Abstract

Exosomes (EXOs) are nano-sized vesicles secreted by most cell types. They are abundant in bio-fluids and harbor specific molecular constituents from their parental cells. Due to these characteristics, EXOs have a great potential in cancer diagnostics for liquid biopsy and personalized medicine. Despite this unique potential, EXOs are not yet widely applied in clinical settings, with two main factors hindering their translational process in diagnostics. Firstly, conventional extraction methods are time-consuming, require large sample volumes and expensive equipment, and often do not provide high-purity samples. Secondly, characterization methods have some limitations, because they are often qualitative, need extensive labeling or complex sampling procedures that can induce artifacts. In this context, novel label-free approaches are rapidly emerging, and are holding potential to revolutionize EXO diagnostics. These methods include the use of nanodevices for EXO purification, and vibrational spectroscopies, scattering, and nanoindentation for characterization. In this progress report, we summarize recent key advances in label-free techniques for EXO purification and characterization. We point out that these methods contribute to reducing costs and processing times, provide complementary information compared to the conventional characterization techniques, and enhance flexibility, thus favoring the discovery of novel and unexplored EXO-based biomarkers. In this process, the impact of nanotechnology is systematically highlighted, showing how the effectiveness of these techniques can be enhanced using nanomaterials, such as plasmonic nanoparticles and nanostructured surfaces, which enable the exploitation of advanced physical phenomena occurring at the nanoscale level.

Highlights

  • In many clinical situations, cancer diagnosis requires single or repeated tissue biopsies of a suspected cancerous region

  • The authors developed an AFM protocol to characterize artificial and natural vesicles mechanics in liquid by AFM imaging: (i) the method allowed them to retrieve extracellular vesicles (EVs) unperturbed geometry and contact angle α in terms of particle height and surface projected radius; (ii) α depends on stiffness and it is roughly independent of EV size; (iii) a deviation from point ii indicates the presence of contaminants; (iv) a calibration curve is provided that allows calculating stiffness from contact angle

  • The results summarized above show that AFM is a versatile and promising tool for the search and validation of novel EXO biomarkers of disease, but it is plagued by severe limitations which are hindering its spread in diagnostics

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Summary

Introduction

Cancer diagnosis requires single or repeated tissue biopsies of a suspected cancerous region. The identification of cancer-specific material in the latter molecular class suggests these nano-sized EVs to be an attractive platform for biomarker development in the field of liquid biopsy and personalized medicine [7,8,9,10,11,12,13] In this context, it is important to recognize that EVs are highly heterogeneous in chemical make-up. Effective conventional techniques exist for EV characterization, such as Western blotting, ELISA, and omics approaches, these methods have some drawbacks, because they are often qualitative and need extensive labeling or complex sampling techniques that can alter the relative ratio of molecular classes In this context, novel label-free approaches are rapidly emerging in EV research. The impact of is systematicallyishighlighted, showing how the effectiveness of effectiveness these techniques can nanotechnology systematically highlighted, showing how the of these be enhanced using nanomaterials, such as plasmonic nanoparticles and nanostructured techniques can be enhanced using nanomaterials, such as plasmonic nanoparticles and surfaces, which enable thewhich exploitation advanced physical and chemical effectsand occurring nanostructured surfaces, enableofthe exploitation of advanced physical chemat the nanoscale level.at the nanoscale level

Extracellular Vesicles Classification and Biogenesis
Exosomes
Microvesicles
Apoptotic Bodies
Exosome Isolation
Ultracentrifugation
Polymer-Based Separation
Size Exclusion Chromatography
Immunoaffinity Techniques
Scattering and Diffraction Provide Unique Information on EV Lipid Bilayer
Aim
Main Findings
Vibrational Spectroscopies for Label-Free Exosome Molecular Profiling in the
Main IR Findings
Exosome Characterization with Raman Spectroscopy
Nanoindentation
Mechanical Findings
AFM Methods
Label-Free Microfluidic Devices for Exosome Isolation
Microfluidics-Based Devices
Electrofluidics-Based Devices
Acoustofluidics-Based Devices
Discussion and Perspectives
Methods
Full Text
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