Emerson Rodrigo da Silva

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.

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.

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.

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

Self-Assembly of Arg-Phe Nanostructures via the Solid-Vapor Phase Method

We report for the first time on the self-assembly of nanostructures composed exclusively of alternating positively charged and hydrophobic amino acids. A novel arginine/phenylalanine octapeptide, RF8, was synthesized. Because the low hydrophobicity of this sequence makes its spontaneous ordering through solution-based methods difficult, a recently proposed solid-vapor approach was used to obtain nanometric architectures on ITO/PET substrates. The formation of the nanostructures was investigated under different preparation conditions, specifically, under different gas-phase solvents (aniline, water, and dichloromethane), different peptide concentrations in the precursor solution, and different incubation times. The stability of the assemblies was experimentally studied by electron microscopy and thermogravimetric analysis coupled with mass spectrometry. The secondary structure was assessed by infrared and Raman spectroscopy, and the arrays were found to assume an antiparallel β-sheet conformation. FEG-SEM images clearly reveal the appearance of fibrillar structures that form extensive homogeneously distributed networks. A close relationship between the morphology and preparation parameters was found, and a concentration-triggered mechanism was suggested. Molecular dynamics simulations were performed to address the thermal stability and nature of intermolecular interactions of the putative assembly structure. Results obtained when water is considered as solvent shows that a stable lamellar structure is formed containing a thin layer of water in between the RF8 peptides that is stabilized by H-bonding.

Steric-induced effects on stabilizing a lamellar structure.

We investigate the behavior of multilamellar phases composed of lecithin and a commercial cosurfactant (Simusol), which is a mixture of ethoxylated fatty acids. Using X-ray scattering and a new procedure to fit the data, relevant parameters characterizing the lamellar structure were determined as a function of membrane composition, varying from 100% of lecithin to 100% of Simulsol. Scattering data illustrating the swelling of the lamellae for different amounts of cosurfactant are presented with the respective behavior of the Caillé parameter. With this experimental approach, we show that the incorporation of ethoxy brushes onto the lipid surface enhances repulsive interactions arising from membrane fluctuations and changes the interactions at the interface between bilayers.

O fenômeno do speckle como introdução à metrologia óptica no laboratório didático

In this work we present the measurement of the linear coefficient of thermal dilatation of aluminium, using the obtained interferogram from Fourier transform over the sum of two displaced speckle images. The achieved result is in good agreement with tabled value and the required components have an accessible cost, turning the experiment an interesting alternative for introduction of optical metrology in teaching laboratory or a project to be developed in experimental subjects.

Structural behaviour and gene delivery in complexes formed between DNA and arginine-containing peptide amphiphiles.

We describe in depth the structure of complexes formed between DNA and two classes of arginine-containing peptide amphiphiles, namely, the lipopeptide PRW-C16 (P = proline, R = arginine, W = tryptophan, C16 = C16 : 0 alkyl chain) and the bolaamphiphile RFL4FR (R = arginine, F = phenylalanine, L = leucine). A combination of X-ray and neutron scattering provided unprecedented insights into the local structure of these complexes. Lipopeptide-based complexes self-assembled into layered structures with large-scale fractal features, hosting DNA in the interstices. Bola-amphiphile scaffolds were characterized by planar structures with DNA strands presumably sandwiched in-between peptide nanotapes. Importantly, complexation did not affect the structural integrity of DNA in either of the two complexes. The bolaamphiphile conjugates displayed high levels of molecular ordering in contrast to the liquid-crystalline features observed in lipopeptide assemblies. Peptide-DNA complexes were assessed for their potential as a means to deliver the reporter vector pEGFP-N1 into SW480 human colon carcinoma cells. Successfully transfected cells expressed green fluorescent protein. The potentiating effect of PRW-C16 on the cellular uptake of ectopic DNA was found to be much greater than that observed with RFL4FR. In contrast to the bolaamphiphile-based conjugate, the liquid-crystalline nature of the lipopeptide complex is likely to play a key role in DNA release and transfection efficiency since these weakly bound structures require lower energy expenditure during disassembly and load release.