Formation of a Fully Anionic Supported Lipid Bilayer to Model Bacterial Inner Membrane for QCM-D Studies.

Supported lipid bilayers (SLBs) on quartz crystals are employed as versatile model systems for studying cell membrane behavior with the use of the highly sensitive technique of quartz crystal microbalance with dissipation monitoring (QCM-D). Since the lipids constituting cell membranes vary from predominantly zwitterionic lipids in mammalian cells to predominantly anionic lipids in the inner membrane of Gram-positive bacteria, the ability to create SLBs of different lipid compositions is essential for representing different cell membranes. While methods to generate stable zwitterionic SLBs and zwitterionic-dominant mixed zwitterionic-anionic SLBs on quartz crystals have been well established, there are no reports of being able to form predominantly or fully anionic SLBs. We describe here a method for forming entirely anionic SLBs by treating the quartz crystal with cationic (3-aminopropyl) trimethoxysilane (APTMS). The formation of the anionic SLB was tracked using QCM-D by monitoring the adsorption of anionic lipid vesicles to a quartz surface and subsequent bilayer formation. Anionic egg L-α-phosphatidylglycerol (PG) vesicles adsorbed on the surface-treated quartz crystal, but did not undergo the vesicle-to-bilayer transition to create an SLB. However, when PG was mixed with 10-40 mole% 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG), the mixed vesicles led to the formation of stable SLBs. The dynamics of SLB formation monitored by QCM-D showed that while SLB formation by zwitterionic lipids followed a two-step process of vesicle adsorption followed by the breakdown of the adsorbed vesicles (which in turn is a result of multiple events) to create the SLB, the PG/LPG mixed vesicles ruptured immediately on contacting the quartz surface resulting in a one-step process of SLB formation. The QCM-D data also enabled the quantitative characterization of the SLB by allowing estimation of the lipid surface density as well as the thickness of the hydrophobic region of the SLB. These fully anionic SLBs are valuable model systems to conduct QCM-D studies of the interactions of extraneous substances such as antimicrobial peptides and nanoparticles with Gram-positive bacterial membranes.

Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers.

Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP-membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications.

Color Space Transformation-Based Algorithm for Evaluation of Thermochromic Behavior of Cholesteric Liquid Crystals Using Polarized Light Microscopy.

Cholesteryl ester liquid crystals exhibit thermochromic properties related to the existence of a twisted nematic phase. When used in applications such as thermal mapping, a color change is often monitored by video cameras. Thus, quantitative methods to evaluate thermochromic behavior (e.g., blue-start, red-start, red-end, color play and bandwidth) from video analysis are desirable. However, obtaining quantitative color measurements from digital images remains a significant technical challenge, especially for highly reflective samples such as liquid crystals (for which ultraviolet-visible (UV-vis) reflectance spectroscopy is typically used). We developed a method to determine thermochromic properties from videos of liquid crystal cooling under polarized light microscopy. We relate observed color transitions to quantifiable changes in the cumulative color difference in the International Commission on Illumination (CIE) L*a*b* color space and validate this method with UV-vis reflectance spectroscopy. The measured thermochromic behavior and associated measurement uncertainties (coefficient of variations) were comparable to UV-vis reflectance measurements.

Thermochromic Fibers via Electrospinning.

Cholesteryl ester liquid crystals exhibit thermochromic properties related to the existence of a twisted nematic phase. We formulate ternary mixtures of cholesteryl benzoate (CB), cholesteryl pelargonate (CP), and cholesteryl oleyl carbonate (COC) to achieve thermochromic behavior. We aim to achieve thermochromic fibers by incorporating the liquid crystal formulations into electrospun fibers. Two methods of incorporating the liquid crystal (LC) are compared: (1) blend electrospinning and (2) coaxial electrospinning using the same solvent system for the liquid crystal. For blend electrospinning, intermolecular interactions seem to be important in facilitating fiber formation since addition of LC can suppress bead formation. Coaxial electrospinning produces fibers with higher nominal fiber production rates (g/hr) and with higher nominal LC content in the fiber (wt. LC/wt. polymer assuming all of the solvent evaporates) but larger fiber size distributions as quantified by the coefficient of variation in fiber diameter than blend electrospinning with a single nozzle. Importantly, our proof-of-concept experiments demonstrate that coaxially electrospinning with LC and solvent in the core preserves the thermochromic properties of the LC so that thermochromic fibers are achieved.