Polyion complex (PIC) nanoparticles for the targeted and passive delivery of antimicrobial ...

149 Pages·2017·19.71 MB·English

by  Ignacio Insua Lopez

Polyion complex (PIC) nanoparticles
for the targeted and passive delivery of
antimicrobial polymers and peptides
by
Ignacio Insua López

A thesis submitted to the
University of Birmingham
for the degree of
DOCTOR OF PHILOSOPHY

School of Chemistry
College of Engineering and Physical Sciences
University of Birmingham
February 2017
University of Birmingham Research Archive

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Abstract
Antibiotic resistance is a serious worldwide threat. Alternative solutions to the
limited pipeline of new antibiotics are urgently needed. Nanotechnology and drug
delivery can be used to develop new therapies from old antimicrobials by control-
ling their distribution in the body.
The goal of this thesis was to investigate the formation and activity of polyion
complex (PIC) nanoparticles as vehicles for the delivery of two cationic antibiot-
ics: poly(ethylene imine), used as model to develop these nanomaterials, and the
FDA-approved antimicrobial peptide polymyxin B. These antibiotics were com-
bined with two types of polyanions to form PIC particles with distinct antimicrobial
properties: 1) Anionic peptides, degradable by bacterial enzymes led to bacteria-
targeted nanoparticles; 2) Acidic polymers assembled particles for passive re-
lease, which tuned the activity of the antibiotic in the absence of a specific trigger.
All the PIC particles prepared were characterised, and their physiological stability
and antimicrobial properties evaluated. To improve the stability and activity of
these nanoparticles, several characteristics of their anionic components were op-
timised: 1) their multivalency, as a function of the peptide’s anionic residues and
polymer’s DP; 2) the acidity of the polymers; and 3) the number of cross-linking
residues in the peptides.

i
This thesis is dedicated to my parents,
for their constant, endless and valuable support over the years.

Esta tesis está dedicada a mis padres,
por su constante, infinito y valioso apoyo durante los años.

“The saddest aspect of life right now is that science gathers knowledge
faster than society gathers wisdom.” – Isaac Asimov.

ii
Acknowledgements
I would like to start by thanking my supervisor, Dr Francisco Fernández-Trillo, for
giving me the opportunity to work in, and actually start, his group. I have learnt a
great deal about science and academic life from Paco, but more importantly, he
has been a good counsellor in scientific and personal matters. I will miss your
cheer-up sessions over coffee or beer when experiments didn’t work.
Big thank you to my two other mentors during these years of PhD: my co-super-
visor Dr Anna Peacock, and my microbiology tutor Dr Anne Marie Krachler.
Thank you to the rest of Paco’s group, past and present, for creating an enjoyable
work atmosphere, specially my UG/PG students (a.k.a. minions) Menisha and
Marion, for their contribution to this thesis; the rest of minions I have mentored
(Maria, Nico, Löic, Louise, Rob, Nada, Greg, Joel and Nathan), for enduring my
supervision; Nico, for his coffee-breaks and help with bacterial work; Zelu, for his
doses of “constructive” criticism; Adam and Oli, for the good times we had during
this time in Birmingham (and Amsterdam); and Daniel, for the commutes to the
lab and your help whenever I needed it.
To my brother from another mother, Xavi. This time in Birmingham would have
never been the same without you. I don’t realise now how much I will miss you,
but I know for sure we’ll still be good friends wherever we go. Thank you and
Mariyuki for the time we lived together.
To my best friend, the person with this strange power that makes my worries
disappear when I am with her. The person I have shared this time in Birmingham
with. In the distance or presence, she was always there: my girlfriend Laura.
To my family (Papá, Mamá, Noe, Paulita y María), for making me feel like I never
left home every time I visit you.
To all other people who have contributed to this thesis, both scientifically and
personally: Dr Marie-Christine Jones for access to DLS, the O’Reilly group in
Warwick, the analytical technicians in our school (Chi, Peter, Allen and Cécile),
my friends in Chemistry: Dr Para, Edgar and Elsa, and to all the “puteros”!
Finally, I would like to acknowledge the editorial work done by the Royal Society
of Chemistry and Elsevier on the published work presented in this thesis.
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Table of contents
PREFACE - Dynamic Light Scattering (DLS): data acquisition and inter- 1
pretation
Overview 1
Instrumentation and analysis parameters 2
Data interpretation 3
References and notes 6
CHAPTER 1 - Polyion complex (PIC) particles: preparation and biomed- 8
ical applications
1.1. Article 10
1.2. Nanotechnology for antimicrobial delivery – PIC nanoparticles 28
CHAPTER 2 - Enzyme-responsive polyion complex (PIC) nanoparticles 40
for the targeted delivery of antimicrobial polymers

2.1. Article 42
2.2. Supplementary information 49
2.3. Additional figures 63
CHAPTER 3 - Elastase-sensitive peptides with higher multivalency form 64
more stable polyion complex (PIC) nanoparticles with compromised sus-
ceptibility to enzymatic degradation
3.1. Article 66
3.2. Supplementary information 74
CHAPTER 4 - Preparation and antimicrobial evaluation of polyion com- 96
plex (PIC) nanoparticles loaded with polymyxin B
4.1. Article 99
4.2. Supplementary information 108
4.3. Composition of commercial polymyxin B 112
iv
CHAPTER 5 - Effect of polyelectrolyte strength and degree of polymeri- 117
sation on the formation, stability and activity of antimicrobial polyion com-
plex (PIC) particles carrying polymyxin B
5.1. Article 119
5.2. Supplementary information 128
CONCLUSIONS 137

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Abbreviations
∆lasAB P. aeruginosa; LasB-knockdown strain
+eV Positive ion mode
a.k.a. Also known as
a.u. Arbitrary units
Ac O Acetic anhydride
2
AcCN Acetonitrile
ACF Autocorrelation function
AFM Atomic force microscopy
CFU Colony forming unit
CMetC Carboxymethyl cellulose
D Diameter
DCM Dichloromethane
D Hydroynamic diameter
H
DIPEA N,N-diisopropylethylamine
DLS Dynamic light scattering
DMEM Dulbecco's modified eagle medium
DMF N,N-Dimethylformamide
DMSO Dimethylsulfoxide
DOX Doxorubicin
DP Degree of polymerisation
DPBS Dulbecco's phosphate-buffered saline
DTBN 5,5'-dithiobis(2-nitrobenzoic acid)
EDT 1,2-ethanedithiol
EDTA Ethylenediaminetetraacetic acid
ESI Electrospray ionisation
Et O Diethylether
2
EtAcO Ethyl acetate
FACS Fluorescence-activated cell sorting
FDA Food and Drug Administration (USA)
Fmoc Fluorenylmethyloxycarbonyl
GPC Gel permeation chromatography
HBTU N,N,Nʹ,Nʹ-tetramethyl-O-(1H-benzotriazol-1-yl)uranium
hexafluorophosphate
HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
HLE Human leukocyte elastase
HPLC High performance liquid chromatography
ISO International Standards Organization (Geneva, Switzerland)
kcps Kilocounts per second
L/B-PEI Linear/Branched-poly(ehtylene imine)
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LasB Elastase B from Pseudomonas aeruginosa
LB Luria Bertani (medium)
m/z Mass/charge ratio
MALDI Matrix-assisted laser desorption/ionisation
MeOH Methanol
MS Mass spectrometry
Mw Molecular weight
MWCO Molecular weight cut-off
NMR Nuclear magnetic resonance
NMVA N-methyl-N-vinylacetamide
NNLS Non-negative least squares
OD Optical density
PAA Poly(acrylic acid)
PAH Poly(allylamine)
PAO1V P. aeruginosa; wild type strain
PBS Phosphate-buffered saline
PDADMA Poly(diallyldimethylammonium chloride)
PdI Polydispersity index
pDNA Plasmid DNA
PEG Poly(ethylene glycol)
PIC Polyion complex
PLL Poly(L-lysine)
Pol-B Polymyxin B
PSS Poly(styrene sulfonate)
PVAm Poly(vinylamine)
r.f.u Relative fluorescence units
R Radius of gyration
g
R Hydrodynamic radius
H
RP Reverse phase
SAXS Small angle X-ray scattering
SC Succinyl casein
SD Standard deviation
siRNA Small interfering RNA
SLS Static light scattering
tBu Tert-butyl
TEM Transmission electron microscopy
TFA Trifluoroacetic acid
TIPS Triisopropylsilane
TOF Time-of-flight
Trt / Trityl Triphenylmethane
UV-Vis Ultraviolet-visible

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Dynamic Light Scattering (DLS):
data acquisition and interpretation

Overview 1,2
DLS is a particle characterisation technique that correlates the fluctuations in the
amount of light scattered by a particle suspension over time with particle size.
Colloidal particles undergo Brownian motion as solvent molecules collide into
them transferring kinetic energy, thus causing the particles to move in random
directions. As such, the intensity of light scattered by a particle suspension will
vary with time as particles in Brownian motion exit and enter the illuminated space
in the sample (i.e. under analysis). As solvent collisions with smaller particles are
more efficient, smaller particles undergo faster Brownian motion. On this basis,
DLS correlates the frequency of the fluctuations in the light scattered by a particle
suspension over time with the diffusion coefficient (D) of the particles – the speed
at which they move. The calculated value of D can then be transformed into a
size parameter known as hydrodynamic diameter (D ) using the Stokes-Einstein
H
equation (Eq. 1); where K represents Boltzmann’s constant, T is the absolute
B
temperature and 𝜂 is the viscosity of the solution:

𝐾 T
%
𝐷 = 𝐸𝑞.1
3 𝜋 𝜂 𝐷
*

The D of a particle is defined as the diameter of a theoretical sphere that diffuses
H
in the same fashion as the particle under analysis (i.e. has the same D). DLS
therefore gives a theoretical size value for the particles under analysis inferred
from the speed at which they diffuse.

The analysis of particle size by DLS offers a rapid, controlled and non-destructive
methodology to probe for differences in nanoparticle formulations and their sta-
bility. For this reason, it has been used throughout this thesis as first characteri-
sation point for the study of nanoparticles.
1