Biomacromolecules 2005, 6, 2895-2913
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Polypeptide Multilayer Films
Donald T. Haynie,* Ling Zhang, Jai S. Rudra, Wanhua Zhao, Yang Zhong, and
Naveen Palath
Bionanosystems Engineering Laboratory, Center for Applied Physics Studies, College of Engineering &
Science, Louisiana Tech University, PO Box 10348, Ruston, Louisiana 71272
Received July 27, 2005; Revised Manuscript Received September 12, 2005
Research on polypeptide multilayer films, coatings, and microcapsules is located at the intersection of several
disciplines: synthetic polymer chemistry and physics, biomaterials science, and nanoscale engineering. The
past few years have witnessed considerable growth in each of these areas. Unexplored territory has been
found at the borders, and new possibilities for technology development are taking form from technological
advances in polypeptide production, sequencing of the human genome, and the nature of peptides themselves.
Most envisioned applications of polypeptide multilayers have a biomedical bent. Prospects seem no less
positive, however, in fields ranging from food technology to environmental science. This review of the
present state of polypeptide multilayer film research covers key points of polypeptides as materials, means
of polymer production and film preparation, film characterization methods, focal points of current research
in basic science, and the outlook for a few specific applications. In addition, it discusses how the study of
polypeptide multilayer films could help to clarify the physical basis of assembly and stability of polyelectrolyte
multilayers, and mention is made of similarities to protein folding studies.
I. Introduction
Projections are that polyelectrolyte multilayer films, coatings, and capsules will be useful for a large variety of
purposes.1-12 Applications could be said to fall into two
general categories, tailoring interactions of a surface with
its environment and fabricating “devices” with defined structural properties. The range of development areas includes
coatings, colloid stabilization, light-emitting or photovoltaic
devices, electrode modification, optical storage and magnetic
films, high charge density batteries, biomaterials, alteration
of biocompatibility, enzyme immobilization, flocculation for
water treatment and paper making, functional membranes,
separations, carriers, controlled release devices, sensors, and
nanoreactors. A key attribute of the preferred method of
preparing multilayer films and capsules is controlled vertical
structuring on the nanometer scale.
A polypeptide multilayer film is defined as a multilayer
film made of polypeptides. In some instances another type
of polymer is involved in the fabrication process, for instance
a chemically modified polypeptide,13 a nonbiological organic
polyelectrolyte,14 or a polysaccharide.15 A polypeptide film
might be deposited to confer specific biofunctionality on a
surface that was otherwise bioinert or to convert a bioactive
surface into one that is not adhesive to cells.16-20
Study of polypeptide multilayer films constitutes a confluence of two more mature streams of inquiry: peptide
structure and function, a significant area of basic research
since about 1905, and polyelectrolyte multilayer films,
developed since the early 1990s (Table 1). Multilayer films
* Corresponding author. Tel: +1 (318) 257.3790 (direct). Fax: +1 (318)
257.2562 (communal). E-mail: [email protected].
of polypeptides are promising for the development of
applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability,
specific biomolecular sensitivity, edibility, environmental
benignity, thermal responsiveness, and stickiness or nonstickiness. Polypeptides are ideally suited for such applications by virtue of their biochemical nature, the control one
can have over chemical structure in various approaches to
polymer synthesis, the ability to control formation of
secondary structure, or the availability of genomic data.
Control over structure and synthesis could also be important
for using designed polypeptides to gain insight on the nature
of polyelectrolyte multilayer film assembly and stability.
Common fabrication concerns in preparing polypeptide
multilayer films, coatings, or capsules are summarized in
Table 2; the same categories pertain to films made of other
kinds of polyelectrolytes. The important point here is that
one can have control over this range of variables even when
polypeptides are selected for more specific reasons, for
example extent of control over polymer synthesis, biocompatibility, or environmental benignity. Given a peptide
design, synthesis is accomplished by a chemical method or
a biological method. The approach to synthesis will depend
on sequence, degree of polymerization, required fidelity with
respect to sequence or length, cost, and production time. If
“natural” cross-linking of the film is required, for example
to stabilize film structure, at least one of the peptides will
feature the amino acid cysteine. The thiol group of cysteine
provides an “inherent” means of cross-linking polypeptides
under mild reaction conditions. A polypeptide multilayer film
or capsule will have surface (“external”) properties which
make it bioactive or nonbioactive. Examples of bioactive
10.1021/bm050525p CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/28/2005
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Haynie et al.
Table 1. Defining Properties of Polypeptides and of LBL Multilayer Films
polypeptides
LBL multilayer films
“designable”
can be produced en masse in bacteria
susceptible to proteolysis
biodegradable
edible
environmentally benign
sequence-specific immunogenicity
predictable R helix/β sheet propensity
fold into proteins in some cases
specific bioactivity in some cases
nm/Å-scale control over thickness
engineered architecture
arbitrary surface area
arbitrary surface shape
simple methodology
environmentally friendly methodology
low-cost methodology
can be used to make capsules
well-suited to an extremely broad range of particles
interesting material properties
Table 2. Variables in Polypeptide Multilayer Film/Coating/Capsule
Fabrication
general area
more specific considerations
polymer synthesis
sequence
length
fidelity
no
yes
bioactive
nonbioactive
active
inactive
small molecules
macromolecules
in vivo
ex vivo
cross-linking
external properties
internal properties
transport
half-life
properties are antimicrobial activity, immunogenicity, and
cytophilicity. Although some antibiotics are small molecules,
and some nonprotein polyelectrolytes are nonimmunogenic
or cytophilic, use of polyeptides enables a great degree of
control over bioactive properties in defined and “natural”
ways. Nonbioactive properties include hydrophobicity, hydrophilicity, and physical protein adsorption. Quite apart from
any biofunctionality a polypeptide might exhibit, the polymer
is a mere chemical on some level. In general, then, bioactive
materials will encompass features of nonbioactive materials.
The film or capsule will be “inactive” or “active” with regard
to “internal” properties. For example, an active film might
feature an entrapped functional enzyme or some other type
of chemically reactive agent. Transport and release properties
of the film or capsule will depend on the choice of peptides
and method of fabrication. The film preparation process could
be optimized for the transport of small molecules which may
or may not be soluble in water or some other solvent, or
release of macromolecules, for example nucleic acids and
peptides. The utility of a polypeptide multilayer film for a
specific application will depend on its half-life with regard
to temperature, pH, ionic strength, solvent, and so on. In
vivo, location of film or capsule deployment will determine
which metabolic mechanisms can affect half-life.
This review does not aim to do justice to the entire topic
of polyelectrolyte multilayer films, much less all of multilayer
films (Figure 1). The more general subject, developed since
the 1960s for colloidal particles and since the 1990s for
polyelectrolytes, is very large indeed, comprising over 2000
papers.21 We offer no apology for providing but partial
coverage of the scientific literature on polyelectrolyte multi-
Figure 1. Position of polypeptide multilayer films in the broader
scheme of things. Further information on the various levels of the
hierarchy can be found in refs 1, 7, 10, 52, and 236-239.
layers. Numerous informative and readable reviews have
already appeared on the subject.1-5,8,10,22 Nor does this review
provide substantial coverage of interesting recent advances
in polypeptide science outside the area of multilayer films,
for example peptides that self-assemble into various types
of nanostructure,23-26 diblock copolypeptides that selfassemble into spherical vesicular assemblies,27 or peptide
block copolymers that self-assemble into fibrils.28 Instead,
the focus of this work is multilayer films made of polypeptides and the unique opportunities related to the choice of
polypeptides for film assembly.
It may seem that our subject is too narrow or immature
for review at this time. Polypeptides, however, are not merely
a type of polyelectrolyte, that is, a polyelectrolyte for which
the linear charge density shows a marked dependence on
pH (a “weak” polyelectrolyte); these biomacromolecules
are of fundamental importance to life as we know it.
Moreover, it would seem rash to assume that what has been
learned about multilayer film fabrication from extensive
study of nonpolypeptide polyelectrolytes, including weak
polyelectrolytes, will form a sufficient basis for predicting
the physical, chemical, and, most importantly, biological
properties of polypeptide multilayer films. Furthermore, a
critical mass of reports on polypeptide multilayers has
appeared in the scientific literature, and the subject has not
been reviewed to date. This review summarizes reports in
the literature and mentions briefly some unpublished data
from our laboratory.
Biomacromolecules, Vol. 6, No. 6, 2005 2897
Polypeptide Multilayer Films
Table 3. Why Study Polypeptide Multilayer Films?
science
technology
physics
“unusual” backbone, role of entropy in adsorption
primary structure, role of different interactions
secondary structure, “inherent” nanoscale organization
chemistry
“inherent” covalent cross-linking
similarity to protein folding and stability
biochemical properties
biology
“inherent” bioactivity
biodegradation
environmental benignity
engineering
coatings
capsules
self-assembly
bio-based materials production
Table 4. Advantages of Polypeptides for Basic Research on
Multilayer Films
large range of different chemical groups in side chains
vast number of different combinations of amino acid in a
relatively short polymer
control over synthesis of polymers
control over contributions of hydrophobicity, hydrophilicity,
and hydrogen bonding potential to film structure and stability
control over secondary structure formation
control over ability to form “natural” cross-links
inherent chirality
One gathers from what has been said thus far that
polypeptide multilayer film development combines knowledge of physics, chemistry, biology, medicine, biotechnology,
and engineering in a way that is basically different from other
polyelectrolytes of interest to multilayer thin film and
microcapsule technology (Table 3). In our view, seeing how
this is so will be key to the realization of interesting and
useful products based on designed polypeptide films and
microcapsules. Reasons to present a review of the subject at
this time, then, are to clarify the scope of this burgeoning
field, summarize what has been achieved thus far, and
suggest possibilities for longer-term development and application.
II. Materials and Methods
Polypeptides. Polymers of amino acids form one of
several classes of biomacromolecule and constitute about half
of the dry mass of a living organism.29 Proteins are the
structural building blocks of materials ranging from hair and
tendons in mammals to the silk produced by insects and
spiders. As to size, the well-known globular protein hemoglobin, for example, has a diameter on the order of
nanometers. The enormous range of possible amino acid side
chains, of which the 20 usual ones are but a small subset,
makes polypeptides particularly promising for exploration
of multilayer film assembly, stability, and function, particularly in a biomedical context (see Table 4). Degree of
polymerization, degree of dispersity, and chemical modification of chain termini or side chains can be controlled,
depending on the method of synthesis or purification
protocol. Polypeptide chirality is important for biofunctionality and characterization of film structure; it could also play
a role in the development of enantioselective films.
medicine
tissue engineering
artificial cells
immunogenicity
edibility/biocompatibility
Considering the 20 usual amino acids alone, there are
∼1041 distinct chemical structures of unmodified 32-mer
peptide. Modern methods of synthesis enable realization in
the laboratory of a large proportion of this vast range of
possibilities. Important for multilayer film and capsule
assembly, some usual amino acid side chains are charged at
neutral pH. Other hydrophilic side chains are polar but
uncharged at neutral pH, and some side chains are hydrophobic. Inclusion of uncharged amino acids in charged
polypeptides will influence polymer assembly behavior and
film stability by forming hydrogen bonds or hydrophobic
interactions.
A key feature of polypeptides is their ability to form
secondary structures. It is known from protein research that
various sequences of amino acid show a preference to adopt
a type of secondary structure, R helix or β sheet.30 Both types
are stabilized by hydrogen bonds which form between
chemical groups in the polymer backbone. The ability of a
peptide to fold into a specific structure, the control one can
have over peptide sequence, and the range of possible ways
of integrating polyelectrolytes with other materials, for
example colloidal particles, together provide a remarkable
range of opportunities for the design of nanoscale materials.
To summarize, hydrophobicity, linear charge density,
propensity to form secondary structure at neutral pH, and
ability to form chemical cross-links can be varied according
to purpose by design of sequence.
Polypeptide Synthesis. There are two basic approaches
to designed polypeptide production: abiotic synthesis and
biotic synthesis. Each has advantages and disadvantages
(Table 5). These make abiotic synthesis, whether solution
phase31,32 or solid phase,33 the preferred approach for most
laboratory studies of film assembly, stability, and functionality, and biotic synthesis the logical option for large-scale
preparation of short peptides or production of long ones.
Nowadays, it is possible to prepare 100-kg quantities of short
peptides by chemical synthesis. Solution-phase synthesis,
though possible, is practically useful for preparation of
homopolypeptides or peptides of defined composition but
indefinite sequence only.31,32 Degree of polymerization in
the solution-phase approach will be determined by the
synthesis conditions, for example duration of reaction and
temperature, which must be worked out by empirical study.
Solid-phase synthesis, by contrast, starts with an NRderivatized amino acid attached to an insoluble resin via a
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Haynie et al.
Table 5. Advantages and Disadvantages of Approaches to Polypeptide Synthesis
approach
advantages
disadvantages
abiotic
does not require design of peptide-encoding genes
does not require knowledge of molecular biology
yields product contaminated relatively little by
nontarget peptides
allows incorporation of nonnatural amino acids,
expanding repertoire of building blocks
in principle, can synthesize virtually any sequence
of natural amino acids
generally 100% efficient coupling of monomers
can prepare very large quantities of material by
conventional methods
attainable economy of scale in production, translating
into a relatively low cost per unit mass
cannot synthesize all possible sequences equally well
does not provide 100% efficient coupling of monomers
cannot prepare uniform samples of peptides above
about 75 residues
usually not suitable for preparation of large quantities
of material
requires design of peptide-encoding genes
biotic
suitable linker molecule.33 The NR protecting group is
removed in a deprotection step, and the next amino acid in
the chain (also NR-protected) becomes coupled. The process
is imperfect, but certain methods, for instance double
coupling, give good overall efficiency. The deprotection/
coupling cycle is repeated until the desired sequence of amino
acids is generated. The peptide-linker support is cleaved,
yielding the peptide and side chain protecting groups. Finally,
the protecting groups are removed. Solid-phase synthesis has
made it possible to obtain useful amounts of a specific
peptide on a routine basis. Abiotic approaches permit study
of polypeptides containing nonnatural amino acids.
As to biotic synthesis, small-scale synthesis can readily
be done in a research laboratory, and 75 kl fermenters exist
for the recombinant production of peptides in E. coli. A gene
encoding the peptide of interest is inserted into a DNA
expression vector, which is taken up by the host cell in a
process called transformation.235 Following induction of gene
expression, macromolecular machines within the host cell
“read” the “instructions” encoded in the recombinant gene
and synthesize the corresponding peptide as though it were
its own. Recombinant gene-expressing host cells are cultured
and then lysed to extract the recombinant peptide of interest,
which is then purified by chromatography and characterized.
Biotic synthesis is attractive because it can be a highly costeffective means of achieving routine production of large
quantities of material, and in principle it will allow a much
higher upper bound on designed peptide length than solidphase synthesis. Moreover, just as with solid-phase synthesis,
biotic approaches are useful for production of sequence
variants by design, permitting comparison with the original
sequence of physical, chemical, and biological properties by
appropriate methods.
Clearly, these technological capabilities in peptide synthesis will be crucial for translating the potential of polypeptide multilayer films into commercial products.
Multilayer Films. Layer-by-layer assembly (LBL) is a
method of making a multilayer thin film from oppositely
charged species,1,6,7,34,35 deposited in succession on a solid
support (Figure 2). The method has attracted interest because
it is simple and considerably more versatile than other
techniques of thin film preparation, for example LangmuirBlodgett deposition. The basic principle of assembly, Coulombic attraction and repulsion, is far more general than the
requires expertise in molecular biology
requires separation of target peptide from bacterial
contaminants
involves proteases which may degrade target peptides,
particularly unstructured ones
type of adsorbing species or surface area or shape of
support.34 Film assembly can be described as the kinetic
trapping of charged species from solution on a surface.1
Multilayer film formation is possible because of charge
reversal on the film surface after each adsorption step.36-51
Surface charge thus depends on the last adsorbed layer,
permitting a degree of control over surface and interface
properties. A high density of charge in the adsorbing species
will result not only in strong attraction between particles in
neighboring layers but also in strong repulsion between likecharged particles in the same layer. That is, electrostatics
both drives film assembly and limits it. Several layers of
material applied in succession create a solid, multilayer
coating. Each layer can have a thickness on the order of
nanometers, enabling the design and engineering of surfaces
and interfaces at the molecular level. Subtle changes in
organization and composition can influence film structure
and functionality. The layering process is repetitive and can
be automated, important for control over the process and
commercialization prospects. Constituents of a film could
be bioactive or bioresponsive materials. Advantages of LBL
over other methods of film production are summarized in
Table 6.
Since the early 1990s there has been considerable interest
in making multilayer films from linear ionic polymers.1,3
Such films are being developed for a variety of applications: for example, contact lens coatings, sustained-release
drug delivery systems, biosensors, and functionally advanced
materials with various electrical, magnetic, and optical
properties.1,2,4-8,10,11,22,35,52 Many different polyelectrolytes
have been studied in this context. Examples are poly(styrene
sulfonate) (PSS), poly(allylamine hydrochloride) (PAH),
poly(acrylic acid) (PAA), and poly(diallyldimethylammonium chloride) (PDADMAC). These polymers are called
“common” or “conventional” in view of their ready availability from commercial sources and their having been
studied extensively. Polyelectrolyte structure, however, would
appear to have little effect on whether LBL is possible if
the ionic groups are accessible. The polymer chains, once
assembled into a multilayer film, tend to become interpenetrated,53,54 whether strong polyelectrolytes or weak
ones.1 Besides synthetic polymers, “natural” polyelectrolytes
such as nucleic acids, proteins, polysaccharides, and charged
nano-objects such as virus particles and membrane fragments
Polypeptide Multilayer Films
Biomacromolecules, Vol. 6, No. 6, 2005 2899
Figure 2. Polypeptide multilayer film fabrication. Bottom: schematic diagram of LBL. Top: corresponding experimental data. Oppositely charged
polypeptides in solution (bottom left) are adsorbed consecutively onto a solid support (bottom center), for example a quartz slide, yielding a
multilayer film (bottom right). Loosely bound material is rinsed off in water or buffer. The film could be dried after each adsorption step for
measurement. The deposition process results in short peptides going from a random coil conformation in solution (top left) to a β sheet in the
film (top right). Some R helix might be present, depending on peptide sequence and solution conditions. Generally, 20 min is sufficient for most
binding sites on the film to become saturated with the oppositely charge polypeptide (top center), when the linear density of charge is above 0.5,
the concentration of material in solution is on the order of 1 mg/mL, and the temperature is about 25 °C. Evidently, the amount of material on
the resonator did not change much during deposition of the negative peptide, but deposition was none the less important for reversing the
surface charge density of the film and enabling subsequent adsorption of the positive peptide.
have been assembled into multilayer films.55 The present
work is focused on polypeptides, a type of weak polyelectrolyte (Figure 1).
Weak Polyelectrolyte LBL. The linear charge density of
a weak polyelectrolyte is “tunable” by simple adjustment of
pH. Many such polymers are essentially fully charged at
neutral pH. The pKa of ionizable groups in a weak polyelectrolyte, however, will be sensitive to the local electronic
environment, and the net charge can shift significantly from
the solution value on formation of a polyelectrolyte complex
or film.56-63 Extensive study by Rubner and colleagues has
revealed key aspects of the LBL assembly behavior of
“conventional” weak polyelectrolytes,52 e.g., PAA and PAH.
Fabrication of films from these polymers represents a type
of molecular-level blending process. Control of the type and
extent of blending enables manipulation of the bulk and
surface properties of the resulting film. Weak polyelectrolytes
thus afford great latitude for controlling internal and surface
material composition, thickness, molecular organization, ionic
“cross-link” density, molecular conformation, wettability,
swelling behavior, surface properties, and reactive functional
groups. Possible molecular conformations in polypeptide
films will include not only the “flat” and “loopy” structures
of conventional polyelectrolyte adsorption64 but also R helices
and β sheets. The pH sensitivity of weak polyelectrolytes
enables changes in film morphology after film preparation.
Such changes can be reversible or irreversible.
In the case of PAA and PAH at pH 7, where the linear
charge density is high, the thickness of an adsorbed layer,
about 3-5 Å, is independent of polymer molecular weight
over a range of about 3 orders of magnitude: 3000-106
g/mol.59 Adsorbed polymer chains are “flat”. If the pH of
the dipping solutions is increased or decreased, however, a
dramatic increase in layer thickness results. At pH 5, for
instance, the thickness of a PAH-PAA bilayer is about 125
Å. Under such conditions, the polymer chains form “loops”
and layer thickness scales as molecular weight to the 0.3
power. This has been explained as a decrease in the surface
charge density onto which fully charged polyelectrolytes are
deposited.65 The adsorbed layer thickness increases with
increasing surface roughness. Conditions which promote
adsorption of “loopy” structures, therefore, lead to deposition
of correspondingly large amounts of polyelectrolyte.
Control of internal and surface composition of weak
polyelectrolyte films is achieved by control of the amount
of polyelectrolyte adsorbed. In this way, surface wettability
can be controlled.2,57 Another approach is to alter the charge
density of the adsorbed polyelectrolyte by changing the pH
of the solution of the other polyion. This alters the surface
charge while keeping the internal structure similar, enabling
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Haynie et al.
Table 6. Advantages of LBL over Other Approaches to Thin Film Production
general advantage
diverse range of materials suitable for deposition
applicable further particulars
nonpolyelectrolytes
polyelectrolytes
deposition on surfaces of almost any kind or shape
nonbiological surfaces
(e.g. plates, stents, contact lenses)
colloids (e.g. micron-sized particles
of calcium carbonate)
biological “cells”
(e.g. red blood cells or viruses)
numerous control variables for deposition
pH
concentration
adsorption time
ionic strength
solvent composition
temperature
broad pre- and post-fabrication processing potential
pH
ionic strength
temperature
nonbiological polymers
biological polymers
astronomical number of possible layer architectures
environmentally friendly production process
low-cost production process
creation of “designer” films using the same polyions.
Assembly of polyelectrolytes of different charge density
allows nonstoichiometric pairing of polyions. The result in
the case of PAA and PAH is a swellable film. Such films
can bind metal cations from aqueous solution by ion
exchange with the protons of the PAA carboxylic acid
groups.66
A final point about weak polyelectrolyte films is pH-driven
reorganization of morphology. Under certain circumstances,
notably when the internal film structure is characterized
by fully charged, “loopy”, randomly arranged chains, reorganization can result in the formation of micropores. For
example, exposing a pH 3.5 PAA/pH 7.5 PAH film to pH
2.4 for 15 s and then rinsing with water leads to phase
separation and micropore formation.58 As discussed below,
the phenomenon has been exploited to “load” polyelectrolyte
microcapsules with various soluble molecules, including
enzymes.
Alternatives to LbL. Recently, alternatives to the repetitive assembly of layers by dipping1 have been developed
for the fabrication of ionic polymer films. We include them
here because they could prove important for the commercial
prospects of such films. An iterative spraying method of film
assembly has been introduced by Schlenoff et al.67 The use
of spin-coaters has been demonstrated by Hong et al.68,69 and
Wang et al.70 More recently, the Strasbourg polyelectrolyte
film group71,72 has shown that continuous and simultaneous
spraying of polyanion and polycation solutions73,74 onto a
vertically oriented charged surface can create a uniform film
that grows continuously with spraying time. The vertical
orientation enables continuous drainage of excess polyion
and solvent. Spraying of PAH and poly(L-glutamic acid)
(PLGA) in this way yields films where the thickness grows
linearly in time, whereas successive deposition of poly(L-
lysine) (PLL) and of PLGA in LBL results in films which
grow exponentially in thickness with layer number. The
growth regime of polyelectrolytes is therefore not only a
function of the polyelectrolytes involved but also of the
assembly method. The method outlined in the cited work
by the Strasbourg group71,72 might not seem to fit the general
theme of multilayer films. In fact, however, “multilayer”
films tend to be somewhat amorphous, with neighboring
layers interpenetrating extensively.54 In the present context,
then, layer is perhaps best defined as “a thickness increment
following an adsorption step”, and multilayer as “multiple
thickness increments applied in succession”. The extent of
interpenetration and of polymer diffusion throughout the film
will depend substantially on the choice of polyelectrolyte.
Film Characterization. Experimental Techniques. Various tools are used to characterize polyelectrolyte multilayer
films. The emphasis here is on physical techniques. Of
course, one might be no less interested in chemical properties
or biofunctionality, perhaps especially in the case of polypeptide films. Examples of such properties include redox
potential and immunogenicity. Methods of characterizing
chemical properties and biofunctionality, however, are not
discussed here, as they will be more specifically related to
target applications than basic film properties. The polyelectrolyte LBL literature is too large to provide a comprehensive account of references in which the methods are used.
The reader is therefore referred to cited reviews on polyelectrolyte multilayer films for specific examples. We do,
however, provide references to papers in which the indicated
methods have been used to study polypeptide multilayer
films.
Selection of method will of course depend on the film
property of interest. Quartz crystal microbalance (QCM), an
acoustic technique, provides information on mass change and
Polypeptide Multilayer Films
kinetics of polyelectrolyte adsorption by way of change in
resonant frequency.75-81 If film density is known, QCM can
also measure film thickness. Dissipative QCM is used to
study viscoelastic properties of hydrated films.13,17,19,82-85 The
streaming potential method is used to characterize electrical
properties of a film surface. The basic principle is to measure
pressure and potential difference on both sides of a capillary.13,17,19,82,86 UV-vis spectroscopy is a relatively inexpensive means of measuring the optical mass of assembled
polyelectrolytes in terms of absorbance increase per layer
or disassembled material in terms of absorbance decrease.77,79
In some cases, UV spectroscopy can provide useful information on the structure in terms of the position of the absorbance
peak maximum. Fourier transform infrared spectroscopy
(FTIR) can measure the optical mass of assembled material
by detecting chemical bond vibrations, in attenuated total
reflection mode14,83,84,87-89 or not.80 Information is obtained
on specific functional groups, specific ion pairings, or
substrate-surface interactions. Surface plasmon resonance
spectroscopy can measure the rate of absorption, optical film
thickness, dielectric constant, and anisotropy. Ellipsometry18,77,85,90,91 and optical waveguide light-mode spectroscopy17,19,82,84,86-89,92 can measure the optical thickness of
a film and its refractive index, the former by reflection and
change in polarization of light on reflection. The amount of
material adsorbed is calculated from the thickness and
refractive index. Mechanical measurement of the thickness
can be made by profilometry.91 X-ray reflectometry is used
to measure the total thickness and roughness of a film and
to assess film stratification and the extent of blending of
layers. The method is limited, however, by generally poor
contrast between adjacent layers and fluctuations in composition.93 Deuteration of polymers can help. X-ray reflectometry
experiments are rather inconvenient to do, and they provide
no information on molecular conformation in the film.
Circular dichroism spectrometry (CD),75,77,79-81,91 by contrast,
and to a lesser extent FTIR spectroscopy, enable a moderately
accurate determination of film secondary structure content,
important when polypeptides are involved. The far-UV CD
signal is particularly sensitive to conformation of the
polypeptide backbone; different secondary structures have
more distinctive spectral signatures in CD than FTIR.
Scanning electron microscopy is useful for study of a film
surface, but it requires coating the sample with a thin layer
of metal. Atomic force microscopy (AFM) is a surface probe
measurement tool used to characterize film surface morphology, roughness, and thickness.19,77,79,82,86,89,91,92 AFM
can be used to study dry films and wet ones. Confocal
fluorescence microscopy has been used to monitor the
diffusion of polypeptides with multilayer films,15 study the
biodegradation of films,93 and visualize polypeptide microcapsules.94,95
Computational Approaches. It must be assumed that
computer-based approaches will someday be advantageous
for the development of polyelectrolyte multilayer thin film
technology. Groundwork in the general area has already
begun to appear in the scientific literature. Messina and coworkers, for example, have done Monte Carlo simulations
of polyelectrolyte LBL film assembly on a spherical charged
Biomacromolecules, Vol. 6, No. 6, 2005 2901
Table 7. Computational Study of Polypeptide LBL
step
further particulars
1. polypeptide design
contour length
net charge at neutral pH
hydrophilic surface area,
hydrophobic surface area
amino acid composition
specific sequence
2. simulation context
vacuum, dubiously realistic, “fast”
implicit solvent, somewhat realistic,
“moderately fast”
explicit solvent, most realistic, “slow”
3. data analysis
time needed to reach equilibrium
potential energy
backbone root-mean-square-deviation
radius of gyration
number of hydrogen bond
between chains
dipole moment
particle and a uniformly charged surface.96-98 The simulations are based on the assumption that the final film structure
is at equilibrium, even though polyelectrolyte adsorption
under normal conditions is nearly irreversible.36,99-103 Molecular dynamics (MD) simulations, by Panchagnula et al.,
are consistent with the view that multilayer formation is
driven by electrostatic interactions, attraction initiating
adsorption and repulsion limiting it.104 Strongly charged
polyelectrolytes gave better surface properties for the controlled buildup of a multilayer film than weakly charged
polyelectrolytes, consistent with experimental studies on
conventional polyelectrolytes1,52,105 and designed polypeptides,76,78-81 and with the known importance of formation
of the first layer for multilayer buildup.22 Polyelectrolytes
of a high degree of polymerization showed an increased
tendency to blend layers at long simulation times, similar to
recent experimental results on polypeptides of different
lengths and structures.79
Important for commercialization of polypeptide multilayer
films, MD simulations can also be used to study some aspects
of the peptide design process. Such analysis will provide
insight on polyelectrolyte complexation and the relationship
between electrostatic interactions, hydrophobic interactions,
hydrogen bond formation, secondary structure, and film
stability. Simulations could also help to understand the
internal structure of an LBL film. Various concerns of
research design in this new area are summarized in Table 7.
A series of MD simulations on designed peptides have been
done to test the role of differences in amino acid sequence
on aspects of peptide interaction.106 The initial structure in
each case was a parallel or antiparallel β sheet with standard
bond angles, selected on the basis of the known secondary
structure content of polypeptide films (Figure 2).75,77,79 The
results show that the primary structure can have a major
impact on the interaction energy in general (Figure 3) and
the number of hydrogen bonds between strands in particular,
especially when the charge density is high and the electrostatic interactions between side chains extensive. These
conclusions are consistent with corresponding experimental
studies.106 The ability to compare simulations and experi-
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Haynie et al.
tions. The same types of interactions stabilize the native
structure of a protein. Some of the nonelectrostatic contributions to film stability might be particularly important in weak
polyelectrolytes, especially in a pH regime where the charge
density of one of the polymers is low. It seems, then, that
polypeptides could be useful in research aimed at determining
the physical basis of polyelectrolyte film assembly and
stability, because approaches to material production, mentioned above, enable an exceptional degree of control over
details of polymer structure. The chirality of polypeptides
will make them particularly useful for study of internal film
structure by CD spectrometry.
Figure 3. Simulated interaction between models of designed peptides
also studied experimentally. The peptide designs were (lysine)31tyrosine (P1), (glutamate)31-tyrosine (N1), (lysine-valine)15-lysinetyrosine (P2), and (glutamate-valine)15-glutamate-tyrosine (N2).
Number of backbone hydrogen bonds is shown for P1-N1, P2-N1,
and P2-N2 after minor moving window smoothing. After 1000 ps
there are about 5, 2, and 17 backbone hydrogen bonds, respectively.
MD simulations were done using the CHARMM package (Accelrys)
and the all-atom CHARMM22 force field. The generalized Born
method was used to model solvent effects. The pH was 7.4. The
energy of the peptide complex was minimized prior to MD. Then the
system was heated from 240 to 350 K for 10 ps and equilibrated for
30 ps. The trajectory was monitored for 1 ns. Conformation samples
were collected every 0.5 ps. Analysis of the potential energy profile
indicated that the system was in equilibrium well prior to 1 ns of
simulation. A higher temperature than that of the experiments was
chosen for the simulations to increase the contrast in stability between
the studied peptide pairs. Simulation data can be compared with
experimental results to develop a means of predicting the interaction
between peptides and some film properties. See Figure 6.
ments directly will doubtless be advantageous for advancing
the field of polypeptide films.
III. Results
Five areas of research on physical properties of polypeptide
multilayer films are discussed here: How do the physical
properties of chosen polypeptides influence whether assembly
is possible at all and the ability of a film to remain intact
under given conditions? What is the basic character of
polypeptide deposition, and what is the underlying mechanism? What is the internal structure of a polypeptide film?
What is the surface morphology of a polypeptide film? How
can polypeptides suitable for LBL be used to form microcapsules? A brief description of recent work in each area
follows.
Physical Basis of Polyelectrolyte Film Assembly and
Stability. Several review-like works on this broad and active
area of research have appeared in the recent edited volume
by Decher and Schlenoff.7 Other helpful works are refs 1,
4, 5, 9, 10, and 108-118. We are interested in whether
polypeptide studies can help to understand polyelectrolyte
film assembly and stability; ad hoc reference only will be
made to reports on polyelectrolyte films considered more
generally.
A basic conclusion of extensive work in the general area
is that Coulombic interactions provide the main driving force
for polyelectrolyte multilayer film assembly. Various other
types of interactions, however, can participate in the assembly
process, notably hydrogen bonding and hydrophobic interac-
Knowledge of protein structure119 could help to understand
the structure of polypeptides in a multilayer film. Regions
of hydrophobic and hydrophilic surface on a peptide
molecule will affect its interaction with other molecules, as
in proteins, though specific details might differ. The solventaccessible nonpolar surface area of a side chain can
contribute thermodynamically favorable hydrophobic contacts to film structure, even if the side chain features an
ionizable group and is charged at neutral pH, as in a folded
protein. Hydrogen bonding in and between peptides will
influence film organization and stability. It seems probable
that all hydrogen bonding potential in a polypeptide film must
be satisfied, whether in the backbone or side chains, as in
the core of a folded globular protein; hydrogen bond donors
have a partial positive charge; acceptors, a partial negative
one. The requirement of electroneutrality in a multilayer film
will probably be met mostly by polyion charge compensation,
as there is a favorable increase in entropy on release of small
counterions to the solution. Some water molecules and
counterions might remain in the film during assembly and
on drying in order to meet the requirements of the hydrogen
bonding potential, as in proteins, but the relative content of
water and counterions will ordinarily be small.120 In any case,
hydrogen bond formation in and between peptides in a film
will give rise to two main types of secondary structures, the
R helix and the β pleated sheet, as in proteins.88 This too
will affect film structure and gross mechanical properties.
Secondary structure formation in PLL or in PLGA in
aqueous solution will depend on degree of polymerization.121
Recent polypeptide multilayer film experiments have shown
that more material is deposited when “long” PLL molecules
are adsorbed onto a solid support than “short” ones under
identical conditions.75 Molecular weights ranged over 3
orders of magnitude in this study, from 1.5 to 222 kDa.
Similar behavior is found with “conventional” polyelectrolytes,122-127 for instance poly(ethyleneimine) and PSS.11 A
longer chain in solution has more ways of associating with
previously formed layers than a shorter one; an adsorbed
longer chain will provide more binding sites than a shorter
one (Figure 4). “Short” chains of PLL and PLGA (<∼4 kDa)
are poorly suited to multilayer film assembly at neutral pH.75
It may be that shorter chains are more readily released from
the film to the solvent than longer ones when the linear
charge density is high; interpolyelectrolyte complexes of
short chains are more soluble than complexes of long
chains.128 “Short” PLL chains, however, do exhibit film
assembly under identical conditions, if the oppositely charged
Polypeptide Multilayer Films
Biomacromolecules, Vol. 6, No. 6, 2005 2903
Figure 4. Schematic view of the length-dependence of polypeptide self-assembly. Generally, short peptides deposit less extensively than long
peptides in LbL, particularly when the linear charge density is high. Various explanations are possible. Long peptides might have some charged
groups sufficiently far from the surface not to be “neutralized” by it and therefore be available to bind oppositely charge polypeptides. In the
present context, these charge are outside a Gaussian surface in which the net enclosed charge is zero. Short peptides might be too closely
associated with the surface for there to be additional charges outside the Gaussian surface. Alternatively, short peptides may bind too loosely
to the surface not to form a complex with oppositely charged peptides and return to solution during a subsequent deposition step. The character
of interactions between individual residues in long peptides and the surface might be the same as in short peptides, but the greater number of
interactions in the long peptide will make it adhere more strongly to the surface. The situation is similar in proteins, in which a large number of
individually weak interactions collectively make a significant contribution to the overall free energy difference between the native state and
denatured conformations.
polymer is long enough, though less material is deposited
from solution than when both PLL and PLGA are “long”.75
Chemical cross-linking of polyelectrolytes in multilayer
films has been studied as a means of stabilizing film
structure, increasing mechanical strength, and influencing
mass transport.129-139 Various approaches are used to achieve
cross-linking, for example heat-induced and photoinduced
approaches and chemical treatment. Such methods, however,
are generally irreversible, favorable for some applications
but reducing the ability of a film to respond to environmental
conditions.
The free thiols in cysteine-containing polypeptides seem
particularly advantageous for “natural” film cross-linking
(Figure 5). A disulfide bond can be formed under mild
oxidizing conditions between two amino acids which have
a thiol group in their side chains, cysteine and some
nonstandard amino acids.29 Disulfide cross-linking is reversible: a disulfide bond can be broken in the presence of a
suitable reducing agent, for example the small biomolecule
glutathione. Disulfide bonds confer increased thermostability
on the folded structure of many secreted proteins and
enzymes. A notable example is the well-studied antibacterial
protein lysozyme.140 Polypeptides designed to include cysteine can form disulfide bonds between polymer chains in a
multilayer film.76 This markedly decreases the rate of film
disintegration or extent of disruption of film structure at
extremes of pH, in organic solvents, or in a water at elevated
temperature.79-81,91,94 Disulfide cross-linking could be particularly useful for peptide-based microencapsulation or
sensitivity to the reducing potential of the surrounding
environment.94 It could also be used to “tune” the mechanical
properties of a multilayer film intended for tissue culture.
Another possibility would be to use cysteine-containing and
suitably charged peptides for stimulated release from a film
of specific compounds bound to the peptides by disulfide
linkages.
Short, “matched” peptide pairs yield denser and more
stable films than long, “unmatched” pairs.79,91,106 Here,
Figure 5. Schematic view of disulfide bond stabilization of a
polypeptide multilayer film. The film is made of polypeptides containing
the amino acid cysteine. Under oxidizing conditions, disulfide bonds
form between polymers, altering mechanical properties of the film at
neutral pH. Cross-links also limit film disassembly in an extreme pH
environment, curbing the ability of electrostatic repulsion to drive film
constituents apart. No disulfide bonds are formed under reducing
conditions. Disulfide bond formation is reversible. Exposure of a crosslinked film to reducing conditions thus results in rupture of the disulfide
bonds. If the cross-links are broken under extreme conditions, film
disassembly will occur. Irreversible cross-linking of a polypeptide film
can be achieved in other ways, e.g., with glutaraldehyde. The diagram
is based in Figure 3 in ref 76.
matching refers to the degree of polymerization and linear
charge density. The stability of films made of matched
polypeptides can be rather different under identical conditions, depending on peptide amino acid composition.106 For
example, films made of (KVKV)7KVKY and (EVEV)7EVEY
at neutral pH exhibit barely detectable disassembly by UV
spectroscopy at pH 4, whereas films of (KVKS)7KVKY and
(EVEN)7EVEY, having identical charge density to the other
peptides but a smaller net hydrophobic surface area, disassemble under the same conditions with first-order kinetics
and a time constant of about 35 min (Figure 6). This would
suggest that hydrophobic interactions can play a significant
role in stabilizing polypeptide films, similar to protein
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Biomacromolecules, Vol. 6, No. 6, 2005
Figure 6. Disassembly behavior of two designed polypeptide films
at mildly acidic pH. The peptide sequences are shown. The charge
density is approximately the same in the two cases at neutral pH;
the number of acidic (glutamic acid, E) side chains equals that of
basic (lysine, K) side chains. Serine (S) and asparagine (N) are polar
but uncharged; valine (V) is hydrophobic. The film containing neither
S nor N exhibits practically no disintegration over a 180-min observation period. By contrast, the film containing S and N disassembles
with first-order kinetics and a time constant of about 35 min at 25 °C.
After 180 min, about 60% of the original film remains. This suggests
that the dissociation constant of glutamic acid is different in the two
cases, that hydrophobic interactions contribute substantially to film
stability, or both. Simulations of peptide complexation might help to
predict polypeptide film behavior. See Figure 3.
thermostability and that film stability in a chosen environment will be a matter of film design.
Growth Mode. There are two main modes of growth of
polyelectrolyte multilayer films: “linear” and “nonlinear”.
The latter can be “supralinear” (in some cases “exponential”)
or “sublinear”. The various modes are distinguished by
degree of change in amount of polyelectrolyte deposited per
adsorption step. Linear growth is displayed by various
combinations of polyelectrolyte, for example PAH/PSS,
PAH/poly(vinyl sulfonate) (PVS) and PDDA/PSS; nonlinear
growth by other combinations, for example PLL/alginate,
PLL/hyaluronan, and PLL/PLGA.15,82,86,87 Linear growth is
typical when polyelectrolyte charge density is high and ionic
strength is low. In such cases, the thickness increment of an
adsorption step will be small. The resulting film will be
dense, and the interactions of molecules in a layer with the
rest of the film are largely constrained to neighboring layers.1
Polyelectrolytes from solution will interact mostly with
constituents of the film surface and less with molecules more
deeply buried.
Exponential growth, by contrast, is said to result from
diffusion of polyelectrolyte molecules into and out from a
film, the amount of migrating polyion being proportional to
film thickness.15 A loose and inhomogeneous film will favor
inward polyelectrolyte diffusion and therefore layer blending
and supralinear growth.86 Charge mismatch between the
polyanions and polycations in a film could increase film
roughness with layer number and thus play a role in
supralinear growth.3 Such growth could also conceivably be
attributable to formation of a complex gel on the film
surface.16 In any case, weak polyelectrolytes can display
linear or exponential growth.
Growth mode can be influenced by adjusting the pH or
ionic strength of polyelectrolyte solution.8,52,141-145 The latter
Haynie et al.
applies to strong and weak polyelectrolytes, the former
mostly to weak polyelectrolytes.1,53 The growth mode can
also depend on the substrate.146-149 Secondary structure
content, density, thickness, and surface morphology of a
polypeptide film can vary over a small range of pH when
an ionizable group is titrated.18,19,75,77,85,89,91
Information on mode of polypeptide film growth can be
found in refs 15, 18, 75, 76, 78-81, 85, 88, and 91. As to
PLL and PLGA, some evidence would suggest that the
thickness of the wet film grows linearly, whereas the mass
deposited grows supralinearly.85 The mass deposited in a wet
film, however, must be calculated with some caution, as the
Sauerbrey equation is strictly valid for rigid films only, and
interpretation of ellipsometric thickness depends on assumptions regarding refractive index. 50-100 kDa PLL and 3070 kDa PLGA molecules show two stages of film assembly
by thickness, refractive index, and mass deposition measurements.18,85 In the first, the polymers would appear to adsorb
in a “flat” conformation and with a minimum of “loops”
and “tails”, resulting in thin layers. Once film thickness is
large enough, however, the substrate has a negligible effect
on growth and layers become thicker. The character of PLL/
PLGA film assembly correlates with structural features of
PLL and PLGA in solution.75 For instance, thicker films are
formed when one of the polypeptides is in the helical state.
It can be difficult to ascribe a specific cause to some observed
effects, though, because structural features of PLL and PLGA
in solution depend on charge density.
Drying and measurement of physical properties in air does
not impair continuation of growth of films of PLL and
PLGA,18,75,77,90 nor of films of designed polypeptides.76,78-81,91,106 The study by Halthur et al. has found that
no mass is lost from PLL/PLGA films on drying and that
the thickness and refractive index return to original values
on rewetting.18 This implies that drying of a polypeptide film
does not result in irreversible changes in film structure, even
if the film is left in air overnight. Presumably, more or less
the same will be true of non-PLL/PLGA polypeptide films.
The behavior of various short designed polypeptides is in
some respects rather different from that of higher molecular
weight PLL and PLGA. Certain cysteine-containing 32mer
peptides, for example, show linear adsorption behavior under
all conditions studied thus far.76,79-81,91 Other 32mer peptides
of similar design behave likewise.106 This suggests that films
of short, designed peptides can be quite dense, perhaps
similar to an organic crystal; this view is supported by
independent experiments on perturbation of film structure
by adsorption of long strong or weak polyelectrolytes.79
Remarkably, the same 32mer peptide can show linear growth
or supralinear growth at low ionic strength, depending on
the design of the oppositely charged 32mer peptide.106 This
suggests that the physical basis of linear versus nonlinear
film growth is still not well understood.
Polypeptides in multilayer films will interact with each
other in all of the “conventional” ways: electrostatic
interactions, hydrophobic interactions, and hydrogen bonds.
They will also form R helices and β sheets, adding degrees
of freedom to film design. Secondary structure formation is
expected to affect the migration of polymers in a film and
Polypeptide Multilayer Films
so influence growth mode. The secondary structure could
also affect the tendency of small chiral molecules to pass
through the film and, perhaps, the tendency of chiral structure
to be induced in “loaded” molecules, e.g., protoporphyrins.
The tendency of a peptide to adopt a secondary structure
will depend on its degree of polymerization121 and amino
acid sequence.30
Thickness. Polyelectrolyte film thickness depends on
humidity.150 Film mechanical properties will depend on
whether the film is wet or dry. There is a large decrease in
thickness of PLL/PLGA films assembled at pH 7.4 in 100
mM NaCl on drying with nitrogen.18 Wet films approximately 50 nm thick shrink to about 1.6 nm when dried,
according to ellipsometry. The water content is estimated at
60-70% in wet PLL/PLGA films.85 10 layer films of dry
PLL/PLGA have a thickness of 8.5 nm on titanium,18 13.5
nm on silica,18 17 nm on oxidized silicon,77 and 20 nm on a
modified gold surface.151 Conditions were similar in these
examples but not identical. In any case, dry film thickness
is roughly 1-2 nm per layer. Halthur et al. have found that
rapid heating from 25 to 37 °C followed by cooling to 25
°C results inasmuch as 25% mass loss in wet PLL/PLGA
films.18 There was practically no mass loss, however, on slow
heating between these temperatures, despite swelling by as
much as 8%. On pH shift from 7.4 to 3.9, the thickness of
wet films decreased by 10-20%, the minimum occurring at
pH 5. Comparison of the pH-dependence data with the
known helix-coil behavior of PLGA would suggest that the
film undergoes a sort of phase transition in this process,
related to the titration of the carboxylic acid side chains.
Thickness depends substantially on the degree of polymerization when peptide adsorption is carried out under
otherwise identical conditions.75,90 See Figure 4. Assuming
that thickness scales linearly with adsorbed mass when the
film is dry, which is to assume that film density is constant,
films of 222.4 kDa PLL and 50.3 kDa PLGA are about 70%
thicker than ones of 48.1 kDa PLL and 17.0 kDa PLGA and
about 300% thicker than ones of 222.4 kDa PLL and 1.53.0 kDa PLGA for the same number of layers.75 Only
comparatively limited assembly of 3.8 kDa PLL and 1.53.0 kDa PLGA is possible on quartz.75,90 These experiments
were done at pH 7.4, where the linear charge density of PLL
and of PLGA is high, and the ionic strength was low. A
more direct measure of dry PLL/PLGA film thickness has
been made by ellipsometry.77,90 The average thickness per
layer of films assembled at pH 7.4 and low ionic strength is
3-fold greater for 84.0 kDa PLL and 84.6 kDa PLGA than
for 14.6 kDa PLL and 13.6 kDA PLGA, polymers 6-fold
shorter.90 Another 3-fold increase in thickness was found on
adding 500 mM NaCl to 10 mM buffer. Films of 84.0 kDa
PLL and 84.6 kDa PLGA assembled at pH 7.4 and 150 mM
NaCl were 17.6 nm thick for 10 layers and 22.0 nm thick
for 12 layers.77 The results resemble independent work on
assembly of nonpolypeptide polyelectrolytes.22,52
Internal Structure. Amino acid residues in a polypeptide
chain are connected by peptide bonds. The rigidity and
planarity of a peptide bond places severe limits on the
degrees of freedom of the polymer backbone.29 This is crucial
for formation of a secondary structure at a thermal energy
Biomacromolecules, Vol. 6, No. 6, 2005 2905
in the range of water in the liquid state at atmospheric
pressure. Once backbone geometry is suitable, hydrogen
bonds will form between backbone donors and acceptors,
stabilizing an R helix or β sheet. The energy of these
hydrogen bonds, however, will be comparable to those in
water; elements of secondary structure in small globular
proteins are stabilized against thermal denaturation by
packing onto a hydrophobic core. In an R helix, all
backbone-backbone hydrogen bonds are made between
residues in the same polypeptide chain; in a β sheet, both
intramolecular and intermolecular backbone hydrogen bonds
are possible. Several hydrogen-bonded β strands could be
arranged either parallel or antiparallel to each other. β sheet,
then, is favored from a configurational point of view for a
given length of polypeptide.79
The internal structure of “ordinary” polyelectrolyte multilayers, such as ones made of the “flexible” polyelectrolytes
PAH and PSS, is said to be “fuzzy” and “disordered”.1,152
There is considerable overlapping of individual layers;
polymers are entangled. Such films are generally isotropic.
Multilayers of polypeptides, by contrast, can be anisotropic.11
The degree of order and anisotropy will depend on the extent
and type of secondary structure formation, which will depend
on the length of polymer and solution conditions121 and
whether the substrate is texturized.153,154 The secondary
structure has been discussed in the context of pure polyelectrolyte multilayer films as providing “local internal order”
to otherwise “fuzzy” structures.87
Some amino acid sequences can form an R helix or a β
sheet depending on the context. Examples of switching
between structures are known from protein folding and
amyloid fibril research.155-163 The most probable conformation of a polypeptide in aqueous solution, particularly a short
one, will in most cases be a random coil. Results of recent
experiments on long polymers of lysine and glutamic acid75
and short designed peptides80 are consistent with theory.121
A short peptide must forfeit a comparatively large fraction
of its degrees of freedom to form a hydrogen bond. pH will
govern the content of β sheet or R helix in PLL or in PLGA
in solution75 or in a multilayer film.77 At neutral pH, these
peptides are in a coil state in solution,75,88 but on formation
of a multilayer film, the polymers adopt a β sheet conformation,77,79,83,84,87,88,90,154 similar to the structure of polypeptide
complexes in solution.88 The secondary structure content of
PLL/PLGA films made at neutral pH is stable against pH
change in the range 5-10.77,88 R helix becomes more
apparent, however, at the expense of β sheet under conditions
where the side chains of glutamic acid or of lysine deionize,
in solution or in a film.18,75,77 The same conditions promote
film disassembly,77,88 owing to thermal fluctuations and
electrostatic repulsion. This is consistent with the denaturation of most proteins at extremes of pH, e.g., chicken-type
lysozymes.140,164 pH sensitivity could be important for
controlling the responsiveness of a polypeptide film to
environmental conditions.165
β sheet is the predominant secondary structure in films of
designed polypeptides formed at neutral pH.76,79-81,91 This
is similar to PLL/PLGA films and consistent with a very
early study in this area.166 β sheet is apparently “universally”
2906
Biomacromolecules, Vol. 6, No. 6, 2005
favored by thermodynamics in a multilayer film when the
charge density of the polypeptides is high; kinetic trapping
of the polymers in the adsorption process is apparently
followed by a relatively slower kinetic process in which a
large proportion of β sheet is formed. The secondary structure
in a film is stabilized by interactions within the element of
structure, namely hydrogen bonds, but probably more by
interactions with the polypeptide structure in the surrounding
environment, as in proteins, amyloid fibrils, and other such
structures.
Various means besides pH change are available for
controlling the secondary structure content of a polypeptide
film. One is choice of polyelectrolyte in the outermost layer.88
Adsorption changes the local electrostatic environment of
the assembled polymers and can bring about significant
overall structure reorganization. Results of Boulmedais et
al. from FTIR experiments on wet PLL/PLGA films have
been interpreted as oscillations in the secondary structure
content on change in the outermost layer during successive
peptide adsorption steps88 (this is not found in dry PLL/
PLGA films77,79,90). Film structure composition appeared to
change even more dramatically when a strong polyelectrolyte
was deposited on the polypeptide film surface.88 It was
concluded that the β sheet structure in a PLL/PLGA film
was completely destroyed by adsorption of PSS, a strong
polyelectrolyte, onto the film surface. Independent experiments provide a different view and have revealed that the
nature and extent of structure change on PSS adsorption will
depend substantially on the chosen polypeptides for otherwise
constant assembly conditions.79 These experiments compared
the behavior of PLL/PLGA films with ones made of designed
32mer peptides. The latter were practically unaffected by
adsorption of PSS, whereas the former were substantially
altered with regard to secondary structure content but not
completely destroyed. Differences in chemical structure
translate into differences in film density and packing, making
the film more or less susceptible to perturbation by the
surrounding environment. Dry films of the designed 32mers
are quasicrystalline.
The secondary structure content of a film can be altered
by changing the composition of the polypeptide adsorption
solutions. Debreczeny et al. have found that the secondary
structure content of multilayers of PLL and PLGA mixed
with poly(L-aspartic acid) depends on the percentage of
PLGA in the polyanion solution.83 Separate work has shown
that the same 32mer polypeptide can assemble under identical
conditions into a film with a high or low percentage of β
sheets, depending on the sequence of the oppositely charged
32mer peptide.106 This finding underscores the importance
of the surrounding environment of peptide for inducing
structure formation.
Temperature too can influence secondary structure content
of a polypeptide film.88 Experiments suggest that a slow rise
in temperature from 26 to 89 °C of a wet PLL/PLGA film
results in a ca. 10% increase in R helix at the expense of β
sheet, as judged by FTIR. This is hard to understand, given
the well-known behavior of proteins in aqueous solution.
Moreover, if the key FTIR bands shift with temperature in
the absence of any structural change, it is not clear how one
Haynie et al.
would account for the effect when estimating secondary
structure content. Further doubts are raised by experiments88
which suggest an increase in β sheet content on rapid heating
of a wet PLL/PLGA film from 26 to 89 °C. Independent
work has shown that a large temperature increase can have
relatively little effect on dry film structure, judging by CD
and depending on the peptides involved. This resembles the
behavior of some unusually thermostable proteins167 and,
perhaps more accurately, the behavior of protein powders.168,169 The secondary structure content of films of
designed 32mers is dominated by β sheet at room temperature, wet or dry, and there is practically no change in
structure content on heating to 95 °C when the film is dry.81
When the film is wet, slow disintegration is evident at this
temperature. Such behavior is in good agreement with
extensive thermodynamic studies of many different proteins
in aqueous solution.29,170
Given that secondary structure formation is a unique
characteristic of polypeptides, it seems appropriate to
compare tools for study of the “internal” structure of peptide
films. FTIR and CD are the main ones for this purpose. In
FTIR, the amide I band is decomposed into absorption bands
representing different secondary structures.83,84,87 The approach is complicated by extensive overlapping of absorption
bands, making determination of secondary structure content
difficult at best. There can be little to differentiate the spectra
of two very different proteins in the folded state.171 CD, by
contrast, can provide extensive information on secondary
structures in peptides.29,75,77,79,80,82,91,154,172-174 CD measures
the differential absorption of right- and left-circularly polarized light. In the far-UV region of the spectrum, 180-260
nm, the signal is very sensitive to the average conformation
of the polypeptide backbone,29 whether the peptide is in
solution or in a film.166 A set of reference spectra and some
assumptions underlie spectral analysis.175 The reference
spectra generally are proteins or peptides for which the
secondary structure content is known by an independent
means, for example X-ray crystallography. The basic principle of analysis is that a CD spectrum can be regarded as a
linear combination of spectra of distinct secondary structures.
By this approach, one can estimate the fractional content of
R helix, β sheet, β turn, and random coil with considerable
accuracy. The transition in a PLL/PLGA film from β sheet
to R helix on pH shift is readily monitored by CD and
analyzed by deconvolution.77 Comparison of the various
physical methods mentioned in this work would suggest that
CD is the most informative one for obtaining information
on the conformation of polymers in a polyelectrolyte thin
film. Moreover, it would appear from a broad survey of the
polyelectrolyte multilayer film literature that CD analysis of
polypeptide films77,80,81,106 has yielded the most detailed
experiment-based view to date of polymer structures in a
polyelectrolyte multilayer film.
Surface Properties. Hydrophilicity and Hydrophobicity.
Hydrophilic polymers are used to modify the surface of a
wide variety of substrates. Biomedical applications include
contact lenses, drug delivery systems, biological adhesives,
anti-thrombogenic coatings, soft tissue replacement, and
permanent implants. Hydrophobic surfaces too are being
Polypeptide Multilayer Films
Figure 7. Schematic diagram showing how a designed polypeptide
can be used to control surface wettability. It is assumed that the
substrate for film assembly has a net negative surface density of
charge. In both cases, the peptides are assumed to be in the β sheet
conformation, based on the experimentally determined tendency of
peptides in a multilayer film, and the sheet is assumed to be parallel
to the substrate. When a polypeptide chain has adopted the β sheet
conformation, the side chains of successive amino acid residues point
in opposite directions, above and below the plane of the sheet. The
filled triangles represent hydrophobic side chains. By this approach,
both peptide design and design of film architecture are required to
bring about reversal of surface properties. The peptides used to
convert the charged surface into a hydrophobic one could also be
used to convert a hydrophobic surface into a charged one, by
reversing the order of peptide assembly.
developed to provide an additional means of controlling
wettability.
Adsorption of polypeptides onto a surface can make it
hydrophilic or hydrophobic according to amino acid composition (Figure 7). LBL offers the additional advantage of
nanometer-scale thick coatings without limitation of shape,
size, or composition of substrate. Typical LBL films grown
on hydrophobic surfaces, however, are “patchy” and inhomogeneous in the absence of more extreme surface
modification,3 for example oxidation or plasma treatment.
Supports made of polyolefins or poly(tetrafluoroethylene)
have yielded a poor LBL result.122,129,131,176-179 Designed
polypeptides would appear to provide a path around the
roadblock.
Experiments have shown that the polypeptides (lysine/
valine)n and (glutamate/valine)n self-assemble in LBL into
multilayer films on quartz.106 Lysine is positively charged
at neutral pH, glutamate negative, valine hydrophobic.
Growth of 32mers is linear. CD spectroscopy has revealed
that the films are dominated by a random coil structure. β
sheet and β turn, however, are more prevalent than R helix.
The amino side chains in a β strand point above or below
the plane of the sheet, alternating in succession. In a β strand
of (lysine-valine)n or of (glutamate-valine)n, then, the charged
side chains point in one direction; the hydrophobic ones, in
the other. Such peptides could be advantageous for forming
a “precursor” layer for assembly of a biodegradable LBL
film of any desired biochemical or biodegradable species,
obviating oxidation, plasma treatment, some other type of
surface modification, or hydrophobic modification of polyions.178,180 The value of such films as coatings will probably depend on the strength of adhesion on the initial
layer.2,129,131,148,181,182
Morphology. Planar polyelectrolyte multilayers of
PDADMAC/PSS exhibit rough and inhomogeneous surfaces.
Addition of salt to the assembly solutions decreases final
surface roughness.144 Multilayers of PAA/PSS on silicon
wafers show granular structures on the surface after a certain
number of layers are formed.183 The surface of a PLL/PVS
Biomacromolecules, Vol. 6, No. 6, 2005 2907
film on a texturized silicon substrate displays “wormlike”
nanoscopic structures.11 These are thought to arise from the
assembly of stiff helical rods of PLL. Orientation of the
wormlike structures depends on the direction of grooves in
the texturized substrate. Separate studies have shown that
surface morphology can vary over a small range of pH.
Gergely et al.89 and others75 have measured the amount of
material adsorbed in PLL/PLGA film formation in the range
pH 3.0-10.5. The rate of polypeptide buildup is substantially
greater at extremes of pH than in neutral solution, with
thickness increasing with decreasing linear charge density.
This would suggest that the mechanisms underlying buildup
will depend on pH, influencing the degree of ionization of
polymers and therefore the relative importance of side
chain-side chain interactions on the film surface, as well
as surface roughness. At high pH, partial ionization of PLL
may yield incomplete charge compensation in the polymer
adsorption process, leading to formation of a precipitate at
the solution-film interface. Richert et al.19 and others75 have
found a transition from very thin and dense films at
intermediate pH values, where both polypeptides are highly
ionized, to much thicker and hydrated films at acidic and at
basic pH, where hydrophobic interactions become relatively
more important. Zhong et al. have found that the ionization
state of thiol plays a key role in the adsorption behavior of
short designed cysteine-containing polypeptides near neutral
pH.91 The adsorbed mass of these polypeptides, film density,
refractive index, and surface morphology vary substantially
over a range of about 1.5 pH units. At pH 7.4 the films are
dense and smooth, while at pH 8.9 they are more diffuse
and comparatively rough.
Encapsulation. The repetitive coating of charged colloidal
particles with charged linear polyions is a simple but useful
concept. This was first done by Keller et al. on silica
microparticles.184 The idea has been developed for silica
particles and negatively charged latex particles which are
readily dissolved under appropriate acidic conditions.185-188
Calcium carbonate colloidal particles are also suited to the
purpose. In all cases, polyelectrolyte is added to a suspension
of oppositely charged microparticles in solution. After some
time, polymer adsorption saturates. Isolation of the polymercoated microparticles is achieved by centrifugation or a
similar means. Repeated adsorption of oppositely charged
polyions and isolation results in a polyelectrolyte multilayer
surrounding the microparticle core.
Polymer microspheres constitute a class of interesting
structures for applications in encapsulation, separation, and
biomedicine. Polyelectrolyte microcapsules can be used as
“microcontainers” to encapsulate specific materials. Proteins
of suitable charge can be incorporated into the polyelectrolyte
shell during assembly53 or, in some cases, “loaded” after
assembly.188 Drugs, enzymes, and other materials of interest
can be trapped in polymeric films or microcapsules fabricated
by LBL.12,51,189-191 Slow release of compounds through the
film is possible,187,189 the rate depending on the number of
layers and the choice of polymers. Hollow microcapsules
of peptides might be particularly interesting if the secondary
structure and other properties can be controlled.
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Biomacromolecules, Vol. 6, No. 6, 2005
The mechanism of reversible capsule permeability is not
understood. It may involve segregation or expansion of the
polyelectrolyte network under “loading” conditions, for
example 1:1 mixture of water and ethanol. Such changes
can encompass defects in the polyion shell, enabling pores
larger than a protein molecule to form. The possibility of
such pore formation was first demonstrated by Rubner and
colleagues in their investigation of weak polyelectrolytes.58
Morikawa et al. have synthesized anionic R-helical poly(γbenzyl-L-glutamate) and investigated the emulsion-templated
self-assembly of these polymers into hollow microspheres
on rapid evaporation of the organic phase.192 The capsules
are stable after air-drying and could serve as supermolecular
delivery vehicles for hydrophobic or water-soluble molecules.
Designed polypeptides present further possibilities for encapsulation and sustained release of chemical compounds
in a biological or medical context. Design of peptides enables
control over degree of polymerization (important for specification of drug formulation), primary structure (important
for susceptibility to proteolysis, etc.), charge properties
(important for controlled assembly), film density (important
for release of small compounds), biocompatibility, immunogenicity, and other properties.78,79,91,95 Polypeptide design
could be oriented toward the trigger of release of chemical
compounds from a film or capsule by a specific physical,
chemical, or biological stimulus, for example a change in
redox potential.94
Various approaches are being developed for “tuning”
polypeptide film or capsule longevity. For example, designed
polypeptides incorporating cysteine can be used to form
multilayer thin films and microcapsules, which subsequently
can be cross-linked under mild reaction conditions and
thereby made more stable.76,79-81,91,94 Besides being “natural”,
disulfide cross-linking is fully reversible. Another approach
involves susceptibility of polypeptide film constituents to
proteolysis (J.S.R. & D.T.H., unpublished results). Proteases
are enzymes which can hydrolyze the peptide bond.193 Some
proteases recognize certain amino acids or sequences.
Trypsin, for example, a pancreatic enzyme, is specific for a
bulky hydrophobic residue preceding the peptide bond
cleaved. Susceptibility of designed polypeptides to proteolysis thus could be a design feature of polypeptide multilayer
film engineering, making a film (or capsule) more or less
likely to degrade after a certain amount of time on exposure
to certain proteases. It is hardly speculation to say that one
could “tune” the longevity of a polypeptide microcapsule
by encapsulating proteases which recognize specific sequences in the film peptides and adjusting the concentration
of protease, number of peptide layers, and film density.
IV. Discussion
Some of the areas of technology development discussed
here are currently being investigated by LBL researchers.
Others appear eminently suitable for development by LBL
but have not been explored. All could be developed as
specific applications of polypeptide multilayer film technology. The following discussion is highly introductory in
nature, and some areas are covered in greater depth than
others.
Haynie et al.
Enantiomeric Separations. Chirality is key to molecular
recognition in biology.194 The pharmaceutical industry’s need
for single enantiomer drugs has spurred the development of
techniques for preparative-scale separation of chiral molecules.195 Membrane-based separations have attracted much
attention due to operational simplicity and low cost. All
amino acids (except glycine) are chiral; a polypeptide chain
is chiral.29 Polyelectrolyte multilayer films fabricated from
polypeptides thus are optically active, enabling analysis by
CD.77,79-81,90,91 Maruyama et al. have reported that membranes modified with ∼1-µm thick poly(amino acid)-bearing
amphiphilic side chains show permeation rate ratios above
8 for R-amino acid enantiomers.196 The chiral selectivity of
PLGA has been probed in a series of experiments by Higashi
et al.197,198 and shown to increase as R helix content
increases.199 PLGA films have also been reported to exhibit
favorable enantioselective binding affinity for D-isomers, with
L-isomers permeating more quickly.200 Multilayer films of
left-handed- and right-handed-PLL and -PLGA show high
permeation rates and promising selectivity.201 It seems
probable that substantially more could be achieved in this
area of development by optimizing the structure of the
assembling peptides for specific separation characteristics.
Antimicrobial Films. Multicellular organisms produce a
range of antimicrobial peptides.202 Examples are defensins
and hystatins. Antimicrobial peptides are candidates for
addressing the public health concern of ever-increasing
antibiotic resistance. It has become a priority to overcome
certain pharmacological limitations and develop antimicrobial
peptides into therapeutics that can be used in different
applications at reasonable cost.
Antimicrobial peptides, regardless of source organism,
could be combined with LBL to develop a new class of
biodegradable, edible, and antimicrobial films, coatings, and
related products, for instance artificial cells. The antibiotic
peptide nisin, for example, has been shown to suppress the
growth of L. monocytogenes after deposition on a silica
surface.203 Antimicrobial coatings which could be used as
food wraps have been developed by incorporating nisin and
the protein lactoferrin in a poly(vinylidene chloride) copolymer film.204 Similar studies on multilayer films involving
lysozyme or histatin-5 and PLGA have shown reduced
proliferation of M. luteus or C. albicans, respectively (J.S.R.,
Komal Dave, and D.T.H., unpublished data). Antiadhesive
multilayer films made of PLL and pegylated-PLGA reduce
adsorption of serum proteins and E. coli, reducing pathological consequences.13 Multilayer films functionalized by the
insertion of defensin inhibit growth of infectious pathogens.205 Polypeptide multilayer thin films incorporating
antimicrobial peptides therefore are considered promising to
protect implants, catheters, needles, surgical tools, tubes, and
all kinds of materials from proliferation of microorganisms.
The ability to control the primary structure of a peptide will
be important to future work in this direction.
Artificial Skin Grafts. Major burn accidents involve
extensive damage to the skin.206 Immediate coverage is
needed to limit loss of fluid and aid tissue repair and
regeneration. The structural and functional properties of an
ideal skin substitute should closely match autograft skin.207
Polypeptide Multilayer Films
Plasticity of the substitute preparation procedure and its
composition provide added value for coverage, minimizing
rejection and activation of the inflammatory response.208
Polypeptides or modified polypeptides could be useful for
preparing artificial skin grafts by LBL, as the method
provides a simple means of producing films without limit
to size, shape, and composition and polypeptides are inherently biocompatible. The usefulness of polypeptide LBL
films in this context might depend on the presence of an
“inert” plasticizer to decrease film brittleness. Preliminary
data show that the presence of 15% glycerol in the assembly
buffers does not impair fabrication of a polypeptide multilayer film (J. S. R. & D. T. H., unpublished results). It seems
likely that control over peptide structure and film architecture
will be important for controlling the physical, chemical, and
biological properties of a polypeptide skin graft.
Cell and Tissue Culture. The extracellular matrix (ECM)
consists of various proteins secreted by cells: fibronectin,
laminin, vitronectin, and collagen.209 Molecular composition
of the ECM depends on cell type and extracellular signals
regulating cell behavior. Attachment of a cell to the ECM
determines cell shape and maintains cell function and tissue
integrity. The nature of a cell’s physical contacts with its
surroundings depends on the stability, organization, and
composition of the ECM. Attachment is mediated by
transmembrane heterodimeric protein receptors called integrins. In tissue engineering, substrate or scaffold requirements
include biocompatibility, biodegradability, cell adhesion, and
sufficient mechanical strength to withstand long-term cell
culture in vitro.
The physical and chemical character of a surface can affect
properties of the secreted ECM and, therefore, the behavior
of cells cultured on the surface. LBL provides a means of
controlling charge density, surface energy, and roughness
of a surface, as well as temperature sensitivity, porosity,
rigidity, and bioactivity. This could be important for the
design of biodegradable supports for three-dimensional cell
growth210,211 or controlled drug delivery.212-214 Related works
in the multilayer film literature include refs 17, 93, 151, and
215-224. A summary by Richert et al. of some recent cell
culture studies done on polyelectrolyte multilayer films
appears in ref 19.
A bioactive coating can be prepared by functionalization
of a polyelectrolyte multilayer. Possibilities include adsorption of a certain protein, e.g., fibronectin,225 or covalent
coupling of a certain peptide, e.g., a synthetic R-melanocortin
derivative.92 The ability to produce synthetic peptides at high
purity with relative ease presents interesting opportunities
for well-defined film “functionalization”. The design of
surfaces for more controlled biofunctionality, for instance
proliferation or nonproliferation of a particular cell line, is
of considerable scientific and industrial interest. An example
is provided by the arginine-glycine-aspartate (RGD)
sequence which occurs in a surprising number of different
ECM and platelet adhesion proteins, notably fibronectin, and
is a key cell recognition signal for integrins. Features of cell
adhesion vary with the flanking residues of the RGD peptide
and distribution of the signal on a surface. Peptides encompassing RGD can be designed and used to prepare polypep-
Biomacromolecules, Vol. 6, No. 6, 2005 2909
tide multilayer films, and the films can be sterilized by
treatment with 70% ethanol and dried in a sterile laminar
flow hood.20 Work in this direction has already appeared in
the scientific literature. For example, Picart et al. have studied
the adhesion of osteoblasts on RGD-grafted multilayer films
of PLL and PLGA.226 Related works include Richert et al.,19
Jessel et al.,227 and Salloum et al.,224 which discuss adhesion
of chondrosarcoma cells, adhesion of monocytes, and adhesion of aortic smooth muscle cells, respectively, on multilayer
films.
Control of Immunogenicity. Immunogenicity of a peptide
is maximized, according to one rule, by keeping the predicted
hydrophobicity low and the predicted hydophilicity, backbone flexibility, surface accessibility, and odds of β turn
formation high.228 Length too is a factor in determining the
immune response of a peptide. Most peptide antigens are
12-16 residues long, though peptides 9 residues or shorter
can be effective immunogens.229 It seems that immunogenicity might be minimized by standing the maximization
approach on its head. Work in this direction could be
important for the development of some kinds of in vivo
applications of LBL films and microcapsules.
Zheng et al. have suggested that the immunogenicity of
peptides designed for LBL might be minimized by basing
the amino acid sequences on solvent-exposed regions in the
folded states of proteins from the same organism.78 Design
elements 7 residues in length, called “sequence motifs,” have
been identified in protein-encoding regions of the human
genome for optimizing certain physical, chemical, and
biological properties of structures made by LBL. Peptide
designs based on these motifs have been shown suitable for
multilayer film assembly. For intravenous applications,
human blood proteins might be a particularly rich source of
motifs for peptide design. Microcapsules manufactured from
such peptides might be useful for the development of
artificial red blood cells or drug delivery systems. Initial
results in this direction are promising.94
Biodegradable Films of Controlled Wettability. Polypeptides made of usual amino acids are inherently biodegradable.
Control of peptide structure and film architecture enables
control of solubility in aqueous solvent and the hydrophobicity of an adsorbed layer (Figure 6). This translates into
control of film wettability. Peptides therefore are suitable
for the preparation of biodegradable films of controlled
wettability. Such films could be useful in a wide variety of
practical applications.22
Implant Technology. Halthur et al. have suggested that
a PLL/PLGA multilayer film, which is both biocompatible
and biodegradable, might make a useful coating for an
implant.18 Design of peptides takes the technology a step
further, enabling “peptide-inherent” cross-linking of polymers
when cysteine is present and control of film density106 and
conferring specific bioactivity when certain sequences are
included, e.g., RGD. Peptide design and film architecture
could be optimized for implant performance in specific
tissues.
Artificial Cells and Drug Delivery Systems. Various
synthetic polypeptides have been investigated for use in drug
delivery and as artificial cells.230 The subject is very large,
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Biomacromolecules, Vol. 6, No. 6, 2005
and scant coverage only can be provided here. The rate of
degradation can be controlled by selection of amino acid
composition, sequence, and chemical modification. A recent
study reports two types of polypeptide microcapsule: Glu
residues in the main chain and Glu residues in the graft chains
of a positively charged main chain. Both show pH-responsive
release of encapsulated FITC-dextran. The release rate is
lower at pH 3.0 than pH 7.5.231 Glucose oxidase has been
encapsulated in multilayer films of PLL and PLGA, using
colloidal particles of calcium carbonate as templates for film
assembly.95 Enzyme activity is retained following encapsulation and dissolution of the core by addition of EDTA. The
polypeptide “membrane” is permeable to small molecules
involved in the activity assay.
Peptide inherent, reversible “locking” of polypeptide
multilayer microcapsules has been achieved by use of
designed polypeptides with cysteine residues.94 Similar to
the behavior of disulfide bonded proteins, e.g., lysozyme,140,165
cross-linked polypeptide microcapsules have a more stable
structure at acidic pH and in organic solvent than noncrosslinked ones. The ability to control pH responsiveness
and stability suggests that designed polypeptide microcapsules could be useful for drug delivery. Inherent biocompatibility and low or negligible immunogenicity of
certain designed polypeptides could make them ideal for such
applications.78 Highly immunogenic peptides also suitable
for microcapsule fabrication could be useful in vaccine
development.
Jessel et al. have reported on the build-up of polypeptide
multilayer coatings with antiinflammatory properties based
on the embedding of piroxicam-cyclodextrin complexes232
and on the use of pyridylamino-β-cyclodextrin as a molecular
chaperone for lipopolysaccharide embedded in a multilayered
polyelectrolyte architecture.233 These examples give some
idea of the potential of polypeptide multilayers for drug
delivery.
There are many possible therapeutic applications of
polymeric artificial cells.234 Examples include encapsulation
of transplanted islet cells to treat diabetic patients and
artificial red blood cells in oxygen therapeutics. Recent
advances in biotechnology, molecular biology, nanotechnology, and polymer chemistry are opening up exciting possibilities in this field. Polypeptide-based approaches are very
promising for reasons outlined in this review.
V. Conclusion
Researchers have merely scratched the surface of what
can be done with polypeptide multilayer films. There is great
promise for the development of useful films and multifunctional coatings. Developments in synthetic chemistry and
biotechnology are likely to prove crucial to commercialization efforts, many of which are likely to be aimed toward
applications in medicine and biotechnology.
Acknowledgment. We thank Yuri Lvov for discussions
on LBL, Bingyun Li for assistance with graphics, and Sujay
Bhad for comments on the manuscript. Work on polypeptide
multilayer films and microcapsules has been supported in
part by a Nanotechnology Exploratory Research award from
Haynie et al.
the National Science Foundation (DMI-0403882), a seed
grant from the Center for Entrepreneurship and Information
Technology, a project grant from Artificial Cell Technologies, Inc., an enhancement grant from the Louisiana Space
Consortium (Louisiana NASA EPSCoR, Project R127172),
and the 2002 Capital Outlay Act 23 of the State of Louisiana
(Governor’s Biotechnology Initiative).
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