Conjugated polymers containing cyanoindanone and selenophene fragments, their preparation methods and applications

Abstract

This invention discloses a conjugated polymer containing cyanoindanone and selenophene fragments, its preparation method, and its application, relating to the field of photosensitizer technology. This invention synthesizes a novel conjugated polymer containing cyanoindanone and selenophene fragments as a photosensitizer and systematically studies its performance in ROS generation and photothermal properties. The results show that it has good penetration, targeting, and low dark toxicity, and can realize PDT / PTT combined therapy.

Description

Technical Field

[0001] This invention relates to the field of photosensitizer technology, specifically to a conjugated polymer containing cyanoindanone and selenophene fragments, its preparation method, and its application. Background Technology

[0002] Reactive oxygen species (ROS) naturally present in living organisms perform defense functions. When intracellular ROS levels exceed normal ranges, cells trigger mechanisms such as apoptosis, necrosis, and autophagy, leading to cell death. Photodynamic therapy (PDT) is based on the interaction of photosensitizers (PS) with substrates under specific wavelengths of light to produce H₂O₂, ·OH, and O₂. - ·、 1 O2 and other ROS components selectively damage target tissues. There are two pathways for PS to generate ROS: Type I ROS generated by electron transfer and Type II ROS generated by energy transfer. Type I ROS occurs when triplet PS reacts directly with intracellular substances through hydrogen or electron transfer, producing reactive free radicals; Type II ROS occurs when the triplet state of PS transitions from its ground state, and the energy excited during the triplet state is transferred to O2, producing highly cytotoxic singlet oxygen (O2). 1 Type II PDS is O2-dependent and limited in the hypoxic tumor microenvironment (TME). PDT utilizes the potential of ROS for treatment, directing the harmful effects of ROS to cancer cells, thereby minimizing damage to surrounding healthy tissue. PDT is widely chosen due to its selectivity. Similar to type I PDT, photothermal therapy (PTT) uses the photothermal effect of photothermal agents (PTA) to convert absorbed light energy into heat, causing thermal burns to the tumor, and is also oxygen-independent. Furthermore, nanomaterial-based photothermal therapy can be activated by near-infrared (NIR) irradiation, penetrating deep into tissues, making it a non-invasive and highly effective treatment technique.

[0003] PS, as one of the core elements of PDT, still has some shortcomings: (1) Porphyrin compounds, as traditional organic PS, have problems such as unstable properties and inability to accurately locate, which greatly reduces the therapeutic effect; (2) Metal complex photosensitizers combine metal ions and organic ligands, have good photostability, and can continuously induce photodynamic therapy. However, the introduction of metal ions will enhance dark toxicity; (3) Small molecule photosensitizers have limited penetration ability due to their relatively short excitation wavelength, and cannot be applied to the treatment of deep tumors.

[0004] Currently, small-molecule photosensitizers are widely studied due to their well-defined composition, structural diversity, and tunable photophysical properties. However, the ROS generation efficiency of small-molecule PS still needs improvement. Recent studies have found that conjugated polymers have higher ROS generation efficiency compared to small-molecule photosensitizers. Conjugated polymers have attracted widespread attention due to their excellent light absorption and efficient energy transfer capabilities. Currently, conjugated polyelectrolytes and hydrophobic conjugated polymer nanoparticles are used in optical imaging, photothermal therapy, and biosensing. Because conjugated polymers can sensitize oxygen molecules to generate ROS under light irradiation, they can also be used for antibacterial and anticancer applications. Furthermore, their larger delocalized electronic structure allows excitons to migrate along the polymer backbone or hop between chains, promoting efficient energy transfer to PS. Therefore, conjugated polymer PS has great therapeutic potential in cancer treatment. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a conjugated polymer containing cyanoindanone and selenophene fragments and a method for preparing the same. The obtained conjugated polymer is expected to be applied to PDT / PTT combination therapy.

[0006] The technical problem to be solved by this invention is achieved by the following technical solution: One objective of this invention is to provide a conjugated polymer, abbreviated as PYT3, with the following structural formula:

[0007] Where n represents the degree of aggregation, which ranges from 2 to 100.

[0008] A second objective of this invention is to provide a method for preparing a conjugated polymer, comprising the following steps: S1, Compound 1, reacts with 2-(6-bromo-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malonitrile to give Compound 2; S2 and compound 2 undergo a coupling reaction with 2,5-bis(trimethyltin)selenophene to obtain a conjugated polymer.

[0009] The synthesis route is as follows:

[0010] Furthermore, the catalyst for the condensation reaction includes, but is not limited to, at least one of pyridine, piperidine, and boron trifluoride ether.

[0011] Furthermore, the molar ratio of compound 1 to 2-(6-bromo-3-oxo-2,3-dihydro-1H-indene-1-yl)malononitrile is 1:(2~6).

[0012] Furthermore, the coupling reaction is carried out in the presence of a palladium catalyst. Even further, the palladium catalyst includes, but is not limited to, at least one of tetrakis(triphenylphosphine)palladium, palladium dichloride (bis(triphenylphosphine)), and bis(dibenzylacetone)palladium.

[0013] Furthermore, the molar ratio of compound 2 to 2,5-bistrimethyltinylselenophene is 1:(1~1.5).

[0014] A third objective of this invention is to provide the application of the conjugated polymer in photosensitizers.

[0015] The fourth objective of this invention is to provide the application of the conjugated polymer in the preparation of PDT / PTT combination therapy drugs for tumor treatment.

[0016] The object of the present invention is to provide the use of the conjugated polymer in the preparation of antibacterial agents.

[0017] The reasons for introducing the cyanoindanone fragment into the conjugated polymer in this invention are as follows: (1) The strong electron-withdrawing ability of cyanoindanone can achieve near-infrared absorption; (2) Cyanoindanone has been shown to have a tendency to tail-tail stacking, and studies have shown that tail-tail stacking helps ROS generation; (3) The strong electron-withdrawing ability of cyanoindanone promotes intramolecular charge transfer, which is beneficial to the generation of type I ROS.

[0018] The reasons for introducing selenophene fragments into the conjugated polymer in this invention are as follows: Introducing selenophene fragments effectively reduces the band gap. Selenium atoms have low electronegativity (2.55) and stronger electron-donating ability, thereby extending the effective conjugation length and reducing the optical band gap. The larger and more easily polarized Se atoms, along with strong intermolecular Se-Se interactions, facilitate interchain charge transfer, thereby enhancing hole mobility and increasing the generation of type I reactive oxygen species.

[0019] The beneficial effects of this invention are as follows: This invention synthesizes a novel conjugated polymer containing cyanoindanone and selenophene fragments as a photosensitizer, and systematically studies its performance in ROS generation and photothermal properties. The results show that it has good penetration, targeting and low dark toxicity, and can realize PDT / PTT combined therapy. Attached Figure Description

[0020] Figure 1 The hydrogen NMR spectrum of PYT3; Figure 2 The UV-Vis absorption spectrum of PYT3 in aqueous phase; Figure 3 The fluorescence emission spectrum of PYT3 in aqueous phase; Figure 4 This is a scanning electron microscope (SEM) image of PYT3 in an aqueous phase. Figure 5 This is a particle size distribution diagram of PYT3 in the aqueous phase; Figure 6 A mixed solution of PYT3 and DCFH-DA was tested at a wavelength of 800 nm and a power of 1.0 W / cm². 2 The fluorescence emission spectrum (a) and fluorescence intensity (F-F0) curves (b) under laser irradiation are shown in Figure 1. Figure 7 A mixed solution of PYT3 and DMPO was tested at a wavelength of 800 nm and a power of 1.0 W / cm². 2 Electron paramagnetic resonance (EPR) spectrum under laser irradiation; Figure 8 For wavelength 800 nm, power 1.0 W / cm 2 A bar chart (a) shows the temperature-time variations of PYT3 aqueous solutions at different concentrations (100, 150, and 200 µg / mL) under laser irradiation; and the bar chart (a) shows the temperature-time variations of PYT3 aqueous solutions at different concentrations (0.8, 1.0 W / cm²) at a wavelength of 800 nm. 2 Temperature-time curve of PYT3 aqueous solution (100 µg / mL) under laser irradiation (b); Figure 9 To use a wavelength of 800 nm and a power of 1.0 W / cm 2 The temperature-time curve of PYT3 aqueous solution (150 µg / mL) after laser irradiation for 10 min followed by cooling for 13 min is shown in (a). The laser was used at a wavelength of 800 nm and a power of 1.0 W / cm². 2 The laser was subjected to three irradiation / cooling cycles (irradiation for 10 min followed by cooling for 13 min), and the temperature-time curve of the PYT3 aqueous solution with a concentration of 150 µg / mL was obtained (b). Figure 10 For wavelength 800 nm, power 1.0 W / cm 2 Infrared thermal images of PYT3 aqueous solution (150 µg / mL) under laser irradiation as irradiation time change; Figure 11 This is a staining image of live / dead cells in HepG2 cells after treatment with PYT3 aqueous solution (150 µg / mL). Detailed Implementation

[0021] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments and illustrations.

[0022] Example 1

[0023] Synthesis of PYT3: Under nitrogen protection, compound 1 (0.2 g, 0.15 mmol), 2-(6-bromo-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile (0.25 g, 0.91 mmol), pyridine (1 mL), and chloroform (45 mL) were added to a reaction flask, and the mixture was stirred at 65 °C for 12 h. After stirring was stopped, the reaction solution was cooled to room temperature and poured into methanol. The mixture was filtered, and the filter cake was subjected to silica gel column chromatography with dichloromethane and petroleum ether in a volume ratio of 1:2 as eluent to give compound 2 in 72% yield.

[0024] S2. Under argon protection, compound 2 (87.5 mg, 0.05 mmol), 2,5-bis(trimethyltinylselenophene) (23 mg, 0.05 mmol), and toluene (5 mL) were added to the reaction flask, followed by tetra(triphenylphosphine)palladium (3 mg). The reaction mixture was stirred at 120 °C for 12 h. After stirring was stopped, the reaction solution was extracted successively with methanol, n-hexane, and chloroform. The organic phase was collected, concentrated under reduced pressure to remove the solvent, and dried to obtain PYT3 in 93% yield.

[0025] Example 2

[0026] Structural characterization of PYT3: PYT3 was dissolved in deuterated chloroform, and the proton NMR spectrum was measured using a nuclear magnetic resonance spectrometer (400 MHz). Figure 1 As shown. By Figure 1 It can be seen that the present invention successfully synthesized the compound PYT3.

[0027] The UV-Vis absorption spectra of PYT3 aqueous solution (150 μg / mL) were determined using a UV-Vis spectrophotometer, a fluorescence spectrometer, a scanning electron microscope, and a particle size analyzer, respectively. Figure 2 ), fluorescence emission spectrum ( Figure 3 ), micromorphology ( Figure 4 ) and particle size distribution ( Figure 5 ).

[0028] Depend on Figure 2 It can be seen that the PYT3 aqueous solution has a broad absorption peak between wavelengths of 732 and 900 nm, with the maximum absorption at 790 nm.

[0029] Depend on Figure 3 It is known that PYT3 aqueous solution exhibits a broad emission peak between 790 and 900 nm under 790 nm excitation. The long-wavelength absorption of PYT3 enables it to penetrate deeper into tissues, and in bioimaging, long-wavelength excitation can reduce tissue autofluorescence, improve the signal-to-noise ratio, and enhance imaging contrast.

[0030] Depend on Figure 4It can be seen that PYT3 exists in the aqueous phase as a spherical dispersion.

[0031] Depend on Figure 5 It is known that PYT3 has an average particle size of 180.6 nm and a small polymer dispersibility index (PDI). The smaller size is more conducive to cell uptake.

[0032] Example 3

[0033] Determination of ROS generation capacity of PYT3: 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) is a typical ROS probe that can be oxidized to the fluorescent substance 2-(2,7-dichloro-9,9a-dihydro-6-hydroxy-8,3-oxo-3H-xanthan-9-yl)benzoic acid (DCF) in the presence of ROS.

[0034] To determine the photodynamic effect of PYT3, this invention uses DCFH-DA as a probe to test the ROS generation capacity of PYT3. The specific steps include: (1) under dark conditions, weigh 0.0024 g of DCFH-DA and dissolve it in a centrifuge tube containing 2.5 mL of dimethyl sulfoxide (DMSO) to prepare a DCFH-DA solution with a concentration of 2 mmol / L; (2) weigh 0.04 g of sodium hydroxide (NaOH) and dissolve it in 100 mL of deionized water to prepare a NaOH solution with a concentration of 0.01 mol / L; (3) weigh 0.1 mmol of PYT3 and disperse it in 100 mL of deionized water to prepare a PYT3 solution with a concentration of 1 mmol / L; (4) under dark conditions, add 800 μL of NaOH solution to 100 μL of DCFH-DA solution, activate for 30 min, add 4.1 mL of deionized water, and then add 10 μL of PYT3 solution. The sample was irradiated once with a laser at a wavelength of 800 nm and a power of 1.0 W / cm², for 30 seconds each time, for a total of 4 irradiations. The fluorescence emission spectrum was measured immediately after each irradiation. Figure 6 a), and plot the fluorescence intensity (F-F0) versus time curve ( Figure 6 b), where F is the real-time fluorescence intensity and F0 is the initial fluorescence intensity.

[0035] Depend on Figure 6 It can be seen that with the increase of irradiation times, the fluorescence peak at 532 nm under 488 nm excitation corresponds to the fluorescence intensity of DCFH-DA oxidized by ROS. The increase in fluorescence intensity here indicates that the ROS content increases with increasing light exposure. This proves that PYT3 generates ROS under specific wavelength excitation.

[0036] Example 4

[0037] Determination of the ·OH generating ability of PYT3: Using an electron paramagnetic resonance (EPR) spectrometer, with 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as a spin electron trap, the ability of PYT3 to generate ·OH was evaluated. The specific steps included: (1) measuring a PYT3 aqueous solution (1 mg / mL) and DMPO in a volume ratio of 10:1 and adding them to a 2.5 mL centrifuge tube wrapped in tin foil, ensuring that the resulting mixed solution was completely protected from light; (2) placing the mixed solution into the EPR spectrometer under dark conditions and starting the instrument to obtain an EPR spectrum; (3) taking another mixed solution, irradiating it under an 808 nm wavelength laser for 30 s, and then placing it into the EPR spectrometer, starting the instrument to obtain an EPR spectrum, and detecting whether free radicals were generated.

[0038] Depend on Figure 7 It can be seen that, compared with the PYT3 aqueous solution and DMPO mixture under dark conditions, the EPR spectrum of the mixture under light conditions showed an obvious 1:2:2:1 peak shape, which can be identified as the peak shape of ·OH, proving that ·OH was generated in the PYT3 aqueous solution under light conditions.

[0039] Example 5

[0040] Photothermal performance measurement of PYT3: PYT3 aqueous solutions (100 µg / mL, 150 µg / mL, and 200 µg / mL) were irradiated with a laser at a wavelength of 800 nm and a power of 1.0 W / cm², and the irradiation time and corresponding solution temperature were recorded. Figure 8 a) By Figure 8 As can be seen from a, with the increase of illumination time, the photothermal activity of PYT3 aqueous solutions of all three concentrations increases, and the photothermal activity level is positively correlated with the concentration, which proves the generation of photothermal activity.

[0041] PYT3 aqueous solution (100 µg / mL) was irradiated with lasers of 800 nm wavelength and different powers (0.8, 1.0 W / cm2). The irradiation time and the corresponding solution temperature were recorded, and temperature-time curves were plotted. Figure 8 b). By Figure 8 b shows that the photothermal effect increases with the increase of illumination time, proving the photothermal effect of PYT3 aqueous solution.

[0042] A PYT3 aqueous solution (150 µg / mL) was irradiated with a laser at a wavelength of 800 nm and a power of 1.0 W / cm². The irradiation time and the corresponding solution temperature were recorded, and a temperature-time curve was plotted. Figure 9a); and perform three irradiation / cooling cycles (irradiation for 10 min followed by cooling for 13 min), record the laser irradiation time and the corresponding solution temperature, and plot the temperature-time curve. Figure 9 b).

[0043] Depend on Figure 9 As can be seen from a, during the first 600 seconds of illumination, the temperature of the PYT3 aqueous solution gradually increased, reaching a peak of 48℃ at 600 seconds. Then, when the illumination stopped, the solution gradually returned to its original temperature.

[0044] Depend on Figure 9 As shown in b, after three irradiation / cooling cycles, the PYT3 aqueous solution underwent a heating-cooling cycle. The maximum heating and heating-cooling trends did not change, and the photothermal performance did not change, proving the stability of its photothermal effect.

[0045] Infrared thermal images were captured by irradiating a PYT3 aqueous solution (150 µg / mL) with a laser at a wavelength of 800 nm and a power of 1.0 W / cm². Figure 10 ).

[0046] Depend on Figure 10 It can be seen that under the infrared camera, the PYT3 aqueous solution reached 50℃ after 2 minutes of illumination, and the photothermal effect was still very obvious after 10 minutes, indicating that the photothermal effect of PYT3 continued to be generated.

[0047] Example 6

[0048] PYT3's in vitro therapeutic capabilities G2 cells were incubated with PYT3 aqueous solution (150 µg / mL) for 15 min. Untreated cells were washed away with PBS. Then, PI / AM staining reagent was added, and the cell viability was observed using a confocal scanning microscope. The staining principle is as follows: the esterase activity of live cells converts non-fluorescent Calcein-AM into green fluorescent Calcein, which remains intracellularly. Simultaneously, the membrane rupture of dead cells allows the red fluorescent dye PI to enter and bind to nucleic acids, thus achieving dual-color, synchronous, and intuitive identification of live and dead cells. Under a confocal microscope, green represents live cells and red represents dead cells in the PI / AM image. Depend on Figure 11 It was found that the cells were all alive before light exposure, but all cells died after 8 minutes of light exposure. This demonstrates the phototoxicity of PYT3.

[0049] In summary, the PYT3 of this invention uses non-fullerene as the acceptor and indanone as the donor, and introduces a selenophene-tin salt with electron transport capabilities, which has the ability to generate type I ROS and has a very stable photothermal effect, and has the potential for PDI / PTT. The biotherapeutic ability of PYT3 has been confirmed by cell experiments.

[0050] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A conjugated polymer, abbreviated as PYT3, has the following structural formula: ； in, n represents the degree of aggregation, ranging from 2 to 100.

2. The method for preparing the conjugated polymer according to claim 1, characterized in that, Includes the following steps: S1, Compound 1, reacts with 2-(6-bromo-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malonitrile to give Compound 2; S2 and compound 2 undergo a coupling reaction with 2,5-bistrimethyltinylselenophene to obtain a conjugated polymer; The synthesis route is as follows: 。 3. The preparation method according to claim 2, characterized in that: The catalyst for the condensation reaction is selected from at least one of pyridine, piperidine, and boron trifluoride ether.

4. The preparation method according to claim 2, characterized in that: The molar ratio of compound 1 to 2-(6-bromo-3-oxo-2,3-dihydro-1H-indene-1-yl)malononitrile is 1:(2~6).

5. The preparation method according to claim 2, characterized in that: The coupling reaction is carried out in the presence of a palladium catalyst.

6. The preparation method according to claim 5, characterized in that: The palladium catalyst is selected from at least one of tetra(triphenylphosphine)palladium, palladium dichloride (bis(triphenylphosphine)), and bis(dibenzylideneacetone)palladium.

7. The preparation method according to claim 2, characterized in that: The molar ratio of compound 2 to 2,5-bistrimethyltinylselenophene is 1:(1~1.5).

8. The use of the conjugated polymer of claim 1 or the conjugated polymer prepared by any one of claims 2 to 7 in photosensitizers.

9. The use of the conjugated polymer of claim 1 or the conjugated polymer prepared by any one of claims 2 to 7 in the preparation of PDT / PTT combination therapy for tumors.

10. The use of the conjugated polymer of claim 1 in the preparation of antibacterial agents.

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