NCAC 2013 Fall Symposia Student Speaker Abstracts
Student Speaker 1
Title: Use of automated high content fluorescence imaging and in silico models for in vitro assessment of
nanomaterial toxicity in Balb/c3T3 cells.
Authors: Georgina Harris1, Taina Palosaari2, Milena Mennecozzi2, David Asturiol2, Jean-Michel
Gineste2, Luis Saavedra2, Roman Liska2, Anne Milcamps2, Maurice Whelan2
1
Center for Alternatives to Animal Testing (CAAT), Johns Hopkins Bloomberg School of Public Health,
Baltimore, MD, USA. 2JRC - European Commission, Institute for Health and Consumer Protection,
Systems Toxicology Unit, Via E. Fermi 2749 TP202, I-21027 Ispra (VA), Italy
There are an increasing number of studies using in vitro approaches to determine the possible cellular
effects of nanomaterials. We propose automated high content fluorescence-based imaging combined with
in silico models, as a useful quantitative tool to screen nanomaterials for
their adverse toxicological effects on cells in culture. Additionally, the technical challenges and
pitfalls associated with automated high throughput/content in vitro nanotoxicity testing are considered.
Over 20 nanomaterials (including titanium oxides, zinc oxides, silicon dioxides, iron oxides, carbon nanotubes) were tested in a series of assays, measuring three endpoints relevant for safety assessment,
namely, cell viability, reactive oxygen species production and DNA double-strand breaks. In silico
analysis was performed to compare the toxicity of the nanomaterials relative to physico-chemical
characteristics. The reliability and relevance of the results obtained is discussed in the context of the
potential of this approach to advance safety assessment science in the support of regulatory decision
making specific to nanomaterials.
Student Speaker 2
Title: Short- and Long-Term Effects of Commercially Available Gold Nanoparticles in Rodents
Authors: Javiera Bahamonde,* Bonnie Brenseke,*† Matthew Chan,* and M. Renee Prater*§
*Virginia Tech, Blacksburg, VA; †Campbell University School of Osteopathic Medicine, Buies Creek,
NC; §Edward Via College of Osteopathic Medicine, Blacksburg, VA
Gold nanoparticles (GNPs) are being intensely investigated for their potential use in biomedical
applications. Nanotoxicity studies are urgently needed to validate their safety in clinical practice. The
objective of this research was to assess the acute and chronic effects of a single exposure to commercially
available GNPs in rats and mice. Gold nanoparticles were purchased and independently characterized.
Animals received either GNPs (1000 mg/kg) or phosphate buffered saline intravenously. Subsets of
animals were euthanized 1, 7, 14, 21, 28 days or 20 weeks post-exposure and samples were collected. The
physicochemical properties of the purchased GNPs were in good agreement with the information
provided by the supplier. Important differences in GNP-induced immune responses were identified when
comparing mice and rats. Liver microgranulomas and a transient increase in serum levels of interleukin18 were observed in GNP-exposed mice. No such alterations were found in rats. Higher accumulation of
GNPs in spleen and longer fecal excretion were observed in rats. In the long-term, exposure to GNPs
incited chronic inflammation in mice, with persistent microgranulomas in liver, spleen, and lymph nodes,
as well as further increased serum levels of interleukin-18. Impairment of body weight gain was also
observed in the GNP-exposed group. In conclusion, GNPs can incite a robust macrophage response in
mice. However, considering the mildness of the toxic effects identified despite the high dose selected for
the study, GNPs continue to have great potential for biomedical uses.
Student Speaker 3
Title: Toxicity and allergy responses in mice following pulmonary exposure to nanoparticle silver
Authors: CE McLoughlin1, S. Anderson1, K. Anderson1, D. Schwegler-Berry1, BT Chen1, JR Roberts1,
1
NIOSH, Morgantown, WV
Expansive commercial use of silver nanoparticles (AgNP) raises the concern of effects following
respiratory exposure in manufacturing workers. Previous work on AgNP has shown dose-dependent lung
toxicity with inflammation and alterations in lung immune parameters in rodents. The goal of the current
study is to characterize effects of AgNP for potential modulation of respiratory allergy in an ovalbumin
(OVA)-induced allergy model in BALB/c mice. Range-finding (RF) studies were conducted in mice
exposed to physiological dispersion medium (DM), 6.1g (LO), 18.2g (ME), or 73g (HI) AgNP. 20
nm diameter AgNP with 0.3% wt polyvinylpovidone coating (NanoAmor, Inc.), were suspended and
sonicated before pharyngeal aspiration (PA) on day 0. Airway hyperreactivity was measured as PenH.
Whole lung lavage (WLL) fluid and cells, lymph node lymphocytes (LN), and serum were collected for
analysis of parameters of lung injury, inflammation, immunophenotyping, and allergic response. Results
indicated a dose-dependent injury and inflammation by day 10 which began to resolve by day 29 with no
changes in PenH with AgNP alone. For the allergy model, animals received i.p. injections of OVA +
aluminum hydroxide gel (alum) during the sensitization phase on days 1 and 10. To elicit an OVAspecific response, 2 PA challenges of OVA were given on days 19 and 29. The second challenge was
given immediately prior to PenH measurement. PenH, WLL and LN cell number were increased in OVA
animals, and the responses were enhanced by both AgME and AgHI exposure. Results indicate potential
for AgNP to exacerbate allergic response in the lung.
Student Speaker 4
Title: Laser 3D printing with nanoscale resolution: improving biocompatibility and mitigating toxicity
from photoinitiators.
Authors: Peter Petrochenko1, 2, Roger Narayan2, Peter Goering1, Aleksandr Ovsianikov3
1
Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, USFDA,
Silver Spring, Maryland, USA. 2 Joint Dept of Biomedical Engineering, University of North Carolina at
Chapel Hill, NC, USA. 3 Institute of Materials Science and Technology, Vienna University of Technology
(TU Wien), Favoritenstrasse 9-11, Vienna, Austria
Three-dimensional (3D) structural features of a scaffold critically influence cellular functions and the
overall performance of a scaffold material. Recent developments in laser-assisted 3D printing using twophoton polymerization (2PP) have allowed for previously unattainable submicron resolution and opened a
number of possibilities for creating 3D cell scaffolds. However, a significant barrier to using 2PP for
biological applications exists due to the toxic nature of photoinitiators required for radical polymerization
using light energy. In this study we demonstrate two main approaches for creating nanotextured porous
3D scaffolds. The first involves printing the scaffold beforehand, removing residual toxic substances and
seeding cells afterwards. The second, more complex approach, involves using lower laser energy and
trapping cells directly in a collagen matrix. Our results from the first method indicate that stable scaffolds
with porosities of over 60% can be custom printed to fit standard well plates. Cells grown on 3D scaffolds
exhibit increased growth and proliferation compared to smooth 2D scaffold controls. Scaffolds
additionally adsorb larger amounts of proteins due to their larger surface area and allow cells to attach in
multiple planes and completely infiltrate the porous scaffolds. Preliminary results from the second
approach show some dead cells in encapsulated regions. In order to improve cytocompatibility 3 possible
antioxidants: Trolox (water-soluble vitamin E), vitamin C, and glutathione; and collagenase are used to
mitigate the toxic effects of photoinitiators. Preliminary improved cytotoxicity and encapsulation results
are demonstrated. 2PP is shown to be a promising technique for fabricating custom 3D scaffolds from a
computer model with potential for in situ cell encapsulation.