Amphiphilic polymer-photo-thermal agent composite material as well as preparation method and application thereof

Abstract

The invention discloses an amphiphilic polymer-photothermal agent composite material as well as a preparation method and application thereof, belongs to the field of light diagnosis and treatment, and provides a positively charged NIR-II region efficient photosensitizer # imgabs0 #. The positive charge design is beneficial to long-time circulation of the nanoparticles in vivo, and tumor site enrichment is promoted through the adsorption effect with a negative cell membrane. The # imgabs 1 # forms small-size nano-particles through single molecule self-assembly, and the molar absorption coefficient # imgabs 2 # is remarkably improved. The confinement fluorescence enhancement effect limits the movement of photosensitizer molecules, reduces the interaction with external molecules, improves the fluorescence brightness, optimizes the intramolecular charge distribution, and enhances the photo-thermal performance. Due to the unique design, dual enhancement of fluorescence and photothermal effects is realized, and a powerful tool is provided for fluorescence and photothermal dual guidance treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of photodiagnosis and treatment, and in particular to an amphiphilic polymer-photothermal agent composite material and a preparation method and application thereof. Background Art

[0002] Phototherapy technology combines fluorescence imaging diagnosis with photothermal therapy of deep tumors and is an emerging medical field. Due to its minimally invasive nature, high spatiotemporal resolution, high biosafety, and high anti-tumor ability, it plays a significant and increasingly important role in precision tumor treatment. As the core of phototherapy technology, the development of high-performance photosensitizers is crucial to the development of phototherapy research. In particular, photosensitizers with absorption and emission characteristics in the near-infrared II region (NIR-II, 1000~1700 nm) have attracted widespread attention in recent years. They have higher signal-to-noise ratio, deeper penetration depth, and lower light absorption, scattering, and autofluorescence interference in biological tissues, making them ideal for fluorescence imaging and photothermal therapy of deep tumors.

[0003] In order to obtain efficient NIR-II photosensitizers, researchers usually adopt multiple strategies to optimize their optical properties. However, despite many efforts, most NIR-II photosensitizers still have some problems to date. In particular, organic small molecule photosensitizers with narrow band gaps tend to have longer conjugated hydrophobic structures, which leads to strong intramolecular charge transfer and easily interacts with external molecules, thereby increasing non-radiative energy losses and reducing fluorescence intensity. In addition, in highly polar solvents, these photosensitizers are prone to aggregation, leading to the occurrence of fluorescence quenching. At the same time, increasing the fluorescence intensity of the photosensitizer may reduce its photothermal performance. How to maintain a good photothermal therapy effect while improving the fluorescence brightness is a difficult problem that needs to be solved urgently.

[0004] There are two key bottlenecks in tumor treatment, namely, tumor capillary extravasation and the permeability of solid tumors, which directly affect the delivery effect of anti-tumor nanophotosensitizers. The traditional EPR effect is not only weak in human tumors, but also the heterogeneity of tumors makes the efficacy of nanophotosensitizers based on the EPR effect limited. The blood vessels and lymphatic vessels inside the tumor are squeezed and deformed, making it difficult to perfuse nanoparticles. In addition, when nanophotosensitizers extravasate from blood vessels into the extracellular matrix of tumor cells, the dense extracellular matrix and increased fluid pressure in the tumor tissue interstitial space will also hinder the penetration of nanoparticles into the tumor tissue.

[0005] At present, the development of fluorescent materials with high fluorescence quantum yield (QY) and high photothermal conversion efficiency (PCE) is becoming more and more Photosensitizers are challenging. And current methods increase QY while reducing PCE, and vice versa. Therefore, how to balance the relationship between the two and develop photosensitizers with both high QY and high PCE is a key issue. Photosensitizers are of great significance for the research of light diagnosis and treatment of cancer, which requires researchers to conduct more in-depth research and exploration in molecular design, material synthesis and optical performance optimization. Summary of the invention

[0006] The purpose of the present invention is to solve the problems in the prior art of photosensitizers, such as decreased fluorescence intensity and molar extinction coefficient, caused by the large particle size of nanoparticles and weak confinement effect. In order to reduce the above-mentioned problems of poor tumor permeability and photothermal therapy effect, an amphiphilic polymer-photothermal agent composite material and its preparation method and application are provided, which show excellent therapeutic effect in deep osteosarcoma PTT.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] An amphiphilic polymer-photothermal agent composite material, the structural formula is as follows:

[0009]

[0010] Wherein, m is 1.4%~1.6%, n is 98.4%~98.6%, which are the repetition times of the structural unit and are calculated based on the molecular weight measured by GPC gel permeation chromatography.

[0011] A method for preparing an amphiphilic polymer-photothermal agent composite material comprises the following steps:

[0012] 1) julolidine and N,N-dimethylformamide are mixed and added into Vilsmeier-Haack reagent for mixed reaction to obtain intermediate product S1;

[0013] 2) The intermediate product S1 and 4-bromoacetophenone are mixed with a sodium hydroxide aqueous solution and ethanol to react to obtain an intermediate product S2;

[0014] 3) The intermediate product S2, nitromethane, N,N-diisopropylethylamine and methanol are mixed and reacted to obtain the intermediate product S3;

[0015] 4) The intermediate product S3, ammonium acetate and n-butanol are mixed and the temperature is raised to react to obtain the intermediate product S4;

[0016] 5) After mixing the intermediate product S4, N,N-diisopropylethylamine and dichloromethane, boron trifluoride etherate is added to react to obtain the intermediate product S5;

[0017] 6) Under nitrogen protection, the intermediate product S5, 4-pyridinephenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, 1,4-dioxane and water are mixed and the temperature is raised to react to obtain the intermediate product S6;

[0018] 7) Under nitrogen protection, the intermediate product S6 is mixed with 1-bromododecane and chloroform, and the temperature is raised to react to obtain the intermediate product WS5;

[0019] 8) Under nitrogen protection, 2-(dimethylamino)ethyl methacrylate is reacted with 1,2-dibromoethane and ethanol to obtain intermediate product A1;

[0020] 9) Under nitrogen protection, methanol was added to 2-(dimethylamino)ethyl methacrylate and intermediate product A1, and then the above solution was mixed with 2,2-bipyridine, cuprous bromide and ethyl isobromobutyrate to obtain the intermediate product ;

[0021] 10) The intermediate product Mix with WS5 and N, N-dimethylformamide, and heat to react to obtain the product amphiphilic polymer-photothermal agent composite material .

[0022] In step 3), the reaction temperature is 60-70°C.

[0023] In step 4), the reaction temperature is 100-110°C.

[0024] In step 6), the reaction temperature is 70-80°C.

[0025] In step 7), the reaction temperature is 80-90°C.

[0026] In step 9), the reaction temperature is 30-40°C.

[0027] In step 10), the reaction temperature is 90-100°C.

[0028] The application of the amphiphilic polymer-photothermal agent composite material is used to prepare anti-tumor drugs.

[0029] The application of the amphiphilic polymer-photothermal agent composite material is used to prepare photodiagnostic agents for fluorescence imaging diagnosis and tumor photothermal therapy.

[0030] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0031] The present invention provides a positively charged NIR-II region efficient photosensitizer based on a special organic small molecule structure , where the positive charge design not only helps the nanoparticles to maintain long-term circulation in the body, but also promotes the enrichment of nanoparticles at the tumor site by regulating their adsorption capacity with negatively charged cell membranes. In addition, Smaller nanoparticles are formed through the self-assembly of single molecules, and this structural feature significantly improves its value. Thanks to the confined fluorescence enhancement effect, The movement of the photosensitizer molecules is restricted, reducing the interaction with external molecules, thereby significantly improving the fluorescence brightness. At the same time, this special structure also optimizes the charge distribution within the molecule and enhances the photothermal performance. The unique design achieves dual enhancement of fluorescence and photothermal effects, providing a powerful tool for tumor treatment under the dual guidance of fluorescence and photothermal effects.

[0032] Furthermore, the present invention It exhibits strong absorption characteristics in the NIR-II region, and its fluorescence QY and PCE both reach high levels. It has excellent biocompatibility and membrane permeability, especially in acidic aqueous solution, it presents a positive charge, which enables it to be effectively enriched in the tumor microenvironment. The small size of the drug makes it easier to penetrate into solid tumors, overcoming the permeability problem in tumor treatment. Under the guidance of near-infrared zone II imaging, the deep-seated osteosarcoma showed an excellent synergistic treatment effect. This result not only verifies As a high-performance photosensitizer, it has great potential in tumor photothermal therapy and provides a new way to solve the energy allocation problem that depends on two competing photophysical processes (fluorescence and photothermal conversion). Through a series of in-depth experimental characterization and demonstration, the system revealed This study has laid a solid foundation for further exploration of its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 For the present invention Performance test results; among them, Figure 1 The a in DLS and TEM results, scale bar = 200 nm; Figure 1 b in Absorption and fluorescence spectra; Figure 1 The c in the equation is the same concentration Fluorescence imaging of Figure 1 The d in the figure is different concentrations. The fluorescence intensity of Figure 1 The e in Photothermal heating curve (1064 nm laser); Figure 1 The f in zata potential; Figure 1 The g in and fluorescence imaging of ICG; Figure 1 The h in and ICG fluorescence stability ; Figure 1 The i in and photothermal stability diagram of ICG; Figure 1 The j in Fluorescence imaging in PBS buffer solution, fetal bovine serum protein solution, bovine serum albumin and whole blood solution ; Figure 1 The k in Fluorescence intensity in PBS buffer solution, fetal bovine serum protein solution, bovine serum albumin and whole blood solution ; Figure 1 The l in The electron cloud distribution and HOMO and LUMO energy levels of the monomer analogues.

[0034] Figure 2 For the present invention The results of cell experiments; among them, Figure 2 The a in Cellular uptake in 143B cells; Figure 2 b in the figure represents different concentrations At 1064 nm laser Survival rate of 143B cells under irradiation ; Figure 2 c shows the fluorescence imaging of 143B cells stained with calcein AM and EthD-1. , Scale bar = 100 μm; Figure 2 Figure d shows the results of flow cytometry detection of 143B cell apoptosis.

[0035] Figure 3 For the present invention In vivo NIR-II fluorescence imaging results; among them, Figure 3 The a in the formula is injection Whole-body fluorescence imaging under 808 nm excitation (1000 nm~1300 nm LP); Figure 3 b in the sentence stands for injection Fluorescence intensity under 808 nm excitation (1000 nm~1300 nm LP); Figure 3 Injection time: 0.5~120 h Fluorescence imaging of Figure 3 Injection time is 0.5~120 hours The fluorescence intensity of Figure 3 The e in it stands for injection 12-hour fluorescence imaging of tumor, spleen, kidney, liver, heart, and lung; Figure 3 f in the figure is the fluorescence intensity of the tumor, spleen, kidney, liver, heart, and lung 12 h after injection of PWS5@NPs; Figure 3 g in the figure is the fluorescence imaging of the tumor leg; Figure 3 h in is the fluorescence intensity of the tumor leg.

[0036] Figure 4 For the present invention The results of laser thermal treatment; among them, Figure 4 a in the figure is the experimental schedule in mice. Figure 4 b in A group of photothermal heating images, Figure 4 c in the figure shows the X-ray images of osteosarcoma mice on days 1, 3, 5, 7, 9 and 11. Figure 4 The d in the figure shows the change in tumor size of mice after 11 days of photothermal treatment. ; Figure 4 e in the figure is a photo of the tumor after mouse dissection and Figure 4 f is the tumor weight.

[0037] Figure 5 For the present invention The results of laser-mediated thermal effect anti-tumor mechanism; among them, Figure 5 a in the figure shows the results of H&E and TUNEL staining of tumor tissues of mice in each group, scale bar = 100 μm; Figure 5 The b in the figure is the classic apoptosis result. Figure 5 The c in the equation is the result of pyroptosis. Figure 5 Figure d in the figure is the Western blot detection result of the relevant proteins. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Conventional instruments and equipment in the art are used in the following examples. The experimental methods in the following examples in which specific conditions are not specified are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Various raw materials are used in the following examples, and unless otherwise specified, conventional commercial products are used, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, if not otherwise specified, "%" represents weight percentage, "part" represents weight part, and ratio represents weight ratio.

[0039] The present invention provides a method for preparing an amphiphilic polymer-photothermal agent, comprising the following steps:

[0040] Step 1: Synthesis of compound S1

[0041] Add N,N-dimethylformamide to phosphorus oxychloride to obtain a Vilsmeier-Haack reagent under nitrogen protection; mix julolidine and N,N-dimethylformamide and add them to the Vilsmeier-Haack reagent, then perform a first reaction, cool to room temperature after the first reaction, pour the mixture into ice water to quench the reaction, and then filter and dry to obtain an intermediate product S1;

[0042] Step 2: Synthesis of compound S2

[0043] The intermediate product S1, 4-bromoacetophenone, sodium hydroxide aqueous solution and ethanol are uniformly mixed to carry out a second reaction. After the second reaction is completed, the mixed solution of the second reaction product is poured into ice water to quench the reaction, and then filtered and dried to obtain an intermediate product S2;

[0044] Step 3: Synthesis of compound S3

[0045] The intermediate product S2, nitromethane, N,N-diisopropylethylamine and methanol are uniformly mixed, and then the temperature is raised to carry out a third reaction, and the solution of the third reaction product is poured into a saturated sodium chloride aqueous solution to stop the reaction, and then extracted, dried, filtered and purified to obtain an intermediate product S3;

[0046] Step 4: Synthesis of compound S4

[0047] The intermediate product S3, ammonium acetate and n-butanol are mixed and mixed, and the temperature is raised to carry out a fourth reaction, followed by vacuum concentration and suction filtration, and the solid is separated and washed to obtain an intermediate product S4;

[0048] Step 5: Synthesis of compound S5

[0049] After mixing the intermediate product S4, N,N-diisopropylethylamine and dry dichloromethane, add boron trifluoride etherate, stir at room temperature in the dark to carry out the fifth reaction, dilute the fifth reaction product with ice water, extract, dry, filter, concentrate and purify to obtain the intermediate product S5;

[0050] Step 6: Synthesis of compound S6

[0051] Under nitrogen protection, a solution obtained by mixing the intermediate product S5, 4-pyridinephenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, 1,4-dioxane and water is heated to carry out a sixth reaction, and after the sixth reaction is completed, the solution is cooled to room temperature, and then concentrated, filtered and purified to obtain an intermediate product S6;

[0052] Step 7: Synthesis of compound WS5

[0053] Under nitrogen protection, the intermediate product S6 is mixed with 1-bromododecane and chloroform, and the temperature is raised to carry out the seventh reaction. After the seventh reaction is completed, the mixture is cooled to room temperature, concentrated, filtered and purified to obtain WS5;

[0054] Step 8: Synthesis of Compound A1

[0055] Under nitrogen protection, 2-(dimethylamino)ethyl methacrylate, 1,2-dibromoethane and ethanol are uniformly mixed to carry out an eighth reaction. After the eighth reaction is completed, the mixture is concentrated and filtered to obtain A1;

[0056] Step 9: Compound Synthesis

[0057] Under nitrogen protection, methanol was added to 2-(dimethylamino)ethyl methacrylate and intermediate product A1, and the above liquid was mixed with 2,2-bipyridine, cuprous bromide, and ethyl isobromobutyrate to carry out the ninth reaction. After the ninth reaction, the product was concentrated, filtered, and purified to obtain ;

[0058] Step 10: Compounds Synthesis

[0059] The intermediate product Mix with WS5 and N,N-dimethylformamide, heat up to carry out the tenth reaction, stir at low temperature and dialyze after the tenth reaction to obtain ;

[0060] In the step 1, the molar ratio of phosphorus oxychloride, N,N-dimethylformamide and julolidine is ; The temperature of the first reaction is 20~30℃; The time of the first reaction is 3~4h.

[0061] In the step 2, the molar ratio of the intermediate product S1 and 4-bromoacetophenone is 1:1; and the temperature of the second reaction is 25-30°C.

[0062] In the step three, the molar ratio of the intermediate product S2 to N,N-diisopropylethylamine is 5:5.5; and the temperature of the third reaction is 60-70°C.

[0063] In the step 4, the molar ratio of the intermediate product S3 to ammonium acetate is 1:15; and the temperature of the fourth reaction is 100-110°C.

[0064] In the step 5, the molar ratio of the intermediate product S4, N,N-diisopropylethylamine and boron trifluoride ether is .

[0065] In the step 6, the molar ratio of the intermediate product S5, 4-pyridinephenylboronic acid, tetrakis(triphenylphosphine)palladium and potassium carbonate is The temperature of the sixth reaction is 70-80° C.; the volume ratio of 1,4-dioxane to water is 5:1.

[0066] In the step seven, the molar ratio of the intermediate product S6 to 1,2-dibromoethane is 1:1; and the temperature of the seventh reaction is 80-90°C.

[0067] In the step eight, the molar ratio of 2-(dimethylamino)ethyl methacrylate to 1,2-dibromoethane is 1:1; and the temperature of the eighth reaction is 25-30°C.

[0068] In the step nine, the molar ratio of the 2,2-bipyridine, cuprous bromide, intermediate A1, 2-(dimethylamino)ethyl methacrylate, and ethyl isobromobutyrate is: ; The temperature of the ninth reaction is 30-40°C.

[0069] In the step 10, the intermediate product The mass ratio of WS5 to WS5 is 1:1; the temperature of the tenth reaction is 90-100°C.

[0070] Among them, through the synergistic effect of "receptor engineering strategy" and "confined fluorescence enhancement effect", The excited state energy of the molecule is finely controlled, which not only significantly improves its fluorescence brightness but also ensures the maintenance of high fluorescence quantum yield. The change of charge properties under neutral and weak acid conditions, that is, slightly negative charge under neutral conditions and positive charge under weak acid conditions, gives it a unique advantage when circulating in the body. Charge reversal occurs, and the positive charge enables it to be tightly adsorbed on the negatively charged tumor blood vessel wall, achieving efficient enrichment of the photosensitizer in the tumor area. At the same time, this charge reversal mechanism also ensures The long circulation characteristics in the body provide a solid foundation for the subsequent integration of tumor diagnosis and treatment. The small size design promotes its deep penetration into solid tumors, allowing the photosensitizer to act more effectively on tumor tissues, compensating for the inherent defects of low PCE photosensitizers.

[0071] also, As self-assembled nanoparticles, the polymer structure inside them not only stabilizes the molecular aggregation state, but also further enhances the fluorescence brightness through the confined fluorescence enhancement effect. Compared with pure PWS5 molecules, On the basis of maintaining the original optical properties, it shows more superior photophysical properties. The improvement of these properties is mainly attributed to the molecular interactions within the nanoparticles and the restriction of molecular motion by confined space. Under the guidance of NIR-II imaging, It demonstrated the ability to quickly light up the blood vessels throughout the mouse body, and its liver retention rate was extremely low, and a large number of nanoparticles could be precisely enriched in the tumor site. Through the photothermal conversion effect, It can achieve efficient deep tumor treatment at low concentrations and induce apoptosis and pyroptosis of tumor cells. In short, through sophisticated molecular design and nanoparticle preparation, It not only optimizes the optical properties of the photosensitizer, but also realizes an integrated strategy for tumor diagnosis and treatment with high brightness and high photothermal performance, providing a new and powerful tool for the synergistic photodynamic and photothermal treatment of tumors.

[0072] Example 1

[0073] Preparation of compound S1: Phosphorus oxychloride Slowly drip dry N,N-dimethylformamide 100 mL round-bottom flask. 2 After stirring for 2 h under the protection of and dry DMF (10 mL) were slowly added to the flask containing the Vilsmeier-Haack reagent. After that, it was stirred at 30 °C for 4 h. After cooling to room temperature, the mixture was poured into ice water (100 mL) to quench the reaction. Then, the precipitate was filtered to obtain a light yellow solid. Finally, the solid was dried in a vacuum oven overnight to obtain compound S1 (yield 78%). 1 H NMR (500 MHz, CDCl 3 ) δ / ppm 9.60 (s, 1H), 7.29 (s, 2H), 3.29 (t, J = 5.0, 4H), 2.77 (t, J = 10.0,4H), 1.97 (m, 4H). 13 C NMR (126 MHz, CDCl 3 ) δ / ppm 190.1, 147.9, 129.5, 124.1,120.4, 50.0, 27.6, 21.3.

[0074] Preparation of compound S2: Compound S1 (2.00 g, 10 mmol), 4-bromoacetophenone (2.00 g, 10 mmol) and aqueous sodium hydroxide solution were added. Slowly add to a 100 mL round-bottom flask containing ethanol (20 mL). Stir the mixture at room temperature for 24 h, then pour the reaction mixture into ice water (100 mL) to quench the reaction, stir for another 2 h, and filter the precipitate to obtain a red solid. Subsequently, dry the red solid product in a vacuum drying oven overnight to obtain compound S2 (yield 80%). 1 H NMR (500 MHz, CDCl 3 ) δ / ppm 7.85 (d, J = 10.0 Hz, 2H),7.69 (d, J = 20.0 Hz, 1H), 7.59 (d, J = 10.0 Hz, 2H), 7.18 (d, J = 20.0 Hz, 1H), 7.11 (s, 2H), 3.26 (t, J = 5.0 Hz, 4H), 2.76 (t, J = 5.0 Hz, 4H), 1.97(m, 4H). 13 C NMR (126 MHz, CDCl 3 ) δ / ppm 189.3, 147.0, 145.5, 138.1, 131.6,129.9, 128.4, 126.8, 121.3, 121.0, 114.9, 108.1, 105.9, 50.0, 27.7, 21.5. IT-TOF / MS: Calcd for [M + H] + 382.0728, found: 382.0804.

[0075] Preparation of compound S3: Compound S2 (1.00 g, 5 mmol), nitromethane (1.00 mL) and N,N-diisopropylethylamine Slowly add to the methanol The reaction mixture was stirred at 70 °C for 24 h, and then the reaction solution was poured into a beaker containing saturated sodium chloride aqueous solution (10 mL) to stop the reaction, and then extracted with ethyl acetate (30 mL). The obtained organic solution was dried over anhydrous sodium sulfate and filtered to obtain a crude product, which was purified by column chromatography (silica gel, ethyl acetate / petroleum ether = 1 / 10) to obtain a yellow solid (yield 67%). 1 H NMR (500 MHz, CDCl 3 ) δ / ppm 7.76 (d, J= 15.0 Hz, 2H), 7.59 (d, J = 10.0 Hz, 2H),6.63 (s, 2H), 4.72 (t, J = 5.0 Hz, 1H), 4.60 (t, J = 5.0 Hz,1H), 3.98 (t, J =10.0 Hz, 1H). 3.38 (m, 2H). 3.10 (t, J = 5.0 Hz, 4H), 2.70 (t, J = 5.0 Hz,4H), 1.97 (m, 4H). 13 C NMR (126 MHz, CDCl 3 ) δ / ppm 196.5, 142.5, 135.3, 132.0,129.6, 128.6, 125.8, 125.4, 121.8, 80.0, 49.9, 41.9, 38.6, 27.7, 21.9, 14.8.IT-TOF / MS: Calcd for [M + H] + 443.0892, found: 443.0965.

[0076] Preparation of compound S4: Compound S3 (1.00 g, 1.0 mmol) and ammonium acetate (NH 4 OAc, 1.16 g, 15.0 mmol) was added to a 100 mL round-bottom flask containing n-butanol (n-BuOH, 20 mL), stirred at 110 °C for 24 h, cooled to room temperature, and then concentrated in vacuo to 5 mL and filtered. The separated solid was washed with ethanol (2×5 mL) to obtain a blue-black solid (yield 37%). 1 H NMR (500 MHz, CDCl 3 ) δ / ppm 7.77 (d, J = 15.0 Hz, 4H), 7.63(d, J = 10.0 Hz, 4H), 7.56 (s, 4H), 6.94 (s, 2H), 5.30 (s, 1H), 3.22 (t, J =5.0 Hz, 8H), 2.76 (t, J = 10.0 Hz, 8H), 2.01 (m, 8H). 13 C NMR (126 MHz, CDCl3 )δ / ppm 156.1, 150.5, 143.9, 138.8, 131.8, 128.7, 125.5, 124.5, 123.3, 121.7,115.8, 49.9, 27.9, 21.8, 1.0.

[0077] Preparation of compound S5: 2 Under the protection of BF, compound S4 (80 mg, 0.11 mmol) and DIPEA (0.2 ml, 1.1 mmol) were added to a 100 mL round-bottom flask containing dry dichloromethane (DCM, 20 mL). 3 •Et 2 O (0.28 mL, 2.0 mmol) was slowly dripped into the solution, stirred at room temperature in the dark for 24 h, then diluted with ice water (20 mL) and extracted with DCM (3×20 mL). The organic solution was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel, dichloromethane / petroleum ether = 1 / 1) to obtain a solid with metallic luster (yield 84%). 1 H NMR (500 MHz, CDCl 3 ) δ / ppm 7.86 (d, J = 10.0 Hz, 4H), 7.60 (s, 4H), 7.56 (d, J =10.0 Hz, 4H), 6.69 (s, 2H), 3.30 (t, J = 5.0 Hz, 8H), 2.77 (t, J = 5.0 Hz,8H), 2.00 (m, 8H). 13 C NMR (126 MHz, CDCl 3 ) δ / ppm 156.1, 150.5, 143.9, 138.8,131.8, 128.7, 125.5, 124.5, 123.3, 121.7, 115.8, 49.9, 30.9, 29.7, 27.9,24.4, 21.8, 11.2, 1.4. MALDI-TOF / MS: [M] + calcd: 845.1530, found: 845.1960.

[0078] Preparation of compound S6: 2 Under protection, compound S5 , 4-pyridinephenylboronic acid , Tetrakis(triphenylphosphine)palladium , potassium carbonate and 1,4-dioxane / water (10 mL, 5 / 1) were added to a 50 mL round-bottom flask. The mixture was stirred at 80 °C for 24 h, then the reaction was cooled to room temperature, the solvent was concentrated in vacuo to 5 mL, and the crude product was filtered. The crude product was purified by column chromatography (silica gel, methanol / dichloromethane = 1 / 50) to obtain a blue-black solid (yield 61%). 1 HNMR (500 MHz, CDCl 3 ) δ / ppm 8.67 (d, J = 5.0 Hz, 4H), 8.13 (d, J = 5.0 Hz,4H), 7.70 (s, 4H), 7.64 (d, J = 5.0 Hz, 4H), 7.55 (d, J = 5.0 Hz, 4H), 6.78(s, 2H) 3.31 (t, J = 5.0 Hz, 8H), 2.78 (t, J = 5.0 Hz, 8H), 2.01 (m, 8H). 13 CNMR (126 MHz, CDCl 3 ) δ / ppm 153.1, 150.2, 148.4, 148.2, 147.6, 143.5, 141.0,138.1, 135.9, 135.0, 134.9, 131.6, 130.3, 128.0, 127.5, 126.9, 121.8, 121.5,121.3, 121.1, 111.4, 51.4, 31.9, 30.2, 29.7, 27.9, 22.7, 22.1, 13.4. MALDI-TOF / MS: [M] + calcd: 841.3876, found: 841.7387.

[0079] Preparation of compound WS5: 2 Under protection, S6 (50 mg, 0.06 mmol) and 1-bromotidecaproide (9.9 mg, 0.06 mmol) were added to a chloroform (CHCl 3, 10 mL) in a round-bottom flask, stirred at 90 °C for 24 h, cooled to room temperature, and the solvent was concentrated in vacuo. Finally, WS5 was purified by column chromatography (silica gel, methanol / dichloromethane = 1 / 25) to obtain a blue-green solid (yield 36%). 1 H NMR (500 MHz, d 6 -DMSO) δ / ppm 9.13 (d, J=10.0, 2H), 8.68 (m,4H),8.27 (m, 6H), 7.96 (d, J=10.0, 2H), 7.83 (s, 2H), 7.70 (d, J=25.0, 4H), 7.23(d, J=15.0, 2H), 4.58 (t, J=10.0, 2H), 3.51 (s,8H), 2.71 (s, 8H), 1.93 (s,10H) ,1.27 (d, J=30.0,18H), 0.84 (t, J=5.0, 3H). 13 C NMR (126MHz, CDC1 3 ) δ / pm145.0, 130.6, 130.2, 129.3, 127.9, 127.1, 124.8, 121.4, 120.7, 114.6, 77.2,70.7, 50.4, 32.0, 31.6, 29.8, 29.7, 29.6, 29.5, 29.2, 28.2, 26.3, 22.8, 21.8,14.3. MALDI-TOF / MS: [M-2Br] + calcd:1011.149, found:1011.055.

[0080] Preparation of compound A1: 2 Under the protection of 2-(dimethylamino)ethyl methacrylate (1.0 g, 6.4 mmol) and 1,2-dibromoethane (1.2 g, 6.4 mmol) were added to a reaction bottle containing ethanol (20 mL) and stirred at 25 °C for 24 h. After the reaction was completed, it was cooled to room temperature and the solvent was concentrated by rotary evaporation. Finally, the concentrated reaction solution was dispersed in n-hexane and filtered to obtain white solid compound A1 (yield 76%). 1 H NMR (500 MHz, d 6 -DMSO) δ / ppm 6.09 (s, 1H), 5.77 (s, 1H), 4.54 (s, 2H), 3.84 (d, J= 55.0, 6H), 3.17 (d, J = 10.0, 6H),1.92 (s, 3H). 13 C NMR (126 MHz, d 6 -DMSO) δ / ppm 165.8, 135.3, 126.7, 63.1,62.2, 58.1, 50.7, 40.0, 22.8, 17.9.

[0081] Preparation of compound PA1@NPs: 2,2-bipyridine (0.071 g, 0.45 mmol) and cuprous bromide (0.033 g, 0.23 mmol) were added to reaction bottle 1 and vacuum / flushed with N2 repeatedly. 2 The reaction was repeated three times to remove oxygen from the reaction flask. Meanwhile, ethyl 2-(dimethylamino)methacrylate (4.28 g, 27.4 mmol), A1 (1.27 g, 6.9 mmol) and methanol (2.0 mL) were added to reaction flask 2 and vacuum / flushed with N2. 2 The cycle was repeated 3 times to remove oxygen from the reaction bottle. Then, the solution in reaction bottle 2 was injected into reaction bottle 1, and the vacuum / N flushing was repeated. 2 The reaction was continued for 30 minutes. Finally, ethyl isobromobutyrate (0.033 g, 0.17 mmol) was slowly injected into the reaction bottle 1. The reaction bottle was placed in a 30 °C oil bath. After 4 hours of reaction, the reaction bottle was placed in liquid nitrogen and THF (100 mL) was added. Finally, the copper ions in the reaction solution were removed by a neutral alumina short column, and the filtrate was concentrated and precipitated with precooled n-hexane to obtain PA1@NPs (yield 11%). The number average molecular weight (Mn) of PA1@NPs was 22988, and the polydispersity index (PDI) was 1.26.

[0082] Preparation of compound PWS5@NPs: Compound PA1@NPs (2.00 g) and WS5 (2.00 g) were slowly added to a 20 mL round-bottom flask containing DMF (5 mL). Stirred at 90 °C for 24 hours, the reaction solution was poured into ice water (20 mL) and continued to stir for 2 hours. The reaction solution was then placed in a dialysis bag (MWCO: 3500 Da) and dialyzed with deionized water for 36 hours to obtain PWS5@NPs (yield 10%). The Mn of PWS5@NPs is 24573 and the PDI is 1.30.

[0083]

[0084] The present invention conducted relevant characterization, performance and therapeutic effect testing experiments on PWS5@NPs.

[0085] (1) Particle size characterization: Accurately weigh the vacuum-dried PWS5@NPs (1 mg) and place it in 1 mL of deionized water. After stirring for 24 hours, a 1 mg / mL PWS5@NPs mother solution was obtained. Next, 20 µL of the test solution was dissolved in 2 mL of deionized water. After ultrasonication for 30 minutes, the particle size was tested using DLS. The saved data was plotted using Origin. In addition, 5 µL of the DLS test solution was added dropwise to a common copper grid and allowed to stand naturally at room temperature for 1 day before TEM testing.

[0086] (2) Absorption spectrum test

[0087] Preparation of test solution series 1: 100 µL of PWS5@NPs stock solution (1 mg / mL) was placed in 900 µL of deionized water to prepare a PWS5@NPs test solution with a concentration of 10 µg / mL; 400 µL of PWS5@NPs stock solution (1 mg / mL) was placed in 1600 µL of deionized water to prepare a PWS5@NPs test solution with a concentration of 20 µg / mL; 600 µL of PWS5@NPs stock solution (1 mg / mL) was placed in 1400 µL of deionized water to prepare a PWS5@NPs test solution with a concentration of 30 µg / mL; 800 µL of PWS5@NPs stock solution (1 mg / mL) was placed in 1200 µL of deionized water to prepare a PWS5@NPs test solution with a concentration of 40 µg / mL; 1000 µL of PWS5@NPs stock solution (1 mg / mL) was placed in 1000 µL of deionized water to prepare a PWS5@NPs test solution with a concentration of 50 µg / mL. The PWS5@NPs test solution with a concentration of 50 µg / mL was prepared in µL of deionized water.

[0088] The absorption spectrum of test liquid series 1 was tested using a spectrophotometer. The test range covered 700 nm~1300 nm. After scanning the solvent baseline, the sample test was started and the relevant data was recorded. The absorption spectrum data of test liquid series 1 was processed using Origin software, and the Ɛ of PWS5@NPs was obtained after linear fitting. Since test liquid series 1 has absorption in NIR-II, an 808 nm excitation light source was used to test the fluorescence spectrum of test liquid series 1, and the data was recorded and saved. The fluorescence spectrum data of test liquid series 1 was normalized using Origin software to obtain the absorption / emission wavelength of PWS5@NPs.

[0089] (3) In vitro fluorescence brightness test

[0090] PWS5@NPs solutions of 100 µg / mL, 200 µg / mL, 300 µg / mL, 400 µg / mL and 500 µg / mL were prepared, and then the above test solutions of different concentrations were placed together in the NIR-II in vivo imager, and the EP tube was imaged by fluorescence using an 808 nm excitation light source, a 1000 nm LP filter and an exposure time of 20 ms. The fluorescence imaging results were calibrated with image J software for fluorescence intensity and analyzed with Origin software.

[0091] (4) Photothermal properties test

[0092] Preparation of test solution series 2: Take a 5 mL EP tube, numbered PWS5@NPs, add 200 µL of PWS5@NPs to it, and then add 800 µL of deionized water to prepare a 0.20 mg / mL test solution. Take 100 µL of test solution series 2 and place it in a 200 µL EP tube, and place the EP tube directly under the infrared thermal imager. The above test samples were irradiated with a 1064nm laser for 10 minutes, and the laser power density was 0.23W / cm 2 After the sample cooled naturally to room temperature, the camera was stopped and the above data were recorded. The data were analyzed using Origin software, and the thermal coupling time lifetime of PWS5@NPs was obtained after linear fitting, which was used for the subsequent calculation of the PCE of PWS5@NPs.

[0093] (5) Zata potential test

[0094] Accurately weigh the vacuum-dried PWS5@NPs (1 mg) and place them in 1 mL of deionized water. Stir for 24 hours to obtain a 1 mg / mL PWS5@NPs mother solution. Take 20 µL of the test solution and drop it into ultrapure water (2 mL) at pH 5, 6, 7, 8, and 9, respectively. After ultrasonication for 30 minutes, place it in DLS to test the zata potential of PWS5@NPs. The saved data is plotted using Origin.

[0095] (6) Photothermal stability test

[0096] 0.2 mg / mL ICG and PWS5@NPs solutions were prepared respectively and tested using NIR-II in vivo imager and infrared thermal imager. In vivo imaging used an 808 nm excitation light source, a 1000 nm LP filter, an exposure time of 30 ms, and continuous laser irradiation of the sample for 120 minutes. Fluorescence images at different time points (0, 10, 20, and 30 minutes) were obtained, and the fluorescence intensity of the sample was calibrated using image J and the data was processed using Origin software. In addition, the infrared thermal imager used a 1064 nm laser light source, and the thermal stability curves of ICG and PWS5@NPs were obtained by turning the laser on and off 5 times.

[0097] (7) Density functional theory calculations

[0098] The O3LYP functional and def2-svp basis set were used to calculate the electron cloud distribution and energy level states, including HOMO and LUMO, of PWS5@NPs analogs (polymer PWS5@NPs are difficult to calculate) using Gaussian 16.

[0099] (8) Fluorescence brightness test simulating the in vivo environment

[0100] PBS buffer solution, fetal bovine serum protein solution, bovine serum albumin and whole blood solution were prepared to simulate the in vivo environment. 10 µL of PWS5@NPs (1 mg / mL) were added to 20 µL of PBS buffer solution, fetal bovine serum protein solution, bovine serum albumin and whole blood solution, respectively. After standing for 1 minute, the solution was placed in a NIR-II in vivo imager for fluorescence imaging. The in vivo imaging used an 808 nm excitation light source, a 1000 nm LP filter and an exposure time of 30 ms.

[0101] (9) Cell experiments

[0102] The 143B (human osteosarcoma) cells used in this experiment were cultured under standard conditions (95% humidity, 5% CO 2 , 37°C) and harvested by digestion with 0.25% (w / v) trypsin. Before further experiments, cells were plated at 2 × 104 cells / cm 2 The cells were seeded at a density of 100 μg / mL in 96-well or 6-well plates in DMEM medium supplemented with 10% fetal bovine serum (Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin (HyClone).

[0103] Cell recovery: Take out the cells from the liquid nitrogen tank and thaw them at 37°C. Add 20 mL of DMEM culture medium containing 10% Gibco, 100 U / mL penicillin and 100 μg / mL HyClone to a T75 culture dish, then add the thawed cell solution and place in a 95% humidity, 5% CO 2 and cultured in an incubator at 37°C.

[0104] Cell passaging and seeding: Remove the DMEM from the culture dish, add PBS to wash once to further clean the DMEM, then remove the PBS, leaving only cells that are completely attached to the wall. Add 2 mL (2 mL for T75 culture dish, 1 mL for T25 culture dish) of trypsin (0.25% concentration) and place in a 37°C incubator for digestion for 2 minutes, then add more than 2 mL of DMEM to terminate digestion, and use a pipette to take the solution into a 15 mL centrifuge tube, mix and centrifuge (1000 r, 5 minutes). Then use a pipette to remove the supernatant, add the required DMEM containing 10% Gibco, 100 U / mL penicillin and 100 μg / mL HyClone, mix well, and take the required cell solution for seeding. Another part of the cell solution is placed in a new culture dish for passaging.

[0105] Cell viability test of PWS5@NPs: 143B cells were added to a 96-well plate (n = 6), and then different concentrations of PWS5@NPs (0, 2, 5, 10, 20, 50, and 100 μg / mL) were added. After the drug and cells were incubated for 4 hours, the power density was 0.23 W / cm 2 The cells were irradiated with 808 nm laser or 1064 nm laser for 5 minutes and then returned to the cell culture incubator for another 4 hours. Finally, the cell viability was determined using PrestoBlue cell viability reagent. Specifically, PrestoBlue reagent and DMEM were prepared at a ratio of 1:9 and 100 μL was added to each well. Incubate at 37°C, 5% CO 2 The cells were incubated for 1 hour under the same conditions, and the absorbance at 570 nm was recorded using a TECAN SPARK microplate reader. The cell viability was calculated by correcting the inactivation of the treated cells with the inactivation of the control cells.

[0106] Test of the cellular endocytosis mechanism of PWS5@NPs: Drug entry into cells: The passaged 143B cells (1.5 × 105) were seeded in 5 confocal dishes (1.5 mL of DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 µg / mL streptomycin) and cultured in a 37°C constant temperature incubator for 24 hours until the cell density reached about 30%-40%. Then, fresh DMEM medium was replaced, and PBS, chlorpromazine (5 µg / mL), filipin (5 µg / mL), cytochalasin D (5 µg / mL) and wortmannin (0.5 µg / mL) were added and incubated with the cells for 1 hour. Subsequently, 20 µL of PWS5@NPs (1 mg / ml) was added and cultured in a 37°C constant temperature incubator for another 1 hour. After the culture medium was aspirated, it was washed twice with sterile PBS solution, new PBS was added, and imaging was performed using a NIR-II fluorescence microscope. The excitation wavelength of PWS5@NPs is 808 nm, and the emission wavelength is 1000 nm~1300 nm.

[0107] PWS5@NPs live-death staining experiment: 143B cells in a 96-well plate (n = 6) were incubated with 50 μg / mL PWS5@NPs for 4 h and then irradiated with 808 nm laser or 1064 nm laser (0.23 W / cm 2 After another 4-hour incubation, the cells were washed three times with PBS and double-stained with calcein-AM and isodimethylenediamine-1 (EthD-1) to detect live and dead cells. Using a 530 nm excitation filter, live cells showed green fluorescence, while using a 645 nm excitation filter, dead cells showed red fluorescence. Cells were imaged using a fluorescence microscope (EVOS FL Auto 2).

[0108] Apoptosis detection experiment of PWS5@NPs: Cell apoptosis was detected using annexin V-FITC apoptosis detection kit according to the manufacturer's instructions. 143B cells were incubated with 50 μg / mL PWS5@NPs for 4 hours in a 6-well plate and then illuminated with 808 nm laser or 1064 nm laser. Irradiation was continued for 5 minutes. After irradiation, the cells were returned to the cell culture incubator for another 2 hours. The cells were collected, washed with PBS and resuspended in annexin V binding buffer, and incubated with annexin V-FITC / PI in the dark for 15 minutes. Flow cytometry analysis was performed within 1 hour, and the proportion of cells in different quadrants was analyzed using quadrant statistics. The cells in the lower left quadrant represent healthy cells, the cells in the lower right quadrant represent apoptotic cells, and the cells in the upper right quadrant represent necrotic or post-apoptotic necrotic cells.

[0109] (10) In vivo NIR-II fluorescence imaging

[0110] Genetically defective nude mice were selected as experimental mice. All mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and were 4-6 weeks old and could be used for experiments. Normal non-tumor nude mice were selected as experimental subjects, and PWS5@NPs (100 µL, 1 mg / mL) were injected through the tail vein. Then the nude mice were placed in a dark box for in vivo fluorescence imaging. The nasal cavity of the nude mice was placed in an air anesthesia tube to keep quiet. The in vivo imaging software was opened, and after deducting background interference, the exposure time was set to 150 ms, the excitation wavelength was set to 808 nm, and the filters were used to 1000 nm LP and 1300 nm LP. The fluorescence imaging images under different filter channels were recorded, and the fluorescence intensity of the blood vessels of the nude mice was calibrated with image J software, and then further analysis was performed with Origin software.

[0111] In order to study the distribution of PWS5@NPs in mice, nude mice with tumors were selected as experimental subjects. An orthotopic osteosarcoma model was established by injecting 143B cells into the bone marrow cavity of the left tibia of mice. The tumor volume of the mice reached about 80 mm 3 ~100mm 3 At 1 pm, drug delivery was achieved by tail vein injection of PWS5@NPs (100 µL, 1 mg / mL), and then the nude mice were placed in a dark box for in vivo imaging and anesthetized through a gas anesthesia tube. The in vivo imaging software was opened, and after background interference was subtracted, the exposure time was set to 80 ms, the excitation wavelength was set to 808 nm, and the filter 1000 nm LP was used. Then, the fluorescence images and signal intensity monitoring of the tumor sites of mice were performed at different time intervals (0.5, 1, 2, 6, 12, 24, 48, 72, 96, and 120 hours) after drug injection. In addition, the mice were dissected at different time points (12 and 120 hours), and the tumor tissues and major organs (heart, liver, spleen, kidney, and lung) of the mice were collected for fluorescence imaging and biodistribution analysis.

[0112] (11) Photothermal therapy

[0113] Twelve mice were randomly divided into two groups: PBS group and PWS5@NPs + 1064 nm laser group. The laser activity of PWS5@NPs (100 µL, 0.5 mg / mL) at 1064 nm (0.5 W / cm 2The photothermal efficacy of osteosarcoma mice was observed under continuous irradiation of laser (60 kV, 10 min). The temperature of the tumor site during PTT, as well as the body weight and tumor growth every day after PTT were recorded. After five PTTs, the treatment was stopped to observe the photothermal efficacy of osteosarcoma mice. On the day before PTT, X-ray (60 kV, 10 ms) was used for imaging. After days 1, 3, 5, 7, 9, and 11, X-ray imaging was used to verify the photothermal efficacy. In addition, the thermal imaging images and temperature values ​​of the tumor site at 0, 1, 2, and 3 minutes of 1064 nm laser irradiation were recorded, and the mice were weighed regularly during PTT to monitor weight changes.

[0114] After 11 days of treatment, H&E staining was performed on tumor tissues and major organs to verify the biosafety of PWS5@NPs. The collected tumor tissues and skin at the PTT treatment site were fixed in 4% paraformaldehyde solution, then 5 µm sections were cut and stained with H&E. To further evaluate the antitumor effect between different groups, TUNEL in situ apoptosis detection was performed on tumor tissues by immunofluorescence. In addition, Western blot experiments were performed. Cells were frozen in RIPA buffer (Beyotime) for 30 min, and the lysate was centrifuged at 20,000 rpm for 20 min. The supernatant was transferred and the protein concentration was determined using a BCA protein assay kit (Pierce). Equal amounts of total protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to immobilon-FL PVDF membranes (Millipore). After blocking with blocking buffer (Odyssey, LI-CORBiosciences) at room temperature for 1 h, the immobilon-FL PVDF membranes were incubated with the designated primary antibodies overnight at 4°C. Immobilon-FL PVDF membranes were incubated with secondary antibodies labeled with fluorescent markers in the 700 nm or 800 nm channels for 1 hour at room temperature in the dark. Immunoreactive bands were scanned and imaged using the LI-COR Odyssey infrared imaging system. Tubulin was used as a loading control for the total fraction.

[0115] like Figure 1 As shown in Figure 2, in the range of 700-1000 nm, especially above 1000 nm, PWS5@NPs has a high absorption capacity and a molar absorption coefficient of It is much higher than similar molecules, ensuring efficient light energy absorption and conversion. At the same time, the fluorescence brightness of PWS5@NPs is high, and it continues to increase with increasing concentration, without aggregation-induced quenching, which provides advantages for its application in fluorescence imaging. In terms of photothermal performance, PWS5@NPs has a fast heating rate and the maximum temperature is sufficient for tumor ablation. Although its photothermal conversion efficiency (PCE) is relatively low, The product of PCE shows that its photothermal performance is still excellent. In addition, PWS5@NPs also exhibit excellent fluorescence stability and thermal stability, and the difference in fluorescence brightness in different solutions simulating in vivo environments is small, indicating that it may have good fluorescence stability in animals.

[0116] like Figure 2 As shown in the figure, the in vitro cell experiment fully verified the photoinduced thermal effect tumor killing mechanism of PWS5@NPs. By testing the cell viability of different concentrations of PWS5@NPs after 1064 nm laser irradiation for 5 minutes (power density of 0.23 W / cm²), it was found that tumor cell death was significant, especially at a concentration of 50 µg / mL and above, the cell death rate was accelerated. It is worth noting that the endocytosis rate of PWS5@NPs is extremely high, and it can enter 143B cells in large quantities in a short period of time, which provides a guarantee for its efficient tumor killing ability. In the absence of laser irradiation, cell activity is basically unaffected, indicating that PWS5@NPs has good safety. In addition, under laser irradiation, PWS5@NPs can effectively induce apoptosis of 143B cells, with a low proportion of necrotic cells and a high proportion of apoptotic cells of 91.88%. These results clearly show that PWS5@NPs can efficiently induce cell apoptosis under laser irradiation and achieve autonomous programmed cell death.

[0117] like Figure 3 As shown in the figure, NIR-II region in vivo fluorescence imaging accurately depicts the dynamic distribution of PWS5@NPs in mice. 12 hours after injection, PWS5@NPs significantly accumulated in the tumor area and widely distributed in the liver and spleen, demonstrating its excellent tumor targeting ability and in vivo imaging performance. In addition, PWS5@NPs have excellent interstitial penetration in the tumor site, further verifying its potential in tumor diagnosis and treatment.

[0118] like Figure 4 As shown in the figure, under the precise guidance of NIR-II imaging, tumor photothermal therapy evaluation was performed on tumor-bearing mice. 12 hours after tail vein injection of PWS5@NPs, under 1064 nm laser irradiation, even if the drug concentration was only 0.5 mg / mL, the temperature of the tumor area still rose sharply, jumping from basal body temperature to 46.7 ℃ within 3 minutes. After several treatments, compared with the PBS control group, the tumor volume in the PWS5@NPs group was greatly reduced, and the growth was significantly curbed. During the entire course of treatment, the mice had stable signs and intact skin, which strongly confirmed the high efficiency and safety of PWS5@NPs in tumor photothermal therapy, especially under low concentration conditions, it still showed excellent photothermal conversion efficiency, opening up a new way for the treatment of deep tumors.

[0119] like Figure 5As shown in Figure 2, the antitumor effect of PWS5@NPs in tumor-bearing mice was evaluated in depth. Under 1064 nm laser irradiation, it exerted a significant antitumor effect by inducing cell apoptosis and pyroptosis, and its mechanism involved increasing the Bax / Bcl2 ratio, promoting the expression of cleaved Caspase 3 and PARP1, and significantly increasing the levels of cleaved Caspase 1 and Gasdermin D.

[0120] The present invention not only demonstrates the potential of the photosensitizer PWS5@NPs with high fluorescence brightness and excellent photothermal conversion efficiency for tumor photothermal therapy under the guidance of NIR-II imaging, but also deeply explores the dual mechanism of action of "receptor engineering strategy" and "confined fluorescence enhancement effect" in improving the optical properties and tumor enrichment ability of the photosensitizer. Specifically, by introducing some positively charged polymers, the present invention successfully regulates the charge distribution of PWS5@NPs, which not only enhances its molecular polarity, but also optimizes its electrostatic interaction with the cell membrane, thereby achieving efficient enrichment of the photosensitizer at the tumor site. In addition, the application of the confined fluorescence enhancement effect significantly improves the fluorescence brightness of PWS5@NPs, making it perform well in NIR-II imaging. At the same time, the small size of PWS5@NPs enables it to penetrate into solid tumors more easily, achieving high enrichment and strong penetration of tumor tissues. Therefore, the precise design and preparation of PWS5@NPs not only optimizes its optical properties, but also enhances its application effect in tumor treatment, providing a new strategy for solving complex tumor treatments that rely on multiple treatment mechanisms.

[0121] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention, such as adjusting the specific value of the charge density or optimizing the molecular structure to further enhance the therapeutic effect, etc., shall fall within the protection scope of the claims of the present invention.

Claims

1. An amphiphilic polymer-photothermal agent composite material, characterized in that: The structural formula is as follows: Among them, m is 1.4%~1.6%, n is 98.4%~98.6%, which is the number of repetitions of the structural unit.

2. The method for preparing the amphiphilic polymer-photothermal agent composite material according to claim 1, characterized in that: The following steps are involved: 1) julolidine and N,N-dimethylformamide are mixed and added into Vilsmeier-Haack reagent for mixed reaction to obtain intermediate product S1; 2) The intermediate product S1 and 4-bromoacetophenone are mixed with a sodium hydroxide aqueous solution and ethanol to react to obtain an intermediate product S2; 3) The intermediate product S2, nitromethane, N,N-diisopropylethylamine and methanol are mixed and reacted to obtain the intermediate product S3; 4) The intermediate product S3, ammonium acetate and n-butanol are mixed and the temperature is raised to react to obtain the intermediate product S4; 5) After mixing the intermediate product S4, N,N-diisopropylethylamine and dichloromethane, boron trifluoride etherate is added to react to obtain the intermediate product S5; 6) Under nitrogen protection, the intermediate product S5, 4-pyridinephenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, 1,4-dioxane and water are mixed and the temperature is raised to react to obtain the intermediate product S6; 7) Under nitrogen protection, the intermediate product S6 is mixed with 1-bromododecane and chloroform, and the temperature is raised to react to obtain the intermediate product WS5; 8) Under nitrogen protection, 2-(dimethylamino)ethyl methacrylate is reacted with 1,2-dibromoethane and ethanol to obtain intermediate product A1; 9) Under nitrogen protection, methanol was added to 2-(dimethylamino)ethyl methacrylate and intermediate product A1, and then the above solution was mixed with 2,2-bipyridine, cuprous bromide and ethyl isobromobutyrate to obtain the intermediate product ; 10) The intermediate product Mix with WS5 and N, N-dimethylformamide, and heat to react to obtain the product amphiphilic polymer-photothermal agent composite material .

3. The preparation method according to claim 2, characterized in that: In step 3), the reaction temperature is 60-70°C.

4. The preparation method according to claim 2, characterized in that: In step 4), the reaction temperature is 100-110°C.

5. The preparation method according to claim 2, characterized in that: In step 6), the reaction temperature is 70-80°C.

6. The preparation method according to claim 2, characterized in that: In step 7), the reaction temperature is 80-90°C.

7. The preparation method according to claim 2, characterized in that: In step 9), the reaction temperature is 30-40°C.

8. The preparation method according to claim 2, characterized in that: In step 10), the reaction temperature is 90-100°C.

9. The use of an amphiphilic polymer-photothermal agent composite material according to claim 1, characterized in that: Used for preparing anti-tumor drugs.

10. The use of an amphiphilic polymer-photothermal agent composite material according to claim 1, characterized in that: Used for the preparation of phototherapeutic agents for fluorescence imaging diagnosis and tumor photothermal therapy.

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