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.
Figure 3
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The amino acid cysteine has a highly reactive thiol group in its side chain. Under oxidizing conditions, two cysteine side
chains will form a disulfide crosslink, whereas under reducing conditions a disulfide bond will break (25). Disulfide bonds
are therefore a way of controlling PMN stability (14, 40–41). Thiol reactivity is potentially useful for drug delivery from
a PMN in at least two ways. In one approach, bioactive molecules are trapped within a disulfide crosslinked film under oxidizing
conditions, for instance the extracellular environment (see Figure 3). The crosslinks are severed when the PMN enters a reducing
environment, as can be found, for example, inside cellular endosomes or lysosomes. The more acidic pH can cause the PMN to
disintegrate, which then releases the encapsulated bioactive molecules. In a second approach (42), bioactive molecules are
conjugated to the peptides of the PMN by disulfide bonds. The bioactive molecules are then released from the PMN under reducing
conditions.
Functional PMNs
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.
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