Sandoval Sergio, Mendez Natalie, Alfaro Jesus G, Yang Jian, Aschemeyer Sharraya, Liberman Alex, Trogler William C, Kummel Andrew C
University of California, San Diego, Moores Cancer Center, Department of Bioengineering, CalIT2 Nanomedicine Laboratory, La Jolla, California 92093, United States.
University of California, San Diego, Department of Nanoengineering, Chemical Engineering, and Material Science, La Jolla, California 92093, United States.
J Biomed Opt. 2015 Aug;20(8):88003. doi: 10.1117/1.JBO.20.8.088003.
A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900 μg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization.
设计了一种测量内吞作用的定量方法，以评估靶向纳米颗粒平台的细胞摄取和特异性。开发了一种简单的N -羟基琥珀酰亚胺酯共轭技术，用荧光报告分子异硫氰酸荧光素和叶酸或聚乙二醇（PEG）对100纳米的中空二氧化硅纳米壳颗粒进行功能化。使用扫描电子显微镜和透射电子显微镜对功能化纳米壳进行表征，并用紫外-可见光谱法定量纳米壳表面功能化叶酸的最大量。利用荧光显微镜和共聚焦显微镜在体外研究了人宫颈癌HeLa细胞和人包皮成纤维细胞（HFF-1）的内吞程度。开发了一种简单的荧光比率分析方法来量化内吞作用与表面粘附。与PEG功能化纳米壳相比，叶酸功能化的纳米壳显示出癌细胞内吞作用增强。荧光比率分析表明，粘附在癌细胞上的叶酸功能化二氧化硅纳米壳中有95%被内吞，而当分别用200和900μg的PEG功能化纳米壳粘附在细胞表面并发生内吞作用时，只有27%。此外，叶酸功能化纳米壳的内吞作用被证明具有癌细胞选择性，同时不影响正常细胞。所开发的荧光比率分析是一种简单快速的验证/确认方法，通过使用内部对照进行归一化来量化数据集之间的细胞摄取。
