A nano-aggregate based on near-infrared two-region aggregation-induced emission material and a preparation method and application thereof

Abstract

This invention proposes a nanoaggregate based on a near-infrared II aggregation-induced emission (AIE) material, its preparation method, and its application, belonging to the field of medical technology. The nanoaggregate of this invention is self-assembled from an AIE material and an amphiphilic carrier material, a DSPE-PEG series polymer. This nanoaggregate exhibits AIE properties in the near-infrared II region and can downregulate the expression of heat shock protein 70 through a proteasome-dependent mechanism. In vitro and in vivo experiments show that this nanoaggregate can achieve complete tumor ablation under low-temperature photothermal therapy at 43-45℃ without the need for combined use with exogenous heat shock protein 70 inhibitors, while simultaneously achieving near-infrared II fluorescence imaging guidance. This invention has the advantages of being single-component, low-toxicity, and integrating imaging and therapy, and has broad application prospects in the field of low-temperature photothermal therapy for tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a nano-aggregate based on near-infrared II aggregation-induced emission material, its preparation method and application. Background Technology

[0002] Low-temperature photothermal therapy (LT-PTT) is a tumor ablation strategy that operates under mild temperature conditions (typically below 45°C). Compared to traditional high-temperature photothermal therapy (typically above 50°C), LT-PTT significantly reduces nonspecific thermal damage to healthy tissue surrounding the tumor and decreases treatment-induced severe inflammatory responses, thus demonstrating good biocompatibility and promising prospects for clinical translation.

[0003] However, the efficacy of LT-PTT is severely limited by the intrinsic stress adaptation mechanisms of tumor cells. Under heat stress, tumor cells rapidly upregulate the expression of heat shock proteins (especially heat shock protein 70, HSP70). HSP70, as a key molecular chaperone, helps maintain protein homeostasis and inhibits apoptosis signaling pathways, thereby endowing tumor cells with strong thermolerance. This heat stress protective mechanism makes tumor cells resistant to mild heating (<45°C), severely weakening the therapeutic effect of LT-PTT. Therefore, effectively inhibiting the HSP70-related heat shock response is key to achieving highly efficient LT-PTT.

[0004] To overcome the heat resistance mediated by HSP70, existing technologies typically employ multi-component nanodelivery systems to co-deliver photothermal agents with HSP70 inhibitors (such as the small molecule inhibitor 17-AAG) or gene silencing agents (such as HSP70 siRNA). During laser irradiation and heat generation, the inhibitor or silencing agent is released to block the protective function of HSP70. For example, studies have constructed liposomes or polymer nanoparticles simultaneously loaded with photothermal agents and HSP70 inhibitors, attempting to achieve a synergistic effect of photothermal therapy and heat shock suppression.

[0005] However, the aforementioned multi-component co-delivery strategies suffer from several technical drawbacks in practical applications. First, due to the different physicochemical properties of photothermal agents and HSP inhibitors, their co-encapsulation leads to complex and unstable nanoparticle-based drug delivery systems, prone to problems such as premature drug leakage and significant batch-to-batch variations in loading rates. Second, the two components often exhibit different pharmacokinetic behaviors in vivo, making it difficult to synchronize release rates and tumor-targeting distribution, resulting in mismatched pharmacokinetics and reduced synergistic therapeutic effects. Furthermore, many traditional HSP70 inhibitors (such as 17-AAG) have poor water solubility and significant off-target toxicity, potentially causing systemic adverse reactions, further limiting their clinical translational potential.

[0006] Therefore, developing a single-component photothermal therapeutic agent that can simultaneously achieve efficient photothermal conversion and intrinsic HSP70 inhibition, thereby enhancing tumor sensitization to low-temperature photothermal activity without the need for combined exogenous inhibitors, is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a nano-aggregate based on near-infrared II aggregation-induced emission materials, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a nano-aggregate based on a near-infrared II aggregation-induced emission material, wherein the nano-aggregate is self-assembled from an aggregation-induced emission material (aDTP-TPA) and an amphiphilic carrier material; The aggregation-induced emission material is a compound with a donor-acceptor-donor structure, and its structural formula is: ; The amphiphilic carrier material is selected from the DSPE-PEG series polymers.

[0009] Furthermore, the aggregation-induced emission material uses benzobisthiadiazole as the core acceptor unit and triphenylamine as the terminal donor unit, and the two are connected by a dialkyl-substituted dithiophene-pyrrole π-bridge.

[0010] Furthermore, the mass ratio of the aggregation-induced emission material to the amphiphilic carrier material is 1:3.

[0011] Furthermore, the amphiphilic carrier material is DSPE-PEG-2000.

[0012] The present invention also provides a method for preparing the above-mentioned nanoaggregates based on near-infrared II aggregation-induced emission materials, comprising the following steps: (1) The aggregation-induced light-emitting material and the amphiphilic polymer are dissolved together in an organic solvent to form an organic phase; (2) Under ultrasonic conditions, the organic phase is added dropwise to the aqueous phase, and self-assembly occurs to form an emulsion; (3) Remove the organic solvent from the emulsion to obtain the nano-aggregates based on near-infrared II aggregation-induced emission materials.

[0013] Furthermore, the volume ratio of the organic solvent to the aqueous phase is 1:10.

[0014] Furthermore, the ratio of the organic solvent to the aggregation-induced luminescence material is 1 mL: 1 mg.

[0015] Furthermore, in step (3), the organic solvent is removed by evaporation of nitrogen gas.

[0016] Furthermore, it also includes a step of sterile filtration using a 0.22 μm filter membrane.

[0017] The present invention also provides the application of the above-mentioned nanoaggregates based on near-infrared II aggregation-induced emission materials in the preparation of drugs for low-temperature photothermal therapy of tumors.

[0018] Furthermore, the hypothermic photothermal therapy is a single-agent therapy without the combination of exogenous heat shock protein 70 inhibitors.

[0019] Furthermore, the treatment temperature of the low-temperature photothermal therapy is 43℃-45℃.

[0020] Furthermore, the nanoaggregates are used to perform the low-temperature photothermal therapy under the guidance of near-infrared II fluorescence imaging.

[0021] Furthermore, the low-temperature photothermal therapy includes the following steps: (a) Applying the nanoaggregates to a subject; (b) Using the near-infrared II aggregation-induced emission properties of the nanoaggregates, fluorescence imaging of the tumor site is performed to determine the peak time of drug enrichment; (c) During the peak drug enrichment time, the tumor site is irradiated with a laser to control the local temperature of the tumor at 43-45°C, thereby achieving tumor ablation.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects: (1) Compared with the existing multi-component nanodelivery system that requires the co-encapsulation of photothermal agents with HSP70 inhibitors or gene silencing agents, the present invention provides a nanoaggregate based on a single-component near-infrared II aggregation-induced emission material (aDTP-TPA), which realizes the combination of photothermal conversion and HSP70 downregulation function, and completely avoids the system complexity, pharmacokinetic mismatch and off-target toxicity of exogenous inhibitors caused by multi-component co-delivery.

[0023] (2) This invention significantly improves the safety and imaging-guided precision of tumor treatment. Due to the use of a single-component strategy, the systemic toxicity and off-target effects of traditional HSP inhibitors (such as 17-AAG) are avoided. At the same time, low-temperature photothermal therapy significantly reduces the risk of thermal damage to surrounding healthy tissues compared to traditional high-temperature photothermal therapy (>50°C). Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The following are the performance test results of aDTP-TPA and aDTP-TPA NAs used in Example 1, where a represents the chemical structure of aDTP-TPA and the DFT-optimized ground-state geometry and key dihedral angles. b shows the relationship between the calculated recombination energy and the normal mode wavenumber; the inset shows the contributions of bond length, bond angle, and dihedral angle. c shows the normalized absorption and photoluminescence spectra of aDTP-TPA in THF. d shows the PL spectra of aDTP-TPA in THF / water mixtures with different water volume fractions. e shows the photostability of aDTP-TPA NAs: the normalized NIR-II fluorescence intensity of aDTP-TPA NAs and ICG under continuous laser irradiation; the inset shows representative NIR-II images of aDTP-TPA NAs and ICG. f shows the photothermal stability of aDTP-TPA NAs and ICG after five laser on / off cycles. g shows the temperature rise curves of aDTP-TPA NAs at different NA concentrations. h represents the heating curves of aDTP-TPA NAs at different power densities. i represents the linear fit of the cooling data used to calculate the photothermal conversion efficiency (η).

[0025] Figure 2 The results of particle size and morphology characterization of aDTP-TPA NAs are shown in the inset, with TEM images as the inset. Figure 3 The results of particle size stability determination for aDTP-TPA NAs are shown in the inset, which includes solar and fluorescence photographs of the nanoparticles. Figure 4Normalized absorption / PL spectra of aDTP-TPA NAs; Figure 5 The following are spectroscopic measurements of the relative fluorescence quantum yield (PLQY) of aDTP-TPA NAs using IR26 as a reference. (A) Absorption spectra of reference dye IR26 at different concentrations (6.70 - 20.0 μmol / L). (B) Fluorescence emission spectra of reference dye IR26 at different concentrations. (C) Linear fitting plot of the integrated fluorescence intensity of IR26 and its absorbance at 808 nm. (D) Absorption spectra of aDTP-TPA NAs at different concentrations (18.0 - 36.1 μmol / L). (E) Fluorescence emission spectra of aDTP-TPA NAs at different concentrations. (F) Linear fitting plot of the integrated fluorescence intensity of aDTP-TPA NAs and its absorbance at 808 nm. Figure 6 The results of in vitro LT-PTT efficacy and HSP70 downregulation experiments in 4T1 cells are shown. a) Calcein-AM / PI stained CLSM images after different treatments. b) Cell viability after incubation with different concentrations of aDTP-TPA NAs with or without 808 nm irradiation. c) CLSM immunofluorescence images of cell nuclei stained with HSP70 and DAPI. Scale bar: 50 μm. d) Corresponding to… Figure 6 Quantitative analysis of average fluorescence intensity in c. e represents Western blot analysis of HSP70; GAPDH was used as an internal control for sample loading. f represents quantitative analysis of HSP70 optical density after GAPDH normalization.

[0026] Figure 7 Flow cytometry analysis results of 4T1 cells co-stained with Calcein-AM / PI after different treatments; Figure 8 The results of the study on the aDTP-TPA-mediated downregulation mechanism of HSP70 are shown below. a) Western blot analysis of HSP70 in 4T1 cells after treatment with aDTP-TPA NAs for 0-36 hours. b) Western blot analysis results after incubation with different concentrations of aDTP-TPA NAs for 24 hours. c) The docking model of aDTP-TPA with HSP70 (PDB: 4PO2). d) Western blot analysis results of HSP70 in cells with and without MG-132. e) Quantitative analysis of HSP70 optical density after GAPDH normalization.

[0027] Figure 9The results of Western blot analysis of HSP70 protein in 4T1 cells treated with aDTP-TPA (dissolved in DMF) for different times and concentrations are shown in Figure a. The results of Western blot analysis of HSP70 in 4T1 cells after treatment with aDTP-TPA for 0-36 hours are shown in Figure b. The results of Western blot analysis of HSP70 in 4T1 cells after incubation with different concentrations of aDTP-TPA for 24 hours are shown in Figure b. Figure 10 The chemical structures of the reference molecules (T-TPA and 3T-TPA) are shown. Figure 11 The results of the Western blot analysis of HSP70 protein levels in 4T1 cells after treatment with T-TPA and 3T-TPA are shown in the following figures: a) Western blot analysis of HSP70 in 4T1 cells after treatment with T-TPA and 3T-TPA for 0-36 hours; b) Western blot analysis of HSP70 in 4T1 cells after treatment with T-TPA NAs and 3T-TPA NAs for 0-36 hours; c) Western blot analysis results after incubation with different concentrations of T-TPA and 3T-TPA for 24 hours; d) Western blot analysis results after incubation with different concentrations of T-TPA NAs and 3T-TPA NAs for 24 hours. Figure 12 Western blot (a) and quantitative analysis (b) results of HSP70 expression in 4T1 cells after treatment with CQ (chloroquine); Figure 13 This section presents the results of in vivo NIR-II imaging-guided cryophotothermal therapy and its mechanism validation. A) shows a schematic timeline of tumor inoculation, intravenous administration of aDTP-TPA NAs, imaging, LT-PTT, and biochemical / histochemical analysis in mice bearing 4T1 tumors. B) shows in vivo NIR-II fluorescence images acquired at specified time points after intravenous injection of aDTP-TPA NAs, reflecting changes over time. C) shows the quantitative analysis results of NIR-II fluorescence intensity in the tumor region over time. D) shows the ex vivo biodistribution of major organs and tumors collected 48 hours after injection; the inset shows the corresponding NIR-II fluorescence images. E) shows representative infrared thermal images during 808 nm laser irradiation. F) shows real-time tumor temperature curves during irradiation. G) shows tumor growth curves after 11 days following different treatments. H) shows a photograph of tumor resection on day 11; dashed circles indicate no tumor masses were observed in the aDTP-TPA NAs+NIR group. i shows representative immunofluorescence images of HSP70 (red) and cell nuclei (DAPI, blue) staining in tumor sections, scale bar: 100 μm. j shows the quantitative analysis results of relative HSP70 fluorescence intensity in tumor sections.

[0028] Figure 14 Changes in relative body weight of mice in different treatment groups; Figure 15 Images of H&E stained sections of the heart, liver, spleen, lungs, and kidneys collected for the four treatment groups. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise stated, all chemical reagents used in this invention were purchased from certified commercial suppliers and used directly without further purification. DSPE-PEG-2000 was purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). The CCK-8 assay kit, cell viability / cytotoxicity assay kit (Calcein AM / PI), and Annexin V-FITC / PI apoptosis assay kit were provided by Beyotime Biotechnology. Anti-Hsp70 antibody (Proteintech, 10995-1-AP), anti-GAPDH antibody (Proteintech, 60004-1-Ig), HRP-labeled goat anti-rabbit IgG (H+L) (Proteintech, SA00001-2), DAPI (Invitrogen, D3571), MG-132 (MCE, HY-13259), CQ (chloroquine, MCE, HY-17589A), DMEM medium (Gibco, C11965500BT), FBS (fetal bovine serum, MIKX, MK1123-500C), 0.25% trypsin-EDTA (Gibco, 25200-072), penicillin-streptomycin mixture (10,000 U / mL, Gibco, 15140122). Eight-week-old female BALB / c mice were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. All other chemicals were purchased from Dieckmann (Hong Kong) Chemical Co., Ltd. and used directly without further purification. Solvents used in synthesis were purchased from VWR Chemicals and used directly without further purification.

[0035] In the performance testing section, absorption spectra were acquired using a PerkinElmer LAMBDA 365 UV / Vis spectrometer; photoluminescence (PL) spectra were measured using a HORIBA Fluoro-Max-4 fluorescence spectrometer. Dynamic light scattering (DLS) data were recorded using a Zetasizer Nano ZS (Malvern, UK). Nuclear magnetic resonance (NMR) spectra were measured using a Bruker AVIII 400 MHz NMR spectrometer. Mass spectrometry data were acquired using a Water's Xevo G2-XS Top instrument. Transmission electron microscopy (TEM) images were collected on a FEI-TALOS-F200X. Confocal images were captured using a laser scanning confocal microscope (Leica). Thermal images were recorded using a FLIR T420 infrared thermal imager. Single-cell suspensions were detected using a CytoFLEX (Beckman) instrument. In vivo fluorescence imaging was acquired using an in vivo fluorescence imaging system from Yingrui Suzhou NIR-Optics Technology Co., Ltd.

[0036] The aggregation-induced emission material (aDTP-TPA) used in the embodiments of the present invention was prepared according to the following method: Under nitrogen (N2) protection, anhydrous toluene (5 mL) was added to a mixture of 4-(4,5-dihexyl-6-(trimethyltinyl)-4H-dithienro[3,2-b:2',3'-d]pyrrolo-2-yl)-N,N-diphenylamine (278 mg, 0.36 mmol), 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (50 mg, 0.14 mmol) and Pd(PPh3)2Cl2 (35 mg, 0.014 mmol). The reaction mixture was stirred at 110 °C for 12 hours. Then, the reaction mixture was cooled and poured into an aqueous solution of KF (potassium fluoride). The mixture was extracted three times with diethyl ether, and the organic phases were combined, washed successively with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 1:1) to give a brown solid product (146 mg, yield 75%), which is the aggregation-induced emission material (aDTP-TPA). The specific reaction procedure is as follows: .

[0037] The technical solution of the present invention will be further illustrated by the following embodiments.

[0038] Example 1 A method for preparing nanoaggregates (aDTP-TPA NAs) based on near-infrared II aggregation-induced emission materials includes the following steps: Weigh aDTP-TPA (1 mg) and DSPE-PEG-2000 (3 mg) in a mass ratio of 1:3, dissolve them in 1 mL of tetrahydrofuran (THF), mix the solutions under continuous sonication, and gradually add them dropwise into 10 mL of ultrapure water. Sonicate the emulsion, then completely remove the solvent under a nitrogen stream. The resulting dispersion is sterilely filtered (0.22 μm) and concentrated to obtain an aDTP-TPA NAs solution (1 mg / mL), which is stored at 4 °C for later use.

[0039] Performance testing methods Test subjects: aDTP-TPA NAs or aDTP-TPA in Example 1.

[0040] Specific testing methods: (1) Calculation of quantum yield of aDTP-TPA nanoaggregates To determine the quantum yield of aDTP-TPA nanoaggregates, the aDTP-TPA NAs solution (1 mg / mL) from Example 1 and a 2 mM IR-26 solution were used. Equal volumes of these two solutions were then added to their respective solvents, with a final volume of 3 mL. The absorbance of the aDTP-TPA nanoaggregates and the IR-26 solution was measured at a specified excitation wavelength using a spectrophotometer, and the absorbance data were carefully recorded. Subsequently, both solutions were uniformly excited at this wavelength, and their respective fluorescence emission spectra were recorded. The integrated fluorescence intensity (i.e., the area under each emission curve) was calculated as a key step in assessing their quantum yield. The reference quantum yield of IR-26 was 0.05%. QY was then calculated using the formula (see original formula), where θsample and θref represent the refractive indices of DCE and THF, respectively.

[0041] (2) Photothermal activity and photothermal conversion efficiency To evaluate the stability of aDTP-TPA NAs, solutions of aDTP-TPA NAs and ICG at the same concentration were exposed to 808 nm laser irradiation and subjected to repeated heating-cooling cycles, with photothermal curves recorded. To evaluate the photothermal performance of aDTP-TPA NAs, a laser irradiation of 0.15 W / cm² was used. 2 Solutions with concentrations of 42, 84, 126, 168, and 210 μg / mL were prepared under the same laser intensity. All samples were exposed to an 808 nm laser under uniform conditions, and thermal distribution and images were captured using a thermal imager. At the same concentration of 50 μg / mL, the effects of 0.15, 0.25, 0.35, 0.45, and 0.55 W / cm² were investigated. 2Effect of different laser intensities. To measure the photothermal conversion efficiency, aDTP-TPA nanoaggregates (50 μg / mL) were irradiated with an 808 nm laser for 300 seconds, and then the solution was cooled to room temperature. During this process, the solution temperature was recorded every 1 second. The photothermal conversion efficiency (η) was calculated according to formula (1), and the final measured photothermal conversion efficiency of aDTP-TPANAs was 39.7%.

[0042] Formula (1) in, h A Let A be the heat transfer coefficient and A be the surface area of ​​the container. T Max The highest steady-state temperature, T Surr For ambient temperature, Q Dis The solvent and the quartz sample cell absorb the heat generated by the light. I The incident laser power, A The absorbance of the sample at 808 nm is given.

[0043] (3) CCK8 detection Biocompatibility of aDTP-TPA NAs was assessed in 293T and 4T1 cells, respectively. Cells were cultured in 96-well plates (8 × 10⁶ cells per well). 3 Cells were added to 100 μL of DMEM medium. Continuous concentrations (0, 20, 40, 60, 80, 100, 150, and 200 μg / mL) of aDTP-TPA NAs were added to the medium, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After another 24 hours of incubation, CCK-8 reagent was added, and the cells were analyzed using a microplate reader 1.5 hours later. To investigate the cryogenic killing effect of aDTP-TPA NAs on 4T1 and 293T cells, different concentrations of aDTP-TPA NAs were added. The groups irradiated at 808 nm were monitored using an infrared camera, and the temperature was controlled at 44°C. The experiment was divided into four groups: (1) 4T1-aDTP-TPA NAs (NIR-); (2) 4T1-aDTP-TPA NAs (NIR+); (3) 293T-aDTP-TPA NAs (NIR-); (4) 293T-aDTP-TPA NAs (NIR+).

[0044] (4) Calcein-AM / PI staining of live / dead cells 4T1 cells were loaded at a rate of 1×10⁴ cells per well. 5Cells were seeded at a concentration of 100 μg / mL in 48-well plates. After incubation for 12 hours, aDTP-TPA NAs were added and exposed for 24 hours. The experiment was divided into five groups: (1) PBS (NIR-), (2) PBS (NIR+), (3) aDTP-TPA NAs (NIR-), (4) aDTP-TPA NAs (NIR+), and (5) 44°C. Subsequently, the cells were irradiated with an 808 nm laser for 15 minutes and the temperature was maintained at 44°C using an infrared thermal imager. Cell viability was assessed by co-staining with propidium iodide (PI) and Calcein-AM for 30 minutes. Fluorescence microscopy analysis was performed using specific excitation and emission settings to quantify live and dead cell populations.

[0045] (5) Annexin V-FITC / PI staining of apoptotic / necrotic cells The experimental grouping and treatment were basically the same as those for live / dead staining described above. After irradiation and maintenance at 44°C for 15 minutes, the cells were cultured for another 24 hours, the culture medium was discarded, and Annexin V-FITC and PI were added for staining. The cells were immediately observed under a fluorescence microscope. For flow cytometry analysis, the cells were digested, centrifuged, stained using the same procedure, and then analyzed by flow cytometry.

[0046] (6) Western blot 4T1 cells were seeded and cultured in six-well plates. Five experimental groups were set up: (1) PBS (NIR-), (2) PBS (NIR+), (3) aDTP-TPA NAs (NIR-), (4) aDTP-TPA NAs (NIR+), and (5) 44℃. After incubation for 12 hours with fresh medium containing 100 μg / mL aDTP-TPA NAs, the cells were treated with 808 nm laser, with group (4) maintained at 44℃. Samples were collected 6 hours after treatment. In the concentration gradient experiment, the concentration was set from 0 to 100 μg / mL. In the time gradient experiment, cells were collected from 0 to 36 hours. Subsequently, total protein was extracted from the collected cells, quantified by BCA method, and then subjected to SDS-PAGE electrophoresis and PVDF membrane transfer. After blocking, the cells were incubated with anti-HSP70 primary and secondary antibodies in sequence, and finally the protein bands were detected by enhanced chemiluminescence (ECL+) system.

[0047] (7) Immunofluorescence staining 4T1 cells were seeded and treated in confocal culture dishes. After laser irradiation (maintained at 44°C), cells were fixed with 4% paraformaldehyde and blocked with 5% BSA. After incubation with anti-HSP70 antibody at room temperature for 1 hour, the cells were washed and incubated with secondary antibody labeled with 488 fluorescence. Finally, the cell nuclei were stained with DAPI for 5 minutes. Fluorescence images were acquired using CLSM.

[0048] (8) Molecular docking of aDTP-TPA with HSP70 The X-ray crystal structure of 4PO2 (PDB:4PO2) was retrieved from protein databases. Docking simulations were performed using OpenBabel, AutoDockTools (ADT3), and AutoDockVina. The optimal pose was selected to analyze interactions, and protein-ligand interaction maps were generated using PyMOL.

[0049] (9) Tumor model All procedures were approved by the Animal Ethics and Welfare Committee of the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. A 4T1 tumor model was established by subcutaneous injection of 4T1 cells into BALB / c mice. The tumor volume reached approximately 100 mm. 3 In vivo experiments were conducted at that time.

[0050] (10) NIR-II imaging aDTP-TPA NAs (100 μL, 500 μg / mL) were injected via tail vein. NIR-II region imaging was performed at different time points (0-48 hours) using a near-infrared camera equipped with a 1000-1500 nm filter, with an 808 nm laser as the excitation source. Major organs and tumors were collected after 48 hours for ex vivo imaging.

[0051] (11) Temperature monitoring and photothermal therapy 24 hours after intravenous injection (imaging data showed the most significant drug enrichment at this time), an 808 nm laser (1W / cm²) was used. 2 The tumor site was irradiated, and the local temperature was monitored using an infrared thermal imager and maintained at 43℃-45℃ for 10 minutes. After treatment, the tumor size and body weight of the mice were recorded.

[0052] (12) In vivo toxicity assessment On day 12 post-injection, mice were euthanized and major organs (heart, liver, spleen, lung, kidney, and tumor) were collected, fixed, sectioned, and stained with H&E. Tumor tissue was frozen sectioned and stained with HSP70 antibody for immunofluorescence. In addition, blood was collected for biosafety marker analysis (WBC, ALT, AST, etc.).

[0053] (13) Statistical analysis All experiments were repeated at least three times, and results are expressed as mean ± standard deviation (SD). (See figure) , , These represent P<0.05, P<0.01, and P<0.001, respectively.

[0054] Performance test results 1. Basic Representation Figure 1 The following are the performance test results of aDTP-TPA and aDTP-TPA NAs used in Example 1, where a is a schematic diagram of the chemical structure of aDTP-TPA and the DFT-optimized ground-state geometry and key dihedral angles. b shows the relationship between the calculated recombination energy and the normal mode wavenumber; the inset shows the contributions of bond length, bond angle, and dihedral angle. c shows the normalized absorption (UV) and photoluminescence (PL) spectra of aDTP-TPA in THF (tetrahydrofuran). d shows the performance of aDTP-TPA at different water volume fractions (f w PL spectrum of THF / water mixture.

[0055] like Figure 1 As shown in Figure a, aDTP-TPA has a donor-acceptor-donor (DAD) structure, consisting of triphenylamine (TPA, the donor) and benzobisthiadiazole (BBT, the acceptor), connected by a dialkyl-substituted dithienro[3,2-b:2′,3′-d]pyrrole (aDTP) π-bridge. This design introduces steric hindrance and produces a conformationally distorted framework. Density functional theory (DFT) optimization yielded a dihedral angle of 50.4° between the BBT and DTP bridge and 26.6° between the DTP and TPA, consistent with the nonplanar structure that suppresses intermolecular π-π stacking and mitigates aggregation-induced luminescence quenching (ACQ). To reasonably explain the nonradiative energy dissipation associated with photothermal conversion, a recombination energy decomposition analysis was performed. Figure 1 As shown in Figure b), excited-state relaxation is mainly dominated by intramolecular motion, with torsional contribution accounting for 49.04% of the total recombination energy, exceeding the components of bond angle (32.57%) and bond length (18.39%). These results indicate that torsional relaxation provides an efficient non-radiative decay pathway, which is beneficial for photothermal heat generation. Figure 1 As shown in Figure c, aDTP-TPA exhibits a maximum absorption at 823 nm, with an absorption tail extending to approximately 1000 nm, enabling efficient excitation in the near-infrared region. The emission peak is located at 1106 nm, supporting its application in NIR-II fluorescence imaging. AIE characteristics were verified in a THF / water mixture. Figure 1 In the middle (d), due to active intramolecular motion, in pure THF (f) w The PL signal is weak in (=0); while increasing the water volume fraction to f w When the concentration is 90%, it leads to an approximately 10-fold increase in PL, which is consistent with the mechanism of restricted intramolecular motion (RIM) in the aggregated state.

[0056] 2. Characterization of Nanoaggregates (NAs) For biological applications, in Example 1, aDTP-TPA was mixed with DSPE-PEG-2000 to obtain water-dispersible nanoaggregates (aDTP-TPA NAs). The resulting NAs were spherical in shape with an average hydrodynamic diameter of approximately 140 nm. Figure 2 The inset shows the particle size and morphology characterization results of aDTP-TPA NAs, with TEM images, and they exhibit good colloidal stability for at least 13 days. Figure 3 The results of particle size stability measurements for aDTP-TPA NAs are shown in the inset, which includes solar and fluorescence photographs of the nanoparticles. Simultaneously, measurements revealed that aDTP-TPA NAs retained the NIR-II optical characteristics of aDTP-TPA. Figure 4 The normalized absorption / PL spectrum of aDTP-TPA NAs is shown. The PL quantum yield (ΦPL) of aDTP-TPA NAs is 0.61%. Figure 5 The graphs show the relative fluorescence quantum yield (PLQY) of aDTP-TPA NAs with IR26 as a reference. A represents the absorption spectra of the reference dye IR26 at different concentrations (6.70–20.0 μmol / L). B represents the fluorescence emission spectra of the reference dye IR26 at different concentrations. C is a linear fit between the integrated fluorescence intensity (PL intensity) of IR26 and its absorbance at 808 nm. D represents the absorption spectra of aDTP-TPA NAs at different concentrations (18.0–36.1 μmol / L). E represents the fluorescence emission spectra of aDTP-TPA NAs at different concentrations. F is a linear fit between the integrated fluorescence intensity of aDTP-TPA NAs and its absorbance at 808 nm. The fluorescence quantum yield of aDTP-TPA NAs is higher than that of IR26.

[0057] Figure 1 In Figure e, the photostability test results of aDTP-TPA NAs and indocyanine green (ICG) are shown, revealing the normalized NIR-II fluorescence intensity of aDTP-TPA NAs and ICG under continuous laser irradiation. The inset shows representative images of DTP-TPA NAs and ICG. Figure f shows the photothermal stability of aDTP-TPA NAs and ICG after five laser on / off cycles. Figure g shows the temperature rise curves of aDTP-TPA NAs at different NA concentrations. Figure h shows the temperature rise curves of aDTP-TPA NAs at different power densities. Figure i shows the linear fit of the cooling data used to calculate the photothermal conversion efficiency (η). It can be seen that compared to indocyanine green (ICG), aDTP-TPA NAs exhibit significantly improved photostability under continuous irradiation. Figure 1 (e), and maintain a consistent heating curve during repeated laser on / off cycles ( Figure 1 (f). Temperature increase depends on NA concentration ( Figure 1 (g) and power density ( Figure 1 (h). Based on cooling analysis ( Figure 1 (i), where η was determined to be 39.70%. Taken together, these results establish aDTP-TPA NAs as a robust NIR-II photothermal diagnostic platform for image-guided LT-PTT.

[0058] 3. In vitro cell experiments The efficacy of in vitro photothermal therapy is based on its excellent optical and photothermal properties. The performance of aDTP-TPANAs was evaluated in 4T1 cells. Cell viability was assessed by confocal laser scanning microscopy (CLSM) using Calcein-AM (live cells, green) and propidium iodide (PI, dead cells, red) co-staining.

[0059] Figure 6 The results of in vitro LT-PTT efficacy and HSP70 downregulation experiments in 4T1 cells.

[0060] Figure 6 Image a shows CLSM images after Calcein-AM / PI staining under different treatments (scale bar: 200 μm). It can be seen that the 44℃ heating control group showed very little cell death, while the aDTP-TPA NAs+NIR group showed significant cell death. In the absence of laser irradiation, aDTP-TPA NAs exhibited negligible dark toxicity, displaying dominant green fluorescence. To simulate LT-PTT conditions, the laser power density was adjusted via feedback control (monitored by an optical power meter) to maintain a constant temperature of 44℃ during irradiation. Under these mild heating conditions, the 44℃ PBS control group resulted in very little cell death, consistent with its resistance to mild high temperatures. In contrast, treatment with aDTP-TPA NAs and irradiation at 808 nm (0.25 W cm⁻¹) significantly reduced cell death. -2 Cells irradiated for 15 minutes (at a temperature maintained at approximately 44 °C) showed obvious PI staining, indicating sensitization to mild heating.

[0061] Figure 6 In Figure b, cell viability was determined after incubation with different concentrations of aDTP-TPA NAs with and without 808 nm irradiation. Quantitative CCK-8 assays further confirmed the concentration-dependent phototoxicity under irradiation, even at concentrations as high as 40 μg / mL. -1 It maintains low dark toxicity at concentrations of [specific concentration].

[0062] Figure 7The flow cytometry (Annexin V-FITC / PI) analysis results of 4T1 cells co-stained with Calcein-AM / PI after different treatments show that the total apoptosis rate of the aDTP-TPA NAs + NIR group was 83.7%.

[0063] The above data demonstrate that aDTP-TPA NAs can achieve highly efficient tumor cell killing under mild thermotherapy.

[0064] 4. HSP70 downregulation mechanism (in vitro) Figure 6 Image c shows a CLSM immunofluorescence image of HSP70 (green) and DAPI-stained cell nuclei (blue). Scale bar: 50 μm. Image d corresponds to... Figure 6 Quantitative analysis of mean fluorescence intensity (MFI) of HSP70 (mean ± standard deviation, n=3). e is Western blot analysis of HSP70; GAPDH was used as an internal control for sample loading. f is quantitative analysis of HSP70 optical density after GAPDH normalization (mean ± standard deviation, n=3).

[0065] The mechanism of HSP70 downregulation: HSP70 is rapidly induced by thermal stress and contributes to heat resistance under mild high temperatures. Therefore, we examined whether aDTP-TPA NAs regulate HSP70 accumulation during LT-PTT. Figure 6 The results showed that immunofluorescence staining revealed a strong increase in HSP70 signal upon heating at 44°C, while under similar mild heating conditions, the aDTP-TPA + NIR group exhibited a significantly reduced HSP70 fluorescence. Figure 6 (c, d). Western blot analysis further supports these observations. Figure 6 (e, f): Heating at 44 °C alone significantly increased HSP70 protein levels, while aDTP-TPA treatment attenuated this upregulation during irradiation. Notably, aDTP-TPA also reduced basal HSP70 abundance in the absence of irradiation (NIR−), indicating an intrinsic HSP70 downregulation effect beyond photothermal heating. Taken together, these data suggest that aDTP-TPA NAs sensitize tumor cells to LT-PTT by inhibiting HSP70-related heat shock protection.

[0066] Figure 8 This study investigated the mechanism of HSP70 downregulation mediated by aDTP-TPA NAs, where a represents the amount of aDTP-TPA NAs (100 μg / mL). -1Western blot analysis of HSP70 in 4T1 cells after 0-36 hours of treatment showed a time-dependent decrease; GAPDH was used as an internal control for sample loading. b represents the concentrations of aDTP-TPA NAs (0-100 μg / mL) with different concentrations. -1 Western blot analysis after 24 hours of incubation showed dose dependence. c shows the docking model of aDTP-TPA with HSP70 (PDB: 4PO2), highlighting the predicted binding pocket near Lys451. d shows Western blot analysis of HSP70 in cells treated as instructed, with or without MG-132, supporting the involvement of the proteasome. e shows the optical density quantification of HSP70 normalized to GAPDH (mean ± standard deviation, n = 3).

[0067] Figure 8 The results showed that when aDTP-TPA NAs (100 μg mL) were used... -1 After treatment, Western blot showed that the abundance of HSP70 decreased in a time-dependent manner, reaching a maximum decrease of 42% at 24 hours. Figure 8 (a) Further dose increases over 24 hours showed a concentration-dependent effect, at 100 μg / mL. -1 HSP70 decreased by up to 64% ( Figure 8 (b) To determine whether the activity originates from the molecular structure or is due to the formation of nanoaggregates (NAs), parallel experiments were performed using molecular state aDTP-TPA dissolved in DMF. Subsequent molecular docking using HSP70 (PDB: 4PO2) showed that aDTP-TPA can be accommodated in a pocket near Lys451, with an estimated binding energy of -5.4 kcal / mol. -1 ( Figure 8 (c), which provides a structural hypothesis for target binding. To investigate the clearance pathway, pathway inhibitors were used, and the proteasome inhibitor MG-132 largely restored HSP70 levels in cells treated with aDTP-TPA ( Figure 8 (d, e).

[0068] Figure 9Western blot analysis results of HSP70 protein in 4T1 cells treated with aDTP-TPA (dissolved in DMF) at different times and concentrations are presented. In Figure a, Western blot analysis of HSP70 in 4T1 cells after 0-36 hours of treatment with aDTP-TPA is shown. In Figure b, Western blot analysis results after 24 hours of incubation with different concentrations of aDTP-TPA are presented. It can be seen that molecular aDTP-TPA produced a similar time- and concentration-dependent decrease in HSP70, and exhibited a larger maximum decrease (up to 85%) than aDTP-TPA NA formulations. These results demonstrate that aDTP-TPA itself is the cause of HSP70 downregulation.

[0069] Figure 10 To obtain the chemical structures of reference molecules (T-TPA and 3T-TPA), T-TPA (or 3T-TPA) (1 mg) and DSPE-PEG-2000 (3 mg) in a mass ratio of 1:3 were weighed and dissolved in 1 mL of tetrahydrofuran (THF). The solutions were mixed under continuous sonication and gradually added dropwise to 10 mL of ultrapure water. The emulsion was then sonicated and the solvent was completely removed under a nitrogen stream. The resulting dispersion was sterilely filtered (0.22 μm) and concentrated to obtain T-TPA NPs (or 3T-TPA NPs) (solution (1 mg / mL), which was stored at 4 °C for subsequent testing.

[0070] Figure 11 The results of the Western blot analysis of HSP70 protein levels in 4T1 cells after treatment with T-TPA and 3T-TPA are shown in the following figures: a) Western blot analysis of HSP70 in 4T1 cells after treatment with T-TPA and 3T-TPA for 0-36 hours; b) Western blot analysis of HSP70 in 4T1 cells after treatment with T-TPA NAs and 3T-TPA NAs for 0-36 hours; c) Western blot analysis results after incubation with different concentrations of T-TPA and 3T-TPA for 24 hours; and d) Western blot analysis results after incubation with different concentrations of T-TPA NAs and 3T-TPA NAs for 24 hours.

[0071] It can be seen that despite sharing related skeletal motifs, neither of these analogues caused a detectable decrease in HSP70 abundance in 4T1 cells, whether tested as molecular solutions or nanoparticle formulations. This contrast highlights a strict structure-activity relationship and is consistent with selective regulation of HSP70 abundance rather than a nonspecific protein homeostatic stress response.

[0072] Figure 12The results of Western blot (a) and quantitative analysis (b) of HSP70 expression in 4T1 cells treated with CQ (chloroquine) show that the lysosomal inhibitor chloroquine did not prevent the reduction of HSP70.

[0073] In summary, the above results support the conclusion that the decrease in HSP70 abundance induced by aDTP-TPA is based on a proteasome-dependent mechanism.

[0074] 5. In vivo imaging and treatment Figure 13 Results of in vivo NIR-II imaging-guided cryophotothermal therapy and its mechanism validation. Table a shows a schematic timeline of tumor inoculation, intravenous administration of aDTP-TPA NAs, imaging, LT-PTT, and biochemical / histochemical analysis in mice bearing 4T1 tumors. Table b shows in vivo NIR-II fluorescence images over time at specified time points (0-48 hours) after intravenous injection of aDTP-TPA NAs. Table c shows quantitative analysis of NIR-II fluorescence intensity in the tumor region over time, showing the highest enrichment at 24 hours post-injection. Table d shows the ex vivo biodistribution of major organs and tumors collected 48 hours post-injection; the inset shows the corresponding NIR-II fluorescence images. Table e shows representative infrared thermal images during 808 nm laser irradiation. Table f shows the real-time tumor temperature curve during irradiation (tumor temperature in the aDTP-TPA NAs group was maintained at approximately 44°C through infrared thermal imaging-guided laser power density feedback control, while heating in the PBS+NIR group was negligible under the same irradiation settings). g shows tumor growth curves at 11 days after different treatments (mean ± standard deviation, n=5). h shows a photograph of the tumor removed on day 11; dashed circles indicate no tumor masses were observed in the aDTP-TPANAs+NIR group. i shows representative immunofluorescence images of HSP70 (red) and cell nuclei (DAPI, blue) staining in tumor sections (scale bar: 100 μm). j shows quantitative analysis of relative HSP70 fluorescence intensity in tumor sections, showing a decrease in HSP70 signal in the aDTP-TPANAs+NIR group compared to the pure heated control (PBS+NIR) (mean ± standard deviation, n=3). P<0.0001).

[0075] It can be seen that after intravenous injection, the NIR-II signal at the tumor site gradually increases over time, reaching its peak at 24 hours after injection. Figure 13 (b, c) This is consistent with EPR (high permeability and retention of solid tumors)-mediated enrichment. Ex vivo imaging after 48 hours further confirmed its preferential enrichment in tumors relative to major organs. Figure 13 (d), which provides a favorable distribution map for image-guided therapy.

[0076] In vivo LT-PTT was evaluated in mice bearing 4T1 tumors in four groups: PBS (dark control), PBS+NIR, aDTP-TPA NAs (dark control), and aDTP-TPA NAs+NIR. Irradiation at 808 nm was performed 24 hours post-injection, based on the imaging window. Infrared thermography showed that during irradiation, the tumor temperature in the aDTP-TPA NAs group increased and remained at approximately 44°C. Figure 13 (e, f) , while the heating generated in the PBS+NIR group was negligible. Tumor surface temperature was monitored by infrared thermal imaging, and laser power density was adjusted in real time to maintain it at approximately 44±1℃ for 10-15 minutes. Tumor growth monitoring showed that under mild temperature conditions, the aDTP-TPA NAs+NIR group achieved complete tumor ablation, while the control group showed rapid tumor progression (e, f). Figure 13 (g, h).

[0077] To assess whether HSP70 regulation also occurs in vivo, tumor sections were analyzed by immunofluorescence staining. Figure 13 (i). Compared to the dark control group, the PBS+NIR group showed an increase in HSP70 signal, consistent with the heat shock response induced by mild photothermal stress. Figure 13 (j). Conversely, tumors from mice treated with aDTP-TPA NAs showed significantly reduced HSP70 signaling, including the aDTP-TPA NAs+NIR group exposed to approximately 44°C. These results are consistent with in vitro observations and support the in vivo inhibition of HSP70-related heat shock protection by aDTP-TPA NAs, thereby promoting effective LT-PTT.

[0078] 6. Safety assessment Changes in relative body weight of mice in different treatment groups are shown in the figure. Figure 14 As can be seen, no significant weight loss was observed during treatment, indicating good systemic tolerability. Finally, systemic biosafety was assessed by hematology, serum biochemistry, and histology. Major hematological and biochemical indicators (including liver and kidney function markers) remained within the normal range and were comparable to those in the PBS group.

[0079] Images of H&E stained sections of the heart, liver, spleen, lungs, and kidneys collected from the four treatment groups (scale bar: 100 μm) are shown below. Figure 15 It can be seen that H&E staining of major organs showed no obvious inflammation or tissue damage, while tumors from the aDTP-TPA NAs+NIR group showed extensive necrosis, which is consistent with the effect of local treatment.

[0080] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A nano-aggregate based on near-infrared II aggregation-induced emission material, characterized in that, The nanoaggregates are self-assembled from aggregation-induced light-emitting materials and amphiphilic carrier materials; The aggregation-induced emission material is a compound with a donor-acceptor-donor structure, and its structural formula is: ； The amphiphilic carrier material is selected from the DSPE-PEG series polymers.

2. The nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 1, characterized in that, The mass ratio of the aggregation-induced emission material to the amphiphilic carrier material is 1:

3.

3. The nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 1, characterized in that, The amphiphilic carrier material is DSPE-PEG-2000.

4. A method for preparing nanoaggregates based on near-infrared II aggregation-induced emission materials as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) The aggregation-induced emission material and the amphiphilic polymer are dissolved together in an organic solvent to form an organic phase; (2) Under ultrasonic conditions, the organic phase is added dropwise to the aqueous phase, and self-assembly occurs to form an emulsion; (3) Remove the organic solvent from the emulsion to obtain the nano-aggregates based on near-infrared II aggregation-induced emission materials.

5. The method for preparing nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 4, characterized in that, The volume ratio of the organic solvent to the aqueous phase is 1:10; The ratio of the organic solvent to the aggregation-induced light-emitting material is 1 mL: 1 mg.

6. The method for preparing nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 4, characterized in that, In step (3), the organic solvent is removed by evaporation of nitrogen gas.

7. The use of a nanoaggregate based on near-infrared II aggregation-induced emission material as described in any one of claims 1-3 in the preparation of a medicament for low-temperature photothermal therapy of tumors.

8. The application of the nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 7 in the preparation of drugs for low-temperature photothermal therapy of tumors, characterized in that, The aforementioned low-temperature photothermal therapy is a single-agent therapy without the combination of exogenous heat shock protein 70 inhibitors.

9. The application of the nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 7 in the preparation of drugs for low-temperature photothermal therapy of tumors, characterized in that, The treatment temperature for the low-temperature photothermal therapy is 43℃-45℃.

10. The application of the nanoaggregates based on near-infrared II aggregation-induced emission materials according to claim 7 in the preparation of drugs for low-temperature photothermal therapy of tumors, characterized in that, The nanoaggregates are used to perform the low-temperature photothermal therapy under the guidance of near-infrared II fluorescence imaging.

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