Aim:We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches. Materials & methods: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment. Results: Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl--cyclodextrin (caveolinmediated endocytosis). A significant methyl--cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy. Conclusion: We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology.
Keywords:In recent years, various types of nanoparticles (NPs; <100 nm in all three dimensions, ISO TS 27687:2008) have been designed for potential biomedical and pharmaceutical applications, such as vehicles for targeted drug delivery, nucleic acids, biomedical imaging or biosensing [1][2][3]. Therefore, an increasing number of research studies have focused on the interaction of NPs with biocellular systems [4,5]. To analyze different types of NP-cell interactions, such as cellular NP uptake, it is essential to have valuable techniques for visualization and quantification, which are mainly based on spectroscopy or microscopy [6].The method of choice for the intracellular detection of NPs depends on the characteristics of the particles (fluorescence, size, and structure or electron density) and on the cellular structure of interest. Advantages of spectroscopy and different microscopic techniques have been reviewed in detail elsewhere [6,7]. In brief, spectroscopy such as inductively coupled plasma-mass spectroscopy [8] allows the quantification and detection of chemical NP elements within the cell, but does not provide any further information on the NP location within the cell. Fluorescence or laser scanning microscopy (LSM) allows real-time analysis or co-localization studies with fluorescently labeled structures [9]. Transmission electron microscopy (TEM) provides the highest resolution and allows quantification of NPs in sub cellular structures [10].