Biospectroscopy of Nanodiamond-Induced Alterations in Conformation of Intra- and Extracellular Proteins: A Nanoscale IR Study.

The toxicity of nanomaterials raises major concerns because of the impact that nanomaterials may have on health, which remains poorly understood. We need to explore the fate of individual nanoparticles in cells at nano and molecular levels to establish their safety. Conformational changes in secondary protein structures are one of the main indicators of impaired biological function, and hence, the ability to identify these changes at a nanoscale level offers unique insights into the nanotoxicity of materials. Here, we used nanoscale infrared spectroscopy and demonstrated for the first time that nanodiamond-induced alterations in both extra- and intracellular secondary protein structures lead to the formation of antiparallel ß-sheet, ß-turns, intermolecular ß-sheet, and aggregation of proteins. These conformational changes of the protein structure may result in the loss of functionality of proteins and in turn lead to adverse effects.

Lorentz contact resonance spectroscopy for nanoscale characterisation of structural and mechanical properties of biological, dental and pharmaceutical materials.

Scanning probe microscopy has been widely used to obtain topographical information and to quantify nanostructural properties of different materials. Qualitative and quantitative imaging is of particular interest to study material-material interactions and map surface properties on a nanoscale (i.e. stiffness and viscoelastic properties). These data are essential for the development of new biomedical materials. Currently, there are limited options to map viscoelastic properties of materials at nanoscale and at high resolutions. Lorentz contact resonance (LCR) is an emerging technique, which allows mapping viscoelasticity of samples with stiffness ranging from a few hundred Pa up to several GPa. Here we demonstrate the applicability of LCR to probe and map the viscoelasticity and stiffness of 'soft' (biological sample: cell treated with nanodiamond), 'medium hard' (pharmaceutical sample: pMDI canister) and 'hard' (human teeth enamel) specimens. The results allowed the identification of nanodiamond on the cells and the qualitative assessment of its distribution based on its nanomechanical properties. It also enabled mapping of the mechanical properties of the cell to demonstrate variability of these characteristics in a single cell. Qualitative imaging of an enamel sample demonstrated variations of stiffness across the specimen and precise identification of enamel prisms (higher stiffness) and enamel interrods (lower stiffness). Similarly, mapping of the pMDI canister wall showed that drug particles were adsorbed to the wall. These particles showed differences in stiffness at nanoscale, which suggested variations in surface composition-multiphasic material. LCR technique emerges as a valuable tool for probing viscoelasticity of samples of varying stiffness's.

Biological impact of nanodiamond particles - label free, high-resolution methods for nanotoxicity assessment.

Current methods for the assessment of nanoparticle safety that are based on 2D cell culture models and fluorescence-based assays show limited sensitivity and they lack biomimicry. Consequently, the health risks associated with the use of many nanoparticles have not yet been established. There is a need to develop in vitro models that mimic physiology more accurately and enable high throughput assessment. There is also a need to set up new assays that offer high sensitivity and are label-free. Here we developed 'mini-liver' models using scaffold-free bioprinting and used these models together with label-free nanoscale techniques for the assessment of toxicity of nanodiamond produced by laser-assisted technology. Results showed that NDs induced cytotoxicity in a concentration and exposure-time dependent manner. The loss of cell function was confirmed by increased cell stiffness, decreased cell membrane barrier integrity and reduced cells mobility. We further showed that NDs elevated the production of reactive oxygen species and reduced cell viability. Our approach that combined mini-liver models with label-free high-resolution techniques showed improved sensitivity in toxicity assessment. Notably, this approach allowed for label-free semi-high throughput measurements of nanoparticle-cell interactions, thus could be considered as a complementary approach to currently used methods.

Dose enhancement and cytotoxicity of gold nanoparticles in colon cancer cells when irradiated with kilo- and mega-voltage radiation.

Despite major advances in the field of radiotherapy, healthy tissue damage continues to constrain the dose that can be prescribed in cancer therapy. Gold nanoparticles (GNPs) have been proposed as a solution to minimize radiation-associated toxicities by enhancing the radiation dose delivered locally to tumor cells. In the current study, we investigated the application of third-generation GNPs in two-dimensional (2D) and three-dimensional (3D) cell cultures and whether there is synergy between the nanoparticles and kilo- or mega-voltage radiation to cause augmented cytotoxicity. The 10-nm GNPs were found to be nontoxic in both 2D and 3D in vitro cultures of colon cancer cells at concentrations of up to 10-25 µg/ml. There was a significant increase in cell survival fraction reduction following exposure to 1 Gy of kilo-voltage (18.3%) and 2 Gy of mega-voltage (35.3%) radiation when the cells were incubated with 50 µg/ml of GNPs. The biocompatibility of the GNPs combined with their substantial synergy with radiation encourages further investigations into their application in targeted cancer treatment.