Interaction of Tau construct K18 with model lipid membranes.

One of the hallmarks of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, resulting from the aggregation of the tubulin associated unit protein (Tau), which holds a vital role in maintaining neuron integrity in a healthy brain. The development of such aggregates and their deposition in the brain seem to correlate with the onset of neurodegeneration processes. The misfolding and subsequent aggregation of the protein into paired helical filaments that further form the tangles, lead to dysfunction of the protein with neuronal loss and cognitive decline. The aggregation of the protein then seems to be a causative factor of the neurodegeneration associated with AD. The hypothesis of an involvement of the membrane in modulating the misfolding and assembly of Tau into paired helical filaments attracts increasing interests. To provide more insight about how lipids can modulate the interactions with Tau, we have conducted a comprehensive Atomic Force Microscopy (AFM) study involving supported lipid bilayers of controlled compositions with the Tau microtubule-binding construct K18. Particularly, the effects of zwitterionic and negatively charged phospholipids on the interaction have been investigated. Deleterious solubilization effects have been evidenced on fluid zwitterionic membranes as well as an inability of K18 to fragment gel phases. The role of negative lipids in the aggregation of the peptide and the particular ability of phosphatidylinositol-4,5-bisphosphate (PIP2) in inducing K18 fibrillization on membranes are also reported.

Very Long Chain Lipids Favor the Formation of a Homogeneous Phase in Stratum Corneum Model Membranes.

The stratum corneum (SC), the outermost layer of mammal epidermis, acts as a barrier dictating the rate of absorption of exogenous molecules through the skin, as well as to prevent excessive water loss from the body. The SC consists of protein-rich corneocytes embedded into a complex lipid mixture. The lipid fraction is mainly constituted of an equimolar mixture of ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol), forming a solid phase in the intracellular space; this lipid phase is supposed to play a fundamental role in the SC barrier function. An unusual characteristic of this biological membrane is that its lipids generally bear very long acyl chains, with the 24-carbon long ones being the most abundant. In this work, we used Raman microspectroscopy and infrared spectroscopy to study the influence of the acyl chain length on the lipid mixing properties in SC model membranes. Our results revealed that the combination of ceramides and FFA bearing a very long chain is required for the formation of homogeneous lipid mixtures, while lipids with shorter chains (16-carbon and 20-carbon atom long) lead to domains with micrometer dimensions. It is proposed that the biological machinery necessary for acyl chain elongation occurring at the mammalian skin level is required to inhibit lipid phase separation, a critical feature in the proper barrier functioning.

Lipid selectivity in detergent extraction from bilayers.

Despite numerous studies on detergent-induced solubilization of membranes and on the underlying mechanisms associated with this process, very little is known regarding the selectivity of detergents for lipids during their extraction from membranes. To get insights about this phenomenon, solubilization of model bilayers prepared from binary lipid mixtures by different detergents was examined. Three commonly used detergents were used: the non-ionic Triton X-100 (TX), the negatively-charged sodium dodecylsulfate (SDS), and the positively-charged n-dodecyltrimethylammonium chloride (DTAC). Two model membranes were used in order to identify if specific intermolecular interactions can lead to lipid selectivity: bilayers made of a binary mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and of a binary mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Therefore, it was possible to describe systems presenting a combination of detergents bearing different charges with bilayers with different polymorphic propensities and charge. In conditions for which partial solubilization was observed, the composition of the extracted lipid phase was quantified with Liquid Chromatography coupled to Mass Spectrometry to elucidate whether a lipid selectivity occurred in the solubilization process. On one hand, it is found that repulsive or attractive electrostatic interactions did not lead to any lipid selectivity. On the other hand, POPE was systematically less extracted than POPC, regardless of the detergent nature. We propose that this lipid selectivity is inherent to the molecular shape of POPE unsuited for micelles curvature properties.

Microfluidic diffusional sizing probes lipid nanodiscs formation.

The biophysical characterisation of membrane proteins and their interactions with lipids in native membrane habitat remains a major challenge. Indeed, traditional solubilisation procedures with detergents often causes the loss of native lipids surrounding membrane proteins, which ultimately impacts structural and functional properties. Recently, copolymer-based nanodiscs have emerged as a highly promising tool, thanks to their unique ability of solubilising membrane proteins directly from native membranes, in the shape of discoidal patches of lipid bilayers. While this methodology finally set us free from the use of detergents, some limitations are however associated with the use of such copolymers. Among them, one can cite the tedious control of the nanodiscs size, their instability in basic pH and in the presence of divalent cations. In this respect, many variants of the widely used Styrene Maleic Acid (SMA) copolymer have been developed to specifically address those limitations. With the multiplication of new SMA copolymer variants and the growing interest in copolymer-based nanodiscs for the characterisation of membrane proteins, there is a need to better understand and control their formation. Among the techniques used to characterise the solubilisation of lipid bilayer by amphipathic molecules, cryo-TEM, 31P NMR, DLS, ITC and fluorescence spectroscopy are the most widely used, with a consensus made in the sense that a combination of these techniques is required. In this work, we propose to evaluate the capacity of Microfluidic Diffusional Sizing (MDS) as a new method to follow copolymer nanodiscs formation. Originally designed to determine protein size through laminar flow diffusion, we present a novel application along with a protocol development to observe nanodiscs formation by MDS. We show that MDS allows to precisely measure the size of nanodiscs, and to determine the copolymer/lipid ratio at the onset of solubilisation. Finally, we use MDS to characterise peptide/nanodisc interaction. The technique shows a promising ability to highlight the pivotal role of lipids in promoting interactions through a case study with an aggregating peptide. This confirmed the relevance of using the MDS and nanodiscs as biomimetic models for such investigations.

Determination of n-alkane partitioning within phosphatidylethanolamine Lα/HII phases.

Alkanes are known to promote the fluid lamellar (Lα)-to-inverted hexagonal (HII) phase transition of different phospholipids. In this work, we studied the interaction of decane and tetradecane with self-assemblies formed of 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE), using sequential 2H and 31P solid-state NMR spectroscopy. This technique allowed calculating the partitioning constant of the alkanes between the Lα and HII phases of POPE. Our results show that both alkanes are preferentially distributed in the HII phase compared to the Lα phase. In the HII phase, both alkanes display a very high conformational disorder, consistent with their location in the intercylinder voids. This preferential partitioning in the HII phase is more pronounced for tetradecane than for decane. This finding is proposed to be associated with a less energetically favored insertion (limited solubility) of tetradecane in the lamellar phase. This proposition is supported by the observation that tetradecane experiences more conformational freedom than its shorter analog in POPE bilayers, suggesting that it is located, on average, closer to the middle of the bilayers. It is also established that the increase in size of the intercylinder voids, by the addition of 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) in the HII cylinders, enhances the partitioning of decane in the HII phase compared to the Lα phase. These findings propose handles to modulate the balance of the relative Lα/HII phase stability.

Membrane domain modulation of Aß1-42 oligomer interactions with supported lipid bilayers: an atomic force microscopy investigation.

Alzheimer's disease is a devastating pathology affecting an increasing number of individuals following the general rise in life expectancy. Amyloid peptide Aß1-42 has been identified as one of the main culprits of the disease. The peptide has been shown to have major effects on lipid membranes, including membrane fragmentation. The membrane composition has been identified as a factor that plays a pivotal role in regulating peptide/membrane interactions and several results suggest that lipid domains, or rafts, can promote peptide-induced membrane damage. In this work, we examined the effects of lipid segregation on the membrane-perturbing ability of Aß1-42 and an oligomeric mutant (G37C), a peptide that shares common features with the suspected toxic intermediates involved in the neurodegeneration process. Atomic force microscopy (AFM) was used to determine the impact of these peptides on the supported lipid bilayers of various compositions. In 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (DOPC/DPPC/cholesterol) and DOPC/sphingomyelin/cholesterol ternary mixtures, two systems exhibiting liquid-liquid phase separations, it was shown that Aß1-42 and G37C exclusively aggregated on liquid-disordered-phase domains, creating large deposits and even causing membrane fragmentation for the latter composition. Cholesterol and ganglioside GM1, the two most documented lipids in the context of Alzheimer's disease, are also considered to play a crucial role in promoting detrimental interactions with amyloid peptides. We show that, in model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, the presence of either cholesterol or GM1 in a proportion of 10 mol%, a content supposed to lead to domain formation, favoured the association of both Aß1-42 and G37C, leading to a harmful membrane fragmentation. The AFM results established that the presence of domains favoured membrane perturbations induced by the amyloid peptides. It is proposed that lipid packing defects at the domain interface could act as adsorption and nucleation sites for the amyloid peptides. The more extensive bilayer perturbations induced by G37C compared to Aß1-42 supported this hypothesis, indicating that oligomers that cannot mature to the fibril state can present considerable toxicity.

Evidence of hydrocarbon nanodrops in highly ordered stratum corneum model membranes.

The stratum corneum (SC), the top layer of skin, dictates the rate of both water loss through the skin and absorption of exogenous molecules into the body. The crystalline organization of the lipids in the SC is believed to be a key feature associated with the very limited permeability of the skin. In this work, we characterized the organization of SC lipid models that include, as in native SC, cholesterol, a series of FFAs (saturated with C16-C24 chains), as well as a ceramide bearing an oleate chain-linked to a very long saturated acyl chain [N-melissoyl-oleoyloxy hexacosanoyl-D-erythro-sphingosine (Cer EOS)]. The latter is reported to be essential for the native SC lipid organization. Our 2H-NMR, infrared, and Raman spectroscopy data reveal that Cer EOS leads to the formation of highly disordered liquid domains in a solid/crystalline matrix. The lipid organization imposes steric constraint on Cer EOS oleate chains in such a way that these hydrocarbon nanodroplets remain in the liquid state down to -30°C. These findings modify the structural description of the SC substantially and propose a novel role of Cer EOS, as this lipid is a strong modulator of SC solid/liquid balance.

Design and Characterization of a Therapeutic Non-phospholipid Liposomal Nanocarrier with Osteoinductive Characteristics To Promote Bone Formation.

Sterosomes are recently developed types of non-phospholipid liposomes formed from single-chain amphiphiles and high content of sterols. Although sterosomes presented significantly increased stability compared to conventional phospholipid liposomes, current sterosome biomaterials are not truly bioactive and have no intrinsic therapeutic effects. The purpose of this study was to develop a sterosome formulation with osteoinductive properties by an effective selection of sterol, one of the sterosome components. Oxysterols are oxidized derivatives of cholesterol and are known to stimulate osteogenesis and bone formation. Thus, 20S-hydroxycholesterol (Oxy), one of the most potent oxysterols for bone regeneration, was examined as a promising candidate molecule to form fluid lamellar phases with a single-chain amphiphile, namely, stearylamine (SA). First, the optimal composition was identified by investigating the phase behavior of SA/Oxy mixtures. Next, the capacity of the optimized SA/Oxy sterosomes to promote osteogenic differentiation of bone marrow stromal cells was assessed in vitro in a hydrogel environment. Furthermore, we explored the effects of osteogenic oxysterol sterosomes in vivo with the mouse critical-sized calvarial defect model. Our results showed that SA/Oxy sterosomes induced osteogenic differentiation in vitro and enhanced calvarial healing without delivery of additional therapeutic agents, indicating their intrinsic bone-forming potential. This study suggests a promising non-phospholipid liposomal platform with osteoinductive properties for delivery of small molecular drugs and/or other therapeutic genes for enhanced bone formation.

Ceramide-C16 Is a Versatile Modulator of Phosphatidylethanolamine Polymorphism.

Ceramide-C16 (CerC16) is a sphingolipid associated with several diseases like diabetes, obesity, Parkinson disease, and certain types of cancers. As a consequence, research efforts are devoted to identify the impact of CerC16 on the behavior of membranes, and to understand how it is involved in these diseases. In this work, we investigated the impacts of CerC16 (up to 20 mol %) on the lipid polymorphism of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), using differential scanning calorimetry, and sequential 2H and 31P solid-state nuclear magnetic resonance spectroscopy. A partial phase diagram is proposed. The results indicate that the presence of CerC16 leads to an upshift of the temperature of the gel-to-liquid crystalline (Lß - Lα) phase transition, leading to a large Lß/Lα phase coexistence region where gel-phase domains contain ∼35 mol % CerC16. It also leads to a downshift of the temperature of the lamellar-to-inverted hexagonal (L - HII) phase transition of POPE. The opposite influence on the two-phase transitions of POPE brings a three-phase coexistence line when the two transitions overlap. The resulting HII phase can be ceramide enriched, coexisting with a Lα phase, or ceramide depleted, coexisting with a Lß phase, depending on the CerC16 proportions. The uncommon capability of CerC16 to modulate the membrane fluidity, its curvature propensity, and the membrane interface properties highlights its potential as a versatile messenger in cell membrane events.

Stealth nanocarriers based sterosomes using PEG post-insertion process.

Sterosomes (STEs), a new and promising non-phospholipidic liposome platform based on palmitic acid (PA) and cholesterol (Chol) mixtures, need to have polyethylene glycol (PEG) chains grafted to their surface in order to obtain long-circulating nanocarriers in the blood stream. A post-insertion method was chosen to achieve this modification. The post-insertion process of PEG-modified distearoylphosphoethanolamine (DSPE-PEG) was monitored using the zeta potential value of STEs. Various conditions including PEG chain length and the DSPE-PEG/PA-Chol ratio, were explored. Zeta potential of STEs changed from about -40mV for non-modified STEs to values close to 0mV by the end of the process, i.e. for PEG-modified STEs. The kinetics of DSPE-PEG insertion and the stability of the resulting PEG-modified STEs were not considerably influenced, within the investigated range, by changes in PEG chain lengths and in DSPE-PEG/PA-Chol proportion. The post-insertion of PEG chains reduced in vitro complement activation as well as in vitro macrophage uptake compared to the non-modified STEs. Moreover, longer blood circulation time in mice was established for PEG-modified STEs intravenously injected compared to non-modified STEs. These results establish that post-insertion process of PEG chains to STEs is a promising strategy for developing long-term circulating drug delivery nanocarriers.

Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions.

Oxygen-depleted hypoxic regions in the tumour are generally resistant to therapies. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour of magnetotactic bacteria, Magnetococcus marinus strain MC-1 (ref. 4), can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals, tend to swim along local magnetic field lines and towards low oxygen concentrations based on a two-state aerotactic sensing system. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in severe combined immunodeficient beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

Effect of Saturated Very Long-Chain Fatty Acids on the Organization of Lipid Membranes: A Study Combining (2)H NMR Spectroscopy and Molecular Dynamics Simulations.

Little is known about the interaction of very long-chain saturated fatty acids (VLCFAs) with biological membranes. However, this could play an important role on interleaflet interactions and signal transduction mechanisms in cells. The aim of this work is to determine how VLCFA structurally adapts in fluid phospholipid bilayers, since both species must exhibit a significant hydrophobic mismatch. The membrane organization has been described by means of (2)H NMR and molecular dynamics simulations. Our results show that the protonation state affects the position and order of free fatty acids (FFAs) in phospholipid membranes. It was shown that the protonated FFA-C24 carboxyl group is located slightly under the POPC head group and therefore its acyl chain can interact with the lipids of the opposite leaflet. This interdigitation of the end of the acyl chain causes a second plateau observed in SC-D profiles, a very unusual feature in lipid systems.

Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner than a specific lipid-peptide affinity. The present work on the lipid extraction by melittin and citrullinated melittin with model membranes emphasizes the complex relation between the affinity, the lipid extraction/membrane fragmentation, and the lipid specificity.

Melittin-Induced Lipid Extraction Modulated by the Methylation Level of Phosphatidylcholine Headgroups.

Protein- and peptide-induced lipid extraction from membranes is a critical process for many biological events, including reverse cholesterol transport and sperm capacitation. In this work, we examine whether such processes could display specificity for some lipid species. Melittin, the main component of dry bee venom, was used as a model amphipathic α-helical peptide. We specifically determined the modulation of melittin-induced lipid extraction from membranes by the change of the methylation level of phospholipid headgroups. Phosphatidylcholine (PC) bilayers were demethylated either by substitution with phosphatidylethanolamine (PE) or chemically by using mono- and dimethylated PE. It is shown that demethylation reduces the association of melittin with membranes, likely because of the resulting tighter chain packing of the phospholipids, which reduces the capacity of the membranes to accommodate inserted melittin. This reduced binding of the peptide is accompanied by an inhibition of the lipid extraction caused by melittin. We demonstrate that melittin selectively extracts PC from PC/PE membranes. This selectivity is proposed to be a consequence of a PE depletion in the surroundings of bound melittin to minimize disruption of the interphospholipid interactions. The resulting PC-enriched vicinity of melittin would be responsible for the observed formation of PC-enriched lipid/peptide particles resulting from the lipid efflux. These findings reveal that modulating the methylation level of phospholipid headgroups is a simple way to control the specificity of lipid extraction from membranes by peptides/proteins and thereby modulate the lipid composition of the membranes.

Evolution and function of mammalian binder of sperm proteins.

Binder of sperm (BSP) proteins are ubiquitous among mammals and have been extensively investigated over the last three decades. They were first characterized in bull seminal plasma and have now been identified in more than 15 different mammalian species where they represent a superfamily. In addition to sharing a common structure, BSP proteins share many characteristics. They are expressed by seminal vesicles and epididymides, interact with similar ligands and bind to the outer leaflet of sperm membranes via an interaction with choline phospholipids. In addition to playing a major role in sperm capacitation, they are implicated as molecular chaperones in sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume and possibly in the interaction between sperm and oocytes, making them crucial multifunctional proteins. Furthermore, BSP proteins can bind to egg yolk low-density lipoproteins and milk components, an interaction important for the protection of sperm during semen preservation in liquid or frozen state. Our current knowledge of BSP proteins strongly emphasizes their fundamental importance in male fertility and in the optimization of semen preservation techniques. Much work is still ahead in order to fully understand all the mysteries of BSP proteins.

Chain Length of Free Fatty Acids Influences the Phase Behavior of Stratum Corneum Model Membranes.

The skin, the largest organ of the human body, forms a flexible interface between our internal and external environment that protects our organism from exogenous compounds as well as excessive water loss. The stratum corneum (SC), the outermost layer of mammal epidermis, is mainly responsible for the skin impermeability. The SC is formed by corneocytes embedded in a lipid matrix, which is mostly constituted of ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol), organized in two coexisting crystalline lamellar phases. This arrangement of lipids is crucial to skin barrier function. The aim of this paper is to determine the impact of FFA chain length on the phase behavior of SC model lipid membranes using solid-state deuterium NMR and IR spectroscopy. We studied ternary mixtures of N-lignoceroyl-d-erythro-sphingosine (Cer24), cholesterol, and palmitic (FFA16) or lignoceric (FFA24) acid in an equimolar ratio. This proportion replicates the lipid composition found in the SC lipid matrix. Our studies revealed that the phase behavior of Cer24/FFA/Chol ternary mixtures is strongly affected by the length of the FFA. We found the formation of phase-separated crystalline lipid domains when using palmitic acid whereas the use of lignoceric acid results in a more homogeneous mixture. In addition, it was observed that mixtures with lignoceric acid form a gel phase, a very unusual feature for SC model mixtures.

SN-38 active loading in poly(lactic-co-glycolic acid) nanoparticles and assessment of their anticancer properties on COLO-205 human colon adenocarcinoma cells.

SN-38 is a highly effective drug against many cancers. The development of an optimal delivery system for SN-38 is extremely challenging due to its low solubility and labile lactone ring. Herein, SN-38 encapsulated in poly(D,L-lactide-co-glycolide) nanoparticles (NPs) is introduced to enhance its solubility, stability and cellular uptake. SN-38-loaded NPs prepared by spontaneous emulsification solvent diffusion (SESD) method had an average diameter of 310 nm, a zeta potential of -9.69 mV and a loading efficiency of 71%. They were able to protect the active lactone ring of SN-38 against inactivation under physiological condition. A colorectal adenocarcinoma cell line (COLO-205) was used to assess the NPs effects on cytotoxicity and cellular uptake. Result showed a significant decreased cell proliferation and cell apoptosis. These results suggest that these SN-38-loaded NPs can be an effective delivery system for the treatment of colon cancer and potentially for other types of cancers.

Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen.

BACKGROUND: Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general. METHODS: Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting. RESULTS: Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, ß-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1). CONCLUSIONS: These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

Nonphospholipid fluid liposomes with switchable photocontrolled release.

We created novel nonphospholipid photosensitive liposomes from a mixture of a monoacylated azobenzene amphiphile (AzoC10N(+)) and cholesterol sulfate (Schol). This system belongs to the family of sterol-enriched nonphospholipid liposomes that were shown to form stable large unilamellar vesicles (LUVs) with enhanced impermeability. Fluid bilayers were successfully prepared from AzoC10N(+)/Schol (25/75 molar ratio) mixtures, and LUVs could be derived at room temperature using standard extrusion methods. The isomerization process of the bilayer-inserted AzoC10N(+) was characterized. Leakage from these liposomes could be induced by the photoconversion of AzoC10N(+) from its trans form to its cis form. This photocontrolled release from fluid liposomes contrasts with the case of phospholipid-based azo-containing liposomes, which are generally required to be in the gel phase to be photosensitive. It is proposed that the very high degree of conformational order of the monoalkylated amphiphile and the tight packing of the hydrophobic core of the AzoC10N(+)/Schol liposomes make them responsive to the presence of the bulky cis azo isomer. Interestingly, the liposome impermeability could be fully restored by the photoisomerization of the cis form back to the trans form, providing a sharp on-and-off control of payload release. In addition, these nonphospholipid liposomes display a very limited passive release. Therefore, it is shown that AzoC10N(+)/Schol LUVs can be used as nanocontainers, whose content can be released by light in a controlled and switchable manner.