Data from: Gramicidin and chlorhexidine encapsulated in bicontinuous microemulsions: Antimicrobial activity performance and their impact on self-assembly
Data files
Feb 05, 2025 version files 450.12 KB
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Fig_1.xlsx
91.03 KB
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Fig_2.xlsx
16.24 KB
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Fig_S5.xlsx
314.31 KB
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README.md
6.18 KB
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Table_4.xlsx
22.37 KB
Abstract
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.
README: 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
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 (< 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.
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.