Carlo Diaferia

Surface engineering critically influences the analytical performance, manufacturability, and translational potential of electrochemical nucleic acid biosensors. Here, we introduce a peptide hydrogel (HG)-based surface engineering strategy that replaces conventional Au−thiol functionalization with a single supramolecular biointerface for disposable electrochemical biosensors. An HG based on Fmoc-FF/Fmoc-FFKK-Amide (1:2, v/v) enabled the direct immobilization of DNA probes onto disposable screen-printed electrodes, integrating electrode functionalization and probe immobilization within a single material and eliminating the multistep workflow associated with conventional immobilization strategies. The resulting platform required approximately 45 min for fabrication while reducing the consumption of costly surface-functionalization reagents, yielding disposable sensors with an estimated fabrication cost of approximately €0.60 per device. Following target hybridization, electrochemical measurements could be performed directly without additional surface-processing or labeling steps. As a proof of concept, the biosensor enabled amplification-free detection of miRNA-107 over the 0.1−1000 nM concentration range, achieving detection limits of 0.3 nM in PBS and 0.6 nM in undiluted human serum with a relative standard deviation below 4%. More broadly, this work establishes supramolecular peptide HG beyond passive scaffolds, positioning them as functional biointerfaces for the next generation of portable, cost-effective, and user-friendly electrochemical nucleic acid biosensors.

Nanogels based on cationic Nap-tripeptides as potential nanoplatforms for the delivery of therapeutic short interfering RNA (siRNA).

Non-covalent forces are the primary drivers of biomolecular interactions. They also represent a key factor in the unexpected tendency for proteins, peptides, and even individual amino acids to self-assemble into precise supramolecular assemblies, often characterized by a β-rich, amyloid-like structure. Studies carried out in the last few decades have shown that peptides/proteins self-assembly not only has structural consequences but also generates spectroscopic properties whose origin remains debated. Here, we investigated the spectroscopic properties of four peptides, GAIIGL, NNQQ, SSTSAA, and GNNQQNG (a derivative of the frequently studied GNNQQNY peptide), devoid of aromatic residues, whose crystallographic characterization has been seminal in elucidating the basis of the aggregation process. The spectroscopic behavior of these peptides was analyzed in two different media and in the solid state, highlighting the conditions that favor UV–visible fluorescence emission. These investigations have also prompted the use of this fluorescence as a potential diagnostic tool in nanomedicine. Importantly, the identification of intrinsic fluorescence signatures in these amyloid-like assemblies may support the development of label-free optical approaches for the early detection and monitoring of aggregation-related pathological processes. For all peptides, the emitted fluorescence spans a rather wide range of wavelengths. Although centered in the blue region, a significant signal is also observed in the green region, independent of their physical state. The structural analysis of the solid used to collect the spectroscopic data reveals features related to the packings in the crystal state. This observation suggests that their three-dimensional crystal structures may serve as reliable models for studies aimed at correlating structural and spectroscopic features.

Ultrashort peptide-based hydrogels represent an attractive class of supramolecular soft materials due to their minimalistic design, chemical versatility, and potential for translational applications. Classical Fmoc-dipeptides, particularly Fmoc-FF, are well established as efficient low-molecular-weight hydrogelators; however, controlling their aqueous solubility and gelation behavior across physiologically relevant conditions remains a key design challenge for injectable and in situ forming materials. Here, we report a pH-responsive injectable hydrogel based on a dipeptide incorporating the unnatural amino acid Fmoc-β-(3-pyridyl)-l-alanine (Fmoc-3-Pal-OH). Introduction of the pyridyl moiety provides a well-defined protonation–deprotonation equilibrium that acts as a molecular switch to regulate the supramolecular self-assembly. Deprotonation in the pH range 6.0–8.0 promotes spontaneous hydrogel formation under mild conditions, while protonation at acidic pH induces a controlled network disassembly. The hydrogel system was comprehensively characterized as a function of the concentration and buffer conditions using fluorescence spectroscopy, circular dichroism, Fourier-transform infrared spectroscopy, scanning electron microscopy, and rheology. pH modulation enables fine control over nanofibrillar organization and viscoelastic properties, yielding mechanically stable hydrogels with tunable stiffness values comparable to those of soft biological tissues. The pH-dependent assembly behavior was further exploited to regulate drug release. Encapsulation of curcumin as a hydrophobic model compound demonstrated high loading capacity and sustained release under neutral conditions, while acidic pH triggered an accelerated release through protonation-induced network collapse. Overall, the results achieved by this research open the way to a simple and robust molecular design strategy to overcome key limitations of conventional Fmoc-based hydrogelators and highlight the potential of protonation-controlled ultrashort peptide assemblies as adaptable polymer-like networks for stimuli-responsive soft materials and drug delivery applications.

Peptide-based materials represent a rapidly growing field in nanotechnology, bridging bottom-up self-assembly and top-down approaches for the development of functional nanostructures. Among these systems, peptide-based nanogels (NGs), namely nanogels in which peptides assume a structural role, have emerged as a promising class of injectable formulations. Typically characterized by a core–shell architecture, these systems are closely related to peptide hydrogels in terms of structural organization. This review provides a state-of-the-art overview of peptides used as core structural elements for NG formulation, focusing on the peptide building blocks employed, the main formulation methodologies, and their current applications, with particular emphasis on pharmaceutical ones. Their potential as drug delivery systems and stimuli-responsive platforms for controlled and targeted release is also reported. For clarity, the reported formulations are classified according to the chemical nature of the core-structuration peptide, distinguishing systems based on Fmoc-FF from those derived from other primary sequences, including Boc-protected tripeptides, dehydropeptides, and chemically crosslinked peptide assemblies.

This review explores the advancements and applications of β-hairpin peptide hydrogels, starting from the paradigmatic case of MAX1 and its highly versatile analogue MAX8. MAX1 (H-VKVKVKVKV^DPPTKVKVKVKV-NH₂) has been identified as the first synthetic β-hairpin peptide for the preparation of stimuli-responsive peptide-based hydrogels. At low ionic strength or neutral pH, MAX1 remains unfolded and soluble. However, under physiological conditions, it folds into a β-hairpin structure, then producing a self-supporting matrix within minutes. The formed gel is shear-thinning and self-healing, making it suitable for injectable therapies. To explore MAX1 molecular space and enhance its practical clinical use, the primary sequence was engineered via chemical modification, with specific single amino acid substitution and relative net charge alteration. This approach generates MAX1 analogues, differing in gelation kinetics, mechanical response and biological performances. The β-hairpin peptide hydrogels are categorized into five different groups: MAX1, MAX1 analogues, MAX8, MAX8 analogues and non-MAX peptides sequences. Collectively, the review outcomes demonstrate the use of β-hairpin peptide matrices as tunable platforms for the development of predictable and stable biomaterials for advanced tissue engineering and drug delivery applications.

There is currently a growing interest in understanding the origins of intrinsic fluorescence as a way to design noninvasive probes for biophysical processes. In this regard, understanding how pH influences fluorescence in nonaromatic biomolecular assemblies is key to controlling their optical properties in realistic cellular conditions. Here, we combine experiments and theory to investigate the pH-dependent emission of solid-state l-Lysine (Lys). Lys aggregates prepared at different pH values using HCl and H₂SO₄ exhibit protonation- and counterion-dependent morphology and fluorescence, as shown by microscopy and steady-state measurements. We find an enhancement in the fluorescence moving from acidic to basic conditions. To uncover the molecular origin of these trends, we performed nonadiabatic molecular dynamics simulations on three Lys crystal models representing distinct protonation states. Our simulations indicate that enhanced protonation under acidic conditions facilitates nonradiative decay via proton transfer, whereas basic conditions favor radiative decay. Our combined experimental-theoretical work highlights pH and counterion identity as key factors tuning fluorescence in Lys assemblies, offering insights for designing pH responsive optical materials based on nonaromatic amino acids.

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. The efficacy and safety of lipoplexes was previously demonstrated in vitro in halting variant expression in the peripheral blood of patients. Preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before clinical investigations can be undertaken, according to regulatory authorities in Europe. In the light of the foregoing, this study aims to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed specific siRNA duplexes for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 h post-transfection, reaching higher significant knockdown after 72 h. Lipoplexes efficiently suppress pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre-clinical platform for testing of in vivo lipoplexes in CRISPR-engineered NOD/ShiLtJ mice carrying the R619W mutation.

Peptide-based nanogels (NGs) represent a cutting-edge class of nanoscale drug delivery systems. Due to their structural properties, NGs platforms can encapsulate and protect therapeutic agents (e.g. peptides, proteins, and nucleic acids), while allowing for controlled and stimuli-responsive release. These pharmacokinetic and pharmacodynamic features can be specifically tuned by including peptide functional elements as NG components. This study explores the formulation, decoration strategies, and structural properties of NGs derived from mixed hydrogel (HG) matrices of Fmoc-diphenylalanine (Fmoc-FF) with cationic amphiphilic peptides (CAPs). CAPs, composed by cationic hexapeptide (GK)3 sequence decorated at its N-terminus with alkyl chain, were found able to confer a net positive charge to Fmoc-FF NGs. Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 NGs were obtained using polysorbate 80 (TWEEN 80) and sorbitane monooleate 80 (SPAN 80) as colloidal stabilizing surfactants and characterized in terms of size, secondary structure, superficial charge and shelf stability by Dynamic Light Scattering (DLS), Circular Dichroism (CD), Fourier Transform Infrared (FT-IR) and Small-Angle X-ray Scattering (SAXS) technique. Different formulative routes were used and mutually compared to encapsulate or adsorb AlexaFluor 430 (succinimidyl ester), used as model of an anionic, pharmaceutical agent. In vitro experiments demonstrated good cytocompatibility of these systems and the release of AlexaFluor 430 was also evaluated.