Cellulose nanocrystals (CNCs) are a next-generation cellulose product with many unique properties including applications in the food industry as a food additive, food coating, and in food-contact packaging material. While CNC is anticipated to be safe due to its similarity to the many forms of cellulose currently used as food additives, special consideration is given to it as it is the first manufactured form of cellulose that is nanoscale in both length and width. A proactive approach to safety has been adopted by manufacturers to demonstrate CNC safety toward responsible commercialization. As part of the safety demonstration, in vivo and in vitro testing strategies were commissioned side-by-side with conventional cellulose, which has been safely used in food for decades. Testing included a 90-day rodent feeding study as well as additional physical, chemical, and biological studies in vitro that follow European Food Safety Authority (EFSA) guidance to demonstrate the safe use of novel food ingredients. The strategy includes assessment of neat materials side-by-side with simulated digestion, mimicking conditions that occur along the gastrointestinal tract as well as intracellularly. An intestinal co-culture model examined any potential toxicological effects from exposure to either pristine or digested forms of CNC including cytotoxicity, metabolic activity, membrane permeability, oxidative stress, and proinflammatory responses. None of the studies demonstrated any toxicity via oral or simulated oral exposure. These studies demonstrate that CNC produced by InnoTech Alberta is similarly safe by ingestion as conventional cellulose with a no-observed-adverse-effect level of 2085.3 (males) and 2682.8 (females) mg/kg/day.

Engineered nanoparticles are utilized as drug delivery carriers in modern medicine due to their high surface area and tailorable surface functionality. After in vivo administration, nanoparticles distribute and interact with biomolecules, such as polar proteins in serum, lipid membranes in cells, and high ionic conditions during digestion. Electrostatic forces and steric hindrances in a nanoparticle population are disturbed and particles agglomerate in biological fluids. Little is known about the stability of nanoparticles in relation to particle surface charge. Here, we compared three different surface-stabilized silver nanoparticles (50 nm) for intracellular agglomeration in human hepatocellular carcinoma cells (HepG2). Nanoparticles stabilized with branched polyethyleneimine conferred a positive surface charge, particles stabilized with lipoic acid conferred a negative surface charge, and particles stabilized with polyethylene glycol conferred a neutral surface charge. Particles were incubated in fetal bovine serum, simulated lung surfactant fluid, and simulated stomach digestion fluid. Each nanoparticle system was characterized via microscopic (transmission electron, fluorescence, and enhanced darkfield) and spectroscopic (hyperspectral, dynamic light scattering, and ultraviolet-visible absorption) techniques. Results showed that nanoparticle transformation included cellular internalization, agglomeration, and degradation and that these changes were dependent upon surface charge and incubation matrix. Hyperspectral analyses showed that positively charged silver nanoparticles red-shifted in spectral analysis after transformations, whereas negatively charged silver nanoparticles blue-shifted. Neutrally charged silver nanoparticles did not demonstrate significant spectral shifts. Spectral shifting indicates de-stabilization in particle suspension, which directly affects agglomeration intracellularly. These characteristics are translatable to critical quality attributes and can be exploited when developing nano-carriers for nanomedicine.

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (μM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

Gold nanoparticles (AuNPs) used in pharmaceutical treatments have been shown to effectively deliver a payload, such as an active pharmaceutical ingredient or image contrast agent, to targeted tissues in need of therapy or diagnostics while minimizing exposure, availability, and accumulation to surrounding biological compartments. Data sets collected in this field of study include some toxico- and pharmacodynamic properties (e.g., distribution and metabolism) but many studies lack information about adsorption of biological molecules or absorption into cells. When nanoparticles are suspended in blood serum, a protein corona cloud forms around its surface. The extent of the applications and implications of this formed cloud are unknown. Some researchers have speculated that the successful use of nanoparticles in pharmaceutical treatments relies on a comprehensive understanding of the protein corona composition. The work presented in this paper uses a suite of data analytics and multi-variant visualization techniques to elucidate particle-to-protein interactions at the molecular level. Through mass spectrometry analyses, corona proteins were identified through large and complex datasets. With such high-output analyses, complex datasets pose a challenge when visualizing and communicating nanoparticle-protein interactions. Thus, the creation of a streamlined visualization method is necessary. A series of user-friendly data informatics techniques were used to demonstrate the data flow of protein corona characteristics. Multi-variant heat maps, pie charts, tables, and three-dimensional regression analyses were used to improve results interpretation, facilitate an iterative data transfer process, and emphasize features of the nanoparticle-protein corona system that might be controllable. Data informatics successfully highlights the differences between protein corona compositions and how they relate to nanoparticle surface charge.

There is momentum in biomedical research to improve the structure and function of in vitro intestinal models that better represent human biology. To build a more comprehensive model, three human cell-types were co-cultured and characterized: i.e., HT29-MTX (intestinal mucous-producing goblet cells), Caco-2 (colon epithelial cells), and Raji B (lymphocytes). Raji B cells transformed a subpopulation of Caco-2 epithelial cells into phagocytic and transcytotic immune-supporting microfold cells (M-cells). A suite of bioassays was implemented to investigate steady-state barrier integrity and cellular communication. The model demonstrated a potentiating effect in metabolism and pro-inflammatory markers. Barrier integrity and cell seeding density seem to play a role in the reliability of endpoint readouts. Microscopic analysis elucidated the importance of multi-cell biomimicry. The data show that monocultures do not have the same characteristics inherent to triple cell culture models. Multiple cell types in an in vitro model produce a better representation of an intact organ and aid in the ability to assess immunomodulatory effects of nanomaterials designed for cancer theranostics after ingestion. As many national and international agencies have stressed, there is a critical need to improve alternative-to-animal strategies for pharmaceuticals in an effort to reduce animal testing.