Lipoprotein particles exhibit distinct mechanical properties

Lipoproteins (LPs) are micelle-like structures with a similar size to extracellular vesicles (EVs) and are therefore often co-isolated, as intensively discussed within the EV community. LPs from human blood plasma are of particular interest as they are responsible for the deposition of cholesterol ester and other fats in the artery, causing lesions, and eventually atherosclerosis. Plasma lipoproteins can be divided according to their size, density and composition into chylomicrons (CM), very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Here, we use atomic force microscopy for mechanical characterization of LPs. We show that the nanoindentation approach used for EV analysis can also be used to characterize LPs, revealing specific differences between some of the particles. Comparing LPs with each other, LDL exhibit a higher bending modulus as compared to CM and VLDL, which is likely related to differences in cholesterol and apolipoproteins. Furthermore, CM typically collapse on the surface after indentation and HDL exhibit a very low height after surface adhesion both being indications for the presence of LPs in an EV sample. Our analysis provides new systematic insights into the mechanical characteristics of LPs

Active probing of the mechanical properties of biological and synthetic vesicles

BACKGROUND: The interest in mechanics of synthetic and biological vesicles has been continuously growing during the last decades. Liposomes serve as model systems for investigating fundamental membrane processes and properties. More recently, extracellular vesicles (EVs) have been investigated mechanically as well. EVs are widely studied in fundamental and applied sciences, but their material properties remained elusive until recently. Elucidating the mechanical properties of vesicles is essential to unveil the mechanisms behind a variety of biological processes, e.g. budding, vesiculation and cellular uptake mechanisms. SCOPE OF REVIEW: The importance of mechanobiology for studies of vesicles and membranes is discussed, as well as the different available techniques to probe their mechanical properties. In particular, the mechanics of vesicles and membranes as obtained by nanoindentation, micropipette aspiration, optical tweezers, electrodeformation and electroporation experiments is addressed. MAJOR CONCLUSIONS: EVs and liposomes possess an astonishing rich, diverse behavior. To better understand their properties, and for optimization of their applications in nanotechnology, an improved understanding of their mechanical properties is needed. Depending on the size of the vesicles and the specific scientific question, different techniques can be chosen for their mechanical characterization. GENERAL SIGNIFICANCE: Understanding the mechanical properties of vesicles is necessary to gain deeper insight in the fundamental biological mechanisms involved in vesicle generation and cellular uptake. This furthermore facilitates technological applications such as using vesicles as targeted drug delivery vehicles. Liposome studies provide insight into fundamental membrane processes and properties, whereas the role and functioning of EVs in biology and medicine is increasingly elucidated

Membrane vesicles: Examination of biophysical properties with atomic force microscopy

Extracellular vesicles (EVs) are not only intensively studied to increase our fundamental knowledge on their functioning, but also for diagnosis, therapeutics and drug delivery purposes. To improve the current and potential applications of EVs, a fundamental understanding of their stability, structure, and function is crucial. Such studies can be conducted at the single particle level to gain biological and physical information about the vesicles and the particle to particle variability. A suitable technique to investigate EVs under near- to physiological conditions is atomic force microscopy (AFM). Operated in liquid, it provides images of the EVs while mechanical properties of the particles can be obtained as well. Here we present our approach and the latest results in studying the structure and mechanics of these particles

Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol

Both natural as well as artificial vesicles are of tremendous interest in biology and nanomedicine. Small vesicles (<200 nm) perform essential functions in cell biology and artificial vesicles (liposomes) are used as drug delivery vehicles. Atomic Force Microscopy (AFM) is a powerful technique to study the structural properties of these vesicles. AFM is a well-established technique for imaging at nanometer resolution and for mechanical measurements under physiological conditions. Here, we describe the procedure of AFM imaging and force spectroscopy on small vesicles. We discuss how to image vesicles with minimal structural disturbance, and how to analyze the data for accurate size and shape measurements. In addition, we describe the procedure for performing nanoindentations on vesicles and the subsequent data analysis including mechanical models used for data interpretation

Nanomechanical probing of extracellular vesicles, liposomes and lipoproteins

Mechanical characterization involves testing the hardness of various materials ranging from metals, plastics and composites to biological materials by application and measurement of load and deformation. While most of the above-mentioned materials are of larger sizes, biological systems often are submicrometer-sized, i.e. extracellular vesicles (EVs). EVs are small membrane-enclosed spheres secreted by cells and they are present in most of human body fluids. Despite their known roles, among others, in the creation of metastasis-favoring environments, and in intercellular communication, the knowledge about the mechanical properties of EVs is fairly limited, mainly because of their small size. Atomic force microscopy (AFM)-based nanoindentation - the method of choice in my thesis - allows for the mechanical characterization of single submicrometer-sized particles under close-to physiological environments. EVs in hereditary spherocytosis, the role of calcium-sensor proteins in membrane fusion, the function of tetraspanin CD63 in sorting cholesterol to HeLa-EVs as well as a potential effect of EV storage on their mechanical properties, inherent properties of lipoproteins (the main carriers of cholesterol and phospholipids within the human body) and the influence of oxygen on the mechanics of cervical cancer EVs were investigated. Next to better and deeper understanding of physiological processes within the human body, diseases and protein functions, mechanical characterization can drive forward the design of targeted drug delivery vehicles, and minimal-invasive diagnostics

Lipoprotein particles exhibit distinct mechanical properties

Lipoproteins (LPs) are micelle-like structures with a similar size to extracellular vesicles (EVs) and are therefore often co-isolated, as intensively discussed within the EV community. LPs from human blood plasma are of particular interest as they are responsible for the deposition of cholesterol ester and other fats in the artery, causing lesions, and eventually atherosclerosis. Plasma lipoproteins can be divided according to their size, density and composition into chylomicrons (CM), very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Here, we use atomic force microscopy for mechanical characterization of LPs. We show that the nanoindentation approach used for EV analysis can also be used to characterize LPs, revealing specific differences between some of the particles. Comparing LPs with each other, LDL exhibit a higher bending modulus as compared to CM and VLDL, which is likely related to differences in cholesterol and apolipoproteins. Furthermore, CM typically collapse on the surface after indentation and HDL exhibit a very low height after surface adhesion both being indications for the presence of LPs in an EV sample. Our analysis provides new systematic insights into the mechanical characteristics of LPs

Atomic Force Microscopy: An Introduction

Imaging of nano-sized particles and sample features is crucial in a variety of research fields. For instance in biological sciences, where it is paramount to investigate structures at the single particle level. Often two-dimensional images are not sufficient and further information such as topography and mechanical properties are required. Furthermore, to increase the biological relevance, it is desired to perform the imaging in close to physiological environments. Atomic force microscopy (AFM) meets these demands in an all-in-one instrument. It provides high-resolution images including surface height information leading to three-dimensional information on sample morphology. AFM can be operated both in air and in buffer solutions. Moreover, it has the capacity to determine protein and membrane material properties via the force spectroscopy mode. Here we discuss the principles of AFM operation and provide examples of how biomolecules can be studied. By including new approaches such as high-speed AFM (HS-AFM) we show how AFM can be used to study a variety of static and dynamic single biomolecules and biomolecular assemblies