Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan-Vesicular Affinity Probes.

Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV-mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan-vesicular affinity ligands for both EVs and their non-canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV-associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin-lacking Red Blood Cell-derived EVs, overcoming limitations associated with antibody-based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first-ever peptide-based application in SiMoA technology.

Naphthalene Diimide-Tetraazacycloalkane Conjugates Are G-Quadruplex-Based HIV-1 Inhibitors with a Dual Mode of Action.

Human immunodeficiency virus 1 (HIV-1) therapeutic regimens consist of three or more drugs targeting different steps of the viral life cycle to limit the emergence of viral resistance. In line with the multitargeting strategy, here we conjugated a naphthalene diimide (NDI) moiety with a tetraazacycloalkane to obtain novel naphthalene diimide (NDI)-tetraazacycloalkane conjugates. The NDI inhibits the HIV-1 promoter activity by binding to LTR G-quadruplexes, and the tetraazacycloalkane mimics AMD3100, which blocks HIV entry into cells by interfering with the CXCR4 coreceptor. We synthesized, purified, and tested the metal-free NDI-tetraazacycloalkane conjugate and the two derived metal-organic complexes (MOCs) that incorporate Cu2+ and Zn2+. The NDI-MOCs showed enhanced binding to LTR G4s as assessed by FRET and CD assays in vitro. They also showed enhanced activity in cells where they dose-dependently reduced LTR promoter activity and inhibited viral entry only of the HIV-1 strain that exploited the CXCR4 coreceptor. The time of addition assay confirmed the dual targeting at the different HIV-1 steps. Our results indicate that the NDI-MOC conjugates can simultaneously inhibit viral entry, by targeting the CXCR4 coreceptor, and LTR promoter activity, by stabilizing the LTR G-quadruplexes. The approach of combining multiple targets in a single compound may streamline treatment regimens and improve the overall patient outcomes.

Computation meets experiment: identification of highly efficient fibrillating peptides.

Self-assembling peptides are of huge interest for biological, medical and nanotechnological applications. The enormous chemical variety that is available from the 20 amino acids offers potentially unlimited peptide sequences, but it is currently an issue to predict their supramolecular behavior in a reliable and cheap way. Herein we report a computational method to screen and forecast the aqueous self-assembly propensity of amyloidogenic pentapeptides. This method was found also as an interesting tool to predict peptide crystallinity, which may be of interest for the development of peptide based drugs.

Particle profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

Acid⋠⋠⋠Amide Supramolecular Synthon for Tuning Amino Acid-Based Hydrogels' Properties.

Invited for the cover of this issue is the Laboratory of Supramolecular and Bio-Nanomaterials, coordinated by Pierangelo Metrangolo, at the Politecnico di Milano, Italy. The image depicts the co-crystal formed by N-Fmoc-pentafluorophenylalanine and benzamide, which is also involved in the formation of their mixed hydrogels. Read the full text of the article at 10.1002/chem.202301743.

Acid⋠⋠⋠Amide Supramolecular Synthon for Tuning Amino Acid-Based Hydrogels' Properties.

Supramolecular hydrogels formed by the self-assembly of N-Fmoc-l-phenylalanine derivatives are gaining relevance for several applications in the materials and biomedical fields. In the challenging attempt to predict or tune their properties, we selected Fmoc-pentafluorophenylalanine (1) as a model efficient gelator, and studied its self-assembly in the presence of benzamide (2), a non-gelator able to form strong hydrogen bonds with the amino acid carboxylic group. Equimolar mixtures of 1 and 2 in organic solvents afforded a 1 : 1 co-crystal thanks to the formation of an acid⋅⋅⋅amide heterodimeric supramolecular synthon. The same synthon occurred in the transparent gels formed by mixing the two components in 1 : 1 ratio in aqueous media, as revealed by structural, spectroscopic, and thermal characterizations performed on both the co-crystal powder and the lyophilized hydrogel. These findings revealed the possibility of modulating the properties of amino acid-based hydrogels by involving the gelator in the formation of a co-crystal. Such a crystal engineering-based approach is shown also to be useful for the time-delayed release of suitable bioactive molecules, when involved as hydrogel coformers.

Cell Penetrating Peptides: Classification, Mechanisms, Methods of Study, and Applications.

Cell-penetrating peptides (CPPs) encompass a class of peptides that possess the remarkable ability to cross cell membranes and deliver various types of cargoes, including drugs, nucleic acids, and proteins, into cells. For this reason, CPPs are largely investigated in drug delivery applications in the context of many diseases, such as cancer, diabetes, and genetic disorders. While sharing this functionality and some common structural features, such as a high content of positively charged amino acids, CPPs represent an extremely diverse group of elements, which can differentiate under many aspects. In this review, we summarize the most common characteristics of CPPs, introduce their main distinctive features, mechanistic aspects that drive their function, and outline the most widely used techniques for their structural and functional studies. We highlight current gaps and future perspectives in this field, which have the potential to significantly impact the future field of drug delivery and therapeutics.

Novel Arginine- and Proline-Rich Candidacidal Peptides Obtained through a Bioinformatic Approach.

Antimicrobial resistance is a major public health concern worldwide. Albeit to a lesser extent than bacteria, fungi are also becoming increasingly resistant to antifungal drugs. Moreover, due to the small number of antifungal classes, therapy options are limited, complicating the clinical management of mycoses. In this view, antimicrobial peptides (AMPs) are a potential alternative to conventional drugs. Among these, Proline-rich antimicrobial peptides (PrAMPs), almost exclusively of animal origins, are of particular interest due to their peculiar mode of action. In this study, a search for new arginine- and proline-rich peptides from plants has been carried out with a bioinformatic approach by sequence alignment and antimicrobial prediction tools. Two peptide candidates were tested against planktonic cells and biofilms of Candida albicans and Candida glabrata strains, including resistant isolates. These peptides showed similar potent activity, with half-maximal effective concentration values in the micromolar range. In addition, some structural and functional features, revealing peculiar mechanistic behaviors, were investigated.

Hybrid Peptide-Agarose Hydrogels for 3D Immunoassays.

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, 3D droplet microarrays, where hydrogels are used as matrices to stably entrap biomolecules onto analytical surfaces, potentially provide relevant advantages over conventional 2D assays, such as increased loading capacity, lower nonspecific binding, and enhanced signal-to-noise ratio. Here, we describe a hybrid hydrogel composed of a self-assembling peptide and commercial agarose (AG) as a suitable matrix for 3D microarray bioassays. The hybrid hydrogel is printable and self-adhesive and allows analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of SARS-CoV-2 infection.

Epitope Mapping on Microarrays Highlights a Sequence on the N Protein with Strong Immune Response in SARS-CoV-2 Patients.

In SARS-CoV-2 pandemic scenario, the identification of rapid methods to detect antibodies against coronavirus has been a wide and urgent issue. Epitope mapping on peptide microarrays is a rapid way to identify sequences with a high immunoreactivity. The process begins with a proteome-wide screening, based on immune affinity; the use of a high-density microarray is followed by a validation phase, where a restricted panel of probes is tested using peptide microarrays; peptide sequences are immobilized through a click-based strategy.COVID-19-positive sera are tested and immuno-domains regions are identified on SARS-CoV-2 spike (S), nucleocapsid (N) protein, and Orf1ab polyprotein. An epitope on N protein (region 155-171) provided good diagnostic performance in discriminating COVID-19-positive vs. healthy individuals. Using this sequence, 92% sensitivity and 100% specificity are reached for IgG detection in COVID-19 samples, and no cross-reactivity with common cold coronaviruses is detected. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

Membrane-Sensing Peptides for Extracellular Vesicle Analysis.

Analytical platforms for small extracellular vesicle (sEV) high-throughput analysis are highly desirable. These bionanoparticles present fairly distinctive lipid membrane features including high curvature, lipid-packing defects, and a relative abundance in lipids. sEV membrane could be considered as a "universal" marker, complementary or alternative to traditional surface-associated proteins. Here, we describe the use of membrane-sensing peptides as a new, highly efficient ligand to directly integrate sEV capturing and analysis on a microarray platform.

Emergence of Elastic Properties in a Minimalist Resilin-Derived Heptapeptide upon Bromination.

Bromination is herein exploited to promote the emergence of elastic behavior in a short peptide-SDSYGAP-derived from resilin, a rubber-like protein exerting its role in the jumping and flight systems of insects. Elastic and resilient hydrogels are obtained, which also show self-healing behavior, thanks to the promoted non-covalent interactions that limit deformations and contribute to the structural recovery of the peptide-based hydrogel. In particular, halogen bonds may stabilize the ß-sheet organization working as non-covalent cross-links between nearby peptide strands. Importantly, the unmodified peptide (i.e., wild type) does not show such properties. Thus, SDSY(3,5-Br)GAP is a novel minimalist peptide elastomer.

Composite Peptide-Agarose Hydrogels for Robust and High-Sensitivity 3D Immunoassays.

Canonical immunoassays rely on highly sensitive and specific capturing of circulating biomarkers by interacting biomolecular baits. In this frame, bioprobe immobilization in spatially discrete three-dimensional (3D) spots onto analytical surfaces by hydrogel encapsulation was shown to provide relevant advantages over conventional two-dimensional (2D) platforms. Yet, the broad application of 3D systems is still hampered by hurdles in matching their straightforward fabrication with optimal functional properties. Herein, we report on a composite hydrogel obtained by combining a self-assembling peptide (namely, Q3 peptide) with low-temperature gelling agarose that is proved to have simple and robust application in the fabrication of microdroplet arrays, overcoming hurdles and limitations commonly associated with 3D hydrogel assays. We demonstrate the real-case scenario feasibility of our 3D system in the profiling of Covid-19 patients' serum IgG immunoreactivity, which showed remarkably improved signal-to-noise ratio over canonical assays in the 2D format and exquisite specificity. Overall, the new two-component hydrogel widens the perspectives of hydrogel-based arrays and represents a step forward towards their routine use in analytical practices.

Photoluminescent nanocluster-based probes for bioimaging applications.

In the continuous search for versatile and better performing probes for optical bioimaging and biosensing applications, many research efforts have focused on the design and optimization of photoluminescent metal nanoclusters. They consist of a metal core composed by a small number of atoms (diameter < 2-3 nm), usually coated by a shell of stabilizing ligands of different nature, and are characterized by molecule-like quantization of electronic states, resulting in discrete and tunable optical transitions in the UV-Vis and NIR spectral regions. Recent advances in their size-selective synthesis and tailored surface functionalization have allowed the effective combination of nanoclusters and biologically relevant molecules into hybrid platforms, that hold a large potential for bioimaging purposes, as well as for the detection and tracking of specific markers of biological processes or diseases. Here, we will present an overview of the latest combined imaging or sensing nanocluster-based systems reported in the literature, classified according to the different families of coating ligands (namely, peptides, proteins, nucleic acids, and biocompatible polymers), highlighting for each of them the possible applications in the biomedical field.

Fibril Structure Demonstrates the Role of Iodine Labelling on a Pentapeptide Self-Assembly.

Iodination has long been employed as a successful labelling strategy to gain structural insights into proteins and other biomolecules via several techniques, including Small Angle X-ray Scattering, Inductively Coupled Plasma Mass Spectrometer (ICP-MS), and single-crystal crystallography. However, when dealing with smaller biomolecular systems, interactions driven by iodine may significantly alter their self-assembly behaviour. The engineering of amyloidogenic peptides for the development of ordered nanomaterials has greatly benefitted from this possibility. Still, to date, iodination has exclusively been applied to aromatic residues. In this work, an aliphatic bis-iodinated amino acid was synthesized and included into a custom pentapeptide, which showed enhanced fibrillogenic behaviour. Peptide single crystal X-ray structure and powder X-ray diffraction on its dried water solution demonstrated the key role of iodine atoms in promoting intermolecular interactions that drive the peptide self-assembly into amyloid fibrils. These findings enlarge the library of halogenated moieties available for directing and engineering the self-assembly of amyloidogenic peptides.

Solid-Phase Synthesis of Gly-Ψ[CH(CF3)NH]-Peptides.

The solid-phase synthesis of Gly-Ψ[CH(CF3)NH]-peptides is presented. In order to achieve this goal, the synthesis of Gly-Ψ[CH(CF3)NH]-dipeptides having the C-terminus unprotected, the N-terminus protected as Fmoc- or Teoc-, and possibly side chain functionalities protected with acid-labile protecting groups has been developed. A selected small library of six peptidomimetics, encompassing analogues of biological relevant peptides, have been obtained in high purity.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region.

A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

Self-Assembling Peptide Hydrogels for 3D Microarrays.

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, hydrogels have emerged as appealing matrices to locally confine biomolecules onto sensing surfaces under solution mimetic conditions, preserving their structural integrity and function. Here, we describe the application of a self-assembling peptide hydrogel as a suitable matrix for 3D microarray bioassays. The hydrogel is printable and self-adhesive and allows for fast analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of arbovirus infection.

Organic Cages as Building Blocks for Mechanically Interlocked Molecules: Towards Molecular Machines.

The research on systems able to perform controllable motions under external stimuli arises great interest in the scientific community. Over the years, a library of innovative devices has been produced, classified in different categories according to the molecular or supramolecular level of motion. This minireview aims to highlight some representative studies, in which organic cages are used as building blocks for mechanically interlocked molecules, and in which intramolecular motions are triggered by external input. However, the application of organic cages in the construction of molecular machines is hardly achieved. A good compromise must actually be reached, between flexibility and rigidity of the cage's framework for an effective control of the intra- and/or intermolecular motion in the final mechanical device. Our final goal is to stimulate researchers' curiosity towards cage-like molecules, so that they take on the challenge of converting a cage into a molecular machine.