Qualifying Exam Manuscript

Quantification of carbon nanotube induced inflammation and toxicity in mice brain

Darwin Yang

Advisor:
Markita Landry

Exam Date:
September 27, 2017

Committee:
Roya Maboudian (Chair)
Wenjun Zhang
Ali Mesbah
Lydia Sohn
Abstract

Introduction

The study of the brain on the micron scale is focused on observing interactions between neurons to better
understand the larger, behavioral phenomena that emerge.

A promising new direction in the study of neurotransmitters in the live brain involves the use of carbon
nanomaterials as electrochemical and optical sensors.
Single-walled carbon nanotubes (SWNTs) in particular show promise in the detection of
neurotransmitters. Once non-covalently modified with a polymer such as single stranded DNA (ssDNA),
SWNTs become well dispersed in aqueous solution and exhibit optical activity in the near infrared
spectrum. This photoluminescence is advantageous over traditional fluorophore fluorescence in that it is
non-photobleachable, shows a high Stokes’ shift, and occurs in a region known as the near infrared
window. In this wavelength range, absorbance of photons by water and scattering by biological material
is minimal, allowing for high transmittance in tissue.1 Specific polymer coronas on the nanotube surface
can impart both selective affinities to molecules and signal transduction that manifests as changes in
photoluminescence.
In particular, SWNTs suspended with single stranded DNA consisting of six G and T nucleotide
base repeats, (GT)6, develop a photoluminescent turn on response to the neurotransmitter, dopamine.
Given with the unique optical properties of SWNTs, the (GT)6 SWNT sensor has been proposed for use in
live mice brains to study region specific dopamine signaling between neurons.

While carbon nanotubes are not directly toxic to neurons, they have been shown to induce higher
neurotoxicity in regions of the brain richer in microglia.2 Microglia are the native macrophages of the
brain, responsible for mediating systemic reactions to pathogenic and foreign material in the brain. In
response to environmental stimuli, microglia respond by differentially expressing one of two expression
profiles: the M1 inflammtory phenotype or the M2 anti-inflammatory phenotype.3 These states are
associated with distinct patterns in gene expression and secretion of signaling proteins (Fig 1). One
particular protein of interest is induced nitric oxide synthase (iNOS) which synthesizes the reactive
nitrogen species, nitric oxide (NO). iNOS expression is very closely linked to activation of

NO is involved in inflammatory signaling pathways and has been shown to cause nonspecific cytotoxicity
at high concentrations in brain tissue. Commented [DY1]: citation

Membrane bound protein receptors of microglia respond to structural motifs belonging to pathogens such
as bacteria and viruses, inducing an immune response in which signaling proteins known as cytokines are
released by the cell, spreading this inflammatory response to neighboring microglia. The goal of this work
is to examine and quantify the degree of the inflammatory response induced by carbon nanotubes, to a

1
Chio, L., Yang, D., & Landry, M. (2017). Surface Engineering of Nanoparticles to Create Synthetic Antibodies,
1575, 363–380. https://doi.org/10.1007/978-1-4939-6857-2
2
Bussy, C., Al-Jamal, K. T., Boczkowski, J., Lanone, S., Prato, M., Bianco, A., & Kostarelos, K. (2015). Microglia
Determine Brain Region-Specific Neurotoxic Responses to Chemically Functionalized Carbon Nanotubes. ACS
Nano, 9(8), 7815–7830. https://doi.org/10.1021/acsnano.5b02358
3
Tang, Y., & Le, W. (2016). Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. Molecular
Neurobiology, 53(2), 1181–1194. https://doi.org/10.1007/s12035-014-9070-5
canonical response from the known endotoxin, LPS. The metrics for comparison are release and
expression of cytokines responsible for cell to cell signaling of inflammation.

Conversely, chronically high NO concentrations have been linked to neurotoxicity45 and diminished
dopamine concentration.6

Research studies involving large, multi-walled carbon nanotubes with highly oxidized surfaces can induce
cytokine release in brain tissue.7

Therefore, in studying the adverse effects of nanomaterials in the brain, an important factor to examine is
the systemic immune response mediated by microglia.
To model this effect, we use the SIM-A9 cell line. This cell line was developed from a prenatal
mouse microglia clone which immortalized spontaneously. Subsequent analysis showed the cell line
exhibited many microglial characteristics, such as expression of CD68—a microglia and macrophage
specific membrane protein—and inflammatory response to stimuli such as lipopolysaccharide (LPS)—an
endotoxin derived from gram negative bacteria cell membrane used to provoke an inflammatory response
from immune cells.8 Since it possesses the cellular mechanisms responsible for inflammation, the SIM-
A9 cell line is a good model for gauging the inflammatory response of carbon nanotubes compared to a
canonical response.

Two directions of study arise: 1) the effect of biology on the sensor, 2) the effect of the sensor on
the biology. The former refers to the manner to which biomolecules affect the dopamine sensor, both
structurally and functionally. It is imperative that the ability to selectively react to dopamine is maintained
over a relevant timescale. Herein, I look at the interaction between enzymes that degrade single stranded
DNA and the (GT)6 DNA on the nanotube surface.
Next, it is equally vital to gauge the degree to which carbon nanotubes perturb the local biological
environment. The interaction between SWNTs and cells is gauged using cytokine biomarkers.

Methods

SWNT suspension and characterization

Single stranded (GT)6 DNA and 5’ thiol modified (GT)6 were used as obtained from IDT. The
thiol modification allowed for PEGylation via maleimide PEG, average MW = 5 kDa (Sigma Aldrich).
Briefly, a 100x excess of solid maleimide PEG (5000 Da average PEG molecular weight, Sigma Aldrich)
was dissolved in a 0.5 mg/mL solution of thiolated DNA in phosphate buffered solution (PBS) with 1
mM TCEP to prevent formation of disulfide bonds. The solution was incubated a room temperature for 24

4
Garry, P. S., Ezra, M., Rowland, M. J., Westbrook, J., & Pattinson, K. T. S. (2015). The role of the nitric oxide
pathway in brain injury and its treatment - From bench to bedside. Experimental Neurology, 263, 235–243.
https://doi.org/10.1016/j.expneurol.2014.10.017
5
Block, M. L., Zecca, L., & Hong, J.-S. (2007). Microglia-mediated neurotoxicity: uncovering the molecular
mechanisms. Nature Reviews Neuroscience, 8(1), 57–69. https://doi.org/10.1038/nrn2038
6
Wegener, G., Volke, V., & Rosenberg, R. (2000). Endogenous nitric oxide decreases hippocampal levels of
serotonin and dopamine in vivo. British Journal of Pharmacology, 130(3), 575–80.
https://doi.org/10.1038/sj.bjp.0703349
7
Bardi, G., Nunes, A., Gherardini, L., Bates, K., Al-Jamal, K. T., Gaillard, C., … Kostarelos, K. (2013).
Functionalized carbon nanotubes in the brain: Cellular internalization and neuroinflammatory responses. PLoS
ONE, 8(11). https://doi.org/10.1371/journal.pone.0080964
8
Nagamoto-Combs, K., Kulas, J., & Combs, C. K. (2014). A Novel Cell Line from Spontaneously Immortalized
Murine Microglia. J Neurosci Methods, (Serres 1985), 759–785. https://doi.org/10.1146/annurev-cellbio-092910-
154240.Sensory
hours with gentle shaking to allow for the reaction to run to completion. Polyacrylamide gel
electrophoresis was performed to ensure complete PEGyation.
Solid HiPco SWNT (NanoIntegris) was added to a 1 mg/mL solution of ssDNA a 2:1 mass ratio.
This mixture was sonicated using a Cole Parmer ultrasonicator with 5 V power output for 10 min. The
resulting suspension was centrifuged at 16,100×g to remove unsuspended, solid nanotubes. Free DNA
was then removed from solution through centrifugal filtration with a 100 kDa mass cutoff filter (Millipore
Amicon). Three consecutive filtrations were performed to ensure complete removal of unbound DNA
before recovery of sample. The final concentration was determined using the absorbance at 632 nm and a
known extinction coefficient.9
Suspensions were diluted to 5 mg/L and 100 µL was placed in wells of a 96-well plate for
fluorescence characterization. Fluorescence spectra were taken using 721 nm excitation across the
wavelength range from 800 to 1400 nm. Response to dopamine was probed by collecting spectra before
and after adding 10 µL of 1 mM dopamine.

Single molecule microscopy

Single molecule microscopy using the cyanine fluorophore Cy5, was used to evaluate the
integrity of the nanotube’s DNA corona in response to nucleases that catalyze the hydrolysis of
nucleotides. SWNTs were suspended with 5’ Cy5 tagged (GT)15 DNA (IDT) following the same protocol
as detailed above. Presence of Cy5 conjugate prevented sequences shorter than 30 bases from suspending
nanotubes in aqueous solution.
(GT)15-Cy5 SWNT were surface immobilized in microfluidic channels using a BSA-biotin and
NeutrAvidin surface immobilization protocol (Fig XXa).10 Cy5 was imaged using a total internal
fluorescent reflectance (TIRF) illumination to increase signal to background ratio. Concentration of
DNA-SWNT was tuned such that well dispersed fluorescent immobilized signals were observed. An
oxygen scavenging imaging buffer was used to promote photostability and minimize bleaching effects
from oxygen.11 Initial images of each channel were collected, sampling across random areas of each
channel to account for distributions in surface coverage.
S1 nuclease (Promega) was diluted to 100 nM and 1 μM concentrations. SIM-A9 cell lysate was
prepared by adding Native lysis Buffer (Abcam) to a pellet of cells at a ratio of 0.5 mL buffer to 1×107
cells. The cells were dispersed and the mixture was put on ice for 30 min. The resulting solution was
clarified by centrifugation at 10,000×g for 10 min. The total protein concentration was determined by UV
absorbance to be approximately 7.2 mg/mL. S1 nuclease or lysate were flowed through channels and
incubated at room temperature for 15 min followed by flow of imaging buffer to remove enzymes and
reproduce imaging conditions. Again, single frame captures were collected across the fluid channels.
A separate control was run to test catalytic activity of S1 nuclease and cell lysate which utilized
5’ biotinylated (GT)15-Cy5 in lieu of the nanotube suspension (Fig XXb). Regions of interest
corresponding to one or more Cy5 molecules were identified using MATLAB image processing. A count
per field of view of these ROIs was determined for each image capture, taken either before or after
enzymatic digestion.

nIR imaging of SWNT internalization

SIM-A9 cells were cultured with DMEM/F12 media supplemented with 10% fetal bovine serum,
5% horse serum, and 1% antibiotics. Cells were plated at a density of 0.1×106 cells per well in a 12-well
plate. Cells were incubated at 37°C and 5% CO2 overnight to allow the cells to adhere to the surface.

9
Bisker, G., Dong, J., Park, H. D., Iverson, N. M., Ahn, J., Nelson, J. T., … Strano, M. S. (2016). Protein-targeted
corona phase molecular recognition. Nature Communications, 7, 10241. https://doi.org/10.1038/ncomms10241
10
Chio, L., Yang, D., & Landry, M. (2017). Surface Engineering of Nanoparticles to Create Synthetic Antibodies,
1575, 363–380. https://doi.org/10.1007/978-1-4939-6857-2
11
Rasnik, I., McKinney, S. A., & Ha, T. (2006). Nonblinking and long-lasting single-molecule fluorescence
imaging. Nature Methods, 3(11), 891–893. https://doi.org/10.1038/nmeth934
Complete media was replaced with sera free media prior to SWNT incubation. Both (GT)6 and PEG-
(GT)6 SWNT suspensions in PBS were added to respective wells to a final concentration of 1 mg/L. After
the desired incubation time (0.5, 1, 2, or 4 hours), cell supernatant was removed and replaced with fresh
sera free media. Live cells were imaged on a custom epifluorescence microscope setup. Brightfield
transmitted light images were taken using LED illumination and an Axiocam CMOS camera. Near-
infrared fluorescence images were collected with a Princeton Instruments InGaAs array under 721 nm
illumination. Transmitted light and nIR images were overlaid using MATLAB. Areas of nanotube
fluorescence were colocalized to corresponding cells. The mean intensity values from these regions of
interest were used to quantify the amount of nanomaterial internalized by each cell.

LDH cytotoxicity assay

Lactate dehydrogenase (LDH) is a cytosolic enzyme involved in cellular metabolism. Damage to
the cell membrane (e.g. cell death) causes release of the enzyme into the surrounding media. The
concentration of extracellular LDH can then be related to the enzymatic activity of the cell supernatant
using a colorimetric reaction involving NADH—a product of LDH activity—which catalyzes the reaction
of tetrazolium salt to formazan (red). Damage to cells induced by a stimulus such as SWNT, was
determined through comparison of formazan concentration to that of controls exhibiting: 100% viability
or no perturbation, and 0% viability (addition of lysis buffer). Hydrogen peroxide (1 mM, Sigma Aldrich)
was used as a positive toxicity control.
SIM-A9 cells were plated in a 96 well plate at a density of 10,000 cells per well.

Inflammation biomarker assays

In response to pathogen associated
Cells were plated in a 96 well plate at 40,000 cells per well and incubated at 37°C, 5% CO2
overnight to allow cells to adhere. Media was replaced with 120 μL sera free DMEM/F12. 5 μL aliquots
of SWNT, LPS or PBS (control) were added to corresponding wells. Samples were incubated for 24
hours to allow for analyte concentrations in cell supernatant to increase above detectable limits. 110 μL
from each well was taken and spun on a benchtop centrifuge to remove any cells or debris. From this, 100
μL was taken for assays of signaling proteins and reactive nitrogen species.

The concentration of nitric oxide produced by microglia upon activation was indirectly assayed
using the Griess test, a colorimetric reaction involving the nitrite ion (NO2-), a relatively inert end product
of nitric oxide. Cells were plated in a 96-well plate at 40,000 cells per well. Suspensions of SWNTs were
added to sera free media alongside lipopolysaccharide (Sigma Aldrich) at a concentration of 5 ng/mL,
serving as a positive control for inflammation.

RT-qPCR
Gene expression levels were evaluated through quantitative analysis of the concentration of gene
specific mRNA relative to a housekeeping gene. The reference gene, phosphoglycdrate kinase (Pgk1),
was selected due to its homogeneous expression level not only in response to external stimuli, but also
across different regions of the mouse brain.12
SIM-A9 cells are cultured in the same manner as described above. Lipopolysaccharide or SWNTs
are added to wells at a final concentration of 5 ng/mL or 5 mg/L respectively in sera free media. The same
volume of PBS was added to a separate well to serve as a control with which to reference results. After 4
hours incubation, media was removed and the adherent cells were washed with PBS before total RNA
extraction using a glass fiber and guanidium based kit (Thermo Fisher). The isolated nucleic acids were
treated with DNAse for 15 min at 37°C to digest genomic DNA that may otherwise be amplified in

12
Boda, E., Pini, A., Hoxha, E., Parolisi, R., & Tempia, F. (2009). Selection of reference genes for quantitative real-
time RT-PCR studies in mouse brain. Journal of Molecular Neuroscience, 37(3), 238–253.
https://doi.org/10.1007/s12031-008-9128-9
downstream PCR steps. RT-qPCR was performed in two steps: 1) nonspecific conversion of RNA to
complementary, double-stranded DNA using random DNA primers (Bio-Rad), 2) quantitative PCR using
primers specific to target genes and dye Sybr green (Thermo Fisher) which becomes photoactive once
intercalated in double stranded DNA. Primers were designed for the genes: tumor necrosis factor α
(TNFα), induced nitric oxide synthase (iNOS), interleukin 1β (IL-1β), interleukin 6 (IL-6), and the
housekeeping gene (Pgk1). Table XXX contains a list of all primer sequences and melting temperatures.
Each gene was run in triplicate. Bio-Rad CFX software logged the fluorescence intensity of each
sample with respect to PCR cycle number, a measure of the total DNA content. Change in expression
between housekeeping and experimental genes and samples was calculated from these curves using ΔΔCt
analysis.

Results and Discussion

Suspension of nanotubes with DNA was characterized by fluorescence spectrometry.

Unsuspended SWNTs do not exhibit photoluminescence due to nonradiative energy transfer between
adjacent nanotubes in a bundle. Both of the nanotube suspensions show photoluminescence from 721 nm
excitation and responsiveness to 100 μM dopamine (Fig XXX) indicating well dispersed SWNTs. Single
molecule microscopy of Cy5 tagged (GT)15 showed that ssDNA adsorbed on the nanotube surface is
protected from the effects of biological nucleases; both concentrated S1 nuclease and cell lysate
containing intracellular SIM-A9 proteins (Fig
XXX). Furthermore, (GT)6 SWNT incubated with
cell lysate for 4 hours retained sensitivity to 100 µM
dopamine (Fig XXX). Therefore, extracellular
conditions in the brain which exhibit orders of
magnitude lower nuclease activity will likely have
minimal activity on our nanotube sensors.

Next, I look at the effects of SWNTs on

microglia. SWNTs with various modifications have
been observed to internalize within mammalian
cells13, including macrophages14. One desired
feature of a synaptic neurotransmitter sensor is that
it remains in the extracellular space of the brain
rather than being sequestered within cells. To gauge
this effect in both PEGylated and non-PEGylated
suspensions, I utilized the innate near infrared
photoluminescence of single-walled carbon
nanotubes.
Figure XX: Internalization of (GT)6 (a) and PEG-(GT)6
SWNT into SIM-A9 cells. Nanotube nIR fluorescence shown
in red. c) Mean fluorescence intensity signal from individual
cells

13
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Smith, B. R., Ghosn, E. E. B., Rallapalli, H., Prescher, J. A., Larson, T., Herzenberg, L. A., & Gambhir, S. S.
(2014). Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery.
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 Both DNA SWNT suspensions
internalized readily into SIM-A9 cells over the
course of hours (Fig XXX). The non-
PEGylated (GT)6 sensor showed a higher
degree of internalization at each time step,
measured using the mean fluorescence
intensity of nanotubes colocalized with cells
(Fig XXXb). Furthermore, control experiments
show that PEG-(GT)6 SWNT not only has a
higher initial intensity, but also a higher
relative intensity than (GT)6 SWNT in
response to SIM-A9 cell lysate (Fig XXXc).
This indicates that the difference in signal
measured across the two samples must be due
to higher quantities of internalized
Figure XX: RT-qPCR gene expression fold change of mouse brain slices
incubated with 5 mg/L (GT)6 or PEG-(GT)6 SWNT for 4 hours at room nanomaterial. This presents an advantage of
temperature. PEGylation of the adsorbed DNA as the
neuronal phenomena under study discussed
above all occur in the extracellular, synaptic space. Therefore, uptake of the sensor by neighboring glia
cells will undeniably decrease the maximum photoluminescence change signal that can be attained.
Despite, the spontaneous internalization of nanomaterial on the order of hours, no cytotoxic
effects were observed (Fig XXX). This indicates that nanotubes do not induce cell death over a time span
relevant to dopamine release, a result consistent with the literature.

Future Work

While the magnitude of the immune response induced by SWNTs is small compared to that of a
traditional inflammatory agent, the full response is likely not contained to the realm of inflammation. The
hydrophobic surface of carbon nanotubes nonspecifically interacts with a diverse array of biomolecules
such as proteins and nucleic acids.15 Therefore, to capture the full extent of the SWNT mediated effect, a Commented [DY2]: NEED CITATION
broader, more thorough search is required. Transcriptomic sequencing using next-generation sequencing
(NGS) techniques is capable of probing changes in expression levels of a
Carbon nanomaterials also differ from molecules like LPS in that degradation of SWNTs is
believed to very slow, on the order of weeks. Therefore, a transient of study comparing expression Commented [DY3]: Need citation
profiles of SWNT induced cells to LPS may reveal differing timescales of microglial activation leading to
effects of chronic inflammation.
In tandem with the continued study of how carbon nanotubes affect the complex
microenvironment of the brain, we can consider methods by which to further mitigate the inflammatory
response measured herein. PEGylation of the DNA offered some benefits particularly in preventing
nanotube uptake. One possible avenue in improving the sensor is in heavier PEGylation of the nanotube
surface. DNA packing on the surface of the nanotube is relatively inefficient compared to amphiphilic
molecules such as phospholipids which form neat, micelle-like structures in the nanotube corona.16
Furthermore, the surfactant sodium dodecylbenzenesulfonate (SDBS) has been used to fill void space
between neighboring DNA molecules on a SWNT surface, inducing a wavelength shift in the nanotube
fluorescence spectra. Phospholipids may also exhibit this effect, with or without a corresponding
chromatic shift. Initial evidence suggests that incubation of (GT)6 wrapped SWNTs with a PEGylated

15
NEED CITATION
16
Wu, Y., Hudson, J. A. S., Lu, Q., Moore, J. M., Mount, A. S., Rao, A. M., … Ke, P. C. (2006). Coating single-
walled carbon nanotubes with phospholipids. Journal of Physical Chemistry B, 110(6), 2475–2478.
https://doi.org/10.1021/jp057252c
phospholipid (16:0 DSPE) does so, protecting the nanotube surface from further adsorption of SDBS (Fig
XXX). This suspension further retains responsivity to dopamine, indicating stability of the DNA in
response to the presence of phospholipid.

References

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