215
advanced “omics” approaches, later discussed in Sect. 8.4.5 (Suzuki et al. 2010;
Calvano et al. 2014).
8.4.3 Proinflammatory Activity and Immunological Response
NMs hold the capacity to interact with the immune system and induce immunological responses (Shegokar et al. 2018). To study NM-induced immunotoxicity, several
in vitro assays have been developed. Some of them, as hemolysis and granulocyte
macrophage colony-forming units (CFU-GM), have been already standardized by
American Society for Testing and Materials International for immunotoxicity evaluation of NMs (Dobrovolskaia 2017).
However, immune response in cells is triggered by the release of cytokines,
mainly expressed by macrophages, that play a key role in the regulation of the
immune response, inflammatory reaction, and phagocytosis (Omar et al. 2015).
Production and release of cytokines are well-known responses of immune system
and represent a widely accepted way to estimate immunostimulation of substances.
Depending on types and levels of cytokines, it is possible to disclose the mechanisms of immunostimulation. Whole blood, peripheral blood mononuclear cells
(PBMC), and human-derived MM-6-cell line are the most common cell culture
models for analyzing cytokines. In particular, whole blood cultures are widely used
in the drug development industry, and they are recommended by the European Food
Safety Authority Scientific Committee (Dobrovolskaia and McNeil 2013). Cytokine
production and release are usually assessed by ELISA (Gunsolus and Haynes 2016).
ELISA takes advantage of antibody/antigen recognition for detecting specific proinflammatory and antiinflammatory cytokines such as IL-8, IL-1β, IL-6, and TNF-α
(Ledur et al. 1995). By means of enzyme-conjugates antibodies such as horseradish
peroxidase and alkaline phosphatase, it is possible to quantify released cytokines by
recording fluorimetric, colorimetric, or luminescent signals (Ledur et al. 1995). The
release of proinflammatory cytokines could also be assessed by monocyte-derived
dendritic cell maturation assay. Indeed, dendritic cells, relevant for their antigenpresenting activity, maturate following exposure to inflammatory cytokines (e.g.,
TNF-α) or pathogen-associated molecular patterns (PAMPs) (e.g., bacterial LPS).
This assay is not only useful to screen nanocarrier interference on DC maturation
process, but it could also be used to study their potential cytotoxicity (Sousa et al.
2017). Besides, dendritic cells maturation could be applied to nanoparticle-based
vaccine formulation in vitro study (Knuschke et al. 2013; Tomić et al. 2014). The
involvement of specific mechanisms in modulating NM-induced immunotoxicity
can also be investigated by analyzing differentially expressed genes by microarrays
(Love et al. 2012).
To complement cytokines analysis, hemolysis, complement activation, and
thrombogenicity testing are also recommended. In vitro hemolysis tests showed a
good correlation with in vivo tests, independently from blood origin species and
anticoagulant (Dobrovolskaia and McNeil 2013). Complement activation is the part
8 In Vitro Methodologies for Toxicological Assessment of Drug Delivery Nanocarriers
advanced “omics” approaches, later discussed in Sect. 8.4.5 (Suzuki et al. 2010;
Calvano et al. 2014).
8.4.3 Proinflammatory Activity and Immunological Response
NMs hold the capacity to interact with the immune system and induce immunological responses (Shegokar et al. 2018). To study NM-induced immunotoxicity, several
in vitro assays have been developed. Some of them, as hemolysis and granulocyte
macrophage colony-forming units (CFU-GM), have been already standardized by
American Society for Testing and Materials International for immunotoxicity evaluation of NMs (Dobrovolskaia 2017).
However, immune response in cells is triggered by the release of cytokines,
mainly expressed by macrophages, that play a key role in the regulation of the
immune response, inflammatory reaction, and phagocytosis (Omar et al. 2015).
Production and release of cytokines are well-known responses of immune system
and represent a widely accepted way to estimate immunostimulation of substances.
Depending on types and levels of cytokines, it is possible to disclose the mechanisms of immunostimulation. Whole blood, peripheral blood mononuclear cells
(PBMC), and human-derived MM-6-cell line are the most common cell culture
models for analyzing cytokines. In particular, whole blood cultures are widely used
in the drug development industry, and they are recommended by the European Food
Safety Authority Scientific Committee (Dobrovolskaia and McNeil 2013). Cytokine
production and release are usually assessed by ELISA (Gunsolus and Haynes 2016).
ELISA takes advantage of antibody/antigen recognition for detecting specific proinflammatory and antiinflammatory cytokines such as IL-8, IL-1β, IL-6, and TNF-α
(Ledur et al. 1995). By means of enzyme-conjugates antibodies such as horseradish
peroxidase and alkaline phosphatase, it is possible to quantify released cytokines by
recording fluorimetric, colorimetric, or luminescent signals (Ledur et al. 1995). The
release of proinflammatory cytokines could also be assessed by monocyte-derived
dendritic cell maturation assay. Indeed, dendritic cells, relevant for their antigenpresenting activity, maturate following exposure to inflammatory cytokines (e.g.,
TNF-α) or pathogen-associated molecular patterns (PAMPs) (e.g., bacterial LPS).
This assay is not only useful to screen nanocarrier interference on DC maturation
process, but it could also be used to study their potential cytotoxicity (Sousa et al.
2017). Besides, dendritic cells maturation could be applied to nanoparticle-based
vaccine formulation in vitro study (Knuschke et al. 2013; Tomić et al. 2014). The
involvement of specific mechanisms in modulating NM-induced immunotoxicity
can also be investigated by analyzing differentially expressed genes by microarrays
(Love et al. 2012).
To complement cytokines analysis, hemolysis, complement activation, and
thrombogenicity testing are also recommended. In vitro hemolysis tests showed a
good correlation with in vivo tests, independently from blood origin species and
anticoagulant (Dobrovolskaia and McNeil 2013). Complement activation is the part
8 In Vitro Methodologies for Toxicological Assessment of Drug Delivery Nanocarriers