Biomacromolecules 2005, 6, 2895-2913
2895
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
2896
Biomacromolecules, Vol. 6, No. 6, 2005
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
2898
Biomacromolecules, Vol. 6, No. 6, 2005
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
2900
Biomacromolecules, Vol. 6, No. 6, 2005
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-
2902
Biomacromolecules, Vol. 6, No. 6, 2005
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 (
