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

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Recent Advances in the Label-Free Characterization of Exosomes for Cancer Liquid Biopsy: from Scattering and Spectroscopy to Nanoindentation and Nanodevices nanomaterials Review Recent Advances in the Label-Free Characterization of Exosomes for Cancer Liquid Biopsy: From Scattering and Spectroscopy to Nanoindentation and Nanodevices Riccardo Di Santo 1, Sabrina Romanò 1,2, Alberto Mazzini 1,2 , Svetlana Jovanovi´c 3 , Giuseppina Nocca 1,4 , Gaetano Campi 5,6 , Massimiliano Papi 1,2 , Marco De Spirito 1,2,* , Flavio Di Giacinto 1,2,† and Gabriele Ciasca 1,2,*,† 1 Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Roma, Italy; [email protected] (R.D.S.); [email protected] (S.R.); [email protected] (A.M.); [email protected] (G.N.); [email protected] (M.P.); fl[email protected] (F.D.G.) 2 Dipartimento di Neuroscienze, Sezione di Fisica, Università Cattolica Del Sacro Cuore, 00168 Roma, Italy 3 “Vinˇca”Institute of Nuclear Sciences—National Institute of the Republic of Serbia, University of Belgrade, 11000 Belgrade, Serbia; [email protected] 4 Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy 5 Rome International Centre Materials Science Superstripes RICMASS, via dei Sabelli 119A, 00185 Rome, Italy; [email protected] 6 Institute of Crystallography, CNR, via Salaria Km 29. 300, Monterotondo Stazione, 00016 Roma, Italy * Correspondence: [email protected] (M.D.S.); [email protected] (G.C.) † These authors contributed equally to this work. Citation: Di Santo, R.; Romanò, S.; Mazzini, A.; Jovanovi´c,S.; Nocca, G.; Campi, G.; Papi, M.; De Spirito, M.; Abstract: Exosomes (EXOs) are nano-sized vesicles secreted by most cell types. They are abundant Di Giacinto, F.; Ciasca, G. Recent in bio-fluids and harbor specific molecular constituents from their parental cells. Due to these char- Advances in the Label-Free acteristics, EXOs have a great potential in cancer diagnostics for liquid biopsy and personalized Characterization of Exosomes for medicine. Despite this unique potential, EXOs are not yet widely applied in clinical settings, with two Cancer Liquid Biopsy: From main factors hindering their translational process in diagnostics. Firstly, conventional extraction Scattering and Spectroscopy to methods are time-consuming, require large sample volumes and expensive equipment, and often Nanoindentation and Nanodevices. do not provide high-purity samples. Secondly, characterization methods have some limitations, Nanomaterials 2021, 11, 1476. https:// because they are often qualitative, need extensive labeling or complex sampling procedures that can doi.org/10.3390/nano11061476 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 Academic Editor: Tambet Teesalu purification, and vibrational spectroscopies, scattering, and nanoindentation for characterization. Received: 29 April 2021 In this progress report, we summarize recent key advances in label-free techniques for EXO pu- Accepted: 27 May 2021 rification and characterization. We point out that these methods contribute to reducing costs and Published: 2 June 2021 processing times, provide complementary information compared to the conventional characteri- zation techniques, and enhance flexibility, thus favoring the discovery of novel and unexplored Publisher’s Note: MDPI stays neutral EXO-based biomarkers. In this process, the impact of nanotechnology is systematically highlighted, with regard to jurisdictional claims in showing how the effectiveness of these techniques can be enhanced using nanomaterials, such as published maps and institutional affil- plasmonic nanoparticles and nanostructured surfaces, which enable the exploitation of advanced iations. physical phenomena occurring at the nanoscale level. Keywords: exosome; extracellular vesicles; liquid biopsies; label-free; biofluids; microfluidics; nan- odevice; SAXS; FTIR; AFM; personalized medicine Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons In many clinical situations, cancer diagnosis requires single or repeated tissue biopsies Attribution (CC BY) license (https:// of a suspected cancerous region. This procedure is invasive and often associated with creativecommons.org/licenses/by/ pain, discomfort, and risk for the patients. Additionally, the tissue region that needs to be 4.0/). Nanomaterials 2021, 11, 1476. https://doi.org/10.3390/nano11061476 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, 1476 2 of 48 sampled can be highly heterogeneous, thus leading to ambiguous conclusions, hardly ac- cessible, or even completely inaccessible by surgery. These drawbacks limit the frequencies with which a region can be sampled to check for cancer, thus hindering the possibility to perform accurate diagnoses, especially in the early stages of the pathology. Liquid biopsy offers a promising diagnostic alternative, because it relies on the analysis of biofluids, such as blood, saliva, and urine [1–6]. Widely used biomarkers in liquid biopsy include cell-free nucleic acids, such as DNA, mRNA, and miRNA, circulating tumor cells, and ex- tracellular vesicles (EVs), which are nanosized lipid vesicles secreted by most cell types. 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 [7–13]. In this context, it is important to recognize that EVs are highly heterogeneous in chem- ical make-up. Three main classes of EVs differing in size can be distinguished, namely, exosomes (EXOs), microvesicles (MVs), and apoptotic bodies (ABs) [14–18]. Aside from size differences, the three EV types can be distinguished because of different biogenic mechanisms. A large research effort in this area has led to the discovery of a wide number of potential cancer biomarkers, mostly based on EXOs. Notably, EXOs are often shed by tumor cells in higher numbers in comparison to normal cells, because tumorigenesis affects many pathways regulating EV release. A higher EXO concentration is thus associated with increasing tumor mass or severity, making EXO-based biopsy attractive as a prognostic biomarker [1–5,7–13,19–23]. Despite this huge diagnostic potential, EXOs have still not been widely applied in clinical settings. In this regard, two main motivations can be highlighted: (i) the first related to extraction and purification methods; (ii) the second related to EV characterization and downstream analysis. Conventional isolation methods mostly rely on time-consuming ultracentrifugation steps and require specialized personnel, working with expensive equipment [24]. Ad- ditionally, ultracentrifugation needs large sample volumes and often does not produce high-purity samples. Other purification methods have been developed so far, including size exclusion chromatography, polymer-based precipitation, and immunocapture approaches. On the one hand, these methods avoid the use of expensive equipment; on the other hand, they are plagued by several limitations including long operation times, unknown contaminants in commercial kits, and the problem of often being restricted to EXOs with a single antigen. Apart from extraction methods, a change in the paradigm of EXO analysis is also required to further stimulate their translational process in diagnostics. Although 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. These methods include nanodevices for EV purification, and vibrational spectroscopies, scattering, and nanoindentation for EV characterization. The potential advantages and dis- advantages of these techniques and their key characteristics in EXO science are summarized in Figure1. Nanomaterials 2021,, 11,, 1476x FOR PEER REVIEW 3 of 4849 Figure 1. Key characteristics of label-free techniques in EV science. In this review, wewe discussdiscuss thethe recentrecent scientificscientific andand technologicaltechnological advancesadvances in thesethese label-free techniques,techniques, highlighting highlighting their their complementary complementary role role compared compared to conventional to conventional and andmore more established established methods methods in EV in science. EV science. We stress We the stress flexibility the flexibility of these methodsof these methods and how andthis flexibilityhow this providesflexibility fertileprovides ground fertile for ground the discovery for the of discovery novel and of unexplored novel and cancerunex- ploredbiomarkers cancer in EXO-basedbiomarkers liquidin EXO-based biopsy. Inliquid this process,biopsy. In the this impact process, of nanotechnology the impact of nanotechnologyis systematically is highlighted, systematically showing highlighted, how the showing effectiveness how the of effectiveness these techniques of these can techniquesbe enhanced can using be enhanced nanomaterials, using suchnanomateri as plasmonicals, such nanoparticles as plasmonic and nanoparticles nanostructured and nanostructuredsurfaces,
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