**3. Difficulties in nanomaterial cytotoxicity analysis: Aggregates, protein corona and NP degradation**

Nanomaterials are intensively studied as promising candidates for biomedical applications (e.g., targeted delivery of therapeutic drugs and medical imaging) with a purpose of eventual human administration [34]. NP design for medical applications should not only meet requirements, such as biocompatibility and biodegradability, but also site-specific delivery, long blood circulation and high cargo loading capacity [35]. Different nanomaterials show unique physical and chemical properties that depend, among others, on the type of materials (e.g., Au or Ag, Fe3 O4 , graphene and graphene oxide), hydrodynamic size, surface charge and aggregation behaviour and have been found to interact, often immediately (within seconds), after contact with biological systems, such as blood or tissue [34, 36, 37]. Nanoparticle aggregation via electrostatic screening can occur in complex aqueous mixtures of cell culture media that contain electrolytes, proteins, lipids and metabolites (highly ionic environment) [11, 38].

NPs at higher concentrations tend to form aggregates (agglomerates) under artificial conditions of *in vitro* cell cultures [16]. Many experiments found that NPs that form aggregates were not as cytotoxic as the same NPs at lower concentrations. Lower concentrations of NPs resulted in better internalisation and biodistribution in the circulatory system and organs [3]. Aggregation process is caused by magnetic attraction forces (types 1, 2 and 4), hydrophobic-hydrophobic interactions (for type 1) or hydrogen bonding between hydroxyl groups [39]. Different types of nanomaterials exhibit different tendency to form aggregates in PBS and culture media. CNTs have a strong tendency to agglomerate due to van der Waals interactions [40]. Metal oxides display higher tendency to form agglomerates in comparison to MWCNT. Metal oxides differ in size but were of similar size in PBS environment; thus, it was concluded that surface chemistry and/or the environment had a more significant effect on the aggregate formation process [14]. The size of aggregates may be dependent on the concentration and they tend to be slightly larger in culture media than in PBS. Moreover, monovalent and divalent cations may affect aggregate formation. Adsorption of media components, serum proteins and Ca2+ on nanoparticle surface determines NP aggregations and size distribution [14]. Agglomeration leads to cytotoxicity reduction, because of lower availability of inorganic NPs in contact with cells. In addition, the size of aggregates prevents their cellular internalisation [39]. Studies based on silica nanoparticles indicated that minimization of NP aggregation could be obtained by introducing an optimum balance of inert (e.g., methyl phosphonate) and active (e.g., hydroxyl and aldehyde) functional groups to the surface [41].

LDH assay results should be verified by calibration curves in the presence of different SWCNT concentrations (in the range of 5–100 μgml−1) at two wavelengths, 580 and 490 nm, for each

Smith et al. [31] presented a simple protocol modification of the LDH analysis, which included membered additional conditional-specific controls. This modification enables accurate simultaneous measurement of the effects of death and growth inhibition. The additional step provides quantitative information that can be useful in applications such as drug discoveries [31]. Another approach in LDH assay analysis was proposed in the experiments of Chan et al. [32]. Modification of the LDH protocol allows to detect necrosis, including secondary

In addition, calcein AM (CAM), Live/Death, neutral red, CellTiter®, Aqueous One (96 AQ), Alamar Blue (AB), CellTiter-Blue® (CTB), CytoToxOne™, and flow cytometry were used to determine their utility in nanoparticle toxicity evaluation. In the cited study, it was found that the results of the assay that depended on direct staining of cells were difficult to interpret, because of dye interactions with NPs. The 96 AQ assay proved optimal for NP analysis. The results were not significantly altered by interactions between the test factor and reagents in the assay [16]. Herzog et al. [33] suggested the clonogenic assay to determine real cytotoxic effect on cell cultures due to the false results (positive or negative) that may occur in NP testing. The clonogenic assay (colony formation assay) is based on the ability of a single cell to form a colony. The latter study was based on the ability of A549, BEAS-2B (normal human bronchial epithelial cells) and HaCaT (normal human keratinocytes) cells to form colonies after 7 (for HaCaT cells) and 10 (for A549 and BEAS-2B) days of incubation with SWCNT (HiPco®). The EC50 comparison showed that the A549 cell line was more resistant than the other two lines. On the other hand, the analysis based on colony size showed that A549 was more sensitive than HaCaT cells. Although the clonogenic assay provided more accurate results than colorimetric tests, it did not become popular because it was too time-consuming for rapid toxicity screening [19, 33].

**3. Difficulties in nanomaterial cytotoxicity analysis: Aggregates,** 

Nanomaterials are intensively studied as promising candidates for biomedical applications (e.g., targeted delivery of therapeutic drugs and medical imaging) with a purpose of eventual human administration [34]. NP design for medical applications should not only meet requirements, such as biocompatibility and biodegradability, but also site-specific delivery, long blood circulation and high cargo loading capacity [35]. Different nanomaterials show unique physical and chemical properties that depend, among others, on the type of materials

aggregation behaviour and have been found to interact, often immediately (within seconds), after contact with biological systems, such as blood or tissue [34, 36, 37]. Nanoparticle aggregation via electrostatic screening can occur in complex aqueous mixtures of cell culture media that contain electrolytes, proteins, lipids and metabolites (highly ionic environment) [11, 38].

, graphene and graphene oxide), hydrodynamic size, surface charge and

**protein corona and NP degradation**

O4

(e.g., Au or Ag, Fe3

LDH assay. This procedure more accurately determines cellular toxicity values [30].

necrosis [32].

184 Cytotoxicity

The protein layer of several nanometres on particle surface is called protein corona and it can be divided into a peripheral *soft* corona (SC)—dynamic protein exchanges with the surrounding medium—and a *hard* corona (HC)—a layer of more or less temporal constant composition (**Figure 2**, **Table 2**) [34, 42, 43]. In blood plasma, the surface of nanoparticles mainly adsorbs proteins, but some minor traces of lipids have also been found in the corona structure. Adsorption of proteins on the nanoparticle surface is the result of protein-nanoparticle binding affinities and protein-protein interactions. *Hard* corona interacts directly with the nanomaterial surface. *Soft* corona proteins interact with the hard corona via weak proteinprotein interactions. Interestingly, the corona on the NP surface does not completely mask the nanomaterial surface or its functional groups [43]. The formation of protein corona and its thickness is a parameter that is also dependent on protein concentration, temperature, duration of particle-protein interaction, serum concentration and shear stress [34, 44].

Protein corona formation strongly affects cellular uptake mechanism, cell-nanoparticle interactions, intracellular location as well as cellular response (e.g., biocompatibility) [34, 35, 44]. The protein corona on the NP surface is hypothesised to hinder interactions of nanoparticle ligands and the targets on the cell surface [44, 47].

The study of Mirshafiee et al. [44] found that the protein corona formed on BCN-NPs (NPs functionalized with bicyclononyne) incubated in medium with 10% serum and 100% serum consisted of abundant proteins, such as chain A, a novel allosteric mechanism in haemoglobin, fetuin, haemoglobin foetal subunit beta or apolipoprotein A-II precursor. It was also reported that ≥88% of proteins in BCN-NP coronas had a molecular weight below 30 kDa. Even relatively low molecular weight proteins created corona that significantly reduced NP

**Figure 2.** Structure of protein corona [34, 42, 43, 45].

targeting efficiency [44]. Single-walled carbon nanotubes (HiPco®) were also found to interact with cell culture medium and its components. Casey et al. described that SWCNT interacted with the medium via physisorption through van der Waals forces [26, 48].

tendency to form aggregates was mostly dependent on physicochemical properties of NPs. However, Tenzer et al. [49] did not describe negative effects of AmSil30 precoated with the protein plasma corona. Biological effects of protein-NPs were analysed using two lines: HeLa and U937 [43, 50]. The study conducted by Maiorano et al. [50] demonstrated that AuNPs incubated in two different culture media (DMEM and RPMI) exhibited different protein coronas. RPMI-treated NPs had less prominent protein coronas and, as a consequence, induced stronger toxicity of HeLa and U937 cells [50]. The study of Gräfe et al. [34] reported that the presence of the protein corona reduced the interaction of human brain microvascular endothelial cells (HBMEC) with magnetic nanoparticles coated with PEI (polyethylenimine) dur-

(↑koff)

response

Nanoparticle-induced pathological effects, such as cell death, coagulation, thrombocytosis or cytotoxicity, are also dependent on the type of NPs, but selected cellular model is also crucial in this kind of experiments [49, 51]. For example, polystyrene-based NPs (PS) with different

The composition of protein corona was analysed using various methods and it was demonstrated that albumin, immunoglobulin G (IgG), fibrinogen and apolipoproteins were present in

both exposed cell lines (HeLa and hMSCs). NPs with PS-NH<sup>2</sup>

**Hard corona Soft corona**

Tightly bound proteins Loosely bound proteins

Directly interacting with surface of nanoparticles Protein-protein interaction

Lower dissociation rate of proteins with nanoparticles

Stable on nanoparticle surface; able to influence the

**Table 2.** Characteristic features of hard (HC) and soft corona (SC) [46].

(↓koff)

functional response

Large binding energy adsorption (↑|ΔGads|) Low binding energy adsorption (↓|ΔGads|)

groups coated with the serum protein were effectively taken up by

and PS-SO3

Higher dissociation rate of proteins with nanoparticles

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Fleeting on nanoparticles; irrelevant for the functional

groups showed

ing 30 min of incubation [34].

lower uptake by both cell lines [51].

PS-COOH and PS-PO3

The process of protein corona formation has a decisive influence on nanoparticle-induced toxicity. For example, silica nanoparticles (AmSil30) precoated with human plasma caused lower cell-death induction in primary human endothelial cells and microvascular endothelial cell line (ISO-HAS1). The resulting effect was dependent on the time of corona formation. The most significant effect was recorded for the early corona, but prolonged incubation with plasma (>30 min) did not counteract membered toxicity [49]. In another example, thrombocytes were used to study the protein corona effect on the biological model. In the latter study, nanoparticles exposed to human plasma for 0.5 min did not activate thrombocytes to form aggregates due to the presence of the plasma protein corona [49]. The impact of protein corona formation on cellular uptake and dispersion state of nanoparticles after exposure to plasma was also investigated. It was found that NPs were monodispersed after short-time exposure (<10 min), whereas aggregates started to form during prolonged exposure (>30 min), but the


**Table 2.** Characteristic features of hard (HC) and soft corona (SC) [46].

targeting efficiency [44]. Single-walled carbon nanotubes (HiPco®) were also found to interact with cell culture medium and its components. Casey et al. described that SWCNT interacted

The process of protein corona formation has a decisive influence on nanoparticle-induced toxicity. For example, silica nanoparticles (AmSil30) precoated with human plasma caused lower cell-death induction in primary human endothelial cells and microvascular endothelial cell line (ISO-HAS1). The resulting effect was dependent on the time of corona formation. The most significant effect was recorded for the early corona, but prolonged incubation with plasma (>30 min) did not counteract membered toxicity [49]. In another example, thrombocytes were used to study the protein corona effect on the biological model. In the latter study, nanoparticles exposed to human plasma for 0.5 min did not activate thrombocytes to form aggregates due to the presence of the plasma protein corona [49]. The impact of protein corona formation on cellular uptake and dispersion state of nanoparticles after exposure to plasma was also investigated. It was found that NPs were monodispersed after short-time exposure (<10 min), whereas aggregates started to form during prolonged exposure (>30 min), but the

with the medium via physisorption through van der Waals forces [26, 48].

**Figure 2.** Structure of protein corona [34, 42, 43, 45].

186 Cytotoxicity

tendency to form aggregates was mostly dependent on physicochemical properties of NPs. However, Tenzer et al. [49] did not describe negative effects of AmSil30 precoated with the protein plasma corona. Biological effects of protein-NPs were analysed using two lines: HeLa and U937 [43, 50]. The study conducted by Maiorano et al. [50] demonstrated that AuNPs incubated in two different culture media (DMEM and RPMI) exhibited different protein coronas. RPMI-treated NPs had less prominent protein coronas and, as a consequence, induced stronger toxicity of HeLa and U937 cells [50]. The study of Gräfe et al. [34] reported that the presence of the protein corona reduced the interaction of human brain microvascular endothelial cells (HBMEC) with magnetic nanoparticles coated with PEI (polyethylenimine) during 30 min of incubation [34].

Nanoparticle-induced pathological effects, such as cell death, coagulation, thrombocytosis or cytotoxicity, are also dependent on the type of NPs, but selected cellular model is also crucial in this kind of experiments [49, 51]. For example, polystyrene-based NPs (PS) with different PS-COOH and PS-PO3 groups coated with the serum protein were effectively taken up by both exposed cell lines (HeLa and hMSCs). NPs with PS-NH<sup>2</sup> and PS-SO3 groups showed lower uptake by both cell lines [51].

The composition of protein corona was analysed using various methods and it was demonstrated that albumin, immunoglobulin G (IgG), fibrinogen and apolipoproteins were present in the corona of all the analysed nanoparticles [43]. Corona identification and composition analysis (**Table 3**) provide not only information about its complexity, conditions of PC formation and physicochemical features but also data on toxicity, cellular interactions and uptake, targeting and finally the usefulness in nanomedicine [46].

For example, Urbas et al. [52] demonstrated that three types of nanoparticles, NPs-GO, Fe3 O4 , and GO-Fe<sup>3</sup> O4 , displayed the ability to deplete various quantities of serum proteins from culture media (**Figure 3**). Graphene oxide and nanocomposite GO-Fe3 O4 showed an increase in protein adsorption from culture medium. The results of the bicinchoninic acid (BCA) assay indicated different capacities of NPs to adsorb proteins in cell cultures [52].

Protein corona composition is also known to affect nanoparticle-cell interactions and biological fate of nanomaterials in cells. Gunawan and co-authors characterised the term 'biological fate' as describing the subcellular localisation of NPs and the distribution of NPs to specific organs *in vivo* [53]. An interesting study performed by Lesniak et al. [54] showed that silica (SiO<sup>2</sup> ) nanoparticles (50 nm) exposed to biological fluids (e.g., serum) mediated the interaction of NPs (at 100 μgmL−1 concentration) with A549 cells. Silica nanoparticles showed different degree and process of internalisation during incubation with the A549 cell line in complete (with 10% foetal bovine serum) and in serum-free medium. NP integration was higher in serum-free medium with accumulation in lysosomes and some of NPs localised free in the cytosol. On the contrary, NPs in complete medium (in the presence of a well-developed corona) were never observed free in the cytoplasmic matrix, but similar to serum-free medium, silica nanoparticles were found to accumulate in lysosomes. Lesniak et al. [54] observed that nanoparticles showed higher tendency to adhere to the cell membrane in serum-free conditions and concluded that the initial stronger adhesion could have partly contributed to higher uptake efficiency. Moreover, the presence of free NPs in the cytosol might be caused by perturbation of the early uptake pathway in cells exposed to serum-free medium (rather than an endogenously regulated cellular process) [53, 54].

on PEG-SWCNTs due to the surface charge and the conformation of surface functional groups (PEG); this resulted in higher accumulation of the aforementioned NPs in the liver compared to the spleen [55]. Poly(D,L-lactide)-based NPs showed interaction of surface functional group (covalently conjugated with apoB100 antibody) with LDL and were highly accumulated by liver macrophages [56]. Solid lipid nanoparticles (SLNs) modified with PEG induced the ABC phenomenon (accelerated blood clearance) upon repeated injections in mice and beagles.

**Figure 3.** Protein adsorption onto tested NPs after 48-h incubation period in complete cell growth medium [52].

The application of polyethyleneglycol (PEG) for nanoparticle modifications reduces (but not totally suppresses) nonspecific protein corona formation [35, 51]. On the other hand, zwitterionic

The use of different nanomaterials for biomedical applications is indispensably associated with wide physico-chemical and biocompatibility analyses. The analysis of the effect of nanomaterials on different types of cells in various experimental conditions is an essential step in assessing the response of biological models (*in vitro* and/or *in vivo*) to direct contact with NPs [2]. On the other hand, cells/cell culture conditions as well as living system/biological fluids also affect morphological and physico-chemical properties of nanomaterials. Interesting results were obtained in the degradation process of sandwich-like mesoporous silica flake

) nanomaterial (developed as anticancer drug system) exposed to PSB solution for 24,

flake analysis was visible as large holes [**Figure 4b**-**d**]. The intensity of mesoporous

rial was degraded already after 24-h incubation in PBS [**Figure 4a**]. Another deformation found

silica flake degradation was time-dependent—the degree of deformation was associated with the size of holes formed in the nanoflake structure. The appearance of shapeless silica agglomerates was an additional result of the degradation process. Ninety-six-hour incubation caused deformation holes in silica nanoflakes that reached the point of total destruction of NPs [58]. Evidence of nanostructure biodegradation of the sandwich-like mesoporous silica flakes has also been confirmed in another study. After 48-h incubation, the whole surface of silica nanoflakes was covered with cavities and was entirely destroyed [59]. The mechanism of silica dissolution is based on two simultaneous processes—degradation and re-deposition of silica

[**Figure 4**] showed that the porous structure of nanomate-

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Moreover, PEGylated SLNs promoted liver/spleen uptake of NPs [57].

NPs were described to lack the protein corona [51].

48 and 96 h. TEM analysis of mSiO<sup>2</sup>

(mSiO<sup>2</sup>

in mSiO<sup>2</sup>

Other results described various biological responses of different cell types to NPs with protein corona layers [53]. Single-walled carbon nanotubes preferentially bound IgM relative to IgG


**Table 3.** Analytical methods for corona evaluation [46].

the corona of all the analysed nanoparticles [43]. Corona identification and composition analysis (**Table 3**) provide not only information about its complexity, conditions of PC formation and physicochemical features but also data on toxicity, cellular interactions and uptake, targeting

For example, Urbas et al. [52] demonstrated that three types of nanoparticles, NPs-GO, Fe3

protein adsorption from culture medium. The results of the bicinchoninic acid (BCA) assay

Protein corona composition is also known to affect nanoparticle-cell interactions and biological fate of nanomaterials in cells. Gunawan and co-authors characterised the term 'biological fate' as describing the subcellular localisation of NPs and the distribution of NPs to specific organs *in vivo*

(50 nm) exposed to biological fluids (e.g., serum) mediated the interaction of NPs (at 100 μgmL−1 concentration) with A549 cells. Silica nanoparticles showed different degree and process of internalisation during incubation with the A549 cell line in complete (with 10% foetal bovine serum) and in serum-free medium. NP integration was higher in serum-free medium with accumulation in lysosomes and some of NPs localised free in the cytosol. On the contrary, NPs in complete medium (in the presence of a well-developed corona) were never observed free in the cytoplasmic matrix, but similar to serum-free medium, silica nanoparticles were found to accumulate in lysosomes. Lesniak et al. [54] observed that nanoparticles showed higher tendency to adhere to the cell membrane in serum-free conditions and concluded that the initial stronger adhesion could have partly contributed to higher uptake efficiency. Moreover, the presence of free NPs in the cytosol might be caused by perturbation of the early uptake pathway in cells exposed to

, displayed the ability to deplete various quantities of serum proteins from cul-

O4

O4 ,

showed an increase in

) nanoparticles

and finally the usefulness in nanomedicine [46].

ture media (**Figure 3**). Graphene oxide and nanocomposite GO-Fe3

indicated different capacities of NPs to adsorb proteins in cell cultures [52].

[53]. An interesting study performed by Lesniak et al. [54] showed that silica (SiO<sup>2</sup>

serum-free medium (rather than an endogenously regulated cellular process) [53, 54].

**Feature Techniques for PC analysis**

Binding affinity/stoichiometry and

**Table 3.** Analytical methods for corona evaluation [46].

protein interaction

(TGA)

simulation

Other results described various biological responses of different cell types to NPs with protein corona layers [53]. Single-walled carbon nanotubes preferentially bound IgM relative to IgG

Isolation of NPs-PC Centrifugation, size exclusion chromatography (SEC), magnetic separation/ magnetic flow field fractionation (MgFFF) PC structure analysis Dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), transmission electron microscopy (TEM) Protein quantitation Bicinchoninic acid (BCA) assay, Bradford assay, thermogravimetric analysis

PC composition One-dimensional gel electrophoresis (1-DE or SDS-PAGE), two-dimensional

gel electrophoresis (2-DE), mass spectrometry (MS)

Fluorescence correlation spectroscopy (FCS), size exclusion chromatography (SEC), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), quartz crystal microbalance (QCM), Z-potential measurement, *in silico*

and GO-Fe<sup>3</sup>

188 Cytotoxicity

O4

**Figure 3.** Protein adsorption onto tested NPs after 48-h incubation period in complete cell growth medium [52].

on PEG-SWCNTs due to the surface charge and the conformation of surface functional groups (PEG); this resulted in higher accumulation of the aforementioned NPs in the liver compared to the spleen [55]. Poly(D,L-lactide)-based NPs showed interaction of surface functional group (covalently conjugated with apoB100 antibody) with LDL and were highly accumulated by liver macrophages [56]. Solid lipid nanoparticles (SLNs) modified with PEG induced the ABC phenomenon (accelerated blood clearance) upon repeated injections in mice and beagles. Moreover, PEGylated SLNs promoted liver/spleen uptake of NPs [57].

The application of polyethyleneglycol (PEG) for nanoparticle modifications reduces (but not totally suppresses) nonspecific protein corona formation [35, 51]. On the other hand, zwitterionic NPs were described to lack the protein corona [51].

The use of different nanomaterials for biomedical applications is indispensably associated with wide physico-chemical and biocompatibility analyses. The analysis of the effect of nanomaterials on different types of cells in various experimental conditions is an essential step in assessing the response of biological models (*in vitro* and/or *in vivo*) to direct contact with NPs [2]. On the other hand, cells/cell culture conditions as well as living system/biological fluids also affect morphological and physico-chemical properties of nanomaterials. Interesting results were obtained in the degradation process of sandwich-like mesoporous silica flake (mSiO<sup>2</sup> ) nanomaterial (developed as anticancer drug system) exposed to PSB solution for 24, 48 and 96 h. TEM analysis of mSiO<sup>2</sup> [**Figure 4**] showed that the porous structure of nanomaterial was degraded already after 24-h incubation in PBS [**Figure 4a**]. Another deformation found in mSiO<sup>2</sup> flake analysis was visible as large holes [**Figure 4b**-**d**]. The intensity of mesoporous silica flake degradation was time-dependent—the degree of deformation was associated with the size of holes formed in the nanoflake structure. The appearance of shapeless silica agglomerates was an additional result of the degradation process. Ninety-six-hour incubation caused deformation holes in silica nanoflakes that reached the point of total destruction of NPs [58].

Evidence of nanostructure biodegradation of the sandwich-like mesoporous silica flakes has also been confirmed in another study. After 48-h incubation, the whole surface of silica nanoflakes was covered with cavities and was entirely destroyed [59]. The mechanism of silica dissolution is based on two simultaneous processes—degradation and re-deposition of silica

comprehensive than the sum of its parts. "*Phenomics*" is a part of complex technologies that also include transcriptomics, proteomics and metabolomics. PMs give a possibility to screen

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Phenotype MicroArrays™ (PMs) are a combination of microplate reader (that can measure OD every 1 min over few hours and provide information about kinetics of carbon energy reactions in a selected cell model) and microscopic modules equipped with fluorescence, brightfield, colour brightfield and phase contrast microscopy (for scanning changes in cell morphology during experiments). Phenotypic assays deliver more information and provide better understanding of the metabolic and cytotoxic effect of test substances [65]. Multiplex arrays can generate information on the use of energy pathways (based on the application of different nutrition analyses, PM-M1 to M4), effects of ions (PM-M5), hormones, metabolic effectors (PM-M6 to M8) and anti-cancer agents (PM-M11 to M14) (**Table 4**), cell number, cell health (based on cell health monitoring using phase contrast microscopy and kinetic determination of cellular energy) and apoptotic induction (via cell subpopulation analysis—examination of the increase in circularity due to cell shrinkage and cytoplasm condensation and lower phase signal exhibition) [65]. PMs can be used in genotype/phenotype analyses, cell line characterisation, metabolic reprogramming, cellular phenotype stability, Warburg effect, cell differentiation or bioprocess development [64]. Well-characterised model cell lines (e.g., HepG2, C3A, Colo205, A549, PC-3, IMR90, HL-60 or CEM) with defined metabolic properties can be used with the PM system to determine specific effects of nanomaterials on selected cell lines and to accurately identify the mechanism involved in the NP effect (e.g., mitochondrial toxicology) on the living system [66]. Array wells coated with different substances and combined with the redox assay (MA or MB redox dyes to measure cell energy [NADH] changes) are used for phenotypic determination. Comparison of two cell lines is visualised by bioinformatic software

thousands of cellular phenotypes in real time [64].

that highlights differences in recorded phenotypes (**Figure 5**) [64, 67].

For example, Phenotype MicroArrays™ (PM-M TOX1 Plate Energetic Substrate Assay, 96-well microplate coated with eight different oxidisable carbon sources—each of the eight nutrition sources coated on one of eight rows on a microplate) give the possibility to screen cell-based energetic phenotype in a target cell model, for example, the MDA-MB-231 RFP breast cancer cell line, using different cellular nutrition sources (e.g., α-D-glucose, inosine, D-galactose, D-glucose-1-phosphate, xylitol, α-ketoglutaric acid, D,L-β-hydroxybutyric acid or pyruvic acid). This kind of multiplex analysis provides information on cell morphology, metabolic activity (metabolic pathway activity), sensitivity in response to particular energetic additives and the final cellular genetic background characterisation. The addition of an apoptotic agent (e.g., oridonin), chemical inhibitor or stimulator provides an opportunity to evaluate the potential mechanism regulating the energy pathway [65, 66]. Another example of PM application was presented by Bochner et al. [68]. Based on four phenotypic assays (PM-M1 to M4, containing 367 substrate nutrients), different human cancer cell lines, including HepG2/C3A, HepG2, Colo 205, A549, PC-3, HL-60 and CCRF-CEM and two murine white and brown adipocyte cell lines were analysed to determine energy-producing pathways. The results showed that human cancer cell lines exhibited distinct metabolic activity profiles. Moreover, white and brown adipocyte cell lines also had different profiles of energetic activity; metabolic fingerprints were established in all cell lines [68]. Similarly, human endothelial cells from the coronary artery

**Figure 4.** Transmission electron microscopy of mSiO<sup>2</sup> ('contr' – control sample) and mSiO<sup>2</sup> incubated in PBS, for 24 (a), 48 (b) and 96 h (c, d) [58].

on nanoparticle surface. Moreover, the effect of "self-healing" defects between both Si─O─Si bonds of double-linked Si atoms explains the very low rate of dissolution at the point of zero net proton charge (PZPC) of the surface [60].

A similar effect of PSB incubation on mesoporous silica nanospheres was observed by Yamada and co-workers [61], as these authors found that the mSiO<sup>2</sup> porous structure was degradable after 2-day incubation in PBS. After 3-day incubation, mSiO<sup>2</sup> displayed size and shape degradation with the final shape deformation and collapse of structures [61]. Another study based on core-shell magnetic mesoporous silica nanoparticles presented comparable results. Silica mesoporous hollow shells immersed in PBS for 2 days displayed structure deformation and additional cavities, whereas 8-day incubation showed complete degradation and coagulation resulting in new structure formation [62]. The erosion of mesoporous silica nanosphere structure modified with titanium dioxide was also observed in contact with *Streptomyces* cells. After 24-h incubation, mesoporous silica shell structure was degraded with simultaneous appearance of agglomerates, which clearly demonstrated that nanomaterial structure and composition could be affected by living cells [63].
