Anfotericina Liposomal: Una aproximación fisicoquímica al conocimiento de su mecanismo de acción

The analysis of surface pressure ()-area (A) isotherms and Cs -1- (compressional modulus- surface pressure) curves corresponding to mixed monolayers of amphotericin B (AmB) and dipalmitoyl phosphatidyl serine (DPPS) spread on subphases of different pH shows the existence of significant interactions between the film-forming components. The formation of a stable complex at the air/water interface, composed of two horizontally oriented AmB molecules and one DPPS molecule in the vertical position, is postulated. Ionic attractive forces and hydrogen bonds between AmB and DPPS polar groups are thought to be responsible for the mutual interactions, which vary with the subphase pH due to the different ionization state of the polar groups implicated in the interaction (carboxylic and amino groups of AmB and phosphate, carboxylic and amino groups of DPPS). By applying the Crisp phase rule to the phase transitions of the mixed monolayers, the miscibility of the film-forming components is discussed. The existence of AmB/phospholipid interactions could explain the action of AmB in the fungi membranes, as well as the low toxicity of AmB when is administered in liposomal form.El análisis de las isotermas presión superficial () - área molecular (A), así como el de las curvas del módulo de compresibilidad (Cs-1) en función de la presión superficial correspondientes a monocapas mixtas de anfotericina B (AmB) y dipalmitoil fosfatidil serina (DPPS) extendidas sobre subfases de diferentes pH, muestra la existencia de importantes interacciones entre ambos componentes, sugiriendo la formación de complejos estables en la interfase aire/agua constituidos por dos moléculas de AmB orientadas horizontalmente y una de DPPS en posición vertical. Las atracciones de tipo iónico y el establecimiento de enlaces de hidrógeno entre los grupos polares de los componentes son los responsables de las interacciones mutuas, las cuales dependen del pH de la subfase como consecuencia del diferente estado de ionización de los grupos implicados en la interacción (grupos carboxílico y amino de la AmB y fosfato, carboxílico y amino del DPPS). La aplicación de la regla de las fases a las transiciones de fase de las monocapas mixtas permite conocer la miscibilidad del complejo con el componente en exceso (AmB o DPPS) cuando la proporción de éste en la mezcla excede a la requerida para la formación del complejo de estequiometría 2:1. La existencia de interacciones AmB/fosfolípido permite formular una interpretación acerca del mecanismo de acción de la AmB sobre la membrana celular de los hongos, así como de su menor toxicidad sobre el huésped cuando se administra en forma de liposomas

New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines

Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called “proton sponge” effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccinesThis work was supported by the Spanish "Ministerio de Ciencia, Innovación y Universidades" (Project PID2019–109517RB-I00)S

Study of the interaction of GB virus C/Hepatitis G virus fusion peptides belonging to the E2 protein with phospholipid Langmuir monolayers

In order to determine the ability of 1,2-dipalmitoyl phosphatidylcholine (DPPC) and 1,2-dioleoyl phosphatidylglycerol (DOPG) to host peptide sequences belonging to the E2 protein of GBV virus C/Hepatitis G virus, the behaviour of Langmuir monolayers formed by these phospholipids and E2 (12-26), E2 (354-363) and E2 (chimeric) peptide sequences was analysed from data of surface pressure (π) versus area per molecule (A) isotherms, compression modulus (Cs-1), excess Gibbs energy of mixing (ΔGexc) and total Gibbs energy of mixing (ΔGmix). Three different behaviours were observed. Mixed films of E2 (12-26) with DPPC or DOPC showed negative values for the excess thermodynamic functions, and thus attractive interactions between mixed films components are greater than in ideal films. Mixtures of E2 (354-363) with DPPC or DOPG, exhibited positive values of excess functions, evidencing weaker interactions in the mixed films in relation to those of pure components. Finally, positive and negative excess functions were observed in E2 (chimeric)/DPPC or DOPG mixed films, depending on their composition. In short, the interaction between the phospholipids used in this work as models of cell membranes and E2 peptides varies with the type of phospholipid and the nature of the peptide (size, bulky, hydrophobicity and electric charge)

Anfotericina Liposomal: una aproximación fisicoquímica al conocimiento de su mecanismo de acción

El análisis de las isotermas presión superficial (..) - área molecular (A), así como el de las curvas del módulo de compresibilidad (Cs -1) en función de la presión superficial correspondientes a monocapas mixtas de anfotericina B (AmB) y dipalmitoil fosfatidil serina (DPPS) extendidas sobre subfases de diferentes pH, muestra la existencia de importantes interacciones entre ambos componentes, sugiriendo la formación de complejos estables en la interfase aire/agua constituidos por dos moléculas de AmB orientadas horizontalmente y una de DPPS en posición vertical. Las atracciones de tipo iónico y el establecimiento de enlaces de hidrógeno entre los grupos polares de los componentes son los responsables de las interacciones mutuas, las cuales dependen del pH de la subfase como consecuencia del diferente estado de ionización de los grupos implicados en la interacción (grupos carboxílico y amino de la AmB y fosfato, carboxílico y amino del DPPS). La aplicación de la regla de las fases a las transiciones de fase de las monocapas mixtas permite conocer la miscibilidad del complejo con el componente en exceso (AmB o DPPS) cuando la proporción de éste en la mezcla excede a la requerida para la formación del complejo de estequiometría 2:1. La existencia de interacciones AmB/fosfolípido permite formular una interpretación acerca del mecanismo de acción de la AmB sobre la membrana celular de los hongos, así como de su menor toxicidad sobre el huésped cuando se administra en forma de liposomas

Study of the interaction of GB virus C/Hepatitis G virus fusion peptides belonging to the E2 protein with phospholipid Langmuir monolayers

In order to determine the ability of 1,2-dipalmitoyl phosphatidylcholine (DPPC) and 1,2-dioleoyl phosphatidylglycerol (DOPG) to host peptide sequences belonging to the E2 protein of GBV virus C/Hepatitis G virus, the behaviour of Langmuir monolayers formed by these phospholipids and E2 (12-26), E2 (354-363) and E2 (chimeric) peptide sequences was analysed from data of surface pressure (π) versus area per molecule (A) isotherms, compression modulus (Cs-1), excess Gibbs energy of mixing (ΔGexc) and total Gibbs energy of mixing (ΔGmix). Three different behaviours were observed. Mixed films of E2 (12-26) with DPPC or DOPC showed negative values for the excess thermodynamic functions, and thus attractive interactions between mixed films components are greater than in ideal films. Mixtures of E2 (354-363) with DPPC or DOPG, exhibited positive values of excess functions, evidencing weaker interactions in the mixed films in relation to those of pure components. Finally, positive and negative excess functions were observed in E2 (chimeric)/DPPC or DOPG mixed films, depending on their composition. In short, the interaction between the phospholipids used in this work as models of cell membranes and E2 peptides varies with the type of phospholipid and the nature of the peptide (size, bulky, hydrophobicity and electric charge)