Coordination-driven supramolecular photosensitizer and application thereof in photodynamic therapy

Abstract

The invention designs and synthesizes a tetraphenyl ethylene covalent molecular cage with pyridine functionalization, and an AIE supermolecule photosensitizer is obtained through coordination and the synergistic effect of a subject and an object. The TPE unit enhances the photophysical properties of the photosensitizer and endows the photosensitizer with the AIE characteristic; the molecular cage and tetraphenylporphyrin are combined through a host and a guest, so that self-aggregation of porphyrin in water is further inhibited, ROS generation is promoted, and cancer cells are effectively killed through photocatalysis NADH oxidation assistance.

Description

Technical Field

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

[0002] At present, cancer remains one of the major health problems in the world. Traditional treatment techniques such as surgical resection, chemotherapy and radiotherapy have a series of traumatic and side effects, which greatly limit their clinical applications. Photodynamic therapy (PDT) is an emerging cancer treatment method. It has strong selectivity, targets cancer cells and has few side effects, which greatly enhances the efficacy. Photosensitizers, as an important component of photodynamic therapy, kill cancer cells by producing 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 to achieve ROS enhancement.

[0003] Porphyrin derivatives in biological systems are the core components of important biomacromolecules in photosystems. Among them, the coordination between the central metal of metal porphyrin and amino acid residues stabilizes the chlorophyll molecule, thereby promoting efficient photon capture, energy transfer and electron transfer. The pyridine-functionalized molecular cage coordinates with the central metal of porphyrin, and further cooperates with the host and guest of the molecular cage cavity to stabilize the porphyrin molecule; a new type of AIE bionic photosensitizer molecule can be formed through coordination-driven host-guest assembly, which effectively inhibits the excited state quenching caused by porphyrin self-aggregation, enhances the ROS generation ability, and improves the PDT efficiency. Summary of the invention

[0004] The present invention adopts two steps S N 2 Reaction design and synthesis of a monofunctionalized hexacationic molecular cage based on tetraphenylethylene (TPE) pyridine group for the construction of coordination-driven host-guest assembly AIE biomimetic photosensitizer. Compound 5 with only three benzyl bromide reaction sites was reacted with compound 4 containing four reactive pyridine groups. According to the principle of reaction site matching, the obtained reaction product will have a free pyridine group. Subsequently, the pyridine monofunctionalized molecular 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 the host and the guest after the coordination of the 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, and the free pyridyl group of the obtained reaction product is coordinated with the central metal of tetraphenyl zinc porphyrin. The structural formula is:

[0007]

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

[0009] The present invention constructs a novel AIE supramolecular photosensitizer through the synergistic effect of the molecular cage and the zinc porphyrin coordination host and guest. Compared with the single molecular cage 1 and the zinc porphyrin 2, the complex 3 as a photosensitizer 2 It has a better ROS generation ability and reaches equilibrium after 20 seconds of illumination. Compared with the ROS generated by the molecular cage 1 and zinc porphyrin 2 alone after 20 seconds of illumination, the complex 3 2 The ROS production capacity of the molecule was 4.8 times and 6.6 times that of the single molecular cage 1 and zinc porphyrin 2, respectively. Its ROS production capacity was detected in vivo and in vitro by ROS indicator, and then it was used in photodynamic therapy, achieving good tumor killing effect.

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

[0011] (1) The present invention designs and synthesizes a tetraphenylethylene covalent molecular cage with pyridine functionalization, and obtains an AIE supramolecular photosensitizer through coordination and host-guest synergy. The TPE unit enhances the photophysical properties of the photosensitizer and gives it AIE characteristics; the molecular cage and tetraphenylporphyrin further inhibit the self-aggregation of porphyrin in water through host-guest binding, promoting the generation of R OS.

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

[0013] Compared with the prior art, the present invention constructs a porphyrin-based bionic supramolecular photosensitizer through coordination and host-guest synergy strategies, effectively improving its ROS generation ability, having good biocompatibility and safety, achieving efficient ROS generation both in vivo and in vitro, and being able to effectively kill cancer cells in vivo, thereby improving the photodynamic therapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The complex 3 of the photosensitizer of Example 1 of the present invention is 2 Design and synthesis route.

[0015] Figure 2 The molecular cages 1 and 2 and the complex 3 described in Example 2 of the present invention 2 's NMR image.

[0016] Figure 3 The complex 3 described in Example 3 of the present invention 2 Mass spectrum of .

[0017] Figure 4 The molecular cages 1 and 2 and the complex 3 described in Example 4 of the present invention 2 Monitoring graph of the total amount of ROS generated.

[0018] Figure 5 The complex 3 described in Example 5 of the present invention 2 NMR image of photocatalytic oxidation of NADH.

[0019] Figure 6 The complex 3 described in Example 6 of the present invention 2 Photodynamic therapy diagram.

[0020] Figure 7 The complex 3 of the photosensitizer of Example 1 of the present invention is 2 The structural formula of . DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1

[0023] See also Figure 1 and Figure 7 , molecular cage 1 and complex 3 2 Design and synthesis diagram:

[0024] Through two steps S N 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 to reflux at 90 ° C until all compounds are dissolved. Disperse 4 (100 mg, 0.168 mmol) in 5 mL 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 NH 4 PF 6 Then, the mixture was stirred at room temperature for 12 hours. The precipitate was collected and the mixture was treated with excess H 2 O (3 × 20 mL) and then dried to obtain 6·3PF 6 – Orange solid. 6 – (100 mg, 63 μmol) and tetrabutylammonium iodide (14.67 mg, 13 μmol) were added to a pressure bottle filled with dry MeCN (100 mL) and heated at 90 ° C. 7 (46 mg, 76 μmol) of acetonitrile suspension was added dropwise to it 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 then an excess of NH 4 PF 6 Stir for 12 hours. Collect the precipitate and use excess H 2 O (3 × 20 mL) and dried to obtain a crude product as an orange solid. 2 Cl 2 :MeCN(saturated NH 4 PF 6)=3:1(v:v) as the mobile phase, and purified by silica gel chromatography to obtain pure 1·6PF 6 – (20 mg, yield: 13.5%).

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

[0026] Example 2

[0027] See also Figure 2 , molecular cages 1 and 2 and complex 3 2 NMR images:

[0028] Weigh compounds 1, 2 and 3·6PF respectively 6 – 、3·6Cl - Dissolve in 450 μL CD 3 CN, 3·6Cl - Dissolved in 450 μL D 2 O, ultrasonically dissolve, prepare 0.5 mM solutions and place them in NMR tubes. Test hydrogen spectra by NMR.

[0029] Example 3

[0030] See also Figure 3 , complex 3 2 Mass spectrum:

[0031] Weigh compound 3·6PF 6 – CH 2 SOLUTION IN HPLC 3 CN, it was prepared into a 0.5 mM solution and tested by mass spectrometer.

[0032] Example 4

[0033] See also Figure 4 , molecular cages 1 and 2 and complex 3 2 Monitoring chart of total ROS generation:

[0034] The compound 2,7-dichlorodihydrofluorescein (DCFH) is used as an indicator to detect total reactive oxygen species (ROS). First, prepare DCFH into a 1mM ethanol solution and store it at -20°C. When using the mother liquor of DCFH ethanol solution, it needs to be activated first. Take 2mL of NaOH solution with a concentration of 0.01M, add the above DCFH ethanol solution (1.0mM, 0.5mL), mix well, and place in the dark for 30 minutes. At the same time, take 10mL of PBS and add it to a centrifuge tube, and precool it at 4°C. After the DCFH solution is protected from light for 30 minutes, add it to the precooled PBS solution, mix well, and place in the dark at 4°C for later use. The concentration of DCFH after activation is 40μM. 2 Prepare a 2.0mM stock solution with ultrapure water and add it to a 40μM DCFH solution. Dilute the test solution with PBS solution to make the photosensitizer have a final concentration of 2.0μM. The DCFH group alone was used as a control. Add the above sample solution to a fluorescent cuvette and 2 The generation of ROS was measured under white light irradiation.

[0035] Example 5

[0036] See also Figure 5 , complex 3 2 NMR images of photocatalytic oxidation of NADH:

[0037] Prepare a 2mM deuterated aqueous solution of NADH in a NMR test tube and add 5mol% of 3 2 As photocatalysts, the light power density was 20 mW / cm 2 The NMR changes of NADH oxidation process were monitored under white light irradiation.

[0038] Example 6

[0039] See also Figure 6 , complex 3 2 Photodynamic therapy diagram:

[0040] Intravenous injection of photosensitizer 3 2 At the same time, the control group was injected with PBS, and the treated mice and the control group mice were treated with light at the same time under light conditions. The mice were euthanized after 21 days, and the tumor volume was then 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 that kills cancer cells, promotes NADH oxidation reaction, and effectively destroys the growth environment of cancer cells, thereby killing cancer cells.

Claims

1. A coordination-driven supramolecular photosensitizer, characterized in that: The photosensitizer is constructed by the synergistic effect of the host and the guest after the molecular cage is coordinated with the zinc porphyrin. The compound having only three benzyl bromide reaction sites and the compound having four reactive pyridyl groups are reacted together, and the free pyridyl group in the obtained reaction product is coordinated with the central metal of tetraphenyl zinc porphyrin. The structural formula is:

2. A coordination-driven supramolecular photosensitizer according to claim 1, characterized in that: The preparation process is to firstly prepare tetrapyridyl tetraphenylethylene through two steps of S N 2 reaction to synthesize the molecular cage. First, compound (4) and p-dibenzyl bromide (5) were refluxed at 90°C for 3 days to obtain 6·3PF6– through ion exchange. Subsequently, equimolar amounts of the intermediate 6·3PF6– and compound (7) were reacted 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– was coordinated with tetraphenylzinc porphyrin (2) in acetonitrile in a 1:1 ratio to obtain the complex 3·6PF6–, and water-soluble 3·6Cl– was obtained by ion exchange by adding excess tetrabutylammonium chloride.

3. A coordination-driven supramolecular photosensitizer according to any one of claims 1 or 2, characterized in that: The photosensitizer reaches a balance in ROS production capacity when irradiated for 20 seconds in photodynamic therapy, which is 4.8 times and 6.6 times the ROS production of a single molecular cage and zinc porphyrin when irradiated for 20 seconds.

Images