Near-infrared two-region conjugated small-molecule photothermal agent and preparation method and application thereof

Abstract

The present application relates to the technical field of nanobiomedical photosensitive diagnosis and treatment, in particular to a near-infrared two-region conjugated small-molecule photothermal agent, a preparation method and application thereof, which has a liposome-like molecular structure.The targeted near-infrared two-region conjugated small-molecule photothermal agent contains a D-A-D conjugated main chain, which makes the absorption red-shifted, improves the fluorescence intensity of the molecule, improves the resolution and contrast of in-vivo imaging, and uses zwitterionic groups to make the near-infrared two-region conjugated small-molecule photothermal agent into a liposome-like structure to reduce the fluorescence quenching of the material.The near-infrared two-region conjugated small-molecule photothermal agent has good biocompatibility and small fluorescence quenching after being modified by a liposome dimyristoyl phosphatidylcholine (DMPC), and is successfully applied to NIR-II laser-induced high-contrast NIR-II fluorescence imaging and efficient NIR-II photothermal therapy.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedical photosensitive diagnostic and therapeutic technology, and in particular to a near-infrared II-region conjugated small molecule photothermal agent, its preparation method, and its application. Background Technology

[0002] In the development of cancer diagnosis and treatment technologies, photothermal therapy has attracted much attention due to its unique advantages. Photothermal therapy utilizes photothermal agents to convert light energy into heat energy, destroying tumor tissue through thermal effects. Near-infrared II (NIR-II, 1000-1700nm) photothermal agents have shown great application potential in the biomedical field. Compared with commonly used near-infrared I (NIR-I, 700-900nm) light sources, NIR-II light sources have many significant advantages. They exhibit weaker photon scattering and deeper tissue penetration, effectively reaching deep tumor tissues and reducing damage to normal tissues. Furthermore, NIR-II light has a higher maximum permissible exposure, meaning that higher power light can be used for treatment while ensuring safety, thereby improving treatment efficacy.

[0003] Conjugated small molecules (CSMs) based on donor-receptor-donor (DAD) structures have been widely studied by researchers as photothermal therapeutic agents due to their well-defined structures, excellent biocompatibility, and good photothermal stability. By rationally expanding the degree of π-conjugation and selecting strong donor-receptor (DA) structures with good matching, a small band gap can be effectively achieved, thereby extending the absorption / emission bands of DAD-CSMs into the near-infrared II region, making them more suitable for photothermal therapy of cancer in deep tissues.

[0004] However, the development of near-infrared II activatable conjugated small molecule photothermal agents currently faces numerous challenges. On the one hand, these photothermal agents generally lack fluorescence emission capabilities, severely hindering their application in precision fluorescence imaging (FI)-guided photothermal therapy (PTT). Fluorescence imaging can display the distribution and aggregation of photothermal agents in vivo in real time and visually, providing precise positioning and guidance for photothermal therapy; the lack of fluorescence emission capability makes precise monitoring of the treatment process difficult. On the other hand, existing near-infrared II fluorophores have significant shortcomings in practical applications. As the band gap decreases, non-radiative decay increases, making it difficult to obtain near-infrared II fluorophores with high quantum yield (QY). Moreover, existing near-infrared II fluorophore designs, such as strong DA structures and / or large π-bridges, are highly susceptible to non-radiative decay in physiological environments through water-related tortuous intramolecular charge transfer (TICT) quenching and π-π stacking-induced quenching, significantly reducing quantum yield and thus affecting the efficacy of fluorescence imaging and photothermal therapy.

[0005] Although some unique physical encapsulation methods have been explored to improve the quantum yield of near-infrared II excited fluorophores in aqueous media, these methods still have limitations and cannot fully meet the needs of clinical applications. Therefore, developing a photothermal therapeutic agent that possesses both good near-infrared II absorption and excellent fluorescence performance, as well as highly efficient photothermal properties, has become an important problem that urgently needs to be solved in the field of cancer photothermal therapy. Summary of the Invention

[0006] The purpose of this invention is to provide a near-infrared II conjugated small molecule photothermal agent, its preparation method, and its application, so as to solve the problems of lack of fluorescence emission capability, severe fluorescence quenching, and poor biocompatibility of existing near-infrared II photothermal therapeutic agents, and to achieve high-contrast NIR-II fluorescence imaging and efficient NIR-II photothermal therapy.

[0007] To achieve the above objectives, the present invention provides a near-infrared II conjugated small molecule photothermal agent with a liposome-like molecular structure, the structural formula of which is as follows.

[0008]

[0009] The targeted near-infrared II conjugated small molecule photothermal agent of the present invention contains a DAD conjugated backbone that red-shifts the absorption, increases the fluorescence intensity of the molecule, and improves the resolution and contrast of in vivo imaging. At the same time, zwitterionic groups are used to transform it into a liposome-like structure to reduce fluorescence quenching of the material.

[0010] This invention also provides a method for preparing the above-mentioned near-infrared II region conjugated small molecule photothermal agent, comprising the following steps:

[0011] S1. Under light-protected conditions, organic conjugated small molecule monomer one, organic conjugated small molecule monomer two, and a solvent were mixed to obtain a mixed solution. Nitrogen gas was then introduced into the mixed solution, followed by the addition of a palladium catalyst. Under a protective atmosphere, a Stieler coupling reaction was carried out to obtain compound I.

[0012] The structural formula of compound I is as follows:

[0013]

[0014] S2. Under light-protected conditions, the compound of formula I is first reacted with dimethylamine to obtain a crude product. Then, the crude product is reacted with 1,3-propanesulfonic acid lactone under a protective atmosphere to prepare a near-infrared II region conjugated small molecule photothermal agent.

[0015] Preferably, the ratio of organic conjugated small molecule monomer one, organic conjugated small molecule monomer two, and solvent in S1 is 0.2-1 mM: 0.4-2 mM: 10-50 mL.

[0016] Preferably, the solvent in S1 includes toluene, and the palladium catalyst includes tetra(triphenylphosphine)palladium.

[0017] Preferably, the nitrogen gas is introduced into S1 for 20–30 min, the temperature of the Stieler coupling reaction is 90–120 °C, and the reaction time is 18–30 h.

[0018] Preferably, the molar ratio of compound I in S2 to 1,3-propanesulfonic acid lactone is 0.5-1:10-12.

[0019] Preferably, the reaction of compound I in S2 with dimethylamine is to first react at 0°C for 1 h, and then raise the temperature to 30-60°C and react for 36-60 h.

[0020] Preferably, the reaction temperature of the crude product in S2 with 1,3-propanesulfonic acid lactone is 50-75°C, and the reaction time is 60-90 h.

[0021] This invention also provides the application of the above-mentioned near-infrared second-zone conjugated small molecule photothermal agent in near-infrared second-window fluorescence imaging and photothermal therapy drugs.

[0022] Preferably, in application, a water-soluble photothermal agent is prepared by nanoprecipitation of a near-infrared II conjugated small molecule photothermal agent and a liposome dimyristoyl phosphatidylcholine.

[0023] The near-infrared II conjugated small molecule photothermal agent of the present invention exhibits good biocompatibility and low fluorescence quenching after modification with liposomal dimyristoylphosphatidylcholine (DMPC), and has been successfully applied to NIR-II laser-induced high-contrast NIR-II fluorescence imaging and efficient NIR-II photothermal therapy.

[0024] The beneficial effects of this invention are:

[0025] (1) The near-infrared II conjugated small molecule photothermal agent of the present invention has a liposome molecular structure. The DAD conjugated backbone causes the absorption to redshift, which significantly improves the fluorescence intensity of the molecule, thereby improving the resolution and contrast of in vivo imaging, which helps to detect and locate tumor tissue more accurately. The zwitterionic groups enable the molecule to form a liposome structure, which effectively reduces the fluorescence quenching of the material and ensures the stability and accuracy of fluorescence imaging.

[0026] (2) The near-infrared II conjugated small molecule photothermal agent of the present invention, after modification with liposomal dimyristoylphosphatidylcholine (DMPC), exhibits good biocompatibility, reduces toxicity and side effects on normal tissues, and improves the safety of treatment. This photothermal agent has been successfully applied to NIR-II laser-induced high-contrast NIR-II fluorescence imaging and efficient NIR-II photothermal therapy, providing an effective means for the precise diagnosis and treatment of cancer, and has broad application prospects.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] Figure 1 The nuclear magnetic resonance spectrum of the near-infrared two-region conjugated small molecule photothermal agent prepared in Example 1 of this invention;

[0029] Figure 2 This is the mass spectrum of the near-infrared two-region conjugated small molecule photothermal agent prepared in Example 1 of the present invention;

[0030] Figure 3 This is the normalized absorption spectrum of the near-infrared two-region conjugated small molecule photothermal agent prepared in Example 1 of the present invention;

[0031] Figure 4 The emission spectra of the organic solution and nano-aqueous solution of the near-infrared II conjugated small molecule photothermal agent prepared in Example 1 of this invention are shown.

[0032] Figure 5 The near-infrared II conjugated small molecule photothermal agent nano-aqueous solution prepared in Example 1 of this invention is analyzed at 1064 nm (1.0 W / cm²). -2 Schematic diagram of photothermal heating curve under laser irradiation;

[0033] Figure 6 This is a schematic diagram showing the photothermal stability of the near-infrared II conjugated small molecule photothermal agent prepared in Example 1 of the present invention over five on / off cycles;

[0034] Figure 7 This is a schematic diagram of cell confocal imaging according to the present invention;

[0035] Figure 7 In the diagram, 'a' represents a nuclear staining image obtained using Hurst staining. Figure 7 In the diagram, b represents the bright-field plot of the cell. Figure 7 In the image, 'c' represents the FITC uptake staining pattern. Figure 7 In this context, d represents the merged overlay image;

[0036] Figure 8 This is a schematic diagram showing the test results of the cytotoxicity and photothermal therapy capabilities of the present invention;

[0037] Figure 9 This is a schematic diagram illustrating the cell viability test results obtained by the present invention using live / dead fluorescence analysis and calcein-AM / PI co-staining;

[0038] Figure 9 In the image, 'a' represents a cell staining pattern treated with PBS. Figure 9 In the image, b represents a cell staining pattern after treatment with PBS and near-infrared laser irradiation. Figure 9 In the image, 'c' represents a cell staining pattern treated with a nano-water solution. Figure 9 In the diagram, d represents a cell staining image obtained by treating the cells with a nano-water solution and then irradiating them with a near-infrared laser.

[0039] Figure 10 This is a schematic diagram illustrating the quantitative analysis of cell photothermal effects using flow cytometry in this invention.

[0040] Figure 10 In the diagram, 'a' represents a flow cytometry plot of cells treated with PBS. Figure 10 In the diagram, b represents a flow cytometry plot of cells treated with PBS and irradiated with near-infrared laser. Figure 10 In the figure, c represents the flow cytometry plot of cells treated with nano-water solution. Figure 10 In the figure, d represents a flow cytometry plot of cells treated with a nano-water solution and irradiated with a near-infrared laser. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0042] A near-infrared II conjugated small molecule photothermal agent with a liposome-like molecular structure, the structural formula of which is as follows.

[0043]

[0044] The targeted near-infrared II conjugated small molecule photothermal agent of the present invention contains a DAD conjugated backbone that red-shifts the absorption, increases the fluorescence intensity of the molecule, and improves the resolution and contrast of in vivo imaging. At the same time, zwitterionic groups are used to transform it into a liposome-like structure to reduce fluorescence quenching of the material.

[0045] This invention also provides a method for preparing the above-mentioned near-infrared II region conjugated small molecule photothermal agent, comprising the following steps:

[0046] S1. Under light-protected conditions, organic conjugated small molecule monomer one, organic conjugated small molecule monomer two, and a solvent were mixed to obtain a mixed solution. Nitrogen gas was then introduced into the mixed solution, followed by the addition of a palladium catalyst. Under a protective atmosphere, a Stieler coupling reaction was carried out to obtain compound I.

[0047] The structural formula of compound I is as follows:

[0048]

[0049] S2. Under light-protected conditions, the compound of formula I is first reacted with dimethylamine to obtain a crude product. Then, the crude product is reacted with 1,3-propanesulfonic acid lactone under a protective atmosphere to prepare a near-infrared II region conjugated small molecule photothermal agent.

[0050] Preferably, the ratio of organic conjugated small molecule monomer one, organic conjugated small molecule monomer two, and solvent in S1 is 0.2-1 mM: 0.4-2 mM: 10-50 mL.

[0051] In some embodiments of the present invention, the molar ratio of organic conjugated small molecule monomer one to organic conjugated small molecule monomer two is 1:2.

[0052] In some embodiments of the present invention, the ratio of organic conjugated small molecule monomer one, organic conjugated small molecule monomer two, and solvent is 0.2 mM: 0.4 mM: 10 mL.

[0053] In some embodiments of the present invention, one of the organic conjugated small molecule monomers is 2,5-bis(2-hexyldecyl)-3,6-bis(5-bromothienyl)-pyrrolopyrroledione and 4,8-bis(5-bromo-4-(2-octyldodecyl)thienyl)-benzo[1,2-c;4,5-c']bis[1,2,5]thiadiazole, CAS number 1374601-35-0;

[0054] The organic conjugated small molecule monomer is 2-(-9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)thiophene, CAS number 570414-33-4.

[0055] Preferably, the solvent in S1 includes toluene, and the palladium catalyst includes tetrakis(triphenylphosphine)palladium. The tetrakis(triphenylphosphine)palladium is used only for catalysis and is not a reactant; therefore, its amount is not further limited and can be any amount known to those skilled in the art.

[0056] Preferably, the nitrogen gas is introduced into S1 for 20–30 min, the temperature of the Stieler coupling reaction is 90–120 °C, and the reaction time is 18–30 h.

[0057] In some embodiments of the present invention, the nitrogen gas is introduced for 20 minutes, the temperature of the Stieler coupling reaction is 100°C, and the reaction time is 24 hours.

[0058] In some embodiments of the present invention, the synthetic route of step S1 is as follows:

[0059]

[0060] In some embodiments of the present invention, in step S1, organic conjugated small molecule monomer II is first dissolved in tetrahydrofuran, and n-butyllithium is added dropwise under the protection of nitrogen at -78°C. After thorough stirring, tributyltin is added, heated to room temperature, and stirred for 24 hours. The reaction is then quenched with water, extracted with dichloromethane, and dried with sodium sulfate to obtain a crude product. Then, the crude product is mixed with organic conjugated small molecule monomer I and a solvent under light-protected conditions to obtain a mixed solution.

[0061] In some embodiments of the present invention, after the Stieler coupling reaction is completed in step S1, the product is evaporated to dryness and then purified by column chromatography to obtain compound of formula I. The developing solvent for purification includes petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:1.

[0062] Preferably, the molar ratio of compound I in S2 to 1,3-propanesulfonic acid lactone is 0.5-1:10-12.

[0063] In some embodiments of the present invention, the molar ratio of compound of formula I in S2 to 1,3-propanesulfonic acid lactone is 1:10.

[0064] Preferably, the reaction of compound I in S2 with dimethylamine is to first react at 0°C for 1 h, and then raise the temperature to 30-60°C and react for 36-60 h.

[0065] In some embodiments of the present invention, the reaction of compound I in S2 with dimethylamine is to first react at 0°C for 1 hour, and then raise the temperature to 40°C and react for 48 hours.

[0066] In some embodiments of the present invention, dimethylamine is dissolved in methanol to prepare a dimethylamine solution with a concentration of 25 wt%.

[0067] Preferably, the reaction temperature of the crude product in S2 with 1,3-propanesulfonic acid lactone is 50-75°C, and the reaction time is 60-90 h.

[0068] In some embodiments of the present invention, the reaction temperature of the crude product in S2 with 1,3-propanesulfonic acid lactone is 60°C and the reaction time is 72 h.

[0069] In some embodiments of the present invention, the synthetic route of step S2 is as follows:

[0070]

[0071] In some embodiments of the present invention, after the reaction in step S2 is completed, the product is evaporated and then purified by chromatography to obtain a near-infrared II region conjugated small molecule photothermal agent. The developing solvent for purification includes petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 5:1.

[0072] This invention also provides the application of the above-mentioned near-infrared second-zone conjugated small molecule photothermal agent in near-infrared second-window fluorescence imaging and photothermal therapy drugs.

[0073] Preferably, in application, a water-soluble photothermal agent is prepared by nanoprecipitation of a near-infrared II conjugated small molecule photothermal agent and a liposomal dimyristoylphosphatidylcholine. This invention does not impose any particular limitation on the nanoprecipitation method; any nanoprecipitation method well-known to those skilled in the art can be used.

[0074] The near-infrared II conjugated small molecule photothermal agent of the present invention exhibits good biocompatibility and low fluorescence quenching after modification with liposomal dimyristoylphosphatidylcholine (DMPC), and has been successfully applied to NIR-II laser-induced high-contrast NIR-II fluorescence imaging and efficient NIR-II photothermal therapy.

[0075] Example 1

[0076] This invention provides a method for preparing a near-infrared II region conjugated small molecule photothermal agent, comprising the following steps:

[0077] S1. First, dissolve (2.0 g, 3.48 mM) 2-(-9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)thiophene in tetrahydrofuran. Add (4.18 mM) n-butyllithium dropwise under nitrogen protection at -78 °C. After stirring thoroughly, add (1.1 mL, 4.18 mM) tributyltin. Heat to room temperature and stir for 24 h. Quench the reaction with water, extract with dichloromethane, and dry with sodium sulfate to obtain the crude product.

[0078] Under light-protected conditions, (0.2 mM) 2,5-bis(2-hexyldecyl)-3,6-bis(5-bromothienyl)-pyrrolopyrroledione, 4,8-bis(5-bromo-4-(2-octyldodecyl)thienyl)-benzo[1,2-c;4,5-c']bis[1,2,5]thiadiazole, and (0.4 mM) crude product were added to 10 mL of toluene and completely dissolved to obtain a mixed solution. Nitrogen gas was then bubbled into the resulting mixed solution for at least 20 minutes. 5 mg of tetrakis(triphenylphosphine)palladium was added, and the reaction was carried out under nitrogen protection at 100 °C for 24 hours. After the reaction was completed, the product was evaporated to dryness and then purified by column chromatography (petroleum ether: ethyl acetate = 2:1 as the developing solvent) to obtain compound I.

[0079] S2. Under light-protected conditions, 0.05 mmol of compound I was dissolved in 2 mL of tetrahydrofuran. 2 mL of a 25 wt% dimethylamine solution was gradually added while stirring at 0 °C. After stirring at 0 °C for 1 h, the reaction temperature was raised to 40 °C and stirred for 48 h. The solvent was then evaporated under vacuum to obtain the crude product, which required no further purification. Subsequently, 2 mL of methanol was added to dissolve the product. Then, 0.5 mmol of 1,3-propanesulfonic acid lactone was added dropwise under a nitrogen atmosphere. The reaction was stirred at 60 °C for 72 h. After cooling, the product was evaporated and then purified by column chromatography (using petroleum ether:ethyl acetate = 5:1 as the developing solvent) to obtain a near-infrared II region conjugated small molecule photothermal agent.

[0080] Example 2

[0081] This invention provides a method for preparing a near-infrared II region conjugated small molecule photothermal agent, comprising the following steps:

[0082] S1. First, dissolve (2.0 g, 3.48 mM) 2-(-9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)thiophene in tetrahydrofuran. Add (4.18 mM) n-butyllithium dropwise under nitrogen protection at -78 °C. After stirring thoroughly, add (1.1 mL, 4.18 mM) tributyltin. Heat to room temperature and stir for 24 h. Quench the reaction with water, extract with dichloromethane, and dry with sodium sulfate to obtain the crude product.

[0083] Under light-protected conditions, (0.2 mM) 2,5-bis(2-hexyldecyl)-3,6-bis(5-bromothienyl)-pyrrolopyrroledione, 4,8-bis(5-bromo-4-(2-octyldodecyl)thienyl)-benzo[1,2-c;4,5-c']bis[1,2,5]thiadiazole, and (2 mM) crude product were completely dissolved in 10 mL of toluene to obtain a mixed solution. Nitrogen gas was then bubbled into the resulting mixed solution for at least 20 minutes. 5 mg of tetrakis(triphenylphosphine)palladium was added, and the reaction was carried out under nitrogen protection at 100 °C for 24 hours. After the reaction was completed, the product was evaporated to dryness and then purified by column chromatography (petroleum ether:ethyl acetate = 2:1 as the developing solvent) to obtain compound I.

[0084] S2. Under light-protected conditions, 0.1 mmol of compound I was dissolved in 2 mL of tetrahydrofuran. While stirring at 0 °C, 2 mL of a 25 wt% dimethylamine solution was gradually added. After stirring at 0 °C for 1 h, the reaction temperature was raised to 40 °C and stirred for 48 h. The solvent was then evaporated under vacuum to obtain the crude product, which required no further purification. Subsequently, 2 mL of methanol was added to dissolve the product. Then, 1.2 mmol of 1,3-propanesulfonic acid lactone was added dropwise under a nitrogen atmosphere. The reaction was stirred at 60 °C for 72 h. After cooling, the product was evaporated and then purified by column chromatography (using petroleum ether:ethyl acetate = 5:1 as the developing solvent) to obtain a near-infrared II region conjugated small molecule photothermal agent.

[0085] Example 3

[0086] This invention provides a method for preparing a near-infrared II region conjugated small molecule photothermal agent, comprising the following steps:

[0087] S1. First, dissolve (2.0 g, 3.48 mM) 2-(-9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)thiophene in tetrahydrofuran. Add (4.18 mM) n-butyllithium dropwise under nitrogen protection at -78 °C. After stirring thoroughly, add (1.1 mL, 4.18 mM) tributyltin. Heat to room temperature and stir for 24 h. Quench the reaction with water, extract with dichloromethane, and dry with sodium sulfate to obtain the crude product.

[0088] Under light-protected conditions, (1 mM) 2,5-bis(2-hexyldecyl)-3,6-bis(5-bromothienyl)-pyrrolopyrroledione, 4,8-bis(5-bromo-4-(2-octyldodecyl)thienyl)-benzo[1,2-c;4,5-c']bis[1,2,5]thiadiazole, and (2 mM) crude product were added to 10 mL of toluene and completely dissolved to obtain a mixed solution. Nitrogen gas was then bubbled into the resulting mixed solution for at least 20 minutes. 5 mg of tetrakis(triphenylphosphine)palladium was added, and the reaction was carried out under nitrogen protection at 100 °C for 24 hours. After the reaction was completed, the product was evaporated to dryness and then purified by column chromatography (petroleum ether: ethyl acetate = 2:1 as the developing solvent) to obtain compound I.

[0089] S2. Under light-protected conditions, 0.05 mmol of compound I was dissolved in 2 mL of tetrahydrofuran. 2 mL of a 25 wt% dimethylamine solution was gradually added while stirring at 0 °C. After stirring at 0 °C for 1 h, the reaction temperature was raised to 40 °C and stirred for 48 h. The solvent was then evaporated under vacuum to obtain the crude product, which required no further purification. Subsequently, 2 mL of methanol was added to dissolve the product. Then, 0.5 mmol of 1,3-propanesulfonic acid lactone was added dropwise under a nitrogen atmosphere. The reaction was stirred at 60 °C for 72 h. After cooling, the product was evaporated and then purified by column chromatography (using petroleum ether:ethyl acetate = 5:1 as the developing solvent) to obtain a near-infrared II region conjugated small molecule photothermal agent.

[0090] Application Example 1

[0091] The near-infrared second-zone conjugated small molecule photothermal agent obtained in Example 1 was combined with liposome dimyristoyl phosphatidylcholine (DMPC) to prepare a water-soluble liposome-modified near-infrared second-zone conjugated small molecule photothermal agent via nanoprecipitation. This agent was then applied to near-infrared second-window fluorescence imaging and photothermal therapy drugs.

[0092] Performance testing and characterization

[0093] Nuclear magnetic resonance spectroscopy and mass spectrometry: The near-infrared II conjugated small molecule photothermal agent prepared in Example 1 was subjected to nuclear magnetic resonance spectroscopy and mass spectrometry. The results are as follows: Figure 1 , Figure 2 As shown. From Figure 1 The proton NMR spectrum shows characteristic proton signals identical to those of conjugated small-molecule photothermal agents in the near-infrared II region; from Figure 2 The mass spectra show that the structure is identical to that of the conjugated small-molecule photothermal agent in the near-infrared II region, with a molecular weight of 2408.74. Figures 1 to 2 It can be seen that Example 1 successfully prepared a near-infrared II region conjugated small molecule photothermal agent.

[0094] Absorption spectroscopy test: Using N,N-dimethylformamide as the organic solvent, an organic solution of the near-infrared II region conjugated small molecule photothermal agent prepared in Example 1 was prepared, and its absorption spectrum was tested. The results are as follows: Figure 3 As shown, from Figure 3 The absorption spectrum shows that its maximum absorption peak is located in the 980 nm band. Furthermore, it exhibits absorption in the near-infrared band from 800 to 1150 nm.

[0095] Emission spectroscopy testing: An organic solution of the near-infrared II conjugated small molecule photothermal agent prepared in Example 1 was prepared using N,N-dimethylformamide as the organic solvent. A nano-aqueous solution of the near-infrared II conjugated small molecule photothermal agent and liposome-derived myristoyl phosphatidylcholine (DMPC) prepared in Example 1 was prepared using pure water as the solvent. The emission spectra of the organic solution and the nano-aqueous solution of the near-infrared II conjugated small molecule photothermal agent were tested. The results are as follows: Figure 4 As shown, compared with the emission band of organic solution, the maximum emission band of nano-aqueous solution shifted by 28 nm, indicating that the fluorescence properties of molecules changed after the formation of nano-aqueous solution, and this change is beneficial to reducing fluorescence quenching.

[0096] Photothermal conversion capability and photothermal stability test: The ability to convert near-infrared II light energy into heat energy and the photothermal stability in aqueous solution were studied using a 1064 nm laser to excite the nano-aqueous solution. The results are as follows: Figure 5 and Figure 6 As shown. From Figure 5 The schematic diagram of the photothermal heating curve shows that the maximum temperature change ΔT of the nano-aqueous solution is 31.9℃, indicating that the near-infrared II conjugated small molecule photothermal agent prepared in Example 1 has good photothermal conversion ability and has the potential for photothermal therapy. Figure 6 The schematic diagram of photothermal stability shows that the nano-aqueous solution maintained its excellent photothermal stability during the five laser-switched heating cycles, ensuring the stability and reliability of the photothermal therapy process.

[0097] Intracellular distribution detection: A fluorescent isothiocyanate-labeled liposome-modified near-infrared II conjugated small molecule photothermal agent (FITC@BTFQ / DMPC) cell culture medium was prepared by encapsulating the near-infrared II conjugated small molecule photothermal agent prepared in Example 1 and fluorescein isothiocyanate (FITC) in liposomes (DMPC). The cell culture medium was co-cultured with mouse breast cancer cells to visually demonstrate the intracellular distribution. The results are as follows: Figure 7 As shown in c, FITC green fluorescence is localized within cells, indicating that the near-infrared II conjugated small molecule photothermal agent has been internalized into cells, providing a basis for the role of photothermal therapy at the cellular level.

[0098] Cytotoxicity and photothermal therapy capability tests: The near-infrared II conjugated small molecule photothermal agent and liposomes (DMPC) prepared in Example 1 were configured into cell culture media with a certain concentration gradient. The highest concentration of the near-infrared II conjugated small molecule photothermal agent in the cell culture media was 100 μM / mL. At the same time, a cell culture media prepared with PBS solution was also prepared as a blank control. After culturing mouse breast cancer cells in the above cell culture media for 4 hours, the cell culture media prepared with near-infrared II conjugated small molecule photothermal agent and liposomes (DMPC) were irradiated with near-infrared laser. CCK-8 reagent was added, and the absorbance at 450 nm was measured. The absorbance obtained was converted into cell viability.

[0099] like Figure 8 As shown, the cell culture medium prepared from a near-infrared II conjugated small molecule photothermal agent and liposomes (DMPC) exhibits low cytotoxicity to mouse breast cancer cells without laser irradiation, indicating that the photothermal agent does not significantly interfere with the physiological functions of normal cells when the photothermal effect is not activated. However, it exhibits strong cytotoxicity under laser irradiation. This is because when the nanosolution is irradiated by laser, the photothermal agent molecules absorb light energy and convert it into heat energy, causing a rapid increase in local temperature. Cancer cells are more sensitive to temperature changes than normal cells, and this photothermal effect is highly selective, effectively killing cancer cells without affecting normal cells. This demonstrates that the near-infrared II conjugated small molecule photothermal agent prepared in this invention has good biocompatibility and possesses photothermal therapy capabilities under laser irradiation.

[0100] Cell viability assay and quantitative analysis of photothermal effect: The near-infrared II conjugated small molecule photothermal agent obtained in Example 1 and liposomes (DMPC) were prepared into a 100 μM nano-aqueous solution. Four groups of mouse breast cancer cell samples were set up for the experiment: PBS solution (blank control group), PBS solution laser irradiation group, nano-aqueous solution treatment group, and nano-aqueous solution laser irradiation group. Cell viability was tested by live / dead fluorescence analysis and calcein-AM / PI co-staining. The results are as follows: Figure 9 As shown in Figure ac, mouse breast cancer cells treated with only nano-aqueous solutions or only laser irradiation (PBS solution laser irradiation) showed negligible apoptosis. This indicates that under normal physiological conditions, this near-infrared II conjugated small molecule photothermal agent has minimal impact on normal cell metabolism and function, and will not cause significant damage to normal tissue cells during treatment, greatly reducing side effects and improving treatment safety; conversely, Figure 9Mouse breast cancer cells incubated with 100 μM nano-aqueous solution exhibited severe cell death when irradiated with 1064 nm laser. This is because after the photothermal agent absorbs near-infrared light, the light energy is converted into heat energy, causing the local temperature to rise rapidly. The high temperature destroys the cell membrane structure of cancer cells, triggering cancer cell death. This photothermal effect is highly selective and can effectively kill cancer cells without affecting normal cells, achieving precision treatment.

[0101] The photothermal effect of cells was quantitatively analyzed by flow cytometry, and the results are as follows: Figure 10 As shown in Figure ac, the apoptosis rate of mouse breast cancer cells was less than 7% after treatment with only nano-aqueous solution or only laser irradiation (PBS solution laser irradiation). This indicates that the nano-solution itself has low cytotoxicity in the absence of photothermal synergy, and laser irradiation alone causes little damage to cells, demonstrating that the nano-aqueous solution has good biosafety. Figure 10 In the d-cell model, after irradiation with a 1064nm laser on a nano-solution, the apoptosis rate increased to 96.4%. During photothermal therapy, the near-infrared II laser was efficiently absorbed by the photothermal agent, and the light energy was rapidly converted into heat energy, causing a sharp increase in local temperature. This high-temperature environment directly damages the cell membrane, organelles, and other structures of cancer cells, promoting apoptosis. This significant change fully verifies the powerful photothermal therapeutic effect of this near-infrared II conjugated small molecule photothermal agent under laser excitation.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A near-infrared II-region conjugated small molecule photothermal agent, characterized in that: It has a liposome-like molecular structure, with the following structural formula. 。 2. A method for preparing a near-infrared II conjugated small molecule photothermal agent as described in claim 1, characterized in that: Includes the following steps, S1. Under a light-protected environment, organic conjugated small molecule monomer I, organic conjugated small molecule monomer II and solvent are mixed to obtain a mixed solution. Nitrogen gas is introduced into the mixed solution and palladium catalyst is added. Under a protective atmosphere, the Stieler coupling reaction is carried out to obtain compound I. The organic conjugated small molecule monomer is 2,5-bis(2-hexyldecyl)-3,6-bis(5-bromothienyl)-pyrrolopyrroledione and 4,8-bis(5-bromo-4-(2-octyldodecyl)thienyl)-benzo[1,2-c;4,5-c']bis[1,2,5]thiadiazole; The organic conjugated small molecule monomer is 2-(-9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)thiophene; The structural formula of compound I is as follows: ； S2. Under light-protected conditions, the compound of formula I is first reacted with dimethylamine to obtain a crude product. Then, the crude product is reacted with 1,3-propanesulfonic acid lactone under a protective atmosphere to prepare a near-infrared II region conjugated small molecule photothermal agent.

3. The method for preparing a near-infrared II-region conjugated small molecule photothermal agent according to claim 2, characterized in that: The ratio of organic conjugated small molecule monomer I, organic conjugated small molecule monomer II, and solvent in S1 is 0.2~1mM: 0.4~2mM: 10~50mL.

4. The method for preparing a near-infrared II-region conjugated small molecule photothermal agent according to claim 2, characterized in that: The solvent in S1 includes toluene, and the palladium catalyst includes tetra(triphenylphosphine)palladium.

5. The method for preparing a near-infrared II-region conjugated small molecule photothermal agent according to claim 2, characterized in that: Nitrogen gas is introduced into S1 for 20-30 minutes, the temperature of the Stieler coupling reaction is 90-120℃, and the reaction time is 18-30 hours.

6. The method for preparing a near-infrared II conjugated small molecule photothermal agent according to claim 2, characterized in that: The molar ratio of compound I in S2 to 1,3-propanesulfonic acid lactone is 0.5~1:10~12.

7. The method for preparing a near-infrared II-region conjugated small molecule photothermal agent according to claim 2, characterized in that: The reaction of compound I in S2 with dimethylamine is first carried out at 0°C for 1 hour, and then the temperature is raised to 30~60°C for 36~60 hours.

8. The method for preparing a near-infrared II conjugated small molecule photothermal agent according to claim 2, characterized in that: The reaction temperature of the crude product in S2 with 1,3-propanesulfonic acid lactone is 50~75℃, and the reaction time is 60~90h.

9. An application of the near-infrared II conjugated small molecule photothermal agent as described in claim 1, characterized in that: Applications in the preparation of near-infrared second-window fluorescence imaging drugs and photothermal therapy drugs.

10. The application of the near-infrared II conjugated small molecule photothermal agent according to claim 9, characterized in that: In application, a water-soluble photothermal agent is prepared by nanoprecipitation of a near-infrared II region conjugated small molecule photothermal agent and a liposome dimyristoyl phosphatidylcholine.

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