Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.     

Fluorescein Derivatives as Fluorescent Probes for pH Monitoring along Recent Biological Applications

Potential of hydrogen (pH) is one of the most relevant parameters characterizing aqueous solutions. In biology, pH is intrinsically linked to cellular life since all metabolic pathways are implicated into ionic flows. In that way, determination of local pH offers a unique and major opportunity to increase our understanding of biological systems. Whereas the most common technique to obtain these data in analytical chemistry is to directly measure potential between two electrodes, in biological systems, this information has to be recovered in-situ without any physical interaction. Based on their non-invasive optical properties, fluorescent pH-sensitive probe are pertinent tools to develop. One of the most notorious pH-sensitive probes is fluorescein. In addition to excellent photophysical properties, this fluorophore presents a pH-sensitivity around neutral and physiologic domains. This review intends to shed new light on the recent use of fluorescein as pH-sensitive probes for biological applications, including targeted probes for specific imaging, flexible monitoring of bacterial growth, and biomedical applications.     

Regulation of Lipid Bilayer Ion Permeability by Antibacterial Polymethyloxazoline-Polyethyleneimine Copolymers

Amphiphilic antimicrobial polymers display activity against the outer bacterial cell membrane, triggering various physiological effects. We investigated the regulation of ion transport across the lipid bilayer to understand differences in biological activity for a series of amphiphilic polymethyloxazoline – polyethyleneimine copolymers. The results confirmed that the tested structures were able to increase the permeability of the lipid bilayer (LB) membrane or its rupture. Black lipid membrane (BLM) experiments show that the triggered conductance profile and its character is strongly correlated with the polymer structure and zeta potential. The polymer exhibiting the highest antimicrobial activity promotes ion transport by using a unique mechanism and step-like characteristics with well-defined discreet openings and closings. The molecule was incorporated into the membrane in a reproducible way, and the observed channel-like activity could be responsible for the antibacterial activity of this molecule.     

Synthesis and studies of two proton–receptor fluorescent probes based on 1,8-naphthalimide

Two isomeric proton–receptor fluorescent probes 1 and 2 were designed and synthesised by employing the photoinduced electron transfer (PET) principle of a ‘fluorophore–spacer–receptor’ module. Their photophysical characteristics were investigated in organic solvents and Britton–Robinson buffer/EtOH (1:1, v/v) solution. Probe 1 was sensitive in the stronger acidic region of 2.21–4.56, while probe 2 displayed a good response towards protons within the pH scope of 4.10–6.09. The specificity of the probes toward protons in commonly used buffer solutions and in the presence of metal cations, Na(i ), K(i ), Ca(ii ), Mg(ii ), Al(iii ), Pb(ii ), Fe(iii ), Ni(ii ), Zn(ii ), Cu(ii ), Hg(ii ), Ag(i ), Co(ii ), Cr(iii ), Mn(ii ), and Cd(ii ) ions, was studied by monitoring the changes in their fluorescence intensity. The tested metal ions exerted no noticeable influence. The results obtained show that the synthesised proton–receptor fluorescent probes have the capability to act as pH-sensitive probes for monitoring pH variations in the range 2.21–6.09.     

The production of pH-sensitive photoluminescent carbon nanoparticles by the carbonization of polyethylenimine and their use for bioimaging

A cationic branched polyelectrolyte of large molecular weight, i.e., polyethylenimine (PEI) of 25,000 Da, was used as both a carbon source and a passivating agent to prepare photoluminescent carbon nanoparticles (CNPs) in one step. In contrast to the previously reported pH-insensitive CNPs, these CNPs have a distinct pH-sensitive feature that gives them the potential to serve as a proton sensor in monitoring cell metabolization process with proton release. When incubated with HeLa cells, the CNPs could readily penetrate the cell membrane and exhibit low cytotoxicity and favorable biocompatibility. The CNPs have been used for HeLa cell imaging.     

Nanoscale patterning controls inorganic-membrane interface structure

The ability to non-destructively integrate inorganic structures into or through biological membranes is essential to realizing full bio-inorganic integration, including arrayed on-chip patch-clamps, drug delivery, and biosensors. Here we explore the role of nanoscale patterning on the strength of biomembrane-inorganic interfaces. AFM measurements show that inorganic probes functionalized with hydrophobic bands with thicknesses complimentary to the hydrophobic lipid bilayer core exhibit strong attachment in the bilayer. As hydrophobic band thickness increases to 2-3 times the bilayer core the interfacial strength decreases, comparable to homogeneously hydrophobic probes. Analytical calculations and molecular dynamics simulations predict a transition between a 'fused' interface and a 'T-junction' that matches the experimental results, showing lipid disorder and defect formation for thicker bands. These results show that matching biological length scales leads to more intimate bio-inorganic junctions, enabling rational design of non-destructive membrane interfaces.     

Kinetics of Light-Induced Intramolecular Charge Transfer and Proton Release in Bacteriorhodopsin

Recent advances in understanding the intramolecular charge transfer and proton release by the light-driven proton pump bacteriorhodopsin (bR) are critically reviewed. The focus is on the time-resolved electrical methods, i.e., photocurrent and photovoltage measurements, and on transient absorption experiments with pH-sensitive dyes. Particular attention is paid to the following topics: charge translocation in the low-pH forms of bR (acid-blue and acid-purple); electrogenicity of the 13-cis cycle; results with bR mutants; surface-bound dyes to detect the proton release at specific sites on either side of the protein; the question of kinetic coupling between the deprotonation of the Schiff base and proton release; and rapid long-range migration of protons along the surface of the purple membrane.     

Tuning the Solution Self-Assembly of a Peptide-PEG (Polyethylene Glycol) Conjugate with α-Cyclodextrin

Cyclodextrins are saccharide ring molecules which act as host cavities that can encapsulate small guest molecules or thread polymer chains. We investigate the influence of alpha-cyclodextrin (αCD) on the aqueous solution self-assembly of a peptide-polymer conjugate YYKLVFF-PEG3K previously studied by our group [Castelletto et al., Polym. Chem., 2010, 1, 453–459]. This conjugate comprises a designed amyloid-forming peptide YYKLVFF that contains the KLVFF sequence from Amyloid β peptide, Aβ16-20, along with two aromatic tyrosine residues to enhance hydrophobicity, as well as polyethylene glycol PEG with molar mass 3 kg mol⁻¹. The conjugate self-assembles into β-sheet fibrils in aqueous solution. Here we show that complexation with αCD instead generates free-floating nanosheets in aqueous solution (with a β-sheet structure). The nanosheets comprise a bilayer with a hydrophobic peptide core and highly swollen PEG outer layers. The transition from fibrils to nanosheets is driven by an increase in the number of αCD molecules threaded on the PEG chains, as determined by ¹H NMR spectroscopy. These findings point to the use of cyclodextrin additives as a powerful means to tune the solution self-assembly in peptide-polymer conjugates and potentially other polymer/biomolecular hybrids.     

Tropolone-Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism,Enzymatic Incorporation into DNA and HeLa Cell Internalization

Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future.     

A chelating dendritic ligand capped quantum dot: preparation, surface passivation, bioconjugation and specific DNA detection

Herein we report the synthesis of a new chelating dendritic ligand (CDL) and its use in the preparation a compact, stable and water-soluble quantum dot (QD), and further development of specific DNA sensor. The CDL, which contains a chelative dihydrolipoic acid moiety for strong QD surface anchoring and four dendritic carboxylic acid groups, provides a stable, compact and entangled hydrophilic coating around the QD that significantly increases the stability of the resulting water-soluble QD. A CDL-capped CdSe/ZnS core/shell QD (CDL-QD) has stronger fluorescence than that capped by a monodendate single-chain thiol, 3-mercapto-propionic acid (MPA-QD). In addition, the fluorescence of the CDL-QD can be enhanced by 2.5-fold by treatments with Zn2+ or S2- ions, presumably due to effective passivation of the surface defects. This level of fluorescence enhancement obtained for the CDL-QD is much greater than that for the MPA-QD. Further, by coupling a short single-stranded DNA target to the QD via the CDL carboxylic acid group, a functional QD-DNA conjugate that can resist non-specific adsorption and hybridize quickly to its complementary DNA probe has been obtained. This functional QD-DNA conjugate is suitable for specific quantification of short, labelled complementary probes at the low DNA probe:QD copy numbers via a QD-sensitised dye fluorescence resonance energy transfer (FRET) response with 500 pM sensitivity on a conventional fluorimeter.     

Retarding effects of DNA on the autoxidation of liposomal suspensions

Deoxyribonucleic acid (DNA) is associated with the cell membrane of prokaryotes and the inner nuclear membrane of eukaryotes. The unsaturated fatty acids of phospholipids, which constitute the bilaminar structure of membranes, undergo autoxidation in the presence of O₂. Calf thymus DNA was incubated with methyl archidonate-enriched phosphatidyl choline liposomes in order to study the effect of DNA upon the oxidation of phospholipids while present in their natural in vivo bilayer configuration. DNA retarded the rate of lipid oxidation and the TBA test, but it did not alter the induction period. These results suggest that DNA is scavenging free radicals produced within the phospholipid bilayer.     

磷酸化二氧化硅填充磺化聚醚醚酮质子交换膜的制备及表征

制备了两种磷酸化改性的介孔二氧化硅亚微米球形颗粒,分别为仅外表面接枝磷酸根基团的颗粒(PMPS-Ⅰ)和内外表面均接枝磷酸根基团的颗粒(PMPS-Ⅱ)。颗粒具有均一的尺寸和规则排布的六面体一维贯通孔道。将制备的二氧化硅颗粒与磺化聚醚醚酮(SPEEK)溶液共混制备杂化膜。与填充PMPS-Ⅰ的杂化膜相比,填充PMPS-Ⅱ的杂化膜显示出较好的质子传导性能。当PMPS-Ⅱ的填充量为5%(质量)时,杂化膜在60℃、100%相对湿度下最高质子传导率为0.241 S·cm^-1。研究结果表明,连续贯通的质子传递通道有助于提高杂化膜的质子传导率。     

Going Forward Laterally: Transmembrane Passage of Hydrophobic Molecules through Protein Channel Walls

Regular phospholipid bilayers do not pose efficient barriers for the transport of hydrophobic molecules. The outer membrane (OM) surrounding Gram‐negative bacteria is a nontypical, asymmetric bilayer with an outer layer of lipopolysaccharide (LPS). The sugar molecules of the LPS layer prevent spontaneous diffusion of hydrophobic molecules across the OM. As regular OM channels such as porins do not allow passage of hydrophobic molecules, specialized OM transport proteins are required for their uptake. Such proteins, exemplified by channels of the FadL family, transport their substrates according to a lateral diffusion mechanism. Here, substrates diffuse from the lumen of the β‐barrel laterally into the OM, through a stable opening in the wall of the barrel. In this way, the lipopolysaccharide barrier is bypassed and, by depositing the substrates into the OM, a driving force for uptake is provided. Lateral diffusion through protein channel walls also occurs in α‐helical inner membrane proteins, and could represent a widespread mechanism for proteins that transport and interact with hydrophobic substrates.     

Fluorescence Anisotropy,FT-IR Spectroscopy and 31-P NMR Studies on the Interaction of Paclitaxel with Lipid Bilayers

To understand the bilayer interaction with paclitaxel, fluorescence polarization, Fourier transform infrared spectroscopy (FT-IR) and 31-phosphorus nuclear magnetic resonance (31P-NMR) studies were performed on paclitaxel bearing liposomes. Fluorescence anisotropy of three probes namely, 1,6-diphenyl-1,3,5-hexatriene (DPH), 12-(9-anthroyloxy) stearic acid (12AS) and 8-anilino-1-naphthalene sulfonate (ANS) were monitored as a function of paclitaxel concentration in the unsaturated bilayers. The incorporation of paclitaxel decreased anisotropy of 12AS and ANS probes, while slightly increased anisotropy of DPH. Paclitaxel has a fluidizing effect in the upper region of the bilayer whereas the hydrophobic core is slightly rigidized. FT-IR spectroscopy showed an increase in the asymmetric and symmetric methylene stretching frequencies, splitting of methylene scissoring band and broadening of carbonyl stretching mode. These studies collectively ascertained that paclitaxel mainly occupies the cooperativity region and interact with the interfacial region of unsaturated bilayers and induces fluidity in the headgroup region of bilayer. At higher loadings (>3 mol%), paclitaxel might gradually tend to accumulate at the interface and eventually partition out of bilayer as a result of solute exclusion phenomenon, resulting in crystallization; seed non-bilayer phases, as revealed by 31P-NMR, thereby destabilizing liposomal formulations. In general, any membrane component which has a rigidization effect will decrease, while that with a fluidizing effect will increase, with a bearing on headgroup interactions, partitioning of paclitaxel into bilayers and stability of the liposomes.     

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (r _DPH and r _TMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, r _DPH was decreased, whereas there was no change in r _TMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.     

Bioinspired Design of Lysolytic Triterpenoid–Peptide Conjugates that Kill African Trypanosomes

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). The active TLF ingredient is the primate-specific apolipoprotein L1 (APOL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies, only two are able to counteract the activity of APOL1; this illustrates its evolutionarily optimized design and trypanocidal potency. Herein, we ask whether a synthetic (syn) TLF can be synthesized by using the design principles of the natural TLF complexes but with different chemical building blocks. We demonstrate the stepwise development of triterpenoid–peptide conjugates, in which the triterpenoids act as a cell-binding, uptake and lysosomal-transport modules and the synthetic peptide GALA acts as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis thus demonstrating a proof-of-principle for the bioinspired, forward-design of a synTLF.     

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochrome bo₃ from E. coli at single-molecule level. Liposomes with reconstituted cytochrome bo₃ were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochrome bo₃ in the membrane; in ∼70 % of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a ΔpH (in the presence of the K⁺ ionophore valinomycin), which was stable over at least ∼800 s. No rapid changes in ΔpH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E. coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li et al. J. Am. Chem. Soc. (2015) 137, 16055–16063).     

质子交换膜燃料电池系统低温启动技术研究进展

质子交换膜燃料电池电动汽车具有绿色环保、续航里程长等优点,但在温度较低的环境下存在启动困难甚至失败的问题,这一问题严重制约了质子交换膜燃料电池电动汽车的发展。研究调查了质子交换膜内部结冰的原理,简述了0℃以下低温环境下启动过程对质子交换膜本身、催化层、气体扩散层以及膜电极整体带来不同程度的损伤,重点分析了质子交换膜燃料电池电动汽车低温启动的策略,可大致分为三类：停机吹扫的控制策略、外部辅助加热和无辅助加热。分析表明每种方法都有其各自的优点与缺点,但总的来说单一的启动方法对质子交换膜燃料电池电动汽车低温启动的效果不如多种方法混合使用的效果理想,未来燃料电池电动汽车的低温启动技术将会朝着多种方法共同协助的趋势发展。     

Composite bilayer films with organic compound-triggered bending properties

Organic compounds are widely used in both industry and daily life, and composite bilayer films with organic compound-triggered bending properties are promising for applications of transducers, soft robotics, and so on. Here, a universal and straightforward strategy to generate composite bilayer films with organic compoundtriggered bending properties is demonstrated. The composite bilayer films with organic compound-triggered bending properties are designed with bilayer structures, in which one layer is a porous polymeric membrane with appropriate solubility parameter that matches the value of organic solvents in order to produce prominent affinity to the solvent molecules, and the other layer is reduced graphene oxide membrane stacked on the porous polymeric membrane as an inert layer for restraining the swelling of the polymeric membrane on one side. Guided by matching the solubility parameters between solvent and polymer, a significant bending curvature of 27.3 cm^-1 is obtained in acetone vapor. The results in this study will provide valuable guidance for designing and developing functional composite materials with significant organic compound-triggered bending properties.     

A study on the interaction between ibuprofen and bilayer lipid membrane

Ibuprofen is a well-known nonsteroidal anti-inflammatory drug, which can interact with lipid membranes. In this paper, the interaction of ibuprofen with bilayer lipid membrane was studied by UV-vis spectroscopy, cyclic voltammetry and AC impedance spectroscopy. UV-vis spectroscopy data indicated directly that ibuprofen could interact with lipid vesicles. In electrochemical experiments, ibuprofen displayed a biphasic behavior on bilayer lipid membrane supported on a glassy carbon electrode. It could stabilize the lipid membrane in low concentration, while it induced defects formation, even removed off bilayer lipid membrane from the surface of the electrode with increasing concentration. The mechanism about the interaction between ibuprofen and supported bilayer lipid membrane was discussed.