stimulus-responsive microgels: design, properties and applications · ii stimulus-responsive...

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

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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.

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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.

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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.

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“The pursuit of knowledge begins with the admission of ignorance.”

~Unknown

“It is not enough to know. We must apply.”

~Goethe

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This thesis is dedicated to

my parents

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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)

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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

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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


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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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]


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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+


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


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


[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.


___________________________________________________________________________ - 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.


[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.


[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



___________________________________________________________________________ - 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.


[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


[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.


___________________________________________________________________________ - 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


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



O

HN

O

HN

HN

O



component

OS

OO

SO

O

O O


SO


N




O

HN

O

HN

O

HN

HN

OO

HN

HN

O



component

OS

OO

SO

O

O O

O

OS

OO

SO

O

O O


SO

OO

SO


NN



O

O


O

HN


component

O


component

O

OHHO

O



O

OH



O

O

O

O


O

HN

O

HN


component

O

OH

O


component

O

OHHO

O O

OHHO

O



O

OH

O

OH


Figure 2-1 Structures and functions of the reactants used in free radical

precipitation polymerization for the synthesis of microgels in this work


___________________________________________________________________________ - 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]


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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 -





[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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


Con

duct

ivity

( μS/

cm)

0

2

4

6

8

10

12


pH

050

100150200250300350400450


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.


___________________________________________________________________________ - 60 -

0123456789

10

0 0.5 1 1.5 2Volume of NaoH (mL)

pH

050

100150200250300350400


Con

duct

ivity

( μS/

cm)

0123456789

10

0 0.5 1 1.5 2Volume of NaoH (mL)

pH

050

100150200250300350400


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,


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


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.


___________________________________________________________________________ - 68 -

0.5

1.5

2.5

20 30 40 50 60Temperature (oC)

Rh/

Ro

0.5

1.5

2.5

3.5


Rh/

Ro

(a) (b)

0.5

1.5

2.5


Rh/

Ro

0.5

1.5

2.5

3.5


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


Rh/

R0

(c)

0.5

1.5

2.5


Rh/

Ro

0.5

1.5

2.5

3.5


Rh/

Ro

(a) (b)

0.5

1.5

2.5


Rh/

Ro

0.5

1.5

2.5

3.5


Rh/

Ro

(a) (b)

0.8

1

1.2

1.4

1.6

1.8

2


Rh/

R0

(c)

0.8

1

1.2

1.4

1.6

1.8

2


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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


[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.


[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


[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.


[29] M. J. Snowden, D. Thomas and B. Vincent, Analyst 1993, 118, 1367-1369.



[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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


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)


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 96 -


[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.


[5] Y. B. Zhao, Y. J. Yang, X. L. Yang and H. B. Xu, Journal of Applied Polymer Science

2006, 102, 3857-3861.


[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.


[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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


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


___________________________________________________________________________ - 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


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).


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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

10

20

30

40

50

60


%

(a)

(b)

0

10

20

30

40

50

60

70


%

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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


[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.


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.

Chapter 5

___________________________________________________________________________ - 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.

Hybrid Microgels for Photo-Thermally Induced Drug Release

___________________________________________________________________________ - 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


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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]


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:


___________________________________________________________________________ - 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.


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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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]


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 143 -

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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


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


0

20

40

60

80

100

120

20 25 30 35 40 45

T (oC)

% d

ye


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%


___________________________________________________________________________ - 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-


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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


___________________________________________________________________________ - 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

___________________________________________________________________________ - 162 -

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


___________________________________________________________________________ - 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 -


[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.


___________________________________________________________________________ - 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.


[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


[22] V. T. Pinkrah, M. J. Snowden, J. C. Mitchell, J. Seidel, B. Z. Chowdhry and G. R. Fern,

Langmuir 2003, 19, 585-590.




[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

___________________________________________________________________________ - 166 -

[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


[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.


___________________________________________________________________________ - 167 -

[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.


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

___________________________________________________________________________ - 169 -

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


___________________________________________________________________________ - 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 -


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


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.


___________________________________________________________________________ - 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]


___________________________________________________________________________ - 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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pH

ζ-po

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mV)

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)

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(b)

(c)

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)

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pH

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tent

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mV)

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ζ-po

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ial (

mV)

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Abs

orba

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)

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(d)pH=4.5

pH=7.5pH=6.3

2.5

2.0

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


___________________________________________________________________________ - 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-


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 182 -

(c)

(d)

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pH=6.3

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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

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300 600 900 1200λ (nm)

Abs

orba

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(AU

)

0.5

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2.5

3.5

18 28 38 48 58T (oC)

D/D

o

(b)Before

centrifugation

After centrifugation

(a)

λ

0

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300 600 900 1200λ (nm)

Abs

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(AU

)

0.5

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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


___________________________________________________________________________ - 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.


___________________________________________________________________________ - 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

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Chapter 8


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

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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


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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

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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


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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

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expected that biopolymeric microgels such as hydroxypropyl cellulose and others

will be researched intensively in the near future.

stimulus-responsive microgels: design, properties and applications · ii stimulus-responsive...

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