Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.

Transdermal delivery enhanced by antimicrobial peptides.

Magainin antimicrobial peptide has been shown to increase skin permeability by perturbing stratum corneum lipids in the skin. In this study, we hypothesized that skin permeation enhancement depends on peptide structure. We therefore measured skin permeability enhancement by modified magainin derivitives and 20 different antimicrobial peptides in a formulation containing ethanol and N-lauroyl sarcosine (NLS). We found that modification of magainin structure did not improve skin permeability enhancement. Although all six magainin-based peptides had alpha-helical structure and fluidized stratum corneum lipids, only magainin and a Gly-Ala substituted magainin with NLS and ethanol significantly increased skin permeability. Among the 20 antimicrobial peptides, only magainin itself and a Lys-Leu analog peptide showed enhancement. Overall, this is the first study to survey skin permeability enhancement by antimicrobial peptides. We conclude that over the range of conditions studied here, most antimicrobial peptides did not enhance skin permeability and that magainin peptide provided the optimal structure.

Biochemical enhancement of transdermal delivery with magainin peptide: modification of electrostatic interactions by changing pH.

Magainin is a naturally occurring, pore-forming peptide that has recently been shown to increase skin permeability. This study tested the hypothesis that electrostatic forces between magainin peptides and drugs mediate drug transport across the skin. Electrostatic interaction between positively charged magainin and a negatively charged model drug, fluorescein, was attractive at pH 7.4 and resulted in a 35-fold increase in delivery across human epidermis in vitro when formulated with 2% N-lauroylsarcosine in 50% ethanol. Increasing to pH 10 or 11 largely neutralized magainin's charge, which eliminated enhancement due to magainin. Shielding electrostatic interactions with 1-2M NaCl solution similarly eliminated enhancement. Showing the opposite dependence on pH, electrostatic interaction between magainin and a positively charged anti-nausea drug, granisetron, was largely neutralized at pH 10 and resulted in a 92-fold increase in transdermal delivery. Decreasing to pH 5 increased magainin's positive charge, which repelled granisetron and progressively decreased transdermal flux. Circular dichroism analysis, multi-photon microscopy, and FTIR spectroscopy showed no significant pH effect on magainin secondary structure, magainin deposition in stratum corneum, or stratum corneum lipid order, respectively. We conclude that magainin increases transdermal delivery by a mechanism involving electrostatic interaction between magainin peptides and drugs.