Park Jung Sun, Kim Taeyeon, Kim Dohoon, Jeong Young-Il
Department of Internal Medicine, Chonnam National University Medical School, 42 Jebongro, Gwangju 61469, Korea.
College of Art&Science, University of Pennsylvania, 249 S 36th St., Philadelphia, PA 19104, USA.
Int J Mol Sci. 2021 Nov 15;22(22):12309. doi: 10.3390/ijms222212309.
The aim of this study is to fabricate reactive oxygen species (ROS)-sensitive nanoparticles composed of succinyl β-cyclodextrin (bCDsu), memantine and thioketal linkages for application in Alzheimer's disease, and to investigate the suppression of -methyl-d-aspartate (NMDA) receptor 1 (NMDAR1) in cells. Thioketal diamine was attached to the carboxyl group of bCDsu to produce thioketal-decorated bCDsu conjugates (bCDsu-thioketal conjugates) and memantine was conjugated with thioketal dicarboxylic acid (memantine-thioketal carboxylic acid conjugates). Memantine-thioketal carboxylic acid conjugates were attached to bCDsu-thioketal conjugates to produce bCDsu-thioketal-memantine (bCDsuMema) conjugates. SH-SY5Y neuroblastoma cells and U87MG cells were used for NMDAR1 protein expression and cellular oxidative stress. Nanoparticles of bCDsuMema conjugates were prepared by means of a dialysis procedure. Nanoparticles of bCDsuMema conjugates had small particle sizes less than 100 nm and their morphology was found to be spherical in transmission electron microscopy observations (TEM). Nanoparticles of bCDsuMema conjugates responded to HO and disintegrated or swelled in aqueous solution. Then, the nanoparticles rapidly released memantine according to the concentration of HO. In an in vivo animal imaging study, thioketal-decorated nanoparticles labelled with fluorescent dye such as chlorin e6 (Ce6) showed that the fluorescence intensity was stronger in the brain than in other organs, indicating that bCDsuMema nanoparticles can efficiently target the brain. When cells were exposed to HO, the viability of cells was time-dependently decreased. Memantine or bCDsuMema nanoparticles did not practically affect the viability of the cells. Furthermore, a western blot assay showed that the oxidative stress produced in cells using HO increased the expression of NMDAR1 protein in both SH-SY5Y and U87MG cells. Memantine or bCDsuMema nanoparticles efficiently suppressed the NMDAR1 protein, which is deeply associated with Alzheimer's disease. Fluorescence microscopy also showed that HO treatment induced green fluorescence intensity, which represents intracellular ROS levels. Furthermore, HO treatment increased the red fluorescence intensity, which represents the NMDAR1 protein, i.e., oxidative stress increases the expression of NMDAR1 protein level in both SH-SY5Y and U87MG cells. When memantine or bCDsuMema nanoparticles were treated in cells, the oxidative stress-mediated expression of NMDAR1 protein in cells was significantly decreased, indicating that bCDsuMema nanoparticles have the capacity to suppress NMDAR1 expression in brain cells, which has relevance in terms of applications in Alzheimer's disease.
本研究的目的是制备由琥珀酰β-环糊精（bCDsu）、美金刚和硫酮键组成的活性氧（ROS）敏感纳米颗粒，用于阿尔茨海默病的治疗，并研究其对细胞中N-甲基-D-天冬氨酸（NMDA）受体1（NMDAR1）的抑制作用。将硫酮二胺连接到bCDsu的羧基上，制备硫酮修饰的bCDsu缀合物（bCDsu-硫酮缀合物），并将美金刚与硫酮二羧酸偶联（美金刚-硫酮羧酸缀合物）。将美金刚-硫酮羧酸缀合物连接到bCDsu-硫酮缀合物上，制备bCDsu-硫酮-美金刚（bCDsuMema）缀合物。使用SH-SY5Y神经母细胞瘤细胞和U87MG细胞进行NMDAR1蛋白表达和细胞氧化应激研究。通过透析法制备bCDsuMema缀合物纳米颗粒。bCDsuMema缀合物纳米颗粒的粒径小于100nm，在透射电子显微镜（TEM）观察中发现其形态为球形。bCDsuMema缀合物纳米颗粒对HO有反应，在水溶液中会分解或膨胀。然后，纳米颗粒根据HO的浓度迅速释放美金刚。在体内动物成像研究中，用荧光染料如二氢卟吩e6（Ce6）标记的硫酮修饰纳米颗粒显示，大脑中的荧光强度比其他器官更强，表明bCDsuMema纳米颗粒可以有效地靶向大脑。当细胞暴露于HO时，细胞活力随时间下降。美金刚或bCDsuMema纳米颗粒实际上对细胞活力没有影响。此外，蛋白质免疫印迹分析表明，使用HO在细胞中产生的氧化应激增加了SH-SY5Y和U87MG细胞中NMDAR1蛋白的表达。美金刚或bCDsuMema纳米颗粒有效地抑制了与阿尔茨海默病密切相关的NMDAR1蛋白。荧光显微镜还显示，HO处理诱导了绿色荧光强度，代表细胞内ROS水平。此外，HO处理增加了红色荧光强度，代表NMDAR1蛋白，即氧化应激增加了SH-SY5Y和U87MG细胞中NMDAR1蛋白水平的表达。当在细胞中用美金刚或bCDsuMema纳米颗粒处理时，氧化应激介导的细胞中NMDAR1蛋白的表达显著降低，表明bCDsuMema纳米颗粒具有抑制脑细胞中NMDAR1表达的能力，这在阿尔茨海默病的应用方面具有相关性。
