Peptide-Folding Triggered Phase Separation and Lipid Membrane Destabilization in Cholesterol-Rich Lipid Vesicles.

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

Gold Nanocluster Extracellular Vesicle Supraparticles: Self-Assembled Nanostructures for Three-Dimensional Uptake Visualization.

Extracellular vesicles (EVs) are secreted by the vast majority of cells and are being intensively studied due to their emerging involvement in a variety of cellular communication processes. However, the study of their cellular uptake and fate has been hampered by difficulty in imaging EVs against the cellular background. Here, we show that EVs combined with hydrophobic gold nanoclusters (AuNCs) can self-assemble into supraparticles, offering an excellent labeling strategy for high-resolution electron microscopic imaging in vitro. We have tracked and visualized the reuptake of breast cancer cell-derived EV AuNC supraparticles into their parent cells, from early endocytosis to lysosomal degradation, using focused ion beam-scanning electron microscopy (FIB-SEM). The presence of gold within the EVs and lysosomes was confirmed via DF-STEM EDX analysis of lift-out sections. The demonstrated formation of AuNC EV supraparticles will facilitate future applications in EV imaging as well as the EV-assisted cellular delivery of AuNCs.

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Re-Engineering Extracellular Vesicles as Smart Nanoscale Therapeutics.

In the past decade, extracellular vesicles (EVs) have emerged as a key cell-free strategy for the treatment of a range of pathologies, including cancer, myocardial infarction, and inflammatory diseases. Indeed, the field is rapidly transitioning from promising in vitro reports toward in vivo animal models and early clinical studies. These investigations exploit the high physicochemical stability and biocompatibility of EVs as well as their innate capacity to communicate with cells via signal transduction and membrane fusion. This review focuses on methods in which EVs can be chemically or biologically modified to broaden, alter, or enhance their therapeutic capability. We examine two broad strategies, which have been used to introduce a wide range of nanoparticles, reporter systems, targeting peptides, pharmaceutics, and functional RNA molecules. First, we explore how EVs can be modified by manipulating their parent cells, either through genetic or metabolic engineering or by introducing exogenous material that is subsequently incorporated into secreted EVs. Second, we consider how EVs can be directly functionalized using strategies such as hydrophobic insertion, covalent surface chemistry, and membrane permeabilization. We discuss the historical context of each specific technology, present prominent examples, and evaluate the complexities, potential pitfalls, and opportunities presented by different re-engineering strategies.