A copper arsenite nanoparticle-loaded alginate hydrogel, a preparation method and application thereof

Abstract

The application relates to the technical field of biomedical engineering, and discloses a sodium alginate hydrogel loaded with copper arsenite nanoparticles as well as a preparation method and application thereof. The hydrogel comprises a sodium alginate hydrogel matrix and copper arsenite nanoparticles loaded therein, the copper arsenite nanoparticles comprise Cu2+, arsenite ions and PLGA-PEG-NH2, the sodium alginate hydrogel loaded with the copper arsenite nanoparticles is used for in-situ solidification to form a drug storage after intratumoral injection. The application constructs a nanoparticle self-assembly system based on PLGA-PEG-NH2, uniformly loads a complex formed by arsenite ions and copper ions in the PLGA-PEG-NH2 carrier material, forms copper arsenite nanoparticles with uniform and controllable particle sizes through a self-assembly process, the nanoparticles form stable complexes through coordination complexation between Cu2+ and As3+, and have the characteristics of specific dissociation in a tumor acid microenvironment.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to an alginate hydrogel loaded with copper arsenite nanoparticles, its preparation method, and its application. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide and a leading cause of cancer-related deaths in women. Triple-negative breast cancer, lacking estrogen receptors, progesterone receptors, and HER2 expression, faces significant challenges in clinical treatment due to the absence of effective targeted therapies.

[0003] Arsenic trioxide (ATO), an effective component of the traditional Chinese medicine arsenic trioxide, has shown significant efficacy in the treatment of acute promyelocytic leukemia. Recent studies have also demonstrated its antitumor activity in solid tumors, including breast cancer. ATO primarily induces tumor cell apoptosis by triggering reactive oxygen species (ROS) bursts, depleting glutathione (GSH), and inducing mitochondrial dysfunction. However, the clinical application of ATO faces limitations due to its narrow therapeutic window, high systemic toxicity, and rapid in vivo clearance, restricting its widespread use in the treatment of solid tumors.

[0004] In recent years, copper death (cuproptosis) has attracted widespread attention as a novel mode of programmed cell death. Triggered by excess Cu²⁺, Cu²⁺ is reduced to Cu⁺ via FDX1, directly targeting lipid-acylated enzymes in the tricarboxylic acid cycle (such as DLAT), leading to their oligomerization and loss of function, ultimately causing mitochondrial metabolic collapse and cell death. Studies have shown that copper death can induce intracellular drug delivery (ICD), release damage-associated molecular patterns (DAMPs), activate dendritic cells (DCs) and T cells, thereby stimulating a systemic anti-tumor immune response.

[0005] Currently, ATO and Cu 2+ Research on its combined use in cancer therapy is still in its early stages. Lee et al. reported a copper arsenite nanoparticle (CuAs-PMs) based on PEG-PDOPA copolymer micelles, which utilizes catechol (DOPA) groups to chelate Cu. 2+It forms a complex with arsenite and generates hydroxyl radicals through a Fenton-like reaction after intravenous injection, thereby enhancing oxidative stress to kill tumor cells. However, this study still has the following shortcomings: (1) The carrier PEG-PDOPA used requires multiple chemical synthesis steps, the preparation process is complicated, and the catechol groups are easily oxidized, resulting in poor batch stability; (2) This system relies solely on intravenous injection, and the accumulation at the tumor site mainly depends on the EPR effect, with limited targeting and retention time, resulting in low drug utilization; (3) Its anti-tumor mechanism mainly focuses on oxidative stress-induced apoptosis, without involving copper death and the synergistic effect of ICD, and it has not achieved local long-term sustained release.

[0006] Therefore, developing a hydrogel delivery system that can efficiently co-deliver Cu²⁺ and As³⁺, achieve local tumor sustained release, and induce synergistic effects of copper death and apoptosis is of great significance and practical value for improving the therapeutic effect of breast cancer and reducing systemic toxicity.

[0007] Patent application CN108743545B discloses an alginate-drug-loaded nanoparticle-polycationic microcapsule and its preparation and application, belonging to the field of medicinal chemistry technology. The microcapsule product provided by this invention is a spherical microcapsule with a particle size of 100-1000 micrometers. Its structure consists of two parts: a microcapsule membrane and a core. The microcapsule membrane is a polyelectrolyte composite hydrogel membrane formed by the layer-by-layer self-assembly of alginate, drug-loaded nanoparticles, and polycations. The core is a hydrogel or hydrosol environment containing animal cells.

[0008] The existing technology has failed to achieve efficient co-delivery of Cu²⁺ and As³⁺, local tumor sustained release, and synergistic effects in inducing copper death and apoptosis.

[0009] Therefore, a new technical solution is needed to solve the above-mentioned technical problems. Summary of the Invention

[0010] The present invention aims to provide an alginate hydrogel loaded with copper arsenite nanoparticles, its preparation method, and its applications. A nanoparticle self-assembly system based on PLGA-PEG-NH2 is constructed, in which a complex formed by arsenite ions and copper ions is uniformly loaded onto a PLGA-PEG-NH2 carrier material, forming copper arsenite nanoparticles (CuAs-PM) with uniform and controllable particle size through a self-assembly process. These nanoparticles utilize the coordination complexation between Cu²⁺ and As³⁺ to form a stable complex and possess the characteristic of specific dissociation in the acidic tumor microenvironment. The nanoparticles can achieve synergistic anti-tumor effects of chemotherapy and immunotherapy, wherein As³⁺ induces tumor cell apoptosis, and Cu²⁺ induces copper death and immunogenic cell death; simultaneously, the nanoparticles significantly reduce drug leakage in non-target tissues, thereby effectively reducing the risk of systemic toxicity.

[0011] The technical solution adopted in this invention is: An alginate hydrogel loaded with copper arsenite nanoparticles, the hydrogel comprising: a sodium alginate hydrogel matrix and copper arsenite nanoparticles loaded therein, the copper arsenite nanoparticles comprising Cu²⁺, arsenite ions and PLGA-PEG-NH₂, the sodium alginate hydrogel loaded with copper arsenite nanoparticles being used for in-situ solidification to form a drug reservoir after intratumoral injection.

[0012] By employing the aforementioned materials, a PLGA-PEG-NH2-based nanoparticle self-assembly system was constructed. A complex formed by arsenite ions and copper ions was uniformly encapsulated within the PLGA-PEG-NH2 carrier material, forming uniformly sized and controllable copper arsenite nanoparticles (CuAs-PM) through a self-assembly process. These nanoparticles utilize the coordination complexation between Cu²⁺ and As³⁺ to form a stable complex and possess the characteristic of specific dissociation in the acidic tumor microenvironment. The nanoparticles can achieve synergistic anti-tumor effects of chemotherapy and immunotherapy, where As³⁺ induces tumor cell apoptosis, and Cu²⁺ induces copper death and immunogenic cell death. Simultaneously, these nanoparticles significantly reduce drug leakage in non-target tissues, thereby effectively reducing the risk of systemic toxicity.

[0013] Preferably, the hydrated particle size of the copper arsenite nanoparticles is not greater than 500 nm, the hydrated particle size of the copper arsenite nanoparticles is 300-350 nm, and the polydispersity index (PDI) of the copper arsenite nanoparticles is less than 0.3.

[0014] Preferably, the sodium alginate hydrogel matrix is ​​formed by calcium ion crosslinking, wherein the sodium alginate hydrogel matrix is ​​formed by crosslinking sodium alginate at a concentration of 1-20 mg / mL with calcium chloride at a concentration of 5-60 mg / mL.

[0015] Preferably, the sodium alginate hydrogel matrix is ​​formed by cross-linking sodium alginate at a concentration of 5 mg / mL with calcium chloride at a concentration of 20 mg / mL.

[0016] Preferably, a method for preparing an alginate hydrogel loaded with copper arsenite nanoparticles according to claims 1-4 comprises the following steps: Step S1: Prepare copper arsenite nanoparticles using PLGA-PEG-NH2; Step S2, Preparation of alginate hydrogel: The copper arsenite nanoparticles prepared in step S1 are mixed evenly with sodium alginate solution, and calcium ion solution is added to crosslink and form an alginate hydrogel loaded with copper arsenite nanoparticles. The calcium ion solution is calcium chloride solution.

[0017] Preferably, the preparation of copper arsenite nanoparticles in step S1 includes the following steps: Step 1: Prepare copper arsenite nanoparticles using a double emulsion-solvent evaporation method; Step 2: Mix the Cu²⁺ aqueous phase and the arsenite-containing aqueous phase with the PLGA-PEG-NH₂ oil phase separately and sonicate to form two W / O emulsions; Step 3: Mix the two emulsions and add them to ultrapure water for ultrasonication to form a W / O / W emulsion. Cu²⁺ and arsenite ions form a copper arsenite complex in the inner aqueous phase. Step 4: Remove the oil phase to obtain copper arsenite nanoparticles.

[0018] By employing the above method and using sodium alginate hydrogel as a carrier, a drug reservoir is formed in situ through intratumoral injection, which significantly prolongs the retention time of Cu²⁺ and As³⁺ at the tumor site, achieving long-term sustained release and overcoming the problem of rapid clearance of arsenic agents. The hydrogel has good biocompatibility, a mild preparation process, and can be mass-produced, providing a new strategy for breast cancer treatment that combines novel mechanism and translational feasibility.

[0019] Preferably, Cu in copper arsenite nanoparticles 2+ With As 3+ The molar ratio is 1:0.5 to 1:0.8.

[0020] Preferably, an alginate hydrogel loaded with copper arsenite nanoparticles is used in the preparation of cancer treatment drugs. The hydrogel exerts an anti-tumor effect through the synergistic effect of inducing copper death and apoptosis. The hydrogel induces a systemic anti-tumor immune response by activating ICD. The hydrogel is administered via intratumoral injection, forming a drug reservoir in situ to achieve long-term sustained release of the drug.

[0021] By employing the aforementioned materials, the hydrogel induces copper death and activates ICD through Cu²⁺, while simultaneously inducing apoptosis through As³⁺, forming a synergistic therapeutic model of "copper death-immune activation-apoptosis." The two work synergistically to produce a cascaded amplified anti-tumor effect.

[0022] Compared with the prior art, the present invention has the following advantages: 1. This invention constructs a nanoparticle self-assembly system based on PLGA-PEG-NH2. A complex formed by arsenite ions and copper ions is uniformly encapsulated within a PLGA-PEG-NH2 carrier material. Through a self-assembly process, uniformly sized and controllable copper arsenite nanoparticles (CuAs-PM) are formed. These nanoparticles utilize the coordination complexation between Cu²⁺ and As³⁺ to form a stable complex and possess the characteristic of specific dissociation in the acidic tumor microenvironment. The nanoparticles can achieve synergistic anti-tumor effects of chemotherapy and immunotherapy, where As³⁺ induces tumor cell apoptosis, and Cu²⁺ induces copper death and immunogenic cell death. Simultaneously, these nanoparticles significantly reduce drug leakage in non-target tissues, thereby effectively reducing the risk of systemic toxicity.

[0023] 2. This invention uses sodium alginate hydrogel as a carrier to form a drug reservoir in situ through intratumoral injection, which significantly prolongs the retention time of Cu²⁺ and As³⁺ at the tumor site, achieving long-term sustained release and overcoming the problem of rapid clearance of arsenic agents.

[0024] 3. The hydrogel of the present invention induces copper death and activates ICD through Cu²⁺, and induces cell apoptosis through As³⁺, forming a three-in-one synergistic therapeutic mode of "copper death-immune activation-cell apoptosis". The two work together to produce a cascade amplified anti-tumor effect.

[0025] 4. The hydrogel of the present invention has good biocompatibility and a mild preparation process, and can be mass-produced, providing a new strategy for breast cancer treatment that combines novel mechanism and translational feasibility. Attached Figure Description

[0026] Figure 1 The diagram illustrates the preparation process and formation of the copper arsenite hydrogel (CuAs-PM@ALG) of this invention, and requires color representation.

[0027] Figure 2 The physicochemical properties and in vitro release diagram of the CuAs-PM@ALG hydrogel of this invention require color representation.

[0028] Figure 3 The diagram shows the formulation screening and rheological characterization of the alginate hydrogel of this invention, which requires color representation.

[0029] Figure 4 The in vitro pharmacodynamic evaluation diagram of the copper arsenite nanoparticles (CuAs-PM) of this invention requires color representation.

[0030] Figure 5The Western blot diagram (DLAT, FDX1, HSP70 protein expression) of copper death induced by copper arsenite nanoparticles (CuAs-PM) of this invention is shown in color.

[0031] Figure 6 The detection diagram (CRT exposure, HMGB1 release, ATP release) of the immunogenic cell death induced by the copper arsenite nanoparticles (CuAs-PM) of this invention requires color representation.

[0032] Figure 7 The in vivo antitumor pharmacodynamic evaluation diagram of the CuAs-PM@ALG hydrogel of this invention requires color representation.

[0033] Figure 8 The in vivo safety evaluation diagram of the CuAs-PM@ALG hydrogel of this invention requires color representation.

[0034] Figure 9 The image shows the evaluation of the immune activation effect of the CuAs-PM@ALG hydrogel of this invention, and color coding is required. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to embodiments. The present invention provides an alginate hydrogel (CuAs-PM@ALG) loaded with copper arsenite nanoparticles. The hydrogel forms a drug reservoir in situ through intratumoral injection, sustaining the release of Cu²⁺ and As³⁺, and enhances the antitumor efficacy through the synergistic effect of inducing copper death and apoptosis.

[0036] Example 1: Preparation of copper arsenite nanoparticles (CuAs-PM) Copper arsenite nanoparticles (CuAs-PM) were prepared using a dual emulsion-solvent evaporation technique. 20 mg of PLGA-PEG-NH2 (PLGA molecular weight 5 kDa, PEG molecular weight 5 kDa, purchased from Hunan Huateng Pharmaceutical Co., Ltd.) was dissolved in 2 mL of dichloromethane as the oil phase. Cu(Ac)2 aqueous solution (36 mg / mL) and NaAsO2 aqueous solution (0.1 M, pH 8.0) were prepared separately. 300 μL of the Cu²⁺ aqueous phase was added to 1 mL of the oil phase, and the mixture was sonicated in an ice bath (200 W, 30 s) to form a W / O primary emulsion (emulsion A). Emulsion B was prepared using the AsO2⁻ aqueous phase in the same manner. Emulsion A and emulsion B were mixed, and 10 mL of ultrapure water was added. The mixture was then sonicated (200 W, 60 s) to form a W / O / W double emulsion. Dichloromethane was removed by rotary evaporation (35 °C), the precipitate was collected by centrifugation (12,000 rpm, 10 min), washed twice with ultrapure water, and lyophilized to obtain a white powder (CuAs-PM). Figure 1This is a schematic diagram illustrating the preparation process and hydrogel formation of CuAs-PM according to the present invention.

[0037] Example 2 Characterization of the physicochemical properties of copper arsenite nanoparticles (CuAs-PM) This embodiment characterizes the particle size, potential, morphology, and in vitro release behavior of the CuAs-PM prepared in Example 1. Figure 2 As shown. Dynamic light scattering (DLS) measured the hydrated particle size of CuAs-PM to be 312.93 ± 2.51 nm. Figure 2 A), the polydispersity index (PDI) is 0.261, and the zeta potential is -22.8 ± 0.9 mV ( Figure 2 B). Scanning electron microscopy-X-ray energy dispersive spectroscopy (SEM-EDS) showed that CuAs-PM was rich in Cu, As, O, and C elements, with Cu and As originating from copper arsenite (CuAs), while O and C originated from PLGA-PEG-NH2 (… Figure 2 D). To further observe its microstructure and elemental distribution, transmission electron microscopy (TEM) was used to show that the nanoparticles exhibit a spherical core-shell structure. Figure 2 Energy dispersive spectroscopy (EDS) confirmed that Cu and As are concentrated in the core, while O is distributed in the outer shell. X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) confirmed that Cu is divalent and As is trivalent, and that they form a copper arsenite complex.

[0038] Example 3 Formulation screening and rheological characterization of alginate hydrogels Equal volumes of sodium alginate (ALG, 1–20 mg / mL) and calcium chloride (CaCl2, 5–60 mg / mL) of different concentrations were mixed, and the gelation time, gel volume, mass, and in vitro degradation time were measured. Figure 3 As shown in A and 3B, the higher the ALG concentration and the higher the CaCl2 concentration, the faster the gelation; the gel formed by the combination of 5 mg / mL ALG and 20 mg / mL CaCl2 gels in about 30 seconds at room temperature. Figure 3 A), with a volume of approximately 400 μL, and an in vitro degradation time of approximately 5 days ( Figure 3 (B) is suitable for intratumoral injection. Therefore, this formulation was determined to be optimal.

[0039] The CuAs-PM prepared in Example 1 was dispersed in a 5 mg / mL ALG solution (final concentration 1 mg / mL), stirred until homogeneous, and then an equal volume of 20 mg / mL CaCl2 was added. The mixture was allowed to stand at room temperature for 30 seconds to obtain the CuAs-PM@ALG hydrogel. Figure 3As shown in Figure F, scanning electron microscopy (SEM) reveals that the hydrogel possesses a porous structure of 500–1000 nm, with nanoparticles uniformly distributed as spherical particles of approximately 250 nm on the pore walls. Rheological testing results (37 °C) are also presented. Figure 3 C, 3D, and 3E show that the hydrogel remained in a gel state before and after drug loading (storage modulus G′ > loss modulus G″), and there were no significant changes in the linear viscoelastic region and loss factor, confirming that drug loading has little effect on the mechanical properties of the gel.

[0040] Example 4: In vitro release of copper arsenite hydrogel (CuAs-PM@ALG) The release behavior of As³⁺ from CuAs-PM@ALG hydrogel was determined using the dialysis bag method (PBS, pH 7.4, 37 ℃). Figure 2 As shown in Figure C, compared with the rapid release of free CuAs-PM nanoparticles (45.6% release in 2 h), CuAs-PM@ALG hydrogel exhibits a significant sustained-release characteristic: a cumulative release of 42.05 ± 0.67% in 12 h and 91.85 ± 0.85% in 48 h. These results indicate that hydrogel loading can effectively prolong drug release time and inhibit burst release.

[0041] Example 5: In vitro cellular uptake, cytotoxicity, migration inhibition, and apoptosis induction of copper arsenite nanoparticles (CuAs-PM) Using 4T1 mouse breast cancer cells (purchased from the Cell Bank of the Chinese Academy of Sciences) as a model, the in vitro antitumor activity of CuAs-PM prepared in Example 1 was evaluated. Figure 4 As shown in C and 4D, after incubating cells with free Cu²⁺ (9.25 μM), free NaAsO₂ (5.1 μM), and CuAs-PM (equal As³⁺ concentration) for 24 h, the intracellular Cu content in the CuAs-PM group was measured to be 28.09 ng / 10⁻⁶ by ICP-MS. 6 Cellular and As content was 8.43 ng / 10 6 The cells showed no significant difference from the free ion group, indicating that the nanoparticles can efficiently co-deliver Cu²⁺ and As³⁺.

[0042] The inhibitory effect of CuAs-PM prepared in Example 1 on the proliferation of 4T1 cells and 293T human embryonic kidney cells (purchased from the Cell Bank of the Chinese Academy of Sciences) was detected using the Cell Counting Kit-8 (CCK-8, purchased from Shanghai Adamas Reagent Co., Ltd.). Logarithmic growth phase 4T1 cells and 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at approximately 5 × 10⁶ cells per well. 3Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight to allow cell adhesion. Then, complete culture media containing CuAs-PM, NaAsO2, and Cu²⁺ were added, respectively, and the plates were set to [condition]. 3+ and Cu 2+ The concentration gradients were 0.5, 1, 3, 9, 27, and 50 μM. A negative control group (containing only RPMI 1640 complete medium, without the drug) was also set up. After incubating the 96-well plates in a cell culture incubator for 24 h, 100 μL of CCK-8 working solution was added to each well, and incubation continued for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. The absorbance of the blank control group (containing only medium, without cells) was recorded as Ab; the absorbance of the negative control group was recorded as An; and the absorbance of the sample group was recorded as Ai. Cell viability was calculated according to formula (5-1). All experiments were repeated three times. The IC50 of CuAs-PM against 4T1 cells was determined by the CCK-8 method. 50 The IC50 concentration was 4.54 μM, and the IC50 concentration against 293T cells was [missing information]. 50 10.25 μM ( Figure 4 E and 4F exhibit selective cytotoxicity.

[0043] (5-1) The scratch assay was used to detect the inhibitory effect of CuAs-PM prepared in Example 1 on the migration of 4T1 cells. Horizontal lines were drawn on the back of a 6-well plate using a marker and a ruler. 4T1 cells were revived and allowed to enter the logarithmic growth phase, then inoculated with 5 × 10⁻⁶ cells per well. 5 Cells were seeded evenly into 6-well plates at a density of 10 cells / well. After 12 h, once the cells had fully adhered and reached confluence, a sterile 200 μL pipette tip was used, with the tip perpendicular to the 6-well plate, to make even scratches along the horizontal lines. The culture medium was discarded, and the cells were gently washed with 1×PBS to remove detached cells. A culture medium containing 1% fetal bovine serum was used as a negative control. The test drugs (Cu²⁺, NaAsO₂, CuAs-PM nanoparticles) were prepared in DMEM medium containing 1% fetal bovine serum and co-incubated with the cells for 36 h. During this period, bright-field images of the scratched areas were captured using an Axio Observer Z1 confocal microscope at 0 h, 6 h, 12 h, 24 h, and 36 h. Three fields of view were randomly selected from each well, and quantification was performed using ImageJ software (version 1.52v). The initial scratch area was denoted as S₀, and the scratch area at time t was denoted as S₂. t The wound healing rate was calculated using formula (5-2). The scratch test results showed that ( Figure 4(A, 4B) After 36 h of drug administration, the healing rate was 76% in the control group, 46% in the Cu²⁺ group, 50% in the NaAsO2 group, and only 20% in the CuAs-PM group, indicating that CuAs-PM significantly inhibited cell migration.

[0044] (5-2) To detect the apoptosis-inducing effect of CuAs-PM prepared in Example 1 on 4T1 cells, quantitative analysis was performed using Annexin V-FITC / PI double staining combined with flow cytometry. Logarithmic growth phase 4T1 cells were cultured at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight at 37 °C with 5% CO2 to allow adherence. After aspirating the original culture medium, serum-free medium containing Cu²⁺, NaAsO₂, and CuAs-PM nanoparticles was added, with a blank medium treatment group serving as a negative control. Each group had 3 replicates. After 24 h of drug treatment, cells were collected by trypsin digestion and washed twice with pre-cooled PBS to remove residual culture medium. Cells were resuspended in 100 μL of 1×PBS buffer in each tube, followed by the addition of 5 μL of FITC-labeled Annexin V and 5 μL of propidium iodide (PI). After gentle mixing, the cells were incubated at 4 °C in the dark for 15 min. After incubation, 400 μL of 1×PBS buffer was added, and the cells were immediately analyzed by flow cytometry. The proportion of FITC and PI double-positive cells was analyzed using FlowJo software to determine the percentage of apoptotic cells. Annexin V-FITC / PI flow cytometry results (…) Figure 4 The results (G, 4H) showed that the apoptosis rate of the CuAs-PM group was 44.8%, which was significantly higher than that of the Cu²⁺ group (30.9%) and the NaAsO2 group (36.6%), confirming that CuAs-PM can effectively induce tumor cell apoptosis.

[0045] Example 6 Detection of copper death-related protein expression induced by copper arsenite nanoparticles (CuAs-PM) To verify whether the CuAs-PM nanoparticles prepared in Example 1 exert their antitumor effect through the copper death pathway, Western blot was used to detect the expression levels of key copper death regulatory proteins DLAT, FDX1, and HSP70. 4T1 cells were cultured at 2 × 10⁻⁶ cells / day. 5Cells were seeded per well in 6-well plates and cultured overnight. Then, blank medium (Control), Cu²⁺ (9.25 μM), NaAsO₂ (5.1 μM), and CuAs-PM (As³⁺ 5.1 μM, Cu²⁺ 9.25 μM) were added, and the cells were incubated for 12 h. Cells were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by the BCA method. An equal volume of protein was subjected to SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk for 1 h, and then incubated overnight at 4 °C with anti-DLAT (1:1000), anti-FDX1 (1:1000), anti-HSP70 (1:1000), and internal control GAPDH (1:5000) antibodies. After washing, HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 h. The membrane was then visualized by ECL chemiluminescence.

[0046] Results (see) Figure 5 The results showed that, compared with the Control group, the ~70 kDa monomeric band of DLAT protein was significantly weakened in the CuAs-PM treatment group, indicating that DLAT underwent irreversible oligomerization; FDX1 protein expression was downregulated, suggesting that the upstream regulatory pathway of copper death was activated; HSP70 protein expression was upregulated, reflecting the compensatory response of cells to protein toxicity stress. Similar trends were also observed in the Cu²⁺ and NaAsO2 groups, but the magnitude of the changes was smaller than that in the CuAs-PM group. These results indicate that CuAs-PM can effectively induce copper death in 4T1 cells, and that copper and arsenic ions have a synergistic enhancing effect.

[0047] Example 7 Detection of immunogenic cell death (ICD) markers induced by copper arsenite nanoparticles (CuAs-PM) Given that Example 6 has demonstrated that CuAs-PM can effectively induce copper death in 4T1 cells (manifested as DLAT oligomerization, FDX1 downregulation, and HSP70 upregulation), and that copper death has been reported to trigger immunogenic cell death (ICD), this example further examines ICD-related markers, including cell surface calreticulin (CRT) exposure, extracellular high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP) release, to verify whether CuAs-PM-induced copper death subsequently triggers ICD.

[0048] (1) CRT exposure detection (immunofluorescence): 4T1 cells were cultured in 24-well plates and treated with the same drugs for 12 h. They were fixed with 4% paraformaldehyde for 15 min, washed with PBS, blocked with 3% BSA for 30 min, and incubated overnight at 4°C with anti-CRT primary antibody (1:200). After washing with PBS, FITC-labeled secondary antibody (1:200) was added and incubated at room temperature for 1 h. The nuclei were stained with DAPI, and observed under a fluorescence microscope. Results ( Figure 6A) The results showed that the cell membrane CRT fluorescence in the Control group was weak; partial membrane fluorescence was observed in the Cu²⁺ and NaAsO2 groups; while the cell membrane CRT fluorescence intensity in the CuAs-PM group was significantly enhanced, showing a ring-shaped bright green color, indicating that a large amount of CRT was exposed on the cell surface.

[0049] (2) HMGB1 release assay (ELISA): Cell culture supernatant was collected 12 h after treatment with the above drugs, cell debris was removed by centrifugation, and the concentration of HMGB1 in the supernatant was determined according to the instructions of the mouse HMGB1 ELISA kit. Results ( Figure 6 B) The results showed that the HMGB1 release in the Control group was extremely low (approximately 2.1 ng / mL); the Cu²⁺ group and the NaAsO2 group increased to 8.7 ng / mL and 9.2 ng / mL, respectively; while the HMGB1 concentration in the CuAs-PM group reached 23.5 ng / mL, which was significantly higher than that in the free ion group, indicating that a large amount of HMGB1 was released into the extracellular space.

[0050] (3) ATP release detection (bioluminescence method): Using the same supernatant, the ATP content was measured using an ATP detection kit and a fluorescence / luminescence analyzer. Results ( Figure 6 C) The results showed that the supernatant ATP concentration in the CuAs-PM treatment group (18.6 μM) was approximately 9 times that of the Control group (2.1 μM), which was significantly higher than that in the Cu²⁺ group (5.2 μM) and the NaAsO2 group (6.3 μM).

[0051] The above results confirm that CuAs-PM can effectively induce ICD in tumor cells, manifested as increased CRT membrane exposure, HMGB1 and ATP release, with a significantly stronger synergistic effect than single ions. These DAMPs signaling can further promote DC maturation and T cell activation, thereby activating systemic anti-tumor immunity.

[0052] Example 8: In vivo antitumor pharmacodynamic evaluation of copper arsenite hydrogel (CuAs-PM@ALG) A 4T1 tumor-bearing BALB / c female mouse model was established using 6-week-old, female, SPF-grade BALB / c mice weighing approximately 20 g. 100 μL of 4T1 cell suspension was injected into the left fat pad of the mice. The 4T1 cell suspension was prepared by resuspending healthy 4T1 cells in logarithmic growth phase with PBS at a cell density of 1 × 10⁻⁶ cells / mL. 8 / mL. After inoculation, the growth of the tumor was closely monitored. The long diameter of the tumor was recorded as L and the transverse diameter as W. The tumor volume was calculated according to formula (8-1). When the tumor volume reached approximately 100 mm³, the model mice were randomly divided into 6 groups (n=5): Control (blank control), blank ALG hydrogel, free Cu²⁺ group (6 mg / kg), free NaAsO₂ group (4 mg / kg), CuAs-PM group (isoas³⁺ concentration), and CuAs-PM@ALG group (isoas³⁺ concentration). The copper arsenite hydrogel (CuAs-PM@ALG, 100 μL) prepared in Example 3 was injected intratumorally once every 2 days for a total of 4 times.

[0053] (5-1) like Figure 7 As shown, the CuAs-PM@ALG group had the highest tumor growth inhibition rate, reaching 37.4% ( Figure 7 The tumor growth curves (A, E) were significantly superior to those of the Cu²⁺ group (7.31%), NaAsO₂ group (16.79%), and CuAs-PM group (28.32%) (p<0.01). Figure 7 B) and mouse weight change curve ( Figure 7 C) This further confirms the efficacy and safety of CuAs-PM@ALG. H&E staining of tumor tissue showed extensive necrosis in the CuAs-PM@ALG group, with the highest TUNEL staining positivity rate. Figure 7 D, 7F), Ki67 immunohistochemical staining positive rate was the lowest ( Figure 7 D, 7G).

[0054] Example 9: In vivo safety evaluation of copper arsenite hydrogel (CuAs-PM@ALG) Based on the above efficacy evaluation, the in vivo safety of the copper arsenite hydrogel CuAs-PM@ALG prepared in Example 3 was further investigated. The results showed that the body weight of mice in each group remained stable during treatment. Figure 7 C), H&E staining of major organs (heart, liver, spleen, lungs, kidneys) showed no obvious pathological damage. Figure 8 A); there were no significant differences in serum biochemical indicators (ALT, AST, BUN, CREA) and routine blood count indicators (RBC, WBC, HGB, PLT) among the groups. Figure 8 BI). Furthermore, the CuAs-PM@ALG group mice showed increased WBC counts, combined with upregulated serum IFN-γ, TNF-α, and IL-6 levels (BI). Figure 9 The elevated levels (F, 9G, 9H) suggest that the increase is due to systemic immune activation rather than pathological factors.

[0055] Example 10: Evaluation of the immune activation effect of copper arsenite hydrogel (CuAs-PM@ALG) In vitro experiments have confirmed that CuAs-PM nanoparticles can induce copper death in tumor cells, thereby triggering immunogenic cell death (ICD), manifested as increased CRT membrane exposure, HMGB1, and ATP release. These damage-associated molecular patterns (DAMPs) can promote dendritic cell (DC) maturation and activate adaptive immune responses. To verify the actual effect of this mechanism in vivo, the copper arsenite hydrogel CuAs-PM@ALG prepared in Example 3 was injected intratumorally. It continuously degraded in the tumor microenvironment, releasing CuAs-PM nanoparticles, which also released DAMPs through the copper death-ICD pathway, thereby mobilizing the immune system. Flow cytometry was then used to analyze immune cell subsets in tumor tissue. Figure 9 As shown, compared with the control group, the proportions of CD4⁺T cells and CD8⁺T cells in tumor tissue of the CuAs-PM@ALG group were significantly increased ( Figure 9 C, 9D), regulatory T cells (Tregs, Figure 9 E) and myeloid-derived suppressor cells (MDSCs, Figure 9 A) The proportion of CD80 / CD86, a marker of DC maturation, was significantly reduced, and the expression of CD80 / CD86, a mature marker of DCs, was upregulated. Figure 9 B). The above results indicate that CuAs-PM@ALG hydrogel can effectively remodel the tumor immune microenvironment and activate anti-tumor immune responses.

[0056] Based on the above embodiments, the synergistic antitumor mechanism of the present invention can be summarized as follows: Cu 2+ Reduced to Cu by FDX1 + It induces mitochondrial copper death and activates immunogenic cell death (ICD), releasing DAMPs to promote DC maturation and T cell activation; As 3+ Apoptosis is induced by depletion of glutathione (GSH) and bursts of reactive oxygen species (ROS). The two form a bidirectional synergistic cycle, producing a cascaded amplified antitumor effect.

[0057] In summary, this invention provides an alginate hydrogel (CuAs-PM@ALG) loaded with copper arsenite nanoparticles. This hydrogel can form a drug reservoir in situ via intratumoral injection, achieving synergistic sustained release of Cu²⁺ and As³⁺. It significantly enhances antitumor efficacy through the synergistic effect of inducing copper death and apoptosis, and activates a systemic antitumor immune response. The hydrogel of this invention exhibits good biocompatibility, a mild preparation process, and controllable drug release, making it suitable for preparing drugs for treating breast cancer.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should be included within the scope of protection defined by the claims of the present invention.

Claims

1. An alginate hydrogel loaded with copper arsenite nanoparticles, characterized in that: The hydrogel comprises: a sodium alginate hydrogel matrix and copper arsenite nanoparticles loaded therein, wherein the copper arsenite nanoparticles contain Cu²⁺, arsenite ions and PLGA-PEG-NH₂.

2. The alginate hydrogel loaded with copper arsenite nanoparticles according to claim 1, characterized in that: The hydrated particle size of the copper arsenite nanoparticles is no greater than 500 nm, the hydrated particle size of the copper arsenite nanoparticles is 300-350 nm, and the polydispersity index (PDI) of the copper arsenite nanoparticles is <0.

3.

3. The alginate hydrogel loaded with copper arsenite nanoparticles according to claim 1, characterized in that: The sodium alginate hydrogel matrix is ​​formed by cross-linking calcium ions, and the sodium alginate hydrogel matrix is ​​formed by cross-linking sodium alginate with a concentration of 1-20 mg / mL and calcium chloride with a concentration of 5-60 mg / mL.

4. The alginate hydrogel loaded with copper arsenite nanoparticles according to claim 3, characterized in that: The sodium alginate hydrogel matrix is ​​formed by cross-linking sodium alginate at a concentration of 5 mg / mL with calcium chloride at a concentration of 20 mg / mL.

5. A method for preparing an alginate hydrogel loaded with copper arsenite nanoparticles according to claims 1-4, characterized in that: Includes the following steps: Step S1: Prepare copper arsenite nanoparticles using PLGA-PEG-NH2; Step S2, Preparation of alginate hydrogel: The copper arsenite nanoparticles prepared in step S1 are mixed evenly with sodium alginate solution, and calcium ion solution is added to crosslink and form an alginate hydrogel loaded with copper arsenite nanoparticles. The calcium ion solution is calcium chloride solution.

6. The method for preparing an alginate hydrogel loaded with copper arsenite nanoparticles according to claim 5, characterized in that: The preparation of copper arsenite nanoparticles in step S1 includes the following steps: Step 1: Prepare copper arsenite nanoparticles using a double emulsion-solvent evaporation method; Step 2: Mix the Cu²⁺ aqueous phase and the arsenite-containing aqueous phase with the PLGA-PEG-NH₂ oil phase separately and sonicate to form two W / O emulsions; Step 3: Mix the two emulsions and add them to ultrapure water for ultrasonication to form a W / O / W emulsion. Cu²⁺ and arsenite ions form a copper arsenite complex in the inner aqueous phase. Step 4: Remove the oil phase to obtain copper arsenite nanoparticles.

7. A method for preparing an alginate hydrogel loaded with copper arsenite nanoparticles according to claim 6, characterized in that: Cu in copper arsenite nanoparticles 2+ With As 3+ The molar ratio is 1:0.5 to 1:0.

8.

8. Application of an alginate hydrogel loaded with copper arsenite nanoparticles in the preparation of cancer treatment drugs.

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