André Romero da Silva

In vitro photodynamic activity of chloro(5,10,15,20-tetraphenylporphyrinato)indium(III) loaded-poly(lactide-co-glycolide) nanoparticles in LNCaP prostate tumour cells

In(III)-meso-tetraphenylporphyrin (InTPP) was encapsulated into nanoparticles (smaller than 200 nm) of poly(d,l-lactide-co-glycolide) (PLGA) using the emulsification-evaporation technique. The photodynamic efficacy of InTPP-loaded nanoparticles and its cellular uptake was investigated with LNCaP prostate tumour cells, in comparison with the free InTPP. The effects of incubation time (1-3h), drug concentration (1.8-7.7 micromol/L) and incident light dose (15-45 J/cm(2)) with both encapsulated and free InTPP were studied. The type of cell death induced by the photochemical process using both encapsulated and free InTPP was also investigated. Cell viability was reduced more significantly with increasing values of these effects for InTPP-loaded nanoparticles than with the free drug. The cellular death induced by both encapsulated and free InTPP was preponderantly apoptotic. Confocal laser scanning microscopy data showed that the InTPP-loaded nanoparticles, as well free InTPP, were localized in the cells, and always in the perinuclear region. Encapsulated InTPP was measured by the intensity of fluorescence intensity of cell extracts and was three times more internalized into the cells than was the free InTPP. Electron paramagnetic resonance experiments corroborated the participation of singlet oxygen in the photocytotoxic effect of nanoparticles loaded with InTPP.

Involvement of mitochondria in apoptosis of cancer cells induced by photodynamic therapy

A terapia fotodinâmica (TFD) e um promissor tratamento clinico que emprega a combinacao de luz visivel, oxigenio e uma substância fotossensibilizadora para tratar doencas de carater oncologico. Esse tipo de tratamento induz a morte celular programada (apoptose) em tecido tumoral. No entanto, o mecanismo de inducao de apoptose por TFD nao esta bem caracterizado. Muitos fotossensibilizadores possuem como alvo principal a mitocondria. O tratamento com TFD causa danos nessa organela e induz a apoptose atraves da liberacao de citocromo c, fator de inducao de apoptose, Smac/DIABLO e certas pro-caspases. A liberacao dessas proteinas, a partir da mitocondria, e regulada por uma familia de proteinas pro e antiapoptoticas, conhecida como familia BCL-2. A proposta dessa minirrevisao e apresentar algumas publicacoes recentes que focalizam o envolvimento da mitocondria em apoptose de celulas cancerigenas induzida por TFD.A terapia fotodinâmica (TFD) e um promissor tratamento clinico que emprega a combinacao de luz visivel, oxigenio e uma substância fotossensibilizadora para tratar doencas de carater oncologico. Esse tipo de tratamento induz a morte celular programada (apoptose) em tecido tumoral. No entanto, o mecanismo de inducao de apoptose por TFD nao esta bem caracterizado. Muitos fotossensibilizadores possuem como alvo principal a mitocondria. O tratamento com TFD causa danos nessa organela e induz a apoptose atraves da liberacao de citocromo c, fator de inducao de apoptose, Smac/DIABLO e certas pro-caspases. A liberacao dessas proteinas, a partir da mitocondria, e regulada por uma familia de proteinas pro e antiapoptoticas, conhecida como familia BCL-2. A proposta dessa minirrevisao e apresentar algumas publicacoes recentes que focalizam o envolvimento da mitocondria em apoptose de celulas cancerigenas induzida por TFD.

Photodynamic activity of chloro(5,10,15,20-tetraphenylporphyrinato)indium(III)

A atividade fotodinâmica do cloro(5,10,15,20-tetrafenilporfirinato) de indio(III) (InTPP) in vitro foi investigado para possivel uso em terapia fotodinâmica (PDT). O rendimento quântico de oxigenio singlete do InTPP (FD = 0,72) em DMSO foi maior que da 5,10,15,20-tetrafenilporfirina (TPP) (FD = 0,52). Os sitios de ligacao entre os fotossensibilizadores e albumina bovina (BSA) sao independentes e com celulas vermelhas de sangue humano (RBC) sao cooperativos, com um e quatro sitios de ligacao por molecula, respectivamente. As constantes de associacao com BSA sao (1,15 ± 0,07) × 105 e (2,6 ± 0,1) × 104 L mol-1 e com RBC sao (2,40 ± 0,05) × 107 L mol-1 e (7,2 ± 0,2) × 104 L mol-1 para InTPP e Photofrin®, respectivamente. O InTPP foi mais eficiente do que Photofrin® em fotooxidar L-triptofano (Trp) e BSA quando maiores concentracoes dos fotossensibilizadores foram utilizadas (acima de 14 µmol L-1). O InTPP foi 1,37 a 1,5 vezes mais eficaz em fotooxidar as RBC do que Photofrin®. Nossos resultados indicam que o InTPP pode ser usado para estudos futuros de PDT.

Drug release from microspheres and nanospheres of poly(lactide-co-glycolide) without sphere separation from the release medium

A new technique using diffuse reflectance spectroscopy was developed that enables the monitoring of encapsulated drug release without particle separation from the assayed medium. Studies of chloro(5,10,15,20-tetraphenylporphyrinato)indium(III) (InTPP) release from microspheres and nanospheres of poly(lactide-co-glycolide) (PLGA) were performed using this new technique. The release of InTPP was biphasic, with an initial fast release followed by a second slower release. Mathematical models applied to the release profiles showed that the release of InTPP from the nanospheres was controlled by diffusion, which is to be expected for a substance homogeneously dispersed within the spheres. However, due to the large size distribution of the microspheres loaded with InTPP, the release profiles were irregular, hampering an adequate fit to mathematical models. Confocal analysis of microparticles showed that the InTPP appeared to be homogenously distributed within the microspheres and no preferential distribution of InTPP towards the interior or towards the surface of the spheres was observed.

Extracellular biosynthesis of silver nanoparticles using the cell-free filtrate of nematophagous fungus Duddingtonia flagrans

The biosynthesis of metallic nanoparticles (NPs) using biological systems such as fungi has evolved to become an important area of nanobiotechnology. Herein, we report for the first time the extracellular synthesis of highly stable silver NPs (AgNPs) using the nematophagous fungus Duddingtonia flagrans (AC001). The fungal cell-free filtrate was analyzed by the Bradford method and 3,5-dinitrosalicylic acid assay and used to synthesize the AgNPs in the presence of a 1 mM AgNO3 solution. They have been characterized by UV–Vis spectroscopy, X-ray diffraction, transmission electron microscopy, dynamic light scattering, Zeta potential measurements, Fourier-transform infrared, and Raman spectroscopes. UV–Vis spectroscopy confirmed bioreduction, while X-ray diffractometry established the crystalline nature of the AgNPs. Dynamic light scattering and transmission electron microscopy images showed approximately 11, 38 nm monodisperse and quasispherical AgNPs. Zeta potential analysis was able to show a considerable stability of AgNPs. The N–H stretches in Fourier-transform infrared spectroscopy indicate the presence of protein molecules. The Raman bands suggest that chitinase was involved in the growth and stabilization of AgNPs, through the coating of the particles. Our results show that the NPs we synthesized have good stability, high yield, and monodispersion.

Type II photooxidation mechanism of biomolecules using chloro (5,10,15,20-Tetraphenylporphyrinato) indium (III) as a photosensitizer

The photooxidation mechanism of bovine serum albumin (BSA), L-tryptophan (Trp) and red blood cells (RBC) by chloro(5,10,15,20-tetraphenylporphyrinato)indium(III) (InTPP) was investigated. The photooxidation rate of Trp, BSA and RBC by InTPP was decreased in the presence of NaN3. The presence of D2O increases the photooxidation rate of Trp and BSA and decreases that of RBC. This decrease is probably related to a reduction of the binding constant between InTPP and RBC in the presence of D2O. No significant change in biomolecule fluorescence or in the percent of hemolysis was observed when radical quenchers (ferricyanide, mannitol and dismutase superoxide) were used. Experiments using electron paramagnetic resonance (EPR) show that only 1O2 was generated by InTPP. A mechanistic model based on the preferential oxidation of Trp and BSA by singlet oxygen is proposed. The agreement between the experimental data and the kinetic model gives additional support to the predominance of a mechanism via 1O2 in biomolecule photooxidation by InTPP.

Determination of structural and thermodynamic parameters of bovine α-trypsin isoform in aqueous-organic media

The α-trypsin isoform is a globular protein that belongs to serine-protease family and has a polypeptide chain of 223 amino acid residues, six disulfide bridges and two domains with similar structures. The effects of aqueous-organic solvent (ethanol) in different concentration on the α-trypsin structure have been investigated by spectroscopic techniques and thermodynamic data analysis. The results from spectroscopic measurements, including far-UV Circular Dichroism, UV-vis absorption spectroscopy, intrinsic tryptophan fluorescence and dynamic light scattering (DLS) suggest the formation of partially folded states, instead of aggregate states, at high ethanol concentration (>60% v/v ethanol), with little loss of secondary structure, but with significant tertiary structure changes. The thermodynamic data (Tm and ΔH) suggest a loosening of intramolecular weak interactions, which reflects in a flexibility increase such that the catalytic capacity can be increased or decreased according to the ethanol concentration into the system. Overall results we suggest that in range of 0-60% v/v ethanol/buffer, α-trypsin undergoes reversible multimerization phenomena with catalytic activity. However from 60% v/v ethanol/buffer, population of folded partially states with less catalytic activity are predominant.

Quantification of Inter-particle Spacing Caused by Thiol Self-Assembled Monolayers Using Transmission Electron Microscopy

AbstractNano-surface modification techniques have been increasingly reported in a multitude of applications. However, the metal-ligand interface can be difficult to characterize, mainly due to the inadequacy of analytical methodologies. Here, we analyze this interface using transmission electron microscopy, which can determine the thickness of the linkers. This work presents a comprehensive physical characterization of gold nanoparticles with modified surfaces using conventional transmission electron microscopy. We have successfully demonstrated a simple and reliable protocol for the quantification of inter-particle spacing caused by SAM thiol ligands bound onto AuNPs. This approach is based on the linear correlation of the distance between the gold nanoparticles and the length of 3-mercaptopropionic acid (MPA), 11-mercaptoundecanoic acid (MUA), and 16-mercaptohexadecanoic acid (MHA). Graphical Abstract

Virola oleifera-capped gold nanoparticles showing radical-scavenging activity and low cytotoxicity

The development of effective nanoparticle therapeutics has been hindered by their surface characteristics, such as hydrophobicity and charge. Therefore, the success of biomedical applications with nanoparticles is governed by the control of these characteristics. In this article, we report an efficient green capping method for gold nanoparticles (AuNPs) by a reduction with sodium citrate and capping with Virola oleifera (Vo), which is a green exudate rich in polyphenols and flavonoids. The Vo-capped AuNPs were characterized by UV, DLS, FTIR, Raman, TEM, DPPH, FRAP and their cytotoxicity was evaluated on the viability of Murine macrophage cell. The AuNPs had an average particle size of 15 nm and were stable over a long time, as indicated by their unchanged SPR and zeta potential values. These nanoparticles were assessed for their antioxidant potential using DPPH and FRAP and demonstrated the highest antioxidant activities and low cytotoxicity. We propose that the Virola oleifera-capped AuNPs have potential biomedical applications.

Facile Synthesis of Monodisperse Gold Nanocrystals Using Virola oleifera

AbstractThe development of new routes and strategies for nanotechnology applications that only employ green synthesis has inspired investigators to devise natural systems. Among these systems, the synthesis of gold nanoparticles using plant extracts has been actively developed as an alternative, efficient, cost-effective, and environmentally safe method for producing nanoparticles, and this approach is also suitable for large-scale synthesis. This study reports reproducible and completely natural gold nanocrystals that were synthesized using Virola oleifera extract. V. oleifera resin is rich in epicatechin, ferulic acid, gallic acid, and flavonoids (i.e., quercetin and eriodictyol). These gold nanoparticles play three roles. First, these nanoparticles exhibit remarkable stability based on their zeta potential. Second, these nanoparticles are functionalized with flavonoids, and third, an efficient, economical, and environmentally friendly mechanism can be employed to produce green nanoparticles with organic compounds on the surface. Our model is capable of reducing the resin of V. oleifera, which creates stability and opens a new avenue for biological applications. This method does not require painstaking conditions or hazardous agents and is a rapid, efficient, and green approach for the fabrication of monodisperse gold nanoparticles. Graphical AbstractThe Virola oleifera reduction method for the synthesis of gold nanoparticles (AuNP’s)