Surface Nanomechanics of Biomolecules and Supramolecular Systems

Surface nanomechanics of biomolecules and supramolecular systems is an interdisciplinary and vital area of current research, with implications/applications spanning from synthetic biology to regenerative medicine, from smart surfaces to molecular machines. Biomolecule surface transformations and nanomachinery arise upon “wiring” them onto surfaces and interfaces. Surface confinement of biomolecules is a common feature of biological systems (e.g., cell membranes) and often a mandatory step for translating their properties into real‐world applications (e.g., biosensors). On surfaces biomolecules undergo peculiar transformations and interactions which they do not experience in solution. Such unedited effects open challenges in synthetic systems, for example, by altering or hindering the designed/expected property, but also disclose a wealth of opportunities and surprises. Based on our latest research, this chapter will bring fresh excerpts from the field. It will start with an accessible description of thermodynamics of surface nanomechanics of biomolecules and supramolecular systems and then will show how it can be implemented to gain understanding of grow factor cell signaling, to single out small ligands able to inhibit protein misfolding, to measure energetics of surface confined ferritin during iron loading, and to realize a universal probe for ammine‐based designer drugs

Protein Thin Film Machines

We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fuelled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model

Exploiting Exosomes for Differential Diagnosis of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by a clonal plasma cell proliferation. Usually, all MM are preceded by an asymptomatic premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS). Differential diagnosis requires the evidence of end-organ damage, but recently new biomarkers are emerging to help clinicians to distinguish MM from the premalignant phase. Circulating exosomes in serum seem to be a powerful tool to be analyzed for liquid biopsy, and in this chapter, we show that MM and MGUS exosomes are different in concentration, biological activity, and biochemical markers. These differences seem to be related to the free light chains (FLCs) associated with exosomes and their propathogenic properties. The cellular processing FLC-decorated exosomes and their ability to activate proinflammatory mechanisms are different in MM and MGUS patients. These elements can be evaluated to create an innovative multiparameter panel to monitor MGUS to MM switching

On the interaction and nanoplasmonics of gold nanoparticles and lipoproteins

The extracellular space is nanostructured, populated by heterogeneous classes of nanoparticles, e.g., extracellular vesicles and lipoproteins, which “made by cells for cells'' mediate intercellular, inter-organ, cross-species, and cross-kingdom communication. However, while techniques to study ENP biology in-vitro and in-vivo are becoming available, knowledge of their colloidal and interfacial properties is poor, although much needed. This paper experimentally shows, for the first time, that the aggregation of citrate-capped gold nanoparticles (AuNPs) triggered by lipid vesicle membranes and the related characteristic redshift of the plasmonic signature also applies/extends to lipoproteins. Such interaction leads to the formation of AuNP-lipoprotein hybrid nanostructures and is sensitive to lipoprotein classes and AuNP/lipoprotein molar ratio, paving the way to further synthetic and analytical developments

Exosome-delivered microRNAs promote IFN-α secretion by human plasmacytoid DCs via TLR7

The excessive production of type I IFNs is a hallmark and a main pathogenic mechanism of many autoimmune diseases, including systemic lupus erythematosus (SLE). In these pathologies, the sustained secretion of type I IFNs is dependent on the improper activation of plasmacytoid DCs (pDCs) by self-nucleic acids. However, the nature and origin of pDC-activating self-nucleic acids is still incompletely characterized. Here, we report that exosomes isolated from the plasma of SLE patients can activate the secretion of IFN-α by human blood pDCs in vitro. This activation requires endosomal acidification and is recapitulated by microRNAs isolated from exosomes, suggesting that exosome-delivered microRNAs act as self-ligands of innate single-stranded endosomal RNA sensors. By using synthetic microRNAs, we identified an IFN induction motif that is responsible for the TLR7-dependent activation, maturation, and survival of human pDCs. These findings identify exosome-delivered microRNAs as potentially novel TLR7 endogenous ligands able to induce pDC activation in SLE patients. Therefore, microRNAs may represent novel pathogenic mediators in the onset of autoimmune reactions and potential therapeutic targets in the treatment of type I IFN-mediated diseases

Quantifying the nanomachinery of the nanoparticle-biomolecule interface

A study is presented of the nanomechanical phenomena experienced by nanoparticle-conjugated biomolecules. A thermodynamic framework is developed to describe the binding of thrombin-binding aptamer (TBA) to thrombin when the TBA is conjugated to nanorods. Binding results in nanorod aggregation (viz. directed self-assembly), which is detectable by absorption spectroscopy. The analysis introduces the energy of aggregation, separating it into TBA–thrombin recognition and surface-work contributions. Consequently, it is demonstrated that self-assembly is driven by the interplay of surface work and thrombin-TBA recognition. It is shown that the work at the surface is about −10 kJ mol−1 [mol superscript -1] and results from the accumulation of in-plane molecular forces of pN magnitude and with a lifetime of <1 s, which arises from TBA nanoscale rearrangements fuelled by thrombin-directed nanorod aggregation. The obtained surface work can map aggregation regimes as a function of different nanoparticle surface conditions. Also, the thermodynamic treatment can be used to obtain quantitative information on surface effects impacting biomolecules on nanoparticle surfaces.MIT-IQS Exchange Progra

Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles

7noopenopenRadeghieri, Annalisa; Savio, Giulia; Zendrini, Andrea; Di Noto, Giuseppe; Salvi, Alessandro; Bergese, Paolo; Piovani, GiovannaRadeghieri, Annalisa; Savio, Giulia; Zendrini, Andrea; DI NOTO, Giuseppe; Salvi, Alessandro; Bergese, Paolo; Piovani, Giovann

Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles

Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonistof active clotting proteases. A deficit in AT function leads to AT qualitative deficiency,challenging to diagnose. Here we report that active AT may travel physiosorbed on thesurface of plasma extracellular vesicles (EVs), contributing to form the “EV-proteincorona.” The corona is enriched in specific AT glycoforms, thus suggesting glycosyla-tion to play a key role in AT partitioning between EVs and plasma. Differences in ATglycoform composition of the corona of EVs separated from plasma of healthy andAT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest noveldirections to unravel pathophysiological mechanisms

Comparison of separation methods for immunomodulatory extracellular vesicles from helminths

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth-derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone

Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid

Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells