We quantify the membranolytic activity of antimicrobial peptides on biomimetic lipid vesicles in a multilayer microfluidic total analysis system.

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.

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.

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 μm 2 /s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

DNA nanotechnology makes use of hydrophobically modified constructs to create synthetic membrane protein mimics. However, nucleic acid structures exhibit poor insertion efficiency, leading to a low activity of membrane-spanning DNA protein mimics. It is suggested that non-ionic surfactants improve insertion efficiency, partly by disrupting hydrophobicity-mediated clusters. Here, we employed confocal microscopy and single-molecule transmembrane current measurements to assess the effects of the non-ionic surfactant octylpolyoxyethylene (oPOE) on the clustering behavior and membrane activity of cholesterol-modified DNA nanostructures. Our findings uncover the role of aggregation in preventing bilayer interactions of hydrophobically decorated constructs, and we highlight that premixing DNA structures with the surfactant does not disrupt the cholesterol-mediated aggregates. However, we observed the surfactant’s strong insertion-facilitating effect, particularly when introduced to the sample separately from DNA. Critically, we report a highly efficient membrane-spanning DNA construct from combining a non-aggregating design with the addition of the oPOE surfactant.

A study was undertaken to assess the feasibility of a preoperative fitting test for an implantable hearing aid in a virtual reality (VR) environment. High-resolution spiral computed tomography (CT) of the mastoid bone was performed, and the results of a mastoidectomy were simulated with manual segmentation on a standard medical workstation. CT was also performed on a temporal bone specimen obtained at real mastoidectomy, and the bone margins were segmented automatically with threshold-based techniques. A triangulated surface representation of the bone structures including the mastoid cavity was generated. These data as well as the computer-aided design (CAD) files of the medical devices were transferred into a VR environment. The CAD components of the hearing aid were manipulated to simulate the surgical implantation procedure. Merging CAD data of an implantable hearing aid with CT data of the temporal bone in a VR environment was shown to be a feasible method of providing three-dimensional information for the presurgical determination of fit and mountability. Advances in hardware and software are expected to improve the usability of this method. Although clinical studies are needed, these results may serve as an impetus for exploring the use of low-cost, widely available VR computer equipment in a potentially broad field of clinical applications.

The equilibrium solubility values and dissolution profiles in each of the media (FaSSIF or FaSSIF-V2) produced using methylene chloride and the corresponding SIF Powder are equivalent for the drugs tested representing acid, basic, and neutral compounds. Therefore, it is practical to switch from the more labor-intensive solvent-evaporation method to the use of standardized instant powders for preparing biorelevant media without affecting the solubility and dissolution results.Testing in the two versions of FaSSIF media with different bile salt/lecithin ratios will give an indication on how the drug and formulation could be affected in vivo by the variations in bile salt/lecithin ratio within the range found in human small intestinal fluids.

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 salt and pH levels, 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 across biomimetic liposomes.