Near-infrared two-region fluorescent i / ii type photosensitizer and application thereof

Abstract

The present application relates to a kind of near-infrared two-region fluorescent I / II type photosensitizer and its application, belong to photosensitizer pharmaceutical technical field.The reagent prepared based on the following formula and its derivatives has higher NIR-II brightness, excellent I / II type photodynamic performance and higher photothermal performance, and can realize the diagnosis and phototherapy of tumor under the condition of lower dose and lower laser power.

Description

Technical Field

[0001] This invention relates to a type I / II photosensitizer with near-infrared II fluorescence and its application, belonging to the field of photosensitizer drug technology. Background Technology

[0002] Near-infrared II (NIR-II, 1000-1700nm) fluorescence imaging technology holds great potential in the fields of tumor phototherapy and diagnosis due to its unique advantages. NIR-II fluorescent agents can absorb near-infrared light energy and convert it into heat energy, thereby directly inducing tumor cell death through photothermal therapy (PTT) and further triggering an anti-tumor immune response to inhibit tumor growth and metastasis. Compared with traditional near-infrared I (NIR-I, 700-900nm) fluorescence imaging, NIR-II fluorescence imaging provides more accurate tumor characteristic information (such as location, size, and boundaries) and dynamic in vivo distribution of NIR-II fluorescent agents, enabling precise determination of when and where to perform photothermal therapy. However, to achieve effective anti-tumor effects, the treatment process typically requires high photothermal temperatures exceeding 50°C or even 60°C. Such high temperatures not only cause damage to adjacent normal tissues due to nonspecific heat diffusion but also require the use of high doses of photothermal agents and / or high-power laser densities, significantly limiting its application in clinical tumor treatment.

[0003] One potential solution to alleviate the limitations of traditional photothermal therapy is to utilize mild photothermal therapy (MPTT) at relatively low temperatures (43-45°C). Multiple reports indicate that MPTT not only increases cellular uptake of therapeutic agents but also demonstrates anticancer therapeutic effects. However, compared to traditional photothermal therapy, MPTT's therapeutic efficacy is insufficient because cancer cells upregulate heat shock proteins (HSPs) as a protective mechanism against thermal damage caused by high temperatures. Therefore, to achieve effective tumor ablation, various adjuvant therapy strategies have been employed, including starvation therapy, gas therapy, chemotherapy, RNA interference therapy, autophagy inhibition therapy, immunotherapy, and HSP inhibition therapy, combined with the design of nanoplatforms to compensate for the decline in MPTT efficacy caused by HSP overexpression. However, the formulation complexity and potential low reproducibility of these nanoplatforms pose significant challenges, as they require the integration of multiple components into a single platform. Furthermore, the therapeutic efficacy of these nanosystems heavily depends on the release of adjuvant therapeutic components. Therefore, the development of photothermal agents capable of simultaneously downregulating HSP expression is urgently needed. NIR-II fluorescent agents with photodynamic therapy (PDT) capabilities hold promise for meeting this requirement because reactive oxygen species (ROS) generated during PDT can destroy HSPs, thereby counteracting the protective effect of intracellular HSPs during MPTT. However, currently available NIR-II fluorescent photosensitizers, when applied to MPTT, suffer from insufficient photodynamic properties, making it difficult to effectively improve treatment outcomes.

[0004] Despite significant efforts to endow NIR-II fluorescent agents, particularly organic fluorescent agents, with photodynamic properties due to their tunable optical properties and high biocompatibility, the photodynamic and fluorescence properties of these organic NIR-II fluorescent agents, especially those absorbing wavelengths above 800 nm, are significantly inferior to those of fluorescent photosensitizers absorbing in the visible light region. Two factors significantly influence the photodynamic and fluorescence properties of NIR-II fluorescent photosensitizers. First, due to their hydrophobic nature, most NIR-II fluorescent photosensitizers tend to aggregate in biological environments. This aggregation leads to a significant reduction in ROS and fluorescence due to intermolecular interactions such as π-π stacking and H-aggregation. Second, currently available NIR-II fluorescent photosensitizers are primarily constructed through the conjugation of electron donor and electron acceptor units, often exhibiting severe solvent chemical shifts. With increasing solvent polarity, the excited-state energy of these photosensitizers tends to return to the ground state via nonradiative transitions, converting into heat and thus reducing fluorescence and / or ROS production. Furthermore, a significant portion of the NIR-II photosensitizers reported exhibited only type II photodynamic properties, which are highly oxygen-dependent. Given the prevalence of hypoxia in most solid tumors and the less oxygen-dependent nature of type I photodynamic therapy, endowing NIR-II photosensitizers with type I photodynamic function is crucial for improving in vivo therapeutic efficacy. Therefore, the development of NIR-II photosensitizers that counteract the aforementioned disadvantages is expected to significantly improve their photodynamic and fluorescence properties, ultimately enhancing the efficacy of MPTT.

[0005] Photosensitizers with near-infrared II fluorescence emission have enormous application potential in the field of tumor phototherapy. On one hand, photosensitizers with near-infrared II fluorescence emission can simultaneously serve as photothermal agents and photosensitizers for both photothermal and photodynamic therapy (PDT) of tumors. This combination effectively overcomes the shortcomings of either treatment alone, significantly improving therapeutic efficacy. This is mainly because photothermal transluminal radiation (PTT) can increase blood flow, thereby improving oxygen supply and enhancing the therapeutic effect of photothermal transluminal radiation (PDT); while PDT can interfere with tumor physiology by disrupting the tumor microenvironment, thereby increasing the sensitivity of tumor cells to PTT. On the other hand, thanks to the advantages of NIR-II fluorescence imaging, NIR-II fluorescence emission can provide more accurate information such as tumor location, size, and dynamic distribution of therapeutic agents, thus allowing for more precise decisions on where, when, at what dose, and with what laser power to implement safer and more efficient phototherapy. However, the current general situation of photosensitizers with near-infrared II fluorescence emission is that while they have sufficient photothermal performance, their fluorescence and photodynamic properties are relatively poor, and most are type II photosensitizers.

[0006] Photodynamic properties are closely related to the intersystem crossing rate of photosensitizers. According to the Fermi-Gold Law, increasing the spin-orbit coupling constant and / or decreasing the energy level difference between singlet and triplet states is beneficial to improving the intersystem crossing rate of photosensitizers, thereby enhancing photodynamic properties. Furthermore, most photosensitizers exhibiting near-infrared II fluorescence emission are strongly hydrophobic, and they typically need to be prepared into nanoparticles that can be stably dispersed in biological systems before they can be applied to biological systems. In this process, two main factors affect the near-infrared II fluorescence and photodynamic properties of the resulting phototherapeutic reagents: i) Constructing donor-acceptor structures is an important strategy for designing such molecules; however, the resulting NIR-II fluorescent photosensitizer molecules suffer from severe solvation effects: their fluorescence and photodynamic properties typically decrease significantly with increasing polarity of the surrounding environment. ii) Due to intermolecular interactions (such as H aggregation, π-π stacking, etc.), NIR-II fluorescent photosensitizer molecules are prone to aggregation during the encapsulation of nanoparticles. This aggregation usually leads to a significant reduction in reactive oxygen species and fluorescence production.

[0007] Therefore, there is an urgent need in this field to develop a class of materials that can counteract the adverse effects of solvation and aggregation on fluorescence and photodynamic properties, thereby obtaining high-performance photosensitizers with near-infrared II fluorescence emission for application in the field of tumor phototherapy. Summary of the Invention

[0008] The technical problem solved by this invention is that it utilizes the heavy atom effect to design a class of compounds AS2 with a high intersystem crossing rate. In the process of preparing these compounds into nanoparticles, their unique fluorescence and photodynamic properties are dependent on the solvent effect and aggregation effect, resulting in phototherapeutic reagents with extremely high NIR-II fluorescence brightness, excellent type I and type II photodynamic properties, and considerable photothermal properties.

[0009] To solve the above-mentioned technical problems, the technical solution proposed by this invention is: a near-infrared II fluorescence type I / II photosensitizer, wherein compound AS2 is used to prepare the type I / II photosensitizer, and the structural formula of compound AS2 is any one or more of the following three:

[0010]

[0011] Preferably, the photosensitizer is prepared from compound AS2 into nanoparticles.

[0012] Preferably, the photosensitizer is prepared as follows: AS2 is encapsulated in an amphiphilic polymer using a nanoprecipitation method to prepare water-soluble nanoparticles. The amphiphilic polymer is DSPE-PEG (PEG length is adjustable, PEG molecular weight is 1000-10000), Pluronic F-127, etc., to prepare AS2. RNanoparticles.

[0013] Preferably, AS2 R The nanoparticles were synthesized via a nanoprecipitation method. The mixed solvent used was a combination of chloroform and tetrahydrofuran (1 mL, chloroform to tetrahydrofuran volume ratio R = 0:5, 1:4, 2:3, or 5:0), used to dissolve compounds AS21 mg and DSPE-PEG. 2000 5 mg was then injected into 10 mL of ultrapure water, and the solutions were sonicated for 2 minutes using a probe sonicator. After the organic solvent evaporated at room temperature, the solutions were filtered through a 0.2 μm filter and concentrated using a 30 kDa membrane to obtain AS2. R Nanoparticles.

[0014] Preferably, AS2 R Nanoparticles are AS2 1:4 This refers to nanoparticles prepared when the volume ratio of chloroform to tetrahydrofuran is 1:4.

[0015] Preferably, AS2 1:4 Nanoparticles 1 O2 and O2 ·- It can significantly reduce the expression of heat shock proteins, thereby improving the efficacy of temperature and photothermal therapy (MPTT).

[0016] To solve the above-mentioned technical problems, another technical solution proposed by the present invention is: any of the photosensitizers described herein is used to prepare anti-tumor drugs.

[0017] To solve the above-mentioned technical problems, another technical solution proposed by the present invention is: the photosensitizer described herein is used to prepare a diagnostic agent for tumor MPTT phototherapy guided by NIR-II fluorescence imaging.

[0018] The beneficial effects of this invention are:

[0019] Compared to other types of organic NIR-II fluorescent molecules at the same mass concentration, AS2 R It exhibited higher brightness. Its excellent fluorescence performance can be attributed to the following factors: First, AS2 possesses an antipolarity-dependent fluorescence quantum yield (QY), meaning that the fluorescence QY initially increases and then decreases with increasing solvent polarity. This unique characteristic makes the polarity difference between the microenvironment polarity of AS2 in nanoparticles and that of AS2 exhibiting the highest fluorescence QY smaller than that of traditional NIR-II fluorescent molecules. Second, AS2 in its aggregated state exhibits a certain degree of resistance to aggregation-induced fluorescence quenching, similar to aggregation-induced emission (AIE) characteristics.

[0020] Mild photothermal therapy (MPTT) offers a safe and promising alternative to conventional photothermal therapy for tumors, but its effectiveness is hampered by heat resistance caused by overexpression of heat shock proteins. Various nanoplatforms have been designed to enhance the efficacy of MPTT through combination therapy. However, these nanoplatforms typically face challenges such as complex formulations, poor reproducibility, and dependence on the release of supplemental therapeutic components to achieve optimal therapeutic effects.

[0021] In this invention, a type I / II photosensitizer (AS2) with high-performance NIR-II fluorescence is used. 1:4 As a simple yet effective nanoplatform, it is derived from the molecule AS2 and enhances the MPTT efficacy against tumors without requiring additional therapeutic components. Through the heavy atom effect, AS2... 1:4 As a type I / II photosensitizer, it exhibits high efficiency in the production of 1 O2(Φ Δ =12.4%) and O2 ·- AS2 exhibits superior performance compared to other NIR-II photosensitizers. In vitro and in vivo experimental results show that AS2… 1:4 The generated 1 O2 and O2 ·- It can significantly reduce HSP90 expression, thereby improving the efficacy of MPTT. AS2 1:4 It exhibits excellent phototherapy performance, significant tumor accumulation, and long-term tumor retention, making it a simple yet superior diagnostic agent for NIR-II fluorescence imaging-guided tumor MPTT phototherapy.

[0022] High-performance NIR-II fluorescent type I / II photosensitizers derived from the molecule AS2 (AS2) 1:4 And demonstrated AS2 by destroying HSPs. 1:4 The effectiveness of tumor suppression in MPTT enhanced by PDT.

[0023] AS2 was designed using the heavy atom effect of bromine atoms, based on the theoretical principle that spin-orbit coupling efficiency is proportional to the fourth power of the atomic number. AS2 not only exhibits high fluorescence quantum yield but also... 1 O2 quantum yield (Φ ΔThe inverse dependence of solvent polarity, and compared with AS1, "Near-Infrared-II Fluorophore with Inverted Dependence of Fluorescence Quantum Yield on Polarity as Potent Phototheranostics for Fluorescence Image-Guided Phototherapy of Tumors", also enhanced the ability to resist aggregation-induced quenching (anti-ACQ) in fluorescence.

[0024] Therefore, AS2 1:4 NIR-II brightness ratio AS1 1:4 More than three times higher. Furthermore, AS2 1:4 It can be used as a type I and type II photosensitizer, producing a quantum yield as high as 12.4%. 1 O2 and O2 produced simultaneously ·- Both of these are far superior to other NIR-II fluorescent photosensitizers. In vitro and in vivo experiments have shown that AS2... 1:4 By generating ROS, the expression of HSP90 was significantly reduced, thereby enhancing the efficacy of MPTT.

[0025] Compared to most previously reported nanosystems for enhancing the efficacy of MPTT, AS2 1:4 It can be produced during phototherapy 1 O2 and O2 ·- It directly downregulates HSP90 expression without releasing additional therapeutic components. AS2 1:4 Its significant phototherapy capabilities, large tumor enrichment, and long-term retention in tumor tissue make it a simple yet superior nanoplatform for NIR-II fluorescence imaging-guided PDT-enhanced MPTT tumors. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings.

[0027] Figure 1 This is a graph showing the relationship between AS2 absorption intensity and AS2 concentration in Example 3 of the present invention.

[0028] Figure 2 This is a graph showing the relationship between AS2 absorption intensity and AS2 concentration in Example 3 of the present invention.

[0029] Figure 3 The absorption spectra of AS2 in different polar solvents in Example 3 of the present invention are shown.

[0030] Figure 4The fluorescence quantum yield of AS2 in different polar solvents in Example 3 of this invention is shown.

[0031] Figure 5 f is specified in Embodiment 3 of the present invention DMSO The ratio of fluorescence area to absorption at different concentrations of AS2.

[0032] Figure 6a The absorption attenuation rate of DPBF in Example 3 of this invention is calculated under laser irradiation (650nm, 28mW cm⁻¹). -2 In toluene / DMSO mixtures with different dimethyl sulfoxide components, at low concentrations (≈2 μg / mL) -1 AS2-induced 1 O2 generation; b is the absorption attenuation rate of DPBF in Example 3 of this invention, under laser irradiation (650nm, 28mW cm⁻¹). -2 In toluene / DMSO mixtures with different dimethyl sulfoxide components, a high concentration (≈12 μg / mL) was observed. -1 AS2-induced 1 O2 is generated.

[0033] Figure 7 AS2 in Embodiment 3 of the present invention R Absorption spectrum in aqueous medium.

[0034] Figure 8 AS2 in Embodiment 3 of the present invention R Fluorescence quantum yields in the 800-1400 nm and 1000 nm-1400 nm ranges, respectively.

[0035] Figure 9 AS2 in Embodiment 3 of the present invention R The resulting decomposition rate of ABDA at 378 nm, and the corresponding SiNc in DMF. 1 O2 quantum yield.

[0036] Figure 10a In embodiment 3 of the present invention, in AS2 1:4 In the presence of (absorbance of 0.5, 808 nm), DHR123 under laser irradiation (808 nm, 100 mW cm⁻¹) -2 b) is the fluorescence spectrum under AS2 in Example 3 of this invention; 1:4 The presence of coumarin under laser irradiation (781nm, 100mW cm⁻¹) -2 Changes in fluorescence spectrum under ( ).

[0037] Figure 11 AS2 at 808 nm (absorbance of 0.2) in Embodiment 3 of the present invention 1:4 O2 and DCTBT NPs·- Comparison of generation capabilities.

[0038] Figure 12a AS2 in Embodiment 3 of the present invention 1:4 At 808nm laser (1.0W cm) -2 a) The change after irradiation and natural cooling to room temperature; b) The relationship between the negative natural logarithm of the temperature difference obtained in the cooling stage in Example 3 of the present invention and the cooling time.

[0039] Figure 13 Different concentrations of AS2 in Example 3 of this invention R (0, 2, 5, 10 and 50 μg mL) -1 Temperature rise curve under 808nm laser irradiation.

[0040] Figure 14 In Example 3 of this invention, under laser irradiation with different power densities, the concentration was 10 μg / mL. -1 AS2 1:4 The temperature rise curve.

[0041] Figure 15 In Embodiment 3 of the present invention, an 808nm laser (1.0W cm⁻¹) is used. -2 Under irradiation, through six laser on / off cycles, at a concentration of 10 μg / mL... -1 AS2 1:4 Temperature changes.

[0042] Figure 16 AS2 / BD is used in Embodiment 3 of the present invention. 1:4 Confocal fluorescence images of treated 4T1 cells, using Lyso-Tracker Green and Mito-Tracker Deep Red under the same conditions with AS21: 4 The incubation period was 1 hour, and P represents the Pearson correlation coefficient.

[0043] Figure 17 In Example 3 of this invention, DCFH-DA is used as a fluorescent probe in AS2. 1:4 (20μg mL -1 The production of intracellular ROS in 4T1 cells treated with 4T1 was characterized.

[0044] Figure 18 In Example 3 of this invention, AS2 is used under normal or hypoxic conditions. 1:4 Cell survival rates after treatment with laser irradiation or no irradiation for 4T1 cells.

[0045] Figure 19These are representative fluorescence images of live / dead cells stained with calcein AM (green) and propidium iodide (red) respectively in Example 3 of this invention.

[0046] Figure 20 The cell survival rate of 4T1 cells incubated under various conditions in Example 3 of this invention is shown to study the efficacy of MPTT.

[0047] Figure 21 AS2 was administered intravenously at different time points in Example 3 of this invention. 1:4 NIR-II fluorescence image of the tumor site after excitation at 808 nm.

[0048] Figure 22 The fluorescence intensity of the tumor site at different time points in Example 3 of the present invention is shown.

[0049] Figure 23a c is the weight curve of tumor-bearing mice at different time intervals after receiving phototherapy in Example 3 of the present invention; b is the weight of the tumor removed from the mouse at the end of phototherapy; c is the tumor volume curve of tumor-bearing mice at different time intervals after receiving phototherapy in Example 3 of the present invention.

[0050] Figure 24 The tumor sections stained with H&E and TUNEL in Example 3 of this invention were collected from mice 24 hours after the specified treatment.

[0051] Figure 25a The first image shows the Western blot results of 4T1 tumor lysates collected from mice 24 hours after the specified treatment in Example 3 of this invention. The relative expression of HSP90 was quantified using β-actin as an internal standard; the second image shows the expression of HSP90 in tumors as determined by immunofluorescence staining in Example 3 of this invention.

[0052] Figure 26 The structures of the two isomers after separating the mixture AS2 in Example 3 of the present invention.

[0053] Figure 27a a) represents the partial fluorescence quantum yield of AS2(γ,γ) in different polar solvents in Example 3 of the present invention; b) represents the partial fluorescence quantum yield of AS2(δ,δ) in different polar solvents in Example 3 of the present invention.

[0054] Figure 28 AS2 in Embodiment 3 of the Invention R (γ,γ) and AS2 R Fluorescence quantum yield of (δ,δ) nanoparticles. Detailed Implementation

[0055] Example 1

[0056] Compound AS2 was synthesized via a Knoevenagel condensation reaction.

[0057] According to the reaction formula

[0058]

[0059] The structural formulas of compound AS2 are as follows:

[0060]

[0061] Synthesized via Knoevenagel condensation: 136.4 mg (0.1 mmol) of compound 1 and 136.5 mg (0.5 mmol) of compound 2 were dissolved in 40 mL of CHCl3, followed by the addition of 0.5 mL of pyridine under argon protection. The resulting mixture was stirred at 56 °C for 12 hours. After cooling to room temperature, the reaction mixture was poured into methanol, from which the crude product precipitated, which was then collected by filtration. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 7 / 3) to give AS2 as a deep blue solid, in a yield of 166 mg with a purity of 89%. The structural data of compound AS2 obtained are as follows: ¹H NMR (400MHz, CDCl₃, δ): 9.17 (d, 2.09H), 8.84 (d, 1.14H), 8.57 (d, 0.73H), 8.03 (d, 0.72H), 7.88–7.84 (m, 2.07H), 7.79 (d, 1.25), 4.75 (d, 4H), 3.22 (t, 4H), 2.11 (m, 2H), 1.87 (m, 4H), 1.53 (m, 4H), 1.37–0.75 (m, 110H); ¹³C NMR (100MHz, CDCl3, δ):187.38,186.96,159.74,159.73,159.10,159.09,15 3.61,147.48,147.47,147.46,145.20,145.18,141.36,138.52,138.29,137. 89,137.77,137.62,137.23,136.14,136.13,135.43,134.12,134.07,133.55,133.54,133.51,133.50,130.80,130.75,130.71,130.65,130.19,129.54, 128.18,126.76,126.33,124.59,120.11,120.10,120.03,115.36,115.20,114.99,113.70,113.65,68.83,68.32,55.87,39.28,32.04,32.02,31.97,31. 97,31.29,30.67,29.99,29.94,29.79,29.75,29.74,29.71,29.69,29.65,29.59,29.56,29.49,29.46,29.36,29.33,25.74,22.79,22.74,22.72,14.23.

[0062] Example 2

[0063] AS2R Preparation of nanoparticles:

[0064] AS2 R The nanoparticles were synthesized via a nanoprecipitation method. Weigh 1 mg of AS2 and 5 mg of DSPE-PEG. 2000 The solution was dissolved in a mixed solvent containing chloroform and tetrahydrofuran (1 mL, chloroform to tetrahydrofuran volume ratio R = 0:5, 1:4, 2:3, and 5:0). The resulting solutions were then added sequentially to 10 mL of ultrapure water and sonicated for 2 minutes using a probe sonicator. After the organic solvent evaporated at room temperature, the solution was filtered through a 0.2 μm filter and concentrated using a 30 kDa membrane to obtain AS2. R For further experiments. AS2 R The concentration was determined by UV-vis-NIR measurement.

[0065] Example 3

[0066] (1) Measurement of the extinction coefficient of AS2 in chloroform: First, a concentration of 100 μg / mL of AS2 in chloroform was prepared. -1 A stock solution was prepared. Subsequently, samples of different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 μg mL) were prepared in chloroform using the stock solution. -1 These samples were used to measure absorption spectra. The relationship between AS2 absorption intensity and AS2 concentration was plotted at 737 nm (maximum absorption wavelength) as follows: Figure 1 As shown, the slope represents the mass extinction coefficient of AS2 in chloroform. By multiplying it by the molecular weight of AS2, the molar extinction coefficient was further calculated, confirming that AS2 has a high extinction coefficient of 92.8 L g at 737 nm. - 1 cm -1 Or 173900 Lmol -1 cm -1 Furthermore, the extinction coefficient of AS2 in dimethyl sulfoxide was determined using the same method, as shown below. Figure 2 As shown, the extinction coefficient of AS2 was measured to be 36.0 L g at 794 nm. -1 cm -1 Or 67500L mol -1 cm -1 .

[0067] (2) Measurement of optical properties of AS2 in solvents of different polarities: The absorption and fluorescence spectra of AS2 in solvents of different polarities were measured. In solvents with relatively low polarity, its absorption characteristics, including peak intensity, shape, and position of absorption bands, showed significant similarities, such as... Figure 3 As shown, with increasing solvent polarity, the fluorescence spectrum broadens, accompanied by a redshift of the main peak and enhancement of the shoulder peak. The fluorescence quantum yield of AS2 initially increases from cyclohexane to toluene and then to chloroform, then decreases with further increases in solvent polarity, as shown in the figure. Figure 4 As shown in the figure. This result indicates that the fluorescence quantum yield of AS2 exhibits an inverse relationship with solvent polarity.

[0068] (3) Measurement of the optical properties of AS2 in a toluene / dimethyl sulfoxide mixed solution: First, a stock solution of AS2 in toluene was prepared, and this solution was added one by one to volumetric flasks and diluted with an appropriate amount of toluene. Subsequently, appropriate amounts of dimethyl sulfoxide were added to each flask, and the mixtures were stirred vigorously to form solutions with different dimethyl sulfoxide volume fractions (f). DMSO Solutions containing AS2 (ranging from 0% to 90%) were immediately measured, along with their absorption and fluorescence spectra (excitation wavelength: 730 nm). The specified fi was determined by integrating the fluorescence intensity over the entire spectral range and dividing by the absorbance of AS2 at 730 nm. DMSO The ratio of brightness to absorption, such as Figure 5 As shown, the luminance to absorptivity ratio (B / A) first increases from 0 to 30%, and then decreases as f... DMSO The further increase in [the concentration of phosphorus] leads to a decrease, which is similar to the trend of AS2 fluorescence quantum yield changing from nonpolar to polar solvents. In toluene / dimethyl sulfoxide mixtures, methylene blue in ethanol was used as a reference (Φ [the concentration of phosphorus] decreased further, which is similar to the trend of AS2 fluorescence quantum yield changing from nonpolar to polar solvents.] Δ =49%), under laser irradiation (laser wavelength 650nm, power 28mW cm⁻¹) -2 AS2 was measured. 1 The O2 quantum yield, as shown in Figure 6, varies regardless of the AS2 concentration. 1 O2 quantum yields all show an initial increase followed by a decrease. DMSO The trend is that AS2 increases and then gradually decreases. These results indicate that AS2 in 1 The O2 quantum yield exhibits an inverse relationship with solvent polarity and possesses aggregation-induced quenching properties. All these experiments were conducted at two different concentrations, approximately 2 and 12 μg / mL. -1 .

[0069] (4)AS2 R Optical properties in aqueous medium: First, their ultraviolet-visible-near-infrared (UV-vis-NIR) spectra were measured, such as... Figure 7 As shown. The absorption band in the near-infrared region extends from AS2. 0:5 To AS2 5:0 It becomes sharper, with the maximum absorption wavelength increasing from AS2. 0:5 The 811nm wavelength is red-shifted to 820nm at AS25:0. AS2R It exhibits excellent light-harvesting capabilities in the near-infrared region, and its extinction coefficient is quite high (e.g., AS2). 1:4 The extinction coefficient at 808 nm is approximately 47.8 L g. -1 cm -1 Or 89500L mol -1 cm -1 It is much higher than many other NIR-II fluorescent photosensitizers.

[0070] (5)AS2 R Fluorescence properties: AS2 R It exhibits bright fluorescence in the 800-1400 nm range, with a main peak at approximately 925 nm and a shoulder peak at approximately 1035 nm. The full width at half maximum (FWHM) is also high, as measured by AS2. 0:5 Shrinking from 147nm to AS2 5:0 81nm. For example... Figure 8 As shown, AS2... 0:5 AS2 1:4 AS2 2:3 and AS2 5:0 The fluorescence quantum yields were calculated to be 3.2%, 5.1%, 7.6%, and 7.5%, respectively, while the corresponding quantum yields in the NIR-II region were 1.4%, 1.7%, 2.4%, and 2.4%, respectively. AS2 1:4 It is an ultra-bright NIR-II fluorescent material with great potential in NIR-II fluorescence imaging.

[0071] (6)AS2 R of 1 O2 generation capacity measurement: AS2 was evaluated using 9,10-anthratridimyl-bis(methylene)dimaleic acid (ABDA) as an indicator. R of 1 O2 generation capacity. ABDA (10 μL, 20 mM) dissolved in dimethyl sulfoxide was added to AS2. R In 2 mL, the absorption wavelength was approximately 781 nm, and the absorbance was approximately 0.5. Subsequently, the sample was continuously irradiated with a laser (781 nm, 100 mW cm⁻¹). -2 The absorption spectrum was recorded at specific time intervals. The change in sample absorption intensity with laser irradiation time at a wavelength of 378 nm was plotted, as shown below. Figure 9 As shown, AS2 is calculated based on the decay rate of ABDA. 0:5 AS2 1:4 AS2 2:3 and AS2 5:0 Φ ΔThe percentages were 5.1%, 12.4%, 9.1%, and 6.4%, respectively. To quantitatively determine AS2... R of 1 O2 quantum yield, choosing SiNc as a reference (SiNc in DMF) 1 The O2 quantum yield is 32%. (In the determination...) 1 When O2 is present, SiNc and AS2 R The absorbance at 781 nm was adjusted to 0.5. 781 nm was chosen as the excitation wavelength (based on the significant absorption peak exhibited by SiNc in DMF at this wavelength). AS2 R of 1 The O2 quantum yield is calculated according to the following equation:

[0072]

[0073] Where, Φ Δ yes 1 O2 quantum yield, S is the slope of ABDA absorption intensity (378nm) with irradiation time, and F is the absorbance correction factor, given by F = 1-10. -OD (-OD represents absorbance at 781 nm) is determined, ref and sam represent SiNc and AS2, respectively. R AS2 in organic solvents or mixed solvents Φ Δ It was determined using a similar experimental procedure, but with methylene blue in ethanol as the standard.

[0074] (4) Hydroxyl radicals (·OH) and O2 ·- Detection: Fluorescent probes coumarin and dihydrorhodamine 123 (DHR123) were used as indicators to detect AS2. R ·OH and O2 in aqueous solution ·- The formation of coumarin (10 μL) and DHR123 (10 μL) was carried out by adding them separately to AS2. R In an aqueous solution (2 mL), the absorbance at 808 nm was adjusted to approximately 0.5. Under an 808 nm laser (100 mW cm⁻¹), -2 After irradiation of the solution, the fluorescence spectra of coumarin (excitation wavelength 330 nm) and DHR123 (excitation wavelength 480 nm) were measured. The fluorescence spectra of DHR123 in AS2 were also measured. R Significant fluorescence enhancement was detected in the solution, while no fluorescence enhancement was observed in AS2R solutions containing coumarin, indicating that AS2R can generate O2 under laser irradiation. ·- However, it cannot generate ·OH, as shown in Figure 10. The O2·- generation ability, indicated by the DHR123 fluorescence enhancement factor (I / I0⁻¹), follows the order: AS2 0:5 <AS25:0 <AS2 2:3 <AS2 1:4 Given the lack of quantitative methods to characterize AS2 R O2 is generated ·- To assess AS2's capabilities more intuitively, we employ a comparative method. R O2 ·- Generation potential. To this end, we utilized recently reported technologies capable of generating O2. ·- The type I photosensitizer DCTBT NPs are used as a reference. AS2 1:4 For example, under the same absorbance at 808nm, after 4 minutes, 100mW cm⁻¹ -2 After laser irradiation, AS2 1:4 The fluorescence enhancement factor of DHR123 was more than 16 times higher than that of DCTBT NPs, such as Figure 11 As shown in the figure. This result demonstrates that AS21:4 also possesses excellent properties as a type I photosensitizer.

[0075] (5)AS2 R Photothermal performance measurement: using an 808nm laser (1.0W cm⁻¹) -2 As a light source, AS2 was measured according to previously reported methods. R Photothermal conversion efficiency for AS2 0:5 AS2 1:4 AS2 2:3 and AS2 5:0 The photothermal conversion efficiencies (PCEs) were determined to be 52.0%, 51.5%, 49.8%, and 47.7%, respectively, based on AS2. 1:4 For example, as shown in Figure 12. To evaluate AS2... R The photothermal properties will be affected by the presence of different concentrations of AS2. R (0, 2, 5, 10 and 50 μg mL) -1 When a 96-well plate was placed under irradiation with an 808nm laser, the temperatures reached 30.9, 39.4, 44.2, and 56.8℃, respectively. Figure 13 As shown, in addition, to evaluate the relationship between temperature and laser power density, a concentration of 10 μg / mL was used. -1 AS2 1:4 Exposure to an 808 nm laser at laser power densities of 0.20, 0.33, 0.50, 0.75, and 1.0 W cm⁻¹ -2 ,like Figure 14 As shown, the temperatures of a 10 μg mL⁻¹ suspension after 10 minutes of irradiation reached 26.1, 27.6, 31.4, 38.1, and 44.2 °C, respectively. Meanwhile, in order to study AS2... 1:4 The photothermal stability was assessed at a concentration of 10 μg / mL.-1 AS2 1:4 Exposure to 808nm laser (1.0W cm⁻¹) -2 Irradiate for 10 minutes. Then, turn off the laser and allow the solution to cool naturally to room temperature. The experiment was repeated six times. Figure 15 As shown, AS21:4 exhibits excellent photothermal stability, with almost identical photothermal behavior observed through repeated heating (laser on) and cooling (laser off) cycles. Temperature changes were recorded using an infrared camera (Fortric225s) during the evaluation of photothermal performance.

[0076] (6) Cell culture: 4T1 cells were cultured in a monolayer incubator at 37°C with humidity control containing 5% CO2. The culture medium was RPMI 1640 with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin added.

[0077] (7) Intracellular colocalization assay: 4T1 cells were cultured overnight in a confocal culture dish at 37°C. Subsequently, 4T1 cells were cultured with AS2 cells. 1:4 They were incubated together for 8 hours. Then, Lyso-Tracker Green and Mito-Tracker Deep Red were incubated with AS2 under the same conditions. 1:4 The cells were incubated for 1 hour. They were then washed with PBS and used for fluorescence imaging, such as... Figure 16 As shown, AS2 / BD1:4 can be internalized and distributed throughout the cytoplasm, with some accumulation in specific organelles such as lysosomes and mitochondria. Studies on intracellular ROS production have confirmed that intracellular ROS can only be effectively generated in the presence of AS21:4 and laser irradiation. Neither AS21:4 alone nor laser irradiation alone can generate intracellular ROS.

[0078] (8) Intracellular 1 O2 generation experiment: 4T1 cells were used at 5×10 4 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured for 12 hours. Subsequently, the cells were treated with RPMI 1640 or AS2 [reagents / plates]. 1:4 (20μg mL -1 The cells were incubated for another 12 hours. Then, 10 μM of 2',7'-dichlorofluorescein diacetate (DCFH-DA) was added to the cells, followed by incubation for another 30 minutes. Subsequently, the cells were washed with phosphate-buffered saline (PBS) and exposed to laser irradiation for 5 minutes (808 nm, 0.6 W cm⁻²), while the control group received no laser irradiation. Finally, fluorescent cell images were captured using a fluorescence microscope at 470 nm excitation, as shown below. Figure 17 As shown, the study confirms that only in the presence of AS2 1:4Intracellular ROS can only be effectively generated under conditions of laser irradiation. AS2 alone... 1:4 Neither laser irradiation nor laser irradiation alone can generate intracellular ROS.

[0079] (9) Cytotoxicity assay: AS2 was assessed using the CCK-8 assay. 1:4 In vitro cytotoxicity and phototoxicity to 4T1 cells. 4T1 cells were dispersed in 96-well plates and incubated at 37°C for 24 hours. Subsequently, the culture medium was replaced with one containing AS2. 1:4 Concentration variation (0 to 30 μg mL) -1 The cells were incubated in a culture medium containing phosphate-buffered saline (PBS) for 12 hours, then washed with PBS and subjected to an 808 nm laser (0.6 W cm⁻¹). -2 Irradiate for 5 minutes. Then incubate in the dark for 24 hours. Perform CCK-8 assay according to standard method. In addition, to further study the effect of phototherapy, Calcein-AM / PI cell staining experiment was performed according to the instruction manual. Once laser irradiation is applied, AS2... 1:4 It exhibits significantly reduced cell viability under both normal oxygen and hypoxia conditions, such as Figure 18 As shown, AS2 was also verified by the carcilium AM / propidium iodide staining experiment. 1:4 Phototoxicity, such as Figure 19 As shown, in order to evaluate AS2 1:4 To investigate whether MPTT can effectively kill tumor cells, a study was conducted in which 4T1 cells were treated for 24 hours and divided into six different groups: control group (I); untreated group (II); and untreated group (III). 1:4 Treatment), control group +45℃ (ii), AS2 1:4 (iii), AS2 1:4 +Laser +Ice (iv), AS2 1:4 +Laser(v) and AS2 1:4 +Laser + Vitamin C (Vc; vi). Cells requiring MPTT are incubated at 45°C (simulating MPTT-only conditions), or exposed to laser irradiation for 5 minutes followed by further incubation at 37°C for 24 hours. Figure 20 As shown, the experimental results indicate that, compared with the control group, the control group at +45℃ and AS2 1:4 and AS2 1:4 The cell viability rates for laser and ice treatments were 58.4%, 95.6%, and 28.2%, respectively. In contrast, AS2... 1:4 +Laser-treated cells had a survival rate of 18.4%, the lowest among the groups mentioned above. However, AS2 1:4 The cell survival rate in the laser + vitamin C group was slightly higher than that in AS2. 1:4+Laser group, 23.4%. These results indicate that photodynamic therapy significantly enhances the cytotoxicity of MPTT.

[0080] (10) Near-infrared-II fluorescence imaging of tumors: Female Balb / c mice carrying subcutaneous 4T1 tumors were used for near-infrared-II fluorescence imaging studies. AS2 1:4 (50 μL, 1 mg mL) -1 The drug was administered to mice via tail vein injection. Imaging was performed at predetermined time intervals using a near-infrared-II fluorescence imaging system, such as... Figure 21 As shown. The excitation wavelength was 808 nm, and a 1000 nm long-pass filter was used for signal acquisition. AS2 was injected intravenously. 1:4 (50 μL, 1 mg mL) -1 Following injection, the fluorescence intensity at the tumor site increased rapidly, peaking 24 hours post-injection and maintaining a strong signal until 96 hours post-injection. Figure 22 As shown. This observation indicates that AS2 1:4 It can rapidly accumulate within the tumor and persist for a considerable period. Meanwhile, the fluorescence intensity in other tissues changes from that of injected AS2. 1:4 The fluorescence gradually decreased over the next 12 hours, indicating that the nanoparticles had been cleared from the circulatory system. Therefore, the gradual increase in tumor fluorescence and the decrease in background fluorescence synergistically enhanced the clarity of the tumor boundary. AS2 1:4 Strong and persistent fluorescence at the tumor site will facilitate successful phototherapy. Imaging results indicate that the optimal time window for phototherapy is during AS2 injection. 1:4 Starting 24 hours later, because at this time AS2 1:4 It accumulates most extensively within tumors.

[0081] (11) Intratumoral phototherapy: When the tumor volume reaches approximately 100 mm 3 Mice carrying 4T1 tumors were randomly divided into 5 groups (n=5 per group): PBS, PBS+L, AS2 1:4 AS2 1:4 +L and AS1 1:4 +L+Vc. The therapeutic agent and imaging agent were administered intravenously to mice at the same dose (50 μL, 1 mg / mL). -1 ). In AS2 1:4 A single dose of laser irradiation (600mW cm) was administered 24 hours after injection. -2 (25 minutes), phototherapy including photothermal and photodynamic therapy was performed. Over the following 14 days, body weight and tumor volume were measured, as shown in Figure 23. The results consistently indicated that AS2 1:4 Laser treatment effectively inhibited tumor growth during the observation period.

[0082] (12) Histological examination: After phototherapy, the mice were euthanized, and tumor tissue was removed, preserved in 4% formalin, and then embedded in paraffin for further analysis. Cellular status and apoptosis within the tumor tissue were assessed by H&E staining and terminal transferase UTP end-labeling (TUNEL). H&E staining analysis was performed on major organs (such as the liver and lungs), as shown in the following figures. Figure 24 As shown, the results consistently indicate that in all experimental groups, AS2 1:4 Laser treatment resulted in the highest proportion of apoptotic cells. These results provide strong evidence that AS2... 1:4 As a photodynamic therapy drug, it has considerable potential in cancer treatment.

[0083] (13) Immunoblotting of proteins: In in vitro experiments, 4T1 cells treated with different methods were lysed using RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors. The concentrations of the collected denatured proteins were determined using a BCA protein assay kit (Pierce). Subsequently, samples with equal protein concentrations were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto PVDF membranes (Millipore Corporation, Billerica, MA). After blocking in 5% bovine serum albumin (BSA) for 1 hour, the PVDF membranes were incubated overnight at 4°C with the main antibody anti-HSP90 (1:2000 dilution, Proteintech) and β-actin (1:5000 dilution, Proteintech). The PVDF membranes were then washed three times and incubated at room temperature for 2 hours with peroxidase-conjugated human anti-rabbit or anti-mouse secondary antibody (1:5000 dilution, Proteintech). Protein bands were visualized using an ECL detection kit, detected in a chemiluminescence imager, and analyzed using a densitometer.

[0084] In in vitro experiments, mice treated with different methods were injected with AS2. 1:4Twenty-four hours later, 4T1 tumor tissue was removed and lysed using RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors. The supernatant was collected by centrifugation, and protein concentration was measured. Subsequently, protein concentration was determined using a BCA kit. Denatured proteins were then separated on an 8% SDS-PAGE gel, transferred to a PVDF membrane, and detected with different primary antibodies, including anti-HSP90 and β-actin. Finally, the membrane was incubated for 2 hours at room temperature with peroxidase-conjugated human anti-rabbit or anti-mouse secondary antibodies. Protein bands were visualized using an ECL detection kit, detected in a chemiluminescence imager, and analyzed using a densitometer, as shown in Figure 25. Compared to the PBS group, only AS2 was used. 1:4 No significant changes in HSP90 expression levels were observed in the treated groups, indicating that AS2 1:4 Treatment alone had no effect on HSP90 expression. However, the HSP90 expression level was slightly increased in the PBS + laser treatment group, possibly due to the slight temperature increase during laser irradiation. In contrast, AS2... 1:4 + The laser-treated group showed a significant decrease in HSP90 expression levels, while AS2 1:4 The laser + vitamin C treatment group showed only a moderate decrease in HSP90 expression levels, indicating that ROS plays a crucial role in inhibiting HSP90 expression. These results collectively demonstrate that AS2 1:4 Laser therapy can effectively reduce the expression level of HSP90 in tumors, thereby significantly reducing the temperature tolerance of cancer cells during phototherapy.

[0085] (14) Separation and purification: The mixture AS2 obtained above is further separated and purified to obtain monomers AS2(γ,γ) and AS2(δ,δ), as follows Figure 26 As shown in Figure 27, the optical properties of AS2(γ,γ) and AS2(δ,δ) in different polar solvents remain consistent with those in the mixture. Water-soluble AS2 nanoparticles were formed using the same method. R (γ,γ), AS2 R After (δ,δ), the fluorescence properties remained basically the same, such as Figure 28 As shown.

[0086] The present invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. A type I / II photosensitizer with near-infrared II fluorescence, characterized in that: Compound AS2 is used to prepare type I / II photosensitizers, wherein the structural formula of compound AS2 is any one or more of the following three: ； AS2 was encapsulated in an amphiphilic polymer using a nanoprecipitation method to prepare a water-soluble AS2. R Nanoparticles, wherein the amphiphilic polymer is DSPE-PEG, and the molecular weight of PEG is 1000 to 10000.

2. The type I / II photosensitizer with near-infrared II fluorescence according to claim 1, characterized in that: AS2 R The nanoparticles were synthesized via a nanoprecipitation method. A mixed solvent containing 1 mL of a combination of chloroform and tetrahydrofuran, with a volume ratio R of 0:5, 1:4, 2:3, or 5:0, was used to dissolve 1 mg of compound AS2 and DSPE-PEG. 2000 5 mg was then injected into 10 mL of ultrapure water, and the solutions were sonicated for 2 minutes using a probe sonicator. After the organic solvent evaporated at room temperature, the solutions were filtered through a 0.2 μm filter and concentrated using a 30 kDa membrane to obtain AS2. R Nanoparticles.

3. The type I / II photosensitizer with near-infrared II fluorescence according to claim 2, characterized in that: AS2 R Nanoparticles are AS2 1:4 This refers to nanoparticles prepared when the volume ratio of chloroform to tetrahydrofuran is 1:

4.

4. The type I / II photosensitizer with near-infrared II fluorescence according to claim 3, characterized in that: AS2 1:4 Nanoparticles 1 O2 and O2 •− It can significantly reduce the expression of heat shock proteins, thereby improving the efficacy of temperature and photothermal therapy (MPTT).

5. The photosensitizer according to any one of claims 1-3 is used to prepare an antitumor drug.

6. The photosensitizer according to any one of claims 1-3 is used to prepare a diagnostic agent for NIR-II fluorescence imaging-guided tumor MPTT phototherapy.

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