Colloidal Dispersions of Gramicidin D in Water: Preparation, Characterization, and Differential Cytotoxicity

Abstract

Gramicidin D (Gr) is a natural mixture of channel peptides A-C with minor differences in chemical structure, which are able to span cell membranes as dimers. These Gr channels allow single-file diffusion of cations, thereby disrupting the usual ionic balance in biological cells and inducing cell lysis. The microbicidal activity of Gr using different carriers such as bilayer vesicles or bilayer disks, supported bilayers on silica, or polystyrene nanoparticles has been described. Gr antimicrobial activity was found to depend strongly on its formulation. Preliminary description of self-assembled Gr nanoparticles (Gr NPs) by our group showed a superior antimicrobial performance for these Gr self-assembled nanospheres. In this work, we further characterize Gr colloidal dispersions in aqueous solution over a range of micromolar concentrations from turbidimetry, obedience to the Rayleigh law for light scattered by NPs smaller than the wavelength of the incident light, dynamic light scattering to ascertain the reproducibility of physical characteristics of Gr NPs, and effects of Gr NPs on the cell viability of five different mammalian cell lines in culture over a micromolar range of Gr concentrations (0.5-5.0 μM). Thereby, the differential cytotoxicity of Gr NPs is inferred from the comparison between effects on microbial cell viability and mammalian cell viability. The results suggest that the simple and efficacious formulation of Gr NPs obtained directly from Gr self-assembly in aqueous solution deserves to be further exploited, aiming at systemic biomedical uses of Gr in vivo against infectious diseases and cancers.

Figure 1

U.V. spectra for gramicidin D absorbance as a function of the wavelength of the incident light in CH₃OH (a) or TFE (b) over a range of micromolar Gr concentrations. Absorbance at 282 nm/280 nm as a function of Gr concentration yielded linear fittings with regression coefficients (R) equal to 0.97924/0.99389, respectively (Supporting Information S1). The mean molar absorptivity of Gr in methanol and TFE calculated from these linear fittings was 21,340 ± 1170 and 19,100 ± 611 cm^–1·M^–1, respectively (Supporting Information S1).

Figure 2

Photos of Gr colloidal dispersions in water at 5, 10, 20, 30, 40, and 50 μM Gr.

Figure 3

Turbidity for Gr colloidal dispersions in pure water as a function of the wavelength of incident light (λ) (a) or as a function of λ^–4 (b).

Figure 4

Turbidity at 400 nm for Gr colloidal dispersions in pure water as a function of [Gr].

Figure 5

Reproducibility of self-assembled gramicidin D (Gr) dispersions in water over a micromolar range of Gr concentrations for five different experiments from determinations of physical properties such as mean hydrodynamic diameter Dz (a), polydispersity P (b), zeta potential ζ (c), and conductance G (d). Each dispersion contains 1% in volume of the solvent trifluoroethanol (TFE).