AN ABSTRACT OF THE DISSERTATION OF
Leah C. Wehmas for the degree of Doctor of Philosophy in Toxicology presented on
August 26, 2015.
Title: Nanotoxicology to Nanotherapeutics: Prioritizing Hazard and Anticancer
Efficacy of Nanomaterials using the Embryonic Zebrafish Model
Abstract approved:
______________________________________________________
Robert L. Tanguay
Juliet A. Greenwood
The numerous different types of nanomaterials and paucity of reliable in vivo
information on toxicity has resulted in enduring uncertainties associated with health
and safety risks, thereby slowing progress in nanotherapeutic assessment and
development. This dissertation explores how the embryonic zebrafish model can be
applied to prioritize commonly used nanomaterial (multi-walled carbon nanotubesMWNTs and metal oxide nanoparticles- MO NPs) hazard and anticancer efficacy
bridging the gap between high throughput in vitro assays and low throughput,
mammalian assays to advance glioblastoma drug development and safety.
First, the zebrafish model was utilized to evaluate comprehensively
characterized MWNTs systematically modified to introduce oxygen functional groups
on the surface. Advanced multivariate statistical methods identified MWNT
physicochemical properties that best estimated the probability of observing an adverse
outcome. The properties considered included surface charge, percent surface oxygen,
dispersed aggregate size and morphology and electrochemical activity. Total surface
oxygen and the highly related parameter surface charge, quantified as the point of zero
charge (PZC), were determined as the best predictor of embryonic zebrafish mortality
at 24 hpf.
Second, the embryonic zebrafish model was used to evaluate the toxicity of
seven widely used semiconductor MO NPs made from zinc oxide (ZnO), titanium
dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic salt
water to identify the physicochemical properties associated with toxicity. Significant
agglomeration, elemental composition and dissolution potential were identified as
major drivers of toxicity. Only ZnO caused significant adverse effects and only when
prepared in pure water (point estimate median lethal concentration = 3.5-9.1 mg/L)
despite charge and size similarities with one cerium dioxide and titanium dioxide NP.
This toxicity was life stage dependent. The 24 h toxicity increased greatly (~22.7 fold)
when zebrafish exposures started at the larval life stage compared to the 24 h toxicity
following embryonic exposure. Measurement of ZnO dissolution revealed high levels
of zinc ion (40-89% of total sample) were generated. Exposure to zinc ion equivalents
revealed dissolved Zn2+ may be a major contributor to ZnO toxicity.
Third, an orthotopic zebrafish xenograft screening assay was created to
discover and prioritize the testing of potential nanotherapeutics targeting glioblastoma
proliferation, migration and invasion. Evaluation of the potential anti-cancer efficacy
of zinc oxide nanoparticles (ZnO NP) and the model phosphatidylinositide 3-kinase (PI
3-kinase) inhibitor LY294002, revealed divergent responses in the assay. ZnO NPs
significantly enhanced glioblastoma proliferation by up to 23% and migration/invasion
by 22 to 49% at the periphery of the cell mass (161+ µm) compared to vehicle control.
Exposures of 3.125-6.25 µM LY294002 significantly decreased glioblastoma
proliferation by up to ~34% and 6.25 µM LY294002 significantly inhibited
migration/invasion by 30 and 48% within the glioblastoma cell mass (0-80 µm and 81­
160 µm respectively). Overall, this dissertation demonstrates how novel methodologies
employing the zebrafish model can be used to quickly and efficiently identify
nanomaterial hazard and advance applications in nanomedicine, thereby efficiently
bridging toxicity and therapeutic efficacy assessments.
©Copyright by Leah C. Wehmas
August 26, 2015
All Rights Reserved
Nanotoxicology to Nanotherapeutics: Prioritizing Hazard and Anticancer Efficacy of
Nanomaterials using the Embryonic Zebrafish Model
by
Leah C. Wehmas
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented August 26, 2015
Commencement June 2016
Doctor of Philosophy dissertation of Leah C. Wehmas presented on August 26, 2015
APPROVED:
Co-Major Professor, representing Toxicology
Co-Major Professor, representing Toxicology
Head of the Department of Environmental and Molecular Toxicology
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Leah C. Wehmas, Author
ACKNOWLEDGEMENTS
I sincerely appreciate the support and advice of my friends and family during
the completion of this dissertation. I also thank my major advisors Dr. Robert Tanguay
and Dr. Juliet Greenwood for their mentorship and belief in my abilities. Thank you to
my committee, members of the Greenwood laboratory and members of the Tanguay
laboratory for their guidance throughout my time in graduate school. A special thanks
goes to Dr. Tod Harper Jr., Dr. Josephine Bonventre, Dr. Sean Bugel and Jane Ladu
for their helpful edits and assistance in the revising this dissertation. A final thanks goes
to the National Science Foundation (NSF1134468), the National Institute of Health
Training Grant (NIH T32 ES07060), Oregon State University’s P. F. and Nellie Buck
Yerex Fellowship and other funding in support of my research.
CONTRIBUTION OF AUTHORS
Chapter 2 was written by Leanne M. Gilbertson with major contributions from
Leah C. Wehmas. Leah C. Wehmas conducted all toxicology experiments including
methods optimization and initial data visualization and analyses of toxicity data. All
parts of Chapter 2 associated with introduction of the zebrafish model, discussion of
multi-walled carbon nanotube toxicity data and future recommendations were written
by Leah C. Wehmas. Leanne M. Gilbertson synthesized and characterized the multiwalled carbon nanotube and wrote about the material science while Fjodor Melnikov
provided the multivariate statistical analyses and predictive toxicity modelling.
Additional advice on Chapter 2 was provided by Paul T. Anastas, Robert L. Tanguay
and Julie B. Zimmerman.
Chapter 3 was primarily written by Leah C. Wehmas with advice from Dr.
Robert Tanguay, Dr. Alex Punnoose, Dr. Juliet Greenwood and Dr. Cliff Pereira.
Catherine Anders and Jordan Chess in the Punnoose laboratory synthesized all
nanomaterials and performed initial characterization (XRD and TEM) of the primary
particles. Leah Wehmas conducted DLS, SEM, ICP-OES, all zebrafish toxicity
assessments, interpretation and discussion of results. Dr. Cliff Pereira performed
statistical analyses of the data.
Chapter 4 was primarily written by Leah C. Wehmas with advice from Dr. Juliet
Greenwood, Dr. Robert Tanguay and Dr. Alex Punnoose. Dr. Alex Punnoose provided
the zinc oxide nanoparticles and zinc oxide bulk material. Leah C. Wehmas developed,
optimized and conducted all zebrafish xenograft experiments. Some assistance in data
interpretation was provided by Dr. Cliff Pereira. Leah C. Wehmas acquired and
analyzed all imaging data with software support from Paula Gedraitis and Vipat
Raksakulthai at Molecular Devices.
TABLE OF CONTENTS
Page
1 Introduction ................................................................................................................1
1.1 General overview ................................................................................................1
1.2 Nanomaterials ....................................................................................................2
1.2.1 Background ................................................................................................2
1.2.2 Environment and human health ..................................................................3
1.2.3 Carbon nanotubes, applications, health and safety ....................................4
1.2.4 Metal oxides, appications, health and safety...............................................6
1.3 Glioblastoma .......................................................................................................7
1.3.1 Background and mutations .........................................................................7
1.3.2 Nanomedicine for brain cancer ...................................................................9
1.4 Zebrafish model ...............................................................................................10
1.4.1 Background ..............................................................................................10
1.4.2 Toxicology and drug discovery screening ...............................................11
1.4.3 Cancer model ............................................................................................12
1.5 Specific aims ....................................................................................................13
2 Toward safer multi-walled carbon nanotube design: Establishing a statistical model
that relates surface charge and embryonic zebrafish mortality....................................17
2.1 Abstract ............................................................................................................18
2.2 Introduction ......................................................................................................18
2.3 Methods ............................................................................................................22
2.3.1 MWNT preparation ..................................................................................22
2.3.2 MWNT characterization ...........................................................................23
TABLE OF CONTENTS (Continued)
Page
2.3.3 Toxicity testing .........................................................................................23
2.3.4 Statistical analysis ....................................................................................25
2.4 Results ..............................................................................................................27
2.4.1 Physicochemical properties of the MWNTs ............................................27
2.4.2 MWNT toxicity profiles ...........................................................................28
2.4.3 Exploratory statistics used to inform the multivariate model ..................29
2.5 Discussion ........................................................................................................32
2.6 Conclusions ......................................................................................................36
3 Comparative metal oxide nanoparticle toxicity using embryonic zebrafish.............47
3.1 Abstract .............................................................................................................48
3.2 Introduction .......................................................................................................49
3.3 Materials and Methods......................................................................................52
3.3.1 Nanoparticle synthesis...............................................................................52
3.3.2 Nanoparticle characterization ...................................................................54
3.3.3 Inductively coupled plasma optical emission spectrometry of zinc
dissolution .........................................................................................................55
3.3.4 Zebrafish husbandry .................................................................................56
3.3.5 Zebrafish bioassays ..................................................................................56
3.3.6 Nanoparticle exposures ............................................................................57
3.3.7 Scanning electron microscopy .................................................................58
3.3.8 Statistics ...................................................................................................59
3.4 Results and Discussion .....................................................................................60
TABLE OF CONTENTS (Continued)
Page
3.4.1 Metal oxide nanoparticles with similar primary particle size differ greatly
in hydrodynamic size and charge .......................................................................60
3.4.2 Only zinc oxide nanoparticles demonstrate acute embryonic zebrafish
toxicity ...............................................................................................................61
3.4.3 Zinc oxide nanoparticle dissolution analysis and toxicity .......................64
3.4.4 Zinc oxide exposure results in life stage-dependent zebrafish toxicity ...66
3.5 Conclusions ......................................................................................................67
4 An orthotopic zebrafish xenograft assay to study novel human glioblastoma
therapeutics targeting migration, invasion and proliferation .......................................78
4.1 Abstract ............................................................................................................79
4.2 Introduction ......................................................................................................79
4.3 Materials and Methods .....................................................................................82
4.3.1 Zebrafish husbandry .................................................................................82
4.3.2 U87MG maintanence and fluorescent labeling ........................................83
4.3.3 Glioblastoma transplantation procedure ...................................................84
4.3.4 Imaging and analysis of U87MG behavior ..............................................84
4.3.5 Xenograft exposure assay .........................................................................86
4.3.6 Statistical methods ....................................................................................88
4.4 Results ..............................................................................................................89
4.4.1 Transplantation conditions support glioblastoma growth ........................89
4.4.2 Zinc oxide nanoparticle enhances U87MG proliferation and dispersal ...90
4.4.3 LY294002 inhibits U87MG proliferation and dispersal ..........................91
4.5 Discussion ........................................................................................................92
TABLE OF CONTENTS (Continued)
Page
4.5.1 Orthotopic zebrafish xenograft assay supports study of glioblastoma
biology ...............................................................................................................92
4.5.2 Application of orthotopic zebrafish xenograft assay assists drug
prioritization ......................................................................................................94
4.5.3 Nanotherapeutic assessment .....................................................................94
4.5.4 Chemotherapeutic assessment ..................................................................96
4.5.5 Future directions .......................................................................................98
5 Final Conclusions ...................................................................................................115
Bibliography ..............................................................................................................124
Appendix A................................................................................................................141
A.1 Methods ....................................................................................................141
Appendix B ................................................................................................................145
LIST OF FIGURES
Figure
Page
1.1 Potential for exposure throughout the life cycle of nanomaterials from production
to disposal …………………………………………………………………...14
1.2 Examples of single walled carbon nanotubes and multi-walled carbon nanotubes
………………………………………………………………………………..15
1.3 The role of PTEN in regulating the PI 3-kinase/AKT …..……………………....16
2.1 MWNT representative exposure images ..……………………………………….43
2.2 MWNT toxicity response .……………………………………………………….44
2.3 Correlating MWNT physicochemical properties with toxicity …………………45
2.4 Model relationship between probability of embryo mortality and MWNT point of
zero charge ……………………………………….………………………….46
3.1 Size to charge relationship between metal oxide nanoparticles …………………73
3.2 Zinc oxide nanoparticle, zinc oxide bulk and zinc ion equivalent toxicity …..…74
3.3 Dissolution of zinc oxide nanoparticles and zinc oxide bulk …………………...76
3.4 96 hpf zebrafish zinc oxide nanoparticle and zinc ion equivalent toxicity ……...77
4.1 Microinjection setup, zebrafish placement and glioblastoma implantation site .102
4.2 Position of embryonic zebrafish for image acquisition ………………………..104
4.3 Image analysis for proliferation, migration and invasion ……………………...105
4.4 Diagram depicting the timeline of the orthotopic zebrafish xenograft assay ….106
4.5 Comparison of the effects of exposure to zinc oxide nanoparticles (ZnO NP), bulk
material control and vehicle control on the fold change in human glioblastoma
(U87MG) cell count ………………………………………………………..107
4.6 Quantitative analysis of human glioblastoma (U87MG) cell spread following zinc
oxide nanoparticle (NP), zinc oxide bulk (BULK), or vehicle control exposure
………………………………………………………………………………109
LIST OF FIGURES (Continued)
Figure
Page
4.7 Comparison of the effects of increasing exposure to LY294002 on the fold change
in human glioblastoma (U87MG) cell count data ………………………….111
4.8 Quantitative analysis of human glioblastoma (U87MG) cell spread following
LY294002 or vehicle control exposure ……….……………………………113
A.1 Pearson correlations between the five measured MWNTs properties …..….…144
B.1 Representative TEM images of each metal oxide nanoparticle synthesis method
…………………………………………………..………………………….145
B.2 Heat map of metal oxide nanoparticle toxicity ………………………………..146
B.3 Zebrafish exposed to zinc oxide nanoparticle ..………………………………..148
LIST OF TABLES
Table
Page
2.1
MWNT samples and treatment conditions ……………………….......….39
2.2
MWNT physicochemical characteristics ..……………………………… 40
2.3
24-hour zebrafish mortality model summary …………………….......….41
2.4
Model validation results..…………………………………………..…… 42
3.1
Metal oxide nanoparticle physical characterization …………….......…...71
3.2
Descriptions of time points and endpoints assessed during five-day embryonic
zebrafish bioassay……………………………………………………..…72
4.1
Mortality associated with zinc oxide nanoparticle (NP) exposures ........100
4.2
Mortality associated LY294002 exposures..............................................101
A.1
Developmental and toxic endpoints measured at either 24 or 120 hours post
fertilization (hpf)……....…………………………………………..……143
1. Introduction
1.1 General overview
This dissertation focuses on identifying and prioritizing the environmental, health and
safety risks associated with nanomaterials so that novel applications, especially in the field of
medicine can be realized. Chapter 2 demonstrates how the embryonic zebrafish can be utilized to
develop a model to predict multi-walled carbon nanotube (MWNT) toxicity based on surface
functionalization. The adverse biological response of embryonic zebrafish exposed to MWNTs
modified with oxygen moieties was used to build a multivariate mathematical model predicting
which properties resulted in zebrafish toxicity. In Chapter 2, I explore how information acquired
from this model can be utilized in the design of safer nanomaterials before wide-scale production
to reduce future environmental health and safety problems. Chapter 3 focuses on how size, charge,
agglomeration and dissolution of a similar class of un-functionalized metal oxide nanoparticles
(MO NPs) influence embryonic zebrafish toxicity. In Chapter 3, I further address caveats of the
typical embryo-larval toxicity assay by exploring how MO NP agglomeration and dissolution
complicate the interpretation of aqueous exposure results. Chapter 4 discusses how I developed
and applied an orthotopic embryonic zebrafish xenograft model of glioblastoma to assess the
efficacy of a putative anticancer MO NP (zinc oxide). Chapter 4 also confirms the efficacy of the
embryonic zebrafish xenograft assay by assessing the well-studied phosphatidylinositol 3-kinase
inhibitor LY294002. This chapter demonstrates how the zebrafish xenograft model can be used to
prioritize candidate therapeutics and streamline the anticancer compound development pipeline.
1
1.2 Nanomaterials
1.2.1 Background
For centuries, humans have unknowingly harnessed the unique properties, especially the
interesting optical phenomena and durability, that emerge in materials at the nanoscale
(operationally defined as the size range of 1-100 nm) (NNI, 2011). Not until the mid to late 20th
century were people actually able to visualize materials at the atomic and molecular level and
deliberately manipulate them to understand the unusual chemical, physical and biological
properties that emerge (NNI, 2015). Within the size range of 1-100 nm, quantum effects dictate
particle behavior making physical and chemical properties size dependent and customizable
(Smith et al., 2010), yet some unique biological behaviors have been observed for materials larger
(~400 nm or less) than the traditional 100 nm size definition for nanomaterials (Gradishar et al.,
2005; Kato et al., 2003; O'Neal et al., 2004). Furthermore, the greater surface area to volume
ratio of materials at the nanoscale enhances biological reactivity of insoluble materials (Nel et al.,
2006; Oberdörster et al., 2005) or enhances dissolution of soluble materials compared to larger
bulk material counterparts (Borm et al., 2006).
The field of nanotechnology exploits the novel and tunable traits of nanomaterials for
diverse applications in material science, chemical catalysts, electronics (Liang et al., 2002; Park
et al., 2002), energy storage (Chan et al., 2008), personal care products, environmental remediation
and medicine (Ashley et al., 2011; Cheng et al., 2011; Hanley et al., 2008; Kang et al., 2008a;
Nie et al., 2007). There are also many naturally occurring nanoscale particles or materials
including biological macromolecules and byproducts of combustion or volcanic eruptions
(Oberdörster, et al., 2005). For the purposes of this dissertation, use of the term “nano” as in
2
nanomaterial, nanoparticle or nanotube will refer to engineered nanomaterials unless specifically
stated otherwise.
1.2.2 Environment and human health
Each year more and more products incorporate nanomaterials, especially in the field of
medicine, with no expectations of slowing. This increases the likelihood of exposure throughout
the material lifecycle from production to disposal particularly for nanotherapeutics (Figure 1.1).
Nanomaterials are of a size similar to many cellular structures, organelles, enzymes and nucleic
acids. The numerous functionalities, material compositions, shapes and coatings increases
opportunities for negative interactions with living organisms and the environment (Nel, et al.,
2006; Oberdörster, et al., 2005). Concerns for environment, human health and safety via intended
(i.e. application of personal care products, use of nanoscale medicines or environmental
remediation) or unintended (i.e. occupational exposure and leaching) exposures remain (Figure
1.1).
Therefore, the United States government established the National Nanotechnology
Initiative (NNI) (NNI, 2015). The NNI realized that all socio-economic benefits associated with
nanomaterials and nanotechnology must be counterbalanced with possible environment, human
health and safety impacts (NSET, 2014). Comprehensive risk assessment requires toxicology
testing, yet the unique challenge associated with nanomaterial toxicology or nanotoxicology is that
physical form (i.e. surface area, shape, agglomeration) as well as chemical composition (i.e.
charge, functionalization, band gap) can interact to cause adverse biological responses not
observed by chemicals alone (Maynard et al., 2010; Oberdürster, 2000). To account for these
challenges, nanotoxicology requires additional test considerations not found in traditional
3
chemical based toxicological safety assessments (Maynard, et al., 2010). In recognition of these
problems, the NNI drafted an Environment, Health and Safety Research Strategy outlining
methods to prioritize and acquire the necessary data for accurate nanomaterial risk assessment.
This requires creating better predictive models relating structure and composition to adverse in
vivo biologic response (NNI, 2011), which is necessary to prioritize the testing of the
nanomaterials that possess the greatest environmental health and safety risk. Furthermore, this
information is necessary to create guidelines and support for the design of safe, environmentally
benign nanomaterials. Two common classes of nanomaterial with many industrial and medical
applications, yet also potential environmental health and safety risks are carbon nanotubes and
metal oxide nanoparticles.
1.2.3 Carbon nanotubes, applications, health and safety
Carbon nanotubes (CNTs) are hollow, cylindrical tubes comprised of individual (single­
wall, SWNTs) sheets of graphene or multiple (multi-walled, MWNTs) sheets of graphene creating
a tube within a tube structure (Figure 1.2) (De Volder et al., 2013). The surface may be bare or
functionalized with different chemical groups enhancing solubility and specificity for delivery of
therapeutics. CNTs possess great mechanical strength, electrical properties, large aspect ratio and
thermal traits making them very attractive for material science, electronics, water purification and
medicine (De Volder, et al., 2013; Popov, 2004). Global CNT production is predicted to surpass
12,300 metric tons in 2015, most of which will be comprised of MWNTs (Patel, 2011). The ability
to attach various functional groups to MWNTs, while useful for biomedical applications, may alter
bioavailability or biodistribution and lead to unexpected adverse biological interactions.
4
There are already concerns that the fiber-like characteristics (large aspect ratio) of CNTs
will cause mesothelioma similar to the effects observed with asbestos exposure (Donaldson et al.,
2006). Studies using mammalian cell lines or rodent models demonstrate MWNTs induce
genotoxicity, inflammation, oxidative stress, granulomas and mesotheliomas (Mitchell et al.,
2007; Poland et al., 2008; Sakamoto et al., 2009; Yang et al., 2009; Ye et al., 2009; Zhu et al.,
2007). However, contradictory data exists. The differences in routes of exposure, MWNT size,
dispersal method and level of agglomeration make it difficult to compare studies and create rational
material synthesis guidelines to reduce adverse biological interaction (Johnston et al., 2010;
Maynard, et al., 2010; Oberdörster, 2010). The two general synthesis guidelines gleaned from
these studies are longer MWNTs are generally more toxic than short (Brown et al., 2007;
Johnston, et al., 2010; Poland, et al., 2008) and care should be taken to remove contaminants
leftover from synthesis (Guo et al., 2007; Kagan et al., 2006).
The high production volumes of MWNTs guaranty that some will eventually end up in the
environment during disposal or as waste byproducts. While research exists investigating MWNT
exposure effects on microbiota (Gilbertson et al., 2014; Kang, et al., 2008a; Kang et al., 2008b),
there is also need to understand effects in vertebrates such as fish during sensitive early life stages.
As with experiments in mammalian models, the few studies investigating MWNT toxicity in
developing fish observe some toxicity but these studies utilize disparate base materials and
experimental methods, which may account for differences in experimental outcomes (Asharani et
al., 2008; Cheng et al., 2009; Cheng et al., 2007). To realize the benefits of MWNTs for fields
such as medicine, there is a need to systematically examine how MWNT physicochemical traits
such as surface functionalization contribute to adverse biological response in vivo, which can be
used to prospectively create safer products instead of dealing with health hazards retrospectively.
5
1.2.4 Metal oxides, applications, health and safety
Metal oxides (MO) comprise a common class of nanoparticles of semiconductor materials
with broad applications in personal care products, electronics, energy storage, medicines and
medical imaging (Nel, et al., 2006; Oberdörster, et al., 2005). Some of the most widely used metal
oxide nanomaterials include zinc oxide (ZnO), titanium dioxide (TiO2), cerium dioxide (CeO2)
and tin dioxide (SnO2). While many of these materials initially drew interest due to antimicrobial
traits (Adams et al., 2006; Li et al., 2008; Premanathan et al., 2011; Reddy et al., 2007), they are
now being applied to cancer imaging and therapeutics (Lee et al., 2007a; Lee et al., 2007b; Liong
et al., 2008; Nie, et al., 2007). In vitro studies demonstrate some metal oxide nanoparticles (MO
NPs) such as ZnO and TiO2 cause inflammatory response, reactive oxygen species generation,
apoptosis and genotoxicity, which could be useful in destroying cancer cells if selective (Prasad et
al., 2013). In fact, some TiO2 and ZnO NPs demonstrate preferential cytotoxicity toward cancer
cells making these materials of particular interest as therapeutics (Akhtar et al., 2012; Hanley, et
al., 2008; Thevenot et al., 2008). Alternatively, CeO2 NPs may act as an antioxidant providing
cytoprotective effects (Özel et al., 2013; Schubert et al., 2006; Xia et al., 2008); however,
contradictory data exists and the reactive oxygen species scavenging ability may depend on the
redox potential of CeO2 NPs in various exposure media (Auffan et al., 2009; García et al., 2011;
Lin et al., 2006). Little information is available on the toxicity of SnO2 NPs but the existing data
suggests it is relatively inert (Zhang et al., 2012). While these in vitro experiments are useful in
exploring specific mechanisms of toxicity at the cellular level, nanomaterial studies are especially
challenging to translate to the organismal level (Maynard, et al., 2010). The same problems
associated with MWNT experiments exist with MO NPs making general conclusions about the
class of materials difficult based on differing in vivo data. In vivo exposures to TiO2 and ZnO NPs
6
have measured oxidative stress response and inflammation, sometimes only at high concentrations
(George et al., 2011; Kao et al., 2012; Paterson et al., 2011; Sayes et al., 2006; Xiong et al.,
2011; Yu et al., 2011; Zhang, et al., 2012; Zhu et al., 2009). Additional controversy arises from
the potential of some MO NPs (ZnO) to dissolve and release toxic ions (Adams, et al., 2006;
Brunner et al., 2006; Franklin et al., 2007; Xia, et al., 2008; Yang et al., 2006; Zhu et al., 2008),
the potential for photoactivation-induced toxicity (TiO2) (Bar-Ilan et al., 2012; Gopalan et al.,
2009; Gurr et al., 2005; Sanders et al., 2012; Yeo et al., 2012) and the use of surfactants, which
influence dispersal and confound interpretation of the physicochemical surface properties
influencing toxicity (Maynard, et al., 2010). A better understanding of the physicochemical
properties contributing to MO NP toxicity in vivo while mitigating these confounding factors is
required for safer MO NP design and for the realization of future MO NP biomedical applications.
1.3 Glioblastoma
1.3.1 Background and mutations
Glioblastoma is the most prevalent and deadly primary brain tumor to occur in adults,
accounting for 45.6% of malignant primary brain cancers (Ostrom et al., 2014). Glioblastoma
arises from astrocytes, a major type of glial cell located in the brain and spinal cord that provides
structural support, maintains homeostasis, and regulates neuronal development (Abbott et al.,
2010). Necrotic foci, aberrant cellular morphology and angiogenesis are histological
characteristics defining glioblastoma (DeAngelis, 2001). Approximately 3.19 per 100,000 people
develop this disease, and it typically occurs later in life starting around age 55 (Ostrom, et al.,
2014). Despite a relatively low incidence, the median survival rate for glioblastoma is only 15 mo
(Hayat, 2011), and the considerable health care services associated with this disease creates a
7
significant socioeconomic onus (Kutikova et al., 2007 ). The invasive nature of glioblastoma leads
to high recurrence making even aggressive, multimodal treatment options such as surgery,
radiation and chemotherapy not very effective (Stupp et al., 2005). Several decades of research
into new glioblastoma treatments has not improved the five-year survival rate, which remains
unacceptably poor at ~5% (Ostrom, et al., 2014).
Further complicating successful treatment of glioblastoma is the heterogeneous mutational
spectrum (TCGA, 2008). No one or two gene mutations cause glioblastoma, but there are essential
signaling pathways controlling normal cellular growth and survival that are commonly
dysregulated (TCGA, 2008). Large scale analyses of genomic alterations identified mutations
leading to aberrant activation of receptor tyrosine kinase-RAS pathway, enhanced protein kinase
B (AKT)- phosphatidylinositol 3-kinase (PI 3-kinase) signaling, or inactivation of p53­
retinoblastoma tumor suppressor pathway in 88% of glioblastoma samples (TCGA, 2008). One
common homozygous mutation that results in overactivation of the PI 3-kinase/AKT pathway is
that of PTEN (TCGA, 2008;
Cantley et al., 1999). PTEN acts as a tumor suppressor in
glioblastoma by preventing the dephosphorylation of phosphatidylinositol 3-phosphate (Cantley,
et al., 1999), which mediates the activation of AKT via PI 3-kinase leading to apoptotic and
therapeutic resistance (Figure 1.3) (Mayo et al., 2002).
Another major obstacle to the development of new glioblastoma therapeutics in addition
to therapeutic resistance is the blood brain barrier (Cecchelli et al., 2007). The blood brain barrier
consists of endothelial cells lining capillaries that supply the central nervous system and helps
maintain the electrochemical environment around neurons and axons necessary for synaptic
transmissions (Cecchelli, et al., 2007). The tight junctions, transporters and specific enzymes
present in the endothelial cells of the blood brain barrier create a physical, chemical and metabolic
8
barrier to the central nervous systems (Cecchelli, et al., 2007). Unless actively transported, only
small, hydrophobic molecules around 400-600 Da (~1 nm in diameter assuming a spherical shape)
can pass the blood brain barrier (Pardridge, 1998). Any new glioblastoma therapeutics must be
able to cross this protective barrier passively or actively to inhibit glioblastoma progression and
be effective. The emerging field of nanomedicine is opening new avenues of glioblastoma
treatment, diagnostic imaging and potential for enhanced blood brain barrier translocation through
functionalization and active transport.
1.3.2 Nanomedicine for brain cancer
Development of nanomaterials for biomedical applications as pharmaceuticals and in vivo
imaging and diagnosis has expanded in recent years, opening new avenues of innovation in human
health (Etheridge et al., 2013). The small size (molecular scale), diverse possibilities in material
composition, numerous morphologies and ease in functionalization makes nanomaterials
especially advantageous as targeted anti-cancer agents (Ashley, et al., 2011), enhanced drug
delivery systems (Nie, et al., 2007) or high resolution imaging agents (Smith et al., 2011). For
instance, the larger surface area to volume ratio of materials at the nano scale increases
bioavailability (Oberdörster, et al., 2005), preparation with biocompatible materials like
poly(lacticzoglycolic acid) and coating with polyethylene glycol helps evade the immune system
extending circulation (Gref et al., 1994) and the small size allows extensive penetration and
retention in tumors (Perrault et al., 2009; Wong et al., 2011). Furthermore, functionalization with
peptides specific to receptors such as integrin αvβ3, which are commonly overexpressed around or
on cancer cells or functionalization with aptamers can enhance delivery and site-specific toxicity
while reducing adverse systemic effects (Cheng, et al., 2011; Farokhzad et al., 2006; Hood et al.,
9
2002). Of additional interest to future glioblastoma drug development is that some nanoparticles
can bypass the blood brain barrier through transport along neurons to the brain (Oberdörster et al.,
2004). Furthermore, certain sizes (150-300) or functionalization that activates the low density
lipoprotein or transferrin receptors may enhance nanoparticle transport across the blood brain
barrier (Wohlfart et al., 2012) and delivery to tumors. These examples, illustrate how
nanomaterials will improve glioblastoma therapeutics.
1.4 Zebrafish model
1.4.1 Background
As a useful developmental and molecular model, detailed information on zebrafish biology,
physiology and morphology has been available for several years (Driever et al., 1996; Driever et
al., 1994; Haffter et al., 1996; Kimmel et al., 1995). The conservation of developmental processes
between zebrafish and other vertebrates including humans makes it a valuable and relevant
developmental model (Haffter, et al., 1996). Large scale mutagenesis screening helped elucidate
many of the genes and molecular signaling pathways involved in zebrafish development (Driever,
et al., 1996) and sequencing of the zebrafish genome added to the translational relevance of the
model by identifying 71.4% genome sequence conservation with that of humans (Howe et al.,
2013). Genomic information has helped simplify genetic techniques for quantifying gene
expression, creating transgenics and producing conditional knockouts through antisense
technologies such as morpholinos to study gene function and expression patterns (Zon et al., 2005).
The knowledge base associated with the zebrafish model has made them adaptable to many other
fields including toxicology and drug discovery.
10
1.4.2 Toxicology and drug discovery screening
The wealth of information on zebrafish biology makes it an advantageous model in
understanding the effects (beneficial and adverse) of compound (chemical and nanomaterial)
exposure (Fako et al., 2009; Hill et al., 2005; Zon, et al., 2005). Zebrafish possess a remarkable
capacity for egg production and require only a few months to reach reproductive maturity making
it possible to provide thousands of embryos for experiments not feasible with mammalian models
(Detrich et al., 1999). The small size and quick development, most major organ systems are fully
developed three days post fertilization, makes the zebrafish amenable to rearing in multi-well
plates for moderate-throughput embryo-larval screening assays (Zon, et al., 2005). The
translucency and external development of the embryos allows for non-invasive phenotypic
observation of compound effects using a stereo microscope (Hill, et al., 2005; Zon, et al., 2005).
Coupling the use of specific transgenic fish with fluorescence microscopy can help elucidate
whether a compound is activating or inhibiting molecular processes, thereby identifying a target
of toxicity or target for therapeutic intervention (Zon, et al., 2005). The ease of phenotypic-based
screening allows for the correlating chemical structure or nanomaterial physicochemical
modifications with biological response to create structure activity relationships informative of
hazard (Harper et al., 2011; Knecht et al., 2013).
The short duration of embryo-larval zebrafish assays (3-5 days) puts it on par with the
speed of in vitro studies. However, additional information on biodistribution, biopersistance,
metabolism, excretion and systemic effects can be investigated in embryo-larval zebrafish assays
that would require numerous different in vitro assays with multiple cell types. The many systems
biology based techniques available for use in the zebrafish such as proteomics, transcriptomics
and metabolomics can provide a holistic understanding of the mechanism of action of a compound,
11
whether it be nanomaterial, drug or chemical, which is highly desirable for cross species
comparisons.
1.4.3 Cancer model
Zebrafish possess orthologues with several genes implicated in human diseases (Howe, et
al., 2013) and develop cancers with molecular signatures similar to human cancers (Lam et al.,
2006). Therefore, zebrafish are increasingly used as a model to study human diseases such as
cancer (Feitsma et al., 2008). Cancer transplantation experiments demonstrate embryonic
zebrafish hold promise to study some details of malignant progression that are incredibly
challenging in mammalian models. For instance, research investigating growth, angiogenesis,
migration, invasion and metastases of a variety of human cancers in embryonic zebrafish
noninvasively already exists (Geiger et al., 2008; Haldi et al., 2006; Lal et al., 2012; Nicoli et
al., 2007; Yang et al., 2013). Moreover, this research suggests that these embryonic zebrafish
xenograft models hold promise in investigating the efficacy of chemo or nanotherapeutic
intervention on proliferation, migration and invasion in real time (Cheng, et al., 2011; Geiger, et
al., 2008; Nicoli, et al., 2007; Yang, et al., 2013). The absence of a fully functional immune
system during early zebrafish development (Lam et al., 2004) means these xenograft experiments
can be completed without potentially confounding effects of immunosuppressive chemicals.
Therefore, embryonic zebrafish xenografts can be incorporated into screening for novel chemo or
nanotherapeutics to aid in drug discovery especially for highly invasive diseases like glioblastoma
where little progress in treatments has occurred over the years.
Development of a realistic embryonic zebrafish xenograft assay of glioblastoma to
prioritize therapeutic compound development remains incomplete. Most embryonic zebrafish
12
xenograft studies transplant the cancer cells in the yolk (Geiger, et al., 2008; Haldi, et al., 2006;
Marques et al., 2009; Yang, et al., 2013) or perivitelline space (Nicoli, et al., 2007). However,
research shows that injection site location can significantly alter typical cancer behavior (Lal, et
al., 2012). Transplanting glioblastoma cells into the brain microenvironment recapitulates the
invasive behavior of glioblastoma (Lal, et al., 2012). Moreover, the presence of a blood brain
barrier by three days post fertilization in zebrafish (Jeong et al., 2008) will also help identify
compounds that can traverse the barrier. Therefore, creation of an orthotopic embryonic zebrafish
xenograft assay is necessary to identify and prioritize chemicals and nanomaterials targeting
glioblastoma proliferation, migration and invasion for future therapeutic advancement.
1.5 Specific aims
By addressing three specific aims, this dissertation will reveal how the embryonic zebrafish
model can be applied to prioritize nanomaterial hazard and anticancer efficacy bridging the fields
of nanotoxicology and nanotherapeutics to advance drug development and safety. The specific
aims of this dissertation are to:
Aim 1. Evaluate two common classes of nanomaterials (MWNT and MO NPs) for toxicity
using the embryo-larval zebrafish assay and prioritize further assessment.
Aim 2. Identify physicochemical properties of MWNTs and MO NPs associated with
zebrafish toxicity to inform safe nanomaterial design and predict nanotoxicity
Aim 3. Establish a novel orthotopic embryonic zebrafish xenograft assay to prioritize
efficacy of nanotherapeutic agents for glioblastoma drug development.
13
Figure 1.1. Potential for exposure throughout the life cycle of nanomaterials from production
to disposal
14
Figure 1.2. Examples of single walled carbon nanotubes and multi-walled carbon nanotubes
Image adapted from Raymond M. Reilly J Nucl Med 2007;48:1039-1042.
15
Figure 1.3. The role of PTEN in regulating the PI 3-kinase/AKT pathway
Abbreviations: PTEN- Phosphatase and tensin homolog; AKT-Protein kinase B; PI3K­
Phosphatidylinositol 3-kinase; PIP2- Phosphatidylinositol 4,5-bisphosphate; PIP3­
Phosphatidylinositol (3,4,5)-trisphosphate; P-Phosphorylation; BAD- BCL2-associated agonist of
cell death; mTOR- Mammalian target of rapamycin, NFKB- Nuclear factor kappa-light-chain­
enhancer of activated B cells
16
2. Toward safer multi-walled carbon nanotube design: Establishing a
statistical model that relates surface charge and embryonic zebrafish
mortality
This article is printed with permission from Informa Healthcare License Number: 3677150628039
Leanne M. Gilbertson, Fjodor Melnikov, Leah C. Wehmas, Paul T. Anastas, Robert L. Tanguay,
and Julie B. Zimmerman
Nanotoxicology
Volume 0
Issue 0
17
2.1 Abstract
Given the increased utility and lack of consensus regarding carbon nanotube (CNT)
environmental and human health hazards, there is a growing demand for guidelines that inform
safer CNT design. In this study, the zebrafish (Danio rerio) model is utilized as a stable, sensitive
biological system to evaluate the bioactivity of systematically modified and comprehensively
characterized multi-walled carbon nanotubes (MWNTs). MWNTs were treated with strong acid
to introduce oxygen functional groups, which were then systematically thermally reduced and
removed using an inert temperature treatment. While 25 phenotypic endpoints were evaluated at
24 and 120 hours post-fertilization (hpf), high mortality at 24 hpf prevented further resolution of
the mode of toxicity leading to mortality. Advanced multivariate statistical methods are employed
to establish a model that identifies those MWNT physicochemical properties that best estimate the
probability of observing an adverse outcome. The physicochemical properties considered in this
study include surface charge, percent surface oxygen, dispersed aggregate size and morphology
and electrochemical activity. Of the five physicochemical properties, surface charge, quantified as
the point of zero charge (PZC), was determined as the best predictor of mortality at 24 hpf. From
a design perspective, the identification of this property–hazard relationship establishes a
foundation for the development of design guidelines for MWNTs with reduced hazard.
2.2 Introduction
There are numerous current and projected applications of carbon nanotubes (CNTs)
spanning a range of sectors, including energy, electronics and healthcare (De Volder et al., 2013;
Endo et al., 2008). These applications are inspired by the unique physical and chemical properties
of CNTs, including tensile strength, electronic and thermal conductivity, aspect ratio and
18
antimicrobial activity, to name a few (Endo, et al., 2008; Popov, 2004). Of the two primary classes
of CNTs, single- (SWNT) and multi-walled (MWNT), MWNTs are primarily utilized in high
concentration, low order applications and accounted for 95% of the total 2010 CNT production
value (Patel, 2011). As such, there is increased likelihood of environmental and human exposure
to MWNTs considering the various pathways of release throughout the life cycle (EPA, 2014).
Given the current lack of consensus regarding the MWNT hazard profile, this is potentially of
concern for environmental and human health.
To better understand and resolve the specific material properties that can induce MWNT
environmental and human health hazard, zebrafish (Danio rerio) were selected as the model
organism. Zebrafish are considered a preferred in vivo vertebrate model organism for assessing
the toxicity of nanomaterials for multiple reasons (Fako et al., 2009; Lin et al., 2013; Rizzo et
al., 2013; Usenko et al., 2007). Zebrafish possess genetic parallels with humans enabling
projection and understanding of potential mechanisms of human toxicity (Fako, et al., 2009).
Zebrafish also have a remarkable degree of molecular and physiological conservation with other
vertebrates, particularly during embryonic development (Amsterdam et al., 2004). Exposure to
exogenous compounds during this sensitive life stage can disrupt key developmental processes
such as cell differentiation, organogenesis, apoptosis, proliferation, ion regulation and
neurogenesis, which manifest as craniofacial malformations, edema, disrupted vasculature and
muscles, abnormal evasion response and even death (Harper et al., 2011a; Hill et al., 2005).
Further, zebrafish are favorable for high throughput toxicity screening methodologies as they
facilitate rapid testing of numerous chronic and acute endpoints, simultaneous testing of a range
of concentrations and the potential to probe detailed mechanisms of toxicity (Harper, et al., 2011a).
19
While there is a significant body of research investigating the impacts of CNTs at the
cellular level (Johnston et al., 2010; Kang et al., 2008a; Kang et al., 2008b; Kang et al., 2007;
Liu et al., 2009; Pasquini et al., 2012; Pasquini et al., 2013a; Vecitis et al., 2010) and using
mammalian models (Kane et al., 2008; Kolosnjaj-Tabi et al., 2010; Lam et al., 2006; Lam et al.,
2004; Muller et al., 2005; Nel et al., 2006), those studies pertaining to higher order aquatic
organisms are more disparate (Baun et al., 2008; Scown et al., 2010). In particular, only a handful
of studies, to date, investigate the impact of CNTs on zebrafish (Adenuga et al., 2013; Asharani
et al., 2008; Cheng et al., 2009; Cheng et al., 2012; Cheng et al., 2007), and there are significant
variations in the experimental methodologies. A seminal paper by Cheng et al. (2007) found that
the chorion serves as a protective barrier, preventing the interaction between the CNT aggregates
and the embryo. While they observed delayed hatching, there were no observed developmental or
mortality implications (Cheng, et al., 2007). To facilitate direct exposure between CNTs and the
embryo, two alternative procedures were developed. The first is a microinjection technique, which
is administered at the one-cell stage (Cheng, et al., 2009; Cheng, et al., 2012) or 8-cell stage
(Asharani, et al., 2008). The second method involves enzymatic dechorionation of the embryos 4
hours post-fertilization (hpf), prior to CNT exposure (Mandrell et al., 2012; Truong et al., 2011).
This latter technique is employed in this study as it is automated, quick, cost effective and more
precise than the manual alternative (Mandrell, et al., 2012).
While the current CNT zebrafish literature thoroughly evaluates both lethal and sublethal
endpoints, there is often a lack of comprehensive physicochemical characterization of the
nanomaterials necessary to draw meaningful conclusions or to compare results between studies
(Asharani, et al., 2008; Cheng, et al., 2009; Cheng, et al., 2012; Cheng, et al., 2007). The approach
employed here is unique from those presented in previous CNT zebrafish studies in that the
20
MWNTs: (1) originated from the same starting batch, which eliminates the potential of
confounding variables associated with batch heterogeneity (Jones et al., 2008); (2) were
systematically modified, minimizing the number of properties being varied at one time; and (3)
were comprehensively characterized for those intrinsic and extrinsic properties most pertinent to
their toxicity including surface charge, oxygen content, dispersed aggregate radius and
morphology and electrochemical activity (Fubini et al., 2010; Powers et al., 2007).
The ability to extrapolate relationships between physicochemical properties and observed
trends in adverse outcomes will improve our understanding of the underlying toxicity mechanisms
as well as promote the design of CNTs for enhanced performance and minimized hazard. To
address the current knowledge gap, this study intends to inform the development of property–
hazard relationships for a given set of MWNTs by developing a logistic model that relates MWNT
physicochemical properties and zebrafish toxicity.
The study presented herein has three primary goals: (1) to determine whether the
established correlating trend of MWNT electrochemical activity and bacterial cytotoxicity
(Gilbertson et al., 2014a; Pasquini, et al., 2013a) is maintained for higher trophic organisms (i.e.
zebrafish), (2) to establish a model that relates the observed trend in the adverse effects in zebrafish
with physicochemical properties of MWNTs and (3) to utilize the model as a first step towards
developing a foundational understanding of the underlying mechanism of the bioactivity of
MWNTs. The combined results aim to elucidate the toxic potential of MWNTs and to inform their
future inherently safer design.
21
2.3 Methods
2.3.1 MWNT preparation
Pristine MWNTs were purchased from CheapTubes (Burlington, VT, CCVD, >95 wt%,
10–20 nm diameter, 10–20 μm length; (Cheaptubes, 2014)). These as-received MWNTs were acid
treated in house via reflux in nitric acid (HNO3, 15.7 M) for 2 h. This acid treated (AT 2) MWNT
sample was then annealed (heat treatment under inert conditions) at increased maximum
temperature (400–900 ‡C, 100 ‡C increments) and served as the model training set. A separate set
of MWNTs, purchased from NanoLab Inc. (Waltham, MA 15 ± 5 nm diameter, 5–20 μm
length, >95% purity), was prepared and utilized to confirm the logistic model (Nanolab, 2011).
These as-received MWNTs (NL-AR) were treated by the manufacturer using a sulfuric–nitric acid
mix and then annealed in house at a subset of the previously applied temperatures (400, 600, and
900 ‡C).
Annealing enabled systematic modifications of MWNT properties via reduction of surface
oxygen groups that were added during the acid treatment. The various functional group moieties
(e.g. carboxylic acid, hydroxyl and carbonyl) possess different thermal properties (Figueiredo et
al., 1999b). As the maximum annealing temperature increases, the more labile groups elude the
MWNT surface while the more thermally stable groups remain (illustrated in the schematic in
Table 2.2). The annealing conditions remained consistent for all samples, including heating under
helium (He) at the indicated maximum temperature for 1 h (10‡ per min heating rate). Compiled
treatment conditions for all of the MWNT samples utilized in this study can be found in Table 2.1.
22
2.3.2 MWNT characterization
The physicochemical properties of the MWNT samples used in this study were
comprehensively characterized for sample purity by thermogravimetric analysis (TGA), surface
charge by point of zero charge (PZC), elemental composition by x-ray photoelectron spectroscopy
(XPS), dispersed aggregate size and size distribution by dynamic light scattering (DLS), dispersed
aggregate morphology by static light scattering (SLS) and electrochemical activity by the oxygen
reduction reaction (ORR) using the rotating disc electrode (RDE) method. Dispersed aggregate
morphology is quantified here by the fractal dimension (Df), which is evaluated using the static
light scattering technique, and is representative of the aggregate compactness and shape (Df = 1:
rod-like morphology, Df = 3: sphere-like morphology; (Chen et al., 2004; Schaefer et al., 2003).
Relative MWNT surface charge was determined via PZC using a mass titration method (Noh et
al., 1989; Pasquini, et al., 2013a). The point of zero charge is the pH at which there is a net zero
surface charge, and thus, the values reported in Table 2.2 are pH values obtained using the
previously described titration method. Details on additional characterization methods utilized in
this study can be found in the Appendix A. Table 2.2 contains the compiled characterization data,
including some previously published data associated with our bacterial cytotoxicity study
(Gilbertson, et al., 2014a).
2.3.3 Toxicity testing
At 4-h post-fertilization (hpf), the acellular chorion was enzymatically removed from
tropical 5D zebrafish embryos using pronase following an established protocol described in
Mandrell et al. (2012) (Mandrell, et al., 2012). At 6 hpf, embryos were rinsed and transferred to a
low ionic strength 62.5 µM CaCl2·2H2O (Sigma Aldrich, St. Louis, MO) medium prepared in
23
ultrapure water (Invitrogen, Grand Island, NY) prior to loading (one embryo per well) into 96-well
plates (Falcon, Waltham, MA) containing 0.05 mL of 62.5 µM CaCl2·2H2O. A low ionic strength
62.5 µM CaCl2·2H2O medium was employed in this assay to minimize MWNT aggregation while
maintaining normal zebrafish development. On the day of exposure, MWNTs were suspended in
a 10% dimethylsulfoxide (DMSO; J.T. Baker, Center Valley, PA) solution prepared with ultrapure
water to create a 1 mg/mL MWNT stock. The stock was sonicated indirectly for 30 min via Cup
Horn with a Fisher Scientific 60 Sonic Dismembrator at 20 kHz. Immediately following
sonication, MWNT stock was diluted in CaCl2·2H2O water to prepare a five-fold, 2X MWNT
dilution series ranging from 100 to 0.16 mg/L with DMSO (1%) and CaCl2·2H2O (62.5 µM). At
8 hpf, 0.05 mL of the 2X MWNT suspension was added to 96-well plates for an n = 48 per
exposure concentration. Zebrafish underwent a static non-renewal exposure to 1X MWNT (50–
0.08 mg/L), 0.5% DMSO and 62.5 µM CaCl2·2H2O for 5 days maintained at 28 ± 1 ‡C in the dark.
The concentration range utilized is the standard range for this established method (Adenuga, et al.,
2013; Truong, et al., 2011). At 24 hpf, embryos were visually evaluated by stereomicroscope for
mortality, spontaneous tail flexion, notochord malformations and delayed developmental
progression. At 120 hpf, zebrafish were evaluated for mortality, phenotypic malformations of the
axis, yolk sac, eye, jaw, snout, brain, otic vesicle, somites, heart, circulation, caudal and pectoral
fins, swim bladder and behavioral response to mechanical stimulus (Appendix Table A.1). Results
were recorded using a binary system of 1 for the presence of an endpoint and 0 for absence. For a
more detailed description of the assay, refer to Truong et al. (2011) (Truong, et al., 2011). All
experiments were conducted in accordance with Institutional Animal Care and Use Committee
protocols at Oregon State University.
24
2.3.4 Statistical analysis
2.3.4.1 Model selection and development. All statistical analyses were conducted using the
R version 3.02 statistical software (R_Core_Team, 2013).
2.3.4.2 The logistic model. A logistic model was used to fit the MWNT physicochemical
property and zebrafish toxicity data to provide a means of deriving a relationship between the
probability of zebrafish embryo mortality (p) and specific MWNT properties. The nature of the
relationship between continuous property data and the binomial toxicity outcome suggests the
appropriate use of binomial regression. Binomial models are commonly used for toxicity outcomes
(Morgan, 1992) and have three major mathematical advantages when modeling binary data,
including adequate consideration of inconsistent variance, no necessity for lack of normality
assumptions and no range restrictions imposed by binary response variables. The logistic binomial
model was chosen for this study as it is easily interpretable and there was no available information
on success asymmetry (Collette, 1991).
In this study, logistic regression was used to predict the category of outcome (e.g. dead or
alive) for individual embryos optimizing the model for minimal complexity. Logistic regression
calculates the outcome as odds ratio of embryo death, or probability of death over the probability
of survival according to Equation (1)
ዉ
Lቷቯቱቼ ሤሲዂ ሥ ሲ ቴቶ ረማሥዉኺ ቃ
(1),(Collett, 1991)
ኺ
where pi = p[xi=1] =1-p[xi=0] and xi is the independent variable corresponding to ith observation
in the sample i = 1, 2, …, n. In this case pi or p[xi=1], is the probability of observing a toxic effect
and 1-pi or p[xi=0] is the probability of embryo survival.
25
Five MWNT physicochemical properties were included as potential predictor variables in
the univariate and multivariate analysis to elucidate the toxicity outcome. The affection of percent
surface oxygen, point of zero charge, dispersed aggregate radius, dispersed aggregate morphology,
and electrochemical activity, and their interactions, on zebrafish embryo toxicity is considered. In
logistic regression, these predictor variables are related to the probability of mortality as shown in
Equation 2:
ሲዂ ሲ
ኾ ሠዧሒ ሜዧሓ ዉሓ ሜዧሔ ዉሔ ሜኔሜዧኼ ዉኼ ሡ
(2)(Collett, 1991)
ማሤ ኾ ሠዧሒ ሜዧሓ ዉሓ ሜዧሔ ዉሔ ሜኔሜዧኼ ዉኼ ሡ
where ዀሚ is the intercept coefficient, ዀማ through ዀዄ are parameter coefficients for independent
variables 1 though k, and x1 through xk are measured parameters for the specific MWNT sample
exposed to zebrafish embryo i.
Equation (3), a rearrangement of equations (1) and (2), represents the general form of
logistic regression for the logit probability function.
ዉ
Lቷቯቱቼ ሤቸዂ ሥ ሲ ቴቶ ረማሥዉኺ ቃ ሲ ዀሚ ራ ዀማሺማ ራ ዀሜ ሺሜ ራ ኔ ራ ዀዄ ሺዄ
ኺ
(3)
The parameters of the logistic model ዀሚ though ዀዄ are derived by the method of maximum
likelihood. Under the assumption of the logistic regression, the chosen coefficients maximize the
probability or likelihood of the observed data (Collett, 1991).
2.3.4.3 Model training and selection. The model was trained on a set of seven
systematically treated MWNT (see Table 2.1 for treatment details) with 240 embryonic zebrafish
tested for each MWNT type (48 embryos per concentration, 5 exposure concentrations). An
iterative model selection process was used to derive the most parsimonious model without over
fitting the data. We used Akaike information criterion (AIC), Wald test, and likelihood-ratio test
to compare and select model fits (Fox, 1997). Parsimonious models with clear interpretations were
preferred, when statistical parameters were similar. Z-score tests were used to assess individual
26
parameter contribution to the overall model. Likelihood ratio, Wald, and the more conservative le
Cessie-van Houswelingeen-Copas-Hosme tests (le Cessie et al., 1991) were also used to assess
model differences
2.3.4.4 Model validation. The final multivariate model performance was tested on a distinct
set of four MWNT samples (see Table 2.1 for treatment details) under the same methodological
conditions as previously described for the toxicity evaluation.
2.4 Results
2.4.1 Physicochemical properties of the MWNTs
Various techniques were utilized here to characterize the intrinsic and extrinsic properties
of the MWNT sample set. The data is compiled in Table 2.2, including a schematic that illustrates
the change in surface oxygen groups with increased annealing temperature (Gilbertson et al.,
2014b). There are some identifiable trends in the compiled data. First, there is a systematic
decrease in the percent surface oxygen with increased annealing temperature due to the varying
thermal stability of oxygen functional groups on MWNT surfaces (Kundu et al., 2008). The
corresponding increase in the PZC with increased annealing temperature results from the lower
thermal stability of the more acidic functional groups, including carboxylic acid (COOH) and
hydroxyl (OH), relative to more basic oxygen moieties, such as carbonyl (C=O) containing
anhydride, ester, and quinone (Figueiredo et al., 1999a; Gilbertson, et al., 2014b; Montes-Morán
et al., 2004).
There are no discernable trends in the dispersed aggregate radius or morphology despite
the significant change in surface oxygen and PZC. The addition of oxygen functional groups is
27
commonly used to enhance SWNT dispersion yet, MWNTs are inherently more dispersible than
SWNTs (Hilding et al., 2003). As such, the changes in surface oxygen do not significantly
influence the dispersity of the MWNT samples used in this study. Finally, there is a positive shift
in the half-wave potential, E1/2, of the MWNT annealed at 600 °C, which is indicative of enhanced
activity toward oxygen reduction (Yeager, 1984). It was previously shown that this shift correlates
with the observed trend in E.coli cytotoxicity (Pasquini et al., 2013b). Pasquini, et al. hypothesize
that the combined reduction of carboxylic acid groups and presence of carbonyl groups,
particularly quinone moieties, is optimized around 600 °C and results in a significant increase in
the MWNT electrochemical and antimicrobial activity (Gilbertson, et al., 2014b). The five
characterized properties (percent oxygen, PZC, aggregate radius, aggregate morphology, and
electrochemical activity) are used in the development of a predictive model for zebrafish toxicity.
2.4.2 MWNT toxicity profiles
2.4.2.1 Impact on embryos. During early developmental stages, embryonic zebrafish
interact with the dosed MWNT samples in concentrations ranging from 0.8 to 50 mg/L. Figure
2.1 includes representative images under different concentrations and at various stages of
development. The images indicate that the MWNTs under all concentration conditions aggregate
and settle over time. When immobile, the interaction between embryo and the MWNTs depends
on direct contact between the settled aggregates to the stationary embryo (Figure 2.1). Once
mobile (> 36 hpf), the zebrafish movement within the plate well disrupts the MWNT aggregates
thus, promoting interaction.
28
2.4.2.2 Concentration-response. Exposure to all MWNTs at concentrations below 50 mg/L
induced minimal adverse response. However, several samples halted developmental progression
at the highest exposure concentration (50 mg/L) due to significant mortality prior to 24 hpf. The
high prevalence of mortality at 24 hpf prevented further evaluation and determination of whether
MWNT exposure significantly influenced the manifestation of signature morphological
malformations in the zebrafish indicative of more chronic endpoints. Thus, the statistical model
development utilizes mortality as the most relevant endpoint for MWNT toxicity prediction.
Figure 2.2 shows the mortality at 24 hpf induced by exposure to the seven MWNT samples treated
as previously described.
There is a significant increase in mortality upon dosing at 50 mg/L compared to the other
concentrations. This trend is most notable for the AT 2, AT2-400, and AT2-500 samples, and
diminishes for those samples treated at higher annealing temperatures.
Due to the highly
pronounced differences in mortality at 50 mg/L exposure concentration, and little to no observed
effects at and below 10 mg/L, the development of the logistic model relied exclusively on the 50
mg/L results.
2.4.3 Exploratory statistics used to inform the multivariate model
2.4.3.1 Parameter correlations. In order to create a model for adverse zebrafish effects, we
first explored the linear relationships between five individual MWNT properties and zebrafish
mortality at 24 hpf (Figure 2.3). As evident from Figure 2.3, PZC and percent surface oxygen
result in the highest linear correlation with mortality at 24 hpf (R2 = 0.647 and 0.665, respectively).
This suggests that of the previously described MWNT properties, percent surface oxygen and
surface charge (PZC) are likely to have a significant influence on the observed trend in mortality.
29
The remaining predictors show some linear correlation with observed zebrafish mortality in order
of decreasing correlation: aggregate morphology, aggregate radius, and the electrochemical
activity (E1/2).
2.4.3.2 Parameter relationships. It is expected that certain MWNT properties would
correlate highly with each other. Pearson correlation analysis was used to test property association
and remove highly related properties prior to model development. The compiled correlation plots
for the measured MWNT properties can be found in Figure A.1. As expected, PZC and percent
surface oxygen are highly correlated (R = 0.901). As explained above, this relationship is expected
because the charge distribution on the MWNT surface is dependent on the nature of the surface
functional group (Figueiredo, et al., 1999a; Montes-Morán, et al., 2004). When two highly colinear predictors were available during the model development, only one parameter was selected.
In the case of PZC and percent surface oxygen, PZC was chosen as the more ubiquitous property
that would be more prudent for future model testing and implementation as it is applicable to CNTs
functionalized or doped with elements other than oxygen including nitrogen and sulfur. In
addition, conclusions drawn herein related to PZC are potentially relevant to other classes of
nanomaterials. As such, PZC and the remaining three properties were considered in the model
development as potential predictors for zebrafish toxicity.
2.4.3.3 Statistical model for predicting zebrafish mortality. A derived logistic regression
was used to elucidate the relationship between intrinsic and extrinsic MWNT physicochemical
properties and their influence on the mortality of zebrafish. The final four MWNT properties that
were considered as potential predictors of mortality at 24 hpf include PZC, aggregate size,
30
aggregate morphology, and electrochemical activity. The percent surface oxygen was eliminated
due to co-linearity considerations as described above. The toxicity was assessed with seven
different MWNTs and 48 embryos tested for a given concentration. The final model was selected
based on highest explanatory power and biological relevance, while maintaining preference for
minimal complexity. The logistic model uses PZC as the sole significant predictor (p = 0.000) of
mortality to provide robust, interpretable results, without overfitting. The model and associated
significance tests are provided in Table 2.3.
For each unit increase in PZC, the natural logarithm of toxicity odds ratio decreases by 1.1
(95% CI = 1.35:0.86). The relative decrease in odds ratio for each unit of PZC increase is 0.33
(95% CI = 0.26–0.42). The model significantly improves the understanding of the relationship
between MWNT properties and embryonic zebrafish toxicity. Both the likelihood ratio and le
Cessie-van Houwelingen-Copas-Hosmer unweighted sum of squares tests of significance confirm
the explanatory power of the model (p = 0.000). Higher PZC is associated with lower probability
of mortality at 24 hpf. Figure 2.4 illustrates the model relationship between probability of embryo
mortality and MWNT PZC.
2.4.3.4 Model validation. To validate the model and its ability to predict zebrafish
mortality based on surface charge, four new MWNT samples were prepared and tested as described
in the “Methods” section. These MWNTs were purchased from another vendor and acid treated
under different conditions to ensure robustness across MWNT batches and acid treatment
conditions. The annealing temperatures for the test set include 400, 600, and 900 °C to obtain
MWNT samples from each significant shift in PZC. All other conditions were held constant.
Table 2.4 summarizes the predicted and measured results for the training set.
31
The model was able to predict the mortality at 24 hpf for the MWNT validation sample set
as illustrated by reasonable agreement between the predicted and measured probability in Table
2.4. The data for these four validation samples are also included in Figure 2.4 (circles) and follow
the same trend as the training sample set (diamonds).
2.5 Discussion
As the field of nanotechnology matures, there is an increased demand to resolve the
potential environmental and human health implications resulting from the utilization, handling,
and disposal of nanomaterials. Lessons learned from toxic legacy chemicals have motivated a
paradigm shift and the development of predictive toxicity models that can be used to estimate a
molecule’s hazard potential without investing the time and resources necessary to complete the
suite of conventional in vivo toxicity assessments (e.g. mammalian and aquatic studies) (Connors
et al.; Kostal et al., 2013; Voutchkova-Kostal et al., 2012). While there are added complexities
when applying this approach to nanomaterials, it is imperative to engage in the resolution of a set
of design guidelines to inform the development of nanomaterials with reduced hazard, ultimately
facilitating sustainable growth of the field by preventing unintended consequences.
The study presented herein, is the first to establish a statistical model to explain the
observed adverse response of embryonic zebrafish upon exposure to carbon nanotubes. The model
is informed by comprehensive physicochemical property characterization of systematically
modified MWNTs from the same starting batch, which minimizes the number of intrinsic
properties being altered at one time and avoids batch-to-batch heterogeneity, respectively. The
importance of such an approach has recently been identified as a primary research objective and
32
is intended for simultaneous resolution and optimization of the performance and safety of
nanomaterials (Harper et al., 2011b; Liu et al., 2013; N.N.I., 2011; N.R.C., 2012).
The logistic model based on the analysis of seven MWNT samples and four independent
physicochemical properties is summarized in Table 2.3 and indicates that MWNT surface charge
is the best predictor of zebrafish mortality at 24 hpf. The model was developed on MWNT samples
with PZC values between 3 and 9. Thus, there is high confidence in the relevance of the model
for CNTs with PZC properties within this range.
The current one-parameter model explains the relationship between the MWNT surface
charge and the associated zebrafish toxicity. The relationship explains a large portion of variability
in the toxicity data (McFadden’s Pseudo R2 = 0.32). McFadden’s Pseudo R2 is a measure similar
to R2 in linear regression and is used for maximum likelihood estimates, such as logistic regression.
McFadden’s Pseudo R2 values between 0.2 and 0.4 are indicative of good fit (McFadden, 1974).
The established PZC-mortality relationship is promising from a material design perspective,
providing a tunable parameter that can be impacted by rational design. However, with additional
data (e.g. differently treated MWNTs and additional measured endpoints at MWNT concentrations
between 10 and 50 mg/L), the remaining toxicity variability and the relationship between MWNT
properties can be further explained leading to enhanced predictive power.
In previous studies, using the same sample set, MWNT electrochemical activity was found
to correlate with the observed trend in bacterial cytotoxicity (Gilbertson, et al., 2014b; Pasquini,
et al., 2013b).
Controlled modification of surface oxygen functional groups significantly
influenced the potential for the MWNTs to facilitate redox reactions and thus, the potential to
disrupt healthy cellular function (Gilbertson, et al., 2014b). This finding highlights the important
contribution of the chemical mechanism to cell viability (Liu, et al., 2013). A goal of the current
33
study is to evaluate whether this correlation was maintained for a higher trophic level aquatic
organism, which we found was not the case. This is an increasingly important consideration as
various stake holder communities attempt to reconcile generalizable hazard profiles for all classes
of nanomaterials (N.R.C., 2012; Nel et al., 2013).
The results presented here indicate that MWNT surface charge, not electrochemical
activity, has the greatest influence on zebrafish mortality. Surface charge is known to influence
the aggregation behavior of nanomaterials (Petosa et al., 2010). The available contact area will
increase with enhanced dispersion, including decreased aggregate size and less compact
morphology (Df ² 2). This will enhance contact between the developing embryos and MWNT
samples as well as promote MWNT uptake, both of which can lead to increased lethality. In
addition, enhanced dispersion will promote molecular level interactions necessary for the
production of reactive oxygen species (ROS), which can induce oxidative stress related adverse
developmental outcomes and embryonic mortality. Yet, characterization of the MWNTs used in
this study reveal that dispersed aggregate properties do not play a critical role in the observed
mortality at 24 hpf. Thus, the surface charge must be impacting the bioactivity of the MWNTs
through a mechanism other than aggregation.
The charged nature of the MWNT surface may also influence the interaction with the
organism (Saxena et al., 2007). The pH of the media used in this study is between 6 and 7; the
system was intentionally not buffered in an effort to reduce ionic strength induced aggregation of
the MWNTs (Truong et al., 2012). Therefore, the MWNTs associated with the greatest embryonic
zebrafish mortality at 24 hpf possessed a negatively charged surface (PZC less than ~6). Several
recent studies have explored the impact of nanomaterial surface charge, controlled by
functionalizing with cationic, neutral, or anionic ligands, on observed adverse outcomes using
34
either Daphnia magna or embryonic zebrafish as the in vivo model organism (Bozich et al., 2014;
Harper, et al., 2011b; Lee et al., 2013). Results from these studies elucidate the important role that
surface charge plays in the observed toxicity trends, which is hypothesized to be governed
primarily by the electrostatic interaction between the material and model organism.
Yet,
inconsistencies among the findings remain unresolved due to the complexities that result from
differences in the nanomaterial studied (i.e. Au, Ag, C60, CNTs) and in vivo model organism
utilized. While the importance of nanomaterial surface charge has been established, the underlying
mechanism is not yet elucidated. As such, this is the subject of ongoing research.
The observed disparity in the driving force of viability between bacteria (E.coli ) and
zebrafish for the same MWNT sample set is not entirely surprising considering variations in
methodological exposure conditions and the differences in biological response between bacteria
and zebrafish. That said, analysis and establishment of statistical models for alternative adverse
developmental endpoints, particularly those sensitive to oxidative stress, would provide further
resolution of the mechanism of zebrafish mortality and may reveal the important contribution of
additional MWNT properties. Elucidation of the underlying mechanism of toxicity is the subject
of ongoing research.
A primary limitation of the current study is the sample size. Due to the complexities of
preparing and comprehensively characterizing a controlled sample set as well as the motivation to
compare to previous studies completed at the cellular level, the sample set was limited here to
seven MWNTs. As such, a confirmation sample set of four MWNTs, (also previously evaluated
at the cellular level) was utilized to confirm the significance of the resulting model. With the
model and statistical methods established, further robust analysis can be applied to sample sets of
interest when they become available. Additional data would allow for more complex hypothesis
35
testing with fewer concerns for overfitting during model development. Another limiting factor to
consider in this study is the reduced number of observations of the other adverse endpoints. Since
mortality at 24 hpf was significant for several of the MWNTs, the number of available observations
for the remaining developmental endpoints was reduced. This prevented the ability to draw
significant statistical conclusions for other developmental endpoints.
Future research will focus on resolving the underlying mechanism of toxicity probing
additional endpoints and using more specific assays. In particular, measures of transcriptional,
proteomic, and metabolomics profiles coupled with in situ detection methods can be used to
identify adverse outcome pathways and the primary cellular targets of CNTs. These studies will
provide additional resolution surrounding the contribution of the chemical versus physical
mechanism to zebrafish toxicity. Since significant adverse effects were primarily observed at the
50 mg/L MWNT concentration, it will be important to include concentrations between 10 and 50
mg/L in future studies. The lower concentrations (< 50 mg/L) will also provide insight into the
potential hazard of CNTs at more environmentally relevant concentrations. Finally, the current
data suggests a threshold dose-response curve shape where mortality is observed upon reaching a
given concentration. Analysis of additional concentrations (10-50 mg/L) will further resolve the
shape of this dose-response curve as well as non-mortality effects of MWNTs.
2.6 Conclusions
Significant embryonic zebrafish mortality was observed at 24 hpf and the highest MWNT
concentration studied here, 50 mg/L. The logistic model developed around this data set for seven
systematically treated MWNTs and four independent physicochemical properties indicates that
surface charge, quantified as the point of zero charge, is the most significant predictor of mortality
36
at 24 hpf. This model was confirmed using a separate set of MWNTs purchased from a different
vendor and systematically modified in the same manner. While MWNTs are the subject of this
study, the approach is applicable to all classes of nanomaterials. A similar approach has been
successfully applied to gold nanomaterials (Harper, et al., 2011b). The study exemplifies the
importance of comprehensively characterizing physicochemical properties of nanomaterials used
in toxicological investigations as well as the utility of statistical methods for extrapolating
generalizable relationships between nanomaterial properties and hazard endpoints. Since the
observed toxicity is correlated with a universal property (i.e. PZC) rather than a specific batch of
MWNTs, the findings are intended for comparison across studies, including those utilizing
different cellular or organism models and different carbon nanomaterials. Furthermore, the
approach and findings presented here are intended to inform the development of nanomaterial
hazard profiles. The threshold observed in the concentration-response curve prevented resolution
of the underlying mechanism of mortality due to the lack of observations at other developmental
endpoints. Still, the establishment of the PZC-mortality relationship serves as a first step towards
the development of predictive models to inform safe carbon nanotube design for minimization of
unintended consequences.
Acknowledgements
The authors would like to thank Patrick Kelleher for assisting with the MWNT preparation,
Eva Albalghiti for collecting PZC measurements, and David Goodwin for providing XPS analysis.
37
Declaration of Interest
The authors declare no conflict of interest. The authors alone are responsible for the
content and writing of the paper. This publication was developed under Assistance Agreement
No. RD83558001-0 by the U.S. Environmental Protection Agency and the NIH grant No. T32
ES07060 and P30 ES000210. It has not been formally reviewed by EPA. The views expressed in
this document are solely those of the authors and do not necessarily reflect those of the Agency.
EPA does not endorse any products or commercial services mentioned in this publication.
38
Table 2.1. MWNT samples and treatment conditions
MWNT sample
Acid treatment
Annealing treatment
(Max Temp)
Model training sample set
AT 2
2 hr reflux 70% HNO3
AT2-400
2 hr reflux 70% HNO3
400°C
AT2-500
2 hr reflux 70% HNO3
500°C
AT2-600
2 hr reflux 70% HNO3
600°C
AT2-700
2 hr reflux 70% HNO3
700°C
AT2-800
2 hr reflux 70% HNO3
800°C
AT2-900
2 hr reflux 70% HNO3
900°C
Model validation sample set
NL-AR
H2SO4:HNO3 by manufacturer
NL-400
H2SO4:HNO3 by manufacturer
400°C
NL-600
H2SO4:HNO3 by manufacturer
600°C
NL-900
H2SO4:HNO3 by manufacturer
900°C
39
Table 2.2. MWNT physicochemical characteristics
Compiled characterization data including point of zero charge (PZC) by titration, percent oxygen
(% O) by X-ray photoelectron spectroscopy (XPS), fractal dimension (Df) by static light scattering
(SLS), aggregate radius by dynamic light scattering (DLS), electrochemical activity (E1/2) by
oxygen reduction reaction (ORR) and the rotating disc electrode method (RDE), and a schematic
representation of the distribution and type of functional group with increased annealing
temperature.
MWNT
sample
Point of
zero
charge
(PZC, pH)
Surface
oxygen
(%O)
Morphology
(Df)
Aggregate
radius
Halfwave
potential
(E1/2, V)
Model training sample set
AT 2
3.08
7.6
1.85
37.5
-0.219
AT2-400
4.16
4.6
1.84
48.8
-0.221
AT2-500
4.73
3.8
2.05
57.2
-0.216
AT2-600
6.11
3.5
2.07
37.5
-0.127
AT2-700
6.35
3.0
1.82
39.5
-0.215
AT2-800
6.43
2.4
2.07
92.1
-0.244
AT2-900
9.42
1.3
2.14
48.8
-0.244
8.5
4.4
3.0
1.2
1.24
1.92
1.70
2.42
41.69
51.52
46.33
67.67
-0.235
-0.215
-0.214
-0.238
Model validation sample set
NL-AR
3.1
NL-400
4.9
NL-600
7.4
NL-900
8.6
*This data is previously published (Gilbertson, et al., 2014b)
40
Schematic
representation of
oxygen functional
group distribution
Table 2.3. 24-hour zebrafish mortality model summary
Parameter
Estimate
(Intercept)
5.10
PZC
-1.10
Null Deviance
428.3
Residual Deviance
289.0
Likelihood Ratio Test
139.3
Le Cessie Test1
46.7
DoF - Degrees of Freedom
Akaike information criterion (AIC)=293.02
Std. Error
z-score
0.64
8.02
0.12
-8.85
DoF: 334
DoF: 333
DoF: 1
0.59
-5.944
41
p-value
0.000
0.000
0.000
0.000
Table 2.4. Model validation results
Sample Name
Predicted Probability (CI)
Measured Probability
Number of Embryos tested
Observed # of dead embryos
Expected # of dead embryos
(CI)
NA-AR
NL-400
NL-600
NL-900
0.84
(0.76-0.90)
0.98
47
46
39
(36-42)
0.42
(0.50-0.35)
0.19
47
9
20
(16-23)
0.04
(0.08-0.02)
0.00
47
0
2
(4-1)
0.01
(0.03-0.005)
0.00
48
0
0
(1-0)
42
a.
0 ppm MWNT, 8 hpf
d.
b.
10 ppm MWNT, 8 hpf
e.
120 hpf
c.
50 ppm MWNT, 8 hpf
120 hpf
Figure 2.1. MWNT representative exposure images
Representative images of the zebrafish embryos under varying MWNT concentrations (a–c) and
at different stages of development (d–e). a–c show an increasing the presence of MWNT
aggregates with increased concentration for embryos 8 hpf. The zebrafish shown in d–e are at 120
hpf, with (e) indicating the accumulation of MWNT aggregates at the gills (arrow).
43
Figure 2.2. MWNT toxicity response
Compiled concentration-response curves for seven differently treated MWNTs and embryonic
zebrafish mortality at 24 hpf. MWNT treatment details are outlined in Table 2.1. Briefly, AT 2
MWNTs were acid treated for 2 h and then annealed at 100° increments from 400 to 900 °C (AT2­
400,-500, -600, -700, -800, -900, respectively). Dispersed MWNT samples were pre-loaded to
wells at concentrations ranging 0–50 mg/L prior to adding the dechorionated embryos 6–8 hpf.
Increased mortality at 24 hpf was observed only at 50 mg/L concentration.
44
Figure 2.3. Correlating MWNT physicochemical properties with toxicity
Linear correlations between the five measured MWNTs physicochemical properties and
embryonic zebrafish mortality observed at 24 hpf. The MWNT properties are in the order of
decreasing correlation with the 24 hpf mortality. Surface oxygen (%) and point of zero charge
(PZC) are the best linear predictors of mortality. Dispersion measurements, including fractal
dimension and aggregate radius are minimally correlated with the mortality endpoints. Finally, the
correlation between MWNT reactivity (half-wave potential) and mortality is negligible. The 95%
confidence interval for the linear regression between MWNT properties and mortality is shown in
grey.
45
Figure 2.4. Model relationship between probability of embryo mortality and MWNT point
of zero charge
A graphical representation of the established statistical model showing the effect of PZC on the
24 hpf mortality model when other covariates are held constant. Surface charge (quantified as the
point of zero charge, PZC) is the best single estimator of toxicity and the correlation indicates
that the greater the PZC, the lower the magnitude of toxicity.
46
3. Comparative metal oxide nanoparticle toxicity using embryonic zebrafish
Leah C. Wehmas, Catherine Anders, Jordan Chess, Alex Punnoose, Cliff B. Pereira, Juliet A.
Greenwood and Robert L. Tanguay
Toxicology Reports
Volume 2
Issue 0
47
3.1 Abstract
Engineered metal oxide nanoparticles (MO NPs) are finding increasing utility in the
medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a
better understanding of whole-animal toxicity is required. We compared the toxicity of seven
widely used semiconductor MO NPs made from zinc oxide (ZnO), titanium dioxide, cerium
dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day
embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would
depend on physicochemical properties. Significant agglomeration of MO NPs in aqueous solutions
is common making it challenging to associate NP characteristics such as size and charge with
toxicity. However, data from our agglomerated MO NPs suggests that the elemental composition
and dissolution potential are major drivers of toxicity. Only ZnO caused significant adverse effects
of all MO particles tested, and only when prepared in pure water (point estimate median lethal
concentration = 3.5-9.1 mg/L). This toxicity was life stage dependent. The 24 h toxicity increased
greatly (~22.7 fold) when zebrafish exposures started at the larval life stage compared to the 24 h
toxicity following embryonic exposure. Investigation into whether dissolution could account for
ZnO toxicity revealed high levels of zinc ion (40-89% of total sample) were generated. Exposure
to zinc ion equivalents revealed dissolved Zn2+ may be a major contributor to ZnO toxicity.
3.2 Introduction
Engineering materials at the nanoscale results in unique characteristics valuable for
applications in electronics, personal care products, environmental remediation and medicine (Nel
et al., 2006; Oberdörster et al., 2005). The semiconducting properties of metal oxide nanoparticles
(MO NPs) such as zinc oxide (ZnO) and titanium dioxide (TiO2) make them particularly popular
48
for use in commercially available sunscreens and cosmetics to block ultraviolet radiation when
they are < 50 nm in size (Adams et al., 2006). Engineered MO NPs are finding increasing utility
in the medical field ranging from use as antimicrobial agents (Adams, et al., 2006; Li et al., 2008;
Premanathan et al., 2011; Reddy et al., 2007) to diagnostic imaging (Bae et al., 2011; Hong et
al., 2011; Lee et al., 2007a; Lee et al., 2007b; Liong et al., 2008; Wang et al., 2008; Yu et al.,
2010) and potential cancer treatment (Hanley et al., 2008; Liong, et al., 2008; Ostrovsky et al.,
2009; Premanathan, et al., 2011; Thevenot et al., 2008). While scaling down the size of materials
to the nanometer realm imparts useful traits, they are then within a size range to interact with
biomolecules, such as proteins and nucleic acids, or organelles such as mitochondria, causing
damage that could interfere with biological functions (Nel, et al., 2006; Oberdörster, et al., 2005).
To date, most anti-cancer applications with engineered MO NPs have been demonstrated
using cell lines (Liong, et al., 2008; Ostrovsky, et al., 2009; Thevenot, et al., 2008; Wang, et al.,
2008). Specifically, in vitro studies indicate ZnO nanomaterials generate reactive oxygen species,
perturb calcium homeostasis within the mitochondria, disrupt cellular membranes, induce
apoptosis and generate an inflammatory response (Hanley, et al., 2008; Ostrovsky, et al., 2009;
Sharma et al., 2009; Xia et al., 2008). TiO2 nanomaterials, in the absence of photoactivation,
require high parts per million exposure concentrations to affect gene transcription, cause DNA and
chromosomal damage, and stimulate inflammation (Gurr et al., 2005; Rahman et al., 2002; Sayes
et al., 2006). Upon photoactivation, TiO2 nanomaterials generate more reactive oxygen species
resulting in greater cytotoxicity to mammalian cells and bacteria (Adams, et al., 2006; Li, et al.,
2008). Conversely, some cerium dioxide (CeO2) nanomaterials scavenge reactive oxygen species
enhancing cell survival in the presence of an oxidant (Brunner et al., 2006; Xia, et al., 2008), but
these results are controversial as others have found the opposite effect (García et al., 2011; Lin et
49
al., 2006). Unfortunately, differences in experimental design and exposure concentrations can
make cross study comparisons difficult, and evaluating the toxicity of these materials under culture
conditions with a single or even a few cell types cannot adequately simulate a living dynamic
organism, which can metabolize, sequester and excrete compounds. Before in vivo efficacy of
these MO NPs as medical agents can occur, a better understanding of whole-animal nanomaterial
toxicity is required. This could enable the engineering of safer nanomaterials for therapeutic
applications. Despite a multitude of data on NP toxicity, data gaps still exist and the limited sample
availability, common with nanomaterials under development, make in vivo nanotoxicology
assessment particularly challenging using traditional mammalian models.
The embryonic zebrafish model has emerged as an inexpensive and efficient alternative for
in vivo nanotoxicity screening (Fako et al., 2009; Harper et al., 2008). This is, in part, due to the
high degree of genetic conservation, anatomical and physiological similarity between zebrafish
and humans particularly throughout development. Additionally, the small size, rapid growth and
transparency of zebrafish embryos makes them conducive for moderate to high-throughput
screening methods. Toxicity assays can be conducted in 96-well plates in which morbidity and
mortality are visually assessed over a short duration. Multiple routes of nanoparticle exposure
including epithelial absorption (primary), ingestion, and respiration (gill uptake) can be assessed
along with identification of potential windows of developmental susceptibility to NPs. The small
quantity of test material required to investigate in vivo toxicity in zebrafish is particularly
advantageous.
Our laboratory assesses nanotoxicity using a well-defined five-day embryonic zebrafish
assay (Harper et al., 2011; Harper, et al., 2008; Truong et al., 2011; Usenko et al., 2007).
Research by others assessing MO NP toxicity with the zebrafish model has primarily focused on
50
characterizing ecotoxicological health risks (Bai et al., 2010; Bar-Ilan et al., 2012; Brun et al.,
2014; Chen et al., 2011; Faria et al., 2014; Lee et al., 2007c; Ma et al., 2013; Yu et al., 2011;
Zhao et al., 2013; Zhu et al., 2009). These assays typically employ zebrafish embryos with intact
chorions, an acellular envelope surrounding the embryo, which can obstruct NP uptake in a size
dependent manner and potentially confound interpretation of concentration response results (Bai,
et al., 2010; Lee, et al., 2007c). Furthermore, NPs are frequently coated with various natural
organic matter to mimic aqueous environmental conditions, which will alter bioavailability (Bian
et al., 2011; Blinova et al., 2010; Handy et al., 2008; Keller et al., 2010). Because our laboratory
is interested in these NPs for medical applications, we enzymatically removed the chorion to
mitigate barriers of NP absorption, and we do not coat the MO NPs with natural organic matter.
Zebrafish exhibit a high degree of tolerance to varying water chemistry parameters such as salinity
and pH allowing us to carefully adjust exposure conditions, such as reducing medium salt content
to diminish agglomeration and enhance particle absorption (Kim et al., 2013; Truong et al., 2012).
The objective of these studies was to assess and compare the in vivo toxicity of seven
semiconductor MO NPs made from zinc oxide (ZnO), titanium dioxide (TiO2), cerium dioxide
(CeO2) and tin dioxide (SnO2). In this article, we report the first in vivo toxicity assessment of
these novel MO NPs compared to bulk controls using the embryonic zebrafish assay under two
medium conditions of differing ionic strengths. While these MO NPs possess similar primary mean
diameters and spherical shapes, they differ in physicochemical properties such as band gap,
hydrodynamic size, charge, chemical composition and ionic state of metal ions, and reactive
oxygen species generation. We hypothesized that differences in MO NP toxicity will depend on
these physicochemical properties. In this study we investigated how hydrodynamic size and charge
of uncoated, non-functionalized MO NPs in a waterborne suspension affected zebrafish toxicity.
51
Most MO NPs caused little to no toxicity in our assay under either medium condition except ZnO.
Similar to other aqueous systems, MO NP agglomeration complicates toxicological studies making
it challenging to study primary particle characteristics, as these particles are not well dispersed,
and ions present in the suspension medium can further enhance agglomeration and impede
dispersal. However, it is important to note that agglomeration is an important parameter in particle
hazard assessment. Despite agglomeration of all our MO NPs, particularly in the high ionic
strength embryo medium, we successfully compared how size and charge were associated with the
toxicity of three MO NPs: TiO2 (TC009), CeO2 (QK055) and ZnO, as they created stable
suspension in low ionic strength water. While all three MO NPs possessed similar hydrodynamic
sizes and similar, high positive charges under our assay conditions, only ZnO was significantly
toxic to embryonic zebrafish. This data suggests that for MO NPs suspended in water, elemental
composition or dissolution are principally important for producing toxicity in zebrafish.
3.3 Materials and Methods
3.3.1 Nanoparticle synthesis
All MO NPs were produced through in-house synthesis. Bulk samples were purchased
from commercially available sources. All chemicals used in our synthesis were reagent grade.
They were used without further modification unless otherwise indicated. Synthesis details of each
nanomaterial system are given below.
ZnO NPs: ZnO NPs were synthesized using forced hydrolysis. Briefly 1.0 g of zinc acetate
dehydrate (Zn(CH3COO)2·2H2O) was heated in 100 mL of diethylene glycol containing 0.5 mL
of nanopure water at 160°C for 90 min. The nanoparticles were then rinsed three times with ethanol
and were separated using centrifugation for 20 min at 20,000 rpm after each rinse.
52
CeO2 NPs: CeO2 NPs were also prepared by a forced-hydrolysis process using cerium
chloride as precursor. The cerium precursor was dissolved along with lithium hydroxide in ethanol,
heated to 70°C in a silicon oil bath, and held while stirring for 90 min. After heating, the solution
was mixed with N-heptane to facilitate crystal growth, and allowed to rest for 20-24 h. The
precipitate was centrifuged and washed in ethanol to remove any remaining precursor, and washed
twice in nanopure water to remove any residual hydroxide and ethanol. The final product was dried
in an oven for 24 h at 50°C before being ground to a fine powder using an agate mortar and pestle.
SnO2 NPs: SnO2 NPs designated as UG022 were prepared using Tin (IV) chloride
pentahydrate (SnCl4•5H2O) and urea. The syntheses were carried out at 90°C in nanopure water
for 90 min. The SnO2 NPs designated UG023 were prepared using Tin (IV) acetate (Sn(C2H4OH)4)
precursor. This synthesis was carried out in benzyl alcohol at 100°C for 90 min. Samples were
extracted from solution by centrifugation at 21,000 rpm before drying in an oven at 50°C.
TiO2 NPs: TiO2 NPs were prepared by combining titanium isopropoxide and benzyl
alcohol in a glove box maintained with nitrogen atmosphere at atmospheric pressure. Samples
were stirred for 5 min prior to the addition of 0.5 mL nanopure water along with heating to 150°C.
Temperature was maintained for 24 h. Cooled NPs were centrifuged and washed using ethanol and
nanopure water followed by drying in an oven for 12 h.
All the as-purchased bulk MO samples from multiple commercial sources had a small
fraction of material present in them as <10 nm crystallites. The commercially acquired bulk MO
samples were annealed in air at 800°C for 3 h to sinter any nanocrystals present before using them
as bulk controls.
53
3.3.2 Nanoparticle characterization
All nanoparticle samples were thoroughly characterized using x-ray diffraction (XRD), Xray photoelectron spectroscopy (XPS), inductively coupled plasma-mass spectrometry (ICP-MS),
transmission electron microscopy (TEM), Zeta potential (charge) and hydrodynamic size
measurements. Room temperature XRD spectra were collected with a Philips X’Pert x-ray
diffractometer using a Cu Kα source (λ = 1.5418 Å) in Bragg-Brentano geometry. Briefly, loose
powder samples were leveled in the sample holder to ensure a smooth surface and mounted on a
fixed horizontal sample plane. Rietveld refinement was utilized to obtain lattice parameters and
crystal size using Materials Analysis Using Diffraction (MAUD) software after correction for
instrumental broadening (Lutterotti et al., 1999). High-resolution TEM analysis was carried out
on a JEOL JEM-2100HR microscope with a specified point-to-point resolution of 0.23 nm and an
operating voltage of the microscope was 200 kV. Image processing was carried out using the
Digital Micrograph software from Gatan (Pleasant, California, USA).
The hydrodynamic size distribution and charge of each MO NP was measured using
dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS instrument. The Malvern
Zetasizer Nano ZS measures particles with diameters within 0.3 nm to 10 µm utilizing NonInvasive Back Scatter technology and the Stokes-Einstein relationship to obtain particle size
distributions based on the diffusion of particles traveling by Brownian motion. Charge is measured
using Laser Doppler Micro-electrophoresis, which involves applying an electric field to particles
in suspension causing them to move at a velocity, which is used to calculate electrophoretic
mobility. The Helmholtz-Smoluchowski equation is employed to convert electrophoretic mobility
to charge. We quantified size and charge using 50 mg MO NP per L embryo medium at 0.5%
dimethyl sulfoxide (DMSO; JT Baker, Center Valley, PA, USA) , pH 7 and using 50 mg MO NP
54
per L purified, deionized water (ultrapure water; Invitrogen, Carlsbad, CA, USA) at 0.5% DMSO
(the highest exposure concentrations tested in the embryonic zebrafish bioassay). Each sample was
prepared using the same preparation methods as the zebrafish embryo larval exposure assay, and
the samples were measured four times within 1-2 h of preparation.
3.3.3 Inductively coupled plasma optical emission spectrometry of zinc dissolution
Measurements of dissolved zinc present in ZnO NP suspensions were quantified by
Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) using a Teledyne Leeman
instrument Prodigy (Hudson, NH, USA) and a protocol modified from Poynton et al. (Poynton et
al., 2010). ZnO NP suspensions were diluted in ultrapure water to two exposure concentrations,
0.625 and 10 mg/L. ZnO bulk suspensions were diluted in ultrapure water to 10 mg/L as well. At
0, 6, 24 and 120 h post sample preparation, suspensions were filtered through a 10kD molecular
weight cutoff Macrosep centrifuge filtration unit (PALL Life Sciences; Ann Arbor, MI, USA)
which will retain all particles with a diameter above ~2 nm, thereby collecting what we defined as
the soluble zinc in the filtrate. Unfiltered ZnO NP and bulk suspensions were also collected to
quantify total zinc concentrations to determine the percentage of dissolved zinc present in ZnO NP
suspensions. Both soluble and total zinc solutions were concentrated and dissolved in 35% nitric
acid overnight. Digested samples were then reconstituted to 1x and a final acid concentration of
7% in millipore water treated with chelex 100 prior to ICP-OES analysis. Analyzed samples were
compared to known zinc standards (ULTRA Scientific; N. Kingstown, RI, USA). A known
quantity of zinc (5 mg/L) was also run through our method to determine zinc recovery (101.5%).
55
3.3.4 Zebrafish husbandry
Adult 5D Tropical zebrafish were housed at the Sinnhuber Aquatic Research Laboratory
in accordance with Institutional Animal Care and Use Committee protocols at Oregon State
University. Spawning fish were maintained at 10 fish/L in 100 L tanks recirculated with reverse
osmosis water reconstituted with sea salts (Instant Ocean; United Pet Group, Inc., Blacksburg, VA,
USA) under a 14h light:10h dark photoperiod at 28±1°C. Spawning occurred in the morning, and
eggs were collected by funnel and staged as documented in Kimmel et al., 1995 (Kimmel et al.,
1995).
3.3.5 Zebrafish bioassays
3.3.5.1 Five-day embryonic zebrafish bioassay. A volume of 0.1 mL of the five-fold
nanoparticle and bulk control exposure concentrations, each prepared in ultrapure water and
embryo medium, was transferred to a sterile polystyrene 96-well tissue culture, flat bottom plate
(Falcon, Manassas, VA). At 4 h post fertilization (hpf), the acellular chorion of the zebrafish eggs
was enzymatically digested using pronase and an automated protocol described by Mandrell et al.
2012 (Mandrell et al., 2012). One dechorionated embryo was transferred by glass pipet to each
well of the plates for a minimum of N=32 per exposure concentration of nanoparticle and bulk
control in water and embryo medium. Embryos damaged during loading were excluded from the
experiment. The embryos were maintained in the exposure solutions at 28±1°C for 120 h. The
plates were wrapped in aluminum foil to prevent potential confounding toxicity from particle
exposure to ambient light. At 24 and 120 hpf, the zebrafish were visually inspected by
stereomicroscope for mortality and a suite of morphological abnormalities, which will be referred
to as toxicological endpoints (see Table 3.2 for descriptions). Data was recorded using a binary
56
system in which the presence of an endpoint received a score of 1 while the absence of an endpoint
received a score of 0. A detailed description of this assay may be found in Truong et al. 2011
(Truong, et al., 2011). Typically, we examine 22 specific endpoints; however, exposures in low
ionic strength ultrapure water, at times, resulted in variable and sometimes elevated background
prevalence of malformations, which was date dependent especially for two endpoints equally
across all exposure concentrations including controls. The endpoints were excluded from the
results. This did not confound our toxicity assessments or statistical analyses as we always
compared exposure groups to the respective controls based on the date of an experiment.
3.3.5.2 96 h post fertilization larval zebrafish bioassay. A dilution series of ZnO NP, ZnO
bulk or zinc ion equivalent, each prepared in ultrapure water, was transferred to a sterile
polystyrene 96-well tissue culture, flat bottom plate at 0.1 mL per well. Zebrafish at 96 hpf were
rinsed in water prior to loading into each plate at one fish per well with a minimum of N=24 per
exposure concentration. Plates were wrapped in foil to exclude light and maintained at 28±1°C.
Plates were inspected for malformations and mortality at 1, 2, 6 and 24 h post exposure. Due to
high lethality, we focused on mortality as the most relevant endpoint. Data was recorded as
described in 3.3.5.1.
3.3.6 Nanoparticle exposures
On the day of an exposure, an aliquot of nanoparticle and bulk material control were
suspended in 100% DMSO at a concentration of 10 mg/mL and water bath sonicated (Ultrasonik
NDI; NEY, Inc., Bloomfield, CT, USA) for 20 min at room temperature. Immediately following
sonication, the nanoparticle and bulk stock solutions were diluted to the desired exposure
57
concentrations. For the five-day embryonic zebrafish assay, nanoparticle and bulk were diluted to
50, 10, 2, 0.4 and 0.08 mg/L in ultrapure water and diluted to 50, 10, 2, 0.4 and 0.08 mg/L in
buffered, pH 7-7.2, embryo medium. Embryo medium is a defined synthetic sea solution consisting
of 15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2·2H2O, 0.15 mM KH2PO4, 0.05 mM Na2HPO4 and 1
mM MgSO4·7H2O commonly used to rear zebrafish embryos and conduct embryo-larval toxicity
assays. The vehicle controls consisted of 0.5% DMSO in ultrapure water and 0.5% DMSO in
embryo medium. To test the toxicity of zinc ion present in ZnO NP solutions, we used the ICP­
OES results from 120 h in 10 mg/L ZnO NP to calculate equivalent concentrations using ZnSO 4­
7H2O, which was diluted in ultrapure water to 98.9, 19.8, 4.0, 0.8 and 0.16 mg/L with 0.5% DMSO.
For the 96 hpf zebrafish bioassay, ZnO NP and ZnO bulk were suspended and sonicated as
described above. ZnO NP, ZnO bulk were diluted in ultrapure water to 20, 10, 5, 2.5, 1.25, 0.625
and 0.3125 mg/L in ultrapure water with DMSO maintained at 0.5%. We used the amount of zinc
ion measured by ICP-OES in the 0.625 mg/L ZnO NP sample after 24 h to calculate equivalent
concentrations using ZnSO4·7H2O for the 96 hpf exposures. This resulted in ZnSO4·7H2O
exposures of 42.7, 21.35, 10.68, 5.34, 2.67, 1.33 and 0.67 mg/L with DMSO maintained at 0.5%.
The dissolved zinc present in 0.625 mg/L ZnO NP was selected to calculate the amount of
ZnSO4·7H2O to use in the 96 hpf exposure because we previously found 0.625 mg/L of ZnO NP
resulted in the lowest observable lethality after 24 h.
3.3.7 Scanning electron microscopy
Larval zebrafish at 96 hpf, were exposed to 0.5% DMSO, 0.625 and 10 mg/L ZnO NP and
10 mg/L ZnO bulk in water for ~1-2 h prior to fixation in electron microscopy fixative. Samples
were prepared by placing 10, 96 hpf larvae in 1 mL of fixative consisting of 1% paraformaldehyde
58
in 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer overnight at room temperature.
Samples were rinsed twice in 0.1 M cacodylate buffer for 15 min each prior to dehydration in 30­
100% acetone or ethanol for 10-15 min each. Samples then underwent critical point drying and
were sputter coated with gold-palladium alloy before imaging. Images were acquired at 500x using
an FEI Quanta 600 FEG scanning electron microscope.
3.3.8 Statistics
Within each MO, medium and nanoparticle vs. bulk combination, logistic regression was
used to test for any differences in proportion mortality or proportion affected across the six
concentrations used in each experiment (5 d.f. test). Because of the low incidences at many
concentrations for many of the combinations, exact logistic regression was employed in SAS
(Cary, NC, USA) using PROC LOGISTIC. When there was evidence of concentration effects
(p<0.0001 for ZnO NP and zinc ion in water), median lethal concentration estimates (LC50) and
median effect concentration (mortality combined with malformed) estimates (EC50) were
determined by first dropping the lowest doses until there was no lack of fit to a logit linear in log
concentration model and then using that model to estimate the quantities of interest (LOGIT and
INVERSECL options in SAS PROC PROBIT). Fisher’s Exact analysis was performed for each
ZnO NP and zinc ion in water separated by experimental date (p<0.05 considered significant).
Within each response and experiment date, concentration above vehicle control was compared to
background. Only for 10 and 50 mg/L was the prevalence significantly higher for both ZnO NP
and zinc ion in water for all experiments studied.
59
3.4 Results and Discussion
3.4.1 Metal oxide nanoparticles with similar primary particle size differ greatly in
hydrodynamic size and charge
We characterized MO NP purity through XRD, XPS and ICP-MS. The primary particle
size (crystallite size) of MO NP was obtained by both TEM while hydrodynamic size and charge
were determined using DLS electrophoresis measurements, respectively. Detailed analyses of MO
NPs using XPS confirmed that there were no unexpected elements in the samples and ICP-MS
further revealed that concentrations of common impurities such as the tested Fe, Mn, Co, Ni, Cr
and V ions were below 1 ng/L. TEM analyses demonstrated that all MO NPs exhibited a spherical
shape (Appendix B, Figure B.1), and the average primary particle size of each material type was
within the 2.8-11.6 nm range in diameter (Table 3.1). DLS data acquired for the highest exposure
concentration (50 mg/L at 0.5% DMSO) indicated that none of our MO NPs stayed well suspended
in embryo medium (Figure 3.1, Table 3.1) likely due to the salts present in embryo medium
(Section 3.3.6). All agglomerated significantly with hydrodynamic diameters ranging from ~150­
1000x larger than the corresponding primary particle size. The variability in count rates and
decreasing size trends observed during DLS acquisition indicated particle sedimentation in embryo
medium making size and charge accuracy questionable besides knowing that the particles were
very large. Therefore, we not only exposed zebrafish embryos to MO NPs and bulk control
material prepared in embryo medium but also MO NPs and bulk controls prepared in ultrapure
water to reduce agglomeration, enhance dispersion and maximize bioavailability (Bian, et al.,
2011) for the toxicity assessments. MO NPs suspension in ultrapure water reduced the average
hydrodynamic diameter, but the particles remained large (>~350 nm diameter). However, TiO2
NP (TC009), CeO2 NP (QK055) and ZnO NP were stable enough in ultrapure water suspension to
60
provide precise DLS data (Table 3.1) These three MO NPs were of similar hydrodynamic size
(~353-416 nm) and all three possessed similar, high positive charges especially TiO2 NP (TC009)
and ZnO NP at ~17.1 and 20.2 mV respectively (Table 3.1, Figure 3.1). While CeO2 NP (QK020)
possessed a significantly smaller mean diameter when prepared in water, charge measurements
near neutrality (-3.54 mV) indicated instability of the suspension.
3.4.2 Only zinc oxide nanoparticles demonstrate acute embryonic zebrafish toxicity
Our embryonic zebrafish toxicity assay involves assessing not only lethality from the seven
MO NP and bulk control exposures, but also phenotypic malformations that accompany the
disruption of key developmental processes that occur during the 8 to 120 hpf exposure period. We
visually inspected exposed 24 hpf embryos for mortality, alterations in spontaneous tail flexion,
notochord malformations as well as delayed developmental progression. At 120 hpf, we evaluated
all exposed zebrafish larvae for the presence or absence of mortality, 14 morphological endpoints,
and one behavioral endpoint (Table 3.2). Some of the morphological endpoints assessed include
axial, craniofacial, somite, fin defects and edema indicating disruption of key processes during
organogenesis or in the case of edema, aberrant ion regulation.
Inspection of toxicity data from MO NPs prepared in water or embryo medium compared
to respective bulk controls demonstrated most had little to no significant concentration-dependent
toxicity, NP or media- specific effects (Figure B.2). For ease of analysis, we condensed the toxicity
data into 24 hpf mortality, total mortality, and cumulatively affected zebrafish, which consisted of
any 120 hpf malformation and mortality. Agglomeration certainly limited bioavailability of some
of the MO NPs contributing, at least in part, to the low observed toxicity especially in the exposures
with embryo medium. Yet some MO NP agglomerates, mainly ZnO and TiO2, have been
61
demonstrated to cause toxicity in adult and embryonic zebrafish under similar waterborne assay
conditions as our experiments (Faria, et al., 2014; Xiong et al., 2011; Yu, et al., 2011; Zhu, et
al., 2009). Exact logistic regression of the condensed MO NP data revealed that ZnO NP prepared
in water produced statistically significant concentration-dependent toxicity after five days of
exposure (Figure 3.2). Across three experiments, the point estimate of the LC50 ranged between
3.5-9.1 mg/L while the EC50, represented by mortality combined with malformed, ranged from
0.5-3.51 mg/L. These results coincide well with Zhu et al. who found, in a slightly shorter zebrafish
embryo-larval assay, that ZnO NPs prepared in water had an LC50 of 1.793 mg/L. Unlike Zhu et
al., our ZnO bulk control caused no significant toxicity when prepared in ultrapure water or embryo
medium (Figure 3.2). Two independent studies evaluating the toxicity of ZnO NPs to microalgae
also found no significant difference between the LC50 of ZnO NP and bulk (Aruoja et al., 2009;
Franklin et al., 2007). The magnitude and similarity in dissolution of ZnO NP and bulk led authors
from both studies to conclude toxicity was attributed primarily to dissolved zinc ions. The
discrepancy in our data may be due to the inherently greater total surface area of ZnO NPs
compared to bulk which could result in a higher degree of dissolution (Borm et al., 2006; Yang et
al., 2006).
While ZnO NPs were the only material to cause toxicity in our assay, this toxicity appeared
to be independent of hydrodynamic size or charge. Both TiO2 (TC009) and CeO2 (QK055)
exhibited similar mean hydrodynamic sizes and positive charges when prepared in ultrapure water
(Table 3.1, Figure 3.1), but did not cause a significant toxic response in the embryonic zebrafish
assay (Figure B.2). This result is contrary to some research, which suggests that positively charged
nanooparticles are more likely to induce toxicity than negative or neutrally charged nanoparticles
of similar size (Harper, et al., 2011; Oberdörster, et al., 2005), which was not the case in our assay.
62
However, these studies were conducted with different core materials and their nanoparticles were
covalently functionalized to create charge (Harper, et al., 2011; Oberdörster, et al., 2005) whereas
our MO NPs lacked coating and surface functionalization. The low toxicity associated with
zebrafish exposure to TiO2 NP and TiO2 agglomerates in the absence of photoactivation during
development corroborates other research. For instance, Zhu et al. reported that exposures of up to
10x our highest exposure concentration (500 mg/L) TiO2 NPs with mean diameters ~half the size
of ours had no significant influence on zebrafish hatch or mortality. No data on TiO2 NP charge in
suspension was provided (Zhu et al., 2008). Griffitt et al. also reported little to no lethality
associated with 48 h exposure to both adult and zebrafish fry (<24 h old) using slightly larger TiO2
NPs (687.5 nm) than ours with a highly negative charge (-25.1 mV) (Griffitt et al., 2009). The low
toxicity of CeO2 particles in our assay falls in line with other research which, in some cases,
considers CeO2 NPs as inert, biocompatible (Ispas et al., 2009; Van Hoecke et al., 2009) or
perhaps beneficial with cytoprotective effects (Xia, et al., 2008). However, others have reported
some acute mortality to the aquatic invertebrate Daphnia magna (LC50 = 12 mg/L) (García, et al.,
2011). Differences in CeO2 NP size (6.7 nm diameter), coating with hexamethylenetetramine or
species sensitivity may explain these results. The low biological response associated with both
TiO2 and CeO2 exposure suggests perhaps charge has little influence on embryonic zebrafish acute
toxicity when the particles are in the size range tested in these studies especially without
photoactivation of TiO2. Calculation of the specific surface area of ZnO, TiO2 (TC009) and CeO2
(QK055) NPs using the mean hydrodynamic radius revealed only small differences at 267000,
341000 and 222000 cm2/g respectively. Charge, size and specific surface area were not good
predictors of toxicity in our study. Our results suggest a material specific driver of toxicity amongst
63
our ZnO, TiO2 (TC009) and CeO2 (QK055) NPs, which may be explained by the propensity of
ZnO to dissolve.
3.4.3 Zinc oxide nanoparticle dissolution analysis and toxicity
Several studies attributed the toxicity of ZnO NPs in waterborne assays to dissolved zinc
ions (Aruoja, et al., 2009; Blinova, et al., 2010; Brun, et al., 2014; Deng et al., 2009; Fairbairn
et al., 2011; Franklin, et al., 2007; Heinlaan et al., 2008; Song et al., 2010); therefore, we wanted
to determine the degree of zinc dissolution from our ZnO NPs to differentiate particle toxicity from
ion toxicity in our assay. We utilized ultrafiltration and ICP-OES to separate and quantify what we
defined as the soluble zinc fraction (zinc that filtered through a 10kD membrane) compared to the
total concentration of ZnO NP and bulk (Figure 3.2). We measured dissolution of two
concentrations of ZnO NP: one low (0.625 mg/L) and one high (10 mg/L) and found that there
was a high initial level of soluble zinc present in both nanoparticle samples compared to the total
zinc, ~ 47 and 44% respectively. The percent of soluble zinc remained relatively stable over 24 h
but increased to ~87% after 120 h in the 10 mg/L (nominal) ZnO NP sample. The 10 mg/L
(nominal) ZnO bulk sample generally had a lower percentage of soluble zinc relative to the total,
over all tested time points (Figure 3.2). As with ZnO NP, the percentage of soluble zinc in the bulk
sample increased over time from 26% initially to 66% after 120 h. We predicted the larger ZnO
bulk particles to have lower levels of soluble zinc compared to nanoparticles due to reduced total
surface compared to the nanoparticles (Bian, et al., 2011; Borm, et al., 2006; Yang, et al., 2006).
Our nominal concentration of zinc differed from the total measured by ICP-OES (Figure 3.2). Our
0.625 and 10 mg/L ZnO NP samples should have contained ~0.5 and 8 mg/L zinc, yet we actually
measured ~0.2-0.3 and ~4-5 mg/L total zinc respectively. The actual total zinc in the 10 mg/L bulk
64
sample was also different from the nominal at ~0.93-0.98 mg/L. We adjusted the exposure
concentrations based on the difference between actual and nominal zinc and compared the toxicity
between ZnO bulk and nanoparticles at equivalent concentrations and found the nanoparticles were
still more toxic than the bulk. For instance, the actual concentration of ZnO in the nominal 50
mg/L ZnO bulk exposure was ~6 mg/L. The nominal 10 mg/L ZnO NP exposure was actually ~5
mg/L. By comparing the toxicity results of these two adjusted exposure concentrations, we found
that ~5 mg/L ZnO NP still caused at least 50% mortality while the bulk caused 0% mortality at a
slightly higher exposure concentration (Figure 3.2). Therefore, we could conclude that the ZnO
NP was more toxic than the ZnO bulk under these assay conditions.
Using the 4.5 mg/L of soluble zinc measured at 120 h as a maximum amount of dissolved
zinc present in 10 mg/L ZnO NP sample, we conducted the five-day embryonic zebrafish assay to
understand whether exposure to zinc ion could result in similar toxicity as ZnO NP. We exposed
8 hpf embryos to a five-fold concentration series of Zn ion equivalent (98.9-0.16 mg/L ZnSO4­
7H2O) prepared in ultrapure water. The mortality and total affected zebrafish results were similar
between ZnO NP and the ion equivalent across all tested concentrations. This suggested that the
zinc ions could be the main driver of ZnO NP toxicity in water for our assay (Figure 3.2). Our
results corroborate several other studies. In a freshwater microalgae exposure, Franklin et al.,
demonstrated that ZnO bulk, ZnO NP and ZnCl2 all had similar IC50 values at ~60 µg Zn/L, which
they attributed to the dissolved (0.1 µm filterable) zinc present the in samples (Franklin, et al.,
2007). Heinlaan et al. found that both ZnO NP and ZnSO4-7H2O had similar EC50 values in their
bacterial assay at 1.9 and 1.1 mg/L, respectively (Heinlaan, et al., 2008). Exposure of embryonic
zebrafish by Brun et al. to ZnO NP and equivalent dissolved zinc revealed similar uptake and tissue
distribution as measured by laser ablation ICP-MS (Brun, et al., 2014).
65
3.4.4 Zinc oxide exposure results in life stage-dependent zebrafish toxicity
Due to the unique advantages of the zebrafish embryo-larval test, we can start exposures
at different stages of key developmental processes such as somitogenesis, neurogenesis,
hepatogenesis, etc to understand how the nanoparticle is causing toxicity. Because ZnO and zinc
ion caused significant toxicity in the five-day embryonic zebrafish assay, we wanted to determine
when toxicity was occurring. We exposed 96 hpf zebrafish larvae to ZnO NP, ZnO bulk and Zn
ion equivalent, assuming ~48% of the ZnO NP suspension was dissolved zinc based on ICP-OES
results. While most major organs are developed by this life stage, the gills are still undergoing
maturation. Surprisingly, exposure to the ZnO NPs starting at 96 hpf resulted in a substantial
increase in mortality after 24 h of exposure (LC50 = 2.20 mg/L, Figure 3.4) compared to 24 h
mortality from exposures starting at 8 hpf (~LC50 at 50+ mg/L, Figure 3.2). Exposure to Zn ion
equivalents resulted in a similar life-stage dependent toxicity as ZnO NP, whereas ZnO bulk did
not cause significant mortality compared to vehicle control (Figure 3.4). We also observed the
presence of skin ulcerations along the body axis of ZnO NP and zinc ion exposed larvae within
1-2 h, which were not apparent in the vehicle or bulk controls. The skin ulcerations were similar
in appearance to those observed by Zhu et al., upon exposure of zebrafish to their ZnO NPs (Zhu,
et al., 2008). Scanning electron microscopy images of zebrafish larvae exposed for ~1-2 h prior to
fixation revealed that the ZnO NPs caused external damage over the entire body including gill
primordia, which was not apparent in the vehicle controls (Figure B.3). Ulcerations appeared to
concentrate around neuromasts of the lateral line but it was difficult to discern if this led lethality.
George et al., demonstrated that ZnO NPs are capable of disrupting plasma membrane integrity in
macrophage and epithelial cell lines (George et al., 2009), which may explain why we observed
tissue ulceration. An acridine orange/ethidium bromide assay demonstrated that exposure of
66
isopods to nanoscale ZnO resulted in cell membrane destabilization in the hepatopancreas (Valant
et al., 2009). Xiong et al. found that 5 mg/L ZnO NP induced gill cell shrinkage in adult zebrafish
but not necessarily cell membrane rupture after 96 h of exposure (Xiong, et al., 2011). Perhaps the
ZnO NPs and/or zinc ions present in the exposure disrupted the larval zebrafish epidermal cells
over the entire body axis including gill primordia, which eventually resulted in mortality. Gills are
known to be a primary target of ZnO NPs in oysters (Trevisan et al., 2014). Our results suggest
that later zebrafish life stages may be more sensitive than the embryonic stage, which could
confound predictive toxicity interpretation. Results by Ma and Diamond support this conclusion
(Ma, et al., 2013). However, they attributed their life-stage dependent differences in toxicity to the
presence of the chorion, which impedes nanoparticle bioavailability when exposures start at earlier
life stages. This is different from our assay as we enzymatically remove the chorion. Nevertheless,
it illustrates that we must carefully consider possible limitations of these exposure paradigms in
toxicity assessments.
3.5 Conclusions
The objective of these studies was to assess and compare the in vivo toxicity of seven novel
MO NPs made from ZnO, TiO2, CeO2 and SnO2 that were uncoated and without surface
functionalization using an embryonic zebrafish assay. We hypothesized the physicochemical
properties hydrodynamic size and charge would dictate in vivo toxicity. However, little toxicity
was observed, partly due to MO NP agglomeration, which reduced bioavailability and made size
and charge measurements challenging. Three MO NPs created stable suspension in low ionic
strength ultrapure water: TiO2 (TC009), CeO2 (QK055) and ZnO. While all three MO NPs
67
possessed similar mean hydrodynamic diameters and charges, only ZnO was significantly toxic
with a point estimate LC50 ranging between 3.5-9.1 mg/L.
Further investigation into ZnO toxicity revealed that it was life stage dependent. The 24 h
toxicity increased greatly (~22.7 fold) when zebrafish exposures started at the larval life stage (96
hpf) compared to the 24 h toxicity following embryonic exposure (8 hpf). This finding is especially
important as many laboratories have adopted the five-day embryo-larval zebrafish assay to
evaluate and predict in vivo toxicity, yet we found that testing at earlier life stages may not be the
most sensitive. This sensitivity did not depend on the chorion as we enzymatically remove it for
unimpeded exposure. Therefore, it may be necessary to test some materials like ZnO at several
early time points to identify the most sensitive window for predicting toxicity for health and safety
assessment. Fortunately, we can efficiently examine the factors contributing to adverse biological
interactions while working with material scientists to quickly revise synthesis methods to modify
material properties followed by re-evaluation of biocompatibility (Harper, et al., 2008). This
approach of using rapid in vivo readouts to inform the engineering of safer nanomaterials that
maintain their desirable properties is consistent with the principles of green chemistry and green
nanoscience (McKenzie et al., 2004).
Because ZnO NP toxicity is frequently associated with dissolution (Aruoja, et al., 2009;
Blinova, et al., 2010; Brun, et al., 2014; Deng, et al., 2009; Fairbairn, et al., 2011; Franklin, et
al., 2007; Heinlaan, et al., 2008; Song, et al., 2010), we quantified the amount released zinc ion
(10 kD filterable). We found high levels of zinc ion (40-89% of total sample) were generated in
our ZnO NP suspensions. Exposure of zebrafish to zinc ion equivalents suggested dissolved zinc
ion may be a major contributor to ZnO toxicity at both embryonic and larval zebrafish life stages
when exposures occurred in ultrapure water. Therefore, we conclude that elemental composition
68
and dissolution potential were key drivers of toxicity amongst ZnO, TiO2 (TC009) and CeO2
(QK055) rather than hydrodynamic size and charge in water suspensions.
Another important discovery of these experiments was that a careful understanding of how
assay conditions, such as medium ion composition and strength, are important in interpreting MO
NP results. Conducting our assay using high ionic strength embryo medium reduced bioavailability
through agglomeration of the MO NPs, thereby reducing zebrafish toxicity. This is not necessarily
a unique conclusion as others have seen similar effects testing silver nanoparticles (Kim, et al.,
2013); however, if we had not conducted our assays using ultrapure water as a medium, we may
have erroneously concluded that all our MO NPs including ZnO NP were non-toxic to zebrafish
and not been able to deduce any relationships between toxicity and physicochemical properties.
Caution must be employed in interpretation of data from these waterborne assays especially
regarding potential MO NP agglomeration, sedimentation and/or dissolution.
Acknowledgments
We would like to thank Teresa Sawyer and the Oregon State University Electron
Microscopy Facility for SEM images and Andy Ungerer of the WM Keck Collaboratory for
Plasma Spectrometry at Oregon State University for assistance with ICP-OES. We would also like
to thank Dr. Yasmeen Nkrumah-Elie for advice on ICP-OES sample preparation and Dr. Stacey
Harper for the use of the Malvern Zetasizer Nano ZS. This research was funded by the NIH T32
ES07060, NIH P30 ES000210 and NSF 1134468.
69
Conflicts of Interest
The authors declare no conflict of interest.
70
Table 3.1. Metal oxide nanoparticle physical characterization
Nanoparticle
Material
Primary Size
± SE (nm)
Hydrodynamic
Diameter ± SE
(nm)
Charge ± SE
(mV)
Poly Dispersity
Index ± SE
Mob ± SE
(µmcm/Vs)
in Embryo Medium
TiO2 (TC009)
11.64±0.26
2110±73.2
-15.1±0.370
0.483±0.0327
-1.18±0.0290
TiO2 (TC010)
10.47±0.13
2550±102
-19.2±0.323
0.479±0.0176
-1.50±0.0256
CeO2 (QK020)
2.87±0.12
2960±112
-8.74±0.561
0.512±0.0242
-0.686±0.0440
CeO2 (QK055)
2.77±0.12
2150±107
-3.27±0.207
0.584±0.0139
0.256±0.0161
8.35
1280±79.7
-18.5±0.260
0.366±0.0343
-1.45±0.0199
SnO2 (UG022)
2.99±0.08
1470±149
-12.2±0.852
0.470±0.0372
-0.957±0.665
SnO2 (UG023)
2.87±0.06
2120±129
-13.6±0.232
0.549±0.0531
-1.06±0.0190
ZnO (BI010)
in Ultrapure Water
TiO2 (TC009)
416±15.0
17.1±1.47
0.445±0.0238
1.34±0.115
TiO2 (TC010)
2170±119
-10.6±5.69
0.542±0.0444
-0.830±0.446
CeO2 (QK020)
521±24.7
-3.54±0.921
0.514±0.004
-0.277±0.0721
CeO2 (QK055)
353±23.6
38.8±2.76
0.432±0.0345
3.04±0.216
ZnO (BI010)
400±3.72
20.2±0.748
0.151±0.010
1.59±0.0586
SnO2 (UG022)
1690±151
-8.49±4.08
0.571±0.0349
-0.667±0.320
SnO2 (UG023)
1210±102
-14.2±3.81
0.553±0.0244
-1.11±0.299
71
Table 3.2. Descriptions of time points and endpoints assessed during five-day embryonic
zebrafish bioassay
Abbreviation
MO24
DP24
SM24
NC24
MORT
YSE_
AXIS
EYE_
SNOU
JAW_
OTIC
PE__
BRAI
SOMI
PFIN
PIG_
CIRC
TRUN
SWIM
NC__
AFTD
Assessed Time Point
(hours post fertilization)
Mortality
24
Delayed developmental progression
24
Reduced or excessive frequency of zebrafish spontaneous tail coiling
24
Notochord malformations
24
Total mortality
120
Yolk sac edema
120
Abnormal body axis curvature such as lordosis or scoliosis of the
spine
120
Eye malformations such as large or small eyes
120
Snout malformations
120
Jaw malformations
120
Otic vesicle malformations
120
Pericardial edema
120
Brain malformations such as edema
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Abnormal somite development
120
Pectoral fin malformations
120
Abnormal pigmentation (hypo or hyper pigmentation)
120
Abnormal circulation or vasculature
120
Truncated body
120
Abnormal swim bladder development
120
Notochord malformations
120
Total mortality combined with any malformation at 120
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Endpoint Description
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Figure 3.1. Size to charge relationship between metal oxide nanoparticle
Dynamic light scattering measurements of 50 mg/L metal oxide nanoparticles (MO NPs)
suspended in low ionic strength medium (WATER) and high ionic strength medium (ZEBRAFISH
EMBRYO MEDIUM). Error bars represent standard error of the mean (n = 4) of hydrodynamic
diameter vertically and charge horizontally. Red circle highlights the MO NPs with very similar
sizes and charges that created stable suspensions when prepared in water; however, only ZnO was
the only MO NP to cause significant mortality and malformations in the five-day embryonic
zebrafish bioassay with water as the exposure medium.
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Figure 3.2. Zinc oxide nanoparticle, zinc oxide bulk and zinc ion equivalent toxicity
Toxicity of zinc oxide nanoparticles (ZnO NP) compared to ZnO Bulk and Zn Ion Equivalent
controls with 0.5% DMSO vehicle in five-day embryonic zebrafish bioassay under two medium
conditions (water and embryo medium). The Zn Ion Equivalent represents the approximate amount
of dissolved zinc present in ZnO NP samples as determined by inductively coupled plasma optical
emission spectrometry. Total affected represents the combined percent of zebrafish with mortality
or any morphological malformation(s) at 120 hours post fertilization. For the ZnO NP prepared in
water and embryo medium, the error bars represent the range in mean percent response (3
experiments and 2 experiments respectively, n=32 per experiment per concentration). For the other
exposures, the data represents the percent response of 1 experiment, n = 32 per concentration. For
ZnO NP (*) and Zn Ion Equivalent (#) data, symbol indicates concentrations where percent
prevalence is significantly above background (p<0.05 Fisher’s Exact Test) for all experiments
studied.
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Figure 3.3. Dissolution of zinc oxide nanoparticles and zinc oxide bulk
Dissolved zinc (Zn) present in blanks (0.5% DMSO in ultrapure water), 0.625 and 10 mg/L zinc
oxide nanoparticle (ZnO NP) and 10 mg/L ZnO Bulk suspensions prepared in 0.5% DMSO,
ultrapure water as measured by ICP-OES over different time points. Error bars represent ±SD,
n=3.
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Figure 3.4. 96 hpf zebrafish zinc oxide nanoparticle and zinc ion equivalent toxicity
Larval zebrafish mortality 24 hours post exposure (hpe) to zinc oxide nanoparticle (ZnO NP) (n=24
per concentration) ZnO Bulk (n=24 per concentration) and Zn Ion Equivalent (n=32 per
concentration) prepared in ultrapure water assuming ~48% nanoparticle dissolution. Exposures
started when zebrafish were 96 hours post fertilization (hpf).
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4. An orthotopic zebrafish xenograft assay to study novel human glioblastoma
therapeutics targeting migration, invasion and proliferation
Leah C. Wehmas, Alex Punnoose, Robert L. Tanguay, Juliet A. Greenwood
In preparation for publication
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4.1 Abstract
Glioblastoma is an aggressive brain cancer requiring improved treatment options. Under
existing methods of drug discovery and development, it will take years before new glioblastoma
therapeutics become available to patients. The embryonic zebrafish xenograft models hold
potential for studying human cancer, identifying new treatments and prioritizing drug
development. We have developed an orthotopic zebrafish xenograft screening assay to discover
and prioritize the testing of compounds that impact glioblastoma proliferation, migration and
invasion. We illustrate the utility of our assay by evaluating the potential anti-cancer efficacy of
zinc oxide nanoparticles (ZnO NP) and the selective phosphatidylinositide 3-kinase (PI 3-kinase)
inhibitor LY294002, known to inhibit glioblastoma proliferation and influence migration in cell
culture and rodent xenograft models. Contrary to expectations, ZnO NPs significantly enhanced
glioblastoma proliferation by 15 to 23% and migration/invasion by 22 to 49% at the periphery of
the cell mass (161+ µm) compared to vehicle control. Exposures of 3.125-6.25 µM LY294002
significantly decreased proliferation by up to 34% compared to control with trends toward
concentration dependent effects. Exposure to 6.25 µM LY294002 significantly inhibited
migration/invasion by 25 to 31% within the glioblastoma cell mass (0-80 µm) and by 23 to 48%
in the next distance region (81-160 µm) compared to control. The results of this assay demonstrate
that we have a short duration, relevant and sensitive embryonic zebrafish-based assay to aid
glioblastoma therapeutic development.
4.2 Introduction
Despite 30 years of extensive research into developing drug therapies, the median survival
rate of patients diagnosed with glioblastoma remains bleak at ~15 months (DeAngelis, 2001; Hegi
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et al., 2005). This poor prognosis is due to a combination of the highly invasive nature and
heterogeneous mutational spectrum of this deadly form of primary brain tumor making complete
surgical resection virtually impossible and adjuvant therapies ineffective (TCGA, 2008;
DeAngelis, 2001). There is a dire need to better understand the biological processes contributing
to glioblastoma invasion and progression to aid in the discovery of novel glioblastoma therapeutics
that more effectively treat this aggressive disease. Further complicating new glioblastoma drug
development is that studying invasion in an intact brain microenvironment can be especially
difficult in traditional mammalian models. The opacity of the mammalian head makes directly
observing how potential therapeutics influence individual invading glioblastoma cells problematic
(Geiger et al., 2008; Haldi et al., 2006; Lal et al., 2012; Marques et al., 2009; Nicoli et al., 2007;
Stoletov et al., 2007). Yet, research suggests that migrating cells will drastically alter morphology
and mechanisms of movement in three-dimensional versus two-dimensional environments and
receive signals from the microenvironment that dictate direction making conducting studies on
migration and invasion in an intact brain desirable (Webb and Horwitz, 2003). While investigating
therapeutic efficacy in mammalian models is an important step in the drug development pipeline,
testing during the early stages of discovery can be challenging. This is especially true in the
expanding field of nanomedicine where compound availability may be limited (Fako and
Furgeson, 2009).
Zebrafish xenograft models hold potential for studying human cancer progression,
migration, invasion, metastases and microenvironment interactions to aid in identifying new
treatments (Geiger, Fu and Kao, 2008; Haldi, Ton, Seng and McGrath, 2006; Lal, La Du, Tanguay
and Greenwood, 2012; Nicoli, Ribatti, Cotelli and Presta, 2007; Stoletov et al., 2010). The
embryonic zebrafish model has proved a useful in quickly identifying and prioritizing the
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screening of promising new pharmaceuticals (Zon and Peterson, 2005). The small size, high
fecundity and transparency of the embryonic zebrafish makes it amenable to conducting exposures
in multi- well plates and studying cancer progression non-invasively through microscopy (Geiger,
Fu and Kao, 2008; Haldi, Ton, Seng and McGrath, 2006; Lal, La Du, Tanguay and Greenwood,
2012; Nicoli, Ribatti, Cotelli and Presta, 2007; Stoletov, Kato, Zardouzian, Kelber, Yang, Shattil
and Klemke, 2010). Furthermore, embryonic zebrafish lack a fully functional adaptive immune
system until ~28 days making it possible to inject human cells without rejection (Lam et al., 2004).
Existing embryonic zebrafish xenograft models have already demonstrated that human cancer
cells, including glioma, can grow, divide, metastasize and induce angiogenesis in zebrafish
similarly to rodent xenograft models (Geiger, Fu and Kao, 2008; Haldi, Ton, Seng and McGrath,
2006; Lee et al., 2005b; Lee et al., 2009; Marques, Weiss, Vlecken, Nitsche, Bakkers, Lagendijk,
Partecke, Heidecke, Lerch and Bagowski, 2009; Nicoli, Ribatti, Cotelli and Presta, 2007; Yang
et al., 2013). A particular advantage of the zebrafish xenograft model is that only a small number
of cancer cells are required for xenotransplantation, which better recapitulates the earlier stages of
cancer progression (Haldi, Ton, Seng and McGrath, 2006; Nicoli, Ribatti, Cotelli and Presta,
2007).
Previous embryonic zebrafish xenograft glioma models, often involve transplanting the
cells into the yolk sac or perivitelline space (Geiger, Fu and Kao, 2008; Haldi, Ton, Seng and
McGrath, 2006; Marques, Weiss, Vlecken, Nitsche, Bakkers, Lagendijk, Partecke, Heidecke,
Lerch and Bagowski, 2009; Yang, Cui, Gu, Xu, Yu, Li, Cui, Zhang and Bian, 2013). These
transplant locations lack the complex brain microenvironment, which is filled with glial cells,
neuronal processes, extracellular matrix, and functional blood brain barrier important for studying
brain cancer (Lal, La Du, Tanguay and Greenwood, 2012). By transplanting glioblastoma cells
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into the 48-72 hour post fertilization zebrafish brain, which possess a functioning blood brain
barrier similar to mammalian models (Jeong et al., 2008), our laboratory has developed an efficient
and relevant assay using high content imaging to study glioblastoma progression and prioritize the
development of new glioblastoma therapies.
Our previous studies revealed that genetic knockdown of a protein important in
glioblastoma invasion (calpain 2) reduces glioblastoma invasion by ~90% (Jang et al., 2010).
Implanting these modified cells in the zebrafish brain microenvironment further demonstrated that
knockdown of calpain 2 reduced glioblastoma invasion by 2.9 fold, which was similar to the
reduction observed in organotypic mouse brain tissues (2.3 fold) (Lal, La Du, Tanguay and
Greenwood, 2012). In this report, we expand upon previous research by developing a robust
screening assay to determine if treatment of the zebrafish xenograft model with exogenous
compounds could significantly impact glioblastoma proliferation, migration and invasion. We
illustrate the utility of our assay by testing zinc oxide nanoparticles (ZnO NPs), which demonstrate
selective toxicity toward cancer cells in vitro, for anti-cancer efficacy in vivo (Hanley et al., 2008).
We also evaluate the selective phosphatidylinositide 3-kinase (PI 3-kinase) inhibitor LY294002,
known to inhibit glioblastoma proliferation and influence migration in cell culture and rodent
xenograft models, for effectiveness in this refined zebrafish xenograft assay (Han et al., 2010; Joy
et al., 2003; Su et al., 2003).
4.3 Materials and Methods
4.3.1 Zebrafish husbandry
Adult 5D Tropical strain zebrafish (Danio rerio) were reared at Sinnhuber Aquatic
Research Laboratory utilizing standardized procedures in accordance with Institutional Animal
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Care and Use Committee protocols at Oregon State University (Reimers et al., 2006). Embryos
were staged according to Kimmel et al. and at ~4 hours post fertilization, the chorion was
enzymatically digested using pronase (63.6 mg/ml, >3.5 U/mg, Sigma Aldrich: P5147, St. Louis,
MO) assisted by a custom automated instrument (Kimmel et al., 1995; Mandrell et al., 2012).
Embryos were maintained in 1x strength E2 embryo medium sans methylene blue (ZIRC
protocols, http://zebrafish.org/documents/protocols/pdf/Fish_Nursery/E2_solution.pdf) under a
14h light:10h dark photoperiod at 28±1°C. To inhibit pigment formation, 1 day post fertilization
(DPF) zebrafish embryos were treated with 0.003% w/v N-phenylthiourea (Sigma Aldrich, St.
Louis, MO), which was renewed daily until xenotransplantation occurred. Upon completion of
experiments, zebrafish were euthanized by overdose to buffered Tricaine (MS-222, Sigma Aldrich,
St. Louis, MO).
4.3.2 U87MG maintenance and fluorescent labeling
Human U87MG glioblastoma cells were purchased from the American Type Culture
Collection (Manassas, VA) and grown in Dulbecco’s Modified Eagles Medium (DMEM; CellGro,
Manassas, VA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO) and
1% L-glutamine (CellGro, Manassas, VA). Cells were incubated at 37°C with 5% CO2 and 95%
air in a humidified chamber. Chloromethylbenzamido (CellTrackerTM CM-DiI; Invitrogen, Grand
Island, NY), a fluorescent cell membrane intercalating dye, was used to label the U87MG cells
according to manufacturer’s directions. Briefly, a 50 µg/µL stock was prepared in dimethyl
sulfoxide (DMSO; Sigma Aldrich, St. Louis, MO), and diluted in phosphate buffered saline to a
working concentration of 2 µM. U87MG cells were incubated in 2 µM CM-DiI for two minutes
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at 37°C followed by 15 minutes at 4°C. Cells were resuspended in serum free DMEM at a
concentration of ~10-20 million cells/mL.
4.3.3 Glioblastoma transplantation procedure
Borosilicate capillary tubes with an outer diameter of 1.14 mm and an inner diameter of
0.5 mm (World Precision Instruments, Sarasota, FL) were used to prepare microinjection needles
on a Model P-97 micropipette puller (Sutter Instrument Co., Novato, CA). Prior to transferring
the fluorescently labeled U87 cells to the microinjection needle, phenol red (Sigma Aldrich, St.
Louis, MO) was added to the cell solution at a 1:10 dilution (v/v). At 2 or 3 DPF, dechorionated
zebrafish larvae were anesthetized in 200 mg/L buffered Tricaine, positioned on an 2% agarose
gel (Figure 4.1) and injected with ~50-100, U87MG glioblastoma cells into the hindbrain ventricle
(Figure 4.1.C.) using a ASI MPPI-2 air-driven pressure injector (Applied Scientific Instruments,
Eugene, OR). Background control zebrafish were injected with the same volume of DMEM mixed
with phenol red minus the U87MG cells. Following xenotransplantation, zebrafish were
transferred to fresh embryo medium and kept in a dark, 33±1°C incubator. Active xenografts
exhibiting no phenotypic malformations, such as edema or fin, axial or craniofacial abnormalities
one day post injection were used for the subsequent assays which ended at 7 DPF.
4.3.4 Imaging and analysis of U87MG behavior
Xenografts were anesthetized in 200 mg/L buffered tricaine for imaging. This
concentration of anesthetic was selected based on Kaufmann et al. for complete immobilization of
zebrafish for several hours (Kaufmann et al., 2012). Anesthetized xenografts were transferred to a
96 well imaging plate (Greiner BioOne, Monroe, NC) at one fish per well and embedded in 0.1
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mL of 0.8% (w/v) low melt agarose (Promega, Madison, WI) containing 200 mg/L tricaine. Before
agarose solidified, xenograft heads were centered, dorsal side down, at the bottom of the well
(Figure 4.2.A.). To minimize the duration in low melt agarose and reduce stress, xenografts were
immobilized, embedded and imaged in groups of 20-24 fish at a time.
Images were acquired on an ImageXpress Micro (Molecular Devices, Sunnyvale, CA)
using a 10x objective. The image view frame focused on the most anterior portion of the head to
the developing pharyngeal arches. All 10x images were acquired and analyzed using this frame of
view for consistency (Figure 4.2.B.). The TRITC filter (excitation 543/22 nm, emission 593/40
nm) was used to locate the U87MG cells and focus on the cancer cell mass. One brightfield image
was acquired for locational reference prior to capturing a TRITC z-stack. Typically, a 24 slice zstack at 8 µm per slice was of sufficient breadth within the z plane of the zebrafish brain to image
all U87MG cells. Fluorescent z-stacks were acquired with 30 ms exposures and 2x2 pixel binning
(Figure 4.2.B.). Zebrafish xenografts and background control zebrafish were imaged at 3 or 4 DPF
and 7 DPF using identical acquisition settings.
All images were analyzed using MetaXpress High Content Image Acquisition and Analysis
Software Version 5 (Molecular Devices, Sunnyvale, CA). Each z-stack was processed with
proprietary best focus stack arithmetic similar to maximum projection to create one fluorescent
image (Figure 4.3). The images were analyzed using the Multi-Wavelength Cell Scoring Module
to obtain cell counts. Individual cells were identified based on an image intensity of 100 gray levels
above background with a minimum cell diameter of 4 µm and maximum cell diameter of 10 µm
(Figure 4.3.A.). For cell migration and invasion, individual cells were identified using MultiWavelength Cell Scoring using the same parameters as stated above. A custom MetaXpress journal
located the centroid of the image based on the locations of all identified cells and calculated the
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distance from the image centroid to all cells. Then the number of cells located in concentric circles
between 0-80, 81-160, 161-240, 241-320 and 321+ µm were quantified (Figure 4.3.B.). The
change in cell count obtained from each concentric circle in the final image (7 DPF) of each
xenograft was normalized to the total cell count of the initial image (3 or 4 DPF) to quantify
changes in cell migration/invasion. No autofluorescence signal was identified as cells during the
analysis of the background control zebrafish (Figure 4.3.C.).
4.3.5 Xenograft exposure assay
After imaging, zebrafish were carefully removed from the low melt agarose, rinsed in 1x
embryo medium and transferred to a sterile polystyrene 96-well tissue culture, flat bottom plate
(Falcon, Manassas, VA) at one fish per well. Each well was pre-loaded with 0.1 mL of 1x embryo
medium at 0.003% PTU for the LY294002 exposure or sterile, deionized water (ultrapure water;
Invitrogen, Grand Island, NY) at 0.003% PTU for the ZnO NP exposure. Any xenograft exhibiting
damage from imaging or transfer was euthanized and excluded from the experiment. The timeline
of each assay is presented in Figure 4.4.
Prior to conducting the xenograft assay, exposure concentrations were selected based on
the results of an initial concentration response test. Briefly, un-injected, age matched, larval
zebrafish were exposed to a range of compound under conditions mimicking the xenograft assay
to identify a maximum tolerable exposure concentration, which resulted in insignificant mortality
or phenotypical malformations compared to the vehicle control. The maximum tolerable
concentration was used to set the upper limit of the concentration response series for the xenograft
assay.
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4.3.5.1 Phosphatidylinositol 3 kinase inhibitor LY294002. The selective pan PI 3-kinase
inhibitor LY294002 (Enzo Life Sciences, Farmingdale, NY) was selected as a positive control for
the inhibition of U87MG proliferation in the xenograft assay. LY294002 was dissolved in DMSO
to prepare a 16.2 mM stock. A two-fold dilution series was prepared at 2x (25-6.25 µM
LY294002) with PTU and DMSO maintained at 0.003% and 1% respectively, and 0.1 mL was
transferred to the 96 well plate contain the xenografts. This resulted in a 4 day static nonrenewal,
exposure to 1x (12.5-3.125 µM) LY294002 with 0.003% PTU and vehicle control of 0.5% DMSO.
4.3.5.2 Zinc oxide nanoparticle. ZnO NPs with a primary particle size of 8.37 nm were
synthesized using forced hydrolysis as described previously in Wehmas et al (Wehmas et al.,
2015). One gram of zinc acetate dehydrate and 99.5:0.5 mL diethylene glycol:nanopure water mix
were heated to 160°C for 90 minutes followed by multiple ethanol rinses and separation using
centrifugation for 20 minutes at 20,000 rpm after each rinse. Immediately prior to exposure, an
aliquot of ZnO NP and ZnO bulk material control were suspended in 100% DMSO at a
concentration of 10 mg/mL and water bath sonicated (Ultrasonik NDI; NEY, Inc., Bloomfield,
CT) for 20 minutes at room temperature. The ZnO bulk material acted as a size control because
the primary particle size is in the micrometer range as opposed to the size traditionally defined as
nano (possessing at least one dimension between 1- 100 nm) for the ZnO NPs. A two-fold dilution
series was prepared in sterile, deionized water (ultrapure water; Invitrogen, Grand Island, NY) at
2x for NP and bulk (0.3125, 0.625 1.25 and 2.5 mg/L) with PTU and DMSO maintained at 0.003%
and 1% respectively. 0.1 mL of the 2x NP and bulk was transferred to the 96 well plate contain
the xenografts preloaded in 0.1 mL deionized water at 0.003% PTU. This resulted in a three day
static nonrenewal, exposure to 1x (0.156-1.25 mg/L) ZnO NP and ZnO bulk control with 0.003%
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PTU and vehicle at 0.5% DMSO (Wehmas, Anders, Chess, Punnoose, Pereira, Greenwood and
Tanguay, 2015). To minimize agglomeration with salts present in zebrafish embryo medium, the
ZnO NP exposures were conducted in ultrapure water (Kim et al., 2013; Wehmas, Anders, Chess,
Punnoose, Pereira, Greenwood and Tanguay, 2015). As previously reported in Wehmas et al., this
ZnO NP has a mean hydrodynamic diameter on the order of microns when prepared in zebrafish
embryo medium whereas its mean hydrodynamic diameter is ~400 nm and zeta potential is ~20.2
mV when prepared in ultrapure water (Wehmas, Anders, Chess, Punnoose, Pereira, Greenwood
and Tanguay, 2015).
Following compound addition, the 96 well plates were sealed with Parafilm, wrapped in
aluminum foil to block incidental light and maintained in a 33±1°C incubator until 7 DPF. At 7
DPF, xenografts were assessed for mortality and phenotypic malformations. Living 7 DPF
xenografts were re-imaged to obtain U87MG proliferation and dispersal data as described in
section 4.3.4.
4.3.6 Statistical methods
Mortality data analyzed using Fisher’s Exact Test comparing each treatment to the vehicle
control. Zebrafish xenograft proliferation and migration/invasion data were analyzed by One-Way
Analysis of Variance using Tukey’s multiple comparison procedure. If assumptions of normality
and equal variance were violated, data was analyzed by Kruskal-Wallis One Way Analysis of
Variance on Ranks using a Dunn’s multiple comparison procedure. Analyses were completed
using SigmaPlot version 11 software and significance was defined as p value < 0.05 (SigmaPlot
11.0, San Jose, CA).
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4.4 Results
4.4.1 Transplantation conditions support glioblastoma growth
Our previous studies demonstrated the utility of using an orthotopic zebrafish xenograft
model of glioblastoma to study invasion in real time (Lal, La Du, Tanguay and Greenwood, 2012).
We also established the importance of injection location for studying human glioblastoma cells by
demonstrating that glioblastoma cells implanted in the zebrafish yolk sac do not invade as they do
when transplanted into the brain (Lal, La Du, Tanguay and Greenwood, 2012). To adapt the model
for compound screening against glioblastoma, we decided to inject the glioblastoma cells into the
zebrafish hindbrain between 2-3 DPF. At this time, minimal mortality was observed due to
injection (~1 and ~20% for 2 and 3 DPF respectively) and most major organ systems are fully
developed during this time period including a rudimentary blood brain barrier allowing us to more
closely simulate tests conducted in mammalian models (Jeong, Kwon, Ahn, Kang, Kwon, Park
and Kim, 2008; Kimmel, Ballard, Kimmel, Ullmann and Schilling, 1995). Based on work by
others, we maintained our xenografts at 33°C, which is lower than the standard 37°C for culturing
glioblastoma cells but higher than the typical 28°C for zebrafish maintenance (Geiger, Fu and Kao,
2008; Yang, Cui, Gu, Xu, Yu, Li, Cui, Zhang and Bian, 2013). This temperature caused no
observable zebrafish toxicity yet resulted in adequate human glioblastoma growth and
migration/invasion over the 3-4 day assay period to detect exposure dependent differences between
treatment groups.
Control xenografts maintained at 33°C from 4 to 7 DPF typically demonstrated ~1.6 fold
increases in glioblastoma proliferation whereas controls maintained at the same temperature for 3
to 7 DPF typically demonstrated ~1.3 to 2 fold increases growth (Figures 4.5.A. & 4.7.A. controls).
Cell migration/invasion for experiments conducted from 3 or 4 to 7 DPF display greater movement
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(between 0.66 to 0.84 fold increases compared to control) near the main cell mass (0-80 µm),
which tapers off (between 0.21 to 0.34 fold changes compared to control) as distance from the
center increases to 161-240 µm (Figures 4.6 & 4.8 controls). The relative change in cell spread for
distances 241+ µm were insignificant (less than 0.06 fold) and not depicted in figures.
4.4.2 Zinc oxide nanoparticle enhances U87MG proliferation and dispersal
Using the 3 day xenograft assay, we tested a putative nanotherapeutic (ZnO NPs) that
significantly and preferentially inhibit cancer cell proliferation in the presence of normal human
cells in culture (Hanley, Layne, Punnoose, Reddy, Coombs, Coombs, Feris and Wingett, 2008).
The minimal quantity of compound needed for assessment in our assay made it ideal for evaluating
systemic toxicity as well as the cancer specific efficacy of these ZnO NPs in vivo. Exposure to
ZnO NP and bulk ZnO did not result in significant lethality, but there was a slight material
dependent increase in mortality at ~19% and 6% respectively compared to the vehicle control
(Table 4.1). Exposure of xenografts to ZnO NP slightly enhanced glioblastoma proliferation by 15
to 23% compared to the vehicle control (Figure 4.5). ZnO NP exposure also tended to augment
glioblastoma migration/invasion slightly in both experiments across all distance increments
compared to vehicle and ZnO bulk controls; however, significant increases were only observed
near the perimeter of the cell mass (161-240 µm) (Figure 4.6). At this distance, exposure to ZnO
NP enhanced migration/invasion by 22 to 49% compared to vehicle and by 25 to 59% compared
to the ZnO bulk across the two experiments (Figure 4.6).
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4.4.3 LY294002 inhibits U87MG proliferation and dispersal
To further confirm the utility of our assay, we evaluated the PI 3-kinase inhibitor
LY294002, which is a well-studied compound known to inhibit glioblastoma proliferation,
migration and invasion in cell culture and mammalian models (Han, Yang, Yue, Huang, Liu, Pu,
Jiang, Yan, Jiang and Kang, 2010; Kubiatowski et al., 2001; Shingu et al., 2003; Su, Mayo,
Donner and Durden, 2003). Exposure of zebrafish xenografts to 3.125-12.5 µM LY294002
resulted in significant decreases in glioblastoma proliferation compared to the vehicle control
(Figure 4.7). We observed some concentration dependent decreases in proliferation. While
exposure to 12.5 µM LY294002 caused significant mortality (Table 4.2), 3.125-6.25 µM
LY294002 resulted in minimal xenograft mortality and up to ~33% reduction in glioblastoma
proliferation compared to the control (Figure 4.7). LY294002 significantly reduced cell
migration/invasion in a concentration dependent manner nearer the main cell mass (0-160 µm)
with less influence at further distances (161+ µm) (Figure 4.8). Within the distance range of 0-80
µm, 6.25 µM LY294002 significantly diminished cell spread by 25 to 31% compared to control
across all three experiments (Figure 4.8). The inhibitory effects of 6.25 µM LY294002 on
migration/invasion were more variable at 81-160 µm with decreases in fold change ranging from
23 to 48% compared to control (Figure 4.8).
While 3.125 µM exposure did not always
significantly reduce migration/invasion compared to control, a trend is apparent across all
experiments for all distance increments depicted (Figure 4.8).
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4.5 Discussion
4.5.1 Orthotopic zebrafish xenograft assay supports study of glioblastoma biology
The main objective of this study was to create a robust orthotopic zebrafish xenograft assay
for the prioritization of putative anticancer compounds to advance glioblastoma drug development.
Using this assay, we demonstrate the U87MG glioblastoma cells proliferate and disperse well in
the zebrafish brain microenvironment over the assay period. We observed a 1.3 to 2 fold increase
in growth over a 3 to 4 day period under control conditions (Figure 4.5 and 4.7). Proliferation was
quantified by counting individual cells based on size and fluorescent signal above background
using image-based software analyses. While this automation increases the throughput of
measuring proliferation, migration and invasion in our zebrafish xenograft assay, compression of
z-stacks needed for the image-based analysis likely underestimates the growth and migration of
glioblastoma cells due to a loss of information in the z plane. Despite this limitation, other
researchers have observed similar growth (~1.5 fold) over 4 days using U251 glioblastoma cells
in a different embryonic zebrafish xenograft model (Geiger, Fu and Kao, 2008). However, the 1.5
fold increase assumes that the U251 growth directly corresponds to a 1.5 fold increase in relative
fluorescence as the U251 cells were labeled by stable transfection with a red fluorescent protein
construct (Geiger, Fu and Kao, 2008).
Under control experimental conditions, the U87MG cells also disperse from the main
cancer cell mass (Figure 4.6 and 4.8), which we quantify as a surrogate for invasion and migration.
Measurements of migration and invasion in embryonic zebrafish xenograft models has typically
involved qualitative scoring or simply counting the number of cells to move away from the initial
cell mass with no information on distance (Lee, Rouhi, Dahl Jensen, Zhang, Ji, Hauptmann,
Ingham and Cao, 2009;
Marques, Weiss, Vlecken, Nitsche, Bakkers, Lagendijk, Partecke,
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Heidecke, Lerch and Bagowski, 2009; Yang, Cui, Gu, Xu, Yu, Li, Cui, Zhang and Bian, 2013).
Our method provides a quantitative method to measure changes in cell dispersal over concentric
distances from the center of the cancer mass. This approach is similar to Zhang et al. who measured
chemical inhibition of leukemia stem cell migration in a zebrafish xenograft model (Zhang et al.,
2014). However, Zhang et al. reported the migration results in a way in which the distance
information was lost making it impossible to tell how far the cells metastasized from the main
mass (Zhang, Shimada, Kuroyanagi, Umemoto, Nishimura and Tanaka, 2014).
These approaches make it difficult to compare our results to others reported in the literature
except to say that migration and invasion are observed in some zebrafish xenograft models.
However, our migration/invasion results differ from those reported by Geiger et al. where no
migration or invasion of U251 cells was observed (Geiger, Fu and Kao, 2008). This may be
partially explained by differences in injection site locations between the two experiments (Geiger,
Fu and Kao, 2008). Geiger et al. implanted U251 cells in the embryonic zebrafish yolk sac and Lal
et al. reported that glioblastoma cells do not migrate or invade when injected into the zebrafish
yolk compared to the brain (Lal, La Du, Tanguay and Greenwood, 2012). Differences in cell types
used between the two experiments do not justify the results by Geiger et al. as ongoing experiments
in our laboratory demonstrate that U251 cells grow, migrate and invade similarly to U87MG cells
(data not shown). Another group studying glioblastoma invasion in an embryonic zebrafish
xenograft model only observed invasion of U87 cells implanted in the zebrafish yolk sac after
injecting a subpopulation enriched for stem cells (Yang, Cui, Gu, Xu, Yu, Li, Cui, Zhang and Bian,
2013). This work further exemplifies the importance of injection site location and
microenvironment in influencing realistic glioblastoma behavior (Yang, Cui, Gu, Xu, Yu, Li, Cui,
Zhang and Bian, 2013).
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4.5.2 Application of orthotopic zebrafish xenograft assay assists drug prioritization
Our embryonic zebrafish xenograft model supports reproducible glioblastoma proliferation
and dispersal beneficial to studying the biological processes controlling this deadly disease. We
also illustrate how the assay can be applied to screening potential nanotherapeutic (ZnO NPs) and
chemotherapeutic agents (LY294002) targeting glioblastoma proliferation, migration and
invasion. The subsequent sections illustrate how this short assay successfully combines the rapid
economy of high throughput in vitro assays with the relevance of slower in vivo mammalian assays
for efficient advances in drug development.
4.5.3 Nanotherapeutic assessment
Metal oxide NPs like ZnO NPs initially drew interest in the biomedical community for
their intrinsic antimicrobial effects (Adams et al., 2006; Brayner et al., 2006; Huang et al., 2008;
Reddy et al., 2007; Wei et al., 1994). Researchers quickly realized that the unique physiochemical
properties of metal oxide NPs also demonstrated specific toxicity toward human cancer cells
compared to normal cell controls (Hanley, Layne, Punnoose, Reddy, Coombs, Coombs, Feris and
Wingett, 2008; Ostrovsky et al., 2009; Thevenot et al., 2008). Work by Hanley et al. identified
that ZnO NPs could selectively kill several human cancer cell types with ~28 to 35 fold preferential
toxicity to cancerous T cells relative to normal peripheral blood mononuclear cells in vitro
(Hanley, Layne, Punnoose, Reddy, Coombs, Coombs, Feris and Wingett, 2008). This group
hypothesized that selective toxicity was due to surface-charge interactions with the cancer cells
(Hanley, Layne, Punnoose, Reddy, Coombs, Coombs, Feris and Wingett, 2008). However, it was
not known if this selective toxicity would be conserved in vivo. Because scaling up the production
of nanomaterials can be challenging during the early stages of discovery, we utilized our orthotopic
94
zebrafish xenograft assay of glioblastoma, which requires ~1-2 µg quantities of material per assay,
to assess the anticancer efficacy these ZnO NPs compared to ZnO bulk material control.
ZnO NPs demonstrated relatively high toxicity to zebrafish when prepared in a stable
suspension medium like water (Wehmas, Anders, Chess, Punnoose, Pereira, Greenwood and
Tanguay, 2015). At the non-lethal exposure concentrations tested in our xenograft assay, the ZnO
NPs significantly enhanced glioblastoma proliferation and migration/invasion compared to the
vehicle control (Figure 4.5 & 4.6), which is the opposite of what we hypothesized would occur
based on the in vitro data. However, we also identified that the ZnO NP agglomerated and
underwent considerable dissolution (~47-50%) when prepared in water (data previously published
(Wehmas, Anders, Chess, Punnoose, Pereira, Greenwood and Tanguay, 2015)). Therefore, we
cannot distinguish whether this increase in glioblastoma proliferation is due to a nanoparticle
effect, an ionic effect or a combination of both.
Research in breast and prostate cancer cells with overexpression of zinc ion importers
demonstrates that exposure to small amounts of ionic zinc can enhance cancer cell proliferation
and invasion (Kagara et al., 2007; Li et al., 2007). Lin et al. identified several zinc ion importers
with increased expression in malignant glioma samples and waterborne exposure of embryonic
zebrafish to zinc can result in distribution to the brain (Brun et al., 2014; Lin et al., 2013).
Furthermore, invasion of glioblastoma cells is partly controlled by matrix metalloproteinases
(MMP) which require zinc as cofactor. Malignant gliomas often possess upregulation of MMP2
and MMP9 (Nakagawa et al., 1994; Nakano et al., 1995). Based on this data, we hypothesize that
the ZnO NP exposure could be delivering enough zinc to the glioblastoma in the zebrafish brain
to enhance growth, migration and invasion. Our short, three day assay allowed us to identify that
95
this negatively charged, ~400 nm, uncoated ZnO NP is not a good candidate for future pre-clinical
assessment in mammalian models.
4.5.4 Chemotherapeutic assessment
The results of the ZnO NP exposure demonstrated that our assay could detect exposureinduced increases in glioblastoma proliferation and migration/invasion. To prove that the assay
was sensitive to exposure-induced decreases in glioblastoma proliferation, the PI 3-kinase inhibitor
LY294002 was tested. LY294002 works as a model anti-proliferative and anti-invasive agent
because glioblastomas frequently possess a genomic mutation that results in the loss of tumor
suppressor PTEN expression, thereby escalating PI 3-kinase activation. The PTEN mutation is
present in 30 to 40% of malignant gliomas and U87MG cells (Li et al., 1997; Smith et al., 2001;
Wang et al., 1997). In normal cells, PTEN acts by dephosphorylating phosphatidylinositol (3,4,5)­
trisphosphate (PtdIns (3,4,5)-P3) mediated activation of serine/threonine kinase (AKT), which is
involved in cell proliferation and survival (Cantley and Neel, 1999). Amplified AKT
phosphorylation by PI 3-kinase also augments migration and invasion of glioblastoma (Joy,
Beaudry, Tran, Ponce, Holz, Demuth and Berens, 2003;
Kubiatowski, Jang, Lachyankar,
Salmonsen, Nabi, Quesenberry, Litofsky, Ross and Recht, 2001). Inhibiting PI 3-kinase activity
with LY294002, thereby decrease glioblastoma proliferation, migration and invasion.
For the LY294002 experiments, we injected the glioblastoma cells into the zebrafish one
day earlier to increase the assay duration and sensitivity (Figure 4.4.B.). Exposure to 3.125-6.25
µM LY294002 inhibited glioblastoma proliferation up to ~33% (Figure 4.7) and up to 31 and 48%
at distances between 0-80 and 81-160 µm respectively (Figure 4.8). Several in vitro and in vivo
experiments demonstrate LY294002 inhibits glioblastoma cell proliferation, migration and
96
invasion (Han, Yang, Yue, Huang, Liu, Pu, Jiang, Yan, Jiang and Kang, 2010; Kubiatowski, Jang,
Lachyankar, Salmonsen, Nabi, Quesenberry, Litofsky, Ross and Recht, 2001; Shingu, Yamada,
Hara, Moritake, Osago, Terashima, Uemura, Yamasaki and Tsuchiya, 2003; Su, Mayo, Donner
and Durden, 2003). Our proliferation results are similar to in vivo work by Su et al. where dosing
orthotopic mouse xenografts with LY294002 resulted in ~40% inhibition of glioblastoma growth
compared to controls after 5 days. However, the repeated dosing regimen of Su et al. differed from
our experiment (Su, Mayo, Donner and Durden, 2003).
Another mouse study using a
subcutaneous glioblastoma xenograft model identified intra-tumoral administration of LY294002
significantly reduced tumor volume by ~50% after 12 days (Han, Yang, Yue, Huang, Liu, Pu,
Jiang, Yan, Jiang and Kang, 2010). Besides directly impacting the growth of the glioblastoma cell,
LY294002 can influence the surrounding environment through antiangiogenic effects, which can
also reduce glioblastoma proliferation (Su, Mayo, Donner and Durden, 2003).
The ~33%
reduction in glioblastoma proliferation by LY294002 in our xenograft assay could be due to both
direct PI 3-kinase inhibition as well as indirect reduction in angiogenesis. Despite assay and model
differences, the zebrafish xenografts absorb sufficient LY294002 to significantly inhibit
glioblastoma proliferation similar to mammalian models. Moreover, in vitro research reveals
LY294002 exposure reduces the number of glioblastoma cells that migrate by 20 to 50 at 5-10
µM, which is not much different from the 31 to 49% reductions in migration/invasion observed
upon 6.25 µM exposure from our assay (Han, Yang, Yue, Huang, Liu, Pu, Jiang, Yan, Jiang and
Kang, 2010; Lee et al., 2005a). Interestingly, Joy et al. demonstrated that migrating glioblastoma
cells become more resistant to apoptosis compared to migration restricted cells. This suggests that
identifying therapeutics that inhibit glioblastoma migration and invasion will be extremely
important in treatment (Joy, Beaudry, Tran, Ponce, Holz, Demuth and Berens, 2003).
97
Glioblastoma is an aggressive brain cancer requiring improved treatment options to
increase patient survival. Under existing drug development paradigms, any new glioblastoma
drugs will typically require several years to transition from discovery phase to approved human
use, which demonstrates a dire need to accelerate the drug development process. Incorporation of
embryonic zebrafish assays into the drug development pipeline already promises to enhance
discovery and prioritization of novel pharmaceuticals including nanomaterials. The results of this
assay demonstrate that we have a short duration, relevant and sensitive embryonic zebrafish-based
assay to identify and prioritize novel glioblastoma therapeutic agents.
4.5.5 Future directions
Ongoing research is determining whether typical aggressive/invasive glioblastoma
behavior (i.e. growth patterns, migration and invasion) are conserved using other glioblastoma cell
types in our orthotopic zebrafish xenograft model. Future research will investigate whether ionic
zinc, at exposure concentrations comparable to the dissolved zinc present in the ZnO NP
experiments, significantly enhances glioblastoma proliferation and dispersal like ZnO NP.
Additional research will involve identifying a compound that strictly targets glioblastoma
migration/invasion without the confounding effects of proliferation inhibition. This will be useful
as a control in future assays and in classifying potential therapeutics targeting migration and
invasion. Some matrix metalloproteinase inhibitors have proven effective in selectively inhibiting
glioblastoma dispersal in other zebrafish xenograft assays (Yang, Cui, Gu, Xu, Yu, Li, Cui, Zhang
and Bian, 2013). Use of transgenic fli1:eGFP zebrafish with fluorescent vasculature will aid in
distinguishing between migrating and invading glioblastoma cells. Furthermore, incorporation of
fli1:eGFP zebrafish and high-resolution confocal imaging will help identify compounds that affect
98
angiogenesis as well as glioblastoma proliferation, migration and invasion in real time. Threedimensional reconstruction of confocal images will augment the sensitivity of the zebrafish
xenograft model by providing information about movement and proliferation of cells in the z plane
of the head. Increasing the incubation temperature of the xenografts to 35°C or incorporating heat
tolerant transgenic zebrafish so that xenografts can be maintained at 37°C may increase the
sensitivity of this assay by enhancing glioblastoma growth.
Acknowledgments
The author thanks Jane LaDu and Dr. Sangeet Lal for helping establish the zebrafish
xenograft model, members of the Punnoose laboratory for supplying ZnO NPs and the Sinnhuber
Aquatic Research Laboratory for providing zebrafish. Thanks also goes to Dr. Josephine
Bonventre for assistance with manuscript editing.
Conflicts of Interest
The authors declare no conflict of interest. The authors alone are responsible for the
content and writing of the paper. This research was funded by NSF1134468 and NIH T32
ES07060 and P30 ES000210.
99
Table 4.1. Mortality associated with zinc oxide nanoparticle (NP) exposures
Treatment (mg/L)
N
Mortality (%)
Date
0
Bulk (0.312 - 1.25)
NP (0.312 - 1.25)
0
Bulk (0.312 - 1.25)
NP (0.312 - 1.25)
26
32
32
14
32
32
0
6.25
18.75
0
6.25
18.75
7/1/2013
100
11/30/2012
Table 4.2. Mortality associated LY294002 exposures
Treatment (µM)
N
Mortality (%)
Date
0
3.125
6.25
12.5
0
3.125
6.25
12.5
0
3.125
6.25
33
33
33
33
28
27
27
27
23
24
24
6.06
6.06
3.03
45.45**
0
14.81
0
51.85***
4.35
0
8.33
1/8/2015
12.5
** p value < 0.01
*** p value < 0.001
24
29.17
101
12/12/2014
12/3/2014
102
Figure 4.1. Microinjection setup, zebrafish placement and glioblastoma implantation site
A) Microinjector setup. B) Placement of anesthetized two or three days post fertilization (DPF)
zebrafish on 2% agarose gel for microinjection of human U87MG glioblastoma cells. C) Close up
drawing depicting 2 dpf zebrafish brain (upper) and whole embryo (lower) showing the injection
site (red line) in the hindbrain ventricle near midbrain junction. e – eye, hb – hindbrain, fb –
forebrain and mb – midbrain.
103
Figure 4.2. Position of embryonic zebrafish for image acquisition
A) Xenograft placement in 96-well plate at 2x with heads centered and positioned dorsal side down
in well using dissecting microscope. Images acquired using molecular devices high content imager
with 2x objective. B) Representative image of three day post fertilization (DPF) xenograft (left:
fluorescent 24 slice (8 um per slice) z-stack acquired 30 ms exposure with 2x2 binning and
combined into one all in focus image using best focus stack arithmetic and right: one brightfield
image acquired at 61 ms exposure overlayed with fluorescent image) depicting frame of view that
is acquired and analyzed at 10x for all fish at 3 or 4 DPF and 7 DPF.
104
Figure 4.3. Image analysis for proliferation, migration and invasion
Representative 10x fluorescent images (top) with corresponding brightfield images showing
position of cells in zebrafish head directly below. A) Glioblastoma injected zebrafish imaged at 3
days post fertilization with Multi-Wavelength Cell Scoring Mask (white) used to delineate and
count individual cells. B) Same xenograft as image A with added concentric circles (yellow) used
to quantify cell migration/invasion in 80 µm distances from the centroid of the cell mass (blue
circle) C) Mock injected zebrafish imaged at 3 days post fertilization. No fluorescent cell count
data obtained from zebrafish mock injection which consisted of cell medium only.
105
A
B
Figure 4.4. Diagram depicting the timeline of the orthotopic zebrafish xenograft assay
A) Overview of three day zinc oxide nanoparticle assay. B) Overview of the four day LY294002
assay.
106
107
Figure 4.5. Comparison of the effects of exposure to zinc oxide nanoparticles (ZnO NP), bulk
material control and vehicle control on the fold change in human glioblastoma (U87MG) cell
count
A) Consists of representative 10x images of four zebrafish imaged at 4 and 7 days post fertilization
(DPF). Each image panel consists of a 10x monochromatic fluorescent image with MultiWavelength Cell Scoring mask (white) identifying individual cells (top) and a 10x monochromatic
fluorescent image with brightfield overlay of the same xenograft showing injection placement in
zebrafish brain (bottom). All images were selected with an initial cell count ranging from 51-59
cells. B) The fold change as determined by the fluorescent signal above background from the CMDiI dyed U87MG cell mass in each xenograft from each treatment group from initiation of the
experiment at 4 and 7 DPF. The 0 treatment contained 0.5% DMSO which was maintained across
all groups. The red plus indicates the overall mean of each treatment group. These values were
determined from two independent experiments (n=14-30 per experiment). Significant differences
for all pairwise comparisons between treatments indicated by letters A-B, p < 0.05.
108
109
Figure 4.6. Quantitative analysis of human glioblastoma (U87MG) cell spread following zinc
oxide nanoparticle (NP), zinc oxide bulk (BULK), or vehicle control exposure
The number of migrated cells within concentric 80 µm rings from the centroid of U87MG mass
were measured. Data reported as relative number of cells migrated within each concentric circle
normalized to the total number of cells at 4 DPF (days post fertilization). The 0 treatment contained
0.5% DMSO which was maintained across all groups. The red plus indicates the overall mean of
each treatment group. These values were determined from two independent experiments (left and
right panels). Note changes in y axis scale between the different distance ranges. Significant
differences for all pairwise comparisons between treatments indicated by letters A-B, n=14-30 per
experiment, p < 0.05.
110
Figure 4.7. Comparison of the effects of increasing exposure to LY294002 on the fold change
in human glioblastoma (U87MG) cell count data
A) Consists of representative 10x images of two zebrafish imaged at 3 and 7 days post fertilization
(DPF). Each image panel consists of a 10x monochromatic fluorescent image with MultiWavelength Cell Scoring mask (white) identifying individual cells (top) and a 10x monochromatic
fluorescent image with brightfield overlay of the same xenograft showing injection placement in
111
zebrafish brain (bottom). Each image was selected with an initial cell count of 45 and 54 cells. B)
The fold change as determined by the fluorescent signal above background from the CM-DiI dyed
U87MG cell mass in each xenograft from each treatment group from initiation of the experiment
at 3 days post fertilization to the end at 7 days post fertilization. The 0 treatment contained 0.5%
DMSO which was maintained across all groups. The red plus indicates the overall mean of each
treatment group. These values were determined from three independent experiments (left, middle
and right panels, n= 13-32 per experiment). Significant differences for all pairwise comparisons
between treatments indicated by letters A-B, p < 0.05.
112
113
Figure 4.8. Quantitative analysis of human glioblastoma (U87MG) cell spread following
LY294002 or vehicle control exposure
The number of migrated cells within concentric 80 µm rings from the centroid of U87MG mass
were measured. Data reported as relative number of cells migrated within each concentric circle
normalized to the total number of cells at 3 DPF (days post fertilization). The 0 treatment contained
0.5% DMSO which was maintained across all groups. The red plus indicates the overall mean of
each treatment group. These values were determined from three independent experiments (left,
middle and right panels). Note changes in y axis scale between the different distance ranges.
Significant differences for all pairwise comparisons between treatments indicated by letters A-B,
n= 13-32 per experiment, p < 0.05.
114
5. Final Conclusions
The numerous different types of nanomaterials and paucity of reliable in vivo information
on their toxicity has resulted in enduring uncertainties associated with health and safety risks,
thereby slowing progress in nanotherapeutic assessment and development (Fako and Furgeson,
2009; Oberdörster, 2010). Aggressive, lethal cancers like glioblastoma require advances in
pharmaceutical development. The benefits of targeted drug delivery, selective toxicity, and blood
brain barrier translocational applications of nanomaterial-based medicines hold promise in
improving glioblastoma treatments and is necessary for increasing survival rates (Etheridge et al.,
2013; Nie et al., 2007; Wohlfart et al., 2012; Wong et al., 2011). The studies included in this
dissertation sought to advise nanomaterial environmental health and safety through efficiently
evaluating toxicity while categorizing physicochemical properties (elemental composition, size,
charge and surface functionalization) indicative of adverse biological response in the zebrafish
embryo-larval assay. Through creation of a novel embryonic zebrafish xenograft assay, these
studies also strove to advance applications in nanomedicine via economical in vivo assessment of
nanoparticles demonstrating preferential cancer toxicity in culture. This chapter will summarize
the major findings of the previous chapters in the context of addressing the challenges associated
with nanotoxicology and therapeutic development while considering the specific aims of this
dissertation:
Aim 1. Evaluate two common classes of nanomaterials (MWNT and MO NPs) for toxicity
using the embryo-larval zebrafish assay and prioritize further assessment.
Aim 2. Identify physicochemical properties of MWNTs and MO NPs associated with
zebrafish toxicity to inform safe design and predict nanotoxicity
115
Aim 3. Establish a novel orthotopic embryonic zebrafish xenograft assay to prioritize
efficacy of nanotherapeutic agents for glioblastoma drug development.
The genetic, anatomic and physiologic conservation between the embryonic zebrafish
model and humans originally made it an advantageous model to study developmental biology and
human disease (Driever et al., 1996; Driever et al., 1994; Haffter et al., 1996; Kimmel et al.,
1995). Incorporation of the embryonic zebrafish into pharmaceutical discovery and toxicology
testing has since demonstrated the medium to high-throughput adaptability and predictive power
of this model (Fako and Furgeson, 2009; Hill et al., 2005; Zon and Peterson, 2005). Applying the
model to nanotoxicology assessment enables rapid in vivo biologic readouts under consistent
experimental conditions making comparative assessment and conclusions on nanomaterial­
biologic interactions streamlined.
The results in Chapters 2 and 3 address Aim 1 of this
dissertation through detailed application of the zebrafish embryo-larval assay toward efficient
nanotoxicity assessment of MWNTs and MO NPs. Briefly, seven uncoated MO NPs made from
CeO2, TiO2, SnO2 and ZnO and seven MWNTs (prepared from the same stock and systematically
functionalized with oxygen groups) were tested. Most of the MO NPs and MWNTs caused little
to no significant adverse response. Three MWNTs and one metal oxide (ZnO NP) caused
significant toxicity (LC50 = 3.5-9.1 mg/L), and for the MWNTs, this only occurred at the highest
exposure concentration tested (50 mg/L). Therefore, these nanomaterials were deemed of highest
concern for further investigation in Aim 2.
Unlike traditional toxicology assessments, nanotoxicology experiments must carefully
consider the physical material properties as well as the chemical (Maynard et al., 2010;
Oberdörster, 2010;
Oberdörster et al., 2005). Drawing conclusions from the results of
nanotoxicology studies can be extremely challenging due to variations in experimental design
116
including different exposure concentrations, routes of exposure, sources of nanomaterials,
dispersal methods, etc. (Maynard, Warheit and Philbert, 2010; Oberdörster, 2010). The results in
Chapters 2 and 3 attend to Aim 2 by contemplating the nanomaterial physicochemical properties
within the assay to explain toxic response and develop associations between nanotoxicity and
hazard. One major physicochemical trait influenced in the zebrafish embryo-larval assays was
agglomeration. Significant agglomeration occurred in MWNT and MO NP assessments, a major
caveat in interpreting nanotoxicity results in general (Maynard, Warheit and Philbert, 2010;
Oberdörster, 2010). Agglomeration likely limited the bioavailability of MWNTs and MO NPs,
although opportunities for contact-dependent toxicity still existed in the assay (Bai et al., 2010).
Therefore, low bioavailability may explain the low biologic response for a majority of
nanomaterials assessed. Use of a low ionic strength exposure medium in the MWNT and MO NP
experiments was important in partially addressing the agglomeration problem by creating more
stable suspensions. This highlights the importance of considering experimental conditions in
nanomaterial experiments. For instance, if only the typical high ionic strength zebrafish embryo
medium was used in the MO NP exposures, we may have erroneously concluded that none of the
MO NPs were toxic.
Another physicochemical property influencing the toxicity of MWNTs included total
surface oxygen/point of zero charge. The three MWNTs that demonstrated significant toxicity
using the zebrafish embryo larval assay possessed total surface oxygen greater than 3.5% (point
of zero charge < ~6). Multivariate statistical modeling and additional experiments confirmed that
total surface oxygen and the highly related parameter, point of zero charge, best predicted MWNT
induced zebrafish mortality. Understanding the properties controlling MO NP toxicity was more
challenging. The surfaces were neither functionalized nor coated; therefore, all agglomerated to
117
some extent even in the low ionic strength exposure medium. Of all the MO NP, three
(CeO2:QK055, TiO2:TC009 and ZnO) created stable exposure suspensions in low ionic strength
medium. These three MO NPs possessed similar positive charges and hydrodynamic sizes.
However, only ZnO NP caused toxicity, making it feasible to conclude that positive charge, size
and surface area are not the physicochemical properties predictive of MO NP adverse response.
This conclusion is contrary to some theories present in nanomaterial literature suggesting
positively charged nanomaterials are more toxic than negatively charged or neutral nanomaterials
(Goodman et al., 2004; Harper et al., 2011; Xia et al., 2006; Xia et al., 2008).
However,
additional controversy surrounds interpretation of soluble nanomaterial, like ZnO NP, toxicity
results (Borm et al., 2006; Brunner et al., 2006). This controversy pertains to elucidating whether
the toxicity is due to a nanomaterial specific effect or dissolution of the ion (Bai, Zhang, Tian, He,
Ma, Zhao and Chai, 2010; Brun et al., 2014; Franklin et al., 2007; Zhu et al., 2009). Coating can
influence dissolution and potentially changes the physicochemical properties of the nanomaterial,
which is why all the MO NPs remained uncoated (Blinova et al., 2010; Keller et al., 2010; Nel
et al., 2006). The ZnO NPs dissolved extensively when prepared in low ionic strength medium.
Exposures to equivalent amounts of ionic zinc resulted in similar toxic response in the zebrafish
assay indicating dissolution as the cause of toxicity. The insolubility and lack of toxicity from the
other MO NPs (SnO2, CeO2 and TiO2) rendered the release of toxic ions from those materials not
a major concern. Additional investigation revealed higher ZnO NP toxicity in larval zebrafish
exposures around the time of gill development compared to embryonic exposures. The gill is a
known target of ionic zinc insult and may explain the life-stage dependent ZnO toxicity (Handy et
al., 2008). The embryonic life stage is considered the most sensitive (OECD); therefore, many
zebrafish embryo-larval assays begin during embryonic development (6-8 hpf). However, limiting
118
nanotoxicity assessment to starting during the embryonic life stage may miss sensitive routes of
toxicity, potentially misclassifying nanomaterials like ZnO NP as less of a concern. This
dissertation recommends that embryo-larval nanotoxicity assays incorporate additional tests at
later larval life stages, especially with potentially soluble nanomaterials, to better characterize
hazard.
ZnO demonstrated some of the major challenges associated with nanotoxicology
assessments including how experimental conditions, dissolution, agglomeration, uptake, etc. can
significantly influence experimental outcome. Therefore, very careful characterization (charge,
size, agglomeration, dissolution, surface modifications including functionalization or surfactant
coating, redox capabilities, etc.) of the nanomaterials in the exposure environment remains
challenging but necessary. Unfortunately, in some instances, existing analytical techniques are not
sufficient to fully answer questions about processes like nanomaterial uptake and localization
especially with soluble nanomaterials. Obtaining adequate characterization data for the MO NPs
and measuring MWNT and MO NP absorption could have helped determine the extent to which
bioavailability influenced biologic response. Despite these problems, the combined phenotypic
screening results, in conjunction with examining in situ nanomaterial charge, size and dissolution
potential led to the conclusion that agglomeration/bioavailability, dissolution potential and surface
functionalization with a highly electronegative groups like oxygen are the physicochemical
properties of the tested nanomaterials with greatest influence on zebrafish toxicity. Nonetheless,
the results and conclusions generated during this dissertation will help inform and improve
nanomaterial design. Understanding nanomaterial physicochemical properties will be important in
synthesizing nanomaterials targeting diseases for successful advancements in the field of
nanomedicine.
119
Years of glioblastoma research have resulted in only modest increases in median survival
rates (Van Meir et al., 2010). Nanomedicine holds promise for improving disease diagnosis and
treatment of highly lethal cancers such as glioblastoma. Both MO NPs and MWNTs demonstrate
applications in the field of nanomedicine including imaging, targeted drug delivery and selective
toxicity toward cancer (Cheng et al., 2011; Hanley et al., 2008; Nie, Xing, Kim and Simons,
2007; Thevenot et al., 2008). The interest in assessing MO NPs for toxicity and safety in the
zebrafish embryo-larval assay began when research on ZnO NP demonstrated selective toxicity
toward cancer cells in culture (Hanley, Layne, Punnoose, Reddy, Coombs, Coombs, Feris and
Wingett, 2008). Under existing pharmaceutical development, it will take years of pre-clinical
assessment before novel nanotherapeutics like ZnO NP are adequately tested for safety and
efficacy to improve treatment of glioblastoma (Fako and Furgeson, 2009; Zon and Peterson,
2005). The results in Chapter 4 address Aim 3 of this dissertation by creating and optimizing an
orthotopic zebrafish xenograft assay to quickly assess and prioritize potential glioblastoma
therapeutics in vivo. The results demonstrate that U87MG glioblastoma cells proliferate and
migrate/invade in the zebrafish brain microenvironment with growth increases similar to other
zebrafish xenograft models of glioblastoma (Geiger et al., 2008). Implantation of the glioblastoma
cells within the brain microenvironment results in realistic migration and invasion not observed in
other zebrafish xenograft models in which cells are implanted in the yolk or perivitelline space
(Geiger, Fu and Kao, 2008; Lal et al., 2012). Therefore, this assay can identify nanotherapeutics
agents targeting invasion and migration in addition to proliferation for prioritization of drug
development. Assessment of ZnO NP revealed that it unexpectedly enhanced glioblastoma
proliferation and migration/invasion significantly. These results revealed that the ZnO NP is not a
good candidate for future drug development. Fortunately, the quick turnaround of this assay
120
provides feedback to material scientists that can be incorporated into better nanomaterial design
and synthesis advancing nanotherapeutic creation.
To further establish the utility and relevance of the orthotopic zebrafish xenograft assay,
the model PI 3-kinase inhibitor LY294002, known to inhibit glioblastoma proliferation, migration
and invasion in rodent models, was also evaluated (Han et al., 2010; Joy et al., 2003; Su et al.,
2003). Exposure to 6.25 uM LY294002 significantly inhibited glioblastoma proliferation,
migration and invasion demonstrating that the embryonic zebrafish xenograft model is predictive
and sensitive at detecting exposure dependent decreases in glioblastoma progression. The
LY294002 exposure demonstrates that the model is efficacious in chemotherapeutic prioritization
as well as nanotherapeutic. Within 3-4 days, compounds can be evaluated for efficacy, thereby
bridging economical fast in vitro testing with low throughput in vivo mammalian assays. Some of
the same challenges associated with assessing nanotoxicity exist for assessing nanotherapeutic
efficacy in the zebrafish xenograft assay. Agglomeration and dissolution affect nanomaterial
bioavailability. However, experimental conditions can be maintained across the toxicity and
therapeutic efficacy assessments, which is very important for understanding the results of
nanomaterial testing.
Future research will focus on determining the mechanisms of nanomaterial toxicity, which
is needed to make the results translatable to other species. These studies will require incorporating
methods of quantifying nanomaterial uptake to ascertain targets of toxicity. Incorporating systems
biology approaches to learn about nanomaterial effects at the transcriptional, protein and metabolic
level will also aid in understanding the mode of action. This information will improve the
mathematical model predicting nanomaterial toxicity. Furthermore, the mathematical model can
be applied toward investigating the influence of positively charged functional groups, aspect ratio,
121
shape or other systematically altered physicochemical properties on biological response to predict
nanotoxicity. This structure activity based data will be useful in customizing nanomaterials for
medical applications to reduce adverse systemic effects while enhancing targeted cancer toxicity.
To enhance the translational relevance of the orthotopic zebrafish xenograft model, future research
will require confirming that other glioblastoma cell types respond similarly to U87MG.
Transplanting primary glioblastoma stem cells in the zebrafish brain will also enhance the utility
of the xenograft assay. Applying CRISPR/Cas9 to not only genetically manipulate the expression
of proteins hypothesized to be important in human glioblastoma progression, but also to
manipulate the expression of endogenous zebrafish proteins will also help us understand how
microenvironment is contributing to glioblastoma progression and contributing to therapeutic
resistance.
Overall, this dissertation demonstrates how novel methodologies incorporating the
zebrafish model can be used to quickly and efficiently advance applications in nanomedicine by
bridging toxicity and therapeutic efficacy assessments.
122
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APPENDICES
140
Appendix A
A.1 Methods
A.1.1 MWNT Characterization.
TGA was used to determine the purity of the acid treated starting material and ensure
negligible contribution of any residual metal catalysts to the observed zebrafish response using a
Setsys 16/18 system under flowing air in the temperature range of 200-1000 °C (10 °C/min ramp
rate). A high degree of purity and minimal redox activity was observed for both starting material
samples.(Gilbertson et al., 2014)
XPS was utilized to measure the total surface oxygen content. Method details are outlined
elsewhere.(Gilbertson et al., 2014) A PHI 5600 XPS system (1253.6 eV) with a high energy
electron energy analyzer, operating at constant pass-energy (58.7 eV), scan rate of 0.125 eV/step
(50 ms/step), and consistent spot size was used to collect data at multiple locations on the prepared
sample.
Commercially available CasaXPS software and peak integration was used for
quantification of total oxygen.
Light scattering techniques enabled quantification of the dispersed aggregate size
distribution and morphology, as the fractal dimension, in solution. Although the data was collected
in DI water rather than the biological media, it has been previously shown that the relative trend
of both aggregate size and fractal dimension is maintained in increased ionic strength
solvents.(Pasquini et al., 2013) The relative trend of these properties is more important to inform
the statistical model rather than the absolute value. Details of sample preparation and data
collection are described elsewhere.(Pasquini et al., 2013, Pasquini et al., 2012) Briefly, a multidetector ALV-Gmbh goniometer was used to collect both DLS (90°) and SLS (17-153°) data. For
DLS, 500 10-second measurements were collected for each sample and the raw correlation
141
functions exported and analyzed in MATLAB and Fortran using the CONTIN algorithm.(Pasquini
et al., 2013) The most probable aggregate size (Table 2) was determined from the peak of the
resulting probability distribution as previously described by Pasquini, et al.(Pasquini et al., 2012)
For SLS data collection, multiple iterations were performed for each sample to optimize the linear
fit of the data and the fractal dimension determined from the slope of the best fit line according to
the known relationship between the scattered light intensity, I, and the wave vector, q, such that
ሑዦሑወ ሗሳ ሥኣ኷ .(Pasquini et al., 2013, Pasquini et al., 2012)
The electrochemical activity was determined by measuring the potential for the MWNTs
to promote the reduction of oxygen using the RDE method. Details of this method are outlined
elsewhere.(Gilbertson et al., 2014, Pasquini et al., 2013) Briefly, a sample MWNT ink was
prepared based on literature methods and deposited onto a glassy carbon electrode (GCE).
Experiments were carried out in triplicate or quadruplicate in oxygen saturated alkaline media
using various electrode rotation rates (400, 625, 900, 1600 and 2500 rpm) and a 5 mV s-1 scan rate.
The measured current was plotted against the potential to produce a polarization curve. The
polarization curve was used to determine the half-wave potential (E1/2), which is representative of
the MWNT activity towards the reduction of oxygen. The compiled characterization results (Table
2) were used to develop a logistic model that explains the observed trend in embryonic zebrafish
response to these various MWNT samples.
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Table A.1. Developmental and toxic endpoints measured at either 24 or 120 hours post
fertilization (hpf). Italicized endpoints were chosen to develop the statistical model
Abbreviation
Effect Description
Time Point (hpf)
MO24
DP24
Mortality at 24 hpf
Delayed development progression at 24 hpf
24
24
SM24
NC24
MORT
Abnormal spontaneous movement at 24 hpf
Notochord Malformations at 24 hpf
Mortality at 120 hpf
24
24
120
TOTAL MORT
Total mortality
120
YSE_
Yolk Sac Edema
120
AXIS
EYE_
SNOU
Axis Malformation
Eye Malformations
Snout Malformations
120
120
120
JAW_
OTIC
PE_
Jaw Malformations
Otic Vessicle Malformations
Pericardial Edema
120
120
120
BRAI
Brain Malformations
120
SOMI
Somite Malformations
120
PFIN
CFIN
PIG_
Pectoral Fin Malformations
Cuadal Fin Malformations
Abnormal Pigmentation
120
120
120
CIRC
TRUN
Abnormal Circulation or Circulatory Malformations
Shortened Trunk
120
120
SWIM
Abnormal Swim Bladder
120
NC_
Notochord Malformations at 120 hpf
120
TR_
Abnormal Touch Response
120
ANY
Any Malformation
TOTAL AFFECTED Total Mortality Combined with Any Malformation
120
120
143
Figure A.1. Pearson correlations between the five measured MWNTs properties
Point of zero charge (PZC) and percent surface oxygen (Oxygen) measurements are highly
correlated (R > 0.9). Due to the linearity between PZC and surface oxygen concentration, only one
of the measures was used as a model predictor. Smaller correlations are observed between PZC
and % oxygen with morphology, aggregate radius (Radius), and electrochemical activity (HWP).
144
Appendix B
Figure B.1. Representative TEM images of each metal oxide nanoparticle synthesis method
Abbreviations: ZnO = zinc oxide, CeO2 = cerium dioxide, SnO2 = tin dioxide, TiO2 = titanium
dioxide.
145
Figure B.2. Heat map of metal oxide nanoparticle toxicity
Heat map displaying the percent prevalence of malformations and mortality resulting from a fiveday embryonic zebrafish exposure to a five-fold concentration response series ranging from 0
50 mg/L metal oxide nanoparticles, bulk controls, and, in the case of zinc, a dissolved zinc
equivalent (0–99 mg/L) ionic control (n = 32 except ZnO NP in water where n = 96). Exposures
were conducted in ultrapure water or embryo medium (EM). The color scale above the heat map
indicates the percent prevalence of a particular endpoint assessed in the zebrafish at 24 h post
fertilization (hpf) or 120 hpf. Abbreviations for endpoints assessed at 24 hpf: MO24 = mortality,
DP = delayed developmental progression, SM = reduced or excessive frequency of zebrafish
146
spontaneous tail coiling, and NC24 = notochord malformation. Abbreviations for endpoints
assessed at 120 hpf: YSE = yolk sac edema, AXIS = abnormal body axis curvature, EYE = eye
malformation, SNOU = snout malformation, JAW = jaw malformation, OTIC = otic vesicle
malformation, PE = pericardial edema, BRAIN = brain malformation, SOMI = abnormal somite
development, PFIN = malformed pectoral fins, PIG = hypo or hyper pigmentation,
CIRC = abnormal circulation or circulatory vasculature, TRUN = shorted body axis,
SWIM = abnormal swim bladder development, NC = notochord malformation, MORT = total
mortality, and AFTD = total dead and malformed larvae.
147
Figure B.3. Zebrafish exposed to zinc oxide nanoparticle
Representative scanning electron microscopy images of 96 hpf zebrafish exposed to zinc oxide
nanoparticle (ZnO NP) and control prepared in ultrapure water for ዣ1–2 h prior to fixation.
148