New polymeric nanoparticles to interrupt the ros and rns derived misregulation of cells

New polymeric nanoparticles
to interrupt the ROS- and RNSderived
misregulation of cells

Van Nam DAO
BPharm. (Hons), MPharmSc.

A thesis submitted for the degree of Doctor of Philosophy at


Monash University in 2021
Faculty of Pharmacy and Pharmaceutical Sciences

New polymeric nanoparticles
to interrupt the ROS- and RNS- derived
misregulation of cells

Van Nam DAO
BPharm. (Hons), MPharmSc.

Supervisors:

Dr. John Quinn
Dr. Michael Whittaker
A/ Prof. Erica Sloan

Panel members:

Dr. Angus Johnston
Dr. Betty Exintaris
Dr. Daniel Poole

A thesis submitted for the degree of Doctor of Philosophy at
Monash University in 2021


Faculty of Pharmacy and Pharmaceutical Sciences

Copyright notice
© Van Nam DAO (2021).
I certify that I have made all reasonable efforts to secure copyright permissions for thirdparty content included in this thesis and have not knowingly added copyright content to my
work without the owner's permission.


Abstract
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have critical roles in various
biological processes and signaling pathways. Moreover, perturbations in ROS and RNS levels often
occur with changes in metabolism and health disorders. The application of scavengers, which are
capable of reducing ROS and RNS levels, could potentially protect cells from being damaged by
these reactive molecules. Further, manipulating ROS and RNS levels can also interrupt downstream
communication between impaired cells and other surrounding tissue, as seen in the tumour
microenvironment (TME) wherein cancer cells use these molecules to communicate with supporting
cells. The delivery of H2S and persulfide compounds that mimic cellular defense pathways and offer
inherent ROS scavenging activities is another potential approach to alleviate ROS levels. Further,
the recent development of nanomedicine has enabled researchers to address issues associated with
small molecular drugs, such as nonspecific binding, low tissue permeation and short retention time.
In this thesis, polymer-based scavengers for ROS and RNS have been developed which i) exploit
advanced polymer design, ii) contain novel reactive moieties, iii) scavenge ROS and/or RNS
intracellularly, and iv) confer downstream effects in the biological milieu. Firstly, in Chapter 1, the
background of ROS and RNS production is discussed, including how these species are regulated in
biological systems. Recent advances in development of hydrogen sulfide and persulfide donors, as

well as macromolecular ROS/RNS scavengers, are also described. Subsequently, in a series of
experimental chapters, data are presented about four main categories of materials that have been
developed: linear and brush polymers bearing trisulfide moieties for releasing hydrogen sulfide and
persulfides (Chapter 2 and Chapter 3), polymer carriers for delivering N-acetyl L-cysteine
intracellularly (Chapter 4), star polymers for conjugating a small molecule antioxidant (TEMPO)
(Chapter 5) and nitric oxide scavenging polymers (Chapter 6). The synthesised compounds were
characterised using a range of analytical techniques, including H 2S-specific amperometry. Further,
the impact of the synthesised materials on cell viability, and their biological performance, i.e. ROS RNS scavenging ability, were evaluated on several models, including a naturally elevated ROS level
model which can simulate TME communication (a co-culture of fibroblasts and breast cancer cells,
Chapters 2, 4 and 5), an externally stimulated oxidative stress model (HEK293 treated with H2O2,
Chapter 3), and a naturally elevated RNS model (activated primary and cell line macrophages,


Chapter 6). Where appropriate, other intracellular behaviour of the synthesised compounds was
evaluated, including cell association, co-localisation with subcellular organelles and intracellular H2S
release. Certain key secondary effects associated with polymer treatment were also examined,
including ROS-associated cellular changes (collagen-1 and F-actin expression, Chapter 2), NOlinked phagocytosis activity (Chapter 6), or mitochondrial functions (superoxide anion production and
co-localisation, Chapter 5). The materials developed in the thesis represent novel entities for
manipulating cellular signaling pathways, and are potentially applicable to diverse fields including the
pharmaceutical, polymer and material sciences, as well as oncology and pharmacology.


Publications during enrolment
1. Urquhart MC, Dao NV, Ercole F, Boyd BJ, Davis TP, Whittaker MR, et al. Polymers with
dithiobenzoate end groups constitutively release hydrogen sulfide upon exposure to cysteine and
homocysteine. ACS Macro Lett. 2020;9(4):553-7.

2. Dao NV, Ercole F, Kaminskas LM, Davis TP, Sloan EK, Whittaker MR, et al. Trisulfide-bearing
PEG brush polymers donate hydrogen sulfide and ameliorate cellular oxidative stress.
Biomacromolecules. 2020: 21(12):5292-305.

3. Dao NV, Ercole F, Urquhart MC, Kaminskas LM, Nowell CJ, Davis TP, et al. Trisulfide linked
cholesteryl PEG conjugate attenuates intracellular ROS and collagen-1 production in a breast cancer
co-culture model. Biomaterials Science. 2021. DOI: 10.1039/D0BM01544J


Thesis including published works declaration
I hereby declare that this thesis contains no material which has been accepted for the award of any
other degree or diploma at any university or equivalent institution and that, to the best of my
knowledge and belief, this thesis contains no material previously published or written by another
person, except where due reference is made in the text of the thesis.

This thesis includes 02 original papers published in peer reviewed journals and 0 submitted
publications. The core theme of the thesis is the development of antioxidant polymers and
nanomedicine. The ideas, development and writing up of all the papers in the thesis were the
principal responsibility of myself, the student, working within the Drug Delivery, Disposition and
Dynamics Theme, Monash Institute of Pharmaceutical Sciences under the supervision of Dr John
Quinn, Dr Michael Whittaker and Associate Professor Erica Sloan.

The inclusion of co-authors reflects the fact that the work came from active collaboration between
researchers and acknowledges input into team-based research.

In the case of Chapter 2 and 3, my contribution to the work involved the following:

(If this is a laboratory-based discipline, a paragraph outlining the assistance given during the
experiments, the nature of the experiments and an attribution to the contributors could follow.)


Thesis
Chapter

Publication
Title

Status

Nature and
%
of
student
contribution

the polymers and revise manuscripts.

cholesteryl

80%.

PEG conjugate

design,

intracellular
and

Published

collagen-1

cancer

co-culture

C.

Urquhart

helped

J.

Nowell

provided

collecting

expertise in imaging and analysis and

data

revised manuscript.

and

Yes

synthesise the polymers.
3) Cameron

analysis, and

production in a
breast

2) Matthew

Research

attenuates

ROS

Coauthor(s),
Monash
student

1) Francesca Ercole helped synthesise

Trisulfide linked

2

Co-author name(s), Nature and % of
Co-author’s contribution*

4) Thomas P. Davis, Lisa M. Kaminskas,

preparing

Erica K. Sloan, Michael R. Whittaker

manuscript

and John F. Quinn formulated the idea
of the project, provided supervision, and

model

had input into manuscript preparation.
1) Francesca Ercole provided expertise

Trisulfidebearing

PEG

brush polymers

hydrogen
sulfide

Published
and

ameliorate
cellular
oxidative stress

on synthesis and amperometric sensing

Research

of H2S and revised manuscript.

design,

donate
3

80%.

2) Thomas P. Davis, Lisa M. Kaminskas,

collecting

Erica K. Sloan formulated the idea of the

data

project and revise manuscript.

and

analysis, and

No

3) Michael R. Whittaker and John F.

preparing

Quinn formulated the idea of the project,

manuscript

provided supervision, and had input into
manuscript preparation.

I have renumbered sections of published papers in order to generate a consistent presentation within
the thesis.
Student name:

Van Nam Dao

Date:

22nd April 2021

I hereby certify that the above declaration correctly reflects the nature and extent of the student’s
and co-authors’ contributions to this work. In instances where I am not the responsible author I have
consulted with the responsible author to agree on the respective contributions of the authors.
Main Supervisor name:

Dr John F. Quinn

Date:

22nd April 2021.

Acknowledgements
Doing a PhD is truly a journey, and to get to the end of the journey with this thesis in hand, I have
received enormous support from all the people around me.
First and foremost, I would like to acknowledge Dr John Quinn and Dr Michael Whittaker for being
my main supervisors, who have deep knowledge of not only polymer chemistry but also other
disciplines, ranging from organic chemistry to analytical chemistry and biology. They have spent a
great amount of time and effort in supervising me, and have given me valuable advice whenever I
am in trouble. Thanks for always encouraging me to explore new knowledge without hesitation. I also
would like to thank Associate Professor Erica Sloan for the supervision and useful advice she gave
me while I was working with cancer models. Erica also gave me the opportunity to think critically
when preparing manuscripts for publication.
I would not have been able to finish my PhD without the great support of Dr Francesca Ercole.
Fran has provided me with amazing expertise in hydrogen sulfide, sulfur and polymer chemistry. She
also taught me chemistry techniques that were essential for my PhD and advised me in my personal
life. I would like to express my deepest appreciation to Fran.
I would like to acknowledge Dr Lisa Kaminskas for her advice during the project and for raising
many important points in preparing the manuscript for the co-culture work, Mr Cameron Nowell for
the impressive expertise in imaging and analysis, and Dr Jason Dang for always being nice and
providing great assistance in analysing NMR data and HRMS data without any hesitation. I also want
to acknowledge Dr Jibriil Ibrahim for the assistance in doing lung alveolar macrophage experiments
and Dr Yuhuan Li for guiding me with the very first steps in cell experiments.
I would like to thank my Milestone Review Panel, Dr Angus Johnston, Dr Betty Exintaris and Dr
Daniel Poole for the inputs and advice during my entire PhD. At CBNS and MIPS, I was also assisted
by Professor Thomas Davis, the CBNS centre and D4 Theme. Dr Nghia Truong, Dr Asuntha
Munasinghe and Ms Karen Drakatos were really helpful during my PhD enrolment. Thank you all for
creating an excellent working and research environment.
I also want to acknowledge the Funding from the Vietnamese Government (in the form of VIED
scholarship), the Faculty Sponsorship from MIPS, the support from the Department of Physical
Chemistry and Physics, Hanoi University of Pharmacy and from the Ministry of Education and

Training, Vietnam. Without the funding and support, I would have not been able to pursue my PhD
at Monash University.
Friends are the people who are willing to help whenever I seek assistance, and I would like to
express my acknowledgement to Aadarash for being a very nice neighbour, who also helped me with
many aspects of my life. Thank you Matt, Alex, Ken, John F for all the social chats, lab work support
and your generosity and kindness. I also want to thank Scott for advising me in imaging and your
kindness in lending me chemicals. You all have made my PhD journey better than I expected. I also
got help from all the members in level 4, the Nanomedine team and the CBNS administration team,
May, Aykut, James, Kristian, Meike, Ayaat, Cheng, Erny, Paulina, Jeffiri, Stefan, Nikos, Ximo, Adrian,
Jason W, Loshini, Anne, Sam, Natalie, Charlotte, Moore, Daniel, Rob, Joanne, Carlos, Emily,
Stephanie, Xiaotong, Inin, Song, Elly, Lars, Kwan, Nil.
Mrs Oanh Nguyen and Mr Cuong Nguyen are my closest friends in Australia, who aided me when
I first arrived in Melbourne and always welcome me as a family member. I really appreciate their
kindness and assistance. It had been good experience in sharing house with Gerard, Artur, Yoni and
Lauren, who made my life much easier and meaningful. Thank you Mai Vu, my colleague in both
Vietnam and Australia and other Vietnamese friends, for your great support and encouragement.
Finally and most importantly, I would like to acknowledge my Mother and my Father, who have
always stayed beside me and supported me with their love. My great-grandmother, grandparents,
sister and all members in my beloved family are also so important to me, thanks for their care and
support!

Nam Dao.


Abbreviations
1H, 13C, 19F-NMR

Proton, Carbon and Fluorine nuclear magnetic resonance

6PGD

6-Phosphogluconate dehydrogenase

AIBN

2,2′-Azobis(2-methylpropionitrile)

Akt

Protein kinase B

ALI

Acute lung injury

ALT

Alanine aminotransferase

APUEMA

2-(3-(2-Aminophenyl) ureido)ethyl methacrylate

ARE

Antioxidant response element

Arg

Arginase

ARPE

Human retinal pigmented epithelial cells

AST

Aspartate aminotransferase

BAS

Bovine serum albumin

BH4

Tetrahydrobiopterin

BJ-5ta

Human foreskin fibroblasts

BMDMs

Bone marrow-derived macrophages

C

mPEG-Cholesterol

CAT

Catalase

CBS

Cystathionine β-synthase

CCL

Chemokine (C‐C motif) ligand

CGMP

Cyclic guanosine monophosphate

CHO

Chinese hamster ovary

COSY

Correlation Spectroscopy

CSE

Cystathionine γ-lyase

CSF-1

Colony stimulating factor-1

CXCL

Chemokine (C-X-C motif) ligand

CYS

Cysteine

D

mPEG-SS-Cholesterol

DADS

Diallyl disulfide

DAS

Diallyl sulfide

DATS

Diallyl trisulfide

DCM

Dichloromethane

DLS

Dynamic light scattering

DMEM

Dulbecco’s Modified Eagle’s Medium - high glucose

DMEM-NP

Dulbecco’s Modified Eagle’s Medium - high glucose, HEPES, no phenol red


DMF

Dimethyl formamide

DMSO

Dimethyl sulfoxide

DPBS

Dulbecco’s phosphate buffer saline

EA

Ethyl acetate

ECM

Extracellular matrix

EDP-NAC

Ester disulfide-prodrug N-acetyl cysteine

EGF-R

Epidermal growth factor receptor

eNOS, NOS3

Endothelial nitric oxide synthase

ER

The endoplasmic reticulum system

ETC

Electron transfer chain

FBS

Fetal bovine serum

FCS

Fetal calf serum

Fe-NTA

Ferric nitrilotriacetate

FI

Fluorescence intensity

FMN

Flavin mononucleotide

G6PD

Glucose-6-phosphate dehydrogenase

GDH

Glucose dehydrogenase

GPC

Gel permeation chromatography

GPX

Glutathione peroxidase

GR

Glutathione reductase

GRXo

Glutaredoxin (oxidised)

GRXr

Glutaredoxin (reduced)

GSH

Gluthathione

GSHr

Glutathione (reduced)

H9C2

Cardiomyocytes

HAEC

Human aortic endothelial cells

HASMC

Human aortic smooth muscle cells

HCT116

Human colon carcinoma cells

HEK

Human embryonic kidney

HEX

n-Hexane

HIF-α

Hypoxia-inducible factor

HL60

Human promyelocytic leukemic cell line

HMBC

Heteronuclear multiple bond correlation

HO

Heme oxygenase

HRMS

High resolution mass spectrometry


HSC

Hepatic stellate cells

HSP90

Heat shock protein

HSQC

Heteronuclear single quantum coherence spectroscopy

HUVEC

Human umbilical vein endothelial cells

I/R

Ischemia-reperfusion

Ic

Immune complexes

ICU

Intensive care unit

IDO

Indoleamine 2,3-dioxygenase

IFN-γ

Interferon-γ

IL

Interleukin

INF/AAR

Infarct size per area at risk

iNOS, NOS2

Inducible nitric oxide synthase

IRF

Interferon regulatory factor

J774

Mus musculus ascites reticulum cell line

KYNU

Kynureninase

L-NAME

N-ω-nitro-L-arginine methyl ester hydrochloride

LOX

Lysyl oxidase

LPS

Lipopolysaccharide

LV

Left ventricular

MAPK

Mitogen-activated protein kinase

MCF-10

Normal epithelial cells

MCF-7, MDA-MB231

Carcinogenic breast cell lines

MESPEGMA, M2

{2-[S-(2-(2-methoxyethoxy)ethyl)-trisulfanyl)]-ethoxy}-penta(ethylene glycol)
methacrylate

MFI

Mean fluorescence intensity

MI/R

Myocardial ischemia-reperfusion

MIP-2

Macrophage-inflammatory protein 2

MMPs

Matrix metalloproteinases

MPEGESMA, M1

2-[S-(2-(methoxy(penta(ethylene glycol)))ethyl)trisulfanyl]ethyl methacrylate

MPP+

1-Methyl-4-phenylpyridinium ion

mtNOS

Mitochondrial NOS

NAC

N-acetyl-L-cysteine

NACA

N-acetyl-L-cysteine amide

NADH

Nicotinamide adenine dinucleotide

NADPH

Nicotinamide adenine dinucleotide phosphate


NAM

N-(2-aminophenyl)-8-mercaptooctanamide

NAPMA

N-(2-aminophenyl) methacrylamide hydrochloride

NFIL

Nuclear factor interleukin regulated

NF-kB

Nuclear factor-κB

NIPAM

N-isopropylacrylamide

nNOS, NOS1

Neuronal NOS

NOS

Nitric oxide synthase

NOX

NADPH oxidase

Nrf2

Nuclear factor-E2-related factor

NSAIDs

Nonsteroidal anti-inflammatory drugs

P

mPEG-SSS-mPEG

PAECs

Pulmonary artery endothelial cells

PAVSMCs

Pig pulmonary artery vascular smooth muscle cells

PDGF-βR

Platelet-derived growth factor-β receptor

PEG

Polyethylene glycol

pen/strep

Penicillin/streptomycin

PFP

Pentafluorophenyl

PtAuNRs

Platinum-coated gold nanorods

PTP1B

Protein tyrosine phosphatase 1B

RAFT

Reversible addition-fragmentation chain-transfer

RAW 264.7

Macrophage cell line

RET

Reverse electron transport

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

RPMI

Roswell Park Memorial Institute

RT

Room temperature

SATO

S-aroylthiooxime

SBNO

Strawberry notch homologue factor

SEM

Standard error of the mean

SNAP

S-nitroso-N-acetylpenicillamine

SNP

Sodium nitroprusside

SOCS

Suppressor of cytokine signalling

SOD

Superoxide dismutase

STAT

Signal transducer and activator of transcription

T

mPEG-SSS-Cholesterol


TA1

Trisulfide alcohol 1 : Me(OCH2CH2)6SSS(CH2CH2O)H

TA2

Trisulfide alcohol 2: Me(OCH2CH2)2SSS(CH2CH2O)6H

TBHP

tert-Butyl hydroperoxide

TEA

Triethylamine

TFK

Trifluoromethyl ketone

TGF-β1

Transforming growth factor β1

THF

Tetrahydrofuran

TLC

Thin layer chromatography

TLRs

Toll-like receptors

TME

Tumour microenvironment

TNF-α

Tumor necrosis factor-α

TOCSY

Total Correlation Spectroscopy

TRXo

Thioredoxin (oxidised)

TRXr

Thioredoxin (reduced)

TTF

Tetrathiafuvalene

Tyr

Tyrosine

U2OS

Human osteosarcoma cell line

VEGF

Vascular endothelial growth factor

XDH

Xanthine dehydrogenase

XO

Xanthine oxidase

XOD

Xanthine oxidoreductase

α-SMA

α-smooth muscle actin


Table of Contents
Chapter 1. Literature review

1

1.1. CELLULAR REACTIVE OXYGEN SPECIES AND REACTIVE NITROGEN SPECIES:
MOLECULAR PERSPECTIVES 2
1.1.1. ROS and RNS in biological systems ......................................................................................... 2
1.1.2. Interplay between ROS and RNS ............................................................................................. 4
1.1.3. ROS formation inside the cells .................................................................................................. 5
1.1.3.1. Superoxide anion O2-......................................................................................................... 5
1.1.3.2. Other ROS formation ......................................................................................................11
1.1.4. Nitric oxide and RNS production .............................................................................................12
1.1.5. RNS to regulate ROS generation ............................................................................................14
1.1.5.1. RNS to induce ROS generation ......................................................................................14
1.1.5.2. NO can reduce ROS levels .............................................................................................20
1.1.6. ROS-RNS in cellular redox state .............................................................................................23
1.1.6.1. Redox state: components and regulation .......................................................................23

1.1.6.2. Redox state in cell cycle and functions ...........................................................................25
1.1.6.3. Changes of redox state in oxidative stress .....................................................................26
1.2. RECENT ADVANCES IN PREPARING HYDROGEN SULFIDE AND PERSULFIDE DONORS
AS CYTOPROTECTIVE AGENTS

29

1.2.1. H2S and persulfide introduction to alleviate oxidative stress, inflammation and apoptosis ....30
1.2.1.1. ROS alleviating effects ....................................................................................................30
1.2.1.2. Anti-inflammatory behaviour ...........................................................................................32
1.2.1.3. Anti-apoptotic ..................................................................................................................34
1.2.2. Recent advances in the application of hydrogen sulfide and persulfide donors as cytoprotective
agents...........................................................................................................................................35
1.2.2.1. Donors to alleviate oxidative stress and rescue cell viability ..........................................35
1.2.2.2. Hydrogen sulfide and persulfide donating molecules to alleviate injuries ......................40
1.2.2.3. H2S and persulfide donors as anti-cancer agents ..........................................................45
1.2.2.4. Other recently developed H2S and persulfide donors .....................................................47
1.2.3. Preparation of trisulfide donors ...............................................................................................49
1.2.3.1. Sulfanyl chloride ..............................................................................................................49
1.2.3.3. Using protecting group ....................................................................................................53
1.2.3.4. Thiol exchange ................................................................................................................55
1.2.3.5. Other methods ................................................................................................................55
1.3. MACROMOLECULAR ROS - RNS SCAVENGERS 56


1.3.1. ROS scavengers .....................................................................................................................56
1.3.1.1. ROS responsive carrier ...................................................................................................56
1.3.1.2. Antioxidant loaded nanoparticles ....................................................................................58
1.3.1.3. Combination therapy .......................................................................................................59
1.3.1.4. Inorganic based particles ................................................................................................59

1.3.2. RNS scavengers .....................................................................................................................60
1.3.2.1. NO scavengers ...............................................................................................................60
1.3.2.2. ONOO- scavengers .........................................................................................................62
1.4. HYPOTHESES AND AIMS 64
1.4.1. Hypotheses .............................................................................................................................64
1.4.2. Aims ........................................................................................................................................64
REFERENCES 65
Chapter 2. Trisulfide linked cholesteryl PEG conjugate attenuates intracellular ROS and
collagen-1 production in a breast cancer co-culture model
2.1. INTRODUCTION

82

2.2. EXPERIMENTAL

84

81

2.2.1. Materials ..................................................................................................................................84
2.2.2. Methods ...................................................................................................................................85
2.2.2.1. Cell cultre ........................................................................................................................85
2.2.2.2. Co-culture of fibroblasts and breast cancer cells ............................................................85
2.2.2.3. Cell viability assay ...........................................................................................................85
2.2.2.4. ROS generation detection ...............................................................................................86
2.2.2.5. Immunofluorescence .......................................................................................................86
2.2.2.6. Imaging and analysis ......................................................................................................86
2.3. RESULTS AND DISCUSSION

87

2.3.1. Trisulfide H2S-Donors - Structures, H2S donating ability and synthetic approaches ..............87
2.3.2. Antioxidant activities of H2S releasing polymers in an elevated ROS co-culture model .........90
2.3.2.1. Co-culture conditions promote cellular alteration in normal fibroblasts and cancer
cells ...........................................................................................................................................90
2.3.2.2. Co-culture conditions induce changes in ROS production .............................................92
2.3.2.3. ROS scavenging effects of trisulfide donors T and P .....................................................94
2.3.3. Associated cellular alterations when ROS are mitigated ........................................................98
2.4. CONCLUSION

101

REFERENCES ...........................................................................................................................102
Chapter 3. Trisulfide-bearing PEG brush polymers donate hydrogen sulfide and ameliorate
cellular oxidative stress

107


3.1. INTRODUCTION

108

3.2. EXPERIMENTAL

110

3.2.1. Materials ................................................................................................................................110
3.2.2. Methods .................................................................................................................................111
3.2.2.1. Synthetic Methods.........................................................................................................111

3.2.2.2. Synthesis of POEGMA950-co-PMPEGESMA and POEGMA950-co-PMESPEGMA .......113
3.2.2.3. Measurement of thiol triggering H2S release using an amperometric H2S sensor .......114
3.2.2.4. Characterisation ............................................................................................................114
3.2.2.5. Cell culture ....................................................................................................................115
3.2.2.6. Cell viability ...................................................................................................................115
3.2.2.7. Intracellular H2S releasing tests ....................................................................................116
3.2.2.8. Intracellular ROS measurement ....................................................................................116
3.3. RESULTS AND DISCUSSION

117

3.3.1. Synthesis of asymmetrical trisulfide functionalised monomers .............................................117
3.3.2. Synthesis of PEG brush polymers decorated with trisulfide moieties ...................................119
3.3.3. H2S releasing tests of synthesised polymers using amperometry technique .......................123
3.3.4. Cell viability ...........................................................................................................................126
3.3.5. Intracellular H2S release........................................................................................................129
3.3.6. ROS ameliorating effects of H2S donating macromolecular donors .....................................130
3.4. CONCLUSION

133

REFERENCES 134
Chapter 4. Delivery of N-acetyl-L-cysteine (NAC) via macromolecular approaches for
optimised antioxidant activity 138
4.1. INTRODUCTION

139

4.2. EXPERIMENTAL

140

4.2.1. Materials ................................................................................................................................140
4.2.2. Methods .................................................................................................................................141
4.2.2.1. Synthetic methods.........................................................................................................141
4.2.2.2. Characterisation ............................................................................................................146
4.2.2.3. Cell culture ....................................................................................................................147
4.3. RESULTS AND DISCUSSION

148

4.3.1. Delivery of NAC via disulfide based conjugation ..................................................................148
4.3.2. Delivery of NAC using a trisulfide linkage .............................................................................150
4.3.2.1. Synthesis of NAC trisulfide monomer using esterification chemistry ............................152
4.3.2.2. Synthesis of NAC trisulfide monomer using isocyanate chemistry ...............................152


4.3.2.3. Introducing NAC trisulfide to polymer chain via post-polymerisation modification .......153
4.3.2.4. Synthesis of phthalimide disulfide monomer ................................................................155
4.3.3. Co-delivery of NAC and trisulfide moieties ...........................................................................157
4.3.3.1. Synthesis of NAC disulfide monomer using acryloyl chloride .......................................157
4.3.3.2. Synthesis of NAC disulfide monomer using isocyanate chemistry ...............................159
4.4. CONCLUSION

161

REFERENCES 165
Chapter 5. TEMPO-conjugated star polymers: old compound, new applications
5.1. INTRODUCTION

167

5.2. EXPERIMENTAL

170

166

5.2.1. Materials ................................................................................................................................170
5.2.2. Methods .................................................................................................................................171
5.2.2.1. Synthesis of POEGA arm .............................................................................................171
5.2.2.2. Synthesis of pentafluorophenyl stars (PFP stars) .........................................................171
5.2.2.3. Conjugating TEMPO to star polymer ............................................................................172
5.2.2.4. Labelling star polymers with Cy5 ..................................................................................173
5.2.2.5. Characterisation ............................................................................................................173
5.2.2.6. Cell culture ....................................................................................................................174
5.3. RESULTS AND DISCUSSION

177

5.3.1. Synthesis of TEMPO conjugated star polymers ...................................................................177
5.3.1.1. Synthesis of PFP star polymers ....................................................................................177
5.3.1.2. Modification of stars with ROS scavenging moiety .......................................................179
5.3.2. Cytotoxicity ............................................................................................................................181
5.3.3. ROS scavenging effects of star polymers .............................................................................185
5.3.4. Cell association assay ...........................................................................................................188
5.3.5. TEMPO-conjugated stars as recycled antioxidants ..............................................................192
5.3.5.1. Reduced TEMPO stars to attenuate total ROS levels of cells ......................................192
5.3.5.2. Mitochondrial superoxide anion assay ..........................................................................193
5.3.6. Mitochondrial co-localisation of star polymers ......................................................................195

5.4. CONCLUSION

199

REFERENCES 200
Chapter 6. Nitric oxide scavenging polymers drive changes in the physiological activity of
cells

203

6.1. INTRODUCTION

204

6.1.1. Macrophages and macrophage polarisation .........................................................................204
6.1.2. Phagocytosis in macrophages ..............................................................................................206


6.1.3. Nitric oxide to regulate macrophage in inflammatory associated functions ..........................209
6.1.3.1. NO is elevated in activated macrophages ....................................................................209
6.1.3.2. Nitric oxide and cytokines .............................................................................................210
6.1.3.3. Nitric oxide and macrophage phagocytosis ..................................................................212
6.2. EXPERIMENTAL

213

6.2.1. Materials ................................................................................................................................213
6.2.2. Methods .................................................................................................................................214
6.2.2.1. Synthesis of nitric oxide reactive monomer ..................................................................214
6.2.2.2. Polymer synthesis .........................................................................................................215

6.2.2.3. Treatment of synthesised materials with reactive species............................................215
6.2.2.4. Characterisation ............................................................................................................217
6.2.2.5. Cell cultures and biology assays ...................................................................................218
6.3. RESULTS AND DISCUSSION

220

6.3.1. APUEMA synthesis ...............................................................................................................220
6.3.2. Polymer synthesis .................................................................................................................221
6.3.3. Treatment of polymers with reactive species ........................................................................222
6.3.3.1. Nitric oxide reacting capability ......................................................................................222
6.3.3.2. Nitric oxide scavenging selectivity over other species ..................................................225
6.3.4. Polymeric nitric oxide scavengers for interrupting nitric oxide-associated cellular functions 227
6.3.4.1. Cytotoxicity ....................................................................................................................227
6.3.4.2. Ability in reducing nitric oxide generation......................................................................228
6.3.4.3. Scavengers to interfere macrophage phagocytosis ......................................................229
6.4. CONCLUSION

236

REFERENCES 237
Chapter 7. Conclusions
APPENDIX

240

245

Appendix A - Supporting information for Chapter 2.
Appendix B - Supporting information for Chapter 3.

Appendix C - Supporting information for Chapter 5.


Chapter 1

Literature review

This chapter summarises the key pathways by which reactive oxygen species (ROS) and reactive
nitrogen species (RNS) are produced and regulated in the biological milieu, as well as the cross-talk
between them. The chapter also discusses the key role of the cellular redox state in controlling
cellular functions, and the different mechanisms that maintain redox homeostasis. Recent advances
in the development of ROS- and RNS-scavengers are introduced, with particular focus on
macromolecular- hydrogen sulfide and persulfide donors as a new molecular vehicle for potentially
arresting dysregulated reactive oxygen species.

1


1.1. CELLULAR REACTIVE OXYGEN SPECIES AND REACTIVE NITROGEN SPECIES:
MOLECULAR PERSPECTIVES
1.1.1. ROS and RNS in biological systems
Reactive oxygen species (ROS) are molecules comprised of oxygen that can readily react with other
compounds. ROS can be either radicals containing unpaired electrons (HO ., HO2., O2-.), or nonradicals which are chemically reactive and can be converted to radical ROS (such as H2O2 or O3).1
ROS have critical roles in various biological processes. For instance, ROS are involved in host
defense and an array of signaling cascades in post-translational processing of proteins and gene
expression, with further impacts on cellular processes, such as differentiation, proliferation,
migration, or response to growth factor stimulation.2, 3 Similarly, reactive nitrogen species (RNS) are
reactive compounds containing nitrogen, such as nitric oxide (NO) and peroxynitrite (ONOO -). NO
can react with ROS to form various other RNS species. 4, 5 NO, ONOO- and other RNS are involved
in regulation of the cardiovascular and nervous systems,6 and also present as signal transducers

and protein modification agents (via nitration, nitrosylation etc.) in various biological processes.
These processes can include gene expression, iron homeostasis, ROS production, and cellular
defense against pathogens at higher concentrations.4, 7-9
Due to their high reactivity, ROS and RNS can participate in a wide range of biological reactions,
with potentially deleterious effects on biomolecules. To limit such damaging effects, a natural
scavenging system is utilised to balance with the ROS - RNS producing machinery (mitochondria,
NADPH oxidases, nitric oxide synthase…) and maintain the basal levels of ROS and RNS (Figure
1.1). Superoxide dismutase, catalase, heme oxygenase, glutathione peroxidase, glutathione, vitamin
C, and vitamin E10-13 are some of the antioxidants which can be found intracellularly in different
compartments, as well as extracellularly in plasma. Failure to maintain the balance between the two
systems is a potential reason for abnormal cellular function, and the genesis or progression of health
disorders. Significantly, high concentrations of the reactive species often occur with changes in
metabolism and are linked to a variety of diseases, including hypertension, neurodegenerative
diseases, cancer, and diabetes.2

2


Figure 1.1. Schematic illustration of cellular redox homeostasis. Mito-ETC: mitochondrial transport chain; ER:
the endoplasmic reticulum system; NOX: NADPH (nicotinamide adenine dinucleotide phosphate) oxidase;
NOS: nitric oxide synthase; GPX: glutathione peroxidase; GR: glutathione reductase; GRXo: glutaredoxin
(oxidised); GRXr, glutaredoxin (reduced); GSHr, glutathione (reduced); GSSG, glutathione (oxidised); TRXo,
thioredoxin (oxidised); TRXr, thioredoxin (reduced); XO, xanthine oxidase. Reprinted by permission from
Springer Nature, Nature Reviews Drug Discovery,1 © Copyright 2009.

For instance, markedly lower levels of antioxidants and upregulated ROS concentrations have
been observed in cancer patients compared to healthy controls. 14, 15 Increased levels of NO have
been associated with autoimmune diseases, and also with the processes in transplant rejection and
septic shock.9 In general, excessive levels of ROS and RNS result in oxidative and nitrosative stress
which can damage cellular machinery and cause impaired cellular functions.4, 16 These changes

include an increased frequency of causative reactions such as hydroxylation, lipid peroxidation, DNA
strand breakage (oxidative stress), and nitration, nitrosylation, and DNA deamination (nitrosative
stress). Significantly, the impact is not limited to misregulated cells, but can also spread to the
surrounding healthy counterparts, in which ROS and RNS contribute to global communication. In
particular, cancer cells can regulate the functions of accompanying fibroblasts and force them to
express a cancer phenotype by producing ROS and RNS, an effect known as “field cancerisation”.17

3


As such, downregulation of reactive species levels to ameliorate oxidative and nitrosative stress can
potentially normalise cellular activities and generate therapeutic benefits. However, understanding
the processes of ROS - RNS formation and regulation is of paramount importance in order to design
and apply external scavengers: this will be the main topic of the next sections. Further, the specific
roles of ROS and RNS in some particular conditions will also be described. Specifically, in chapter
2, we will discuss the role of ROS in cancer communication, and in chapter 6, we will present data
about nitric oxide in macrophage regulation.
1.1.2. Interplay between ROS and RNS
Due to their shared high chemical reactivity and ability to modulate cellular signaling pathways, NO
and ROS can exert complementary effects, as has been reported for macrophages, 18 where cells
require high levels for pathogen recognition and digestion. In some situations, ROS and RNS can
stimulate counteracting regulation. For example, while nitric oxide inhibits nuclear factor-kB (NF-kB)
activation by inducing its inhibitor expression and activity, ROS can activate IkB kinase leading to
activation of NF-kB.19 ROS and RNS can also share regulators, as seen with Rac, one protein
component of some NOX isoforms, which has the ability to regulate both NO and O 2-. functions.20
Additionally, NO and ROS can regulate each other in vivo.21, 22 Expression of NOX5 has been
shown to increase eNOS (endothelial nitric oxide synthase) activity and NO release from both bovine
and human endothelial cells.23 Superoxide is also able to stimulate iNOS (inducible nitric oxide
synthase) expression and favour NO production,24 and thus ROS improves NO function. Nitric oxide,
on the other hand, can induce ROS production by interfering with antioxidant enzymes,25 or reduce

ROS production by changing gene expression of ROS producing systems. 26 NO is further known to
exhibit “self-regulating” activity. For instance, when macrophages sense pathogens, NO generated
by eNOS (at basal levels) can activate iNOS expression to boost nitric oxide generation.27 There is
also a feed-forward mechanism for maintaining NO function: endothelial NO stimulates extracellular
SOD (superoxide dismutase) expression in adjacent smooth muscle cells, thus reducing O 2-., and
preventing peroxynitrite formation with deleterious effects. 28 ROS, on the other hand, can stimulate
their production by utilising different ROS producing machinery. ROS escaping from mitochondria
can activate NOX2 and NOX1, facilitating the uncoupling of eNOS and additional ROS generation. 29
Further, the upregulation of certain isoforms of NADPH oxidase in the early stages of vascular

4


disease also resulted in uncoupling of eNOS, as well as oxidative conversion of xanthine
dehydrogenase into xanthine oxidase, facilitating additional production of ROS. 30
The complicated regulatory mechanisms of ROS and RNS in the biological milieu also impact
treatment strategies. For instance, applying classical antioxidants such as ascorbic acid or vitamin
E, either separately or in combination, failed to manage ROS levels in cardiovascular disease, while
using statins with the ability to generate nitric oxide effectively downregulate ROS levels. The effects
originate from the inhibitory impacts of nitric oxide on the NOX systems and superoxide anion
production.26 However, in cancer, nitric oxide is known to regulate mitochondrial dysfunction, and
plays a key role in oxidative spread and “field cancerisation”,17 suggesting potential applications of
both ROS and RNS scavengers in cancer therapy.
As such, understanding the interplay between ROS and NO is crucial to achieving therapeutic
outcomes and better drug design. In the next section, we will focus on the production systems for
ROS and RNS, and some modulating effects of nitric oxide on ROS generation, to provide a closer
view of how ROS levels change upon nitric oxide manipulation.
1.1.3. ROS formation inside the cells
Intracellular ROS are produced mainly in mitochondria and by NADPH oxidases (NOXs). Other
possible sources include enzymes such as xanthine oxidase, cyclooxygenase, lipoxygenase, 23, 31

and uncoupled nitric oxide synthase (NOS).32 From these sources, O2-. is envisaged to be the first
oxidant produced, which is further converted to secondary oxidants, such as H 2O2 or HO..33-35
Consequently, the following section will focus on superoxide anion generation inside the cells with
less attention to other reactive oxygen species.
1.1.3.1. Superoxide anion O2-.
Superoxide anion is produced by the one electron reduction of oxygen. The reduction potential Eh of
this reaction can be written as:

𝐸ℎ = −160 + 61.5 × 𝑙𝑜𝑔

[𝑂2 ]
[𝑂2−. ]

and is estimated to fall in the range of 150-230 mV, indicating the thermodynamically favoured
formation of O2-. in vivo.33, 36 The rate constant of the reaction is recorded as 103 M-1s-1 inferring a

5