Toxicological assessment of nanomaterials: the role of in vitro Raman microspectroscopic analysis.

The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimize the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a 'label-free high content nanotoxicological assessment technique' using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the cellular toxic responses upon exposure to nanoparticles, changes on the identified spectral markers upon exposure to different nanoparticles, cell death mechanisms, and the effects of nanoparticles on different cell lines are summarized. Moreover, 3D toxicity plots of spectral markers, as a function of time and dose, are introduced as new methodology for toxicological analysis based on the intrinsic properties of the biomolecular changes, such as cytoplasmic RNA aberrations, protein and lipid damage associated with the toxic response. The 3D evolution of the spectral markers are correlated with the results obtained by commonly used cytotoxicity assays, and significant similarities are observed between band intensity and percentage viability obtained by the Alamar Blue assay, as an example. Therefore, the developed 3D plots can be used to identify toxicological properties of a nanomaterial and can potentially be used to predict toxicity, which can provide rapid advances in nanomedicine. Graphical Abstract Spectral markers of cytotoxicity as a function of time and dose.

Label-free, high content screening using Raman microspectroscopy: the toxicological response of different cell lines to amine-modified polystyrene nanoparticles (PS-NH2).

Nanotoxicology has become an established area of science due to growing concerns over the production and potential use of nanomaterials in a wide-range of areas from pharmaceutics to nanomedicine. Although different cytotoxicity assays have been developed and are widely used to determine the toxicity of nanomaterials, the production of multi-parametric information in a rapid and non-invasive way is still challenging, when the amount and diversity of physicochemical properties of nanomaterials are considered. High content screening can provide such analysis, but is often prohibitive in terms of capital and recurrent costs in academic environments. As a label-free technique, the applicability of Raman microspectroscopy for the analysis of cells, tissues and bodily fluids has been extensively demonstrated. The multi-parametric information in the fingerprint region has also been used for the determination of nanoparticle localisation and toxicity. In this study, the applicability of Raman microspectroscopy as a 'high content nanotoxicological screening technique' is demonstrated, with the aid of multivariate analysis, on non-cancerous (immortalized human bronchial epithelium) and cancerous cell-lines (human lung carcinoma and human lung epidermoid cells). Aminated polystyrene nanoparticles are chosen as model nanoparticles due to their well-established toxic properties and cells were exposed to the nanoparticles for periods from 24-72 hours. Spectral markers of cellular responses such as oxidative stress, cytoplasmic RNA aberrations and liposomal rupture are identified and cell-line dependent systematic variations in these spectral markers, as a function of the exposure time, are observed using Raman microspectroscopy, and are correlated with cellular assays and imaging techniques.

Modification of the in vitro uptake mechanism and antioxidant levels in HaCaT cells and resultant changes to toxicity and oxidative stress of G4 and G6 poly(amidoamine) dendrimer nanoparticles.

The mechanism of cellular uptake by endocytosis and subsequent oxidative stress has been identified as the paradigm for the toxic response of cationically surface charged nanoparticles. In an attempt to circumvent the process, the effect of increased cellular membrane permeability on the uptake mechanisms of poly(amidoamine) dendrimers generations 4 (G4) and 6 (G6) in vitro was investigated. Immortalised, non-cancerous human keratinocyte (HaCaT) cells were treated with DL-buthionine-(S,R)-sulfoximine (BSO). Active uptake of the particles was monitored using fluorescence microscopy to identify and quantify endosomal activity and resultant oxidative stress, manifested as increased levels of reactive oxygen species, monitored using the carboxy-H2DCFDA dye. Dose-dependent cytotoxicity for G4 and G6 exposure was registered using the cytotoxicity assays Alamar Blue and MTT, from 6 to 72 h. Reduced uptake by endocytosis is observed for both dendrimer species. A dramatic change, compared to untreated cells, is observed in the cytotoxic and oxidative stress response of the BSO-treated cells. The significantly increased mitochondrial activity, dose-dependent antioxidant behaviour and reduced degree of endocytosis for both dendrimer generations, in BSO-treated cells, indicate enhanced permeability of the cell membrane, resulting in the passive, diffusive uptake of dendrimers, replacing endocytosis as the primary uptake mechanism. The complex MTT response reflects the importance of glutathione in maintaining redox balance within the mitochondria. The study highlights the importance of regulation of this redox balance for cell metabolism but also points to the potential of controlling the nanoparticle uptake mechanisms, and resultant cytotoxicity, with implications for nanomedicine.

Acellular reactivity of polymeric dendrimer nanoparticles as an indicator of oxidative stress in vitro.

The need for rapid and cost-effective pre-screening protocols of the toxicological response of the vast array of emerging nanoparticle types is apparent and the emerging consensus on the paradigm of oxidative stress by generation of intracellular reactive oxygen species as a primary source of the toxic response suggests the development of acellular assays to screen for nanoparticle surface reactivity. This study explores the potential of the monoamine oxidase A (MAO-A) enzyme-based assay with polymeric dendrimers as cofactors and serotonin as substrate, which generates H2O2, quantified by the conversion of the Carboxy-H2DCFDA dye to its fluorescent form. A range of generations of both PAMAM (poly(amidoamine)) (G4-G7) and PPI (poly(propylene imine)) (G0-G4) dendritic polymer nanoparticles are used as test particles to validate the quantitative nature of the assay response as a function of nanoparticle physico-chemical properties. The assay is well behaved as a function of dose over low dose ranges and the acellular reaction rate (ARR) is well correlated with the number of surface amino groups for the combined dendrimer series. For each series, the ARR is also well correlated with the previously documented cytotoxicity, although the correlation is substantially different for each series of dendrimers, pointing to the additional importance of cellular uptake rates in the determination of toxicity.

Numerical simulations of in vitro nanoparticle toxicity - the case of poly(amido amine) dendrimers.

A phenomenological rate equation model is constructed to numerically simulate nanoparticle uptake and subsequent cellular response. Polyamidoamine dendrimers (generations 4-6) are modelled and the temporal evolution of the intracellular cascade of; increased levels of reactive oxygen species, intracellular antioxidant species, caspase activation, mitochondrial membrane potential decay, tumour necrosis factor and interleukin generation is simulated, based on experimental observations. The dose and generation dependence of several of these response factors are seen to well represent experimental observations at a range of time points. The model indicates that variations between responses of different cell-lines, including murine macrophages, human keratinocytes and colon cells, can be simulated and understood in terms of different intracellular antioxidant levels, and, within a given cell-line, varying responses of different cytotoxicity assays can be understood in terms of their sensitivities to different intracellular cascade events. The model serves as a tool to interpolate and visualise the range of dose and temporal dependences and elucidate the mechanisms underlying the in vitro cytotoxic response to nanoparticle exposure and describes the interaction in terms of independent nanoparticle properties and cellular parameters, based on reaction rates. Such an approach could be a valid alternative to that of effective concentrations for classification of nanotoxicity and may lay the foundation for future quantitative structure activity relationships and predictive nanotoxicity models.

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined.

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-ß-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.