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 because 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.
Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane ...models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K+ concentration. In combination with microfluidics, we employed our DNA-based K+ sensor for extraction of the permeation coefficient of potassium ions. We measured K+ permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K+ flux in different concentration gradients allows us to estimate the complementary H+ flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K+ transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H+/K+ selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H+/K+ permeability ratio of ∼104.
Antimicrobial peptides (AMPs) are emerging as important players in the fight against antibiotic resistance. In parallel, the field of microfluidics has matured and its benefits are being exploited in ...applications of biomimetics and standardized testing. Membrane models are essential tools extensively utilized in studying the activity and modes of action of AMPs. Here, we describe how the utilization of microfluidic platforms in characterizing membrane active peptides can develop a reliable colorful image that classical techniques have rendered black and white.
Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic ...molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a "memory" to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.
Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane‐targeted ...molecular machineries, many of which operate through energy dissipation. Likewise, man‐made light‐activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well‐defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor‐induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor‐induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out‐of‐equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.
Explore the nuanced relationship between light‐driven molecular motors and lipid membranes in this research. Delve into the continuous interplay, observing how these motors, akin to dynamic springs fueled by light, orchestrate subtle yet fascinating shape transitions in membranes. Observe how these out‐of‐equilibrium systems can modulate area expansion through their action in lipid membranes.
Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a ...detailed understanding of their structure–activity relationships. However, classical characterization assays often lack the ability to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations and related the observed modes to the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner and for assigning specific modes of action to the corresponding antimicrobial mechanisms.
Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit ...multiple targets in bacteria starting with their most exposed regions-their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide's physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.
Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components ...and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residueswhich range in size and chemical compositionwith a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.
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.
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