ABSTRACT
Photodynamic inactivation of
Leishmania
spp. 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 against
Leishmania tropica
promastigotes and axenic amastigotes
in vitro
. The uptake of these PCs by both
Leishmania
stages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation of
Leishmania
spp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitized
Leishmania tropica
strains 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 live
Leishmania tropica
showed 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 tropica
strains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC
50
s) 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 of
Leishmania
spp. for use as vaccines or vaccine carriers.
摘要
光动力灭活
利什曼
的光动力灭活需要光敏剂被细胞摄取,例如被大配体轴向取代的硅(IV)-酞菁(PC)的内吞作用。我们在此报告,当被含氨基的配体取代后,PC(PC1 和 PC2)被内吞,并显示出更强的抗利什曼原虫的效力。
热带利什曼病
原虫和轴丝母细胞的效力
体外
.利什曼原虫和轴丝母细胞对这些多糖的吸收
利什曼病
阶段对这些多氯联苯的吸收均符合预期的饱和动力学。开发了灵敏的检测方法,用于评估光动力灭活利什曼原虫的能力。
利什曼
通过两种方法使其发出荧光:转染原原体以表达绿色荧光蛋白(GFP)和用羧基荧光素琥珀酰亚胺酯(CFSE)加载原原体。PC 敏化的
致敏的
菌株在暴露于红光(波长 ∼ 650 nm)、1 至 2 J cm -2 的通量下后,显微镜下可以看到它们失去了运动性、结构完整性和 GFP/CFSE 荧光。
-2
.荧光定量分析基于活的热带利什曼原虫 GFP/CFSE 的损失。
定量荧光测定
与 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑(MTT)细胞活力测定的结果一致。
热带利什曼病
菌株对 PC1 和 PC2 介导的光失活的敏感性是其宿主细胞或巨噬细胞的 100 倍。
50
s)来判断。用氨基而不是其他配体对 PC 进行轴向替代,似乎可使其利什曼病光解活性提高多达 40 倍。因此,PC1 和 PC2 有可能用于利什曼病光动力疗法和利什曼病的氧化光活化。
利什曼病
用作疫苗或疫苗载体。