模拟生物膜上铅离子毒性机理的研究

以玻碳电极支撑的磷脂双层膜(s-BLM)作为生物膜的模型,Fe(CN)63-/4-为探针分子,利用循环伏安法和交流阻抗技术研究了铅离子与s-BLM的相互作用,结果表明,铅离子与s-BLM之间可以发生比较强烈的相互作用,诱发s-BLM上形成离子通道,并且作用后的s-BLM在10 mmol/L的乙二胺四乙酸(EDTA)溶液中能够自我修复,这表明采用强的鳌合剂从机体组织中将铅夺取,撤走铅离子则通道关闭。     

Disruption of model cell membranes by carbon nanotubes

Carbon nanotubes (CNTs) have one of the highest production volumes among carbonaceous engineered nanoparticles (ENPs) worldwide and are have potential uses in applications including biomedicine, nanocomposites, and energy conversion. However, CNTs possible widespread usage and associated likelihood for biological exposures have driven concerns regarding their nanotoxicity and ecological impact. In this work, we probe the responses of planar suspended lipid bilayer membranes, used as model cell membranes, to functionalized multi-walled carbon nanotubes (MWCNT), CdSe/ZnS quantum dots, and a control organic compound, melittin, using an electrophysiological measurement platform. The electrophysiological measurements show that MWCNTs in a concentration range of 1.6–12 ppm disrupt lipid membranes by inducing significant transmembrane current fluxes, which suggest that MWCNTs insert and traverse the lipid bilayer membrane, forming transmembrane carbon nanotubes channels that allow the transport of ions. This paper demonstrates a direct measurement of ion migration across lipid bilayers induced by CNTs. Electrophysiological measurements can provide unique insights into the lipid bilayer–ENPs interactions and have the potential to serve as a preliminary screening tool for nanotoxicity.     

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.     

The Central PXXP Motif Is Crucial for PMAP-23 Translocation across the Lipid Bilayer

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells.     

Bola型表面活性剂2.Bola化合物囊泡

Bola型两亲化合物分子因具有中部是疏水基,两端为亲水基团的特殊结构,可在水中聚集形成单层类脂膜（MLM）和单分子层囊泡（MLM囊泡）。许多单分子层囊泡具有良好的热稳定性。具有两个不同亲水基团的bola化合物可形成内外表面分别由不同亲水基团组成的不对称单分子层囊泡。Bola化合物可以参与到普通两亲化合物所形成的双层类脂膜中并改变膜的稳定性,还可成为连接双层类脂膜内外的电子或离子通道。     

Supramolecular Cationic Assemblies against Multidrug-Resistant Microorganisms: Activity and Mechanism of Action

The growing challenge of antimicrobial resistance to antibiotics requires novel synthetic drugs or new formulations for old drugs. Here, cationic nanostructured particles (NPs) self-assembled from cationic bilayer fragments and polyelectrolytes are tested against four multidrug-resistant (MDR) strains of clinical importance. The non-hemolytic poly(diallyldimethylammonium) chloride (PDDA) polymer as the outer NP layer shows a remarkable activity against these organisms. The mechanism of cell death involves bacterial membrane lysis as determined from the leakage of inner phosphorylated compounds and possibly disassembly of the NP with the appearance of multilayered fibers made of the NP components and the biopolymers withdrawn from the cell wall. The NPs display broad-spectrum activity against MDR microorganisms, including Gram-negative and Gram-positive bacteria and yeast.     

From anion receptors to transporters

Cystic fibrosis is the most well-known of a variety of diseases termed channelopathies, in which the regulation of ion transport across cell membranes is so disrupted that the threshold of a pathology is passed. The human toll exacted by these diseases has led a number of research groups, including our own, to create compounds that mediate ion transport across lipid bilayers. In this Account, we discuss three classes of synthetic compounds that were refined to bind and transport anions across lipid bilayer membranes. All of the compounds were originally designed as anion receptors, that is, species that would simply create stable complexes with anions, but were then further developed as transporters. By studying structurally simple systems and varying their properties to change the degree of preorganization, the affinity for anions, or the lipophilicity, we have begun to rationalize why particular anion transport mechanisms (cotransport or antiport processes) occur in particular cases. For example, we have studied the chloride transport properties of receptors based on the closely related structures of isophthalamide and pyridine-2,6-dicarboxamide: the central ring in each case was augmented with pendant methylimidazole groups designed to cotransport H(+) and Cl(-). We observed that the more preorganized pyridine-based receptor was the more efficient transporter, a finding replicated with a series of isophthalamides in which one contained hydroxyl groups designed to preorganize the receptor. This latter class of compound, together with the natural product prodigiosin, can transport bicarbonate (as part of a chloride/bicarbonate antiport process) across lipid bilayer membranes. We have also studied the membrane transport properties of calix[4]pyrroles. Although the parent meso-octamethylcalix[4]pyrrole functions solely as a Cs(+)/Cl(-) cotransporter, other compounds with increased anion affinities can function through an antiport process. One example is octafluoro-meso-octamethylcalix[4]pyrrole; with its electron-withdrawing substituents, it can operate through a chloride/bicarbonate antiport process. Moreover, calix[4]pyrroles with additional hydrogen bond donors can operate through a chloride/nitrate antiport process. Thus, increasing the affinity of the receptor in these cases allows the compound to transport an anion in the absence of a cation. Finally, we have studied the transport properties of simple thioureas and shown that these compounds are highly potent chloride/bicarbonate antiport agents that function at low concentrations. In contrast, the urea analogues are inactive. The higher hydrophobicity (reflected in higher values for the logarithm of the water-octanol partition constant, or log P) and lower polar surface areas of the thiourea compounds compared to their urea analogues may provide a clue to the high potency of these compounds. This observation might serve as a basis for designing future small-molecule transporters.     

Is It Possible to Find an Antimicrobial Peptide That Passes the Membrane Bilayer with Minimal Force Resistance? An Attempt at a Predictive Approach by Molecular Dynamics Simulation

There is still no answer to the mechanism of penetration of AMP peptides through the membrane bilayer. Several mechanisms for such a process have been proposed. It is necessary to understand whether it is possible, using the molecular dynamics method, to determine the ability of peptides of different compositions and lengths to pass through a membrane bilayer. To explain the passage of a peptide through a membrane bilayer, a method for preparing a membrane phospholipid bilayer was proposed, and 656 steered molecular dynamics calculations were carried out for pulling 7 amyloidogenic peptides with antimicrobial potential, and monopeptides (homo-repeats consisting of 10 residues of the same amino acid: Poly (Ala), Poly (Leu), Poly (Met), Poly (Arg), and Poly (Glu)) with various sequences through the membrane. Among the 15 studied peptides, the peptides exhibiting the least force resistance when passing through the bilayer were found, and the maximum reaction occurred at the boundary of the membrane bilayer entry. We found that the best correlation between the maximum membrane reaction force and the calculated parameters corresponds to the instability index (the correlation coefficient is above 0.9). One of the interesting results of this study is that the 10 residue amyloidogenic peptides and their extended peptides, with nine added residue cell-penetrating peptides and four residue linkers, both with established antimicrobial activity, have the same bilayer resistance force. All calculated data are summarized and posted on the server.     

Long-term and fast-bactericidal activity of methacrylamide-based copolymer for antibiofilm coatings and antibacterial wipes applications

Bacterial-related infections can be hazardous for human health and the surrounding environment. Traditional antibiotic-based treatments for these infections are increasingly ineffective due to the emergence of antibiotic-resistant bacteria. Antimicrobial peptide mimics have emerged as promising replacements owing to their potency against bacteria and lack of susceptibility to generate resistant cells. Thus, we synthesized a random copolymer, consisting of aminopropyl methacrylamide and benzyl methacrylamide (AB polymer) by random co-polymerization that mimics host–defense antimicrobial peptides. For its use as a coating, the AB polymer is drop-casted onto a cleaned glass substrate and tested for its antibacterial activity toward Escherichia coli and Staphylococcus aureus, wherein almost 99% of antibacterial activity was observed within 5 min. The prepared coating also possessed excellent longevity characteristics of up to 5 weeks. The AB polymer is also able to inhibit biofilm formation as well as disrupt a mature biofilm and can also be employed as an antibacterial wipe for cleaning bacterial contaminated surfaces. Mechanism study through SEM analysis showed that the AB polymer ruptures the bilayer membrane of both bacterial strains, thereby leading to pore formation causing cell death. Cell viability study depicted that 71% of the A549 lung carcinoma epithelial cells are viable compared to 80% on bare glass substrate. Thus, the synthesized AB polymer may be used in a variety of antibacterial applications directly in the form of solution (wipes) or forming a coating (drop casted/spray coated) for battling bacterial colonization.     

动态表面张力破裂磷脂膜的分子动力学模拟

磷脂双层膜在生物传感器、仿生膜和生物膜反应器等领域具有广阔的应用前景。揭示磷脂膜破裂过程规律对于磷脂膜器件设计和应用具有重要的基础意义。以二棕榈酰磷脂酰胆碱(dipalmitoyl phosphatidylcholine, DPPC)和二棕榈酰磷脂酰甘油(dipalmitoyl phosphoglycerol, DPPG)作为磷脂膜组分,采用粗粒化分子动力学模拟研究了磷脂膜组成对其破裂过程的影响规律。首先建立了磷脂膜破裂动力学的临界破裂时间及临界破裂表面张力的识别方法;进而考察了磷脂膜组成对其破裂动力学的影响规律。模拟结果表明随着带负电组分DPPG 含量增加,磷脂膜平均临界破裂时间延迟且分布变宽,即磷脂膜强度提高,磷脂膜破裂呈现非均匀特性。提出了描述动态表面张力作用下磷脂膜破裂过程的"动态"微观对抗理论,由该理论可预期磷脂膜的线张力随着DPPG 含量提高而增强,与分子动力学模拟结果相符。为基于磷脂膜的分子器件的设计提供了数值模拟及理论依据。     

Ion transport through a porphyrin-terminated hybrid bilayer membrane

A hybrid bilayer membrane (HBM) has been prepared on a glassy carbon electrode (GCE). The HBM consists of an inner layer of n-hexadecylamine (HDA) covalently attached at the GCE and an out-layer of tetra-(N-hexadecylpyridiniurnyl)porphyrin (TC₁₆PyP). The HDA was covalently bounded on the carbon surfaces through its primary amine under cyclic voltammetric potential scans forming a self-assembled monolayer (SAM). The TC₁₆PyP forms a second layer on top of the HDA layer due to hydrophobic interaction. The TC₁₆PyP/HDA/GCE assembly has been utilized to study ion transport through hybrid bilayer membranes. The ion transport through this porphyrin-terminated HBM has been found to strongly dependent on pH. It allows negatively charged redox ions, Fe(CN)₆³⁻, to penetrate at pH <6 while repelling positively charged Ru(NH₃)₆³⁺. However, no faradaic current has been detected at pH >6 with either ions. We believe that electrostatic interaction between the redox ions and the charged membrane is the main reason for this pH dependent ion transport.     

Potassium ion transport by gramicidin and valinomycin across a Ag(1 1 1)-supported tethered bilayer lipid membrane

A tethered bilayer lipid membrane (tBLM) was prepared by anchoring a thiolipid monolayer to a Ag(1 1 1) single crystal from a thiolipid solution in ethanol; a diphytanoylphosphatidylcholine monolayer was then self-assembled on top of the thiolipid monolayer from a lipid solution in hexane or ethanol. The thickness of the mixed thiolipid|lipid bilayer was estimated at 7.5 ± 0.5 nm by scratching holes in the tBLM with an AFM tip and determining their height profile and the frequency distribution of height. The effect of the incorporation of gramicidin A and valinomycin upon K⁺ ion transport across the tBLM was investigated by electrochemical impedance spectroscopy. The position of the inflection point of the sigmoidal curve of the in-phase component of the electrode admittance against potential allowed the determination of an approximate scale of the absolute potential difference across the whole Ag(1 1 1)|(aqueous solution) interface, on the basis of a model of the electrified interface.     

Solid-state NMR studies of the structure, dynamics, and assembly of beta-sheet membrane peptides and alpha-helical membrane proteins with antibiotic activities

beta-Sheet antimicrobial peptides and alpha-helical channel-forming colicins are bactericidal molecules that target the lipid membranes of sensitive cells. Understanding the mechanisms of action of these proteins requires knowledge of their three-dimensional structure in the lipid bilayer. Solid-state NMR has been used to determine the conformation, orientation, depth of insertion, oligomerization, mobility, and lipid interaction of these membrane peptides and proteins. We review the NMR methods developed and applied to study the structure and dynamics of these antibiotic membrane proteins. These studies shed light on how these peptides disrupt lipid membranes and provide fundamental insights into the folding of beta-sheet and alpha-helical membrane proteins.     

Supramolecular Chemistry in the Biomembrane

The combination of supramolecular functional systems with biomolecular chemistry has been a fruitful exercise for decades, leading to a greater understanding of biomolecules and to a great variety of applications, for example, in drug delivery and sensing. Within these developments, the phospholipid bilayer membrane, surrounding live cells, with all its functions has also intrigued supramolecular chemists. Herein, recent efforts from the supramolecular chemistry community to mimic natural functions of lipid membranes, such as sensing, molecular recognition, membrane fusion, signal transduction, and gated transport, are reviewed.     

Anesthetics may restructure the hydrogen belts of membranes

Anesthetic molecules can form hydrogen bonds or organize hydrogen-bond networks. It is argued that they affect the neuronal cell membrane not by an amorphous fluidization of the hyprophobic core of the lipid bilayer but by a restructuring of its hydrogen belts, i.e., the regions occupied by the CO and OH groups of the membrane lipids. The structured disturbance of the hydrogen-bond network is translated latitudinally to hydrogen-bonding sites of the proteins of the membrane, causing allosteric changes of ion channels that result in neuronal blocking.     

Conformation, dynamics, and insertion of a noncysteine-containing protegrin-1 analogue in lipid membranes from solid-state NMR spectroscopy

Disulfide-bonded beta-hairpin structures are common among antimicrobial peptides. Disulfide bonds are known to be important for antimicrobial activity, but the underlying structural reason is not well understood. We have investigated the membrane-bound structure of a disulfide-deleted analogue of the antimicrobial peptide protegrin-1, in which the four Cys residues were replaced by Ala. The secondary structure, dynamics, and topology of this Ala-PG1 peptide in the membrane were determined by using magic-angle-spinning NMR spectroscopy. Conformation-dependent (13)C isotropic chemical shifts of multiple (13)C-labeled residues were obtained from 1D cross-polarization and direct-polarization spectra, and from 2D J-coupling-mediated (13)C-(13)C correlation spectra. Most labeled residues exhibited two conformations: a random coil and a beta-sheet structure. The dual-conformation property was present in both anionic lipid bilayers, which mimic the bacterial membrane, and zwitterionic cholesterol-containing bilayers, which mimic the eukaryotic cell membrane. The mobility of the peptide was measured by using a 2D C-H dipolar-shift correlation experiment. The random-coil fraction was highly mobile whereas the beta-sheet component was rigid. (1)H spin diffusion from the lipid chains to the peptide indicates that the beta-sheet component was well inserted into the anionic membrane, but surface bound in the cholesterol-containing neutral membrane. Thus, the removal of disulfide bonds changed some PG-1 molecules to highly mobile random coils that were poorly associated with the lipid membrane, but other molecules retained a beta-sheet conformation and had a similar membrane-binding topology to the parent peptide. Thus, the reduced antimicrobial activity of Ala-PG1 was largely due to the reduced number of insertion-competent beta-sheet molecules, rather than uniformly weakened activity of identically structured peptides.     

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.     

Interactive transport of guanidinylated poly(propylene imine)-based dendrimers through liposomal and cellular membranes

The ability of guanidinylated poly(propylene imine) dendrimers to translocate across lipid bilayers was assessed by employing either a model phosphate-bearing liposomal membrane system or A549 human lung carcinoma cells. Two dendrimer generations, differing in the number of surface guanidinium groups, were employed, while surface acetylation or the use of spacers affected the binding of the guanidinium group to the phosphate moiety and finally the transport efficiency. Following adhesion of dendrimers with liposomes, fusion or transport occurred. Transport through the liposomal bilayer was observed at low guanidinium/phosphate molar ratios, and was enhanced when the bilayer was in the liquid-crystalline phase. For effective transport through the liposomal membrane, an optimum balance between the binding strength and the degree of hydrophobicity of the guanidinylated dendrimer is required. In experiments performed in vitro with cells, efficient penetration and internalization in subcellular organelles and cytosol was observed.     

异丙酚与支撑双层类脂膜作用的电化学研究

以支撑双层类脂膜(s-BLM)作为生物膜模型,采用循环伏安法和交流阻抗技术研究了异丙酚与双层类脂膜的相互作用.结果表明,异丙酚能降低磷脂分子的有序性, 诱发s-BLM 上形成孔洞或缺陷, 且这种相互作用对作用时间、异丙酚浓度以及胆固醇的存在与否具有依赖性.     

Probing and Manipulating the Lateral Pressure Profile in Lipid Bilayers Using Membrane-Active Peptides—A Solid-State 19F NMR Study

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A ¹⁹F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state ¹⁹F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes.