Berenice Venegas

On the lateral structure of model membranes containing cholesterol.

This article summarizes the current view of the sterol superlattice model, which provides a microscopic and molecular description of lateral structure of membranes containing cholesterol, ergosterol, or dehydroergosterol. Special attention is focused on the important, but not yet widely recognized, lessons learned from the studies of sterol superlattices. The major points are: (1) Fine details of cholesterol lateral organization depend on the materials and methods for membrane preparation and on the membrane type. (2) Cholesterol content is extremely important in determining cholesterol lateral organization, and the effect of cholesterol content on membranes should be examined using small cholesterol mole fraction increments. (3) Samples with high vesicle concentrations may need a long time to form sterol superlattices; however, long vesicle incubation in model membrane studies and the existence of sterol superlattice in cells are not mutually exclusive. (4) An increase in cholesterol content does not always condense membranes or make membranes more ordered. (5) The interfaces between regular and irregular regions could play an important role in membrane activities. The last part of this article discusses the use of the knowledge gained from model membrane studies of cholesterol superlattice to investigate membrane lateral organization in cells and to develop new liposome applications.

Bipolar tetraether archaeosomes exhibit unusual stability against autoclaving as studied by dynamic light scattering and electron microscopy

The stability of liposomes made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius against autoclaving has been studied by using dynamic light scattering and transmission electron microscopy. PLFE lipids have structures distinctly different from those derived from eukaryotes and prokaryotes. PLFE lipids are bipolar tetraether molecules and may contain up to four cyclopentane rings in each of the two dibiphytanyl chains. In the pH range 4-10, PLFE-based archaeosomes, with and without polyethyleneglycol- and maleimide-lipids, are able to retain vesicle size, size distribution, and morphology through at least six autoclaving cycles. The cell growth temperature (65 degrees C vs. 78 degrees C), hence the number of cyclopentane rings in the hydrocarbon chains, does not affect this general conclusion. By contrast, at the same pH range, most conventional liposomes made of monopolar diester lipids and cholesterol or pegylated lipids cannot withhold vesicle size and size distribution against just one cycle of autoclaving. At pH<4, the particle size and polydispersity of PLFE-based archaeosomes increase with autoclaving cycles, suggesting that aggregation or membrane disruption may have occurred at extreme acidic conditions during heat sterilization. Under high salt conditions, dye leakage from PLFE archaeosomes due to autoclaving is significantly less than that from pegylated liposomes composed of conventional lipids. The ability to maintain vesicle integrity after multiple autoclaving cycles indicates the potential usefulness of utilizing PLFE-based archaeosomes as autoclavable and durable drug (including genes, peptides, vaccines, siRNA) delivery vehicles.

Radiation-Guided Targeting of Combretastatin Encapsulated Immunoliposomes to Mammary Tumors

PurposeRadiation upregulates expression of endothelial cell adhesion molecules providing a potential avenue for targeting drugs to irradiated tissue. Induced upregulation of E-selectin can be used to target immunoliposomes to solid tumors. The effects of targeting immunoliposomes containing the antivascular drug combretastatin disodium phosphate (CA4P) to irradiated mammary tumors were investigated in this study.MethodsMice bearing transplanted MCa-4 mouse mammary tumors were assigned to one of the factorial treatments permuting the administration of free CA4P, tumor irradiation, CA4P encapsulated liposomes, and CA4P encapsulated immunoliposomes (conjugated with anti-E-selectin). Single and fractionated dosing of radiation and/or CA4P was evaluated.ResultsFor single dose treatments the group that received a single dose of radiation plus a single dose of immunoliposomes showed a significant delay in tumor growth compared to all other treatment groups. Fractionated radiation plus fractionated doses of immunoliposomes resulted in further tumor growth delay; however, it was not significantly different from other fractionated dose treatment groups that combined radiation and CA4P.ConclusionsTargeting of antivascular drugs to irradiated tumors via ligand-bearing liposomes results in significant tumor growth delay. This effect can be further potentiated using a fractionated irradiation dosing schedule combined with fractionated immunoliposome treatments.

Cholesterol superlattice modulates CA4P release from liposomes and CA4P cytotoxicity on mammary cancer cells.

Liposomal drugs are a useful alternative to conventional drugs and hold great promise for targeted delivery in the treatment of many diseases. Most of the liposomal drugs on the market or under clinical trials include cholesterol as a membrane stabilizing agent. Here, we used liposomal CA4P, an antivascular drug, to demonstrate that cholesterol content can actually modulate the release and cytotoxicity of liposomal drugs in a delicate and predictable manner. We found that both the rate of the CA4P release from the interior aqueous compartment of the liposomes to the bulk aqueous phase and the extent of the drugs cytotoxicity undergo a biphasic variation, as large as 50%, with liposomal cholesterol content at the theoretically predicted C(r), e.g., 22.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol for maximal superlattice formation. It appears that at C(r), CA4P can be released from the liposomes more readily than at non-C(r), probably due to the increased domain boundaries between superlattice and nonsuperlattice regions, which consequently results in increased cytotoxicity. The idea that the increased domain boundaries at C(r) would facilitate the escape of molecules from membranes was further supported by the data of dehydroergosterol transfer from liposomes to MβCD. These results together show that the functional importance of sterol superlattice formation in liposomes can be propagated to distal targeted cells and reveal a new, to our knowledge, mechanism for how sterol content and membrane lateral organization can control the release of entrapped or embedded molecules in membranes.

High Vapor Pressure Perfluorocarbons Cause Vesicle Fusion and Changes in Membrane Packing

Perfluorocarbons (PFCs) hold great promise for biomedical applications. However, relatively little is known about the impact of these chemicals on membranes. We used unilamellar vesicles to explore the effects of PFCs on membrane packing and vesicle stability. Four clinically relevant PFCs with varying vapor pressures (PP1, 294 mbar; PP2, 141 mbar; PP4, 9.6 mbar; and PP9, 2.9 mbar) were examined. Microscopy imaging and spectroscopic measurements suggest that PFCs, especially those with high vapor pressures, lead to vesicle fusion within hours. Upon exposure to PP1 and PP2 for 72 h, vesicles retained a spherical shape, but the size changed from approximately 200 nm to approximately 20-40 mum. In addition, membrane packing underwent marked changes during this timeframe. A significant decrease in water content in the lipid polar headgroup regions occurred during the first 1-2-h exposure to PFCs, followed by a steady increase in water content over time. Possible mechanisms were proposed to explain these dramatic structural changes. The finding that chemically inert PFCs exhibited fusogenic activity and marked changes in membrane surface packing is novel, and should be considered when using PFCs for biomedical applications.

Fluorescence Detection of Signs of Sterol Superlattice Formation in Lipid Membranes

There is a significant amount of experimental data, obtained predominantly from fluorescence studies, showing that sterol-containing liposomes can exhibit multiple biphasic changes in membrane properties at specific critical mole fractions of sterol such as 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol%. This can be understood in terms of the sterol regular distribution (e.g., superlattice) model. Here, the authors use excitation generalized polarization of 6-lauroyl-2-dimethylamino-naphthalene fluorescence in fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol unilamellar vesicles to illustrate the experimental procedures and conditions that are required to detect multiple biphasic changes at predicted sterol mole fraction values in liposomal membranes. For this detection, the use of small sterol increments over a wide sterol mole fraction range is essential. Lipid concentration, incubation time, thermal history, and degree of sterol oxidation of liposomal membranes are critical factors. The principles and methodologies described here can be extended to other probes or bioactive molecules, such as enzymes, and can be applied to study sterol lateral organization in multicomponent lipid membranes.