Liposomes are nano-sized spherical vesicles composed of an aqueous core surrounded by one (or more) phospholipid bilayer shells. Owing to their high biocompatibility, chemical composition variability, and ease of preparation, as well as their large variety of structural properties, liposomes have been employed in a large variety of nanomedicine and biomedical applications, including nanocarriers for drug delivery, in nutraceutical fields, for immunoassays, clinical diagnostics, tissue engineering, and theranostics formulations. Particularly important is the role of liposomes in drug-delivery applications, as they improve the performance of the encapsulated drugs, reducing side effects and toxicity by enhancing its in vitro- and in vivo-controlled delivery and activity. These applications stimulated a great effort for the scale-up of the formation processes in view of suitable industrial development. Despite the improvements of conventional approaches and the development of novel routes of liposome preparation, their intrinsic sensitivity to mechanical and chemical actions is responsible for some critical issues connected with a limited colloidal stability and reduced entrapment efficiency of cargo molecules. This article analyzes the main features of the formation and fabrication techniques of liposome nanocarriers, with a special focus on the structure, parameters, and the critical factors that influence the development of a suitable and stable formulation. Recent developments and new methods for liposome preparation are also discussed, with the objective of updating the reader and providing future directions for research and development.

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Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane–membrane interactions and possible future research directions.

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions

G protein-coupled receptors (GPCRs) are a large membrane protein family found in higher organisms, including the human body. GPCRs mediate cellular responses to diverse extracellular stimuli and thus control key physiological functions, which makes them important targets for drug design. Signaling by GPCRs is related to the structure and dynamics of these proteins, which are modulated by extrinsic ligands as well as by intracellular binding partners such as G proteins and arrestins. Here, we review some basics of using nuclear magnetic resonance (NMR) spectroscopy in solution for the characterization of GPCR conformations and intermolecular interactions that relate to transmembrane signaling.

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G Protein-coupled Receptor (GPCR) Reconstitution and Labeling for Solution Nuclear Magnetic Resonance (NMR) Studies of the Structural Basis of Transmembrane Signaling

Chronic low-grade vascular inflammation and endothelial dysfunction significantly contribute to the pathogenesis of cardiovascular diseases. In endothelial cells (ECs), anti-inflammatory or pro-inflammatory signaling can be induced by different patterns of the fluid shear stress (SS) exerted by blood flow on ECs. Laminar blood flow with high magnitude is anti-inflammatory, while disturbed flow and laminar flow with low magnitude is pro-inflammatory. Endothelial mechanosensors are the key upstream signaling proteins in SS-induced pro- and anti-inflammatory responses. Being transmembrane proteins, mechanosensors, not only experience fluid SS but also become regulated by the biomechanical properties of the lipid bilayer and the cytoskeleton. We review the apparent effects of pro-inflammatory factors (hypoxia, oxidative stress, hypercholesterolemia, and cytokines) on the biomechanics of the lipid bilayer and the cytoskeleton. An analysis of the available data suggests that the formation of a vicious circle may occur, in which pro-inflammatory cytokines enhance and attenuate SS-induced pro-inflammatory and anti-inflammatory signaling, respectively.

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Endothelial Cell Plasma Membrane Biomechanics Mediates Effects of Pro-Inflammatory Factors on Endothelial Mechanosensors: Vicious Circle Formation in Atherogenic Inflammation

Integral membrane proteins (IMPs) fulfill important physiological functions by providing cell–environment, cell–cell and virus–host communication; nutrients intake; export of toxic compounds out of cells; and more. However, some IMPs have obliterated functions due to polypeptide mutations, modifications in membrane properties and/or other environmental factors—resulting in damaged binding to ligands and the adoption of non-physiological conformations that prevent the protein from returning to its physiological state. Thus, elucidating IMPs’ mechanisms of function and malfunction at the molecular level is important for enhancing our understanding of cell and organism physiology. This understanding also helps pharmaceutical developments for restoring or inhibiting protein activity. To this end, in vitro studies provide invaluable information about IMPs’ structure and the relation between structural dynamics and function. Typically, these studies are conducted on transferred from native membranes to membrane-mimicking nano-platforms (membrane mimetics) purified IMPs. Here, we review the most widely used membrane mimetics in structural and functional studies of IMPs. These membrane mimetics are detergents, liposomes, bicelles, nanodiscs/Lipodisqs, amphipols, and lipidic cubic phases. We also discuss the protocols for IMPs reconstitution in membrane mimetics as well as the applicability of these membrane mimetic-IMP complexes in studies via a variety of biochemical, biophysical, and structural biology techniques.

Akram Bani Ahmad

Papers

Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins