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Electronic Theses and Dissertations, 2004-2019

2019

Tumor-Targeted Conjugated Polymer Nanoparticles with Encapsulated Iron and Its Biomedical Application for In Vitro Killing of Melanoma Cell Lines Through Ferroptosis Assisted Chemodynamic Therapy (CDT)

Khalaf Jasim University of Central Florida

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STARS Citation Jasim, Khalaf, "Tumor-Targeted Conjugated Polymer Nanoparticles with Encapsulated Iron and Its Biomedical Application for In Vitro Killing of Melanoma Cell Lines Through Ferroptosis Assisted Chemodynamic Therapy (CDT)" (2019). Electronic Theses and Dissertations, 2004-2019. 6875. https://stars.library.ucf.edu/etd/6875 TUMOR-TARGETED CONJUGATED POLYMER NANOPARTICLES WITH ENCAPSULATED IRON AND ITS BIOMEDICAL APPLICATION FOR IN VITRO KILLING OF MELANOMA CELL LINES THROUGH FERROPTOSIS ASSISTED CHEMODYNAMIC THERAPY (CDT) by

KHALAF AHMED JASIM B.S. Tikrit University, 2006 M.Sc. Tikrit University, 2009

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry in the College of Sciences at the University of Central Florida Orlando, Florida

Fall Term 2019

Major Professor: Andre J. Gesquiere

© 2019 Khalaf Jasim

ii ABSTRACT

Melanoma represents one of the most aggressive and lethal forms of skin cancer, with annually rising incidences throughout the world. Although chemotherapy modalities remain the mainstay of treatment, the therapeutic potential of chemotherapy typically is hampered by multidrug resistance (MDR) and nonspecific drug distribution that causes side-effects. To surmount such limitations, novel nanoformulations of low band-gap poly ({4,8-bis [(2-ethylhexyl) oxy] benzo [1,2-b:4,5-b′] dithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl) carbonyl] thieno [3,4- b] thiophenediyl}) (PTB7) and poly [2,1,3-benzothiadiazole- 4,7- diyl [4,4-bis (2-ethylhexyl)- 4H- cyclopenta [2,1-b:3,4-b’] dithiophene-2,6-diyl]] (PCPDTBT) were fabricated by a reprecipitation method. These conjugated polymer nanoparticles were functionalized with the polypeptide endothelin-3 (EDN3-CPNPs) to target melanoma. The combination of EDNRB and EDN3 is unique to melanoma’s endothelin axis in an otherwise healthy body. Therapeutic effects were studied in vitro for photodynamic (PDT) and chemodynamic (CDT) therapy applications. The

PTB7 derived EDN3-CPNPs showed limited PDT effect and were difficult to handle due to challenges with preparation and poor colloidal stability. We, therefore, moved forward with

PCPDTBT as an alternative polymer. Here, we serendipitously discovered that the PCPDTBT derived EDN3-CPNPs efficiently and specifically kill tumor cells that overexpress the endothelin

B receptor agonized by EDN3. We found that tumor cell killing proceeds through ferroptosis, a reactive oxygen species (ROS) mechanism that is not dependent on external activation by, for example, light, as is the case in PDT. The EDN3-CPNPs obtained from PCPDTBT are loaded with iron (Fe3+) that is a residual catalyst of the polymer synthesis. This iron content catalyzes

iii ferroptosis in the cells. The ferroptosis mechanism is also not heavily reliant on oxygen availability and is, therefore, promising for the treatment of hypoxic tumors. The resulting hydroxyl radicals

(•OH) can rapidly oxidize bio-macromolecules, cause damage to DNA, and reduce tumor cell population. The results reported in this dissertation demonstrate that melanoma targeted EDN3-

CPNPs present a new therapeutic avenue. In the future, this approach can be broadened to other tumors by replacing the targeting ligand to address cases where conventional methods are not feasible or no longer effective.

iv ACKNOWLEDGMENT

First and foremost, I would like to sincerely thank my advisor Dr. Andre J. Gesquiere, for welcoming me into his lab and giving me a unique opportunity of research in the field of nanomedicine. He is not only a mentor but also a friend. I appreciate his support, endless patience, excellent guidance, and expertise during the full length of my project. His broad experience and insight have helped to shape my work. I also appreciate his financial assistance, which allowed me to attend the Nano Boston conference that was held on April 22-24, 2019. Moreover, I would like to thank the Ministry of Higher Education and Scientific Research (MOHESR) of Iraq for its financial support through the scholarship program while pursuing my Ph.D. degree at the

Nanoscience Technology Center in the Laboratory of Nanoscale Imaging and Spectroscopy

(LNIS) at University of Central Florida.

I want to extend my appreciation to my candidacy and dissertation committee members Dr.

Andre J. Gesquiere, Dr. Andres Campiglia, Dr. Saleh Naser, Dr. Seth Elsheimer, and Dr. James

Harper for their substantial efforts and valuable suggestions for my candidacy and dissertation. I want to show my gratitude to the research engineers, Mr. Kirk Scammon, and Mr. Mikhail Klimov at the Advanced Materials Processing and Analysis Center for their assistance in STEM and TEM images and analysis experiments. I owe my most profound gratefulness to my laboratory colleagues for their support with the cell culture experiments, beneficial advice about my projects, and for continually offering me excellent scientific advice and positive enthusiasm, Edward Price,

Yasmine Abdellatif, and Ahmed Aboutaleb. I extend my thanks to the colleagues in the Laboratory of Nanoscale Imaging and Spectroscopy (LNIS), Marty Topps (for the contribution of data,

v figures, and discussion to the PTB7 PDT project), Torus Washington, Olivia George, Alondra M

Ortiz Ortiz, and Sajan Shroff. I also thanks Dr. Saleh Abdula Ahmed Al-Jibori, minister of

Ministry of Industry and Minerals of Iraq, a faculty at Chemistry Department and College of

Pharmacy at Tikrit University, Dr. Faiz Muhsen Hamed (Chemistry Department chair), Dr. Emaad

Mohammed Alawsaj,(Chemistry Department undergraduate coordinator), Dr. Fadhil Dawood

Khalid, and. Dr. Mohammed M. Asker, and all faculties and staff at the Chemistry Department at

Tikrit University for assistance with figures and discussion on our work.

I would like to extend my special thanks to Dr. Seth Elsheimer, Dr. James Harper, and Dr.

Mohammed Daoudi for their friendly support and for always making time for me when I needed help, and for the numerous recommendation letters they wrote for me.

Lastly, I want to thank my greatest supporters, my family. I am profoundly thankful and blessed to have you in my life. My father, my mother, my brother, my nephew (Yousif Shehab

Ahmed), and friends thank you for your unwavering love, unconditional encouragement, continuous motivation, and love to allow me to complete my doctoral program smoothly and successfully. Thanks for all your prayers and praise when I needed it the most. I would not have had the opportunity to have this experience without your support and generosity. I hope always to make you proud.

With gratitude,

Khalaf Jasim

vi TABLE OF CONTENTS

LIST OF FIGURES ...... xi

LIST OF TABLES ...... xvii

LIST OF ACRONYMS (or) ABBREVIATION ...... xviii

CHAPTER ONE: INTRODUCTION ...... 1

1.1 Background and Significance ...... 1

1.1.1 Melanoma ...... 1

1.1.2 Environmental Factors and Prevention ...... 3

1.1.3 Melanoma Statistics ...... 6

1.1.4 The Endothelin Axis’ Role in Melanoma ...... 7

1.2 Melanoma Therapies ...... 9

1.2.1 Problems with Chemotherapy ...... 11

1.2.2 Nanoparticles-Assisted Chemotherapy ...... 12

1.2.3 Nanoparticles-Assisted Photodynamic Therapy (PDT) ...... 14

1.2.4 Nanoparticles-Assisted Chemodynamic Therapy (CDT) ...... 16

1.3 Scope of Reported Dissertation Research ...... 24

CHAPTER TWO: MATERIALS AND METHODS ...... 28

2.1 Introduction ...... 28

vii 2.2 Materials ...... 29

2.3 Nanoparticle Fabrication...... 30

2.3.1 Purification of PCPDTBT Polymer ...... 30

2.3.2 Purification of PTB7 Polymer ...... 30

2.3.3 Fabrication of CPNPs Obtained from PCPDTBT Polymer ...... 31

2.3.4 Fabrication of CPNPs Obtained from PTB7 Polymer ...... 32

2.4 Bioconjugation of CPNPs Obtained from PCPDTBT...... 33

2.5 Bioconjugation of CPNPs Obtained from PTB7 ...... 34

2.6 Characterization of Nanoparticles ...... 35

2.6.1 UV-Vis Spectroscopy ...... 35

2.6.2 Fluorescence Spectroscopy ...... 35

2.6.3 Fourier-Transform Infrared Spectroscopy ...... 36

2.6.4 Dynamic Light Scattering (DLS) ...... 36

2.6.5 Transmission Electron Microscopy (TEM) ...... 36

2.6.6 Scanning Transmission Electron Microscopy (STEM) ...... 36

2.7 Cell Culture ...... 37

2.7.1 Cell Viability Determination by MTS Assay (PCPDTBT) ...... 37

2.7.2 PDT and Cell Viability Determination by MTS Assay (PTB7) ...... 38

2.8 Quantitation of Amount of Endothelin Peptide Attached to CPNPs...... 39

viii 2.9 Total Quantitation of Iron Content in Nanoparticles...... 41

2.10 Observation of Cellular Uptake and Localization of Nanoparticles by TEM & STEM ... 42

CHAPTER THREE: POLYMERIC NANOPARTICLES OF PTB7 FOR PHOTODYNAMIC

THERAPY OF MELANOMA SKIN CANCER ...... 44

3.1 Introduction ...... 44

3.2 Results and Discussion ...... 45

3.2.1 Size Measurements ...... 45

3.2.2 MTS Cell Viability Assay without Light ...... 46

3.2.3 MTS Cell Viability Assay with Light ...... 49

3.3 Conclusions ...... 49

CHAPTER FOUR: POLYMERIC NANOPARTICLES WITH ENCAPSULATED IRON FOR

FERROPTOSIS ASSISTED CHEMODYNAMIC THERAPY OF MELANOMA SKIN

CANCER ...... 51

4.1 Introduction ...... 51

4.2 Fabrication of CPNPs ...... 54

4.3 Bioconjugation of CPNPs ...... 60

4.4 Determination of Particle Size, Polydispersity Index (PDI), and ζ-Potential (ZP) ...... 73

4.5 EDN3-CPNP as Anti-Tumor Cell Effect ...... 79

4.6 Ferroptosis as The Tumor Cell Killing Mechanism ...... 84

ix CHAPTER FIVE: CONCLUSIONS ...... 89

APPENDIX A: COPYRIGHT PERMISSION ...... 90

APPENDIX B: COPYRIGHT PERMISSION ...... 93

LIST OF REFERENCES ...... 96

x LIST OF FIGURES

Figure 1: Illustration showing a melanocyte-melanin producing cells (adapted from Google image references)...... 1

Figure 2: Clark's levels...... 3

Figure 3: Illustration shows a cross section of human skin tissue...... 4

Figure 4: A relationship between the incidence of UV rays, people's skin color, and the risk of skin cancer (reproduced from reference).8 ...... 5

Figure 5: Sunscreen filters reflect UV radiation–Illustration (adapted from Google image references)...... 6

Figure 6: Actual and projected cancer incidence rates, United States, 1975 to 2020...... 7

Figure 7: Structures of the endothelin peptides...... 9

Figure 8: Schematic illustration of suggested mechanisms in vivo of limited targeting of nanoparticles on a cell membrane of healthy cells (Right) vs. extensive targeting of cancer cells on the mechanism of active targeting (bottom left) or can be achieved on the mechanism of passive targeting by accumulation in the tumor cells via the enhanced permeability and retention effect

(EPR) (top left)...... 14

Figure 9:Schematic representation of target-activated PDT agents by a specific wavelength, in which the therapeutic agent is a photosensitizer (PS)...... 15

Figure 10: Schematic illustrations of nanoceria-doped SPNs under NIR laser irradiation

(reproduced from reference).52 ...... 16

xi Figure 11: Schematic illustration of intracellular distribution of SPION-micelles, followed by iron release, which is then involved in a Fenton reaction in the presence of H2O2 that is generated from

β-lap (reproduced from reference).107 ...... 20

Figure 12:Schematic illustration of the damage of cancer cells occurred via rMOF-FA nanoparticles (reproduced from reference).94 ...... 21

Figure 13: Schematic illustration internalized of the LHRH peptide coated FePt NPs, causing oxidative damages to proteins, lipids, and DNA (reproduced from reference).108 ...... 22

Figure 14: Schematic illustrating the design and synthesis of magnetic nanoparticles which is then

109 involved in a Fenton reaction in the presence of H2O2 (reproduced from reference)...... 23

Figure 15: Schematic illustration of endocytosis-free nanoparticles vs. active mechanisms internalized into the cells (reproduced from reference).88 ...... 24

Figure 16: Schematic illustrating the biological significance of the Fenton and Haber–Weiss reactions. In the presence of Fe3+ (delivered by EDN3-CPNPs) the Haber-Weiss reaction produces

2+ Fe for the Fenton reaction, in which sufficiently elevated H2O2 in tumor cells is reduced to the highly reactive hydroxyl radical and molecular oxygen is returned as a product...... 26

Figure 17: Raw materials needed for Iron-detection reagent...... 42

Figure 18: Observed z average size distribution for PTB7 NPs...... 46

Figure 19: MTS data for cells with no light exposure...... 48

Figure 20: MTS results for cells with 1 hour of light exposure...... 49

Figure 21: The mechanism of ROS formation proceeds through the iron-mediated Fenton reaction.

...... 52

Figure 22: Synthesis of PCPDTBT monomers...... 53

xii Figure 23: Synthesis of PCPDTBT polymers...... 53

Figure 24: A schematic diagram showing the preparation of low-bandgap polymer-based CPdots via reprecipitation method and their UV−Vis absorption and emission spectra (reproduced from reference).141 ...... 54

Figure 25: Schematic illustration of the formation of conjugated polymer nanoparticles carrying iron (CPNPs and EDN3-CPNPs)...... 56

Figure 26: (A) Schematic illustration of the reprecipitation method, a technique in which a solution of organic material in a good solvent was injected into a very poor solvent for the organic materials.(B) Normalized absorption (blue) and emission (red) spectra of the EDN3-CPNPs in water, CPNPs in water, and PCPDTBT in THF...... 57

Figure 27: Photoluminescence excitation and emission spectroscopies of PCPDTBT in THF. ... 58

Figure 28: Photoluminescence excitation and emission spectroscopies of CPNPs in DI water. .. 58

Figure 29: Photoluminescence excitation and emission spectroscopies of EDN3-CPNPs in DI water...... 59

Figure 30: Schematic representation of the interaction of endothelins with their receptors. The ligands interact with the receptor through its terminal free carboxylic end group, which is unaffected by our coupling chemistry...... 61

Figure 31: Mechanism of bioconjugation the surface of CPNPs by carbodiimide-mediated coupling reaction with EDC and NHS...... 62

Figure 32: Targeting, cellular uptake of EDN3-CPNPs, and iron release using receptor-mediated endocytosis...... 63

xiii Figure 33: Schematic representation of the interactions of endothelins with EDNRA binds most strongly to EDN1 and EDN2 with a very minimum affinity towards EDN3, while EDNRB binds the EDN1, EDN2, and EDN3 with similar affinities. Amino acids sequence in proteins that are identical in both receptors (black circles), different in both receptors (blue circles), identical to each other (green circles), only in ETAR (purple circles), and only in ETBR (red circles)...... 64

Figure 34: FTIR spectra acquired for the raw materials PCPDTBT used in nanoparticle fabrication.

...... 67

Figure 35: FTIR spectra acquired for the raw materials PS-PEO-COOH used in nanoparticle fabrication...... 67

Figure 36: FTIR spectra acquired for the obtained conjugated polymer nanoparticles CPNPs. ... 68

Figure 37: FTIR spectra acquired for the obtained conjugated polymer nanoparticles EDN3-

CPNPs...... 68

Figure 38: (A) Schematic illustration of the treatment procedure for building a standardization curve for BCA assay of three different replicate experiments (B) A principal of the bicinchoninic acid (BCA) assay...... 71

Figure 39: Absorbance plot obtained for bovine serum albumin (BSA) by using microplate procedure...... 72

Figure 40: The proposed mechanism of the bicinchoninic acid (BCA) assay principle...... 73

Figure 41: Stability of suspensions with relation to zeta potential...... 76

Figure 42: (A) Distribution of hydrodynamic diameters of the EDN3-CPNPs in DI water measured by DLS. (B) Diameters of EDN3-CPNPs stored at 4 ⁰C as a function of time, up to 6 months. (C)

Zeta potential measurements of the CPNPs and EDN3-CPNPs in DI water (-34.7 mV and -54.6

xiv mV nm). The zeta potential supports the well-dispersed nature of the nanoparticles. Time- dependent zeta potential measurements of the (D) CPNPs and (E) EDN3-CPNPs in DI water as stored at 4 ⁰C, for up to 6 months. (F) TEM elemental analysis was utilized to verify the presence of iron of the obtained EDN3-CPNPs...... 77

Figure 43: (A) TEM images of EDN3-CPNPs (scale bar is 50 nm) exhibit polycrystalline structure and uncoated PCPDTBT nanoparticles (scale bar is 20 nm). (B) STEM image of EDN3-CPNPs, scale bar is 100 nm. (C) Observation of cellular uptake and localization of EDN3-CPNPs for A375 melanoma cell line (1.4 µg/mL internalized and damaged the nucleus), scale bar is 100 nm. (D)

Localization of EDN3-CPNPs for Malem-3M melanoma cell line (1.4 µg/mL internalized and damaged the nucleus), scale bar is 100 nm. (E) Zoomed-in TEM image shows the nanoparticles localized inside the nuclei of A375 cells (scale bar is 100 nm). (F) Zoomed-in TEM image shows the nanoparticles localized inside the cytoplasm of Malme-3M cell only (scale bar is 100 nm). In this case the EDN3-CPNPs did not cause damage to the nucleus...... 78

Figure 44: In vitro cell cytotoxicity as measured by MTS assay upon different doses of CPNPs

(gray column) and EDN3-CPNPs (black column) in the cell culture medium for a period of incubation of 24 h at 37 °C of (A) A375, (B) Malme-3M, (C) SK-Mel-28; (A−C), (D) Hepa 1−6,

(E) T24; (D, E) as nonmelanoma cell lines, and (F) AML12 as a healthy cell line. Error bars represent the quality deviation of the mean ± s.d. (n ≥ 3). A P-value < 0.05 was considered statistically significant and nonsignificant for P > 0.05. *P < 0.05, and **P < 0.01...... 82

Figure 45: TEM control experiment on untreated cells. (A) A375 and (B) Malme-3M.TEM experiment on treated cells with CPNPs. (C) Malme-3M and (D) A375 cells. (E) and (F) The

xv optical microscope images of A375 cell line incubated with 14 µg/mL EDN3-CPNPs a period of incubation of 24 h in the dark in 75 cm2 cell culture flask...... 83

Figure 46: (A) Schematic illustration of the treatment procedure for building a standard curve for the ferrozine assay of three different replicate experiments. The inset picture shows a yellow colored iron-chelating reagent which was stored in the dark at room temperature. (B) Chemical tool for the colorimetric detection of iron ions and the complex formed with ferrozine...... 86

Figure 47: Calibration curve obtained for FeCl3...... 87

Figure 48: TEM image of blank grid...... 90

Figure 49: TEM elemental analysis was utilized to verify the presence of iron of the obtained

EDN3-CPNPs...... 91

Figure 50: TEM elemental analysis was utilized to verify the presence of iron of the raw material of PCPDTBT...... 91

Figure 51: TEM elemental analysis was utilized to verify the presence of iron of the obtained

CPNPs...... 92

xvi LIST OF TABLES

Table 1: Characteristics of PCPDTBT NPs, CPNPs, and EDN3-CPNPs...... 75

xvii LIST OF ACRONYMS (or) ABBREVIATION

ADCs Antibody–Drug Conjugates

ATCC American Tissue Culture Center

BCA The Bicinchoninic Acid Assay

BSA Bovine Serum Albumin

BT Benzothiadiazole

CDT Chemodynamic Therapy cm Centimeter

CPDT Cyclopentadithiophene

CPNPs Conjugated Polymer Nanoparticles

Cu1+ Cuprous ions

Cu2+ Cupric ions

DI Deionized (DI) water

DLS Dynamic Light Scattering

DMEM Dulbecco's Modified Eagle’s Medium

DMPHEN 2,9-Dimethyl-1,10-phenanthroline

DPBS Dulbecco's Phosphate-Buffered Saline xviii DTIC Dacarbazine

EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

EDN1 Endothelin-1(porcine/human/rat/canine)

EDN2 Endothelin-1(human)

EDN3 Endothelin-3 (human, rat)

EDNRA Endothelin-A Receptors

EDNRB Endothelin-B Receptors

EDTA Ethylenediaminetetraacetic Acid

EPR Enhanced Permeability and Retention Effect

ER- Endothelin receptor negative control

ER+ Endothelin receptor positive control

FBS Fetal bovine serum

Fe2+ Ferrous ion

Fe3+ Ferric ion

FeCl3 Iron (III) Chloride

FTIR Fourier-transform infrared spectroscopy

HBSS Hank’s Balanced Salt Solution

xix HCl Hydrochloric Acid

HPLC High Performance Liquid Chromatography

IR Infrared Spectroscopy

MDR Multi-Drug Resistant mL Milliliter mM Milimolar mm Millimeter

NHS N-Hydroxysuccinimide

•− O2 Superoxide Anion

PBS Phosphate Buffered Saline

PCD Programmed Cell Death

PCPDTBT (Poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-

cyclopenta[2,1-b:3,4-b’] dithiophene-2,6-diyl]])

PDI Polydispersity Index

PDT Photodynamic Therapy

PS-PEO-COOH Polystyrene–graft– [poly (ethylene oxide), carboxylic-acid end-functionalized

PTFE Polytetrafluoroethylene

ROS Reactive Oxygen Species xx RPM Revolutions Per Minute

SODs Superoxide Dismutases

STEM Scanning Transmission Electron Microscopy

TEM Transmission Electron Microscopy

THF Tetrahydrofuran

TME Tumor Microenvironment

UVA Ultraviolet Radiation A

UVB Ultraviolet Radiation B

UVC Ultraviolet Radiation C

UV-Vis Ultraviolet–Visible Spectroscopy

ZP ζ-Potential

μg Microgram

xxi SUMMARY

The dissertation presented herein consists of five chapters. Chapter 1 is an introduction to the research scope discussed in this dissertation, addressing the background and significance of the research. It offers knowledge on various types of proposed melanoma treatments: surgery, radiation therapy (RT), photodynamic therapy (PDT), chemotherapy, and their mechanisms of treating the diseases, as well as their disadvantages and problems. In particular, it focuses on discussions on how these problems have been tackled with the development of novel tumor- targeted polymer nanoparticles carrying iron for chemodynamic therapy (CDT). Then, I comprise the literature review by discussing previous research on my subject. Chapter 2 consists of materials, methods, and preliminary discussions for the methods used in this dissertation research.

Chapter 3 includes results and discussion of the observations made on using polymeric nanoparticles of PTB7 for photodynamic therapy (PDT) of melanoma skin cancer. Chapter 4 contains results and discussion of the observations made on using polymer nanoparticles carrying iron for chemodynamic therapy (CDT) on various cancer cell lines. Chapter 5 finishes off with the conclusions of this dissertation research.

xxii CHAPTER ONE: INTRODUCTION

1.1 Background and Significance

1.1.1 Melanoma

Melanoma is the most aggressive form of skin cancer, with annually rising incidences throughout the world. It is caused by the uncontrolled division of melanocytes (pigment cells) in the basal layer of the epidermis (Figure1) and has limited treatment options.1 Melanoma cells can migrate through the bloodstream and lymph system from an original site to another location.1 The uncontrollable growth can disable normal functions of the body, and in severe cases, cause death.2

Figure 1: Illustration showing a melanocyte-melanin producing cells (adapted from Google image references).

1 Three main attributes categorize melanomas: the thickness of the primary tumor, the spread to lymph nodes, and metastasis that together determine their stage. Wallace H. Clark Jr. discovered the stages at Massachusetts General Hospital and Harvard University in the 1960s. Figure 2 represents Clark's level demonstrating a staging system that describes the depth of melanoma as it grows in the skin. In Clark’s Level I, melanomas have been characterized by having a primary tumor that is un-ulcerated with a thickness less than 1mm and has not spread to the lymph nodes

(the tumor is localized in the epidermis). In Level II, melanomas are characterized by having a primary tumor that is between 1 and 4 mm thick, which may be ulcerated but has not yet spread to the lymph nodes. A Level III melanoma is characterized by having a primary tumor that has spread to nearby lymph nodes (cancer has spread into the papillary-reticular dermal interface through the papillary dermis). Finally, in Level IV, melanomas have been characterized by having spread to distant lymph nodes and the subcutaneous tissue.

2

Figure 2: Clark's levels.

1.1.2 Environmental Factors and Prevention

Ultraviolet (UV) radiation exposure is recognized as a significant environmental risk factor for melanoma.3, 4 However, ultraviolet light in the A spectrum is a longer wavelength, which accounts for approximately 95% of the ultraviolet (UV) radiation reaching the lower atmosphere and the Earth's surface. These UVA rays go into the skin further compared to UVB rays and mutate

DNA via producing reactive oxygen species (ROS) that cause oxidative stress (significant damage to cell structures) and cytotoxic environment (Figure 3).5, 6

3

Figure 3: Illustration shows a cross section of human skin tissue.

The epidermis allows only 17.5% of UVA and 7.4% of UVB to penetrate in black skin, while skin penetration of white skin reaches 55% of UVA and 24% of UVB.7 Figure 4 represents a relationship between the incidence of UV rays, people's skin color, and the risk of skin cancer.8

4

Figure 4: A relationship between the incidence of UV rays, people's skin color, and the risk of skin cancer (reproduced from reference).8

Figure 4 shows how sunscreen filters work to block and absorb UV rays.8 Sunscreen can filter out UV rays by combining two types of ingredients. Inorganic ingredients, such as titanium or zinc oxides, scatter or reflect UV rays by forming a physical barrier. Meanwhile, organic ingredients, such as oxybenzone or octyl methoxycinnamate, absorb and dissipate UV rays as heat.

5

Figure 5: Sunscreen filters reflect UV radiation–Illustration (adapted from Google image references).

1.1.3 Melanoma Statistics

Based on the statistics of 2017, approximately 87,110 individuals were newly diagnosed with cases of melanoma in the United States alone.9, 10 On the other hand, reported cases were nearly 76,100 for the year 2014, with an estimated 9,710 expected deaths.11 Expected new melanoma cases and deaths in 2020, according to The Centers for Disease Control and Prevention, are shown in Figure 6. Thanks to the recent development of early diagnosis and treatment of melanoma, the growth in the number of deaths by melanoma has been suppressed.

6

Figure 6: Actual and projected cancer incidence rates, United States, 1975 to 2020.

Source: Reproduced from The Centers for Disease Control and Prevention. https://www.cdc.gov/cancer/dcpc/research/articles/cancer_2020_incidence.htm

1.1.4 The Endothelin Axis’ Role in Melanoma

Endothelins (EDN) are three structurally similar peptides incorporating 21-amino acids.

EDN1, EDN2, and EDN3, as shown in Figure 7, can bind to two subtypes of G-protein-coupled heptahelical receptors, namely endothelin receptor A and endothelin receptor B (EDNRA and

EDNRB).12, 13 The EDN axis is accepted to assume a role in different malignancies, including melanoma, central nervous system tumors, prostate, ovarian, cervical, and breast carcinomas.14

Four types of EDN receptors have been distinguished: EDNRA, EDNRB, EDNRC, and

EDNRAX. In mammalian animals, just EDNRA and EDNRB exist. Both EDNRA and EDNRB are proteins consisting of 400 amino acids located on the surface of cells.15 The genes for EDN1,

7 EDN2, EDN3, EDNRA, and EDNRB are located on chromosomes 6, 1, 20, 4, and 13, respectively.16 The two most essential receptors share excellent arrangement homology and can be recognized by their specificity. EDNRA binds most strongly to EDN1 compared to EDN2, whereas it shows the lowest affinity to EDN315 [EDN1>EDN2>>EDN3].17 In contrast, EDNRB has an equal affinity for all three endothelin isoforms. EDN1, EDN2, and EDN3 with equal affinity

[EDN1=EDN2=EDN3].18 Endothelin-B receptors (EDNRB) are overexpressed in melanoma and considered as a sign of its development progression as determined from 159 human melanoma cases.19 Past investigations have shown expression of EDNRB in eleven different cell lines of malignant melanoma, though just a single of these cell lines showed additional characterized by expression of EDNRA.20, 21 Reports indicate that EDNRB expression increases in melanoma cells as they progress to the metastatic disease state.22, 23 The endothelin 3 peptide (EDN3) was found to internalize rapidly into the surface of melanoma cells.24 The receptors recognize their corresponding agonist through complementarity-determining region loops, specifically between the EDNRB binding pocket and the N-terminal amine of EDN3, which is deeply buried within the

EDNRB pocket during the interaction.25

8

Figure 7: Structures of the endothelin peptides.

1.2 Melanoma Therapies

There are various types of proposed melanoma treatments: surgery, radiation therapy (RT), photodynamic therapy (PDT), chemotherapy, chemodynamic therapy (CDT), and their combinations.26 The treatment of melanoma depends on the site of the tumor and the stage of the disease. Early-stage that has not grown deeply (not metastasized) to the lymph nodes or the

9 surrounding tissue can be eliminated through chemo-surgical treatment successfully with a 99% survival rate.27, 28 Surgery is the physical removal of the cancerous tissue or tumor from a specific place in the body. Surgical excision removes the melanoma tissue along with some normal tissue around it (called the surgical margin). 29 Surgery is the most invasive method but enables the removal of a large volume of the tumor area during the localized early stage. However, advanced stages (III-IV), highly aggressive malignant skin cancer (metastatic melanoma) are notoriously difficult to eradicate through surgical removal and exhibit survival rates of less than 10% in 5 years. Radiation therapy (RT), performed utilizing an external radiation source, causes dysfunctions DNA in the cancer cells through apoptosis.30, 31 Photodynamic therapy (PDT), on the other hand, is a clinically approved procedure for melanoma treatment. It is a relatively new form of treatment that uses a unique drug, known as a photosensitizer, along with the particular type and a specific wavelength of light which utilizes reactive oxygen species (ROS), produced by photosensitizers. ROS can exert their selective cytotoxic effects on malignant cells resulting in cell death (phototoxicity).26 Chemotherapy is a drug therapy used to destroy cancer cells. It attacks cancer cells with small molecules targeted towards rapid growth or biomarkers that are highly expressed in the tumor area to prevent cells from spreading, dividing to make more cells, and slowing their growth.32, 33

Radiation therapy and chemotherapy are indirect treatments and less invasive.26 Due to their lesser invasiveness, these therapies are often prescribed before surgery or after the surgery as adjuvant therapies.33, 34

10 All these types of therapies target the removal of melanoma cells from the human body.

However, patients still have low response rates and serious adverse effects. Therefore, establishing new methods of treatment of advanced melanoma stages (III-IV) is in high demand.

1.2.1 Problems with Chemotherapy

Chemotherapy is used in clinical practice as conventional therapy for metastatic melanoma.

Even though chemotherapy is less invasive, chemotherapeutic agents damage both malignant and healthy cells due to their non-specificity and patients may show drug resistance.35 Therefore, side effects due to non-targeted distribution of drugs occur that limit the dosage to be used.26 Drug resistance is due to cancer cells’ intrinsic resistance against these drugs, the so-called multidrug resistance (MDR).36 Because of their MDR ability, some cancer cells may survive chemotherapy.37-40 MDR is discussed further in detail on the next page. Thus, this treatment may not completely eradicate the tumor, and cancer patients may relapse after chemotherapy is over.36

Multidrug Drug Resistance (MDR):

The evolution of multidrug resistance (MDR) or chemotherapeutic resistance in melanoma and most cancers has become a significant challenge to successful chemotherapy. It is a complex process that results from the alteration of drug targets and combines multifaceted non-cellular and cellular-based mechanisms. MDR can occur in cancer cells by various mechanisms, including suppression of apoptosis (cell death inhibition), modifications on agent's metabolism (including at the cellular level, e.g., exocytosis), and enhanced DNA repair.41, 42 Due to these MDR abilities, the chemotherapy of cells with MDR is challenging. Temozolomide, vinblastine, and Dacarbazine

11 (DTIC) are the most common drugs that are known to be of use in chemotherapy for malignant melanoma treatment. However, a meager response rate is reported. The response rates to temozolomide, vinblastine, and DTIC were 28%, 9.5%, and 7%, respectively, with 64 patients having been reported in a clinical trial.43 Prospective chemotherapy needs to overcome the problems described above: MDR and side effects, to improve the patient's quality of life as well as to increase the drug efficacy. To completely eradicate the tumor, it is mandatory to overcome the problem of MDR. A simple method to solve the problem of MDR is to increase the amount

(dose) of anti-cancer drugs that are delivered to the tumor areas so that they can override the MDR of tumors. However, merely increasing the dose of drugs causes an increase in the risk of resulting severe side effects. Therefore, instead of increasing the overall treatment of antitumor drugs, it is necessary to improve delivery and develop materials that show less MDR.

1.2.2 Nanoparticles-Assisted Chemotherapy

MDR is a significant obstacle to the successful application of current melanoma therapies.

It is a crucial factor in relapse and metastasis of melanoma. To surmount this problem, nanocarriers-based drug delivery systems have been established as novel strategies for overcoming

MDR. They have significant advantages that targeted tumors and as a result affected the well- controlled release of drugs. Melanoma researchers over the past two decades have reached the point where they smartly designed nanoparticles with tumor targeting ligands. These preferentially accumulate within the tumor region through active and passive targeting to overcome MDR, which can aid successful chemotherapy (Figure 8). Several nanoparticulate drug delivery systems have been reported for nanoparticle assisted chemotherapy. DOX and PTX are the two clinically

12 approved drugs with altered biodistribution.44 Liposomes45, Micelles46, dendrimers47, polymer- drug conjugates48, and nanoparticles44, 49 are the most commonly used nanoparticles for drug delivery. Drugs loaded into nanoparticles improved biodistribution and pharmacokinetic profiles.44 Nanoparticles-drug delivery systems provide a promising solution that allows simultaneous targeting for delivering more than one agent simultaneously at the cellular level.

Primarily, the ideal nanoparticles should rapidly accumulate within targeted cells. This can be obtained by functionalizing the surface of the nanoparticles with specific targeted molecules.

Among these nanoparticles, polymeric-based drug delivery platforms have attracted much attention in recent years. This is attributed to their easy preparation, surface properties, and potential for surface modification. Their tumor accumulation relies on targeting of cancer cells through active targeting (ligand-receptor interaction) or passive targeting by accumulation in the tumor cells via the enhanced permeability and retention (EPR) effect.

13

Figure 8: Schematic illustration of suggested mechanisms in vivo of limited targeting of nanoparticles on a cell membrane of healthy cells (Right) vs. extensive targeting of cancer cells on the mechanism of active targeting (bottom left) or can be achieved on the mechanism of passive targeting by accumulation in the tumor cells via the enhanced permeability and retention effect (EPR) (top left).

1.2.3 Nanoparticles-Assisted Photodynamic Therapy (PDT)

Photodynamic therapy (PDT) is a well-established treatment procedure for plenty of diseases and localized cancer, including melanoma. The mechanism involves the systemic or topical administration of photosensitizer drug, along with application of light to the target site, which causes photo-damage and subsequent tumor cell death (Figure 9). While PDT is minimally

14 invasive, untargeted photosensitizers distribute throughout the patients body, resulting in undesirable adverse effects. 50, 51

Figure 9:Schematic representation of target-activated PDT agents by a specific wavelength, in which the therapeutic agent is a photosensitizer (PS).

A research study reports a development that modulates the phototherapeutic performance of hybrid π-conjugated organic semiconductor PCPDTBT (SPNs). SPNs are self-regulated NIR – absorbing/emitting PDT's efficacy for cancer therapy optimization in vitro (Figure 10). These

SPNs consist of two-components: the nanostructures and the nanoceria. Nanostructures serve as

NIR–absorbing/emitting organic semiconducting polymers. Nanoceria, on the other hand, serves 15 as a smart intraparticle regulator that increases/decreases levels of reactive oxygen species (ROS) production. In a murine mouse model, nanoceria-doped SPNs under NIR laser irradiation at 808 nm, this not only leads to significantly enhanced PDT efficacy but also results in minimum damage towards healthy tissue.52

Figure 10: Schematic illustrations of nanoceria-doped SPNs under NIR laser irradiation (reproduced from reference).52

1.2.4 Nanoparticles-Assisted Chemodynamic Therapy (CDT)

In recent years, a lot of effort has been devoted to the development of reactive oxygen species (ROS)-dependent anticancer therapies53-56, including chemodynamic therapy (CDT).57-59

CDT generates ROS by converting endogenous hydrogen peroxide (H2O2) that is abundant in the tumor microenvironment (TME)60 into hydroxyl radicals (•OH), which kills tumor cells 16 efficiently.61-66 The mechanism of ROS formation proceeds through the iron-mediated Fenton reaction.67 Several nanoparticulate iron delivery systems have been reported for ferroptosis assisted CDT, including iron-based68-76, lipid-based (NP coating to solubilize IONPs, or micelles)

66, 70, 77, 78, silica-based 76, 79, 80, or metal-organic framework derived.68, 70 Iron oxide nanoparticles

• (IONPs) require acidic conditions to disproportionate H2O2 into OH, thus cellular internalization through lysosomal pathways is desirable.81 Silica-based nanoparticles can be either decorated with cargo for iron delivery or iron oxide nanoparticles can be loaded into the silica core. Similarly, polymer-based nanoparticles76, including iron, are loaded into the core.

Nanoparticles tend to remain longer inside tumor cells once internalized due to the lower exocytosis rates compared to small molecule drugs.82-84 Targeted nanoparticles can thus be used to deliver chemotherapeutic agents with the expectation of reduced MDR85, reduced side effects86,

87, and increased treatment efficacy.87

While there is a breadth of nanocarriers for delivery of iron available, only a few have tumor-targeting functionality built-in. These iron delivery nanomaterials thus typically exhibit nonspecific action and may cause significant side effects when applied in vivo, with delivery mainly through the enhanced permeability and retention (EPR) effect.57, 59, 76 The internalization pathway is often not well controlled, although the size of the nanoparticles typically falls in the

10−50 nm range for which endocytosis is common.88 We alleviate these issues herein by designing conjugated polymer nanoparticles (CPNPs) for receptor-mediated endocytosis, which both increases specificity and control of internalization compared to previously reported nanoparticle iron delivery systems. We demonstrate this herein for the case study of melanoma by conjugation

17 CPNPs with endothelin-3 (EDN3), which specifically binds with the melanoma endothelin-B receptors (EDNRB). The CDT effect of our CPNPs is comparable with published iron delivery nanoparticle platforms.

Ferroptosis:

Ferroptosis is an iron-dependent form of non-apoptotic regulated cell death, first identified by Stockwell et al. in 2012.89 It is biochemically, morphologically, and genetically distinct from apoptosis, a popular form of regulated cell death by producing highly toxic •OH though a Fenton reaction.90-92 It has, therefore, been hypothesized that therapeutic doses of iron could selectively suppress cancer cell growth while leaving healthy cells unharmed 90-93, but the approach has remained mostly unexplored.92, 94, 95 Due to mitochondrial malfunction as a significant source of

96- H2O2 and metabolic activity, cancer cells produce a high level of H2O2 compared to normal cells.

101 A high concentration of H2O2 can react with iron in the acidic environment and produce highly toxic •OH through a Fenton reaction.102 The result •OH do not diffuse from generation sites due to high reactivity and a short half-life (10-9s) but instead oxidize any surrounding bio- macromolecules very rapidly.103 Two factors that facilitate the selective killing of cancer cells are the level of H2O2 generation and overexpression of the receptor-ligand interaction located on the cell surface. Among various iron oxide nanoparticles that have been cited for cancer therapy95, 104-

106, a study steered by researchers at the Stanford University School of Medicine (SUSM), accidentally discovered that ultrasmall Ferumoxytol (Feraheme™), an iron oxide nanoparticle with a diameter of less than 5 nm invented over 10 years ago. FDA approved in 2009 and commercially launched by AMAG Pharmaceuticals, Inc. in the U.S., Ferumoxytol is widely used in chronic kidney disease (CKD) patients to treat iron deficiency in adult and to light up tumors 18 so surgeons can see where to cut. It can deliver iron-loaded nanoparticles into tumors, thus increasing their anticancer efficacy. After only 24-48 h of exposure time, high doses of these peptide-coated nanoparticles cause a “wave of destruction,” via ferroptosis , making this as a novel and reactive pathway for melanoma treatment.104 Researchers at SUSM sought to, more definitively, identify the mechanism of a suppressive effect on a broad panel of cancer metastasis in mice. The researchers then examined whether melanoma death is iron-dependent, a known ferroptosis requirement. Collectively, through several examinations, the SUSM researchers demonstrated that the process of cell death was occurring via ferroptosis, a distinct form of programmed cell death that differs from other forms, such as apoptosis, necroptosis, and autosis.

Taken together, the foregoing data determined that nanoparticles treated with an iron chelator, deferoxamine (DFO), almost entirely inhibited cell death, suggesting that iron-loaded nanoparticles may engage ferroptosis in cells. Moreover, in their findings, surface-modifications of nanoparticles with cancer-targeting aMSH peptides, triggered cellular internalization.

Interestingly, they also determined that ferroptosis in the cell lines occurred with no requirement of surface modification with aMSH and delivery occurred through the accumulation of the nanoparticles within tumors via leakiness of tumor vasculature due to the EPR effect.

Other studies report that altering iron metabolism is a well-founded path for cancer therapy.107 For improved therapeutic efficacy in nitroquinoline-oxide (NQO)-overexpressing tumor cells, a study reports that SPION-micelles with an encapsulated anticancer drug (β-lap) were internalized into acidic organelles. The H2O2 generated from β-lap reacts with iron released from the micelles in a pH-dependent manner to produce •OH through a Fenton reaction, which can oxidize any surrounding, as shown in Figure 11. 19

Figure 11: Schematic illustration of intracellular distribution of SPION-micelles, followed by iron release, which is then involved in a Fenton reaction in the presence of H2O2 that is generated from β-lap (reproduced from reference).107

An iron nanoparticle conjugated with folic acid was prepared with an average hydrodynamic diameter of 244.6 nm.94 Within cells in an acidic environment such as that found in 20 cancer tissue, iron releases from the nanoparticle very rapidly. Later, iron reacts with the high levels of H2O2, thus produced by mitochondria mainly found in cancer cells. The resulting highly toxic •OH generated through a Fenton reaction can oxidize proteins, lipids, and DNA. This treatment decreases malignant cell viability with no noticeable effect of a normal cell, as illustrated in Figure 12.

Figure 12:Schematic illustration of the damage of cancer cells occurred via rMOF-FA nanoparticles (reproduced from reference).94

Another study has presented a new therapeutic tool to treat cancer cells through luteinizing hormone-releasing hormone (LHRH) peptide coated FePt NPs. The controlled release of iron 21 within cells in an acidic environment catalyzes H2O2 to produce reactive oxygen species (ROS), which are toxic to tumor cells, as illustrated in Figure 13.108

Figure 13: Schematic illustration internalized of the LHRH peptide coated FePt NPs, causing oxidative damages to proteins, lipids, and DNA (reproduced from reference).108

Another study hypothesized that ferroptosis could be significantly enhanced by increasing the concentrations of all raw materials involved in the Fenton reaction, such as Fe3+, Fe2+, and

H2O2. This study concluded that the acceleration of Fenton reaction inside cells generates ROS that induce cancer cell death, as shown in Figure 14.109

22

Figure 14: Schematic illustrating the design and synthesis of magnetic nanoparticles which is then 109 involved in a Fenton reaction in the presence of H2O2 (reproduced from reference).

Another research study reports the toxicity mechanism induced by nanoparticles that contain iron and/or other metal ions, thus involved in a Fenton reaction. The study compared internalization mechanisms of the nanoparticles when they enter cells in two approaches: endocytosis-free nanoparticles, and through active internalization. This study concluded that an

23 endocytosis pathway where nanoparticles rapidly end up confined in lysosomes might result in more release of relatively toxic ions (e.g., Fe2+/3+, Cd2+, Ag+, and Au1+/3+ ions) in the acidic environment. As a result, increased ROS levels through ferroptosis or apoptosis mechanisms, thus cause DNA and membrane damages, as illustrated in Figure 15.81, 88

Figure 15: Schematic illustration of endocytosis-free nanoparticles vs. active mechanisms internalized into the cells (reproduced from reference).88

1.3 Scope of Reported Dissertation Research

We aimed to eradicate melanoma cancer cells in vitro. With the developed targeted CPNPs, we had success in suppressing tumor cell count, whereas untargeted CPNPs did not show much killing effect of the utilized cancer cells. This novel tumor-targeted (CPNP) system was prepared

24 from poly [2,1,3-benzothiadiazole-4,7-diyl [4,4-bis (2-ethylhexyl) - 4H-cyclopenta [2,1-b:3,4-b’] dithiophene-2,6-diyl]] (PCPDTBT). The CPNPs are around 62 nm in size, tumor-targeting, and deliver iron (as Fe3+) to the intracellular environment to trigger ferroptosis, followed by tumor cell death (Figure 16). In my dissertation, I focus on the improvement of a CPNP formulation. To improve its efficacy, I focused on the most critical factor, which is the cellular uptake efficiency, which occurs through two essential characteristics:

First, the surface properties, such as charge, of the CPNPs is a factor influencing cellular uptake efficiency. The higher the negative charge, the higher the cellular uptake, because the melanoma cell membrane is positively charged. The relationship between the CPNP’s surface and the cellular uptake is further discussed in chapter four.

The second factor in increasing uptake is to add some targeting moieties on the CPNP surface to increase the chance of receptor-mediated endocytosis. Cells that are expressing high amounts of EDNRB on the membrane surface can uptake more ligand coated CPNPs than uncoated.

To analyze these factors, we utilize various analytical methods. The CPNPs are measured using dynamic light scattering (DLS), which gives data on their hydrodynamic size, size distribution, and the zeta potential. Meanwhile, scanning transmission electron microscopy

25 (STEM) and transmission electron microscopy (TEM) were utilized to evaluate the size uniformity of the CPNPs and their location in the cells in the dried state.

Figure 16: Schematic illustrating the biological significance of the Fenton and Haber–Weiss reactions. In the presence of Fe3+ (delivered by EDN3-CPNPs) the Haber-Weiss reaction produces 2+ Fe for the Fenton reaction, in which sufficiently elevated H2O2 in tumor cells is reduced to the highly reactive hydroxyl radical and molecular oxygen is returned as a product.

For the case study of melanoma, we found that our endothelin-3 modified CPNPs (EDN3-

CPNPs) encapsulating iron appropriately targeted melanoma tumor cells, resulted in effective

CDT, and this finally resulted in the increased killing of tumor cells compared to untargeted

CPNPs. We compared the uptake of EDN3-CPNPs to CPNPs and their main difference was in the

26 internalization efficacy. The EDN3-CPNPs showed a rapid internalization of melanoma cells and, therefore, the rapid releasing of iron.

Conversely, unmodified CPNP uptake across the studied cell lines is suggested to be possible due to the attractive interaction of the anionic CPNPs with the relatively positively charged EDNRB receptors with less CDT effect in most cases. Besides, untargeted CPNPs showed a negligible impact on the intracellular environments of healthy cells due to the absence of endothelin-B receptors (EDNRB).

27 CHAPTER TWO: MATERIALS AND METHODS

2.1 Introduction

To make the iron-carrying nanoparticles used in therapeutics more effective towards curing the melanoma cancer, it is necessary to understand how it works in the human body. To fully understand this, it is fundamental to find out its functional properties and mode of action in vitro before introducing them in vivo. This was obtained by growing cell lines in the lab under controlled conditions and conducting experiments on them for in vitro studies. Conjugated polymer nanoparticles (CPNPs) carrying iron as a CDT tool were fabricated, characterized, and observed significant efficacy in targeting and treatment. Functionalization of nanoparticles with endothelin-

3 as targeting agents towards melanoma endothelin-B receptors (EDNRB) expressing cell lines was achieved to make CDT targeted, thus reducing the side effects by avoiding uptake in healthy cells. Nanoparticle-based iron delivery systems contribute to a significant treatment for various cancers. Also, they provide other advantages such as protection from degradation, controlled release of encapsulated iron into tumor sites, increased circulation time, and potent tumor-targeted efficacy. The nanoparticle-encapsulated iron has a broad range of solubility with significantly enhanced cell cytotoxicity. Among these, conjugated polymer-based nanoparticles (CPNPs) demonstrate promising activity against melanoma. The cell viability rate of our CPNPs after CDT was quantified by MTS assay, which is discussed in detail here within. Cell morphology was observed by Transmission Electron Microscopy (TEM) and Scanning Transmission Electron

Microscopy (STEM). The hydrodynamic diameters and zeta potential of the synthesized CPNPs and EDN3-CPNPs were determined by dynamic light scattering (DLS). UV-Vis and Fluorescence 28 spectroscopies were performed to confirm the formation of nanoparticles. All the experimental details have been discussed in the following subsections.

2.2 Materials

All reagents were purchased from common commercial sources. The reagents were used without further purification unless otherwise stated. Poly [2,1,3-benzothiadiazole-4,7-diyl [4,4-bis

(2-ethylhexyl)-4H-cyclopenta [2,1-b:3,4-b’] dithiophene-2,6-diyl]] (PCPDTBT) with a molecular weight 41 000 g/mol was purchased from 1-Material Inc. The polymer was purified to remove low molecular weight polymers. Comb-like polymer (SEOCOOH comb) polystyrene–graft–[poly

(ethyleneoxide),carboxylic-acidend-functionalized] (PS-PEO-COOH) with a molecular weight of

36 500 g/mol (average Mn, PDI 1.3, Mw 47,450 g/mol) was purchased from Polymer Source Inc.

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), endothelin 3 (human, rat), N-

Hydroxysuccinimide (NHS), Iron (III) chloride, and ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4- triazine-4′,4″-disulfonic acid sodium salt) were purchased from Sigma-Aldrich. THF

(tetrahydrofuran, Drisolv) was purchased from EMD Millipore. Acetate Buffer, pH 4.5 was purchased from Ricca Chemical. Sodium L-Ascorbate was purchased from TCI. Neocuproine

(2,9-Dimethyl-1,10-phenanthroline, DMPHEN) was purchased from Alfa Aesar. Hydrochloric acid 37%, was purchased from Acros Organics.

29 2.3 Nanoparticle Fabrication

2.3.1 Purification of PCPDTBT Polymer

To purify PCPDTBT,6 mL of HPLC grade acetone was added to 3 mg of the polymer in a vial. This vial was then heated gently on a hot plate at a low temperature of 20 °C for 15 min. The low molecular weight PCPDTBT remained in the supernatant, while the high molecular weight settled at the bottom. To the settled PCPDTBT, a small amount of approximately 3 mL of chloroform was added. A green solution was observed immediately. To this solution, acetone was added in a 1:5 ratio of chloroform to acetone. To protect the solution from the light, the suspension was transferred to a foil-wrapped 50 mL centrifuge tube. Amicon Ultra-15 centrifugal filters with a molecular weight cutoff of 10 000 kDa were used for filtration. The suspension was centrifuged at 4000 rpm for 10 min at 20 °C. The supernatant was removed by micropipette, and the procedure was repeated until the supernatant showed a light green color (around four repetitions). The purified PCPDTBT was then dried overnight at room temperature before being subjected to CPNPs fabrication.

2.3.2 Purification of PTB7 Polymer

Before using to make nanoparticles, PTB7 polymer was first purified to remove low molecular weight impurities. 3mg of PTB7 was added to a vial with 6 mL of acetone (EMD

Millipore) and heated gently on a hot plate at 50 ⁰C for 30 min. The supernatant of this solution was removed by micropipette, and the remaining settled at the bottom of the tube was transferred to a 50 mL centrifuge tube. 6 mL of chloroform (EMD Millipore) was added, followed by 15 mL 30 of acetone. This mixture was centrifuged (Eppendorf 5810 R) for 10 min at 4000 rpm. The supernatant of this mixture was removed, 15 mL of acetone was then added, followed by 3 mL of chloroform. The solution was again centrifuged at 4000 rpm for 10 min. This process of supernatant removal and addition of acetone and chloroform was repeated twice. After the final supernatant was removed, the remaining settled was left in a fume hood, so trace amounts of solvent would evaporate.

2.3.3 Fabrication of CPNPs Obtained from PCPDTBT Polymer

To obtain a 0.1 mg/mL stock solution of PCPDTBT, 1 mg of purified polymer was dissolved in 10 mL of THF, stirred, and heated at 60 °C for 2 h. The polymer solution was then filtered through a PTFE membrane filter with a 0.2 μm pore size before adding a stock solution.

In contrast, to obtain a 0.02 mg/mL stock solution of PS−PEO−COOH, 1 mg of this polymer was dissolved in 50 mL of THF. Approximately a 23:1 molar ratio solution of PCPDTBT to

PS−PEO−COOH was prepared by adding 10 mL of PCPDTBT stock solution to 2.3 mL of

PS−PEO−COOH stock solution. This mixture was then gently stirred at room temperature for about 30 min. Under vigorous stirring, 1 mL of this mixture was quickly injected into 6 mL of DI water. After injection, stirring was stopped immediately. The THF solvent was then removed under vacuum at room temperature for up to 3 days.

Molarity Calculation

1. PCPDTBT

PCPDTBT molecular weight, M.wt = 41 000 g/mole

31 No. of moles (n) = Wt. (g) / M.wt (g / mole)

M = n / v (L)

−8 M diluted = 4.0 × 10 mole / L

= 2.44 × 10−6 M (concentration of undiluted PCPDTBT {stock solution})

2. PS-PEO-COOH

PS-PEO-COOH molecular weight, M.wt = 47 450 g/mole

n = wt. (g) / M.wt (g/mole)

M = 4.2 × 10−7 mole/L (stock concentration of PS-PEO-COOH (1 mg / 50 mL THF)

3. Mixture of PCPDTBT (10 mL) + PS-PEO-COOH (2.5 mL)

4. Molar ratio PCPDTBT (M dilute) in 12.5 mL to PS-PEO-COOH (M dilute) in 12.5 mL

2.0 × 10-6 0.084 × 10-6

2.0 / 0.084 = 23

Molar ratio 23:1 PCPDTBT: PS-PEO-COOH in 12.5 mL sol.

5. Total mass calculation in EDN3-CPNPs

Total = 6.12 × 10-4 mg/mL of PS-PEO-COOH + 1.34 × 10-2 mg/mL of PCPDTBT +

3.38 ×10-4 mg/mL of EDN-3

Mass of EDN3-CPNPs = 14 µg/mL in water

2.3.4 Fabrication of CPNPs Obtained from PTB7 Polymer

PS-PEO-COOH:PTB7 nanoparticles were prepared using the reprecipitation method.

Approximately 1 mg of PTB7 was dissolved in 3 mL of THF (EMD Millipore) and stirred at 200 rpm for 2 h at 50 °C. Then, the solution was filtered through a 0.2 micrometer nylon syringe filter

32 (VWR). Enough PS-PEO-COOH was added to bring the molar ratio of PS-PEO-COOH and PTB7 to the desired molar ratio. A portion of this solution was removed, and its volume adjusted, so the concentration of PTB7 in solution was 0.1 mg/mL, then left in a tightly sealed vial for a minimum of 2 h while stirring to allow both polymers to dissolve completely. A gas-tight syringe removed

1 mL of this solution with a beveled tip. Prior to injecting the solution, any bubbles drawn into the syringe were removed. The solution was injected by hand at the fastest rate possible into a narrow glass vial containing 5 mL of water stirred at the 1500 rpm. After injection, stirring was immediately stopped, and the stir bar was removed. THF was allowed to evaporate from the solution in a fume hood. Size distribution measurements for the nanoparticles were obtained using dynamic light scattering (Malvern Nano ZS90).

2.4 Bioconjugation of CPNPs Obtained from PCPDTBT

To chemically link endothelin-3 (human, rat) to the CPNPs, the carboxylic groups

(−COOH) at the nanoparticle surface were first activated with EDC-NHS in the dark at room temperature. Then, 200 μL of EDC solution (1mg in 10 mL of DI water) was added to 12 mL of

CPNPs, and the mixture was stirred gently for 5 min. Next, 100 μL of NHS solution (1 mg in 10 mL DI water) was added, and the mixture was stirred for another 30 min. Finally, 350 μL of a freshly prepared solution of endothelin-3 (human, rat) was added, and the mixture was stirred for

12 h. The molar ratio of PS−PEO−COOH to EDN3 was 1:10, while the molar ratio of

PS−PEO−COOH to EDC-NHS was 1:100. These functionalized nanoparticles (EDN3-CPNPs) were purified by centrifugal filtration.

33 To remove any unreacted EDN3, NHS, and EDC from the obtained nanoparticle suspension, 12 mL of EDN3-CPNPs was filtered by centrifugal filtration. Amicon Ultra-15 centrifugal filters with the molecular weight cutoff 30 000 kDa were used for filtration. The nanoparticle solution was spun at 2000 rpm at 20 °C in a swinging bucket rotor for 6 min. For complete removal of unreacted NHS, EDC, and EDN3 from the EDN3-CPNPs solution, two filtrations were needed. After each filtration cycle, approximately 6 mL of filtrate was removed, and the volume of the EDN3-CPNPs suspension was adjusted back to 12 mL by adding a complete growth medium.

2.5 Bioconjugation of CPNPs Obtained from PTB7

PTB7 nanoparticles were functionalized by first adding EDC (Sigma Aldrich) to the solution in at a ratio of 1 mole PS-PEO-COOH for every 10 moles of EDC. The solution was stirred at room temperature for 15 min before NHS (Sigma Aldrich) was added at a ratio of 1 mole

PS-PEO-COOH for every 10 moles NHS. After 15 min, endothelin-3 (Sigma Aldrich) was added to the solution at the ratio of 1 mole PS-PEO-COOH for every 5 moles of endothelin-3. The solution was stirred for 2 h and then purified by centrifuging the solution using a nylon centrifugal filter with a molecular weight cutoff 50 000 kDa (EMD Millipore).

Centrifugal filtration was carried out by spinning the solutions at rates between 1500 and

2000 rpm to reduce the volume to 10% of its original volume while attempting to minimize the aggregation of the nanoparticles. DI water was then used to bring the solution back to its original volume. This process has repeated a minimum of 4 times per solution. After the final round of

34 centrifugal filtration, the concentrated solution was not adjusted back to its original volume and instead saved in a concentrated form for further use.

An alternative purification approach was investigated but never used for cell studies using dialysis membranes with a 50 000 kDa molecular cutoff (Spectrum Labs). Solutions were loaded into five floating dialysis membranes and suspended in 500 mL of DI water. The solutions were left in a fridge for 24 h to prevent the breakdown of the peptides. This procedure was repeated four times without removing the solution from the dialysis membrane.

2.6 Characterization of Nanoparticles

2.6.1 UV-Vis Spectroscopy

Absorption spectroscopy on nanoparticle suspensions in water and the raw polymer material (PCPDTBT) in THF was completed with a 1 cm path length quartz cuvette using an

Agilent 8453 spectrophotometer. We kept the absorbance below 1 to keep within the linear range of Beer’s law.

2.6.2 Fluorescence Spectroscopy

Fluorescence emission spectra were collected following absorption spectroscopy in the same 1 cm path length quartz cuvette with a Nanolog Horiba Jobin Yvon Fluorimeter. Their parameter settings are 687 nm excitation wavelength, slit width 5 nm, and emission wavelength

712-950 nm, slit width 5nm.

35 2.6.3 Fourier-Transform Infrared Spectroscopy

The powders of PCPDTBT, PS−PEO−COOH, CPNPs, and EDN3-CPNPs were characterized by IR spectroscopy. The CPNPs and EDN3-CPNPs were frozen in DI water and lyophilized (freeze-dried) using a FreeZone 4.5 L Freeze-Dry System (Labconco) prior to analysis.

All measurements were acquired over the range 600−3500 cm−1 on a PerkinElmer Spectrum 100

FTIR spectrometer.

2.6.4 Dynamic Light Scattering (DLS)

The hydrodynamic diameters of the synthesized CPNPs and EDN3-CPNPs were determined by dynamic light scattering using a Zeta Particle Size Analyzer (Nano-ZS, Malvern

Instrument).1 mL sample added to the appropriate cuvette without touching transparent areas.

2.6.5 Transmission Electron Microscopy (TEM)

The particle size was also determined by TEM (FEI Tecnai F30 TEM (300 kV) and JEOL

TEM-1011 (100 kV)). The TEM grids used were C-flat 1 μm 200 mesh (cat. no. CF21-50) purchased from Electron Microscopy Sciences. About 2-3 µL was pipetted onto a TEM grid and dried under vacuum.

2.6.6 Scanning Transmission Electron Microscopy (STEM)

STEM observations were conducted on a Zeiss ULTRA-55 FEG SEM (Zeiss, 30 kV) to analyze the size of functionalized nanoparticles. The TEM grids used were C-flat 1 μm 300 mesh 36 (cat. no. CF31-50) purchased from Electron Microscopy Sciences. About 2-3 µL was pipetted onto a TEM grid and dried under vacuum.

2.7 Cell Culture

Cell lines were acquired from the American Tissue Culture Center (ATCC), Malme-3M (a malignant human melanoma cell line) (at fifth passage) was cultured in Dulbecco’s modified

Eagle’s medium (DMEM), Ham’s F-12 media supplemented with 20% heat-inactivated fetal bovine serum (FBS), 1% penicillin−streptomycin, and L-glutamine (100×), all purchased from

Corning. A375 (at fourth passage) and SK-Mel-28 (at second passage) (human melanoma cell lines), AML 12 (mouse liver cell line, noncancerous) (at third passage), Hepa 1−6 (mouse liver cell line, cancerous) (at third passage), and the human urinary bladder cancerous T24 cell line (at fourth passage) were cultured in DMEM medium supplemented with 10% FBS. Cells were grown under standard conditions for 24 h at 37 °C in a humidified incubator in an atmosphere of 5% CO2 and 95% air in a T-75 flask for 4−7 days to reach a confluence of 70−80% before being subjected to any further experimentation.

2.7.1 Cell Viability Determination by MTS Assay (PCPDTBT)

In vitro cell viability was determined with the CellTiter 96® AQueous One Solution

Reagent, which contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS]. Cells in known volume were counted using a hemocytometer. All cells were seeded in 96-well plate with a density of 10 000 cells/well and incubated under standard conditions for 24 h prior to MTS assay. The medium was 37 removed entirely, and the cells were thoroughly washed with 100 µL fresh medium. The medium was then replaced with another 100 µL fresh medium containing appropriate doses (14, 1.4, 0.14, and 0.014 µg/mL) of CPNPs and EDN3-CPNPs, respectively along with control experiment (0 mg/mL) for 24 h. Subsequently, wells were carefully washed twice with fresh medium to remove any uninternalized nanoparticles. The assay was then performed by adding 20 μL of the CellTiter

96 AQueous One Solution Reagent (purchased from Promega) per well. The absorbance was recorded at 490 nm on BioTek ELx808 absorbance microplate reader at time 0 to determine the background signal. The plates were then incubated again for up to 1−4 h protected from light.

Absorbance was then rerecorded at the same wavelength indicated above. All experiments were performed in triplicate. The cell viability (%) was calculated by the following formula, where [A] is the average absorbance:110, 111

[A]490 (sample (treated cells))−[A]490 (blank (no cells)) Cell viability (%) = × 100 ( 1 ) [A]490 (control (untreated cells))−[A]490 (blank (no cells))

Statistical analysis was performed on the data presented in chapter 4. A two-tailed

Student’s t test was used to assess the significant differences between cell viability values observed for nontargeted and targeted CPNPs. A p-value < 0.05 was considered statistically significant.

Outcomes were indicated as *P < 0.05 and **P < 0.01.

2.7.2 PDT and Cell Viability Determination by MTS Assay (PTB7)

100 µL of a given cell line was plated into a well at a density of 500 000 cells/mL. After being given one day to grow with normal media, the media in each well was then changed to

DMEM with FBS or DMEM only. 100 µL of either unfunctionalized nanoparticles, functionalized 38 nanoparticles, or media only was added to the wells and incubated for another 24 h. After this period, the media in each well was again changed.

To assess the effect of light on the cell lines incubated with nanoparticles, well plates with cell lines were removed from the incubator and exposed to the maximum power of a solar simulator for 1 h. The cells were then put back into the incubator for 4 h. After the 4-h incubation period, an MTS assay (Promega) was performed according to manufacturer provided protocol, absorbance measured at 490 nm using a Biotek ELY 808 plate reader as well as a reading at 4 h, the difference between the two is used as the reported absorbance due to MTS.

To evaluate the intrinsic toxicity of these nanoparticles, cells were plated in well plates and incubated with the nanoparticles in the same conditions as cells exposed to light. MTS was added after the cells had been incubated for 24 h with nanoparticles, and the media had been changed.

2.8 Quantitation of Amount of Endothelin Peptide Attached to CPNPs

BioVision’s Extra Sense BCA Protein Assay Kit was performed to assess the amount of endothelin-3 attached per mg of CPNPs. Absorption spectroscopy on the assay was first completed in a 1 cm path length quartz cuvette with an Agilent 8453 Spectrophotometer to confirm the absorption wavelength (562 nm). The microplate reader (BioTek ELx808) could approach this peak absorption with a 570 nm filter. A standard curve was created by plotting the absorbance versus different concentrations of the standard solution expressed in μg/mL, bovine serum albumin

(BSA). Data for the calibration curve were obtained using 8 standard samples consisting of one part of a known concentration of (100 μL BSA standard) and 1 part of BCA Working Reagent

39 (100 μL) per well on a 96-well plate. The instructions from the assay kit were followed for the quantification. The blank consists of DI water. The standard curve was found to be linear over the range of 0−40 μg/mL. The total protein concentration is immediately observable by a color change of the sample from green to purple in proportion to the protein concentration. The absorbance was measured at a wavelength of 570 nm in a BioTek ELx808 absorbance microplate reader.111, 112 The original published protocol was modified for the quantitation of the amount of endothelin peptide attached to the CPNPs.

The assay relies on two chemical reactions: (1) the reduction of cupric ions Cu2+ to cuprous ions Cu1+ and (2) the chelation step of one Cu1+ with two BCA molecules, forming an intense purple BCA-Cu1+-BCA complex, the intensity of which can be measured spectrophotometrically near the maximum absorbance peak 562nm. The amount of complex formed is dependent upon the amount of EDN3 in solution to be quantified. The BCA−copper complex was performed on

96- well plates, which enabled the EDN3 content of a large number of samples to be measured simultaneously with the use of a microtiter plate reader. The BCA assay of our protocol consists of mixing 1 part of the solution to be quantified (14 µg/mL EDN3-CPNPs sample 150 μL) with 1 part of the prepared (BCA Working Reagent 150 μL) per well. The net absorbance at 570 nm is found by subtracting the absorbance (A570) of the blank from the recorded A570 values for the BSA protein standards and EDN3-CPNPs sample. A standard curve created by plotting the net absorbance at 570 nm versus the BSA protein standard different concentrations. The obtained absorbance is directly proportional to the amount of EDN3 in solution to be quantified. The protein concentration of the EDN3-CPNPs sample falls within the working linear range. The protein concentration of the sample was determined by comparison with the known protein standard 40 concentrations of BSA. All experiments were performed in triplicate, and 14 µg/mL of unmodified

CPNPs (without EDN3 on the surface) was applied as a control.

2.9 Total Quantitation of Iron Content in Nanoparticles

The iron content in CPNPs was determined using a ferrozine-based spectrophotometric iron estimation protocol. Ferrozine is as an effective chelator for ferrous iron, but not ferric iron, and assists in forming a Fe2+−ferrozine complex that absorbs strongly at 562 nm.113-116 The original published protocol was modified for the quantification of total iron in CPNPs. For this protocol,

EDN3-CPNPs (5 mL) were dispersed in 1.4 N hydrochloric acid (1 volume nanomaterial/mL) and kept for 2 h at 60 °C in a water bath sonicator to allow the mineralization and the complete dissolution of the EDN3-CPNPs carrying iron. The iron-detection reagent was prepared from 6.5 mM ferrozine, 6.5 mM neocuproine, and 1 M ascorbic acid (Figure 17) dissolved in acetate buffer at pH 4.5 and sonicated in a bath sonicator to accelerate the dissolution of the ferrozine and neocuproine. The resulting yellow-colored solution, 0.5 mL, was then added to 0.5 mL of the dissolved nanoparticle solution and the mixture was shaken continuously for 60 min at room temperature. Data for the calibration curve were obtained using 8 standard samples consisting of one part of a known concentration (FeCl3 standard 100 μL) with 1 part of the prepared Ferrozine

Working Reagent (100 μL) per well (the total volume loaded per well in a 96-well plate is 200 μL in triplicate). The instructions from the assay kit were followed for the quantification. The optical density of the sample was measured at 570 nm using a BioTek ELx808 absorbance microplate reader. The concentration of the iron was calculated by comparing this value with a calibration curve obtained by recording the absorbance of FeCl3 standard solutions in HCl 0.01 N (ranging

41 from 0 to 40 μg/mL). The standard curve was found to be linear over the range of 0−40 μg/mL.

HCl (0.01 N) with the iron-detection reagent was used as a blank.

ferrozine neocuproine ascorbic acid ferric chloride

Figure 17: Raw materials needed for Iron-detection reagent.

2.10 Observation of Cellular Uptake and Localization of Nanoparticles by TEM & STEM

Cells were treated with EDN3-CPNPs (1.4 µg/mL) cultured in 75 cm2 flasks for 24 h. The cells were then thoroughly washed with 5 mL of PBS (phosphate-buffered saline) three times

(purchased from Corning) to remove extracellular nanoparticles. Subsequently, cells were trypsinized with 5 mL of 0.25% trypsin and 2.21 mM EDTA, 1× (obtained from Corning), and incubated at 37 °C for 15 min. The cell suspension was thoroughly mixed (three times). The cells were then settled and collected into a soft pellet. The supernatant was removed using a micropipette without removing the settlement at the bottom of the tube. The pellet was washed with HBSS

(Hank’s Balanced Salt Solution) without phenol red (purchased from Lonza). To the settled cells, a small amount of 4% paraformaldehyde obtained from Electron Microscopy Sciences was applied for 15 min at room temperature. The cell suspension was thoroughly mixed. The fixed cells were then settled and washed with DPBS, Dulbecco’s phosphate-buffered saline (purchased from

Corning). Then, a small drop of the cell suspension (about 2−3 μL) was pipetted onto a TEM grid

42 and dried under vacuum. The TEM grids used were C-flat 1 μm 200 mesh (cat. no. CF21−50) and

300 mesh (cat. no. CF31−50) purchased from Electron Microscopy Sciences. The specimens were viewed using transmission electron microscopy [TEM] (JEOL TEM-1011) and scanning transmission electron microscopy [STEM] (Zeiss ULTRA-55 FEG SEM).

43 CHAPTER THREE: POLYMERIC NANOPARTICLES OF PTB7 FOR PHOTODYNAMIC THERAPY OF MELANOMA SKIN CANCER

3.1 Introduction

Photodynamic therapy or PDT offers an effective treatment to combat melanomas. PDT is a light-based therapy that uses a sensitizer that is applied to cells, then activated by light that releases toxic chemicals to cancer cells or tissues. Often these chemicals are reactive oxygen species (ROS) formed by the transfer of energy or an electron to oxygen, allowing for the generation of singlet oxygen or other ROS. When ROS reach sufficient concentrations, they induce oxidative stress that damages DNA and proteins, eventually leading to cell death, by necrosis or apoptosis.117-119

PDT sensitizers can be either cellular-targeted or non-targeted. Targeted PDT specifically affects malignant tumors with minimum effect on healthy cells. Patients receiving the non-targeted

PDT of Photofrin must avoid sunlight or bright indoor lights for 30 days or more, according to

FDA recommendation.120 This is attributed to the sensitivity of the skin to light through the residual drugs. It has been cited that the nonpolar nature of the Photofrin and many other photosensitizers most likely causes this persistence in the skin. 121 Light sensitivity could persist for a couple of weeks after treatment is completed according to a study of Japanese patients who received PDT.122 To improve patient survival and quality of life in the treatment of melanoma, a drug would quickly clear the body while present within targeted cancer cells. Doshi et al. demonstrated that water-stable folate – CPNPs are explicitly targeted to ovarian cancer cells while having minimal impact on other cell lines.123 The conducting polymers MEHPPV used to prepare

44 Folate-CPNPs to have a maximum absorbance wavelength near 500 nm. Typical skin and melanomas have a maximum absorbance wavelength in the range of 500-600 nm.124, 125 The overlap of maximum absorbance between the skin and Folate-CPNPs prevents the penetration of useful light. This may be a major limiting factor of Folate-CPNPs to melanoma PDT. However, conducting polymers thus have absorbance wavelength above 600 nm would be good candidates for use in PDT. A photoconductive polymer PTB7 with an absorbance wavelength in the 600-700 nm range makes it an attractive candidate for targeted PDT, where the absorbance of the skin begins to weaken.

In the work presented here, water-stable CPNPs composed of PTB7 and PS-PEO-COOH were prepared using the reprecipitation method. The obtained CPNPs were then functionalized with endothelin 3.

3.2 Results and Discussion

3.2.1 Size Measurements

Figure 18 shows the size measurements of PTB7/PS-PEO-COOH NPs were taken after each batch was created. Nanotechnology has shown promise in delivering anticancer drugs, reducing side effects, and enhancing efficiency. Many experiments indicated that a nanoparticle uptake via receptor-mediated endocytosis, highly depends on the size of the nanoparticles. 126-128

Most trials recorded an optimal size in the diameter of the nanoparticles, which is approximately

50 nm at which the highest uptake rate takes place through receptor-mediated endocytosis (RME).

However, there is also a maximum size reported, which is about 200 nm and beyond, at which the 45 cellular uptake does not occur. Our CPNPs showed an optimized size of around 60 nm in diameter.

PTB7 derived EDN3-CPNPs, on the other hand, were difficult to handle due to challenges with preparation and poor colloidal stability.

Figure 18: Observed z average size distribution for PTB7 NPs.

3.2.2 MTS Cell Viability Assay without Light

Malme-3M cells exposed to functionalized PTB7 nanoparticles and unfunctionalized

PTB7 nanoparticles show no significant difference in viability between the two populations, which in turn are both not different from the untreated controls. Functionalized nanoparticles were produced with a 2:1 PTB7 to PS-PEO-COOH molar ratio and the cells were grown in DMEM 46 (Figure 19). It should also be noted that while the Malme-3M cells also appeared to be less viable than the cells exposed to only the FBS media, this difference was not significantly different. It is possible that there was enough free endothelin-3 in the solutions of functionalized nanoparticles to increase the viability of cells that were nutrient-deprived such as those grown in DMEM through reduced apoptosis signaling and enhanced proliferation signaling. Conversely, the cells grown in

FBS would have had normal metabolism and thus could be more susceptible to increased oxidative stress induced by internalized nanoparticles.

In the dark, Malme-3M cells in DMEM were significantly more viable when exposed to

2:1 functionalized NPs than 2:1 unfunctionalized NPs. The cells treated with 2:1 unfunctionalized

NPs were significantly less viable than untreated cells, while Malme-3M cells treated with 2:1 functionalized NPs were not significantly different than untreated NPs. Malme-3M cells showed no significant difference in cell viability between NPs-treated and untreated cells, apart from the cells treated with 2:1 unfunctionalized NPs in DMEM.

When looking at the T24 cell line, it is observed that the cells exposed to 10:1 PTB7: PS-

PEO-COOH NPs in FBS were significantly less viable than the untreated cells. T24 cells exposed to 10:1 NPs were not significantly different than untreated cells, cells exposed to 2:1 NPs in either

DMEM or FBS similarly showed no significant difference in viability from untreated cells.

T24 showed no difference between untreated and treated cells when cells were treated with

2:1 NPs. When treated with 10:1 NPs, all cells treated with 10:1 unfunctionalized NPs were significantly less viable than untreated cells, while only cells treated with 10:1 functionalized NPs in FBS showed significantly reduced viability when compared to untreated cells in FBS.

47 It is possible that T24 cells are more sensitive to PTB7 NPs than Malme-3M cells, it is also possible that NPs with 10:1 PTB7 to PS-PEO-COOH ratios have lower surface charge than those with a 2:1 ratio making them easier to absorb non-specifically into cells.

Overall these findings indicate that the PTB7 nanoparticles are not cytotoxic when PDT is not being applied.

Functionalized MTS No Light Exposure Unfunctionalized Media Only 1.6

1.4

1.2

1.0

0.8

Absorbance 0.6

0.4

0.2

0.0 Malme Malme Malme Malme T-24 2:1 T-24 2:1 T-24 10:1 T-24 10:1 2:1 2:1 FBS 10:1 10:1 FBS DMEM FBS DMEM FBS DMEM DMEM Cell Line and Treatment

Figure 19: MTS data for cells with no light exposure.

48 3.2.3 MTS Cell Viability Assay with Light

For light treated samples, the sample treated with functionalized NPs in DMEM also showed no significant difference between the sample treated with unfunctionalized and functionalized NPs, and the respective controls (Figure 20).

Functionalized MTS 1 Hour of Light Exposure Unfunctionalized 2.0 Media Only

1.8

1.6

1.4

1.2

1.0

0.8 Absorbance

0.6

0.4

0.2

0.0 Malme Malme Malme Malme T-24 2:1 T-24 2:1 T-24 10:1 T-24 10:1 2:1 2:1 FBS 10:1 10:1 FBS DMEM FBS DMEM FBS DMEM DMEM Cell Line and Treatment

Figure 20: MTS results for cells with 1 hour of light exposure.

3.3 Conclusions

In this work, low band-gap poly ({4,8-bis [(2-ethylhexyl) oxy] benzo [1,2-b:4,5-b′] dithiophene-2,6-diyl}{3-fluoro-2-[(2-ethylhexyl) carbonyl] thieno [3,4-b] thiophenediyl}) (PTB7) was made into PTB7 nanoparticles by the reprecipitation method. Then the obtained conjugated 49 polymer nanoparticles (CPNPs) were functionalized with the polypeptide endothelin-3 to form

EDN3-CPNPs, which specifically target melanoma and bladder cells that have overexpression endothelin receptor type b on their cells surfaces towards PDT treatment. Taken together, the results indicate that our approach was not successful. No significant differences between controls and nanoparticle treated samples were found after PDT treatment, and no difference between PDT treatment with targeted and untargeted PTB7 nanoparticles was found. These findings are attributed to the PTB7 derived EDN3-CPNPs were difficult to handle due to challenges with preparation and poor colloidal stability.

50 CHAPTER FOUR: POLYMERIC NANOPARTICLES WITH ENCAPSULATED IRON FOR FERROPTOSIS ASSISTED CHEMODYNAMIC THERAPY OF MELANOMA SKIN CANCER

4.1 Introduction

In this chapter, we report the development and application of novel CPNPs for chemodynamic therapy (CDT). The CPNPs carry iron in the core to catalyze the Fenton reaction in tumor cells to perform CDT (Figure 21) and are tumor-targeted. The nanoparticle design allows for the change of the surface functional groups to target different tumors. The iron in the core of our nanoparticles is a serendipitous outcome of the synthetic process that is used to synthesize the polymer. Iron is a catalyst in the synthetic process and is not readily removed by purification; we, therefore, exploited this advantage in our CPNP-based tumor-targeted CDT. Conjugated polymer nanoparticles have received substantial attention for nanomedicine applications129-131, including light-activated ROS formation in photodynamic therapy (PDT)56, 123, 132-135, but have not been developed for CDT. In work reported herein, the CPNPs carry iron that acts as a catalyst in Fenton chemistry to produce ROS from hydrogen peroxide (H2O2) that is endogenous and elevated for many types of tumors.61, 62, 65 Unlike the PDT method that also produces ROS, ferroptosis-based

CDT does not require external activation by light. In addition, although ferroptosis proceeds through the formation of ROS, the ROS source is endogenous H2O2, not oxygen. Thus, the ferroptosis-based CDT mechanism is not expected to suffer dramatically reduced efficacy in hypoxic tumors.136 CDT is, therefore, a promising avenue for the development of a treatment of multidrug resistant (MDR) or PDT resistant melanomas, in particular for advanced stages (III-IV) of melanoma where conventional methods are not feasible or no longer effective.57, 81, 107, 110

51 Here we report tumor-targeted conjugated polymer nanoparticles prepared from

PCPDTBT (poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4- b′] dithiophene-2,6-diyl]]). The CPNPs are around 62 nm in size, tumor-targeted, and deliver iron

(as Fe3+) to the intracellular environment of tumor cells to trigger ferroptosis-based CDT followed by tumor cell death. For the case study of melanoma, we found that our endothelin-3 modified

CPNPs (EDN3- CPNPs) target melanoma tumor cells and resulted in effective CDT. The results of our work with EDN3-CPNPs show enhanced melanoma tumor targeting and tumor cell killing.

Entering the NPs into the nucleus

NPs localized in nucleus

NPs in the cytoplasm TEM Grid (1.0μm diameter holes) Damaged nucleuses Cells membrane

A375 CPNPs 100 ** EDN3-CPNPs

80 ** 60 ** **

Cell Viabilty Cell 40 % % 20

0 Control 0.014 0.14 1.4 14 Concentrations (mg/mL)

Figure 21: The mechanism of ROS formation proceeds through the iron-mediated Fenton reaction.

Low band gap conjugated copolymer (poly{[4,4-bis(2-ethylhexyl)-cyclopenta-(2,1-b;3,4- b′) dithiophen]-2,6-diyl-alt-(2,1,3-benzothiadiazole)−4,7-diyl}) (PCPDTBT) is based on the cyclopentadithiophene CPDT (donor) and benzothiadiazole BT (acceptor). The preparation of this polymer involves iron (III) chloride as a catalyst, as shown in Figures 22 and Figure 23, 137-140; we, therefore, expected the corresponding CPNPs to be rich in iron content. This warranted

52 investigation of their usefulness as a ferroptosis inducing nanomedicine for melanoma CDT as an alternative to PDT.

Figure 22: Synthesis of PCPDTBT monomers.

Figure 23: Synthesis of PCPDTBT polymers.

53 CPNPs of this PCPDTBT polymer without further surface modifications were prepared recently in aqueous solution using the reprecipitation method Figure 24. The nanoparticles exhibited an aqueous stable colloidal dispersion owing to its negatively charged surface (-30 mV) with a hydrodynamic diameter of approximately 75 nm. These CPNPs were shown to be internalized by macrophages.141

Figure 24: A schematic diagram showing the preparation of low-bandgap polymer-based CPdots via reprecipitation method and their UV−Vis absorption and emission spectra (reproduced from reference).141

4.2 Fabrication of CPNPs

The preparation of PCPDTBT polymer involves iron (III) chloride as a catalyst137, 139, 142,

143; we, therefore, expected the corresponding CPNPs to be rich in iron content. Difficulties with the removal of the iron (III) chloride catalyst from conjugated polymers has been a known issue in conjugated polymer plastic solar cells even after multiple purification steps, supporting the

54 reasonableness of our premise.144-148 This warranted investigation of their usefulness as a ferroptosis-inducing nanomedicine for antitumor CDT towards otherwise difficult to treat tumors.

We prepared PCPDTBT/PS-PEO-COOH composite nanoparticles using the reprecipitation method; a method described previously149 in which a mixture of PCPDTBT and PS-PEO-COOH polymers is dissolved in a good organic solvent (i.e., THF) and then injected rapidly into water

(poor solvent) under vigorous stirring ( Figure 25 and Figure 26A). The resulting nanoparticles have the characteristics of good water dispersibility, low cytotoxicity, and excellent stability in the aqueous medium – at least 6 months at 4 ⁰C (vide infra).

The CPNP was designed to consist of two components: a hydrophobic polymer core and a hydrophilic carboxyl corona for further bioconjugation. PS−PEO−COOH itself consists of a hydrophobic polystyrene backbone and hydrophilic side chains of ethylene oxide terminated with the carboxylic acid. We speculate that throughout the CPNPs formation, the functional groups

(−COOH) and hydrophilic PEO chains extend outside the CPNPs core into the aqueous surroundings, whereas the hydrophobic polystyrene backbones are embedded inside the

CPNPs.129, 150 The change in ζ potential to a more negative value supports this suggestion.

Characterization by UV−Vis and fluorescence spectroscopy (Figure 26B, Figure 27, Figure 28, and Figure 29), gives a clear indication that nanoparticles were formed. The absorption spectrum of molecularly dissolved PCPDTBT in THF as the solvent shows an absorption peak around 721 nm, while broadened peaks were seen for CPNPs and EDN3-CPNPs with 700 nm absorption maximum. The main absorption bands of molecular PCPDTBT are S0 → S1 (λ max = 721 nm) and

S0 → S2 (λ max = 412 nm). Both are observed in our UV−Vis spectra and correlate with previous reports.151, 152 In our work and other publications, the later transition seems less affected by 55 nanoparticle formation, while the first transition is significantly affected.153-155 The spectral broadening and blueshift of nanoparticles compared to molecularly dispersed PCPDTBT indicate aggregation of the polymer chains into nanoparticles, likely in the form of H-aggregates.153, 154, 156

The emission peak of molecularly dissolved PCPDTBT in THF was observed at 760 nm, while

CPNPs and EDN3-CPNPs were found with a significant redshift to 840 and 830 nm, respectively, indicating interchain π−π stacking and thus again suggesting nanoparticle formation.154, 156

Figure 25: Schematic illustration of the formation of conjugated polymer nanoparticles carrying iron (CPNPs and EDN3-CPNPs).

56

A

B 1.0 Abs. spectrum of PCPDTBT in THF 1.0 Abs. spectrum of CPNPs in DI water Abs. spectrum of EDN3-CPNPs in DI water Em. spectrum of EDN3-CPNPs in DI water Em. spectrum of PCPDTBT in THF 0.8 Em. spectrum of CPNPs in DI water 0.8

0.6 0.6

0.4 0.4

0.2 0.2

Normalized Absorption (a.u.) Absorption Normalized Normalized Emission (a.u.) Emission Normalized 0.0 0.0 400 600 800 Wavelength (nm)

Figure 26: (A) Schematic illustration of the reprecipitation method, a technique in which a solution of organic material in a good solvent was injected into a very poor solvent for the organic materials.(B) Normalized absorption (blue) and emission (red) spectra of the EDN3-CPNPs in water, CPNPs in water, and PCPDTBT in THF.

57 PCPDTBT 125000 Ex Em 100000

75000

50000

25000 Normalized Intensity (CPS)

0 400 600 800 1000

Wavelength (nm)

Figure 27: Photoluminescence excitation and emission spectroscopies of PCPDTBT in THF.

120000 CPNPs Ex Em 96000

72000

48000

24000 NormalizedIntensity (CPS)

0 400 600 800 1000

Wavelength (nm)

Figure 28: Photoluminescence excitation and emission spectroscopies of CPNPs in DI water. 58 110000 EDN3-CPNPs

88000 Ex Em

66000

44000

22000 Normalized Intensity (CPS) 0 400 600 800 1000

Wavelength (nm)

Figure 29: Photoluminescence excitation and emission spectroscopies of EDN3-CPNPs in DI water.

59 4.3 Bioconjugation of CPNPs

The choice of the EDN3-targeting ligand was motivated by the fact that endothelin receptor

B (EDNRB) is overexpressed in most human melanomas and is unique to diseased tissues. The

EDN axis is accepted to assume a central role in different malignancies, including melanoma, central nervous system tumors, prostate, ovarian, cervical and breast.14 Reports indicate that

EDNRB expression increases in human carcinoma melanoma cells as they progress to the metastatic disease state.22, 157 EDNRB is particularly engaging as a target for antibody−drug conjugates (ADCs) because of its negligible expression on healthy tissue, localization, and rapid endocytosis.158, 159 The receptors recognize their corresponding agonist through complementarity- determining region loops, specifically between the EDNRB binding pocket and the N-terminal amine of EDN3, which is deeply buried within the EDNRB pocket during the interaction (Figure

32 and Figure 33).25

60

Figure 30: Schematic representation of the interaction of endothelins with their receptors. The ligands interact with the receptor through its terminal free carboxylic end group, which is unaffected by our coupling chemistry. Our strategy for bioconjugation on the surface of CPNPs is based on a carbodiimide- mediated coupling reaction with EDC and NHS.129, 160 For this method, the EDC/NHS solution was added to the CPNPs suspension. The EDN3 peptide solution was then applied. Peptide bonds were formed between the free carboxylic end groups of CPNPs with the primary amine groups of endothelin 3, producing highly selective nanoparticles for targeting of malignant melanoma

(Figure 30 and Figure 31). We recognize the need for the N-terminal amine of EDN3 to be avoided during conjugation to nanoparticles to allow efficient binding between EDN3 and EDNRB. A weakness in our CPNP-EDN3 coupling approach is that the EDC/NHS approach (discussed below) indiscriminately couples free amines of EDN3 to the COOH groups on the nanoparticles.

As a result, it is probable that we obtain EDN3-CPNPs that are heterogeneously decorated. This implies that not every EDN3 that is attached to the nanoparticle surface will be effective at activating EDNRB. An alternative approach to be explored in future work is to use Lys7 or Lys9 as sites for CPNPs conjugation with EDN3, since these are away from the EDNRB binding site 61 and exposed to solvents. Such specific conjugation may be feasible by solid-phase peptide synthesis, potentially in combination with other conjugation techniques.

Figure 31: Mechanism of bioconjugation the surface of CPNPs by carbodiimide-mediated coupling reaction with EDC and NHS.

62

Figure 32: Targeting, cellular uptake of EDN3-CPNPs, and iron release using receptor-mediated endocytosis.

63

Figure 33: Schematic representation of the interactions of endothelins with EDNRA binds most strongly to EDN1 and EDN2 with a very minimum affinity towards EDN3, while EDNRB binds the EDN1, EDN2, and EDN3 with similar affinities. Amino acids sequence in proteins that are identical in both receptors (black circles), different in both receptors (blue circles), identical to each other (green circles), only in ETAR (purple circles), and only in ETBR (red circles).

64 We speculate that the internalization mechanism of EDN3-CPNPs into melanoma cells correlates with previous reports161-163 and involves the following steps : (I) Initially, EDN3-CPNPs internalizes into melanomas (A375, Malme-3M) and human bladder cancer cells (T24) using the

161 endocytosis pathway. The EDN3-CPNPS transport into the malignant cells via receptor- mediated endocytosis through overexpression of endothelin receptor-B (EDNRB) present on the

162 surface of these cells. (II) The EDN3-CPNPS then migrates by fusing cell membranes until they reach an early endosome form. (III) They travel along the increasingly acidic endocytic pathway

(pH range 6.0-5.0) and end up in late endosomes or lysosomes (pH < 5.0).163 (IV) During this process the EDN3-CPNPs release iron as Fe(III), which is converted to Fe(II) via the Haber–Weiss reaction. The Fe(II) then participates in the Fenton reaction to produce hydroxyl radicals (•OH)

• from H2O2 that is naturally and abundantly present in cancer cells. The resulting OH can rapidly oxidize bio-macromolecules, cause damage to DNA, and reduce tumor cell population. (Figure

32).

Conjugation of the peptide to CPNPs was analyzed by FTIR; data shown in Figure 34,

Figure 35, Figure 36, and Figure 37. FTIR spectra of the lyophilized CPNPs and EDN3-CPNPs show a broad and strong peak at 3400 cm-1 corresponding to the O–H stretch of hydrogen-bonded hydroxyls, while this peak is weak as compared with PS-PEO-COOH. The peak at 1090 cm-1 belonging to the C–O stretch of hydroxyls suggests the presence of PS-PEO-COOH. The FTIR spectra also exhibited that the three peaks at 2850-2958 cm−1 resulted from PCPDTBT attributed to C-H bond asymmetric and symmetric stretching vibration. Moreover, the peaks at 1550 and

1360 cm−1 are due to the asymmetric and symmetric deformation vibration of the C-H bond resulted from PCPDTBT, verifying that the CPNPs have been synthesized. Figure 37 shows peaks 65 −1 164-166 at 2360, and 2339 cm that are attributed to atmospheric CO2. New peaks were also observed in the 1750-2250 cm-1 range. These peaks can most likely be attributed to contributions from amino acids present in the EDN3 peptide, suggesting conjugation of endothelin-3 peptides to CPNPs.

Given the complexity of the amino acid mixture in EDN3 (21 amino acids), it is difficult to indicate to which transitions correspond to particular functional groups of EDN3. To the best of our

+ knowledge, the characteristic vibration band of the NH3 group of amino acids and the O-H bond in the acid group appear in the 3000-3500cm−1 range.167-169 The COO− asymmetric and symmetric stretching vibrations of carboxylic acid are observed at 1615 and 1410 cm−1, respectively.167-169

-1 168 −1 The band at 1337-1350 cm is attributed to the CH2 twisting mode. The peak at 2100 cm is attributed to the combination band, which is the result of two fundamental frequencies being excited simultaneously so that the excitation is allowed by symmetry.168, 169 The peak at 1750 cm-

1 is attributed to carbonyl groups present in amides.170 167

66

99.5 PCPDTBT

99.0

) %

( 98.5 98.0 97.5

97.0 C-H

Transmittance Transmittance 96.5 96.0 C-H 95.5 4000 3500 3000 2500 2000 1500 1000 Wavelength(cm-1)

Figure 34: FTIR spectra acquired for the raw materials PCPDTBT used in nanoparticle fabrication.

120 PS-PEO-COOH

) 100

% ( - 80 COO

60

40 Transmittance Transmittance

20 O–H C-H C–O

4000 3500 3000 2500 2000 1500 1000 -1 Wavelength(cm )

Figure 35: FTIR spectra acquired for the raw materials PS-PEO-COOH used in nanoparticle fabrication.

67

105 CPNPs

100 -

) COO %

( 95

90 85 80

Transmittance Transmittance 75 C-H

70 O–H C–O 65 4000 3500 3000 2500 2000 1500 1000 -1 Wavelength(cm ) Figure 36: FTIR spectra acquired for the obtained conjugated polymer nanoparticles CPNPs.

96 EDN3-CPNPs -

) O–H COO %

( 94

92

CO2 90

Transmittance Transmittance 88

86 C-H C–O

4000 3500 3000 2500 2000 1500 1000 -1 Wavelength(cm ) Figure 37: FTIR spectra acquired for the obtained conjugated polymer nanoparticles EDN3- CPNPs.

68 Ligand attachment to CPNPs was further confirmed and quantified by the Extra Sense BCA

Protein Assay.171 BSA is used as a standard for the BCA protein assay at the working range from

0 to 40 μg/mL (150 μL per well in triplicate). The sensitivity of the assay was improved by allowing the color to develop for 1−12 h at 37 °C, rather than the recommended period of up to 2 h. We avoided excessive incubation to prevent the absorbance from exceeding 1 to ensure we remain within the linear range of Beer’s Law. The absorbance was measured at the maximum absorbance (570 nm). We prepared a standard curve by plotting the average blank-corrected 570 nm reading for each BSA standard vs. its concentration in μg/mL (Figure 39). The protein concentration of the EDN3-CPNPs sample fell within the linear working range; hence, the protein concentration of samples was determined in the BCA protein assay by comparison with the standard curve.

Qualitatively, (Figure 38A) wells on the top right show the expected purple color due to the formation of EDN3/Cu2+ complex (as an intermediate that reacts with BCA), confirming our synthesized nanoparticles (14 µg/mL EDN3-CPNPs) contain EDN3 polypeptides. However, wells on the top right containing unmodified CPNPs (without EDN3 groups on CPNPs surface but

-COOH groups present) 14 µg/mL also show the purple color but displayed lower color intensity than the EDN3-CPNPs (Figure 38B). Thus, we next sought to evaluate the reason for the color formation of the unmodified CPNPs with the assay reagent. First, the BCA working reagent was applied to aqueous suspensions of uncoated PCPDTBT NPs (without any -COOH or EDN3 groups present on NPs surface) in the bottom right wells. The result did not show the purple color indicated above, while the wells are still green (color of PCPDTBT nanoparticles), which suggests that -

COOH groups on the surface of the coated CPNPs are probably involved in the color development. 69 Second, the BCA working reagent was applied to a small amount of the raw materials of

PCPDTBT and PS-PEO-COOH powders. The result show PCPDTBT polymer is not soluble in the aqueous BCA working reagent. However, PS-PEO-COOH showed purple color with the BCA working reagent immediately, further indicating that the -COOH group located on the surface of the non-functionalized CPNPs is involved in the color development with the BCA assay (Figure

40).

Quantitatively, due to these findings, we expect not all carboxylic groups on CPNPs are modified by endothelin 3. We, therefore, modified the quantitative analysis to determine the total amount of EDN3 attached to the CPNPs by subtracting the absorbance (A) obtained from CPNPs from the absorbance obtained from EDN3-CPNPs:

(퐴) EDN3 − CPNPs − (A) CPNPs = (A) Sample ( 2 )

The standard BSA absorbance curve of at a wavelength of 570 nm was used to determine the concentration of the endothelin peptide attached to the CPNPs through:

( ) ( ) A Sample− A Sample Blank × Standard Conc. ( 3 ) (A) Standard

From our data and equation 3, the surface conjugated EDN3 concentration for the 14

µg/mL EDN3-CPNPs sample was determined to be 7.65 µg/mL. We also determined the percent yield of EDN3 conjugation to the CPNPs surface by:

Percent yield = Actual yield × 100% ( 4 ) Theoretical yield

70 Knowing that we initially applied 10.00 µg/mL, the percent yield of EDN3 conjugation to the

CPNPs surface is 76.5 ± 1.41%.

A Final BSA concentration µg/mL BCA reagent working sol. applied to: EDN3-CPNPs -2 40 1.4×10 mg/mL

Stock sol. of PCPDTBT CPNPs -2 20 polymer in THF 1.4×10 mg/mL

BCA reagent working sol. applied to:

PCPDTBT NPs without PEOlation 10 (without PS-PEO-COOH on NPs surface)

BCA reagent working sol. applied to: 5 EDN3 (10 µg/mL)

EDN3-CPNPs 2.5 A 1.4×10-2 mg/mL

B CPNPs 1 1.4×10-2 mg/mL

PCPDTBT NPs without 0.5 C PEOlation (without PS-PEO-COOH on NPs surface)

BCA reagent H2O (blank) working sol.

B

Figure 38: (A) Schematic illustration of the treatment procedure for building a standardization curve for BCA assay of three different replicate experiments (B) A principal of the bicinchoninic acid (BCA) assay.

71 0.7 max = 562 nm 1 0.6

0.5 Absorbance (a.u) Visible absorption spectrum of EDN3 -CPNPs complex of BCA reagent

0 0.4 480 500 520 540 560 580 600 620 640 Wavelength (nm)

0.3 y = 0.0151x + 0.0402

O.D. 570 nm 570 O.D. R² = 0.9992 0.2

0.1

0.0 0 10 20 30 40 Concentration (µg/mL)

Figure 39: Absorbance plot obtained for bovine serum albumin (BSA) by using microplate procedure.

72

Figure 40: The proposed mechanism of the bicinchoninic acid (BCA) assay principle.

4.4 Determination of Particle Size, Polydispersity Index (PDI), and ζ-Potential (ZP)

We characterized the size and surface charge of the CPNPs by dynamic light scattering

(DLS), transmission electron microscopy (TEM), scanning transmission electron microscopy

(STEM), and ζ potential measurements. The average diameter and ζ potential of EDN3-CPNPs measured with DLS were around 62 nm and −54.5 mV, respectively (Figure 42A, C) and (Table1).

The EDN3-CPNPs size is monodisperse and ideal for cellular internalization.172 The average 73 diameter and ζ potential of CPNPs we prepared without further functionalization were about 58 nm and −34.2 mV (Figure 42C) and (Table1). A slight increase in the hydrodynamic diameter from 58 to 62 nm from CPNPs to EDN3-CPNPs may be attributed to the −COOH and −COEDN3 surface coatings. Our findings for CPNPs of PCPDTBT without further surface coating and further modifications are in line with previous findings. Our nanoparticles exhibited a negatively charged surface (−26.2 mV) with a hydrodynamic diameter of approximately 18.2 nm (Table 1), which is significantly smaller than the previously reported value of 75 nm with a negatively charged surface

(−30 mV) that was prepared in a similar manner.173 The difference in particle size is likely due to the difference in the ratio of injected volume versus water volume and control of injection speed, which are factors that strongly affect particle size (besides initial polymer concentration).

In addition, more vigorous stirring or sonication during injection also allows for smaller particle sizes. The average size of the EDN3-CPNPs as measured by TEM and STEM exhibits an average diameter at around 62 nm (Figure 43A-D) and (Table 1). More importantly, the EDN3-CPNPs exhibit a comparable average diameter of approximately 62 nm on DLS, TEM, and STEM. The images obtained by TEM and STEM indicate some aggregation, while DLS demonstrated monodisperse particles. This was likely caused by the fact that TEM measured the diameter of the

EDN3-CPNPs after they had been dried on the surface of the TEM grid, whereas DLS measured the diameter in aqueous solution.174-176 Transmission electron microscope (TEM) results confirmed the formation of the nanoparticles with the size around 62 nm. The zoomed-in TEM images suggest a polycrystalline structure.

74 Table 1: Characteristics of PCPDTBT NPs, CPNPs, and EDN3-CPNPs.

Importantly, EDN3-CPNPs demonstrated a substantial increase in negative surface charges compared to the non-functionalized CPNPs. This finding further confirms the presence of endothelin molecules on the nanoparticle surface since the −COOH group of EDN3 will add a negative surface charge. The high negative surface charge contributed to the stability of the suspension and prevented them from aggregation in aqueous solution, typically a value in excess of −30 mV indicates good stability (Figure 41).177-181 The value of the ζ potentials we measured suggest stability of the colloidal CPNPs and EDN3- CPNPs (CPNPs, −34.2 mV = moderate stability; EDN3- CPNPs, −54.5 mV = good stability), respectively.

75

Figure 41: Stability of suspensions with relation to zeta potential.

Indeed, EDN3-CPNPs and CPNPs showed high stability in DI water, for up to 6 months at

4 °C with the absence of aggregation and with consistent ζ potential (Figure 42B, D, E). TEM elemental analysis of EDN3-CPNPs (Figure 42F) confirmed that the nanoparticles contain iron (as

Fe3+), presumably as ferric chloride since the preparation of this polymer involves iron (III) chloride as a catalyst,137, 139, 142, 143.

76 Diameter of FNPs (Pdots) as a function of storage at room temperature (38.0 nm)

45 A 25 14 B 12 40 10 20 8 35 6 30 15 4

2 25 Intensity (%) 10 0 20

Number(%) 0 50 100 150 200 250

Size (nm) 15 Diameter (nm) Diameter 5 10

0 5 0 20 40 60 80 100 120 140 0 1 2 3 4 5 6 Size (nm) Time (month)

250000 -40 C D -35 200000 -30 EDN3-CPNPs CPNPs 150000 -25 -20

100000 -15 Total Counts Total

-10 Zeta potential (mv) 50000 -5

0 0 -150 -100 -50 0 50 100 150 0 1 2 3 4 5 6 Zeta Potential (mV) Time (month) -60 E F -50

-40

-30

Energy (KeV)

-20 Zetapotential (mv) -10

0 0 1 2 3 4 5 6 Time (month)

Figure 42: (A) Distribution of hydrodynamic diameters of the EDN3-CPNPs in DI water measured by DLS. (B) Diameters of EDN3-CPNPs stored at 4 ⁰C as a function of time, up to 6 months. (C) Zeta potential measurements of the CPNPs and EDN3-CPNPs in DI water (-34.7 mV and -54.6 mV nm). The zeta potential supports the well-dispersed nature of the nanoparticles. Time- dependent zeta potential measurements of the (D) CPNPs and (E) EDN3-CPNPs in DI water as stored at 4 ⁰C, for up to 6 months. (F) TEM elemental analysis was utilized to verify the presence of iron of the obtained EDN3-CPNPs.

77

A B

Entering the NPs into Malme-3M cells NPs in the cytoplasm the nucleus C D

NPs localized in nuclei

NPs in the cytoplasm NPs ̴ 60nm TEM Grid (1.0μm diameter holes) TEM Grid Damaged nuclei Cell membranes (1.0 µm diameter holes)

E F NPs in the cytoplasm Cell membrane

Damaged Nucleus NPs localized in nuclei nuclei Figure 43: (A) TEM images of EDN3-CPNPs (scale bar is 50 nm) exhibit polycrystalline structure and uncoated PCPDTBT nanoparticles (scale bar is 20 nm). (B) STEM image of EDN3-CPNPs, scale bar is 100 nm. (C) Observation of cellular uptake and localization of EDN3-CPNPs for A375 melanoma cell line (1.4 µg/mL internalized and damaged the nucleus), scale bar is 100 nm. (D) Localization of EDN3-CPNPs for Malem-3M melanoma cell line (1.4 µg/mL internalized and damaged the nucleus), scale bar is 100 nm. (E) Zoomed-in TEM image shows the nanoparticles localized inside the nuclei of A375 cells (scale bar is 100 nm). (F) Zoomed-in TEM image shows the nanoparticles localized inside the cytoplasm of Malme-3M cell only (scale bar is 100 nm). In this case the EDN3-CPNPs did not cause damage to the nucleus.

78

4.5 EDN3-CPNP as Anti-Tumor Cell Effect

Next, we assessed the CDT effectiveness of CPNPs and EDN3-CPNPs on a normal cell line and five different types of cancer cell lines. We studied three different types of melanoma skin cancer cell lines (A375, Malme-3M, and SK-Mel-28), two different nonmelanoma cell lines (Hepa

1−6 and T24), and one healthy cell line (AML12). These were treated with different doses (14,

1.4, 0.14, and 0.014 µg/mL) of CPNPs and EDN3-CPNPs, and cell viability was tested by MTS assay. The results are shown in 44A-F. We observed that cell killing was higher with targeted

EDN3-CPNPs for A375 and Malme-3M compared to the untargeted CPNPs. These melanoma cells are strongly positive for endothelin receptor B.182 Moreover, for these cell lines, we observed a significant increase in cell death relative to other cell lines (SK-Mel-28, T24, Hepa 1−6, and

AML12). These results suggest effective targeting of EDN3-CPNPs to tumor cells strongly overexpressing EDNRB with dose-dependent tumor cell killing.107, 183 The data in Figure 44A, B suggest that the CDT performance in terms of absolute tumor cell killing with targeted EDN3-

CPNPs is slightly better for A375 compared to Malme-3M, probably because A375 exhibits a somewhat higher expression of the endothelin receptor B on its cell surface compared to Malme-

3M.24, 182, 184 The specificity of the EDN3-CPNPs appears to be better for Malme-3M, however, as suggested by the larger difference in cell viability between treatment with targeted and untargeted

CPNPs.

The human bladder cancer cell (T24) is moderately positive for endothelin receptor B compared to A375 and Malme-3M.185, 186 We observed that for T24, the cellular viability at 14

µg/mL of EDN3-CPNPs demonstrated a decrease in viability to 45% after 24 h. Unlike for A375

79 and Malme-3M, there is no significant difference between targeted and untargeted CPNPs at lower doses. The CDT efficiency is much lower for T24 than for A375 and Malme-3M though. The first result is well explained by the lower EDNRB expression levels of T24. We can only speculate at this time that the lack of differentiation between targeted and untargeted CPNPs in terms of CDT effectiveness is due to the properties of T24, possible metabolic rate, or nonspecific internalization pathways. Nonspecific CPNPs uptake across the studied cell lines is also suggested to be possible due to attractive interaction of the anionic CPNPs (ζ potential −34.7 mV) with the relatively positively charged EDNRB.187, 188 SK-Mel-28 has limited overexpression of EDNRB compared to the tumor cells discussed above.182, 189 Little difference with the normal control (AML12) and non-

EDNRB overexpressing tumor cells was observed for SK-Mel-28 except for the highest applied dose, where a modest CDT response is evident. Hepa 1−6 and AML12 showed no significant difference between EDN3-CPNPs and CPNPs with limited loss of cell viability, reflecting their lack of endothelin receptors.13 We observed that viability decreased similarly to 80% for both cell lines after 24 h for the highest applied dose. Production of minimal amounts of hydrogen peroxide by healthy human cells of AML12 relative to tumor cells in our study is another possible reason for the absence of CDT effectiveness in addition to the lack of EDNRB on the cell surface.62

Morphology changes in TEM images further indicate that CDT occurred in EDN3-CPNPs treated cells, resulting in cell damage. The TEM and STEM images in Figure 43C-F show nanoparticles are localized around and inside the nucleus for A375, and in the cytoplasm for

Malme-3M.190-193 The nanoparticle size determined from these data is approximately 62 nm in agreement with DLS, TEM, and STEM data (Figure 43A, B) and (Table 1). From TEM, we can further observe that, for example, the 1.4 µg/mL dose of EDN3-CPNPs internalized and damaged

80 the nucleus of A375 cells (Figure 43C). A TEM control experiment (Figure 45 A, B) on untreated cells shows that such damage does not occur in the absence of EDN3-CPNPs, indicating that the observed effects are due to CDT. The pristine nuclei appear with dark contrast in the TEM images shown in Figure 45 A, B as expected. We also investigated the EDN3 targeting effect for the nanoparticles to the melanoma cell lines. TEM images in Figure 45 C, D indicate that fewer nanoparticles are present compared to the melanoma-targeted EDN3-CPNPs, although damage to the nucleus is still apparent. These observations are consistent with the data presented in Figure

44.

81 A375 CPNPs * Malme-3M CPNPs A 100 EDN3-CPNPs B 100 EDN3-CPNPs ** **

80 80 ** ** 60 ** 60 **

** Cell Viabilty Cell

40 Viabilty Cell 40

% % % 20 20

0 0 Control 0.014 0.14 1.4 14 Control 0.014 0.14 1.4 14 Concentrations (mg/mL) Concentrations (mg/mL)

SK-Mel-28 CPNPs Hepa 1-6 CPNPs C 100 EDN3-CPNPs D 100 EDN3-CPNPs

80 * 80

60 60 Cell Viabilty Cell

40 Viabilty Cell 40

% % 20 20

0 0 Control 0.014 0.14 1.4 14 Control 0.014 0.14 1.4 14 Concentrations (mg/mL) Concentrations (mg/mL)

T24 CPNPs AML12 CPNPs E 100 EDN3-CPNPs F 100 EDN3-CPNPs

80 * 80 *

60 60 Cell Viabilty Cell

Cell Viabilty Cell 40 40

% % % % 20 20

0 0 Control 0.014 0.14 1.4 14 Control 0.014 0.14 1.4 14 Concentrations (mg/mL) Concentrations (mg/mL)

Figure 44: In vitro cell cytotoxicity as measured by MTS assay upon different doses of CPNPs (gray column) and EDN3-CPNPs (black column) in the cell culture medium for a period of incubation of 24 h at 37 °C of (A) A375, (B) Malme-3M, (C) SK-Mel-28; (A−C), (D) Hepa 1−6, (E) T24; (D, E) as nonmelanoma cell lines, and (F) AML12 as a healthy cell line. Error bars represent the quality deviation of the mean ± s.d. (n ≥ 3). A P-value < 0.05 was considered statistically significant and nonsignificant for P > 0.05. *P < 0.05, and **P < 0.01.

82

A B

C D

E F

≤ 24 hrs

Figure 45: TEM control experiment on untreated cells. (A) A375 and (B) Malme-3M.TEM experiment on treated cells with CPNPs. (C) Malme-3M and (D) A375 cells. (E) and (F) The optical microscope images of A375 cell line incubated with 14 µg/mL EDN3-CPNPs a period of incubation of 24 h in the dark in 75 cm2 cell culture flask.

83

4.6 Ferroptosis as The Tumor Cell Killing Mechanism

To confirm the role of iron in the EDN3-CPNPs-mediated CDT, it is crucial to quantify the iron content in the sample. A ferrozine based assay was used in determining the iron concentration of EDN3-CPNPs based on the absorbance of the ferrozine−Fe2+ complex, the intensity of which can be measured spectrophotometrically at 570 nm. One of the main advantages of the ferrozine assay is that several samples can be measured at once. To estimate the iron concentration of the EDN3-CPNPs suspension, a standard curve of absorbance at 570 nm as a function of Fe3+ concentration was created using 8 known iron standard samples at the linear working range from 0 to 40 μg/mL (Figure 47). FeCl3 standard samples were dissolved in 0.01 N

HCl, followed by the addition of the ferrozine-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine and 1 M ascorbic acid dissolved in acetate buffer at pH 4.5) to form complexes. On the other hand, EDN3-CPNPs suspension of 14 µg/mL was dissolved in 1.4 N hydrochloric acid and kept for 2 h at 60 °C in a water bath sonicator to allow the mineralization and the complete dissolution of the EDN3-CPNPs carrying iron, followed by the addition of an equal volume of the ferrozine detection reagent to form complexes. In contrast, the blank consists of HCl 0.01 N and an iron-detection reagent with no iron. The Fe2+−ferrozine complex was performed on 96-well plates, and the absorbance was measured at 570 nm on a microplate reader (200 μL per well in triplicate). The net absorbance at 570 nm is found by subtracting the absorbance (A570) of the blank from the recorded A570 values for the FeCl3 standard samples and EDN3-CPNPs sample. The iron content of the EDN3-CPNPs sample was then determined by comparing its absorbance to that of a range of standard concentrations of equal volume that had been prepared similar to the EDN3-

84 CPNPs solution. The iron concentration was calculated to be 5.2 µg/mL = 0.033mM for 14 µg/mL

EDN3-CPNPs suspension.

It is noteworthy that the presence of iron (III) is an interfering agent in the analysis of the ferrozine assay. Therefore, ascorbic acid is added as a reducing agent to ensure an entire reduction of the iron is in the divalent. Ascorbic acid reduces the ferric to ferrous ions, which react with the chelator ferrozine to form a deeply colored purple complex with an absorption maximum near 562 nm, allowing for quantification. Next, for the experiments of the microplate, we applied the iron- detection reagent without ascorbic acid to 14 µg/mL EDN3-CPNPs to determine if the formation of the purple complex is possible. The result did not show the typical color, but instead, it gave us a nutty color, confirming this assay works only in the presence of ferrous ions. Thus, the assay depends on the presence of ascorbic acid (Figure 46A, B). This result corroborates that our EDN3-

CPNPs contain ferric ions as ferric chloride because the preparation of this polymer involves iron

(III) chloride as a catalyst,137, 139, 142, 143.

85

Final FeCl3 concentration µg/mL A EDN3-CPNPs 40 1.4×10-2

Iron detection reagent applied to 20 EDN3-CPNPs 1.4× 10-2 mg/mL

Iron detection reagent without ascorbic acid 10 applied to EDN3-CPNPs 1.4× 10-2 mg/mL

5

2.5

1

0.5

Blank HCl 0.01 N with iron-reagent

B

Figure 46: (A) Schematic illustration of the treatment procedure for building a standard curve for the ferrozine assay of three different replicate experiments. The inset picture shows a yellow colored iron-chelating reagent which was stored in the dark at room temperature. (B) Chemical tool for the colorimetric detection of iron ions and the complex formed with ferrozine.

86 3.5 0.6

0.5 max = 562 nm

3.0 0.4

0.3

2.5 0.2 Visible absorption

O.D. 570 nm spectrum of ferrous 0.1 complex of ferrozine 0.0 2.0 500 520 540 560 580 600 620 640 Wavelength (nm)

1.5

y = 0.0759x + 0.0675 O.D. 570 nm 570 O.D. 1.0 R² = 0.9998

0.5

0.0 0 10 20 30 40 Concentration (µg/mL)

Figure 47: Calibration curve obtained for FeCl3.

By contrast, the ferrozine assay in the presence of the ascorbic acid takes on a purple/fuchsia color tone, indicating Fe3+ is present. Besides iron, copper is the only metal that could react with ferrozine to form a colored complex.194 Therefore, neocuproine is also present in the color reagent to prevent copper interference. To investigate whether the ferrozine-based quantitation of iron can be perturbed by a copper divalent cation, an experiment was conducted to determine the intensity of the purple complex formed by applying the iron-detection reagent without neocuproine to 14 µg/mL EDN3-CPNPs. The result did not show any noticeable changes in color intensity and did not alter the absorbance of the Fe2+−ferrozine complex at 570 nm, indicating that copper ions do not exist to interfere with iron quantification. A stable Fe2+−ferrozine complex that forms in aqueous solution between the pH values of 4 and 9 was previously 87 reported194, as such careful sample preparation is crucial. Our results demonstrated the EDN3-

CPNPs contains the iron needed to kill cancer cells via the ferroptosis mechanism. Finally, the resulting CPNPs showed another area of study. It showed reasonable sensitivity towards copper ions. It might represent a promising sensor for copper ions in alkaline environments. This lead to the hypothesis that, in the present copper ions, not only lead to the specific cross-linked formation of the purple complex that integrate copper ions with the protein on the surface of the CPNPs but also lead to the creation of the purple complex with the carboxyl terminated units on the surface of the non-functionalized CPNPs. This task was accomplished by blending CPNPs with

BioVision’s Extra Sense™ BCA Protein Assay Kit.

88 CHAPTER FIVE: CONCLUSIONS

In summary of this dissertation research program, the resulting CPNPs obtained from PTB7 conjugated polymer-functionalized polypeptide endothelin-3 (human, rat) fabricated by a reprecipitation method was investigated. The results demonstrated that there was no significant tumor targeting and treating via photodynamic therapy in vitro. On the other hand, a novel conjugated polymer CPNP that enables tumor-targeted chemodynamic therapy (CDT) is reported.

The CPNP delivers iron to the targeted tumor cells to enable ferroptosis through Fenton chemistry that produces reactive oxygen species (ROS). No activation by external stimuli is needed for ROS production. Our CDT platform was tested using melanoma as the case study. The melanoma- targeted EDN3-CPNPs demonstrated significant CDT with respect to A375 and Malme-3M melanoma. Herein we showed that the melanoma endothelin axis was successfully targeted through the overexpressed EDNRB receptor on the surface of these cancer cells. In comparison, no apparent CDT effect was observed for the healthy cell line AML12 and the non-EDNRB receptor overexpressing tumor Hepa 1−6 except at higher doses. We expect that, due to the flexible design of the CPNP, other tumors can be targeted by exchanging the targeting ligand. Our studies reported herein thus lay the groundwork for a promising new class of nanomedicines. Therefore, it will be useful to explore combined chemodynamic and photodynamic therapy for treating melanoma. Moreover, it is critical to determine whether this finding could have consequences on tumor growth in vivo.

89 APPENDIX A: COPYRIGHT PERMISSION

Figure 48: TEM image of blank grid.

90

Figure 49: TEM elemental analysis was utilized to verify the presence of iron of the obtained EDN3-CPNPs.

Figure 50: TEM elemental analysis was utilized to verify the presence of iron of the raw material of PCPDTBT.

91

Figure 51: TEM elemental analysis was utilized to verify the presence of iron of the obtained CPNPs.

92

APPENDIX B: COPYRIGHT PERMISSION

93

94

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