A dual-drug sequential delivery system, a composite hydrogel, its preparation method, and its applications.

Abstract

This invention belongs to the field of antitumor drug delivery technology, and discloses a dual-drug sequential delivery system, a composite hydrogel, its preparation method, and its applications. This invention achieves sequential release by encapsulating a cobalt (Co)-polyphenol coordination nanozyme (CoNZ) in a hydrogel and integrating it with microfibrils loaded with chloroquine (CQ). This invention provides a promising new approach to improving the treatment of CRPC by initially inducing ROS-mediated oxidative damage, subsequently inhibiting autophagy and blocking the TLR9 / NF-κB signaling pathway, synergistically disrupting the survival mechanisms of tumor cells.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug delivery technology, specifically to a dual-drug sequential delivery system, a composite hydrogel, its preparation method, and its application. Background Technology

[0002] Prostate cancer (PCa) has become a major global public health burden due to its rising incidence and the aging population. Although standard therapies such as surgery, radiotherapy, and androgen deprivation therapy are effective in the early stages of the disease, a significant proportion of patients inevitably progress to castration-resistant prostate cancer (CRPC). CRPC is a lethal stage characterized by strong treatment resistance, high invasiveness, and a high likelihood of widespread metastasis. Current clinical treatments for CRPC have limited efficacy, necessitating the development of novel treatment strategies that can effectively overcome the inherent drug resistance mechanisms of the tumor.

[0003] To address this challenge, reactive oxygen species (ROS)-based therapeutic strategies have garnered significant attention in recent years. These strategies leverage the high levels of endogenous hydrogen peroxide (H2O2) in the tumor microenvironment (TME) to induce irreversible oxidative damage in tumor cells. Chemodynamic therapy (CDT) is particularly noteworthy: using endogenous H2O2 as a substrate, it catalyzes the generation of large amounts of ROS through a Fenton-like reaction, without relying on external stimuli or complex equipment such as photodynamic or acoustic catalysis, thus possessing greater clinical translational potential and practical application advantages. However, the efficacy of CDT in the TME is limited due to insufficient catalytic substrates, enhanced antioxidant defense systems in the tumor cell TME, and adaptive resistance pathways such as protective autophagy.

[0004] Therefore, there is an urgent need to develop new methods that can effectively inhibit the progression of CRPC and overcome treatment resistance, providing new directions for the clinical treatment of tumors. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a dual-drug sequential delivery system, a composite hydrogel, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a dual-drug sequential delivery system comprising a cobalt-polyphenol coordinated nanozyme and a chloroquine-loaded microfiber.

[0007] In a preferred embodiment of the dual-drug sequential delivery system of the present invention, the microfiber is a polylactic acid-glycolic acid copolymer; the cobalt is a cobalt atom; and the polyphenol is epigallocatechin gallate.

[0008] In a second aspect, the present invention provides a composite hydrogel comprising aldehyde-modified hyaluronic acid; wherein the aldehyde-modified hyaluronic acid is encapsulated with a cobalt-polyphenol coordinated nanozyme and chloroquine-loaded fiber patch fragments; wherein the chloroquine-loaded fiber patch fragments comprise chloroquine-loaded microfibers and a carboxymethyl chitosan matrix.

[0009] In a preferred embodiment of the composite hydrogel of the present invention, the microfibers are polylactic acid-glycolic acid copolymers; the cobalt is cobalt atoms; and the polyphenol is epigallocatechin gallate.

[0010] Thirdly, the present invention provides a method for preparing a composite hydrogel, comprising the following steps: (1) Cobalt-polyphenol coordinated nanozymes were synthesized by antisolvent method; (2) Polylactic acid-glycolic acid copolymer is dissolved in hexafluoroisopropanol, and chloroquine is added to obtain an electrospinning solution; the electrospinning solution is electrospinned to generate microfibers, which are dried to obtain CQ@F patches; the CQ@F patches are embedded in a carboxymethyl chitosan matrix, frozen and sliced ​​to obtain chloroquine-loaded fiber patch fragments; (3) Oxidize hyaluronic acid with sodium periodate to generate aldehyde-modified hyaluronic acid; (4) Disperse the cobalt-polyphenol coordinated nanozyme in the aldehyde-modified hyaluronic acid solution to obtain a dispersion; heat and melt the chloroquine-loaded fiber patch fragments to obtain a melt; mix the dispersion and the melt to obtain a mixture, incubate, and obtain the composite hydrogel Co / CQF-CH.

[0011] In a preferred embodiment of the preparation method described in this invention, in step (1), the antisolvent method is as follows: S1. Dissolve polyvinylpyrrolidone in a solvent, add a solution containing Co(NO3)2•6H2O dropwise, stir, and then add a solution containing epigallocatechin gallate to obtain a mixture; S2. Stir the mixture continuously at room temperature for at least 4 h, dialyze to remove unreacted small molecules and residual solvent; freeze-dry to collect the product and obtain cobalt-polyphenol coordinated nanozyme.

[0012] In a preferred embodiment of the preparation method described in this invention, in step (2), the concentration of the polylactic acid-glycolic acid copolymer is 10%-20% (w / v); the final concentration of chloroquine in the electrospinning solution is 0.5%-2% (w / v); the voltage of the electrospinning is 10 kV-15 kV; and the thickness of the slice is 10 μm-50 μm.

[0013] As a preferred embodiment of the preparation method described in this invention, in step (3), the preparation method of the aldehyde-modified hyaluronic acid is as follows: dissolve hyaluronic acid in water, add sodium periodate solution dropwise; react in the dark, then add ethylene glycol to terminate the reaction; dialyze, and freeze dry to obtain aldehyde-modified hyaluronic acid.

[0014] As a further preferred embodiment of the preparation method described in this invention, the temperature of the light-protected reaction is 35 °C-40 °C, and the time is 1 h-3 h.

[0015] As a preferred embodiment of the preparation method described in this invention, in step (4), the incubation temperature is 29.3 °C-40.8 °C and the time is 5 min-30 min; the ratio of carboxymethyl chitosan and aldehyde-modified hyaluronic acid in the mixture is 3% (w / v): (0.8%-2%) (w / v).

[0016] Preferably, the ratio of carboxymethyl chitosan to aldehyde-modified hyaluronic acid in the mixture is 3% (w / v): 1.6% (w / v); and the incubation temperature is 35.09 °C.

[0017] Fourthly, the present invention provides an injectable preparation comprising any one of the dual-drug sequential delivery system, the composite hydrogel, and the composite hydrogel prepared by the preparation method.

[0018] Fifthly, the present invention applies the dual-drug sequential delivery system, the composite hydrogel, the composite hydrogel prepared by the preparation method, and the injection to the delivery of antitumor drugs.

[0019] In a sixth aspect, the present invention applies the dual-drug sequential delivery system, the composite hydrogel, the composite hydrogel prepared by the preparation method, and the injection to the preparation of antitumor drugs.

[0020] In a preferred embodiment of the application described in this invention, the tumor is prostate cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The dual-drug sequential delivery system of this invention combines the nanocatalytic effect of ROS accumulation and oxygen (O2) generation, and further blocks autophagy and NF-κB survival signaling pathways. A polyphenol-metal network-based nanozyme (CoNZ) is introduced to introduce the lysosomal inhibitor chloroquine (CQ). CoNZ is constructed from cobalt (Co) atoms and epigallocatechin gallate (EGCG). CoNZ possesses multiple enzyme catalytic activities, simultaneously inducing strong oxidative stress and alleviating hypoxia in the tumor microenvironment, while depleting the cell's endogenous antioxidant system, leading to damage to key organelles such as mitochondria and triggering CRPC cell apoptosis. EGCG can specifically bind to the complex formed by nuclear import protein α3 and the NF-κB nuclear localization sequence, further enhancing the inhibitory effect on the NF-κB survival signaling pathway. Chloroquine, as a dual inhibitor, can simultaneously block autophagy and the TLR9 / NF-κB signaling pathway, effectively preventing cancer cells from repairing oxidative damage or activating pro-survival signals, thereby significantly enhancing the ROS-mediated cell-killing effect. Synergistically disrupting the survival mechanisms of tumor cells provides a promising new approach to improving the treatment of CRPC.

[0022] 2. The composite hydrogel of this invention encapsulates a cobalt (Co)-polyphenol coordination nanozyme (CoNZ) within the hydrogel and integrates it with microfibrils loaded with chloroquine, achieving sequential release. The preferentially released CoNZ efficiently catalyzes the generation of ROS and oxygen, while simultaneously consuming key antioxidants such as reduced glutathione, leading to organelle damage, inducing apoptosis, and alleviating the hypoxic state of the tumor microenvironment, thereby downregulating the expression of pro-tumor growth factors. The subsequently released CQ effectively inhibits autophagic flux and lysosomal activation of TLR9. Notably, CoNZ specifically binds to the complex formed by nuclear import protein α3 and the NF-κB nuclear localization sequence, blocking its nuclear translocation and further enhancing the inhibitory effect on tumor survival signals. This sequential drug delivery system exhibits significant anti-tumor effects in both in vitro and in vivo models, effectively inhibiting CRPC progression and potentially overcoming treatment resistance, providing a universal platform for the synergistic treatment of various tumors. Attached Figure Description

[0023] Figure 1This is a schematic diagram of a microfiber-hydrogel injection for xenograft therapy of CRPC. In the diagram, (A) Co-EGCG coordination nanozyme (CoNZ) is synthesized and dispersed in OHA solution; (B) CMCS-OHA cross-linked thermoresponsive hydrogel is prepared, which is loaded with CoNZ and CQ-containing PLGA microfibers; (C) CoNZ and CQ are responsively and continuously released in the TME through intratumoral injection of a dual-drug sustained-release system, thereby inducing excessive oxidative stress through a cascade-enhanced multi-enzyme-like catalytic reaction and synergistically inhibiting autophagy and TLR9 / NF-κB signaling pathways, thereby inhibiting the survival and progression of CRPC.

[0024] Figure 2 Synthesis and characterization of CoNZ; Figure 1 shows: (A) Schematic diagram of CoNZ synthesis; (B) Representative TEM image of CoNZ; (C) Hydrodynamic particle size distribution and average diameter (mean ± standard deviation) of CoNZ; (D) Zeta potential (mean ± standard deviation) of CoNZ; (E) XRD pattern of CoNZ; (F) ICP-AES determination of Co and EGCG content in CoNZ; (GI) XPS results of Co 2p (G), O 1s (H) and C 1s (I) in CoNZ, used to analyze elemental valence states and chemical bond characteristics; (J) CoNZ and reference sample in Co... K XANES spectra of the edge; (K)CoNZ and reference sample at R FT-EXAFS spectra of (L)CoNZ in space; R FT-EXAFS raw data and their fitted curves in space; optimized ball-and-stick model of Co-O4 coordination structure in (M)CoNZ; WT-EXAFS contour plots of (NQ)Co foil (N), CoO (O), Co2O3 (P) and CoNZ (Q).

[0025] Figure 3 Characterization of CoNZ; In the figure, (A) UV-Vis absorption spectra of Co(NO3)2, EGCG and CoNZ; (B) Representative TEM image of CoNZ.

[0026] Figure 4 For Co valence state analysis; Co K Absorb energy while absorbing it ( E The relationship between 0 and valence state and its linear fitting results.

[0027] Figure 5 For S3 FT-EXAFS analysis; in the figure, (A) is the FT-EXAFS spectrum of Co foil in R space; (B) and (C) are the FT-EXAFS spectra of CoNZ and Co foil respectively. K FT-EXAFS spectrum of space.

[0028] Figure 6 The figures show various enzyme-like activities of CoNZ. (A) Detection mechanism of POD-like activity; (B) UV-Vis absorption spectra of MB after incubation with different concentrations of CoNZ; (C) Quantification of CoNZ •OH generation efficiency by the absorbance peak at 665 nm; (D) POD-like catalytic reaction pathway and corresponding ΔG curve based on DFT calculations; (E) CAT-like activity detection mechanism; (F) UV-Vis absorption spectra of halogenated oxalool after incubation with different concentrations of CoNZ; (G) Quantification of CoNZ conversion efficiency to H2O2 by the absorbance peak at 570 nm; (H) O2 generation of different concentrations of CoNZ in the presence of 1 mM H2O2; (I) CAT-like catalytic reaction pathway and ΔG curve based on DFT calculations; (J) SOD-like activity detection mechanism; (K) UV-Vis absorption spectra of NBT after incubation with different concentrations of CoNZ; (L) Quantification of CoNZ •O2 generation by the absorbance peak at 560 nm. − Scavenging efficiency; (M) SOD-like catalytic reaction pathway and ΔG curve based on DFT calculation; (N) NOX-like activity detection mechanism; (O) and (P) UV-Vis absorption spectra of halogenated (O) and NBT formazan (P) after incubation with different concentrations of CoNZ; (Q) NOX-like catalytic reaction pathway and ΔG curve based on DFT calculation; To balance calculation efficiency and accuracy, a simplified model with similar chemical properties was used to replace NADPH for simulation; (R) GPX-like activity detection mechanism; (S) UV-Vis absorption spectra of TNB after incubation with different concentrations of CoNZ at pH 8.5; (T) Quantification of CoNZ consumption efficiency of GSH by the absorbance peak at 410 nm; (U) GPX-like catalytic reaction pathway and ΔG curve based on DFT calculation. In a physiological environment containing proton deprivation agents, CH3–S – As a simplified analogue of GSH, it improves computational efficiency while maintaining the validity of the reaction mechanism. All data are expressed as mean ± standard deviation.

[0029] Figure 7 CoNZ-mediated intracellular catalytic reactions inhibit CRPC cell viability; In the figure, (A) FCM analysis of CoNZ uptake in PC-3 cells; (B, C) Representative fluorescence images (B) and FCM results (C) of total ROS levels in PC-3 cells after DCFH-DA staining, with quantitative analysis after 24 hours of treatment with different concentrations of CoNZ; (D, E) Intracellular oxidative stress levels were assessed using [Ru(dpp)3]Cl2 staining, with representative fluorescence images (D) and FCM results (E) of PC-3 cells, and quantitative analysis after 12 hours of co-incubation; (F, G) OxiVision... TMThe probe detected intracellular H2O2 levels, providing fluorescence images (F) and FCM data (G) of PC-3 cells, and quantitative results after 24 hours of treatment with different concentrations; (H, I) intracellular •O2 was assessed by DHE staining. − (H) Representative fluorescence images and FCM analysis (I) of PC-3 cells after co-incubation for 12 hours; (J) Changes in NADPH levels in PC-3 cells after treatment with different concentrations of CoNZ for 12 hours; (K, L) Detection of intracellular GSH levels using NDA staining, representative fluorescence images (K) and FCM results (L) of PC-3 cells after co-incubation for 12 hours; (M) Schematic diagram of the CoNZ-mediated intracellular catalytic cascade; (N) Cell viability assay results of PC-3 cells after treatment with different concentrations of CoNZ for 24 hours and 48 hours. All data are expressed as mean ± standard deviation.

[0030] Figure 8 Cell viability assays for other prostate cancer cell lines; Figure 1 shows the cell viability assays for PC-3M-IE8 cells (A) and RM-1 cells (B) after treatment with different concentrations of CoNZ; data are expressed as mean ± standard deviation.

[0031] Figure 9 Preparation and characterization of CQ@F fragments and hydrogels; Figure 1 shows: (A) Schematic diagram of the preparation of CQ@F fragments and their dispersion in CMCS solution; (B) Representative fluorescence micrograph of PLGA electrospun fibers loaded with Nile Red (left) and macroscopic photograph of the cut fiber sheet (right); (C) Typical SEM images of blank fiber fragment (left) and CQ@F fragment (right) (inset is a low-magnification SEM image); (D) Typical EDS elemental distribution map of CQ@F fragments. (E) FTIR spectra of HA and OHA; (F) Rheological temperature scanning results of CMCS / OHA hydrogels with different formulations (G': storage modulus; G”: loss modulus); (G) Schematic diagram of CH hydrogel preparation based on Schiff base reaction mechanism; (H) FTIR spectra of CMCS, OHA and CH hydrogels; (I) Photograph of inverted vial test during hydrogel formation process; (J) Rheological strain scanning curve of Co / CQF-CH hydrogel; (K) Rheological thixotropic cyclic scanning results of Co / CQF-CH hydrogel; (L) Typical EDS elemental distribution map of Co / CQF-CH hydrogel; (M) In vitro photograph of gel after subcutaneous injection in mice; (N) Cumulative drug release curve of Co / CQF-CH in lipase-containing buffer at pH 5.4; (O) Statistical results of cumulative release rate of Co / CQF-CH. All data are expressed as mean ± standard deviation.

[0032] Figure 10The rheological properties of hydrogels with different formulations are analyzed. The figure shows the rheological strain scanning results (A, C, E, G) and thixotropic test curves (B, D, F, H) of CH (A, B), Co-CH (C, D), F-CH (E, F) and CQ@F-CH (G, H).

[0033] Figure 11 The in vitro degradation behavior of Co / CQF-CH is shown in the figure. (A) Representative photographs of Co / CQ-CH at different conditions and time points; (B) Weight retention rate of Co / CQ-CH in different buffer systems over time. Data are expressed as mean ± standard deviation.

[0034] Figure 12 The therapeutic effect of Co / CQF-CH on CRPC cells and its potential mechanism; In the figure, (A) the synergistic anti-tumor effect of CoNZ and CQ on PC-3 cells after 24 hours and 48 hours of treatment; (B) the survival rate of PC-3 cells after different treatments; (C) live / dead staining fluorescence images of PC-3 cells in different treatment groups; (D) flow cytometry analysis using annexin V-fluorescein isothiocyanate / propidium iodide double staining to show the apoptosis of PC-3 cells after different treatments; (E) quantitative results of the proportion of early apoptotic cells; (F) quantitative results of the proportion of late apoptotic cells; (G, I, J) Western blot detection of the expression levels of apoptosis-related proteins BCL2 and XIAP (G), hypoxia-related proteins HIF1α and VEGF (I), and autophagy-related proteins P62 and LC3B (J); (H) representative DCF fluorescence staining images of ROS levels in PC-3 cells 12 hours after different treatments.

[0035] Figure 13 Effect of chloroquine treatment on PC-3 cell viability; data are expressed as mean ± standard deviation.

[0036] Figure 14The effects of Co / CQF-CH on the NF-κB signaling pathway and its mechanism of action are shown in the figure. (A) TLR9 activation-related proteins, including full-length TLR9 (F-TLR9) and cleaved TLR9 (C-TLR9); (B) key phosphorylated proteins pIκBα and pP65 in the TLR9 / NF-κB pathway; (C) downstream effector proteins TGFβ1 and COX2 associated with CRPC progression in the NF-κB pathway; (D–F) molecular docking analysis, including the three-dimensional docking (D) and two-dimensional interaction diagram (E) of the IPOα3-P50 / P65 NLS complex with EGCG in CoNZ, and the contribution analysis of each individual amino acid residue to the binding free energy (F); (G) representative images of PC-3 cell proliferation and migration capabilities 24 hours after different treatments; (H) quantitative analysis of cell migration using a wound healing assay. Data are expressed as mean ± standard deviation.

[0037] Figure 15 The results are quantitative analysis of key proteins related to the TLR9 / NF-κB signaling pathway; data are expressed as mean ± standard deviation.

[0038] Figure 16 Representative immunoblotting results for MyD88 and IRAK4 protein expression.

[0039] Figure 17 The figure illustrates the antitumor effect of Co / CQF-CH in a CRPC xenograft model. (A) Schematic diagram of the animal experiment process; (B, C) Tumor growth curves showing changes in tumor volume in a single mouse during the experiment (B), and the average tumor growth trend of each group (C); (D, E) Representative in vivo images of mice in each group after treatment (D) and macroscopic morphological images of tumor tissue after dissection (E); (F) Tumor inhibition rate calculated based on tumor weight of each group at the last measurement; (G) Representative pathological analysis of tumor tissue sections, including HE staining, Ki67 immunofluorescence staining, TUNEL fluorescence staining, and BCL2 immunofluorescence staining; (H) Quantitative analysis of the proportion of tumor necrosis area, the proportion of Ki67-positive cells, the proportion of TUNEL-positive cells, and the fold change in BCL2 fluorescence intensity (MFI). Data are expressed as mean ± standard deviation.

[0040] Figure 18The therapeutic mechanism of Co / CQF-CH in a CRPC xenograft model is shown in the figure. (A) Representative immunohistochemical images of 8-OHdG in tumor sections of each group, as well as immunofluorescence staining images of HIF1α, VEGF, LC3B and P62; (B) Quantitative analysis of the expression levels of 8-OHdG, HIF1α, VEGF, LC3B and P62 in tumor tissue; (C) GSEA results show a significant correlation with oxidative stress and autophagy pathways; (D) Representative immunohistochemical images of pP65 in tumor sections of each group, as well as immunofluorescence staining images of COX2 and TGFβ1; (E) Quantitative analysis of the expression levels of pP65, COX2 and TGFβ1 in tumor tissue; (F) GSEA results show a close correlation with TLR signaling pathway and NF-κB cascade reaction. All data are expressed as mean ± standard deviation.

[0041] Figure 19 Figure 1 shows the changes in mouse body weight and serum biochemical parameters; (A) shows the changes in mouse body weight from tumor inoculation to sample harvest; (B–H) show the levels of CK (B), LDH (C), ALT (D), AST (E), ALP (F), CREA (G), and BUN (H) in the peripheral serum of mice in each treatment group. Data are expressed as mean ± standard deviation.

[0042] Figure 20 Histological evaluation of the major organs was conducted; analysis of representative HE-stained sections of the mouse heart, liver, spleen, lungs, and kidneys revealed no significant pathological changes. Detailed Implementation

[0043] This invention develops an injectable microfiber-hydrogel composite system capable of continuously releasing CoNZ and CQ (… Figure 1 B). The CoNZ-loaded hydrogel was constructed by crosslinking a Schiff base that is acid-responsively cleavable between carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (OHA), and encapsulated electrospun fiber fragments loaded with CQ, with lengths in the micrometer range and thicknesses down to the nanometer range. Figure 1 A and B). By optimizing the formulation of the gel precursor solution, this injectable sol rapidly transforms from a viscosity-dominant state to an elasticity-dominant gel state under body temperature conditions, thereby achieving local retention and sustained release of therapeutic drugs in a TME response. Figure 1B and C). The injectable microfiber-hydrogel composite system of the present invention, as a drug delivery platform, enables precise timing control of drug delivery, achieving preferential release of CoNZ followed by delayed release of CQ, resulting in superior therapeutic efficacy compared to single drug delivery systems or the combined use of free drugs. In a CRPC xenograft mouse model, local administration of this dual-drug sequential delivery system significantly improved treatment outcomes (B and C). Figure 1 (C), which can be applied to the treatment of CRPC.

[0044] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0045] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. In the examples, v / v refers to the volume ratio of the substances, and w / v refers to the mass-volume ratio of the substances.

[0046] All cell lines involved in the examples were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin. Normoalyptic culture conditions were maintained in a standard cell culture incubator, providing a constant humidity environment containing 20% ​​oxygen and 5% carbon dioxide at a temperature of 37 °C. Hypoxic culture conditions were established using a three-gas cell culture incubator, with a gas composition of 1% oxygen, 94% nitrogen, and 5% carbon dioxide, also at a temperature of 37 °C.

[0047] Example 1: Preparation of Nanozyme CoNZ (1) Synthesis of nanozyme CoNZ CoNZ was synthesized using the antisolvent method. Figure 2 A) First, 990 mg of polyvinylpyrrolidone (PVP) was dissolved in 75 mL of methanol under continuous stirring. Then, 15 mL of a methanol solution containing 1 g of Co(NO3)2•6H2O was added dropwise. After stirring for 10 min, another 15 mL of a methanol solution containing 250 mg of EGCG was added dropwise under stirring. The resulting mixture was stirred continuously at room temperature for at least 4 hours, followed by dialyzing against ultrapure water using a dialysis bag with a molecular weight cutoff of 1 kDa for 72 hours (with 6 fluid changes during this period) to remove unreacted small molecules and residual solvent. Finally, the product was collected by freeze-drying and stored at -20 °C for later use.

[0048] In Co 2+During the coordination assembly with EGCG, stable metal-polyphenol nanocomplexes are gradually formed through hydrogen bonding and π–π stacking interactions induced by an antisolvent method. This process was preliminarily verified by UV-Vis absorption spectroscopy. Figure 3 A). CoNZ exhibits a characteristic absorption peak at ~280 nm, falling between the typical absorption wavelengths of pure EGCG (~275 nm) and Co(NO3)2 (~300 nm); simultaneously, the absorption peak of Co(NO3)2 disappears at approximately 510 nm, indicating that Co... 2+ CoNZ has been successfully doped into nanocomposite structures. Transmission electron microscopy (TEM) results show that the CoNZ particle size is approximately 45 nm. Figure 2 B and Figure 3 B), slightly smaller than the average hydrodynamic diameter measured by dynamic light scattering (52.2 ± 1.5 nm). Figure 2 The difference (C) may originate from the hydration layer effect. The Zeta potential (ζ) of CoNZ is −15.1 ± 0.9 mV ( Figure 2 D), indicating that it has a certain degree of colloidal stability. The X-ray diffraction (XRD) pattern shows broad and diffuse diffraction peaks, indicating that the material has low crystallinity, consistent with the characteristics of an amorphous structure. Figure 2 E). Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) determined that the Co content was 3.0% of the total mass, and EGCG accounted for 97.0%. Figure 2 F). X-ray photoelectron spectroscopy (XPS) further confirmed the presence of Co: two main peaks appeared in the high-resolution Co 2p spectrum, which were attributed to Co. 2+ Co 2p 1 / 2 and Co 2p 3 / 2 Orbit, accompanied by characteristic satellite peaks ( Figure 2 G), indicating that cobalt exists in a lower oxidation state, endowing CoNZ with reduction-independent rather than oxidation-dominant catalytic activity, thereby promoting the generation of free radicals. The O 1s spectrum shows contributions from Co–O, C–O, and C=O bonds ( Figure 2 H), confirming Co 2+ Successful coordination with EGCG molecules was achieved, and some organic functional groups were retained. The C1s spectrum also clearly identified C–C, C–O, and C=O bond signals originating from EGCG. Figure 2 I), further supporting the structural integrity of the complex.

[0049] Next, using X-ray absorption fine structure (XAFS) measurement technology, and with Co foil, CoO, and Co2O3 as reference samples, the precise coordination structure of the catalytic center in CoNZ was systematically studied. Specifically, Co... K X-ray absorption near edge structure (XANES) spectrum Figure 2 The J-ray discrepancy indicates that the leading peak of CoNZ is located at approximately 7720.7 eV, between that of Co foil and Co₂O₃, and closer to the leading peak of CoO. Based on this, it can be inferred that the Co atoms in CoNZ carry a positive charge, with an average valence state close to +2 ( ). Figure 4 This result is consistent with XPS analysis. Figure 2 G). Fourier-transformed extended X-ray absorption fine structure (FT-EXAFS) spectrum ( Figure 2 Co exhibits a dominant peak at approximately 1.6 Å, attributed to the Co–O coordination shell, consistent with characteristics found in CoO and Co₂O₃. In contrast, no significant Co–Co metallic bond peak was observed at ~2.2 Å, and no signal corresponding to the Co–O–Co bridging structure was detected in the ~2.5–2.9 Å range, indicating that Co exists in CoNZ in a single-atom form. Further EXAFS fitting analysis was conducted to further elucidate the local chemical environment and coordination configuration of Co in CoNZ. R space( Figure 2 L and Figure 5 A) and K space( Figure 5 The fitting results of B and C, combined with the calculated parameters, indicate that the average bond length between Co and O is approximately 2.0 Å, significantly shorter than the Co–Co distance (~2.5 Å) in Co foil. The best fit results show that the coordination number of Co in CoNZ is approximately 4, i.e., a Co–O4 coordination structure is formed. Figure 2 L, Figure 5 A), whose geometry within the polyphenol-metal framework is as follows: Figure 2 As shown in M. Wavelet-transformed (WT) analysis ( Figure 2The N to Q sequence further confirmed the absence of identifiable Co–Co or Co–O–Co scattering pathways, supporting the conclusion that Co exists as an isolated single-atom active site, thereby maximizing the utilization of the catalytic center and enhancing the structural stability of CoNZ. In summary, the above multi-scale characterization results collectively validate the successful synthesis of CoNZ and reveal its excellent redox catalytic potential.

[0050] (2) Determination of the catalytic activity of CoNZ nanozymes for multiple enzyme types The activities of various enzymes in CoNZ were detected using a UV-Vis spectrophotometer to evaluate its enzyme-mimicking catalytic performance.

[0051] Peroxidase (POD) activity was evaluated by degradation of methylene blue (MB). MB solutions were prepared in a 25 mM NaHCO3 / CO2 buffer system (pH ≈ 6.4): CO2 gas was bubbled into the 25 mM NaHCO3 solution until the pH stabilized. Each reaction system contained 100 μL of MB solution (100 μg / mL). –1 100 μL of H₂O₂ (100 mM) and different concentrations of CoNZ were incubated at 37 °C in the dark for 2 hours, and the UV-Vis absorption spectra in the range of 400–800 nm were recorded. • OH generation efficiency was calculated using the following formula: • OH generation rate (%) = (ΔA₀ – ΔA n ) / ΔA0× 100%, where ΔA n This indicates that the CoNZ concentration is n μg mL –1 The decrease in absorbance at 664 nm.

[0052] Catalase (CAT) activity was determined in 10 mM Tris-HCl buffer (pH 7.4) using two complementary methods: one for monitoring H2O2 consumption and the other for direct detection of O2 generation. In the first method, 100 μL of H2O2 (5 mM) was co-incubated with a gradient concentration of CoNZ at 37 °C for 30 min, followed by the addition of working solution containing 90 μL of Amplex Red (AR) solution (1 mM) and 240 μL of horseradish peroxidase (HRP) solution (100 U / mL). –1 Immediately acquire UV-Vis absorption spectra at 300–650 nm. H2O2 conversion rate is quantified using the AR signal, calculated as follows: H2O2 conversion rate (%) = (ΔA0 – ΔA) n ) / ΔA0× 100%, where ΔA nTo achieve a CoNZ concentration of n μg / mL –1 The change in absorbance at 571 nm was measured at that time. The second method uses a dissolved oxygen probe to monitor O2 generation: 7.5 mL of H2O2 (2 mM) was mixed with different concentrations of CoNZ, and the dissolved oxygen level was continuously measured every 15 seconds at room temperature for 90 minutes.

[0053] Superoxide dismutase (SOD) activity was determined in 10 mM PBS buffer (pH 7.4) using the nitroblue tetrazolium (NBT) photoreduction method. Equal volumes of L-methionine (130 mM), riboflavin (200 μM), and NBT (750 μM) solutions (300 μL each) were mixed, followed by the addition of gradient concentrations of CoNZ. All procedures were performed in the dark. The samples were then placed in a UV-Vis chamber and irradiated at 254 nm for 3 minutes. After thorough mixing, UV-Vis spectroscopy was performed at 300–800 nm. The activity was measured using •O2. – The scavenging ability indirectly reflects the SOD-like activity of CoNZ. The H2O2 generation rate is calculated by the following formula: H2O2 generation rate (%) = (ΔA0 – ΔA n ) / ΔA0×100%, where ΔA n To achieve a CoNZ concentration of n μg / mL –1 The decrease in absorbance at 560 nm.

[0054] Glutathione peroxidase (GPX) activity was assessed in 50 mM Tris-HCl buffer (pH 8.5) using the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) method to detect glutathione (GSH) consumption. Each reaction system consisted of 100 μL GSH (1 mM) and 50 μL of CoNZ solution at different concentrations; the control group used 50 μL H2O2 (10 mM) instead of CoNZ solution. After incubation at room temperature for 4 hours, 1 mL of DTNB solution (200 μM) was added and vortexed to mix, and the absorption spectrum in the range of 350–500 nm was measured. The GSH depletion rate was calculated as: GSH depletion rate (%) = (ΔA0 – ΔA n ) / ΔA0× 100%, where ΔA n This indicates that the CoNZ concentration is n μg mL –1 The decrease in absorbance at 412 nm.

[0055] As previously mentioned, H2O2 (using the AR / HRP system) and •O2 were detected in PBS buffer (pH 7.4). – The generation of (using NBT) was used to assess the activity of CoNZ-based nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidases (NOXs). For •O2 – For detection, 300 μL of NADPH solution (1 mM) and 300 μL of NBT solution (750 μM) were mixed with gradient concentrations of CoNZ and incubated at room temperature in the dark for 30 min. The absorption spectra were then recorded at 300–800 nm. For H2O2 detection, 100 μL of NADPH (1 mM) was incubated with different concentrations of CoNZ under the same conditions, followed by the addition of AR / HRP working solution, and the absorption spectra were immediately recorded at 300–650 nm.

[0056] (3) Density functional theory calculation of the catalytic activity of nanozyme CoNZ All density functional theory (DFT) calculations were performed within the DFT framework using the B3LYP method. C, H, O, N, and S atoms were represented using the 6-311G(d,p) basis set, while Co atoms used the LANL2DZ basis set. Long-range van der Waals interactions were considered using the DFT-D3 correction scheme. The optimized structure was obtained by simulating solvation in aqueous solution using a polarization continuum model. Reaction thermodynamics was evaluated using Gibbs free energy (ΔG). All calculations were performed using Gaussian 16 software.

[0057] By evaluating various enzyme-mimicking activities of CoNZ, we examined its ability to convert weakly oxidizing ROS into more potent hydroxyl radicals (•OH), thereby inducing cancer cell death. Peroxidase (POD)-like activity manifests as the conversion of H₂O₂ to •OH via a Fenton-like reaction, triggering severe oxidative stress damage. To assess the •OH generation ability of CoNZ, methylene blue (MB) was used as a probe molecule. Figure 6 A), this molecule can be oxidized by •OH and decolorized to the colorless product LMB (leuco-MB). Experimental results show that as the concentration of CoNZ increases, the solution color and absorbance at 665 nm decrease in a concentration-dependent manner. Figure 6 B). The MB decolorization rate increased from 35% to 70%, indicating that CoNZ possesses efficient and stable POD-like catalytic performance. Figure 6C). To further elucidate its catalytic mechanism, a simplified Co–O4 configuration was obtained based on EXAFS analysis ( Figure 2 M) Construct a model and combine it with density functional theory (DFT) to calculate and simulate the POD-sample reaction pathway of CoNZ. Figure 6 D). In the proposed catalytic pathway, H₂O₂ first adsorbs onto the CoNZ surface to form an *H₂O₂ intermediate (* represents the adsorption site on CoNZ). This process is driven by the coordination of oxygen atoms in H₂O₂ with Co, and the interaction between hydrogen atoms in H₂O₂ and neighboring lattice oxygen atoms. Subsequently, the two oxygen atoms in H₂O₂ gradually dissociate, undergoing a transition state (TS). Although the first two steps are rate-determining steps (RDS), the corresponding ΔGs are 2.2 and 11.5 kcal·mol⁻¹, respectively. –1 However, the subsequent formation of •OH releases a large amount of energy, ΔG being –30.3 kcal·mol⁻¹. –1 This indicates that the entire POD-like catalytic process is thermodynamically highly feasible.

[0058] Meanwhile, CoNZ exhibits significant catalase (CAT)-like activity, effectively catalyzing the decomposition of H2O2 into H2O and O2, thus potentially alleviating the hypoxic state in the TME. The commercial probe AR was used to quantify its CAT-like activity by detecting H2O2 consumption in the presence of HRP: AR reacts with H2O2 to generate resorcinol, producing a detectable red fluorescence signal at 570 nm. Figure 6 E and F). With increasing CoNZ concentration, H2O2 consumption showed a concentration-dependent effect, and the decolorization rate increased from 16% to 52%, indicating a significant enhancement in catalytic efficiency. Figure 6 G). To further verify its complete CAT-like activity, O2 generation was monitored in real time using a dissolved oxygen meter. The results showed that the O2 concentration rose rapidly within 120 seconds and tended to saturate. Figure 6 H). DFT calculations were then performed to reveal the potential mechanism of CoNZ-catalyzed H2O2 decomposition, including the relevant reaction pathways and energy changes, as follows: Figure 6 As shown in Figure I. Calculations show that two H₂O₂ molecules adsorb sequentially onto the CoNZ surface, undergoing two consecutive RDS, with corresponding activation barriers of 2.2 and 0.8 kcal·mol⁻¹, respectively. –1 The reaction then produces one H2O molecule, one O2 molecule, and one *H2O intermediate, at which point ΔG significantly decreases to -17.6 kcal·mol⁻¹. –1Ultimately, two molecules of H₂O and one molecule of O₂ are produced, and the total ΔG for the entire reaction process is –20.9 kcal·mol⁻¹. –1 The ΔG values ​​for the last two steps are both highly negative, indicating that this type of CAT catalytic process is thermodynamically feasible.

[0059] Despite consuming H2O2, CoNZ still exhibits significant superoxide dismutase (SOD)-like activity, capable of converting •O2 into H2O2. – It is effectively converted into H2O2, thus providing substrate for POD and CAT-like catalytic reactions. NBT can be used to detect •O2. – Its relationship with •O2 – The reaction produces blue-violet NBT formazan, which has a measurable absorption peak at 560 nm. Figure 6 J and K). As the CoNZ concentration increases, the solution color gradually lightens, indicating that •O2 – The H2O2 formation efficiency increased accordingly, with the decolorization rate rising from 24% to 88%, further confirming that CoNZ possesses highly efficient SOD simulation capabilities. Figure 6 L). The potential catalytic mechanism was analyzed using DFT calculations, and possible reaction pathways and corresponding ΔG values ​​were obtained. Figure 6 As shown in M. Existing research indicates that •O2 – It can act as a Brønsted base and readily captures protons in aqueous solution to form •O₂H; therefore, the •O₂H radical is used instead of •O₂ in DFT calculations. – Modeling was performed. Calculation results show that the two *•O2H radicals adsorb sequentially onto the CoNZ surface, with ΔG continuously decreasing during the process, ultimately reaching -23.6 kcal·mol⁻¹ when a dual adsorption state is formed. –1 The formation of H₂O₂ requires overcoming two consecutive energy barriers, 0.9 and 5.3 kcal·mol⁻¹, respectively. –1 However, the overall reaction ΔG is still negative (–17.4 kcal·mol⁻¹). –1 Furthermore, the absolute value is relatively large. These results indicate that it is thermodynamically feasible for CoNZ to promote H2O2 accumulation by simulating SOD activity.

[0060] Furthermore, the O2 generated by CoNZ's CAT-like activity can serve as a substrate for the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH), indicating that CoNZ possesses NADPH oxidase (NOX)-like activity. Figure 6Specifically, CoNZ catalyzes the oxidation of NADPH to oxidized nicotinamide adenine dinucleotide phosphate (NADP). + At the same time, it reduces O2 to •O2. – Or H2O2. To verify the catalytic process, the corresponding reaction products were detected using AR and NBT methods, respectively. With increasing CoNZ concentration, the absorbance peaks at 570 nm and 560 nm both showed a concentration-dependent increase. Figure 6 The presence of O and P further confirms that CoNZ possesses NOx-like catalytic activity. DTF analysis results also support that CoNZ can effectively drive NOx-like catalytic reactions. Given the relatively complex molecular structure of NADPH, to balance computational efficiency and accuracy, a reaction center model with similar chemical properties but a simplified structure was chosen for theoretical calculations. For example... Figure 6 As shown in Q, one NADPH molecule and one O2 molecule rapidly adsorb onto the CoNZ surface, and ΔG decreases to –2.7 kcal·mol⁻¹. –1 Subsequently, protonated O2 is formed. − For the intermediate and the reaction complex with the *NADP+ structure, ΔG was further significantly reduced to –42.5 kcal·mol⁻¹. –1 This indicates that the CoNZ-mediated cascade reaction is thermodynamically feasible.

[0061] Besides inducing oxidative damage, CoNZ can also oxidize glutathione (GSH) to oxidized glutathione (GSSG) through glutathione peroxidase (GPX)-like activity, thereby disrupting the cell's oxidative defense mechanisms. GSH is an important antioxidant, and its expression level in cancer cells is significantly higher than in normal cells, in order to cope with stronger oxidative stress, maintain redox homeostasis, and ensure cell survival. GSH levels were measured using the DTNB method; DTNB, as a dithiocyanate reagent, reacts with thiol groups to generate yellow 5-thio-2-nitrobenzoic acid (TNB). Figure 6 R). CoNZ treatment led to a concentration-dependent decrease in GSH levels, eventually approaching the effect of the 1 mM H2O2 treatment group (positive control). Figure 6 S and T). Subsequently, DFT calculations were performed to gain a deeper understanding of the CoNZ-mediated GPX-like catalytic process. Due to the complex molecular structure of glutathione, direct computational modeling is difficult; therefore, methyl thiol (CH3–SH) was used as a simplified analog. Under physiological conditions, the –SH group in glutathione can be rapidly deprotonated to form –S.– Especially in the presence of endogenous proton receptors (such as •O2) − In this case, to simulate this deprotonation process, a GPX-like catalytic reaction was carried out in a solution at pH 8.5; therefore, CH3–S was used. − As a substitute for GSH, it accelerates computation while maintaining reaction efficiency. When a GSH molecule adsorbs onto the CoNZ surface, ΔG rapidly decreases to –33.3 kcal·mol⁻¹. –1 Subsequently, when a second GSH molecule is adsorbed, the reaction energy barrier is extremely low, only 1.3 kcal·mol⁻¹. –1 ( Figure 6 U). Finally, a GSSG molecule forms on the CoNZ surface, with a yield of -36.1 kcal·mol⁻¹. –1 The high negative ΔG desorption indicates that CoNZ can thermodynamically and efficiently consume GSH through its GPX-like activity. Given the crucial role of NADPH in maintaining reduced glutathione levels, it is believed that the synergistic consumption of NADPH and GSH may produce an additive effect, thereby more comprehensively disrupting the oxidative defense system of CRPC cells.

[0062] Example 2: Cellular uptake of the nanozyme CoNZ To quantitatively determine the cellular uptake of CoNZ prepared in Example 1, the nanoparticles were fluorescently labeled with 5-aminofluorescein (5-AF). CoNZ and 5-AF were co-dissolved in methanol and stirred in the dark for 8 hours. The resulting solution was dialyzed in the dark for 72 hours using dialysis tubes with a molecular weight cutoff of 1 kDa. PC-3 cells were seeded in 6-well plates and co-incubated with 5-AF-labeled CoNZ for 2, 4, and 6 hours, respectively. Cell uptake was then analyzed by flow cytometry (FCM).

[0063] PC-3 cells are androgen-independent CRPC cell lines with enhanced oxidative stress tolerance. CoNZ was conjugated with 5-AF to assess its cellular uptake efficiency. FCM results showed that the 5-AF fluorescence signal gradually increased with incubation time, indicating that CoNZ was internalized by 78.2% of PC-3 cells at 2 hours and nearly completely uptaken after 6 hours. Figure 7 A).

[0064] Example 3: Catalytic activity of CoNZ nanozyme in cells for multiple enzyme types Different fluorescent probes were used to monitor the corresponding intracellular substrates or products associated with the enzyme-like activities indicated by CoNZ. PC-3 cells were cultured in 24-well plates, imaged using an inverted fluorescence microscope, and quantitative FCM analysis was performed in 6-well plates. Intracellular global ROS levels were assessed after 24 hours of co-incubation with different concentrations of CoNZ. Cells were then washed and stained with 2',7'-dichlorodihydrofluoresceindiacetate (DCFH-DA, 10 μM) for FCM analysis. Simultaneously, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was performed for fluorescence microscopy imaging. Intracellular H2O2 levels were assessed after 12 hours of co-incubation with different concentrations of CoNZ. Subsequently, the H2O2 levels were measured using the commercially available OxiVision H2O2 sensor. TM Cells were stained with Green (5 μM) and then subjected to fluorescence and bright-field microscopy imaging and quantitative FCM analysis. To assess intracellular O2 production, cells were co-incubated with different concentrations of CoNZ for 12 hours under hypoxic conditions. Subsequently, cells were treated with the hypoxic probe [Ru(dpp)3]Cl2 (10 μg / mL) in a hypoxic environment. −1 After incubation for 4 hours, fluorescence imaging and flow cytometry were performed for quantitative analysis. •O2 was measured after treatment with different concentrations of CoNZ. − The level was used to assess intracellular H2O2 replenishment. After removing uninternalized CoNZ, 10 μM DHE was added to specifically detect •O2. − Residual DHE was then removed, and intracellular •O2 was quantified by flow cytometry. − Signal intensity. DAPI staining was applied when acquiring fluorescence images. Intracellular NADPH levels were determined using a commercial assay kit. Cells were lysed on ice after co-incubating with CoNZ for 12 hours, followed by centrifugation. The supernatant was then incubated with water-soluble tetrazolium-8 from the kit at 37 °C for 30 minutes. Peak absorbance of the UV-Vis spectrum was measured at 450 nm using a microplate reader. A standard curve for NADPH was pre-plotted to calculate the amount of intracellular NADPH. Intracellular glutathione (GSH) levels were detected using NDA (50 μM), a fluorescent probe specific for thiols, after treatment with different concentrations of CoNZ for 12 hours. Quantitative analysis was then performed by flow cytometry (FCM), and fluorescence images were captured.

[0065] Intracellular total ROS levels were detected using the cell membrane permeability probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). In the presence of ROS, DCFH-DA can be hydrolyzed by esterases and further oxidized to 2',7'-dichlorofluorescein (DCF), producing green fluorescence. Fluorescence microscopy images showed that the fluorescence intensity of DCF increased in a gradient with increasing CoNZ concentration, reflecting an increase in ROS levels. Figure 7 B). FCM analysis also showed that the mean fluorescence intensity (MFI) of DCF increased significantly with increasing CoNZ concentration, confirming that CoNZ can induce intracellular ROS accumulation (B). Figure 7 C). To further evaluate the intracellular CAT-like activity of CoNZ under hypoxic conditions, O2 production in PC-3 cells was detected. The oxygen-sensitive fluorescent probe [Ru(dpp)3]Cl2 was used, and its red fluorescence intensity was negatively correlated with O2 concentration. Microscopic observation and FCM analysis both showed that the red fluorescence signal decreased in a concentration-dependent manner with increasing CoNZ concentration, reaching a minimum at a concentration of 100 μg / mL. –1 At that time, the MFI of [Ru(dpp)3]Cl2 decreased significantly ( Figure 7 (D and E) indicate that CoNZ possesses the ability to produce O2 intracellularly, demonstrating its CAT-like activity. To verify the catalytic conversion of H2O2 by CoNZ, the commercial probe OxiVision was used. TM The probe emits green fluorescence in the presence of H₂O₂. Microscopic and FCM results both show that when the CoNZ concentration exceeds 250 μg / mL... –1 At that time, OxiVision TM Fluorescence was significantly reduced, and the H2O2 signal was almost undetectable. Figure 7 (F and G), further confirming that CoNZ can effectively catalyze the decomposition of H2O2. It can be seen that in PC-3 cells, CoNZ can lead to ROS accumulation, increased O2 levels and H2O2 consumption, providing strong evidence that it has dual enzyme mimicry activities similar to POD and CAT in cells.

[0066] Given that both POD-like and CAT-like activities involve H2O2 consumption, further investigation was conducted by detecting intracellular •O2. − The study aimed to assess whether CoNZ promotes H2O2 formation through its SOD-like catalytic activity. Dihydroethidium (DHE) was used as a specific probe, which can be detected by •O2. −Oxidation results in DNA insertion and the generation of red fluorescence. After CoNZ treatment, both fluorescence imaging and FCM analysis showed that the DHE fluorescence signal significantly decreased with increasing CoNZ concentration, indicating that •O2… − It was effectively cleared, thus confirming that CoNZ has SOD-like activity in cells. Figure 7 H and I). Furthermore, biochemical assays of intracellular NADPH showed that high concentrations of CoNZ significantly reduced NADPH levels in PC-3 cells (H and I). Figure 7 This result shows that CoNZ also possesses NOXs-like activity in the intracellular environment.

[0067] In addition to the excessive accumulation of intracellular ROS caused by the combined activities of POD-like, CAT-like, SOD-like, and NOXs-like activities, this study further investigated whether CoNZ possesses GPX-like activity, i.e., disrupting the redox homeostasis of CRPC cells by consuming GSH. GSH contains free sulfhydryl groups and can specifically react with 2,3-naphthalenedicarboxaldehyde (NDA) to generate green fluorescence. Fluorescence microscopy showed that PC-3 cells had high levels of endogenous GSH, consistent with the tolerance characteristics of CRPC cells to oxidative stress. After CoNZ treatment, the GSH fluorescence signal gradually weakened with increasing CoNZ concentration. Figure 7 K). FCM analysis also showed a significant decrease in mean fluorescence intensity (MFI), an effect that could still be observed even at only one-tenth of the concentration required to induce ROS accumulation. Figure 7 L), suggesting that GSH consumption is mainly attributed to the GPX-like activity of CoNZ, rather than to ROS oxidation.

[0068] In summary, these results indicate that CoNZ possesses multiple enzyme catalytic activities and constructs an interconnected redox regulatory network within CRPC cells. Figure 7 M). Through a series of enzymatic reactions induced by CoNZ, the intracellular total ROS level significantly increased due to the generation of highly reactive •OH, while the antioxidant defense system was inhibited. This synergistic effect led to significant oxidative damage in the nucleus, mitochondria, and other key organelles, thus endowing CoNZ with the ability to effectively trigger cell death. To verify this mechanism, the cytotoxicity of CoNZ on CRPC cells was assessed using the CCK-8 assay. The results showed that within the concentration range capable of inducing intracellular ROS accumulation, CoNZ significantly reduced cell viability in a concentration-dependent manner (M). Figure 7 N). The half-maximal inhibitory concentration (IC50) of CoNZ in PC-3 cells after 24 and 48 hours of treatment. 50 ) respectively 1055 μg mL −1 and 750 μg mL −1In addition, in two other representative prostate cancer cell lines, PC-3M-IE8 (… Figure 8 A) and RM-1 ( Figure 8 A similar inhibitory effect was also observed in B), further confirming that CoNZ-induced oxidative stress can effectively overcome the defense mechanisms of prostate cancer cells and induce cell death.

[0069] Example 4: Preparation of CQ@F fragments CQ-loaded fiber (CQ@F) fragments were obtained from chloroquine (CQ)-loaded poly(lactic-co-glycolic acid) (PLGA) microfibers via cryo-slicing. First, a homogeneous electrospinning precursor solution was prepared by dissolving PLGA in hexafluoroisopropanol (HFIP) at a concentration of 15% (w / v). CQ was then added to a final concentration of 1% (w / v), ensuring complete dissolution. The resulting solution was electrospinned under a high-voltage power supply (10–15 kV), with the feed rate precisely controlled by an injection pump. The generated microfibers were collected on a rotating drum and air-dried. The dried CQ@F patches were embedded in a carboxymethylchitosan (CMCS) matrix, frozen, and then sliced ​​into uniform sheets approximately 20 μm thick using an ice cutter. The CMCS solution containing the CQ@F fragments was collected after thawing and used for subsequent experiments.

[0070] To achieve orderly drug release, PLGA was selected as the carrier for the chemical oxygen demand (CQ). Fiber patches were prepared using electrospinning technology and then fragmented and embedded into a CMCS hydrogel. PLGA is a biocompatible and biodegradable polymer material that has been approved for clinical use. PLGA was dissolved in hexafluoroisopropanol (HFIP), and under optimized electrospinning parameters, uniformly morphologically uniform and structurally continuous fiber sheets were successfully prepared. Figure 9 A, B). Subsequently, CQ was added to the HFIP solution of PLGA and electrospinned under the same conditions to obtain CQ-loaded fiber patches (CQ@F). Figure 9 A). The obtained fiber membrane was cut and embedded in the CMCS matrix, and then subjected to low-temperature microdissection to obtain fiber fragments of uniform length. Scanning electron microscopy (SEM) results showed that the average length of the fiber fragments was approximately 20 μm, and the diameter ranged from 0.8 to 1.5 μm. Figure 9C). There was no significant difference in morphology between the blank fiber and the CQ-loaded fiber, indicating that the introduction of CQ did not affect the stability of the fiber structure. Furthermore, elemental mapping analysis showed that the chlorine signal in CQ was uniformly distributed throughout the fiber, confirming the good dispersion of CQ in the fiber fragments. Figure 9 D).

[0071] Example 5: Preparation of Co / CQF-CH hydrogel (1) Preparation of Co / CQF-CH hydrogel The preparation of the Co / CQF-CH composite hydrogel loaded with CoNZ fragments began with the oxidation of hyaluronic acid (HA) with sodium periodate to generate aldehyde-modified HA (OHA). Specifically, HA was dissolved in 400 mL of ultrapure water at a concentration of 1% (w / v), and 45 mL of 10% (w / v) sodium periodate solution was added dropwise under stirring. The reaction was carried out at 37 °C in the dark for 2 hours, followed by the addition of 1 mL of ethylene glycol to terminate the reaction. After stirring for another 2 hours, the product was dialyzed in ultrapure water for 72 hours in a dialysis bag with a molecular weight cutoff of 1 kDa. Finally, the product was freeze-dried to obtain OHA powder, which served as an injectable thermoresponsive hydrogel matrix for loading CoNZ and encapsulating CQ@F fragments.

[0072] To prepare the Co / CQF-CH hydrogel, the CoNZ obtained in Example 1 was first dispersed in an OHA solution. Simultaneously, the CQ@F frozen slices obtained in Example 2 were melted by heating to obtain a CMCS solution containing CQ@F fragments. The two phases were mixed at an optimized volume ratio to form a homogeneous precursor mixture, which was then incubated in a 37 °C water bath for 10 minutes to form a stable hydrogel with a cross-linked network structure, named Co / CQF-CH.

[0073] Chitosan is a natural polysaccharide with good biocompatibility and modifiability, but its solubility is low under neutral or alkaline conditions due to the presence of numerous primary amine groups. Carboxymethylation modification not only improves the biocompatibility of Co / CQF-CH hydrogels but also significantly enhances the water solubility of CMCSs and their ability to form thermoresponsive hydrogels. Commercially available CMCSs with a substitution degree exceeding 80% were used. Hyaluronic acid (HA) is a major component of connective tissue and is widely used clinically. OHA introduces aldehyde groups through HA oxidation, thereby achieving covalent cross-linking with CMCSs. Fourier transform infrared (FTIR) spectroscopy at 1730 cm⁻¹... −1 The characteristic absorption peak appearing at [location] is attributed to the stretching vibration of the aldehyde group (C=O), confirming the successful synthesis of OHA. Figure 9 E).

[0074] CMCS and OHA were blended in different proportions to prepare CMCS / OHA hydrogels, and the optimal formulation was determined by rheological temperature scanning experiments. When the storage modulus (G') was less than the loss modulus (G"), the sample exhibited a sol state dominated by viscosity; conversely, when G' > G"), the sample transformed into a gel state dominated by elasticity. With a fixed CMCS concentration of 3% (w / v), the gelation temperature gradually decreased with increasing OHA concentration. Figure 9 F). Considering both injectability and the need for in-situ gelation within tumors, a formulation consisting of 3% (w / v) CMCS and 1.6% (w / v) OHA was ultimately selected. This formulation gelled at 35.09 °C and was named CH. The cross-linking mechanism primarily stems from the Schiff base reaction between the primary amine groups (–NH2) in CMCS and the aldehyde groups (–CHO) in OHA, forming imine bonds (C=N) and thus constructing a primary hydrogel network. Furthermore, other non-covalent interactions also contribute to enhancing the cross-linking structure, including the protonated primary amine groups (–NH3) in CMCS. + The electrostatic attraction between CMCS and the deprotonated carboxyl group (–COO–) in OHA or CMCS, and the abundant hydrogen bonds formed between the hydroxyl and amino groups in the CMCS and OHA molecules. Figure 9 G). FTIR analysis showed that it was located at 1730 cm in OHA. –1 The aldehyde stretching vibration peak at [location], and the 3000–3500 cm⁻¹ peak in CMCS. –1 The characteristic absorption peaks of primary amine groups within the range essentially disappear in CH4, indicating that these functional groups are effectively consumed during the reaction. Meanwhile, at approximately 1660–1690 cm⁻¹... –1 A new strong absorption peak appears at this point, which is attributed to the stretching vibration of the imine group (C=N) in CH, further confirming the formation of Schiff base bonds during the gelation process. Figure 9 H). The gel transition process of CH was visually verified through an inverted vial experiment: after the sample was mixed, it was heated to 37 °C, and the hydrogel stably adhered to the bottom of the vial and no longer flowed. Figure 9 I). In rheological strain scanning tests, G” of CH is almost parallel to G’ in the low strain region, but when the strain exceeds 300%, G” exceeds G’, exhibiting typical shear-thinning behavior. Figure 10 A). Further rheological and thixotropic studies showed that when the strain recovered from 350% to 1%, the storage modulus of CH was essentially restored, demonstrating good structural recoverability. Figure 10 (B) indicates that the hydrogel possesses excellent plasticity and self-healing ability, enabling it to rapidly reconstruct the three-dimensional network structure after injection, maintain morphological integrity, and reduce mechanical damage to surrounding tissues.

[0075] A dual-drug-loaded injection system was successfully prepared using fiber fragments and injectable hydrogels. An OHA solution containing CoNZ was thoroughly mixed with a molten CMCS solution embedding CQ@F fragments to achieve homogeneous blending and cross-linking. Four functionalized hydrogels were prepared in addition to CH: a CoNZ-loaded hydrogel (Co-CH), a hydrogel containing blank fiber fragments (F-CH), a hydrogel containing CQ@F fragments (CQF-CH), and a composite hydrogel simultaneously loaded with CoNZ and CQ@F fragments (Co / CQF-CH). Rheological testing results showed that all formulations exhibited rheological behavior similar to CH, indicating that the introduction of fiber fragments and nanozymes did not significantly affect the plasticity and structural adaptability of the hydrogels. Figure 9 J, K and Figure 10 C–H). SEM images and elemental mapping analysis further confirmed the presence of CQ@F fiber structures in Co / CQF-CH, and detected obvious Cl and Co signals, indicating the successful integration of the two active ingredients (C–H). Figure 9 L). Subcutaneous injection experiments were then conducted in a mouse model. Results of in vitro observation ( Figure 9 The results (M) show that within one hour after injection, the hydrogel forms initial spherical gel clumps with a rough surface, which gradually transform into regular-shaped, smooth elliptical gel bodies after 24 hours. This dynamic evolution process clearly verifies that the designed hydrogel system possesses good injectability and thermoresponsive in-situ gelation ability.

[0076] (2) Degradation and drug release of Co / CQF-CH hydrogel The degradation behavior of the hydrogel was assessed by placing samples in different degradation solutions: pH 7.4, 6.4, and 5.4 (with or without 2 mg / mL). –1 The degradation rate was assessed by incubation in phosphate buffered solution (PBS) for lipase. Samples were maintained at 37 °C with gentle shaking at 60 rpm. Samples were removed at predetermined time points, photographed, and weighed. Degradation rate was calculated using the formula: Degradation (%) = (W0 – W) t The calculation is performed using W0 × 100%, where W0 is the initial mass and W... t Let be the mass at time t, and plot the degradation curve accordingly.

[0077] Drug release behavior was determined by immersing freshly prepared hydrogels in different degradation solutions. Supernatants were collected at predetermined time points, and an equal volume of fresh release medium was added. Drug concentration was determined using UV-Vis spectrophotometry combined with a pre-established standard curve. The cumulative percentage of drug release was defined as the ratio of the total released amount to the initial drug load and was plotted as a function of time for analysis.

[0078] The imine bonds in the Schiff base structure are subject to pH-responsive degradation under acidic conditions, especially in TME, where the pH value is typically close to 5.0. Besides the pH-responsive mechanism, PLGA molecules are also susceptible to enzymatic hydrolysis due to the high ester bond content. To systematically evaluate the degradation behavior of Co / CQF-CH, in vitro degradation experiments were conducted in buffer solutions at pH 7.4, 6.4, and 5.4, with an additional lipase added in the pH 5.4 group to simulate the enzymatic hydrolysis environment. Co / CQF-CH samples were immersed in the above solutions, and photographic records were taken at multiple time points. Figure 11 A) and quality determination ( Figure 11 B). Degradation curves showed that on day 15, the remaining mass of the sample under pH 5.4 conditions was significantly lower than that under pH 7.4 conditions, and degradation was further accelerated in the presence of lipase. Figure 11 B) indicates that the material has the ability to degrade in response to dual stimulation of TME (low pH and enzyme).

[0079] Based on this, the in vitro drug release behavior was further investigated in a pH 5.4 buffer containing lipase to better reflect the physiological conditions of TME. The results showed that CoNZ and CQ exhibited a time-dependent sequential release pattern: CoNZ was released at a faster rate and to a greater extent initially, while CQ release was relatively delayed. This release sequence is beneficial for initially inducing oxidative stress and mitochondrial dysfunction, thus providing an ideal therapeutic window for subsequent CQ-mediated protective autophagy inhibition. By day 5, the cumulative release of the two drugs tended to be consistent, achieving synchronous release and thus continuously activating the synergistic antitumor effect. Figure 9 N). Analysis of cumulative release rates at different time points further confirmed that early release of CoNZ dominated, and gradually converged with the release rate of CQ over time (N). Figure 9 Therefore, Co / CQF-CH is expected to achieve orderly delivery of two drugs after injection, taking into account both phased intervention and long-term synergistic effects, and producing a sustained cytotoxic effect on CRPC cells.

[0080] Example 6: In vitro antitumor experiment (1) Cytotoxicity test PC-3, PC-3M-IE8, or RM-1 cells were seeded in 96-well or 12-well plates and cultured overnight. For single-drug cytotoxicity assessment, cells were treated with different concentrations of CoNZ or CQ for 24 or 48 hours. For synergistic effect analysis, cell viability was measured by treatment with CoNZ (750 μg / mL for 24 hours). −1 Or 250 μg·mL over 48 hours −1Evaluation was performed after treatment with CQ (40 μM for 24 hours or 20 μM for 48 hours), alone or in combination. To assess the cytotoxicity of the drug-loaded hydrogel, PC-3 cells were treated for 24 hours. Cell viability was then determined using the CCK8 assay, and absorbance at 450 nm was measured.

[0081] (2) Apoptosis experiment To assess the effect of apoptosis induction, PC-3 cells were cultured in 6-well plates and then co-incubated with drug-loaded hydrogels for 12 hours. The proportion of early and late apoptotic cells was then analyzed by FCM using an apoptosis detection kit. For live / dead cell staining, PC-3 cells were first treated with drug-loaded hydrogels in 24-well plates for 24 hours. Then, cells were stained with calcein-AM (live cell marker) and propidium iodide (PI, dead cell marker) at 37 °C for 30 minutes, followed by fluorescence microscopy imaging.

[0082] (3) Western blot experiment PC-3 cells were seeded in 6-well plates and treated with drug-loaded hydrogels for 12 hours, followed by incubation under hypoxic conditions to detect the expression levels of HIF1α and VEGF. After treatment, total cell protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors, and then incubated on ice for 20 minutes after adding phenylmethanesulfonyl fluoride (PMSF, 1 mM). The cells were then centrifuged at 12000 g at 4°C for 20 minutes, and the supernatant was collected and mixed with 5 times the volume of loading buffer. The supernatant was then denatured at 95°C for 15 minutes (or 37°C for 45 minutes) for membrane protein detection. The denatured protein samples were loaded into 4–20% gradient SDS-PAGE gels and separated by electrophoresis at a constant voltage of 80 V in a vertical electrophoresis system until bromophenol blue migrated to the bottom of the gel. Next, the protein was transferred from the gel to a methanol-activated polyvinylidene fluoride (PVDF) membrane using a wet transfer method at 4°C and a constant current of 350 mA via a blot module for 1 hour. After transfer, the PVDF membrane was washed several times with tris-buffered saline with Tween 20 (TBST) and blocked at room temperature for 2 hours in TBST solution containing 5% (w / v) bovine serum albumin (BSA), with gentle shaking to block non-specific binding sites. Subsequently, the membrane was co-incubated with the following primary antibodies: TLR9 (1:1000), pP65 (Ser536) (1:1000), XIAP (1:1000), MyD88 (1:1000), pIκBα (1:10000), IRAK4 (1:2000), p62 / SQSTM1 (1:1000), BCL2 (1:2000), LC3B (1:2000), COX2 (1:5000), HIF1α (1:1000), VEGF (1:1000), TGFβ1 (1:5000), vinculin (1:50000), and GAPDH (1:100000) overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 minutes each time, and then incubated with the corresponding HRP-labeled secondary antibody (1:10000) at room temperature for 2 hours. After washing again, the blots were developed using an enhanced chemiluminescence (ECL) kit, and the chemiluminescence signals were acquired using a multi-mode imaging system. Finally, the grayscale values ​​of the protein blots were quantitatively analyzed using ImageJ (Fiji) software.

[0083] (4) Scratch test Cell migration was assessed using a scratch healing assay. PC-3 cells were seeded in 6-well plates and cultured to approximately 70% confluence. Wounds were created by scratching the cell monolayer using a sterile pipette tip. After removing cell debris, the cells were incubated in serum-free media containing different hydrogels. Microscopic images were taken after co-incubation at 0, 3, 6, 9, 12, and 24 hours, and the wound area was measured using Fiji software. Cell migration was quantified using the following formula: Migration rate (%) = [(A0 – A] t ) / A0] ×100%, where A0 is the initial wound area, A t Let be the wound area at time t.

[0084] The cytotoxicity of CQ to CRPC cells was assessed using the CCK-8 assay. The results showed that the inhibitory effect of CQ on PC-3 cells was concentration-dependent, with IC50 values ​​of 150 μM and 20 μM after 24 and 48 hours of treatment, respectively. Figure 13 Further comparison of the efficacy of CoNZ and CQ monotherapy and combination therapy revealed that after 24 hours of co-incubation, the inhibitory effect in the combination group was slightly higher than the sum of the effects of the two drugs alone, and the synergistic effect was even more significant after 48 hours. Figure 12 A) indicates the presence of significant synergistic cytotoxicity.

[0085] Five control groups were set up in the experiment: untreated group (Ctrl), blank vector group (F-CH), CQ-loaded group (CQF-CH), CoNZ-loaded group (Co / F-CH), and free drug combination group (Co+CQ). CCK-8 results showed that compared with the single-drug loading groups, the Co+CQ group and the Co / CQF-CH group had significantly enhanced inhibitory effects on PC-3 cells, with cell viability decreasing to approximately 40% after 48 hours of treatment. Figure 12 B), and Figure 9 The experimental results of the carrier-free system in group A showed a consistent trend. Furthermore, cytotoxicity was further verified by double staining with calcein-AM (live cell marker) and propidium iodide (PI, dead cell marker). Fluorescence images showed strong red fluorescence of the PI marker in the Co+CQ group and the Co / CQF-CH group, indicating significant cell death; while only a small amount of red signal was observed in the single-drug groups, and almost no dead cells were seen in the Ctrl group and the F-CH group. Figure 12 C).

[0086] Quantitative analysis by flow cytometry showed that the apoptosis rate in the F-CH group was similar to that in the control group (Ctrl group), and was almost negligible. Figure 12 The cell viability data in B were consistent. Co / F-CH mainly induced an increase in early apoptosis, suggesting that apoptosis was initiated by oxidative damage; while CQF-CH mainly induced late apoptosis, consistent with the characteristic that autophagy inhibition promotes the death of damaged cells. Figure 12D~F). The Co / CQF-CH treatment group induced 52.3% early apoptosis and 27.8% late apoptosis, compared to 29.7% and 28.2% late apoptosis in the Co+CQ group. Although the late apoptosis rate in the Co / CQF-CH group was not statistically different from that in the control group, its early apoptosis rate and total apoptosis rate were significantly increased. These results indicate that the hydrogel co-delivery system loaded with fiber fragments can enhance the apoptosis effect under combined treatment by regulating the release kinetics of CoNZ and CQ. Further analysis of the expression levels of apoptosis-related proteins using Western blotting revealed two key endogenous apoptosis inhibitors: B-cell lymphoma 2 protein (BCL2) and X-linked inhibitor of apoptosis protein (XIAP). The results showed that CQF-CH slightly downregulated BCL2 expression, while Co / F-CH significantly reduced BCL2 levels, with the most significant decrease in BCL2 expression observed in the Co / CQF-CH group. Figure 12 (G) indicates that the oxidative stress induced by CoNZ release plays a stronger role in activating apoptosis signaling pathways than CQ-mediated autophagy inhibition. Conversely, XIAP, an inhibitor of caspase activity, was significantly reduced in the Co / CQF-CH group, suggesting that the combination therapy effectively weakened the anti-apoptotic ability of cancer cells.

[0087] ROS levels in PC-3 cells were detected using the DCFH-DA probe. Fluorescence microscopy showed that after 12 hours of treatment with CoNZ-containing groups, the DCF fluorescence intensity of Co / CQF-CH was significantly enhanced and more widely distributed, indicating that CoNZ maintained good ROS generation activity after being loaded into the hydrogel. Figure 12 H). Furthermore, the CQF-CH group also showed a certain degree of increased ROS signal, possibly due to the blockage of intrinsic autophagic flux after CQ release, leading to the accumulation of oxidative stress. To further evaluate whether the oxygen-producing capacity of CoNZ could alleviate hypoxia in the tumor microenvironment, the expression levels of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) were measured. Under normoxic conditions, HIF1α is usually rapidly degraded, while it is stabilized and promotes VEGF expression in a hypoxic environment, thereby driving tumor angiogenesis. Western blot results showed that the HIF1α protein level was significantly reduced in the CoNZ-containing treatment group, while VEGF expression was inhibited (…). Figure 12 (I) This suggests that the released CoNZ effectively alleviated local hypoxia through oxygen production, and may inhibit tumor angiogenesis and growth-stimulating signals. Consistent with the CQ-mediated autophagic flux blocking mechanism, Western blot analysis showed that p62 protein was significantly accumulated in all CQ-treated groups, especially in the Co / CQF-CH group. Figure 12J). Simultaneously, the LC3B-II / LC3B-I ratio significantly increased, particularly in the Co / CQF-CH group, indicating efficient conversion of LC3B-I to LC3B-II and abnormal accumulation of autophagosomes (J). Figure 12 These results confirm that CQ retains its highly efficient autophagy-inhibiting function after successful incorporation into the hydrogel, with the Co / CQF-CH group showing the best results.

[0088] After CQ treatment, C-TLR9 levels decreased in all groups, with the most significant decrease observed in the Co / CQF-CH group. This result suggests that TLR9-mediated intracellular signal transduction was significantly inhibited. Figure 14 A and Figure 15 As upstream molecules in the TLR9 signaling cascade, the expression levels of MyD88 and IRAK4 did not change significantly with C-TLR9 levels, remaining relatively stable. Figure 16 This indicates that it is not directly affected by the downstream TLR9 activation state. In contrast, the phosphorylated IκBα (pIκBα) level shows a trend consistent with that of C-TLR9, suggesting that its degradation process is correspondingly reduced. Figure 14 B). Furthermore, all CQ-containing treatment groups significantly downregulated the expression of phosphorylated p65 (pP65), with the Co / CQF-CH group showing the strongest inhibitory effect, indicating that the entire signaling pathway from TLR9 to NF-κB was effectively blocked. Figure 14 B). Meanwhile, compared to the control group, the expression of COX2 and TGFβ1 (both key effector molecules upregulated after NF-κB activation and closely related to CRPC progression) was significantly reduced in all CQ treatment groups, reaching the maximum inhibition level in the Co / CQF-CH group. Figure 14 C). The above results confirm that the released CQ can effectively inhibit the activity of the TLR9 / NF-κB pro-survival signaling pathway in CRPC cells. Co-CH treatment can also significantly reduce the expression levels of pP65, COX2, and TGFβ1 in PC-3 cells, but has little effect on C-TLR9 levels, suggesting that the inhibitory effect of CoNZ on the NF-κB pathway may not depend on TLR9. To verify this hypothesis, EGCG was used to represent the organic components in CoNZ, and ligand-protein molecular docking analysis was performed on the complex structure (PDBID: 7LF4) of importin α3 (IPOα3) binding to the P50 and P65 nuclear localization sequences (NLS). The docking results showed that there was an interaction between EGCG and IPOα3 (C chain), P65 NLS (D chain), and P50 NLS (F chain), with a total binding affinity of −8.3 kcal mol–1 ( Figure 14D). Three-dimensional (3D) analysis shows that EGCG mainly binds to IPOα3 and P50 through hydrogen bonds, with hydrophobic interactions contributing relatively little; its interaction with P65 is solely through hydrogen bonds. Within IPOα3, EGCG forms two π-cation interactions with Arg306 (R306). Two-dimensional interaction diagrams further confirm the presence of multiple hydrogen bonds and hydrophobic interactions between EGCG and the three protein molecules. Figure 14 E). The three key amino acid residues that contribute the most energy are Tyr298 (Y298) in P65, Trp264 (W264) in IPOα3, and Asp271 (D271). Figure 14 F). In summary, the results indicate that CoNZ can spontaneously bind to the nuclear transporter complex composed of IPOα3, P50, and P65, preventing its translocation to the cell nucleus and thereby inhibiting the expression of NF-κB downstream target genes COX2 and TGFβ1.

[0089] After confirming the inhibition of COX2 and TGFβ1 expression, the migration and proliferation abilities of cells were assessed using a scratch assay. After 24 hours, the scratched areas of PC-3 cells in both the untreated and F-CH treated groups were almost completely closed, indicating strong migration and proliferation activity. Figure 14 G). In contrast, cell wound healing was significantly delayed in the CQF-CH or Co / F-CH treatment groups, with noticeable gaps still visible in the scratched area. Particularly significant was the lowest wound closure in the Co / CQF-CH treatment group, where the scratched area remained essentially cell-free. Figure 14 G and H). Furthermore, the cell density at the wound edge was significantly reduced in this group, suggesting that cell migration and viability were significantly inhibited. Figure 14 G and H).

[0090] In summary, these results indicate that Co / CQF-CH not only induces cell death but also effectively weakens the migration and regeneration potential of surviving cells. This effect may be achieved by downregulating related genes regulated by NF-κB, further supporting the efficacy of Co / CQF-CH in CRPC cell therapy.

[0091] Example 7: In vivo anti-tumor experiment (1) Construction and treatment of subcutaneous PC-3 tumor model in mice Animal experiments establishing the PC-3 subcutaneous tumor model in mice strictly followed the Animal Protection and Use Guidelines of Sun Yat-sen University and were approved by the Institutional Animal Protection and Use Committee of Sun Yat-sen University. Six- to eight-week-old male BALB / c nude mice, weighing 18-22 g, were selected and housed in a specified pathogen-free (SPF) environment, with a 7-day acclimatization period before the experiment. Each mouse was subcutaneously injected with 1×10⁻⁶ ppm in its right back.6 PC-3 cells (suspended in 100 µL PBS) were used to establish a tumor-bearing model. Tumor size was measured periodically using electronic calipers after inoculation. Tumor volume (V) was calculated using the formula V = 0.5 × L × W. 2 The calculation is performed, where L and W represent the major and minor diameters of the tumor, respectively. When the tumor volume reaches approximately 150 mm... 3 Mice were randomly divided into six groups: Ctrl group, F-CH group, CQF-CH group, Co / F-CH group, Co+CQ group, and Co / CQF-CH group. Each group received 100µL of the treatment solution via intratumoral injection, with the needle inserted along the longitudinal axis of the tumor and withdrawn slowly to minimize extravasation. Tumor volume was recorded every 3 days until the experimental endpoint. During monitoring, if the tumor volume exceeded 1500 mm², [further action was taken]. 3 Humane euthanasia was performed on the mice. All mice were euthanized and photographed before sampling. Tumor tissue was then collected, weighed, and photographed for recording. Tumor inhibition rate (%) = (W c - W t ) / W c × 100%, W c W represents the average tumor weight in the control group. t The average tumor weight is given for each treatment group. Three tumor samples were selected from the control group and the Co / CQF-CH group, and immediately cryopreserved in liquid nitrogen for subsequent batch RNA sequencing analysis. The remaining tumor tissues and major organs (heart, liver, spleen, lung, and kidney) were fixed in 4% (w / v) paraformaldehyde (PFA), embedded in paraffin, and then used for immunofluorescence (IF) and immunohistochemistry (IHC) staining.

[0092] To assess biocompatibility and systemic toxicity, mouse body weight changes were recorded every 3 days during treatment. After the experiment, retroorbital venous blood was collected, and serum was obtained by centrifugation at 1000 g for 25 minutes at 4 °C. Blood biochemical parameters were detected using an automated biochemical analyzer. Tissue sections from major organs were stained with hematoxylin-eosin (HE) and subjected to histopathological analysis to assess the presence of inflammatory responses, necrosis, or structural abnormalities, in order to comprehensively determine systemic toxicity and biocompatibility.

[0093] A castration-resistant prostate cancer (CRPC) xenograft model was constructed by subcutaneously inoculating PC-3 cells into BALB / c nude mice to evaluate the in vivo antitumor efficacy of a sequential drug delivery system. The hydrogel was freshly prepared on ice and then injected intratumorally. Figure 17A). The experiment consisted of six groups: control group (Ctrl, G1), F-CH (G2), CQF-CH (G3), Co / F-CH (G4), free Co+CQ (G5), and Co / CQF-CH (G6) for comparative analysis. Tumor volume was measured periodically throughout the tumor growth and treatment process. Compared to the control group, the tumor growth rate in groups G3 and G4 was slower, but still showed a continuous growth trend. Figure 17 B, C). In group G5, tumors were initially suppressed, followed by regeneration, with the final treatment effect similar to the single-drug treatment group. Notably, group G6 had the smallest final tumor volume, demonstrating superior tumor suppression; furthermore, two mice in this group experienced complete tumor regression, further confirming its significant tumor-suppressive ability. Figure 17 D, E). After treatment, the tumor was removed and weighed, and the tumor inhibition rate was calculated (D, E). Figure 17 F). The results showed that the Co / CQF-CH group had the highest tumor inhibition rate (approximately 80%), significantly higher than the CQ monotherapy group (~32%) and the CoNZ group (~18%), and also superior to the free drug combination group (Co+CQ, ~35%). This result was consistent with changes in tumor volume and imaging records. HE staining and IF staining were used for histological analysis of tumor tissue to assess the degree of tissue damage and cellular biological response. Figure 17 G, H). Compared with group G1, no significant pathological changes were observed in group G2, indicating that the hydrogel matrix itself did not significantly interfere with the tissue. The tumor necrosis areas were similar in groups G3 and G4, the necrosis area was significantly expanded in group G5, and the necrosis area was the largest in group G6 (approximately 65%), significantly higher than that in group G5 (approximately 40%). Ki67 is a nuclear protein related to cell proliferation; therefore, Ki67 immunofluorescence staining was used to assess cell proliferation activity. Figure 17 G, H). Monotherapy reduced the proportion of Ki67-positive cells by approximately 50%, while combination therapy further reduced it to 80–90%, suggesting that multiple treatment modalities combined can produce a stronger inhibitory effect on proliferation. To further assess tumor cell apoptosis, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method and BCL2 immunofluorescence staining were used. Figure 17G, H). The G6 group showed the strongest TUNEL positive signal, significantly higher than all single-drug groups and the free combination group, indicating that co-delivery via injectable hydrogel can effectively enhance the pro-apoptotic effect. Meanwhile, the BCL2 expression levels in the G4, G5, and G6 groups were significantly lower than those in the G1, G2, and G3 groups. Interestingly, TUNEL staining showed comparable apoptosis levels in the G3 and G4 groups; however, BCL2 expression was significantly reduced in the G4 group (~60% decrease), G5 group, and G6 group (80–90% decrease), suggesting that CoNZ and CQ may induce apoptosis through different mechanisms: CoNZ mainly activates the apoptotic pathway by inducing oxidative stress damage, while CQ independently exerts its effect by inhibiting protective autophagy. These in vivo observations are highly consistent with the FCM and Western blot results from in vitro experiments. Figure 12 D–G).

[0094] To further elucidate the mechanism of action of in vivo therapy, markers related to oxidative damage, hypoxia, autophagy, and the NF-κB signaling pathway in tumor tissue were detected. Figure 18 Oxidative damage is considered one of the key mechanisms of antitumor efficacy. Therefore, we used IHC to detect 8-hydroxy-2'-deoxyguanosine (8-OHdG), a recognized biomarker of DNA oxidative damage. Figure 18 (A and B). The results showed that the mean optical density (MOD) of 8-OHdG in all treatment groups was higher than that in the control group, with the increase being more significant in the CoNZ-containing treatment group. Notably, the 8-OHdG level in the combined treatment group was approximately 3 times higher than that in the control group, indicating that CoNZ can effectively generate a large amount of ROS and significantly enhance intratumoral oxidative stress through synergistic effects. In addition to directly inducing oxidative damage, CoNZ can also alleviate hypoxia in the tumor microenvironment (TME) by generating O2 in situ. In all CoNZ-containing treatment groups, the fluorescence signal intensity of HIF1α and VEGF decreased by more than 50%, suggesting that oxygen supply in the TME was improved. Figure 18 A and B). To validate the above results, further transcriptome sequencing and bioinformatics analysis were performed. Gene set enrichment analysis (GSEA) showed that in Co / CQF-CH treated CRPC tumors, pathways related to oxidative damage response, hypoxia relief, and downstream VEGF expression regulation were significantly activated or inhibited (…). Figure 18(C) Simultaneously, the GSH metabolic pathway showed a negative enrichment trend, consistent with previous validation results demonstrating CoNZ's ability to mimic GPX and NOX enzyme activities, further illustrating its ability to disrupt the antioxidant defense system of CRPC cells. Furthermore, GSEA results regarding hypoxia and the VEGF pathway were highly consistent with immunofluorescence data, confirming effective inhibition of the HIF1α / VEGF signaling axis. These findings, corroborated by in vitro experimental results, highlight the multiple therapeutic advantages of CoNZ: not only inducing tumor cell death through ROS accumulation but also reducing tumor invasiveness by alleviating hypoxia.

[0095] Subsequently, the autophagy-inhibiting effect of the released CQ was assessed in vivo by analyzing the expression levels of LC3B and p62 in tumor tissue. The results showed that the LC3B and p62 fluorescence signals in the CQ-treated group were significantly stronger than those in the G1, G2, and G4 groups, while the latter three groups showed weaker signals. Figure 18 A and B). In particular, the LC3B signal in group G6 was significantly higher than that in group G3, suggesting that autophagic flux was effectively blocked. GO functional annotation and GSEA of the Reactome pathway further showed that autophagy-related pathways were significantly negatively enriched in Co / CQF-CH treated CRPC tumors ( Figure 18 C), supporting the conclusion that autophagy was effectively inhibited. By examining the effects of loaded CoNZ and CQ on the TLR9 / NF-κB signaling pathway, it was found that compared to the G1 group, the MOD value of pP65 was reduced by at least 50% in all CQ-treated groups, indicating that CQ can significantly inhibit NF-κB activation. Figure 18 (D and E). In addition to pP65, the expression levels of COX2 and TGFβ1 were detected to assess the downstream effects of NF-κB signaling activation. In group G3, the fluorescence signals of COX2 and TGFβ1 were reduced by approximately 25% and 70%, respectively, while in group G6, they were further reduced to approximately 70% and 90%, respectively. Figure 18 This phenomenon (D and E) may be attributed to the synergistic effect of CQ-mediated NF-κB pathway inhibition and the specific binding of the IPOα3-P50 / P65 NLS complex to CoNZ, thereby inhibiting NF-κB activity. Given the crucial role of the NF-κB pathway in CRPC cell survival, its inhibition helps downregulate multiple pro-survival factors, thereby inhibiting tumor progression. GSEA results also showed negative enrichment of multiple NF-κB-related pathways. Furthermore, inhibition of NF-κB signaling also led to negative enrichment of the KEGG cell cycle pathway ( Figure 18 F), consistent with the observed tumor proliferation inhibition effect.

[0096] In summary, histological analysis and GSEA results based on transcriptome sequencing jointly confirmed that CoNZ preferentially released from Co / CQF-CH exhibited the expected multi-enzyme activity in vivo and possessed the ability to bind to the IPOα3-P50 / P65 NLS complex. Subsequent CQ release effectively inhibited lysosomal acidification, thereby blocking the TLR9 / NF-κB signaling pathway and inhibiting protective autophagic flux. The combination therapy demonstrated a more significant antitumor effect, and the drug delivery system based on injectable hydrogels further enhanced therapeutic efficacy. While evaluating therapeutic efficacy, a systematic review of the in vivo biosafety of the in situ injection antitumor therapy strategy is also crucial. In addition to continuous monitoring of tumor volume, body weight changes in each group of mice were recorded throughout the experiment. Figure 19 As shown in Figure A, the body weight of mice in all groups showed a steady increasing trend over time, with no statistically significant differences between groups. Blood biochemical indicators, including creatine kinase (CK), Figure 19 B) Lactate dehydrogenase (LDH), Figure 19 C) Alanine aminotransferase (ALT, Figure 19 D) Aspartate aminotransferase (AST), Figure 19 E), alkaline phosphatase (ALP, Figure 19 F), Creatinine (CREA), Figure 19 G) and blood urea nitrogen (BUN, Figure 19 H), no significant differences were observed among the experimental groups. Furthermore, such as Figure 20 As shown, histopathological evaluation of tissue sections from major organs (including heart, liver, spleen, lung, and kidney) stained with hematoxylin and eosin (HE) revealed no significant abnormalities or necrotic lesions in mice across different treatment groups. These results demonstrate that the dual-drug sequential delivery system constructed in this invention possesses significant anti-tumor efficacy while exhibiting low in vivo toxicity and excellent biosafety. It not only effectively inhibits the progression of CRPC but also overcomes treatment resistance.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A dual-drug sequential delivery system, characterized in that, This includes cobalt-polyphenol coordinated nanozymes and chloroquine-loaded microfibers.

2. The dual-drug sequential delivery system according to claim 1, characterized in that, The microfibers are polylactic acid-glycolic acid copolymers; the cobalt is cobalt atoms; and the polyphenols are epigallocatechin gallate.

3. A composite hydrogel, characterized in that, It includes aldehyde-modified hyaluronic acid; the aldehyde-modified hyaluronic acid is encapsulated with cobalt-polyphenol coordinated nanozymes and chloroquine-loaded fiber patch fragments; the chloroquine-loaded fiber patch fragments include chloroquine-loaded microfibers and a carboxymethyl chitosan matrix.

4. The composite hydrogel according to claim 3, characterized in that, The microfibers are polylactic acid-glycolic acid copolymers; the cobalt is cobalt atoms; and the polyphenols are epigallocatechin gallate.

5. A method for preparing a composite hydrogel, characterized in that, Includes the following steps: (1) Cobalt-polyphenol coordinated nanozymes were synthesized by antisolvent method; (2) Polylactic acid-glycolic acid copolymer is dissolved in hexafluoroisopropanol, and chloroquine is added to obtain an electrospinning solution; the electrospinning solution is electrospinned to generate microfibers, which are dried to obtain CQ@F patches; the CQ@F patches are embedded in a carboxymethyl chitosan matrix, frozen and sliced ​​to obtain chloroquine-loaded fiber patch fragments; (3) Oxidize hyaluronic acid with sodium periodate to generate aldehyde-modified hyaluronic acid; (4) Disperse the cobalt-polyphenol coordinated nanozyme in the aldehyde-modified hyaluronic acid solution to obtain a dispersion; heat and melt the chloroquine-loaded fiber patch fragments to obtain a melt; mix the dispersion and the melt to obtain a mixture, incubate, and obtain the composite hydrogel Co / CQF-CH.

6. The preparation method according to claim 5, characterized in that, In step (1), the antisolvent method is as follows: S1. Dissolve polyvinylpyrrolidone in a solvent, add a solution containing Co(NO3)2•6H2O dropwise, stir, and then add a solution containing epigallocatechin gallate to obtain a mixture; S2. Stir the mixture continuously at room temperature for at least 4 hours, dialyze to remove unreacted small molecules and residual solvent; freeze-dry to collect the product and obtain cobalt-polyphenol coordinated nanozyme.

7. The preparation method according to claim 5, characterized in that, In step (2), the concentration of the polylactic acid-glycolic acid copolymer is 10%-20% (w / v); the final concentration of chloroquine in the electrospinning solution is 0.5%-2% (w / v); the voltage of the electrospinning is 10 kV-15 kV; and the thickness of the slice is 10 μm-50 μm.

8. The preparation method according to claim 5, characterized in that, In step (3), the preparation method of the aldehyde-modified hyaluronic acid is as follows: hyaluronic acid is dissolved in water, and sodium periodate solution is added dropwise; the reaction is carried out in the dark, and then ethylene glycol is added to terminate the reaction; the reaction is dialyzed and freeze-dried to obtain aldehyde-modified hyaluronic acid.

9. The preparation method according to claim 7, characterized in that, The light-protected reaction is carried out at a temperature of 35 °C-40 °C for 1-3 hours.

10. The preparation method according to claim 5, characterized in that, In step (4), the incubation temperature is 29.3°C - 40.8°C and the time is 5 min - 30 min; the ratio of carboxymethyl chitosan and aldehyde-modified hyaluronic acid in the mixture is 3% (w / v): (0.8% - 2%) (w / v).

11. An injectable preparation, characterized in that, It includes any one of the following: the dual-drug sequential delivery system according to claim 1 or 2, the composite hydrogel according to claim 3 or 4, and the composite hydrogel prepared by the preparation method according to any one of claims 5-9.

12. The use of the dual-drug sequential delivery system of claim 1 or 2, the composite hydrogel of claim 3 or 4, the composite hydrogel prepared by any one of claims 5-9, and the injection of claim 10 in the delivery of antitumor drugs.

13. The use of the dual-drug sequential delivery system of claim 1 or 2, the composite hydrogel of claim 3 or 4, the composite hydrogel prepared by the preparation method of any one of claims 5-9, and the injection of claim 10 in the preparation of antitumor drugs.

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