nano-aggregate formation of ab diblock and abc triblock...
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Nano-aggregate Formation of AB Diblock and ABC Triblock
Copolymers in Aqueous Solutions
Anil KHANAL
Department of Energy and Materials Science
Graduate School of Science and Engineering
SAGA UNIVERSITY
September, 2005
Nano-aggregate Formation of AB Diblock and
ABC Triblock Copolymers in Aqueous Solutions
By
Anil KHANAL
Supervisor: Professor Kenichi NAKASHIMA
A Dissertation Presented to the Graduate School of Science and
Engineering of Saga University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
September 2005
Approval
Graduate School of Science and Engineering Saga University
1 Honjo-machi, Saga 840-8502, Japan CERTIFICATE OF APPROVAL
Ph. D. Dissertation
This is to certify that the Ph. D Dissertation of
Anil KHANAL
has been approved by the Examining Committee for the dissertation requirement for the Doctor of Philosophy degree in Chemistry at the
September, 2005 graduation.
Dissertation committee: Supervisor, Prof. Kenichi NAKASHIMA Dept. of Chemistry
Member, Prof. Tohru MIYAJIMA
Member, Prof. Yushi OISHI
Member, Prof. Noboru TAKISAWA
Acknowledgements
First and foremost, I would like to express my deep sense of appreciation and admiration to my supervisor, Professor Dr. Kenichi Nakashima for his constant encouragement, kind support, invaluable guidance and for many more reasons. I express my gratitude to him as well as his wife (Yuki Nakashima) to whom I ever consider my family.
I am deeply indebted to Professor T. Miyajima for his generous support. I would like to thank Professor N. Takisawa for his invaluable advice on the handling of dynamic light scattering during entire periods of my research. I am also thankful to Professor Y. Oishi and his student N. Kawasaki for their advice and suggestion on handling atomic force microscopy.
I also wish to convey my sincere thanks to Professor Y. Tajima（Juntendo University）for his generous assistance in my research work.
I am indebted to Dr. M. Uehara and Dr. H. Nakamura (National Institute of Advanced Industrial Science and Technology, Tosu), who helped me to perform scanning electron microscopy work.
Grateful acknowledgement is made to all my friends at Nakashima lab and others labs especially Y. Li, Y. Takai, H. Fukuma, Y. Takahashi, Y. Nakashima, D. Parajuli, who made my research so intellectually stimulating and enjoyable.
I would also like to thank Japanese Ministry of Education, Science and Culture (Monbusho) and the Committee of Post Graduate Special Joint Program (PSJS) of Saga University, for providing financial support for my doctoral study.
Special thanks are given to Professor Dr. Kedar Nath Ghimire at Tribhuvan University, Nepal due to his constant supports in every respect. I am also deeply grateful to Professor Dr. Mohan Bikram Gewali and Professor Shambhu Thapa at Tribhuvan University, Nepal, for their innovative suggestion and encouragement during my research periods.
My sincere thanks go to Director, Dr. Tika B. Karki, Food research officer, Indra B. Basnet as well as all my friends at Department of Food Technology and Quality Control, Nepal for their moral support.
I would like to thank Professor Dr. Pratap Bahadur (South Gujarat University, India) for his contribution in improving this thesis.
Last, but not least, I would like to thank my parents for their encouragement and moral support. A note of thanks goes to Ms. Nani Maiya Bajracharya, Ashok Khanal, Amit Khanal, Anjir Khanal for their everlasting supports and love.
I would like to dedicate this thesis to my grandmother. Anil KHANAL
Abstract
This work comprises several aspects of polymeric micelles from AB diblock and
ABC triblock copolymers in aqueous solutions.
The first chapter gives a brief and general background of micelles and their
applications in industrial and biological processes.
The 2nd chapter describes the self-assembly of AB diblock copolymers such as
poly(ethylene oxide)-b-polymethacrylate (PEO-b-PMA). The PEO-b-PMA is a double
hydrophilic diblock copolymer (DHBC) which dissolves molecularly in water. However,
when PMA block of DHBC is made insolubilized (hydrophobic) with suitable
counterions such as cationic surfactants, metal ions or polycations through charge
neutralization, it forms micelle-like aggregates having a core of insoluble block. An
important advantage of DHBC micelles over micelles from conventional amphiphilic
block copolymers is that DHBC micelles can incorporate ionic species into the core
domain. In this regard, the nanoaggregates of PEO-b-PMA were fabricated using
chitosan (Ch) or methylglycolchitosan (MGC). These chitosans are polycations and
bind to anionic PMA block of PEO-b-PMA by electrostatic interaction, resulting in
hydrophobically modified PMA block. The incorporation of Ch or MGC into the PMA
block of PEO-b-PMA was monitored electrophoretically and by fluorescence
spectroscopy. The formation of nanoaggregates was confirmed by dynamic light
scattering and atomic force microscopy. The properties of these nanoaggregates depend
upon the concentration of the polymer as well as the concentration of Ch or MGC.
Chitosan exhibits antimicrobial, wound-healing, hypocholesterolemic properties etc.
Thus, formation of nanoaggregates of Ch and its family with PEO-b-PMA seems to
have potential applications in many fields including drug delivery systems because the
micelles with biocompatible hydrophilic shell show low uptake by reticuloendothelial
systems and significantly protect the incorporated drug from fast degradation, blood
clearance and elimination from the body.
However, the nanoaggregates of DHBC have a drawback. When these are used as a
drug vehicle for ionic drugs, they collapsed in the blood stream because the solution of
drug incorporated nanoaggregates gets diluted more than hundred times in the blood
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stream. Thus, it is essential to use another copolymer which exhibits frozen nature so
that it will not collapse in the blood stream. This can to be achieved by the use of ABC
type triblock copolymers, such as polystyrene-b-poly(2-vinyl pyridine)-b
-poly(ethylene oxide) (PS-PVP-PEO). Thus, in chapters 3,4,5 and 6, the micelles of
PS-PVP-PEO have been investigated.
The PS-PVP-PEO micelle has a glassy and hydrophobic PS core, an ionizable
hydrophilic PVP shell and a hydrophilic PEO corona. The micelle has an extended PVP
shell at low pH (<5) due to repulsive forces among the protonated PVP units and a
shrunken PVP shell at high pH (>5) due to the insoluble nature of the deprotonated
PVP shell. Thus, the micelles of PS-PVP-PEO have single-core at low pH and double
core at high pH. These features seem to be utilized to incorporate the anionic species
such as surfactants, drugs, metal salts etc.
In chapter 3, morphological changes of PS-PVP-PEO micelles have been
investigated by binding sodium dodecyl sulfate ( SDS). SDS is an anionic surfactant
and binds to the protonated PVP of PS-PVP-PEO micelles by electrostatic attraction.
The change in the size of SDS/PS-PVP-PEO complexes was detected by dynamic light
scattering techniques and atomic force microscopy while the binding of SDS was
confirmed by zeta-potential measurements. It was found that in the absence of SDS, the
zeta-potential of neat PS-PVP-PEO micelle was +13 mV. The incorporation of SDS
into the protonated PVP blocks of micelles gradually decreased zeta-potential of the
micelles and finally led to 0 mV zeta-potential due to electrical neutralization of the
PVP blocks. This induces conformational change of the PVP shell from an extended to a
shrunken form.
Physicochemical properties of PS-PVP-PEO micelles have been investigated in the
chapter 4 by dynamic light scattering method, zeta-potential measurement, atomic force
microscopy, and fluorescence spectroscopy. The micelles had an average diameter of
about 200 nm under acidic conditions. The addition of dextran sulfate to a micellar
solution under acidic conditions resulted in a significant decrease in the micelle size.
This was due to a conformational change in the PVP block form extended to shrunken
forms after the cationic PVP block was neutralized with dextran sulfate. The release of
an anionic dye (eosin Y) from the micelles was examined using dialysis, and found to
take tens of hours.
II
In chapter 5, new organic-inorganic nanocomplexes were fabricated from the
micelles of PS-PVP-PEO and tungsten compounds such as tungstate (W12-),
undecatungstophosphate (PW117-) and undecatungstosilicate (SiW11
8-) in acidic aqueous
solutions. The size and morphology of the fabricated complexes were characterized by
dynamic light scattering method, atomic force microscopy, scanning electron
microscopy, and fluorescence spectroscopy. PW117- and SiW11,
8- were found to bind
to the micelles not only by electrostatic interaction but also through hydrogen bond
formation etc.
In the chapter 6, studies are reported on the incorporation and release of an anionic
drug, cloxacillin sodium (CLX), from PS-PVP-PEO micelles. CLX exhibits antibiotic
activity and its binding to micelles cancels the repulsive forces among the PVP shell
of micelles. This induces the conformational change of the PVP transforming the shell
from an extended to a shrunken form. Furthermore, release of CLX from
PS-PVP-PEO micelle was investigated in vitro and it is found that the release kinetics
of the CLX is conforms to a model based on the consecutive chemical process and
CLX was retained in the micelle of PS-PVP-PEO at a time scale of several tens of hours
indicating that PS-PVP-PEO micelles can be used for the delivery of CLX, if the
PVP block is replaced by some suitable biocompatible polymer.
The major conclusions are summarized in chapter 7.
III
Contents
Chapter 1. Introduction ------------------------------------------- 1 1.1. Block Copolymers: Their Classification and Synthesis ------------------ 1
1.1.1. Classification of Copolymers ------------------------------------------ 3 1.1.2. Synthetic Methods of Block Copolymers ------------------------------- 4
1.2. Amphiphilic Diblock Copolymers Micelles ------------------------------- 5 1.2.1. Micellization of Amphiphilic Diblock Copolymers-Generalities --- 5
1.2.2. Preparation Techniques 6 1.2.3. Characterization of Amphiphilic Diblock Polymer Micelles - Experimental Techniques ---------------------------------------------------------------------- 8 1.2.4. Dynamics of Micellar Systems --------------------------------------- 14 1.2.5. Solubilization in Micelle ------------------------------------------------ 15 1.2.6. Internal Viscosity of the Micellar Core -------------------------------- 16
1.2.7. Theories -------------------------------------------------------------------- 17 1.2.8. Difference between Polymeric Micelles (PM) and Low Molar Mass
Surfactant Micelles (SM) ------------------------------------------------------ 17 1.2.9. Applications of Polymeric Micelles ---------------------------------- 18
1.3. Micellization of Double Hydrophilic Diblock Copolymers ------------ 20 1.3.1. Background -------------------------------------------------------------- 20
1.3.2 Preparation Techniques --------------------------------------------------- 21 1.3.3. PIC Micelle Formation from PEO-b-PMA -------------------------- 24
1.4. Core-Shell-Corona Micelles from ABC Triblock Copolymers ------ 26 1.4.1. Basic Concepts ---------------------------------------------------------- 26
1.5. References -------------------------------------------------------------------- 29
Chapter 2. Fabrication of Nanoaggregates of Poly (ethylene oxide)-b-Polymethacrylate by Complex Formation with Chitosan or Methylglycolchitosan ------------------------------------------------------- 37
2.1. Introduction -------------------------------------------------------------------- 37
2.2. Experimental Section ------------------------------------------------------ 38 2.2.1. Materials ----------------------------------------------------------------- 38 2.2.2. Preparation of Nanoaggregates --------------------------------------- 39 2.2.3 DLS Measurements ----------------------------------------------------- 40 2.2.4. Zeta-Potential Measurements ----------------------------------------- 40 2.2.5. Fluorescence Spectroscopy ------------------------------------------- 40 2.2.6. Atomic Force Microscopy -------------------------------------------- 41
2.3. Results and Discussion --------------------------------------------------- 41 2.3.1. MGC /PEO-b-PMA System ---------------------------------------- 41 2.3.2. Ch/PEO-b-PMA System ------------------------------------------- 47
2.4. Conclusions --------------------------------------------------------------- 52 2.5. References ------------------------------------------------------------------ 52
Chapter 3. Morphological Change of the Micelle of Poly styrene-b-Poly (2-vinyl pyridine)-b-Poly(ethylene oxide) induced by Binding of Sodium Dodecyl Sulfate -------------------------------- 55 3.1. Introduction --------------------------------------------------------------- 55 3.2. Experimental Section ---------------------------------------------------- 56
3.2.1. Materials -------------------------------------------------------------- 56 3.2.2. Preparation of Micelles --------------------------------------------- 57 3.2.3. DLS Measurements ------------------------------------------------- 57 3.2.4. Atomic Force Microscopy ----------------------------------------- 58 3.2.5. Zeta-Potential Measurements --------------------------------------- 58
3.3. Results and Discussion ---------------------------------------------------- 58 3.3.1. Hydrodynamic Diameter ------------------------------------------- 58
3.3.2. Atomic Force Microscopy ------------------------------------------- 60 3.3.3. Zeta-Potential Measurements ------------------------------------- 61
3.4. Conclusions -------------------------------------------------------------- 63 3.5. References ---------------------------------------------------------------- 64
Chapter 4. Physicochemical Properties of Micelles of Polystyrene -b-Poly(2-vinylpyridine)-b-poly(ethylene oxide) in Aqueous Solutions ------------- 66 4.1. Introduction ---------------------------------------------------------------------- 66 4.2. Experimental Section ---------------------------------------------------------- 68
4.2.1. Materials ------------------------------------------------------------------ 68 4.2.2. Sample Preparation ------------------------------------------------------ 68 4.2.3. Characterization of the Micelle ----------------------------------------- 69
4.3. Results and Discussion ------------------------------------------------------ 69 4.3.1. Detection of Micelle Formation -------------------------------------- 69 4.3.2. Incorporation of Dextran Sulfate (DS) -------------------------------- 72 4.3.3. Release of Organic Dye ------------------------------------------------ 75
4.4. Conclusions -------------------------------------------------------------------- 77 4.5. References --------------------------------------------------------------------- 78
Chapter 5. Fabrication of Organic-Inorganic Nano-complexes Using ABC Type Triblock Copolymers and Polyoxotungstates ------- 80
5.1. Introduction ------------------------------------------------------------------ 81 5.2. Experimental Section ------------------------------------------------------- 82
5.2.1. Materials ---------------------------------------------------------------- 82 5.2.2. Preparation of Organic-Inorganic Nano-complexes ---------------- 82
5.2.3. Zeta-Potential Measurements --------------------------------------- 83
5.2.4. Dynamic Light Scattering (DLS) Measurements ------------------ 83 5.2.5. Atomic Force Microscopy (AFM) ----------------------------------- 84
5.2.6 Scanning Electron Microscopy (SEM) -------------------------------- 84 5.2.7 Fluorescence Spectroscopy -------------------------------------------- 84
5.3. Results and Discussion ----------------------------------------------------- 85 5.3.1. Zeta-Potential of Nano-complexes ------------------------------------ 85
5.3.2. Dynamic Light Scattering (DLS) Measurements ------------------ 87
5.3.3. Morphological Studies -------------------------------------------------- 89
5.3.4. Fluorescence Spectroscopic Study ----------------------------------- 92
5.4. Conclusions ------------------------------------------------------------------ 94
5.5. References --------------------------------------------------------------------- 95
Chapter 6. Incorporation and Release of Cloxacillin Sodium in Micelles of Polystyrene-b-Poly(2-vinyl pyridine)-b-Poly(ethylene oxide)
------------ 98 6.1. Introduction ------------------------------------------------------------ 99 6.2. Experimental Section ----------------------------------------------- 101
6.2.1. Materials ----------------------------------------------------------- 101 6.2.2. Preparation of PS-PVP-PEO Micelle and Incorporation of CLX -- 101 6.2.3. Zeta-Potential Measurements -------------------------------------- 102 6.2.4. DLS Measurements ------------------------------------------------- 102 6.2.5. Fluorescence Spectroscopy ---------------------------------------- 103
6.2.6. Release Experiments ---------------------------------------------- 103 6.3. Results and Discussion -------------------------------------------- 104 6.3.1. Zeta-Potential Measurements ------------------------------------- 104 6.3.2. DLS Measurements ------------------------------------------------ 104
6.3.3. Fluorescence Spectroscopy ----------------------------------------- 106 6.3.4. Release Experiments ------------------------------------------------ 109
6.4. Conclusions -------------------------------------------------------------- 113 6.5. References ----------------------------------------------------------------- 114
Chapter 7. Summary ----------------------------------------------------- 117 Resume -------------------------------------------------------------------- 122
Publications ------------------------------------------------------------ 123
(Chapter 1)
1. Introduction
1.1. Block Copolymers: Their Classification and Synthesis
A polymer is a long, repeating chain of atoms, formed through the linkage of many
molecules called monomers. A high polymer is one in which the number of repeating units
is in excess of about 1000. This number is termed as degree of polymerization (DP). The
molecular weight of polymer is often given by the product of the molecular weight of the
repeating units and DP. The repeating units constituting the polymer molecules are called
constitutional repeat units (or CRU). Depending upon the nature of monomer, a polymer
can be classified as homopolymer and copolymer. A homopolymer consists of a chain of
similar monomer such as poly (ethylene oxide), polystyrene, polyisoprene etc.
Copolymers are defined as polymers composed of two different monomer units and are
classified into four types by monomer placement viz., random copolymers, alternating
copolymers, graft copolymers and block copolymers [1]. The architectures of these four
types of copolymers are demonstrated using two monomer units as A and B as shown in
Fig. 1. Random polymers are the statistical arrangement of copolymer having repeating
units in their backbone. Alternating copolymers are synthesized by the alternate placement
of monomers. Both block and graft copolymers are composed of several segments and
they differ in the inter-segment linkage site as graft copolymers are defined by chemically
linked pair of homopolymers, while block copolymers are composed of terminally
connected structures and thus, graft polymers have a comb type structure as shown in Fig.
1d.ｄｄｄ
1
Monomer unit A Monomer unit B
(a) Random
(b) Alternating
(c) Block
(d) Graft
Fig.1. Different types of copolymers.
2
1.1.1. Classification of Block Copolymers
fThe fascinating area of copolymers research is too big. Thus, we restrict ourselves to
block copolymers with an emphasis on their classification, synthesis and applications.
Block copolymers are classified into several types as shown in Fig.2. The simplest block
copolymers is AB type block copolymer, which is composed of one segment of A unit of
homopolymer with B units of other homopolymer (Fig. 2a). In the second type of
copolymer, both terminals of B unit is connected at the terminal of A unit, and thus, it is
referred to as an ABA type block copolymers ( Fig. 2b). In the third type of block
copolymers, A and B segments are connected many times and such polymers referred to as
multiblock copolymers ( Fig. 2c).
(b) ABA type triblock copolymers (a) AB type diblock copolymers
(d) Star block copolymers (c) (AB)n type multiblock copolymers
Fig. 2. Types of block copolymers.
3
The fourth type of block copolymers is star block copolymers. In this family of block
copolymers, unit A has multi-arm functionality and copolymerizes to form blocks of B and
shows a star-like shape. The number of arms of the star block copolymers depends on the
number of functional groups on block A (Fig. 2d).
1.1.2. Synthetic Methods of Block Copolymers
Block copolymers can be synthesized by a large number of well known
polymerization techniques, including anionic, radical, cationic, ring-opening, photo, group
transfer and Ziegler/Natta polymerization. From the last three decades, extensive work has
been carried out to synthesize the block copolymers. The most widely used technique to
make block copolymers is living polymerization, in which the molecular weight of the
individual blocks (variation of initiator/monomer ratio), the volume ratio (variation of
monomer/monomer ratio), as well as the block arrangement (AB, ABA, BAB), can be
adjusted in a desired manner. The most widely used techniques to synthesize block
copolymers are briefly described below:
(i) Sequential addition of the monomer. This is a widely used method with a possibility to
synthesize AB, ABA by using monofunctional initiating system and BAB and CBABC by
using bifunctional initiator.
(iii) Addition of the monomers at the same time. This method is applicable when
reactivity of the monomers is different and permits a mono and bifunctional initiation [2,3].
Due to different reactivities of monomer, polymerization of second monomer starts just
after the completion of polymerization of more reactive first monomer.
There are different types of initiators, which can induce the living polymerization but
anionic initiators are common due to long history of studies and actual applications to
polymer industries.
(iii) Coupling of homo- or block copolymers. Coupling of terminal functionalized homo-
and block copolymers can be used to synthesize AB, ABA and star-block copolymers.
4
However, a successful synthesis is dependent on several preconditions such as (i) the
coupling reaction proceeds completely without any side-reaction (ii) a 100% end group
functionality of copolymers is a necessary condition to approach a complete coupling
reaction (iii) both types of homopolymers are available in the exact mole ratio of the
functional end groups.
(iv) Site transformation technique. Recently, site transformation technique has been applied
to synthesize the block copolymers in which monomers with completely different
chemical structures can be polymerized by sequential addition with a change in the
polymerization technique [4 -6].
1.2. Amphiphilic Diblock Copolymer Micelles
1.2.1. Amphiphilic Diblock Copolymers Micelles-Generalities
Micro- and nanosized materials with unique morphologies and functions have
become important in medical applications [7-9] as water-miscible carriers [10] and
contaminants removers [11] etc. In this context, Price [12], Piirma [13] surveyed the
colloidal aspects of the block copolymers in solution after the first discovery of micelle by
Merret [14] in grafted natural rubber and found that when the block copolymers are
dissolved in a liquid that is a thermodynamically good solvent for one block and a
precipitant for the other, the copolymer chains may associate reversibly to form micellar
aggregates. For example, when polystyrene-b-poly (ethylene oxide), diblock copolymers
(PS-b-PEO) are dissolved into water, they form micelles because water acts as a bad
solvent for PS block and good solvent for PEO block. Thus, such micelles consist of a more
or less swollen core of the insoluble blocks (PS) surrounded by a flexible fringe of
soluble blocks (PEO) called corona as shown in Fig. 3.
Micelle formation occurs as a result of two forces. One is an attractive force that leads
5
to the association of molecules through hydrophobic interaction while the other, a repulsive
force through hydration, prevents unlimited growth of the micelles to a distinct
macroscopic phase.
PS PEO
PS (Core)
PEO (Corona)
Fig 3. Micelle of PS-b-PEO.
1.2.2. Preparation Techniques
Until 1980s, most studies on block copolymers micelles were reported using organic
selective solvents [15-20]. Typical examples of these are given in the Table 1.
6
Table 1. Some typical micellar systems
Copolymers Solvent (selectively good for)
PS-b-PMMA
PS-b- PI
PS- b-PB-b -PS
PS-b- PDMS
PS-b- hPI
PS-b- hPB- b-PS
PS-b-PVP
p-xylene (PS)
acetone (PMMA)
dimethylformamide (PS)
hexane (PI)
methyl ethyl ketone (PS)
decane (PDMS)
heptane (hPI)
1,4-dioxane (PS)
1,4-dioxane (PS)
toluene (PS)
PS: polystyrene, PMMA: poly(methyl methacrylate), PI: polyisoprene, PB: polybutadiene, PDMS:
poly(dimethylsiloxane), hPI: hydrogenated polyisoprene, hPB: hydrogenated polybutadiene, PVP:
poly(2-vinyl pyridine)
7
Only in last twenty years the effort shifted to micelles formed by hydrophobic/hydrophilic
block copolymers in aqueous media [21-24]. Polymer micelles in aqueous media are of
interest because of their practical application in our daily life.
Block copolymers micellar systems are generally produced by one of the followings
two procedures. In the first technique, the copolymers are molecularly dissolved in a
common solvent e.g. that is ‘good’ for both blocks, and then the conditions such as
temperature or composition of the solvent are changed in the way that requires formation of
micelles. For examples, poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene
oxide), (PEO–b-PPO–b-PEO) forms micelles when molecularly dissolved aqueous
solution of PEO–b-PPO–b-PEO is heated to above room temperature [25]. In the second
techniques, a solid sample of copolymers are directly dissolved in a selective solvent so that
one block of block copolymers does not dissolve and other block dissolves as described
previously for PS-b-PEO systems [26, 27].
However, these techniques are not universal for all block copolymers. If some block
copolymers contain glassy block like, polystyrene or poly(methyl methacylate) with high
mass fraction and molar mass, it will not dissolve in a solvent like water. In this context,
distillation or step-wise dialysis technique is employed [28, 29]. For example, PS-b-PEO
diblock copolymers will not dissolve into water if the mass fraction and molar mass are
higher than 0.2 and 2 × 104 g mol-1, respectively. To obtain micelles from PS-b-PEO, the
copolymer is first dissolved in tetrahydrofuran (THF) and water mixtures. Then, THF is
removed from resulting micellar solution either by azeotropic distillation under reduced
pressure or by dialysis [30].
1.2.3. Characterization of Amphiphilic Diblock Copolymers Micelles—Experimental
Techniques
The micellization of amphiphilic copolymers is similar to the process described for
low molecular weight surfactants. At very low concentrations, the polymer exists only as
single chains. As the concentration increases to reach a critical value called the critical
8
micelle concentration (CMC), polymer chains start to associate and form micelles in such a
way that the hydrophobic part of the copolymer will avoid contact with the aqueous media.
At CMC, an important quantity of solvent can be found inside the micellar core and
micelles are described as loose aggregates which exhibit larger size than micelles formed at
higher concentrations. At these concentrations, the equilibrium will favour micelle
formation; micelles will adopt their low energy state configuration and the remaining
solvent will gradually be released from the hydrophobic core resulting in a decrease in
micellar size.
In general, it can be assumed that block copolymers in a selective solvent form
micelles via a so-called closed association process, characterized by a certain CMC,
below which only molecularly dissolved copolymer is present in solution, usually as
unimers. Above CMC, multimolecular micelles are in equilibrium with the unimers.
Fig. 4 represents the schematic structures of A–B diblock copolymers micelles. Such a
micellar system is characterized by:
1. the equilibrium constant for unimers micelles.
2. the CMC and critical micelle temperature(CMT).
3. the morphology which in the simplest case can be considered as spherical.
4. Mm, the molecular weight of the micelle.
5. Z, the aggregation or association number, e.g. the average number of polymer chains in
a micelle, deduced from Mm and the molecular weight Mu of the unimer with Z=Mm/Mu
(note that different symbols, such as P, f, N, etc are used for Z by different authors).
6. Rg, the radius of gyration of the micelle.
7. Rh, the total hydrodynamic radius of the micelle.
8. the ratio Rg/Rh which is informative of the shape.
9. Rc, the micellar core radius.
10. L, the thickness of the shell (corona) formed by the soluble blocks.
To characterize above properties of micelles different experimental techniques have
been employed as shown in Table 2.
9
“Hairy micelle” L>>Rc
“Crew-cut” L<Rc
Interphase
Fig. 4. Schematic representation of AB diblock copolymers micelles in a selective solvent
of the A block. Rc: core radius; L: shell (corona) thickness.
10
Table 2. Experimental methods for characterization of polymeric micelles
Method Information obtained
(a) Static light scattering
(b) Small-angle X-ray and neutron
scattering
(c) Dynamic light scattering
(d) Cryoelectron microscopy
(e) Sedimentation velocity
(f) Size exclusion chromatography
(g) Stopped flow
(h) Steady-state and time-resolved
fluorescence
(i) Nuclear magnetic resonance
Weight-average molar mass
Radius of gyration
Weight-average molar mass
Radius of gyration
Core radius
Macrolattice structure
Diffusion coefficient
Hydrodynamic radius
Size and shape
Micelles/unimer mass ratio
Dynamics of micellar equilibrium
Kinetics of micelle formation and
dissociation
Dynamics of micellar equilibrium,
Dynamics on segmental level,
Aggregation number
Dynamics on segmental level
11
The physical methods for the characterization of block copolymers micellar systems
have been reviewed extensively by Tuzar [31], Munk [32], Chu and Zhou [33], Hamley
[34] and by Webber [35].
1.2.3.1. Critical Micelle Concentration (CMC)
In principle, the CMC of polymeric surfactants can be determined by a suitable
physical property e.g., osmotic pressure, surface tension that shows sudden changes at or
near the CMC. It is difficult to determine the CMC of the polymeric surfactants by applying
above methods [36] because polymeric surfactants have very low CMCs and diffusion
coefficients.
Although light scattering method can be used to determine the molecular weight and
aggregation number of micelles, the onset of micellization can be detected only if the CMC
falls within the sensitivity of the scattering method which is rarely the case for polymers in
water [37]. Thus, a preferred method to determine the CMC involves the use of fluorescent
probes [37, 38], among which pyrene is the most widely used. Pyrene is a hydrocarbon
with four benzene rings fused together and is sensitive to the polarity of the surrounding
environment [39]. Below CMC, pyrene remains in water, a medium of high polarity.
When micelles are formed, pyrene partitions preferentially toward the hydrophobic domain
afforded by the micellar core and thus, experiences a non-polar environment [39].
Consequently, numerous changes such as an increase in the fluorescence intensity, a change
in the vibrational fine structure of the emission spectra and a red shift of the (0,0) band in
the excitation spectra, are observed.
The apparent CMC can be obtained from the plot of the fluorescence intensity of
pyrene, the I1/I3 ratio from emission spectra or the I333/I338 ratio from excitation spectra,
against concentration; a major change in the slope indicates the onset of micellization [39].
However, the CMC determined by the fluorescence technique needs to be carefully
interpreted for two reasons. First, the concentration of pyrene should be kept extremely low
12
(10−7 M) so that a change in slope can be precisely detected as micellization occurs. Second,
a gradual change in the fluorescence spectrum can sometimes be attributed to the presence
of hydrophobic impurities or association of the probe with individual polymer chains or
premicellar aggregates [39].
1.2.3.2. Morphology
The morphology of block copolymers micelles can be seen by cryo-electron
transmission electron microscopy, atomic force microscopy etc. Most of the micelles
show spherical shape as reported by Price, Esselink and Lam et al [40, 41]. However, the
micelles of various block copolymers show different morphological shape such as spherical,
ellipsoidal, rod-like, vesicles, crew-cut micelles, flower-like micelles etc [42-45]. In this
context, Zhulina and Birshtein [46] and Halperin [47] proposed “star” micelles (Fig. 5a).
These micelles are spherical with small core and expanded corona, and form when the
length of soluble block is considerably longer than that of the insoluble block. Conversely,
Nagarajan et al [48] and Eisenberg et al [49] reported “crew-cut” micelles with large cores
and short coronal “hair”, formed from asymmetric block copolymers with relatively long
insoluble blocks (Fig. 5b).
Fig. 5. Schematic illustration of (a) a star and (b) a crew-cut micelle.
1.2.3.3. Size, Molecular Weight
The dimensions and the molecular weight of block copolymers micelles can be
13
determined by quite a number of techniques, especially scattering and hydrodynamic
characterization techniques as summarized before in Table 2. In general practice, the
hydrodynamic radius Rh is determined by dynamic light scattering techniques. By treating
the micelles as hydrodynamically equivalent spheres and using the Stokes–Einstein relation,
Rh can be evaluated from the translational diffusion coefficient extrapolated to infinite
dilution (D0):
Rh = KT/6πη D0 (1)
where k is the Boltzmann constant; T is the absolute temperature; η is the viscosity of the
solvent. 1.2.4. Dynamics of Micellar Systems Concerning the dynamics of block copolymers in solution, we have to consider the one side
kinetics of micellization, which corresponds to the dynamic of the micellar equilibrium
unimers micelles as well as to the problem of hybridization in micellar systems.
1.2.4. 1. Kinetics of Micellization
As mentioned above micelles are in dynamic equilibrium with individual molecules
（unimers） in solution. Thus, there is constant exchange between micelles and individual
molecules. Additionally, micelles themselves are continuously disintegrating and reforming.
By considering above facts, the kinetics of micellization has been studied by various
techniques such as stopped-flow, temperature-jump, pressure-jump and ultrasonic
absorption [29]. These techniques reveal two relaxation processes involved in micellar
dynamics. The fast relaxation process with relaxation time 1 (generally of the order of
microseconds) is associated with the fast exchange of unimers between micelles and the
surrounding bulk phase. This process is considered as the collision between unimers and
micelles. The second relaxation time 2 (usually of the order of milliseconds to minutes) is
14
attributed to the micelle formation and dissolution process. As the slowest step determines
the rate process, 2 represents the entire process of the formation or disintegration of a
micelle. This slow relaxation time is directly correlated with the average lifetime of a
micelle, and hence the molecular packing in the micelle, which in turn relates to the
stability of a micelle. Micellar dynamics depends on temperature, pressure and
concentration.
Major differences in kinetics of micellization observed in diblock (AB ) and
triblock (ABA ) copolymers could be explained by the fact that the escape of a unimer,
which has to disentangle from the micellar core, might be much easier in a diblock than
in a triblock structure. Indirect methods for obtaining information on the kinetics of
association/dissociation phenomena include size exclusion chromatography (SEC) and
ultracentrifugation techniques. By SEC, for instance, two well-separated peaks are
observed due to the slow dynamic unimer micelle equilibrium. However, no
quantitative conclusions can be obtained from SEC.
1.2.4.2. Micelle Hybridization
Mixing of two different copolymers of the same family is called hybridization.
Micelle hybridization provides valuable information about the kinetics and
thermodynamics of the micellar systems. Thus, in this technique, first two different
copolymers of the same family are micellized separately in a given selective solvent. The
two solutions are mixed and the changes of their properties are followed as a function of
time by convenient technique. In this concern, Cantu et al. [50] used light scattering,
Prochazka et al. [51] followed the fluorescence of labeled copolymers and Munk et al. [52]
used sedimentation techniques.
1.2.5. Solubilization in Micelles
15
One of the most useful properties of micelles is their ability to enhance the aqueous
solubility of hydrophobic substances which otherwise are only sparingly soluble in water.
The enhancement in the solubility arises from the fact that micellar core can serve as
compatible microenvironment for water-insoluble solute molecules. This phenomenon of
enhanced solubility is referred to as ‘solubilization’. Solubilizing capacity of the
hydrophobic core increases with the molecular weight of the polymer and temperature.
More polar hydrophobic compounds incorporate more easily into micelle core as compared
to non-polar ones [53, 54]. The solubilization characteristics of the block copolymers
micelles are usually given in terms of micelle–water partition coefficients which is the ratio
of the concentration of the solubilizate inside the micelle to its concentration in the aqueous
phase. The solubilization capacity can be expressed either in the form of the volume or
mass fraction of the solubilizate in the micellar core, or as the number of moles solubilized
per gram of hydrophobic block.
Due to this solubilization property of the polymeric micelles, hydrophobic drugs can
be incorporated into the core. Thus, micelles act as a vehicle to transport the hydrophobic
drugs. For example, poorly water soluble drug, adriamycin can be incorporated into the
core of poly(ethylene oxide)-b-poly( -benzyl -aspartate) micelles[55].
1.2.6. Internal Viscosity of the Micellar Core
The viscosity of the micellar core influences the physical stability of the micelles as
well as drug release. The internal viscosity (microviscosity) of the micelles can be
determined by using fluorescent probes such as bis(1-pyrenyl-methyl)ether, (dipyme) [56],
1,2-(1,1’-dipyrenyl)propane [57] or 1,6-diphenyl-1,3,5-hexatriene, (DPH) [58]. Dipyme is
sensitive to both polarity and viscosity changes in its local environment. Dipyme contains
pyrenyl groups. Thus, the extent of it intramolecular excimer emission depends upon the
rate of conformational change of pyrenyl groups. In this context, the intensity ratio of
excimer to monomer (IE/IM) can be used to estimate the fluidity of the environment which
surrounds dipyme. In order to know the viscosity of micellar core, dipyme is mixed with
16
micelle. The ratio of IE/IM, then, can be used to calculate the internal viscosity of the
micellar core. A small ratio of IE/IM correlates with a low mobility and a solid-like micellar
core. Recently, Winnik et al. showed with dipyme that the microviscosity of the inner core
of micelles depends on the position of the hydrophobic moiety (random versus end-grafted
chain) [59]. Internal viscosity can also be obtained from the depolarization of DPH [59]
because anisotropy values are directly related to the rotational freedom of DPH; the
higher the local viscosity of the associated DPH region, the higher is the anisotropy values.
Also, 1H-nuclear magnetic resonance (1H NMR) provides some information on the
viscosity of the micellar core [60, 61].
1.2.7. Theories of the Micelles
In the last ten years, several workers have strived to derive relations between structural
parameters of a micelle (CMC, Z, Rc,L,Rh) and basic characteristics of the constituent
block copolymers (molecular weight and composition) using various models and various
mathematetical approaches. In all these theories, the total Gibbs energy G(micelle) of the
micelle is expressed as the sum of several contributions, mainly those related to the core
G(core), the shell G(shell), and the core/shell interface G(interface) :
G(micelle)= G(core)+ G(shell)+ G(interface) (2)
Minimization of G(micelle) with respect to parameters characterizing the micelle leads
correlations between the copolymer and micellar characteristics.
According to the reviews by Gast [62] and Linse [63], these theories are based either
on the scaling concepts derived from Alexander–De Gennes theory or on the mean field
theory developed by Noolandi and Hong [64] and Leibler et al.[65].
1.2.8. Difference between Micelles of Polymeric Micelles and Conventional Surfactant
1. Molar mass of polymeric micelles is at least two orders of magnitude larger than that of
ordinary micelles.
17
2. CMC of polymeric micelles is much smaller than that of surfactant micelles.
3. Kinetics of micelle formation and dissociation are substantially slow processes in
polymeric micelles. They may be even kinetically frozen.
1.2.9. Applications of Polymeric Micelles
It is reported that the therapeutic applications of some anticancer agents are associated
with serious problems. When such a drug without drug vehicle is orally or intravenously
administrated, it reaches into blood vessels where it aggregates due to its hydrophobic
nature. This causes the immobilization of blood vessels resulting in side effects as severe as
respiratory system failure [66]. Overall, the formation of drug aggregates decreases the
bioavailability of drug and even produces the local toxicity in human body [67]. To
overcome theses problems, polymeric micelles can be used as a vehicle to transport the
hydrophobic drug because water insoluble drugs can be incorporated into the core of
micelles by chemical or physical interaction. The hydrophobic core of polymeric micelles
provides a suitable microenvironment for the incorporation of water-insoluble drugs while
the biocompatible hydrophilic shell like PEO, imparts steric stabilization and prevents the
interaction with blood components. This, along with their small size (generally < 100 nm)
reduces clearance by the mononuclear phagocyte system and favours the passive drug
targetting, after intravenous or oral administration [68-72].
Similar to block copolymers, low molecular weight amphiphiles are well known to
form micelles that solubilize hydrophobic drugs. Thus, several studies have been done
regarding the use of surfactant as a drug vehicle. Finally, it was found that for the purpose
of drug delivery many advantages may exist for polymeric micelles, mainly due to the
polymeric nature of these systems. The main advantages of polymeric micelles over
conventional surfactant micelles are summarized as follows:
(i) The tendency for micellization is overall much higher in block copolymers in
comparison to surfactants because thermodynamic stability in polymeric micelles is much
higher than that of low molecular weight surfactant micelles as indicated by CMC. The
CMC of polymeric micelles is often found to be micromolar range in sharp contrasts to low
18
molecular surfactant with millimolar CMC [73].
(ii) When drug incorporated polymeric micelle or surfactant micelle reaches to blood
stream, it will be diluted more than hundred times. In this situation, polymeric micelles will
remain kinetically stable due to low CMC while surfactant micelles will be diluted to
values below CMC and rapidly dissociate. In order to compensate this situation, the
concentration of surfactant micelles should be increased. But, oral administration of such a
high dose of drug incorporated surfactant micelles produces the local toxicity to human
health.
It has been reported that the polymeric micelles exhibit slow dissociation rates (in
hours and days) even below their CMCs and also it has rigid core as evidenced from 1H
NMR and fluorescent probe studies. These conditions make polymeric micelles kinetically
more stable and even diluted below CMC. This situation is sharp contrast to surfactant
micelles, which tend to break up in milliseconds upon dilution.
(iii) Unlike low molecular weight surfactant micelles that typically have mobile cores,
sustained and controlled drug release may be achieved with polymeric micelles. As a result,
the rapid loss of drug with attendant risk of intravascular precipitation of water-insoluble
drug posses less risk. Further, polymeric micelles may potentially act as a vehicle and
influence the pharmacokinetics of drugs in a favourable manner.
In this concern, most of the studies have been carried out on the micelles of pluronic
(PEO-b-PPO-b-PEO) because they can solubilize hydrophobic anticancer drugs like
haloperidol, indomethacin, doxorubicin (DOX), epirubicin and amphotericin B. It has been
observed that physically encapsulated DOX in the micelles of PEO-b-PPO-b-PEO
significantly increased the antitumor effects of the drug in vivo. Thus, a parenteral
formulation of DOX in this micelles has been entered as a phase I clinical trials in Canada.
Besides above application of polymeric micelles, they can be used in dispersants,
emulsifiers, wetting agents, foam stabilizers, flocculants, demulsifiers, viscosity modifiers,
etc., in many industrial and pharmaceutical preparations [74-76]. All of above aspects,
essentially based on the solubilization capacity of block copolymers micellar systems as
reported by Riess et al.[77], Malmsten [78], Arshady [79], and Torchilin [80].
19
In summary, polymer micelles show quite promising tools for the delivery of
non-ionic drugs. However, these can not incorporate ionic drugs due to their non-polar core.
In this context, polyion complex micelles are developed.
1.3. Micellization of Double Hydrophilic DiBlock Copolymers:
1.3.1. Background
It is found that oral or intravenous administration of neat ionic drugs is associated with
several serious problems although they are hydrophilic. These drugs after reaching in blood
stream may interact with some components of blood vessel. This causes the deposition of
drugs in the blood stream without reaching to the affected pathological site. Thus, the
effectiveness of drugs will be reduced due to elimination of these drugs by extraction
mechanism. Thus, it is essential to have some drug vehicle for these drugs. In this context,
the polymeric micelles seem to be a promising tool. However, these polymeric micelles can
not incorporate the ionic drugs because the core is non-polar. Thus, several attempts have
been made in order to solve these problems by using lipoplex and polyplexs [81-84] etc.
However, serious problems still remain in this non-viral vector system especially in vivo
situation because complexes of lipoplex with drug are in the charge-neutralized condition.
This condition causes the complexes insoluble in water or hydrophobic and it is nearly
impossible to transport these complexes in blood capillaries because the complexes may
aggregate in the blood stream.
In order to overcome above demerits, micelles from double hydrophilic diblock
copolymers (DHBCs) seem to be promising tool because these can incorporate ionic drugs
into the core and will not aggregate in the blood stream. In this concern, several
investigations have been done although the first direct synthesis of these block copolymers
was reported as early as 1972 [85]. The DHBCs consist of water soluble blocks of different
chemical nature. In aqueous solution, they behave as unimers like classical polymers or
20
polyelectrolytes, whereas their amphiphilic characteristics, such as surface activity and
micelle formation appear only under the influence external stimuli such as temperature, pH
or ionic strength changes. Micellization of these copolymers can further be induced by
insolubilizing one of the blocks via electrostatic interaction with oppositely charged species
such as polymers, metal derivatives etc.
There are several synthetic strategies towards double hydrophilic diblock copolymers.
The most defined block copolymers with adjustable block lengths can be obtained via
living polymerization (anionic, cationic and group transfer polymerization). However,
the range of suitable monomers is limited. Thus, also the possibility to couple the two
readily synthesized blocks or to modify one block of the block copolymers by polymer
analogous reactions is an advantageous synthetic strategy. In numerous cases, PEO is
used as the hydrophilic block of a DHBC, and many synthesis rely on the chemical nature
of this polymer.
1.3.2. Preparation Techniques of DHBC Micelles:
1.3.2.1. Temperature Variation
Micellization of double hydrophilic diblock can be induced by increasing or
decreasing the temperature. Poly(vinyl alcohol)-b-poly(methyl vinyl ethyl),
(PVA-b-PMVE) forms the micelles at 298 K because above this temperature PMVE block
of PVA-b-PMVE becomes hydrophobic while PVA becomes still soluble in water. Thus,
hydrophilic diblock copolymers which contain PMVE, poly (N-isopropyl acrylamide),
poly((diethylamino)ethylmethacrylate), poly( 2-N-morpholinoethyl methacrylate) blocks
can be prepared by varying the temperature because these blocks become insoluble upon
heating [86-87]. Temperature dependent micellization can be monitored by light scattering
techniques, revealing unimers below and well-defined micelles plus small amounts of
micellar aggregates above the CMT.
21
1.3.2.2. pH Variation
Poly (ethylene oxide)-b-poly(2-vinyl pyridine) (PEO-b-PVP) cannot form micelles at
low pH. But, it forms micelles at pH 5 with PVP core and PEO corona because at this pH
the PVP block becomes deprotonated which makes it hydrophobic [88]. It was found that
the rate of base addition did not influence the micelle size and once the micelles/aggregates
were formed, dilution does not affect the size anymore down to 10–5 mg/ml, whereas for
total polymer concentrations higher than 16 mg/ml, aggregation of the micelles and
precipitation occurred. Similarly, micelles of poly(acrylic acid)-b-poly(2-vinyl pyridine),
(PAA-b -PVP) can be prepared by this method[89].
1.3.2.3. Variation of the Ionic Strength
Weakly basic, poly(2-(N-morpholinoethyl) methacrylate),(PMEMA) homopolymer is
known to be completely soluble in acidic solution due to protonation but can be
precipitated easily by the addition of electrolytes. Hence, a DHBC molecule containing a
PMEMA block can form micelles upon the addition of salts, such as sodium sulfate. This
was demonstrated for poly((dimethylamino)ethyl methacrylate)-b-
poly(2-(N-morpholinoethyl) methacrylate), PDMAEMA-b -PMEMA, which showed
micelle by the addition of electrolytes having the core of PMEMA [90].
1.3.2.4. Complexation
Polyelectrolyte blocks in DHBCs can also become hydrophobic due to charge
neutralization upon complexation with oppositely charged molecules. In such cases, these
polyion complex (PIC) micelles can be formed due to electrostatic interaction. The PIC
shows narrow size distribution and small sizes. PIC micelles combine the
properties of both of block copolymers (micelle formation and enhanced solubility) with
those of polyelectrolyte complexes (pH and salt sensitivity).
22
The first example of the formation of stable and monodisperse PIC micelles was
reported at neutral pH for a mixture of poly(ethylene oxide)-b-poly(lysine), (PEO-b- PLys)
and poly(ethylene oxide)-b-poly( aspartic acid ), (PEO-b-PAsp) having the same
polymerization degree [91]. By means of viscosity measurements, it was found that the
unit ratio PLys to pAsp was equal, i. e., a 1:1 stoichiometry. The dispersity of these
micelles was found to be very low in a large concentration range of 1–10 mg/ml, and
spherical micelles remained stable for weeks with respect to their size. It was shown that
chain length recognition exists between DHBCs with a polycationic or polyanionic block,
respectively, indicating that the PIC micelle formation process is strictly driven by phase
separation [92]. Even in mixtures with different block lengths of PEO-b- PLys and
PEO-b-PAsp, respectively, the resulting micelles showed extremely narrow size
distribution and matched chain lengths of the charged segments, as monitored by GPC.
PIC micelle formation can also be induced by an electrostatic interaction of
PEO-b-PAsp with an equimolar ratio of PLys homopolymer to pAsp units [93]. Again, the
micelles were found to be very stable and monodispersed. Laser doppler electrophoresis
indicated that the micelles were electrically neutral suggesting that the micelle corona is
purely formed by PEO. The micelles were considerably stable at low ionic strength but
dissociated at higher ionic strength due to electrostatic shielding.
Stable micelles of PIC can also be formed between a DHBC molecule and a metal
complex. An example is the complexation of the antitumor active
cis-diamminedichloroplatinum(cisplatin) with PEO-b- PAsp by ligand exchange of the two
chloride ligands by the carboxylate groups of PAsp [94, 95]. As carboxylate ligands show a
low nucleophilicity, complex formation is reversible, thus making this system a promising
candidate for the release of platinum complex as an antitumor drug, especially, as the
platinum complex content in the micelles was found to be as high as 40–50%. For this
system, also a critical chain length of PAsp exists with respect to micelle formation, and the
micelle polydispersity decreased with increasing chain length of the PAsp block. The
CMC of these micelles in water was reported to be extremely low (0.01 mg/ml).
The DHBC analogs to polyelectrolyte-surfactant complexes also received attention
23
recently, as these complexes are water-soluble and combine the virtues of block
copolymers and polyelectrolyte complexes. In this context, many groups reported the
complexes of the sodium salt of poly(ethylene oxide)-b-poly( methacrylic acid), (PEO-
b-PMAA) and cationic single tail surfactants like, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, cetyltrimethylammonium bromide,
dodecylpyridinium chloride and cetylpyridinium bromide [96-99] as well as cationic
double and triple tail surfactants like, didodecyldimethylammonium bromide,
dimethyldioctadecylammonium bromide and trioctylmethylammonium bromide.
1.3.3. PIC Micelle Formation from poly (ethylene oxide)-b-poly(methacrylate)
Poly(ethylene oxide)-b-poly(methacrylate) molecule, (PEO-b-PMA) is double
hydrophilic diblock copolymers (Fig.6). PEO-b-PMA molecules do not form micelles on
dissolving in water. But, it forms micelles when anionic PMA block of the PEO-b-PMA is
insolubilized by some cationic counterions through electrostatic interaction. In this thesis,
we have reported fabrication of PIC micelles from PEO-b-PMA using chitosan and its
family (chapter 2) [100]. Fig. 7 represents schematically the formation of PIC micelles
from PEO-b-PMA and counter cations.
y
x CH 2O C
COO -
CH 3
CH 2 CH 2
PEO PMA
Fig. 6. Structure of PEO-b-PMA.
24
PEO PMA
M+ Counterion
PMA(Core)
PEO(Corona)
Fig.7. Schematic representation of PIC micelle formation.
25
The properties of these PIC micelles strongly depend on the counter ions. The
significance of these new types of PCI micelles is that they can incorporate ionic
compounds into their cores, in quite contrast to conventional polymeric micelles discussed
before in which only hydrophobic species can be incorporated into the core in aqueous
solutions. This property is especially important when PCI micelles are employed as carriers
for ionic drugs like chitosan.
1.4. Core-Shell-Corona Micelles from ABC Triblock Copolymers
1.4.1. Basic Concepts
The drug incorporated micelles of double hydrophilic diblock polymer will be diluted
more than hundred times in the blood stream when they reach to blood stream. This leads to
collapse of micelle within the short intervals of times because the micelle is unfrozen.
This decreases the bioactivity of drug and leads to ineffective treatment. Thus, it is essential
to find out a new micelle which is of frozen nature so that it takes several hours to take
equilibrium between unimers and micelles even it is diluted in the blood stream. There are
several reasons for searching for long-circulating drug carriers. At least three of them seem
to be most important. First, keeping the certain pharmaceuticals or drugs long time in the
blood stream enhances the drug availability. Thus, long-circulating drug carrier may
maintain a required level of drug in the blood for extended time intervals. Second,
long-circulating drug-containing microparticulates or large macromolecular aggregates can
slowly accumulate (so-called enhanced permeability and retention effect, known also as a
‘passive’ targetting or accumulation via an impaired filtration mechanism) in pathological
sites with affected and leaky vasculature (such as tumors, inflammations) and improve or
enhance drug delivery in those areas [101-103]. Third, prolonged circulation can help to
26
achieve a better targetting effect for those targetted (specific ligand-modified) drugs since it
increases the total quantity of targetted drug passing through the target, and the number of
interactions between targetted drugs and their targets. This attempts seems to be achieved
by using ABC triblock copolymers like, polystyrene-b-poly(2-vinyl
pyridine)-b-poly(ethylene oxide),(PS-PVP-PEO). The structure of PS-PVP-PEO is given in
Fig.8.
CH 2 CH CHy
x
N
CH 2 CH 2 CH 2 Oz
Fig. 8. Structure of PS-PVP-PEO.
The micelle of PS-PVP-PEO was investigated in aqueous solution. It contains three
layers: a hydrophobic PS core, a hydrophilic PVP shell and a hydrophilic PEO corona as
shown in Fig.9. The PVP block of this micelle shows an extended conformation at low
pH(<5) due to the presence of repulsive forces among the protonated PVP block while at
high pH (>5) the PVP block show a shrunken conformation due to the presence of
deprotonated PVP blocks. The protonation deprotonation equilibrium tunes the
micelle from single core to double core as shown in the Fig. 9 [104,105]. Thus,
hydrodynamic diameter of PS-PVP-PEO micelles varies from 94 nm to 68 nm on
increasing the pH from low to high in concomitant with the decrease in the thickness of
PVP shell from 8.5 nm to 5.5 nm [104].
27
On decreasing the pH of PS-PVP-PEO micelles in aqueous solution by the addition of
excess of hydrochloric acid increases the hydrodynamic diameter of micelles from 68 nm to
90 nm. But, neutralization of acidic micelles by the addition of equimolar amount of
sodium hydroxide does not restore the initial size of micelles (68 nm). Thus, PS-PVP-PEO
micelle is pH responsive but it shows non-reversible pH cycling. However, PS-PVP-PEO
micelles having shorter chain length of PEO show reversible pH cycling. This can be
explained by the influence of PEO length. If PEO block is very long, loss in entropy of
PEO block can not be balanced by hydrophobic interaction of deprotonated PVP blocks on
adding equimolar amounts of sodium hydroxide. However, such a balance occurs in the
micelles of PS-PVP-PEO having shorter PEO block.
Tuzar et al reported that the micellization of PS-PVP-PEO shows strong tendency to
aggregate due to decrease in solubility of PVP chains in micelle shell [106]. Such
aggregation can be stimulated by stirring, shaking and filtration of micellar solution.
However, this aggregation behavior is lower in acidic solution than that of neutral solution
because tendency of aggregation of protonated PVP is low due to electrostatic repulsion
between the protonated PVP block. In addition, slightly protonated PVP increases the
solubility of PEO. They demonstrated that fluorescence correlation spectroscopy is better
technique to avoid the secondary aggregation, because this technique does not require
filtration.
It seems that the morphology of the PVP blocks of PS-PVP-PEO micelles can be
induced by binding some anionic species into the protonated PVP blocks by electrostatic
interaction at low pH. Binding of anionic species to the protonated PVP blocks of the
PS-PVP-PEO micelles may cancel the repulsive forces among the protonated PVP blocks
which in turn make the PVP blocks insoluble in water [107]. This may cause the
conformational changes of the PVP blocks. In this context, the discussion on morphological
changes of the micelle by binding sodium dodecyl sulfate to the PVP is reported in chapter
3.
The ABC triblock micelles seem to have more complex structure than those of AB or
ABA polymeric micelles. Thus, it is interesting to investigate the physicochemical
properties of these micelles. Thus, physicochemical aspects of these micelles in different
28
aqueous systems are described in chapter 4 [108].
PS PVP PEO
pH>5 pH<5
Fig.9. Effect of pH on PS-PVP-PEO micelles.
29
Nowadays, organic and inorganic nano-hybrids seem to be promising in many fields
including drug delivery systems because some chemotherapeutic agents or drugs are
inorganic in nature. Thus, fabrication of such a nano-hybrids using PS-PVP-PEO micelles
may give a new approach in nanotechnology. Fabrication of nano-hybrid by using
PS-PVP-PEO micelles and polyoxotungstates is investigated in (chapter5) [109].
The PS-PVP-PEO micelle is frozen in nature. Thus, it is interesting to know how long
the incorporated drug will retain in the micelles in different aqueous environment. In this
context, chapter 6 describes the release of cloxacillin sodium from the micelles of
PS-PVP-PEO [110].
Overall, the main motivation for studying ABC triblock copolymer micelles is related
to the presence of a middle nanosized compartment in the micellar structure in addition to
frozen nature. Depending on the investigated systems, this middle compartment can be
either soluble or insoluble in the considered solvent, leading to different types of micelles.
Since the middle micellar compartment can be characterized by its own morphology, size,
physical and chemical properties, the spectrum of application for ABC block copolymers
micelles is broadly enhanced compared to AB or ABA systems. Thus, the field of ABC
triblock micelles is emerging.
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36
(Chapter 2)
Fabrication of Nanoaggregates of Poly (ethylene oxide)-b-Polymethacrylate by Complex Formation with
Chitosan or Methylglycolchitosan
Nanoaggregate formation of poly (ethylene oxide)-b-polymethacrylate (PEO-b
PMA) is induced by chitosan (Ch) or methylglycolchitosan (MGC). The nanoaggregates
are basically obtained by electrical charge neutralization of the anionic PMA block of
the PEO-b-PMA polymer with cationic Ch or MGC, which results in insolubilization of
the PMA block to form the core of the aggregates. Formation of the nanoaggregates was
confirmed by dynamic light scattering, fluorescence spectroscopy, atomic force
microscopy and zeta-potential measurements. The properties of the nanoaggregates
depend upon the concentration of the polymer as well as the concentration of Ch or
MGC. The significance of these aggregates is their ability to incorporate ionic species,
leading them to potential applications as drug carriers and nanoreactors.
2.1. Introduction
In the past decades, extensive attention has been paid to preparation of micelles
from block copolymers. The polymeric micelles have wide applications in drug
delivery systems [1, 2] and separation techniques [3]. On dissolving the amphiphilic
block copolymer into water, its self-assembly occurs to form the polymeric micelles
having a hydrophobic core and a hydrophilic corona [4, 5]. Although the micelles of
amphiphilic block copolymers have various advantages compared to the micelles of low
molecular-weight surfactants, there is a drawback that they cannot incorporate ionic
species into the core because the core is hydrophobic in nature [6].
Recently, steadily increasing attention has been paid to the double hydrophilic
copolymers (DHBCs) because they have a unique self assembling property which leads
them to form polymeric micelles in the presence of some suitable counterions [7-9]. The
37
DHBC cannot form the micelle by itself on dissolving it into water. However, when one
block of it is insolubilized with a suitable counterion such as a surfactant, metal ion or
polyion, it forms a micelle having a core of the insolubilized block. The electrostatic
interaction (or coordinate bond in some cases) between the added counterion and one
block of the DHBC plays a primary role in the micellization process. For example, poly
(ethylene oxide)-b-polymethacrylate (PEO-b-PMA) can form micelles when the
cationic PMA block is insolubilized with a counterion such as poly-L-lysine (PLS) [9].
This nanoaggregate had proved to be stable, monodispersed and able to incorporate
ionic drugs into the core due to its ionic nature. Other examples of DHBC assemblies
have been reported by Kataoka et al. [10-13], Kabanov et al. [14, 15], and Bronstein et
al. [16, 17]. Nanoaggregate formation of DHBCs has been extensively reviewed by
Cölfen [7].
Based on our earlier studies [8, 9, 18, 19] and those of others [20-25], we tried to
fabricate the nanoaggregates using PEO-b-PMA and the chitosan family such as
chitosan (Ch) and methylglycolchitosan (MGC). These chitosans are polycations and
thus may bind to the anionic PMA block of PEO-b-PMA by electrostatic interaction,
resulting in insolubilization of the PMA block. Ch exhibits antimicrobial and
wound-healing properties[26]. Several investigations so far have been attempted
regarding its function in health parameters. These studies include chitosan's
hypocholesterolemic properties [27], as well as its effect on arthrosis [28], ulcers [29],
osteoporosis [30], etc. Thus, formation of nanoaggregates of Ch and its family with
PEO-b-PMA may break fresh ground for drug delivery systems as well as basic
research on nano-science. As we expected, we have obtained the nanoaggregate of
PEO-b-PMA and Ch or MGC. The nanoaggregate formation was confirmed by dynamic
light scattering (DLS) measurements, fluorescence spectroscopy, atomic force
microscopy (AFM) and zeta-potential measurements.
2.2. Experimental Section
2.2.1 Materials
38
PEO-b-PMA was supplied from Polymer Source, Inc; and used as received. The
molecular weight of each block is Mn (PEO ) = 7,500 and Mn (PMA ) = 15,500.
Ch and MGC were purchased from Wako and used without further purification. The
molecular weight of Ch ranges from 3,000 to 30,000, and that of MGC is more than
150,000. The deacetylation degree (DA) of Ch and MGC is 40~50% and 100%,
respectively. Pyrene (Py) was purified by vacuum sublimation.
2.2.2. Preparation of Nanoaggregates
A known amount of PEO-b-PMA stock solution (2 g/L) was titrated with a
solution of Ch or MGC. The amount of added Ch or MGC is expressed by an apparent
degree of neutralization (DN), which is defined as:
DN (%) = DA×(Amounts of counterions in base molar units) ×100 (1)
(Amount of COOH groups in mole units)
We assume that DA = 0.4 for Ch and DA = 1.0 for MGC.
All the solutions were prepared using water purified with the Millipore MiliQ
system. The solutions of counterion/PEO-b-PMA were transparent over the whole range
of DN and stable for several weeks. In the case of the Ch/PEO-b-PMA nanoaggregates
solution, the desired pH is obtained by adding hydrochloric acid or sodium hydroxide.
The pH of the solutions of MGC/PEO-b-PMA was 7.2, where about 70% of the
carboxylic groups in PMA and almost all the amine groups in MGC are ionized [31].
In the Ch/PEO-b-PMA systems, the pH was adjusted to 4.65 to realize a condition
where both the carboxylic groups in PMA and the amino groups in Ch have an ionic
form. However, only 10% of the carboxylic groups have an ionic form at this pH
[31]. In spite of this fact, we did not choose another pH value because most of the
carboxylic groups have a nonionic form at a pH lower than 4.65, while almost all of
the amino groups have a nonionic form at a pH higher than 4.65. For these reasons,
DN does not have its original meaning. However, we use DN just to show the amount
of NH2 groups in the added chitosans relative to that of COOH groups in the PMA
block.
39
2.2.3. DLS Measurements
DLS measurements were carried out with an Otsuka ELS-800 apparatus at a fixed
90˚ scattering angle. Correlation functions were analyzed by a histogram method and
used to determine the diffusion coefficient (D) of the samples. The hydrodynamic radius
(Rh) was calculated from D using the Stokes-Einstein equation:
Rh = kBT / (6πη D), (2)
where kB the is Boltzmann constant, T the absolute temperature, and η the solvent
viscosity.
2.2.4. Zeta-Potential Measurements
The measurements of electrophoretic mobility (EPM) were performed at 25°C
with an Otsuka ELS-8000 apparatus. The zeta-potential of the particles was calculated
from the EPM using the Smoluchowski equation:
µE = ζ ε / η, (3)
where µE is EPM, ζ the zeta-potential, ε the permittivity of the solvent, and η the
viscosity.
2.2.5. Fluorescence Spectroscopy
Steady-state fluorescence spectra of the samples were recorded with a Hitachi
F-6500 fluorescence spectrophotometer (right angle geometry, 1 cm × 1 cm quartz cell).
First a known volume of Py stock solution (6× 10-5 M in methanol) was transferred into
a 10-mL volumetric flask. The solvent was evaporated by gentle heating under a
nitrogen gas stream. A known volume of PEO-b-PMA stock solution was then added to
the volumetric flask containing Py, followed by the addition of counterion solutions.
Finally, hydrochloric acid or sodium hydroxide was added to the volumetric flask to
obtain the desired pH. The concentration of Py was 0.6 µM which is close to the
saturation concentration of Py in water at 22 °C. Py is excited at 334 nm. The band
widths were 3 and 1 nm for excitation and emission, respectively.
40
2.2.6. Atomic Force Microscopy
AFM images were obtained in the dynamic force mode (corresponding to the
tapping mode) with an SPA 300 unit together with an SPI 3700 control station (Seiko
Instruments Industry, Co., Japan) in air. The samples for AFM observations were
prepared by evaporating a drop of the micellar solution on freshly cleaved mica in air.
2.3. Results and Discussion
2.3.1 MGC /PEO-b-PMA System
2.3.1.1. DLS Measurement
Figure 1 shows the dependence of the hydrodynamic diameter (2Rh) of MGC/
PEO-b-PMA nanoaggregates on DN or polymer concentration. It seems from Figure 1a
that the nanoaggregates start to form at DN = 10 %. A further increase in DN does not
cause any significant change in the hydrodynamic diameter of the nanoaggregates.
Figure 1b represents the hydrodynamic diameter of the complexes between
MGC and PEO-b-PMA as a function of the polymer concentration at 100 % DN. The
hydrodynamic diameter rises at a polymer concentration of 0.002 g/L, indicating
nanoaggregate formation. It should be noted here that this value cannot be regarded as
the critical aggregation concentration (cac) of MGC/PEO-b-PMA at 100 % DN,
because DLS does not have sufficient sensitivity to be employed for cac determination.
Therefore, the cac was investigated by fluorescence spectroscopy (see subsection 3.1.3).
A further increase in the concentration of polymer does not cause a significant change
in the hydrodynamic diameter of the complexes.
41
0
50
100
150
200
0 50 100 150 200DN / %
Dia
met
er /
nm
(a)
0
50
100
150
200
0.0001 0.001 0.01 0.1 1[PEO-b-PMA] / g L-1
Dia
met
er /
nm (b)
Fig.1. Dependence of hydrodynamic diameter of the nanoaggregates of
PEO-b-PMA and MGC as a function of (a) DN (concentration of PEO-b-PMA: 0.02
g/L) and (b) the polymer concentration at 100 % DN.
2.3.1.2. Zeta-Potential Measurements
We carried out the zeta-potential measurements in order to confirm the binding of
counterions to the PEO-b-PMA polymer. Figure 2 shows the zeta-potential of
aggregates of PEO-b-PMA with MGC as a function of DN or polymer concentration.
The increase in DN results in a steady increase in zeta-potential (Fig. 2a).
At 100 % DN, the zeta-potential is close to 0 mV, indicating that almost all the
charges of PMA are neutralized by cationic counterions.
Further addition of the counterions above 100% DN causes a change in the sign of
the zeta-potential towards the positive side. This fact suggests further incorporation of
the counterion into the electroneutral complexes of MGC/PEO-b-PMA. The binding of
cationic MGC to the electroneutral MGC/PEO-b-PMA might be due to a hydrogen bond
between the oxygen atom of PEO and the OH group of MGC. When DN > 160%, the
42
zeta-potential shows a downward curve implying that MGC is not bound to
PEO-b-PMA anymore.
-30
-20
-10
0
10
20
30
0 50 100 150 200 250
DN / %
ζ / m
V
(a)
-30
-20
-10
0
10
20
30
0.001 0.01 0.1 1
[PEO-b-PMA] / g L-1
ζ / m
V
(b)
Fig.2. The zeta-potential of the aggregates of PEO-b-PMA and MGC as a function
of (a) DN at constant polymer concentration (0.02 g/L) and (b) the polymer
concentration at fixed DN (100 %).
Figure 2b presents the dependence of the zeta-potential of MGC/PEO-b-PMA
complexes on polymer concentration at a constant 100 % DN. The zeta-potential is
43
found to be almost constant for the concentration range from 0.002 g/L to 0.2 g/L.
This indicates that MGC neutralizes almost all anionic PMA at 100 % DN regardless of
the polymer concentration. Consequently, the electroneutral compound of MGC/PMA is
obtained over the whole region of polymer concentration at 100% DN.
2.3.1.3. Fluorescence Spectroscopy
Because we cannot determine the cac of the counterion/PEO-b-PMA by the DLS
measurements; we tried to determine it by another technique. In this respect, we used
fluorescence spectroscopy. Figure 3 represents the fluorescence spectra of Py in
aqueous solutions of MGC/PEO-b-PMA nanoaggregates.
0
100
200
300
400
350 400 450 500
Concentration of No. PMA-b-PEO (g/L)
1 0.000 2 0.002 3 0.004 4 0.010
4 V
III
I
Wavelenght / nWavelength/nm m
1 I nte
nsity
/ a.
uIn
tens
ity /a
.u.
Fig. 3. Fluorescence spectra of Py (0.6µM) in aqueous solutions of the nanoaggregates of MGC/PEO-b-PMA at different polymer concentrations. DN = 100 % = constant.
44
0
100
200
300
10-5 10-4 10-3 10-2 10-1 100
(a)Fl
uore
scen
ce in
tens
ity
[PEO-b-PMA] / gL-1
0
•
10-5 10-4 10-3 10-2 10-1 1001.71
1.74
1.77
1.80
1.83
1.86(b)
[PEO-b-PMA] / g L-1
I 1/ I3
0
Fig. 4. Dependence of (a) fluorescence intensity and (b) I1/I3 of Py (0.6µM) on the
concentration of nanoaggregates of MGC / PEO-b-PMA. DN = 100 % = constant.
The fluorescence intensity is represented by the peak height of the band V (see text for
details).
45
The band I is a 0-0 band of the S1 →S0 transition, and the bands III and V are
vibronic bands [32]. We note that the fluorescence intensity increases with increasing
polymer concentration. This indicates that Py is effectively encapsulated into the
MGC/PEO-b-PMA nanoaggregates. If Py is located in the nanoaggregates, deactivation
of the fluorescent state by molecular motion is suppressed, resulting in an increase in
the fluorescence quantum yield. We plotted the fluorescence intensity against the
polymer concentration (Fig. 4a). The fluorescence intensity is represented by the peak
height of the band V because it is known that the intensity of this band is not much
affected by the polarity of the environment [32]. We noted a sharp increase in the
intensity at 0.002 g/L. This value corresponds to the concentration at which the DLS
signal rises (Fig. 1b).
Another useful fluorescence parameter of Py is the intensity ratio between bands I
and III (so-called I1/I3 ratio). This parameter has been widely used to determine the
CMC of conventional detergent micelles [32-33] and polymeric micelles [34-36]
because the I1/I3 ratio is sensitive to solvent polarity: e.g., it is about 1.87 in water but
is 0.61 in hexane [33]. When Py is added to micellar solutions, it is incorporated into
the hydrophobic domain of the micelles. Incorporation of Py into the hydrophobic
domain of the micelles gives a lower value of I1/I3 than that in the aqueous bulk phase.
Thus, the I1/I3 ratio is employed for determination of the CMC. The I1/I3 ratio was
also used for detecting the cac of the aggregates between PEO-b-PMA and CTAC [18,
37]. The dependence of I1/I3 on the concentration of MGC/PEO-b-PMA is presented
in Fig. 4b. The value of I1/I3 remains fairly constant at lower concentrations of the
nanoaggregates, being very close to that obtained in pure water. Above a concentration
of 0.002 g/L, the I1/I3 value shows a pronounced fall with the concentration. By
combining these fluorescence data with the DLS data, we conclude that the cac of the
nanoaggregates at 100 % DN is close to 0.002 g/L.
2.3.1.4. AFM Characterization
The AFM measurement was performed to confirm the formation of
nanoaggregates of MGC/PEO-b-PMA. Figure 5 represents an AFM image of the
nanoaggregates of MGC /PEO-b-PMA at a polymer concentration of 0.002 g/L.
46
Fig. 5. AFM image of the nanoaggregates of MGC/PEO-b-PMA.
This picture provides strong evidence of nanoaggregate formation. The averaged
diameter of the nanoaggregates is calculated to be 79 nm. This value is smaller than that
obtained by DLS measurements. The difference can be understood by taking into
account the fact that AFM gives a number-averaged diameter while DLS measurements
give a z-averaged diameter. In addition to this, deswelling of the micelle corona also
occurs in AFM measurements which leads to a decrease in average diameter of the
nanoaggregates.
2.3.2. Ch/PEO-b-PMA System
2.3.2.1. DLS Measurements
The hydrodynamic diameter of the nanoaggregates of Ch/PEO-b-PMA is shown in
Figure 6 as a function of DN and PEO-b-PMA concentration. In Figure 6a, the
hydrodynamic diameter of the nanoaggregates of Ch/PEO-b-PMA is plotted against DN
at a fixed polymer concentration of 0.02 g/L. The hydrodynamic diameter rises at 10%
DN, indicating nanoaggregate formation. Afterwards, the hydrodynamic diameter
decreases continuously with increasing DN. The hydrodynamic diameter decreases from
154 nm to 71 nm when DN is increased from 10% to 80%. This seems to be due to
47
the fact that, at lower DN, there is still repulsive force in the PMA block because the
anionic PMA block of PEO-b-PMA is partially neutralized by cationic Ch.
Consequently, the core of Ch/PMA will be swollen with water. On increasing the DN,
the repulsive force in PMA decreases continuously with the electrical neutralization
process, and this directs the cores of complexes to become more hydrophobic and
compact. A Similar dependence of the hydrodynamic diameter of the PEO-b-PMA
nanoaggregates on DN was observed by our group in the systems where
cetyltrimethylammonium chloride [19] and cationic anesthetics [18] were employed as
counter ions.
0
50
100
150
200
0.0001 0.001 0.01 0.1 1
[PEO-b-PMA] /gL-1
Dia
met
er /
nm
(b)
0
50
100
150
200
0 20 40 60 80
DN /%
Dia
mete
r/nm
(a)
Fig. 6. Dependence of hydrodynamic diameter of the nanoaggregates of
PEO-b-PMA and Chitosan as a function of (a) DN (concentration of PEO-b-PMA: 0.02
g/L) and (b) the polymer concentration at 40 % DN.
48
Figure 6b represents the size of the nanoaggregates as a function of polymer
concentration at 40% DN. There is a sharp increase in the diameter within the range of
0.001 g / L to 0.002 g /L, indicating nanoaggregate formation. On increasing the
polymer concentration from 0.1 g/L to 0.5 g/L, the diameter of the nanoaggregates
abruptly increases from 109 nm to 155 nm. This might be due to secondary aggregation
at higher polymer concentration.
2.3.2.2. Fluorescence Spectroscopy
Figure 7 represents the emission spectra of Py in the presence and absence of
Ch/PEO-b-PMA aggregates. We note, as in the case of the MGC/PEO-b-PMA system,
that the fluorescence intensity increases with increasing polymer concentration. The
increase in the fluorescence intensity indicates the encapsulation of Py into the
nanoaggregates.
0
100
200
300
400
350 400 450 500
V
1
4 Concentration of NO. PMA-b-PEO (g/L)
1 0.000 2 0.002 3 0.010 4 0.040
III
I
Wavelenght / nmWavelength/nm
Inte
sity
/ a.
u
Fig. 7. Fluorescence spectra of Py (0.6 µM) in aqueous solutions of the
nanoaggregates of Ch/PEO-b-PMA at different polymer concentrations. DN = 40 %=
constant.
49
10-3 10-2 10-1
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
I 1 / I 3
[PEO-b-PMA] / gL-1
(b)
0
•
10-3 10-2 10-10
50
100
150
200
250
300
[PEO-b-PMA] / gL-1
Fluo
resc
ence
inte
nsity
(a)
0
Fig. 8. Dependence of (a) fluorescence intensity and (b) I1/I3 of Py (0.6 µM )
fluorescence on the concentration of nanoaggregates of Ch/PEO-b-PMA. DN = 40 %=
constant. The fluorescence intensity is represented by the peak height of the band V (see
text for details).
50
Figures 8a and 8b respectively illustrate plots of the fluorescence intensity and the
I1/I3 ratio of Py as a function of concentration of the polymer. However, the change in
the fluorescence intensity and I1/I3 ratio are too gradual to be employed for cac
determination. Therefore, we could not obtain the cac value at 40 % DN. The cac
determination at higher DN will be done in future work.
2.3.2.3. AFM Characterization
Nanoaggregates of Ch/PEO-b-PMA are also confirmed by AFM. Figure 9
represents the AFM image of the nanoaggregates of Ch/PEO-b-PMA at a polymer
concentration of 0.002 g/L. This picture evidences the formation of complexes of Ch
and the polymer. The averaged diameter of the nanoaggregates is calculated to be 82
nm. This value is smaller than that obtained by DLS measurements. The difference
seems to be due to the same reasons as described for the nanoaggregates of
MGC/PMA-PEO (see subsection 3.1.4).
Fig. 9. AFM image of the nanoaggregates of Ch/ PEO-b-PMA.
51
2.4. Conclusions
We have fabricated the nanoaggregates of PEO-b-PMA by insolubilizing the
anionic PMA block with a cationic counterion Ch or MGC. The formation of these
nanoaggregates was confirmed by various instrumental techniques such as dynamic
light scattering, zeta-potential measurements, fluorescence spectroscopy and atomic
force microscopy.
In the MGC/PEO-b-PMA system, the nanoaggregates have hydrodynamic
diameters ranging from 121 nm to 105 nm at different concentrations of PEO-b-PMA
when DN= 100 %. The zeta-potential of these nanoaggregates has a negative value at
DN < 100 % but reaches almost 0 mV at 100 % DN, indicating the binding of cationic
MGC into the anionic PMA block of PEO-b-PMA. The fluorescence intensity of Py
increases with the increasing concentration of PEO-b-PMA at constant DN.
Concomitantly, the I1/I3 ratio of Py fluorescence decreases. These changes in the
fluorescence parameters support formation of the nanoaggregates. AFM measurements
gave clear images of almost spherical nanoaggregates.
In the Ch/PEO-b-PMA system, DLS measurements revealed that the
hydrodynamic diameter of the nanoaggregates is about 110 nm when the polymer
concentration is lower than 0.1 g/L and DN is fixed at 40%. However, the diameter
abruptly increases from 110 nm to 155 nm when the polymer concentration is increased
over 0.1 g/L. This change suggests formation of secondary aggregates. AFM and
fluorescence spectroscopy provided concrete evidence of the nanoaggregate formation
between Ch and PEO-b-PMA.
2.5. References
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2. K. Kataoka, G.S. Kwon, M. Yokoyama, T. Okano, Y. Sakurai, J.
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Macromolecules, 1992, 25, 644.
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8. Y. Li, Y.-K. Gong, K. Nakashima, Y. Murata, Langmuir, 2002, 18,
6727.
9. Y. Li, L.-J. Ding, H. Nakamura, K. Nakashima, J. Colloid. Int.
Sci., 2003, 264, 561.
10. A. Harada, K. Kataoka, Macromolecules, 1995, 28, 5294.
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Katayose, Macromolecules, 1996, 29, 8556.
13. A. Harada, K. Kataoka, J. Macromol. Sci. Pure Appl. Chem. A, 1997,
34, 2119.
14. T.K. Bronich, A.V. Kabanov, V.A. Kabanov, K. Yu, A. Eisenberg,
Macromolecules, 1997,30, 3519.
15. T.K. Bronich, A.M. Popov, A. Eisenberg, V.A. Kabanov, A.V.
Kabanov, K. Yu, Langmuir, 2000,16, 481.
16. L.M. Bronstein, S.N. Sidorov, A.Y. Gourkova, P.M. Valetsky, J.
Hartmann, M. Breulmann, H. Colfen, M. Antonietti, Inorg. Chim. Acta,
1998, 280, 348.
17. L.M. Bronstein, S.N. Sidorov, P.M. Valetsky, J. Hartmann, H.
Colfen, M. Antonietti, Langmuir, 1999, 15, 6265.
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2000, 281, 562.
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Kabanov, A.V. Kabanov, Langmuir, 1999,14, 6101.
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54
(Chapter 3)
Morphological Change of the Micelle of Poly styrene-b-Poly (2-vinyl pyridine)-b-Poly(ethylene oxide)
induced by Binding of Sodium Dodecyl Sulfate.
Morphological change of micelle of polystyrene-b-poly(2-vinyl
pyridine)-b-poly(ethylene oxide) (PS-PVP-PEO) polymer was induced by binding
sodium dodecyl sulfate (SDS) to the PVP block in acidic aqueous solutions. The change
in the size of SDS/PS-PVP-PEO complexes was detected by dynamic light scattering
measurements and atomic force microscopy, and the binding of SDS was confirmed by
zeta-potential measurements. When the micelle was free from SDS in acidic aqueous
solutions, the hydrodynamic diameter of the micelle was 216 nm, reflecting the
extended conformation of the PVP block due to the repulsion between protonated
pyridine units. As the cationic PVP block was electrically neutralized with anionic SDS,
the diameter was gradually reduced in concomitant with the decrease in zeta-potential,
and finally reached 175 nm when the PVP block was completely neutralized. The
decrease in the diameter shows the morphological change of the PVP block from
extended to shrunken forms. Further addition of SDS did not cause the changes of the
diameter nor zeta-potential. This indicates that SDS was not bound to the PS-PVP-PEO
polymer after the PVP block was fully neutralized, and that the hydrophobic binding of
SDS to the polymer was negligible due to the low concentration of SDS.
3.1. Introduction
Association of block copolymers in a selective solvent, which is poor for one block
and good for the other block, can produce structures like micelles or physical
networks[1-3]. Such structures find potential applications both in industry and basic
55
research. There have been a lot of works on the micelles of AB diblock copolymers and
ABA triblock copolymers [4-9]. However, little is known about self association of ABC
triblock copolymers, although they are precursors of very complex self-assembled
struc
resulting in conformational
chan
d the
binding of SDS to the polymer was confirmed by electrophoresis measurements.
3.2. Experimental Section
.2.1. Materials
as purchased from TCI (guaranteed grade) and used without
further purification.
tures in the bulk [10].
Recently, core-shell-corona (CSC) aqueous micelles of polystyrene-b-poly(2-vinyl
pyridine)-b-poly(ethylene oxide) (PS-PVP-PEO) triblock polymer have been
reported[11-12]. They have a frozen PS core, an intermediate PVP shell, and PEO
corona. One of the interesting properties of the micelles is that the morphology is pH
sensitive because the solubility of the PVP in water depends upon pH[13]. The PVP
block is water-soluble at pH <5 and water-insoluble at pH >5 due to the
protonation-deprotonation equilibrium in pyridine ring,
ge between extended and shrunken forms.
Based on earlier studies by us[14,15] and other groups[16-26], we consider that
we may bind water-soluble protonated PVP block of PS-PVP-PEO micelles with some
suitable anion. This may induce the morphological change of the micelles by
insolubilizing the PVP block. Thus, we tried to bind anionic surfactant, sodium dodecyl
sulfate (SDS), to the PS-PVP-PEO micelle at low pH. Such a study has not been
reported yet. We have succeeded in inducing the morphological change of the micelles
by binding SDS to the PVP block at low pH. The conformational change was observed
by dynamic light scattering (DLS) and atomic force microscopy (AFM), an
3
PS-PVP-PEO was supplied from Polymer Source Inc, and used as received. The
number-averaged molecular weights of each block are 14100 (PS), 12300 (PVP), and
35000 (PEO). SDS w
56
3.2.2. Preparation of Micelle
ired value of apparent degree of neutralization (DN), which is
defined by:
.2.3. DLS Measurements
Hydrodynamic radius (Rh) was
is Boltzmann constant, T the absolute temperature, and η the solvent
iscosity.
First, known amount of PS-PVP-PEO was dissolved in dilute hydrochloric acid
(0.01 M). Then, this solution was heated at 65°C for two hours. The solution was cooled
and transferred into a volumetric flask to give the final concentration of 0.1 g/L. The
solution was stirred slowly for several days until clear solution was obtained (stock
solution). The final pH of the stock solution was around 2. The micellar solutions were
obtained by diluting the stock solution with water. Desired pH was obtained with 0.1M
sodium hydroxide. Known amounts of SDS were added into the PS-PVP-PEO micellar
solutions to get a des
DN(%) = (1)
(Amount of pyridine in mole unit).
(Amount of SDS in mole unit) ×100
All these experiments were performed below the critical micelle concentration
(CMC) of SDS (CMC = 8×10-3M). All the solutions were prepared using water
purified with Millipore MiliQ system. The solution of SDS/PS-PVP-PEO was
transparent over the whole ranges of the degree of neutralization.
3
DLS measurements were carried out with Otsuka ELS-800 apparatus at fixed 90˚
scattering angle. Correlation functions were analyzed by a histogram method and used
to determine the diffusion coefficient (D) of the samples.
calculated from D by using the Stokes-Einstein equation:
Rh =kBT/(6πη D), (2)
Where kB
v
57
3.2.4. AFM Measurements
repared by evaporating a drop of the micellar
solution on freshly cleaved mica in air.
.2.5. Zeta-potential Measurements
-potential of the particles was calculated from the
EPM using Smoluchowski equation:
where µE is EPM, ζ the zeta-potential, ε the permittivity of liquid, and η the viscosity.
3.3. Results and Discussion
.3.1. Hydrodynamic Diameter
ynamic diameter is reached at 40 % DN and afterwards the size is not
muc
AFM images were obtained in dynamic force mode with a SPA 300 unit together
with a SPI 3700 control station (Seiko Instruments Industry, Co., Japan) in the air. The
samples for AFM observations were p
3
The measurements of electrophoresis mobility (EPM) were performed at 25°C with
Otsuka ELS-8000 apparatus. The zeta
µE=ζε/η (3)
3
Figure 1 shows the dependence of the hydrodynamic diameters (2Rh) of the
SDS/PS-PVP-PEO nanoaggregates on DN at various pH in acidic conditions. When we
add SDS to the PS-PVP-PEO micelle, the hydrodynamic diameter decreases with
increasing amount of SDS (that is, DN), and finally reaches minimum at 100 % DN.
Further addition of SDS does not bring about significant change in the value of
hydrodynamic diameter. If we look at the change more closely, we note the difference in
the effect of pH. At pH 2 and 3, the hydrodynamic diameter decreases from 216 nm to
175 nm on increasing DN from 0 % to 100 %. After 100 % DN, almost the same value of
hydrodynamic diameter (ca.175 nm) is observed both at pH 2 and 3. At pH 4, however, the
minimum hydrod
h changed.
The change of hydrodynamic diameter can be attributed to the morphological
58
change of PVP block. When the pH of the solution is 2 and 3, PVP block has
completely cationic form in the absence of SDS, and has extended conformation due to
the repulsion between positive charges of pyridine unit. When we add SDS to the
micellar solution, the positive charge of pyridine unit is neutralized with sulfate group
of SDS bound to the polymer. Thus the repulsion between the cationic pyridine units is
decreased and the solubility of PVP block is also decreased, resulting in the
conformational change of PVP block from extended to shrunken forms, which in turn
ggregates of SDS/PS-PVP-PEO on DN. Concentration of PS-PVP-PEO: 0.025 g/L.
175
200
225
gives the decreased hydrodynamic diameter.
175
200
225
0 50 100 150 200175
200
Diam
eter
/ nm
DN/ %
(a) pH 2
(b) pH 3
(c) pH 4
Fig.1. Dependence of hydrodynamic diameter of the a
59
It should be noted here that the change in the hydrodynamic diameter from 216 nm to
175 nm (the size change: 41 nm) at pH 2 and 3 is not surprising because the chain
length of the PVP block is calculated to be 80 nm. The contribution of the PVP block
to the hydrodynamic diameter is 160 nm under the assumption of fully extended
structure, and this value is much larger than the change in the size of the micelle. The
micelle has a hydrodynamic diameter of 216 nm at pH 2 and 3 in the absence of SDS.
However, the micelle has different hydrodynamic size of 192 nm at pH 4 in the absence
of SDS, because PVP block is partially protonated and it has somewhat shrunken form.
Hence, addition of SDS at this pH gave different behavior of morphological changes as
compared to that at pH 2 and 3. The hydrodynamic size reaches the minimum value at
the earlier stage of DN (i.e. 40 % DN) in sharp contrast to the cases of pH 2 and 3,
where the minimum values are attained at 100 % DN.
3.3.2. AFM
To obtain further evidence of the morphological change, we carried out AFM
measurements (Fig. 2).
(b) (a)
Fig. 2. AFM images of the micelles of PS-PVP-PEO (a) and SDS/PS-PVP-PEO (100 % DN) (b) at pH 2.
60
Figure 2a shows the AFM image of PS-PVP-PEO micelle at pH 2. The micelle has an
average diameter of 150 nm in the absence of SDS. In the presence of SDS at the level
of 100 % DN, we obtained an average diameter of 110 nm (Fig. 2b). This gives a
strong support for the morphological change of PS-PVP-PEO micelle by addition of
SDS.
3.3.3. Zeta-Potential
In order to confirm the binding of SDS to the micelle of PS-PVP-PEO, we carried
out zeta-potential measurements. Figure 3 represents a plot of zeta-potential of the
complexes as a function of DN at various pHs in acidic conditions. The increase in DN
resulted in continuous decrease in zeta-potential values: from 13 to 0 mV at pH 2 and
from 11 to 0 mV at pH 3 by successive addition of SDS. Importantly, at 100 % DN,
the zeta-potential was 0 mV at pH 2 or pH 3, indicating all the positive charge of
pyridine unit is completely neutralized by anionic SDS. Consequently, electroneutral
complex of SDS/PS-PVP-PEO is obtained. This fact indicates one-to-one stoichiometric
binding of SDS to the pyridine unit of the PVP block at these pH.
Below the 100 % DN, the zeta-potential of PS-PVP-PEO and its complex with
SDS have a little different value between pH 2 and pH 3. The zeta-potential is 8 mV at
pH 2 and 4.72 mV at pH 3 when DN is 60 %. Such difference is found even in 0 %
DN, where zeta-potential is 13 mV at pH 2 and 11 mV at pH 3 due to direct
proportionality of zeta-potential on charge density which itself is pH dependent. In the
case of pH 4, however, the zeta-potential greatly differs from that in pH 2 or pH 3: even
in 0 % DN, the zeta-potential is 2 mV. This indicates that the PVP block has extremely
low positive charge at pH 4. This is understood by the fact that deprotonation of the
pyridine groups occurs at pH 3.5 when we increase the pH of the solutions. Thus, the
pyridine unit of PVP block is partially protonated at pH 4. As a result, at pH 4, we
observed the zero zeta-potential at 40 % DN, in sharp contrast to the cases of pH 2 and
pH 3 where zero zeta-potential is observed at 100 % DN.
It is interesting to note that zeta-potential remains constant after 100 % DN at pH 2
and pH 3, and also after 40 % DN at pH 4. This implies that SDS is not incorporated
61
into the electroneutral complex of SDS/PS-PVP-PEO.
0
4
8
12
0
4
8
12
0 50 100 150 2000
4
8
12
DN/%
ζ / m
V
(a) pH 2
(b) pH 3
(c) pH 4
Fig. 3. Dependence of zeta-potential of the aggregates of SDS/PS-PVP-PEO on DN. Concentration of PS-PVP-PEO: 0.025 g/L.
62
This may be surprising because SDS is known to bind to PEO by hydrophobic
interaction. However, the concentration of SDS is about 0.097 mM at 200 % DN which
is too low for SDS to bind to the PEO chain. It is also interesting to compare the
behavior of hydrodynamic diameter (Fig. 1) with that of zeta-potential (Fig.3). If we
look at the hydrodynamic diameter at pH 2 and pH 3, it decreases slowly when DN is
increased from 0 % to 50 %, but drastically decreases after that. In contrast,
zeta-potential shows monotonous decreases over the DN range from 0 % to 100 %.
This difference implies that some critical value of deprotonation exists for the structural
change of the PVP block to occur. A similar example was reported for the
conformational transition between extended and compact forms in polymethacrylic acid
in aqueous solutions [27].
Finally, we mention the salt effect due to the addition of hydrochloric acid and
sodium hydroxide. To examine it, we prepared two kinds of aqueous micellar solutions
at pH 2 and pH 3: no sodium chloride was added in one kind of solutions, and sodium
chloride was added at the concentration of 0.097 mM in the other kind of solutions. The
concentration of 0.097 mM was selected because it corresponds to 200 % DN when the
polymer concentration is 0.025 g/L. We obtained similar hydrodynamic diameters as
well as zeta potentials in the two kinds of solutions both at pH 2 and pH 3. Therefore,
we can safely conclude that the salt effect is negligible in the present study.
3.4. Conclusion
We have succeeded in inducing morphological change of PS-b-PVP-b-PEO
micelles by binding SDS to the PVP block at various pHs in acidic conditions. The
change of morphology was detected by DLS measurements and AFM as indicated by
the decreased micelle diameter. The incorporation of SDS was confirmed by
zeta-potential measurements as a decrease in positive zeta-potential down to zero mV.
This is the first example of the morphological change of PS-b-PVP-b-PEO micelle that
is brought about by the binding of surfactants to the protonated pyridine units.
63
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V. Kabanov, Langmuir, 1998, 14, 6101.
19. A.V. Kabanov, T.K. Bronich, V.A. Kabanov, K. Yu, A. Eisenberg, J. Am.
Chem. Soc., 1998, 120, 9941.
20. T.K. Bronich, A.M. Popov, A. Eisenberg, V.A. Kabanov, A.V. Kabanov,
Langmuir, 2000, 16, 481.
21. L. Bronstein, M. Sedlak, J. Hartmann, M. Breulmann, H. Cölfen, M.
Antonietti, Polym. Mater. Sci. Eng., 1997, 76, 54.
22. L.M. Bronstein, S.N. Sidorov, A.Y. Gourkova, P.M. Valetsky, J. Hartmann,
M. Breulmann, H. Cölfen, M. Antonietti, Inorg. Chim. Acta., 1998, 280, 348.
23. L. Bronstein, M. Antonietti, P. valetsky, Nanopart. Nanostruct., 1998, 145.
24. S.N. Sidorov, L.M. Bronstein, P.M. Valetsky, J. Hartmann, H. Cölfen, H.
Schnablegger, M. Antonietti, J. Colloid Interface Sci., 1999, 212, 197.
25. J.F. Gohy, S.K. Varshney, S. Antoun, R. Jerome, Macromolecules, 2000, 33,
9298.
26. L.M. Bronstein, S.N. Sidorov, P.M. Valetsky, J. Hartmann, H. Cölfen, M.
Antonietti, Langmuir, 1999, 15, 625.
27. K. Nakashima, Y. Fujimoto, T. Anzai, J. Dong, H. Sato, Y. Ozaki, Y. Bull.
Chem. Soc. Jpn., 1999, 72, 1233.
65
(Chapter 4)
Physicochemical Properties of Micelles of Polystyrene-b-Poly(2-vinylpyridine)-b-Poly(ethylene oxide) in Aqueous
Solutions
Micelles of polystyrene-b-poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PS-b-
P2VP-b-PEO) triblock copolymer were prepared in aqueous solutions. The
physicochemical properties of the micelles were investigated by dynamic light
scattering, zeta-potential measurements, atomic-force microscopy, and fluorescence
spectroscopy. The micelles had an average diameter of about 200 nm under acidic
conditions. The addition of dextran sulfate to a micellar solution under acidic
conditions resulted in a significant decrease in the micelle size. This was due to a
conformational change in the P2VP block form extended to shrunken forms after the
cationic P2VP block was electrically neutralized with negative dextran sulfate. The
release of anionic dye (eosin Y) from the micelle particles was studied by a dialysis
method, and proved to take tens of hours.
4.1. Introduction
Recently, polymeric micelles from double hydrophilic block copolymers in
aqueous solutions have attracted much attention [1-5]. In these micelles, complexation
with a substrate through an electrostatic interaction or a coordination reaction can
reverse the hydrophilicity of one block into hydrophobicity, resulting in forming the
micelle core. For example, poly(ethylene oxide-b-methacrylate) (PEO-b-PMA) forms
micelles if the PMA block is insolubilized in water by complexation with cationic
surfactants or metal ions[4,5]. One of the advantages of double hydrophilic block
copolymer micelles, compared with conventional polymeric micelles formed from
66
amphiphilic block copolymers[6], is that they can incorporate ionic species into the core
domain. This property is especially important when polymeric micelles are employed as
nano-reactors for ionic reactions, or as carriers for ionic drugs[1].
From the viewpoint of delivery systems for ionic drugs, another property is
required in vivo: i.e. the micelle should not collapse after being highly diluted in the
blood stream. For such a requirement, there seem to be two hopeful ways. One is to
employ polymeric micelles with a cross-linked core[7-9], and the other is to use frozen
micelles[10,11], in which the exchange of unimers between the micelle and the
aqueous bulk phase takes a long time scale of hours or more. In this respect, polymeric
micelles having both ionic and frozen inner layers are anticipated. This attempt seemed
to be achieved by the use of ABC type triblock copolymers, in which the A and B
blocks formed a frozen core and an ionic shell, respectively, and the C block formed
a corona.
Very recently, three groups investigated core-shell-corona micelles of the poly
styrene-b-poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PS-b-P2VP-b-PEO) triblock
copolymer, which seems to fulfill the requirements described above[12-15]. Gohy et al.
reported on a pH-sensitive morphological change in the core-shell-corona aqueous
micelles of the PS-b-P2VP-b-PEO triblock copolymer[12,13]. According to them, the
volume of the P2VP shell is significantly increased at a pH lower than 5, because this
block is protonated at a low pH, resulting in an extension of the P2VP chain due to a
repulsion between protonated pyridyl groups. Stepanek et al. studied the micellization
behavior of the PS-b-P2VP-b-PEO triblock copolymer, mainly using fluorescence
correlation spectroscopy (FCS), and demonstrated that the micelle was easily
transformed to the secondary aggregates of the micelle[14]. In their experiments, the
secondary aggregation was provoked by stirring, shaking, and filtration of micellar
solutions. They stressed the advantage of FCS compared to light-scattering techniques
because the former does not need a filtration process. Khanal et al. found that the PS-
P2VP-PEO micelle undergoes a significant size change by adding sodium dodecyl
sulfate (SDS) under acidic conditions[15]. The diameter of the micelle decreases with
increasing SDS concentration. They ascribed the decrease in the diameter to a
conformational change in the P2VP block from extended to shrunken structures. The
67
binding of anionic SDS to the protonated P2VP block decreases the repulsion between
the P2VP chains, resulting in a shrinkage of the P2VP domain.
In the present work, we investigated the physicochemical properties of the PS-
b-P2VP-b-PEO micelle, while focusing on the ionic interactions in the P2VP shell. We
found by dynamic light scattering (DLS) measurements that the PS-b-P2VP-b-PEO
micelle showed a significant size change by incorporating dextran sulfate into the P2VP
shell. From dialysis, followed by Vis-absorption spectroscopy, we found that the
retention of an anionic dye (Eosin Y) in the micelle continued for at least tens of hours.
4.2. Experimental Section
4.2.1. Materials
PS-b-P2VP-b-PEO (Polymer Source Inc.) was used as supplied. The molecular
weights was nM (PS) = 14100, nM (PVP) = 12300, and nM (PEO) = 35000. The
distribution of the molecular weights was: WM / nM = 1.04 for PS, 1.06 for PS-b-P2VP,
and 1.08 for PS-b-P2VP-b-PEO. Pyrene (Py, Aldrich) was purified by vacuum
sublimation. Naphthalene (N, zone-refined) from TCI was used as supplied. Sodium
dextran sulfate (DS, Wako, MW=5000) and Eosin Y (EY, Wako) were used without
further purification.
4.2.2. Sample Preparation
A weighed polymer sample was dissolved in an acidic aqueous solution, and then
heated at 65 ˚C for ca. 2 h. After being cooled, the solution was transferred to a
volumetric flask to give a final concentration of 0.1 g L-1. The solution was agitated
with a magnetic stirrer at room temperature for several days until a clear solution was
obtained (stock solution). The pH of the thus obtained stock solution was ca. 3. A
known amount of the stock solution was transferred to a 10 mL volumetric flask. Then,
a fluorescence probe and/or other chemicals were added to the flask as needed, followed
by dilution with neutral, acidic or alkaline water to give a desired concentration. The
sample solutions were kept at room temperature for one night before the measurements.
68
4.2.3. Characterization of the Micelle
DLS was measured with an Otsuka ELS-800 at a fixed 90˚ scattering angle. The
hydrodynamic diameter (Dh) was obtained by a cumulant method. The zeta-potential
of the particles was calculated based on the electrophoresis mobility, which was
measured with an Otsuka ELS-8000 apparatus. Fluorescence spectra were recorded on
a Jasco FP-6500 spectrofluorometer. Absorption spectra were recorded on a Jasco
Ubest-50 spectrophotometer. Atomic force microscopy (AFM) images were obtained in
dynamic force mode ( corresponding to the tapping mode ) with a SPA 300 unit
together with a SPI 3700 control station (Seiko Instruments Industry Co. Japan) in air.
The samples for AFM observations were prepared by evaporating a drop of the micellar
solutions on freshly cleaved mica in air.
4.3. Results and Discussion
4.3.1. Detection of Micelle Formation
The micelle formation of the same polymer has already been described in the
literature [12-14]. However, we tried to detect micelle formation in our own sample,
because the sample preparation procedure was different from that in the literature.
Figure 1 shows Dh of the micelles as a function of the polymer concentration at pH 3. A
constant diameter of about 185 nm was obtained when the polymer concentration was
above 0.005 g L-1. The polydispersity of the micelle diameter was about 0.1 in the
analysis by the cumulant method. It should be noted that the diameter in a low polymer
concentration region (c < 0.005 gL-1) was somewhat smaller than that in a high-
concentration region. The reason is currently unclear. One of the possible reasons is
that the reliability of the measurements is comparatively low in this region, due to low
scattering intensity.
Micelle formation was also confirmed by an analysis of the vibronic fine structure
of pyrene fluorescence (Fig. 2). It is well known that the polarity of the
microenvironment of pyrene can be probed by the I1/I3 ratio in its fluorescence
69
spectrum[16,17]. A decrease in the I1/I3 ratio with increasing polymer concentration
indicates a preferential solubilization of pyrene into the micelles. The I1/I3 ratio at the
polymer concentration above 0.02 g/L is 1.3, which is an intermediate value between
those in PS film (1.05) and P2VP film (1.49)[18]. This fact implies that pyrene is
located at the interface between the PS and P2VP layers. Micelle formation was also
confirmed by AFM measurements. Figure 3 represents an AFM image for a sample at
pH 3. The micelles were close to spherical particles with average diameters of 130 nm
at pH 3. In DLS measurements, we obtained a diameter of 185 nm at pH 3. The average
diameter in AFM measurements was smaller than that in DLS. This is reasonable
because AFM yields a number-averaged diameter, whereas DLS yields a z-averaged
diameter. The AFM data provide strong evidence for micelle formation.
It should be noted here that the micelle size is not well coincident with those
reported by Gohy et al.[12,13] and Stepanek et al.[14]. For example, Gohy et al.
obtained Dh = 69 nm at pH>5 for the same polymer as ours. The difference among the
groups seems to be due to the fact that the size of a frozen micelle is strongly dependent
on the sample-preparation procedure, because the micelle formation equilibrium was
freezed; that is, the micelles were not in the thermodynamical equilibrium states in
every case. We dissolved the sample directly into water, while Gohy et al. prepared it
by dialysis of the organic solution against water.
I 1/I 3
Dia
met
er/ n
m
Concentration/gL-1
Fig. 1. Hydrodynamic diameter of the micelles (○) and I1/I3 ratio of pyrene (●) as a
function of the polymer concentration at pH 3. The zero value in the diameter at low
polymer concentrations (c < 0.002 gL-1) indicates that the scattering intensity was too
weak to give reliable hydrodynamic diameter.
70
0
100
200
300
400
500
600
350 400 450 500
Wavelength/nm
Inte
nsity
/a.u
No Conc./g L-1
0 01 0.00052 0.0013 0.0024 0.0035 0.0046 0.0057 0.0088 0.019 0.0210 0.0311 0.05
11
0
І
III
Fig. 2. Fluorescence spectra of Py in micellar solutions of PS-b-P2VP-b-PEO at pH 3.
Concentration of Py is fixed at 0.6 µM. Py is excited at 335 nm. Band widths are 5 nm
and 1 nm in excitation and emission sides, respectively.
Fig. 3. AFM image of the micelles of PS-b-P2VP-b-PEO (0.025 g L-1) at pH 3.
71
4.3.2 Incorporation of DS
We were interested in incorporating of DS into the micelle particles of PS-b-P2VP-b-
PEO, because DS is known to have anticoagulant activity [19]. The incorporation of
DS was monitored by the change in the micellar size, since we found in a previous
study that the PS-b-P2VP-b-PEO micelle shows a significant decrease in size at pH<5
if the protonated P2VP block is electrically neutralized with an anionic surfactant,
sodium dodecyl sulfate (SDS)[15]. The decrease in the size of the PS-b-P2VP-b-PEO
micelle is explained by a conformational change of the P2VP block from extended to
shrunk forms. Therefore, we expected that we could observe a similar change when
DS was used instead of SDS. The change in the micellar size is schematically shown
in Fig. 4. To express the amount of added DS, we introduced an apparent degree of
neutralization (DN), which is defined by:
(Amount of sulfate group in mole unit) ×100
(Amount of pyridyl group in mole unit)
DN（%）= .
Figure 5a shows the dependence of Dh of the DS/PS-b-P2VP-b-PEO nanoaggregates
on DN at pH 3. When we added DS to the PS-b-P2VP-b-PEO micelle, the hydrodynamic
diameter decreased with increasing amount of DS (i.e. DN), and finally reached a
minimum value at 100% DN. Further addition of DS did not bring about a significant
change in Dh. It should be noted here that Dh at DN = 0% is 205 nm, which is significantly
larger than that in Fig. 1. The difference seems to originate from the fact the micelle has a
frozen nature due to a glassy PS core[10,11]. It is known that frozen micelles are in the
states of the local minima of the Gibbs free energy, and the transition from the local
72
minima to the real minimum is kinetically frozen[20]. Therefore, it is not surprising that
the micelle size differs from sample to sample if they are prepared in a different batch.
pH < 5
PS PVP PEO
Single-layered-core micelle
×-×-× (DS )
Double-layered-core micelle
Fig. 4. Schematic illustration of morphological change in the micelle of PS-b-P2VP-
b-PEO induced by addition of DS.
73
160
170
180
190
200
210
0 50 100 150 200
DN / %
Dia
met
er /n
m
ζ
/ m
V
-202468
1012
0 50 100 150 200
DN /%
(b)
Fig. 5. Dependence of hydrodynamic diameter (a) and zeta-potential (b) of DS/PS-b-
P2VP-b-PEO on DN (i.e. added DS) at pH 3. Concentration of PS-b-P2VP-b-PEO is
fixed at 0.025 g/L.
In order to confirm the binding of DS to the micelle of PS-b-P2VP-b-PEO, we
carried out zeta-potential measurements. Figure 5b represents a plot of the zeta-
potential of the complexes as a function of DN at pH 3. The successive addition of DS
resulted in a continuous decrease in the zeta-potential from 11 to 0 mV. Importantly, at
74
100% DN, the zeta-potential was 0 mV, indicating that the positive charge of the
pyridine unit was completely neutralized by anionic DS. The zeta-potential was not
decreased after DN was 100%, indicating that no further binding of DS occurred. These
facts indicate that the interaction between the PS-b-P2VP-b-PEO micelle particle and
DS is dominated by an electrostatic interaction, and that one-to-one stoichiometric
binding of the sulfate group of DS to the pyridine unit of the P2VP block takes place.
4.3.3. Release of Organic Dye
The release of a small organic molecule from the PS-b-P2VP-b-PEO micelle is
interesting in view of the controlled release of medicine. We carried out an experiment
by a dialysis method [21], using EY as a representative of small organic molecules.
Because EY is an organic dye with a carboxylic group, it takes an anionic form under
basic conditions. EY was incorporated into the micelles of PS-b-P2VP-b-PEO in an
aqueous solution. The initial concentrations of EY and PS-b-P2VP-b-PEO in the
micellar solution were 15 µmol/L and 0.01 g/L, respectively. These concentrations
correspond to 80% DN under the assumption that the carboxylic group of EY and the
pyridyl group of P2VP are fully ionized. A dialysis bag containing 10 mL of the micellar
solution was put into 300 mL of water, which had a known pH adjusted with HCl or
NaOH. Then, the concentration of EY inside the dialysis bag was monitored at an
adequate time interval by visible absorption spectroscopy.
Figure 6 shows the change in the absorbance of EY at pH 3 as a function of time.
The concentration of EY inside the dialysis bag seems to decrease exponentially.
Therefore, we tried to analyze the curve by a kinetic model, as shown in Scheme 1:
Scheme 1.
K1
DW1 K2
DW2 , DM
where DM stands for a dye molecule (i.e. EY) incorporated into the micelle. DW1 and
DW2 denote the dye molecule in aqueous bulk phases inside and outside of the dialysis
bag, respectively; k1 is a rate constant for the dye to exit from the micelle to the
75
aqueous phase inside the dialysis bag, and k2 is a rate constant for the dye to diffuse
from the inside to the outside of the dialysis bag across the membrane. For simplicity,
we assume that the first step in Scheme 1 is the rate-determining step (i.e. k1 << k2).
Then, we obtain a first-order kinetic equation,
d ([DM] t /dt = k1 [DM]t, (2)
where [DM]t indicates the molar concentration of DM at time t. The solution of this
equation is
[DM]t = [DM]0 exp(-k1t). (3)
As we monitored, the absorbance (A) of the dye molecules inside the dialysis bag,
the absorbance at time t, is given by
At = ε ([DM]t + [DW1]t ) , (4)
where ε stands for the molar extinction coefficients of the dye. Under the
assumption that the first step in Scheme 1 is the rate-determining step, [DW1]t in Eq.4 is
negligible (i.e. [DW1]t = 0). From Eqs.3 and 4, we obtain:
At = ε [DM]t = ε [DM]0 exp(-k1t), (5)
ln (At/ A0) = - k1t, (6)
where A0 is the absorbance at time 0 (i.e. A0 = ε [DM]0) .
0.00
0.05
0.10
0 10 20 30 40 50 60 70 80
Dialysis time/h
Abs
orba
nce
Fig. 6. Dependence of the absorbance of EY on dialysis time at pH 3.
76
In Fig. 7, ln (At/A0) is plotted against t. The plot gives a rate constant of 0.0387 h-1,
although the plot deviates from a straight line to some extent. Here, we define the
release time (τ) as the reciprocal of k. Then, we obtain τ = 25.8 h. The obtained release
time indicates that the retention of EY in the micelle is long enough for the micelle to be
employed for a controlled release. Therefore, the results imply potential applications of
this type of core-shell-corona micelles to drug-delivery systems.
-2.5
-2
-1.5
-1
-0.5
0
0 10 20 30 40
Time/h
ln A
t / A
0
50
Fig. 7. Plot of ln(At /A0) against dialysis time. This plot is based on the data in Fig. 6.
4.4. Conclusions
We have revealed that the micelle of the PS-b-P2VP-b-PEO triblock copolymer
has an ability to incorporate ionic molecules into the inner layers of the micelle particle.
77
It was also shown that the release time of EY from the micelle particles is on the order
of 10 h. Although the PS-b-P2VP-b-PEO micelle may not be suitable for clinical use,
because of a possible toxicity of the P2VP block, the strategy of the present study will
be applicable to developing practical drug carriers by replacing the P2VP block with a
cationic biocompatible polymer.
4.5. References
1. H. Cölfen, Macromol. Rapid Commun., 2001,22,219.
2. A.V. Kabanov, T.K. Bronich, V.A. Kabanov, K. Yu, A. Eisenberg, J. Am.
Chem. Soc., 1998, 120, 9941.
3. A. Harada, K. Kataoka, Science, 1999,283, 65.
4. Y. Li, Y.-K. Gong, K. Nakashima, Y. Murata, Langmuir, 2002,18, 6727.
5. Y. Li, K. Nakashima, Langmuir, 2003, 19, 548.
6. Z. Tuzar, P. Kratochvíl, Surface and Colloid Science, Plenum Press: New York,
1993, Chapter 1.
7. A. Guo, G. Liu, J. Tao, Macromolecules, 1996, 29, 2487.
8. R.S. Underhill, G. Liu, Chem. Mater., 2000, 12, 3633.
9. Y. Kakizawa, A. Harada, K. Kataoka, J. Am. Chem. Soc., 1999,121, 11247.
10. Y. Wang, R. Balaji, R.P. Quirk, W. L. Mattice, Polym. Bull., 1992, 28, 333.
11. R. Xu, M.A. Winnik, G. Riess, B. Chu, M.D. Croucher, Macromolecules,
1992, 25, 644.
12. J.F. Gohy, N. Willet, S. Varshney, J.X. Zhang, R. Jérôme, Angew. Chem.
Int. Ed., 2001, 40, 3214.
13. J.F. Gohy, N. Willet, S. Varshney, J.X. Zhang, R. Jérôme, e-Polymer,2002,
no. 035.
14. M. Stepanek, J. Humpolickova, K. Prochazka, M. Hof, Z. Tuzar, M.
Spirkova, T. Wolff, Collect. Czech. Chem. Commun., 2003, 68, 2120.
15. A. Khanal, Y. Li, N. Takisawa, N. Kawasaki, Y. Oishi, K. Nakashima,
Langmuir, 2004, 20, 4809.
16. D.C. Dong, M.A. Winnik, Photochem. Photobiol., 1982,35, 17.
17. K. Kalyanasundaram, J.K. Thomas, J. Am. Chem. Soc., 1977, 99, 2039.
78
18. K. Nakashima, M.A. Winnik, K.H. Dai, E.J. Kramer, J. Washiyama,
Macromolecules, 1992, 25, 6866.
19. Y. Tajima, Biomed. Res. Trace elements, 2002, 13, 46.
20. P. Munk, “Solvents and Self-Organization of Polymers,” ed. by S. E. Webber, P.
Munk, and Z. Tuzar, Kluwer Academic Publishers, Dordrecht,1995, pp. 19-32.
21. N. Nishiyama, M. Yokoyama, T. Aoyagi, T. Okano, Y. Sakurai, K.
Kataoka, Langmuir, 1999,15, 377.
79
(Chapter 5)
Fabrication of Organic-Inorganic Nano-complexes Using
ABC Type Triblock Copolymers and Polyoxotungstates
New organic-inorganic nano-complexes were produced from a micelle of tri-block
polymer, polystyrene-b-poly (2-vinylpyridine)-b-poly (ethylene oxide) (PS-PVP-PEO)
and tungsten compounds such as tungstate (W12-), undecatungstophospate (PW11
7-) and
undecatungstosilicate (SiW118-) in acidic aqueous solutions. The size and morphology of
the complexes were characterized by measurements of dynamic light scattering, atomic
force microscopy, and scanning electron microscopy. This complex is assembled mainly
by the charge interaction between the inorganic polyanions and the positively charged
PVP block in the PS-PVP-PEO molecule, which was confirmed by zeta-potential and
fluorescence spectroscopic studies. In the absence of the inorganic anions, the zeta-
potential of the micelle was +11 mV at pH 3 due to the positive charge of the PVP block.
When the inorganic anion was mixed with the PS-PVP-PEO micelle, decrease in the
zeta-potential due to charge neutralization occurred with incorporation of inorganic
anions into the PS-PVP-PEO micelle. The minimum zeta-potential was 0, -33, and -35
mV for W12-/PS-PVP-PEO, PW11
7-/PS-PVP-PEO, and SiW118-/PS-PVP-PEO complexes,
respectively. Excess in negative charge occurred in the latter two complexes indicates
that PS-PVP-PEO molecules bound PW117- and SiW11
8- by forces other than charge
interaction. In addition, the incorporation of an inorganic polyanion into the micelle
gave a new morphology of the micelle. In the absence of the polyanion, the PS-PVP-
PEO micelles showed an extended conformation due to repulsive forces working among
the positively charged PVP blocks. Addition of polyanion induced shrunken forms of
the micelles, since the charge repulsion was cancelled by the polyanion. This feature
may be useful to develop a new type of functioning micelle.
80
5.1. Introduction
The synthesis of chemically and physically well-defined nano-particles is a great
interest in various fields of science: e.g., applications of nano-sized materials have been
eagerly investigated in pharmacology, bioengineering, medical chemistry, and industry
such as ceramics. Making a polymeric micelle is one of the promising ways in nano-
technologies, and several different methodologies using chelating agents [1-4], ionic
substances [5, 6], or surfactant [7-12] have been developed to control the size and
morphology of the nano-particles for the past few decades.
Nowadays, various hybrid micelles composed by organic and inorganic materials
are fabricated to break fresh ground of nano-science [13-18]. However, only a quite few
products using ABC type triblock copolymers have been known until today [19, 20].
Recently, Jérôme et al. reported that a gold salt was incorporated into chemically well-
defined “core-shell-corona”-type micelles which were made by polystyrene-b-poly(2-
vinylpyridine)-b-poly(ethylene oxide) (PS-PVP-PEO) triblock copolymers in aqueous
solution [19, 20]. Structurally, these micelles have a “frozen” PS core, positively
charged PVP shell as an intermediate layer, and outer PEO corona under acidic
conditions [19-23]. The whole micelle structure showed an extended conformation at
pH < 5 due to the repulsive forces working among positively charged PVP blocks.
Addition of some anionic substances into this micelle cancelled the repulsive forces
electrically, and therefore the PVP block turned insoluble to water. This process induced
a conformational change of the micelle from an extended form to shrunken one.
By using this feature of PS-PVP-PEO micelle binding anionic substances, we can
fabricate novel organic-inorganic hybrid nano-complexes. In this study, inorganic
polyanions such as undecatungstophosphate ([PW11O39]7-, PW117-) and
undecatungstosilicate ([SiW11O39]8-, SiW118-) were used as a counteranion. These two
compounds are well known as “Keggin”-structural polyoxotungtates (POTs) with many
biochemical activities [24] such as a photocatalyst, anti-viral agent [25, 26], and
anticoagulant [27, 28]. Recently, it was found that POTs have a strong sensitizing effect
on methicillin-resistant Staphylococcus aureus to β-lactam antibiotics [24, 29-32].
81
Therefore, some new functioning micelles can be formed from such a bioactive material
by using molecular hybrid techniques. The nano-complexes thus prepared were
characterized by measurements on zeta-potential, dynamic light scattering (DLS),
atomic force microscopy (AFM), and scanning electron microscopy (SEM).
5.2. Experimental
5.2.1. Materials
PS-PVP-PEO was supplied from Polymer Source Inc. (Dorval, Canada), and used
without further purification. The number-averaged molecular weights of PS, PVP, and
PEO block are 14100, 12300, and 35000, respectively. Sodium tungstate (Na2WO4, W12-
), dodecatungstophosphoric acid (H3[PW12O40], PW12) and dodecatungstosilicic acid
(H4[SiW12O40], SiW12) were obtained from Wako Pure Chemicals (Osaka, Japan).
Highly purified water by Millipore MiliQ system was used throughout the experiments.
PW117- and SiW11
8- were prepared by partial hydrolysis of PW12 and SiW12, and they
were purified by recrystalization prior to the experiments [29-33].
5.2.2. Preparation of Organic-Inorganic Nano-complexes
After PS-PVP-PEO was dissolved in 0.01M HCl, the mixture was heated at 65oC
for 2 hours. The solution was cooled and the concentration was adjusted 0.1 g/L with
water. The solution was slowly stirred for several days until the appearance turned to
clear (stock solution, pH= ～2). Working solutions to make micelles were prepared by
diluting the stock solution with water, and pH was adjusted to a desired level by adding
0.1M NaOH, if necessary.
Upon adding tungsten compounds to the PS-PVP-PEO micelles, it is convenient if
the amount of added compound is expressed by charge basis. Thus, a parameter called
“apparent degree of neutralization (ADN)” was tentatively defined by the following
formula:
82
ADN(%) =
(Normality of pyridine unit).
(Normality of tungsten compounds) ×100
The numerator was calculated by (molarity of added anionic compound) x (anionic
valence): e.g., 1 mM PW117- is expressed as (1 x 7 =) 7 mN. The denominator was
estimated from the pyridine content in PS-PVP-PEO micelles.
All the solution used in the experiments (i.e., tungsten compounds and PS-PVP-
PEO micelles) were stable and transparent under the experimental conditions for several
weeks.
5.2.3. Zeta-Potential Measurements
Electrophoretic mobility (EPM) of the test sample was measured at 25°C with
ELS-8000 (Otsuka, Tokyo, Japan). The zeta-potential of the sample was calculated from
the EPM using the following Smoluchowski’s equation:
µE = ζ ε / η ,
where µE is EPM, ζ the zeta-potential, ε the permittivity of solvent, and η the viscosity
of solvent.
5.2.4. Dynamic Light Scattering (DLS) Measurements
DLS was measured with ELS-800 (Otsuka, Tokyo, Japan) at fixed 90˚ scattering
angle. Correlation functions were analyzed by a histogram method and used to
determine the diffusion coefficient (D) of the nano-complexes in the test sample.
Hydrodynamic radius (Rh) was calculated from D by using the following Stokes-
Einstein’s equation:
Rh = kB T / (6 πη D),
83
where kB is Boltzmann constant, T the absolute temperature, and η the solvent
viscosity.
5.2.5. Atomic Force Microscopy (AFM)
AFM images were obtained in dynamic force mode (corresponding to the
tapping mode ) with SPA 300 unit and SPI 3700 control station (Seiko Instruments
Industry, Tokyo, Japan) in the room air. The samples for AFM observations were
prepared by evaporating a drop of the test sample on a mica plate freshly cleaved in air.
5.2.6. Scanning Electron Microscopy (SEM)
SEM techniques were carried out with S-5200, Type-SK2 electron microscope
(Hitachi High Technologies, Tokyo, Japan). The samples were prepared by evaporating
a drop of the test sample solution on the glass plate under vacuum conditions.
5.2.7. Fluorescence Spectroscopy
Pyrene (Py) was used as a fluorescent probe. A small portion of a methanol
solution of Py was taken into a volumetric flask, and methanol was gradually
evaporated by heating under nitrogen gas stream. Then, the solution of the PS-PVP-
PEO complexes mixed with tungsten compounds was added, and the final concentration
of Py in the test sample was kept constant at 0.6 µM, which is near to its saturation level
in water at 22° C. Steady-state fluorescence spectra of the air-equilibrated samples (in 1
cm × 1 cm quartz cell) were recorded with FP-6500 fluorescence spectrophotometer
(JASCO, Tokyo, Japan) at right angle geometry. The band widths of the light
wavelength were 3 nm and 1 nm for excitation (334 nm) and emission, respectively.
84
5.3. Results and Discussion
5.3.1. Zeta-Potential of Nano-complexes
Micelles prepared in this report are unique in the following features: (i) This is a
molecular hybrid of both organic and inorganic compounds. (ii) The micelle structure is
maintained by charge interaction in addition to hydrophilic-hydrophobic dynamics. (iii)
POTs with a high anionic valency (-7 ~ -8) present in a small molecular diameter
(~10�) are incorporated in the core part. This may consolidate the structure of the
micelle, and therefore, it is important to know how POTs interact with the micelles.
Fig. 1 shows the effect of counteranion on the zeta-potential of the nano-complexes.
In the absence of the inorganic anions, the zeta-potential of the micelle was +11 mV at
pH 3 due to the positive charge of the PVP block in the PS-PVP-PEO micelle. When
W12- was used as a counteranion (Fig. 1a), the zeta-potential of the micelle was
continuously decreased as ADN increased. However, the zeta-potential did not become
0 mV at 100% ADN, and an excess amount of W12- (ADN = 220%) was required to
cancel the positive charge. This means that: (i) The affinity of W12- to the PVP block
was relatively weak, and only ～50% of W12- could be bound by the PVP block at most.
(ii) Steric hindrance occurred, thereby preventing W12- from binding with the PVP block
effectively. Further addition of W12- (to ～300% ADN) caused almost no change in the
zeta-potential from ～0 mV, which did not turn to negative. This suggests that the
interaction between W12- and the PS-PVP-PEO micelle is mainly maintained by a
charge interaction.
When PW117- (Fig. 1b) or SiW11
8- (Fig. 1c) was used as a counteranion, the zeta-
potential of the micelle was steeply decreased to ～0 mV at 100% ADN. This means
that the affinity of POTs to the PVP block was high, and most of them were bound to
the PS-PVP-PEO micelles. However, additional POTs (100〜800% ADN) caused
further decrease in the zeta-potential, which turned to negative and finally steadied
down -30〜-40mV. Excess in negative charge occurred in these complexes even after
electrical neutralization was completed.
85
0
2
4
6
8
10
12
0 50 100 150 200 250 300
DN / %
ζ / m
V
(a)
-40
-30
-20
-10
0
10
20
0 200 400 600 800
DN / %
ζ / m
V
-40
-30
-20
-10
0
10
20
0 200 400 600 800DN / %
ζ / m
V
(b)
(c)
Figure 1. Dose-dependency of counteranions in zeta-potential of PS-PVP-PEO
micelle. Zeta-potential of PS-PVP-PEO micelles was measured at pH 3 with
counteranions: (a) W12-, (b) PW11
7-, and (c) SiW118-
. Concentration of PS-PVP-PEO
was 0.025 g/L.
86
This indicates that PS-PVP-PEO molecules bound PW117- and SiW11
8- by forces other
than charge interaction. It is well known that POTs can bind PEO to form a quantitative
(but not strictly stoichiometiric) complex, and this property has been applied to
quantification of nonionic detergents [34]. Although the detailed mechanism of the
interaction has not been elucidated yet, it is possible that: (i) The O-atoms in the PEO
block were partially protonated to form oxonium cation, where POTs interacted by
charge. (ii) POTs polarized non-polar part of the PS-PVP-PEO molecule (e.g., the PEO
block) with their very high negative charge and bound the dipoles. (iii) Hydrogen
bonding was made between the O-atoms in the POT molecule and H-atoms in the PS-
PVP-PEO molecule. (iv) The PS-PV molecule formed an inclusion (clathrate)
compound with POTs as a guest like crown ether [35].
5.3.2. Dynamic Light Scattering (DLS) Measurements
In order to measure the change of hydrodynamic diameter of the PS-PVP-PEO
micelles upon charge neutralization, DLS of the micelles was measured. The results are
shown in Fig. 2, and the hydrodynamic diameter (2Rh) of the micelle was found to be
around 200～230 nm at pH 3 in the absence of counteranion.
When W12- was used as a counteranion (Fig. 2a), the hydrodynamic diameter of the
micelle continuously decreased to ～170 nm as ADN increased. This suggests that the
micelle structure was shrunken due to the repulsive forces working among the PVP
blocks were weakened by charge neutralization, as shown elsewhere [19, 21, 22].
However, the diameter did not minimize at 100% ADN, and an excess amount of W12-
(ADN = 220%) was required to do so. This means that the affinity of W12- to the PS-
PVP-PEO micelles was relatively weak and/or steric hindrance occurred, as described
in the former section.
When PW117- (Fig. 2b) or SiW11
8- (Fig. 2c) was used as a counteranion, the
hydrodynamic diameter of the micelle became smaller (120～130 nm) than that of W12-
/PS-PVP-PEO complex. Although W12- induced only ～20% reduction in the micelle
size at the most, PW117- and SiW11
8- gave ～40% reduction.
87
125
145
165
185
205
225
0 200 400 600 800
DN / %
Dia
met
er /
nm
170
180
190
200
210
0 50 100 150 200 250 300
DN / %
Dia
met
er /
nm
(a)
(b)
130
150
170
190
210
230
0 200 400 600 800
DN / %
Dia
met
er /
nm
(c)
Figure 2. Dose-dependency of counteranions in hydrodynamic diameter of PS-PVP-
PEO micelles. Hydrodynamic diameter of PS-PVP-PEO micelles was measured at pH 3
with counteranions: (a) W12-, (b) PW11
7-, and (c) SiW118-
. Concentration of PS-PVP-
PEO was 0.025 g/L.
88
This means that charge neutralization of the micelles strongly occurred when the
micelles were mixed with POTs. In addition, ADN giving the minimum hydrodynamic
diameter on the micelles (350～400%) was almost equivalent to that inducing the
lowest zeta-potential on them (Fig. 1b and Fig. 1c), suggesting that morphological
changes of the micelles were closely related with the charge.
It is noteworthy that the PS-PVP-PEO micelles still bound PW117- and SiW11
8- with
continuously shrinking micelle size even after the zeta-potential turned to negative. This
means that POT molecules were accumulated into the micelles against electrical
repulsion. Although excess in positive charge maintained extended micelle
conformation with a larger hydrodynamic diameter, overloading the negative charge by
POTs rather caused further shrinking of the micelle size. This means that the negative
charge induced by POTs did not work to separate PS-PVP-PEO molecules each other,
rather POTs gathered them to the center of the micelle like cross-linking glue. This is
probably because PS-PVP-PEO molecules bound PW117- and SiW11
8- by forces other
than charge interaction, as described in the former section.
The assay mixture inevitably contains a little amount of NaCl as a result of
neutralization (pH adjustment) of HCl with NaOH. However, there was almost no effect
on hydrodynamic diameters and zeta-potentials, when 0.388 mM NaCl (equivalent
NaCl concentration in PW117- (or SiW11
8-)/PS-PVP-PEO complex solution at ADN =
800 %) alone was mixed into in the PS-PVP-PEO micelle solution. Therefore, it was
confirmed that the salt effect is negligible in the assay system.
5.3.3. Morphological Studies
In the former two sections, it was suggested that the size of the PS-PVP-PEO
micelles decreased, when the positive charge in the PVP block was neutralized by POTs.
In order to confirm this, the micelles were morphologically characterized with AFM
(Fig. 3) and SEM (Fig. 4) techniques.
89
(a)
(b)
(c)
Figure 3. AFM images of PS-PVP-PEO micelle. (a) PS-PVP-PEO alone, (b) PW117-
/PS-PVP-PEO complex at ADN = 350%, and (c) SiW118-/PS-PVP-PEO complex at
ADN = 400%.
90
(a)
(b)
(c)
Figure 4. SEM images of PS-PVP-PEO micelle. (a) PS-PVP-PEO alone, (b) PW117-
/PS-PVP-PEO complex at ADN = 350%, and (c) SiW118-/PS-PVP-PEO complex at
ADN = 400%. Ten gradients correspond to 500 nm in the Fig. (a), and to 1µm in
Figs.(b) and (c), respectively.
91
In AFM images, the average diameter of the PS-PVP-PEO micelle at pH 3 was 130
nm in the absence of the tungsten compounds (Fig. 3a). In the presence of PW117-
/SiW118-, it was reduced to ～80 nm at ADN = 350/400%, respectively (Fig. 3b and Fig.
3c). Although the micelle size was calculated smaller than the results of DLS study, it
was confirmed that POTs induced ～40% reduction in the micelle size.
In SEM images, the mean size of the micelle was estimated as 110 nm in the
absence of the tungsten compounds (Fig. 4a). In the presence of PW117-/SiW11
8-, the
micelle size was reduced to ～75 nm at ADN = 350/400%, respectively (Fig. 4b and Fig.
4c). In this case, the size reduction ratio induced by POTs was ～35%.
It was observed that the morphology and size of the micelles were almost same in
both AFM and SEM studies. The size reduction ratio induced by POTs was in good
accordance with the results of DLS study. However, the hydrodynamic diameter given
by the DLS study was larger than the size measured by AFM/SEM technique. This is
probably due to that AFM/SEM yields a number-averaged size whereas DLS does a z-
averaged one. In addition, the hydration layers also counts in DLS measurements.
5.3.4. Fluorescence Spectroscopic Study
Pyrene (Py) is a well-known fluorescent probe of which fluorescence intensity is
easily affected by various kinds of molecular interaction with many factors [36, 37].
Therefore, the degree of molecular integration can be estimated from the signal intensity
when Py is mixed with the micelle solution. Fig. 5 shows fluorescence profile of 0.6 µM
Py solution with PW117-/PS-PVP-PEO nano-complex. As ADN was increased, the
fluorescence intensity of Py decreased continuously and was finally minimized at ADN
= 350 %. Further addition of PW117- caused almost no change, and excimer of Py was
not formed as far as judged from the pattern of fluorescent spectra.
The inner filtering effect of Py molecule is known to be relatively small, and Py is
preferentially accommodated in polymeric micelles in aqueous solutions [38]. It was
confirmed that much higher concentration of PW117- (in mM order) was required to
induce the fluorescence quenching on Py molecules in the bulk phase of an aqueous
92
solution (data not shown). Therefore, it was considered that the decrease in Py
fluorescence shown in Fig. 5 is mainly attributable to the fluorescence quenching by
interaction with PW117- molecules accumulated in the core part of the micelles. In other
words, it was confirmed that PW117- is incorporated into the micelle of PS-PVP-PEO
indeed, forming an organic-inorganic nano-complex in aqueous solutions.
0
20
40
60
80
100
120
140
160
360 370 380 390 400 410 420 430 440 450Wavenght / nmWavelength/nm
Inte
nsity
/ a.
u
ADN
30 %
100 %
148 %
178 %
213%
245 %
350 %
Figure 5. Fluoresence specrta of Py in PS-PVP-PEO micelles. The fluorescence of Py
(0.6µM) with PW117-/PS-PVP-PEO complex was measured in aqueous solutions
(excitation = 334 nm). Concentration of PS-PVP-PEO was 0.025 g/L, and the amount of
PW117- is expressed as ADN.
From the Stern-Volmer analysis (Fig. 6) on the fluorescence quenching of Py
induced by PW117-, a relatively higher Stern-Volmer constant (Ksv= 1.45× 105 M-1) was
calculated (the inner filter effect caused by PW117- was eliminated by a conventional
method [39]). This means that both Py and PW117- are highly concentrated into the PS-
PVP-PEO micelles.
93
0
2
4
6
8
0 10 20 30 40 50
M
F0 / F
[PW11] / µPW11 /µM
Figure 6. Stern-Volmer plot for the quenching of Py flourescence in PW117-/PS-PVP-
PEO complex. The extent of fluorescence quenching of Py caused by PW117- in the
aqueous solution was plotted.
5.4. Conclusion
We have succeeded in fabricating the organic-inorganic nano-complexes from
tungsten compounds (W12-, PW11
7-, and SiW118-) and PS-PVP-PEO triblock copolymer.
The complexes were formed mainly by charge interaction between the cationic PVP
block of the micelle and the counteranions. However, zeta-potential study and
fluorescent analysis suggested that some other binding forces worked between PS-PVP-
PEO micelles and POTs.
94
The addition of POTs into the PS-PVP-PEO micelles caused a significant
morphological change in the micelles, and they turned shrunken with 〜40% size
reduction ratio. This means that the structure of the micelles became tight, and the
micelles could hold various materials tightly and thickly, as suggested by the high
Stern-Volmer constant of a fluorescent probe. This feature may be useful to develop a
new type of functioning micelle.
5.5. References
1. W.R. Fischer, U. Schwertmann, Clays Clay Mine, 1975, 33.
2. R.S. Sapiesko, E. Matijevic, J. Colloid Interface Sci., 1980, 74,405.
3. R.M. Cornell, Clays Clay Miner., 1985, 33,219.
4. M.E. Tadros, I. Mayes, J. Colloid Interface sci., 1979, 72,245.
5. T. Ishikawa, E. Matijevic, Colloid Polym. Sci,1991, 269,179.
6. T. Ishikawa, E. Matijevic, J. Colloid Interface Sci., 1988,123, 122.
7. K. Kandori, I. Horii, A. Yasukawa, T. Ishikawa, J. Mater. Sci., 1995, 30, 2145.
8. A. Chittofrati, E. Matijevic, Colloids Surf., 1990, 48, 65.
9. H. Sakai, H. Kawahara, M. Shimazaki, M. Abe, Langmuir,1998, 14, 2208.
10. N.R. Jana, L. Gearheart, C.J. Murphy, Langmuir, 2001, 17, 6782.
11. N. Pinna, K. Weiss, H. Sack-Kongehl, W. Vogel, J. Urban, M.P. Pileni,
Langmui, 2001, 17, 7982.
12. A. Filankembo, P. Andre, I. Lisiecki, C. Petit, T. Gulik-Krzywicki, B.W.
Ninham, M.P. Pileni, Colloids Surf A: Physicochem. Eng. Aspects, 2000, 174, 221.
13. L.M. Bronstein, S.N. Sidorov, P.M. Valetsky, Langmuir, 1999,15, 6256.
14. S. Förster, M. Antonietti, Adv. Mater.,1998, 10, 195.
15. G. Wegne, P. Baum, M. Müller, J. Norwig, K. Landfester, Macromol. Symp.,
2001, 175, 349.
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16. H. Cölfen, L. Qi, Chem. Eur. J., 2001,7,106.
17. H. Cölfen, Macromol. Rapid Commun., 2001,22, 219.
18. L.M. Bronstein, S.N. Sidorov, A.Y. Gourkova, P.M. Valetsky, J. Hartmann,
M. Breulmann, H. Cölfen, M. Antonietti, Inorg. Chim. Acta,1998, 280, 348.
19. J.F. Gohy, N. Willet, S. Varshney, J.X. Zhang, R. Jérôme, Angew Chem. Int.
Ed., 2001, 40, 3214.
20. L. Lei, J.F. Gohy, N. Willet, S.K. Varshney, J.X. Zhang, R. Jérôme,
Macromolecules, 2004, 37,1089.
21. A. Khanal, Y. Li, N. Takisawa, N. Kawasaki, Y. Oishi, K. Nakashima,
Langmuir, 2004, 20, 4809.
22. J.F. Gohy, N. Willet, S. Varshney, J.X. Zhang, R. Jérôme, e-Polymer, 2002,
No 35.
23. M. Stepanek, J. Humpolickova, K. Prochakza, M. Hof, Z. Tuzar, M.
Spirkova, T. Wolff, Collect. Czech. Chem. Commun., 2003, 68,121.
24. Y. Tajima, (2005) Mini Rev. Med. Chem. 5: in press.
25. D. Li, Y. Guo, C. Hu, C. Jiang, E. Wang, Journal of Molecular Catalysis A:
Chemical, 2004 ,207,183.
26. Y. Yang, Y. Gao, C. Hun, E. Wang, Applied Catalysis A: General, 2003, 252,
305.
27. J.H. Bragdon, R.J. Havel, Science, 1954, 120, 113.
28. Y. Tajima, R. Shizuka, S. Oshitani, H. Amagai, Thromb. Res.,1990, 57, 697.
29. Y. Tajima, J. Inorg. Biochem.,1997, 68, 93.
30. T. Yamase, N. Fukuda, Y. Tajima, Biol. Pharm. Bull., 1996, 19, 459.
31.N. Fukuda, T. Yamase, Y. Tajima, Biol. Pharm. Bull., 1999, 22, 463.
32. Y. Tajima, Biol. Pharm. Bull., 2001, 24, 1079.
33. Y. Tajima, J. Biochem. Biophys. Methods, 1999, 38,217.
34. C.B. Shaffer, F.H. Dritchfield, Anal. Chem.,1947,19, 32.
96
35. M.J. Harris, (1992) Introduction to biotechnical and applications of poly(ethylene
glycol) in: Biotechnical and Biomedical Applications, Plenum Press New York, London,
chap 1, pp 4.
36. K. Kalyanasundaram, J.K. Thomas, J. Am. Chem. Soc.,1977, 99, 2039.
37. D.C. Dong, M.A. Winnk, Photochem. Photobiol.,1982, 35, 17.
38. M. Wilhelm, C-L. Zhao, Y. Wang, R. Xu, M.A. Winnik, J-L. Mura,G.
Riess, M.D. Croucher, Macromolecules,1991,24, 1033.
39. J.R. Lakowicz, (1983) Plenum Press, New York.
97
(Chapter 6)
Incorporation and Release of Cloxacillin Sodium in Micelles of Polystyrene-b-Poly(2-vinyl
pyridine)-b-Poly(ethylene oxide)
A novel drug carrier system was constructed from anionic drug cloxacillin sodium
(CLX) and micelle of polystyrene-b-poly(-2-vinyl pyridine)-b-poly( ethylene oxide)
(PS-PVP-PEO) by incorporating the former into the micelle of the latter. The
incorporation of CLX into the micelle of PS-PVP-PEO was confirmed by
zeta-potential measurements, dynamic light scattering (DLS), and fluorescence
spectroscopy. In the absence of the anionic drug, CLX, the zeta-potential of neat
PS-PVP-PEO micelle was +13 mV under aqueous acidic condition due to the presence
of positive charges in the PVP unit. The addition of CLX into the micelle of
PS-PVP-PEO decreased the zeta-potential of the micelle smoothly and finally led to a
minimum zeta-potential around 0 mV. This fact shows that the added CLX is
effectively incorporated into the PS-PVP-PEO micelle by electrostatic attraction. In
concomitant with the decrease in zeta-potential, a decrease in hydrodynamic diameter
from 94 nm to 69 nm was observed on addition of CLX to the PS-PVP-PEO micellar
solution. This fact also indicates the incorporation of CLX into the PS-PVP-PEO
micelles because the binding of CLX to the PVP block of the micelles induces a
conformational change from an extended to a shrunken form due to the cancellation of
the repulsive force in the PVP blocks by CLX. Fluorescence quenching of pyrene by
CLX gave additional evidence for the effective bindings of CLX to the PS-PVP-PEO
micelles. Further, release of CLX from the nanoaggregates of CLX/PS-PVP-PEO
was investigated in vitro. It was found that the release kinetics of the CLX is
conformed to a model based on the consecutive chemical kinetics.
98
6.1. Introduction
Micro- and nanosized materials with unique morphology and functions have
become more and more important in medical applications [1-3], water-miscible
carriers [4] and contaminants removers [5] etc. In this respect, a new method was
employed recently to manufacture the nano-structured particles through the
self-assembly of amphiphilic AB diblock or ABA triblock copolymers in aqueous
solutions [6-13]. Nowadays, these polymeric micelles with core-shell architecture
seem to have potential applications in many fields including drug delivery systems
because micelles with biocompatible hydrophilic shell show low uptake by
reticuloendothelial systems even if they have a nonbiocompatible core [14] and
significantly protect the incorporated drug from fast degradation, blood clearance and
elimination from the body [15,16].
A few works have been reported so far by considering amphiphilic ABC triblock
polymers [17,18] although they may form more complex nanostructures and the
interactions of drugs with the nanostructures may break a fresh ground of nano-science
as found in other nano-structured particles [19- 24]. Recently, Jérôme et al. reported
the incorporation of gold salt into the well defined core-shell-corona micelle from
amphiphilic triblock polymer of polystyrene-b-poly(-2-vinyl pyridine)-b-poly(ethylene
oxide) (PS-PVP-PEO); Fig.1a [17,18]. This micelle has a glassy and hydrophobic PS
core, an ionizable hydrophilic PVP shell and a hydrophilic PEO corona. The micelle
has an extended PVP shell at low pH (<5) due to repulsive forces among the protonated
PVP units. Addition of the anionic gold salt into this micelle cancels the positive
charge of the PVP unit, leading to a morphological change of the PVP shell from an
extended to a shrunken form [17,18]. A similar morphological change of the
PS-PVP-PEO micelles was brought about by changing pH because the PVP block
undergoes deprotonation at high pH (>5) [25, 26]. We also succeeded in inducing a
similar morphological change of PS-PVP-PEO micelle by incorporating sodium
dodecylsulfate [27], dextran sulfate [28] or polyoxotungstates [29].
These behaviors seem to be utilized to construct novel nanoaggregates which can
be utilized for drug delivery. In this study, we tried to incorporate an anionic drug,
99
cloxacillin sodium (CLX), into the protonated PVP block of PS-PVP-PEO micelle to
investigate a possibility of the micelle as a drug carrier. CLX (Fig.1b) is known as a
penicillinase-resistant, semi-synthetic penicillin, and β-lactam antibiotic [30]. It has
an antibacterial activity against penicillinase-producing strains of gram positive cocci,
particularly staphylococcal species. It is widely used for the treatment of several
bacterial infections in human and animal diseases such as endocarditis, deep-seated
abscesses, and osteomyelitis [31-33]. The formation of nanoaggregates of
CLX/PS-PVP-PEO was confirmed by zeta-potential, dynamic light scattering (DLS)
and fluorescence spectroscopy. Then, release kinetics of CLX from the
nanoaggregates of CLX/PS-PVP-PEO was investigated in order to know how long
CLX is retained in the micelles of PS-PVP-PEO.
CH 2 CH CHy
x
N
CH 2 CH 2 CH 2 Oz
(a) PS-PVP-PEO
(b) CLX
Fig.1. Structures of PS-PVP-PEO and CLX.
100
6. 2. Experimental
6.2.1. Materials
PS-PVP-PEO was supplied from Polymer Source Inc. (Canada), and used
without further purification. The number-averaged molecular weights of the PS, PVP,
and PEO block are 14100, 12300, and 35000, respectively. Cloxacillin sodium salt
monohydrate and pyrene (Py) were purchased from Aldrich (Japan). Py was purified
by vacuum sublimation and CLX was used as received. Dimethylformamide (DMF)
was purchased from Sigma-Aldrich (Germany) and used without further purification.
Water was purified by Millipore MiliQ system.
6.2.2. Preparation of PS-PVP-PEO Micelle and Incorporation of CLX
Micelle of PS-PVP-PEO was prepared as described by Jérôme et al. [17, 25].
First, 0.1 g of PS-PVP-PEO was dissolved into the mixtures of 0.5 g water and 4.4 g
DMF. The solution was stirred for several days until PS-PVP-PEO polymer was
completely dissolved. Then, the solution was dialyzed against water until the solvent
was free from DMF. The solution was transferred into a 100 mL volumetric flask to
obtain a stock solution of the polymeric micelle (1 g/L). The pH of the stock solution
was adjusted to 3 with 0.1 M HCl. The stock solution was kept more than 24 hours
before it was further diluted to get a desired concentration. The pH of the sample
solution was adjusted with 0.01 M HCl or 0.01 M NaOH.
CLX was added into the micellar solution of PS-PVP-PEO at pH 3 or pH 7. The
amount of added CLX was expressed by “apparent degree of neutralization (DN)”
which is defined by the following formula:
101
DN(%) =
(Molarity of pyridine unit ).
(1)
(Molarity of added CLX) ×100
The solutions of the aggregates (i.e., CLX /PS-PVP-PEO) were stable and
transparent for several weeks under the experimental conditions.
6.2.3. Zeta-Potential Measurements
Electrophoretic mobility (µE) of the samples was measured at 25°C with
ELS-8000 (Otsuka, Japan). The zeta-potential of the samples was calculated from µE
using following Smoluchowski’s equation:
µE =ζ ε / η , (2)
where ζ is the zeta-potential, ε the permittivity of solvent, and η the viscosity of
solvent.
6.2.4. DLS Measurements
DLS was measured with ELS-800 (Otsuka, Japan) at fixed 90˚ scattering angle.
Correlation functions were analyzed by a histogram method and used to determine the
diffusion coefficient (D) of the nano-aggregates in the sample. Hydrodynamic radius
(Rh) was calculated from D by using Stokes-Einstein’s equation:
Rh=kBT/ (6πηD), (3)
where kB is Boltzmann constant, T the absolute temperature, and η the solvent
viscosity.
102
6.2.5. Fluorescence Spectroscopy
Stock solution of Py (30 µM) was prepared in methanol. 0.1 mL of the stock
solution was transferred into a 5 mL volumetric flask and the solvent was removed by
gentle heating under nitrogen gas stream. Then, known amounts of the micellar
solution of PS-PVP-PEO and CLX were added into the volumetric flask containing Py.
Py was solubilized in a solution of CLX/PS-PVP-PEO by keeping it for 60 h at room
temperature. The final concentration of Py in the samples was kept constant at 0.6
µM, which is near to its saturation level in water (0.8µM [34]]) at 22° C. Steady-state
fluorescence spectra were recorded on FP-6500 fluorescence spectrophotometer
(JASCO, Japan) at right angle geometry. The band widths were 3 nm and 1 nm for
excitation (334 nm) and emission, respectively.
6.2.6. Release Experiments
Release rate of CLX from the nanoaggregates of CLX/PS-PVP-PEO micelle was
monitored by UV-Vis absorption spectroscopy using Jasco Ubest-50 UV-Vis
spectrophotometer. First, 10 mL solution of the CLX / PS-PVP-PEO nanoaggregates
at 100% DN was put inside a dialysis tube. The concentration of PS-PVP-PEO was
0.7 g/L and the pH of the nanoaggregate solution was maintained at 3 or 7. The
dialysis tube containing the nanoaggregates solution was then immersed into 435 mL of
water with the same pH as that of the nanoaggregates solution. A required amount of
solution outside the dialysis tube was taken off at definite intervals of time to measure
the amount of CLX which was released from the nanoaggregates of CLX /PS-PVP-PEO.
The UV absorbance of CLX was recorded at 210 nm. The solution taken off for UV
absorption measurements was immediately returned to the dialysis tube after the UV
measurements. The release kinetics was analyzed by a non-linear least square
method using Graph Pad Prism software.
103
6. 3. Results and Discussion
6.3.1. Zeta-Potential Measurements
The nano-aggregates were assembled by binding of anionic CLX into the
protonated PVP block of the PS-PVP-PEO micelle by charge interaction. Thus,
the construction of nanoaggregates can be confirmed by measuring the zeta-potential at
different DN values. Figure 2a represents a plot of zeta-potential of the CLX /
PS-PVP-PEO complexes as a function of DN at pH 3. When the micelle of
PS-PVP-PEO was free from CLX at pH 3, the PVP was protonated as indicated by +13
mV zeta-potential. For the fabrication of the nanoaggregates of CLX/PS-PVP-PEO,
the anionic drug CLX was successively added into the micellar solution. This led to
the incorporation of the anionic drug into the protonated PVP block by electrostatic
attraction. This process is reflected in the progressive decrease in the zeta-potential
of the PVP block of the micelle particles. Consequently, 0 mV zeta-potential was
observed at 160% DN. This result clarifies the formation of electro-neutral
nanoaggregate of CLX / PS-PVP-PEO. Beyond 160% DN, CLX did not bind to the
electro-neutral compound of CLX/PS-PVP-PEO as indicated by almost no change in
zeta-potential. It should be noted here why 160% DN is necessary for reaching 0 mV
zeta-potential. This can be understood by considering pKa of CLX. As the pKa of
CLX is 2.7, about 67 % of CLX is ionized at pH 3. Therefore only 67% of the added
CLX was effective for neutralizing the PVP block of PS-PVP-PEO.
6.3. 2. DLS Measurements
Figure 2b represents a plot of the hydrodynamic diameter of CLX/PS-PVP-PEO
against DN at pH 3. In the absence of CLX, the hydrodynamic diameter of the
micelle was 94 nm. On increasing DN, the hydrodynamic diameter continuously
decreased and finally reached a value of 69 nm at 160 % DN. Further addition of
CLX did not bring about any significant change in the hydrodynamic diameter.
104
This change can be accounted by considering a conformational change in the
protonated PVP block of the PS-PVP-PEO micelle.
0
2
4
6
8
10
12
0 50 100 150 200 250 300DN /%
ζ / m
V
(a)
65
70
75
80
85
90
95
0 50 100 150 200 250 300
DN / %
Dia
met
er /
nm
(b)
Fig. 2. Dependence of zeta-potential (a) and hydrodynamic diameter (b) of
CLX/PS-PVP-PEO nanoaggregates on DN at pH 3.
105
In the absence of CLX, the micelle had +13 mV zeta-potential at pH 3 indicating
positive charges in the PVP block. These positive charges exerted the repulsive force
among the pyridine units and gave an extended conformation to the protonated PVP
block. When CLX was added to the micellar solution, the positive charge of pyridine
unit was cancelled by the negative charge of a carboxylate ion of CLX. This process
reduced the repulsive forces between the PVP blocks and decreased the solubility of
the PVP blocks. Consequently, conformational change of the PVP blocks from
extended to shrunken forms occurred, which in turn gave the decreased hydrodynamic
diameter of the micelle. Such conformational changes have been reported already
after incorporating some anionic species (e.g., sodium dodecylsulfate, dextran sulfate
and polyoxotungstates) into the PS-PVP-PEO micelle [27-29]. Importantly, the
constant hydrodynamic diameter of CLX/PS-PVP-PEO nanoaggregates started at
160 % DN where 0 mV zeta-potential was observed. After 160 % DN, CLX does not
seem to be incorporated to the complexes as indicated by the almost constant
hydrodynamic diameter and 0 mV zeta-potential.
6.3. 3. Fluorescence Spectroscopy
Figures 3a and 3b represent fluorescence spectra of 0.6 µM Py in
CLX/PS-PVP-PEO nanoaggregates at pH 3 and 7, respectively. From Fig. 3, it is
evident that on increasing DN, the fluorescence intensity of Py decreases continuously
both at pH 3 and 7. This is due to the quenching of Py fluorescence by CLX.
However, the quenching of Py fluorescence is more significant at pH 3 than at pH 7.
Quantitative discussion on the quenching is given later based on the Stern-Volmer
equation. Recently, we reported that Py is located at the interface between PS and
PVP domains in the micelle of PS-PVP-PEO [28]. This observation agrees well with
the results of the present quenching experiments. If Py was located inside PS core or
inside PEO corona in the PS-PVP-PEO micelles, the CLX molecules bound to the PVP
block would not effectively quench the Py fluorescence.
106
(a) DN DN DN DN DN DN DN DN DN
0
50
100
150
350 400 450 500DN / %
Inte
sity/
a.u
0%20%40%60%80%100%120%140%160%
DN
(b)
0
50
100
150
200
250
300
350 400 450 500
DN / %
Inte
sity/
a.u
0%
20%
60%
80%
100%
140%
180% DN
DN
DN
DN
DN
DN
Fig. 3. Fluoresence spectra of Py (0.6µM) in CLX/PS-PVP-PEO nanoaggregates
at pH 3 (a) and pH 7 ( b). Concentration of PS-PVP-PEO was 0.7 g/L, and the
amount of CLX is expressed by DN. The samples were excited at 334 nm.
107
1
6
11
16
21
26
0 0.0005 0.001 0.0015 0.002 0.0025 0.003
[CLX]/M
0
(a)
F/F
F 0/F
1.5
1
(b)
0 0.0005 0.001 0.0015 0.002 0.0025 0.003
[CLX]/ M
FF0
//F
F 0
F 0/F
Fig. 4. Stern-Volmer plots for the quenching of Py flourescence by CLX
in the CLX/PS-PVP-PEO nanoaggregates at pH 3 (a) and pH 7 ( b). The plots
are based on the data in Fig.3.
108
Stern–Volmer analysis has been done using equation 4 to make a quantitative
discussion on the quenching of Py fluorescence by CLX in the CLX/PS-PVP-PEO
nanoaggregates.
F0/F=1+Ksv [Q] (4)
where, F0 and F are the fluorescence intensity of Py in the absence and presence of
CLX, respectively, [Q] is the concentration of CLX, and Ksv is the Stern–Volmer
constant.
The Figure 4a represents the Stern-Volmer plot at pH 3. The inner filter effect
of CLX was eliminated from the observed intensity by a conventional method [35].
The Stern-Volmer constant was found to be 7.4×103 M-1. Figure 4b shows the
Stern-Volmer analysis on the fluorescence quenching of Py by CLX at pH 7. The
Stern-Volmer constant obtained is 2.5 ×102 M-1. The Stern-Volmer constant at pH 3 is
higher in comparison to that at pH 7. On the other hand , it was confirmed from the
experiments in polymer-free aqueous solutions that the quenching rate constant for
Py-CLX pair does not depend on pH. These facts suggest that at pH 3, CLX was
bound to PS-PVP-PEO micelle by stronger attraction than at pH 7. The weaker
attraction between CLX and the PVP block at pH 7 can be explained on the basis of
the properties of PVP block of the micelle. The PVP block is protonated at pH <5
and deprotonated at pH >5 [26]. Hence, the micelle contains neutral PVP unit at pH
7 which leads to no electrostatic attraction between the added CLX and the micelle.
At pH 7, the added CLX seems to be bound to the micelle only by hydrophobic force.
6.3.4. Release Experiments
Some of the polymeric micelles can be used as vehicles to transport drugs
[14-16]. In this case, release kinetics of the drugs from the micelles is an important
subject. Thus, we carried out the release experiments for CLX/PS-PVP-PEO system
because PS-PVP-PEO may be used for a drug delivery of CLX if the PVP block is
replaced by some suitable biocompatible polymer.
109
Figures 5a and 5b, respectively, show the absorbance of CLX outside the dialysis
tube at pH 3 and 7 as a function of time ( see “ Experimental Section” for experimental
details).
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30 40 50 60 70
Time/h
(a)
At/A
∞
A t/ A
∞
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30 40 50 60 70
Time/h
∞
(b)
At/A
A t/ A
∞
Fig. 5. Plots of the absorbance of CLX against time at pH 3 (a) and pH 7 (b).
The solid lines indicate fitting curves based on eq. 12 (see text for details).
110
To analyze Fig. 5, we proposed a release kinetics of the drug based upon the
concept of consecutive chemical kinetics. First, the drug incorporated in the
PS-PVP-PEO micelle is released from the micelle to aqueous bulk phase inside the
dialysis bag (Fig. 6). Then, it comes out from the dialysis bag through the membrane
to the aqueous phase outside the dialysis tube. These processes can be shown by the
following scheme:
k1 DW1
k2
DW2. DM (5)
In this scheme, DM denotes a CLX molecule incorporated in the micelle, and
DW1 and DW2 denote the CLX molecule in aqueous bulk phases inside and outside
the dialysis tube, respectively. k1 is a rate constant for the drug to exit from the
micelle to the aqueous bulk phase inside the dialysis bag, and k2 is a rate constant for
the drug to diffuse from the inside to the outside of the dialysis bag across the
membrane.
Dialysis membrane Micellar solution
Fig. 6. Schematic illustration for the drug release experiments. The solid circle
represents a particle of the polymer micelle.
111
Now, let us suppose that the amount of DM at time zero is a. At time t, x amount
of the drug is released from the micelle to yield y amount of DW2. Then at time t, we
have,
[DM]t= (a-x)/V,
[DW2]t=y/V, (7)
[DW1]t=(x-y)/V, (8)
where [DM]t, [DW2]t, and [DW1]t are the concentrations of DM ,DW2 and DW1 at time t,
respectively, and V is a total volume of the system. The solution of this type of
kinetic equation is given in the literature [36]:
a V
(k2-k1)
1+ k1exp (-k2t) -k2exp (-k1t) ,
(6)
(9)
=[DW2]t
The absorbance of CLX in the outside of the dialysis bag is proportional to [DW2]t.
Therefore, we have:
(k2-k1) , k1exp (-k2t) -k2exp (-k1t)
1 + (10)
At= εl [DW2]t =
ε l a V
where ε is molar extinction coefficient of CLX and l is an optical path length of the
sample cell.
Equation 10 can be written as:
εla
At= {1+αexp (-k2t) -βexp (-k1t)}, b V
(11)
112
where, α=k1/(k2-K1), β= k2/(k2-K1). The absorbance of DW2 at t= ∞，A∞ ，can be
expressed by A∞ = εlab/V, where b is constant. It should be noted here that the
value of A∞ can not be obtained by giving t = ∞ in eq. 11, because some amount of
CLX is partitioned in the micelle even at t = ∞. This feature is different from that of
usual consecutive chemical kinetics. To obtain A∞, we continued the dialysis until At
reached a plateau value. Dividing eq. 11 by A∞ on the both hand side, then eq.12 is
obtained:
At/A∞= {1+αexp (-k2t) -βexp (-k1t)}/b, (12)
According to eq. 12, we calculated k1 and k2 by non-linear least square method.
The results of the calculation are shown in Fig. 5 with solid lines. The rate constants
obtained are: k1= 0.046 h-1, k2 = 1.1 h-1 at pH 3, and k1= 0.074 h-1, k2=1.1 h-1 at pH 7.
These values seem to be reasonable by considering the protonated and deprotonated
nature of the PVP blocks. At pH 3, CLX is more strongly bound to the micelle of
PS-PVP-PEO by electrostatic attraction. Hence, the incorporated CLX is released
more slowly from the micelle at pH 3. Thus, we obtained a lower value of k1 at pH 3
in comparison to pH 7. The almost equal value of k2 at both pH is also reasonable
because k2 is a rate constant for the drug to diffuse from the inside to the outside of the
dialysis bag across the membrane, and it does not seem to depend upon the pH of
the solution. Here, we define a retention time (tR) of the drug in the micelle as a
reciprocal of k1. Then, we obtain tR = 22 h at pH 3 and tR = 14 h at pH 7. These
values indicate that CLX is retained in the PS-PVP-PEO at these time scales.
6. 4. Conclusion We have succeeded in incorporating an anionic drug CLX into the micelle of
PS-PVP-PEO in aqueous solutions. Addition of CLX into the micellar solution at pH
3 decreases the zeta-potential of the neat micelle from 13 mV to 0 mV. This fact
clearly indicates that the electrostatic interaction between the anionic drug and the
protonated PVP block of the micelle plays an important role during the
incorporation of CLX into the micelle. The decrease in hydrodynamic diameter of
113
the micelle from 94 nm to 69 nm was also observed on addition of CLX to the
micellar solution of PS-PVP-PEO. This fact further supports the formation of
nanoaggregates of CLX and PS-PVP-PEO micelle.
Furthermore, the incorporation of CLX into the micelles of PS-PVP-PEO was
confirmed by fluorescence spectroscopy. A higher value of Stern-Volmer constant
was obtained at pH 3 than at pH 7. This fact clearly demonstrates the stronger
interaction between the added CLX and the PS-PVP-PEO micelle at pH 3.
In the releases experiments of CLX, the value of k1 is found to be 0.046 h-1 at
pH 3 and 0.074 h-1 at pH 7. These rate constants seem to be reasonable on the basis
of the protonation and deprotonation equilibrium of the PVP block of the micelle.
Due to the protonated nature of the PVP block at pH 3, the incorporated CLX is
released more slowly from the micelle of PS-PVP-PEO than at pH 7 because at pH 3,
the anionic drug CLX is bound to the protonated PVP block by electrostatic
interaction while at pH 7, there is no such a strong interaction between CLX and the
micelle. From the k1 values obtained, we realize that CLX is retained in the
micelle of PS-PVP-PEO at a time scale of several tens of hours when the solution of
the CLX/PS-PVP-PEO aggregates is diluted with large amount of water.
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116
(Chapter 7)
Summary
The motivation of this work is to describe different aspects of micelles of AB
diblock and ABC triblock copolymers in aqueous solutions. A brief introduction and
broad applications of polymeric micelles have been described in chapter 1 by focusing
especially their use in delivery systems for hydrophobic drugs. The polymeric micelles
forms at lower CMC and are kinetically more stable than those formed by low
molecular weight surfactants. Due to these properties, polymeric micelles show many
advantages over surfactant micelles. These polymeric micelles can solubilize
hydrophobic drugs into their cores. Thus, the incorporated drugs are protected from fast
degradation.
Some chemotherapeutic agents and anticancer drugs are associated with some
serious problems due to their ionic nature. In the case of oral administration of such a
neat drug always interacts with some component of blood vessels. This decreases the
effectiveness of the drugs. As a result, the drug gets degraded (or eliminated) from the
body in a short time interval. In order to circumvent these demerits, polyion complex
(PIC) micelle of double hydrophilic diblock copolymers (DHBC) seems to be
promising tools because this micellar core can incorporate ionic drugs by electrical
neutralization. In such micelle, the incorporated drug is surrounded by a hydrophilic,
biocompatible and neutral poly(ethylene oxide). Hence, the drugs will not interact with
blood vessel due to steric stabilization effect of PEO. In this context, chapter 2 describes
the fabrication of nanoaggregates of poly (ethylene oxide)-b-polymethacrylate,
(PEO-b-PMA) and chitosan(Ch) and its derivative methylglycolchitosan(MGC).
Chitosan possesses antimicrobial, wound-healing and hypocholesterolemic properties.
Ch/PEO-b-PMA nanoaggregates have been fabricated by electrical neutralization of
anionic PMA blocks of PEO-b-PMA with cationic Ch. This leads to insolubilization of
the PMA blocks which in turn changes to the core. These PIC micelles have properties
of both amphiphilic block copolymers micelles and polyelectrolyte complexes. Thus,
117
these PIC micelles have clear advantage over the conventional polymeric micelles
because the later can not incorporate ionic drugs due to their non-polar core. The
incorporation of Ch or MGC into the PMA block of PEO-b-PMA was monitored by
zeta-potential measurements and fluorescence spectroscopy. The formation of
nanoaggregates was confirmed by dynamic light scattering and atomic force microscopy.
It has been found that the properties of these nanoaggregates depend upon the
concentration of the polymer as well as on the concentration of Ch or MGC.
Although micelles of DHBC have above features, they have a drawback due to
unfrozen nature. Hence, these micelles get collapsed in the blood stream when used as a
drug vehicle to transport the ionic drugs because the drug incorporated micellar solution
will be diluted more than hundred times in the blood stream. Thus, it is essential to use
another copolymer which exhibits frozen nature so that it will not collapse in the blood
stream even if it is diluted more than hundred times. In addition, there are several
reasons for searching long-circulating drug carriers. At least three of them seem to be
the most important. First, keeping the certain pharmaceuticals or drugs long time in the
blood stream enhances the drug availability. Thus, a long-circulating drug carrier may
maintain required level of drug in the blood for extended time intervals. Second,
long-circulating drug-containing nanoaggregates can slowly accumulate (so-called
enhanced permeability and retention effect, known also as a ‘passive’ targetting or
accumulation via an impaired filtration mechanism) in pathological sites with affected
and leaky vasculature (such as tumors, inflammations) and improve (or enhance) drug
delivery in those areas. Third, prolonged circulation can help to achieve a better
targetting effect for those targetted (specific ligand-modified) drugs since it increases
the total quantity of targetted drug passing through the target, and the number of
interactions between targetted drugs and their targets. This attempt seemed to be
achieved by the use of ABC type triblock copolymers, such as poly
styrene-b-poly(2-vinyl pyridine)-b-poly(ethylene oxide), (PS-PVP-PEO) in aqueous
systems. In this context, micelles of PS-PVP-PEO have been investigated in
chapters 3,4,5 and 6.
PS-PVP-PEO forms the micelles in water. These core-shell-corona micelles have
three compartments, glassy core of PS, ionizable shell of PVP and hydrophilic corona of
PEO. Out of these three compartments, the PVP block can be tuned to desired
118
morphology under different conditions. The PVP blocks can be made hydrophobic or
hydrophilic by changing the pH of solution. At low pH(<5), the PVP block of
PS-PVP-PEO terpolymer is protonated and becomes hydrophilic while at high pH(>5),
the PVP blocks gets deprotonated and becomes hydrophobic. This protonation and
deprotonation equilibrium also induces conformational change of the PVP from
extended to shrunken forms. Hence, hydrodynamic diameter of micelles tunes from 94
nm to 69 nm on increasing pH from low to high.
The PS-PVP-PEO micelles may bind to anionic surfactant because they have
protonated PVP blocks at low pH. In this context, anionic surfactant sodium dodecyl
sulfate(SDS) was incorporated into the PS-PVP-PEO micelles as described in the
chapter 3. The incorporation of SDS into the protonated PVP blocks induced the
morphological changes of the PVP blocks because incorporated SDS cancels the
positive charges of the PVP blocks by electrical neutralization which in turn makes
PVP block insoluble in water. The formation of nanoaggregates of SDS/PS-PVP-PEO
was detected by dynamic light scattering and atomic force microscopy while the
binding of SDS to the protonated PVP blocks was confirmed by zeta-potential
measurements. The zeta-potential of neat PS-PVP-PEO micelle was +13 mV.
Incorporation of SDS into the protonated PVP led to 0 mV zeta-potential as well
decreased the hydrodynamic diameter of micelles from 217 nm to 175 nm. It is
surprising to note here that SDS did not bind to the PS-PVP-PEO micelles after the PVP
block was fully neutralized. This implies that hydrophobic binding of SDS to the
polymer was negligible due to the low concentration of SDS.
It is interesting to investigate the physicochemical properties of ABC triblock
copolymer micelles because they may show different properties than those of the
micelles of AB or ABA due to more complex structure of ABC micelles. Thus, chapter 4
describes the physicochemical properties of PS-PVP-PEO micelles. It is found that the
micelles had an average diameter of about 200 nm under acidic conditions. From
fluorescence spectroscopy, it was found that pyrene was accommodated at the interface
between the PS and PVP blocks domain. Addition of anionic drug, dextran sulfate (DS)
having anticoagulant properties to a micellar solution under acidic conditions resulted in
significant decrease in the micelle size as well as zeta-potential. The zeta-potential of
neat micelles was +11 mV and incorporation of DS into the micelles led to around 0 mV
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zeta-potential. The hydrodynamic diameter of DS/PS-PVP-PEO nanoaggregates was
found to be 165 nm where 0 mV zeta-potential was recorded. This revealed that DS was
incorporated to the protonated PVP blocks of the micelles by electrical neutralization.
From the release experiments, it was found that anionic dye (eosin Y) retained in the
micelles for several tens of hours.
Nowadays, emerging interest is given in the fabrication of nano-hybrids especially
between organic and inorganic complexes because these complexes may give a new
approach to nanotechnology. In this context, chapter 5 describes fabrication of
nano-hybrids of PS-PVP-PEO micelles and tungsten compounds such as tungstate
(W12-), undecatungstophosphate (PW11
7-) and undecatungstosilicate (SiW118-) in acidic
aqueous solutions. These polyoxotungstates exhibit photocatalytic, antiviral and
anticoagulant activities as well as it show strong sensitizing effect on
methicillin-resistant Staphylococcus aureus to β-lactam antibiotics. The size and
morphology of the fabricated complexes of tungsten compounds and PS-PVP-PEO
micelles were characterized by dynamic light scattering, atomic force microscopy,
scanning electron microscopy and fluorescence spectroscopy. In the case of PW117-
and SiW11,8- they bound to the micelles not only by charge intercation but also other
forces because recorded zeta-potential after the formation of electroneutral complexes
of PW117-/PS-PVP-PEO showed around -33 mV. Although the detailed mechanism of
the interaction has not been elucidated yet, it is possible that: (i) The O-atoms in the
PEO block were partially protonated to form oxonium cation, where POTs interacted by
charge. (ii) POTs polarized non-polar part of the PS-PVP-PEO molecule (e.g., the PEO
block) with their very high negative charge and bound the dipoles. (iii) Hydrogen
bonding was made between the O-atoms in the POT molecule and H-atoms in the
PS-PVP-PEO molecule. (iv) The PS-PVP-PEO molecule formed an inclusion (clathrate)
compound with POTs as a guest like crown ether.
As mentioned above that, PS-PVP-PEO micelles have frozen core. Thus, it is
interesting to find out release kinetics of drug from the micelles because such kinetics
will be helpful for drug formulation. Thus, chapter 6 reports the release of an anionic
drug, cloxacillin sodium (CLX) from the micelles of PS-PVP-PEO. The cloxacillin
exhibits antibiotic activitiy and is used for the treatment of several diseases in human as
well as in animals. Incorporating CLX into the micelle of PS-PVP-PEO decreased the
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zeta-potential of the neat micelle from 13 mV to 0 mV. This fact clearly indicates that
the electrostatic interaction between the anionic drug and the protonated PVP block of
the micelle plays an important role during the incorporation. The decrease in
hydrodynamic diameter of the micelle from 94 nm to 69 nm was also observed on
addition of CLX to the micellar solution of PS-PVP-PEO. This fact supports the
formation of nanoaggregates of CLX and PS-PVP-PEO micelle.
Furthermore, the incorporation of CLX into the micelles of PS-PVP-PEO was
confirmed by fluorescence spectroscopy. A higher value of Stern-Volmer constant was
obtained at pH 3 than at pH 7. This fact clearly demonstrates stronger interaction
between the added CLX and the PS-PVP-PEO micelle at pH 3.
From the release experiments, it is found that the release kinetics of the CLX
conformed to a model based on the consecutive chemical kinetics and CLX retained
in the micelle of PS-PVP-PEO at a time scale of several tens of hours indicating that
PS-PVP-PEO micelles can be used as a vehicle for the delivery of CLX, if the PVP
block is replaced by some suitable biocompatible polymer.
Overall, the micelles of ABC triblock copolymers seem to be promising because
these contain three layers out of which the middle layer (PVP) can be made soluble
(protonated), or insoluble (deprotonated) depending upon the investigation condition. In
addition, these micelles show broad potential applications in comparison to the micelles
of AB diblock or ABA triblock copolymers because ABC triblock copolymers show
more complex chemical structure in bulk state and may give well-controlled aggregate
than that of the AB diblock or ABA triblock copolymers in water.
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Resume Name Anil Khanal Nationality Nepali Date of birth April 24, 1970 Sex and Marital status Male, Single Academic Qualification: 2002-2005 – Ph D, Saga University, Japan. 1993-1995 - M. Sc.,Tribhuvan University, Nepal. 1991-1993 - B. Sc., Amrit science college (Tribhuvan University), Nepal. 1989-1991 - I. Sc., Amrit science college (Tribhuvan University), Nepal. Position: 11/1995 - till date - Chemist in Department of Food Technology and Quality Control, Ministry of Agriculture, Katmandu, Nepal. Experiences: 1. Preparation and characterization of nanoaggregates (e.g. polymer
micelle) using block copolymers. 2. Analysis of various food commodities and related research works. Conferences: 1. International Polymer Conference, Japan (2005). 2. New Trends in Supramolecular Chemistry 2nd workshop, Japan (2005). 3. Kyushu Colloid, Japan (2005). 4. World Polymer Congress, Macro2004, France (2004). 5. Kyushu Colloid Colloquium International, Japan (2004). 6. Physicochemical Intercollegiate, Japan (2003).
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Publications 1. “Incorporation and release of cloxacillin sodium in micelles of
Polystyrene-b-Poly(2-vinyl pyridine)-b-Poly(ethylene oxide)” J. Controlled Release, in press (2005). A. Khanal, K. Nakashima.
2. “Fabrication of nanoaggregates of poly (ethylene oxide)-b-polymethacrylate by complex formation with chitosan or methylglycolchitosan", Colloids Surf., A: Physiochem. Eng. Asp.,in press (2005). A. Khanal, Y. Nakashima, Y. Li, K. Nakashima, N. Kawasaki and Y. Oishi.
3. “Fabrication of organic-inorganic nano-complexes using ABC type triblock copolymer and polyoxotungstates”, Colloid. Polym. Sci., in press (2005). A. Khanal, K. Nakashima, N. Kawasaki, Y. Oishi, M. Uehara, H. Nakamura, Y. Tajima.
4. “Physicochemical properties of micelles of poly(styrene-b-2-vinylpyridine-b-ethylene oxide) in aqueous solutions”, Bull. Chem. Soc. Jpn., 2005, 78, 529-533., Y. Li, A. Khanal, N. Kawasaki, Y. Oishi, K. Nakashima.
5. “Morphological change of the micelle of poly(Styrene)-b -poly(2-Vinyl Pyridine)-b-poly(Ethylene Oxide) induced by binding of Sodium dodecyl sulfate and dextran sulfate”, Macro (2004),e-polymer A. Khanal, Y. Li, N. Takisawa and K. Nakashima.
6. “Physicochemical properties of polymeric micelle with frozen core, ionic shell and water-soluble corona in aqueous solutions”, Macro (2004),e-polymer, Y. Li, A. Khanal, Y. Oishi, and Y. Tajima.
7. “Morphological change of the micelle of poly(styrene)-poly(2-vinylpyridine)-poly(ethylene oxide) induced by binding of sodium dodecyl sulfate” Langmuir, 2004, 20, 4809-4812., A. Khanal, Y. Li, N. Takisawa, N. Kawasaki, Y. Oishi, K. Nakashima.
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