Vesicular and Planar Membranes of Archaea Lipids: Unusual Physical Properties and Biomedical Applications.

Liposomes and planar membranes made of archaea or archaea-like lipids exhibit many unusual physical properties compared to model membranes composed of conventional diester lipids. Here, we review several recent findings in this research area, which include (1) thermosensitive archaeosomes with the capability to drastically change the membrane surface charge, (2) MthK channel's capability to insert into tightly packed tetraether black lipid membranes and exhibit channel activity with surprisingly high calcium sensitivity, and (3) the intercalation of apolar squalane into the midplane space of diether bilayers to impede proton permeation. We also review the usage of tetraether archaeosomes as nanocarriers of therapeutics and vaccine adjuvants, as well as the biomedical applications of planar archaea lipid membranes. The discussion on archaeosomal therapeutics is focused on partially purified tetraether lipid fractions such as the polar lipid fraction E (PLFE) and glyceryl caldityl tetraether (GCTE), which are the main components of PLFE with the sugar and phosphate removed.

Polar Lipid Fraction E from Sulfolobus acidocaldarius and Dipalmitoylphosphatidylcholine Can Form Stable yet Thermo-Sensitive Tetraether/Diester Hybrid Archaeosomes with Controlled Release Capability.

Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from -48 mV at 37 °C to -16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid 'flip-flop'. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell's cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42-44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions.

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division.

Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

Liposomes Containing Lipid-Soluble Zn(II)-Bis-dipicolylamine Derivatives Show Potential To Be Targeted to Phosphatidylserine on the Surface of Cancer Cells.

Here we used a lipid-soluble Zn(II)-bis-dipicolylamine derivative as a membrane component to develop liposomal carriers that have potential to be targeted to phosphatidylserine (PS) rich surfaces on cancer cells and to preferentially kill cancer cells without using anticancer drugs. This DPA derivative (abbreviated as DPA-Cy3[22,22]) contains the fluorophore cyanine 3 (Cy3) and two 22-carbon chains that can be anchored into liposomal membrane bilayers. DPA-Cy3[22,22]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) unilamellar vesicles (∼150 nm) showed selective binding to PS-containing liposomes as demonstrated by anion exchange chromatography. This binding does not result in vesicle fusion or aggregation. Flow cytometry showed that DPA-Cy3[22,22]/POPC liposomes have preferential binding to MCF-7 breast cancer cells over MCF-12A noncancer cells due to 3-7 times more PS exposures on MCF-7. The extent of liposome binding with MCF-7 cells was increased by two times after cells were pretreated with the apoptotic inducer camptothecin, which increased PS exposure to the cell surface. Moreover, our flow cytometry data also suggest that local cell membrane perturbations may occur upon liposome binding and internalization. This implies that DPA-Cy3[22,22]/POPC liposomes alone may have a PS-dependent cytotoxic effect. This assertion was supported by the cell proliferation assay, which showed that 9.1 mol % DPA-Cy3[22,22]/POPC liposomes exert cytotoxicity on MCF-7 cells 3.5 times higher than that on MCF-12A cells. These results indicate that DPA-Cy3[22,22]-containing liposomes hold great promise as efficient nano drug carriers.

Concentration-Induced J-Aggregate Formation Causes a Biphasic Change in the Release of trans-Combretastatin A4 Disodium Phosphate from Archaeosomes and the Subsequent Cytotoxicity on Mammary Cancer Cells.

Combretastatin A4 disodium phosphate (CA4P) is a fluorescent, water-soluble prodrug able to induce vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose. As a continued effort to develop efficient liposomal CA4P to treat solid tumor, we herein investigate the physical and spectroscopic properties of CA4P in aqueous solution and the mechanism of CA4P release from archaeal tetraether liposomes (archaeosomes). We found that cis-CA4P can be photoisomerized to trans-CA4P. This photoisomerization results in an increase in fluorescence intensity. Both cis- and trans-CA4P undergo fluorescence intensity self-quenching after they reach a critical concentration Cq (∼0.15-0.25 mM). Moreover, both cis- and trans-CA4P in buffer exhibit a red shift in their excitation spectrum and an increase in excitation spectrum band sharpness with increasing concentration, which can be attributed to the formation of J-aggregates. The onset of the dramatic change in excitation maximum occurs at concentrations close to Cq, suggesting that the self-quenching arises from extensive J-aggregate formation and that, when CA4P concentration exceeds Cq, J-aggregate formation begins to increase sharply. Our data also suggest that the extent of J-aggregate formation plays a critical role in CA4P release from tetraether archaeosomes and in the subsequent cytotoxicity on cultured human breast cancer MCF-7 cells. The drug leakage and cytotoxicity rate constants vary with the initial CA4P concentration entrapped inside archaeosomes in a biphasic manner, reaching a local maximum at 0.25-0.50 mM. A mechanism based on the concept of J-aggregate formation has been proposed to explain the biphasic changes in drug release and cytotoxicity with increasing drug concentration. Tetraether archaeosomes are extraordinarily stable and relatively nontoxic to animals; thus, they are promising nano drug carriers. The results obtained from this study pave the way for future development of archaeosomal CA4P to treat solid tumors.

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 drug's 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 MßCD. 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.

On physical properties of tetraether lipid membranes: effects of cyclopentane rings.

This paper reviews the recent findings related to the physical properties of tetraether lipid membranes, with special attention to the effects of the number, position, and configuration of cyclopentane rings on membrane properties. We discuss the findings obtained from liposomes and monolayers, composed of naturally occurring archaeal tetraether lipids and synthetic tetraethers as well as the results from computer simulations. It appears that the number, position, and stereochemistry of cyclopentane rings in the dibiphytanyl chains of tetraether lipids have significant influence on packing tightness, lipid conformation, membrane thickness and organization, and headgroup hydration/orientation.

Physical properties of archaeal tetraether lipid membranes as revealed by differential scanning and pressure perturbation calorimetry, molecular acoustics, and neutron reflectometry: effects of pressure and cell growth temperature.

The polar lipid fraction E (PLFE) is a major tetraether lipid component in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Using differential scanning and pressure perturbation calorimetry as well as ultrasound velocity and density measurements, we have determined the compressibilities and volume fluctuations of PLFE liposomes derived from different cell growth temperatures (T(g) = 68, 76, and 81 °C). The compressibility and volume fluctuation values of PLFE liposomes, which are substantially less than those detected from diester lipid membranes (e.g., DPPC), exhibit small but significant differences with T(g). Among the three T(g)s employed, 76 °C leads to the least compressible and most tightly packed PLFE membranes. This temperature is within the range for optimal cell growth (75-80 °C). It is known that a decrease in T(g) decreases the number of cyclopentane rings in archael tetraether lipids. Thus, our data enable us to present the new view that membrane packing in PLFE liposomes varies with the number of cyclopentane rings in a nonlinear manner, reaching maximal tightness when the tetraether lipids are derived from cells grown at optimal T(g)s. In addition, we have studied the effects of pressure on total layer thickness, d, and neutron scattering length density, ρ(n), of a silicon-D(2)O interface that is covered with a PLFE membrane using neutron reflectometry (NR). At 55 °C, d and ρ(n) are found to be rather insensitive to pressure up to 1800 bar, suggesting minor changes of the thickness of the membrane's hydrophobic core and headgroup orientation upon compression only.

Compressibilities and volume fluctuations of archaeal tetraether liposomes.

Bipolar tetraether lipids (BTLs) are abundant in crenarchaeota, which thrive in both thermophilic and nonthermophilic environments, with wide-ranging growth temperatures (4-108°C). BTL liposomes can serve as membrane models to explore the role of BTLs in the thermal stability of the plasma membrane of crenarchaeota. In this study, we focus on the liposomes made of the polar lipid fraction E (PLFE). PLFE is one of the main BTLs isolated from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using molecular acoustics (ultrasound velocimetry and densimetry), pressure perturbation calorimetry, and differential scanning calorimetry, we have determined partial specific adiabatic and isothermal compressibility, their respective compressibility coefficients, partial specific volume, and relative volume fluctuations of PLFE large unilamellar vesicles (LUVs) over a wide range of temperatures (20-85°C). The results are compared with those obtained from liposomes made of dipalmitoyl-L-α-phosphatidylcholine (DPPC), a conventional monopolar diester lipid. We found that, in the entire temperature range examined, compressibilities of PLFE LUVs are low, comparable to those found in gel state of DPPC. Relative volume fluctuations of PLFE LUVs at any given temperature examined are 1.6-2.2 times more damped than those found in DPPC LUVs. Both compressibilities and relative volume fluctuations in PLFE LUVs are much less temperature-sensitive than those in DPPC liposomes. The isothermal compressibility coefficient (ß(T)(lipid)) of PLFE LUVs changes from 3.59 × 10(-10) Pa(-1) at 25°C to 4.08 × 10(-10) Pa(-1) at 78°C. Volume fluctuations of PLFE LUVs change only 0.25% from 30°C to 80°C. The highly damped volume fluctuations and their low temperature sensitivity, echo that PLFE liposomes are rigid and tightly packed. To our knowledge, the data provide a deeper understanding of lipid packing in PLFE liposomes than has been previously reported, as well as a molecular explanation for the low solute permeation and limited membrane lateral motion. The obtained results may help to establish new strategies for rational design of stable BTL-based liposomes for drug/vaccine delivery.

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.

Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function.

Recent attempts at rebuilding the myocardium using stem cells have yielded disappointing results. The lack of a supporting vasculature may, in part, explain these disappointing findings. However, concerns over possible side effects have hampered attempts at revascularizing the infarcted myocardium using systemic delivery of proangiogenic compounds. In this study, we develop the technology to enhance the morphology and function of postinfarct neovasculature. Previously, we have shown that the up-regulated expression of endothelial cell adhesion molecules in the myocardial infarction (MI) region provides a potential avenue for selectively targeting drugs to infarcted tissue. After treatment with anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF), changes in cardiac function and vasculature post-MI were quantified in a rat MI model. Targeted delivery of VEGF to post-MI tissue resulted in significant increase in fractional shortening and improved systolic function. These functional improvements were accompanied by a 21% increase in the number of anatomical vessels and a 74% increase in the number of perfused vessels in the MI region of treated animals. No significant improvements in cardiac function were observed in untreated, systemic VEGF-treated, nontargeted liposome-treated, or blank immunoliposome-treated animals. Targeted delivery of low doses of proangiogenic compounds to post-MI tissue results in significant improvements in cardiac function and vascular structure.

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.

Sterol superlattice affects antioxidant potency and can be used to assess adverse effects of antioxidants.

We have developed a fluorescence method to examine how membrane sterol lateral organization affects the potency of antioxidants, and used this information to evaluate possible adverse effects of lipid-soluble antioxidants seen in recent clinical studies. In the presence of an antioxidant, the lag time (tau) produced during free radical-induced sterol oxidation in lipid vesicles reflects the potency of the antioxidant. The ascorbic acid-induced tau value varies with sterol mol% in a biphasic manner, showing a minimum at the critical sterol mole fraction for maximal superlattice formation (C r), in ascorbic acid concentrations

Critical factors for detection of biphasic changes in membrane properties at specific sterol mole fractions for maximal superlattice formation.

Here we use the excitation generalized polarization (GPex) of 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan) fluorescence in fluid cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine large unilamellar vesicles to explore the experimental conditions that would be required in order to detect a biphasic change in membrane properties at specific sterol mole fractions (Cr) (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol %) for maximal sterol superlattice formation. Laurdan's GPex changes with sterol content in an alternating manner, showing minima (termed as GPex dips) at approximately Cr. GPex dips are detectable if the vesicles are preincubated for a sufficient time period and protected from sterol oxidation. In most cases, vesicles with a higher lipid concentration take a longer time to show a GPex dip at Cr. The depth of the GPex dip increases with increasing incubation time and eventually reaches a plateau, at which the maximum area covered by superlattices is expected to be achieved. However, if the vesicles are not protected against sterol oxidation, the GPex dips are attenuated or obliterated. These effects can be attributed to the increased inter-bilayer lipid exchange and the increased vesicle-vesicle interactions present at high lipid (vesicle) concentrations as well as the decreased interactions between oxysterols and phospholipids. These possible explanations have been incorporated into a kinetic model that is able to calculate the effects of sterol oxidation and lipid concentration on the depth of the GPex dip. The depth of the GPex dip, the required incubation time for the dip formation, and the lipid concentration dependence of the GPex dip vary with Cr, suggesting different physical properties for different sterol superlattices. To detect a biphasic change in membrane properties at Cr, one should also use small sterol mole fraction increments over a wide range, keep all of the vesicles in the same sample set under the same thermal history, and consider lipid concentration, probe type, and Cr value. These results improve our mechanistic understanding of sterol superlattice formation and explain why some studies, especially those requiring high lipid concentrations, did not detect a biphasic change in membrane properties at Cr.

Fluorometric assay for detection of sterol oxidation in liposomal membranes.

The authors have developed a fluorescence assay to measure the rate and extent of sterol oxidation in lipid bilayers. Dehydroergosterol (DHE), a fluorescent cholesterol analog, is used as a probe and at the same time as a membrane component. The assay can also be performed on bilayers containing a mixture of sterols including DHE and nonfluorescent sterols, such as cholesterol and ergosterol. The fluorescence intensity of DHE decreases on oxidation, so the rate and extent of free radical- or enzyme-induced sterol oxidation can be measured as a function of temperature and membrane composition. For the studies, two-component (e.g., phosphatidylcholine (PC)/DHE) and multicomponent (e.g., DHE/PC/bovine-brain sphingomyelin) large unilamellar vesicles were used, and sterol oxidation was initiated either by the peroxy radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride or by the enzyme cholesterol oxidase. The data gathered from this assay may be used to examine the effects of water- and lipid-soluble antioxidants on membrane sterol oxidation produced by free radicals. This assay can be used to test the potency of antioxidants and pro-oxidants, and can be used to determine whether unknown substances demonstrate antioxidant activity against sterol oxidation. The assay can also be used as a tool to examine the effect of sterol lateral organization on sterol oxidation (in the presence or absence of antioxidants). In agreement with the sterol regular distribution model, it is found that both free radical- and enzyme-induced sterol oxidation vary with membrane sterol content in a well defined alternating manner.

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.

Fluorescence studies of lipid regular distribution in membranes.

This article reviews the use of fluorescent lipids and free probes in the studies of lipid regular distribution in model membranes. The first part of this article summarizes the evidence and physical properties for lipid regular distribution in pyrene-labeled phosphatidylcholine (PC)/unlabeled PC binary mixtures as revealed by the fluorescence of pyrene-labeled PC. The original and the extended hexagonal superlattice model are discussed. The second part focuses on the fluorescence studies of sterol regular distributions in membranes. The experimental evidence for sterol superlattice formation obtained from the fluorescent sterol (i.e. dehydroergosterol) and non-sterol fluorescent probes (e.g. DPH and Laurdan) are evaluated. Prospects and concerns are given with regard to the sterol regular distribution. The third part deals briefly with the evidence for polar headgroup superlattices. The emphasis of this article is placed on the new concept that membrane properties and activities, including the activities of surface acting enzymes, drug partitioning, and membrane free volume, are fine-tuned by minute changes in the concentration of bulky lipids (e.g. sterols and pyrene-containing acyl chains) in the vicinities of the critical mole fractions for superlattice formation.

Design, fabrication, and characterization of archaeal tetraether free-standing planar membranes in a PDMS- and PCB-based fluidic platform.

The polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius contains exclusively bipolar tetraether lipids, which are able to form extraordinarily stable vesicular membranes against a number of chemical, physical, and mechanical stressors. PLFE liposomes have thus been considered appealing biomaterials holding great promise for biotechnology applications such as drug delivery and biosensing. Here we demonstrated that PLFE can also form free-standing "planar" membranes on micropores (∼100 µm) of polydimethylsiloxane (PDMS) thin films embedded in printed circuit board (PCB)-based fluidics. To build this device, two novel approaches were employed: (i) an S1813 sacrificial layer was used to facilitate the fabrication of the PDMS thin film, and (ii) oxygen plasma treatment was utilized to conveniently bond the PDMS thin film to the PCB board and the PDMS fluidic chamber. Using electrochemical impedance spectroscopy, we found that the dielectric properties of PLFE planar membranes suspended on the PDMS films are distinctly different from those obtained from diester lipid and triblock copolymer membranes. In addition to resistance (R) and capacitance (C) that were commonly seen in all the membranes examined, PLFE planar membranes showed an inductance (L) component. Furthermore, PLFE planar membranes displayed a relatively large membrane resistance, suggesting that, among the membranes examined, PLFE planar membrane would be a better matrix for studying channel proteins and transmembrane events. PLFE planar membranes also exhibited a sharp decrease in phase angle with the frequency of the input AC signal at ∼1 MHz, which could be utilized to develop sensors for monitoring PLFE membrane integrity in fluidics. Since the stability of free-standing planar lipid membranes increases with increasing membrane packing tightness and PLFE lipid membranes are more tightly packed than those made of diester lipids, PLFE free-standing planar membranes are expected to be considerably stable. All these salient features make PLFE planar membranes particularly attractive for model studies of channel proteins and transmembrane events and for high-throughput drug screening and artificial photosynthesis. This work can be extended to nanopores of PDMS thin films in microfluidics and eventually aid in membrane-based new lab-on-a-chip 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.

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.