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.

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.

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.

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.

Injectable Tripeptide/Polymer Nanoparticles Supramolecular Hydrogel: A Candidate for the Treatment of Inflammatory Pathologies.

Supramolecular peptide-based hydrogels attract great attention in several fields, i.e., biomedicine, catalysis, energy, and materials chemistry, due to the noncovalent nature of the self-assembly and functional tunable properties defined by the amino acid sequence. In this work, we developed an injectable hybrid supramolecular hydrogel whose formation was triggered by electrostatic interactions between a phosphorylated tripeptide, Fmoc-FFpY (F: phenylalanine, pY: phosphorylated tyrosine), and cationic polymer nanoparticles made of vinylimidazole and ketoprofen (poly(HKT-co-VI) NPs). Hydrogel formation was assessed through inverted tube tests, and its fibrillary structure, around polymer NPs, was observed by transmission electron microscopy. Interestingly, peptide self-assembly yields the formation of nontwisted and twisted fibers, which could be attributed to ß-sheets and α-helix structures, respectively, as characterized by circular dichroism and infrared spectroscopies. An increase of the elastic modulus of the Fmoc-FFpY/polymer NPs hybrid hydrogels was observed with peptide concentration as well as its injectability property, due to its shear thinning behavior and self-healing ability. After checking their stability under physiological conditions, the cytotoxicity properties of these hybrid hydrogels were evaluated in contact with human dermal fibroblasts (FBH) and murine macrophages (RAW 264.7). Finally, the Fmoc-FFpY/polymer NPs hybrid hydrogels exhibited a great nitric oxide reduction (∼67%) up to basal values of pro-inflammatory RAW 264.7 cells, thus confirming their excellent anti-inflammatory properties for the treatment of localized inflammatory pathologies.

Constructing a Facile Biocomputing Platform Based on Smart Supramolecular Hydrogel Film Electrodes with Immobilized Enzymes and Gold Nanoclusters.

Herein, fluorescent gold nanoclusters (AuNCs) and horseradish peroxidase (HRP) were simultaneously embedded into self-assembled dipeptide supramolecular films of N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) on the surface of ITO electrodes (Fmoc-FF/AuNCs/HRP) by using a simple single-step process. In the films, both the fluorescence property of AuNCs and the bioelectrocatalytic property of HRP were well maintained and could be reversibly regulated by pH-sensitive structural changes in the Fmoc-FF hydrogel films. Cu(II)/EDTA in the solution could lead to the aggregation/disaggregation of AuNCs and further quenching/dequenching the fluorescence signal from the films. Meanwhile, the blue complexes formed by Cu(II) and EDTA could produce a UV-vis signal in the solution. In addition, the coordinated Cu(II) in the films enhanced the electrocatalytic capacity toward the reduction of H2O2 and could switch the current signal. A biomolecular logic circuit was built based on the smart film electrode system by using pH, the concentrations of EDTA, Cu(II) and H2O2 as inputs, while the fluorescence intensity (FL), current (I) and UV-vis extinction (E) of the solution as outputs. Various logic devices were fabricated using the uniform platform, consisting of an encoder/decoder, demultiplexer, dual-transfer gate, keypad lock, digital comparator, half adder, and controlled NOT (CNOT) gate. Specifically, an electronic three-value logic gate, gullibility (ANY) gate, was first mimicked in this biocomputing system. This work not only demonstrated the construction of a new type of multivalued logic gate by using a dipeptide micromolecular matrix but also provided a new approach for designing sophisticated biologic functions, establishing smart multianalyte biosensing or fabricating biology information processing through the use of a simple film system.

Supramolecular tripeptide self-assembly initiated at the surface of coacervates by polyelectrolyte exchange.

Spatial control of supramolecular self-assembly can yield compartmentalized structures, a key feature for the design of artificial cells. Inducing self-assembly from and on compartments is still a challenge. Polyelectrolyte complex coacervates are simple model droplet systems able to reproduce the basic features of membrane-less organelles, appearing in cells. Here, we demonstrate the supramolecular self-assembly of a phosphorylated tripeptide, Fmoc-FFpY (Fmoc: fluorenyl-methoxycarbonyl; F: phenyl alanine, pY: phosphorylated tyrosine), on the surface of poly(l-glutamic acid)/poly(allylamine hydrochloride) (PGA/PAH) complex coacervate microdroplets. The phosphorylated peptides self-assemble, without dephosphorylation, through ion pairing between the phosphate groups of Fmoc-FFpY and the amine groups of PAH. This process provides spontaneous capsules formed by an amorphous polyelectrolyte complex core surrounded by a structured peptide/PAH shell. Similar fibrillar Fmoc-FFpY self-assembled structures are obtained at the interface between the peptide solution and a PGA/PAH polyelectrolyte multilayer, a complex coacervate in the thin film or "multilayer" format. In contact with the peptide solution, PAH chains diffuse out of the coacervate or multilayer film and complex with Fmoc-FFpY at the solution interface, exchanging any PGA with which they were associated. Self-assembly of Fmoc-FFpY, now concentrated by complexation with PAH, follows quickly.