Sources of Variability in Nanoparticle Uptake by Cells

Understanding how nano-sized objects are taken up by cells is important for applications within medicine (nanomedicine), as well as to avoid unforeseen hazard due to nanotechnology (nanosafety). Even within the same cell population, one typically observes a large cell-to-cell variability in nanoparticle uptake, raising the question of the underlying cause(s). Here we investigate cell-to-cell variability in polystyrene nanoparticle uptake by HeLa cells, with generalisations of the results to silica nanoparticles and liposomes, as well as to A549 and primary human umbilical vein endothelial cells. We show that uptake of nanoparticles is correlated with cell size within a cell population, thereby reproducing and generalising previous reports highlighting the role of cell size in nanoparticle uptake. By repeatedly isolating (using fluorescence-activated cell sorting) the cells that take up the most and least nanoparticles, respectively, and performing RNA sequencing on these cells separately, we examine the underlying gene expression that contributes to high and low polystyrene nanoparticle accumulation in HeLa cells. We can thereby show that cell size is not the sole driver of cell-to-cell variability, but that other cellular characteristics also play a role. In contrast to cell size, these characteristics are more specific to the object (nanoparticle or protein) being taken up, but are nevertheless highly heterogeneous, complicating their detailed identification. Overall, our results highlight the complexity underlying the cellular features that determine nanoparticle uptake propensity

Particulate airborne impurities

The cumulative effects of air pollutants are of principal concern in research on environmental protection in Sweden. Post-industrial society has imposed many limits on emitted air pollutants, yet the number of reports on the negative effects from them is increasing, largely due to human activity in the form of industrial emissions and increased traffic flows. Rising concerns over the health effects from airborne particulate matter (PM) stem from in vitro, in vivo, and cohort studies revealing effects of mostly negative nature. Full insight into the health effects from PM can only be achieved through practical investigation of the mode of toxicity from distinct types of particles and requires techniques for their identification, monitoring, and the production of model fractions for health studies. To this effect, comprehensive collection and chemical analysis of particulates at the origin of emission was performed in order to provide clearer insight into the nature of the particulates at exposure and add detail to aid risk assessment. Methods of capturing particles and analyzing their chemical nature were devised using scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). Furthermore, taking the approach of in vitro cytotoxicity testing, nanoparticles of types typical to automotive emissions, were synthesized and extensively characterized using SEM-EDS, X-ray diffraction (XRD), transmission electron microscopy (TEM),dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). The produced model magnetite and palladium nanoparticles were found to induce toxicity in human pulmonary epithelial cells (A549 and PBEC) as well as impact severely on immunological and renal cells (221 B- and 293T-cells) in a dose-dependent manner

Deriving a Provisional Tolerable Intake for Intravenous Exposure to Silver Nanoparticles Released from Medical Devices

Silver nanoparticles (AgNP) are incorporated into medical devices for their anti-microbial characteristics. The potential exposure and toxicity of AgNPs is unknown due to varying physicochemical particle properties and lack of toxicological data. The aim of this safety assessment is to derive a provisional tolerable intake (pTI) value for AgNPs released from blood-contacting medical devices. A literature review of in vivo studies investigating critical health effects induced from intravenous (i. v.) exposure to AgNPs was evaluated by the Annapolis Accords principles and Toxicological Data Reliability Assessment Tool (ToxRTool). The point of departure (POD) was based on an i. v. 28-day repeated AgNP (20 nm) dose toxicity study reporting an increase in relative spleen weight in rats with a 5% lower confidence bound of the benchmark dose (BMDL05) of 0.14 mg/kg bw/day. The POD was extrapolated to humans by a modifying factor of 1,000 to account for intraspecies variability, interspecies differences and lack of long-term toxicity data. The pTI for long-term i. v. exposure to 20 nm AgNPs released from blood-contacting medical devices was 0.14 μg/kg bw/day. This pTI may not be appropriate for nanoparticles of other physicochemical properties or routes of administration. The methodology is appropriate for deriving pTIs for nanoparticles in general

Analysis of the Influence of Cell Heterogeneity on Nanoparticle Dose Response

Understanding the effect of variability in the interaction of individual cells with nanoparticles on the overall response of the cell population to a nanoagent is a fundamental challenge in bionanotechnology. Here, we show that the technique of time-resolved, high-throughput microscopy can be used in this endeavor. Mass measurement with single-cell resolution provides statistically robust assessments of cell heterogeneity, while the addition of a temporal element allows assessment of separate processes leading to deconvolution of the effects of particle supply and biological response. We provide a specific demonstration of the approach, in vitro, through time-resolved measurement of fibroblast cell (HFF-1) death caused by exposure to cationic nanoparticles. The results show that heterogeneity in cell area is the major source of variability with area-dependent nanoparticle capture rates determining the time of cell death and hence the form of the exposure–response characteristic. Moreover, due to the particulate nature of the nanoparticle suspension, there is a reduction in the particle concentration over the course of the experiment, eventually causing saturation in the level of measured biological outcome. A generalized mathematical description of the system is proposed, based on a simple model of particle depletion from a finite supply reservoir. This captures the essential aspects of the nanoparticle–cell interaction dynamics and accurately predicts the population exposure–response curves from individual cell heterogeneity distributions

Development of a nanomaterial bio-screening platform for neurological applications

Nanoparticle platforms are being intensively investigated for neurological applications. Current biological models used to identify clinically relevant materials have major limitations, e.g. technical/ethical issues with live animal experimentation, failure to replicate neural cell diversity, limited control over cellular stoichiometries and poor reproducibility. High-throughput neuro-mimetic screening systems are required to address these challenges. We describe an advanced multicellular neural model comprising the major non-neuronal/glial cells of the central nervous system (CNS), shown to account for ~99.5% of CNS nanoparticle uptake. This model offers critical advantages for neuro-nanomaterials testing while reducing animal use: one primary source and culture medium for all cell types, standardized biomolecular corona formation and defined/reproducible cellular stoichiometry. Using dynamic time-lapse imaging, we demonstrate in real-time that microglia (neural immune cells) dramatically limit particle uptake in other neural subtypes (paralleling post-mortem observations after nanoparticle injection in vivo), highlighting the utility of the system in predicting neural handling of biomaterials

Cytotoxicity in the Age of Nano: The Role of Fourth Period Transition Metal Oxide Nanoparticle Physicochemical Properties

A clear understanding of physicochemical factors governing nanoparticle toxicity is still in its infancy. We used a systematic approach to delineate physicochemical properties of nanoparticles that govern cytotoxicity. The cytotoxicity of fourth period metal oxide nanoparticles (NPs): TiO2, Cr2O3, Mn2O3, Fe2O3, NiO, CuO, and ZnO increases with the atomic number of the transition metal oxide. This trend was not cell-type specific, as observed in non-transformed human lung cells (BEAS-2B) and human bronchoalveolar carcinoma-derived cells (A549). Addition of NPs to the cell culture medium did not significantly alter pH. Physiochemical properties were assessed to discover the determinants of cytotoxicity: (1) point-of-zero charge (PZC) (i.e., isoelectric point) described the surface charge of NPs in cytosolic and lysosomal compartments; (2) relative number of available binding sites on the NP surface quantified by X-ray photoelectron spectroscopy was used to estimate the probability of biomolecular interactions on the particle surface; (3) band-gap energy measurements to predict electron abstraction from NPs which might lead to oxidative stress and subsequent cell death; and (4) ion dissolution. Our results indicate that cytotoxicity is a function of particle surface charge, the relative number of available surface binding sites, and metal ion dissolution from NPs. These findings provide a physicochemical basis for both risk assessment and the design of safer nanomaterials

Development of Spectroscopic Methods for Dynamic Cellular Level Study of Biochemical Kinetics and Disease Progression

One of the current fundamental objectives in biomedical research is understanding molecular and cellular mechanisms of disease progression. Recent work in genetics support the stochastic nature of disease progression on the single cell level. For example, recent work has demonstrated that cancer as a disease state is reached after the accumulation of damages that result in genetic errors. Other diseases like Huntingtons, Parkinsons, Alzheimers, cardiovascular disease are developed over time and their cellular mechanisms of disease transition are largely unknown. Modern techniques of disease characterization are perturbative, invasive and fully destructive to biological samples. Many methods need a probe or enhancement to take data which alters the biochemistry of the cells and may not be a true representations of cellular mechanisms. Current methods of characterizing disease progression cannot measure dynamics of a process but rely on an average state of a system at a fixed endpoint. They track cellular changes at a population level that rely on static ensemble averages that compare the same population at different time points or populations exposed to different stimuli. Ensemble averaging obscures spatiotemporal and dynamic molecular and cellular mechanism information by only measuring changes before and after disease transitions which neglects mechanistic information. This type of snap shot measurement contains no information regarding the transition into a disease state. The use of an ensemble averages ignores single cell level changes by assuming cells in a population are similar. In reality individual cell-to-cell variability in the same cell population can cause one cell to transition to disease state while another cell does not. Fluctuations are indicators of disease and if cellular processes are not studied spatiotemporally then key molecular changes are undetected. If the path to disease progression is known on an individual cell level, then treatments can be modified to alleviate or prevent disease through early detection. The aim of this thesis is to quantitatively and dynamically measure a biomedical sample on the single cell level without destroying or manipulating it significantly to characterize cellular mechanisms. The technique developed uses microRaman Spectroscopy to analyze molecular signatures of single cells and compare differences between signatures of cells in different populations

Physicocehmical Properties and Pre-Exposure Processes Govern Gold Nanoparticle Accumulation in Freshwater Species

Monitoring the distribution and subsequent effects of nanoparticle (NP) contaminants in aquatic ecosystems will be pivotal to developing regulations that minimize their environmental footprint. Regulators are in a unique position to take a proactive role in shaping how we produce and consume nanomaterials as opposed to the reactive role they have had to adopt with other contaminants. Over the last few decades, researchers have made great strides in describing the fate, behavior, and toxicity of NPs in environmental systems. Recent initiatives have made the transition to scenarios with greater environmental relevance, yet important aspects of fate and behavior remain unexplored. The goal of this dissertation research was to fill in several of those gaps, emphasizing relationships between gold NP characteristics, water chemistry and biodynamic parameters that will contribute to development of robust fate and behavior models. Daphnia magna and Pimephales promelas were used as model organisms to differentiate the impact of characteristics and water chemistry on two unrelated species residing in a common aquatic habitat. Uptake and elimination rate constants were derived empirically for D. magna exposed to anionic spheres (4, 20 and 30 nm core diameter) anionic rods (18 x 58 nm) and cationic rods (18 x 58 nm) in moderately hard water (MHW). Size and surface charge greatly affected the uptake and elimination rate constant while shape had a relatively minor influence on accumulation. Multiple linear regression models revealed that D. magna favor accumulation of larger cationic NPs at high concentration exposures and larger anionic NPs at low concentration exposures. D. magna and P. promelas were then challenged with cationic and zwitterionic NPs in MHW and wastewater (WW) that represented a direct release scenario and a WWTP release scenario, respectively. Surface charge influenced not only the biodynamics in MHW exposures for both D. magna and P. promelas but also dictated the interactions between the NP and the wastewater components. Cationic NPs transformed in the presence of WW including an increase in size and a slight decrease in surface charge while zwitterionic NPs were unaffected. The influences of these transformations were species specific as D. magna experienced a significant decrease in the uptake rate constant while neither uptake nor elimination was affected in P. promelas. Finally, we exposed P. promelas to a nano-pharmaceutical (doxorubicin-NP) and the free pharmaceutical (doxorubicin) to determine if the NP altered the distribution and accumulation patterns of the pharmaceutical. The intestine was the primary site of doxorubicin accumulation and the total accumulated content was not significantly affected by the form of the pharmaceutical. Despite a lack of statistical significance, several trends in my data suggest that nano-medicines do not behave like a standard pharmaceutical and, therefore, warrant further investigation to define its environmental impact. Overall my data argue for prioritization of particle characteristics in risk assessment and inclusion of transformative pre-release processes in fate and behavior model development. At the moment releases of NPs into the environment are well below toxic thresholds. Yet as the popularity of nanotechnology further penetrates all aspects of society, engineered NPs will form a larger presence in environmental systems that could give rise to serious environmental consequences. Proactive regulation of NPs aided by comprehensive modeling initiatives are of paramount importance to making sure we use this technology responsibly or else we risk adding another name to the dubious pantheon of legacy contaminants