DOI: 10.5061/dryad.0000000dh
title: Data from: Gramicidin and chlorhexidine encapsulated in bicontinuous microemulsions: Antimicrobial activity performance and their impact on self-assembly

description: 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.
description: Bicontinuous microemulsion (BME) samples were analyzed at room
 temperature (22±1<sup>o</sup>C) 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, <em>I(Q)</em>, 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,
 <em>drtsans</em>, and accessed through Jupyter notebook
 interface. The resultant <em>I(Q) vs. Q</em> 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 <em>Q</em> range of 0.01 to 0.7
 Å<sup>-1</sup>. 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
 W<sub>IV</sub> system. One hundred µl of washed overnight
 grown bacteria were aseptically spread on the surface of the skin (1.5 x 1
 cm<sup>2</sup>) 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 37<sup>o</sup>C for 24-48 h and then bacterial
 colonies were enumerated. Each experiment was carried out thrice and
 assayed in duplicate.
description: # Data from: Gramicidin and chlorhexidine encapsulated in bicontinuous
 microemulsions: Antimicrobial activity performance and their impact on
 self-assembly
 [https://doi.org/10.5061/dryad.0000000dh](https://doi.org/10.5061/dryad.0000000dh) #### Description of the data and file structure **Fig1.xlsx and FigS5.xlsx** contain small-angle neutron and x-ray scattering (SANS and SAXS, respectively) samples. The former were collected on the Bio-SANS instrument at Oak Ridge National Laboratory (ORNL) during the Spring of 2024 (ORNL IPTS 32383). Fig2.xlsx contains SANS data for the AOT/Polysorbate 95 Winsor-IV system sample series in the presence of gramicidin after subtraction of the Teubner-Strey model (Eq. S1; model parameters given in Tables 2 and S1). Table_4.xlsx contains the skin bioassay raw data that were used to calculate average values and standard deviation for each treatment. ### Files and variables #### File: Fig\_1.xlsx **Description:** Data for Figure 1 of the main paper: SANS data: effect of gramicidin (G) and chlorhexidine (CH) incorporation on the structure of BMEs for the three systems: Polysorbate 80 Winsor-IV system and AOT/Polysorbate 85 Winsor-IV and -III systems for Fig1a, Fig1b, and Fig1c worksheets, respectively. The *Fig1bporod* worksheet contains data for the most of the samples identified in the Fig1b worksheet except for 0.1% gramicidin, but with a truncated *Q* range and more datapoints within the *Q* range present. (The data for Fig1b and Fig1bporod were collected as separate runs on the BioSANS instrument.) The latter data was used for performing Porod analysis (Eq S5). Row 1: ID number. Gramicidin (G) and chlorhexidine (CH) concentrations given in Row 2, ORNL ID numbers (IPTS 32383) given in Row 3, and parameters defined in Row 4. For each worksheet, *Q* (abscissa) and error for *Q* (given in Columns B and C, respectively) are shared by all samples on the given worksheet. The curves contained in the figures are not included herein. They were generated from the Teubner-Strey (TS) model (Eq. S2), with model parameters given in **Tables 2 and S1**, except for Fig 1C, 1% gramicidin, for which the Schultz polydisperse sphere form factor model was applied at low-*Q* (&lt; 0.02 /Å); model parameters given in **Table 3**. (The latter concentration assumes exclusive partitioning of gramicidin to the middle, bicontinuous phase.) **Figure 1** data and curves were multiplied by a constant to improve visualization, with the constants given in the figure caption. Multiplication by a constant was not included in the spreadsheet. Data were also employed to generate the data contained in the Guinier Plot (**Figure S4**), with the background, *b*, subtracted from *I(Q)* for the ordinate. ##### Variables * Q (scattering angle, abscissa parameter; inverse Angstroms) * Error for *Q* * I(Q) (scattered intensity, ordinate parameter; inverse cm) * Error for I(Q) #### File: Fig\_2.xlsx **Description:** Data for **Figure 2** of the main paper: SANS Data for the AOT/Polysorbate 85 Winsor-IV system in the presence of gramicidin, after subtraction of the Teubner-Strey model data, at low-*Q*. Curves represent prolate and oblate ellipsoid models for 0.75% plus 50% and 0.4% gramicidin/0.4% chlorhexidine, respectively. Parameter values for the prolate and oblate ellipsoid models are given in **Table 3**. Row 1: ID number. Gramicidin (G) and chlorhexidine (CH) concentrations given in Row 2, ORNL ID numbers (IPTS 32383) given in Row 3, and parameters defined in Row 4. The curves contained in the figures are not included herein; model type and parameters given in **Table 3**. Figure curves were multiplied by a constant to improve visualization, with the constants given in the figure caption. Multiplication by a constant was not included in the spreadsheet. "N/A" refers to data "not available". ##### Variables * Q (scattering angle, abscissa parameter; inverse Angstroms) * Error for Q * *I(Q)* (scattered intensity, ordinate parameter; inverse cm) * Error for I(Q) #### File: Table\_4.xlsx **Description:** Data for **Table 4**: Recovered bacterial counts (log colony-forming units, log CFU) from artificial skin treated with BMEs containing antimicrobial peptides and antiseptic compared to control treatments (Polysorbate 80 Winsor-IV system at 22C). Row 1: Sample ID; Row 2: microorganism; Row 3: treatment; Row 4: exposure time. Rows 5 and 6 (green color): average and standard deviation values contained in Table 4, calculated through the individual data taken for colonies, given in rows 7-24 (blue color) ##### Variables * Microorganism * Treatment * Bacterial counts #### File: Fig\_S5.xlsx **Description:** Data for **Figure S5**: SAXS data: effect of gramicidin (G) and chlorhexidine (CH) incorporation on the structure of BMEs for the Polysorbate 80 Winsor-IV system using D2O and H2O as solvent and AOT/Polysorbate 85 Winsor-IV system (100% H2O as solvent) for *FigS5a*, *FigS5b*, and *FigS5c* worksheets, respectively. Row 1: ID number. Gramicidin (G) and chlorhexidine (CH) concentrations given in Row 2, parameters defined in Row 3, and data in Rows 4-984. For each worksheet, *Q* (abscissa) (given in Column B, is shared by all samples on the given worksheet. The curves contained in the figures are not included herein. They were generated from the Teubner-Strey (TS) model (Eq. S1), with model parameters given in **Table S2**. **Figure S5** data and curves were multiplied by a constant to improve visualization, with the constants given in the figure caption. Multiplication by a constant was not included in the spreadsheet. NOTE: a few values of I(Q) were negative, which occurred because of scatter in the data and from subtraction of the baseline and empty SAXS capillary (red font). These values should not be used in any subsequent workup, such as plotting or model fitting. "N/A" refers to data "not available". ##### Variables * Q (scattering angle, abscissa parameter; inverse Angstroms) * I(Q) (scattered intensity, ordinate parameter; inverse cm) * Error for I(Q) ## Code/software All data uploaded to this repository were prepared using Microsoft Excel.