(cache) Construction of Biomacromolecular Nanosheets and Their Biomedical Application as a Wound Dressing Material 生体高分子から

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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

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Promoter: Prof. Dr. Shinji Takeoka

Referees: Prof. Dr. Nobuhito Goda

Prof. Dr. Satoshi Tsuneda

Assist. Prof. Dr. Arianna Menciassi

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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

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Contents

Preface

Chapter 1 Principle, Design and Construction of

Molecular Organized Nanomaterials

1. Introduction

2. Fundamental Aspects of Self-Assembly and Self-Organization

2-1. Molecular Organizations Derived from Self-Assembly

2-2. Principles of Self-Assembly

2-3. Principles of Self-Organization

3. Principles of Intermolecular and Surface Interactions

3-1. Classification of Intermolecular Interactions

3-2. Molecular Organization of Intermacromolecular Complexes

3-3. Intermolecular and Surface Interactions

3-4. Van der Waals Interactions

4. Functional Ultra-Thin Films Based on Molecular Organizations

4-1. Nanomatrials Science Developed by Soft-Nanotechnology

4-2. Functional Ultra-Thin Films on the Static Interface

4-3. Modulation for the Integrated Surface

4-4. Analytical Tools for the Ultra-Thin Films

5. Polymer Nanosheets Utilized in the Biomedical Field

5-1. Biomedical Application of the Functional Ultra-Thin Films

5-2. Functional Films Exfoliated to Free-Standing Nanosheets

5-3. Shape Consideration on the Drug Carrier: Beyond Particles

5-4. Micro-Patterned Nanosheets as Potential DDS Carriers

References

Chapter 2 Construction of Biomacromolecular Nanosheets

in the Development of Nano-Adhesive Plasters

1. Introduction

2. Construction of Free-Standing Biomacromolecular Nanosheets

2-1. Polysaccharide Nanosheets Obtained by “Sacrificial Layer Method”

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2-2. ECM-Nanosheets Obtained by “Supporting Film Method”

3. Structural Characterization of Biomacromolecular Nanosheets

3-1. Surface Characterization by AFM (Polysaccharides Nanosheets)

3-2. Structural Characterization by FT-IR (Polysaccharides Nanosheets)

3-3. Surface Characterization by AFM (ECM-Nanosheets)

4. Ubiquitous Transference of Biomacromolecular Nanosheets

4-1. Development of “Nano-Adhesive Plaster”

4-2. Ubiquitous Transfer onto the Biological Tissue Surface

4-3. Ubiquitously Preparative Cell Culture Environment

5. Summary

References

Chapter 3 Optical Property of the Biomacromolecular Nanosheets

through Macro/Microscopic Surface Characterization

1. Introduction

2. Thin Film Interference Theory and Structural Color

2-1. Structural Colors in Nature

2-2. Fundamental Optical Processes

3. Microscopic Surface Morphology of Polysaccharide Nanosheets

3-1. Spin-Coating Assisted Structural Color Changes

3-2. Surface Characterization by AFM

3-3. Surface Characterization by SEM

4. Analysis of the Structural Colors of Polysaccharide Nanosheets

4-1. Sequential Structural Color Changes

4-2. Analysis of the Structural Color Changes

5. Summary

References

Chapter 4 Evaluation of Adhesive and Mechanical Property

of the Biomacromolecular Nanosheets

1. Introduction

2. Water Mediated Preparation of Polysaccharide Nanosheets

2-1. Total Preparation in Aqueous Condition

2-2. Structural Characterization

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3. Physical Adhesive Property of Polysaccharide Nanosheets

3-1. Micro-Scratching Test

3-2. Evaluation of Physical Adhesive Property

4. Mechanical Property of Polysaccharide Nanosheets

4-1. Bulge Test

4-2. Evaluation of Mechanical Property-1 (1 mm hole)

4-3. Evaluation of Mechanical Property-2 (6 mm hole)

5. Summary

References

Chapter 5 Wound Dressing Effect of the Biomacromolecular Nanosheets

Integrated for Pleural Tissue-Defect Repair

1. Introduction

2. Biology and Management of Wounds

2-1. Wound Biology

2-2. Classification of Wound Healing Process

2-3. Chronic Wounds and Effective Wound Management

2-4. Wound Dressing Materials

3. Physiological Property of Polysaccharide Nanosheets

3-1. Morphological Analysis of Blood Compatibility

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

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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

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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

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Chapter 1

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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

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Chapter 1

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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

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.

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Chapter 1

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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

. 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

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

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Chapter 1

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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, T

S or D, T

S, T

D, T

D, B

Materials, optoelectronics

Microfabrication, sensors, nanoelectronics

Biomembranes, emulsions

Displays

Band gap materials, molecular sieves

Models of crack propagation

Electronic circuits

Micofabrication

Biological osciilations

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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

. (b) Self-assembled peptide-amphiphile nanofibers

. (c) An array of

millimeter-sized polymeric plates assembled at a water/perfluorodecalin interface by

capillary interactions

. (d) An optical micrograph of a cell with fluorescently labeled

cytoskeleton and nucleus

. (e) Reaction diffusion waves in a Belousov-Zabatinski

reaction in a 3.5-inch Petri dish

. (f) A simple aggregate of three mm-sized rotating

magnetized disks via vortex-vortex interactions

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

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structure. Biological self-assembly can thus be described by a series of principles that

are often (but not always) obeyed

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 T∆Stranslation is based exclusively on considerations of concentration and is

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provided only as an approximation. The value for T∆Sconformation is of smaller magnitude

than T∆Stranslation 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

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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

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

, liquid crystals

, colloids

, micelles

, emulsions

, phase-separated

polymers

, Langmuir-Blodgett films

, and self-assembled monolayers

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

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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 T∆S 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

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Chapter 1

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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.

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

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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,

……Cl

. According to the point charge model, the energy is described by the

Coulomb law.

Ees = ZA•ZB•e2

( 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

(Drij

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

0.1

in water

0.1

Interactions

Ion-Ion

Proton acceptor

– Proton doner

Dipole – Dipole

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(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

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

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providing different insights and information are categorized here (Fig. 1-6)

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,

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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

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

for a sphere near a surface as

W(D) = -π

Cρ2

R/6D,

or for two flat surfaces as

W(D) = -πCρ2

R/12D

where ρ is a number of atomic density. The resulting interaction laws for some common

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geometries are shown in Fig. 1-7, given in terms of the conventional Hamaker constant

A = -π

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.

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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)

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

”: 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

Subatomic

Particles

Subatomic

Particles

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

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materials. The second strategy is “top-down

”: 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

. 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

Fig. 1-9 Langmuir-Blodgett technique.

(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

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

, 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

antireflective coatings

, antistatic coatings, stickiness or non-stickiness

, surface

induced nucleation

, antifouling

, hydrophilicity or hydrophobicity, biocompatibility

antibacterial properties, molecular recognition, chemical sensing or biosensing

microchannel flow control

…).

(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)

. The formation of polymer brushes via SIP was a novel

approach utilizing functional polymers such as pH-, photo-, and thermo-responsive

polymers.

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 nm

15 - 50 nm

25 -100 nm

1 μm

100 nm - 10 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.

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

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).

Fig. 1-13 (a) Schemas of the Hyarulonic Acid (HA) and Poly-lysine (PLL) layering

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).

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).

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).

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.

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

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

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

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

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

Page 37

Chapter 1

- 29 -

drugs, recognition proteins, enzymes, genes, etc

. 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

and/or dodecapeptide

(H12) derived from fibrinogen as a recognition site for activated platelets

. 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

Building block:

albumin

Biocompatible barrier:

Poly(ethylene glycol) (PEG)

Recognition site:

dodecapeptide (H12)

H12-PEG-polyAlb

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)

. 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

. They are excellent tools to study the immobilization of proteins such

as redox proteins

, enzymes

, 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.

particle 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

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

, an electrophotography method

, a

micromold method and gravity

, a micromold method and a lateral capillary force

, a

patterned Au film, and a drying process of a colloidal solution onto the patterned Au

film

. 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).

(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

. 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)

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

5 μm

Page 41

Chapter 1

- 33 -

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Bioconjugate Chem. 2005, 16, 1589. (c) Y. Okamura et al., Transfusion 2007, 47,

1254. (d)Y. Okamura et al., Transfus. Med. 2008, 18, 158.

50. Y. Okamura, M. Handa, H. Suzuki, Y. Ikeda, S. Takeoka. J. Artif. Organs 2006, 9,

251.

51. A. Ulman, An Introduction to Ultrathin Organic Films from Langmuir–Blodgett to

Self-Assembly, Academic Press, San Diego, 1991.

52. D. E. Khoshtariya, J. Wei, H. Liu, H. Yue, D. H. Waldeck. J. Am. Chem. Soc. 2003,

125, 7704.

53. E. E. Ferapontova, S. Shipovskov, L. Gorton. Biosens. Bioelectron. 2007, 22, 2508.

54. D. Niwa, Y. Yamada, T. Homma, T. Osaka. J. Phys. Chem. B 2004, 108, 3240.

55. Y. Masuda, W. S. Seo, K. Koumoto. Jpn. J. Appl. Phys. 2000, 39, 4596.

56. H. Fudouzi, M. Kobayashi, M. Egashira, N. Shinya. Adv. Powder Technol. 1997, 8,

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251.

57. W. J. Wen, N. Wang, D. W. Zheng, C. Chen, K. N. Tu. J. Mater. Res. 1999, 14, 1186.

58. Y. Sun, G. C. Walker. J. Phys. Chem. B 2002, 106, 2217.

59. Q. Guo, C. Arnoux, R. E. Palmer. Langmuir 2001, 17, 7150.

60. Y. Masuda, K. Tomimoto, K. Koumoto. Langmuir 2003, 19, 5179.

61. Y. Okamura, S. Utsunomiya, H. Suzuki, D. Niwa, T. Osaka, S. Takeoka. Colloids

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

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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

. 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

. 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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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

. Recent studies in LbL assembly using biopolymer revealed the utility

of polysaccharides for biomedical applications such as drug delivery and tissue

engineering

. 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

. 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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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

) (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

. 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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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

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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)

. 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

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

. 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).

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

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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

) polyelectrolytes

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),

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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,

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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main driving force building the nanosheet structure was confirmed to be electrostatic

interaction and hydrogen bonding

. For the nanosheet, the asymmetric (band at 1627

-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

. 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.

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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

(a)

(b)

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[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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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

, 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

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

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

cells/mL)

500 μm

(a)

(b)

(c)

1 cm

Page 65

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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

). 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.

Page 66

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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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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

. 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)

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.

Page 73

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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

. 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

. 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

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

(= 90

-85

), 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

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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’

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).

fle

ctivity (-

.))

Wavelength (nm)

400

500

600

700

Page 78

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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)

. Moreover, the angles of

refraction (θ1, θ2) were almost perpendicular to the substrate surface, so that cosθ1 and

θ1

θ2

mλ = 2(n1d1cosθ1+n2d2cosθ2)

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

Page 84

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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

, 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

. However,

polysaccharides are generally brittle because of high crystallinity introduced by inter-

and intra-molecular secondary bonding

. 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

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

. 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

) 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.

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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

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

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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

. 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.

Page 89

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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

. 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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We utilized the micro-scratch test (Fig. 4-4)

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

N/m)

showed over 7 times higher than that of the 1482-nm one (0.02 x 10

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,

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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

50 100 150 200

Thickness (nm )

ritic

l lo

)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

250 500 750 1000 1250 1500 1750

Thickness (nm)

ritic

l lo

)

(a)

(b)

(c)

Page 92

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- 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

)/(4 × h × d), ε = (2 × d

)/(3 × a

), E = σ /ε

Pressure gauge

Air

Steel chamber

Nanosheet

Syringe pump

Page 93

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- 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

. 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

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

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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

, and considerably smaller than the

Page 95

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- 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

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

. Generally, ultra-thin polymer films of tens-of-nm thickness show a

glass-transition temperature lower than the corresponding bulk

. 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

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- 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)

fle

tio

: d

(μ

)

0.0

2.0

3.0

4.0

5.0

200

400

600

800

1000

1200

1.0

1400

0.005

Strain: ε (-)

tre

: σ

)

0.010

0.000

120

4.0 kPa

1 mm

Page 97

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- 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

. 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

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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

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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

. 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

. 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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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)

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

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)

Page 102

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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

Keratinocytes

Neutrophil

Fibroblast

Daremal scar

(collagen)

Scab

Keratinocytes

migration

Fibroblast

Neutrophil

Macrophage

Blood vessel

Exposure of thromboplastic

tissue elements

Fibroblast

Resident monocyte

Platelet

Red blood cell

Dermis

SQ Fat

Epidermis

Scab

Fibroblast

Neutrophil

Macrophage

Page 103

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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

and inhibit wound healing. In addition, exudate

from chronic wound differs from acute wound fluid with relatively higher levels of

tissue destructive proteinase enzymes

and therefore more corrosive. The smell and

Page 104

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staining caused by exudate can also have a negative impact on a patient’s general

health and quality of life

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)

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)

, type of material

employed to produce the dressing (e.g. hydrocolloid, alginate, collagen)

and the

physical form of the dressing (ointment, film, foam, gel)

. 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

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classified into primary, secondary and island dressings

. 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

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?

Impermeable, increased stimulation of repair?

Characteristics/function

Page 106

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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

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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[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

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

, 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

. Therefore, the very weak pro-coagulant

Page 109

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

and/or

the heparin-induced thrombocytopenia (HIT)

; 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

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

Page 110

Chapter 5

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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

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)

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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.

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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

tin

)

tin

)

(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)

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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

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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

(a)

(b)

(c)

Tissue adhesion

Macrophages

Fibrin sheet

Air space

Page 116

Chapter 5

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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

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Chapter 6

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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.

Bacterial infection impairs healing through several mechanisms.

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

organisms per gram of tissue are present in the wound.

For

examples, skin grafts on open wounds are likely to fail if quantitative culture shows

more than 10

organisms/g tissue, which provides further evidence that bacterial load

impacts repair.

Similarly, well-vascularized muscle flaps heal open wounds

successfully if bacterial loads are not greater than 10

organisms/g tissue.

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

, 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

) 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

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[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

respectively. Subsequently, the cecal puncture was sealed using either a polysaccharide

nanosheet (20 × 20 mm

) 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

. 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

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

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

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

. 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)

Time after operation (days)

ility

)

100

Time after operation (days)

ility

)

100

(b)

(c)

( x

04 )

Nanosheet

PVA

200

400

600

800

1000

1200

1400

1600

(d)

Perforation

Page 125

Chapter 6

- 117 -

model: Time-course study of murine viability after sealing the (a) 0.5 mm

and (b) 0.8

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

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

-fold by PBS, placed by a spiral platter on brain

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Chapter 6

- 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

, 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

. In contrast with the 0.5 mm

sized puncture,

the WBC count was decreased in the control mice after 0.8 mm

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

)

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

) 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

. 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

and (b) 0.8 mm

puncture holes using the polysaccharide nanosheet (solid bar) or

control PVA film (blank bar). (c) TNF-α counts for 0.8 mm

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)

)

Nanosheet

PVA

100

150

200

250

300

350

400

( x

/μ

)

Time after operation (days)

(b)

(a)

Time after operation (days)

( x

/μ

)

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[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)

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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)

100

250

200

150

(-)

Nanosheet (+)

Nanosheet (-)

Page 130

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- 122 -

[In vitro transmembrane assay for evaluation of bacterial permeability through the

polysaccharide nanosheet]

The free-standing polysaccharide nanosheet (20 × 20 mm

) 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

(i.e. 0.8 x 3 mm

). 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

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

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

. 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

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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.

NH2

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

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

(in the case of 12.5 μg of TC was deposited on

the polysaccharide LbL film sized 1 cm

), 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

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

PVAc

100

Time (min)

f d

)

(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

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

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

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

ility

)

100

1 2

5 6

Time (day)

****

PVAc-(TC)LbL

(TC)LbL

PVAc-LbL

(×

)

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

, drug delivery

, reversible thickness control

antirefrection control

, permeability control

, and ion-selectivity

. 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

, 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

, 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

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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

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

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

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

) 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

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

400

500

600

700

Wavelength (nm)

itta

)

ii)

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

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- 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)

. 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

. The radicals or the active species, generated through a reversible

redox process catalyzed by a transition metal complex (Mt

–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

Monomer

Termination

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

Bromo initiation

ATRP of NIPAM

PMPDEA, CuBr

MeOH/water

TEA

THF

NH2

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

200

400

600

800

1000

1200

1400

x 104

TS070108100.spe

Binding Energy (eV)

-C

-O

-N

-C

-B

-S

Spectrum Skip Auto by 1

500

1000

1500

2000

2500

3000

TS070108100.spe

Binding Energy (eV)

c/s

-Br3d

(a)

LbL

Br-LbL

0.1 0.2

0.3 0.4

0.5

0.6

100

NIPAM concentration (M)

llip

tric

)

100

120

140

Polymerization time (hrs)

llip

tric

)

(c)

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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.

due to the effect of monomer concentration in the reactant in order to produce

concentrated pNIPAM brushes.

Spectrum Skip Auto by 1

200

400

600

800

1000

1200

1400

0.5

1.5

2.5

x 104

TS070108103.spe

Binding Energy (eV)

-C

-O

-N

-C

-S

(c)

Wavelength (cm-1)

(a)

3500 3000 2500 2000 1500 1000

s. (-; a

LbL

pNIPAM-LbL

(b)

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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

. 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

) 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

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- 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

-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)

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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

. 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

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

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

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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

C, b) 32.0

C, c) 40.0

C and d) 60.0

C, and decrement to e) 23.3

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)

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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

C. Then, it was recorded at 40

C and 60

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

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

C. With increasing the temperature up to 40

C, the contact angles became

71 + 2 degree. The angle was almost recovered as 65 + 3 degree after cooling down to

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

C and b) 40

C, and c)

mean contact angles in the function of different water temperatures. i.e. Water contact

(a)

(b)

(c)

t a

)

25 oC

40 oC

Page 157

Chapter 7

- 149 -

angles were measured three times at different positions in the order of 25

C (left), 40

C (middle) and 25

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

C to 40

C, which was

smaller than that we obtained in the previous report such as 45 degree

; 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

. 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

. 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

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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)

Page 159

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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

C on the

air-water interface and slightly flexible with creased surface (Fig. 7-14a). As the water

temperature was increased to 40

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

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

C, b) 40

C, c) 25

C and d) 40

C. The temperature was

changed consequently in the order of a)-d).

1 cm

(a)

(b)

(c)

(d)

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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

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

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

Page 161

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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

C to 40

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

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

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)

Page 162

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- 154 -

The pNIPAM-nanosheet (1 cm x 2 cm) was a) suspending in the water at 25

C and b)

floating-up on the air-water interface at 40

C. c) Schemas explaining the effect of

anionic surfactants. The pNIPAM-nanosheet was suspending at both 25 (< LCST) and

C (> LCST) when the concentration of SDS was over critical micelle concentration

(CMC). However, the pNIPAM-nanosheet was floating-up at 40

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

. 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

Page 163

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

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Chapter 8

Conclusions and Future Prospects

1. Conclusions

2. Future Prospects

References

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Chapter 8

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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)

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Chapter 8

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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

, 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

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Chapter 8

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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)

. 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

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Chapter 8

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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

. Several efforts to

control delivery release systems have already been reported using polymer based

nano-particles encapsulating drugs

. 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

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References

1. G. C. Vitale, B. R. Davis, T. C. Tran, Am. J. Surg. 2005, 190, 228.

2. A. J. Swiston et al., Nano Lett. 2008, 8, 4446.

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