Stimuli-responsive polymer brushes for on-chip cell adhesion control

155 Pages·2017·9.68 MB·English

by  Siddhartha Varma

Stimuli-responsive polymer brushes for on-chip cell
adhesion control
Siddhartha Varma
To cite this version:
Siddhartha Varma. Stimuli-responsive polymer brushes for on-chip cell adhesion control. Biological
Physics [physics.bio-ph]. Université Grenoble Alpes, 2016. English. ￿NNT: 2016GREAY057￿. ￿tel-
01588155￿
HAL Id: tel-01588155
https://theses.hal.science/tel-01588155
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THÈSE
Pour obtenir le grade de
DOCTEUR DE LA COMMUNAUTÉ UNIVERSITÉ
GRENOBLE ALPES
Spécialité : Physique pour le vivant
Arrêté ministériel : 7 août 2006

Présentée par
Siddhartha VARMA

Thèse dirigée par Lionel BUREAU et
codirigée par Delphine DEBARRE

préparée au sein du Laboratoire Interdisciplinaire de Physique
dans l'École Doctorale de Physique

Brosses de polymères stimuli-
sensibles pour le contrôle de
l'adhésion cellulaire

Thèse soutenue publiquement le 10 octobre 2016,
devant le jury composé de :
Mme Elisabeth CHARLAIX
Professeur Université Grenoble Alpes, LIPhy Grenoble, présidente
Mme Marie-Pierre VALIGNAT
Maître de conférence Université Aix-Marseille, LAI Marseille, rapporteur
M Jean DAILLANT
Directeur général Synchrotron Soleil, Gif-sur-Yvette, rapporteur
M Thierry CHARITAT
Professeur Université de Strasbourg, ICS, examinateur
M Laurent HEUX
Directeur de recherche CNRS, CERMAV Grenoble, examinateur

“If you want to shine like a sun, first burn like a sun” - A.P.J abdul Kalam
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Chapter 1
Acknowledgements
First and foremost, I would like to express my deepest gratitude to my supervisors Dr.
Lionel Bureau and Dr. Delphine Debarre for their continuous support during my Ph.D.
study. Their valuable time, constructive advice, and willingness to share their knowledge
have been stimulating in all the time of my research and writing of the thesis. The en-
thusiasm they had for research was always mitivational for me, even during tough times
during my Ph.D. I thank them for welcoming me in the lab as an intern, and providing
me a chance to learn and contribute towards the development of the project. Without
their precious support, it would not have been possible to conduct this research.
I would like to thank my thesis committee members starting from Dr. Marie-Pierre
Valignat and Dr. Jean Daillant for evaluating my thesis and providing their insightful
comments. I would like to thank the other jury members Professor Elizabeth Charlaix,
Dr. Thierry Charitat, and Dr. Laurent Heux for accepting to judge my Ph.D. Thesis.
I would like to thank all the faculties at my masters level in India, as well a France
for providing me with the rich scientific background. A word of special thanks goes to
Professor S. Annapoorni and Professor Jean Claude Moutet for exposing me with the
research opportunities in France. I would like to thank Dr. Dinesh Rangappa for con-
stantly supporting me uptil now in my research endeavours. I will always be indebted
to your valuable inputs. I would like to thank Dr Jrme Fortage and Dr. Marie-Nolle
Collomb for their contribution towards the experimental characterization and analytical
discussions. Thank you to Dr. Claire Boisset and her group for their help with the GPC
analyses.
I thank the Agence Nationale de la Recherche (ANR) for their financial support dur-
ing this work. I would like to thank the administrative department for their assistence
throughout my stay at LIPhy. My time in Grenoble and LIPhy was made enjoyable in
large part due to the many colleagues and friends I had. I am grateful to all my collegues
for all the fun we had, for backing me up whenever I felt low, and for their love and
support towards the fulfillment of my thesis. Thank you very much for your support and
friendship.
Lastly, I would like to thank my family for all their love and encoragement. For my
parents and grandparents who raised me with the ability to pursue what I wanted and
their constant support in my life. Thank you for always believing in me. Thank you
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Siddhartha Varma
Laboratoire Interdisciplinaire De Physique (LIPhy)
10thOctober 2016.
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Abstract - R´esum´e en fran¸cais
The aim of the current Ph.D thesis was to successfully design stimuli responsive polymer
brushes in order to allow dynamic cell-substrate adhesive interactions.
For this purpose, Atom Transfer Radical Polymerization (ATRP) and Activators Re-
generated by electron Transfer (ARGET)-ATRP were used in order to prepare thermo
responsive Poly(N-isopropylacrylamide) (PNIPAM) brushes. Both the methods were ap-
plied under varying surface densities and polymerization times, and the kinetics of the
brush growth using both the protocols was investigated. A well controlled chain growth
was reported under ARGET-ATRP protocol, in contrast to the ATRP method. The
above tested protocol was used to grow PNIPAM brushes that were patterned via deep
UV photoablation strategy to design thermoresponsive patterned substrates for protein
adsorption studies. The substrates showed excellent adhesive properties and reusability
with long term storage capacity.
The conformational changes of PNIPAM brushes, grown via the ARGET-ATRP pro-
tocol, were probed by an originally built set-up based on Reflection Interference Contrast
Microscopy (RICM). RICM allowed to estimate the optical response of the brushes as a
function of their height profile making it an interesting tool for brush characterization.
The response of the brush was studied as a function of brush grafting density and chain
length. The results provided a unique evidence of non-monotonic structural changes in
the brushes, across the Local Critical Solution Tempearature (LCST) of the polymer.
RICM was employed to achieve the challenging task of estimating the molecular param-
eters of the brush and understanding the physical origin of the phenomenon of thermal
hysteresis in a polymer brush.
Stimuli Responsive Polymers, sensitive to non-invasive stimuli, were synthesized with
an aim to address dynamic single cell adhesion studies at their physiological conditions.
Free Radical Polymerization and ARGET-ATRP protocol were used to design two photo-
thermo-responsive poly(DMA-AZAA) and poly(DMA-NIPAM-AZAA) polymers. The
conformational changes of the designed polymers were investigated at length by varying
the overall composition of monomers in the system. Both the polymers were water solu-
ble that enabled an easy characterization. The DMA-NIPAM-AZAA terpolymer solution
showed a sharp phase separation at 37◦C that could be reversibly switched under light
irradiation, making it compatoble for cell adhesion studies.
Le but de cette th`ese de doctorat ´etait de concevoir des brosses de polym`eres stimuli-
sensibles afin de controˆler dynamiquement les interactions adh´esives entre une cellule et
son substrat.
Pour cela, nous avons utilis´e la polym´erisation radicalaire par transfert d’atomes
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(ATRP) initi´ee en surface, et sa variante permettant de r´eg´en´erer in situ le catalyseur de
polym´erisation (ARGET-ATRP), pour pr´eparer des brosses thermo-sensibles de poly(N-
isopropylacrylamide) (PNIPAM). Les deux m´ethodes ont ´et´e appliqu´ees pour diff´erentes
densit´essurfaciquesettempsdepolym´erisation,etlescin´etiquesdecroissancedelabrosse
a` l’aide des deux protocoles ont ´et´e ´etudi´es. Une croissance de chaˆıne bien controˆl´ee a ´et´e
observ´ee avec le protocole ARGET-ATRP, mais pas avec la m´ethode ATRP. Le protocole
test´e ci-dessus a ´et´e utilis´e pour fabriquer des brosses de PNIPAM qui ont ´et´e pattern´ees
par l’interm´ediaire d’une strat´egie d’ablation aux UV profonds, afin de concevoir des
substrats permettant de contrˆoler spatialement l’adsorption de prot´eines. Ces substrats
ont montr´e d’excellentes propri´et´es adh´esives, sont r´eutilisables et peuvent se stocker sur
de longues p´eriodes.
Les changements conformationnels de brosses PNIPAM ont ´et´e sond´es graˆce a` un
dispositif original mis en place sur la base d’un microscope en r´eflexion a` contraste
d’interf´erences (RICM). La technique RICM a permis d’estimer la r´eponse optique des
brosses en fonction de leur profil de hauteur, ce qui en fait un outil int´eressant pour leur
caract´erisation. La r´eponse de la brosse a´et´e´etudi´ee en fonction de sa densit´e de greffage
et de la longueur de chaˆıne. Les r´esultats ont fourni une preuve unique de l’existence d’un
ph´enom`ene de s´eparation de phase verticale, donnant lieu a` des changements structurels
non-uniformes dans les brosses lors du passage de la temp´erature inf´erieure de solubilit´e
du PNIPAM dans l’eau. Le RICM a ´et´e utilis´e pour r´ealiser la tˆache complexe d’estimer
les param`etres mol´eculaires de la brosse et la compr´ehension de l’origine physique du
ph´enom`ene d’hysteresis thermique dans une brosse de polym`ere.
De nouveaux polym`eres stimuli-sensibles ont ´et´e synth´etis´es dans le but d’obtenir des
syst`emes d’int´erˆet pour les ´etudes biologiques en conditions physiologiques. Nous avons
con¸cusdiff´erentsco-polym`eresphoto-thermo-sensiblesa`based’acrylamidesetd’acrylates.
Leschangementsdeconformationdespolym`erescon¸cusont´et´e´etudi´esend´etailenfaisant
varier la composition globale des monom`eres dans le syst`eme. Nous avons identifi´e une
composition de ter-polym`eres dont les solutions aqueuses ont montr´e une s´eparation de
phase`a37◦Cquipeutˆetrer´eversiblesousirradiationlumineuse,cequilarendcompatible
pour les ´etudes d’adh´esion cellulaire.
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Contents
1 Acknowledgements 3
2 Introduction 11
2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Cells and their environment . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.1 Cell signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.2 Cell adhesion in biology . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.3 Cell adhesion and spreading . . . . . . . . . . . . . . . . . . . . . 13
2.2.4 Cellular forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 Cell-substrate interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4 Tissue engineering and material science . . . . . . . . . . . . . . . . . . . 16
2.5 Scaffolds mimicking the extra cellular matrix . . . . . . . . . . . . . . . . 17
2.6 Cell patterning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.6.1 Imposing geometric restrictions to study cellular behaviour . . . . 19
2.6.2 Suppressing non-specific cell substrate interactions . . . . . . . . . 20
2.6.3 Patterning cell adsorption and adhesion . . . . . . . . . . . . . . 21
2.7 Smart materials for cell adhesion control . . . . . . . . . . . . . . . . . . 23
2.7.1 Self assembled monolayers (SAMs) . . . . . . . . . . . . . . . . . 24
2.7.2 Stimuli responsive polymeric surfaces . . . . . . . . . . . . . . . . 27
2.8 Aims of the present work . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Bibliography 37
3 PNIPAM Brushes: Design and Patterning 47
3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.1 Polymer brushes . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.2 Design strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.2.3 Chain growth protocols following the “grafting from” approach . . 50
3.2.4 Surface patterning methods . . . . . . . . . . . . . . . . . . . . . 53
3.3 Protocols for brush growth . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3.2 Preparation of the samples . . . . . . . . . . . . . . . . . . . . . . 55
3.3.3 Brush growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3.4 Varying the brush grafting density . . . . . . . . . . . . . . . . . 56
3.4 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.4.1 Characterization of brushes . . . . . . . . . . . . . . . . . . . . . 57
3.4.2 Effect of polymerization time with ATRP . . . . . . . . . . . . . . 58
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