An Integrated Microfluidic Platform for Quantifying Drug Permeation across Biomimetic Vesicle Membranes

Abstract

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological pH and salt concentrations, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin and ciprofloxacin across biomimetic liposomes.

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.…”

Biophysical characterization reveals the similarities of liposomes produced using microfluidics and electroformation

“…The dimensions of our junction are scaled up by a factor of 2 compared to the original design to obtain larger liposomes than typically possible with the originally published chip design (14). The scaled-up channels furthermore lead to higher flow rates and higher liposome production rates than the original device (19). However, if operated in a high flow rate regime, the vesicles often only have approx.…”

“…The microfluidic chips were fabricated from polydimethylsiloxane (PDMS) using established photo-and soft lithography techniques (19). A master mold of the structures of the microfluidic chip was produced by spin coating a thin layer of SU-8 2025 (MicroChem, Newton, MA) on a 4inch silicon wafer (University Wafer, Boston, MA).…”

“…Given the advantages of lab-on-chip techniques for drug development studies (17), we integrated OLA with a platform for the characterization of membrane active antimicrobials (18), as well as with a platform for the quantification of drug permeation across membranes (19). In the field of synthetic biology, mechanical division (20) and membrane tension mediated growth (21) of OLA vesicles, as well as coacervate formation within liposomes have been shown (22).…”