Bruna Renata Casadei

Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C12E8) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C12E8 DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C12E8 DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C12E8 treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C12E8 to the study of DRMs.

Transdermal delivery of butamben using elastic and conventional liposomes

Abstract Gel formulations containing the local anesthetic butamben (BTB) encapsulated in either conventional (BTBLUV) or elastic (BTBLUV-EL) liposomes were prepared and characterized, and then evaluated in terms of their skin permeability. Parameters measured included vesicle size and surface charge, BTB fluorescence anisotropy, encapsulation efficiency, partition coefficient and liposomal membrane organization. Encapsulation efficiencies and membrane/water partition coefficients were determined using a phase separation. The partition coefficients of the elastic and conventional formulations were 2025 ± 234 and 1136 ± 241, respectively. The sizes of the elastic and conventional liposomes did not change significantly (p > 0.05) following incorporation of the anesthetic. As expected, the elastic liposomes presented order parameters that were lower than those of the conventional liposomes, as determined by electron paramagnetic resonance with a 5-stearic acid nitroxide probe incorporated into the bilayer. After 8 h, the fluxes into the receiving solution (µg/cm2/h) were 6.95 ± 1.60 (10% BTB), 23.17 ± 6.09 (10% BTBLUV) and 29.93 ± 6.54 (10% BTBLUV-EL). The corresponding time lags (h) were 1.90 ± 0.48, 1.23 ± 0.28 and 1.57 ± 0.38, respectively. The permeability coefficients (10−3 cm/h) were 1.02 ± 0.23, 2.96 ± 0.77 and 4.14 ± 0.9, for 10% BTB, 10% BTBLUV and 10% BTBLUV-EL, respectively. The results demonstrate that anesthetic access through the skin can be considerably enhanced using liposomal gel formulations, compared to plain gel formulations.

Development of egg PC/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine

Abstract Context: Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required. Objective: This study therefore aimed to provide extended RVC release and increased upload using modified liposomes. Materials and methods: Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07 mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5 + (NH4)2SO4 and pH 7.4 + (NH4)2SO4) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4). Results and discussion: An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5in) or pH 7.4 with 250 mM (NH4)2SO4 in the inner aqueous core (LMVV 7.4in + ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ∼70%) and sustained release (∼25 h). Conclusion: The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Abstract Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4 °C and 37 °C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.

Encapsulation of ropivacaine in a combined (donor-acceptor, ionic-gradient) liposomal system promotes extended anesthesia time

Ropivacaine is a local anesthetic with similar potency but lower systemic toxicity than bupivacaine, the most commonly used spinal anesthetic. The present study concerns the development of a combined drug delivery system for ropivacaine, comprised of two types of liposomes: donor multivesicular vesicles containing 250 mM (NH4)2SO4 plus the anesthetic, and acceptor large unilamellar vesicles with internal pH of 5.5. Both kinds of liposomes were composed of hydrogenated soy-phosphatidylcholine:cholesterol (2:1 mol%) and were prepared at pH 7.4. Dynamic light scattering, transmission electron microscopy and electron paramagnetic resonance techniques were used to characterize the average particle size, polydispersity, zeta potential, morphology and fluidity of the liposomes. In vitro dialysis experiments showed that the combined liposomal system provided significantly longer (72 h) release of ropivacaine, compared to conventional liposomes (~45 h), or plain ropivacaine (~4 h) (p <0.05). The pre-formulations tested were significantly less toxic to 3T3 cells, with toxicity increasing in the order: combined system < ropivacaine in donor or acceptor liposomes < ropivacaine in conventional liposomes < plain ropivacaine. The combined formulation, containing 2% ropivacaine, increased the anesthesia duration up to 9 h after subcutaneous infiltration in mice. In conclusion, a promising drug delivery system for ropivacaine was described, which can be loaded with large amounts of the anesthetic (2%), with reduced in vitro cytotoxicity and extended anesthesia time.

Transdermal Delivery Of Butamben Using Elastic And Conventional Liposomes.

Abstract Gel formulations containing the local anesthetic butamben (BTB) encapsulated in either conventional (BTBLUV) or elastic (BTBLUV-EL) liposomes were prepared and characterized, and then evaluated in terms of their skin permeability. Parameters measured included vesicle size and surface charge, BTB fluorescence anisotropy, encapsulation efficiency, partition coefficient and liposomal membrane organization. Encapsulation efficiencies and membrane/water partition coefficients were determined using a phase separation. The partition coefficients of the elastic and conventional formulations were 2025 ± 234 and 1136 ± 241, respectively. The sizes of the elastic and conventional liposomes did not change significantly (p > 0.05) following incorporation of the anesthetic. As expected, the elastic liposomes presented order parameters that were lower than those of the conventional liposomes, as determined by electron paramagnetic resonance with a 5-stearic acid nitroxide probe incorporated into the bilayer. After 8 h, the fluxes into the receiving solution (µg/cm2/h) were 6.95 ± 1.60 (10% BTB), 23.17 ± 6.09 (10% BTBLUV) and 29.93 ± 6.54 (10% BTBLUV-EL). The corresponding time lags (h) were 1.90 ± 0.48, 1.23 ± 0.28 and 1.57 ± 0.38, respectively. The permeability coefficients (10−3 cm/h) were 1.02 ± 0.23, 2.96 ± 0.77 and 4.14 ± 0.9, for 10% BTB, 10% BTBLUV and 10% BTBLUV-EL, respectively. The results demonstrate that anesthetic access through the skin can be considerably enhanced using liposomal gel formulations, compared to plain gel formulations.