Gramicidin and chlorhexidine encapsulated in bicontinuous microemulsions: Antimicrobial activity performance and their impact on self-assembly

The utility of bicontinuous microemulsions (BMEs) as carriers of the antimicrobial peptide (AMP) gramicidin D and antiseptic chlorhexidine was investigated for possible topical delivery to chronic wounds. The two water-insoluble solutes dissolved in pre-formed one-phase BMEs of Water/ Polysorbate 80/ Limonene/ Ethanol/ Glycerol and Water/ Aerosol-OT (AOT)/ Polysorbate 85/ Isopropyl Myristate and an AOT/ Polysorbate 85 Winsor-III system, achieving gramicidin and chlorhexidine concentrations of 1.0 (wt)% and 0.5% individually and 0.5% and 0.3% in mixtures at 22oC, respectively. Small-angle neutron scattering measurements demonstrated that both solutes decreased surfactant interfacial activity and increased interfacial fluidity for the Polysorbate 80 system. For the AOT/ Polysorbate systems, ellipsoidal aggregates consisting of gramicidin and likely adsorbed surfactant and oil formed, while chlorhexidine enhanced the surface activity of surfactants. According to bioassays performed on artificial skin, the incorporation of melittin, gramicidin, and chlorhexidine enhanced the bioactivity of BMEs for 24 h treatment against relevant antibiotic-resistant bacteria found on skin relative to controls. Yet, BME treatments were less effective than aqueous melittin control, in contrast to well diffusion bioassays performed previously. The results reflect the strong impact of AMPs and antiseptics on BME structure and dynamics and the complexity of formulating BMEs for optimal antimicrobial activity.

Methods
Bicontinuous microemulsion (BME) samples were analyzed at room temperature (22±1oC) for their structure using the Bio-SANS instrument at the High Flux Isotope Reactor Facility at Oak Ridge National Laboratory, Oak Ridge, TN, USA,  US Department of Energy facility.  (Heller et al., 2014). Instrument settings are given in the main paper. Scattering intensity, I(Q), was obtained by azimuthally averaging the processed two-dimensional images, which were normalized to incident beam monitor counts, and corrected for detector dark current, pixel sensitivity and empty cell background. Reduction of SANS measurements was performed using ORNL-developed reduction software, drtsans, and accessed through Jupyter notebook interface. The resultant I(Q) vs. Q data were analyzed through fitting with a nonlinear general scattering law based on form and structure factors, implemented in a Igor Pro software package prepared by personnel at the Center for Neutron Research of the (U.S.) National Institute of Standards, NCNR. Analysis of SANS data was conducted using the Teubner-Strey (TS) model commonly employed for BMEs. Guinier and Porod analyses were also performed using Microsoft Excel. For the AOT/Polysorbate 85 Winsor-IV BME system, an additional scattering feature was observed in the low-Q region. After subtraction of the TS model, the remaining "excess" data was fit using ellipsoidal or spherical models.

SAXS measurements were carried out at 23º C on a Xeuss 3.0 instrument from Xenocs (Santa Barbara, CA, USA) equipped with D2+ MetalJet X-ray source (Ga Ka, 9.2 keV, wavelength [λ] = 1.341 Å). The instrument measured across a Q range of 0.01 to 0.7 Å-1. A volume of 100 µL of a BME sample was added to a 0.1 mm quartz capillary tube and sealed with epoxy resin. The tubes were placed vertically in the sample holder and aligned perpendicularly to the directions of the x-ray beam in transmission mode.  All data were normalized and reduced according to the manufacturer’s recommendations. Baseline scattering and the scattering contribution of the empty capillary were subtracted from the collected SAXS data.

The skin bioassay described below was applied to BMEs formed by the Polysorbate 80 WIV system. One hundred µl of washed overnight grown bacteria were aseptically spread on the surface of the skin (1.5 x 1 cm2) kept within a sterile petri dish within a Biosafety hood. These samples were then treated with either 100 µl of BME or control solution for a contact time of 10 min or 24 h at room temperature The bacteria were recovered by repeated pipetting using 9.9 ml sterile PBS initially and subsequently ten-fold diluted in sterile PBS, before surface-spread plating on sterile TSA plates. These plates were incubated at 37oC for 24-48 h and then bacterial colonies were enumerated. Each experiment was carried out thrice and assayed in duplicate.