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Applications of Micro- and Nanoparticles in Activating Photodynamic Therapeutic Agents within Deep-seated
Targets
By Erkinay Abliz
B.A in Physics, July 1997, Xinjiang Normal University, P. R. China
M.A in Physics, July 2004, City College of New York
A dissertation submitted to
The Faculty of The School of Engineering and Applied Sciences
of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy
January 31, 2012
Dissertation directed by
Jason M. Zara
Associate Professor of Engineering and Applied Science
ii
The School of Engineering and Applied Science of The George Washington University certifies
that Erkinay Abliz has passed the Final Examination for the degree of Doctor of Philosophy as of
July 29, 2011. This is the final and approved form of the dissertation.
Erkinay Abliz
Dissertation Research Committee:
Jason Zara, Professor of Electrical Engineering, George Washington University, Dissertation Director
Murray Loew, Professor of Electrical Engineering, George Washington University, Committee Member
Shahrokh Ahmadi, Professor of Electrical Engineering, George Washington University, Committee Member
Darrel Tata, Research Scientist, US Food and Drug Administration, Committee Member
Ilko Ilev, Research Scientist, US Food and Drug Administration, Committee Member
“Applications of micro- and nanoparticles in activating photodynamic therapeutic agents within deep-seated targets”
iii
Abstract of Dissertation
“Applications of micro- and nanoparticles in activating photodynamic therapeutic agents within deep-seated targets”
Photodynamic Therapy (PDT) is a therapeutic method that uses photo-sensitizers that can
be preferentially localized in pathological tissue [1-3]. The dominant mode of
photodynamic therapy (PDT) action is through the generation of reactive oxygen species
(ROS). When the photo-sensitizer in tissue is excited by light, it interacts with molecular
oxygen and transfers its energy to molecular oxygen to create highly reactive oxygen in its
singlet state in tissues. Despite being non-invasive and having excellent selectivity for
diseased tissue, PDT has not yet gained general clinical acceptance, largely due to the
inherent limitations of light transport and penetration which restrict external light from
activating photo-agents within target volumes deep inside the body. The photo-sensitizers
that are approved for PDT treatment in oncology are found to maximally absorb light in the
violet region of the visible spectrum, around 400 nm, and blood is a very strong absorber at
this wavelength. Thus, the photo-agent’s absorption characteristics inherently limit the
effectiveness of PDT applications to target-sites that are shallow in depth, 2 – 3mm. For
this reason, the clinical application of PDT has been limited to skin lesions, superficial
solid tumors, or endoscopically-accessible regions [5].
One of the worldwide approved photo-sensitizers in oncology, Photofrin II, is known to
have good selectivity towards diseased tissue, and its major sub-cellular target is known to
be mitochondria [1-3]. In this work, both X-ray down-converting (DC) and Infrared up-
converting (UC) particles were studied as platforms to generate visible luminescence to
iv
activate the photo-sensitizer Photofrin II. Specifically, I have investigated DC particles
composed of gadolinium oxysulfide doped with terbium (GdO2S: Tb) and UC particles
composed of sodium yttrium fluoride co-doped with ytterbium and thulium (NaYF4:
Yb/Tm).
The DC and UC particles were tested in a cellular-like medium; the test tube with the DC
particles was then irradiated with 120 keV X-rays, while the test tube with the UC particles
was irradiated with a 980 nm laser. The ROS generation for each test tube was quantified
by measuring the change in the absorbance of Vitamin C. In vitro studies on human
glioblastoma cell lines were then conducted to investigate the possible cellular toxicity of
these DC and UC particles through cell viability assays and an endotoxin detection assay.
The therapeutic effectiveness of these particles via Photofrin II activation was also
evaluated on in vitro human cancer cells through measurement of ROS levels and cell
viability assays. Theoretical modeling of the experiment was generated using both
analytical technique and Monte Carlo Modeling of light transport.
The results obtained from cellular-like medium showed that both submicron- to micron-
sized DC and UC particles have great potential to activate Photofrin II and to generate
substantial levels of ROS. Specifically, the results on in vitro cellular studies have shown
that 20 micron-sized DC particles have great potential to activate Photofrin II in deep
seated targets and to generate substantial levels of ROS and no potential cell toxicity was
observed. However, the UC particles tested (50 nm) were shown to be toxic to the cell
lines. The cell killing does not appear to be due to the particles' efficiency in activating the
photo-sensitizer, but rather appears to be due to toxicity of the particles.
v
Table of Contents
Abstract of Dissertation .................................................................................................................. iii
Table of Contents ...............................................................................................................................v
List of Figures ................................................................................................................................. vii
List of Tables ......................................................................................................................................x
List of Acronyms ...............................................................................................................................xi
Chapter1: Introduction .....................................................................................................................1
Chapter 2: Background.....................................................................................................................4
2.1. Mechanism of PDT ................................................................................................................ 4
2.2. Mechanism of Tumor Destruction ....................................................................................... 6
2.2.1. Cellular Effects................................................................................................................ 7
2.2.2. Vascular Effects .............................................................................................................. 7
2.2.3. Reaction of the Immune System.................................................................................... 8
2.3. Photosensitizer ....................................................................................................................... 8
2.4. Tumor Oxygenation............................................................................................................. 10
Chapter 3: Micro- and Nanoparticles Induced Visible Luminescence to Activate
Photosensitizers within Deep- Seated Tumors ..............................................................................12
3.1. Review of X-ray Production and Down Converting (DC) Micro-Particles .................... 13
3.1.1. X-Ray Generation......................................................................................................... 14
3.1.2. X-Ray Down-Converting Particles.............................................................................. 16
3.2. Review of Up Converting (UC) Nanoparticles .................................................................. 17
Chapter 4 : Experimental Quantification of ROS Generation....................................................22
4.1. Spectroscopic Characterization of Photofrin II, Up-converting (UC), and Down-Converting (DC) Particles.......................................................................................................... 22
4.2. Experimental Quantification of ROS Generation from DC and UC Particles .............. 33
vi
4.3. ROS Generation from Photofrin II Activated by 405 nm and 633 nm Lasers............... 35
4.4. ROS generation from X-ray down-converters .................................................................. 36
4.5. ROS Generation from IR Up-convertors .......................................................................... 39
Chapter 5: Safety Evaluation of “Rare-earth” Based Materials and Therapeutic Efficacy on
Selective Cancer Cell Lines.............................................................................................................41
5.1. Cell Maintenance, Cellular Metabolic Activity Measurement Techniques.................... 41
5.2. Therapeutic Efficacy and Cell Toxicity Results of X-ray DC Particles on Selective Cancer Cell Lines........................................................................................................................ 43
5.3. Therapeutic Efficacy and Cell Toxicity Results of Infrared UC Particles on Selective Cancer Cell Lines........................................................................................................................ 47
Chapter 6 : Theoretical modeling of ROS generation ..................................................................59
6.1. X-ray absorption coefficients of the test medium components ........................................ 60
6.2. Analytical modeling of X-ray absorbed dose and generated fluorescence light in the test medium in the presence of X-ray down convertors ................................................................. 62
6.3. Statistical Modeling: Quantifying fluorescent light fluence distribution using Monte Carlo Modeling. .......................................................................................................................... 66
6.4. Theoretical quantification of amount of ROS generation................................................ 81
Chapter 7: Conclusion.....................................................................................................................88
References.........................................................................................................................................92
Appendix...........................................................................................................................................96
vii
List of Figures
Figure.2.1.Modes of Photodynamic killing.............................................................................................................5
Figure 2.2.The Jablonski energy diagram photosensitizing process...................................................................6
Figure.3.1.Abrosption coefficients of whole blood...............................................................................................13
Figure.3.2. Two types of X-ray production...........................................................................................................16
Figure.3.3.Principal UC processes for Lanthanide doped crystals ....................................................................19
Figure.3.4.Proposed energy transfer mechanism showing UC processes .........................................................20
Figure.4.1.Spectroscopic characterization of Photofrin II .................................................................................23
Figure.4.2. Spectroscopic characterization of X-ray of DC particles.................................................................25
Figure.4.3.X-ray induced light emission intensity dependence on X-ray photon energy.. ...............................26
Figure.4.4.Experimental set up for measuring IR induced light emission spectrum of UC particles.............27
Figure.4.5.Emission spectrum of UC particles in response to 980nm laser excitation .....................................28
Figure.4.6.Emission intensity profile of peak values of UC particles.................................................................30
Figure.4.7.Quantum yield measurement system..................................................................................................32
Figure.4.8.Interaction of Vitamin C with resulting ROS in dehydroascorbic acid ..........................................33
Figure.4.9.Absorption spectrum of unoxidized Vitamin C in PBS. ...................................................................34
Figure 4.10.Unoxidized Vitamin C absorbance in PBS as a function of concentration ..................................34
Figure.4.11.ROS production from photo ii due to 633 nm and 405nm lasers ...................................................35
Figure.4.12.Experimental set up for measuring ROS generation from X-ray DC particles............................37
Figure.4.13.Comparision of ROS production from Photofrin II between activation through DC particles
and X-rays alone .....................................................................................................................................................37
Figure.4.14.Comparison of ROS production from Photo II between activation by 9mW/cm2 He-Ne laser
and through the X-ray induced luminescence......................................................................................................38
Figure.4.15.Experimental set up for measuring ROS generation from IR UC particles ...............................39
viii
Figure.4.16.ROS generation from UC particles...................................................................................................39
Figure.5.1.Structures of MTS tetrazolium and its formazan product. ..............................................................43
Figure.5.2.X-ray exposure set up and measurement of cell viability 48 hours post exposure. ........................44
Figure.5.3.Human Glioblastoma cellular metabolic activity through MTS measurements taken 48 Hrs after
a 15 Min diagnostic X-ray exposure. ....................................................................................................................45
Figure.5.4.Assessment on the potential cellular influence of Gd2O2S: Tb particles on human glioblastoma
cell lines. ..................................................................................................................................................................47
Figure.5.5.Infrared laser exposure set up and measurement of cell viability 48 hours post exposure . .........48
Figure.5.6.Normalized Human Glioblastoma cellular metabolic activity through MTS measurements taken
48 Hrs after a 5 Min of laser exposure (5mg/ml). ................................................................................................49
Figure 5.7. Normalized Human Glioblastoma cellular metabolic activity through MTS measurements taken
48 Hrs after a 5 Min of laser exposure (0.5mg/ml). .............................................................................................50
Figure.5.8.Human Glioblastoma cellular metabolic activity through MTS measurements taken 48 Hrs after
different exposure times and laser intensity ........................................................................................................51
Figure.5.9.Normalized Human Glioblastoma cellular metabolic activity after 135 sec of laser(980nm)
exposure ..................................................................................................................................................................53
Figure.5.10.Typical standard curve for LAL assay . ...........................................................................................57
Figure.6.1.Mass-energy absorption and attenuation coefficients at different X-ray photon energies for
Gadolinium Oxysulfide... .......................................................................................................................................61
Figure. 6.2. Excitation beam profile .....................................................................................................................68
Figure.6.3.Fluorescent photons are created at the point of photon absorption ................................................71
Figure 6.4.Deflection of a photon by a scattering event. .....................................................................................72
Figure.6.5.Internal reflectance and transmittance. .............................................................................................74
Figure.6.6.Excitation photon tracking flow chart ...............................................................................................76
Figure.6.8.Fluorescence photon tracking flow chart. ..........................................................................................78
ix
Figure 6.9.Results of X-ray photon simulation ....................................................................................................79
Figure 6.9.Results of infrared photon simulation ................................................................................................80
x
List of tables
Table.2.1. Clinically approved photosensitizers in oncology.................................................................................9
Table.5.1.Absorption values of dc particles at 405 nm using LAL assay. ..........................................................58
Table.6.1.Physical properties of components of test medium at 120 keV X-ray exposure...............................61
Table.6.2.Comparision outcome between experiment and theory .....................................................................84
Table.6.3.Connection chart between theoretical modeling and experimental measurements for DC particles.
..................................................................................................................................................................................85
Table.6.4.Connection chart between theoretical modeling and experimental measurements for UC
particles……………………………………………………………………………………………………………86
xi
List of Acronyms
PDT – Photodynamic Therapy
ROS – Reactive Oxygen Species
IR – Infrared
UC – Up-conversion
DC – Down-conversion
ESA – Excited state absorption,
ETA – Energy transfer up-conversion
PA – Photon avalanche
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Chapter1: Introduction
Photodynamic Therapy (PDT) is a minimally invasive therapeutic method that uses
photosensitizers that can be preferentially localized in pathological tissue. The ultimate
result of the absorption of photons by the photosensitizer in the presence of ground triplet-
state molecular oxygen results in the generation of highly cytotoxic species, including
singlet oxygen ( 21O ), free radicals and peroxides, that attack the sub-cellular components
of the targeted cells [1-3]. There is a general consensus in the literature that greater than
90% of cell killing is via generation of singlet oxygen. The singlet oxygen has a very short
lifetime (<40 μs) in the water based biological environment and a very short free-diffusion
radius of action (< 20 nm). Consequently, the damaged area is essentially confined within
the tissue that contains the photosensitizer and then exposed to light with the appropriate
wavelength [3].
Photodynamic therapy (PDT) has been used for many years to treat many different
diseases, including macular degeneration, several skin disorders, and several types of
cancers [2]. Compared to current treatments, such as surgery, radiation therapy and
chemotherapy, PDT is relatively non-invasive, may be more accurately targeted, and is not
subject to the total-dose limitations associated with radiotherapy. Despite these advantages,
PDT has not yet gained general clinical acceptance. All of the photosensitizers that are
approved for PDT treatment in oncology absorb light in the visible spectral regions below
640 nm, preventing access to more deeply seated tumors due to strong light absorption by
blood in this wavelength range. As a result, the clinical application of PDT is limited to
skin lesions, superficial solid tumors, or endoscopically accessible regions [4]. One of the
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world-wide approved photosensitizers, Photofrin II, is known to have good selectivity
towards diseased tissue and its major sub-cellular target is known to be mitochondria.
Recently, there has been interest in the development of nanoparticles-based photosensitizer
delivery system that is comprised of photo-agent molecules directly anchored at the surface
of the nanoparticles. These nanoparticles are fabricated with an inorganic core, and absorb
either incident X- ray photons (down-conversion) or multiple infrared photons (up-
conversion) and then relax to emit visible light at specific wavelengths determined by the
material composition of the nanoparticles. The attached photo-sensitizer molecules could
be activated directly through emitted light from the core of the nanoparticles or through
direct-energy transfer schemes, resulting in copious reactive oxygen species (ROS)
production [6-8]. However, the sub-cellular distribution of the photo-agent when ligated to
the nanoparticles is expected to be vastly different than the free photo-agent. Free Photofrin
II is known to have a high affinity for the mitochondria [1-3][9], whereas, the Photofrin II
bound to nanoparticles is expected to be concentrated within the lysosomes of the cells[10]
.
As far as Photofrin II is concerned, the mitochondrion is its critical sub-cellular target from
which apoptotic signals are delivered [1-3]. The dominant cell killing mechanism of action
for Photofrin II is through the generation of singlet oxygen. Singlet oxygen molecules are
very short lived (< 40 �s with a free diffusion path length of < 20 nm). Therefore due to the
very short singlet oxygen's diffusion path length, it becomes crucial that the Photofrin
reaches the mitochondrial targets. In order to ensure that Photofrin II reaches
mitochondrial target and effectively contributes to singlet oxygen generation, in my
experimental design, the Photofrin II is separated from the nanoparticles. �
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In this dissertation, I shall begin Chapter 2 by providing background on the topics essential
to the research: mechanisms of PDT, mechanisms of tumor destruction, mechanism of
singlet oxygen generation and photosensitizer followed by background on micro/
nanoparticles in Chapter 3. In Chapter 4, I will describe results of spectroscopic
characterization of Photofrin II, up-converting (UC) and down-converting (DC) particles;
experimental quantification of ROS generation from X-ray induced DC luminescence and
IR induced UC luminescence in cellular like medium. Chapter 5 includes experimental
results on safety and effectiveness of rare earth based UC and DC particles in activating
Photofrin II on human glioblastoma cell lines. Chapter 6 describes steps and results of
theoretical modeling of ROS generation from Photofrin II activated through UC and DC-
induced fluorescence. Finally Chapter 7 summarizes results, conclusions and proposed
future work.
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Chapter 2: Background
Three critical elements are required for the photodynamic therapy process to occur: a
photosensitizer agent that can be localized into pathological tissue and activated by light; a
narrowband light exposure with wavelengths corresponding to the main excitation peak
known as the “Soret” peak of the photosensitizer, and the presence of molecular oxygen [1-
4]. The main controllable parameters that can influence the outcome of PDT treatment are
the photo-agent’s concentration and the light dose. For localized tumors it is also important
to examine subtle parameters such as drug bio-distribution, localization, aggregation,
oxygen supply and consumption and tissue optical properties, to enhance therapeutic
efficacy, shorten treatment time, and eliminate skin photosensitization completely [2].
2.1. Mechanism of PDT
The cell killing mechanism in PDT is known to be predominantly through enhanced
generation of reactive oxygen species (ROS) through type 1 and type 2 mechanistic
pathways as shown in Figure.2.1 below[11]. Upon visible light activation, the excited
photo-agent either transfers its excited electrons to molecular oxygen forming superoxide
anions O2- and H2O2 (type 1 pathway), or transfers its excited energy onto the ground
(triplet) state 23O , resulting in an excited oxygen molecule in its singlet state 2
1O (type 2
pathway). There is a general consensus in the literature that greater than 90% of cell killing
is via type 2 pathways [11] .
��������������������������������������������������������������������������������������������������������������������������������������������������������5
Figure.2.1. Modes of Photodynamic killing. h�: Photon energy at given frequency �, P: photosensitizer, S: substrate, from[11]
Figure.2.2 is a diagram of the energy level pathway for a photosensitizer in the
photosensitizing process [12]. The molecules are excited from the ground state 1P , to the
excited singlet state 1P� , with a probability proportional to the product of the absorption
coefficient a� and irradiance �. Once in the 1P� state, the molecule can relax by
fluorescent photon emission (with quantum yield fl� ) or intersystem cross to the first
triplet state, 3P� (quantum yield isc� ). From the triplet state, the molecule can either relax
by phosphorescent photon emission (quantum yield ph� ), or be quenched by interaction
with a ground state triplet oxygen molecule 23O , to produce excited singlet state
oxygen 21O . Photo bleaching of the molecule can come directly from 1P
� or 3P� or
Type I
�
��
�����
�����
223*1
*3
OPOP
SPSP
Ph 1� ��� *3P Type II
)(21
211
23*3
OSSO
OPOP
����
�����
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from 1P , 1P� and 3P
� in combination with 21O , or from photosensitizer intermolecular
interaction, resulting in destruction of the photosensitizer molecule.
Figure.2.2. The Jablonski energy level diagram for a photosensitizer molecule in the photosensitizing process (indicated by dotted lines). 1P : ground state photosensitizer in its singlet state, 1P
� : excited
photosensitizer in its singlet state, a� :Absorption coefficient of the tissue, �: Irradiance,
fl� fluorescence quantum yield, ph� :phosphorescence quantum yield, isc� :inter-system crossing,
3P� :metastable triplet state photosensitizer, 2
3O ground state triplet molecular oxygen, 21O :excited
singlet oxygen, modified from [12].
2.2. Mechanism of Tumor Destruction
PDT mediates tumor destruction by three mechanisms: direct cytotoxic effects of free
radicals and oxidation products on tumor cells, damage to the tumor-associated vasculature,
and activation of immune response against tumor cells [1][2][4][13]. These three PDT
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effects influence each other, and the combination of all three of these factors is required for
long-term tumor control.
2.2.1. Cellular Effects
PDT induces cell death via either apoptotic or necrotic pathways. The drug incubation time
prior to the administration of light influences the mode of cell death: longer incubation
times (1 day) result in apoptosis and shorter incubation times (1 hour) result in necrosis [1-
2]. As discussed earlier, the highly reactive 21O has a short lifetime (<40μs) and short
radius of action (<20 ns). For lipophilic and anionic sensitizers this will damage all
membranes including plasma, mitochondrial, and lysosomal membranes and also
membranes of the nucleus and endoplasmic reticulum [2] [12]. There is evidence that the
inactivation of membrane transport systems, plasma membrane depolarization and the
inhibition of DNA repair enzymes may precede inactivation of mitochondrial enzymes; the
latter is often the key event leading to cell death [2][12]. �
2.2.2. Vascular Effects
The effects of PDT on micro-vascular structures are rapid and dramatic and the
consequences of this vascular damage for the tumor microenvironment are severe. It has
been shown that Photofrin-PDT at high fluence rates can protect normal skin
microvasculature while severely damages tumor vasculature and kills tumor cells.
Occlusion of the tumor- surrounding vasculature can contribute to tumor control by
depriving nutrients and retarding the vascular resupply of the tumor [14-19].
��������������������������������������������������������������������������������������������������������������������������������������������������������8
2.2.3. Reaction of the Immune System
PDT has also been shown to trigger an inflammatory response and enhance specific anti-
tumor responses. Infiltration of lymphocytes, leukocytes, and macrophages into PDT
treated tissue is an indication of activation of the immune system in response to PDT. The
strength of the inflammatory response varies with the photosensitizer. For instance,
Photofrin II-PDT induces a strong inflammatory response and rapid influx of neutrophils,
which is critical to long-term tumor control [12-13]. Tumor tissue disruption is a direct
effect of PDT, whereas the immune response is required to eliminate the surviving cells.
2.3. Photosensitizer
Photosensitizers which undergo efficient intersystem crossing into the excited triplet state,
and whose triplet state is long- lived enough to allow adequate time for interaction with
oxygen, produce high yields of singlet oxygen [12][20]. Most photosensitizers in clinical
use have triplet-state quantum yields from 40 % to 60% [19]. Table.1 shows the list of
clinically approved photosensitizers in oncology, many of which were introduced in the
1980s and 1990s [12]. However, new generation PDT photosensitizers are continually
being discovered and investigated. Photosensitizers can be categorized by their chemical
structures and origins. In general, they can be divided into three broad families: (i)
porphyrin-based photosensitizers (e.g., Photofrin, ALA/ PpIX, BPD-MA), (ii) chlorophyll-
based photosensitizers (e.g., chlorins, purpurins, bacteriochlorins), and (iii) dyes (e.g.,
phtalocyanine, napthalocyanine). Most of the currently approved clinical photosensitizers
belong to the porphyrin family. Traditionally, the porphyrin photosensitizers and those
photosensitizers developed in the late 1970s and early 1980s are called first generation
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photosensitizers (e.g., Photofrin). Porphyrin derivatives or synthetics made since the late
1980s are called second generation photosensitizers (e.g., ALA, and Photofrin II). Third
generation photosensitizers generally refer to the modifications such as biologic conjugates
(e.g., antibody conjugate, liposome conjugate) and with built-in photo quenching or
bleaching capability [12][19].
The general guidelines for comparing different photosensitizers are based on:(i)low dark
toxicity but strong photo toxicity, (ii) good selectivity towards target cells, (iii) longer
excitation wavelength allowing deeper light penetration, (iv) biocompatibility and rapid
removal from normal healthy tissues of the body, and (v) different routes of administration.
There are only a few photosensitizers in oncology that have received official approval
around the world [2] [3] [19]. Table.2.1 below lists a few photosensitizers that are used
worldwide [14] .
Table.2.1. Clinically approved photosensitizers in oncology, from [13].
Photofrin is the first photosensitizer approved by health agencies worldwide for the
treatment of cancer [21-27]. Photofrin II is commercially available from Axcan Pharma,
Inc. and has the longest clinical history and patient track record [12]. Canada approved
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Photofrin II applications in 1993 for the treatment of bladder cancer. Photofrin II was
approved in Japan in 1994 (for early stage lung cancer). It was approved by the U.S. FDA
for clinical phase II trials in December 1995 esophageal cancer, and in 1998, it was
approved for the treatment of early non-small cell lung cancer. In August 2003 the FDA
approved its use for Barrett's esophagus, and endobronchial lesions. It is also being
considered as a potential therapy against Kaposi’s sarcoma, psoriasis and cancers of the
head, brain, neck and breast and early-stage cervical cancers [20-22]. A major sub- cellular
target for Photofrin II is known to be mitochondria. Photofrin II is also shown to have great
selectivity toward diseased tissue [1]. In general, Photofrin II doses range from 1 to 2 mg
per kilogram of patient’s body mass. Patients are known to become susceptible to severe
burns from bright light exposure including the sunlight during Photofrin II treatment,
therefore, patients and their family need to be educated prior to receiving the Photofrin II to
take appropriate precautions such as wearing clothing that covers the body completely.
Patients shouldn’t remain in dark room during the day either as photo bleaching by low-
level light enhances clearance of the drug from the skin [2].
The mechanisms of action of Photofrin-mediated PDT include vascular endothelial cell
damage with hypoxia and thrombosis, ischemic tumor cell necrosis, and intense local
inflammation associated with immune response [23].
2.4. Tumor Oxygenation
Tissue oxygen supply is another important factor that affects PDT treatment outcome. Any
reduction of oxygen supply reduces the amount of 21O generation, causing negative
outcome of PDT treatment. Reduction can arise from many different sources such as
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preexisting tumor hypoxia, vascular damage, and through rapid photochemical oxygen
depletion during PDT treatment which is governed by the intensity of light exposure [2].
Because of deteriorating diffusion geometry, structural abnormalities of tumor micro
vessels and disturbed microcirculation, solid tumors are prone to develop hypoxic regions
within the tumor volume. With photosensitizers including Photofrin II that can constrict
and occlude vessels, blood-flow obstruction can be remarkably large, restricting oxygen
supply to the tumor [2].
Photochemical oxygen depletion will result if the rate of photodynamic oxygen
consumption is faster than that rate of oxygen resupply from the vasculature. The oxygen
depletion is found to depend upon: 1) the tissue concentration of the photosensitizer and its
absorption coefficient value at the wavelength of excitation, 2) the intensity of light (i.e.,
fluence rate), and 3) the vascular supply of the tissue. If the first two parameters are high,
and the third parameter is low, 21O generation will be fast and oxygen depletion occurs
rapidly [2]. Photobleaching is the photochemical destruction of the photosensitizer.
Destruction of the photosensitizer through photobleaching will reduce the occurrence of
oxygen depletion [2].
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Chapter 3: Micro- and Nanoparticles Induced Visible Luminescence to Activate Photosensitizers within Deep-
Seated Tumors �
Compared to current cancer treatments, such as surgery, radiation, and chemotherapy, PDT
is considered to be minimally invasive. Due to the photo-agent’s high degree of selectivity
in the diseased tissue, the PDT strategy offers a greater capability to accurately target and
destroy the target of interest, and is not subject to the total-dose limitations associated with
radiotherapy [4]. Despite these advantages, PDT has not yet gained general clinical
acceptance. Photofrin II has its primary excitation maxima near 400 nm. However, human
blood is the dominant absorber near 400 nm as well. Thus at the present time, the
absorption of the surrounding tissue of the light needed to excited the Photofrin II
inherently limits the use of PDT applications to the target-sites which are shallow in depth
(~ 2 – 3mm). Consequently, the clinical applications of PDT have been limited to skin
lesions, superficial solid tumors, or endoscopically accessible regions. To increase light
penetration depth, PDT treatments are traditionally made with a red He-Ne laser at 633nm
wavelength, where oxygenated blood is known to absorb considerably less (three orders of
magnitude) than at 400 nm, as shown Figure. 3.1 [28].
�
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Figure.3.1. Absorption coefficients of whole blood
RED = oxy-hemoglobin, BLUE = deoxy-hemoglobin, from[28]
For these reasons, additional strategies need to be designed to activate photodynamic
agents within deep-seated tumor locations in the body. One possibility to reach in deep-
seated tumors is the use of “soft” energy diagnostic X-rays and infrared lasers as non-
invasive tools to produce visible light emission from “rare earth” particles. These particles
are fabricated with an inorganic core, and absorb either incident X- ray photons (down-
conversion) or multiple infrared photons (up-conversion) then relax to emit visible light.
Photosensitizer molecules could be activated directly through emitted light from the
particles [6-8]. Section 3.1 provides background on X-ray generation and X-ray down
converting particles; Section 3.1 provides background on Infrared up converting particles.
3.1. Review of X-ray Production and Down Converting (DC) Micro-
Particles
In this section principles of X-ray generation, and background on micron size DC particles
will be summarized.
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3.1.1. X-Ray Generation
X-rays are produced when electrons (initially at rest) are accelerated under high electric
potential difference between cathode and anode plates within a vacuum (X-ray) tube, and
converting the kinetic energy of the accelerated electrons into electromagnetic radiation as
a result of collisional and radiative interactions [29, 30]. The following events are required
to produce X-rays. First, free electrons are required in the evacuated environment of the X-
ray tube insert for electrical conduction between the electrodes; The next step involves
application of high-voltage differential (50-150 kV) by X-ray generator to the cathode and
anode plates in order to accelerate the electrons to the electrically positive anode plate; X-
rays are produced through the interaction between the highly energized electrons and anode
plates (i.e., targets). Generally, targets are made of Tungsten due to its high atomic number
(Z=74) and very high melting point. These properties facilitate efficient X-ray production
and allow the target to tolerate the high-power deposition of the x-ray generation process
without being destroyed [29].
There are two possible interactions of electrons with the target, resulting in the production
of Bremsstrahlung (breaking radiation) and characteristic radiation, as shown in Figure 3.2
(a). The Bremsstrahlung X-rays result from the conversion of kinetic energy to
electromagnetic radiation when the incident electrons are decelerated through the
interaction in the vicinity of the target nucleus. Closer interactions between the nucleus
and the electrons cause greater decelerations and result in higher X-ray energy. X-ray
energy is at a maximum when electrons give up all their kinetic energy when stopped by
target nuclei and are determined by the peak potential difference. The spectrums of X-rays
are produced with minimum number at peak energy, and linearly increasing in number with
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decreasing energy (see unfiltered Bremsstrahlung spectrum) [31] . It was theoretically
formulated by Kramer that when the electron’s velocity vector is perpendicular to its
deceleration vector during the collision within the anode atoms, the spectral X-ray intensity
distribution as a function of wavelength � is given by[32, 33]:
)/1}.(1/{... 200 �� ZiVCI (3.1)
Where Z is the atomic number of the anode material and i is the X-ray tube current. �o = hc
/ (eVo), where h is the Planck’s constant and c = speed of light. �o is known as the “cut-off”
wavelength. Where Vo is the electric plate potential difference.
Characteristic X-rays result when the incident electron interacts with the target atom and
removes the electron from its innermost K shell (Figure 3.2(b)). Because now the atom is
energetically unstable, electrons from the other shells (L, M, N, and O) will make the
transition to fill the K-vacancy. As a result, a discrete energy X-ray photon is created with
energy equal to the difference in binding energies (for Tungsten, binding energies of the K,
L, M,). The emitted X-ray energies are characteristic of the element (Tungsten) [29]. These
characteristic X-rays add mono-energetic peaks to continuous spectrums. (See Figure.3.2
(b)).
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(a) (b)�
Figure.3.2. X-ray production; ( a) X-ray production by energy conversion. Events 1, 2, and 3 depict
incident electrons interacting in the vicinity of the target nucleus, resulting in Bremsstrahlung x-rays with the emission of a continuous energy spectrum of x-ray photons. Event 4 demonstrates
characteristic radiation emission, where an incident electron with energy greater than the K-shell binding energy collides with and ejects the inner electron creating an unstable vacancy. An outer shell electron transitions to the inner shell and emits an x-ray with energy equal to the difference in binding
energies of the outer electron shell and K shell that are “characteristic” of tungsten. (b) Bremsstrahlung and characteristic radiation spectra are shown for a tungsten anode with x-ray tube
operation at 80, 100,120, and 140 kVp and equal tube current, from [29].
3.1.2. X-Ray Down-Converting Particles
Down conversion is a process in which the absorption of a high frequency photon (X-ray)
yields to emission of output radiation in the visible range [34-38]. The phosphor Gd2O2S:
Tb has been widely used in radiographic intensifying screens (scintillating screens) in
medical imaging systems such as X-ray fluoroscopy, X-ray Computed Tomography, Single
Photon Emission Tomography, and Positron Emission Tomography due to its high
absorption of X-ray energy and efficiency in converting it into visible light [34-37]. X-ray
scintillation of Gd2O2S requires some minute levels of crystal lattice packing defects for
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significant visible light emission to occur. Small lattice packing defects are achieved
through the introduction of a second rare-earth element dopant such as Tb [34-36].
3.2. Review of Up Converting (UC) Nanoparticles
Up conversion (UC) is a nonlinear process in which successive absorption of two or more
near infrared wavelength photons leads to the emission of output radiation at shorter
wavelength within the visible range, through intermediate long-lived energy states [39-41].
Lanthanide-doped phosphor UC nanoparticles were first utilized by Zijlman and coworkers
to study biological recognition events in which submicron-size phosphor crystals (0.2–0.4
μm) surface labeled with antibodies were utilized as a novel luminescent reporter for the
sensitive detection of antigens in tissue sections or on cell membranes [39]. The UC
technique significantly minimizes background auto-fluorescence, photo-bleaching, and
photo-damage to the biological specimens and offers remarkable sample penetration depths
that are much higher than those obtained by UV or visible excitation [41]. UC processes
can be induced by low power (intensity is about 1mW/cm2), cost-effective, continuous
wave lasers. This is advantageous because low power lasers are required for biological
applications in order to minimize surrounding tissue damage [41].
In recent years, biological applications of UC nanoparticles have been rapidly expanded to
in vitro detection, in vivo imaging, molecular sensing, and drug delivery [41-46]. Inorganic
crystals exhibit UC luminescence when lanthanide dopants are added to the crystalline host
lattice in low concentrations. Efficient UC only occurs by using a small number of well-
selected dopant–host combinations. Rare earth fluorides are regarded as excellent host
lattices for up-conversion luminescence of lanthanide dopants due to their high refractive
��������������������������������������������������������������������������������������������������������������������������������������������������������18
index and low phonon energy and ability to exhibit adequate thermal and environmental
stability. Among the investigated fluorides, NaYF4 has been found to be one of the most
efficient UC host lattices and has attracted more attention in the field of materials science
over the past two decades. The dopants are in the form of localized luminescent centers.
The dopant ion radiates upon its excitation to a higher energetic state obtained from the
non-radiative transfer of the energy from another dopant ion. The ion that emits the
radiation is called an activator, while the donor of the energy is the sensitizer [41].
UC processes are mainly divided into three broad classes: excited state absorption (ESA),
energy transfer up conversion (ETU), and photon avalanche (PA). All these processes
involve the sequential absorption of two or more photons (Fig.3.3). Figure.3.3(a) shows
ESA in which excitation takes the form of successive absorption of pump photons by a
single sensitizer. The first pump photon promotes the dopant from the ground state (G) into
a metastable state (E1). The second photon promotes the same excited dopant from E1 state
to higher energy state E2. Optical transition from E2�G results in higher energy photon
emission. Figure.3.3 (b) shows the ETU process in which each of two neighboring ions
populates the E1 level by absorbing a pump phonon of the same energy. One of the ions is
promoted to the upper emitting E2 while the other ion relaxes back to ground state G by
non-radiative energy transfer. Figure.3.3 (c) shows a PA process in which the meta-stable
level population is established through the inverse population of the E1 level by non-
resonant ground state absorption (GSA) followed by resonant ESA to populate the upper
visible emitting level E2.Cross relaxation energy transfer then occurs between the excited
ion and neighboring ground state ion causing both ions to occupy the E1 level. Then the
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two ions populate E2 to further initiate cross relaxation, then strong UC emission is
produced followed by an exponential increase in the E2 level population by ESA [41].
Figure.3.3. Principal UC processes for lanthanide-doped crystals :(a) excited state absorption, (b) energy transfer up conversion,(c) photon avalanche. The dashed/dotted, dashed, and full arrows
represent photon excitation, energy transfer, and emission processes, respectively, from [41].
Requirements for efficient Lanthanide (La) luminescent bioprobes are: (i) water solubility,
(ii) large thermodynamic stability, (iii) inertness, (iv) intense absorption above 330nm, (v)
efficient energy transfer into La ion (vi) coordination cavity minimizing non-radiative
deactivations, (vii) long excited state life time, (viii) ability to conjugate with bio-active
molecules while retaining its photo physical properties without altering the bio-affinity of
the host [40, 41, 46, 47] .
The mechanism of up conversion for the Yb3+, Er3+ or Yb3+, Tm3+ co-doped nano-
crystals has been extensively studied, and is illustrated in Figure.3.4. The absorber Yb3+
��������������������������������������������������������������������������������������������������������������������������������������������������������20
ions absorb NIR light, followed by the energy transfer to the emitter Er3+ or Tm3+ ions
that then emit visible light. Although the emitter can be excited directly, co-doping of the
absorber with ions such as Yb3+ in the nanocrystals usually generates stronger up
conversion fluorescence, because Yb3+ ions have a broad and strong absorption at �980
nm (the absorption cross-section of Yb3+ is 10 times larger than that of Er3+) [41].
Figure.3.4. Proposed energy transfer mechanisms showing the UC processes in Er3+, Tm3+, and Yb3+ doped crystals under 980-nm diode laser excitation. The dashed-dotted, dashed, dotted, and full arrows
represent photon excitation, energy transfer, multiphonon relaxation, and emission processes, respectively. Only visible and NIR emissions are shown here, from [41]
The fluorescence quantum yield (QY) can be defined as the ratio of photons absorbed to
photons emitted. It gives the probability of deactivation of the excited state through the
process of fluorescence.
QY = no. of photons emitted / no. of photons absorbed (3.2)
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The UC particle investigated in this work, NaYF4: Yb/Tm, is relatively new to the UC field
of study and its optical and physical properties vary greatly based on how they are
synthesized, and upon their surface modification properties [41, 48, 49]. It was reported
previously that introduction of an inert crystalline shell of an undoped material around each
doped nanocrystal can increase luminescence efficiency up to 30 times [49].
Determination the quantum yield of UC nanoparticles are very difficult because standards
that show up-conversion property are not available and there have not been any reports
until very recently. It was determined by Boyer et al that quantum yield of various sizes of
NaYF4 particles vary from 0.005% to 0.3 % [50].
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Chapter 4 : Experimental Quantification of ROS Generation
�
This chapter describes the ROS generation from Photofrin II through the activation of X-
ray DC particles, and infrared UC particles in a cellular-like medium. The ROS generation
was quantified by measuring the change in the absorbance of Vitamin C at 266 nm. This
vitamin C essay works due to the fact that when Vitamin C is oxidized it has no absorbance
at 266 nm, so absorbance decreases with ROS generation. In order to understand and
illustrate how the UC and DC particles’ physical properties are related to the ROS
generation from Photofrin II, I also measured the spectral characteristics of both
Gd2O2S:Tb and NaYF4:Yb/Tm particles and of Photofrin II.
4.1. Spectroscopic Characterization of Photofrin II, Up-converting
(UC), and Down-Converting (DC) Particles
I measured the spectral characteristics of both DC (Gd2O2S:Tb) and UC(NaYF4:Yb/Tm)
particles and of Photofrin II to understand how these particles’ physical properties affect
Photofrin II excitation. The light absorption spectrum of Photofrin II was measured using a
Shimadzu, Inc. (UV-3101PC) scanning absorption spectrophotometer (Figure.4.1 (a)). The
emission and excitation spectral characteristics of Photofrin II were measured using a
Photon Technology International, Inc double monochromator fluorescence scanning
spectrophotometer (Figure.4.1 (b) and Figure.4.1 (c)). The Photofrin II solution was
prepared at a physiologically relevant concentration of 20 �g /ml in Ca+2 free and Mg+2 free
Dulbecco’s PBS (In general Photo II dose ranges from 1 to 2 mg per kilogram of patient’s
body mass. Considering a patient with 70 Kg of body weight with 5000 ml of blood,
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physiologically relevant concentrations range from 14 to 28 �g /ml). For all of these
measurements the samples were placed in a 4 ml quartz cuvette. Figure.4.1 (a), Figure.4.1
(b), and Figure.4.1(c) are the absorption, normalized emission and excitation spectrum of
Photofrin II respectively.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
Wavelength (nm)
Nor
malized
Em
ission
397nm
325 nm504 nm
537nm559nm
We can see from Figure.4.1 that Photofrin II has an absorption maximum at 366 nm
(Figure.4.1 (a)), an emission peak of 612 nm, (Figure.4.1 (b)) and a main excitation peak
(c)�
a� b
0
0.2
0.4
0.6
0.8
1
1.2
200 250 300 350 400 450 500 550 600 650 700Wavelength (in nm)
Abs
orpt
ion
(OD
)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800
Wavelength (nm)
Nor
mal
ized
em
issi
on
366 nm 612 nm
675 nm
(b)(a)�
Figure.4.1. Spectroscopic characterization of Photofrin II. (a)Absorption spectrum of Photofrin II in Dulbecco's PBS without Ca/Mg.[ Photo II ] = 10μg/ml. (b) Normalized Photofrin II emission
spectrum in DPBS.[Photo II] = 20μg/ml, Excitation wavelength = 400nm. (c) Normalized Photofrin II excitation spectrum in DPBS. [Photo II] = 20 μg/ml, emission measured at 612nm, from [50].
��������������������������������������������������������������������������������������������������������������������������������������������������������24
(i.e., the Soret band) near 400 nm (Figure.4.1(c)). These results are of interest because they
allow us to understand the connection between the activated amount of Photofrin II and
the resulting amount of ROS generation. Since Photofrin II has Soret a band near 400 nm,
we expect the highest amount of Photofrin II activation and ROS generation when it is
excited with a 400 nm source.
In order to measure the emission spectrum of the Gd2O2S:Tb particles, they were placed
into a 15 ml polystyrene test tube in powder form and irradiated with diagnostic X-rays
operating with a constant X-ray tube current of 20 mA and X-ray tube potential differences
of 130 keV. The X-ray induced spectrum from the particles was measured using an Ocean
Optics, Inc. fiber optic spectrometer spanning the wavelength range from 200 nm to 900
nm (Figure.4.2 (a) is the experimental set up and Figure.4.2 (b) is the result). The beam
spot size was 10 cm. The sample was 1 meter away from the source and 0.5 meters away
from the collimator.
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
200 250 300 350 400 450 500 550 600 650 700 750 800
Wavelength (in nm)
544nm
493nm588nm
622nm
Figure.4.2. Measuring X-ray induced light emission spectrum from Gd2O2S:Tb particles. (a) Experimental set up for measuring X-ray induced light emission spectrum from Gd2O2S:Tb particles.
(b) Normalized X-ray induced light emission spectrum from Gd2O2S:Tb particles with X-ray tube settings of 130kVp operating with a current of 20mA, from [50].
Figure.4.2 (b) shows the emission spectrum of Gd2O2S: Tb particles. We could see that
Gd2O2S: Tb particles have their emission peak at 544 nm. Since the Gd2O2S: Tb emission
peak of 544 nm does not match the Photofrin II Soret band of 397 nm; we expect only
partial activation of Photofrin II when excited by emission from Gd2O2S: Tb particles.
In order to understand how the luminescence intensity is related to X-ray energy,
Gd2O2S:Tb particles, in powder form, were placed into a 15 ml polystyrene test tube and
Spectrometer�
X�ray�generator�
Collimator�
Beam�spot�size� Test�tube
(a)�
(b)�
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irradiated with diagnostic X-rays operating with a constant X-ray tube current of 20 mA
and various X-ray tube potential differences ranging from 10 keV to 130 keV. We
measured the X-ray induced emission intensity at 544 nm wavelength as a function of X-
ray energy using a Newport, Inc. light power meter (The experimental set up was similar to
the Figure.4.2(a), however the power meter was replaced with the spectrometer).The power
meter was placed in line with and 1cm away from the polystyrene test tube.
y = -1E-12x6 + 6E-10x5 - 1E-07x4 + 8E-06x3 - 0.0002x2 + 0.0017xR2 = 1
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80 100 120 140
Maximium X-ray Photon Energy (in kVp)
Inte
nsity
(uW
/cm̂
2)
Figure.4.3. X-ray induced light emission intensity dependence on the maximum X-ray photon energy (with a fixed tube current of 20mAs) from rare-earth (Gd2O2S:Tb) particles, from [50].
�We could see from Figure.4.3 that, above 60 keV of X-ray excitation energy, there is a
relatively linear relationship between the X-ray energy and fluorescence intensity measured
at 544 nm emission wavelength. From this result we expect that the amount of Photofrin II,
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which is activated at 544 nm, and the generated ROS, to increase with increased X-ray
energy.
In order to understand the emission characteristics of NaYF4:Yb/Tm particles in pellet
form, 0.5 cm in diameter (since the materials are lyophilized there is a water-soluble
portion that has been freeze-dried, each pellet has 0.5 mg of nanocrystal) were placed on a
horizontal stage 1 cm away from laser source and irradiated with 980 nm laser with an
intensity ranging from 150 to 1000 mW/cm2 (Figure.4.4 shows the experimental setup).
The infrared laser-induced emission spectrum from the NaYF4: Yb/Tm particles were then
collected by using an Ocean Optics, Inc. fiber optic spectrometer over the wavelength
ranged from 200 to 900 nm (results are shown in Figure.4.5). The fiber tip was
perpendicular to the laser beam direction.
Figure.4.4. Experimental set up for measuring IR induced light emission spectrum of NaYF4:Yb/Tm up- convertors. P: particles in pellet form.
Figure.4.5 (a) shows the emission spectrum of the NaYF4: Yb/Tm up convertors. Figure.4.5
(b) is the same emission spectrum of the NaYF4: Yb/Tm particles not including 802 nm
peaks in order to intensify other emission peaks.
Laser�Diode�Spectrometer�
Signal�acquisition�
UV�IR�fiber�
P
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Intensity profile of particles ecxited with 980nm Laser
700900
1100130015001700190021002300
200 300 400 500 600 700 800
Emission wavelength(nm)
Fluo
resc
ence
in
tens
ity(A
rbita
ry u
nits
)
644nm
475nm
450nm
360nm
Figure.4.5. Emission spectrum of the NaYF4:Yb/Tm up converter(a) Emission profile of NaYF4:Yb/Tm in response to 980nm laser excitation including 802 nm emission peak;(b) Emission profile of
NaYF4:Yb/Tm in response to 980nm laser excitation not including 802 nm emission peak
We could see from Figure.4.5 that NaYF4: Yb/Tm particles have several emission peaks
(360 nm, 450 nm, 475 nm, 644 nm, and 802 nm) in UV-VIS-NIR. Since emission peak
(b)�
Infrared induced light emission spectrum from NaYF4:Yb/Tm particles with 980nm laser operating at
950mW/cm^2
7002700470067008700
10700127001470016700
200 400 600 800Emission wavelength (nm)
Fluo
resc
ence
inte
nity
(Arb
itary
un
its)
802nm
644nm475nm
450nm361nm
(a)�
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values from these particles are different from the Photofrin II Soret band of 400 nm, we
expect there will be a lower amount of Photofrin II activation and ROS generation when
activated by NaYF4: Yb/Tm particles compared to activation by a 400 nm source.
I attempted to obtain the absorption spectrum of NaYF4: Yb/Tm particles in PBS solution
using a Shimadzu, Inc. (UV-3101PC) scanning absorption spectrophotometer. However, I
was not able to obtain the absorption spectrum due to the strong and inevitable absorbance
of water around 1000 nm. We expect that these particles would exhibit an absorption peak
at 980 nm since they only emit fluorescence when they are excited with 980 nm laser.
In order to see how the laser excitation power is related to emission intensity, the
NaYF4:Yb/Tm particles in pellet form (0.5cm in diameter, since the materials are
lyophilized there is a water-soluble portion that has been freeze-dried, each pellet has 0.5
mg of nanocrystal) were placed on a horizontal stage 1 cm away from Laser source and
irradiated with a 980 nm laser with an intensity ranging from 150 to 1000 mW/cm2(setup
shown in Figure.4.4). The infrared laser induced emission spectrum from the NaYF4:
Yb/Tm particles were measured using an Ocean Optics, Inc. fiber optic spectrometer over
the wavelength range 200-900 nm. Figure.4.6 (a) shows emission intensity profile of peak
values in response to various laser intensities. Figure.4.6 (b) is the same emission intensity
profile of the NaYF4: Yb/Tm particles, not including values at 802 nm in order to more
clearly show the variations in the other intensity values.
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Fluorescence intensity profile of NaYF4:Yb/Tm particles in response to different 980 laser excitation intensity
700
1200
1700
2200
2700
150 250 350 450 550 650 750 850 950
980nm laser Intensity (mW/cm^2)
Fluo
resc
ence
In
tens
ity(A
rbita
ry u
nits
)
265nm349nm360nm450nm475nm644nm
Fluorescence intensity profile of NaYF4:Yb/Tm particles in response to different 980 laser excitation intensity
02000400060008000
10000120001400016000
150 250 350 450 550 650 750 850 950980nm laser intensity(mW/cm^2)
Fluo
resc
ence
inte
nsity
(A
rbita
ry u
nits
)
265nm349nm360nm450nm475nm644nm802nm
�
Figure.4.6. Emission intensity profile of peak values,(a)Fluorescence intensity profile of NaYF4:Yb/Tm in response to different 980nm laser excitation intensity including 802 nm emission
peak;(b)Fluorescence intensity profile of NaYF4:Yb/Tm in response to different 980nm laser excitation intensity not including 802 nm emission peak.
��������������������������������������������������������������������������������������������������������������������������������������������������������31
We could see from Figure.4.6 that there is a relatively linear relationship between the
Infrared excitation power (intensity) and fluorescence intensity when the excitation
intensity is lower than 1W/cm2.
I attempted to measure fluorescence intensity in absolute units using a Newport, Inc. light
power meter placed 1cm away from the pellet and perpendicular to the laser beam direction
(the experimental set up is similar to Figure.4.4, a power meter was replaced with
spectrometer), but I wasn’t able to measure fluorescence intensity in absolute units due to
very weak fluorescence signal. In a previously published paper it was described how very
high fluorescence intensity was achieved in absolute units as a function of excitation power
(they reported 40 mW of fluorescence emission for 200 mW excitation power)[48]. In that
particular experimental setting the particles in solution were placed in a cuvette and
irradiated with 980 nm laser on one side. Then the fluorescence intensity was collected
from the other side. It seems that the achieved result may have been produced by intensity
of the excitation laser. In fact I believe it was caused by the geometry of their setup.
I also attempted to make quantum yield (QY) measurements using the system described in
Figure.4.7. The system was composed of a barium sulfate coated integrating sphere, UV-
VIS transmitting optical filter, Cuvette holder, and 980 nm Laser source. The sample was
held in a quartz cuvette located in the center of the integrating sphere. The sample was
excited with a 980 nm laser diode. The light was delivered to the entrance port using a high
efficiency fiber and was collimated to a beam diameter of 1 mm and directed on the
sample. The emission intensities were measured using a Newport, Inc. light power meter
with and without the UV-VIS transmitting optical filter in order to separate the
fluorescence signal from the excitation signal.
��������������������������������������������������������������������������������������������������������������������������������������������������������32
Figure.4.7. Quantum Yield measurement system. F: UV-VIS transmitting optical filter
I was not able obtain Quantum Yield using the system described in Figure.4.7. This is
believed to be due to the fact that the fluorescence intensity was very weak compared to the
excitation light power and therefore the integrating sphere and optical detectors were not
sensitive enough to measure the fluorescence intensity.
As has been described earlier, only measuring the values of relative units is not adequate
for evaluating the effectiveness of these particles and makes it difficult to theoretically
predict the efficiency of these particles in Photofrin II activation. Determination of the
quantum yield of UC nanoparticles is also very difficult because standards that show up-
conversion properties are not available and there have not been any reports until very
recently. It was determined by Boyer et al that quantum yield of various sizes of NaYF4
particles vary from 0.005% to 0.3 % [51]. Since my Quantum Yield measurements were
not successful, I had used 0.1% for my modeling.
Integrating�sphereIR�laser�
source F
Spectrometer
��������������������������������������������������������������������������������������������������������������������������������������������������������33
4.2. Experimental Quantification of ROS Generation from DC and
UC Particles
Quantification of ROS generation was made through Beer’s Law and the change in
absorbance of un-oxidized Vitamin C at 266 nm (Figure.4.8). This assay is made possible
due to the fact that oxidized Vitamin C has no absorbance at 266 nm, i.e., its molar
extinction coefficient (�) is zero at 266 nm, whereas unoxidized Vitamin C has a large � of
~15,400 M-1cm-1 in PBS.
Fig.4.8. Interaction of Vitamin C with ROS resulting in dehydroascorbic acid, modified from[51]
Vitamin C with 100 �M/ml concentration in Ca+2 free and Mg+2 free Dulbecco’s PBS were
placed in a 4 ml quartz cuvette and its light absorption spectra were measured using
Shimadzu, Inc. (UV-3101PC) scanning absorption spectrophotometer. Figure.4.9 shows
that unoxidized Vitamin C has its highest absorption peak at 266 nm.
+ H2O ROS
Vitamin C (Unoxidized) Ascorbic Acid
Vitamin C (Oxidized)
Dehydroascorbic Acid
��������������������������������������������������������������������������������������������������������������������������������������������������������34
Absorption spectrum of Vitamin C in PBS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
225 235 245 255 265 275 285 295 305 315 325
Wavelength (nm)
Abs
orba
nce (O
D)
266nm
Figure.4.9. Absorption spectrum of unoxidized Vitamin C in PBS.
In order to quantify how the Vitamin C concentration is related to its light absorption
properties, Vitamin C solutions were prepared at concentrations ranging from 10 – 100uM
in Ca+2 free and Mg+2 free Dulbecco’s PBS. For all of the above measurements the
samples were placed in a 4 ml quartz cuvette. Figure.4.10 shows the absorbance of vitamin
C as a function of concentration.
Figure.4.10. Unoxidized Vitamin C absorbance in PBS as a function of concentration.
From Figure.4.10 we could see the linear relationship between the Vitamin C concentration
and absorbance collected at 266 nm. Therefore, the reduction of unoxidized Vitamin C
concentration due to ROS generation and interaction can be directly evaluated by taking
��������������������������������������������������������������������������������������������������������������������������������������������������������35
ratios: �C = {�A/Ao}* Co, where Ao and Co are initial absorbance and concentration of
Vitamin C, respectively, at time t = 0.
4.3. ROS Generation from Photofrin II Activated by 405 nm and
633 nm Lasers
Figure.4.1.c showed the Photofrin II excitation spectrum and its Soret band that peaks at
397 nm. To see how ROS generation from Photofrin II excited at 400 nm differs from 633
nm excitation, which is currently utilized clinically, ROS generation from Photofrin II was
measured using both 405 nm and 633 nm lasers. Photofrin II (5 mg/ml) and Vitamin C (100
μM/ml) in Dulbecco’s PB solution were placed in a 4 ml quartz cuvette and irradiated with
405 nm and 633 nm lasers. The spot size was 1 cm and the power was 0.587 mW for both
lasers. The ROS generation was quantified by measuring the change in the absorbance of
Vitamin C. Figure.4.11 shows ROS production from Photofrin II using lasers operating at
405 nm and 633 nm.
�
Fig.4.11. ROS production from Photo II due to 633nm and 405 nm lasers operating at 0.587mW
��������������������������������������������������������������������������������������������������������������������������������������������������������36
As shown in Figure.4.11, by comparing values of ROS generation, we can see that
Photofrin II has produced 8.5 times greater ROS (by looking rate of change, 0.1905 vs.
0.0226) when irradiated near its main (Soret band) excitation peak at ~400 nm as compared
to the same laser power at the clinical wavelength of 633 nm.
4.4. ROS generation from X-ray down-converters
Figure.4.12 is the experimental setup for the measurement of ROS generation from the X-
ray down convertors. Photofrin II (20�g/ml) and Vitamin C (100 μM/ml) in Dulbecco’s PB
solution were placed into a 15 ml polystyrene test tube with and without the particles and
irradiated with diagnostic 120 keV X-rays operating with a constant X-ray tube current of
20 mA. The spot size was 10 cm and the sample was 1 meter away from the source and 0.5
meters away from the collimator. The ROS generation was quantified by measuring the
change in the absorbance of Vitamin C using Shimadzu, Inc. (UV-3101PC) scanning
absorption spectrophotometer. Figure.4.13 shows ROS production from Photofrin II with
particles and without X-rays, with X-rays and without particles, and with X-rays and
particles both present.
��������������������������������������������������������������������������������������������������������������������������������������������������������37
Figure.4.12. Experimental set up for measuring ROS generation from X-ray down convertors.
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Exposure time (minutes)
Cha
nge
in V
itam
in C
conc
entr
atio
n(uM
)
dark+Vitamin C
dark+Photo II+particles+Vit C
X-rays+PhotoII+Vit C
X-rays+PhotoII+particles+Vit C
[Photo II]=10ug/ml,[Vit C]=100uM, [GdO2S2:Tb]=10mg/ml
Figure.4.13. Comparison of ROS production from Photo II between activation through X-ray induced Luminescence and X-rays alone.
We can see that there is greater ROS generation when X-ray and particles are both present.
I believe the ROS generation from X-rays alone is due to the formation of H2O2 through
the Type 1 mechanistic pathway.
Spectrometer�Signal�Acquisition�
X�ray�generator�
Collimator�
Beam�spot�size� Test�tube
��������������������������������������������������������������������������������������������������������������������������������������������������������38
In order to see how comparable our ROS generation from X-ray DC particles to the
clinically used 633 nm laser, ROS generation from Photofrin II was measured using a 633
nm laser. Photofrin II (10� g/ml) and Vitamin C (100 μM/ml) in Dulbecco’s PB solution
were placed in a 4 ml quartz cuvette and irradiated with 633 nm laser. The beam spot size
was 1 cm and the laser power was set to 9 mW. The ROS generation was quantified by
measuring the change in the absorbance of Vitamin C. Figure.4.14 shows ROS production
from Photofrin II using 633 nm laser operating at 9 mW.
Comparision of ROS generatiofrom Re-He lase+PhotoII to X-ray+Photo II + particles
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
Exposure time (minutes)
RO
S (in
uM
)
Dark
He-Ne laser+PhotoII
X-rays+PhotoII+particles+Vit C
Figure.4.14. Comparison of ROS production from Photo II between activation by 9mW/cm2 He-Ne
laser and through the X-ray induced luminescence.
By looking at Figure.4.14, we could see that the rate of ROS generation through the X-ray
induced luminescence is comparable to that generated by a 9 mW/cm2 He-Ne laser.
��������������������������������������������������������������������������������������������������������������������������������������������������������39
4.5. ROS Generation from IR Up-convertors
Figure.4.15 is the experimental setup for the measurement of ROS generation from the IR
up-convertors. The Photofrin II (10 mg/ml) and Vitamin C (100 μM/ml) in Dulbecco’s PB
solution were placed in 4 ml quartz cuvette and irradiated with a 980 nm laser, with the
particles (10 mg/ml) and without the particles. The laser spot size was 1 cm and power
measured was 830 mW.
Figure.4.15. Experimental set up for measuring ROS generation from IR up- convertors. Q: quartz cuvette.
Figure.4.16 shows ROS production from Photofrin II when there are particles with no laser
exposure and particles with the 980 nm laser exposure.
�
Figure.4.16. ROS generation from NaYF4:Yb/Tm particles. N=2
Laser Diode
Signal acquisition
Q
Spectrometer
[Photo II]=10ug/ml,[Vit C]=100uM,[NaYF4:Yb,Tm]]=10mg/ml
-20
-10
0
10
20
30
40
50
0 5 10 15 20 25
Exposure time(minutes)
Cha
nge
in V
it C
co
ncen
trat
ion(
uM )
dark averageexp avarage
��������������������������������������������������������������������������������������������������������������������������������������������������������40
As shown in Figure.4.16, a rapid increase in the rate of ROS generation was seen in the
first five minutes of IR exposure which then leveled off. The ROS generation was 32 times
greater than that of ROS generation from GdO2S2:Tb particles. The significant difference
in ROS generation from Infrared UC particles and X-ray DC particles is important because
developing the most efficient technique for ROS generation depends on accurate selection
of the most appropriate particles for Photofrin II activation.
In this chapter, I attempted to measure ROS generation from X-ray DC particles, and
infrared UC particles in cellular like medium. The ROS generation was quantified by
measuring the change in the absorbance of Vitamin C. In order to understand and illustrate
how the UC and DC particles’ physical properties are related to the ROS generation from
Photofrin II, I also measured the spectral characteristics of both Gd2O2S:Tb and
NaYF4:Yb/Tm particles and of Photofrin II.
The summary of the results include: Photofrin II has a main excitation peak (i.e., the Soret
band) near 400 nm (Figure 4.1.(c))and it produced 8.5 times greater ROS when irradiated
near its main (Soret band) excitation peak at ~400 nm as compared to the same laser power
at the clinical wavelength of 633 nm; There was greater ROS generation when X-rays and
particles are both present for Photofrin II activation and the rate of ROS generation through
the X-ray induced luminescence was found comparable to that generated by a 9 mW/cm2
He-Ne laser; The ROS generation from Infrared NaYF4:Yb,Tm up-converting particles
were found to be 32 times greater than that of ROS generation from Gd2O2S:Tb down-
converting particles.�
�
�
��������������������������������������������������������������������������������������������������������������������������������������������������������41
Chapter 5: Safety Evaluation of “Rare-earth” Based Materials and Therapeutic Efficacy on Selective Cancer
Cell Lines �
As discussed in Chapter 4, significant ROS generation results were recorded (through the
vitamin C assay) when the DC (Gd2O2S: Tb) and UC (NaYF4: Yb/Tm) particles were
irradiated in DPBS in presence of Photofrin II (20μg/ml). The results showed that both
submicron- to micron-sized DC and UC particles have great potential to activate Photofrin
II and to generate substantial levels of ROS. As the next step in this investigation, I
investigated the therapeutic efficacy of these particles in activating Photofrin II on in vitro
human brain cancer cells. In addition, the possible cellular toxicity of the DC and UC
particles was also investigated. Section 5.1 describes techniques used for cell preparation,
cell line maintenance, and cellular metabolic activity measurements; Section 5.2 describes
the results of the therapeutic efficacy of DC particles on the human glioblastoma cancer
cell lines and their cell toxicity evaluation; And section 5.3 describes the results of the
therapeutic efficacy of the UC particles on the human glioblastoma cancer cell lines and
their cell toxicity evaluation.
5.1. Cell Maintenance, Cellular Metabolic Activity Measurement
Techniques
Cell line Maintenance: Human malignant (brain cancer) glioblastoma cells were
purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown
and maintained in T-75 flasks under incubation conditions of 5% CO2 at 37oC. The
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adherent cells were maintained in ATCC formulated DMEM / F12 growth medium with
10% of fetal bovine serum and 50 units/ml of penicillin and streptomycin antibiotics.
Cell preparation: When the actively dividing glioblastoma cells reached 50 – 60%
confluence within the T-75 flasks, the cells were trypsinized and brought into suspension.
The cells were then spun down and the (trypsin) supernate was discarded. The cells were
re-suspended in their respective fresh growth media at an initial working concentration of
10 K/ml. The cell suspension was then transferred into single wells of 96-well plates with a
transfer volume of 0.1 ml (or 1,000 cells seeded per selected well). The cells were seeded
into every other well in order to minimize possible overlap in the X-ray or laser light
exposure. The cells within the 96 well plates were returned back into the incubator for
approximately 40 hours before the X-ray or laser light exposure.
Measuring cellular metabolic activity: The metabolic response of glioblastoma cells to
X-ray/infrared laser exposure was assessed with a non-radioactive colorimetric cell
metabolic assay (Tetrazolium compound (MTS), Promega, Madison, WI) in duplicate
(control and exposed), three days after the X-ray/Infrared Laser exposure treatments. On
the day of measurement, the 96-well plates were removed from the incubator and 20 μl of
the MTS solution were added to each cell containing well. The plates were then returned to
the incubator for a two hour incubation period.
Functionally, the MTS readily permeates through the cell membrane and is metabolized
and converted into formazan by living cells (Figure.5.1). Conversion into formazan induces
a maximum change in absorption at 490 nm wavelength.
��������������������������������������������������������������������������������������������������������������������������������������������������������43
Two hours after the addition of MTS, absorption measurements were made at 490 nm with
a 96 well plate reader. The average absorbance value at 490 nm of the treated cell’s
metabolic activity was computed with standard deviations, and X-ray treated, or infrared
treated cell’s metabolic activity were computed and normalized relative to the sham
exposed (control) metabolic activity.
�
Figure.5.1. Structures of MTS tetrazolium and its formazan product, from:http://www.promega.com/tbs/tb245/tb245.pdf
��
5.2. Therapeutic Efficacy and Cell Toxicity Results of X-ray DC
Particles on Selective Cancer Cell Lines
Therapeutic efficacy: Figure.5.2 is experimental setup for the measurement of cellular
metabolic activity in response to X-ray exposure. Equal numbers of human glioblastoma
cells (103 / well) were seeded in six central wells of 96 well plates for X-ray exposure. The
seeded glioblastoma cells were incubated for 12 hours overnight prior to Photofrin II
incubation. After an additional 24 hours of Photo II incubation, the 96 well plates selected
for X-ray treatments were incubated with Gd2O2S: Tb particles (5mg/ml) for 4 hours and
��������������������������������������������������������������������������������������������������������������������������������������������������������44
thereafter X-ray exposed. All X-ray exposures were done in the dark at room temperature.
Cells were exposed to a 120 keV X-ray beam for 15 minutes. Beam spot size was 10 cm.
The sample was 1 meter away from the source and 0.5 meters away from the collimator.
The irradiated cells with their sham exposed counterparts were then returned to the
incubator (and incubated in the dark) for an additional 48 hours and assayed for cell
viability using the MTS assay. Results of Glioblastoma cell viability due to the 15 minute
diagnostic X-ray exposure are shown in Figure.5.3.
Figure.5.2. X-ray exposure set up and measurement of cell viability 48 hours post exposure.
Cellular�metabolic�activity�analysis�
Signal�Acquisition�
X�ray�generator�
Collimator�
Beam�spot�size�i i
96�well�plates
48�hours�incubation�
��������������������������������������������������������������������������������������������������������������������������������������������������������45
Normalized Glioblastoma Cell Viability After 15 Min Diagnostic X-ray Exposure (120kVp, 20mAs) , [Photofrin II]=20ug/ml, [Gd2O2S:Tb]=5mg/ml, MTS Incubation Time 2 Hrs
020406080
100120140
cells
cells
+part
icles
cells
+part
icles
+Pho
to2
cells
+Pho
to2
treatment conditions
% o
f cel
l via
bilit
y re
lativ
e to
con
trol
dark
X-rayexposure
Figure.5.3. Normalized Human Glioblastoma cellular metabolic activity through MTS measurements taken 48 Hrs after a 15 Min diagnostic X-ray exposure (120kVp, 20mAs) [Photofrin II] =20�g/ml,
[Gd2O2S: Tb] =5mg/ml MTS incubation time 2 Hrs. N = 3, Avg. + SD, from [50].
As shown Figure.5.3, approximately 20% suppression in the cellular metabolic activity was
realized from the X-ray alone and X-rays with Photo II treatment conditions. Interestingly,
the presence of Gd2O2S:Tb particles without the Photo II appears to confer protection
against the ionizing radiation as no reduction in the cellular metabolic activity was
observed.�This conferred protection is mechanistically conceivable due to the fact that the
density of Gd2O2S:Tb is 7.44 fold greater than water [31], and the 20 μm size particles
sink down on top of the glioblastoma cells, surround the periphery of cells, and fill the
empty spaces between cells, thus forming a “shield” barrier surrounding the cells. The
Gd2O2S: Tb attenuation coefficient is approximately 70 fold greater than the brain tissue
��������������������������������������������������������������������������������������������������������������������������������������������������������46
for a 120 keV photons (taking the density of glioblastoma cells ~ 1g/cm3 and density of
Gd2O2S:Tb ~ 7g/cm3 ). Severe suppression (> 90% relative to controls) in the metabolic
activity of human glioblastoma cells due to the presence of clinically relevant concentration
of ([20 μg/ml]) Photo II, with Gd2O2S:Tb particles ([5mg/ml]), and (120 keV) diagnostic
X-ray exposure was observed.
Potential cell toxicity determination of Gd2O2S: Tb particles: The human glioblastoma
cell suspension was transferred into single wells of 96-well plates with a transfer volume of
0.1 ml or 1000 cells/well. Cell toxicity of Gd2O2S: Tb particles on these glioblastoma cell
lines was assessed 48 hours after the particle treatment (5 mg/ml concentration, 20 μm in
size) through the MTS assay. On the day of measurement, the 96-well plates were removed
from the incubator and 20 micro-liters of the MTS solution was added to each cell
containing well. The plates were then returned to the incubator for a two hour incubation
period. The particles’ absorbance values at 490 nm were subtracted from the average
absorbance value at 490 nm of the particle treated cell’s metabolic activity. Figure.5.4
shows the cellular effects of Gd2O2S:Tb particles by themselves on the human glioblastoma
cell lines.
��������������������������������������������������������������������������������������������������������������������������������������������������������47
Figure.5.4. Assessment on the potential cellular influence of 5 mg/ml Gd2O2S: Tb particles on human glioblastoma. Human glioblastoma cell were co-incubated with 5mg/ml of Gd2O2S: Tb for 48 Hrs and their cellular metabolic activity was determined through the MTS assay. N = 3, Avg. + SD, from [50].
We could see from Figure.5.4 that there is no remarkable change in the cellular metabolic
activity when human glioblastoma cell lines were treated with Gd2O2S: Tb particles
relative to control.
5.3. Therapeutic Efficacy and Cell Toxicity Results of Infrared UC
Particles on Selective Cancer Cell Lines
Therapeutic efficacy: Figure.5.5 is the experimental setup to measure cellular metabolic
activity in response to infrared laser exposure. Equal numbers of human glioblastoma cells
(103 / well) were seeded in every other wells of 96 well plates for infrared laser exposure.
The seeded glioblastoma cells were incubated for 12 hours overnight prior to Photo II
incubation. After an additional 24 hours of Photo II incubation, the 96 well plates selected
for laser treatments were incubated with NaYF4: Yb/Tm and thereafter laser exposed for 5
010
2030
4050
6070
8090
100110
Control Control + Particles
% o
f cel
lula
r met
abol
ic a
ctiv
ity re
lativ
e to
con
trol
��������������������������������������������������������������������������������������������������������������������������������������������������������48
minutes. All laser exposures were done in the dark at room temperature. The beam
direction was perpendicular to the 96 well plates and beam spot size was 0.5 cm. The
irradiated cells with their sham exposed (control) counterparts were then returned to the
incubator (and incubated in the dark) for an additional 48 hours and assayed for cell
viability using the MTS assay.
Figure.5.5. Infrared laser exposure set up and measurement of cell viability 48 hours post exposure.
Figure.5.6 shows the results of cellular metabolic activity measurements of the UC
particles at the particle concentration of 5mg/ml.
��������������������������������������������������������������������������������������������������������������������������������������������������������49
Figure.5.6. Normalized Human Glioblastoma cellular metabolic activity through MTS measurements taken 48 Hrs after 5 Min of 980nm Laser exposure [Photofrin II] =20�g/ml, [NaYF4: Yb/Tm] =5mg/ml
MTS incubation time 2 Hrs. N = 3, Avg. + SD.
We can see from Figure.5.6 that complete shutdown of cellular metabolic activity resulted
including the background (dark condition) at all the particle treated conditions at a
concentration of 5 mg/ml. Then particle concentration was reduced into 0.5 mg/ml.
Figure.5.7 shows the results of the human glioblastoma cell viability due to the combined
treatment of Photo II with NaYF4: Yb/Tm particles (0.5mg/ml) with 5 minutes of 980 nm
laser exposure.
0
20
40
60
80
100
120
cells cells+UC particles cells+UC particles+Photo II
Treatment conditions
% o
f cel
lula
r met
abilic
activ
ity
rela
tive
to c
ontrol
darkLaser exp
��������������������������������������������������������������������������������������������������������������������������������������������������������50
MTS results of 980 nm laser exposure Exposure time:5minutes, [Photofrin II]=20ug/ml,
[NaYF4:Ym,Tm]=0.5mg/ml
0
20
40
60
80
100
120
cells
cells+
partic
les
cells+
partic
les+P
hoto2
cells+
Photo2
Treatment conditions
% o
f cel
l via
bilit
y re
lativ
e to
co
ntro
ldarklaser exp
Figure.5.7. Normalized Human Glioblastoma cellular metabolic activity through MTS measurements taken 48 Hrs after a 5 Min of laser exposure (980nm, 1982mW/cm^2) [Photofrin II] =20μg/ml,
[NaYF4: Yb/Tm] =0.5mg/ml, MTS incubation time 2 Hrs. N = 3, Avg. + SD.
As shown in Figure.5.7, while 50% reduction in human glioblastoma cell viability in all
particle only treated conditions was observed, the dramatic reduction (>90%) in all laser
exposed conditions was observed.
We also investigated amount of optimum laser intensity and exposure times in order to
ensure that they don’t contribute to the cell metabolic activity measurement. Equal numbers
of human glioblastoma cells (103 / well) were seeded in 96 well plates for infrared laser
exposure. After 48 hours of incubation, the 96 well plates selected for laser treatments were
laser exposed for 60,135, and 300 seconds at different laser intensities. All laser exposures
��������������������������������������������������������������������������������������������������������������������������������������������������������51
were done in the dark at room temperature. The experimental set up was the same as
Figure.5.5. The irradiated cells were then returned to the incubator for additional 48 hours
and assayed for cell viability using the MTS assay (Figure.5.8).
MTS results of Glios at different Laser exposure times
0.0000.1000.2000.3000.4000.5000.6000.7000.8000.9001.000
contr
ol
5min,
237m
w/cm2
5min,
849m
W/cm
2
5min,
1415
mW/cm2
5min,
1982
mW/cm2
2min1
5sec
,1982
mW/cm
2
1min,
1982
mW/cm2
exposure times and laser intensity
ave
Figure.5.8. Human Glioblastoma cellular metabolic activity through MTS measurements taken 48 Hrs after different exposure times and laser intensity, MTS incubation time 2 Hrs. N = 3, Avg. + SD.
As shown in Figure.5.8, there is similar cellular metabolic activity response relative to
control condition when the cells are irradiated with 980 nm laser with the 1415 mW/cm2
intensity with 5 minutes of exposure time and 1982 mW/cm2 intensity with 2 minutes and
��������������������������������������������������������������������������������������������������������������������������������������������������������52
15 seconds exposure time. I choose 1982 mW/ cm2 intensity with 2 minutes and 15 seconds
exposure time as optimal laser parameters due to shorter exposure time.
To ensure cell suppression is mainly from Photofrin II activation through the visible light
from UC particles, next step was to evaluate cellular metabolic response to UC particles
using optimal laser parameters with reduced particle concentration and reduced Photofrin II
concentration.
Equal numbers of human glioblastoma cells (103 / well) were seeded in wells of 96 well
plates for Infrared laser exposure. The seeded glioblastoma cells were incubated for 12
hours overnight prior to Photo II incubation. After an additional 24 hours of Photo II
incubation, the 96 well plates selected for laser treatments were incubated with
NaYF4:Yb/Tm (0.2 mg/ml) and thereafter laser exposed for 2 minutes and 15 seconds
with a 1982 mW/cm2 laser intensity. All laser exposures were done in the dark at room
temperature. The experimental set up is same as Figure.5.5. The irradiated cells with their
sham exposed counterparts were then returned to the incubator (and incubated in the dark)
for an additional 48 hours and assayed for cell viability using the MTS assay. Figure.5.9 is
the result of cellular metabolic response to 1983 mW/cm2 intensity 980 nm laser exposure
for 2 minutes and 15 seconds.
��������������������������������������������������������������������������������������������������������������������������������������������������������53
0
20
40
60
80
100
120
cells
only
cells+
partic
les
cells
+partic
les+Pho
to II
cells+
Photo
II
Treatment conditions
% o
f cel
l via
bilit
y re
lativ
e to
con
trol
darklaser exp
Fgure.5.9. Normalized Human Glioblastoma cellular metabolic activity through MTS measurement taken 48 Hrs after 135 sec of laser exposure (980nm, 1982mW/cm2) [Photofrin II]=15μg/ml,
[NaYF4:Yb/Tm]=0.2mg/ml, MTS incubation time 2 Hrs. N = 3, Avg. + SD.
We can see from Figure.5.9 that the laser exposure of UC particles (0.2mg/ml) did not
contribute to the Photofrin II activation compared to its sham exposed (control) condition.
We can also see that reducing the amount of Photofrin II concentration (15 μg/ml)
resulted in increased cell metabolic activity.
The results of therapeutic efficacy of UC particles had shown that there is severe cell
suppression (>99%) when NaYF4:Yb/Tm particles were used at desired concentration(5
μg/ml) ; 50% cell suppression when the particle concentration was decreased to 0.5 mg/ml
and that the cell suppression wasn’t due to Photofrin II activation through laser induced
luminescence from particles. In order to investigate the source of cell suppression, I
��������������������������������������������������������������������������������������������������������������������������������������������������������54
decided to carry extensive studies on toxicity measurements of the NaYF4: Yb/Tm
particles.
Potential cell toxicity determination of NaYF4: Yb/Tm particles: At first, the cell
toxicity evaluation of NaYF4: Yb/Tm particles on the human glioblastoma cell lines was
assessed using the same MTS assay technique as for the Gd2O2S: Tb particles. The cell
suspension was transferred into single wells of 96-well plates with a transfer volume of 0.1
ml or 1000 cells/well. Cell toxicity of NaYF4: Yb/Tm particles on these glioblastoma cell
lines was assessed 48 hours after the particle treatment (5 mg/ml concentration, 50 nm in
size) through the MTS assay. On the day of measurement, the 96-well plates were removed
from the incubator and 20 μl of the MTS solution was added to each cell containing well.
The plates were then returned to the incubator for a two hour incubation period. The
particles’ absorbance values at 490 nm are subtracted from the average absorbance value at
490 nm of the particle treated cell’s metabolic activity.
Potential cellular influences of 5 mg/ml of NaYF4: Yb/Tm on human glioblastoma cell
lines had resulted in severe suppression (100%) in metabolic activity of the cells (The
results are the same as Figure.5.6).
Nano-enabled drugs and diagnostics present challenges for regulatory agencies such as the
US Food and Drug Administration (FDA). At this present time, the FDA does not have
specific guidance documents, but has recently published recommendations on the subject.
The present medical regulations are expected to apply to nano-enabled drugs and
diagnostics. Additional regulations are required, when considering nanoparticles
��������������������������������������������������������������������������������������������������������������������������������������������������������55
It has been also recently reported (by the FDA Nanotechnology Characterization Group)
that as the nanoparticles size decreases, the particles have tendency to be more toxic (from:
“Agency Nanotechnology Draft Guidance” CDRH Nanotech Reviewer Network (NRN)
Meeting, CDRH/OSEL Presentation).
In addition, since physiochemical properties of nanomaterials are different from those of
their bulk counterparts, their interaction with biological systems is expected to be different.
The effects may vary between different kinds of nanoparticles, depending on chemical
composition, size, and shape.
Contamination of nanoparticles may cause misleading results in toxicity screens
(nanoformulations that are not inherently toxic may appear to be so due to contamination)
and in efficacy tests for certain applications. Testing for endotoxin contamination and
pyrogenicity which examines the ability of the nanoparticles to cause fever are also critical
in vitro assessments before moving on to animal studies and clinical use.
New studies have shown that Endotoxin contamination is a significant hurdle to the
preclinical development of nanoparticles formulations. The large surface areas and high
reactivity of nanoparticles along with the fact that nanoparticles are frequently synthesized
on (dirty) equipment causes endotoxins contamination to be common among many
nanoparticles formulations undergoing preclinical characterization. In recent studies,
endotoxin contamination of gold nanoparticles was shown to be associated with undesired
inflammatory reactions, while purified gold nanoparticles did not cause an inflammatory
response. Endotoxin has been shown to cause tumor regression and was proposed as a drug
in clinical oncology trials (later discontinued owing to severe immunotoxicity).
��������������������������������������������������������������������������������������������������������������������������������������������������������56
Since endotoxins may influence the results of toxicity and efficacy studies, it is important
to identify the source of cell toxicity (endotoxins, or toxic particles) before such studies in
order to avoid misinterpretation of study results.
The LAL assay is an enzyme-based assay with a working time of 45 minutes. The LAL
assay is intended for the quantitative measurement of endotoxins in culture medium,
buffers, plasma, serum and other solutions. Bacterial endotoxin, like lipopolysaccharide
(LPS), is a fever-producing by-product of gram-negative bacteria commonly known as
pyrogen. The principle of the test is based on the fact that bacteria cause intravascular
coagulation in the American horseshoe crab, Limulus Polyphemus. The agent responsible
for the clotting phenomena resided in the crab's amoebocytes, or circulating blood cells and
that pyrogen (bacterial endotoxin) triggered the turbidity and gel-forming reaction
enzymatically. Thus, endotoxins cause an opacity and gelation in Limulus amebocyte
lysate (LAL), which is based on an enzymatic reaction. The simplicity and economy of the
LAL chromogenic endpoint assay encourages the testing of various biologicals (including
sera), devices, (air) filters and tissue culture medium for the presence of harmful levels of
Endotoxin [52].
Endotoxin contamination was assessed with the in vitro limulus amoebocyte lysate (LAL)
assay. Samples at different concentrations and standards were incubated with LAL reagent.
The absorbance at 405 nm was measured with a spectrophotometer. A standard curve was
obtained by plotting the absorbance (linear) versus the corresponding concentrations of the
E. coli standards (log). The endotoxin concentrations of samples, which are run
concurrently with the standards, were determined from the standard curve.
��������������������������������������������������������������������������������������������������������������������������������������������������������57
In another study, the NaYF4: Yb/Tm particles were tested for endotoxin contamination
and the particles were found to be endotoxin free. Figure.5.10 shows the standard curve for
the detection of presence of Endotoxin. It shows the typical absorbance values at 405nm for
different Endotoxin concentrations present. Endotoxin free water has absorbance value of
0.105. Table.5.1 shows the results of absorbance values obtained for NaYF4: Yb/Tm
particles at different concentrations and they found to have similar (smaller ) absorbance
values to endotoxin free water which shows our sample is free of Endotoxin.
LAL chromogenic endpoint essaystandard curve
0.000
0.500
1.000
1.500
2.000
2.500
3.000
0.00 0.20 0.40 0.60 0.80 1.00 1.20
Endotoxin (EU/ml)
Abs
orpt
ion
at 4
05 n
m
�
Figure.5.10. Standard curve for LAL assay (Water has an absorption value of 0.105).
��������������������������������������������������������������������������������������������������������������������������������������������������������58
Table.5.1. Absorption values at 405 nm of NaYF4: Yb/Tm particles at different concentration using LAL assay
Sample
concentration
Mean absorbance at
405nm
Stock (1 mg/ml) 0.068
10X (0.1 mg/ml) 0.089
20X (0.05 mg/ml) 0.114
40X (0.025 mg/ml) 0.098
The results of endotoxin study on NaYF4: Yb/Tm particles showed that there is no
detectable endotoxin, so it suggests that the particles are toxic itself.
Conclusion: The results on in vitro cellular studies have shown that 20 micron-sized DC
particles have great potential to activate Photofrin II in deep seated targets and to generate
substantial levels of ROS and no potential cell toxicity was observed. However, the UC
particles were shown to be toxic to the cell lines. The cell killing through ROS generation
appears not to have been due to the particles' efficiency in activating the photo-sensitizer,
but rather due to toxicity of the particles.
��������������������������������������������������������������������������������������������������������������������������������������������������������59
Chapter 6 : Theoretical modeling of ROS generation �
PDT depends on the amount of light delivered (L), the amount of photosensitizing drug
(S0) in the tissue, and the amount of oxygen (O2) in the tissue. Absorption of light converts
S0 into an activated drug (S*). Reaction of S* with oxygen yields oxidizing radicals
(primarily singlet oxygen, as discussed in Chapter 1, the cell killing mechanism in PDT is
known to be predominantly through enhanced generation of reactive oxygen species
(ROS). Greater than 90% ROS generation is through the generation of excited oxygen
molecule in its singlet state 21O ,). A fraction (f) of these radicals attacks critical sites within
the cell causing an accumulated oxidative damage (A). When the accumulated damage
exceeds a threshold, A > A th, then cell death occurs [53].
The aim of this chapter is to estimate the amount of excitation light deposited and
fluorescent light produced by down-converting (DC)/ up-converting (UC) particles, in
response to X-ray radiation dose/infrared laser irradiation, and to assess activation of the
photosensitizer as well as the theoretical effectiveness of the produced singlet oxygen.
As for X-ray DC particles, the amount of deposited X-ray radiation dose and generated
fluorescent light in the test medium will be quantified using both analytical and statistical
methods. The analytical modeling is based on the assumption that all of the particles are
uniformly distributed, and they all receive the same X-ray energy due to the high
penetration depth of X-rays at 120 keV. To ensure the results of analytical modeling were
not contingent upon these specific assumptions, I created a statistical model in which the
photon direction and photon absorption of the sample are determined randomly.
��������������������������������������������������������������������������������������������������������������������������������������������������������60
As for Infrared UC particles, the analytical modeling of X-ray DC particles cannot be used
due to existence of significant amount of absorption and scattering of the Infrared light by
the all the sample components. However, I used the same statistical modeling as X-ray DC
particles for quantifying amount of absorbed infrared light and generated fluorescent light.
Section 6.1 provides information on the physical properties of sample components; section
6.2 describes the steps for analytical modeling of the fluorescence light generation from X
ray DC particles; section 6.3 describes steps and results of statistical modeling of
fluorescence light generation from UC and DC particles respectively; section 6.4 describes
theoretical results of amount of singlet oxygen generated.
6.1. X-ray absorption coefficients of the test medium components
Prior to quantifying the amount of fluorescent light generated, the physical properties of the
materials present in the test sample need to be known. Our test media is composed of
water, polystyrene test tube, Gd2O2S: Tb particles and air. This section provides
information on the physical properties of the materials that are present in the test sample for
X-ray DC particles. Figure.6.1 and Table.6.1 show X-ray mass absorption and attenuation
coefficients and some physical properties for water, dry air, Gd2O2S, and polystyrene at
120 keV X-ray exposure [5]. From Figure.6.1 and Table.6.1, we can note that Gd2O2S has
137 times stronger absorbance compare to water, and 341 times compared to polystyrene.
��������������������������������������������������������������������������������������������������������������������������������������������������������61
The values given on Table.6.1 are used in theoretical modeling.
X-ray attenuation coefficients from NIST
Figure.6.1. Mass-energy absorption and attenuation coefficients at different X-ray photon energies for Gadolinium Oxysulfide, from: http://physics.nist.gov/PhysRefData/XrayMassCoef/tab4.html
Table.6.1. Physical Properties of several materials at 120 keV X-ray exposure
Air Water Polystyrene Gadolinium
Oxysulfide
Mass attenuation
coefficient 0.1467 cm2/g 0.16262 cm2/g 0.15536 cm2/g
1.94304
cm2/g
Mass energy
absorption coefficient 0.023934 cm2/g 0.06332 cm2/g 0.024304 cm2/g 1.16872 cm2/g
Density 0.001204g/cm3 1 g/cm3 1.05 g/cm3 7.44 g/cm3
Attenuation coefficient 0.0001766 cm-1 0.16262 cm-1 0.163128 cm-1 14.459 cm-1
Absorption coefficient 0.0000288 cm-1 0.06332 cm-1 0.0255192 cm-1 8.695 cm-1
��������������������������������������������������������������������������������������������������������������������������������������������������������62
6.2. Analytical modeling of X-ray absorbed dose and generated
fluorescence light in the test medium in the presence of X-ray
down convertors
Quantifying fluorescence intensity as a function of X-ray absorbed dose: The analytical
modeling is based on the assumption that all of the DC particles are uniformly distributed,
and they all receive same X-ray energy due to the high penetration depth of X-rays at 120
keV[34]. The amount of X-ray dose deposited and the generated amount of fluorescent
light will be found as a function of X-ray absorption efficiency, intrinsic conversion
efficiency, and molecular weight of the DC particles and the X-ray exposure rate.
Absorbed dose, also known as total ionizing dose (TID), is a measure of the energy
deposited in a medium by ionizing radiation. It is equal to the energy deposited per unit
mass of medium. The SI unit for absorbed dose is Gray (Gy) and is defined as:
1Gy=1J/kg
Another unit for absorbed dose is rad, which represents the absorption of 100 ergs of
energy per gram of absorbing material.
1rad=100ergs/g= 210� J/kg
1Gy=100 rad
In the presence of full charged particle equilibrium, the absorbed dose to air is given by
[54]:
��������������������������������������������������������������������������������������������������������������������������������������������������������63
)()./(10876.0)/(97.33)./(10`58.2).()/( 24 RXRkgJCJ
RkgCRXkgJDair
�� ����
(6.1)
Where the X(R) is the exposure in roentgens. The SI unit for exposure is C/kg.
(1R=2.58 kgC /10 4�� ).
Since 1 rad= kgJ /10 2� :
�)(radDair (0.876 Rrad
).X(R) (6.2)
We can see from Equation (6.2) that roentgen–to-rad conversion factor for air, under the
condition of electronic equilibrium is 0.876.
In the presence of full charged particle equilibrium, the absorbed dose (D) to a medium can
be calculated from the energy flux and the weighted mean mass energy absorption
coefficient, ��en [13]:
D= )( ��en (6.3)
The dose to the air is related to the dose to the medium by the following relationship [13]:
;.)()(
.)()( A
DD
airen
meden
air
med
airen
meden
air
med
����
����
�
�
(6.4)
Where air is the energy fluence at point in air and med is the energy fluence at the same
point when a material other than air (medium) is interposed in the beam. A is the
��������������������������������������������������������������������������������������������������������������������������������������������������������64
transmission factor that equals the ratio air
med
at the point of interest and it is close to 0.99
for soft tissue and it will approaches to 1 when the beam energy decreases to orthovoltage
range (100 to 350 keV supplied by x-ray generators used for radiation therapy).
From equations (6.3) and (6.4) we can obtain the relationship between exposure to air and
absorbed dose to a medium.
;].)()(
)876.0[(.)()(
XRradDD
airen
medenair
airen
medenmed ��
������
�� (6.5)
The term in brackets is represented by the symbol medf and is called the roentgen-to-radian
conversion factor. We can see in Equation (6.5) that this factor depends on the mass energy
absorption coefficient of the medium relative to the air. Thus, the f factor is a function of
the medium composition as well as the photon energy.
So the equation becomes [54]:
medD = medf .X (6.6)
The total absorbed dose for a given mass m will be:
medD = medf .X. m (6.7)
From the NIST website, for X-ray energy of 120KeV, ( airen )�� =0.023934 cm2/g,
( Gden )�� =1.16872 cm2 /g, the f factor is 42.78.
��������������������������������������������������������������������������������������������������������������������������������������������������������65
Substituting the values I have used for my experiment to the equation (6.7), ( medf =42.78
rad/R=0.4278 J/ (kg R), m=60 mg=0.06 kg, X� =192 mR/s =0.192 R/s , t=15 min=900 sec),
we could calculate expected X-ray dose absorbed by our sample to be:
medD = medf .X. m= tXmfmed .. � = 4.435 J
The rate of X-ray energy that is converted to fluorescence light by particles then could be
written as:
XmfmdtdD
dtdE
medcmed
c�.... �� �� (6.8)
Where the c� is the Intrinsic Conversion Efficiency of the Gd2O2S: Tb particles. Intrinsic
Conversion Efficiency ( c� ) is defined as the fraction of absorbed X-ray energy into light
within the mass of the particles.
The Intrinsic Conversion Efficiency, ICE ( c� ) of a Gd2O2S: Tb crystal in absorbing and
converting one X-ray energy photon into numerous visible light photons is reported in the
literature to be dependent on the percent of Tb doping, and the size of the crystals, it
typically ranges within 15 – 20% [34-37]. For example, we could calculate for a Gd2O2S:
Tb crystal having an ICE value of 15%, which corresponds to 20 μm size, one absorbed
120 keV X-ray photon will yield 7895 green wavelength photons (544 nm wavelength),
with each photon having an energy value of 2.28 eV(equations 6.9 and 6.10). This
Quantum Yield value is used when excitation photons are absorbed and fluorescence
photons are generated.
��������������������������������������������������������������������������������������������������������������������������������������������������������66
)10.6.(7895%15*28.2
120*
)9.6.(28.2544*
c ���
���
eVkeV
EEN
eVnmhhE
g
xg
g
�
Where gE and are the energy and wavelength of the green wavelength photon emitted by
the Gd2O2S: Tb particles, h is the Planck’s constant, xE is the energy of the X-ray photon.
gN is the number of photons emitted at 544 nm wavelength.
Substituting experimental values to the equation (6.8) the rate of X-ray energy that is
absorbed and converted to fluorescent light found to be 4.928 mJ/s .
Where 0m is the molecular weight of the particle, and X� is the x-ray exposure rate.
Total amount of X-ray energy that is absorbed and converted to fluorescent light by a given
mass m will be:
mXfE medc ..�� = tXmfmedc .�� (6.11)
Substituting experimental values to the equation (6.11), I found total amount of
fluorescence light generated to be 0.6653J. This energy level would provide ROS
generation (1.0002 �M) on the level that was measured experimentally (0.9529 �M).
6.3. Statistical Modeling: Quantifying fluorescent light fluence
distribution using Monte Carlo Modeling.
The stochastic numerical Monte Carlo (MC) model provides a basis for simulating photon
propagation in a homogeneous medium with random scatterers and absorbers[55]. This
��������������������������������������������������������������������������������������������������������������������������������������������������������67
modeling is used for modeling of X-ray and infrared laser energy deposited in the test
medium. It is also used for quantifying the amount of fluorescence light fluence rate
generated from X-ray DC particles and infrared UC particles. Fluorescence will depend on:
(1)the fluence rate distribution of the excitation light, (2) the product of the absorption
coefficient and quantum yield of fluorophores, and (3) the attenuation of the fluorescence
light by absorption and scattering in sample[56]. My modeling includes the following
steps:
1) Generating random numbers.
I will use random number generator function to create random numbers that are uniformly
distributed between 0 and 1. Random numbers are used to determine original photon
position, photon step size, and probability of photon scattering, absorption, transmission,
and reflection.
2) Computing light distribution produced by a finite radius, collimated excitation
beam.
Photons will be launched uniformly orthogonal to the sample surface from the (X-
ray/Infrared) source in the x-y-plane within the beam radius R, as shown figure 6.2. The
radial magnitude r, the angle � is chosen such that (r, �) define the launch point:
randomRr � (6.12)
�=2(random) (6.13)
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Figure.6.2. Excitation beam profile. Beam radius:R, radial magnitude:r, angle:�, X and Z are thickness
of the sample in x and z directions, 1n and 2n are the refractive indexes of the air and the sample.�
Positions x and y are chosen based on r and �:
x=rcos(�) (6.14)
y=rsin(�)
3) Moving the photon at the air –sample interface:
Photons will be either transmitted through the sample or reflected back from the sample at
the air-tissue interface.
Since the photons are injected orthogonally, the specular reflectance is specified by:
��������������������������������������������������������������������������������������������������������������������������������������������������������69
221
221
)()(
nnnnRsp �
�� (6.15)
Where 1n and 2n are the refractive indexes of the air and the tissue respectively. For my
modeling, ;11 �n ;4.12 �n
If spR >random, the photon is reflected back.
If spR <random, the photon is transmitted.
4) Determining step size of the photons.
The movement of each photon is variable and distance corresponds to a photon travels
from a scattering event to the next scattering or absorbing event. The step size of the
photon while the photon is inside the sample is given by [55]:
�s = -ln (random)/ ( sa �� � ). (6.16)
Where a� , and s� are mean absorption, and scattering coefficients of the sample at
excitation wavelength. They are found by :
)(...........)()()( 33
3
2
21
1
12 na
n
naaaa
cccc�
��
��
��
�� ����� (6.17)
)(...........)()()( 33
32
2
21
1
1ns
n
nssss
cccc�
��
��
��
�� �����
��������������������������������������������������������������������������������������������������������������������������������������������������������70
Where 1c , 2c ,… nc are concentrations of the components of the sample, 1� , 2� , n� are
densities of the components, 1a� , 2a� , na
� and 1s� , 2s� , ns� are absorption and scattering
coefficients of the components at excitation wavelength. For my modeling, water, and
particles are main absorbers and scatterers of excitation light (n=2).
Photon direction is set by the angle of scattering from the original direction of propagations
to the new direction of propagation which will be discussed in step 6.
5) Recording photon absorption
Probability of photon absorption is computed by:
If randomsa
a �� ���
, photon takes the new step
If randomsa
a �� ���
, Photon is absorbed and is terminated (update the absorption at this
point and fluorescent photons are created only for absorbed photons by particles)
(Figure.6.3).
��������������������������������������������������������������������������������������������������������������������������������������������������������71
Figure.6.3. Fluorescent photons are created at the point of photon absorption by particles
6) Photon scattering
Once the photon has taken a step and moved to the new position and is not absorbed, it is
ready to be scattered (Figure.6.4). The selection of the deflection angle is calculated by
[55]:
)cos(� =���������])
211(1[
21 2
22
�gggg
g ���
�� if g>0 (6.18)
12 �� �������������������������������������������������������������if g=0
The azimuthal angle, , is calculated by
=2 (random) (6.19)
Excitation�photon
Fluorescent�photon
Excitation�photon�absorption�and�fluorescent�photon�is�generated
��������������������������������������������������������������������������������������������������������������������������������������������������������72
Figure. 6.4.Deflection of a photon by a scattering event. The angle of deflection, �, azimuthal angle, �
(Modified from [55])
Once we calculate the deflection and azimuthal angle, the new trajectory of the photon
),,( zyx ��� ��� is calculated from the old trajectory ),,( zyx ��� , the deflection angle � and the
azimuthal angle [54]:
���������� cos)sincos(
1sin
2 xyzxz
x ���
�� �
��������
�� cos)sincos(1sin
2 yxzy
z
y ���
�� (6.20)
������ cos1cossin 2zzz ����� �
��������������������������������������������������������������������������������������������������������������������������������������������������������73
If the angle is close to normal ( ),9999999.0�z� then the following is formula is used:
��� cossin��x �
��� sinsin��y (6.21)
zzz ���� /cos���
7) When photons hit the boundary
When photons hit the boundary they will be either transmitted or reflected back to the
tissue (Figure.6.5).
The internal reflectance is calculated by Fresnel’s law
� � ])(tan)(tan
)(sin)(sin
[21
2
2
2
2
ti
ti
ti
tiiR
����
����
���
���
� (6.22)
Where i� and t� are the angles of incidence and transmittance respectively.
If � �iR � <random number, then the photon exits the tissue and terminated.
If � �iR � >random number, then the photon is reflected.
��������������������������������������������������������������������������������������������������������������������������������������������������������74
Figure.6.5.Internal reflectance and transmittance: Blue lines indicate transmittance, i� and t� are the angles of incidence and transmittance, green lines indicate internal reflectance, X and Z are thickness
of the sample in x and z directions, because of symmetry y direction is not shown here.
In both cases the actual position of escape needs to be calculated using foreshortened step
size:
z -boundary:
���
���
�
����
����
z
zzZs
zs
�
� (6.23)
x-boundary:
���
���
�
����
�����
x
xxXs
xXs
�
�2/
2/
(6.24)
y -boundary:
���
���
�
����
�����
y
y
yYs
yYs
�
�2/
2/
(6.25)
),,(),,(
zyx
zyx���
�
),,(),,(
zyx
zyx���
�),,(
),,(
zyx
zyx���
�
),,(),,(
zyx
zyx���
�
),,()2,,(
zyx
zZyx��� �� �
),,(),,(
zyx
zyx��� �
� �
),,(),,(
zyx
zyxX����
� �
),,(),,(
zyx
zyxX����
�� �
i�
Z�
t�
��X/2�
X/2�
��������������������������������������������������������������������������������������������������������������������������������������������������������75
The reflected photon will also have a new position and trajectory. The new position is
computed as following:
Outside top of the surface (z<0): substitute z with –z;
Outside slab at bottom of the surface (z>0): substitute z with 2Z-z;
Outside slab at right side of the surface(x>X/2): substitute x with X-x;
Outside slab at left side of the surface(x<-X/2): substitute x with -X-x;
Outside slab at outward direction of the surface(y>Y/2): substitute y with Y-y;
Outside slab at inward direction of the surface(y<-Y/2): substitute y with –Y-y;
And in call cases the corresponding trajectory is reversed.
A summary of the excitation photon tracking is shown in Figure.6.6.
��������������������������������������������������������������������������������������������������������������������������������������������������������76
Figure.6.6. Excitation photon tracking flow chart.
9) Fluorescence:
Fluorescent photons are created at the same location where excitation photons are absorbed
by particles. At the point of creation, the photon at the fluorescence wavelength is given a
direction assuming isotropic generation. Step size �s is given by equation (6.16) at
emission wavelength. As a fluorescence photon encounters sample, the probability of its
absorption is determined according to .)()(
)(
flsfla
fla
����
Start
Initialize�Photon�
Update�Reflectance�
Move�Photon�at�a�Variable�Step�
Photon�in�Sample?
Is�photon�absorbed?
Change�Photon�Direction�
No�
Yes�
Yes�
No�
No�
Yes�
Internally�Reflected?�
Get�Photon�Position�and�Direction�
Update�Reflection�
Store�location�of�absorption�event�
Return�to�main�program�
Call�Fluorescent�Function
�Last�No
Yes End�Program�
��������������������������������������������������������������������������������������������������������������������������������������������������������77
Where )(),( flsfla �� are the total absorption and scattering coefficients of the sample at
the emission wavelength, they are found by (6.17) at the emission wavelength.
(10)Computation of fluorescent light fluence at certain point
The fluence rate )(rn� per unit input for small volume �V(r) is given by [55]:
)()()(
)(fla
an NrV
rNr�
��
� (6.26)
Where )(rNa is the number of photons absorbed in �V(r), N is the total number of photons
in the simulation, )( fla � is the absorption coefficient of the sample at the emission
wavelength.
A summary of the fluorescence photon tracking is shown in Figure.6.7.
�
��������������������������������������������������������������������������������������������������������������������������������������������������������78
Figure.6.7. Flourescence photon tracking flow chart.
11) Results
For X-ray DC particles, 101012.1 � photons/s (120 keV photon energy with 192 R/s
exposure rate) need to be simulated. Because of computer memory, I launched 61012.1 �
photons/s and multiplied the number of absorbed photons by 10,000. For infrared UC
particles, 181082.3 � photons/s (840 mW/cm^2 laser intensity with 1.27 eV photon energy)
need to be simulated. Because of computer memory, I launched 61032.3 � photons/s and
multiplied the number of absorbed photons by 1210 . The distribution of absorbed photons,
generated fluorescence photons, and fluence rates are shown below.
Start at x,y,z of absorbtion
Isotropic generation of fluorescence
Move Photon at a Variable Step
Update Photon Weight Due to Absorption
Photon in Sample?
Weight Too Small?
Survive Roulette?
Change Photon Direction
No
Yes
Yes Yes
Main function
No
No
No
Yes
Internally Reflected?
Get Photon Position and Direction
Update remitted fluorescence
Return to main function, initialize new excitation photon
��������������������������������������������������������������������������������������������������������������������������������������������������������79
Figure.6.8. Results of X-ray photon simulation. (a) Fluence rate distribution of X-ray photons in Y/2 position. (b) Distribution of absorbed X-ray photons in Y/2 position. (c) Fluence rate distribution of generated fluorescence photons in Y/2 position. (d) Distribution of absorbed fluorescence photons in
Y/2 position
��������������������������������������������������������������������������������������������������������������������������������������������������������80
Figure.6.9. Results of infrared photon simulation. (a) Fluence rate distribution of infrared photons in Y/2 position. (b) Distribution of absorbed infrared photons in Y/2 position. (c) Fluence rate distribution of generated fluorescence photons in Y/2 position. (d) Distribution of absorbed fluorescence photons in
Y/2 position
We could see from Figures 6.8 and 6.9 that there strongest absorption of excitation photons
occurred at the air-sample boundary and is attenuated over the sample depth. For X-ray
irradiation, the water and DC particles both contributed to the strong absorption. As for
��������������������������������������������������������������������������������������������������������������������������������������������������������81
infrared illumination, the strong absorption was mainly due to the water. Since X-ray DC
particles have quantum yield of 7895, it generated strongest fluorescence as opposed to UC
particles.
6.4. Theoretical quantification of amount of ROS generation
PDT depends on the amount of light delivered (L), the amount of photosensitizing drug
(S0) in the tissue, and the amount of oxygen (O2) in the tissue. Absorption of light converts
S0 into an activated drug (S*). Reaction of S* with oxygen yields oxidizing radicals
(primarily singlet oxygen, as discussed in Chapter 1, the cell killing mechanism in PDT is
known to be predominantly through enhanced generation of reactive oxygen species
(ROS). Greater than 90% ROS generation is through the generation of excited Oxygen
molecule in its singlet state 21O ,). A fraction (f) of these radicals attacks critical sites within
the cell causing an accumulated oxidative damage (A). When the accumulated damage
exceeds a threshold, A > A th, then cell death occurs.
The amount of light provided for the drug activation is found by:
231061000�
�hcc
L � [moles/liter] (6.27)
Where
is the fluence rate of light [W/cm2] or [J/(cm2 s)],
/(hc) is number of photons per J of energy [ph/J],
is the photon wavelength in [cm],
c is the speed of light, 3.0x1010 [cm/s],
��������������������������������������������������������������������������������������������������������������������������������������������������������82
h is Planck's constant, 6.6x10-34 [J s],
there are 1000 cm3 per liter,
there are 6x1023 photons per mole of photons.
The rate constant for drug activation is
131 10141.9 ��� ck � ])/([ 11 �� litermoless (6.28)
Where
c is the speed of light, 3.0x1010 [cm/s],
� is the extinction coefficient of the Photofrin II, 310047.3 � ])/([ 11 �� litermolescm , which
is found using absorption spectrometer.
The rate of production of activated drug is:
01* LSk
dtdS
� ])/([ 1 litermoless � (6.29)
Total amount of activated drug in per unit volume is :
23001 1061000*�
��hc
TSTLSkS �� )/( litermoles (6.30)
Where T is light exposure time (300 sec for X-ray DC particles and for infrared UC
particles).
The rate of singlet oxygen generation is:
��������������������������������������������������������������������������������������������������������������������������������������������������������83
�� ��� ���� 230
*2
1
1061000.][][
hcS
dtSd
dtOd (6.31)
The total amount of singlet oxygen generated is found by:
�� ��� ���� 2302
1
1061000.*][
hcTSSO (6.32)
The singlet oxygen quantum yield, �� , also termed as quantum efficiency, is defined as the
number of 1O2 molecules generated for each photon absorbed by a photosensitizer. It is a
key property of a photosensitizing agent. The production of 1O2 by photosensitization
involves four steps: (1) Absorption of light by the photosensitizer; (2) Formation of the
photosensitizer triplet state; (3) Trapping of the triplet state by molecular oxygen within its
lifetime; (4) Energy transfer from the triplet state to molecular oxygen state [57]. The
published values of �� show considerable variations with the solvent, reaction conditions,
and the measurement techniques. The review by Redmond and Gamlin (1999) gives a
range of published singlet oxygen quantum yields in biologically relevant media between
0.19 and 0.89 at 540nm excitation wavelength [58]. I used 0.24 for my modeling.
Substituting light fluence rate values to X-ray DC particles and infrared UC particles into
equation (6.27), and using equation (6.31), I attempted to theoretically quantify amount of
singlet oxygen generated. Table.6.2 shows the final results of theoretical calculation and
comparison outcome with experimental results.
��������������������������������������������������������������������������������������������������������������������������������������������������������84
Table.6.2. Comparison outcome between experiment and theory
Experimental ROS generation
Theoretical prediction of ROS generation
Comparison outcome between experiment and theory
Analytical Modeling 1.0002 �M
95% match X-ray DC particles
0.9529�M
Monte Carlo Modeling 0.400218 �M
42% match
Infrared UC particles
31.443 �M Monte Carlo Modeling 0.00000774 �M
Doesn’t match with experimental results
Table.6.3 and Table.6.4 are the Connection charts between theoretical modeling and
experimental measurements for X-ray DC particles and infrared UC particles.
��������������������������������������������������������������������������������������������������������������������������������������������������������85
Table.6.3.Connection chart between theoretical modeling and experimental measurements for X-ray DC particles
Analytical Modeling Monte Carlo Modeling Experimental validation
What needs to be quantified
In this model, it is assumed that 1)all the particles and drug molecules are uniformly distributed in test medium, 2) All the particles receive the same X-ray dose and all the drug molecules receive the same amount of fluorescent light. 3) X-ray absorption from polystyrene test tube is negligible compare to particle absorption.
In this model, it is assumed that 1) All the particles and drug molecules are uniformly distributed in test medium. 2)X-rays and fluorescent light photon propagation (absorption, scattering) simulations are based on the random walks that photons make as they travel through sample, which are chosen by statistically sampling the probability distributions for step size and angular deflection per scattering event.
Not applicable
1)Amount of X-ray Energy Deposited in the test medium
Found as a function of: 1) X-ray absorption efficiency of the particles (from literature) 2) X-ray exposure rate (experimental set up) 3) Particle concentration (experimental set up)
Required parameters: 1) Dimensions of the test medium (experimental set up) 2) X-ray absorption and scattering coefficients of the sample components (from literature)
Not applicable
2)Amount of Fluorescence light generated
Found as a function of : 1)Amount of X-ray absorbed dose (from step 1) 2) Intrinsic conversion efficiency (converting absorbed X-ray energy into fluorescence) of DC particles.
(from literature)
Required parameters: 1)Dimensions of the test medium (experimental set up) 2) X-ray, and infrared absorption and scattering coefficients of the sample components (from literature) 3) Quantum yield of the Gd2O2S: Tb particles (from literature)
Not applicable
4)Amount of singlet Oxygen generation
found as a function of: 1)Amount of photosensitizer activated (from step 3) 2) Photosensitizer singlet oxygen quantum yield (from literature).
found as a function of: 1)Amount of photosensitizer activated (from step 3) 2) Photosensitizer singlet oxygen quantum yield (from literature).
Experimental results shows amount of singlet oxygen generated through the change in the absorbance of Vitamin C. Analytical modeling well matches with experimental results (>90%). Monte Carlo modeling 40% matches with the experimental results.
��������������������������������������������������������������������������������������������������������������������������������������������������������86
Table.6.4.Connection chart between theoretical modeling and experimental measurements for infrared UC particles
Monte Carlo Modeling: In this model, it is assumed that 1) All the particles and drug molecules are uniformly distributed in test medium. 2)Infrared and fluorescent light photon absorption, scattering simulations are based on the random walks that photons make as they travel through sample, which are chosen by statistically sampling the probability distributions for step size and angular deflection per scattering event.
Experimental validation
What needs to be Quantified
required parameters Required parameters that need experimental quantification
Amount of absorbed Infrared energy in test medium
1)Dimensions of the test medium(experimental set up) 2) Infrared attenuation coefficients of the sample components (from literature)
Infrared attenuation coefficients of the NaYF4:Yb,Tm particles (wasn’t successful due to strong absorption of water)
Not applicable
Amount generated fluorescence light by the UC particles
1)Dimensions of the test medium(experimental set up)
2)Quantum yield of the NaYF4:Yb,Tm particles ((from literature)
Quantum yield of the NaYF4:Yb,Tm particles(not successful due to very weak fluorescence)
Not applicable
Amount of photosensitizer activated
Found as a function of: 1) Amount of light provided (from previous step), 2) Extinction coefficient of the photosensitizer, 3) Concentration of ground state photosensitizer (experimental set up).
Extinction coefficient of the photosensitizer
Not applicable
Amount of singlet Oxygen generation
Found as a function of: 1)Amount of photosensitizer
activated (from previous step) 2) Photosensitizer singlet
oxygen quantum yield (from literature).
Experimental results show amount of
21O generated through the change in the absorbance of Vitamin C. Theoretical modeling doesn’t predict experimental results due to the toxicity of particles.
��������������������������������������������������������������������������������������������������������������������������������������������������������87
In this chapter I attempted to theoretically quantify amount of light provided by DC and
UC particles and ROS generation.
My results have shown that the analytical modeling is sufficient to predict the X-ray
absorbed dose, generated fluorescence light amount, and the ROS generation. The Monte
Carlo modeling demonstrates the need for further modification.
As for Infrared UC particles, theoretical modeling does not predict our experimental results
of ROS generation. I had also shown in Chapter 5 that the cell killing through ROS
generation form UC particles were not to due to the particles' efficiency in activating the
photo-sensitizer, but rather due to toxicity of the particles.
��������������������������������������������������������������������������������������������������������������������������������������������������������88
Chapter 7: Conclusion �
Despite being non-invasive and having excellent selectivity for diseased tissue, PDT has
not yet gained general clinical acceptance, largely due to the inherent light transport and
penetration limitations which restrict light sources outside the body from activating photo-
agents within target volumes deep inside the body. The photo-sensitizers that are approved
for PDT treatment in oncology are found to maximally absorb light in the violet region of
the visible spectrum, around 400 nm, and blood is a very strong absorber at this
wavelength. Thus, the photo-agent’s absorption characteristics inherently limit the
effectiveness of PDT applications to target-sites which are shallow in depth, 2 – 3 mm. For
this reason, the clinical application of PDT has been limited to skin lesions, superficial
solid tumors, or endoscopically-accessible regions. One of the world-wide approved photo-
sensitizers in oncology, Photofrin II, is known to have good selectivity towards diseased
tissue, and its major sub-cellular target is known to be mitochondria [1-3].
In my research work, both X-ray down-converting (DC) and Infrared up-converting (UC)
particles were studied as viable platforms of generating visible luminescence to activate the
photo-sensitizer Photofrin II. Specifically, I have investigated DC particles composed of
gadolinium oxysulfide doped with terbium (GdO2S: Tb) and UC particles composed of
sodium yttrium fluoride co-doped with ytterbium and thulium (NaYF4: Yb/Tm).
The DC and UC particles were tested in a cellular-like medium; the results obtained
showed that both submicron- to micron-sized DC and UC particles have great potential to
activate Photofrin II and to generate substantial levels of ROS.
��������������������������������������������������������������������������������������������������������������������������������������������������������89
In vitro studies on human glioblastoma cell lines were then conducted to investigate the
possible cellular toxicity of these DC and UC particles through cell viability assays and
Endotoxin detection assay. The therapeutic effectiveness of these particles via Photofrin II
activation was also evaluated on in vitro human cancer cells through measurement of ROS
levels and cell viability assays. Theoretical modeling of the experiment was generated
using both Analytical technique and Monte Carlo Modeling of light transport.
Severe suppression (> 90% relative to controls) in the metabolic activity of human
glioblastoma cells due to the presence of clinically relevant concentration of ([20 μg/ml])
Photo II, with Gd2O2S:Tb particles ([5mg/ml]), and (120 kVp) diagnostic X-ray exposure
was observed.
At first the therapeutic efficacy of the UC particles was investigated at the particle
concentration of 5 mg/ml, it resulted in complete cell death including the control condition.
Then the particle concentration was reduced into 0.2 mg/ml. The dramatic reduction (>50%
including control) in the human glioblastoma cell viability due to the NaYF4: Yb/Tm only
treatment was observed.
The results on in vitro cellular studies have shown that 20 micron-sized DC particles have
great potential to activate Photofrin II in deep seated targets and to generate substantial
levels of ROS. No potential cell toxicity was observed. However, the UC particles were
shown to be toxic to the cell lines. The cell killing through ROS generation was not due to
the particles' efficiency in activating the photo-sensitizer, but rather due to toxicity of the
particles.
��������������������������������������������������������������������������������������������������������������������������������������������������������90
DC particles GdO2S:Tb have been utilized for several decades in X-ray imaging as
scintillators to convert the X-ray photons into visible photons, and their optical and
physical properties have been well quantified. However, the rare earth UC particles NaYF4:
Yb/Tm are relatively new and its optical and physical properties vary greatly based on how
it is synthesized. Different sources published different quantum yield values of NaYF4:
Yb/Tm particles and no significant cell toxicity was reported as opposed to our
investigation. In literature published to date, biocompatibility of UC nano-particles has
been explored to limited extent. Quantum yield efficiency and bio-compatibility need to be
greatly improved in order for NaYF4: Yb/Tm particles to be used in biological tissues as a
light source.
In summary, the work described in this project offers a number of contributions: (a) ROS
yield was significantly greater when the Photofrin II excitation wavelength was near the
Photofrin II Soret band; (b) Gd2O2S:Tb particles revealed a great potential to activate
Photofrin II in deep seated targets and induce severe cell suppression (>90%) through ROS
generation; And (c) NaYF4:Yb/Tm particles’ toxicity at a desired level of concentration
and the cell killing ability through the ROS generation was due to the toxicity of the
particles and not because of the particles' efficiency in activating the photo-sensitizer.
(i)shifting the “rare-earth” based particles visible emission band closer towards the
Photofrin II Soret (400 nm) band in order to obtain greater levels of oxidative
stress.(ii)reducing the size of the DC particles for future in-vivo studies in animals (20
micron DC particles size are too big and would cause damage to kidneys and the liver),
dropping the size down to 500 nm is recommended ; (iii) increasing the fluorescence
quantum yield by increasing the surface area of the UC nanoparticles ( it is recommended
��������������������������������������������������������������������������������������������������������������������������������������������������������91
to increase the size of the UC nanoparticles from 50 nm to 250 nm);and (iv) further
research needs to be undertaken in reducing the cellular toxicity of the UC particles for it to
be applicable to in-vivo animal models and much needs to be done in terms of
biocompatibility before using them in clinical settings.
�
�
�
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References
1. Snyder, J.W., et al., Photodynamic therapy: a means to enhanced drug delivery to tumors. 2003/12/18 ed. Cancer Res. Vol. 63. 2003. 8126-31.
2. Thoma J.Doughterty, J.G.L., Photodynamic Therapy(PDT) and Clinical applications, in Biopphotonics Handbook,CRC press. 2003. p. Ch 38.
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Appendix
MatLab�code�for�Monte�Carlo�modeling�of�distribution�of�excitation�light�
function simpleMC()%MatLab code for Monte Carlo modeling of distribution of excitation light and propagation of fluorescence light%This code generates absorption,and fluence profile of excitation/fluorescence light %(Erkinay Abliz)clear all;close all;clc;%%function Ab = Propagation_of_ex-light(mu_a_ex,mu_s_ex,N,E_photon)N = 1.12e+6; % Number of photons launched (3.82e+18 for IR)mu_a_ex=.079872+0.06332; %X-ray absorption coefficient of fluorophore in the test medium(water+particles)mu_s_ex=0.1077432;%X-ray scattering coefficient of the test medium(water+particles)mu_a_em = 0.0006+0.00023;mu_s_em = 0.003;Re=.079872/(.079872+0.06332);%ratio of absorbed photons that is converted to fluorescence light% Rf=0.00023/(0.0006+0.00023);%ratio of absorbed photons that is absorbed by photofrin IIQY=7895;%(0.001 for IR) %number of fluoresnce photons generated for each absorbed photonZ=1; %thicknes of the sample are in cmX=4;Y=1;beam_radius=2;%in cm (0.5 for IR)n1=1; %Refractive index of the airn2=1.4;%Refractive index of the sampleEx_photon=1.92261276e-14;%(2.018743398e-19 for IR) in Joules corresponds to 120 keV(1.26eV for IR) photon energyFE_photon=3.652964244e-19; %in Joules corresponds to 2.28 keV photon energy%grid size in x,y,z dimensionsdx=0.025;dz=0.025;dy=0.025;%Number of bins in x,y,z directionsBins_z = Z/dz;Bins_x= X/dx;Bins_y= Y/dy;%Blank absorption matrix before the absorption A = zeros(Bins_x+1,Bins_y+1,Bins_z+1); F = zeros(Bins_x+1,Bins_y+1,Bins_z+1); ex_Fluence = zeros(Bins_x+1,Bins_y+1,Bins_z+1); em_Fluence = zeros(Bins_x+1,Bins_y+1,Bins_z+1);Beam = zeros(N,2);
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%%%%%%%%%%Photon transmits or reflects from the boundary%%%%%%% Rsp=(n1-n2)^2/(n1+n2)^2;%specular reflectance N = N - N*Rsp;%number of photons that enters the tissue%Launching photon from origin for n=1:N% p = int32(n/N)*100;% disp([int2str(p) '% completed']) W = 1;% photon is alive ri= beam_radius*sqrt(rand); % Choosing launch positions based on RND phi = 2.0*pi*(rand); x = ri*cos(phi); y = ri*sin(phi);
%%%%%%When the beam radius is large,some portion of the beam doesn't hit the tissue%%%%%
if abs(x) > X/2 || abs(y) > Y/2 W = 2;
end%When beam hits and enters the tissue:
Beam(n,1) = x; Beam(n,2) = y;
z = 0; ux = 0; %No x-axis trajectory */ uy = 0; %No y-axis trajectory */ uz = 1.0; % All trajectory is along z-axis
%%%%%%When photon is inside the tissue%%%%%%%%%%
[A,x,y,z,W,ex_Fluence] = scatter2(W,A,mu_a_ex,mu_s_ex,n1,n2,x,y,z,... dx,dy,dz,Bins_x,Bins_y,ux,uy,uz,X,Y,Z,Ex_photon,ex_Fluence);
if W == 0for iter = 1
W = 1; [F,x,y,z,W,em_Fluence] = scatter2(W,F,mu_a_em,mu_s_em,n1,n2,... x,y,z,dx,dy,dz,Bins_x,Bins_y,... ux,uy,uz,X,Y,Z,FE_photon,em_Fluence);
endend
end
A_x_z(:,:) = A(:,Bins_y/2,:); F_x_z(:,:) = Re*QY*F(:,Bins_y/2,:);
EM_Fluence_x_z(:,:) =Re*QY*em_Fluence(:,Bins_y/2,:);EX_Fluence_x_z(:,:) = ex_Fluence(:,Bins_y/2,:);
f1 = figure(1);
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imshow(A_x_z,[]); set(f1,'Colormap',colormap(jet)); title('Distribution of absorbed excitation photons') xlabel('Sample thickness in beam directon');ylabel('Sample height')
f2 = figure(2);imshow(EX_Fluence_x_z,[]); set(f2,'Colormap',colormap(jet));title('Fluence rate of ecxitation light') xlabel('Sample thickness in beam directon');ylabel('Sample height')
f3=figure(3);imshow(F_x_z,[]); set(f3,'Colormap',colormap(jet));title('Distribution of absorbed fluorescence photons')xlabel('Sample thickness in beam directon');ylabel('Sample height')f4=figure(4);imshow(EM_Fluence_x_z,[]); set(f4,'Colormap',colormap(jet));title('Fluence rate of fluorescence light')xlabel('Sample thickness in beam directon');ylabel('Sample height')
function [A,x,y,z,W,Fluence] = scatter2(W,A,mu_a,mu_s,n1,n2,x,y,z,dx,dy,dz,Bins_x,Bins_y,ux,uy,uz,X,Y,Z,E_photon,Fluence)
while W == 1; Albedo=mu_a/(mu_a+mu_s);
if Albedo>rand; i = (round(x/dx)+1) + (Bins_x/2); j = (round(y/dy)+1) + (Bins_y/2); k = (round(z/dz)+1);
A(i,j,k)=A(i,j,k)+1; % Photon is absorbed
dV=dx*dy*dz; %The volume of the i th element Source(i,j,k) = (A(i,j,k)*E_photon) /(dV);%Energy density Fluence(i,j,k) = Source(i,j,k)/mu_a;%fluence rate for the grid element
%A(i,j,k)=R*(A(i,j,k)); W = 0;
else%photon takes new step%%scatter%%%%%%%%%%%%%scatter%%%%%%%%%
S = 1;while S > 1
S=-log(rand)/(mu_a+mu_s); %step sizeend
phi = 2.0*pi*(rand); h=2*(rand)-1; th=acos(h);
if uz>0.99999; ux=sin(th)*cos(phi); uy=sin(th)*sin(phi); uz=(uz/abs(uz))*cos(th);
else ux=ux*cos(th)+(sin(th)*(ux*uz*cos(phi)-uy*sin(phi)))/sqrt(1-uz^2);
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uy=uy*cos(th)+(sin(th)*(uy*uz*cos(phi)+ux*sin(phi)))/sqrt(1-uz^2); uz=uz*cos(th)-sin(th)*cos(phi)*sqrt(1-uz^2);
end x=x+S*ux; y=y+S*uy; z=z+S*uz;
end%%%%%%%Photon hits the boundary%%%%if abs(x) >= X || abs(y) >= Y || z >= 2*Z || z <= -Z
W = 2;endif abs(x) >= X/2 || abs(y) >= Y/2 || z >= Z || z <= 0%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%if uz>0
th_i=acos(uz);end
if uz<0 th_i=pi-acos(uz);
endif ux>0
th_i=acos(ux);end
if ux<0 th_i=pi-acos(ux);
endif uy>0
th_i=acos(uy);end
if uy<0 th_i=pi-acos(uy);
end%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
th_t=asin((n2/n1)*sin(th_i)); R=((sin(th_i-th_t))^2)/(2*(sin(th_i+th_t))^2)+((tan(th_i-th_t))^2)/(2*(tan(th_i+th_t))^2);
crit_angle=asin(n1/n2);if R>=rand||th_i>crit_angle % internally reflected
if z>=Z z=2*Z-z; uz=-uz;
elseif z<=0 z=-z; uz=-uz;
endif x<=-X/2
x=-X-x; ux=-ux;
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elseif x>=X/2 x=X-x; ux=-ux;
endif y<=-Y/2;
y=-Y-y; uy=-uy;
elseif y>=Y/2 y=Y-y; uy=-uy;
endelse W=2;end
endend
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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