Functionalization with ApoE-derived peptides enhances the interaction with brain capillary endothelial cells of nanoliposomes binding amyloid-beta peptide

Nanoliposomes containing phosphatidic acid or cardiolipin are able to target in vitro with very high affinity amyloid-β (Aβ), a peptide whose overproduction and progressive aggregation in the brain play a central role in the pathogenesis of Alzheimer's disease. However, the presence of the blood–brain barrier (BBB) severely limits the penetration of either drugs or drug vehicles (nanoparticles) to the brain. Therefore, there is a need to develop and design approaches specifically driving nanoparticles to brain in a better and effective way. The aim of the present investigation is the search of a strategy promoting the interaction of liposomes containing acidic phospholipids with brain capillary endothelial cells, as a first step toward their passage across the BBB. We describe the preparation and physical characterization of nano-sized liposomes decorated with peptides derived from apolipoprotein E and characterize their interaction with human immortalized brain capillary cells cultured in vitro (hCMEC/D3). For this purpose, we synthesized two ApoE-derived peptides (the fragment 141–150 or its tandem dimer) containing a cysteine residue at the C-terminus and decorated NL by exploiting the cysteine reaction with a maleimide-group on the nanoparticle surface. NL without ApoE functionalization did not show either relevant membrane accumulation or cellular uptake, as monitored by confocal microscopy using fluorescently labeled nanoliposomes or quantifying the cell-associated radioactivity of isotopically labeled nanoliposomes. The uptake of nanoliposomes by cell monolayers was enhanced by ApoE-peptide-functionalization, and was higher with the fragment 141–150 than with its tandem dimer. The best performance was displayed by nanoliposomes containing phosphatidic acid and decorated with the ApoE fragment 141–150. Moreover, we show that the functionalization of liposomes containing acidic phospholipids with the ApoE fragment 141–150 scarcely affects their reported ability to bind Aβ peptide in vitro. These are important and promising features for the possibility to use these nanoliposomes for the targeting of Aβ in the brain districts.     

新型磁性纳米脂质复合物对肝癌细胞和肝细胞的作用研究

目的:研究新型磁性纳米脂质复合物对肝癌细胞和肝细胞的作用。方法:将肝癌细胞(Hep-G2)和肝细胞(L-02)根据加入的不同浓度新型纳米脂质复合物,各自分为空白对照组、不同浓度含铁脂质体组、空脂质体组,用MTT法观察各组细胞毒性,筛选出适合的浓度和时间;用普鲁士蓝染色、电镜检测观察各组细胞吞噬情况;用原子分光光度计观察各组细胞内铁含量;用细胞磁共振观察新型纳米脂质复合物体外显影效果。结果:新型磁性纳米脂质复合物含铁浓度在0.238μg/ml以上时,24小时有较强的细胞毒性,对肝癌细胞和肝细胞的抑制率均超过40%,故以下实验选择含铁浓度在0.238μg/ml以下;肝癌细胞和肝细胞与各浓度新型磁性纳米脂质复合物共同培养,24小时普鲁士蓝染色最高阳性率分别为5.5%和1.25%,24小时细胞内铁含量最高值分别为0.675pg/cell和0.460pg/cell;24小时电镜观察肝癌细胞和肝细胞空白对照组均未见颗粒样物质,各含铁脂质体组和空脂质体组均可见颗粒样物质;24小时肝癌细胞和肝细胞磁共振感兴趣区均未见明显信号表达。结论:新型磁性纳米脂质复合物含铁浓度在0.238μg/ml以下时,24小时细胞毒性较小;肝癌细胞对新型磁性纳米脂质复合物的吞噬作用稍高于肝细胞对其的吞噬;进入细胞内的新型磁性纳米脂质复合物含铁量低,体外显影效果不佳。