Development of a bis-pyrene phospholipid probe for fluorometric detection of phospholipase A2 inhibition.

In this work, we report the design, synthesis, and application of a bis-pyrene phospholipid probe for detection of phospholipase A2 action through changes in pyrene monomer and excimer fluorescence intensities. Continuous fluorometric assays enabled detection of the activities of multiple PLA2 enzymes as well as the decrease in catalysis by PLA2 from honey bee venom caused by the inhibitor p-bromo phenacylbromide. Thin-layer chromatography and mass spectrometry analysis were also used to validate probe hydrolysis by PLA2. Mass spectrometry data also supported cleavage of the probe by phospholipase C and D enzymes, although changes in fluorescence were not observed in these cases. Nevertheless, the bis-pyrene phospholipid probe developed in this work is effective for detection of PLA2 enzyme activity through an assay that enables screening for inhibitor development.

Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism.

We report the use of clickable monoacylglycerol (MAG) analogs as probes for the labeling of glycerolipids during lipid metabolism. Incorporation of azide tags onto the glycerol region was pursued to develop probes that would label glycerolipids, in which the click tag would not be removed through processes including acyl chain and headgroup remodeling. Analysis of clickable MAG probes containing acyl chains of different length resulted in widely variable cell imaging and cytotoxicity profiles. Based on these results, we focused on a probe bearing a short acyl chain (C4 -MAG-N3 ) that was found to infiltrate natural lipid biosynthetic pathways to produce click-tagged versions of both neutral and phospholipid products. Alternatively, strategic blocking of the glycerol sn-3 position in probe C4 -MEG-N3 served to deactivate phospholipid tagging and focus labeling on neutral lipids. This work shows that lipid metabolic labeling profiles can be tuned based on probe structures and provides valuable tools for evaluating alterations to lipid metabolism in cells.

Copper-responsive liposomes for triggered cargo release employing a picolinamide-lipid conjugate.

In this work, we report triggered content release from liposomes brought about by copper chelation to a synthetic lipid switch containing a picolinamide headgroup. Fluorescence-based dye-leakage assays showcase release of carboxyfluorescein dye cargo upon copper treatment and control of liposomal release based on copper abundance. Our results additionally show that this platform is selective for copper and is accompanied by significant changes to liposome properties upon treatment with this ion.

Metabolite-Responsive Liposomes Employing Synthetic Lipid Switches Driven by Molecular Recognition Principles.

The ability to exert control over lipid properties, including structure, charge, function, and self-assembly characteristics is a powerful tool that can be implemented to achieve a wide range of biomedical applications. Examples in this arena include the development of caged lipids for controlled activation of signaling properties, metabolic labeling strategies for tracking lipid biosynthesis, lipid activity probes for identifying cognate binding partners, approaches for in situ membrane assembly, and liposome triggered release strategies. In this Account, we describe recent advancements in the latter area entailing the development of stimuli-responsive liposomes through programmable changes to lipid self-assembly properties, which can be harnessed to drive the release of encapsulated contents toward applications including drug delivery. We will focus on an emerging paradigm involving liposomal platforms that are sensitized toward chemical agents ranging from metal cations to small organic molecules that exhibit dysregulation in disease states. This has been achieved by developing synthetic lipid switches that are designed to undergo programmed conformational changes upon the recognition of specific target analytes. These structural alterations are leveraged to perturb the packing of lipids within the membrane and thereby drive the release of encapsulated contents.We provide an overview of the inspiration, design, and characterization of liposomes that selectively respond to wide-ranging target analytes. This series of studies began with the development of calcium-responsive liposomes utilizing a lipid switch inspired by sensors including indo-1. Following this successful demonstration, we next showed that the selectivity of the lipid switch could be altered among different metal cations by producing a liposomal platform for which release is induced through zinc binding. Our next goal was to develop metabolite-responsive liposomes in which switching is driven by molecular recognition events involving phosphorylated small molecules. In this work, screening of lipid switches designed to interact with phosphorylated metabolites led to the identification of liposomal formulations that selectivity release contents in the presence of adenosine triphosphate (ATP). Finally, we were able to modulate the metabolite selectivity by rationally designing a modified lipid switch structure that is activated through complexation of inositol-(1,4,5)-trisphosphate (IP3). These projects show the progression of our approaches for liposome release triggered by molecular recognition principles, building from ion-responsive lipid switches to structures that are activated by small molecules. These "smart" liposomal platforms provide an important addition to the toolbox for controlled cargo release since they respond to ions or small molecules that are commonly overproduced by diseased cells.

Zinc Triggered Release of Encapsulated Cargo from Liposomes via a Synthetic Lipid Switch.

Liposomes are effective nanocarriers due to their ability to encapsulate and deliver a wide variety of therapeutics. However, therapeutic potential would be improved by enhanced control over the release of drug cargo. Zinc ions provide exciting new targets for stimuli-responsive lipid design due to their overly abundant concentrations associated with diseased cells. Herein, we report zinc-triggered release of liposomal contents exploiting synthetic lipid switches designed to undergo conformational changes in the presence of this ion. Initially, Nile red leakage assays were conducted that validated successful dose-dependent triggering of release using zinc-responsive lipids (ZRLs). In addition, dynamic light scattering and confocal microscopy experiments showed that zinc treatment led to morphological changes in lipid nanoparticles only when ZRLs were present in formulations. Next, zinc-binding experiments conducted in a solution (NMR, MS) or membrane (zeta potential) context confirmed ZRL-Zn complexation. Finally, polar cargo release from liposomes was achieved. The results from these wide-ranging experiments using four different compounds indicated that zinc-responsive properties varied based on ZRL structure, providing insights into the structural requirements for activity. This work has established zinc-responsive liposomal platforms toward the development of clinical triggered release formulations.

Demolish and Rebuild: Controlling Lipid Self-Assembly toward Triggered Release and Artificial Cells.

The ability to modulate the structures of lipid membranes, predicated on our nuanced understanding of the properties that drive and alter lipid self-assembly, has opened up many exciting biological applications. In this Perspective, we focus on two endeavors in which the same principles are invoked to achieve completely opposite results. On one hand, controlled liposome decomposition enables triggered release of encapsulated cargo through the development of synthetic lipid switches that perturb lipid packing in the presence of disease-associated stimuli. In particular, recent approaches have utilized artificial lipid switches designed to undergo major conformational changes in response to a range of target conditions. On the other end of the spectrum, the ability to drive the in situ formation of lipid bilayer membranes from soluble precursors is an important component in the establishment of artificial cells. This work has culminated in chemoenzymatic strategies that enable lipid manufacturing from simple components. Herein, we describe recent advancements in these two unique undertakings that are linked by their reliance on common principles of lipid self-assembly.