In situ interaction between the hormone 17α-ethynylestradiol and the liquid-ordered phase composed of the lipid rafts sphingomyelin and cholesterol.

Hormone treatments are frequently associated with cardiovascular diseases and cancers in women. Additionally, the detrimental effects of their presence as contaminants in water remain a concern. The transport of hormones through cell membranes is essential for their biological action, but investigating cell permeability is challenging owing to the experimental difficulty in dealing with whole cells. In this paper, we study the interaction of the synthetic hormone 17α-ethynylestradiol (EE2) with membrane models containing the key raft components sphingomyelin (SM) and cholesterol (Chol). The models consisted of Langmuir monolayers and giant unilamellar vesicles (GUVs) that represent bilayers. EE2 induced expansion of SM monolayers upon interacting with the non-hydrated amide group of SM head, but it had practically no effect on SM GUVs because these group are not available for interaction in bilayers. In contrast, EE2 interacted with hydrated phosphate group (PO2-) and amide group of SM/Chol mixture monolayer, which could explain the loss in phase contrast of liquid-ordered GUVs suggesting pore formation. A comparison with reported EE2 effects on GUVs in the fluid phase, for which no loss in phase contrast was observed, indicates that the liquid-ordered phase consisting of lipid rafts is relevant to be associated with the changes on cell permeability caused by the hormones.

Chemical and morphological effects of the contraceptive hormone 17 α-ethynylestradiol on fluid lipid membranes.

The lack of studies involving the effects in human health associated with the chronic ingestion of pollutants lead to the path of investigating the action of these compounds in cell membrane models. We demonstrated the interaction (causes and consequences) of the hormone 17 α-ethinylestradiol (EE2) with lipid monolayers (prepared as Langmuir films) and bilayers prepared as small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs). Both fluidity and majority chemical composition of real plasma cell membrane were guaranteed using the phospholipid 1-palmitoil-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Surface pressure-mean molecular area (π-A) isotherms and PM-IRRAS measurements highlighted the strong interaction of EE2 with POPC monolayers, leading the hormone to remain at the air/water interface and promoting its penetration into the phospholipid hydrophobic chains. In the case of bilayers, the entrance of the hormone inside the SUV is likely facilitated by their high curvature. In GUVs, EE2 was responsible for changes in the spherical shape, forming structures like buds and lipid protrusions. The set of results indicates the strong effects of EE2 on fluid membranes, which is an important feature to predict its damage in human cells.

Langmuir-Schaefer Perylene Derivative Films: Influence of the Molecular Chemical Structure on the Supramolecular Arrangement.

Since the optical and electrical properties of organic thin films devices depend on their supramolecular arrangement and the molecular chemical structure, the understanding of such characteristics is essential for the optimization of these devices. In this study, we determine the supramolecular arrangement of thin films produced using the Langmuir-Schaefer (LS) technique and explain how its supramolecular arrangement is affected by the molecular chemical structure using two perylene derivatives: bis-butylimide (BuPTCD) and bis-phenethylimide (PhPTCD). The optical absorption measurements reveal that both films grow homogeneously and indicate that the presence of H aggregates (forbidden emission) is higher for BuPTCD LS film than for PhPTCD LS film. Atomic force microscopic analysis shows that the PhPTCD LS film is rougher than the BuPTCD film. In addition, FTIR analyses indicate that both films have head-on molecular organization. XRD patterns reveal that both the BuPTCD LS film and the PhPTCD LS film are crystalline, but that crystallinity is more prevalent in the BuPTCD LS film. Thus, the results show that the difference presented in the chemical structures leads the films to have different supramolecular arrangements, with consequences for their optical properties.

Near-infrared/visible-emitting nanosilica modified with silylated Ru(II) and Ln(III) complexes.

Three luminescent silica-based nanohybrids were fabricated by grafting of silylated Ru(II) and Nd/Yb(III) complexes onto mesoporous silica nanoparticles obtained by microemulsion method. The prepared nanohybrids were characterized by Fourier transform-Raman spectroscopy, solid state-nuclear magnetic resonance, high resolution-transmission electron microscopy and scanning and transmission electron microscopy techniques. The chemical integrity and the grafting of all complexes inside MSNs nanopores as well as a good distribution of metal complexes onto MSNs surface were achieved for all nanohybrids. Photophysical results revealed that by monitoring the excitation on Ru(II) moieties from SiO 2 -RuNd and SiO 2 -RuYb nanohybrids, the sensitization of NIR-emitting Nd/Yb(III) ions were successfully detected via energy transfer processes. Energy transfer rates (k EnT) of 0.20 × 107 and 0.11 × 107 s-1 and efficiencies of energy transfer (η EnT) of 40% and 27.5% were obtained for SiO 2 -RuNd and SiO 2 -RuYb nanohybrids, respectively. These results confirm the preparation of promising dual (near-infrared/visible)-emitting silica-based nanohybrids as new nanotools for applications as nanosensores and nanomarkers.

Luminescent nanohybrids based on silica and silylated Ru(II)-Yb(III) heterobinuclear complex: new tools for biological media analysis.

Lanthanide (Ln) complexes emitting in the near-infrared (NIR) region have fostered great interest as upcoming optical tags owing to their high spatial and temporal resolution emission as well deeper light penetration in biological tissues for non-invasive monitoring. For use in live-cell imaging, lanthanide complexes with long-wavelength absorption and good brightness are especially critical. Light-harvesting ligands of Ln complexes are typically excited in the ultraviolet region, which in turn trigger simultaneously autofluorescence and long-exposition damage of living systems. The association of d-metalloligands rather than organic chromophores enables the excitation of NIR-emitting Ln complex occurs in the visible region. Taking advantage of the long-lived excited states and intense absorption band in the ultraviolet (UV) to NIR region of Ru(II), we successfully design a dual-emitting (in the visible and NIR region) d-f heterobinuclear complex based on Ru(II) metalloligand and Yb(III) complex. In addition, we developed luminescent nanohybrids by grafting of Ru(II)-Yb(III) heterobinuclear complexes containing silylated ligands on the surface of mesoporous and dense silica matrix. The nanomarkers were successfully applied for imaging of murine melanoma B16-F10 and neonatal human dermal fibroblast HDFn cell cultures by one-photon or two-photon absorption using laser scanning confocal microscopy. Great cellular uptake, low cytotoxicity and the possibility to achieve visible and NIR emission via two-photons excitation show that the nanohybrids are remarkable markers for in vitro and a potential tool for in vivo applications.

pH and Charge Effects Behind the Interaction of Artepillin C, the Major Component of Green Propolis, With Amphiphilic Aggregates: Optical Absorption and Fluorescence Spectroscopy Studies.

Brazilian green propolis is one of the bee products most consumed in the world to prevent diseases, owing antioxidant, antimicrobial, anti-inflammatory and antitumor activities. The major component of Brazilian green propolis is Artepillin C (ArtC), a cinnamic acid derivative with two prenylated groups that improve the affinity of the compound for lipophilic environment. Here, we have employed optical absorption and fluorescence techniques to draw conclusions on how ArtC interacts with amphiphilic aggregates commonly used as model membranes having different charges in the polar head group. Optical absorption spectra were representative of the protonation state of ArtC, dictated by the local pH at the surface of micelles and lipid vesicles. Fluorescence results showed that, in the presence of micelles and vesicles, the polarizability around ArtC was modified, compared to the value in aqueous medium, and the molecule should be located preferentially on the surface region of the model membranes, with an enhanced interaction with the less ordered state of the lipid vesicles.

Interaction of Artepillin C with model membranes: Effects of pH and ionic strength.

Artepillin C is the major constituent of green propolis, one of the most consumed products in popular medicine owing to its therapeutic effects, including antitumor activity. Artepillin C differs from other cinnamic acid derivatives due to the presence of two prenylated groups in its structure, believed to enhance access to the cell membrane and resulting in pharmacological activity. The membrane outer leaflet of tumor cells is exposed to an acidic extracellular environment, which could modulate the protonation state of antitumor drugs and hence their interaction with the cell membrane. Herein, we investigated the interaction of Artepillin C with Langmuir monolayers and giant unilamellar vesicles (GUVs) of 1,2­dipalmitoyl­sn­glycerol­3­phosphocholine (DPPC) used as model membranes, in physiological and acidic environments. We observed that protonation of the carboxyl group of Artepillin C is essential for the interaction, with larger shifts induced in the surface pressure isotherms of DPPC monolayers in comparison with deprotonated Artepillin C. Also observed was a decrease in lipid packing inferred from the compressibility modulus and Brewster angle microscopy (BAM) images for monolayers on acidic subphases. Results with microscopy techniques on GUVs confirmed that. Artepillin C causes a curvature stress of the lipid bilayer only in its neutral state, causing the GUVs to burst. The stronger effects of neutral Artepillin C on both monolayers and GUVs were maintained when the ionic strength was increased. Taken together, the results indicate that Artepillin C may have preferential attachment to a more acidic environment which might be an important feature for its antitumor activity.

Photoluminescent properties in perylene PVD films: Influence of molecular aggregates and supramolecular arrangement.

Organic thin films are at the forefront of basic studies and applications in the field of physics, chemistry, biochemistry and materials science. For example, the intrinsic supramolecular arrangement, or simply the formation of aggregates may alter the optical and electrical properties, which would impact the potential applications of the material. Here, an attempt is made to correlate the molecular structures of two perylene derivatives, bis butylimido perylene (BuPTCD) and bis phenethylimido perylene (PhPTCD), with their film formation, in particular, the supramolecular arrangement and the photoluminescent properties. Emission spectra show that the PhPTCD has a radiative efficiency (RE) higher than that for BuPTCD when both are in solutions (monomers). Complementary, regarding PVD films, UV-Vis absorption measurements reveal that PhPTCD forms, predominantly, J aggregates, which are responsible for perylene derivative emission. However, BuPTCD PVD films are found to provide higher RE than PhPTCD PVD film. This apparent controversy could be explained considering other features such as crystallinity and molecular organization. The PVD film of BuPTCD is crystalline while PhPTCD PVD film is amorphous; BuPTCD has an edge-on while PhPTCD has a face-on molecular organization in PVD films.

Optical absorption and fluorescence spectroscopy studies of Artepillin C, the major component of green propolis.

The bioactivity of propolis against several pathogens is well established, leading to the extensive consumption of that bee product to prevent diseases. Brazilian green propolis, collected by the species Apis mellifera, is one of the most consumed in the world. The chemical composition of green propolis is complex and it has been shown that it displays antioxidant, antimicrobial, anti-inflammatory and antitumor activities, especially due to the high content of Artepillin C. The molecule is a derivative of cinnamic acid with two prenylated groups, responsible for the improvement of the affinity of the compound for lipophilic environment. A carboxylic group (COOH) is also present in the molecule, making it a pH-sensitive compound and the pH-dependent structure of Artepillin C, may modulate its biological activity related to interactions with the cellular membrane of organisms and tissues. Molecular properties of Artepillin C on aqueous solution were examined by optical absorption, steady state and time-resolved fluorescence spectroscopies. Acid-base titration based on the spectral position of the near UV absorption band, resulted in the pKa value of 4.65 for the carboxylic group in Artepillin C. In acidic pH, below the pKa value, an absorption band raised around 350nm at Artepillin C concentration above 50µM, due to aggregation of the molecule. In neutral pH, with excitation at 310nm, Artepillin C presents dual emission at 400 and 450nm. In pH close to the pKa, the optical spectra show contribution from both protonated and deprotonated species. A three-exponential function was necessary to fit the intensity decays at the different pHs, dominated by a very short lifetime component, around 0.060ns. The fast decay resulted in emission before fluorescence depolarization, and in values of fluorescence anisotropy higher than could be expected for monomeric forms of the compound. The results give fundamental knowledge about the protonation-deprotonation state of the molecule, that may be relevant in processes mediated by biological membranes.

Interaction of Artepillin C with model membranes.

Green propolis, a mixture of beeswax and resinous compounds processed by Apis mellifera, displays several pharmacological properties. Artepillin C, the major compound in green propolis, consists of two prenylated groups bound to a phenyl group. Several studies have focused on the therapeutic effects of Artepillin C, but there is no evidence that it interacts with amphiphilic aggregates to mimic cell membranes. We have experimentally and computationally examined the interaction between Artepillin C and model membranes composed of dimyristoylphosphatidylcholine (DMPC) because phosphatidylcholine (PC) is one of the most abundant phospholipids in eukaryotic cell membranes. PC is located in both outer and inner leaflets and has been used as a simplified membrane model and a non-specific target to study the action of amphiphilic molecules with therapeutic effects. Experimental results indicated that Artepillin C adsorbed onto the DMPC monolayers. Its presence in the lipid suspension pointed to an increased tendency toward unilamellar vesicles and to decreased bilayer thickness. Artepillin C caused point defects in the lipid structure, which eliminated the ripple phase and the pre-transition in thermotropic chain melting. According to molecular dynamics (MD) simulations, (1) Artepillin C aggregated in the aqueous phase before it entered the bilayer; (2) Artepillin C was oriented along the direction normal to the surface; (3) the negatively charged group on Artepillin C was accommodated in the polar region of the membrane; and (4) thinner regions emerged around the Artepillin C molecules. These results help an understanding of the molecular mechanisms underlying the biological action of propolis.

Miltefosine-loaded lipid nanoparticles: Improving miltefosine stability and reducing its hemolytic potential toward erythtocytes and its cytotoxic effect on macrophages.

The toxic effects of miltefosine on the epithelial cells of the gastrointestinal tract and its hemolytic action on erythrocytes have limited its use as an antileishmanial agent. As part of our search for new strategies to overcome the side effects of miltefosine during the treatment of leishmaniasis, we have developed stable miltefosine-loaded lipid nanoparticles in an attempt to reduce the toxic effects of the drug. We have evaluated lipid nanoparticles containing varying amounts of miltefosine and cholesterol, prepared by sonication, in terms of their physicochemical properties, preliminary stability, hemolytic potential toward erythrocytes, and cytotoxicity to macrophages and to promastigote and amastigote forms of Leishmania (L.) chagasi. Miltefosine loading into lipid nanoparticles was 100% for low drug concentrations (7.0 to 20.0mg/mL). Particle size decreased from 143nm (control) to between 43 and 69nm. From fluorescence studies, it was observed that the presence of miltefosine and cholesterol (below 103µM) promoted ordering effects in the phospholipid region of the nanoparticles. The formulation containing 15mg/mL miltefosine was stable for at least six months at 4°C and in simulated gastrointestinal fluids, and did not promote epithelial gastrointestinal irritability in Balb/C mice. When loaded into lipid nanoparticles, the hemolytic potential of miltefosine and its cytotoxicity to macrophages diminished, while its antiparasitic activity remained unaltered. The results suggested that miltefosine-loaded lipid nanoparticles may be promising for the treatment of leishmaniasis and might be suitable for oral and parenteral use.

Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake.

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, ß and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy.

Static and time-resolved fluorescence of tryptophan and ortho-aminobenzoic acid was used to investigate the interaction of the synthetic antimicrobial peptide L1A (IDGLKAIWKKVADLLKNT-NH2) with POPC and POPC:POPG. N-acetylated (Ac-L1A) and N-terminus covalently bonded ortho-aminobenzoic acid (Abz-L1A-W8V) were also used. Static fluorescence and quenching by acrylamide showed that the peptides adsorption to the lipid bilayers was accompanied by spectral blue shift and by a decrease in fluorescence quenching, indicating that the peptides moved to a less polar environment probably buried in the lipidic phase of the vesicles. These results also suggest that the loss of the N-terminus charge allowed deeper fluorophore insertion in the bilayer. Despite the local character of spectroscopic information, conclusions can be drawn about the peptides as a whole. The dynamic behaviors of the peptides are such that the mean intensity lifetimes, the long correlation time and the residual anisotropy at long times increased when the peptides adsorb in lipid vesicles, being larger in anionic vesicles. From the steady-state increase in fluorescence intensity and anisotropy, we observed that the partition coefficient of peptides L1A and its Abz analog in both types of vesicles are higher than the acetylated analog; moreover, the affinity to the anionic vesicle is higher than to the zwitterionic.

Comparison between cucurbiturils and ß-cyclodextrin interactions with cholesterol molecules present in Langmuir monolayers used as a biomembrane model.

Specific surface techniques can probe the interaction of cholesterol (Chol) with substances that are able to host and/or sequester this biomolecule, provided that the additives are properly assembled at the interface. Reports on inclusion complexes of Chol with ß-cyclodextrins exist in the literature. Here we compare the interaction of ß-cyclodextrin and cucurbiturils with Chol present in Langmuir phospholipid (dipalmitoylphosphatidylcholine, DPPC) monolayers, used as a biomembrane model. Cucurbiturils, CB[n], comprise macrocyclic host molecules consisting of n glycoluril units. Classic surface pressure curves, dilatational surface viscoelasticity measurements, and fluorescence emission spectra and images obtained by time-resolved fluorescence of the corresponding Langmuir-Blodgett films have shown that homologues with 5 and 6 glycoluril units, CB[5] and CB[6], do not form inclusion complexes. Higher-order homologues, such as CB[7], are likely to complex with Chol with changes in the minimum molecular areas recorded for DPPC/Chol monolayers, the fluorescence decay lifetimes, and the dilatational surface viscosities of the monolayers generated in the presence of these molecules. Moreover, we proof the removal of cholesterol from the biomimetic interface in the presence of CB[7] by means of fluorescence spectra from the subphase support of monolayers containing fluorescent-labeled Chol.