Formation and morphologies of novel self-assembled micelles from chitosan derivatives

We successfully synthesized N-phthaloyl-carboxymethylchitosan (CMPhCh) from chitosan. CMPhCh could be self-assembled to form various morphologies of crew-cut micelle-like aggregates using a mixed solution of water and N,N-dimethylformamide (DMF). The results of scanning electron microscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS) revealed that the morphologies of the aggregates exhibited vesicles, vesicle-encapsulating vesicles, onion-like vesicles, and large compound micelles (LCM). Their morphologies were changed with varying the concentration of CMPhCh, the ratio of DMF in the mixture, and the self-assembly process. In relatively high CMPhCh concentration and content of DMF, CMPhCh was self-assembled to form the onion-like vesicles with a thin wall and 80-240 nm diameter. For the first time, TEM observation provided straightforward evidence of the onion-like vesicles prepared from natural polymer.     

Phase diagrams,mechanisms and unique characteristics of alternating-structured polymer self-assembly via simulations

Alternating-structured polymers(ASPs), like alternating copolymers, regular multiblock copolymers and polycondensates, are very important polymer structures with broad applications in photoelectric materials. However, their self-assembly behaviors,especially the self-assembly of alternating copolymers, have not been clearly studied up to now. Meanwhile, the unique characteristics therein have not been systematically disclosed yet by both experiments and theories. Herein, we have performed a systematic simulation study on the self-assembly of ASPs with two coil alternating segments in solution through dissipative particle dynamics(DPD) simulations. Several morphological phase diagrams were constructed as functions of different impact parameters. Diverse self-assemblies were observed, including spherical micelles, micelle networks, worm-like micelles, disklike micelles, multimicelle aggregates, bicontinuous micelles, vesicles, nanotubes and channelized micelles. Furthermore, a morphological evolutionary roadmap for all these self-assemblies was constructed, along with which the detailed molecular packing models and self-assembly mechanisms for each aggregate were disclosed. The ASPs were found to adopt a folded-chain mechanism in the self-assemblies. Finally, the unique characteristics for the self-assembly of alternating copolymers were revealed especially, including(1) ultra-fine and uniform feature sizes of the aggregates;(2) independence of self-assembled structures from molecular weight and molecular weight distribution;(3) ultra-small unimolecular aggregates. We believe the current work is beneficial for understanding the self-assembly of alternating structured polymers in solution and can serve as a guide for the further experiments.     

A colloidal route to fabricate hierarchical sticky and non-sticky substrates

Ginsenoside Ro (Ro), a natural anionic biosurfactant derived from ginseng, has been found to markedly increase the solubility of saikosaponin a (SSa), which is the active ingredient of Radix Bupleuri. SSa is minimally soluble in water. To determine the mechanism by which Ro solubilizes SSa, the self-assembly behavior of Ro and the phase behavior of blended Ro and SSa systems were studied by mesoscopic dynamics (MesoDyn) and dissipative particle dynamics (DPD) simulations. The simulation results show that Ro can form vesicles via the closure of oblate membranes. At low concentrations, SSa molecules are solubilized in the palisade layer of the Ro vesicles. At high concentrations, they interact with Ro molecules to form mixed vesicles with Ro adsorbing on the surfaces of the vesicles. The evaluation of the SSa solubilization process reveals that, at low concentrations, Ro aggregates preferentially to form vesicles, which then absorb SSa into themselves. However, at high concentrations, SSa first self-aggregates and then dissolves. This is because the solubilization behavior of Ro shifts the precipitation-dissolution equilibrium in the direction of dissolution. These results of the simulations are consistent with those of transmission electron microscopy (TEM) and dynamic light scattering (DLS).     

Supra-assembly of siliceous vesicles

We report a new approach to produce macroporous ( approximately 110 nm in diameter) ordered siliceous foams (MOSF) by using block copolymers as templates in the absence of any organic cosolvent. The fine three-dimensional honeycomb structure of MOSF was determined by electron tomography. A formation mechanism of MOSF that spans from the atomic to macroscopic scale is proposed, which involves the cooperative self-assembly of unilamellar vesicles followed by the supra-assembly of vesicles. The fusion of soft vesicles finally leads to MOSF with well-ordered and defined honeycomb structures.     

Lipid membrane formation by vesicle fusion on silicon dioxide surfaces modified with alkyl self-assembled monolayer islands

Using atomic force microscopy, we have investigated the formation of the dipalmitoylphosphatidylcholine (DPPC) membrane by the vesicle fusion method on SiO2 surfaces modified with self-assembled monolayer (SAM) islands of octadecyltrichlorosilane (OTS) with sizes comparable to those of the vesicles. OTS-SAM islands with various sizes and coverages can be constructed on the SiO2 surfaces prepared by thermal oxidation followed by partial hydroxylation in a H2O2/H2SO4 solution. When vesicles are sufficiently smaller than the SiO2 domains, DPPC bilayers and DPPC/OTS layers form on the SiO2 and OTS domains, respectively. However, the adhesion of larger vesicles onto SiO2 is prevented by the OTS islands; therefore only DPPC/OTS layers form without formation of DPPC bilayers on the SiO2 domains. On surfaces with domains on the scale of tens to hundreds of nanometers, the relative size between the hydrophilic domains and the vesicles becomes an important factor in the membrane formation by the fusion of vesicles.     

Permeability and conductivity of red blood cell templated polyelectrolyte capsules coated with supplementary layers

The permeability of ions and small polar molecules through polyelectrolyte multilayer capsules templated on red blood cells was studied by means of confocal microscopy and electrorotation. Capsules were obtained by removing the cell after polyelectrolyte multilayer formation by means of NaOCl treatment. This procedure results in cross-linking of poly(allylamine hydrochloride) (PAH) molecules and destroying poly(styrene sulfonate) (PSS) within the multilayer. Capsules are obtained being remarkably different from layer-by-layer (LbL) capsules. These capsules are rather permeable for low as well as for high molecular weight species. However, upon adsorption of extra polyelectrolyte layers the permeability decreased remarkably. The assembly of six supplementary layers of PAH and PSS rendered the capsule almost impermeable for fluorescein. Resealing by supplementary layers is a potential means for filling and release control. By means of electrorotation measurements, it was shown that the capsule walls obtained isolating properties in electrolyte solutions. Conclusions are drawn concerning the mechanism of permeability through cell templated polyelectrolyte multilayer capsules.     

Size of supramolecular SNARE complex: membrane-directed self-assembly

Full length v-SNARE protein in lipid vesicles when exposed to t-SNARE-reconstituted lipid membrane results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores, and establishing continuity between the opposing bilayers. It is known that smaller vesicles fuse more efficiently than larger ones, and hence the curvature of secretory vesicles may dictate the potency and efficacy of their fusion at the cell plasma membrane. The diameter of t- and v-SNARE vesicles may, therefore, reflect the size of the t-/v-SNARE complex formed. In the present study, this hypothesis was tested, and results from the study demonstrate that the size of the t-/v-SNARE complex is directly proportional to the vesicle diameter (R2 = 0.9725).     

Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

Layer-by-layer assembly of ordinary and composite coordination multilayers

Coordination self-assembly of bishydroxamate-based metal-organic multilayers on gold employing a layer-by-layer (LbL) approach was investigated. It is shown that the solution chemistry of the participating metal ion has a marked influence on the composition and properties of the multilayers. Use of Ce4+ and particularly zirconium(IV) acetylacetonate (Zr(acac)4) solutions in the ion-binding step of multilayer construction leads to multilayers with a near-stoichiometric metal ion-to-ligand ratio, suggesting a structure close to that predicted by a simple coordination self-assembly scheme. On the other hand use of a ZrCl4 solution as the source of metal ions in the multilayer construction leads to a multilayer with greater thickness and a large excess of Zr(IV), evenly distributed between the organic layers. In the latter case, a ratio of ca. 1:2 between the excess Zr and oxygen, as well as long-term Zr4+ binding experiments showing deposition of ZrO2, suggest the formation of a zirconia-type nanophase between the bishydroxamate organic repeat units during multilayer self-assembly. Hence, while the multilayer prepared using Zr(acac)4 solution appears to represent a "true" coordination-based structure, the one prepared using ZrCl4 is best described as a composite organic-ceramic multilayer. Composite multilayers prepared in this way display different properties from those of the stoichiometric ones, such as improved dielectric behavior and higher stiffness. Even greater mechanical stability is obtained with multilayers constructed using alternate binding of ZrCl4 and Ce4+. The concept of LbL formation of coordination-based composite organic-ceramic structures may be useful in obtaining nanometer-scale structures with tunable properties.     

杂臂星型嵌段共聚物在溶液中自组装形成囊泡结构的计算机模拟

利用耗散粒子动力学模拟方法, 研究了杂臂星型嵌段共聚物A_m(B_n)₂在溶液中自组装形成囊泡的行为. 主要分析了自组装过程、亲水分枝和疏水分枝的长度及分子构型对组装结构的影响. 结果表明, 杂臂星型聚合物在溶液中会自组装形成碟状胶束, 之后弯曲闭合形成囊泡. 当亲水部分的分枝较短时, 易于形成囊泡结构; 在可形成囊泡结构的条件下, 双分子层囊泡膜的厚度随分枝长度的增加而增加. 与构成相近的线型嵌段共聚物相比, 杂臂星型嵌段共聚物更易形成囊泡结构, 且形成的囊泡结构较薄.     

Lipid-based mechanisms for vesicle fission

Shape transformations and topological changes of lipid vesicles, such as fusion, budding, and fission, have important chemical physical and biological significance. In this paper, we study the fission process of lipid vesicles. Two distinct routes are considered that are both based on an asymmetry of the lipid distribution within the membrane. This asymmetry consists of a nonuniform distribution of two types of lipids. In the first mechanism, the two types of lipids are equally distributed over both leaflets of the membrane. Phase separation of the lipids within both leaflets, however, results in the formation of rafts, which form buds that can split off. In the second mechanism, the asymmetry consists of a difference in composition between the two monolayers of the membrane. This difference in composition yields a spontaneous curvature, reshaping the vesicle into a dumbbell such that it can split. Both pathways are studied with molecular dynamics simulations using a coarse-grained lipid model. For each of the pathways, the conditions required to obtain complete fission are investigated, and it is shown that for the second pathway, much smaller differences between the lipids are needed to obtain fission than for the first pathway. Furthermore, the lipid composition of the resulting split vesicles is shown to be completely different for both pathways, and essential differences between the fission pathway and the pathway of the inverse process, i.e., fusion, are shown to exist.     

2005年我国内地学者高分子科学基础研究进展概述

简要介绍了2005年度我国内地学者在高分子主要前沿领域基础研究的进展，涉及的领域主要包括特殊构筑高分子的合成、高分子结构表征、光电功能高分子、高分子自组装与超分子聚合物、高分子微纳结构与纳米复合体系、类细胞膜融合分裂的实时观测和生物医用高分子等。     

Characterization of pore formation by streptolysin O on supported lipid membranes by impedance spectroscopy and surface plasmon resonance spectroscopy

We report the study of the interactions of bacterial toxin streptolysin O (SLO) and cholesterol-containing membranes using electrochemical impedance and surface plasmon resonance (SPR) spectroscopy at low hemolytic units on a novel supported membrane interface. The detailed understanding of the process will aid significantly the construction of nanoscale transport channels for biosensing applications. Cholesterol-containing egg PC vesicles, pristine and incubated with SLO toxin, were fused onto a hexyl thioctate (HT)-modified gold substrate. The charge-transfer resistance of the resulting lipid membrane, which is related to the formation of the transmembrane pores, is measured with the aid of an electroactive probe. Impedance spectra were collected over a range of 0.1-100 kHz, and the obtained complex resistance was fit to an equivalent circuit. The charge-transfer resistance decreases for increasing SLO concentration, following a first-order exponential decay. The two-part membrane interface was further characterized with SPR spectroscopy. For the hexyl thioctate support layer, an equivalent monolayer thickness of 1.3 nm was determined. This value suggests a loosely packed structure of the monolayer on gold, presenting an ideal platform for permeability studies. A comparative study on the fusion behavior of vesicles with and without SLO induced pores revealed no substantial difference for the two systems, indicating that the pore formation has no adverse effect on the integrity of the vesicles. The resulting lipid membrane thickness from pre-perforated lipids was found to be 3.2 nm, suggesting that one leaflet is knocked off during the fusion process and a hybrid membrane is formed. A slightly higher thickness value of 3.4 nm was obtained for membranes from non-perforated vesicles. Deposition of lipids and subsequent incubation with SLO, as monitored by SPR, shows that the HT surface chemistry allows partial insertion of the toxin into the membrane, indicating unique properties as compared to the previously explored long-chain alkylthiols.     

Counterion-controlled transition of a cationic gemini from submicroscopic to giant vesicles

While much is known about the self-assembly of lipids on nanoscale, our understanding of their biologically relevant mesoscale organization remains incomplete. Here, we show for a cationic gemini lipid a sharp and reversible transition from small vesicles with an average diameter of approximately 40 nm to giant vesicles (GVs) with an average diameter of approximately 11 microm. This transition is dependent on proper [NaCl] and specific temperature. Below this transition and in the vicinity of the air/water interface, a series of mesoscale morphological transitions was observed, revealing complex structures resembling biological membranes. On the basis of microscopy experiments, a tentative [NaCl] versus temperature shape/size phase diagram was constructed. To explain this unprecedented transition, we propose a novel mechanism whereby a specific interaction of Cl(-) counterion with the cationic gemini surfactant initiates the formation of a commensurate solute counterion lattice with low spontaneous curvature. In keeping with the high bending rigidity of NaCl crystal, this tightly associated ionic lattice enslaves membrane curvature and the mesoscale 3-D organization of this lipid.     

Proton diffusion across membranes of vesicles of poly(styrene-b-acrylic acid) diblock copolymers

A study of proton diffusion across membranes of block copolymer vesicles in dilute solution is described. The vesicles were formed by the self-assembly of a diblock copolymer of poly(styrene-b-acrylic acid) (PS(310)-b-PAA(36), where the numbers represent the degree of polymerization for individual blocks). A pH gradient was created across the vesicle membrane with the interior pH (pH(in)) of ca. 2.9 and the exterior pH (pH(out)) of ca. 8.5. The permeability of the polystyrene (PS) membrane was tuned by the addition of different amounts of dioxane (0-40 wt %) to the external aqueous solution. Proton concentrations in the solution outside of the vesicles were followed by monitoring the spectrum of a pH-sensitive fluorescent dye, namely 8-hydroxypyrene-1,3,6-trisulfonate. After the start of the experiment, the proton concentrations increase linearly with the square root of time, while the slopes of the lines increase with dioxane content. To calculate the diffusion coefficients of the protons across the vesicular membrane, the concentration data were fitted using a model, which describes the diffusion of species across the membrane of a reservoir. The apparent diffusion coefficient (D*, which equals the true diffusion coefficient multiplied by the partition coefficient of protons between PS and water) increases from 1.1 x 10(-18) cm(2)/s at 7 wt % dioxane in the external solution to 1.2 x 10(-14) cm(2)/s at 40 wt %. The increase of D* with dioxane content is related to its plasticization of the PS membrane, which can be used as a gating mechanism.     

Membrane fusion of giant unilamellar vesicles of neutral phospholipid membranes induced by La3+

Membrane fusions of vesicles of biomembranes play various important roles in cells, but their mechanisms are unclear and controversial. In the present study, we found that 30 microM to 1 mM La3+ induced membrane fusion of two giant unilamellar vesicles (GUVs) composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoleoylphosphatidylethanolamine (DPOPE). We succeeded in observing a process of this membrane fusion in detail. First, two GUVs became strongly associated, with a partition membrane between them composed of two bilayers, one from each GUV. Then, the partition membrane was suddenly broken at one site on its edge. The area of this breakage site gradually spread, until it was completely separated from the GUV to complete the membrane fusion. Here, we propose a new model (i.e., the partition breakage model) for the mechanism of La3+ -induced membrane fusion of GUVs.     

Study of Organic Self-Assembled Nanosystems by Means of High-Frequency ESR/ENDOR: The Case of Oil Asphaltenes

An approach to investigate self-assembly of oil disperse systems based on high-field electron nuclear double resonance has been suggested. As exemplified by asphaltenes, the oil components most prone to self-assembly, the formation of planar rather than multilayer structures has been directly evidenced experimentally. The suggested method can be applied to elucidate the self-assembly mechanism in wide range of organic objects.     

Effect of polyethylene glycol-2000 on amino acid surfactant-based vesicles

Polyethylene glycol (PEG)-modified amino acid surfactant-based vesicles were prepared to improve the stability and cellular delivery of drugs. The vesicles comprised PEG-2000, sodium N-lauroylsarcosinate hydrate (SNLS), 1-decanol, and deionized water. The complex showed vesicular structures that were almost the same as the original vesicles, and their size distribution was (100–150 nm). Transmission electron microscopy (TEM) results revealed that no fusion occurred at 1.25 wt.% PEG concentration. The steric hindrance present among the vesicles prevented aggregation of the particles. No visual phase separation was observed for 6 months at room temperature 28?ºC. At higher molar concentration of PEG, fusion and wrinkling occurred owing to the association of PEG chains. The decreasing bending curvature led to the formation of fused vesicles with multilayer structure, as revealed by TEM and differential scanning calorimetry (DSC).     

Electrochemical characterization of polyelectrolyte/gold nanoparticle multilayers self-assembled on gold electrodes

Polyelectrolyte/gold nanoparticle multilayers composed of poly(l-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized gold nanoparticles (Au NPs) were built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode modified with a first layer of MSA. The assemblies were characterized using UV-vis absorption spectroscopy, cyclic and square-wave voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. Charge transport through the multilayer was studied experimentally as well as theoretically by using two different redox pairs [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+/2+). This paper reports a large sensitivity to the charge of the outermost layer for the permeability of these assemblies to the probe ions. With the former redox pair, dramatic changes in the impedance response were obtained for thin multilayers each time a new layer was deposited. In the latter case, the multilayer behaves as a conductor exhibiting a strikingly lower impedance response, the electric current being enhanced as more layers are added for Au NP terminated multilayers. These results are interpreted quite satisfactorily by means of a capillary membrane model that encompasses the wide variety of behaviors observed. It is concluded that nonlinear slow diffusion through defects (pinholes) in the multilayer is the governing mechanism for the [Fe(CN)(6)](3-/4-) species, whereas electron transfer through the Au NPs is the dominant mechanism in the case of the [Ru(NH(3))(6)](3+/2+) pair.     

Electrophysiological Characterization of Transport Across Outer‐Membrane Channels from Gram‐Negative Bacteria in Presence of Lipopolysaccharides

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we present an approach using fusion of native outer membrane vesicles (OMVs) into a planar lipid bilayer, allowing characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion surprisingly revealed only single or few channel activities. The asymmetry of the OMVs translates after fusion into the lipid membrane with the lipopolysaccharides (LPS) dominantly present at the side of OMV addition. Compared to the conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles for functional and structural studies of membrane channels in the native membrane.