Bilayer Interior Research Articles

Capacity for increased surface area in the hydrophobic core of β-sheet peptide bilayer nanoribbons.

Amphipathic peptides with amino acids arranged in alternating patterns of hydrophobic and hydrophilic residues efficiently self‐assemble into β‐sheet bilayer nanoribbons. Hydrophobic side chain functionality is effectively buried in the interior of the putative bilayer of these nanoribbons. This study investigates consequences on self‐assembly of increasing the surface area of aromatic side chain groups that reside in the hydrophobic core of nanoribbons derived from Ac‐(XKXE)2‐NH2 peptides (X = hydrophobic residue). A series of Ac‐(XKXE)2‐NH2 peptides incorporating aromatic amino acids of increasing molecular volume and steric profile (X = phenylalanine [Phe], homophenylalanine [Hph], tryptophan [Trp], 1‐naphthylalanine [1‐Nal], 2‐naphthylalanine [2‐Nal], or biphenylalanine [Bip]) were assessed to determine substitution effects on self‐assembly propensity and on morphology of the resulting nanoribbon structures. Additional studies were conducted to determine the effects of incorporating amino acids of differing steric profile in the hydrophobic core (Ac‐X1KFEFKFE‐NH2 and Ac‐(X1,5KFE)‐NH2 peptides, X = Trp or Bip). Spectroscopic analysis by circular dichroism (CD) and Fourier transform infrared (FT‐IR) spectroscopy indicated β‐sheet formation for all variants. Self‐assembly rate increased with peptide hydrophobicity; increased molecular volume of the hydrophobic side chain groups did not appear to induce kinetic penalties on self‐assembly rates. Transmission electron microscopy (TEM) imaging indicated variation in fibril morphology as a function of amino acid in the X positions. This study confirms that hydrophobicity of amphipathic Ac‐(XKXE)2‐NH2 peptides correlates to self‐assembly propensity and that the hydrophobic core of the resulting nanoribbon bilayers has a significant capacity to accommodate sterically demanding functional groups. These findings provide insight that may be used to guide the exploitation of self‐assembled amphipathic peptides as functional biomaterials.

Molecular organization in hydroperoxidized POPC bilayers

Lipid hydroperoxides are the primary reaction products of lipid oxidation, a natural outcome of life under oxygen. While playing a major role in cell metabolism, the microscopic origins of the effects of lipid hydroperoxidation on biomembranes remain elusive. Here we probe the polar structure of partially to fully hydroperoxidized bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by a combination of environment-sensitive fluorescent probes and coarse-grained Martini numerical simulations. We find that the inserted organic hydroperoxide group -OOH migrates preferentially to the surface for bilayers with small fractions of hydroperoxidized lipids, but populates also significantly the bilayer interior for larger fractions. Our findings suggest that by modifying the intimate polarity of biomembranes, lipid peroxidation will have a significant impact on the activity of transmembrane proteins and on the bio-medical efficiency of membrane active molecules such as cell-penetrating and antimicrobial peptides.

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states.

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study, we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium toward the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

Thermostable Fluorescent Capsules with the Cross‐Linked Heterocyclic Polymer Shell from Poly(pyrrole‐phenosafranin)

AbstractThe present investigation describes the synthesis of capsules with a poly(pyrrole‐phenosafranin) cross‐linked shell. The capsules are prepared in a water solution by the facile single‐step chemical oxidative polymerization of monomers within hydrophobic interior of lipid bilayers that act as temporary scaffolds created by liposomes from 1,2‐dilauroyl‐sn‐glycero‐3‐phosphatidylcholine. The polypyrrole links phenosafranin units via amino groups, thus new thermostable nanocapsules are obtained without additional cross‐linking agents. Their spherical shape is confirmed by electron microscopy. Wide spectral characteristics of capsules by using different analytical methods provide detailed information about their structures.

Discerning Membrane Steady-State Oxygen Flux by Monte Carlo Markov Chain Modeling.

Molecular oxygen (O2) permeability coefficients for lipid bilayers have previously been estimated using both electron paramagnetic resonance (EPR) oximetry and molecular dynamics simulation data. Yet, neither technique captures the fluxes that exist physiologically. Here, the dynamic steady state is modeled using a stochastic approach built on atomic resolution molecular dynamics simulation data. A Monte Carlo Markov chain technique is used to examine membrane-level fluxes of oxygen in lipid-water systems. At steady state, the concentration of oxygen is found to be higher inside the model membranes than in surrounding water, consistent with the known favorable partitioning of O2 toward the lipid phase. Pure phospholipid 1-palmitoyl,2-oleoyl-phosphatidylcholine (POPC) bilayers accrue ~40% more O2 molecules at steady state than POPC/cholesterol bilayers (1:1 molecular ratio) mimicking the red blood cell membrane. Steady-state levels of oxygen were reached inside both bilayer types within the same timeframe, but depletion of oxygen from the bilayer interior occurred 17% faster for POPC than for POPC/cholesterol. Likewise, first-order rate constants estimated for accrual to steady state were the same for POPC and POPC/cholesterol, at 190μs-1, while first-order rate constants for depletion of the accrued O2 from the bilayers differed, at 95μs-1 for POPC and 81μs-1 for POPC/cholesterol (lower by 15%). These results are consistent with prior experiments in red blood cells (RBCs) with varying membrane cholesterol content, in which additional cholesterol slowed oxygen uptake and release. Further work is needed to understand whether differences in RBC membrane cholesterol content would affect the delivery of oxygen to tissues.

Site-specific peroxidation modulates lipid bilayer mechanics

Peroxidation of plasma membranes, characterized by oxidative attack of lipidic carbon–carbon double-bonds in unsaturated fatty acids, has been identified as an important biochemical event in multiple pathological conditions, including neurodegenerative diseases, atherosclerosis, diabetes, preeclampsia, aging, cancer, etc. Changes to the lipid bilayer structure as a result of lipid peroxidation may lead to lipid membrane malfunction, and consequently initiate further downstream biochemical cascades. However, how lipid peroxidation modulates the mechanical properties of lipid membranes remains largely controversial. In this study, we investigate the peroxidation of lipids with polyunsaturated fatty acid tails using molecular dynamics simulations. By systematically varying the oxidation site, we find that lipid peroxidation alters the biophysical properties of bilayer membrane in a peroxidation site-specific manner. Specifically, our results suggest that peroxidation at sites in the bilayer interior disturbs and softens the membrane, whereas peroxidation at sites near the membrane–water interface results in a more ordered and stiffer membrane. Such a peroxidation site-specific modulation of lipid membrane mechanics provides an explanation for the contradictory results obtained in previous experiments. Our study paves the way for an improved understanding of the initiation of the downstream cellular dysfunction caused by lipid peroxidation.

The helix 0 of endophilin modifies membrane material properties and induces local curvature

The amphipathic helix 0 of endophilin (i.e., H0-Endo) is important to membrane binding, but its function of curvature generation remains controversial. We used electron paramagnetic resonance (EPR) spectroscopy to study effects of H0-Endo on membrane material properties. We found that H0-Endo reduced lipid chain mobility and increased bilayer polarity, i.e., making the bilayer interior more polar. Lipid-dependent examination revealed that anionic lipids augmented the effect of H0-Endo, while cholesterol had a minimal impact. Our EPR spectroscopy of magnetically aligned bicelles showed that as the peptide-to-lipid ratio increased, the lipid chain orientational order decreased gradually, followed by a sudden loss. We discuss an interfacial-bound model of the amphipathic H0-Endo to account for all EPR data. We used atomic force microscopy and fluorescence microscopy to explore membrane morphological changes. We found that H0-Endo caused the formation of micron-sized holes in mica-supported planar bilayers. Hole formation is likely caused by two competing forces – the adhesion force exerted by the substrate represses bilayer budging, whereas the line tension originating from peptide clustering has a tendency of destabilizing bilayer organization. In the absence of substrate influences, membrane curvature induction was manifested by generating small vesicles surrounding giant unilamellar vesicles. Our results of membrane perforation and vesiculation suggest that the functionality of H0-Endo is more than just coordinating membrane binding of endophilin.

Structural Superiority of Guanidinium-Rich, Four-Armed Copolypeptides: Role of Multiple Peptide-Membrane Interactions in Enhancing Bacterial Membrane Perturbation and Permeability.

The development of novel antimicrobials is a top priority to address the growing epidemic of multidrug-resistant pathogens. Since cationic nonamphiphilic star-shaped antimicrobials are promising molecular scaffolds that provide a high charge density in binding anionic bacterial bilayers, this research aimed to further increase their membrane perturbation capability by introducing guanidinium groups to the antimicrobials via enhancing membrane insertion. In particular, computational simulation and experimental investigations revealed that our designed guanidinium-rich alternating copolypeptide, four-armed poly(arginine-alt-glycine), can interact with both the headgroups and unsaturated tails of phospholipids in bacterial membranes through multiple interactions, including electrostatic, cation-π, and T-shaped π-π interactions, allowing it to penetrate deeper inside the biologically inaccessible high-energy barrier of the hydrophobic lipid bilayer interior to cause membrane permeabilization and precipitation of the bacterial cytoplasm. Furthermore, glycine was observed to have a unique effect in enhancing the performance of arginine-based copolypeptide. Four-armed poly(arginine-alt-glycine) exhibited broad-spectrum antimicrobial activity, high bactericidal efficiency, and negligible hemolysis. The in vivo antibacterial performance of the copolypeptide was superior to that of doxycycline in a mouse model of Pseudomonas aeruginosa skin infection, accompanied by negligible local and systemic toxicity. Our results demonstrate that this guanidinium-rich, nonamphiphilic, star-shaped structure may promote the development of next-generation antimicrobials.

‐sitosterol and gentisic acid loaded 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine liposomal particles

The aim of the present study was the examination of the impact of -sitosterol andgentisic acid on the characteristics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) liposomal particles: (a) bilayer permeability (fluorescence spectroscopy),(b) particle size, polydispersity index (PDI) and zeta potential (photon correlationspectroscopy) and (c) thermal properties (differential scanning calorimetry). -sitosterol induced the increase of liposomal bilayer rigidity, due to rearranging ofthe phospholipid chains, while gentisic acid enhanced the membrane fluidity, dueto the reduced orderliness and the increase of phospholipid dynamics. The inclusionof -sitosterol in liposomes caused a significant increase in particle diameterand PDI, while the encapsulation of gentisic acid did not have influence on particlesize distribution. Apart from that, the presence of -sitosterol resulted in thesignificant zeta potential increase, and thus a better stability of liposomal spheres(in the absence and in the presence of gentisic acid). -sitosterol decreased maintransition temperature (Tm) and phase transition enthalpy (H), and caused thedisappearance of the pre-transition peak as well, whereas the presence of gentisicacid produced a slight decrease in Tm and increase of H. Therefore, gentisic acidhad more favourable, stabilizing interactions with phospholipids than -sitosterol.Thus, it can be concluded that -sitosterol is located in the bilayer interior betweenphospholipids acyl chains, and gentisic acid is incorporated near the outer leaflet ofthe phospholipid membrane, next to the polar head groups. -sitosterol and gentisicacid loaded DPPC liposomal particles have a potential to be used in food andpharmaceutical products, due to the important individual and possible synergisticbeneficial health properties of -sitosterol and gentisic acid.

Permeation of nanoparticles across the intestinal lipid membrane: dependence on shape and surface chemistry studied through molecular simulations.

Nanoparticles are being explored for topical and oral drug delivery applications as they can cross various biological barriers, for example, the intestinal epithelium. The ability of nanoparticles to cross barriers depends on their morphological and surface properties such as size, surface chemistry and shape, among others. The effect of nanoparticle size on their membrane permeability has been well studied both experimentally and theoretically. However, less attention has been given to understand the role of nanoparticle shape in their translocation across biological barrier membranes. Here, we report on the influence of the nanoparticle's shape, surface chemistry and concentration on their permeation across a human intestinal apical cell membrane model. A representative multicomponent lipid bilayer model of the human intestinal apical membrane was built. The free energy of permeation of nanoparticles across the model lipid bilayer was calculated using multiple umbrella sampling simulations. The interaction of these nanoparticles with the model lipid bilayer was captured using extensive microsecond unrestrained molecular dynamics simulations. We observed that: (a) irrespective of the surface chemistry, the efficacy of nanoparticle penetration across the lipid layer was in the order of rod > disc > sphere; (b) irrespective of the shape, apolar and nonpolar nanoparticles were found to locate in the interior of the lipid bilayer, whereas charged and polar nanoparticles were either adsorbed on the lipid headgroups or remained in the water layer; (c) apolar and nonpolar disc shaped nanoparticles had higher efficacy in permeation across the lipid bilayer as compared to disc and sphere shaped nanoparticles; and (d) at a higher concentration of nanoparticles, sphere and disc shaped nanoparticles exhibited more agglomeration as compared to rod shaped nanoparticles. Based on these outcomes, a few nanoparticles were designed which penetrated readily into the lipid layer and these nanoparticles were also able to co-deliver a therapeutic protein inside the lipid layer. The apical model lipid membrane and protocols used in this study can thus be utilized for the in silico design of nanoparticles for the oral delivery of therapeutics.

Deciphering and Controlling Structural and Functional Parameters of the Shells in Vesicle-Templated Polymer Nanocapsules.

Vesicle-templated nanocapsules are prepared by polymerization of hydrophobic acrylic monomers and cross-linkers in the hydrophobic interior of self-assembled bilayers. Understanding the mechanism of capsule formation and the influence of synthetic parameters on the structural features and functional performance of nanocapsules is critical for the rational design of functional nanodevices, an emerging trend of application of the nanocapsule platform. This study investigated the relationship between basic parameters of the formulation and synthesis of nanocapsules and structural and functional characteristics of the resulting structures. Variations in the monomer/surfactant ratio, temperature of polymerization, and the molar fraction of the free-radical initiators were investigated with a multipronged approach, including shell thickness measurements using small-angle neutron scattering, evaluation of the structural integrity of nanocapsules with scanning electron microscopy, and determination of the retention of entrapped molecules using absorbance and fluorescence spectroscopy. Surprisingly, the thickness of the shells did not correlate with the monomer/surfactant ratio, supporting the hypothesis of substantial stabilization of the surfactant bilayer with loaded monomers. Decreasing the temperature of polymerization had no effect on the spherical structure of nanocapsules but resulted in progressively lower retention of entrapped molecules, suggesting that a spherical skeleton of nanocapsule forms rapidly, followed by filling the gaps to create the structure without pinholes. Lower content of initiators resulted in slower reactions, outlining the baseline conditions for practical synthetic protocols. Taken together, these findings provide insights into the formation of nanocapsules and offer methods for controlling the properties of nanocapsules in viable synthetic methods.

Structure and Function in Antimicrobial Piscidins: Histidine Position, Directionality of Membrane Insertion, and pH-Dependent Permeabilization.

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.

Ionizable amino lipid interactions with POPC: implications for lipid nanoparticle function.

Lipid nanoparticles (LNPs) composed of ionizable cationic lipids are currently the leading systems for siRNA delivery in liver disease, with the major limitation of low siRNA release efficacy into the cytoplasm. Ionizable cationic lipids are known to be of critical importance in LNP structure and stability, siRNA entrapment, and endosomal disruption. However, their distribution inside the LNPs and their exact role in cytoplasmic delivery remain unclear. A recent study [Kulkarni et al., On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA, ACS Nano, 2018, 12(5), 4787-4795] on LNP-siRNA systems containing the ionizable lipid DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2 segregates from other components and forms an amorphous oil droplet in the core of LNPs. In this paper, we present evidence supporting the model proposed by Kulkarni et al. We studied KC2 segregation in the presence of POPC using molecular dynamics simulation, deuterium NMR, SAXS, and cryo-TEM experiments, and found that neutral KC2 has a high tendency to separate from POPC dispersions. KC2 confinement, upon raising the pH during the formulation process, could result in rearrangement of the internal structure of LNPs. As interactions between cationic KC2 and anionic endosomal lipids are thought to be a key factor in cargo release, KC2 confinement inside the LNP may be responsible for the observed low release efficacy.

Orientation of nitro-group governs the fluorescence lifetime of nitrobenzoxadiazole (NBD)-labeled lipids in lipid bilayers.

Nitrobenzoxadiazole (NBD) labeled lipids are popular fluorescent probes of membrane structure and dynamics, and have been widely used in both model systems and living cells. Irrespective of attachment to the lipid head group or hydrocarbon chains, the NBD fluorophore generally adopts a transverse bilayer location near the host lipid carbonyl/glycerol moieties. Still, considerable variability is observed in the measured fluorescence lifetimes, indicating that overall fluorophore location is not the determinant of NBD fluorescence properties. Combining fluorescence experiments and molecular dynamics simulations, we show that for two almost identical NBD probes, significant differences in fluorophore orientation and fluorescence lifetime are observed. Integrating these findings with literature data, we demonstrate a correlation between NBD orientation and fluorescence lifetime. The latter is longer when the NBD nitro group is predominantly oriented towards the bilayer interior, compared to probes for which it points to the water medium.

Insight into the Dynamics of Different Fluorophores in the Interior of Aerosol OT Lamellar Structures in the Presence of Sugars: From Picosecond-to-Femtosecond Study.

It is well reported that sugar molecules provide different types of stabilization to biomembranes both in vitro as well as in vivo. In the present article, our focus is to investigate the interactions of two sugar molecules (sucrose and sucralose) with the lamellar structures of aerosol OT (AOT). We have attempted the structural characterization of the lamellae in the presence and absence of sugars with the aid of dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), Fourier transformed infrared spectroscopy (FTIR), polarized optical microscopy (POM), and cryogenic-transmission electron microscopic (cryo-TEM) techniques. In this study, we have chosen three Coumarin dyes, which show a wide variation of hydrophobicity and performed a combination of steady-state and time-resolved fluorescence measurements to unveil the inner detail of the location of the sugars and their specific interactions with the lamellar structures. Our study reveals that sucrose molecules are present in the interfacial region with a major population whereas the most probable location of sucralose is the interior of the AOT bilayer. Therefore, sucralose molecules probably penetrate the bilayer by decreasing the efficient packing of AOT. The important essence of this study is the location and the interactions of sucralose with the lamellae which may provide a future direction to the transportation of the drug molecules in the biomembrane.

Different mechanisms on the stabilization of POPC membrane by trehalose upon varied mechanical stress

Trehalose has been proven to be able to protect the membrane from being damaged by dehydration. Since dehydration is accompanied by large mechanical stress, it is meaningful to study the effect of trehalose on the membrane subject to lateral stress for understanding its protective mechanism. In present work, molecular simulations were performed on the POPC bilayers with different amount of trehalose under lateral pressure and tension. The results indicate that trehalose invariably induces increase in the area per lipid Axy and decrease in bilayer thickness hz and ordering degree |SCD|, which mean improved structure of membrane. However, the main roles trehalose plays are slightly different in stabilizing compressed and expanded membrane. Under tensile stress, the water replacement effect plays a leading role. Trehalose reaches as deep as water into the bilayer. Hydrogen bonds (H-bonds) forming between trehalose and lipid compensate the reduction of water-lipid H-bonds. More trehalose will replace more water to form H-bonds with lipid and help to hold the structure together through abundant H-bond bridges of various patterns. Under lateral pressure, the replacement of water is limited since trehalose only interacts with the bilayer surface zone. The sum of water-lipid (W-L) and trehalose-lipid (T-L) H-bond reduces upon addition of trehalose. Nevertheless, the penetration depth of water increases in the presence of trehalose while WL H-bonds become less. At the same time, WL H-bonds lifetime is significantly extended. These facts suggest that water forming H-bonds with phosphate and carbonyl oxygen and locating in bilayer interior is trapped by trehalose which is closer to surface and has longer H-bonds lifetime. It can be speculated that the residual water maintains the effective hydration level and the low ordering degree of membrane. This effect is similar to Water entrapment which takes place in the stabilization of protein by trehalose. Moreover, the presence of trehalose increases the viscidity of the system and makes it easy to vitrify. The membrane becomes stiffer and has more resistance to deformation from the perspective of Ka. The vitrification mechanism is not excluded whether the membrane is compressed or stretched. Our findings could provide some insights in the design of protecting agents and processes.

Sphingomyelin Stereoisomers Reveal That Homophilic Interactions Cause Nanodomain Formation

Sphingomyelin is an abundant lipid in some cellular membrane domains, such as lipid rafts. Hydrogen bonding and hydrophobic interactions of the lipid with surrounding components such as neighboring sphingomyelin and cholesterol (Cho) are widely considered to stabilize the raft-like liquid-ordered (Lo) domains in membrane bilayers. However, details of their interactions responsible for the formation of Lo domains remain largely unknown. In this study, the enantiomer of stearoyl sphingomyelin (ent-SSM) was prepared, and its physicochemical properties were compared with the natural SSM and the diastereomer of SSM to examine possible stereoselective lipid-lipid interactions. Interestingly, differential scanning calorimetry experiments demonstrated that palmitoyl sphingomyelin, with natural stereochemistry, exhibited higher miscibility with SSM bilayers than with ent-SSM bilayers, indicating that the homophilic sphingomyelin interactions occurred in a stereoselective manner. Solid-state 2H NMR revealed that Cho elicited its ordering effect very similarly on SSM and ent-SSM (and even on the diastereomer of SSM), suggesting that SSM-Cho interactions are not significantly affected by stereospecific hydrogen bonding. SSM and ent-SSM formed gel-like domains with very similar lateral packing in SSM/Cho/palmitoyloleoyl phosphatidylcholine membranes, as shown by fluorescence lifetime experiments. This observation can be explained by a homophilic hydrogen-bond network, which was largely responsible for the formation of gel-like nanodomains of SSMs (or ent-SSM). Our previous study revealed that Cho-poor gel-like domains contributed significantly to the formation of an Lo phase in sphingomyelin/Cho membranes. The results of the study presented here further show that SSM-SSM interactions occur near the headgroup region, whereas hydrophobic SSM-Cho interactions appeared important in the bilayer interior for Lo domain formation. The homophilic interactions of sphingomyelins could be mainly responsible for the formation of the domains of nanometer size, which may correspond to the small sphingomyelin/Cho-based rafts that temporally occur in biological membranes.

Nanoparticles Self-Assembly within Lipid Bilayers.

Coarse-grained molecular dynamics simulations are used to model the self-assembly of small hydrophobic nanoparticles (NPs) within the interior of lipid bilayers. The simulation results reveal the conditions under which NPs form clusters and lattices within lipid bilayers of planar and spherical shapes, depending on the NP–lipid coupling strengths. The formation of nanopores within spherical bilayers with self-assembled planar NPs is also described. These observations can provide guidance in the preparation of functional bio-inorganic systems.

Dimeric camptothecin derived phospholipid assembled liposomes with high drug loading for cancer therapy.

In this study, a newly liposomal formulation of camptothecin (CPT) based on the dimeric camptothecin glycerophosphorylcholine (di-CPT-GPC) prodrug was developed. The di-CPT-GPC prodrug was synthesized through the heterogeneous conjugation of camptothecin-20 succinate with glycerophosphorylcholine. It undergoes assembly to form liposomes without any excipient through the thin film hydration method, which, confirmed by dynamic light scattering (DLS), have an average diameter of approximately 165 ± 5 nm. Observations on cryogenic transmission electron microscopy (cryo-TEM) demonstrated that the liposomes possess a typical multilamellar vesicle structure with a bilayer thickness of approximately 4 nm. The liposomes with a CPT loading up to 62 wt% maintained good stability in simulated physiological fluid. This can be attributed to the protection of the liposomes having CPT groups sequestered within the bilayer interior. Moreover, the in vitro release behavior of di-CPT-GPC liposomes was monitored using different media. The results showed that the liposomes could dissociate and sustainably release free active form CPT in a weak acidic environment. In vitro anticancer activity tests indicated that di-CPT-GPC liposomes had comparable cytotoxicity to the parent drug against MCF-7, HeLa and HepG-2 cells. Finally, a preliminary in vivo antitumor evaluation revealed that the liposomes inhibited tumor growth. Taken together, the di-CPT-GPC assembled liposomes with high drug loading could be a promising nanoformulation of CPT.

Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay

Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To understand this complex and dynamic system, artificial lipid bilayer membranes have served as unprecedented tools for experimental examination, in which some aspects of biomembrane features have been extracted, and to which various methodologies have been applied. Among the lipid bilayers involving liposomes, planar lipid bilayers and nanodiscs, recent developments of lipid bilayer methods and the results of our channel studies are reviewed herein. Principles and techniques of bilayer formation are summarized, which have been extended to the current techniques, where a bilayer is formed from lipid-coated water-in-oil droplets (water-in-oil bilayer). In our newly developed method, termed the contact bubble bilayer (CBB) method, a water bubble is blown from a pipette into a bulk oil phase, and monolayer-lined bubbles are docked to form a bilayer through manipulation by pipette. An asymmetric bilayer can be readily formed, and changes in composition in one leaflet were possible. Taking advantage of the topological configuration of the CBB, such that the membrane's hydrophobic interior is contiguous with the surrounding bulk organic phase, oil-dissolved substances such as cholesterol were delivered directly to the bilayer interior to perfuse around the membrane-embedded channels (membrane perfusion), and current recordings in the single-channel allowed detection of immediate changes in the channels' response to cholesterol. Chemical and mechanical manipulation in each monolayer (monolayer technology) allows the examination of dynamic channel-membrane interplay.