H-aggregation type I photosensitizer as well as preparation method and application thereof

Abstract

The invention discloses an H-aggregation type I photosensitizer as well as a preparation method and application thereof. The structural formula of the H-aggregation type I photosensitizer disclosed by the invention is # imgabs0. The H-aggregation type I photosensitizer disclosed by the invention is characterized in that aminophenylboronic acid and 5-bromothieno [3, 2-b] thiophene-2-formaldehyde are subjected to a reaction to obtain an intermediate product NBSCHO, and the NBSCHO and 3-(dicyanomethylene) indanone IC are subjected to a reaction to obtain the H-aggregation type I photosensitizer. The photosensitizer disclosed by the invention has a D-pi-A type molecular structure, and due to a well-designed plane characteristic between a pi structure and an A structure, the photosensitizer has relatively strong pi-pi accumulation capacity, and formation of H-aggregation accumulation of the photosensitizer is driven, so that the active oxygen generation effect of molecules, especially generation of I type active oxygen, is improved. The material can efficiently generate I-type active oxygen under the irradiation of white light or near-infrared laser, so that tumor cells are efficiently killed, and an efficient photodynamic therapy effect is shown.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical diagnosis and treatment, and particularly relates to an H-aggregated type I photosensitizer, a preparation method thereof and an application thereof. Background Art

[0002] In recent years, photodynamic therapy (PDT) has attracted much attention in the field of cancer treatment due to its advantages such as non-invasiveness, high spatiotemporal controllability, and low systemic toxicity. PDT relies on the cytotoxic type I (superoxide anion O2 ·- and hydroxyl radical OH·) and type II (singlet oxygen 1 O2 and reactive oxygen species ROS) generated by photosensitizers (PSs) under light irradiation. Currently, the dominant type II PDT involves the excitation energy transfer (EET) of photosensitizers (PSs) to surrounding oxygen to generate singlet oxygen, but the hypoxic environment of tumors seriously affects its effect. And type I ROS generates toxic free radicals by the photosensitizer transferring electrons to surrounding oxygen or substrates, and can effectively overcome the hypoxic dilemma. Therefore, it is of great significance to develop type I PSs with reduced oxygen dependence.

[0003] Unfortunately, due to the lack of a general molecular design method, type I photosensitizers (PSs) have been reported much less frequently than type II PSs. This is mainly because the excited-state energy transfer competes with the electron transfer (ET) process, resulting in a decrease in the electron transfer efficiency and limiting the development of type I PSs. Therefore, enhancing the electron transfer ability between PSs and substrates has become the key to designing type I photosensitizer molecules. In recent years, molecular engineering, supramolecular engineering, or aggregate engineering for constructing type I photosensitizers have emerged continuously, mainly divided into the following categories: First, through molecular engineering strategies such as donor engineering, acceptor engineering, and cationization, the electronic structure and its distribution of the photosensitizer are regulated to strengthen the intramolecular charge transfer and separation ability of the photosensitizer; Second, an electron-rich environment is constructed to make it easier for the photosensitizer to obtain electrons from the environment, thereby initiating the type I ROS generation process; Third, by introducing electron acceptor molecules, the separation of holes and electrons between molecules is promoted, thereby enhancing the electron transfer ability of the supramolecule. It is not difficult to find that these methods are all aimed at promoting the intramolecular or intermolecular charge transfer (CT) and charge separation (CS) abilities, thus significantly improving their electron transfer efficiency with substrates and enhancing the generation of type I ROS. H-aggregates are a type of aggregate mainly formed by face-to-face stacking between molecules, which are driven by intermolecular interactions such as dipole-dipole interactions, π-π interactions, and hydrogen bonds between molecules. The H-type exciton coupling in H-aggregates has the characteristics of suppressing singlet (S1) fluorescence radiation and promoting excited-state deactivation channels such as intersystem crossing and thermal relaxation. Therefore, it can effectively enhance the ISC channel of molecules and promote the conversion of molecules from the singlet state to the triplet state (T1). Therefore, the H-aggregation strategy has become an effective strategy for designing efficient room-temperature phosphorescence (RTP) materials. And the generation of both ROS and phosphorescence requires the molecule to transform into T1. Therefore, designing photosensitizer molecules as H-aggregates has the potential to increase the generation of T1 and promote the generation of ROS.

[0004] More importantly, the highly overlapping π planes between molecules in H-aggregates cause a large overlap of the electronic orbits of the molecules, endowing them with excellent intermolecular charge transfer and separation abilities, which is beneficial to the generation of type I ROS. Therefore, due to the advantages of promoted triplet generation and charge separation ability, H-aggregates have great potential in constructing efficient type I PSs. However, few studies and reports have been conducted on type I PSs developed by the H-aggregation strategy. Therefore, designing photosensitizers with H-aggregation is a very promising photosensitizer design idea. Summary of the Invention

[0005] In view of the problems existing in current photosensitizers, the purpose of the present invention is to provide an H-aggregated type I photosensitizer; the photosensitizer material of the present invention has a D-π-A structure, can be excited by white light or 660nm excitation light, and efficiently produces ROS. Under light excitation, the photosensitizer can generate a large amount of type I reactive oxygen (hydroxyl free radicals, superoxide anions) efficiently, thereby efficiently killing tumor cells.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned H-aggregation type I photosensitizer.

[0007] Another object of the present invention is to provide the application of the above-mentioned H-aggregation type I photosensitizer.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] An H-aggregated type I photosensitizer, the structural formula is as follows:

[0010]

[0011] Wherein, R is independently a C1-C30 alkyl group, a phenyl group, a C1-C30 alkylphenyl group, or a C1-C30 alkoxyphenyl group.

[0012] The H-aggregation type I photosensitizer of the present invention has a D-π-A structure, an aromatic amine is a donor group, thieno[3,2-b]thiophene is a π bridge, and 3-(dicyanomethylene)indone is an acceptor.

[0013] Preferably, the structural formula of the H-aggregated type I photosensitizer is as follows:

[0014]

[0015] Here, n is independently an integer of 0 to 29, and m is independently an integer of 0 to 30.

[0016] Further preferably, the structural formula of the H-aggregated type I photosensitizer is as follows:

[0017]

[0018] The H-aggregated type I photosensitizer of the present invention has an intramolecular C=O···S interaction between the thiophene and the carbonyl group of the receptor motif, which induces a good planar configuration within the molecule. Taking MTBSIC as an example, its intramolecular interaction and molecular configuration are as follows: Figure 30 shown.

[0019] The stacking structure of the H-aggregated type I photosensitizer crystal of the present invention is a reverse dislocation parallel H-aggregation with π-π stacking as the main stacking method. The schematic diagram of the stacking method and the representative crystal structure (MTBSIC) are as follows:Figure 31 as shown

[0020] The preparation method of the above-mentioned H-aggregated type I photosensitizer comprises the following steps:

[0021] Reacting aminophenylboronic acid NBOH with 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO to obtain an intermediate NBSCHO, and reacting NBSCHO with 3-(dicyanomethylene)indanone IC to obtain the H-aggregated type I photosensitizer NBSIC;

[0022]

[0023] Wherein, each R is independently a C1-C30 alkyl group, a phenyl group, a C1-C30 alkylphenyl group, or a C1-C30 alkoxyphenyl group.

[0024] Preferably, the preparation method of the H-aggregated type I photosensitizer comprises the following steps:

[0025] (1) Adding aminophenylboronic acid NBOH, 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO, potassium carbonate, and tetrakis(triphenylphosphine)palladium into a mixed solvent of THF and water under a protective atmosphere, refluxing and reacting, and purifying to obtain the intermediate NBSCHO;

[0026] (2) Adding the intermediate NBSCHO and 3-(dicyanomethylene)indanone IC into a solvent and a catalyst, refluxing and reacting, and purifying to obtain the H-aggregated type I photosensitizer NBSIC.

[0027] More preferably, in step (1), the molar ratio of aminophenylboronic acid NBOH, 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO, potassium carbonate, and tetrakis(triphenylphosphine)palladium is 1.1-1.5:1:2-10:0.01-0.05.

[0028] More preferably, in step (1), the volume ratio of THF to water in the mixed solvent is 5-3:1;

[0029] More preferably, in step (1), the molar volume ratio of 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO to the mixed solvent is 1 mmol:1-15 mL.

[0030] More preferably, in step (1), the reflux reaction temperature is 60-80 °C and the time is 8-24 h;

[0031] More preferably, in step (1), the protective atmosphere is nitrogen.

[0032] Further preferably, in step (2), the solvent is one or more of dichloromethane, chloroform, ethanol, and acetic anhydride;

[0033] Further preferably, in step (2), the molar volume ratio of the intermediate NBSCHO to the solvent is 1 mmol: 1 - 10 mL.

[0034] Further preferably, in step (2), the catalyst is one or more of triethylamine, pyridine, and piperidine;

[0035] Further preferably, in step (2), the molar ratio of the catalyst to the intermediate NBSCHO is 0 - 0.05: 1.

[0036] Further preferably, in step (2), the molar ratio of the intermediate NBSCHO to 3-(dicyanomethylene) indanone IC is 1: 1.0 - 1.4;

[0037] Further preferably, in step (2), the temperature of the reflux reaction is 30 - 120 °C, and the time is 2 - 24 h.

[0038] Application of the above H-aggregated type I photosensitizer in the preparation of cancer cell photodynamic therapy reagents. The photosensitizer of the present invention has a high ability to generate type I reactive oxygen species, can effectively generate type I ROS under light irradiation, and is applied to the photodynamic therapy of cancer cells.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The photosensitizer of the present invention has a strong ability to generate type I ROS and can be excited by white light or 660 nm near-infrared excitation light.

[0041] (2) The photosensitizer of the present invention has a simple structure and is easy to synthesize. Description of the Drawings

[0042] Figure 1 Are the molecular structures of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0043] Figure 2 Are the synthetic routes of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0044] Figure 3 Are the absorption spectra of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC in THF.

[0045] Figure 4 Is the absorption spectrum of photosensitizer TSIC in different THF and water mixed solutions.

[0046] Figure 5Absorption spectra of photosensitizer TBSIC in different THF / water mixed solutions.

[0047] Figure 6 Absorption spectra of photosensitizer MTSIC in different THF / water mixed solutions.

[0048] Figure 7 Absorption spectra of photosensitizer MTBSIC in different THF / water mixed solutions.

[0049] Figure 8 Absorption spectra of photosensitizer TSIC in a THF / water mixed solution with a ratio of 10:90 and in pure THF.

[0050] Figure 9 Absorption spectra of photosensitizer TBSIC in a THF / water mixed solution with a ratio of 10:90 and in pure THF.

[0051] Figure 10 Absorption spectra of photosensitizer MTSIC in a THF / water mixed solution with a ratio of 10:90 and in pure THF.

[0052] Figure 11 Absorption spectra of photosensitizer MTBSIC in a THF / water mixed solution with a ratio of 10:90 and in pure THF.

[0053] Figure 12 Nanoparticle sizes of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0054] Figure 13 Absorption spectra of nanoparticles of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0055] Figure 14 Absorption spectra of nanoparticles, aggregates, and molecular monomers of photosensitizer TSIC.

[0056] Figure 15 Absorption spectra of nanoparticles, aggregates, and molecular monomers of photosensitizer TBSIC.

[0057] Figure 16 Absorption spectra of nanoparticles, aggregates, and molecular monomers of photosensitizer MTSIC.

[0058] Figure 17 Absorption spectra of nanoparticles, aggregates, and molecular monomers of photosensitizer MTBSIC.

[0059] Figure 18 Crystal packing structure diagram of photosensitizer MTBSIC shown.

[0060] Figure 19 It is a graph of the magnification of the fluorescence intensity of the DCFH solution under white light irradiation by photosensitizers TSIC, TBSIC, and Ce6.

[0061] Figure 20 It is a graph of the magnification of the fluorescence intensity of the DHR123 solution under white light irradiation by photosensitizers TSIC, TBSIC, and Ce6.

[0062] Figure 21 It is a graph of the magnification of the fluorescence intensity of the HPF solution under white light irradiation by photosensitizers TSIC, TBSIC, and Ce6.

[0063] Figure 22 It is a graph of the magnification of the decrease in the absorption intensity of the ABDA solution under white light irradiation by photosensitizers TSIC, TBSIC, and Ce6.

[0064] Figure 23 It is a graph of the magnification of the fluorescence intensity of the DCFH solution under 660 nm laser irradiation by photosensitizers MTSIC, MTBSIC, and Ce6.

[0065] Figure 24 It is a graph of the magnification of the fluorescence intensity of the DHR123 solution under 660 nm laser irradiation by photosensitizers MTSIC, MTBSIC, and Ce6.

[0066] Figure 25 It is a graph of the magnification of the fluorescence intensity of the HPF solution under 660 nm laser irradiation by photosensitizers MTSIC, MTBSIC, and Ce6.

[0067] Figure 26 It is a graph of the magnification of the decrease in the absorption intensity of the ABDA solution under 660 nm laser irradiation by photosensitizers MTSIC, MTBSIC, and Ce6.

[0068] Figure 27 It is a schematic diagram of the enhancement of type I reactive oxygen species generation by H-aggregated photosensitizers.

[0069] Figure 28 It is the dark toxicity of MTSIC nanoparticles and MTBSIC nanoparticles to 4T1 cells.

[0070] Figure 29 It is the phototoxicity of MTSIC nanoparticles and MTBSIC nanoparticles to 4T1 cells under light irradiation.

[0071] Figure 30 It is the intramolecular interaction and molecular configuration of the photosensitizer MTBSIC in the examples.

[0072] Figure 31Schematic diagram of the stacking mode of the stacking structure of the type I photosensitizer crystal of the H-aggregation type and the representative crystal structure (MTBSIC). Detailed implementation mode

[0073] In order to better understand the technical solution of the present invention, the implementation mode of the present invention will be described in detail below with reference to the drawings and embodiments.

[0074] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and the experimental materials used in the following embodiments can be obtained through conventional commercial channels unless otherwise specified.

[0075] Example 1

[0076] Synthesis of H-aggregation type white light absorbing type I photosensitizer TBSIC.

[0077] (1) 5-Bromothieno[3,2-b]thiophene-2-carbaldehyde (1 mmol, 1 eq), 4-borotriphenylamine (1.1 eq), K2CO3 (3 eq) and tetrakis(triphenylphosphine)palladium(0) (0.05 eq) were placed in a two-necked round bottom reaction flask. After purging with nitrogen three times, a THF / water mixed solvent (12 mL) with a deoxygenated volume ratio of 3:1 was added. The mixture was refluxed and stirred at 80 °C for 12 hours under nitrogen protection. The progress of the reaction was detected by thin layer chromatography. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was concentrated by rotary evaporation, extracted with dichloromethane and washed with water three times, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (dichloromethane / petroleum ether) to obtain the intermediate TBSCHO.

[0078] (2) TBSCHO (1 mmol, 1 eq) and 3-(dicyanomethylene)indanone (1.05 eq) were dissolved in ethanol (10 mL). After dropping three drops of piperidine, the mixture was stirred at 80 °C for 8 hours and then cooled to room temperature. The obtained solution was filtered to collect the filter cake, which was slightly washed with ethanol and then recrystallized by heating and refluxing with dichloromethane / ethanol to obtain a dark brown solid TBSIC. Its molecular structural formula is as Figure 1 shown, and the synthesis process is as Figure 2 shown.

[0079] Example 2

[0080] Synthesis of H-aggregation type near-infrared absorbing type I photosensitizer MTBSIC.

[0081] (1) 4,4'-Dimethoxy-4”-boronic acid triphenylamine (1.2 eq), 5-bromothieno[3,2-b]thiophene-2-carbaldehyde (1 eq, 1 mmol), tetrakis(triphenylphosphine)palladium(0) (0.05 eq), and potassium carbonate (3 eq) were added to a two-necked round-bottom reaction flask. After purging with nitrogen three times, a deoxygenated mixed solvent of THF / water with a volume ratio of 3:1 (12 mL) was added. The mixture was heated under reflux at 80 °C for 12 h under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was rotary evaporated and then recrystallized with tetrahydrofuran and water. The obtained filter cake was dissolved in an appropriate amount of dichloromethane, and then the dichloromethane solution was filtered through diatomaceous earth and silica gel. The obtained filtrate was added with an appropriate amount of ethanol for recrystallization, and the orange solid intermediate MTBSCHO was obtained by filtration.

[0082] (2) Subsequently, MTBSCHO (1 mmol, 1 eq) and 3-(dicyanomethylene)indanone (1.1 eq) were dissolved in acetic anhydride (10 mL). The mixture was stirred at 80 °C for 8 h and then cooled to room temperature. After rotary evaporation, the residue was slurried with ethanol and an appropriate amount of chloroform at 50 °C for 1 h. Then, it was filtered while hot, and the filter cake was washed with ethanol and recrystallized with dichloromethane and ethanol to obtain a golden-brown needle-like solid MTBSIC. Its molecular structural formula is as shown in Figure 1 and the synthesis equation is as shown in Figure 2 .

[0083] Comparative Example 1

[0084] Synthesis of near-infrared absorbing photosensitizer TSIC.

[0085] (1) Referring to step (1) of Example 1, 5-bromothiophene-2-carbaldehyde was used instead of 5-bromothieno[3,2-b]thiophene-2-carbaldehyde in the reaction, and the post-treatment was the same to prepare the intermediate TSCHO;

[0086] (2) Referring to step (2) of Example 1, TSCHO was used instead of TBSCHO as the raw material in the reaction, ethanol was used as the solvent, and three drops of piperidine were added. After the reaction was completed as monitored by thin-layer chromatography, the reaction solution was cooled to room temperature, and the filter cake was washed with ethanol after filtration. After collecting the filter cake, it was dissolved in dichloromethane and recrystallized with an excess of n-hexane. The filter cake was washed with n-hexane after filtration to obtain a golden-green needle-like solid TSIC. Its molecular structural formula is as shown in Figure 1 and the synthesis route is as shown in Figure 2 .

[0087] Comparative Example 2

[0088] Synthesis of near-infrared absorbing photosensitizer MTSIC.

[0089] (1) Referring to step (1) of Example 2, replace the reaction raw materials with 4-bromo-4',4'-dimethoxytriphenylamine and 5-formyl-2-thiopheneboronic acid to prepare the intermediate MTSCHO, and the purification method is the same;

[0090] (2) Referring to step (2) of Example 2, after the reaction is completed, cool it to room temperature and then add a large amount of ethanol to precipitate the crude product. After filtering and collecting the filter cake, dissolve it in dichloromethane, add an excess of petroleum ether for recrystallization, filter to obtain a solid, then dissolve it in dichloromethane again, add an excess of ethanol for recrystallization, and filter to obtain a dark brown powdery product MTSIC. Its molecular structural formula is as Figure 1 shown, and the synthesis steps are as Figure 2 shown.

[0091] Example 3

[0092] UV-visible absorption spectra and fluorescence emission spectra of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0093] The solution absorption of TSIC, TBSIC, MTSIC, and MTBSIC was respectively measured at room temperature using a UV spectrophotometer model UV-2600 produced by Shimadzu, Japan. The solvent was THF for all, and the molecular concentration was 10 μM. The data collection range was from 300 to 900 nm. The absorption values were normalized. The fluorescence emission of TSIC, TBSIC, MTSIC, and MTBSIC was respectively measured at room temperature using a fluorescence spectrometer model Horiba Fluoromax-4 produced by Shimadzu Corporation, Japan. The solvent used was THF, and the molecular concentration was 10 μM. The fluorescence emission values were normalized.

[0094] Figure 3 are the UV-visible absorption spectra of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC. The absorption difference between TBSIC and TSIC is only 2 nm, and the absorption difference between MTBSIC and MTSIC is also only 2 nm, indicating that extending the π unit from thiophene to bithiophene has little effect on the absorption of the molecule in the monomer state. However, the MTSIC molecule shows a 33-nm absorption red shift compared to the TSIC molecule, and MTBSIC also shows a 33-nm absorption red shift compared to TBSIC, indicating that as the donor strength increases, the photosensitizer shows a red shift trend. The materials all have strong absorption in the near-infrared region.

[0095] Example 4

[0096] Aggregation behavior test of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0097] The solution absorptions of TSIC, TBSIC, MTSIC, and MTBSIC were respectively measured at room temperature using a UV-visible spectrophotometer (model UV-2600) produced by Shimadzu, Japan. The solvents were THF and water, and the ratios of THF to water were 10 / 0, 9 / 1, 8 / 2, 7 / 3, 6 / 4, 5 / 5, 4 / 6, 3 / 7, 2 / 8, and 1 / 9. The molecular concentration was 10 μM. The data collection range was from 300 to 900 nm.

[0098] Figure 4 It is the UV-visible absorption spectrum of the photosensitizer TSIC in THF / water mixed solvents with different ratios. Obviously, the absorption wavelength of TSIC molecules did not change significantly when water was gradually added.

[0099] Figure 5 It is the UV-visible absorption spectrum of the photosensitizer TBSIC in THF / water mixed solvents with different ratios. Obviously, the absorption wavelength of TBSIC molecules showed a blue shift when excessive water was added.

[0100] Figure 6 It is the UV-visible absorption spectrum of the photosensitizer MTSIC in THF / water mixed solvents with different ratios. Obviously, the absorption wavelength of MTSIC molecules did not change significantly when water was gradually added.

[0101] Figure 7 It is the UV-visible absorption spectrum of the photosensitizer MTBSIC in THF / water mixed solvents with different ratios. Obviously, the absorption wavelength of MTBSIC molecules showed a blue shift when excessive water was added.

[0102] Figure 8 It is the comparative UV-visible absorption spectrum of the photosensitizer TSIC in pure THF and 10 / 90 THF / water mixed solvent. TSIC did not show a change in the absorption spectrum, indicating the formation of amorphous aggregates.

[0103] Figure 9 It is the comparative UV-visible absorption spectrum of the photosensitizer TBSIC in pure THF and 10 / 90 THF / water mixed solvent. The aggregates formed by TBSIC had a significant blue shift compared to the monomers, indicating the formation of H-aggregates by TBSIC.

[0104] Figure 10 It is the comparative UV-visible absorption spectrum of the photosensitizer MTSIC in pure THF and 10 / 90 THF / water mixed solvent. MTSIC did not show a change in the absorption spectrum, indicating the formation of amorphous aggregates.

[0105] Figure 11It is a comparative diagram of the ultraviolet-visible absorption spectra of photosensitizer MTBSIC in pure THF and a 10 / 90 THF / water mixed solvent. The aggregates formed by MTBSIC have an obvious blue shift compared to the monomers, indicating that MTBSIC forms H-aggregates.

[0106] Example 5

[0107] Testing of nanoparticles of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0108] Dissolve 1 mg of photosensitizer and 2 mg of DSPE-PEG 2000 in 1 mL of tetrahydrofuran. After ensuring its full dissolution by ultrasonic bath treatment for 30 minutes, add the THF solution to 10 mL of ultrapure water under probe sonication conditions and continue sonication for 2 minutes. Then, place the mixture in a fume hood and stir overnight in the dark to volatilize THF. Filter through a 0.22 μm polyvinylidene fluoride (PVDF) syringe filter to obtain a transparent nanoparticle solution. Use a Zetasizer Nano-S90 dynamic light scattering instrument from Malvern Instruments to measure the particle sizes of TSIC, TBSIC, MTSIC, and MTBSIC nanoparticles.

[0109] Figure 12 It is the particle size test of nanoparticles of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC. Their particle sizes are all relatively small and are all about 35 nm.

[0110] Example 6

[0111] Ultraviolet-visible absorption spectrum test of nanoparticles of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC.

[0112] Using a UV-2600 ultraviolet spectrophotometer produced by Shimadzu of Japan, the solution absorptions of TSIC, TBSIC, MTSIC, and MTBSIC nanoparticles were respectively tested at room temperature. The solvent is water. The molecular concentration is 10 μM. The data collection range is from 300 to 900 nm.

[0113] Figure 13 It is the absorption spectrum of nanoparticles of photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC. Obviously, the H-aggregated photosensitizer TBSIC is bluer than the photosensitizer TSIC, and the H-aggregated photosensitizer MTBSIC is bluer than the photosensitizer MTSIC.

[0114] Figure 14 It is a comparative test of the absorption spectra of nanoparticles of photosensitizer TSIC with its monomers and aggregates. Its absorption wavelength is the same as that of the amorphous aggregates and monomers, indicating that it maintains amorphous aggregation inside the nanoparticles.

[0115] Figure 15 It is the comparison test of the absorption spectra of photosensitizer TBSIC nanoparticles with their monomers and aggregates. The absorption of the nanoparticles of H-aggregated photosensitizer TBSIC is similar to that of its H-aggregates, and there is a large blue shift compared with the monomers, indicating that H-aggregation is maintained inside the nanoparticles.

[0116] Figure 16 It is the comparison test of the absorption spectra of photosensitizer MTSIC nanoparticles with their monomers and aggregates. Its absorption wavelength is the same as that of the amorphous aggregates and monomers, indicating that amorphous aggregation is maintained inside the nanoparticles.

[0117] Figure 17 It is the comparison test of the absorption spectra of photosensitizer MTBSIC nanoparticles with their monomers and aggregates. The absorption of the nanoparticles of H-aggregated photosensitizer MTBSIC is similar to that of its H-aggregates, and there is a certain blue shift compared with the monomers, indicating that H-aggregation is maintained inside the nanoparticles.

[0118] Experimental Example 7

[0119] Single crystals of the obtained MTBSIC molecules were grown, and the stacking structure was tested using an X-ray single crystal diffractometer. Figure 18 It is the single crystal structure of MTBSIC, and its stacking form shows that it is a typical H-aggregate with reverse dislocation parallel stacking.

[0120] Example 8

[0121] According to the photosensitizers TSIC, TBSIC, MTSIC, and MTBSIC nanoparticles, they were divided into two groups for ROS tests. The first group is the photosensitizers that absorb white light: the generation of ROS by TSIC and H-aggregated photosensitizer TBSIC under white light irradiation. The second group is the photosensitizers that absorb near-infrared light: the generation of ROS by MTSIC and H-aggregated photosensitizer MTBSIC under 660 nm laser irradiation.

[0122] (1) Total ROS generation test:

[0123] First, 2′,7′-dichlorodihydrofluorescein (DCFH) was used as an indicator to detect the total ROS generation ability. A 10 mM DCFH-DA stock solution was prepared with absolute ethanol. Take 0.1 mL of the stock solution, add 0.9 mL of absolute ethanol and 4 mL of 0.01 M NaOH solution, and then add 15 mL of PBS to obtain a DCFH solution with a final concentration of 50 μM, which was used in the dark at room temperature. In a quartz cuvette, add 200 μL of the photosensitizer stock solution (10 μM photosensitizer stock solution), 200 μL of the DCFH solution, and 1600 μL of the PBS solution to prepare a final solution of 2 mL, where the final concentration of the photosensitizer solution is 1 μM and the final concentration of DCFH is 5 μM. Irradiate with white light or 660 nm laser light, the power of the white light is 50 mW / cm 2 , and the power of the laser is 0.5 W / cm 2 . The total irradiation time is 90 s, and the fluorescence intensity is measured every 10 s. The change in fluorescence intensity was monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 488 nm, the excitation and emission slits were set to 1 nm, and the luminescence intensity at 525 nm was recorded. The same test method was also applicable to the test of the control photosensitizer Ce6.

[0124] (2) Superoxide anion generation test

[0125] Dihydrorhodamine 123 (DHR123) was used as an indicator to detect the generation ability of superoxide anion (O2 ·- ). A 10 mM DHR123 stock solution was prepared with DMF, and a DHR123 solution with a final concentration of 50 μM was obtained using ultrapure water. In a quartz cuvette, add 200 μL of the photosensitizer stock solution (10 μM photosensitizer stock solution), 200 μL of the DHR123 solution, and 1600 μL of the PBS solution to prepare a photosensitizer solution with a final concentration of 1 μM and a final concentration of DHR123 of 5 μM. Irradiate with white light or 660 nm laser light, the power of the white light is 50 mW / cm 2 , and the power of the laser is 0.5 W / cm 2 . The total irradiation time is 90 s, and the fluorescence intensity is measured every 10 s. The change in fluorescence intensity was monitored using a Horiba Fluoromax-4 fluorescence spectrometer. Excitation was performed at 488 nm, the luminescence intensity at 525 nm was recorded, and the excitation and emission slits were set to 1 nm. The same test method was also applicable to the test of the control photosensitizer Ce6.

[0126] (3) Hydroxyl radical generation test

[0127] Using hydroxyphenyl fluorescein (HPF) as an indicator to detect the generation ability of hydroxyl radicals (OH·). Prepare a 5 mM HPF stock solution with DMF, and use ultrapure water to prepare an HPF solution with a final concentration of 50 μM. Add 200 μL of the photosensitizer stock solution (10 μM photosensitizer stock solution), 200 μL of the HPF solution, and 1600 μL of the PBS solution to a quartz cuvette to configure a photosensitizer solution with a final concentration of 1 μM and an HPF final concentration of 5 μM. Irradiate with white light or 660 nm laser light. The power of the white light is 50 mW / cm 2 , and the power of the laser is 0.5 W / cm 2 . The total irradiation time is 90 s, and the fluorescence intensity is measured every 10 s. Use a Horiba Fluoromax-4 fluorescence spectrometer to monitor the change in fluorescence intensity. Excite at 480 nm, record the emission intensity at 515 nm, and set the excitation and emission slits to 1 nm. The same test method is also applicable to the test of the control photosensitizer Ce6.

[0128] (4) Singlet oxygen generation test:

[0129] Using 9,10-anthracenediylbis(methylene)-dimalonic acid (ABDA) as an indicator to detect the generation ability of singlet oxygen ( 1 O2). Prepare a 10 mM ABDA stock solution with DMF, and use ultrapure water to prepare an ABDA solution with a final concentration of 50 μM. Add 500 μL of the photosensitizer stock solution (50 μM photosensitizer stock solution) and 10 μL of ABDA to a quartz cuvette to configure a photosensitizer solution with a final concentration of 10 μM. Irradiate with white light or 660 nm laser light. The power of the white light is 50 mW / cm 2 , and the power of the laser is 0.5 W / cm 2 . The total irradiation time is 5 minutes, and the interval time is 1 minute. Use a UV-2600 ultraviolet spectrophotometer from Shimadzu, Japan, to monitor the change in the ABDA absorption spectrum and plot a graph at the maximum absorption wavelength of 420 nm. The absorption background of the photosensitizer itself is also deducted during the test.

[0130] Figure 19 It is a multiple graph of the fluorescence intensity change of the DCFH solution after white light irradiation of the white light photosensitizer TSIC, the H-aggregated white light photosensitizer TBSIC, and the commercial photosensitizer Ce6 at different times. The H-aggregated white light photosensitizer TBSIC has a stronger total ROS generation ability than the white light photosensitizer TSIC under white light irradiation.

[0131] Figure 20It is a multiple graph of the fluorescence intensity change of the DHR123 solution of the white light photosensitizer TSIC, the H-aggregated white light photosensitizer TBSIC, and the commercial photosensitizer Ce6 after white light irradiation at different times. The H-aggregated white light photosensitizer TBSIC has a stronger ability to generate superoxide anions than the white light photosensitizer TSIC under white light irradiation.

[0132] Figure 21 It is a multiple graph of the fluorescence intensity change of the HPF solution of the white light photosensitizer TSIC, the H-aggregated white light photosensitizer TBSIC, and the commercial photosensitizer Ce6 after white light irradiation at different times. The H-aggregated white light photosensitizer TBSIC has a stronger ability to generate hydroxyl radicals than the white light photosensitizer TSIC under white light irradiation.

[0133] Figure 22 It is a multiple graph of the absorption intensity change of the ABDA solution of the white light photosensitizer TSIC, the H-aggregated white light photosensitizer TBSIC, and the commercial photosensitizer Ce6 after white light irradiation at different times. The H-aggregated white light photosensitizer TBSIC has a slightly weaker ability to generate singlet oxygen than the white light photosensitizer TSIC under white light irradiation.

[0134] Figure 23 It is a multiple graph of the fluorescence intensity change of the DCFH solution of the near-infrared photosensitizer MTSIC, the H-aggregated near-infrared photosensitizer MTBSIC, and the commercial photosensitizer Ce6 after 660nm laser irradiation at different times. The H-aggregated near-infrared photosensitizer MTBSIC has a stronger ability to generate total ROS than the near-infrared photosensitizer MTSI under 660nm laser irradiation.

[0135] Figure 24 It is a multiple graph of the fluorescence intensity change of the DHR123 solution of the near-infrared photosensitizer MTSIC, the H-aggregated near-infrared photosensitizer MTBSIC, and the commercial photosensitizer Ce6 after 660nm laser irradiation at different times. The H-aggregated near-infrared photosensitizer MTBSIC has a stronger ability to generate superoxide anions than the near-infrared photosensitizer MTSI under 660nm laser irradiation.

[0136] Figure 25 It is a multiple graph of the fluorescence intensity change of the HPF solution of the near-infrared photosensitizer MTSIC, the H-aggregated near-infrared photosensitizer MTBSIC, and the commercial photosensitizer Ce6 after 660nm laser irradiation at different times. The H-aggregated near-infrared photosensitizer MTBSIC has a stronger ability to generate hydroxyl radicals than the near-infrared photosensitizer MTSI under 660nm laser irradiation.

[0137] Figure 26Fold change graphs of the fluorescence intensity of the HPF solutions of the near-infrared photosensitizer MTSIC, the H-aggregated near-infrared photosensitizer MTBSIC, and the commercial photosensitizer Ce6 after being irradiated with 660 nm laser at different times. The H-aggregated near-infrared photosensitizer MTBSIC has a slightly weaker singlet oxygen generation ability than the near-infrared photosensitizer MTSI under 660 nm laser irradiation.

[0138] Based on the ROS test results in this section, H-aggregated photosensitizers exhibit better total ROS generation ability and type I ROS generation ability than amorphous aggregated photosensitizers. Figure 27 A schematic diagram showing the enhancement of type I ROS by this H-aggregated molecule is presented.

[0139] Example 9

[0140] The near-infrared absorbing photosensitizer MTSIC and the H-aggregated near-infrared absorbing photosensitizer MTBSIC were selected for subsequent cytotoxicity experiments to verify their photodynamic therapy effects.

[0141] Cytotoxicity experiments were carried out using 4T1 cells as a cancer cell model. The 4T1 cells were seeded in 96-well plates at a density of 2.5×10 3 cells per well. After culturing for 24 hours, the culture media containing 0, 1, 2, 5, 10, 20 μM of MTSIC and MTBSIC were added to the 96-well plates containing 4T1 cells. Subsequently, in the phototoxicity group, after co-incubating the 4T1 cells with MTSIC and MTBSIC for 12 h, the old culture media were aspirated, and the 4T1 cells were washed 3 times with PBS. Then, the cells were irradiated with a 660 nm laser (0.2 W / cm 2 ) for 10 min and then cultured for another 12 h. Subsequently, the old culture media were aspirated, the 4T1 cells were washed 3 times with PBS, the culture media containing 0.5 mg / mL MTT were added, and the cells were cultured in the dark for 3 h. After carefully aspirating the culture media, 100 μL of DMSO was added to each well. Finally, the 96-well plates were shaken for 5 min using a multi-functional microplate reader, and the absorbance values of each well at 570 nm were measured.

[0142] In the dark toxicity group, after adding the culture media containing 0, 1, 2, 5, 10, 20 μM of MTSIC and MTBSIC to the 96-well plates containing 4T1 cells and co-incubating for 24 h, the 4T1 cells were washed 3 times with PBS, the culture media containing 0.5 mg / mL MTT were added, and the cells were cultured in the dark for 3 h. After carefully aspirating the culture media, 100 μL of DMSO was added to each well. Finally, the 96-well plates were shaken for 5 min using a multi-functional microplate reader, and the absorbance values of each well at 570 nm were measured.

[0143] Figure 28Dark toxicity killing of 4T1 cancer cells by near-infrared absorbing photosensitizer MTSIC and H-aggregated near-infrared absorbing photosensitizer MTBSIC at different concentrations. Apparently, neither the near-infrared absorbing photosensitizer MTSIC nor the H-aggregated near-infrared absorbing photosensitizer MTBSIC showed dark toxicity.

[0144] Figure 29 Phototoxicity killing of 4T1 cancer cells by near-infrared absorbing photosensitizer MTSIC and H-aggregated near-infrared absorbing photosensitizer MTBSIC at different concentrations. Apparently, the H-aggregated near-infrared absorbing photosensitizer MTBSIC achieved more significant killing of 4T1 cancer cells compared to the near-infrared absorbing photosensitizer MTSIC.

[0145] The embodiments described above are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. An H-aggregated type I photosensitizer, characterized in that, The structural formula is as follows: Wherein, each R is independently a C1-C30 alkyl group, a phenyl group, a C1-C30 alkylphenyl group, or a C1-C30 alkoxyphenyl group.

2. The H-aggregated type I photosensitizer according to claim 1, wherein The structural formula is as follows: Wherein, each n is independently an integer from 0 to 29, and each m is independently an integer from 0 to 30.

3. The preparation method of the type I photosensitizer of H-aggregation type according to any one of claims 1 to 2, characterized in that, It includes the following steps: Reacting aminophenylboronic acid NBOH with 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO to obtain an intermediate NBSCHO, and reacting NBSCHO with 3-(dicyanomethylene)indanone IC to obtain an H-aggregated type I photosensitizer NBSIC; Wherein, each R is independently a C1-C30 alkyl group, a phenyl group, a C1-C30 alkylphenyl group, or a C1-C30 alkoxyphenyl group.

4. The preparation method according to claim 3, wherein It includes the following steps: (1) Add aminophenylboronic acid NBOH, 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO, potassium carbonate, and tetrakis(triphenylphosphine)palladium into a mixed solvent of THF and water under a protective atmosphere, reflux and react, and purify to obtain the intermediate NBSCHO; (2) Add the intermediate NBSCHO and 3-(dicyanomethylene)indanone IC into a solvent and a catalyst, reflux and react to obtain an H-aggregated type I photosensitizer NBSIC.

5. The preparation method according to claim 4, characterized in that In step (1), the molar ratio of aminophenylboronic acid NBOH, 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO, potassium carbonate, and tetrakis(triphenylphosphine)palladium is 1.1-1.5:1:2-10:0.01-0.05: In step (1), the volume ratio of THF to water in the mixed solvent is 5-3:1; In step (1), the molar volume ratio of 5-bromothieno[3,2-b]thiophene-2-carbaldehyde Br-BS-CHO to the mixed solvent is 1 mmol:1-15 mL.

6. The preparation method according to claim 4, characterized in that In step (1), the reflux reaction temperature is 60-80 °C and the time is 8-24 h; In step (1), the protective atmosphere is nitrogen.

7. The preparation method according to claim 4, characterized in that In step (2), the solvent is one or more of dichloromethane, chloroform, ethanol, and acetic anhydride; In step (2), the molar volume ratio of the intermediate NBSCHO to the solvent is 1 mmol:1-10 mL.

8. The preparation method according to claim 4, characterized in that In step (2), the catalyst is one or more of triethylamine, pyridine, and piperidine; In step (2), the molar ratio of the catalyst to the intermediate NBSCHO is 0-0.05:

1.

9. The preparation method according to claim 4, characterized in that In step (2), the molar ratio of the intermediate NBSCHO to 3-(dicyanomethylene)indanone IC is 1:1.0-1.4; In step (2), the reflux reaction temperature is 30-120 °C and the time is 2-24 h.

10. Use of the H-aggregated type I photosensitizer according to any one of claims 1-2 in the preparation of a cancer cell photodynamic therapy reagent.

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