hypoxia. Furthermore, the isoelectric point (PI) of biocompatible CAT is 5.8–6.4, close to the pH of acidic tumor microenvironments (TME), which may endow it with controllable regulation under pathological conditions. Besides hypoxia, another key factor in the phototherapy treatment process (PDT) is finding the optimal therapeutic window for light irradiation. Currently, using near-infrared (NIR) fluorescence imaging (650–900 nm) to guide PDT has become a prerequisite for precision medicine, mainly due to the reduced autofluorescence and deep penetration capabilities of NIR fluorescence, which facilitates in vivo imaging.

[0004] Quantum dots (QDs) are a promising class of fluorophores with high quantum yield, tunable spectra, and high photostability. Compared to the most studied QDs containing toxic elements such as Cd, Pb, or Hg, Ag2SQDs, with their negligible toxicity and good biocompatibility, have been considered ideal candidates for in vivo fluorescence imaging in recent years. Furthermore, near-infrared Ag2SQDs also exhibit photothermal effects, enabling photothermal therapy (PTT). Currently, clinical research focuses on combining PDT with other therapies such as chemotherapy and PTT, which has proven more effective than single-therapy approaches. However, photoactivators for PDT or PTT encapsulated in traditional nanocarriers may exhibit self-aggregation and premature leakage during in vivo application, ultimately affecting efficacy. In addition, hydrophobicity, systemic toxicity, and controlled drug release during chemotherapy are also key challenges. Therefore, the orderly integration of component modules to maximize their functional advantages while maintaining high safety is a crucial task for achieving multimodal synergistic cancer therapy.

[0005] The development of "bottom-up" self-assembly nanotechnology provides an effective means to organize multiple components into a single nanosystem to establish a highly ordered architecture. Currently, nature has utilized proteins as multifunctional building blocks, generating a dazzling array of materials through supramolecular self-assembly to perform important functions. Inspired by the wisdom of nature, significant efforts have been made to utilize self-assembly to construct novel biomimetic nanomaterials. Compared to traditional encapsulated nanocarriers, direct self-assembly of functional subjects can effectively reduce the introduction of non-functional molecules, further improving biosafety and increasing the potential for clinical translation. Molecular self-assembly is typically mediated by weak non-covalent interactions, particularly hydrophobic interactions, electrostatic interactions, hydrogen bonds, and metal coordination interactions. Among these, electrostatic interactions are widely used due to their excellent long-range and dynamically reversible properties. However, single-charge electrostatic assemblies are often fragile and unstable, especially under complex in vivo conditions. In contrast, multi-charge-driven electrostatic assembly not only retains the dynamic controllability of supramolecular assembly but also maintains considerable stability, making it suitable for biomedical applications. Furthermore, the difference between specific TMEs (lower pH values) and normal physiological environments offers opportunities for the controlled regulation of biomolecules such as proteins, primarily due to variations in protein surface charge under different pH conditions. Simultaneously, the reversibility of electrostatic interactions may lead to disintegration in response to specific stimuli (such as acidic TMEs), potentially facilitating controlled drug release. Currently, most research on CAT proteins focuses on their oxygenation capacity, while investigations into the correlation between CAT surface charge and physiological and pathological microenvironments are scarce, which is significant for electrostatic assembly and regulation. On the other hand, fluorescent QDs, due to their ligand-tunable properties, can generate multi-charge effects, making them promising substrates for driving protein electrostatic assembly.

[0006] Therefore, there is an urgent need to develop TME-responsive protein nanoassemblies based on multi-level electrostatic interactions to achieve effective imaging-guided combination therapy and inhibit tumors. Summary of the Invention

[0007] The present invention provides a method for preparing electrostatically self-assembled nanocomposite materials and their applications, aiming to solve the problems existing in the above-mentioned background art.

[0008] To achieve the above-mentioned technical objectives, the present invention mainly adopts the following technical solutions:

[0009] This invention provides a method for preparing a multi-charge electrostatic self-assembled nanocomposite material, comprising the following steps:

[0010] (1) Synthesis of CAT-Ce6: Ce6-NHS solution was added dropwise to CAT solution and the reaction was stirred in the dark to obtain crude CAT-Ce6. Then, the crude CAT-Ce6 was purified to obtain pure CAT-Ce6.

[0011] (2) Synthesis of negatively charged Ag2S-3MPAQDs: Under an inert atmosphere, 3-mercaptopropionic acid, which was completely dissolved at room temperature, was mixed with deionized water. Then AgNO3 solution was added, and the pH was adjusted to make the solution colorless and transparent. The reaction was continued under magnetic stirring in an oil bath to obtain a crude product Ag2S-3MPAQDs in a brownish-yellow solution. Then Ag2S-3MPAQDs crude product was purified to obtain negatively charged Ag2S-3MPAQDs.

[0012] (3) Synthesis of positively charged Ag2S-NH2QDs: EDC and NHS were added to Ag2S-3MPAQDs prepared in step (2), mixed evenly at room temperature, stirred and reacted in the dark, then six-armed amino PEG was added, and the reaction was continued to be stirred in the dark until complete. After dialysis, the positively charged Ag2S-NH2QDs were obtained by freeze drying.

[0013] (4) Preparation of Ag2S@CAT-Ce6@Oxa nanocomposite material: Ag2S-NH2QDs solution and CAT-Ce6 solution were ultrasonically mixed to obtain a mixed solution. Oxa was dissolved in a mixture of methanol and DMSO and added dropwise to the mixed solution. The mixture was stirred at room temperature in the dark to obtain crude Ag2S@CAT-Ce6@Oxa. The crude Ag2S@CAT-Ce6@Oxa was purified to obtain pure Ag2S@CAT-Ce6@Oxa nanoparticles.

[0014] In a preferred embodiment of the present invention, in step (1), the Ce6-NHS solution is prepared by the following method: EDC and NHS are dispersed in dimethyl sulfoxide, then mixed evenly with Ce6 at room temperature, and then stirred and reacted in a dark room temperature environment to obtain an activated Ce6-NHS solution.

[0015] In a preferred embodiment of the present invention, in step (1), the purification of crude CAT-Ce6 is carried out by the following method: after centrifuging the crude CAT-Ce6, the supernatant is taken and dialyzed in different gradient PBS buffers with continuous magnetic stirring using a dialysis bag. Then, the dialyzed CAT-Ce6 is centrifuged again, and the final supernatant is freeze-dried to obtain pure powdered CAT-Ce6.

[0016] Furthermore, the molecular weight cutoff of the dialysis bag is 10 kDa.

[0017] In a preferred embodiment of the present invention, in step (2), the pH adjuster is 2 mol·L⁻¹. -1 NaOH solution or 2 mol·L -1 The solution contains CH3COOH, and the pH of the solution is adjusted to 7.2-7.8 using the pH adjuster until the solution is colorless and transparent.

[0018] In a preferred embodiment of the present invention, in step (2), the crude Ag2S-3MPAQDs is purified by the following method: the crude Ag2S-3MPAQDs is centrifuged, the supernatant is washed and mixed with ultrapure water, then centrifuged and ultrafiltered, and the purified Ag2S-3MPAQDs is then sealed and stored in the dark at 4°C.

[0019] Preferably, in step (2), the ultrafiltration tube used during centrifugal ultrafiltration is an ultrafiltration tube with a molecular cutoff of 10 kDa, namely Millipore Amico Ultra.

[0020] In a preferred embodiment of the present invention, in step (4), the weight ratio of the Ag2S-NH2QDs solution and the CAT-Ce6 solution is 1:7.5-7.5:1, and the pH of the mixed solution is 7.4.

[0021] In a preferred embodiment of the present invention, in step (4), the crude Ag2S@CAT-Ce6@Oxa is purified by the following method: the crude Ag2S@CAT-Ce6@Oxa is centrifuged to remove free CAT-Ce6, Ag2S-NH2QDs and Oxa, and finally pure Ag2S@CAT-Ce6@Oxa nanoparticles are obtained.

[0022] Furthermore, the present invention also provides the application of the multi-charge electrostatic self-assembled nanocomposite material prepared by the preparation method described above in the preparation of antitumor drugs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Self-assembly technology: This invention introduces surface-amined Ag2S quantum dots as positively charged connecting groups, initiating multi-charge electrostatic interactions with CAT under physiological conditions. This enables self-assembly into layered, ordered nanostructures, further reducing the introduction of non-functional molecules and improving biocompatibility. In the hypoxic environment of tumors, the reversibility of this electrostatic assembly can also lead to its disintegration in response to specific stimuli (such as acidic TME), facilitating controlled drug release and improving therapeutic efficacy.

[0025] Solution for Hypoxic Tumors: This invention proposes a strategy using catalase (CAT) to decompose hydrogen peroxide (H2O2) within tumors into oxygen. Since solid tumors contain high levels of H2O2, utilizing the highly efficient catalytic action of CAT to convert it into oxygen is an attractive option. Furthermore, CAT's isoelectric point (PI) is between 5.8 and 6.4, close to the acidic pH of the tumor microenvironment (TME), allowing for controllable regulation under pathological conditions. Therefore, this invention utilizes CAT as an oxygen-supplementing element to effectively alleviate tumor hypoxia in vivo, providing a better environment for treatments such as photodynamic therapy.

[0026] In vivo imaging guidance: This invention uses near-infrared QDs as fluorescent probes to guide treatment through in vivo imaging. This imaging method has excellent penetration depth and spatial resolution, enabling precise localization and observation of tumors.

[0027] Multimodal synergistic therapy: This invention combines the advantages of photodynamic therapy (PDT), photothermal therapy (PTT), and chemotherapy to form a multimodal synergistic therapy strategy that can effectively inhibit tumor growth and prolong survival time.

[0028] Controlled Release: This invention utilizes an electrostatic assembly to achieve controlled drug release and slow disintegration of the nanocomposite. This method can, to some extent, achieve sustained drug release, improving efficacy and reducing side effects.

[0029] Biocompatibility: The nanocomposition candidates of the present invention all have intrinsic biocompatibility and good biocompatibility, and have broad application prospects in vivo. Attached Figure Description

[0030] Figure 1 A schematic diagram of Ag2S@CAT-Ce6@OxaNPs assembly provided in the embodiments of the present invention, and a diagram of PDT / PTT synergistic chemotherapy effect guided by near-infrared imaging through disassembly in a stimulating slightly acidic environment;

[0031] Figure 2The construction and characterization of Ag2S@CAT-Ce6@OxaNPs are shown, including: a) analysis of the normalized extinction spectra of free CAT and CAT-Ce6 by UV-Vis spectroscopy; b) TEM and high-resolution transmission electron microscopy (HRTEM) images of Ag2SQDs (inset). c) XRD patterns of Ag2SQDs (red curve) and standard Ag2SQDs (JCPDS card number 14-0072, black curve); d) 1, 2, 3, 4, 5 are zeta potential analyses of Ag2S-3MPA, Ag2S-NH2, CAT, CAT-Ce6, and Ag2S@CAT-Ce6@OxaNPs, respectively; e, f) Histograms and TEM images of the assembled Ag2S@CAT-Ce6@OxaNPsQDs at pH=7.4, corresponding to the DLS size distribution; g) Average size distribution and stable distribution of Ag2S@CAT-Ce6@OxaNPs at different time points in DMEM, 10mM PBS, and 10% FBS; h) Molecular docking model of Oxa with four residues (VAL55, SER337, MET339, and ALA345) on CAT.

[0032] Figure 3 Characterization of the disassembly and assembly process of Ag2S@CAT-Ce6@OxaNPs, including: a) Calculation of the isoelectric point of protein CAT at pH = 5.5, 6.5, and 7.4. b) Heatmaps showing the dependence on different pH and ionic strengths. c) Particle size distribution of Ag2S@CAT-Ce6@OxaNPs over time at pH = 6.5 (red line) and comparison with the particle size distribution at pH = 7.4 (gray line). A graph showing the size distribution of Ag2S@CAT-Ce6@OxaNPs over time during the disassembly process at pH = 7.4 and pH = 6.5, measured by DLS. d) Release frequency of Oxa from Ag2S@CAT-Ce6@OxaNPs over time under simulated physiological (pH = 7.4) and acidic TME (pH = 6.5) conditions. e) Fluorescence spectra of Ag2S@CAT-Ce6@OxaNPs before and after assembly. f) The depolymerization process of Ag2S@CAT-Ce6@Oxa NPs was observed by TEM in PBS buffer solution at pH 6.5. Images i, ii, and iii correspond to TEM images at depolymerization time points of 0, 10, and 30 min, respectively.

[0033] Figure 4The graphs show the evaluation of in vitro PDT and PTT performance, including: a) oxygen production by Ag2S@CAT-Ce6@OxaNPs in H2O2 solutions of different concentrations; b) oxygen production and consumption levels of Ag2S@CAT-Ce6@OxaNPs solutions before and after light irradiation; c) oxygen production curves of Ag2S@CAT-Ce6@OxaNPs in H2O2 (100 μM) solutions at different pH values; d) singlet oxygen produced by Ag2S@CAT-Ce6@OxaNPs or Ag2S@BSA-Ce6@OxaNPs under normal and hypoxic conditions with H2O2, measured by SOSG fluorescence under 650 nm laser irradiation; and e) Ag2S@CAT-Ce6@OxaNPs in 0.3 W cm⁻¹ -2 f) Photothermal stability of Ag2S@CAT-Ce6@OxaNPs under 600nm laser irradiation; g) Photothermal curves of Ag2S@CAT-Ce6@OxaNPs under 600nm laser irradiation at different power densities; g) Photothermal images of Ag2S@CAT-Ce6@OxaNPs at different time intervals within 3 minutes under 600nm laser irradiation, with a laser power of 0.3Wcm. -2 .

[0034] Figure 5 To validate the in vitro cellular uptake and cytotoxicity of Ag2S@CAT-Ce6@OxaNPs, the following data were analyzed: a) fluorescence image of HT29 cells uptake of Ag2S@CAT-Ce6@OxaNPs over time under laser confocal fluorescence microscopy; d) quantitative fluorescence analysis using ImageJ software after incubation with Ag2S@CAT-Ce6@OxaNPs for different time periods (scale bar: 25 μm); b) detection of singlet oxygen after SOSG, acting as a probe, entered the cells under laser irradiation following incubation with different nanoparticles; e) various parameters shown in figure b. The corresponding quantitative fluorescence analysis was performed for each group, with a scale bar of 50 μm; c) the relative viability of HT29 cells after treatment with Ag2SQDs and f) Ag2S@CAT-Ce6@OxaNPs for 24 hours, with error estimation calculated based on triplicate samples; g) the cell viability of HT29 cells treated with Ag2S@ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@OxaNPs under hypoxic conditions with 650 nm light irradiation (Ce6 concentration: 12 μg / ml; 650 nm laser power density: 300 mW / cm²). -2 Irradiation time: 5 minutes;

[0035] Figure 6To evaluate the in vitro synergistic therapeutic effect of Ag2S@CAT-Ce6@OxaNPs on HT29 tumor cells; where a) under normoxic and hypoxic conditions, HT29 cells were irradiated with PBS, Ag2S@ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@OxaNPs under 650nm laser irradiation for live / dead staining; and b) the corresponding quantitative fluorescence analysis bar graphs, where green signals of Calcein-AM represent live cells and red signals of propidium iodide (PI) represent dead cells (Ce6 concentration: 12μg / mL); c, d) under hypoxic conditions, HT29 cells were irradiated with PBS, Ag2S@ce6, Ag2S@CAT-Ce6, and... Western blot analysis of HIF-1α protein expression in HT29 cells treated with Ag2S@CAT-Ce6@OxaNPs. Western blot analysis was performed three times, and data are expressed as mean ± standard error (n=3), where n represents the number of independent samples. Statistical analysis was performed using one-way ANOVA. NS: no statistical significance, *p<0.05, **P<0.01, ***P<0.001; e) After treatment with PBS, Ag2S@ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@OxaNPs under 650nm laser irradiation, Annexin V-FITC / PI staining was performed, followed by flow cytometry analysis to assess the cell death mechanism under hypoxic conditions.

[0036] Figure 7 This image shows a synergistic therapy guided by in vivo near-infrared imaging of Ag2S@CAT-Ce6@OxaNPs. a) Schematic diagram of the BALB / c xenograft colon cancer nude mouse model and the mouse anti-tumor treatment regimen mediated by in vivo near-infrared light-triggered combined therapy; b) Infrared thermography of HT29 tumor nude mice injected with PBS (pH 7.4, 50 mM) and Ag2S@CAT-Ce6@OxaNPs (2 mg / ml, 200 μL); c) Infrared thermography of the control group and Ag2S@CAT-Ce6@OxaNPs after intravenous injection. Following synergistic treatment with Ag2S@Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@OxaNPs, tumor fluorescence images of HT29 tumor-bearing nude mice at different treatment times; d) photographs of colon cancer tumors collected on day 14 after the end of treatment in all groups; e) tumor mass of HT29 tumor-bearing mice in each group after treatment (n = 3 biologically independent samples); f) weight of colon cancer tumors collected from the first treatment to the final day 14; and g) weight changes of HT29 tumor-bearing nude mice throughout the treatment process.

[0037] Figure 8 HE staining results for major organs and tumors are used for biosafety assessment. Detailed Implementation

[0038] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Example 1: Preparation of Ag2S@CAT-Ce6@Oxa nanomaterials

[0040] (1) Synthesis of CAT-Ce6

[0041] EDC (0.96 mg) and NHS (0.63 mg) were pre-dispersed in DMSO (100 μL) and then mixed thoroughly with Ce6 (2.0 mg) at room temperature, followed by stirring in the dark at room temperature for 0.5 h. The activated Ce6-NHS was then added dropwise to a solution of CAT (5 mg, 1.0 mL), and finally stirred in the dark for 12 h. To obtain CAT-Ce6, the crude product was obtained by centrifuging the product at 14000 rpm for 10 min, and then dialyzing the supernatant in different gradient PBS buffers with continuous magnetic stirring for 24 h using a dialysis bag (10 kDa). The purified CAT-Ce6 was then centrifuged at 10000 rpm for 5 min, and the final supernatant was freeze-dried to obtain powdered CAT-Ce6.

[0042] Activated Ce6-NHS was added dropwise to a solution of BAS (5 mg, 1.0 mL), and the mixture was stirred in the dark for 12 hours. The purification procedure was the same as that for CAT-Ce6, and the final product was obtained by freeze-drying.

[0043] (2) Synthesis of negatively charged Ag₂S⁻³MPAQDs

[0044] After 3-mercaptopropionic acid was completely dissolved at room temperature, 228 μL (25 mM) was taken and mixed with 90 mL of deionized water in a necked flask and stirred for 30 minutes under argon atmosphere. Then, AgNO3 solution (5 mM, 10 mL) was added, and the mixture was sonicated and then stirred with NaOH (2 mol·L⁻¹). -1 ) and CH3COOH solution (2 mol·L -1The pH was adjusted to approximately 7.5 until the solution became colorless and transparent. The mixture was then reacted at 90°C for 12 hours with magnetic stirring in an oil bath to obtain a brownish-yellow solution as the crude product Ag₂S-3MPAQDs. The product was centrifuged at 10,000 rpm for 10 minutes. The supernatant was washed and mixed with ultrapure water, then ultrafiltered three times (using a 10 kDa ultrafiltration tube, Millipore Amico Ultra). The purified Ag₂S-3MPAQDs were then stored sealed and protected from light at 4°C.

[0045] The prepared Ag2S-3MPAQDs were further mixed with EDC (0.96 mg) and NHS (0.63 mg) at room temperature and stirred in the dark for 0.5 hours. Then, hexa-arm aminoPEG was added and stirred in the dark for 12 hours. After dialysis, the mixture was lyophilized to obtain positively charged Ag2S-NH2QDs. The concentration of Ag2S-NH2QDs was measured by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0046] (3) Preparation of Ag2S@CAT-Ce6@Oxa nanomaterials

[0047] Ag2S@CAT-Ce6@Oxa was prepared via a self-assembly strategy. Ag2S-NH2QDs and CAT-Ce6 solutions were ultrasonically mixed at different weight ratios (1:7.5–7.5:1) for 0.5 hours. Oxa (0.5 mg, 100 μL) was then dissolved in a mixture of methanol and DMSO beforehand and added dropwise to the above mixture (pH = 7.4, 10 mM). The mixture was stirred for two hours in the dark at room temperature. The purified Ag2S@CAT-Ce6@OxaNPs were then centrifuged at 14000 r / min for 10 minutes to remove free CAT-Ce6, Ag2S-NH2QDs, and Oxa, finally yielding Ag2S@CAT-Ce6@OxaNPs. When the weight ratio of Ag2S-NH2QDs solution to CAT-Ce6 solution was 1:7.5 and the Oxa content was 0.5 mg / ml, the loaded Oxa content was detected to be 10% ± 0.3% of the initial content. 1 mL of Ag2S-NH2QDs solution was taken and Oxa (0.5 mg, 100 μL) was added and stirred for two hours to obtain Ag2S@Oxa.

[0048] Ag2S@Ce6 was obtained by mixing Ag2S-NH2QDs solution with Ce6 (12 μg / mL) and stirring for two hours. Ag2S@CAT-Ce6 was obtained by mixing Ag2S-NH2QDs solution and CAT-Ce6 solution at a weight ratio of 1:7.5 and stirring for two hours. Ag2S@BSA-Ce6@Oxa was obtained by mixing Ag2S-NH2QDs solution and BSA-Ce6 solution at a weight ratio of 1:7.5, with an Oxa content of (0.5 mg / mL) and stirring at room temperature for two hours.

[0049] This invention discloses a smart near-infrared QDs-mediated protein nanosystem (Ag2S@CAT-Ce6@Oxa) based on a multi-charge electrostatic self-assembly strategy, which can specifically respond to TME and be used for imaging-guided synergistic PDT / PTT / chemotherapy, such as... Figure 1 As shown, the protein CAT, with its mottled negatively charged surface, was selected as an oxygenation scaffold and then chemically conjugated with chloroethylene (Ce6) via zero-length crosslinking to serve as a PS. Subsequently, surface-amined Ag2S-NH2QDs were introduced as positively charged linking groups, initiating multi-charge electrostatic interactions with CAT under physiological conditions. This not only allowed for self-assembly into a layered, ordered nanostructure but also enabled the effective loading of the CRC-specific anticancer drug oxaliplatin (Oxa) during assembly. The acidic TME, similar to the PI of CAT, weakened the charge effect that could induce nanosystem disintegration, leading to the release of Oxa while maintaining fluorescence imaging, PDT, and PTT capabilities.

[0050] The intelligent therapeutic agent nanosystem of the present invention has several outstanding features:

[0051] 1) Enhanced PDT can be achieved both in vitro and in vivo by greatly alleviating tumor hypoxia and Ce6 activity;

[0052] 2) Visualizing the accumulation of nanoparticles through near-infrared fluorescence imaging to guide further phototherapy is highly beneficial for precise and efficient treatment;

[0053] 3) Encapsulating anticancer drugs in an electrostatic assembly not only solves the problems of hydrophobicity and directional delivery, but more importantly, by adjusting the charging effect intensity of CAT through the pH characteristics of TME, the slow disintegration of the electrostatic assembly can be effectively controlled, which is of great significance for achieving sustained drug release.

[0054] 4) Each candidate nano-component possesses inherent biocompatibility, giving it good biocompatibility and promising broad application prospects in vivo.

[0055] 5) The synergistic effect of PDT and PTT, combined with TME-mediated controlled chemotherapy of Oxa, can significantly inhibit tumor growth and prolong life in mice.

[0056] Therefore, the present invention provides a simple method for fabricating a unique type of therapeutic nanosystem composed of reactive functional proteins that can improve cancer treatment by alleviating tumor hypoxia under the guidance of fluorescence imaging.

[0057] Test case

[0058] Experimental Example 1: Physicochemical Properties of Nanocomposite Material (Ag2S@CAT-Ce6@Oxa)

[0059] CAT, a potent enzyme, can trigger the decomposition of endogenous H2O2 in tumors, thereby generating oxygen. To address tumor hypoxia, the natural protein CAT was introduced as a crucial component of the assembly pattern, and the size distribution of free CAT was first measured using DLS. The binding of CAT to PSs not only solves the water solubility problem of PSs but also improves the efficacy of PDT. To achieve this, conventional PS, Ce6, and CAT were covalently modified by forming amide bonds. The UV-Vis absorption spectrum of CAT-Ce6 is shown below. Figure 2 As shown in Figure a, the covalent modification of CAT-Ce6 results in a redshift of its characteristic peak at 660 nm compared to free Ce6, confirming the successful conjugation of CAT and Ce6. Figure 2 As shown in d(iii, iv), under simulated physiological conditions (pH = 7.4), the zeta potential of CAT itself is -29.2 mV, while that of the modified CAT-Ce6 is -37.7 mV. Since the covalent modification of CAT-Ce6 consumes a large number of amino groups on the protein surface, CAT-Ce6 exhibits a strong negative charge under physiological conditions. This not only indicates that the covalent modification of CAT-Ce6 is successful, but also provides a good opportunity for electrostatic assembly.

[0060] First, another functional assembly building block, Ag2S-3MPAQDs, was prepared via a simple one-step synthesis method to obtain negatively charged QDs. TEM image ( Figure 2 As shown in Figure b), the prepared Ag₂S-3MPAQDs are monodisperse, with an average size of approximately 2.59 ± 0.65 nm, as determined by measuring the diameter of 303 nanoparticles. Notably, high-resolution transmission electron microscopy (HRTEM) characterization... Figure 2 Figure b (inset) shows high crystallinity with a sharp lattice edge of d = 0.2604 nm, which likely corresponds to the (-121) crystal plane of Ag₂S. The crystal structure of the Ag₂SQDs was then further examined by XRD. Figure 2As shown in Figure c, the position and relative intensity of the diffraction peaks (red curve) are consistent with the monoclinic plane of Ag₂S (black line, JCPDS card number 14-0072), indicating the presence of Ag₂S nanocrystals. To fabricate a structure with multiple positively charged particles on its surface and further improve the biocompatibility of Ag₂S-3MPAQDs, 6ARM-PEG-NH₂ with multiple amino-phase linkages was introduced and covalently modified onto the surface of Ag₂S-3MPAQDs via chemical crosslinking. The significant change in zeta potential from -67.6 mV of the initially synthesized Ag₂S-3MPAQDs to 64.2 mV of Ag₂S-NH₂QDs verifies the successful preparation of positively charged Ag₂S-NH₂QDs. Figure 2 in d,i,ii).

[0061] Due to the charge properties of the obtained multi-component patterns, a self-assembly process is then carried out via multi-charge electrostatic interactions to construct integrated nanosystems. Analysis of the equivalent surfaces shows that, under simulated physiological conditions (pH = 7.4), negative charges dominate, which provides a strong theoretical basis for the feasibility of protein assembly under electrostatic interactions. Figure 3 (a) To further optimize the assembly conditions, we adjusted the ionic strength to modulate charge interactions, thereby controlling the ordered assembly of CAT and Ag2S-NH2QDs.

[0062] TEM and DLS results for Ag2S@CAT-Ce6@OxaNPs showed sizes of approximately 100 nm and 126 nm, respectively. The larger size observed on DLS is due to the measurement of the hydrated particle size. The consistency between the TEM and DLS results confirms that, under physiological conditions, driven by the multivalent charge effect, multi-component primitive units can self-assemble into uniform spherical nanoparticles. On the other hand, we used DLS to record the particle size changes in the mixed solution at different time points. We found that the particle size in the aqueous solution increased continuously over time, finally stabilizing at a stable value after two hours, consistent with the TEM results.

[0063] To further verify the stability of the assembled Ag2S@CAT-Ce6@OxaNPs in different solutions, the nanoparticles were dissolved in different buffer solutions (10 mM PBS, 10% FBS, DMEM), and the size of Ag2S@CAT-Ce6@OxaNPs in different buffer solutions was measured by DLS over 0 to 24 hours. The size of the nanoparticles remained essentially unchanged in all three buffer solutions, indicating that these self-assembled NPs possess high stability. Figure 2 f, g).

[0064] Experimental Example 2: pH-responsive disassembly properties of nanocomposite material (Ag2S@CAT-Ce6@OxaNPs)

[0065] DLS can be used to measure the hydrodynamic size distribution of particles in solution. The obtained Ag2S@CAT-Ce6@OxaNPs solution was adjusted to approximately pH 6.5. First, the size of the nanoparticles in the solution was monitored using DLS at different times (0, 10, 20, 30, 40, 50, 60 min). TEM, on the other hand, is a high-resolution imaging technique that uses an electron beam to observe the structure and morphology of materials. It can observe the shape and distribution of Ag2S@CAT-Ce6@OxaNPs at the nanoscale. To better provide information on particle size, shape, and aggregation state, nanoparticle solutions at different times were prepared for TEM, and the morphology and distribution of the nanoparticles were then observed under a transmission electron microscope.

[0066] Through changes in electrical potential at different pH levels ( Figure 3 a) and heatmap ( Figure 3 The charge data at different pH values ​​in section b) indicate that the simulated tumor's slightly acidic environment is close to the protein's isoelectric point, weakening the charge effect and making it insufficient to maintain electrostatic assembly. This may lead to a slow disassembly, thereby achieving sustained drug release. Based on this simulation result, we conducted further experimental investigations.

[0067] To further investigate the pH-dependent depolymerization behavior of Ag2S@CAT-Ce6@Oxa nanoparticles, we simulated an acidic tumor microenvironment (pH = 6.5) and allowed the obtained Ag2S@CAT-Ce6@Oxa nanoparticles to stand in this environment for 1 hour. Under physiological conditions, the assembled Ag2S@CAT-Ce6@Oxa NPs maintained a stable pH value. Figure 3 (c: 125 nm). The particle size reduction in aqueous solution at pH 6.5 indicates that the assembled Ag2S@CAT-Ce6@Oxa NPs exhibit slow decomposition over time. To visually observe the changes in the decomposition morphology, TEM observation was performed on the decomposition process, and the results show... Figure 3 In the first step, uniform spherical nanoparticles initially measuring approximately 100 nanometers were gradually broken down into particles approximately 10 nanometers in size, consistent with previous DLS results. To further investigate the changes in fluorescence signal of the nanoparticles after decomposition, the fluorescence spectra of the assembled Ag2S@CAT-Ce6@OxaNPs before and after decomposition were measured. Figure 2 The results from the study showed that the fluorescence signal of the nanoparticles did not change before and after decomposition, indicating that the nanoparticles can still maintain a good fluorescence signal after decomposition.

[0068] During the decomposition process of this system, the complex biological environment may cause a slow release of the drug Oxa bound to the protein. To further investigate the release process of the chemotherapeutic drug Oxa from Ag2S@CAT-Ce6@Oxa nanoparticles, we measured the absorbance of the characteristic peak at 250 nm of the oxaliplatin UV spectrum of the assemblies at different stages and calculated their drug release concentrations. Figure 3 As shown in Figure d, under normal simulated physiological conditions (pH = 7.4), Ag2S@CAT-Ce6@Oxa nanoparticles remained stable for an extended period without drug leakage. In contrast, under slightly acidic conditions (pH = 6.5), the characteristic absorption peak corresponding to Oxa gradually increased at 250 nm, indicating that Oxa was continuously released as the Ag2S@CAT-Ce6@Oxa nanoparticles decomposed. Furthermore, it was observed that the continuous release could last for more than 30 minutes, reaching a state of complete release. All these results demonstrate that the pH-responsive decomposition of Ag2S@CAT-Ce6@Oxa nanoparticles under acidic conditions can effectively induce sustained drug release. Example 3: Oxygen production and photodynamic properties of the nanocomposite material (Ag2S@CAT-Ce6@Oxa)

[0069] The production of O2 by Ag2S@CAT-Ce6@OxaNPs was validated by the catalytic performance of catalase on the nanoparticles. H2O2 solutions of different concentrations (10, 25, 50, 100 μM) and Ag2S@CAT-Ce6@OxaNPs solutions were prepared. These solutions were exposed to a 650 nm laser for 3 minutes. Oxygen content was measured using a dissolved oxygen meter to evaluate the catalytic activity of the nanoparticles for hydrogen peroxide. The same experimental procedures were repeated under different pH conditions to evaluate the catalytic activity of the nanoparticles at different pH values. The nanoparticles used in the experiment were Ag2S@CAT-Ce6@OxaNPs, with catalase at 500 μg / mL and Ce6 at 12 μg / mL. Catalase is a naturally occurring protein that, as an effective catalase, can effectively catalyze the decomposition of H2O2 into water and oxygen.

[0070] To evaluate the ability of Ag2S@CAT-Ce6@OxaNPs to generate singlet oxygen under normal and low oxygen conditions, the experimental procedure was as follows. BSA-Ce6@Ag2SQDs@OxaNPs, CAT-Ce6@Ag2SQDs@OxaNPs (Ce6: 12 μg / mL), and SOSG dissolved in methanol (3 μM) were incubated in a buffer solution containing the same concentration of hydrogen peroxide. The samples were then subjected to laser irradiation (650 nm, 300 mW / cm²) under both nitrogen-purifying and non-nitrogen-purifying conditions. 2The singlet state of oxygen produced by the sample under different conditions was measured. H₂O₂ (100 μM) was chosen in the experiment to simulate the hydrogen peroxide concentration in an oxygen-consuming tumor environment. Singlet oxygen can be detected by SOSG, and the fluorescence of SOSG can be measured within the detection limit of the spectrometer. The ability to generate singlet oxygen was determined by evaluating the fluorescence signal of SOSG at 528 nm under 494 nm excitation.

[0071] The experimental results show that even at low H2O2 levels, Ag2S@CAT-Ce6@Oxa nanoparticles maintain their catalytic activity in decomposing H2O2 into O2, and the amount of singlet oxygen produced increases with increasing H2O2 concentration (100 μM). Figure 4 (a) Furthermore, pH values ​​within the physiological range had no significant effect on the catalytic efficiency of Ag2S@CAT-Ce6@OxaNPs. Figure 4 (c) However, we noted that the catalytic activity of Ag2S@CAT-Ce6@OxaNPs was stronger when the pH was below 6.5, indicating that Ag2S@CAT-Ce6@OxaNPs still maintained good catalytic activity even in acidic TME. To verify the feasibility of PDT using Ag2S@CAT-Ce6@OxaNPs, we measured the change in oxygen content at different illumination intervals under a 660nm LED lamp to demonstrate the oxygen consumption during the PDT process (c). Figure 4 (b) We found that the oxygen concentration in the Ag2S@CAT-Ce6@Oxa system decreased significantly with increasing illumination time, with the highest oxygen consumption rate at 15 minutes of illumination. This determined the optimal illumination time for subsequent experiments in cells and biomass. Example 4: Photothermal properties of the nanocomposite material (Ag2S@CAT-Ce6@OxaNPs)

[0072] In the experiment, 1 ml of Ag2S@CAT-Ce6@OxaNPs aqueous solution was added to an EP tube, and then different power densities (0, 50, 100, 150, 300 mW / cm²) were applied. 2 The Ag2S@CAT-Ce6@OxaNPs were irradiated with a 650nm laser for 3 minutes. Temperatures at different time points were recorded using an FTIR infrared imager. For the thermal cycling curve testing of Ag2S@CAT-Ce6@OxaNPs, the experimental evaluation was performed using a 650nm laser (300mW / cm²). 2 Irradiate the sample for 3 minutes, then stop the laser irradiation and record the temperature at different time points using a temperature detector.

[0073] According to previous literature, Ag2SQDs possess certain photothermal conversion properties, which are attributed to the strong absorption of quantum dots in the near-infrared biological window. The in vitro photothermal conversion properties of Ag2S@CAT-Ce6@OxaNPs under 600 nm irradiation were investigated. The photothermal conversion was performed when the laser power density ranged from 0 to 0.3 W / cm². -2 When the temperature changes, it increases significantly. This is at a laser power density of 0.3 W / cm². -2 At that time, the temperature of the Ag2S@CAT-Ce6@Oxa aqueous solution can rapidly reach approximately 60°C within three minutes. Figure 4 In the middle (f), under the same conditions, the temperature rise of deionized water is negligible. Furthermore, at 0.3Wcm... -2 Under high laser power, the rapid temperature rise of Ag2S@CAT-Ce6@Oxa (200 μg / ml) was monitored using an infrared thermal imager, such as... Figure 4 As shown in g. These results directly demonstrate that Ag2S@CAT-Ce6@Oxa can efficiently and rapidly convert light energy into heat energy under laser irradiation. To evaluate the photothermal stability of Ag2S@CAT-Ce6@Oxa, the temperature curves of Ag2S@CAT-Ce6@Oxa aqueous solution under laser irradiation were recorded over five cycles of heating and cooling. Figure 4 (e). The results showed that the solution temperature did not change significantly during each test cycle, indicating that Ag2S@CAT-Ce6@Oxa can serve as a stable light absorber with good photothermal effects in the near-infrared biological window for cancer photothermal therapy.

[0074] Experimental Example 5: Cellular Uptake Capacity of Nanocomposite Material (Ag2S@CAT-Ce6@Oxa)

[0075] The most important application of nanomaterials in biological systems is low cytotoxicity. HT29 cells were cultured in confocal culture dishes with Ag2S@CAT-Ce6@OxaNPs (Ce6: 12ug / ml) added to fresh culture medium and cultured overnight. After 0, 30, 60, and 90 minutes of culture, the cells were washed three times with sterile PBS. Then, DAPI (200 μL) was added to each culture dish for staining, and after 10 minutes of treatment, the cells were washed three times again with sterile PBS. Finally, the uptake of nanoparticles by the cells was observed using a confocal laser scanning microscope (CLSM). Figure 5As shown in Figure c, after co-culturing Ag2SQDs with cells at a high concentration of 250 μg / mL for 48 hours, the cell viability remained above 90%, indicating that the obtained Ag2SQDs exhibited low cytotoxicity and good biocompatibility, consistent with previous reports. We immediately followed the same method to investigate the cytotoxicity of the assembled Ag2S@CAT-Ce6@Oxa against colon cancer cells (HT29) for subsequent applications in a range of related biological fields. When co-cultured with Ag2S@CAT-Ce6@OxaNPs, over 85% of HT29 cells survived, even with a Ce6 concentration as high as 16 ppm in the nanoparticles. Figure 5 (f). The results showed that the prepared composition, Ag2S@CAT-Ce6@Oxa nanoparticles, still exhibited low toxicity and satisfactory biocompatibility. To further investigate the optimal time required for Ag2S@CAT-Ce6@Oxa NPs to enter cells, since the quantum dots contained in the assembled Ag2S@CAT-Ce6@Oxa NPs possess a certain near-infrared imaging capability, this could be obtained by measuring their intracellular fluorescence intensity. The reagent was incubated with HT29 cells for different times (0, 30, 60, and 90 minutes), and then the cells were imaged under a confocal microscope. Furthermore, as the co-existence time of the nanoparticles with the cells increased, the intracellular fluorescence intensity became stronger, such as... Figure 5 As shown in Figure a, Ag2S@CAT-Ce6@Oxa nanoparticles exhibited more efficient cellular uptake after 90 minutes of co-existence with cells, consistent with subsequent quantitative fluorescence intensity results. Figure 5 (d) This means that cells can effectively absorb Ag2S@CAT-Ce6@Oxa NPs, which to some extent provides an optimal time window for subsequent PDT treatment at the cellular level.

[0076] Experimental Example 6: Intracellular Antitumor Properties of Nanocomposite Material (Ag2S@CAT-Ce6@Oxa)

[0077] To verify the improved PDT efficiency of Ag2S@CAT-Ce6@Oxa, a series of experiments were conducted using HT29 cells. HT29 cells were grown in confocal disks and cultured for 24 hours to achieve appropriate cell density. Next, different nanoparticles (PBS, Ag2S-Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@Oxa NPs, with a Ce6 concentration of 12 μg / mL) were added and incubated with the cells for 4 hours to ensure sufficient contact between the nanoparticles and the cells. After incubation, the cells were placed under laser irradiation (650 nm, 300 mW / cm²). 2Cells were subjected to a 3-minute induction process to excite the photosensitizer within the nanoparticles, generating singlet oxygen. Then, 10 μM SOSG probe was added and allowed to react with singlet oxygen for 10 minutes. Finally, the cells were washed three times with PBS, and changes in SOSG fluorescence intensity were observed using laser confocal microscopy to determine the generation and distribution of singlet oxygen.

[0078] To observe the viability of HT29 cells, they were treated with different agents including PBS, Ag2S-Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@OxaNPs, with a Ce6 concentration of 12 μg / mL. Treatment time was 12 hours, followed by induction at 650 nm and 300 mW / cm². 2 The cells were irradiated with a laser for 3 minutes. After 4 hours of incubation, they were stained with Calcein-AM / PI for 30 minutes. The labeled cells were then washed three times with PBS and imaged using CLSM. The excitation wavelengths for Calcein-AM and PI were 488 nm and 561 nm, respectively.

[0079] Intracellular singlet oxygen production was tested on HT29 cells. These cells were pre-incubated with different nanoparticles for 4 hours: PBS group, Ag2S@Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@OxaCAT nanoparticle group (Ce6 equivalent concentration: 12 μg / mL). The cells were then irradiated with a laser (300 mW / cm²). -2 (10 min), SOSG was used as a fluorescent singlet oxygen probe and co-cultured with cells for laser scanning confocal microscopy imaging. Figure 5 In the control group (b), almost no fluorescence was observed, while the Ag2S@Ce6 group without CAT showed a paler green fluorescence compared to the control group. Meanwhile, both the Ag2S@CAT-Ce6 and Ag2S@CAT-Ce6@Oxa groups showed bright green fluorescence. Furthermore, the fluorescence intensity of the cells in the four groups was quantified. Figure 5 (e) The results showed that the groups treated with Ag2S@CAT-Ce6 and Ag2S@CAT-Ce6@Oxa nanoparticles exhibited enhanced singlet oxygen production. The comparable singlet oxygen production in both groups was attributed to the peroxidase alleviating the hypoxic environment, thereby improving PDT efficiency.

[0080] Prior to this, the low cytotoxicity of the nanoparticles was verified. Phototoxicity was determined using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) method upon irradiation with a 650 nm near-infrared laser at different Ce6 concentrations. Figure 4As shown in Figure g, both the Ag2S@CAT-Ce6 and Ag2S@CAT-Ce6@Oxa groups exhibited considerable phototoxicity against HT29 cells. It should be noted that the phototoxicity of the Ag2S@CAT-Ce6 and Ag2S@CAT-Ce6@Oxa groups was higher than that of the Ag2SCe6 group.

[0081] Case Study 7: Evaluation of Therapeutic Effects under Normoxist and Hypoxic Conditions

[0082] To simulate the effect of killing tumor cells in vitro, the PDT efficiency of Ag2S@CAT-Ce6@Oxa was evaluated using calcein-AM / PI. Figure 6 (a) Under normal oxygen conditions, compared with the PBS group, Ag2S@Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@Oxa showed some degree of red and green fluorescence (red represents live cells, green represents dead cells). Furthermore, under normal oxygen or hypoxic conditions, Ag2S@CAT-Ce6@Oxa NPs had more dead cells than other groups, which is consistent with... Figure 6 The fluorescence intensity analysis results calculated by b are consistent, indicating that with the help of the self-assembly system, the Ag2S@CAT-Ce6@Oxa nanoparticles in the cell can still indirectly provide oxygen well even under hypoxic conditions, thereby enhancing the killing effect of specific laser-triggered PDT on colon cancer tumor cells.

[0083] Apoptosis detection in HT29 cells from different treatment groups can be quantitatively studied. Four groups were set up under laser irradiation: control group, Ag2S@Ce6 group, Ag2S@CAT-Ce6 group, and Ag2S@CAT-Ce6@Oxa group. Except for the control group, all groups showed a certain number of apoptotic cells, while the Ag2S@CAT-Ce6@Oxa group induced approximately 60.79% apoptosis, indicating that the phototherapy effect of near-infrared induced Ag2S@CAT-Ce6@Oxa mainly leads to cell death through apoptosis rather than necrosis. Figure 6(e). We then specifically investigated how HIF-1α levels changed in colon cancer cells in a hypoxic environment after incubation with nanoparticles containing or without CAT. HIF-1α expression levels in tumors are induced by hypoxia and can be analyzed using Western blot as an indicator of hypoxia. As the results showed, HIF-1α expression levels were significantly increased in the Ag2S@Ce6 group compared to the control group, but significantly decreased compared to the Ag2S@Ce6 and Ag2S@CAT-Ce6@Oxa groups. Furthermore, there was almost no difference in HIF-1α expression levels between Ag2S@CAT-Ce6 and Ag2S@CAT-Ce6@Oxa. This suggests that carefully designed Ag2S@CAT-Ce6@Oxa nanoprobes can effectively resist aerobic environments, improve the efficacy of PDT, and thus have a good killing effect on tumor cells. Figure 6 (c, d)

[0084] Experimental Example 8: In vivo near-infrared imaging-guided synergistic PDT / PTT / chemotherapy

[0085] Inspired by the outstanding photothermal conversion capabilities of Ag2S@CAT-Ce6@Oxa nanoparticles in vitro, we investigated the in vivo photothermal response (PTT) after intravenous injection of these particles. First, a colon cancer transplantation tumor model was established in the axilla of nude mice, and the nude mice carrying BALB / cHT29 tumors were divided into a control group and an Ag2S@CAT-Ce6@Oxa group. In the experiment, when the tumor diameter of the nude mice reached 5-6 mm, they were first divided into groups. Then, different drugs were administered intravenously via the tail vein, including the control group (Ag2S: 1 mg / kg), Ag2S@Oxa, Ag2S-Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@Oxa NPs (main component content: Oxa: 5 mg / kg, Ce6: 1 mg / kg, 200 μL). Twelve hours after injection, mice were anesthetized (using pentobarbital at a concentration of 6 mg / mL, dose of 10 μL / g) and then tumor fluorescence imaging was performed using the IVISLumina series XR system. During this process, the laser wavelength was set to 600 nm, and the emission and collection wavelengths were set to 800 nm to assess the accumulation time of nanoparticles in the tumor and to evaluate the synergistic therapeutic effect.

[0086] When the tumor volume of a colon cancer mouse reaches 60 mm 3Mice were randomly divided into 5 groups. All groups received intravenous injections of control group, Ag2S@Oxa, Ag2S-Ce6, Ag2S@CAT-Ce6, and Ag2S@CAT-Ce6@Oxa NPs (Ag2S: 1 mg / kg / ml, Oxa: 5 mg / kg, Ce6: 1 mg / kg, 200 μL) on days 0, 3, 9, and 10. Body weight and tumor volume were recorded regularly throughout the treatment period, every 4-5 days. Tumor volume was calculated using the following formula: V = length × width × height × π / 6. Subcutaneous tumor growth inhibition value (TGI) was calculated using the following formula: TGI = (1 - B / A) × 100% (A: mean tumor weight in the control group; B: mean tumor weight in the treatment group). Treatment was discontinued when the mice's body weight decreased by 20-25% or the tumor diameter at any size was less than 20 mm.

[0087] Nude mice were injected with PBS and Ag2S@CAT-Ce6@OxaNPs, respectively. Twelve hours later, they were irradiated with a NIR-I laser for 3 minutes. The real-time temperature of the tumor area after laser irradiation was monitored using an infrared thermal imager. Figure 7 (g). In mice injected with Ag2S@CAT-Ce6@Oxa, the temperature at the tumor site rapidly increased by 29.6℃ within the first 3 minutes, and then remained at 59.5℃. However, after NIR-I laser irradiation, the tumor temperature in the PBS group fluctuated slightly, eventually remaining at 31.4℃, essentially consistent with the initial tumor temperature. These results indicate that Ag2S@CAT-Ce6@Oxa nanoparticles can significantly increase local tumor temperature under near-infrared irradiation, demonstrating good in vivo photothermal capabilities.

[0088] To investigate the in vivo synergistic therapeutic effect of Ag2S@CAT-Ce6@Oxa nanoparticles, a nude mouse model of BALB / cHT29 tumors was constructed. On day 8 after subcutaneous injection of HT29 cell clusters into nude mice, the subcutaneous colon cancer tumors grew to 50-100 mm. 3 This enables in vivo synergistic therapy experiments to be conducted under the guidance of near-infrared imaging. Figure 7(a) Nude mice carrying constructed BALB / cHT29 tumors were divided into five treatment groups, each consisting of three mice, and monitored for 14 days in each group. The five treatment groups included: (i) a pure Ag2SQDs control group, (ii) Ag2S@OxaNPs, (iii) Ag2S@Ce6NPs, (iv) Ag2S@CAT-Ce6NPs, and (v) Ag2S@CAT-Ce6@OxaNPs (specific values: Ce6 3.0 mg / kg, Oxa 5 mg / kg). The in vivo therapeutic effects of the nanoparticles in each group were evaluated at different time points with the aid of a small animal imaging system, with tail vein injection performed the night before imaging. To investigate the enrichment time of Ag2S@CAT-Ce6@Oxa nanoparticles at the tumor site in nude mice, in vivo fluorescence imaging was performed to monitor the fluorescence signal at the tumor site after tail vein injection of the nanoparticles. As time progressed, fluorescence signals at the tumor sites were monitored, reaching their peak 12 hours after the nanoparticles entered the bloodstream, before halving to half by 24 hours. Mice treated with Ag2S@CAT-Ce6@Oxa nanoparticles were immediately sacrificed, and their major organs were imaged in vitro. Results showed fluorescence signals in the tumors and liver, while other major organs, such as the spleen, kidneys, heart, and lungs, showed very low fluorescence signals. We then quantified the fluorescence signals at different time intervals at the mouse tumor sites, finding that the fluorescence signal peaked 12 hours after injection, consistent with previous results, thus determining the optimal phototherapy time for the next laser treatment.

[0089] Tumor sites in nude mice were irradiated with a 660 nm laser (300 mW / cm) for 10 minutes. In vivo fluorescence imaging was performed using the IVIS Spectrum small animal in vivo optical imaging system to record tumor progression on days 1, 4, 10, and 14 after PDT treatment. Figure 7 (d). Figure 7 The relative tumor growth is shown in Figure 1. We noted that tumor growth was most inhibited in group V, indicating the highest antitumor efficacy in the Ag2S@CAT-Ce6@Oxa nanoparticle treatment group. Compared to the control group, the Ag2S@Oxa, Ag2S@Ce6, and Ag2S@CAT-Ce6 groups all showed some degree of tumor inhibition. Finally, we resected and weighed the tumors in all treatment groups after day 14. Figure 7(b, e). The results showed that the tumor mass of the Ag2S@CAT-Ce6 and Ag2S@CAT-Ce6@Oxa groups was significantly lower than that of other groups, especially the Ag2S@CAT-Ce6@Oxa group. Fluorescence images and in vivo mouse tumor photographs both pointed to the same result: the tumors in this group were the smallest and had the lowest mass among all groups. The next two groups, Ag2S@OxaNPs and Ag2S@Ce6NPs, showed some inhibitory effect on tumor growth compared to the control group. This may be because their Ag2S quantum dots contain chemotherapeutic drugs or photosensitizers, producing some therapeutic effect. Compared with single-component treatment, the tumor inhibition effect was not very good. However, after treatment with assembled Ag2S@CAT-Ce6@Oxa nanoparticles containing both chemotherapeutic drugs and photosensitizers, images of tumor detachment and quantitative analysis of tumor mass showed that in vivo PDT / PTT synergistic chemotherapy with Ag2S@CAT-Ce6@Oxa nanoparticles was the most effective, achieving the best anti-tumor effect.

[0090] Experimental Example 9: Biosafety Assessment

[0091] Overall, since the mice maintained a healthy weight, none of the treated mice exhibited any significant systemic toxicity. Figure 7 (g). To evaluate the systemic toxicity of the five treatment groups to tumor-bearing mice during treatment, major organs such as the heart, liver, spleen, lungs, kidneys, and tumors from each group were collected for histological examination. Figure 8 As shown, histological examination revealed some disordered hepatocytes and thickened pulmonary interstitial tissue in the Ag2S@Oxa group during HE staining, but no significant damage was found in major organs. The liver and lung damage observed with Ag2S@Oxa may be due to the toxicity of free chemotherapeutic drugs in mice. In contrast, Ag2S@CAT-Ce6@Oxa, by slowly releasing chemotherapeutic drugs in an acidic TME, can better exert the anticancer effect of PDT / PTT synergistic chemotherapy while maintaining good biocompatibility. In conclusion, all these results suggest that Ag2S@CAT-Ce6@Oxa NPs may be a promising nanomedicine for near-infrared imaging-guided PDT / PTT synergistic chemotherapy in the treatment of colon cancer, exhibiting better safety and efficacy compared to the clinical use of tail vein-dependent Oxa.

[0092] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a multi-charge electrostatically self-assembled nanocomposite material, characterized in that, Comprising the following steps: (1) Synthesis of CAT-Ce6: dropwise add Ce6-NHS solution to catalase CAT solution, stir the reaction in the dark to obtain crude CAT-Ce6, then purify the crude CAT-Ce6 to obtain pure CAT-Ce6; (2) Synthesis of negatively charged Ag2S-3MPA QDs: under the protection of inert atmosphere, mix 3-mercaptopropionic acid completely dissolved at room temperature with deionized water, then add AgNO3 solution, adjust the solution color to be colorless and transparent by using a pH adjuster, then continue the reaction under magnetic stirring in an oil bath pot to obtain crude Ag2S-3MPA QDs in the form of brownish yellow solution, then purify the crude Ag2S-3MPA QDs to obtain negatively charged Ag2S-3MPA QDs; (3) Synthesis of positively charged Ag2S-NH2 QDs: add EDC and NHS to Ag2S-3MPA QDs prepared in step (2), mix uniformly at room temperature, stir the reaction in the dark, then add six-armed amino PEG, continue the stirring reaction in the dark until completion, then freeze-dry after dialysis to obtain positively charged Ag2S-NH2 QDs; (4) Preparation of Ag2S@CAT-Ce6@Oxa nanocomposites: ultrasonically mix Ag2S-NH2 QDs solution and CAT-Ce6 solution to obtain a mixed solution, dissolve oxaliplatin Oxa in a mixture of methanol and DMSO, then dropwise add the solution to the mixed solution, stir at room temperature in the dark to obtain crude Ag2S@CAT-Ce6@Oxa, purify the crude Ag2S@CAT-Ce6@Oxa to obtain pure Ag2S@CAT-Ce6@Oxa nanoparticles.

2. The method of claim 1, wherein: In step (1), the Ce6-NHS solution is prepared by dispersing EDC and NHS in dimethyl sulfoxide, then mixing with Ce6 uniformly at room temperature, then stirring the reaction in the dark at room temperature to obtain activated Ce6-NHS solution.

3. The method of claim 1, wherein: In step (1), the crude CAT-Ce6 is purified by centrifuging the crude CAT-Ce6, taking the supernatant, dialyzing in different gradient PBS buffer under continuous magnetic stirring, then centrifuging the dialyzed CAT-Ce6, and finally obtaining powder CAT-Ce6 pure product through freeze-drying treatment steps.

4. The method of claim 3, wherein: The molecular weight cut-off of the dialysis bag is 10 kDa.

5. The method of claim 1, wherein: In step (2), the pH regulator is a 2 mol / L NaOH solution or a 2 mol / L CH3COOH solution, and the pH of the solution is adjusted to 7.2-7.8 using the pH regulator until the solution is colorless and transparent. -1 In step (2), the pH regulator is a 2 mol / L NaOH solution or a 2 mol / L CH3COOH solution, and the pH of the solution is adjusted to 7.2-7.8 using the pH regulator until the solution is colorless and transparent. -1 In step (2), the pH regulator is a 2 mol / L NaOH solution or a 2 mol / L CH3COOH solution, and the pH of the solution is adjusted to 7.2-7 6. The method of claim 1, wherein: In step (2), the crude Ag2S-3MPA QDs are purified by centrifuging the crude Ag2S-3MPA QDs, washing the supernatant with ultrapure water, mixing and centrifuging ultrafiltration, then sealing and storing the purified Ag2S-3MPA QDs at 4°C in the dark.

7. The method of claim 6, wherein the method further comprises: In step (2), the ultrafiltration tube used is an ultrafiltration tube with a molecular weight cut-off of 10 kDa, Millipore Amico Ultra.

8. The method of claim 1, wherein: In step (4), the weight ratio of Ag2S-NH2 QDs solution to CAT-Ce6 solution is 1:7.5-7.5:1, and the pH of the mixed solution is 7.

4.

9. The method of claim 1, wherein: In step (4), the Ag2S@CAT-Ce6@Oxa crude product is purified by the following method: the Ag2S@CAT-Ce6@Oxa crude product is centrifuged to remove free CAT-Ce6, Ag2S-NH2 QDs and Oxa, and finally the Ag2S@CAT-Ce6@Oxa nanoparticle pure product is obtained.

10. The use of the polyelectrostatically self-assembled nanocomposite prepared by the preparation method according to any one of claims 1-9 in the preparation of an antitumor drug.

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