(502a) Effects of Synthetic Linear and Cyclic Peptide Analogs On Liposome Phase Behavior, Transport, and Morphology

Cell penetrating peptides (CPP) are small cationic peptides that possess the ability to traverse plasma membranes, which makes them candidates as molecular transporters and therapeutic agents. Many CPPs exhibit antimicrobial activity and can disrupt bacterial membranes that are anionic in nature. We have recently reported a series of short novel linear peptide analogues (LPAs), synthesized as Arg-Cn-Arg-Cn-Lys where Cn represents the alkyl linkage separating the cationic residues (n= 4 to 11), as molecular transporters for drug delivery (Ye et al., J Med. Chem. 50, 2007, 3604). To understand LPA and newly-synthesized cyclic peptide analogs (CPA) membrane interaction mechanisms and guide the design of new peptides, we are examining how these peptides disrupt lipid phase behavior, alter membrane permeability, and restructure zwitterionic (DPPC) and mixed zwitterionic/anionic liposomes (DPPC/DPPG and DPPC/DPPS). Isothermal titration calorimetry (ITC) is used to examine the binding of the peptides with the bilayers. For the series, LPA-C4, C7 and C11, LPA-C11 exhibits strong binding to fluid DPPC and DPPC/DPPG bilayers and endothermic effects signifying lipid disordering and distribution, whereas, LPA C4 and C7 displayed weaker exothermic binding. Similarly, differential scanning calorimetry (DSC) shows a decrease in melting temperature and an elimination of the pre-transition in the lipid bilayers with the addition of LPA-C11, while LPA-C4 and C7 showed little effect. While all LPAs bound to the lipids via electrostatic attractions between the cationic residues and the lipid phosphate groups (DPPC, DPPG, and DPPS) and charge attraction between the cationic residues and glycerol headgroups (DPPG and DPPS), our calorimetric studies show that the length of the hydrophobic linkage controls binding and lipid perturbation. Carboxyflurescein permeability studies were conducted to determine if lipid disruption increased membrane leakage. LPA-C11 revealed highest permeability with anionic lipids, consistent with DSC and binding results. Structural and morphological changes of the liposomes were detected by cryogenic transmission electron microscopy (cryo-TEM). In general, LPA-C4 and C7 led to small changes in liposome structure, while LPA-C11 caused liposome fusion and produced large unenclosed liposomes in DPPC and DPPC/DPPG, , For DPPC/DPPS, the addition of all LPAs caused the morphological change from spherical liposomes to disks.. We attribute this to non-ideal mixing between DPPC and DPPS, which was not observed between DPPC and DPPG. Hence, the LPA peptides can bind tightly and restructure both zwitterionic and zwitterionic/anionic membranes, but lead to significant morphological changes when the membrane contains domains rich in zwitterionic or anionic lipids. Currently we are exploring a variety of cyclic peptides consisting of eight alternating amino acids containing electrostatic and hydrophobic residues. We hypothesize that these cyclic peptides will bind strongly to lipid bilayers and can self-assemble within the membrane to function as membrane-spanning channels or pores. A collaborative investigation on the antimicrobial properties possessed by these linear and cyclic peptide analogs will also be presented.