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Protein-inspired multilayer nanofilms: science, technology and medicine
Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 150 – 157 www.nanomedjournal.com
Cellular Nanomedicine
Protein-inspired multilayer nanofilms: science, technology and medicine Donald T. Haynie, BS, PhD,4 Ling Zhang, MS, Wanhua Zhao, BS, PhD, Jai S. Rudra, BS Artificial Cell Technologies, Inc., New Haven, Connecticut, USA Received 2 June 2006; accepted 19 July 2006
Abstract
The field of polypeptide multilayer nanofilm research flourishes where study of protein structure and function shares a border with development of polyelectrolyte multilayers. The soil is fertile for creative input and promises a harvest of interesting results: the structure of a film can be predetermined on a layer-by-layer (LBL) basis, a huge variety of polypeptide sequences can be realized in large quantities by modern methods of synthesis, and the fabrication process is environmentally benign. In electrostatic LBL assembly, multilayer film assembly is driven primarily by coulombic interactions, but hydrophobic interactions and hydrogen bonds also contribute to film formation and stability, the amount depending on polypeptide design. Most peptides suitable for LBL assembly form films with a large percentage of b-sheet at neutral pH; it would appear that b-sheet is favored over a-helix in this context by the contribution to entropy of the number of ways of forming a b-sheet from a single polypeptide chain. Film thickness and roughness depend rather substantially on amino acid composition. Promising applications of the polypeptide multilayer film platform technology include coatings for medical implant devices, scaffolds for tissue engineering, coatings for targeted drug delivery, artificial cells for oxygen therapeutics, and artificial viruses for immunization. In each case peptide structure is tailored to the application. Here we summarize recent results of experimental studies and computational work from our laboratory, showing how the study of protein structure has inspired the design of polypeptide films and pointing out new opportunities for technology development. This work also provides a brief introduction to polypeptide structure and multilayer films. D 2006 Published by Elsevier Inc.
Key words:
Artificial cell; Biomimetics; Coating; Drug delivery; Edibility; Film; Immunogenicity; Nanotechnology; Polypeptide; Tissue engineering
Polypeptides and multilayer nanofilms Polypeptide multilayer films are bintegratedQ nanostructures of two belementsQ: polypeptide chains and multilayer films. The latter element represents bdesignQ, and the former represents bnatureQ. Investigation of the two more fundamental areas has proceeded essentially independently—until now. All proteins comprise at least one polypeptide chain. Knowledge of the structure and function of proteins has inspired the design of peptides, a type of weak polyelectrolyte, for fabrication of a special type of polyelectrolyte multilayer films. A brief review of polypeptides and 4 Corresponding author. Artificial Cell Technologies, Inc., 5 Science Park, Suite 13, New Haven, Connecticut 06511. E-mail address: [email protected] (D.T. Haynie). 1549-9634/$ – see front matter D 2006 Published by Elsevier Inc. doi:10.1016/j.nano.2006.07.008
multilayer films is provided here. Our group has provided broader coverage elsewhere [1], and a summary of the physics of polypeptide multilayer films also exists [2]. Polypeptides (and prizes) The polypeptide belementQ represents bnatureQ. First described by E. Fischer about a century ago [3], polypeptides form one of four classes of biological macromolecules—the others being nucleic acids, polysaccharides, and phospholipids [4]. The first determination of the chemical structure of a bioactive peptide, cow insulin, was achieved by F. Sanger (Nobel laureate, Chemistry, 1958) in the early 1950s. A key feature of the amino acid subunits of a polypeptide is their structure (Figure 1, A). An amino group, a carboxyl group, a hydrogen atom, and a variable side
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Fig 1. Polypeptide structure. A, Fischer diagram of an amino acid. R signifies the side chain. There are 20 usual amino acid types. B, Stick model of a 32residue peptide. The amino terminus is at the left. The polymer in extended conformation has a length of about 10 nm.
chain, R, constitute the substituents of the central a-carbon. There are 20 usual side chains in nature. The number of possible bnon-naturalQ side chains is practically unlimited, and scores of novel ones are available from commercial sources for abiotic peptide synthesis. Amino acids are chiral: each of the bnaturalQ ones has a right-handed form (d isomer) and a left-handed form (l isomer)—with the exceptionglycine, which has just a hydrogen atom for its R side chain. All amino acids in all proteins in all known organisms are l isomers. There are four hierarchical levels of peptide-based structures, and they were first defined by K. LinderstrbmLang in the 1950s [5]. Primary structure denotes the sequence of amino acid residues in a polypeptide chain (Figure 1, B). The most common types of secondary structure, a-helices and b-sheets, were predicted by L. Pauling (Nobel laureate, Chemistry, 1954) and R. Corey in the early 1950s and visualized in atomic-resolution protein structures shortly thereafter. Helices and sheets are characterized in part by distinctive patterns of hydrogen bonds (relatively weak electrostatic interactions) formed between chemical groups in the polypeptide backbone. Nearly all known protein structures comprise at least one a-helix or b-sheet [6]; secondary structures can also form in peptides that do not fold into proteins [7]. Tertiary structure refers to the relative spatial orientation of secondary structure elements and the noncovalent contacts between amino acids, as in the small muscle protein myoglobin. Quaternary structure is the relationship between polypeptide chains when more than one is involved in forming the overall protein structure, as in the blood protein hemoglobin, a structural relative of myoglobin. When properly folded and biologically
functional, a protein is said to be in the native state. Folded protein structure was first visualized at atomic resolution in the late 1950s and early 1960s: sperm whale myoglobin and horse heart hemoglobin by J. Kendrew and M. Perutz (Nobel laureates, Chemistry, 1962), respectively, at Cambridge University, and hen egg white lysozyme by D.C. Phillips at Oxford University. Since then, the number of protein structures determined at or near atomic resolution has risen exponentially with each passing year. A disulfide bond is a bnaturalQ type of covalent cross-link that can form between cysteine side chains. Disulfide bonds stabilize protein structure by reducing the entropy of the polymer chain, particularly in a denatured state (for example, [8]). Ribonuclease A is a small protein with four native disulfide bonds. In the early 1960s C. Anfinsen (Nobel laureate, Chemistry, 1972) showed that all the binstructionsQ required for the ribonuclease polypeptide to go from a denatured state in which no disulfide bonds were intact to native protein structure in water were bencodedQ in the amino acid sequence. In some cases, at least, acquisition of biologically functional protein structure from a disordered conformation is driven by a minimizing the Gibbs free energy of the protein–solvent system. Genome sequencing efforts have revealed that the myriad of DNA-encoded polypeptide chains in the different living organisms on Earth fold into a comparatively tiny number of qualitatively different structures [6]. Several broad structural classes of proteins have been identified. In globular proteins (for example, myoglobin), the polypeptide folds into a highly compact, ball-like shape. Fibrous proteins, found in hair, tendons, and spider silk, tend to be elongated. Elastin, for example, found in skin and lung, can be stretched without
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Fig 2. Sequential steps of layer-by-layer assembly.
tearing. Formed of loose and unstructured polypeptide chains, this protein provides the elasticity needed to fulfill its biological role. Membrane proteins (such as the insulin receptor) are embedded in the plasma membrane of a cell. Proteins can also be classified according to function [6]. Enzymes, such as lysozyme, catalyze covalent bond formation or rupture in specific chemical reactions. Structural proteins provide mechanical support for cells and tissues. Transport proteins, such as hemoglobin, carry small molecules and ions. Motor proteins generate force, causing muscle contraction and allowing movement in the case of skeletal muscle myosin. Signaling proteins and peptides, such as insulin, are chemical signals that control physiological function, often by binding noncovalently to receptor proteins embedded in the plasma membrane, which transmit the signal inside or outside the cell. Storage proteins bind ions or metabolites and store them. Gene-regulatory proteins switch gene expression on and off in response to environmental cues. Antibodies bind specific antigens and stimulate the immune system to protect the body from an invading pathogen. Proteins are nanometer-scale machines of remarkable functional variety. Details of protein structure and function, revealed by decades of study, form a library of bmotifsQ for the design of polypeptides for multilayer film assembly. The tiny size of proteins and their ability to self-organize structure, self-recognize other molecules, and function repetitively with exquisite specificity, inspired much of the conceptual content of the now-famous lecture by R. Feynman (Nobel laureate, Physics, 1965), bPlenty of Room at the BottomQ, given in 1959 [9]. (The lecture’s title would seem to be a pun on the title of a 1959 British film, bRoom at the TopQ [10]. The story is about a money- and status-seeking blue-collar worker who is willing to do whatever it might take to climb the ladder to success (become an essential component of the complex network of interactions that characterize a living organism). Simone Signoret won the Oscar for best actress for her role in the
film as the epitome of sexy, womanly vulnerability [11]—an element of the seductive vision that motivates the sojourn of the atom from bobscurityQ to bstardomQ. The worker seems to signify for Feynman the proverbial talented but destitute science or engineering scholar who will do just about anything to achieve success, get a degree and find a position. This search for the means to secure a good post is aided by nature herself, who inspires the investigation of her structure and function. At the time the film was current, celebrities bound for Hollywood arrived more often than not by train and disembarked not in working-class Los Angeles but in pleasant Pasadena [12]. Little imagination is needed to guess that an evening with Simone was the dream of many a science or engineering student at the then virtually all-male California Institute of Technology, and that she was the subject of a good deal of boff-topicQ chat. Feynman’s lecture is now generally taken as the bgenesisQ of nanotechnology (for example, [13]). His thinking on the matter may have been stimulated by knowledge of or interaction with other notables at Caltech, for instance, T. Morgan (chromosome theory of heredity), L. Pauling (genetic basis of sickle-cell anemia), and M. Delbrqck (bacterial virus structure and function) (Nobel laureate, Medicine or Physiology, 1969). Multilayer films The polyelectrolyte multilayer film belementQ of a polypeptide film represents bdesignQ. The method of thinfilm preparation known as layer-by-layer assembly (LBL; Figure 2), was first described in the mid-1960s by R. Iler [14], a chemist with the DuPont Company, although conceptual seeds are evident in work of I. Langmuir (Nobel laureate, Chemistry, 1932) from the early 1940s [15]. Iler died before his work on multilayer films could be developed, and U.S. patents credited to him expired around the close of the 1980s. Study of polyelectrolyte LBL was first developed in the early 1990s by G. Decher, now at Universite´ Louis Pasteur
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[16]. Polymer adsorption in the formation of each layer is driven by electrostatic attraction and limited by electrostatic repulsion. Charge neutralization in a film occurs by bintrinsicQ charge compensation (oppositely charged polyelectrolyte) or bextrinsicQ compensation (counterions) [16-18]. Repeated alteration of the net charge of the assembling species results in a multilayer film. There is nanometer-scale control over film thickness. LBL is conceptually simple and environmentally friendly, it can be automated, and it can be achieved by bdippingQ of the substrate into polymer solutions [16] or by bsprayingQ [19]. Cross-linking of polyelectrolytes in a film has been developed in a variety of ways for stabilization of film structure (summarized in [20]). The linear charge density of a bstrongQ polyelectrolyte is practically independent of pH in the usual range. Charges on such polymers in a multilayer film are generally well matched: small salt ions are excluded from the bulk film, and layers are thin and dense [21,22]. Salt concentration plays a key role in determining film thickness for a given solvent and number of layers [23,24]. Layer thickness varies roughly linearly with salt concentration [25,26]. There seems to be a lower bound on charge density for selflimited polymer adsorption [27,28]. Strong polyelectrolyte multilayer films are not well stratified, but interpenetration occurs in just a few layers [16,29]. bWeakQ polyelectrolyte LBL was first developed in the latter half of the 1990s by M. Rubner at Massachusetts Institute of Technology [30]. The average charge per unit length of a weak polyelectrolyte can vary considerably with pH, particularly near the pKa of polymer side chains [31-33]. The linear charge density of adsorbing polymers and therefore the surface charge of a polyelectrolyte film can be adjusted by changing the degree of polymer ionization. It thus is possible to control layer thickness, level of layer interpenetration, and surface wettability of a film. In the poly(acrylic acid)/poly(allylamine hydrochloride) system, for example, film thickness changes from 5 to 80 2 on a small change in pH [33], and the advancing water contact angle of the surface can vary from 0 to 50 deg [32]. Substantial and irreversible changes in film morphology can be realized by acidic pH treatment, including preparation of a uniformly microporous film [34]. Change of environmental pH can also result in history-dependent swelling behavior [35]. Salt type and ionic strength influence film structure by changing the local electrostatic environment [36,37]. Polyelectrolytes that are readily available from commercial sources have been studied extensively. They are therefore called bconventionalQ polyelectrolytes, whether strong or weak. Polypeptide multilayer films bNature and designQ: knowledge of peptide and protein structure and function is fused with knowledge of polyelectrolyte multilayer films to develop polypeptide multilayer
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nanofilms. Combining these belementsQ yields a unique class of interesting structures with a broad range of useful properties, particularly for biomedical applications. Surprisingly, very little research on polypeptide multilayer films appears in the scientific literature before 2004 [1], and the first work involving designed peptides [38] was published in the same year. Science Polypeptide structure Polypeptides are weak polyelectrolytes. Knowledge of peptide structure and function is extensive, and conventional weak polyelectrolyte LBL has been studied for about a decade. Nevertheless, the physical properties of a polypeptide film cannot be predicted from knowledge of amino acid sequence alone; the polymer self-assembly process in LBL is complex. Of course, bulk film properties must be attributable to details of interactions between constituent molecules, but it is unclear how the one will determine the other in any specific case; particle behavior in supramolecular structures is less well understood than other areas of physics. Predictions of the chemical and biological properties of a polypeptide film will be all the more speculative, even if environmental benignity can generally be assumed, except in cases of specific sequence information. Many peptide sequences are known to form amyloid fibrils [39], but a sequence selected at random is unlikely to threaten the life of an organism. Simple combinatorics says there are 3220 6 ~1041 chemically distinct polypeptides 32 residues long, considering the 20 usual amino acids alone. For comparison, Big Bang theory would suggest that the universe is a mere ~1027 nanoseconds old. Importantly for commercial prospects of polypeptide multilayer films, a large proportion of the possible peptide structures can be realized by at least one approach to synthesis. Moreover, in may cases large quantities of material can be prepared by solid-phase synthesis or genetic engineering of bacteria [40,41]. Research on polypeptides for LBL may be described as bbasicQ or bappliedQ. In both cases a more or less essential linear charge density polyelectrolyte criterion applies for self-limited multilayer film growth and control of film thickness. A polypeptide, or any polyelectrolyte for that matter, must have a sufficiently high net charge and relatively broad charge distribution for LBL. These constraints shrink the space of polypeptide structures suitable for typical multilayer film assembly. Let us assume that the minimum absolute charge density is 0.5 per amino acid residue. Examples of sequences consistent with the criterion are (KVKGKCKV)3KVKGKCKY and (EVEGECEV)3EVEGECEY, which are positive and negative, respectively, at neutral pH because of the ionization properties of lysine (K) and glutamic acid (E) [38]. Net charge is a more important criterion than charge distribution for LBL, but clustering of ionized or hydrophobic residues might
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make the polypeptide prone to aggregation in an aqueous medium [42]. Glycine (G) hinders secondary structure formation in polymers before film assembly, valine (V) promotes formation of hydrophobic interactions between polymers, cysteine (C) allows bnaturalQ and reversible crosslink formation between polymers, and tyrosine (Y) is useful for UV spectroscopic detection [38,43-46]. As to applied research, a useful example is provided by biomedicine. Charge density and distribution are insufficient sequence selection criteria for peptides and peptide structures intended for use in the bloodstream; in addition, the peptides must not stimulate proliferation of splenocytes, activate the complement system, or spur platelet aggregation, and immunogenicity should be controlled. One approach to realizing the desired immune properties of a designed peptide is to take a cue from nature and select sequences encoded by the genome of the organism in which the peptide or peptide structure will be used [46], particularly blood proteins. Film properties Research activity on polypeptide multilayer films in our group falls into two categories: experimental studies and computational work. All calculations are interpreted with reference to the corresponding experiments. The main areas of experimental effort have been film assembly [38,44-49] and disassembly [38,43,45,49], internal structure [45,48,49], and surface morphology [44,48,49]. The same studies have sought to reveal the role in peptide assembly of linear charge density [44,46,47,49,50], polymer length [47], and amino acid composition and sequence [45,46,49,50]. Figure 3, A shows how assembly behavior can depend on degree of polymerization [47], and Figure 3, B illustrates disassembly of a designed polypeptide multilayer film [38]. The secondary structure content of a poly(llysine)/poly(l-glutamic acid) film prepared at pH 7.4 is 1/20 a-helix, 1/5 b-sheet, 3/10 b-turn, and the rest random coil Fig 3. Results of experimental studies of polypeptide multilayer films. A, Length dependence of peptide deposition. Decrease in resonant frequency is proportional to increase in film mass. The peptide mass range was 3 kd (bsmallQ) to over 200 kd (blargeQ). B, Reversible disulfide bond stabilization of a film at acidic pH. Disulfide bonds are formed under oxidizing conditions. C, Dependence of mass density on peptide properties. The line is a linear fit to all data points. It nearly passes through the origin. D, Dependence of surface roughness on peptide properties. The line indicates the trend only. Frequency shift is proportional to mass deposited under the conditions of the experiment. P1, (KKKK)7KKKY; N1, (EEEE)7EEEY (N1); P2, (KVKV)7KVKY; N2, (EVEV)7EVEY; P3, (KVKS)7KVKY; N3, (EVEN)7EVEY. P1 and N1 have a high linear charge density, P2, N2, P3, and N3 have a low linear charge density, P2 and N2 are hydrophobic, P3 and N3 are hydrophilic. P = positive; N = negative. All these peptides are 32mers (see reference [50]). A, reprinted from Haynie DT, Balkundi S, Palath N, Chakravarthula K, Dave K. Polypeptide multilayer films: role of molecular structure and charge. Langmuir. 2004;20:4540-7. Copyright 2004 American Chemical Society. B, reprinted with permission from Li B, Haynie DT. Multilayer biomimetics: reversible covalent stabilization of a nanostructured biofilm. Biomacromolecules. 2004;5:1667-70. Copyright 2004 American Chemical Society.
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[48]. Film density (Figure 3, C) and surface roughness (Figure 3, D) also depend on peptide sequence [49,50]. Computational studies initiated by our group have been designed to complement experimental work on polypeptide multilayer nanofilms. Database development and data mining projects have been undertaken to develop algorithms for sequence design and for extraction of peptide sequence motifs from translated genomic data [46]. Each unique sequence motif is a sort of bsuper amino acid residueQ for the design of peptides for multilayer film fabrication, a type of structural unit of a larger scale structure. Molecular dynamics simulations of designed peptides [50] are based on the premise that physical properties of interpolyelectrolyte complexes will provide insight on interaction between oppositely charged polyelectrolytes in a multilayer film [51]. Technology Specific applications of the polypeptide film platform technology divide broadly into two areas: inside the body and outside the body. Some of the applications mentioned here are being developed by Artificial Cell Technologies, Inc. In vivo applications Polypeptide microcapsules are promising for the development of artificial cells [45,52-54], for example, for oxygen therapeutics or drug delivery (Figure 4). Degradation rate, pH responsiveness, capsule wall density, and permeability can be controlled by amino acid composition, sequence, disulfide cross-linking, method of peptide assembly, and chemical modification of peptides. We have encapsulated glucose oxidase in a polypeptide microcapsule and subsequently demonstrated enzyme activity [53]. We have also encapsulated hemoglobin and demonstrated responsiveness to oxygen tension [54]. Polypeptide microcapsules could also be useful for targeted and sustainedrelease drug delivery. Control over amino acid sequence permits control over immunogenicity. Certain artificial cell applications will require minimized immunotoxicity and controlled immunogenicity. We have outlined a method of amino acid sequence selection from genomic information to accelerate achievement of the design criteria [46]. Maximization of the immune response, however, could also be of therapeutic value, as in a vaccine. The polypeptide capsule platform technology is suited to this application too. The approach features the advantage of simultaneous presentation of multiple antigenic sites and possible encapsulation of antiviral agents. Antimicrobial peptides address the public health concern of growing resistance of bacteria to certain chemical compounds. Such peptides could be integrated with LBL to develop a new class of antimicrobial films, coatings, and related products. Antimicrobial activity is conferred on a poly(vinylidene chloride) copolymer film by incorporation of the antibacterial peptide nisin [55]. Antiadhesive multilayer films made of poly(L-lysine) (PLL) and pegylated-
Fig 4. Polypeptide multilayer film-based artificial cell visualized by confocal fluorescence microscopy. A, Artificial cell. Scale bar = 3.85 m. B, Fluorescence intensity profile. Reprinted with permission from Palath N, Bhad S, Montazeri R, Guidry CA, Haynie DT. Polypeptide multilayer nanofilm artificial red blood cells. J Biomed Mater Res. In press 2006. Copyright 2006 Wiley Interscience.
PLGA (poly(L-glutamic acid)) have been shown to reduce adsorption of serum proteins and Escherichia coli [56], and films functionalized by the insertion of defensin inhibit growth of infectious pathogens, reducing pathological consequences [57]. Vascular implants, catheters, needles, surgical tools, and foods can be protected or preserved by coating them with antimicrobial peptides.
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Ex vivo applications Polypeptide multilayer films can be used to control the cytophilicity of a surface by promoting biocompatibility, biodegradability, cell adhesion, or mechanical strength to withstand long-term cell culture in vitro. This is important for the design of scaffolds for cell growth with control over temperature sensitivity, porosity, and rigidity. A bioactive coating can be prepared by functionalization of a multilayer film to favor proliferation of a unique cell line or a range of cell lines. Adhesion of osteoblasts, chondrosarcoma cells, monocytes, and aortic smooth muscle cells on multilayer films has been reported [58-61]. This research area is of considerable scientific and industrial interest, and the ability to design polypeptides for LBL will promote it. The restructuring of water on hydrophobic surfaces is entropically unfavorable and translates into limited surface wettability. The primary structure of a polypeptide is key to controlling its solubility in an aqueous solvent and the hydrophobicity of a multilayer film. Polypeptide films of controlled wettability are important for a variety of practical applications, for example, contact lens coatings. The operational simplicity and low cost of membranebased separations have attracted much attention for preparative-scale separation of chiral molecules. Modified membranes with amphiphilic side chains exhibit permeation rate ratios above 8 for a-amino acid enantiomers [62], and chiral selectivity and enantioselective binding affinity for d isomers increase with secondary structure [63]. Multilayer films fabricated from charged polypeptides are optically active, permitting analysis by circular dichroism spectrometry, and they exhibit high permeation rates and encouraging selectivity [64]. Substantial advances in separations could be made by optimizing the structure of polypeptides and using polypeptide multilayer films for specific separation characteristics [65]. Summary Nanotechnology has been inspired by knowledge of the structure and function of proteins, nature’s own nanoscale machines. Polypeptide nanofilms combine principles of protein structure and polyelectrolyte film fabrication. These films display a range of properties, depending on design of amino acid sequence, and they are suited to the development of a variety of practical applications, notably medicine. References [1] Haynie DT, Zhang L, Rudra JS, Zhao W, Zhong Y. Polypeptide multilayer films. Biomacromolecules 2005;6:2895 - 913. [2] Haynie DT. Physics of polypeptide multilayer films. J Biomed Res B Appl Biomater 2006;78B:243 - 52. [3] Fruton JS. Proteins, enzymes, genes: the interplay of chemistry and biology. New Haven (Conn)7 Yale University Press; 1999. p. 186 - 9. [4] Voet D, Voet JG. Biochemistry. 2nd ed. New York7 John Wiley; 1995.
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Cellular Nanomedicine
Protein-inspired multilayer nanofilms: science, technology and medicine Donald T. Haynie, BS, PhD,4 Ling Zhang, MS, Wanhua Zhao, BS, PhD, Jai S. Rudra, BS Artificial Cell Technologies, Inc., New Haven, Connecticut, USA Received 2 June 2006; accepted 19 July 2006
Abstract
The field of polypeptide multilayer nanofilm research flourishes where study of protein structure and function shares a border with development of polyelectrolyte multilayers. The soil is fertile for creative input and promises a harvest of interesting results: the structure of a film can be predetermined on a layer-by-layer (LBL) basis, a huge variety of polypeptide sequences can be realized in large quantities by modern methods of synthesis, and the fabrication process is environmentally benign. In electrostatic LBL assembly, multilayer film assembly is driven primarily by coulombic interactions, but hydrophobic interactions and hydrogen bonds also contribute to film formation and stability, the amount depending on polypeptide design. Most peptides suitable for LBL assembly form films with a large percentage of b-sheet at neutral pH; it would appear that b-sheet is favored over a-helix in this context by the contribution to entropy of the number of ways of forming a b-sheet from a single polypeptide chain. Film thickness and roughness depend rather substantially on amino acid composition. Promising applications of the polypeptide multilayer film platform technology include coatings for medical implant devices, scaffolds for tissue engineering, coatings for targeted drug delivery, artificial cells for oxygen therapeutics, and artificial viruses for immunization. In each case peptide structure is tailored to the application. Here we summarize recent results of experimental studies and computational work from our laboratory, showing how the study of protein structure has inspired the design of polypeptide films and pointing out new opportunities for technology development. This work also provides a brief introduction to polypeptide structure and multilayer films. D 2006 Published by Elsevier Inc.
Key words:
Artificial cell; Biomimetics; Coating; Drug delivery; Edibility; Film; Immunogenicity; Nanotechnology; Polypeptide; Tissue engineering
Polypeptides and multilayer nanofilms Polypeptide multilayer films are bintegratedQ nanostructures of two belementsQ: polypeptide chains and multilayer films. The latter element represents bdesignQ, and the former represents bnatureQ. Investigation of the two more fundamental areas has proceeded essentially independently—until now. All proteins comprise at least one polypeptide chain. Knowledge of the structure and function of proteins has inspired the design of peptides, a type of weak polyelectrolyte, for fabrication of a special type of polyelectrolyte multilayer films. A brief review of polypeptides and 4 Corresponding author. Artificial Cell Technologies, Inc., 5 Science Park, Suite 13, New Haven, Connecticut 06511. E-mail address: [email protected] (D.T. Haynie). 1549-9634/$ – see front matter D 2006 Published by Elsevier Inc. doi:10.1016/j.nano.2006.07.008
multilayer films is provided here. Our group has provided broader coverage elsewhere [1], and a summary of the physics of polypeptide multilayer films also exists [2]. Polypeptides (and prizes) The polypeptide belementQ represents bnatureQ. First described by E. Fischer about a century ago [3], polypeptides form one of four classes of biological macromolecules—the others being nucleic acids, polysaccharides, and phospholipids [4]. The first determination of the chemical structure of a bioactive peptide, cow insulin, was achieved by F. Sanger (Nobel laureate, Chemistry, 1958) in the early 1950s. A key feature of the amino acid subunits of a polypeptide is their structure (Figure 1, A). An amino group, a carboxyl group, a hydrogen atom, and a variable side
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Fig 1. Polypeptide structure. A, Fischer diagram of an amino acid. R signifies the side chain. There are 20 usual amino acid types. B, Stick model of a 32residue peptide. The amino terminus is at the left. The polymer in extended conformation has a length of about 10 nm.
chain, R, constitute the substituents of the central a-carbon. There are 20 usual side chains in nature. The number of possible bnon-naturalQ side chains is practically unlimited, and scores of novel ones are available from commercial sources for abiotic peptide synthesis. Amino acids are chiral: each of the bnaturalQ ones has a right-handed form (d isomer) and a left-handed form (l isomer)—with the exceptionglycine, which has just a hydrogen atom for its R side chain. All amino acids in all proteins in all known organisms are l isomers. There are four hierarchical levels of peptide-based structures, and they were first defined by K. LinderstrbmLang in the 1950s [5]. Primary structure denotes the sequence of amino acid residues in a polypeptide chain (Figure 1, B). The most common types of secondary structure, a-helices and b-sheets, were predicted by L. Pauling (Nobel laureate, Chemistry, 1954) and R. Corey in the early 1950s and visualized in atomic-resolution protein structures shortly thereafter. Helices and sheets are characterized in part by distinctive patterns of hydrogen bonds (relatively weak electrostatic interactions) formed between chemical groups in the polypeptide backbone. Nearly all known protein structures comprise at least one a-helix or b-sheet [6]; secondary structures can also form in peptides that do not fold into proteins [7]. Tertiary structure refers to the relative spatial orientation of secondary structure elements and the noncovalent contacts between amino acids, as in the small muscle protein myoglobin. Quaternary structure is the relationship between polypeptide chains when more than one is involved in forming the overall protein structure, as in the blood protein hemoglobin, a structural relative of myoglobin. When properly folded and biologically
functional, a protein is said to be in the native state. Folded protein structure was first visualized at atomic resolution in the late 1950s and early 1960s: sperm whale myoglobin and horse heart hemoglobin by J. Kendrew and M. Perutz (Nobel laureates, Chemistry, 1962), respectively, at Cambridge University, and hen egg white lysozyme by D.C. Phillips at Oxford University. Since then, the number of protein structures determined at or near atomic resolution has risen exponentially with each passing year. A disulfide bond is a bnaturalQ type of covalent cross-link that can form between cysteine side chains. Disulfide bonds stabilize protein structure by reducing the entropy of the polymer chain, particularly in a denatured state (for example, [8]). Ribonuclease A is a small protein with four native disulfide bonds. In the early 1960s C. Anfinsen (Nobel laureate, Chemistry, 1972) showed that all the binstructionsQ required for the ribonuclease polypeptide to go from a denatured state in which no disulfide bonds were intact to native protein structure in water were bencodedQ in the amino acid sequence. In some cases, at least, acquisition of biologically functional protein structure from a disordered conformation is driven by a minimizing the Gibbs free energy of the protein–solvent system. Genome sequencing efforts have revealed that the myriad of DNA-encoded polypeptide chains in the different living organisms on Earth fold into a comparatively tiny number of qualitatively different structures [6]. Several broad structural classes of proteins have been identified. In globular proteins (for example, myoglobin), the polypeptide folds into a highly compact, ball-like shape. Fibrous proteins, found in hair, tendons, and spider silk, tend to be elongated. Elastin, for example, found in skin and lung, can be stretched without
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Fig 2. Sequential steps of layer-by-layer assembly.
tearing. Formed of loose and unstructured polypeptide chains, this protein provides the elasticity needed to fulfill its biological role. Membrane proteins (such as the insulin receptor) are embedded in the plasma membrane of a cell. Proteins can also be classified according to function [6]. Enzymes, such as lysozyme, catalyze covalent bond formation or rupture in specific chemical reactions. Structural proteins provide mechanical support for cells and tissues. Transport proteins, such as hemoglobin, carry small molecules and ions. Motor proteins generate force, causing muscle contraction and allowing movement in the case of skeletal muscle myosin. Signaling proteins and peptides, such as insulin, are chemical signals that control physiological function, often by binding noncovalently to receptor proteins embedded in the plasma membrane, which transmit the signal inside or outside the cell. Storage proteins bind ions or metabolites and store them. Gene-regulatory proteins switch gene expression on and off in response to environmental cues. Antibodies bind specific antigens and stimulate the immune system to protect the body from an invading pathogen. Proteins are nanometer-scale machines of remarkable functional variety. Details of protein structure and function, revealed by decades of study, form a library of bmotifsQ for the design of polypeptides for multilayer film assembly. The tiny size of proteins and their ability to self-organize structure, self-recognize other molecules, and function repetitively with exquisite specificity, inspired much of the conceptual content of the now-famous lecture by R. Feynman (Nobel laureate, Physics, 1965), bPlenty of Room at the BottomQ, given in 1959 [9]. (The lecture’s title would seem to be a pun on the title of a 1959 British film, bRoom at the TopQ [10]. The story is about a money- and status-seeking blue-collar worker who is willing to do whatever it might take to climb the ladder to success (become an essential component of the complex network of interactions that characterize a living organism). Simone Signoret won the Oscar for best actress for her role in the
film as the epitome of sexy, womanly vulnerability [11]—an element of the seductive vision that motivates the sojourn of the atom from bobscurityQ to bstardomQ. The worker seems to signify for Feynman the proverbial talented but destitute science or engineering scholar who will do just about anything to achieve success, get a degree and find a position. This search for the means to secure a good post is aided by nature herself, who inspires the investigation of her structure and function. At the time the film was current, celebrities bound for Hollywood arrived more often than not by train and disembarked not in working-class Los Angeles but in pleasant Pasadena [12]. Little imagination is needed to guess that an evening with Simone was the dream of many a science or engineering student at the then virtually all-male California Institute of Technology, and that she was the subject of a good deal of boff-topicQ chat. Feynman’s lecture is now generally taken as the bgenesisQ of nanotechnology (for example, [13]). His thinking on the matter may have been stimulated by knowledge of or interaction with other notables at Caltech, for instance, T. Morgan (chromosome theory of heredity), L. Pauling (genetic basis of sickle-cell anemia), and M. Delbrqck (bacterial virus structure and function) (Nobel laureate, Medicine or Physiology, 1969). Multilayer films The polyelectrolyte multilayer film belementQ of a polypeptide film represents bdesignQ. The method of thinfilm preparation known as layer-by-layer assembly (LBL; Figure 2), was first described in the mid-1960s by R. Iler [14], a chemist with the DuPont Company, although conceptual seeds are evident in work of I. Langmuir (Nobel laureate, Chemistry, 1932) from the early 1940s [15]. Iler died before his work on multilayer films could be developed, and U.S. patents credited to him expired around the close of the 1980s. Study of polyelectrolyte LBL was first developed in the early 1990s by G. Decher, now at Universite´ Louis Pasteur
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[16]. Polymer adsorption in the formation of each layer is driven by electrostatic attraction and limited by electrostatic repulsion. Charge neutralization in a film occurs by bintrinsicQ charge compensation (oppositely charged polyelectrolyte) or bextrinsicQ compensation (counterions) [16-18]. Repeated alteration of the net charge of the assembling species results in a multilayer film. There is nanometer-scale control over film thickness. LBL is conceptually simple and environmentally friendly, it can be automated, and it can be achieved by bdippingQ of the substrate into polymer solutions [16] or by bsprayingQ [19]. Cross-linking of polyelectrolytes in a film has been developed in a variety of ways for stabilization of film structure (summarized in [20]). The linear charge density of a bstrongQ polyelectrolyte is practically independent of pH in the usual range. Charges on such polymers in a multilayer film are generally well matched: small salt ions are excluded from the bulk film, and layers are thin and dense [21,22]. Salt concentration plays a key role in determining film thickness for a given solvent and number of layers [23,24]. Layer thickness varies roughly linearly with salt concentration [25,26]. There seems to be a lower bound on charge density for selflimited polymer adsorption [27,28]. Strong polyelectrolyte multilayer films are not well stratified, but interpenetration occurs in just a few layers [16,29]. bWeakQ polyelectrolyte LBL was first developed in the latter half of the 1990s by M. Rubner at Massachusetts Institute of Technology [30]. The average charge per unit length of a weak polyelectrolyte can vary considerably with pH, particularly near the pKa of polymer side chains [31-33]. The linear charge density of adsorbing polymers and therefore the surface charge of a polyelectrolyte film can be adjusted by changing the degree of polymer ionization. It thus is possible to control layer thickness, level of layer interpenetration, and surface wettability of a film. In the poly(acrylic acid)/poly(allylamine hydrochloride) system, for example, film thickness changes from 5 to 80 2 on a small change in pH [33], and the advancing water contact angle of the surface can vary from 0 to 50 deg [32]. Substantial and irreversible changes in film morphology can be realized by acidic pH treatment, including preparation of a uniformly microporous film [34]. Change of environmental pH can also result in history-dependent swelling behavior [35]. Salt type and ionic strength influence film structure by changing the local electrostatic environment [36,37]. Polyelectrolytes that are readily available from commercial sources have been studied extensively. They are therefore called bconventionalQ polyelectrolytes, whether strong or weak. Polypeptide multilayer films bNature and designQ: knowledge of peptide and protein structure and function is fused with knowledge of polyelectrolyte multilayer films to develop polypeptide multilayer
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nanofilms. Combining these belementsQ yields a unique class of interesting structures with a broad range of useful properties, particularly for biomedical applications. Surprisingly, very little research on polypeptide multilayer films appears in the scientific literature before 2004 [1], and the first work involving designed peptides [38] was published in the same year. Science Polypeptide structure Polypeptides are weak polyelectrolytes. Knowledge of peptide structure and function is extensive, and conventional weak polyelectrolyte LBL has been studied for about a decade. Nevertheless, the physical properties of a polypeptide film cannot be predicted from knowledge of amino acid sequence alone; the polymer self-assembly process in LBL is complex. Of course, bulk film properties must be attributable to details of interactions between constituent molecules, but it is unclear how the one will determine the other in any specific case; particle behavior in supramolecular structures is less well understood than other areas of physics. Predictions of the chemical and biological properties of a polypeptide film will be all the more speculative, even if environmental benignity can generally be assumed, except in cases of specific sequence information. Many peptide sequences are known to form amyloid fibrils [39], but a sequence selected at random is unlikely to threaten the life of an organism. Simple combinatorics says there are 3220 6 ~1041 chemically distinct polypeptides 32 residues long, considering the 20 usual amino acids alone. For comparison, Big Bang theory would suggest that the universe is a mere ~1027 nanoseconds old. Importantly for commercial prospects of polypeptide multilayer films, a large proportion of the possible peptide structures can be realized by at least one approach to synthesis. Moreover, in may cases large quantities of material can be prepared by solid-phase synthesis or genetic engineering of bacteria [40,41]. Research on polypeptides for LBL may be described as bbasicQ or bappliedQ. In both cases a more or less essential linear charge density polyelectrolyte criterion applies for self-limited multilayer film growth and control of film thickness. A polypeptide, or any polyelectrolyte for that matter, must have a sufficiently high net charge and relatively broad charge distribution for LBL. These constraints shrink the space of polypeptide structures suitable for typical multilayer film assembly. Let us assume that the minimum absolute charge density is 0.5 per amino acid residue. Examples of sequences consistent with the criterion are (KVKGKCKV)3KVKGKCKY and (EVEGECEV)3EVEGECEY, which are positive and negative, respectively, at neutral pH because of the ionization properties of lysine (K) and glutamic acid (E) [38]. Net charge is a more important criterion than charge distribution for LBL, but clustering of ionized or hydrophobic residues might
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make the polypeptide prone to aggregation in an aqueous medium [42]. Glycine (G) hinders secondary structure formation in polymers before film assembly, valine (V) promotes formation of hydrophobic interactions between polymers, cysteine (C) allows bnaturalQ and reversible crosslink formation between polymers, and tyrosine (Y) is useful for UV spectroscopic detection [38,43-46]. As to applied research, a useful example is provided by biomedicine. Charge density and distribution are insufficient sequence selection criteria for peptides and peptide structures intended for use in the bloodstream; in addition, the peptides must not stimulate proliferation of splenocytes, activate the complement system, or spur platelet aggregation, and immunogenicity should be controlled. One approach to realizing the desired immune properties of a designed peptide is to take a cue from nature and select sequences encoded by the genome of the organism in which the peptide or peptide structure will be used [46], particularly blood proteins. Film properties Research activity on polypeptide multilayer films in our group falls into two categories: experimental studies and computational work. All calculations are interpreted with reference to the corresponding experiments. The main areas of experimental effort have been film assembly [38,44-49] and disassembly [38,43,45,49], internal structure [45,48,49], and surface morphology [44,48,49]. The same studies have sought to reveal the role in peptide assembly of linear charge density [44,46,47,49,50], polymer length [47], and amino acid composition and sequence [45,46,49,50]. Figure 3, A shows how assembly behavior can depend on degree of polymerization [47], and Figure 3, B illustrates disassembly of a designed polypeptide multilayer film [38]. The secondary structure content of a poly(llysine)/poly(l-glutamic acid) film prepared at pH 7.4 is 1/20 a-helix, 1/5 b-sheet, 3/10 b-turn, and the rest random coil Fig 3. Results of experimental studies of polypeptide multilayer films. A, Length dependence of peptide deposition. Decrease in resonant frequency is proportional to increase in film mass. The peptide mass range was 3 kd (bsmallQ) to over 200 kd (blargeQ). B, Reversible disulfide bond stabilization of a film at acidic pH. Disulfide bonds are formed under oxidizing conditions. C, Dependence of mass density on peptide properties. The line is a linear fit to all data points. It nearly passes through the origin. D, Dependence of surface roughness on peptide properties. The line indicates the trend only. Frequency shift is proportional to mass deposited under the conditions of the experiment. P1, (KKKK)7KKKY; N1, (EEEE)7EEEY (N1); P2, (KVKV)7KVKY; N2, (EVEV)7EVEY; P3, (KVKS)7KVKY; N3, (EVEN)7EVEY. P1 and N1 have a high linear charge density, P2, N2, P3, and N3 have a low linear charge density, P2 and N2 are hydrophobic, P3 and N3 are hydrophilic. P = positive; N = negative. All these peptides are 32mers (see reference [50]). A, reprinted from Haynie DT, Balkundi S, Palath N, Chakravarthula K, Dave K. Polypeptide multilayer films: role of molecular structure and charge. Langmuir. 2004;20:4540-7. Copyright 2004 American Chemical Society. B, reprinted with permission from Li B, Haynie DT. Multilayer biomimetics: reversible covalent stabilization of a nanostructured biofilm. Biomacromolecules. 2004;5:1667-70. Copyright 2004 American Chemical Society.
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[48]. Film density (Figure 3, C) and surface roughness (Figure 3, D) also depend on peptide sequence [49,50]. Computational studies initiated by our group have been designed to complement experimental work on polypeptide multilayer nanofilms. Database development and data mining projects have been undertaken to develop algorithms for sequence design and for extraction of peptide sequence motifs from translated genomic data [46]. Each unique sequence motif is a sort of bsuper amino acid residueQ for the design of peptides for multilayer film fabrication, a type of structural unit of a larger scale structure. Molecular dynamics simulations of designed peptides [50] are based on the premise that physical properties of interpolyelectrolyte complexes will provide insight on interaction between oppositely charged polyelectrolytes in a multilayer film [51]. Technology Specific applications of the polypeptide film platform technology divide broadly into two areas: inside the body and outside the body. Some of the applications mentioned here are being developed by Artificial Cell Technologies, Inc. In vivo applications Polypeptide microcapsules are promising for the development of artificial cells [45,52-54], for example, for oxygen therapeutics or drug delivery (Figure 4). Degradation rate, pH responsiveness, capsule wall density, and permeability can be controlled by amino acid composition, sequence, disulfide cross-linking, method of peptide assembly, and chemical modification of peptides. We have encapsulated glucose oxidase in a polypeptide microcapsule and subsequently demonstrated enzyme activity [53]. We have also encapsulated hemoglobin and demonstrated responsiveness to oxygen tension [54]. Polypeptide microcapsules could also be useful for targeted and sustainedrelease drug delivery. Control over amino acid sequence permits control over immunogenicity. Certain artificial cell applications will require minimized immunotoxicity and controlled immunogenicity. We have outlined a method of amino acid sequence selection from genomic information to accelerate achievement of the design criteria [46]. Maximization of the immune response, however, could also be of therapeutic value, as in a vaccine. The polypeptide capsule platform technology is suited to this application too. The approach features the advantage of simultaneous presentation of multiple antigenic sites and possible encapsulation of antiviral agents. Antimicrobial peptides address the public health concern of growing resistance of bacteria to certain chemical compounds. Such peptides could be integrated with LBL to develop a new class of antimicrobial films, coatings, and related products. Antimicrobial activity is conferred on a poly(vinylidene chloride) copolymer film by incorporation of the antibacterial peptide nisin [55]. Antiadhesive multilayer films made of poly(L-lysine) (PLL) and pegylated-
Fig 4. Polypeptide multilayer film-based artificial cell visualized by confocal fluorescence microscopy. A, Artificial cell. Scale bar = 3.85 m. B, Fluorescence intensity profile. Reprinted with permission from Palath N, Bhad S, Montazeri R, Guidry CA, Haynie DT. Polypeptide multilayer nanofilm artificial red blood cells. J Biomed Mater Res. In press 2006. Copyright 2006 Wiley Interscience.
PLGA (poly(L-glutamic acid)) have been shown to reduce adsorption of serum proteins and Escherichia coli [56], and films functionalized by the insertion of defensin inhibit growth of infectious pathogens, reducing pathological consequences [57]. Vascular implants, catheters, needles, surgical tools, and foods can be protected or preserved by coating them with antimicrobial peptides.
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Ex vivo applications Polypeptide multilayer films can be used to control the cytophilicity of a surface by promoting biocompatibility, biodegradability, cell adhesion, or mechanical strength to withstand long-term cell culture in vitro. This is important for the design of scaffolds for cell growth with control over temperature sensitivity, porosity, and rigidity. A bioactive coating can be prepared by functionalization of a multilayer film to favor proliferation of a unique cell line or a range of cell lines. Adhesion of osteoblasts, chondrosarcoma cells, monocytes, and aortic smooth muscle cells on multilayer films has been reported [58-61]. This research area is of considerable scientific and industrial interest, and the ability to design polypeptides for LBL will promote it. The restructuring of water on hydrophobic surfaces is entropically unfavorable and translates into limited surface wettability. The primary structure of a polypeptide is key to controlling its solubility in an aqueous solvent and the hydrophobicity of a multilayer film. Polypeptide films of controlled wettability are important for a variety of practical applications, for example, contact lens coatings. The operational simplicity and low cost of membranebased separations have attracted much attention for preparative-scale separation of chiral molecules. Modified membranes with amphiphilic side chains exhibit permeation rate ratios above 8 for a-amino acid enantiomers [62], and chiral selectivity and enantioselective binding affinity for d isomers increase with secondary structure [63]. Multilayer films fabricated from charged polypeptides are optically active, permitting analysis by circular dichroism spectrometry, and they exhibit high permeation rates and encouraging selectivity [64]. Substantial advances in separations could be made by optimizing the structure of polypeptides and using polypeptide multilayer films for specific separation characteristics [65]. Summary Nanotechnology has been inspired by knowledge of the structure and function of proteins, nature’s own nanoscale machines. Polypeptide nanofilms combine principles of protein structure and polyelectrolyte film fabrication. These films display a range of properties, depending on design of amino acid sequence, and they are suited to the development of a variety of practical applications, notably medicine. References [1] Haynie DT, Zhang L, Rudra JS, Zhao W, Zhong Y. Polypeptide multilayer films. Biomacromolecules 2005;6:2895 - 913. [2] Haynie DT. Physics of polypeptide multilayer films. J Biomed Res B Appl Biomater 2006;78B:243 - 52. [3] Fruton JS. Proteins, enzymes, genes: the interplay of chemistry and biology. New Haven (Conn)7 Yale University Press; 1999. p. 186 - 9. [4] Voet D, Voet JG. Biochemistry. 2nd ed. New York7 John Wiley; 1995.
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