Fluorescence of Aggregated Aromatic Peptides for Studying the Kinetics of Aggregation and Hardening of Amyloid-like Structures.

The capability of amyloid-like peptide fibers to emit intrinsic-fluorescence enables the study of their formation, stability and hardening through time-resolved fluorescence analysis, without the need for additional intercalating dyes. This approach allows the monitoring of amyloid-like peptides aggregation kinetics using minimal sample volumes, and the simultaneous testing of numerous experimental conditions and analytes, offering rapid and reproducible results. The analytical procedure applied to the aromatic hexapeptide F6, alone or derivatized with PEG (polyethylene glycol) moiety of different lengths, suggests that aggregation into large anisotropic structures negatively correlates with initial monomer concentration and relies on the presence of charged N- and C-termini. PEGylation reduces the extent of aggregates hardening, possibly by retaining water, and overall impacts the final structural properties of the aggregates.

Hybrid PNA-peptide hydrogels as injectable CEST-MRI agents.

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH2, named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Fmoc-FF hydrogels and nanogels for improved and selective delivery of dexamethasone in leukemic cells and diagnostic applications.

Dexamethasone (DEX) is a synthetic analogue of cortisol commonly used for the treatment of different pathological conditions, comprising cancer, ocular disorders, and COVID-19 infection. Its clinical use is hampered by the low solubility and severe side effects due to its systemic administration. The capability of peptide-based nanosystems, like hydrogels (HGs) and nanogels (NGs), to serve as vehicles for the passive targeting of active pharmaceutical ingredients and the selective internalization into leukemic cells has here been demonstrated. Peptide based HGs loaded with DEX were formulated via the "solvent-switch" method, using Fmoc-FF homopeptide as building block. Due to the tight interaction of the drug with the peptidic matrix, a significant stiffening of the gel (G' = 67.9 kPa) was observed. The corresponding injectable NGs, obtained from the sub-micronization of the HG, in the presence of two stabilizing agents (SPAN®60 and TWEEN®60, 48/52 w/w), were found to be stable up to 90 days, with a mean diameter of 105 nm. NGs do not exhibit hemolytic effects on human serum, moreover they are selectively internalized by RS4;11 leukemic cells over healthy PBMCs, paving the way for the generation of new diagnostic strategies targeting onco-hematological diseases.

Hybrid peptide-PNA monomers as building blocks for the fabrication of supramolecular aggregates.

Advantages like biocompatibility, biodegradability and tunability allowed the exploitation of peptides and peptidomimetics as versatile therapeutic or diagnostic agents. Because of their selectivity towards transmembrane receptors or cell membranes, peptides have also been identified as suitable molecules able to deliver in vivo macromolecules, proteins or nucleic acids. However, after the identification of the homodimer diphenylalanine (FF) as an aggregative motif inside the Aß1-42 polypeptide, short and ultrashort peptides have been studied as building blocks for the fabrication of supramolecular, ordered nanostructures for applications in biotechnological, biomedical and industrial fields. In this perspective, many hybrid molecules that combine FF with other chemical entities have been synthesized and characterized. Two novel hybrid derivatives (tFaF and cFgF), in which the FF homodimer is alternated with the peptide-nucleic acid (PNA) heterodimer "g-c" (guanine-cytosine) or "a-t" (adenine-thymine) and their dimeric forms (tFaF)2 and (cFgF)2 were synthesized. The structural characterization performed by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopies highlighted the capability of all the FF-PNA derivatives to self-assemble into ß-sheet structures. As a consequence of this supramolecular organization, the resulting aggregates also exhibit optoelectronic properties already reported for other similar nanostructures. This photoemissive behavior is promising for their potential applications in bioimaging.

Self-Assembled Materials Based on Fully Aromatic Peptides: The Impact of Tryptophan, Tyrosine, and Dopa Residues.

Peptides are able to self-organize in structural elements including cross-ß structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-ß that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Multicomponent Peptide-Based Hydrogels Containing Chemical Functional Groups as Innovative Platforms for Biotechnological Applications.

Multicomponent hydrogels (HGs) based on ultrashort aromatic peptides have been exploited as biocompatible matrices for tissue engineering applications, the delivery of therapeutic and diagnostic agents, and the development of biosensors. Due to its capability to gel under physiological conditions of pH and ionic strength, the low molecular-weight Fmoc-FF (Nα-fluorenylmethoxycarbonyl-diphenylalanine) homodimer is one of the most studied hydrogelators. The introduction into the Fmoc-FF hydrogel of additional molecules like protein, organic compounds, or other peptide sequences often allows the generation of novel hydrogels with improved mechanical and functional properties. In this perspective, here we studied a library of novel multicomponent Fmoc-FF based hydrogels doped with different amounts of the tripeptide Fmoc-FFX (in which X= Cys, Ser, or Thr). The insertion of these tripeptides allows to obtain hydrogels functionalized with thiol or alcohol groups that can be used for their chemical post-derivatization with bioactive molecules of interest like diagnostic or biosensing agents. These novel multicomponent hydrogels share a similar peptide organization in their supramolecular matrix. The hydrogels' biocompatibility, and their propensity to support adhesion, proliferation, and even cell differentiation, assessed in vitro on fibroblast cell lines, allows us to conclude that the hybrid hydrogels are not toxic and can potentially act as a scaffold and support for cell culture growth.

Development of cationic peptide-based hydrogels loaded with iopamidol for CEST-MRI detection.

Peptide-based hydrogels have been recently investigated as materials for biomedical applications like tissue engineering and delivery of drugs and imaging agents. Among the synthetic peptide hydrogelators, the cationic hexapeptides Ac-K1 and Ac-K2 were proposed as scaffolds for bioprinting applications. Here, we report the formulation of Ac-K1 and Ac-K2 hydrogels loaded with iopamidol, an iodinated contrast agent clinically approved for X-ray computed tomography, and more recently identified as an efficient CEST-MRI probe. Iopamidol-loaded hydrogels were soft, injectable and non-toxic both in vitro (on three tumor cell lines: GL261, TS/A and 3T3-NIH) and in vivo (in Balb/c mice inoculated with TS/A breast cancer cells). The in vitro CEST-MRI study evidenced the typical features of the CEST pattern of iopamidol, with a CEST contrast higher than 50%. Due to their injectability and good ability to retain the contrast agent, the herein investigated systems can be considered as promising candidates for the development of smart MRI detectable hydrogels.

Self-assembled aggregates based on cationic amphiphilic peptides: structural insight.

Short and ultra-short peptides have recently emerged as suitable building blocks for the fabrication of self-assembled innovative materials. Peptide aggregation is strictly related to the amino acids composing the sequence and their capability to establish intermolecular interactions. Additional structural and functional properties can also be achieved by peptide derivatization (e.g. with polymeric moieties, alkyl chains or other organic molecules). For instance, peptide amphiphiles (PAs), containing one or more alkyl tails on the backbone, have a propensity to form highly ordered nanostructures like nanotapes, twisted helices, nanotubes and cylindrical nanostructures. Further lateral interactions among peptides can also promote hydrogelation. Here we report the synthesis and the aggregation behaviour of four PAs containing cationic tetra- or hexa-peptides (C19-VAGK, C19-K1, C19-K2 and C19-K3) derivatized with a nonadecanoic alkyl chain. In their acetylated (Ac-) or fluorenylated (Fmoc-) versions, these peptides previously demonstrated the ability to form biocompatible hydrogels potentially suitable as extracellular matrices for tissue engineering or diagnostic MRI applications. In the micromolar range, PAs self-assemble in aqueous solution into nanotapes, or small clusters, resulting in high biocompatibility on HaCat cells up to 72 hours of incubation. Moreover, C19-VAGK also forms a gel at a concentration of 5 wt%.

A Comprehensive Analysis of the Intrinsic Visible Fluorescence Emitted by Peptide/Protein Amyloid-like Assemblies.

Amyloid aggregation is a widespread process that involves proteins and peptides with different molecular complexity and amino acid composition. The structural motif (cross-ß) underlying this supramolecular organization generates aggregates endowed with special mechanical and spectroscopic properties with huge implications in biomedical and technological fields, including emerging precision medicine. The puzzling ability of these assemblies to emit intrinsic and label-free fluorescence in regions of the electromagnetic spectrum, such as visible and even infrared, usually considered to be forbidden in the polypeptide chain, has attracted interest for its many implications in both basic and applied science. Despite the interest in this phenomenon, the physical basis of its origin is still poorly understood. To gain a global view of the available information on this phenomenon, we here provide an exhaustive survey of the current literature in which original data on this fluorescence have been reported. The emitting systems have been classified in terms of their molecular complexity, amino acid composition, and physical state. Information about the wavelength of the radiation used for the excitation as well as the emission range/peak has also been retrieved. The data collected here provide a picture of the complexity of this multifaceted phenomenon that could be helpful for future studies aimed at defining its structural and electronic basis and/or stimulating new applications.

Differently N-Capped Analogues of Fmoc-FF.

Short and ultra-short peptides have been recently envisioned as excellent building blocks for the formulation of hydrogels with appealing properties. Due to its simplicity and capability to gel under physiological conditions, Fmoc-FF (Nα -fluorenylmethoxycarbonyl-diphenylalanine), remains one of the most studied low molecular-weight hydrogelators. Since its first identification in 2006, a plethora of its analogues were synthetized and investigated for the fabrication of novel supramolecular materials. Here we report a description of the Fmoc-FF analogues in which the aromatic Fmoc group is replaced with other substituents. These analogues are distinguished into five different classes including derivatives: i) customized with solid phase peptide synthesis protecting groups; ii) containing non-aromatic groups, iii) containing aromatic groups, iv) derivatized with metal complexes and v) containing stimuli-responsive groups. The morphological, mechanical, and functional effects caused by this modification on the resulting material are also pointed out.

Caveolin-Mediated Internalization of Fmoc-FF Nanogels in Breast Cancer Cell Lines.

INTRODUCTION: Hydrogel nanoparticles, also known as nanogels (NGs), have been recently proposed as alternative supramolecular vehicles for the delivery of biologically relevant molecules like anticancer drugs and contrast agents. The inner compartment of peptide based NGs can be opportunely modified according to the chemical features of the cargo, thus improving its loading and release. A full understanding of the intracellular mechanism involved in nanogel uptake by cancer cells and tissues would further contribute to the potential diagnostic and clinical applications of these nanocarriers, allowing the fine tuning of their selectivity, potency, and activity. The structural characterization of nanogels were assessed by Dynamic Light Scattering (DLS) and Nanoparticles Tracking Analysis (NTA) analysis. Cells viability of Fmoc-FF nanogels was evaluated by MTT assay on six breast cancer cell lines at different incubation times (24, 48, and 72 h) and peptide concentrations (in the range 6.25 × 10-4 ÷ 5·10-3 × wt%). The cell cycle and mechanisms involved in Fmoc-FF nanogels intracellular uptake were evaluated using flow cytometry and confocal analysis, respectively. Fmoc-FF nanogels, endowed with a diameter of ~130 nm and a zeta potential of ~-20.0/-25.0 mV, enter cancer cells via caveolae, mostly those responsible for albumin uptake. The specificity of the machinery used by Fmoc-FF nanogels confers a selectivity toward cancer cell lines overexpressing the protein caveolin1 and efficiently performing caveolae-mediated endocytosis.

Ultrashort Cationic Peptide Fmoc-FFK as Hydrogel Building Block for Potential Biomedical Applications.

Fmoc-diphenylalanine (Fmoc-FF) is a low-molecular-weight peptide hydrogelator. This simple all-aromatic peptide can generate self-supporting hydrogel materials, which have been proposed as novel materials for diagnostic and pharmaceutical applications. Our knowledge of the molecular determinants of Fmoc-FF aggregation is used as a guide to design new peptide-based gelators, with features for the development of improved tools. Here, we enlarge the plethora of Fmoc-FF-based hydrogelated matrices by studying the properties of the Fmoc-FFK tripeptide, alone or in combination with Fmoc-FF. For multicomponent matrices, the relative weight ratios between Fmoc-FFK and Fmoc-FF (specifically, 1/1, 1/5, 1/10, and 1/20 w/w) are evaluated. All the systems and their multiscale organization are studied using different experimental techniques, including rheology, circular dichroism, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM). Preliminary profiles of biocompatibility for the studied systems are also described by testing them in vitro on HaCaT and 3T3-L1 cell lines. Additionally, the lysine (K) residue at the C-terminus of the Fmoc-FF moiety introduces into the supramolecular material chemical functions (amino groups) which may be useful for modification/derivatization with bioactive molecules of interest, including diagnostic probes, chelating agents, active pharmaceutical ingredients, or peptide nucleic acids.

Peptide-Based Hydrogels and Nanogels Containing Gd(III) Complexes as T1 Relaxation Agents.

New peptide-based hydrogels incorporating Gd(III) chelates with different hydration states, molecular structures and overall negative charges ([Gd(BOPTA)]2−), [Gd(DTPA)]2−, and ([Gd(AAZTA)]−) were prepared and characterized. N-terminal Fmoc- or acetyl-derivatized hexapeptides (K1, K2 and K3) containing five aliphatic amino acids (differently ordered Gly, Ala, Val, Leu and Ile) and a charged lysine at the amidated C-terminal were used for the formation of the hydrogels. Particular attention was paid to the investigation of the morphological and rheological properties of the nanoparticles, in addition to the assessment of the ability (relaxivity) of the confined complexes to accelerate the longitudinal relaxation rate of the water protons localized in the polymeric network. The relaxivity values at high magnetic fields (>0.5 T) of the paramagnetic hydrogels appear to be more than five times higher than those of isolated chelates in an aqueous solution, reaching a value of 25 mmol−1 s−1 for Fmoc-K2+[Gd(BOPTA)]2− at 0.5 T and 310 K. Furthermore, an interesting trend of decrease of relaxivity with increasing the degree of rigidity of the hydrogel was observed. The type of interactions between the various complexes and the polymeric network also plays a key role in influencing the relaxivity values of the final materials. Nanogels were also obtained from the submicronization of the hydrogel containing [Gd(BOPTA)]2− chelate. Circular dichroism, dynamic light scattering and relaxometric investigations on these nanoparticles revealed the formation of nanogels endowed with higher relaxivities (r1 = 41 mM−1 s−1 at 0.5 T MHz and 310 K) than the corresponding hydrogels.

Incorporation of PEG Diacrylates (PEGDA) Generates Hybrid Fmoc-FF Hydrogel Matrices.

Generated by a hierarchical and multiscale self-assembling phenomenon, peptide-based hydrogels (HGs) are soft materials useful for a variety of applications. Short and ultra-short peptides are intriguing building blocks for hydrogel fabrication. These matrices can also be obtained by mixing low-molecular-weight peptides with other chemical entities (e.g., polymers, other peptides). The combination of two or more constituents opens the door to the development of hybrid systems with tunable mechanical properties and unexpected biofunctionalities or morphologies. For this scope, the formulation, the multiscale analysis, and the supramolecular characterization of novel hybrid peptide-polymer hydrogels are herein described. The proposed matrices contain the Fmoc-FF (Nα-fluorenylmethyloxycarbonyl diphenylalanine) hydrogelator at a concentration of 0.5 wt% (5.0 mg/mL) and a diacrylate α-/ω-substituted polyethylene-glycol derivative (PEGDA). Two PEGDA derivatives, PEGDA 1 and PEGDA2 (mean molecular weights of 575 and 250 Da, respectively), are mixed with Fmoc-FF at different ratios (Fmoc-FF/PEGDA at 1/1, 1/2, 1/5, 1/10 mol/mol). All the multicomponent hybrid peptide-polymer hydrogels are scrutinized with a large panel of analytical techniques (including proton relaxometry, FTIR, WAXS, rheometry, and scanning electronic microscopy). The matrices were found to be able to generate mechanical responses in the 2-8 kPa range, producing a panel of tunable materials with the same chemical composition. The release of a model drug (Naphthol Yellow S) is reported too. The tunable features, the different topologies, and the versatility of the proposed materials open the door to the development of tools for different applicative areas, including diagnostics, liquid biopsies and responsive materials. The incorporation of a diacrylate function also suggests the possible development of interpenetrating networks upon cross-linking reactions. All the collected data allow a mutual comparison between the different matrices, thus confirming the significance of the hybrid peptide/polymer-based methodology as a strategy for the design of innovative materials.

Fmoc-Diphenylalanine Hydrogels: Optimization of Preparation Methods and Structural Insights.

Hydrogels (HGs) are tri-dimensional materials with a non-Newtonian flow behaviour formed by networks able to encapsulate high amounts of water or other biological fluids. They can be prepared using both synthetic or natural polymers and their mechanical and functional properties may change according to the preparation method, the solvent, the pH, and to others experimental parameters. Recently, many short and ultra-short peptides have been investigated as building blocks for the formulation of biocompatible hydrogels suitable for different biomedical applications. Due to its simplicity and capability to gel in physiological conditions, Fmoc-FF dipeptide is one of the most studied peptide hydrogelators. Although its identification dates to 15 ago, its behaviour is currently studied because of the observation that the final material obtained is deeply dependent on the preparation method. To collect information about their formulation, here are reported some different strategies adopted until now for the Fmoc-FF HG preparation, noting the changes in the structural arrangement and behaviour in terms of stiffness, matrix porosity, and stability induced by the different formulation strategy on the final material.

Multicomponent Hydrogel Matrices of Fmoc-FF and Cationic Peptides for Application in Tissue Engineering.

In the last years, peptide-based hydrogels are being increasingly used as suitable matrices for biomedical and pharmaceutical applications, including drug delivery and tissue engineering. Recently, the synthesis and the gelation properties of a small library of cationic peptides, containing a Lys residue at the C-terminus and derivatized with an Fmoc group or with the fluorenyl methoxycarbonyl-diphenylalanine (FmocFF) at the N-terminus are derived. Here, it is demonstrated that the combination of these peptides with the well-known hydrogelator FmocFF, in different weight/weight ratios, allows the achievement of seven novel self-sorted hydrogels, which share similar peptide organization of their supramolecular matrix. Rheological and relaxometric characterization highlight a different mechanical rigidity and water mobility in the gels as demonstrated by the storage modulus values (200 Pa < G' < 35 000 Pa) and by relaxometry, respectively. In vitro studies demonstrate that most of the tested mixed hydrogels do not disturb significantly the cell viability (>95%) over 72 h of treatment. Moreover, in virtue to its capability to strongly favor adhesion, spreading and duplication of 3T3-L1 cells, one of the tested hydrogel may be eligible as synthetic extracellular matrix.

Solid-state optical properties of self-assembling amyloid-like peptides with different charged states at the terminal ends.

The self-assembling of small peptides not only leads to the formation of intriguing nanoarchitectures, but also generates materials with unexpected functional properties. Oligopeptides can form amyloid-like cross-ß assemblies that are able to emit intrinsic photoluminescence (PL), over the whole near-UV/visible range, whose origin is still largely debated. As proton transfer between the peptide chain termini within the assembly is one of the invoked interpretations of this phenomenon, we here evaluated the solid state PL properties of a series of self-assembled hexaphenylalanine peptides characterized by a different terminal charge state. Overall, our data indicate that the charge state of these peptides has a marginal role in the PL emission as all systems exhibit very similar multicolour PL associated with a violation of the Kasha's rule. On the other hand, charged/uncharged ends occasionally produce differences in the quantum yields. The generality of these observations has been proven by extending these analyses to the Aß16-21 peptide. Collectively, the present findings provide useful information for deciphering the code that links the spectroscopic properties of these assemblies to their structural/electronic features.

Peptide-based hydrogels as delivery systems for doxorubicin.

Hydrogels (HGs) and nanogels (NGs) have been recently identified as innovative supramolecular materials for many applications in biomedical field such as in tissue engineering, optoelectronic, and local delivery of active pharmaceutical ingredients (APIs). Due to their in vivo biocompatibility, synthetic accessibility, low cost, and tunability, peptides have been used as suitable building blocks for preparation of HGs and NGs formulations. Peptide HGs have shown an outstanding potential to deliver small drugs, protein therapeutics, or diagnostic probes, maintaining the efficacy of their loaded molecules, preventing degradation phenomena, and responding to external physicochemical stimuli. In this review, we discuss the possible use of peptide-based HGs and NGs as vehicles for the delivery of the anticancer drug doxorubicin (Dox). This anthracycline is clinically used for leukemia, stomach, lung, ovarian, breast, and bladder cancer therapy. The loading of Dox into supramolecular systems (liposomes, micelles, hydrogels, and nanogels) allows reducing its cardiotoxicity. According to a primary sequence classification of the constituent peptide, doxorubicin-loaded systems are here classified in short and ultra-short peptide-based HGs, RGD, or RADA-peptide-based HGs and peptide-based NGs.

The Introduction of a Cysteine Residue Modulates The Mechanical Properties of Aromatic-Based Solid Aggregates and Self-Supporting Hydrogels.

Peptide-based hydrogels, originated by multiscale self-assembling phenomenon, have been proposed as multivalent tools in different technological areas. Structural studies and molecular dynamics simulations pointed out the capability of completely aromatic peptides to gelificate if hydrophilic and hydrophobic forces are opportunely balanced. Here, the effect produced by the introduction of a Cys residue in the heteroaromatic sequence of (FY)3 and in its PEGylated variant was evaluated. The physicochemical characterization indicates that both FYFCFYF and PEG8-FYFCFYF are able to self-assemble in supramolecular nanostructures whose basic cross-ß motif resembles the one detected in the ancestor (FY)3 assemblies. However, gelification occurs only for FYFCFYF at a concentration of 1.5 wt%. After cross-linking of cysteine residues, the hydrogel undergoes to an improvement of the rigidity compared to the parent (FY)3 assemblies as suggested by the storage modulus (G') that increases from 970 to 3360 Pa. The mechanical properties of FYFCFYF are compatible with its potential application in bone tissue regeneration. Moreover, the avalaibility of a Cys residue in the middle of the peptide sequence could allow the hydrogel derivatization with targeting moieties or with biologically relevant molecules.

Fluorescence Emission of Self-assembling Amyloid-like Peptides: Solution versus Solid State.

Analysis of the intrinsic UV-visible fluorescence exhibited by self-assembling amyloid-like peptides in solution and in solid the state highlights that their physical state has a profound impact on the optical properties. In the solid state, a linear dependence of the fluorescence emission peaks as a function of excitation wavelength is detected. On the contrary, an excitation-independent emission is observed in solution. The present findings constitute a valuable benchmark for current and future explanations of the fluorescence emission by amyloids.