Abstract
Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.
Highlights
The fact that natural and engineered nanoparticles (NPs) have novel properties and the potential to access isolated parts of the body, including the brain, creates a coherent interest to develop nanomaterials for biomedical applications such as imaging, diagnostics and therapeutics
By Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), e.g. determination of hydrodynamic radii of the particles, we show that these intracellular interactions are highly dynamic and performed by monodisperse NPs
In order to define the properties of particles used in the current analyses, transmission electron microscopy (TEM) was employed to determine the size of PS NPs and silica NPs in detail
Summary
The fact that natural and engineered nanoparticles (NPs) have novel properties and the potential to access isolated parts of the body, including the brain, creates a coherent interest to develop nanomaterials for biomedical applications such as imaging, diagnostics and therapeutics. At their target organ, NPs effectively enter cells by endocytosis [1,2,3,4], the underlying in-depth mechanisms of cross-membrane translocation are still largely unknown. Consistent with this, understanding the biological interface of nanomaterials requires biophysical methods to analyse the properties and behavior of NPs within cells [8]. Characterization of NPs in solution conventionally involves analyses of features such as hydrodynamic radius (Rh) and zeta potential by means of dynamic light scattering
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