Leaflet by Leaflet Synergistic Effects of Antimicrobial Peptides on Bacterial and Mammalian Membrane Models

Antimicrobial peptides (AMPs) offer significant hope in the fight against antibiotic resistance. Operating via a mechanism different from that of antibiotics, they target the microbial membrane and ideally should damage it without impacting mammalian cells. Here, the interactions of two AMPs, magainin 2 and PGLa, and their synergistic effects on bacterial and mammalian membrane models were studied using electrochemical impedance spectroscopy, atomic force microscopy (AFM), and fluorescence correlation spectroscopy. Toroidal pore formation was observed by AFM when the two AMPs were combined, while individually AMP effects were confined to the exterior leaflet of the bacterial membrane analogue. Using microcavity-supported lipid bilayers, the diffusivity of each bilayer leaflet could be studied independently, and we observed that combined, the AMPs penetrate both leaflets of the bacterial model but individually each peptide had a limited impact on the proximal leaflet of the bacterial model. The impact of AMPs on a ternary, mammalian mimetic membrane was much weaker.

S2 the fabrication of gold arrays, the PS microsphere of 1 μm diameter were drop cast into the goldcoated silicon wafers by utilizing the gravity assisted technique to form a highly ordered array.
Then, electrochemical deposition was used to deposit gold onto the electrode around the array to 50% of the sphere height. 3,4 Finally, the top interpore surface of the gold array electrodes were selectively modified with self-assembled monolayer (SAM) of 1 mM 6-mercapto-1-hexanol (MH). This SAM is required to promote bilayer stability. Finally, the PS sphere template was removed by washing with THF and ethanol to yield top surface SAM modified gold arrays. The electrodes are sonicated in buffer and kept in contact with working buffer until use.
To fabricate substrates for microscopy, optically transparent PDMS microcavity arrays were prepared by drop casting 4.6 μm PS microsphere onto a mica sheet. PDMS was poured across mica sheet and curing was completed at 90°C for approximately 1 h until the PDMS becomes hard. The resulting PDMS substrate was peeled off the mica and the resulting microcavity array was sonicated in tetrahydrofuran (THF) for about 15 m to remove the PS sphere templates. Prior to filling the cavities with PBS buffer of pH 7.4, the substrate was treated by oxygen plasma for 5 m to render it hydrophilic and then sonicated in PBS buffer for 15 m.
For both gold and polymer arrays the same bilayer deposition procedure was applied; lipid monolayer transfer was accomplished using Langmuir-Blodgett deposition. Usually, 50 μL of lipids (1 mg/mL in chloroform) was added dropwise onto the subphase (Milli Q water, 18.2 MΩ.cm) at room temperature (20  1C), and chloroform was then evaporated for 7 m. The monolayers were subjected 4 cycles of compression/decompression at a barrier speed of 20 mm/min not exceeding the final surface pressure (Π) of 35 mN m -1 . Then, the monolayer is compressed up to 32 mN m -1 and held for at least 300 seconds before transfer. Lipid monolayers were transferred onto the hydrophilic gold and PDMS substrates vertically up at a speed of 5 and 10 mm/m respectively. For the FCS study, we labelled the lower leaflet as well by DOPE-ATTO532 which is doped into the lipid compositions and injected during the Langmuir-Blodget technique by which monolayer has been formed. Then, the monolayer formed substrates were immersed in a 0.25 mg/ml liposome solution, and the fusion was allowed to occur for 1 to 1.5 h to form bilayers. The substrate was maintained in contact with liposome solution throughout the fusion. The substrate was washed gently with working buffer (PBS pH 7.4) to remove any unfused liposomes and was kept in contact with this buffer while being transferred to the electrochemical chamber. Similarly, for the PDMS substrate, the chamber was washed 3 to 4 times with working S3 buffer (PBS pH 7.4) and was kept in contact with this solution until the experiment was completed to prevent the bilayer drying.

Liposome preparation
Briefly, in this work, liposome fusion was used to form the distal lipid leaflet of microcavity supported lipid bilayers (MSLBs). To prepare the liposomes, stock solutions of all liposome components such as DOPC, brain sphingomyelin and cholesterol 10 mg/ml each, and E. coli extract (1 mg/ml) were prepared in chloroform and stored in sealed glass vials at -20°C.  8.0E5 1.0E6

Electrochemical impedance spectroscopy
The electrochemical measurements were performed with a CH760A potentiostat (CH Instruments, USA). A standard 3-electrode cell comprised of gold microcavity suspended bilayer as a working electrode, an Ag/AgCl (1 M KCl) reference electrode and a platinum wire auxiliary electrode.
However, herein probe free method is used to avoid interactions with drug in main EIS data.

Atomic Force Microscopy:
Lipid bilayers were imaged using atomic force microscopy (AFM) with a Veeco Bioscope II system coupled with Zeiss Axiovert inverted optical microscope IX70 from Nanotech House in   that is, the waist of the exciting laser beam. ω was measured for each excitation wavelength using a reference solution of free dye for which the diffusion coefficient is known. The ω was determined by calibration using reference dyes; ATTO655 (AttoTEC, GmbH) for a 640 nm laser and ATTO532 for a 532 nm laser at 20°C in water.
The lateral diffusion of the free ATTO655 and ATTO532, e.g., unbound dye in PBS buffer, were calculated by fitting the ACFs obtained in for 10 nM solution of each molecule to a 3D model (eq. 3). Equation 3 includes a κ term, which defines the shape of the confocal volume.

Experimental section for Surface Enhanced Raman Spectroscopy (SERS) Measurements
The gold-microcavity-supported lipid bilayers in PBS at pH 7.4 were executed with Raman spectroscopy with a confocal microscope (Horiba, U.K.) and LabSpec software, LabSpec 5.45.09.
A laser with 785 nm was used for excitation through a 600 μm pinhole equipped with a dispersion grating with 1200 grooves/mm. A 50× (air, NA:0.75) objective was used for both excitation and detection. The spectra of lipid bilayer were collected using 1 % laser, 0.1 mW (to avoid any damage to the bilayer) with an exposure time of 4 s and accumulation for 6 s. The instrument was calibrated using a Si (100) wafer calibrated to its standard peak at 520.6 cm −1 and the Rayleigh line before the measurement of the sample. The spectra of the powder form of the lipid were collected using a flat gold substrate with 10% laser power and 100 μm pinhole.