RESUMO
Resealing of membrane pores is crucial for cell survival. Membrane surface charge and medium composition are studied as defining regulators of membrane stability. Pores are generated by electric field or detergents. Giant vesicles composed of zwitterionic and negatively charged lipids mixed at varying ratios are subjected to a strong electric pulse. Interestingly, charged vesicles appear prone to catastrophic collapse transforming them into tubular structures. The spectrum of destabilization responses includes the generation of long-living submicroscopic pores and partial vesicle bursting. The origin of these phenomena is related to the membrane edge tension, which governs pore closure. This edge tension significantly decreases as a function of the fraction of charged lipids. Destabilization of charged vesicles upon pore formation is universal-it is also observed with other poration stimuli. Disruption propensity is enhanced for membranes made of lipids with higher degree of unsaturation. It can be reversed by screening membrane charge in the presence of calcium ions. The observed findings in light of theories of stability and curvature generation are interpreted and mechanisms acting in cells to prevent total membrane collapse upon poration are discussed. Enhanced membrane stability is crucial for the success of electroporation-based technologies for cancer treatment and gene transfer.
Assuntos
Membrana Celular/química , Sobrevivência Celular/genética , Bicamadas Lipídicas/química , Lipídeos/química , Cálcio/farmacologia , Membrana Celular/genética , Detergentes/farmacologia , Campos Eletromagnéticos/efeitos adversos , Eletroporação , Humanos , Bicamadas Lipídicas/efeitos da radiação , Porosidade/efeitos dos fármacos , Porosidade/efeitos da radiação , Propriedades de SuperfícieRESUMO
Membrane-modification effects, induced by ultraviolet (UV) irradiation in diacetylenic liposomes, were analyzed upon contact with cells, biological membranes, and proteins. Liposomes formulated with mixtures of unsaturated 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine and saturated 1,2-dimyristoyl-sn-glycero-3-phosphocholine, in a 1:1 molar ratio, were compared with those that were UV-irradiated and analyzed in several aspects. Membrane polymerization inherence on size stability was studied as well as its impact on mitochondrial and microsomal membrane peroxidation induction, hemolytic activity, and cell viability. Moreover, in order to gain insight about the possible irradiation effect on interfacial membrane properties, interaction with bovine serum albumin (BSA), lysozyme (Lyso), and apolipoprotein (apoA-I) was studied. Improved size stability was found for polymerized liposomes after a period of 30 days at 4°C. In addition, membrane irradiation had no marked effect on cell viability, hemolysis, or induction of microsomal and mitochondrial membrane peroxidation. Interfacial membrane characteristics were found to be altered after polymerization, since a differential protein binding for polymerized or nonpolymerized membranes was observed for BSA and Lyso, but not for apoA-I. The substantial contribution of this work is the finding that even when maintaining the same lipid composition, changes induced by UV irradiation are sufficient to increase size stability and establish differences in protein binding, in particular, reducing the amount of bound Lyso and BSA, without increasing formulation cytotoxicity. This work aimed at showing that the usage of diacetylenic lipids and UV modification of membrane interfacial properties should be strategies to be taken into consideration when designing new delivery systems.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/farmacologia , Lipossomos/química , Lipossomos/farmacologia , Polimerização/efeitos da radiação , Ligação Proteica/efeitos da radiação , Animais , Apolipoproteína A-I/metabolismo , Bovinos , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Dimiristoilfosfatidilcolina/química , Di-Inos/química , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/efeitos da radiação , Peroxidação de Lipídeos/efeitos dos fármacos , Lipossomos/metabolismo , Lipossomos/efeitos da radiação , Lipossomos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Muramidase/metabolismo , Tamanho da Partícula , Fosfatidilcolinas/química , Albumina Sérica/metabolismo , Raios UltravioletaRESUMO
We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid headgroup. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.
Assuntos
Luz , Bicamadas Lipídicas/química , Estresse Oxidativo , Fosfatidiletanolaminas/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Lipossomas Unilamelares/química , Simulação por Computador , Fluorescência , Bicamadas Lipídicas/efeitos da radiação , Microscopia de Fluorescência , Modelos Químicos , Oxigênio/química , Fosfatidilcolinas/química , Fosfatidilcolinas/efeitos da radiação , Fosfatidiletanolaminas/efeitos da radiação , Fármacos Fotossensibilizantes/efeitos da radiação , Porfirinas/efeitos da radiação , Fatores de Tempo , Lipossomas Unilamelares/efeitos da radiaçãoRESUMO
While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure-Staphylococcus aureus alpha-hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the alpha-hemolysin pore, we analyze the blockage of the channel by the crown/K(+) complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.