建立体外共培养血脑屏障模型评价纳米粒的胞吞转运和毒性(英文)

Establishment of coculture model of blood-brain barrier in vitro for nanoparticle's transcytosis and toxicity evaluation

目的建立大鼠脑毛细血管内皮细胞(BCECs)和星形胶质细胞共培养模型,评价纳米粒的跨血脑屏障(BBB)转运和对内皮细胞紧密连接的毒性。方法采用复乳/溶媒蒸发法制备载荧光探针6-香豆素的聚乙二醇-聚乳酸纳米粒。首先,分别从新生鼠大脑分离培养BCECs和星形胶质细胞并进行免疫组化鉴定。然后,将BCECs接种于细胞培养池微孔膜的上面,将星形胶质细胞接种于膜下面建立共培养模型。分别测定14C-蔗糖和纳米粒的渗透系数。结果载6-香豆素纳米粒的平均重均粒径为(102.4±6.8)nm,zeta电位为(-16.81±1.05)mV。BCECs的VIII因子表达呈阳性;星形胶质细胞的胶质原纤维酸性蛋白表达呈阳性。模型的跨内皮细胞电阻值为(313±23)Ω.cm2。扫描电镜和透射电镜观察发现,共培养模型中的BCECs形成紧密连接。纳米粒的质量浓度低于200μg.mL-1时,不影响14C-蔗糖渗透系数的改变,表明其不会影响BBB内皮细胞的紧密连接。10μg.mL-1载6-香豆素纳米粒的渗透系数为0.29×10-3cm.m in-1。结论该大鼠BBB模型与体内情况接近,适合用于评价纳米粒的脑内转运和毒性。

Aim 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. Methods 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. Results 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） Ω·cm^2. 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 μg·mL^-1 did not impact the integrity of BBB endothelial tight junctions. The permeability of 10 μg·mL^-1 6-coumarin labeled nanoparticle was 0.29 ×10^-3cm·min^-1 Conclusion 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.