Wendel A. Alves

L-Diphenylalanine Microtubes As a Potential Drug-Delivery System: Characterization, Release Kinetics, and Cytotoxicity

Microtubes obtained from the self-assembly of L-diphenylalanine (FF-MTs) were evaluated as potential vehicles for drug delivery. The biological marker Rhodamine B (RhB) was chosen as a model drug and conjugated to the peptide arrays during self-organization in the liquid phase. Microscopy and X-ray studies were performed to provide morphological and structural information. The data revealed that the cargo was distributed either in small aggregates at the hydrophobic surface of the FF-MTs or homogeneously embedded in the structure, presumably anchored at polar sites in the matrix. Raman spectroscopy revealed notable shifts of the characteristic RhB resonance peaks, demonstrating the successful conjugation of the fluorophore and peptide assemblies. In vitro assays were conducted in erythrocytes and fibroblast cells. Interestingly, FF-MTs were found to modulate the release of the load. The release of RhB from the FF-MTs followed first-order kinetics with a steady-state profile, demonstrating the potential of these carriers to deliver drugs at constant rates in the body. Cytotoxicity investigations revealed high cell viability up to concentrations of 5 mg mL(-1), demonstrating the low toxicity of the FF-MTs.

Electrochemical Determination of Dopamine Based on Self-Assembled Peptide Nanostructure

Self-assembled peptide nanostructures are electronically insulating as are most biomaterials derived from natural amino acids. To obtain additional properties and increase the applicability of peptide nanomaterials, some chemical modifications can be performed and materials can be functionalized to form hybrid compounds. In this work, we described the formation of L-diphenylalanine nanotubes (PNTs) with cyclic-tetrameric copper(II) species containing the ligand (4-imidazolyl)ethylene-2-amino-1-ethylpyridine [Cu(4)(apyhist)(4)](4+) in the Nafion membrane on a vitreous carbon electrode surface. This copper complex has been studied as structural and functional models for the active centers of copper containing redox enzymes. Scanning electron microscopy was used to confirm the formation of the nanostructures. The electrochemical properties of the PNT-[Cu(4)(apyhist)(4)](4+)/Nafion film on a glassy carbon electrode were characterized using cyclic voltammetry and square-wave voltammetry and showed high electrocatalytic activity toward the oxidation of dopamine (DA). The detection sensitivity was found to be enhanced by the use of copper(II) complex in the PNTs/Nafion films. Under the optimum conditions, the square-wave voltammetry peak height was linearly related to the DA concentration over two concentration intervals, viz., 5.0-40 μmol L(-1) and 40-1000 μmol L(-1). The detection limit was 2.80 μmol L(-1) (S/N = 3), and ascorbic acid did not interfere with the DA detection. These results suggested that this hybrid bioinorganic system provides an attractive advantage for a new type of electrochemical sensors. The detection sensitivity was found to be enhanced by use of PNTs.

The adsorption of 2‚ 2’: 6’‚ 2’’-terpyridine‚ 4’-(5-mercaptopentyl)-2‚ 2’: 6’‚ 2’’-terpyridinyl‚ and perchlorate on silver and copper surfaces monitored by SERS

Abstract The adsorption of 2,2′:6′,2″-terpyridine on Ag and Cu electrode surfaces has been studied by using surface enhanced Raman spectroscopy (SERS). Two types of surface complexes have been found on a silver electrode: the first one is stable near the open circuit potential, having bonding between tpy and silver active site analogous to the AgN bonding in the [AgI(tpy)Cl]·2H2O complex; and the second one is stable in the −0.1 to −0.5 V (Ag/AgCl) potential range, where the interaction between tpy and silver active site is similar to the CuN bonding in the [CuII(tpy)Cl2] complex. The SERS spectra of 4′-(5-mercaptopentyl)-2,2′:6′,2″-terpyridinyl (tpy–SH) have shown an increase in the relative intensity of the mercaptopentyl moiety bands and a band at 310 cm−1, assigned to the AgS stretching, indicating an adsorption through the S atom. In addition, using the Raman results of different copper perchlorate salts obtained with different hydration degrees, it has been possible to characterize the adsorption of perchlorate anion on silver electrode and the kind of coordination in the [CuII(tpy)(H2O)(ClO4)]ClO4 and [CuII(tpy)(H2O)(ClO4)2] complexes.

Structural and photophysical properties of peptide micro/nanotubes functionalized with hypericin.

Hypericin is a photosensitizer with promising applications in photodynamic therapy (PDT) for cancer and infectious diseases treatments. Herein, we present a basic research study of L-diphenylalanine micro/nanotubes (FF-NTs) functionalized with hypericin. The system has special properties according to the hypericin concentration, with direct consequences on both morphological and photophysical behaviors. A clear dependence between the size of the tubes and the concentration of hypericin is revealed. The generation of reactive oxygen species (ROS) is found to be improved by ∼57% in the presence of FF-NTs, as indirectly measured from the absorbance profile of 1,3-diphenylisobenzofuran (DPBF). In addition, when hypericin appears conjugated with FF-NTs, the characteristic fluorescence lifetime is significantly boosted, demonstrating the role of FF-NTs to enhance the photophysical properties and stabilizing the fluorophore in excited states. Electron paramagnetic resonance allows the proposition of a mechanism for the generation of ROS. Molecular dynamics simulations bring new insights into the interaction between hypericin and peptide assemblies, suggesting the spatial organization of the fluorophore onto the surface of the supramolecular structures as a key element to improve the photophysical properties reported here.

Self-Assembled Arginine-Capped Peptide Bolaamphiphile Nanosheets for Cell Culture and Controlled Wettability Surfaces.

The spontaneous assembly of a peptide bolaamphiphile in water, namely, RFL4FR (R, arginine; F, phenylalanine; L, leucine) is investigated, along with its novel properties in surface modification and usage as substrates for cell culture. RFL4FR self-assembles into nanosheets through lateral association of the peptide backbone. The L4 sequence is located within the core of the nanosheets, whereas the R moieties are exposed to the water at the surface of the nanosheets. Kinetic assays indicate that the self-assembly is driven by a remarkable two-step process, where a nucleation phase is followed by fast growth of nanosheets with an autocatalysis process. The internal structure of the nanosheets is formed from ultrathin bolaamphiphile monolayers with a crystalline orthorhombic symmetry with cross-β organization. We show that human corneal stromal fibroblast (hCSF) cells can grow on polystyrene films coated with films dried from RFL4FR solutions. For the first time, this type of amphiphilic peptide is used as a substrate to modulate the wettability of solid surfaces for cell culture applications.

The effects of water molecules on the electronic and structural properties of peptide nanotubes

The self-assembly of short amino acid chains appears to be one of the most promising strategies for the fabrication of nanostructures. Their solubility in water and the possibility of chemical modification by targeting the amino or carboxyl terminus give peptide-based nanostructures several advantages over carbon nanotube nanostructures. However, because these systems are synthesized in aqueous solution, a deeper understanding is needed on the effects of water especially with respect to the electronic, structural and transport properties. In this work, the electronic properties of L-diphenylalanine nanotubes (FF-NTs) have been studied using the Self-Consistent Charge Density-Functional-based Tight-Binding method augmented with dispersion interaction. The presence of water molecules in the central hydrophilic channel and their interaction with the nanostructures are addressed. We demonstrate that the presence of water leads to significant changes in the electronic properties of these systems decreasing the band gap which can lead to an increase in the hopping probability and the conductivity.

Self-Assembly of a Designed Alternating Arginine/Phenylalanine Oligopeptide

A model octapeptide peptide consisting of an alternating sequence of arginine (Arg) and phenylalanine (Phe) residues, namely, [Arg-Phe]4, was prepared, and its self-assembly in solution studied. The simple alternating [Arg-Phe]4 peptide sequence allows for unique insights into the aggregation process and the structure of the self-assembled motifs. Fluorescence and UV-vis assays were used to determine critical aggregation concentrations, corresponding to the formation of oligomeric species and β-sheet rich structures organized into both spheroidal aggregates and highly ordered fibrils. Electron and atomic force microscopy images show globular aggregates and long unbranched fibers with diameters ranging from ∼4 nm up to ∼40 nm. Infrared and circular dichroism spectroscopy show the formation of β-sheet structures. X-ray diffraction on oriented stalks show that the peptide fibers have an internal lamellar structure, with an orthorhombic unit cell with parameters a ∼ 27.6 Å, b ∼ 9.7 Å, and c ∼ 9.6 Å. In situ small-angle X-ray scattering (SAXS) shows the presence of low molecular weight oligomers in equilibrium with mature fibers which are likely made up from 5 or 6 intertwined protofilaments. Finally, weak gel solutions are probed under gentle shear, suggesting the ability of these arginine-rich fibers to form networks.

A nonenzymatic biosensor based on gold electrodes modified with peptide self-assemblies for detecting ammonia and urea oxidation.

We have developed a nonenzymatic biosensor for the detection of ammonia and urea oxidation based on the deposition of peptide microstructures onto thiolated gold electrodes. FF-MNSs/MCP/Au assemblies were obtained by modifying gold substrates with 4-mercaptopyridine (MCP), followed by coating with l,l-diphenylalanine micro/nanostructures (FF-MNSs) grown in the solid-vapor phase. Benzene rings and amide groups with peptide micro/nanostructures interact with synthetic NH4(+) receptors through cation-π and hydrogen bonding. AuOH clusters on the Au surface provided the catalytic sites. The application of a predetermined concentration of analytes at the peptide interfaces activated the catalytic sites. We observed a relationship between the stability of films and the crystal structure of peptides, and we organized the FF-MNSs into an orthorhombic symmetry that was the most suitable assembly for creation of our biosensors. At 0.1 mol L(-1) NaOH, these FF-MNSs/MCP/Au electrodes have electrocatalytic properties regarding ammonia and urea oxidation that are comparable to those of enzyme-based architectures. Under optimal conditions, the electrocatalytic response is proportional to the ammonia and urea concentration in the range 0.1-1.0 mmol L(-1). The sensitivity was calculated as 2.83 and 81.3 μA mmol L(-1) cm(-2) for ammonia and urea, respectively, at +0.40 V (vs SCE). Our detection method is easy to follow, does not require a mediator or enzyme, and has strong potential for detecting urea via nonenzymatic routes.

Bioinspired peptide nanostructures for organic field-effect transistors.

Peptide-based nanostructures derived from natural amino acids are superior building blocks for biocompatible devices as they can be used in a bottom-up process without the need for expensive lithography. A dense nanostructured network of l,l-diphenylalanine (FF) was synthesized using the solid-vapor-phase technique. Formation of the nanostructures and structure-phase relationship were investigated by electron microscopy and Raman scattering. Thin films of l,l-diphenylalanine micro/nanostructures (FF-MNSs) were used as the dielectric layer in pentacene-based field-effect transistors (FETs) and metal-insulator-semiconductor diodes both in bottom-gate and in top-gate structures. Bias stress studies show that FF-MNS-based pentacene FETs are more resistant to degradation than pentacene FETs using FF thin film (without any nanostructures) as the dielectric layer when both are subjected to sustained electric fields. Furthermore, it is demonstrated that the FF-MNSs can be functionalized for detection of enzyme-analyte interactions. This work opens up a novel and facile route toward scalable organic electronics using peptide nanostructures as scaffolding and as a platform for biosensing.

The role of water and structure on the generation of reactive oxygen species in peptide/hypericin complexes

Hybrid associates formed between peptide assemblies and fluorophores are attractive mainly because of their unique properties for biomedical applications. Recently, we demonstrated that the production of reactive oxygen species (ROS) by hypericin and their stability in excited states are enhanced upon conjugation with l,l‐diphenylalanine microtubes (FF‐MNTs). Although the detailed mechanisms responsible for improving the photophysical properties of ROS remain unclear, tentative hypotheses have suggested that the driving force is the growth of overall dipolar moments ascribed either to coupling between aligned H2O dipoles within the ordered structures or to the organization of hypericin molecules on peptide interfaces. To provide new insights on ROS activity in hypericin/FF‐MNTs hybrids and further explore the role of water in this respect, we present results obtained from investigations on the behavior of these complexes organized into different crystalline arrangements. Specifically, we monitored and compared the photophysical performance of hypericin bound to FF‐MNTs with peptides organized in both hexagonal (water‐rich) and orthorhombic (water‐free) symmetries. From a theoretical perspective, we present the results of new molecular dynamics simulations that highlight the distinct hypericin/peptide interaction at the interface of FF‐MNTs for the different symmetries. As a conclusion, we propose that although water enhances photophysical properties, the organization induced by peptide structures and the availability of a hydrophobic environment surrounding the hypericin/peptide interface are paramount to optimizing ROS generation. The findings presented here provide useful basic research insights for designing peptide/fluorophore complexes with outstanding technological potential. Copyright