Thatyane M. Nobre

Interaction of chitosan and mucin in a biomembrane model environment.

Chitosans have been widely exploited in biological applications, including drug delivery and tissue engineering, especially owing to their mucoadhesive properties, but the molecular-level mechanisms for the chitosan action are not known in detail. It is believed that chitosan could affect the mucus by interacting with the proteins mucins, in a process mediated by the cell membrane. In this study we used Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) as simplified membrane models to investigate the interplay between the activity of mucins and chitosan. Surface pressure and surface potential measurements were performed with DMPA monolayers onto which chitosan and/or mucin was adsorbed. We found that the expanding effect from mucin was considerably reduced when chitosan was injected after mucin had been adsorbed on the DMPA monolayer. The results were consistent with the formation of complexes between mucin and chitosan, thus highlighting the importance of electrostatic interactions. Furthermore, chitosan could remove mucin that was co-deposited along with DMPA in Langmuir-Blodgett (LB) films, which could be ascribed to molecular-level interactions between chitosan and mucin inferred from the FTIR spectra of the LB films. In conclusion, the results with Langmuir and LB films suggest that electrostatic interactions are crucial for the mucoadhesive mechanism, which is affected by the complexation between chitosan and mucin.

Chitosan as a removing agent of beta-lactoglobulin from membrane models.

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers, whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms, Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC), the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property.

Molecular view of the interaction between iota-carrageenan and a phospholipid film and its role in enzyme immobilization.

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Disruption of Saccharomyces cerevisiae by Plantaricin 149 and investigation of its mechanism of action with biomembrane model systems

The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity data, we determined that 2.5 muM is the critical concentration at which Pln149a will disrupt a negative DPPG monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain in the membrane, promoting its disruption.

A new strategy to investigate the toxicity of nanomaterials using Langmuir monolayers as membrane models

Abstract Nanomaterials such as carbon nanotubes (CNTs) and nanoparticles have received enormous attention in analytical areas for their potential applications as new tools for biotechnology and life sciences. Most of these possible applications involve the use of CNTs and related materials as vehicles for drug delivery and/or gene therapy. In this study, we introduce a methodology to evaluate the interactions between CNTs/dendrimers nanoconjugates and phospholipid biomembrane models, using the Langmuir film balance technique. Our main goal was to elucidate the action of engineered nanomaterials in cell membranes, at the molecular level, using a membrane model system. The penetration of single-walled carbon nanotubes (SWCNTs)/polyamidoamine dendrimer nanocomplexes into dipalmitoylphosphatidylcholine monolayers was pronounced, as revealed by adsorption kinetics and surface pressure measurements. These findings suggest that SWCNTs were able to interact even at high surface pressure values, ∼30 mN/m. Therefore, the results confirm that the presence of the nanomaterial affects the packing of the synthetic membranes. We believe the methodology introduced here may be of great importance for further nanotoxicity studies.

The specificity of frutalin lectin using biomembrane models.

Frutalin is a homotetrameric alpha-d-galactose (d-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for d-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of d-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level.

Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions

Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal membrane enzyme responsible for the final step in the synthesis of triacylglycerides. Although DGATs from a wide range of organisms have nearly identical sequences, there is little structural information available for these enzymes. The substrate binding sites of DGAT1 are predicted to be in its large luminal extramembranous loop and to include common motifs with acyl-CoA cholesterol acyltransferase enzymes and the diacylglycerol binding domain found in protein kinases. In this study, synthetic peptides corresponding to the predicted binding sites of DGAT1 enzyme were examined using synchrotron radiation circular dichroism spectroscopy, fluorescence emission and adsorption onto lipid monolayers to determine their interactions with substrates associated with triacylglyceride synthesis (oleoyl-CoA and dioleoylglycerol). One of the peptides, Sit1, which includes the FYxDWWN motif common to both DGAT1 and acyl-CoA cholesterol acyltransferase, changes its conformation in the presence of both substrates, suggesting its capability to bind their acyl chains. The other peptide (Sit2), which includes the putative diacylglycerol binding domain HKWCIRHFYKP found in protein kinase C and diacylglycerol kinases, appears to interact with the charged headgroup region of the substrates. Moreover, in an extended-peptide which contains Sit1 and Sit2 sequences separated by a flexible linker, larger conformational changes were induced by both substrates, suggesting that the two binding sites may bring the substrates into close proximity within the membrane, thus catalyzing the formation of the triacylglyceride product.

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid-rich extract and membrane models

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA‐ KAAGQAALGAL‐NH2, DS 01) with phospholipid (PL) monolayers comprising (i) a lipid‐rich extract of Leishmania amazonensis (LRE‐La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid‐air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE‐La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE‐La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright

Molecular-Level Modifications Induced by Photo-Oxidation of Lipid Monolayers Interacting with Erythrosin

Incorporation into cell membranes is key for the action of photosensitizers in photomedicine treatments, with hydroperoxidation as the prominent pathway of lipid oxidation. In this paper, we use Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as cell membrane models to investigate adsorption of the photosensitizer erythrosin and its effect on photoinduced lipid oxidation. From surface pressure isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) data, erythrosin was found to adsorb mainly via electrostatic interaction with the choline in the head groups of both DOPC and DPPC. It caused larger monolayer expansion in DOPC, with possible penetration into the hydrophobic unsaturated chains, while penetration into the DPPC saturated chains was insignificant. Easier penetration is due to the less packed DOPC monolayer, in comparison to the more compact DPPC according to the monolayer compressibility data. Most importantly, light irradiation at 530 nm made the erythrosin-containing DOPC monolayer become less unstable, with a relative surface area increase of ca. 19%, in agreement with previous findings for bioadhesive giant vesicles. The relative area increase is consistent with hydroperoxidation, supporting the erythrosin penetration into the lipid chains, which favors singlet oxygen generation close to double bonds, an important requirement for photodynamic efficiency.