6.2.3 In Vitro Cell Models in Drug Screening
A significant chapter of Raman-based protocols for cellular investigations is in vitro
drug screening. The potential of Raman spectroscopy in this field has been well
established over the last decade, especially in understanding of chemotherapeutic
drugs’ actions. Raman spectroscopy gives an opportunity to determine the cellular
response to a given compound and also to follow the uptake and intracellular
accumulation of drugs and potentially identify cell-resistant strains [109]. Raman
spectroscopy together with the protocol of cell cloning also proved to be especially
useful for clinicians to better tailor the treatment to each individual patient [110].
Among a number of compounds tested with the use of Raman-based methodology
such as cisplatin, vincristine, and nilotinib, a substantial interest has been put on
doxorubicin (DOX) and its analogues [111, 112]. DOX belongs to the group of
compounds named anthracyclines, which exhibit cytostatic activity and are widely
used in chemotherapy. Despite the relatively well-established usage of DOX in
anti-tumor treatment, its mechanism of action is not fully elucidated, but it is known
that it includes such events as intercalation into nuclear DNA, DNA alkylation,
inhibition of DNA synthesis or formation of free radicals, altogether leading to cell
apoptosis [113, 114]. DOX-based therapy is complicated by its cardiotoxicity
which can lead to severe cardiac complications even years after administration. This
duality of DOX activity leads to a large number of investigations that aim on
assessing both the activity and cardiotoxicity effects but also synthesis of analogues
and derivatives of DOX [115]. Structurally, anthracyclines possess a tetracycline
ring structure with a daunosamine group attached by a glycosidic linkage. This
tetracycle is a chromophore group giving the absorption band at 480 nm (p−p
*
transition). This feature gives rise to the Raman spectrum of DOX acquired at the
488-nm excitation wavelength exhibiting a very strong fluorescence background
(Fig. 6.6) [116]. The non-resonant spectra of DOX can be recorded at the 785-nm
excitation wavelength, where the characteristic spectral profile is present with
several marker bands (Fig. 6.6). Considering the above facts, spectral features at
both wavelengths are suitable for investigations focused on DOX–cell interactions.
The potential of imaging in assessment of nuclear accumulation of anthracyclines in
endothelial cells was demonstrated with the use of the 488-nm excitation [117]. The
maximum of DOX fluorescence coincides with the range in which the increased
background in DOX-treated cells is elevated. This enables for the analysis of
changes in cell spectral profile that occurs after treatment with DOX but also for
imaging of its distribution inside the cell (Fig. 6.6). Investigations performed at the
785-nm excitation reveal similar information on the distribution of the drug based,
however, on the characteristic doxorubicin Raman marker bands [112]. However,
spectra collected with the use of the 785-nm excitation enable for more specific
investigations of the modes of interaction of the drug, as, in the absence of the
strong fluorescence background, the drug itself as well as the biochemical cell
environment can be monitored in parallel. On the other hand, for fast imaging with
better spatial resolution, application of the 488-nm excitation is preferable.
176
K. Czamara et al.
A significant chapter of Raman-based protocols for cellular investigations is in vitro
drug screening. The potential of Raman spectroscopy in this field has been well
established over the last decade, especially in understanding of chemotherapeutic
drugs’ actions. Raman spectroscopy gives an opportunity to determine the cellular
response to a given compound and also to follow the uptake and intracellular
accumulation of drugs and potentially identify cell-resistant strains [109]. Raman
spectroscopy together with the protocol of cell cloning also proved to be especially
useful for clinicians to better tailor the treatment to each individual patient [110].
Among a number of compounds tested with the use of Raman-based methodology
such as cisplatin, vincristine, and nilotinib, a substantial interest has been put on
doxorubicin (DOX) and its analogues [111, 112]. DOX belongs to the group of
compounds named anthracyclines, which exhibit cytostatic activity and are widely
used in chemotherapy. Despite the relatively well-established usage of DOX in
anti-tumor treatment, its mechanism of action is not fully elucidated, but it is known
that it includes such events as intercalation into nuclear DNA, DNA alkylation,
inhibition of DNA synthesis or formation of free radicals, altogether leading to cell
apoptosis [113, 114]. DOX-based therapy is complicated by its cardiotoxicity
which can lead to severe cardiac complications even years after administration. This
duality of DOX activity leads to a large number of investigations that aim on
assessing both the activity and cardiotoxicity effects but also synthesis of analogues
and derivatives of DOX [115]. Structurally, anthracyclines possess a tetracycline
ring structure with a daunosamine group attached by a glycosidic linkage. This
tetracycle is a chromophore group giving the absorption band at 480 nm (p−p
*
transition). This feature gives rise to the Raman spectrum of DOX acquired at the
488-nm excitation wavelength exhibiting a very strong fluorescence background
(Fig. 6.6) [116]. The non-resonant spectra of DOX can be recorded at the 785-nm
excitation wavelength, where the characteristic spectral profile is present with
several marker bands (Fig. 6.6). Considering the above facts, spectral features at
both wavelengths are suitable for investigations focused on DOX–cell interactions.
The potential of imaging in assessment of nuclear accumulation of anthracyclines in
endothelial cells was demonstrated with the use of the 488-nm excitation [117]. The
maximum of DOX fluorescence coincides with the range in which the increased
background in DOX-treated cells is elevated. This enables for the analysis of
changes in cell spectral profile that occurs after treatment with DOX but also for
imaging of its distribution inside the cell (Fig. 6.6). Investigations performed at the
785-nm excitation reveal similar information on the distribution of the drug based,
however, on the characteristic doxorubicin Raman marker bands [112]. However,
spectra collected with the use of the 785-nm excitation enable for more specific
investigations of the modes of interaction of the drug, as, in the absence of the
strong fluorescence background, the drug itself as well as the biochemical cell
environment can be monitored in parallel. On the other hand, for fast imaging with
better spatial resolution, application of the 488-nm excitation is preferable.
176
K. Czamara et al.