Marcela Manrique-Moreno

Effects of the nonsteroidal anti-inflammatory drug naproxen on human erythrocytes and on cell membrane molecular models

Naproxen, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of lipids found in the outer and inner moieties of the erythrocyte and most cell membranes, respectively. This report presents evidence that naproxen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes it has been observed that the drug induced shape changes, forming echinocytes at a concentration as low as 10microM; b) X-ray diffraction showed that naproxen strongly interacted with DMPC multilayers; in contrast, no perturbing effects on DMPE multilayers were detected; c) differential scanning calorimetry (DSC) data showed a decrease in the melting temperature (T(m)) of DMPC liposomes, which was attributed to a destabilization of the gel phase, effect that was less pronounced for DMPE. These experimental results were observed at concentrations lower than those reported for plasma after therapeutic administration. This is the first time in which the structural effects of naproxen on the human erythrocyte membrane have been described.

Microsolvation of dimethylphosphate: a molecular model for the interaction of cell membranes with water

We present an exhaustive stochastic search of the quantum conformational spaces of the (CH(3)O)(2)PO(2)(-) + nH(2)O (n = 1,2,3) systems. We uncover structural, conformational and energetic features of the problem. As in the isolated species, clusters containing the gauche-gauche (gg) conformation of dimethylphosphate (DMP(-)) are energetically preferred, however, contributions from hydrated gauche-anti (ga) and anti-anti (aa) monomers cannot be neglected because such structures are quite common and because they are close in energy to those containing the gg monomer. At least seven distinct types of O∙∙∙H-O-H contacts lead to DMP(-) ↔ water interactions that are always stabilizing, but not strong enough to induce significant changes in the geometries of either DMP(-) or water units. Our results lead us to postulate DMP(-) to be a suitable model to study explicit and detailed aspects of microsolvation of cell membranes.

Human cells and cell membrane molecular models are affected in vitro by the nonsteroidal anti-inflammatory drug ibuprofen.

This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10μM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300μM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300μM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied.

Structural effects of the Solanum steroids solasodine, diosgenin and solanine on human erythrocytes and molecular models of eukaryotic membranes.

This report presents evidence that the following Solanum steroids: solasodine, diosgenin and solanine interact with human erythrocytes and molecular models of their membranes as follows: a) X-ray diffraction studies showed that the compounds at low molar ratios (0.1-10.0mol%) induced increasing structural perturbation to dimyristoylphosphatidylcholine bilayers and to a considerable lower extent to those of dimyristoylphosphatidylethanolamine; b) differential scanning calorimetry data showed that the compounds were able to alter the cooperativity of dimyristoylphosphatidylcholine, dimyristoylphosphatidylethanolamine and dimyristoylphosphatidylserine phase transitions in a concentration-dependent manner; c) in the presence of steroids, the fluorescence of Merocyanine 540 incorporated to the membranes decreased suggesting a fluidization of the lipid system; d) scanning electron microscopy observations showed that all steroids altered the normal shape of human erythrocytes inducing mainly echinocytosis, characterized by the formation of blebs in their surfaces, an indication that their molecules are located into the outer monolayer of the erythrocyte membrane.

Biophysical study of the non-steroidal anti-inflammatory drugs (NSAID) ibuprofen, naproxen and diclofenac with phosphatidylserine bilayer membranes

Non-steroidal anti-inflammatory drugs (NSAIDs) represent an effective pain treatment option and therefore one of the most sold therapeutic agents worldwide. The study of the molecular interactions responsible for their physiological activity, but also for their side effects, is therefore important. This report presents data on the interaction of the most consumed NSAIDs (ibuprofen, naproxen and diclofenac) with one main phospholipid in eukaryotic cells, dimyristoylphosphatidylserine (DMPS). The applied techniques are Fourier-transform infrared spectroscopy (FTIR), with which in transmission the gel to liquid crystalline phase transition of the acyl chains in the absence and presence of the NSAID are monitored, supplemented by differential scanning calorimetry (DSC) data on the phase transition. FTIR in reflection (ATR, attenuated total reflectance) is applied to record the dependence of the interactions of the NSAID with particular functional groups observed in the DMPS spectrum such as the ester carbonyl and phosphate vibrational bands. With Förster resonance energy transfer (FRET) a possible intercalation of the NSAID into the DMPS liposomes and with isothermal titration calorimetry (ITC) the thermodynamics of the interaction are monitored. The data show that the NSAID react in a particular way with this lipid, but in some parameters the three NSAID clearly differ, with which now a clear picture of the interaction processes is possible.

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. I) biophysical characterization of the interaction mechanisms with bacterial model membranes.

Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO2(-) stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. II) anti-bacterial and anti-endotoxic effects.

Antimicrobial peptides (AMPs) are important components of the innate immune system of animals, plants, fungi and bacteria and are recently under discussion as promising alternatives to conventional antibiotics. We have investigated two cecropin-like synthetic peptides, Gm1, which corresponds to the natural overall uncharged Galleria mellonella native peptide and ΔGm1, a modified overall positively charged Gm1 variant. We have analysed these peptides for their potential to inhibit the endotoxin-induced secretion of tumour necrosis factor-α (TNF-α) from human mononuclear cells. Furthermore, in a conventional microbiological assay, the ability of these peptides to inhibit the growth of the rough mutant bacteria Salmonella enterica Minnesota R60 and the polymyxin B-resistant Proteus mirabilis R45 was investigated and atomic force microscopy (AFM) measurements were performed to characterize the morphology of the bacteria treated by the two peptides. We have also studied their cytotoxic properties in a haemolysis assay to clarify potential toxic effects. Our data revealed for both peptides minor anti-inflammatory (anti-endotoxin) activity, but demonstrated antimicrobial activity with differences depending on the endotoxin composition of the respective bacteria. In accordance with the antimicrobial assay, AFM data revealed a stronger morphology change of the R45 bacteria than for the R60. Furthermore, Gm1 had a stronger effect on the bacteria than ΔGm1, leading to a different morphology regarding indentations and coalescing of bacterial structures. The findings verify the biophysical measurements with the peptides on model systems. Both peptides lack any haemolytic activity up to an amount of 100μg/ml, making them suitable as new anti-infective agents.

Antimicrobial activity and interactions of cationic peptides derived from Galleria mellonella cecropin D-like peptide with model membranes.

Antimicrobial peptides are effector molecules of the innate immune system against invading pathogens. The cationic charge in their structures has a strong correlation with antimicrobial activity, being responsible for the initial electrostatic interaction between peptides and the anionic microbial surface. This paper contains evidence that charge modification in the neutral peptide Gm cecropin D-like (WT) improved the antimicrobial activity of the modified peptides. Two cationic peptides derived from WT sequence named as ΔM1 and ΔM2, with net charge of +5 and +9, respectively, showed at least an eightfold increase in their antimicrobial activity in comparison to WT. The mechanism of action of these peptides was investigated using small unilamellar vesicles (SUVs) as model membranes. To study permeabilization effects of the peptides on cell membranes, entrapped calcein liposomes were used and the results showed that all peptides induced calcein release from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) SUVs, whereas in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), POPC/POPG and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG SUVs, only ΔM1 and ΔM2 induced a notable permeabilization. In addition, interactions of these peptides with phospholipids at the level of the glycerol backbone and hydrophobic domain were studied through observed changes in generalized polarization and fluorescence anisotropy using probes such as Laurdan and DPH, respectively. The results suggest that peptides slightly ordered the bilayer structure at the level of glycerol backbone and on the hydrophobic core in 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) SUVs, whereas in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/DMPG SUVs, only ΔM1 and ΔM2 peptides increased the order of bilayers. Thus, peptides would be inducing clustering of phospholipids creating phospholipid domains with a higher phase transition temperature.

Morphological Effects and Antioxidant Capacity of Solanum crispum (Natre) In Vitro Assayed on Human Erythrocytes

In order to gain insight into the molecular mechanism of the antioxidant properties of Solanum crispum, aqueous extracts of its leaves were assayed on human erythrocytes and molecular models of its membrane. Phenolics and alkaloids were detected by HPLC–MS. Scanning electron and defocusing microscopy showed that S. crispum changed erythrocytes from the normal shape to echinocytes. These results imply that molecules present in the aqueous extracts were located in the outer monolayer of the erythrocyte membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that S. crispum preferentially interacted with DMPC bilayers. Experiments regarding its antioxidant properties showed that S. crispum neutralized the oxidative capacity of HClO on DMPE bilayers; defocusing microscopy and hemolysis assays demonstrated the protective effect of S. crispum against the oxidant effects of HClO on human erythrocytes.

Antioxidant Capacity of Gallic Acid in vitro Assayed on Human Erythrocytes

Gallic acid (GA) is a polyphenol present in many plants. This study was aimed to investigate the molecular interaction of GA with the human erythrocyte membrane and to determine its antioxidant capacity. The molecular interaction with the membrane of human red cells and the antioxidant property was assayed on both human red cells and molecular models of its membrane. Observations by optical, scanning electron, and defocusing microscopy demonstrated that GA is capable to convert red cells from their normal biconcave shape to crenated echinocytes. This result indicates that GA molecules are positioned in the outer monolayer of the red cell membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were selected as classes of phospholipids found in the outer and inner monolayers of the red cell membrane, respectively. X-ray diffraction and differential scanning calorimetry showed that GA was preferentially bound to DMPC bilayers. Experiments related to the antioxidant capacity of GA indicated that this compound offsets HClO oxidative capacity on DMPE bilayers. In addition, optical, scanning, defocusing microscopy, and hemolysis assays confirmed the protective capacity of GA against HClO deleterious effects on human red cells. As a conclusion, GA would be capable to block the access of oxidants into the lipid bilayer, and thus avoid their access into red cells.