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Polypeptide Multilayer Nanofilms in Drug Delivery
Polypeptide multilayer nanofilms (PMNs) have recently been developed as multifunctional biomaterials (14). Surprisingly, perhaps, the earliest work on PMNs was for optical coatings (15) and electrochemical electrode coatings (16). It was later proposed to make PMNs out of derivatives of poly(L-lysine) in which "functional" peptides, for example hormone molecules, were grafted onto monomer side chains via the free amino group (17). PMNs are polyelectrolyte multilayer films made largely if not entirely of polypeptides (14).
Polypeptides are interesting for biomaterials development because they are "natural." Peptides constitute half of the dry mass of an organism and they are biodegradable. Moreover, peptides can exhibit extremely high biological specificity and their breakdown products are generally not only benign, but indeed useful as food (18). Furthermore, polypeptides can be designed; an astronomical number of chemically different peptide structures can be realized, and a large number of different peptides can be prepared in huge quantities by various methods (19–20). Peptides designed for PMN fabrication can be protein inspired (21).
Polyelectrolyte multilayer films were first studied systematically by Decher and colleagues (22–23). Polyelectrolytes suitable for multilayer film fabrications can be classified as "strong" or "weak," depending on how they are affected by pH. "Strong" polyelectrolytes show limited dependence on pH, "weak" polyelectrolytes the opposite (24). Polypeptides are weak polyelectrolytes. The isoelectric point of a peptide, the pH at which its net charge equals 0, is a strong function of amino acid composition (25). The author's research group has performed studies to determine how the PMN architecture can be altered with changing pH.
Several charges are needed on a polymer for it to overcome the dissipative tendency of thermal energy and bind to a surface. This resembles the length requirement of an oligonucleotide to prime polymerization of the complementary DNA strand. Relatively little heat is exchanged on binding of a polyelectrolyte to an oppositely charged surface, so ΔG≈–TΔS, where G is free energy, T is temperature (in degrees Kelvin), and S is entropy. In other words, adsorption is largely driven by an increase in entropy. This increase is as a result of the release of small counterions to solution as side-chain charges in the newly adsorbed polymers form ion pairs with side chains of opposite polarity on the surface of the nanofilm. A useful compendium of essays on experimental and theoretical aspects of multilayer films is available (26), as is a brief summary of the physics of nanofilm fabrication (34).
It should be noted that the polyelectrolyte film fabrication method outlined above places no strict geometrical requirement for the substrate. Indeed, these films have been built on substrates of varying surface roughness and shape. Spherical particles in the nanometer to micrometer range have attracted considerable attention (27–28). There are three basic approaches with spherical particles: coating microparticles when the particles are the material of interest (29), trapping a material of interest that has been adsorbed onto template particles (30), and creating hollow spherical shells by dissolving the "dummy" template particles after nanofilm fabrication for subsequent "loading" and "release" of molecules of interest (31–33). All these approaches present interesting possibilities for systemic or local drug delivery.
The work of the author's research group has been comprehensive, encompassing the physics, chemistry, and biology of multilayer nanofilms made of polypeptides (14, 21, 34). With regard to physics, we have investigated how the electric charge, hydrophobic surface, and side-chain hydrogen bonds of peptide structures contribute to PMN assembly, structure, and stability (35). Amino acid side chains have rather different chemical properties. With regard to biology, we have investigated how PMN designs influence cell behavior in vitro and in vivo (36–37). We have been especially interested in the extent to which we can control cellular phenotype in vitro or foreign body tissue reactions in vivo by encoding biological information in peptides for PMN fabrication.
We have also explored coding regions of the human genome for sequences that are suitable for PMN fabrication (38). These sequences will generally have a large number of charged side chains per residue and a large net charge at neutral pH. Both conditions are needed for control over peptide solubility and binding affinity in PMN. Such sequence information is also potentially useful for controlling the immunogenicity of individual peptides and PMNs. PMNs intended for contact with blood, for instance, could be designed using sequence information from known blood proteins, tested experimentally, and modified as needed to achieve the desired properties.
Our chemical inquiries have focused on structural alterations of PMNs with changes in pH. Naturally, pH will influence the architecture and behavior of a PMN. The choice of peptides that go into the PMN therefore must take into account their behavior in environments of various acidities, so that the desired architecture can be achieved. At neutral pH, acidic side chains (for example, the carboxylic acid group of glutamic acid) and basic side chains (for example, the amino group of lysine) are ionized with high probability. The tendency to be ionized at a given pH is even higher inside a PMN. At neutral pH, and in the absence of proteases, a PMN is in essence indefinitely stable (39). In contrast, at acidic pH, glutamic acid side chains become protonated. This raises the electric potential of the entire nanofilm, increases the electrostatic repulsion between positively-charged peptides, and creates a net flow of small negative counterions into the film. The overall effect is gradually to delaminate the PMN. The rate and overall extent of delamination can be controlled by changing the structure of peptides used for PMN fabrication. Control over PMN stability in vivo is potentially useful for delivery of encapsulated drugs.
PMN stability can be controlled in at least two more ways: by incorporating recognition sequences in peptides and by peptide crosslinking (14). Various proteases are known to recognize specific amino acid sequences. Such information could be used to determine the overall susceptibility of a peptide or PMN design to proteolysis and therefore the stability of the peptide or PMN in a specific biochemical environment. Peptides for PMN fabrication could be made of nonnatural amino acids to limit proteolysis.
PMNs can be "functionalized" in various ways (14). We have been interested in how knowledge of various cell-receptor signals in extracellular matrix proteins could be used to alter the cytophilicity of PMNs (36–37). We have encoded various receptor signals into peptides, incorporated these peptides into PMNs, and then seeded cells on these PMNs in vitro. We have investigated how the resulting films influence cell adhesion, morphology, proliferation, and differentiation. We have found that some cell lines are considerably more sensitive than others to PMN architecture and thickness such as primary cells versus transformed cells. Such knowledge may prove useful when drug makers design applications of the PMN technology.
Cell sensitivity to PMN structure has suggested that PMNs could be useful for drug delivery ex vivo or in vitro in cell and tissue culture, and in vivo in localized delivery from an implantable medical device (14, 37). The development of ex vivo methods will be crucial for the future of stem cell therapeutics and regenerative medicine. Localized delivery of drugs from implant devices may increase the usefulness of implants such as glucose sensors.
In conclusion, substantial progress has been made in the development of the PMN platform technology in the past few years. From multiple angles the prospects for commercializing the technology look good. The technology is promising for biomaterials development, and it has good potential to influence development of a variety of approaches to drug delivery. The technology could also be useful for development of multifunctional biomaterials. For instance, a variety of bioactive peptides could be used simultaneously to functionalize a PMN for different purposes, or various drugs could be encapsulated simultaneously for co-delivery. It is possible that other basic themes for technology development have yet to be identified. It is certain in any case that many variations on the known themes wait to be explored.
Donald T. Haynie, PhD , is vice-president of research and chief scientific officer at Artificial Cell Technologies, Inc., 5 Science Park at Yale,
New Haven, CT 06511, tel. 1 203.772.3430, firstname.lastname@example.org
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