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Extracellular vesicles (EVs) are lipid vesicles secreted by all cells into the extracellular space and act as nanosized biological messengers among cells. They carry a specific molecular cargo, composed of lipids, proteins, nucleic acids, and carbohydrates, which reflects the state of their parent cells. Due to their remarkable structural and compositional heterogeneity, characterizing EVs, particularly from a biochemical perspective, presents complex challenges. In this context, mid-infrared (IR) spectroscopy is emerging as a valuable tool, providing researchers with a comprehensive and label-free spectral fingerprint of EVs in terms of their specific molecular content. This review aims to provide an up-to-date critical overview of the major advancements in mid-IR spectroscopy of extracellular vesicles, encompassing both fundamental and applied research achievements. We also systematically emphasize the new possibilities offered by the integration of emerging cutting-edge IR technologies, such as tip-enhanced and surface-enhanced spectroscopy approaches, along with the growing use of machine learning for data analysis and spectral interpretation. Additionally, to assist researchers in navigating this intricate subject, our manuscript includes a wide and detailed collection of the spectral peaks that have been assigned to EV molecular constituents up to now in the literature.

Extracellular vesicles (EVs) are abundantly released into the systemic circulation, where they show remarkable stability and harbor molecular constituents that provide biochemical information about their cells of origin. Due to this characteristic, EVs are attracting increasing attention as a source of circulating biomarkers for cancer liquid biopsy and personalized medicine. Despite this potential, none of the discovered biomarkers has entered the clinical practice so far, and novel approaches for the label-free characterization of EVs are highly demanded. In this regard, Fourier Transform Infrared Spectroscopy (FTIR) has great potential as it provides a quick, reproducible, and informative biomolecular fingerprint of EVs. In this pilot study, we investigated, for the first time in the literature, the capability of FTIR spectroscopy to distinguish between EVs extracted from sera of cancer patients and controls based on their mid-IR spectral response. For this purpose, EV-enriched suspensions were obtained from the serum of patients diagnosed with Hepatocellular Carcinoma (HCC) of nonviral origin and noncancer subjects. Our data point out the presence of statistically significant differences in the integrated intensities of major mid-IR absorption bands, including the carbohydrate and nucleic acids band, the protein amide I and II bands, and the lipid CH stretching band. Additionally, we used Principal Component Analysis combined with Linear Discriminant Analysis (PCA-LDA) for the automated classification of spectral data according to the shape of specific mid-IR spectral signatures. The diagnostic performances of the proposed spectral biomarkers, alone and combined, were evaluated using multivariate logistic regression followed by a Receiving Operator Curve analysis, obtaining large Areas Under the Curve (AUC = 0.91, 95% CI 0.81-1.0). Very interestingly, our analyses suggest that the discussed spectral biomarkers can outperform the classification ability of two wid

All living systems are maintained by a constant flux of metabolic energy and, among the different reactions, the process of lipids storage and lipolysis is of fundamental importance. Current research has focused on the investigation of lipid droplets (LD) as a powerful biomarker for the early detection of metabolic and neurological disorders. Efforts in this field aim at increasing selectivity for LD detection by exploiting existing or newly synthesized probes. However, LD constitute only the final product of a complex series of reactions during which fatty acids are transformed into triglycerides and cholesterol is transformed in cholesteryl esters. These final products can be accumulated in intracellular organelles or deposits other than LD. A complete spatial mapping of the intracellular sites of triglycerides and cholesteryl esters formation and storage is, therefore, crucial to highlight any potential metabolic imbalance, thus predicting and counteracting its progression. Here, we present a machine learning assisted, polarity-driven segmentation which enables to localize and quantify triglycerides and cholesteryl esters biosynthesis sites in all intracellular organelles, thus allowing to monitor in real-time the overall process of the turnover of these non-polar lipids in living cells. This technique is applied to normal and differentiated PC12 cells to test how the level of activation of biosynthetic pathways changes in response to the differentiation process.

Background: Extracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required. Results: In this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 mu m. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 mu m) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 mu m, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs d

Red blood cells (RBCs) are characterized by a remarkable elasticity, which allows them to undergo very large deformation when passing through small vessels and capillaries. This extreme deformability is altered in various clinical conditions, suggesting that the analysis of red blood cell (RBC) mechanics has potential applications in the search for non-invasive and cost-effective blood biomarkers. Here, we provide a comparative study of the mechanical response of RBCs in patients with Alzheimer's disease (AD) and healthy subjects. For this purpose, RBC viscoelastic response was investigated using atomic force microscopy (AFM) in the force spectroscopy mode. Two types of analyses were performed: (i) a conventional analysis of AFM force-distance (FD) curves, which allowed us to retrieve the apparent Young's modulus, E; and (ii) a more in-depth analysis of time-dependent relaxation curves in the framework of the standard linear solid (SLS) model, which allowed us to estimate cell viscosity and elasticity, independently. Our data demonstrate that, while conventional analysis of AFM FD curves fails in distinguishing the two groups, the mechanical parameters obtained with the SLS model show a very good classification ability. The diagnostic performance of mechanical parameters was assessed using receiving operator characteristic (ROC) curves, showing very large areas under the curves (AUC) for selected biomarkers (AUC > 0.9). Taken all together, the data presented here demonstrate that RBC mechanics are significantly altered in AD, also highlighting the key role played by viscous forces. These RBC abnormalities in AD, which include both a modified elasticity and viscosity, could be considered a potential source of plasmatic biomarkers in the field of liquid biopsy to be used in combination with more established indicators of the pathology.

Exosomes (EXOs) are considered an exceptionally promising source of cancer biomarkers for personalized medicine and liquid biopsy. Despite this potential, the EXOs translation process in diagnostics is still at its birth, and the development of reliable and reproducible methods for their characterization is highly demanded. Fourier Transform Infrared (FTIR) Spectroscopy is perfectly suited for this purpose, as it can provide a label-free biochemical profile of EXOs in terms of lipid, protein, and nucleic acid content. Here we evaluated the applicability of FTIR spectroscopy to the study of cancer-derived EXOs as a function of cell differentiation. For this purpose, we used N-acetyl-L-Cysteine (NAC) to induce a controlled differentiation in human colon carcinoma cells from a proliferative mesenchymal morphology to a less invasive epithelial phenotype, as measured with fluorescence and electron microscopy. EXOs derived from cells with different phenotypes showed significant variation in the relative intensity of the amide I-II and CH-stretching bands in the mid-IR range, indicating the spectroscopic lipid/protein ratio as an effective classification parameter. Additionally, we showed that different cell phenotypes are associated with a shape modification in these spectral bands that can be automatically detected by combining Principal Component Analysis (PCA) with Linear Discriminant Analysis (LDA). On the one hand, our study confirms that an FTIR analysis of EXOs allows scientists to precisely detect modifications occurring at the parental cell level; on the other hand, it unveils a set of effective spectral biomarkers able to monitoring cell changes from a mesenchymal to an epithelial phenotype, a clinically valuable piece of information considering that the epithelial-to-mesenchymal transition is a key step in the metastatic process.

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.

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.

During the transition period, high-yielding dairy cows suffer from negative energy balance, intense lipomobilization and impaired lipid metabolism; this metabolic condition can lead to overburdened triglycerides accumulation in the liver, known as liver lipidosis, which has been associated to impaired fertility in dairy cows. The mechanisms of this impairment can be in principle correlated with the presence and the extent of ectopic fat depots. However, current methods for evaluating fat accumulation in liver and in the reproductive tract suffer from low resolution, sensitivity, and specificity. Confocal microscopes are equipped with Gallium arsenide phosphide detectors, thus enabling the acquisition of intense signals from tissue biopsies. This method could differentiate whether fat deposition occurred without requiring sample sectioning. Here, we examined with this technique liver, uterine and ovarian samples of heifers and regularly slaughtered repeat breeder and overconditioned dairy cows, to quantify lipid droplets and depots at a submicrometer scale with high specificity. With the aid of this technique, we found lipid depots in uterine and ovarian specimens. Moreover, we found that the size and number of depots increased with the degree of liver lipidosis. Further studies are needed to elucidate the relationship between the severity and extent of these deposits and the fertility of lactating dairy cows. Since tissues other than liver display different characteristic lipid droplet distributions, this technique can be potentially employed to shed new light on the pathogenesis of lipidosis and to assess new risk factors for infertility.

Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts. We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique. The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings. This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms.

In physiological conditions, red blood cells (RBCs) are capable of dramatic deformations when passing through the microvasculature. This extreme deformability is closely related to the RBC biconcave shape, to the fluidic nature of the haemoglobin and the cell membrane structure, primarily consisting of a phospholipid bilayer with an underlying two-dimensional spectrin network. In many pathological and inflammatory conditions, the shape and the extreme deformability of erythrocytes appear to be significantly altered. These findings have stimulated intense research towards the search and validation of novel erythrocyte-based mechanical biomarkers, useful for disease diagnosis and therapy monitoring. In this study, we investigated with Atomic Force Microscopy (AFM) the mechanical properties of erythrocytes obtained from a 68 years old cirrhotic man diagnosed with spur cell anaemia and cold agglutinated disease, before and after liver transplantation. Mechanical changes are compared with ultrastructural alterations as studied by scanning electron microscopy and discussed according to confocal fluorescence microscopy results, showing possible alterations induced by the cirrhotic environment at the level of the RBCs cytoskeletal organisation and lipidic composition. Taken together, the results here presented show that liver transplantation not only contributes to restoring the proper RBC morphology, but it also induces recovery of the physiological viscous behaviour of cells, further stressing the relevance of viscous and dissipative forces in determining the RBC biomechanical response.