388
H. J. Byrne et al.
medium in vitro, and with the cytotoxic assays themselves [152, 152]. the underlying principles of internalisation and distribution of nanoparticles within cells and
any associated toxicity to humans are still relatively poorly understood. however,
there are numerous studies that have demonstrated the ability of nanoparticles to
cross membranes and internalise within many different cell types [153–155]. In
order to assess potential risks, but also benefits in terms of nanomedicine, a greater
understanding of migration of nanoparticles intra- and inter-cellularly and essentially from one part of the body to another, along with information on the translocation
of nanoparticles within organelles, is urgently required.
Among the main challenges faced by nanotoxicologists are the detection and
identification of nanoparticles that have crossed the cell membrane and monitoring of their intracellular trafficking. Imaging cells exposed to fluorescently labelled
nanoparticles using confocal fluorescence microscopy is one of the most common
ways in which to ensure nanoparticles can be tracked and monitored as they enter
and localise within cells [156–158]. however, not all nanoparticles can be easily
fluorescently labelled. Furthermore, there have been reports that labelled nanoparticles can release the dye into the surrounding biological environment, and so the distribution of fluorescence within the cell does not necessarily represent the presence
or subcellular distribution of the nanoparticles [159–161]. Furthermore, it is not
clear that the transport mechanisms of smaller nanoparticles, fluorescently labelled
with anionic moieties, are the same as their unlabelled counterparts. transmission
Electron microscopy can also be employed to visualise nanoparticles within cells,
but significant sample processing (fixing and ultramicrotoming) is required and
only particles with sufficient electronic contrast to the cellular environment can be
visualized [162, 163]. thus, there is a requirement for a sensitive technique to localise and identify nanoparticles internalised in cells, ideally based on their chemical
composition, rather than fluorescence labels or electronic contrast. Identification of
their local environment (e.g. endosomes, lysosomes, mitochondria) could aid in elucidating their intracellular trafficking and interaction mechanisms, and the resulting
changes in cellular metabolism.
Raman spectroscopy potentially offers a label free probe of nanoparticles within
cells, which can potentially analyse their local environment, and ultimately changes
in the cellular metabolism which can be correlated with cytotoxic responses [164],
oxidative stress, or inflammation. Kneipp et al. have previously demonstrated the
use of Surface Enhanced Raman Scattering from gold nanoparticles and nanoaggregates to probe the environment of the subcellular compartments through which
they are trafficked [165, 166]. they have also demonstrated the use of molecular
labelled nanoparticles as more specific probes of the local environment [167–169].
however, the uptake rates and mechanisms as well as the subsequent trafficking
may be specific to the nanoparticle type, size and surface chemistry. Furthermore,
the molecular specificity of the surface enhancement process is not well understood.
therefore, a truly label free method for monitoring and characterising the cellular
uptake and subcellular localisation of nanoparticles in general is still required.
It has been demonstrated that, based on the spectroscopic fingerprint of polystyrene, Raman spectroscopy, coupled with routine multivariate analyses such as
H. J. Byrne et al.
medium in vitro, and with the cytotoxic assays themselves [152, 152]. the underlying principles of internalisation and distribution of nanoparticles within cells and
any associated toxicity to humans are still relatively poorly understood. however,
there are numerous studies that have demonstrated the ability of nanoparticles to
cross membranes and internalise within many different cell types [153–155]. In
order to assess potential risks, but also benefits in terms of nanomedicine, a greater
understanding of migration of nanoparticles intra- and inter-cellularly and essentially from one part of the body to another, along with information on the translocation
of nanoparticles within organelles, is urgently required.
Among the main challenges faced by nanotoxicologists are the detection and
identification of nanoparticles that have crossed the cell membrane and monitoring of their intracellular trafficking. Imaging cells exposed to fluorescently labelled
nanoparticles using confocal fluorescence microscopy is one of the most common
ways in which to ensure nanoparticles can be tracked and monitored as they enter
and localise within cells [156–158]. however, not all nanoparticles can be easily
fluorescently labelled. Furthermore, there have been reports that labelled nanoparticles can release the dye into the surrounding biological environment, and so the distribution of fluorescence within the cell does not necessarily represent the presence
or subcellular distribution of the nanoparticles [159–161]. Furthermore, it is not
clear that the transport mechanisms of smaller nanoparticles, fluorescently labelled
with anionic moieties, are the same as their unlabelled counterparts. transmission
Electron microscopy can also be employed to visualise nanoparticles within cells,
but significant sample processing (fixing and ultramicrotoming) is required and
only particles with sufficient electronic contrast to the cellular environment can be
visualized [162, 163]. thus, there is a requirement for a sensitive technique to localise and identify nanoparticles internalised in cells, ideally based on their chemical
composition, rather than fluorescence labels or electronic contrast. Identification of
their local environment (e.g. endosomes, lysosomes, mitochondria) could aid in elucidating their intracellular trafficking and interaction mechanisms, and the resulting
changes in cellular metabolism.
Raman spectroscopy potentially offers a label free probe of nanoparticles within
cells, which can potentially analyse their local environment, and ultimately changes
in the cellular metabolism which can be correlated with cytotoxic responses [164],
oxidative stress, or inflammation. Kneipp et al. have previously demonstrated the
use of Surface Enhanced Raman Scattering from gold nanoparticles and nanoaggregates to probe the environment of the subcellular compartments through which
they are trafficked [165, 166]. they have also demonstrated the use of molecular
labelled nanoparticles as more specific probes of the local environment [167–169].
however, the uptake rates and mechanisms as well as the subsequent trafficking
may be specific to the nanoparticle type, size and surface chemistry. Furthermore,
the molecular specificity of the surface enhancement process is not well understood.
therefore, a truly label free method for monitoring and characterising the cellular
uptake and subcellular localisation of nanoparticles in general is still required.
It has been demonstrated that, based on the spectroscopic fingerprint of polystyrene, Raman spectroscopy, coupled with routine multivariate analyses such as