Modifying Membranotropic Action of Antimicrobial Peptide Gramicidin S by Star-like Polyacrylamide and Lipid Composition of Nanocontainers

Abstract

Gramicidin S (GS), one of the first discovered antimicrobial peptides, still shows strong antibiotic activity after decades of clinical use, with no evidence of resistance. The relatively high hemolytic activity and narrow therapeutic window of GS limit its use in topical applications. Encapsulation and targeted delivery may be the way to develop the internal administration of this drug. The lipid composition of membranes and non-covalent interactions affect GS's affinity for and partitioning into lipid bilayers as monomers or oligomers, which are crucial for GS activity. Using both differential scanning calorimetry (DSC) and FTIR methods, the impact of GS on dipalmitoylphosphatidylcholine (DPPC) membranes was tested. Additionally, the combined effect of GS and cholesterol on membrane characteristics was observed; while dipalmitoylphosphatydylglycerol (DPPG) and cerebrosides did not affect GS binding to DPPC membranes, cholesterol significantly altered the membrane, with 30% mol concentration being most effective in enhancing GS binding. The effect of star-like dextran-polyacrylamide D-g-PAA(PE) on GS binding to the membrane was tested, revealing that it interacted with GS in the membrane and significantly increased the proportion of GS oligomers. Instead, calcium ions affected GS binding to the membrane differently, with independent binding of calcium and GS and no interaction between them. This study shows how GS interactions with lipid membranes can be effectively modulated, potentially leading to new formulations for internal GS administration. Modified liposomes or polymer nanocarriers for targeted GS delivery could be used to treat protein misfolding disorders and inflammatory conditions associated with free-radical processes in cell membranes.

Keywords: Fourier transform infrared spectroscopy FTIR; antimicrobial peptide; differential scanning calorimetry DSC; drug delivery; gramicidin S; lipid membranes; nanocontainers.

Figure 1

Chemical structures of decapeptide gramicidin S (GS), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatydylglycerol (DPPG), cholesterol (Chol), and cerebroside (Cer, R1, and R2 are fatty acid residues) and schematic representation of star-like polyanionic dextran-polyacrylamide copolymer D-g-PAA(PE).

Figure 2

Dependences of melting temperature of DPPC (T_m) on GS concentration (c, mol %). Solid circles are plotted for the initial DSC peak and GS monomer binding; data attributed to the binding of GS oligomers are marked with open circles. Means ± SDs for 3–5 independent preparations are shown.

Figure 3

Effect of Chol content on thermodynamic parameters of GS binding with the DPPC membrane. The peak temperatures of Chol-enriched (lower four points with correspondent approximate curve, marked T_m*) and Chol-depleted lipid phases (upper five points with correspondent approximate curve, marked T_m) are plotted against Chol concentration in the DPPC membrane containing GS at 5 mol %. Data from one representative experiment out of three independent preparations.

Figure 4

Values of enthalpies of DSC peaks of DPPC membrane (showed as “membrane”) in the presence of GS and/or polymer D-g-PAA(PE) (showed as “polymer”). Empty columns correspond to the enthalpies of GS “oligomer” peaks. Data are shown for GS content 5 mol %. Means ± SDs of 3–5 independent preparations are shown. Significant changes with p < 0.05 are indicated with * over solid lines for the effect of GS oligomer binding and over dotted line for the effect of D-g-PAA(PE) polymer.

Figure 5

Dependences of T_m (panel a) and T_m* (panel b) on GS content in DPPC membranes prepared on water subphase (■), 100 мM CaCl₂ (●), and 200 мM CaCl₂ (▲).