Lu Wei, Tan Yu-zhen, Jiang Xin-guo
Department of Pharmaceutics, School of Pharmacy, Shanghai Medical School, Fudan University, China.
Yao Xue Xue Bao. 2006 Apr;41(4):296-304.
A method of coculture of brain capillary endothelial cells (BCECs) and astrocytes of rats was used to evaluate nanoparticle's blood-brain barrier (BBB) transcytosis and toxicity at the endothelial tight junction.
A lipophilic fluorescent probe, 6-coumarin, was incorporated in poly (ethyleneglycol)-poly (lactide) nanoparticle using double emulsion/solvent evaporation method. BCECs and astrocytes were firstly isolated from brain of newborn rats and characterized by their morphology and immunocytochemistry staining, separately. Subsequently, a coculture model with BCECs on the top of micro-porous membrane of cell culture insert and astrocytes on the bottom side was established. The permeability of 14C-labeled sucrose and nanoparticle were determined, separately.
The mean weight-based diameter of 6-coumarin loaded nanoparticles was (102.4 +/- 6.8) nm, with zeta potential of (-16.81 +/- 1.05) mV. BCECs were positive for factor VIII staining and glial fibrillary acidic protein was expressed in astrocytes. The transendothelial electrical resistance reached up to (313 +/- 23) omega x cm2. The tight junction between BCECs in the coculture model could be visualized by both scanning electron microscopy and transmission electron microscopy. The unchanged paracellular transport of sucrose proved that nanoparticle with concentration lower than 200 microg x mL(-1) did not impact the integrity of BBB endothelial tight junctions. The permeability of 10 microg x mL(-1) 6-coumarin labeled nanoparticle was 0.29 x 10(-3) cm x min(-1).
This in vitro experimental model of rat BBB was close to resemble the in vivo situation for examination of the permeability of nanoparticle and toxicity evaluation.
采用大鼠脑毛细血管内皮细胞（BCECs）与星形胶质细胞共培养的方法，评估纳米颗粒的血脑屏障（BBB）转胞吞作用及其在内皮紧密连接处的毒性。
采用复乳/溶剂挥发法将亲脂性荧光探针6-香豆素载入聚（乙二醇）-聚（丙交酯）纳米颗粒中。首先从新生大鼠脑中分离出BCECs和星形胶质细胞，并分别通过形态学和免疫细胞化学染色进行鉴定。随后，建立细胞培养插入物微孔膜顶部为BCECs、底部为星形胶质细胞的共培养模型。分别测定14C标记蔗糖和纳米颗粒的通透性。
负载6-香豆素的纳米颗粒基于重量的平均直径为（102.4±6.8）nm，ζ电位为（-16.81±1.05）mV。BCECs对因子VIII染色呈阳性，星形胶质细胞中表达胶质纤维酸性蛋白。跨内皮电阻高达（313±23）Ω·cm2。共培养模型中BCECs之间的紧密连接可通过扫描电子显微镜和透射电子显微镜观察到。蔗糖的细胞旁转运未改变，证明浓度低于200μg·mL-1的纳米颗粒不会影响BBB内皮紧密连接的完整性。10μg·mL-1 6-香豆素标记纳米颗粒的通透性为0.29×10-3 cm·min-1。
该大鼠BBB体外实验模型接近体内情况，可用于纳米颗粒通透性检测和毒性评估。
