Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol

Abstract

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.

Recent Advances in the Label-Free Characterization of Exosomes for Cancer Liquid Biopsy: From Scattering and Spectroscopy to Nanoindentation and Nanodevices

“…Cantilever deflection is recorded thanks to an optical setup coupled with high-precision piezoelectric actuators (Figure 6a). Aside from imaging purposes, the same cantilever can be used as a sensitive mechanical probe to indent the sample, acquiring spatially resolved images of its mechanical properties [168][169][170][171][172][173][174]. This operation mode is often referred to as force spectroscopy, and it is remarkably flexible, allowing scientists to measure a wide range of mechanical parameters, such as stiffness, Young's modulus, adhesion force, work adhesion, hysteresis, dissipation, and relaxation times [113,[175][176][177][178][179][180].…”

“…A typical shape of a correct indentation curve on exosomes (d) together with the mechanical signature of EXO membrane tethering (e). Reprinted with permission from ref [174]. Copyright Frontiers (2020).…”

“…Currently, the AFM mechanical characterization of EXOs appears to be still in its infancy, with only a few papers published on the subject [174,190,[192][193][194]. Among these papers, most of them cover mainly methodological aspects, also attempting to establish a shared methodology for EXO characterization, thus reducing the aforementioned variability (see Table 5).…”

Comprehensive label-free characterization of extracellular vesicles and their surface proteins

“…For differentiation between the species detected on the surface, topography and elasticity images have been used. Particles with a height of >8 nm above the background that present with a clearly delineated lower elasticity than the substrate have been classified as EVs, as previously described using tapping mode phase imaging (Sharma et al, 2010) and force-distance curve-based deformation imaging (Vorselen et al, 2020). Other particles were designated as non-vesicular particles (NVPs).…”

“…A more advanced method for measuring particle number combines SPR with atomic force microscopy (AFM; Obeid et al (2017)). The latter presents a promising first strategy to obtain more detailed information about EV properties (Vorselen et al, 2020). Nevertheless, ensuring that the counted particles are indeed EVs and that these EVs are therapeutically active (e.g.…”