Photothermal agent capable of effectively absorbing near-infrared light and having high photothermal conversion efficiency as well as preparation method and application of photothermal agent

Abstract

The invention provides a photo-thermal agent which can effectively absorb near-infrared light and has high photo-thermal conversion efficiency, and a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The photo-thermal agent capable of effectively absorbing near-infrared light and having high photo-thermal conversion efficiency is composed of a plurality of photo-thermal agent monomers, the structural formula of the photo-thermal agent monomers is # imgabs0, the photo-thermal conversion efficiency of the photo-thermal agent is as high as 90.2%, and the photo-thermal conversion efficiency of obtained nanoparticles is also as high as 78.0% and is far higher than that of a commercial photo-thermal agent indocyanine green (ICG).

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, a preparation method of the photothermal agent, and an application of the photothermal agent. Background Art

[0002] With the progress of the times and the improvement of people's living standards, more and more unhealthy lifestyles have led to a significant increase in the cancer incidence rate. Cancer has become a major global public health problem. At present, the traditional methods for treating cancer mainly include surgical treatment, chemotherapy, and radiotherapy. Surgical treatment is to remove solid tumors through invasive surgery. Surgical treatment requires incising the wound to remove the tumor site, which brings great pain to patients and cannot completely remove the lesion. It often needs to be removed repeatedly, seriously reducing the patient survival rate. Radiotherapy generally uses radiation to irradiate tissues at a local position, causing large-area cell death and tumor atrophy. In principle, radiotherapy can completely kill cancer cells, but high-energy rays will also kill normal cells and even endanger important organs near the tumor, causing great harm to the human body. Chemotherapy inhibits the growth and reproduction of tumor cells through high-dose chemotherapy drugs, thereby interfering with the life activities of tumor cells and finally killing tumor cells. Chemotherapy has large toxic and side effects, and long-term use will make cancer cells develop drug resistance, and cancer is likely to relapse or metastasize, and the treatment effect is limited.

[0003] In recent years, some new treatment methods have emerged continuously. Among them, photothermal therapy has attracted much attention from researchers. Photothermal therapy mainly relies on the action of photothermal conversion reagents to generate local high temperatures. When the tumor temperature reaches above 45 °C, it can cause apoptosis and necrosis of cells. At the same time, it is found that in addition to the direct toxicity to tumor cells, photothermal therapy also has the potential to induce immunogenic cell death of tumor cells and promote tumor immunotherapy. Photothermal conversion reagents are the key to this treatment method. A high-quality photothermal conversion reagent should have: (1) must have a strong light absorption ability; (2) preferably can absorb near-infrared light; (3) have a high photothermal conversion efficiency (PCE). Unfortunately, manufacturing a photothermal conversion reagent that can effectively absorb near-infrared light and has a high PCE usually requires a wide conjugate structure and enhanced electron delocalization. However, this excessive distortion of the structure inadvertently reduces their light absorption ability, resulting in poor output performance in bioimaging and phototherapy applications.

[0004] Therefore, there is an urgent need to study how to manufacture a high-quality photothermal conversion reagent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency (PCE). Summary of the Invention

[0005] In view of this, to solve the problem that there is currently no photothermal conversion reagent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, on the one hand, the present invention provides a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, and this photothermal agent can effectively absorb near-infrared light and has a high photothermal conversion efficiency.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, which is composed of several photothermal agent monomers, and the structural formula of the photothermal agent monomer is:

[0008]

[0009] On the other hand, the present invention provides a preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, including the following steps:

[0010] Step (1): Add p-bromobenzaldehyde and 2-methylpyrrole to anhydrous dichloromethane, add trifluoroacetic acid, react under nitrogen protection at room temperature. After the reaction is completed, add 2,3-dichloro-5,6-dicyanobenzoquinone to the system, stir and then add triethylamine to react, add boron trifluoride diethyl ether to react. After the reaction is completed, add saturated sodium chloride solution to quench the reaction, extract with dichloromethane, and purify by column chromatography to obtain intermediate BO-2;

[0011] Step (2): Add BO-2 and 4-diphenylaminobenzaldehyde to anhydrous ethanol, add a catalytic amount of piperidine and acetic acid to the system and reflux to react. After the reflux reaction is completed, purify by column chromatography to obtain product BO-4;

[0012] Step (3): Dissolve BO-4 in tetrahydrofuran, drop it into rapidly stirred ultrapure water, remove tetrahydrofuran, and centrifuge to obtain nanoparticles BO-4NPs of BO-4.

[0013] Preferably, in step (1), the molar ratio of p-bromobenzaldehyde, 2-methylpyrrole, 2,3-dichloro-5,6-dicyanobenzoquinone, and boron trifluoride diethyl ether is 1.0:3.0 - 25.0:1.0 - 3.0:6.0 - 9.0:6.0 - 12.0.

[0014] Preferably, in step (2), the molar ratio of BO-2 and 4-diphenylaminobenzaldehyde is 1.0:3.0.

[0015] Preferably, in step (3), the concentration of BO-4 is 5.0 - 7.5 mg / mL.

[0016] Preferably, in step (1), the total reaction time is 11.0 - 15.0 hours;

[0017] In step (2), the reflux temperature is 70 - 75 °C and the time is 11.0 - 15.0 hours.

[0018] Preferably, in step (3), the volume of ultrapure water is 30.0 - 40.0 mL.

[0019] In a third aspect, the present invention provides the use of the above-mentioned photothermal agent or the photothermal agent prepared by the preparation method of the photothermal agent in photoacoustic imaging.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned photothermal agent or the photothermal agent prepared by the preparation method of the photothermal agent in photothermal imaging.

[0021] In a fifth aspect, the present invention provides the use of the above-mentioned photothermal agent or the photothermal agent prepared by the preparation method of the photothermal agent in a photothermal agent for in vivo tumor treatment.

[0022] The present invention has the following beneficial effects compared with the prior art:

[0023] (1) The photothermal agent BO-4 prepared by the above-mentioned photothermal agent or the preparation method of the photothermal agent provided by the present invention can not only effectively absorb near-infrared light but also has a high photothermal conversion efficiency. The photothermal conversion efficiency is as high as 90.2%, and the photothermal conversion efficiency of the obtained nanoparticles BO-4NPs is also as high as 78.0%, which is far higher than that of the commercial photothermal agent indocyanine green (ICG).

[0024] (2) The present invention first uses bromophenyl and triphenylamine as highly efficient rotating molecular rotors to synthesize a photothermal conversion reagent, and its synthesis method is simple and efficient.

[0025] (3) The present invention has good biocompatibility and has a light-controlled release property.

[0026] (4) When the photothermal agent of the present invention is used in a photothermal agent for in vivo tumor treatment, the tumor ablation effect is obvious. After two treatments, the tumor site is basically completely disappeared, and the growth of metastatic tumors is significantly inhibited.

[0027] (5) When the present invention is used in photoacoustic imaging or photothermal imaging, it has good photoacoustic imaging and photothermal imaging capabilities. Description of the Drawings

[0028] Figure 1 1H-NMR spectra of BO-0, BO-1, BO-2, BO-3, and BO-4 of the present invention 1 1H-NMR spectra.

[0029] Figure 2 13C-NMR spectra of BO-0, BO-1, BO-2, BO-3, and BO-4 of the present invention 13 13C-NMR spectra.

[0030] Figure 3 Mass spectrometry characterization of BO-0, BO-1, BO-2, BO-3, and BO-4 of the present invention, HR-MS spectrum.

[0031] Figure 4 Fluorescence spectra and UV absorption spectra of BO-0, BO-1, BO-2, BO-3, BO-4, BO-0 NPs, BO-1 NPs, BO-2 NPs, BO-3 NPs, and BO-4 NPs of the present invention in aqueous solution. Among them, A) shows the fluorescence spectra and UV absorption spectra of BO-0 and BO-0 NPs in aqueous solution; B) shows the fluorescence spectra and UV absorption spectra of BO-1 and BO-1 NPs in aqueous solution; C) shows the fluorescence spectra and UV absorption spectra of BO-2 and BO-2 NPs in aqueous solution; D) shows the fluorescence spectra and UV absorption spectra of BO-3 and BO-3 NPs in aqueous solution; E) shows the fluorescence spectra and UV absorption spectra of BO-4 and BO-4 NPs in aqueous solution;

[0032] Figure 5 Heating, cooling, and fitting of BO-0, BO-1, BO-2, BO-3, and BO-4 of the present invention in aqueous solution. Among them, A), B), C), D), and E) correspond to BO-0, BO-1, BO-2, BO-3, and BO-4.

[0033] Figure 6 Fluorescence spectra of BO-0, BO-1, BO-2, BO-3, and BO-4 of the present invention at the same concentration in aqueous solution.

[0034] Figure 7 Photodynamic diagrams of BO-4 and BO-4 NPs of the present invention. Among them, A) shows the singlet oxygen generation ability diagram of BO-4 and BO-4 NPs under white light excitation; B) shows the singlet oxygen generation ability diagram of BO-4 and BO-4 NPs at the optimal excitation (using ABDA as the singlet oxygen scavenger and MB as the control); C) shows the reactive oxygen species generation ability diagram of BO-4 and BO-4 NPs under white light excitation; D) shows the reactive oxygen species generation ability diagram of BO-4 and BO-4 NPs at the optimal excitation (using DCFH as the reactive oxygen species scavenger and MB as the control).

[0035] Figure 8 Dynamic light scattering and SEM images of BO-4 NPs. Among them, A) is the dynamic light scattering diagram; B) is the SEM image.

[0036] Figure 9This is the photoacoustic imaging diagram of BO-4 NPs of the present invention. Among them, A) is the linear relationship diagram between the concentration of BO-4 NPs and the photoacoustic intensity; B) is the photoacoustic intensity diagram of ten scans of BO-4 NPs at 100 μg·mL-1; C) is the ultrasonic imaging and photoacoustic imaging diagram of ten scans of BO-4 NPs at 100 μg·mL-1.

[0037] Figure 10 This is the photothermal property test diagram of BO-4 and BO-4 NPs of the present invention. Among them, A) is the heating curve of BO-4 (0, 6, 8, 10, 20, 30 μM) irradiated by laser (730 nm, 1.0 W·cm -2 ) for 15 min; B) is the heating curve of 10 μM BO-4 irradiated by lasers with different powers (730 nm, 0.2, 0.4, 0.6, 0.8, 1.0 W·cm -2 ) for 15 min; C) is the ultraviolet spectrum of BO-4 before and after 3 minutes of light irradiation; D) is the heating curves of BO-4 NPs with different concentrations (0, 60, 80, 100, 150, 200 μg·mL -1 ) irradiated by laser (808 nm, 1.0 W·cm -2 ) for 15 min; E) is the heating curve of 100 μg·mL -1 BO-4 NPs irradiated by lasers with different powers (808 nm 0.2, 0.4, 0.6, 0.8, 1.0 W·cm -2 ) for 15 min; F) is the ultraviolet spectrum of BO-4 NPs before and after 3 minutes of light irradiation; G) is the fitting of the cooling curve of BO-4 NPs; H) is the photothermal cycle stability of BO-4 and BO-4 NPs.

[0038] Figure 11 This is the cytotoxicity analysis, live / dead cell staining, flow cytometry analysis of apoptosis and bright field image of cells of BO-4 NPs of the present invention. Among them, A) is the cytotoxicity analysis; B) is the live / dead cell staining; C) is the flow cytometry analysis of apoptosis; D) is the bright field image of cells.

[0039] Figure 12 This is the photoacoustic imaging diagram of BO-4 NPs of the present invention.

[0040] Figure 13 This is the photothermal therapy diagram of BO-4 NPs of the present invention. Among them, A) is the schematic diagram of the treatment; B) is the thermal imaging diagrams of the PBS + laser group and the BO-4 NPs + laser group under laser irradiation at different times; C) is the change of tumor volume over time in different treatment groups; D) is the change of mouse body weight; E) is the quantification result of the temperature at the tumor site; F) is the H&E staining of the tumor site; G) is the tumor picture after the treatment; H) is the H&E staining of the main organs of normal mice and mice injected with BO-4 NPs 48 hours later.

[0041] Figure 14 This is the immunotherapy diagram of the BO-4 NPs of the present invention. Among them, A) is the treatment schematic diagram; B) is the body weight of the mice; C) is the growth of the control tumor, primary tumor and metastatic tumor; D) is the content of CD+4 T cells and HMGB1 in the control tumor, primary tumor and metastatic tumor; E) is the H&E staining of the control tumor, primary tumor and metastatic tumor and the immunohistochemical diagram of the content of CD+4 T cells and HMGB1 therein; F) is the H&E staining of the treated mice and untreated mice. Detailed implementation mode

[0042] Fabricating a photothermal conversion reagent that can effectively absorb near-infrared light and has a high PCE usually requires the inclusion of extensive conjugated structures and enhanced electron delocalization. However, this excessive distortion of the structure inadvertently reduces their light absorption ability, resulting in poor output performance in bioimaging and phototherapy applications. Then, how can a high-quality photothermal conversion reagent be fabricated to effectively absorb near-infrared light and simultaneously have a high PCE? From the perspective of photophysical principles, the photothermal effect is closely related to non-radiative dissipation after absorbing photon energy, so competitive processes such as radiative transitions and the formation of triplet excited states should be reduced. Therefore, we believe that the "efficient molecular motor" strategy has the potential to significantly improve the non-radiative conversion efficiency of photothermal conversion agents. In addition, J-aggregates represent a unique class of highly ordered molecular arrangements, characterized by head-to-tail slip stacking, and this molecular configuration leads to photon coupling, which in turn leads to spectral red-shift and enhanced light absorption. Taken together, we propose a comprehensive and general method based on the strategic utilization of "efficient molecular motors" and J-aggregates. We call this innovation the "efficient molecular motor matrix", aiming to synergistically enhance the strong absorption of near-infrared light and the PCE of traditional photothermal conversion reagents.

[0043] In this paper, a set of five BODIPY derivatives (labeled BO-0, BO-1, BO-2, BO-3, and BO-4) was carefully designed and synthesized. This involved incorporating varying amounts of molecular rotors into BODIPY, known for its remarkable molar absorption coefficient and exceptional photostability, with the goal of creating photothermal conversion agents with excellent PCEs. The PCEs of these derivatives were evaluated under consistent conditions, revealing a direct correlation between enhanced photothermal performance and an increased number of "efficient molecular motors." To address these issues, we proposed a comprehensive and versatile approach, the "efficient molecular motor matrix" strategy. This strategy strategically utilizes a combination of efficient molecular rotation and J-aggregates. Molecular rotation amplifies nonradiative transitions, significantly improving photothermal conversion efficiency, while J-aggregates induce a red-shift in the molecular spectrum, enhancing light absorption. Of particular note is BO-4, which consists of a near-infrared-absorbing donor-acceptor-donor (DAD) molecule and exhibits exceptional light absorption, with a molar absorption coefficient as high as 2.63×10 ^4 M ^-1 cm ^-1 , and achieved an extraordinary conversion efficiency of up to 90.2%. Using the nanoprecipitation method, BO-4 showed an extremely strong tendency to self-assemble into J-aggregate nanoparticles (NPs). This self-assembly process leads to structural reorganization, manifested as a significant red shift in the spectrum and translated into higher light absorption levels, utilizing the inherent properties of J-aggregate formation. In vitro and in vivo experiments further confirmed that BO-4 NPs were able to generate heat under 808nm laser irradiation, almost completely eliminating primary tumors in tumor-bearing mice after two systemic PTT treatments, and significantly inhibiting the growth of metastatic tumors in vitro. Therefore, we believe that this study provides a general method of "high-efficiency molecular motor matrix" to enhance photothermal conversion efficiency, thereby paving the way for clinical applications in future cancer treatments.

[0044] The technical solution of the present invention will be clearly described in detail below with reference to specific embodiments.

[0045] Example 1

[0046] Preparation and characterization of BO-0, BO-1, BO-2, BO-3, and BO-4:

[0047] The synthetic routes of BO-0, BO-1, BO-2, BO-3 and BO-4 are as follows:

[0048]

[0049] Preparation of BO-0:

[0050] 3,5-Dimethyl-2-pyrrolecarboxaldehyde (0.12 g, 0.91 mmol) and 2,4-dimethylpyrrole (0.10 mL, 1.10 mmol) were added to 20.00 mL of anhydrous dichloromethane, and 1 drop of trifluoroacetic acid (TFA) was added. The reaction was carried out under nitrogen protection at room temperature for 8 hours.

[0051] After the reaction was completed, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ, 0.20 g, 0.91 mmol) was added to the system. After stirring for 5 min, triethylamine (TEA, 0.38 mL, 2.73 mmol) was added and the reaction was carried out for 1 hour. Boron trifluoride diethyl etherate (0.67 mL, 5.46 mmol) was added and the reaction was carried out for 2 hours. Then, saturated sodium chloride solution was added to quench the reaction. The mixture was extracted with dichloromethane and purified by column chromatography. 0.067 g of solid was obtained. The yield was 29.67%.

[0052] As Figure 1 shown, the 1 1H NMR (600 MHz, DMSO) δ 7.67 (s, 1H), 6.20 (s, 2H), 2.42 (s, 6H), 2.27 (s, 6H).

[0053] As Figure 2 shown, the 13 13C NMR (151 MHz, DMSO) δ 9.10, 11.45, 14.74, 46.21, 119.44, 122.67, 142.40, 156.23. HR-MS m / z: [M] calculated for C 13 H 15 BF2N2, 248.13; [M + H] + measured, 249.13.

[0054] Preparation of BO-1:

[0055] p-Bromobenzaldehyde (0.30 g, 1.62 mmol) and 2,4-dimethylpyrrole (0.50 mL, 4.86 mmol) were added to 20.00 mL of anhydrous dichloromethane, and 1 drop of trifluoroacetic acid (TFA) was added. The reaction was carried out under nitrogen protection at room temperature for 8 hours. After the reaction was completed, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ, 0.36 g, 1.62 mmol) was added to the system. After stirring for 5 min, triethylamine (TEA, 0.67 mL, 4.86 mmol) was added and the reaction was carried out for 1 hour. Boron trifluoride diethyl etherate (1.23 mL, 9.72 mmol) was added and the reaction was carried out for 2 hours. Then, saturated sodium chloride solution was added to quench the reaction. The mixture was extracted with dichloromethane and purified by column chromatography. 0.40 g of solid was obtained. The yield was 61.41%.

[0056] AsFigure 1 As shown, for BO-1 1 1H NMR (600 MHz, DMSO) δ 7.77 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 6.20 (s, 2H), 2.45 (s, 6H), 1.38 (s, 6H).

[0057] As Figure 2 shown, for BO-1 13 13C NMR (151 MHz, DMSO) δ 172.26, 155.67, 146.19, 143.09, 140.89, 133.67, 132.81, 130.95, 130.68, 123.15, 122.04, 84.90, 26.02, 14.68. HR-MS m / z: [M] calculated for C 19 H 18 BBrF2N2, 402.07; [M+H] + measured, 403.22.

[0058] Preparation of BO-2:

[0059] p-Bromobenzaldehyde (0.50 g, 3.33 mmol) and 2-methylpyrrole (0.84 mL, 9.99 mmol) were added to 20.00 mL of anhydrous dichloromethane, 1 drop of trifluoroacetic acid (TFA) was added, and the reaction was carried out under nitrogen protection at room temperature for 8 hours. After the reaction was completed, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ, 0.76 g, 3.33 mmol) was added to the system, stirred for 5 min, then triethylamine (TEA, 2.80 mL, 19.98 mmol) was added and the reaction was carried out for 1 hour. Boron trifluoride diethyl etherate (3.70 mL, 29.97 mmol) was added and the reaction was carried out for 2 hours, then saturated sodium chloride solution was added to quench the reaction. Extracted with dichloromethane and purified by column chromatography. 0.43 g of solid was obtained. The yield was 39.80%.

[0060] As Figure 1 shown, for BO-2 1 1H NMR (600 MHz, DMSO) δ 7.76 (d, J = 6.5 Hz, 2H), 7.52 (d, J = 6.4 Hz, 2H), 6.81 (s, 2H), 6.48 (s, 2H), 2.56 (s, 6H).

[0061] As Figure 2 shown, for BO-2 1313C NMR (151 MHz, DMSO) δ 157.95, 141.25, 133.90, 132.73, 132.03, 131.09, 124.55, 120.68, 46.21, 15.01, 9.12. HR-MS m / z: [M] calculated for C 17 H 14 BBrF2N2, 374.04; [M + H] + measured, 375.04

[0062] Preparation of BO-3:

[0063] BO-2 (0.14 g, 0.36 mmol) and 4-diphenylaminobenzaldehyde (0.10 g, 0.36 mmol) were added to absolute ethanol, and a catalytic amount of piperidine and acetic acid were added to the system. The reaction was refluxed for 12 hours. Purification by column chromatography gave 0.011 g of solid. The yield was 4.86%.

[0064] As Figure 1 shown, the 1 1H NMR (600 MHz, DMSO) δ 7.77 (dd, J = 8.3, 3.1 Hz, 3H), 7.68 (d, J = 16.3 Hz, 1H), 7.57 - 7.50 (m, 4H), 7.41 (s, 1H), 7.37 (t, J = 7.8 Hz, 3H), 7.27 (d, J = 8.8 Hz, 1H), 7.15 (t, J = 7.4 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 4.5 Hz, 1H), 6.82 (d, J = 4.1 Hz, 1H), 6.76 (d, J = 4.0 Hz, 1H), 6.47 (dd, J = 15.5, 4.1 Hz, 2H), 1.23 (s, 3H).

[0065] As Figure 2 shown, the 13 13C NMR (151 MHz, DMSO) δ 157.94, 149.20 146.84, 141.25, 138.68, 133.90, 133.04, 132.73, 132.03, 131.07, 129.64, 129.29, 15.02. HR-MS m / z: [M] calculated for C 36 H 27 BBrF2N3, 629.14; [M + 2 H] + measured, 631.13

[0066] Preparation of BO-4:

[0067] Add BO-2 (0.25 g, 0.67 mmol) and 4-diphenylaminobenzaldehyde (0.55 g, 2.00 mmol) into absolute ethanol, add a catalytic amount of piperidine and acetic acid to the system. React under reflux for 12 hours. Purify by column chromatography. Obtain 0.17 g of solid. The yield is 30.44%.

[0068] As Figure 1 shown, the 1 1H NMR (600 MHz, DMSO) δ 7.77 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 16.2 Hz, 2H), 7.53 (dd, J = 19.0, 8.5 Hz, 6H), 7.43 (d, J = 16.1 Hz, 2H), 7.36 (t, J = 7.9 Hz, 8H), 7.24 (d, J = 4.5 Hz, 2H), 7.14 (t, J = 7.4 Hz, 4H), 7.10 (d, J = 7.7 Hz, 8H), 6.96 (d, J = 8.6 Hz, 4H), 6.87 (d, J = 4.4 Hz, 2H).

[0069] As Figure 2 shown, the 13 13C NMR (151 MHz, DMSO) δ 154.83, 149.03, 146.84, 137.64, 136.13, 135.58, 133.25, 132.82, 132.08, 130.16, 129.86, 129.13, 125.54, 124.64, 121.95, 116.55. HR-MS m / z: [M] calculated for C 55 H 40 BBrF2N4, 884.2497; [M] measured, 884.2487.

[0070] Take 30.0 mg each of BO-0, BO-1, BO-2, BO-3, and BO-4 and dissolve them in tetrahydrofuran (4.0 mL) respectively. Drop them into rapidly stirred ultrapure water (40.0 mL). After stirring for 5 minutes, blow off the tetrahydrofuran with hot air and centrifuge to obtain nanoparticles BO-0NPs, BO-1NPs, BO-2NPs, BO-3NPs, and BO-4NPs.

[0071] As Figure 3 shown, the mass spectrometry characterization of BO-0, BO-1, BO-2, BO-3, and BO-4 is shown.

[0072] Example 2

[0073] BO-0, BO-1, BO-2, BO-3, and BO-4 in Example 1 were dissolved in dimethyl sulfoxide (DMSO) to prepare a 1.0 mM stock solution.

[0074] BO-0 NPs, BO-1 NPs, BO-2 NPs, BO-3 NPs, and BO-4 NPs were dispersed in ultrapure water to prepare a 300 μg·mL -1 stock solution.

[0075] The fluorescence spectra and ultraviolet absorption spectra of BO-0, BO-1, BO-2, BO-3, BO-4, BO-0 NPs, BO-1 NPs, BO-2 NPs, BO-3 NPs, and BO-4 NPs in aqueous solution were recorded using a fluorescence spectrometer and an ultraviolet-visible absorption spectrometer, respectively ( Figure 4 A), B), C), D), and E) in). As shown by the spectral absorption data of BO-0, BO-1, BO-2, BO-3, and BO-4 in aqueous solution, their respective absorption peaks appeared at 501, 504, 509, 604, and 735 nm. Similarly, the fluorescence spectra also showed a red shift from 512 to 828 nm. This systematic long-wave shift was attributed to the extension of the conjugated structure by introducing bromophenyl and TPA moieties into the molecular framework. Due to the significant absorption ability of the BODIPY core, these compounds all exhibited a relatively large molar absorption coefficient (MAC, ≈10 ^3 -10 ^4 L·mol ^-1 ·cm ^-1 ). Although these molecules exhibited a relatively high MAC, with the increase in the number of rotors in the molecular structure, an increase in the photothermal conversion efficiency was observed ( Figure 5 A), B), C), D), and E) in), the fluorescence signal gradually weakened ( Figure 6 ), and BO-4, which had the highest conversion efficiency, and BO-4 NPs hardly produced reactive oxygen species ( ​ A), B), C), and D) in). These phenomena indicated a gradual decrease in radiative transitions and an increase in non-radiative transitions, which was in perfect agreement with the "efficient molecular motor" strategy. After the formation of NPs, significant red shifts occurred in both the absorption and fluorescence spectra of BO-4 NPs. The maximum absorption peak shifted to 783 nm, and there was a strong absorption at 808 nm, while other BO-0 to BO-2 NPs did not have these characteristics. This shift of BO-4 NPs could be attributed to the enhanced intermolecular interaction after self-assembly into J-aggregates, demonstrating the feasibility of the "efficient molecular motor matrix" strategy. Dynamic light scattering experiments showed that the average hydrodynamic diameter of BO-4 NPs was 43 nm ( ​ A) in), and scanning electron microscopy could observe that BO-4 NPs were spherical with a diameter of 33 nm​ in B)). At the same time, BO-4 NPs have good photoacoustic imaging ability, showing a good linear relationship with concentration and good stability ( ​ ).

[0076] Example 3

[0077] To investigate the photothermal conversion ability of BO-4 and BO-4 NPs, first, the heating curves of BO-4 (0, 6, 8, 10, 20, 30 μM) irradiated by laser (730 nm, 1.0 W·cm -2 ) for 15 min were explored ( ​ in A)), and second, the heating curves of 10 μM BO-4 irradiated by lasers with different powers (730 nm, 0.2, 0.4, 0.6, 0.8, 1.0 W·cm -2 ) for 15 min were explored ( ​ in B)). For BO-4 NPs, the heating curves of different concentrations of BO-4 NPs (0, 60, 80, 100, 150, 200 μg·mL -1 ) irradiated by laser (808 nm, 1.0 W·cm -2 ) for 15 min were first explored ( ​ in D)). Second, the heating curves of 100 μg·mL -1 BO-4 NPs irradiated by lasers with different powers (808 nm, 0.2, 0.4, 0.6, 0.8, 1.0 W·cm -2 ) for 15 min were investigated ( ​ in E)). By fitting the cooling curve of BO-4 NPs ( ​ in G)) and calculating through the photothermal conversion formula, it can be known that the photothermal conversion efficiency of monomer BO-4 is as high as 90.2% at the optimal concentration, and the photothermal conversion efficiency of BO-4 NPs is 78.0%, which is much greater than that of the commercial photothermal agent indocyanine green (ICG).

[0078] The photothermal cycling stabilities of BO-4 and BO-4 NPs were investigated respectively. The results show that both of them have good cycling stability during five on / off cycles of the laser ( ​ in H)). On the contrary, the photothermal conversion ability of ICG decreases significantly after the first cycle, and it can be seen from the figure that the stability of BO-4 NPs is slightly better than that of BO-4. The ultraviolet spectra of both before and after 3 minutes of light irradiation were measured respectively ( ​ in C) and F)), and the ultraviolet absorption spectra basically overlap, which also indicates that the molecular structures of BO-4 and BO-4 NPs do not change after laser irradiation, and both have good stability.

[0079] Example 4

[0080] The cytotoxicity of this material against 4T1 cells under light and non-light conditions was studied by the MTT assay. First, the 4T1 cells were allowed to adhere and grow for 24 hours, then co-cultured with a series of BO-4NPs solutions at different concentrations for 24 hours, and finally the cell viability was examined using the standard MTT assay procedure. The phototoxicity experiment was similar, with the main difference being that an 808 nm laser with a power of 1.0 W·cm -2 was used to irradiate for 15 min, and then the cell viability was examined by the MTT method. As can be seen from ​ A) in, under non-light conditions, BO-4NPs had good biocompatibility, and even at high concentrations, the 4T1 cells could still maintain a high activity; however, after applying light, the cell viability decreased sharply with the increase in the material concentration, indicating that this material could rapidly and effectively kill cancer cells under laser induction.

[0081] Example 6

[0082] To further investigate the inhibitory effect of BO-4NPs on tumor cells, Calcein AM (live cell indicator) and PI (dead cell indicator) were used in this experiment to distinguish live cells and dead cells. Live cells produce green fluorescence after being stained with Calcein AM, and dead cells produce red fluorescence after being stained with PI. As shown in ​ B) in, only green fluorescence could be observed in the cells incubated with BO-4NPs without light irradiation, and only red fluorescence could be observed after light irradiation. For the cells treated with PBS, only green fluorescence could be observed whether with or without light irradiation. From the cell apoptosis experiment ( ​ C) in), the same result could also be observed. From the bright-field cell images ( ​ D) in), it was known that the way of cell death induced by BO-4NPs was apoptosis and necrosis.

[0083] Example 7

[0084] To investigate the photoacoustic imaging performance of BO-4NPs in tumor-bearing mice, a tumor-bearing mouse model was first constructed. Male BALB / c nude mice (3 weeks old, 20 - 25 g) purchased were injected with 4T1 tumor cells in the right forelimb. When the tumor volume reached 100 - 300 mm 3 , BO-4NPs were injected into the mice at a concentration of 100 μg·mL -1 by intratumoral injection of 50 μL, and the photoacoustic signals in the tumor area were detected. The experimental results are shown in ​ . As can be seen from the figure, the photoacoustic signal gradually became stronger and then weaker over time.

[0085] Example 8

[0086] To investigate the tumor treatment ability of BO-4 NPs at the in vivo level, a tumor-bearing mouse model was first constructed. Male BALB / c nude mice (3 weeks old, 20 - 25 g) purchased were injected with 4T1 tumor cells into the right forelimb. When the tumor volume reached 100 - 300 mm 3 , the tumor-bearing mice were randomly divided into 3 groups, namely: (1) PBS + laser group; (2) BO-4 NPs group; (3) BO-4 NPs + laser group. PBS, BO-4 NPs (100 μg·mL -1 ) 50 μL were respectively injected into the tumor site. Each mouse in the light irradiation group was irradiated with 808 nm laser at 1.0 W·cm -2 for 10 min. The body weight and tumor size of the mice were recorded daily starting from the administration. The results are shown as ​ . ​ A) in ​ is a schematic diagram of the treatment. B) in ​ is a thermal imaging diagram of the PBS + laser group and the BO-4 NPs + laser group under laser irradiation at different times. It can be seen from the quantitative results of the temperature at the tumor site ( ​ E)) that the temperature at the tumor site of the PBS + laser group basically remained at about 43 °C, and the temperature of the BO-4 NPs + laser group gradually increased with the increase of the irradiation time, reaching 69.8 °C at 10 min. After 5 days of treatment, the tumors of the mice basically disappeared. ​ C) in ​ shows the change of tumor volume of different treatment groups over time. It can be seen that the BO-4 NPs + laser group can significantly inhibit the growth of tumors. ​ D) in ​ shows that there was no obvious change in the body weight of the mice during the treatment, indicating that due to its light-controlled release characteristics, systemic toxicity was avoided. After the treatment, the tumor tissues of the mice in the PBS + laser group, BO-4 NPs group, and BO-4 NPs + laser group were sectioned and stained with H&E ( ​ F)). The results showed that there were no obvious differences in the tumor cells within the group. By comparing between different groups, it was found that the tumor cells in the PBS + laser group and the BO-4 NPs group still had the typical morphological characteristics of tumor cells such as abnormal enlargement of the cell nucleus, disordered arrangement, and crowding, but obvious necrosis of the tumor cells could be seen in the BO-4 NPs + laser group.

[0087] To investigate the ability of BO-4 NPs to synergistically regulate immunity, a tumor-bearing mouse model was first constructed. Male BALB / c nude mice (3 weeks old, 20 - 25 g) purchased were injected with 4T1 tumor cells into the left forelimb to simulate the primary tumor. Three days later, 4T1 tumor cells were injected into the left forelimb to simulate the metastatic tumor. When the tumor volume in the left forelimb reached 100 - 300 mm 3The mice were divided into two groups: one was the experimental group, which was injected with BO-4NPs and irradiated with laser, and the other was the control group, which did not receive any treatment. The body weight of the mice and the volume of the tumors on both sides were recorded during the treatment period. ​ As shown, ​ A) is a treatment diagram. The weight of mice hardly changed during the treatment period ( ​ In B), the control tumor grew rapidly, the primary tumor basically disappeared after two treatments, and the growth of metastatic tumors was significantly inhibited ( ​ C)). After the treatment, the primary tumors and metastatic tumors of the control and treatment groups were removed and subjected to H&E and immunohistochemistry. The results showed that the expression of CD4+ T cells and high mobility group box 1 (HMGB1) in the distal tumors and metastatic tumors of the treated mice was significantly upregulated compared with the control group ( ​ D) and E)) and H&E staining of the main organs of mice after treatment and untreated tumor mice ( ​ In Figure F), compared to treated mice, untreated mice showed lesions in all major organs except the heart. This suggests that BO-4NPs trigger immunogenic cell death through photothermal therapy, activating the immune system.

[0088] The above are only preferred embodiments of the present invention; however, the scope of protection of the present invention is not limited thereto. Any person skilled in the art who, within the technical scope disclosed by the present invention, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present invention shall be covered by the scope of protection of the present invention.

Claims

1. A photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, characterized in that, It is composed of several photothermal agent monomers, and the structural formula of the photothermal agent monomer is:

2. A preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, characterized in that, It includes the following steps: Step (1): Add p-bromobenzaldehyde and 2-methylpyrrole into anhydrous dichloromethane, add trifluoroacetic acid, react under nitrogen protection at room temperature. After the reaction is completed, add 2,3-dichloro-5,6-dicyanobenzoquinone to the system, stir and then add triethylamine to react, add boron trifluoride diethyl etherate to react. After the reaction is completed, add saturated sodium chloride solution to quench the reaction, extract with dichloromethane, and purify by column chromatography to obtain intermediate BO-2; Step (2): Add BO-2 and 4-diphenylaminobenzaldehyde into anhydrous ethanol, add a catalytic amount of piperidine and acetic acid to the system and reflux to react. After the reflux reaction is completed, purify by column chromatography to obtain product BO-4; Step (3): Dissolve BO-4 in tetrahydrofuran, drop it into rapidly stirred ultrapure water, remove tetrahydrofuran, and centrifuge to obtain nanoparticles BO-4NPs of BO-4.

3. The preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency according to claim 2, characterized in that, In step (1), the molar ratio of p-bromobenzaldehyde, 2-methylpyrrole, 2,3-dichloro-5,6-dicyanobenzoquinone, and boron trifluoride diethyl etherate is 1.0:3.0 - 25.0:1.0 - 3.0:6.0 - 9.0:6.0 - 12.

0.

4. The preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency according to claim 2, characterized in that, In step (2), the molar ratio of BO-2 and 4-diphenylaminobenzaldehyde is 1.0:3.

0.

5. The preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency according to claim 2, characterized in that, In step (3), the concentration of BO-4 is 5.0 - 7.5 mg / mL.

6. The preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency according to claim 2, characterized in that, In step (1), the total reaction time is 11.0 - 15.0 hours; In step (2), the reflux temperature is 70 - 75 °C and the time is 11.0 - 15.

7. A preparation method of a photothermal agent that can effectively absorb near-infrared light and has a high photothermal conversion efficiency, according to any one of claims 2-6, characterized in that, ​ ​ ​ ​

Images