Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones

Abstract

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug – we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.

On-chip density-based purification of liposomes

“…For example, a "feeder channel" that flows trigger molecules (e.g., adenosine triphosphate as an energy source or melittin peptides to form a membrane pore) can be fused to the post-hole channel in order to trigger a specific response in the liposomes. Such a system would be ideal, for example, to study the dynamics of proteins crucially involved in the bacterial cell division (FtsZ, 5 MinCDE 56 ), antibiotic transport across the lipid bilayer, 12 or attempting a controlled growth of liposomes via external supply of lipids. 6 We believe that the density-based separation of 1-octanol droplets from liposomes, presented here, will make OLA more user-friendly and accessible for a broad range of experiments.…”

“…[1][2][3][4][5][6][7][8][9] Liposomes have already found applications in the medical field, acting as carriers for drug and gene delivery, 10,11 and in bioanalytical systems, especially in integrated microfluidic devices. 12,13 We recently reported a new microfluidic method, Octanol-assisted Liposome Assembly (OLA), to produce liposomes in a controlled and efficient manner. 14 There are a number of important advantages of OLA over existing bulk methods (extrusion, 15 hydration, 16 and electroformation 17 ) and microfluidic methods: [18][19][20][21][22][23] (1) The use of a biocompatible organic solvent that enables an efficient and quick de-wetting of the double-emulsion droplets within a few minutes.…”

Antibiotic transport kinetics in Gram-negative bacteria revealed via single-cell uptake analysis and mathematical modelling

“…As a first approximation, we assume that diffusion through the LPS-lipid bilayer is negligible ( 4~0 ) in comparison to porin-mediated transport (3). Furthermore, we postulate that ofloxacin molecules, like other fluoroquinolones (18,19), diffuse across the inner membrane lipid bilayer (rate constant 3 ) and that the efflux of drug molecules from the periplasm to the external medium follows 15 Michaelis-Menten kinetics with maximal rate and Michaelis constant (14). Finally, parameters , and denote the volumes of the outer membrane, periplasm and cytoplasm, respectively (Table S2).…”

“…The camera was controlled using Manager 1.4 (30). We chose to always dissolve the ofloxacin in PBS to ensure that the pH conditions remained 35 uniform during drug exposure across all experiments and metabolic conditions; it is well known that pH regulates the charge state of fluoroquinolones, which affects their membrane permeabilities (18,19). The LED was triggered by the camera to ensure that the cells were only exposed to the excitation light during image acquisition.…”

Characterization of lipid composition and diffusivity in OLA generated vesicles

“…Unilamellar liposomes of several microns in diameter, termed Giant Unilamellar Vesicles (GUVs), are especially widespread in the fields of biophysics and synthetic biology, where they are used in the bottom up construction of synthetic cells [ 4 , 5 ]. GUVs have also been used as model membranes for drug transport studies across lipids [ 6 , 7 ] and for studying antibiotic transport facilitated by bacterial porins [ 8 ]. Others have used GUVs as micro containers for chemical reactions [ 9 ].…”