Near-infrared two-region micromolecule photothermal agent based on quinonoid structure and preparation method and application thereof

Abstract

The invention discloses a near-infrared two-region micromolecule photothermal agent based on a quinonoid structure as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the photothermal agent, by introducing a quinone structure, the light absorption efficiency and the photothermal conversion efficiency in an NIR-II window are remarkably improved, and the photothermal agent is suitable for photothermal therapy with the wavelength of 1064 nm. The preparation method comprises a multi-step organic synthesis reaction, and finally a dark brown solid BPT-FNC is obtained. In addition, the invention further provides a preparation method of the nano aggregate, and the biocompatibility and the tumor targeting property of the nano aggregate are enhanced by coating the micromolecular photothermal agent with the polymer nano shell. The nano aggregate has application potential in preparation of a tumor photothermal therapy reagent. Experimental results show that the BPT-FNC NAs shows excellent photo-thermal performance and biological safety under 1064nm laser irradiation, can induce pyroptosis of cells and activate immune response, and provides an innovative solution for deep tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a near-infrared second-zone small molecule photothermal agent based on a quinone structure, and a preparation method and application thereof. Background Art

[0002] Photothermal therapy (PTT) is an emerging non-invasive cancer treatment that converts light energy into localized high temperatures through photothermal agents (PTAs) to achieve tumor ablation. Compared with the first near-infrared window (NIR-I, 700-900nm), the second near-infrared window (NIR-II, 1000-1700nm) has a higher tissue penetration depth and lower background interference, and therefore has become a research hotspot. However, the current research and development of small molecule photothermal agents suitable for the NIR-II window, especially the 1064nm wavelength, faces significant challenges. Existing photothermal agents are mostly based on conjugated polymers. Although red-shifted absorption can be achieved by extending the π-conjugated system, their clinical translation and application are limited due to problems such as structural heterogeneity, poor synthetic reproducibility, and poor biocompatibility. In contrast, small molecule photothermal agents have significant advantages due to their well-defined molecular structure, high chemical controllability, and potential biocompatibility and biodegradability. However, their absorption performance in the NIR-II window is still limited due to limited conjugation length and insufficient intramolecular charge transfer (ICT) properties. In recent years, quinoidal structures have become a promising design strategy due to their ability to increase the degree of π electron delocalization, reduce the energy difference between the HOMO (highest occupied molecular orbital) and the LUMO (lowest unoccupied molecular orbital) (HOMO-LUMO energy gap), and stabilize open-shell singlet states or multi-radical states. However, the application of small molecule photothermal agents based on the quinoid structure under 1064nm excitation is not yet mature, and their structure-performance relationship is still not fully understood. Therefore, an innovative design strategy is urgently needed to develop small molecule photothermal agents with efficient NIR-II absorption, high photothermal conversion efficiency, and good biocompatibility to meet the needs of deep-seated tumor treatment. Summary of the Invention

[0003] To address the above technical issues, the present invention proposes a near-infrared II window small molecule photothermal agent based on a quinone-like structure, as well as its preparation method and application. By introducing a quinone-like structure, a new small molecule photothermal agent has been developed. By increasing the degree of π electron delocalization, reducing the HOMO-LUMO energy gap, and stabilizing open-shell singlet or free radical states, its light absorption and photothermal conversion efficiency in the NIR-II window are significantly improved. This enables highly efficient deep-seated tumor treatment while also taking into account biocompatibility and immunotherapy synergy, providing an innovative solution for tumor treatment.

[0004] To achieve the above object, the present application provides the following technical solutions.

[0005] One of the purposes of the present application is to provide a quinoid structure-based near-infrared two-region small molecule photothermal agent (BPT-FNC), the chemical structural formula of which is shown as formula (1):

[0006]

[0007] The present application designs a new type of small molecule photothermal agent BPT-FNC through quinoid structure engineering, which has an open shell single state ground state and a thermally accessible three state, and shows significant NIR-II absorption characteristics. After the molecule is prepared into a nano aggregate, it shows high efficient photothermal conversion performance under 1064nm laser irradiation. In addition, the present application also overcomes the shortcomings of the prior art by inducing pyroptosis and immunogenic cell death of the photothermal agent, and realizes efficient deep tumor photothermal treatment and immune activation.

[0008] The second purpose of the present application is to provide a preparation method of the quinoid structure-based near-infrared two-region small molecule photothermal agent, comprising the following steps:

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

[0010] 2) FN-Br and malononitrile are dissolved in anhydrous dichloromethane, and a solution of titanium tetrachloride is added dropwise at 0℃ under nitrogen protection. The reaction mixture is stirred at 0℃ for 1 hour, and then anhydrous pyridine is added dropwise under argon protection at the same temperature. Continue to stir at 0℃ for 1 hour, then quench the reaction with water, and extract the mixture with dichloromethane. The combined organic layer is dried over anhydrous Na2SO4, filtered, vacuum concentrated, and the crude product is purified by silica gel column chromatography with hexane / dichloromethane as the eluent to obtain yellow solid product FNC-Br (2-(3-bromo-6,7-difluoro-4-oxonaphthalen-1(4H)-methylene) malononitrile);

[0011] 3) BPT-Sn, FNC-Br and Pd(PPh3)2Cl2 were dissolved in anhydrous toluene, and the resulting mixture was stirred at 120℃ overnight, after cooling to room temperature, the mixture was poured into a 10wt% aqueous potassium fluoride solution, and extracted with ether, the combined organic layers were washed with water and brine in turn, then dried with anhydrous Na2SO3, filtered, concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography with dichloromethane / hexane as eluent to obtain BPT-FNC, a small molecule photothermal agent, as a dark brown solid.

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

[0013] Further, in step 2), the ratio of the amount of use 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] The third object of the present application is to provide a nanoaggregate comprising the small molecule photothermal agent based on quinoid structure in the near-infrared second region.

[0016] The fourth object of the present application is to provide a preparation method of the nanoaggregate, comprising the following steps: coating the small molecule photothermal agent based on quinoid structure in the near-infrared second region with a polymer nanoshell.

[0017] The fifth object of the present application is to provide an application of the nanoaggregate in the preparation of a tumor photothermal treatment reagent.

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

[0019] The present invention addresses three key challenges in the development of small molecule photocatalytic materials (PTAs) for cancer treatment. First, to overcome the scarcity of small molecule PTAs that can be excited at a wavelength of 1064 nm, a Quine-type molecular design strategy is introduced by gradually designing electron-deficient acceptor units. This approach enables the construction of BPT-FNCs with open-shell diradical characteristics, narrow optical band gaps, and strong absorption in the near-infrared-II region (especially at a wavelength of 1064 nm). Second, to ensure safe and effective deep tissue treatment, the present invention formulates BPT-FNCs into biocompatible nanoaggregates that exhibit excellent colloidal stability, tumor targeting, and a strong photothermal effect under low-power 1064 nm laser irradiation. This performance is maintained even under simulated tissue conditions, highlighting the potential of BPT-FNCs in non-invasive and deep-penetrating photothermal therapy (PTT). Third, in addition to thermal ablation, the present invention also demonstrated that BPT-FNC-mediated photothermal conversion (PTT) can activate cell pyroptosis and trigger the release of immunogenic DAMPs, which together induce cell apoptosis (ICD) and stimulate systemic anti-tumor immune responses, effectively connecting local tumor destruction with immune activation. In summary, the present invention has established a multifunctional nanoplatform that combines quinone radical engineering, clinically safe deep tissue photothermal conversion (PTT), and immunostimulatory cell death. This result not only deepens the understanding of the structure and function of quinone-type PTA, but also provides a promising design blueprint for the development of dual-functional photodiagnostic agents for precision cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 This is a reaction flow chart for the synthesis of FN-Br in Example 1;

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

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

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

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

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

[0027] Figure 7 This is a reaction flow chart for the synthesis of BPT-FI in Comparative Example 1;

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

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

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

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

[0032] Figure 12 This is a reaction flow chart for the synthesis of BPT-FN in Comparative Example 1;

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

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

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

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

[0037] Figure 17 The electronic structures and optical properties of the compounds in Example 1 (BPT-FNC), Comparative Example 1 (BPT-FI), and Comparative Example 2 (BPT-FN) are shown in Figure 1, where a is the HOMO-LUMO distribution and energy levels 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, a is the concentration of BPT-FI, BPT-FN and BPT-FNC in THF solution (5×10-4 M) ESR spectra measured in solution; b ESR spectra measured in nanoscale aggregates (1 mg / mL) for BPT-FI, BPT-FN and BPT-FNC; c ESR spectra measured in solid state (10 mg) for BPT-FI, BPT-FN and BPT-FNC; d VT-ESR spectra recorded in the range of 230 K to 310 K for 10 mg of BPT-FNC solid; e Corresponding fitting of the spectra in d using Bleaney-Bowers equation, indicating a thermally accessible triplet state; f Normalized ESR signal intensity of solid state BPT-FNC under white light irradiation (20 mW / cm 2 ) at different times, inset is the corresponding ESR spectra; g Schematic vertical energy diagram of excited state BPT-FNC, showing a thermally accessible triplet state of the diradical (right) relative to the singlet ground state of the diradical (left);

[0039] Figure 19 are the optical properties and photothermal performance of BPT-FI NAs, BPT-FN NAs and BPT-FNC NAs in Example 2; wherein, a are the normalized absorption and emission spectra of BPT-FI NAs in aqueous dispersion; b are the normalized absorption and emission spectra of BPT-FN NAs in aqueous dispersion; c are the normalized absorption and emission spectra of BPT-FNC NAs in aqueous dispersion; d are the hydrodynamic size distribution of BPT-FI NAs, BPT-FN NAs and BPT-FNC NAs measured by dynamic light scattering (DLS); e are the colloidal stability of BPT-FN NAs in water, PBS and DMEM respectively within two weeks; f are the temperature rise curves of BPT-FI NAs, BPT-FN NAs and BPT-FNC NAs solutions (50 μg / mL, the prepared 1 mg / mL NAs stock solution was diluted with ultrapure water to 50 μg / mL) under 1064 nm laser irradiation (0.7 W / cm 2 ); g, h are the temperature change and the corresponding infrared thermal imaging map of BPT-FNC NAs at different concentrations under 1064 nm laser irradiation (0.7 W / cm 2 ); i, j are the temperature rise and infrared thermal imaging map of BPT-FNC NAs at a fixed concentration of 50 μg / mL under different 1064 nm laser power densities; k is the photothermal stability of BPT-FNC NAs in five laser on / off heating and cooling cycles (1064 nm, 0.7 W / cm 2 ); 1 is the photothermal conversion efficiency of BPT-FNC NAs;

[0040] Figure 20BPT-FNC NAs induced 4T1 tumor cell killing in vitro; wherein, a is 4T1 cells treated with different concentrations (0μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL) of BPT-FNC NAs, with or without 1064nm laser irradiation (1W / cm 2 b is the change of cell viability when normal cells (L929 and HEK293T) were treated with BPT-FNC NAs at different concentrations (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL), showing low cytotoxicity; c is the change of cell viability when normal cells (L929 and HEK293T) were treated with BPT-FNC NAs at different concentrations (0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL), showing low cytotoxicity; 2 d, 5 minutes) after treatment with PBS or BPT-FNCNAs, 4T1 cells were stained for live / dead (Calcein-AM / PI), scale bar: 50 μm; d, under the same PTT conditions, 4T1 cells were treated with PBS or BPT-FNCNAs, and then stained with Annexin V-FITC / PI; e, after laser treatment, 4T1 cells were stained with MTDR / GreenNuc TM Figure 3: Western blot analysis of mitochondrial potential and nuclear integrity of 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of pyroptosis-related protein expression in 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of pyroptosis-related protein expression in 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of pyroptosis-related protein expression in 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of mitochondrial potential and nuclear integrity of 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of mitochondrial potential and nuclear integrity of 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of mitochondrial potential and nuclear integrity of 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of mitochondrial potential and nuclear integrity of 4T1 cells treated with BPT-FNC NAs and laser irradiation. Figure 3: Western blot analysis of mitochondrial potential and nuclear integrity of 4T1 cells treated with BPT-FNC NAs and laser irradiation. 2 Quantification of LDH release in the culture medium of 4T1 cells after treatment with PBS or BPT-FNC NAs at 4°C (5 min), indicating membrane disruption and cytotoxicity;

[0041] Figure 21Figure 2 shows the NIR-II fluorescence imaging and PTT performance of BPT-FNC NAs in tumor-bearing BALB / c nude mice; a is the NIR-II fluorescence imaging of nude mice 3 days after intravenous injection of BPT-FNC NAs, showing tumor accumulation; b is the quantitative analysis of the fluorescence intensity of the tumor in a, confirming the preferential accumulation of BPT-FNC NAs; c is a schematic diagram of the PTT process in nude mice; d is the NIR-II fluorescence imaging of nude mice 3 days after intravenous injection of BPT-FNC NAs, showing the accumulation of tumors; 2 ) under PBS and BPT-FNC NAs, the temperature curves of the tumors of mice treated with PBS and BPT-FNC NAs over time; e is the statistical analysis of the temperature increase of the tumors in the BPT-FNC NAs and PBS groups under 1064 nm laser irradiation; f is a representative image of the tumors in each group on the 10th day after treatment, scale bar: 1 cm; g is the change in tumor volume of nude mice during the treatment period; h is the monitoring of the body weight of mice during the entire treatment period, indicating the safety of the system (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, lung, and kidney after different treatments. Scale bar, 200 μm.

[0044] Figure 24 Figure 3 Biochemical analysis and blood cell count analysis of mice in different treatment groups after photothermal therapy; RBC-red blood cell; 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 ± SD, and are derived from n = 3 biologically independent animals.

[0045] Figure 25 The therapeutic effects of BPT-FNC NAs under 1064 nm and 808 nm laser irradiation in tumor-bearing BALB / c mice; a is a schematic diagram of the in vivo deep tissue PTT setup using chicken breast tissue to simulate the deep tumor environment; b is a NIR-IIPTT photograph of a tumor-bearing mouse covered with 3 mm thick chicken tissue; c is a diagram of the BPT-FNC NAs under 1064 nm and 808 nm laser irradiation (0.7 W / cm 2 ) is an infrared thermal image of the tumor site covered with 3 mm thick chicken tissue; d is an infrared thermal image of the tumor site covered with 3 mm thick chicken tissue under 1064 nm and 808 nm laser irradiation (0.7 W / cm 2) temperature increase curve of BPT-FNC NAs; e is a representative tumor photo of each treatment group after 12 days; f is the change in tumor volume from day 0 to day 12 in mice treated with PBS, BPT-FNC NAs+1064nm and BPT-FNC NAs+808nm; g is the final weight of the tumor in each group on day 12 after treatment; h is the body weight of mice monitored during the 12-day treatment period, indicating biosafety; ij is PTT and cell pyroptosis-activated immune cells: (i) CD4 and (j) CD11c staining of tumors extracted from different groups. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting 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 terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The present invention designed and synthesized three molecules (BPT-FI, BPT-FN and BPT-FNC), each of which contains a mature electron-rich central core BPT. The first molecule BPT-FI is synthesized using 5,6-difluoro-1H-indene-1,3(2H)-dione (FI) as the acceptor unit, the second compound BPT-FN uses 6,7-difluoronaphthalene-1,4-dione (FN) as the acceptor unit, providing a structurally isomeric BPT-FI substitute with different electronic properties, and the third derivative BPT-FNC is synthesized using 2-(6,7-difluoro-4-oxonaphthalene-1(4H)-methylene)malononitrile (FNC) as the end group, and four cyano substituents are introduced to enhance the electron-withdrawing strength. All compounds are synthesized by 1 H and 13 The structure of the product was confirmed by C NMR spectroscopy and high-resolution mass spectrometry (HRMS).

[0052] Unless otherwise specified, the "room temperature" in the present invention refers to 25±2°C.

[0053] The raw materials used in the present invention are all purchased from the market.

[0054] The technical solution of the present invention is further illustrated by the following examples.

[0055] Example 1

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

[0057] 1) Synthesis of 2-bromo-6,7-difluoronaphthalene-1,4-dione (FN-Br) (e.g. Figure 1 At 40°C, N-bromosuccinimide (3.56 g, 20.0 mmol, 2.0 equivalents) was dissolved in a mixed solution of water (40 mL) and acetic acid (20 mL). A solution of 6,7-difluoronaphthalen-1-ol (1.80 g, 10.0 mmol, 1.0 equivalents) 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 resulting precipitate was collected by filtration; the crude solid was purified by silica gel column chromatography with hexane / dichloromethane (2:1, v / v) as eluent to give FN-Br (1.97 g, 72%) as a yellow solid. 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-oxonaphthalene-1(4H)-methylene)malononitrile (FNC-Br) (e.g. Figure 1 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 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; stirring was continued at 0°C for 1 hour, then the reaction was quenched with water, and the mixture was extracted with dichloromethane. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography using hexane / dichloromethane (2:1, v / v) as eluent to obtain 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 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 at 120°C overnight. After cooling to room temperature, the mixture was poured into a 10 wt% aqueous potassium fluoride (KF) solution and extracted with ether. The combined organic layers were washed sequentially 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 BPT-FNC (94 mg, 56% yield), a dark brown solid. 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 Figure 3-Figure 6 .

[0060] Comparative Example 1

[0061] A preparation method of a near-infrared second region small molecule photothermal agent (BPT-FI) based on a quinone structure (such as Figure 7 ), comprising the following steps:

[0062] BPT-CHO (100.0 mg, 0.097 mmol, 1.0 equiv) and 5,6-difluoro-1H-indene-1,3(2H)-dione (89 mg, 0.49 mmol, 5.0 equiv) 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 at 60°C overnight. After cooling to room temperature, the mixture was poured into methanol and the resulting precipitate was collected by filtration. The crude product was purified by silica gel column chromatography with dichloromethane / hexane (2:1, v / v) as eluent to give BPT-FI (99 mg, 75%) as a dark blue solid. 1H NMR (500 MHz, CDC13) δ 8.24 (s, 2H), 7.78 (ddd, J = 10.3, 7.9, 6.7 Hz, 4H), 4.86 - 4.73 (m, 4H), 3.24 (t, J = 7.8 Hz, 4H), 2.13 (p, J = 6.9 Hz, 2H), 1.93 (p, J = 7.7 Hz, 4H), 1.51 (ddd, J = 15.1, 8.5, 6.0 Hz, 4H), 1.41 (p, J = 6.7 Hz, 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, 4.2 Hz, 6H), 0.68 (td, J = 7.3, 3.4 Hz, 6H). 13 C NMR (126 MHz, CDC13) δ 188.27, 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, 113.06, 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 (471 MHz, CDC13) δ -124.23 (dt, J = 19.0, 7.6 Hz), -124.59 (dt, J = 18.9, 7.5 Hz). HRMS: m / z = 1355.5206 ([M+H] + calcd. for C 76 H 87 F4N4O4S5 + :1355.5262). Details are shown in Figures 8-11 .

[0063] Comparative Example 2

[0064] A preparation method of a near-infrared two-zone small-molecule photothermal agent (BPT-FN) based on a quinonoid structure, comprising the following steps:

[0065] 1) Synthesis of 2-bromo-6,7-difluoronaphthalene-1,4-dione (FN-Br) (reaction flow chart as shown in Figure 1At 40°C, N-bromosuccinimide (3.56 g, 20.0 mmol, 2.0 equivalents) was dissolved in a mixed solution of water (40 mL) and acetic acid (20 mL). A solution of 6,7-difluoronaphthalen-1-ol (1.80 g, 10.0 mmol, 1.0 equivalents) 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 resulting precipitate was collected by filtration. The crude solid was purified by silica gel column chromatography using hexane / dichloromethane (2:1, v / v) as eluent to obtain a yellow solid product, FN-Br (1.97 g, 72%).

[0066] 2) Synthesis of BPT-FN (e.g. Figure 12 Under nitrogen, 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 at 120°C overnight. After cooling to room temperature, the mixture was poured into a 10 wt% aqueous solution of potassium fluoride (KF) and extracted with ether. The combined organic layers were washed with water and brine, dried over anhydrous Na2SO3, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with dichloromethane / hexane (2:1, v / v) as eluent to give BPT-FN (111 mg, 71% yield) as a dark green solid. 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 Figure 13-16 .

[0067] 1. Density functional theory (DFT) calculations were used to investigate the geometric conformations and electronic structures 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 structures 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 in the figure, all compounds mainly adopt a coplanar conformation, which is driven by the rigid BTP core and the non-covalent S···O interactions between the thiophene ring and the acceptor unit. Frontier molecular orbital (FMO) analysis shows that the LUMO of BTP-FI is delocalized throughout the skeleton, while the LUMO of BTP-FN is located on the terminal acceptor, indicating stronger intramolecular charge transfer (ICT). Therefore, the HOMO-LUMO energy gap narrows from 2.29 eV in BTP-FI to 1.70 eV in BTP-FN. The introduction of a cyano group in BTP-FNC further reduces the orbital energy and reduces the HOMO-LUMO energy gap to 1.34 eV. To verify these results, the photophysical properties of the three molecules were measured in tetrahydrofuran (THF) solution ( Figure 17(bd in Figure 1). 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. The corresponding emission peaks are observed at 691 nm, 1021 nm, and 1118 nm, respectively. These trends are consistent with the calculated results, confirming a gradual decrease in the band gap and a red shift in the absorption / emission peaks across the entire series.

[0069] 2. To elucidate how structural differences associated with quinone-type 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), electron spin resonance (ESR) measurements were quantitatively performed on the three compounds in solution, nanoaggregates, and solid state. The nanoaggregates were prepared as follows: 1 mg of BPT-FNC and 3 mg of Pluronic F-127 were dissolved in 1 mL of tetrahydrofuran (THF) and sonicated. The 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 stream, and the resulting mixture was transferred to a dialysis membrane (MWCO: 3500 Da) and dialyzed against fresh MilliQ water for 24 hours. To ensure complete removal of unreacted cRGD, fresh 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: 10,000 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, Figure 18 As can be seen from the ac in Figure 3, BTP-FNC exhibits a clear ESR signal in all states, indicating the presence of open-shell radicals. In contrast, BTP-FN only shows a weak ESR signal in the solid state, while BTP-FI does not detect any form of signal. These findings are consistent with the generally accepted principle that the quinoid structure in organic semiconductors stabilizes unpaired electrons through delocalized π conjugation and supports diradical resonance structures, thereby promoting the formation of free radicals. Therefore, BTP-FN has stronger radical characteristics than BTP-FI, which is attributed to its quinonized acceptor unit, and the radical stability is enhanced after aggregation. For BTP-FNC, the introduction of four cyano groups further increases the electron defect, which can achieve radical stabilization even in solution, which is reflected in its consistently strong ESR response.

[0071] In order to further explore the free radical properties of BPT-FNC, the present invention carried out variable temperature ESR (VT-ESR) measurements in the solid state. Figure 18 As shown in Figure d, the ESR signal intensity gradually increases with the temperature increasing from 230K to 310K, indicating the existence of thermally accessible triplet states (Tt) and open-shell singlet diradical ground states. This behavior was further confirmed by superconducting quantum interference device (SQUID) measurements, which showed that the molar magnetic susceptibility was positively correlated with temperature, which is consistent with the thermal layout of the Tt state. The VT-ESR data were fitted with the Bleaney-Bowers equation to obtain a relatively small singlet-triplet energy gap (ΔE S0-Tt ), is -1.85kcal / mol( Figure 18 e), which supports the thermal accessibility of triplet species. To determine whether the observed diradical behavior is 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 min of continuous illumination, confirming that the diradical property of BPT-FNC is intrinsic rather than photoinduced.

[0072] Collectively, these data confirm that BPT-FNC exhibits intrinsic open-shell diradical character in the singlet ground state, which is attributed to its Quinone structure, which enhances the D-A interaction and promotes π electron delocalization ( Figure 18 g in ). This observation, together with the weaker spin coupling than in conventional closed-shell molecules, facilitates the S0-Tt The S0→Tt transition is controlled by the S1-T1 Controlled photoexcited 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 , indicating 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, which may open up an efficient non-radiative decay pathway, thereby improving the photothermal performance of BPT-FNC.

[0073] Example 2

[0074] In view of the good potential of BPT-FI, BPT-FN and BPT-FNC as PTA, the present invention encapsulated them in the amphiphilic copolymer Pluronic F-127 to form water-dispersible nanoaggregates (NA) suitable for biological applications. The specific preparation method includes the following steps: the preparation method of the nanoaggregate state 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) respectively, and sonicated. The solution is then mixed with 9 mL of deionized water and sonicated for 2 minutes at an output power of 75 W using an ultrasonic probe (VCX150, Sonics); subsequently, 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, and 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: 10,000 Da) to obtain BPT-FI NAs, BPT-FN NAs and BPT-FNC NAs, respectively.

[0075] Figure 19 The optical properties and photothermal performance of BPT-FI NAs, BPT-FN NAs and BPT-FNC NAs in Example 2. Figure 19 As can be seen from a in the figure, BPT-FI 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-FN NAs are red-shifted to 715 nm and 1044 nm, respectively ( Figure 19 It is worth noting that the absorption peak of BPT-FNC NAs is significantly red-shifted, with a peak at 971 nm and an absorption range extending to above 1300 nm, with a strong absorption at 1064 nm ( Figure 19 c in the figure). Its emission peak reaches 1308 nm, which is one of the longest emission wavelengths reported in pure organic systems. Dynamic light scattering (DLS) measurements show that the average hydrodynamic diameter of BPT-FI NAs is 154.7 nm, that of BPT-FN NAs is 140.5 nm, and that of BPT-FNCNAs is 113.4 nm ( Figure 19 Stability assessment in water, phosphate-buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) showed that the hydrodynamic diameter remained almost unchanged over several weeks ( Figure 19 e), indicating that it has excellent colloidal stability.

[0076] In order to evaluate the photothermal conversion capability of NAs, a 1064 nm laser (0.7 W / cm 2 ) irradiated the aqueous dispersion and monitored the corresponding temperature changes. At a concentration of 50 μg / mL, only BPT-FNC NAs showed a significant temperature increase, reaching 55°C within 5 minutes ( Figure 19 f), which is attributed to their strong absorption at 1064 nm. In contrast, BPT-FI NAs and BPT-FN NAs, which have no absorption in the NIR-II region, exhibited negligible temperature increase 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 ( Figure 19 At 100 μg / mL, the temperature increased to 60°C within 5 minutes, while even at 12.5 μg / mL, the temperature reached 45°C. In contrast, the PBS control group showed only a slight increase from 26.8°C to 30°C. In addition, the temperature changes at different laser power densities were evaluated using 50 μg / mL BPT-FNC NAs nanoparticles. Figure 19 As shown in Figure 1i and Figure 19j, as the laser power increases, the final temperature also increases. The solution at 0.9W / cm 2 When it reaches 70℃, at 0.5W / cm 2 The temperature of the PBS control group remained at around 30°C, independent of the power density. To evaluate the photothermal stability, the same solution was subjected to five consecutive heating-cooling cycles under 1064nm irradiation. In each cycle, the temperature repeatedly exceeded 57°C and returned to baseline after the laser was turned off, with no significant degradation in performance ( Figure 19 k in the figure), demonstrating its excellent thermal and photostability. Finally, the photothermal conversion efficiency (PCE) of BPT-FNC NAs was calculated to be 31.5% ( Figure 19 l), highlighting its great potential for NIR-II photothermal applications.

[0077] Application Example 1-In vitro photothermal anti-tumor experiment

[0078] In view of the excellent photothermal properties of BPT-FNC NAs, the present invention systematically evaluated its in vitro anti-tumor effect.

[0079] Figure 20 BPT-FNC NAs induced 4T1 tumor cell killing in vitro. Figure 20As shown in Figure a, without laser irradiation, BPT-FNC NAs showed negligible cytotoxicity, demonstrating their inherent biocompatibility under normal conditions. However, under 1064 nm laser (1 W / cm 2 ) irradiated for 5 minutes, the mortality rate of 4T1 cancer cells incubated with BPT-FNC NAs increased significantly. Notably, as the concentration of BPT-FNC NAs increased from 0 to 50 μg / mL, cell viability decreased by 98%, highlighting its strong phototoxicity and anti-tumor 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, which further demonstrated the excellent biocompatibility and selective toxicity of nucleoside analogs (NAs) ( Figure 20 b) in the above example.

[0080] In order to explore its mechanism of killing tumor cells, the present invention performed Calcein-AM / PI staining. Figure 20 As shown in Figure c, 4T1 cells treated with BPT-FNC NAs and laser irradiation showed strong PI (red) fluorescence, but no Calcein-AM (green) signal, indicating extensive cell death. Annexin V-FITC staining further showed that only in the BPT-FNC NAs+laser group (BPT-FNC+L, Figure 20 In the d, strong green and red fluorescence was shown, which is consistent with cell pyroptosis or apoptosis, while the control group showed no signal, which confirmed the biosafety of unirradiated BPT-FNC NAs. Notably, cell membrane swelling, a hallmark of cell pyroptosis, was also observed (indicated by the white arrow). To further explore the underlying mechanism of cell death, the present invention evaluated mitochondrial damage and activation of cell pyroptosis. MitoTrackerTM Deep Red (MTDR) staining showed that mitochondrial membrane potential (MMP) was significantly reduced only in the BPT-FNC+L group, indicating that laser activation induced mitochondrial-specific damage ( Figure 20 e in the figure). In contrast, all other groups showed intact mitochondrial signals, indicating that mitochondrial damage was specifically induced by BPT-FNCNAs under laser irradiation. In addition, Western blot analysis showed that Gasdermin D (GSDMD) was cleaved within 0.5 hours after irradiation and continued to cleave for up to 2 hours ( Figure 20 f), indicating that the pyroptosis pathway is activated. Taken together, these results indicate 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 might also induce ICD. To test this hypothesis, we examined marker DAMPs, including calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1) release, adenosine triphosphate (ATP) secretion, and lactate dehydrogenase (LDH) leakage. Figure 20 As shown in Figure 3, the surface exposure of CRT in the BPT-FNC+L group was significantly enhanced, while the intracellular level of HMGB1 was reduced, indicating active release. Western blot analysis confirmed these trends ( Figure 20 i) and extracellular HMGB1 was detected in the culture medium, confirming its release after treatment. Further analysis showed that ATP release plays a key role in inducing cell death. The extracellular ATP level in the BPT-FNC+L group was significantly increased ( Figure 20 j in the figure), accompanied by a corresponding decrease in intracellular ATP ( Figure 20 k), indicating active secretion. In addition, the LDH level in the culture medium increased ( Figure 20 l) in Figure 1 further demonstrates membrane disruption and loss of cell integrity.

[0082] Together, these results demonstrate that BPT-FNC NAs, combined with 1064 nm laser irradiation, induce CRT exposure, HMGB1 and ATP release, and LDH leakage, hallmark features of ICD. Thus, BPT-FNC NAs not only induce pyroptosis-mediated cancer cell death but also promote the release of immunogenic signals, suggesting their potential to stimulate anticancer immune responses.

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

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

[0085] Figure 21 The NIR-II fluorescence imaging and PTT performance of BPT-FNC NAs in tumor-bearing BALB / c nude mice were investigated. 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 of the initial time point. This cumulative pattern indicates effective tumor targeting and identifies 24 hours after injection as the optimal time for PTT. Thereafter, the signal steadily decreased, falling to less than half of the peak intensity within 72 hours, indicating that the nanoparticles had been effectively cleared systemically. Quantitative analysis of fluorescence intensity over three days ( Figure 21 b) further confirmed the time-dependent 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 group (BPT-FNC), and BPT-FNC NAs plus laser group (BPT-FNC+L). After intravenous injection of BPT-FNC NAs or PBS, the mice were treated with 1064 nm laser (0.3 W / cm 2 , 10 minutes) to irradiate the tumor. 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, which was sufficient to effectively ablate the tumor. In contrast, the PBS+L group only slightly increased by about 3°C, confirming that the photothermal effect is a specific effect of BPT-FNCNAs ( Figure 21 To evaluate the efficacy and safety of the treatment, tumor volume and body weight were monitored over 10 days. Tumor size was measured every two days, and images of the treated tumor tissue were taken ( Figure 21 f). Tumors in mice in the PBS, PBS+L, and BPT-FNC groups continued to grow, whereas tumors in the BPT-FNC+L group almost completely regressed within 4 days, with no recurrence observed throughout the 10-day study ( Figure 21 It is worth noting that no significant changes in body weight were observed in any of the groups ( Figure 21 h), which indicates that BPT-FNC NAs have extremely low systemic toxicity and excellent in vivo biosafety.

[0087] 2. To evaluate the biodistribution of BPT-FNC NAs, major organs were resected and fluorescence imaging was performed. Strong fluorescence signals were detected in tumors, 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 22BPT-FNC NAs were shown to accumulate in tumors by enhancing the enhanced permeability and retention (EPR) effect, and were cleared by the liver and spleen. Statistical analysis showed that the fluorescence intensity in the tumor was comparable to that in the liver and spleen, demonstrating the excellent tumor targeting and diagnostic ability of BPT-FNC NAs in vivo. H&E staining of major organs showed no morphological abnormalities or signs of inflammation in all treatment groups Figure 23 ). In addition, 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 have excellent biocompatibility, supporting their potential for safe and effective photodiagnosis and therapy applications.

[0088] Example 3 - Advantages of NIR-II excitation-based photothermal immunotherapy in deep tumor treatment

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

[0090] Figure 25 BPT-FNC NAs were based on tumor-bearing BALB / c mice, and the therapeutic effect of BPT-FNC NAs under 1064 nm and 808 nm laser irradiation. After intravenous injection of BPT-FNC NAs, a 3 mm thick chicken tissue section was used to cover the tumor area to simulate a deep tissue environment Figure 25 (a in and b in 25). Notably, two BPT-FNC NA solutions were prepared by the present application to have the same absorbance at 808 nm and 1064 nm wavelengths to eliminate the influence of different light absorption rates on the results. The tumor was then irradiated with 808 nm or 1064 nm laser, with a power density of 0.7 W / cm 2 In the group receiving 1064 nm laser treatment, the tumor temperature rapidly increased to 58.2°C, significantly higher than the 43.3°C in the 808 nm laser group Figure 25 (c-d in). The higher temperature increase obtained by 1064 nm laser irradiation highlights its superior biological tissue penetration ability and can effectively heat deep tumors. More importantly, a power density of 0.7 W / cm 2 complies with the maximum permissible exposure (MPE) of 1064 nm laser (1.0 W / cm 2 ), while exceeding the maximum permissible exposure (MPE) of 808 nm laser (0.33 W / cm 2 ), highlighting the safety and tissue penetration of NIR-II light enhancement.

[0091] The therapeutic effect of NIR-II excitation was further evaluated by monitoring tumor growth. The BPT-FNC NAs+808nm group showed only slight tumor inhibition in the first four days after treatment, but tumor growth recovered thereafter, which was very similar to the results of the PBS control group. In contrast, 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 This remarkable therapeutic effect was mainly attributed to the deeper tissue penetration and more efficient photothermal heating achieved by NIR-II excitation. Importantly, no significant changes in body weight were observed in all treatment groups during the experimental period, confirming the biosafety 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 the activation of ICDs. To determine whether this effect can also stimulate anti-tumor immunity in vivo, the present invention evaluated the activation of key immune cell populations. Specifically, the CD4 T cells representing both adaptive and innate immune responses were evaluated. + T cells and CD11c + Infiltration of dendritic cells (DCs). Figure 25 As shown in Figures i and j in 25, both immune cell types were significantly increased in the BPT-FNC+1064nm group compared with the PBS group and the BPT-FNC+808nm group, indicating that the immune activation effect was significantly enhanced after treatment.

[0092] Taken together, these results clearly demonstrate that the 1064 nm excitable BPT-FNC nanoplatform offers significant advantages over conventional 808 nm excitable systems, including deeper tissue penetration, higher irradiation safety, and superior immune response activation.

[0093] Based on the above, the present invention proposes a three-step tumor elimination mechanism based on BPT-FNC nanoparticles. First, under 1064nm laser irradiation, BPT-FNC NAs generate localized photothermal energy, leading to mitochondrial dysfunction and caspase-1 activation, which in turn cleaves GSDMD and releases its N-terminal fragment (GSDMD-N); secondly, GSDMD-N inserts into the plasma membrane to form pores, inducing cell pyroptosis, which is characterized by cell swelling, membrane rupture, and the release of immunogenic DAMPs such as ATP, HMGB1, and CRT; thirdly, 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 functionality of BPT-FNC NAs as a photothermally activated nanoplatform, which can mediate precise tumor ablation while stimulating powerful anti-tumor immunity, providing great prospects 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 changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A near-infrared second-zone small molecule photothermal agent based on a quinone structure, characterized in that: Its chemical structure is shown in formula (1):

2. A method for preparing a near-infrared second-region small molecule photothermal agent based on a quinone structure according to claim 1, characterized in that: The following steps are involved: 1) At 40° C., N-bromosuccinimide was dissolved in a mixed solution of water and acetic acid, and a solution of 6,7-difluoronaphthalene-1-ol dissolved in acetic acid 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, and the resulting precipitate was collected by filtration. The crude solid was purified by silica gel column chromatography using hexane / dichloromethane as eluent to obtain a yellow solid product FN-Br; 2) FN-Br and malononitrile were dissolved in anhydrous dichloromethane. Titanium tetrachloride solution was added dropwise at 0°C under nitrogen protection. The reaction mixture was stirred at 0°C for 1 hour. Anhydrous pyridine was then added dropwise at the same temperature under argon protection and 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 in vacuo. The crude product was purified by silica gel column chromatography using hexane / dichloromethane as eluent to obtain a yellow solid product, FNC-Br. 3) BPT-Sn, FNC-Br, and Pd(PPh3)2Cl2 were dissolved in anhydrous toluene, and the resulting mixture was stirred at 120°C overnight. After cooling to room temperature, the mixture was poured into a 10 wt% aqueous potassium fluoride solution and extracted with ether. The combined organic layers were washed sequentially with water and brine, then dried over anhydrous Na2SO3, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane / hexane as the eluent to obtain BPT-FNC, a dark brown solid, a small molecule photothermal agent.

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

1.

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

5. The method for preparing the near-infrared second region small molecule photothermal agent based on 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 second zone small molecule photothermal agent based on quinone structure as described in claim 1.

7. A method for preparing nano-aggregates according to claim 6, characterized in that: The following steps are involved: The near-infrared second zone small molecule photothermal agent based on the quinone structure is coated with a polymer nanoshell.

8. Use of the nanoaggregate according to claim 6 in preparing a tumor photothermal therapy agent.

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