A photothermal reagent, a photothermal reagent nanoparticle and a preparation method thereof

Abstract

The present invention belongs to the field of biomedical technologies, and particularly relates to a photothermal reagent, a photothermal reagent nanoparticle and a preparation method thereof. The photothermal reagent is NSQ2, and its molecular structural formula is as follows: The present invention designs and synthesizes a squarylium photothermal reagent with a donor-acceptor-donor molecular configuration. Through "dipole-dipole counteraction" and intermolecular interactions provided by triphenylethylene, NSQ2 can form a precise H-aggregated dimer through supramolecular self-assembly. After encapsulating NSQ2 into polymer nanoparticles, a precise H-aggregated dimer can still be formed. Under the irradiation of a near-infrared second-region laser (1064 nm, 1 W cm-2) for NSQ2 NPs, due to the exciton coupling of its H-aggregated excited state, the non-radiative transition rate is greatly increased, and the light energy is efficiently converted into heat energy to the greatest extent. Therefore, NSQ2 NPs have an ultra-high photothermal conversion efficiency and can induce complete tumor ablation in tumor-bearing mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of photothermal therapy, and particularly relates to a photothermal reagent, a photothermal reagent nanoparticle and a preparation method thereof. Background Art

[0002] Photothermal therapy is a method for physically treating tumors. It uses visible light or near-infrared light to generate local high heat by irradiating a photothermal reagent to ablate cancer cells. Compared with traditional cancer treatment methods such as chemotherapy, radiotherapy, and surgery, photothermal therapy has the advantages of high spatio-temporal precision, low invasiveness, and no toxicity to normal tissues. Among them, photothermal reagents with near-infrared second-region (1000-1700 nm) response have attracted much attention because of their higher penetration depth than the near-infrared first region or the visible light region and higher maximum allowable exposure rate. Small molecule organic photothermal reagents have received increasing attention because they exhibit better biocompatibility and biodegradability compared to inorganic photothermal reagents.

[0003] In order to obtain ideal therapeutic performance, a photothermal reagent needs to have a high photothermal conversion efficiency. In the practical application of photothermal reagents, encapsulating small molecules into polymer nanoparticles is a prerequisite for photothermal therapy applications because it improves the photostability, blood circulation time, tumor targeting ability, and tumor permeability of photothermal reagents. However, the arrangement of molecular aggregates inside the formed nanoparticles is uncontrollable, so the molecular packing configuration cannot be determined as clearly as that of its crystal by single crystal x-ray crystallography. Therefore, the precise molecular packing structure can be analyzed precisely and quantitatively, so as to establish a structure-function relationship and achieve a high photothermal conversion efficiency to realize excellent therapeutic performance.

[0004] Squaraine dyes have received increasing attention because of their typical cyanine-like photophysical properties, that is, they have strong and narrow absorption in the red part of the visible light spectrum and the near-infrared region, and at the same time have strong fluorescence. In addition, their molecular structure is easy to functionalize, which makes their application in the fields of bioimaging and host-guest systems attractive. Squaraine also exhibits excellent p-type semiconductor performance in organic thin film transistors and solar cells. However, despite such a wide range of applications, the research on the self-assembly of squaraine into dye aggregates is still less, and the relationship between structure and function is often accidental and unpredictable. Therefore, it is of great significance to develop squaraine photothermal reagents with precise molecular packing structures, establish a structure-function relationship, and achieve a high photothermal conversion efficiency to realize excellent therapeutic performance. Summary of the Invention

[0005] To address the above problems, the present invention provides a photothermal reagent, photothermal reagent nanoparticles, and their preparation methods and applications. First, a squarylium photothermal reagent (NSQ2) with a donor (D)-acceptor (A)-donor (D) molecular configuration was designed and synthesized. Due to its unique molecular structure, NSQ2 can form precise H-aggregated dimers through "dipole-dipole counteraction" and intermolecular interactions provided by triphenylethylene. After encapsulating NSQ2 into polymer nanoparticles (NSQ2 NPs), precise H-aggregated dimers can still be formed. Under near-infrared II (NIR-II) laser irradiation (1064 nm, 1 W cm -2 ), due to the exciton coupling of its H-aggregated excited state, the non-radiative transition rate is greatly increased, maximizing the efficient conversion of light energy into heat energy. Therefore, NSQ2 NPs have an ultra-high photothermal conversion efficiency (93.1%). After injecting NSQ2 NPs into mice via the tail vein, after laser irradiation at a wavelength of 1064 nm (1 W cm -2 ), it can induce complete tumor ablation in tumor-bearing mice.

[0006] To solve the above technical problems of the present invention, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to provide a photothermal reagent, and the photothermal reagent is NSQ2, and its molecular structural formula is as follows:

[0008] .

[0009] The second object of the present invention is to provide a synthesis method of a photothermal reagent, including the following steps:

[0010] S1, Dissolve 1,8-naphthalimide in chloroform, slowly add liquid bromine under an ice-water bath condition, and continue to stir at room temperature for 48 hours after the addition is complete. Then pour the saturated aqueous sodium thiosulfate solution into the reaction mixture; filter the resulting precipitate, wash it with water to obtain the compound of formula (2);

[0011] S2, Dissolve the compound of formula (2) obtained in S1, 1-bromo-2-octyldodecane, and potassium carbonate in acetonitrile. After refluxing the mixture for 72 h, evaporate the solvent under reduced pressure, and extract the mixture with ethyl acetate and water; dry the organic layer with sodium sulfate and filter; evaporate the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography to obtain the compound of formula (3);

[0012] S3. Add the compound of formula (3), triphenylethylene borate, cesium carbonate, and tetrakis(triphenylphosphine)palladium into 1,4-dioxane / water respectively, stir and react at 90 °C for 12 h under argon atmosphere. After the reaction is completed, cool the reaction mixture to room temperature, evaporate the solvent under reduced pressure, and extract the obtained mixture with ethyl acetate and water; dry the organic layer with sodium sulfate and filter; evaporate the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography to obtain the compound of formula (4);

[0013] purify it to obtain the compound of formula (4);

[0014] S4. Dropwise add the tetrahydrofuran solution of methylmagnesium chloride into the anhydrous tetrahydrofuran solution of the compound of formula (4) under ice-water bath condition. After the addition is completed, heat the reaction system to 60 °C and stir for 2 h. After the reaction is completed, cool it to 0 °C, add 0.5 - 1 mL of water, and then add 1 - 3 mL of 70% perchloric acid solution to the reaction mixture; then extract the obtained dark blue solution with dichloromethane and water; dry the organic layer with sodium sulfate and filter, evaporate the solvent under reduced pressure to obtain the crude product, and the crude product is a blue-black solid to obtain the compound of formula (5);

[0015] S5. Heat and reflux the compound of formula (5) and the toluene / n-butanol solution of propanedicyanine-squaric acid in a Dean-Stark apparatus for 2 h. After the reaction is completed, evaporate the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography to obtain NSQ2;

[0016] The reaction formulas are as follows:

[0017]

[0018] Furthermore, in S1, the molar ratio of the 1,8-naphthalimide to liquid bromine is 1:4 - 6.

[0019] Furthermore, in S2, the molar ratio of the compound of formula (2), 1-bromo-2-octyldodecane, and potassium carbonate is 1:(2 - 4):(4 - 6), and the mobile phase of the column chromatography is analytical pure petroleum ether:ethyl acetate (V / V) = 40 - 60:1.

[0020] Furthermore, in S3, the molar ratio of the triphenylethylene borate, the compound of formula (3), cesium carbonate, and tetrakis(triphenylphosphine)palladium is 1:1:3:0.05; the volume ratio of the reaction solvent 1,4-dioxane / water is 4:1; the mobile phase of the column chromatography is analytical pure petroleum ether:ethyl acetate (V / V) = 20 - 40:1.

[0021] Furthermore, in S4, the concentration of the tetrahydrofuran solution of methylmagnesium chloride is 1 mol / L, and the molar ratio of methylmagnesium chloride to the compound of formula (4) is 2 - 4:1.

[0022] Furthermore, in S5, the molar ratio of the formula (5) and malondicyanine-squaric acid is 1:1; the volume ratio of the reaction solvent toluene / n-butanol is 1:1; and the mobile phase of the column chromatography is analytical grade petroleum ether: ethyl acetate (V / V) = 4-6:1.

[0023] The third object of the present invention is to provide a photothermal reagent nanoparticle (NSQ2 NPs) prepared using the photothermal reagent described in claim 1 as a raw material.

[0024] The fourth object of the present invention is to provide a method for preparing photothermal agent nanoparticles, comprising the following steps:

[0025] NSQ2 is dissolved in tetrahydrofuran, and the tetrahydrofuran solution of NSQ2 is added to an ultrapure aqueous solution containing Pluronic F-127 under ultrasonic treatment. The resulting dispersion is ultrasonically treated for 30-40 minutes to obtain a colloidal dispersion; the colloidal dispersion is rotary evaporated in a rotary evaporator at a water bath temperature of 35°C for 3-10 minutes to remove tetrahydrofuran, and the resulting colloidal dispersion is first dialyzed in ultrapure water for 2-4 days. During the dialysis process, the ultrapure water is replaced every 4-6 hours, and then the dispersion is dialyzed with physiological saline, and the solution in the dialysis bag is collected to obtain nanoparticles of the photothermal reagent (NSQ2 NPs).

[0026] Further, the tetrahydrofuran solution of NSQ2 and the ultrapure aqueous solution containing Pluronic F-127 are

[0027] The volume ratio of the two solutions is 1:4-6; the molar ratio of the NSQ2 and Pluronic F-127 is 1:1; and the dialysis bag is a regenerated cellulose dialysis bag 3500.

[0028] Beneficial effects of the present invention:

[0029] 1. Due to its unique molecular structure, NSQ2 supramolecular self-assembly can form precise H-aggregation dimers through the intermolecular interaction provided by "dipole-dipole hedge" and triphenylethylene. After NSQ2 is encapsulated into polymer nanoparticles (NSQ2 NPs), precise H-aggregation dimers can still be formed. NSQ2 NPs can be irradiated with near-infrared laser in the second region (NIR-Ⅱ) (1064nm, 1W cm -2 ) under the condition of 1064 nm (1W cm -2)After irradiation with a laser of [wavelength], it can induce complete tumor ablation in tumor-bearing mice.

[0030] 2. The "H-aggregation" strategy in the second near-infrared region provides a new platform for the further design of photothermal therapy agents for cancer treatment. Brief Description of the Drawings

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 is the synthesis route of the second near-infrared H-aggregation photothermal reagent NSQ2;

[0033] Figure 2 a) in [figure] is the ultraviolet / visible / near-infrared absorption spectrum of NSQ2 in different solvents; Figure 2 b) in [figure] is the ultraviolet / visible / near-infrared absorption spectrum of NSQ2 in a tetrahydrofuran-water mixed solvent with different water volume fractions; Figure 2 c) and d) in [figure] are the concentration-dependent ultraviolet / visible / near-infrared absorption spectrum and its normalized absorption spectrum of NSQ2 in water / tetrahydrofuran 97:3 at 25 °C;

[0034] Figure 3 a) in [figure] is the particle size distribution of NSQ2 NPs and its transmission electron microscope image; Figure 3 b) in [figure] is the ultraviolet / visible / near-infrared absorption spectrum of NSQ2 NPs in water; Figure 3 c) in [figure] is the photothermal heating curve of NSQ2 NPs with different concentrations (5 - 80 μM) under laser irradiation at 1064 nm (1 W cm -2 ) wavelength; Figure 3 d) in [figure] is the study of the photothermal stability of NSQ2 NPs in five heating-cooling processes;

[0035] Figure 4 is a snapshot of amorphous NSQ2 NPs obtained by molecular dynamics simulation;

[0036] Figure 5 is a schematic diagram of the interaction between NSQ2 molecules;

[0037] Figure 6 a) and b) in [figure] are HUVEC and B16-F10 cells incubated with NSQ2 NPs at different concentrations in the dark and NIR-II light (1064 nm, 1 W cm -2, cell survival rate after irradiation (6 min); Figure 6 In c), the uptake of nanoparticles by B16-F10; Figure 6 In d), after irradiation with near-infrared laser at 1064 nm (1 W cm -2 ), dead / live cell staining of B16-F10;

[0038] Figure 7 In a) and b), thermal infrared (IR) images of B16-F10 tumor-bearing mice and the temperature change curves of the corresponding tumors after intravenous injection of NSQ2 NPs or saline in mice and irradiation with 1064 nm 1 Wcm -2 laser for 6 minutes; Figure 7 In c) and d), the changes in tumor volume of mice after different treatments; Figure 7 In e), the changes in body weight of mice after different treatment methods; Figure 7 In f), TUNEL staining of tumor tissues with different treatment methods (scale bar: 100 μm). Detailed implementation methods

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the specification and specific implementation methods. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] Example 1: Preparation of the compound of formula (2)

[0041] Dissolve 1,8-naphthalimide (10 mmol, 1.69 g) in chloroform, and slowly add liquid bromine (50 mmol, 8 g) under ice-water bath conditions. After the addition is complete, continue stirring for 48 h, and then pour saturated aqueous sodium thiosulfate solution (50 mL) into the reaction mixture. Filter the resulting precipitate and wash it with water to obtain the compound of formula (2) as a yellow solid (2.23 g, 89.9%).

[0042] Example 2: Preparation of the compound of formula (3)

[0043] Dissolve the compound of formula (2) (5 mmol, 1.24 g), 1-bromo-2-octyldodecane (15 mmol, 5.4 g), and potassium carbonate (25 mmol, 3.4 g) in 50 mL of acetonitrile. After refluxing the mixture for 72 h, evaporate the solvent under reduced pressure. Extract the mixture three times with 50 mL each of ethyl acetate and water. Dry the organic layer over sodium sulfate and filter. Evaporate the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography. The mobile phase for column chromatography is analytical grade petroleum ether:ethyl acetate (V / V) = 50:1 to obtain the compound of formula (3) as a yellow oily liquid (2.12 g, 80.1%).

[0044] The 1H NMR and 13C NMR spectra of the compound of formula (3) are as follows: 1 H NMR (400 MHz, CDCl3- d ): δ 8.10 (d, J = 8.3 Hz, 1H), 8.05 (d, J = 7.0 Hz, 1H), 7.76 (t, J = 7.6 Hz,1H), 7.61 (d, J = 7.5 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 3.75 (d, J = 7.4 Hz,2H), 1.93 (p, J = 6.3 Hz, 1H), 1.32 (q, J = 5.9 Hz, 8H), 1.23 (d, J = 16.7Hz, 23H), 0.86 (q, J = 6.5 Hz, 6H). 13 C NMR (101 MHz, CDCl3- d ): δ 167.61, 139.69,131.10, 130.11, 129.57, 128.82, 126.87, 126.09, 124.99, 113.76, 106.10,44.76, 37.24, 31.89, 31.84, 31.58, 29.92, 29.59, 29.55, 29.50, 29.31, 29.25,26.49, 22.67, 22.63, 14.10, 14.09.

[0045] Example 3: Preparation of the compound of formula (4)

[0046] Triphenylvinyl borate (1 mmol, 385 mg), the compound of formula (3) (1 mmol, 529 mg), cesium carbonate (3 mmol, 975 mg) and tetrakis(triphenylphosphine)palladium (0.05 mmol, 57.8 mg) were separately added to 25 mL of 1,4-dioxane / water with a volume ratio of 4:1. The reaction was stirred at 90 °C for 12 h under argon. After the reaction was completed, it was cooled to room temperature, and the solvent was removed by evaporation under reduced pressure. The resulting mixture was extracted three times with 50 mL each of ethyl acetate and water. The organic layer was dried over sodium sulfate and filtered. The solvent was removed by evaporation under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography. The mobile phase of the column chromatography was analytical grade petroleum ether:ethyl acetate (V / V) = 30:1, and the compound of formula (4) was obtained as a dark yellow oily liquid.

[0047] The 1H NMR and 13C NMR spectra of the compound of formula (4) are as follows: 1 H NMR (400 MHz, CDCl3- d ): δ 7.96–7.86 (dd, 2H), 7.51–7.45 (m, 1H), 7.20 (d, J = 7.3 Hz, 1H), 7.17 (s,5H), 7.13–7.05 (m, 5H), 6.99–6.92 (m, 5H), 6.69 (d, J = 7.3 Hz, 1H), 3.74 (s,2H), 1.95 (s, 1H), 1.34 (d, J = 5.6 Hz, 7H), 1.27 (d, J = 16.9 Hz, 24H),0.92–0.85 (m, 6H). 13 C NMR (101 MHz, CDCl3- d ): δ 168.42, 143.71, 143.68, 143.37,143.26, 139.22, 137.98, 135.28, 131.79, 131.47, 130.75, 130.61, 130.01,128.63, 128.26, 127.84, 127.81, 127.48, 126.82, 126.75, 126.56, 126.41,125.30, 123.79, 105.17, 44.69, 37.37, 31.93, 31.89, 31.68, 29.99, 29.64,29.61, 29.56, 29.35, 29.29, 26.53, 22.70, 22.67, 14.14.

[0048] Example 4: Preparation of the compound of formula (5)

[0049] A solution of methylmagnesium chloride in tetrahydrofuran (1 mol / L, 3 mL) was added dropwise to a solution of the compound of formula (4) (1 mmol, 704 mg) in anhydrous tetrahydrofuran under an ice-water bath. After the addition was complete, the reaction system was heated to 60 °C and stirred for 2 hours. After the reaction was completed, it was cooled to 0 °C, 0.5 mL of water was added, and then 2 mL of perchloric acid solution with a mass concentration of 70% was added to the reaction mixture. The resulting dark blue solution was then extracted three times with dichloromethane and water, 50 mL each time. The organic layer was dried over sodium sulfate, filtered, and the solvent was removed by evaporation under reduced pressure to obtain a crude product, which was a blue-black solid, the compound of formula (5). The obtained crude product was directly used for the next step without purification.

[0050] Example 5: Preparation of the photothermal reagent NSQ2

[0051] A 20 mL toluene / n-butanol solution of the compound of formula (5) (1 mmol, 803 mg) and malononitrile-squaric acid (1 mmol, 291 mg) was heated to reflux in a Dean - Stark apparatus for 2 hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (analytical pure petroleum ether: ethyl acetate = 5:1) to obtain the photothermal reagent NSQ2 as a black solid.

[0052] 1H NMR, 13C NMR and high-resolution mass spectrometry analysis of the photothermal reagent NSQ2 are as follows: 1 H NMR(400 MHz, CDCl3- d ): δ 8.64 (d, J = 7.5 Hz, 2H), 8.13 (d, J = 8.2 Hz, 2H), 7.82(t, J = 7.8 Hz, 2H), 7.55 (dd, J = 18.6, 7.6 Hz, 10H), 7.45 (t, J = 7.3 Hz,2H), 7.13 (dd, J = 31.2, 7.5 Hz, 2H), 6.76 (d, J = 12.1 Hz, 2H), 4.05 (dd, J= 18.0, 7.4 Hz, 4H), 2.19 (m, J = 6.5, 6.0 Hz, 2H), 1.45–1.38 (m, 10H), 1.23(d, J = 5.5 Hz, 52H), 0.85 (t, J = 6.7 Hz, 12H). 1313C NMR (101 MHz, CDCl3- d ): δ 174.42, 167.67, 162.96, 151.84, 144.13, 143.62, 143.44, 143.06, 140.86, 138.24, 137.85, 132.21, 131.45, 130.80, 130.78, 130.00, 129.22, 129.21, 127.90, 127.87, 127.61, 126.92, 126.69, 125.55, 117.84, 108.60, 94.10, 48.93, 44.39, 37.91, 31.91, 31.87, 31.44, 29.99, 29.64, 29.62, 29.60, 29.57, 29.51, 29.35, 29.29, 26.32, 22.68, 22.67, 14.12. TOF-MS m / z: NSQ2 theoretical [M+H] + 13 111 H1 25 N4O + : 1529.9848, found: 1529.9716.

[0053] The target compound was synthesized by the analysis of 1H NMR, 13C NMR and high-resolution mass spectrometry.

[0054] The synthetic route of the photothermal reagent NSQ2 is shown in Figure 1 .

[0055] Example 6: Preparation of nanoparticles of the photothermal reagent NSQ2 (NSQ2 NPs)

[0056] The photothermal reagent NSQ2 was dissolved in tetrahydrofuran (1 mL, 1.53 mg / mL), and under sonication, it was added to ultrapure water (5 mL, 2.2 mg / mL) containing Pluronic F-127. The resulting dispersion was sonicated for 30 min to obtain a colloidal dispersion; the colloidal dispersion was rotary evaporated (water bath temperature was 35 °C), and after removing tetrahydrofuran, dialysis was carried out. During dialysis, ultrapure water was changed every 6 hours, and finally the dispersion was dialyzed with physiological saline. The dialysis bag was a regenerated cellulose dialysis bag with a molecular weight cut-off of 3500. Finally, the solution in the dialysis tube was collected, which was the nanoparticles of the photothermal reagent NSQ2 (NSQ2 NPs).

[0057] Test Example 1

[0058] The ultraviolet-visible-near-infrared absorption spectrum of NSQ2 obtained in Example 5 was measured. As can be seen from Figure 2 a) therein, NSQ2 has a strong absorption in the NIR-II region, and its molar extinction coefficient in n-hexane is as high as 1.53×10 5 M -1 cm -1 . The long absorption wavelength in the near-infrared region for photothermal therapy is one of the important prerequisites for in vivo applications. Compared with the near-infrared first region, the near-infrared second region can excite the photoactivity of photothermal therapy, enabling it to penetrate deeper into tissues and causing less tissue damage. As can be seen from Figure 2 b) therein, as the water content in the tetrahydrofuran / water mixed solvent gradually increases, the maximum absorption wavelength of NSQ2 gradually blue-shifts, showing H-aggregation. In addition, the aggregation process of NSQ2 was also studied. By using a mixed solvent of water / tetrahydrofuran (97 / 3) and gradually increasing the concentration of NSQ2, the maximum absorption peak of H-aggregation gradually appears in its ultraviolet-visible-near-infrared absorption spectrum. Through spectral calculation, the aggregation number n = 2 can be obtained. Therefore, it can be concluded that NSQ2 can form H-aggregated dimers in water.

[0059] NSQ2 was prepared into nanoparticles using Pluronic F-127. During this process, the hydrophobic part self-assembled and aggregated in the core, and the hydrophilic PEG chains formed a shell, forming water-soluble nanoparticles. The size and morphology of NSQ NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) respectively. As shown in Figure 3 a) therein, the DLS data of the nanoparticles show that their hydrated particle size is 120 nm, while the TEM images show that the nanoparticles are spherical with a diameter of 90 nm. The smaller size measured by TEM may be due to the shrinkage of the hydration layer in the dried TEM sample. As is well known, nanoparticles with diameters between 10 and 200 nm can enhance their permeability and retention effects and accumulate at the tumor site. Therefore, these nanoparticles with an average diameter of 100 nm are suitable for in vivo anti-tumor therapy.

[0060] The ultraviolet-visible-near-infrared absorption spectrum of NSQ2 NPs obtained in Example 6, as shown in Figure 3 b) therein, shows a significant H-band at its maximum absorption wavelength, and the molar extinction coefficient at the absorption peak is recorded as 1.02×10 5 M -1 cm -1 . Due to its strong absorption in the NIR-II region, it is particularly useful for biomedical applications. Next, the photothermal conversion of nanoparticles NSQ2 Ns in water was carried out. As shown in Figure 3As shown in c), it is the photothermal conversion of NSQ2 NPs nanoparticles with different concentrations (5 - 80 μM) under the laser irradiation (1 W cm -2 ) at a wavelength of 1064 nm. The results show that when the concentration is 80 μM, the photothermal conversion is the highest, and the temperature increase ΔT is 59 °C. As shown in Figure 3 d), it is the study of the photothermal stability of NSQ2 NPs during five heating-cooling processes. The results show that even after 5 heating and cooling cycles under continuous laser irradiation, NSQ2 NPs still exhibit excellent thermal and photostability.

[0061] Test Example 2

[0062] The ultraviolet absorption spectrum of the NSQ2 NPs obtained in Example 6 was analyzed, and it was found that the ultraviolet absorption spectrum of NSQ2 NPs is mainly the H-band. The process of forming its nanoparticles was studied in this invention. According to the results of molecular dynamics simulation, as shown in Figure 4 , during the self-assembly process of NSQ2, H-aggregated dimers were formed. It shows that after NSQ2 NPs are excited by a laser at a wavelength of 1064 nm, due to the exciton coupling of its H-aggregated excited state, the non-radiative transition rate is greatly increased, and the light energy is maximally converted into heat energy, resulting in its ultra-high photothermal conversion efficiency (93.1%). Figure 5 The intermolecular interaction of NSQ2 is given. The left figure shows the intermolecular hydrogen bond interaction between the benzene hydrogen and cyano group of triphenylethylene, and the right figure shows that the dispersion force generated by the "dipole-dipole counteraction" of the NSQ2 molecular core causes it to form H-aggregation.

[0063] Test Example 3

[0064] The phototoxicity of the NSQ2 NPs obtained in Example 6 to normal cells HUVEC and cancer cells B16-F10 was detected by the MTT method. The cytotoxicity of NSQ2 NPs to the two cell lines was evaluated under dark conditions. Figure 6 a) and b) in it are the HUVEC and B16-F10 cells incubated with different concentrations of NSQ2 NPs under dark and NIR-II light (1064 nm, 1 W cm -2, cell viability after (6 min) irradiation. The results showed that under dark conditions, even with a high concentration of NSQ2 NPS, almost no cytotoxicity was observed, indicating that the nanoparticles have good biocompatibility; in contrast, under light conditions, as the concentration of NSQ2 NPs increased, cell viability decreased significantly, indicating that the photothermal effect of NPs in cells can effectively kill cancer cells through low-intensity NIR-II radiation. Next, the uptake of NSQ2 NPs by tumor cells was studied. A hydrophobic red-emitting dye was loaded into the core of the nanoparticles for fluorescence tracking, Figure 6 As shown in c) of Figure 6 , confocal laser scanning microscopy (CLSM) showed strong red fluorescence in the cytoplasm after incubation, indicating that NSQ2 NPs were effectively taken up by cancer cells. To visually show the effectiveness of the photothermal therapy of NSQ2 NPs, calcein AM (green) and propidium iodide (red) dyes were used for live cell staining. Green fluorescence represents live cells, and red indicates dead cells. As expected, as Figure 6 shown in d) of Figure 6 , NSQ2 NPs induced complete destruction of B16-F10 cells after NIR-II laser irradiation (1064 nm, 0.3 W cm -2 , 6 min), and the cells showed obvious red fluorescence. However, in the group with only light irradiation and the group with only NSQ2 NPs used, only green fluorescence was observed, indicating that NSQ2 NPs have a strong killing effect on cancer cells under laser irradiation, leading to the death of cancer cells.

[0065] Test Example 4

[0066] The in vivo photothermal therapy effect of the NSQ2 NPs obtained in Example 6 was tested using B16-F10 C57BL / 6 tumor-bearing mice. The anti-tumor effect of NSQ2 NPs was studied using NIR-II laser (1 W cm -2 ). As Figure 7 shown in a) and b) of Figure 7 , after 6 minutes of laser irradiation, in the group injected with NSQ2 NPs nanoparticles, the tumor temperature increased significantly to 58.3 °C, higher than the minimum temperature required for tumor hyperthermia (45 °C). The tumor temperature treated with normal saline changed gently and only rose to 36.1 °C after laser irradiation. It shows that NSQ2 NPs exhibit good photothermal properties during the in vivo photothermal conversion process. The photothermal therapy effect of each group was evaluated by monitoring the tumor volume every other day for 20 days after treatment, Figure 7 as shown in c) and d) of Figure 7 are the tumor volumes of mice after different treatment methods. The groups with only normal saline, only NSQ2 NPs, and light irradiation alone could not inhibit tumor growth, and the tumor volume increased by an average of 12 - 16 times. These results are consistent with the in vitro phototoxicity experiment results, confirming that NSQ2 NPs have good photothermal therapy effects under low-intensity laser irradiation. As Figure 7In Figure e), the weight of all mice in the control and experimental groups increased slowly, indicating that the treatment had no systemic toxic effects on mice, which also confirmed that NSQ2 NPs also showed excellent physiological safety in vivo. The TdT-mediated dUTP nick-end labeling (TUNEL) method was used to analyze the apoptosis of tumor cells in tumor biopsy tissues, showing that the NSQ2 NPs + laser treatment group could effectively promote tumor cell apoptosis and inhibit tumor growth; this was not observed in the other three control groups, see Figure 7 (e)

[0067] In summary, the present invention prepares a new photothermal agent for cancer treatment. A cyanine photothermal agent (NSQ2) with a molecular configuration of donor (D)-acceptor (A)-donor (D) was synthesized. Due to the unique structure of the NSQ2 molecule, its "dipole-dipole hedge" and the intermolecular interaction provided by triphenylethylene can make the NSQ2 supramolecular self-assemble to form a precise H-aggregated dimer. The NSQ2 dimer is irradiated by near-infrared laser in the second region (1064 nm, 1W cm -2 ), due to the exciton coupling of its H-aggregated excited state, the non-radiative transition rate is greatly increased, resulting in the efficient conversion of light energy into heat energy. Importantly, after encapsulating NSQ2 into polymer nanoparticles, precise H-dimers can still be formed. Therefore, NSQ2 NPs show an ultra-high photothermal conversion efficiency of 93.1%. After tail vein injection of NSQ2 NPs, at 1064 nm (1 W cm -2 ) laser irradiation can induce complete tumor ablation in tumor-bearing mice.

[0068] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Other changes or modifications can be made within the knowledge of ordinary technicians in this field. It is not necessary and impossible to list all the embodiments here. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A photothermal reagent, characterized in that, The photothermal reagent is NSQ2, and its molecular structural formula is as follows: 。 2. The preparation method of the photothermal reagent according to claim 1, wherein, It includes the following steps: S1. Dissolve 1,8-naphthalimide in chloroform, slowly add liquid bromine under an ice-water bath condition. After the addition is complete, continue to stir at room temperature for 48 hours, then pour the saturated aqueous sodium thiosulfate solution into the reaction mixture; filter the resulting precipitate, wash it with water to obtain the compound of formula (2); S2. Dissolve the compound of formula (2) obtained in S1, 1-bromo-2-octyldodecane, and potassium carbonate in acetonitrile. After the mixture refluxes for 72 h, evaporate the solvent under reduced pressure, and extract the mixture with ethyl acetate and water; dry the organic layer with sodium sulfate and filter; evaporate the solvent under reduced pressure to obtain a crude product, and purify it by silica gel column chromatography to obtain the compound of formula (3); S3. Add the compound of formula (3), 1,2,2-triphenylethyleneboronic acid pinacol ester, cesium carbonate, and tetrakis(triphenylphosphine)palladium to 1,4-dioxane / water respectively, stir and react at 90 °C for 12 h under an argon atmosphere. After the reaction is complete, cool to room temperature, evaporate the solvent under reduced pressure, and extract the resulting mixture with ethyl acetate and water; dry the organic layer with sodium sulfate and filter; evaporate the solvent under reduced pressure to obtain a crude product, and purify it by silica gel column chromatography to obtain the compound of formula (4); S4. Dropwise add the tetrahydrofuran solution of methylmagnesium chloride to the anhydrous tetrahydrofuran solution of the compound of formula (4) under an ice-water bath condition. After the addition is complete, raise the temperature of the reaction system to 60 °C and stir for 2 hours. After the reaction is complete, cool to 0 °C, add 0.5 - 1 mL of water, and then add 1 - 3 mL of 70% perchloric acid solution to the reaction mixture; then extract the resulting dark blue solution with dichloromethane and water; dry the organic layer with sodium sulfate and filter, evaporate the solvent under reduced pressure to obtain a crude product, and obtain the compound of formula (5) as a blue-black solid for the crude product; S5. Heat the toluene / n-butanol solution of the compound of formula (5) and cyanine-squaric acid under reflux in a Dean-Stark apparatus for 2 h. After the reaction is completed, evaporate the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography to obtain NSQ2; the structural formula of the cyanine-squaric acid is: ; Among them, the reaction formula is as follows: 。 3. The preparation method according to claim 2, wherein In S1, the molar ratio of 1,8-naphthalimide to liquid bromine is 1:4 - 6.

4. The preparation method according to claim 2, characterized in that, In S2, the molar ratio of the compound of formula (2), 1-bromo-2-octyldodecane, and potassium carbonate is 1:(2 - 4):(4 - 6), and the mobile phase of the column chromatography is analytical pure petroleum ether:ethyl acetate = 40 - 60:1 by volume ratio.

5. The preparation method according to claim 2, characterized in that, In S3, the molar ratio of 1,2,2-triphenylethyleneboronic acid pinacol ester, the compound of formula (3), cesium carbonate, and tetrakis(triphenylphosphine)palladium is 1:1:3:0.05; the volume ratio of the reaction solvent 1,4-dioxane / water is 4:1; the mobile phase of the column chromatography is analytical pure petroleum ether:ethyl acetate = 20 - 40:1 by volume ratio.

6. The preparation method according to claim 2, characterized in that, In S4, the concentration of the tetrahydrofuran solution of methylmagnesium chloride is 1 mol / L, and the molar ratio of methylmagnesium chloride to the compound of formula (4) is 2 - 4:

1.

7. According to the preparation method described in claim 2, characterized in that, In S5, the molar ratio of the compound of formula (5) to malonocyanine-squaric acid is 1:1; the volume ratio of the reaction solvent toluene / n-butanol is 1:1; the mobile phase of the column chromatography is analytical pure petroleum ether:ethyl acetate = 4 - 6:1 by volume ratio.

8. A photothermal reagent nanoparticle prepared from the photothermal reagent described in claim 1 as a raw material.

9. The preparation method of the photothermal reagent nanoparticles according to claim 8, wherein, It includes the following steps: Dissolve NSQ2 in tetrahydrofuran. Under ultrasonic treatment, add the tetrahydrofuran solution of NSQ2 to the ultrapure aqueous solution containing Pluronic F-127. Ultrasonically treat the resulting dispersion for 30 - 40 min to obtain a colloidal dispersion. Rotate and evaporate the colloidal dispersion in a rotary evaporator at a water bath temperature of 35 °C for 3 - 10 min to remove tetrahydrofuran. Dialyze the resulting colloidal dispersion in ultrapure water for 2 - 4 days. During dialysis, change the ultrapure water every 4 - 6 h. Then dialyze the dispersion with physiological saline and collect the solution in the dialysis bag to obtain the nanoparticles of the photothermal reagent.

10. The preparation method according to claim 9, wherein, The volume ratio of the tetrahydrofuran solution of NSQ2 to the ultrapure aqueous solution containing Pluronic F-127 is 1:4 - 6; the molar ratio of NSQ2 to Pluronic F-127 is 1:1; the dialysis bag is a regenerated cellulose dialysis bag 3500.

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