218
following xenobiotic exposure can be detected by monitoring metabolite variations
and can be studied via a metabolomics approach. Metabolomics can provide a fast
screening of a wide range of metabolites (biomarkers) that are related to welldefined pathways or processes, therefore giving mechanistic insight into NMs toxicity. The main techniques applied in metabolomic studies are liquid chromatography
coupled with mass spectrometry and nuclear magnetic resonance spectroscopy (Lv
et al. 2015).
Moreover, advanced microspectroscopy techniques are gaining importance for
the evaluation of physiological and pathological events. For instance, infrared
microspectroscopy, as a noninvasive and label-free technique, is considered a fast
and informative multiscreening platform to study complex systems such as cells and
tissues, allowing the characterization of the most important cellular components
(proteins, lipids, nucleic acids, and carbohydrates), as well as their possible modifications, both at a compositional and a structural level. As an example, the gathered
details obtained with this technique can provide information of membrane fluidity
and composition, protein content and structure, and nucleic acid structural alterations. Thus, infrared microspectroscopy can overcome the limitations of classical
methods, providing more accurate information for the toxicological evaluation of
NMs and other xenobiotics (Bunaciu et al. 2014).
8.5 Challenges of Toxicological In Vitro Testing
As previously discussed, the physicochemical properties which make NMs interesting systems for the biomedical field are also correlated to their high reactivity and
biological activity. Also, several experimental challenges, not only dependent on
their physicochemical properties, emerge when studying NM-induced toxicity
(Fig. 8.4). Thus, the safety assessment of NMs is often more complex than safety
assessment of bulk materials.
Bulk material chemical and biological purity are relevant parameters for nanomedicine production and activity, as well as in hazard characterization. For instance,
the presence of hazardous and bioactive contaminants, such as endotoxins, can
induce inflammatory signaling mediators and other immunological responses,
endotoxin shock, and even tissue injury (Azhdarzadeh et al. 2015). Thus, endotoxin
content should be evaluated before immunogenicity studies. If the presence of
endotoxins is detected, they can be removed by sterilization methods, such as filtration, autoclaving, or irradiation (Peters et al. 2017). However, these conventional
techniques can interfere with and alter NMs physicochemical properties, and, like a
cycle, affect their efficacy (Vetten et al. 2014). Therefore, in the pharmaceutical
industry more advanced techniques for endotoxin removal such as several types of
chromatography and ultrafiltration are commonly performed. For instance, affinity
chromatography Polymyxin B columns are very effective for endotoxin removal
due to their very high binding affinity for lipid A, which is one of the main components of LPS. However, these facts only reveal that there is still no efficient and
M. C. Teixeira et al.
following xenobiotic exposure can be detected by monitoring metabolite variations
and can be studied via a metabolomics approach. Metabolomics can provide a fast
screening of a wide range of metabolites (biomarkers) that are related to welldefined pathways or processes, therefore giving mechanistic insight into NMs toxicity. The main techniques applied in metabolomic studies are liquid chromatography
coupled with mass spectrometry and nuclear magnetic resonance spectroscopy (Lv
et al. 2015).
Moreover, advanced microspectroscopy techniques are gaining importance for
the evaluation of physiological and pathological events. For instance, infrared
microspectroscopy, as a noninvasive and label-free technique, is considered a fast
and informative multiscreening platform to study complex systems such as cells and
tissues, allowing the characterization of the most important cellular components
(proteins, lipids, nucleic acids, and carbohydrates), as well as their possible modifications, both at a compositional and a structural level. As an example, the gathered
details obtained with this technique can provide information of membrane fluidity
and composition, protein content and structure, and nucleic acid structural alterations. Thus, infrared microspectroscopy can overcome the limitations of classical
methods, providing more accurate information for the toxicological evaluation of
NMs and other xenobiotics (Bunaciu et al. 2014).
8.5 Challenges of Toxicological In Vitro Testing
As previously discussed, the physicochemical properties which make NMs interesting systems for the biomedical field are also correlated to their high reactivity and
biological activity. Also, several experimental challenges, not only dependent on
their physicochemical properties, emerge when studying NM-induced toxicity
(Fig. 8.4). Thus, the safety assessment of NMs is often more complex than safety
assessment of bulk materials.
Bulk material chemical and biological purity are relevant parameters for nanomedicine production and activity, as well as in hazard characterization. For instance,
the presence of hazardous and bioactive contaminants, such as endotoxins, can
induce inflammatory signaling mediators and other immunological responses,
endotoxin shock, and even tissue injury (Azhdarzadeh et al. 2015). Thus, endotoxin
content should be evaluated before immunogenicity studies. If the presence of
endotoxins is detected, they can be removed by sterilization methods, such as filtration, autoclaving, or irradiation (Peters et al. 2017). However, these conventional
techniques can interfere with and alter NMs physicochemical properties, and, like a
cycle, affect their efficacy (Vetten et al. 2014). Therefore, in the pharmaceutical
industry more advanced techniques for endotoxin removal such as several types of
chromatography and ultrafiltration are commonly performed. For instance, affinity
chromatography Polymyxin B columns are very effective for endotoxin removal
due to their very high binding affinity for lipid A, which is one of the main components of LPS. However, these facts only reveal that there is still no efficient and
M. C. Teixeira et al.