A squarylium-based type i photosensitizer, and a preparation method and application thereof

Abstract

The present application relates to a kind of I type photosensitizer based on squarylium and its preparation method and application, belong to the preparation of chemical material and biomedical nanomaterial technical field.The I type photosensitizer provided by the present application, its molecular structure is with tetraphenyl ethylene substituted indoline derivative as donor, with square acid as acceptor.The rotor structure of tetraphenyl ethylene can effectively avoid the close π-π stacking between molecules.The I type photosensitizer of the present application has the advantages of high molar absorption coefficient, good light stability and low biological toxicity, and also shows the efficient I type active oxygen (·OH) generation capacity and photo-thermal conversion capacity (54.2%), therefore it can be used for photodynamic / photothermal combined antitumor and antibacterial treatment.In photodynamic / photothermal combined treatment, after using laser irradiation, the I type photosensitizer can simultaneously generate a large amount of active oxygen (·OH) and heat, which can efficiently kill tumor cells and bacteria.

Description

A Type I photosensitizer based on squaric acid cyanine, its preparation method and application Technical Field

[0001] This invention belongs to the field of chemical material preparation and biomedical nanomaterial technology, and more specifically, relates to a type I photosensitizer based on squaric acid cyanine, its preparation method and application. Background Technology

[0002] Phototherapy strategies, due to their advantages such as being non-invasive, having controllable timing and location, and minimizing drug resistance and side effects, have become a highly effective cancer treatment method that has emerged in recent years. These strategies mainly include photodynamic therapy (PDT) and photothermal therapy (PTT). Specifically, in PDT, under the excitation of a laser of a specific wavelength, a photosensitizer in a triplet state reacts with surrounding substrates (such as O2 or H2O) to produce toxic reactive oxygen species (ROS) that kill cancer cells. Based on the type of ROS produced, photosensitizers are classified into type I (generating ·OH or ·O2). - ) and Type II (generating) 1 There are two main types of photosensitizers: type I and type II. Currently, most organic photosensitizers primarily generate type II reactive oxygen species (ROS), which are highly oxygen-dependent. The hypoxic characteristics of the microenvironment affect their therapeutic efficacy. Compared to type II photosensitizers, type I photosensitizers are less oxygen-dependent and have greater therapeutic potential in hypoxic microenvironments. Therefore, the development of type I photosensitizers is of great significance. PTT (photothermal phototransduction) refers to a therapeutic strategy that converts light into heat. Molecules in the excited state return to the ground state through non-radiative transitions, releasing heat and inducing a local temperature rise, thereby killing cells. Compared to single PDT or PTT, combined PDT / PTT therapy can effectively reduce the heat required for treatment and address issues such as insufficient ROS sensitivity in some cancer cells, thus significantly reducing the survival rate of heat-resistant cells and the side effects of high-dose drugs.

[0003] Currently used organic photosensitizers (such as porphyrins and phthalocyanines) have rigid structures, poor solubility under physiological conditions, and strong aggregation characteristics, which easily lead to a significant reduction in the efficiency of reactive oxygen species generation, resulting in poor photodynamic sterilization effects. Therefore, existing technologies require further improvement and development. Summary of the Invention

[0004] This invention solves the technical problems of existing photosensitizers, such as complex preparation methods, difficulty in modification, poor biocompatibility, decreased reactive oxygen species generation capacity due to aggregation, and limited functionality. It provides a type I photosensitizer, its preparation method, and its applications. The type I photosensitizer uses a tetraphenylethylene-substituted indoline derivative as the donor and squaric acid as the acceptor. This type I photosensitizer has advantages such as simple synthesis, low biotoxicity, good light / thermal stability, and excellent reactive oxygen species generation capacity and photothermal conversion efficiency.

[0005] According to a first aspect of the present invention, a type I photosensitizer based on squaric acid cyanine is provided, wherein the type I photosensitizer uses a tetraphenylethylene-substituted indoline derivative as a donor and squaric acid as an acceptor; the structural formula of the type I photosensitizer is shown in Formula I:

[0006]

[0007] R1 is selected from CH3, CH2SO3Na and CH2SO3K; R2 is selected from H, OCH3, -N(CH3)2 and -N(C6H5)2.

[0008] According to another aspect of the present invention, a method for preparing the aforementioned type I photosensitizer based on squaricine is provided, comprising the following steps:

[0009] (1) Dissolve the compound of formula II and 3,4-dihydroxy-3-cyclobutene-1,2-dione in a mixed organic solvent, wherein the mixed organic solvent is a mixture of benzene-based organic solvent and alcohol-based organic solvent;

[0010] (2) The mixed solvent obtained in step (1) is reacted at 100-140℃. After the reaction is complete, it is purified. When R1 is CH2SO3Na or CH2SO3K, it is also necessary to undergo sodium ionization or potassium ionization to obtain the type I photosensitizer based on squaric acid cyanine with the structural formula shown in Formula I.

[0011]

[0012] R1 is selected from CH3, CH2SO3Na and CH2SO3K; R2 is selected from H, OCH3, -N(CH3)2 and -N(C6H5)2.

[0013] Preferably, the benzene-based organic solvent is benzene, toluene, or xylene, and the alcohol-based organic solvent is propanol, n-butanol, or n-pentanol.

[0014] Preferably, the molar ratio of the compound of formula II to 3,4-dihydroxy-3-cyclobutene-1,2-dione is (2-2.4):1.

[0015] Preferably, the concentration of the compound of formula II in the mixed organic solvent is 1 mg / mL to 10 mg / mL.

[0016] Preferably, in step (2), the reaction time is 8-24 hours.

[0017] According to another aspect of the present invention, the application of the aforementioned type I photosensitizer based on squaric acid cyanine is provided for the preparation of photodynamic and photothermal combined antitumor agents or photodynamic and photothermal combined antibacterial agents, wherein the type I photosensitizer based on squaric acid cyanine is assembled to obtain core-shell structured nanoparticles.

[0018] Preferably, the type I photosensitizer based on squaric acid cyanine and the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide are added to an organic solvent miscible with water, and then added to water under ultrasonic conditions. After ultrasonic treatment, the type I photosensitizer based on squaric acid cyanine and the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide are assembled to obtain core-shell structured nanoparticles. Then, the organic solvent is removed by dialysis to obtain a mixed system of nanoparticles and water.

[0019] Preferably, the mass ratio of the type I photosensitizer based on squaric acid cyanine to the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide is 1:(2-20).

[0020] Preferably, the concentration of nanoparticles in the mixed system is 0.1 mg / mL to 0.2 mg / mL.

[0021] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0022] (1) Squamousine has strong absorption in the visible / near-infrared region, high molar absorptivity, low biotoxicity, ease of synthesis, and good chemical / photostability. In addition, tetraphenylethylene has a propeller-like conformation in its structure. This unique propeller structure is more likely to stack in a disordered manner under physiological conditions, thereby effectively inhibiting strong intramolecular / intermolecular π-π stacking. Moreover, this rotor structure can still maintain local rotation / vibration even during aggregation, which is conducive to dissipating excited state energy through non-radiative pathways. Therefore, introducing tetraphenylethylene into the squamousine system can effectively avoid the decrease in reactive oxygen species generation caused by aggregation.

[0023] (2) The type I photosensitizer nanoparticles prepared by the present invention can effectively avoid the problem of reduced reactive oxygen species production capacity due to aggregation under physiological conditions, and exhibit enhanced reactive oxygen species production capacity due to aggregation, and have good photodynamic properties.

[0024] (3) The active oxygen generated by the type I photosensitizer nanoparticles prepared in this invention is ·OH, which can effectively avoid the decline of photodynamic performance in hypoxic environments and has more advantages in practical applications.

[0025] (4) The type I photosensitizer nanoparticles prepared by the present invention have excellent photothermal conversion capabilities. The photothermal conversion efficiencies of the type I photosensitizers TPE-SQ6 NPs and TPE-SQ7 NPs of the present invention were calculated to be 52.5% and 54.2%, respectively. Therefore, they can be applied to photodynamic / photothermal combined therapy.

[0026] (5) The type I photosensitizer nanoparticles prepared by this invention have good photo / thermal stability. After undergoing four heating / cooling cycles, their photothermal conversion ability remained almost unchanged, indicating that the fluorescent probe has good photo / thermal stability.

[0027] (6) The type I photosensitizer nanoparticles prepared by this invention have excellent biocompatibility and low toxicity, and have great application potential in the biomedical field.

[0028] (7) The type I photosensitizer nanoparticles prepared in this invention are used for photodynamic / photothermal combined anti-tumor and antibacterial purposes. Under laser irradiation, they can generate a large amount of active oxygen (·OH) and heat, thus effectively killing tumor cells and bacteria, showing great application prospects in phototherapy. Attached Figure Description

[0029] Figure 1 is the proton spectrum of the type I photosensitizer TPE-SQ6 of this invention.

[0030] Figure 2 is the hydrogen spectrum of the type I photosensitizer TPE-SQ7 of this invention.

[0031] Figure 3 is a schematic diagram of nanoparticles prepared by encapsulating the type I photosensitizer of the present invention with F127.

[0032] Figure 4 shows the absorption spectrum (a) and average particle size change (b) of the Type I photosensitizer nanoparticles of the present invention after being placed for different times.

[0033] Figure 5 shows the fluorescence enhancement factor at 525 nm wavelength as a function of laser light (635 nm, 0.50 W cm⁻¹) with or without the addition of DCFH (20 μM) as the reactive oxygen species indicator, and with or without the addition of the type I photosensitizer of this invention (5 μM) and commercial photosensitizers Ce6 and RB. -2 A comparison graph of the irradiation time variation curves, where the excitation wavelength is 488nm.

[0034] Figure 6 shows the relative absorption intensity of ABDA (25 μM) at 378 nm wavelength with and without the addition of the type I photosensitizer of this invention (5 μM) as a function of laser light (635 nm, 0.50 W cm⁻¹). -2 ) Curve showing the change in irradiation time.

[0035] Figure 7 shows the effect of DMPO as a free radical scavenger, and the type I photosensitizer (5 μM) of this invention under laser (635 nm, 0.50 W cm⁻¹) conditions.-2 EPR image after 5 minutes of irradiation.

[0036] Figure 8 shows the effect of using NBT (25 μM) as O2. - Indicator, with or without the addition of the type I photosensitizer of this invention (5 μM), used with a laser (635 nm, 0.50 W cm⁻¹). -2 A bar chart comparing the relative absorption intensity at 260nm before and after 5 minutes of irradiation.

[0037] Figure 9 shows the effect of using a laser (635nm, 0.50W cm⁻¹). -2 Irradiation, temperature rise curves of aqueous solutions of different concentrations of the type I photosensitizers of this invention, TPE-SQ6NPs(a) and TPE-SQ7NPs(b), are shown.

[0038] Figure 10 shows the photostability curves of the type I photosensitizers TPE-SQ6 NPs, TPE-SQ7 NPs and ICG (40 μM) of the present invention after four heating-cooling cycles.

[0039] Figure 11 is a bar chart showing the cell viability of NIH-3T3 cells after co-incubation with different concentrations of the type I photosensitizer of this invention, TPE-SQ6 NPs (a) and TPE-SQ7 NPs (b), for 24 h, using the CCK-8 assay.

[0040] Figure 12 shows Staphylococcus aureus (top) and Escherichia coli (bottom) in the presence / absence of the type I photosensitizers of this invention, TPE-SQ6NPs and TPE-SQ7NPs (20 μM) and / or laser (635 nm, 0.5 W cm⁻¹). -2 Photographs of bacterial colonies on agar plates under certain conditions.

[0041] Figure 13 shows Staphylococcus aureus (top) and Escherichia coli (bottom) in the presence / absence of the type I photosensitizers of this invention, TPE-SQ6NPs and TPE-SQ7NPs (20 μM) and / or laser (635 nm, 0.5 W cm⁻¹). -2 A bar chart showing bacterial survival rates under various conditions.

[0042] Figure 14 shows Staphylococcus aureus (top) and Escherichia coli (bottom) in the presence / absence of the type I photosensitizers of this invention, TPE-SQ6NPs and TPE-SQ7NPs (20 μM) and / or laser (635 nm, 0.5 W cm⁻¹). -2 Scanning electron microscope images under certain conditions.

[0043] Figure 15 is a bar chart showing the survival rate of 4T1 cells under normoxic and hypoxic conditions with different concentrations of the type I photosensitizer TPE-SQ7 NPs of this invention added.

[0044] Figure 16 is a bar chart showing the survival rate of 4T1 cells under normoxic and hypoxic conditions in different treatment groups.

[0045] Figure 17 shows photographs of double staining experiments of dead / live cells in 4T1 cells under normoxic and hypoxic conditions in different treatment groups.

[0046] Figure 18 shows laser scanning confocal microscopy images of different treatment groups under normoxic and hypoxic conditions.

[0047] Figure 19 shows the curves of mouse body weight change (a) and mouse tumor growth (b) in different treatment groups.

[0048] Figure 20 shows (a) a bar chart of tumor quality and (b) a photograph of the tumors in mice in different treatment groups after the treatment. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0050] This invention provides a type I photosensitizer, which uses a tetraphenylethylene-substituted indoline derivative as a donor and squaric acid as an acceptor; its structural formula is as follows:

[0051]

[0052] R1 is selected from one of CH3, CH2SO3Na and CH2SO3K; R2 is selected from one of H, OCH3, -N(CH3)2 and -N(C6H5)2.

[0053] According to another aspect of the present invention, a method for preparing the aforementioned type I photosensitizer based on squaricine is provided, comprising the following steps:

[0054] (1) Synthesis of TPE-SQ6, a type I photosensitizer based on squaricine.

[0055] 1-Butyl-2,3,3-trimethyl-5-(tetraphenylyl)-3H-indole and 3,4-dihydroxy-3-cyclobutene-1,2-dione were dissolved in an organic solvent A consisting of benzene and alcohols. Organic solvent A was a mixture of organic solvents B and C, where organic solvent B was benzene, toluene, or xylene, and organic solvent C was propanol, n-butanol, or n-pentanol. The reaction was carried out at 100-140°C under an inert gas atmosphere. After complete reaction, extraction and separation were performed. The resulting organic phase was dried, and the solvent was removed by rotary evaporation under reduced pressure. The product was purified by column chromatography and dried to obtain the type I photosensitizer TPE-SQ6 based on squaric acid cyanine, as shown in Formula III.

[0056]

[0057] (2) Synthesis of TPE-SQ7, a type I photosensitizer based on squaricine.

[0058] 1-(4-Butylsulfonate)-2,3,3-trimethyl-5-(tetraphenylyl)-3H-indole and 3,4-dihydroxy-3-cyclobutene-1,2-dione are dissolved in an organic solvent A consisting of benzene and alcohols. Organic solvent A is a mixture of organic solvent B and organic solvent C, where organic solvent B is benzene, toluene, or xylene, and organic solvent C is propanol, n-butanol, or n-pentanol. The reaction is carried out at 100-140°C under an inert gas atmosphere. After the reaction is complete, the reaction solvent is removed by rotary evaporation under reduced pressure, followed by column chromatography. The obtained product is dissolved in organic solvent D, which is a mixture of organic solvent E and organic solvent F. Organic solvent E is dichloromethane, trichloromethane, or tetrahydrofuran, and organic solvent F is methanol, ethanol, or acetone. A saturated aqueous solution of NaHCO3 or a saturated aqueous solution of Na2CO3 is then added, and after thorough stirring, the mixture is filtered under reduced pressure and dried to obtain the type I photosensitizer TPE-SQ7 based on squaric acid cyanine, as shown in Formula IV.

[0059]

[0060] Preferably, in step (1), the molar ratio of 1-butyl-2,3,3-trimethyl-5-(tetraphenyl)-3H-indole and 3,4-dihydroxy-3-cyclobutene-1,2-dione is 1:(1~1.2), and the concentration of 1-butyl-2,3,3-trimethyl-5-(tetraphenyl)-3H-indole in the mixed solution of organic solvent A is 1 mg / mL~10 mg / mL.

[0061] Preferably, in step (2), the molar ratio of 1-(4-butylsulfonate)-2,3,3-trimethyl-5-(tetraphenyl)-3H-indole and 3,4-dihydroxy-3-cyclobutene-1,2-dione is 1:(1~1.2), and the concentration of 1-(4-butylsulfonate)-2,3,3-trimethyl-5-(tetraphenyl)-3H-indole in organic solvent A is 1 mg / mL to 10 mg / mL.

[0062] According to another aspect of the present invention, the aforementioned type I photosensitizer based on squaric acid cyanine is provided for use in the preparation of photodynamic / photothermal combined antitumor and antibacterial agents.

[0063] Preferably, the type I photosensitizer based on squaric acid cyanine and the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide are added to an organic solvent miscible with water, added to water under ultrasonic conditions, and then subjected to ultrasonic treatment, so that the type I photosensitizer based on squaric acid cyanine and the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide self-assemble to obtain core-shell structured nanoparticles (NPs), and then the organic solvent is removed by dialysis to obtain nanoparticles.

[0064] Preferably, the mass ratio of the type I photosensitizer based on squaric acid cyanine to the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide is 1:(5-20); and the concentration of the nanoparticles is 0.1 mg / mL to 0.2 mg / mL.

[0065] The preparation method of the type I photosensitizer provided by this invention is shown in the following formula:

[0066]

[0067] The preparation method includes the following steps: Compound 1 and 3,4-dihydroxy-3-cyclobutene-1,2-dione are subjected to a condensation reaction to obtain type I photosensitizer TPE-SQ6. Similarly, compound 2 is subjected to a condensation reaction with 3,4-dihydroxy-3-cyclobutene-1,2-dione to obtain the product, which is then ionized with a saturated sodium bicarbonate aqueous solution to obtain type I photosensitizer TPE-SQ7.

[0068] This invention relates to a method for preparing a type I photosensitizer based on squaricine, comprising the following steps:

[0069] (1) A certain amount of compound 1 and 3,4-dihydroxy-3-cyclobutene-1,2-dione were dissolved in an organic solvent A containing benzene and alcohols. Organic solvent A was a mixture of organic solvent B and organic solvent C, where organic solvent B was benzene, toluene, or xylene, and organic solvent C was propanol or n-butanol. The reaction was carried out at 100-140℃ for a certain time (8-24 h) under an inert gas atmosphere. After complete reaction, the mixture was purified to obtain TPE-SQ6, a type I photosensitizer based on squaricocyanine, with the structural formula shown in Formula I.

[0070] (2) A certain amount of compounds 2 and 3,4-dihydroxy-3-cyclobutene-1,2-dione are dissolved in an organic solvent A containing benzene and alcohols. Organic solvent A is a mixture of organic solvent B and organic solvent C, where organic solvent B is benzene, toluene, or xylene, and organic solvent C is propanol or n-butanol. The reaction is carried out at 100-140℃ for a certain time (8-24 h) under inert gas protection. After complete reaction, the product is purified, and a saturated NaHCO3 aqueous solution or a saturated Na2CO3 aqueous solution is added. After stirring at room temperature for a certain time, the mixture is filtered. The upper filter cake is then washed with deionized water and dried to obtain the type I photosensitizer TPE-SQ7 based on squaricocyanine, as shown in Formula I.

[0071] Take type I photosensitizer TPE-SQ6 or TPE-SQ7 and a commercially available triblock polymer of ethylene oxide-propylene oxide-ethylene oxide (F127) to prepare an organic solvent of a certain concentration (due to differences in solubility, TPE-SQ6 is soluble in tetrahydrofuran, while TPE-SQ7 is soluble in N,N-dimethylformamide). Under ultrasonic conditions, quickly add the solvent to water, and continue ultrasonication for 2 minutes after the addition is complete. Then transfer the solvent to a dialysis bag (Mw = 3500 Da) and dialyze for 24 hours, changing the water every 3-4 hours during the process. This yields type I photosensitizer nanoparticles.

[0072] The type I photosensitizer provided by this invention is used for photodynamic / photothermal combined antibacterial treatment. The type I photosensitizer and surfactant F127 are self-assembled into nanoparticles, which are then internalized by bacteria. Even at a low concentration (20 μM), it can efficiently kill bacteria and cancer cells, showing good photodynamic / photothermal combined therapeutic effects and showing good application prospects in anti-tumor and antibacterial applications.

[0073] Example 1: Preparation of Type I photosensitizers TPE-SQ6 and TPE-SQ7

[0074] The specific steps are as follows:

[0075] (1) Synthesis of type I photosensitizer TPE-SQ6:

[0076] Under N2 protection, compound 1 (0.20 g, 0.30 mmol) and squaric acid (0.015 g, 0.14 mmol) were added sequentially to a Dean-Stark apparatus, followed by the injection of solvent-dried toluene (4 mL) and n-butanol (4 mL). The reaction was heated to reflux for 24 h and then cooled to room temperature. After removing the solvent using a rotary evaporator, the crude product was purified by column chromatography on silica gel (eluent: dichloromethane: ethyl acetate = 10:1) to obtain product TPE-SQ6 as a blue-green solid (0.15 g, 94.7%). Figure 1 shows the 1H NMR spectrum of the type I photosensitizer TPE-SQ6 prepared in this example. Specifically: 1 H NMR (600MHz, CDCl3, δ): 7.51 (s, 2H, ArH), 7.49 (d, J = 8.2Hz, 2H, ArH), 7.35 (d, J = 8.3Hz, 4H, ArH), 7.17-7.02 (m, 34H, ArH), 6.99 (d, J = 8.3Hz, 2H, ArH), 5.98(s,2H,-C=CH-),3.99(s,4H,-NCH2-),1.81(s,12H,-C(CH3)2),1.62- 1.55(m,4H,-CH2-), 1.47(qui,J=7.6Hz,4H,-CH2-), 0.99(t,J=7.4Hz,6H). 13 C NMR (100MHz, CDCl3, δ):169.8,143.6,142.7,141.9,141.2,140.4,138.3,136.6,131.9,131.4,127.8,12 7.7,127.6,126.5,126.4,126.0,120.8,109.5,87.0,49.3,43.7,29.2,27.2,20.4,13.9.HR-ESI-MS:m / z calcd.for C 86 H 76 N2O2: 1169.59796 [M+H + ],found1169.59819[M+H + ].

[0077] (2) Synthesis of type I photosensitizer TPE-SQ7

[0078] Under N2 protection, compound 2 (0.10 g, 0.16 mmol) and squaric acid (0.008 g, 0.07 mmol) were added sequentially to a Dean-Stark apparatus, followed by the injection of solvent-dried toluene (3 mL) and n-butanol (3 mL). The reaction was heated under reflux for 24 h and then cooled to room temperature. After removing the solvent by rotary evaporation under reduced pressure, the crude product was purified by column chromatography on silica gel (eluent: dichloromethane:methanol = 10:1). Subsequently, the obtained product was dissolved in THF (10 mL), and methanol (2 mL) and saturated sodium bicarbonate aqueous solution (10 mL) were added. The mixed solution was stirred at room temperature for 2 h and then filtered under reduced pressure. The upper layer product was washed with water several times to obtain product TPE-SQ7 as a dark green solid (0.07 g, 60.1%). Figure 1 shows the 1H NMR spectrum of the type I photosensitizer TPE-SQ7 prepared in this example. Specifically: 1 H NMR (600MHz, CDCl3, δ): 7.82 (s, 2H, ArH), 7.61 (d, J = 8.3Hz, 2H, ArH), 7.54 (d, J = 8.4Hz, 4H, ArH), 7.39 (d, J = 8.5Hz, 2H, ArH ),7.23-7.10(m,18H,ArH),7.08-6.95(m,16H,ArH),5.81(s,2H,-C=CH-),4.09(s,4H,-NCH2-),2.50-2.46(m,4H,-CH2SO3 – ),1.85-1.75(m,4H,-CH2-),1.71(s,12H,-C(CH3)2),1.26(m,4H,-CH2-). 13 C NMR (100MHz, CDCl3, δ): 178.7,169.3,143.8,143.7,142.8,142.4,142.3,141.1,140.6,138.2,135.5,131.7,131.2,13 1.1,128.5,128.3,128.3,127.2,127.1,126.2,120.8,111.2,86.9,51.3,49.3,43.6,27.0,26.4,23.1.HR-ESI-MS:m / z calcd.for C 86 H 74 N2O8S2Na2:663.24488[m / z],found663.24504[m / z].

[0079] Example 2: Preparation method of type I photosensitizer TPE-SQ6 and TPE-SQ7 nanoparticles

[0080] Dissolve 1.0 mmol of TPE-SQ6 or TPE-SQ7 and 15 mg of the commercially available surfactant poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F127) in 1.0 mL of organic solvent (due to differences in solubility, TPE-SQ6 is soluble in tetrahydrofuran, i.e., THF, while TPE-SQ7 is soluble in N,N-dimethylformamide, i.e., DMF). After the sample is completely dissolved, quickly add it to 10 mL of deionized water. During this process, continuously sonicate using a probe sonicator for 2 min (output power: 50%) to form NPs. Then, transfer the solution to a dialysis bag (MWCO: 3500 Da) and dialyze with deionized water for 24 h. Replace the deionized water every 3-4 h to completely remove THF or DMF. After dialysis, uniformly sized ratiometric fluorescent probe nanoparticles are obtained. A schematic diagram of the preparation method is shown in Figure 3.

[0081] Example 3: Long-term stability of type I photosensitizer TPE-SQ6 and TPE-SQ7 nanoparticles

[0082] Two 5mL sample vials were taken, and 0.3mL of solutions of TPE-SQ6 and TPE-SQ7 nanoparticles obtained in Example 2 (the concentration of the original fluorescent probe solution was 0.1mM) were added to each vial. Then, 2.7mL of aqueous solution was added, and the mixture was stirred at room temperature for 2 minutes. The absorption spectra and average particle size were then measured using a UV-Vis spectrophotometer and a particle size analyzer, respectively (see Figure 4). The results show that the absorption spectra and average particle size of the aqueous solutions of TPE-SQ6 and TPE-SQ7 nanoparticles remained almost unchanged after 15 days, demonstrating excellent long-term stability.

[0083] Example 4: Evaluation of the photodynamic properties of type I photosensitizer TPE-SQ6 and TPE-SQ7 nanoparticles

[0084] (1) Detection of total reactive oxygen species (ROS)

[0085] 2',7'-Dichlorofluorescein diacetic acid (DCFH-DA) was used as an indicator to detect ROS formation. After DCFH-DA reacts chemically with ROS, the green fluorescence gradually increases, with a maximum emission wavelength of 525 nm. The specific method is as follows: Take 0.5 mL of DCFH-DA, 10... -3 An ethanol solution of M) was added to a solution with a concentration of 10. -2 The solution was added to 2 mL of NaOH aqueous solution and stirred at room temperature for 30 min. Then 10 mL of PBS (1×, pH = 7.4) solution was added to obtain a concentration of 4×10⁻⁶. -5M's 2',7'-dichlorodihydrofluorescein (DCFH) solution should be stored protected from light until use. Take 1.5 mL of the above DCFH (4 × 10⁻⁶) solution. -5 The M) solution was transferred to a 4 mL sample vial, and then 0.15 mL of TPE-SQ6 NPs or TPE-SQ7 NPs (10 mL each) from Example 2 was added. -4 M) and PBS (1.35 mL, 1×, pH = 7.4) solution. After thorough mixing, its fluorescence spectrum was measured. Then, a laser (635 nm, 0.5 W cm⁻¹) was used. -2 Irradiation was performed for 30 seconds each time, and the fluorescence spectrum was recorded. Irradiation lasted for a total of 5 minutes. The ratio of the fluorescence intensity at 525 nm after irradiation to the initial fluorescence intensity (I / I0⁻¹) was calculated as a function of irradiation time, as shown in Figure 5. The results showed that the fluorescence intensity of the DCFH probe at 525 nm significantly increased with increasing laser irradiation time, indicating that TPE-SQ6 and TPE-SQ7 NPs have good ROS generation ability. Furthermore, compared with commercial photosensitizers dihydroporphyrin e6 (Ce6) and Rose bengal (RB), the type I photosensitizers TPE-SQ6 and TPE-SQ7 NPs of this invention exhibit superior ROS generation ability.

[0086] (2) Singlet oxygen 1 O2) detection

[0087] 9,10-Anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) was used as an indicator for detection. 1 The generation of O2, 1 O2 reacts with ABDA, causing a decrease in its characteristic absorption peak at 378 nm. The specific method involves preparing a solution with a concentration of 5 × 10⁻⁶. -5 M is prepared with an ABDA aqueous solution, and then 1.5 mL of this solution is transferred to a 4 mL sample vial. Subsequently, TPE-SQ6 NPs or TPE-SQ7 NPs (0.15 mL, 10 mL) from Example 2 are added. -4 M) and 1.35 mL of PBS (1×, pH = 7.4) solution. After thorough mixing, its absorption spectrum was measured. Then, a laser (635 nm, 0.5 W cm⁻¹) was used. -2 The system was irradiated with laser light for 30 seconds each time, and the absorption spectrum was recorded. The irradiation time was 5 minutes in total. The ratio of the absorbance at 378 nm after irradiation to the initial absorbance (A / A0) was calculated as a function of irradiation time, as shown in Figure 6. The results show that after laser irradiation, the ratio of the absorbance at 378 nm to the initial absorbance did not change significantly, indicating that there was no significant change in the absorbance of the laser in the system. 1 The generation of O2 indicates that TPE-SQ6 and TPE-SQ7 NPs are not type II photosensitizers.

[0088] (3) Detection of hydroxyl radicals (·OH)

[0089] Electron paramagnetic resonance (EPR) testing: TPE-SQ6 NPs or TPE-SQ7 NPs were tested using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as a free radical scavenger in a laser (635 nm, 0.5 W cm⁻¹). -2 Whether free radicals will be generated under irradiation. The specific method is as follows: First, take 15 μL of TPE-SQ6 NPs or TPE-SQ7 NPs from Example 2, 10... -4 M) was transferred to a 1.5 mL centrifuge tube, followed by the addition of 285 μL of deionized water and 30 μL of DMPO. After thorough mixing, this was used as a control group for testing. Then, a laser (635 nm, 0.5 W cm⁻¹) was used. -2 The solution was irradiated for 5 minutes before testing, forming the experimental group (see Figure 7). The test results showed that after 5 minutes of laser irradiation, both TPE-SQ6 and TPE-SQ7 NPs containing DMPO produced obvious signal peaks in aqueous solutions with a ratio of 1:2:2:1, indicating that the generated ROS type was ·OH, thus proving that TPE-SQ6 and TPE-SQ7 are type I photosensitizers.

[0090] (4) Superoxide anion free radical (·O2) - ) detection

[0091] Nitrotetrazole blue chloride (NBT) was used as an indicator to detect the presence of O2. - The generation of O2 - It will react with NBT and cause a decrease in its characteristic absorption peak at 260 nm. The specific method is as follows: prepare a solution with a concentration of 5 × 10⁻⁶. -5 M is an NBT aqueous solution, and then 1.5 mL of the above solution is transferred to a 4 mL sample vial. Subsequently, TPE-SQ6 NPs or TPE-SQ7 NPs (0.15 mL, 10 mL) from Example 2 are added. -4 M) and 1.35 mL of PBS (1×, pH = 7.4) solution. After thorough mixing, its absorption spectrum was measured. Then, a laser (635 nm, 0.5 W cm⁻¹) was used. -2 After irradiating the solution for 5 minutes, its absorption spectrum was recorded. The ratio of the absorbance at 260 nm after irradiation to the initial absorbance (A / A0) was calculated as a function of irradiation time, as shown in Figure 8. The test results show that after irradiation with laser for 5 minutes, the ratio of the absorbance at 260 nm to the initial absorbance in the solution did not change significantly, indicating that no O2 was generated in the system. - .

[0092] Example 5: Evaluation of the photothermal properties of type I photosensitizer TPE-SQ6 and TPE-SQ7 nanoparticles

[0093] The TPE-SQ6 and TPE-SQ7 NPs from Example 2 were diluted to different concentrations (0, 5, 10, 20, and 40 μM) using deionized water, and 1 mL of each was transferred to a 1.5 mL centrifuge tube. The solutions were then subjected to laser treatment (635 nm, 0.5 W cm⁻¹). -2 Under irradiation by a laser (635nm, 0.50W cm⁻¹), temperature changes were recorded using a thermal imager. Recordings were taken every 30 seconds, with a total irradiation time of 5 minutes for each group. Deionized water was used as a control group under the same conditions (see Figure 9). The test results show that under laser (635nm, 0.50W cm⁻¹) conditions... -2 Under irradiation, the temperatures of their aqueous solutions rose rapidly, and the higher the concentration, the faster the temperature rose. Furthermore, after 5 minutes of irradiation, the solution temperatures of TPE-SQ6 and TPE-SQ7 NPs (40 μM) rose to 50.9 °C and 55 °C, respectively, indicating that TPE-SQ6 and TPE-SQ7 NPs possess excellent photothermal conversion capabilities.

[0094] Example 6: Evaluation of the photostability of type I photosensitizers TPE-SQ6 and TPE-SQ7 nanoparticles

[0095] Prepare 40 μM TPE-SQ6 and TPE-SQ7 NPs (as in Example 2), and transfer 1 mL of each to a 1.5 mL centrifuge tube. [The solution was then subjected to a laser (635 nm, 0.5 W cm⁻¹)]. -2 Under illumination, data were recorded every 30 seconds using a thermal imager, with a total illumination time of 5 minutes per group. After illumination was stopped, a cycle was completed until the solution returned to its initial temperature. Four cycles were recorded (see Figure 10). The test results showed that after four heating / cooling cycles, the temperature change curves of both NPs showed almost no decay. In contrast, the photothermal conversion ability of indocyanine green (ICG) decreased significantly under the same conditions, indicating that TPE-SQ6 and TPE-SQ7 NPs possess good photostability.

[0096] Example 7: Evaluation of the dark cytotoxicity of type I photosensitizers TPE-SQ6 and TPE-SQ7 nanoparticles

[0097] The biocompatibility of TPE-SQ6 and TPE-SQ7 NPs was evaluated using the CCK-8 assay (Cell Counting Kit-8). Specific experimental procedures: Different concentrations of TPE-SQ6 and TPE-SQ7 NPs from Example 2 (0, 2.5, 5, 10, and 20 μM) were added to NIH-3T3 cells and incubated for 24 h. Cell viability was observed (see Figure 11). The results showed that when the NP concentration increased to 20 μM, the cell viability still exceeded 90%, indicating that both NPs have good biocompatibility and low dark cytotoxicity, which will be beneficial for their biomedical applications.

[0098] Example 8: Type I photosensitizers TPE-SQ6 and TPE-SQ7 nanoparticles for combined photodynamic / photothermal antibacterial action.

[0099] Using Staphylococcus aureus and Escherichia coli as representatives of Gram-positive and Gram-negative bacteria, respectively, the photodynamic / photothermal combined antibacterial effects of the TPE-SQ6 and TPE-SQ7 nanoparticles prepared in Example 2 were systematically studied. Specific experimental steps: First, single colonies (first generation) of each of the two bacteria were taken and inoculated into Luria-Bertani (LB) medium, and cultured overnight in a shaker at 120 rpm and 37°C to obtain the second generation. Subsequently, the second generation was diluted 100-fold with LB medium and transferred to a shaker at 37°C for 4 hours. The absorbance (OD) of the two bacterial suspensions at 600 nm was measured using a UV-Vis spectrophotometer. 600 nm Adjust the concentration to 0.5. Then, take an appropriate amount of bacterial suspension (OD). 600 nm =1.0) was added to a diluted solution of TPE-SQ6 and TPE-SQ7 NPs (20 μM), stabilized for 1 min, and then subjected to laser treatment (635 nm, 0.50 W cm⁻¹). -2 Irradiation for 15 minutes was used as the experimental group. Bacteria without TPE-SQ6 NPs, TPE-SQ7 NPs, and light irradiation served as the control group. Additionally, bacterial suspensions treated only with NPs or lasers served as the comparative experimental group. The resulting bacterial suspensions were then diluted 10... 4100 μL of the diluted bacterial suspension was inoculated onto LB agar medium and incubated at 37°C for 18 h. The culture dishes were photographed, and the number of colonies formed was counted (Figures 12 and 13). The test results showed that neither the control group nor the experimental group exhibited significant bactericidal activity. However, when bacteria were treated with TPE-SQ6 NPs (20 μM) or TPE-SQ7 NPs (20 μM) and irradiated with a laser for 15 min, Staphylococcus aureus was completely killed, achieving an antibacterial rate of 100%, while the antibacterial rates against Escherichia coli reached 95.7% and 98.4%, respectively. Furthermore, the scanning electron microscopy results of the antibacterial experiment are shown in Figure 14. Test results showed that in the control and experimental groups, the bacteria maintained clear edges and intact morphology. However, when treated with TPE-SQ6 NPs or TPE-SQ7 NPs and irradiated with a laser, the bacteria suffered severe damage and ruptured (as indicated by the arrows in the figure), ultimately leading to bacterial death. These results demonstrate that both TPE-SQ6 and TPE-SQ7 NPs possess excellent photodynamic / photothermal combined antibacterial capabilities, showing promising application prospects in broad-spectrum antibacterial applications.

[0100] Example 9: Evaluation of the cytotoxicity of type I photosensitizer TPE-SQ7 nanoparticles against 4T1 cells

[0101] To evaluate the photodynamic / photothermal combined therapeutic effect of the TPE-SQ7 NPs prepared in Example 2, 4T1 cells were cultured in 96-well plates for 24 hours under normoxic (21% O2) or hypoxic (1% O2) conditions. Then, under normoxic (21% O2) or hypoxic (1% O2) conditions, the culture medium was replaced with 100 μL of DMEM containing different concentrations of TPE-SQ7 NPs, and incubated for 4 hours. Afterward, the cells were replaced with fresh DMEM sealed in an anaerobic bag (1% O2) and treated with a 635 nm laser (0.5 W cm⁻¹). -2Irradiation for 5 minutes. After further incubation for 12 hours, cell viability was examined using the Cell Counting Kit-8 (CCK-8) assay (Figure 15). Furthermore, under the same conditions, cell viability was compared between different treatment groups (control group, laser group, TPE-SQ7 NPs group, Ce6+ laser group, and TPE-SQ7 NPs+laser group) (Figure 16). Simultaneously, related double staining experiments of dead / live cells are shown in Figure 18. The test results show that TPE-SQ7 nanoparticles exhibit concentration-dependent phototoxicity; at a concentration of 30 μM, the survival rate of 4T1 cells under normoxic conditions was only 15.2%, while the survival rate under hypoxic conditions was 18.9%, indicating that the type I photosensitizer TPE-SQ7 NPs of this invention can still effectively kill 4T1 cells under hypoxic conditions. Furthermore, the double staining experiment of dead / live cells is shown in Figure 17, further demonstrating that TPE-SQ7 NPs still have excellent killing effects on 4T1 cells under hypoxic conditions.

[0102] Example 10: Evaluation of reactive oxygen species generation in mouse breast cancer cells by type I photosensitizer TPE-SQ7 nanoparticles

[0103] Using dichlorodihydrofluorescein-acetoacetate (DCFH-DA) as a reactive oxygen species indicator, 4T1 cells were cultured in 96-well plates for 8 hours, then transferred to anaerobic bags at 37°C for another 4 hours and divided into 5 groups: (1) control group, (2) laser group, (3) TPE-SQ7 NPs group, (4) Ce6+ laser group, and (5) TPE-SQ7 NPs+ laser group. A 2 mm agarose gel was inserted into each well, and DCFH-DA probes were added and incubated for 30 minutes. Then, different groups were treated, and the cells were sealed in anaerobic bags at 37°C (1% O2) for another 30 minutes. Subsequently, a 635 nm laser (0.5 W cm⁻¹) was used. -2 Irradiation was performed for 5 minutes. After incubation for 0.5 hours, the cells were washed three times with PBS and observed under a fluorescence microscope (Figure 18). The results showed that the light-irradiated TPE-SQ7 NPs produced bright green fluorescence in both normoxic and hypoxic 4T1 cells, indicating that TPE-SQ7 NPs can effectively generate reactive oxygen species (ROS) in 4T1 cells. In contrast, the commercial photosensitizer Ce6 only generates ROS under normoxic conditions. Furthermore, the control group, the laser group, and unirradiated TPE-SQ7 NPs did not show green fluorescence, indicating that the source of ROS generation is TPE-SQ7 NPs, and that light irradiation is a necessary condition for the photosensitizer to generate ROS.

[0104] Example 11: Photodynamic / photothermal combined antitumor therapy of type I photosensitizer TPE-SQ7 nanoparticles in mice

[0105] The TPE-SQ7 NPs prepared in Example 2 were injected into 4T1 tumor-bearing mice via tail vein injection, with PBS injection serving as the control group. The 4T1 tumor-bearing mice were randomly divided into five groups (n=3 per group): (1) control group, (2) laser group, (3) TPE-SQ7 NPs group, (4) Ce6+ laser group, and (5) TPE-SQ7 NPs+ laser group. For groups (2), (4), and (5), 12 hours after injection, a 635nm laser (0.5W cm⁻¹) was used. -2 The tumor site was irradiated for 5 minutes. The weight and tumor volume of the mice were measured every other day until day 16, when the tumors of the 4T1 tumor-bearing mice were dissected and photographed. The results of photodynamic / photothermal combined antitumor treatment of the tumors in 4T1 tumor-bearing mice are shown in Figures 19 and 20. The test results show that during treatment, the TPE-SQ7 NPs + laser group had a significant inhibitory effect on tumor growth, achieving a tumor inhibition rate of 94.4%, demonstrating a very efficient photodynamic / photothermal combined treatment effect. In contrast, the commercially available type II photosensitizer Ce6 + light irradiation group showed a poor effect in inhibiting tumor growth; in the hypoxic environment of the tumor, the oxygen-dependent type II photodynamic antitumor effect was not ideal. Furthermore, other groups failed to inhibit tumor growth.

[0106] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of type I photosensitizers based on squaricine in the preparation of photodynamic and photothermal combined anti-breast cancer tumor agents, characterized in that, The type I photosensitizer based on squaric acid cyanine is assembled to obtain core-shell structured nanoparticles; the type I photosensitizer based on squaric acid cyanine uses a tetraphenylethylene-substituted indoline derivative as a donor and squaric acid as an acceptor; the structural formula of the type I photosensitizer is shown in Formula I: Formula I; wherein R1 is selected from one of CH3, CH2SO3Na and CH2SO3K; R2 is selected from one of H, OCH3, -N(CH3)2 and -N(C6H5)2.

2. The application as described in claim 1, characterized in that, The type I photosensitizer based on squaric acid cyanine and the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide were added to an organic solvent miscible with water, and then added to water under ultrasonic conditions. After ultrasonic treatment, the type I photosensitizer based on squaric acid cyanine and the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide were assembled to obtain core-shell structured nanoparticles. Then, the organic solvent was removed by dialysis to obtain a mixed system of nanoparticles and water.

3. The application as described in claim 2, characterized in that, The mass ratio of the type I photosensitizer based on squaric acid cyanine to the triblock polymer of ethylene oxide-propylene oxide-ethylene oxide is 1:(2~20).

4. The application as described in claim 2, characterized in that, The concentration of nanoparticles in the hybrid system is 0.1 mg / mL to 0.2 mg / mL.

5. The application as described in claim 1, characterized in that, The type I photosensitizer based on squaric acid cyanine is prepared by the following steps: (1) dissolving compound of formula II and 3,4-dihydroxy-3-cyclobutene-1,2-dione in a mixed organic solvent, wherein the mixed organic solvent is a mixture of benzene-based organic solvent and alcohol-based organic solvent; (2) heating the mixed solvent obtained in step (1) at 100-140 °C. o C is reacted, and after the reaction is complete, purification is performed. When R1 is CH2SO3Na or CH2SO3K, it is also necessary to perform sodium ionization or potassium ionization treatment accordingly to obtain a type I photosensitizer based on squaric acid cyanine with the structural formula shown in Formula I. Formula II; wherein R1 is selected from one of CH3, CH2SO3Na and CH2SO3K; R2 is selected from one of H, OCH3, -N(CH3)2 and -N(C6H5)2.

6. The application as described in claim 5, characterized in that, The benzene-based organic solvent is benzene, toluene, or xylene, and the alcohol-based organic solvent is propanol, n-butanol, or n-pentanol.

7. The application as described in claim 5 or 6, characterized in that, The molar ratio of compound II to 3,4-dihydroxy-3-cyclobutene-1,2-dione is (2-2.4):

1.

8. The application as described in claim 5 or 6, characterized in that, The concentration of compound II in mixed organic solvents is 1 mg / mL to 10 mg / mL.

9. The application as described in claim 5 or 6, characterized in that, In step (2), the reaction time is 8-24 h.

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