Detection of Cell Membrane Interactions with Lipid-functionalized Single-walled Carbon Nanotubes

Abstract

Membrane-active molecules are of great importance to drug delivery and antimicrobials applications. While the ability to prototype new membrane-active molecules has improved greatly with the advent of automated chemistries and rapid biomolecule expression techniques, testing methods are still limited by throughput, cost, and modularity. Existing methods suffer from feasibility constraints of working with pathogenic, living cells and by intrinsic limitations of model systems. Herein, we demonstrate an abiotic sensor that uses semiconducting single-walled carbon nanotubes (SWNT) as near infrared fluorescent transducers to report membrane interactions. This sensor is comprised of SWNT aqueously suspended in a phospholipid monolayer; these SWNT probes are very sensitive to solvent access (changes in permittivity) and thus report morphological changes to the membrane by modulation of fluorescent signal where binding and disruption are reported as brightening and attenuation, respectively. This mechanism is first demonstrated with chemical and physical membrane-disruptive agents including ethanol, sodium dodecyl sulfate, and application of electrical pulses. Known cell-penetrating and antimicrobial peptides are then used to demonstrate how the dynamic response of these sensors can be deconvoluted to evaluate different, parallel mechanisms of interaction. Lastly, SWNT functionalized in several different bacterial lipopolysaccharides (P aeruginosa, K pneumoniae, and E coli) are used to evaluate a panel of known membrane-disrupting antimicrobials to demonstrate that drug selectivity can be assessed by suspension of SWNT with different membrane materials.

Characterizing the Interactions of Cell Membrane-Disrupting Peptides with Lipid-Functionalized Single-Walled Carbon Nanotube Systems for Antimicrobial Screening

“…Four systems were built with POPC lipids placed in cylindrical arrangement around SWNTs (Figure S1, Table S1). These four systems contained 9:1, 15:1, 20:1 and 30:1 POPC to SWNT mass density ratios, where 30:1 ratio corresponds to the mass density ratio used in previous experimental preparations 16 . Modeled systems contained single SWNTs because POPC-SWNT conjugates are presumed to emit light and thus perform sensing only when containing single SWNTs, whereas conjugates with bundled SWNTs have quenched emission [47][48][49] .…”

“…Interactions of Peptides with POPC Bilayers. Phospholipid-functionalized SWNTs could be a novel nanotechnology tool capable of discovering new molecules that disrupt membranes 16 . To have this capability, the optical signal emitted by SWNTs in the presence of candidate molecules should be proportional to the structural disruption of the lipid corona on SWNT and, importantly, to the disruption that the candidate molecules cause in the actual membrane bilayers.…”

“…Next, we examined how two of the properties calculated in simulations correlate with the experimental nIR emission change of lipid-wrapped SWNTs after addition of disruptor molecules 16 . Figure S6 shows the correlations between the experimentally measured SWNT nIR emission change and two properties from simulations, namely, the fractions of time that disruptor molecules bind to POPC-SWNT, and the water-SWNT contact area change due to addition of disruptors.…”

“…In case of the POPC-wrapped SWNTs binding to antimicrobial peptides, the addition of small concentrations of these peptides was shown to primarily decrease the intensity of the SWNT emission 16 . While the optical signals of lipid-wrapped SWNTs in response to addition of antimicrobial peptides were determined experimentally 16 , no structural studies of these systems have been reported to date, and therefore, the actual structural changes in lipid corona that induce the decrease in the SWNT fluorescence emission intensity are unknown. Furthermore, it is unclear if the peptide-induced structural changes in lipid-SWNTs are qualitatively and quantitatively related to the structural changes these peptides make in bilayer membranes made of the same lipids.…”

“…New technologies that address some of these limitations would be helpful and could become a part of the toolbox for antimicrobial discovery efforts. Recently, an abiotic system based on lipid-wrapped single-walled carbon nanotube (SWNT) conjugates has been proposed and investigated as a new optical sensing platform for screening of antimicrobial compounds 16 . This platform uses SWNTs as near infrared (nIR) fluorescent transducers to report lipid interactions with antimicrobial compounds that take place on the SWNT surface.…”