Evaluation of Quatsome Morphology, Composition, and Stability for Pseudomonas aeruginosa Biofilm Eradication.

Biofilm infections are a major cause of food poisoning and hospital-acquired infections. Quaternary ammonium compounds are a group of effective disinfectants widely used in industry and households, yet their efficacy is lessened when used as antibiofilm agents compared to that against planktonic bacteria. It is therefore necessary to identify alternative formulations of quaternary ammonium compounds to achieve an effective biofilm dispersal. Quaternary ammonium amphiphiles can form vesicular structures termed "quatsomes" in the presence of cholesterol. In addition to their intrinsic antimicrobial properties, quatsomes can also be used for the delivery of other types of antibiotics or biomarkers. In this study, quatsomes were prepared from binary mixtures of cholesterol and mono- or dialkyl-quaternary ammonium compounds; then, the integrity and stability of their vesicular structure were assessed and related to monomer chain number and chain length. The quatsomes were used to treat Pseudomonas aeruginosa biofilms, showing effective antibiofilm abilities comparable to those of their monomers. A systematic liquid chromatography-mass spectrometry method for quantifying quatsome vesicle components was also developed and used to establish the significance of cholesterol in the quatsome self-assembly processes.

Formation of Oxidation- and Acid-Sensitive Assemblies from Sterols and a Quaternary Ammonium Ferrocene Derivative: Quatsome- and Onion-like Vesicles and Extended Nanoribbons.

Quatsomes are a class of nonphospholipid vesicles in which bilayers are formed from mixtures of quaternary ammonium (QA) amphiphiles and sterols. We describe the formation of oxidation and acid-sensitive quatsome-like vesicles and other bilayer assemblies from mixtures of a ferrocenylated QA amphiphile (FTDMA) and several cholesterol derivatives. The influence of the sterol and the preparation method (extrusion or probe sonication) on the stability and morphology of the resulting vesicles is explored; a variety of structures can be obtained from small (ca. 30 nm) spherical unilamellar and oligolamellar quatsome-like vesicles to large (ca. 200 nm) multilamellar onion-like vesicles to extended nanoribbons many micrometers long. FTDMA-sterol vesicles undergo drastic shifts in vesicle and membrane structure when treated with a chemical oxidant (Frémy's salt), a feature previously observed in liposomes containing FTDMA and now confirmed in nonphospholipid vesicles. Size distributions of spherical quatsome-like vesicles obtained from cryo-TEM are examined to estimate the membrane bending rigidity, and a hypothesis is presented to explain the underlying mechanism of the profound membrane alterations observed as a consequence of ferrocene oxidation.

EDTA-Gradient Loading of Doxorubicin into Ferrocene-Containing Liposomes: Effect of Lipid Composition and Visualization of Triggered Release by Cryo-TEM.

Efficient delivery of therapeutic compounds to their sites of action has been a ubiquitous concern throughout the history of human medicine. The tumor microenvironment offers a variety of endogenous stimuli that may be exploited by a responsive nanocarrier, including heterogeneities in redox potential. In the early stages of the design of such responsive delivery systems, it is necessary to develop a comprehensive understanding of the biophysical mechanism by which the stimulus response occurs, as well as how the response may change from the inclusion of cargo compounds. We describe the optimization of lipid compositions for liposomes containing synthetic ferrocene-appended lipids to achieve highly efficient loading of doxorubicin via an ethylenediaminetetraacetic acid (EDTA) gradient. Liposomes containing ferrocenylated phospholipid are shown to be unstable to the loading conditions, while those including a ferrocenylated alkylammonium amphiphile obtain a near-quantitative loading efficiency. Calorimetric studies demonstrate that this instability is the consequence of the relative degree of lipid hydrolysis that occurs under the acidic loading conditions. Drug-loaded liposomes of the optimized composition are studied by cryo-TEM; the presence of doxorubicin aggregates is observed inside vesicles, and doxorubicin release, as well as the changes in membrane structure resulting from oxidant treatment, is also observed by cryogenic transmission electron microscopy (cryo-TEM). These results further demonstrate the potential of ferrocene lipids in the design of redox-responsive nanocarriers and begin to explore their possible role as probes of membrane dynamics.

Redox-Triggered Disassembly of Nanosized Liposomes Containing Ferrocene-Appended Amphiphiles.

We report a redox-responsive liposomal system capable of oxidatively triggered disassembly. We describe the synthesis, electrochemical characterization, and incorporation into vesicles of an alternative redox lipid with significantly improved synthetic efficiency and scalability compared to a ferrocene-appended phospholipid previously employed by our group in giant vesicles. The redox-triggered disassembly of both redox lipids is examined in nanosized liposomes as well as the influence of cholesterol mole fraction on liposome disassembly and suitability of various chemical oxidants for  in vitro disassembly experiments. Electronic structure density functional theory calculations of membrane-embedded ferrocenes are provided to characterize the role of charge redistribution in the initial stages of the disassembly process.