Photochemical reaction long afterglow luminescence photosensitizer material, nanoparticle and preparation method and application thereof

Abstract

The application discloses a kind of photochemical reaction long afterglow luminescence photosensitizer materials, nanoparticles and preparation method and application thereof.The photochemical reaction long afterglow luminescence photosensitizer materials are obtained by reacting 3,4-disubstituted maleic anhydride derivatives with isoluminol or isoluminol derivatives containing amino group;The substituent group of the 3,4-disubstituted maleic anhydride derivative is R1 and R2.The photochemical reaction long afterglow luminescence photosensitizer materials of the application realize high signal-to-noise ratio in vivo by photochemical reaction long afterglow luminescence, long-life fluorescent imaging, and have important application prospect in efficient clinical PDT treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and specifically relates to a class of photochemical reaction long-afterglow luminescence photosensitizer materials, nanoparticles, their preparation methods and applications. Background Technology

[0002] Fluorescence, with its high sensitivity and high resolution, is widely used for real-time visualization of biomedical processes. However, its penetration depth is quite limited due to the requirements for excitation light and the autofluorescence of biological structures. Although red and near-infrared fluorescent materials have been extensively developed to reduce tissue autofluorescence, achieving high penetration depth remains a significant challenge, especially considering the insufficient penetration of excitation light. In contrast, long-persistence luminescence typically has a higher penetration depth than fluorescence. Furthermore, photochemical long-persistence luminescence is independent of excitation light and avoids background autofluorescence, thus holding great promise for imaging deep tissues.

[0003] Afterglow luminescent materials can store excitation energy and emit persistent fluorescence, and have been widely used in various cutting-edge research fields, including but not limited to optoelectronic devices, anti-counterfeiting, information encryption, analytical detection, and bioimaging. Inorganic long-afterglow metallic luminescent materials, due to structural defects or complex impurities forming traps, typically have extremely long lifetimes, but face the challenge of potential cytotoxicity after long-term use. Conversely, organic long-afterglow phosphorescence exhibits advantages such as better biocompatibility, rich molecular designability, and functional tunability, but also suffers from limitations in crystal engineering, host-guest doping, and hydrogen bonding, making them unsuitable for biomedical applications, as they typically occur in crystals or thin films. In contrast, another type of persistent luminescence, called photochemically activated long-afterglow systems, is prepared by integrating a photosensitizer (PS), an energy buffer unit, and a suitable fluorescent unit. Its long-afterglow luminescence mechanism involves the oxidation of the energy buffer unit by singlet oxygen generated by the PS, producing an unstable high-energy intermediate, releasing energy, and transferring it to a suitable luminescent body to produce persistent luminescence. Therefore, this novel material can achieve long-persistent luminescence in solution or ordinary environments, rather than under harsh conditions, showing broad application potential in fields such as bioimaging, immunochromatography, and information encryption. However, few studies have focused on developing long-persistent luminescence-guided photoluminescence (LAL-PDT) technology based on photochemically activated long-persistent luminescence mechanisms. This may be attributed to the ROS generated by PS in the LAL-PDT system, which is mainly used to oxidize the energy buffer unit to generate persistent fluorescence, but the relatively small amount of ROS is insufficient to inhibit tumor growth through the PDT process. Therefore, the high ROS efficiency of PS may be key to constructing an effective LAL-PDT system, which is expected to achieve satisfactory long-persistent fluorescence imaging and good PDT results (Pu K, et al., Nat Biotechnol, 2017, 35, 1102). Summary of the Invention

[0004] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a class of photochemical reaction long afterglow luminescence photosensitizer materials, nanoparticles, their preparation methods and applications.

[0005] The primary objective of this invention is to provide a class of photochemical reaction long-afterglow luminescence photosensitizer materials and their nanoparticles.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned photochemical reaction long afterglow luminescence photosensitizer material and its nanoparticles, which is simple and effective, uses readily available raw materials, and produces stable products.

[0007] Another object of the present invention is to provide the application of the aforementioned photochemical reaction long-afterglow luminescence photosensitizer material and its nanoparticles in in vivo high signal-to-noise ratio imaging and efficient tumor treatment. This application achieves high signal-to-noise ratio tissue imaging and high reactive oxygen species generation efficiency for efficient tumor PDT through photochemical reaction long-afterglow luminescence.

[0008] Another objective of this invention is to establish a model for in vivo tumor treatment using the above-mentioned photosensitizer material, in order to demonstrate that the photosensitizer material of this invention can be used to prepare a drug for tumor treatment.

[0009] The objective of this invention is achieved by at least one of the following technical solutions.

[0010] The present invention provides a type of photochemically reactive long-afterglow luminescence photosensitizer material, the general structural formula of which is shown below:

[0011]

[0012] Where x is any integer between 0 and 4 (0, 1, 2, 3, 4), y is any integer between 0 and 2 (0, 1, 2), and R1 and R2 are independently one of the aromatic hydrocarbons or derivative groups of aromatic hydrocarbons.

[0013] Further, the aromatic hydrocarbon group is one of phenyl, naphthyl, tetraphenylvinyl, 4-methoxyphenyl, diethylaminophenyl, dimethylaminophenyl, carbazolephenyl, phenthiazinephenyl, phenoxazinephenyl, bithienyl, thienocyclopentadienyl, 9,10-dihydro-9,9-dimethylacridyl, 9,10-dihydro-9,9-diphenylacridyl, diphenylamino, triphenylamino, 4,4'-dimethoxytriphenylamino, diphenylaminothienyl, 4,4'-dimethoxydiphenylaminothienyl, 4,4'-(2,1,3-benzothiadiazole-4,7-diyl)bis[N,N-diphenyl]aniline or naphthamidophenyl.

[0014] Furthermore, R1 and R2 are independently one of the following groups:

[0015]

[0016] The method for preparing the above-mentioned photochemical reaction long-afterglow luminescence photosensitizer material provided by the present invention includes the following steps:

[0017] The 3,4-disubstituted maleic anhydride derivative is reacted with isoluminol or an amino-containing isoluminol derivative to obtain the photochemical reaction long afterglow luminescence photosensitizer material; the substituents of the 3,4-disubstituted maleic anhydride derivative are R1 and R2.

[0018] Further, the molar ratio of the 3,4-disubstituted maleic anhydride derivative to isoluminol or amino-containing isoluminol ranges from 1:1 to 1:2. In this invention, the molar ratio of the 3,4-disubstituted maleic anhydride derivative to isoluminol or N-(4-aminobutyl)-N-ethyl isoluminol is 1:1.2.

[0019] Furthermore, the reaction is carried out at a temperature of 140-180°C for 8-16 hours.

[0020] Furthermore, the reaction is carried out in an organic solvent;

[0021] More preferably, the organic solvent is DMF; the volume molar ratio of the organic solvent to the 3,4-disubstituted maleic anhydride derivative is 20-40 mL: 1 mmol;

[0022] Furthermore, the reaction is carried out under the catalysis of an alkaline catalyst;

[0023] More preferably, the alkaline catalyst is triethylamine; the volume molar ratio of the organic solvent to the 3,4-disubstituted maleic anhydride derivative is 0.05-0.2 mL: 1 mmol.

[0024] Furthermore, the preparation method of the 3,4-disubstituted maleic anhydride derivative is as follows: using squaric acid and thionyl chloride as raw materials, and N,N-dimethylformamide as a catalyst, squaric acid acyl chloride is obtained by heating reaction. Then, it is reacted with various aromatic hydrocarbon derivatives by Friedel-Crafts reaction to obtain 3,4-disubstituted squaric acid derivatives. Subsequently, the 3,4-disubstituted squaric acid derivatives undergo photocatalytic oxidation cyclization reaction under ultraviolet light irradiation to obtain 3,4-disubstituted maleic anhydride derivatives.

[0025] More preferably, the molar ratio of squaric acid to thionyl chloride is between 1:2 and 1:50. In this invention, the molar ratio of squaric acid to thionyl chloride used is 1:31.2.

[0026] More preferably, the molar ratio of squaric acid chloride to aromatic hydrocarbon derivative is between 1:2 and 1:3. In this invention, the molar ratio of squaric acid chloride to aromatic hydrocarbon derivative is 1:2.2.

[0027] More preferably, the Friedel-Crafts reaction between the squaric acid acyl chloride and the aromatic hydrocarbon derivative takes 5-12 hours, and the reaction time used in this invention is 10 hours.

[0028] More preferably, the amount of the 3,4-disubstituted squaric acid derivative is arbitrary. In this invention, the amount of the 3,4-disubstituted squaric acid derivative used is 3.4 mol.

[0029] The present invention provides a type of photochemical reaction long-afterglow luminescence photosensitizer nanoparticle material, comprising the above-mentioned photochemical reaction long-afterglow luminescence photosensitizer material and a coating material; wherein the photochemical reaction long-afterglow luminescence photosensitizer material is coated inside the coating material.

[0030] Furthermore, the coating material is DSPE-PEG. 1000-3000 .

[0031] The preparation method of the above-mentioned photochemical reaction long-afterglow luminescence photosensitizer nanoparticle material provided by the present invention includes the following steps:

[0032] The photochemical reaction long-afterglow luminescence photosensitizer material and the coating material are dissolved in an organic solvent, then dropped into water, and ultrasonically treated to remove the organic solvent, thereby obtaining the photochemical reaction long-afterglow luminescence photosensitizer nanoparticle material.

[0033] The present invention relates to the application of the above-mentioned photochemical reaction long-afterglow luminescence photosensitizer materials and photochemical reaction long-afterglow luminescence photosensitizer nanoparticle materials in the preparation of in vivo high signal-to-noise ratio imaging reagents and highly efficient tumor treatment (anti-tumor) drugs.

[0034] This invention develops a photochemical reaction long-afterglow luminescence photosensitizer material. This type of luminescent probe can be activated by singlet oxygen, and this type of long-afterglow luminescence photosensitizer can efficiently generate singlet oxygen under illumination, thereby achieving photochemical reaction long-afterglow luminescence. This property has potential applications not only in high signal-to-noise ratio, high sensitivity and deeper penetration bioimaging, but also has very important application prospects in efficient and non-invasive clinical PDT treatment.

[0035] The preparation method provided by this invention introduces isoluminol or isoluminol derivatives as the energy storage unit of a photochemical reaction long-afterglow luminescence photosensitizer material. Isoluminol or isoluminol derivatives are coupled with maleic anhydride fluorescent derivatives to synthesize a class of photochemical reaction long-afterglow luminescence photosensitizer materials. This type of material uses singlet oxygen or sodium hypochlorite as the afterglow luminescence initiator, which reacts chemically with isoluminol or isoluminol derivatives to generate a high-energy intermediate. The high-energy intermediate then undergoes intramolecular electron exchange with a fluorescent group to achieve chemical excitation of a high-energy state, realizing photochemical reaction long-afterglow luminescence. Different fluorescent groups can be introduced to achieve photochemical reaction long-afterglow luminescence with different colors. Furthermore, studies have found that this type of material can efficiently generate singlet oxygen under illumination, becoming an initiator for photochemical reaction long-afterglow luminescence and activating it, thereby achieving photochemical reaction long-afterglow luminescence. This enables high signal-to-noise ratio and long-lifetime fluorescence imaging in vivo, and the high singlet oxygen yield has significant application prospects in clinical PDT treatment.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] (1) The photochemical reaction long afterglow luminescence photosensitizer material provided by the present invention is a new photosensitizer material with long afterglow luminescence performance. This material can overcome the problems of poor tumor treatment effect caused by fluorescence aggregation quenching effect and aggregation-induced ROS reduction in the prior art.

[0038] (2) The photochemical reaction long afterglow luminescence photosensitizer material provided by the present invention can be activated by the singlet oxygen generated by itself to achieve long afterglow luminescence, thus realizing photochemical reaction long afterglow luminescence. It is a new type of photochemical reaction long afterglow luminescence photosensitizer material.

[0039] (3) The photochemical reaction long afterglow luminescence photosensitizer material provided by the present invention can achieve high signal-to-noise ratio and long-life fluorescence imaging in vivo.

[0040] (4) The photochemical reaction long afterglow luminescence photosensitizer material provided by the present invention has good killing effect on cancer cells and has high curative effect on mouse live tumors. Therefore, it can be used to prepare drugs for treating tumors. Attached Figure Description

[0041] Figure 1 Fluorescence and afterglow spectra of photochemical reaction long-afterglow luminescent photosensitizers (TMI and TMA) nanoparticles;

[0042] Figure 2 The image shows the screening results of activators for photochemical reaction long-afterglow luminescence photosensitizers (TMI and TMA);

[0043] Figure 3Figure (B) shows the ROS yield of TMA and TMI detected using the DCFH-DA fluorescent probe, and Figure (C) shows the singlet oxygen yield of TMA and TMI detected using the ABDA fluorescent probe.

[0044] Figure 4 Figure showing the toxicity assessment results of TMA (A) and TMI (B) on HeLa cells;

[0045] Figure 5 The results of the in vitro photochemical reaction of TMA and TMI with long afterglow luminescence tissue penetration depth are shown in Figure (A) and the results of the in vitro fluorescence penetration depth of TMA and TMI are shown in Figure (B).

[0046] Figure 6 The image shows the long afterglow luminescence results of the in vitro photochemical reaction of TMA and TMI nanoparticles.

[0047] Figure 7 Long-afterglow luminescence imaging of the in vivo photochemical reaction of TMA and TMI nanoparticles;

[0048] Figure 8 Figure (A) shows the body weight of mice in the control group and after photodynamic therapy, and Figure (B) shows the tumor growth inhibition of mice in the control group and after photodynamic therapy. Detailed Implementation

[0049] The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0050] The synthetic routes for the photochemical reaction long-afterglow luminescence photosensitizer materials in Examples 1-6 are as follows:

[0051]

[0052] Example 1

[0053] Preparation of a photochemically reactive long-afterglow luminescence photosensitizer material (singlet oxygen photosensitizer, labeled PMA in Example 1)

[0054]

[0055] a) 3,4-Dichlorocyclo-3-ene-1,2-dione (2): Squamous acid (1g, 8.78mmol) was added to a 100ml two-necked flask, followed by thionyl chloride (20mL, 274mmol). The mixture was heated to 60℃, and then 0.1mL of N,N-dimethylformamide was added. The reaction was allowed to proceed for 6 hours. After the reaction was completed, excess thionyl chloride was removed by rotary evaporator, the product was collected, and the next step of the reaction was carried out directly.

[0056] b) Synthesis of 3,4-bis(4-methoxyphenyl)cyclo-3-ene-1,2-dione (3-1): Aluminum trichloride (2.58 g, 19.32 mmol) was added to a 100 mL two-necked flask, and nitrogen was circulated three times (10 min each time) under vacuum-nitrogen conditions. Then, compound (2) obtained in step (a) was dissolved in 20 mL of dichloromethane and injected into the reaction flask. Anisole (2.09 g, 19.32 mmol) was then added, and the mixture was heated to 55 °C and reacted for 10 h. After the reaction was completed, the mixture was extracted with dichloromethane / water (the volume ratio of dichloromethane to water was 1 / 1), dried over anhydrous magnesium sulfate for 2 h, and purified by column chromatography to obtain a yellow-pink compound (1.94 g) with a yield of 75%. 1 H NMR (400MHz, CDCl3) δ8.11 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 3.92 (s, 3H).

[0057] c) Synthesis of compound 3,4-bis(4-methoxyphenyl)furan-2,5-dione (4-1): 3,4-bis(4-methoxyphenyl)cyclo-3-ene-1,2-dione (SQ-PhOCH3) (1 g, 3.40 mmol) was added to a 100 mL dry round-bottom flask, followed by the addition of 45 mL tetrahydrofuran to a two-necked flask. The mixture was irradiated with a 410 nm UV lamp for 6 h. After the reaction was stopped, the solvent was removed using a rotary evaporator, and the mixture was purified by column chromatography to obtain 0.74 g of a golden yellow solid, with a yield of 70%. 1 H NMR (500MHz, CDCl3) δ7.56 (d, J = 9.0 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 3.85 (s, 3H).

[0058] d) Synthesis of 6-((4-(3,4-bis(4-methoxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)(ethyl)amino)-2,3-dihydro-o-phenyldiazine-1,4-dione (PMA): Compound 4-1 (310.0 mg, 1 mmol) and N-(4-aminobutyl)-N-ethyl isoluminol (331.2 mg, 1.2 mmol) were added to a two-necked flask. Then, 20 mL of DMF and 0.1 mL of triethylamine were injected as catalysts, and the mixture was heated to reflux at 160 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature. The mixture was purified by column chromatography to obtain a yellow-green solid (539.6 mg), with a yield of 95%. 1 H NMR (400MHz, CDCl3) δ8.09(d,J=8.9Hz,1H),7.48(d,J=8.6Hz,4H),7.30(s,1H),7.14(d,J=8.6Hz,1H) ,6.87(d,J=8.6Hz,4H),3.82(s,6H),3.70(t,J=6.1Hz,2H),3.52(dd,J=15.6,7.2Hz,4H),1.76(s,5H). 13 C NMR(101MHz, CDCl3)δ171.38(s),160.73(s),134.04(s),131.44(s),121.30(s),114.13(s),55 .31(s),37.77(s),29.66(s),26.17(s),24.53(s),14.13(s),11.97(s).HRMS(m / z):591.2222.

[0059] Example 2

[0060] Preparation of a photochemically reactive long-afterglow luminescence photosensitizer material (singlet oxygen photosensitizer, labeled PMI in Example 2)

[0061]

[0062] The synthesis route is as follows:

[0063] a) The synthesis steps of intermediates 2, 3-1, 4-1 are the same as those in Example 1.

[0064] b) Synthesis of compound 6-(3,4-bis(4-methoxyphenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,3-dihydro-o-phenyldiazine-1,4-dione (PMI): Compound 4-1 (310.0 mg, 1 mmol) and isoluminol (212.4 mg, 1.2 mmol) were added to a two-necked flask. Then, 20 mL of DMF and 0.1 mL of triethylamine were injected as catalysts, and the mixture was heated to reflux at 160 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature. The mixture was purified by column chromatography to obtain a yellow solid (445.7 mg), with a yield of 95%. 1 H NMR (400MHz, CDCl3) δ13.68 (s, 1H), 12.58-12.21 (m, 1H), 8.35 (s, 1H), 8.23 ​​(d, J = 8.3Hz, 1H), 7 .94(d,J=7.9Hz,1H),7.52(dd,J=20.4,8.5Hz,4H),6.88(dd,J=20.1,8.6Hz,4H),3.84(s,6H). 13 C NMR (126MHz, CDCl3) δ169.25(s), 160.94(s), 134.38(s), 131.70(s), 120.92(s), 114.15(s), 55.33(s). HRMS(m / z): 470.1349.

[0065] Example 3

[0066] Preparation of a photochemically reactive long-afterglow luminescence photosensitizer material (singlet oxygen photosensitizer, labeled CMA in Example 3)

[0067]

[0068] The synthesis route is as follows:

[0069] a) The synthesis steps of intermediate 2,3-2,4-2 are the same as in Example 1, except that the aromatic substituent anisole is replaced with 9-phenylcarbazole.

[0070] b) Synthesis of compound 6-((4-(3,4-bis(9-phenyl-9H-carbazole-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)(ethyl)amino)-2,3-dihydro-o-phenyldiazine-1,4-dione (CMA): Compound 4-2 (580.2 mg, 1 mmol) and N-(4-aminobutyl)-N-ethyl isoluminol (331.2 mg, 1.2 mmol) were added to a two-necked flask. Then, 40 mL of DMF and 0.1 mL of triethylamine were injected as catalysts, and the mixture was heated to reflux at 160 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature. The mixture was purified by column chromatography to obtain an orange-red solid (821.5 mg), with a yield of 98%.

[0071] Example 4

[0072] Preparation of a photochemically reactive long-afterglow luminescence photosensitizer material (singlet oxygen photosensitizer, labeled CMI in Example 4)

[0073]

[0074] The synthesis route is as follows:

[0075] a) The synthesis steps of intermediate 2,3-2,4-2 are the same as in Example 1, except that the aromatic substituent anisole is replaced with 9-phenylcarbazole.

[0076] b) Synthesis of compound 6-(3,4-bis(9-phenyl-9H-carbazol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,3-dihydro-o-phenyldiazine-1,4-dione (CMI): Compound 4-2 (580.2 mg, 1 mmol) and isoluminol (212.4 mg, 1.2 mmol) were added to a two-necked flask. Then, 35 mL of DMF and 0.1 mL of triethylamine were injected as catalysts, and the mixture was heated to reflux at 160 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature. The mixture was purified by column chromatography to obtain an orange-red solid (724.4 mg), with a yield of 98%.

[0077] Example 5

[0078] Preparation of a photochemically reactive long-afterglow luminescence photosensitizer material (singlet oxygen photosensitizer, labeled TMA in Example 5)

[0079]

[0080] The synthesis route is as follows:

[0081] a) The synthesis steps of intermediates 2,3-3,4-3 are the same as in Example 1, except that the aromatic substituent anisole is replaced with triphenylamine.

[0082] b) Synthesis of compound 6-((4-(3,4-bis(4-(diphenylamino)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyl)(ethyl)amino)-2,3-dihydro-o-phenyldiazine-1,4-dione (TMA): Compound 4-3 (584.2 mg, 1 mmol) and N-(4-aminobutyl)-N-ethyl isoluminol (331.2 mg, 1.2 mmol) were added to a two-necked flask. Then, 40 mL of DMF and 0.1 mL of triethylamine were injected as catalysts, and the mixture was heated to reflux at 160 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature. The product was purified by column chromatography to obtain a red solid (825.5 mg), with a yield of 98%. 1 H NMR (500MHz, CDCl3) δ8.09(d,J=8.9Hz,1H),7.49-7.41(m,4H),7.13(d,J=7.6Hz,8H),7.10-7.03(m,5H),6.96(d ,J=8.8Hz,4H),3.68(d,J=6.4Hz,2H),3.58-3.41(m,4H),1.75(s,6H),1.33-1.18(m,6H),0.88(t,J=7.0Hz,2H). 13 C NMR(101MHz,DMSO)δ171.22(s),148.80(s),146.74(s),133.46(s),131.24(s),130.23(s),125.78(s) ,124.74(s),121.89(s),120.59(s),31.43(s),22.54(s),14.44(s),12.24(s).HRMS(m / z):865.3479.

[0083] Example 6

[0084] Preparation of a photochemically reactive long-afterglow luminescence photosensitizer material (singlet oxygen photosensitizer, labeled TMI in Example 6)

[0085]

[0086] The synthesis route is as follows:

[0087] a) The synthesis steps of intermediates 2,3-3,4-3 are the same as in Example 1, except that the aromatic substituent anisole is replaced with triphenylamine.

[0088] b) Synthesis of compound 6-(3,4-bis(4-(diphenylamino)phenyl)-2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2,3-dihydro-o-phenyldiazine-1,4-dione (TMI): Compound 4-3 (584.2 mg, 1 mmol) and isoluminol (212.4 mg, 1.2 mmol) were added to a two-necked flask. Then, 35 mL of DMF and 0.1 mL of triethylamine were injected as catalysts, and the mixture was heated to reflux at 160 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature. The mixture was purified by column chromatography to obtain a dark red solid (669.2 mg), with a yield of 90%. 1 HNMR (500MHz, CDCl3) δ8.21 (s, 2H), 7.99 (d, J = 7.2Hz, 1H), 7.42 (d, J = 8.8Hz, 4H), 7.35 (t, J = 7.9Hz, 8H), 7.18-7.06 (m, 12H), 6.86 (d, J = 8.8Hz, 4H). 13 CNMR(101MHz, CDCl3)δ169.40(s),149.35(s),146.76(s),133.51(s),131.05(s), 129.53(s),125.73(s),124.19(s),121.18(s),120.72(s).HRMS(m / z):744.2602.

[0089] Example 7

[0090] Fluorescence emission and afterglow emission spectra of singlet oxygen-type photosensitizer materials and long-afterglow luminescent nanoparticles were tested.

[0091] First, we utilize the polymer DSPE-PEG 2k The photosensitizer materials (TMI and TMA) were coated into water-soluble nanoparticles. The specific procedure was as follows: 4 mg of the photosensitizer material (TMI or TMA) was mixed with 8 mg of DSPE-PEG. 2k Dissolve in 4 mL of tetrahydrofuran, then add 1 mL of the mixed solution dropwise to 9 mL of ultrapure water. After sonicating for 3 minutes using a cell disruptor, stir overnight at room temperature to remove the tetrahydrofuran. Concentrate using an ultrafiltration tube to obtain water-soluble photosensitizer materials (TMI and TMA) nanoparticles.

[0092] We then used fluorescence emission spectroscopy to measure the fluorescence emission spectra of TMA and TMI nanoparticles (excitation wavelength 486 nm), utilizing singlet oxygen (…). 1 O2 was used as an activator, and its afterglow emission spectrum was measured. Figure 1As shown, the fluorescence spectra and afterglow spectra of TMA and TMI are in good agreement. The fluorescence and afterglow emission of TMA are around 660 nm, while TMI, due to its stronger intramolecular charge transfer effect, has a significant redshift in emission compared to TMA, redshifting to around 678 nm.

[0093] Example 8

[0094] Screening of activators for singlet oxygen-type photosensitizer materials and long afterglow luminescent materials

[0095] Figure 2 This is a graph showing the activator screening results for photochemical reaction long-afterglow luminescence photosensitizers (TMI and TMA). TMI and TMA were respectively reacted with seven activators (Ca... 2+ Mg 2+ Fe 2+ H2O2, OH· 1 O2, ClO - The two activated photochemical reaction molecules with long afterglow luminescence were detected using an ultra-weak chemiluminescence detector. The detection results are as follows: Figure 2 As shown. The two luminescent materials react with the first five activators (Ca). 2+ Mg 2+ Fe 2+ There was no response from H2O2 and OH·, but once added 1 O2 and ClO - The luminescence signal immediately increased, and ClO - Activation allows for a longer, more vibrant afterglow.

[0096] Example 9

[0097] Reactive oxygen species and singlet oxygen species testing based on photochemical reaction-based long-afterglow luminescence photosensitizer materials (TMI and TMA).

[0098] We measured the reactive oxygen generation efficiency of this type of material. Figure 3Figure B shows the results of evaluating the total ROS generation efficiency of photochemically reactive long-afterglow luminescent photosensitizer nanoparticles (TMI and TMA) using the DCFH-DA probe. Under white light irradiation, the fluorescence intensity of DCFH-DA in TMI rapidly increased to nearly 130 times, much greater than that of TMA (50 times), indicating that both TMI and TMA molecules have strong reactive oxygen species (ROS) generation efficiencies, with TMI exhibiting a higher ROS generation efficiency than TMA. Simultaneously, we also measured the ROS generation efficiency of TMH, finding that maleimide structures with only alkyl chains do not possess ROS generation capabilities. Only after coupling with isoluminol derivatives does a strong intramolecular charge transfer effect form, promoting intersystem crossing processes and inducing ROS generation in the photosensitizer materials TMI and TMA, resulting in their photochemical afterglow properties. Figure 3 In the figure, C represents the use of a singlet oxygen probe (ABDA) to evaluate the type of reactive oxygen species generated by the photosensitizer as singlet oxygen and the singlet oxygen generation efficiency. Under white light irradiation, TMI and TMA molecules exhibit high singlet oxygen generation efficiencies, and the singlet oxygen generation efficiency of nanoparticles is significantly lower than that of small molecules. This suggests that the singlet oxygen generated by nanoparticles may be consumed through another pathway. Combined with the structural characteristics of the photosensitizer molecules, the singlet oxygen generated by nanoparticles may be used to induce the photochemical reaction of the photosensitizer molecules and its long-afterglow luminescence properties. Figure 3 In the diagram, DCFH-DA represents pure DCFH-DA with illumination, RB represents RB+DCFH-DA with illumination, TMI NPs represents TMI NPs+DCFH-DA with illumination, TMA NPs represents TMANPs+DCFH-DA with illumination, TMI represents TMI+DCFH-DA with illumination, and TMA represents TMA+DCFH-DA with illumination. Figure 3 In the C, ABDA represents pure ABDA plus illumination, RB represents RB+ABDA plus illumination, TMI represents TMI+ABDA plus illumination, TMI NPs represents TMI NPs+ABDA plus illumination, TMA represents TMA+ABDA plus illumination, and TMA NPs represents TMA NPs+ABDA plus illumination.

[0099] Example 10

[0100] Toxicity assessment of photochemical reaction-based long-afterglow luminescence photosensitizer materials (TMI and TMA)

[0101] Using 4T1 cells as a model, this invention investigated the toxicity of photosensitizers.

[0102] After culturing 4T1 cells in 96-well plates for 24 hours, they were first incubated with PBS solutions containing different concentrations of TMI and TMA (0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, and 64 μM) for 20 minutes. Then, 100 μL of DMEM (10% fetal bovine serum) was added, and the cells were cultured for another 24 hours. Finally, MTT was added, and the cells were cultured for 4 hours. The UV absorption intensity at 570 nm was measured to investigate their dark toxicity. Additionally, another 96-well plate of cells was subjected to white light (50 mW cm⁻¹). -2 After irradiation for 10 minutes, the cells were cultured for 12 hours, then MTT was added and the cells were cultured for 4 hours. The phototoxicity was studied by measuring the ultraviolet absorption intensity at 570 nm. Figure 4 Figure A shows the toxicity assessment results of TMA on 4T1 cells. TMA exhibits almost no dark toxicity to the cells, demonstrating good biocompatibility. However, it showed some phototoxicity at a photosensitizer concentration of 64 μM. Under the same conditions, TMI also showed little dark toxicity and good biocompatibility; however, under light irradiation, cell viability decreased to 50% at a TMI concentration of 16 μM, and almost all cells died at 64 μM. Figure 4 The result (B) indicates that TMI is significantly more effective than TMA in killing 4T1 cells. TMI molecules exhibit lower cell viability and stronger phototoxicity, consistent with the experimental results regarding ROS production capacity. Figure 3 (A) in the middle.

[0103] Example 11

[0104] In vitro tissue penetration depth simulation study of photosensitizers TMI and TMA with long afterglow luminescence properties due to photochemical reaction

[0105] This experiment uses pure ham as an experimental model to simulate tissue. This invention studies the in vitro tissue penetration depth of TMI and TMA, long-afterglow luminescent molecules in the photochemical reaction of this system.

[0106] Each layer of ham slices is 4mm thick, produced by adding an external activator, singlet oxygen (…). 1 O2) was used to study the penetration depth of long-afterglow luminescence from the in vitro photochemical reaction of molecular TMI and TMA. Figure 5 In study A), by progressively stacking ham slices to increase the depth of the simulated tissue, the results showed that TMI could penetrate up to 3.2 cm, while TMA could penetrate up to 2.4 cm. A comparison was made using fluorescence (468 nm laser) to study the penetration depth. Figure 5 Results B) show that only weak fluorescence can be seen when the simulated tissue depth is 4 mm, indicating that photochemical reaction long afterglow luminescence has a better application prospect than ordinary fluorescence in tissue penetration depth imaging, and TMI has a stronger luminescence intensity than TMA, resulting in better imaging effect.

[0107] Example 12

[0108] In vitro photochemical reaction long afterglow luminescence evaluation

[0109] This invention studies the in vitro photochemical reaction of TMI and TMA molecules as long-afterglow luminescent photosensitizers. A PBS group was used as the control group, and nanoparticles of TMI and TMA were used as the experimental group. FL fluorescence (468nm laser) imaging results showed that TMA had a stronger fluorescence intensity than TMI, which is consistent with... Figure 5 The experimental results for B were the same. In the absence of light, the fluorescence of all three substances was not emitted. However, when tested with 200mW cm⁻¹, the fluorescence was consistent with that of B. -2 The three substances were continuously illuminated with a white light for 30 seconds, and then the light source was turned off before their fluorescence intensity was collected. PBS did not show any fluorescence before or after illumination, while TMI and TMA showed obvious fluorescence signals after 30 seconds of continuous illumination. Figure 6 This indicates that under white light irradiation, the singlet oxygen produced by TMI and TMA can act as an activator for long-afterglow luminescence in their own photochemical reactions, thus exhibiting a significant luminescence signal. Furthermore, the luminescence signal of TMA is stronger than that of TMI. Combined with the singlet oxygen yield results (…), Figure 3 Based on B), it can be inferred that the yield of singlet oxygen detected by TMA is lower than that of TMI. This may be because more of the singlet oxygen produced by TMA is used to activate long-afterglow luminescence, thus exhibiting a lower singlet oxygen yield. Therefore, TMA exhibits a stronger long-afterglow luminescence signal.

[0110] Example 13

[0111] Long-afterglow imaging assessment of in vivo photochemical reactions

[0112] A tumor model was established in mice by subcutaneously implanting 4T1 cells. The tumor size in the subcutaneous mouse tumor was 100 mm². 3 Subsequently, in vivo fluorescence imaging and long-persistence imaging of photochemical reactions of TMI and TMA nanoparticles in mice were evaluated. The concentration of both TMI and TMA nanoparticles was 4 mM (based on the molar amounts of TMI and TMA), and the intratumoral injection dose was 10 μL per mouse. After injection, fluorescence imaging was first performed using a 468 nm laser on an animal imaging system. The imaging results showed that fluorescence was observed not only at the tumor site but also in other areas due to the autoluminescence of mouse hair, exhibiting a significantly low signal-to-noise ratio. Next, long-persistence imaging of the photochemical reaction at the tumor site was performed using a white light lamp (200 mW cm⁻¹). -2 Irradiate the tumor site continuously for 30 seconds, then immediately turn off the white light source and perform afterglow imaging. Figure 7Based on the imaging results in (A) and (B), TMA nanoparticles can only perform afterglow imaging for 60 seconds, while TMI nanoparticles can perform imaging for up to 120 seconds, indicating that TMI has a longer photochemical afterglow lifetime. In addition, regardless of whether it is TMA or TMI, photochemical afterglow imaging only shows obvious fluorescence at the tumor site and there is no hair autofluorescence interference, indicating that photochemical afterglow imaging has a higher signal-to-noise ratio.

[0113] Example 14

[0114] Tumor phototoxicity assessment of TMI and TMA nanoparticles

[0115] Using 4T1 cells as a model, a tumor animal model was established by subcutaneously implanting them into mice. When the subcutaneous tumor size in mice reached 50 mm... 3 Subsequently, the efficacy of TMI and TMA nanoparticles in photodynamic therapy of mouse tumors was evaluated. Next, this invention used TMI and TMA nanoparticles as photosensitizers to perform photodynamic therapy on solid tumors on the surface of 4T1 tumor-bearing nude mice. The concentration of both TMI and TMA nanoparticles was 2 mM (based on the molar amounts of TMI and TMA), and the intratumoral injection dose was 50 μL / mouse; after administration, white light therapy (200 mW cm⁻¹) was performed on the tumor site. -2 The white light therapy was administered for 15 minutes, a total of three times. After the treatment cycle, tumor volume changes in the mice were recorded every two days. Figure 8 As shown in B, the tumors in the control group of mice that did not undergo photodynamic therapy proliferated rapidly, while the growth of tumors was effectively inhibited after photodynamic therapy, indicating that TMI and TMA nanoparticles still have good effects in in vivo tumor treatment. Figure 8 In the diagram, PBS represents pure PBS without light, PBS+L represents PBS with light, TMA NPs represents TMA NPs without light, TMA NPs+L represents TMA NPs with light, TMI NPs represents TMINPs without light, and TMI NPs+L represents TMI NPs with light.

[0116] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A class of photochemical reaction long-persistent luminescence photosensitizer materials, characterized in that, The general structural formula is as follows: Where x is 4, y is any integer between 0 and 2, and R1 and R2 are independently one of the following groups: 。 2. The preparation method of the photochemical reaction long-afterglow luminescence photosensitizer material according to claim 1, characterized in that, Includes the following steps: 3,4-disubstituted maleic anhydride derivatives , Isoluminol or amino-containing isoluminol derivatives reacting to obtain said photochemically reactive long-persistent luminescence photosensitizer material; said R is independently selected from R1 or R2.

3. The method for preparing the photochemical reaction long-afterglow luminescence photosensitizer material according to claim 2, characterized in that, The molar ratio of the 3,4-disubstituted maleic anhydride derivative to isoluminol or an amino-containing isoluminol derivative ranges from 1:1 to 1:

2.

4. The preparation method of the photochemical reaction long afterglow luminescence photosensitizer material according to claim 3, characterized in that, The reaction is carried out at a temperature of 140-180 °C for 8-16 h.

5. The method for preparing the photochemical reaction long-afterglow luminescence photosensitizer material according to claim 3, characterized in that, The reaction is carried out in an organic solvent, namely DMF, and the volume molar ratio of the organic solvent to the 3,4-disubstituted maleic anhydride derivative is 20-40 mL: 1 mmol. The reaction was carried out under the catalysis of a basic catalyst, namely triethylamine; the volume molar ratio of the basic catalyst to the 3,4-disubstituted maleic anhydride derivative was 0.05-0.2 mL:1 mmol.

6. A class of photochemical reaction long afterglow luminescence photosensitizer nanoparticle materials, characterized in that, It includes the photochemical reaction long-afterglow luminescence photosensitizer material and the coating material as described in claim 1; the photochemical reaction long-afterglow luminescence photosensitizer material is coated inside the coating material.

7. The photochemical reaction long-persistent luminescence photosensitizer nanoparticle material according to claim 6, wherein, The coating material is DSPE-PEG 1000-3000 .

8. The use of the photochemical reaction long-afterglow luminescence photosensitizer material according to claim 1 and the photochemical reaction long-afterglow luminescence photosensitizer nanoparticle material according to any one of claims 6-7 in the preparation of imaging reagents and antitumor drugs.

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