阅读说明：本技术 一种可用于双光子光动力治疗的多功能生物探针及其制备方法和用途 (Multifunctional biological probe for two-photon photodynamic therapy and preparation method and application thereof ) 是由 田肖和 曹洪志 吴大俊 吴杰颖 李丹丹 张琼 马文 李胜利 田玉鹏 于 2019-12-26 设计创作，主要内容包括：本发明公开了一种可用于双光子光动力治疗的多功能生物探针及其制备方法和用途,其中多功能生物探针的结构式如下：<Image he="345" wi="700" file="DDA0002337541190000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明通过分子设计合成了一种具有优异的双光子活性、较低暗毒性及较高光毒性,并能同时靶向线粒体及脂滴两种细胞器的小分子U-TsO,鉴于U-TsO具有优异的综合性能,可作为一种用于双光子光动力学治疗的生物探针。(The invention discloses a multifunctional bioprobe for two-photon photodynamic therapy and a preparation method and application thereof, wherein the structural formula of the multifunctional bioprobe is as follows: the invention synthesizes the small molecule U-TsO which has excellent two-photon activity, lower dark toxicity and higher phototoxicity and can simultaneously target two organelles of mitochondria and lipid droplets through molecular design, and the U-TsO can be used as a biological probe for two-photon photodynamic therapy in view of excellent comprehensive performance.)

1. A multifunctional biological probe for two-photon photodynamic therapy, which is abbreviated as U-TsO, is characterized by having the following structural formula:

2. a method for preparing the multifunctional bioprobe of claim 1, characterized by comprising the steps of:

adding dried NaH into a Schlenk bottle, adding redistilled DMF, fully dissolving the NaH and the DMF, and sealing the DMF in an oil way; completely dissolving U-OH in redistilled DMF, slowly dripping into the system, and stirring and dispersing uniformly at normal temperature; then weighing B1, adding into a reaction bottle, slowly heating to 65 ℃, and reacting for 12 h; and cooling to room temperature after the reaction is finished, pouring the reaction solution into a large amount of ice water, adjusting the pH value to 6-7 by using a diluted HCl solution, repeatedly extracting by using dichloromethane, combining extract liquor, adding anhydrous magnesium sulfate, drying, performing suction filtration, evaporating the solvent to obtain an oily substance, and performing column chromatography separation to obtain a red oily substance U-TsO.

3. The method of claim 2, wherein:

the eluent for column chromatography was ethyl acetate: petroleum ether is 1:2, v/v.

4. Use of the multifunctional bioprobe of claim 1, characterized in that: is used as a biological probe in the process of two-photon photodynamic therapy.

5. Use according to claim 4, characterized in that:

the multifunctional biological probe can simultaneously target two organelles of mitochondria and lipid droplets, namely has dual-targeting property of the mitochondria and the lipid droplets.

Technical Field

The invention relates to a multifunctional biological probe for two-photon photodynamic therapy, a preparation method and application thereof.

Background

Photodynamic therapy (PDT) is a very efficient, minimally invasive cancer treatment and has attracted considerable attention in recent years. For example, BODIPY derivatives and porphyrin ring derivatives, etc., which can generate Reactive Oxygen Species (ROS), have been applied in photodynamic therapy, however, their relatively large molecular weight tends to result in poor water solubility. In addition, have d⁸Complexes such as Ru (II) and Ir (III) with electronic configurations have higher photodynamic therapeutic effect, because the complexes are more abundant in intersystem crossing and easier to cross to a T1 state, and further easily react with oxygen to form ROS, but the compounds generally have higher dark toxicity and lower metabolic rate in cells.

Notably, most of the reported photodynamic therapy probes are limited to targeting a single organelle. This greatly limits the timeliness of treatment, as cell death, such as Autophagic Cell Death (ACD), apoptosis, or necrosis, involves the damage and action of multiple organelles (e.g., lysosomes, endoplasmic reticulum, etc.). Therefore, a multiorganelle targeting molecule targeted for photodynamic therapy would be advantageous to induce stronger cytotoxicity.

Compared with single photon laser, the two-photon laser has the advantages of longer wavelength, lower energy, stronger tissue penetrating capacity and the like, has smaller damage to organisms, can cause small damage to normal tissue cells under the condition of good treatment effect by two-photon photodynamic treatment, is a novel and safe tumor treatment mode, and has wide application prospect.

Disclosure of Invention

The invention provides a multifunctional biological probe for two-photon photodynamic therapy, a preparation method and application thereof, wherein a small molecule U-TsO which has excellent two-photon activity, lower dark toxicity and higher phototoxicity and can simultaneously target mitochondria and lipid droplet two organelles is synthesized through molecular design.

The invention discloses a multifunctional biological probe for two-photon photodynamic therapy, which is abbreviated as U-TsO and has the following structural formula:

the preparation method of the multifunctional bioprobe comprises the following steps:

adding 0.6g (0.024mol) of dried NaH into a 250mL Schlenk bottle, measuring 15mL of redistilled DMF, fully dissolving and sealing oil; 4.0g (8.65mmol) of U-OH is completely dissolved in 10mL of redistilled DMF, slowly and dropwise added into the system, stirred for 30min at normal temperature, then 15.5 g (0.02mol) of B15 is weighed and added into a reaction bottle, and the temperature is slowly increased to 65 ℃ for reaction for 12 h; after the reaction, the reaction mixture was cooled to room temperature, poured into a large amount of ice water, the pH of the reaction mixture was adjusted to 6 to 7 with a diluted HCl solution, repeatedly extracted with dichloromethane, the extracts were combined, dried over anhydrous magnesium sulfate, filtered under suction, and the solvent was evaporated to dryness to give an oil, and a red oil, U-TsO (4.1g), was obtained by column chromatography (ethyl acetate/petroleum ether ═ 1: 2). Yield: 71.9 percent.

The synthetic route of the invention is as follows:

the multifunctional biological probe of the invention is used as a biological probe in the process of two-photon photodynamic therapy.

The multifunctional biological probe can simultaneously target two organelles of mitochondria and lipid droplets, namely has dual-targeting property of the mitochondria and the lipid droplets.

The invention has the beneficial effects that:

1. the U-TsO has a good rigid plane, effectively limits molecular torsion, reduces energy loss of non-radiative transition, has higher fluorescence quantum yield (32.24 percent), and is easier to generate ROS (reactive oxygen species) to cause cell death. As shown in fig. 1 and 3.

2. The U-TsO has double targeting properties of mitochondria and lipid droplets, namely the U-TsO can simultaneously target the mitochondria and the lipid droplets in cells, thereby being beneficial to enhancing the photodynamic therapy effect of light. As shown in fig. 4, 6 and 7.

3. The U-TsO contains an ether oxygen chain, has low dark toxicity, has an oil-water separation coefficient logP of 0.87667, and is easy to penetrate a biological membrane to enter a cell or an organelle. As shown in fig. 1 (a).

4. The U-TsO has a two-photon absorption effect, the two-photon fluorescence signal is strongest at the wavelength of 680nm, and compared with single-photon photodynamic therapy, the two-photon photodynamic therapy not only has high penetrability, but also has less damage to normal cells. As shown in fig. 2 and 5.

5. The U-TsO raw material is easy to obtain and the synthesis is simple. The similar multifunctional biological dye which can be used for two-photon photodynamic therapy does not exist, and the method has strong commercial value.

Drawings

FIG. 1(a) shows phototoxicity and dark toxicity of U-TsO, (b) shows that U-TsO enhances the fluorescence intensity of DCFH-DA under laser irradiation, wherein a shows that U-TsO has low dark toxicity, i.e., low cytotoxicity, and strong phototoxicity, and b shows that U-TsO has high ROS-generating ability, i.e., strong phototoxicity.

FIG. 2 shows a two-photon absorption cross section of U-TsO and its verification. The U-TsO has better two-photon effect.

FIG. 3 shows the (a) bright field pattern and (b) DCFH-DA (c) confocal fluorescence pattern of U-TsO cells incubated with 5. mu.M U-TsO under different laser irradiation times. The results show that U-TsO in the cells also has the effect consistent with that of in vitro experiments, and ROS is easy to generate, so that the fluorescence of DCFH-DA is enhanced, and the DCFH-DA can be used for photodynamic therapy.

FIG. 4 is a fluorescent plot of the co-localization of U-TsO intracellularly with mitochondrial commercial dyes and lipid droplet commercial dyes. As can be seen from the co-localization plot, U-TsO can simultaneously target mitochondria and lipid droplets in cells.

FIG. 5(a) is a graph showing the photodynamic therapy effect of U-TsO under the irradiation of a single photon light source laser for a certain period of time, (b) is a graph showing the photodynamic therapy effect of U-TsO under the irradiation of a two photon light source laser for a certain period of time, and (c) is a partially enlarged view of a cell bright field. The results show that the cell nucleus membrane deformation, nuclear bubbling and membrane bubbling after the U-TsO incubation are obvious cell death phenomena under the laser irradiation for a certain time, and single and double photons have obvious photodynamic treatment effects.

FIG. 6 is a graph of fluorescence of U-TsO after photodynamic therapy, co-localization of U-TsO with a lysosomal commercial dye. As can be seen, most of the lipid droplets coalesce with lysosomes to form autophagosomes, suggesting that ROS production in lipid droplets induces apoptosis of autophagic cells.

FIG. 7(a) shows the mitochondrial change of U-TsO incubated cells before and after photodynamic treatment and (b) shows the average mitochondrial length. Indicating that after photodynamic therapy the morphology of mitochondria changed significantly, breaking from a long strip into fragments and expanding to a circle, and the average length was also significantly reduced from 5.04 μm to 1.36 μm. Indicating that the U-TsO targets mitochondria and produces a photodynamic therapeutic effect.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.