Fishing to Design Inherently Safer Nanoparticles - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 277

characterization of data (i.e., purity, concentration, ligand size, core size, particle size,
etc.). Without knowing the basic structural and purity information, it is not possible to
define the structure variable in SAR. Therefore, insufficiently characterized materials
are of no value for SAR studies.
20.3 TIER 1: RAPID TOXICITY SCREENING
20.3.1 Waterborne Exposure
Initially, all nanoparticles are processed through a rapid screening assay. The first step
of this assay is to remove the embryonic chorion, an acellular envelope that acts as a
protective barrier, at 6 h post fertilization (hpf) via enzymatic digestion. Removing the
chorion eliminates a potential barrier to uptake from the surrounding water. Embryos
are then exposed to a standard range of seven nanoparticle concentrations (fivefold
serial dilutions) plus a vehicle/water control with the highest concentration defined by
the limit of solubility. If a solvent is used, dimethyl sulfoxide (DMSO) is preferred
based on previous studies indicating that DMSO is compatible with embryonic
development (Harper, 2007; Isaacson et al., 2007; Usenko et al., 2007, 2008;
Harper, 2008b). All concentrations utilizing a solvent are prepared with less than
1% DMSO in embryo media. Embryos are exposed to appropriate nanoparticle
concentrations at 8hpf to ensure coverage of gastrulation and organogenesis, the
periods of development that are most conserved among vertebrates. A total of 12
animals ( n ¼ 12) are exposed to each concentration, with one individual per well in a
96-well plate with 100 mL of exposure solution. Thus, for any given nanoparticle, the
first step of waterborne exposure will require one 96-well plate.
After waterborne exposure, each embryo is evaluated at two time points: 24 and
120 hpf. At 24 hpf, embryos are evaluated for viability, developmental progression,
and spontaneous movement (the earliest behavior in zebrafish). At 120 hpf, larval
morphology (body, axis, eye, snout, jaw, otic vesicle, notochord, heart, brain, somites,
fin, morphometrics) and behavioral end points (motility and tactile response) are
evaluated and scored in a binary fashion (i.e., for each developmental end point, a
score of normal or abnormal is recorded) according to well-established methods
(Andreasen et al., 2002; Tanguay, 2003; Brent, 2004; Haendel et al., 2004). If more
than 10%mortality occurs in the control group, then the assessment for that particular
nanoparticle must be repeated. Any nanoparticle that induces adverse effects will
proceed to tier 2, in which the potential cause of observed effects is investigated.
20.3.2 Statistical Analysis
Embryos are scored in a binary fashion and a Fisher's exact test is used to provide a
proportional statistical analysis of differences between control and nanoparticle-
exposed groups. The one-sided Fisher's exact test is used to calculate the sample size
of 12 embryos per treatment, and since embryos are exposed individually, a well
represents an individual test subject. This provides 80% power to detect differences