SPRi analysis of molecular interactions of mApoE-functionalized liposomes as drug delivery systems for brain diseases.

The application of liposomes (LPs) to central nervous system disorders could represents a turning point in the therapy and quality of life of patients. Indeed, LPs have demonstrated their ability to cross the blood-brain barrier (BBB) and, as a consequence, to enhance the therapeutics delivery into the brain. Some approaches for BBB crossing involve the modification of LP surfaces with biologically active ligands. Among them, the Apolipoprotein E-modified peptide (mApoE) has been used for several LP-based nanovectors under investigation. In this study, we propose Surface Plasmon Resonance imaging (SPRi) for the characterization of multifunctionalized LPs for Glioblastoma treatment. LPs were functionalized with mApoE and with a metallo-protease sensitive lipopeptide to deliver and guarantee the localized release of an encapsulated drug in diseased areas. The SPRi analysis was optimized in order to evaluate the binding affinity between LPs and mApoE receptors, finding that mApoE-LPs generated SPRi signals referred to interactions between mApoE and receptors mainly present in the brain. Moreover, a significant binding between LPs and VCAM-1 (endothelial receptor) was observed, whereas LPs did not interact significantly with peripheral receptors expressed on monocytes and lymphocytes. SPRi results confirmed not only the presence of mApoE on LP surfaces, but also its binding affinity, thanks to the specific interaction with selected receptors. In conclusion, the high sensitivity and the multiplexing capability associated with the low volumes of sample required and the minimal sample preparation, make SPRi an excellent technique for the characterization of multifunctionalized nanoparticles-based formulations.

Multiplexing Biosensor for the Detection of Extracellular Vesicles as Biomarkers of Tissue Damage and Recovery after Ischemic Stroke.

The inflammatory, reparative and regenerative mechanisms activated in ischemic stroke patients immediately after the event cooperate in the response to injury, in the restoration of functions and in brain remodeling even weeks after the event and can be sustained by the rehabilitation treatment. Nonetheless, patients' response to treatments is difficult to predict because of the lack of specific measurable markers of recovery, which could be complementary to clinical scales in the evaluation of patients. Considering that Extracellular Vesicles (EVs) are carriers of multiple molecules involved in the response to stroke injury, in the present study, we have identified a panel of EV-associated molecules that (i) confirm the crucial involvement of EVs in the processes that follow ischemic stroke, (ii) could possibly profile ischemic stroke patients at the beginning of the rehabilitation program, (iii) could be used in predicting patients' response to treatment. By means of a multiplexing Surface Plasmon Resonance imaging biosensor, subacute ischemic stroke patients were proven to have increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) and translocator protein (TSPO) on the surface of small EVs in blood. Besides, microglia EVs and endothelial EVs were shown to be significantly involved in the intercellular communications that occur more than 10 days after ischemic stroke, thus being potential tools for the profiling of patients in the subacute phase after ischemic stroke and in the prediction of their recovery.

Raman Spectroscopy Characterization of Multi-Functionalized Liposomes as Drug-Delivery Systems for Neurological Disorders.

The characterization of nanoparticle-based drug-delivery systems represents a crucial step in achieving a comprehensive overview of their physical, chemical, and biological features and evaluating their efficacy and safety in biological systems. We propose Raman Spectroscopy (RS) for the characterization of liposomes (LPs) to be tested for the control of neuroinflammation and microglial dysfunctions in Glioblastoma multiforme and Alzheimer's disease. Drug-loaded LPs were functionalized to cross the blood-brain barrier and to guarantee localized and controlled drug release. The Raman spectra of each LP component were used to evaluate their contribution in the LP Raman fingerprint. Raman data analysis made it possible to statistically discriminate LPs with different functionalization patterns, showing that each molecular component has an influence in the Raman spectrum of the final LP formulation. Moreover, CLS analysis on Raman data revealed a good level of synthetic reproducibility of the formulations and confirmed their stability within one month from their synthesis, demonstrating the ability of the technique to evaluate the efficacy of LP synthesis using small amount of sample. RS represents a valuable tool for a fast, sensitive and label free biochemical characterization of LPs that could be used for quality control of nanoparticle-based therapeutics.

Biochemical Characterization of Human Salivary Extracellular Vesicles as a Valuable Source of Biomarkers.

Extracellular vesicles (EVs) are natural nanoparticles secreted under physiological and pathological conditions. Thanks to their diagnostic potential, EVs are increasingly being studied as biomarkers of a variety of diseases, including neurological disorders. To date, most studies on EV biomarkers use blood as the source, despite different disadvantages that may cause an impure isolation of the EVs. In the present article, we propose the use of saliva as a valuable source of EVs that could be studied as biomarkers in an easily accessible biofluid. Using a comparable protocol for the isolation of EVs from both liquid biopsies, salivary EVs showed greater purity in terms of co-isolates (evaluated by nanoparticle tracking analysis and Conan test). In addition, Raman spectroscopy was used for the identification of the overall biochemical composition of EVs coming from the two different biofluids. Even considering the limited amount of EVs that can be isolated from saliva, the use of Raman spectroscopy was not hampered, and it was able to provide a comprehensive characterization of EVs in a high throughput and repeatable manner. Raman spectroscopy can thus represent a turning point in the application of salivary EVs in clinics, taking advantage of the simple method of collection of the liquid biopsy and of the quick, sensitive and label-free biophotonics-based approach.