Peptide-based packaging systems show great potential as safer drug delivery systems. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. Here, we describe a set of 15 & 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. These peptides undergo supramolecular self-assembly and form solvent-filled, bilayer delimited spheres with 50–200 nm diameters as confirmed by TEM, STEM and DLS. Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these all-peptide structures are stabilized potentially by both hydrophobic interactions and hydrogen bonds and remain intact at low micromolar concentrations and higher temperatures. A linear peptide lacking the branch point showed no self-assembly properties. We have observed that these peptide vesicles can trap fluorescent dye molecules within their interior and are taken up by N/N 1003A rabbit lens epithelial cells grown in culture. These assemblies are thus potential drug delivery systems that can overcome some of the key limitations of the current packaging systems.

In a recent article (Gudlur et al., (2012) PLOS ONE, 7 (9) e45374) we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These poly-cationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. In this report we examine the lipid-like properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4° C. Having the ability to prepare stable uniform nano-scale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK4-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity.

Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor α 1 -subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45±6 μM from that of 210±70 μM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Towards obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membraneanchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.

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