The Controlled ROS Production And Intracellular Distribution Of Corannulene

Photodynamic therapy(PDT)is one of the most promising,non-invasive methods for treating various premalignant and malignant diseases.PDT usually involves three key elements: photosensitizer(PS),light source and tissue oxygen.Recently,the development of new PSs has aroused great interest to scientists,for example,carbon materials.Corannulene(Cor),belonging to carbon materials,has been proved to be a potential PS,which generates ROS including hydrogen peroxide,hydroxyl radicals,and superoxide.The poor aqueous solubility of Cor severely limits its application in biological field.Two vehicles were employed to enhance its water solubility in our project,which were methoxy poly(ethylene glycol)and gamma-cyclodextrin to obtain two water-soluble Cor systems,namely ?-CD/Cor complexes and mPEG-Cor micelles.The effect of Cor vehicles on ROS production by the two Cor formulations was investigated in aqueous solution and at cell level,and the ability of targeting mitochondria and distribution of Cor in cells were examined.The ?-CD/Cor complexes and mPEG-Cor micelles were prepared according to the methods available in our laboratory.The characterization of the two formulations was detected and compared,including UV spectra,drug loading,particle size and morphology,and stability in PBS.ROS generated by two types of water-soluble Cor systems was detected and compared in aqueous solution.The ROS-sensitive probe(DCFH-DA)was selected to measure the total ROS production.Hydrogen peroxide and superoxide produced by the both Cor systems were also analyzed by the fluorescence analysis assisted with superoxide-sensitive fluorescent probe(DHE)and Hydrogen peroxide assay kit.Hydroxyl radical production of the two kinds of Cor systems was verified by electron spin spectroscopy.Therefore,irrespective of the vehicle type,Cor could produce superoxide,hydrogen peroxide,and hydroxyl radicals upon the ultraviolet light irradiation.PEGylation was less efficient than the cyclodextrin complexation with regard to ROS production in aqueous solution.ROS production generated by the two types of Cor systems was detected and compared at the cellular level.The high performance liquid chromatography was used to detect the degree of uptake of the two formulations in human prostate cancer cells,and there was no significant difference between them.The total ROS and superoxide production in cells were measured using DCFH-DA probe and DHE probe by a confocal laser scanning microscope(CLSM).The experimental results were consistent with the above results of ROS and superoxide production in aqueous solution.The mitochondrial ROS was compared by MitoSOX red probe,which proved that the more accumulation of the ?-CD/Cor than mPEG-Cor in mitochondria.The cell viability of the two Cor formulations was compared by the standard MTT assay method and ?-CD/Cor complexes yielded a more desirable in vitro photodynamic anti-cancer effect than mPEG-Cor micelles.The ability of targeting mitochondria and Cor distribution in the cells were investigated.The co-localization was analyzed by CLSM between fluorescence-labeled Cor with mitochondria,lysosomes,endoplasmic reticulum,and Golgi apparatus with the help of a variety of fluorescent probes.The co-localization analysis between planar perylene(Per)and mitochondria was performed as the control.The results showed that the degree of Cor/mitochondria co-localization was higher than that of Per,proving that the electron delocalization induced by curvature of Cor enhanced the accumulation of Cor in mitochondria to a certain degree.Cor existed in the main organelles,including mitochondria,lysosomes,Golgi apparatus,and endoplasmic reticulum.There was a significant Cor accumulation in the endoplasmic reticulum compared to other organelles;the detailed mechanism remains to be further investigated.In summary,the current work highlighted the importance of Cor vehicles in realizing controlled ROS production.Compared to the PEGylation approach,the cyclodextrin complexation method was more effective in generating ROS by Cor in vitro and promoting the Cor mitochondrial accumulation.Herein,Cor could be a potential PS with ROS production controlled by Cor concentration,light irradiation and the modification method.In addition,the intracellular distribution of Cor was also analyzed.Cor existed in the main organelles,and the electron delocalization induced by curvature of Cor enhanced its accumulation in mitochondria,which could potentially enhance the PDT efficiency with Cor as a photosensitizer.The current project emphasized the importance of dipole in the pharmaceutical application of carbon materials.