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Construction of Biomacromolecular Nanosheets and Their Biomedical Application as a Wound Dressing Material 生体高分子から
Page 1
Construction of Biomacromolecular Nanosheets and
Their Biomedical Application as a Wound Dressing Material
生体高分子からなるナノシートの構築と
創傷被覆材としての医用応用
February 2009
早稲田大学大学院先進理工学研究科
生命医科学専攻 生体分子集合科学研究
Toshinori FUJIE
藤枝 俊宣

Page 2
Promoter: Prof. Dr. Shinji Takeoka
Referees: Prof. Dr. Nobuhito Goda
Prof. Dr. Satoshi Tsuneda
Assist. Prof. Dr. Arianna Menciassi

Page 3
Preface
In the development of nanobiotechnology, novel clinical interventions have been emerged
out, represented by drug delivery systems, regenerative medicine, etc. One of the technological
efforts in the surgical field is tissue engineering, which fabricate the tailor-made type of
transplantable biological tissues outside of the patient body in the use of artificial cellular matrix.
However, such technology has also high risks such as contaminative infection due to long
periods of cell culture. Therefore, innovative biomaterials are urgently needed for the minimal
invasive repair of surgical defects, particularly in the field of emergency medication.
Recent developments in materials science have led us to discover a method for producing
free-standing polymer nanosheets. The huge size-aspect ratio of such nanosheets exhibits unique
property such as robustness, physical adhesion and high flexibility. The author focuses on their
unique physical property, and envisaged the biomedical application of the polymer nanosheets
(biomacromolecular nanosheets) in the development of novel wound dressing materials.
This thesis is consisted of eight chapters. The first chapter deals with the core principles
of nanomaterials science and nanotechnology in the basis of self-assembly and self-organization
system, and then introduced the fundamental aspects of organic (macro)molecules organized
nanomaterials; in particular, focusing on two-dimensional nanostructures such as nanosheets.
Then, fundamental aspects of the free-standing biomacromolecular nanosheets with tens-of-nm
thick is described throughout Chapter 2-4, including design, construction, characterization,
evaluation of adhesive and mechanical property, and applications with the ubiquitous
transference of the biomacromolecular nanosheets onto biological interfaces. In Chapter 5 and 6,
the author demonstrated the practically biomedical application of the biomacromolecular
nanosheets as wound dressing materials, integrated for typical tissue-defects, where the author
revealed the clinical benefits of the biomacromolecular nanosheets for wound healing.
Moreover, the surface modification techniques of the biomacromolecular nanosheets are
introduced, envisaging the functional nanosheets in Chapter 7. Finally, the conclusion and the
future prospects of this thesis are described in Chapter 8.
As the biomedical application of the polymer nanosheets, the author is convinced that
the integration of the biomacromolecular nanosheets for tissue-defects should be an alternative
and innovative intervention in the next generation of the surgical operation.
Toshinori Fujie

Page 4
-i-
Contents
Preface
Chapter 1 Principle, Design and Construction of
Molecular Organized Nanomaterials
1. Introduction
1
2. Fundamental Aspects of Self-Assembly and Self-Organization
3
2-1. Molecular Organizations Derived from Self-Assembly
3
2-2. Principles of Self-Assembly
5
2-3. Principles of Self-Organization
7
3. Principles of Intermolecular and Surface Interactions
10
3-1. Classification of Intermolecular Interactions
10
3-2. Molecular Organization of Intermacromolecular Complexes
11
3-3. Intermolecular and Surface Interactions
12
3-4. Van der Waals Interactions
14
4. Functional Ultra-Thin Films Based on Molecular Organizations
16
4-1. Nanomatrials Science Developed by Soft-Nanotechnology
16
4-2. Functional Ultra-Thin Films on the Static Interface
17
4-3. Modulation for the Integrated Surface
20
4-4. Analytical Tools for the Ultra-Thin Films
22
5. Polymer Nanosheets Utilized in the Biomedical Field
23
5-1. Biomedical Application of the Functional Ultra-Thin Films
23
5-2. Functional Films Exfoliated to Free-Standing Nanosheets
26
5-3. Shape Consideration on the Drug Carrier: Beyond Particles
28
5-4. Micro-Patterned Nanosheets as Potential DDS Carriers
30
References
33
Chapter 2 Construction of Biomacromolecular Nanosheets
in the Development of Nano-Adhesive Plasters
1. Introduction
38
2. Construction of Free-Standing Biomacromolecular Nanosheets
39
2-1. Polysaccharide Nanosheets Obtained by “Sacrificial Layer Method”
39

Page 5
-ii-
2-2. ECM-Nanosheets Obtained by “Supporting Film Method”
41
3. Structural Characterization of Biomacromolecular Nanosheets
45
3-1. Surface Characterization by AFM (Polysaccharides Nanosheets)
45
3-2. Structural Characterization by FT-IR (Polysaccharides Nanosheets)
46
3-3. Surface Characterization by AFM (ECM-Nanosheets)
48
4. Ubiquitous Transference of Biomacromolecular Nanosheets
50
4-1. Development of “Nano-Adhesive Plaster”
50
4-2. Ubiquitous Transfer onto the Biological Tissue Surface
53
4-3. Ubiquitously Preparative Cell Culture Environment
55
5. Summary
57
References
58
Chapter 3 Optical Property of the Biomacromolecular Nanosheets
through Macro/Microscopic Surface Characterization
1. Introduction
62
2. Thin Film Interference Theory and Structural Color
62
2-1. Structural Colors in Nature
62
2-2. Fundamental Optical Processes
63
3. Microscopic Surface Morphology of Polysaccharide Nanosheets
65
3-1. Spin-Coating Assisted Structural Color Changes
65
3-2. Surface Characterization by AFM
66
3-3. Surface Characterization by SEM
67
4. Analysis of the Structural Colors of Polysaccharide Nanosheets
67
4-1. Sequential Structural Color Changes
67
4-2. Analysis of the Structural Color Changes
69
5. Summary
72
References
73
Chapter 4 Evaluation of Adhesive and Mechanical Property
of the Biomacromolecular Nanosheets
1. Introduction
76
2. Water Mediated Preparation of Polysaccharide Nanosheets
76
2-1. Total Preparation in Aqueous Condition
76
2-2. Structural Characterization
79

Page 6
-iii-
3. Physical Adhesive Property of Polysaccharide Nanosheets
81
3-1. Micro-Scratching Test
81
3-2. Evaluation of Physical Adhesive Property
82
4. Mechanical Property of Polysaccharide Nanosheets
84
4-1. Bulge Test
84
4-2. Evaluation of Mechanical Property-1 (1 mm hole)
85
4-3. Evaluation of Mechanical Property-2 (6 mm hole)
88
5. Summary
89
References
90
Chapter 5 Wound Dressing Effect of the Biomacromolecular Nanosheets
Integrated for Pleural Tissue-Defect Repair
1. Introduction
92
2. Biology and Management of Wounds
93
2-1. Wound Biology
93
2-2. Classification of Wound Healing Process
93
2-3. Chronic Wounds and Effective Wound Management
95
2-4. Wound Dressing Materials
96
3. Physiological Property of Polysaccharide Nanosheets
98
3-1. Morphological Analysis of Blood Compatibility
98
3-2. Evaluation of Blood Coagulation Activity
100
4. Polysaccharide Nanosheets Integrated for Tissue-Defect Repair 102
4-1. Treatment of Visceral Pleural Defect
102
4-2. Evaluation of Biological Mechanical Strength
103
4-3. Wound Healing Effect
104
5. Summary
108
References
109
Chapter 6 Treatment of Bacterial Peritonitis in Gastrointestinal
Perforation with the Biomacromolecular Nanosheets
1. Introduction
112
2. Polysaccharide Nanosheets Integrated for Gastrointestinal Perforation 112
2-1. Wound Infection Affecting Repair
112
2-2. Murine Cecal Puncture Model
113

Page 7
-iv-
2-3. Evaluation of Murine Viability
115
3. Immunological Response Repaired by Polysaccharide Nanosheets 118
3-1. Evaluation of Immunological Response
118
3-2. Evaluation of Bacterial Growth Inhibition
120
4. Development of Antibiotics Loaded Polysaccharide Nanosheets 122
4-1. Antibiotics for Surgical Treatment
122
4-2. Construction of TC Loaded Polysaccharide Nanosheets
123
4-3. Anti-Inflammatory Effect
125
5. Summary
127
References
128
Chapter 7 Development of the Surface Modification Techniques
for the Functional Biomacromolecular Nanosheets
1. Introduction
130
2. Selective Surface Modification of the Nanosheets with Particles 130
2-1. Selective Surface Modification with Different Latex Beads
130
2-2. LBs Modified Nanosheets (ϕ: 2 μm)
132
2-3. LBs Modified Nanosheets (ϕ: 200 nm)
134
2-4. Hetero-Surface Modified Nanosheets
135
3. Construction of Thermo-Responsive Free-Standing Nanosheets 136
3-1. Polymer Brushes Obtained by ATRP
136
3-2. Optimization of the Polymerization Conditions
138
3-3. Surface Characterization of the pNIPAM-Nanosheets
143
3-4. Evaluation of Thermo-Responsive Property
147
4. Hydrodynamics of Free-Standing pNIPAM-Nanosheet
149
4-1. Thermo-Responsive Behavior in the Free-Standing States
149
4-2. Hydrodynamic Transformation Mediated by Surfactants
152
5. Summary
154
References
156
Chapter 8 Conclusions and Future Prospects
1. Conclusions
158
2. Future Prospects
159
2-1. Endoscopic Surgery with Magnetic Nanosheets
159

Page 8
-v-
2-2. Cell / Tissue Surface Engineering Nanosheets
160
2-3. Biological Facial Fastener
160
2-4. Drug Delivery Devices
161
References
162
Academic achievement
Acknowledgement

Page 9
Chapter 1
- 1 -
Chapter 1
Principle, Design and Construction of Molecular Organized
Nanomaterials
1. Introduction
2. Fundamental Aspects of Self-Assembly and Self-Organization
3. Principles of Intermolecular and Surface Interactions
4. Functional Ultra-Thin Films Based on Molecular Organizations
5. Polymer Nanosheets Utilized in the Biomedical Field
References

Page 10
Chapter 1
- 2 -
1. Introduction
Nature exploits self-assembly and self-organization of soft materials in many
ways, for example, to produce cell membranes, biopolymer fibers and viruses.
Membranes are the most common cellular structures in living body (Fig. 1-1) where
they are involved in almost all aspects of cellular activity ranging from simple
mechanical functions such as motility, food entrapment, and transport to highly specific
biochemical processes such as energy transduction, immunological recognition, nerve
conduction, and biosynthesis, carried by the synergetic assembly of chemical reaction
1
.
Fig. 1-1 Functional membranes in living body.
Mankind is now able to design bio-inspired materials at the nanoscale whether through
atom-by-atom or molecule-by-molecule methods (top-down) or through self-assembly
and organization (bottom-up). The latter method encompasses nanotechnology.

Page 11
Chapter 1
- 3 -
In this chapter, the author describes general statements about molecular
organizations for designing macromolecular organized nanomaterials, particularly
focusing on two-dimensional objects such as film, sheet and membrane. In Section 1.2,
general statements about molecular organizations are described through the overview
about molecular self-assembly and organization including their fundamental aspects. In
Section 1.3, interfacial science, one of the most important principles for constructing
molecular organizations, is explained based on intermolecular and surface interactions
In Section 1.4 and 1.5, general statements about nano (structured) materials is described
including recent development of nanotechnology. Then, fundamental aspect of ultra-thin
films (nanosheets), particularly organic nanosheets, is described by depicting their
recent research highlight from fundamental to application.
2. Fundamental Aspects of Self-Assembly and Self-Organization
2-1. Molecular Organizations Derived from Self-Assembly
Self-assembling is the autonomous organization of components into patterns or
structures without human intervention
2
. Although “Self-assembly” is not a formalized
subject, it can be controlled by proper design of the components if definitions of the
term “self-assembly” is limited to process that involve pre-existing components are
reversible. Self-assembly is the most relevant to nanostructures: that is the spontaneous
assembly of molecules into structured, stable, non-covalently joined aggregate
3
.
Molecular self-assembly combines features of each of the preceding strategies to make
large, structurally well-defined assemblies of atoms: (i) formation of well-defined
molecules of intermediate structural complexity through sequential covalent synthesis;
(ii) formation of large, stable structurally defined aggregates of these molecules through
hydrogen bonds, van der Waals interactions, or other noncovalent links; and (iii) use of

Page 12
Chapter 1
- 4 -
multiple copies of one or several of the constituent molecules, or of a polymer, to
simplify the synthetic task.
There are two main kinds of self-assembly: static and dynamic although some of
the systems include definitions of the term “self-organization”. Static self-assembly (S)
involves systems that are at global or local equilibrium and do not dissipate energy
(Table 1-1; Fig. 1-2 a-c). For example, molecular crystals are formed by static
self-assembly; so are most folded, globular proteins. In static self-assembly, formation
of the ordered structure may require energy (for example in the form of stirring), but
once it is formed, it is stable.
In dynamic self-assembly (D), the interactions responsible for the formation of
structures or patterns between components only occur if the system is dissipating energy
(Table 1-1; Fig. 1-2 d-f). The patterns formed by competition between reaction and
diffusion in oscillating chemical reactions are simple examples; biological cells are
much more complex ones. Moreover, two further variants of self-assembly are defined.
In templated self-assembly (T), interactions between the components and regular
features in their environment determine the structures that form. Crystallization on
surfaces that determine the morphology of the crystal is one example; crystallization of
Table 1-1 Examples of self-assembly (S, static, D, dynamic, T, templated, B, biological)
System
Type
Applications/importance
Atomic, ionic, and molecular crystals
Phase-separated and ionic layered polymers
Self-assembled monolayers (SAMs)
Lipid bilayers and black lipd films
Liquid crystals
Colloidal crystals
Bubble rafts
Macro- and mesoscopic structures
Fluidic self-assembly
“Light matter”
Oscillating and reaction-diffusion reactions
Bacterial colonies
S
S
S, T
S
S
S
S
S or D, T
S, T
D, T
D
D, B
Materials, optoelectronics
Microfabrication, sensors, nanoelectronics
Biomembranes, emulsions
Displays
Band gap materials, molecular sieves
Models of crack propagation
Electronic circuits
Micofabrication
Biological osciilations

Page 13
Chapter 1
- 5 -
colloids in three-dimensional optical fields is another. The characteristic of biological
self-assembly (B) is the variety and complexity of the functions that it produces.
Fig. 1-2 Examples of static and dynamic self-assembly. (a) Crystal structure of
ribosome
4
. (b) Self-assembled peptide-amphiphile nanofibers
5
. (c) An array of
millimeter-sized polymeric plates assembled at a water/perfluorodecalin interface by
capillary interactions
6
. (d) An optical micrograph of a cell with fluorescently labeled
cytoskeleton and nucleus
7
. (e) Reaction diffusion waves in a Belousov-Zabatinski
reaction in a 3.5-inch Petri dish
8
. (f) A simple aggregate of three mm-sized rotating
magnetized disks via vortex-vortex interactions
9
.
2-2. Principles of Self-Assembly
The single feature common to all of these biological structures is the reliance
upon non-covalent self-assembly of preformed and well-defined subassemblies to
obtain the final structure, rather than the creation of a single, large, covalently linked
a)
b)
c)
d)
e)
f)

Page 14
Chapter 1
- 6 -
structure. Biological self-assembly can thus be described by a series of principles that
are often (but not always) obeyed
3
:
1) Self-assembly involves association by many weak, reversible interactions to obtain a
final structure that represents a thermodynamic minimum. Incorrect structural units are
rejected in the dynamic, equilibrium assembly. This equilibration allows high fidelity in
the process.
2) Self-assembly occurs by a modular process. The formation of stable subassemblies
by sequential covalent processes precedes their assembly into the final structure. This
mechanism allows for efficient assembly from the preformed units.
3) Only a small number of types of molecules are normally involved in modular
self-assembly. Consequently, a limited set of binding interactions is required to cause
the final structure to form. This principle minimizes the amount of information required
for a particular structure.
4) Self-assembly often displays positive co-operativity.
5) Complementarities in molecular shape provide the foundation for the association
between components. Shape-dependent association based on van der Waals and
hydrophobic interactions can be made more specific and stronger by hydrogen bonding
and electrostatic interactions.
Because self-assembled structures represent thermodynamic minima, because
they are formed by reversible association of a number of individual molecules, an
because the enthalpies of the interactions holding molecules together are relatively weak,
the interplay of enthalpy and entropy (∆H and ∆S) in their formation is more important
than in synthesis based on formation of covalent bonds (Fig. 1-3). Thereby, the values
of ∆H vary widely depending on the type of molecular interactions that are involved.
The value for TStranslation is based exclusively on considerations of concentration and is

Page 15
Chapter 1
- 7 -
provided only as an approximation. The value for TSconformation is of smaller magnitude
than TStranslation but the sum of many contributions, resulting from freezing
conformations around many bonds in a large, flexible molecule, can make loss of
conformational entropy significant in the thermodynamics of self-assembly processes.
Fig. 1-3 Types of thermodynamic issues that ate involved in molecular self-assembly.
If there are a number of particles associating, and if a number of conformationally
mobile sections of the participating molecules are frozen on aggregation, the sum of
these unfavorable entropic terms can be significant. These considerations suggest that
molecules designed for self-assembly should be as rigid as is consistent with achieving
good intermolecular contact between the interacting surfaces and that the area of
contacting molecular surface be made large. The criteria of rigidity and multipoint
contact are also relatively easily met by using networks of hydrogen bonds in
non-aqueous solvents, and these systems have, in consequence, been extensively
examined as models for self-assembly.
2-3. Principles of Self-Organization
A wide variety of phenomena are regarded as self organization in the various

Page 16
Chapter 1
- 8 -
scientific disciplines, and the definitions applied differ. The different steps in the
evolution of matter from the development of the universe up to the formation of
biological macromolecules and to the origin of life are understood as a chain of
fundamental processes of self organization. The following selection is taken from the
relevant literature
10
.
CHEMISTRY: Self-organization = well-defined structures result spontaneously from
the components of a system by non-covalent forces (self-assembly), for example, in
liquid crystals, micelles, oscillating reactions.
BIOLOGY: Self-organization = a spontaneous building-up of complex structures which
takes place under adequate environmental conditions solely on the basis of the
respective molecular property, namely, without the effect of external factors, for
example, protein folding, formation of lipid double layers, morphogenesis.
PHYSICS: Self-organization = spontaneous formation of new three-dimensional and
temporal structures in complex systems which results from the cooperative effect of
partial systems, for example, ferromagnetism, superconductivity, convection cells.
The non-covalent bond as a required connection between atoms and relies instead
on weaker and less directional bonds, such as ionic bonds, hydrogen bonds, and van der
Waals interactions, to organize atoms, ions, or molecules into structures. Molecular
crystals
11
, liquid crystals
12
, colloids
13
, micelles
14
, emulsions
15
, phase-separated
polymers
16
, Langmuir-Blodgett films
17
, and self-assembled monolayers
18
represent
examples of types of structures prepared with self-organization. The distinguishing
feature of these methods is self-organization. The molecules or ions adjust their own
positions to reach a thermodynamic minimum; the chemist does not specify these

Page 17
Chapter 1
- 9 -
positions.
The ordered state is distinguished by the fact that individual molecules are located
at restricted three-dimensional regions, for example, a lattice site in a crystal or the
position in the three-dimensional structure of a protein. A localization is always
accompanied by a decrease of the number of realizable states and hence a loss of
entropy. Temperature plays always an important role in the case of phase transitions
between different order states because of the contribution TS to the free energy.
Besides temperature, further external fields E may influence the degree of order and the
phase transitions. The field strength and temperature at which the phase transitions take
place can be depicted schematically in phase diagrams (Fig. 1-4). The critical
temperature Tc above which the system is disordered is indicated on the temperature
axis. Phase transitions from the disordered phase (U) to different ordered phases (L, H)
may take place below the critical temperature. These phase transitions are accompanied
by a self-organization of the system. The stability range of the different phases can be
taken from the phase diagram. Different ordered structures for one and the same
material can be produced by varying the temperature and field strength. This variability
has a favorable effect on the production and optimization of materials.
Fig. 1-4 Transition from a disordered phase (U) to ordered phases (H, L) by variation of

Page 18
Chapter 1
- 10 -
the temperature T and the external field E. Tc is the critical temperature above which
ordered phase are not accessible. The transition from U→H, L corresponds to a
self-organization.
10
3. Principles of Intermolecular and Surface Interactions
3-1. Classification of Intermolecular Interactions
Intermolecular interactions were introduced for the first time by van der Waals in
1873: he thus attempted to explain the deviation of the real gas from the ideal gas. In
order to apply the ideal gas law equation to the behavior of real gases, allowance should
be made for the attractive and repulsive forces occurring between molecules. From that
time on, the dipole moment theory by Debye and the dispersion energy or induced
dipole theory by London were the main driving forces of the research about
intermolecular interactions. When two different molecules (A and B) approach, the
molecular energy is changed by the following phenomena (Table 1-2):
(1) overlap of electron clouds of A and B
(2) exchange of electrons
(3) electron transfer or delocalization of localized electrons
(4) changes of the electronic states of A and B (the polarization may be due to the
change in the electron distribution and/or mixing of excited states.
(5) electrostatic interactions (between atomic nucleus of A (B) and electron of B (A),
between two electrons (of A and B) and between two atomic nucleus (of A and B)
(6) dipole-dipole interactions when A and B have dipole moments. These factors never
act individually in real cases; i.e. some factors are observed at the same time
cooperatively or concertedly, or one phenomenon induces another one. These factors
must therefore be discussed into Coulomb forces, hydrogen-bonding forces, van der

Page 19
Chapter 1
- 11 -
Waals forces, charge transfer forces, there are other interactions such as ion-dipole and
solvophobic interactions.
3-2. Molecular Organization of Intermacromolecular Complexes
The Coulomb force acts between two charged molecules, for example,
Na
+
……Cl
-
. According to the point charge model, the energy is described by the
Coulomb law.
Ees = ZA•ZB•e2
/R
( ZA and ZB are valencies of charges of A, B and R is the difference between A and B)
This interaction is characterized by comparatively long-range and relatively strong
forces, being about several tens of kcal/mol and differing from other interaction forces.
In polyelectrolyte systems, the theory is adjusted either to the point charge model
assuming a distribution of point charges on the polymer chain or to the dipole-ion
theory considering an ion pair as a dipole. Their potential energies are expressed as
Uij = qiqj/Drij
Uij = [(μi•μj)-3(μi•rij)(μj•rij)/rij
2
(Drij
3
).
The formation process of polyelectrolyte complexes may be divided into three main
classes (Fig. 1-5):
(1) primary complex formation
Table 1-2 Classification of covalent and non-covalent bond
Classification
Length (nm)
Energy (kcal/mol)
Covalent bond
Electrostatic interaction
Hydrogen bond
Van derWaals interaction (per atom)
0.15
0.25
0.30
0.35
in vacuum
90
80
4
0.1
in water
90
3
1
0.1
Interactions
Ion-Ion
Proton acceptor
– Proton doner
Dipole – Dipole

Page 20
Chapter 1
- 12 -
(2) reformation process within intracomplexes
(3) intercomplex aggregation process.
The first step is realized through secondary binding forces such as Coulomb forces
immediately after mixing oppositely charged polyelectrolyte solutions. This reaction is
very rapid. The second step proceeds within the order of an hour involving the
formation of new bonds and/or the correction of the distortions of the polymer chains.
The third step involves aggregation of secondary complexes mainly through
hydrophobic interactions. Such aggregation is influenced by many factors, e.g. the
structure of the polymer components and the complex conditions.
Fig. 1-5 Schematic representation of the intermacromolecular complexes
19
.
3-3. Intermolecular and Surface Interactions
As any force measurement with respect to intermolecular interactions is in the
below-10-eV category, we should be worrying a variety of data. Therefore, it is better to
draw upon whatever relevant data exists as the situation arises. According to the type of
information on intermolecular and surface forces, different types of measurements

Page 21
Chapter 1
- 13 -
providing different insights and information are categorized here (Fig. 1-6)
20
.
Fig. 1-6 Different types of measurements on the forces between particles and surfaces;
(a) Adhesion measurements (xerography, aerosols, crop dusting and particle adhesion).
(b) Peeling measurements (adhesive tapes and crack propagation). (c) Direct
measurements of forces as a function of distance D (testing theories of intermolecular
forces). (d) Contact angle measurements (detergency, mineral separation processes
using froth flotation, nonstick pans and waterproofed fibers). (e) Equilibrium
thicknesses of thin free films (soap films and foams). (f) Equilibrium thickness if thin
adsorbed films (wetting of hydrophilic surfaces by water, adsorption of molecules from
vapor and drainage of liquid layers). (g) Interparticle spacing in liquids (colloids, paints,

Page 22
Chapter 1
- 14 -
ink and pharmaceutical dispersions). (h) Sheet-like particle spacing in liquids (clay and
soil swelling behavior, microstructure of soaps and biological membrane interactions).
(i) Coagulation studies (basic experimental technique for testing the stability of
colloidal preparations).
3-4. Van der Waals Interactions
Van der Waals interactions play a central role in all phenomena involving
intermolecular interactions, for while they are not as strong as electrostatic or hydrogen
bonding interactions, they are always present. When we consider the long-range
interactions between macroscopic particles and surfaces in liquids we should find that
the two most important forces are the van der Waals and electrostatic interactions and
that at shorter distance (below 1 to 3 nm) salvation forces often dominate over both. Let
us simplify that the interactions is nonretarted and additive. An interatomic van der
Waals pair potential of the form is given
w = -C/r
6
where w is an interaction free energy (J), C is an interaction constant and r is distance
between bodies; one may sum the energies of all the atoms in one body with all the
atoms in the other. Thus, following formulas are obtained in the “two-body” potential
for an atom near a surface as
w(D) = -πCρ/6D
3
for a sphere near a surface as
W(D) = -π
2
Cρ2
R/6D,
or for two flat surfaces as
W(D) = -πCρ2
R/12D
2
where ρ is a number of atomic density. The resulting interaction laws for some common

Page 23
Chapter 1
- 15 -
geometries are shown in Fig. 1-7, given in terms of the conventional Hamaker constant
A = -π
2
Cρ1ρ2.
Typical values for the Hamaker contants of condensed phases, whether solid or liquid,
are about 10
-19
J for interactions in vacuo. The total interaction between any two
surfaces must also include the van der Waals interaction potential is largely insensitive
to variations in electrolyte concentration and pH, and so may be considered as fixed for
any particular solute-solvent system.
Fig. 1-7 Non-retarded van der Waals interaction free energies between bodies of
different geometries calculated on the basis of pairwise additivity.

Page 24
Chapter 1
- 16 -
4. Functional Ultra-Thin Films Based on Molecular Organizations
4-1. Nanomatrials Science Developed by Soft-Nanotechnology
The phrase “nano (structured) materials,” implies two important ideas: i) that at
least some of the property-determining heterogeneity in materials occurs in the size
range of nanostructures (1–100 nm), and ii) that these nanostructures might be
synthesized and distributed (or organized), at least in part, by design (Fig 1-8)
21
.
Fig. 1-8 Nanomaterials science in the complexity as a function of length scale.
Two broad strategies are commonly employed for generating nanomaterials. The
first is “bottom-up
22
”: that is, to use the techniques of molecular synthesis, colloid
chemistry, polymer science, and related areas to make structures with nanometer
dimensions. These nanostructures are formed in parallel and can sometimes be nearly
identical, but usually have no long-range order when incorporated into extended
Functional
Biomolecular
Complex
Polymers &
Supramolecular
Objects
Molecules
Molecules
Subatomic
Particles
Subatomic
Particles
Atoms
Atoms
<< 0.0001 nm
≈ 0.0001 nm
0.4 - 2.0 nm
1.0 - 50 nm
10 - 500 nm
500 - 5000 nm
100000000 nm
Biomacromolecules
& Supramolecular
Objects
High Polymers
&
Nanomaterials
Microtechnology
Cellular Life
Artificial
Intelligence
Biological
Intelligence
Although essentially identical constituents
(with repect to atoms & small molecules)
bio-functionality arises from:
Control of surface functionality (proteins)
Control of molecular orientation
Control of nanoscale arrangement

Page 25
Chapter 1
- 17 -
materials. The second strategy is “top-down
2
”: that is, to use the various methods of
lithography to pattern materials. Currently, the maximum resolution of these patterns is
significantly coarser than the dimensions of structures formed using bottom-up methods.
Materials science needs an accessible strategy to bridge these two methods of formation,
and to enable the fabrication of materials with the fine resolution of bottom-up methods
and the longer-range and arbitrary structure of top-down processes. This bridging
strategy is “self-assembly
2, 23
” and “self-organization
10, 24
”: that is, to allow structures
(in principle, structures of any size, but especially nanostructures) synthesized
bottom-up to organize themselves into regular patterns or structures by using local
forces to find the lowest-energy configuration, and to guide this molecular organizations
using templates fabricated top-down.
4-2. Functional Ultra-Thin Films on the Static Interface
Several techniques have been developed to construct the functional interface for
the materials, utilizing self-assembly and self-organization of (macro)molecules (See
below). For example, the Langmuir-Blodgett film, the layer-by-layer (LbL) film and
self-assembled monolayer (SAM) were constructed on the various interfaces such as not
only solid substrate (glass, gold, mica, polymer SiO2 substrate) but also liquid interface
(water). Therefore, obtained ultra-thin films energetically gain the favorable conditions
owing to the stable and static interface in the self-organization process. Nonetheless,
self-assembly is also important to construct the functional films, which can be
modulated with the molecularly encoded information.
Langmuir-Blodgett (LB) Technique
The molecularly controlled fabrication of nanostructured films has been

Page 26
Chapter 1
- 18 -
dominated by the conceptually elegant Langmuir-Blodgett (LB) technique (Fig.1-9), in
which monolayers are formed on a water surface and subsequently transferred onto a
solid support
25
. Donor and accepter dyes in different layers of LB films provided direct
proof of distance dependent Förster energy transfer on the nanoscale. It is the first true
nanomanipulations that allows for mechanical handling of individual molecular layers
such as separation and contact formation with ångstrom precision
26
.
Fig. 1-9 Langmuir-Blodgett technique.
27
(a) Scheme of a Langmuir-Blodgett trough as
the moveable barrier reduces the area available to the monolayer. (b) Idealized pressure
vs area isotherm indicating phase transitions of the monolayer.
Layer-by-Layer Deposition
One novel methodology for the fabrication of nano-scale materials involving a
wide variety of macromolecules is a layer-by-layer (LbL) technique (Fig. 1-10)
21, 28
.
Fig. 1-10 The layer-by-layer deposition technique
28
.

Page 27
Chapter 1
- 19 -
The LbL method involves alternative adsorption of oppositely charged
polyelectrolytes by different non-covalent linking such as electrostatic interactions,
hydrogen bonding or hydrophobic interactions. Template assisted assembly is much
faster than self-assembly/chemical modification cycles whose outcome is often
uncertain or difficult to predict. For the case of LbL-deposition, it can be tailored to
even allow multimaterial assembly of several compounds without special chemical
modifications
29
, thus giving access to multilayer films whose complex functionality can
fall into the two following categories:
(1) Tailoring of surface interactions: Every object interacts with its environment via its
surface. Thus all properties depending on this interaction are dictated by surface
functionality which can be tailored for many needs (e.g. corrosion protection
30
,
antireflective coatings
31
, antistatic coatings, stickiness or non-stickiness
32
, surface
induced nucleation
33
, antifouling
34
, hydrophilicity or hydrophobicity, biocompatibility
35
,
antibacterial properties, molecular recognition, chemical sensing or biosensing
36
,
microchannel flow control
37
…).
(2) Fabrication of surface based devices: The sequence of deposition of different
materials defines the multilayer architecture and thus the device properties. One may
call this knowledge based (or programmed, or directed, or controlled, or template
assisted) assembly, in contrast to self-assembly. It leads to property engineering by
controlling the mostly one-dimensional spatial arrangement of functionality in
multimaterial layered nanocomposites, which means literally the nanoscopic assembly
of hundreds of different materials in a single device using environmentally friendly,
ultra-low-cost techniques.

Page 28
Chapter 1
- 20 -
Self-Assembled Monolayers (SAMs)
Self Assembled Monolayers (SAMs) based on constituent molecules, such as
thiols and silanes (Fig. 1-11). For SAMs, synthetic chemistry is used only to construct
the basic building blocks, and weaker intermolecular bonds such as van der Waals
bonds are involved in arranging and binding the blocks together into a structure. This
weak bonding makes solution, and hence reversible, processing of SAMs possible. Thus,
solution processing and manufacturing of SAMs offer the enviable goal of mass
production with the possibility of error correction at any stage of assembly.
Fig. 1-11 Self-assembled monolayers consisted of a thiol molecule.
4-3. Modulation for the Integrated Surface
Molecular organizations have been widely accepted and utilized for recent
nanomaterials science and nanotechnology. As one of the most novel nanotechnology by
molecular organizations, it is noteworthy to mention about nano/micro lithography
(Table 1-3). The soft lithography refers to a collection of techniques for creating
microstuructures and nanostuructures based on printing, molding and embossing. The
techniques were developed as an alternative to photolithography and electron-beam
lithography such as ‘stamp (template) fabrication’, and share the name ‘soft
lithography’ because they are based on using a patterned elastmeric polymer as a mask,
stamp or mold, to pattern ‘soft materials’.

Page 29
Chapter 1
- 21 -
Stimuli-responsive surface is one of the attractive research keywords in the
material science, which has been facilitated by surface-initiated polymerization (SIP)
techniques (Fig. 1-12)
38
. The formation of polymer brushes via SIP was a novel
approach utilizing functional polymers such as pH-, photo-, and thermo-responsive
polymers.
39
The “grafting-from” or SIP approach has the benefit of placing the
initiating groups directly on the surface. This allows good control over the grafting
process while various polymerization techniques have been developed for the
preparation of polymer brushes.
Fig. 1-12 (a) Schematic illustration of different process used for the attachment of
polymers to surfaces: (a) “grafting to”; (b) grafting via incorporation of surface-bound
monomeric units; (c) grafting from/surface-initiated polymerization.
Table 1-3 State-of-the-art techniques for Nano/Micro lithography
Miniature Size
100 nm - 10 cm
10 -100 nm
10 -100 nm
> 10 nm
15 - 50 nm
25 -100 nm
1 μm
100 nm - 10 μm
> 1 μm
> 1 μm
> 1 μm
Name
Soft lithography
Micro-contact printing (μCP)
Nano-transfer printing (nTP)
Dip pen lithography (DPN)
Stamp (template) fabrication
Blockcopolymer lithography
Nano-imprint lithography (NIL)
STM lithography
Electron-beam lithography
Ion-beam lithography
Micro engineering
UV-photolithography
Electrochemical etching
Photo oxidation
Notes
PDMS stamp
Free-solvent printing process
Mutiple pens such as AFM tips
Blockcopolymer templates
stamped by quarts mold

Page 30
Chapter 1
- 22 -
Traditional free radical polymerization creates the polymer brushes layer of
thicknesses up to 100 nm with high grafting density involving immobilized initiators
(azo-, peroxide- or photo-initiators) on the surface. However, this chemistry offers poor
control ability over the homogenous brush length around ten-of-nanometers scale.
40
This disadvantage can be overcome by the use of controlled living polymerization
chemistry, such as atom transfer radical polymerization (ATRP) which is more suited
for the preparation of homogenous polymer brush surface on the polymer nanosheets.
4-4. Analytical Tools for the Ultra-Thin Films
As the development nanotechnology, several analytical tools have been employed
for specific investigation of structural and interfacial property of the nanomaterials.
These tools are based on the surface science in the use of various probes such as liquid,
particle, stylus, light, ions, X-ray, electron beam and neutron beam (Table 1-4).
Table 1-4 Analytical tools for nanomaterials
Analytical method
Probe
Liquid
Particle
Stylus
Light
Ions
X-ray
Electron beam
Neutron beam
Static contact angle
Dynamic contact angle
Zeta potential measurement
Atomic force microscopy (AFM)
Lateral force microscopy (LFM)
Chemical force microscopy (CFM)
Kelvin force microscopy (KFM)
Surface force measurement
Infrared reflection absorption spectroscopy (IRAS)
Second harmonic generation (SHG)
Second frequency generation (SFG)
Ellipsometry
UV-visible spectroscopy
Fluorescence microscopy
Secondary ion mass spectroscopy (SIMS)
X-ray photoelectron spectroscopy (XPS)
X-ray reflectivity (XR)
Low energy electron diffraction (LEED)
Reflection high energy electron diffraction
Transmittance electron diffraction (TED)
Neutron reflectivity (NR)
Information
Surface free energy
Surface roughness, Density
Zeta potential
Surface roughness
Molecular cohesion
Adsorption force
Surface potential
Repulsive and attractive force
Chemical groups and bonds
Molecular orientation
Crystallinity
Thickness, Refractive index
Density, Molecular orientation
Chemical groups
Fragmental ions
Elements and chemical groups
Thickness, Density, Roughness
Crystal structure, Orientation
Crystal structure, Orientation
Crystal structure, Orientation
Thickness, Density, Roughness

Page 31
Chapter 1
- 23 -
5. Functional Ultra-Thin Films / Nanosheets Integrated for the Nanobiotechnology
5-1. Biomedical Application of the Functional Ultra-Thin Films
Recent highlights in the biomedical research have been outcome from
regenerative medicine represented by tissue engineering, drug delivery systems (DDS)
and cell transplantation. With respect to the biomedical application of the nanosheets,
they are utilized as the novel nanomaterials such as tissue scaffold and artificial barrier
on the cell surface, synoptic results are shown as follows.
Tissue engineering Approaches
The ease of controlling the structure of LbL films possibly allows us to
manipulate adhesion, differentiation, proliferation, and even function of the attached
cells at the molecular level, which will eventually allow for their application in the
tissue engineering (Fig. 1-13).
41
Fig. 1-13 (a) Schemas of the Hyarulonic Acid (HA) and Poly-lysine (PLL) layering
a)
b)
c)

Page 32
Chapter 1
- 24 -
approach to pattern co-cultures. Patterned co-cultures of (b) embryonic stem cells with
NIH-3T3 fibroblasts and (c) AML12 hepatocyte cells with NIH-3T3 fibroblasts. Scale
bars represent 200 μm.
As shown in Fig. 1-12a, non-biofouling HA micropatterns can prevent the
adsorption of fibronectin proteins and subsequent seeding of a cell type A. In the next
step, ionic adsorption of poly(L-lysine) onto HA patterns was used to switch the HA
surfaces from cell-repulsive to cell-adherent, thereby facilitating the adhesion of a
second cell type B. To adopt this strategy, Khademhosseini et al successfully fabricated
patterned co-cultures of embryonic stem cells with NIH-3T3 fibroblasts (Fig. 1-12b) or
AML12 hepatocyte cells with NIH-3T3 fibroblasts (Fig. 1-12c).
42
The fact that such
co-cultures remained stable for at least five days further confirmed that the patterned
LbL technique can potentially provide a valuable tool for studying cell–cell interactions,
maintaining cells in culture, and engineering organs for the tissue engineering.
Hollow Capsules for Drug Delivery Applicaiton
The biofunctionalization of nano- and microparticles with a specific cell-targeting
ligand offers the potential for delivery of therapeutic-loaded particles to a particular site
or specific cell type in the body. Targeted delivery has several advantages over
nonspecific or passive delivery, including the introduction of a high local concentration
of the therapeutic at the target site and the reduction of undesirable toxic effects of the
drug on other tissues and cells.
Particles that have attracted particular interest as potential drug-delivery carriers
are polymer capsules formed by the layer-by-layer (LbL) assembly technique. Polymer
capsules have the potential to perform the dual role of protecting the body from the

Page 33
Chapter 1
- 25 -
potentially harmful side effects of the therapeutic, while also protecting the therapeutic
from degradation by the body. These capsules are typically formed by the sequential
deposition of interacting polymers onto colloidal particle templates (core/shell particles),
followed by removal of the core/template (Fig. 1-14a). Recently, Caruso et al utilized
the targeting specificity of the clinically relevant huA33 mAb to demonstrate the
specific targeting of LbL particles. The ability of huA33 mAb immobilized on LbL
core/shell particles and polymer capsules to target A33 antigen-expressing LIM1215
colorectal cancer cells has been investigated (Fig. 1-14b).
43
Fig. 1-14 (a) Schematic illustration to demonstrate the formation of core/shell particles
and capsules biofunctionalized with huA33 mAb. (b) Selective uptake of huA33
mAb-coated 500 nm fluorescent core/shell particles by LIM1215 cells.
Surface Improvement of Cells for the Minimum Invasive Transplantation
There have been several problems such as infection and antigenecity, resulting in
the rejection response of patient body. To overcome these problems, cell surface
engineering has been developed by Iwata et al, where they coated the surface of
transplantable islets of Langerhans by using biocompatible amphophilic polymers such
core/shell particle
hollow capsule
core decomposition
huA33 antibody-coated core-shell particles and capsules
cell with
A33 antigen
cell without
A33 antigen
cell with blocked
A33 antigen
biofunctionalization
10 μm

Page 34
Chapter 1
- 26 -
as PEG-conjugated phospholipids (Fig. 1-15).
44
Fig. 1-15 Microscopic images of islets encapsulated by ultra-thin layer-by-layer
membranes of PVA anchored to PEG-conjugated phospholipids.
5-2. Functional Films Exfoliated to Free-Standing Nanosheets
Convenient fabrication or manipulation of nanoscale materials will significantly
enhance the potential applicability of nanomaterials. Ultra-thin films with a sheet-like
structure of nanometer thickness are namely called ‘nanosheets’. Removal of template
such as substrates and core, free-standing nanostructures are obtained (Fig. 1-16).
Fig. 1-16 Basic construction concept of free-standing nanostructures.
a)
b)
Static ultra-thin film
Template: substrate
Free-standing nanosheet
Template: core
Core-shell particle
Hollow particle

Page 35
Chapter 1
- 27 -
It has been reported that the nanosheets possessed unexpectedly compliant and robust
structures from micro-/nanomechanical studies
45
although their size aspect ratio is
10-100 times bigger compared with the conventional bulk films with micrometer sized
thickness. Furthermore, it is attracted nanomaterials because of their potential functions
that are not available in bulk composites (e.g. high flexibility and transparent). Several
fabrication techniques of the free-standing nanosheets are summarized as following
(Table 1-5).
Up to date, fundamental characteristics are focused on the free-standing
nanosheets, rather than application of them. On behalf of nanosheets research, Tsukruk
et al should be described because they demonstrated the application of nanosheets for
the nano/micro sensing device. It has been suggested that these nanocomposite
free-standing LbL nanosheets can be exploited for the fabrication of thermal sensors
with a sensitivity better than 1 mK, which is critical for high-resolution thermal imaging,
with a compact size for portable applications. The mechanical sensitivity measured for
these films is close to 1 nmPa
–1
, which is also promising for sensitive acoustic
microsensing. They have demonstrated that LbL nanosheets (less than 70 nm in
Exfoliation
Solvent assisted transfer
Sacrificial layer
Supporting film
Sacrificial layer
Electroetching
Water evaporation
Sacrificial layer
Sacrificial layer
Sacrificial layer
Solvent assisted transfer
Solvent assisted transfer
Electroetching
Sacrificial layer
Table 1-5 Series of free-standing nanosheets
Film procedure
Organic
Spin-coating
Layer-by-Layer (LbL)
Cross-linked LbL
Spin-coating assisted LbL
Self-assembled monolayer
Direct surfactant-assembly
LbL alternating spray
Langmuir-Blodgett
Cross-linked resins
Filtration of nanofibers
Inorganic
Electropolishing
Silicon electrodepositon
Hybrid
Sol/gel interpenetrating network
References
Forrest (1996)
Kotov (2000)
Whitesides (2003)
Tsukruk (2004)
Eck (2005)
Ichinose (2005)
Decher (2006)
Miyashita (2006)
Kunitake (2007)
Ichinose (2007)
Kruse (2007)
Striemer (2007)
Kunitake (2006)
Materials
Conventional polymers
Polyelectrolytes
Polyelectrolytes
Polyelectrolytes
Biphenylthiol
Surfactants
Polyelectrolytes
Amphiphilic polymers
Thermal cross-linkable resins
Nanofibers
Tantalum oxide
Silicon
Organic/inorganic polymers

Page 36
Chapter 1
- 28 -
thickness) can be suspended over large microfabricated arrays of pre-fabricated
microcavities (Fig. 1-17). Thus, arrays of gas microcavities sealed with a flexible
membrane can be fabricated
46
.
Considering the wide-spreading applications of the conventional static nanosheets
on the solid substrate (for sensing, controlled release and optical detection device), it is
challenging for material scientists to reveal the unique nature of the free-standing
nanosheets and demonstrate the practical application of them.
Fig. 1-17 (a-c) Thermo-optical behavior of the freely suspended nanosheet at various
temperatures below (a), at (b), and above (c) ambient temperature, along with the
corresponding cross sections of the deflected nanosheet.
5-3. Shape Consideration on the Drug Carrier: Beyond Particles
In recent years, much attention has been paid to a minimally invasive treatment in
clinical aspects in the development of novel biomaterials. For example, in internal
medicine, drug delivery systems (DDS) has been developed as a new pharmacological
approach to improve the efficacy and safety of drugs, and wound dressings in surgery
have also been attracting many researchers to fabricate biomaterials with non-viral
compounds. In DDS, vesicles, micelles, emulsions, and biodegradable nanoparticles
have been extensively studied as carriers for biologically active substances such as
c)
b)
a)

Page 37
Chapter 1
- 29 -
drugs, recognition proteins, enzymes, genes, etc
47
. There are two concepts for the
development of DDS, passive and active targeting systems. In the latter case,
recognition proteins such as antibodies and various ligands are conjugated to the surface
of the carriers to target the tissue epitopes or specific cells.
For example, biocompatible and biodegradable nanoparticles are developed
carrying recombinant fragments of platelet membrane proteins
48
and/or dodecapeptide
(H12) derived from fibrinogen as a recognition site for activated platelets
49
. These
nanoparticles specifically recognize the bleeding site or activated platelets showing a
potential applicability as platelet substitutes (Fig. 1-18).
Fig. 1-18 Platelet substitutes: (a) Schematic representation of H12-PEG-polyAlb and (b)
their hemostatic ability evaluated by tail bleeding time of thrombocytopenic rats at three
hours after administration.
In the approach to the conjugation of high and low molecular weight molecules
such as glycoprotein (GP) Ibα and dodecapeptide to the surface of the particle together,
the activity of dodecapeptide was suppressed by the steric hindrance of the GPIbα, and
particle surface
a)
Building block:
albumin
Biocompatible barrier:
Poly(ethylene glycol) (PEG)
Recognition site:
dodecapeptide (H12)
H12-PEG-polyAlb
b)

Page 38
Chapter 1
- 30 -
found that a spacer such as a poly(ethylene glycol) chain was needed in the conjugation
of the peptides (Fig. 1-19a)
50
. On the other hand, sheet-shaped carriers, having both
obverse and reverse surfaces have several advantages over spherical-shaped carriers
because they have a larger contact area for targeting, bilateral structures leading
hetero-functionality by surface modification and unique dynamics caused of high
flexibility (Fig. 1-19b).
Fig. 1-19 Schematic representation of surface-morphological difference between (a)
particle- and (b) sheet-shaped drag carries.
5-4. Micro-Patterned Nanosheets as Potential DDS Carriers
Self-assembled monolayers (SAM/SAMs) have been widely applied to control
physical and chemical properties of the surfaces of glass, quartz, SiO2/Si wafers, or
silica particles
51
. They are excellent tools to study the immobilization of proteins such
as redox proteins
52
, enzymes
53
, and immunoglobulins using covalent or noncovalent
bonds such as ionic or hydrogen bonds, van der Waals interaction, and hydrophobic
interaction with the various terminal groups of SAM.
a)
particle surface
b)
sheet surface
sheet surface

Page 39
Chapter 1
- 31 -
Generally, it is easy to construct patterned SAMs with uniform sizes and shapes
on silicon oxide or gold substrates using a conventional photolithography processes
54
.
This approach is used for the electrochemical analysis of proteins immobilized by
adsorption on the substrates. On the other hand, two-dimensional patterns with steady
repeatability in the particle array have also been achieved by site-selective deposition
using chemical bonding or electrostatic interaction
55
, an electrophotography method
56
, a
micromold method and gravity
57
, a micromold method and a lateral capillary force
58
, a
patterned Au film, and a drying process of a colloidal solution onto the patterned Au
film
59
. Using the patterned SAMs as a template for fabrication of the nanosheet, we
proposed a novel method to fabricate a free-standing nanosheet having heterosurfaces
by a combination of four processes (Fig. 1-20).
Fig. 1-19 Fabrication processes of free-standing nanosheets having hetero-surfaces. (1)
specific adsorption and two-dimensional cross-linking on a patterned
octadecyltrimethoxysilane SAM region (ODS-SAM); (2) surface-modification (obverse
side) of the resulting nanosheet; (3) detachment from the ODS-SAM using surfactant
and water-soluble sacrificial layer; and (4) surface-modification (reverse side).
Si
O
O
O
(n-octadecyltrimethoxysilane;ODS)
(3) Detachment
(2) Surface modification
(obverse side)
(4) Surface modification
(reverse side)
(1) Adsorption & Cross-linking
SiO2 substrate
ODS
photoresist /
UV lithography
O2 plasma
ashing
Patterned ODS-SAM

Page 40
Chapter 1
- 32 -
Furthermore, a novel method for deposition of a close-packed particle
monolayer onto a patterned hydrophilic SAM was also recently reported using a liquid
mold of which the drying process was also described
60
. There is no report on the
preparation of free-standing nanosheets composed of macromolecules or
nanoparticle-based having uniform micrometer shape, nanometer thickness, and
heterogenous surfaces, for use as sheet-shaped carriers (Fig. 1-21)
61
.
Fig. 1-21 Free-standing nanoparticle-fused nanosheets: a) Photo and b) SEM image of
latex bead-fused nanosheets transferred from the ODS-SAM to the PAA film. c) SEM
image of free-standing latex bead-fused sheets after dissolution of the resulting PAA
film. d) Images of TRITC and FITC-labelled latex bead-fused sheets using confocal
laser fluorescence microscopy.
20 μm
1 cm
a)
b)
c)
5 μm
d)

Page 41
Chapter 1
- 33 -
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37. S. L. R. Barker, D. Ross, M. J. Tarlov, M. Gaitan, L. E. Locascio Anal. Chem. 2000,
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38. a) R. C. Advincula, W. J. Brittain, K. C. Caster, J. Rühe (Eds.), Polymer Brushes:

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Synthesis, Characterization, Applications, Wiley-VCH, Weinheim, 2003. b) K.
Matyjaszewski, J. Xia, Chem. Rev. 2001, 101, 2921.
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Macromoleucles 2008, 41, 429.
40. P. Uhlmann et al., Prog. Org. Coat. 2006, 55, 168.
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Surf. A 2008, 318, 184.

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Chapter 2
Construction of Biomacromolecular Nanosheets in the
Development of Nano-Adhesive Plasters
1. Introduction
2. Construction of Free-Standing Biomacromolecular Nanosheets
3. Structural Characterization of Biomacromolecular Nanosheets
4. Ubiquitous Transference of Biomacromolecular Nanosheets
5. Summary
References

Page 46
Chapter 2
- 38 -
1. Introduction
Convenient fabrication or manipulation of nano-scale materials will significantly
enhance the potential applicability of nanotechnology. One novel methodology for the
fabrication of nanoscale materials involving a wide variety of macromolecules is the
layer-by-layer (LbL) technique
1
. The LbL method involves alternative adsorption of
oppositely charged polyelectrolytes by different non-covalent linking such as
electrostatic interactions, hydrogen bonding, or hydrophobic interactions
1a–d
.
Application of LbL-based materials has been explored in several fields, such as
electrochemical devices, chemical sensors, nanomechanical sensors, nanoscale
chemical/biological reactors, and as a drug-delivery system
2
. Nonetheless, potential
applications of the LbL method ubiquitously apply to both the liquid and gas phases.
Therefore, the ubiquitous manipulation of LbL-based nanocomposites is critical to the
development of further functions in nanotechnology.
In this chapter, the author focused on the convenient manipulation of the polymer
nanosheet involving ubiquitous transfer from the liquid-solid surface to the air-solid
surface using a water-soluble sacrificial membrane. This technique of transferring the
polymer nanosheet is the potential advantage of transferring the ultra-thin films from
the conventional solid substrate to any surfaces which are not always solid interface
such as human skin or organs. Therefore, this method was exploited to the construction
of a new biomedical material named as a ‘nano-adhesive plaster’ consisting of
polysaccharides (i.e., polysaccharide nanosheet) or extracellular matrix components
such as collagen and hyaluronic acid (i.e., ECM-nanosheet). Nano-adheisve plaster
ubiquitously released the biomacromolecular nanosheets onto the surface of skin, organ
and cell culture dish.

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- 39 -
2. Construction of Free-Standing Biomacromolecular Nanosheets
2-1. Polysaccharide Nanosheets Obtained by “Sacrificial Layer Method”
In order to fabricate the polysaccharide nanosheet, chitosan and sodium alginate
(Na alginate) were used, which have amino and carboxylic groups as cationic and
anionic polyelectrolytes at ambient pH. These polysaccharides are used in biomedical
fields such as wound dressing and artificial skin because of their biocompatibility and
biodegradability
3
. Recent studies in LbL assembly using biopolymer revealed the utility
of polysaccharides for biomedical applications such as drug delivery and tissue
engineering
4
. Therefore, polysaccharides were selected as building blocks of the
nanosheet. The methodology for the fabrication of the polysaccharide nanosheet utilizes
the SA-LbL method (Fig. 2-1).
Fig. 2-1 Preparative scheme of the free-standing polysaccharide nanosheets by the
sacrificial layer method.
Each polysaccharide was prepared in an aqueous solution containing 0.5 M NaCl in
order to weaken the electrostatic interaction between the polyelectrolytes, thereby
forming a flat smooth surface
5
. The nanosheet with 10.5 pairs of polysaccharide layers
was prepared on a silicon wafer covered with an acetone-soluble photoresist sacrificial
layer; 2-μm thick, composed of a novolac resin and a photoactive compound). Upon
exfoliation of the polysaccharide nanosheet, the sacrificial layer was placed in acetone
Photoresist
SiO2 substrate
Na Alginate
Chitosan
SA-LbL method (4500 rpm, 15 s)
Dipping
(r.t., 20 min)
Release in acetone

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- 40 -
and the transparent nanosheet on the substrate was gradually detached from the edges of
the substrate. After 20 min, the polysaccharide nanosheet was fully detached without
distorting the shape and size of the substrate (approximately 4 cm
2
) (Fig. 2-2a). The
resulting free-standing nanosheet floating in acetone was then washed by exchanging
the acetone three times. This polysaccharide nanosheet was quite stable in acetone or
phosphate buffer saline (PBS, pH 7.4) solution (if exchanged acetone with PBS) for
more than three months. Besides, it could be scooped with a wire loop just as described
by Kunitake et al. using a nanosheet consisting of an organic/inorganic interpenetrating
network
6
. Furthermore, no crack was observed on the nanosheet in the air (Fig. 2-2b).
However, once the nanosheet sustained by the wire loop was damaged with a needle, it
was ruptured and overwhelmed immediately. These results demonstrated that the
nanosheet can be treated in a dried state provided it is sustained by the frame. However,
its configuration (e.g., shape and size) was restricted by the supporting frame size.
Fig. 2-2 Micrograph of a polysaccharide nanosheet: a) detached from the substrate and
floating in acetone or b) in the air supported by a wire loop.
[Materials]
The biodegradable polyelectrolytes, chitosan (Mw = 88 kDa) and sodium alginate
(Na Alginate, Mw = 106 kDa) were purchased from Nacalai Tesque., Inc. (Kyoto, Japan).
OFPR-800 LB photoresist (200 cP) and polyvinyl alcohol (PVA, Mw = 22 kDa) were
(a)
(b)
1 cm

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- 41 -
purchased from Tokyo Ohka Kogyo Co. Ltd. (Kanagawa, Japan) and Kanto Chemical
Co., Inc. (Tokyo, Japan), respectively. Silicon wafers purchased from KST World Co.
(Fukui, Japan), cut to a size of 20 mm x 20 mm, were immersed in a mixture of sulfuric
acid/hydrogen peroxide (3/1) for 10 min and then thoroughly rinsed with deionized
(D.I.) water (18 MΩ cm). A CaF2 substrate for FTIR analysis was purchased from
Sigma Koki Co., LTD. (Tokyo, Japan). Luminescent pigment was purchased from
Sinloihi Co., LTD. (Kanagawa, Japan). Chitosan (1 mg/mL, 1 % acetic acid, 0.5 M
NaCl) and Na alginate (1 mg/mL, 0.5 M NaCl) solutions were prepared with D.I. water.
[Preparation of the polysaccharide nanosheets]
All routines for nanosheet fabrication were conducted in a cleanroom (class
10000 conditions) to avoid contamination. The free-standing polysaccharide nanosheet
was fabricated by a spin-assisted layer-by-layer (SA-LbL) method. A 150 μL solution of
the polyelectrolyte was dropped onto the substrates and then the substrate was rotated at
4500 rpm (rpm = revolutions per minute) for 15 s. Then the substrate was rinsed twice
with D.I. water and dried by spinning (ca. 30 s). According to the above conditions, the
nanosheets were prepared in the following steps: a) spin-coating the photoresist layer
(800 rpm, 3 s and 7000 rpm, 20 s); b) deposition of the chitosan layer (20 min) by
physical adsorption; c) repetition of the chitosan and Na alginate multi-layering by the
SA-LbL method (4500 rpm, 15 s for each polyelectrolyte); d) termination of the
SA-LbL at the chitosan spin-coating stage; e) immersion of the resulting polysaccharide
nanosheet on the substrate for dissolution of the underlying resist layer in acetone.
2-2. ECM-Nanosheets Obtained by “Supporting Film Method”
The extracellular matrix (ECM), once thought to function only as a scaffold to

Page 50
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- 42 -
maintain tissue and organ structure, regulates many aspects of cell behavior, including
cell proliferation and growth, survival, change in cell shape, migration, and
differentiation. It is a complex assembly of many proteins and polysaccharides forming
an elaborated meshwork within tissues (Fig. 2-3)
7
. The primary components are fibrous
structural proteins (e.g. collagens, laminins, fibronectin, vitronectin and elastin),
specialized proteins (e.g. growth factors, small maticellular proteins and small
integrin-binding glycoproteins) and proteoglycans. Therefore, construction of the
biomacromolecular nanosheets derived from ECM components are challenging for not
only understanding the physiological meaning of ECM but also biomedical application
of the polymer nanosheets.
Fig. 2-3 Schematic representation of the ECM structure
8
.
In order to fabricate the ECM-nanosheets, LbL method was chosen, using
collagen and hyaluronic acid as a polycation and polyanion pairs of LbL films. The
components of collagen and hyaluronic acid are major components of ECM, which are
often applied for the artificial cell matrix. Because collagen is a fibrous protein
consisted of repeating Gly-X-Y amino acids (typically, X and Y are Pro and hydroxyl
Pro), the film morphology is affected by the centrifugical force in the SA-LbL method.

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Therefore, the conventional dipping LbL process was employed to construct the stable
LbL film composed of collagen and hyaluronic acid (Fig. 2-4).
Fig. 2-4 Preparative scheme of the free-standing ECM-nanosheets by the supporting
film method.
Each polyelectrolyte was prepared 0.5 mg/mL in an acetate buffer solution (pH
4.7), thereby inducing a stable electrostatic interaction between layers. The nanosheet
with 7.5 layer pairs consisted of Col and HA was prepared on the SiO2 substrate. The
ECM-nanosheet was obtained by ‘the supporting film method’ reported by the
Whitesides group
9
. This method allows the water-mediated collection of the
free-standing polymer nanosheets by peeling a dried bilayered film from a SiO2
substrate. Particularly, this methodology is suitable for a collection of protein included
nanosheets because the procedure does not employ any organic solvent. The bilayered
film consists of the polymer nanosheets supported by a water-soluble thin film such that
the interaction between the bilayered components is higher than that between the
polymer nanosheet and the SiO2 substrate, thereby facilitating the removal of the film.
The resulting bilayered film composed of the ECM-nanosheet (7.5 layer pairs) and an
approximately 70 μm thick PVA membrane was easily peeled off from the edge of the
SiO2 substrate with tweezers and can be released into an aqueous solution by dissolution
of the PVA, resulting in the free-standing ECM-nanosheet (Fig. 2-5).
HA
Col
Acetate
buffer
Acetate
buffer
PVA (10 wt%)
Peel-off
Polycation: Collagen (Col) (Mw: 300 kDa, 0.5 mg/mL)
Polyanion: Hyaluronic acid (HA): Mw: 1400 kDa, 0.5 mg/mL
Dry in vacuo
Dipping LbL
Release in PBS

Page 52
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- 44 -
Fig. 2-5 Micrograph of a free-standing ECM-nanosheet (inside of the dashed circle)
after dissolution of the water-soluble supporting film.
[Materials]
Collagen (Col, Mw = 300 kDa) was purchased from Koken, Inc. (Tokyo, Japan)
and hyaluronic acid sodium salt (HA, Mw = 1400 kDa) were kindly donatecd from
Shiseido, Inc. (Tokyo, Japan). Polyvinyl alcohol (PVA, Mw = 22 kDa) were purchased
from Kanto Chemical Co., Inc. (Tokyo, Japan). Silicon wafers purchased from KST
World Co. (Fukui, Japan), cut to a size of 40 mm x 40 mm, were immersed in a mixture
of sulfuric acid/hydrogen peroxide (3/1) for 10 min and then thoroughly rinsed with
deionized (D.I.) water (18 MΩ cm). Col (0/5 mg/mL, pH 4.7 in the acetate buffer) and
HA (1 mg/mL, pH 4.7 in the acetate buffer) solutions were prepared with D.I. water.
[Preparation of the ECM-nanosheets]
All routines for nanosheet fabrication were conducted in a cleanroom (class
10000 conditions) to avoid contamination. The free-standing ECM-nanosheet was
fabricated by a dipping LbL method. The SiO2 substrate was immersed in a petri-dish
filled with a 10 mL Col solution (0.5 mg/mL) for 3 min, and then the surface of the

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substrate was thoroughly rinsed and immersed in an acetate buffer (pH 4.7) for 1.5 min.
Next, the substrate was immersed in a HA solution (0.5 mg/mL, pH 4.7) for 3 min, and
rinsed with the acetate buffer in the same manner as Col step. According to the above
conditions, the free-standing ECM-nanosheets were prepared in the following steps: a)
repetition of Col and HA multi-layering by the dipping LbL method (3 min for each
polyelectrolyte) and rinse with the acetate buffer (pH 4.7) after each layering; b)
termination of LbL in a Col stage and drying the surface by N2 flow; 3) casting a
supporting layer of a 10 wt% PVA solution on the multilayered substrate for over 12 hrs
until the PVA membrnae was dry; 4) The bilayered film of the ECM-nanosheet and PVA
membrane was peeled off the SiO2 substrate with tweezers. Another such bilayered film
was immersed in water or PBS (pH 7.4) to obtain free-standing ECM-nanosheets.
3. Structural Characterization of Biomacromolecular Nanosheets
3-1. Surface Characterization by AFM (Polysaccharides Nanosheets)
For the morphological study of the polysaccharide nanosheet surface, the
free-standing nanosheet floating in acetone was transferred onto a fresh silicon wafer
and observed by atomic force microscopy (AFM) (Fig. 2-6a and b).
Fig. 2-6 AFM images of the edge of the transferred polysaccharide nanosheet on a SiO2
substrate: (a) top view and (b) cross-sectional image.
(b)
(a)

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From the cross-sectional analysis of the polysaccharide nanosheet edge, the thickness
was estimated to be 30.2 + 4.3 nm (Fig. 2-6b). This thickness is in good agreement with
that determined for the nanosheet prepared on the substrate (30.7 + 4.5 nm), as shown
by an ellipsometric analysis (Fig. 2-7). Therefore, the thickness of the nanosheet was
maintained before and after transference. Because the nanosheet was a LbL film directly
assembled on the silicon wafer by 10.5 pairs of the polysaccharides, the thickness of
one pair of the polyelectrolytes was calculated to be approximately 2.9 nm. This
thickness is in good agreement to that of previously reported LbL films fabricated by
the SA-LbL method using similar molecular weight (i.e., ~10
5
) polyelectrolytes
10
.
Figure 2-7. Ellipsometric thickness of the polysaccharide films as a function of the
number of polysaccharide layer pairs.
3-2. Structural Characterization by FT-IR (Polysaccharides Nanosheets)
The polysaccharide nanosheet was characterized by FT-IR spectroscopy. Samples
were prepared as follows: chitosan was dissolved in a 1 N hydrochloric acid solution
(Figure 2-8a), Na alginate aqueous solution (Figure 2-8b), the polysaccharide nanosheet
(Figure 2-8c), the polysaccharide LbL film, composed of chitosan and Na alginate
directly assembled in the same number of layer pairs as the nanosheet (Figure 2-8d),

Page 55
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and 2.2 μm of photoresist used as a sacrificial layer (Figure 2-8e), were dried on a
calcium fluoride (CaF2) substrate and then analyzed by FTIR spectroscopy.
Figure 2-8. FTIR spectra of various films: (a) chitosan hydrochloride, (b) Na alginate,
(c) polysaccharide nanosheet, (d) polysaccharide LbL film and (e) photoresist.
The vibration bands of the polysaccharide nanosheet were in complete agreement
to those of the LbL film, particularly in the range of the fingerprint region from 1800 to
1300 cm
-1
. Additionally, the 2.2 μm thickness of the photoresist is represented by the
vibration mode of a novolac resin and a photoactive compound (diazonaphthoquinone),
corresponding to the stretching vibration of aromatic methylene (band at 3045 cm
-1
) and
nitrile (band at 2120 cm
-1
). However, these signals were hardly present in the vibration
bands of the nanosheet. From comparison of the bands attributed to the vibration mode
among chitosan hydrochloric acid, Na alginate and the polysaccharides nanosheet, the

Page 56
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- 48 -
main driving force building the nanosheet structure was confirmed to be electrostatic
interaction and hydrogen bonding
11
. For the nanosheet, the asymmetric (band at 1627
cm
-1
) and symmetric (band at 1523 cm
-1
) N-H bending vibrations of non-acylated
2-aminoglucose primary amines derived from the chitosan homo-polymer were
completely absent. This observation suggests that the –NH3
+
of chitosan had reacted
with the –COO
-
of the alginate by electrostatic interaction. Moreover, the band around
3000 cm
-1
(the O-H stretching vibration modes of polysaccharide) of the nanosheet
became broader than that of the chitosan and alginate homo-polymers, which suggested
intermolecular hydrogen bonding between chitosan and alginate was enhanced over the
intramolecular hydrogen bonds of each homo-polymer.
3-3. Surface Characterization by AFM (ECM-Nanosheets)
Thickness of the ECM-nanosheets and their surface morphology was analyzed by
surface profiler and AFM by transferring the free-standing nanosheet in water onto a
silicon wafer. The thickness of the ECM-nanosheet showed an exponential curve in the
function of layer pairs of Col and HA (Fig. 2-9a). One of the reasons for the mechanism
of exponential growth curve is that the adsorbed amount of hyaluronic acid is generally
smaller than the adsorbed amount of Col in the LbL matrix even if it varies with the
layers
12
. HA most likely forms networks composed of double stranded helixes that are
one or two helixes in width. Since the molecular dimension for an HA helix is
approximately 5 Å, HA is adsorbed in the form of flat sheets of HA double helixes. The
Col, however, has notably higher molecular dimensions, and its lowest structural unit is
the microfibril that has a width of 4 nm and approximate length of 500 nm. It might
therefore adsorb with the microfibriles lying in a side-by-side configuration.

Page 57
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Fig. 2-9 Exponential growth curve of the thickness of the ECM-nanosheets as a
function of the number of Col and HA layer pairs measured by a surface profiler.
In the case of 65 nm thick ECM-nanosheet (7.5 layer pairs), formation of the
microfibrils originated from Col was observed on the ECM-nanosheet from AFM
images (Fig. 2-10a). These Col related microfibrils were almost maintained after
exfoliation from the SiO2 substrate (Fig. 2-10b). The size of microfibrils were less than
500 nm in width and approximately 10 μm in size, respectively. It is suggested that each
microfibril is composed of Cols intermolecularly complexed with HA.
Fig. 2-10 The AFM images of the ECM-nanosheets on the SiO2 substrate (a) before and
(b) after exfoliation of the nanosheets.
5.0 μm
5.0 μm
(a)
(b)

Page 58
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- 50 -
[Characterization of biomacromolecular nanosheets]
The biomacromolecular nanosheets were photographed using a digital camera
OLYMPUS C-5050 ZOOM (Olympus Co., Tokyo, Japan). Surface morphology was
observed by AFM in a tapping mode (MFP-3D-BIO, Asylum Research Co., Santa
Barbara, CA and NanoScale Hybrid Microscope, Keyence Co., Tokyo, Japan). A stokes
ellipsometer (Gaertner Scientific Co., Skokie, IL) was also used to measure the
thickness of the nanosheet. The wide-range surface roughness of the PVA membrane as
well as that of the nanosheets was measured with a surface profiler α-step (KLA-Tencor
Corp., San Jose, CA). Characterization of the free-standing nanosheet was confirmed by
FT/IR-410 (JASCO Corp., Tokyo, Japan).
4. Ubiquitous Transference of Biomacromolecular Nanosheets
4-1. Development of “Nano-Adhesive Plaster”
In order to transfer the polysaccharide nanosheet from the surface of one substrate
to another without distorting the overall shape, a hydrophilic sacrificial membrane was
incorporated between the polysaccharide nanosheet and the substrate. The author named
this three-layered composite film a ‘nano-adhesive plaster’ because it was envisaged
that the nanosheet could attach to skin and the substrate was subsequently peeled off by
the dissolution of the sacrificial membrane using water (Fig. 2-11a).
The PVA membrane was chosen as a suitable material for the sacrificial layer
because it is a water-soluble polymer that does not adversely affect the skin. Owing of
its flexibility, silicon rubber was chosen as a substrate for the PVA membrane. The PVA
membrane was prepared by spin-coating of a 20 wt% PVA aqueous solution on a
polypropylene (PP) substrate. The PVA membrane, approximately 1.2-μm thickness
(measured by surface profiler), was then spontaneously released from the

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acetone-insoluble PP substrate within a few seconds by immersion in acetone. The
free-standing PVA membrane was robust and compliant in acetone when picked up with
tweezers. It was also tested that several flexible polymeric substrates that are insoluble
or poorly-soluble in acetone such as polyethylene terephthalate, silicon rubber and
polyvinylchloride. However, PVA was repelled by all of them because the intermediate
hydrophilic/hydrophobic surface of the PP substrate would be suitable. The
free-standing PVA membrane floating in acetone was scooped and then transferred onto
the silicone rubber substrate.
With the resulting PVA-silicone rubber substrate, the polysaccharide nanosheet,
modified with a small amount of commercialized luminescent pigment for ease of
visibility in the dark, was scooped onto the air-solid surface. As a result, three kinds of
free-standing sheets with different thickness were assembled to fabricate the
nano-adhesive plaster; silicone rubber on a milliscale (1.0 mm), PVA membrane on a
micronscale (1.2 μm) and a polysaccharide nanosheet on a nanoscale (30 nm) (Fig.
2-11b). A luminescent-labeled nanosheet was clearly observed in the dark as a square
shape and no cracks or deformations were observed in the nanosheet over a period of a
few months when stored at the ambient temperature. Although the surface roughness of
the PVA membrane was much higher (RMS: 129 + 98 nm) than that of the
polysaccharide nanosheet, any deformation caused by the surface roughness was not
found in the polysaccharide nanosheet, suggesting that the polysaccharide nanosheet
was stable and fitted on the surface of the PVA membrane with good flexibility.
Moreover, the surface roughness of the PVA membrane did not affect that of the
polysaccharide nanosheet when the polysaccharide nanosheet was released from the
silicone rubber because the PVA membrane was dissolved by water easily.

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Fig. 2-11 a) Schematic illustration showing the fabrication of a nano-adhesive plaster. b)
Three kinds of free-standing floating in acetone, photographed in the dark; the surface
of polysaccharide nanosheet was modified with luminescent pigment for ease of
visibility.
[Fabrication of Nano-Adhesive Plaster]
For fabrication of a nano-adhesive plaster, a free-standing PVA membrane as a
sacrificial layer was prepared by spin-coating (800 rpm, 3 s and 7000 rpm, 20 s) of a
PVA aqueous solution (20 wt %) on a polypropylene (PP) substrate (5 cm x 5 cm). The
PVA layer was released in acetone and transferred onto a piece of flexible silicone
rubber. The polysaccharide nanosheet adsorbed on the substrate, before immersion in
acetone, was immersed into an aqueous dispersion of commercialized luminescent
pigment for 20 min. A small amount of luminescent pigment was present on the surface
of the nanosheet. Then the luminescent pigment-labeled nanosheet was released in
acetone from the substrate and transferred onto the surface of the PVA-silicone rubber.
Sequentially, the nanosheet was transferred onto the human skin by dissolution of the
PVA membrane with 500 μL of water.
Spin-coating of
sacrificial layer
PVA membrane
released in acetone
polyvinyl alcohol (PVA)
Silicon rubber
Polypropylene
substrate
Luminescent pigments
labeled nanosheet
Polysaccharide nanosheet
scooped in acetone
Nano-adhesive plaster
Nanosheet released from the
silicon rubber with water
skin
PVA membrane adsorbed on a
silicone rubber as a sacrificial layer
(a)
3 cm
Silicone rubber
Nanosheet
PVA membrane
(b)

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- 53 -
4-2. Ubiquitous Transfer onto the Biological Tissue Surface
Transference to the Skin
To test the practical applications of the nano-adhesive plaster, it was attached onto
the skin at the right arm of a human subject. Prior to transference of the polysaccharide
nanosheet from the PVA-silicone rubber substrate onto the skin, the contour on the
nanosheet surface was observed due to differences in reflectivity (Fig. 2-13a arrow).
The polysaccharide nanosheet was then released from the PVA-silicone rubber substrate
within a few seconds by the dissolution of the PVA layer with a drop of 500 μL D.I.
water through a micropipette. The nanosheet on the skin was barely visible from the top
view under visible light (Fig. 2-13b). Luminescent signals from the modified nanosheet
confirmed that the shape and size of the polysaccharide nanosheet were preserved on
the skin. Furthermore, luminescent regions were barely detected on the removed
silicone rubber side, demonstrating the successful transference of the nanosheet onto the
skin (Fig. 2-13c).
Fig. 2-13 A nano-adhesive plaster on the human skin (a) before the polysaccharide
nanosheet was released from the silicone rubber and (b) after release from the silicone
rubber or (c) same image as (b) except captured in the dark.
(a)
(b)
(c)

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Moreover, the configuration of the polysaccharide nanosheet was stable for at
least 24 hrs, despite perspiration from the skin and the possibility of being rinsed away
by washing with soap. These results indicated that the polysaccharide nanosheet was
released from the silicone rubber by dissolution of the PVA layer with a drop of water
and completely transferred onto the skin. The polysaccharide nanosheet was no longer
visible on the skin, perhaps because the surface relief of the skin perfectly matched that
of the flexible sheet at the nanometer scale. Considering the high biocompatibility and
biodegradability of the LbL ultra-thin films composed of chitosan and Na alginate were
reported by some research groups
13
, the free-standing polysaccharide nanosheet will be
applied in biological systems.
Transference to the Organ
The nano-adhesive plaster was also employed onto the cecum of a living rat. The
adhesion of the polysaccharide nanosheet supported by the water-soluble PVA
membrane was observed from the luminescent signals (Fig. 2-14a). Then the PVA
membrane was dissolved with a few mL drop of saline through the syringe.
Luminescent signals from the modified nanosheet confirmed that the shape and size of
the polysaccharide nanosheet were preserved on the cecum. Furthermore, overall shape
of the polysaccharide nanosheet was completely fitted on the surface relief of the organ
due to high flexibility of the nanosheet and occurrence of the firm tissue-adhesion due
to polysaccharide nanosheet was not observed (Fig. 2-14b). These results indicated
that the polysaccharide nanosheet was successfully transferred onto the tissue surface by
dissolution of the PVA membrane and quite stable on the tissue surface. The author
succeeded into transference of the polysaccharide nanosheet not only onto the skin
surface but also tissue surface. Nonetheless, it would be prospected that the

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nano-adhesive plaster was potentially applicable for the unique surgical or cosmetic
dressing materials.
Fig. 2-14 Nano-adhesive plaster on the rat cecum: a) before and b) after dissolution of
supported PVA membrane. The surface of the nanosheet was modified with luminescent
pigments.
4-3. Ubiquitously Preparative Cell Culture Environment
The ECM-nanosheet is also ubiquitously transferred onto another interface by
using water-soluble supporting film. The author demonstrated the transference of the
ECM-nanosheet onto the polystyrene petri-dish. The HepG2 were cultured in order to
confirm the cell-adhesion property on the ECM-nanosheet. The ECM-nanosheet
supported by the PVA membrane was cut into the desired shape, which was then
transferred onto the petri-dish by dissolution of PVA with water. When the HepG2 cells
were cultured for 10 days, the cells were specifically adhered on the surface of the
ECM-nanosheet whereas the cell adhesion was not observed on the intact petri-dish (Fig.
2-15a). Magnified images showed that the interface between the ECM-nanosheet
overlaid regions was dramatically distinguished by the morphology of the cell adhesion
(Fig. 2-15b and c), thereby the cell adhesion property of the ECM-nanosheet was
confirmed. It is noteworthy that the biomacromolecular nanosheets composed of the
(a)
(b)

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polysaccharides such as chitosan, Na alginate and HA did not show the cell-adhesion
property. Therefore, incorporation of the Col into the biomacromolecular nanosheets is a
useful way to integrate the bio-interface on the nanosheets, which will be applicable for
the construction of the building blocks for the tissue-engineering
14
.
Fig. 2-15 HepG2 cells adhered on the surface of the ECM-nanosheet overlaid on the
petri-dish. Macroscopic image (a) and magnified image, (b) and (c) of the interface
between the HepG2 adhered ECM-nanosheet and the intact petri-dish correspining to
the dashed circle regions in (a).
[HepG2 cell culture]
HepG2 (Human hepatocellular carcinoma cell line) were cultured 10% fetal
bovine serum, penicillin streptomycin solution (x 100) (Wako Pure Chemical Industries,
Ltd., Osaka, Japan) in a humid atmosphere, in 5% CO2 at 37
o
C. After reaching
confluence, the cells were dissociated with 0.05 wt% trypcin–0.53 mmol/L EDTA4Na
solution with phenol red (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The
ECM-nanosheet supported by the PVA membrane was attached on the 60-mm
polystyrene petri-dish (BD Falcon, Co., Franklin Lakes, NJ), and PVA was dissolved by
distilled water and dried. The dish was filled with the HepG2 cells (1 x 10
5
cells/mL)
500 μm
500 μm
(a)
(b)
(c)
1 cm

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and cultured for 10 days, which was observed by an optical microscopy (Olympus, Co.,
Tokyo, Japan).
5. Summary
The author successfully constructed two kinds of biomacromolecular nanosheets
(tens-of-nm thick) with a high aspect ratio of size to thickness (>10
6
). Ubiquitous
transference of the polysaccharide nanosheet from the silicone rubber substrate onto a
human skin or an organ surface was demonstrated by fabricating a three-layered
nano-adhesive plaster. Furthermore, the transferred ECM-nanosheets onto the cell
culture dish showed the cell-adhesion property derived from the incorporation of Col
into the structure. By ubiquitous transference of the free-standing biomacromolecular
nanosheets, ultrathin films have been freed from the conventional solid substrate and the
advantages of the functional ultra-thin films at various interfaces.

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- 58 -
REFERENCES
1. a) Y. Lvov, G. Decher, H. Mohwald, Langmuir 1993, 9, 481. b) Y. Lvov, K. Ariga, I.
Ichinose, T. Kunitake, J. Am. Chem. Soc. 1995, 117, 6117. c) G. Decher, Y. Lvov, J.
Schmitt, Thin Solid Films 1994, 244, 772. d) V. V. Tsukruk, V. N. Bliznyuk, D Visser,
A. L. Campbell, T. J. Buning, W. W. Adams, Macromolecules 1997, 30, 6615. e) G.
Decher, Science 1997, 277, 1232. f) G. Decher, J. B. Schlenoff, Multilayer Thin
Films: Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim,
2003.
2. a) T. Serizawa, M. Tamaguchi, M. Akashi, Biomacromolecules 2002, 3, 724. b) L.
Zhai, F. C. Cobeci, R. E. Cohen, M. F. Rubner, Nano Lett. 2004, 4, 1349. c) K. Sano,
H. Sasaki, K. Shibata, J. Am. Chem. Soc. 2006, 128, 1717. d) C. Jiang, V. V. Tsukruk,
Adv. Mater. 2006, 18, 829. e) Z. Tang, Y. Wang, P. Podsiadlo, N. A. Kotov, Adv.
Mater. 2006, 18, 3203.
3. a) M. N. V. R. Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. J. Domb,
Chem. Rev. 2004, 104, 6017. b) H. Yi, L. Wu, W. E. Bentley, R. Ghodssi, G. W.
Rubloff, J. N. Culver, G. F. Payne, Biomacromolecules 2005, 6, 2881. c) I. Liao, A. C.
A. Wan, E. K. F. Yim, K. W. Leong, J. of Controlled Release. 2005, 104, 347.
4. a) T. Serizawa, M. Yamaguchi, M. Akashi, Biomacromolecules 2002, 3, 724. b) Z.
Tang, Y. Wang, P. Podsiadlo, N. A. Kotov, Adv. Mater. 2006, 18, 3203.
5. a) S. L. Clark, M. F. Motague, P. T. Hammond, Macromolecules 1997, 30, 7237. b) J.
Cho, H. Jang, B. Yeom, H. Kim, R. Kim, S. Kim, K. Char, F. Caruso, Langmuir 2006,
22, 1356.
6. R. Vendamme, S. Onoue, A. Nakao, T. Kunitake, Nature Mater. 2006, 5, 494.
7. W. P. Daley, S. B. Peters, M. Larsen, J. Cell Sci. 2007, 121, 255.
8 B. Alberts et al., Molecular Biology of the Cell, Garland Science, New York, 2002.
9. A. D. Stroock, R. S. Kane, M. Weck, S. J. Metallo, G. M. Whitesides, Langmuir 2003,
19, 2466.
10. a) J. Cho, K. Char, J. Hong, K. Lee, Adv. Mater. 2001, 13, 1076. b) J. Cho, K. Char,
Langmuir 2004, 20, 4011. c) C. Jiang, S. Markutsya, V. V. Tsukruk, Adv. Mater. 2004,
16, 157.
11. M. G. Sankalia, R. C. Mashru, J. M. Sankalia, V. B. Sutariya, Eur. J.

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Pharm.Biopharm. 2007, 65, 215-232.
12. J. Johansson, T. Halthur, M. Herranen, L. Sderberg, U. Elofsson, J. Hilborn,
Biomacromolecules 2005, 6, 135.
13. a) Y. Yang, Q. He, L. Duan, Y. Cui, J. Li, Biomaterials 2007, 28, 3083. b) A. L.
Hillberg, M. Tabrizian, Biomacromolecules 2006, 7, 2742.
14. a) R. Langer, J. P. Vacanti, Science 1993, 14, 920. b) K. Nishida et al., N. Engl. J.
Med. 2004, 351, 1187.

Page 68

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Chapter 3
Optical Property of the Biomacromolecular Nanosheets
through Macro/Microscopic Surface Characterization
1. Introduction
2. Fundamental Principle of Thin Film Interference and Structural Color
3. Microscopic Surface Morphology of Polysaccharide Nanosheets
4. Analysis of the Structural Colors of Polysaccharide Nanosheets
5. Summary
References

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- 62 -
1. Introduction
In this chapter, the surface morphological property of the biomacromolecular
nanosheets was investigated using several analytical methods such as atomic force
microscope, scanning electron microscopy and spectroscopy. In the basis of ‘thin film
interference theory’, it was known that the stepwise increments of the ultra-thin
multilayer showed sequential structural color changes. The author adopted this
phenomenon to the sequential assembly of free-standing polysaccharide nanosheets
onto the SiO2 substrate, which will be a fundamental and essential finding to utilize the
free-standing nanosheets for further application such as a dressing material.
2. Thin Film Interference Theory and Structural Color
2-1. Structural Colors in Nature
Coloring in nature mostly comes from the inherent colors of materials, but it
sometimes has a purely physical origin, such as diffraction or interference of light. The
latter, called structural color or iridescence, has long been a problem of scientific
interest. Recently, structural colors have attracted great interest because their
applications have been rapidly progressing in many fields related to vision, such as the
paint, automobile, cosmetics, and textile industries. As the research progresses, however,
it has become clear that these colors are due to the presence of surprisingly minute
microstructures, which are hardly attainable even by ultramodern nanotechnology.
Fundamentally, most of the structural colors originate from basic optical processes
represented by thin-film interference, multilayer interference, a diffraction grating effect,
photonic crystals, light scattering, and so on (Fig. 3-1). However, to enhance the
perception of the eyes, natural creatures have produced various designs, in the course of
evolution, to fulfill simultaneously high reflectivity in a specific wavelength range and

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the generation of diffusive light in a wide angular range. At a glance, these two
characteristics seem to contradict each other in the usual optical sense, but these
seemingly conflicting requirements are realized by combining appropriate amounts of
regularity and irregularity of the structure.
Fig. 3-1 Hierarchy of the structure is contributing to structural colors
1
. The most
fundamental scale is that giving the interference of light around 0.2 μm. The regular
structures contributing to the light interference are retained within a range indicated as a
gray zone. If this range is narrow, the diffraction of light plays an important role,
whereas if broad, the geometrical optics dominates. In either case, this range helps to
produce diffusive light. The hierarchy in a larger scale indicates the irregularity at
various levels and possibly adds the macroscopic texture to the structural color.
2-2. Fundamental Optical Processes
Thin-film interference is one of the simplest structural colors and is widely
distributed in nature. Consider a plane wave of light that is incident on a thin layer of
thickness d and refractive index nb at the angle of refraction θb (Fig. 3-2). Then, the
reflected light beams from the two interfaces interfere with each other. In general, the

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condition of interference differs whether the thin layer is or is not attached to a material
having a higher refractive index. The former is the case for antireflective coatings on
glass, while a typical example of the latter is a soap bubble. The condition of
interference for light with wavelength λ, under which the reflection is enforced
(constructive interference), becomes an equation (1)
1
:
mλ = 2(nbdcosθb)
(1)
where m is an integer for antireflective coatings, while it is a half integer for the
soap-bubble case. Typical examples of the calculations for both cases are shown in Fig.
3-2. It is clear that the reflectivity is relatively low and changes smoothly with
wavelength. Thus, the thin-film interference shows only weak dependence on the
wavelength. It is easily understood from the eq. (1) that the wavelength showing a
maximum reflectivity changes continuously to a shorter wavelength as the incident
angle is increased. Thus, one of the characteristics of structural colors, that the color
changes with viewing angle, is reproduced.
Fig. 3-2 Thin-film interference; (a) configuration, (b) and (c) reflectivity from a thin
film (n = 1.25) with a thickness of 100 nm (b) in air and (c) attached to a material with a
higher refractive index (n = 1.5). Solid and dashed limes are the calculated curves.

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3. Microscopic Surface Morphology of Polysaccharide Nanosheets
3-1. Spin-Coating Assisted Structural Color Changes
During the spin-coated fabrication of the SA-LbL film, a structural color change
was observed on the surface of the SiO2 substrate as the film thickness increased. It was
found that the structural color was controlled by changing the number of layers of the
nanosheet adsorbed on the substrate (Fig. 3-3). With a bare SiO2 substrate surface, we
noted a bronze-yellow color at an observation angle of 85
o
. Soon after placing a
free-standing polysaccharide nanosheet on another SiO2 substrate from acetone, it was
observed that the structural color of the sample started to change from bronze-yellow,
gradually turning to red, as the remaining acetone was aspirated. Interestingly, once the
nanosheet was adsorbed on the substrate and dried in the air, it remained attached to the
substrate, even when immersed in acetone again. Furthermore, it possessed a large
contact area, with quite a flat and smooth surface, and its extreme thinness made it very
flexible.
Fig. 3-3 The microscopic images of structural colors for the individual polysaccharide
LbL films. Each picture was taken under optical microscope with respect to the
thickness of the LbL films. Scale bar of pictures shows all 50 μm.
0 nm (silver bronze)
35 nm (yellow)
58 nm (pale red)
85 nm (blue)
114 nm (light blue)
136 nm (green)

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3-2. Surface Characterization by AFM
For the morphological study of the polysaccharide nanosheet surface, the
free-standing nanosheet floating in acetone was transferred onto a fresh silicon wafer
and observed by atomic force microscopy (AFM). Large-scale (90 μm x 90 μm)
topographic images shown in Fig. 3-4 revealed that the nanosheet surface was as
smooth and flat as the silicon wafer surface without any corrugations and wrinkles.
These topographical results were obtained due to the high-speed horizontal diffusion of
the polymers during spin-coating
2
. Furthermore, scanning the surface morphology of
the polysaccharide nanosheet with a surface profiler in the range of 500 μm showed the
significant difference in surface roughness (root-mean squared (RMS) values) of 1.9 +
0.7 nm (0.5 M NaCl) and 7.1 + 2.4 nm (0 M NaCl) although the significant difference in
thickness of the polysaccharide nanosheet was not found. Hence, the relatively high
ionic strength (0.5 M NaCl) of the polyelectrolyte aqueous solution during fabrication
(i.e., using the SA-LbL method) presumably weakened the electrostatic interactions of
the polymers, generating a flat smooth surface nanosheet in the overall morphology of
the polysaccharide nanosheet
3
.
Fig. 3-4 AFM 3-dimensional image of the edge of the transferred polysaccharide
nanosheet on a SiO2 substrate.

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3-3. Surface Characterization by SEM
The microscopic picture is obtained by scanning electron microscopy (SEM). SEM
observations of the polysaccharide nanosheets were undertaken using a HITACHI
S-4300 (Hitachi Co., Tokyo, Japan). Samples on a porous alumina membrane (Anodisc
®
,
Whatman Ltd., Maidstone, UK) were coated with a platinum layer using an
ion-sputtering coater (HITACHI E-1045; 18 mA, 40 s., Hitachi Co.). The
polysaccharide nanosheet was transparent and tightly adhered on the porous alumina
disk that underlaied pores were confirmed (Fig. 3-5). The thickness of the
polysaccharide nanosheet film, measured using a surface profiler, was determined to be
39 ± 0.9 nm with 10.5 layer pairs of polysaccharides.
Fig. 3-5 SEM image of the polysaccharide nanosheet with a thickness of 39 nm on a
porous alumina disk.
4. Analysis of the Structural Colors of Polysaccharide Nanosheets
4-1. Sequential Structural Color Changes
When one or more 30-nm polysaccharide nanosheets was overlaid on the original
nanosheet, it was observed that the sequential unique structural colors on the surface of
the substrates shown in Fig. 3-6, depending on the number of overlaid nanosheets: red
1 μm
35-nm polysaccharide nanosheet
Alumina disk

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in the single layer (Fig. 3-6a), blue in the double layers (Fig. 3-6b) and green in the
triple layers (Fig. 3-6c).
Fig. 3-6 Micrographs of (a) red in the single, (b) blue in the double and (c) green in the
triple layered polysaccharide nanosheets adsorbed on a SiO2 substrate.
To further analyze the structural color changes, it was obtained that the UV-Vis
reflective spectrum from the SiO2 substrate surface where different numbers of
polysaccharide nanosheets were adsorbed. The author used an incident light angle of 5
o
(= 90
o
-85
o
), corresponding to the observation angle of Fig. 3-5, so that the visible color
change could be accurately compared with the UV-Vis reflective spectrum. As the
number of adsorbed nanosheets increased, the maximum wavelength for each spectrum,
shown in Fig. 3-7, lengthened also; it was identified that the wavelengths as 400 and
700 nm in the single layer, 420 and 700 nm in the double layers, and 530 nm in the
triple layers, respectively. These values corresponded to the structural colors of the
overlaid nanosheets shown in Fig. 3-6. Evidently, while the air-solid interface remained
smooth and flat, the structural color at the air-solid interface formed by the adhesion of
the nanosheets changed upon each nanometer-scale stepwise increment in the total
thickness.
Single : red
Double : blue
Triple : green
a)
b)
c)

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Fig. 3-7 UV–vis reflective spectra between 400 and 700 nm of (a) single, (b) double and
c) triple polysaccharide nanosheets adsorbed on an SiO2 substrate, measured at the same
observation angle as in Fig. 3-6.
4-2. Analysis of the Structural Color Changes
The author applied ‘thin film interference theory’
4
to explain the changes
observed in the colors of the sequential assembled polysaccharide nanosheets layers on
the SiO2 substrate. It is expected that, generally, a flat and smooth ultra-thin film on a
substrate, having an optical thickness less than the wavelength of visible light (< 800
nm) will show such a structural color change due to optical interference effects. The
color of the samples, made up of two kinds of thin films, is described by the following
eq. (2):
mλ = 2(n1d1cosθ1+n2d2cosθ2)
(2)
where m, λ, n1 and n2, d1 and d2, θ1 and θ2 are defined as the order of the dominant
reflection, the wavelength of the dominant reflection, the refractive index, the layer
thickness, and the angle of refraction of the light through the film 1 and 2, respectively
(Fig. 3-8).
R
e
fle
ctivity (-
(a
.u
.))
Wavelength (nm)
400
500
600
700
a
b
c

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- 70 -
Fig. 3-8 Schematic representation of the thin film interference theory.
This equation indicates that the reflected light wavelength is proportional to the film
thickness when the refractive index of the thin film and the substrate are known and the
light incident angle is fixed. For this case, it is assigned that the polysaccharide
nanosheet as the film 1, and as film 2, the SiO2 layer of the silicon wafer substrate;
ellipsometry showed that d2 and n2 had values of 200 nm and 1.46. Hence, it should be
able to verify the variation of λ with the thickness of the polysaccharide nanosheet, d1.
The maximum in the reflected wavelength in the visible wavelength region
between 400-800 nm was plotted against the total thickness of the overlaid 30 nm
polysaccharide nanosheets on the SiO2 substrate (Fig. 3-9, triangles). In order to better
evaluate the structural color changes with film thickness, the author also produced the
polysaccharide LbL films which were directly prepared on the SiO2 substrate by the
SA-LbL method and observed under the optical microscope at the same observation
angle of Fig. 3-6; their wavelength maxima was then also plotted (Fig. 3-9, circles). The
lines in Fig. 3-9 represent theoretical predictions on the basis of eq. (2), using refractive
index values found by ellipsometry of 1.52 (n1) for the polysaccharide nanosheet and
1.46 (n2) for the bare SiO2 layer that was 200 nm thick (d2)
5
. Moreover, the angles of
refraction (θ1, θ2) were almost perpendicular to the substrate surface, so that cosθ1 and
d1
d2
θ1
θ2
n1
n2
mλ = 2(n1d1cosθ1+n2d2cosθ2)
λ
Film 1
Film 2

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- 71 -
cosθ2 should equal 1. Hence eq. (2) can be rewritten as follows:
mλ = 3.04d1 + 588
(3)
where d1 and m are the ellipsometric thickness of the overlaid polysaccharide nanosheet
or the polysaccharide LbL film and the order of the interference, respectively. For m = 1
eq. (3) predicts the theoretical line that is shown overlaid on the plots in Fig. 3-9 of data
for a maximum wavelength between 588 and 800 nm, and in the case of m = 2 the line
corresponds to the data between 400 and 522 nm.
Fig. 3-9 Maximum reflectivity of the polysaccharide nanosheet or LbL film plotted
against ellipsometric thickness. Solid lines show theoretical predictions, based on thin
film interference theory.
The good fit of the reflectivity data to these lines suggests that the stepwise
thickness increment, created by overlaying the free-standing 30-nm nanosheet on the
SiO2 substrate, was the main determinant of the shift of the dominant reflective
wavelength. Furthermore, this agreement indicates that the surface of the polysaccharide
nanosheet was quite flat and smooth, and that each overlaid nanosheet must have been
closely adsorbed onto the one underlying it. Furthermore, it was able to obtain LbL

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ultra-thin films with a desired nanometer thickness by overlaying the nanosheets
themselves, without employing a conventional LbL process, such as multi-layering of
polyelectrolytes. In addition, whatever charged surface faced the other, the
polysaccharide nanosheets was overlaid, individually; the main driving force of the
adhesion between the nanosheets must have been the van der Waals interaction, rather
than electrostatics.
5. Summary
The optical characteristics of the polysaccharide nanosheets were analyzed on the
basis of ‘thin film interference theory’ by making stepwise increments in thickness
through sequentially overlaying free-standing 30-nm polysaccharide nanosheets onto a
SiO2 substrate. This result clarified that the surface of the polysaccharide nanosheet was
macroscopically quite flat, smooth and physically adhesionable onto the desired surface
including the nanosheets themselves.

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References
1. S. Kinoshita, S. Yoshioka, ChemPhysChem, 2005, 6, 1442.
2. a) J. Cho, K. Char, J. Hong, K. Lee, Adv. Mater. 2001, 13, 1076. b) J.Cho, K. Char,
Langmuir 2004, 20, 4011. c) C. Jiang, S. Markutsya, V. V. Tsukruk, Adv. Mater. 2004,
16, 157.
3. a) S. L. Clark, M. F. Motague, P. T. Hammond, Macromolecules 1997, 30, 7237. b) J.
Cho et al., Langmuir 2006, 22, 1356.
4. a) C.L. Schauer et al., Thin Solid Films 2003, 434, 250. (b) J.A. Radford, T. Alfrey Jr.,
W.J. Schrenk, Polym. Eng. Sci. 1973, 13, 216.
5. M.D. Cathell, C.L. Schauer, Biomacromolecules 2007, 8, 33.

Page 82

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Chapter 4
Evaluation of Adhesive and Mechanical Property of the
Biomacromolecular Nanosheets
1. Introduction
2. Water Mediated Preparation of Polysaccharide Nanosheets
3. Physical Adhesive Property of Polysaccharide Nanosheets
4. Mechanical Property of Polysaccharide Nanosheets
5. Summary
References

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Chapter 4
- 76 -
1. Introduction
Considering the biomedical application of the biomacromolecular nanosheets as a
wound dressing material, not only surface property but also structural properties such as
robustness, flexibility and adhesiveness should be investigated, which is the attractive
properties of the nanosheets, derived from the film’s huge size-aspect ratio. In this
chapter, versatile methodology of constructing biocompatible polysaccharide
nanosheets was described without using organic solvents. Then, fundamental structural
properties were evaluated by micro-scratching test and bulge test, envisaging the
biomedical application of the polysaccharide nanosheets for sheet-shaped biomaterials..
2. Water Mediated Preparation of Polysaccharide Nanosheets
2-1. Total Preparation in Aqueous Condition
A polysaccharide nanosheet was fabricated by a spin-coating assisted
layer-by-layer (SA-LbL) method
1
, using chitosan and sodium alginate (Na alginate)
polyelectrolyte solutions on a SiO2 substrate. Chitosan and Na alginate were chosen for
the components of the polymer nanosheet because of their biocompatibility and
biodegradability, which are typical for components of wound-dressings
2
. However,
polysaccharides are generally brittle because of high crystallinity introduced by inter-
and intra-molecular secondary bonding
3
. Therefore, we focused on the SA-LbL
approach because this method produced a versatile ultra-thin film with a flat and smooth
surface by multi-layering of the polysaccharides in close to single molecule layers free
from deformation. In order to obtain a free-standing polysaccharide nanosheet from a
substrate, we previously used a sacrificial layer in order to detach the polysaccharide
nanosheet from the substrate, which can be applicable until approximately 9 cm
2
when
the thickness is tens of nanometer.

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In order to obtain a large nanosheet, we chose to adopt the ‘supporting film’
method
4
. This method allows the convenient collection of free-standing polymer
nanosheets by peeling a dried bilayered film from a SiO2 substrate. The bilayer film
consists of the polymer nanosheets supported by a water-soluble thin film such that the
interaction between the bilayer components is higher than that between the polymer
nanosheet and the SiO2 substrate, thereby facilitating the removal of the film. Following
the method shown in the schema (Fig. 4-1a), a 10 wt% concentrated poly(vinyl alcohol)
(PVA) aqueous solution was cast on a polysaccharide covered substrate and dried for 12
hrs until a robust PVA film was obtained. The resulting bilayered film composed of the
polysaccharide nanosheet with the thickness of several tens of nanometers and an
approximately 70 μm thick PVA film was easily peeled off from the edge of the SiO2
substrate with tweezers and can be released into an aqueous solution by dissolution of
the PVA film. This water-mediated release methodology is suitable for a wound dressing
because the procedure does not employ any organic solvent.
Fig. 4-1 Schematic representation of the fabrication method for a free-standing
polysaccharide nanosheet using the water-soluble supporting PVA film.
[Materials]
The biodegradable polyelectrolytes, chitosan (Mw = 88 kDa) and sodium alginate (Na
SA-LbL of chitosan and Na alginate
(4500 rpm, 15s)
SiO2 substrate
PVA cast
(r.t., 12 hrs)
Dissolution of PVA in PBS
Peeling-off of (nanosheet + PVA)
film with tweezers

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Alginate, Mw = 106 kDa), were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).
Poly(vinyl alcohol) (PVA, Mw = 22 kDa) was purchased from Kanto Chemical Co., Inc.
(Tokyo, Japan). Silicon wafers (SiO2 substrates) purchased from KST World Co. (Fukui,
Japan) cut into a proper size (typically 4 cm
2
) were immersed in a mixture of sulfuric
acid/hydrogen peroxide (3/1) for a 10 min wash, and were then thoroughly rinsed with
deionized (D.I.) water (18 MΩ cm).
[Preparation of polysaccharide nanosheets]
All preparation routines for polysaccharide nanosheet fabrication were conducted in a
clean room (class 10000 conditions) to avoid contamination. Chitosan (1 mg/mL, 1 %
(v/v) acetic acid) and Na alginate (1 mg/mL) solutions were prepared with D.I. water. A
1 mL solution of chitosan as a first layer was dropped onto the SiO2 substrate and then
the substrate was rotated at 4500 rpm (rpm = revolutions per minute) for 15-20 s. Then,
the substrate was rinsed twice with D.I. water and dried by spinning (ca. 30 s). Then,
nanosheets were prepared via the following steps: 1) repetition of chitosan and Na
alginate multi-layering by a SA-LbL method (4500 rpm, 15 s for each polyelectrolyte)
and rinsing by water spin-coating after each layering; 2) termination of SA-LbL in a
chitosan spin-coating stage and drying the surface by N2 flow; 3) casting of a supporting
layer of a 10 wt% PVA aqueous solution on the polysaccharide multilayered substrate
for over 12 hrs until the PVA film was dry; 4) The bilayered film of the polysaccharide
nanosheet and PVA used for wound repair of beagle dogs was peeled off the SiO2
substrate with tweezers. Another such bilayered film was immersed in water or PBS
(pH 7.4) in order to obtain the polysaccharide nanosheet, which was then scooped onto
a bare SiO2 substrate. Then the polysaccharide nanosheet on the substrate was left until
the water was aspirated in a desiccator.

Page 87
Chapter 4
- 79 -
2-2. Structural Characterization
The overall shape of the bilayered film corresponded to that of the substrate (Fig.
4-2a). As the bilayered film was gently immersed into a petri dish filled with a
phosphate buffer saline (PBS: pH7.4, 37
o
C) solution, the water-soluble PVA layer was
immediately dissolved to obtain an ultra-thin film in a free-standing state that was
approximately 4 cm in diameter, corresponding in size and shape to those of the original
SiO2 substrate (Fig. 4-2b). The resulting free-standing nanosheet was transparent, very
flexible and not swelled in aqueous condition; it could be flapped by manipulating with
tweezers. Moreover, it is the largest non-covalently bonded free-standing polymer
nanosheet obtained in aqueous condition, which has ever been reported as far as we
know. We next microscopically observed the surface morphology of the polysaccharide
nanosheet consisting of 20.5 layer pairs of polysaccharides deposited on a porous
alumina membrane (Anodisc
®
) using a scanning electron microscope (SEM) and found
a smooth and flat surface sheet that was 75 nm thick (Fig. 4-2b inset).
Fig. 4-2 Polysaccharide nanosheet obtained by supporting film method. (a) Bilayered
film, consisting of a 75-nm polysaccharide LbL ultra-thin film and a 70 μm
water-soluble PVA supporting film, peeled from a SiO2 substrate. (b) A free-standing
75-nm polysaccharide nanosheet floating in PBS approximately 4 cm in diameter,
retaining the shape of the SiO2 substrate. Inset shows a SEM cross-sectional view of the
2 cm
(a)
2 cm
(b)
1 μm

Page 88
Chapter 4
- 80 -
75-nm polysaccharide nanosheet on an alumina porous membrane (the white arrows
indicate the locations of the polysaccharide nanosheet).
The specific thickness of the nanosheet was measured using a probe-type
scanning surface profiler on the SiO2 substrate after three exchanges of PBS, resulting
in a value of 35 ± 2.3 nm when we used 10.5 layer pairs of polysaccharides in the
preparation, and corresponds to the thickness before PVA casting (i.e., 35 ± 2.1 nm,
measured by the surface profiler). The root-mean-square surface roughness was as quite
low, at 2.4 ± 0.52 nm. A single layer pair of the nanosheet was calculated to be
approximately 3.3 nm by dividing the thickness the number of layer pairs
5
. This value
indicates that, each polysaccharide was assembled inside of the nanosheet in almost a
single molecular layer. Following this result, we prepared several thicknesses of
polysaccharide nanosheets by controlling the number of layers during SA-LbL, the
thickness being proportional to the number of layer pairs respectively (Fig. 4-3).
Fig. 4-3 Thickness of the polysaccharide nanosheets before (●) and after (▲)
detachment from the SiO2 substrate, measured by surface-profiler as a function of the
number of polysaccharide layer pairs.

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- 81 -
Although the typical cast polysaccharide film that was over 1 micrometer in
thickness tended to be brittle, the free-standing polysaccharide nanosheets were highly
flexible. This is due to the appearance of a “liquid-like” layer inside of the nanosheet,
resulting from the relaxation of the individual polymer chains on decreasing the
thickness of the film
23
. Moreover, a polysaccharide nanosheet mounted on a wire loop
was very stable for at least three month in dry conditions, and was quite stable with
respect to maintaining its shape and thickness in PBS (pH 7.4) over three months.
Therefore, we had succeeded in the versatile fabrication of a free-standing
polysaccharide nanosheet using a water-soluble supporting film.
[Characterization]
The surface morphology, such as thickness and root-mean-square (RMS) roughness,
was analyzed by a α-step surface profiler (KLA-Tencor Corp., San Jose, CA) and the
overall morphology of the polysaccharide nanosheet was photographed using a
OLYMPUS C-5050 ZOOM digital camera (Olympus Co., Tokyo, Japan). The SEM
observation of the polysaccharide nanosheets was undertaken with HITACHI S-4300
at an accelerating voltage of 10 kV, using samples on a porous alumina membrane
(Anodisc
®
, Whatman Ltd., Maidstone, UK) coated with a platinum layer (ca. 40 nm
thickness) using an ion-sputtering coater HITACHI E-1045. (Hitachi Ltd., Tokyo,
Japan).
3. Physical Adhesive Property of Polysaccharide Nanosheets
3-1. Micro-Scratching Test
In order to determine the optimum thickness of the polysaccharide nanosheet for
in vivo tissue-defect repair usage, the micro-scrach test and bulge test was performed.

Page 90
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- 82 -
We utilized the micro-scratch test (Fig. 4-4)
6
for the evaluation of the macroscopically
adhesive property of the ultra-thin films such as nanosheets.
The micro-scratch tester (CSR-2000, Rhesca Co., Tokyo, Japan) was utilized for
evaluation of the physical adhesive property of the polysaccharide nanosheet. We
prepared various thicknesses of the nanosheets from 39 to 1482 nm. The micro-scratch
tester employs a diamond stylus which oscillates parallel to the surface of the
polysaccharide nanosheet on the SiO2 substrate. The adhesive failure of the nanosheet
peeled by the stylus was detected as ‘critical load’ of the polysaccharide nanosheet
where the critical load is a relative value of the adhesive force.
Fig. 4-4 Schematic representation of the micro-scratching test and its working principle.
3-2. Evaluation of Physical Adhesive Property
The physical Adhesive property of the polysaccharide nanosheets did not show
significant differences between before and after the detachment (Figure 4-5a). The
critical load of the polysaccharide nanosheets was drastically increased as the thickness
was decreased below 200 nm; the critical load of the 39-nm thickness (0.15 x 10
6
N/m)
showed over 7 times higher than that of the 1482-nm one (0.02 x 10
6
N/m). Moreover,
the microscopic observation revealed the different trail after scratching among the
200-nm thickness such as ‘cut-off (1482 nm)’ and ‘drawn (77 nm)’ like trails (Fig. 4-5b,

Page 91
Chapter 4
- 83 -
5c and Fig. 6). This suggested that the elasticity of thin films should be changed in the
thickness among 200 nm. Therefore, we selected the top-three adhesive polysaccharide
nanosheets for the evaluation of the mechanical property.
Fig. 4-5 Evaluation of adhesive properties of the polysaccharide nanosheets. (a) The
relationship between the critical load against the different thickness of the
polysaccharide nanosheet between before (circle) and after (triangle) detachment of the
nanosheet (Inset: magnification of the graph under the 200 nm thickness). (b) and (c)
Microscopic observation of the polysaccharide nanosheets after micro-scratch test.
Black arrows show the direction of the stylus on the nanosheet, and dashed arrows
indicate the detached points from the SiO2 substrate. Scale bars show 100 μm.
[Evaluation of physical adhesive properties]
The micro-scratch tester (CSR-2000, Rhesca Co., Tokyo, Japan) was utilized for
evaluation of the physical adhesive property of the polysaccharide nanosheet. We
prepared various thicknesses of the nanosheets from 39 to 1482 nm. The micro-scratch
tester employs a diamond stylus of 25 μm in radius, and the stylus is forced to oscillate
with amplitude of 100 μm parallel to the surface of the polysaccharide nanosheets
adhered on the SiO2 substrate, where the adhesive failure of the nanosheet peeled by the
0.00
0.05
0.10
0.15
0.20
0
50 100 150 200
Thickness (nm )
C
ritic
a
l lo
a
d
in
g
(1
06
N
/m
)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0
250 500 750 1000 1250 1500 1750
Thickness (nm)
C
ritic
a
l lo
a
d
in
g
(1
06
N
/m
)
(a)
(b)
(c)

Page 92
Chapter 4
- 84 -
stylus was detected as the critical load of the polysaccharide nanosheet.
4. Mechanical Property of Polysaccharide Nanosheets
4-1. Bulge Test
The bulge test was used for evaluation of the mechanical strength for the
polysaccharide nanosheet. A free-standing polysaccharide nanosheet floating in water
was scooped onto a steel plate with either a 1 mm or 6 mm diameter circular hole in the
middle. The plate covered with the polysaccharide nanosheet was then fixed onto a
custom-made steel chamber by firm adhesion. The pressure applied to the
polysaccharide nanosheet through the circular hole of the plate was monitored by a
digital pressure gauge (Keyence Co., Tokyo, Japan), and the deflection of the
polysaccharide nanosheet was viewed from the side by a stereomicroscope (Olympus
Co., Tokyo, Japan) until distortion was apparent (Fig. 4-6).
Fig. 4-6 Schematic representation of the bulge test.
Each measurement for a polysaccharide nanosheet of a particular thickness was
performed at least three times. In order to determine the ultimate tensile strength, the
ultimate tensile elongation and the elastic modulus of the polysaccharide nanosheets,
the following equations have been used:
σ = (P × a
2
)/(4 × h × d), ε = (2 × d
2
)/(3 × a
2
), E = σ /ε
Pressure gauge
P
h
a
d
Air
Steel chamber
Nanosheet
Syringe pump

Page 93
Chapter 4
- 85 -
where σ, P, a, h, d, ε and E represent tensile stress, applied pressure, radius of the
circular hole of the steel plate, thickness, deflection, tensile strain and elastic modulus
about the polysaccharide nanosheet, respectively.
4-2. Evaluation of Mechanical Property-1 (1 mm hole)
The bulge test has been frequently used for the evaluation of the mechanical
strength of ultra-thin films
7
. Following the micro-scratch test, the author prepared three
types of polysaccharide nanosheets with different thickness of 35, 75 and 114 nm,
which were transferred onto steel plates with either 1 mm or 6 mm diameters circular
holes in the center. It is noteworthy that the polysaccharide nanosheet easily adhered
onto the steel plate without using a chemical adhesive. The nanosheet on the steel plate
was then placed in a custom-made steel chamber. As a pressure was applied to the
polysaccharide nanosheet through the circular hole of the plate, a deflection of the
nanosheet was monitored from a side-view of the plates until distortion was occurred.
During the bulge test, the monitoring environment was kept at the ambient conditions
(temperature: 25
o
C, humidity: 37%) because the molecular dynamics of polyelectrolytes
are influenced by temperature and humidity; high humidity in the bulge test such as
measurement under hydro-pressure showed the exaggerate expansion of the
polysaccharide nanosheet due to incorporation of water into the polysaccharide
nanosheet, resulted in the false evaluation of the potential elastic modulus of the
nanosheet.
The relationship between pressure and deflection was non-linear and the overall
deflection of the polysaccharide nanosheet was damped by an increment of the
thickness. This suggested the elasticity of the polysaccharide nanosheet was dependent
on the total film thickness (Fig. 4-7a). Then, the values derived from the

Page 94
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- 86 -
pressure-deflection curve were converted into a stress-strain curve. From the initial
elasticity of the stress-strain curve (Fig. 4-7b), the ultimate tensile strength (σmax),
elongation (εmax) and elastic modulus (E) were calculated for the different thickness of
the polysaccharide nanosheets (Table 4-1).
Fig. 4-7 Evaluation of mechanical properties of the polysaccharide nanosheets for the 1
mm hole. a and b, Pressure-deflection and stress-strain curves using a 1 mm diameter
circular hole for various thicknesses of polysaccharide nanosheets (open circle: 35 nm,
open square: 75 nm, open triangle: 114 nm).
The elastic modulus of the 35 nm polysaccahride nanosheet was 1.1 ± 0.4 GPa, a
similar value to that (E = 1.5 GPa) of the polymer nanosheet composed of
poly(allylamine hydrochloride) and poly(sodium styrenesulphonate) with the same
thickness (35 nm) reported by the Tsukruk group
8
, and considerably smaller than the

Page 95
Chapter 4
- 87 -
typical elastic modulus of a cellulose film (E = 15 GPa) fabricated from the similarly
structured molecule chitosan with the thickness of over 1 micrometer. This result
suggested that tens-of-nm thick polymer nanosheet was quite flexible due to the low
elastic modulus. Furthermore, the elastic modulus of the 35 nm polysaccharide
nanosheet was similar to that of tendon in the biological tissues (E = 0.7 GPa). In Table
4-2, the mechanical properties of another biological tissues were summarized
9
.
As the thickness of the polysaccharide nanosheet was increased, the elastic
modulus recorded closer values (75 nm: E = 8.1 ± 2.5 GPa, 114 nm: E = 11.0 ± 1.6
GPa). These results are in good agreement with the report by the Rubner group where
the elastic modulus of ultra-thin films composed of poly(allylamine hydrochloride) and
poly(sodium styrenesulphonate) reached a plateau value when the thickness was close
to 70 nm
10
. Generally, ultra-thin polymer films of tens-of-nm thickness show a
glass-transition temperature lower than the corresponding bulk
22
. This reflects a specific
interfacial property of the ultra-thin film, such as unrestricted macromolecular mobility,
and is probably the reason why the such nanosheets have low elastic moduli.
Corresponding to this assumption, as the thickness of the nanosheet was increased,
some of the 114 nm polysaccharide nanosheets were spontenuously detached from the

Page 96
Chapter 4
- 88 -
steel plate before breaking during the bulge test, which strongly suggested that thickness
and flexibility derived from the tens-of-nm thickness and low elastic modulus has
relationship with the physical adhesive propety.
4-3. Evaluation of Mechanical Property-2 (6 mm hole)
The author performed a bulge test using a 75 nm polysaccahride nanosheet
overlapped on a 6 mm diameter hole because it was capable of the adhesiveness over
114 nm as well as robustness over 35 nm in the thickness. The elastic modulus of this
nanosheet, 9.6 ± 3.1 GPa (σmax: 159 MPa, εmax: 6.5 %) was similar to that of the same
film on the 1 mm diamater hole. The S.D. value of the 6 mm modulus was slightly
larger than that of 1 mm due to the difficulty of maintaing a homogenous in-flow
pressure to the nanosheet for the large hole. Furthermore, no distortion of the 75 nm
film was found until a pressure of 4.5 kPa (Fig. 4-8a and 8b).
Fig. 4-8 Evaluation of mechanical properties of the polysaccharide nanosheets for the 6
mm hole. (a) Sequential macroscopic cross-sectional views of the deflected 75-nm
polysaccharide nanosheet when different pressures were applied through the 6 mm
diameter hole. (g) A corresponding pressure-deflection curve (Inset: stress-strain curve).
2.0 kPa
0.0 kPa
(a)
(b)
Pressure: P (kPa)
D
e
fle
c
tio
n
: d
m
)
0.0
2.0
3.0
4.0
5.0
0
200
400
600
800
1000
1200
1.0
1400
0.005
Strain: ε (-)
S
tre
s
s
: σ
(M
P
a
)
0.010
0.000
0
40
80
120
4.0 kPa
1 mm

Page 97
Chapter 4
- 89 -
This is an important result because a pressure of 30 cmH2O (approximately 3
kPa) is a clinically important criteria in tissue-defect repair applications. This pressure is
a physiologically expected typical value in normal respiration through lung, and has
been noted in connection with wound dressings such as fibrin sheets
11
. Hence, as the 75
nm film was stable to pressures well over this value, it was decided that the 75 nm
polysaccharide nanosheet was appropriate for a clinical application using beagle dogs.
5. Summary
The author constructed the free-standing polysaccharide nanosheets under totally
aqueous conditions, exploiting the combination of SA-LbL and supporting films. The
micro-scratch test and the bulge test indicated that the polysaccharide nanosheets with
tens-of-nm thickness had a sufficient physical adhesive property and mechanical
strength for clinical use. As a result, the polysaccharide nanosheet with the thickness of
75 nm was a suitable candidate for the clinical application as a wound dressing material.

Page 98
Chapter 4
- 90 -
References
1. a) J. Cho, K. Char, Langmuir 2004, 20, 4011. b) C. Jiang, S. Markutsya, V. V.
Tsukruk, Adv. Mater. 2004, 16, 157.
2. a) J. S. Boateng, K. H. Matthews, H. N. Stevens, G. M. Eccleston, J. Pharm. Sci.
2008, 97, 2892. b) H. J. Kim et al., J. Biomater. Sci. Polym. Ed. 1999, 10, 543.
3. a) M. N. V. R. Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. J. Domb,
Chem. Rev. 2004, 104, 6017. b) H. Yi, L. Wu, W. E. Bentley, R. Ghodssi, G. W.
Rubloff, J. N. Culver, G. F. Payne, Biomacromolecules 2005, 6, 2881. c) I. Liao, A. C.
A. Wan, E. K. F. Yim, K. W. Leong, J. of Controlled Release. 2005, 104, 347.
4. A. D. Stroock, R. S. Kane, M. Weck, S. J. Metallo, G. M. Whitesides, Langmuir 2003,
19, 2466.
5. J. Cho, K. Char, J. Hong, K. Lee, Adv. Mater. 2001, 13, 1076.
6. S. Baba, T. Midorikawa, T. Nakano, Appl. Surf. Sci. 1999, 144, 344.
7. a) J. J. Vlassak, W. D. Nix, J. Mater. Res. 1992, 7, 3242. b) S. Markutuya, C. Jiang, Y.
Pikus, V. V. Tsukruk, Adv. Funct. Mater. 2005, 15, 771. c) H. Watanabe, T. Ohzono, T.
Kunitake, Macromolecules 2007, 40, 1369.
8. C. Jiang, S. Markutsya, Y. Pikus, V. V. Tsukruk, Nature Mater. 2004, 3, 721.
9. R. P. Lanza, R. Langer, J. Vacanti, Priciples of Tissue Engineering, Academic Press,
California, 2000.
10. A. J. Nolte, M. F. Rubner, R. E. Cohen, Macromolecules 2005, 38, 5367.
11. a) T. Fabian, J. A. Federico, R. B. Ponn, Ann. Thorac. Surg. 2003, 75, 1587. b) H.
Miyamoto et al., Jpn. J. Thorac. Cardiovasc. Surg. 2003, 51, 232. c) P. Hollaus, N.
Pridun, J. Cardiovasc. Surg. (Torino) 1994, 35, 169.

Page 99
Chapter 5
- 91 -
Chapter 5
Wound Dressing Effect of the Biomacromolecular Nanosheets
Integrated for Pleural Tissue-Defect Repair
1. Introduction
2. Biology and Management of Wounds
3. Physiological Property of Polysaccharide Nanosheets
4. Polysaccharide Nanosheets Integrated for Tissue-Defect Repair
5. Summary
References

Page 100
Chapter 5
- 92 -
1. Introduction
Surgical repair for tissue defects is basically achieved by three fundamental
maneuvers; suture, plication, and overlapping. Despite their high reliabilities for wound
repair, suture and plication are often accompanied by a volume reduction of the repaired
organs. For example, the conventional repair of a pleural defect by suture and plication
usually reduces the volume of pulmonary tissue, thereby decreasing respiratory
functions. Pulmonary air leakage due to visceral pleural injury is one of the most
common postoperative complications after thoracic surgery. It might be caused by the
prolonged placement of a drainage tube and/or longer hospitalization, and the worst of
all; it may lead to thoracic empyema. Therefore, the tight and firm repair of a pleural
injury/defect is really important to prevent air leakage
1
. Nevertheless, it is sometimes
practically difficult to suture or plicate a large defect or fragile tissue of the
emphysematous lung. Overlapping is therefore seen as an ideal maneuver to repair a
pleural defect, because it simply seals the injured surface without reducing the tissue
volume of the injured lung. Some investigators have demonstrated the efficacy of fibrin
glue (sheet) composed of fibrin-glue-coated collagen fleece, a typical adhesive material,
for the repair of a visceral pleural defect
2
. However, this material will cause severe
pleural adhesion. In a case of high-risk patients with respiratory failure, such a severe
pleural adhesion might further deteriorate pulmonary dysfunctions, a serious
complication for the compromised patients. Therefore, a novel tissue sealant that does
not cause tissue adhesion is required.
In this chapter, the biomedical application of biomacromolecular nanosheets was
described as newly constructed wound dressing materials. The clinical benefits of sheet
type nano-biomaterials were revealed by exploiting their properties such as physical
adhesiveness, flexibility and robustness.

Page 101
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- 93 -
2. Biology and Management of Wounds
2-1. Wound Biology
The overlapping segments of the repair process are conceptually defined as
inflammation, proliferation, and remodeling. During the inflammatory phase,
hemostasis occurs and an acute inflammatory infiltrate ensues. The proliferative phase
is characterized by fibroplasia, granulation, contraction, and epithelialization. The final
phase is remodeling, which is commonly described as scar maturation (Fig. 5-1)
3
.
Fig. 5-1 Temporal relationship of repair stages and cellular infiltrates into the wound.
2-2. Classification of Wound Healing Process
Wound healing progresses through a series of interdependent and overlapping
stages in which a variety of cellular and matrix components act together to reestablish
the integrity of damaged tissue and replacement of lost tissue. The wound healing
process has been reviewed and described by Schultz
4
as comprising five overlapping
stages that involve complex biochemical and cellular processes. These are described as
hemostasis, inflammation, migration, proliferation and remodeling (or maturation)
phases (Fig. 5-2)
5
.

Page 102
Chapter 5
- 94 -
Fig. 5-2 Schematic representation of the phases of wound healing process, which is
classified as (a) hemostasis, (b) inflammation, (c) migration, (d) proreiferation, and (e)
remodeling phases, respectively.
Hemostasis phase
Immediately after tissue injury, hemostasis is stimulated by platelet degranulation
and exposure of tissue thromboplastic agents (Fig. 5-2a). The clot dries to form a scab
and provides strength and support to the injured tissue. Hemostasis, therefore, plays a
protective role as well as contributing to successful healing.
Inflammation phase
Within 24 hours, a neutrophil efflux into the wound occurs (Fig. 5-2b). The
neutrophils scavenge debris, bacteria, and secrete cytokines for monocyte and
lymphocyte attraction and activation. Keratinocytes begin migration when a provisional
matrix is present.
Migration phase
At 2 to 3 days after tissue injury, the macrophage becomes the predominant
b)
Keratinocytes
Neutrophil
Fibroblast
e)
Daremal scar
(collagen)
d)
Scab
Keratinocytes
migration
Fibroblast
Neutrophil
Macrophage
a)
Blood vessel
Exposure of thromboplastic
tissue elements
Fibroblast
Resident monocyte
Platelet
Red blood cell
Dermis
SQ Fat
Epidermis
c)
Scab
Fibroblast
Neutrophil
Macrophage

Page 103
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- 95 -
inflammatory cell type in clean, noninfected wounds (Fig. 5-2c). These cells then
regulate the repair process by secretion of a myriad of growth factors, including types
that induce fibroblast and endothelial cell migration and proliferation.
Proliferation phase
Fibroblasts are activated and present at the wound by 3 to 5 days after injury (Fig.
5-2d). These cells secrete matrix components and growth factors that continue to
stimulate healing. Keratinocyte migration (epiboly) begins over the new matrix.
Migration starts from the wound edges as well as from epidermal cell nests at sweat
glands and hair follicles in the center of the wound.
Remodeling phase
Scar formation is the outcome of healing in the case of postnatal skin (Fig. 5-2e).
Scar is composed of densely packed, disorganized collagen fiber bundles. Remodeling
occurs up to 1 to 2 years after injury and consists of further collagen cross-linking and
regression of capillaries, which account for the softening of scar and its color change
from red to white.
2-3. Chronic Wounds and Effective Wound Management
Although most wounds will heal uneventfully, problems can sometimes occur,
that lead to failure of the wound to heal or a prolonged healing time. Failure of a wound
to heal within the expected time frame usually results in a chronic wound. A chronic
wound fails to heal because the orderly sequence of events is disrupted at one more of
the stages of wound healing. Excessive production of exudates can cause maceration of
healthy skin tissue around the wound
6
and inhibit wound healing. In addition, exudate
from chronic wound differs from acute wound fluid with relatively higher levels of
tissue destructive proteinase enzymes
7
and therefore more corrosive. The smell and

Page 104
Chapter 5
- 96 -
staining caused by exudate can also have a negative impact on a patient’s general
health and quality of life
8
.
Several factors apart from the choice of wound dressings need to be considered to
ensure successful wound healing. In the case of chronic wounds, underlying factors
such as disease, drug therapy and patient circumstance must all be reviewed and
addressed before a particular wound dressing is applied. Table 5-1 describes factors to
be considered in the choice of wound dressings based on their performance
characteristics (functions)
9
.
2-4. Wound Dressing Materials
Dressings are classified in a number of ways depending on their function in the
wound (debridement, antibacterial, occlusive, absorbent, adherence)
10
, type of material
employed to produce the dressing (e.g. hydrocolloid, alginate, collagen)
11
and the
physical form of the dressing (ointment, film, foam, gel)
12
. Dressings are further
Table 5-1 Desirable characteristic of wound dressings
Desirable Characteristics
Clinical significance to wound healing
Debridement (wound cleaning)
Provide or maintain a moist wound environment
Absorption. Removal of blood and excess exudate
Gaseous exchange (water vapor and air)
Prevent infection: Protect the wound from bacterial
invasion
Provision of thermal insulation
Low adherence. Protects the wound from trauma
Cost effective Low frequency of dressing change
Enhances migration of leucocytes into the wound bed and supports the
accumulation of enzymes. Necrotic tissue, foreign bodies and particles
prolong the inflammatory phase and serve as a medium for bacterial
growth.
Prevents desiccation and cell death, enhances epidermal migration,
promotes angiogenesis and connective tissue synthesis and supports
autolysis by rehydration of desiccated tissue.
In chronic wounds, there is excess exudate containing tissue degrading
enzymes that block the proliferation and activity of cells and break down
extracellular matrix materials and growth factors, thus delaying wound
healing. Excess exudate can also macerate surrounding skin.
Permeability to water vapour controls the management of exudate. Low
tissue oxygen levels stimulate angiogenesis. Raised tissue oxygen
stimulates epithelialisation and fibroblasts.
Infection prolongs the inflammatory phase and delays collagen synthesis,
inhibits epidermal migration and induces additional tissue damage.
Infected wounds can give an unpleasant odour.
Normal tissue temperature improves the blood flow to the wound bed and
enhances epidermal migration.
Adherent dressings may be painful and difficult to remove and cause
further tissue damage.
Dressing comparisons based on treatment costs rather than unit or pack
costs should be made (cost-benefit-ratio). Although many dressings are
more expensive than traditional materials, the more rapid response to
treatment may save considerably on total cost.

Page 105
Chapter 5
- 97 -
classified into primary, secondary and island dressings
13
. Dressings which make
physical contact with the wound surface are referred to as primary dressings while
secondary dressings cover the primary dressing. Island dressings possess a central
absorbent region that is surrounded by an adhesive portion. Other classification criteria
include traditional dressings, modern and advanced dressings, skin replacement
products and wound healing devices. There have been many studies conducted relating
to specific dressings
14
.
Classification criteria can be useful in the selection of a given dressing but many
dressings fit all the criteria. For example an occlusive dressing may also be a
hydrocolloid. In this section, dressings are classified according to traditional or modern
(moist wound environment) dressings (Table 5-2). Modern dressings are discussed
under the type of material (hydrocolloid, alginate, hydrogel) employed to produce the
dressing and the physical form (film, foam) of the dressing.
Table 5-2 Desirable characteristic of wound dressings
Desirable Characteristics
Composition
Gauze
Calcium-alginates
Impregnated gauzes
Films
Foams
Hydrogels
Hydrocolloids
Absorptive powders and pastes
Silicone
Mechanical vacuum
Dermal matrix replacements
Dermal living replacements
Skin living replacement
Woven cotton fibers
Seaweed polymer that forms a gel when adsorbs fluid
Fine mesh fabric (silicone, nylon) with dermal
porcine collagens
Plastic (polyurethane), semipermeable
Hydrophilic (wound side) and hydrophobic (outer
side), semipermeable
Water (96%) and polymer (polyethyleneoxide)
Hydrophilic colloidal particles and adhesives
Starch copolymers, hydrocolloidal particles
Silicone sheets
Vacuum, sponge, plastic film
Acellular matrix
Absorbable matrix populated with fibroblasts
Collagen matrix populated with human fibroblasts
with an outer layer of human keratinocytes
Permeable with desiccation, debridement,
painful removal
Absorbs exudate, nonadherent, nonirritating,
requires a cover dressing (permeable)
Nonadherent, semipermeable
Allows water vapor permeation, adhesive
Necrotic/exudative wounds
Aqueous environment, requires secondary
dressing, no adherence, not recommended if
infection is present, semipermeable
Absorbs exudate, used as a filler, good for
deep wounds
Sheet induces a localized electromagnetic
field, decreases scar formation?
Sponge confirms to wound and vacuum
removes edema fluid, stimulation of repair?
Permeable, increased stimulation of repair?
Permeable, increased stimulation of repair?
Impermeable, increased stimulation of repair?
Characteristics/function

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3. Physiological Property of Polysaccharide Nanosheets
3-1. Morphological Analysis of Blood Compatibility
As shown in the previous Chapter 4, a micro-scratch test and a bulge test
indicated that the polysaccharide nanosheet with tens-of-nm thickness has sufficient
adhesive property and mechanical strength for clinical use. The author optimized the
property of the nanosheet such as thickness in 75 nm, critical load in 9.1 x 10
4
N/m, and
elastic modulus in 9.6 GPa for clinical usage.
The polysaccharide nanosheet in the form of a bilayered film with water-soulble
supporting film can be transferred onto the visceral pleural defect site of a beagle dog
by dissolution of the PVA film. Upon the practical biomedical application of
polysaccharide nanosheets, it had been afraid that bleeding from the defect site must be
a general feature of a surgical wound. However, because of its robust and flexible
structure, the polysaccharide nanosheet not only inhibited permeation of blood
components from the defect site but also regulated the position of blood clots
accumulated along the interface of the nanosheet, which should induce/stimulate the
subsequent growth position of the fibroblast cells and angiogenesis in the tissue-defect
sites. To test whether this was so, human blood was dropped onto a polysaccharide
nanosheet prepared on a poly(propylene) mesh filter. Although blood cells, mainly
erythrocytes, easily permeated inside the fibrous mesh (Fig. 5-2a), they stagnated on the
surface of the polysaccharide nanosheet without distortion and penetration into the
nanosheet as well as hemolysis of blood cells (Fig. 5-2b). Additionally, it was observed
that some platelets were partially adhered on the surface of the nanosheet with
spreading pseudopodia (Fig. 5-2c).
This polysaccharide nanosheet may act not only as a barrier but also as a scaffold
for in-flow blood cells in a healing process of hemostasis. It is noteworthy that the

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polysaccharide nanosheet provides the hetero-interface for the tissue-defect sites
whether to be repaired or not by working as a separator against incoming blood flow,
where the subsequent growing position of fibroblast cells will be clearly determined by
the position of formulated blood clots. Moreover, considering that primary mechanical
strength of the tissue-defect sites repaired by nanosheet may be fragile, the formation of
normal blood clots will reinforce the tissue-defect repaired sites.
Fig. 5-2 Physiological effect of the polysaccharide nanosheet contacting blood cells. (a)
SEM image of blood cells flowing into PP fibrous mesh. (b) SEM image of blood cells
deposited on the surface of a 75-nm polysaccharide nanosheet overlapping PP fibrous
mesh. (c) Magnified SEM image of the dashed square region in b. The physiological
morphology of activated platelets was observed with spreading pseudopodia where the
polysaccharide nanosheet was acting as a scaffold for platelet adhesion.
(a)
(c)
(b)

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- 100 -
[SEM observation of the polysaccharide nanosheet in contact with blood]
A 2 μL of blood (donated from T.F. with his permission) mixed with a 10-fold volume
of 3.8% (w/v) sodium citrate was dropped on the surface of a polysaccharide nanosheet
which covered a albumin adsorbed polypropylene prefilter (30 μm porous filter:
Millipore Co., Billerica, MA). The sample was fixed with 1% (v/v) glutaraldehyde in
0.1 M phosphate buffer (pH 7.4) for 30 min, post-fixed with 1% (w/v) osmium tetroxide
in the same buffer for 30 min, and dehydrated with a graded ethanol series. After drying
the sample in a Hitachi ES-2020 freeze-dryer using t-butyl alcohol, SEM observation
was undertaken using a HITACHI S-4500 at an accelerating voltage of 10 kV, where the
sample on the filter was coated with an osmium tetroxide layer (ca. 30 nm thickness)
using NL-OPC80 ion-sputtering coater. (Nippon Laser & Electronics Lab, Nagoya,
Japan).
3-2. Evaluation of Blood Coagulation Activity
Lee-White test
15
was undertaken to judge the blood pro-coagulation property of
the polysaccharide nanosheet by observing the substrate surface (See Supplementary
Information). The polysaccharide nanosheet was overlaid on the surface of a
polypropylene (PP) substrate where whole blood showed poor coagulation. After 30
minutes of incubation in whole human blood and gentle rinsing with PBS, some blood
clots were observed at the interface of the substrate covered with the polysaccharide
nanosheet, while few clots were observed at the native PP surface (Fig. 5-3).
Interestingly, it was not obtained such a strong blood pro-coagulation activity reported
by the Akashi group
15
, where they used the dipping chitosan LbL films. This difference
should be caused of the surface roughness between spin-coating with low RMS and
dipping with high RMS in the LbL system
16
. Therefore, the very weak pro-coagulant

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Chapter 5
- 101 -
activity without using an anti-coagulant agent such as heparin is a clinical advantage
because the post-operative embolism due to the strong pro-coagulant activity
17
and/or
the heparin-induced thrombocytopenia (HIT)
18
; one of the considerably major problems
in the clinical situation, can be escaped in the use of the polysaccharide nanosheet.
Fig. 5-3 Blood pro-coagulation activity of the polysaccharide nanosheet overlaid on a
polypropylene substrate before (left panel) and after (right panel) contacting blood. The
area overlaid by the polysaccharide nanosheet is shown inside the dashed square.
[Blood coagulation assay: Lee-White Test]
A polypropylene (PP) substrate formed by cutting a commercial PP sheet into a 1 cm x
5 cm piece was partially covered with the polysaccharide nanosheet. Then the substrate
was immersed in a petri dish filled with whole blood and incubated at 37
o
C. After
incubation for 30 min, the substrate was taken out of the dish, and the surface was
gently rinsed with PBS. Strong blood pro-coagulation activity was not observed at the
interface of the substrate covered with the polysaccharide nanosheet.
PP substrate
Polysaccharide
nanosheet

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- 102 -
4. Polysaccharide Nanosheets Integrated for Tissue-Defect Repair
4-1. Treatment of Visceral Pleural Defect
The author demonstrated a practical working test of the 75 nm polysaccharide
nanosheet for an in vivo visceral pleural defect model of beagle dogs. The
polysaccharide nanosheet, or a fibrin sheet used as a positive control, was placed onto a
pleural defect area prepared by an 3.2 cm
2
aorta punch on the right anterior, middle and
posterior lobes. The 75 nm polysaccharide nanosheet was placed on a supporting PVA
film (70 μm in thickness) because the free-standing polysaccharide nanosheet itself was
too fragile in the air. Then the PVA film was dissolved with a PBS solution (Fig. 5-4a)
until the letter ‘P’ written on the surface of PVA was removed (Fig. 5-4b). As the PVA
film dissolved, the underlying polysaccharide nanosheet fitted onto the curvature of the
remaining tissue, remaining fully overlapping on the pleural defect without the use of
any chemical adhesive. After a drying for a few minutes, the polysaccharide nanosheet
was completely assimilated to the tissue surface.
Fig. 5-4 Tissue-defect repair using a polysaccharide nanosheet. (a) Schematic
explanation of the intervention and (b) the bilayered film adhered on the pleural
(a)
6 mm
pleura
Sealing with the (nanosheet + PVA) film
Tissue-defect by punch
Nanosheet adhered on the
defect after dissolution of PVA
alveolo
18 mm
(b)

Page 111
Chapter 5
- 103 -
tissue-defect site. The capital P was written on the surface of the PVA membrane in
order to distinguish the bilateral side.
4-2. Evaluation of Biological Mechanical Strength
The pressure resistance of the repaired site was determined at 5 min, 3 hrs and 24
hrs after repair. The airway pressure at which the air leakage occurred was measured
using a manometer and termed ‘bursting pressure’, which was individually measured
for each repaired lobe, while the bronchi of the other two lobes were clamped. The
highest airway pressure applied was 60 cmH2O because air leakage could occur from
the intact pulmonary hilum at pressures above 60 cmH2O. At 5 min after repair, the
polysaccharide nanosheet showed a lower bursting pressure (31.7 ± 10.3 cmH2O) than
the fibrin sheet (45.0 ± 5.5 cmH2O) (Fig. 5-5a).
The bursting pressure of the polysaccharide nanosheet was slightly lower than
that found in the bulge test (ca. 45 cmH2O for the 6 mm diameter hole prepared on the
steel substrate). The reason for this discrepancy in the nanosheet bursting pressure
might be due to the repeated stretch and/or expansion of pulmonary surface by
ventilation. After 3 hrs after the repair, the outline of the square shaped polysaccharide
nanosheet assimilating to the tissue surface could be faintly seen (Fig. 5-5b, indicated
by arrows), and the mechanical durability was dramatically increased. The bursting
pressure of the polysaccharide nanosheet reached 56.7 ± 6.1 cmH2O at 3 hrs after repair.
The mechanical strength of the polysaccharide nanosheet was probably reinforced
by the deposition of blood cell components such as erythrocytes and platelets within
fibrin networks. As this deposition occurs relatively quickly, the most significant
increment in the mechanical strength was observed at 3 hrs after repair compared to that
at 5 min, and it was not further increased at 24 hrs.

Page 112
Chapter 5
- 104 -
Fig. 5-5 Visceral pleural defect repair using a polysaccharide nanosheet. (a)
Time-course changes in bursting pressure after repair using the polysaccharide
nanosheet (black bars) or the fibrin sheet (white bars) (* p<0.05, the polysaccharide
nanosheet vs. the fibrin sheet. **p<0.05, at 5 min vs. 3 hrs after repair). The units of the
vertical axis on the left and right hand sides are cmH2O and kPa, respectively. (b)
Polysaccharide nanosheet with a thickness of 75 nm secured the repaired defect when
pressurized by over 50 cmH2O pressure at 3 hrs after repair. The region indicated by
arrows showed the polysaccharide sealed area.
4-3. Wound Healing Effect
A further analysis of the wound healing after tissue-defect repair was undertaken
by a histological examination for investigating the different healing mechanism between
the polysaccharide nanosheet and the fibrin sheet. Tissue specimens for histological
examination were obtained at 5 min, 3 hrs, 3 days and 7 days after repair. The specimen
containing the repairing lesion in each lobe was fixed with formalin followed by
paraffinization and then stained with hematoxylin-eosin.
Polysaccharide Nanosheets
It is noteworthy that the wound healing after treatment with the polysaccharide
(b)
5 min
3 hours
24 hours
Times after operation
B
u
rs
tin
g
p
re
s
s
u
re
(c
m
H
2O
)
0
10
20
30
40
50
60
70
0
1
2
3
4
5
6
B
u
rs
tin
g
p
re
s
s
u
re
(k
P
a
)
*
**
**
7
(a)

Page 113
Chapter 5
- 105 -
nanosheet was quite distinct from that with the fibrin sheet. Although it was difficult to
observe the polysaccharide nanosheet overlapped on the pleural defect, the formation of
flat-shaped blood clots localized along the polysaccharide nanosheet was clearly
observed in the region of the defects at 5 min and 3 hrs after repair (Fig. 5-6a and 5-6b).
This finding suggested that blood cells initially deposited under the polysaccharide
nanosheet just like the image shown in Figure 3b, which were transformed to stable
blood clots by persistent leakage of blood discharge from alveolar capillaries without
producing blood clots on the outside of pleura. At 3 days after repair, fibroblasts were
grown as surrounding the blood clots with replacing preformed clots (Fig. 5-6c). At 7
days after repair, angiogenesis was observed where the blood clots were originally
formulated under the polysaccharide nanosheet, and any sequence of wound healing
process was never found on the outside of the polysaccharide nanosheet (Fig. 5-6d),
resulted in the no occurrence of postsurgical adhesive lesion. At 30 days after repair, the
original tissue-defect site was no longer confirmed and further hospitalization was not
required. Following these findings, it was found that the polysaccharide nanosheet was
not only working as an interfacial separator between the in- and outside of the pleura
but also providing the flat and stable scaffold for wound healing process.
Fig. 5-6 Representative histological findings at different time points after repair
Fibroblast
(c)
(d)
Angiogenesis
Nanosheet
(b)
Blood retention
Nanosheet
Blood retention
(a)

Page 114
Chapter 5
- 106 -
(magnification 4×) using the polysaccharide nanosheet. Panels correspond to the
polysaccharide nanosheet at 5 min (a), 3 hrs (b), 3 days (c) and 7 days (d) after repair.
Fibrin Sheets
In contrast to the polysaccharide nanosheet, repair of the pleural defect by the
fibrin sheet exhibited large vacant air spaces at 3 hrs because the thick fibrin sheet was
not so flexible to densly overlap the defect site, which caused detachment from the
lesion of the pleural defect due to influx of discharge into the interface between the
pleura and the fibrin sheet, resulting in the random retention of blood components (Fig.
5-7a). At 3 days repaired by the fibrin sheet, random growth of fibroblasts was observed
as well as induction of inflammatory tissue reactions, such as an emergence of
macrophages (Fig. 5-7b). Furthermore, it is a critically important clinical issue that the
fibrin sheet also strongly adhered onto the chest wall (Fig. 5-7c) because severe pleural
adhesion might reduce a respiratory function and may cause a reoccurrence of
pneumothorax. This is because that the solid fibrin sheet cannot densely overlap the
tissue-defect without bonding agents such as fibrinogen and thrombin, resulting in the
large air space in the tissue repaired site. Therefore, the influx of blood components /
discharge through the air space produced the further adhesive region outside of alveolo
with another tissue surface, whereas the flexible polysaccharide nanosheet densely and
physically overlapped the tissue-defect site. The polysaccharide nanosheet has very
desirable properties: physical adhesiveness due to flexibility and sufficient mechanical
strength without chemical adhesive such as fibrin sheet. Thus, repair by overlapping a
tissue-defect with the polysaccharide nanosheet has a significant advantage in
maintaining the function of the remaining lung against sustained ventilation and the
pressure from respiration and bleeding.

Page 115
Chapter 5
- 107 -
Fig. 5-7 Representative histological findings at different time points after repair
(magnification 4×) using the fibrin sheets. Panels correspond, respectively, to the fibrin
sheets at 3 hrs (a), 3 days (b) and 7 days (c) after repair. Strong adhesion between the
fibrin sheet and the chest wall (CW) was observed.
[Visceral pleural defect repair]
Twenty-four male beagle dogs weighing 10 kg on average were intubated after
anesthesia with ketamine (10 mg/kg, i.m.), pancuronium (0.1 mg/kg, i.v.) and sodium
pentobarbital (25 mg/kg, i.v.). The cuffed endotracheal tube was connected to a
mechanical respirator (Model SN-480-6, Shinano Inc., Tokyo, Japan) and the animals
were ventilated with room air (a tidal volume of 15 mL/kg and a respiratory rate of 10
times/min). The body temperature was stabilized at 39 °C by monitoring the rectal
temperature. The dogs were placed in the left lateral position and a right thoracotomy
was performed. A pleural defect was made on the anterior, middle and posterior surfaces
of each lobe with the airway pressure maintained at 10 cmH2O. The defect was a 6 mm
circle with a depth of 2 mm made using an aorta punch (Scanlan
®
, St. Paul, MN, USA).
Bleeding vessels were cauterized if needed, but sites where hemostasis was difficult to
achieve were not used. The pleural defects were repaired using either the polysaccharide
nanosheet or fibrin glue-coated collagen fleece (i.e., fibrin sheet, TachoComb
®
, CSL
Behring, Tokyo, Japan). An 18 × 18 mm piece of each sheet was applied over the defect.
Fibrin sheet
CW
Fibrin sheet
(a)
(b)
(c)
Tissue adhesion
Macrophages
Fibrin sheet
Air space

Page 116
Chapter 5
- 108 -
At 5 min, 3 hrs and 24 hrs after repair, pressure was applied from the airway and the
bursting pressure of the applied sheets was measured with a manometer (ADInstrument
BPamp, Utah Medical Products Inc., Utah, UT).
[Histological analysis]
Histological specimens were also obtained from the dogs at 3 hrs, 3 days, and 7days
after repair. All data are shown as the mean ± standard deviation. Statistical analyses
were performed using an unpaired t-test with p<0.05 set as the level of statistical
significance. All animal experiments were approved by the Animal Research Committee
of the National Defense Medical College.
5. Summary
The author succeeded in the construction of a free-standing polysaccharide
nanosheet under totally aqueous conditions exploiting the SA-LbL method. Furthermore,
it was found that the polysaccharide nanosheet with the thickness of 75 nm was a
suitable candidate for repairing the visceral pleural defect without any loss of
respiratory function of the lung, as determined by a bulge test (Chapter 4) and in vivo
study. The flexible property of the soft nano-biomaterial built from secondary
interactions is utilized for a minimally invasive treatment of the tissue-defect repair
without post-surgical adhesion. The applications of polymer nanosheets are expected to
include not only respiratory surgery, but also tissue repair of other organs. The desirable
mechanical property of the polymer nanosheet can be easily modulated by changing the
polymer and thickness. Therefore, combination with other functionally biocompatible
polymers will spread the more applicability of the polymer nanosheet for a novel wound
dressing of tissue-defect repair from soft to hard organs.

Page 117
Chapter 5
- 109 -
References
1. a) H. L. Porte et al., Ann. Thorac. Surg. 2001, 71, 1618. b) M. Kawamura et al., Eur.
J. Cardiothorac. Surg. 2005, 28, 39.
2. M. Gika, M. Kawamura, Y. Izumi, K. Kobayashi, Interact. Cardiovasc. Thorac. Surg.
2007, 6, 12.
3. M. Li et al., Wounds: Biology, Pathology, and Management, Springer, New York,
2003.
4. G. S. Schultz, Molecular regulation of wound healing: Acute and chronic wounds:
Nursing management, Mosby, St. Louis, MO, 1999.
5. J. S. Boateng, K. H. Matthews, H. N.E. Stevens, G. M. Eccleston, J. Pharma. Sci.
2008, 97, 2892.
6. K. Cutting, R. J. White, J. Wound Care 2002, 11, 275.
7. W. Y. J. Chen, A. A. Rogers, M. J. Lydon, J. Invest. Dermatol. 1992, 99, 559.
8. A. Hareendran et al., J. Wound Care 2005, 14, 53.
9. a) M. Rothe, V. Falanga, Arch. Dermatol. 1989, 125, 1390. b) S. Thomas, Wounds
and wound healing in: Wound management and dressings, Pharmaceutical Press,
London, 1990.
10. S. K. Purner, M. Babu, Burns 2000, 26, 54.
11. D. Queen, H. Orsted, H. Sanada, G. Sussman, Int. Wound J. 2004, 1, 59.
12. A. F. Falabella, Dermatol. Ther. 2006, 19, 317.
13. L. van Rijswijk, J. Wound Care 2006, 15, 11.
14. D. A. Morgan, Hosp. Pharmacist. 2002, 9, 261.
15. a) T. Serizawa, M. Yamaguchi, T. Matsuyama, M. Akashi, Biomacromolecules 2000,
1, 306. b) T. Serizawa, M. Yamaguchi, M. Akashi, Biomacromolecules 2002, 3, 724.
16. J. Seo, J. L. Lutkenhaus, J. Kim, P. T. Hammond, K. Char, Langmuir 2008, 24,
7995.
17. E. K. F. Yim, I. Liao, K. W. Leong, Tissue Eng. 2007, 13, 423.
18. Z. K. Baldwin, A. L. Spitzer, V. L. Ng, A. H. Harken, Surgery 2008, 143, 305.

Page 118

Page 119
Chapter 6
- 111 -
Chapter 6
Treatment of Bacterial Peritonitis in Gastrointestinal
Perforation with the Biomacromolecular Nanosheets
1. Introduction
2. Polysaccharide Nanosheets Integrated for Gastrointestinal Perforation
3. Immunological Response Repaired by Polysaccharide Nanosheets
4. Development of Antibiotics Loaded Polysaccharide Nanosheets
5. Summary
References

Page 120
Chapter 6
- 112 -
1. Introduction
Gastrointestinal perforation is a major cause of bacterial peritonitis that usually
leads to severe sepsis. Postoperative anastomotic breakdown, which is one of the major
complications after gastrointestinal surgery, also causes bacterial peritonitis.
Therapeutic treatment by suture for perforated/leaked lesion is crucial. However, such
procedures are often technically difficult because the perforated lesions are usually quite
fragile. Thus, several therapeutic approachs for gastrointestinal perforation or
anastomotic breakdown has been developed to replace conventional suturing
1, 2
. In the
previous Chapters, the author demonstrated successful construction of a wound dressing
material composed of the polysaccharide nanosheet, which is semi-absorbable and
physical adhesive property of this material is 7.5 times more strongly than that of
conventional films with over 1 μm thick due to a huge size-aspect ratio, and has a
flexible and robust macromolecular organization with an elastic modulus of an
approximate 1.3 GPa similar value to tendon in the living body, which will reinforce the
defected tissue by sealing.
In this chapter, the author investigated the sealing effect of the polysaccharide
nanosheet as a wound dressing using experimental bacterial peritonitis made by murine
cecal puncture. The author found that this ultra-thin nanosheet tightly overlapped the
perforated lesion without any bonding agents, thereby increasing survival rates and
improving bacterial peritonitis. Moreover, loading drugs such as antibiotics on the
nanosheet was also studied as the second generation of the functional nanosheets.
2. Polysaccharide Nanosheets Integrated for Gastrointestinal Perforation
2-1. Wound Infection Affecting Repair
Wound infection is an imbalance between immune response and bacterial

Page 121
Chapter 6
- 113 -
growth.
3
Bacterial infection impairs healing through several mechanisms.
4
At the
wound site, acute and chronic inflammatory infiltrates slow fibroblast proliferation and
thus slow ECM synthesis and deposition. Although the exact mechanisms are not
known, sepsis causes systemic effects that can impede the repair processes.
Open wounds invariably become colonized by bacteria. The wound has no
protective barrier to prevent bacterial adherence in the exposed dermis or subcutaneous
fat and muscle. Colonization does not preclude healing. However, if bacterial infection
occurs, then healing can be not only delayed but also stopped. A threshold number of
bacteria in the wound appear to be necessary to overcome host resistance and cause
clinical wound infection. Bacterial contamination results in clinical infection and delays
healing if more than 10
5
organisms per gram of tissue are present in the wound.
5
For
examples, skin grafts on open wounds are likely to fail if quantitative culture shows
more than 10
5
organisms/g tissue, which provides further evidence that bacterial load
impacts repair.
6
Similarly, well-vascularized muscle flaps heal open wounds
successfully if bacterial loads are not greater than 10
5
organisms/g tissue.
7
These
studies demonstrate that high levels of bacteria inhibit the normal healing processes.
2-2. Murine Cecal Puncture Model
The polysaccharide nanosheet was fabricated by a spin-coating assisted
layer-by-layer (SA-LbL) method using chitosan and sodium alginate (Na Alginate)
polyelectrolytes. According to the micro-scratch test
8
, the polysaccharide nanosheet
with the thickness of 39 nm had a strong physical adhesive property without utilizing
chemical bonding agents. Either the polysaccharide nanosheet with the thickness of 39
nm supported by a PVA film (70 μm thickness) or the control PVA film alone (70 μm
thickness) was placed onto a cecal perforated lesion (0.5 or 0.8 mm
2
) made by needle

Page 122
Chapter 6
- 114 -
puncture (Fig. 6-1a and 6-1b). The polysaccharide nanosheet was placed with the
supporting PVA film because the free-standing polysaccharide nanosheet itself
spontaneously shrinks in air. Thereafter, both of the PVA film between the
polysaccharide nanosheet and control was dissolved by dropwise addition of PBS
solution until the letter ‘P’ written on the surface was washed away (Fig. 6-1c). As the
supporting PVA film was dissolved, the underlying polysaccharide nanosheet was
observed tightly overlapping the tissue surface of the perforated lesion without any
bonding agents. However, the punctured hole appeared again for the control PVA film
due to dissolution by the addition of PBS (Fig. 6-1d).
Fig. 6-1 The polysaccharide nanosheet was applied to a murine cecal puncture model:
(a) cecal defect site punctured with a needle (dashed circle line showing an actual
punctured site), (b) sealing with the polysaccharide nanosheet supported by a
water-soluble PVA film where the letter ‘P’ was written in order to distinguish the PVA
side, (c) dissolution of the supporting PVA film with saline, and (d) after dissolution of
the PVA film.
(a)
(b)
(c)
(d)

Page 123
Chapter 6
- 115 -
[Murine Cecal puncture model and sealing with polysaccharide nanosheet]
All experiments were performed under the approval of the National Defense
Medical College Laboratory Animal Resource Protocols. Male C57BL/6 mice were
studied (8 weeks old, weight 20 g; Japan SLC, Hamamatsu, Japan). Under deep ether
anesthesia, the anterior abdominal wall of mice was shaved, and a small incision made
to expose the cecum. The cecum was punctured once with an 18- or 21-gauge needle
causing a defect of 0.8 or 1.0 mm diameter, corresponding to an area of 0.5 or 0.8 mm
2
,
respectively. Subsequently, the cecal puncture was sealed using either a polysaccharide
nanosheet (20 × 20 mm
2
) supported by a PVA film or a PVA film alone (i.e., no
nanosheet) as a control. The cecum was then returned to the peritoneal cavity, and the
abdomen was closed. Histological specimens were also obtained from the mice at 2
weeks after repair.
2-3. Evaluation of Murine Viability
Previous reports on murine cecal ligation and puncture have demonstrated that the
size of cecal defect caused by needle puncture significantly affected the severity of
intra-abdominal infection and mouse mortality
9
. The author used this animal model to
evaluate the sealing effect of the polysaccharide nanosheet on two different sizes of
cecal puncture. The mice were subjected to cecal puncture sized at either 0.5 or 0.8 mm
2
.
The perforated lesions were then overlapped with either a polysaccharide nanosheet or
the control PVA film. Percentage survival rate was then monitored. Overlapping
treatment with the polysaccharide nanosheet rescued 100% of the mice subjected to 0.5
mm
2
cecal puncture, while the control mice only showed 80% survival rate (Fig. 6-2a).
On the other hands, survival rate for mice subjected to a 0.8 mm
2
cecal puncture showed
over two-fold increase of survival rate (90%) over the control PVA film group (40%)

Page 124
Chapter 6
- 116 -
(Fig. 6-2b). This is because the overlapped PVA film was dissolved away by PBS or
postoperative body fluid.
The author also examined the number of viable bacteria in mouse peritoneal
lavage 1 day after cecal puncture sized to 0.8 mm
2
. Overlapping treatment with the
polysaccharide nanosheet decreased over 10-fold viable bacterial counts against control
group in the peritoneal lavage of the perforated mice (Fig. 6-2c). This result suggests
that the bacterial peritonitis was a major reason for the death and the polysaccharide
nanosheet overlapping the perforated lesion afford significant protection against
bacterial infection. Histological analysis of murine cecum was undertaken after
treatment by the polysaccharide nanosheet at 2 weeks. Instead of the accumulation of
fibroblasts around the tissue-defect site, lipocyte or fibroblasts bridged the tissue-defect
site (Fig. 6-2d).
Fig. 6-2 Clinical effect of the polysaccharide nanosheet on a murine cecal puncture
(a)
0
1
2
3
4
5
6
7
8
Time after operation (days)
M
o
u
s
e
v
ia
b
ility
(%
)
20
40
60
80
100
0
0
1
2
3
4
5
6
7
8
Time after operation (days)
M
o
u
s
e
v
ia
b
ility
(%
)
20
40
60
80
100
0
(b)
**
(c)
C
F
U
( x
1
04 )
Nanosheet
PVA
200
400
600
800
1000
1200
1400
1600
0
*
(d)
Perforation

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Chapter 6
- 117 -
model: Time-course study of murine viability after sealing the (a) 0.5 mm
2
and (b) 0.8
mm
2
puncture holes with a polysaccharide nanosheet (circle) or water-soluble PVA film
alone as a control (triangle). (c) The number of intraperitorial bacterium at 1 day after
the treatment using a polysaccharide nanosheet (solid bar) or control PVA film (white
bar) for the 0.8 mm
2
hole. Data shown are mean ± SE from 10 mice in each study group.
** P<0.01, * P<0.05 vs other group. (d) A histological specimen of the tissue-defect site
(murine cecum) sealed with the polysaccharide nanosheet at 2 weeks after repair
(hematoxylin-eosin staining, magnification 4×).
Any accumulation of fibroblasts was not observed on the outside of the muscular
wall, resulting in the prevention of the postsurgical adhesion with other organs.
Considering that postoperative adhesive ileus is one of the most common and
distressing complications of peritonitis, most of the murine cecum underwent very little
postsurgical adhesion with other organs after treatment with the polysaccharide
nanosheet. In the conventional surgical intervention, suturing or chemical bonding
agents were often required to securely seal the tissue-defect site although inflammatory
response caused post-surgical adhesion was sometimes remained as serious problems.
However, the polysaccharide nanosheet can physically overlap and adhere on the
tissue-defect site, resulting in the non serious inflammatory response during repair.
[Viable bacterial counts in the peritoneal lavage]
Peritoneal lavage was collected from the mice 1 day after surgical intervention.
Under deep ether anesthesia, 1 mL of PBS was intraperitoreally injected into the mouse,
and then 0.5 mL of peritoneal lavage was aseptically collected. The bacterial
suspensions were serially diluted 10
4
-fold by PBS, placed by a spiral platter on brain

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- 118 -
heart infusion agar plates, and incubated at 37°C for 24 h. The number of bacteria was
then counted according to the observed colonies on the agar plates.
3. Immunological Response Repaired by Polysaccharide Nanosheets
3-1. Evaluation of Immunological Response
The author examined the change of WBC count after cecal puncture in the mice
treated with the polysaccharide nanosheet or the control PVA film. After cecal puncture
sized to 0.5 mm
2
, the control mice showed an increase in WBC count due to
intra-abdominal bacterial infection. However, overlapping treatment with the
polysaccharide nanosheet suppressed the increase in WBC in the subjected mice (Fig.
6-3a), which displayed an improvement of bacterial peritonitis.
The severe form of bacterial peritonitis was also investigated, which is induced by
the larger cecal puncture sized at 0.8 mm
2
. In contrast with the 0.5 mm
2
sized puncture,
the WBC count was decreased in the control mice after 0.8 mm
2
cecal puncture,
suggesting the occurrence of serious bacterial peritonitis. However, overlapping
treatment with the polysaccharide nanosheet prevented the WBC decrease with severe
peritonitis (Fig. 6-3b). The control mice with the smaller cecal puncture (0.5 mm
2
)
showed an increase in WBC count after the procedure, resulting from the host immune
response to bacteria emerging from the perforated cecum. However, mice given the
larger cecal puncture (0.8 mm
2
) showed a decrease in WBC count, resulting from host
immune suppression/dysfunction due to excessive stimulation by a large amount of
bacteria. However, overlapping treatment with the polysaccharide nanosheet was
effective even after the supporting film had dissolved for both the mild to moderate case
of peritonitis as well as a severe case of peritonitis. Actually, the overlapping treatment
by the polysaccharide nanosheet almost completely suppressed the bacterial growth in

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Chapter 6
- 119 -
the abdominal cavity of mice suffering from cecal perforation, suggesting an inhibition
of bacterial penetration through the polysaccharide nanosheet.
The author also examined serum TNF levels in the mice 24 h after cecal puncture
sized to 0.8 mm
2
. Overlapping treatment with the polysaccharide nanosheet
significantly suppressed the elevation of serum TNF level in cecal-punctured mice
compared to that with the control PVA film alone (Fig. 6-3c). Therefore, physically
overlapping treatment with the polysaccharide nanosheet improved immunological
dysfunction such as TNF-induced multi-organ injuries resulting from intra-abdominal
infection. Treatment using the polysaccharide nanosheet suppressed an increase in WBC
count during minor infection and suppressed leukocyte activity during serious infection,
which is supported by decrease of the elevation of serum TNF level observed during
severe peritonitis.
Fig. 6-3 Immunological response after treatment with a polysaccharide nanosheet on
murine cecal puncture model: Time-course study of leukocyte counts for the (a) 0.5
mm
2
and (b) 0.8 mm
2
puncture holes using the polysaccharide nanosheet (solid bar) or
control PVA film (blank bar). (c) TNF-α counts for 0.8 mm
2
hole at 1 day after
treatment using a polysaccharide nanosheet (solid bar) or control PVA film (white bar).
Data shown are mean ± SE from 5 mice in each study group. * P<0.05 vs other group.
(c)
T
N
F
(p
g
/m
L
)
Nanosheet
PVA
*
0
50
100
150
200
250
300
350
400
W
B
C
( x
1
03
L
)
2
4
10
0
6
8
1
2
3
7
Time after operation (days)
(b)
*
*
*
(a)
2
4
10
0
6
8
1
2
3
7
Time after operation (days)
*
*
W
B
C
( x
1
03
L
)

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Chapter 6
- 120 -
[Measurements of WBC count and serum TNF level]
Blood samples were obtained from the retro-orbital plexus of mice at 1, 2, 3 and 7
days after surgical intervention. Serum samples were stored at -80°C until assayed.
White blood cells (WBC) in the murine whole blood were counted with a PEC-170
hematology analyzer (Beckman Coulter, Hialeah, FL). Serum TNF levels were
measured using cytokine-specific ELISA kits (Endogen, Woburn, MA).
3-2. Evaluation of Bacterial Growth Inhibition
The author confirmed that the polysaccharide nanosheet densely adhered on the
surface of transmembrane (TM) as expressing interference color (Fig. 6-4a). Then, the
TM with the polysaccharide nanosheet was set for the in vitro Transwell Membrane
®
(TM) assay (Fig. 6-4b).
Fig. 6-4 Evaluation of anti-bacterium permeability of a polysaccharide nanosheet: (a)
optical microscope image of Transwell Membrane
®
(TM) covered with the
polysaccharide nanosheet where the pore covered with the nanosheet was reflecting the
light compared with the uncovered TM (inset). (b) Schematic illustration of an
experimental setup.
The number of bacteria passing through the TM that was sealed with the polysaccharide
nanosheet was significantly lower than that through the TM alone (Fig. 6-5a).
30 μm
(a)
Polysaccharide nanosheet
E. coli (1 x 106)
mesh (ϕ: 3 μm)
FBS (600 μL)
FBS (100 μL)
(b)

Page 129
Chapter 6
- 121 -
Microscopic observations of the surface of the TM after culturing for 6 h showed few
microorganisms on the polysaccharide nanosheet side (Fig. 6-5b, left panel) although
numerous organisms were observed on the reverse mesh side (Fig. 6-5b, right panel).
Taken together, these results suggest that the polysaccharide nanosheet was working as
a physical barrier for bacterial permeation in the physiological condition, resulting in
the improvement of the in vivo survival ratio for the murine cecal puncture. Consistent
with in vivo findings, the polysaccharide nanosheet also inhibited bacterial permeability
using an in vitro transmembrane assay. As most of the suspended E. coli did not
translocate through the polysaccharide nanosheet, the nanosheet acted as an efficient
barrier. Thus, protection against the spread of bacteria by the polysaccharide nanosheet
might prevent the mice from deteriorating during perforating peritonitis.
Fig. 6-5 Anti-bacterium permeability effects of the polysaccharide nanosheet: (a) The
number of E. coli passing through the Transwell Membrane
®
covered with (solid bar)
and without (blank bar) a polysaccharide nanosheet after 6 hours incubation, and (b)
observation of the nanosheet (left) and mesh (right) side at the same time as (a) under an
optical microscope. (a) Data shown are mean ± SE from five individual experiments. *
P<0.05 vs other group. (b) Representative data are shown from five individual
experiments with similar results.
Nanosheet side
10 μm
Mesh side
10 μm
(b)
*
(a)
50
100
250
0
200
150
C
F
U
x
1
05
(-)
Nanosheet (+)
Nanosheet (-)

Page 130
Chapter 6
- 122 -
[In vitro transmembrane assay for evaluation of bacterial permeability through the
polysaccharide nanosheet]
The free-standing polysaccharide nanosheet (20 × 20 mm
2
) was scooped onto the
outside of a Transwell Membrane
®
(TM) purchased from Corning Co., Inc. (Corning,
NY), with a pore size of 3 μm. Because the commercialized TM pore size was
unsuitable for evaluation of E. coli permeability, the author made three additional pores
with a total area of 2.4 mm
2
(i.e. 0.8 x 3 mm
2
). The polysaccharide nanosheet-attached
to TM was positioned across a well filled with 100 and 600 μL of 10% FBS-RPMI 1640
(without antibiotic) in the inner and outer compartments, respectively. A 10 μL
suspension of E. coli (1 x 10
5
cells/μL) was carefully added to the inner compartment
and then cultured for 6 h. The number of bacteria in the outer well that penetrated
through the polysaccharide nanosheet-attached TM was subsequently determined.
Control TM was also made using three additional pores with a total area of 2.4 mm
2
,
without the polysaccharide nanosheet. Microscopic observation of the surface of the
polysaccharide nanosheet-attached TM was performed after staining for E. coli (Gram
staining).
4. Development of Antibiotics Loaded Polysaccharide Nanosheets
4-1. Antibiotics for Surgical Treatment
Treatment of the closed, infected wound depends on whether fluid or necrotic
tissue is present. If no fluid is draining or loculated, then cellulitis can be successfully
treated with appropriate antibiotics
10
. The wound should be opened, sutures removed,
irrigated, and debrided if pus or necrotic tissue is present. Appropriate antibiotic
administration following wound cultures treats surrounding cellulitis. Signs of wound
infection include fever, tenderness, erythema, edema, and drainage. Therefore, loading

Page 131
Chapter 6
- 123 -
antibiotics on the polysaccharide nanosheet should be a desirable combination for a
wound dressing material such as not only overlapping but also antibacterial effect on the
gastrointestinal perforation. In particular, the author used tetracycline (TC) for the
loading agents on the nanosheets because it can be soluble in methanol which does not
dissolve the nanosheets, and quantitatively traceable with a fluorescence (Fig. 6-6).
Fig. 6-6 Chemical structure of the tetracycline with fluorescent property.
4-2. Construction of TC loaded Polysaccharide Nanosheets
The TC loaded polysaccharide nanosheet was obtained by the following scheme
(Fig. 6-7). The TC dissolved in methanol was dropped and dried on the surface of
polysaccharide LbL films supported by the PVA film (named TC-LbL film). In order to
stable embedment of TC on the LbL film, poly(vinylacetate) film was spin-coated on
the surface of the TC-LbL film as a hydrophobic barrier, resulting in three layered
structure of a PVAc-TC-LbL film with the thickness of approximately 200 nm.
Fig. 6-7 Preparative scheme of the tetracycline loaded polysaccharide nanosheet.
OH
O
HO
NH2
O
O
OH
OH
N
OH
Ex: 380 nm, Em: 510 nm
SiO2 substrate
Spin coating LbL of Chitosan and
Na alginate (4500 rpm, 20 s)
Casting PVA (10wt%)
SiO2 substrate
SiO2 substrate
Drying
Drying
SiO2 substrate
Deposition TC solution (10μL)
SiO2 substrate
Spin-coating PVAc (4000 rpm, 20 s)
Peeling off and reversing a sheet
PVAc-TC-LbL film
PVA Film
LbL

Page 132
Chapter 6
- 124 -
This film was characterized by SEM (Fig. 6-8a) and conforcal laser microscopy
(CLSM) (Fig. 6-8b); that showed the thickness of PVAc-TC-LbL was approximately
200 nm and the middle layer of TC (colored in blue) was sandwiched by PVAc (labeled
with carboxyfluorescein, colored in green) and LbL (labeled with TRITC, colored in
red) layers. By utilizing the fluorescent property of TC, the loading density of TC on the
LbL film was estimated to be 6.2 μg/cm
2
(in the case of 12.5 μg of TC was deposited on
the polysaccharide LbL film sized 1 cm
2
), which can be easily adjusted by initially
dropped volume of the TC/methanol solution. Moreover, the hydrophobic barrier of
PVAc layer effectively prevented to dissolve TC on the film at least less than 60 min.,
confirmed by the kinetic release profile of TC in the physiological condition (pH 7.4, 37
o
C) (Fig. 6-8c). This is good enough to keep the TC layer inside of the film during
deposition of the film onto the tissue-defect site.
Fig. 6-8 Characterization of the TC loaded polysaccharide nanosheet. (a) SEM image
and (b) CLSM image. (c) Physiological stability of TC on the nanosheets covered with
the different thickness of PVAc hydrophobic layer: 0 nm (▲), 50 nm (■), 100 nm (●).
250 nm
Nanosheet
LbL
TC
PVAc
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
Time (min)
P
e
rc
e
n
ta
g
e
o
f d
ru
g
re
le
a
s
e
(%
)
(a)
(b)
(c)
SiO2
Nanosheet
Glass

Page 133
Chapter 6
- 125 -
[Materials]
Materials for preparation of the polysaccharide nanosheets were described in the
previous chapter. Tetracyclin (TC: Mw = 481Da) was purchased from Wako Co., Inc.
Osaka, Japan), which was dissolved into 5 mg/mL by methanol. poly(vinylacetate)
(PVAc: Mw = 98 kDa) was purchased from Sigma-Aldrich Co., Inc. Tokyo, Japan),
which was dissolved into 20 mg/mL by acetone.
[Preparation and characterization of the TC loaded polysaccharide nanosheets]
The polysaccharide nanosheets with the thickness of 40 nm supported by PVA
membrane was prepared as described in the previous chapter. On the surface of the
polysaccharide nanosheet (back side of the polysaccharide LbL film), 10 μL of TC
solution was cast and dried for overnight. As a hydrophobic barrier preventing
dissolution of TC by exudates, PVAc was spin-coated on the surface of the prepared
TC-LbL film. Structural characterization of the PVAc-TC-LbL film was undertaken by
SEM (HITACHI S-4300; Hitachi Co., Tokyo, Japan) with a platinum layer using an
ion-sputtering coater (HITACHI E-1045; 18 mA, 40 s., Hitachi Co., Tokyo, Japan), and
CLSM (FluoView, Olympus Co., Tokyo, Japan). Physiological stability of the TC on the
LbL film was evaluated by tracing the fluorescent signals of TC (Ex: 380 nm, Em: 510
nm) in the PBS (pH 7.4, 37
o
C).
4-3. Anti-Inflammatory Effect
Anti-inflammatory effect of the polysaccharide nanosheet loading TC was
evaluated by the murine cecal puncture model. This time, the punctured site on the
murine cecum was produced by sticking with 18 G needle (0.8 mm
2
puncture), resulting
in the leakage of digests. The perforated lesions were then overlapped with the

Page 134
Chapter 6
- 126 -
PVAc-TC-LbL film (Table 6-1, 1), and percentage survival rate was monitored. The
control samples of a TC-LbL film (Table 6-1, 2: without hydrophobic barrier) and a
PVAc-LbL film (Table 6-1, 3: without antibiotics) were also examined for the
percentage survival rate.
Overlapping treatment with the sample film 1 (Fig. 6-9a) rescued 100% of the
murine cecal puncture model, while the control 2 and 3 rescued 70% and 50 % at 7 days
after operation (Fig. 6-9b). The number of mice peritoneal bacteria in the film 1 was
also significantly decreased less than 0.1 % of those in the control film 2 and 3 after 1
day (Fig. 6-9c).
Fig. 6-9 Anti-inflammatory effect of the polysaccharide nanosheets loading antibiotics;
(a) Macroscopic image of the PVAc-TC-LbL film 1 on the murine cecum, illuminated
by black light. (b) Time-course study of murine viability after sealing the 0.8 mm
2
Table 6-1 Property of the prepared nanosheets for in vivo test
Entry
Thickness (nm)
TC (μg/cm2)
PVAc-TC-LbL film: 1
TC-LbL film: 2
PVAc-LbL film: 3
177 ± 9
69 ± 6
142 ± 4
6.2 ± 0.53
5.6 ± 0.27
0
M
ic
e
v
ia
b
ility
(%
)
0
20
40
60
80
100
0
1 2
3
4
5 6
7
Time (day)
****
PVAc-(TC)LbL
(TC)LbL
PVAc-LbL
0
5
10
15
20
25
30
2
3
1
C
F
U
1
04
)
*
TC-LbL
Cecum
Needle
(18 G)

Page 135
Chapter 6
- 127 -
puncture hole with the sample film 1 (black), the control film 2 (gray) or the control
film 3 (white). (c) The number of intraperitorial bacterium at 1 day after the treatment
using the sample film 1 (black), the control film 2 (gray) or the control film 3 (white).
Data shown are mean ± SE from 10 mice in each study group. ** P<0.01, * P<0.05 vs
other group.
Considering the physical adhesive property of polysaccharide nanosheet as well
as the antimicrobial property of antibiotics, the sample 1 should be stably adhered on
the perforated lesion, and then released TC which protected bacterial peritonitis. On the
other hands, the control film 2 could not release enough amount of TC to express the
anti-inflammatory effect. This is because the TC layer without hydrophobic barrier layer
of PVAc was not stable on the surface of the LbL film against the wet condition such as
inside of the body. Moreover, the control film 3 did not show neither sufficient adhesive
property nor anti-inflammatory effect due to the higher film thickness over 100 nm and
the absence of antibiotics in the structure.
5. Summary
The author investigated the potential efficacy of the polysaccharide nanosheet as a
wound dressing material for the gastrointestinal perforation in the use of experimental
bacterial peritonitis made by murine cecal puncture. The polysaccharide nanosheet
densely overlapped the perforated lesion without any adhesive agents, resulting in the
prevention of bacterial penetration. Thereby, usage of the polysaccharide nanosheet
increased survival rates. Moreover, loading antibiotics on the surface enhanced their
anti-inflamatory effect. The clinical benefit of nanosheet-type biomaterial was revealed,
which will replace the conventional surgical interventions.

Page 136
Chapter 6
- 128 -
References
1. T. B. Reece, T. S. Maxey, I. L. Kron, Am. J. Surg. 2001, 182, 40S.
2. a) T. Fabian, J. A. Federico, R. B. Ponn, Ann. Thorac. Surg. 2003, 75, 1587. b) P.
Hollaus, N. Pridun, J. Cardiovasc. Surg. (Torino) 1994, 35, 169. c) V. M. Nivasu, T.
T. Reddy, S. Tammishetti, Biomaterials, 2004, 25, 3283. d) Y. Murakami et al., J.
Biomed. Mater. Res. A. 2007, 80, 421. e). S. Ohya, H. Sonoda, Y. Nakayama, T.
Matsuda, Biomaterials 2005, 26, 655.
3. M. C. Robson, Clin. Plast. Surv. 1979, 6, 493.
4. M. C. Robson, Surg. Clin. North Am. 1997, 77, 637.
5. M. C. Robson, B. D. Stenberg, J. P. Heggers, Clin. Plast. Surg. 1990, 17, 485.
6. M. C. Robson, T. J. Krizek, J. Trauma 1973, 13, 213.
7. M. C. Murphy, M. C. Robson, J. P. Heggers, M. Kadowaki, J. Surg. Res. 1986, 41,
75.
8. S. Baba, T. Midorikawa, T. Nakano, Appl. Surf. Sci. 1999, 144, 344.
9. S. Ono et al., Am. J. Surg. 2001, 182, 491.
10. M. Li et al., Wounds: Biology, Pathology, and Management, Springer, New York,
2003.

Page 137
Chapter 7
- 129 -
Chapter 7
Development of the Surface Modification Techniques for the
Functional Biomacromolecular Nanosheets
1. Introduction
2. Selective Surface Modification of Nanosheets with Micro/Nano-Particles
3. Construction of Thermo-Responsive Free-Standing Nanosheets
4. Hydrodynamic Transformation of Free-Standing pNIPAM-Nanosheet
5. Summary
References

Page 138
Chapter 7
- 130 -
1. Introduction
The surface modification of nanomaterials is expected to enhance their potential
application for friction control
1
, drug delivery
2
, reversible thickness control
3
,
antirefrection control
4
, permeability control
5
, and ion-selectivity
6
. In particular, when
novel functional macromolecules such as synthetic polymers, proteins, DNA and
polysaccharides are fabricated on a substrate, the materials may possess novel
functionality, as a result of a cooperative effect of the modified functional substances
and interface of the substrate. Since the approach to fabricating free-standing nanosheets
has been established
7
, the next target would be to enhance the functionality of the
nanosheets by modifying their surfaces through chemical or physical conjugation. The
nanosheets are quite transparent and flexible, which obstruct both the precise
modification of their surfaces and the analysis of the modified surface.
In the first part of this chapter, the author focused on the structural colors of
polymer nanosheets where the conjugates or sculptures on the surface of a polymer
nanosheet should influence the optical path length
8
, and developed a selective surface
modification of a free-standing polymer nanosheet by physical adsorption of polymer
micro/nano-meter sized particles. In the second part of this chapter, the author
constructed the functionally stimuli-responsive polymer brushes composed of
poly(N-isopropylacrylamide) (pNIPAM) on the surface of the polymer nanosheet by
means of surface-initiated polymerization.
2. Selective Surface Modification of the Nanosheets with Particles
2-1. Selective Surface Modification with Different Latex Beads
The author performed selective surface modification of polysaccharide

Page 139
Chapter 7
- 131 -
nanosheets with micro/nano-particles using the water-soluble sacrificial layer (Fig. 7-1).
Fig. 7-1 Latex beads modification on the hetero surface of a polysaccharide nanosheet.
For preparation of the sacrificial layer, the water-solubility of PVA was exploited,
which was poorly soluble in acidic conditions, and easily dissolved at around pH 7. As
particles for the modification of the surface of the nanosheet, latex beads (LBs) with
diameters of 2 μm and 200 nm were selected because of their ease of visibility and
discrimination. Then, to prepare a firm PVA-SiO2 substrate, a PVA aqueous solution (10
wt%, 2 mL) was cast and dried on the SiO2 substrate (2 cm x 2 cm) in a desiccator for
12 hrs. The selective surface modification was carried by the following procedure. First,
the free-standing polysaccharide nanosheet, released from the substrate in acetone as
described above, was transferred onto the PVA-SiO2 substrate. Then, the transferred
nanosheet was gently immersed in a dispersion of LBs (ϕ: 200 nm, 2.3 x 10
10
particles/mL, pH 4 adjusted with phthalic acid) for 15 min, resulting in the physical
adsorption of LBs onto the obverse side of the nanosheet. After being taken out of the
pH 4 solution, the substrate was inverted, and re-immersed in water around pH 7 so that
the obverse-modified nanosheet might be released by dissolving the PVA layer. This
nanosheet was transferred onto another PVA-SiO2 substrate, with the unmodified,
Releasing in water
Releasing in water
Adsorption of latex beads (200 nm)
Nanosheet scooped by PVA-SiO2
substrate in acetone
Adsorption of latex beads (2 μm)
Scooped by PVA-SiO2 substrate

Page 140
Chapter 7
- 132 -
reverse side of the nanosheet kept uppermost. Then, the prepared substrate was
immersed into a dispersion of the second LBs (ϕ: 2 μm, 2.3 x 10
7
particles/mL, pH 4)
for modification of the reverse side.
[Materials & Characterization]
Biodegradable polyelectrolytes, chitosan (Mw = 88 kDa, deacetylation degree >
80%) and sodium alginate (Na Alginate, Mw = 106 kDa, an approximate Glucuronic /
Mannuronic ratio of 1 / 1.3), were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).
Acetone-soluble photoresist (OFPR-800 LB) and poly(vinyl alcohol) (PVA, Mw = 22
kDa) were purchased from Tokyo Ohka Kogyo Co. Ltd. (Kanagawa, Japan) and Kanto
Chemical Co., Inc. (Tokyo, Japan), respectively. Silicon wafers (200 nm-thick SiO2
substrates, boron doped, crystal face 100, p type), purchased from KST World Co.
(Fukui, Japan), were cut into a proper size (typically 4 cm
2
) and immersed in a mixture
of sulfuric acid/hydrogen peroxide (3/1) for a 10 min washing and then thoroughly
rinsed with deionized (D.I.) water (18 MΩ cm). Latex beads (diameter: 200 nm and 2
μm) were purchased from Polysciences Co., Inc. (CA, USA). The hetero-surface
modified polysaccharide nanosheet was released in water and subjected to observation
with a digital microscope (Keyence Co., Osaka, Japan) or a scanning electron
microscope (SEM, Hitachi Ltd., Tokyo, Japan).
2-2. LBs Modified Nanosheets (ϕ: 2 μm)
The author observed a change in the optical properties of the nanosheet when the
each surface was modified with either micro or nano-particles. The use of latex beads
with diameters of 200 nm and 2 μm enabled us to easily distinguish the surface

Page 141
Chapter 7
- 133 -
morphological change of the nanosheet by microscopic observation.
One edge of a nanosheet, one side of which was covered with 2 μm-LBs, was
diagonally folded to make the unmodified side face outward, as shown in the schematic
image (Fig. 7-2a). Optical microscopy of the border region of the nanosheet showed that
the left region, where the nanosheet had directly adhered on the substrate, exhibited a
red color, while the 2 μm-LBs were transparent. On the other hand, the folded area was
blue as expected; however, the color immediately surrounding the latex beads was red.
The red color was observed when the distance between two latex beads was less than
approximately 2 μm. It was suggested that the latex beads were sandwiched between
two layers, so that a space existed between the upper and the lower layers of the
nanosheet, where the beads were thus clustered together. In this state, the new red color
most likely originated from the lower monolayer reflection only, because a blue color
would require the whole thickness of the doubled, combined layer on the substrate.
Fig. 7-2 Optical microscopic image and schematic diagram at the edge (a) and bulk (b)
region of polysaccharide nanosheet modified with 2 μm-LBs.
The critical distance (approximately 2 μm) between the latex beads where the red
color was observed probably reflected the flexibility of the nanosheet; thus, these
differences could allow an evaluation of the elasticity of polymer nanosheets by
20 μm
Unfolded area
Folded area
ϕ 2 μm
polysaccharide
nanosheet
20 μm
a)
b)

Page 142
Chapter 7
- 134 -
utilizing the structural color obtained from LBs with various diameters. It should be
specifically noted that, in the bulk area of the folded 2 μm-LBs nanosheet, there were
approximately twice as many LBs in the folded region of the nanosheet as in the
unfolded, or single layer (Fig. 7-2b). Apparently, the beads were selectively adsorbed on
the obverse surface of the nanosheet.
2-3. LBs Modified Nanosheets (ϕ: 200 nm)
The author also attempted a one-sided surface modification of the polysaccharide
nanosheet with 200 nm-LBs. The unique structural color caused by the localization of
the LBs shown in Fig. 5 was not observed in the case of 200 nm-LBs. The diameter of
these beads, less than the optical wavelength, was too small to observe under an optical
microscope, so that the structural color observed was blue. In macroscopic images, the
optical interference was observed on the glass substrate where a 200-nm LBs nanosheet
was overlaid, while that was not found where the unmodified nanosheet was overlaid
(Fig. 7-3a).
Fig. 7-3 Morphological change in the polysaccharide nanosheets modified (i) with or
(ii) without 200 nm-LBs on a glass substrate: a) optical micrograph and b) transmittance
spectra. Line (iii) shows the spectrum of the glass substrate.
i) 200 nm-LBs nanosheet
ii) nanosheet
a)
400
500
600
700
85
86
87
88
89
90
91
92
93
94
95
Wavelength (nm)
T
ra
n
s
m
itta
n
c
e
(%
)
i)
ii)
b)
iii)

Page 143
Chapter 7
- 135 -
The region containing a nanosheet coated with 200 nm-LBs yielded a turbid surface,
due to a decrease in light transmittance compared to an unmodified surface. This effect
occurred because the 200 nm-LBs dispersed on the surface of the polysaccharide
nanosheet scattered the light passing through the nanosheet on the glass substrate (Fig.
7-3b). Therefore, the turbidity of the polymer nanosheet might be controllable by the
adsorption of small particles such as LBs on the surface of the polymer nanosheet.
2-4. Hetero-Surface Modified Nanosheets
Using the same procedure as for the selective surface modification of 2 μm-LBs,
LBs with different diameters were adsorbed onto the hetero-surface of the
polysaccharide nanosheet (obverse: 200 nm-LBs, reverse: 2 μm-LBs). The 200 nm-LBs
was adsorbed onto the obverse surface of the polysaccharide nanosheet prior to
adsorbing 2 μm-LBs onto the reverse side (Fig. 7-1), and scooped the resulting
hetero-surface modified polysaccharide nanosheet onto another SiO2 substrate for SEM
observation. Fig. 7-4 shows that the 200 nm-LBs were clearly observable, because they
had adsorbed on the obverse-side, whereas only vague outlines of the 2 μm-LBs,
adsorbed on the reverse-side, appeared. No rupture or corrugation was noted even in
vacuo, although the nanosheet was approximately 30 nm thick. The surface contour of
the 2 μm-LBs proves high flexibility to the monolayer of polysaccharide nanosheet
covering them, as depicted in Fig. 7-2. Moreover, it was seen that the overall contour of
the 2 μm-LBs became clearer when they were separated by a distance of more than
approximately 2 μm. This result corresponded well with the critical distance between
the LBs found to affect the structural color discussed earlier. These data indicate that the
polysaccharide nanosheet was very flexible and mechanically strong enough to enfold

Page 144
Chapter 7
- 136 -
the 2 μm objects. Further, a selective hetero-surface modification of the polysaccharide
nanosheet could successfully be accomplished with different sizes of LBs.
Fig. 7-4 SEM image and schematic diagram of polysaccharide nanosheet modified with
200 nm and 2 μm-LBs, respectively.
3. Construction of Thermo-Responsive Free-Standing Nanosheets
3-1. Polymer Brushes Obtained by ATRP
The author focused on the ultra-thin and flexible free-standing polymer
nanosheets, and envisaged to functionalize the surface with thermo-responsive polymer
brushes via an atom transfer radical polymerization (ATRP)
9
. ATRP has its root in atom
transfer radical addition, which targets the formation of 1:1 adducts of alkyl halides and
alkenes also catalyzed by transition metal complexes. A general mechanism for ATRP is
shown in Fig. 7-5
10
. The radicals or the active species, generated through a reversible
redox process catalyzed by a transition metal complex (Mt
n
–Y/Ligand, where Y may be
a ligand or the counterion), which undergoes a one-electron oxidation with concomitant
abstraction of the (pseudo)halogen atom, X, from a dormant species, R-X. This process
occurs with a rate constant of activation, kact, and deactivation kdeact. Then, polymer
chains grow by the addition of the intermediate radicals to monomers in a manner
2 μm
ϕ 200 nm
ϕ 2 μm
polysaccharide nanosheet

Page 145
Chapter 7
- 137 -
similar to conventional radical polymerization, with the rate constant of propagation kp.
Termination reactions (kt) also occur in ATRP, mainly through radical coupling and
disproportionation: however, in a well-controlled ATRP, no more than a few percent of
the polymer chains undergo termination. Therefore, an ATRP will have not only a small
contribution of terminated chains, but also a uniform growth of all the chains, which is
accomplished through fast initiation and rapid reversible deactivation.
Fig. 7-5 Reaction mechanism of an atom transfer radical polymerization (ATRP).
In the present study, polysaccharide nanosheets with the thickness of tens of
nanometerswere employed, which was composed of chitosan (polycation) and sodium
alginate (polyanion). These polysaccharides potentially have chemically reactive groups
in their own chemical structures such as amino and hydroxide groups, so that various
ATRP initiators can be coupled with them in the presence of base
11, 12
. As described in
the scheme (Fig. 7-6), the polysaccharide LbL films was prepared by the SA-LbL
method, where the surface was functionalized with pNIPAM brushes by ATRP. A
free-standing polysaccharide nanosheet bearing pNIPAM-brushes (pNIPAM-nanosheet)
was obtained by a supporting film method. Hydrodynamic properties of the resulting
pNIPAM-nanosheet suspended in water were macroscopically investigated such as a
thermo-responsive behavior and surface hydrophobicity.
R-Br +
Cu(I)Br /
Ligand
kact
kdeact
R・ +
Br-Cu(II)Br /
Ligand
kp
Monomer
Termination
kt

Page 146
Chapter 7
- 138 -
Fig. 7-6 Preparative procedure for a free-standing polysaccharide nanosheet bearing
poly(N-isopropylacrylamide) brushes as a stimuli-responsive surface.
3-2. Optimization of the Polymerization Conditions
The polysaccharide LbL film was directly prepared on the surface of the SiO2
substrate by a SA-LbL method, resulting in the ellipsometric thickness of 41 + 0.33 nm
with the 10.5 layer pairs of polysaccharides. The thickness was proportional to the
number of the layer pairs. Then, the prepared polysaccharide film on the substrate was
placed in the flask, and a 2-BIB initiator in a liquid state was reacted with hydroxide or
amino groups in polysaccharides producing ester or amide linkage. The surface
functionalization by 2-BIB was confirmed by an XPS analysis. The narrow scan mode
showed the presence of Br3d at 72 eV originating from the brominated initiator. This
peak was not observed in the LbL film prior to its functionalization (Fig. 7-7a).
The NIPAM was then polymerized on the polysaccharide LbL surface in the
different polymerization conditions such as concentration and polymerization time.
Effect of the conditions was evaluated from the ellipsometric thickness of the films
including a pNIPAM brushes and LbL films. As the NIPAM monomer concentration
was increased, the thickness was proportionally increased. At a NIPAM concentration of
Br
Br
Br
Bromo initiation
ATRP of NIPAM
NH
C
O
PMPDEA, CuBr
MeOH/water
Br
C
Br
O
TEA
THF
OH
NH2
OH
SiO2
Peel-off with tweezers
Release in water
Dry in vacuo
PVA cast

Page 147
Chapter 7
- 139 -
0.5 M, the total film thickness reached 88 nm (thickness of pNIPAM brushes; 47 nm)
(Fig. 7-7b). Then, we changed the polymerization time by fixing the concentration of
0.5 M. In one hour polymerization, the total film thickness became constant at 124 nm
(pNIPAM brushes thick is 83 nm), and no further growth of the thickness was observed
(Fig. 7-7c). However, the surface became inhomogeneous in the excess reaction over 1
hour. Therefore, the total film thickness was optimized in 88 nm (pNIPAM brushes; 47
nm, polysaccharide LbL film; 41 nm) where the polymerization condition was 0.5 M of
the NIPAM concentration and 0.5 hrs of the polymerization time.
Fig. 7-7 Spectroscopic analysis of pNIPAM-nanosheet; a) a XPS spectrum of bromo
functionalized surface (red: polysaccharide nanosheet, blue: the one after bromo
initiation), ellipsometric thickness of the pNIPAM-nanosheet on the SiO2 substrate in
the function of b) NIPAM monomer concentration and c) polymerization time.
Spectrum Skip Auto by 1
0
200
400
600
800
1000
1200
1400
0
1
2
3
4
5
6
7
x 104
TS070108100.spe
Binding Energy (eV)
c
/s
-C
K
LL
-O
K
LL
-O
K
LL
-O
1s
-N
1s
-C
1s
-B
r3
p
-B
r3
d
-S
i2
s
-S
i2
p
Spectrum Skip Auto by 1
62
64
66
68
70
72
74
76
78
80
82
84
0
500
1000
1500
2000
2500
3000
TS070108100.spe
Binding Energy (eV)
c/s
-Br3d
(a)
LbL
Br-LbL
40
0
0.1 0.2
0.3 0.4
0.5
0.6
30
50
60
70
80
90
100
NIPAM concentration (M)
E
llip
s
o
m
e
tric
th
ic
k
n
e
s
s
(n
m
)
b)
0
1
2
3
4
5
0
20
40
60
80
100
120
140
Polymerization time (hrs)
E
llip
s
o
m
e
tric
th
ic
k
n
e
s
s
(n
m
)
(c)

Page 148
Chapter 7
- 140 -
The FT-IR spectra showed the strong methylene stretch centered at 3000 cm
-1
and
the amide I carbonyl backbone stretch centered at 1650 cm
-1
derived from the isopropyl
and amide group of pNIPAM brushes although the asymmetric (1600 cm
-1
) and
symmetric (1400 cm
-1
) stretch derived from the carboxylate anion group of the
polysaccharide LbL film were hindered (Fig. 7-8a).
Fig. 7-8 Spectroscopic analysis of pNIPAM-nanosheet; (a) FT-IR spectra of
pNIPAM-nanosheet on the SiO2 substrate (red: polysaccharide nanosheet, blue:
pNIPAM-nanosheet). (b) XPS survey scan of pNIPAM-nanosheet on the SiO2 substrate.
The lack of Cu and Br peaks is indicative of the complete removal of the ATRP catalyst.
(c) GPC chromatograph of pNIPAM polymer. Polydispersity (PDI) was slightly higher
due to the effect of monomer concentration in the reactant in order to produce
concentrated pNIPAM brushes.
Spectrum Skip Auto by 1
0
200
400
600
800
1000
1200
1400
0
0.5
1
1.5
2
2.5
3
x 104
TS070108103.spe
Binding Energy (eV)
c
/s
-C
K
LL
-O
K
LL
-O
K
LL
-O
1s
-N
1s
-C
1s
-S
i2
s
-S
i2
p
(c)
Wavelength (cm-1)
(a)
3500 3000 2500 2000 1500 1000
A
b
s. (-; a
.u
.)
LbL
pNIPAM-LbL
(b)

Page 149
Chapter 7
- 141 -
Additionally, XPS clarifying the elemental composition of the
pNIPAM-nanosheet on the SiO2 substrate determines the degree of polymerization and
the inclusion of CuBr salts during ATRP. The survey scan of the pNIPAM-nanosheet
showed only the peaks of carbon, oxygen, nitrogen, and silicon (Fig. 7-8b). The absence
of Cu2p peak indicated that the catalyst was successfully removed after
post-polymerization rinsing.
To obtain the possible molecular weight of the pNIPAM polymer consisting
brushes, the ATRP was carried out by adding a small amount of a free initiator in the
reaction mixture
13
. The use of the free initiator together with ATRP has been previously
shown to be advantageous for an approximate estimation of the molecular weight of
polymers. Therefore, GPC was performed for the free pNIPAM polymer grown from
the free initiator after ATRP of pNIPAM brushes. In the 0.5 M NIPAM concentration, a
small portion of the free initiator (2-bromoisobutyricacidethylester) was added into the
reaction solution. After polymerization, the free pNIPAM polymer was purified by
ion-exchange column (Amberlyst) to remove Cu2+ ions, and elution was precipitated in
diethyl ether. The GPC chromatograph showed an average Mn of pNIPAM as 85,000
g/mol although a polydispersity indedx (PDI) was slightly higher (PDI; 2.59) than that
of our previous results (PDI; 1.40) (Fig. 7-8c), because of the effect of the high
monomer concentration to build the condensed pNIPAM brushes.
[Materials]
Polyelectrolytes; chitosan (Mw; 88 kDa, deacetylation degree; > 80%) and sodium
alginate (Na Alginate, Mw; 106 kDa, an approximate Glucuronic / Mannuronic ratio; 1 /
1.3), were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Poly(vinyl alcohol)
(PVA, Mw = 22 kDa) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).

Page 150
Chapter 7
- 142 -
Silicon wafers (200 nm-thick SiO2 substrates, boron doped, crystal face 100, p type),
purchased from KST World Co. (Fukui, Japan), were cut into a proper size (typically 4
cm
2
) and immersed in a mixture of sulfuric acid/hydrogen peroxide (3/1) for a 10 min
washing and then thoroughly rinsed with deionized (D.I.) water (18 MΩ cm).
Triethylamine,
2-bromoisobutyrylbromide
(2-BIB),
CuBr
and
N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA) were used as received from
Tokyo Chemical Industry (TCI) Co. (Tokyo, Japan). N-isopropylacrylamide (NIPAM),
purchased from TCI, was purified by recrystalization from hexane. Sodium
N-dodecylsulfate (SDS) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).
[Preparation of the pNIPAM-nanosheet with the polymerization of NIPAM]
Surface functionalizaition of the polysaccharide LbL film was undertaken with a
bromo initiator as following reported by Husson groups. The polysaccharide LbL film
on the SiO2 substrate was immersed into a 50 mL flask containing 25 mL of the dry
THF solution of 0.5 mL triethylamine. The flask was placed in an ice bath, and the
contents were gently stirred. Then, 0.5 mL of a 2-BIB initiator in liquid was slowly
added in droplets to the solution under N2 atmosphere. The flask purged with N2 gas
was removed from the ice bath, and the reaction in the flask was allowed to continue at
r.t. overnight. After the reaction, the functionalized substrate by the initiator was
removed from the flask by tweezers. It was rinsed with THF and D.I. water, and dried in
vacuo. for overnight.
The ATRP condition for NIPAM polymerization was performed by basically
following to our previous report. We prepared three Shlenk flasks for ATRP. The first
Shlenk flask was charged with 25.4 mg (0.176 mmol) of CuBr and 111 μL (0.530
mmol) of PMDETA under N2 atmosphere. A second Shlenk flask was charged with 2 g

Page 151
Chapter 7
- 143 -
(17.7 mmol) of NIPAM dissolved in 17.5 mL water and 17.5 mL methanol. As the
concentration of NIPAM was changed from 0.1 to 0.5 M, the volumes of CuBr and
PMDETA were adjusted in the stoichiometric mixing ratio corresponding to the NIPAM
molar ratio. Alternatively, the surface-functionalized LbL substrate was placed in a third
Shlenk flask. Then, the mixture in the first shlenk flask was transferred via cannula to
the second Shlenk flask, which was finally transferred to the third flask containing the
LbL substrate. The polymerization was performed at r.t. for 0.5 to 4 hours. After
polymerization, the substrate was removed and thoroughly rinsed with D.I. water and
methanol, and dried in vacuo. for overnight. Finally, the pNIPAM-nanosheet was
obtained using a water-soluble supporting film. A 10 wt% PVA aqueous solution was
casted on the pNIPAM-nanosheet on the SiO2 substrate and dried in vacuo. Then, the
bilayered film of the pNIPAM-nanosheet and PVA as a water-soluble supporting film
was peeled from the SiO2 substrate with tweezers. As the bilayered film was immersed
in D.I. water, the free-standing pNIPAM-nanosheet was obtained, and photographed
using a digital camera (OLYMPUS C-5050 ZOOM, Olympus Co., Tokyo, Japan) for
macroscopic observation.
3-3. Surface Characterization of the pNIPAM-Nanosheets
FT-IR imaging was performed in order to analyze the pNIPAM brushes on the
nanosheet macroscopically. Formation of the homogenous pNIPAM brushes was
essential to produce the thermo-responsive surface. FT-IR imaging is one of the
powerful tools for obtaining spatially and temporally-resolved chemical and structural
information. The images of pNIPAM brushes sliced at 1650 cm
-1
(Fig. 7-9a) showed
uniform and remarkable signals (green) of the amide I carbonyl backbone stretch,
corresponding to the pNIPAM brushes. However, such signals were not detected in the

Page 152
Chapter 7
- 144 -
certain regions such as the spots in the absence of chemical signals (arrows) and
carboxylated regions with strong signals (reddish dots inside of a dashed circle) with the
comparison of the slice at 1600 cm
-1
(Fig. 7-9b). This suggested that the pNIPAM
brushes were not grown from the carboxylate anion groups of the LbL film.
Fig. 7-9 Surface characterization of pNIPAM-nanosheet; IR-images of the
pNIPAM-nanosheet in spatial area of 176 μm × 176 μm for a) 1650 cm
-1
and b) 1600
cm
-1
. The arrows and dashed circle regions corresponding between a) and b).
The AFM images showed the microscopically morphological difference of the
nanosheet before and after polymerization. The nanosheet before polymerization
possessed a highly smooth surface with the thickness of 44.7 nm and the surface
roughness of 0.495 nm in RMS (root mean square) value (Fig. 7-10a). In contrast, the
surface of the pNIPAM-nanosheet after polymerization possessed the rougher surface
with homogenous polymer brushes with the total film thickness of 107.6 nm and the
surface roughness of 0.775 nm in a RMS value (Fig. 7-10b). Under the same ATRP
conditions, the pNIPAM brushed surface was also obtained on the monolayer of the
chitosan spin-coated surface with the thickness of 7.9 nm and the RMS of 1.92 nm (Fig.
7-10c and d), while the homogenous growth of pNIPAM brushes was not obtained
(a)
(b)

Page 153
Chapter 7
- 145 -
because the amount of reactive groups (-OH and –NH2) for the bromo initiation and
ATRP were much less than the polysaccharide LbL film. Therefore, the polysaccharide
LbL film is a quite useful platform for building up the polymer brushes.
Fig. 7-10 AFM images of pNIPAM-nanosheet a) before and b) after polymerization.
AFM images of pNIPAM brushed chitosan monolayer; c) surface morphology with the
RMS value of 1.92 nm and d) cross-sectional line profile of a polymer brush between
blue and red arrows, indicating 7.9 nm in height difference.
[Characterization of the pNIPAM-nanosheet]
Ellipsometry was applied to determine the thickness of the polysaccharide LbL
film. All measurements were conducted using a null ellipsometer operating in a
polarizer-compensator-sample-analyzer (Multiskop, Optrel, Berlin) mode. As a light
7.97 nm
(a)
(b)
(c)
(d)

Page 154
Chapter 7
- 146 -
source, a He-Ne laser (λ = 632.8 nm) was applied, and the angle of incidence was set to
60
o
. A multilayer flat model was used to calculate LbL thicknesses from the
experimentally measured ellipsometric angles of ∆ and ψ, assuming a refractive index
of 1.50 and 1.46 for the LbL films and 1.5 nm-thick SiO2 layer, respectively. Then the
film thickness was calculated using a fitting program (Elli, Optrel).
X-ray photoelectron spectroscopy (XPS) was carried out on a PHI 5700
instrument with monochromatic Al Kα X-ray source (1486.6 eV) incident at 90
o
relative
to the axis of a hemispherical energy analyzer. The spectrometer was operated both at
high and low resolutions with pass energies of 23.5 eV and 187.85 eV, respectively.
Photoelectrons were collected at a takeoff angle of 45
o
from the surface, and an analyzer
spot diameter of 1.1 mm. The survey spectra were collected from 0 to 1400 eV, and the
high-resolution spectrum were obtained for C1s, O1s, S2p, N1s and Au4f. All spectra
were collected at room temperature with a base pressure of 1 x 10
-8
. Electron binding
energies were calibrated with respect to the C1s line at 284.5 eV.
FT-IR imaging was performed on a Digilab Stingray imaging system consisting of
a Digilab FTS 7000 spectrometer, a UMA 600 microscope, and 32 × 32 mercury
cadmium telluride IR imaging focal plane array (MCT-FPA) image detector with an
average spatial area of 176 μm × 176 μm in a transmission mode. An 8 cm
-1
nominal
spectral resolution and an undersampling ratio (UDR) of 4 for the imaging were set up
and spectral data was collected with 1240 scans. All image processing and data
extraction were obtained using the software packages Win-IR Pro 3.4.
Atomic force microscopy (AFM, Agilent 5500 AFM/SPM System, Agilent
Technologies, CA) was used to investigate surface morphologies and surface analysis.
The AFM measurements were carried out with a piezoscanner (maximum scan size: 9×9
μm2
) at room temperature. Commercially available tapping mode tips (TAP300, Silicon

Page 155
Chapter 7
- 147 -
AFM Probes, TedPella, Inc., CA) were used on cantilevers with a resonance frequency
in the range of 290-410 kHz. All images (AFM topography, Tapping mode) were
filtered, and analyzed by using SPIP (Scanning Probe Image Processor, Imagemet.com).
A temperature controller (321 Autotuning, Lakeshore) was used for temperature-driven
in-situ AFM scanning.
3-4. Evaluation of Thermo-Responsive Surface of pNIPAM-Nanosheet
The temperature-driven in-situ AFM scanning was utilized to evaluate the
thermo-responsive properties of the pNIPAM-nanosheet (Fig. 7-11).
Fig. 7-11 Stimuli-responsive surface of pNIPAM-nanosheet monitored under thermal
temperature-driven in-situ AFM scanning for stepwise increment of temperature; a) 23.3
o
C, b) 32.0
o
C, c) 40.0
o
C and d) 60.0
o
C, and decrement to e) 23.3
o
C after d).
23.3°C
Rrms = 1.05 nm
(a)
Rrms = 0.932 nm
32.0°C
(b)
Rrms = 0.812 nm
40.0°C
(c)
23.3°C
Rrms = 1.07 nm
(e)
60.0°C
Rrms = 0.592 nm
(d)

Page 156
Chapter 7
- 148 -
This method was performed to specifically observe the morphological change in
the same position of the sample surface corresponding to in-situ temperature change.
Morphological changes in the surface and roughness of the pNIPAM-nanosheet were
analyzed where the scanning region was fixed during the observation. At first, the RMS
value of 1.05 nm was recorded at 25
o
C. Then, it was recorded at 40
o
C and 60
o
C (over
the LCST of pNIPAM) of 0.812 nm and 0.592 nm, respectively. It indicates that the
surface morphology was getting smoother with temperature increment. Moreover, the
surface roughness was recovered (RMS value is 1.07 nm) after cooling down to 25
o
C.
Additionally, we measured the mean water contact angles of the
pNIPAM-nanosheet by changing the temperature in the similar way to in situ AFM
scanning. The mean contact angle of the pNIPAM-nanosheet was recorded as 59 + 8
degree at 25
o
C. With increasing the temperature up to 40
o
C, the contact angles became
71 + 2 degree. The angle was almost recovered as 65 + 3 degree after cooling down to
25
o
C (Fig. 7-12).
Fig. 7-12 Water contact angles of the pNIPAM-nanosheet with thermal-responsive
surface; typical photographs of water contact angles at a) 25
o
C and b) 40
o
C, and c)
mean contact angles in the function of different water temperatures. i.e. Water contact
(a)
(b)
(c)
30
35
40
45
50
55
60
C
o
n
ta
c
t a
n
g
le
(d
e
g
re
e
)
65
70
75
80
25 oC
25 oC
40 oC

Page 157
Chapter 7
- 149 -
angles were measured three times at different positions in the order of 25
o
C (left), 40
o
C (middle) and 25
o
C (right).
Therefore, it was found that the pNIPAM-nanosheet on the substrate showed a
thermo-responsive property induced by a coil-to-globular transition of pNIPAM brushes.
The difference of the contact angles was 12 degree from 25
o
C to 40
o
C, which was
smaller than that we obtained in the previous report such as 45 degree
11
; pNIPAM
brushes on the LbL film composed of poly(allylamine hydrochloride) (PAH) and
poly(acrylic acid) (PAA). This difference would be occurred due to the hydrophlicity of
the underlayered LbL film where the LbL film of PAH and PAA is 20 degree and that of
polysaccharides is 62 degree.
4. Hydrodynamics of Free-Standing pNIPAM-Nanosheet
4-1. Thermo-Responsive Behavior in the Free-Standing States
In general, the coil-to-globular transition of pNIPAM brushes on the solid surface
is a more gradual transition than that observed in a dilute aqueous solution due to the
difference in the configuration of water molecules around pNIPAM
14
. Therefore, the
flexible free-standing pNIPAM nanosheet obtained by removal of the solid substrate
will enable to enhance the thermo-responsive property of the pNIPAM brushes because
of the increment of the water accessibility to pNIPAM. In order to peel off the
pNIPAM-nanosheet from a SiO2 substrate, the supporting film method was applied in
the mean of a water-soluble supporting film
15
. This method allows the convenient
collection of the free-standing nanosheet by peeling a dried bilayered film from the SiO2
substrate because the interaction between the bilayered film composed of the
pNIPAM-nanosheet and the water-soluble supporting film is higher than that between

Page 158
Chapter 7
- 150 -
the pNIAPM-nanosheet and the SiO2 substrate. Afterwards, the pNIPAM-nanosheet can
be released into an aqueous solution by dissolving of the PVA film.
Following to the method shown in the schema (Fig. 7-6), a 10 wt% concentrated
poly(vinyl alcohol) (PVA) aqueous solution was cast on a pNIPAM-nanosheet on the
SiO2 substrate. Then, the substrate was dried in vacuo. until a robust PVA film was
obtained. The resulting bilayered film was composed of the pNIPAM-nanosheet with 88
nm thick and the PVA film with 70 μm thick, and was easily peeled off from the edge of
the SiO2 substrate by picking up with tweezers (Fig. 7-13a). The surface of the SiO2
substrate was quite smooth (Fig. 7-13b) and the backside of the nanosheet showed a
mosaic structure, in which each mosaic piece was a comparable size to that of pNIPAM
brushes, as shown in Fig. 7-10b (Fig. 7-13c). These results suggested that the
pNIPAM-nanosheet was completely transferred to the PVA film with keeping an overall
structure prepared on the SiO2 substrate.
Fig. 7-13 Peeling-off using the supporting film method; a) macroscopic image of a
bilayered film (1 cm x 2 cm) composed of pNIPAM-nanosheet and supporting PVA film
peeled with tweezers. AFM images of b) the SiO2 substrate after peeling-off and c) the
reverse side of the pNIPAM-nanosheet.
When the bilayered film was dropped into the D.I. water, the free-standing
pNIPAM-nanosheet was immediately appeared on the air-water interface by dissolution
(b)
(c)
(a)
(b)
(c)

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- 151 -
of the PVA film. After three-times exchange of the aqueous medium with the fresh D.I.
water, the free-standing pNIPAM-nanosheet was colored in pale blue at 25
o
C on the
air-water interface and slightly flexible with creased surface (Fig. 7-14a). As the water
temperature was increased to 40
o
C, the color of the pNIPAM-nanosheet was turned to
yellow. Furthermore, the flexible surface was no longer seen but solicited at the
air-water interface (Fig. 7-14b). Interestingly, the cycle of these morphological
transformation in color and flexibility was reversibly observed at the air-water interface,
corresponding to the temperature change; transparently pale blue with flexible surface at
25
o
C (Fig. 7-14c), and turbid yellow with solid surface (Fig. 7-14d).
Fig. 7-14 Morphological transformation of a free-standing pNIPAM-nanosheet (1 cm x
2 cm) in water at a) 25
o
C, b) 40
o
C, c) 25
o
C and d) 40
o
C. The temperature was
changed consequently in the order of a)-d).
1 cm
(a)
(b)
(c)
(d)

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- 152 -
In Chapter 3, the structural colors of the polysaccharide nanosheets were
evaluated where the structural color was principally consequent of the increment of their
thicknesses. Therefore, the color changes of the free-standing pNIPAM-nanosheet can
be explained as the increment of the thickness of the pNIPAM-nanosheet. The accurate
estimation of the thickness was quite difficult in this experiment because the specific
data of the refractive indexes and the optical thickness should be required in the
free-standing states of the pNIPAM-nanosheet. However, considering the reversible
change of the surface roughness and surface wettability as shown in Fig. 7-11 and Fig.
7-12, the surface morphology of the pNIPAM-nanosheet and swellability was reversibly
changed by the coil-to-globular transition of the pNIPAM brushes. This result explains
the morphological transformation of the free-standing nanosheets as following: the
pNIPAM at 25
o
C is extended with hydrophilic states; thereby the surface morphology
of the pNIPAM nanosheet is flexible and transparent with bearing water molecules on
the surface. On the other hands, the pNIPAM at 40
o
C is collapsed with hydrophobic
states; therefore the surface morphology of the pNIPAM nanosheet was solid and turbid
with hydrophobically organized pNIPAM brushes. Hence, the morphological
transformation of the pNIPAM-nanosheet was induced by the thermo-responsive
property of the pNIPAM brushes.
4-2. Hydrodynamic Transformation Mediated by Surfactants
The effect of an anionic surfactant on the pNIPAM-nanosheet was evaluated
because the interfacial hydrodynamics of pNIPAM was highly dependent on the
concentration of SDS. Two different concentrations of a SDS medium were prepared
among the critical micelle concentration (CMC: 8 mM) of SDS for dispersion of the
pNIPAM-nanosheet. In the case of 16 mM SDS over CMC, the free-standing pNIPAM

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- 153 -
nanosheet was gradually sunk into the medium and suspended. However, no
morphological transformation was observed at this concentration although the
temperature was increased from 25
o
C to 40
o
C. This suggested that the surface of
pNIPAM-nanosheet was covered with SDS micelles, to reduce the thermo-responsive
hydrodynamic behavior of pNIPAM brushes. On the other hands, in the case of 0.16
mM SDS under CMC, the pNIPAM-nanosheet at 25
o
C was stably suspended in the
SDS medium (Fig. 7-15a). Interestingly, the pNIPAM-nanosheet was gradually floated
up and finally localized at the air-water interface with the structural color of yellow as
the temperature increased to 40
o
C (Fig. 7-15b).
Fig. 7-14 Hydrodynamic effect of anionic surfactant SDS on the pNIPAM-nanosheet.
(a)
(b)
(c)
SDS
[SDS] < CMC
(Floating-up)
T < LCST
pNIPAM
T > LCST
[SDS] > CMC
(Suspending)
(Suspending)

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- 154 -
The pNIPAM-nanosheet (1 cm x 2 cm) was a) suspending in the water at 25
o
C and b)
floating-up on the air-water interface at 40
o
C. c) Schemas explaining the effect of
anionic surfactants. The pNIPAM-nanosheet was suspending at both 25 (< LCST) and
40
o
C (> LCST) when the concentration of SDS was over critical micelle concentration
(CMC). However, the pNIPAM-nanosheet was floating-up at 40
o
C when that was under
CMC.
This result strongly suggested that the SDS concentration in the medium was
critical to control the hydrodynamics of the free-standing pNIPAM-nanosheet.
Previously, Napper et al. indicated the ability of SDS molecules interacted into
pNIPAM brushes by varying the SDS concentration
16
. They showed that the SDS
concentration over CMC strongly interrupted the coil-to-globular transition of pNIPAM
due to the hydrophobic interaction of SDS molecules into the pNIPAM brushes.
However, such transition was not interrupted by the SDS molecules under CMC.
According to their results, the free-standing pNIPAM nanosheet can be suspended with
the addition of SDS. Particularly, the shape transformation of the pNIPAM-nanosheet
was occurred as long as SDS concentration is under CMC because such concentration
enables the nanosheet suspend but not interrupt the coil-to-globular transition of
pNIPAM brushes (Fig. 7-15c). Hence, the SDS concentration under CMC was a critical
value for inducing thermo-responsive property of the pNIPAM-nanosheet by LCST,
such as suspending and floating-up of hydrodynamics among LCST of pNIPAM.
5. Summary
The bilateral surfaces of a free-standing polysaccharide nanosheet were
selectively modified with different sizes of latex beads. These surface modifications

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Chapter 7
- 155 -
were analyzed on the basis of the optical properties of the nanosheets on the SiO2
substrate, in particular their as structural colors, which offered a unique identification
method for surface modification. Moreover, the utilization of poly(vinyl alcohol) as a
water-soluble sacrificial layer permitted further conjugation, because the entire surface
modification procedure could be performed in aqueous conditions. Design of
biocompatible free-standing nanosheets selectively modified with different ligands or
different particles containing bioactive substances will enable a controlled release in a
dual or multi release mechanism.
Thermo-responsive polymer brushes of pNIPAM were successfully constructed
on the surface of a polysaccharide nanosheet by the combination of a SA-LbL method,
atom transfer radical polymerization and a water-soluble supporting film method, which
was confirmed by ellipsometry, IR-imaging, in situ temperature-controlled AFM and
contact angle measurements. The morphological transformation of the free-standing
pNIPAM-nanosheet was clarified through the reversible structural color change on the
air-water interface. Moreover, the thermo-responsive hydrodynamic behavior of the
pNIPAM-nanosheet, giving an effect on changing the shape, was also observed by
controlling the concentration of SDS. It was clarified that the free-standing polymer
nanosheet was an attractive platform for building the stimuli-responsive surface.
Therefore, other functionalized surfaces (for examples, pH-, light- and magnetic
sensitive surface) will be applicable in the use of stimuli-responsive polymer brushes.

Page 164
Chapter 7
- 156 -
References
1. D. P. Chang, J. E. Dolbow, S. Zauscher, Langmuir 2007, 23, 250.
2. J. H. Kim, T. R. Lee, Drug Dev. Res. 2006, 67, 61.
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17, 4092.
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9. R. C. Advincula, W. J. Brittain, K. C. Caster, J. Rühe (Eds.), Polymer Brushes:
Synthesis, Characterization, Applications, Wiley-VCH, Weinheim, 2003.
10. K. Matyjaszewski, J. Xia, Chem. Rev. 2001, 101, 2921.
11. A. Carlmark, E. Malmström, J. Am. Chem. Soc. 2002, 124, 900.
12. N. Singh, J. Wang, M. Ulbricht, S. R. Wickramasinghe, S. C. Husson, J. Membr. Sci.
2008, 309, 64.
13. T. M. Fulghum, N. C. Estillore, C-D. Vo, S. P. Armes, R. C. Advincula,
Macromoleucles 2008, 41, 429.
14 Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; O’Brien, M. J.; Lopez, G. P.
Langmuir 2003, 19, 2545-2549.
15. A. D. Stroock, R. S. Kane, M. Weck, S. J. Metallo, G. M. Whitesides, Langmuir
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16. P. W. Zhu, D. H. Napper, Langmuir 1996, 12, 5992.

Page 165
Chapter 8
- 157 -
Chapter 8
Conclusions and Future Prospects
1. Conclusions
2. Future Prospects
References

Page 166
Chapter 8
- 158 -
1. Conclusions
In this thesis, the author described the biomedical application of the
biomacromolecular nanosheets as newly constructed wound dressing nanomaterials.
The notable characteristics obtained from the respective study are summarized below.
The author is convinced that the integration of the biomacromolecular nanosheets will
work as an alternative intervention for the conventional surgical treatment.
1. Fundamental methodology to construct the free-standing biomacromolecular
nanosheet with tens-of-nm thick is established, utilizing layer-by-layer methods, which
involves the ubiquitous transference of the biomacromolecular nanosheets onto various
interfaces such as biological tissues and cell culture environment. (Chapter 2)
2. The surface property of the nanosheet is characterized in the basis of ‘thin film
interference theory’, which clarifies that the polysaccharide nanosheets possess flat,
smooth and physically adhesive surface. (Chapter 3)
3. The physical adhesive and mechanical property of the polysaccharide nanosheets are
investigated, which optimize the suitable thickness of the polysaccharide nanosheets for
clinical application as a wound dressing material. (Chapter 4)
4. The polysaccharide nanosheet is applied for the tissue-defect repair, which reveals the
flexible and physical adhesive property of the nanosheets effectively works in a wound
healing process, resulting in the no occurrence of post-surgical adhesion. (Chapter 5)
5. The polysaccharide nanosheet is applied for the treatment of bacterial peritonitis,
where the combination of antibiotics with the nanosheets not only enhances viability but
also inhabits intraperitorially bacterial growth. (Chapter 6)
6. Two kinds of surface modification techniques are developed for the functionalzation
of the polysaccharide nanosheets, by means of physical adsorption using micro/nano
particles selectively on the hetero-surface of the nanosheet, and chemically
polymerization resulting in the thermo-responsive surface. (Chapter 7)

Page 167
Chapter 8
- 159 -
2. Future Prospects
The author summarized the unique properties of the biomacromolecular
nanosheets as robust, flexible and physically adhesive sheet-type nanomaterials.
Focusing on these properties, the author simply used the nanosheets for wound dressing
materials in the surgical repair of pleural defect and gastrointestinal peritonitis. Beyond
the fundamentally biomedical application of the nanosheets, the next target should be
the “functional nanosheets”, which will be adequate for the active matters in the
nanobiotechnology such as remote-control, surface engineering and controlled release.
The author described the future prospects of the functional nanosheets below.
2-1. Endoscopic Surgery with Magnetic Nanosheets
One of the state-of-the-art technologies in the recent surgical intervention is the
endoscopic surgery
1
, which repaired affected site using the various modules attached at
the head of the endoscope. Most of the operation using endoscopic surgery does not
require abdominal operations; it is minimally invasive for the patient. However, there
have been still some problems to treat with the delicate incisions or defects due to the
limits of the field of view. Considering high flexibility of the nanosheets, the nanosheets
can be passing through the endoscope by folding as small as possible, and expanded
after evacuate the endscope (Fig. 8-1). In particular, creation of the magnetic property
on the nanosheets (magnetic nanosheets) will enable the magnetic nanosheets
remote-controllablly to land and adhere on the wound site.
Fig. 8-1 Remote-controllable nanosheets driven by magnetic field.
Internal lens
Suspend in the organ
Wound
Magnetic Field
EXPAND!!
Magnetic
Nanosheet

Page 168
Chapter 8
- 160 -
2-2. Cell / Tissue Surface Engineering Nanosheets
A cell’s surface composition determines its interactions with the environment,
ability to communicate with other cells, and trafficking to tissues. Recently, Rubner et al.
reported how to use conventional photolithographic patterning techniques to engineer
novel patch heterostructures containing both a payload component (superparamagnetic
nanoparticles in the present study) and a cell-adhesive face that partially comprises
hyaluronic acid (Fig. 8-3)
2
. This approach has potential for broad applications of the
cell/tissue surface engineering in bioimaging, cellular functionalization, immune system
and tissue engineering, and cell-based therapeutics.
Fig. 8-3 Cell surface functionalization by nanopatches.
2-3. Biological Facial Fastener
As described in the Chapter 7, hetero-surface availability for the surface
modification is one of the unique characters of the free-standing sheet-type materials,
which gives separated dual-functional surfaces in one structure. For example, the
nanosheets can possess different nature of adhesive properties on the dual interfaces; the
one is a physical adhesive interface derived from the ultra-thin thickness of the
nanosheets, and the other is a chemical/biological adhesive interface derived from
recognition ligands such as peptide, origosaccharides and synthetic functional polymer
brushes. Such nanosheets with hetero-adhesive properties will be effective as biological
facial fasteners for a crushed wound, in the clinical management of traumatic and burn
injury by skin-grafting operation (Fig. 8-2).
25 μm

Page 169
Chapter 8
- 161 -
Fig. 8-2 Nanosheets as the biological facial fastener to combine tissues.
2-4. Drug Delivery Devices
Many types of implantable controlled delivery devices are in various stages of
production and clinical evaluation. These devices have been designed to release drugs at
various dosages and for both intermittent and continuous delivery
3
. Several efforts to
control delivery release systems have already been reported using polymer based
nano-particles encapsulating drugs
4
. They can modulate the release period by changing
the biodegradability of the polymer composition. Combination of such nano-particles
inside or surface of the nanosheets will enable to release multi-drugs on demand of
periods, which can be applicable for the implantable controlled delivery devices.
Fig. 8-4 (a) Schematic representation of multi-drugs release from implantable
nanosheets, (b) containing different biodegradability of nano-particles in the structure.
Physi-sorption
(van der Waals)
Transplanted tissue
(e.g. Artificial skin)
Chemi-sorption
(Ligand-Receptor)
Nanosheet with molecular
recognition sites
Densely Fastened
Tissues
Tissue Surface
Drugs
Base nanosheet
Nano-particle shells with
different degradability
(a)
(b)
Implantable nanosheet

Page 170
Chapter 8
- 162 -
References
1. G. C. Vitale, B. R. Davis, T. C. Tran, Am. J. Surg. 2005, 190, 228.
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3. S. Sengupta et al., Nature 2005, 436, 568.
4. D. A. LaVan, T. McGuire, R. Langer, Nature Biotechnol. 2003, 21, 1184.

Page 171
Academic achievement
(List of Publication)
(1) T. Fujie, M. Kinoshtia, S. Shono, Y. Okamura, D. Saitoh, S. Takeoka. “Sealing
effect of a polysaccharide nanosheet for murine perforating peritonitis”, Surgery,
(submitted).
(2) T. Fujie, N. Matsutani, M. Kinoshtia, Y. Okamura, A. Saito, S. Takeoka. “Adhesive,
flexible and robust polysaccharide nanosheet integrated for tissue-defect repair”,
Nature Nanotechnol., (submitted).
(3) Y. Okamura, S. Takeoka, K. Eto, I. Maekawa, T. Fujie, H. Maruyama, Y. Ikeda, M.
Handa. “Development of fibrinogen γ-chain peptide-coated, adenosine
diphosphate-encapsulated liposomes as a synthetic platelet substitute”, J. Thromb.
Haemost., (in press).
(4) T. Fujie, Y. Okamura, S. Takeoka. “Selective surface modification of free-standing
polysaccharide nanosheet with micro/nano-particles identified by structural color
changes”, Colloids Surf., A., 334, 28-33 (2009).
(5) Y. Okamura, T. Fujie, T. Nogawa, H. Maruyama, Y. Ikeda, M. Handa, S. Takeoka.
“Hemostatic effects of polymerized albumin particles carrying fibrinogen γ-chain
dodecapeptide as platelet substitutes in severely thrombocytopenic rabbits”, Transfus.
Med., 18, 158-166 (2008).
(6) T. Fujie, Y. Okamura, S. Takeoka. “Ubiquitous transference of free-standing
polysaccharide nanosheet in the development of nano-adhesive plaster”, Adv. Mater.,
19, 3549-3553 (2007).
(7) Y. Okamura, T. Fujie, H. Maruyama, M. Handa, Y. Ikeda, S. Takeoka. “Prolongation
effects of hemostatic ability of poly(ethylene glycol)-modified polymerized albumin
particles carrying fibrinogen-γ chain dodecapeptide”, Transfusion, 47, 1254-1262
(2007).
(Preprints on International Symposium)
(1) S. Takeoka, Y. Okamura, T. Fujie, Y. Fukui. “Development of biodegradable
nanosheets as nano-adhesive plaster”, Pure Applied Chemistry, 80, 2259-2271
(2008).
(2) S. Takeoka, Y. Fukui, T. Fujie, Y. Okamura. “Novel polymeric nanosheets for
geronic applications”, Gerontechnology, 47, 220-224 (2008).
(3) S. Takeoka, T. Fujie, Y. Okamura. “Manipulation of free-standing polysaccharide
nanosheets and their application on a nano-adhesive plaster”, Polym. Prep. (Am.

Page 172
Chem. Soc., Div. Polym. Chem.), 48(2), 976-977 (2007).
(4) Y. Okamura, T. Fujie, M. Handa, Y. Ikeda, S. Takeoka. “Hemostatic effects of
polymerized albumin nano-particles carrying fibrinogen-γ chain dodecapeptide as
platelet substitutes”, Polym. Prep. (Am. Chem. Soc., Div. Polym. Chem.), 47(2),
854-855 (2006).
(Reviews)
(1) 木下学, 藤枝俊宣. 「超薄膜ナノシートの外科への応用」, 医学のあゆみ, 228(2),
187-189 (2009).
(2) 岡村陽介, 藤枝俊宣, 武岡真司. 「生体吸収性高分子ナノシートの構築と新しい
医用材料としての展開」, 化学工業, 59(11), 825-831 (2008).
(3) 藤枝俊宣, 岡村陽介, 武岡真司. 「医用材料としての高分子超薄膜(ナノシート)
の開発」, コンバーテック, 427(10), 137-143 (2008).
(4) 岡村陽介, 藤枝俊宣, 半田誠, 池田康夫, 武岡真司. 「血小板代替物の開発の
現状」, 人工血液, 13(4), 155-160 (2006).
(Patent)
(1) 武岡真司, 岡村陽介, 藤枝俊宣, 宇都宮沙織, 後藤隆弘. 「薄膜状高分子構造
体とその調製方法」PCT/JP2007/071437.
(Press release)
(1) 武岡真司, 藤枝俊宣. 「ナノ絆創膏開発」日刊工業新聞, 2007 年 12 月 4 日, 第
1 面,
(International Symposium)
(1) T. Fujie, Y. Okamura, S. Takeoka. “Discrimination of hetero-surface for selective
modificaiton of the free-standing polysaccharide nanosheet in the utilization of
structural color”, American Chemical Society 235th National Meeting & Exposition,
New Orleans, 2008. 4.
(2) T. Fujie, S. Takeoka. “Fabrication of a free-standing polysaccharide nanosheet in
application of nano-adhesive plaster”, The 1st Global COE International Symposium
on ‘Practical Chemical Wisdom’, Tokyo, 2007. 12.
(3) T. Fujie, Y. Okamura, S. Takeoka. “Fabrication of a free-standing polysaccharide
nanosheet in application of nano-adhesive plaster, 12th IUPAC International

Page 173
Synposium on MacroMolecular Complexes”, Fukuoka, 2007. 8.
(4) T. Fujie, Y. Okamura, M. Handa, Y. Ikeda, S. Takeoka. “Prolonged hemostatic ability
of poly(ethylene glycol)-modified polymerized albumin particles carrying
fibrinogen-gamma chain dodecapeptide”, XX1st Congress of the International
Society on Thrombosis and Haemostasis, Geneva, 2007. 7.
(5) T. Fujie, S. Takeoka. “Construction of layer-by-layer nanosheets consisting of
sodium alginate and chitosan”, The 4th 21 COE International Symposium on
‘Practical Nano-Chemistry’, Tokyo, 2006. 12.
(6) T. Fujie, Y. Okamura, S. Takeoka. “Prolongation effects of hemostatic ability of
poly(ethylene glycol)-modified polymeric albumin particles carrying fibrinogen-γ
chain dodecapeptide”, 3rd IUPAC-sponsored International Symposium on Macro-
and Supramolecular Architectures and Materials (MAM-06) : Practical
Nano-Chemistry and Novel Approaches, Tokyo, 2006. 5.
(Domestic Symposium)
(1) 藤枝俊宣, 松谷哲行, 木下学, 岡村陽介, 武岡真司. 「多糖ナノシートを用い
た胸膜欠損モデルイヌへの創傷被覆効果」第 57 回高分子討論会(2008 年 9 月,
大阪).
(2) 藤枝俊宣, 木下学, 岡村陽介, 松谷哲行, 庄野聡, 野上弥志郎, 斎藤大蔵, 武
岡真司. 「マウス穿孔性腹膜炎に対する超薄膜ナノシートを用いた被覆対策」
第 108 回日本外科学会定期学術集会(2008 年 5 月, 長崎),
(3) 藤枝俊宣, 岡村陽介, 武岡真司. 「ナノ絆創膏」開発に向けた自己支持性多糖
ナノシートの物性評価」第 88 回日本化学会春季年会(2008 年 3 月, 東京),
(4) 藤枝俊宣, 岡村陽介, 武岡真司. 「ナノ絆創膏開発に向けた交互積層法による
自己支持性多糖ナノシート」第16回ポリマー材料フォーラム(2007年11月, 東
京),
(5) 藤枝俊宣, 岡村陽介, 半田誠, 池田康夫, 武岡真司. 「重篤な血小板減少症モ
デル動物を用いた H12 ペプチド結合ポリエチレングリコール修飾アルブミン
重合体の止血能評価」第 14 回日本血液代替物学会(2007 年 6 月, 東京),
(6) 藤枝俊宣, 岡村陽介, 武岡真司. 「交互積層法による多糖ナノシートの構築と
ナノ絆創膏としての応用」第 56 回高分子学会年次大会(2007 年 5 月, 京都),
(7) 藤枝俊宣, 岡村陽介, 武岡真司. 「任意の膜厚を有する(アルギン酸/キトサン)
ナノシートの作製」第 28 回日本バイオマテリアル学会(2006 年 11 月, 東京),
(8) 藤枝俊宣, 岡村陽介, 半田誠, 池田康夫, 武岡真司. 「H12 ペプチド結合アル
ブミン重合体の PEG 修飾と止血能の延長効果」第 29 回日本血栓止血学会学
術集会(2006 年 11 月, 栃木)

Page 174
Acknowledgement
The presented thesis is the collection of the author’s studies, which have been carried out
under the direction of Prof. Dr. Shinji Takeoka at the Department of Life Science and Medical
Bioscience, Waseda University during 2007–2009. The author expresses the greatest
acknowledgement to Prof. Dr. Shinji Takeoka for his valuable suggestions, helpful discussions,
and continuous encouragement throughout this work. The author also expresses his sincere
gratitude to Prof. Dr. Nobuhito Goda, Prof. Dr. Satoshi Tsuneda and Assist. Prof. Dr. Arianna
Menciassi for the efforts as members of the judging committee for the doctorial thesis.
The author would like to express special thanks to the members of National Defense
Medical College. In particular, grateful acknowledgement is made to Prof. Dr. Daizoh Saitoh,
Assist. Prof. Dr. Manabu Kinoshita, Dr. Satoshi Shono and Dr. Noriyuki Matsutani for their
valuable suggestions.
The author gratefully acknowledges to Prof. Dr. Rigoberto C. Advincula and the
laboratory members for his advice and encouragement. In particular, sincere acknowledgement
is made to Dr. Jin Young Park, Miss. Nicel C. Estillore and Miss. Maria Celeste R. Tria.
The author expresses his sincere gratitude to Prof. Dr. Hiroyuki Nishide for his helpful
and valuable suggestions. The author also acknowledges to Dr. Teruyuki Komatsu, Dr. Hiromi
Sakai, Dr. Keitaro Sou and Dr. Akito Nakagawa for the advice, remarks, and discussions.
The author acknowledges to his mentor from bachelor to doctorial Course, Dr. Yosuke
Okamura for the discussions with full of passion not only on his research activity but also on his
way of life. His attitude towards science always inspired the author.
The author expresses his acknowledgement to Dr. Satoshi Arai, Dr. Tomoyasu Atoji, Dr.
Shinsuke Ishihara, Dr. Ichiro Takemura, Dr. Yosuke Obata, Miss. Izumi Sato, Dr. Masami Shoji,
Mr. Fumiaki Kato, Mr. Takeshi Ibe, and Mr. Atsushi Murata.
The author expresses the remarkable thanks to the member of Team Nanosheets, Mr.
Daisuke Niwa, Mr. Yoshihito Fukui, Mr. Kouki Kabata, Mr. Sho Furutate and Mr. Akihiro Saito.
Without their tremendous efforts and powers, the author would not accomplish his work.
All members in the Laboratory of Biomolecular Assembly and the Laboratory of Polymer
Chemistry offered kind assistance for which the author would like to thank deeply. The author
acknowledges to the Yoshida Foundation Scholarship and Global COE Program, MEXT.
Finally, the author expresses his deepest gratitude to his family, Mr. Fumitada Fujie, Mrs.
Nobuko Fujie, Mr. Hiroyuki Fujie, Mr. Shigefumi Fujie, Mrs. Kana Fujie and Miss. Yuko
Ohmura for their affectionate contributions.
January, 2009
Toshinori Fujie