A coordination-driven supramolecular photosensitizer and its application in photodynamic therapy

Abstract

This study designed and synthesized a pyridine-functionalized tetraphenylethylene covalent molecular cage, creating an AIE supramolecular photosensitizer through coordination and host-guest synergy. The TPE unit enhances the photophysical properties of the photosensitizer, imparting AIE characteristics. The molecular cage and tetraphenylporphyrin, through host-guest binding, further inhibit porphyrin self-aggregation in water, promoting ROS generation and effectively killing cancer cells through photocatalytic NADH oxidation.

Description

Technical Field

[0001] The present invention specifically relates to the field of detection, in particular to an AIE supramolecular photosensitizer for generating reactive oxygen species to achieve efficient photodynamic anticancer performance. Background Art

[0002] Currently, cancer remains one of the major health problems worldwide. Traditional treatment techniques such as surgical resection, chemotherapy, and radiotherapy have a series of traumatic and side effects, which greatly limit their clinical application. Photodynamic therapy (PDT) is an emerging cancer treatment method. Due to its strong selectivity, targeted cancer cell targeting, and few side effects, it greatly enhances the efficacy. Photosensitizers, as an important component of PDT, kill cancer cells by generating reactive oxygen species (ROS) under light irradiation. Traditional porphyrin photosensitizers have excellent light absorption properties and good biocompatibility and are widely used in tumor targeted therapy. However, their aggregation-induced quenching (ACQ) effect in aqueous media leads to fluorescence quenching, which seriously affects their therapeutic effect in vivo. Therefore, there is an urgent need for aggregation-induced emission (AIE)-type photosensitizers that can inhibit the aggregation-induced quenching of porphyrins in water and achieve ROS enhancement.

[0003] Porphyrin derivatives in biological systems are core components of important biomacromolecules in photosystems. The coordination between the central metal of the metalloporphyrin and amino acid residues stabilizes the chlorophyll molecule, thereby promoting efficient photon capture, energy transfer, and electron transfer. Pyridine-functionalized molecular cages coordinate with the central metal of the porphyrin, further synergizing with the host-guest interaction within the molecular cage cavity to stabilize the porphyrin molecule. Coordination-driven host-guest assembly can form novel AIE biomimetic photosensitizer molecules, effectively inhibiting excited-state quenching caused by porphyrin self-aggregation, enhancing ROS generation, and improving PDT efficiency. Summary of the Invention

[0004] The present invention is carried out through two steps S N A monofunctionalized hexacationic molecular cage based on tetraphenylethylene (TPE) pyridyl groups was synthesized by reaction design to construct a coordination-driven host-guest assembly AIE biomimetic photosensitizer. Compound 5, which has only three benzyl bromide reactive sites, was reacted with compound 4, which contains four reactive pyridyl groups. Based on the principle of reactive site matching, the reaction product contains a free pyridyl group. Subsequently, the monofunctionalized pyridyl cage coordinated with the central metal of tetraphenylzinc porphyrin to form an AIE biomimetic supramolecular photosensitizer.

[0005] The present invention provides the following technical solutions:

[0006] The present invention provides a coordination-driven supramolecular photosensitizer. The photosensitizer is constructed through the synergistic effect of host and guest after the coordination of a molecular cage and zinc porphyrin. The photosensitizer is prepared by reacting a compound having only three benzyl bromide reaction sites with a compound containing four reactive pyridyl groups. The free pyridyl group of the obtained reaction product is coordinated with the central metal of tetraphenylzinc porphyrin. The structural formula is:

[0007]

[0008] During the preparation, tetrapyridyl tetraphenylethylene is firstly prepared by two steps of S N 2. Reaction synthesis of molecular cage 1. First, compound 4 reacted with p-dibenzyl bromide 5 at 90 ° C for 3 days, and 6·3PF6 was obtained after ion exchange. – ; Then, an equimolar amount of compound 6·3PF6 – The reaction was carried out with compound 7 in the presence of 0.2 equivalents of tetrabutylammonium iodide at 110 °C for 3 days, and pure 1·6PF6 was obtained by column chromatography. – ; Finally, 1·6PF6 – The complex 3·6PF6 was obtained by 1:1 coordination with tetraphenylzinc porphyrin 2 in acetonitrile. – , water-soluble 3·6Cl was obtained by adding excess tetrabutylammonium chloride for ion exchange. – .

[0009] This invention constructs a novel AIE supramolecular photosensitizer through the synergistic effect of a molecular cage and a zinc porphyrin coordinated host-guest. Compared to molecular cage 1 and zinc porphyrin 2 alone, complex 32, as a photosensitizer, has a superior ROS production capacity, reaching equilibrium after 20 seconds of illumination. Compared to the ROS generated by molecular cage 1 and zinc porphyrin 2 alone after 20 seconds of illumination, the ROS production capacity of complex 32 is 4.8 times and 6.6 times that of molecular cage 1 and zinc porphyrin 2 alone, respectively. Its ROS production capacity was tested in vitro and in vivo using a ROS indicator, and it was subsequently used in photodynamic therapy, achieving a good tumor killing effect.

[0010] Based on the above technical solution, the present invention has the following beneficial effects:

[0011] (1) This invention designs and synthesizes a pyridine-functionalized tetraphenylethylene covalent molecular cage, which is used to obtain an AIE supramolecular photosensitizer through coordination and host-guest synergy. The TPE unit enhances the photophysical properties of the photosensitizer, endowing it with AIE characteristics. The molecular cage and tetraphenylporphyrin, through host-guest binding, further inhibit the self-aggregation of porphyrin in water and promote the generation of ROS.

[0012] (2) The present invention constructs a novel supramolecular photosensitizer with efficient ROS generation capability and applies it to photodynamic therapy, effectively generating ROS and killing cancer cells through photocatalytic NADH oxidation assistance.

[0013] Compared with the existing technology, the present invention constructs a porphyrin-based biomimetic supramolecular photosensitizer through the strategy of coordination and host-guest collaboration, effectively improving its ROS generation ability, with good biocompatibility and safety, achieving efficient ROS generation both in vivo and in vitro, and can effectively kill cancer cells in vivo, improving the effect of photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The invention relates to the design and synthesis route of the complex 32 of the photosensitizer in Example 1.

[0015] Figure 2 1 and 2 and the complex 32 according to Example 2 of the present invention.

[0016] Figure 3 This is the mass spectrum of the complex 32 described in Example 3 of the present invention.

[0017] Figure 4 This is a monitoring diagram of the total amount of ROS generated by the molecular cages 1 and 2 and the complex 32 described in Example 4 of the present invention.

[0018] Figure 5 This is the NMR image of the photocatalytic oxidation of NADH by the complex 32 described in Example 5 of the present invention.

[0019] Figure 6 This is a diagram of the photodynamic therapy of the complex 32 described in Example 6 of the present invention.

[0020] Figure 7 is the structural formula of the complex 32 involving the photosensitizer in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0022] Example 1

[0023] See also Figure 1 and Figure 7 , Design and synthesis diagram of molecular cage 1 and complex 32:

[0024] Through two steps SN 2 Reaction synthesis of molecular cage 1, weigh 5 (533.6 mg, 2.02 mmol) into a two-necked flask filled with dry MeCN (50 mL), and heat under reflux at 90 ° C until all compounds are dissolved. Disperse 4 (100 mg, 0.168 mmol) in 5 mL of dry MeCN and add it to the solution of 5 in batches, and reflux at 90 ° C for three days. After the reaction, the mixture was cooled to room temperature, the precipitate was collected and washed with excess acetone (3×40 mL) and centrifuged to obtain a crude product as an orange solid. The crude product was dissolved in deionized water (25 mL) and excess NH4PF6 was added, followed by stirring at room temperature for 12 hours. The precipitate was collected and washed with excess H2O (3×20 mL) and then dried to obtain 6·3PF6 – Orange solid. – (100 mg, 63 μmol) and tetrabutylammonium iodide (14.67 mg, 13 μmol) were added to a pressure bottle containing dry MeCN (100 mL) and heated at 90°C. A suspension of 7 (46 mg, 76 μmol) in acetonitrile was added dropwise in batches and heated to 110°C for three days. The mixture was then cooled to room temperature, and the precipitate was collected and washed with excess MeCN (3 × 40 mL) to obtain a crude product as an orange solid. The crude product was dissolved in water (25 mL) and excess NH4PF6 was added and stirred for 12 hours. The precipitate was collected, washed with excess H2O (3 × 20 mL) and dried to obtain a crude product as an orange solid. The crude product was purified by silica gel chromatography using CH2Cl2:MeCN (saturated NH4PF6) = 3:1 (v:v) as the mobile phase to obtain pure 1·6PF6. – (20 mg, yield: 13.5%).

[0025] Subsequently, pure 1·6PF6 – (20 mg, 8.3 μmol) was dissolved in MeCN, and zinc tetraphenylporphyrin (2) (5.7 mg, 8.4 μmol) was added. After stirring at room temperature for 12 h, the zinc porphyrin was completely dissolved. The MeCN was removed by drying to obtain a black solid 3·6PF6 – (19.6 mg, yield 95.2%). – To a solution of 20 mg, 6.5 μmol of tetrabutylammonium chloride (20 mg, 6.5 μmol) in MeCN (3 mL) was added an excess of tetrabutylammonium chloride (19.4 mg, 70 μmol) and stirred for 12 h. The precipitate was collected and washed with excess MeCN (3 × 5 mL) to obtain 3·6Cl - The product was obtained as a black solid (13 mg, yield: 81.2%).

[0026] Example 2

[0027] See also Figure 2 , NMR images of molecular cages 1 and 2 and complex 32:

[0028] Weigh compounds 1, 2 and 3·6PF6 respectively – 、3·6Cl - Dissolved in 450 μL CD3CN, 3·6Cl - Dissolve in 450 μL of D2O, sonicate, and prepare 0.5 mM solutions in NMR tubes. Analyze hydrogen spectra using NMR.

[0029] Example 3

[0030] See also Figure 3 , mass spectrum of complex 32:

[0031] Weigh compound 3·6PF6 – Dissolve in CH3CN for HPLC to prepare a 0.5 mM solution, and test the mass spectrum by mass spectrometer.

[0032] Example 4

[0033] See also Figure 4 , Monitoring diagram of the total amount of ROS generated by molecular cages 1 and 2 and complex 32:

[0034] The compound 2,7-dichlorodihydrofluorescein (DCFH) was used as an indicator to detect total reactive oxygen species (ROS). First, a 1 mM solution of DCFH in ethanol was prepared and stored at -20°C. When using a stock solution of DCFH in ethanol, activation is required. Add 2 mL of a 0.01 M NaOH solution to the above-mentioned 1.0 mM DCFH ethanol solution (0.5 mL), mix thoroughly, and incubate in the dark for 30 minutes. Simultaneously, add 10 mL of PBS to a centrifuge tube and pre-chill at 4°C. After the DCFH solution has been protected from light for 30 minutes, add it to the pre-chilled PBS solution, mix thoroughly, and store in the dark at 4°C until ready to use. The activated DCFH concentration is 40 μM. A 2.0 mM stock solution was prepared using ultrapure water and added to a 40 μM DCFH solution. The test solution was diluted with PBS to a final photosensitizer concentration of 2.0 μM. A DCFH-only group served as a control. The above sample solution was added to the fluorescence cuvette and the light power density was 20mW / cm 2 The generation of ROS was measured under white light irradiation.

[0035] Example 5

[0036] See also Figure 5 , NMR images of complex 32 for photocatalytic oxidation of NADH:

[0037] A 2mM deuterium aqueous solution of NADH was prepared in a nuclear magnetic test tube, and 5 mol% of 32 was added as a photocatalyst. 2 The nuclear magnetic resonance changes of NADH oxidation process were monitored under white light irradiation.

[0038] Example 6

[0039] See also Figure 6 , Photodynamic therapy diagram of complex 32:

[0040] The photosensitizer 32 was injected intravenously into mice bearing 4T1 breast tumors. At the same time, the control group was injected with PBS. The treated mice and the control mice were treated with light at the same time under light conditions. The mice were euthanized after 21 days, and the tumor volume was measured.

[0041] Experimental comparison shows that the present invention constructs a new type of AIE supramolecular photosensitizer through the synergistic effect of molecular cages and zinc porphyrin coordination host and guest, which has better ROS generation ability, produces reactive oxygen species that kill cancer cells, promotes NADH oxidation reaction, effectively destroys the growth environment of cancer cells, and thus kills cancer cells.

Claims

1. A coordination-driven supramolecular photosensitizer, characterized in that: Its structural formula is: or .

2. The method for preparing a coordination-driven supramolecular photosensitizer according to claim 1, wherein: The photosensitizer is constructed through the synergistic effect of the host and guest after the molecular cage is coordinated with the zinc porphyrin. The compound with only three benzyl bromide reaction sites and the compound containing four reactive pyridyl groups are reacted together. The free pyridyl group in the obtained reaction product is coordinated with the central metal of tetraphenyl zinc porphyrin. During the preparation, the tetrapyridyl tetraphenylethylene is firstly prepared through two steps of S N 2. Reaction synthesis of molecular cages. First, compound 4,4',4''-((2-(4-methoxyphenyl)ethylene-1,1,2-triyl)tris(benzene-4,1-diyl))tripyridine (4) and p-dibenzyl bromide (5) were refluxed at 90 °C for 3 days, and 6·3PF6 was obtained after ion exchange. – ; Subsequently, an equimolar amount of the intermediate 6·3PF6 – The reaction was carried out with compound 1,1,2,2-tetrakis(4-(pyridin-4-yl)phenyl)ethylene (7) at 110 °C for 3 days in the presence of 0.2 equivalents of tetrabutylammonium iodide, and pure 1·6PF6 was obtained by column chromatography. – ; Finally, 1·6PF6 – The complex 3·6PF6 was obtained by 1:1 coordination with tetraphenylzinc porphyrin (2) in acetonitrile. – , obtained by adding excess tetrabutylammonium chloride for ion exchange, the reaction process is as follows: 。 3. The use of a coordination-driven supramolecular photosensitizer according to claim 1, characterized in that: Application of the photosensitizer in preparing a photodynamic therapy agent.

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