Near-infrared two-zone photothermal diagnosis and treatment agent, and preparation method and application thereof

Abstract

This invention discloses a near-infrared II photothermal therapeutic agent, its preparation method, and its application, belonging to the field of optical diagnostics and treatment technology. To address the problems of insufficient light absorption, fluorescence quenching, and inadequate molecular structure optimization in existing photothermal therapeutic agents, this invention proposes a molecular design strategy of "central plane + peripheral twisting," developing a novel NIR-II photothermal therapeutic agent, 4TPE-TB, which is a near-infrared II photothermal therapeutic agent. This invention enhances the light absorption capacity of the near-infrared II photothermal therapeutic agent by strengthening the conjugation of the molecular core, and utilizes the peripheral twisted structure to suppress intermolecular stacking, significantly improving photothermal conversion efficiency and fluorescence performance, thereby achieving a comprehensive improvement in the therapeutic agent's performance.

Description

Technical Field

[0001] This invention belongs to the field of optical diagnostic and therapeutic technology, and particularly relates to a near-infrared II photothermal diagnostic agent, its preparation method and application. Background Technology

[0002] Photothermal diagnostic and therapeutic technologies, combining photothermal therapy (PTT) and fluorescence imaging (FLI) for precise cancer diagnosis and treatment, have become a cutting-edge, non-invasive, and highly controllable treatment approach. However, developing photothermal agents suitable for the second near-infrared window (NIR-II, 1000-1700 nm) still faces many challenges. Traditional NIR-II dyes, such as indocyanine green (ICG), despite their strong absorption and fluorescence properties, are prone to intermolecular π-π stacking in aqueous solutions, leading to fluorescence quenching and reduced photothermal conversion efficiency. Furthermore, while aggregation-induced emission (AIE) molecules exhibit excellent resistance to fluorescence quenching in the aggregated state, existing AIE molecules have limited absorption range and low absorption coefficients (ε) in the second near-infrared window, restricting their optical and thermal output capabilities. To address these issues, it is necessary to optimize upstream photon absorption capacity and regulate downstream energy utilization pathways through molecular design. While existing D-π-A-π-D molecular structures can suppress intermolecular π-π stacking, the steric hindrance between the core and the π-bridge may reduce the molecular conjugation length, leading to insufficient redshift in absorption and low fluorescence brightness. Therefore, an innovative molecular design strategy is urgently needed to achieve a balance between fluorescence emission and photothermal conversion while enhancing molecular absorption capacity, for the development of high-performance NIR-II photothermal therapeutic agents. Summary of the Invention

[0003] Existing photothermal therapeutic agents suffer from the following problems: First, existing molecules have low light absorption efficiency in the NIR-II window, making it difficult to achieve efficient photothermal output and fluorescence imaging; second, traditional dyes are prone to aggregation quenching in aqueous solutions, leading to reduced fluorescence emission efficiency; and finally, existing molecular designs have failed to achieve a balance between enhancing molecular conjugation and suppressing intermolecular π-π stacking, limiting their overall performance. Therefore, to address the problems of insufficient light absorption, fluorescence quenching, and inadequate molecular structure optimization in existing photothermal therapeutic agents, this invention proposes a "central plane + peripheral twisting" molecular design strategy, developing a novel NIR-II photothermal therapeutic agent, 4TPE-TB, which is a near-infrared II photothermal therapeutic agent. This invention improves the light absorption capacity of the near-infrared II photothermal therapeutic agent by enhancing the conjugation of the molecular core and suppresses intermolecular stacking through the peripheral twisted structure, significantly improving photothermal conversion efficiency and fluorescence performance, thereby achieving a comprehensive improvement in the therapeutic agent's performance.

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

[0005] This invention provides a near-infrared II photothermal therapeutic agent, the structural formula of which is as follows:

[0006]

[0007] Furthermore, the average particle size of the near-infrared II photothermal therapeutic agent is less than 120 nm.

[0008] The near-infrared II photothermal therapeutic agent provided by this invention is a photothermal therapeutic agent based on a "central plane + peripheral twisting" molecular design strategy. It comprises a benzothiadiazole core and at least two tetraphenylethylene peripheral units, and its molecular structure has the ability to enhance molecular conjugation and inhibit intermolecular stacking. The near-infrared II photothermal therapeutic agent of this invention emits fluorescence in the near-infrared II window (NIR-II, 1000-1700 nm) and exhibits a high fluorescence quantum yield.

[0009] This invention also provides a method for preparing the above-mentioned near-infrared II photothermal therapeutic agent, using (4-(2,2-bis(4-hexoxy)phenyl)-1-phenylvinyl)phenyl)tributyltinane as the starting material, and preparing compound 7 through π-bridge connection of 2,3-dibromothiophene; compound 7 is reacted with trimethyltin chloride in an organic solvent under the catalysis of trimethyltin chloride to obtain compound 8; compound 8 is mixed with 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole), and then synthesized by coupling reaction in a composite catalytic system of Pd2(dba)3 and P(o-tol)3 to form the near-infrared II photothermal therapeutic agent (4TPE-TB); wherein, the structural formula of compound 7 is as follows. The structural formula of compound 8 is as follows: The present invention also provides the application of the above-mentioned near-infrared II photothermal diagnostic agent in the preparation of cancer cell therapeutic drugs.

[0010] The present invention also provides the application of the above-mentioned near-infrared II photothermal diagnostic agent in the preparation of drugs that enhance anti-tumor immune responses.

[0011] This invention also provides a therapeutic diagnostic nanoparticle, comprising an amphiphilic polymer with a post-modifiable maleimide group as the encapsulation shell and the aforementioned near-infrared II photothermal diagnostic agent as the core. The amphiphilic polymer can be used to encapsulate the near-infrared II photothermal diagnostic agent and enhance its water solubility and biocompatibility.

[0012] Furthermore, the therapeutic diagnostic nanoparticles are surface-modified with cyclic RGD peptides to enhance their targeting of tumor cells.

[0013] The present invention also provides the application of the above-mentioned therapeutic and diagnostic nanoparticles in the preparation of cancer cell therapeutic drugs.

[0014] The present invention also provides the application of the above-mentioned therapeutic and diagnostic nanoparticles in the preparation of drugs that enhance antitumor immune responses.

[0015] The present invention also provides the application of the above-mentioned near-infrared II photothermal diagnostic agent or the above-mentioned therapeutic diagnostic nanoparticles in the field of fluorescence imaging for non-therapeutic purposes.

[0016] The therapeutic and diagnostic nanoparticles of this invention have a photothermal conversion efficiency of ≥50% under 808nm laser irradiation.

[0017] The method for treating cancer cells using the aforementioned therapeutic diagnostic nanoparticles includes the following steps:

[0018] To bring the target cancer cells into contact with the therapeutic diagnostic nanoparticles;

[0019] The nanoparticles convert light energy into heat energy to induce apoptosis in cancer cells by irradiating the target cancer cells with near-infrared laser.

[0020] The treatment process of cancer cells is monitored using fluorescence imaging technology, which is selected from confocal microscopy, photoacoustic microscopy, or near-infrared imaging.

[0021] In the method of using the above-mentioned therapeutic diagnostic nanoparticles to treat cancer cells, it is also possible to activate the immune system through photothermal effects.

[0022] The method for using the above-mentioned therapeutic diagnostic nanoparticles to inhibit tumor growth or eliminate tumors in mammals includes the following steps:

[0023] Applying therapeutic and diagnostic nanoparticles to mammals;

[0024] Locate the tumor site using imaging techniques;

[0025] When a near-infrared laser is used to irradiate a tumor site, the therapeutic diagnostic nanoparticles convert light energy into heat energy, raising the local temperature to above 45°C to stop tumor growth or achieve tumor elimination.

[0026] In this invention, photothermal therapy can activate the immune system, thereby further enhancing the systemic anti-tumor immune response.

[0027] The photothermal diagnostic agents or therapeutic nanoparticles of the present invention can achieve anti-tumor treatment through the synergistic effect of photothermal therapy and immune activation.

[0028] Compared with the prior art, the present invention has the following advantages and technical effects:

[0029] This invention proposes a molecular design strategy of "central planarity + peripheral twisting" to construct a near-infrared (NIR-II) phototherapy agent, 4TPE-TB, addressing the challenges related to upstream and downstream processes of excited-state energy. The design principle of 4TPE-TB is based on two key principles. First, for upstream extension, the planarized molecular central framework enhances intramolecular conjugation, thereby achieving longer excitation wavelengths and higher photon absorption. Second, for downstream regulation, introducing multiple twisted TPE units into the peripheral structure minimizes intermolecular stacking, prevents fluorescence quenching during aggregation, and maintains intramolecular motion for efficient photothermal conversion. Therefore, 4TPE-TB NPs exhibit excellent performance in achieving enhanced and longer-wavelength photon absorption while balancing fluorescence emission and photothermal conversion capabilities. These properties enable high-resolution near-infrared (NIR-II) fluorescent probes (FLI) and effective photothermal therapy (PTT) for cancer. Furthermore, 4TPE-TB NPs can initiate a systemic immune response, promoting durable anti-tumor immunity. In summary, this invention provides a simple and effective method for designing high-performance NIR-II phototherapy agents and optimizes the input and output of excited-state energy, providing a multifunctional platform for realizing NIR-II excitation light irradiation (FLI) and photothermal therapy (PTT), and paving the way for the development of multimodal imaging-guided photothermal therapy systems for clinical applications. Attached Figure Description

[0030] 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:

[0031] Figure 1 For compound 2 in CDCl3 1 H NMR spectrum (298K).

[0032] Figure 2 For compound 2 in CDCl3 13 C NMR spectrum (298K).

[0033] Figure 3 2TPE-oTB in CDCl3 1 H NMR spectrum (298K).

[0034] Figure 4 2TPE-oTB in CDCl3 13 C NMR spectrum (298K).

[0035] Figure 5 For compound 4 in CDCl3 1 H NMR spectrum (298K).

[0036] Figure 6 For compound 4 in CDCl3 13 C NMR spectrum (298K).

[0037] Figure 7 For compound 2TPE-mTB in CDCl3 1 H NMR spectrum (298K).

[0038] Figure 8 For compound 2TPE-mTB in CDCl3 13 C NMR spectrum (298K).

[0039] Figure 9 For compound 7 in CDCl3 1 H NMR spectrum (298K).

[0040] Figure 10 For compound 7 in CDCl3 13 C NMR spectrum (298K).

[0041] Figure 11 4TPE-TB in CDCl3 1 H NMR spectrum (298K).

[0042] Figure 12 4TPE-TB in CDCl3 13 C NMR spectrum (298K).

[0043] Figure 13 ESI-MS plot of 2TPE-oTB.

[0044] Figure 14 ESI-MS plot of 2TPE-mTB.

[0045] Figure 15 ESI-MS image of 4TPE-TB.

[0046] Figure 16The calculation and characterization results of three nanoparticles, 2TPE-oTB, 2TPE-mTB, and 4TPE-TB, are shown in Figure a. a represents the ground-state structure of the three molecules optimized at the B3LYP / 6-31G(d) level; b represents the calculated HOMO and LUMO of the three molecules; ce represents the contributions of bond length, bond angle, and dihedral angle of 2TPE-oTB, 2TPE-mTB, and 4TPE-TB to the total recombination energy, respectively. Inset: Comparison of the optimized structures calculated for the ground-state (blue) and excited-state (red) electronic states; f and g represent the absorption and emission spectra of 2TPE-oTB, 2TPE-mTB, and 4TPE-TB in a 10 μM THF solution, respectively; h summarizes the photophysical properties of the three nanoparticles.

[0047] Figure 17 This document presents a schematic diagram of the nanoparticle preparation process and characterization results. Figure a shows the process flow diagram for preparing 2TPE-oTB, 2TPE-mTB, and 4TPE-TB nanoparticles using DSPE-PEG2000-Mal as the surfactant and cyclic RGD as the post-modifier. Figure b shows the DLS analysis results of these three nanoparticles in PBS. Figure c shows the stability analysis results of the size changes of these three nanoparticles after 30 days of placement in PBS at 4°C. Insets: photographs of 2TPE-oTB nanoparticles (left), 2TPE-mTB nanoparticles (middle), and 4TPE-TB nanoparticles (right). Figure d shows the stability analysis results of the nanoparticles at 80°C. The photobleaching analysis results of the above three nanoparticles and ICG under 8nm laser irradiation for 60 minutes were obtained; e represents the normalized absorption spectrum of the three nanoparticles 2TPE-oTB, 2TPE-mTB, and 4TPE-TB, with colored numbers representing the maximum absorption peak of NPs and dashed lines representing the wavelength of 980nm; f represents the normalized fluorescence spectrum of the three NPs, with colored numbers representing the maximum emission peak of NPs; g represents the product of the absorbance (A980) of the three NPs at 980nm and its fluorescence quantum yield (Φ), representing the brightness of the NPs (NPs concentration: 0.1mg / mL); h represents the 808nm laser (0.8W / cm²). 2 The photothermal conversion behavior of three NPs with PBS under irradiation; i represents the laser beam at 808 nm (0.8 W / cm²). 2 Photothermal stability of three types of NPs under irradiation in 5 heating-cooling cycles; j represents the PCE calculation results of 4TPE-TB NPs.

[0048] Figure 18The results of in vitro cell experiments with 4TPE-TB NPs are shown. Image a is a confocal laser scanning microscopy (CLSM) image showing cell uptake of 4TPE-TB NPs (scale bar: 20 μm); image b shows different concentrations of 4TPE-TB NPs and different irradiations (dark or 808 nm laser, 1 W / cm²). 2 Effects of 10 minutes on cell viability; c represents different treatments (PBS, PBS+L, 4TPE-TB NPs (50μM) and 4TPE-TB NPs (50μM)+L; L: 808nm laser, 1W / cm 2 CLSM images of live / dead 4T1 cells 24 hours after irradiation (scale bar: 50 μm).

[0049] Figure 19 The image shows NIR-II imaging results of 4TPE-TB NPs, where a represents the imaging results of different concentrations (0.05, 0.1, 0.2, 0.4 mg / mL) using a 980 nm laser with an LP 1300 filter (500 ms). -1 The images presented are as follows: a) NIR-II images of 2TPE-oTB NPs, 2TPE-mTB NPs, 4TPE-TB NPs, and ICG in PBS; b) Quantitative brightness calculation results of 2TPE-oTB NPs, 2TPE-mTB NPs, 4TPE-TB NPs, and ICG under the above conditions; c) NIR-II images of 4TPE-TB NPs and ICG in glass capillaries with different thicknesses of fat emulsion (0, 1, 2, 3, 4 mm) using a 980 nm laser with an LP1300 filter (500 ms); d) Quantitative brightness values ​​of 4TPE-TB NPs and ICG at different fat emulsion thicknesses; e) In vivo fluorescence angiography of BALB / c nude mice using 4TPE-TB NPs, with a laser wavelength of 980 nm and an LP1300 filter. 1300, exposure time 700ms; f is the cross-sectional fluorescence intensity distribution of abdominal blood vessels (blue line in e); g is the cross-sectional fluorescence intensity distribution of cecal blood vessels (red line in e); h is the biodistribution of 4TPE-TB NPs in tumor-bearing mice after intravenous injection at different times, with pink dashed circles representing tumors; i is the quantitative brightness value of the tumor at different times.

[0050] Figure 20 The results of in vivo photothermal therapy using 4TPE-TB NPs on a BalB / c female mouse xenograft 4T1 mammary tumor model are shown. In this figure, 'a' represents the injection of PBS or 4TPE-TB NPs (1 mg / mL, 200 μL, 1 W / cm²) into the mice. 2After irradiation at 808 nm, the temperature changes at the tumor site were observed; b and c showed the tumor weight images and corresponding quantitative values ​​collected after PTT treatment in the PBS, PBS+L, 4TPE-TB NPs, and 4TPE-TB NPs+L groups, respectively; d and e showed the tumor volume and body weight curves of mice after PTT treatment in the PBS, PBS+L, 4TPE-TB NPs, and 4TPE-TB NPs+L groups, respectively; f showed the H&E and TUNEL staining results of tumor sections after different treatments in the PBS, PBS+L, 4TPE-TB NPs, and 4TPE-TB NPs+L groups.

[0051] Figure 21 Images of H&E staining of major organs, including the kidney, liver, lung, spleen, heart, intestines, and brain, after processing with PBS and 4TPE-TB NPs under both light and dark conditions. Scale bars are 100 μm.

[0052] Figure 22 In the figure, a represents the immunofluorescence staining of CD3 / CD4, CD3 / CD8, and CD80 / CD86 in tumor sections after photothermal treatment with 4TPE-TB NPs, with a scale bar of 50 μm; b and d represent the statistical data of CD80 / CD86, CD3 / CD8, and CD3 / CD4 positive cells in tumor sections after photothermal treatment with 4TPE-TB NPs, respectively. *** represents P<0.001. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Unless otherwise specified, the room temperature in this invention is 25±2℃.

[0057] All raw materials used in the embodiments of this invention were purchased commercially. As an example, DSPE-PEG2000 and DSPE-PEG-Mal were both purchased from Sigma-Aldrich.

[0058] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0059] The technical solution of the present invention will be further illustrated by the following embodiments.

[0060] Example 1

[0061] This embodiment provides a method for preparing a near-infrared II photothermal therapeutic agent, the route of which is as follows:

[0062]

[0063] The specific steps are as follows:

[0064] Synthesis of 2-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4-dodecylthiophene (compound 2):

[0065] Under N2 protection, a mixture of 4,4'-(2-(4-bromophenyl)-2-styrene-1,1-diyl)bis(methoxybenzene) (compound 1, 1.0 g, 2.1 mmol), tributyl(4-dodecylthiophene-2-yl)stanane (200 mg, 2.5 mmol), Pd2(dba)3 (97 mg, 0.11 mmol), and P(o-tol)3 (258 mg, 0.85 mmol) was added to anhydrous toluene (10 mL). The mixture was stirred at 110°C for 12 hours; then, the reaction mixture was cooled and poured into an aqueous KF solution. The mixture was extracted three times with diethyl ether, and the organic phases were combined. The mixture was washed successively with water and brine (saturated brine, the same below), dried over Na2SO4, concentrated under reduced pressure, and the residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 5:1 (volume ratio, the same below)) to give a pale yellow oily product (981 mg, yield 72%), which is compound 2. 1HNMR (500MHz, CDCl3) δ7.37(d,J=8.5Hz,2H),7.17-7.10(m,4H),7.08(dd,J=7.9,1.8Hz,2H),7.02(t,J=8.9Hz,4H),6.97(d,J=8.8Hz,2H),6.85(d,J= 1.3Hz,1H),6.68(dd,J=10.8,8.7Hz,4H),3.78(d,J=4.0Hz,6H),2.61(t,J =7.7Hz,2H),1.69-1.61(m,2H),1.40-1.26(m,18H),0.92(t,J=6.9Hz,3H). 13 C NMR (126MHz, CDCl3) δ192.70,158.17,158.09,144.28,144.18,143.88,143.45, 140.30,138.71,136.36,136.33,132.63,132.19,131.85,131.48,127.74,126. 18,124.81,124.22,119.22,113.14,113.00,55.11,31.96,30.65,30.50,29.71 ,29.68,29.63,29.51,29.39,29.35,22.73,14.17.ESI-MS: m / z=643.3612([M+H] + calcd.for C 44 H 51 O2S + :643.3605).

[0066] Synthesis of 5-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-3-dodecylthiophen-2-yl)trimethylstannane (compound 3):

[0067] Under nitrogen protection at -78°C, n-butyllithium (0.62 mL, 2.4 M hexane solution, 1.50 mmol) was added to an anhydrous THF (12 mL) solution of compound 2 (800 mg, 1.25 mmol). The mixture was stirred at the same temperature (i.e., -78°C) for 1 hour. Then, trimethyltin chloride (1.6 mL, 1.0 M hexane solution, 1.62 mmol) was added, and the reaction was heated to room temperature and stirred overnight. The reaction mixture was quenched with KF aqueous solution, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product (i.e., compound 3). It can be used without further purification.

[0068] Synthesis of 2TPE-oTB:

[0069] Under a nitrogen atmosphere, a mixture of compound 3 (297 mg, 0.37 mmol), 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (50 mg, 0.14 mmol), Pd2(dba)3 (13 mg, 0.014 mmol), and P(o-tol)3 (35 mg, 0.11 mmol) was added to anhydrous toluene (5 mL). The reaction mixture was stirred at 110 °C for 12 hours. The reaction mixture was then cooled and poured into an aqueous KF solution, and the mixture was extracted three times with diethyl ether. The combined organic phases were washed successively with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 1:1) to give 161 mg of a deep blue solid product (77% yield), which was 2TPE-oTB. 1 H NMR (500MHz, CDCl3) δ7.49(d,J=8.2Hz,4H),7.44(s,2H),7.21-7.13(m,6H),7.13-7.05(m,8H),7.01(dd,J=15.7,8.7Hz,8H),6.70(dd ,J=15.7,8.8Hz,8H),3.80(d,J=14.7Hz,12H),2.59(t,J=7.8Hz,4H),1.65(p,J=7.5Hz,4H),1.36-1.08(m,36H),0.90(t,J=7.0Hz,6H). 13 C NMR (126MHz, CDCl3) δ158.26,158.15,153.25,146.91,145.67,144.09,140.57, 138.71,136.31,136.29,132.67,131.92,131.76,131.52,128.53,127.80,126. 25,125.27,125.06,116.15,113.18,113.03,55.13,31.94,30.46,30.34,29.68 ,29.65,29.62,29.53,29.48,29.38,22.72,14.17.ESI-MS: m / z=1475.6483([M] + calcd.for C 94 H 98 N4O4S4:1475.65O4).

[0070] Synthesis of 2-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-3-dodecylthiophene (compound 4):

[0071] Under N2 protection, a mixture of 4,4'-(2-(4-bromophenyl)-2-styrene-1,1-diyl)bis(methoxybenzene) (compound 1, 1.0 g, 2.1 mmol), tributyl(3-dodecylthiophene-2-yl)stanane (200 mg, 2.5 mmol), Pd2(dba)3 (97 mg, 0.11 mmol), and P(o-tol)3 (258 mg, 0.85 mmol) was added to anhydrous toluene (10 mL). The reaction mixture was stirred at 110 °C for 12 hours. Then, the reaction mixture was cooled and poured into an aqueous KF solution. The mixture was extracted three times with diethyl ether, and the organic phases were combined, washed successively with water and brine, dried over Na2SO4, concentrated under reduced pressure, and the residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 5:1) to give a colorless oily product (927 mg, yield 68%), which is compound 4. 1 H NMR (400MHz, CDCl3) δ7.23-7.03(m,10H),7.03-6.93(m,5H),6.67(dd,J=8.7,3.7Hz,4H),3. 77(s,6H),2.67-2.59(m,2H),1.64-1.54(s,2H),1.36-1.22(m,18H),0.91(t,J=6.7Hz,3H). 13 C NMR (101MHz, CDCl3) δ158.20,158.10,144.16,143.22,140.43,138.78,138.60, 137.77,136.34,136.29,132.65,132.62,132.47,131.48,131.43,129.65,128.4 4,127.74,126.17,123.31,113.05,113.01,55.10,31.95,30.94,29.73,29.67, 29.62,29.52,29.47,29.38,28.78,22.71,14.15.ESI-MS: m / z=1476.6483([M+H] + calcd.for C 94 H 98 N4O4S4:1476.6504).ESI-MS:m / z=643.3609([M+H] + calcd.for C 44 H51 O2S + :643.3605).

[0072] Synthesis of (5-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4-dodecylthiophen-2-yl)trimethylstannane (compound 5):

[0073] Under nitrogen protection at -78°C, n-butyllithium (0.62 mL, 2.4 M hexane solution, 1.50 mmol) was added to an anhydrous THF (12 mL) solution of compound 4 (800 mg, 1.25 mmol). The mixture was stirred at the same temperature for 1 hour, then trimethyltin chloride (1.6 mL, 1.0 M hexane solution, 1.62 mmol) was added. The mixture was heated to room temperature and stirred overnight. The reaction mixture was quenched with KF aqueous solution, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product, which could be used without further purification. The crude product obtained was compound 5.

[0074] Synthesis of 2TPE-mTB:

[0075] Under a nitrogen atmosphere, anhydrous toluene (5 mL) was added to a mixture of compound 5 (297 mg, 0.37 mmol), 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (50 mg, 0.14 mmol), Pd2(dba)3 (13 mg, 0.014 mmol), and P(o-tol)3 (35 mg, 0.11 mmol). The reaction mixture was stirred at 110 °C for 12 hours. Then, the reaction mixture was cooled and poured into an aqueous KF solution. The mixture was extracted three times with diethyl ether. The combined organic phases were washed successively with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 1:1) to give a brown solid (161 mg, yield 77%), which is 2TPE-mTB. 1 H NMR (500MHz, CDCl3) δ8.86(s,2H),7.39(d,J=8.2Hz,4H),7.23-7.10(m,14H),7.05(d,J=8.8Hz,4H),7.01(d,J=8.7Hz,4H),6.73(d,J=8.8H z,4H),6.69(d,J=8.8Hz,4H),3.80(d,J=6.6Hz,12H),2.81(t,J=7.9Hz,4H),1.82-1.72(m,4H),1.47-1.21(m,36H),0.90(t,J=6.8Hz,6H).13 C NMR (126MHz, CDCl3) δ158.28,158.14,151.14,144.18,144.06,143.87,140.65,14 0.05,138.75,136.36,136.28,135.75,132.72,132.68,132.25,131.65,131.55,1 28.28,127.81,126.25,113.18,113.04,112.97,55.14,31.97,30.98,29.78,29.7 4,29.72,29.68,29.56,29.42,29.08,22.73,14.17.ESI-MS: m / z=1476.6644([M+H] + calcd.for C 94 H 99 N4O4S4:1476.6578).

[0076] Synthesis of 2,3-bis(4-(2,2-bis(4-hexyloxy)phenyl)-1-phenylvinyl)phenyl)thiophene (compound 7):

[0077] Under N2 protection, a mixture of (4-(2,2-bis(4-hexoxy)phenyl)-1-phenylvinyl)phenyl)tributyltinane (compound 6, 1.7 g, 2.1 mmol), 2,3-dibromothiophene (200 mg, 0.83 mmol), Pd2(dba)3 (38 mg, 0.041 mmol), and P(o-tol)3 (101 mg, 0.33 mmol) was added to anhydrous toluene (5 mL). The reaction mixture was stirred at 110 °C for 12 hours. Then, the reaction mixture was cooled and poured into an aqueous KF solution. The mixture was extracted three times with diethyl ether, and the organic phases were combined, washed successively with water and brine, dried over Na2SO4, concentrated under reduced pressure, and the residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 2:1) to give a viscous yellow oil (713 mg, yield 75%), which is compound 7. 1 H NMR (400MHz, CDCl3) δ7.28 (d, J=5.0Hz, 1H), 7.25-6.97 (m, 27H), 6.79-6.61 (m, 8H ),4.07-3.84(m,8H),1.91-1.74(m,8H),1.58-1.33(m,24H),1.07-0.87(m,12H). 13C NMR (101MHz, CDCl3) δ157.82,157.80,157.75,144.18,144.06,143.76,143.21,140.72,140.52,138.89 ,138.69,138.56,137.88,136.23,136.16,136.12,136.09,134.26,132.73,132.69,132.07,131.49,13 1.37,131.32,130.34,128.60,128.47,127.79,127.76,126.20,126.15,123.78,113.63,113.56,113.5 1,77.47,77.15,76.83,67.87,31.74,31.72,29.39,25.85,22.70,14.17.ESI-MS: m / z=1145.6487([M+H] + calcd.for C 80 H 89 O4S + :1145.6477).

[0078] Synthesis of (4,5-bis(4-(2,2-bis(4-(hexoxy)phenyl)-1-phenylvinyl)phenyl)thiophen-2-yl)trimethylstannane (compound 8):

[0079] Under nitrogen protection at -78°C, n-butyllithium (0.31 mL, 2.4 M hexane solution, 0.73 mmol) was added to an anhydrous THF (6 mL) solution of compound 7 (700 mg, 0.61 mmol). The mixture was stirred at the same temperature for 1 hour, then trimethyltin chloride (0.80 mL, 1.0 M hexane solution, 0.80 mmol) was added. The mixture was heated to room temperature and stirred overnight. The reaction mixture was quenched with KF aqueous solution, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to obtain the crude product, which could be used without further purification. The crude product was compound 8.

[0080] Synthesis of 4TPE-TB:

[0081] Under a nitrogen atmosphere, anhydrous toluene (5 mL) was added to a mixture of compound 8 (464 mg, 0.36 mmol), 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (50 mg, 0.14 mmol), Pd2(dba)3 (13 mg, 0.014 mmol), and P(o-tol)3 (35 mg, 0.11 mmol). The reaction mixture was stirred at 110 °C for 12 hours. The reaction mixture was then cooled and poured into a KF aqueous solution. The mixture was extracted three times with diethyl ether. The combined organic phases were washed successively with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (stationary phase: silica gel; eluent: n-hexane:dichloromethane = 1:2) to give a brown solid (285 mg, yield 81%), which was 4TPE-TB. 1 H NMR (500MHz, CDCl3) δ9.01 (s, 2H), 7.26-7.20 (m, 8H), 7.20-7.09 (m, 20H), 7.06 (d, J = 7.9Hz, 4H), 7.03-6.96 ( m,20H),6.74-6.60(m,16H),3.97-3.82(m,16H),1.85-1.68(m,16H),1.53-1.24(m,48H),0.98-0.84(m,24H). 13 C NMR (126MHz, CDCl3) δ157.83,157.80,157.73,151.09,144.35,144.29,144.18,143.99,14 3.50,140.84,140.50,139.21,138.84,138.62,136.17,136.14,136.05,135.92,134.10,1 32.72,132.66,131.76,131.45,131.43,128.60,128.43,127.77,126.19,126.12,113.57, 113.51,112.93,67.86,31.66,29.33,25.79,22.64,14.09.ESI-MS: m / z=2503.1965([M+Na] + calcd.for C 166 H 174 N4O8S4Na + :2503.2141).

[0082] All synthesized molecules and their intermediates were produced using... 1 H NMR, 13Comprehensive characterization was performed using C10 NMR and high-resolution mass spectrometry (HRMS). Detailed spectral data for all compounds are available as follows: Figure 1-15 As shown, the successful synthesis and structural integrity of the designed molecule are confirmed.

[0083] The optimized ground state configuration is calculated based on density functional theory (DFT). Figure 16 In Figure a), the results show that the dihedral angle between the BBT core and the thiophene π-bridge in 2TPE-oTB is approximately 47°, significantly larger than the dihedral angles observed in 2TPE-mTB (~0°) and 4TPE-TB (~2°). This increased molecular planarity enhances intramolecular charge transfer (ICT), which should increase the molar absorptivity of both 2TPE-mTB and 4TPE-TB and cause a redshift in the absorption / emission wavelengths. Notably, while both 2TPE-mTB and 4TPE-TB primarily exhibit a planar central region, it is speculated that the more crowded TPE portion on the periphery of 4TPE-TB may play a role in suppressing π-π stacking. This structural arrangement may contribute to maintaining the robust emission of 4TPE-TB in its aggregated state. Figure 16 Figure b shows that the band gaps (ΔEg) between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) of 2TPE-oTB, 2TPE-mTB, and 4TPE-TB are 1.47, 1.28, and 1.22 eV, respectively. Notably, the smallest ΔEg observed in 4TPE-TB can be attributed to more efficient conjugation and a stronger ICT effect promoted by more TPE moieties. Furthermore, the recombination energies (λ) of these three molecules were analyzed to reveal the contribution of intramolecular motion to photoexcited nonradiative decay. Figure 16 (Ce). The λ values ​​of 2TPE-oTB, 2TPE-mTB, and 4TPE-TB are 10653, 1560, and 1287 cm⁻¹, respectively. -1 The calculated root mean square deviations (RMSDs) for the ground state and excited state configurations were 1.63 and 0.29, respectively. Overall, from a theoretical perspective, 4TPE-TB consists of a coplanar center and a highly distorted periphery, yet it still possesses a rigid structure, suggesting that 4TPE-TB may have the potential to serve as a multifunctional reagent for enhancing photon absorption.

[0084] 1. Photophysical properties

[0085] The photophysical properties of these three molecules in solution were systematically evaluated. For example... Figure 16 As shown in Figure f, all compounds exhibit significant charge-transfer absorption peaks with absorption tails extending into the near-infrared region. Among the three, 2TPE-oTB shows the largest blue-shift absorption, with a peak at 669 nm and an ε of 1.7 × 10⁻⁶. 4 M-1 cm -1 Conversely, the absorption spectrum of 2TPE-mTB showed a significant redshift, with a peak at 809 nm and an increase in ε to 2.4 × 10⁻⁶. 4 M -1 cm -1 Notably, 4TPE-TB exhibits the largest redshift absorption peak at 813 nm, with an absorption tail extending over 1000 nm. Furthermore, its molar absorptivity is significantly higher than the other two molecules, almost doubling to 4.0 × 10⁻⁶. 4 M -1 cm -1 . Figure 16 The fluorescence spectra of 2TPE-oTB, 2TPE-mTB, and 4TPE-TB, as shown in Figure g, exhibit maximum emission peaks at 975 nm, 999 nm, and 1003 nm, respectively. This fluorescence trend is consistent with the observed absorption spectra. Compared to 2TPE-oTB, 2TPE-mTB and 4TPE-TB show almost overlapping absorption and fluorescence spectra, with a smaller wavelength redshift and Stokes shift (Δλ), which is consistent with the calculated results. Figure 16 As shown in Figure h, the experimental data indicate that 4TPE-TB does indeed exhibit excellent photophysical properties, including enhanced molar absorbance and longer excitation and emission wavelengths, which should improve the "upstream" process of excited state energy.

[0086] 2. Preparation and characterization of nanoparticles

[0087] To improve the biocompatibility and targeting of the aforementioned phototherapy agents, nanoparticles were prepared using the amphiphilic copolymer DSPE-PEG2000-Mal with post-modifiable maleimide groups as the encapsulation matrix. The specific preparation method is as follows: 1 mg of 4TPE-TB, 1 mg of DSPE-PEG2000, and 1 mg of DSPE-PEG-Mal were dissolved in 1 mL of tetrahydrofuran (THF) and sonicated. Then, this solution was mixed with 9 mL of deionized water and sonicated for 2 minutes using an ultrasonic probe (VCX150, Sonics) at 75 W output power. Subsequently, THF was evaporated under a nitrogen flow to obtain maleimide-functionalized nanoparticles. The obtained maleimide-functionalized nanoparticles were then combined with cRGD peptide (purchased from Xi'an Ruixi Biotechnology Co., Ltd.): cRGD stock solution (5 × 10⁻⁶) was added to the cRGD peptide. -2M, 30 μL) was added to the maleimide-functionalized nanoparticle solution, and stirred for 12 hours. The mixture was then transferred to a dialysis membrane (MWCO: 3500 Da) and dialyzed against fresh MilliQ water for 24 hours (the MilliQ water was changed every 4 hours to ensure complete removal of unreacted cRGD). 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 4TPE-TB nanoparticles (4TPE-TB NPs). 2TPE-oTB NPs and 2TPE-mTB NPs were obtained using the same method. A schematic diagram of the above nanoparticle synthesis process is shown below. Figure 17 As shown in Figure a. To enhance the precise tumor-tracking capability of nanoparticles, cyclic RGD peptides (cRGD) were functionalized onto the surface of nanoparticles via a click reaction. Figure 17 As shown in Figure b, the average hydrodynamic diameters of the obtained 2TPE-oTB NPs, 2TPE-mTB NPs, and 4TPE-TB NPs, as determined by dynamic light scattering (DLS), were 116 nm, 91 nm, and 93 nm, respectively. Furthermore, they exhibited a narrow polydispersity index (PDI) of less than 0.2, indicating a uniform size distribution. To assess the stability of the nanoparticles, the change in their hydrodynamic diameter over time was monitored in phosphate-buffered saline (PBS, pH = 7.4). The average particle size of the three nanoparticles remained consistent over one month, indicating excellent stability under physiological conditions. Figure 17 (c) Furthermore, the photostability of the nanoparticles was evaluated by measuring their fluorescence emission under prolonged illumination and comparing the results with those of commercially available ICG (purchased from TCI (TCI (Shanghai) Chemical Industry Development Co., Ltd.)). When exposed to 808 nm laser irradiation, the ICG almost completely quenched its fluorescence within 5 minutes. Figure 17 (d). In stark contrast, even after one hour of continuous illumination, the fluorescence intensity of these three nanoparticles did not decrease significantly.

[0088] The absorption and emission properties of the nanoparticles were then evaluated. The absorption peaks of all three types of nanoparticles showed a redshift compared to their respective solution states. Figure 17 (e). Specifically, the maximum absorption peaks of 2TPE-oTB NPs, 2TPE-mTB NPs, and 4TPE-TB NPs are located at 698 nm, 829 nm, and 855 nm, respectively. Notably, both 2TPE-mTB and 4TPE-TB nanoparticles show absorption at 808 nm and 980 nm, respectively, while 2TPE-oTB nanoparticles show no response to excitation at 980 nm. Fluorescence spectroscopy ( Figure 17 Further, in section f), it is shown that compared to 2TPE-oTB nanoparticles (976 nm), the emission wavelengths of 2TPE-mTB nanoparticles and 4TPE-TB nanoparticles (1102 nm and 1037 nm, respectively) are red-shifted into the near-infrared II window. Notably, 4TPE-TB NPs exhibit a higher fluorescence signal than 2TPE-mTB NPs, indicating resistance to fluorescence quenching upon aggregation. This is because the twisted edges of 4TPE-TB suppress intermolecular stacking. Besides the fluorescence quantum yield (Φ), the absorbance of NPs is also an important factor affecting NIR-II brightness. Figure 17 Figure g shows the fluorescence intensity of the three NPs (0.1 mg / mL) under 980 nm excitation, defined as the product of Φ and the absorbance at that wavelength (A980). The 4TPE-TB NPs exhibited the best NIR-II fluorescence intensity, which was 6 times and 23 times higher than that of the 2TPE-mTB NPs and 2TPE-oTB NPs, respectively.

[0089] 3. Photothermal performance

[0090] Furthermore, the photothermal properties of nanoparticles are evaluated by monitoring temperature changes during laser irradiation. For example... Figure 17 As shown in h, at a power density of 0.8 W / cm² 2 Under 808 nm laser irradiation, the 2TPE-oTB nanoparticles exhibited the smallest temperature rise. In contrast, both the 2TPE-mTB and 4TPE-TB nanoparticles showed rapid and significant temperature rises, reaching 80 °C within 3 minutes, highlighting their efficient photothermal conversion capabilities. Furthermore, the nanoparticles underwent five consecutive heating-cooling cycles under 808 nm laser irradiation to evaluate their photothermal stability. The nanoparticles maintained stable performance without significant degradation, demonstrating their excellent thermal stability. Figure 17 (i). It is worth noting that the calculated PCE of 4TPE-TB NPs is 50%, which is likely due to the strong intramolecular motion of the four TPE units. Figure 17 (j). Overall, 4TPE-TB NPs outperform the other two classes of NPs as phototherapeutic agents, exhibiting superior performance in enhanced absorption at longer excitation wavelengths, higher NIR-II brightness, and efficient photothermal conversion. These results validate effective molecular design, enabling simultaneous modulation of upstream and downstream processes of excited-state energies in NIR-II materials, making 4TPE-TB NPs a highly promising candidate material for multimodal applications.

[0091] 4. In vitro cytotoxicity assessment and cell imaging

[0092] The fluorescence and photothermal properties of 4TPE-TB NPs were utilized to explore their application in cancer therapy. Initially, the fluorescence properties of 4TPE-TB NPs were used to track the uptake of NPs by tumor cells. Figure 18 As shown in Figure a, after 4 hours of incubation with 4T1 cells, a large accumulation of 4TPE-TB NPs was observed, mainly located in the cytoplasm. To assess the biocompatibility of 4TPE-TB NPs, in vitro cytotoxicity studies and cell imaging were performed. The results showed that at concentrations up to 50 μg / mL, the NPs exhibited almost no cytotoxicity under dark conditions. Figure 18 (b). However, when exposed to 808nm laser irradiation (1W / cm²), 2 After 24 hours, cell viability decreased in a concentration-dependent manner, decreasing by almost 100% at a concentration of 20 μg / mL.

[0093] Confocal laser scanning microscopy (CLSM) images of 4T1 cells stained with propidium iodide (PI) and calcein AM further validated this. Figure 18 (c) Cells in the 4TPE-TB NPs group maintained strong green fluorescence, indicating that 4TPE-TB NPs are non-toxic under dark conditions. Conversely, irradiation with an 808 nm laser (1 W / cm²) resulted in cell toxicity. 2 Cells treated with 4TPE-TB NPs for 10 minutes showed red fluorescence after 24 hours, indicating that the photothermal effect of 4TPE-TB NPs led to a large number of cell deaths. The control experiment without NPs showed negligible effect on cell viability, further confirming the powerful PTT efficacy of 4TPE-TB NPs. These in vitro cell experiments highlight the excellent biocompatibility and strong photothermal cancer cell killing ability of 4TPE-TB NPs.

[0094] In vitro and in vivo near-infrared II imaging excited at 5.980 nm

[0095] The near-infrared-II imaging performance of nanoparticles (NPs) was evaluated using an in vivo imaging system (IVIS), with ICG as a reference. A 980 nm excitation laser was employed to achieve deeper imaging. Figure 19 As shown in Figure a, only 2TPE-mTB NPs and 4TPE-TB NPs exhibited detectable near-infrared II fluorescence signals under a 1300 nm long-pass filter (LP1300), while 2TPE-oTB NPs and ICG showed negligible fluorescence under the same conditions. This result is consistent with the calculated near-infrared II brightness of the nanoparticles above. Quantitative fluorescence intensity analysis showed that the fluorescence intensity of 4TPE-TB NPs increased with increasing concentration (0.05-0.4 mg / mL), highlighting its excellent fluorescence performance under physiological conditions. Figure 19(b) Besides brightness, spatial resolution and penetration depth are also key evaluation metrics for fluorescence angiography (FLI). Therefore, using fat emulsion as a biomimetic tissue medium, the imaging performance of nanoparticles (NPs) at different depths (0-4 mm) was evaluated to assess their imaging depth. 4TPE-TB NPs and ICG were dispersed in mouse serum and injected into glass capillaries, which were then immersed in fat emulsion solutions at different depths. Figure 19 As shown in Figure c, 4TPE-TB NPs achieved high-fidelity imaging of glass capillaries, while the ICG group showed almost no fluorescence signal even without fat emulsion coverage (0 mm). Figure 19 Signal quantization in the middle d further confirmed that 4TPE-TB NPs can still retain fluorescence signals at tissue thicknesses up to 3 mm.

[0096] Based on the good penetration depth observed in vitro, the application potential of 4TPE-TB NPs in in vivo fluorescence angiography was further explored using BALB / c female nude mice as a model. 200 μL of 4TPE-TB NPs (1 mg / mL) were injected intravenously, followed by imaging in the NIR-II window using a 980 nm laser. Figure 19 As shown in Figure e, high-resolution NIR-II angiography was successfully achieved. The abdominal blood vessels of the mouse are clearly visible. Figure 19 The blue line in the middle (e) shows that the vessel diameter measured from the angiography image is approximately 0.17 mm and 0.15 mm. Figure 19 (f). Notably, the superior penetration depth of 4TPE-TB NPs enables clear imaging of the cecal structure. Figure 19 (The red line in the middle of the image). Furthermore, the vascular structures on the intestinal serosa showed four distinct fluorescence peaks, corresponding to vascular branch diameters of 0.18 mm, 0.13 mm, 0.20 mm, and 0.16 mm, respectively. Figure 19 (g). These results demonstrate that 4TPE-TB NPs can achieve high-resolution imaging to assess microvessels.

[0097] Furthermore, to evaluate the potential of 4TPE-TB NPs in tumor tracing, in vivo imaging of tumor sites in 4T1 tumor-bearing mice was performed. Bright fluorescent signals were observed at the tumor sites 12 hours after intravenous injection of 4TPE-TB NPs. Figure 19 (h). Fluorescence intensity peaked at 24 hours, exhibiting a high signal-to-noise ratio and clear tumor boundaries. Notably, strong fluorescence signals could still be detected at the tumor site for up to 72 hours post-injection. Figure 19(i) indicates that 4TPE-TB NPs have a prolonged retention time and stable tumor accumulation. To confirm the specific accumulation of 4TPE-TB NPs in tumors, mice were euthanized 72 hours after injection, and their tumors and major organs (e.g., liver, spleen, kidney, lung, and heart) were collected for near-infrared-II fluorescence imaging. Tumors exhibited significantly higher near-infrared-II fluorescence intensity. These findings highlight the superior tumor targeting ability and accumulation efficiency of 4TPE-TB NPs, as well as their excellent NIR-II imaging performance.

[0098] 6. Effects of internal phototherapy

[0099] To verify the anticancer potential and phototherapy efficacy of 4TPE-TB NPs in vivo, experiments were conducted using 4T1 tumor-bearing mice. The photothermal performance of 4TPE-TB NPs was first evaluated by monitoring tumor temperature during laser irradiation (photothermal imaging (PTI)). Figure 20 As shown in Figure a, the tumor temperature of mice treated with 4TPE-TB NPs (1 mg / mL, 200 μL) increased rapidly, even after irradiation with an 808 nm laser (1 W / cm²). 2 Under laser irradiation, the temperature of the tumor exceeded 50°C within one minute and reached approximately 60°C within five minutes. In contrast, the temperature change in the PBS+L group mice was negligible. These results indicate that 4TPE-TB NPs effectively accumulate at the tumor site under laser irradiation and generate sufficient heat to induce hyperthermia in the tumor.

[0100] To evaluate the antitumor efficacy of 4TPE-TB NPs, mice were randomly assigned to four groups: PBS group, PBS+L group, 4TPE-TB NPs group, and 4TPE-TB NPs+L group. Mice receiving 4TPE-TB NPs were administered a single dose via tail vein injection. For the laser therapy groups, the 4TPE-TB NPs+L group and the PBS+L group received three irradiations of 808nm laser for the first three days of the entire treatment period. Tumor size was measured every two days for 15 days to assess treatment outcomes. Figure 20 As shown in Figures b and c, the 4TPE-TB NPs+L group exhibited significant tumor growth inhibition compared to the other three groups. Notably, the tumors in this group were completely eradicated within 5 days, and no signs of recurrence were observed in the subsequent 10 days. In contrast, tumor growth remained unaffected in the PBS, PBS+L, and 4TPE-TB NPs groups, with no significant inhibition observed. Figure 20 (d). Notably, no significant changes in body weight were observed in any of the 15-day study groups. Figure 20(e) indicates that 4TPE-TB NPs are well-tolerated and biocompatible. These results strongly demonstrate the superior photothermal efficacy and antitumor activity of 4TPE-TB NPs, highlighting their potential as a highly effective and biocompatible cancer therapeutic agent.

[0101] To elucidate the mechanism of tumor elimination during phototherapy, tumor tissue was stained with hematoxylin and eosin (H&E) and truncation-end labeling of terminal deoxynucleotidyl transferase (dUTP) (TUNEL). Figure 20 (f). Compared with the three control groups, the 4TPE-TB NPs+L treatment group showed significant tumor cell destruction and tissue damage, indicating that photothermal therapy (PTT) has a powerful anticancer effect. Furthermore, TUNEL staining confirmed that the combined use of 4TPE-TB NPs and laser irradiation effectively induced tumor cell apoptosis, further validating its therapeutic mechanism. The in vivo safety of 4TPE-TB NPs was also assessed. Major organs were collected from mice treated with NPs and then subjected to H&E staining. No pathological abnormalities were detected in the organ samples from the 4TPE-TB NPs+L treatment group. Figure 21 The results indicate that 4TPE-TB NPs possess excellent biocompatibility. These results suggest that 4TPE-TB NPs have the potential for photothermal therapy under fluorescence imaging guidance, with minimal systemic toxicity.

[0102] 7. Assessment of immune response

[0103] To investigate whether 4TPE-TB NPs-mediated photothermal therapy can induce an immune response, immunofluorescence staining was used to analyze the proportions of immune cells such as CD80+ / CD86+ dendritic cells, CD3+ / CD8+ cytotoxic T cells, and CD3+ / CD4+ helper T cells in tumor tissue sections from different treatment groups. Figure 22 (a) Mature dendritic cells (DCs) migrate to tumor-draining lymph nodes and activate T cells through physical interactions between DCs and T cells. Significantly enhanced fluorescence of CD80+ / CD86+ dendritic cells in tumor tissue indicates that more dendritic cells are recruited to the tumor. Quantitative analysis showed that the number of CD80+ / CD86+ dendritic cells (DCs) in the 4TPE-TB NPs+L group was more than three times higher than that in the control groups (PBS+L and 4TPE-TB NPs). Figure 22 (b) Then, the inventors studied the distribution of activated T cells within tumor tissue. CD3+ / CD8+ and CD3+ / CD4+ T cells (which play roles in cancer cell killing and triggering immune responses, respectively) were significantly increased in the 4TPE-TB NPs+L group compared to other groups. The density of CD3+ / CD8+ T cells was approximately seven times higher (b). Figure 22(c) The number of CD3+ / CD4+ T cells was about five times higher. Figure 22 (d). These results demonstrate that 4TPE-TB NPs-mediated PTT not only effectively destroys primary tumors but also activates a robust immune response. This immune activation may contribute to systemic anti-tumor immunity, thereby reducing the likelihood of tumor recurrence. Therefore, these findings suggest that the dual role of 4TPE-TB NPs in direct tumor ablation and immune system activation enhances their potential as a powerful therapeutic platform for cancer treatment.

[0104] 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 compound for use as a near-infrared II photothermal diagnostic agent, characterized in that, The structure is as follows: 。 2. A method for preparing the compound for near-infrared II photothermal diagnostic and therapeutic agents according to claim 1, characterized in that, Starting with (4-(2,2-bis(4-hexoxy)phenyl)-1-phenylvinyl)phenyl)tributyltinane, compound 7 was prepared via a π-bridge of 2,3-dibromothiophene. Compound 7 was then reacted with trimethyltin chloride in an organic solvent under trimethyltin chloride catalysis to yield compound 8. Compound 8 was then mixed with 4,7-dibromobenzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) and coupled in a Pd2(dba)3 and P(o-tol)3 composite catalytic system to synthesize the compound for near-infrared II photothermal diagnostic agents. The structural formula of compound 7 is as follows: The structural formula of compound 8 is as follows: .

3. The use of the compound as described in claim 1 for use as a near-infrared II photothermal diagnostic agent in the preparation of cancer cell therapeutic drugs.

4. The use of a compound as described in claim 1 for use as a near-infrared II photothermal diagnostic agent in the preparation of drugs that enhance antitumor immune responses.

5. A therapeutic and diagnostic nanoparticle, characterized in that, The encapsulation shell is an amphiphilic polymer with a post-modifiable maleimide group, and the core is the compound of claim 1 for near-infrared II photothermal therapy.

6. The therapeutic and diagnostic nanoparticles according to claim 5, characterized in that, The therapeutic and diagnostic nanoparticles are surface-modified with cyclic RGD peptides.

7. The use of a therapeutic diagnostic nanoparticle as described in any one of claims 5-6 in the preparation of a cancer cell therapeutic drug.

8. The use of a therapeutic diagnostic nanoparticle as described in any one of claims 5-6 in the preparation of a drug that enhances antitumor immune response.

9. The use of a compound for near-infrared II photothermal diagnostic agents as described in claim 1, or the use of therapeutic diagnostic nanoparticles as described in any one of claims 5-6, in the preparation of contrast agents for fluorescence imaging.

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