Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves

DEVELOPMENT OF A NOVEL NANOSENSOR FOR THE STUDY OF BIOMOLECULAR INTERACTIONS

The evaluation and quantification of molecular interactions is of paramount importance in modern biology and molecular medicine. Therefore, there is a continuous exploration for new methodologies capable to detect and to measure binding affinities during reversible molecular interactions. This work is devoted to explore a new tool based on the high sensitivity that the measurement of the scattered light intensity offers when the binding occurs on the surface of index-matched colloids. Static light scattering is not a traditional technique to study molecular association because the binding of insulated ligands and receptors in dilute solutions produce negligible increment of the scattered light, while mesoscopic particles hosting multiple receptors or ligands, including real bacteria, typically scatter too much light compared with the contributions due to molecular adhesion on their surface. This difficulty can be overcome by supporting the receptors on nano-scale latex spheres whose refractive index closely matches the one of water. As \u201cPhantom Nanoparticles\u201d (PNPs), we have used highly hydrophobic monodisperse spherical fluoroelastomer colloids, with radius R = 39 \uc5} 1 nm, and whose refractive index, at our working temperature (30\uc5\ue3C) and wavelength (633 nm), is np0 = 1.3248 (under the same conditions the refractive index of water is nW = 1.3319). Surfactants added to a PNP dispersion readily adsorb on their hydrophobic surfaces, generating a self-assembled monolayer which can be easily equipped with molecular hydrophilic end groups of various kind, including well-known receptors and/or ligands. This label-free method has been assessed through the precise determination of the binding constant of the antibiotic vancomycin with the tripeptide L-Lys-D-Ala-D-Ala and of the vancomycin dimerization constant. We have enlightened the role of bidentate effect and molecular hindrance in the activity of this glycopeptide. After this success first result, an accurate determination of the optimal properties of nanoparticles employed has been performed by comparative experiments and through theoretical evaluation (CHAPETR 3). The effects of size, refractive index, electric charge, and dilution on the reliability and accuracy of the method has been evaluated. Quite surprisingly, perfect index matching and minimal size (i.e., maximum surface), which is almost attained in one of the colloids here employed, do not represent the ideal conditions. Rather, we show that a nanoparticle radius of 100 nm and a refractive index slightly below that of water yields the best signal/background amplitude. We also show that repulsive interactions can lead to artifacts in the adsorption isotherm, thus indicating that electrostatic stabilization should be kept at a minimum. Successively, the particles, already optically phantom, have also been made biologically \u201cinvisible\u201d through PEG coating and decorated by interacting proteins, thus providing a mean to investigate the biological properties of proteins (CHAPETR 4). Avidin decorated Phantom Nanoparticles have been prepared and were employed to detect interactions between different kinds of biotinylated proteins. Using this approach, biotinylated protein A was anchored on the surface of the nanoparticles, and were exploited as a functional probe for the rapid, quantitative, picomolar detection of human IgG antibodies. We used Phantom Nanoparticles to evaluate substrate recognition by Streptomyces PMF Phospholipase D inactivated mutants (CHAPETR 5). The use of this specific technique seems to have some peculiar advantages over other methods in the case of phospholipidsacting enzymes. In fact, the substrate (or a substrate analog) can be organized onto the surface of Phantom Nanoparticles at a desired concentration with optimal display of the polar head group, while the hydrophobic chains result packed into the surfactant monolayer, thus limiting the occurrence of non-specific interactions. The last part of this work is focused on an attempt toward the stable functionalization of Phantom Nanoparticles (CHAPETR 6). Diacetylene surfactants spontaneously adsorb on the nanoparticles and then they are polymerized by exposure to UV light. So far, the stability of the amphiphilic coating around the nanoparticle solely depended on weak hydrophobic interactions. The attachment of the polymer to the particle surface, because of the numerous contact points, is highly stable and can be improved further by crosslinking of the polymer shell. Huns generating a quantifiable number of functional groups suitable for covalent receptor anchorage. All these observations, demonstrate the feasibility of this new technique, which makes it possible to easily generate different synthetic receptors, and highlight this technique as a versatile novel method to study, both qualitatively and quantitatively, of molecular recognition processes. The work described in this thesis is partially published in: Morasso C., Bellini T., Monti D., Bassi M., Prosperi D., Riva S.: Dispersed phantom scatterer technique reveals subtle differences in substrate recognition by phospholipase D inactive mutant; ChemBioChem. Submitted, currently under review Prosperi D., Morasso C., Tortora P., Monti D., Bellini T.: Avidin decorated core-shell nanoparticles for biorecognition studies by elastic light scattering; ChemBioChem. 2007 (8): 1021-1028 (Impact factor: 3,446). Prosperi D., Morasso C., Mantegazza F., Buscaglia M., Houg L., Bellini T.: Phantom nanoparticles as probes of biomolecular interaction; Small. 2006 (8-9):1060-1067 (Impact factor: 6,408)

Raman analysis reveals biochemical differences in plasma of Crohn's Disease patients

Backgrounds and Aims: There is no accurate and reliable circulating biomarker to diagnose Crohn's disease [CD]. Raman spectroscopy is a relatively new approach that provides information on the biochemical composition of samples in minutes and virtually without any sample preparation. We aimed to test the use of Raman spectroscopy analysis of plasma samples as a potential diagnostic tool for CD. Methods: We analysed by Raman spectroscopy dry plasma samples obtained from 77 CD patients [CD] and 45 healthy controls [HC]. In the dataset obtained, we analysed spectra differences between CD and HC, as well as among CD patients with different disease behaviours. We also developed a method, based on principal component analysis followed by a linear discrimination analysis [PCA-LDA], for the automatic classification of individuals based on plasma spectra analysis. Results: Compared with HC, the CD spectra were characterised by less intense peaks corresponding to carotenoids [p <10-4] and by more intense peaks corresponding to proteins with \u3b2-sheet secondary structure [p <10-4]. Differences were also found on Raman peaks relative to lipids [p = 0.0007] and aromatic amino acids [p <10-4]. The predictive model we developed was able to classify CD and HC subjects with 83.6% accuracy [sensitivity 80.0% and specificity 85.7%] and F1-score of 86.8%. Conclusions: Our results indicate that Raman spectroscopy of blood plasma can identify metabolic variations associated with CD and it could be a rapid pre-screening tool to use before further specific evaluation

Raman spectroscopy reveals biochemical differences in plasma derived extracellular vesicles from sporadic amyotrophic lateral sclerosis patients

Sporadic Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease for which there is no validated blood based biomarker. Extracellular vesicles (EVs) have the potential to solve this unmet clinical need. However, due to their heterogeneity and complex chemical composition, EVs are difficult to study. Raman spectroscopy (RS) is an optical method that seems particularly well suited to address this task. In fact, RS provides an overview of the biochemical composition of EVs quickly and virtually without any sample preparation. In this work, we studied by RS small extracellular vesicles (sEVs), large extracellular vesicles (lEVs) and blood plasma of sporadic ALS patients and of a matched cohort of healthy controls. The obtained results highlighted lEVs as a particularly promising biomarker for ALS. In fact, their Raman spectra show that sporadic ALS patients have a different lipid content and less intense bands relative to the aromatic aminoacid phenylalanine

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies. © 2020 American Chemical Society

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies. © 2020 American Chemical Society