applications of micro- and nanoparticles in activating

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] .

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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

��

��

��6

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].

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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

��15

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)).

��16

(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

��17

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

��19

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)

��21

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].

�

�

�

�

�

�

�

�

�

�

�

��22

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,

��23

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.

��25

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)�

��26

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,

��27

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

��28

�

�

�

�

�

�

�

�

�

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)�

��29

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.

�

��30

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�


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

��42

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�


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


% 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


% 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)

��68

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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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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applications of micro- and nanoparticles in activating

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