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Multifunctional flourocarbon-conjugated mesoporous silica nanoparticles of varied morphologies to enhance theranostic effects in breast cancer

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Multifunctional flourocarbon-conjugated mesoporous silica nanoparticles of varied morphologies to enhance theranostic effects in breast cancer
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Nelson, Anna Laura M. ( author )
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English
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Nanobiotechnology ( lcsh )
Breast -- Cancer ( lcsh )
Drug delivery systems ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Breast cancer is one of the most prevalent carcinomas in the United States affecting over one million women each year [44]. Current chemotherapy treatments can be especially harsh on the body due to the immense cytotoxicity of the chemo agents and the lack of a targeting modality. Over the past decade, researchers have been utilizing mesoporous silica nanoparticles (MSN) to serve as a drug delivery vehicle in an effort to increase therapeutic effects. Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor tyrosine kinase which is overexpressed in about 25-30 percent of all human breast cancers [15]. Here, we propose the use of Herceptin (anti-HER2 monoclonal antibody, Genentech) as a targeting moiety, in combination with MSN, to develop a targeted drug delivery vehicle for HER2+ breast cancer. Mesoporous silica nanoparticles were chosen to serve as the drug delivery vehicle as they are readily functionalizable, have a porous structure suitable for drug delivery [54, 62]. MSN have been successfully utilized as a platform for multifunctional drug delivery imaging systems. It has previously been shown that MSN-Herceptin produces high-quality ultrasound images; MSN were also shown to aggregate at the site of a tumor tissue and have a longer duration within the body, unlike gas microbubbles. [42]. Gas microbubbles, the current clinical gold standard for ultrasound contrast agents which utilize a liquid perfluorocarbon (PFC) emulsed within a microbubble, have numerous limitations including a low shelf-life and a low systemic circulatory duration [55]. Although amorphous MSN have proven to increase therapeutic and diagnostic effects in breast cancer cells, alternative morphologies may produce greater results. By comparing MSN (e.g. spherical, amorphous, and tube) and with the conjugation of a fluorocarbon, we hope to optimize both the drug delivery efficiency and ultrasound contrast, maximizing the therapeutic and diagnostic effects. A multifunctional, hybrid polymer-nanoparticle system was developed utilizing polyethhylene glycol(PEG), a highly biocompatible polymer, and a perfluorocarbon conjugated to the surface. FT-IR was used to confirm the successful conjugation of each. Preliminary ultrasound images depict a distinct trend between merely the nanoparticle alone versus the hybrid-polymer nanoparticle with a fluorocarbon conjugated to the surface. It was evident that the fluorocarbon conjugated particles emit a higher pixel intensity than its counterpart. Furthermore, all nanoparticles had a clear binding preference for HER2 overexpressing breast cancer cells. In summary, by conjugating a PFC to the surface of the MSN and by analyzing morphology of the particles, both the therapeutic and diagnostic effects were shown to be enhanced.
Thesis:
Thesis (M.S.) - University of Colorado Denver.
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Includes bibliographic references
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Department of Bioengineering
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by Anne Laura M. Nelson.

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Full Text
MULTIFUNCTIONAL FLOUROCARBON-CONJUGATED MESOPOROUS
SILICA NANOPARTICLES OF VARIED MORPHOLOGIES TO ENHANCE
THERANOSTIC EFFECTS IN BREAST CANCER
by
ANNA LAURA, M. NELSON
Bachelor of Science, Furman University, 2012
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Science
Bioengineering
2015


This thesis for the Master of Science degree by
Anna Laura, M. Nelson
has been approved for the
Bioengineering Program
by
Daewon Park, Advisor
Daewon Park, Chair
Bolin Liu
Kendall Hunter
October 19, 2015
n


Nelson, Anna Laura, M. (M.S., Bioengineering)
Multifunctional Flourocarbon-conjugated mesoporous silica nanoparticles of varied
morphologies to enhance theranostic effects in breast cancer
Thesis directed by Assistant Professor Daewon Park
ABSTRACT
Breast cancer is one of the most prevalent carcinomas in the United States af-
fecting over one million women each year [44]. Current chemotherapy treatments can
be especially harsh on the body due to the immense cytotoxicity of the chemo-agents
and the lack of a targeting modality. Over the past decade, researchers have been
utilizing mesoporous silica nanoparticles (MSN) to serve as a drug delivery vehicle
in an effort to increase therapeutic effects. Human epidermal growth factor recep-
tor 2 (HER2) is a transmembrane receptor tyrosine kinase which is overexpressed in
about 25-30 percent of all human breast cancers [15]. Here, we propose the use of
Herceptin@(anti-HER2 monoclonal antibody, Genentech) as a targeting moiety, in
combination with MSN, to develop a targeted drug delivery vehicle for HER2+ breast
cancer.
Mesoporous silica nanoparticles were chosen to serve as the drug delivery vehicle
as they are readily functionalizable, have a porous structure suitable for drug delivery
[54, 62], MSN have been successfully utilized as a platform for multifunctional drug
deli very/imaging systems. It has previously been shown that MSN-Herceptin pro-
duces high-quality ultrasound images; MSN were also shown to aggregate at the site
of a tumor tissue and have a longer duration within the body, unlike gas microbub-
bles [42], Gas microbubbles, the current clinical gold standard for ultrasound contrast
agents which utilize a liquid perfluorocarbon (PFC) emulsed within a microbubble,
have numerous limitations including a low shelf-life and a low systemic circulatory
duration [55]. Although amorphous MSN have proven to increase therapeutic and di-


agnostic effects in breast cancer cells, alternative morphologies may produce greater
results. By comparing MSN (e.g. spherical, amorphous, and tube) and with the
conjugation of a fluorocarbon, we hope to optimize both the drug delivery efficiency
and ultrasound contrast, maximizing the therapeutic and diagnostic effects.
A multifunctional, hybrid polymer-nanoparticle system was developed utilizing
polyethhylene glycol(PEG), a highly biocompatible polymer, and a perfluorocarbon
conjugated to the surface. FT-IR was used to confirm the successful conjugation
of each. Preliminary ultrasound images depict a distinct trend between merely the
nanoparticle alone versus the hybrid-polymer nanoparticle with a fluorocarbon con-
jugated to the surface. It was evident that the fluorocarbon conjugated particles emit
a higher pixel intensity than its counterpart. Furthermore, all nanoparticles had a
clear binding preference for HER2 overexpressing breast cancer cells. In summary,
by conjugating a PFC to the surface of the MSN and by analyzing morphology of the
particles, both the therapeutic and diagnostic effects were shown to be enhanced.
The form and content of this abstract are approved. I recommend its publication.
Approved: Dae won Park
IV


ACKNOWLEDGMENT
I would like to thank several individuals for their continuous support throughout my
experience.
Firstly, I would like to thank my advisor, Dr. Daewon Park for his guidance
throughout my Masters career. This was an amazing opportunity and I have learned
immeasurable skills and lessons in lab technique. I would also like to thank my
other committee members, Dr. Kendall Hunter and Dr. Bohn Liu, for their support,
dedication and willingness towards assisting and offering their expert advice and
resources.
I want to especially thank all of the amazing members of the Translational Bio-
materials Research Laboratory: Melissa Laughter, James Bardill, and David Lee. My
experience would not have been the same without you all.
Several other individuals have helped contribute to my project. First, I would like
to thank Brisa Pena for her continued help and teachings, especially her help with the
fluorescent microscopy. Miranda Intrator, a fellow lab member, helped tremendously
with her guidance and support. I would also like to thank Dr. Trewyns lab at
University of Colorado-School of Mines and the Electron Microscopy lab for their
generous help throughout.
Lastly, I want to reach out to my family. You all know I think youre amazing. I
could not have completed this thesis without all of your love and support throughout
the entire process.
v


Declaration of original work
by
Anna Laura Nelson
This masters thesis was independently composed and authored by myself, using
the support from my advisor, committee members, lab members, fellow students,
and the Department of Bioengineering. The research and ideas presented in this
document originated from the Translational Biomaterials Research Laboratory under
the guidance of Dr. Daewon Park. All resources and funds were provided by Dr.
Daewon Park and the Department of Bioengineering.
Anna Laura Nelson
vi


TABLE OF CONTENTS
Tables......................................................................... x
Figures ...................................................................... xi
Chapter
1. Introduction................................................................ 1
1.1 HER2+ breast cancer.................................................. 2
1.2 Current clinical screening modalities for breast cancer.............. 5
1.3 Current clinical cancer treatments................................... 6
1.3.1 Effects of chemotherapy and drug resistance.................... 6
1.3.2 Monoclonal antibodies.......................................... 7
1.3.3 Herceptin resistance........................................... 9
1.3.4 Personalized therapy may reduce drug resistance................ 9
1.4 Objective of this study............................................. 10
1.4.1 Aims of this study............................................ 11
1.5 Thesis layout....................................................... 11
2. Literature review.......................................................... 12
2.1 Targeted drug delivery modalities .................................. 12
2.1.1 Effect of particle size and morphology ....................... 14
2.1.2 Passive and active targeting.................................. 16
2.2 Ultrasound contrast agents.......................................... 17
2.2.1 Gas microbubbles ............................................. 18
2.2.2 Effect of microbubble size on ultrasound contrast............. 19
2.2.3 Perfluorocarbon emulsed nanoparticles......................... 20
2.2.4 Limitations to gas microbubbles and PFC-emulsed nanoparticles 20
2.3 Pegylation.......................................................... 21
3. Preliminary data and proposed drug delivery Modality....................... 23
3.1 Preliminary work ................................................... 23
vii


3.2 Proposed drug delivery modality..................................... 24
4. Materials and methodology............................................... 27
4.1 Materials........................................................... 27
4.2 MSN preparation .................................................... 27
4.2.1 MSN amorphous preparation..................................... 27
4.2.2 MSN spherical synthesis....................................... 27
4.2.3 MSN tube synthesis............................................ 28
4.2.4 MSN surface hydroxylation..................................... 28
4.2.5 HDI conjugation............................................... 28
4.2.6 Pegylation.................................................... 29
4.2.7 Flourocarbon conjugation...................................... 29
4.2.8 FITC conjugation ............................................. 29
4.2.9 Herceptin conjugation......................................... 30
4.3 Structural Characterization......................................... 30
4.4 Electron microscopy................................................. 30
4.5 Zeta sizer.......................................................... 31
4.6 Ultrasound apparatus................................................ 31
4.6.1 Quantifying pixel intensity................................... 31
4.7 Cell preparation.................................................... 32
4.8 Fluorescent microscopy.............................................. 32
5. Main results and discussion ............................................ 33
5.1 Fluorocarbon conjugation............................................ 33
5.2 MSN characterization................................................ 36
5.2.1 MSN modification analysis by FT-IR.......................... 36
5.2.2 Structural characterization ................................. 39
5.3 Ultrasound imaging.................................................. 42
5.3.1 Conclusions from ultrasound imaging analysis.................. 46
viii


5.4 In vitro studies.................................................... 49
5.4.1 Qualitative remarks on in vitro studies.................... 55
6. Conclusion and proposed future work...................................... 57
6.1 Conclusive remarks.................................................. 57
6.2 Proposed future work................................................ 58
References.................................................................... 60
Appendix
A. Zeta-sizer plots........................................................... 66
IX


TABLES
Table
5.1 The table below depicts the average size of each morphology nanoparticle
concluded from the zeta-sizer. Each NP-HER was pegylated and fluo-
rocarbon conjugated prior to Herceptin (HER) conjugation. Thus, the
MSN-HER depicts the value after all conjugations, MSN-OH-HDI-PEG-
FC-CDI-HER.................................................. 41
5.2 Mean and Standard Deviations for MSN........................ 46
5.3 P-values between each morphology MSN and their fluorocarbon-conjugated
counterparts. P-values are reported for both the F-test and t-tests com-
pleted between each of these groups................................. 46
5.4 The table depicts the incubation time for MSN+cells and seeding density
of HER2 overexpressing cells in each well.............................. 49
x


FIGURES
Figure
1.1 The four members of the HER family of receptors and their respective
signaling pathways. Image A), depicts the dimerization of HER2/HER
family of receptors, which in turn activates various signaling cascades, in-
cluding PI3K/Akt. Image B). and C). depict the monoclonal antibody,
Trastuzumab, binding to the HER2 receptor and its effects can be ob-
served in images E). and F) [28]...................................... 4
1.2 The sensitivity and specihcty of mammography, ultrasound and mam-
mography plus ultrasound. It is evident that by utilizing both screening
modalities, both specificity and sensitivity increase [5]............. 5
1.3 Mechanisms of action for the combined treatment of Trastuzumab and
chemotherapeutic agents. Following treatments, angiogenisis is then in-
hibited, ADCC is initiated and several phenotypic changes are noted [44], 8
2.1 Depiction of disparate targeted drug delivery vehicles a), polymeric
nanogel, b). polymeric micelle, c). gold nanoparticle, d). iron oxide
nanoparticle, e). siRNA entrapped in a liposomal vessel, f). stimuli-
responsive capped MSN [38]................................................. 13
2.2 Effect of particle size on extravasation of blood stream. Smaller particles
tend to have a lower retention within tumor tissue, whereas larger particles
may not be able to traverse the gap between endothelial junctions [21], . 15
2.3 Passive vs. active targeting of drug delivery vehicles. The targeted
nanoparticles have an antibody conjugated on the surface in order to ac-
tively target a known recpetor on malignant cells, whereas non-targeted
particles rely on enhanced permeability and retention effect to target ma-
lignant cells. [21]........................................................ 17
2.4 Common properties of ultrasound contrast agents [33]....................... 19
xi


2.5 The degradation of Optison@microbubbles following the introduction of
a gas-deficient buffer solution after time points a). 0 minutes, b). 1.5
minutes, c). 4 minutes and d). 6 minutes.[48] ............................ 22
3.1 Preliminary work depicting the MPI emitted by amorphous silica nanopar-
ticles [42]............................................................. 23
3.2 Preliminary in vitro studies displaying the colocalization of amorphous
MSN with HER 2 overexpressing breast cancer cell line [42]........... 24
3.3 Proposed mechanism of action of our designed drug delivery vehicle. . 26
5.1 Chemical structure of MSN-OH-FC....................................... 33
5.2 Chemical structure of MSN-OH-HDI-PEG-FC............................... 34
5.3 Images taken of amorphous MSN a), before pegylation and with a higher
molar ratio of the FC used, and b). after pegylation, with a lower molar
ratio of the FC used.............................................. 35
5.4 FT-IR spectra taken of amorphous MSN. Region A depicts confirmation
of free hydroxyl groups following surface hydroxylation and pegylation. B
depicts HDI conjugation and C, D confirm pegylation............... 37
5.5 FT-IR spectra of amorphous MSN. Region A was found to correlate with
fluorocarbon conjugation. Region B depicts C=C bonds found in both
the fluorocarbon and CDI. CDI was further confirmed through regions C
and D, depicting C-N and C=N bonds................................... 38
5.6 Electron microscopy images of MSN for structural characterization anal-
ysis. A-B). TEM images of spherical MSN, C). SEM image of spherical
MSN, D-E). TEM images of amorphous MSN, F). SEM image of amor-
phous MSN, G-H). TEM of tube MSN, I). SEM of tube MSN........ 40
5.7 Ultrasound images taken of each nanoparticle (amorphous, spherical and
tubular) with and without a fluorocarbon conjugated to the surface at
disparate concentrations.......................................... 44
xii


5.8 Overall comparison of the MSN morphologies conjugated to fluorocarbon
(A) and without fluorocarbon conjugation (B)........................... 45
5.9 Analysis of the pixel intensities from the ultrasound studies produced by
A), spherical MSN B). amorphous MSN and C). tube MSN. All image
analysis was performed in Image J. (*) represents statistical significance
between groups......................................................... 47
5.10 Schematic of the in vitro 24-well plate assay. A-FC-HER represents amor-
phous MSN-fluorocarbon and Herceptin conjugated. Similarly, S rep-
resents spherical and T represents tube. A-HER represents amorphous
MSN-Herceptin conjugated (without fluorocarbon)........................ 50
5.11 Images above were taken using Fluorescent microscopy and display all
six MSN samples (shown in Figure 5.10) when incubated with cells at a
density of 100,000 cells/well. Each MSN sample was incubated for either
30 minutes or 2 hours at a concentration of 25/ig/well.Each particle was
labeled with FITC (green) and the cells were stained with DAPI...... 51
5.12 Images above were taken using Fluorescent microscopy and display all
six MSN samples (shown in Figure 5.10) when incubated with cells at a
density of 50,000 cells/well. Each MSN sample was incubated for either
30 minutes or 2 hours at a concentration of 25/ig/well. Each particle was
labeled with FITC (green) and the cells were stained with DAPI...... 52
5.13 Zoomed in images of the MSN + 50K cells/well depicted in Figure 5.12. 53
A.l Zeta-sizer plot performed on spherical MSN. Performed in triplicate. . 66
A.2 Zeta-sizer plot performed on spherical MSN-Her.Performed in triplicate. 66
A.3 Zeta-sizer plot performed on amorphous MSN.Performed in triplicate. . 67
A.4 Zeta-sizer plot performed on amorphous MSN-Her.Performed in triplicate. 67
A.5 Zeta-sizer plot performed on tube MSN.Performed in triplicate........... 67
A.6 Zeta-sizer plot performed on tube MSN-Her.Performed in triplicate. ... 68
xiii


LIST OF ABBREVIATIONS
APTMS 3-aminopropyltrimethoxysilane
ADCC antibody dependent cell cytotoxicity
CSC cancer stem cell
CDI carbonyl diimidazole
CTAB N-cetyltrimethylammonium bromide
DMF dimethylformamide
FITC fluorescein isothiocyanate
FC fluorocarbon
FTIR fourier transform infrared spectroscopy
HC1 hydrochloric acid
HD I hexamethylene diisocyanate
HER2 human epidermal growth factor receptor-2
HER3 human epidermal growth factor receptor-3
IGF-1R insulin-like growth factor 1-receptor
MPI mean pixel intensity
MSN mesoporous silica nanoparticles
MW molecular weight
NP nanoparticle
NK natural killer cell
PFC perfluorocarbon
PBS phosphate buffered solution
PEG polyethylene glycol
PTEN phosphatase and tensin homolog
TEOS tetraethoxysilane
THF tetrahydrofuran
US ultrasound
xiv


1. Introduction
Cancer continues to be a leading cause of death affecting millions worldwide.
Breast cancer is one of the most prevalent carcinomas in the United States affecting
over one million women each year [45]. Current chemotherapy treatments can be
especially harsh on the body due to the immense cytotoxicity of the chemo-agents
and the lack of a targeting modality. Over the past decade, researchers have been
utilizing mesoporous silica nanoparticles (MSN) to serve as a drug delivery vehicle in
an effort to increase therapeutic effects.
Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor
tyrosine kinase which is overexpressed in about 25-30 percent of all human breast can-
cers [15]. An overexpression of HER2 could result in a poor prognosis as lower overall
survival rates have been noted in metastatic breast cancer [46]. However, many meth-
ods are being utilized to directly target and thus downregulate these oncoproteins.
Trastuzumab (Herceptin@, Genentech) is an anti-HER2 monoclonal antibody that
is currently used in clinical practice for patients with HER2-overexpressing (HER2+)
breast cancer. Here, we propose the use of Herceptin as a targeting moiety, in com-
bination with MSN, to develop a targeted drug delivery vehicle for HER2+ breast
cancer.
MSN have been successfully utilized as a platform for multifunctional drug deliv-
ery/imaging systems. For example, MSN conjugated to a fluorophore have been used
for near-infrared fluorescence (NIRF) imaging [63], and MSN alone can be used as
an ultrasound contrast agent for ultrasound imaging. Ultrasound was chosen as the
desired screening modality, as it is relatively inexpensive and can mitigate the risk
of radiation. Mammography serves as the current clinical standard for detection of
breast cancer, however mammography has several setbacks including its high levels
of ionization, its high false positives and its difficulty in detecting malignancies in
dense breast tissues [8, 34], MSNs have been shown to have a longer duration within
1


the body as opposed to gas microbubbles, the current clinical standard in ultrasound
contrast agents [42], It has previously been shown that MSN-Herceptin produces
high-quality ultrasound images; MSN was also shown to aggregate at the site of a
tumor tissue, unlike gas microbubbles [42], Furthermore, a fluorocarbon was conju-
gated to the outer surface of the MSN in order to test its ability to enhance pixel
intensity in ultrasound. The nanoparticles were tested separately but also with a
fluorocarbon conjugated to the surface to determine an optimal multifunctional drug
delivery vehicle.
Although spherical MSN have proven to increase therapeutic and diagnostic ef-
fects in breast cancer cells in vitro, alternative morphologies may produce even greater
results. By varying the morphology of these MSN (e.g. spherical, core-shell, tubu-
lar), we hope to optimize both the drug delivery efficiency and ultrasound contrast,
maximizing the therapeutic and diagnostic effects of the MSN-Herceptin multifunc-
tional platform. These alternative morphologies will be tested in their ability to
emit ultrasound contrast alone but also compared to their fluorocarbon-conjugated
counterpart.
1.1 HER2+ breast cancer
Human epidermal growth factor receptor 2 is part of a receptor tyrosine kinase
family of transmembrane proteins on breast cells. HER2 can function normally when
expressed at low levels and serves to promote growth when appropriate. However,
aberrant expression of HER2 is frequently seen in about 25-30 % of all malignant
breast cancers and is associated with a poor prognosis [28, 58] HER2 positive
breast cancer has been found to be correlated with higher rates of return and overall
survival [52],
The most frequent genetic aberration promoting constitutive cell proliferation and
ultimately tumor formation in breast tissue is HER2 gene amplification [29]. This
gene amplification further results in the HER2 overexpression on breast epithelium.
2


An activated HER family of receptors promote proliferation and cell survival
as a result from the binding of various ligands (typically growth factors) to their
target receptor, triggering the activation of several downstream signaling pathways
[29]. After the ligand is bound to the extracellular domain of a HER receptor, a
conformational change occurs leading to the heterodimerization or homodimerization
with other receptors in its family (HER3, HER4, etc.) [58, 28, 29]. As HER2 does
not bind any of the aforementioned ligands, it serves to activate the other receptors
via heterodimerization [29]. This activation then triggers the phosphorylation of
the downstream effector molecules located on the intracellular domain of the HER
receptors, specifically the tyrosine residues [29]. Upon the phosphorylation of these
downstream molecules, a signaling cascade is then initiated.
Several signaling pathways are initiated following the dimerization of HER2, in-
cluding PI3K/Akt, Ras/MAPK, and the mTOR pathways [29, 26, 40, 47].As HER2-
positive breast cancer inherently has a high expression level of HER2, these pathways
are thus overactive and possibly uncontrolled as the negative feedback loops are dereg-
ulated [29]. It has been found that this dimerization serves as the oncogenic driver,
deregulating the activation of the previously listed signaling pathways. Hence, if this
dimerization were then targeted for inactivation via Trastuzumab, thus the signal-
ing pathways would inherently be downregulated [29].PI3K/Akt pathway has been
shown to inhibit cell death, further promoting cell survival and proliferation [28, 26].
As PI3K is activated, it then binds and phosphorylates PIP2 (phosphatidylinositol-
4,5-bispphosphate) into PIP3 (phosphatidylinositol-3,4,5-triphosphate), located on
the inner plasma membrane [47]. After the phosphorylation of PIP3, the proto-onco-
protein, Akt (protein kinase B) is then activated [40]. This activation of Akt leads to
disparate effects downstream including evading of apoptosis. Following this protein
activation, ultimately the inhibition of the caspase cascade has been reported, thus
leading to cell survival [40]. Hence, it is evident that the malignant phenotype would
3


favor constitutive activation of this given pathway in order to obtain the aforemen-
tioned advantageous characteristics [29].
Figure f.f: The four members of the HER family of receptors and their respective
signaling pathways. Image A), depicts the dimerization of HER2/HER family of
receptors, which in turn activates various signaling cascades, including PI3K/Akt.
Image B). and C). depict the monoclonal antibody, Trastuzumab, binding to the
HER2 receptor and its effects can be observed in images E). and F) [28].
Aberrant overexpression of HER2 has been correlated with promoting carcinogen-
esis and thus serves as a poor prognostic factor [58]. Not only does HER2 overexpres-
sion occur in the primary tumor site, but it also occurs in the secondary metastatic
site [58]. Thus, this receptor serves as an excellent biomarker to target for inhibiting
tumor proliferation and thus tumor progression in HER2 + breast cancer.
4


1.2 Current clinical screening modalities for breast cancer
There are several imaging modalities, which are currently used for the clinical
detection breast cancer including mammography, MRI and high-frequency breast ul-
trasound [36]. Mammography is utilized as the current clinical standard for the
detection of breast cancer; however clinicians are progressively becoming more aware
of the disparate setbacks.
Mammography has difficulty detecting malignant tumors in younger women with
a denser breast tissue and shows to have a low sensitivity for such women [8, 34],
Magnetic resonance imaging (MRI) may serve as a more attractive imaging modality
for denser breast tissue yet proves to be more expensive [34], MRI, in comparison to
mammography and breast ultrasound, has also been shown to maintain a high sensi-
tivity, specificity and positive predictive value for detecting breast cancer, especially
to those with a genetic predisposition [36].
Specificity
1.0 0 8 0.6 0.4 0.2 0
Figure 1.2: The sensitivity and specihcty of mammography, ultrasound and mam-
mography plus ultrasound. It is evident that by utilizing both screening modalities,
both specificity and sensitivity increase [5].
5


Ultrasound serves as an attractive screening modality as it is relatively inexpen-
sive, renders images in real-time and can mitigate the risk of radiation, as opposed
to screening via mammography [57]. Ultrasound is frequently used to render live im-
ages, thus is utilized clinically to direct biopsies and thus has high potential to serve
as a prevalent screening modality [61]. As it is also moveable, it has the capability
to image a patient bedside, which proves to be a unique characteristic as compared
to alternative imaging modalities. However, further augmentation of this particular
modality is immensely needed as there continues to be numerous pitfalls when utiliz-
ing ultrasound as a primary screening method, including high rates of false positives
and negatives [57].
1.3 Current clinical cancer treatments
1.3.1 Effects of chemotherapy and drug resistance
Chemotherapy has been utilized for treating cancer for several decades. Chemother-
apeutic agents typically inhibit cell proliferation by targeting disparate proteins in-
volved in the cell cycle, and thus further promoting apoptosis. As these chemother-
apeutic agents target and inhibit the cell cycle, a normal cell process, there prove to
be debilitating side effects from these drugs. From the patients perspective, the most
frequently reported side effects include: hair loss, nausea and fatigue amongst a few
[4]-
Chemotherapy has been known to eradicate most cancer cells, yet could pos-
sibly leave remaining a more resistant cancer cell. Drug resistance is a frequently
occurring obstacle in current clinical treatment for cancer as many patients are diag-
nosed with malignancies more than once [23]. It has been postulated that following
chemotherapy, persisting cancer stem cells could potentially have the capacity to pro-
mote regeneration of a tumor [18]. The derivation of such cancer stem cells (CSC)
has yet to be deciphered. Two key hypotheses of origination include 1) if the CSC
originates from a malignant transformation of a normal stem cell or 2) if the CSC
6


originates from a malignant cell, which de-differentiates into acquiring stem cell-like
characteristics [18].
When assessing a clinical perspective, there are several posed mechanisms for
developing drug resistance including elimination of the drug or developing a mutation
of the drugs target [23]. Frequently, cells which develop a resistance for one drug often
have resistance toward multiple drugs (i.e. multidrug resistant) [18]. Thus, these
remaining CSC could potentially promote a new tumor growth following treatment
of chemotherapy, ultimately leading to developing a colony of multidrug resistant
tumor cells.
1.3.2 Monoclonal antibodies
Herceptins high affinity for HER2 is a direct cause from the two antigen-specific
sites which target the extracellular domain of HER2 [28, 53]. Following the binding of
Herceptin to its receptor, there are several phenotypic changes including: inhibition
of dimerization, antibody-dependent cell cytotoxicity and induction of cell apoptosis
and inhibition of angiogenesis [28].
Perhaps the most prevalent mechanism in which Herceptin mitigates cell signaling
is the inhibition of HER2/HER3 dimerization. Furthermore, as Herceptin alleviates
the HER2 heterodimerization, its downstream signaling pathway, the PI3K/Akt path-
way, is thus suppressed. This in turn inhibits cell growth and survival, further pro-
moting cell apoptosis [60]. It has also been suggested that the protein (Phosphatase
and tensin homolog) PTEN may be involved in Herceptins ability to downregulate
the PI3K/Akt pathway by regulating the intracellular levels of phosphatidylinositol-
3,4,5triphosphate in cells [12, 6]
Antibody dependent cell cytotoxicity (ADCC) proves to be an imperative mech-
anism of action for how monoclonal antibodies ultimately inhibit tumor formation.
ADCC involves the recognition of the Fc region on an antibody by a receptor located
on the surface of a natural killer cell (an effector cell of immune system) [16]. Fol-
7


lowing the binding of the Fc region to the receptor, the NK cells release proteins and
proteases, causing the lysis of the targeted cell [16]. It has been found that following
the treatment of Herceptin, an increase of NK cells inhitrating the tumor was ob-
served [3]. Furthermore, these NK cells demonstrated an expression of the FcyRIII
receptor (CD16), confirming the cells were lysed via the ADCC mechanism (as CD16
is the receptor on NK cells responsible for binding to the Fc region) [3].
Figure 1.3: Mechanisms of action for the combined treatment of Trastuzumab and
chemotherapeutic agents. Following treatments, angiogenisis is then inhibited, ADCC
is initiated and several phenotypic changes are noted [44],
Lastly, angiogenesis, the formation of new blood vessels from pre-existing ones,
was found to also play a key role in the inhibition of tumor growth following the
treatment of Herceptin. Herceptin was shown to mitigate the blood vessels diameter
and volume, yet did not affect the length of the vessel [30]. These findings were
compared to breast tumors without Herceptin treatment. Thus, as angiogenesis was
significantly reduced, tumor growth was then slowed and more normal vasculature
8


was observed [30].
1.3.3 Herceptin resistance
Majority of the patients who initially respond to Herceptin acquire resistance on
average a year following the initial treatment. The mechanisms of resistance have
yet to be elucidated, yet several potential mechanisms include: steric hindrance of
the targeted receptor-ligand, heterodimerization of HER2/insulin-like growth factor-1
receptor (IGF-IR), and constitutively active signaling [46, 45].
The first possible mechanism for developing Herceptin resistance would involve
the steric hindrance of a glycoprotein, MUC4, which binds to HER2 further inhibiting
the binding of Herceptin and HER2. Furthermore, as MUC4 essentially acts as a
ligand for HER2, it thus activates its downstream signaling pathway and effecter
molecules [45].
Crosstalk between IGF-IR and HER2 were shown to also activate the HER2
downstream pathway. After inhibiting IGF-IR in Herceptin resistant cell lines via
utilizing a monoclonal antibody specific for that particular receptor, Herceptin sensi-
tivity was resumed [46]. Both aforementioned mechanisms result in the activation of
the downstream signaling pathways, PI3K/Akt, which plays a key role in Herceptin
resistance. As trastuzumab acts to inhibit the activation of this pathway, constitutive
activation of PI3K/Akt (caused by listed mechanisms) can then ultimately lead to
evading apoptosis of the targeted malignant cells [45].
1.3.4 Personalized therapy may reduce drug resistance
Genome sequencing has contributed to a greater understanding of cellular aber-
rations in many diseases including cancer. These enlightenments have led to the
development of targeted drugs, such as monoclonal antibodies or tyrosine kinase in-
hibitors, which are specific to cell biomarkers [24], Thus, this more personalized
treatment requires the tumor characterization of each patient. Furthermore, as there
9


exists breast cancer tumors which do not overexpress HER2, the response rate to
treating these tumors with Herceptin would be minimal.
As previously stated, acquired drug resistance continues to be a significant clin-
ical limitation to chemotherapy but also monoclonal antibodies, such as Herceptin.
The mechanisms of chemotherapeutic and Herceptin drug resistance were discussed
previously. It was contributed to that of a tumors immense heterogeneity of genetic
aberrations that a tumor can continue to proliferate despite treatments [24]. Gonza-
lez et. al (2013) proposed a solution to mitigate this inherent challenge could be a
combinatorial treatment including all targeted molecular therapies fitting the tumor
profile.
Utilizing personalized therapy exhibits great potential for overcoming initial
chemotherapeutic and Herceptin resistance. As this drug delivery vehicle serves to
target the malignant cells, it theoretically has the capacity to increase the thera-
peutic effects of undergoing these harsh treatments. The non-malignant cells should
theoretically not be receiving chemotherapeutic treatment and thus should remain
with normal function. Drug delivery could potentially upregulate the immune system
when compared to current chemotherapy treatment. By utilizing a vehicle, it could
inhibit the immune cells from being attacked by the chemotherapeutic agents.
Furthermore, by utilizing a drug delivery vehicle with combinatorial treatments
targeting the entirety of a tumors genetic aberrations, the tumor can theoretically
be attacked on multiple levels.
1.4 Objective of this study
The main objective of the study was to develop and optimize a multifunctional
drug delivery vehicle to serve as an ultrasound contrast agent and to deliver chemoa-
gents at the site of a tumor. HER2 + breast cancer was chosen as the model for this
vehicle. Three different morphologies of mesoporous silica nanoparticles were utilized
(spherical, hexagonal rod and amorphous) and compared in order to decipher which
10


morphology has the highest capacity to increase therapeutic and diagnostic results.
Furthermore, a fluorocarbon was chosen to enhance the pixel intensity and was conju-
gated to the surface of each morphology MSN. All three morphologies were compared
to each other as well as their fluorocarbon conjugated counterparts.
1.4.1 Aims of this study
The objectives of this study can be summarized as follows:
Specific aim 1: Develop three separate morphologies of MSN (Spherical, hexago-
nal tube and amorphous) and successfully conjugate a fluorocarbon to the surface.
Specific aim 2: Quantify the mean pixel intensities produced by each MSN with
and without fluorocarbon conjugation.
Specific aim 3: Observe any preferential binding of the nanoparticles and HER2+
cells, or any differences in endocytosis of the nanoparticle by the HER2+ cells.
1.5 Thesis layout
Chapter 1 contains an introduction to the scope and main objectives of this thesis.
Chapter 2 presents an overview of targeted drug delivery vehicles, current ultra-
sound contrast agents and their proposed mechanisms, passive vs. active targeting of
drug delivery modalities and Pegylation.
Chapter 3 reviews preliminary data and introduces the proposed design and de-
velopment of this multifunctional drug delivery vehicle.
Chapter 4 covers materials and all methodologies including the synthesis and
surface modifications and all experimental designs used in this study.
Chapter 5 contains results and discussion.
Chapter 6 comprises the conclusions and suggestions for future work.
11


2. Literature review
2.1 Targeted drug delivery modalities
Drug delivery modalities have been discovered, synthesized and utilized to deliver
drugs more efficiently over the course of the last few decades [20]. For the scope of this
thesis, a background on drug delivery vehicles utilized for cancer treatments will be
discussed. Targeted cancer therapeutics were developed to amplify the drug uptake
of the malignant cells and to mitigate drug uptake of non-malignant cells, further
maximizing therapeutic efficiency [1].
Nanoparticles are characterized as less than 1 micron in size and have vast for-
mulations in materials as well as modifications. Several drug delivery vehicles have
been developed from disparate materials, including liposomes, polymer-based, mi-
celles, dendrimers and silicon oxide nanoparticles amongst other materials [1, 59]. As
these nanoparticles are capable of several chemical modifications, many drug deliv-
ery developments have been geared toward synthesizing multifunctional modalities.
Nanoparticles also have the capability to accumulate at the site of the tumor through
enhanced permeability and retention effects as well as through active targeting via
antibodies (explained further in detail below). Surface modifications allow for these
nanoparticles to have prolonged systemic circulation times as well as inhibit nonspe-
cific protein binding [11], Due to this nonspecific protein binding, it has been shown
that a hybrid nanoparticle-polymer system allows for a 10 to 100 fold increase of
drug reaching the tumor as opposed to free chemotherapeutic agents alone [59]. Fur-
thermore, nanoparticles are internalized by the malignant cells through endocytosis,
increasing the killing capacity of the chemoagents reaching the nucleus [59]. There are
several nanoparticles which are currently undergoing clinical investigation, including
liposomal drug delivery systems (i.e. Doxil, Abraxane) or the multifunctional
iron oxide nanoparticles, which serve as an MRI contrast agent as well as drug carrier
(Combidex) [20].
12


Figure 2.1: Depiction of disparate targeted drug delivery vehicles a), polymeric
nanogel, b). polymeric micelle, c). gold nanoparticle, d). iron oxide nanoparticle, e).
siRNA entrapped in a liposomal vessel, f). stimuli-responsive capped MSN [38].
Mesoporous silica nanoparticles (type MCM-41) were first synthesized in the early
2000s and has since been utilized and optimized to serve as an efficient drug delivery
vehicle [56]. Silica has been viewed as an advantageous material as its relatively sim-
plistic condensation reactions can be easily functionalized, it has a porous structure
suitable for drug delivery, is scalable and is cost-effective [54, 62, 56]. There have
been several current studies on optimizing the silica nanoparticles size, morphology,
porosity and surface characteristics [54], In another study, it was determined that
mesoporous silica nanoparticles with higher surface areas serve to be a more ideal
platform as they were shown to have longer systemic circulatory half-lives as well as
13


have the higher capacity to undergo surface modifications [27].
2.1.1 Effect of particle size and morphology
There are several parameters which can determine the systemic circulatory half-
life as well as successful endocytosis of malignant cells as opposed to non-malignant
cells including size, shape, charge and surface hydrophilicity [21, 20, 1]. Thus, by
manipulating these parameters, the ability of the nanoparticles to be internalized in
malignant tissues can be enhanced.
It has been shown that the size of the nanoparticles has an immense effect on its
capability to extravasate the blood stream as well as particle uptake [1, 21]. Smaller
particles have a lower retention within the tumor tissue and thus have the capability
to target non-malignant cells; however, this can be mitigated by utilizing active tar-
geting [1], On the contrary, larger particles (>200 nm) are recognized more readily
by the mononuclear phagocytic system (MPS) and can be removed from the blood
via macrophages. Particle size also affects how the particles are cleared within the
body. It has been found that (non-functionalized) particles of 1-5 microns tend to
accumulate in the liver whereas larger particles than such tend to get trapped in
capillaries [13].
Disparate particle sizes not only have an immense effect on targeting the tu-
mor tissue, but also have an impact on cell internalization. It was found that silica
nanoparticles with a diameter of 50 nm was the most likely to be successfully endo-
cytosed when compared to silica nanoparticles of size 30 nm, 110 nm, 170 nm and
280 nm [39]. It was thus reported that the optimum particle size for cell uptake was
roughly 50 nm, but was also dependent on the surface charge of the particles [39].
Another study observed that despite all nanoparticles between the range of 2-100 nm
having an impact on cell signaling process, the particles with a diameter of 40 and 50
nm displayed the greatest impact [31]. Alternatively, it has also been reported that
the most efficient size for endocytosis was roughly 200 nm and that particles larger
14


Figure 2.2: Effect of particle size on extravasation of blood stream. Smaller particles
tend to have a lower retention within tumor tissue, whereas larger particles may not
be able to traverse the gap between endothelial junctions [21].
than 1 micron were observed to have minimal cellular uptake [54],
MSNs when endocytosed, were shown to undergo dissolution within the lysosome
[11]. It was also determined that the concentration of Silica within the culture media
increased over time, indicating that the cells were consistently excreting Silica ion.
Several studies indicated that mesoporous silica nanoparticles degrade rapidly within
the first two days followed by a slow degradation [11],
It has been shown that particle shape also takes effect both on cell internalization
and extravasation of the blood stream. It was shown that cylindrical cationic PEG-
based particles at all sizes had a higher internalization ( 75%) as opposed to cubic
cationic PEG-based particles [25]. It was also shown that the cylindrical particles
15


with a diameter of 150 nm and height of 450 nm had a faster internalization rate as
opposed to the more symmetrical cylindrical particles (d=200 nm, h=200nm) [25].
In this study, the long rod particles ( 450 nm) were shown to be internalized at a
faster rate than both the shorter rod particles ( 240 nm) and spherical particles ( 100
nm) [27]. Champion et al. concluded that the internalization rate of disparate shaped
particles was determined by where the macrophages initially attached to the particles.
Furthermore, it was determined that an ellipse was endocytosed more swiftly at the
pointed ends as opposed to the flat surface of the particle [13].
2.1.2 Passive and active targeting
Nanoparticles have the capability of undergoing both passive and active targeting.
As blood vessels in tumors tend to be of irregular shape and size, this leaky and dilated
characteristic can be utilized to enhance the permeability of these nanoparticles.
Furthermore, in tumor vasculature the endothelial junctions have been shown to be
larger in size depending on tumor type and grade. This allows for the nanoparticles
to be able to extravasate from the blood stream into the tumor more easily [1]. Many
particles which extravasate into the tumor have an enhanced retention rate as tumor
tissues have a higher tendency to lack lymphatic vessels (or they are shown to be non-
functional) [7]. This enhanced permeability and retention effect has been utilized for
cancer therapeutics and has been shown that encapsulated chemotherapeutic agents
accumulate within a tumor at a rate of 10-100 times higher as opposed to the free
chemotherapeutic [1].
Nanoparticles can be used for active targeting by conjugating a biomarker specifi-
cally seeking a known location [7]. Known biomarkers, such as monoclonal antibodies,
once conjugated to a delivery vehicle will result in a higher drug concentration in the
malignant tumor tissue as opposed to the non-malignant tissue [1]. Furthermore,
active targeting can promote receptor-mediated endocytosis as well as antibody de-
pendent cell cytotoxicity, which will allow for the internalization of the particle, and
16


$ 3$C
Targeted NPs
Non-targeted NPs
Figure 2.3: Passive vs. active targeting of drug delivery vehicles. The targeted
nanoparticles have an antibody conjugated on the surface in order to actively target
a known recpetor on malignant cells, whereas non-targeted particles rely on enhanced
permeability and retention effect to target malignant cells. [21],
thus the drug will either be released at the surface or through endocytosis [3]. Ac-
tive targeting has the potential to enhance early detection as they can then serve
as a noninvasive imaging technique. Furthermore, once successful binding of the
antibody-vehicle device to the desired location, detection of a particular type or stage
(if metastatic) of disease can then be determined [7, 43]. Furthermore, a more mul-
tifunctional, targeted-nanoparticle system, also duo-ing as a contrast agent, could
potentially serve as a noninvasive detection of pathology [7].
2.2 Ultrasound contrast agents
As previously stated, ultrasound could potentially serve as an attractive imaging
modality due to it eliminating the risk of radiation, its relatively cost-effectiveness
and its ability to produce images in real-time [57]. Essentially, an ultrasound image
is the analysis of the acoustic waves being received back after being transmitted
through tissues. Differences in tissues produce disparate acoustic echoes back to the
17


receiver and thus an acoustic image can be detected [41]. Ultrasound has been known
to have several limitations, such as image distortion, low resolution and low contrast
[35, 55]. These distortions, though, could potentially be alleviated with increasing the
contrast of the targeted tissue [35, 55]. Furthermore, contrast agents were designed to
produce a density mismatch between the contrast agent and the surrounding tissues
[48]. There are several developed methods (which will be discussed in greater detail
below) for increasing the contrast of the targeted tissue including gas microbubbles
and perfluorocarbon-emulsed nanoparticles.
2.2.1 Gas microbubbles
Gas microbubbles, the current clinical standard as ultrasound contrast agents,
were first discovered in the late 1960s and were proven to enhance vascular circuitry
lasting only a few minutes before the bubbles were then dissolved [9]. Since then,
many pharmaceutical companies are focusing on optimizing these microbubbles and
have made significant progress in various fields including cardiology and radiology
[22].
Gas microbubbles have a diameter ranging from 1-7 microns, which causes their
resonance frequencies to he within the US frequency range used for clinical practice,
around 2-10 MHz [49, 9]. Microbubbles are comprised of an outer shell (denatured
albumin, phospholipids or surfactants) which encapsulate various gases (e.g. perflu-
orocarbon) [49, 17]. The variation in pixel intensity between the microbubble con-
trast agents and its surrounding tissues is derived from the microbubbles harmonic
vibration, whereas tissues are nearly incompressible [17, 9]. Furthermore, these mi-
crobubbles are then highly echogenic due to this difference in material properties
between itself (compressed gas) and its adjacent tissues. Disparate frequencies are
then produced and detected [7].
It has been found that despite the stark contrast in impedance from the second
harmonic imaging of tissue/gas mismatch, there continues to be a great limitation for
18


Contrast agent characteristics
Size
-microbubble (mean diameter 1-4 mm)
-nanoparticle (mean diameter <1 mm)
Gas composition
-Air or nitrogen
-Sulfur hexafluoride
-Perfluorocarbons (C3F8,C4F10)
Shell composition
-lipid or other lipid-like surfactant
-protein (albumin)
-biocompatible polymers
Current Opinion in Biotechnology
Figure 2.4: Common properties of ultrasound contrast agents [33].
the gas microbubbles. These gaseous bubbles or particles have been found to have a
limited shelf -life of less than one week when stored at 4C [55]. Thus, a more stable
solution has the potential to be a more realistic application.
2.2.2 Effect of microbubble size on ultrasound contrast
The effect of particle size for either microbubble or nanoparticle contrast agents
have recently been studied in order to discover an optimal size producing an impedance
mismatch as well as an optimal circulatory duration within the body. Many current
clinical ultrasound contrast agents were found to have a high polydispersity, such as
Dehnity(Lantheus Medical Imaging, USA), having a range of microbubbles from
one to ten microns [51]. Sirsi et al, following the successful isolation of microbubbles
between specific ranges (1-2 microns, 4-5 microns, 6-8 microns) observed the effects
of the contrast emitted in an in vivo model utilizing a high frequency ultrasound
modality. It was found that the contrast increased with increasing microbubble size.
The duration of the microbubbles in circulation also increased with size [51]. For
practical clinical purposes, an optimal size must be obtained which maximizes this
acoustic backscatter, yet is able to successfully traverse capillaries without causing
any occlusions [48].
19


2.2.3 Perfluorocarbon emulsed nanoparticles
Fluorocarbon nanoparticles have been widely used as an enhanced ultrasound
contrasting agent, however all prior systems have used an emulsion-based system
typically utilizing a liquid, volatile perfluorocarbon [64, 55]. Similarly with gas mi-
crobubbbles, the impedance mismatch between the gas nanoparticles and the sur-
rounding tissues gives rise to the enhanced ultrasound images [55]. Above a certain
pressure applied via the ultrasound, these liquid perfluorocarbon-emulsed nanoparti-
cles are vaporized into their highly echogenic gaseous state [14]. Nanoparticles filled
with perfluorocarbon liquid are known to have a longer lifespan within the blood
stream as compared to gas microbubbles, which have a relatively shorter lifespan
[32, 35],
In other studies elucidating this mechanism, it was found that this ultrasound
vaporization provokes a response propelling the particles forward, at a rate depen-
dent on the characteristics of the fluorocarbon chosen [32], While fluorocarbons or
perfluorocarbons are composed of disparate properties, like boiling point, which may
ultimately decipher its expansion, motion and thus velocity; it has been determined
that fluorocarbons increase pixel intensity in ultrasound due in part to this bullet-like
propulsion [32],
2.2.4 Limitations to gas microbubbles and PFC-emulsed nanoparticles
As previously stated, there exists several limitations to current clinical US con-
trast agents. The first limitation would be the relatively short shelf-life of gas mi-
crobubbles, depending on the material used for encapsulation (i.e. lipid shell vs. solid
shell) [55, 22], Microbubbles composed with lipid shells tend to be less stable, yet
were found to be highly echogenic [22], It was found that these lipid microbubbles,
when undergoing acoustic waves, the shell expands, ruptures, reseals, compresses,
20


buckles and respreads at every cycle [22], This mechanism of action, albeit produc-
ing highly contrast images, is also limited to a relatively short systemic circulatory
lifespan.
Optison (GE Healthcare) is one of the current FDA approved PFC emulsed
ultrasound contrast agent composed of a albumin-coated hollow shell, encapsulat-
ing octafluoropropane [22, 48]. This fluorocarbon-emulsed bubble was found to
have a much longer circulation time than its initial air-encapsulated counterpart
(Albunex), due to the lower solubility properties of the FC [48].This character-
istic was concluded to allow for a higher retention of gas microbubbles in aqueous
solution for a longer duration [48]. Despite this observed increase in ciculatory dura-
tion, the circulation lifespan was still found to be less than fO minutes (Figure 2.5)
after applied pressure from acoustic energy [48]. Figure 2.5 below depicts this re-
duction in the Optison@gas microbubbles following the application of a gas-deficient
buffer solution (was stated to be identical results as for applying acoustic waves) over
several time points.
2.3 Pegylation
By conjugating polyethylene glycol (PEG) on the nanoparticles, the biocompat-
ibility has been shown to be greatly increased as the particles are not affected by
non-specific protein binding [ft]. Polyethylene glycol is an FDA approved hydrophilic
polymer, which is frequently used to modify the surface of nanoparticles [56]. By con-
jugating PEG, the systemic circulatory half-life has been shown to be vastly increased
as opsonization is delayed or prevented, and thus the particles are not rapidly cleared
by the reticuloendothelial system (RES) [ft, 56]. It was also shown that by function-
alizing the surface of the particles with PEG, the degradation rate was slower when
compared to non-functionalized nanoparticles [11]. Unmodified silica nanoparticles
were shown to lose its structural characteristics (i.e. porosity) following 1 month of
the study [11].
21


Figure 2.5: The degradation of Optisonmicrobubbles following the introduction of
a gas-deficient buffer solution after time points a). 0 minutes, b). 1.5 minutes, c). 4
minutes and d). 6 minutes. [48]
With the addition of PEG comes great biocompatibility enhancements, but also
has other known benefits for clinical applications. It has been found that by conju-
gating a certain percentage of polyethylene glycol, the nanoparticle-polymer system
can redisperse in solution following lyophilization more easily than its non-pegylated
counterpart [55].
22


3. Preliminary data and proposed drug delivery Modality
3.1 Preliminary work
Preliminary work has been completed in Dr. Daewon Parks Translational Bio-
materials Research Laboratory performed by A. Milgroom, M. Intrator, and K. Mad-
haven. All preliminary work utilized amorphous MCM-41 MSNs.
Mesoporous silica nanoparticles have been shown to be biocompatible and also
have shown to emit ultrasound contrast. In preliminary studies, polyacrylamide phan-
toms and the amorphous nanoparticles were imaged with various concentrations of
nanoparticles (0.1 mg/mL, 0.3 mg/mL, 0.5 mg/mL and 1.0 mg/mL). Polyacrylamide
was chosen as a phantom for its similarity to soft tissue. Preliminary work performed
on amorphous MSN exhibited higher pixel intensities given higher MSN concentra-
tions within the phantoms.
Figure 3.1: Preliminary work depicting the MPI emitted by amorphous silica nanopar-
ticles [42],
The cellular interaction between amorphous MSN-Herceptin-FITC was observed
using fluorescent microscopy. Comparing the interaction between HER2+ and HER2-
cell lines can reveal the efficiency of MSN-Herceptin-FITC and its preference toward
binding to HER2 overexpressing cell lines. Preliminary work on amorphous MSN has
23


confirmed the binding and internalization of the MSN in HER2+ cells with few at-
tachments to HER2- cells [3]. Flow cytometry was performed to quantify the binding
efficiency of MSN-Herceptin-FITC to HER2+ and HER2- cell lines for amorphous
MSN.
a
Figure 3.2: Preliminary in vitro studies displaying the colocalization of amorphous
MSN with HER 2 overexpressing breast cancer cell line [42].
By further optimizing the morphology and further functionalizing these MSNs,
an optimal multifunctional drug delivery vehicle can then be deciphered. Spherical,
tube and amorphous morphologies were tested and compared for their abilities to
enhance pixel intensity as well as to test for preferential binding towards HER2+
breast cancer cells. Furthermore, the conjugation of a fluorocarbon was tested for its
ability to further augment the pixel intensity in ultrasound imaging.
3.2 Proposed drug delivery modality
Mesoporous silica nanoparticles were chosen to serve as the drug delivery vehi-
cle for their relative ease in synthesis, they are readily functionalizable and have a
porous structure suitable for drug delivery [54, 62], It was posed to vary the shape
of these silica particles and compare each in its ability to serve as an ultrasound
contrast agent as well as compare endocytosis rates between them. Furthermore, as
24


it was previously found that MSN are slightly echogenic alone, it was proposed to
conjugate a fluorocarbon on the surface of each particle in order to further enhance
this echogenicity [42],
It was predicted that the fluorocarbon conjugated particles would follow a similar
mechanism to the PFC-emulsed nanoparticles, as previously noted. Current ultra-
sound contrast agents which utilize a liquid PFC report a phase-transition following
the introduction of acoustic waves to its highly echogenic gaseous state [55, 64], Fur-
thermore, this impedance mismatch between the tissue/gas results in a higher MPI
[8]. As previously stated, the short shelf-life (less than one week when stored at 4C)
of these gaseous particles poses a significant limitation [55].
In order to further increase the shelf-life of these particles, yet still preserve the
higher contrast produced by these enhanced particles, it was determined to conjugate
a PFC to the surface of the MSN. It has previously been reported that freeze-drying
gaseous particles (to increase the microbubbles shelf-life) significantly reduces their
echogenic factor and were observed to collapse or fuse during the process [55]. By
conjugating the fluorocarbon to the surface of the particles, the shelf-life would in-
herently increase as the particles no longer have a gaseous core which would have the
potential to collapse.
Not only would conjugating the fluorocarbon to the surface potentially enhance
the shelf-life of the particles, but it was also posed to enhance the duration of systemic
circulation time within the body. Gaseous microbubbles have been reported to have
a relatively short lifespan in circulation, lasting only a few minutes [9]. By circum-
venting a gaseous core, the particles could enhance vascular circuitry. Several studies
indicated that mesoporous silica nanoparticles degrade rapidly within the first two
days followed by a slow degradation [11], This degradation rate is inherently slower
than the rapid degradation of gas micrbubbles.
25


Trastuzumab serves as the targeting modality to
target Human epidermal growth factor receptor-2
KEY
Trastuzumab
P mesoporous silica nanoparticles
Doxorubicin (chemo-agent)
rHuman epidermal growth factor receptor-2
HER2
HER2 overexpressed
Breast cancer cell
plasma membrane
Figure 3.3: Proposed mechanism of action of our designed drug delivery vehicle.
Not only will this proposed drug delivery vehicle have the potential to enhance
diagnostic effects, but it was proposed to combine both passive and active targeting of
the malignant cells, which could enhance the therapeutic effects as well. Herceptin, or
Trastuzumab, was chosen as the antibody to be chemically conjugated to the surface
of the nanoparticles. Thus, the antibody would serve to actively target the malignant
cells. By combining active and passive targeting, the proposed modality would have
the potential to mitigate or alleviate drug resistance.
It was first proposed to conjugate the fluorocarbon to the surface of hydroxylated
MSN. A limitation was reached with this step due to the immense hydrophobicity
of the MSN-FC particles. This limitation is discussed in detail in the Results and
Discussion portion of this thesis. To circumvent this limitation, the particles were
Pegylated prior to conjugating the fluorocarbon.
26


4. Materials and methodology
4.1 Materials
MCM-41 type hexagonal MSNs, hexamethylene diisocyanate (HDI), anhydrous
dimethylformamide (DMF), anhydrous toluene and fluoroscein isothiocyanate (FITC)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-cetyltrimethylammonium
bromide (CTAB), 2 M sodium hydroxide (NaOH), tetraethyl orthosilicate (TEOS),
Polyethylene glycol (PEG) MW 1000 and MW 4000, carbonyldiimadazole (CDI), an-
hydrous toluene, anhydrous methanol and were purchased from Alfa Aesar (Ward Hill,
MA, USA). IX Phosphate buffered saline (PBS), Trypsin 0.5 %, DMEM F12 media
and penicillin/streptomyocin and anhydrous diethyl ether were purchased from Fisher
Scientific (Pittsburgh, PA, USA). Pentafluorophenylpropyl-trimethoxysilane was pur-
chased from Gelest. 3-Aminopropyltrimethoxysilane (APTMS) was purchased from
TCI. Hydrochloric Acid (HC1) was purchased from BDI.Trastuzumab, SKBR3 cell
line and Hoechst stain were generous donations.
4.2 MSN preparation
4.2.1 MSN amorphous preparation
MCM-41 type hexagonal MSNs were dispersed in 200 proof ethanol at 5 mg/mL.
The solution was then sonicated for 10 minutes and were filtered using a FRIT flask.
4.2.2 MSN spherical synthesis
In a 1000 mL round bottom flask, CTAB (1 g, 0.00274 mol) was dissolved in
480 mL nanopure water followed by the addition of 2 M NaOH (3.5 mL, 0.007 mol).
The solution was heated to 80 C and rapidly stirred until there is a vortex to the
bottom of the flask. Next, TEOS (9.07 mL, 0.041 mol) was added dropwise into the
solution. The solution was reacted for 2 hours at 80 C with vigorous stirring. The
solution was then filtered using a FRIT flask and was washed with nanopure water
and anhydrous methanol. To remove the surfactant molecule, CTAB, dried spherical
27


MSN were placed in a 250 mL flask and dissolved in 100 mL anhydrous methanol.
The solution was then sonicated for 15 minutes. Next, HC1 (1 mL) was added to the
solution. This was then refluxed for 18 hours at 65 C and rapidly stirred until there
is a vortex to the bottom of the flask. The solution was then filtered using a FRIT
flask and was washed with nanopure water and anhydrous methanol.
4.2.3 MSN tube synthesis
Tube-shaped MSN were synthesized using a similar method as spherical MSN.
In a 500 mL round bottom flask, CTAB (1 g,0.00274 mol) was dissolved in 120 mL
nanopure water followed by the addition of 2M NaOH (1.75 mL, 0.0035 mol). The
solution was reacted at room temperature with vigorous stirring. TEOS (2.5, 0.0112
mol) was added dropwise into the solution. The solution was reacted for 24 hours
at room temperature with vigorous stirring. The solution was then filtered using a
FRIT flask and was washed with anhydrous methanol. The same method for tube
MSN surfactant removal was followed as described in spherical MSN portion.
4.2.4 MSN surface hydroxylation
To hydroxylate the surface of the MSNs, the following procedure was followed:
Dried surfactant-removed MSN were dissolved in nanopure water (200 mL/ g MSN).
The solution was then sonicated for 10 minutes, and reacted for 4 hours at 70 C with
moderate stirring. The solution was then filtered using a FRIT flask and was washed
with nanopure water and anhydrous methanol.
4.2.5 HDI conjugation
Surface hydroxylated, surfactant-removed MSN were dissolved in 5 mL anhydrous
DMF. The solution was then reacted with excess hexamethylene diisocyanate (HDI)
(5 mL, 0.03 mol) at 60 C C for 24 hours with moderate stirring. The solution was
then precipitated in cold anhydrous ether. Once the precipitates fell to the bottom of
the flask, the ether was removed by pouring off the supernatant. Cold anhydrous ether
28


was then added again. The MSN solution was then sonicated for 10 minutes. This
step was repeated twice (supernatant discarded, anhydrous ether added, sonication).
The dispersed MSN solution was then transferred to a 50 mL falcon tube and was
centrifuged for 5 minutes at 5000 rpm. The supernatant was then removed.
4.2.6 Pegylation
The dried MSN-HDI conjugated particles were dissolved in 5 mL anhydrous DMF.
Simultaneously in another flask, PEG (1000 MW) (5 g, 0.005 mol) was dissolved in
2 mL anhydrous DMF and was heated to 60 C C for 5 minutes or until completely
dissolved. Afterward, the MSN solution was then added to the excess PEG solution
and was then reacted at 60 C C for 24 hours. The solution was then filtered using
a FRIT flask and washed with anhydrous toluene. *For tube MSN synthesis, PEG
4000 MW was used.
4.2.7 Flourocarbon conjugation
The dried MSN-HDI conjugated, PEGylated particles were then dissolved in (100
mL) of anhydrous toluene. The solution was then sonicated for 15 minutes. Immedi-
ately following, the fluorocarbon (pentafluorophenylpropyl-trimethoxysilane (5 mM)
was added. The solution was refluxed for 24 hours at 95 C. The solution was then
filtered using a FRIT flask and was washed with nanopure water and anhydrous
toluene.
4.2.8 FITC conjugation
FITC was utilized as a fluorophore to fluoresce the MSNs for fluorescent mi-
croscopy. First, FITC (2.0 mg) was dissolved in 500 n L anhydrous DMF. Next,
APTMS (5.0 n L) was added to the FITC solution. The solution was reacted for 30
minutes at room temperature with moderate stirring. FITC-APTMS was grafted to
the MSN for further surface functionalization. First, the MSN were dissolved in 100
mL of anhydrous toluene. The solution was then sonicated for 15 minutes to further
29


disperse the particles. The FITC-APTMS solution was then added dropwise to the
MSN/toluene solution and was reacted for 24 hours at 80 C with moderate stirring.
The solution was filtered using a FRIT flask and gave rise to orange precipitates.
4.2.9 Herceptin conjugation
For the coupling of Herceptin to the MSNs, first the remaining hydroxyl groups
from the pegylated MSNs were reacted with carbonyldiimadazole (CDI) at a 1:1
ratio of MSN:CDI. The solution was reacted at 60 C C for 24 hours with moderate
stirring. Following the reaction, the MSN-CDI solution was filtered using a FRIT
flask and washed with anhydrous toluene. Second, a solution was made of 0.5 mg/mL
Herceptin in IX PBS. Next, the dried MSN-CDI particles (4mg/mL) were then added
to the Herceptin solution and were reacted for 24 hours at room temperature with
moderate stirring. Next, the solution was then centrifuged for 10 minutes at 3000
rpm. The supernatant was then decanted. This step was repeated two times more
(centrifugation and removal of supernatant). MSN-Herceptin were stored in IX PBS
at 60 C.
4.3 Structural Characterization
Structural characterization of the MSN after all conjugations were obtained us-
ing fourier transform infrared spectroscopy. 10 mg of each MSN sample (after each
conjugation) were dissolved in 200 fi L anhydrous THF. Each sample was sonicated
for further dispersion. An aliquot of each sample was placed on a polyethylene IR
card (International Crystal Laboratories). The FTIR spectra were collected on an
Nicolet 6700 FT-IR spectrometer from ThermoElectron Corporation.
4.4 Electron microscopy
To confirm the morphology of synthesized MSN, images were taken using scan-
ning and transmission electron microscopy. SEM and TEM was taken at University
30


of Colorado Anschutz Medical Campus and University of Colorado Mines. All trans-
mission electron microscopy samples were prepared by taking an aliquot of the MSN
and dispersing in 1 mL 200 proof ethanol. The sample was sonicated for 10 minutes.
Immediately following, one drop of the solution was placed on the carbon grid and
let dry in the oven for 1 minute. TEM imaging performed using a Hitachi H-7650
operated at 60kV or JEOL JSM-1400+ operated at 120kV. For scanning electron
microscopy, the dried MSN (powder) was mounted on SEM stub with double-sided
carbon tape Sputter Coat with Gold/Palladium (Leica EM ACE200) for 30 seconds.
SEM imaging was performed with SEM (JEOL JSM-6010LA).
4.5 Zeta sizer
Nanoparticle samples (lOmg) were dispersed in 1 mL of dH20. A Malvern Zeta
Sizer 2000 (Malvern Intruments, USA) was used to perform this assay.
4.6 Ultrasound apparatus
Single-pulse ultrasound measurements were obtained using Non-Destructive Test-
ing (NDT) with a frequency of 6.6 MHz at a depth of 2.5 cm. (GE-Panametric). All
transducers were Accuscan type S immersion transducers with point target focus
(PTF). Reciever signal was acquired on an Inhniium 8000 High Performance Oscillo-
scope (Agilent Technologies).
4.6.1 Quantifying pixel intensity
The main purpose of the ultrasound assay was to quantify the average pixel
intensity for each of the synthesized nanoparticles, with and without the fluorocarbon
conjugation. The MSN of varying concentrations (0 mg/mL, 0.5 mg/mL, 1.0 mg/ml,
2.0 mg/mL, 5.0 mg/mL) were dissolved in 5 mL PBS. Each solution was sonicated for
10 mintues followed by placing the samples within dialysis tubing (Spectra/Por 4, 25
mm, MWCO 12000-14000). Immediately following the addition of the samples, the
dialysis tubings were then placed in a PBS bath on top of an agar gel. Agar served
31


as a tissue-mimicking phantom and also served to reduce extraneous noise emitted
into the dialysis tubing. The agar gels were made by autoclaving 3% agar in dH20 at
250 C F for 30 minutes. The liquid agar was then poured in plastic containers and
allowed to cool, forming a gel. Ultrasound images were taken at room temperature.
Three images were taken of each sample at each concentration and were analyzed
using Image J to discover the mean pixel intensity. Each image was 150 x 100 pixel
squares with an area of 150,000.
4.7 Cell preparation
SKBR3 cells were prepared in a solution of DMEM F12 media with 10% fetal
bovine serum (FBS) and l%penicillin/streptomyocin. All cell assays were completed
in triplicate. The cells were plated on a 24 well plate at a seeding density of 50,000
cells/well and 100,000 cells/well.
4.8 Fluorescent microscopy
The nanoparticles were seeded at 25 n g/well. The nanoparticle/cell solution was
then placed on a shaker and then incubated for 30 minutes while another 24 well
plate with the same concentrations of both cells and nanoparticles were incubated for
2 hours. Following the incubation periods, the nanoparticle/cells were washed with
PBS IX. The cells were then fixed for 15 minutes using a 10% Formalin solution.
Following the fixative, the cells were washed with PBS IX and then were stained
with Hoechst at a 1:2,000 dilution for 10 minutes. Live-dead staining was performed
using ethidium homodimer-15.1 (EthD-1). The FITC-labeled nanoparticle and the
stained cells were observed under a fluorescent microscope.
32


5. Main results and discussion
The results and discussion section consists of two components:
-Limitations of conjugating a fluorocarbon directly to the silica nanoparticles
-Characterization, ultrasound imaging results and in vitro work regarding all
three silica particles (sphere, amorphous, and hexagonal rod)
5.1 Fluorocarbon conjugation
Mesoporous silica nanoparticles are synthesized through a series of condensation
reactions. CTAB serves as the surfactant molecule and thus forms a template for the
TEOS to form a mineral like substance surrounding them. Surfactant or template
removal allows for the MSN to acquire a porous structure. It is through this porous
structure that allows a chemo-agent to potentially be loaded via diffusion.
The silica nanoparticles were originally conjugated to surface-hydroxylated nanopar-
ticles by means previously mentioned in the methodology section, yet conjugated at
a higher molar concentration than listed. It was previously posed to conjugate 50
mM of the fluorocarbon to the surface of the hydroxylated particles. One limitation
was reached when the MSN-FC were dispersed in IX PBS solution; the particles were
found to be highly hydrophobic and thus would not disperse within the PBS buffer
solution following sonication for 10 minutes. Highly hydrophobic pharmaceuticals
are problematic in regard to administering via injectable systems due to their low
solubility [2], Thus, the development of a more hydrophilic device could circumvent
this occlusion.
Figure 5.1: Chemical structure of MSN-OH-FC.
33


It was found to increase the hydrophilicity of the particles by manipulating two
modifications. 1). by conjugating PEG, a hydrophilic polymer, the surface hy-
drophilicity of the polymer can then be increased and 2). by decreasing the amount
of the molar concentration of the perfluorocarbon utilized thus the surface hydropho-
bicity of the particles will then decrease. By Pegylating the particles, not only will
this method increase the hydrophilicity and thus allow for the particles to disperse
in PBS solution, it also increases the biocompatibility of the particles. As previously
mentioned, the literature states that pegylating the particles will prevent opsonins
from tagging the particles for clearance by the reticuloendothelial system [11, 56].
hexamethylene diisocyanate
MSN---O'

polyethylene glycol
Figure 5.2: Chemical structure of MSN-OH-HDI-PEG-FC.
34


It was proposed to react the surface hydroxylated MSN with hexamethylene di-
isocyanate (HDI) in order to functionalize the surface with an isocyanate group. The
surface isocyanate group will then react with PEGs hydroxyl groups. The fluoro-
carbon, pentafluorophenylpropyltrimethoxysilane, reacted in a similar mechanism as
aminopropyl-trimethoxysilane (APTMS) to the outer surface PEG hydroxyl groups.
The initial nanoparticle-FC synthesis can be seen in Figure 5.1, followed by the
nanoparticle-PEG-FC synthesis in Figure 5.2. The differences in dispersing the parti-
cles in dH20 can visibly be seen in the figure below (Figure 5.3). These modifications
were further validated by FTIR as seen below.
Amorphous MSN-OH-HDI-PEG-FC
5 mM FC
Figure 5.3: Images taken of amorphous MSN a), before pegylation and with a higher
molar ratio of the FC used, and b). after pegylation, with a lower molar ratio of the
FC used.
35


5.2 MSN characterization
5.2.1 MSN modification analysis by FT-IR
Following each MSN modification, Fourier Transform Infrared Spectroscopy (FT-
IR) was taken to confirm successful conjugation techniques. In FT-IR, the absorbance
of differing atoms covalently bonded can be seen at certain wavelengths, and thus
able to display successful conjugation. All spectra displayed were taken utilizing
amorphous MSN as the model to suggest successful conjugations. The spectra of
amorphous MSN was taken and can be seen below (Figure 5.4, Figure 5.5). FT-IR
was utilized to further confirm each modification was successfully conjugated subse-
quently to the surface of the particles. Thus, following each modification an aliquot
of the sample was taken for FT-IR analysis and then compared to confirm successful
conjugation. The spectra after HDI conjugation, Pegylation, FC conjugation, CDI
and Herceptin conjugations can be viewed below.
As previously mentioned, the particles were first hydroxylated. By examining the
region around 3378 cm"1, the presence of surface hydroxyl groups can be confirmed.
The wavenumber which correlates to the free O-H groups can be viewed at site A of
Figure 5.4[50]. The surface hydroxyl groups can be seen in both the MSN-HDI and
MSN-HDI-PEG particles. This further suggests that following both surface hydrox-
ylation and following pegylation, free hydroxyl groups reside on the surface of the
particles. We can then confirm HDI conjugation by examining the region 2275 cm'1.
This region correlates with an isocyanate stretch (N=C=0). This stretch can visibly
be seen in the FT-IR spectra B for both MSN-HDI and MSN-HDI-PEG. Thus, the
conjugation of HDI can be confirmed.
To further confirm the conjugation of PEG,the regions 1362 and 1287 cm"1 can be
analyzed. These wavenumbers are correlated to the C-0 stretching [50]. In the MSN-
HDI-PEG spectrum, an obvious shift is found further signifying the presence of C-0
36


bonds (C). The regions of 966 and 849 can also be examined for their correlation to
C-C stretching [50]. This region can be observed at Figure 5.4 D, further indicating
the presence of the C-C bonds. Given the appearance of the free hydroxyl group
(A), the C-0 stretching (C), and the C-C bonds (D), it can be determined that
PEG was conjugated to the MSN-HDI particles. The conjugation of the fluorocarbon
Figure 5.4: FT-IR spectra taken of amorphous MSN. Region A depicts confirmation
of free hydroxyl groups following surface hydroxylation and pegylation. B depicts
HDI conjugation and C, D confirm pegylation.
can be confirmed by viewing regions 1050 and 1132 cm"1. This region is associated
with C-F stretch modes. There is a clear peak at this region seen below at site
A of Figure 5.5. Furthermore, the regions 1566 cm'1 can be analyzed to examine
the C=C bonds. There is a distinct peak (B) at this region further signifying the
successful conjugation of the fluorocarbon to the MSN-HD1-PEG particles. Following
37


----MSN-HD4-PEG-FC
MSN HM-PEG-FC-COI
Figure 5.5: FT-IR spectra of amorphous MSN. Region A was found to correlate
with fluorocarbon conjugation. Region B depicts C=C bonds found in both the
fluorocarbon and CDI. CDI was further confirmed through regions C and D, depicting
C-N and C=N bonds.
the fluorocarbon conjugation, CDI was then reacted to the remaining surface hydroxyl
groups. Similarly, the region 1566 cm"1 can be analyzed for C=C bonds. Once
again there is a distinct peak for MSN-HDI-PEG-FC-CDI at peak (B) at this region,
confirming the presence of CDI. For further confirmation, the regions 1200,1650 cm'1
can be examined for C-N and C=N bonds respectively. These can be observed at
C and D respectively in Figure 5.5. Thus, the conjugation of CDI was found to be
validated.
38


5.2.2 Structural characterization
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)
were utilized to confirm the morphology of synthesized mesoporous silica nanopar-
ticles. Images of spherical, amorphous, and hexagonal rod shaped nanoparticles are
displayed below (Figure 5.6). TEM images of spherical nanoparticles were taken at
the University of Colorado school of Mines; all other TEM images were taken at the
University of Colorado-Anschutz Medical campus. The images A-C depict a success-
ful synthesis of spherical MCM-41 of 150-200 nm in diameter with good porosity, as
indicated by the porous membrane. Surfactant removal step of MSN synthesis allows
for the porosity of the nanoparticles. The SEM image (Image C) of the synthesized
spherical nanoparticles display some dispersity in size of each particle. Images D-F
were taken of the amorphous nanoparticles. Images D/E depict the disparate mor-
phologies and sizes seen in the amorphous sample. The SEM images (represented in
Image F) reveal a much larger agglomerate of greater than 10 um. Furthermore, the
amorphous particles appear to have a distinct porous structure, as shown by the red
arrow in Image D. The hexagonal rod shaped nanoparticles can be seen in images
G-I. The particles appear to be larger than the spherical nanoparticles with a length
of about 1-2 microns and a diameter of about 1 micron. Image H displays the high
resolution TEM image of the particles porosity. The particles dispersity can be seen
in the SEM image (Image I) and depicts a relatively rod shape. It is evident that
some residual surfactant remains on the surface of the particles, as shown by the red
arrow in Image I.
To further characterize the particle size, the size of each morphology both before
and after conjugations were taken and performed with the Zeta-sizer. All zeta-sizer
plots can be viewed in Appendix A. The spherical nanoparticles exhibited a relatively
39


Figure 5.6: Electron microscopy images of MSN for structural characterization anal-
ysis. A-B). TEM images of spherical MSN, C). SEM image of spherical MSN, D-E).
TEM images of amorphous MSN, F). SEM image of amorphous MSN, G-H). TEM
of tube MSN, I). SEM of tube MSN.
heterogeneous size when imaged with SEM, which was confirmed with the zeta-sizer
data. According to the latter assay, the spherical particles were found to be on average
~900 nm with the range of dispersity from 100 nm to well over 1 micron. This range
is visibly apparent in the SEM image in Figure 5.6, image C.
The amorphous particles were found to have a wide array of sizes from 100 nm to
well over 1 um, as shown in images D/E. These amorphous particles could be reported
to even have a higher diameter of over 10 microns, as seen in Image F; however, this
image could depict an agglomeration of smaller particles. The zeta-sizer data was
inconclusive as the average particles were reported to be between 7 and 8 microns,
40


yet the reported peaks were found to be between 1 and 5 microns. This limitation
will be discussed further below.
Table 5.1: The table below depicts the average size of each morphology nanoparticle
concluded from the zeta-sizer. Each NP-HER was pegylated and fluorocarbon conju-
gated prior to Herceptin (HER) conjugation. Thus, the MSN-HER depicts the value
after all conjugations, MSN-OH-HDI-PEG-FC-CDI-HER.
MSN C avg 1 C avg 2 C avg 3
Amorphous 7878 8566 7342
Sphere 917 968 853
Tube 949.6 1097 1083
Amorphous-HER 7569 6535 6255
Sphere-HER 4922 4360 4400
Tube-HER 6802 6660 6923
It is observed that the average size of amorphous MSN when compared to the
average size of amorphous MSN-HER does not appear to increase as visibly seen
with both spherical and tubular MSN. This observation could be due to the high
heterogeneous dispersity displayed in both the electron microscopy as well as the
zeta-sizer plots (shown in Appendix A). It is apparent that there is a very diverse
range of amorphous particle size from <100 nm to well over 10 microns.
The tubular nanoparticles were found to be relatively homogeneous in size when
viewed through electron microscopy. The tube particles were observed to have 2 mi-
cron length by 1 micron diameter. This size was further confirmed utilizing TEM. The
zeta-sizer information revealed similar data with an average of ~1 micron; however a
high dispersity was noted with peaks ranging from ~300 nm to ~5 microns.
41


There were several limitations reached with this particular method. Due to the
relatively dense nature of the silica particles, it was apparent that they were settling
towards the bottom of the cuvette. It was also apparent that the particles were quite
disperse in size and exuded immense disparities, especially the amorphous particles.
There were peaks ranging from 200 nm to peaks well over 1 micron. It is apparent
that the particles may even have larger peaks than the light scattering (LS) results
concluded and thus the zeta-average was noteably higher than the reported peaks.
Also, LS may not be the most accurate method to analyze the size of tubular-
shaped or amorphous particles, since LS assumes that each sample has similar refrac-
tive trends as spheres. This may distort the results of the data to some extent.
5.3 Ultrasound imaging
Following the subsequent conjugations, an ultrasound assay was performed to
test the performance of each morphology particle to serve as an ultrasound contrast
agent. Many studies have imaged nanoparticles for ultrasound imaging utilizing vari-
ous methods. In previous work, the nanoparticles were dispersed in a tissue-mimicking
scaffold. This scaffold could be developed using disparate materials including poly-
acrylamide and agar, as both have shown to simulate soft tissue [42, 10]. However, it
was found that the striations in agar produced great noise in ultrasound and thus we
were unable to determine accurately the pixel intensity produced by the nanoparticles
as opposed to the extraneous noise. It was then determined to utilize another method
[37] performed with dialysis tubing immersed in PBS. By utilizing this method, we
were able to isolate the pixel intensity exuded by the nanoparticles without extraneous
influence from the other materials.
Ultrasound images were taken of each particle synthesized both with and without
fluorocarbon conjugation. The particles which had no fluorocarbon conjugation were
synthesized, followed the reaction for removal of surfactant before imaging. The
particles were dispersed in PBS at various concentrations, then placed in dialysis
42


tubing on top of a tissue-mimicking medium. The raw ultrasound images can be
viewed in Figure 5.7 below.
Three images were obtained for each sample prepared. After obtaining the ultra-
sound images, a 100x150 area section was analyzed within the dialysis tubing. Each
section was analyzed in Image J and the mean pixel intensity was then obtained.
This process was repeated at least three times for each MSN type, to have a sample
size of at least n=9 images analyzed per group.
All statistical information was obtained utilizing the software StatPlus. After
acquiring 6 separate sample groups (sphere MSN, sphere MSN-FC, amorphous MSN,
amorphous MSN-FC, tube MSN, and tube MSN-FC), their variances were exam-
ined between the separate groups. Each MSN was only tested between itself and its
fluorocarbon-conjugated counterpart at each concentration. A F-test was utilized to
measure the variances between the groups. If the null was rejected, then unequal
variances were assumed. The F-test p-values between each group are reported be-
low (Table 5.3). Depending on the F-test results, either a two-tailed t-test assuming
equal or unequal variances was then performed on each morphology compared to
their fluorocarbon conjugated counterpart. Statistical significance was considered at
p<0.05.
The averaged data concluded from the Image J analysis of the ultrasound images
was then plotted and can be viewed in Figure 5.8 below. The results for comparing
the three morphologies without a fluorocarbon conjugation can be viewed in Figure
5.8 B, and with the fluorocarbon conjugation in Figure 5.8 A. It is apparent that all
morphologies (with and without fluorocarbon conjugation) share the same general
trend: the mean pixel intensity (MPI) increases with increasing concentration of
nanoparticles. This is consistent with the preliminary work performed on amorphous
nanoparticles [42],
43


Amorphous Amorphous Spherical Spherical Tube
MSN MSN-FC MSN MSN-FC Tube MSN MSN-FC
0.5
mg/mL
1.0
mg/mL
2.0
mg/mL
5.0
mg/mL
Figure 5.7: Ultrasound images taken of each nanoparticle (amorphous, spherical and
tubular) with and without a fluorocarbon conjugated to the surface at disparate
concentrations.
It is apparent that the spherical-FC particles and amorphous-FC particles appear
to follow a very similar trend, whereas the tube-FC particles emitted a more variable
MPI at each concentration (Figure 5.8 A). Tube-FC particles displayed a trend of
plateauing following 1.0 mg/mL (Figure 5.8A). All statistical information including
p-values, mean and standard deviation can be viewed in Table 5.2, Table 5.3.
When observing the nanoparticles without conjugations (Figure 5.8 B), various
results are seen at each concentration. Both amorphous MSN and tube MSN were
found to emit a higher averaged MPI at each concentration than the spherical MSN.
Tubular MSN appears to increase in MPI more rapidly, yet was observed to plateau
following concentrations over 1 mg/mL.
44


A
70
Spherical MSN fC
Amorphous MSN-FC
Tube MSN FC
Spherical MSN
Amorphous MSN
* Tube MSN
Figure 5.8: Overall comparison of the MSN morphologies conjugated to fluorocarbon
(A) and without fluorocarbon conjugation (B).
The MSN were then plotted to compare each MSN to their fluorocarbon-
conjugated counterpart (Figure 5.9). It was evident that the amorphous MSN-FC
conjugated particles were all reported (except at 0.5 mg/mL) to have a higher MPI
than the amorphous MSN alone (Figure 5.9 B). Similarly, the spherical MSN-FC
particles displayed a similar trend as the amorphous MSN particles. The spherical
MSN-FC particles all exuded a higher average MPI as opposed to the spherical MSN
(Figure 5.9 A). This trend was also observed when analyzing the tube MSN-FC and
tube MSN MPIs. It was found that the tube MSN alone had a higher average MPI
at the lowest concentration of 0.5 mg/mL, yet all other concentrations concluded to
lower MPI than its fluorocarbon-conjugated counterpart (Figure 5.9 C).
45


Table 5.2: Mean and Standard Deviations for MSN
Sphere MSN Sphere- -FC Amorphi DUS Amor-FC Tube MSN Tube- FC
Cone. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.
0.5 8.565 2.675 15.85 7.12 17.96 1.20 15.58 3.02 16.73 7.87 7.06 4.24
1.0 12.27 4.34 22.98 7.32 19.81 2.56 24.68 5.75 23.28 8.12 35.83 5.46
2.0 14.40 4.90 35.62 8.83 23.44 3.41 33.70 4.66 25.09 5.33 42.37 2.86
5.0 20.25 6.05 54.27 4.94 36.52 4.34 47.43 7.42 28.33 6.48 42.23 6.98
Table 5.3: P-values between each morphology MSN and their fluorocarbon-conjugated
counterparts. P-values are reported for both the F-test and t-tests completed between
each of these groups.
Sphere MSN & Sphere MSN-FC Amor MSN & Amor MSN-FC Tube MSN & Tube MSN-FC
Cone. F-test t-test F-test t-test F-test t-test
0.5 0.00902 0.00207 0.01768 0.05336 0.081 0.00215
1.0 0.08806 0.00015 0.0342 0.04038 0.262 0.00048
2.0 0.0348 <0.0001 0.398 <0.0001 0.0971 <0.0001
5.0 0.579 <0.0001 0.150 0.0022 0.839 0.00047
Statistical significance was reported and can be seen in Figure 5.9, labeled as (*)
or listed in Table 5.3.
5.3.1 Conclusions from ultrasound imaging analysis
The fluorocarbon-conjugated particles were predicted to emit a higher MPI as
opposed to their non-fluorocarbon conjugated counterparts. This general trend was
confirmed for all morphologies and can be viewed in Figure 5.9. It was predicted that
the fluorocarbon conjugated particles would follow a similar mechanism to the PFC-
emulsed nanoparticles, however the explicit mechanism remains unsurfaced. Current
ultrasound contrast agents which utilize a liquid PFC report a phase-transition follow-
ing the introduction of acoustic waves to its highly echogenic gaseous state [55, 64],
Furthermore, this impedance mismatch between the tissue/gas results in a higher
MPI [8]. As previously stated, the short shelf-life (less than one week when stored at
4C) of these gaseous particles poses a significant limitation [55].
46


A 70
B
60
0
0 1 2 3 4 S 6
COftMBtHtWO
Figure 5.9: Analysis of the pixel intensities from the ultrasound studies produced by
A), spherical MSN B). amorphous MSN and C). tube MSN. All image analysis was
performed in Image J. (*) represents statistical significance between groups.
In order to further increase the shelf-life of these particles, yet still preserve the
higher contrast produced by these enhanced particles, it was determined to conjugate
a PFC to the surface of the MSN. It has previously been reported that freeze-drying
gaseous particles significantly reduces their echogenic factor and were observed to
collapse or fuse during the process [55]. By conjugating the fluorocarbon to the
surface of the particles, the shelf-life would inherently increase as the particles no
longer have a gaseous core which would have the potential to collapse.
As Figure 5.9 displays slightly higher MPI in the amorphous (5.9 B), spherical
(5.9 B) and tube (5.9 C) plots further signifying the acoustic properties of the flu-
orocarbon could potentially be maintained. Despite this general trend noted, both
47


amorphous and tube MSN at 0.5 mg/mL concentration produced either no statistical
difference or significantly lower MPI than both their fluorocarbon counterpart. How-
ever, all other concentrations above 0.5 mg/mL display the fluorocarbon conjugated
MSN emit a statistically significant higher MPI than the particles alone. As previ-
ously reported, each MSN alone produced contrast; yet, as found, their fluorocarbon-
conjugated counterpart further enhances this contrast.
Not only would conjugating the fluorocarbon to the surface potentially enhance
the shelf-life of the particles, but it was also posed to enhance the duration of sys-
temic circulation time within the body. Gaseous microbubbles have been reported
to have a relatively short lifespan in circulation, lasting only a few minutes [9]. By
circumventing a gaseous core, the particles could enhance vascular circuitry.
Furthermore, the MPI emitted from each particle could be correlated to the size
of the particles themselves. It has previously been reported that the MPI increases
with increasing microbubble size [51]. As gaseous microbubbles emit a much higher
MPI (and due to a disparate mechanism) as opposed to solid nanoparticles, this
finding may hold a small or negligible influence. It was also postulated that with
modifying the surface of the MSN, the size will inherently increase and thus may
have some influence on ultrasound contrast. However, Figure 5.8 B displays the two
largest particle morphologies emitting a much greater MPI at all concentrations. Ta-
ble 5.1 depicts the average size of the particles found from DLS. All particles showed
a greater increase in size (>1 micron increase) following the sequence of conjugations
to successfully PEGylate, Fluorocarbon and Herceptin conjugate each MSN. It was
apparent that the larger, conjugated MSN of all morphologies increased in MPI (Fig-
ure 5.9 A,B,C). Yet this argument does not prove true for amorphous MSN, as the
particle average size was reported to be consistent even following all conjugations
(Table 5.1). It was concluded that this could be due in part to the high dispersity
of all amorphous MSN, thus distorting the averaged value. Furthermore, amorphous
48


MSN-FC compared to amorphous MSN was shown to display statistical significance
at all concentrations except 0.5 mg/mL. Thus, if the size of the amorphous MSN
stays consistent following all conjugations and yet produces a higher MPI following
this FC conjugation, then it can be determined that the higher MPI produced was
due to the FC conjugation.
5.4 In vitro studies
The HER2 overexpressing breast cancer cells were seeded at a density of 50,000
cells/well and 100,000 cells/well in separate 24 well plates. Six samples of particles
were tested and the schematic of the 24 well plate can be viewed below (Figure 5.10).
Two plates of each density were incubated for 30 minutes following the addition of
each MSN at a concentration of 25/ig/well. Following 30 minutes, all cells were then
fixed and stained with DAPI (nucleus). This process was completed again, yet with
a 2 hour incubation period after introducing the MSN (also at a concentration of
25/ig/well). Incubation periods of 30 minutes was chosen based off prior literature
and an extension of the time was chosen to find any differences in cellular endocytosis
following a 30 minute incubation period [42],
Table 5.4: The table depicts the incubation time for MSN+cells and seeding density
of HER2 overexpressing cells in each well.
Plate # Incubation time Seeding density
1 30 min 100,000 cells/well
2 30 min 50,000 cells/well
3 2 hr 100,000 cells/well
4 2 hr 50,000 cells/well
Following the alloted incubation time and the hxing/staining techniques (de-
scribed in Method section), the cells were then images using Fluorescent microscopy.
49


Figure 5.10: Schematic of the in vitro 24-well plate assay. A-FC-HER represents amor-
phous MSN-fluorocarbon and Flerceptin conjugated. Similarly, S represents spherical
and T represents tube. A-F1ER represents amorphous MSN-Flerceptin conjugated
(without fluorocarbon).
The images are displayed below in Figures 5.11 and 5.12 for plates seeded at 100,000
cells/well and 50,000 cells/well respectively.
It is apparent that even following only a 30 minute incubation period of each
MSN with the cells, it can be observed that there is co-localization of the fluorescently-
labeled green MSN with the HER2 overexpressing breast cancer cells (Figure 5.11/12).
The morphology of cells depicted following 30 minute incubation period appear to
remain round and globular. It is apparent that after 30 minute incubation period,
not all spherical-PEG-FC-Herceptin labeled particles attached to the surface of the
cells as the red arrow depicts (Figure 5.12 A, Figure 5.13 A). It is also apparent that
50


Amorphous Sphere Tube Amorphous Sphere Tube
MSN'PEG-FGHerceptm MSN-PEG-FC-Herceptin MSK-PEG-FC-llerceptin MSN-Herccptin MSN-Herceptin MSN-Herceptin
Figure 5.11: Images above were taken using Fluorescent microscopy and display all
six MSN samples (shown in Figure 5.10) when incubated with cells at a density of
100,000 cells/well. Each MSN sample was incubated for either 30 minutes or 2 hours
at a concentration of 25/ig/well.Each particle was labeled with FITC (green) and the
cells were stained with DAPI.
51


Figure 5.12: Images above were taken using Fluorescent microscopy and display all
six MSN samples (shown in Figure 5.10) when incubated with cells at a density of
50,000 cells/well. Each MSN sample was incubated for either 30 minutes or 2 hours
at a concentration of 25/ig/well. Each particle was labeled with FITC (green) and
the cells were stained with DAPI.
52


Sphere
MSN-PEG-FC-Herceptin
Tube
MSN-Herceptin
Tube
MSN-Herceptin
Figure 5.13: Zoomed in images of the MSN + 50K cells/well depicted in Figure 5.12.
53


arrow (5.13) B points out a cell which has three groups of MSN colocalized to the
nucleus. The cell does not appear round, globular in shape as the surrounding cells
appear. This suggests that the tube-Her particles were successfully endocytosed by
the cell and were able to promote cell apoptosis, as the cell membrane appears to be
lysed.
Following 2 hour incubation period, it was apparent that all MSN samples were
co-localized with the surface of the cells, suggesting Herceptin binding to the HER2
receptor. The row of 10X images depict a larger representation of the wells as opposed
to the 40X rows. Following the 2 hour incubation period, more lysed cells were noted.
Arrow C depicts a cell co-localized with three groups tube-MSN-Fler yet has a similar
shape of cell as does arrow B depict. This once again could suggest a cell lysing
following the endocytosis of the tube-MSN-Fler. Similarly, as arrow D points out,
an agglomerarion of three cells (image below displays three nuclei) appears to have
undergone death as well. Flowever, no co-localization of green particles is visible.
As previously noted, the characteristics of both the particle size and morphology
have an effect on cell internalization. It was reported that the smaller particles with an
average diameter of ~50 nm were endocytosed at a higher rate as opposed to various
sized particles ranging from 2-100 nm [31, 39]. In another study, the morphology
of particles was found to have a greater effect on cell internalization. In this study,
it was concluded that more symmetrical shaped particles (i.e. a cube or a cylinder
with same diameter and height size) were endocytosed at a lesser rate than their
unsymmetrical counterparts [25]. Similarly, in another study, it was found that long
rod MSN particles (>450nm) were endocytosed at a higher rate than their shorter
counterpart [13]. It was also observed that the rod shaped MSN endocytosed at a
greater rate than spherical shaped MSN. Furthermore, Champion et. al concluded
that particles with a larger surface area were reported to be internalized moreso.
54


5.4.1 Qualitative remarks on in vitro studies
Furthermore, only qualitative remarks can be made on the endocytosis rate of
each morphology MSN-PEG-FC-Herceptin versus their non-fluorocarbon conjugated
counterparts. It was apparent that the tube-PEG-FC-Herceptin in both the 50K
and 100K images depicted particle agglomeration (and almost coating) near the cell
surface, suggesting preferential binding of the synthesized drug delivery modality.
By utilizing fluorescent microscopy, we are unable to determine if cells are endo-
cytosing the particles or if they are merely residing on the surface. It is assumed
that to some extent the particles will undergo receptor-mediated endocytosis via the
Trastuzumab binding to the HER2 receptor. It was also assumed that the cells will
undergo receptor-mediated endocytosis (RME) of the particles to a greater extent
following the 2 hour incubation period.
It was quite evident that the MSN-PEG-FC-Her labeled particles did not inhibit
the preferential binding of Herceptin to its receptor, HER2. This further signifies that
our synthesized device will be able to bind to the HER2 overexpressing breast cancer
cells even following the series of conjugations. Furthermore, it was also apparent that
the fluorocarbon conjugated particles acted in similar fashion to that of their non-
FC conjugated counterpart. Thus, the MSN-PEG-FC-Her particles displayed similar
binding after 30 minutes of incubation as well as 2 hours.
It was also apparent that in both the tube MSN-Her 30 min and 2 hr incubation
periods, both reported cell death (as shown by arrows B and C). This can then
suggested a higher rate of RME following the introduction of tube MSN to the cells.
It is also apparent that the tube morphology MSN may suggest an enhanced (or
swifter) rate of RME as there was cell death reported after the 30 minute incubation
period.
55


Despite these observations, more quantitative results needs to be performed in
order to quantify which shape particle produces the highest rate of therapeutic alle-
viation.
56


6. Conclusion and proposed future work
6.1 Conclusive remarks
The design of this study was conducted in order to enhance both therapeutic
and diagnostic effects utilizing HER2 overexpressing breast cancer as a model for the
device. Current clinical treatments for cancer lack a targeting modality and remains
to be highly toxic to all cells, including non-malignant. Drug delivery modalities
are progressing into becoming multifunctional, serving to enhance diagnostic imagery
as well as therapeutic effects. However, current FDA-approved UCA have several
limitations including short shelf-life and a short systemic circulatory duration [55].
To circumvent these shortcomings, a fluorocarbon-conjugated MSN was chosen with
varied morphologies to further enhance both therapeutic and diagnostic effects.
MSNs were successfully synthesized and structurally characterized by both elec-
tron microscopy and LS. All morphologies were shown to have a wide range of size
dispersity. Following the characterization, the particles successfully underwent a se-
ries of conjugations in order to conjugate a Fluorocarbon to the outer surface. It has
previously been shown that a gaseous perfluorocarbon microbubble emits a higher
pixel intensity in ultrasound imaging through the tissue/gas impedance mismatch [8].
Similarly, a liquid perfluorocarbon-emulsed nanoparticle emits a higher MPI through
phase-transitioning into its highly echogenic gaseous state following the introduction
of acoustic waves [55, 64], It was proposed that the shelf-life of an ultrasound contrast
agent can be increased by utilizing a solid particle (instead of a bubble) and through
conjugating a fluorocarbon to the surface.
The three morphology MSN were tested to determine their MPI at various con-
centrations along with their FC conjugated counterpart. A general trend was seen
which depicted the FC conjugated particles emitted a higher MPI as compared to
the non-FC conjugated MSN. All concentrations tested (except 0.5 mg/ml) were re-
ported to have statistically significant differences. Tube and amorphous MSN alone
57


were shown to have a higher contrast than the spherical MSN at all concentrations.
To test the targeted MSNs capability of being endocytosed to their targeted cells, a
in vitro assay was performed. The binding preference of the MSN-PEG-FC-Her par-
ticles were observationally analyzed to have a similar binding rate with the MSN-Her
particles. It was suggested that some particles were successfully being endocytosed
by the cells, which then underwent apoptosis. This can be concluded as some cells
with colocalized green particles appear to have a lysed cell membrane. However, ad-
ditional cell studies need to be performed to further confirm the cell lysing due to the
endocytosis of the nanoparticles.
In summary, a multifunctional, hybrid polymer-NP system was successfully devel-
oped to enhance contrast in ultrasound as well as to serve as a targeted, drug delivery
modality. Three morphologies were tested in their capability for ultrasound contrast
and rate of endocytosis. It can be suggested that tube and amorphous MSN emit a
higher contrast alone and all morphologies share a general trend with the conjugation
of a fluorocarbon. The in vitro cell study suggests that tube MSN may have a faster
rate of endocytosis as there are several dead cells noted following only 30 minutes of
treatment.
6.2 Proposed future work
1. Optimization of particles
The optimization of the silica nanoparticles is necessary in order to develop a
highly translational multifunctional drug delivery vehicle. It would be ideal for ex-
travasation of the blood stream for a reduction of the particles to be a size of less than
1 micron. Thus, the particles would be able to traverse the gap between endothelial
cells as well as have the potential to have a higher rate of endocytosis. A desired
particle would be under 1 micron following the series of conjugations. Thus, further
optimization would be needed.
58


1. Frequency Sweep
To further understand the mechanism of how the FC conjugated particles are
emitting a higher contrast, it was determined to begin with a frequency sweep. Cur-
rent clinical ultrasound performs at frequencies between 1-10 MHz, thus the sweep
would be within this range. Albeit the developed system is not a gas bubble, it may
have a resonance frequency to which it produces the highest ultrasound contrast.
When excited via acoustic waves near resonance frequency, the bubble is excited for a
longer duration resulting in nonlinear oscillations and harmonic scattering [19]. Fur-
thermore, its solid scattering counterpart does not have a resonance frequency and
thus is excited equally [19]. By utilizing a frequency sweep, it could be determined
if the FC conjugated to the surface has a resonance frequency, or if the particles are
merely solid scatterers.
2. In vitro Live \ Dead staining
To quantify how many cells are alive or dead following the treatment of each MSN.
The cell study would be performed as described in the Methodology section, yet with
the addition of the stain before imaging. We could then determine the therapeutic
effects of each morphology MSN with or without the fluorocarbon. Furthermore, the
MSNs capability to be endocytosed by the cells and to promote apoptosis (via ADCC
and inhibition of signaling pathways), could be determined.
3. Breast Cancer Animal Model
The next stage of the study (following optimization of the proposed drug delivery
vehicles) would be to analyze tumor growth/reduction in mice following a series of
injections of each particle. The capacity of each system to emit ultrasound contrast
would further be examined as well as its ability to reduce the tumor size.
59


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65


APPENDIX A. Zeta-sizer plots
Figure A.l: Zeta-sizer plot performed on spherical MSN. Performed in triplicate.
Are Dotffeto* by Mm4y
SO
5
e
£
<|J-
0 1
Sire (d nm)
Record 19 Sphere MSN~Her 1 Record 20 Sphere IISN-Her 2.
Record 21 Sphere MSN-Hcr 3________________________________________
Figure A.2: Zeta-sizer plot performed on spherical MSN-Fler.Performed in triplicate.
66


feurte titt by Wm4y
100
£ 80
1 GO
| *0
f 20
0
0 1
3 >
I/i
1 10 100 1000 10000
Se [ Record 10 Sigma MSN 1
Record U Srgma USN2
Record 1S Sigma MSN 3-
Figure A.3: Zeta-sizer plot performed on amorphous MSN.Performed in triplicate.
Stze DatrtutoA by Wervuy
Record 10 Smjito MSN-Her 1 Record 11 Srgma WiU-*ier 2
Record 12 S*jma MSN-Her 3_______________________________________
Figure A.4: Zeta-sizer plot performed on amorphous MSN-Her.Performed in tripli-
cate.
Figure A.5: Zeta-sizer plot performed on tube MSN.Performed in triplicate.
67


S 60
SO
0
30
JO
10
flJ-
0 1
10 too
$rc
1000
. I
Li
10000
Record ? tubeUSH-Mer I
Record 8 tubeMSN-Her 2
Record 0 tubeMSW-Herl]
Figure A.6: Zeta-sizer plot performed on tube MSN-Her.Performed in triplicate.
68


Full Text

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MULTIFUNCTIONALFLOUROCARBON-CONJUGATEDMESOPOROUS SILICANANOPARTICLESOFVARIEDMORPHOLOGIESTOENHANCE THERANOSTICEFFECTSINBREASTCANCER by ANNALAURA,M.NELSON BachelorofScience,FurmanUniversity,2012 Athesissubmittedtothe FacultyoftheGraduateSchoolofthe UniversityofColoradoinpartialfulllment oftherequirementsforthedegreeof MasterofScience Bioengineering 2015

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ThisthesisfortheMasterofSciencedegreeby AnnaLaura,M.Nelson hasbeenapprovedforthe BioengineeringProgram by DaewonPark,Advisor DaewonPark,Chair BolinLiu KendallHunter October19,2015 ii

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Nelson,AnnaLaura,M.M.S.,Bioengineering MultifunctionalFlourocarbon-conjugatedmesoporoussilicananoparticlesofvaried morphologiestoenhancetheranosticeectsinbreastcancer ThesisdirectedbyAssistantProfessorDaewonPark ABSTRACT BreastcancerisoneofthemostprevalentcarcinomasintheUnitedStatesaffectingoveronemillionwomeneachyear[44].Currentchemotherapytreatmentscan beespeciallyharshonthebodyduetotheimmensecytotoxicityofthechemo-agents andthelackofatargetingmodality.Overthepastdecade,researchershavebeen utilizingmesoporoussilicananoparticlesMSNtoserveasadrugdeliveryvehicle inaneorttoincreasetherapeuticeects.Humanepidermalgrowthfactorreceptor2HER2isatransmembranereceptortyrosinekinasewhichisoverexpressedin about25-30percentofallhumanbreastcancers[15].Here,weproposetheuseof Herceptin R anti-HER2monoclonalantibody,Genentechasatargetingmoiety,in combinationwithMSN,todevelopatargeteddrugdeliveryvehicleforHER2+breast cancer. Mesoporoussilicananoparticleswerechosentoserveasthedrugdeliveryvehicle astheyarereadilyfunctionalizable,haveaporousstructuresuitablefordrugdelivery [54,62].MSNhavebeensuccessfullyutilizedasaplatformformultifunctionaldrug delivery/imagingsystems.IthaspreviouslybeenshownthatMSN-Herceptinproduceshigh-qualityultrasoundimages;MSNwerealsoshowntoaggregateatthesite ofatumortissueandhavealongerdurationwithinthebody,unlikegasmicrobubbles[42].Gasmicrobubbles,thecurrentclinicalgoldstandardforultrasoundcontrast agentswhichutilizealiquidperuorocarbonPFCemulsedwithinamicrobubble, havenumerouslimitationsincludingalowshelf-lifeandalowsystemiccirculatory duration[55].AlthoughamorphousMSNhaveproventoincreasetherapeuticanddiiii

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agnosticeectsinbreastcancercells,alternativemorphologiesmayproducegreater results.BycomparingMSNe.g.spherical,amorphous,andtubeandwiththe conjugationofauorocarbon,wehopetooptimizeboththedrugdeliveryeciency andultrasoundcontrast,maximizingthetherapeuticanddiagnosticeects. Amultifunctional,hybridpolymer-nanoparticlesystemwasdevelopedutilizing polyethhyleneglycolPEG,ahighlybiocompatiblepolymer,andaperuorocarbon conjugatedtothesurface.FT-IRwasusedtoconrmthesuccessfulconjugation ofeach.Preliminaryultrasoundimagesdepictadistincttrendbetweenmerelythe nanoparticlealoneversusthehybrid-polymernanoparticlewithauorocarbonconjugatedtothesurface.Itwasevidentthattheuorocarbonconjugatedparticlesemit ahigherpixelintensitythanitscounterpart.Furthermore,allnanoparticleshada clearbindingpreferenceforHER2overexpressingbreastcancercells.Insummary, byconjugatingaPFCtothesurfaceoftheMSNandbyanalyzingmorphologyofthe particles,boththetherapeuticanddiagnosticeectswereshowntobeenhanced. Theformandcontentofthisabstractareapproved.Irecommenditspublication. Approved:DaewonPark iv

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ACKNOWLEDGMENT Iwouldliketothankseveralindividualsfortheircontinuoussupportthroughoutmy experience. Firstly,Iwouldliketothankmyadvisor,Dr.DaewonParkforhisguidance throughoutmyMaster'scareer.ThiswasanamazingopportunityandIhavelearned immeasurableskillsandlessonsinlabtechnique.Iwouldalsoliketothankmy othercommitteemembers,Dr.KendallHunterandDr.BolinLiu,fortheirsupport, dedicationandwillingnesstowardsassistingandoeringtheirexpertadviceand resources. IwanttoespeciallythankalloftheamazingmembersoftheTranslationalBiomaterialsResearchLaboratory:MelissaLaughter,JamesBardill,andDavidLee.My experiencewouldnothavebeenthesamewithoutyouall. Severalotherindividualshavehelpedcontributetomyproject.First,Iwouldlike tothankBrisaPenaforhercontinuedhelpandteachings,especiallyherhelpwiththe uorescentmicroscopy.MirandaIntrator,afellowlabmember,helpedtremendously withherguidanceandsupport.IwouldalsoliketothankDr.Trewyn'slabat UniversityofColorado-SchoolofMinesandtheElectronMicroscopylabfortheir generoushelpthroughout. Lastly,Iwanttoreachouttomyfamily.YouallknowIthinkyou'reamazing.I couldnothavecompletedthisthesiswithoutallofyourloveandsupportthroughout theentireprocess. v

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Declarationoforiginalwork by AnnaLauraNelson Thismastersthesiswasindependentlycomposedandauthoredbymyself,using thesupportfrommyadvisor,committeemembers,labmembers,fellowstudents, andtheDepartmentofBioengineering.Theresearchandideaspresentedinthis documentoriginatedfromtheTranslationalBiomaterialsResearchLaboratoryunder theguidanceofDr.DaewonPark.AllresourcesandfundswereprovidedbyDr. DaewonParkandtheDepartmentofBioengineering. AnnaLauraNelson vi

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TABLEOFCONTENTS Tables........................................x Figures.......................................xi Chapter 1.Introduction...................................1 1.1HER2+breastcancer..........................2 1.2Currentclinicalscreeningmodalitiesforbreastcancer........5 1.3Currentclinicalcancertreatments...................6 1.3.1Eectsofchemotherapyanddrugresistance..........6 1.3.2Monoclonalantibodies......................7 1.3.3Herceptinresistance.......................9 1.3.4Personalizedtherapymayreducedrugresistance.......9 1.4Objectiveofthisstudy.........................10 1.4.1Aimsofthisstudy........................11 1.5Thesislayout...............................11 2.Literaturereview................................12 2.1Targeteddrugdeliverymodalities...................12 2.1.1Eectofparticlesizeandmorphology.............14 2.1.2Passiveandactivetargeting...................16 2.2Ultrasoundcontrastagents.......................17 2.2.1Gasmicrobubbles........................18 2.2.2Eectofmicrobubblesizeonultrasoundcontrast.......19 2.2.3Peruorocarbonemulsednanoparticles.............20 2.2.4LimitationstogasmicrobubblesandPFC-emulsednanoparticles20 2.3Pegylation................................21 3.PreliminarydataandproposeddrugdeliveryModality...........23 3.1Preliminarywork............................23 vii

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3.2Proposeddrugdeliverymodality....................24 4.Materialsandmethodology...........................27 4.1Materials.................................27 4.2MSNpreparation............................27 4.2.1MSNamorphouspreparation..................27 4.2.2MSNsphericalsynthesis.....................27 4.2.3MSNtubesynthesis.......................28 4.2.4MSNsurfacehydroxylation...................28 4.2.5HDIconjugation.........................28 4.2.6Pegylation.............................29 4.2.7Flourocarbonconjugation....................29 4.2.8FITCconjugation........................29 4.2.9Herceptinconjugation......................30 4.3StructuralCharacterization.......................30 4.4Electronmicroscopy...........................30 4.5Zetasizer.................................31 4.6Ultrasoundapparatus..........................31 4.6.1Quantifyingpixelintensity....................31 4.7Cellpreparation.............................32 4.8Fluorescentmicroscopy.........................32 5.Mainresultsanddiscussion..........................33 5.1Fluorocarbonconjugation........................33 5.2MSNcharacterization..........................36 5.2.1MSNmodicationanalysisbyFT-IR..............36 5.2.2Structuralcharacterization...................39 5.3Ultrasoundimaging...........................42 5.3.1Conclusionsfromultrasoundimaginganalysis.........46 viii

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5.4Invitrostudies..............................49 5.4.1Qualitativeremarksoninvitrostudies.............55 6.Conclusionandproposedfuturework.....................57 6.1Conclusiveremarks...........................57 6.2Proposedfuturework..........................58 References ......................................60 Appendix A.Zeta-sizerplots.................................66 ix

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TABLES Table 5.1Thetablebelowdepictstheaveragesizeofeachmorphologynanoparticle concludedfromthezeta-sizer.EachNP-HERwaspegylatedanduorocarbonconjugatedpriortoHerceptinHERconjugation.Thus,the MSN-HERdepictsthevalueafterallconjugations,MSN-OH-HDI-PEGFC-CDI-HER.................................41 5.2MeanandStandardDeviationsforMSN..................46 5.3P-valuesbetweeneachmorphologyMSNandtheiruorocarbon-conjugated counterparts.P-valuesarereportedforboththeF-testandt-testscompletedbetweeneachofthesegroups.....................46 5.4ThetabledepictstheincubationtimeforMSN+cellsandseedingdensity ofHER2overexpressingcellsineachwell..................49 x

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FIGURES Figure 1.1ThefourmembersoftheHERfamilyofreceptorsandtheirrespective signalingpathways.ImageA.depictsthedimerizationofHER2/HER familyofreceptors,whichinturnactivatesvarioussignalingcascades,includingPI3K/Akt.ImageB.andC.depictthemonoclonalantibody, Trastuzumab,bindingtotheHER2receptoranditseectscanbeobservedinimagesE.andF[28].......................4 1.2Thesensitivityandspecictyofmammography,ultrasoundandmammographyplusultrasound.Itisevidentthatbyutilizingbothscreening modalities,bothspecicityandsensitivityincrease[5]...........5 1.3MechanismsofactionforthecombinedtreatmentofTrastuzumaband chemotherapeuticagents.Followingtreatments,angiogenisisistheninhibited,ADCCisinitiatedandseveralphenotypicchangesarenoted[44].8 2.1Depictionofdisparatetargeteddrugdeliveryvehiclesa.polymeric nanogel,b.polymericmicelle,c.goldnanoparticle,d.ironoxide nanoparticle,e.siRNAentrappedinaliposomalvessel,f.stimuliresponsivecappedMSN[38].........................13 2.2Eectofparticlesizeonextravasationofbloodstream.Smallerparticles tendtohavealowerretentionwithintumortissue,whereaslargerparticles maynotbeabletotraversethegapbetweenendothelialjunctions[21]..15 2.3Passivevs.activetargetingofdrugdeliveryvehicles.Thetargeted nanoparticleshaveanantibodyconjugatedonthesurfaceinordertoactivelytargetaknownrecpetoronmalignantcells,whereasnon-targeted particlesrelyonenhancedpermeabilityandretentioneecttotargetmalignantcells.[21]................................17 2.4Commonpropertiesofultrasoundcontrastagents[33]...........19 xi

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2.5ThedegradationofOptison R microbubblesfollowingtheintroductionof agas-decientbuersolutionaftertimepointsa.0minutes,b.1.5 minutes,c.4minutesandd.6minutes.[48]...............22 3.1PreliminaryworkdepictingtheMPIemittedbyamorphoussilicananoparticles[42]....................................23 3.2Preliminaryinvitrostudiesdisplayingthecolocalizationofamorphous MSNwithHER2overexpressingbreastcancercellline[42]........24 3.3Proposedmechanismofactionofourdesigneddrugdeliveryvehicle...26 5.1ChemicalstructureofMSN-OH-FC.....................33 5.2ChemicalstructureofMSN-OH-HDI-PEG-FC...............34 5.3ImagestakenofamorphousMSNa.beforepegylationandwithahigher molarratiooftheFCused,andb.afterpegylation,withalowermolar ratiooftheFCused..............................35 5.4FT-IRspectratakenofamorphousMSN.RegionAdepictsconrmation offreehydroxylgroupsfollowingsurfacehydroxylationandpegylation.B depictsHDIconjugationandC,Dconrmpegylation...........37 5.5FT-IRspectraofamorphousMSN.RegionAwasfoundtocorrelatewith uorocarbonconjugation.RegionBdepictsC=Cbondsfoundinboth theuorocarbonandCDI.CDIwasfurtherconrmedthroughregionsC andD,depictingC-NandC=Nbonds....................38 5.6ElectronmicroscopyimagesofMSNforstructuralcharacterizationanalysis.A-B.TEMimagesofsphericalMSN,C.SEMimageofspherical MSN,D-E.TEMimagesofamorphousMSN,F.SEMimageofamorphousMSN,G-H.TEMoftubeMSN,I.SEMoftubeMSN.......40 5.7Ultrasoundimagestakenofeachnanoparticleamorphous,sphericaland tubularwithandwithoutauorocarbonconjugatedtothesurfaceat disparateconcentrations............................44 xii

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5.8OverallcomparisonoftheMSNmorphologiesconjugatedtouorocarbon AandwithoutuorocarbonconjugationB...............45 5.9Analysisofthepixelintensitiesfromtheultrasoundstudiesproducedby A.sphericalMSNB.amorphousMSNandC.tubeMSN.Allimage analysiswasperformedinImageJ.*representsstatisticalsignicance betweengroups.................................47 5.10Schematicoftheinvitro24-wellplateassay.A-FC-HERrepresentsamorphousMSN-uorocarbonandHerceptinconjugated.Similarly,SrepresentssphericalandTrepresentstube.A-HERrepresentsamorphous MSN-Herceptinconjugatedwithoutuorocarbon.............50 5.11ImagesaboveweretakenusingFluorescentmicroscopyanddisplayall sixMSNsamplesshowninFigure5.10whenincubatedwithcellsata densityof100,000cells/well.EachMSNsamplewasincubatedforeither 30minutesor2hoursataconcentrationof25 g/well.Eachparticlewas labeledwithFITCgreenandthecellswerestainedwithDAPI.....51 5.12ImagesaboveweretakenusingFluorescentmicroscopyanddisplayall sixMSNsamplesshowninFigure5.10whenincubatedwithcellsata densityof50,000cells/well.EachMSNsamplewasincubatedforeither 30minutesor2hoursataconcentrationof25 g/well.Eachparticlewas labeledwithFITCgreenandthecellswerestainedwithDAPI.....52 5.13ZoomedinimagesoftheMSN+50Kcells/welldepictedinFigure5.12.53 A.1Zeta-sizerplotperformedonsphericalMSN.Performedintriplicate...66 A.2Zeta-sizerplotperformedonsphericalMSN-Her.Performedintriplicate.66 A.3Zeta-sizerplotperformedonamorphousMSN.Performedintriplicate...67 A.4Zeta-sizerplotperformedonamorphousMSN-Her.Performedintriplicate.67 A.5Zeta-sizerplotperformedontubeMSN.Performedintriplicate......67 A.6Zeta-sizerplotperformedontubeMSN-Her.Performedintriplicate....68 xiii

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LISTOFABBREVIATIONS APTMS3-aminopropyltrimethoxysilane ADCCantibodydependentcellcytotoxicity CSCcancerstemcell CDIcarbonyldiimidazole CTABN-cetyltrimethylammoniumbromide DMFdimethylformamide FITCuoresceinisothiocyanate FCuorocarbon FTIRfouriertransforminfraredspectroscopy HClhydrochloricacid HDIhexamethylenediisocyanate HER2humanepidermalgrowthfactorreceptor-2 HER3humanepidermalgrowthfactorreceptor-3 IGF-1Rinsulin-likegrowthfactor1-receptor MPImeanpixelintensity MSNmesoporoussilicananoparticles MWmolecularweight NPnanoparticle NKnaturalkillercell PFCperuorocarbon PBSphosphatebueredsolution PEGpolyethyleneglycol PTENphosphataseandtensinhomolog TEOStetraethoxysilane THFtetrahydrofuran USultrasound xiv

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1.Introduction Cancercontinuestobealeadingcauseofdeathaectingmillionsworldwide. BreastcancerisoneofthemostprevalentcarcinomasintheUnitedStatesaecting overonemillionwomeneachyear[45].Currentchemotherapytreatmentscanbe especiallyharshonthebodyduetotheimmensecytotoxicityofthechemo-agents andthelackofatargetingmodality.Overthepastdecade,researchershavebeen utilizingmesoporoussilicananoparticlesMSNtoserveasadrugdeliveryvehiclein aneorttoincreasetherapeuticeects. Humanepidermalgrowthfactorreceptor2HER2isatransmembranereceptor tyrosinekinasewhichisoverexpressedinabout25-30percentofallhumanbreastcancers[15].AnoverexpressionofHER2couldresultinapoorprognosisasloweroverall survivalrateshavebeennotedinmetastaticbreastcancer[46].However,manymethodsarebeingutilizedtodirectlytargetandthusdownregulatetheseoncoproteins. TrastuzumabHerceptin R ,Genentechisananti-HER2monoclonalantibodythat iscurrentlyusedinclinicalpracticeforpatientswithHER2-overexpressingHER2+ breastcancer.Here,weproposetheuseofHerceptinasatargetingmoiety,incombinationwithMSN,todevelopatargeteddrugdeliveryvehicleforHER2+breast cancer. MSNhavebeensuccessfullyutilizedasaplatformformultifunctionaldrugdelivery/imagingsystems.Forexample,MSNconjugatedtoauorophorehavebeenused fornear-infrareduorescenceNIRFimaging[63],andMSNalonecanbeusedas anultrasoundcontrastagentforultrasoundimaging.Ultrasoundwaschosenasthe desiredscreeningmodality,asitisrelativelyinexpensiveandcanmitigatetherisk ofradiation.Mammographyservesasthecurrentclinicalstandardfordetectionof breastcancer,howevermammographyhasseveralsetbacksincludingitshighlevels ofionization,itshighfalsepositivesanditsdicultyindetectingmalignanciesin densebreasttissues[8,34].MSNshavebeenshowntohavealongerdurationwithin 1

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thebodyasopposedtogasmicrobubbles,thecurrentclinicalstandardinultrasound contrastagents[42].IthaspreviouslybeenshownthatMSN-Herceptinproduces high-qualityultrasoundimages;MSNwasalsoshowntoaggregateatthesiteofa tumortissue,unlikegasmicrobubbles[42].Furthermore,auorocarbonwasconjugatedtotheoutersurfaceoftheMSNinordertotestitsabilitytoenhancepixel intensityinultrasound.Thenanoparticlesweretestedseparatelybutalsowitha uorocarbonconjugatedtothesurfacetodetermineanoptimalmultifunctionaldrug deliveryvehicle. AlthoughsphericalMSNhaveproventoincreasetherapeuticanddiagnosticeffectsinbreastcancercellsinvitro,alternativemorphologiesmayproduceevengreater results.ByvaryingthemorphologyoftheseMSNe.g.spherical,core-shell,tubular,wehopetooptimizeboththedrugdeliveryeciencyandultrasoundcontrast, maximizingthetherapeuticanddiagnosticeectsoftheMSN-Herceptinmultifunctionalplatform.Thesealternativemorphologieswillbetestedintheirabilityto emitultrasoundcontrastalonebutalsocomparedtotheiruorocarbon-conjugated counterpart. 1.1HER2+breastcancer Humanepidermalgrowthfactorreceptor2ispartofareceptortyrosinekinase familyoftransmembraneproteinsonbreastcells.HER2canfunctionnormallywhen expressedatlowlevelsandservestopromotegrowthwhenappropriate.However, aberrantexpressionofHER2isfrequentlyseeninabout25-30%ofallmalignant breastcancersandisassociatedwithapoorprognosis[28,58].HER2positive breastcancerhasbeenfoundtobecorrelatedwithhigherratesofreturnandoverall survival[52]. Themostfrequentgeneticaberrationpromotingconstitutivecellproliferationand ultimatelytumorformationinbreasttissueisHER2geneamplication[29].This geneamplicationfurtherresultsintheHER2overexpressiononbreastepithelium. 2

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AnactivatedHERfamilyofreceptorspromoteproliferationandcellsurvival asaresultfromthebindingofvariousligandstypicallygrowthfactorstotheir targetreceptor,triggeringtheactivationofseveraldownstreamsignalingpathways [29].AftertheligandisboundtotheextracellulardomainofaHERreceptor,a conformationalchangeoccursleadingtotheheterodimerizationorhomodimerization withotherreceptorsinitsfamilyHER3,HER4,etc.[58,28,29].AsHER2does notbindanyoftheaforementionedligands,itservestoactivatetheotherreceptors viaheterodimerization[29].Thisactivationthentriggersthephosphorylationof thedownstreameectormoleculeslocatedontheintracellulardomainoftheHER receptors,specicallythetyrosineresidues[29].Uponthephosphorylationofthese downstreammolecules,asignalingcascadeistheninitiated. SeveralsignalingpathwaysareinitiatedfollowingthedimerizationofHER2,includingPI3K/Akt,Ras/MAPK,andthemTORpathways[29,26,40,47].AsHER2positivebreastcancerinherentlyhasahighexpressionlevelofHER2,thesepathways arethusoveractiveandpossiblyuncontrolledasthenegativefeedbackloopsarederegulated[29].Ithasbeenfoundthatthisdimerizationservesastheoncogenicdriver, deregulatingtheactivationofthepreviouslylistedsignalingpathways.Hence,ifthis dimerizationwerethentargetedforinactivationviaTrastuzumab,thusthesignalingpathwayswouldinherentlybedownregulated[29].PI3K/Aktpathwayhasbeen showntoinhibitcelldeath,furtherpromotingcellsurvivalandproliferation[28,26]. AsPI3Kisactivated,itthenbindsandphosphorylatesPIP2phosphatidylinositol4,5-bispphosphateintoPIP3phosphatidylinositol-3,4,5-triphosphate,locatedon theinnerplasmamembrane[47].AfterthephosphorylationofPIP3,theproto-oncoprotein,AktproteinkinaseBisthenactivated[40].ThisactivationofAktleadsto disparateeectsdownstreamincludingevadingofapoptosis.Followingthisprotein activation,ultimatelytheinhibitionofthecaspasecascadehasbeenreported,thus leadingtocellsurvival[40].Hence,itisevidentthatthemalignantphenotypewould 3

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favorconstitutiveactivationofthisgivenpathwayinordertoobtaintheaforementionedadvantageouscharacteristics[29]. Figure1.1:ThefourmembersoftheHERfamilyofreceptorsandtheirrespective signalingpathways.ImageA.depictsthedimerizationofHER2/HERfamilyof receptors,whichinturnactivatesvarioussignalingcascades,includingPI3K/Akt. ImageB.andC.depictthemonoclonalantibody,Trastuzumab,bindingtothe HER2receptoranditseectscanbeobservedinimagesE.andF[28]. AberrantoverexpressionofHER2hasbeencorrelatedwithpromotingcarcinogenesisandthusservesasapoorprognosticfactor[58].NotonlydoesHER2overexpressionoccurintheprimarytumorsite,butitalsooccursinthesecondarymetastatic site[58].Thus,thisreceptorservesasanexcellentbiomarkertotargetforinhibiting tumorproliferationandthustumorprogressioninHER2+breastcancer. 4

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1.2Currentclinicalscreeningmodalitiesforbreastcancer Thereareseveralimagingmodalities,whicharecurrentlyusedfortheclinical detectionbreastcancerincludingmammography,MRIandhigh-frequencybreastultrasound[36].Mammographyisutilizedasthecurrentclinicalstandardforthe detectionofbreastcancer;howevercliniciansareprogressivelybecomingmoreaware ofthedisparatesetbacks. Mammographyhasdicultydetectingmalignanttumorsinyoungerwomenwith adenserbreasttissueandshowstohavealowsensitivityforsuchwomen[8,34]. MagneticresonanceimagingMRImayserveasamoreattractiveimagingmodality fordenserbreasttissueyetprovestobemoreexpensive[34].MRI,incomparisonto mammographyandbreastultrasound,hasalsobeenshowntomaintainahighsensitivity,specicityandpositivepredictivevaluefordetectingbreastcancer,especially tothosewithageneticpredisposition[36]. Figure1.2:Thesensitivityandspecictyofmammography,ultrasoundandmammographyplusultrasound.Itisevidentthatbyutilizingbothscreeningmodalities, bothspecicityandsensitivityincrease[5]. 5

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Ultrasoundservesasanattractivescreeningmodalityasitisrelativelyinexpensive,rendersimagesinreal-timeandcanmitigatetheriskofradiation,asopposed toscreeningviamammography[57].Ultrasoundisfrequentlyusedtorenderliveimages,thusisutilizedclinicallytodirectbiopsiesandthushashighpotentialtoserve asaprevalentscreeningmodality[61].Asitisalsomoveable,ithasthecapability toimageapatientbedside,whichprovestobeauniquecharacteristicascompared toalternativeimagingmodalities.However,furtheraugmentationofthisparticular modalityisimmenselyneededastherecontinuestobenumerouspitfallswhenutilizingultrasoundasaprimaryscreeningmethod,includinghighratesoffalsepositives andnegatives[57]. 1.3Currentclinicalcancertreatments 1.3.1Eectsofchemotherapyanddrugresistance Chemotherapyhasbeenutilizedfortreatingcancerforseveraldecades.Chemotherapeuticagentstypicallyinhibitcellproliferationbytargetingdisparateproteinsinvolvedinthecellcycle,andthusfurtherpromotingapoptosis.Asthesechemotherapeuticagentstargetandinhibitthecellcycle,anormalcellprocess,thereproveto bedebilitatingsideeectsfromthesedrugs.Fromthepatientsperspective,themost frequentlyreportedsideeectsinclude:hairloss,nauseaandfatigueamongstafew [4]. Chemotherapyhasbeenknowntoeradicatemostcancercells,yetcouldpossiblyleaveremainingamoreresistantcancercell.Drugresistanceisafrequently occurringobstacleincurrentclinicaltreatmentforcancerasmanypatientsarediagnosedwithmalignanciesmorethanonce[23].Ithasbeenpostulatedthatfollowing chemotherapy,persistingcancerstemcellscouldpotentiallyhavethecapacitytopromoteregenerationofatumor[18].ThederivationofsuchcancerstemcellsCSC hasyettobedeciphered.Twokeyhypothesesoforiginationinclude1iftheCSC originatesfromamalignanttransformationofanormalstemcellor2iftheCSC 6

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originatesfromamalignantcell,whichde-dierentiatesintoacquiringstemcell-like characteristics[18]. Whenassessingaclinicalperspective,thereareseveralposedmechanismsfor developingdrugresistanceincludingeliminationofthedrugordevelopingamutation ofthedrugstarget[23].Frequently,cellswhichdeveloparesistanceforonedrugoften haveresistancetowardmultipledrugsi.e.multidrugresistant[18].Thus,these remainingCSCcouldpotentiallypromoteanewtumorgrowthfollowingtreatment ofchemotherapy,ultimatelyleadingtodevelopingacolonyofmultidrugresistant tumorcells. 1.3.2Monoclonalantibodies Herceptin'shighanityforHER2isadirectcausefromthetwoantigen-specic siteswhichtargettheextracellulardomainofHER2[28,53].Followingthebindingof Herceptintoitsreceptor,thereareseveralphenotypicchangesincluding:inhibition ofdimerization,antibody-dependentcellcytotoxicityandinductionofcellapoptosis andinhibitionofangiogenesis[28]. PerhapsthemostprevalentmechanisminwhichHerceptinmitigatescellsignaling istheinhibitionofHER2/HER3dimerization.Furthermore,asHerceptinalleviates theHER2heterodimerization,itsdownstreamsignalingpathway,thePI3K/Aktpathway,isthussuppressed.Thisinturninhibitscellgrowthandsurvival,furtherpromotingcellapoptosis[60].IthasalsobeensuggestedthattheproteinPhosphatase andtensinhomologPTENmaybeinvolvedinHerceptinsabilitytodownregulate thePI3K/Aktpathwaybyregulatingtheintracellularlevelsofphosphatidylinositol3,4,5triphosphateincells[12,6] AntibodydependentcellcytotoxicityADCCprovestobeanimperativemechanismofactionforhowmonoclonalantibodiesultimatelyinhibittumorformation. ADCCinvolvestherecognitionoftheFcregiononanantibodybyareceptorlocated onthesurfaceofanaturalkillercellaneectorcellofimmunesystem[16].Fol7

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lowingthebindingoftheFcregiontothereceptor,theNKcellsreleaseproteinsand proteases,causingthelysisofthetargetedcell[16].Ithasbeenfoundthatfollowing thetreatmentofHerceptin,anincreaseofNKcellsinltratingthetumorwasobserved[3].Furthermore,theseNKcellsdemonstratedanexpressionoftheFcyRIII receptorCD16,conrmingthecellswerelysedviatheADCCmechanismasCD16 isthereceptoronNKcellsresponsibleforbindingtotheFcregion[3]. Figure1.3:MechanismsofactionforthecombinedtreatmentofTrastuzumaband chemotherapeuticagents.Followingtreatments,angiogenisisistheninhibited,ADCC isinitiatedandseveralphenotypicchangesarenoted[44]. Lastly,angiogenesis,theformationofnewbloodvesselsfrompre-existingones, wasfoundtoalsoplayakeyroleintheinhibitionoftumorgrowthfollowingthe treatmentofHerceptin.Herceptinwasshowntomitigatethebloodvesselsdiameter andvolume,yetdidnotaectthelengthofthevessel[30].Thesendingswere comparedtobreasttumorswithoutHerceptintreatment.Thus,asangiogenesiswas signicantlyreduced,tumorgrowthwasthenslowedandmorenormalvasculature 8

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wasobserved[30]. 1.3.3Herceptinresistance MajorityofthepatientswhoinitiallyrespondtoHerceptinacquireresistanceon averageayearfollowingtheinitialtreatment.Themechanismsofresistancehave yettobeelucidated,yetseveralpotentialmechanismsinclude:sterichindranceof thetargetedreceptor-ligand,heterodimerizationofHER2/insulin-likegrowthfactor-1 receptorIGF-IR,andconstitutivelyactivesignaling[46,45]. TherstpossiblemechanismfordevelopingHerceptinresistancewouldinvolve thesterichindranceofaglycoprotein,MUC4,whichbindstoHER2furtherinhibiting thebindingofHerceptinandHER2.Furthermore,asMUC4essentiallyactsasa ligandforHER2,itthusactivatesitsdownstreamsignalingpathwayandeecter molecules[45]. CrosstalkbetweenIGF-IRandHER2wereshowntoalsoactivatetheHER2 downstreampathway.AfterinhibitingIGF-IRinHerceptinresistantcelllinesvia utilizingamonoclonalantibodyspecicforthatparticularreceptor,Herceptinsensitivitywasresumed[46].Bothaforementionedmechanismsresultintheactivationof thedownstreamsignalingpathways,PI3K/Akt,whichplaysakeyroleinHerceptin resistance.Astrastuzumabactstoinhibittheactivationofthispathway,constitutive activationofPI3K/Aktcausedbylistedmechanismscanthenultimatelyleadto evadingapoptosisofthetargetedmalignantcells[45]. 1.3.4Personalizedtherapymayreducedrugresistance Genomesequencinghascontributedtoagreaterunderstandingofcellularaberrationsinmanydiseasesincludingcancer.Theseenlightenmentshaveledtothe developmentoftargeteddrugs,suchasmonoclonalantibodiesortyrosinekinaseinhibitors,whicharespecictocellbiomarkers[24].Thus,thismorepersonalized treatmentrequiresthetumorcharacterizationofeachpatient.Furthermore,asthere 9

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existsbreastcancertumorswhichdonotoverexpressHER2,theresponserateto treatingthesetumorswithHerceptinwouldbeminimal. Aspreviouslystated,acquireddrugresistancecontinuestobeasignicantclinicallimitationtochemotherapybutalsomonoclonalantibodies,suchasHerceptin. ThemechanismsofchemotherapeuticandHerceptindrugresistancewerediscussed previously.Itwascontributedtothatofatumor'simmenseheterogeneityofgenetic aberrationsthatatumorcancontinuetoproliferatedespitetreatments[24].Gonzalezet.alproposedasolutiontomitigatethisinherentchallengecouldbea combinatorialtreatmentincludingalltargetedmoleculartherapiesttingthetumor prole. Utilizingpersonalizedtherapyexhibitsgreatpotentialforovercominginitial chemotherapeuticandHerceptinresistance.Asthisdrugdeliveryvehicleservesto targetthemalignantcells,ittheoreticallyhasthecapacitytoincreasethetherapeuticeectsofundergoingtheseharshtreatments.Thenon-malignantcellsshould theoreticallynotbereceivingchemotherapeutictreatmentandthusshouldremain withnormalfunction.Drugdeliverycouldpotentiallyupregulatetheimmunesystem whencomparedtocurrentchemotherapytreatment.Byutilizingavehicle,itcould inhibittheimmunecellsfrombeingattackedbythechemotherapeuticagents. Furthermore,byutilizingadrugdeliveryvehiclewithcombinatorialtreatments targetingtheentiretyofatumor'sgeneticaberrations,thetumorcantheoretically beattackedonmultiplelevels. 1.4Objectiveofthisstudy Themainobjectiveofthestudywastodevelopandoptimizeamultifunctional drugdeliveryvehicletoserveasanultrasoundcontrastagentandtodeliverchemoagentsatthesiteofatumor.HER2+breastcancerwaschosenasthemodelforthis vehicle.Threedierentmorphologiesofmesoporoussilicananoparticleswereutilized spherical,hexagonalrodandamorphousandcomparedinordertodecipherwhich 10

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morphologyhasthehighestcapacitytoincreasetherapeuticanddiagnosticresults. Furthermore,auorocarbonwaschosentoenhancethepixelintensityandwasconjugatedtothesurfaceofeachmorphologyMSN.Allthreemorphologieswerecompared toeachotheraswellastheiruorocarbonconjugatedcounterparts. 1.4.1Aimsofthisstudy Theobjectivesofthisstudycanbesummarizedasfollows: Specicaim1:DevelopthreeseparatemorphologiesofMSNSpherical,hexagonaltubeandamorphousandsuccessfullyconjugateauorocarbontothesurface. Specicaim2:QuantifythemeanpixelintensitiesproducedbyeachMSNwith andwithoutuorocarbonconjugation. Specicaim3:ObserveanypreferentialbindingofthenanoparticlesandHER2+ cells,oranydierencesinendocytosisofthenanoparticlebytheHER2+cells. 1.5Thesislayout Chapter1containsanintroductiontothescopeandmainobjectivesofthisthesis. Chapter2presentsanoverviewoftargeteddrugdeliveryvehicles,currentultrasoundcontrastagentsandtheirproposedmechanisms,passivevs.activetargetingof drugdeliverymodalitiesandPegylation. Chapter3reviewspreliminarydataandintroducestheproposeddesignanddevelopmentofthismultifunctionaldrugdeliveryvehicle. Chapter4coversmaterialsandallmethodologiesincludingthesynthesisand surfacemodicationsandallexperimentaldesignsusedinthisstudy. Chapter5containsresultsanddiscussion. Chapter6comprisestheconclusionsandsuggestionsforfuturework. 11

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2.Literaturereview 2.1Targeteddrugdeliverymodalities Drugdeliverymodalitieshavebeendiscovered,synthesizedandutilizedtodeliver drugsmoreecientlyoverthecourseofthelastfewdecades[20].Forthescopeofthis thesis,abackgroundondrugdeliveryvehiclesutilizedforcancertreatmentswillbe discussed.Targetedcancertherapeuticsweredevelopedtoamplifythedruguptake ofthemalignantcellsandtomitigatedruguptakeofnon-malignantcells,further maximizingtherapeuticeciency[1]. Nanoparticlesarecharacterizedaslessthan1microninsizeandhavevastformulationsinmaterialsaswellasmodications.Severaldrugdeliveryvehicleshave beendevelopedfromdisparatematerials,includingliposomes,polymer-based,micelles,dendrimersandsiliconoxidenanoparticlesamongstothermaterials[1,59].As thesenanoparticlesarecapableofseveralchemicalmodications,manydrugdeliverydevelopmentshavebeengearedtowardsynthesizingmultifunctionalmodalities. Nanoparticlesalsohavethecapabilitytoaccumulateatthesiteofthetumorthrough enhancedpermeabilityandretentioneectsaswellasthroughactivetargetingvia antibodiesexplainedfurtherindetailbelow.Surfacemodicationsallowforthese nanoparticlestohaveprolongedsystemiccirculationtimesaswellasinhibitnonspecicproteinbinding[11].Duetothisnonspecicproteinbinding,ithasbeenshown thatahybridnanoparticle-polymersystemallowsfora10to100foldincreaseof drugreachingthetumorasopposedtofreechemotherapeuticagentsalone[59].Furthermore,nanoparticlesareinternalizedbythemalignantcellsthroughendocytosis, increasingthekillingcapacityofthechemoagentsreachingthenucleus[59].Thereare severalnanoparticleswhicharecurrentlyundergoingclinicalinvestigation,including liposomaldrugdeliverysystemsi.e.Doxil R ,Abraxane R orthemultifunctional ironoxidenanoparticles,whichserveasanMRIcontrastagentaswellasdrugcarrier Combidex R [20]. 12

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Figure2.1:Depictionofdisparatetargeteddrugdeliveryvehiclesa.polymeric nanogel,b.polymericmicelle,c.goldnanoparticle,d.ironoxidenanoparticle,e. siRNAentrappedinaliposomalvessel,f.stimuli-responsivecappedMSN[38]. MesoporoussilicananoparticlestypeMCM-41wererstsynthesizedintheearly 2000sandhassincebeenutilizedandoptimizedtoserveasanecientdrugdelivery vehicle[56].Silicahasbeenviewedasanadvantageousmaterialasitsrelativelysimplisticcondensationreactionscanbeeasilyfunctionalized,ithasaporousstructure suitablefordrugdelivery,isscalableandiscost-eective[54,62,56].Therehave beenseveralcurrentstudiesonoptimizingthesilicananoparticlessize,morphology, porosityandsurfacecharacteristics[54].Inanotherstudy,itwasdeterminedthat mesoporoussilicananoparticleswithhighersurfaceareasservetobeamoreideal platformastheywereshowntohavelongersystemiccirculatoryhalf-livesaswellas 13

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havethehighercapacitytoundergosurfacemodications[27]. 2.1.1Eectofparticlesizeandmorphology Thereareseveralparameterswhichcandeterminethesystemiccirculatoryhalflifeaswellassuccessfulendocytosisofmalignantcellsasopposedtonon-malignant cellsincludingsize,shape,chargeandsurfacehydrophilicity[21,20,1].Thus,by manipulatingtheseparameters,theabilityofthenanoparticlestobeinternalizedin malignanttissuescanbeenhanced. Ithasbeenshownthatthesizeofthenanoparticleshasanimmenseeectonits capabilitytoextravasatethebloodstreamaswellasparticleuptake[1,21].Smaller particleshavealowerretentionwithinthetumortissueandthushavethecapability totargetnon-malignantcells;however,thiscanbemitigatedbyutilizingactivetargeting[1].Onthecontrary,largerparticles > 200nmarerecognizedmorereadily bythemononuclearphagocyticsystemMPSandcanberemovedfromtheblood viamacrophages.Particlesizealsoaectshowtheparticlesareclearedwithinthe body.Ithasbeenfoundthatnon-functionalizedparticlesof1-5micronstendto accumulateintheliverwhereaslargerparticlesthansuchtendtogettrappedin capillaries[13]. Disparateparticlesizesnotonlyhaveanimmenseeectontargetingthetumortissue,butalsohaveanimpactoncellinternalization.Itwasfoundthatsilica nanoparticleswithadiameterof50nmwasthemostlikelytobesuccessfullyendocytosedwhencomparedtosilicananoparticlesofsize30nm,110nm,170nmand 280nm[39].Itwasthusreportedthattheoptimumparticlesizeforcelluptakewas roughly50nm,butwasalsodependentonthesurfacechargeoftheparticles[39]. Anotherstudyobservedthatdespiteallnanoparticlesbetweentherangeof2-100nm havinganimpactoncellsignalingprocess,theparticleswithadiameterof40and50 nmdisplayedthegreatestimpact[31].Alternatively,ithasalsobeenreportedthat themostecientsizeforendocytosiswasroughly200nmandthatparticleslarger 14

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Figure2.2:Eectofparticlesizeonextravasationofbloodstream.Smallerparticles tendtohavealowerretentionwithintumortissue,whereaslargerparticlesmaynot beabletotraversethegapbetweenendothelialjunctions[21]. than1micronwereobservedtohaveminimalcellularuptake[54]. MSNswhenendocytosed,wereshowntoundergodissolutionwithinthelysosome [11].ItwasalsodeterminedthattheconcentrationofSilicawithintheculturemedia increasedovertime,indicatingthatthecellswereconsistentlyexcretingSilicaion. Severalstudiesindicatedthatmesoporoussilicananoparticlesdegraderapidlywithin thersttwodaysfollowedbyaslowdegradation[11]. Ithasbeenshownthatparticleshapealsotakeseectbothoncellinternalization andextravasationofthebloodstream.ItwasshownthatcylindricalcationicPEGbasedparticlesatallsizeshadahigherinternalization75%asopposedtocubic cationicPEG-basedparticles[25].Itwasalsoshownthatthecylindricalparticles 15

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withadiameterof150nmandheightof450nmhadafasterinternalizationrateas opposedtothemoresymmetricalcylindricalparticlesd=200nm,h=200nm[25]. Inthisstudy,thelongrodparticles450nmwereshowntobeinternalizedata fasterratethanboththeshorterrodparticles240nmandsphericalparticles100 nm[27].Championetal.concludedthattheinternalizationrateofdisparateshaped particleswasdeterminedbywherethemacrophagesinitiallyattachedtotheparticles. Furthermore,itwasdeterminedthatanellipsewasendocytosedmoreswiftlyatthe pointedendsasopposedtotheatsurfaceoftheparticle[13]. 2.1.2Passiveandactivetargeting Nanoparticleshavethecapabilityofundergoingbothpassiveandactivetargeting. Asbloodvesselsintumorstendtobeofirregularshapeandsize,thisleakyanddilated characteristiccanbeutilizedtoenhancethepermeabilityofthesenanoparticles. Furthermore,intumorvasculaturetheendothelialjunctionshavebeenshowntobe largerinsizedependingontumortypeandgrade.Thisallowsforthenanoparticles tobeabletoextravasatefromthebloodstreamintothetumormoreeasily[1].Many particleswhichextravasateintothetumorhaveanenhancedretentionrateastumor tissueshaveahighertendencytolacklymphaticvesselsortheyareshowntobenonfunctional[7].Thisenhancedpermeabilityandretentioneecthasbeenutilizedfor cancertherapeuticsandhasbeenshownthatencapsulatedchemotherapeuticagents accumulatewithinatumoratarateof10-100timeshigherasopposedtothefree chemotherapeutic[1]. Nanoparticlescanbeusedforactivetargetingbyconjugatingabiomarkerspecicallyseekingaknownlocation[7].Knownbiomarkers,suchasmonoclonalantibodies, onceconjugatedtoadeliveryvehiclewillresultinahigherdrugconcentrationinthe malignanttumortissueasopposedtothenon-malignanttissue[1].Furthermore, activetargetingcanpromotereceptor-mediatedendocytosisaswellasantibodydependentcellcytotoxicity,whichwillallowfortheinternalizationoftheparticle,and 16

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Figure2.3:Passivevs.activetargetingofdrugdeliveryvehicles.Thetargeted nanoparticleshaveanantibodyconjugatedonthesurfaceinordertoactivelytarget aknownrecpetoronmalignantcells,whereasnon-targetedparticlesrelyonenhanced permeabilityandretentioneecttotargetmalignantcells.[21]. thusthedrugwilleitherbereleasedatthesurfaceorthroughendocytosis[3].Activetargetinghasthepotentialtoenhanceearlydetectionastheycanthenserve asanoninvasiveimagingtechnique.Furthermore,oncesuccessfulbindingofthe antibody-vehicledevicetothedesiredlocation,detectionofaparticulartypeorstage ifmetastaticofdiseasecanthenbedetermined[7,43].Furthermore,amoremultifunctional,targeted-nanoparticlesystem,alsoduo-ingasacontrastagent,could potentiallyserveasanoninvasivedetectionofpathology[7]. 2.2Ultrasoundcontrastagents Aspreviouslystated,ultrasoundcouldpotentiallyserveasanattractiveimaging modalityduetoiteliminatingtheriskofradiation,itsrelativelycost-eectiveness anditsabilitytoproduceimagesinreal-time[57].Essentially,anultrasoundimage istheanalysisoftheacousticwavesbeingreceivedbackafterbeingtransmitted throughtissues.Dierencesintissuesproducedisparateacousticechoesbacktothe 17

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receiverandthusanacousticimagecanbedetected[41].Ultrasoundhasbeenknown tohaveseverallimitations,suchasimagedistortion,lowresolutionandlowcontrast [35,55].Thesedistortions,though,couldpotentiallybealleviatedwithincreasingthe contrastofthetargetedtissue[35,55].Furthermore,contrastagentsweredesignedto produceadensitymismatchbetweenthecontrastagentandthesurroundingtissues [48].Thereareseveraldevelopedmethodswhichwillbediscussedingreaterdetail belowforincreasingthecontrastofthetargetedtissueincludinggasmicrobubbles andperuorocarbon-emulsednanoparticles. 2.2.1Gasmicrobubbles Gasmicrobubbles,thecurrentclinicalstandardasultrasoundcontrastagents, wererstdiscoveredinthelate1960sandwereproventoenhancevascularcircuitry lastingonlyafewminutesbeforethebubbleswerethendissolved[9].Sincethen, manypharmaceuticalcompaniesarefocusingonoptimizingthesemicrobubblesand havemadesignicantprogressinvariouseldsincludingcardiologyandradiology [22]. Gasmicrobubbleshaveadiameterrangingfrom1-7microns,whichcausestheir resonancefrequenciestoliewithintheUSfrequencyrangeusedforclinicalpractice, around2-10MHz[49,9].Microbubblesarecomprisedofanoutershelldenatured albumin,phospholipidsorsurfactantswhichencapsulatevariousgasese.g.peruorocarbon[49,17].Thevariationinpixelintensitybetweenthemicrobubblecontrastagentsanditssurroundingtissuesisderivedfromthemicrobubblesharmonic vibration,whereastissuesarenearlyincompressible[17,9].Furthermore,thesemicrobubblesarethenhighlyechogenicduetothisdierenceinmaterialproperties betweenitselfcompressedgasanditsadjacenttissues.Disparatefrequenciesare thenproducedanddetected[7]. Ithasbeenfoundthatdespitethestarkcontrastinimpedancefromthesecond harmonicimagingoftissue/gasmismatch,therecontinuestobeagreatlimitationfor 18

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Figure2.4:Commonpropertiesofultrasoundcontrastagents[33]. thegasmicrobubbles.Thesegaseousbubblesorparticleshavebeenfoundtohavea limitedshelf-lifeoflessthanoneweekwhenstoredat4 C[55].Thus,amorestable solutionhasthepotentialtobeamorerealisticapplication. 2.2.2Eectofmicrobubblesizeonultrasoundcontrast Theeectofparticlesizeforeithermicrobubbleornanoparticlecontrastagents haverecentlybeenstudiedinordertodiscoveranoptimalsizeproducinganimpedance mismatchaswellasanoptimalcirculatorydurationwithinthebody.Manycurrent clinicalultrasoundcontrastagentswerefoundtohaveahighpolydispersity,suchas Denity R LantheusMedicalImaging,USA,havingarangeofmicrobubblesfrom onetotenmicrons[51].Sirsietal,followingthesuccessfulisolationofmicrobubbles betweenspecicranges-2microns,4-5microns,6-8micronsobservedtheeects ofthecontrastemittedinaninvivomodelutilizingahighfrequencyultrasound modality.Itwasfoundthatthecontrastincreasedwithincreasingmicrobubblesize. Thedurationofthemicrobubblesincirculationalsoincreasedwithsize[51].For practicalclinicalpurposes,anoptimalsizemustbeobtainedwhichmaximizesthis acousticbackscatter,yetisabletosuccessfullytraversecapillarieswithoutcausing anyocclusions[48]. 19

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2.2.3Peruorocarbonemulsednanoparticles Fluorocarbonnanoparticleshavebeenwidelyusedasanenhancedultrasound contrastingagent,howeverallpriorsystemshaveusedanemulsion-basedsystem typicallyutilizingaliquid,volatileperuorocarbon[64,55].Similarlywithgasmicrobubbbles,theimpedancemismatchbetweenthegasnanoparticlesandthesurroundingtissuesgivesrisetotheenhancedultrasoundimages[55].Aboveacertain pressureappliedviatheultrasound,theseliquidperuorocarbon-emulsednanoparticlesarevaporizedintotheirhighlyechogenicgaseousstate[14].Nanoparticleslled withperuorocarbonliquidareknowntohavealongerlifespanwithintheblood streamascomparedtogasmicrobubbles,whichhavearelativelyshorterlifespan [32,35]. Inotherstudieselucidatingthismechanism,itwasfoundthatthisultrasound vaporizationprovokesaresponsepropellingtheparticlesforward,ataratedependentonthecharacteristicsoftheuorocarbonchosen[32].Whileuorocarbonsor peruorocarbonsarecomposedofdisparateproperties,likeboilingpoint,whichmay ultimatelydecipheritsexpansion,motionandthusvelocity;ithasbeendetermined thatuorocarbonsincreasepixelintensityinultrasounddueinparttothisbullet-like propulsion[32]. 2.2.4LimitationstogasmicrobubblesandPFC-emulsednanoparticles Aspreviouslystated,thereexistsseverallimitationstocurrentclinicalUScontrastagents.Therstlimitationwouldbetherelativelyshortshelf-lifeofgasmicrobubbles,dependingonthematerialusedforencapsulationi.e.lipidshellvs.solid shell[55,22].Microbubblescomposedwithlipidshellstendtobelessstable,yet werefoundtobehighlyechogenic[22].Itwasfoundthattheselipidmicrobubbles, whenundergoingacousticwaves,theshell"expands,ruptures,reseals,compresses, 20

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bucklesandrespreads"ateverycycle[22].Thismechanismofaction,albeitproducinghighlycontrastimages,isalsolimitedtoarelativelyshortsystemiccirculatory lifespan. Optison TM GEHealthcareisoneofthecurrentFDAapprovedPFCemulsed ultrasoundcontrastagentcomposedofaalbumin-coatedhollowshell,encapsulatingoctauoropropane[22,48].Thisuorocarbon-emulsedbubblewasfoundto haveamuchlongercirculationtimethanitsinitialair-encapsulatedcounterpart Albunex R ,duetothelowersolubilitypropertiesoftheFC[48].Thischaracteristicwasconcludedtoallowforahigherretentionofgasmicrobubblesinaqueous solutionforalongerduration[48].Despitethisobservedincreaseinciculatoryduration,thecirculationlifespanwasstillfoundtobelessthan10minutesFigure2.5 afterappliedpressurefromacousticenergy[48].Figure2.5belowdepictsthisreductionintheOptison R gasmicrobubblesfollowingtheapplicationofagas-decient buersolutionwasstatedtobeidenticalresultsasforapplyingacousticwavesover severaltimepoints. 2.3Pegylation ByconjugatingpolyethyleneglycolPEGonthenanoparticles,thebiocompatibilityhasbeenshowntobegreatlyincreasedastheparticlesarenotaectedby non-specicproteinbinding[11].PolyethyleneglycolisanFDAapprovedhydrophilic polymer,whichisfrequentlyusedtomodifythesurfaceofnanoparticles[56].ByconjugatingPEG,thesystemiccirculatoryhalf-lifehasbeenshowntobevastlyincreased asopsonizationisdelayedorprevented,andthustheparticlesarenotrapidlycleared bythereticuloendothelialsystemRES[11,56].ItwasalsoshownthatbyfunctionalizingthesurfaceoftheparticleswithPEG,thedegradationratewasslowerwhen comparedtonon-functionalizednanoparticles[11].Unmodiedsilicananoparticles wereshowntoloseitsstructuralcharacteristicsi.e.porosityfollowing1monthof thestudy[11]. 21

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Figure2.5:ThedegradationofOptison R microbubblesfollowingtheintroductionof agas-decientbuersolutionaftertimepointsa.0minutes,b.1.5minutes,c.4 minutesandd.6minutes.[48] WiththeadditionofPEGcomesgreatbiocompatibilityenhancements,butalso hasotherknownbenetsforclinicalapplications.Ithasbeenfoundthatbyconjugatingacertainpercentageofpolyethyleneglycol,thenanoparticle-polymersystem canredisperseinsolutionfollowinglyophilizationmoreeasilythanitsnon-pegylated counterpart[55]. 22

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3.PreliminarydataandproposeddrugdeliveryModality 3.1Preliminarywork PreliminaryworkhasbeencompletedinDr.DaewonParksTranslationalBiomaterialsResearchLaboratoryperformedbyA.Milgroom,M.Intrator,andK.Madhaven.AllpreliminaryworkutilizedamorphousMCM-41MSNs. Mesoporoussilicananoparticleshavebeenshowntobebiocompatibleandalso haveshowntoemitultrasoundcontrast.Inpreliminarystudies,polyacrylamidephantomsandtheamorphousnanoparticleswereimagedwithvariousconcentrationsof nanoparticles.1mg/mL,0.3mg/mL,0.5mg/mLand1.0mg/mL.Polyacrylamide waschosenasaphantomforitssimilaritytosofttissue.Preliminaryworkperformed onamorphousMSNexhibitedhigherpixelintensitiesgivenhigherMSNconcentrationswithinthephantoms. Figure3.1:PreliminaryworkdepictingtheMPIemittedbyamorphoussilicananoparticles[42]. ThecellularinteractionbetweenamorphousMSN-Herceptin-FITCwasobserved usinguorescentmicroscopy.ComparingtheinteractionbetweenHER2+andHER2celllinescanrevealtheeciencyofMSN-Herceptin-FITCanditspreferencetoward bindingtoHER2overexpressingcelllines.PreliminaryworkonamorphousMSNhas 23

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conrmedthebindingandinternalizationoftheMSNinHER2+cellswithfewattachmentstoHER2-cells[3].Flowcytometrywasperformedtoquantifythebinding eciencyofMSN-Herceptin-FITCtoHER2+andHER2-celllinesforamorphous MSN. Figure3.2:Preliminaryinvitrostudiesdisplayingthecolocalizationofamorphous MSNwithHER2overexpressingbreastcancercellline[42]. ByfurtheroptimizingthemorphologyandfurtherfunctionalizingtheseMSNs, anoptimalmultifunctionaldrugdeliveryvehiclecanthenbedeciphered.Spherical, tubeandamorphousmorphologiesweretestedandcomparedfortheirabilitiesto enhancepixelintensityaswellastotestforpreferentialbindingtowardsHER2+ breastcancercells.Furthermore,theconjugationofauorocarbonwastestedforits abilitytofurtheraugmentthepixelintensityinultrasoundimaging. 3.2Proposeddrugdeliverymodality Mesoporoussilicananoparticleswerechosentoserveasthedrugdeliveryvehiclefortheirrelativeeaseinsynthesis,theyarereadilyfunctionalizableandhavea porousstructuresuitablefordrugdelivery[54,62].Itwasposedtovarytheshape ofthesesilicaparticlesandcompareeachinit'sabilitytoserveasanultrasound contrastagentaswellascompareendocytosisratesbetweenthem.Furthermore,as 24

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itwaspreviouslyfoundthatMSNareslightlyechogenicalone,itwasproposedto conjugateauorocarbononthesurfaceofeachparticleinordertofurtherenhance thisechogenicity[42]. Itwaspredictedthattheuorocarbonconjugatedparticleswouldfollowasimilar mechanismtothePFC-emulsednanoparticles,aspreviouslynoted.CurrentultrasoundcontrastagentswhichutilizealiquidPFCreportaphase-transitionfollowing theintroductionofacousticwavestoitshighlyechogenicgaseousstate[55,64].Furthermore,thisimpedancemismatchbetweenthetissue/gasresultsinahigherMPI [8].Aspreviouslystated,theshortshelf-lifelessthanoneweekwhenstoredat4 C ofthesegaseousparticlesposesasignicantlimitation[55]. Inordertofurtherincreasetheshelf-lifeoftheseparticles,yetstillpreservethe highercontrastproducedbytheseenhancedparticles,itwasdeterminedtoconjugate aPFCtothesurfaceoftheMSN.Ithaspreviouslybeenreportedthatfreeze-drying gaseousparticlestoincreasethemicrobubble'sshelf-lifesignicantlyreducestheir echogenicfactorandwereobservedtocollapseorfuseduringtheprocess[55].By conjugatingtheuorocarbontothesurfaceoftheparticles,theshelf-lifewouldinherentlyincreaseastheparticlesnolongerhaveagaseouscorewhichwouldhavethe potentialtocollapse. Notonlywouldconjugatingtheuorocarbontothesurfacepotentiallyenhance theshelf-lifeoftheparticles,butitwasalsoposedtoenhancethedurationofsystemic circulationtimewithinthebody.Gaseousmicrobubbleshavebeenreportedtohave arelativelyshortlifespanincirculation,lastingonlyafewminutes[9].Bycircumventingagaseouscore,theparticlescouldenhancevascularcircuitry.Severalstudies indicatedthatmesoporoussilicananoparticlesdegraderapidlywithinthersttwo daysfollowedbyaslowdegradation[11].Thisdegradationrateisinherentlyslower thantherapiddegradationofgasmicrbubbles. 25

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Figure3.3:Proposedmechanismofactionofourdesigneddrugdeliveryvehicle. Notonlywillthisproposeddrugdeliveryvehiclehavethepotentialtoenhance diagnosticeects,butitwasproposedtocombinebothpassiveandactivetargetingof themalignantcells,whichcouldenhancethetherapeuticeectsaswell.Herceptin,or Trastuzumab,waschosenastheantibodytobechemicallyconjugatedtothesurface ofthenanoparticles.Thus,theantibodywouldservetoactivelytargetthemalignant cells.Bycombiningactiveandpassivetargeting,theproposedmodalitywouldhave thepotentialtomitigateoralleviatedrugresistance. Itwasrstproposedtoconjugatetheuorocarbontothesurfaceofhydroxylated MSN.Alimitationwasreachedwiththisstepduetotheimmensehydrophobicity oftheMSN-FCparticles.ThislimitationisdiscussedindetailintheResultsand Discussionportionofthisthesis.Tocircumventthislimitation,theparticleswere Pegylatedpriortoconjugatingtheuorocarbon. 26

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4.Materialsandmethodology 4.1Materials MCM-41typehexagonalMSNs,hexamethylenediisocyanateHDI,anhydrous dimethylformamideDMF,anhydroustolueneanduorosceinisothiocyanateFITC werepurchasedfromSigma-AldrichSt.Louis,MO,USA.N-cetyltrimethylammonium bromideCTAB,2MsodiumhydroxideNaOH,tetraethylorthosilicateTEOS, PolyethyleneglycolPEGMW1000andMW4000,carbonyldiimadazoleCDI,anhydroustoluene,anhydrousmethanolandwerepurchasedfromAlfaAesarWardHill, MA,USA.1XPhosphatebueredsalinePBS,Trypsin0.5%,DMEMF12media andpenicillin/streptomyocinandanhydrousdiethyletherwerepurchasedfromFisher ScienticPittsburgh,PA,USA.Pentauorophenylpropyl-trimethoxysilanewaspurchasedfromGelest.3-AminopropyltrimethoxysilaneAPTMSwaspurchasedfrom TCI.HydrochloricAcidHClwaspurchasedfromBDI.Trastuzumab,SKBR3cell lineandHoechststainweregenerousdonations. 4.2MSNpreparation 4.2.1MSNamorphouspreparation MCM-41typehexagonalMSNsweredispersedin200proofethanolat5mg/mL. Thesolutionwasthensonicatedfor10minutesandwerelteredusingaFRITask. 4.2.2MSNsphericalsynthesis Ina1000mLroundbottomask,CTABg,0.00274molwasdissolvedin 480mLnanopurewaterfollowedbytheadditionof2MNaOH.5mL,0.007mol. Thesolutionwasheatedto80 Candrapidlystirreduntilthereisavortextothe bottomoftheask.Next,TEOS.07mL,0.041molwasaddeddropwiseintothe solution.Thesolutionwasreactedfor2hoursat80 Cwithvigorousstirring.The solutionwasthenlteredusingaFRITaskandwaswashedwithnanopurewater andanhydrousmethanol.Toremovethesurfactantmolecule,CTAB,driedspherical 27

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MSNwereplacedina250mLaskanddissolvedin100mLanhydrousmethanol. Thesolutionwasthensonicatedfor15minutes.Next,HClmLwasaddedtothe solution.Thiswasthenreuxedfor18hoursat65 Candrapidlystirreduntilthere isavortextothebottomoftheask.ThesolutionwasthenlteredusingaFRIT askandwaswashedwithnanopurewaterandanhydrousmethanol. 4.2.3MSNtubesynthesis Tube-shapedMSNweresynthesizedusingasimilarmethodassphericalMSN. Ina500mLroundbottomask,CTABg,0.00274molwasdissolvedin120mL nanopurewaterfollowedbytheadditionof2MNaOH.75mL,0.0035mol.The solutionwasreactedatroomtemperaturewithvigorousstirring.TEOS.5,0.0112 molwasaddeddropwiseintothesolution.Thesolutionwasreactedfor24hours atroomtemperaturewithvigorousstirring.Thesolutionwasthenlteredusinga FRITaskandwaswashedwithanhydrousmethanol.Thesamemethodfortube MSNsurfactantremovalwasfollowedasdescribedinsphericalMSNportion. 4.2.4MSNsurfacehydroxylation TohydroxylatethesurfaceoftheMSNs,thefollowingprocedurewasfollowed: Driedsurfactant-removedMSNweredissolvedinnanopurewatermL/gMSN. Thesolutionwasthensonicatedfor10minutes,andreactedfor4hoursat70 Cwith moderatestirring.ThesolutionwasthenlteredusingaFRITaskandwaswashed withnanopurewaterandanhydrousmethanol. 4.2.5HDIconjugation Surfacehydroxylated,surfactant-removedMSNweredissolvedin5mLanhydrous DMF.ThesolutionwasthenreactedwithexcesshexamethylenediisocyanateHDI mL,0.03molat60 CCfor24hourswithmoderatestirring.Thesolutionwas thenprecipitatedincoldanhydrousether.Oncetheprecipitatesfelltothebottomof theask,theetherwasremovedbypouringothesupernatant.Coldanhydrousether 28

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wasthenaddedagain.TheMSNsolutionwasthensonicatedfor10minutes.This stepwasrepeatedtwicesupernatantdiscarded,anhydrousetheradded,sonication. ThedispersedMSNsolutionwasthentransferredtoa50mLfalcontubeandwas centrifugedfor5minutesat5000rpm.Thesupernatantwasthenremoved. 4.2.6Pegylation ThedriedMSN-HDIconjugatedparticlesweredissolvedin5mLanhydrousDMF. Simultaneouslyinanotherask,PEGMWg,0.005molwasdissolvedin 2mLanhydrousDMFandwasheatedto60 CCfor5minutesoruntilcompletely dissolved.Afterward,theMSNsolutionwasthenaddedtotheexcessPEGsolution andwasthenreactedat60 CCfor24hours.Thesolutionwasthenlteredusing aFRITaskandwashedwithanhydroustoluene.*FortubeMSNsynthesis,PEG 4000MWwasused. 4.2.7Flourocarbonconjugation ThedriedMSN-HDIconjugated,PEGylatedparticleswerethendissolvedin mLofanhydroustoluene.Thesolutionwasthensonicatedfor15minutes.Immediatelyfollowing,theuorocarbonpentauorophenylpropyl-trimethoxysilanemM wasadded.Thesolutionwasreuxedfor24hoursat95 C.Thesolutionwasthen lteredusingaFRITaskandwaswashedwithnanopurewaterandanhydrous toluene. 4.2.8FITCconjugation FITCwasutilizedasauorophoretouorescetheMSNsforuorescentmicroscopy.First,FITC.0mgwasdissolvedin500 LanhydrousDMF.Next, APTMS.0 LwasaddedtotheFITCsolution.Thesolutionwasreactedfor30 minutesatroomtemperaturewithmoderatestirring.FITC-APTMSwasgraftedto theMSNforfurthersurfacefunctionalization.First,theMSNweredissolvedin100 mLofanhydroustoluene.Thesolutionwasthensonicatedfor15minutestofurther 29

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dispersetheparticles.TheFITC-APTMSsolutionwasthenaddeddropwisetothe MSN/toluenesolutionandwasreactedfor24hoursat80 Cwithmoderatestirring. ThesolutionwaslteredusingaFRITaskandgaverisetoorangeprecipitates. 4.2.9Herceptinconjugation ForthecouplingofHerceptintotheMSNs,rsttheremaininghydroxylgroups fromthepegylatedMSN'swerereactedwithcarbonyldiimadazoleCDIata1:1 ratioofMSN:CDI.Thesolutionwasreactedat60 CCfor24hourswithmoderate stirring.Followingthereaction,theMSN-CDIsolutionwaslteredusingaFRIT askandwashedwithanhydroustoluene.Second,asolutionwasmadeof0.5mg/mL Herceptinin1XPBS.Next,thedriedMSN-CDIparticlesmg/mLwerethenadded totheHerceptinsolutionandwerereactedfor24hoursatroomtemperaturewith moderatestirring.Next,thesolutionwasthencentrifugedfor10minutesat3000 rpm.Thesupernatantwasthendecanted.Thisstepwasrepeatedtwotimesmore centrifugationandremovalofsupernatant.MSN-Herceptinwerestoredin1XPBS at60 C. 4.3StructuralCharacterization StructuralcharacterizationoftheMSNafterallconjugationswereobtainedusingfouriertransforminfraredspectroscopy.10mgofeachMSNsampleaftereach conjugationweredissolvedin200 LanhydrousTHF.Eachsamplewassonicated forfurtherdispersion.AnaliquotofeachsamplewasplacedonapolyethyleneIR cardInternationalCrystalLaboratories.TheFTIRspectrawerecollectedonan Nicolet6700FT-IRspectrometerfromThermoElectronCorporation. 4.4Electronmicroscopy ToconrmthemorphologyofsynthesizedMSN,imagesweretakenusingscanningandtransmissionelectronmicroscopy.SEMandTEMwastakenatUniversity 30

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ofColoradoAnschutzMedicalCampusandUniversityofColoradoMines.AlltransmissionelectronmicroscopysampleswerepreparedbytakinganaliquotoftheMSN anddispersingin1mL200proofethanol.Thesamplewassonicatedfor10minutes. Immediatelyfollowing,onedropofthesolutionwasplacedonthecarbongridand letdryintheovenfor1minute.TEMimagingperformedusingaHitachiH-7650 operatedat60kVorJEOLJSM-1400+operatedat120kV.Forscanningelectron microscopy,thedriedMSNpowderwasmountedonSEMstubwithdouble-sided carbontapeSputterCoatwithGold/PalladiumLeicaEMACE200for30seconds. SEMimagingwasperformedwithSEMJEOLJSM-6010LA. 4.5Zetasizer Nanoparticlesamplesmgweredispersedin1mLofdH2O.AMalvernZeta Sizer2000MalvernIntruments,USAwasusedtoperformthisassay. 4.6Ultrasoundapparatus Single-pulseultrasoundmeasurementswereobtainedusingNon-DestructiveTestingNDTwithafrequencyof6.6MHzatadepthof2.5cm.GE-Panametric.All transducerswereAccuscantypeSimmersiontransducerswithpointtargetfocus PTF.RecieversignalwasacquiredonanInniium8000HighPerformanceOscilloscopeAgilentTechnologies. 4.6.1Quantifyingpixelintensity Themainpurposeoftheultrasoundassaywastoquantifytheaveragepixel intensityforeachofthesynthesizednanoparticles,withandwithouttheuorocarbon conjugation.TheMSNofvaryingconcentrationsmg/mL,0.5mg/mL,1.0mg/ml, 2.0mg/mL,5.0mg/mLweredissolvedin5mLPBS.Eachsolutionwassonicatedfor 10mintuesfollowedbyplacingthesampleswithindialysistubingSpectra/Por4,25 mm,MWCO12000-14000.Immediatelyfollowingtheadditionofthesamples,the dialysistubingswerethenplacedinaPBSbathontopofanagargel.Agarserved 31

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asatissue-mimickingphantomandalsoservedtoreduceextraneousnoiseemitted intothedialysistubing.Theagargelsweremadebyautoclaving3%agarindH2Oat 250 CFfor30minutes.Theliquidagarwasthenpouredinplasticcontainersand allowedtocool,formingagel.Ultrasoundimagesweretakenatroomtemperature. Threeimagesweretakenofeachsampleateachconcentrationandwereanalyzed usingImageJtodiscoverthemeanpixelintensity.Eachimagewas150x100pixel squareswithanareaof150,000. 4.7Cellpreparation SKBR3cellswerepreparedinasolutionofDMEMF12mediawith10%fetal bovineserumFBSand1%penicillin/streptomyocin.Allcellassayswerecompleted intriplicate.Thecellswereplatedona24wellplateataseedingdensityof50,000 cells/welland100,000cells/well. 4.8Fluorescentmicroscopy Thenanoparticleswereseededat25 g/well.Thenanoparticle/cellsolutionwas thenplacedonashakerandthenincubatedfor30minuteswhileanother24well platewiththesameconcentrationsofbothcellsandnanoparticleswereincubatedfor 2hours.Followingtheincubationperiods,thenanoparticle/cellswerewashedwith PBS1X.Thecellswerethenxedfor15minutesusinga10%Formalinsolution. Followingthexative,thecellswerewashedwithPBS1Xandthenwerestained withHoechstata1:2,000dilutionfor10minutes.Live-deadstainingwasperformed usingethidiumhomodimer-15.1EthD-1.TheFITC-labelednanoparticleandthe stainedcellswereobservedunderauorescentmicroscope. 32

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5.Mainresultsanddiscussion Theresultsanddiscussionsectionconsistsoftwocomponents: -Limitationsofconjugatingauorocarbondirectlytothesilicananoparticles -Characterization,ultrasoundimagingresultsandinvitroworkregardingall threesilicaparticlessphere,amorphous,andhexagonalrod 5.1Fluorocarbonconjugation Mesoporoussilicananoparticlesaresynthesizedthroughaseriesofcondensation reactions.CTABservesasthesurfactantmoleculeandthusformsatemplateforthe TEOStoformaminerallikesubstancesurroundingthem.Surfactantortemplate removalallowsfortheMSNtoacquireaporousstructure.Itisthroughthisporous structurethatallowsachemo-agenttopotentiallybeloadedviadiusion. Thesilicananoparticleswereoriginallyconjugatedtosurface-hydroxylatednanoparticlesbymeanspreviouslymentionedinthemethodologysection,yetconjugatedat ahighermolarconcentrationthanlisted.Itwaspreviouslyposedtoconjugate50 mMoftheuorocarbontothesurfaceofthehydroxylatedparticles.Onelimitation wasreachedwhentheMSN-FCweredispersedin1XPBSsolution;theparticleswere foundtobehighlyhydrophobicandthuswouldnotdispersewithinthePBSbuer solutionfollowingsonicationfor10minutes.Highlyhydrophobicpharmaceuticals areproblematicinregardtoadministeringviainjectablesystemsduetotheirlow solubility[2].Thus,thedevelopmentofamorehydrophilicdevicecouldcircumvent thisocclusion. Figure5.1:ChemicalstructureofMSN-OH-FC. 33

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Itwasfoundtoincreasethehydrophilicityoftheparticlesbymanipulatingtwo modications.1.byconjugatingPEG,ahydrophilicpolymer,thesurfacehydrophilicityofthepolymercanthenbeincreasedand2.bydecreasingtheamount ofthemolarconcentrationoftheperuorocarbonutilizedthusthesurfacehydrophobicityoftheparticleswillthendecrease.ByPegylatingtheparticles,notonlywill thismethodincreasethehydrophilicityandthusallowfortheparticlestodisperse inPBSsolution,italsoincreasesthebiocompatibilityoftheparticles.Aspreviously mentioned,theliteraturestatesthatpegylatingtheparticleswillpreventopsonins fromtaggingtheparticlesforclearancebythereticuloendothelialsystem[11,56]. Figure5.2:ChemicalstructureofMSN-OH-HDI-PEG-FC. 34

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ItwasproposedtoreactthesurfacehydroxylatedMSNwithhexamethylenediisocyanateHDIinordertofunctionalizethesurfacewithanisocyanategroup.The surfaceisocyanategroupwillthenreactwithPEGshydroxylgroups.Theuorocarbon,pentauorophenylpropyltrimethoxysilane,reactedinasimilarmechanismas aminopropyl-trimethoxysilaneAPTMStotheoutersurfacePEGhydroxylgroups. Theinitialnanoparticle-FCsynthesiscanbeseeninFigure5.1,followedbythe nanoparticle-PEG-FCsynthesisinFigure5.2.ThedierencesindispersingtheparticlesindH2OcanvisiblybeseeninthegurebelowFigure5.3.Thesemodications werefurthervalidatedbyFTIRasseenbelow. Figure5.3:ImagestakenofamorphousMSNa.beforepegylationandwithahigher molarratiooftheFCused,andb.afterpegylation,withalowermolarratioofthe FCused. 35

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5.2MSNcharacterization 5.2.1MSNmodicationanalysisbyFT-IR FollowingeachMSNmodication,FourierTransformInfraredSpectroscopyFTIRwastakentoconrmsuccessfulconjugationtechniques.InFT-IR,theabsorbance ofdieringatomscovalentlybondedcanbeseenatcertainwavelengths,andthus abletodisplaysuccessfulconjugation.Allspectradisplayedweretakenutilizing amorphousMSNasthemodeltosuggestsuccessfulconjugations.Thespectraof amorphousMSNwastakenandcanbeseenbelowFigure5.4,Figure5.5.FT-IR wasutilizedtofurtherconrmeachmodicationwassuccessfullyconjugatedsubsequentlytothesurfaceoftheparticles.Thus,followingeachmodicationanaliquot ofthesamplewastakenforFT-IRanalysisandthencomparedtoconrmsuccessful conjugation.ThespectraafterHDIconjugation,Pegylation,FCconjugation,CDI andHerceptinconjugationscanbeviewedbelow. Aspreviouslymentioned,theparticleswerersthydroxylated.Byexaminingthe regionaround3378cm -1 ,thepresenceofsurfacehydroxylgroupscanbeconrmed. ThewavenumberwhichcorrelatestothefreeO-HgroupscanbeviewedatsiteAof Figure5.4[50].ThesurfacehydroxylgroupscanbeseeninboththeMSN-HDIand MSN-HDI-PEGparticles.Thisfurthersuggeststhatfollowingbothsurfacehydroxylationandfollowingpegylation,freehydroxylgroupsresideonthesurfaceofthe particles.WecanthenconrmHDIconjugationbyexaminingtheregion2275cm -1 ThisregioncorrelateswithanisocyanatestretchN=C=O.Thisstretchcanvisibly beseenintheFT-IRspectraBforbothMSN-HDIandMSN-HDI-PEG.Thus,the conjugationofHDIcanbeconrmed. TofurtherconrmtheconjugationofPEG,theregions1362and1287cm -1 canbe analyzed.ThesewavenumbersarecorrelatedtotheC-Ostretching[50].IntheMSNHDI-PEGspectrum,anobviousshiftisfoundfurthersignifyingthepresenceofC-O 36

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bondsC.Theregionsof966and849canalsobeexaminedfortheircorrelationto C-Cstretching[50].ThisregioncanbeobservedatFigure5.4D,furtherindicating thepresenceoftheC-Cbonds.Giventheappearanceofthefreehydroxylgroup A,theC-OstretchingC,andtheC-CbondsD,itcanbedeterminedthat PEGwasconjugatedtotheMSN-HDIparticles.Theconjugationoftheuorocarbon Figure5.4:FT-IRspectratakenofamorphousMSN.RegionAdepictsconrmation offreehydroxylgroupsfollowingsurfacehydroxylationandpegylation.Bdepicts HDIconjugationandC,Dconrmpegylation. canbeconrmedbyviewingregions1050and1132cm -1 .Thisregionisassociated withC-Fstretchmodes.Thereisaclearpeakatthisregionseenbelowatsite AofFigure5.5.Furthermore,theregions1566cm -1 canbeanalyzedtoexamine theC=Cbonds.ThereisadistinctpeakBatthisregionfurthersignifyingthe successfulconjugationoftheuorocarbontotheMSN-HDI-PEGparticles.Following 37

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Figure5.5:FT-IRspectraofamorphousMSN.RegionAwasfoundtocorrelate withuorocarbonconjugation.RegionBdepictsC=Cbondsfoundinboththe uorocarbonandCDI.CDIwasfurtherconrmedthroughregionsCandD,depicting C-NandC=Nbonds. theuorocarbonconjugation,CDIwasthenreactedtotheremainingsurfacehydroxyl groups.Similarly,theregion1566cm -1 canbeanalyzedforC=Cbonds.Once againthereisadistinctpeakforMSN-HDI-PEG-FC-CDIatpeakBatthisregion, conrmingthepresenceofCDI.Forfurtherconrmation,theregions1200,1650cm -1 canbeexaminedforC-NandC=Nbondsrespectively.Thesecanbeobservedat CandDrespectivelyinFigure5.5.Thus,theconjugationofCDIwasfoundtobe validated. 38

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5.2.2Structuralcharacterization TransmissionelectronmicroscopyTEMandscanningelectronmicroscopySEM wereutilizedtoconrmthemorphologyofsynthesizedmesoporoussilicananoparticles.Imagesofspherical,amorphous,andhexagonalrodshapednanoparticlesare displayedbelowFigure5.6.TEMimagesofsphericalnanoparticlesweretakenat theUniversityofColoradoschoolofMines;allotherTEMimagesweretakenatthe UniversityofColorado-AnschutzMedicalcampus.TheimagesA-CdepictasuccessfulsynthesisofsphericalMCM-41of150-200nmindiameterwithgoodporosity,as indicatedbytheporousmembrane.SurfactantremovalstepofMSNsynthesisallows fortheporosityofthenanoparticles.TheSEMimageImageCofthesynthesized sphericalnanoparticlesdisplaysomedispersityinsizeofeachparticle.ImagesD-F weretakenoftheamorphousnanoparticles.ImagesD/Edepictthedisparatemorphologiesandsizesseenintheamorphoussample.TheSEMimagesrepresentedin ImageFrevealamuchlargeragglomerateofgreaterthan10um.Furthermore,the amorphousparticlesappeartohaveadistinctporousstructure,asshownbythered arrowinImageD.Thehexagonalrodshapednanoparticlescanbeseeninimages G-I.Theparticlesappeartobelargerthanthesphericalnanoparticleswithalength ofabout1-2micronsandadiameterofabout1micron.ImageHdisplaysthehigh resolutionTEMimageoftheparticlesporosity.Theparticlesdispersitycanbeseen intheSEMimageImageIanddepictsarelativelyrodshape.Itisevidentthat someresidualsurfactantremainsonthesurfaceoftheparticles,asshownbythered arrowinImageI. Tofurthercharacterizetheparticlesize,thesizeofeachmorphologybothbefore andafterconjugationsweretakenandperformedwiththeZeta-sizer.Allzeta-sizer plotscanbeviewedinAppendixA.Thesphericalnanoparticlesexhibitedarelatively 39

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Figure5.6:ElectronmicroscopyimagesofMSNforstructuralcharacterizationanalysis.A-B.TEMimagesofsphericalMSN,C.SEMimageofsphericalMSN,D-E. TEMimagesofamorphousMSN,F.SEMimageofamorphousMSN,G-H.TEM oftubeMSN,I.SEMoftubeMSN. heterogeneoussizewhenimagedwithSEM,whichwasconrmedwiththezeta-sizer data.Accordingtothelatterassay,thesphericalparticleswerefoundtobeonaverage 900nmwiththerangeofdispersityfrom100nmtowellover1micron.Thisrange isvisiblyapparentintheSEMimageinFigure5.6,imageC. Theamorphousparticleswerefoundtohaveawidearrayofsizesfrom100nmto wellover1um,asshowninimagesD/E.Theseamorphousparticlescouldbereported toevenhaveahigherdiameterofover10microns,asseeninImageF;however,this imagecoulddepictanagglomerationofsmallerparticles.Thezeta-sizerdatawas inconclusiveastheaverageparticleswerereportedtobebetween7and8microns, 40

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yetthereportedpeakswerefoundtobebetween1and5microns.Thislimitation willbediscussedfurtherbelow. Table5.1:Thetablebelowdepictstheaveragesizeofeachmorphologynanoparticle concludedfromthezeta-sizer.EachNP-HERwaspegylatedanduorocarbonconjugatedpriortoHerceptinHERconjugation.Thus,theMSN-HERdepictsthevalue afterallconjugations,MSN-OH-HDI-PEG-FC-CDI-HER. MSN avg1 avg2 avg3 Amorphous787885667342 Sphere917968853 Tube949.610971083 Amorphous-HER756965356255 Sphere-HER492243604400 Tube-HER680266606923 ItisobservedthattheaveragesizeofamorphousMSNwhencomparedtothe averagesizeofamorphousMSN-HERdoesnotappeartoincreaseasvisiblyseen withbothsphericalandtubularMSN.Thisobservationcouldbeduetothehigh heterogeneousdispersitydisplayedinboththeelectronmicroscopyaswellasthe zeta-sizerplotsshowninAppendixA.Itisapparentthatthereisaverydiverse rangeofamorphousparticlesizefrom < 100nmtowellover10microns. Thetubularnanoparticleswerefoundtoberelativelyhomogeneousinsizewhen viewedthroughelectronmicroscopy.Thetubeparticleswereobservedtohave2micronlengthby1microndiameter.ThissizewasfurtherconrmedutilizingTEM.The zeta-sizerinformationrevealedsimilardatawithanaverageof 1micron;howevera highdispersitywasnotedwithpeaksrangingfrom 300nmto 5microns. 41

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Therewereseverallimitationsreachedwiththisparticularmethod.Duetothe relativelydensenatureofthesilicaparticles,itwasapparentthattheyweresettling towardsthebottomofthecuvette.Itwasalsoapparentthattheparticleswerequite disperseinsizeandexudedimmensedisparities,especiallytheamorphousparticles. Therewerepeaksrangingfrom200nmtopeakswellover1micron.Itisapparent thattheparticlesmayevenhavelargerpeaksthanthelightscatteringLSresults concludedandthusthezeta-averagewasnoteablyhigherthanthereportedpeaks. Also,LSmaynotbethemostaccuratemethodtoanalyzethesizeoftubularshapedoramorphousparticles,sinceLSassumesthateachsamplehassimilarrefractivetrendsasspheres.Thismaydistorttheresultsofthedatatosomeextent. 5.3Ultrasoundimaging Followingthesubsequentconjugations,anultrasoundassaywasperformedto testtheperformanceofeachmorphologyparticletoserveasanultrasoundcontrast agent.Manystudieshaveimagednanoparticlesforultrasoundimagingutilizingvariousmethods.Inpreviouswork,thenanoparticlesweredispersedinatissue-mimicking scaold.Thisscaoldcouldbedevelopedusingdisparatematerialsincludingpolyacrylamideandagar,asbothhaveshowntosimulatesofttissue[42,10].However,it wasfoundthatthestriationsinagarproducedgreatnoiseinultrasoundandthuswe wereunabletodetermineaccuratelythepixelintensityproducedbythenanoparticles asopposedtotheextraneousnoise.Itwasthendeterminedtoutilizeanothermethod [37]performedwithdialysistubingimmersedinPBS.Byutilizingthismethod,we wereabletoisolatethepixelintensityexudedbythenanoparticleswithoutextraneous inuencefromtheothermaterials. Ultrasoundimagesweretakenofeachparticlesynthesizedbothwithandwithout uorocarbonconjugation.Theparticleswhichhadnouorocarbonconjugationwere synthesized,followedthereactionforremovalofsurfactantbeforeimaging.The particlesweredispersedinPBSatvariousconcentrations,thenplacedindialysis 42

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tubingontopofatissue-mimickingmedium.Therawultrasoundimagescanbe viewedinFigure5.7below. Threeimageswereobtainedforeachsampleprepared.Afterobtainingtheultrasoundimages,a100x150areasectionwasanalyzedwithinthedialysistubing.Each sectionwasanalyzedinImageJandthemeanpixelintensitywasthenobtained. ThisprocesswasrepeatedatleastthreetimesforeachMSNtype,tohaveasample sizeofatleastn=9imagesanalyzedpergroup. AllstatisticalinformationwasobtainedutilizingthesoftwareStatPlus.After acquiring6separatesamplegroupssphereMSN,sphereMSN-FC,amorphousMSN, amorphousMSN-FC,tubeMSN,andtubeMSN-FC,theirvarianceswereexaminedbetweentheseparategroups.EachMSNwasonlytestedbetweenitselfandits uorocarbon-conjugatedcounterpartateachconcentration.AF-testwasutilizedto measurethevariancesbetweenthegroups.Ifthenullwasrejected,thenunequal varianceswereassumed.TheF-testp-valuesbetweeneachgrouparereportedbelowTable5.3.DependingontheF-testresults,eitheratwo-tailedt-testassuming equalorunequalvarianceswasthenperformedoneachmorphologycomparedto theiruorocarbonconjugatedcounterpart.Statisticalsignicancewasconsideredat p < 0.05. TheaverageddataconcludedfromtheImageJanalysisoftheultrasoundimages wasthenplottedandcanbeviewedinFigure5.8below.Theresultsforcomparing thethreemorphologieswithoutauorocarbonconjugationcanbeviewedinFigure 5.8B,andwiththeuorocarbonconjugationinFigure5.8A.Itisapparentthatall morphologieswithandwithoutuorocarbonconjugationsharethesamegeneral trend:themeanpixelintensityMPIincreaseswithincreasingconcentrationof nanoparticles.Thisisconsistentwiththepreliminaryworkperformedonamorphous nanoparticles[42]. 43

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Figure5.7:Ultrasoundimagestakenofeachnanoparticleamorphous,sphericaland tubularwithandwithoutauorocarbonconjugatedtothesurfaceatdisparate concentrations. Itisapparentthatthespherical-FCparticlesandamorphous-FCparticlesappear tofollowaverysimilartrend,whereasthetube-FCparticlesemittedamorevariable MPIateachconcentrationFigure5.8A.Tube-FCparticlesdisplayedatrendof plateauingfollowing1.0mg/mLFigure5.8A.Allstatisticalinformationincluding p-values,meanandstandarddeviationcanbeviewedinTable5.2,Table5.3. WhenobservingthenanoparticleswithoutconjugationsFigure5.8B,various resultsareseenateachconcentration.BothamorphousMSNandtubeMSNwere foundtoemitahigheraveragedMPIateachconcentrationthanthesphericalMSN. TubularMSNappearstoincreaseinMPImorerapidly,yetwasobservedtoplateau followingconcentrationsover1mg/mL. 44

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Figure5.8:OverallcomparisonoftheMSNmorphologiesconjugatedtouorocarbon AandwithoutuorocarbonconjugationB. TheMSNwerethenplottedtocompareeachMSNtotheiruorocarbonconjugatedcounterpartFigure5.9.ItwasevidentthattheamorphousMSN-FC conjugatedparticleswereallreportedexceptat0.5mg/mLtohaveahigherMPI thantheamorphousMSNaloneFigure5.9B.Similarly,thesphericalMSN-FC particlesdisplayedasimilartrendastheamorphousMSNparticles.Thespherical MSN-FCparticlesallexudedahigheraverageMPIasopposedtothesphericalMSN Figure5.9A.ThistrendwasalsoobservedwhenanalyzingthetubeMSN-FCand tubeMSNMPI's.ItwasfoundthatthetubeMSNalonehadahigheraverageMPI atthelowestconcentrationof0.5mg/mL,yetallotherconcentrationsconcludedto lowerMPIthanitsuorocarbon-conjugatedcounterpartFigure5.9C. 45

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Table5.2:MeanandStandardDeviationsforMSN SphereMSNSphere-FCAmorphousAmor-FCTubeMSNTube-FC Conc.MeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D. 0.58.5652.67515.857.1217.961.2015.583.0216.737.877.064.24 1.012.274.3422.987.3219.812.5624.685.7523.288.1235.835.46 2.014.404.9035.628.8323.443.4133.704.6625.095.3342.372.86 5.020.256.0554.274.9436.524.3447.437.4228.336.4842.236.98 Table5.3:P-valuesbetweeneachmorphologyMSNandtheiruorocarbon-conjugated counterparts.P-valuesarereportedforboththeF-testandt-testscompletedbetween eachofthesegroups. SphereMSN&SphereMSN-FCAmorMSN&AmorMSN-FCTubeMSN&TubeMSN-FC Conc.F-testt-testF-testt-testF-testt-test 0.50.009020.002070.017680.053360.0810.00215 1.00.088060.000150.03420.040380.2620.00048 2.00.0348 < 0.00010.398 < 0.00010.0971 < 0.0001 5.00.579 < 0.00010.1500.00220.8390.00047 StatisticalsignicancewasreportedandcanbeseeninFigure5.9,labeledas* orlistedinTable5.3. 5.3.1Conclusionsfromultrasoundimaginganalysis Theuorocarbon-conjugatedparticleswerepredictedtoemitahigherMPIas opposedtotheirnon-uorocarbonconjugatedcounterparts.Thisgeneraltrendwas conrmedforallmorphologiesandcanbeviewedinFigure5.9.Itwaspredictedthat theuorocarbonconjugatedparticleswouldfollowasimilarmechanismtothePFCemulsednanoparticles,howevertheexplicitmechanismremainsunsurfaced.Current ultrasoundcontrastagentswhichutilizealiquidPFCreportaphase-transitionfollowingtheintroductionofacousticwavestoitshighlyechogenicgaseousstate[55,64]. Furthermore,thisimpedancemismatchbetweenthetissue/gasresultsinahigher MPI[8].Aspreviouslystated,theshortshelf-lifelessthanoneweekwhenstoredat 4 Cofthesegaseousparticlesposesasignicantlimitation[55]. 46

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Figure5.9:Analysisofthepixelintensitiesfromtheultrasoundstudiesproducedby A.sphericalMSNB.amorphousMSNandC.tubeMSN.Allimageanalysiswas performedinImageJ.*representsstatisticalsignicancebetweengroups. Inordertofurtherincreasetheshelf-lifeoftheseparticles,yetstillpreservethe highercontrastproducedbytheseenhancedparticles,itwasdeterminedtoconjugate aPFCtothesurfaceoftheMSN.Ithaspreviouslybeenreportedthatfreeze-drying gaseousparticlessignicantlyreducestheirechogenicfactorandwereobservedto collapseorfuseduringtheprocess[55].Byconjugatingtheuorocarbontothe surfaceoftheparticles,theshelf-lifewouldinherentlyincreaseastheparticlesno longerhaveagaseouscorewhichwouldhavethepotentialtocollapse. AsFigure5.9displaysslightlyhigherMPIintheamorphous.9B,spherical .9Bandtube.9Cplotsfurthersignifyingtheacousticpropertiesoftheuorocarboncouldpotentiallybemaintained.Despitethisgeneraltrendnoted,both 47

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amorphousandtubeMSNat0.5mg/mLconcentrationproducedeithernostatistical dierenceorsignicantlylowerMPIthanboththeiruorocarboncounterpart.However,allotherconcentrationsabove0.5mg/mLdisplaytheuorocarbonconjugated MSNemitastatisticallysignicanthigherMPIthantheparticlesalone.Aspreviouslyreported,eachMSNaloneproducedcontrast;yet,asfound,theiruorocarbonconjugatedcounterpartfurtherenhancesthiscontrast. Notonlywouldconjugatingtheuorocarbontothesurfacepotentiallyenhance theshelf-lifeoftheparticles,butitwasalsoposedtoenhancethedurationofsystemiccirculationtimewithinthebody.Gaseousmicrobubbleshavebeenreported tohavearelativelyshortlifespanincirculation,lastingonlyafewminutes[9].By circumventingagaseouscore,theparticlescouldenhancevascularcircuitry. Furthermore,theMPIemittedfromeachparticlecouldbecorrelatedtothesize oftheparticlesthemselves.IthaspreviouslybeenreportedthattheMPIincreases withincreasingmicrobubblesize[51].Asgaseousmicrobubblesemitamuchhigher MPIandduetoadisparatemechanismasopposedtosolidnanoparticles,this ndingmayholdasmallornegligibleinuence.Itwasalsopostulatedthatwith modifyingthesurfaceoftheMSN,thesizewillinherentlyincreaseandthusmay havesomeinuenceonultrasoundcontrast.However,Figure5.8Bdisplaysthetwo largestparticlemorphologiesemittingamuchgreaterMPIatallconcentrations.Table5.1depictstheaveragesizeoftheparticlesfoundfromDLS.Allparticlesshowed agreaterincreaseinsize > 1micronincreasefollowingthesequenceofconjugations tosuccessfullyPEGylate,FluorocarbonandHerceptinconjugateeachMSN.Itwas apparentthatthelarger,conjugatedMSNofallmorphologiesincreasedinMPIFigure5.9A,B,C.YetthisargumentdoesnotprovetrueforamorphousMSN,asthe particleaveragesizewasreportedtobeconsistentevenfollowingallconjugations Table5.1.Itwasconcludedthatthiscouldbedueinparttothehighdispersity ofallamorphousMSN,thusdistortingtheaveragedvalue.Furthermore,amorphous 48

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MSN-FCcomparedtoamorphousMSNwasshowntodisplaystatisticalsignicance atallconcentrationsexcept0.5mg/mL.Thus,ifthesizeoftheamorphousMSN staysconsistentfollowingallconjugationsandyetproducesahigherMPIfollowing thisFCconjugation,thenitcanbedeterminedthatthehigherMPIproducedwas duetotheFCconjugation. 5.4Invitrostudies TheHER2overexpressingbreastcancercellswereseededatadensityof50,000 cells/welland100,000cells/wellinseparate24wellplates.Sixsamplesofparticles weretestedandtheschematicofthe24wellplatecanbeviewedbelowFigure5.10. Twoplatesofeachdensitywereincubatedfor30minutesfollowingtheadditionof eachMSNataconcentrationof25 g/well.Following30minutes,allcellswerethen xedandstainedwithDAPInucleus.Thisprocesswascompletedagain,yetwith a2hourincubationperiodafterintroducingtheMSNalsoataconcentrationof 25 g/well.Incubationperiodsof30minuteswaschosenbasedopriorliterature andanextensionofthetimewaschosentondanydierencesincellularendocytosis followinga30minuteincubationperiod[42]. Table5.4:ThetabledepictstheincubationtimeforMSN+cellsandseedingdensity ofHER2overexpressingcellsineachwell. Plate#IncubationtimeSeedingdensity 130min100,000cells/well 230min50,000cells/well 32hr100,000cells/well 42hr50,000cells/well Followingtheallotedincubationtimeandthexing/stainingtechniquesdescribedinMethodsection,thecellswerethenimagesusingFluorescentmicroscopy. 49

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Figure5.10:Schematicoftheinvitro24-wellplateassay.A-FC-HERrepresentsamorphousMSN-uorocarbonandHerceptinconjugated.Similarly,Srepresentsspherical andTrepresentstube.A-HERrepresentsamorphousMSN-Herceptinconjugated withoutuorocarbon. TheimagesaredisplayedbelowinFigures5.11and5.12forplatesseededat100,000 cells/welland50,000cells/wellrespectively. Itisapparentthatevenfollowingonlya30minuteincubationperiodofeach MSNwiththecells,itcanbeobservedthatthereisco-localizationoftheuorescentlylabeledgreenMSNwiththeHER2overexpressingbreastcancercellsFigure5.11/12. Themorphologyofcellsdepictedfollowing30minuteincubationperiodappearto remainroundandglobular.Itisapparentthatafter30minuteincubationperiod, notallspherical-PEG-FC-Herceptinlabeledparticlesattachedtothesurfaceofthe cellsastheredarrowdepictsFigure5.12A,Figure5.13A.Itisalsoapparentthat 50

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Figure5.11:ImagesaboveweretakenusingFluorescentmicroscopyanddisplayall sixMSNsamplesshowninFigure5.10whenincubatedwithcellsatadensityof 100,000cells/well.EachMSNsamplewasincubatedforeither30minutesor2hours ataconcentrationof25 g/well.EachparticlewaslabeledwithFITCgreenandthe cellswerestainedwithDAPI. 51

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Figure5.12:ImagesaboveweretakenusingFluorescentmicroscopyanddisplayall sixMSNsamplesshowninFigure5.10whenincubatedwithcellsatadensityof 50,000cells/well.EachMSNsamplewasincubatedforeither30minutesor2hours ataconcentrationof25 g/well.EachparticlewaslabeledwithFITCgreenand thecellswerestainedwithDAPI. 52

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Figure5.13:ZoomedinimagesoftheMSN+50Kcells/welldepictedinFigure5.12. 53

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arrow.13BpointsoutacellwhichhasthreegroupsofMSNcolocalizedtothe nucleus.Thecelldoesnotappearround,globularinshapeasthesurroundingcells appear.Thissuggeststhatthetube-Herparticlesweresuccessfullyendocytosedby thecellandwereabletopromotecellapoptosis,asthecellmembraneappearstobe lysed. Following2hourincubationperiod,itwasapparentthatallMSNsampleswere co-localizedwiththesurfaceofthecells,suggestingHerceptinbindingtotheHER2 receptor.Therowof10Ximagesdepictalargerrepresentationofthewellsasopposed tothe40Xrows.Followingthe2hourincubationperiod,morelysedcellswerenoted. ArrowCdepictsacellco-localizedwiththreegroupstube-MSN-Heryethasasimilar shapeofcellasdoesarrowBdepict.Thisonceagaincouldsuggestacelllysing followingtheendocytosisofthetube-MSN-Her.Similarly,asarrowDpointsout, anagglomerarionofthreecellsimagebelowdisplaysthreenucleiappearstohave undergonedeathaswell.However,noco-localizationofgreenparticlesisvisible. Aspreviouslynoted,thecharacteristicsofboththeparticlesizeandmorphology haveaneectoncellinternalization.Itwasreportedthatthesmallerparticleswithan averagediameterof 50nmwereendocytosedatahigherrateasopposedtovarious sizedparticlesrangingfrom2-100nm[31,39].Inanotherstudy,themorphology ofparticleswasfoundtohaveagreatereectoncellinternalization.Inthisstudy, itwasconcludedthatmoresymmetricalshapedparticlesi.e.acubeoracylinder withsamediameterandheightsizewereendocytosedatalesserratethantheir unsymmetricalcounterparts[25].Similarly,inanotherstudy,itwasfoundthatlong rodMSNparticles > 450nmwereendocytosedatahigherratethantheirshorter counterpart[13].ItwasalsoobservedthattherodshapedMSNendocytosedata greaterratethansphericalshapedMSN.Furthermore,Championet.alconcluded thatparticleswithalargersurfaceareawerereportedtobeinternalizedmoreso. 54

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5.4.1Qualitativeremarksoninvitrostudies Furthermore,onlyqualitativeremarkscanbemadeontheendocytosisrateof eachmorphologyMSN-PEG-FC-Herceptinversustheirnon-uorocarbonconjugated counterparts.Itwasapparentthatthetube-PEG-FC-Herceptininboththe50K and100Kimagesdepictedparticleagglomerationandalmostcoatingnearthecell surface,suggestingpreferentialbindingofthesynthesizeddrugdeliverymodality. Byutilizinguorescentmicroscopy,weareunabletodetermineifcellsareendocytosingtheparticlesoriftheyaremerelyresidingonthesurface.Itisassumed thattosomeextenttheparticleswillundergoreceptor-mediatedendocytosisviathe TrastuzumabbindingtotheHER2receptor.Itwasalsoassumedthatthecellswill undergoreceptor-mediatedendocytosisRMEoftheparticlestoagreaterextent followingthe2hourincubationperiod. ItwasquiteevidentthattheMSN-PEG-FC-Herlabeledparticlesdidnotinhibit thepreferentialbindingofHerceptintoitsreceptor,HER2.Thisfurthersigniesthat oursynthesizeddevicewillbeabletobindtotheHER2overexpressingbreastcancer cellsevenfollowingtheseriesofconjugations.Furthermore,itwasalsoapparentthat theuorocarbonconjugatedparticlesactedinsimilarfashiontothatoftheirnonFCconjugatedcounterpart.Thus,theMSN-PEG-FC-Herparticlesdisplayedsimilar bindingafter30minutesofincubationaswellas2hours. ItwasalsoapparentthatinboththetubeMSN-Her30minand2hrincubation periods,bothreportedcelldeathasshownbyarrowsBandC.Thiscanthen suggestedahigherrateofRMEfollowingtheintroductionoftubeMSNtothecells. ItisalsoapparentthatthetubemorphologyMSNmaysuggestanenhancedor swifterrateofRMEastherewascelldeathreportedafterthe30minuteincubation period. 55

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Despitetheseobservations,morequantitativeresultsneedstobeperformedin ordertoquantifywhichshapeparticleproducesthehighestrateoftherapeuticalleviation. 56

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6.Conclusionandproposedfuturework 6.1Conclusiveremarks Thedesignofthisstudywasconductedinordertoenhanceboththerapeutic anddiagnosticeectsutilizingHER2overexpressingbreastcancerasamodelforthe device.Currentclinicaltreatmentsforcancerlackatargetingmodalityandremains tobehighlytoxictoallcells,includingnon-malignant.Drugdeliverymodalities areprogressingintobecomingmultifunctional,servingtoenhancediagnosticimagery aswellastherapeuticeects.However,currentFDA-approvedUCAhaveseveral limitationsincludingshortshelf-lifeandashortsystemiccirculatoryduration[55]. Tocircumventtheseshortcomings,auorocarbon-conjugatedMSNwaschosenwith variedmorphologiestofurtherenhanceboththerapeuticanddiagnosticeects. MSN'sweresuccessfullysynthesizedandstructurallycharacterizedbybothelectronmicroscopyandLS.Allmorphologieswereshowntohaveawiderangeofsize dispersity.Followingthecharacterization,theparticlessuccessfullyunderwentaseriesofconjugationsinordertoconjugateaFluorocarbontotheoutersurface.Ithas previouslybeenshownthatagaseousperuorocarbonmicrobubbleemitsahigher pixelintensityinultrasoundimagingthroughthetissue/gasimpedancemismatch[8]. Similarly,aliquidperuorocarbon-emulsednanoparticleemitsahigherMPIthrough phase-transitioningintoitshighlyechogenicgaseousstatefollowingtheintroduction ofacousticwaves[55,64].Itwasproposedthattheshelf-lifeofanultrasoundcontrast agentcanbeincreasedbyutilizingasolidparticleinsteadofabubbleandthrough conjugatingauorocarbontothesurface. ThethreemorphologyMSNweretestedtodeterminetheirMPIatvariousconcentrationsalongwiththeirFCconjugatedcounterpart.Ageneraltrendwasseen whichdepictedtheFCconjugatedparticlesemittedahigherMPIascomparedto thenon-FCconjugatedMSN.Allconcentrationstestedexcept0.5mg/mlwerereportedtohavestatisticallysignicantdierences.TubeandamorphousMSNalone 57

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wereshowntohaveahighercontrastthanthesphericalMSNatallconcentrations. TotestthetargetedMSN'scapabilityofbeingendocytosedtotheirtargetedcells,a invitroassaywasperformed.ThebindingpreferenceoftheMSN-PEG-FC-HerparticleswereobservationallyanalyzedtohaveasimilarbindingratewiththeMSN-Her particles.Itwassuggestedthatsomeparticlesweresuccessfullybeingendocytosed bythecells,whichthenunderwentapoptosis.Thiscanbeconcludedassomecells withcolocalizedgreenparticlesappeartohavealysedcellmembrane.However,additionalcellstudiesneedtobeperformedtofurtherconrmthecelllysingduetothe endocytosisofthenanoparticles. Insummary,amultifunctional,hybridpolymer-NPsystemwassuccessfullydevelopedtoenhancecontrastinultrasoundaswellastoserveasatargeted,drugdelivery modality.Threemorphologiesweretestedintheircapabilityforultrasoundcontrast andrateofendocytosis.ItcanbesuggestedthattubeandamorphousMSNemita highercontrastaloneandallmorphologiesshareageneraltrendwiththeconjugation ofauorocarbon.TheinvitrocellstudysuggeststhattubeMSNmayhaveafaster rateofendocytosisasthereareseveraldeadcellsnotedfollowingonly30minutesof treatment. 6.2Proposedfuturework 1.Optimizationofparticles Theoptimizationofthesilicananoparticlesisnecessaryinordertodevelopa highlytranslationalmultifunctionaldrugdeliveryvehicle.Itwouldbeidealforextravasationofthebloodstreamforareductionoftheparticlestobeasizeoflessthan 1micron.Thus,theparticleswouldbeabletotraversethegapbetweenendothelial cellsaswellashavethepotentialtohaveahigherrateofendocytosis.Adesired particlewouldbeunder1micronfollowingtheseriesofconjugations.Thus,further optimizationwouldbeneeded. 58

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1.FrequencySweep TofurtherunderstandthemechanismofhowtheFCconjugatedparticlesare emittingahighercontrast,itwasdeterminedtobeginwithafrequencysweep.Currentclinicalultrasoundperformsatfrequenciesbetween1-10MHz,thusthesweep wouldbewithinthisrange.Albeitthedevelopedsystemisnotagasbubble,itmay havearesonancefrequencytowhichitproducesthehighestultrasoundcontrast. Whenexcitedviaacousticwavesnearresonancefrequency,thebubbleisexcitedfora longerdurationresultinginnonlinearoscillationsandharmonicscattering[19].Furthermore,itssolidscatteringcounterpartdoesnothavearesonancefrequencyand thusisexcitedequally[19].Byutilizingafrequencysweep,itcouldbedetermined iftheFCconjugatedtothesurfacehasaresonancefrequency,oriftheparticlesare merelysolidscatterers. 2.InvitroLive n Deadstaining ToquantifyhowmanycellsarealiveordeadfollowingthetreatmentofeachMSN. ThecellstudywouldbeperformedasdescribedintheMethodologysection,yetwith theadditionofthestainbeforeimaging.Wecouldthendeterminethetherapeutic eectsofeachmorphologyMSNwithorwithouttheuorocarbon.Furthermore,the MSN'scapabilitytobeendocytosedbythecellsandtopromoteapoptosisviaADCC andinhibitionofsignalingpathways,couldbedetermined. 3.BreastCancerAnimalModel Thenextstageofthestudyfollowingoptimizationoftheproposeddrugdelivery vehicleswouldbetoanalyzetumorgrowth/reductioninmicefollowingaseriesof injectionsofeachparticle.Thecapacityofeachsystemtoemitultrasoundcontrast wouldfurtherbeexaminedaswellasitsabilitytoreducethetumorsize. 59

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APPENDIXA.Zeta-sizerplots FigureA.1:Zeta-sizerplotperformedonsphericalMSN.Performedintriplicate. FigureA.2:Zeta-sizerplotperformedonsphericalMSN-Her.Performedintriplicate. 66

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FigureA.3:Zeta-sizerplotperformedonamorphousMSN.Performedintriplicate. FigureA.4:Zeta-sizerplotperformedonamorphousMSN-Her.Performedintriplicate. FigureA.5:Zeta-sizerplotperformedontubeMSN.Performedintriplicate. 67

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FigureA.6:Zeta-sizerplotperformedontubeMSN-Her.Performedintriplicate. 68