Al-Qahtani Ahmed, Alkahtani Saad, Kolli Bala, Tripathi Pankaj, Dutta Sujoy, Al-Kahtane Abdullah A, Jiang Xiong-Jie, Ng Dennis K P, Chang Kwang Poo
Department of Infection and Immunity, Research Center, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
Antimicrob Agents Chemother. 2016 Mar 25;60(4):2003-11. doi: 10.1128/AAC.01879-15. Print 2016 Apr.
Photodynamic inactivation ofLeishmaniaspp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency againstLeishmania tropicapromastigotes and axenic amastigotesin vitro The uptake of these PCs by bothLeishmaniastages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation ofLeishmaniaspp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitizedLeishmania tropicastrains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm(-2) Quantitative fluorescence assays based on the loss of GFP/CFSE from liveLeishmania tropicashowed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.Leishmania tropicastrains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation ofLeishmaniaspp. for use as vaccines or vaccine carriers.
利什曼原虫属的光动力灭活需要细胞摄取光敏剂，例如轴向被庞大配体取代的硅（IV）-酞菁（PC）的内吞作用。我们在此报告，当用含氨基的配体取代时，PC（PC1和PC2）被内吞，并在体外对热带利什曼原虫前鞭毛体和无菌无鞭毛体显示出更高的效力。正如预期的那样，这两个利什曼原虫阶段对这些PC的摄取遵循饱和动力学。开发了灵敏的测定方法，通过两种方式使利什曼原虫属发出荧光来评估其光动力灭活：转染前鞭毛体以表达绿色荧光蛋白（GFP）并使其负载羧基荧光素琥珀酰亚胺酯（CFSE）。在显微镜下可以看到，经PC敏化的热带利什曼原虫菌株在暴露于波长约为650 nm的红光、通量为1至2 J cm(-2) 后失去运动能力、结构完整性以及GFP/CFSE荧光。基于活的热带利什曼原虫中GFP/CFSE损失的定量荧光测定表明，PC1和PC2剂量依赖性地使两个阶段对光灭活敏感，这与3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）细胞活力测定结果一致。从这些细胞的估计50%有效浓度（EC50）判断，热带利什曼原虫菌株对PC1和PC2介导的光灭活的敏感性比其宿主细胞或巨噬细胞高100倍以上。用氨基而非其他配体对PC进行轴向取代似乎可使其对利什曼原虫的光解活性提高多达40倍。因此，PC1和PC2可能可用于利什曼病的光动力治疗以及用作疫苗或疫苗载体的利什曼原虫属的氧化光灭活。
