Interaction of dicentrinone, an antitrypanosomal aporphine alkaloid isolated from Ocotea puberula (Lauraceae), in cell membrane models at the air-water interface.

In the present work, the oxoaporphine alkaloid dicentrinone was isolated, for the first time, from leaves of Ocotea puberula (Lauraceae). This alkaloid exhibited antiparasitic activity against trypomastigote forms of Trypanosoma cruzi (IC50 of 16.4 ± 1.7 µM), similar to the positive control benznidazole (IC50 of 18.7 ± 4.1 µM), reduced mammalian cytotoxicity (CC50 > 200 µM), and a selectivity index (SI) higher than 12. These results were correlated with the effects observed using cellular membrane models, represented by 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), in Langmuir monolayers. Dicentrinone was incorporated in the films, submitted to lateral compression, and characterized by tensiometry. As observed in compression-decompression and time-stability curves, dicentrinone expanded the lipid monolayers, decreased the compressional modulus of the film, and reduced the stability of the monolayer. Brewster Angle Microscopy and interfacial Infrared Spectroscopy showed that dicentrinone causes the monolayers to be segregated in phases, and to increase the number of gauche/trans conformers ratio for the lipid acyl methylene groups, indicating configurational disorder. As a result, dicentrinone caused a disturbance in the cell membrane models, altering the physicochemical properties of the lipid surface such as thermodynamic, rheological, morphological, and structural aspects. These results can be useful to understand the interactions between dicentrinone and lipid biological surfaces at the molecular level.

Is alkaline phosphatase biomimeticaly immobilized on titanium able to propagate the biomineralization process?

Tissue-nonspecific alkaline phosphatase (TNAP) is a key enzyme in the biomineralization process as it produces phosphate from a number of phospho-substrates stimulating mineralization while it also inactivates inorganic pyrophosphate, a potent mineralization inhibitor. We have previously reported on the reconstitution of TNAP on Langmuir monolayers as well as proteoliposomes. In the present study, thin films composed of dimyristoylphosphatidic acid (DMPA) were deposited on titanium supports by the Langmuir-Blodgett (LB) technique, and we determined preservation of TNAP's phosphohydrolytic activity after incorporation into the LB films. Increased mineralization was observed after exposing the supports containing the DMPA:TNAP LB films to solutions of phospho-substrates, thus evidencing the role of TNAP on the growth of calcium phosphates after immobilization. These coatings deposited on metallic supports can be potentially applied as osteoconductive materials, aiming at the optimization of bone-substitutes integration in vivo.

Chitosan effects on monolayers of zwitterionic, anionic and a natural lipid extract from E. coli at physiological pH.

Langmuir monolayers are used to simulate the biological membrane environment, acting as a mimetic system of the outer or the inner membrane leaflet. Herein, we analyze the interaction of membrane models with a partially N-acetylated chitosan (Ch35%) possessing a quasi-ideal random pattern of acetylation, full water solubility up to pH ≈ 8.5 and unusually high weight average molecular weight. Lipid monolayers containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethalonamine (DPPE), dipalmitoyl phosphatidyl glycerol (DPPG) or E. coli total lipid extract were spread onto subphases buffered at pH 4.5 or 7.4. The incorporation of Ch35% chitosan caused monolayer expansion and a general trend of decreasing monolayer rigidity with Ch35% concentration. Due to its relatively high content of N-acetylglucosamine (GlcNAc) units, Ch35% interactions with negatively charged monolayers and with E. coli extract were weaker than those involving zwitterionic monolayers or lipid rafts. While the smaller interaction with negatively charged lipids was unexpected, this finding can be attributed to the degree of acetylation (35%) which imparts a small number of charged groups for Ch35% to interact. Chitosan properties are therefore determinant for interactions with model cell membranes, which explains the variability in chitosan bactericide activity in the literature. This is the first study on the effects from chitosans on realistic models of bacterial membranes under physiological pH.

The role of size, charge, and cholesterol of cell membrane models in interactions with graphene oxide.

The growing in manufacturing and applications of graphene oxide (GO), a two-dimensional nanomaterial, highlights the need for a better understanding of its environmental impact and toxicity. This work investigates the interaction of GO with cell membrane models as an indication for GO's potential harmfulness. A wide range of biologically-relevant membrane parameters (size, charge and, cholesterol content) and simple optical techniques were used to evaluate the outcome of interactions of vesicular cell membrane models with GO. Loss of membrane integrity was found to be positively correlated with electrostatic attraction and negatively correlated with cholesterol content. The size of vesicle-GO aggregates increased as a function of initial vesicle size, while cholesterol content was found to have a negligible effect on aggregation. Interestingly, charged vesicles reduced vesicle-GO aggregate size either by electrostatic repulsion of negatively charge vesicles or by GO folding following attachment of positively charge vesicles. Overall, by examining how key biologically-relevant parameters of membrane models affect interactions with GO, we have augmented the understanding of the potential threats of GO towards biological cell and to the environment.

Incorporation of dehydrodieugenol, a neolignan isolated from Nectandra leucantha (Lauraceae), in lipid Langmuir monolayers as biomembrane models.

Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.

Evaluation of the effects in cellular membrane models of antitrypanosomal poly-thymolformaldehyde (PTF) using Langmuir monolayers.

The polymerization of bioactive compounds may be interesting because the supramolecular structures formed can boost biological action on microorganism membranes. In the present work, poly-thymolformaldehyde (PTF) activity, prepared by condensation of thymol and formaldehyde, was evaluated against trypomastigote forms of Trypanosoma cruzi and related with the physicochemical changes provided by the incorporation of the compound in protozoan cell membrane models. PTF exhibited an EC50 value of 23.4 µg/mL and no toxicity against mammalian cells (CC50 > 200 µg/mL). To understand the molecular action of PTF as an antiprotozoal candidate, this compound was incorporated in Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG) as a model for parasite cell membranes. PTF shifted DPPG surface pressure-area isotherms to higher areas, indicating its incorporation in the lipid films. Additionally, it changed the thermodynamic, compressional, structural, and morphological properties of the floating monolayers, decreasing the collapse pressure, reducing the surface elasticity, and segregating molecules at the interface, forming domains with different reflectivities. Infrared spectroscopy showed that the lipid films with PTF presented an increased rate of gauche/all-trans conformers for the methylene groups from the acyl chains, indicating molecular disorder. Therefore, these results show that PTF alters the physicochemical properties of DPPG monolayers as a model for protozoa cell membranes, which can enhance the comprehension of the parasitic action of PTF against T. cruzi.

A bactericide peptide changing the static and dilatational surface elasticity properties of zwitterionic lipids at the air-water interface: Relationship with the thermodynamic, structural and morphological properties.

In this paper, we studied how different hydrophilicity degrees of the polar groups of the lipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) influence the interaction of the antibiotic peptide vancomycin (VC), affecting the physicochemical properties of the monolayers, including thermodynamic, rheological, structural and morphological ones. Lipid Langmuir monolayers were prepared at air-water interfaces with VC aqueous solution as subphase and characterized with tensiometry, Brewster angle microscopy, infrared spectroscopy, dilatational, and interfacial shear rheology. The presence of PC or PE groups as polar head groups of the phospholipid monolayers modulated the interaction of VC adsorbing from the aqueous subphase since for DPPC, vancomycin condenses the monolayer, making it less stable, fluid, and more disordered. In contrast, for DPPE, vancomycin expands the monolayer, making it more stable, keeping the compressibility, and leading to the formation of interfacial aggregates, which are not observed for DPPC. We concluded thatelectrostatic interactions induced the insertion of the peptide into the polar heads of the monolayers (DPPE), while hydrophobic interactions, in addition to ion-dipole interactions, induced the adsorption of the peptide onto the polar head of the monolayers (DPPC).

Consequences of the exposure to bisphenol A in cell membrane models at the molecular level and hamster ovary cells viability.

The inadequate disposal and the difficulty in its removal from water treatment systems have made the endocrine disruptor bisphenol A (BPA) a significant hazard for humans and animals. The molecular-level mechanisms of BPA action are not known in detail, which calls for systematic investigations using cell membrane models. This paper shows that BPA affects Langmuir monolayers and giant unilamellar vesicles (GUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) used as membrane models, in a concentration-dependent manner and with effects that depend on BPA aggregation. BPA increases DPPC monolayer fluidity in surface pressure isotherms upon interacting with the headgroups through hydrogen bonding, according to polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). In DPPC GUVs, BPA induced wrinkling and distortion in the spherical shape of the vesicles, but this was only observed for fresh solutions where it is not aggregated. BPA also decreased the viability of hamster ovary cells (CHO) in in vitro experiments. In contrast, aged, aggregated BPA solutions did not affect the GUVs and even increased CHO viability. These results may be rationalized in terms of size-dependent effects of BPA, which may be relevant for its endocrine-disrupting effects.

The prion fragment PrP106-127 adopts a secondary structure typical of aggregated fibrils in langmuir monolayers of brain lipid extract.

Understanding protein aggregation is essential to unveil molecular mechanisms associated with neurodegenerative diseases such as Alzheimer's, Huntington's and spongiform encephalopathy, particularly to determine the role of interaction with cell membranes. In this study, we employ Langmuir monolayers as cell membrane models to mimic interaction with the peptide KTNMHKHMAGAAAAGAVVGGLG-OH, a fragment from the human prion protein including residues 106-127, believed to be involved in protein aggregation. Using in situ polarization-modulated infrared reflection adsorption spectroscopy (PM-IRRAS) for Langmuir monolayers and FTIR for solid films, we found that PrP106-127 adopts mainly ß-sheets, random coils and ß-turns in Langmuir monolayers and in Langmuir-Blodgett (LB) and cast films. This also applies to monolayers and solid films made with PrP106-127 and a brain total lipid extract (BTLE). In contrast, some α-helices are observed in the secondary structure of PrP106-127 in monolayers, and especially in solid films, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In summary, in a model representing brain cells (BTLE), the secondary structure of PrP106-127 is typical of fiber aggregates, while aggregation is unlikely if PrP106-127 interacts with a membrane model (DOPC) characteristic of mammalian cells.

Lipopolysaccharides and peptidoglycans modulating the interaction of Au naparticles with cell membranes models at the air-water interface.

Understanding the interactions between nanoparticles and biological surfaces is of great importance for many areas of nanomedicine and calls for detailed studies at the molecular level using simplified models of cellular membranes. In this paper, water-dispersed polyvinylpyrrolidonestabilized gold nanoparticles (AuNPs) were incorporated in floating monolayers of selected lipids at the air-water interface as cell membrane models. Surface pressure-area isotherms showed the condensation of glycoside-free lipid monolayers, suggesting their adsorption on the nanoparticle surface through the hydrophilic head groups. On the other hand, monolayers containing glycoside derivatives expanded upon AuNPs incorporation, pointing that the supramolecular structure formed should facilitate the incorporation of these nanoparticles in cellular membranes. These findings can be therefore correlated with the possible toxicity, microbicide and antitumorigenic effects of these nanoparticles in lipidic surfaces of erythrocyte and microbial membranes.

Evidence of photoinduced lipid hydroperoxidation in Langmuir monolayers containing Eosin Y.

Photodynamic therapy (PDT) efficiency depends on many factors including the incorporation of the photosensitizer (PS) in cell membranes and possible lipid hydroperoxidation. In this study, we show that hydroperoxidation may be photoinduced when eosin Y is incorporated into Langmuir monolayers that serve as cell membrane models. This occurs for Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which have unsaturation in their hydrophobic chains. In contrast, light irradiation had no effect on monolayers of saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Evidence of hydroperoxidation was obtained from the area increase in eosin-containing DOPC and POPC monolayers upon irradiation, which was accompanied by a decrease in monolayer thickness according to grazing incidence X-ray off-specular scattering (GIXOS) data. Furthermore, the changes in polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) induced by irradiation were consistent with hydroperoxide migration toward the lipid hydrophilic heads.. In summary, this combination of experimental methods allowed us to determine the effects of eosin Y interaction with cell membrane models under irradiation, which may be associated with the underlying mechanisms of eosin Y as photosensitizer in PDT.

Vibrational spectroscopy for probing molecular-level interactions in organic films mimicking biointerfaces.

Investigation into nanostructured organic films has served many purposes, including the design of functionalized surfaces that may be applied in biomedical devices and tissue engineering and for studying physiological processes depending on the interaction with cell membranes. Of particular relevance are Langmuir monolayers, Langmuir-Blodgett (LB) and layer-by-layer (LbL) films used to simulate biological interfaces. In this review, we shall focus on the use of vibrational spectroscopy methods to probe molecular-level interactions at biomimetic interfaces, with special emphasis on three surface-specific techniques, namely sum frequency generation (SFG), polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and surface-enhanced Raman scattering (SERS). The two types of systems selected for exemplifying the potential of the methods are the cell membrane models and the functionalized surfaces with biomolecules. Examples will be given on how SFG and PM-IRRAS can be combined to determine the effects from biomolecules on cell membrane models, which include determination of the orientation and preservation of secondary structure. Crucial information for the action of biomolecules on model membranes has also been obtained with PM-IRRAS, as is the case of chitosan removing proteins from the membrane. SERS will be shown as promising for enabling detection limits down to the single-molecule level. The strengths and limitations of these methods will also be discussed, in addition to the prospects for the near future.