MMP-2-responsive targeted brain glioma synergistic diagnosis and treatment and immune activation nanoprobe and preparation method and application thereof

Abstract

The application discloses an MMP-2 responsive targeted brain glioma synergistic diagnosis and treatment and immune activation nano probe and a preparation method and application thereof, and relates to the technical field of biological medicine. The probe is formed by assembling an Angiopep-2 targeting peptide, an MMP-2 response sequence, a chemotherapeutic drug and a photosensitizer I-BODIPY, has a hydrodynamic diameter of 10-50 nm, and has excellent biocompatibility. The probe is combined with an LRP1 receptor through the Angiopep-2 to cross the blood-brain barrier, is specifically cut by MMP-2 in a tumor microenvironment to release drugs at a fixed point, integrates the functions of chemotherapy, photothermal / photodynamic therapy and fluorescence imaging, synchronously activates immunogenic cell death and paraptosis, and reshapes an anti-tumor immune microenvironment. The probe can realize precise targeting of brain glioma, synergistic diagnosis and treatment, immune activation and tumor staging evaluation, significantly prolongs the survival period of tumor-bearing mice, and provides a new scheme for glioblastoma diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an MMP-2 responsive targeted glioma synergistic diagnosis and treatment and immune activation nanoprobe, its preparation method and application. Background Technology

[0002] Glioblastoma (GBM), classified as grade IV glioma by the World Health Organization, is the most common malignant tumor of the central nervous system (CNS), accounting for approximately 50.1% of all cases. Current treatments for GBM primarily include surgery, medication, radiation therapy, and electric field therapy. Temozolomide, a chemotherapy drug, is used throughout the entire treatment process, from initial diagnosis to relapse, and is the systemic treatment with the most robust evidence-based support and the clearest survival benefit according to guidelines and evidence-based medicine. However, temozolomide hydrolyzes in systemic circulation to 3-methyl(triazalenoyl)imidazol-4-carboxamide (MTIC). The high polarity of MTIC makes it difficult to cross the blood-brain barrier (BBB), significantly reducing treatment efficacy. Furthermore, the immunosuppressive microenvironment presents a significant challenge to GBM treatment. Therefore, achieving effective drug delivery across the BBB and improving the immunosuppressive microenvironment are core requirements for improving the current state of GBM treatment.

[0003] In recent years, nanotechnology-based drug delivery systems have provided new directions for the treatment of GBM (genomic leukemia). Among these, peptides have been widely used due to their excellent biocompatibility, high affinity, ease of synthesis and modification, and diverse functions. Peptides can self-assemble into well-defined nanostructures through their inherent aggregation capabilities, serving as drug carriers to improve penetration efficiency. Furthermore, targeted peptides can undergo receptor-mediated endocytosis, facilitating nanosystems to cross the blood-brain barrier (BBB) ​​and specifically accumulate in tumor tissues and cells. To avoid off-target toxicity, stimulus-response disassembly strategies have emerged. These strategies leverage the precise coding characteristics of peptide sequences to introduce response sites, combining them with tumor-specific highly expressed enzymes (such as MMP-2 / 9 and Cathepsin B) as substrates to achieve targeted drug activation and release. Simultaneously, to address the heterogeneity and invasiveness of brain tumors, multimodal phototherapy (integrating PTT, PDT, and fluorescence imaging) has attracted widespread attention. Its "one molecule, multiple functions" strategy, due to its well-defined structure and excellent biocompatibility, is superior to traditional multi-component integration models and has become a new direction for development.

[0004] While cancer immunotherapy represents a novel paradigm, its response rate is low in GBM (tumor-bound tumors). The core reason is that GBM are often "immune desert" cold tumors, where anti-tumor immunity is blocked during the antigen presentation stage. In clinical practice, low immunogenicity often stems from insufficient antigen presentation, such as impaired IFN-γ signaling leading to downregulation of MHC-I expression and impaired dendritic cell (DC) maturation, rather than insufficient mutation numbers. Inducing immunogenic cell death (ICD) can fill the antigen presentation gap by exposing damage-associated molecular patterns (DAMPs), but ICD inducers lack specificity, hindering clinical translation. Furthermore, the enrichment of immunosuppressive cells (TAMs, MDSCs, etc.) in the tumor microenvironment further suppresses the immune response; therefore, reshaping the immune microenvironment is crucial for the transformation of cold tumors into "hot" tumors. Paraapoptosis, as an atypical programmed cell death, can enhance immunogenicity by inducing endoplasmic reticulum stress and ROS activation to release DAMPs, providing a new pathway to overcome immune resistance, but it has not yet been effectively combined with existing strategies.

[0005] Existing technologies still face multiple bottlenecks: peptide-based nanodelivery systems have limited cross-BBB capabilities, insufficient precision in synergistic disassembly and targeting in response to stimuli, and off-target toxicity is not completely eliminated; multimodal phototherapy and immunotherapy have low integration, making it impossible to achieve efficient synergy in drug delivery, diagnosis, and immune activation; the immune activation potential of ICD and paraaptosis has not been linked, making it difficult to construct an immune response of "rapid ignition + continuous antigen supply," and thus unable to effectively overcome GBM resistance and immunosuppression.

[0006] In summary, there is an urgent need to develop an integrated solution that combines cross-BBB, precise targeting, collaborative diagnosis and treatment, and efficient immune activation to overcome the multiple challenges in GBM treatment and provide a new path for GBM diagnosis and treatment. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide an MMP-2-responsive targeted glioma synergistic diagnosis and immune activation nanoprobe and its application, in order to solve the problems existing in the prior art.

[0008] This invention designs a novel, specific fluorescent nanoprobe (APZT) that integrates diagnostic and therapeutic functions through peptide self-assembly. The fluorescent molecule I-BODIPY is coupled to a tandem peptide composed of a matrix metalloproteinase 2 (MMP-2) responsive peptide and an Angiopep-2 targeting peptide via a click chemistry reaction. Chemotherapy drugs are simultaneously added to the self-assembly system, encapsulating them within a hydrophobic cavity during assembly, ultimately resulting in a micelle structure with one hydrophilic end and the other hydrophobic end. The hybrid self-assembled nanoparticles (APZT) can recognize low-density lipoprotein-associated receptor 1 (LRP-1), thereby promoting BBB transport and targeting GBM. Due to the aggregation and fluorescence quenching properties of BODIPY, APZT exhibits a PTT effect in its assembled state due to non-radiative transitions. Once it reaches the GBM lesion site, the responsive peptide is cleaved by the highly expressed MMP-2, causing the system to disassemble. On one hand, Angiopep-2 is released, blocking LRP-1-mediated efflux from the lumen and enhancing the retention of chemotherapy drugs at the lesion site. On the other hand, with the MMP-2-triggered disassembly, the fluorescent molecule BODIPY is instantaneously "unlocked," and the fluorescence intensity increases in parallel with the tumor malignancy and MMP-2 level, enabling in-situ visualization and grading of the lesion. Simultaneously, the activated BODIPY generates a large amount of reactive oxygen species under external light irradiation, initiating photodynamic therapy and achieving integrated diagnosis and treatment. In addition, PDT induces mitochondrial oxidative stress by activating BODIPY aggregated in mitochondria, generating a large amount of ROS, which subsequently induces endoplasmic reticulum stress, phosphorylating the eukaryotic translation initiation factor e1F2a, and ultimately controlling the release / exposure of DAMPs. PDT can also directly induce endoplasmic reticulum stress in cells, causing a large amount of Ca2+ to be released into the cytoplasm and mitochondria, leading to an increase in mitochondrial ROS. This vicious cycle between endoplasmic reticulum stress and mitochondrial disorder causes changes in cell morphology and accelerates the para-apoptotic death pathway. APZT utilizes ROS-Ca... 2+ The axis triggers a dual cell death pathway of immunogenic death and paraapoptotic death. Through a synergistic strategy of "rapid ICD ignition + continuous antigen supply from paraapoptosis", it crosses the BBB, precisely targets the lesion site, overcomes drug resistance, and simultaneously activates innate and adaptive immunity, providing a new paradigm for multimodal phototherapy-immunotherapy combined with GBM.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an MMP-2 responsive targeted glioma synergistic diagnosis and immune activation nanoprobe, the nanoprobe comprising micelle APZ and an antitumor drug, wherein the antitumor drug is encapsulated in the center of the micelle APZ, and the micelle APZ is self-assembled from monomers with the structural formula shown in (I). ; Wherein, R is the amino acid sequence as shown in SEQ ID NO. 1.

[0010] Furthermore, the antitumor drug is at least one selected from temozolomide, carmustine, lomustine, semustine, procarbazine, vincristine, etoposide, teniposide, cisplatin, and carboplatin.

[0011] Furthermore, the hydrodynamic diameter of the nanoprobe is 10-50 nm, and the zeta potential is 0.1-2.0 mV.

[0012] Furthermore, the antitumor drug is temozolomide.

[0013] In a second aspect, the present invention provides the application of any of the above-described nanoprobes in the preparation of glioma diagnostic and therapeutic drugs or in the preparation of glioma staging assessment products.

[0014] Furthermore, the integrated diagnosis and treatment includes in vivo fluorescence imaging, photothermal therapy, photodynamic therapy, and chemotherapy.

[0015] In a third aspect, the present invention provides the application of the aforementioned APZ micelles in glial imaging of the brain.

[0016] In a fourth aspect, the present invention provides a method for preparing the above-described nanoprobe, comprising the following steps: (1) Dissolve I-BODIPY and azide-terminated polypeptide in DMSO, add CuSO4 and tris(3-hydroxypropyltriazolylmethyl)amine, then add aminoguanidine and sodium ascorbate in DMSO solution, and stir at room temperature for 20-40 min. (2) Add the antitumor drug dissolved in DMSO to the system obtained in (1), continue stirring and slowly add phosphate buffer, stir and then purify by gradient dialysis to obtain the nanoprobe; The structural formula of the I-BODIPY is as follows: .

[0017] Further, the mass ratio of I-BODIPY to the azide-terminated polypeptide in step I is 1:2~5; the mass ratio of I-BODIPY to CuSO4 and tris(3-hydroxypropyltriazolylmethyl)amine is 1:0.01~0.02:0.20~0.30; the mass ratio of I-BODIPY to aminoguanidine and sodium ascorbate is 1:3~4:5~7 respectively; and the mass ratio of I-BODIPY to the antitumor drug is 1:2~5.

[0018] Furthermore, the gradient dialysis purification is performed by gradient dialysis using 30, 50, and 100 mM EDTA solutions, with each concentration dialyzed three times, followed by three dialysis cycles in ultrapure water.

[0019] The beneficial effects of this invention include at least the following: (1) The primary advantage of the multifunctional nanoprobe APZT provided by this invention lies in its efficient crossing of the blood-brain barrier and precise targeted delivery to tumors. The targeting peptide Angiopep-2 it carries can specifically bind to the LRP1 receptor highly expressed by brain capillary endothelial cells, and gently penetrate the blood-brain barrier through receptor-mediated endocytosis, solving the core problem that traditional chemotherapy drug temozolomide is difficult to penetrate the BBB. After entering the tumor microenvironment, the PLGVR sequence embedded in APZT can be specifically cleaved by the tumor-highly expressed MMP-2, triggering the disassembly and site-specific release of temozolomide and photosensitizer BODIPY. At the same time, the free targeting peptide can block LRP1-mediated drug efflux, significantly increasing the level of drug accumulation in the brain, and showing extremely strong targeting selectivity for U87-MG glioblastoma cells with high MMP-2 expression, greatly reducing off-target toxicity.

[0020] (2) The APZT provided by this invention integrates multimodal synergistic diagnostic and therapeutic functions of photothermal therapy (PTT), photodynamic therapy (PDT), chemotherapy, and fluorescence imaging, significantly improving tumor killing efficiency. Under near-infrared laser irradiation, APZT can rapidly heat up to above 50°C, and its photothermal performance does not significantly decrease after 5 cycles, achieving highly efficient photothermal killing. The BODIPY released after MMP-2 activation can generate a large amount of singlet oxygen, exerting a strong photodynamic therapy effect, and forming a synergistic chemotherapy effect with the encapsulated temozolomide, reducing the survival rate of tumor cells under laser irradiation to 30%. At the same time, the fluorescence signal of APZT is specifically activated after MMP-2 triggering, which can monitor drug delivery, disassembly process, and tumor distribution in real time, achieving "diagnosis and treatment synchronization" and providing visual support for the evaluation of treatment effect.

[0021] (3) The nanoprobe provided by this invention innovatively activates both immunogenic cell death (ICD) and para-apoptotic cell death pathways, effectively reshaping the anti-tumor immune microenvironment; APZT generates oxidative stress through mitochondrial targeting, promoting calreticulin (CRT) membrane exposure, high-mobility group box 1 (HMGB1) efflux and adenosine triphosphate (ATP) secretion, significantly enhancing the immunogenicity of tumor cells, thereby activating dendritic cell maturation and mobilizing CD4 + / CD8 + T cell proliferation breaks the "immune desert" dilemma of GBM. Simultaneously, APZT can target the endoplasmic reticulum to induce endoplasmic reticulum stress, through ROS-Ca... 2+Axial positive feedback loop induces cell vacuolization, providing an alternative death pathway for apoptotic drug-resistant tumors. Vacuolated cells continuously break down and release intracellular antigens, forming an immune response of "rapid ignition + continuous antigen supply", effectively inducing immune memory and inhibiting tumor recurrence.

[0022] (4) The APZT provided by this invention exhibits significant anti-tumor efficacy and excellent biocompatibility in in vivo experiments. In the U87-MG subcutaneous tumor-bearing nude mouse model, the tumor volume and weight in the APZT treatment group were significantly smaller than those in the PBS group and the free TMZ group. In the orthotopic brain tumor model, it can significantly delay tumor progression, prolong the survival time of tumor-bearing mice to more than twice that of the control group, and the weight of mice remains stable without significant decrease. Histological analysis shows that the tumor size is the smallest after APZT treatment, while there is no significant damage to major organs such as the heart, liver, spleen, lungs, and kidneys. Blood indicators are not significantly different from those of the control group. It can be stably maintained in physiological environments such as DMEM, PBS, and 10% FBS, with outstanding biocompatibility and good potential for clinical translation.

[0023] (5) The APZT provided by this invention also has the function of tumor staging assessment, expanding the clinical application scenarios. Based on the MMP-2 responsive fluorescence activation characteristics, the degree of tumor malignancy is positively correlated with the fluorescence intensity of APZT. Through in vivo non-invasive fluorescence imaging, GBM of different malignant degrees can be quickly distinguished, achieving accurate tumor staging. The imaging results are highly consistent with histological analysis, and can provide a reference for the formulation of individualized clinical treatment plans without invasive operation. At the same time, it conforms to the 3R principle of experimental animals and has unique value in the field of integrated tumor diagnosis and treatment. Attached Figure Description

[0024] Figure 1 Preparation and characterization of APZT. (a) MS spectrum of BODIPY. (b) LC-MS spectrum of APZT. (c) Representative TEM image of APZT (scale bar: 50 nm). (d) Hydrodynamic diameter of APZT. (e) Zeta potential of different particles. (f) UV-Vis absorption spectra of different samples. (g) Average size distribution of APZT NPs at different time points in DMEM, PBS and 10% FBS. (h) Under 650 nm laser irradiation, different power densities (50-300 mW) cm -2 (i) Temperature variation of APZT NPs under 5 on / off irradiation cycles of 650 nm laser irradiation. (j) Temperature variation of PBS, BODIPY and APZT NPs exposed to 300 mW laser at different time points. cm-2 Photothermal imaging for up to 5 minutes under a 650 nm laser. (k) Schematic diagram of APZT micelles disassembling into TMZ and BODIPY under enzyme stimulation. (l) TEM image of MMP-2-induced APZT disassembly (scale bar: 30 nm, 100 nm). (m) Cumulative drug release curves of TMZ in PBS (pH=7.4) containing different concentrations of MMP-2. (n) UV-Vis spectral changes of APZT micelles after the addition of MMP-2, indicating enzyme-triggered disassembly. (o) Detection of APZT groups using DPBF probes under laser irradiation (300 W cm⁻²). 1 O2 generation. (p) Singlet oxygen generation (SOG) in the enzymatic response of APZT was assessed in PBS with or without MMP-2 under 695 nm laser irradiation using the SOSG probe.

[0025] Figure 2 For in vitro uptake, endosomal cell escape, BBB penetration, and tumor targeting of APZT. (a) CLSM images of HCMEC / d3 cells and U87-MG cells incubated with APZT for different time periods (scale bar: 100 μm). (bc) Quantification of U87-MG cells incubated with APZT by FCM. (d) CLSM images of U87-MG cells treated with APZT (green) stained with lysosomal tracking agent (red) and Hochest 33342 (blue) (scale bar: 100 μm). (e) Lysosomal colocalization coefficients of BODIPY with APZT in U87-MG cells at different time points. (f) Schematic diagram of the constructed in vitro BBB model used to assess the stimulus-responsive disassembly and BBB penetration ability of APZT. (g) TEER measurements confirm tight junction formation in the BBB model. (hk) Representative flow cytometry histograms of APZT uptake in HCMEC / d3 and U87-MG cells, with each histogram followed by quantitative analysis of the corresponding mean fluorescence intensity. (l) APZT crosses the BBB via intraluminal LRP1 and releases Ang2 after being cleaved by tumor-enriched MMP-2 to block intraluminal LRP1 efflux and increase brain drug levels. (m) CLSM images (scale bar: 50 μm) of U87 cells after pretreatment with different concentrations of MMP-2 followed by co-incubation with APZT for 1 hour. (n) CLSM images (scale bar: 100 μm) of U87-MG, A549, and HeLa cells after incubation with APZT in serum-free medium for 6 hours.

[0026] Figure 3 The in vitro antitumor activity of APZT. (a) Representative fluorescence micrographs of U87-MG cells after DCFH-DA staining and co-incubation with different treatment groups (scale bar: 200 μm). (b) Quantitative analysis of the MFI of DCFH-DA by ImageJ. (c) Flow cytometry and (d) Quantitative analysis of the MFI of DCFH-DA. (e) CCK-8 assay to determine the cell viability of PBS, TMZ, APZ, and APZT NPs under 650 nm laser irradiation for 5 min and without irradiation. (f) CCK-8 assay to determine the cell viability of different concentrations of APZT NPs under 650 nM laser irradiation for 5 min and without irradiation. (g) U87-MG cells treated with PBS, TMZ, APZ, and APZT for 8 hours, followed by CLSM staining with cadherin-AM (green, cell viability) and PI (red, cell death) (scale bar: 200 μm) and corresponding quantitative fluorescence analysis (h). (i) Apoptosis assays performed on NPs treated with PBS, TMZ, APZ, and APZT under 650 nm laser irradiation by Annexin V / PI staining. (j) Corresponding quantitative results.

[0027] Figure 4 Immunostimulation response of APZT NPs to U87 cells. (a) Immunofluorescence staining showing the expression of CRT and (c) HMGB1 in U87-MG cells (scale bar: 50 μm). (b) Quantitative analysis of MFI of CRT in a. (d) Quantitative analysis of MFI of HMGB1 in c. (ef) Quantitative analysis of CRT exposure level using flow cytometry. (g) Quantitative analysis of adenosine triphosphate (ATP) in U87-MG cells after different treatments. (h) Western blot analysis showing the expression levels of HMGB1 and CRT in U87-MG cells after specified treatments. (i) Cross-heatmap and cluster analysis of DEGs (q<0.05 and |FC|>1). (j) Volcano plot of differentially expressed genes (DEGs) between APZT and control groups. (k) Cluster heatmap of gene expression. The DEGs identified from the APZT group compared to the control group are shown. Rows (genes) and columns (samples) are hierarchically clustered based on expression similarity. (l) Chord diagram visualization of pathway enrichment analysis of the DEGs. (m) Protein-protein interaction (PPI) network of the target genes. (n) GSEA showed significant activation of the ICD pathway in the APZT group compared to the PBS control group.

[0028] Figure 5 To investigate the mechanism of APZT-induced ICD. (a) Schematic diagram of the APZT-induced ICD response in U87-MG cells. (b) CLSM co-localization image of APZT NPs and Mito-Tracker probe (scale bar: 50 μm). (c) Representative fluorescence micrographs of U87-MG cells stained with DCFH-DA after co-incubation with different concentrations of APZT (scale bar: 200 μm). (d) Quantitative analysis of MFI of DCFH-DA by Image J. (e) Measurement of U87-MG cell membrane permeability by LDH after treatment with PBS, TMZ, and APZT. (f) CLSM image of U87-MG cells stained with JC-1 (scale bar: 50 μm). (g) Flow cytometry quantification of mitochondrial membrane potential of U87 cells under different treatments. (h) Immunofluorescence staining showed the expression of p-PERK and p-eIf2α in U87-MG cells. (i) Quantification of TNF-α and (j) IL-6 secretion in U87-MG cells using an enzyme-linked immunosorbent assay (ELISA) kit (n=3). (k) Representative flow cytometry analysis of mature DCs after different treatments (CD80+CD86+).

[0029] Figure 6 Activation of the paraapoptotic death pathway. (a) Schematic diagram of the paraapoptotic death pathway induced by APZT in U87-MG cells. (b) CLSM co-localization image of APZT NPs and ER-Tracker probe (scale bar: 50 μm). (c) Cytoplasmic Ca in U87-MG cells. 2+ Representative confocal images. Visualize Ca using Rhod-2 (red). 2+ (d) Cell nuclei stained with Hochest 33342 (blue) (scale bar: 50 μm). Confocal microscopy imaging shows that APZT-treated cells exhibit significant vacuolation compared to the untreated control (arrow pointing) (scale bar: 25 μm). (e) Changes in ER morphology after treatment. Confocal image of U87-MG cells stained with ER-Tracker (ER, green) (scale bar: 25 μm).

[0030] Figure 7RNA-Seq analysis of APZT nanoparticles. (a) Gene set enrichment analysis (GSEA) identified specific pathway enrichment patterns in each group, with results displayed as color-coded feature spectra representing each comparison group. (b) KEGG compound analysis, with bar colors representing pathway categories. (c) KEGG enrichment analysis of metabolites. (d) Selected enriched gene ontology (GO) terms and pathways; this chart shows significantly enriched biological processes, molecular functions, and signaling pathways obtained through comparative transcriptome analysis. (e) Heatmap of differentially expressed genes (DEGs) related to calcium ion transport, oxidative stress, unfolded protein response (UPR), and immune response in U87-MG cells after different treatments. (f) GSEA enrichment map of differentially expressed genes in the APZT group. (g) Comparison of calcium transport, immunogenic cell death (ICD), and T cell-mediated immune response scores between the two groups.

[0031] Figure 8 The in vivo anti-glioma efficacy of APZT in nude mouse models. (a) Progress graph of the U87-MG tumor-bearing BALB / c nude mouse model treated with APZT. (b) Monitoring of body weight changes in animal models. (c) Comparison of tumor growth inhibition curves. (d) Tumor weight collected from U87-MG tumor-bearing mice 24 days after treatment. (e) Images obtained after tumor resection following different treatments. (f) Infrared thermographic images of U87-MG tumor-bearing mice after intravenous injection of PBS and APZT nanoparticles followed by 650 nm laser irradiation for 5 minutes. (g) Temperature change curves of U87-MG tumor-bearing mice under the above treatments. (h) Time series diagram of the orthotopic U87-MG tumor-bearing BALB / c nude mouse model treated with APZT. (i) Survival rate of mice in different treatment groups, (j) Body weight change curves, and (k) Representative H&E staining images.

[0032] Figure 9To demonstrate the activation of anti-tumor immune responses by APZT in tumor-bearing mice. (a) Schematic diagram of experimental design: Mice were orthotopically inoculated with GL261-Luc cells on day 0, followed by six rounds of intravenous administration combined with laser irradiation starting from day 10. All biological samples were collected on day 30 for subsequent analysis. (b) In vivo chemiluminescence imaging of C57BL / 6 mice orthotopically bearing GL261-Luc glioblastoma treated intravenously with PBS, TMZ, or APZT (treatment times were day 11, 14, 17, 20, and 23 post-inoculation). (c) Quantitative chemiluminescence intensity analysis of GBM tumor burden in mice. (d) Curves of mouse body weight change after different treatments. (e) Survival curves of mice (n=3 per group). (f) Representative digital images of tumor tissue stained with Ki67, CRT, HMGB1, and TUNEL in each group. (g) Representative flow cytometry contour plots and (hi) quantitative analysis of splenic infiltrating CD4+ and CD8+ T cells after treatment with PBS, free TMZ, and APZT. (j) Mouse tumor re-challenge experiment using GL261-Luc cells. (k) Representative images of immunodeficient nude mice and black mice before and after therapeutic re-challenge.

[0033] Figure 10 For tumor staging assessment based on APZT-responsive fluorescence activation of MMP-2. (a) Schematic diagram of the disassembly and assembly mechanism of APZT after in vivo. (b) In vivo fluorescence imaging of subcutaneous U87 MG glioblastoma-bearing mouse models of different malignancies after a single tail vein injection of APZT. (c) Quantitative fluorescence intensity analysis of GBM tumors in subcutaneous xenograft mouse models. (d) Representative H&E stained images of tumor sections from two groups. (e) Non-invasive in vivo fluorescence monitoring and (g) quantitative analysis of orthotopic GL261 glioblastoma models of different malignancies 20 minutes after tail vein injection of APZT. (f) Fluorescence images (IVIS imaging) and quantitative analysis of the brain and major organs 1 hour after injection (h). (i) H&E stained images of whole brain sections from representative mice in the above treatment groups.

[0034] Figure 11 The synthetic route for I-BODIPY (B4) is shown.

[0035] Figure 12 This is a schematic diagram of APZT assembly.

[0036] Figure 13 The figure shows the characterization results of APZ.

[0037] Figure 14 This is the drug standard curve for temozolomide.

[0038] Figure 15 To assess blood-brain barrier formation by measuring changes in resistance at different times using a cell endothelial resistance meter.

[0039] Figure 16 Quantitative fluorescence analysis of APZT recovery after the addition of exogenous MMP-2.

[0040] Figure 17 The fluorescence quantification was performed after APZT was co-incubated with cells expressing different levels of MMP-2. Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0043] The following specific embodiments illustrate the solution proposed in this invention: Example 1 I. Experimental Methods Preparation of I-BODIPY (B4): Synthetic route as follows Figure 11 As shown.

[0044] (1) Synthesis of compound BODIPY 1 (B1) Under argon protection, 2,4-dimethylpyrrole (760 mg, 8 mmol) and 4-hydroxybenzaldehyde (488 mg, 4 mmol) were dissolved in 200 mL of dry DCM (dichloromethane). Four drops of trifluoroacetic acid were added, and the reagent bottle containing the mixture was wrapped with aluminum foil and stirred slowly at room temperature for 4 h. Then, 2,3-dichloro-5,6-dicyanobenzoquinone (884 mg, 4 mmol) was added to the solution, and the mixture was stirred for another 20 min. The reaction mixture was then treated with triethylamine (6 mL) for 5 min, followed by slow dropwise addition of boron trifluoride diethyl ether (6.4 mL) to the mixture using a syringe, and the reaction was continued for 40 min. After the reaction was completed, the resulting dark brown mixture was washed with ultrapure water (3 × 20 mL) and brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure, and then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 9, v / v) to give red crystals B1 (yield: 32%). 1H NMR (400 MHz, CDCl3): δ 7.12 (d, J = 7.8 Hz, 2H), 6.95 (d, J = 7.8 Hz, 2H), 5.98 (s, 2H), 2.55 (s, 6H), 1.44 (s, 6H); HRMS m / z: C 19 H 19 BF2N2O [M+Na] + calcdfor 363.1645 found 363.1609. (2) Synthesis of compound BODIPY 2 (B2) B1 (400 mg, 96 mmol) and 3-bromopropyne (205 mg, 140 mmol) were dissolved in acetone (50 mL) containing anhydrous potassium carbonate (132 mg, 77 mmol), and the reaction solution was refluxed for 40 h. After the addition of ultrapure water, the product was extracted with DCM, and the extract was dried and the solvent was evaporated. The crude product was purified by silica gel column chromatography using DCM / n-hexane (3:2) as the eluent to give red solid B2 (yield: 50%). 1 H NMR (400 MHz, CDCl3): δ 7.19 (d, J =7.7 Hz, 2H), 7.09 (d, J = 7.5 Hz, 2H), 6.16 - 5.97 (m, 2H), 4.76 (s, 2H), 2.63- 2.55 (m, 7H), 1.42 (s, 6H); HRMS m / z: C 22 H 21 BF2N2O [M+H] + calcd for 379.1793found 379.1777. (3) Synthesize compound BODIPY 3 (B3) B2 (215 mg, 0.57 mmol) and iodine (335 mg, 1.31 mmol) were added to 15 mL of anhydrous ethanol solution. Iodic acid (230 mg, 1.31 mmol) was dissolved in 1.5 mL of water and added to the mixture. The reaction was stirred at room temperature. After all the starting materials were consumed, 10 mL of saturated sodium thiosulfate aqueous solution was added, and the product was extracted into an organic phase DCM (3 × 30 mL). The extract in the organic phase was dried, and the solvent was evaporated using a rotary evaporator. The product was then purified by silica gel column chromatography (DCM / n-hexane = 1 / 3, v / v) to give red solid B3 (yield: 70%). 1 HNMR (400 MHz, CDCl3): δ 7.16 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 4.78(s, 2H), 2.64 (s, 6H), 2.57 (s, 1H), 1.43 (s, 6H); HRMS m / z: C 22 H 19 BF2I2N2O [M] + calcd for 629.9648 found 629.9669. (4) Synthesis of compound I-BODIPY (B4) B3 (200 mg, 0.32 mmol) and 4-methoxybenzaldehyde (173.2 mg, 1.27 mmol) were dissolved in toluene (29 mL), and acetic acid (glacial acetic acid) (0.6 mL, 11.52 mmol) and piperidine (0.9 mL, 7.8 mmol) were added. The resulting mixture was heated under reflux for several hours. The solvent was then concentrated under reduced pressure, and the residue was diluted with ultrapure water (20 mL) and extracted with CDM. The extract was dried over anhydrous sodium sulfate, the solvent was evaporated, and the extract was purified by silica gel column chromatography (DCM / n-hexane = 1 / 1, v / v) to give green solid B4 (yield: 60%). 1 H NMR (400 MHz, CDCl3): δ 8.13 (d, J = 16.6Hz, 2H), 7.71 – 7.52 (m, 6H), 7.20 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 6.95 (d, J= 8.8 Hz, 2H), 4.79 (s, 2H), 3.86 (s, 6H), 2.58 (t, J = 2.4 Hz, 1H),1.49 (s, 6H); HRMS m / z: C 38 H 31 BF₂I₂N₂O₃[M+Na] + calcd for 889.0383 found 889.0334. Preparation and characterization of APZT: 1 mg I-BODIPY and 3 mg azide-terminated peptide were pre-dissolved in 1.25 mL and 500 µL of DMSO, respectively. 0.01 mg CuSO4 and 0.27 mg tris(3-hydroxypropyltriazolylmethyl)amine were pre-mixed in 180 µL of DMSO solution. Then, aminoguanidine (3.455 mg) and sodium ascorbate (6.191 mg) dissolved in 600 µL of DMSO solution were added to this system. All components were then added sequentially, and the mixture was stirred at room temperature for 30 minutes. Next, 3 mg TMZ (temozolomide) was dissolved in 1.25 mL of DMSO solution, and the mixture was stirred for another 30 minutes to form system 1. After stirring, system 1 solution was slowly added dropwise to 11.2425 mL of phosphate buffer solution (100 mM, pH=7), and the mixture was stirred for another hour. After the reaction, gradient dialysis was performed using EDTA solutions of different concentrations (100, 50, and 30 mM), with each concentration dialyzed three times, followed by three rounds of dialysis in ultrapure water. Finally, the morphology of APZT was observed by transmission electron microscopy, and the particle size and zeta potential of the samples were determined using dynamic light scattering.

[0045] TMZ-loaded APZT drug loading and release performance: APZT was synthesized according to the aforementioned method, and its TMZ loading capacity was subsequently evaluated. First, TMZ solutions with concentrations of 0, 5, 10, 15, 20, 25, 30, 35, and 40 μg / ml⁻¹ were prepared to establish a standard calibration curve. Then, APZT was subjected to gradient dialysis in EDTA buffer solutions of 100, 50, and 30 mM, with the dialysate changed every two hours for a total of three changes, followed by three rounds of dialysis in ultrapure water. The TMZ content in the dialysate was quantified using UV-Vis spectroscopy to calculate the APZT drug loading. The encapsulation efficiency of the nanoparticle APZT was calculated using the following formula.

[0046] EE (%) = [ m (loaded drug) / m [(initial total drug)]*100% MMP-2-triggered APZT disassembly accompanied by fluorescence activation and ROS generation: To evaluate the responsive fluorescence properties of MMP-2, APZT micelles were co-incubated with human MMP-2 enzyme at 37°C for 24 hours. The morphology and particle size changes of APZT were observed using transmission electron microscopy, and the absorbance changes of APZT before and after the addition of MMP-2 were monitored using a UV-Vis spectrophotometer. For the detection of MMP-2-responsive reactive oxygen species, a DPBF probe was used: After adding the DPBF probe to the APZT solution, laser irradiation was performed for 1 to 8 minutes (1, 2, 3, 4, 5, 6, 7, 8 minutes), and the absorbance of the sample solution was measured at 417 nm using a UV-Vis spectrophotometer. Furthermore, to directly observe singlet oxygen generation, SOSG was used as a detection reagent, and the changes in SOSG fluorescence signal in the APZT samples before and after the addition of MMP-2 were monitored within 20 minutes under 660 nm laser irradiation using a microplate reader.

[0047] Construction of a monolayer Transwell model and evaluation of the blood-brain barrier penetration performance of nanoparticles: The blood-brain barrier penetration ability of APZT was evaluated using a Transwell cell culture system (3412, Corning). This model was constructed by co-culturing HCMEC / d3 and U87 MG cells within the Transwell system to build an in vitro blood-brain barrier. The specific procedure was as follows: Transwell chambers with a central porous polycarbonate membrane were placed in each well of a 6-well plate. HCMEC / d3 cells were added at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 10% in the upper chamber of a Transwell and cultured for more than 7 days in medium containing 10% fetal bovine serum to form a complete monolayer barrier. U87 MG cells (2 × 10⁶ cells per well) 5 On day 6, HCMEC / d3 and U87 MG cells were seeded into the corresponding lower chamber. After day 8, the original culture medium in the upper chamber was discarded, and materials (PBS, TMZ, and APZT) were added and transferred to the wells seeded with U87 cells for co-incubation. HCMEC / d3 and U87 MG cells were collected at 1, 2, and 6 hours for subsequent flow cytometry analysis.

[0048] Immunogenic cell death characterization: U87 MG cells were seeded in culture dishes and incubated overnight. 100 μL of different samples were added to treat cells for 12 hours to investigate their effects. To assess the expression and localization of specific proteins, cells were thoroughly washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 at room temperature, and then blocked with immunostaining blocking solution. Cells were then incubated overnight at 4°C with a primary antibody targeting the target protein, followed by incubation with a secondary antibody at room temperature in the dark for 1 hour. Nuclear staining was performed using Hoechst 33342, and the expression and distribution of cytoplasmic calreticulin (CRT) and high-mobility group box 1 (HMGB1) were observed using confocal microscopy, with quantitative analysis performed using ImageJ software. The ATP content in the cell supernatant was detected using ELISA according to the manufacturer's instructions. Furthermore, cells were collected, incubated with anti-CRT antibody, and analyzed by flow cytometry.

[0049] In vitro subcellular targeting ability study of APZT: Cells were seeded in confocal culture dishes and co-incubated with APZT overnight at 37°C. For organelle-specific staining, cells were treated with ER Tracker (endoplasmic reticulum) and Mito Tracker (mitochondria), respectively; all samples were stained with Hoechst 33342 for nuclear staining. The colocalization of APZT with each organelle was analyzed using ImageJ software.

[0050] Immunocellular activation assay: U87 MG cells were seeded in six-well plates and treated with different methods. After treatment, U87 MG cells were collected and co-cultured with RAW264.7 macrophages in six-well plates for 24 hours. After co-culture, cells were collected and stained with CD80-FITC and CD86-PE antibodies. After incubation at room temperature in the dark for 30 minutes, flow cytometry analysis was performed.

[0051] Animal Models: All animal experiments were conducted in accordance with the "Guidelines for Laboratory Animal Care and Use" and approved by the Animal Experiment Ethics Committee of Hainan Medical University. Experimental animals were housed in a specific pathogen-free environment with a 12-hour light / dark cycle, maintaining an ambient temperature of 20±3℃ and relative humidity between 40% and 50%. Free access to water and food was provided throughout the study. To establish a glioblastoma model, female BALB / c-nu and C57BL / 6J mice (6-8 weeks old, 20-22 grams) were anesthetized by inhaling oxygen containing 1-5% isoflurane. U87 MG cells and GL-261 cells labeled with luciferase suspended in PBS were slowly injected into the mouse brain using the anterior fontanelle as a reference point, following stereotactic coordinates (2.5 mm lateral, 2.5 mm posterior, 2 mm depth). After injection, the skin was sutured, and the mice were returned to their original cages. Postoperatively, the animals' general health, neurological signs, and wound healing were observed daily.

[0052] Treatment efficacy evaluation and immune mechanism investigation: The U87 MG-Luc model was used to evaluate the treatment efficacy. Mice were randomly divided into three groups and received intravenous injections of PBS, TMZ, or APZT, respectively, every three days for a total of six doses. Tumor progression during treatment was monitored by bioluminescence imaging: All mice were intraperitoneally injected with 150 mg / kg before each imaging session. -1 D-fluorescein potassium solution (100 μL) was injected, and images were taken using a small animal in vivo imaging system 10 minutes later. Tumor burden was quantified by total fluorescence intensity. Tumor size, body weight, and overall survival of mice were continuously monitored. Mice were sacrificed at the experimental endpoint, and brain tissue was collected for histological (H&E) and immunohistochemical analysis. Major organs were also harvested for H&E staining to assess potential systemic toxicity. Spleens were cut into rice-grain-sized tissue blocks to observe immune activation. Cells were separated by centrifugation after mechanical grinding, and then labeled with CD3, CD4, and CD8 antibodies and analyzed by flow cytometry.

[0053] APZT's MMP-2 responsive fluorescence activation was used for tumor staging assessment: After establishing a U87 MG subcutaneous tumor model, mice with different tumor volumes were selected for in vivo imaging. APZT was injected via the tail vein, and imaging began 20 minutes after injection. Images were continuously acquired at 5-minute intervals to monitor the fluorescence recovery and metabolic clearance of APZT in tumors of different malignant degrees. Subsequently, an orthotopic GL261-Luc glioma model was established using the same method. The tumor was initially graded using bioluminescence imaging, followed by real-time fluorescence imaging after APZT injection via the tail vein. Mice were immediately sacrificed after imaging, and major organs were collected for in vitro fluorescence assessment. In addition, subcutaneous tumor and brain tissue were collected after each imaging session for hematoxylin-eosin staining, and histological analysis was used to verify the in vivo grading results.

[0054] Statistical Analysis: All values ​​in the report are the averages of at least three biological replicates derived from independent biological experiments with consistent results. Data are presented as mean ± standard deviation and analyzed using GraphPad Prism 10.0 software; other data were processed using Origin 2021 software. Significance assessment of multiple comparisons was performed using one-way ANOVA combined with Tukey's post-hoc test, with significance levels set as follows: p < 0.0001, *p < 0.001, p < 0.01, *p < 0.05.

[0055] II. Experimental Results 1. Preparation and characterization of APZT The synthetic route of APZT is shown above. First, an I-BODIPY photosensitizer (B4) based on heavy atom effects and electron donor group modification was selected. This material, modified with alkyne groups, exhibits different properties in generating reactive oxygen species and near-infrared fluorescence imaging. Simultaneously, a functional peptide sequence TFFYGGSRGKRNNFKTEEYGGGPLGVRK(N3) (SEQ ID NO. 1) modified with an azide was designed. This sequence consists of a targeting peptide Angiopep-2 sequence and a peptide sequence PLGVR. The peptide sequence PLGVR is the corresponding substrate for matrix metalloproteinase-2 (MMP-2). Three glycine residues were introduced between the two peptide sequences to separate the two functional regions, and an azide was introduced at the terminal position to couple with the organic functional molecule. This sequence was characterized by MS (Figure 1a).

[0056] Subsequently, the two components are linked through a click chemistry reaction of azide and alkynyl groups, and the hydrophilic polypeptide sequence undergoes supramolecular self-assembly with the hydrophobic organic fluorescent molecule BODIPY. Figure 12 Through various interactions such as hydrophilicity / hydrophobicity, hydrogen bonding, and π-π stacking, an outward hydrophilic end and an inward hydrophobic end are formed. Simultaneously, the chemotherapy drug temozolomide is pre-added to the assembly system and encapsulated within a hydrophobic cavity during self-assembly. A multifunctional nanoprobe (referred to as APZT) integrating fluorescence imaging, PDT, PTT, and chemotherapy functions is constructed. For comparison, nanoparticles without TMZ encapsulation are defined as APZ. Transmission electron microscopy (TEM) further observes the morphology of APZ and APZT. The results show that APZ (… Figure 13 ) and APZT have spherical morphology and uniform size distribution ( Figure 1 c). The average particle sizes of APZ and APZT, measured by dynamic light scattering (DLS), are approximately 20 nm and 30 nm, respectively. Figure 1d), with Zeta potentials (mV) of 0.3 mV and 1.4 mV, respectively. Compared to free BODIPY, APZT exhibits detectable absorption peaks at 380 nm and 665 nm in the UV-Vis absorption spectrum. Similarly, compared to free TMZ, APZT also shows a smaller absorption peak at 325 nm (see [reference needed]). Figure 1 f). These results confirm the successful conjugation of the BODIPY portion and the effective load on the TMZ.

[0057] 2. Performance verification of APZT Subsequently, the performance of APZT was verified. Stability tests showed that the micelles could maintain their original size under different environments (DMEM medium, pH=7.4 PBS, 10% FBS) (Figure 1g). Nanoparticles exhibit photothermal effects before stimulus-response disassembly due to non-radiative transitions under near-infrared irradiation; therefore, the photothermal properties of APZT were systematically studied. First, the effect of different laser power densities on the heat generation efficiency of APZT was investigated. A significant correlation was observed between the increase in laser power density and the resulting temperature increase, indicating that APZT possesses good photothermal performance. Figure 1 h). The photothermal performance of APZT is highly stable and can be repeated for at least five cycles without significant performance degradation (Figure 1i). Subsequently, infrared thermal imaging was used to detect the effect of exposure to 300mW. cm -2 Under 650 nm laser irradiation, APZT rapidly heated to over 50 °C within 5 minutes (Fig. 1j). These results confirm the excellent photothermal properties of APZT. Next, the disassembly behavior of MMP-2 was investigated. The PLGVR motif in APZT was cleaved by MMP-2, which is highly expressed in the tumor microenvironment, initiating the disassembly of nanoparticles accompanied by the release of TMZ and BODIPY (Fig. 1k). To investigate the MMP-2 response efficacy, MMP-2 (2 μg mL⁻¹) was added to PBS at pH 7.4 to simulate the high level of this enzyme in the serum of cancer patients. First, TEM was used to observe the morphological changes of APZT before and after the addition of MMP-2. After MMP-2 treatment, spherical nanoparticles disappeared, and a small number of aggregates appeared (Fig. 1l). Subsequently, the disassembly of APZT was further verified by monitoring the release of TMZ. According to the standard curve of free TMZ in the UV-Vis spectrum (…),… Figure 14The encapsulation efficiency (EE) of TMZ calculated using Eq was 23.3%. APZT was incubated in PBS buffer with different concentrations of MMP-2. Under physiological conditions, as shown in Figure 1m, the drug release curve was exceptionally slow, with only 8.4% of TMZ released from APZT within 24 hours. However, under 10mM MMP-2 conditions, the release rate of TMZ was significantly accelerated, with 86.69% of TMZ released within 24 hours. Compared to APZT, APZT treated with MMP-2 exhibited significantly enhanced absorbance, confirming its successful disassembly (Figure 1n). Furthermore, APZT achieved efficient recovery of BODIPY fluorescence signal and singlet oxygen generation under MMP-2 activation. Subsequently, the photodynamic therapy function of APZT was verified. APZT and MMP-2 were co-incubated at 37°C for 1 hour, and after adding a DPBF probe, the mixture was irradiated with a 650 nm laser. The absorbance was recorded every minute, showing that the singlet oxygen production of APZT steadily increased with the extension of irradiation time. Figure 1 o). Subsequently, SOSG was used as 1 O2 monitoring probes, monitoring fluorescence intensity at 525 nm, showed that compared to the control group, the fluorescence of the MMP-2 treated group continuously increased for more than 20 minutes (Figure 1p). These results confirm that APZT has efficient in vitro production... 1 The ability of O2. Based on these findings, micelles exhibit a strong photothermal effect when they remain stable and in their aggregated state, but under MMP-2-triggered disassembly, they effectively activate photodynamic properties.

[0058] 3. In vitro uptake and lysosomal escape of APZT Cellular uptake of APZT was evaluated in human U87-MG glioma cells, which have been shown to overexpress low-density lipoprotein receptor-associated protein-1 (LRP1) in the literature. To verify the in vitro uptake of APZT by U87 cells, PBS, TMZ, and APZT were added to U87 cells and incubated for different times. Cellular uptake was studied using confocal laser scanning microscopy (CLSM) and flow cytometry. CLSM images showed that co-incubation with APZT for 6 hours exhibited the strongest intracellular fluorescence, while HCMEC / d3 cells cultured with APZT showed no fluorescence (Fig. 2a). The abundant MMP-2 in tumor cells specifically cleaved the PLGVR motif in APZT, triggering the disassembly of the nanoprobe and the release of the fluorophore BODIPY. This observation further indicates that the nanoparticles maintain their structural integrity when crossing the blood-brain barrier. Similarly, the flow cytometry results were consistent with the CLSM results (Figs. 2b and 2c). When U87 cells were co-incubated with APZT at different concentrations for 4 hours, it was found that cellular uptake increased in a concentration-dependent manner.

[0059] Following endocytosis, the trapped BODIPY and TMZ must escape the lysosomes and enter the cytoplasm to perform their biological functions. To monitor endoderm escape, U87 cells were cultured with APZT, lysosomes were labeled with Lyso-Tracker Red staining, and the spatial distribution of the two fluorophores was tracked using CLSM imaging. At 30 minutes, a weak recovery of fluorescence was observed. After 1 hour, BODIPY fluorescence co-localized with lysosomes. At 2 hours, the green signal increased significantly, clearly separating from the red lysosome dots, accompanied by a significant decrease in the co-localization coefficient, demonstrating that APZT successfully escaped the lysosomes and entered the cytoplasm. Figure 2 d and 2e).

[0060] 4. Performance evaluation of APZT in crossing the blood-brain barrier and targeting tumors Low-density lipoprotein receptor-associated protein-1 (LRP1) is overexpressed in brain capillary endothelial cells, and the targeting peptide Angiopep-2 can specifically bind to LRP1. Numerous studies have shown that nanomedicines associated with Angiopep-2 function cross the blood-brain barrier (BBB) ​​more effectively and enter the central nervous system (CNS) at significantly higher doses. To evaluate the ability of APZT to cross the BBB and target brain tumors, a monolayer Transwell system was established as an in vitro BBB model. Briefly, human cortical microvascular endothelial cells / D3 (HCMEC / d3) were cultured in the superior vena cava, while human GBM tumor cells (U-87MG) were cultured in the inferior vena cava, separated by a porous membrane (Fig. 2f). After 7 days, HCMEC / d3 cells formed a dense layer with no obvious gaps. To functionally validate the integrity of tight junctions, transendothelial resistance (TEER) was monitored using an epithelial voltammeter equipped with chopstick electrodes on a vertical flow cleaning bench. The TEER value gradually increased, reaching 200 Ω by day 7. cm 2 The value stabilized and became consistent with the established in vitro BBB baseline (Figure 2g and 2g). Figure 15 ).

[0061] Subsequently, APZT was added to the superior chamber and co-incubated with HCMEC / D3 cells for 1 h, 2 h, and 6 h. Cells were then collected, and the uptake in the superior and inferior chambers at each time point was quantified by flow cytometry. Figure 2 As shown in h and 2i, no significant fluorescence recovery was observed in the HCMEC / D3 monolayer, while the fluorescence intensity of U87 cells gradually increased over time (Figs. 2j and 2k). These data indicate that APZT can effectively cross the blood-brain barrier via LRP-1-mediated transcytosis while maintaining endothelial integrity, thus ensuring gentle access to tumor cells. Once inside the tumor microenvironment, APZT is degraded in response to the high MMP-2 activity expressed by glioma cells, releasing BODIPY and restoring fluorescence.

[0062] Angiopep-2 is a two-stage active targeting peptide. After APZT crosses the blood-brain barrier via LRP-1-mediated transcytosis, the targeting peptide continues to guide the nanosystem to the tumor site. Once the nanoparticles reach the tumor site, locally overexpressed MMP-2 specifically cleaves the PLGVR sequence embedded in the APZT micelles, leading to micelle disassembly and release of the coated BODIPY and TMZ; thus, the extinguished fluorescence is restored (Fig. 21). To evaluate this MMP-2-stimulated response to fluorescence recovery in vitro, different concentrations of exogenous MMP-2 were first added to cells and co-incubated with APZT. A positive correlation was observed between fluorescence recovery and MMP-2 concentration (Fig. 2m and 2m). Figure 16 Cell lines with different MMP-2 expression levels were used: A549 human lung cancer cells overexpressing MMP-2, U87MG human glioblastoma cells, and HeLa human cervical cancer cells with low MMP-2 expression. APZT was co-incubated with each cell type for 6 hours to allow sufficient time for enzyme digestion. Confocal microscopy showed that MMP-2-positive A549 and U87MG cells exhibited significantly higher intracellular fluorescence intensity than MMP-2-negative HeLa cells. Furthermore, U87MG cells showed stronger fluorescence than A549 cells due to APZT carrying a peptide that specifically targets U87 cells. Exposure to the same dose of APZT significantly reduced the number of U87 cells compared to the other two groups, demonstrating APZT's excellent targeting ability (Figure 2n and 2n). Figure 17 ).

[0063] To further validate this targeting ability, CCK-8 cytotoxicity assays were performed on U87MG, A549, HeLa, and 4T1 cells. APZT showed limited cytotoxicity against HeLa and 4T1 cells. In MMP-2 overexpressing A549 cells, micellar disassembly released some chemotherapeutic drugs and fluorescent molecules, causing mild cytotoxicity. Under the combined influence of high MMP-2 expression and targeting specificity, U87MG cells exhibited the most significant cytotoxic response—a result consistent with confocal observations. Furthermore, after photodynamic therapy under external light irradiation, U87MG cells showed the best therapeutic effect among the four cell lines.

[0064] LRP1 exhibits bidirectional transport properties in the blood-brain barrier endothelium: its luminal (blood-facing) side mediates the uptake of nanoparticles from the blood to the brain, while its luminal (brain-facing) side mediates clearance from the brain back to the blood, thus achieving bidirectional transport. However, after MMP-2 cleaves APZT, the A2-targeting peptide (LRP1 ligand) is released and excreted, thereby blocking LRP1-mediated luminal outflow, increasing the drug concentration accumulated in the brain, and improving therapeutic efficacy.

[0065] 5. In vitro antitumor properties of APZT The in vitro PDT effect of APZT was explored by assessing intracellular ROS levels in U87 cells using a 2',7'-dichlorodihydrofluorescein diacetic acid (DCFH-DA) probe. Under laser irradiation, CLSM images revealed a strong green fluorescence signal of DCF converted from ROS-oxidized H2DCF-DA in APZT-treated cells, demonstrating that the significant intracellular ROS production was triggered by laser irradiation (Figs. 3a and 3b). Flow cytometry further confirmed these results: the APZT group showed significantly higher MFI values ​​than the PBS and TMZ groups (Figs. 3c and 3d).

[0066] Subsequently, the PDT toxicity of APZT in U87 cells was investigated using the Cell Counting Kit-8 (CCK-8) assay. Cells were co-incubated with the drug and then photodynamic therapy was performed with a 650 nm laser for 5 minutes. As shown in Figure 3e, laser-induced cell damage to PBS-treated cells was negligible, indicating that the laser intensity was appropriate. In contrast, under light irradiation, TMZ, APZ, and APZT all exhibited varying degrees of cytotoxicity to U87 cells compared to the PBS group. Among them, APZT-treated cells showed the lowest survival rate in a significantly dose-dependent manner, an increase that can be partly attributed to enhanced ROS production within the cells (Figure 3f). The figure shows the half-maximal inhibitory concentration (IC50) of the drug. 50 The inhibitory effect was 10 mM. This inhibitory effect was further supported by cell scratch assays, which showed that APZT combined with laser treatment most effectively inhibited U87 cell migration. Therefore, this IC50 value of APZT is [not specified]. 50The values ​​were used as drug concentrations for subsequent cell experiments. Cells treated with APZ (the control group without TMZ) showed high cell viability in the absence of laser irradiation. APZT induced 60% cell death in the absence of laser irradiation, primarily due to the tumor cell-killing effect of TMZ. Importantly, cell viability decreased to 30% under laser irradiation, indicating that laser-induced ROS and the chemotherapeutic agent TMZ synergistically produced an effective inhibitory effect on tumor cells. Subsequently, HCMEC / D3 cells were designated as the control group for drug-induced dark cell toxicity assessment. TMZ exhibited dose-dependent toxicity. In contrast, both APZ and APZT showed excellent safety. This finding also confirms that APZT can cross the blood-brain barrier while maintaining its structural integrity without causing significant damage. Further analysis included live / dead staining of U87 cells after phototherapy. Confocal images showed significantly more cell death in the APZT-NP treatment group, highlighting the precise targeting and therapeutic efficacy of the nanoparticles on U87 cells (Fig. 3g, 3h).

[0067] To monitor the ROS-induced apoptosis phase induced by PDT activation, U87 cells were incubated with different NPs under laser irradiation or without irradiation, followed by flow cytometry analysis using Annexin V fluorescein isothiocyanate (FITC) (green) and propidium iodide (PI) (red) staining. Live cells, early apoptotic cells, and late apoptotic / necrotic cells were identified as Annexin V-FITC- / PI-, Annexin V-FITC+ / PI-, and Annexin V-FITC+ / PI+ populations, respectively. Among the different treatments, APZT induced the highest percentage of early and late apoptotic cells triggered by laser irradiation (approximately 65%) (Figures 3i and 3j). In summary, these findings confirm that APZT triggers substantial ROS production under laser irradiation and significantly enhances its in vitro photodynamic properties by utilizing the heavy atom effect of BODIPY iodide, resulting in excellent antitumor efficacy.

[0068] 6. Immune stimulation response of APZT nanoparticles in U87 cells Extensive evidence from basic and clinical studies suggests that PDT can induce ICD, including the release of DAMPs composed of calreticulin (CRT), extracellular adenosine triphosphate (ATP), and high-mobility group box 1 (HMGB1). DAMP signaling promotes the formation of mature dendritic cells (DCs), which can effectively present antigens and mobilize the host's acquired immune system. Therefore, it is hypothesized that APZT-mediated PDT may promote ICD effects. To confirm this hypothesis, the expression of specific DAMPs in different treatment groups was assessed in U87 cells. After 8 hours of co-incubation with APZT, immunofluorescence showed significant CALR accumulation on the U87 plasma membrane (Fig. 4a). Flow cytometry analysis also showed significant CALR positive signaling in the APZT-treated group (Fig. 4e), highlighting the effective ability of APZT to drive CALR translocation in glioblastoma cells. Simultaneously, HMGB1 immunostaining revealed that, compared to PBS or TMZ-treated controls, APZT induced a significant efflux of HMGB1 from the nucleus to the cytoplasm, followed by extracellular release (Fig. 4c), thus strongly initiating an immune response. Furthermore, cells treated with APZT exhibited the highest and most significant enhancement in ATP secretion, 2.9 times and 11.7 times that of the PBS and TMZ controls, respectively (Fig. 4g). Western blot analysis yielded similar results (Fig. 4h). In conclusion, the above evidence collectively suggests that APZT is an effective and promising inducer of immunogenic cell death.

[0069] To further verify the activation of immunogenic cell death after APZT treatment, RNA sequencing and transcriptional analysis were performed on cell samples from the control group and the APZT combined with phototherapy group. Differential gene ring heatmap ( Figure 4 i) The differentially expressed genes between the two groups were visually displayed. The selection criteria for differentially expressed genes in this analysis were |log2FC|>1 and FDR-corrected q-value<0.05 (or adj.P.Val<0.05). Subsequently, differential expression analysis was performed on the differentially expressed genes between the APZT group and the control group. Figure 4 The volcano plot showed that, compared to the control group, the APZT group had 2683 differentially expressed genes, including 1554 significantly upregulated genes (q < 0.05 and FC > 1) and 1129 significantly downregulated genes (q < 0.05 and FC < -1). Based on this result, target genes related to immunogenic death were screened, and the expression differences of these genes were visually displayed using a heatmap. Figure 4(k) In the APZT group, multiple hallmark ICD-related genes showed synergistic and significant upregulation after treatment. Among them, the upregulation of NLRP3 is a key early event in the occurrence of ICD. S100A5 is a classic DAMP, which is highly expressed and released in tumor cells. It can directly activate innate immunity by binding to receptors such as RAGE on immune cells, further amplifying the inflammatory microenvironment. The significantly increased expression level of MX1, a classic effector gene of type I interferon, indicates the amplification of downstream interferon signaling. TNFSF10 can promote the release of DAMP signals from dying cells and enhance the maturation of dendritic cells and their antigen cross-presentation ability. In addition, several key negative immune regulatory genes showed consistent downregulation. Among these factors: CISH downregulation, besides inhibiting T cell receptor and cytokine signaling, enhanced the response potential of effector T cells; TNFAIP3 (A20) downregulation weakened the negative feedback of the NF-κB pathway, potentially prolonging and amplifying the inflammatory alarm triggered by ICD; SNAI1 downregulation suggested weakened tumor EMT properties, which is beneficial for improving immune cell infiltration and recognition; and DUSP1 downregulation may have relieved the inhibition of key activation signaling pathways in macrophages and T cells. This positive and negative regulatory pattern together confirms the occurrence of ICD in the APZT treatment group. Figure 4 j, k). These synergistically changing genes functionally converge on two core biological processes (j, k). Figure 4 The pathways involved in APZT therapy include two main pathways: one is the 'immune and inflammatory response' pathway, which directly drives anti-tumor immunity; the other is the 'cell adhesion, migration, and extracellular matrix' pathway, which supports immune cell infiltration and function. The co-enrichment of these two pathways indicates that APZT therapy not only systematically activates immune signaling but also simultaneously remodels the tumor's physical microenvironment to support immune attack. To further clarify the regulatory relationships between these genes, a protein-protein interaction network was constructed. Figure 4 Further gene set variation analysis confirmed at the pathway activity level that pathways related to cytoplasmic DNA sensing, NLRP3 inflammasome assembly, antigen processing and presentation, and inflammatory response were specifically activated in the treatment group. Figure 4 In summary, multi-omics data from multiple dimensions, including gene expression, pathway enrichment, and network regulation, collectively demonstrate that APZT treatment can effectively induce immunogenic cell death.

[0070] 7. Mechanism study of APZT-induced ICD Studies have shown that photodynamic therapy can induce oxidative stress in specific subcellular organelles by activating organelle-associated photosensitizers. Colocalization assays showed that after co-incubation with APZT, the green fluorescence of BODIPY within APZT significantly overlapped with the red fluorescence of mitochondrial probes, confirming the widespread accumulation of APZT in mitochondria (Fig. 5b). To verify mitochondrial oxidative stress, DCFH-DA was used as a probe to expose cells to different concentrations of APZT, resulting in bright, concentration-dependent green fluorescence, demonstrating that higher APZT levels induce significant ROS production (Fig. 5c). Once oxidative stress is initiated, elevated mitochondrial ROS impairs mitochondrial structure and function, leading to a decrease in mitochondrial membrane potential (ΔΨm). Confocal laser scanning microscopy showed that U87 cells treated with APZT exhibited the lowest mitochondrial membrane potential compared to the PBS and TMZ treatment groups (Fig. 5f), which was also confirmed by flow cytometry (Fig. 5g). Furthermore, LDH release assays confirmed oxidative stress (Fig. 5e), indicating that APZT significantly impaired cell membrane integrity and disrupted intracellular homeostasis.

[0071] Studies have shown that ROS generated by oxidative stress can directly oxidize proteins, leading to impaired protein structure and function. Misfolded or unfolded proteins accumulate in the endoplasmic reticulum (ER) and competitively bind to ER molecular chaperones (BiP, which binds immunoglobulins), causing BiP to dissociate from ER stress sensors. PERK kinase dimerizes and cross-phosphorylates, becoming activated, and activated PERK phosphorylates the eukaryotic translation initiation factor e1IF2a. Therefore, confocal imaging was performed on U87 cells treated with different methods. The results showed that PERK was effectively activated in the APZT group, and p-PERK expression increased, resulting in a significant increase in phosphorylated eIF2a (p-eIF2a) expression in the APZT group compared to PBS and TMZ (Figure 5h). Phosphorylated eIF2a controls the release / exposure of DAMPs during ER stress by regulating translation initiation and activating downstream stress response genes.

[0072] In summary, these results indicate that the ICD-induced effect in U87 cells is partly due to the release of photosensitizer BODIPY after APZT cleaves in the tumor microenvironment where MMP2 is highly expressed. BODIPY then accumulates in the mitochondria and generates a large amount of ROS under photodynamic induction, thereby causing cellular oxidative stress. The ROS subsequently generated by cellular oxidative stress will cause endoplasmic reticulum stress, leading to eIF2a phosphorylation and ultimately the release of DAMPs molecules.

[0073] Dendritic cells (DCs) are specialized antigen-presenting cells that play a crucial role in recognizing damage-associated molecular patterns (DAMPs) related to immunogenic cell death (ICD) and in the subsequent processing of tumor antigens. This study analyzed DC activation and maturation by detecting relevant cytokines and surface costimuli. First, the expression of TNF-α and IL-6 was examined. TNF-α is one of the cytokines secreted during DC activation, while IL-6 can relieve the tumor immunosuppressive microenvironment and promote the maturation of surface costimuli. Enzyme-linked immunosorbent assay (ELISA) results showed that TNF-α expression was increased and IL-6 expression was significantly decreased in the APZT group, providing a favorable environment for DC maturation. A binary co-incubation system was used, co-incubating U87 cells and RAW cells, followed by exposure to PBS, TMZ, and APZT for 24 h, respectively, to verify the effect of ICD on DC maturation. After cell culture, flow cytometry analysis of biomarkers of mature DCs revealed that, compared with the control group, the expression of CD80 and CD86 in the APZT group was significantly upregulated (Figure 5k). These results together indicate that APZT can effectively promote the maturation of DCs.

[0074] 8. Activation of the paraapoptotic death pathway Spatially and functionally, the endoplasmic reticulum and mitochondria interact through multiple contact sites to ensure coordinated cellular function; these contact sites are known as mitochondrial-associated endoplasmic reticulum membranes (MAMs). MAMs are deeply involved in the regulation of the intracellular microenvironment, particularly reactive oxygen species (ROS) and calcium. 2+ Exchange. Endoplasmic reticulum colocalization experiments revealed that APZT can target not only mitochondria but also the endoplasmic reticulum, accumulating in large quantities within it (Figure 6b). Since the accumulation of exogenous substances in the endoplasmic reticulum can induce endoplasmic reticulum stress, APZT can also directly induce endoplasmic reticulum stress.

[0075] The downstream effect of endoplasmic reticulum stress is Ca 2+ This flow from the endoplasmic reticulum to the mitochondria via the MAMs also causes an increase in mitochondrial ROS. Using mitochondrial Ca... 2+ The indicator Rhod-2AM was used to study intracellular Ca2+ after different treatments. 2+ The distribution of [the data]. For example... Figure 6 As shown in Figure c, the brightest red fluorescence was observed in the APZT group, with an intensity 50 times higher than that of the control group. (The last sentence appears to be incomplete and unrelated to the preceding text. It likely refers to Ca in mitochondria.) 2+ The outbreak causes an increase in ROS, which in turn exacerbates endoplasmic reticulum stress and Ca2+. 2+ This outflow, this continuous stimulation, effectively prolongs the endoplasmic reticulum stress time, preventing the timely processing of unfolded proteins, ultimately leading to cell death. Specifically, mitochondrial Ca2+... 2+Overload inhibits ATP synthesis, leading to osmotic imbalance, cell swelling, organelle vacuolation, and ultimately, non-membrane rupture lysis and death. Microscopic observation showed that TMZ-treated U87 cells maintained a morphology similar to the PBS control, while APZT-treated cells exhibited abundant vacuolation (Fig. 6d). ER-Tracker staining further revealed extensive ER vacuolation and tissue-free morphology in the APZT group (Fig. 6e). Flow cytometry confirmed that APZT-treated U87 cells exhibited more severe late apoptosis or necrosis compared to PBS and TMZ-treated U87 cells. Overall, these results indicate that APZT induces the paraapoptotic death pathway.

[0076] Studies have shown that most gliomas have developed resistance to caspase-dependent apoptosis pathways, while the paraapoptotic death pathway can provide an alternative death pathway for "apoptotically resistant" tumors. The above experiments validate that ROS-Ca... 2+ The axis is a co-trigger of the paraapoptotic death pathway and the immunogenic death pathway. ICD first rapidly exposes DAMP molecules, igniting DCs and CTLs. Vacuolated cells continue to disintegrate after 24–48 h, entering the paraapoptotic death phase, releasing mtDNA and cytoplasmic proteins, maintaining inflammatory signals, forming multiple pulses of antigen-adjuvant-cytokine, and significantly enhancing the generation of memory T cells in the body.

[0077] 9. RNA transcriptome analysis of APZT nanoparticles To delve deeper into the underlying mechanisms of APZT-induced ICD, transcriptome data were analyzed from multiple perspectives. Gene set condensation analysis (GSEA) revealed activation characteristics specific to APZT treatment, including cellular stress and damage, exposure and release of DAMPs, sensing and alarm mechanisms of the innate immune system, and activation of adaptive immune responses. Figure 7 a). This further corroborates the reliability of the aforementioned experimental results. Differential metabolite pathway enrichment analysis based on the Kyoto Encyclopedia of Genetics and Genomes (KEGG) database showed significant enrichment of pathways related to 'cell growth and death', 'signal transduction', 'protein folding and degradation', and 'energy metabolism'. Figure 7(b) This enrichment pattern functionally links the cell death patterns observed in the aforementioned experiments to stress signaling, suggesting that APZT treatment may trigger immunogenic cell death by interfering with these core processes. Further analysis revealed that key metabolic sensing and regulatory pathways, including PPAR signaling and AMPK signaling, were significantly inhibited in the APZT treatment group, indicating that the energy homeostasis maintenance mechanisms of tumor cells were disrupted in response to treatment stress. This strongly supports the sustained and intense activation of unfolded protein responses in the calcium ion and ROS cycling pathways mentioned earlier. This persistent endoplasmic reticulum stress, exceeding cellular compensatory capacity, directly triggers the 'paraapoptotic death pathway' characterized by endoplasmic reticulum and mitochondrial vacuolation, pushing tumor cells towards an irreversible death endpoint. On the other hand, the treatment directly intervened in the oncogenic signaling network and may have improved the immune microenvironment. The downregulation of transcriptional misregulation in cancer directly confirms the reversal effect of treatment on oncogenic transcriptional programs. More importantly, the inhibition of the TGF-β signaling pathway has significant immunological implications; its weakened signaling suggests that the immunosuppressive state mediated by it, including T cell function suppression, regulatory T cell induction, and recruitment of myeloid-derived suppressor cells, may be relieved. This removes a key obstacle to the effective functioning of effector immune cells (such as cytotoxic T cells) activated via ICDs. Figure 7 c). In summary, APZT therapy not only initiates anti-tumor immunity by activating immunogenic death-related pathways (such as antigen presentation and cytokine storm) (Figure S), but also drives tumor cells towards immunogenic death at a deeper level by inhibiting key metabolic support, oncogenic signaling, and immunosuppressive pathways, ensuring the effective execution of the immune response. Furthermore, GO analysis confirmed that differentially expressed genes are mainly enriched in biological processes related to endoplasmic reticulum stress, mitochondrial dysfunction, calcium ion transport activation, immunogenic death, innate immune response, and adaptive immune response. Figure 7 d).

[0078] The aforementioned pathway analysis system revealed the biological functional landscape reshaped by APZT treatment. To anchor these functional changes to specific executive genes, this invention generated a heatmap of differentially expressed genes in key pathways. The results showed that genes such as the mitochondrial antioxidant enzyme SOD2 and the metabolic enzyme PYCR1 were significantly upregulated, while the expression of stress regulators such as HIF1A and FOXO1 was also significantly altered. These genes synergistically mapped to the process of 'cellular oxidative stress,' confirming at the gene expression level that APZT treatment successfully placed tumor cells in a strong redox crisis, laying the molecular basis for subsequent cell death. Gene set enrichment assay (GSVA) ​​was used to detect the enrichment of DEGS in gene set units to further reveal the biological mechanism of synergistic therapy. The results also showed that APZT treatment induced significant gene changes related to unfolded protein binding, calcium ion transport activity, Toll-like receptor signaling pathways, and immune responses. Furthermore, GSVA enrichment scores were performed on APZT and the control group, revealing that the APZT group had higher scores for calcium ion transport, immunogenic death, and T cell-mediated immunity than the control group. The above results indicate that APZT activates immunogenic death through photodynamic endoplasmic reticulum stress. Endoplasmic reticulum stress promotes intracellular calcium ion and ROS cycling, which in turn hinders cell self-repair. This process induces the paraapoptotic death pathway, ultimately leading to cell membrane rupture, leakage of contents, and irreversible cell death. It also triggers a strong anti-tumor immune response in tumors.

[0079] 10. In vivo antitumor effect of APZT in nude mouse models Prior to conducting in vivo tumor treatment studies, the biosafety of APZT was evaluated in healthy female BALB / c-nu mice. Mice were intravenously injected with 5 mg / kg APZT. During a 20-day health observation period, there was no significant difference in body weight between the APZT group and the control group. Blood parameters in the APZT group were not significantly different from those in the control group. Subsequently, after confirming successful model establishment, the efficacy of APZT in inhibiting tumor growth was evaluated using a U87-MG subcutaneous tumor-bearing nude mouse model. Treatment began 7 days after tumor inoculation and was repeated every 3 days; 1 hour after drug injection, the mice were irradiated with a 650 nm laser for 5 minutes. Figure 8a) During the 24-day treatment period, tumor volume and body weight of mice were measured every 2 days (Fig. 8b). As shown in Fig. 8c, the PBS group did not significantly inhibit tumor growth, and the free TMZ group showed moderate tumor-suppressive efficacy. The APZT group achieved significant tumor inhibition. In addition, the tumors excised from the mice were weighed, and the mass and volume showed similar trends (Fig. 8d). Digital images of the tumors further confirmed the significant tumor inhibition after APZT treatment (Fig. 8e). Simultaneously, the photothermal efficacy of APZT in subcutaneous tumors was evaluated in vivo. One hour after tail vein injection, the tumors were irradiated with a 650 nm laser (500 mW cm⁻²) for 5 minutes, and the temperature of the tumor area was recorded using an infrared thermal imaging camera (Fig. 8f). As shown in Fig. 8g, compared with the control group, the temperature of the tumor area treated with APZT was significantly enhanced by up to 50 °C, demonstrating highly efficient photothermal capabilities in vivo.

[0080] To further evaluate the efficacy of APZT against orthotopic brain tumors, U87-MG cells were injected into the right brain of 8-week-old BALB / c-nu mice. The antitumor efficacy of APZT was examined using the treatment regimen shown in Figure 8h. On day 10 post-implantation, GBM tumor mice received intravenous injections of PBS, free TMZ, and APZT, respectively. Brain tumor development was monitored every 7 days using IVIS. Compared with the PBS and free TMZ treatment groups, tumor growth in APZT-treated mice was slow. Importantly, free TMZ failed to elicit an effective antitumor response in mice carrying U87-MG tumors due to drug resistance mechanisms and insufficient brain accumulation. When its targeting motif was cleaved, the target peptide released by APZT was rapidly cleared from circulation, thereby interrupting LRP-1-mediated drug efflux and significantly prolonging the retention time of released chemotherapeutic agents and photosensitizers within the tumor, ultimately improving therapeutic efficacy. Bioluminescent quantitative results further confirmed that APZT significantly inhibited tumor proliferation. Importantly, APZT treatment significantly prolonged the survival of U87-MG tumor-bearing mice, which was longer than that of TMZ and control mice (Fig. 8i), and their body weight remained stable (Fig. 8j), consistent with the prolonged mouse survival. Whole-brain histological analysis using hematoxylin and eosin (H&E) staining showed that APZT resulted in the smallest tumor size among all treatments. Figure 8 This is consistent with bioluminescent imaging. H&E-stained major organ sections did not show significant damage caused by these nanoparticles, highlighting their excellent biocompatibility.

[0081] 11. APZT activates anti-tumor immune responses in tumor-bearing mice. To further demonstrate that APZT can induce personalized anti-tumor-specific immune responses, an orthotopic GBM model was established using C57BL / 6 immunocompetent mice. The drug treatment process is shown in Figure 9a. As expected, APZT, exhibiting the weakest tumor bioluminescence intensity, inhibited tumor cell growth (Figure 9b). Quantitative bioluminescence results showed that APZT had the strongest anti-tumor effect (Figure 9c). Notably, PBS-treated mice showed a sharp decrease in body weight, reflecting the increasing severity of damage as the brain tumor rapidly progresses. In contrast, APZT-treated mice showed a relatively mild decrease in body weight during treatment (Figure 9d). Notably, APZT prolonged the survival of mice; treated BALB / c-nu mice lived longer than control mice (Figure 9e). The effective tumor-suppressive effect of APZT was further confirmed by H&E staining of brain slices (Figure 9b). The significant therapeutic effect of nanomedicine on brain slices was confirmed by Ki-67 immunohistochemical staining and transferase-mediated dUTP nick-end labeling (TUNEL) staining (Fig. 9f). IHC consistently found that APZT induced HMGB1 efflux and surface CRT exposure in primary tumors of immunocompetent C57 mice—similar findings to those in nude mouse brain slices—thus confirming the induction of immunogenic cell death. Next, to determine whether these DAMPs induced dendritic cell phagocytosis and subsequent antigen presentation to activate cytotoxic T lymphocyte activation, the population of CD4+ / CD8+ T cells in the spleen was analyzed. As shown in Fig. 9g, the APZT group exhibited the highest proportion of CD4+ / CD8+ T cells in the spleen. These results indicate that APZT effectively activates cytotoxic T cells, thereby initiating a systemic immune response.

[0082] In addition to immune activation, the efficacy of APZT in inducing immune memory was also investigated. A 45-day immune re-challenge experiment compared the memory T cell responses and protective efficacy in immunocompetent C57BL / 6 mice and T-cell-deficient BALB / c-nu mice (Figure 9j). First, GL-261 cells were treated with APZT for 12 hours. Subsequently, the treated cells were injected into the left side of the mice. After 21 days of monitoring, fresh GL-261 cells were injected again into the right side of the mice. Tumor development was then monitored daily. Tumors were observed in the nude mice between days 8 and 10 post-re-challenge. Simultaneously, APZT delayed tumor development in C57 mice (Figure 9k). In summary, APZT significantly activated anti-tumor immunity and induced immune memory to prevent tumor recurrence.

[0083] 12. Tumor staging assessment using the MMP-2 responsive fluorescence activation properties of APZT. To adhere to the 3R principles of ethical research and the use of laboratory animals, mice with tumors of varying malignancy were selected from previous experiments to evaluate APZT's tumor staging ability and stimulus-response fluorescence activation performance. Initially, two nude mice with subcutaneous tumors were selected, exhibiting significant differences in tumor severity. Fluorescence signals were detectable 20 minutes after tail vein injection of APZT, peaking within one hour and then gradually decreasing (Fig. 10b). Comparative analysis between the two groups revealed a positive correlation between tumor malignancy and APZT fluorescence intensity (Fig. 10c), a finding further supported by HE staining results (Fig. 10d). To validate these findings, experiments were repeated using mice with tumors in situ. Tumor localization was first performed using bioluminescence imaging, followed by APZT injection. Varying degrees of fluorescence recovery were observed in vivo (Fig. 10e), and in vitro organ imaging showed that APZT exhibited strong tumor-targeting specificity, with effective disintegration occurring only at the tumor site (Fig. 10f). These experimental results confirm that APZT can undergo MMP-2-mediated stimulus-responsive disassembly, and the degree of disassembly is positively correlated with tumor malignancy, thereby enabling accurate tumor staging assessment. This was confirmed by the brain slice results of each group of mice (Figure 10g).

[0084] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0085] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A MMP-2-responsive targeted nanoprobe for the synergistic diagnosis and treatment of gliomas and immune activation, characterized in that, The nanoprobe includes micelle APZ and an anti-tumor drug, wherein the anti-tumor drug is encapsulated in the center of the micelle APZ, and the micelle APZ is self-assembled from monomers with the structural formula shown in (I); , Wherein, R is the amino acid sequence as shown in SEQ ID NO.

1.

2. The nanoprobe according to claim 1, characterized in that, The antitumor drug is at least one of temozolomide, carmustine, lomustine, semustine, procarbazine, vincristine, etoposide, teniposide, cisplatin, and carboplatin.

3. The nanoprobe according to claim 1, characterized in that, The nanoprobe has a hydrodynamic diameter of 10-50 nm and a zeta potential of 0.1-2.0 mV.

4. The nanoprobe according to claim 1, characterized in that, The antitumor drug in question is temozolomide.

5. The use of the nanoprobe according to any one of claims 1 to 4 in the preparation of a drug for the integrated diagnosis and treatment of glioma or in the preparation of a product for the staging assessment of glioma.

6. The application according to claim 5, characterized in that, The integrated diagnosis and treatment includes in vivo fluorescence imaging, photothermal therapy, photodynamic therapy, and chemotherapy.

7. The application of the APZ micelles of claim 1 in glial imaging of the brain.

8. The method for preparing the nanoprobe according to claim 1, characterized in that, Includes the following steps: (1) Dissolve I-BODIPY and azide-terminated polypeptide in DMSO, add CuSO4 and tris(3-hydroxypropyltriazolylmethyl)amine, then add aminoguanidine and sodium ascorbate in DMSO solution, and stir at room temperature for 20-40 min. (2) Add the antitumor drug dissolved in DMSO to the system obtained in (1), continue stirring and slowly add phosphate buffer, stir and then purify by gradient dialysis to obtain the nanoprobe; The structural formula of the I-BODIPY is as follows: 。 9. The method according to claim 8, characterized in that, The mass ratio of I-BODIPY to the azide-terminated polypeptide in step I is 1:2~5; the mass ratio of I-BODIPY to CuSO4 and tris(3-hydroxypropyltriazolylmethyl)amine is 1:0.01~0.02:0.20~0.30; the mass ratio of I-BODIPY to aminoguanidine and sodium ascorbate is 1:3~4:5~7; and the mass ratio of I-BODIPY to the antitumor drug is 1:2~5.

10. The method according to claim 8, characterized in that, The gradient dialysis purification was performed by using 30, 50, and 100 mM EDTA solutions for gradient dialysis, with each concentration dialyzed three times, followed by three dialysis cycles in ultrapure water.

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