A near-infrared two-region small-molecule photothermal agent based on a quinoid structure, a preparation method and application thereof

Abstract

The application discloses a kind of near-infrared two-zone small molecule photothermal agent based on quinone structure and its preparation method and application, belong to the field of biological medicine.The photothermal agent by introducing quinone structure, significantly improve the light absorption and photothermal conversion efficiency in NIR-II window, suitable for 1064nm wavelength photothermal treatment.Its preparation method includes multi-step organic synthesis reaction, finally obtain dark brown solid BPT-FNC.In addition, the application also provides a kind of nano aggregate preparation method, by polymer nanoshell coating small molecule photothermal agent, enhance its biocompatibility and tumor targeting.The nano aggregate has application potential in the preparation of tumor photothermal treatment reagent.Experimental results show that BPT-FNC NAs under 1064nm laser irradiation shows excellent photothermal performance and biological safety, can induce cell pyroptosis and activate immune response, provides innovative solution for deep tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a near-infrared II region small molecule photothermal agent based on a quinone structure, its preparation method, and its application. Background Technology

[0002] Photothermal therapy (PTT) is an emerging non-invasive cancer treatment that uses photothermal agents (PTAs) to convert light energy into localized high temperatures, thereby ablating tumors. Compared to the first near-infrared window (NIR-I, 700-900 nm) and the second near-infrared window (NIR-II, 1000-1700 nm), PTT offers greater tissue penetration depth and lower background interference, making it a hot research topic. However, the development of small-molecule photothermal agents suitable for the NIR-II window, especially at a wavelength of 1064 nm, faces significant challenges. Existing photothermal agents are mostly based on conjugated polymers. Although redshift absorption can be achieved by extending the π-conjugated system, their clinical translation is limited by structural heterogeneity, poor synthetic reproducibility, and poor biocompatibility. In contrast, small-molecule photothermal agents possess significant advantages due to their well-defined molecular structures, high chemical controllability, and potential biocompatibility and biodegradability. However, their absorption performance in the NIR-II window remains limited due to finite conjugation length and insufficient intramolecular charge transfer (ICT) characteristics. In recent years, quinoidal structures have emerged as a promising design strategy due to their ability to increase π-electron delocalization, reduce the energy gap between HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) (HOMO-LUMO band gap), and stabilize open-shell singlet states or multiple radical states. However, the application of quinoline-based small-molecule photothermal agents under 1064 nm excitation is still immature, and the understanding of their structure-performance relationship is insufficient. Therefore, an innovative design strategy is urgently needed to develop small-molecule photothermal agents that combine high NIR-II absorption, high photothermal conversion efficiency, and good biocompatibility to meet the needs of deep tumor treatment. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention proposes a near-infrared II (NIR-II) small-molecule photothermal agent based on a quinone structure, its preparation method, and its applications. By introducing a quinone structure, a novel small-molecule photothermal agent is developed. This agent significantly improves light absorption and photothermal conversion efficiency in the NIR-II window by increasing π-electron delocalization, reducing the HOMO-LUMO band gap, and stabilizing open-shell singlet states or free radical states. This enables highly efficient deep tumor treatment while also considering biocompatibility and synergistic effects with immunotherapy, providing an innovative solution for tumor treatment.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] One of the objectives of this invention is to provide a near-infrared II small molecule photothermal agent (BPT-FNC) based on a quinone structure, the chemical structure of which is shown in formula (1):

[0006]

[0007] This invention utilizes quinone structural engineering to design a novel small-molecule photothermal agent, BPT-FNC. This molecule possesses an open-shell singlet ground state and a thermally accessible triplet state, exhibiting significant NIR-II absorption characteristics. When this molecule is fabricated into nanoaggregates, it demonstrates highly efficient photothermal conversion performance under 1064 nm laser irradiation. Furthermore, this invention also overcomes the shortcomings of existing technologies by inducing pyroptosis and immunogenic cell death through the photothermal agent, achieving highly efficient photothermal therapy and immune activation for deep tumors.

[0008] A second objective of this invention is to provide a method for preparing the near-infrared II small molecule photothermal agent based on the quinone structure, comprising the following steps:

[0009] 1) At 40 °C, N-bromosuccinimide (NBS) was dissolved in a mixed solution of water and acetic acid. Under a nitrogen atmosphere, a solution of 6,7-difluoronaphth-1-ol dissolved in acetic acid was added dropwise. The reaction mixture was stirred at 40 °C for 2 hours. After cooling to room temperature, the mixture was poured into water, and the precipitate was collected by filtration. The crude solid was purified by silica gel column chromatography using hexane / dichloromethane as the eluent to give the yellow solid product FN-Br (2-bromo-6,7-difluoronaphth-1,4-dione).

[0010] 2) FN-Br and malononitrile were dissolved in anhydrous dichloromethane. Under nitrogen protection, titanium tetrachloride solution was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 1 hour. Then, under argon protection, anhydrous pyridine was added dropwise at the same temperature. The mixture was stirred at 0°C for another hour. The reaction was then quenched with water and the mixture was extracted with dichloromethane. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography using hexane / dichloromethane as the eluent to give the yellow solid product FNC-Br(2-(3-bromo-6,7-difluoro-4-oxonaphth-1(4H)-methylene)malononitrile).

[0011] 3) Dissolve BPT-Sn, FNC-Br and Pd(PPh3)2Cl2 in anhydrous toluene. Stir the resulting mixture overnight at 120°C. After cooling to room temperature, pour the mixture into a 10wt% potassium fluoride aqueous solution and extract with diethyl ether. Wash the combined organic layers with water and brine in sequence, then dry with anhydrous Na2SO3, filter, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography with dichloromethane / hexane as the eluent to obtain dark brown solid BPT-FNC, i.e., small molecule photothermal agent.

[0012] Further, in step 1), the molar ratio of N-bromosuccinimide to 6,7-difluoronaphth-1-ol is 2:1.

[0013] Further, in step 2), the ratio of FN-Br, malononitrile, titanium tetrachloride solution and anhydrous pyridine is 1g:290mg:4.4mL:0.44mL.

[0014] Further, in step 3), the molar ratio of BPT-Sn, FNC-Br and Pd(PPh3)2Cl2 is 0.12∶0.35∶0.012.

[0015] A third objective of this invention is to provide a nano-aggregate comprising the aforementioned near-infrared II small molecule photothermal agent based on a quinone structure.

[0016] The fourth objective of this invention is to provide a method for preparing the nano-aggregates, comprising the following steps: coating the near-infrared II small molecule photothermal agent based on a quinone structure with a polymer nanoshell.

[0017] The fifth objective of this invention is to provide an application of the aforementioned nanoaggregates in the preparation of photothermal therapy reagents for tumors.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] This invention addresses three key challenges in developing small-molecule photocatalytic materials (PTA) for cancer therapy. First, to overcome the scarcity of small-molecule PTAs excitable at 1064 nm, a quine-type molecular design strategy is introduced through the stepwise design of electron-deficient acceptor units. This approach enables the construction of BPT-FNCs with open-shell biradical characteristics, narrow optical band gaps, and strong absorption in the near-infrared II region (especially at 1064 nm). Second, to ensure safe and effective deep tissue therapy, this invention formulates BPT-FNCs into biocompatible nanoaggregates that exhibit excellent colloidal stability, tumor targeting, and a powerful photothermal effect under low-power 1064 nm laser irradiation. These properties are maintained even under simulated tissue conditions, highlighting the potential of BPT-FNCs for non-invasive and deep-penetrating photothermal therapy (PTT). Third, in addition to thermal ablation, this invention also demonstrates that BPT-FNC-mediated photothermal conversion (PTT) can activate pyroptosis and trigger the release of immunogenic DAMPs. These DAMPs collectively induce apoptosis (ICD) and stimulate systemic anti-tumor immune responses, effectively linking local tumor destruction with immune activation. In summary, this invention establishes a multifunctional nanoplatform that combines quinone-type free radical engineering, clinically safe deep tissue photothermal conversion (PTT), and immunostimulatory cell death. These results not only deepen our understanding of the structure and function of quinone-type PTAs but also provide a promising design blueprint for developing bifunctional phototherapeutic agents for precision cancer immunotherapy. Attached Figure Description

[0020] 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:

[0021] Figure 1 This is a flowchart of the reaction process for synthesizing FN-Br in Example 1;

[0022] Figure 2 This is a flow chart of the reaction process for synthesizing BPT-FNC in Example 1;

[0023] Figure 3 For BPT-FNC in Example 1 1 H NMR spectrum;

[0024] Figure 4 For BPT-FNC in Example 1 19 F NMR spectrum;

[0025] Figure 5 For BPT-FNC in Example 1 13 C NMR spectrum;

[0026] Figure 6 The HRMS spectrum of BPT-FNC in Example 1;

[0027] Figure 7 The reaction flow diagram for the synthesis of BPT-FI in Comparative Example 1 is shown.

[0028] Figure 8 For BPT-FI in Comparative Example 1 1 HNMR spectrum;

[0029] Figure 9 For BPT-FI in Comparative Example 1 19 F NMR spectrum;

[0030] Figure 10 For BPT-FI in Comparative Example 1 13 C NMR spectrum;

[0031] Figure 11 The HRMS spectrum of BPT-FI in Comparative Example 1;

[0032] Figure 12 The reaction flow chart for the synthesis of BPT-FN in Comparative Example 1 is shown.

[0033] Figure 13 For BPT-FN in Comparative Example 2 1 HNMR spectrum;

[0034] Figure 14 For BPT-FN in Comparative Example 2 19 F NMR spectrum;

[0035] Figure 15 For BPT-FN in Comparative Example 2 13 C NMR spectrum;

[0036] Figure 16 The HRMS spectrum of BPT-FN in Comparative Example 2;

[0037] Figure 17 The electronic structure and optical properties of the compounds in Example 1 (BPT-FNC), Comparative Example 1 (BPT-FI), and Comparative Example 2 (BPT-FN) are shown, where a is the HOMO-LUMO distribution and energy level of BPT-FI, BPT-FN, and BPT-FNC calculated using DFT (B3LYP / 6-31G(d,p)); b, c, and d are the absorption and photoluminescence (PL) spectra of the three compounds in THF solution.

[0038] Figure 18 For ESR characterization and excited-state analysis, where a represents BPT-FI, BPT-FN, and BPT-FNC in THF solution (5 × 10⁻⁶).-4 b) ESR spectra measured in M); b) ESR spectra of BPT-FI, BPT-FN, and BPT-FNC in nano-aggregate state (1 mg / mL); c) ESR spectra of BPT-FI, BPT-FN, and BPT-FNC in solid state (10 mg); d) VT-ESR spectra of 10 mg BPT-FNC solid recorded in the range of 230 K to 310 K; e) Fitting the spectrum in d using the Bleaney-Bowers equation, indicating that it is a thermally accessible triplet state; f) White light irradiation at different times (20 mW / cm²). 2 The normalized ESR signal intensity of solid-state BPT-FNC is shown in the inset, with the corresponding ESR spectrum; g is a schematic vertical energy diagram of excited-state BPT-FNC, showing the thermally accessible diradical triplet state (right) relative to the diradical singlet ground state (left);

[0039] Figure 19 The optical and photothermal properties of BPT-FI NAs, BPT-FNN NAs, and BPT-FNC NAs in Example 2 are shown below; where a is the normalized absorption and emission spectrum of BPT-FI NAs in an aqueous dispersion; b is the normalized absorption and emission spectrum of BPT-FNN NAs in an aqueous dispersion; c is the normalized absorption and emission spectrum of BPT-FNC NAs in an aqueous dispersion; d is the hydrodynamic size distribution of BPT-FI NAs, BPT-FNN NAs, and BPT-FNC NAs measured by dynamic light scattering (DLS); e is the colloidal stability of BPT-FNNAs in water, PBS, and DMEM over two weeks; f is the value of BPT-FNNAs irradiated by 1064 nm laser (0.7 W / cm²). 2 Temperature rise curves of BPT-FI NAs, BPT-FN NAs, and BPT-FNC NAs solutions (50 μg / mL, prepared by diluting a 1 mg / mL NAs stock solution with ultrapure water to 50 μg / mL); g and h represent the values ​​of 1064 nm laser irradiation (0.7 W / cm²). 2 Temperature changes and corresponding infrared thermographs of BPT-FNC NAs at different concentrations under different 1064nm laser power densities at a fixed concentration of 50μg / mL; i and j represent the temperature rise and infrared thermographs of BPT-FNC NAs at different 1064nm laser power densities; k represents the temperature rise of BPT-FNC NAs under five laser on / off heating and cooling cycles (1064nm, 0.7W / cm²). 2 The photothermal stability of ) is represented by l, where l is the photothermal conversion efficiency of BPT-FNC NAs.

[0040] Figure 20BPT-FNC NAs were used to induce the killing of 4T1 tumor cells in vitro; where a represents the treatment of 4T1 cells with different concentrations (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL) of BPT-FNC NAs, with or without 1064 nm laser irradiation (1 W / cm²). 2 b) Changes in cell viability under different concentrations (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL) of BPT-FNC NAs, showing low cytotoxicity; c) Changes in cell viability under 1064 nm laser irradiation (1 W / cm²). 2 After treatment with PBS or BPT-FNC NAs for 5 minutes, 4T1 cells were stained for live / dead cells (Calcein-AM / PI). Scale bar: 50 μm; d: Annexin V-FITC / PI staining of 4T1 cells after treatment with PBS or BPT-FNC NAs under the same PTT conditions; e: staining of 4T1 cells after laser treatment using MTDR / GreenNuc TM Staining was used to assess mitochondrial potential and nuclear integrity in 4T1 cells; f shows Western blot analysis of pyroptosis-related protein expression in 4T1 cells treated with BPT-FNC NAs and laser irradiation; g shows immunofluorescence staining of CRT to assess ICD-related DAMP exposure in 4T1 cells after treatment; h shows immunofluorescence staining of HMGB1 to assess ICD-related DAMP exposure in 4T1 cells after treatment; i shows Western blot analysis of CRT and HMGB1 expression in 4T1 cells treated with PBS or BPT-FNC NAs under laser irradiation; j shows ATP release in cell lysates after treatment, indicating TCD-related danger signals; k shows ATP release in cell lysates after treatment, indicating TCD-related danger signals; l shows ATP release in cell lysates after treatment of 4T1 cells under 1064 nm laser irradiation (1 W / cm²). 2 After treatment with PBS or BPT-FNC NAs for 5 minutes, the release of LDH in 4T1 cells was quantified to indicate membrane disruption and cytotoxicity.

[0041] Figure 21NIR-II fluorescence imaging and PTT performance of BPT-FNC NAs in tumor-bearing BALB / c nude mice; where a) is NIR-II fluorescence imaging of nude mice 3 days after intravenous injection of BPT-FNC NAs, showing tumor accumulation; b) quantitative analysis of the fluorescence intensity of the tumor in a), confirming preferential accumulation of BPT-FNC NAs; c) schematic diagram of the PTT process in nude mice; d) under 1064nm laser irradiation (0.3W / cm²). 2 (e) Temperature curves of tumors in mice treated with PBS and BPT-FNC NAs over time; (f) Statistical analysis of tumor temperature increase in the BPT-FNC NAs and PBS groups under 1064nm laser irradiation; (f) Representative images of tumors in each group on day 10 after treatment (scale bar: 1cm); (g) Tumor volume changes in nude mice during treatment; (h) Monitoring of mouse body weight throughout the treatment period, indicating system safety (n=3).

[0042] Figure 22 NIR-II fluorescence imaging and fluorescence intensity of major organs 24 hours after intravenous injection of BPT-FNC NAs;

[0043] Figure 23 H&E staining of major organs such as heart, liver, spleen, lungs and kidneys after different treatments. Scale bar, 200 μm.

[0044] Figure 24 Biochemical and hematological analyses were performed on mice in different treatment groups after photothermal therapy. RBC - red blood cells; HGB - hemoglobin; MCH - mean corpuscular hemoglobin concentration; MCV - mean corpuscular volume; ALT - alanine aminotransferase; AST - aspartate aminotransferase; BUN - blood urea nitrogen; CREA - creatinine. Data are expressed as mean ± standard deviation and were obtained from n = 3 biologically independent animals.

[0045] Figure 25 The therapeutic effects of BPT-FNC NAs on tumor-bearing BALB / c mice under 1064nm and 808nm laser irradiation are shown in Figure a. Figure a is a schematic diagram of the in vivo deep tissue PTT setup using chicken breast tissue to simulate a deep tumor environment; Figure b is a description of NIR-IIPTT images of tumor-bearing mice covered with 3mm thick chicken tissue; Figure c shows the effects of 1064nm and 808nm laser irradiation (0.7W / cm²). 2 d) is an infrared thermal image of a tumor site covered with 3 mm thick chicken tissue; d) is an infrared thermal image of a tumor site covered with 3 mm thick chicken tissue under 1064 nm and 808 nm laser irradiation (0.7 W / cm²). 2(i) Temperature rise curve of BPT-FNC NAs; (e) Representative tumor images of each treatment group after 12 days; (f) Tumor volume changes from day 0 to day 12 in mice treated with PBS, BPT-FNC NAs+1064nm and BPT-FNC NAs+808nm; (g) Final weight of tumors in each group on day 12 after treatment; (h) Monitored body weight of mice during the 12-day treatment period to indicate biosafety; (ij) PTT and pyroptosis activated immune cells: (i) CD4 and (j) CD11c staining of tumors extracted from different groups. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0050] 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.

[0051] This invention designed and synthesized three molecules (BPT-FI, BPT-FN, and BPT-FNC), each containing a mature electron-rich BPT core. The first molecule, BPT-FI, was synthesized using 5,6-difluoro-1H-inden-1,3(2H)-dione (FI) as the acceptor unit. The second compound, BPT-FN, used 6,7-difluoronaphthyl-1,4-dione (FN) as the acceptor unit, providing a structurally isomer of BPT-FI with different electronic properties. The third derivative, BPT-FNC, was synthesized using 2-(6,7-difluoro-4-oxonaphthyl-1(4H)-methylene)malononitrile (FNC) as the terminal group, and four cyano substituents were introduced to enhance electron-withdrawing strength. All compounds were synthesized via... 1 H and 13 Its structure was fully characterized by C10 NMR spectroscopy and high-resolution mass spectrometry (HRMS).

[0052] Unless otherwise specified, "room temperature" in this invention refers to 25±2℃.

[0053] All raw materials used in this invention were purchased from the market.

[0054] The technical solution of the present invention will be further illustrated by the following embodiments.

[0055] Example 1

[0056] A method for preparing a near-infrared II small molecule photothermal agent (BPT-FNC) based on a quinone structure includes the following steps:

[0057] 1) Synthesis of 2-bromo-6,7-difluoronaphthyl-1,4-dione (FN-Br) (e.g.) Figure 1 As shown): N-bromosuccinimide (3.56 g, 20.0 mmol, 2.0 equivalent) was dissolved in a mixed solution of water (40 mL) and acetic acid (20 mL) at 40 °C. 6,7-difluoronaphthyl-1-ol (1.80 g, 10.0 mmol, 1.0 equivalent) dissolved in acetic acid (20 mL) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred at 40 °C for 2 hours. After cooling to room temperature, the mixture was poured into water (100 mL), and the precipitate was collected by filtration. The crude solid was purified by silica gel column chromatography using hexane / dichloromethane (2:1, v / v) as the eluent to give a yellow solid product FN-Br (1.97 g, 72%). 1 H NMR (500MHz, CDCl3) δ8.01 (dd, J=9.7, 7.3Hz, 1H), 7.91 (dd, J=9.5, 7.3Hz, 1H), 7.58 (s, 1H). 13C NMR (126MHz, CDCl3) δ180.20,175.96,155.50,155.40,155.14,155.04,153.42,153.32,153.0 7,152.96,140.43,140.11,129.71,129.66,128.68,128.64,117.49,117.33,116.51,116.36. 19 F NMR (471MHz, CDCl3) δ-124.84,-125.47.HRMS: m / z=320.9479([M+H] + calcd.for C 13 H4BrF2N2O2 + :320.9470).

[0058] 2) Synthesis of 2-(3-bromo-6,7-difluoro-4-oxonaphth-1(4H)-methylene)malononitrile (FNC-Br) (e.g.) Figure 1 As shown): FN-Br (1.00 g, 3.66 mmol, 1.0 equivalent) and malononitrile (290 mg, 4.39 mmol, 1.2 equivalent) were dissolved in anhydrous dichloromethane (20 mL). Under nitrogen protection, titanium tetrachloride solution (1 M dissolved in anhydrous dichloromethane, 4.4 mL, 1.2 equivalent) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour, and then anhydrous pyridine (0.44 mL, 1.5 equivalent) was added dropwise at the same temperature under argon protection. The mixture was stirred at 0 °C for another 1 hour, and then the reaction was quenched with water. The mixture was extracted with dichloromethane, and the combined organic layers were dried over anhydrous Na2SO4, filtered, concentrated under vacuum, and the crude product was purified by silica gel column chromatography with hexane / dichloromethane (2:1, v / v) as the eluent to give the yellow solid product FNC-Br (529 mg, 45%). 1 H NMR (500MHz, CDCl3) δ8.75 (dd, J = 10.9, 6.8 Hz, 1H), 8.39 (s, 1H), 8.19 (t, J = 8.8 Hz, 1H). 13 C NMR (126MHz, CDCl3) δ174.54,154.60,154.57,154.50,152.58,152.53,152.43,150.27,138.45,134.69,128.51,1 28.47,126.71,126.67,126.65,126.61,119.08,119.06,118.93,118.91,116.77,116.59,113.21,112.08,85.89. 19F NMR (471MHz, CDCl3) δ-123.18,-123.55.HRMS: m / z=1355.5206([M+H] + calcd.for C 76 H 87 F4N4O4S5 + :1355.5262).

[0059] 3) Synthesis of BPT-FNC (e.g.) Figure 2 As shown: BPT-Sn (150 mg, 0.12 mmol, 1.0 equiv), FNC-Br (111 mg, 0.35 mmol, 3.0 equiv), and Pd(PPh3)2Cl2 (8 mg, 0.012 mmol, 0.1 equiv) were dissolved in anhydrous toluene (10.0 mL). The reaction mixture was stirred overnight at 120 °C. After cooling to room temperature, the mixture was poured into a 10 wt% potassium fluoride (KF) aqueous solution and extracted with diethyl ether. The combined organic layers were washed successively with water and brine, dried over anhydrous Na2SO3, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane / hexane (2:1, v / v) as the eluent to obtain a dark brown solid BPT-FNC (94 mg, yield 56%), i.e., a small molecule photothermal agent. 1 H NMR (500MHz, CDCl3) δ8.78 (dd, J=11.0, 6.6Hz, 2H), 8.23 ​​(t, J=8.7Hz, 2H), 8.07 (s, 2H),4.72(t,J=7.0Hz,4H),3.13(t,J=8.3Hz,4H),2.16-2.07(m,2H),2.02(q,J=8.0 Hz,4H),1.60(d,J=7.4Hz,4H),1.45(t,J=7.8Hz,4H),1.33(d,J=14.5Hz,32H),1.02 (s,12H),0.92(t,J=6.6Hz,6H),0.74(q,J=4.2Hz,6H),0.69(dd,J=7.3,4.1Hz,6H). 13C NMR (126MHz, CDCl3) δ181.19,154.66,154.56,154.31,154.20,152.59,152.49,152.22,152.12,149.97,1 47.64,145.95,141.98,137.54,136.88,132.80,130.57,128.94,128.87,127.27,127.22,126.62,118.47, 118.32,116.10,115.93,114.61,113.41,112.73,81.77,55.37,40.14,31.98,31.02,30.03,29.75,29.72,29.70,29.66,29.62,29.45,29.41,29.37,29.19,27.64,23.16,23.13,22.80,22.75,14.19,13.79,10.17. 19 F NMR (471MHz, CDCl3) δ-124.54,-125.35.HRMS: m / z=1451.5486([M+H] + calcd.for C 82 H 87 F4N4O2S5 + (1451.5405). See details. Figures 3-6 .

[0060] Comparative Example 1

[0061] A method for preparing a near-infrared II small molecule photothermal agent (BPT-FI) based on a quinone structure (e.g.) Figure 7 (As shown), including the following steps:

[0062] BPT-CHO (100.0 mg, 0.097 mmol, 1.0 equivalent) and 5,6-difluoro-1H-indene-1,3(2H)-dione (89 mg, 0.49 mmol, 5.0 equivalent) were dissolved in chloroform (5.0 mL) in a round-bottom flask under nitrogen protection. Anhydrous pyridine (0.5 mL) was then added, and the reaction mixture was stirred overnight at 60 °C. After cooling to room temperature, the mixture was poured into methanol, and the precipitate was collected by filtration. The crude product was purified by silica gel column chromatography using dichloromethane / hexane (2:1, v / v) as the eluent to give a dark blue solid BPT-FI (99 mg, 75%). 1H NMR (500MHz, CDCl3) δ8.24 (s, 2H), 7.78 (ddd, J = 10.3, 7.9, 6.7Hz, 4H), 4.86-4.73 ( m,4H),3.24(t,J=7.8Hz,4H),2.13(p,J=6.9Hz,2H),1.93(p,J=7.7Hz,4H),1.51(dd d,J=15.1,8.5,6.0Hz,4H),1.41(p,J=6.7Hz,4H),1.36-1.16(m,32H),1.15-0.91(m ,12H),0.92-0.86(m,6H),0.78(td,J=7.4,4.2Hz,6H),0.68(td,J=7.3,3.4Hz,6H). 13 C NMR (126MHz, CDCl3) δ188.27,187.57,156.03,154.02,153.87,151.13,147.55,144.37 ,139.16,139.13,139.09,137.56,137.46,134.21,133.31,132.49,128.91,120.99,11 3.06, 112.07, 111.90, 111.74, 55.49, 40.30, 31.93, 30.81, 29.75, 29.65, 29.63, 29.55, 29.45, 29.36, 29.28, 27.60, 27.57, 23.21, 22.84, 22.71, 14.15, 13.74, 10.26, 10.21. 19 F NMR(471MHz, CDCl3)δ-124.23(dt,J=19.0,7.6Hz),-124.59(dt,J=18.9,7.5Hz).HRMS:m / z=1355.5206([M+H] + calcd.for C 76 H 87 F4N4O4S5 + (1355.5262). See details. Figures 8-11 .

[0063] Comparative Example 2

[0064] A method for preparing a near-infrared II small molecule photothermal agent (BPT-FN) based on a quinone structure includes the following steps:

[0065] 1) Synthesis of 2-bromo-6,7-difluoronaphthalene-1,4-dione (FN-Br) (reaction flow diagram as follows) Figure 1As shown): N-bromosuccinimide (3.56 g, 20.0 mmol, 2.0 equivalent) was dissolved in a mixed solution of water (40 mL) and acetic acid (20 mL) at 40 °C. 6,7-difluoronaphthyl-1-ol (1.80 g, 10.0 mmol, 1.0 equivalent) dissolved in acetic acid (20 mL) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred at 40 °C for 2 hours, cooled to room temperature, and the mixture was poured into water (100 mL). The precipitate was collected by filtration. The crude solid was purified by silica gel column chromatography using hexane / dichloromethane (2:1, v / v) as the eluent to give a yellow solid product, FN-Br (1.97 g, 72%).

[0066] 2) Synthesis of BPT-FN (e.g.) Figure 12 As shown): Under nitrogen protection, BPT-Sn (150 mg, 0.12 mmol, 1.0 equiv), FN-Br (95 mg, 0.35 mmol, 3.0 equiv) and Pd(PPh3)2Cl2 (8 mg, 0.012 mmol, 0.1 equiv) were dissolved in anhydrous toluene (10.0 mL), and the reaction mixture was stirred overnight at 120 °C. After cooling to room temperature, the mixture was poured into a 10 wt% potassium fluoride (KF) aqueous solution and extracted with diethyl ether. The combined organic layers were washed successively with water and brine, dried over anhydrous Na2SO3, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography with dichloromethane / hexane (2:1, v / v) as eluent to give a dark green solid BPT-FN (111 mg, yield 71%). 1 H NMR (500MHz, CDCl3) δ8.06 (dd, J=9.6, 7.2Hz, 2H), 7.97 (dd, J=9.6, 7.3Hz, 2H), 7.18 ( s,2H),4.79-4.64(m,4H),3.07-3.00(m,4H),2.13(p,J=6.5Hz,2H),2.01-1.91(m,4H ),1.57-1.49(m,4H),1.43(td,J=8.4,4.5Hz,4H),1.38-1.24(m,32H),1.22-0.97(m, 12H), 0.92(t,J=6.9Hz,6H), 0.74(td,J=7.5,3.2Hz,6H), 0.68(td,J=7.3,3.7Hz,6H). 13C NMR (126MHz, CDCl3) δ182.75,181.87,155.50,155.39,155.08,154.97,153.42,153.31,153.01,1 52.91,147.64,144.44,141.69,140.07,137.04,132.85,132.26,130.23,129.84,128.41,126.88 ,124.86,116.86,116.71,115.63,115.48,112.17,112.15,55.27,40.10,31.94,30.08,29.94,29.73,29.68,29.65,29.62,29.38,29.08,27.65,23.16,22.79,22.72,14.14,13.74,10.15,10.13. 19 F NMR (471MHz, CDCl3) δ-125.83,-126.78.HRMS: m / z=1355.5211([M+H] + calcd.forC 76 H 87 F4N4O4S5 + (1355.5262). See details. Figures 13-16 .

[0067] 1. Density functional theory (DFT) was used to calculate and investigate the geometric conformation and electronic structure of the three compounds in Example 1 (BPT-FNC), Comparative Example 1 (BPT-FI), and Comparative Example 2 (BPT-FN).

[0068] Figure 17 The electronic structure and optical properties of the compounds in Example 1 (BPT-FNC), Comparative Example 1 (BPT-FI), and Comparative Example 2 (BPT-FN) are as follows: Figure 17 As shown in a, all compounds predominantly adopt a coplanar conformation, driven by a rigid BTP core and non-covalent S···O interactions between the thiophene ring and the acceptor unit. Frontier molecular orbital (FMO) analysis revealed that the LUMO of BTP-FI is delocalized across the entire backbone, while the LUMO of BTP-FN is located at the terminal acceptor, indicating stronger intramolecular charge transfer (ICT). Consequently, the HOMO-LUMO band gap narrows from 2.29 eV in BTP-FI to 1.70 eV in BTP-FN. Introducing a cyano group into BTP-FNC further lowers the orbital energy and reduces the HOMO-LUMO band gap to 1.34 eV. To validate these results, the photophysical properties of these three molecules were measured in tetrahydrofuran (THF) solution. Figure 17(bd in the text). The maximum absorption wavelength of BTP-FI is 622 nm, that of BTP-FN is 633 nm, and that of BTP-FNC is 829 nm. The absorption wavelength of BTP-FNC extends into the near-infrared II region. Corresponding emission peaks were observed at 691 nm, 1021 nm, and 1118 nm, respectively. These trends are consistent with the calculated results, confirming that the band gap gradually decreases and the absorption / emission peaks redshift throughout the series.

[0069] 2. In order to clarify how structural differences related to quinone structural features affect the photophysical properties of the three compounds in Example 1 (BPT-FNC), Comparative Example 1 (BPT-FI), and Comparative Example 2 (BPT-FN), the electron spin resonance (ESR) measurements of the three compounds were performed quantitatively in solution, nano-aggregates, and solid state. The preparation method of the nano-aggregates was as follows: 1 mg BPT-FNC and 3 mg Pluronic F-127 were dissolved in 1 mL of tetrahydrofuran (THF) and sonicated. This solution was then mixed with 9 mL of deionized water and sonicated for 2 minutes using an ultrasonic probe (VCX150, Sonics) at 75 W output power. Subsequently, the THF was evaporated under a nitrogen flow, and the resulting mixture was transferred to a dialysis membrane (MWCO: 3500 Da) and dialyzed in fresh MilliQ water for 24 hours. To ensure complete removal of unreacted cRGD, the MilliQ water was replaced every 4 hours. The resulting suspension was then filtered through a 0.2 μm syringe filter and concentrated to 1 mL using a sterile Millipore Amicon Ultra-3 15 mL ultrafiltration centrifuge tube (molecular weight cutoff: 10000 Da) to obtain BPT-FNC nanoaggregates (BPT-FNC NAs). BPT-FI NAs and BPT-FN NAs were obtained using the same method.

[0070] Figure 18 For ESR characterization and excited-state analysis, from Figure 18 As can be seen from the ac values, BTP-FNC exhibits a significant ESR signal in all states, indicating the presence of open-shell radicals. In contrast, BTP-FN shows a weaker ESR signal only in the solid state, while BTP-FI shows no signal of any kind. These findings are consistent with the accepted principle that quinone structures in organic semiconductors stabilize unpaired electrons through delocalized π-conjugation and support bisradical resonance structures, thereby promoting radical formation. Therefore, BTP-FN has stronger radical properties than BTP-FI, attributed to its quinone-modified acceptor unit, which enhances radical stability upon aggregation. For BTP-FNC, the introduction of four cyano groups further increases electronic defects, enabling radical stabilization even in solution, as reflected in its consistently strong ESR response.

[0071] To further investigate the radical properties of BPT-FNC, this invention performed variable-temperature ESR (VT-ESR) measurements in the solid state. Figure 18 As shown in d, the ESR signal intensity gradually increases with temperature from 230 K to 310 K, indicating the existence of a thermally accessible triplet (Tt) state and an open-shell singlet biradical ground state. Measurements using a superconducting quantum interference device (SQUID) further confirm this behavior, showing a positive correlation between molar magnetic susceptibility and temperature, consistent with the thermal configuration of the Tt state. Fitting the VT-ESR data using the Bleaney-Bowers equation yields a relatively small singlet-triplet bandgap (ΔE). S0-Tt ), which is -1.85 kcal / mol ( Figure 18 (e) This supports the thermal accessibility of triplet species. To determine whether the observed biradical behavior was photoinduced, additional ESR measurements were performed under white light or 808 nm laser irradiation. Figure 18 As shown in f, the ESR signal remained unchanged after 10 minutes of continuous illumination, confirming that the biradical properties of BPT-FNC are inherent and not photoinduced.

[0072] In summary, these data confirm that BPT-FNC exhibits an inherent open-shell biradical character in the singlet ground state, attributed to its Quine structure, which enhances the DA interaction and promotes π-electron delocalization. Figure 18 (g in the text). This observation, coupled with weaker spin coupling than traditional closed-shell molecules, facilitates the generation of ΔE. S0-Tt The controlled S0→Tt transition, rather than the transition caused by ΔE S1-T1 Controlled photoexcitation of the S1→T1 transition. Excited-state calculations show that the S1 and T1 energy levels are 1.22 eV and 0.72 eV, respectively, which corresponds to a relatively large ΔE of 0.50 eV. S1-T1 This indicates that the S1→T1 intersystem crossing efficiency is low. In contrast, the energy required for the S0→Tt transition obtained by VT-ESR is only 0.08 eV, indicating that the triplet state is thermally activated from the ground state. This may open up a highly efficient non-radiative decay pathway, thereby improving the photothermal performance of BPT-FNC.

[0073] Example 2

[0074] Given the good potential of BPT-FI, BPT-FN and BPT-FNC as PTAs, the present invention encapsulates them in the amphiphilic copolymer Pluronic F-127 to form water-dispersible nanoaggregates (NAs) suitable for biological applications. The specific preparation method includes the following steps: The preparation method of the nano-aggregates is as follows: 1 mg of BPT-FI, BPT-FN and BPT-FNC and 3 mg of Pluronic F-127 are dissolved in 1 mL of tetrahydrofuran (THF), and sonicated. Then, the solution is mixed with 9 mL of deionized water and sonicated for 2 minutes using an ultrasonic probe (VCX150, Sonics) at an output power of 75 W. Subsequently, the THF is evaporated under a nitrogen flow, and the resulting mixture is transferred to a dialysis membrane (MWCO: 3500 Da) and dialyzed in fresh MilliQ water for 24 hours. To ensure complete removal of unreacted cRGD, fresh MilliQ water is replaced every 4 hours. The resulting suspension is then filtered through a 0.2 μm syringe filter and concentrated to 1 mL using a sterile MilliporeAmicon Ultra-3 15 mL ultrafiltration centrifuge tube (molecular weight cutoff: 10000 Da) to obtain BPT-FI NAs, BPT-FN NAs and BPT-FNC NAs, respectively.

[0075] Figure 19 The optical and photothermal properties of the BPT-FI NAs, BPT-FN NAs, and BPT-FNC NAs in Example 2 are described. From... Figure 19 As can be seen from 'a', BPT-FIN NAs exhibit a broad absorption band, with a maximum at 556 nm and an emission peak at 760 nm. In contrast, the absorption and emission peaks of BPT-FIN NAs show redshifts at 715 nm and 1044 nm, respectively. Figure 19 (b) It is worth noting that the absorption peak of BPT-FNC NAs shows a significant red shift, with a peak value at 971 nm and an absorption range extending above 1300 nm, including strong absorption at 1064 nm. Figure 19 (c) Its emission peak reaches 1308 nm, which is one of the longest emission wavelengths reported in purely organic systems. Dynamic light scattering (DLS) measurements show that the average hydrodynamic diameter of BPT-FIN NAs is 154.7 nm, BPT-FNC NAs is 140.5 nm, and BPT-FNC NAs is 113.4 nm. Figure 19 Stability assessments in water, phosphate-buffered saline (PBS), and Duchene modified Eagle medium (DMEM) showed that the hydrodynamic diameter remained virtually unchanged over several weeks. Figure 19 The value of e) indicates that it has excellent colloidal stability.

[0076] To evaluate the photothermal conversion capability of NAs, a 1064nm laser (0.7W / cm²) was used. 2 The aqueous dispersion was irradiated, and the corresponding temperature changes were monitored. At a concentration of 50 μg / mL, only BPT-FNC NAs showed a significant temperature increase, reaching 55 °C within 5 minutes. Figure 19 The high concentration of BPT-FNCNAs (f) is attributed to their strong absorption at 1064 nm. Conversely, BPT-FNCNAs and BPT-FNNAs, which have no absorption in the NIR-II region, exhibit negligible temperature increases under the same conditions. The photothermal properties of BPT-FNCNAs were then systematically investigated. Under 1064 nm laser irradiation, dispersions of different concentrations showed concentration-dependent heating (f). Figure 19 The temperature rose to 60°C within 5 minutes at 100 μg / mL, while it reached 45°C even at 12.5 μg / mL. In contrast, the PBS control group showed only a slight increase from 26.8°C to 30°C. Furthermore, temperature changes at different laser power densities were evaluated using 50 μg / mL BPT-FNC NAs nanoparticles. Figure 19 As shown in i in 19 and j in 19, the final temperature increases with increasing laser power, and the solution reaches a temperature of 0.9 W / cm². 2 When it reaches 70℃, at 0.5W / cm 2 The temperature reached 50°C. The PBS control group maintained a temperature of around 30°C, independent of power density. To assess photothermal stability, the same solution was subjected to five consecutive heating-cooling cycles under 1064 nm radiation. In each cycle, the temperature repeatedly exceeded 57°C and returned to baseline after laser shutdown, with no significant performance degradation. Figure 19 The excellent thermal and optical stability of BPT-FNC NAs was demonstrated by the k value. Finally, the photothermal conversion efficiency (PCE) of BPT-FNC NAs was calculated to be 31.5%. Figure 19 The 'l' in the text highlights its enormous potential in NIR-II photothermal applications.

[0077] Application Example 1 - In Vitro Photothermal Antitumor Experiment

[0078] Given the excellent photothermal properties of BPT-FNC NAs, this invention systematically evaluated their in vitro antitumor effects.

[0079] Figure 20 BPT-FNC NAs were used to induce 4T1 tumor cell killing in vitro. Figure 20As shown in a, BPT-FNC NAs exhibited negligible cytotoxicity in the absence of laser irradiation, demonstrating their inherent biocompatibility under normal conditions. However, under 1064 nm laser (1 W / cm²) conditions, the cytotoxicity decreased significantly. 2 Five minutes after irradiation, the mortality rate of 4T1 cancer cells incubated with BPT-FNC NAs significantly increased. Notably, cell viability decreased by 98% as the concentration of BPT-FNC NAs increased from 0 to 50 μg / mL, highlighting its potent phototoxicity and antitumor effects under laser irradiation. In contrast, normal cells, including L929 and HEK293T, maintained a high survival rate of over 98% even at the highest concentration of 50 μg / mL, further demonstrating the excellent biocompatibility and selective toxicity of nucleoside analogues (NAs). Figure 20 (b)

[0080] To investigate its mechanism of killing tumor cells, this invention performed Calcein-AM / PI staining. For example... Figure 20 As shown in c, 4T1 cells treated with BPT-FNC NAs and laser irradiation exhibited strong PI (red) fluorescence but no Calcein-AM (green) signal, indicating widespread cell death. Annexin V-FITC staining further showed that only in the BPT-FNC NAs + laser group (BPT-FNC + L, Figure 20 The d-cells showed strong green and red fluorescence, consistent with pyroptosis or apoptosis, while the control group showed no signal, confirming the biosafety of unirradiated BPT-FNC NAs. Notably, cell membrane swelling, a hallmark of pyroptosis (indicated by white arrows), was also observed. To further investigate the potential mechanisms of cell death, this invention assessed mitochondrial damage and pyroptosis activation. MitoTracker™ Deep Red (MTDR) staining showed a significant decrease in mitochondrial membrane potential (MMP) only in the BPT-FNC+L group, indicating that laser activation induced mitochondrial-specific damage (MMP). Figure 20 (e). In contrast, all other groups showed intact mitochondrial signals, indicating that mitochondrial damage was specifically induced by BPT-FNCNAs under laser irradiation. Furthermore, Western blot analysis showed that Gasdermin D (GSDMD) cleaved within 0.5 hours after irradiation and persisted for up to 2 hours. Figure 20 The result in f) indicates that the pyroptosis pathway is activated. In summary, these results demonstrate that BPT-FNC NAs can induce mitochondrial dysfunction and trigger pyroptosis under 1064 nm laser irradiation, thus providing an effective and selective strategy for photothermal cancer therapy.

[0081] Given the association between pyroptosis and the release of immunogenic signals, it was hypothesized that BPT-FNC NAs combined with laser irradiation therapy might also induce ICD. To test this hypothesis, hallmark DAMPs were examined, including calreticulin (CRT) exposure, high-mobility group box 1 (HMGB1) release, adenosine triphosphate (ATP) secretion, and lactate dehydrogenase (LDH) leakage. Figure 20 As shown in gh, the BPT-FNC+L group exhibited significantly enhanced CRT surface exposure, while intracellular HMGB1 levels decreased, indicating active release. Western blot analysis confirmed these trends. Figure 20 The study also detected extracellular HMGB1 in the culture medium, confirming its release after treatment. Further analysis showed that ATP release plays a crucial role in inducing cell death. Extracellular ATP levels were significantly increased in the BPT-FNC+L group (i). Figure 20 (j) is accompanied by a corresponding decrease in intracellular ATP ( Figure 20 The presence of k in the culture medium indicates active secretion. Furthermore, elevated LDH levels in the culture medium ( Figure 20 The (l) further demonstrates membrane rupture and loss of cell integrity.

[0082] In summary, these results confirm that BPT-FNC NAs, combined with 1064 nm laser irradiation, can induce CRT exposure, HMGB1 and ATP release, and LDH leakage—all hallmarks of ICDs. Therefore, BPT-FNC NAs not only induce pyroptosis-mediated cancer cell death but also promote the release of immunogenic signals, indicating their potential to stimulate anti-cancer immune responses.

[0083] Application Example 2 - In vivo photothermal therapy in a nude mouse tumor model

[0084] 1. BPT-FNC NAs showed excellent performance in vitro, and their potential for in vivo diagnosis and treatment of 4T1 tumors in BALB / c nude mice was further investigated. To determine the optimal time window for PTT, BPT-FNC NAs were administered intravenously, and fluorescence imaging was performed at different time points using an in vivo imaging system (IVIS).

[0085] Figure 21 NIR-II fluorescence imaging and PTT performance of BPT-FNC NAs in tumor-bearing BALB / c nude mice. Figure 21As shown in Figure a, no fluorescence signal was detected before injection, indicating a clear imaging background. After injection, fluorescence at the tumor site gradually increased, reaching a peak at 24 hours, with the signal intensity more than four times that at the initial time point. This cumulative pattern indicates effective tumor targeting and identifies 24 hours post-injection as the optimal time for PTT (post-injection fluorescence detection). Subsequently, the signal steadily decreased, falling to less than half of the peak intensity within 72 hours, indicating that the nanoparticles were effectively cleared systemically. Quantitative analysis of fluorescence intensity over three days (…) Figure 21 b) further confirms the time-varying accumulation and clearance kinetics of BPT-FNC NAs in tumors, supporting their potential as diagnostic imaging agents and therapeutic nanoplatforms.

[0086] Next, the efficacy of fluorescence imaging (FLI)-guided PTT using BPT-FNC NAs was evaluated. Figure 21 (c) Mice were randomly divided into four groups: PBS group, PBS plus laser group (PBS+L), BPT-FNC NAs (BPT-FNC) group, and BPT-FNC NAs plus laser group (BPT-FNC+L). After intravenous injection of BPT-FNC NAs or PBS, mice were treated with a 1064nm laser (0.3W / cm²). 2 The tumor was irradiated for 10 minutes. Infrared thermal imaging was used to monitor the tumor temperature in real time. Figure 21 (d) In the BPT-FNC+L group, the tumor temperature rapidly rose to 54.8°C within 2 minutes and stabilized at around 58°C, sufficient to effectively ablate the tumor. In contrast, the PBS+L group only increased slightly by about 3°C, confirming that the photothermal effect is a specific effect of BPT-FNCNAs. Figure 21 (e). To assess treatment efficacy and safety, tumor volume and body weight were monitored over 10 days. Tumor size was measured every two days, and tumor tissue was imaged post-treatment. Figure 21 (f) In mice in the PBS, PBS+L, and BPT-FNC groups, tumors continued to grow, while tumors in the BPT-FNC+L group almost completely regressed within 4 days, and no recurrence was observed throughout the 10-day study. Figure 21 (g in the original text). It is noteworthy that no significant changes in body weight were observed in any of the groups. Figure 21 The h in the figure indicates that BPT-FNC NAs have extremely low systemic toxicity and excellent in vivo biocompatibility.

[0087] 2. To assess the biodistribution of BPT-FNC NAs, major organs were removed and fluorescence imaging was performed. Strong fluorescence signals were detected in the tumor, spleen, and liver. Figure 22 NIR-II fluorescence imaging and fluorescence intensity of major organs 24 hours after intravenous injection of BPT-FNC NAs. Figure 22This indicates that BPT-FNC NAs accumulate in tumors through enhanced permeability and retention (EPR) effects and are cleared via the liver and spleen. Statistical analysis showed that the fluorescence intensity in tumors was comparable to that in the liver and spleen, demonstrating the excellent tumor targeting and diagnostic capabilities of BPT-FNC NAs in vivo. H&E staining of major organs showed no morphological abnormalities or signs of inflammation in any of the treatment groups. Figure 23 Furthermore, biochemical analysis showed no significant changes in key indicators, and routine blood tests confirmed that blood components were not affected by BPT-FNC nanoparticles or laser irradiation. Figure 24 These results indicate that BPT-FNC nanoparticles possess excellent biocompatibility, supporting their potential for safe and effective phototherapy applications.

[0088] Application Example 3 - Advantages of NIR-II-stimulated photothermal immunotherapy in the treatment of deep tumors

[0089] To further demonstrate the advantages of NIR-II-induced photothermal immunotherapy in treating deep tumors, chicken breast tissue was used to cover 4T1 tumors to simulate the tumor environment.

[0090] Figure 25 To illustrate the therapeutic effects of BPT-FNC NAs under 1064nm and 808nm laser irradiation in tumor-bearing BALB / c mice. Following intravenous injection of BPT-FNC NAs, a 3mm thick slice of chicken tissue was placed over the tumor area to simulate the deep tissue environment. Figure 25 (a in 25 and b in 25). It is noteworthy that this invention prepared two BPT-FNC NA solutions with identical absorbance at wavelengths of 808 nm and 1064 nm to eliminate the influence of different light absorption rates on the results. The tumor was then irradiated with either 808 nm or 1064 nm laser light at a power density of 0.7 W / cm². 2 In the group receiving 1064nm laser treatment, the tumor temperature rapidly increased to 58.2℃, significantly higher than the 43.3℃ in the 808nm laser group. Figure 25 (cd in the text). The higher temperature rise achieved by 1064nm laser irradiation highlights its superior ability to penetrate biological tissues and effectively heat deep tumors. More importantly, 0.7W / cm 2 The power density perfectly matches the maximum effective radiation (MPE) of a 1064nm laser (1.0W / cm²). 2 This exceeds the maximum effective radiation (MPE) of an 808nm laser (0.33W / cm²). 2 This highlights the safety and tissue penetration of NIR-II light enhancement.

[0091] The efficacy of NIR-II stimulation was further evaluated by monitoring tumor growth. The BPT-FNC NAs + 808nm group showed only mild tumor suppression in the first four days after treatment, but tumor growth subsequently resumed, very similar to the results in the PBS control group. Conversely, the BPT-FNC NAs + 1064nm group achieved complete inhibition of tumor growth, and no recurrence was observed during the 12-day treatment period. Figure 25 (e.g. in the example). This significant therapeutic effect is primarily attributed to the deeper tissue penetration and more effective photothermal heating achieved by NIR-II excitation. Importantly, no significant changes in body weight were observed in any of the treatment groups during the experiment, confirming the biocompatibility of BPT-FNC NAs under irradiation. Figure 25 (h) The above in vitro studies showed that the photothermal effect of BPT-FNCNAs can induce the release of signature DAMPs, consistent with ICD activation. To determine whether this effect can also stimulate in vivo anti-tumor immunity, this invention evaluated the activation of key immune cell populations. Specifically, CD4+, representing adaptive and innate immune responses, was evaluated. + T cells and CD11c + Dendritic cell (DC) infiltration. For example... Figure 25 As shown in i in 25 and j in 25, compared with the PBS group and the BPT-FNC+808nm group, the BPT-FNC+1064nm group showed a significant increase in both immune cell types, indicating a significant enhancement in immune activation after treatment.

[0092] In summary, these results clearly demonstrate that the 1064nm excitable BPT-FNC nanoplatform has significant advantages over the traditional 808nm excitable system, including deeper tissue penetration, higher radiation safety, and better immune response activation.

[0093] Based on the above, this invention proposes a three-step tumor ablation mechanism based on BPT-FNC nanoparticles. First, under 1064nm laser irradiation, BPT-FNC NAs generate local photothermal energy, leading to mitochondrial dysfunction and caspase-1 activation, which in turn cleaves GSDMD and releases its N-terminal fragment (GSDMD-N). Second, GSDMD-N inserts into the plasma membrane to form pores, inducing pyroptosis, characterized by cell swelling, membrane rupture, and the release of immunogenic DAMPs, such as ATP, HMGB1, and CRT. Third, these DAMPs promote the maturation of dendritic cells (DCs) and the activation of T cells, thereby initiating a systemic anti-tumor immune response. In summary, these results highlight the dual function of BPT-FNC NAs as a photothermally activated nanoplatform, capable of mediating precise tumor ablation while simultaneously stimulating a robust anti-tumor immune response, offering great promise for synergistic photothermal immunotherapy in cancer treatment.

[0094] 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 near-infrared II region small molecule photothermal agent based on a quinone structure, characterized in that, Its chemical structural formula is shown in formula (1): (1)。 2. A method for preparing a near-infrared II small molecule photothermal agent based on a quinone structure as described in claim 1, characterized in that, Includes the following steps: 1) At 40°C, N-bromosuccinimide was dissolved in a mixed solution of water and acetic acid. Under a nitrogen atmosphere, a solution of 6,7-difluoronaphthyl-1-ol dissolved in acetic acid was added dropwise. The reaction mixture was stirred at 40°C for 2 hours. After cooling to room temperature, the mixture was poured into water, and the precipitate was collected by filtration. The crude solid was purified by silica gel column chromatography using hexane / dichloromethane as eluent to give a yellow solid product, FN-Br. The structural formula of FN-Br is: ; 2) FN-Br and malononitrile were dissolved in anhydrous dichloromethane. Under nitrogen protection, titanium tetrachloride solution was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 1 hour. Then, under argon protection, anhydrous pyridine was added dropwise at the same temperature. Stirring was continued at 0°C for 1 hour. The reaction was then quenched with water, and the mixture was extracted with dichloromethane. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography using hexane / dichloromethane as eluent to obtain a yellow solid product, FNC-Br. The structural formula of FNC-Br is: ; 3) Dissolve BPT-Sn, FNC-Br and Pd(PPh3)2Cl2 in anhydrous toluene. Stir the resulting mixture overnight at 120°C. After cooling to room temperature, pour the mixture into a 10wt% potassium fluoride aqueous solution and extract with diethyl ether. Wash the combined organic layers with water and brine in sequence, then dry with anhydrous Na2SO3, filter, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography with dichloromethane / hexane as the eluent to obtain dark brown solid BPT-FNC, i.e., small molecule photothermal agent. The structural formula of BPT-Sn is: ; The structural formula of the BPT-FNC is: .

3. The preparation method of the near-infrared II small molecule photothermal agent based on the quinone structure according to claim 2, characterized in that, In step 1), the molar ratio of N-bromosuccinimide to 6,7-difluoronaphth-1-ol is 2:

1.

4. The preparation method of the near-infrared II small molecule photothermal agent based on the quinone structure according to claim 2, characterized in that, In step 2), the ratio of FN-Br, malononitrile, titanium tetrachloride solution and anhydrous pyridine is 1g:290mg:4.4mL:0.44mL.

5. The preparation method of the near-infrared II small molecule photothermal agent based on the quinone structure according to claim 2, characterized in that, In step 3), the molar ratio of BPT-Sn, FNC-Br and Pd(PPh3)2Cl2 is 0.12:0.35:0.

012.

6. A nanoaggregate, characterized in that, It includes the near-infrared II region small molecule photothermal agent and polymer nanoshell based on the quinone structure as described in claim 1.

7. A method for preparing nanoaggregates as described in claim 6, characterized in that, Includes the following steps: The near-infrared II region small molecule photothermal agent based on a quinone structure is coated with a polymer nanoshell.

8. The use of the nanoaggregate as described in claim 6 in the preparation of a tumor photothermal therapy reagent.

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