Lipid droplet-targeted NIR-I photothermal agent and its application in hypoxic tumor treatment

Abstract

The application discloses a kind of lipid droplet targeted NIR-I photothermal agent and its application in hypoxic tumor treatment, belong to the technical field of photothermal therapy.The NIR-I photothermal agent of the application is a kind of organic photothermal material based on donor-acceptor structure, with strong electron-deficient and rigid planar structure dithiophene pyrrolone sub benzodifuran diketone (BTPDBDF) as acceptor unit, respectively with triphenylamine and tetraphenyl ethylene as donor unit, with strong intramolecular charge transfer, absorption spectrum reaches near infrared region one.Simultaneously, the material is prepared into water-soluble nanoparticles by nano precipitation method, and the biocompatibility and targeting are improved.The nano preparation has the ability of specific targeting cell lipid droplet, and shows significant heating performance and high photothermal conversion efficiency under 808 nm laser irradiation, and shows good near-infrared light capturing capacity, so as to facilitate effective photothermal therapy under the condition of tumor hypoxia.

Description

Technical Field

[0001] This invention belongs to the field of photothermal therapy technology, specifically relating to a lipid droplet-targeted NIR-I photothermal agent and its application in the treatment of hypoxic tumors. Background Technology

[0002] Since tumors are generally in a hypoxic state, treatment methods that rely on the generation of reactive oxygen species, such as type II photosensitizers, have inherent limitations. Therefore, providing a new strategy that is not limited by oxygen and can effectively kill tumor cells under hypoxic conditions has become an urgent technical problem to be solved.

[0003] Photothermal therapy is a tumor treatment strategy based on photothermal conversion materials. Its core mechanism involves using near-infrared light to excite a photothermal reagent, efficiently converting light energy into heat energy, thereby achieving local thermal ablation of diseased tissue. Compared to type II photosensitizer therapy, which relies on reactive oxygen species, this strategy offers advantages such as minimal invasiveness, high spatiotemporal selectivity, low systemic toxicity, low risk of inducing tolerance, and no oxygen dependence. With the expansion of the near-infrared optical window and the development of novel photothermal materials, photothermal therapy has seen continuous improvements in tissue penetration depth and biosafety. In the near-infrared band, light has higher tissue-allowable exposure energy and can reduce photon scattering and tissue autofluorescence, thus significantly improving the imaging signal-to-noise ratio and treatment accuracy, providing a technological foundation for precision diagnosis and treatment of tumors.

[0004] Meanwhile, photothermal reagents can achieve precise positioning at the subcellular level. Through multiple synergistic mechanisms such as specifically targeting and destroying the energy metabolism center of tumor cells, and inducing lipid peroxidation, they can synergistically improve treatment efficiency, enhance tumor selectivity, and reduce systemic toxicity. Summary of the Invention

[0005] This invention provides a lipid droplet-targeting NIR-I photothermal agent and its application in hypoxic tumor therapy through molecular design using donor-receptor (DA) structures. The NIR-I photothermal agent of this invention exhibits strong absorption, a high molar absorptivity, and high photothermal conversion efficiency in the near-infrared window, and can specifically target lipid droplets, making it suitable for near-infrared photothermal imaging and photothermal therapy of tumors in hypoxic environments.

[0006] The lipid droplet-targeted NIR-I photothermal agent of this invention has the following structure:

[0007]

[0008] The preparation method of the lipid droplet-targeted NIR-I photothermal agent of the present invention includes the following steps:

[0009] Step 1: Thiophene-pyrrole dione, benzodifuran dione, piperidine, and acetic acid were mixed uniformly and heated to carry out the first step reaction. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain a solid. The solid was washed with methanol, dried, and finally purified by column chromatography to obtain the intermediate product S1, the structure of which is shown below:

[0010]

[0011] Step 2: Under an inert gas atmosphere and in the dark, S1, N-bromosuccinimide, and tetrahydrofuran were mixed and heated to carry out the second step reaction. After the reaction was completed, the reaction solution was poured into water to quench the reaction. Finally, the mixture was extracted with dichloromethane, dried, and concentrated. Then, it was purified by column chromatography to obtain the intermediate product S2, the structure of which is shown below:

[0012]

[0013] Step 3:

[0014] 3a. Under an inert gas atmosphere, S2, triphenylamine 4-borate, potassium carbonate aqueous solution, tetrahydrofuran, and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) (Pd-132) were mixed uniformly and heated to react. After the reaction was completed, the reaction solution was poured into water to quench the reaction, followed by extraction with dichloromethane, drying and concentration, and finally purification by column chromatography to obtain the final product W1, the structural formula of which is shown below:

[0015]

[0016] 3b. Under an inert gas atmosphere, S2, 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene, tetratetraphenylphosphine palladium, potassium carbonate aqueous solution, tetrabutylammonium bromide, and anhydrous toluene were mixed and heated to a uniform temperature for reaction. After the reaction was completed, the reaction solution was poured into water to quench the reaction, followed by extraction with dichloromethane, drying and concentration, and finally purification by column chromatography to obtain the final product W2, the structural formula of which is shown below:

[0017] .

[0018] In specific implementation, in step 1, the molar ratio of thienopyrrole dione, benzodifuran dione and piperidine is 2:1:0.2; the reaction temperature of step 1 is 120℃ and the reaction time is 18 h.

[0019] In specific implementation, in step 2, the molar ratio of S1 to N-bromosuccinimide is 1:7; the reaction temperature in step 2 is 65℃, and the reaction time is 24 h.

[0020] In specific implementation, in step 3a, the molar ratio of S2, 4-boronic acid triphenylamine, potassium carbonate and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) is 1:2.5:5:0.2; the reaction temperature in 3a is 65℃ and the reaction time is 24 h.

[0021] In specific implementation, in step 3b, the molar ratio of S2, 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene, potassium carbonate, tetratriphenylphosphine palladium and tetrabutylammonium bromide is 1:2.5:5:0.2:0.2; the reaction temperature in 3b is 90℃ and the reaction time is 24 h.

[0022] The present invention relates to the application of lipid droplet-targeted NIR-I photothermal agent in the preparation of hypoxic tumor therapeutic drug formulations.

[0023] The present invention relates to the application of lipid droplet-targeted NIR-I photothermal agent in the preparation of a photothermal combined therapy for tumor diseases.

[0024] In practical applications, the NIR-I photothermal agent and the amphiphilic polymer DSPE-PEG5000 are prepared into water-soluble nanoparticles by nanoprecipitation, thereby improving their biocompatibility and targeting.

[0025] The lipid droplet-targeted NIR-I photothermal agent of this invention is suitable for near-infrared photothermal imaging and lipid droplet-targeted tumor photothermal therapy.

[0026] The molecules W1 and W2 synthesized in this invention are only soluble in organic solvents, which can damage cells and prevent subsequent biological experiments. Therefore, W1 and W2 synthesized in this invention can only achieve good biocompatibility when used in the form of nanomaterials. The testing section characterized the basic photophysical properties of W1, W2, and nanoparticles, while the specific application performance is demonstrated by the nanoparticles.

[0027] The beneficial effects of this invention are reflected in:

[0028] This invention provides lipid droplet-targeted NIR-I photothermal agents, namely W1 and W2. These photothermal agents utilize a strongly electron-deficient dithiophene-pyrrolidone-ylidene difurandione (BTPDBDF) with a rigid planar structure as the acceptor unit, and triphenylamine and tetraphenylethylene as the donor units, respectively. They exhibit strong intramolecular charge transfer and their absorption spectra reach the near-infrared region I. The maximum absorption peaks of W1 and W2 are located at 843 nm and 784 nm, respectively, with molar absorptivity of 6.235 × 10⁻⁶. 4 L mol -1 cm -1 and 6.067 × 10 4 L mol -1 cm-1 Both have absorption wavelengths that are highly matched with 808 nm lasers, and their extremely high molar absorption coefficients indicate that they have strong light-capturing capabilities in the corresponding near-infrared band, thus providing a key guarantee for efficient photothermal tumor therapy.

[0029] This invention further prepares water-soluble nanoparticles by combining the photothermal reagent with the amphiphilic polymer DSPE-PEG5000 via a nanoprecipitation method, thereby improving their biocompatibility. The resulting nanoparticles can specifically target lipid droplets, and their photothermal conversion efficiencies reach 65.68% (W1 NPs) and 61.62% (W2 NPs), respectively. In a tumor-bearing mouse model, after exposure to an 808 nm laser (1 W / cm²), the nanoparticles were successfully used. 2 After irradiation, both types of nanoparticles effectively increased the temperature at the tumor site and achieved tumor ablation; among them, W1 NPs, with a redder absorption spectrum and superior photothermal performance, exhibited a more significant therapeutic effect. This invention provides a clear technical solution and practical basis for developing efficient and targeted organic small molecule photothermal formulations. Attached Figure Description

[0030] Figure 1 For intermediate product S1 in CDCl3 1 H nuclear magnetic resonance spectrum.

[0031] Figure 2 For intermediate product S1 in CDCl3 13 C10 NMR spectrum.

[0032] Figure 3 For intermediate product S2 in CDCl3 1 H nuclear magnetic resonance spectrum.

[0033] Figure 4 For intermediate product S2 in CDCl3 13 C10 NMR spectrum.

[0034] Figure 5 For W1 in CDCl3 1 H nuclear magnetic resonance spectrum.

[0035] Figure 6 For W1 in CDCl3 13 C10 NMR spectrum.

[0036] Figure 7 The image is a MALDI-TOF MS plot of W1.

[0037] Figure 8 For W2 in CDCl3 1 H nuclear magnetic resonance spectrum.

[0038] Figure 9For W2 in CDCl3 13 C10 NMR spectrum.

[0039] Figure 10 The image is a MALDI-TOF MS plot of W2.

[0040] Figure 11 The figures show the photophysical properties of W1, W2, W1 NPs, and W2 NPs of the present invention. Figure (a) shows the UV-Vis-NIR absorption spectra of W1 and W2; Figures (b) and (c) show the hydrodynamic diameters and morphologies of W1 NPs and W2 NPs measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively; Figure (d) shows the UV-Vis-NIR absorption spectra of W1 NPs and W2 NPs; Figure (e) shows the fluorescence emission spectra of W1 NPs and W2 NPs at short wavelengths; and Figure (f) shows the hydrodynamic diameters of W1 NPs and W2 NPs after 14 days in PBS.

[0041] Figure 12 The figures show the in vitro photothermal properties of the W1 NPs and W2 NPs of the present invention. Figure (a) is an in vitro photothermal imaging image of the W1 NPs and W2 NPs; Figures (b) and (c) are the photothermal conversion efficiency of the W1 NPs and W2 NPs, respectively; Figures (d) and (e) are the photothermal stability of the W1 NPs and W2 NPs, respectively.

[0042] Figure 13 Figure (a) shows the cytotoxicity test results of W1 NPs and W2 NPs according to the present invention; and Figure (b) shows the cytotoxicity test results of W2 NPs.

[0043] Figure 14 The images show the lipid droplet targeting maps and hypoxic and normoxic cell killing capabilities of W1 NPs and W2 NPs of the present invention. Figure (a) shows the lipid droplet targeting map and hypoxic environment creation map of W1 NPs; Figure (b) shows the live-death experiment of W1 NPs and W2 NPs.

[0044] Figure 15 This is a schematic diagram illustrating the photothermal therapy process of W1 NPs and W2 NPs on mouse tumors according to the present invention. Figure (a) shows W1 NPs, W2 NPs, and the control group under an 808 nm laser (1 W / cm²). 2 (a) In vivo photothermal imaging under irradiation; (b) Tumor volume of a mouse after dissection 15 days after photothermal therapy; (c) Photograph of the tumor. Detailed Implementation

[0045] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0046] Unless otherwise specified, all raw materials used in this invention are obtained through commercial purchase. For example, DSPE-PEG5000 was purchased from Energie.

[0047] Any aspects of this invention not described in detail are conventional methods in the field.

[0048] The preparation method of the lipid droplet-targeted NIR-I photothermal agent of the present invention is as follows:

[0049]

[0050]

[0051] The specific steps are as follows:

[0052] Step 1: Synthesis of compound S1

[0053] Thiophene-2-pyrroledione (2.74 g, 6.32 mmol), benzodifurandione (0.60 g, 3.16 mmol), and piperidine (63.00 µL, 0.64 mmol) were added to 50 mL of acetic acid solution (36%–38% by mass) and stirred at 120 °C for 18 h. After the reaction was complete, the mixture was cooled to room temperature and filtered. The solid was washed with methanol and then purified by silica gel column chromatography using eluent (PE:CH2Cl2 = 4:1) to obtain a black-green solid, which was compound S1. The molar ratio of thieno-2-pyrroledione, benzodifurandione, and piperidine was 2:1:0.2.

[0054] Step 2: Synthesis of compound S2

[0055] Compound S1 (1.00 g, 0.98 mmol) was added to a 40.00 mL THF solution under an inert gas atmosphere and in the dark. NBS (1.22 g, 6.86 mmol) was added in portions, and the reaction was carried out at 65 °C for 24 h. After the reaction was complete, the reaction solution was quenched in water, extracted three times with CH2Cl2, and the organic phase was washed with water and brine, then dried over anhydrous Na2SO4. Purification by silica gel column chromatography with eluent (PE:CH2Cl2 = 1:1) yielded a blackish-green solid, compound S2. The molar ratio of compound S1 to N-bromosuccinimide was 1:7.

[0056] Step 3: Synthesis of W1

[0057] Under inert gas conditions, compound S2 (0.42 g, 0.36 mmol), triphenylamine 4-borate (0.26 g, 0.89 mmol), and 3.00 mL of potassium carbonate solution were added to 9.00 mL of THF solution. Bubbling was performed for 15 min, followed by the addition of 0.06 g of Pd-132. The reaction was carried out at 65 °C for 24 h. After the reaction was complete, the reaction solution was quenched in water, extracted three times with CH2Cl2, and the combined organic layers were washed three times with water. The mixture was then dried over anhydrous Na2SO4 and concentrated. The product was purified by silica gel column chromatography using an eluent (PE:CH2Cl2 = 1:1), yielding a black solid, designated W1. This indicates that the molar ratio of compound S2, triphenylamine 4-borate, potassium carbonate, and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) was 1:2.5:5:0.2.

[0058] Step 4: Synthesis of W2

[0059] Under inert gas conditions, compound S2 (0.30 g, 0.26 mmol), 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene (0.30 g, 0.64 mmol), 1.30 mL potassium carbonate solution, and 5.00 mg tetrabutylammonium bromide were added to 5.00 mL dry toluene solution. After bubbling for 15 min, 15.00 mg tetra-triphenylphosphine palladium was added, and the reaction was carried out at 90 °C for 24 h. After the reaction was completed, the reaction solution was quenched in water, extracted three times with CH2Cl2, and the combined organic layers were washed three times with water. The mixture was then dried over anhydrous Na2SO4 and concentrated. The product was purified by silica gel column chromatography with eluent (PE:CH2Cl2 = 1:1) to obtain a green solid, W2. That is, the molar ratio of S2, 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene, potassium carbonate, tetra-triphenylphosphine palladium and tetrabutylammonium bromide is 1:2.5:5:0.2:0.2.

[0060] Step 5: Preparation of W1 NPs

[0061] 1 mg of W1 and 5 mg of DSPE-PEG5000 were dissolved in a THF:CHCl3 solution (v / v = 4:1) and sonicated until homogeneous. The mixture was then added dropwise to 10 mL of deionized water. The solution was then evaporated under N2 protection in a fume hood with stirring. Larger nanoparticles were then filtered through a 0.45 µM filter and finally lyophilized for later use.

[0062] Step 6: Preparation of W2 NPs

[0063] 1 mg of W2 and 5 mg of DSPE-PEG5000 were dissolved in a THF solution and sonicated until homogeneous. The mixture was then added dropwise to 10 mL of deionized water. The solution was then evaporated by stirring in a fume hood under N2 protection. Larger nanoparticles were then filtered through a 0.45 µM filter and finally lyophilized for later use.

[0064] The lipid droplet-targeted NIR-I photothermal agent of this invention uses dithiophene-pyrrolidone-ylidene-difurandione (BTPDBDF), which has a strong electron-deficient and rigid planar structure, as the acceptor unit, and triphenylamine and tetraphenylethylene as the donor units, respectively. It exhibits strong intramolecular charge transfer and its absorption spectrum reaches the near-infrared region I. The maximum absorption peaks of W1 and W2 are located at 843 nm and 784 nm, respectively, with molar absorptivity of 6.235 × 10⁻⁶. 4 L mol -1 cm -1 and 6.067 ×10 4 L mol -1 cm -1 Both have absorption wavelengths that are highly matched with 808 nm lasers, and their extremely high molar absorption coefficients indicate that they have strong light-capturing capabilities in the corresponding near-infrared band, thus providing a key guarantee for efficient photothermal tumor therapy.

[0065] This invention further prepares water-soluble nanoparticles by combining the photothermal reagent with the amphiphilic polymer DSPE-PEG5000 via a nanoprecipitation method, thereby improving their biocompatibility. The resulting nanoparticles can specifically target lipid droplets, and their photothermal conversion efficiencies reach 65.68% (W1 NPs) and 61.62% (W2 NPs), respectively. In a tumor-bearing mouse model, after irradiation with an 808 nm laser (1 W / cm²), both types of nanoparticles can effectively increase the temperature at the tumor site and achieve tumor ablation; among them, W1 NPs, with a redder absorption spectrum and superior photothermal performance, exhibits a more significant therapeutic effect. This invention provides a clear technical solution and practical basis for developing efficient and targeted organic small molecule photothermal agents.

[0066] Example 1:

[0067] Synthesis of compound S1: Thiophene-pyrrole dione (2.74 g, 6.32 mmol), benzodifuran dione (0.60 g, 3.16 mmol), and piperidine (63.00 µL, 0.64 mmol) were added to 50 mL of acetic acid solution, and the mixture was stirred at 120 °C for 18 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The solid was washed with methanol, and then purified by silica gel column chromatography with eluent (PE:CH2Cl2 = 4:1) to obtain 2.10 g of a blackish-green solid, with a yield of 57%. 1 H NMR (400MHz, Chloroform-d) δ 8.90 (s, 2H), 7.62 (d, J = 5.0 Hz, 2H), 6.67 (d, J = 5.1Hz, 2H), 3.62 (d, J = 7.2 Hz, 4H), 1.82 (s, 2H), 1.37- 1.13 (m, 64H), 0.84(t, J = 6.8 Hz, 12H). 13 C NMR (101 MHz, CDCl3) δ 168.89, 168.32, 154.38,150.12, 138.74, 130.76, 124.17, 115.63, 114.35, 110.30, 108.24, 45.24, 36.11,30.89, 30.85, 30.49, 28.92, 25.42, 13.10.

[0068] Synthesis of compound S2: Compound S1 (1.00 g, 0.98 mmol) was added to a 40.00 mL THF solution under light-protected conditions, followed by the addition of NBS (1.22 g, 6.86 mmol) in portions. The reaction was carried out at 65 °C for 24 h. After the reaction was completed, the reaction solution was quenched in water, extracted three times with CH2Cl2, and the organic phase was washed with water and brine, then dried over anhydrous Na2SO4. The product was purified by silica gel column chromatography with eluent (PE:CH2Cl2 = 1:1) to obtain 1.00 g of a blackish-green solid, with a yield of 87%. 1H NMR (400 MHz, Chloroform-d) δ 8.72 (s, 2H), 6.68 (s, 2H), 3.63 (d, J = 6.9 Hz, 4H), 1.80 (s, 2H), 1.25 (s, 105H), 0.91- 0.78 (m, 12H). 13 C NMR (151 MHz, CDCl3) δ 169.46, 168.85, 154.36, 151.06, 130.30, 125.10,115.86, 115.83, 115.01, 109.12, 46.38, 37.35, 32.00, 31.97, 31.55, 30.05,29.75, 29.72, 29.70, 29.65, 29.43, 29.40, 26.54, 22.76, 14.19.

[0069] Synthesis of W1: Under inert gas conditions, compound S2 (0.42 g, 0.36 mmol), triphenylamine 4-borate (0.26 g, 0.89 mmol), and 3.00 mL of potassium carbonate solution were added to 9.00 mL of THF solution. Bubbling was performed for 15 min, followed by the addition of 0.06 g of Pd-132. The reaction was carried out at 65 °C for 24 h. After the reaction was complete, the reaction solution was quenched in water, extracted three times with CH2Cl2, and the combined organic layers were washed three times with water. The mixture was then dried over anhydrous Na2SO4 and concentrated. The product was purified by silica gel column chromatography using eluent (PE:CH2Cl2 = 1:1) to a black solid of 0.15 g. The yield was 28%. 1 H NMR (400 MHz, Chloroform-d) δ 8.77 (s, 2H), 7.42 (s, 4H), 7.29 (dd, J = 15.3, 7.7 Hz, 14H), 7.12 (d, J = 7.8 Hz, 20H), 6.93 (s, 4H), 6.64 (s, 2H), 3.64 (s, 4H), 1.82 (s,2H), 1.36- 1.16 (m, 111H), 0.84 (m, J = 6.8 Hz, 12H). 13C NMR (151 MHz, CDCl3)δ 169.84, 155.82, 150.87, 149.23, 146.61, 129.55, 126.81, 125.58, 124.31,121.34, 108.37, 105.58, 46.06, 37.26, 31.92, 31.89, 31.51, 31.48, 30.04,29.71, 29.65, 29.61, 29.37, 26.47, 22.69, 14.14.MALDI-TOF / MS: [M] + -calcd: 1507.82, Found: 1507.7673.

[0070] Synthesis of W2: Under inert gas conditions, compound S2 (0.30 g, 0.26 mmol), 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene (0.30 g, 0.64 mmol), 1.30 mL potassium carbonate solution, and 5.00 mg tetrabutylammonium bromide were added to 5.00 mL dry toluene solution. After bubbling for 15 min, 15.00 mg tetra-triphenylphosphine palladium was added, and the reaction was carried out at 90 °C for 24 h. After the reaction was completed, the reaction solution was quenched in water, extracted three times with CH2Cl2, and the combined organic layers were washed three times with water. The mixture was then dried over anhydrous Na2SO4 and concentrated. The product was then purified by silica gel column chromatography with eluent (PE:CHCl3 = 1:1) to obtain 0.23 g of green solid, with a yield of 53%. 1 H NMR (400 MHz, Chloroform-d) δ 8.79 (s,2H), 7.23 (d, J = 8.1 Hz, 4H), 7.14 (s, 4H), 7.04 (m, 25H), 6.98 (d, J = 8.1Hz, 6H), 6.51 (s, 2H), 3.60 (s, 4H), 1.79 (s, 2H), 1.36- 1.16 (m, 92H), 0.85(m, J = 5.0Hz, 12H). 13C NMR (151 MHz, CDCl3) δ 169.56, 155.87, 150.82,145.55, 143.52, 143.32, 142.19, 140.01, 132.09, 131.43, 131.32, 127.94,127.74, 127.04, 126.88, 126.80, 125.08, 115.04, 108.54, 46.24, 37.20, 31.93,31.89, 31.45, 30.06, 29.72, 29.66, 29.60, 29.37, 26.47, 26.45, 22.70, 14.15.MALDI-TOF / MS:[M] + -calcd: 1681.89, Found: 1681.8699.

[0071] All synthesized molecules and their intermediates were produced using... 1 H NMR, 13 Comprehensive characterization was performed using C10 NMR and MALDI-TOF MS. Detailed spectroscopic data for all compounds are available as follows: Figure 1-10 As shown, the successful synthesis and structural integrity of the designed molecule are confirmed.

[0072] Example 2: Performance Testing

[0073] 1. Photophysical properties of W1, W2, W1 NPs and W2 NPs

[0074] This invention provides organic small-molecule photothermal reagents W1 and W2 with near-infrared absorption properties, designed based on a donor-acceptor (DA) structure. The target molecules are synthesized using dithienenopyrrolidone-ylidene difuranedione (BTPDBDF) as the acceptor unit and triphenylamine and tetraphenylethylene as donor units, respectively, through a three-step reaction involving Knoevenagel condensation, bromination, and Suzuki coupling. The structures were confirmed by 1H NMR, 1C NMR, and high-resolution mass spectrometry (see [link to article]). Figures 1-10 ).

[0075] Figure 11 Figure (a) shows the UV-Vis-NIR absorption spectra of W1 and W2 in chloroform. The maximum absorption wavelength of W1 (λ) max Located at 843 nm, the molar absorption coefficient (ε) is 6.24 × 10⁻⁶. 4 L mol -1 cm -1 ; λ of W2 max Located at 784 nm, ε is 6.07 × 10⁻⁶. 4 L mol-1 cm -1 This indicates that both possess efficient light-harvesting capabilities. W1 exhibits a redshift of approximately 59 nm compared to W2, attributed to the stronger electron-donating ability of TPA, which further enhances the push-pull electron effect of the DAD structure and reduces the band gap. To suit biological system research, a nanoprecipitation method was used, employing the amphiphilic polymer DSPE-PEG5000 as a carrier to encapsulate W1 and W2 into nanoparticles (W1 NPs and W2 NPs) with good water dispersibility and biocompatibility, respectively. Characterization by transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed that the obtained nanoparticles have uniform morphology and are spherical, with hydrodynamic diameters of 142.1 nm and 165.6 nm, respectively. Figure 11 (b and c) are suitable for accumulation in tumor tissue through high permeability and retention (EPR) effects. After 14 days of continuous monitoring in PBS, no significant change in nanoparticle size or precipitation was observed. Figure 11 (f) This confirms its excellent long-term colloidal stability. Further testing of the optical properties of the nanoparticles in water was conducted. Figure 11 As shown in Figure d, the maximum absorption peaks of W1 NPs and W2 NPs are located at 763 nm and 680 nm, respectively, showing a significant blue shift compared to their absorption in chloroform, suggesting that the change in aggregation state after encapsulation affects their light absorption behavior. Fluorescence spectroscopy analysis shows ( Figure 11 (e) W1 NPs exhibit significant fluorescence emission at short wavelengths, while W2 NPs show no significant emission; this fluorescence property makes W1 NPs a potential optical probe that can be used for subsequent cell targeting and real-time tracking.

[0076] 2. In vitro photothermal properties

[0077] Based on the advantages of near-infrared light in terms of tissue penetration depth, safety, and the ability to use higher laser power, this invention uses the W1 NPs and W2 NPs as photothermal conversion materials and systematically evaluates their photothermal performance. Under an 808 nm laser (1 W / cm²), 2 The temperature changes of aqueous solutions of NPs with different concentrations under irradiation are as follows: Figure 12 As shown in Figure a, the irradiation time increased, reaching a stable state within 10 minutes. At a concentration of 100 µM, the final temperatures of W1 NPs and W2 NPs reached 49℃ and 48℃, respectively. The calculated photothermal conversion efficiencies of W1 NPs and W2 NPs were 65.68% and 61.62%, respectively. Figure 12 In cases b and c), the stability is higher than that of most reported organic small-molecule photothermal materials. To assess stability, five laser-switching cycles were performed on the NPs solution. Figure 12As shown in d and e, after multiple heating-cooling cycles, their heating capacity did not decrease significantly, proving that they have good photothermal stability and meet the requirements of subsequent in vivo and in vitro experiments.

[0078] 3. In vitro studies of nanoparticles

[0079] MTT assays were performed to evaluate the cytotoxicity of W1 NPs and W2 NPs. The results showed that ( Figure 13 In cases a and b), under light-protected conditions, even at concentrations as high as 200 μg / mL, the two types of nanoparticles had minimal impact on cell viability, indicating negligible dark toxicity. Under 808 nm laser irradiation, cell viability decreased significantly with increasing nanoparticle concentration, and the viability of the W1NPs treatment group was consistently lower than that of the W2NPs group, confirming that W1NPs possess stronger photoinduced cell-killing efficacy. Next, to clarify the intracellular localization of the nanoparticles of this invention, co-localization microscopy (CLMS) was used for analysis. Figure 14 As shown in Figure a, after co-incubating HepG2 cells with W1 NPs (10 µM) for 3 hours, the cells were stained with BODIPY 493 / 503. The blue fluorescence signal of W1 NPs highly overlapped with the green signal of BODIPY, confirming their excellent lipid droplet targeting ability. The cell microenvironment was verified using a hypoxia probe, and the photothermal killing effect was assessed using Calcein-AM / PI double staining. Figure 14 As shown in Figure b, under light-protected conditions, regardless of hypoxia or normoxic conditions, the PBS, W1 NPs, and W2 NPs treatment groups all exhibited strong Calcein-AM green fluorescence, with no PI red fluorescence, indicating that NPs had no significant cytotoxicity in the absence of light. Under 808 nm laser irradiation, the green fluorescence of the W1 NPs and W2 NPs treatment groups decreased, while the PI red fluorescence increased. The red fluorescence of the W1 NPs group was more uniform and intense, indicating a more thorough photothermal killing effect. MTT experiments further confirmed this. Figure 13 (a) and (b): Under light-protected conditions, even at a concentration of 200 μg / mL, the two NPs had minimal impact on cell viability, and their dark toxicity was negligible. Under 808 nm laser irradiation, cell viability decreased significantly with increasing NP concentration, and the viability of the W1 NPs group was consistently lower than that of the W2 NPs group, indicating that the W1 NPs had stronger photoinduced cell killing efficacy.

[0080] 4. In vivo studies of nanoparticles

[0081] To evaluate the in vivo antitumor effects of W1 NPs and W2 NPs, a tumor-bearing mouse model was constructed and randomly divided into five groups (n=5 per group): PBS group, W1 NPs group, W2 NPs group, W1 NPs + laser group, and W2 NPs + laser group. The experimental procedure is as follows: Figure 15 As shown in Figure a. Under 808 nm laser illumination, infrared thermal imaging shows ( Figure 15 In the W1 and W2 NPs treatment groups, the tumor site temperature increased significantly over time, stabilizing after approximately 10 minutes. Tumor volume monitoring results ( Figure 15 As shown in b), only the W1NPs+laser group showed complete inhibition of tumor growth, ultimately resulting in complete ablation, and no recurrence within 15 days post-treatment; the tumors in the other groups continued to grow. Tumor images are shown below. Figure 15 As shown in c.

[0082] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A lipid droplet-targeted NIR-I photothermal agent, characterized in that... Selected from compounds with the following structures: 。 2. The preparation method of the lipid droplet-targeted NIR-I photothermal agent according to claim 1, characterized in that... Includes the following steps: Step 1: Thiophene-pyrrole dione, benzodifuran dione, piperidine, and acetic acid were mixed uniformly and heated to carry out the first step reaction. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain a solid. After washing and drying, the solid was purified by column chromatography to obtain the intermediate product S1, with the following structural formula: ； Step 2: Under an inert gas atmosphere and in the dark, S1, N-bromosuccinimide, and tetrahydrofuran were mixed and heated to carry out the second step reaction. After the reaction was completed, the reaction solution was poured into water to quench the reaction. Finally, the mixture was extracted with dichloromethane, dried, and concentrated, and purified by column chromatography to obtain the intermediate product S2, with the following structural formula: ； Step 3: 3a. Under an inert gas atmosphere, S2, triphenylamine 4-borate, potassium carbonate aqueous solution, tetrahydrofuran, and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) were mixed and heated to a uniform temperature for reaction. After the reaction was completed, the reaction solution was poured into water to quench the reaction. Then, it was extracted with dichloromethane, dried, and concentrated. Finally, it was purified by column chromatography to obtain product W1, with the following structural formula: ； 3b. Under an inert gas atmosphere, S2, 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene, tetratetraphenylphosphine palladium, potassium carbonate aqueous solution, tetrabutylammonium bromide, and anhydrous toluene were mixed and heated to a uniform temperature for reaction. After the reaction was completed, the reaction solution was poured into water to quench the reaction. Subsequently, it was extracted with dichloromethane, dried, and concentrated. Finally, it was purified by column chromatography to obtain product W2, with the structural formula shown below: 。 3. The preparation method according to claim 2, characterized in that: In step 1, the molar ratio of thienopyrrole dione, benzodifuran dione and piperidine is 2:1:0.2; the reaction temperature of step 1 is 120℃ and the reaction time is 18 h.

4. The preparation method according to claim 2, characterized in that: In step 2, the molar ratio of S1 to N-bromosuccinimide is 1:7; the reaction temperature in step 2 is 65℃, and the reaction time is 24 h.

5. The preparation method according to claim 2, characterized in that: In step 3a, the molar ratio of S2, 4-boronic acid triphenylamine, potassium carbonate and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium(II) is 1:2.5:5:0.2; the reaction temperature in step 3a is 65°C and the reaction time is 24 h.

6. The preparation method according to claim 2, characterized in that: In step 3b, the molar ratio of S2, 1-(4-phenylboronic acid pinacol ester)-1,2,2-tristyrene, potassium carbonate, tetratriphenylphosphine palladium and tetrabutylammonium bromide is 1:2.5:5:0.2:0.2; the reaction temperature in step 3b is 90℃ and the reaction time is 24h.

7. The use of the lipid droplet-targeted NIR-I photothermal agent of claim 1 in the preparation of hypoxic tumor therapeutic drug formulations.

8. The application according to claim 7, characterized in that: The NIR-I photothermal agent and the amphiphilic polymer DSPE-PEG5000 were prepared into water-soluble nanoparticles by nanoprecipitation, thereby improving their biocompatibility and targeting properties.

9. The use of the lipid droplet-targeted NIR-I photothermal agent according to claim 1 in the preparation of a photothermal combined therapy for tumor diseases.

10. The application according to claim 9, characterized in that: The NIR-I photothermal agent and the amphiphilic polymer DSPE-PEG5000 were prepared into water-soluble nanoparticles by nanoprecipitation, thereby improving their biocompatibility and targeting properties.

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