Time Resolved, High-throughput Microscopy in Nanotoxicological Assessment Matthew J. Ware1,2, Neenu Singh1, Kennith E. Meissner3, Paul Rees1, Biana Godin2* and Huw D. Summers1* 1 Centre for Nanohealth, College of Engineering, Swansea University, Swansea, UK 2 Department of Nanomedicine, Houston Methodist Research Institute, Houston, Texas, USA 3 Interdisciplinary Program of Toxicology, Texas A&M University, College Station, Texas, USA *Shared seniority Biomedical Engineering Society Meeting, Seattle 2013 Results Conclusions Introduction (A) (B) Dose for NPs in in vitro systems is more dynamic, more complicated and less comparable between particle types than it is for soluble chemicals Cellular dose is a function of diffusion, sedimentation and agglomeration rates which are determined by systemic and particle properties and define the transport rate of NPs to cells residing within the culture flask and particle-cellular interactions upon contact [2]. Toxicological assessment of nanomaterials poses numerous important challenges. Currently, the dose-response curve, measured at a single randomly-chosen time point, is the gold standard for nanotoxicological evaluation, similarly to molecularly dispersed solutes. However when considering cell membrane-nanoparticle interactions, other considerations regarding cell dosimetry must apply, due to a number of biological and physico-chemical processes involved. Surprisingly, the direct measure of individual cell dosimetry has been largely overlooked in favor of using overall metrics of exposure. Here we present several techniques which evaluate time-response in addition to dose-response using high-throughput systems which are able to achieve accurate target cell dosimetry and an increased competence in identifying particle kinetics, biological processes and cell heterogeneity. A mathematical model correlating the heterogeneity in biological response to mechanisms of nanoparticle (NP) arrival and the available cell area is developed. Fig 2: Percentage cell death as a function of dose and time. (A) Pseudo colored DRAQ7 signals at 0, 12, and 24 hours for 4.5 and 45nM doses in 2ml media. (B) The cumulative number of DRAQ7 signals between 0-24 hours. Dying and/or dead cells are labeled by DRAQ7 as it passively diffuses through cytoplasmic membranes damaged by PEI-QDs and through nuclear membranes to eventually bind with adenine-thymine rich repeats in DNA at which point it becomes fluorescent. Higher doses of 18 and 45nM induced 100% cytotoxicity within 4 hours. Lower doses of between 2-6nM induced 5-80% within 24 hours. A deceleration in the rate of cell death at lower doses was observed Fig 1: Membranal Saturation. HFF-1 cells exposed to (A) 6nM PEI-QDs in 2ml media for 3 hours at 20X magnification and (B) 3nM PEI-QDs in 2ml media for 24 hours observed at 50,000x magnification. A high affinity between PEI-QDs and the cell membrane was observed and at higher doses complete membranal saturation occurred. Degradation of the cell membrane was a major mechanism of cell death in this system. We propose the safety assessment of NP must include: a complete characterization of the NP under investigation in conditions similar to the biological environment direct experimental measurements of the particle kinetics and their impact on cellular dose cellular dose content and cell heterogeneity quantification of the studied particle the introduction of time as an additional variable which allows successive observations to be made of the physical and biological processes acting on the same cell population a more thorough methodology provided in dose-response manuscripts which outline careful descriptions of the test system, including media constituents, temperature, dimensions of the wells and exactly how the NP were mixed in the media. Fig 3: Surface accumulation as a function of dose and time. Surface accumulation is measured in fluorescence intensity at the PEI-QDs wavelength. The rate of delivery of NP dose to the cells will impact the magnitude and timing of cellular responses. The arrival of PEI-QDs at the bottom of the culture well (Fig 3) and to the cells (Fig 4) as a function of time suggested a non-linear rate of influx, which was caused by the fixed supply of PEI-QDs in the media depleting over time. At low but still toxic concentrations of NPs used in typical experiments the depletion of the number of NPs available for interaction with the cell population completely invalidates a linear arrival approximation, an effect which is pertinent to all dose-response based assays. , transfer rate to cell ND, dose accumulation particle reservoir Na = f (Conc., Vol., time) Commonly assumed, infinite reservoir with constant rate of delivery, f Depletion of particles from a finite reservoir of initial concentration, C and volume, V: Ncell= CV[1-exp(-t/τ)] Ncell= CVft Methods CdSe/ZnS quantum dots (QD) (Zeta Potential=18.6 ±5.7mV, size(d)=51.39nm ±17.30nm) were chosen to demonstrate the techniques seen in this work due to their popularity within the scientific community, photoluminescent properties and their ability to enter living cells [1]. Highly branched polyethylenimine (PEI), which is a cationic polymer (MW 25,000), was attached to the QD surface to induce cytotoxicity. Time-response based measurements formed the basis of the nanotoxicological study of BrPEI-QDs when exposed to Human Foreskin Fibroblast (HFF-1) cells. In-Cell Analyzer 2000 microscope (INCELL) (GE Healthcare) and DRAQ7TM (Biostatus), which is a far-red fluorescent marker for necrosis, was used to quantify cumulative cell death over time. ImageJ 1.46r software (National Institutes of Health, USA) was used to analyze images obtained from INCELL to evaluate particle arrival rate and cell-dose variation due to biological heterogeneity. These measures were based on the quantification of pixel intensity on the PEI-QDs channel (Ex/Em 350/568nm) whereas single cell areas were manually segmented and measured using brightfield images. BIOLOGICAL HETEROGENIETY Fig 4: Model and experimental data of PEI-QDs accumulation over time. Model is represented by smooth lines and experimental data, represented by circled lines,. Consideration of the varied depletion in the NP supply over time leading to a non-linear rate of dose arrival to the cells can be incorporated into the current understanding of dose arrival. Single cell-dose variation arises from cell area heterogeneity. Smaller cells were seen to be less prone to cytotoxicity possibly due to a decreased area for NP interaction, an effect which has obvious connotations for many therapeutic techniques, especially in cancer therapies. acell = area of a single cell A = total surface of the well τ is the mean uptake time per particle [1] Ncell=(acell/A) CV[1-exp(-t/τ)] Fig 6: Percentage cell death experimental data (red) and model (blue) References [2] td = time to death acell = area of single cell  Wiethoff, C.M., et al., The potential role of proteoglycans in cationic lipid-ediated gene delivery: studies of the interaction of cationic lipid-dna complexes with model glycosaminoglycans. journal of biological chemistry, 2001. 276(35): p. 32806-32813. Teeguarden, J.G., et al., Particokinetics In Vitro: Dosimetry Considerations for In Vitro Nanoparticle Toxicity Assessments. Toxicological Sciences, 2007. 95(2): p. 300-312. Fig 5: HFF-1 cell area distribution (n=680). Population wide area distribution was used to model population wide cell death distribution (Fig 6)