Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Sci Rep. 2018 Jan 12;8(1):638. doi: 10.1038/s41598-017-18947-x.
Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates were investigated in terms of PCI assisted cellular activity. It is found that tetramethylrhodamine and Alexa Fluor 555 conjugated octaarginine PNA upon irradiation exhibit more than ten-fold increase in antisense activity in the HeLa pLuc705 luciferase splice correction assay. An analogous fluorescein conjugate did not show any significant enhancement due to photobleaching, and neither did an Alexa Fluor 488 conjugate. Using fluorescence microscopy a correlation between endosomal escape and antisense activity was demonstrated, and in parallel a correlation to localized formation of ROS assigned primarily to singlet oxygen was also observed. The results show that tetramethylrhodamine (and to lesser extent Alexa Fluor 555) conjugated octaarginine PNAs are as effectively delivered to the cytosol compartment by PCI as by chloroquine assisted delivery and also indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore.
光化学内化 (PCI) 是一种基于生成光诱导活性氧 (ROS) 的细胞内药物递送方法，这种 ROS 会破坏内体膜，从而将药物释放到细胞质中。在我们的研究中，研究了一系列反义荧光团八聚精氨酸肽核酸 (PNA) 缀合物在 PCI 辅助细胞活性方面的作用。结果发现，四甲基罗丹明和 Alexa Fluor 555 缀合的八聚精氨酸 PNA 在照射下，在 HeLa pLuc705 荧光素酶剪接校正测定中，反义活性增加了十倍以上。类似的荧光素缀合物由于光漂白没有显示出任何显著增强，而 Alexa Fluor 488 缀合物也没有。通过荧光显微镜观察到内体逃逸与反义活性之间存在相关性，同时也观察到与主要归因于单线态氧的局部 ROS 形成之间存在相关性。结果表明，四甲基罗丹明（和较小程度的 Alexa Fluor 555）缀合的八聚精氨酸 PNA 通过 PCI 有效递送到细胞质区室，与氯喹辅助递送一样，并且还表明有效的光动力内体逃逸强烈依赖于光化学单线态氧形成的量子产率、光稳定性以及生色团的亲脂性。
