Engineering Amino Acid and Peptide Supramolecular Architectures through Fluorination.

Fluorinated non-natural amino acids are attracting considerable research interest, especially in the biomedical field and in materials science, thanks to their ability to self-assemble into peculiar supramolecular structures. The conformational changes induced by the presence of fluorine atoms obviously affect their functions, as well as the biological activity of the deriving peptides and proteins. Here, we will briefly describe the main effects of fluorination on the aggregation behavior of such building blocks, focusing in particular on their improved tendency to form fibrils, and gels therefrom. Our aim is to underline the promising potential of fluorination as a tool to affect the self-assembly features of amino acids, both when used alone and when inserted into polypeptide sequences. The ability of fluorine to influence physical, chemical, and structural properties of these substrates offers the possibility to engineer bioinspired materials with specific and tunable functions.

FM19G11-loaded nanoparticles modulate energetic status and production of reactive oxygen species in myoblasts from ALS mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Considerable evidence indicates that early skeletal muscle atrophy plays a crucial role in the disease pathogenesis, leading to an altered muscle-motor neuron crosstalk that, in turn, may contribute to motor neuron degeneration. Currently, there is no effective treatment for ALS, highlighting the need to dig deeper into the pathological mechanisms for developing innovative therapeutic strategies. FM19G11 is a novel drug able to modulate the global cellular metabolism, but its effects on ALS skeletal muscle atrophy and mitochondrial metabolism have never been evaluated, yet. This study investigated whether FM19G11-loaded nanoparticles (NPs) may affect the bioenergetic status in myoblasts isolated from G93A-SOD1 mice at different disease stages. We found that FM19G1-loaded NP treatment was able to increase transcriptional levels of Akt1, Akt3, Mef2a, Mef2c and Ucp2, which are key genes associated with cell proliferation (Akt1, Akt3), muscle differentiation (Mef2c), and mitochondrial activity (Ucp2), in G93A-SOD1 myoblasts. These cells also showed a significant reduction of mitochondrial area and networks, in addition to decreased ROS production after treatment with FM19G11-loaded NPs, suggesting a ROS clearance upon the amelioration of mitochondrial dynamics. Our overall findings demonstrate a significant impact of FM19G11-loaded NPs on muscle cell function and bioenergetic status in G93A-SOD1 myoblasts, thus promising to open new avenues towards possible adoption of FM19G11-based nanotherapies to slow muscle degeneration in the frame of ALS and muscle disorders.

Structural Insights on the Role of Halogen Bonding in Protein MEK Kinase-Inhibitor Complexes.

Kinases are enzymes that play a critical role in governing essential biological processes. Due to their pivotal involvement in cancer cell signaling, they have become key targets in the development of anti-cancer drugs. Among these drugs, those containing the 2,4-dihalophenyl moiety demonstrated significant potential. Here we show how this moiety, particularly the 2-fluoro-4-iodophenyl one, is crucial for the structural stability of the formed drug-enzyme complexes. Crystallographic analysis of reported kinase-inhibitor complex structures highlights the role of the halogen bonding that this moiety forms with specific residues of the kinase binding site. This interaction is not limited to FDA-approved MEK inhibitors, but it is also relevant for other kinase inhibitors, indicating its broad relevance in the design of this class of drugs.

Design of fluorinated stealth poly(ε-caprolactone) nanocarriers.

The covalent functionalization of polymers with fluorinated moieties represents a promising strategy for the development of multimodal systems. Moreover, polymer fluorination often endows the resulting nanocarriers with improved colloidal stability in the biological environment. In this work, we developed fluorinated pegylated (PEG) biodegradable poly(ε-caprolactone) (PCL) drug nanocarriers showing both high colloidal stability and stealth properties, as well as being (19F)-Nuclear Magnetic Resonance (NMR) detectable. The optimized nanocarriers were obtained mixing a PEG-PCL block copolymer with a nonafluoro-functionalized PCL polymer. The role of PEGylation and fluorination on self-assembly and colloidal behavior of the obtained nanoparticles (NPs) was investigated, as well as their respective role on stealth properties and colloidal stability. To prove the feasibility of the developed NPs as potential 19F NMR detectable drug delivery systems, a hydrophobic drug was successfully encapsulated, and the maintenance of the relevant 19F NMR properties evaluated. Drug-loaded fluorinated NPs still retained a sharp and intense 19F NMR signal and good relaxivity parameters (i.e., T1 and T2 relaxation times) in water, which were not impaired by drug encapsulation.

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.

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.

Fluoride Anions: Unexploited but Effective Halogen Bond Acceptors.

Due to their high electron density, fluoride anions can be considered the most effective halogen bond (HaB) acceptors among the halides. However, under common experimental conditions, F- uncommonly acts as HaB acceptor, expectedly as it is present in hydrated form. Herein we report that under specific crystallization conditions a hydrogen bond-free F- functioning as donor of electron density can be obtained, with the formed HaBs constituting the driving force of the observed crystal packings. Computations confirm the strength of these HaBs compared to analogous interactions involving other halides.

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.

Pivotal role of the protein corona in the cell uptake of fluorinated nanoparticles with increased sensitivity for 19F-MR imaging.

In vivo cell tracking by non-invasive imaging technologies is needed to accelerate the clinical translation of innovative cell-based therapies. In this regard, 19F-MRI has recently gained increased attention for unbiased localization of labeled cells over time. To push forward the use of 19F-MRI for cell tracking, the development of highly performant 19F-probes is required. PLGA-based NPs containing PERFECTA, a multibranched superfluorinated molecule with an optimal MRI profile thanks to its 36 magnetically equivalent fluorine atoms, are promising 19F-MRI probes. In this work we demonstrate the importance of the surface functionalization of these NPs in relation to their interaction with the biological environment, stressing the pivotal role of the formation of the protein corona (PC) in their cellular labelling efficacy. In particular, our studies showed that the formation of PC NPs strongly promotes the cellular internalization of these NPs in microglia cells. We advocate that the formation of PC NPs in the culture medium can be a key element to be used for the optimization of cell labelling with a considerable increase of the detection sensitivity by 19F-MRI.

Flattened and Wrinkled Encapsulated Droplets: Shape Morphing Induced by Gravity and Evaporation.

We report surprising morphological changes of suspension droplets (containing class II hydrophobin protein HFBI from Trichoderma reesei in water) as they evaporate with a contact line pinned on a rigid solid substrate. Both pendant and sessile droplets display the formation of an encapsulating elastic film as the bulk concentration of solute reaches a critical value during evaporation, but the morphology of the droplet varies significantly: for sessile droplets, the elastic film ultimately crumples in a nearly flattened area close to the apex while in pendant droplets, circumferential wrinkling occurs close to the contact line. These different morphologies are understood through a gravito-elastocapillary model that predicts the droplet morphology and the onset of shape changes, as well as showing that the influence of the direction of gravity remains crucial even for very small droplets (where the effect of gravity can normally be neglected). The results pave the way to control droplet shape in several engineering and biomedical applications.

Superfluorinated Extracellular Vesicles for In Vivo Imaging by 19F-MRI.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.

Systematic Study of Podand Molecules for Synergistic Halogen and Hydrogen Bond-Driven Anion Recognition in the Solid State.

The increasing demand of species for the efficient capture and sensing of anions benefits from a systematic study of anion binding capabilities in the solid state. This work reports a detailed crystallographic study of ten structurally related podands and shows that these charged receptors bind anions with a combination of charge-assisted halogen and hydrogen bonds. Computational tools helped in highlighting the role of the different involved interaction and afforded possible design principles for the design of improved podands.

Templated Out-of-Equilibrium Self-Assembly of Branched Au Nanoshells.

Out-of-equilibrium self-assembly of metal nanoparticles (NPs) has been devised using different types of strategies and fuels, but achieving finite 3D structures with a controlled morphology through this assembly mode is still rare. Here, a spherical peptide-gold superstructure (PAuSS) is used as a template to control the out-of-equilibrium self-assembly of Au NPs, obtaining a transient 3D-branched Au-nanoshell (BAuNS) stabilized by sodium dodecyl sulphate (SDS). The BAuNS dismantles upon SDS concentration gradient equilibration over time in the sample solution, leading to NPs disassembly and regression to PAuSS. Notably, BAuNS assembly and disassembly promotes temporary interparticle plasmonic coupling, leading to reversible and tunable changes of their plasmonic properties, a highly desirable behavior in the development of optoelectronic nanodevices.

Optimization of superfluorinated PLGA nanoparticles for enhanced cell labelling and detection by 19F-MRI.

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emergent imaging technique for molecular imaging and cell tracking. Lack of intrinsic 19F signals in tissues allows unambiguous in vivo detection of exogenous fluorinated probes, complementary to the anatomical and multiparametric information obtained by standard 1H-MRI. However, the intrinsic low sensitivity of MRI technique requires the need of designing increasingly effective fluorinated tracers. PERFECTA, with its 36 magnetically equivalent 19F atoms and a designed branched molecular structure, represents an excellent superfluorinated tracer. In this paper, we report the development of PERFECTA loaded PLGA NPs stabilized by different coatings as promising 19F-MRI probes. The results clearly show the optimal cellular uptake of the produced colloidally stable PERFECTA loaded PLGA NPs without impact on cells viability. Importantly, NPs stabilization with the anionic surfactant sodium cholate (NaC) clearly enhances NPs internalization within cells with respect to PVA-coated NPs. Moreover, the optimized NPs are characterized by shorter T1 relaxation times with respect to other PERFECTA formulations that would allow the increase of 19F-MRI sensitivity with fast imaging acquisitions.

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.

Synthesis and Linker-Controlled Self-Assembly of Dendritic Amphiphiles with Branched Fluorinated Tails.

Amphiphiles containing fluorinated segments tend to aggregate in the aqueous solution into structure of lower curvature than their hydrocarbon analogs due to their larger diameter. A benefit of supramolecular structures incorporating fluorine moieties is their high electron density, which can be viewed in cryo-TEM with better contrast than their hydrogenated forms. A modular approach has been developed for the synthesis of a new family of nonionic branched amphiphiles consisting of oligoglycerol units (G2) as the hydrophilic part and a branched fluorinated (F27) hydrophobic part. The design of this hydrophobic moiety allows to achieve a higher fluorine density than the previously used straight-chain perfluoroalkanes. Two different chemical approaches, amide, and triazole, are used to link the hydrophilic and hydrophobic segments. In addition, the aggregation behavior is investigated by dynamic light scattering (DLS) and cryo-TEM. The measurements prove the formation of multivesicular (MVVs) and multilamellar (MLVs) vesicles as well as smaller unilamellar vesicles. Further, the cell viability test proves the low cell toxicity of these nanoarchitectures for potential biomedical applications.

High-resolution crystal structure of a 20 kDa superfluorinated gold nanocluster.

Crystallization of atomically precise nanoclusters is gaining increasing attention, due to the opportunity of elucidating both intracluster and intercluster packing modes, and exploiting the functionality of the resulting highly pure crystallized materials. Herein, we report the design and single-crystal X-ray structure of a superfluorinated 20 kDa gold nanocluster, with an Au25 core coated by a shell of multi-branched highly fluorinated thiols (SF27) resulting in almost 500 fluorine atoms, i.e., ([Au25(SF27)18]0). The cluster shows a switchable solubility in the fluorous phase. X-ray analysis and computational studies reveal the key role of both intracluster and intercluster F···F contacts in driving [Au25(SF27)18]0 crystal packing and stabilization, highlighting the ability of multi-branched fluorinated thiols to endow atomically precise nanoclusters with remarkable crystallogenic behavior.

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.