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Stimulus-Responsive Microgels:
Design, Properties and Applications
By
Mallika Das
A thesis submitted in conformity with the requirements for the degree
Doctor of Philosophy
Department of Chemistry University of Toronto
2008
© Copyright by Mallika Das, 2008
ii
Stimulus-Responsive Microgels:
Design, Properties and Applications
Mallika Das
Doctor of Philosophy
Department of Chemistry University of Toronto
2008
Abstract
Materials science today is a multidisciplinary effort comprising an accelerated
convergence of diverse fields spanning the physical, applied, and engineering sciences. This
diversity promises to deliver the next generation of advanced functional materials for a wide
range of specific applications. In particular, the past decade has seen a growing interest in the
development of nanoscale materials for sophisticated technologies. Aqueous colloidal
microgels have emerged as a promising class of soft materials for multiple biotechnology
applications. The amalgamation of physical, chemical and mechanical properties of microgels
with optical properties of nanostructures in hybrid composite particles further enhances the
capabilities of these materials. This work covers the general areas of responsive polymer
microgels and their composites, and encompasses methods of fabricating microgel-based drug
delivery systems for controlled and targeted therapeutic applications.
iii
The first part of this thesis is devoted to acquainting the reader with the fundamental
aspects of the synthesis, functionalization and characteristic properties of stimulus-responsive
microgels constructed from poly(N-isopropylacrylamide) (poly(NIPAm)) and other functional
comonomers. In particular, the role of electrostatics on the swelling-deswelling transitions of
polyampholyte microgels upon exposure to a range of environmental stimuli including pH,
temperature, and salt concentration are discussed. The templated synthesis of bimetallic gold
and silver nanoparticles in zwitterionic microgels is also described.
The latter part of this thesis focuses on the rational development of microgel-based
drug delivery systems for controlled and targeted drug release. Specifically, the development
of a biofunctionalized, pH-responsive drug delivery system (DDS) is illustrated, and shown to
effectively suppress cancer cells when loaded with an anticancer agent. In another chapter, the
design of tailored hybrid particles that combine the thermal response of microgels with the
light-sensitive properties of gold nanorods to create a DDS for photothermally-induced drug
release is discussed. The photothermally-triggered volume transitions of hybrid microgels
under physiological conditions are reported, and their suitability for the said application
evaluated. In another component of this work, it is explicitly shown that electrostatic
interactions were not needed to deposit gold nanorods on poly(NIPAm)-derived particles,
thereby eliminating the need for incorporation of charged functional groups in the microgels
that are otherwise responsible for large, undesirable shifts and broadening of the phase
transition.
iv
Acknowledgements I first wish to express my deepest appreciation to my supervisor, Professor Eugenia
Kumacheva, for teaching, advising and supporting me throughout my work. I also have deep
gratitude towards Dr. S Xu for being a great mentor in my early years as a graduate student.
I am most grateful for having had the opportunity to work with a team of
exceptionally intelligent and wonderful people in Professor Kumacheva’s group. Thanks to
Dr. Chantal Paquet, Lindsey Fiddes, Ivan Gorelikov, Ilya Gourevich, Daniele Fava, Minseok
Seo, Patrick Lewis, Dr. Hong Zhang, Dr. Hung Pham, Zhihong Nie, Dr. Lora Field, Dr. Alla
Petukova, Andrew Paton, Wei Li, Patrick Lewis, Ethan Tumarkin and Alexandra Chestakova.
I owe a lot to my collaborators at the Institute for Biomaterials and Biomedical
Engineering and at the Princess Margaret Hospital, who have helped me develop my work.
Thanks to Sawitri Mardyani, Professor Warren Chan, David Gwiercer, Dr. Eduardo
Moriyama, Dr. Robert Weersink and Professor Brian Wilson. I am also grateful to Professor
Mitchell A. Winnink for being on my Supervisory committee, and for his valuable insights
and helpful discussion.
I wish to thank my family and friends who have been supportive and kind throughout
all my years as a graduate student. Special thanks to Dr. Wesley Whitnall, Dr. Sean Clapham,
Dr. Diane Clapham, Dr. Darren Anderson, Marco, Dr. Nikhil Gunari, my parents, and my
sister, Dipika.
I would also like to thank the following organizations for financial support: the
University of Toronto, the Martin Moskovits Graduate Scholarship in Science and
Technology, the F.E. Beamish Graduate Scholarship in Science and Technology, and
NSERC.
v
“The pursuit of knowledge begins with the admission of ignorance.”
~Unknown
“It is not enough to know. We must apply.”
~Goethe
vi
This thesis is dedicated to
my parents
vii
Table of Contents
Overview…………………………………………………………………………... 1
Chapter 1 Introduction to Polymer Microgels……………………………… 8
1.1 Definition of Microgels……………………………………………... 8
1.2 Classifications of Microgels ……………………………………… 9
1.2.1 Classification based on Crosslinking…………………………….. 9
1.2.1.1 Physically Crosslinked Microgels……………………………….. 9
1.2.1.2 Chemically Crosslinked Microgels……………………………… 10
1.2.2 Classification based on Response…………………………………... 11
1.3 Thermoresponsive poly(N-isopropylacrylamide) poly(NIPAm)…….. 11
1.3.1 Solution behavior of poly(NIPAm)………………………………… 11
1.3.2 Poly(NIPAm) Macrogels…………………………………………… 12
1.3.3 Poly(NIPAm) Microgels……………………………………………. 14
1.4 Preparation of Microgels…………………………………………….. 15
1.5 Characterization of Microgels……………………………………….. 15
1.6 Stimuli-Responsive Properties of Microgels………………………… 16
1.6.1 Effect of Temperature………………………………………………. 16
1.6.2 Effect of pH and Ionic Strength……………………………………. 16
1.6.3 Effect of Solvents…………………………………………………... 17
1.7 Applications of Microgels……………………………………………. 18
1.7.1 Microgels as Microreactors…………………………………………. 18
1.7.2 Microgels as Photonic Crystals…………………………………….. 20
1.7.3 Microgels as Microlenses…………………………………………... 21
1.7.4 Microgels for Drug Delivery……………………………………….. 22
1.8 Conclusions…………………………………………………………... 23
1.9 References……………………………………………………………. 25
Chapter 2 Materials and Methods.................................................................... 33
2.1 Preparation of Microgels…………………………………………....... 33
2.1.1 Reagents…………………………………………………………… 33
2.1.2 Synthesis of Microgels…………………………………………….. 35
viii
2.1.3 Purification of Microgels…………………………………………... 37
2.2 Particle Characterization…………………………………………….. 38
2.2.1 Particle Size………………………………………………………… 38
2.2.2 Electrokinetic Potential…………………………………………….. 40
2.2.3 Scanning Electron Microscopy……………………………………... 42
2.3 Preparation of Gold Nanorods……………………………………….. 44
2.3.1 Synthesis of Gold Nanorods……………………………………….. 44
2.3.2 Characterization of Gold Nanorods………………………………… 45
2.4 References……………………………………………………………. 47
Chapter 3 From Polyampholyte to Polyelectrolyte Microgels……………... 48
3.1 Introduction………………………………………………………… 48
3.2 Research Objectives……………………………………………….. 49
3.3 Background………………………………………………………… 50
3.4 Experimental Procedure……………………………………………… 52
3.4.1 Synthesis and Characterization of Microgels………………………. 52
3.4.2 Quantitative Determination of Charged Groups in Microgels……... 53
3.5 Results………………………………………………………………... 57
3.5.1 Effect of pH………………………………………………………… 58
3.5.2 Effect of Ionic Strength…………………………………………….. 62
3.5.3 Effect of Temperature……………………………………………… 63
3.5.4 Effect of Solvent…………………………………………………… 65
3.6 Discussion…………………………………………………………… 67
3.6.1 Effect of pH and Ionic Strength…………………………………… 68
3.6.2 Effect of Temperature …………………………………………… 70
3.6.3 Effect of Solvent………………………………………………….... 71
3.7 Conclusions………………………………………………………….. 73
3.8 References 75
Chapter 4 Zwitterionic Sulfobetaine Microgels……………………………. 78
4.1 Introduction………………………………………………………….. 78
4.2 Research Objectives…………………………………………………. 80
ix
4.3 Experimental………………………………………………………… 81
4.3.1 Materials…………………………………………………………… 81
4.3.2 Synthesis of zwitterionic sulfobetaine microgels………………….. 82
4.3.3 Characterization and Instrumentation…………………………… 83
4.4 Results……………………………………………………………….. 84
4.4.1 Size of Zwitterionic poly(NIPAm-SPP) microgels……………….. 84
4.4.2 Effect of pH on swelling…………………………………………… 84
4.4.3 Effect of temperature………………………………………………. 85
4.4.4 Effect of salts………………………………………………………. 86
4.5 Conclusion…………………………………………………………… 88
4.6 References……………………………………………………………. 90
Chapter 5 Biofunctionalized pH-responsive Microgels for
Cancer Cell Targeting……………………………………………… 92
5.1 Introduction…………………………………………………………... 92
5.2 Background………………………………………………………… 94
5.2.1 pH-mediated drug release………………………………………….. 94
5.2.2 Cancer treatment and intracellular drug delivery………………… 95
5.2.3 Biofunctionalized stimulus-responsive microgels in drug delivery… 97
5.3 Research objectives…………………………………………………... 98
5.4 Experimental…………………………………………………………. 99
5.4.1 Synthesis of microgels……………………………………………….. 99
5.4.2 Particle Characterization…………………………………………….. 100
5.4.3 Drug and dye uptake into microgels…………………………………. 100
5.4.4 Conjugation of transferrin and albumin to loaded microgels……… 101
5.4.5 Rhodamine-loaded microgel assay………………………………….. 101
5.4.6 Doxorubicin-loaded microgel assay………………………………… 101
5.5 Results and Discussion……………………………………………………. 102
5.5.1 pH response of microgels…………………………………………… 102
5.5.2 Loading and pH-induced release of rhodamine dye……………….. 103
5.5.3 Biofunctionalization of microgels……………………………………. 104
5.5.4 Intracellular uptake of bioconjugated microgels……………………. 106
5.5.6 In Vitro studies of uptake and release using an anticancer drug…… 107
5.5.6.1 Quantitative determination of drug uptake by microgels……… 108
x
5.5.6.2 pH-dependent release of drug from microgels………………….. 110
5.5.6.3 In vitro test of cell viability…………………………………….. 112
5.6 Conclusion………………………………………………………………… 114
5.7 References………………………………………………………………….. 115
Chapter 6 Hybrid Microgels for Photothermally-Induced Drug Release…….. 118
6.1 Introduction………………………………………………………………… 118
6.2 Hybrid microgels loaded with gold nanorods ……………………………... 119
6.3 Tuning the thermal response of microgels…………………………………. 120
6.4 Research objectives……………………………………………………… 123
6.5 Experimental……………………………………………………………….. 124
6.5.1 Materials……………………………………………………………… 124
6.5.2 Synthesis of microgels……………………………………………….. 124
6.5.3 Synthesis of gold nanorods………………………………………… 124
6.5.4 Preparation of hybrid microgels……………………………………… 125
6.5.5 Characterization of microgel properties……………………………… 125
6.5.6 Characterization of photothermally induced transitions……………… 126
6.6 Results………………………………………………………………………. 126
6.6.1 Copolymerization of NIPAm with acidic functionalities…………….. 127
6.6.2 Microgels with interpenetrated network structure……………………. 130
6.6.3 Copolymerization with hydrophobic comonomers………………….. 132
6.7 Discussion on the VPTTs of the synthesized microgels…………………… 128
6.8 Incorporation of gold nanorods into microgels…………………………….. 135
6.9 Thermally-induced volume transitions of hybrid microgels……………….. 137
6.10 Photothermally-induced volume transitions of hybrid microgels…………. 138
6.11 Current research on thermally-induced drug release…………………….… 139
6.12 Loading pure and hybrid microgels with a model compound.....………… 142
6.13 In vitro release of rhodamine from hybrid microgels……………………. 143
6.14 Visualization of loading and release of dye in microgels …………… 146
6.15 Real-time, photothermally-induced release……………………………….. 149
6.16 Conclusions and outlook………………………………………………… 151
6.17 References…………………………………………………………………. 153
xi
Chapter 7 Sequestering Gold Nanorods into Polyampholyte Microgels……. 157
7.1 Introduction………………………………………………………………… 157
7.2 Research Objectives……………………………………………………….. 159
7.3 Experimental………………………………………………………………. 160
7.3.1 Synthesis of microgels…………………………………………….. 160
7.3.2 Preparation of Gold Nanorods …………………………………… 160
7.3.3 Preparation of hybrid microgels……………………………………. 160
7.3.4 Characterization……………………………………………………. 161
7.4 Results …………………………………………………………………… 161
7.4.1 Properties of pure microgels and pure gold nanorods…………….. 161
7.4.2 Sequestration of CTAB-stabilized Au NRs onto microgels……… 163
7.4.3 Sequestration of polyelectrolyte-coated Au NRs onto microgels… 165
7.4.4 Properties of hybrid microgels with CTAB-stabilized Au NRs……. 169
7.5 Conclusions…………………………………………………………………. 172
7.6 References………………………………………………………………….. 173
Chapter 8 Summary and Future Outlook…………………………………… 175
8.1 Summary…………………………………………………………………… 175
8.2 Future outlook……………………………………………………………… 177
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List of Figures
Overview
Figure 1 Representation of growing interest in the field of polymer microgels for drug delivery
applications from years 1997 to 2007 inclusive, in the form of number of publications per year. Data
collected from Web of science and Sci-Finder Scholar
………………………………………………………………………………………………………......2
Figure 2 Size ranges of polymer microgels for different modes of drug administration. 17
…………………………………………………………………………………………………………..3
Chapter 1
Figure 1-1 Schematic representation of the conformational, ‘cage-like’ arrangement of water
molecules around poly(NIPAm) at temperatures below the LCST of ca. 31oC. The polymer is highly
solvated due to hydrogen bonding between water molecules and amide residues of poly(NIPAm).
……………………………………………………………………………………………………… 14
Figure 1-2 Schematic illustration of the structural rearrangement of water molecules around poly
(NIPAm) during the volume phase transition. At temperatures above the LCST, the hydrogen bonds
between water molecules and amide residues break and an entropically-favored release of water from
the polymer network occurs.
……………………………………………………………………………………………………… 14
Figure 1-3 Schematic depiction of the temperature-induced phase transition in poly (NIPAm) chains
and gels. Diagram is not to scale.
……………………………………………………………………………………………………… 15
Figure 1-4 Scheme of synthesis of NPs within microgels.[34]
……………………………………………………………………………………………………… 21
Chapter 2
Figure 2-1 Structures and functions of the reactants used in free radical precipitation polymerization
for the synthesis of microgels in this work
…………………………………………………………………………………………………………35
xiii
Figure 2-2 Scheme of microgel synthesis by redox polymerization. All the monomers are dissolved in
water and the solution is heated to 70 °C with surfactant sodium dodecyl sulfate (SDS). The
polymerization is initiated by a free-radical initiator potassium persulfate (KPS). Comonomers with
different functionalities can also be polymerized in the microgel
………...…………….............................................................................................................................37
Figure 2-3 Precipitation polymerization. After initiation the oligoradical grows to a critical length
before collapsing on itself to form a precursor particle. The precursor particle continues to grow either
by aggregating with other precursor particles or with growing oligomers, and eventually the microgel
particle precipitates out of solution
…………………………………………………………………………………………………………38
Figure 2-4 Schematic layout of dynamic light scattering (DLS) setup. The sample is illuminated and
the scattered light intensity is detected at 90o from the laser source, and fed to the autocorrelator. The
generated autocorrelator function is then used to calculate the diffusion coefficient
…………………………………………………………………………………………………………40
Figure 2-5 Schematic representation of the electrical double layer that surrounds stable colloidal
particles
…………………………………………………………………………………………………………42
Figure 2-6 Schematic illustration of scanning and transmission electron microscope
…………………………………………………………………………………………………………44
Figure 2-7 Synthetic scheme showing preparation and growth mechanism of Au NRs as adapted from
the method of El Sayed
…………………………………………………………………………………………………………47
Figure 2-8 Absorbance spectra of gold nanorods with aspect ratio of 4.3 in the pure dispersion (---)
and in hybrid microgels (-). Inset shows the shift in absorbance with change in aspect ratio46
…………………………………………………………………………………………………………48
Chapter 3
xiv
Figure 3-1.Schematic representation of swelling properties of polyelectrolyte and polyampholyte
microgels. (a) Anionic PE microgels. Ionization of the anionic groups at high pH and resultant
electrostatic repulsion between them causes microgel swelling. (b) Cationic PE microgels. At low pH,
electrostatic repulsion between ionized cationic groups causes microgel swelling. (c) Polyampholyte
(PA) microgels. The PA microgels are swollen at low and high pH values, due to repulsion between
charged cationic and anionic groups, respectively. In the interim pH region, PA microgels have
zwitterionic properties and contract due to electrostatic attraction between the oppositely charged
groups. For simplicity counterions are omitted.
…………………………………………………………………………………………………………53
Figure 3-2 Representative potentiometric (top) and conductometric (bottom) titration curves of poly
(NIPAm-AA) microgel (0.2wt%) titrated against NaOH.
…………………………………………………………………………………………………………57
Figure 3-3 Representative potentiometric (top) and conductometric (bottom) titration curves of
polyampholyte microgels (AA/VI = 2) titrated against NaOH, to determine the number of acidic
groups…………………………………………………………………………………………………59
Figure 3-4. Variation in Rh/R0 (a,b) and electrokinetic potential (ζ-potential) (a’, b’) of PE microgels
as a function of pH: (a,a’) poly(NIPAm-AA), R0 = 75 nm; (b,b’) poly(NIPAm-VI), R0 = 143 nm. The
dashed curves are given for eye guidance.
…………………………………………………………………………………………………………61
Figure 3-5. Effect of pH on the variation in Rh/R0 (a-d) and ζ-potential (a’-d’) for polyampholyte
microgels in 0.01M KCl solution at 25oC: (a, a’) PA-0.46, R0 = 79 nm ; (b, b’) PA-0.9, R0 = 73.8 nm;
(c, c’) PA-1.25, R0 = 59.6 nm; (d, d’) PA-1.65 R0 = 57.2 nm. Dashed lines are drawn as eye guidelines.
The horizontal dashed line demarks ζ-potential = 0
…………………………………………………………………………………………………………64
Figure 3-6 (a) Variation in normalized hydrodynamic radius (Rh/R0) as a function of KCl
concentration for polyelectrolyte microgels: (◆) poly (NIPAm-AA), pH=7.0, T = 25oC, R0 = 22.6 nm;
(■) poly (NIPAm-VI), pH=4.0, T = 25oC, R0 = 91 nm (b) Variation in normalized hydrodynamic
radius (Rh/R0) as a function of KCl concentration for polyampholyte microgels: (◆) PA-0.46, R0 = 24.5
xv
nm (■) PA-0.9), R0 = 44.2 nm (▲) PA-1.25,R0 = 28.6 nm (×) PA-1.65, R0 = 24.5 nm; pH=pI, T =
25oC. R0’s
…………………………………………………………………………………………………………65
Figure 3-7. Variation in microgel size as a function of temperature: (a) poly (NIPAm-AA) microgels,
(■) pH=3.5, R0 =50 nm ( ) pH =7.0, R0 =69.5 nm; (b) poly(NIPAm-VI) microgels, (■) pH=4.0, R0
=63.9 nm ( ) pH=7.5, R0 =52.6nm; (c) PA microgels with various compositions at corresponding pI
values, ( ) PA-0.46, R0 =49.8 nm; ( ) PA-1.65, R0 = 35.9 nm; (□) PA-1.25, R0 =42.4 nm. Rh is the
hydrodynamic radius of microgels at a particular temperature and R0 is the minimum Rh observed just
before aggregation of PA microgels. All microgels were studied in 0.1M KCl solution. Dashed lines
are given for eye guidance.
…………………………………………………………………………………………………………67
Figure 3-8. Variation in Rh/R0 of microgels in mixed solvents. (a) poly(NIPAm-AA), R0 =72.1 nm; (b)
poly(NIPAm-VI), R0 =122 nm; (c) PA-0.46, pI=5.8, R0 =79 nm; (d) PA-0.9, pI=5.6, R0 =73.8 nm;
(e) PA-1.65, pI=4.75, R0 =57.2 nm, ( )pH=4.0, (□) pH=pI, (▲) pH=7.5; (f) Variation in ζ-potential
of PA microgels in mixed solvents at the isoelectric point (determined in aqueous solutions): ( )PA-
0.46, ( ) PA-0.9, (•) PA-1.65.
…………………………………………………………………………………………………………70
Chapter 4
Figure 4-1 Chemical structure of monomers used in the present work. (a) N-isopropylacrylamide (b)
N,N-Dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine, SPP c) N-N’-
methylene-bis-acrylamide, BIS.
…………………………………………………………………………………………………………86
Figure 4-2. Variation of hydrodynamic diameters Dh as a function of the pH for zwitterionic
microgels poly(NIPAm-SPP). Solid lines are drawn for eye guideline. (■) NS1(▲) NS2 (♦)NS3 (X)
NS4
…………………………………………………………………………………………………………89
Figure 4-3. Variation in (a) hydrodynamic diameters Dh and (b) normalized hydrodynamic diameters
Dh/D0 as a function of temperature for zwitterionic microgels in water. D0 is the hydrodynamic
diameter of microgels at 50 °C. The particles were dispersed in water at pH=7. Solid lines serve as eye
guideline. (♦) NS0 (■) NS1 (▲) NS2 (X) NS3
…………………………………………………………………………………………………………90
xvi
Figure 4-4. Effect of concentration of (a) KCl and (b) CdCl2 on the volume phase transition of poly
(NIPAm-SPP) zwitterionic microgels containing 3.068% SPP; (♦)10-5M (□)10-3M (▲)5x10-1M (○)10-
1M (◊) 1 M. (c) Onset of the VPTT as a function of salt concentration. (d) Initial hydrodynamic radius
of microgels at 15oC in salt solutions.
…………………………………………………………………………………………………………92
Chapter 5
Figure 5-1 Schematic representation of the use of the receptor-mediated endocytosis pathway for the
targeted delivery of a drug. The pH-responive DDS is exposed to the intracellular pH-gradient as it
progresses through the endocytic environment. This pH gradient can employed as a trigger to promote
controlled drug release into the cytosol.
………………………………………………………………………………………………………..102
Figure 5-2 Conceptual diagram of proposed biofunctionalized, pH-responsive drug delivery system
for intracellular cancer cell targeting.
……………………………………………………………………………………………… ……….105
Figure 5-3 Variation in normalized hydrodynamic diameter of microgel particles as a function of pH
where D0 is the smallest diameter of microgel particle in the range studied. D0=142.3nm All
measurements were taken at 25 oC in 0.01M KCl. The average hydrodynamic diameter of the
microgels was ca. 110 and 156 nm at pH= 4.5 and pH=7.4, respectively.
………………………………………………………………………………………………………..109
Figure 5-4 Chemical structure of Rhodamine 6G- hydrochloride. The dye has a pKa value of 8.3,
making it positively charged at pH=7.4.
…………………………………………………………………………………………………..........109
Figure 5-5 Fluorescence images of R6G-loaded microgels at pH 7.4 (a) and at pH=4.5 (b) The net
uptake of R6G (expressed as a percentage of the total amount of R6G added at the start of the exp)
was 33.5%
…………………………………………………………………………………………………..........110
Figure 5-6 Scheme depicting bioconjugation of carboxylic acid functionalized microgels using
carbodiimide coupling.
…………………………………………………………………………………………………..........112
xvii
Figure 5-7 Differential interference contrast (DIC) (left) and epifluorescent (right) images of HeLa
cells after 24 hours incubation with R6G-loaded microgel-DDSs not conjugated to any protein (a),
conjugated to albumin (b) and conjugated to transferrin (c). R6G is released from transferrin-
conjugated microgels due to change in pH during RME. 20x objective N.A. = 0.4, λex = 480 +/- 40
nm (100 W Hg lamp), λem = 535 nm.
…………………………………………………………………………………………………..........112
Figure 5-8 Chemical structure of the anticancer drug, Doxorubicin. The red compound is
weakly basic and has a pKa value of 8.3.
…………………………………………………………………………………………………..........115
Figure 5-9 Loading capacity (left columns) and association efficiency (right columns) of
Doxorubicin in poly (NIPAm-AA) microgel particles at 37oC in 0.01M PBS at pH 7.4 for
(a) 0.1 and (b) 0.2wt% microgel dispersion.
…………………………………………………………………………………………………..........117
Figure 5-10 Percentage cumulative release of Dox from microgels (LC of 45.8%) at 37oC at
different pH values: ( ) pH=7.4 (■) pH=4.5
…………………………………………………………………………………………………..........118
Figure 5-10. Viability of HeLa cells after incubation for 36h with different systems: (a) Transferrin-
conjugated Dox-loaded microgels; (b) Dox-loaded microgels in solution with free transferrin (no
conjugation); (c) Albumin-conjugated Dox-loaded microgels; (d) Plain Dox-loaded microgels (no
conjugation); (e) Transferrin-conjugated plain microgels (no Dox);(f) HeLa cells only.
…………………………………………………………………………………………………..........120
Chapter 6
Figure 6-1. Variation in hydrodynamic diameter of poly(NIPMAm-UA) (U5) ( ) and poly
(NIPMAm) (Δ) microgels as a function of temperature in 0.01 M PBS pH=7.4. The incorporation of
UA in the poly (NIPAm) microgel results in a slight increase in the volume phase transition
temperature.
…………………………………………………………………………………………………..........136
(d)
xviii
Figure 6-2. Variation in hydrodynamic diameter of poly(NIPAm-MA) ( ) and poly(NIPAm) (Δ)
microgels as a function of temperature in 0.01 M PBS pH=7.4. The increase in the VPTT is caused by
the hydrophilicity of the charged carboxylic acid groups at neutral pH. …………………………………………………………………………………………………..........137
Figure 6-3. Variation in hydrodynamic diameter of poly(NiPAm-NIPMAm)/PAA IPN ( ) and and
poly(NIPAm-NIPMAm) (Δ) microgels as a function of temperature in 0.01 M PBS pH=7.4.
………………………………………………………………………………………………………..139
Figure 6-4. Variation in hydrodynamic diameter of poly(NiPAm-AA-BMA) ( ) and and
poly(NIPAm-BMA) (Δ) microgels as a function of temperature in 0.01 M PBS pH=7.4.
…………………………………………………………………………………………………..........140
Figure 6-5 TEM images of (a) hybrid poly(NIPAm-MA) microgels. Scale bar is 2 μm. Inset shows a
single NR-loaded 200 nm microgel particle. (b) Poly(NIPAm)/PAA IPN hybrid microgels. Scale bar
is 300nm.
…………………………………………………………………………………………………..........144
Figure 6-6 Absorption spectra of gold NRs prior to (black line) and following NR incorporation in
poly(NIPAm-MA) (yellow line) and poly(NIPAm-NIPMAm)-PAA IPN4 (red line) microgels.
…………………………………………………………………………………………………..........145
Figure 6-7. Variation in deswelling ratios, D/D0, of NR-free (Δ) and NR-loaded (■) microgels in PBS
at pH=7.4. (a) poly(NIPAm-MA) microgels (Series M2, Table 1, Chapter 3); (b) poly(NIPAm-
NIPMAm)/ PAA IPN microgels (Series IPN4, Table 1). D and D0 are the hydrodynamic diameters of
the corresponding microgels in buffer solution of pH= 7.4, at the temperature of interest and at room
temperature, respectively.
…………………………………………………………………………………………………..........146
Figure 6-8. Variation in deswelling ratio, V/V0 where V0 and V are the volumes of microgel at 25oC
and at temperature, T respectively, as a function of the number of laser on and laser off events of
pure(♦) and hybrid (■) microgels respectively. (a) M2 poly (NIPAM-MA)
…………………………………………………………………………………………………..........148
xix
Figure 6-9 Scheme showing plausible use of hybrid microgels in light-induced drug delivery systems.
The hybrid microgels are loaded with gold nanorods tuned to absorb in the near IR, the spectral range
that is ideal for biomedical applications since it can penetrate body tissues. Laser irradiation of the
NRs results in non-radiative energy transfer and local heating of the polymer network, thereby
triggering a deswelling transition, which can promote the release of a loaded drug.
…………………………………………………………………………………………………..........151
Figure 6-10 Loading capacity(LC) and Association Efficiency (AE) of R6G in pure and hybrid
microgel dispersions (0.1 wt% microgel).
…………………………………………………………………………………………………..........153
Figure 6-11. Amount of R6G dye released from and remaining within hybrid microgels (0.1 wt%
microgels) dispersed in 0.01M PBS at pH=7.4 as a function of temperature. (a) Poly(NIPAm-MA), LC
57.2% (b) Poly (NIPAm-NIPMAm), LC 48.6% (c) Poly (NIPAm-NIPMAm)/PAA IPN, LC 51.4%
…………………………………………………………………………………………………..........154
Figure 6-12 Fluorescence images of pure poly(NIPAM-MA) microgels loaded with Rhodamine 6G
(LC=57.2%) in 0.01M PBS buffer at different temperatures. Scale bar is 10�m. (a) T=24oC (b) T=
37oC (c) T =40oC
…………………………………………………………………………………………………..........156
Figure 6-13 Fluorescence images of hybrid poly(NIPAM-NIPMAm) microgels loaded with
Rhodamine 6G (LC = 48.6%) in 0.01M PBS buffer at different temperatures. Scale bar is 2μm. (a)
T=24oC (b) T =40oC
…………………………………………………………………………………………………..........157
Figure 6-14 Fluorescence intensity of Rhodamine 6G loaded in poly (NIPAm-MA) and poly(NIPAm-
NIPMAm) microgels at room temperature and at 40oC. Increase in temperature corresponded to a
decrease in fluorescence intensity in both microgel systems. Intensity of pure R6G solution did not
change with temperature in the present temperature range studied.
…………………………………………………………………………………………………..........159
Figure 6-15 Fluorescence images of hybrid poly(NIPAm-MA) microgels loaded with Rhodamine 6G
(LC = 49.5%) in 0.01M PBS buffer before laser irradiation T=37oC (left) and after laser irradiation,
T=37oC, right. Scale bar is 2μm.
…………………………………………………………………………………………………..........160
xx
Chapter 7
Figure 7-1 Variation in hydrodynamic diameter (a) and electrokinetic potential (b) of poly(NIPAm-
AA-VI) microgels plotted as a function of pH. Variation in electrokinetic potential (c) and
absorbance spectra (d) of NRs measured at different pH values
…………………………………………………………………………………………………..........174
Figure 7-2 Transmission electron microscopy images of hybrid poly(NIPAm-AA-VI) microgels
loaded with gold NRs at different pH values: (a) pH=4.5 (b) pH~pI=6.3 (c) pH=7.5. Scale bar is 800
nm. Scale bar for insets is 150 nm. The amount of Au in each system as determined from inductively
coupled plasma studies was 11.9, 9.7 and 10.8 mg/L at pH values of 4.5, 6.3 and 7.5 respectively.
…………………………………………………………………………………………………..........176
Figure 7-3 Fragments of transmission electron micrographs of hybrid poly (NIPAm-AA-VI)
microgels loaded with polyelectrolyte-coated gold NRs at different pH values: (a) pH=4.5 (b)
pH~pI=6.3 (c) pH=7.5 Scale bar is 800 nm. Scale bar for insets is 150 nm.
…………………………………………………………………………………………………..........178
Figure 7-4 TEM images of (a) neutral poly(NIPAm-NIPMAm) microgels at pH=7 and (b) cationic
poly(NIPAm-VI) microgels at pH=4.5 loaded with Au nanorods.
…………………………………………………………………………………………………..........179
Figure 7-5 Variation in (a) hydrodynamic diameter and (b) ζ-potential of hybrid microgels loaded
with NRs as a function of pH. (c) Variation in normalized hydrodynamic diameter, D/D0, of pure ( )
and hybrid (♦) microgels plotted as a function of pH, where D0 is the smallest size of microgels
obtained in the range studied. (d) Absorbance spectra of gold NRs loaded in polyampholyte microgels
at different pH values.
…………………………………………………………………………………………………..........182
Figure 7-6 (a) Temperature-induced variation in normalized hydrodynamic diameter, D/D0, of pure
(open symbols) and hybrid (filled symbols) microgels at pH=4.5(♦), pH =7.5 (▲) and pH=6.3(■) (b)
Absorbance spectra of hybrid microgels before and after centrifugation at 4000 RPM and temperature
=40oC.
…………………………………………………………………………………………………..........183
xxi
List of Tables
Chapter 3
Table 3-1 Compositions and characteristics of polyelectrolyte and polyampholyte microgels
………………………………………………………………………………………………….....54
Chapter 4
Table 4- 1Formulations used in microgel synthesis and the hydrodynamic diameter of the
corresponding particles
………………………………………………………………………………………………….....87
Chapter 5
Table 5-1. pH values in different tissue and cellular environments.[23]
…………………………………………………………………………………………………...100
Chapter 6
Table 6-2 Thermoresponsive polymers with phase transition temperstures that fall between
30 and 40 oC.
…………………………………………………………………………………………………...149
xxii
List of Abbreviations
AA Acrylic Acid
AE Association Efficiency
BIS N-N’-methylene-bis-acrylamide
BMA Butylmethacrylate
CTAB Cetyltrimethylammoniumbromide
DIC Differential Interference Contrast
DLS Dynamic Light Scattering
DOX Doxorubicin
DDS Drug Delivery System
EPR Enhanced Permeation and Retention
ICP Inductively Coupled Plasma
IPN Interpenetrated Network
KPS Potassium Persulfate
LC Loading Capacity
LCST Lower Critical Solution Temperature
MA Maleic Acid
NIPAm N-isopropylacrylamide
NIPMAm N-isopropylmethacrylamide
NP(s) Nanoparticle(s)
NR(s) Nanorod(s)
PA Polyampholyte
PAA Polyacrylic Acid
PBS Phosphate Buffered Saline
PCS Photon Correlation Spectroscopy
PE Polyelectrolyte
IEP Isoelectric Point
RME Receptor-Mediated Endocytosis
RPM Revolutions Per Minute
R6G Rhodamine 6G hydrochloride
SDS Sodium Dodecylsulfate
SEM Scanning Electron Microscopy
xxiii
SPP N,N-Dimethyl-N-(3-methacrylamidopropyl)-N-(3-
sulfopropyl) ammonium betaine
STEM Scanning Transmission Electron Microscopy
UA Undecanoic Acid
VI 1,4-Vinylimidazole
VPT Volume Phase Transition
VPTT Volume Phase Transition Temperature
xxiv
Publications during PhD Study
Das, M., Sanson N., Fava D., Kumacheva E., Microgels Loaded with Gold Nanorods:
Photothermally Triggered Volume Transitions Under Physiological Conditions,
Langmuir 2007, 23, 196-201
Das, M., Zhang, H., Kumacheva, E. Microgels: Old Materials with New Applications,
Annual Review of Materials Research, 2006 36, 117-142
Das, M., Marydani, S., Chan, W.C.W., Kumacheva, E., Biofunctionalized pH-
responsive microgels for cancer cell targeting: Rational design, Advanced Materials,
2006 18, 80-83
Das, M., Kumacheva, E., From Polyelectrolyte to Polyampholyte Microgels:
Comparison of Swelling Properties, Colloid and Polymer Science, 2006 , 283, 1073-
1084
Das, M., Morduokovski, L., Kumacheva, E., Sequestering gold nanorods into
polyampholyte microgels, Advanced Materials, 2008 (in press)
Papers in Progress
Das, M., Sanson,N., Kumacheva,E., Zwitterionic Microgels as Templates for the
Synthesis of Bimetallic Nanoparticles- (submitted at time of writing)
Das, M., Giewercer, D. , Sanson, N., Fava, D., Weersink, R., Wilson, B.,
Kumacheva,E., Photothermally-Induced Drug Release from Hybrid Microgels (in
progress)
Overview
___________________________________________________________________________ - 1 -
Overview
Hydrogels are crosslinked polymeric networks which absorb and retain large
amounts of water.[1] The characteristic network structure of hydrogels is
responsible for their unique ability to undergo abrupt volume changes in response
to environmental stimulii such as change in pH,[2] temperature[3] or ionic
strength.[4] Depending on the nature of the incorporated functional groups,
polymer hydrogels may be classified as neutral,[5] cationic,[6-8] anionic,[9]
amphiphilic[10, 11] or zwitterionic[12] gels. Electrostatic repulsion or attraction
between charged groups distributed throughout the hydrogel network results in
increased swelling or deswelling of the elastic polymer network in aqueous media.
Hydrogels may also be classified by size: macrogels are bulk gels ranging anywhere
from a millimeter to a few cm.[13] Colloidally stable hydrogel particles that range
from 100nm to several hundred microns in size are called microgels.
Microgels have increasingly become recognized as environmentally
responsive systems that have great potential in ‘smart’, ‘controlled’ and
‘regulated’ applications. In particular, they have rapidly gained importance as
carriers for therapeutic drugs and diagnostic agents. Figure 1 illustrates the
growing research interest in microgels for drug delivery applications over the past
Overview
___________________________________________________________________________ - 2 -
decade. The porous polymer network structure of synthetic microgels provides an
ideal reservoir for loaded drugs, protects them from environmental degradation
and hazards, and offers a template for the post-synthetic modification or
vectorization of the drug carriers.
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 20070
10
20
30
40
50
60
70
80
1
Year
Num
ber o
f Pub
licat
ions
Figure 1. Representation of growing interest in the field of polymer microgels for drug
delivery applications from years 1997 to 2007 inclusive, in the form of number of
publications per year. Data collected from Web of science and Sci-Finder Scholar
Microgels may also be rendered sensitive to physiological conditions. A
responsive drug delivery system is one of the most recognized technologies for
intelligent drug release. It must be able to regulate drug release in response to
external biological, physical or chemical stimuli. Targeting character may be
achieved by functionalizing microgels with receptor-specific ligands.[14, 15]
Typically, these biofunctionalized microgels can travel through the bloodstream,
target diseased tissues outside the bloodstream and be taken up by intracellular
compartments of targeted cells. Major requirements for an effective drug delivery
Overview
___________________________________________________________________________ - 3 -
system (DDS) include small size, extended circulating time, and reduced
interaction with serum proteins to prevent renal clearance.[16]
In particular, the dimension of drug carriers is an important determinant in
the release kinetics in addition to polymer molecular weight, porosity, and drug
distribution within the particles. Furthermore, the particle size determines both
the route of drug administration and the pathway of drug uptake to the targeted
tissues. Polymeric microgels with controlled size, size distribution and morphology
have already found a variety of applications in pharmaceutical and biomedical
sciences. Typical microgel sizes range from 0.1 μm to 10μm. Particles with sizes
smaller than ca. 500nm are sometimes referred to as nanogels. The size ranges of
microgels with their corresponding routes of drug administration are shown in
Figure 1. All hydrogel particles in this work are in the submicron size range, but
are referred to as microgels.
Delivery
Ocular
Nasal
Pulmonary
Oral
Intratumoral
Intramuscular
Intravenous
Transdermal
1 μm 2 μm 5 μm 10 μm 20 μm 400 μm
Delivery
Ocular
Nasal
Pulmonary
Oral
Intratumoral
Intramuscular
Intravenous
Transdermal
1 μm 2 μm 5 μm 10 μm 20 μm 400 μm1 μm 2 μm 5 μm 10 μm 20 μm 400 μm
Figure 2 Size ranges of polymer microgels for different modes of drug administration. 17
Overview
___________________________________________________________________________ - 4 -
Research objectives
The work presented herein describes the synthesis and behavior of stimuli-
responsive polymer microgels in different environments, with respect to various
factors including polymer composition, change in temperature, pH, ionic strength,
salt concentration, and solvent quality. Furthermore, the functional roles of
microgels as regulatory components of potential biomedical, diagnostic and drug
release applications were explored. Specifically, biofunctionalized, pH-responsive
microgels were shown to act as effective DDSs for cancer cell targeting. The
temperature-induced volume phase transitions of several microgel systems were
tuned to make them appropriate for use in controlled release biomedical
applications. Hybrid microgels doped with gold nanorods were shown to have
potential use in light-induced drug targeting and release.
Chapter 1 provides a brief introduction to polymer microgels and their
current applications. This chapter provides insight on how the unique stimuli-
responsive properties of polymer microgels may be manipulated and tailored for
specific responsive and sensory applications. Chapter 2 describes the materials and
methods used in the present work. In Chapter 3, a detailed study of ternary
polyampholyte microgels and polyelectrolyte microgels containing weak acidic and
basic groups is presented, with respect to their compositions and environmentally-
responsive behavior, and, with special focus on the electrostatic interactions
between the charged functionalities. In Chapter 4, the swelling response of a
binary polyampholyte microgel functionalized with a zwitterionic monomer with
strong acidic and basic groups, is reported, and shown to exhibit polyelectrolyte
behavior. These zwitterionic microgels were used as templates for the in-situ
synthesis of bimetallic gold and silver nanoparticles.
Overview
___________________________________________________________________________ - 5 -
The rational design of a biofunctionalized pH-responsive DDS for cancer cell
targeting is discussed in Chapter 5. Cytotoxicity studies revealed that this drug-
loaded DDS enhanced cancer cell suppression compared to several control systems.
In Chapter 6 the development of a DDS for light-induced release of a drug from
poly(NIPAm)-based microgels is described. The various synthetic routes we
explored in order to tailor the thermally-responsive volume transitions of microgels
to be sharp and large within physiologically useful conditions are presented. The
preparation of hybrid microgels by sequestering gold nanorods into the
aforementioned microgel systems is described, their photothermally-triggered
volume transitions under physiological conditions is reported, and their potential
applications for thermally and photothermally-triggered drug release is
demonstrated. The results of studies evaluating the influence of coulombic forces
on the successful physical incorporation of gold NRs in poly(NIPAm)-based microgels
are presented in Chapter 7. It was determined that electrostatics alone are not the
governing interaction that enable poly(NIPAm)-based microgels to be loaded with
gold nanorods. These findings are important because electrostatic and hydrophobic
interactions are of fundamental importance to the performance of microgels as
carriers for DDSs. Hence all properties of the interacting components of stimuli-
responsive microgels must be better understood for realizing DDSs with high
performance capacities. Finally, Chapter 8 remarks on the future outlook of this
work.
Overview
___________________________________________________________________________ - 6 -
References
[1] B. R. Saunders and B. Vincent, Advances in Colloid and Interface Science 1999, 80, 1-25.
[2] S. Bhattacharya, F. Eckert, V. Boyko and A. Pich, Small 2007, 3, 650-657.
[3] R. Pelton, Advances in Colloid and Interface Science 2000, 85, 1-33.
[4] A. E. Routh and B. Vincent, Journal of Colloid and Interface Science 2004, 273, 435-441.
[5] M. Andersson and S. L. Maunu, Journal of Polymer Science Part B-Polymer Physics
2006, 44, 3305-3314.
[6] K. S. Kim and B. Vincent, Polymer Journal 2005, 37, 565-570.
[7] V. T. Pinkrah, A. E. Beezer, B. Z. Chowdhry, L. H. Gracia, V. J. Cornelius, J. C. Mitchell,
V. Castro-Lopez and M. J. Snowden, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2005, 262, 76-80.
[8] M. J. Molina, M. R. Gomez-Anton and I. F. Pierola, Journal of Physical Chemistry B
2007, 111, 12066-12074.
[9] T. Hoare and R. Pelton, Langmuir 2004, 20, 2123-2133.
[10] K. Ogawa, A. Nakayama and E. Kokufuta, Langmuir 2003, 19, 3178-3184.
[11] H. Ni, H. Kawaguchi and T. Endo, Macromolecules 2007, 40, 6370-6376.
[12] S. Nayak and L. A. Lyon, Abstracts of Papers of the American Chemical Society 2003,
226, U397-U398.
[13] M. J. Murray and M. J. Snowden, Advances in Colloid and Interface Science 1995, 54,
73-91.
[14] S. Nayak, H. Lee, J. Chmielewski and L. A. Lyon, Journal of the American Chemical
Society 2004, 126, 10258-10259.
[15] M. Das, S. Mardyani, W. C. W. Chan and E. Kumacheva, Advanced Materials 2006, 18,
80-83.
Overview
___________________________________________________________________________ - 7 -
[16] K. S. Kim and N. B. Graham, Journal of Industrial and Engineering Chemistry 1998, 4,
221-225.
Introduction to Polymer Microgels
___________________________________________________________________________ - 8 -
Chapter 1
Introduction to Polymer Microgels
1.1 Definition of microgels
Polymer microgels are crosslinked colloidal particles with a network
structure that are swollen in a suitable solvent.[1] Aqueous colloidal microgels
(where the solvent is water) are referred to as hydrogels. The past decade has seen
microgels receive increasing attention in theoretical studies on soft matter[2] and in
applied fields.[3-7] In particular, they have rapidly gained importance in materials
science fields owing to their potential applications in drug delivery,[5, 8-28]
sensing,[18, 29-31] the fabrication of photonic crystals,[13, 32-34] template-based
synthesis of inorganic nanoparticles,[33, 35-41] and separation and purification
technologies.[42-45]
Chapter 1
___________________________________________________________________________ - 9 -
The vast array of applications that microgels are suitable for arises from
their stimulus-responsive nature, that is, their ability to undergo reversible volume
phase transitions in response to external stimuli such as a change in pH,[46-50]
temperature,[46, 51-54] ionic strength of the surrounding medium,[50, 55, 56] quality of
solvent,[57, 58] and the action of an external electromagnetic field.[37, 59-62] The
swelling and deswelling transitions of stimulus-responsive microgels are governed
by the imbalance between repulsive and attractive forces acting within the
particles: swelling occurs when intra-particle ionic repulsion and osmotic forces
exceed attractive forces, such as hydrogen bonding, Van der Waals interactions,
hydrophobic and specific interactions, e.g., biotin-streptavidin binding.
1.2 Classifications of microgels
Microgels are best classified in two ways. Firstly, they may be grouped
according to the chemical or physical nature of the cross-links that are responsible
for their network structure and finite size. Secondly, they may be sorted by their
specific responsive properties, as determined by the types of functional groups
within the particle and the polymer composition.
1.2.1 Physically-crosslinked microgels
In physically crosslinked microgels, network formation occurs via non-
covalent attractive forces such as hydrophobic [63-66] or ionic interactions.[67, 68] The
latter is more prevalent. This physical gelation is ideal for biodegradable systems
that can reversibly go from the solution state to the gel state. Physically cross-
linked microgels have been used for the encapsulation of drugs, cells and proteins,
which are released upon dissolution of the polymer network.
Introduction to Polymer Microgels
___________________________________________________________________________ - 10 -
Physically crosslinked systems are also extremely sensitive to many factors
and may lose stability and fall apart to yield individual polymer molecules under
particular conditions. These factors include polymer composition, temperature,
ionic strength of the medium, as well as the concentrations of the polymer and
cross-linking agent. For example, ionically cross-linked microgels may disintegrate
upon a change in salt concentration.
Physically crosslinked microgels are often constructed from biopolymers.
For example, chitosan particles or their derivatives can be obtained by cross-linking
the polymer either with multifunctional inorganic compounds, such as sodium
tripolyphosphate[26, 69] or with an oppositely charged polymer, such as DNA. Typical
examples of other physically crosslinked biopolymeric microgels include
alginate,[67] dextran, agarose,[70] and carrageenan.[68, 71]
1.2.2 Chemically cross-linked microgels
Chemically cross-linked microgels are relatively more stable than their
physically crosslinked counterparts due to their covalent nature. These microgels
usually maintain a permanent structure unless a labile functionality has been
intentionally added to the network.
Covalently crosslinked microgels are typically synthesized by
copolymerizing monomers in the presence of a multifunctional crosslinking agent.
For example, poly (2-hydroxyethyl methacrylate) is a widely studied microgel
synthesized by polymerizing 2-hydroxy methacrylate with ethylene glycol
dimethacrylate. [72]
Microgels in the size range of 100–1000 nm are typically obtained by free-
radical polymerization[73] or condensation polymerization.[74] A wide variety of
Chapter 1
___________________________________________________________________________ - 11 -
monomers including, e.g., styrene,[75] methyl methacrylate,[76] methacrylic acid,[77,
78] divinylbenzene,[79] ethyleneglycoldimethacrylate,[80] N-isopropylacrylamide,[49, 51,
52, 81] N-isopropylmethacrylamide,[82, 83] t-butylacrylamide,[11] and N-
diethylacrylamide[84] have been used for microgel synthesis.
1.2.3 Classification based on response
Microgels may also be classified as stimuli-responsive or non-responsive
gels. Non-responsive microgels simply swell upon absorption of water whereas
stimulus-responsive microgels swell or deswell in response to one or more subtle
changes in the environment and are therefore called ‘smart’ materials. These
include changes in temperature, pH, electric field, magnetic field and specific
biomolecules/enzymes. Multiresponsive microgels are responsive to several of
these environmental stimulii. The microgels studied in this work are derivatives of
the thermosensitive, water-soluble monomer, N-isopropylacrylamide (NIPAm).
1.3 Thermoresponsive poly(N-isopropylacrylamide) systems
Poly (NIPAm) is a well known thermo-responsive polymer that has been
widely used to prepare temperature-responsive hydrogels. It is typically
synthesized by free radical redox polymerizations, details of which are provided in
Chapter 2. The following section briefly reviews the unique temperature-responsive
properties of poly (NIPAm) systems.
Introduction to Polymer Microgels
___________________________________________________________________________ - 12 -
1.3.1 Solution behavior of poly(NIPAm)
The solution behavior of a polymer in a solvent depends on polymer-
solvent, polymer-polymer, and solvent-solvent interactions. At low temperatures,
poly(NIPAm) is highly solvated due to hydrogen bonding between the amide
residues on the polymer chain and the water molecules. Furthermore, there is a
‘cage-like’ conformational arrangement of water molecules around the isopropyl
groups (Figure 1-1).[85, 86] This structural arrangement is termed the ‘hydrophobic
effect’.[1] Hence at low temperatures, the polymer-solvent interactions are
stronger than the polymer-polymer interactions and poly (NIPAm) exists in a
random coil state. At elevated temperatures, the hydrogen bonds between the
polymer and the water molecules are broken, leading to an entropically favored
expulsion of water from the polymer network. Consequently the polymer-polymer
interactions become stronger than the polymer-solvent interactions, resulting in
phase separation as the polymer assumes a globule conformation. Figure 1-2 shows
the temperature-induced coil to globule transition of poly(NIPAm). The
temperature at which this phase transition occurs is called the Lower Critical
Solution Temperature (LCST). For poly (NIPAM) the LCST occurs at 32oC in water.[87]
1.3.2 Thermodynamic origin of the phase transition
The LCST of poly(NIPAm) is an entropically driven transition. Heskins and
Guillet[88] first propsed the thermodynamic origin of the LCST. The Gibbs free
energy of the system is given by the following equation:
ΔGm = ΔHm – TΔSm Equation 1
where ΔGm is free energy of mixing, ΔHm is the enthalpy change of mixing, T is
temperature in Kelvin and ΔSm is the entropy change on mixing. At low
Chapter 1
___________________________________________________________________________ - 13 -
temperatures, formation of hydrogen bonds between NIPAm and water reduce the
free energy of mixing (ΔGm) as the enthalpic contribution (ΔHm) is negative.
Structured water around the poly(NIPAm) leads to a loss in entropy (negative ΔSm
term) and a positive entropic contribution. As T increases the positive entropic
contribution to the free energy grows. When the positive entropic contribution
dominates over the enthalpic contribution, phase separation begins.
1.3.3 Poly(NIPAm) macrogels
In bulk poly(NIPAm) macrogels, the LCST of the parent polymer manifests
as the Volume Phase Transition Temperature (VPTT). Below the VPTT, the
macrogels remain in their most swollen, hydrophilic state. Above the VPTT, the
gels deswell going from the swollen, hydrophilic state to the shrunken (relatively
hydrophobic) state. The VPTT depends on several factors: the hydrophobic-
hydrophilic balance, solvency effects and the crosslinking density. The deswelling
rate of hydrogels is inversely proportional to the square of it’s smallest dimension.
Gotoh et al. have shown that gels with a large pore size, and hence faster
deswelling rate can be obtained by polymerizing poly(NIPAm) gels at temperatures
higher than the LCST of the polymer.[84]
Introduction to Polymer Microgels
___________________________________________________________________________ - 14 -
Figure 1-1 Schematic representation of the conformational, ‘cage-like’ arrangement of
water molecules around poly(NIPAm) at temperatures below the LCST of ca. 31oC. The
polymer is highly solvated due to hydrogen bonding between water molecules and
amide residues of poly(NIPAm).
OHN
NHO
H
OH
H
OH
H
OH
HO
HH
OH
HO
H
H O
H
HO
H
H O
H
H
OH
HO
H
HO
HH
O H
OHN
NHO
H
OH
HO
H
H O
H
HO
H HO
H
HO
H
H O
H
HO
H
H O
H
H
OH
HO
H
HO
HH
O H
Increase in Temperature
T ~ 32oC
OHN
NHO
H
OH
H
OH
H
OH
HO
HH
OH
HO
H
H O
H
HO
H
H O
H
H
OH
HO
H
HO
HH
O H
OHN
NHO
H
OH
HO
H
H O
H
HO
H HO
H
HO
H
H O
H
HO
H
H O
H
H
OH
HO
H
HO
HH
O H
OHN
NHO
H
OH
H
OH
H
OH
HO
HH
OH
HO
H
H O
H
HO
H
H O
H
H
OH
HO
H
HO
HH
O H
OHN
NHO
H
OH
HO
H
H O
H
HO
H HO
H
HO
H
H O
H
HO
H
H O
H
H
OH
HO
H
HO
HH
O H
Increase in Temperature
T ~ 32oC
Figure 1-2 Schematic illustration of the structural rearrangement of water molecules
around poly (NIPAm) during the volume phase transition. At temperatures above the
LCST, the hydrogen bonds between water molecules and amide residues break and an
entropically-favored release of water from the polymer network occurs.
Chapter 1
___________________________________________________________________________ - 15 -
Figure 1-3 Schematic depiction of the temperature-induced phase transition in poly
(NIPAm) chains and gels. Diagram is not to scale.
1.3.3 Poly(NIPAm) microgels
Colloidal microgels constructed from poly (NIPAm) range from 50 nm to 5
μm in size and exhibit similar properties to their macrogel counterparts, i.e., they
undergo a volume phase transition (VPT) at the LCST of poly(NIPAm). The VPTT of
the microgels is affected by cross-linking density, solvent nature and composition,
and the nature of the functional groups in the copolymer.[87] Microgels posses
Introduction to Polymer Microgels
___________________________________________________________________________ - 16 -
several advantages over bulk gels: small size and volume, high surface area, faster
response to stimuli and high diffusivity.
1.4 Preparation of microgels
Microgels can be synthesized by a variety of techniques: precipitation
polymerization, miniemulsion polymerization and microemulsion polymerization.
However, the typical synthesis of poly(NIPAm) microgels specifically uses free-
radical precipitation polymerization of NIPAm crosslinked with N,N-methylene-
bisacrylamide (BIS).[89] The cross-linking agent is vital because it prevents the
microgel from dissolving in water at low temperatures.[74] A description of the
synthetic procedure is provided in Chapter 2.
1.5 Characterization of microgels
Several techniques are used to characterize microgels. They include light
scattering, differential scanning calorimetry, fluorometry, small-angle neutron
scattering, UV-VIS spectrophotometry, rheology and NMR. Dynamic light scattering
(DLS) has been used most often to study the solution behavior of microgels. The
temperature-induced volume phase transition of poly (NIPAm) microgels can be
followed by detecting the scattered light. A dilute dispersion of microgels appears
transparent because at T < VPTT the microgels are swollen with water and the
contrast in refractive indices of the polymer and the solvent is small. At T > VPTT,
the expulsion of water from the particles causes an increase in refractive index
contrast between the polymer and the solvent, and the dispersion appears turbid.
Details of this experimental technique are provided in Chapter 2.
Chapter 1
___________________________________________________________________________ - 17 -
1.6 Stimuli-responsive properties of microgels
Microgels are responsive to pH, temperature, ionic strength, action of
electric and magnetic fields, and solvent composition. However, only those
properties pertinent to the applications described in this dissertation are
summarized below.
1.6.1 Effect of temperature
The origin of the thermoresponsive properties of polyNIPAm microgels were
discussed above. At T < VPTT the microgels are individually swollen with water and
at T > VPTT the microgels deswell due to expulsion of water from the microgel
interior.[74] The VPTT of polyNIPAm microgels is slightly higher than the LCST of
linear poly(NIPAm).[90] This shift in the transition temperature results from
increased heterogeneity in the lengths of subchains in the microgels. At T > VPTT,
the regions with longer subchains collapse before the regions with shorter
subchains do, due to the greater magnitude of hydrophobic forces. Thus different
regions undergo the phase transition at slightly different temperatures.
The VPTT of polyNIPAm-based microgels can be shifted by copolymerization
with other reactive functional monomers, due to alteration of the hydrophobic-
hydrophilic balance in the polymer. This effect is discussed in some detail in
Chapter 5. Typically, incorporation of hydrophilic species increases and broadens
the phase transition temperature. Conversely, incorporation of hydrophobic groups
generally decreases the phase transition temperature.
Introduction to Polymer Microgels
___________________________________________________________________________ - 18 -
1.6.2 Effect of pH and ionic strength
Copolymerization of NIPAm with ionic monomers such as acrylic acid,[32]
methacrylic acid,[91] vinyl pyridine,[46, 75] and vinyl imidazole[92] yields microgels
with tunable, multiresponsive properties. Of particular interest is the
functionalization of poly(NIPAm) microgels with carboxylic acid groups, generally
incorporated by copolymerization of NIPAm with acrylic acid (AA) or methacrylic
acid (MAA). The resulting polyelectrolyte microgels undergo volume transitions in
response to change in temperature, pH, and ionic strength.
The dependence of swelling behavior on pH and ionic strength in
polyelectrolyte microgels largely originates from electrostatic interactions between
the ionic groups. For example, poly(NIPAm-AA) microgels undergo a sharp increase
in size at pH ~4.5 due to deprotonation of the carboxylic acids and the resultant
electrostatic repulsion between the negatively charged carboxylate residues (pKa
of AA ~4.25).[50] An increase in ionic strength of the medium causes a decrease in
microgel size: Introduction of an inert electrolyte screens the repulsive interactions
that enhance swelling and results in a deswelling transition. [21]
The concentration of electrolyte in a dispersion of pure poly(NIPAm)
microgels affects colloidal stability of the particles. [93] Saunders and coworkers
observed that at a particular temperature, particles flocculate under higher ionic
strength.[54] This is because cations or anions disrupt the structured water
molecules around poly(NIPAm) at a certain salt concentration and break the H-
bonds.
Thermodynamically speaking, free ions alter the entropic contribution to
the chi parameter within the Flory-Rehner theory. The magnitude of this
contribution is dictated by the position of the salt in the hoffmeister series.
Chapter 1
___________________________________________________________________________ - 19 -
1.6.3 Effect of solvents
Poly(NIPAm) microgels show interesting behavior in mixed solvents due to
cononsolvency of poly(NIPAm).[94] The LCST of poly(NIPAm) decreases with
increasing methanol concentrations until a concentration of 55% methanol is
reached, beyond which, the LCST increases sharply.[95] The same effect is observed
for microgels in mixed solvents and was first observed by McPhee et al.[96] The
mechanism for cononsolvency is explained by the formation of a disordered
tetrahedral arrangement of water molecules about the alcohol that breaks the
existing hydrogen-bonded network in alcohol-water mixtures. This phenomenon is
called clathrate-hydrate formation. In pure aqueous dispersions, water molecules
assume a structured arrangement around hydrophobic isopropyl groups and form
hydrogen bonds with the amide residues of the poly(NIPAm) chain. The addition of
alcohol results in the removal of the water molecules solvating NIPAm to form
clathrate hydrates. This process not only disrupts the existing hydrogen-bonded
network, but also facilitates hydrophobic interactions between isopropyl groups,
subsequently causing microgel shrinkage. At higher volume fractions of alcohol,
when no more water molecules are available for clathrate hydrate formation, the
alcohol can directly interact with the poly(NIPAm), i.e., polymer-solvent
interactions increase, and the microgels swell again.
1.7 Applications of microgels
Over the past decade, microgels have rapidly gained momentum as
intelligent materials due to their stimulus-responsive nature. Several new
applications of microgels have arisen. These include their uses as microreactors for
the synthesis of inorganic nanoparticles (NPs) with predetermined properties, as
Introduction to Polymer Microgels
___________________________________________________________________________ - 20 -
building blocks of photonic crystals, as tunable optical lenses, and as carriers for
targeted drug delivery. The section below briefly charts the functional roles and
properties of microgels in the context of these applications.
1.7.1 Microgels as microreactors
Recently, template-based synthesis of nanoparticles (NPs) in dendrimers,[97,
98] block copolymer micelles,[99-101] star block copolymers,[102, 103] and
polyelectrolyte multilayers[104, 105] has attracted much attention. In comparison
with other polymer template systems, microgels serve as ideal microreactors for NP
formation due to their simple synthesis, easy functionalization, and relatively large
size comparable to the wavelength of visible light. The last feature is important for
optical applications of microgels.
Zhang et al used poly(N-isopropylacrylamide-acrylic acid-2-hydroxyethyl
acrylate) [poly(NIPAm-AA-HEA)] microgels with hydrodynamic diameter of 200-600
nm as templates for the synthesis of three exemplary types of NPs: semiconductor,
metal, and magnetic nanoparticles.[35] They optimized the reaction conditions and
microgel compositions to obtain NPs with optical properties that remained
unpreturbed in the microgel host.
Chapter 1
___________________________________________________________________________ - 21 -
Figure 1-4 Scheme showing synthesis of NPs within microgels.[35]
The introduction of acrylic acid into the microgels was motivated by the
need for anionic groups for sequestering metal cations in the microgel interior.
Copolymerization of NIPAm with HEA at [AA]/[NIPAm] = 0.36 decreased the
microgel void size at pH < 4.3, [35] spatially separated the nucleation sites of the
NPs, and enhanced the compatibility of hybrid microgels with a hydrophobic shell.
Figure 1-3 shows a schematic of NP synthesis in microgels. In the first step of stage
1, carboxylic groups of AA were ionized at high pH. The precursor cations were
then introduced into the dispersion and sequestered by the poly(NIPAm-AA-HEA)
microgels. Stage 2 was determined by the type of NPs to be synthesized. In the
case of CdS particles, an aqueous solution of Na2S was slowly introduced into the
dispersion. Silver NPs and nanoclusters were synthesized by the reduction of Ag+
Introduction to Polymer Microgels
___________________________________________________________________________ - 22 -
ions with a reducing agent, NaBH4, and by the use of UV irradiation, respectively.
Magnetic NPs were synthesized by the oxidation of Fe2+ ions.
1.7.2 Microgels as photonic crystals
Hybrid microgels are excellent examples of materials with structural
hierarchy. Coupling of structure- and composition-dependent properties of both
polymer microgels and inorganic nanoparticles opens new avenues in the
production of “smart” materials with many degrees of freedom in controlling their
performance. Hybrid microgels containing NPs that are either synthesized in situ or
preformed, have potential applications as functional building blocks for the
fabrication of photonic crystals. In some applications of photonic materials, the use
of microgels is impeded by the softness of microgel particles (which interferes with
crystallization of colloid particles), the hydrophilic nature of the particles, and
polymer sensitivity to external stimuli (that is, by the very same features that
make microgels useful for other applications). Kumacheva et al circumvented these
limitations by encapsulating hybrid poly(NIPAm-AA-HEA) microgels with a dense
hydrophobic shell of a copolymer of methyl methacrylate, butyl acrylate, and
acrylic acid (MMA-BA-AA).[13] The narrow polydispersity, negative charge, and
smooth surface of the these hybrid core-shell particles carrying CdS and Ag NPs in
their cores favored their self-assembly into colloid crystals.
In contrast with the colloidal crystals, described above, Lyon et al. [11]
reported color-tunable colloidal crystals formed by the assembly of
thermoresponsive poly(NIPAm-AA) microgels. Upon centrifugation, the microgels
assembled into a close-packed colloidal crystalline array that displayed striking
irridescence. The Bragg diffraction was modulated by a change in temperature.
Chapter 1
___________________________________________________________________________ - 23 -
The microgels underwent a reversible order-disorder phase transition upon crossing
the VPTT of the particles. At room temperature the system was in the ordered
state and featured a sharp Bragg diffraction peak in the transmission spectrum,
whereas above 32oC, the system behaved as a disordered turbid fluid. Upon
cooling, the microgel dispersion spontaneously reordered with a degree of order
that was equal to or greater than that of the original crystal. Below the VPTT, the
position, breadth and intensity of the diffraction peak, underwent small changes,
whereas above the VPPT, the diffraction peak disappeared due to crystal
disordering. The remarkable tendency of the crystal to reorder allowed the
material to survive extensive physical and chemical manipulation.
Lyon et al.[11] went on to show that the thermoresponsiveness and
associated change in size of the microgels in these colloid crystals could be used to
create color tunability. The size of microgels, and hence the lattice constant of the
colloid crystal and the wavelength of the resulting Bragg peak, were controlled by
carefully modulating the temperature around the range of the VPTT (between 30-
34oC) during particle centrifugation. In this manner, colloid crystals of a
predetermined and tunable color were formed.
1.7.3 Microgels as microlenses
Microgels have also been employed in the fabrication of micro-optical
arrays with dynamically tunable focal lengths. Lyon et al.[106] reported the
fabrication of ordered microlens arrays via the electrostatically driven assembly of
poly(NIPAm-AA) microgels on glass substrates functionalized with
aminopropyltrimethoxysilane. At pH = 6.5 the electrostatic attraction between the
anionic carboxylate groups of the microgels and the amine groups on the substrate
Introduction to Polymer Microgels
___________________________________________________________________________ - 24 -
enabled binding of the particles to the surface. The lensing ability of the microgels
spread on the substrate originated from their hemispherical shape and the
refractive index contrast between the contracted microgel and the medium. A
higher refractive index contrast resulted in a lens with shorter focal length and
improved lens power.
More recently, Lyon et al. [107] reported the fabrication of arrays of photo-
switchable microlenses. These arrays were fabricated by depositing poly(NIPAm-AA)
microgels onto a surface coated with gold nanoparticles. The system was locally
heated by its irradiation with λ = 532 nm photons (the surface plasmon modes of
the Au NPs). Plasmon excitation of the NPs resulted in energy transfer to the
microgels in the form of heat to the microgel particles. The modulation of the focal
length of the microlens arrays was investigated by their illumination with laser light
of various powers at different temperatures and pH values. The microlens arrays
were reported to exhibit enhanced focusing abilities when laser light excitation of
the Au NPs resulted in the heating of the poly(NIPAm-AA) microgels to a
temperature greater than their VPTT. Given their inherently swift deswelling
response, simple fabrication techniques, and the dynamic tunability of focal
length, microgel-based microlens arrays are promising devices for the future
development of micro-optics technologies.
1.7.4 Microgels for drug delivery
One of the key areas of intensive research is the application of microgels in
controlled drug delivery. The open network structure of microgels can be used to
incorporate small molecules such as drugs in their interiors while their large
swelling-deswelling transitions may be employed as physico-chemo-mechanical
Chapter 1
___________________________________________________________________________ - 25 -
triggers to direct release of the drugs. In addition to pH, ionic strength, or
temperature-triggered volume transitions, microgels loaded with a drug can
interact with biological components or events such as enzymatic processes that
would activate the release of the drug. Functionalization of microgels allows one to
tune their volume transitions in physiologically relevant conditions. Furthermore,
by attaching receptor-specific proteins to the microgel surface, one can achieve
selective targeting ability designed to treat specific diseases or specific tumor
cells.
The primary triggers that are used in microgel-based drug delivery systems
are pH and temperature. Langer et al.[108] and Frechet et al.[5] reported pH-
triggered nonspecific release of a drug from submicron-sized microgel particles to
the macrophages. These particles, however, were too large to reach tumor sites.
Lyon et al.[10] reported folate-mediated cell targeting with 270nm-sized
poly(NIPAm) microgels that exhibited temperature-dependent cytotoxicity. This
cytotoxicity was attributed to aggregation of particles in the cytosol at elevated
temperatures. Soppimath et al. [109] reported poly(NIPAm-co-dimethylacrylamide-
co-undecanoic acid) microgels that were stable at pH = 7.4 and 37oC but that
aggregated in an acidic environment, triggering the release of drug molecules.
These particles, however, were not bioconjugated or tested in the cell
environment.
We have demonstrated the use of two types of biofunctionalized, pH-
responsive, drug-loaded microgels for targeted intracellular delivery to HeLa
cancer cells[110] poly(NIPAm-AA) and the biopolymeric, chitosan-based microgels.[26]
For the former system, we used the pH-triggered deswelling of the microgel,
leading to the forced expulsion of the drug from its interior, whereas for the latter
Introduction to Polymer Microgels
___________________________________________________________________________ - 26 -
system, pH-induced swelling favored diffusion-controlled release of the drug.
Biopolymeric microgels like chitosan and carrageenan are increasingly being
investigated as more desirable drug carriers owing to their biocompatibility and
reduced cytotoxicity.
Hybrid microgels with photothermally modulated volume transitions also
have promising applications in drug delivery. To induce photothermal transitions,
typically photosensitive moieties like dyes or metal nanoparticles[111-116] are
incorporated within microgels and irradiated at their resonance wavelengths.
Conversion of light energy to heat through nonradiative relaxation causes hydrogel
heating and, for polymers with a lower critical solution temperature (LCST), leads
to microgel deswelling. For applications of photothermally-responsive microgels as
drug delivery carriers, it is critical that the resonance wavelengths of the
photosensitive moieties occur in the spectral range from 800 nm to 1200 nm,
known as the water window, since this range can penetrate body tissues.
1.8 Conclusions
Stimulus-responsive polymer microgels swell and shrink reversibly upon
exposure to various environmental stimuli such as change in pH, temperature, ionic
strength or magnetic fields. Their responsive properties make them ideal
candidates for smart materials. In particular, temperature and pH-sensitive
poly(NIPAm)-based microgels are of interest for biological and optical applications
and have been researched extensively in the past decade. Significant areas of
development include the use of microgels for the templated synthesis of inorganic
nanoparticles with pre-determined properties, as optically active materials
including lenses and photonic crystals, and as primary carriers in site-specific and
Chapter 1
___________________________________________________________________________ - 27 -
controlled drug delivery systems. Facile synthesis and functionalization of microgel
particles provide a broad range of variables for tuning their properties and
favorably distinguishes them from other particulate polymer materials used for
similar applications.
Introduction to Polymer Microgels
___________________________________________________________________________ - 28 -
1.9 References for Chapter 1
[1] B. R. Saunders and B. Vincent, Advances in Colloid and Interface Science 1999, 80, 1-25.
[2] H. Senff, W. Richtering, C. Norhausen, A. Weiss and M. Ballauff, Langmuir 1999, 15,
102-106.
[3] B. Jeong and A. Gutowska, Trends in Biotechnology 2002, 20, 360-360.
[4] Y. Ogawa, K. Ogawa, B. L. Wang and E. Kokufuta, Langmuir 2001, 17, 2670-2674.
[5] N. Murthy, M. C. Xu, S. Schuck, J. Kunisawa, N. Shastri and J. M. J. Frechet,
Proceedings of the National Academy of Sciences of the United States of America 2003, 100,
4995-5000.
[6] J. Mrkic and B. R. Saunders, Journal of Colloid and Interface Science 2000, 222, 75-82.
[7] L. M. Liz-Marzan, D. J. Norris, M. G. Bawendi, T. Betley, H. Doyle, P. Guyot-Sionnest,
V. I. Klimov, N. A. Kotov, P. Mulvaney, C. B. Murray, D. J. Schiffrin, M. Shim, S. Sun and
C. Wang, Mrs Bulletin 2001, 26, 981-+.
[8] L. Bromberg, M. Temchenko and T. A. Hatton, Langmuir 2002, 18, 4944-4952.
[9] V. C. Lopez, J. Hadgraft and M. J. Snowden, International Journal of Pharmaceutics
2005, 292, 137-147.
[10] S. Nayak, H. Lee, J. Chmielewski and L. A. Lyon, Journal of the American Chemical
Society 2004, 126, 10258-10259.
[11] C. M. Nolan, C. D. Reyes, J. D. Debord, A. J. Garcia and L. A. Lyon,
Biomacromolecules 2005, 6, 2032-2039.
[12] M. V. S. Varma, A. M. Kaushal and S. Garg, Journal of Controlled Release 2005, 103,
499-510.
[13] S. Q. Xu, J. G. Zhang, C. Paquet, Y. K. Lin and E. Kumacheva, Advanced Functional
Materials 2003, 13, 468-472.
Chapter 1
___________________________________________________________________________ - 29 -
[14] V. Alakhov, G. Pietrzynski, K. Patel, A. Kabanov, L. Bromberg and T. A. Hatton,
Journal of Pharmacy and Pharmacology 2004, 56, 1233-1241.
[15] B. G. De Geest, C. Dejugnat, E. Verhoeven, G. B. Sukhorukov, A. M. Jonas, J. Plain, J.
Demeester and S. C. De Smedt, Journal of Controlled Release 2006, 116, 159-169.
[16] B. G. De Geest, B. G. Stubbe, A. M. Jonas, T. Van Thienen, W. L. J. Hinrichs, J.
Demeester and S. C. De Smedt, Biomacromolecules 2006, 7, 373-379.
[17] J. X. Gu, F. Xia, Y. Wu, X. Z. Qu, Z. Z. Yang and L. Jiang, Journal of Controlled
Release 2007, 117, 396-402.
[18] T. Hoare and R. Pelton, Macromolecules 2007, 40, 670-678.
[19] P. F. Kiser, G. Wilson and D. Needham, Journal of Controlled Release 2000, 68, 9-22.
[20] A. Jalil and H. Uludag, Materialwissenschaft Und Werkstofftechnik 2004, 35, 972-979.
[21] V. T. Pinkrah, A. E. Beezer, B. Z. Chowdhry, L. H. Gracia, V. J. Cornelius, J. C.
Mitchell, V. Castro-Lopez and M. J. Snowden, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2005, 262, 76-80.
[22] I. Lynch, P. de Gregorio and K. A. Dawson, Journal of Physical Chemistry B 2005, 109,
6257-6261.
[23] M. Malmsten, Soft Matter 2006, 2, 760-769.
[24] J. P. K. Tan and K. C. Tam, Journal of Controlled Release 2007, 118, 87-94.
[25] S. V. Vinogradov, Current Pharmaceutical Design 2006, 12, 4703-4712.
[26] H. Zhang, S. Mardyani, W. C. W. Chan and E. Kumacheva, Biomacromolecules 2006, 7,
1568-1572.
[27] C. M. Nolan, L. T. Gelbaum and L. A. Lyon, Biomacromolecules 2006, 7, 2918-2922.
[28] S. M. Standley, I. Mende, S. L. Goh, Y. J. Kwon, T. T. Beaudette, E. G. Engleman and J.
M. J. Frechet, Bioconjugate Chemistry 2007, 18, 77-83.
Introduction to Polymer Microgels
___________________________________________________________________________ - 30 -
[29] V. Lapeyre, I. Gosse, S. Chevreux and V. Ravaine, Biomacromolecules 2006, 7, 3356-
3363.
[30] J. B. Qu, L. Y. Chu, M. Yang, R. Xie, L. Hu and W. M. Chen, Advanced Functional
Materials 2006, 16, 1865-1872.
[31] J. R. Retama, B. Lopez-Ruiz and E. Lopez-Cabarcos, Biomaterials 2003, 24, 2965-2973.
[32] C. D. Jones and L. A. Lyon, Macromolecules 2000, 33, 8301-8306.
[33] S. Q. Xu, J. G. Zhang and E. Kumacheva, Composite Interfaces 2003, 10, 405-421.
[34] L. A. Lyon, J. D. Debord, S. B. Debord, C. D. Jones, J. G. McGrath and M. J. Serpe,
Journal of Physical Chemistry B 2004, 108, 19099-19108.
[35] J. G. Zhang, S. Q. Xu and E. Kumacheva, Journal of the American Chemical Society
2004, 126, 7908-7914.
[36] J. G. Zhang, S. Q. Xu and E. Kumacheva, Advanced Materials 2005, 17, 2336-+.
[37] D. Suzuki and H. Kawaguchi, Langmuir 2005, 21, 8175-8179.
[38] J. Kim, N. Singh and L. A. Lyon, Biomacromolecules 2007, 8, 1157-1161.
[39] M. Schierhorn and L. M. Liz-Marzan, Nano Letters 2002, 2, 13-16.
[40] P. Ulanski, W. Pawlowska, S. Kadlubowski, A. Henke, R. Gottlieb, K. F. Arndt, L.
Bromberg, T. A. Hatton and J. M. Rosiak, Polymers for Advanced Technologies 2006, 17,
804-813.
[41] Y. Lu, Y. Mei, M. Drechsler and M. Ballauff, Angewandte Chemie-International Edition
2006, 45, 813-816.
[42] P. Nilsson and P. Hansson, Journal of Physical Chemistry B 2005, 109, 23843-23856.
[43] L. Bromberg, M. Temchenko and T. A. Hatton, Langmuir 2003, 19, 8675-8684.
[44] P. Li and A. K. SenGupta, Reactive & Functional Polymers 2000, 44, 273-287.
[45] R. Barreiro-Iglesias, C. Alvarez-Lorenzo and A. Concheiro, Journal of Controlled
Release 2001, 77, 59-75.
Chapter 1
___________________________________________________________________________ - 31 -
[46] K. S. Kim and B. Vincent, Polymer Journal 2005, 37, 565-570.
[47] Y. M. Mohan, K. Lee, T. Premkumar and K. E. Geckeler, Polymer 2007, 48, 158-164.
[48] G. Nisato, J. P. Munch and S. J. Candau, Langmuir 1999, 15, 4236-4244.
[49] T. Hoare and R. Pelton, Langmuir 2004, 20, 2123-2133.
[50] M. J. Snowden, B. Z. Chowdhry, B. Vincent and G. E. Morris, Journal of the Chemical
Society-Faraday Transactions 1996, 92, 5013-5016.
[51] P. J. Dowding, B. Vincent and E. Williams, Journal of Colloid and Interface Science
2000, 221, 268-272.
[52] M. Andersson and S. L. Maunu, Colloid and Polymer Science 2006, 285, 293-303.
[53] X. M. Ma, Y. J. Cui, X. Zhao, S. X. Zheng and X. Z. Tang, Journal of Colloid and
Interface Science 2004, 276, 53-59.
[54] E. Daly and B. R. Saunders, Langmuir 2000, 16, 5546-5552.
[55] T. Lopez-Leon, A. Elaissari, J. L. Ortega-Vinuesa and D. Bastos-Gonzalez,
Chemphyschem 2007, 8, 148-156.
[56] M. J. Snowden, D. Thomas and B. Vincent, Analyst 1993, 118, 1367-1369.
[57] M. J. Molina, M. R. Gomez-Anton and I. F. Pierola, Journal of Physical Chemistry B
2007, 111, 12066-12074.
[58] S. Dragan, L. Ghimici and C. Wandrey, Macromolecular Symposia 2004, 211, 107-119.
[59] S. Bhattacharya, R. A. Moss, H. Ringsdorf and J. Simon, Langmuir 1997, 13, 1869-1872.
[60] B. Brugger and W. Richtering, Advanced Materials 2007, 19, 2973-+.
[61] D. Duracher, A. Elaissari and C. Pichot, Journal of Polymer Science Part a-Polymer
Chemistry 1999, 37, 1823-1837.
[62] C. Menager, O. Sandre, J. Mangili and V. Cabuil, Polymer 2004, 45, 2475-2481.
[63] A. Omari, G. Chauveteau and R. Tabary, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2003, 225, 37-48.
Introduction to Polymer Microgels
___________________________________________________________________________ - 32 -
[64] W. de Carvalho and M. Djabourov, Rheologica Acta 1997, 36, 591-609.
[65] T. Nishikawa, K. Akiyoshi and J. Sunamoto, Journal of the American Chemical Society
1996, 118, 6110-6115.
[66] N. Morimoto, T. Endo, M. Ohtomi, Y. Iwasaki and K. Akiyoshi, Macromolecular
Bioscience 2005, 5, 710-716.
[67] C. Ouwerx, N. Velings, M. M. Mestdagh and M. A. V. Axelos, Polymer Gels and
Networks 1998, 6, 393-408.
[68] H. Zhang, E. Tumarkin, R. Peerani, Z. Nie, R. M. A. Sullan, G. C. Walker and E.
Kumacheva, Journal of the American Chemical Society 2006, 128, 12205-12210.
[69] S. M. Kuo, G. C. C. Niu, S. J. Chang, C. H. Kuo and M. S. Bair, Journal of Applied
Polymer Science 2004, 94, 2150-2157.
[70] D. Bulone and P. L. S. Biagio, Biophysical Journal 1990, 57, A256-A256.
[71] J. Ortiz and J. M. Aguilera, Food Science and Technology International 2004, 10, 223-
232.
[72] G. M. Eichenbaum, P. F. Kiser, A. V. Dobrynin, S. A. Simon and D. Needham,
Macromolecules 1999, 32, 4867-4878.
[73] M. Das, H. Zhang and E. Kumacheva, Annual Review of Materials Research 2006, 36,
117-142.
[74] R. Pelton, Advances in Colloid and Interface Science 2000, 85, 1-33.
[75] A. Loxley and B. Vincent, Colloid and Polymer Science 1997, 275, 1108-1114.
[76] I. Kaneda and B. Vincent, Journal of Colloid and Interface Science 2004, 274, 49-54.
[77] H. Ni, H. Kawaguchi and T. Endo, Macromolecules 2007, 40, 6370-6376.
[78] J. Xu, F. Zeng, S. Z. Wu, X. X. Liu, C. Hou and Z. Tong, Nanotechnology 2007, 18.
[79] T. K. Bronich, S. Bontha, L. S. Shlyakhtenko, L. Bromberg, T. A. Hatton and A. V.
Kabanov, Journal of Drug Targeting 2006, 14, 357-366.
Chapter 1
___________________________________________________________________________ - 33 -
[80] H. Tobita and Y. Yoshihara, Journal of Polymer Science Part B-Polymer Physics 1996,
34, 1415-1422.
[81] M. L. Christensen and K. Keiding, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2005, 252, 61-69.
[82] I. Berndt and W. Richtering, Macromolecules 2003, 36, 8780-8785.
[83] I. Berndt, J. S. Pedersen, P. Lindner and W. Richtering, Langmuir 2006, 22, 459-468.
[84] T. Gotoh, Y. Nakatani and S. Sakohara, Journal of Applied Polymer Science 1998, 69,
895-906.
[85] M. Bradley and B. Vincent, Langmuir 2005, 21, 8630-8634.
[86] H. Ringsdorf, J. Venzmer and F. M. Winnik, Macromolecules 1991, 24, 1678-1686.
[87] J. Huang and X. Y. Wu, Journal of Polymer Science Part a-Polymer Chemistry 1999, 37,
2667-2676.
[88] M. Heskins and J. E. Guillet, Journal of Macromolecular Science. Part A, Pure &
Applied Chemistry 1968, 2, 1441-1455.
[89] D. J. Gan and L. A. Lyon, Journal of the American Chemical Society 2001, 123, 7511-
7517.
[90] C. Z. Wu, S. , Macromolecules 1997, 30, 574-576.
[91] K. S. Kim, M. H. Kim and S. H. Cho, Journal of Industrial and Engineering Chemistry
2005, 11, 736-742.
[92] B. Isik, Advances in Polymer Technology 2003, 22, 246-251.
[93] A. F. Routh and B. Vincent, Langmuir 2002, 18, 5366-5369.
[94] H. M. Crowther and B. Vincent, Colloid and Polymer Science 1998, 276, 46-51.
[95] F. M. Winnik, H. Ringsdorf and J. Venzmer, Macromolecules 1990, 23, 2415-2416.
[96] W. McPhee, K. C. Tam and R. Pelton, Journal of Colloid and Interface Science 1993,
156, 24-30.
Introduction to Polymer Microgels
___________________________________________________________________________ - 34 -
[97] K. Sooklal, L. H. Hanus, H. J. Ploehn and C. J. Murphy, Advanced Materials 1998, 10,
1083-+.
[98] B. I. Lemon and R. M. Crooks, Journal of the American Chemical Society 2000, 122,
12886-12887.
[99] M. Moffitt, L. McMahon, V. Pessel and A. Eisenberg, Chemistry of Materials 1995, 7,
1185-1192.
[100] M. Moffitt and A. Eisenberg, Chemistry of Materials 1995, 7, 1178-1184.
[101] Q. Wang, Y. B. Zhao, Y. J. Yang, H. B. Xu and X. L. Yang, Colloid and Polymer
Science 2007, 285, 515-521.
[102] M. Filali, M. A. R. Meier, U. S. Schubert and J. F. Gohy, Langmuir 2005, 21, 7995-
8000.
[103] J. H. Youk, M. K. Park, J. Locklin, R. Advincula, J. Yang and J. Mays, Langmuir 2002,
18, 2455-2458.
[104] F. Caruso, M. Spasova, A. Susha, M. Giersig and R. A. Caruso, Chemistry of Materials
2001, 13, 109-116.
[105] J. W. Ostrander, A. A. Mamedov and N. A. Kotov, Journal of the American Chemical
Society 2001, 123, 1101-1110.
[106] J. H. Kim and T. R. Lee, Chemistry of Materials 2004, 16, 3647-3651.
[107] S. M. Kim JS, Lyon LA. , Angew. Chem. Int. Ed. 2005., 44:, 1333--1336.
[108] D. A. LaVan, D. M. Lynn and R. Langer, Nature Reviews Drug Discovery 2002, 1, 77-
84.
[109] K. S. Soppimath, D. C. W. Tan and Y. Y. Yang, Advanced Materials 2005, 17, 318-+.
[110] M. Das, S. Mardyani, W. C. W. Chan and E. Kumacheva, Advanced Materials 2006,
18, 80-83.
[111] S. Nayak and L. A. Lyon, Chemistry of Materials 2004, 16, 2623-2627.
Chapter 1
___________________________________________________________________________ - 35 -
[112] A. Suzuki and T. Tanaka, Nature 1990, 346, 345-347.
[113] C. E. Reese, A. V. Mikhonin, M. Kamenjicki, A. Tikhonov and S. A. Asher, Journal of
the American Chemical Society 2004, 126, 1493-1496.
[114] C. Wang, N. T. Flynn and R. Langer, Advanced Materials 2004, 16, 1074-+.
[115] M. Q. Zhu, L. Q. Wang, G. J. Exarhos and A. D. Q. Li, Journal of the American
Chemical Society 2004, 126, 2656-2657.
[116] C. D. Jones and L. A. Lyon, Journal of the American Chemical Society 2003, 125, 460-
465.
Materials and Methods
___________________________________________________________________________ - 36 -
Chapter 2
Materials and Methods
The present chapter covers the basic synthetic and characterization
methods used in this work. The standard synthetic procedure for preparation of
microgels by free radical precipitation polymerization is discussed. A brief account
of the instrumental methods used for particle characterization is also provided.
2.1 Preparation of microgels
2.1.1 Reagents
N-isopropylacrylamide (NIPAm), acrylic acid (AA), vinylimidazole(VI),
maleic acid(MA), undecanoic acid (UA), butylmethacrylate (BMA), potassium
persulfate (KPS), N,N-methylenebisacrylamide (BIS), were purchased from Aldrich
(Canada) and used as received. Deionized water with a resistance of 18.2 MΩ
(Millipore Milli-Q) was used. Figure 3-1 shows the structures and functions of the
reactants used for microgel synthesis in this work.
Chapter 2
___________________________________________________________________________ - 37 -
N-isopropylacrylamide (NIPAm) Major monomer
Structure Name Function
O
HN
O
HN
HN
O
N,N- methylenebisacrylamide (BIS) Crosslinker
Neutral Thermo-responsive
component
OS
OO
SO
O
O O
OPotassium persulfate (KPS) Anionic Initiator
SO
OO Sodium dodecyl sulfate (SDS) Anionic surfactant
N
Cetyltrimethylammoniumbromide(CTAB) Cationic surfactant
Butylmethacrylate (BMA) Comonomer
O
O
hydrophobic component
O
HN
N-isopropylmethacrylamide (NIPMAm) Neutral ComonomerThermo-responsive
component
O
OH Acrylic acid (AA) Anionic ComonomerpH-responsive
component
O
OHHO
O
pH-responsive component
Maleic acid (MA) Anionic Comonomer
O
OH
Undecanoic acid (UA) Anionic Comonomer
N-isopropylacrylamide (NIPAm) Major monomer
Structure Name Function
O
HN
O
HN
HN
O
N,N- methylenebisacrylamide (BIS) Crosslinker
Neutral Thermo-responsive
component
OS
OO
SO
O
O O
OPotassium persulfate (KPS) Anionic Initiator
SO
OO Sodium dodecyl sulfate (SDS) Anionic surfactant
N
Cetyltrimethylammoniumbromide(CTAB) Cationic surfactant
N-isopropylacrylamide (NIPAm) Major monomer
Structure Name Function
O
HN
O
HN
O
HN
HN
OO
HN
HN
O
N,N- methylenebisacrylamide (BIS) Crosslinker
Neutral Thermo-responsive
component
OS
OO
SO
O
O O
O
OS
OO
SO
O
O O
OPotassium persulfate (KPS) Anionic Initiator
SO
OO
SO
OO Sodium dodecyl sulfate (SDS) Anionic surfactant
NN
Cetyltrimethylammoniumbromide(CTAB) Cationic surfactant
Butylmethacrylate (BMA) Comonomer
O
O
hydrophobic component
O
HN
N-isopropylmethacrylamide (NIPMAm) Neutral ComonomerThermo-responsive
component
O
OH Acrylic acid (AA) Anionic ComonomerpH-responsive
component
O
OHHO
O
pH-responsive component
Maleic acid (MA) Anionic Comonomer
O
OH
Undecanoic acid (UA) Anionic Comonomer
Butylmethacrylate (BMA) Comonomer
O
O
O
O
hydrophobic component
O
HN
O
HN
N-isopropylmethacrylamide (NIPMAm) Neutral ComonomerThermo-responsive
component
O
OH
O
OH Acrylic acid (AA) Anionic ComonomerpH-responsive
component
O
OHHO
O O
OHHO
O
pH-responsive component
Maleic acid (MA) Anionic Comonomer
O
OH
O
OH
Undecanoic acid (UA) Anionic Comonomer
Figure 2-1 Structures and functions of the reactants used in free radical
precipitation polymerization for the synthesis of microgels in this work
Materials and Methods
___________________________________________________________________________ - 38 -
2.1.2 Synthesis of microgels
The microgels in this work were prepared by free radical precipitation
polymerization. In this method the primary monomer, NIPAm, and the cross-linker,
BIS are dissolved in ca. 90mL of deionized water, together with comonomers and
stabilizing surfactant at a concentration below the critical micelle concentration
(CMC). The solution is added to a three-necked, jacketed round bottomed flask
connected to a circulating water bath and equipped with a mechanical stirrer,
nitrogen inlet and condenser. The reaction mixture is purged with nitrogen for one
hour to remove any dissolved oxygen that otherwise retards the polymerization.
The solution is then heated to 70 °C, under a gentle stream of nitrogen gas. The
initiator, potassium persulfate (KPS), is dissolved in 10mL of deionized water and
added to the heated solution. The reaction is allowed to proceed for at least four
hours under continuous mechanical stirring at 300 rpm for the duration of the
polymerization. At the end of the reaction the solution is cooled and filtered.
Figure 2-2 depicts the synthetic scheme for preparation of microgels. This process
usually yields monodisperse microgels. Note that originally we followed the
procedures of Vincent [1]and Pelton [2] for the purification of monomers prior to
microgel synthesis. However, we observed no difference in the microgel
composition or size, based on light scattering data, titrations and scanning electron
microscopy (SEM) imaging. Thereafter, we used monomers as received.
Chapter 2
___________________________________________________________________________ - 39 -
O NH O OH
OHO
O
O
OH
OH
O
NH
K2S2O8
70oCx y
O NH O OH
OHO
O
O
OH
OH
O
NH
K2S2O8
70oCx y
Figure 2-2. Scheme of microgel synthesis by redox polymerization. All the monomers
are dissolved in water and the solution is heated to 70°C with surfactant sodium
dodecyl sulfate (SDS). The polymerization is initiated by a free-radical initiator
potassium persulfate (KPS). Comonomers with different functionalities can also be
polymerized in the microgel.
Figure 2-3 illustrates the mechanism of precipitation polymerization. Particle
formation occurs by homogeneous nucleation [2]. Polymerization is carried out at
elevated temperature for two reasons. Firstly, sulfate radicals which initiate the
polymerization are generated at high temperatures. Secondly, after initiation, the
water-soluble oligomers grow until they reach a critical chain length. Beyond this
critical length, the growing chain collapses to form precursor particles. The chain
collapses because the polymerization temperature is higher than the LCST of the
polymer and hence it phase separates. The precursors may then either aggregate
with other precursor particles or deposit onto an existing, colloidally stable
microgel particle. The growing polymer particles typically achieve colloidal
stability with the aid of surfactants and electrostatic stabilization provided by the
ionic groups originating from the initiator. Surfactant is used to prepare smaller
microgels, because in this case, the precursor particles must be stabilized earlier in
the reaction. [3]
Materials and Methods
___________________________________________________________________________ - 40 -
Figure 2-3 Precipitation polymerization. After initiation the oligoradical grows to a
critical length before collapsing on itself to form a precursor particle. The precursor
particle continues to grow either by aggregating with other precursor particles or with
growing oligomers, and eventually the microgel particle precipitates out of solution.
2.1.3 Purification of microgels
Microgel polymerizations often leave a significant amount of linear or
slightly branched polymer, called sol. Sol can be effectively removed by repeated
centrifugation, decantation, and redispersion of the microgels in water. Unreacted
monomer and excess surfactant can be removed by dialysis against daily changes of
water for 14 days. Note that dialysis even over extended periods of time is not
always sufficient to remove all linear polymers or sol from microgel dispersions.
However, a combination of dialysis and repeated centrifugation (4 times or more),
decantation and redispersion techniques at appropriate pH values can effectively
remove linear polymers, from a dispersion of microgels ca. 200nm in diameter.
In the present work, microgels less than 200nm in size were typically
purified by dialysis for 14-21 days against daily changes of deionized water.
(Spectra/Por, MWCO: 12-14,000). Microgels larger than 200nm in size were purified
by repeated centrifugation (up to four times) at 10,000 RPM (25,000G) for 30mins
Chapter 2
___________________________________________________________________________ - 41 -
at room temperature in a temperature-controlled centrifuge, and redispersed in
water or buffer media depending on the requirement.
2.2 Particle characterization
2.2.1 Particle size
Particle dimensions of all samples in this thesis were determined by photon
correlation spectroscopy (PCS), also known as dynamic light scattering (DLS). All
experiments were carried out on a Protein Solutions DynaPro-MS/X. The schematic
layout of the instrument is shown in Figure 2-4. The source is a semiconductor laser
of λ = 832.4 nm. The laser light illuminates the sample through an optical fiber,
and the scattered light is collected by an avalanche photodiode, placed at 90° to
the source. The fluctuations in scattered light intensity are collected, and the
signal fed to the autocorrelator, where the data is used to plot the autocorrelation
function. The time-dependent fluctuations in the scattered intensity of light are
directly related to the rate of diffusion of the particle through the solvent. Hence
the decay of the autocorrelation function is used to calculate the diffusion
coefficient, D.
Materials and Methods
___________________________________________________________________________ - 42 -
Laserλ=832.4nm
AutocorrelatorCPU
Scattered light
Incident light
Sample
Transmitted light
Scattering angle = 90o
PhotodiodeDetector
Laserλ=832.4nm
AutocorrelatorCPUCPU
Scattered light
Incident light
Sample
Transmitted light
Scattering angle = 90o
PhotodiodeDetector
Figure 2-4. Schematic layout of dynamic light scattering (DLS) setup. The diagram is
not to scale. The sample is illuminated and the scattered light intensity is detected at
90o from the laser source, and fed to the autocorrelator. The generated autocorrelator
function is then used to calculate the diffusion coefficient.
Assuming that the particles have random Brownian motion, the
hydrodynamic radius (Rh) of the particles can be calculated from the diffusion
coefficient using the Stokes-Einstein equation,
DTkR b
h πη6= Equation 1
where kb is the Boltzman constant, T is the temperature in Kelvin, and η is the
solvent viscosity .
Chapter 2
___________________________________________________________________________ - 43 -
2.2.2 Particle charge and electrokinetic potential
The electrokinetic potential (ζ-potential or surface charge) of all microgels
in this work was measured to ensure colloidal stability and to verify that ionic
functional groups were successfully incorporated during copolymerization. All
measurements of electrokinetic potential were carried out on the Zetasizer
3000HSA (Malvern Instruments).
Like most colloids dispersed in an aqueous phase, microgels carry charged
groups at the surface, which originate from the initiator fragment or from the
surfactant. This surface charge finds stability by having hydrated counterions from
the aqueous phase spread over the particle surface. These hydrated ions form a
rigid sphere that is stationary with respect to the colloid. A second diffuse layer of
mobile ions forms on top of the rigid sphere and is generally responsible for the
colloid stability of the system. Unlike the inner (Stern) layer in which the
counterions remain fixed at the particle surface, the ions in the outer (Guoy-
Chapman) layer are displaced as the particle moves through the dispersion. The
loss of counterions from the diffuse layer induces a charge at the slipping plane.
When a voltage is applied to this particle solution, the charge at this slipping plane
is termed the zeta potential. The greater the absolute value of zeta potential, the
greater is colloidal stability.
Collectively, the inner and outer shell of ions surrounding the colloid is
termed the electric double layer and is described by the Boltzmann equation:
( )[ ] rrRxpsr /Re −Ψ=Ψ κ Equation 2
where ψr is the potential at a distance r from the center of the particle, R is the
spherical radius of the particle and ψs is the potential just outside the layer of
Materials and Methods
___________________________________________________________________________ - 44 -
bound ions at the beginning of the so-called diffuse layer. Therefore κ is the
exponential constant relating potential with distance away from the particle and is
found to vary according to Equation 3
kTIeN orA εεκ /22 = Equation 3
where NA is the Avogadro number, e is the elementary charge, I is the ionic
strength, ε0 is the permittivity of free space, εr is the relative permittivity of the
medium, k is the Boltzmann constant and T is the temperature. It should be noted
that κ has units of inverse length and for this reason 1/κ gives a measure of the
thickness of the double layer.
-
+
Particle surfaceStern layerSlipping plane
Ψ(X)
Ψ(O)
1/e
1/κ
Zeta-potential
Diffuse layer
Distance/nmDebyelength
+
++++
++++
---
------
-
--
-
+
+
+
+
+
+
+
+
--
++
Particle surfaceStern layerSlipping plane
Ψ(X)
Ψ(O)
1/e
1/κ
Zeta-potential
Diffuse layer
Distance/nmDebyelength
++
++++++++
++++++++
------
------------
--
----
--
++
++
++
++
++
++
++
++
Figure 2-5 Schematic representation of the electrical double layer that surrounds
stable colloidal particles
Chapter 2
___________________________________________________________________________ - 45 -
In this method, electrokinetic potential is obtained by measuring particle
mobility in electrophoresis experiments. A Laser Doppler Velocimeter (LDV) applies
an electrical field of known strength across the sample, through which a laser is
then passed. Charged particles in the dispersion will migrate to the oppositely
charged electrode with a velocity proportional to the magnitude of the zeta
potential. The changing velocity of the moving particle will then induce a
frequency shift in the incident laser beam. The measured electrophoretic
mobility, μe may be calculated by using either the Huckel or Smoluchowski
approximations of 1 or 1.5 respectively for Henry's Function, f(ka ) according to the
following relation,
ηζεμ ⋅
⋅⋅⋅=
3)(2 ka
e
f Equation 4
where ε is the dielectric constant of the sample, η is the viscosity of the liquid
phase, .and ζ is the zeta potential.
Limitations of electrokinetic potential measurements
While zetapotential measurements according to Henry’s methods are fairly
accurate for hard latex particles, they are somewhat ambiguous for soft, highly
swollen particles like microgels due to the absence of a well-defined slipping
plane. Nevertheless, the measurement of electrokinetic potential provides an
estimated indication of the relative differences in surface charge of soft particles
and is used in this work. A more accurate treatment of surface charge of soft
particles is found in Oshima’s theory.[4] Electrophoretic mobility values which scale
with electrokinetic potential but do not assume any specific interfacial geometry
are an oft used alternative to discard any ambiguity.
Materials and Methods
___________________________________________________________________________ - 46 -
2.2.3 Scanning electron microscopy
Detailed information on the theory of scanning electron microscopy (SEM)
and scanning transmission electron microscopy (STEM) can be found elsewhere.[5-7]
SEM and STEM both work through a very similar process. Briefly, a tightly focused
electron beam is directed towards a sample and then scanned through the x, and y
coordinates, similar to the way an electron beam is scanned over the pixels of a
cathode ray tube. The difference in the two techniques lies primarily in the
placement of the detectors. A schematic showing the basic set up for both SEM
and STEM is shown in Figure 2-6
Figure 2-6. Schematic Illustration of Scanning and Transmission Electron Microscope
SEM relies on the detection of secondary electrons that are emitted from a
sample after the impact of an electron from the primary beam. The electron from
the primary beam has sufficient energy to knock an electron out of the valence
Chapter 2
___________________________________________________________________________ - 47 -
shell of an atom that it strikes. This electron will have a much lower energy than
those in the primary beam, and they will be emitted in all directions. Because of
the lower energy of the secondary electrons they will not be able to penetrate any
significant depth of material, and as a result the information garnered from the
SEM primarily gives information about the surface of the sample. The secondary
electron detector is typically placed above the sample, off to one side. The
placement of the detector in this position is what provides the shadows and depth
that are seen in the micrographs. If the secondary electrons are required to pass
through another portion of the sample on their way to the detector, they will be
absorbed or scattered, resulting in a dark spot, or shadow. However if the path for
the secondary electron to the detector is clear a large number of electrons will be
counted, resulting in a bright spot.
STEM differs in that the detector is placed directly behind the sample.
Whereas SEM detects emitted electrons, the STEM detects electrons from the
primary beam that have passed through the sample without being absorbed or
scattered. This allows it to give information about the interior of the sample, but
also limits the use of this technique to relatively thin samples.
2.3 Preparation of gold nanorods
2.3.1 Synthesis of gold nanorods
Gold nanorods were synthesized following the procedure outlined by El
Sayed et al.[8] scaled-up to prepare 100 ml of nanorods suspension in water. Figure
2-7 illustrates the synthesis and growth mechanism of this procedure. A gold seed
solution was prepared by reduction with sodium borohydride (0.5 ml, 10 mM in ice-
cold water) of HAuCl4 (0.12 ml, 5 mM) in 2.5 ml of cetyl trimethyl ammonium
Materials and Methods
___________________________________________________________________________ - 48 -
bromide (CTAB) solution (0.2 M in water). For the preparation of a growth solution,
25 ml of a 0.2 M CTAB solution were mixed with 25 ml of a 0.2 M benzyl dodecyl
BLA BLA (BDAC) solution. To this solution, 5 ml of HAuCl4 (5 mM in water), 2.8 ml of
silver nitrate (4 mM in water) and 40 ml of water were added. Upon addition of 1
ml of 0.8 M ascorbic acid, the dark yellow solution turned colorless. The last step
of the nanorods synthesis was the addition of 1 ml of 5-minute-aged seed solution
to the growth solution. This route allowed for the preparation of gold nanorods
with plasmon bands up to 840 nm. The nanorods were purified by three rounds of
centrifugation at 10000 rpm for 30 min each round. At the end of each round, the
supernatant was discarded and the precipitated nanorods were re-dispersed in
deionized water.
Chapter 2
___________________________________________________________________________ - 49 -
Seed Solution:
Nanorods with aspect ratio to absorb less than 850 nm:
Nanorods with aspect ratio to absorb over 850 nm:
CH3(CH2)15 NCH3CH3
CH3
Br-
CTAB
+ HAuCl4NaBH4 °°°° °
°°°°°
°°°
°
Au seeds ~ 4nm diameter
°°° °
CH3(CH2)15 NCH3CH3
CH3
Br-
CTAB
AgNO3HAuCl4 ascorbic acid
°°°° °°°°° °
°°°
°
Au seeds
Au Nanorods
+
CH3(CH2)15 N
CH3
CH3
Cl-
BDAC
Seed Solution:
Nanorods with aspect ratio to absorb less than 850 nm:
Nanorods with aspect ratio to absorb over 850 nm:
CH3(CH2)15 NCH3CH3
CH3
Br-
CTAB
+ HAuCl4NaBH4 °°°° °
°°°°°
°°°
°
Au seeds ~ 4nm diameter
°°° °
CH3(CH2)15 NCH3CH3
CH3
Br-
CTAB
AgNO3HAuCl4 ascorbic acid
°°°° °°°°° °
°°°
°
Au seeds
Au Nanorods
+
CH3(CH2)15 N
CH3
CH3
Cl-
BDAC
Figure 2-7 Synthetic scheme showing preparation and growth mechanism of gold
nanorods as adapted from the method of El Sayed.[8]
2.3.2 Characterization of gold nanorods
The excitation of surface plasmons by light is denoted by surface plasmon
resonance (SPR). Surface plasmons are surface electromagnetic waves that
propagate in a parallel fashion along a metal/dielectric or a metal/vacuum
interface. Because these waves are at the boundary of the metal and the external
medium (air for example), their oscillations are extremely sensitive to any change
in the ‘boundary’, such as the adsorption of molecules onto the metal surface.
Surface plasmons are typically excited by the incidence of an electron or light
Materials and Methods
___________________________________________________________________________ - 50 -
beam in the infra-red or ultra-violet region. Typical metals that support surface
plasmon are Au or Ag, but other metals can also support plasmon generation such
as copper and titanium.
400 600 800 1000 1200 1400 16000.0
0.5
1.0
1.5
2.0
2.5
400 600 800 1000 1200
a
Abs
orba
nce,
AU
Wavelength, nm
b
dc
Figure 2-8. Absorbance spectra of gold nanorods with aspect ratio of 4.3 in the
pure dispersion (---) and in hybrid microgels (-). Inset shows the shift in absorbance
with change in aspect ratio.
Gold NRs in this work were characterized by their UV-VIS absorption
spectra. Figure 2-8 shows the absorbance spectra of pure dispersion of NRs and, of
NRs loaded in poly(NIPAm-AA) microgels. The peaks occurring at 500 and 800 nm
correspond to the surface plasmons of the cross-sectional end and the long surface
of the NRs respectively. Increase in the aspect ratio (which is also the increase in
length in this case) of the NRs leads to red-shift in the absorbance spectra. The NRs
remain stable in the solution of excess CTAB for three months.
Aspect ratios
a 2
b 2.5
c 4.3
d 6
Chapter 2
___________________________________________________________________________ - 51 -
2.4 References for Chapter 2
[1] M. J. Snowden, B. Z. Chowdhry, B. Vincent and G. E. Morris, Journal of the Chemical
Society-Faraday Transactions 1996, 92, 5013-5016.
[2] R. Pelton, Advances in Colloid and Interface Science 2000, 85, 1-33.
[3] M. Andersson and S. L. Maunu, Journal of Polymer Science Part B-Polymer Physics
2006, 44, 3305-3314.
[4] T. Hoare and R. Pelton, Polymer 2005, 46, 1139-1150.
[5] J. Goldstein, D. Newbury, D. Joy, C. Lyman, P. Echlin, E. Lifshin, L. Sawyer and J.
Michael, Scanning Electron Microscopy and X-Ray Microanalysis, Kluwer Academic/Plenum
Publishers, New York, 2003, p.
[6] L. Reimer, Scanning Electron Microscopy: Physics of Image Formation and
Microanalysis, Springer-Verlag, Heidelberg, 1998, p.
[7] R. E. Lee, Scanning Electron Microscopy and X-Ray Microanalysis, P T R Prentice Hall,
Inc., Englewood Cliffs, 1993, p.
[8] B. Nikoobakht and M. A. El-Sayed, Chemistry of Materials 2003, 15, 1957-1962.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 52 -
Chapter 3
From Polyelectrolyte to Polyampholyte
Microgels
3.1 Introduction
Polymer microgels functionalized with ionic groups are called
polyelectrolyte (PE) microgels.[1, 2] When both cationic and anionic groups are
present along the polymer chain, the particles are referred to as polyampholyte
(PA) microgels.[3-5] The presence of oppositely charged groups in the polymer
network make the properties of PA microgels very different from those of PE
microgels.[5] From a scientific perspective, PA microgels are fascinating systems,
due to the multiple electrostatic interactions acting in parallel with, or competing
against each other within the microgel interior. In the last decade, significant
Chapter 3
___________________________________________________________________________ - 53 -
efforts have focused on understanding the swelling behavior of macroscopic PA
gels[6-11], whose response times are on the order of several hours. The
characteristic response time, τ, of a gel is given by τ = l2/π2D where l is the
characteristic linear size of the gel, and D is the diffusion coefficient of the
network,[12] It follows that microgels offer significantly faster response times than
their macrscopic counterparts, often on the order of seconds and even fractions of
seconds.[13, 14]
Ogawa and coworkers reported the results of systemic studies of the
response of PA microgels to variations in temperature, pH, and salt concentration
in the dispersion medium.[15] Although the authors did not observe any
polyampholyte behavior in KCl solutions, they did observe enhanced colloid
stability for microgels with similar numbers of acidic and basic groups, relative to
their stability in pure water. Conversely, PA microgels with an excess of cationic or
anionic groups were reported to exhibit greater stability in pure water, but
aggregated at higher salt concentrations.[15] The authors attributed their results to
intra- and inter-particle interactions which were not solely dependent on
electrostatic interactions but originated from hydrogen bonding and hydrophobic
association, as well. Nayak and Lyon showed that PA microgels synthesized by
copolymerization of NIPAm, AA and N-(3-Aminopropyl) methacrylamide displayed
zwitterionic behavior in a particular pH range.[16] Temperature-dependent volume
transitions of the PA microgels with balanced compositions of oppositely charged
groups were found to be sharper in the zwitterionic pH range than at a non-
zwitterionic pH,[16] due to the closer proximity of anionic and cationic charges that
permitted ion pair formation and facilitated the expulsion of water from the
microgel interior.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 54 -
3.2 Research objectives
While the aforementioned works provided valuable insight into the
behavior of PA microgels, a comprehensive study of the similarities and differences
between the swelling behavior of PE and PA microgels has not been reported,
though properties of macroscopic PA gels have been extensively studied by
Tanaka.[9, 17] and others.[8, 18] Furthermore, the effect of the compositions of the
dispersion medium on the swelling behavior of PA microgels has not previously
been considered. The effect of solvent is important in two respects. Firstly,
osmotic interactions between the functional groups compete with, or enhance the
interactions of the polymer with the solvent. Secondly, the strength of the
electrostatic forces acting in the microgels depends on the dielectric constant of
the solvent and the extent of dissociation of ionic groups. The present work
endeavours to compare PE and PA microgels and highlights the differences and
similarities in their swelling response to variations in pH, temperature, ionic
strength and solvent composition.
3.3 Background
Volume transitions in polymer gels result from competing attractive and
repulsive interactions, namely polymer rubber elasticity and osmotic swelling.[9, 19-
22] Other governing factors include H-bonding, hydrophobic forces, van der Waals
forces, coulombic interactions, osmotic pressure due to the counter ions, and
specific forces (e.g., biotin-strepavidin interactions).[21-24] Although all the forces
acting in PE microgels are present in PA microgels, the coulombic interactions in PA
and PE microgels are fundamentally different. A schematic of the electrostatically
driven volume transitions in PE and PA microgels is depicted in Figure 3-1. When a
Chapter 3
___________________________________________________________________________ - 55 -
change in pH leads to ionization of the acidic or basic groups (Figures 3-1a and 3-
1b, respectively) the resulting electrostatic repulsion between the like charges
causes the network to swell, leading to an increase in particle size. Figure 3-1c
shows a schematic of the reversible swelling-deswelling transitions of PA microgels.
Figure 3-1 Schematic representation of swelling properties of polyelectrolyte and
polyampholyte microgels. (a) Anionic PE microgels. Ionization of the anionic groups at
high pH and resultant electrostatic repulsion between them causes microgel swelling.
(b) Cationic PE microgels. At low pH, electrostatic repulsion between ionized cationic
groups causes microgel swelling. (c) Polyampholyte (PA) microgels. The PA microgels
are swollen at low and high pH values, due to repulsion between charged cationic and
anionic groups, respectively. In the interim pH region, PA microgels have zwitterionic
properties and contract due to electrostatic attraction between the oppositely charged
groups. For simplicity counterions are omitted.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 56 -
At low pH and high pH, the extent of swelling is enhanced by repulsion between
protonated cationic groups and deprotonated anionic groups respectively. In the
interim region of pH (the ‘zwitterionic region’) a large fraction of both cationic and
anionic groups in the PA microgels exist in their charged state. Hence ion pairing
between them dominates over repulsion between unpaired like charges, leading to
microgel shrinkage and a net reduction in charge. The pH at which the net charge
of the PA microgels is zero, that is, when the positive and negative charges in the
microgels are exactly balanced, is defined as the isoelectric point (pI), and
corresponds to their smallest size in the zwitterionic regime.
3.4 Experimental Procedure
3.4.1 Synthesis and characterization of microgels
Anionic and cationic PE microgels were obtained by free radical
polymerization of N-isopropylacrylamide with acrylic acid (AA) and KPS initiator, or
vinylimidazole (VI) and V50 initiator respectively. PA microgels with different
compositions were obtained by copolymerization of N-isopropylacrylamide with
various amounts of AA and VI, using BIS as crosslinker, and KPS as initiator and SDS
as stabilizing agent. The reaction mixture was adjusted to a pH value of 9 with
potassium hydroxide to ensure that acrylic acid was deprotonated and that VI
remained neutral during the polymerization, so as to help stability. The
concentration of NIPAm and KPS in the reaction mixture was kept constant at 82.3
± 1.3 mol % and 0.1 mol% while the ratios of AA/VI were changed as shown in Table
3-1.After polymerization was complete the microgel dispersion was purified by
dialysis against daily changes of deionized water for 21 days and centrifugation
Chapter 3
___________________________________________________________________________ - 57 -
under ionized conditions and temperature 4oC. Microgel particles were
characterized by DLS and measurements of electrokinetic potential.
Table 3-1 Compositions and characteristics of polyelectrolyte and polyampholyte
microgels
Monomer compositions in Functional groups/particle** reaction mixture* mol% Nacidic Nbasic Nacidic/Nbasic
[NIPAM] [AA] [VI] x 10 4 x 10 4 Rh (nm)*** pIPE-NAA 83.2 21.9 0 33.6 75PE-NVI 82.4 0 17.4 37.9 143PA-0.46 83.6 7.3 5.6 3.9 8.5 0.46 79 5.81PA-0.90 83.3 8.7 4.5 34.3 38.6 0.90 74 5.61PA-1.25 81.1 11.3 4.3 23.4 18.7 1.25 60 5.24PA-1.65 82.7 11.5 2.2 8.8 5.3 1.65 57 4.75
* The concentrations of SDS and BIS in the reaction mixture were 0.05 and 3.5-4 mol
%, respectively
** Data obtained from conductometric titration.
*** Hydrodynamic radius, Rh was measured at room temperature in 0.01M KCl
solution. For PE and PA microgels, Rh was measured at pH = pka and pH = pI, respectively.
3.4.2 Quantitative determination of charged groups in microgels
Simultaneous potentiometric and conductometric titrations with NaOH or
HCl were performed to estimate the amounts of AA and VI residues in the PE
microgels, respectively.
Conductometric and potentiometric titration of polyelectrolyte microgels
At the beginning of the titration, the dispersion of PE microgels were
acidified or alkalized to ensure that AA or VI groups were in their uncharged state.
Figure 3-2 shows that the representative conductivity and potentiometric titration
curves of poly(NIPAm-AA) microgels titrated against NaOH are in reasonable
agreement with each other. Three regions were observed in the conductivity
titration curves of PE microgels, plotted as the variation in conductivity of the
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 58 -
system versus volume of titrant. In the first region, the conductivity of the
dispersion decreased with neutralization of excessive H+ or OH- ions that were
present due to pre-acidification or pre-alkalization. In the second region,
ionization of acidic or basic groups in the polymer caused an increase in
conductivity, and hence the slope of curve conductivity vs volume of titrant. In the
third region, the slope was equal to the theoretical conductivity of the titrant
(NaOH or HCl).
0
2
4
6
8
10
12
0 0.5 1 1.5 2Volume of NaOH (mL)
pH
050
100150200250300350400450
0 0.5 1 1.5 2Volume of NaOH (mL)
Con
duct
ivity
( μS/
cm)
0
2
4
6
8
10
12
0 0.5 1 1.5 2Volume of NaOH (mL)
pH
050
100150200250300350400450
0 0.5 1 1.5 2Volume of NaOH (mL)
Con
duct
ivity
( μS/
cm)
Figure 3-2 Representative potentiometric (top) and conductometric (bottom) titration
curves of poly (NIPAm-AA) microgel (0.2wt%) titrated against NaOH.
Chapter 3
___________________________________________________________________________ - 59 -
The intersection of the extrapolated lines drawn as tangents to the
titration curve in the first and the third regions yielded the equivalence point and
provided the number of consumed moles of H+ or OH- as
Moles of H+ = [(NHCl x V HCl) – (NNaOH x V NaOH(consumed))]
Moles of OH- = [(NNaOH x V NaOH) – (NHCl x V HCl(consumed))]
where V is the volume of NaOH or HCl given in mL.
Conductometric and potentiometric titration of polyampholyte microgels
The potentiometric (top) and conductometric (bottom) titration curves for
PA microgels are shown in Figure 3-3. Acidification of the PA microgels to pH=2.5
prior to titration ensured that the AA residues were uncharged and that VI groups
were protonated thereby minimizing the interactions between oppositely charged
groups. Since the values of pKa for AA and VI are 4.26 and 6.94 respectively, both
functional groups are charged in this pH range. Hence there is some ambiguity in
differentiating whether AA or VI groups are being titrated in this region. However,
due to the lower pKa value of AA and, because the titrant was added slowly and the
solution was permitted to stir for several minutes (upto 20 mins for each data
point) to reach an equilibrium, AA groups were titrated first. When titrating against
NaOH, the first two regions of the titration curves of PA microgels were similar to
that of PE microgels; in the third region conductivity slightly decreased with
neutralization of protonated VI residues and in the fourth region conductivity
increased due to the presence of free OH- - ions. The intercepts of the slopes to
the titration curves in the second and third regions, and, in the third and fourth
regions yielded the end points with respect to COOH and =NH+ groups, respectively.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 60 -
0123456789
10
0 0.5 1 1.5 2Volume of NaoH (mL)
pH
050
100150200250300350400
0 0.5 1 1.5 2Volume of NaOH (mL)
Con
duct
ivity
( μS/
cm)
0123456789
10
0 0.5 1 1.5 2Volume of NaoH (mL)
pH
050
100150200250300350400
0 0.5 1 1.5 2Volume of NaOH (mL)
Con
duct
ivity
( μS/
cm)
Figure 3-3 Representative potentiometric (top) and conductometric (bottom) titration
curves of 0.2 wt% dispersion of polyampholyte microgels (AA/VI = 2) titrated against
NaOH, to determine the number of acidic groups.
Although, some interaction between the charged VI and AA groups in the
zwitterionic pH regime may have occurred, the end points obtained from both
conductometric and potentiometric titrations were verified by comparison with the
pH at the isoelectric point as determined from electrokinetic potential
Chapter 3
___________________________________________________________________________ - 61 -
measurements of the microgels. We found reasonable agreement between all three
values.
The number of –COO – and/or ≡NH+ groups in the microgels was calculated as34
NCOO- = [(moles of H+) NAv ]/ number of particles per unit volume or
NNH+= [(moles of OH-) NAv]/number of particles per unit volume (1)
where NAv is the Avogadro number, NAv = 6.023 x 10 23 molecules mol–1.
The number of particles per unit volume was estimated as V/ vi, where V is
the total volume of particles per unit volume of dispersion (mL) and vi is the mean
volume of a particle. The values V and vi were found as
V = )()(
1−gmLgW
ρ (3.2)
where W is the mass of ‘wet’ microgel in a dialysed dispersion, isolated by filtering
microgels (pore size 0.22 μm) at pI and ρ is density. Since the microgel particles were
highly swollen in water, we assumed ρ = 1.0 g mL –1.
The value of vi, was determined by
vi = 4/3 (π Rh3) (3.3)
The data thus obtained from titrations and measurements of electrokinetic
potential is presented in Table 3-1, along with the molar ratio of acidic/basic
groups, the isoelectric point of PA microgels, and microgel size.
3.5 Results
After synthesis, microgels with different fractions of AA and VI had
different dimensions. To compare the effect of the variation of pH, ionic strength,
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 62 -
temperature and ethanol concentration in the dispersion medium on microgel
swelling, the change in particle size was studied. The data collected is presented
herein as the variation in normalized hydrodynamic radius of microgels (Rh/R0),
where Rh is the average hydrodynamic radius of particles at a given pH, salt
concentration, temperature, or solvent composition, and R0 is the smallest
hydrodynamic radius in each set of measurements.
3.5.1 Effect of pH
0 2 4 6 8 10pH
1.9
1.4
0.9
Rh/R
0
(a)
0 2 4 6 8 10pH
0
-10
-20
-30
-40
-50
ζ (m
V)(a’)
0 2 4 6 8 10pH
0.9
1.1
1.3
1.5
Rh/R
0
(b)
0 2 4 6 8 10pH
80
60
40
20
0
ζ (m
V)
(b’)
0 2 4 6 8 10pH
1.9
1.4
0.9
Rh/R
0
(a)
0 2 4 6 8 10pH
1.9
1.4
0.9
Rh/R
0
0 2 4 6 8 10pH
1.9
1.4
0.90 2 4 6 8 10
pH
1.9
1.4
0.9
Rh/R
0
(a)
0 2 4 6 8 10pH
0
-10
-20
-30
-40
-50
ζ (m
V)(a’)
0 2 4 6 8 10pH
0
-10
-20
-30
-40
-50
ζ (m
V)
0 2 4 6 8 10pH
0
-10
-20
-30
-40
-500 2 4 6 8 10
pH
0
-10
-20
-30
-40
-50
ζ (m
V)(a’)
0 2 4 6 8 10pH
0.9
1.1
1.3
1.5
Rh/R
0
(b)
0 2 4 6 8 10pH
0.9
1.1
1.3
1.5
Rh/R
0
0 2 4 6 8 10pH
0.9
1.1
1.3
1.5
0 2 4 6 8 10pH
0.9
1.1
1.3
1.5
Rh/R
0
(b)
0 2 4 6 8 10pH
80
60
40
20
0
ζ (m
V)
(b’)
0 2 4 6 8 10pH
80
60
40
20
0
ζ (m
V)
0 2 4 6 8 10pH
80
60
40
20
00 2 4 6 8 10
pH
80
60
40
20
0
ζ (m
V)
(b’)
Figure 3-4. Variation in Rh/R0 (a,b) and electrokinetic potential (ζ-potential) (a’, b’) of
PE microgels as a function of pH: (a,a’) poly(NIPAm-AA), R0 = 75 nm; (b,b’) poly(NIPAm-
VI), R0 = 143 nm. The dashed curves are given for eye guidance.
Figure 3-4 shows the variation in the normalized microgel size (left), Rh/R0
and electrokinetic potential (right) of PE microgels. In Figure 3-4a the value of
Chapter 3
___________________________________________________________________________ - 63 -
Rh/Ro of the anionic poly (NIPAm-AA) microgels remained almost constant at
1.0<pH<4.0 while in the range 4.0<pH<5.5 a steep swelling transition occurred.
The increase in size was attributed to electrostatic repulsion between –COO- groups
(pKa of AA = 4.25).[15] The particles continued to swell at pH > 5.5, ultimately
reaching a 100% increase in size compared to that at low pH. The variation in
electrokinetic potential (Figure 3-4a’) correlated with the change in microgel size
in the same pH range. At pH < 4.0, ζ-potential was close to zero, implying that
most of the -COOH groups of AA were not dissociated whereas in the range
4.0<pH<9.0, the value of �-potential reduced to reach the value of ca. -46 mV.
Figures 3-4b and 3-4b’ show the variation in size and electrokinetic
potential respectively, of cationic poly(NIPAm-VI) microgels as a function of pH.
The microgels rapidly swelled with increasing acidity in the range 4.0<pH<6.5
(Figure 3-4b). The swelling was ascribed to repulsion between the protonated
imidazole groups at pH<7.0 (pKa of VI is 6.99)[25, 26]. The dependence of ζ-potential
on pH (Figure 3-4b’) followed the trend in the variation of particle size with the
maximum value of ζ-potential observed at pH ≈ 4.0, highlighting yet again the
dominance of coulombic forces on variation in microgel size. Microgel shrinkage in
the range 2.0<pH<4.0 was attributed to the increased ionic strength of the
medium.
Figure 3-5 shows the variation in Rh/R0 and ζ-potential as a function of pH
for PA microgels with different fractions of cationic and anionic residues. Figure 3-
5a-d (left column) shows that all PA microgels displayed a similar trend: strong
shrinking at 4.0<pH<7.0 (the largest contraction occurring at the isoelectric point),
and two swelling regions on either side of the isoelectric point (pI) (ζ-potential =
0). Since the values of pKa of AA and VI are 4.25[27, 28] and 6.99,[25] respectively, in
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 64 -
the range corresponding to microgel shrinkage the particles carried both positive
and negative charges, that is, showed zwitterionic behavior (corroborated by ζ-
potential measurements, Figure 3-5 a’-d’). Increase in the molar ratio of
AA(anionic) to VI (cationic) residues had two consequences: the shift of pI towards
lower values of pH, and the change in the shape of the curve Rh/R0. The former
effect arose because the number of COO- groups outnumbered the number of NH≡+
groups in the zwitterionic regime. Hence increased acidity was required to
protonate the excess COO- groups (that were not neutralized by NH≡+ moieties) to
reach the isoelectric point. The latter feature revealed itself in the different
extents of swelling of the PA microgels with different compositions. For example,
at pH=4.0, the higher content of VI in PA-0.46 compared to PA-1.65 was reflected
by the values of Rh/R0 of ca. 2.5 and 1.5, respectively (left ‘humps’ in Figures 3-5a
and 3-5d, respectively). Similarly, at pH=7.0, the higher AA content in PA-1.65 vs
PA-0.46 resulted in Rh/R0 values of ca. 2.6 and 1.0, respectively (right “humps” in
Figures 3-5d and 3-5a, respectively). Thus, the swelling profile of PA microgels with
a large fraction of VI resembled that of cationic PE microgels (Figure 3-4b).
Likewise, the swelling curve of microgels with a large fraction of AA (Figure 3-5d)
resembled the anionic PE microgels (Figure 3-4a). By contrast, PA microgels with
more symmetric compositions (PA-0.9 and PA-1.25) showed relatively similar
extents of swelling on either side of the pI: two distinct swelling regions with a
maximum ratio Rh/R0 of ca. 2.5 (Figures 3-5b and 3-5c).
Chapter 3
___________________________________________________________________________ - 65 -
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
-40
-20
0
20
0 2 4 6 8 10pH
Η (m
V)0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
-30
-15
0
15
30
0 2 4 6 8 10pH
Η(m
V)-40
-20
0
20
40
0 2 4 6 8 10pH
Η (m
V)
-40
-20
0
20
0 2 4 6 8 10pH
Η(m
V)
(b) (b')
(a) (a')
(c) (c')
(d) (d')
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
-40
-20
0
20
0 2 4 6 8 10pH
Η (m
V)0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
-30
-15
0
15
30
0 2 4 6 8 10pH
Η(m
V)-40
-20
0
20
40
0 2 4 6 8 10pH
Η (m
V)
-40
-20
0
20
0 2 4 6 8 10pH
Η(m
V)
(b) (b')
(a) (a')
(c) (c')
(d) (d')
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
-40
-20
0
20
0 2 4 6 8 10pH
Η (m
V)0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
-30
-15
0
15
30
0 2 4 6 8 10pH
Η(m
V)-40
-20
0
20
40
0 2 4 6 8 10pH
Η (m
V)
-40
-20
0
20
0 2 4 6 8 10pH
Η(m
V)
(b) (b')
(a) (a')
(c) (c')
(d) (d')
ζ (m
V)ζ
(mV)
ζ (m
V)ζ
(mV)
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
-40
-20
0
20
0 2 4 6 8 10pH
Η (m
V)0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
-30
-15
0
15
30
0 2 4 6 8 10pH
Η(m
V)-40
-20
0
20
40
0 2 4 6 8 10pH
Η (m
V)
-40
-20
0
20
0 2 4 6 8 10pH
Η(m
V)
(b) (b')
(a) (a')
(c) (c')
(d) (d')
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
-40
-20
0
20
0 2 4 6 8 10pH
Η (m
V)0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
-30
-15
0
15
30
0 2 4 6 8 10pH
Η(m
V)-40
-20
0
20
40
0 2 4 6 8 10pH
Η (m
V)
-40
-20
0
20
0 2 4 6 8 10pH
Η(m
V)
(b) (b')
(a) (a')
(c) (c')
(d) (d')
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
-40
-20
0
20
0 2 4 6 8 10pH
Η (m
V)0.5
1
1.5
2
2.5
3
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
0.5
1
1.5
2
2.5
0 2 4 6 8 10pH
Rh
/Ro
-30
-15
0
15
30
0 2 4 6 8 10pH
Η(m
V)-40
-20
0
20
40
0 2 4 6 8 10pH
Η (m
V)
-40
-20
0
20
0 2 4 6 8 10pH
Η(m
V)
(b) (b')
(a) (a')
(c) (c')
(d) (d')
ζ (m
V)ζ
(mV)
ζ (m
V)ζ
(mV)
Figure 3-5. Effect of pH on the variation in Rh/R0 (a-d) and ζ-potential (a’-d’) for
polyampholyte microgels in 0.01M KCl solution at 25oC: (a, a’) PA-0.46, R0 = 79 nm ; (b,
b’) PA-0.9, R0 = 73.8 nm; (c, c’) PA-1.25, R0 = 59.6 nm; (d, d’) PA-1.65 R0 = 57.2 nm.
Dashed lines are drawn as eye guidelines. The horizontal dashed line demarks ζ-
potential = 0
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 66 -
3.5.2 Effect of salt concentration
The effects of electrolyte concentration on the swelling properties of PE
and PA microgels were examined by diluting microgel dispersions with KCl solutions
of different concentration and measuring the change in particle size. Figure 3-6a
shows the variation in normalized size of poly(NIPAm-AA) microgels (pH=7.0) and
poly(NIPAm-VI) microgels (pH=4.0) as a function of electrolyte concentration. In
both dispersions, increase in the concentration of KCl from 10-5 to 2.0 M resulted in
contraction of the microgels. The total shrinkage observed for poly(NIPAm-AA) and
poly(NIPAm-VI) microgels was ca. 75% and 50%, respectively. Such polyelectrolyte
behavior was typical of polyelectrolytes in salt solutions.[29, 30] No particle
aggregation was noticed up to KCl concentration of 1M.
aggr
egat
ion
00.5
11.5
22.5
33.5
44.5
0.000001 0.0001 0.01 1log[KCl](M)
Rh/R
0
0
5
10
15
20
0.000001 0.0001 0.01 1log[KCL] (M)
Rh/R
0
PA-0.46PA-0.9PA-1.25PA-1.65
(a) (b)
aggr
egat
ion
00.5
11.5
22.5
33.5
44.5
0.000001 0.0001 0.01 1log[KCl](M)
Rh/R
0
0
5
10
15
20
0.000001 0.0001 0.01 1log[KCL] (M)
Rh/R
0
PA-0.46PA-0.9PA-1.25PA-1.65
aggr
egat
ion
00.5
11.5
22.5
33.5
44.5
0.000001 0.0001 0.01 1log[KCl](M)
Rh/R
0
0
5
10
15
20
0.000001 0.0001 0.01 1log[KCL] (M)
Rh/R
0
PA-0.46PA-0.9PA-1.25PA-1.65
(a) (b)
Figure 3-6 (a) Variation in normalized hydrodynamic radius (Rh/R0) as a function of KCl
concentration for polyelectrolyte microgels: (◆) poly (NIPAm-AA), pH=7.0, T = 25oC, R0
= 22.6 nm; (■) poly (NIPAm-VI), pH=4.0, T = 25oC, R0 = 91 nm (b) Variation in
normalized hydrodynamic radius (Rh/R0) as a function of KCl concentration for
polyampholyte microgels: (◆) PA-0.46, R0 = 24.5 nm (■) PA-0.9), R0 = 44.2 nm (▲) PA-
1.25,R0 = 28.6 nm (×) PA-1.65, R0 = 24.5 nm; pH=pI, T = 25oC. R0’s
Chapter 3
___________________________________________________________________________ - 67 -
The temperature-induced volume transitions of PA microgels were studied
at their respective pI values. Figure 3-6b shows the swelling profiles of PA
microgels with different compositions as a function of KCl concentration. At salt
concentrations below 0.005M, no significant change in microgel size was observed.
All PA microgels displayed a notable swelling peak at higher salt concentrations
indicating antipolyelectrolyte behaviour.
No discernable correlation between the asymmetric and symmetric
compositions of PA microgels and their swelling profiles in salt solutions was
observed in the present work. Instead, we found that increasing AA content in PA
microgels led to larger swelling ratios in the range 0.005M<[KCl]< 0.4M. For
example, the values of Rh/R0 for PA-0.46 (lowest AA content) and PA-1.65 (highest
AA content) were 1.7 and 18, respectively. Note that this substantially large
increase in microgel size did not occur due to particle flocculation since the light
scattering data showed relatively narrow size distributions and negligible change in
scattering intensity. At higher concentrations of KCl, the microgels aggregated
presumably due to reduced electrostatic repusion between them.
3.5.3 Effect of temperature
Figures 3-7 shows the variation in hydrodynamic radius of PE microgels as a
function of temperature. For each system, measurements were conducted at two
critical values of pH: one that rendered the microgels ionic and more hydrophilic,
and the other that made them almost neutral and less hydrophilic. Note that all
VPTTs measured in the current work were determined by monitoring the change in
hydrodynamic radii of microgels.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 68 -
0.5
1.5
2.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
0.5
1.5
2.5
3.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
(a) (b)
0.5
1.5
2.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
0.5
1.5
2.5
3.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
(a) (b)
0.8
1
1.2
1.4
1.6
1.8
2
20 30 40 50Temperature (oC)
Rh/
R0
(c)
0.8
1
1.2
1.4
1.6
1.8
2
20 30 40 50Temperature (oC)
Rh/
R0
(c)
0.5
1.5
2.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
0.5
1.5
2.5
3.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
(a) (b)
0.5
1.5
2.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
0.5
1.5
2.5
3.5
20 30 40 50 60Temperature (oC)
Rh/
Ro
(a) (b)
0.8
1
1.2
1.4
1.6
1.8
2
20 30 40 50Temperature (oC)
Rh/
R0
(c)
0.8
1
1.2
1.4
1.6
1.8
2
20 30 40 50Temperature (oC)
Rh/
R0
(c)
Figure 3-7. Variation in microgel size as a function of temperature: (a) poly (NIPAm-AA)
microgels, (■) pH=3.5, R0 =50 nm ( ) pH =7.0, R0 =69.5 nm; (b) poly(NIPAm-VI)
microgels, (■) pH=4.0, R0 =63.9 nm ( ) pH=7.5, R0 =52.6nm; (c) PA microgels with
various compositions at corresponding pI values, ( ) PA-0.46, R0 =49.8 nm; ( ) PA-
1.65, R0 = 35.9 nm; (□) PA-1.25, R0 =42.4 nm. Rh is the hydrodynamic radius of
microgels at a particular temperature and R0 is the minimum Rh observed just before
aggregation of PA microgels. All microgels were studied in 0.1M KCl solution. Dashed
lines are given for eye guidance.
Figure 3-7a shows that the value of Rh/R0 for poly(NIPAm-AA) microgels in
the entire temperature range studied was significantly larger at pH=7.0 than at
pH=3.5. The volume phase transition temperature (VPTT) was ca. 30 oC at pH=3.5
and 55 oC at pH=7.0. The shift in VPTT at pH= 3.5 to a value lower than that of
Chapter 3
___________________________________________________________________________ - 69 -
homopolymer poly(NIPAm) microgels (ca. 32 oC)[31, 32] was due to the decreased
hydrophilicity of AA.[33, 34] At pH = 7.0, the shift of VPTT to a higher value than that
of homopolymer poly(NIPAm) microgels (ca. 32 oC)[32] was attributed to the
increased hydrophilicity of ionized AA segments and the electrostatic repulsion
between them. Similarly, for poly(NIPAm-VI) microgels (Figure 3-7b), protonation
of the imidazole groups and augmented hydrophilicity of the microgels shifted the
VPTT to ca. 35 oC at pH=4.0 (versus 31 oC at pH=7.5).
Figure 3-5c shows the temperature dependent change in size of PA
microgels with different compositions, determined at their respective pI values.
Note that all PA microgels coagulated with increase in temperature, in contrast to
PE microgels which showed no coagulation in the temperature range studied.
Symmetric PA microgels displayed greater colloidal stability than asymmetric PA
microgels: coagulation occurred above 48oC in the former, compared to above 37oC
in the latter systems. Below the temperature at which the loss of colloid stability
was observed, the PA microgels featured gradual de-swelling. The asymmetric PA-
0.46 and PA-1.65 microgels underwent ca. 23% and 30% shrinkage, respectively,
upon heating from 25 to 37oC while the symmetric PA-1.25 microgels showed a
comparatively larger reduction in size of ca. 43 % at 48 oC.
3.5.4 Effect of solvent.
Figure 3-8a-e shows the variation in normalized size of PE and PA microgels
at different pH values (corresponding to charged and neutral microgel states) as a
function of the volume fraction of ethanol, � added to the aqueous medium. In
Figure 3-8a for poly (NIPAm-AA) microgels, a clear minimum in Rh /R0 was observed
at φ = 0.5. The extent of swelling of these microgels in the deprotonated state (pH
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 70 -
= 7.5) was three times larger than in the protonated state (pH = 4.0).
Contrastingly, poly(NIPAm-VI) microgels (Figure 3-8b), remained shrunken in both
protonated and deprotonated states at φ ≤ 0.4 while in the region 0.4<φ<0.5 the
value of Rh/R0 abruptly increased. The increase was steeper at pH= 4.0,
corresponding to the ionized state of the microgels. For larger values of φ the
particles shrank at pH = 4.0 but continued to swell at pH=7.5.
Figure 3-8 c-e shows the change in Rh/R0 for the PA microgels with highly
asymmetric and symmetric compositions as a function of φ. A marked swelling
maximum was observed at φ ≈ 0.5 in all PA microgels, irrespective of the pH range.
Consider first PA-0.46, the microgel with asymmetric composition (Figure 3-6c).
Since in this system VI was present in the largest and AA in the smallest amount, its
swelling behavior was anticipated to resemble that of poly(NIPAm-VI) microgels.
Contrary to expectations, at pH=4.0, (when imidazole groups were protonated),
the maximum extent of swelling was relatively small (Rh/R0 ≈ 1.5) while at pH =
pI(aq) = 5.8 and at pH = 7.0, the values of Rh/R0 were significantly larger (ca. 6.0
and 4.5, respectively). For asymmetric PA-1.65 microgels (enriched with AA) the
maximum extent of swelling was expected to occur at pH =7.5, similar to
poly(NIPAm-AA) microgels. Instead, Figure 3-8e shows that the extents of swelling
at all pH values were in close proximity to each other (3.5 < Rh/R0 < 4.0).
For the symmetric PA-0.9 microgel (Figure 3-8d), the value of Rh/R0 at pH =
4.0 was ca. 1.8 (greater than that for PA-0.46 but smaller than that of PA-1.65).
Overall the extent of swelling of the PA microgels in mixed solvents at pH=4.0 and
φ ≈ 0.5 correlated with the amount of AA present in the system: an increase in AA
content apparently led to greater swelling. However, this effect could not be
Chapter 3
___________________________________________________________________________ - 71 -
01234567
0 0.2 0.4 0.6 0.8 1(φ) ethanol
Rh/
Ro
01234567
0 0.2 0.4 0.6 0.8 1(φ) ethanol
Rh/
Ro
0
1
2
3
4
5
0 0.2 0.4 0.6 0.8 1(φ) ethanol
Rh/
Ro
01234567
0 0.2 0.4 0.6 0.8 1(φ) ethanol
Rh/
Ro
0
1
2
3
4
5
0 0.2 0.4 0.6 0.8 1 (φ) ethanol
Rh/
Ro
-8
-6
-4
-2
0
2
0 0.2 0.4 0.6 0.8 1(φ) ethanol
ζ (m
V)
(a) (b)
(c) (d)
(e) (f)
Figure 3-8. Variation in Rh/R0 of microgels in mixed solvents. (a) poly(NIPAm-AA), R0
=72.1 nm; (b) poly(NIPAm-VI), R0 =122 nm; (c) PA-0.46, pI=5.8, R0 =79 nm; (d) PA-
0.9, pI=5.6, R0 =73.8 nm; (e) PA-1.65, pI=4.75, R0 =57.2 nm, ( )pH=4.0, (□) pH=pI, (�)
pH=7.5; (f) Variation in ζ-potential of PA microgels in mixed solvents at the isoelectric
point (determined in aqueous solutions): ( )PA-0.46, ( ) PA-0.9, (•) PA-1.65.
attributed to coulombic interactions alone because AA groups were only partially
ionized at pH= 4.0.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 72 -
At the values of pH close to pI all PA microgels showed significant swelling
at φ ≈ 0.5 (molar fraction of ethanol is 0.24). This phenomenon was unexpected
because at pI, the microgels were believed to be in their most compact state due
to electrostatic attraction between the oppositely charged groups. Since the
isoelectric point for the mixed solvent medium differed from that in water, we
examined the effect of addition of ethanol on ionization of VI and AA groups by
measuring ζ-potential of the PA microgels at pI (aq) as a function of φ (Figure. 3-
8d). The small increase in the magnitude of zetapotential at φ = 0.5 (-4 to -7 mV)
(Figure3-8f), was not sufficient to explain the magnitude of the observed changes
in size of the PA microgels.
3.6 Discussion
The variation in degree of swelling for PE microgels was consistent with the
state and nature of the ionic groups in binary copolymers. For example, at pH<4.3
(below pKa of AA) the anionic poly(NIPAm-AA) microgels remained in a shrunken
state while at pH>4.3, they swelled due to repulsion between the deprotonated
carboxylic acid groups and their increased hydrophilicity.[30] Similarly, the cationic
poly(NIPAm-VI) microgels were in a shrunken state at pH >7.0 (above pKa of VI)
whilst at pH<7.0, protonation of the imidazole groups and subsequent repulsion
between them resulted in microgel swelling. The swelling of PA microgels upon
change in pH appeared as a seeming combination of responses of PE microgels.
Outside the zwitterionic window, the PA microgels carried a substantial number of
similarly charged groups; repulsion between the like charges caused swelling at
high and low pH values, similar to PE microgels. Contrastingly, in the zwitterionic
window, the PA microgels shrank due to ion pairing between AA and VI residues.
Chapter 3
___________________________________________________________________________ - 73 -
The discussion focuses on the volume changes of PA microgels in the zwitterionic
region with emphasis on the effects of microgel composition and ion pairing. Note
that in this regime, ion coupling between the oppositely charged groups did not
rule out the existence of charged groups excluded from ion pairing.
It is also worthwhile to mention that the dramatic changes in size we
observed for the different microgels were partly due to the low crosslinking density
of the microgels. While the distribution of functional groups throughout the
microgels is also important and has some bearing both on swelling extent and value
of pI, the low cross-linking density in the microgels allows for large swelling
capacity.
3.6.1 Effect of pH and ionic strength.
In the zwitterionic window (4.3<pI<7.0), the PA microgels shrank due to
effective ion coupling between COO-- and =NH+-groups (Figure 3-5a-d). The
fractions of positively and negatively charged groups in the microgels played an
important role in swelling behavior. The swelling profiles of the microgels with a
large fraction of AA or VI fragments had asymmetric shapes with greater swelling at
either high or low pH regions respectively: any contraction that occurred due to
limited ion pairing was over-ruled by repulsion between the excess numbers of like
charges at these pH localities. The swelling profiles of these ‘asymmetric’ PA
microgels were similar to those of PE microgels. PA microgels with almost equal
amounts of AA and VI showed more symmetric swelling profiles with relatively
similar degrees of swelling in pH regions above and below pI.
In order to obtain a ‘symmetric’ pH-dependent, volume response for the PA
microgels, the molar ratios of the ionic AA and VI comonomers in the batch
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 74 -
reaction were adjusted, to account for their differing reactivity. A random
distribution of ionic groups throughout the particle was desirable to maximize the
probability that the two monomers would be in sufficiently close proximity to
experience intrachain interactions between them. The reactivity ratios of NIPAm
(monomer 1) and AA (monomer 2) in water were estimated to be r1=0.571 and
r2=0.320 from the kinetc rate constants for homo-propagation and cross-
propagation reactions, reported by others.[35] In general, ionization of acidic
monomers yields carboxylate species which have relatively lower reactivity
compared to the acrylamide.[36] The VI species has been reported to react faster
than NIPAm,[37] but there has been no report to date on the absolute relative
reactivity of VI to AA species. However, the available literature qualitatively
indicates that the VI species is more reactive than AA. Indeed, consideration of the
initial monomer compositions in the reaction mixtures, the titration data, and
swelling profiles of poly(NIPAm-AA-VI) microgels in this work indicated that the
reactivity of VI with respect to NIPAm in water is greater than that of AA.
Increase in salt concentration caused shrinkage of the PE microgels due to
the screening of electrostatic repulsion between like charges. In contrast, PA
microgels showed antipolyelectrolyte behaviour: the addition of salt disturbed the
electrostatic intra- and interchain attractions between oppositely charged ionic
groups, causing microgel swelling. Contrary to expectations, there was no
discernable correlation between the number of ion pairs (Table 3-1) in the PA
microgels and their swelling ratios. Instead, increasing AA content dominated the
swelling ratio of PA microgels. This result was in agreement with previous reports
on the enhanced swelling of poly(NIPAm-AA) microgels with increasing AA content
in electrolyte solutions; the hydrophilicity of the AA residues facilitated
Chapter 3
___________________________________________________________________________ - 75 -
conformational rearrangement of the polymer chains and in turn, enhanced the
degree of microgel swelling.[30] However, at sigificantly higher ionic strengths,
antipolyelectrolyte behavior was observed and microgel swelling occurred due to
suppressed electrostatic attraction between oppositely charged groups.
3.6.2 Effect of temperature
Both PE and PA microgels underwent the temperature-induced shrinkage at
higher temperatures in comparison with poly(NIPAm) homopolymers (VPTT =31oC)
in their ionized states due to the presence of the hydrophilic AA and VI segments.
The loss in colloidal stability of PA microgels at higher temperatures resulted from
hydrophobic interactions between the particles, consistent with the behavior of PA
microgels reported by Ogawa and coworkers.[15]
The ‘symmetric’ PA microgels exhibited a broader temperature-dependent
volume transition and were more stable to de-swelling than the ‘asymmetric’ PA
microgels, due to the presence of a larger number of hydrophilic COO- and ≡NH+
ions in the zwitterionic window (Table 3-1). Previous reports reasoned that the
larger ion-pair content in the symmetric PA systems acted as ‘physical cross-links.’
Hence symmetric PA microgels were already in a highly shrunken state at room
temperature and their extent of de-swelling upon heating was limited.[15]
Admittedly, at pI, the PA microgels assume their smallest size at room temperature
(in relation to their size at other pH values), due to electrostatic attraction
between the oppositely charged groups. Nevertheless, not all charged groups are
able to form ion pairs due to the constraints imposed by polymer chain
connectivity. We believe that in our work, the greater temperature-induced
shrinkage of symmetric PA microgels occurred due to the larger number of ion pairs
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 76 -
formed: contraction of the polymer chains brought the charged AA and VI residues
closer to each other, enabling previously unpaired charges to couple and enhancing
the electrostatic attraction between the already paired groups. Our experiments
followed a trend similar to that observed of Lyon and coworkers[16] who reported
that PA microgels underwent greater shrinkage at zwitterionic pH than at non-
zwitterionic pH, although the volume phase transitions in our systems were
broader.
3.6.2 Effect of solvent
Introduction of ethanol to aqueous dispersions of PE and PA microgels gave
rise to two coexistent phenomena: change in electrostatic interactions and
solvency-related effects. For electrostatic effects, two competing factors must be
considered. Firstly, the strength of the electrostatic effects in ethanol-water
mixtures may increase, since coulombic interactions are stronger in ethanol than in
water (the dielectric constants of ethanol and water are 24.3 and 81, respectively).
Simultaneously, the net effective charge of the microgels may diminish due to
reduced polarity of the medium and altered pKa values of the ionic groups. The
quality of the solvent affects the monomer-monomer and monomer-solvent
interactions, the natures of which vary with change in polymer and solvent
compositions. Cononsolvency occurs when the mixed medium is a poorer solvent
for the particles than either of its pure components, while cosolvency occurs when
the mixed medium is a superior solvent for the particles than either of its pure
components.[38] Winnik and coworkers have reported the cononsolvency behavior
of poly(NIPAm) in methanol-water mixtures due to the formation of clathrate
hydrates.[38] Vincent and Tanaka confirmed that cononsolvency behavior was
Chapter 3
___________________________________________________________________________ - 77 -
preserved in poly (NIPAm-AA) microgels containing a small amount of AA at
0.4<φ<0.6 in ethanol-water mixtures.[39] However, for larger concentrations of AA,
poly (NIPAm-AA) microgels showed cosolvency behavior, much like that shown by
AA homopolymer gels.[40] In our work, cationic and anionic PE microgels showed
different behavior in the range of ethanol concentration 0.4<φ<0.6: poly(NIPAm-VI)
microgels swelled while poly(NIPAm-AA) microgels shrank. For both PE microgels,
the greatest extent of swelling occurred at pH corresponding to their charged
states: at pH=7.5 for poly(NIPAm-AA) and at pH=4.0 for poly(NIPAm-VI). Thus for
poly(NIPAm-VI) electrostatic repulsion between the charged ≡NH+ groups enhanced
swelling, while for poly(NIPAm-AA) microgels, repulsion between the –COO- -groups
counteracted the deswelling of the system. The pH dependence of the variation in
Rh/R0 indicated the importance of electrostatic effects in microgel swelling, in
addition to that of solvent quality. The swelling properties of PA microgels also
originated from the aforementioned solvency and electrostatic effects. The former
involved competing solvency (governed by charged AA and VI residues) and
cononsolvency (governed by NIPAm residues) effects.31 The latter could enhance
contraction of the PA microgels in the zwitterionic regime due to stronger
electrostatic attraction between the oppositely charged groups. Such behavior was
observed by Tanaka for bulk PA gels exposed to mixtures of water and ethanol. [17]
In our work, all PA microgels showed a swelling maximum at φ=0.5,
irrespective of microgel composition or pH value, indicating the overriding
influence of solvent quality on microgel swelling. In particular, the notably strong
swelling of all PA microgels at pH ≈ pI was unexpected: since the microgels were in
their most compact state in the zwitterionic regime, they were expected to resist
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 78 -
swelling in this region. The small deviation of electrokinetic potential from zero at
φ = 0.5 and pH ≈ pI, (Figure3-8f) was insufficient to explain the magnitude of the
observed changes in Rh/R0 on the basis of electrostatic interactions. Since at φ =
0.5, poly(NIPAm-VI) microgels showed a swelling maximum, the swelling of PA
microgels at φ=0.5 may have been governed by imidazole-solvent interactions. This
justification could also explain the decrease in the value of Rh/R0 from ca. 6 for PA-
0.46 (Figure3-8c) to ca. 3.5 for PA-1.65 (Figure 3-8e) with decreasing VI content in
the PA microgels. However, the pH- and composition-dependent variation in Rh/R0
for the PA microgels indicated that this was not entirely the case: the values of
Rh/R0 for poly (NIPAm-AA) (Figure 3-8a) and poly (NIPAm-VI) (Figure 3-8b) at pH=7.5
and φ=0.5 were ca. 3.5 and 2.7, respectively, implying that, the swelling of all PA
microgels at pH= 7.5 was driven by both AA-solvent and VI-solvent interactions.
Furthermore, we recall that for PE microgels at φ=0.5, repulsion between the ≡NH+-
groups favored swelling at pH=4.0. However, at φ = 0.5, Rh/R0 for PA-0.46 (microgel
with highest content of VI) was lower at pH=4.0 than at pH=7.5. In fact, at pH=4.0
and φ=0.5, decreasing content of VI led to a progressive increase in the degree of
swelling. Even more surprising was the fact that this increased degree of swelling
occurred along with increase in AA content. The latter behavior was also
unexpected because the contribution of AA to swelling at pH=4.0 was significantly
smaller than that of VI (as determined from the pH-dependent variation in swelling
of PE microgels, (Figure 3-8a,b).
3.7 Conclusions
We examined the swelling behavior of PE and PA microgels in response to
the variation in pH, ionic strength, temperature, and solvent composition. PA
Chapter 3
___________________________________________________________________________ - 79 -
microgels with an excess of either a cationic or an anionic group showed pH-
dependent swelling behavior much like that of their PE counterparts. PA microgels
with symmetric compositions exhibited swelling at both low and high pH ranges. To
obtain equivalent degrees of swelling (‘symmetric’ swelling) in PA microgels at low
and high values of pH, the composition of the reaction mixture was tuned to
account for the different reactivities of the comonomers. In KCl solutions, PA
microgels showed antipolyelectrolyte behavior: they swelled with increasing
electrolyte concentration. The temperature-dependent volume phase transitions
of both PE and PA microgels shifted to higher values than that of poly(NIPAm) due
to the hydrophilicity of ionized AA and VI groups. Ion-pairing between charged AA
and VI groups increased the extent of the temperature-induced deswelling in PA
microgels with symmetric composition. The solvent-dependent swelling behavior of
PE and PA microgels showed that competing electrostatic and solvency interactions
determined their swelling response. The influence of electrostatic effects on PE
microgel swelling behavior in ethanol-water mixtures was evident from their
increase in size at pH values corresponding to the ionic states of AA and VI groups.
However, solvency effects dominated the swelling behavior of all PA microgels,
which showed a swelling maximum at φ=0.5, irrespective of microgel composition
or pH value.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 80 -
References for Chapter 3
[1] V. T. Pinkrah, M. J. Snowden, J. C. Mitchell, J. Seidel, B. Z. Chowdhry and G. R. Fern,
Langmuir 2003, 19, 585-590.
[2] F. Grohn and M. Antonietti, Macromolecules 2000, 33, 5938-5949.
[3] S. Neyret and B. Vincent, Polymer 1997, 38, 6129-6134.
[4] B. H. Tan, P. Ravi and K. C. Tam, Macromolecular Rapid Communications 2006, 27,
522-528.
[5] M. Das and E. Kumacheva, Colloid and Polymer Science 2006, 284, 1073-1084.
[6] J. P. Baker, D. R. Stephens, H. W. Blanch and J. M. Prausnitz, Macromolecules 1992, 25,
1955-1958.
[7] S. E. Kudaibergenov and V. B. Sigitov, Langmuir 1999, 15, 4230-4235.
[8] G. Nisato, J. P. Munch and S. J. Candau, Langmuir 1999, 15, 4236-4244.
[9] A. E. English, S. Mafe, J. A. Manzanares, X. H. Yu, A. Y. Grosberg and T. Tanaka,
Journal of Chemical Physics 1996, 104, 8713-8720.
[10] S. Wen and W. T. K. Stevenson, Colloid and Polymer Science 1993, 271, 38-49.
[11] M. Antonietti, Angewandte Chemie-International Edition in English 1988, 27, 1743-
1747.
[12] T. Tanaka and D. J. Fillmore, Journal of Chemical Physics 1979, 70, 1214-1218.
[13] L. Bromberg, M. Temchenko, V. Alakhov and T. A. Hatton, Langmuir 2005, 21, 1590-
1598.
[14] J. M. D. Heijl and F. E. Du Prez, Polymer 2004, 45, 6771-6778.
[15] K. Ogawa, A. Nakayama and E. Kokufuta, Langmuir 2003, 19, 3178-3184.
[16] S. Nayak and L. A. Lyon, Polymer Preprints 2003, 44, 679-680.
[17] T. Tanaka, Physical Review Letters 1978, 40, 820-823.
[18] L. Y. Chen, Y. M. Du and R. H. Huang, Polymer International 2003, 52, 56-61.
Chapter 3
___________________________________________________________________________ - 81 -
[19] A. Fernandez-Nieves, A. Fernandez-Barbero, B. Vincent and F. J. de las Nieves, Journal
of Chemical Physics 2003, 119, 10383-10388.
[20] Y. Takeoka, A. N. Berker, R. Du, T. Enoki, A. Grosberg, M. Kardar, T. Oya, K. Tanaka,
G. Q. Wang, X. H. Yu and T. Tanaka, Physical Review Letters 1999, 82, 4863-4865.
[21] B. R. Saunders and B. Vincent, Advances in Colloid and Interface Science 1999, 80, 1-
25.
[22] B. R. Saunders and B. Vincent, Journal of the Chemical Society-Faraday Transactions
1996, 92, 3385-3389.
[23] K. Kratz, T. Hellweg and W. Eimer, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2000, 170, 137-149.
[24] B. R. Saunders, H. M. Crowther and B. Vincent, Macromolecules 1997, 30, 482-487.
[25] M. J. Molina, M. R. Gomez-Anton and I. F. Pierola, Journal of Polymer Science Part B-
Polymer Physics 2004, 42, 2294-2307.
[26] C. Luca, S. Racovita, V. Neagu and M. I. Avadanei, Reactive & Functional Polymers
2007, 67, 1440-1447.
[27] T. Tamura, H. Uehara, K. Ogawara, S. Kawauchi, M. Satoh and J. Komiyama, Journal of
Polymer Science Part B-Polymer Physics 1999, 37, 1523-1531.
[28] T. Hoare and R. Pelton, Langmuir 2006, 22, 7342-7350.
[29] M. J. Snowden, D. Thomas and B. Vincent, Analyst 1993, 118, 1367-1369.
[30] M. J. Snowden, B. Z. Chowdhry, B. Vincent and G. E. Morris, Journal of the Chemical
Society-Faraday Transactions 1996, 92, 5013-5016.
[31] M. Andersson and S. L. Maunu, Colloid and Polymer Science 2006, 285, 293-303.
[32] H. M. Crowther, B. R. Saunders, S. J. Mears, T. Cosgrove, B. Vincent, S. M. King and
G. E. Yu, Colloids and Surfaces a-Physicochemical and Engineering Aspects 1999, 152, 327-
333.
From Polyampholyte to Polyelectrolyte Microgels
___________________________________________________________________________ - 82 -
[33] G. Bokias, G. Staikos and I. Iliopoulos, Polymer 2000, 41, 7399-7405.
[34] M. J. Tiera, G. R. dos Santos, V. A. D. Tiera, N. A. B. Vieira, E. Frolini, R. C. da Silva
and W. Loh, Colloid and Polymer Science 2005, 283, 662-670.
[35] T. Hoare and D. McLean, Macromolecular Theory and Simulations 2006, 15, 619-632.
[36] T. Hoare and D. McLean, Journal of Physical Chemistry B 2006, 110, 20327-20336.
[37] H. S. Bisht, L. Wan, G. Z. Mao and D. Oupicky, Polymer 2005, 46, 7945-7952.
[38] H. Ringsdorf, J. Simon and F. M. Winnik, Macromolecules 1992, 25, 7306-7312.
[39] T. Amiya, Y. Hirokawa, Y. Hirose, Y. Li and T. Tanaka, Journal of Chemical Physics
1987, 86, 2375-2379.
[40] F. Ikkai, N. Masui, T. Karino, S. Naito, K. Kurita and M. Shibayama, Langmuir 2003,
19, 2568-2574.
Chapter 4
___________________________________________________________________________ - 83 -
Chapter 4
Zwitterionic Sulfobetaine Microgels
Acknowledgements: This work was conducted in collaboration with Dr. Nicolas Sanson who
synthesized several of the microgels and performed some of the characterization experiments.
4.1 Introduction
Polyampholyte microgels carry both positive and negative charges in a
broad range of physicochemical conditions.[1-5] These microgels exhibit rich
phenomenology in their stimuli responsive properties due to the complex
interactions between their oppositely charged functional groups.[6] The pH-
dependent volume transitions in ionically functionalized microgels are governed by
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 84 -
the relative numbers and types of ionized groups. In PA microgels functionalized
with weak acidic (AA) and basic (VI) groups, the pH-induced change in size of
exhibits two peaks at low and high pH.[4, 7] These peaks correspond to the most
charged states of the cationic and anionic groups, respectively. In the intermediate
pH range, the formation of ion couples between the oppositely charged groups
leads to microgel deswelling.[7, 8] In contrast, PA microgels functionalized with
strong acidic (sulfonate) and basic (quarternary ammonium) groups[9] are not pH-
sensitive since they are charged in the entire pH range, but are sensitive to swell in
salt solutions.[10]
The swelling capacity of pure poly(NIPAm) microgels in aqueous solutions
reduces upon the addition of salt. PE microgels, like poly(NIPAm-AA) also deswell in
salt solutions due to screening of charge repulsion between functional groups in the
polymer network. This deswelling of PE microgels in the presence of free
electrolyte is termed polyelectrolyte behavior. Contrastingly, polyampholyte
systems are known to exhibit antipolyelectrolyte behavior that is characterized by
swelling and expansion of the polymer in the presence of salt solutions. This
swelling is attributed to shielding of intra- and inter-macromolecular electrostatic
interactions by the free salt. PA hydrogels are reported to show antipolyelectrolyte
behavior only in a very narrow composition at nearly net-zero charge densities. At
non-zero charge densities, polyampholyte hydrogels often show polyelectrolyte
behavior. Copolymerization of a zwitterionic monomer with poly (NIPAm) could
afford microgels capable of simultaneously retaining high swelling capacity
together with reversible thermo-sensitivity, in salt solutions.
Polyampholyte microgels are synthesized by i) statistical copolymerization
of cationic and anionic monomers[3, 7], from alternating copolymers of cationic and
Chapter 4
___________________________________________________________________________ - 85 -
anionic origin [11], and (iii) by copolymerizing monomers containing both the
cationic and anionic functional groups (zwitterions).[12, 13] The latter group of
polymers has an equal number of anionic and cationic species on the same
monomer units, and is referred to herein as zwitterionic species.
In polyampholyte microgels obtained by statistical copolymerization, the
distribution of charged groups within the microgels is determined by the reactivity
of comonomers and the solubility of the corresponding polymers.[7] It has been
shown that depending on these two factors the microgel can have a uniform, a
gradient, or a core-shell structure, each with different swelling/deswelling
profiles.[14] These structural differences cast some ambiguity over the
interpretation of the volume transitions in polyampholyte microgels since their
swelling properties are to a large extent explained by the interactions of oppositely
charged groups that are in close proximity. This problem is alleviated in systems
containing an equal number of cationic and anionic groups. Polymeric betaines are
a class of monomers that can introduce an equal number of charged cationic and
anionic groups when copolymerized with NIPAm.
An excellent review on polymeric betains was recently provided by
Laschewsky et al.[15] Macroscopic betain hydrogels synthesized by copolymerizing N-
isopropylacrylamide and N,N-Dimethyl-N-(3-methacrylamidopropyl)-N-(3-
sulfopropyl) ammonium betaine (SPP) were reported by Cai and Gupta.[16] The
authors did not observe antipolyelectrolyte behavior in the presence of a
monovalent salt but found a notable degree of swelling of the hydrogel in the
presence of divalent salts. Furthermore, at high salt concentrations exceeding 1 M,
the temperature-induced volume transition typical for poly(NIPAm) was
suppressed. No explanation for the different behavior of the zwitterionic hydrogels
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 86 -
was provided in this work. However, this behavior may be attributed to the
increasing solubility of SPP in electrolyte solutions. Sulfobetaines are only sparingly
soluble in water due to the formation of polyelectrolyte complexes built from ionic
crosslinks. Addition of free electrolyte disrupts these crosslinks, increasing polymer
solubility and allowing chain expansion.
4.2 Research objectives
Sulfobetaines are a class of polymers that contain both a strong
quarternary ammonium and a strong sulfonate group separated by several
methylene units.[17, 18] Both of these ionic groups remain charged in the entire
range of pH. Thus, in contrast to PA microgels containing weak acidic and basic
groups, microgels functionalized with sulfobetaines do not show pH-dependent
swelling behavior, but are expected to swell in salt solutions, irrespective of pH of
the medium. Hence sulfobetaine-functionalized microgels have potential
applications as ion scavengers in the entire range of pH and may serve as ideal
microreactors for one-step, in situ synthesis of composite nanoparticles.
To date, no report describing the synthesis and the properties of
zwitterionic betaine microgels exists. In the present work, zwitterionic microgels
of NIPAm copolymerized with N,N-Dimethyl-N-(3-methacrylamidopropyl)-N-(3-
sulfopropyl)ammonium betaine (SPP) were synthesized in various volume ratios by
free radical precipitation polymerization. Incorporation of SPP monomers carrying
both cationic and anionic groups afforded microgels with equal number of
oppositely charged groups. The effect of microgel composition on the swelling
transitions of poly (NIPAm-SPP) microgels in response to changes in temperature,
pH and electrolyte concentrations was investigated.
Chapter 4
___________________________________________________________________________ - 87 -
4.3 Experimental
4.3.1 Materials
N-isopropylacrylamide (NIPAm), N,N’-methylene-bis-acrylamide BIS (a
crosslinking agent), potassium persulfate KPS (initiator) were purchased from
Aldrich Chemical Co. (Canada) and used as received. N,N-Dimethyl-N-(3-
methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine (SPP) was a gift of
RASCHIG company. The structure of monomers used in the copolymerization are
shown in Figure 4-1.
NIPAm SPP BIS
Figure 4-1 Chemical structure of monomers used in the present work. a) N-
isopropylacrylamide b) N,N-Dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)
ammonium betaine, SPP c) N-N’-methylene-bis-acrylamide, BIS.
4.3.2 Synthesis of zwitterionic poly(NIPAm-SPP) microgels
Poly(NIPAm-SPP) microgels were prepared by free radical precipitation
polymerization and purified by dialysis for one week against deionized water
H2C CH
C O
NH
CHH3C CH3
H2C C
CH3
C O
NH
(CH2)3
NH3C CH3
(CH2)3
SO3
H2C CH
C O
NH
CH2
NH
C O
CHH2C
(a) (b) (c)
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 88 -
(twice daily changes of water). Further purification was carried out by
centrifugation at 18000G for 30 mins at room temperature in a temperature-
controlled centrifuge. The sample codes and the recipes used for the microgel
synthesis are provided in Table 4-1. A poly(NIPAm) microgel (Sample NS0) was used
as a control system. The microgels coagulated when the concentration of SPP in
the reaction mixture exceeded 4.2 wt %.
Table 4- 1Formulations used in microgel synthesis and the hydrodynamic diameter
of the corresponding particles
4.3.3 Characterization of properties of microgels
The hydrodynamic diameter, Dh, of the microgels was measured using
photon correlation spectroscopy (Zetasizer 3000HS, Malvern, UK) with a 10 mW
laser operating at 633 nm. Experiments were carried out at room temperature at
the scattering angle of 90°. A CONTIN statistical method was used to convert the
measured correlation data into a particle size distribution. The temperature-
dependent variation of microgel size was determined in the temperature range
from 15 to 60 °C by photon correlation spectroscopy setup (PCS, Protein Solutions
Composition (mol %) Sample
NIPAm SPP BIS KPS
Dh at 25°Ca
(nm)
NS0 100 0 4.43 2.97 646
NS1 98.02 1.98 3.88 2.55 682
NS2 96.92 3.08 3.98 2.65 739
NS3 96.32 3.68 4.06 2.66 817
a Hydrodynamic diameters were determined by Dynamic Light Scattering
Chapter 4
___________________________________________________________________________ - 89 -
Inc.) equipped with a temperature controller. At each temperature, the solutions
were stabilized for 20 min.
4.4 Results and discussion
4.4.1 Size of poly(NIPAm-SPP) microgels
Table 4-1 shows that the hydrodynamic diameter of poly (NIPAm-SPP)
microgels was larger than that of pure poly(NIPAm) microgels. Furthermore, the
size of the zwitterionic microgels gradually increased with increasing concentration
of SPP monomer in the reaction mixture. This effect was surprising because SPP is
only sparingly soluble in water, due to the formation of ionic complexes between
its oppositely charged groups. Hence its incorporation into poly(NIPAm) was
expected to cause an increase in intra-particle electrostatic attraction between
the ionic groups and result in microgel shrinkage. The increase in size is most likely
a consequence of the osmotic swelling by mobile counterions within the particle.
4.4.2 Effect of pH
We further examined the effect of pH on the swelling properties of poly
(NIPAm-SPP) microgels in the range 3 ≤ pH ≤ 10 (Figure 4-2). Expectedly, the size of
particles did not notably change in the entire range of pH studied, regardless of the
microgel composition. This result differed from the appearance of the two swelling
peaks at low and high pH measured for statistical polyampholyte microgels,
functionalized with weak acid and base moieties.[7] The microgels in the present
work however, retained their zwitterionic form in the entire pH range due to the
strongly ionised quaternary ammonium and sulfonate species on the SPP groups.
Consequently, any ion pairing between the cationic and anionic groups did not
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 90 -
depend on pH. The positive and negative charges were almost compensated: in the
whole pH range, the value of ξ-potential (electrophoretic mobility) did not exceed
- 3 mV, similar to the poly(NIPAm) microgels. The small negative ξ-potential
originated from the presence of the anionic initiator, potassium persulfate on the
polymer chains.
400
800
1200
1600
2 4 6 8 10 12pH
D (n
m)
NS1NS2NS3NS4
Figure 4-2. Variation of hydrodynamic diameters Dh as a function of the pH for
zwitterionic microgels poly(NIPAm-SPP). Solid lines are drawn for eye guideline. (■)
NS1(▲) NS2 (♦)NS3 (X) NS4
4.4.3 Effect of temperature
Figure 4-3 shows the dependence of the hydrodynamic diameter Dh and the
normalized change in hydrodynamic diameter Dh/D0 of the poly (NIPAm-SPP)
microgels as a function of temperature where D0 is the hydrodynamic diameter of
particles in the shrunken state at 50 °C. For all zwitterionic microgels the (VPTT)
was in the range of temperatures between from 32 to 34 °C, that is, slightly higher
than for the poly(NIPAm) microgels. The volume transition was broader and showed
a substantially smaller degree of shrinkage than the poly (NIPAm) microgels. For
example, between 29 and 45oC, NS2 and NS0 microgels underwent 75 and 89 %
Chapter 4
___________________________________________________________________________ - 91 -
reduction in volume respectively. This effect was counter-intuitive: it could be
expected that with increasing concentration of SPP, ion coupling would result in a
stronger deswelling transition of the microgels in water. Apparently, increasing
hydrophilicity of the zwitterionic microgels overbalanced the trend to ion coupling.
For NS4 with its higher SPP content (4.11 mol %), no microgel shrinkage was
observed in the range of temperature 15 °C ≤ T ≤ 60 °C.
Figure 4-3. Variation in (a) hydrodynamic diameters Dh and (b) normalized
hydrodynamic diameters Dh/D0 as a function of temperature for zwitterionic microgels
in water. D0 is the hydrodynamic diameter of microgels at 50 °C. The particles were
dispersed in water at pH=7. Solid lines serve as eye guideline. (♦) NS0 (■) NS1 (▲) NS2
(X) NS3
4.4.4 Effect of salts
We further studied the effect of electrolytes on the properties of poly
(NIPAm-SPP) microgels by measuring the size of microgels in aqueous solutions of
monovalent and divalent salts at different concentrations. Figure 4-4 shows the
volume phase transition behaviors of zwitterionic microgels containing 3.068% SPP
in monovalent (KCl, Figure 4-5a) and divalent (CdCl2, Figure4-5b) salt solutions. All
microgels showed two similar trends. First, with increasing temperature the size of
250
400
550
700
850
10 20 30 40 50T (oC)
D (n
m)
NS0NS1NS2NS3
0.8
1.2
1.6
2
2.4
10 20 30 40 50T (oC)
D/D
0
NS0NS1NS2NS3
250
400
550
700
850
10 20 30 40 50T (oC)
D (n
m)
NS0NS1NS2NS3
0.8
1.2
1.6
2
2.4
10 20 30 40 50T (oC)
D/D
0
NS0NS1NS2NS3
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 92 -
200
250
300
350
400
450
10 20 30 40 50T (oC)
Rh
(nm
)
10-5M10-3M5*10-1M10-1M1M
200
250
300
350
400
450
10 20 30 40 50T (oC)
Rh
(nm
)
10-3M10-5M5*10-1M0.1M1M
(a) (b)
200
250
300
350
400
450
10 20 30 40 50T (oC)
Rh
(nm
)
10-5M10-3M5*10-1M10-1M1M
200
250
300
350
400
450
10 20 30 40 50T (oC)
Rh
(nm
)
10-3M10-5M5*10-1M0.1M1M
200
250
300
350
400
450
10 20 30 40 50T (oC)
Rh
(nm
)
10-5M10-3M5*10-1M10-1M1M
200
250
300
350
400
450
10 20 30 40 50T (oC)
Rh
(nm
)
10-3M10-5M5*10-1M0.1M1M
(a) (b)
Figure 4-4. Effect of concentration of a)KCl and b)CdCl2 on the volume phase
transition of poly (NIPAm-SPP) zwitterionic microgels containing 3.068% SPP; (♦)10-5M
(□)10-3M (▲)5x10-1M (○)10-1M (◊) 1 M. (c) Onset of the VPTT as a function of salt
concentration. (d) Initial hydrodynamic radius of microgels at 15oC in salt solutions.
microgels sharply decreased at the VPTT. Secondly, with salt concentration
increasing from 10-5 M to 1 M the value of VPTT shifted to lower values. No obvious
trend in the change of microgel size as a function of electrolyte concentration was
observed. Indeed, only a ca. 12.5 % reduction in volume of microgels was observed
upon increase in KCl concentration from 10-5 M to 1 M (Figure 4-4a). These results
are similar with those observed for NIPAm-SPP hydrogels where the authors
300
320
340
360
380
400
420
0.0001 0.01 1[Salt]
Rh
at 1
5o C (n
m)
[KCl][CdCl2]
10
15
20
25
30
35
0.00001 0.001 0.1 10
[Salt]
Ons
et o
f LC
ST (o C
)
[KCl][CdCl2]
VPTT
(c) (d)
300
320
340
360
380
400
420
0.0001 0.01 1[Salt]
Rh
at 1
5o C (n
m)
[KCl][CdCl2]
10
15
20
25
30
35
0.00001 0.001 0.1 10
[Salt]
Ons
et o
f LC
ST (o C
)
[KCl][CdCl2]
VPTT
10
15
20
25
30
35
0.00001 0.001 0.1 10
[Salt]
Ons
et o
f LC
ST (o C
)
[KCl][CdCl2]
VPTT
(c) (d)
Chapter 4
___________________________________________________________________________ - 93 -
reported the total loss of temperature sensitivity at high salt concentrations. They
explained this behavior by the fact that the added KCl can not break the interchain
or intergroup association.[16]
In both cases, onset of the VPTT shifted to lower temperatures with
increase in salt concentration. This decrease was gradual up to 0.1M electrolyte
concentration, but sharper at higher salt concentrations. Based on the results in
Figure 4-4, no obvious antipolyelectrolyte behavior was observed for the
zwitterionic microgels in the presence of both monovalent and divalent salts.
These results were unexpected since the solubility of the copolymers of SPP are
favored by the addition of low molecular weight electrolyte such as KCl due to the
suppressed intra- or intermolecular association in pure water.[19-21] Instead we
observed pure polyelectrolyte behavior: a decrease in microgel size and a lower
VPTT with increasing salt concentration, more prominent with increasing content
of the zwitterionic SPP in the microgels. In other words, the zwitterionic,
polyampholyte microgels behaved like polyelectrolyte systems at all salt
concentrations, in spite of carrying an equal number of cationic and anionic groups.
Lee and coworkers[12] previously reported the polyelectrolyte behavior of
zwitterionic sulfobetaine hydrogels in saline solutions of 10-5 to 10-1 M
concentrations, but observed antipolyelectrolyte behavior at salt concentrations
exceeding 0.5M, in contrast with results in our work. The unexpected
polyelectrolyte behavior shown by the zwitterionic sulfobetaine microgels in the
present work may be a result of the difference in binding affinities of the
ammonium and sulfonate residues to the respective counterions of the free
electrolyte. The quarternary ammonium ion is known to bind more strongly to the
Cl- than the sulfonate does to a metal cation.[19] Hence, with increasing
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 94 -
concentration of electrolyte, it is plausible that the SPP residue becomes relatively
anionic, and the zwitterionic microgels therefore show increasingly polyanionic
behavior.
4.5 Conclusion and outlook
Zwitterionic PA microgels of poly(NIPAm-SPP) attained a larger size with
increasing content of SPP. This was probably due to the increased hydrophilicity of
the polymer at the relatively low concentrations of SPP in the microgel. At low
concentrations, any ion-coupling between the oppositely charged residues was
negated by the hydrophilicity imparted to the polymer by the charged groups. No
variation in microgel size occurred with change in pH as was expected due to the
permanent charge carried by the quarternary ammonium and the sulfonate groups.
The temperature responsive swelling and deswelling transitions of
poly(NIPAm-SPP) microgels were retained in monovalent and divalent salt solutions.
The behavior of zwitterionic microgels in monovalent and divalent salt solutions
was found to resemble that of pure polyelectrolytes. Increase in the salt
concentration of the aqueous medium led to a decrease in the onset of the VPTT of
poly(NIPAm-SPP) microgels in both KCl and CdCl2 solutions. This behavior was
unexpected and may be due to the stronger binding affinity of the ammonium to
the Cl- than the sulfonate to the metal cation in the range of concentration
studied. From the results of these studies, we conclude that negligible ion-coupling
occurs between the charged SPP groups, in the range of salt concentrations
studied.
The persistent presence of the oppositely charged sulfonate and ammonium
groups in poly(NIPAm-SPP) microgels at all pH values is useful for the sequestration
Chapter 4
___________________________________________________________________________ - 95 -
of metal ions. It follows that the zwitterionic sulfobetaine microgels may serve as
ideal microreactors for the synthesis of nanoparticle composites. However, the
synthesis of such composites may be limited by the small content of charged groups
in the microgels.
Zwitterionic Sulfobetaine Microgels
___________________________________________________________________________ - 96 -
4.5 References for Chapter 4
[1] J. P. Baker, D. R. Stephens, H. W. Blanch and J. M. Prausnitz, Macromolecules 1992, 25,
1955-1958.
[2] S. E. Kudaibergenov and A. Ciferri, Macromolecular Rapid Communications 2007, 28,
1969-1986.
[3] S. Neyret and B. Vincent, Polymer 1997, 38, 6129-6134.
[4] K. Ogawa, A. Nakayama and E. Kokufuta, Langmuir 2003, 19, 3178-3184.
[5] Y. B. Zhao, Y. J. Yang, X. L. Yang and H. B. Xu, Journal of Applied Polymer Science
2006, 102, 3857-3861.
[6] G. Nisato, J. P. Munch and S. J. Candau, Langmuir 1999, 15, 4236-4244.
[7] M. Das and E. Kumacheva, Colloid and Polymer Science 2006, 284, 1073-1084.
[8] S. Nayak and L. A. Lyon, Abstracts of Papers of the American Chemical Society 2003,
226, U397-U398.
[9] A. G. Didukh, R. B. Koizhaiganova, G. Khamitzhanova, L. A. Bimendina and S. E.
Kudaibergenov, Polymer International 2003, 52, 883-891.
[10] M. B. Huglin and J. M. Rego, Macromolecules 1993, 26, 3118-3126.
[11] B. H. Tan, P. Ravi and K. C. Tam, Macromolecular Rapid Communications 2006, 27,
522-528.
[12] W. F. Lee and P. L. Yeh, Journal of Applied Polymer Science 1999, 74, 2170-2180.
[13] W. Xue, S. Champ and M. B. Huglin, European Polymer Journal 2001, 37, 869-875.
[14] T. Hoare and D. McLean, Journal of Physical Chemistry B 2006, 110, 20327-20336.
[15] S. Kudaibergenov, W. Jaeger and A. Laschewsky in Polymeric betaines: Synthesis,
characterization, and application, Vol. 201 2006, pp. 157-224.
[16] W. Cai and R. B. Gupta, Journal of Applied Polymer Science 2003, 88, 2032-2037.
[17] W. F. Lee and C. F. Chen, Polymer Gels and Networks 1998, 6, 493-511.
Chapter 4
___________________________________________________________________________ - 97 -
[18] K. Kabiri, S. Faraji-Dana and M. J. Zohuriaan-Mehr, Polymers for Advanced
Technologies 2005, 16, 659-666.
[19] S. E. Kudaibergenov, Polyampholytes: Synthesis, Characterization and Application,
Kluver Academic, New York, 2002, p.
[20] S. E. Kudaibergenov, W. Jaeger and A. Laschewsky, Advance in Polymer Science 2006,
201, 157-224.
[21] J. S. Lowe, B. Z. Chowdhry, J. R. Parsonage and M. J. Snowden, Polymer 1998, 39,
1207-1212.
Chapter 5
___________________________________________________________________________ - 98 -
Chapter 5
Biofunctionalized pH-responsive Polymer
Microgels for Cancer Cell Targeting
Acknowledgements: This work was conducted in collaboration with Sawitri Marydani in
Professor Warren Chan’s Group at the Institute of Biomaterials and Biomedical Engineering,
University of Toronto. Bioconjugation and cytotoxicity experiments on Hela cells were
conducted in Prof. Warren Chan’s Lab.
5.1 Introduction
Advances in the development of novel therapeutic molecules have been
limited by a lack of effective delivery technologies.The design of polymer-based
drug delivery sytems (DDSs) for targeted and controlled release of therapeutic
agents is of growing interest in the materials, chemical, biomedical and
pharmaceutical disciplines. The integration of these two properties into DDSs can
dramatically improve therapeutic efficiency whilst reducing toxic side effects.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 99 -
The function of most polymeric, particulate DDSs is to reduce
immunogenicity, degradation and toxicity, while improving circulation time.
Generally, the polymeric carrier must be water-soluble, non-toxic and non-
immunogenic at all stages of the delivery, including a safe excretion. If the
polymer is non-degradable as is often the case (e.g., polymethacrylates), then the
size of the microgels must be below the renal threshold so it does not acculumulate
in the body. If the polymer is degradable, the toxicity of the degradation products
must be considered as well.
While macromolecular carriers (polymer chains and polymer-peptide
conjugates) have shown some promise [1-10] as DDSs, they have serious drawbacks,
namely, insubstantial protection of the drug from the body’s defense mechanisms
and potentially low drug doses delivered to the target site. Particulate or micro-
reservoir, polymer-based drug delivery systems by contrast provide a means for
delivering drugs to diseased sites in high doses, protecting drugs from enzymatic
degradation, and inhibiting the delivery of drugs to healthy tissues. Some of the
useful particulate polymer-based DDSs include liposome-based [11] or micelle-
based[12-14] DDSs.
Stimuli-responsive microgels whose open network structures allow for the
incorporation of drugs, are an important subcategory of particulate DDSs.[15-19]
Besides carrying out passive functions as drug carriers, stimuli-responsive microgels
can carry out more active roles such as the release of a drug or biomolecule upon
an external stimulus.[15] In addition to pH-, ionic strength-, or temperature-
triggered volume transitions, microgels loaded with a drug can interact with
biological components or events (e.g., enzymatic processes) that can trigger the
release of a loaded drug. Furthermore, the relatively facile synthesis and
Chapter 5
___________________________________________________________________________ - 100 -
functionalization of polymeric microgels affords several advantages: their size may
be manipulated from 100 nanometers to several micrometers; their volume phase
transitions can be tuned to occur within relevant physiological conditions; and their
surfaces can be conjugated to receptor-specific biomolecules to attain selective
targeting ability designed to treat specific diseases or tumor cells.
5.2 Background
5.2.1 pH-mediated drug release
Application of stimuli-responsive microgels to drug delivery systems
requires the response of a system to be tailored to the specific properties of the
targeted medium. Oral delivery of any drug must obviously take into account the
pH change along the gastro-intestinal tract from acidic (pH~2-3) in the stomach to
weakly basic (pH~5-8) in the intestine.[20] However, numerous other subtle pH
gradients occur in various tissues and organs of the human body. For instance,
cancerous and inflamed or wounded tissues are reported to have a pH more acidic
than that of the physiological pH of 7.4.[21] The same is true for different cellular
compartments[22], as is illustrated in Table 5-1.[23] It follows that these biologically
occurring pH-gradients can trigger a physicochemical response that translates to a
change in conformation, stability, solubility, or hydrophobic-hydrophilic balance in
the polymeric DDSs and directs the release of a loaded drug towards a specific
cellular or tissue region, thereby enhancing drug efficacy.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 101 -
Table 5-1. pH values in different tissue and cellular environments.[23]
Tissue/Cellular Compartment pH
Blood 7.35-7.45
Stomach 1.0-3.0
Duodenum 4.8-8.2
Colon 7.0-7.5
Early endosome 6.0-6.5
Late endosome 5.0-6.0
Lysosome 4.5-5.0
Golgi 6.4
Tumor, extracellular 6.5-7.2
Ionisable polymers with pKa values that fall between 3 and 10 make good
candidates for pH-responsive polymeric DDSs. Common examples of the active
functional groups in these polymers include carboxylic acid-based polyanions,
cationic amines, poly(ethyleneimine), modified chitosan and phosphines. [23]
5.2.2 Cancer Treatment and Intra-Cellular Drug Delivery
One of the main impediments to effective and improved cancer treatment
remains in the non-specific distribution of administered drugs resulting in low
tumor concentration and systemic toxicity. The main factors prohibiting the
effective distribution of anti-cancer agents to tumor sites include the highly
Chapter 5
___________________________________________________________________________ - 102 -
disorganized tumor vasculature, high blood viscosity in the tumor and high
interstitial pressure within the tumor tissue.[24] Recently, several strategies aimed
at enhancing tumor targeting were explored, including drug modifications and the
development of advanced carriers of anticer agents.[6, 9, 25, 26] For example, the
enhanced permeation and retention (EPR) effect is a phenomenon characterised by
enhanced accumulation of macromolecular or particulate drugs in the tumor aided
by the increased permeability caused by the defective, leaky vasculature
characteristic of tumor tissues.[27] Exploitation of the EPR effect[27-30] by DDSs has
significantly enhanced feasible drug concentrations in tumor sites while approaches
involving the sophisticated design of polymeric, stimuli-responsive DDSs have
improved therapeutic efficiency. For example, the pH-triggered delivery of a drug
using polymer-drug (prodrug) conjugates with an acid-labile linker has been shown.
The drug is released at the target site by the pH-triggered cleavage of the
conjugate bond, upon entering the extracellular tissue or being internalized into
tumor cells. Reported polymer-drug conjugatesinclude poly(ethylene glycol)(PEG),8
dextran,9 N-(2-ydroxypropyl)methacrylamide copolymer.10
Cellular uptake of polymeric DDSs usually occurs by fluid-phase pinocytosis
or receptor-mediated endocytosis (RME).[31] A schematic showing the intracellular
uptake of a pH-responsive DDS via RME is shown in Figure 5-1. Post-internalization,
the DDS remains in the vesicles as it progresses from the early endosomes, to late
endosomes, and finally through to lysosomes before being eliminated from the
tumor. During the uptake process, the polymer drug carrier is subjected to the
intracellular pH gradient spanning pH~7.4 (extracellular) to pH~5.5, which can
provide the stimulus required to release a drug into the cytoplasm before
elimination from the cellular compartment.[32] The primary academic challenge in
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 103 -
intracellular drug delivery is to promote endosomal escape of the drug from the
delivery vehicle to the cytoplasm. Some strategies that have been used to date
include the use of dendritic polymers, lytic peptides and pH-sensitive polymers.[23,
32, 33] The latter is of particular interest.
Figure 5-1 Schematic representation of the use of the receptor-mediated endocytosis
pathway for the targeted delivery of a drug. The pH-responive DDS is exposed to the
intracellular pH-gradient as it progresses through the endocytic environment. This pH
gradient can employed as a trigger to promote controlled drug release into the cytosol.
5.2.3 Biofunctionalized microgels for drug delivery
Polymer microgels are ideal components of particulate DDSs. They have
several important advantages over other particulate carriers, namely, stability,
ease of synthesis, good control over particle size, and easy functionalisation
providing stimulus-responsive behavior. Several works on the use of pH-responsive
microgels for drug delivery have been reported.[15, 34-36] Langer et al. [37] and
Frechet et al. [15] reported the pH-triggered non-specific release of a drug from
microgels to the macrophages, the pH-responsive particles used in their
Not to scale
pH-responsive microgel
Nucleus
Cell membrane pH~ 7.4
pH ~6.5
3
4 5
1 2
6
pH~ 5
endosome
lysosome
Drug loading
Bioconjugation
Chapter 5
___________________________________________________________________________ - 104 -
experiments were too large to reach tumor sites. Yang et al.[11] reported polymer
core-shell microgels that were stable at pH=7.4 and 37oC, but deformed and
precipitated in an acidic environment, triggering the release of the drug molecules.
These particles however were not tested in the cellular environment.
The introduction of targeting ligands onto drug carriers reduces unwanted
toxicity, and enhances therapeutic performance. Typically, the targeting ligand is
chosen such that it binds selectively to specific cell receptors that are
overexpressed in tumor cells. Examples of different targeting species that have
been used include sugars, peptides and folic acid. Lyon et al. [19] reported folate-
mediated cell targeting with microgels of diameter ca. 270 nm that exhibited
temperature-dependent cytotoxicity. However, this cytotoxicity was only induced
at 37 oC, which is very close to the temperature of healthy tissues.
5.3 Research objectives
In this work, we employed the rational design of a microgel-based drug
delivery system for cytosolic drug delivery to tumor cells. This strategy was
inspired by recent discoveries on characteristics of tumors, [12] which provide an
excellent guide for the design of DDSs capable of selectively targeting diseased
cells and releasing drugs only in specific biological conditions. We implemented the
following criteria in the design of effective particulate DDSs for cancer therapy:
(1) The presence of appropriate functional groups for conjugation with
targeting species which can selectively transport the microgels to a diseased site
(2) Small (below 200 nm) size of microgels to maximize extravasation into
tumors
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 105 -
(3) A release mechanism induced by biological stimuli such as change in pH
or interactions with enzymes, ions or proteins.
(4) Incorporation of the drug into the microgel by physical means, as
opposed to its covalent attachment (which may potentially alter the drug’s
effectiveness).
We used pH-responsive microgels which possessed all the aforementioned
design requirements. Figure 5-2 shows the general scheme outlining the
modification and function of our drug delivery system. The microgels were
synthesized to obtain particles with diameter of ca. 150 nm, similar to that of
typical viruses. The drug was incorporated into the microgels by a diffusion-driven
process, aided by electrostatic interactions between the microgel and the drug. We
conjugated receptor-specific molecules onto the surface of the microgels for
targeting diseased cells. Finally, we demonstrated the feasibility of this microgel-
based DDS to deliver small organic molecules and anticancer drugs into cancer cells
via receptor mediated endocytosis (RME).
5.4 Experimental
5.4.1 Synthesis of microgels
Poly (N-isopropylacrylamide-acrylic acid) [poly (NIPAm-AA)] microgel
particles were synthesized by free radical precipitation polymerization. The
composition of the reaction mixture was as follws: 1.2 g (84.6 mol%) NIPAm, 0.01 g
(15.4 mol%) AA, 0.0520 g (3.5 mol%) BIS, 0.02 g (0.06%) SDS and 0.015 g (1.2 mol%)
KPS in 100 g of water. The resulting microgels were purified by dialysis against daily
changes of water for 21 days. The dialysed microgels were subjected to repeated
(up to four times) centrifugation and redispersion in deionized water at 30,000 X G
and 24oC for 30 minutes in a temperature-controlled centrifuge.
Chapter 5
___________________________________________________________________________ - 106 -
Figure 5-2 Conceptual diagram of proposed biofunctionalized, pH-responsive
drug delivery system for intracellular cancer cell targeting.
5.4.2 Particle characterization
Particle sizes were determined by photon correlation spectroscopy (PCS,
Protein Solutions Inc.) equipped with a temperature control. All solutions used for
PCS measurements were diluted with de-ionized water and adjusted to the desired
pH with HCl and/or NaOH while monitoring with an Ecomet pH meter. For
temperature-dependent measurements, the sample was allowed to equilibrate for
20 minutes at the desired temperature before data collection. Electrokinetic
potential of the particles was measured using the Zetasizer 3000 HS (Malvern
Instruments).
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 107 -
5.4.3 Drug and dye uptake into particles
Drug and dye uptake experiments were tested in 0.01M phosphate buffered
saline (PBS) at pH 7.4. 100 μL of the purified microgel dispersion was mixed
together with 100μL of 10-4 M R6G in 5mL of 0.01M at pH 7.4 and allowed to
equilibrate overnight on a rotary mixer at room temperature. The dye-loaded
microgel dispersions were then separated from the suspension by
ultracentrifugation at room temperature (in a temperature-controlled centrifuge)
and the supernatant solution was diluted as necessary to quantitatively determine
loading capacity by UV/VIS spectrometry. Absorbance readings were taken at
530nm and 464 nm for R6G and Dox respectively. The residual drug or dye
concentrations in the supernatant were then calculated based on calibration curves
obtained from drug/dye solutions of known concentrations in PBS at pH 7.4. For
Dox infusions, 50 μL of 10-4M Dox solutions were mixed with 50 and 100 μL of
purified microgel samples, and diluted to 5mL of 0.01M PBS (pH 7.4).
5.4.4 Conjugation of transferrin and albumin to loaded gels
The conjugation of transferrin and albumin to the microgels was performed
through the same process. A 10mg/ml stock solution of the proteins was made.
20μLof this solution was then mixed with 50μL of loaded microgels. Then, at least
10 fold molar excess of 1-Ethyl-3-(2-dimethylaminopropyl) carbodiimide
hydrochloride was added to mediate the formation of an amide bond between
carboxylic groups on the gel and amino groups on the protein. The reaction was
left to proceed for at least two hours.
5.4.5 R6G-loaded gels assay
Chapter 5
___________________________________________________________________________ - 108 -
HeLa cells were grown on coverslips in 100mm tissue culture dishes until
50% confluency. R6G- loaded hydrogel particles, conjugated to transferrin, were
dispersed in high glucose DMEM, supplemented with 10% fetal bovine serum, 1 %
penicillin, and 1% amphotericin B. For the controls, we used bare R6G-loaded
microgel particles, particles conjugated to bovine serum albumin, and particles in
solution with but not conjugated to transferrin. All microgels maintained their
colloidal stability. Cells were incubated overnight. Coverslips were washed with
10mM PBS. The cells were fixed with 3-4% paraformaldehyde followed by 3 washes
with PBS. The cells were examined at 20X magnification through differential
interference contrast and epifluorescence.
5.4.6 Dox-loaded gels assay
HeLa cells were grown in 100mm tissue culture dishes until 80-100%
confluency. Dox-loaded hydrogel particles, conjugated to transferrin, were
dispersed in high glucose DMEM, supplemented with 10% fetal bovine serum, 1 %
penicillin, and 1% amphotericin B. For the controls, we used bare Dox-loaded
hydrogel particles, particles conjugated to bovine serum albumin, and particles in
solution with but not conjugated to transferrin. Cells were incubated for 36 hours.
The cells were then trypsinized and stained with trypan blue. The numbers of live
and dead cells were counted under the microscope.
5.5 Results and discussion
5.5.1 pH-response of microgels
The volume phase transition temperature (VPTT) of the copolymer particles
was above the body temperature of 37oC. At pH≈7.0 the carboxylic groups of AA
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 109 -
were ionized (ζ-potential = -46mV) while at pH ≈ 4.0 they were largely protonated
and carried only a weak charge (ζ-potential = -1.2 mV). Figure 5-3 shows the
variation of microgel size in the range 3.0 < pH < 8.0. At pH ≈ 7.0 the microgels
were 50% larger in size than at pH ≈ 4.0. The increase in microgel size at pH ≈ 7.0
occurred due to electrostatic repulsion between the deprotonated carboxylic acid
moieties and the consequent increase in hydrophilicity of the polymer. [14-15]
0.8
1.2
1.6
2
2.4
2.5 4.5 6.5 8.5pH
D/D
0
Figure 5-3 Variation in normalized hydrodynamic diameter of microgel particles as a
function of pH where D0 is the smallest diameter of microgel particle in the range
studied. D0=142.3nm All measurements were taken at 25 oC in 0.01M KCl. The average
hydrodynamic diameter of the microgels was ca. 110 and 156 nm at pH= 4.5 and
pH=7.4, respectively.
5.5.2 Loading and release of rhodamine dye
Rhodamine 6G (R6G), a commercially available fluorescent dye was
introduced into the swollen, negatively charged microgel particles in 0.01M
phosphate buffered saline (PBS) at pH = 7.4, by physical mixing. The chemical
structure of R6G is shown in Figure 5-4. R6G is a weakly basic dye with a pKa value
Chapter 5
___________________________________________________________________________ - 110 -
of ~8.3, making it positively charged at pH=7.4. Electrostatic attraction between
the positively charged R6G and the negatively charged microgels assisted the
diffusion-driven loading of microgel particles with R6G.
OHN
HN
O
O
Cl
Figure 5-4 Chemical structure of Rhodamine 6G- hydrochloride. The dye has a pKa
value of 8.3, making it positively charged at pH=7.4.
(b)(a) (b)(a)
Figure 5-5 Fluorescence images of R6G-loaded microgels at pH 7.4 (a) and at pH=4.5
(b) The net uptake of R6G (expressed as a percentage of the total amount of R6G
added at the start of the exp) was 33.5%
Figure 5-5 a shows an optical microscopy image of the microgels loaded
with R6G at pH = 7.4. The presence of discrete bright spots on the dark background
indicated that the dye was localized in the microgel particles. Figure 5-5 b shows
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 111 -
the R6G-loaded microgels at pH=4.5. The diffuse fluorescence signal in the
background points to the release of R6G from the microgel interior into the
continuous medium upon microgel deswelling. Protonation of the carboxylic groups
resulted in suppression of both the repulsive electrostatic forces that caused
swelling, and the attractive electrostatic forces that maintained the dye within the
microgel. Note that the values of pH used in these model release experiments were
typical of those in the extracellular matrix (pH=7.4) and lysosome (pH=4.5) (final
intra-cellular point of a molecule undergoing receptor mediated endocytosis before
entry into the cytoplasm).[38]
5.5.4 Biofunctionalization of microgels
In the next stage, HeLa cancer cells were chosen to study the intra-cellular
uptake of the pH-responsive, R6G-loaded microgel DDSs in vitro. This model cell
line was chosen because it has been fully characterized for intracellular delivery
through RME, using the targeting, iron-carrying protein, transferrin.[39]
The R6G-loaded microgel particles were conjugated to apo-transferrin via
carbodiimide coupling. Apo-transferrin is a tertiary protein which binds selectively
to the transferrin receptors on the surface of HeLa cells, enabling endocytosis.[39,
40] The mechanism of transferrin binding to its receptor has not been clearly
elucidated. It is however believed that the binding mechanism is related to the
conformational rearrangement that occurs as the N-lobe of the transferrin binds to
the cell receptor.[38, 41] The bioconjugation reaction was carried out in phosphate-
buffered media of pH 7.4, with the aid of the coupling agent, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC). The optimal pH conditions
for this reaction is in fact between pH 4.7-6, but it can also be successfully carried
Chapter 5
___________________________________________________________________________ - 112 -
out at pH 7.4, albeit with reduced reaction efficiency. It is plausible that some
amount of the apo-transferrin may have physically adhered to the microgel
surface.
Figure 5-6 Scheme depicting bioconjugation of carboxylic acid functionalized microgels
using carbodiimide coupling.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 113 -
(a)
(c)
(b)
(a)
(c)
(b)
Figure 5-7 Differential interference contrast (DIC) (left) and epifluorescent (right)
images of HeLa cells after 24 hours incubation with R6G-loaded microgel-DDSs not
conjugated to any protein (a), conjugated to albumin (b) and conjugated to transferrin
(c). R6G is released from transferrin-conjugated microgels due to change in pH during
RME. 20x objective N.A. = 0.4, λex = 480 +/- 40 nm (100 W Hg lamp), λem = 535 nm.
5.5.5 Intracellular uptake of bioconjugated microgels
The targeting efficiency of the pH-responsive microgel DDSs was assessed
with the aid of control experiments. Figure 5-7 shows differential interference
contrast (left column) and corresponding epifluorescent (right column) images of
HeLa cells after 24 h incubation with R6G-loaded microgels not conjugated
(d)
Chapter 5
___________________________________________________________________________ - 114 -
to any protein (a), conjugated to the non-endocytic protein, albumin (b),
and conjugated to transferrin (c). A strong luminescence signal was observed only
for HeLa cells incubated with transferrin-conjugated microgels (Figure 5-7c) in
contrast with the control systems (Figures 5-7a and 5-7b) where only a weak
luminescence was observed. These observations confirmed that the release of R6G
into the cytoplasm was due to transferrin-targeting to HeLa cells and pH-triggered
deswelling of microgels upon exposure to the acidic lysosomal environment.
By measuring the luminescence intensity per cell we estimated that the
transferrin-conjugated microgels delivered over 3 and 100 times more R6G to the
cells than the albumin-conjugated and bare microgels, respectively. The slightly
enhanced fluorescence for cells incubated with albumin-conjugated microgels (as
compared to the bare microgels) was attributed to non-specific binding of albumin
to HeLa cells.
5.5.6 In Vitro studies of uptake and release of an anticancer drug
Doxorubicin (Dox), also known as Adriyamycin, is a widely used anticancer
drug. It is a DNA-interacting drug that is believed to inhibit the replication process
by intercalating with DNA.[42, 43] Dox is commonly used to treat some leukemias,
and cancers of the bladder, breast, lung, ovaries, thyroid, and stomach to name a
few. Dox is known to have a range of acute side effects from nausea, vomiting and
hair loss to myelosupression and cardiotoxicity. These side effects and its red color
have earned Dox the nickname ‘red devil’. Thus targeted delivery is especially
advantageous for this drug.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 115 -
Figure 5-8 Chemical structure of the anticancer drug, Doxorubicin. The red
compound is weakly basic and has a pKa value of 8.3.
Dox is a weakly basic drug, with a pKa value of ~8.22.{Sturgeon, 1977 #191}
Therefore at pH = 7.4, Dox is positively charged enabling the electrostatically-
driven incorporation of the drug into the microgel interior.
5.5.6.1 Quantitative determination of drug uptake by microgels
The amount of drug taken up by microgels at 37oC in PBS media at pH 7.4
was evaluated using the following relations:
Loading Capacity = Weight of loaded drug
Weight of microgel particleLoading Capacity =
Weight of loaded drug
Weight of microgel particle
Association Efficiency = Weight of loaded drug
Weight of total initial amount of drug added to microgel dispersion
Association Efficiency = Weight of loaded drug
Weight of total initial amount of drug added to microgel dispersion
The loading capacity (LC) as the term suggests, is an indication of the
amount of drug that may be incorporated into the particle. The association
efficiency (AE) determines the percentage efficiency of drug uptake in the carrier,
relative to the total initial amount of drug introduced into the microgel dispersion.
Chapter 5
___________________________________________________________________________ - 116 -
Together, these parameters express the suitability of the polymer particle as a
potential drug reservoir and indicate the performance of drug loading. These
factors are especially important considerations for formulations in which the
loading of the drug has been achieved primarily by a diffusion-driven process.
In the present work, both the diffusion-driven process, and electrostatic
interactions were utilized to load the drug into microgel particles. The
concentration of Doxorubicin in 0.01M PBS buffer was determined by UV/VIS
spectrometry. The loading capacity and association efficiency of Dox-microgel
formulations as a function of initial drug concentrations are shown in Figure 5-9 a
and b. In general, increasing the initial concentration of Dox resulted in an increase
in the LC of microgels due to the stronger concentration gradient between the
buffer medium and the interior of the microgel particles. Conversely, the AE
progressively decreased with increasing Dox concentration presumably due to the
approach of the maximum capacity of drug loading. The maximum loading
capacities for Dox in the microgels were 45.8 and 56.7% for 0.1 and 0.2 wt%
microgel dispersions respectively. The relatively higher AE observed for the
dispersion containing more microgel particles (Figure 5-9 b) was attributed to
enhanced ionic interactions due to the augmented presence of deprotonated
carboxylic acid groups that could attract positively charged Dox at pH 7.4.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 117 -
0
10
20
30
40
50
60
0.01 0.02 0.03 0.05Concentration of Dox (mg/mL)
%
(a)
(b)
0
10
20
30
40
50
60
70
0.01 0.02 0.03 0.05Concentration of Dox (mg/mL)
%
0
10
20
30
40
50
60
0.01 0.02 0.03 0.05Concentration of Dox (mg/mL)
%
(a)
(b)
0
10
20
30
40
50
60
70
0.01 0.02 0.03 0.05Concentration of Dox (mg/mL)
%
Figure 5-9. Loading capacity (left columns) and association efficiency (right
columns) of Doxorubicin in poly (NIPAm-AA) microgel particles at 37oC in 0.01M
PBS at pH 7.4 for (a) 0.1 and (b) 0.2wt% microgel dispersion.
Chapter 5
___________________________________________________________________________ - 118 -
5.5.6.2 Effect of pH on drug release from microgels
The effect of pH on the release of Dox from microgels was investigated in
PBS media at 37oC. Figure 5-10 shows the cumulative release of Dox over a period
of 5 days at pH 7.4 and at pH 4.5. The release profiles obtained at both pH values
were characterized by an initial burst of rapid drug release within the first day,
which subsequently leveled out thereafter as the system reached equilibrium. At
pH=7.4, 40.3% of drug was released at the end of the first day, compared with
59.5% at pH =4.5. The slightly greater amount of Dox released at pH=4.5 than at
pH=7.4 was due to the pH-induced shrinkage of Dox-loaded microgels upon being
exposed to acidic media. After 5 days, a maximum release of 50.1% and 68.8% were
observed at pH= 7.4 and pH=4.5 respectively.
0
10
20
30
40
50
60
70
80
90
0 1 2 3 4 5 6Time (days)
% C
umul
ativ
e R
elea
se
Figure 5-10. Percentage cumulative release of Dox from microgels (LC of 45.8%)
at 37oC at different pH values: ( ) pH=7.4 (■) pH=4.5
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 119 -
5.5.6.3 In Vitro test of cell viability
We further examined the cytotoxicity of bioconjugated, pH-responsive
microgels loaded with Doxorubicin (Dox). We compared the viability (percentage of
survived cells) of HeLa cells after 36 h incubation with transferrin-conjugated Dox-
loaded microgels with that of several control systems. Cell viability was assessed
using a Trypan Blue exclusion assay. In such an assay, cells with an intact
membrane are able to exclude the dye while cells with a damaged membrane will
take up the dye. The percentage of viable cells is given by
% viable cells = Number of unstained cells/ Total number of cells X100
Figure 5-11(a) shows that HeLa cells incubated with transferrin-conjugated
Dox-loaded microgels had viability of 28.4 % (that is, mortality of 72.6%). The
control systems included Dox-loaded microgels in solution with, but not conjugated
to transferrin (b), albumin-conjugated Dox-loaded microgels (c), Dox-loaded non-
conjugated microgels in buffer saline solution (d), transferrin-conjugated microgels
in absence of Dox (e) and plain HeLa cells (f). These systems showed cell mortality
of 30.6, 27.0, 33.8, 24.3, and 32.3%, respectively.
Chapter 5
___________________________________________________________________________ - 120 -
0
20
40
60
80
100
a b c d e f
% C
ell V
iabi
lity
Figure 5-11 Viability of HeLa cells after incubation for 36h with different systems: a)
Transferrin-conjugated Dox-loaded microgels; b) Dox-loaded microgels in solution with
free transferrin (no conjugation); c) Albumin-conjugated Dox-loaded microgels; d) Plain
Dox-loaded microgels (no conjugation); e) Transferrin-conjugated plain microgels (no
Dox); f) HeLa cells only.
The mortality values of HeLa cells in all the control systems were
significantly lower than those for transferrin-conjugated Dox-loaded microgels and
clearly indicated that biofunctionalized pH-responsive microgels were successfully
taken up by the cells and carried the chemotherapeutic agent into the cytosol.
The enhanced cell suppression in the transferrin-conjugated system is testament to
the targeting ability achieved by bioconjugation. In addition, the pH-induced
deswelling of microgels at pH=4.5 may promote a faster diffusion-driven release of
Dox from the acidic endocytic vesicles.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 121 -
5.6 Conclusions and future outlook
In summary, the rational design of a targeted, pH-responsive drug delivery
system was demonstrated. Bio-conjugated pH-responsive microgels, offer an
effective approach for highly specific targeting of cancer cells. Exposure of the
drug or dye-loaded microgel particles to the intra-cellular pH-gradient during the
receptor-mediated endocytosis process was utilized for targeted delivery of organic
molecules (including an anticancer drug) into cancer cells. In the future,
experiments will be extended to in vivo systems and to biopolymer-based and
biodegradable microgels.
Although the results of the work presented by others and herein are
promising for the development of advanced DDSs, the challenges are manifold.
Stimulus-responsive DDSs like the pH-sensitive microgels described in this work are
susceptible to the fluctuations in environmental conditions in the blood stream like
pH and ionic strength, leading to premature volume transitions and release. While
vectorization of the DDS can help lessen this problem, still others persist. Foremost
are the interactions of the drug carrier with serum proteins, salts and enzymes that
can form complexes. The reticuloendothelial system (RES), which is the body’s
immune response consisting primarily of macrophages attacks the DDSs and can
efficiently seize them from circulation. As well, non-specific renal clearance of the
DDS does occur in many instances. Therefore, vital requirements for enhancing the
performance of DDSs include minimizing their interactions with serum proteins and
extending circulation time. One approach that has found appreciable success is to
protect the surface of the carrier with grafted or adsorbed polymers like
polyethyleneglycol (PEG), which can inhibit their interactions with biological
species until reaching the target site.
Chapter 5
___________________________________________________________________________ - 122 -
5.7 References
[1] R. Duncan, Nature Reviews Drug Discovery 2003, 2, 347-360.
[2] Y. Katayama, T. Sonoda and M. Maeda, Macromolecules 2001, 34, 8569-8573.
[3] Y. Chau, R. F. Padera, N. M. Dang and R. Langer, International Journal of Cancer 2006,
118, 1519-1526.
[4] A. Mitra, A. Nan, J. C. Papadimitriou, H. Ghandehari and B. R. Line, Nuclear Medicine
and Biology 2006, 33, 43-52.
[5] R. J. Christie and D. W. Grainger, Advanced Drug Delivery Reviews 2003, 55, 421-437.
[6] K. Ulbrich and V. Subr, Advanced Drug Delivery Reviews 2004, 56, 1023-1050.
[7] T. Etrych, M. Jelinkova, B. Rihova and K. Ulbrich, Journal of Controlled Release 2001,
73, 89-102.
[8] R. Duncan, S. Gac-Breton, R. Keane, R. Musila, Y. N. Sat, R. Satchi and F. Searle,
Journal of Controlled Release 2001, 74, 135-146.
[9] M. J. Vicent and R. Duncan, Trends in Biotechnology 2006, 24, 39-47.
[10] P. C. A. Rodrigues, U. Beyer, P. Schumacher, T. Roth, H. H. Fiebig, C. Unger, L.
Messori, P. Orioli, D. H. Paper, R. Mulhaupt and F. Kratz, Bioorganic & Medicinal
Chemistry 1999, 7, 2517-2524.
[11] P. F. Kiser, G. Wilson and D. Needham, Journal of Controlled Release 2000, 68, 9-22.
[12] M. F. Francis, M. Cristea and F. M. Winnik, Pure and Applied Chemistry 2004, 76,
1321-1335.
[13] G. S. Kwon and K. Kataoka, Advanced Drug Delivery Reviews 1995, 16, 295-309.
[14] M. Yokoyama, G. S. Kwon, T. Okano, Y. Sakurai, T. Seto and K. Kataoka, Bioconjugate
Chemistry 1992, 3, 295-301.
Biofunctionalized pH-Resp[onsive Microgels for Cancer Cell Targeting
___________________________________________________________________________ - 123 -
[15] N. Murthy, M. C. Xu, S. Schuck, J. Kunisawa, N. Shastri and J. M. J. Frechet,
Proceedings of the National Academy of Sciences of the United States of America 2003, 100,
4995-5000.
[16] L. Bromberg, M. Temchenko and T. A. Hatton, Langmuir 2002, 18, 4944-4952.
[17] S. V. Vinogradov, T. K. Bronich and A. V. Kabanov, Advanced Drug Delivery Reviews
2002, 54, 135-147.
[18] G. M. Eichenbaum, P. F. Kiser, A. V. Dobrynin, S. A. Simon and D. Needham,
Macromolecules 1999, 32, 4867-4878.
[19] S. Nayak, H. Lee, J. Chmielewski and L. A. Lyon, Journal of the American Chemical
Society 2004, 126, 10258-10259.
[20] W. N. Charman, C. J. H. Porter, S. Mithani and J. B. Dressman, Journal of
Pharmaceutical Sciences 1997, 86, 269-282.
[21] L. E. Gerweck and K. Seetharaman, Cancer Research 1996, 56, 1194-1198.
[22] S. Simon, D. Roy and M. Schindler, Proceedings of the National Academy of Sciences of
the United States of America 1994, 91, 1128-1132.
[23] D. Schmaljohann, Advanced Drug Delivery Reviews 2006, 58, 1655-1670.
[24] L. H. Reddy, Journal of Pharmacy and Pharmacology 2005, 57, 1231-1242.
[25] R. Pola, M. Pechar, K. Ulbrich and A. F. Fres, Journal of Bioactive and Compatible
Polymers 2007, 22, 602-620.
[26] C. Li, Advanced Drug Delivery Reviews 2002, 54, 695-713.
[27] K. Greish, Journal of Drug Targeting 2007, 15, 457-464.
[28] S. Modi, J. P. Jain, A. J. Domb and N. Kumar, Current Pharmaceutical Design 2006, 12,
4785-4796.
[29] T. Minko, S. S. Dharap, R. I. Pakunlu and Y. Wang, Current Drug Targets 2004, 5, 389-
406.
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___________________________________________________________________________ - 124 -
[30] H. Maeda, K. Greish and J. Fang in The EPR effect and polymeric drugs: A paradigm
shift for cancer chemotherapy in the 21st century, Vol. 193 2006, pp. 103-121.
[31] H. Sato, Y. Sugiyama, A. Tsuji and I. Horikoshi, Advanced Drug Delivery Reviews 1996,
19, 445-467.
[32] V. P. Torchilin, Annual Review of Biomedical Engineering 2006, 8, 343-375.
[33] A. K. Patri, J. F. Kukowska-Latallo and J. R. Baker, Advanced Drug Delivery Reviews
2005, 57, 2203-2214.
[34] K. S. Soppimath, T. M. Aminabhavi, A. M. Dave, S. G. Kumbar and W. E. Rudzinski,
Drug Development and Industrial Pharmacy 2002, 28, 957-974.
[35] C. Alvarez-Lorenzo and A. Concheiro, Journal of Controlled Release 2002, 80, 247-257.
[36] K. S. Soppimath, A. R. Kulkarni and T. M. Aminabhavi, Journal of Controlled Release
2001, 75, 331-345.
[37] D. M. Lynn, M. M. Amiji and R. Langer, Angewandte Chemie-International Edition
2001, 40, 1707-1710.
[38] L. A. Bareford and P. W. Swaan, Advanced Drug Delivery Reviews 2007, 59, 748-758.
[39] Z. M. Qian, H. Y. Li, H. Z. Sun and K. Ho, Pharmacological Reviews 2002, 54, 561-587.
[40] E. Wagner, D. Curiel and M. Cotten, Advanced Drug Delivery Reviews 1994, 14, 113-
135.
[41] P. Ponka and C. N. Lok, International Journal of Biochemistry & Cell Biology 1999, 31,
1111-1137.
[42] F. Zunino and G. Capranico, Anti-Cancer Drug Design 1990, 5, 307-317.
[43] E. Ferrazzi, J. M. Woynarowski, A. Arakali, D. E. Brenner and T. A. Beerman, Cancer
Communications 1991, 3, 173-180.
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___________________________________________________________________________ - 125 -
Chapter 6
Hybrid Microgels for Photo-Thermally
Induced Drug Release
Acknowledgements: Thanks to Daniele Fava and Dr. Nicolas Sanson for synthesis of Au NRs
and poly (NIPAm-AA-BMA), and poly (NIPAm-NIPMAm)/PAA IPN microgels respectively.
Special thanks to David Gwiercer, Dr. Eduardo Moriyama, Dr. Robert Weersink and
Professor Brian Wilson at Princess Margaret Hospital for their assistance in confocal
imaging.
6.1 Introduction
The incorporation of functional nanostructures comprising semiconductors,
noble metals, biominerals and metal oxides into polymer microgels has opened new
avenues for materials with advanced structural and functional properties.[1] These
hybrid microgels have applications in the template-based synthesis of NPs[2], in the
Chapter 6
___________________________________________________________________________ - 126 -
fabrication of photonic crystals[3], and in sensory optical devices.[4] Kawaguchi and
coworkers reported the fabrication of hybrid microgels that exhibited multiple
brilliant colors due to the inter-particle interactions of surface plasmon resonance
using bimetallic Au/Ag nanoparticles.[5] Lyon and coworkers reported the assembly
of colloidal crystals comprising thermoresponsive core/shell microgels containing
localized Au nanoparticles[5] and showed that the hybrid microgels retained their
temperature sensitivity and narrow polydispersity. These colloidal crystals are
examples of tunable optical materials that contain refractive index periodicity on
multiple length scales.
Specifically, the photothermally modulated volume transitions of polymer
microgels may have promising implications for site-specific, light-induced drug
release and photo-dynamic therapy. Typically, such volume transitions are induced
by irradiating photosensitive moieties such as dyes or metal nanoparticles that
have been embedded in the thermally reversible polymer matrix, at their
resonance wavelengths. Conversion of light energy to heat through nonradiative
relaxation causes hydrogel heating and, for polymers with an LCST, results in their
deswelling.
6.2 Hybrid microgels doped with Au nanorods (NRs)
Hybrid microgels doped with Au nanorods (NRs) are prime examples of
materials with structural hierarchy.[6] For applications of thermoresponsive gels as
drug delivery carriers, the photosensitive species must strongly absorb in the
spectral range 800 nm < λ < 1200 nm or the “water window” as this is the region
that can effectively penetrate body tissues. The biocompatibility of Au makes it a
desirable material for use in hybrid microgels for drug release applications. Gold
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 127 -
NRs exhibit strong anisotropy, responsible for two, well-separated plasmon
resonance bands corresponding to the longitudinal and transverse axes in their
UV/VIS spectra.[7] The absorption wavelength of gold NRs may be tuned by changing
their aspect ratio.[8] Our group previously showed the photothermally-induced,
reversible deswelling of microgels loaded with gold NRs designed to absorb near IR
light.[9] At pH = 4.0, following irradiation at �� 809 nm, hybrid poly-(NIPAm-AA)
microgels underwent a volume transition at 33 °C. These temperature and pH
values did not correspond to the physiological conditions of pH=7.4 and the
temperature of 37-41 °C. In fact, at pH =7.4, no transition up to 60 °C was
observed for microgels containing AA in concentrations as small as 3 mol %: a very
small amount of AA in the microgels caused a large shift in the volume phase-
transition temperature (VPTT).
6.3 Tuning the thermal response of microgels
Poly(NIPAm) microgels functionalized with reactive groups have immense
technological potential. The coupling of the thermal phase transition of
poly(NIPAm) with the chemical reactivity and pH responsiveness of functional
acidic and basic groups has permitted the design of a range of environmentally
sensitive devices such as sensors,[10] rheology modifiers[11], and drug delivery
vehicles.[12, 13] The successful implementation of these applications however, relies
on the designer’s ability to control and predict the swelling responses of the
microgels upon being subjected to the desired stimulus with respect to i) the
definition and breadth of the volume phase transition, ii) the temperature of the
Chapter 6
___________________________________________________________________________ - 128 -
transition, and, iii) the deswelling ratio. These responses depend on the synergetic
effects of several important factors:
1. The interplay of hydrophobic and hydrophilic interactions in the
polymer network and the surrounding solution.
Generally, increased hydrophobicity in the poly(NIPAm) network
encourages phase separation and decreases the transition temperature whereas
increased hydrophilicity increases the same. For example, copolymers of NIPAm
and the hydrophilic dimethylacrylamide (DMAAm) show an increasing LCST with
increasing content of DMAAm.[14] Similarly, the LCST of NIPAm copolymerized with
the hydrophobic isopropylmethacrylate (iPMA) decreases with increase in iPMA
content.[15]
2. Electrostatic interactions that originate from the ionic functional
groups in the polymer, the nature of the counter-ions, and, the pH and
ionic strength of the dispersion medium.
Many examples of NIPAm copolymerized with acidic and/or basic
comonomers have been reported.[16-23] These thermoresponsive copolymer
microgels are also able to respond to changes in pH and ionic strength in addition
to changes in temperature. While multiresponsive microgels are advantageous for
many applications, in certain scenarios, they pose additional difficulties. For
instance, while the incorporation of acrylic acid (AA) in poly(NIPAm) microgels
introduces charged functional groups and pH-sensitivity, it also causes a large pH-
dependent shift in the volume phase transition temperature (VPTT), often placing
it outside the relevant range for many bio-oriented applications. Vincent and
coworkers showed that poly(NIPAm-AA) microgels containing 5% of AA in the
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 129 -
reaction mixture showed a VPTT of about 34oC at pH of 3.5, while at pH 7, the
transition occurred at 60oC. Kumacheva et al showed that poly(NIPAm-AA)
microgels containing 3mol% AA in the feed showed a transition of about 31oC at
pH=4, but did not observe any transition at pH=7.4.[9] Furthermore, potentiometric
and conductometric titrations revealed that in general only 10% of the total AA
monomer introduced to the reaction flask was incorporated in the NIPAm-AA
microgels showing that a very small amount of AA can create a sizeable shift in the
transition temperature. Similarly, NIPAm microgels functionalized with basic
moieties show a significant increase in the VPTT at low pH due to the increased
hydrophilicity introduced by the charged ionic groups.
3. The spatial separation and distribution of the functional groups
responsible for the different sensitivities throughout the microgel.
The distribution of functional groups in microgels obtained by random
copolymerization is governed by the reactivity ratios of the different comonomers
and cannot be controlled.[17] However, knowledge of the kinetic constants of the
different reactants may allow one to predict the radial distribution of the
functional groups in the microgel. Hoare et al.[24] showed that both the radial and
chain distributions of functional groups significantly impact the swelling and
electrophoretic behavior of multiresponsive microgels. Broadening of the
temperature-induced phase transition of functionalized NIPAm-based microgels
most often results from blockiness in the polymer network, caused by the differing
reactivity ratios of the comonomers and the associated interruption of the
poly(NIPAm) chain. The aforementioned drawbacks of random copolymerization of
NIPAm, may be overcome if the regions of different sensitivity are spatially
Chapter 6
___________________________________________________________________________ - 130 -
separated in the polymer network and possess defined domains. For spherical,
colloidal microgels, a core-shell structure is ideally suited to achieve this property.
Jones and Lyon[25] were the first to report microgels of a core-shell structure. They
studied NIPAm-based microgels where either the core or the shell was
copolymerized with AA, thereby introducing pH sensitivity to a specific region.
Berndt and Richtering reported the synthesis of dually temperature-responsive
core-shell microgels comprising a poly(NIPAm) core and a poly(NIPMAm) shell.[26]
They observed two transitions for the core-shell microgels, corresponding closely
with the phase transition temperatures of the two different polymers in the
system. An alternative approach that may be used to prevent large deviations of
the transition temperature upon the incorporation of ionic moieties is to physically
incorporate the relevant functional groups in the microgels during the synthesis.
For example, Hu and coworkers reported the formation of interpenetrated
networks of polyAA with poly(NIPAm) microgels.[27]
6.4 Research objectives
The aim of this work was to develop a drug delivery system (DDS) for
photothermally-triggered drug release under specific conditions, suitable for
biological applications. Specifically, we envisioned that the temperature responsive
nature of poly(NIPAm)-derived microgels and the optical properties of gold
nanorods (NRs) may be combined to yield a photothermally-responsive entity.
While both the thermo-responsive and optical properties of the hybrid composite
system can be harnessed for applications in DDSs, several important criteria must
be met for such carriers to be used in practice. We defined the following criteria
for our proposed DDS as follows:
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___________________________________________________________________________ - 131 -
1. The temperature-induced transition must occur in the narrow,
physiologically relevant range of 38-42oC at pH=7.4 in buffer
media.
2. The extent of deswelling must be sufficiently large to trigger
release of a loaded drug.
3. To sequester positively charged NRs, microgels must contain
negatively charged functional groups.
4. The dispersion of hybrid microgels loaded with Au NRs must be
stable in the physiological environment and upon cyclical heating
and cooling.
5. The Au NRs must remain within the microgels when subjected to
repeated irradiation-triggered deswelling-swelling transitions.
We used different approaches to tune the temperature-induced transition
of multiresponsive microgels according to the criteria outlined above. The
strategies explored were (i) the synthesis of microgels with an interpenetrating
network (IPN)structure; (ii) counterbalancing hydrophilicity of charged acidic
groups by copolymerizing NIPAm and acrylic acid with a hydrophobic monomer; (iii)
copolymerization of NIPAm with different types of acidic monomers that temper
the hydrophilic/hydrophobic properties of the microgels. We further explored the
suitability of the aforementioned hybrid microgels as DDSs for photo-thermally
triggered release applications.
Chapter 6
___________________________________________________________________________ - 132 -
6.5 Experimental
6.5.1 Materials
N-isopropylacrylamide (NIPAm), N-isopropylmethacrylamide (NIPMAm),
maleic acid (MA), undecanoic acid (UA), butyl methacrylate (BMA), polyacrylic acid
(PAA, Mw=2000g/mol), crosslinking agent N-N’-methylene-bis-acrylamide (BIS),
and initiator potassium persulfate (KPS) were purchased from Aldrich Chemical Co.
(Canada) and used as received.
6.5.2 Synthesis of microgels
All microgels were prepared via free radical precipitation polymerization.
The proportions of reactants in various reaction mixtures are given in Table 3-1.
Following synthesis, the microgels were purified several times by centrifugation
(10000 rpm, 30 min at 4°C) and redispersed in 0.01M phosphate buffered saline
(PBS) solution of pH 7.4.
For the synthesis of poly (NIPAm-UA) microgels, undecanoic acid was first
reacted with NaOH to yield the water-soluble salt form, prior to incorporation in
the reaction mixture. For the preparation of poly(NIPAm-NIPMAm)-PAA IPN
microgels, PAA was added to the aqueous reaction mixture prior to the injection of
the initiator.
6.5.3 Synthesis of gold nanorods
Gold NRs were synthesized following the procedure developed by
Nikoobakht and El Sayed,[28] as described in Chapter 2, and were scaled up to
obtain a 100 mL dispersion of the NRs. This route allowed for the preparation of
gold NRs with plasmon bands centered at l=840 nm. The NRs were purified via
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 133 -
three 30-min-long centrifugation cycles at 10 000 rpm. At the end of each
centrifugation cycle, the supernatant was removed, and the precipitated NRs were
redispersed in deionized (DI) water.
6.5.4 Preparation of hybrid microgels
Microgels were loaded with NRs by mixing the purified microgel and NR
dispersions under continuous stirring in the ratio of 2:1, respectively. The
dispersion of NRs was added dropwise to the microgel dispersion under constant
stirring.
6.5.5 Characterization of microgel properties
Variation in microgel size as a function of temperature was measured using
a photon correlation spectroscopy setup (PCS, Protein Solutions Inc.) equipped with
a temperature controller. All measurements were conducted in phosphate buffered
saline at pH=7.4 unless otherwise specified. Electrophoretic mobility of microgels
were measured on the Zetasizer 3000HS (Malvern Instruments) to determine the
charge carried by the microgels.
Presence of PAA in the microgels was verified by Fourier transform infrared
(FTIR) spectroscopy. The spectrum of IPNs were similar to that of pure poly(NIPAm)
except that a new band, characteristic of absorption of carboxylic groups appeared
at 1738 cm-1. Samples of the microgels in an acid form were dried in air and
embedded in KBr pellets. Experiments were carried out using a Perkin-Elmer
PARAGON 500 FTIR instrument. Each sample was analyzed between 400 and 4000
cm-1, at 32 scans and a resolution of 4 cm-1.
Chapter 6
___________________________________________________________________________ - 134 -
Loading of microgels with NRs was examined by scanning transmission
electron microscopy (TEM) (Hitachi HD 2000).
6.5.5 Characterization of photothermally-induced transitions
Photothermally triggered change in microgel size was measured using a PCS
setup at 632.8 nm (Zetasizer 3000HS, Malvern Instruments), modified to
accommodate the pump laser. The hybrid microgels dispersed in PBS buffer at pH =
7.4 were heated to 36 °C and then irradiated at �= 809 nm (surgical laser
CW/pulsed, 1.5 W power). The temperature of the microgel dispersion was
monitored with a thermocouple probe placed inside the cuvette.
6.6 Results
Several series of experiments were carried out to find the microgel that
best satisfied our desired criteria. For each microgel system we verified the
temperature range and sharpness of temperature-induced transitions. The best
representative results for each of the microgel series are shown in Figures 6-1 to 6-
4. Other results such as the temperature range of the volume transition and extent
of microgel shrinkage are shown in Table 6-1. Microgels with the best performance
(based on the criteria described above) were loaded with gold NRs and examined
under TEM.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 135 -
Table 6-1 Recipes for microgel synthesis
Series Microgels NIPAM NIPMAM AA BMA UA MA PAA KPS BIS mol% D Transition range(mol) (mol) (mol) (mol) (mol) (mol) (mol) (mol) (mol) acid (nm) (oC)
A NIPAM 1.25x10-2 1.67x10-4 124
B1 NIPAM-BMA-AA 4.12x10-3 1.25x10-4 8.44x10-5 1.85x10-4 3.36x10-4 2.00% 27-52B2 NIPAM-BMA-AA 4.12x10-3 1.67x10-4 8.44x10-5 1.85x10-4 3.36x10-4 3.00% 27-52B3 NIPAM-BMA-AA 4.1x10-3 3.33x10-4 8.44x10-5 1.85x10-4 3.36x10-4 4.00% 25-48B4 NIPAM-BMA-AA 4.1x10-3 8.33x10-4 8.44x10-5 1.85x10-4 3.36x10-4 8.00%
U1 NIPAM-UA 7.8x10-3 4.2x10-4 2.2x10-4 3.36x10-4 5.00% 427 27-33U2 NIPAM-UA 4.2x10-3 4.2x10-4 2.2x10-4 3.36x10-4 10.00% 538.2 23-35U3 NIPAM-UA 2.4x10-3 4.2x10-4 2.2x10-4 3.36x10-4 15.00% 567 21-32U4 NIPMAM-UA 7.9x10-3 4.2x10-4 2.2x10-4 3.36x10-4 5.00% 271.9 39-46U5 NIPMAM-UA 3.7x10-3 4.2x10-4 2.2x10-4 3.36x10-4 10.00% 300.9 37-43U6 NIPMAM-UA 2.1x10-3 4.2x10-4 2.2x10-4 3.36x10-4 15.00% 358.7 33-45
M1 NIPAM-MA 8x10-3 4.2x10-4 2.2x10-4 3.36x10-4 5.00% 413.7 31-33M2 NIPAM-MA 4.2x10-3 4.2x10-4 2.2x10-4 3.36x10-4 10.00% 624.2 38-40M3 NIPAM-MA 2.4x10-3 4.2x10-4 2.2x10-4 3.36x10-4 15.00% 671.3 40-44
N2 NIPAM-NIPMAM 4.4x10-3 5.5x10-3 2.2x10-4 3.36x10-4 775.6 37-41
IPN3 NIPAM-NIPMAM PAA 3.52x10-3 3.12x10-3 1x10-4 2.22x10-4 3.43x10-4 255 35-42IPN4 NIPAM-NIPMAM PAA 2.67x10-3 3.93x10-3 1.02x10-4 2.22x10-4 3.49x10-4 266 38-45
6.6.1 Copolymerization of NIPAm with different acidic
functionalities
Copolymerization of NIPAm with monoprotic, undecanoic acid.
We examined the effect of copolymerization of NIPAm with undecanoic
acid on the onset of microgel deswelling and the sharpness of volume-temperature
transitions. We built our work on the results of Yang and coworkers who found no
change in the LCST of poly(NIPAm) in the linear copolymer of NIPAm and UA. No
shift in LCST occurred due to the hydrophobicity of UA. [29]
We copolymerized NIPAm with UA in various compositions (Table 1, series U1-U3)
and examined the volume-temperature transitions of the corresponding microgels
at pH=7.4. Table 6-1 shows the change in microgel size as a function of
temperature for microgels with different fractions of UA. The best result was
achieved for the microgels of series U1 synthesized at molar ratio of 100/5
Chapter 6
___________________________________________________________________________ - 136 -
50
100
150
200
250
300
20 25 30 35 40 45 50
T(oC)
Dh
(nm
)
D(
)
50
100
150
200
250
300
20 25 30 35 40 45 50
T(oC)
Dh
(nm
)
D(
)
Figure 6-1. Variation in hydrodynamic diameter of poly(NIPMAm-UA) (U5) ( ) and poly
(NIPMAm) (Δ) microgels as a function of temperature in 0.01 M PBS pH=7.4. The
incorporation of UA in the poly (NIPAm) microgel results in a slight increase in the
volume phase transition temperature.
[NIPAm]/[UA] in the reaction mixture. These microgels underwent ca. 95%
reduction in volume in the temperature range from 37 to 45oC. The hydrophobicity
of the undecanoic chain nullified any increase in the VPTT due to the hydrophilic
COOH groups.
To shift the temperature-induced volume transition to the narrower target
range of 38-42oC we replaced the NIPAm with N-isopropylmethacrylamide
(NIPMAm). The structure of NIPMAm is similar to that of NIPAm, except for the
presence of a methyl group on the �-carbon that restricts free rotation of the main
chain and inhibits hydrophobic interactions.[26, 30] As a result, the LCST of NIPMAm
occurs at 42-44oC, almost 10oC higher than that of pure NIPAm.[31] Poly (NIPMAm-
UA) microgels of different compositions showed VPTTs in the temperature range
from 27 to 35oC (TABLE 6-1). In particular, Figure 6-1 shows that poly(NIPMAm-UA)
microgels containing 5mol % UA (U4) underwent a 91% volume reduction between
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 137 -
39 and 41oC. However, the transition range of poly(NIPMAm-UA) microgels was
broader than that of poly(NIPAm-UA), presumably due to the inhibited hydrophobic
interactions in the former case.
Copolymerization of NIPAm with diprotic maleic acid
Since the VPTT of poly(NIPAm) depends on the conformational arrangement
of water molecules around the amide residues,[32] the interruption of poly(NIPAm)
chain segments by other functional groups typically results in deviation from, and
broadening of, the VPTT of poly(NIPAm). The use of a multifunctional ionic group
can help to increase the net charge density and thereby amplify the change in size
during the swelling-deswelling transition whilst introducing fewer interruptions in
the NIPAm chain segments, hence limiting the broadening effect on the volume-
temperature transition.
Accordingly, we attempted to modulate the volume-temperature transitions of
microgels by copolymerizing NIPAm with maleic acid (MA), a diprotic carboxylic
acid with two pKa values of 1.9 and 6.08.[33, 34] The presence of two charged –COO-
groups on the functional monomer (instead of one as in acrylic acid) simultaneously
acts to increase the local charge density and magnitude of intrachain repulsion
between the ionic groups, and to curtail the relative extent of broadening of the
phase transition. Figure 6-2 shows the thermally-induced transitions of
poly(NIPAm) (reference system) and poly(NIPAm-MA) microgels polymerized at
10/90 mol% [MA]/[NIPAm] in the reaction mixture . The latter system displayed ca.
74% decrease in particle diameter in the desired temperature range from 38 to
40oC in buffer media at pH=7.4, which translates to a 98.2% change in microgel
volume. This dramatic change in particle size within the narrow temperature range
Chapter 6
___________________________________________________________________________ - 138 -
100
200
300
400
500
600
700
20 25 30 35 40 45 50
T (oC)
Dh
(nm
)
100
200
300
400
500
600
700
20 25 30 35 40 45 50
T (oC)
Dh
(nm
)
Figure 6-2. Variation in hydrodynamic diameter of poly(NIPAm-MA) ( ) and
poly(NIPAm) (Δ) microgels as a function of temperature in 0.01 M PBS pH=7.4. The
increase in the VPTT is caused by the hydrophilicity of the charged carboxylic acid
groups at neutral pH.
of this transition, in buffer media made this poly(NIPAm-MA) microgel a most
promising candidate for our proposed DDS.
6.6.2 Microgels with interpenetrating network structures
While the copolymerization of acidic functionalities with NIPAm is an
effective route to tuning volume-temperature transitions of microgels, the varying
reactivity of the monomers limits control over the final composition of microgels,
the distribution of functional groups within the particles and influences their
swelling behavior.[24, 35, 36]We explored an alternative method for incorporating
acidic functional groups in the microgels by preparing microgels from two polymers
physically bonded into an interpenetrating network (IPN) structure. Xia et al. [27]
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 139 -
previously reported the synthesis of poly(NIPAm) microgels interpenetrated with
poly(AA). The primary advantages of using IPNs over randomly copolymerized
microgels is the facile incorporation of a large number of functional groups within
the particle without any accompanying shift in the VPTT of poly(NIPAm).[37]
100
150
200
250
300
350
400
450
500
550
20 25 30 35 40 45 50
T (oC)
Dh
(nm
)
Figure 6-3. Variation in hydrodynamic diameter of poly(NiPAm-NIPMAm)/PAA IPN ( )
and and poly(NIPAm-NIPMAm) (Δ) microgels as a function of temperature in 0.01 M PBS
pH=7.4.
In the present work, the targeted temperature range was attained by
polymerizing one of the polymers of the IPN from a copolymer of NIPAm and
NIPMAm. The IPN microgels were obtained by copolymerizing NIPAm and NIPMAm in
the presence of PAA. Presence of PAA in the microgels was confirmed by FTIR
measurements that showed a new band at 1738 cm-1, characteristic for absorption
by carboxylic groups. Furthermore, the �-potential of the poly(NIPAm-NIPMAm) IPN
microgels was -23.2 mV versus only several millivolts for poly(NIPAm-NIPMAm)
microgels. Increasing concentration of PAA led to a decrease in microgel size, in
contrast with earlier observations by Hu and Xia.[27] Presently, we are unable to
explain this difference. However, we speculate that both the significantly lower
Chapter 6
___________________________________________________________________________ - 140 -
molecular weight of the PAA used in the present work and the use of a different
synthetic procedure from that used by Hu and coworkers may be responsible for
the different observations. The observed smaller microgel size of the IPN systems
in comparison to the reference corresponding poly(NIPAm-NIPMAm) particles may
be due to the formation of microdomains resulting from hydrophobic interactions
between short PAA chains and poly(NIPAm) segments in the network. Note that no
shift in VPPT compared to the host poly(NIPAm-NIPMAm) microgels was observed
for all IPN microgels, irrespective of PAA content.
Figure 6-3 shows the variation in size of poly(NIPAm-NIPMAm) microgels
synthesized at a 41/59 [NIPAm]/[NIPMAm] molar ratio and that for the
corresponding IPN poly-(NIPAm-NIPMAm)-PAA particles. The poly(NIPAm-
NIPMAm)/PAA IPN microgels shrank from ca. 250 to ca. 150 nm in the temperature
range of 41-47 °C, corresponding to a 78.4% decrease in volume. The deswelling
ratio of poly (NIPAm- NIPMAm)/PAA IPNs was smaller than that of pure poly(NIPAm-
NIPMAm) microgels.
6.6.3 Copolymerization with hydrophobic comonomers
Generally, copolymerization of NIPAm with a more hydrophobic monomer
shifts the onset of microgel shrinkage to lower temperatures. For example, Feil et
al[32] showed that the microgels synthesized from NIPAm, , AA and butyl
methacrylate (BMA) had VPTTs in the range from 35oC to 40oC in PBS solution of
pH=7.4. These results illustrated how the hydrophobicity of BMA counterbalanced
the hydrophilicity of AA at pH=7.4, and higher values of volume phase transitions in
poly(NIPAm-AA) (caused by the hydrophilic nature of the acrylic acid at pH=7.4)
were counterbalances by the hydrophobic nature of BMA.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 141 -
Here, we synthesized poly(NIPAm-BMA-AA) microgels at a constant molar
ratio [NIPAm]/[BMA] while the content of AA in the reaction mixture varied from 2
to 8 mol %. The recipes used for the microgel synthesis are given in Table 6-1
(series B). The initial size of poly(NIPAm-AA-BMA) microgels and the temperature of
volume transition increased with increasing AA content in the reaction mixture. On
the other hand, with increasing concentration of AA, the extent of microgel
shrinkage as a function of temperature decreased. The volume-temperature
transitions were broad and spanned a range of ca. 10-12 degrees. However, unlike
for poly(NIPAm-AA) microgels, all volume-temperature transitions started well
below 60oC at pH 7.4 in 0.01M PBS.
200
250
300
350
400
20 25 30 35 40 45 50T(oC)
Dh
(nm
)
200
250
300
350
400
20 25 30 35 40 45 50T(oC)
Dh
(nm
)
Figure 6-4. Variation in hydrodynamic diameter of poly(NiPAm-AA-BMA) ( ) and
and poly(NIPAm-BMA) (Δ) microgels as a function of temperature in 0.01 M PBS
pH=7.4.
Figure 6-4 shows the variation in hydrodynamic diameter of poly (NIPAm-
BMA-AA) microgels (Series B, Table 6-1) with the VPTT closest to the desirable
Chapter 6
___________________________________________________________________________ - 142 -
temperature range. As a reference, on the same graph we show the swelling
behavior of poly(NIPAm-BMA) microgels. In the temperature range from 25 to 50oC,
the change in microgel diameter was ~130 nm, that is ca. a 13 % of the original
microgel size. We attribute the breadth of the deswelling transition and the poor
shrinkage of the microgels to the presence of BMA: due to the higher
hydrophobicity and reactivity polyBMA may cause it to segregate towards the
center of the microgel, increasing the rigidity of the polymer and, thus reducing
the overall extent of microgel shrinkage.
Note that for microgels of series B1and B2 (Table 6-1) with 2 and 3 mol % of
AA content in the reaction mixture, notable shrinkage started at ca. 35 oC whereas
for series B3 and B4 the transition began at 41 oC. Hence in principle, the onset of
transition may be shifted to the desired range of 38-42 oC by tuning the
concentration of AA. Nevertheless, the broad volume-temperature transitions for
poly(NIPAm-BMA-AA) microgels did not make this approach promising in view of our
desired criteria.
6.7 Discussion on the phase transitions of synthesized microgels
We tuned the temperature-induced volume phase transitions of NIPAm-
based microgels to yield sharper transitions in the physiologically relevant range
spanning 38-42oC in 0.01M phosphate buffered saline solution of pH=7.4 . Starting
with the well known poly (NIPAm-AA) microgel system, we showed that
incorporation of a hydrophobic moiety like BMA reduces the temperature of the
transition to our targeted range of 35-45oC in PBS at pH=7.4. However, the
deswelling ratios for these microgels, though notable, were not large.
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Exploration of functional carboxylic acids other than AA led us to realize
narrower and sharper transitions with larger deswelling ratios in the microgels
containing UA and MA. Both microgels showed a decrease in the VPTT compared
with poly (NIPAm-AA) microgels. For poly(NIPAm-UA), this decrease was ascribed to
the increased hydrophobicity of the polymer chain, while for poly(NIPAm-MA), the
diprotic MA, the reduction in VPTT was believed to be a consequence of the
functional-group distribution. For example, for poly (NIPAm-AA) microgels no
transitions were observed in PBS at pH=7.4 while poly (NIPAm-MA) showed narrow
and sharp transitions between 38-40oC. Hoare and coworkers showed that AA
groups are relatively uniformly distributed throughout poly(NIPAm-AA)
microgels.[17] This implies that poly(NIPAm) segments are interrupted at frequent
intervals in the polymer chain, and together with the increased hydrophilicity of
AA, explains the broad nature of the transition at neutral pH. In contrast, MA, with
its much lower relative reactivity,[24, 38] is believed to be distributed in a gradient
fashion through the particle, with the majority being located in the peripheral
region. This explains why the nature of the volume phase transition for
poly(NIPAm-MA) microgels remains almost as sharp as that of pure poly(NIPAm).
The increase in the VPTT relative to poly(NIPAm) is endorsed by the hydrophilicity
of the deprotonated carboxylic acids at pH=7.4 while the large volume change
results from the increased charge density in the locale of the functional groups,
gifted by the diprotic nature of MA. It follows that apart from knowledge of the
hydrophobic/hydrophilic and ionic characters of the comonomers, insight into their
relative reactivity values and chain functional group distributions can facilitate
their rational selection for copolymerization with poly(NIPAm), according to the
target application.
Chapter 6
___________________________________________________________________________ - 144 -
6.8 Incorporation of gold nanorods into microgels
The two microgel systems that displayed the largest deswelling transitions
over the narrowest temperature range within the physiologically useful
temperature bracket were selected to explore the incorporation of NRs within the
microgels; namely, poly (NIPAm-MA) (series M2) and poly(NIPAm-NIPMAm)/PAA IPN
(Series IPN4). Gold NRs were effectively loaded into the microgels simply by
physically stirring the microgel and NR dispersions together. Figure 6-5a and b
shows TEM images of the poly(NIPAm-MA) microgels and IPNs of poly(NIPAm-
NIPMAm)/ PAA loaded with gold NRs, respectively. All the NRs appeared to be
located on the microgels while an extremely small amount was observed in the free
interstitial space. The distribution of NRs on the microgels was homogeneous in
both systems.
(a) (b)(a) (b)
Figure 6-5 TEM images of (a) hybrid poly(NIPAm-MA) microgels. Scale bar is 2 μm. Inset
shows a single NR-loaded 200 nm microgel particle. (b) Poly(NIPAm)/PAA IPN hybrid
microgels. Scale bar is 300nm.
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___________________________________________________________________________ - 145 -
Figure 6-6 Absorption spectra of gold NRs prior to (black line) and following NR
incorporation in poly(NIPAm-MA) (yellow line) and poly(NIPAm-NIPMAm)-PAA IPN4 (red
line) microgels.
We further tested the absorption properties of gold NRs following their
deposition onto poly(NIPAm-MA) and poly-(NIPAm-NIPMAm)/PAA microgels. Figure
6-6 shows that, for both systems, the absorption properties of the gold NRs did not
change significantly upon incorporation within the microgel interior. A slight (up to
20 nm) red shift was caused by the change in the dielectric constant of the
environment surrounding the NRs.
6.9 Thermally-induced volume phase transitions of hybrid
microgels
Following the incorporation of NRs in the microgels, we examined
temperature-induced variations in the size of hybrid poly-(NIPAm-MA) and
poly(NIPAm-NIPMAm)/PAA IPN microgels. Figure 6-7 shows the relative change in
microgel size, D/D0, where D0 is the hydrodynamic diameter of the corresponding
microgel at 15°C in buffer solution at pH= 7.4.
Chapter 6
___________________________________________________________________________ - 146 -
0
1
2
3
4
5
6
7
20 25 30 35 40 45
T (oC)
D/D
0
0
1
2
3
4
5
6
7
20 25 30 35 40 45 50
T(oC)
D/D
0
(a) (b)
0
1
2
3
4
5
6
7
20 25 30 35 40 45
T (oC)
D/D
0
0
1
2
3
4
5
6
7
20 25 30 35 40 45 50
T(oC)
D/D
0
0
1
2
3
4
5
6
7
20 25 30 35 40 45
T (oC)
D/D
0
0
1
2
3
4
5
6
7
20 25 30 35 40 45 50
T(oC)
D/D
0
(a) (b)
Figure 6-7. Variation in deswelling ratios, D/D0, of NR-free (Δ) and NR-loaded ( )
microgels in PBS at pH=7.4. (a) poly(NIPAm-MA) microgels (Series M2, Table 1, Chapter
3); (b) poly(NIPAm-NIPMAm)/ PAA IPN microgels (Series IPN4, Table 1). D and D0 are the
hydrodynamic diameters of the corresponding microgels in buffer solution of pH= 7.4,
at the temperature of interest and at room temperature, respectively.
Figure 6-7a shows the deswelling behavior of NR-free and hybrid poly(NiPAm-
MA) microgels in buffer solution at pH = 7.4. At 25 oC, the hybrid poly(NIPAm-MA)
microgels were ca. 7% smaller in diameter than NR-free microgels. This difference,
though very small, was attributed to the higher ionic strength of the hybrid system
and the physical cross-linking of the negatively charged microgels with the
positively charged gold NRs. Upon heating, the hybrid microgels shrank to a slightly
smaller size. However, both the relative deswelling ratios and the VPTs of pure and
hybrid microgels was very similar. The transition occurred in the targeted range of
38-41oC.
We found that hybrid poly (NIPAm-NIPMAm)/PAA IPN microgels coagulated
in a PBS buffer when heated above 40 �C. Therefore, the behavior of NR-loaded IPN
microgels was examined in water at pH = 7.0. Figure 6-7b shows the variation in
the deswelling ratio D/D0 of NR-free and NR-loaded poly (NIPAm-NIPMAm)/PAA
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 147 -
microgels in water. The trends were similar to those shown in Figure 6-7a.: hybrid
microgels were slightly smaller in size but displayed very similar deswelling
behavior to their NR-free counterparts. However, the deswelling extent of hybrid
poly(NIPAm-NIPMAm)/PAA IPN microgels was significantly smaller than that of the
poly(NIPAm-MA) microgels.
6.10 Photothermally-triggered volume transitions of hybrid
microgels
We further explored the potential applications of the hybrid microgels with
respect to their photo-thermally triggered volume phase transitions. The hybrid
poly(NIPAm-MA) microgels dispersed in PBS, pH=7.4 were heated to 36 oC and
repetitively irradiated at λ = 809 nm. The duration of irradiation was approximately
a minute, and the time interval between the irradiation cycles was approximately 3
min. In parallel, we measured the temperature of the dispersion. Note that the
irradiation wavelength and the absorbance wavelength of NRs loaded in microgels
were not precisely matched (within 20nm or so). Following irradiation, we observed
a rapid (within a minute) change in particle size. Figure 6-8 shows the reduction in
volume of hybrid poly(NIPAm-MA) microgels doped with gold NRs of 78±4% (that is,
about 80% of the volume reduction induced by heating hybrid microgels to 40 °C
(Figure 6- 7a).
Chapter 6
___________________________________________________________________________ - 148 -
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10n
V/V
o
Figure 6-8. Variation in deswelling ratio, V/V0 where V0 and V are the volumes
of microgel at 25oC and at temperature, T respectively, as a function of the
number of laser on and laser off events of pure(♦) and hybrid (■) microgels
respectively. (a) M2 poly (NIPAM-MA)
Using the results of steady-state swelling experiments, we estimate the
temperature of the microgel particles to be 40 °C. We emphatically note that
following irradiation, the temperature of the bulk dispersion remained at 36 °C.
Contrastingly, the control experiment showed that the dispersion of pure microgels
underwent a shrinkage of only 25 ±1 % in volume upon illumination at λ = 810 nm.
The strong shrinkage observed in the hybrid system resulted from the local
heating of microgels, following conversion of light energy to heat by the gold
nanorods. The laser on and laser off cycles were repeated several times. The
persistent, reversible nature of the photothermally induced de-swelling/swelling
transitions of hybrid microgels indicated that the NRs remained within the
poly(NIPAm-MA) microgels during the heating cycles.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 149 -
6.11 Current research on thermally-induced drug release
The temperature-induced phase transitions in thermoresponsive polymer
microgels rely on several biologically relevant interactions, namely Van der waals
forces, hydrophobic interactions, repulsive and attractive ionic interactions and
hydrogen bonding. Most biomedical applications rely on the changes from room
temperature to body temperature in order to induce a change in these
physicochemical properties e.g., gelation, particularly in topical applications and in
injectable biodegradable scaffolds. In vitro applications in cell culture also use the
stimulated swelling and shrinking of hydrogels with their change in surface
properties. However, applications of microgels for internal medicine require the
induced response to be dramatic upon subtle variations in temperature, within the
narrow physiological temperature range of 37-40oC.
Table 6-2 shows a number of thermoresponsive polymers relevant to
biomedical applications since their phase transitions fall between 30-40oC.[39] It
should however be noted that the transition temperature is strongly dependent on
several key factors including molecular weight, solvent quality and salt
concentration.
Table 6-2
Polymer
Phase Transition Temperature in Aqueous
Media
poly(NIPAm) 32-34 oC
Poly (N,N-diethylacrylamide) 30-34 oC
Poly (methyl vinyl ether) 37 oC
Poly (N-vinylcaprolactam) 30-50 oC
Chapter 6
___________________________________________________________________________ - 150 -
Lyon and coworkers have previously shown the thermally-modulated
release of insulin and doxorubicin from layer by layer (LbL) assembled poly(NIPAM-
AA) microgel thin films.[40-42] The release mechanism however was related to
gradual partitioning of the loaded insulin as a result of repeated heat cycles that
the microgel thin film was subjected to. More recently, Lyon and coworkers showed
the thermally-triggered release of insulin from microgels at temperatures above
the VPTT. In these systems, the deswelling transition acts as a trigger that
squeezes the loaded drug into the surrounding medium, much like a sponge. [41]
Some groups have shown the thermally-induced, diffusion-driven release of a drug
as a result of the increased permeability of poly(NIPAm) microgels upon occurrence
of the swelling transition that accompanies a drop in temperature.[43] In this case,
the drug is effectively entrapped within the microgel in its shrunken state and is
released upon swelling. For both scenarios, the release is triggered by the sharp
change in the swollen or shrunken state of the microgel at the VPTT.
In the present work, the former approach was used. We proposed to
employ hybrid polymer microgels doped with gold nanorods for the application of
photothermally triggered dye/drug release. Fig 6-9 illustrates our proposed
concept.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 151 -
Figure 6-9 Scheme showing plausible use of hybrid microgels in light-induced drug
delivery systems. The hybrid microgels are loaded with gold nanorods tuned to absorb
in the near IR, the spectral range that is ideal for biomedical applications since it can
penetrate body tissues. Laser irradiation of the NRs results in non-radiative energy
transfer and local heating of the polymer network, thereby triggering a deswelling
transition, which can promote the release of a loaded drug.
In our design, the VPTTs of the hybrid microgels are tuned to occur in the
physiologically relevant ranges as described earlier. Upon irradiation in the near
IR, absorption of light by the gold NRs results in non-radiative energy transfer to
the temperature-sensitive microgel and results in local heating, subsequently
causing microgel shrinkage. A loaded, water-soluble drug may then be ‘squeezed’
out of the microgel and released into the surrounding medium. Biofuntionalization
of the hybrid microgels can also be used to lend them targeting ability for sepecific
cancer cells.
Chapter 6
___________________________________________________________________________ - 152 -
6.12 Loading pure and hybrid microgels with a model compound
Several different pure and hybrid microgel systems (Series listed in Table 6-
1) were loaded by physical mixing in 0.01M PBS at pH=7.4 with R6G at room
temperature. The suspensions of dye-loaded microgels were isolated from their
supernatants by centrifugation at 20oC. Figure 6-10 summarizes the loading
capacity and association efficiency (see Chapter 5) of R6G in three of these
microgel systems: poly(NIPAm-MA) or M2, poly(NIPAm-NIPMAm) and poly (NIPAm-
NIPMAm)/PAA IPN. Pure poly (NIPAm-MA) microgels had the maximum LC values of
57.2%, followed by 53.4% and 45.3% for the IPN and neutral microgels respectively.
Presumably, the enhanced loading of positively charged R6G dye into the
negatively charged microgels, compared to the neutral microgels, was reflective of
the electrostatic attractions between the former pair. Expectedly, both the LC and
AE values of pure microgels were higher by ca. 10% than that of hybrid microgels
for all three systems. This indicated that the NRs present in hybrid microgels
occupied some sites available to R6G in the pure systems, and reduced the
permeability of the microgels to the dye. Furthermore, partial charge
compensation of the anionic microgels by the cationic NRs may also have
diminished the loading of cationic R6G dye into the hybrid particles.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 153 -
0
10
20
30
40
50
60
70
NIPAm-MA NIPAm-NIPMAm IPN PAA/-NIPAm-NIPMAm
%
LC of pure microgelsLC of hybrid microgelsAE of pure microgelsAE of hybrid microgels
Figure 6-10 Loading capacity(LC) and Association Efficiency (AE) of R6G in pure and
hybrid microgel dispersions (0.1 wt% microgel).
6.13 In-vitro temperature-induced release from hybrid microgels
The temperature-triggered release of R6G from NR-loaded microgels was
examined. Three hybrid microgel systems were loaded with R6G by physical mixing
in PBS at pH=7.4, and allowed to equilibrate over 24 hours. The absorbance of the
dispersion was measured. Subsequently, 3mL aliquots of the dispersion were taken,
and centrifuged under 20,000 XG at relevant temperatures for 30 minutes. Each
sample was immediately removed and separated from the supernatant. The
absorbance intensities of both the precipitate and the supernatant were measured
for each sample, and the concentration of R6G present was calculated with the
help of a calibration curve. Figure 6-11 shows the summarized results of these
experiments for three microgel systems.
Chapter 6
___________________________________________________________________________ - 154 -
0
20
40
60
80
100
120
20 25 30 35 40 45T (oC)
% d
ye
% dye remaining% dye released
0
20
40
60
80
100
120
20 25 30 35 40 45
T (oC)
% d
ye
% dye remaining% dye released
0
20
40
60
80
100
120
20 25 30 35 40 45T (oC)
% d
ye
dye remainingdye released
(a)
(b)
(c)
0
20
40
60
80
100
120
20 25 30 35 40 45T (oC)
% d
ye
% dye remaining% dye released
0
20
40
60
80
100
120
20 25 30 35 40 45
T (oC)
% d
ye
% dye remaining% dye released
0
20
40
60
80
100
120
20 25 30 35 40 45T (oC)
% d
ye
dye remainingdye released
(a)
(b)
(c)
Figure 6-11. Amount of R6G dye released from and remaining within hybrid microgels
(0.1 wt% microgels) dispersed in 0.01M PBS at pH=7.4 as a function of temperature. (a)
Poly(NIPAm-MA), LC 57.2% (b) Poly (NIPAm-NIPMAm), LC 48.6% (c) Poly (NIPAm-
NIPMAm)/PAA IPN, LC 51.4%
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 155 -
The release profiles for dye remaining within the microgels and for dye
released are complementary in all three systems, within reasonable scatter (±
4.7%). Recall that poly(NIPAm-MA) (Figure 6-12 a) microgels had shown both the
sharpest volume transition and the largest deswelling ratio of all the microgel
systems. However, 18.9% and 35.1% of the loaded dye was released at 37 and 40oC
respectively. Thus almost half of the total amount of released dye escaped from
the microgel between 25 and 37oC, i.e., before the onset of the sharp volume
transition. Nevertheless, there was an incremental amount of R6G released
between 37 and 40oC, corresponding somewhat with the VPTT of the microgels.
The cumulative amount of R6G dye released from the neutral poly(NIPAM-
NIPMAm) microgels was 9.1% and 37.2% at 37 and 40oC, respectively. Although, a
notable amount of R6G was released before the onset of the VPT, a significantly
larger amount of the dye was released during the actual deswelling transition. The
IPN microgel network released 17.1% and 47.2% of R6G at 37 and 40oC. The slightly
greater amount of released dye for the poly(NIPAm-NIPMAm)/PAA IPN system,
compared to the neutral microgels was most likely due to the presence of
hydrophilic PAA channels throughout the hydrophobic network at the transition
temperature, which encouraged escape of the dye.
6.14 Visualization of loading and release of dye
The fluorescence of R6G was employed to visually test the loading ability of
the microgels. All pure and hybrid microgels with VPTTs falling within the
physiologically useful temperature range were loaded with R6G and imaged. The
representative results are presented herein. Fig 6-12 shows images of dispersions
of pure R6G-loaded poly(NIPAm-MA) microgels in PBS at room temperature, body
Chapter 6
___________________________________________________________________________ - 156 -
temperature and above body temperature (above the transition temperature)
taken under the confocal microscope (Zeiss), equipped with a heating stage. The
temperature at which each image was obtained was allowed to equlibrate for 15
mins.
NMA T=24oC NMA T=40oCNMA T=37oCNMA T=24oC NMA T=40oCNMA T=37oC
Figure 6-12 Fluorescence images of pure poly(NIPAM-MA) microgels loaded with
Rhodamine 6G (LC=57.2%) in 0.01M PBS buffer at different temperatures. Scale
bar is 10μm. (a) T=24oC (b) T= 37oC (c) T =40oC
At room temperature, the dye was successfully trapped within the
microgels as supported by the bright spots on the dark background. At 37oC, just
below the onset of the temperature-induced volume transition, the dye remained
trapped within the microgel interior, but the relatively brighter appearance of the
‘spots’ indicated that the fluorescence intensity coming from the microgels had
increased. At 40oC (above the VPTT), the dye appeared to remain confined to the
microgels and the fluorescence intensity of the bright spots had further increased.
These results showed that although some dye was probably released upon
temperature-induced microgel shrinkage, the majority remained entrapped within
the particles. Electrostatic attraction between positively charged R6G and the
negatively charged microgels at pH=7.4 may have inhibited the temperature-
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 157 -
induced release of the dye from poly(NIPAm-MA) microgels. Similar results were
obtained for hybrid poly(NIPAm-MA) microgels.
NIPAM-NIPMAM T=40oCNIPAM-NIPMAM T=24oC
Figure 6-13 Fluorescence images of hybrid poly(NIPAM-NIPMAm) microgels
loaded with Rhodamine 6G (LC = 48.6%) in 0.01M PBS buffer at different
temperatures. Scale bar is 2μm. (a) T=24oC (b) T =40oC
Figure 6-13 shows images of hybrid poly(NIPAm-NIPMAm) microgels loaded
with R6G (LC =35.1%) at 25 and 40oC. At room temperature, discrete bright spots
were observed on a relatively dark background, indicating the high affinity of R6G
for the microgel particles. However, the noticeable fluorescence intensity in some
areas of the largely dark background, suggested that some dye had rapidly diffused
away from the microgel particles upon being introduced into the dispersion
medium. At 40oC (above the VPTT) the fluorescence intensity of the bright spots
had increased considerably, but the dye appeared to remain entrapped within the
microgels, much like in the previous case. Several other pure and hybrid systems
loaded with R6G including poly(NIPAm-NIPMAm)/PAA IPN, poly(NIPMAm-UA) and
zwitterionic poly(NIPAm-SPP) microgels yielded similar images. The fluorescence
Chapter 6
___________________________________________________________________________ - 158 -
intensity of the loaded microgels (bright spots) was consistently observed to
increase with rise in temperature, but no system conclusively illustrated rapid,
temperature-induced release of the dye upon crossing the VPTT. A plausible
explanation for the increase in fluorescence intensity from the microgels was the
increase in concentration of R6G per unit volume upon microgel shrinkage, given
that the dye remained strongly-bound to the polymer network during the
deswelling transition.
We considered the possibility of fluorescence quenching, which may have
occurred if the concentration of loaded dye within the particles was sufficiently
large. The fluorescence intensity of pure R6G solution and R6G loaded in two
microgel systems (0.01mg/mL) at room temperature and 40oC was determined.
Solutions of R6G dispersed in PBS or water showed absolutely no difference in
intensity at the different temperatures studied (25, 37, and 40oC). However, R6G
loaded in microgel systems showed a slight decrease in fluorescence intensity with
increasing temperature, (Figure 6-14) in complete contradiction of the
aforementioned imaging results. Presently, we are unable to explain these
observations.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 159 -
0.00E+00
5.00E+05
1.00E+06
1.50E+06
2.00E+06
2.50E+06
3.00E+06
3.50E+06
525 575 625 675⎝ (nm)
Inte
nsity
(AU
)
NMA 40 DEGN-NM 40 DEGNMA 25 DEGN-NM 25 DEG
Figure 6-14 Fluorescence intensity of Rhodamine 6G loaded in poly (NIPAm-MA) and
poly(NIPAm-NIPMAm) microgels at room temperature and at 40oC. Increase in
temperature corresponded to a decrease in fluorescence intensity in both microgel
systems. Intensity of pure R6G solution did not change with temperature in the present
temperature range studied.
6.15 Real-time, photothermally-induced release studies
While cumulative release profiles showed that up to ca. 40% of loaded R6G
was released from microgels after centrifugation for 30 mins at temperatures in
the vicinity of the VPT, real-time images qualitatively implied that the majority of
the dye appeared to be entrenched within the interior, irrespective of the microgel
system, even after resting for 15 mins above the VPTT. Thus, we were unable to
conclusively visualize thermally-triggered, rapid release of the dye from microgels.
Nevertheless, we sought to witness the real-time, laser-induced release of dye or
drug from the microgel dispersion, hoping that the focusing of the laser beam on a
small area may yield a different response. We were encouraged by reports that
near-IR induced shrinkage of poly(NIPAm) gel was much more rapid than that
Chapter 6
___________________________________________________________________________ - 160 -
offered by ambient heating (seconds compared to mins),[44, 45] because the former
was not accompanied by the well known hydrophobic ‘skin’ layer formation at the
gel surface,[44, 45] that disturbs the rapid deswelling of the network and could
inhibit the release of a molecular payload. The R6G-loaded hybrid microgel
dispersions were therefore heated under the confocal microscope, to a
temperature just below the VPT, and irradiated with an 808nm CW laser (1mW) for
a period of 5 mins. The temperature in the dispersion medium was measured with
the help of a thermocouple. Figure 6-15 shows optical microscopy images of
dispersions of R6G-loaded, hybrid poly(NIPAm-MA) microgels, before and after laser
irradiation. Similar to the cases observed upon thermal heating (above), laser
irradiation also resulted in increased fluorescence intensity of the bright spots, and
rapid, laser-induced release of the dye was not observed.
T = 37oC
Before laser irradiation
T = 37oCT = 40oC upon
After laser irradiation
T = 37oC
Before laser irradiation
T = 37oCT = 40oC upon
After laser irradiation
Figure 6-15 Fluorescence images of hybrid poly(NIPAm-MA) microgels loaded with
Rhodamine 6G (LC = 49.5%) in 0.01M PBS buffer before laser irradiation T=37oC (left)
and after laser irradiation, T=37oC, right. Scale bar is 2μm.
Our various attempts to visualize real-time photothermally triggered
release of R6-G from different hybrid microgels were unsuccessful. Note that real-
time visualization of laser -induced, rapid release of dye from hybrid microgels
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 161 -
presented some technical challenges, as there is considerable difficulty in focusing
a narrow laser beam on a fixed, small area in the dispersion of microgels since,
they are in random motion. However, recently, Shitani et al.[44] were able to show
the real-time, photothermally-triggered release of R6G-labelled dextran from bulk
NIPAm hydrogels embedded with Au NRS. Their findings are indicative that the
premise of photo-thermally triggered release of a drug from poly (NIPAm)-based
microgels is credible.
6.16 Conclusions and outlook
Several microgel systems possessing a temperature-induced volume phase
transition in biologically-useful conditions (0.01M phosphate buffered media,
pH=7.4, 37-42oC) were synthesized. The narrowest and sharpest temperature-
induced transition was obtained for poly(NIPAm-MA) microgels containing 10mol%
MA in the reaction mixture (M2). This microgel underwent a massive 98% decrease
in volume between 38 and 40oC. Another promising system was the poly (NIPAM-
NIPMAm)/PAA IPN microgel system, which showed an 84% reduction in volume
between 37 and 44oC.
Photothermally-triggered volume transitions of hybrid microgels showed
that Au NRs remained strongly bound to the microgels, through several heating and
cooling cycles. The VPTT of pure and hybrid microgels were approximately the
same in both systems, showing that the sequestering of gold NRs did not
significantly alter the hydrophobic-hydrophilic balance in the host polymer
microgels.
Hybrid microgels were successfully loaded with R6G at room temperature in
buffer media. In-vitro release of R6G from three hybrid microgel systems was
Chapter 6
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shown upon heating above the VPTT for 30 mins. However, in all three systems,
more than 60% of R6G remained trapped within the microgel interiors.
Several factors may be considered in further pursuing efforts to see
photothermally triggered release of the dye from the microgel. Firstly, it must be
noted that the diffusion-driven rate of release from the microgel interior is smaller
than the rate of shrinkage of the microgel. Hence the comparative kinetics of
diffusion and microgel shrinkage must be considered. Furthermore, poly(NIPAm)
copolymers are well-known to form a thick, hydrophobic ‘skin’ on their surface
when they collapse, inhibiting the transport of loaded materials to the exterior of
the microgels.[45]
The partition coefficient of the loaded dye or drug must also be considered
since the polymer-drug interaction is a determinant factor in the release kinetics of
the DDS.[46-48] Hoare et al.[49] have recently published an excellent work in which
they studied the interactions of water-soluble drugs of different charges and
relative hydrophobicities with carboxylic acid functionalized, NIPAm-based
microgels with different functional group distributions. They found that both the
radial distribution of carboxylic functional groups and the hydrophobicities of the
cationic drugs strongly affect drug partitioning between the solution and microgel
phases. Hence it is expected that the functional group distribution and the relative
hydrophobicity of the drug will have considerable impact on loading capacity and
release profiles of microgel-based DDSs.
The method of uptake may also affect release kinetics. Lyon and
coworkers[41] found that drugs loaded into microgels via the ‘breathing in’
technique, i.e., swollen from the shrunken to the swollen state in the solution of
the drug are more firmly entrenched within the polymer network and not as rapidly
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 163 -
released as those loaded by physically mixing a dispersion of microgels in the
swollen state with a solution of the drug. All the aforementioned points are
relevant to the realization of efficient photothermally-responsive drug delivery
systems.
Chapter 6
___________________________________________________________________________ - 164 -
6.17 References for Chapter 6
[1] J. H. Kim and T. R. Lee, Langmuir 2007, 23, 6504-6509.
[2] J. G. Zhang, S. Q. Xu and E. Kumacheva, Journal of the American Chemical Society
2004, 126, 7908-7914.
[3] S. Q. Xu, J. G. Zhang, C. Paquet, Y. K. Lin and E. Kumacheva, Advanced Functional
Materials 2003, 13, 468-472.
[4] S. M. Kim JS, Lyon LA. , Angew. Chem. Int. Ed. 2005., 44:, 1333--1336.
[5] D. Suzuki and H. Kawaguchi, Langmuir 2005, 21, 8175-8179.
[6] S. Q. Xu, J. G. Zhang and E. Kumacheva, Composite Interfaces 2003, 10, 405-421.
[7] J. D. Debord, S. Eustis, S. B. Debord, M. T. Lofye and L. A. Lyon, Advanced Materials
2002, 14, 658-662.
[8] J. Perez-Juste, I. Pastoriza-Santos, L. M. Liz-Marzan and P. Mulvaney, Coordination
Chemistry Reviews 2005, 249, 1870-1901.
[9] I. Gorelikov, L. M. Field and E. Kumacheva, Journal of the American Chemical Society
2004, 126, 15938-15939.
[10] J. Kim, S. Nayak and L. A. Lyon, Journal of the American Chemical Society 2005, 127,
9588-9592.
[11] L. Valette, J. P. Pascault and B. Magny, Macromolecular Materials and Engineering
2002, 287, 52-61.
[12] S. M. Standley, I. Mende, S. L. Goh, Y. J. Kwon, T. T. Beaudette, E. G. Engleman and J.
M. J. Frechet, Bioconjugate Chemistry 2007, 18, 77-83.
[13] V. C. Lopez, S. L. Raghavan and M. J. Snowden, Reactive & Functional Polymers 2004,
58, 175-185.
[14] N. J. Flint, S. Gardebrecht and L. Swanson, Journal of Fluorescence 1998, 8, 343-353.
Hybrid Microgels for Photo-Thermally Induced Drug Release
___________________________________________________________________________ - 165 -
[15] X. M. Ma, J. Y. Xi, X. B. Huang, M. Zhao and X. Z. Tang, Materials Letters 2004, 58,
3400-3404.
[16] R. Yoshida, K. Omata, K. Yamaura, M. Ebata, M. Tanaka and M. Takai, Lab on a Chip
2006, 6, 1384-1386.
[17] T. Hoare and R. Pelton, Langmuir 2004, 20, 2123-2133.
[18] M. J. Garcia-Salinas, M. S. Romero-Cano and F. J. de las Nieves, Journal of Colloid and
Interface Science 2002, 248, 54-61.
[19] M. Hinge, Colloid Journal 2007, 69, 342-347.
[20] P. C. A. Rodrigues, U. Beyer, P. Schumacher, T. Roth, H. H. Fiebig, C. Unger, L.
Messori, P. Orioli, D. H. Paper, R. Mulhaupt and F. Kratz, Bioorganic & Medicinal
Chemistry 1999, 7, 2517-2524.
[21] V. T. Pinkrah, A. E. Beezer, B. Z. Chowdhry, L. H. Gracia, V. J. Cornelius, J. C.
Mitchell, V. Castro-Lopez and M. J. Snowden, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2005, 262, 76-80.
[22] V. T. Pinkrah, M. J. Snowden, J. C. Mitchell, J. Seidel, B. Z. Chowdhry and G. R. Fern,
Langmuir 2003, 19, 585-590.
[23] M. J. Snowden, B. Z. Chowdhry, B. Vincent and G. E. Morris, Journal of the Chemical
Society-Faraday Transactions 1996, 92, 5013-5016.
[24] T. Hoare and D. McLean, Journal of Physical Chemistry B 2006, 110, 20327-20336.
[25] C. D. Jones and L. A. Lyon, Macromolecules 2000, 33, 8301-8306.
[26] I. Berndt and W. Richtering, Macromolecules 2003, 36, 8780-8785.
[27] X. H. Xia and Z. B. Hu, Langmuir 2004, 20, 2094-2098.
[28] B. Nikoobakht and M. A. El-Sayed, Chemistry of Materials 2003, 15, 1957-1962.
[29] K. S. Soppimath, D. C. W. Tan and Y. Y. Yang, Advanced Materials 2005, 17, 318-+.
[30] I. Berndt, J. S. Pedersen, P. Lindner and W. Richtering, Langmuir 2006, 22, 459-468.
Chapter 6
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[31] D. Duracher, A. Elaissari and C. Pichot, Journal of Polymer Science Part a-Polymer
Chemistry 1999, 37, 1823-1837.
[32] H. Feil, Y. H. Bae, F. J. Jan and S. W. Kim, Macromolecules 1993, 26, 2496-2500.
[33] R. A. Weiss-Malik, F. J. Solis and B. L. Vernon, Journal of Applied Polymer Science
2004, 94, 2110-2116.
[34] B. G. De Geest, J. P. Urbanski, T. Thorsen, J. Demeester and S. C. De Smedt, Langmuir
2005, 21, 10275-10279.
[35] M. L. Christensen and K. Keiding, Colloids and Surfaces a-Physicochemical and
Engineering Aspects 2005, 252, 61-69.
[36] T. Hoare and R. Pelton, Abstracts of Papers of the American Chemical Society 2002,
224, U497-U497.
[37] X. H. Xia, Z. B. Hu and M. Marquez, Journal of Controlled Release 2005, 103, 21-30.
[38] T. Hoare and D. McLean, Macromolecular Theory and Simulations 2006, 15, 619-632.
[39] D. Schmaljohann, Advanced Drug Delivery Reviews 2006, 58, 1655-1670.
[40] C. M. Nolan, M. J. Serpe and L. A. Lyon, Macromolecular Symposia 2005, 227, 285-
294.
[41] C. M. Nolan, L. T. Gelbaum and L. A. Lyon, Biomacromolecules 2006, 7, 2918-2922.
[42] M. J. Serpe, K. A. Yarmey, C. M. Nolan and L. A. Lyon, Biomacromolecules 2005, 6,
408-413.
[43] S. H. Qin, Y. Geng, D. E. Discher and S. Yang, Advanced Materials 2006, 18, 2905-+.
[44] A. Shiotani, T. Mori, T. Niidome, Y. Niidome and Y. Katayama, Langmuir 2007, 23,
4012-4018.
[45] D. J. Gan and L. A. Lyon, Journal of the American Chemical Society 2001, 123, 7511-
7517.
Hybrid Microgels for Photo-Thermally Induced Drug Release
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[46] J. Y. Wu, S. Q. Liu, P. W. S. Heng and Y. Y. Yang, Journal of Controlled Release 2005,
102, 361-372.
[47] C. Khoury, T. Adalsteinsson, B. Johnson, W. C. Crone and D. J. Beebe, Biomedical
Microdevices 2003, 5, 35-45.
[48] C. M. Nolan, C. D. Reyes, J. D. Debord, A. J. Garcia and L. A. Lyon,
Biomacromolecules 2005, 6, 2032-2039.
[49] T. Hoare and R. Pelton, Langmuir 2008, 24, 1005-1012.
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 168 -
Chapter 7
Sequestering Gold Nanorods into
Microgels
Acknowledgements: Gold nanorods utilized in this work were kindly synthesized by Daniele
Fava and Leo Mordoukhovski.
7.1 Introduction
Polymer microgels undergo stimuli-responsive volume transitions, which
enable their use in the fabrication of ‘smart’, ‘tunable’ materials and switchable’
devices. [1-5] Recently, the use of hybrid polymer-inorganic microgels comprising
semiconductor quantum dots or nanoparticles of noble metals has opened new
Chapter 7
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avenues in the synthesis and fabrication of materials with advanced properties.[6-15]
In particular, the sequestration of gold nanoparticles by temperature-responsive
polymer microgels paved the way to photothermally-triggered swelling-deswelling
transitions of the polymer host.[16-20] Light absorbed by the gold nanoparticles led
to non-radiative energy transfer to the microgels, heating them to a temperature
above the volume-temperature transition.[17,18] Several potential applications arose
from the photothermally-triggered swelling-deswelling transitions of hybrid
microgels. For example, Lyon et al[19] reported the fabrication of photo-switchable
arrays of microlenses using temperature responsive microgels of N-
isopropylacrylamide (NIPAm) copolymerized with acrylic acid (AA) that were
brought in contact with gold nanoparticles (NPs). The system was irradiated at λ =
532 nm (the surface plasmon modes of the Au nanoparticles), which led to energy
transfer to the microgels in the form of heat and the shrinkage of the particles.
Suzuki et a[14] reported reversible color changes in temperature responsive
microgels loaded with gold and bimetallic gold-silver nanoparticles, that occurred
due to changing interactions between the NPs when microgels underwent swelling-
deswelling transitions.
Alternatively, photothermally induced volume-temperature transitions of
polymer microgels doped with Au particles can be used for the release of drugs
incorporated in the interior of the particles. Following irradiation, the drug may be
‘squeezed out’ and released to the surrounding medium.[16,17] The biocompatibility
of Au makes it a desirable material for use in hybrid microgels for drug release. For
biomedical applications of photothermally triggered drug release, it is imperative
to use irradiation wavelengths in the spectral range of 800-1200 nm (commonly
referred to as the ‘water window’) since they can penetrate body tissues. The use
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 170 -
of gold nanorods (NRs) as photosensitizers allows one to use the water-window
wavelengths of irradiation: the longitudinal plasmonic peak of the NRs can be
conveniently positioned in the desired spectral range by varying the length of the
NRs.[21]
An important design criterion for the application of NR-loaded microgels in
biomedical applications is the existence of strong interactions between the
microgel host and the NRs. This factor determines not only the loading capacity of
the NRs in the microgels but also the performance of the microgel drug carrier:
upon deswelling and release of the drug, the NRs must remain in the microgels.
Currently, electrostatic attractions between the positively charged NRs and the
negatively charged microgels are accepted to be the driving force for the
sequestering of NRs by microgels.[9] Typically, NRs carry a positive charge (due to
the stabilization with cationic surfactants) such as cetyltrimethylammonium
bromide (CTAB) which can also be further increased by coating NRs with a layer of
cationic polyelectrolyte.[21] Negatively charged microgels were obtained by
copolymerizing a host polymer e.g., poly(NIPAm) with anionic monomers such as
acrylic, methacrylic, or maleic acid[17] or by forming interpenetrating networks
with anionic polymers.[22] The use of acidic residues imposes limitations on the
microgel design. Typically, at biological pH values the incorporation of these
functionalities in microgels leads to the broadening and shift of the phase
transition temperatures and adds a further complexity to polymer-drug
interactions. [23-25]
Chapter 7
___________________________________________________________________________ - 171 -
7.2 Research objectives
In the current study, we examined the role of electrostatic interactions in
the sequestration of gold NRs into temperature responsive poly(NIPAm)-based
microgels. We used polyampholyte poly(N-isopropylacrylamide-co-acrylic acid-co-
vinylimidazole) poly(NIPAm-AA-VI) microgel particles as a model system, whose
varying number of charged groups at different pH values allowed us to explore the
role of electrostatic forces on the sequestering of NRs into microgels. For
comparison sake, poly(NIPAm-AA) and poly(NIPAm-VI) microgels were also doped
with Au nanorods. The affinity of the Au NRs for the microgels was qualitatively
and quantitatively characterized with the aid of light scattering, electrophoretic
mobility measurements, inductively coupled plasma and STEM.
7.3 Experimental
7.3.1 Synthesis of microgels
Details of the synthetic procedure for the copolymerization of poly(NIPAm-
AA-VI) polyampholyte microgels is described in Chapters 2 and 3. The microgels
were prepared via free radical precipitation polymerization at monomer weight
ratio AA/VI= 2 in the presence of 4 wt% of the crosslinking agent, N-
N,methylenebisacrylamide. After polymerization the microgel dispersion was
purified by dialysis against deionized water for 14 days (daily changes of water,
Spectra/Por Membrane, MWCO: 12-14,000), followed by centrifugation at 11,000
RPM for 30 mins, and redispersion in aqueous solution, adjusted to requisite pH for
measurements.
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 172 -
7.3.2 Preparation of gold nanorods (NRs)
Gold nanorods were synthesized following the procedure outlined by El
Sayed et al.[26] scaled-up to prepare 100 mL of NR suspension in water. The NRs
were purified by three rounds of centrifugation at 6000 rpm for 30 min each round.
At the end of each round, the supernatant was discarded and the precipitated
nanorods were re-dispersed in deionized water.
7.3.3 Preparation of hybrid microgels.
Hybrid microgels were prepared by the dropwise addition of the purified
dispersion of NRs to a purified dispersion of microgels in volume ratio 2:1,
respectively, under constant stirring. The value of pH of the microgel dispersion
and that of the dispersion of NRs was adjusted to the desired value by adding HCl
or NaOH solutions prior to doping.
7.3.4 Characterization
Particle dimensions were determined by photon correlation spectroscopy
(PCS, Protein Solutions Inc.). The hydrodynamic radii of the microgels were
calculated based on the measured diffusion coefficients by using the Stokes-
Einstein equation. Measurements of electrokinetic potential of the microgels were
conducted using the Zetasizer 3000HSA (Malvern instruments, U.K.). Prior to data
collection, each sample was equilibrated for 10 min at the desired temperature.
Hybrid microgels doped with Au NRs were imaged by scanning transmission electron
microscopy without centrifugation on the Hitachi HD-2000
The amount of Au metal content per unit volume of the dispersion of hybrid
microgels was determined by inductively coupled plasma atomic emission
Chapter 7
___________________________________________________________________________ - 173 -
spectroscopy (Optima 3000 ICP-AES). Hybrid microgel dispersions were prepared at
three different pH values as described above and centrifuged for 30 min at 4000
rpm. The precipitated hybrid particles were redispersed in a known volume of
water with the corresponding pH and filtered through a 0.45 μm filter, prior to
being subjected to plasma.
7.4 Results
7.4.1 Properties of pure microgels and pure gold nanorods
Poly(NIPAm-AA-VI) polyampholyte microgels were synthesized using the
procedure described elsewhere.[1] Gold nanorods stabilized with cetyl trimethyl
ammonium bromide (CTAB) were synthesized following the procedure reported by
El Sayed et al. [26] The mean diameter and length of the NRs were 8 and 48 nm,
respectively. Prior to the studying the loading of polymer microgels with gold NRs,
the properties of the individual components at 2.0<pH<10 were examined.
Figure 7-1 shows the variation in size (a) and electrokinetic potential (b) of
poly(NIPAm-AA-VI) microgels as a function of pH. At low pH the swelling maximum
appeared due to repulsive interactions between the protonated positively charged
amino group. At high pH swelling occurred due to repulsive interactions between
the deprotonated negatively charged carboxylic groups. In the interim range of
4.5<pH<8.0, mutual attraction and partial charge compensation between the
oppositely charged groups led to strong shrinkage and reduced zetapotential
respectively.[1, 27-29] The strong shrinking correlated with the variation in the
electrokinetic potential of the microgels and the values of pKa of AA and VI of 4.25
and 6.99, respectively.[25, 27]
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 174 -
Figure 7-1c shows the variation in electrokinetic potential, (ζ-potential), of
the NRs plotted as a function of pH. The NRs remained charged in the entire range
of pH values studied in the present work. The drop in ζ-potential at pH>8 occurred
100
150
200
250
300
2 4 6 8 10pH
Dh
(nm
)
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Abs
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2.5
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1.5
1.0
0.5
0
2.5
2.0
1.5
1.0
0.5
0
(a)
(b)
(c)
Figure 7-1 Variation in hydrodynamic diameter (a) and electrokinetic potential (b) of
poly(NIPAm-AA-VI) microgels plotted as a function of pH. Variation in electrokinetic
potential (c) and absorbance spectra (d) of NRs measured at different pH values
most likely due to partial charge compensation of CTAB by the increased
concentration of hydroxide ions at alkaline pH.
The absence of aggregation of the NRs following the change in pH of their
aqueous dispersion was verified by TEM imaging and by examining their
Chapter 7
___________________________________________________________________________ - 175 -
aggregation-dependent absorption spectra.[21] Figure 7-1d shows the absorbance
spectra of the dispersion of gold NRs in three solutions of pH=4.5, 6.3, and 7.5. All
spectra revealed two characteristic plasmonic peaks corresponding to the
transverse and longitudinal plasmon bands of Au NRs.[21, 30-32] The position of the
longitudinal absorbance band (peak position at 942 nm for all three curves) did not
show any shift under different pH conditions, indicating that no aggregation
occurred between the NRs. The absence of aggregation was also confirmed by
imaging NRs using transmission electron microscopy (TEM).
7.4.2 Sequestration of CTAB-stabilized gold nanorods into microgels
Loading of NRs into microgels was carried out by the dropwise addition of the
dispersion of NRs to the dispersion of polyampholyte microgels in volume ratio 2:1,
respectively. Figure 7-2 shows typical TEM images of poly(NIPAm-AA-VI) microgels
loaded with NRs at three pH values, namely, pH = 4.5, pH=6.3, and pH =7.5,
corresponding to the positively charged, almost neutral (close to isoelectric point),
and negatively charged microgels respectively. Surprisingly, irrespective of the pH
of the medium, the TEM images indicated qualitatively comparable sequestering of
ca. 80-90 NRs per microgel particle. No NRs were observed in the surrounding
medium.
In principle, the variation in pH can affect NR loading for two reasons.
Firstly, the swelling-deswelling transitions of the microgels, at 2.0<pH<10 (Fig. 7-
1a) change both the surface area and the polymer network’s mesh size. Both
factors may change the loading capacity of NRs in microgels. Close inspection of
the TEM images shows that the deposition of NRs occurred mostly on the surface of
the microgels, since their relatively large size most likely prohibited their
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 176 -
Figure 7-2 Transmission electron microscopy images of hybrid poly(NIPAm-AA-VI)
microgels loaded with gold NRs at different pH values: (a) pH=4.5 (b) pH~pI=6.3 (c)
pH=7.5. Scale bar is 800 nm. Scale bar for insets is 150 nm. The amount of Au in each
system as determined from inductively coupled plasma studies was 11.9, 9.7 and 10.8
mg/L at pH values of 4.5, 6.3 and 7.5 respectively.
migration into the particle interior. A similar observation was made by Liz-Marzán
and coworkers.[21] Secondly, the variation in microgel charge in the low, interim,
and high pH regions is expected to influence the sequestration of NRs into the
particles, and the number of positively charged NRs deposited on the cationic
Chapter 7
___________________________________________________________________________ - 177 -
microgels (pH =4.5) should be significantly smaller that that for the anionic
microgels (pH =7.5).
The amount of gold in the microgels was analyzed from inductively coupled
plasma (ICP) studies, following the removal of ‘free’ or loosely attached NRs by
centrifuging the hybrid microgel dispersion at 4000 rpm and 25oC, in a
temperature-controlled centrifuge. ICP results showed that the amount of Au
present in each system was comparable, irrespective of the pH: 11.9, 9.7, and 10.8
mg/L at pH of 4.5, 6.3 and 7.5 respectively. The slightly smaller amount of gold in
microgels at pH=6.3 was caused by their significant shrinkage in the zwitterionic pH
range, and the resulting decrease in surface area available for deposition of the
NRs.
7. 4.3 Sequestration of polyelectrolyte-coated gold nanorods into
microgels
To further elucidate the nature of sequestering of NRs by the
polyampholyte microgels we coated CTAB-stabilized NRs with two polyelectrolyte
layers (still keeping NRs cationic) and examined the uptake of polyelectrolyte-
coated NRs by the PA microgels at different pH values. The NRs were coated with a
layer of negatively charged poly(styrene sulfonate) (MW=70,000) and positively
changed poly(dimethyl ammoniumchloride) (MW = 100,000). The resulting values of
ζ-potential of the NRs were 53, 49, and 52 mV at pH of 4.5, 6.3, and 7.5
respectively.
Polyelectrolyte-coated NRs were mixed with the polyampholyte microgels
at the three pH values studied. The TEM images in Figure 7-3 show a striking
difference in the loading of polyelectrolyte-coated NRs compared to CTAB-
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 178 -
stabilized NRs into microgels. Expectedly, at pH=4.5 positively charged,
polyelectrolyte-coated gold NRs showed no affinity for the positively charged
Figure 7-3 Fragments of transmission electron micrographs of hybrid poly (NIPAm-AA-VI)
microgels loaded with polyelectrolyte-coated gold NRs at different pH values: (a) pH=4.5 (b)
pH~pI=6.3 (c) pH=7.5 Scale bar is 800 nm. Scale bar for insets is 150 nm.
microgels and accumulated in the intervening medium. At pH =6.3 most of the NRs
remained in the intervening medium. At pH = 7.5, the NRs were sequestered by the
Chapter 7
___________________________________________________________________________ - 179 -
negatively charged PA microgels, though in smaller amounts compared to the
uptake of CTAB-stabilized NRs.
Hence the affinity of the gold NRs towards the positively charged and
neutral microgels was altered upon coating them with a cationic polyelectrolyte
layer. This result indicated that for the sequestering of the CTAB-coated NRs,
electrostatic forces alone were not the defining factor.
It is worthwhile to mention that in earlier studies we observed qualitatively
similar sequestering of CTAB-coated NRs by other neutral and cationic acrylamide-
based microgels, including poly(NIPAm), poly(N-isopropylmethacrylamide),
poly(NIPAm-VI), and poly(N-isopropylacrylamide-N,N-dimethyl-N-(3-
methacrylamidopropyl)-N-(3-sulfopropylammoniumbetaine). Figure 7-4 shows
images of several acrylamide-derived microgels loaded with Au NRs. Note also that
that we were unable to dope hydroxypropyl cellulose microgels with the Au NRs.
(a)
(b)
(a)
(b)
Figure 7-4 TEM images of (a) neutral poly(NIPAm-NIPMAm) microgels at pH=7 and (b)
cationic poly(NIPAm-VI) microgels at pH=4.5 loaded with Au nanorods.
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 180 -
Uptake of anionic, citrate-stabilized gold nanoparticles by negatively
charged poly(NIPAm-methacrylic acid) microgels was earlier reported by Mohwald
et al.[33] and ascribed to the physical entrapment of the nanoparticles in the
interior of microgels. The authors found that the sequestering capacity was
suppressed for large nanoparticles with sizes comparable or greater than the mesh
size of the microgels. Similarly, in the present work, large NR dimensions precluded
their entrapment and most of the NRs deposited in the surface of microgels, similar
to the work of Liz-Marzan.[21] The reported sequestering of Au nanoparticles into
polyurethane microspheres has been ascribed Au and N coordination.[34] It is known
that centrifugation of NRs results in some loss of CTAB from the bilayer, [21] and
may lead to increased exposure of the NR- surface to the polymer. While the
coordination of acrylamides to hard transition metal ions through the oxygen atom
is known, there is no substantial evidence to support N-coordination modes of
metal ion-acrylamide complexes.[35] Coordination of ionic gold to the nitrogen atom
of acrylamides under extremely basic conditions is an unlikely possibility, and in
our work, we have elemental gold in moderately acidic or basic pH media.
The affinity of CTAB-coated Au NRs for the PA microgels at the pH values
corresponding to swelling maxima and at pH~pI was the same. The fact that both
neutral and positively charged microgels can be doped equally well with Au NRs
suggests that electrostatics alone is not the governing force driving the doping
process. Presently, the reason for the successful sequestration of NRs into
acrylamide microgels is not clearly understood. However, since only the CTAB-
coated NRs showed complete sequestration into the PA microgels irrespective of
pH, the forces driving the loading were caused by either gold or CTAB interaction
with the microgels.
Chapter 7
___________________________________________________________________________ - 181 -
7.4.4 Properties of hybrid microgels
We further examined the variation in size and ζ-potential of the NR-loaded
hybrid microgels (Figure 7-5a,b and c). The size of the hybrid microgels at all the
pH values was ca. 15 % smaller than those of pure microgels, as illustrated in Figure
7-5c due to the formation of ‘pseudo crosslinks’ generated by attractive
interactions between the NRs and the microgels. These interactions likely include
electrostatic interactions between the NRs and carboxylic acid groups in the
microgels at pH=7.5, specific interactions between gold nanorods, CTAB and the
polymer such as the possible coordination of gold to acrylamide, or hydrophobic
interactions between the CTAB and hydrophobic isopropyl groups in NIPAm.
At all pH values, the ζ-potential of the hybrid microgels was ca. 6 mV more
positive than that of pure microgels, due to the presence of positively charged NRs.
Absorbance spectra of NRs loaded in hybrid microgels (Figure 7-5c) showed a small
red shift compared to pure NRs, from 942 to 943, 943 and 944 nm at pH values of
4.5, 6.3 and 7.5 respectively. The small shift was caused by the change in
dielectric constant of the medium surrounding the NRs and not by the coupling of
the plasmonic properties of the NRs.
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 182 -
(c)
(d)
0
0.05
0.1
0.15
0.2
0.25
300 600 900 1200⎯ (nm)
Abs
orba
nce
(AU
) pH=4.5pH=7.5
pH=6.3
λ
2.5
2.0
1.5
1.0
0.5
0
100
150
200
250
300
2 4 6 8 10pH
D (n
m)
-30
-20
-10
0
10
20
30
2 4 6 8 10
pH
ζ-po
tent
ial (
mV)
0.5
1
1.5
2
2.5
3
2 4 6 8 10pH
D/D
o
(a)
(b)
(c)
(d)
0
0.05
0.1
0.15
0.2
0.25
300 600 900 1200⎯ (nm)
Abs
orba
nce
(AU
) pH=4.5pH=7.5
pH=6.3
λ
2.5
2.0
1.5
1.0
0.5
0
100
150
200
250
300
2 4 6 8 10pH
D (n
m)
-30
-20
-10
0
10
20
30
2 4 6 8 10
pH
ζ-po
tent
ial (
mV)
0.5
1
1.5
2
2.5
3
2 4 6 8 10pH
D/D
o
(a)
(b)
(c)
(d)
0
0.05
0.1
0.15
0.2
0.25
300 600 900 1200⎯ (nm)
Abs
orba
nce
(AU
) pH=4.5pH=7.5
pH=6.3
λ
2.5
2.0
1.5
1.0
0.5
00
0.05
0.1
0.15
0.2
0.25
300 600 900 1200⎯ (nm)
Abs
orba
nce
(AU
) pH=4.5pH=7.5
pH=6.3
λ
2.5
2.0
1.5
1.0
0.5
0
2.5
2.0
1.5
1.0
0.5
0
100
150
200
250
300
2 4 6 8 10pH
D (n
m)
-30
-20
-10
0
10
20
30
2 4 6 8 10
pH
ζ-po
tent
ial (
mV)
0.5
1
1.5
2
2.5
3
2 4 6 8 10pH
D/D
o
100
150
200
250
300
2 4 6 8 10pH
D (n
m)
-30
-20
-10
0
10
20
30
2 4 6 8 10
pH
ζ-po
tent
ial (
mV)
0.5
1
1.5
2
2.5
3
2 4 6 8 10pH
D/D
o
(a)
(b)
Figure 7-5 Variation in (a) hydrodynamic diameter and (b) ζ-potential of hybrid
microgels loaded with NRs as a function of pH. (c) Variation in normalized
hydrodynamic diameter, D/D0, of pure ( ) and hybrid (♦) microgels plotted as a
function of pH, where D0 is the smallest size of microgels obtained in the range
studied. (d) Absorbance spectra of gold NRs loaded in polyampholyte microgels at
different pH values.
Furthermore, we confirmed that microgels loaded with NRs remained
temperature responsive at different pH values. Figure 7-6a shows the variation in
the hydrodynamic size of pure and hybrid microgels as a function of temperature.
At pH=4.5 and at pH=7.52, the absolute size of both the pure and hybrid PA
microgels was larger than at pH~pI=6.3.
Chapter 7
___________________________________________________________________________ - 183 -
0
0.5
1
1.5
2
2.5
300 600 900 1200λ (nm)
Abs
orba
nce
(AU
)
0.5
1.5
2.5
3.5
18 28 38 48 58T (oC)
D/D
o
(b)Before
centrifugation
After centrifugation
(a)
λ
0
0.5
1
1.5
2
2.5
300 600 900 1200λ (nm)
Abs
orba
nce
(AU
)
0.5
1.5
2.5
3.5
18 28 38 48 58T (oC)
D/D
o
(b)Before
centrifugation
After centrifugation
(a)
λ
Figure 7-6 (a) Temperature-induced variation in normalized hydrodynamic diameter,
D/D0, of pure (open symbols) and hybrid (filled symbols) microgels at pH=4.5(♦), pH
=7.5 (▲) and pH=6.3(■) (b) Absorbance spectra of hybrid microgels before and after
centrifugation at 4000 RPM and temperature =40oC.
The volume-temperature transition curves of the hybrid microgels very
closely followed the trends of the corresponding pure microgels, indicating that
microgels loaded with gold NRs retained their temperature-responsive properties,
irrespective of the pH of the medium. Absorbance spectra of hybrid microgels
loaded with gold NRs before and after centrifugation at 40oC are shown in Figure 7-
5b. The decrease in absorbance intensity that was observed for the centrifuged
sample indicated that some NRs were lost to the supernatant after centrifugation
at elevated temperature.
7.4 Conclusions and outlook
In summary, we explicitly showed that electrostatic interactions are not
the governing force driving sequestration of gold nanorods into NIPAm-derived
microgels as was previously believed, although they are thought to have some
influence on the loading process. The affinity of gold NRs for the polyampholyte
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 184 -
microgels at pH values corresponding to their cationic, anionic and close-to-neutral
states were comparable. These results have two important implications. First, they
show that strong binding forces of gold NRs and/or CTAB to polyacrylamide
microgels overcome electrostatic repulsion between the nanoparticles and
microgels. Second, in order to sequester gold nanorods, the synthesis of
temperature-responsive polyacrylamide microgels may not require the
incorporation of anionic functional groups and thus the undesirable shift and
broadening of the volume-temperature transitions can be avoided.
Chapter 7
___________________________________________________________________________ - 185 -
References for Chapter 7
[1] M. Das, E. Kumacheva, Colloid. Polym. Sci. 2006, 284, 1073-1084.
[2] M. A. Alam, M. A. J. Miah, H. Ahmad, Colloid. Polym. Sci. 2007, 285, 715-720.
[3] H. M. Crowther, B. R. Saunders, S. J. Mears, T. Cosgrove, B. Vincent, S. M. King, G. E.
Yu, Colloids Surf., A 1999, 152, 327-333.
[4] M. Andersson and S. L. Maunu, Colloid. Polym. Sci. 2006, 285, 293-303.
[5] Y. J. Zhang, Y. Guan, S. Q. Zhou, Biomacromolecules 2006, 7, 3196-3201
[6] S. Bhattacharya, F. Eckert, V. Boyko, A. Pich, Small 2007, 3, 650-657.
[7] A.J. Pich, H. J-P. Adler, Polym. Int. 2007, 56, 291-307.
[8] Y. J. Gong, M. Y. Gao, D. Y. Wang, H. Mohwald, Chem. Mater. 2005, 17, 2648-2653.
[9] M. Das, H. Zhang, E. Kumacheva, Annu. Rev. Mater. Res. 2006, 36, 117-142.
[10] J.G. Zhang, S. Q. Xu, E. Kumacheva, JACS 2004, 126, 7908-7914
[11] D.B. Shenoy, G. B. Sukhorukov, Macromol. Biosci. 2005, 5, 451-458.
[12] S. Q. Xu, J. G. Zhang, C. Paquet, Y. K. Lin and E. Kumacheva, Adv. Funct. Mater.
2003, 13, 468-472.
[13] Y. Lu, Y. Mei, M. Ballauf, M. Dreschler, J. Phys. Chem. B 2006, 110, 3930-3937.
[14] D. Suzuki, H. Kawaguchi, Langmuir 2006, 22, 3818-3822.
[15] N. Singh, L. A. Lyon, Chem. Mater. 2007, 19, 719-726.
[16] J.H Kim, T.R. Lee, Drug Dev. Res. 2006 67, 61-69.
[17] M. Das, N. Sanson, D. Fava, E. Kumacheva, Langmuir 2007, 23, 196-201
[18] I. Gorelikov, L. M. Field and E. Kumacheva, J. Am. Chem. Soc. 2004, 126, 15938-
15939.
[19] S. M. Kim JS, LA. Lyon, Angew. Chem. Int. Ed. 2005, 44, 1333-1336.
[20] D. Suzuki, H. Kawaguchi, Langmuir 2005, 21, 8175-8179.
Sequestering Gold Nanorods into Microgels
___________________________________________________________________________ - 186 -
[21] M. Karg, I. Pastoriza-Santos, J. Perez-Juste, T. Hellweg, L. M. Liz-Marzan, Small 2007,
3, 1222-1229.
[22] X. H. Xia, Z. B. Hu, Langmuir 2004, 20, 2094-2098.
[23] T. Hoare, R. Pelton, J. Phys. Chem. B 2007, 111, 1334-1342.
[24] T. Hoare, D. McLean, J. Phys. Chem. B 2006, 110, 20327-20336.
[25] K. Ogawa, A. Nakayama, E. Kokufuta, Langmuir 2003, 19, 3178-3184.
[26] B. Nikoobakht, M. A. El-Sayed, Chem. Mater. 2003, 15, 1957-1962
[27] B. R. Saunders, Langmuir 2004, 20, 3925-3932.
[28] M. J. Snowden, B. Z. Chowdhry, B. Vincent, G. E. Morris, J. Chem. Soc. Faraday
Trans. 1996, 92, 5013-5016.
[29] S. P. Nayak, L. A. Lyon, Polymer Prepr. 2003, 44, 679-681.
[30] P. K. Jain, S. Eustis, M. A. El-Sayed, J. Phys. Chem. B 2006, 110, 18243-18253.
[31] B. N. Khlebtsov, N. G. Khlebtsov, J. Phys. Chem. C 2007, 111, 11516-11527.
[32] J. Perez-Juste, I. Pastoriza-Santos, L. M. Liz-Marzan and P. Mulvaney, Coord. Chem.
Rev. 2005, 249, 1870-1901.
[33] M. Kuang, D. Y. Wang, H. Mohwald, Adv. Funct. Mater. 2005, 15, 1611-1616.
[34] S. Phadtare, A. Kumar, V. P. Vinod, C. Dash, D. V. Palaskar, M. Rao, P. G. Shukla, S.
Sivaram, M. Sastry, Chem. Mater. 2003, 15, 1944-1949.
[35] K.B. Girma, V. Lorenz, S. Blaurock, F. T. Edelmann, Coord. Chem. Rev. 2005, 249,
1283-1293
Summary and Future Outlook
___________________________________________________________________________ - 187 -
Chapter 8
Summary and Future Outlook
8.1 Summary
The overall goal of the work in this dissertation was to study the design,
properties and applications of composite stimuli-responsive microgels. In
particular, their role as suitable particulate carriers for controlled and targeted
drug delivery systems was explored.
The swelling response of several polyelectrolyte (PE) and polyampholyte
(PA) microgels functionalized with acrylic acid and vinylimidazole, with respect to
changes in pH, ionic strength, temperature, solvent and polymer compositions were
discussed in Chapter 3. The PA microgels underwent swelling at high and low pH
values, and shrinkage in the intermediate pH range. In KCl solutions, all PA
Chapter 8
___________________________________________________________________________ - 188 -
microgels showed antipolyelectrolyte behavior. The temperature-dependent
volume phase transitions of both PE and PA microgels shifted to higher values than
that of poly(NIPAm) due to the hydrophilicity of ionized AA and VI groups. The
solvent-dependent swelling behavior of PE and PA microgels showed that
competing electrostatic and solvency interactions determined their swelling
response.
In Chapter 4, the results of synthesis and characterization of a series of
zwitterionic sulfobetaine microgels were presented. Although these microgels
contained equal amounts of strong, oppositely charged groups, no polyampholyte
behavior was observed in monovalent and divalent salt solutions. The unexpected
polyelectrolyte behavior displayed by the zwitterionic microgels is possibly a result
of the different binding affinities of the charged sulfonate and ammonium residues
to their respective counterions of the free electrolyte, and indicates the necessity
for considering all factors in the local environment of microgels when drafting their
design for a particular application. The zwitterionic sulfobetaine microgels have
potential applications as pH-stable microreactors for inorganic, metal,
nanocomposites.
The use of pH-responsive poly(NIPAm-AA) microgels for controlled and
targeted intra-cellular drug release was illustrated by the results shown in Chapter
5. The functional AA groups were used to bioconjugate a targeting, receptor-
specific protein, apotransferrin to the microgels, thereby enabling them to target
cancer cells, and be taken up by receptor-mediated endocytosis. Exposure of the
microgel-based DDS to the intracellular pH-gradient triggered release of the drug.
Cytotoxicity studies showed that the bioconjugated, pH-responsive microgels
Summary and Future Outlook
___________________________________________________________________________ - 189 -
effectively enhanced cancer cell suppression upon being loaded with an anticancer
drug.
The design and suitability of a DDS for photothermally-triggered drug
release under specific conditions, suitable for biological applications was described
in Chapter 6. The volume phase transition temperature of thermoresponsive
polyNIPAm microgels was tuned to occur in biologically useful conditions (T =35-
40oC, PBS pH=7.4) by copolymerization with various functional monomers. The
temperature responsive properties of the tuned poly(NIPAm)-derived microgels
were combined with the optical properties of gold nanorods (NRs) to yield hybrid
microgels that were shown to be photothermally responsive. Rhodamine 6G, a red
dye was successfully loaded into the hybrid microgels and in-vitro release profiles
reported.
In Chapter 7 the role of coulombic interactions in the sequestration of gold
nanorods (NRs) by microgels is reported. The affinity of gold NRs for NIPAm-derived
polyampholyte microgels was shown to be the same, regardless of the pH of the
medium. The sequestration of gold NRs into the particles was equally efficient for
the cationic, anionic and close-to-neutral states of the microgels, showing that
electrostatic interactions alone are not the crucial force driving the loading
process. In particular, the successful doping of positively charged NRs into
positively charged microgels revealed that strong binding forces between the gold
NRs and polyacrylamide microgels outweighed any electrostatic repulsion between
them. It was concluded that in order to sequester gold nanorods, the synthesis of
temperature-responsive polyacrylamide microgels may not require the
incorporation of anionic functional groups and thus the undesirable shift and
broadening of the volume-temperature transitions can be avoided.
Chapter 8
___________________________________________________________________________ - 190 -
8.2 Future outlook
The past decade has seen burgeoning research in the field of stimuli-
responsive polymer microgels for varied applications including their use as
microreactors for the synthesis of inorganic nanoparticles with predetermined
properties, as building blocks of photonic crystals, as tunable optical lenses, and as
components of controlled and targeted drug release systems.
Facile synthesis and functionalization of microgel particles offer a broad
range of variables for tuning their properties and favorably distinguish them from
other particulate polymer materials used for similar applications. Hybrid microgels
are excellent examples of materials with structural hierarchy. Coupling of the
structure- and composition-dependent properties of polymer microgels together
with inorganic nanoparticles opens new avenues in the production of “smart”
materials with many degrees of freedom in controlling their performance. It is
anticipated that the realization of new horizons in the applications of microgels as
advanced polymer materials will depend critically on the use of polymers with
conductive, optically limiting, photoactive, or other specific properties.
In particular, specific and vivid applications of microgels as intelligent
carriers of drugs have been identified and demonstrated with a focus on site-
specific, stimuli-triggered intracellular drug delivery. The environmental sensitivity
of microgels is advantageous for such applications, and may be tailored to achieve
a response under biological pH and temperature. However, the successful
realization of such drug delivery systems is subject to numerous critical
considerations. The porous network of the microgels is one issue of contention
because it allows for the nonspecific diffusion of drugs to healthy tissues before
reaching the target organ. Biofunctionalizing receptor-specific ligands to the
Summary and Future Outlook
___________________________________________________________________________ - 191 -
microgel surface can lend targeting ability to microgel-based DDSs and reduce this
unwanted toxicity. Another possible solution is to functionalize microgels with
specific groups that generate attractive forces between the microgel and the drug
before it reaches the target site. However, these attractive forces must be
overcome upon reaching the target, in order to enable release of the drug. It
follows that the nature and extent of various, specific interactions between the
loaded drug and the polymer particle must be considered at all stages of the
delivery process, when rationally designing an effective, microgel-based DDS.
These interactions may include ionic interactions, hydrophobic interactions,
partition coefficient of the drug and solubility parameters.
The stimuli-responsive properties of microgel-based DDSs that are
employed in their environmentally-triggered release mechanisms, themselves pose
challenges. The presence of salts, proteins and enzymes in biological environments
can alter the delicate balance of forces acting within the microgels and result in
undesirable, premature volume transitions, or, in the case of ionically crosslinked
microgels, even result in microgel collapse. Such problems may be alleviated by
protecting the surface of microgel particles with grafted or adsorbed polymers
[e.g., poly(ethylene oxide)] that inhibit interactions with biological species, until
reaching the target site.
Biopolymeric microgels are increasingly being investigated as more
desirable drug carriers owing to their biocompatibility and reduced cytotoxicity.
Several additional challenges are associated with the use of biomicrogels.
Reproducibility of the results obtained for biomicrogels may be problematic
because of their reliance on naturally occurring materials. As well, t.he purification
of biopolymeric systems is sometimes rigorous and time consuming. However, it is
Chapter 8
___________________________________________________________________________ - 192 -
expected that biopolymeric microgels such as hydroxypropyl cellulose and others
will be researched intensively in the near future.