Lavado, Andrea Sofia Caetano das Neves

337 Pages·2017·26.63 MB·English

by  Christine Middleton

Porphyrinic-nanoplatforms: controlled
intracellular generation of reactive
oxygen species in human mesenchymal
stem cells
Andrea Sofia Caetano das Neves Lavado MSc
Thesis submitted to the University of
Nottingham for the degree of Doctor of
Philosophy
July, 2014
Dedication
I dedicate this thesis to my beloved parents
Frederico and Alzira
With all my heart
Abstract
Reactive Oxygen Species (ROS) are known as important intracellular signaling
molecules. These are also well known for their role in oxidative stress and
cellular damage, leading to their involvement in several pathologies. Despite the
widespread postulation of ROS mechanisms, little is actually known about the
immediate response in living cells to the generation of these highly reactive com-
pounds. The development of nanoplatforms incorporating photosensitizers would
permit the generation of ROS at specific sub-cellular locations and determine the
in situ cellular response.
The work presented in this thesis describes the development of porphyrinic
nanoplatforms for the controlled generation of ROS and investigates their impact
on the surface marker expression of human Mesenchymal Stem Cells (hMSCs).
Surface tailoring of polyacrylamide nanoparticles with alkyne and amine func-
tionalities were exploited to achieve stable reactive chemical groups for further
conjugation. Nanoplatforms surface was also modulated with trimethylam-
monium functionalities for the development of nanosystems for sub-cellular
targeting and facilitated uptake. Physicochemical characterisation of alkyne and
alkyne/trimethylammonium functionalised constructs showed sizes in the range of
40 nm with a positive surface charge. Alkyne/trimethylammonium nanosystems
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were found to be stable over long periods of time, whilst amino functionalised
nanosystems were found to be prone to aggregation.
Mechanisms of conjugation were exploited to create covalent linkage of por-
phyrinic photosensitizers to mono and dually functionalised constructs. Con-
jugation through ”click chemistry” allowed stable coupling with alkyne and
alkyne/trimethylammonium nanosystems. To overcome aggregation associated
with amino functionalised nanoplatforms, porphyrin conjugated monomers were
synthesised which resulted in stable polyacrylamide nanoparticles. The developed
conjugated nanosystems showed final sizes in the range 40-100 nm, while conjug-
ates with surface charges greater than + 20 mV have led to sizes higher than 100
nm.
The effect of surface charge on cellular delivery was investigated and nanosys-
tems with a surface charge in the range + 13 mV to + 18 mV proved optimal in
terms of cell delivery and viability. It was found that highly charged nanosystems
(above + 20 mV) remained attached to the cellular membrane and had a negative
effect on cell viability. In addition, intracellular co-localisation studies showed
preferential mitochondrial targeting of the delivered nanosystems.
Production of ROS in nanoparticle treated hMSCs was achieved by exposure
to light at wavelength of 575 nm. For porphyrin conjugated nanosystems a single
light dosage resulted in a ”blast zone” in the irradiated area where significant
production of hydrogen peroxide was also observed. Titration of the amount of
porphyrin conjugated at the surface of nanoparticles resulted in systems with dif-
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ferent levels of ROS production. Control of ROS generation allowed development
of a nanoplatform that was used to expose cells to repeated exposure of ROS over
a time period of 100 minutes.
The surface marker expression of hMSCs treated with porphyrin conjugated
nanosystems was investigated. In the absence of light the surface marker ex-
pression of hMSCs was maintained, positive for CD29 and CD105 and negative for
CD34 and CD45. Increased generation ROS in hMSCs did not produce alterations
in the surface marker expression of cells, and over two generations of treated cells
(light and nanoparticles) no changes were detected in surface marker expression.
The developed nanoplatforms have the potential to be applied as a tool to in-
vestigate the cellular mechanisms and metabolism associated with different levels
of oxidative stress. In addition, these nanosystems could also represent an innov-
ative platform for theranostic applications (drug delivery/diagnostic).
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Acknowledgements
I would like to thank both of my supervisors Dr Jonathan Aylott and Dr Weng
Chan for their guidance and support throughout this thesis.
I would like to acknowledge the financial support from the EPSRC.
I would like to express my gratitude to Dr Rhodri Jones for his patience, held and
guidance. I would like to thank to my collaboration, Dr Francesca Giuntini, Dr
Ross W Boyle, Dr Andrew Beeby and Miss Gery Rosser.
I would like to thanks to Amer Alhaj Zen for his friendship and collaboration in
this work. Also thank you to Peter Magennis for his help.
Thanks also to my friends and colleagues in D38 - Veeren Chauhan, Erin Wik-
antyasning, Arran Basra, Arpan Desai, Robby Pineda, Leo Marques, Leigh-Anne
Carroll, Rosie Adsley, for all their help and suggestions, and also for making our
lab a truly great place in which to work. Thanks for the golden hours and the
golden moments!
Thanks to all my friends and colleagues from CBS, Glen Kirkham, Fabio Rui,
Adam Taylor, Gavin Morris, Stephanie Strohbucker, Sivaneswary Genapathy,
Robert Hampson for all their help.
Thankyou, toPaulCoolingandChristineGrainger-Boultbyfortheincrediblehelp
and patience throughout these four years. Also thank you to Emma King and Ian
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Ward for all their help with the microscopy. Also thank you to all my colleagues
and friends from Boots Science Building.
A very special thank you to my friends Ivan Lafayette, Daniela Alves and Andrea
Goncalves, for their friendship, patience and encouragement. To my dear friend
Susana Moleirinho thanks for all the encouragement and friendship.
A very special thank you to my dearest parents and best friends in the whole
world, Frederico e Alzira for the incredible support, love and guidance you have
always given me. I could not have asked for more supportive parents, and would
never have reached this point without your unwavering confidence in me.
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Contents
List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
List of Figures xi
List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx
List of Tables xx
List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1 General Introduction 1
1.1 Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Photosensitizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Photosensitizers Characteristics . . . . . . . . . . . . . . . . 3
1.2.2 Photosensitized Reactions . . . . . . . . . . . . . . . . . . . 5
1.2.2.1 Photosensitization Type I Reactions . . . . . . . . 6
1.2.2.2 Photosensitization Type II Reactions . . . . . . . . 8
1.3 Photosensitizers Applications . . . . . . . . . . . . . . . . . . . . . 10
1.3.1 Photodynamic therapy . . . . . . . . . . . . . . . . . . . . . 11
1.4 Porphyrins as photosensitizers . . . . . . . . . . . . . . . . . . . . . 13
1.4.1 Generations of porphyrinic based photosensitizers . . . . . . 16
1.4.2 Effects of photosensitizers. . . . . . . . . . . . . . . . . . . . 18
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1.5 Nanotechnology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.5.1 Polyacrylamide nanoparticles . . . . . . . . . . . . . . . . . 20
1.5.2 Polyacrylamide nanoparticles . . . . . . . . . . . . . . . . . 20
1.5.2.1 Polyacrylamide nanoparticles and photosensitizers . 21
1.6 Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1.7 Thesis Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2 Instrumentation materials and methods 31
2.1 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.1 Size Characterization of nanoparticles. . . . . . . . . . . . . 31
2.1.1.1 Dynamic Light Scattering (DLS). . . . . . . . . . . 31
2.1.1.2 Disc Centrifuge (CPS) . . . . . . . . . . . . . . . . 32
2.1.2 Surface characterization of nanoparticles: Zeta Potential. . . 34
2.1.3 Fluorescence Characterization . . . . . . . . . . . . . . . . . 36
2.1.4 Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . 37
2.1.5 Flow cytometry (FCM). . . . . . . . . . . . . . . . . . . . . 37
2.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.2.2 Exploiting the surface chemistry of polyacrylamide nano-
particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.2.2.1 Synthesis of N-propargyl acrylamide (III) . . . . . 41
2.2.2.2 Synthesis of polyacrylamide nanoparticles (PANPs) 42
2.2.2.3 Amino Functionalised Polyacrylamide Nano-
particles (AmNPs) . . . . . . . . . . . . . . . . . . 43
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