Single Walled Carbon Nanohorns as Photothermal Absorbers, and Incorporation of Spatial Digital ...

115 Pages·2013·7.44 MB·English

Single Walled Carbon Nanohorns as Photothermal Absorbers, and Incorporation of Spatial
Digital Image Analysis into Cancer Diagnostics and Therapy

Jon Robert Whitney

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in
partial fulfillment of the requirements for the degree of

Doctor of Philosophy
In
Mechanical Engineering

Marissa Nichole Rylander
John Robertson
Christopher Rylander
Mark Paul
Richard Walker

April 3, 2013
Blacksburg, Virginia

Keywords: Nanotechnology, Cancer, Photothermal, Digital Image Analysis
Single Walled Carbon Nanohorns as Photothermal Absorbers, and Incorporation of Spatial
Digital Image Analysis into Cancer Diagnostics and Therapy

Jon Whitney

ABSTRACT

Background: Photothermal therapy is an actively researched cancer treatment alternative due to
its potential as a minimally invasive treatment with fewer health complications than high energy
radiation therapies. However, traditional experimental analysis methods used to assess
photothermal therapy lack spatial assessment needed to assess photothermal therapy.

Goals: The first goal is to determine the therapeutic impact of combining Single Walled Carbon
Nanohorns (SWNHs) with photothermal therapy. The second is to advance the measurement
tools used to assess photothermal therapy by developing viability measurement methods which
incorporate detailed quantitative spatial information

Methods: Photothermal therapy was tested with and without SWNHs in in vitro cell monolayers,
in vitro tissue phantoms, and ex-vivo tissue. Digital image analysis methods were developed
which allowed for spatial viability information to be obtained.

Results: The inclusion of SWNHs dramatically increased the temperatures reached in each
experiment. Digital image analysis methods were shown to quantify spatial viability with a high
degree of accuracy and precision in 2D and 3D. Experimental data indicated that there were
areas of collateral damage (partially treated tissue) surrounding areas of completely treated tissue
ranging which were between 46% and 78% of the completely treated volume. In each case the
heat transfer properties of the experimental system had a large impact on the area of treatment.
Variation in the temperature and viability response of photothermal therapy for specific laser and
nanoparticle treatment parameters was quantified.

Conclusions: This research has brought an experimental cancer treatment procedure from
experiments in cell monolayers to tests in ex-vivo tissue to analyze viability response. The
strengths of photothermal therapy such as its minimally invasive nature, and effectiveness at
killing cells were experimentally demonstrated. This research has also developed the tools
necessary to assess the spatial impact in vitro and lay the foundations for assessing spatial impact
in vivo. These tools may be used to assess other treatments beyond photothermal therapy, and
serve as a basis for improving the analysis of biological systems both in vitro and in vivo.
Table of Contents
Abstract ........................................................................................................................................... ii

List of Figures ................................................................................................................................ iv

Acknowledgements ....................................................................................................................... vii

Attributions .................................................................................................................................. viii

Chapter 1- Introduction ....................................................................................................................1

Chapter 2 – Summary of Specific Aims ..........................................................................................4

Chapter 3 – Specific Aim 1 ............................................................................................................11
SWNH impact on Photothermal therapy

Chapter 4 – Specific Aim 2 ............................................................................................................26
2D Spatial Viability Measurement

Chapter 5 – Specific Aim 3 ............................................................................................................41
3D Spatial Viability Measurement

Chapter 6 – Specific Aim 4 ............................................................................................................58
Arrhenius Comparison of Experimental Models

Chapter 7 – Specific Aim 5 ............................................................................................................82
Ex Vivo Spatial Damage Assessment

Chapter 8 – Conclusions ...............................................................................................................96

Chapter 9 – Non-dimensional Analysis .........................................................................................97

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List of Figures.

Figure 1.1 Electron microscopy images of SWNHs. ........................................................13

Figure 1.2 Absorbance of concentrations ranging between 0 and 0.1 mg/ml of SWNH
dissolved in DI water. .......................................................................................................18

Figure 1.3 Attenuation coefficient as a function of SWNH concentration in media for
varying wavelength. .........................................................................................................19

Figure 1.4 Mass attenuation coefficient of SWNHs in media as a function of wavelength.
............................................................................................................................................19

Figure 1.5 Maximum temperature measured during laser irradiation with a thermocouple
8 mm away from the incident laser beam. .......................................................................20

Figure 1.6 Trypan blue staining of RENCA cells measured 24 hours following varying
treatment conditions. .........................................................................................................21

Figure 1.7 Percent Viability of cells observed by Vi-Cell in 24 well well. .....................22

Figure 2.1 Diagram illustrating the BEAST method. ....................................................28

Figure 2.2 Stages in the image analysis process. .............................................................31

Figure 2.3 Example of spatial temperature and viability mapping. .................................32

Figure 2.4 Description of temperature analysis .................................................................33

Figure 2.5 Measured viability and digital viability maps. ...............................................35

Figure 2.6 Viability response as a function of radial distance and temperature. ..............36

Figure 2.7 Viability analysis accuracy ...............................................................................37

Figure 3.1 Diagram of phantom irradiation experimental setup using a ytterbium fiber
laser with a wavelength of 1064 nm and irradiance of 3.8 W/cm2. .................................44

Figure 3.2 The SPECTRL digital image analysis process. ..............................................45

Figure 3.3 Illustration of analysis structure. ....................................................................46

Figure 3.4 Illustration displaying the radial measurement of the size of the kill zone
transition zone and the untreated viability. ......................................................................47

Figure 3.5 Fluorescent image mosaic of treated area in phantom. ...................................49
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Figure 3.6 Normalized percent viability (solid lines) and temperature (dashed lines) as a
function of radial distance for varying durations of laser exposure. ................................50

Figure 3.7 Kill, transition, and kill + transition zone volumes calculated from vertical
DVR analysis. ..................................................................................................................52

Figure 3.8 Demonstration and validation of 3D viability measurement. .........................53

Figure 3.9 Volume of kill and transition zones and comparison between projected 2D
analysis and measured 3D analysis. .................................................................................54

Figure 4.1 The method used to generate isothermal and photothermal Arrhenius
parameters. ......................................................................................................................62

Figure 4.2 Methodology used to obtain the spatial comparison between Arrhenius
generated spatial viability predictions, and spatially measured viability response. ........65

Figure 4.3 Viability measured response and predictions based on the best fit Arrhenius
parameters for each experimental system. .........................................................................68

Figure 4.4 Predicted viability response demonstrating the impact of cellular
microenvironment on Arrhenius parameter generated viability predictions. ..................69

Figure 4.5 Predicted viability for both cell monolayers and phantoms demonstrating the
impact of heating method on Arrhenius parameter generated viability. ..........................71

Figure 4.6 Temperature as a function of the Natural Log of the rate reaction constant k. 73

Figure 4.7 Comparison of measured viability response with 2D Arrhenius predictions
made using calculated Arrhenius parameters at 10, 20, 40, 60, and 80 seconds of laser
irradiation. ........................................................................................................................74

Figure 4.8 Comparison of Arrhenius model predictions using spatial photothermal
phantom temperature data to create 2D viability response predictions and measured
viability response in a photothermally treated phantom. ..................................................75

Figure 4.9 Quantitative analysis of the accuracy of Arrhenius parameters from each
cellular microenvironment and heating method to predict the viability response in
photothermally treated phantoms. ....................................................................................77

Figure 4.10 Reaction rate, k, as a function of temperature for in vitro water bath heated
RWPE-1 and PC3 cell lines based on Arrhenius parameters determined by Rylander et. al
[149]. ................................................................................................................................80

Figure 5.1 Radial temperature at 180 seconds as a function of experimental condition and
bladder. .............................................................................................................................87
v
Figure 5.2 Illustration of bladder treatment and SWNH injection. ..................................88

Figure 5.3 Relationship between peak temperature and histologically determined burn
severity. ............................................................................................................................90

Figure 5.4 H&E stained samples with digitally identified regions of tissue damage
highlighted in dark green. ................................................................................................92

Figure 5.5 Statistical correlation between histologically determined burn size and
measured damage area from digital image analysis. .......................................................93

Figure 6.1 Thermal fluid vortices observed as a result of photothermal heating .............97

Figure 6.2 Illustration of physical setup for six well dish irradiation ................................99

Figure 6.3 Control volume analysis for heat loss ............................................................103

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I would like to thank my advisor Dr. Nichole Rylander for all her support while
conducting this research. I would like to thank my committee members for
providing feedback in producing this work. I would like to thank Dr. John
Robertson for providing clinical expertise and encouragement vital to this
research. I would like to thank my lab mates who have helped me develop my
skills in a biology research lab, as well as improving my own academic
standards. I would like to thank Oak Ridge National Laboratory for providing
the Single Walled Nanohorns used in this research as well as technical feedback.
I would also like to thank all of the undergraduate researchers who have helped
drive this research forward, especially Bill Carswell who has been an invaluable
asset to this team. Other undergraduate research members who have contributed
to this work are Katie Sherman, Amanda Rodgers, Alex Simon, Callie Zawaski,
Erica Harvie, Scott Hopkins, Tom Gilmartin, Rose Phillips, Abbi Jo Bonanno,
Thao Do, Mary Kyle Manson, and Taylor Young.

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Several colleagues aided in the writing and research used in this dissertation.

Dr. Marissa Nichole Rylander is the primary advisor and committee chair for this research. She
provided extensive guidance for research and writing in each chapter of this dissertation.

Chapter 3: SWNH impact on Photothermal therapy

Saugata Sarkar conducted absorption and scattering measurements of SWNHs, and
assisted in editing the manuscript

Jianfei Zhang functionalized SWNHs used in experiments.

Thao Do, Taylor Young, and Mary Kyle Manson assisted conducting viability
measurements. Dr. Thomas Campbell, Dr. Alex Puretzky, Dr. Christopher Rouleau, Dr.
Karen More, and Dr. David Geohegan provided the SWNHs used. Dr. David Geohegan
and Dr. Alex Puretzky provided feedback and editing for the manuscript.

Chapter 4: 2D Spatial Viability Measurement

Tom Gillmartin and Rose Phillips conducted viability measurements, spectrum analysis,
and thermal analysis critical to this work.

Amanda Rodgers provided extensive editorial assistance creating this manuscript.

Erica Harvie helped formulate computer code to align temperature and viability data

William Carswell provided editorial feedback.

Dr. David Geohegan provided SWNHs needed for all photothermal work.

Dr. Chris Rylander provided editorial feedback.

Chapter 5: 3D Spatial Viability Measurement

Matt Dewitt improved the phantom creation process and obtained viability and
temperature response for multiple data points. Provided editorial feedback.

Bryce Whited provided editorial feedback.

William Carswell developed several critical components of the SPECTRL method,
investigated causes of cell death in phantoms, and provided editorial feedback.

Alex Simon investigated vertical temperature distributions in phantoms.

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Callie Zawaski helped create computer code to align temperature and viability data.

Scott Hopkins helped run viability experiments, as well as improve cell survival in
phantoms.

Chapter 6: Arrhenius Comparison of Experimental Models

William Carswell provided extensive computer coding, intellectual, and editorial
feedback in this work.

Katie Sherman obtained viability and temperature data necessary in this work.

Chapter 7: Ex Vivo Spatial Damage Assessment

William Carswell provided editorial feedback, as well extensive computer coding to
develop the viability analysis methods used.

Katie Sherman assisted with conducting and dissecting ex vivo studies, imaging, and
editorial feedback.

Emily Brandon assisted with ex vivo studies, refinement of bladder management, and
injection methods.

Dr. John Robertson provided critical feedback, and evaluated histological samples to
determine viability response, so it could be compared with digital analysis.

Chapter 8: Non-Dimensional Analysis

Dr. Mark Paul provided extensive feedback interpreting non-dimensional parameters and
identifying goals for non-dimensional work, as well as editorial feedback.

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Background
There are over 500,000 cancer deaths in the US annually [1]. A variety of techniques
are used to eliminate tumors from patients including chemotherapy, tumor resection, and
radiation treatments. However, prognosis remains poor despite active research.
Photothermal therapy is an actively researched method which delivers therapeutic heat to
eliminate tumors and prevent recurrence. Photothermal therapy is a minimally invasive
alternative to tumor resection which has the potential to be less damaging to surrounding
healthy tissue than resection or radiation therapy. In photothermal therapy, a region of
tissue is irradiated such that light is absorbed and heat is generated within the targeted
area. The treatment boundaries are therefore defined by thermal diffusion within the
tissue. In order to achieve selectivity, nanoparticles such as Single Walled Carbon
Nanotubes (SWNTs) [2-4], Multi-Walled Carbon Nanotubes (MWNTs) [5-9], Single
Walled Carbon Nanohorns (SWNHs) [10, 11], and Gold nanoshells [12-14] are used as
photoabsorbers to deliver therapeutic heat directly to tumors. Nanoparticles may be
added to tumors through injection, selective uptake, or active antibody targeting. Once
inside a tumor, the high absorption of nanoparticles produces locally generated heat
under Near Infra-Red radiation between 800 and 1064 nm wavelengths [9, 10, 15-17].
The result is selective heating with well-defined treatment boundaries within the tissue.
There are two main components to understanding the mechanism of photothermal
therapy. The first is heat generation and thermal diffusion through tissue which is
regulated by Penne’s bioheat equation:
∂T
Equation 1. ρc =∇⋅[k(T)∇T]−ω(T)c (T −T )+Q(r!,t) [18]
t t ∂t b b a
!"
where ! is the density of the tissue, c is the heat capacitance, is the change in
t t
!#
temperature over time, k(T) is the temperature dependent thermal conductivity, T is
temperature, " is the blood perfusion constant, c is the heat capacitance of the blood,
b b
(T-T ) is the difference in temperature between the blood inlet and outlet, and Q is the
a
heat generation within the tissue volume due to an applied source. Heterogeneity in the
thermal conductivity, heat capacitance, density, and blood perfusion all change the way
heat is conducted through tissue [19]. Therefore the heat diffusion through tissue, and
thus the treatment volume, may be uneven; compromising selectivity.
The second component of photothermal therapy is the biological response to thermal
exposure. This is especially important because cells exposed to high temperatures (above
42 °C) for extended time periods undergo a variety of changes, including protein
unfolding, destruction of organelles, DNA destruction, and tearing of the cellular
membrane [20]. This process is fundamentally chemical, and has therefore been modeled
using the Arrhenius method, resulting in a predictive model of cellular viability in
response to thermal exposure [20]. The Arrhenius model calculates the rate of a chemical
reaction based off of temperature and chemical properties. When applied to cellular
thermal damage, the model assumes that a cellular damage event is caused by a chemical
structure in the cell (organelle, protein, etc.) reaching an activation energy threshold
1