from block copolymer synthesis to bacterial adhesion studies

175 Pages·2017·6.11 MB·English

by  Nicolas Cottenye

Antimicrobial surfaces based on self-assembled
nanoreactors: from block copolymer synthesis to
bacterial adhesion studies
Nicolas Cottenye
To cite this version:
NicolasCottenye. Antimicrobialsurfacesbasedonself-assemblednanoreactors: fromblockcopolymer
synthesis to bacterial adhesion studies. Other. Université de Haute Alsace - Mulhouse; Universität
Basel. Philosophisch-naturwissenschaftliche Fakultät, 2010. English. ￿NNT: 2010MULH5351￿. ￿tel-
00598560￿
HAL Id: tel-00598560
https://theses.hal.science/tel-00598560
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

Antimicrobial surfaces based on self-assembled nanoreactors: from
block copolymer synthesis to bacterial adhesion study.

Inauguraldissertation / Manuscrit de These

Zur Erlangung der Würde eines / en vue d’obtenir le

Doktorat der Philosophie
vorgelegt der
Doctorat en Chimie
Philosophisch-
de L’Université de Haute Alsace
Naturwissenschaftlichen Fakultät

der Universität Basel

Auf der Basis eines Cotutelle-vertrags/ dans le cadre d’un accord de cotutelle.

Von/ par

Nicolas Cottenye

Aus/né à Roubaix (Frankreich/France)

Basel, 2010

.

Original document stored on the publication server of the University of Basel: edoc.unibas.ch

This work is licenced under the agreement „Attribution Non-Commercial No Derivatives – 2.5 Switzerland―. The complete text may be
viewed here: creativecommons.org/licenses/by-nc-nd/2.5/ch/deed.en
2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von

Prof. Dr. Wolfgang Meier, supervisor
Dr. K. Anselme, supervisor
Dr. L.Ploux, supervisor
Prof. Dr. Marcus Textor, referee
Dr. Helmut Schlaad, referee

Basel, den 14 Dezember 2010

Dekan
Prof. Dr. Martin Spiess
2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

PhD titles.

English.
Antimicrobial surfaces based on self-assembled nanoreactors: from block copolymer synthesis to bacterial
adhesion studies

Français.
Surfaces antimicrobiennes basées sur l‘auto-assemblage de nanoréacteurs: de la synthèse du copolymère à
l‘étude de l‘adhésion bactérienne.

Keywords.

English.
Polymer, amphiphile, vesicle, bacteria, biofilm, antimicrobial, surfaces

Français.
Polymère, amphiphile, vésicule, bactérie, biofilm, antimicrobien, surfaces

Laboratories.
Department of Chemistry
University of Basel
Klingelbergstrasse 80
CH-4056 Basel, Switzerland
Tel: [phone]
Fax: [phone]

IS2M (CNRS LRC7228)
Institut de Science des Matériaux de Mulhouse
15, rue Jean Starcky - BP 2488
68057 Mulhouse cedex, France
Tel: [phone]
Fax: [phone]
2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

Abstracts.
English.
The aim of this work is to develop a new strategy for the prevention of biofilm growth. For this purpose, we
prepared bioactive surfaces resulting from the surface-immobilization of nanoreactors self-assembled from
amphiphilic poly(isobutylene)-block-oligonucleotide copolymers. The block copolymer was synthesized and
characterized via appropriate complementary techniques. Self-assembly into vesicles allowed the functional
encapsulation of enzymes, as assayed through enzyme activity monitoring, leading to a prodrug-drug system. The
self-assembled structures were specifically immobilized on surfaces via base pairing between the oligonucleotide
block of the copolymer and the surface tethered complementary nucleotide sequence.
Using E.coli strains, we first observed an influence of the two density of oligonucleotides immobilized on
the surface on the number of adherent bacteria. This influence may be due to an effect of surface charge density.
We then confirmed the well-known role of curli in biofilm cohesion, and we showed gene over-expression
associated with curli production on oligonucleotide-modified surfaces. We demonstrated that gene over-
expression does not depend on the topographical features of the surface or on the composition of the nucleotide
sequences used in this study. Finally, we demonstrated that the presence of the vesicular structure is able to
produce strong anti-adhesive properties of the surface. We assume, from observations of bacterial response in
dynamic conditions, that this effect is due to increased bacterial motility on the surface, leading to a high
detachment rate. Which is further confirms by a comparable bacterial response observed on agar hydrogel of
different hardnesses. This result provides a preliminary outcome, paving the way to new approaches to
antimicrobial strategies.
2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

Français.
Ce travail a pour but le développement d‘une nouvelle classe de surfaces destinée à la lutte contre la
formation de biofilms. Ces surfaces se basent sur l‘immobilisation de nanoréacteurs, résultant de l‘auto-
assemblage d‘un copolymère amphiphile poly(isobutylène)-bloc-oligonucléotide sous forme de vésicules
encapsulant une enzyme. Le rôle de cette enzyme est de produire des agents antimicrobiens à partir de précurseurs
inactifs. Le copolymère a été synthétisé et caractérisé par des techniques complémentaires et le maintien de
l‘activité enzymatique a été démontré. Les surfaces bioactives sont finalement obtenues par l‘immobilisation des
nanoréacteurs grâce aux capacités d‘hybridation des séquences nucléiques complémentaires.
Nous avons d‘abord montré l‘influence des deux densités d‘oligonucléotides utilisées sur le nombre
d’E.coli adhérentes, due probablement aux variations de charges surfaciques associées. Nous avons ensuite
confirmé l‘influence des curli sur la cohésion des biofilms et mis en évidence une surexpression de la production
de curli en présence d‘oligonucléotides sur les surfaces, et ce, indépendamment de la séquence et de la
topographie considérées. Finalement, nous avons observé un effet antiadhésif lié aux structures vésiculaires
indépendant de la topographie de la surface. L‘étude complémentaire, menée en mode de culture dynamique, a
conduit à envisager que les propriétés mécaniques de la surface affectent la rétention des bactéries en modifiant
leur mobilité sur la surface. L‘analogie avec des résultats obtenus sur des hydrogels d‘agar de différentes duretés
conforte cette hypothèse. Ces résultats posent les fondements permettant d‘envisager de nouvelles stratégies dans
la lutte contre la formation des biofilms.
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

Acknowledgment.

A number of people helped and were involved in this work more or less directly. I wish to thank them all. I
wish in particular to cite some of them for their invaluable help and contribution.
The committee: Prof. Dr. M. Textor, Prof. Dr. H. Schlaad and Prof. Dr. T. Pfohl are acknowledged for
having agreed to consider and report on this thesis.
My supervisors: Prof. Dr. W. Meier, Dr. K. Anselme, Dr. L. Ploux and Dr. C. Vebert-Nardin are
acknowledged for their help, discussions, sympathy and, moreover, their patience and compliance with my
dramatic writing skills.

Collaborators are thanked for having shared their unique skills, discussions and sympathy: Dr A. Ponche
and P. Fioux (XPS), Dr. S. Rigolet (solid state NMR), Dr. O. Onaca (molecular biology), Dr. K. Mougin
(ellipsometry and contact angle measurements), G. Persy (TEM), Dr. A. Ziegler (CD spectroscopy) and M.Inglins
for critical review of this thesis.
Trainee students are acknowledged for their contribution as the small hands of some parts of this work: J.
Sonnet, L. Costato and M. I. Syga.

My close colleagues: J. Böhmler and K. Langowska are thanked for their everyday contribution, discussion
and happiness (or not…), my Basler ―Menschen‖ office roommate for a cool work atmosphere and the Mulhouse
office girls for more girly but still cool ambiance.
All people from the labs in Basel and Mulhouse: colleagues, administrative staff, technicians, etc... I
enjoyed my work in those labs, thanks to you.

Finally I would like to provide more personal thanks to my wife, Sandra, who always supports me in all my
choices; thanks my love.

To close this acknowledgment section, since nothing is possible without a little bit of money, I would like
to thank the financial supports of this work:
The SNSF (200020-121822), the NCCR-NANO and the “CRUS stipend fur cotutelle de thèse, Programme
2010”.
2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

Content.
PHD TITLES. .....................................................................................................................................................................................4
ENGLISH............................................................................................................................................................................... 4
FRANÇAIS. ........................................................................................................................................................................... 4
KEYWORDS. .....................................................................................................................................................................................4
ENGLISH............................................................................................................................................................................... 4
FRANÇAIS. ........................................................................................................................................................................... 4
LABORATORIES..............................................................................................................................................................................4
ABSTRACTS. .....................................................................................................................................................................................5
ENGLISH............................................................................................................................................................................... 5
FRANÇAIS. ........................................................................................................................................................................... 6
ACKNOWLEDGMENT. ..................................................................................................................................................................7
CONTENT. .........................................................................................................................................................................................8
GENERAL INTRODUCTION. .................................................................................................................................................... 12
BIOFILMS AND THEIR CONTROL. .......................................................................................................................................12
AMPHIPHILES, VESICLES, AND THEIR APPLICATIONS. ......................................................................................................15
SCOPE OF THE THESIS. .......................................................................................................................................................17
REFERENCES. .....................................................................................................................................................................19
PART I: POLYMERS, NANOREACTORS, AND HYBRIDIZATION FOR CONTROLLED SURFACE
PREPARATION.............................................................................................................................................................................. 22
A. Introduction. ..................................................................................................................................................................... 23
B. State of the art. .................................................................................................................................................................. 25
B.1 Amphiphilic block copolymers, amphiphilic ―chimeras‖ their properties and synthesis. ..............................................25
B.2 Nanoreactors: Definition, description and limitations. ................................................................................................28
B.3 Prodrug-drug system. ................................................................................................................................................31
B.4 Immobilization of vesicles to surfaces: possible strategies. .........................................................................................33
B.5 Oligonucleotide hybridization. ..................................................................................................................................35
B.6 Vesicles mechanical properties. .................................................................................................................................36
C. Materials and methods. .................................................................................................................................................... 37
C.1 Oligonucleotide-based amphiphilic block copolymer synthesis...................................................................................37
C.1.a Materials. ....................................................................................................................................................... 37
C.1.b Solid phase synthesis. ..................................................................................................................................... 38
C.2 Chemical analysis of the diblock copolymer. .............................................................................................................38
C.2.a Nuclear magnetic resonance (NMR). ............................................................................................................... 38
C.2.b Fourier Transform Infrared Spectroscopy (FTIR)............................................................................................. 39
C.2.c Elementary analysis. ....................................................................................................................................... 39
C.3 UV-Vis spectroscopy for polymer concentration quantification. .................................................................................39
C.4 Self-assembly of the diblock copolymer. ...................................................................................................................39
C.5 Self-assembly characterisation and chain configuration. .............................................................................................39
2010 Nicolas Cottenye
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Antimicrobial surface based on self-assembled nanoreactors: from block copolymer synthesis to bacterial adhesion study

C.5.a Circular Dichroism Spectrocscopy (CD). ......................................................................................................... 39
C.5.b Transmission Electron Microscopy (TEM). ..................................................................................................... 40
C.5.c Scanning Electron Microscopy (SEM). ............................................................................................................ 40
C.5.d Dynamic Light Scattering (DLS)..................................................................................................................... 40
C.6 Nanoreactor preparation and characterization.............................................................................................................40
C.6.a Materials. ....................................................................................................................................................... 40
C.6.b Nanoreactor preparation.................................................................................................................................. 41
C.6.c Nanoreactor purification. ................................................................................................................................ 41
C.6.d Other samples and sample names. ................................................................................................................... 42
C.6.e Polymer and enzyme concentration determination............................................................................................ 43
C.6.f Enzyme activity monitoring and concentration determination from Michaelis-Menten kinetics measurement by
fluorescence spectroscopy........................................................................................................................................ 44
C.7 Vesicle Labeling. ......................................................................................................................................................45
C.8 Oligonucleotide modified surfaces.............................................................................................................................45
C.8.a Materials. ....................................................................................................................................................... 45
C.8.b Oligonucleotide self-assembled monolayer (SAM) modified surfaces............................................................... 45
(i) Protocol for ―low density‖ oligonucleotide modified surfaces. ...................................................................45
(ii) Protocol for―high density‖oligonucleotide modified surfaces. ...................................................................46
C.8.c Type of oligonucleotide modified surfaces....................................................................................................... 46
C.9 Vesicle modified surfaces..........................................................................................................................................47
C.9.a Surfaces on the immobilization of vesicles through nonspecific base pairing..................................................... 47
C.9.b Surfaces resulting from the immobilization of vesicles though hybridization. ................................................... 47
C.9.c Reference surfaces. ......................................................................................................................................... 47
C.10 Surface characterization. .........................................................................................................................................48
C.10.a Fluorescence microscopy. ............................................................................................................................. 48
C.10.b X-ray Photoelectron Spectroscopy (XPS). ..................................................................................................... 48
C.10.c Contact angle measurements. ........................................................................................................................ 48
C.10.d Ellipsometry. ................................................................................................................................................ 49
C.10.e Atomic force microscopy (AFM)................................................................................................................... 49
C.10.f Quartz crystal microbalance with dissipation monitoring QCM-D. .................................................................. 49
CHAPTER 1. BLOC COPOLYMER SYNTHESIS AND CHARACTERIZATION...........................................................................51
1.1. Results and discussion ...........................................................................................................................................51
1.1.a. Chemical analysis. ..................................................................................................................................51
1.1.b. Self-assembly and chain configuration.....................................................................................................56
1.2. Conclusion............................................................................................................................................................60
CHAPTER 2. NANOREACTOR PREPARATION AND CHARACTERIZATION...........................................................................62
2.1. Results and discussion ...........................................................................................................................................63
2.1.a. Nanoreactor preparation and purification: step by step characterization.....................................................63
2.1.b. Nanoreactor activity, Michaelis-Menten kinetics and encapsulation efficiency. .........................................67
2.1.c. Proteinase K experiment. ........................................................................................................................69
2.2. Conclusion............................................................................................................................................................70
2010 Nicolas Cottenye