Specific interactions of complementary mono- and multivalent guests with recognition-induced polymersomes

We have explored the interactions of mono- and multivalent guests with Recognition-Induced Polymersomes (RIPs) formed from complementary random copolymers featuring diamidopyridine and thymine functionality. Addition of monovalent guests featuring imide functionality to these RIPs induced a temporary swelling of the vesicles, followed by dissociation of the vesicles due to competitive binding of the guest. Conversely, multivalent thymine-functionalized nanoparticle guests were rapidly incorporated into the RIPs, inducing a contraction of RIP diameter over time. These mono- and multivalent interactions were extremely specific: highly analogous control systems showed no interaction with the RIP structures. Taken together, these studies demonstrate highly selective molecular "lock and key" control over higher-order assembly and recognition processes.     

剧毒性Ⅱ型核糖体失活蛋白蓖麻毒素和相思子毒素的检测鉴定方法研究进展

Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,该类毒素大都具有一对二硫键连接的A-B链结构特征,B链具有半乳糖结合特性,能够与真核细胞膜表面受体特异性结合,将具有N-糖苷酶活性的A链导入细胞,与核糖体特定位点发生脱嘌呤作用使核糖体失活,最终通过抑制蛋白质合成而展现出细胞毒性。Ⅱ型RIPs毒素毒性极强,来源于植物的蓖麻毒素(ricin)和相思子毒素(abrin)的毒性分别是神经性毒剂维埃克斯(Vx)的385倍和2885倍。同时,该类毒素来源广泛、易于制备、稳定性好,成为一类潜在化生恐怖战剂,受到国内外广泛关注,其中蓖麻毒素作为唯一的蛋白毒素被收录于禁止化学武器公约禁控清单。近年来发生的多次蓖麻毒素邮件恐怖事件,进一步促进了有关Ⅱ型RIPs毒素的准确、灵敏、快速的检测鉴定技术的发展。剧毒性Ⅱ型RIPs毒素的检测鉴定方法主要涉及免疫分析法为代表的特异性识别和生物质谱分析为主的定性定量检测方法,以及基于脱嘌呤反应活性和细胞毒性的毒素活性检测方法。基于抗原-抗体作用的免疫检测法及基于寡核苷酸适配体的特异性识别检测法具有速度快、灵敏度高的优势,但对于复杂样品中高度同源蛋白的检测,易产生假阳性结果。随着生物质谱技术的快速发展,电喷雾离化(ESI)或基质辅助激光解吸离化(MALDI)等技术广泛应用于蛋白质的准确鉴定,不仅能够提供蛋白毒素的准确分子量和结构序列信息,而且能够实现准确定量。酶解质谱法是应用最为广泛的检测鉴定方法,通过酶解肽指纹谱分析,实现蛋白毒素的准确鉴定;对于复杂样品中蛋白毒素的分析,通过多种蛋白酶酶解策略获得丰富的特异性肽段标志物,然后进行肽段标志物的靶向质谱分析从而获得准确的定性及定量信息,方法有效提升了Ⅱ型RIPs毒素鉴定的准确度和灵敏度。免疫分析法和生物质谱法能够准确鉴定Ⅱ型RIPs毒素,但无法识别毒素是否还保持毒性。对于Ⅱ型RIPs毒素的活性分析,主要包括基于N-糖苷酶活性的脱嘌呤反应测定法和细胞毒性测定法,两种方法均可实现毒素毒性的简便、快速、灵敏的分析检测,是Ⅱ型RIPs毒素检测方法的有效补充。由于该类毒素的高度敏感性,国际禁止化学武器组织(OPCW)对相关样品中Ⅱ型RIPs毒素的分析提出了唯一性鉴定的技术要求。该文引用了Ⅱ型RIPs毒素及其检测方法相关的70篇文献,综述了以上Ⅱ型RIPs毒素的结构性质、中毒机理及典型剧毒性Ⅱ型RIPs毒素检测方法的研究进展,对不同检测方法的特点和应用潜力进行了总结,并结合OPCW对Ⅱ型RIPs毒素唯一性鉴定的技术需求,展望了未来Ⅱ型RIPs毒素检测技术研究的发展趋势。     

Molecular exchange through the vesicle membrane of siloxane surfactant in water/glycerol mixed solvents

The effect of glycerol on the permeability of vesicle membranes of a siloxane surfactant, the block copolymer polyethyleneoxide-b-polydimethylsiloxane-polyethyleneoxide, (EO)15-(DMS)15-(EO)15, was studied with freeze-fracture transmission electron microscopy (FF-TEM) and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) spectroscopy. The FF-TEM results show that, in pure water, the surfactant can form small vesicles with diameters of less than 25 nm, as well as a few multilamellar vesicles with diameters larger than 250 nm. Gradual substitution of water with glycerol to a glycerol content of 40% leads to significant structural transformations: small vesicles are gradually swollen, and large multilamellar vesicles disappear. A glycerol content of 60% results in the complete disintegration of the vesicles into membrane fragments. PFG-NMR measurements indicate that the vesicle membrane does not represent an effective barrier for water molecules on the NMR time scale; hence, the average residence time of water in the encapsulated state is below tau b = 2 ms. In contrast, the average residence time of glycerol molecules in the encapsulated state can be as large as tau b = 910 ms. The permeability of the vesicle membrane increases with increasing glycerol concentration in the solvent: At a concentration of 40%, the residence time tau b is lowered to approximately 290 ms. After vesicle destruction at higher glycerol concentrations, a small glycerol fraction is still bound by membrane fragments that are formed after the disintegration of the vesicles.     

Synthesis and characterizations of amorphous carbon nanotubes by pyrolysis of ferrocene confined within AAM templates

Amorphous carbon nanotubes (a-CNTs) are synthesized by pyrolysis of ferrocene confined in the nanopores of the anodic alumina membrane (AAM) and characterized by field emission scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), electron energy-loss spectroscopy (EELS), and Raman spectroscopy. It is shown that the a-CNT has an ultrathin amorphous wall (approximately 3 nm) and a relatively large diameter (approximately 50 nm), and is capsulated with iron oxide nanoparticles. It is found that the growth of the a-CNTs is governed mainly by the template limitation effect. Electrical transport measurements on individual a-CNTs demonstrate that the a-CNT may be connected with electrodes via either ohmic or Schottky contacts, and the resisitivity of the a-CNTs was measured to be 4.5 x 10(-3) Omega cm.     

Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.     

Colloidal adhesion of phospholipid vesicles: high-resolution reflection interference contrast microscopy and theory

High-resolution reflection interference contrast microscopy (HR-RICM) was developed for probing the deformation and adhesion of phospholipid vesicles induced by colloidal forces on solid surfaces. The new technique raised the upper limit of the measured membrane–substrate separation from 1 to 4.5 μm and improved the spatial resolution of the heterogeneous contact zones. It was applied to elucidate the effects of wall thickness, pH and osmotic stress on the non-specific adhesion of giant unilamellar vesicles (ULV) and multilamellar vesicles (MLV) on fused silica substrates. By simultaneous cross-polarization light microscopy and HR-RICM measurements, it was observed that ULV with the wall thickness of a single bilayer would be significantly deformed in its equilibrium state on the substrate as the dimension of its adhesive–cohesive zone was 29% higher than the theoretical value of a rigid sphere with the same diameter. Besides, electrostatic interaction was shown as a significant driving force for vesicle adhesions since the reduction in pH significantly increased the degree of deformation of adhering ULV and heterogeneity of the adhesion discs. The degree of MLV deformation on the solid surfaces was significantly less than that of ULV. When the wall thickness of vesicle increased, the dimension of contact zone was reduced dramatically due to the increase of membrane bending modulus. Most important, the adhesion strength of colloidal adhesion approached that of specific adhesion. Finally, the increase of osmotic stress led to the collapse of adhering vesicles on the non-deformable substrate and raised the area of adhesive contact zone. To interpret these results better, the equilibrium deformation of adhering vesicle was modeled as a truncated sphere and the adhesion energy was calculated with a new theory.     

Ni(OH)2纳米管的制备、表征及电化学性能

以多孔氧化铝为模板, 在不同溶液浓度下, 用化学沉积法制备了氢氧化镍纳米管. 采用XRD, SEM, TEM和HRTEM等手段, 对产物的物相、表面形貌及微结构进行了表征. 结果表明所得产物是高纯度的氢氧化镍纳米管, 外径约为180～220 nm, 管壁厚20～30 nm. 将所制备的氢氧化镍纳米管制成电极, 其电化学性能测试表明, Ni(OH)₂纳米管的中空结构特点, 能够有效地提高镍电极的充电效率、放电比容量、高倍率及高温放电性能. 机理分析表明中空结构的Ni(OH)₂纳米管对于提高碱性二次电池的综合性能有着极为重要的意义.     

Slow Reorganization of Small Phosphatidylcholine Vesicles upon Adsorption of Amphiphilic Polymers

Static or dynamic light scattering measurements were performed in parallel, on dilute mixtures of DPPC/DPPA vesicles (typical radius 60 nm) and hydrophobically modified polymers. This technique gave evidence of the slow kinetics involved in both the reorganization of an adsorbed polymer layer and the membrane breakage. Hours, or sometimes days, were required in order to follow the variation of both the hydrodynamic radius and the scattering intensity at intermediate stages. Images of the intermediate species were collected using freeze-fracture electron microscopy (FFEM). Comparison of different polymers (of varying molecular weight or structure) revealed the prime importance of hydrophobicity on the disruption of membranes. Although the presence of a few percent of pendant alkyl chains along the polymer backbone induced adsorption to membranes, only the association with the more hydrophobic ones (>25 mol% of pendant octyl groups) resulted in small mixed objects of micellar size (radius about 10 nm). The drop of the mean radius of intermediate structures formed upon the vesicle breakage was also sensitive to temperature. A tentative mechanism was proposed on the basis of kinetics and FFEM studies. Copyright 2001 Academic Press.     

Extruded vesicles of dioctadecyldimethylammonium bromide and chloride investigated by light scattering and cryogenic transmission electron microscopy

Combined dynamic and static light scattering (DLS, SLS) and cryogenic transmission electron microscopy (cryo-TEM) were used to investigate extruded cationic vesicles of dioctadecyldimethylammonium chloride and bromide (DODAX, X being Cl(-) or Br(-)). In salt-free dispersions the mean hydrodynamic diameter, D(h), and the weight average molecular weight, M(w), are larger for DODAB than for DODAC vesicles, and both D(h) and M(w) increase with the diameter (varphi) of the extrusion filter. NaCl (NaBr) decreases (increases) the DODAB (DODAC) vesicle size, reflecting the general trend of DODAB to assemble as larger vesicles than DODAC. The polydispersity index is lower than 0.25, indicating the dispersions are rather polydisperse. Cryo-TEM micrographs show that the smaller vesicles are spherical while the larger ones are oblong or faceted, and the vesicle samples are fairly polydisperse in size and morphology. They also indicate that the vesicle size increases with phi and DODAB assembles as larger vesicles than DODAC. Lens-shaped vesicles were observed in the extruded preparations. Both light scattering and cryo-TEM indicate that the vesicle size is larger or smaller than phi when phi is smaller or larger than the optimal phi approximately 200 nm.     

Lipid bilayer membrane with atomic step structure: supported bilayer on a step-and-terrace TiO2(100) surface

The formation of a supported planar lipid bilayer (SPLB) and its morphology on step-and-terrace rutile TiO 2(100) surfaces were investigated by fluorescence microscopy and atomic force microscopy. The TiO 2(100) surfaces consisting of atomic steps and flat terraces were formed on a rutile TiO 2 single-crystal wafer by a wet treatment and annealing under a flow of oxygen. An intact vesicular layer formed on the TiO 2(100) surface when the surface was incubated in a sonicated vesicle suspension under the condition that a full-coverage SPLB forms on SiO 2, as reported in previous studies. However, a full-coverage, continuous, fluid SPLB was obtained on the step-and-terrace TiO 2(100) depending on the lipid concentration, incubation time, and vesicle size. The SPLB on the TiO 2(100) also has step-and-terrace morphology following the substrate structure precisely even though the SPLB is in the fluid phase and an approximately 1-nm-thick water layer exists between the SPLB and the substrate. This membrane distortion on the atomic scale affects the phase-separation structure of a binary bilayer of micrometer order. The interaction energy calculated including DLVO and non-DLVO factors shows that a lipid membrane on the TiO 2(100) gains 20 times more energy than on SiO 2. This specifically strong attraction on TiO 2 makes the fluid SPLB precisely follow the substrate structure of angstrom order.     

Visualization of the nanoscale pattern of recently-deposited cellulose microfibrils and matrix materials in never-dried primary walls of the onion epidermis

For more than 10 years epidermal cell layers from onion scales have been used as a model system to study the relationship between cellulose orientation, cell growth and tissue mechanics. To bring such analyses to the nanoscale, we have developed a procedure for preparing epidermal peels of onion scales for atomic force microscopy to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall, with minimal disturbance and under conditions very close to the native state of the cell wall. The oriented, multilayer distribution of cellulose microfibrils, approximately ~3 nm wide, is readily observed over extended lengths, along with other features such as the distribution of matrix substances between and on top of microfibrils. The microfibril orientation and alignment appear more dispersed in younger scales compared with older scales, consistent with reported values for mechanical and growth anisotropy of whole epidermal sheets. These results open the door to future work to relate cell wall structure at the nm scale with larger-scale tissue properties such as growth and mechanical behaviors and the action of cell wall loosening agents to induce creep of primary cell walls.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

Supported lipid bilayers on biocompatible polysaccharide multilayers

Formation of supported lipid bilayers on soft polymer cushions is a useful approach to decouple the membrane from the substrate for applications involving membrane proteins. We prepared biocompatible polymer cushions by the layer-by-layer assembly of two polysaccharide polyelectrolytes, chitosan (CHI) and hyaluronic acid, on glass and silicon substrates. (CHI/HA)(5) films were characterized by atomic force microscopy, giving an average thickness of 57 nm and roughness of 25 nm in aqueous solution at pH 6.5. Formation of zwitterionic lipid bilayers by the vesicle fusion method was attempted using DOPC vesicles at pH 4 and 6.5 on (CHI/HA)(5) films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; a combination of fluorescence and AFM indicated that this was attributable to formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. By contrast, more uniform bilayers with mobile lipids were produced at pH 4. Fluorescence recovery after photobleaching gave diffusion coefficients that were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. These results demonstrate that polysaccharides provide a useful alternative to other polymer cushions, particularly for applications where biocompatibility is important.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Exocytosis of a single bovine adrenal chromaffin cell: the electrical and morphological studies

Exocytosis of a single bovine adrenal chromaffin cell, triggered by histamine stimulation, was investigated via the electric responses detected with single-walled carbon-nanotube field-effect transistors (SWCNT-FET) and the morphological changes acquired by atomic force microscopy (AFM). Secretion of chromogranin A (CgA), stored in the vesicles of a single chromaffin cell, can be monitored in situ by the antibody against CgA (CgA-antibody) functionalized on the SWCNT-FET devices. The SWCNT-FET can further discriminate the amount of released CgA with different levels of histamine stimulations. The AFM morphological studies on a chromaffin cell indicate that the depression structures on the cell surface, caused by the histamine-evoked exocytotic fusion pores, appeared much more frequently than those without histamine stimulation or with the pretreatment of mepyramine before histamine stimulation. The vesicle diameters are about 50 nm calculated from the obtained three-dimensional AFM images. In comparison, the fusion pores of chromaffin cells stimulated by high-K (+) buffer solution were also investigated to have a wider-ranging distribution of vesicle diameters of 60-260 nm. This work demonstrates that the combination of novel techniques, SWCNT-FET and AFM, can provide further insights into the fundamental properties of exocytosis in neuroendocrine cells.     

可聚合囊泡体系(烯丙基-二甲基-十二烷基溴化胺和十二烷基硫酸钠)在溶液中的盐效应

高稳定的囊泡广泛用于制作生物模型、药物输送以及合成纳米材料的模板。获得高稳定囊泡结构的重要方法之一是用聚合反应固定囊泡结构。作为可聚合囊泡制备的前期基础工作,研究了一种可聚合的囊泡体系：1-丙烯基－2,2,二甲基－十二烷基溴化胺（ADDB）和ADDB与十二烷基磺酸钠（SDS）的等摩尔比混合体系。该囊泡体系即使在高浓度盐水中也能够自发地形成均相的囊泡溶液。在聚合之前,采用动态激光光散射（DLS）、冷冻蚀刻透射电镜（FF-TEM）技术研究了可聚合囊泡的盐效应。DLS测试发现没有盐存在时,囊泡大小为83 nm,盐的浓度增加到250 mmol/L时,囊泡尺寸增大到250 nm。然而继续增大盐浓度到1000 mmol/L, 囊泡尺寸减小到180nm. FF-TEM结果发现盐浓度小于150 mM时, 单个囊泡为70 nm左右,然而明显存在囊泡的絮凝与融合;当盐浓度增加到400 mM时,单个囊泡尺寸减小到20 nm. 因此DLS 观测到囊泡尺寸增大的原因是由于囊泡的絮凝与融合;而尺寸减小的原因是由于在高盐浓度下,盐屏蔽了带电颗粒之间的静电相互作用,在熵增的驱使下,大囊泡变成小囊泡。     

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with ^tzG and ^thG, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs’ substrate recognition features.     

光交联固化的聚合物囊泡用于降温触发的药物释放

The stability of nanocarriers in physiological environments is of importance for biomedical applications. Among the existing crosslinking approaches for enhancing the structural integrity and stability, photocrosslinking has been considered to be an ideal crosslinking chemistry, as it is non-toxic and cost-effective, and does not require an additional crosslinker or generate by-products. Meanwhile, most current temperature-responsive nanocarriers are designed and synthesized for drug release by increasing temperature. However, heating may induce cell damage during triggered drug release. Therefore, lowering temperature-triggered nanocarriers need to be developed for drug delivery and safe drug release during therapeutic hypothermia. In this study, we prepared an amphiphilic block copolymer, poly(ethylene oxide)-block-poly[N-isopropyl acrylamide-stat-7-(2-methacryloyloxyethoxy)-4-methylcoumarin]-block-poly(acrylic acid) [PEO₄₃-b-P(NIPAM₇₁-stat-CMA₈)-b-PAA₁₃], by reversible addition fragmentation chain transfer (RAFT) polymerization. Successful synthesis of the polymer was verified by proton nuclear magnetic resonance (¹H NMR) and size exclusion chromatography (SEC). The copolymers self-assembled into vesicles in aqueous solution, with the P(NIPAM-stat-CMA) block forming an inhomogeneous membrane and the PEO chains and PAA chains forming mixed coronas. The cavity of this vesicle could be utilized to load hydrophilic drugs. The CMA groups could undergo photocrosslinking and enhance the stability of vesicles in biological applications, and the PNIPAM moiety endowed the vesicle with temperature-responsive properties. Upon decreasing the temperature, the vesicles swelled and released the loaded drugs. The size distribution and morphology of the vesicles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) experiments. After staining with phosphotungstic acid, the hollow morphology of the vesicles with a phase-separated inhomogeneous membrane was observed by TEM and SEM. The DLS results showed that the hydrodynamic diameter of the vesicles was 208 nm and the polydispersity was 0.075. The size of the vesicles observed by TEM was between 180 and 200 nm, which was in accordance with that measured by DLS. To verify the drug loading capacity and controlled release ability of the vesicle, a water-soluble antibiotic was encapsulated in the vesicles. The experimental results showed that the drug loading content was 10.4% relative to the vesicles and the drug loading efficiency was approximately 32.7%. For vesicles containing the same amount of antibiotics, the release rate at 25 ℃ was 35% higher than that at 37 ℃ after 12 h in aqueous solution. Overall, this photocrosslinked vesicle with temperature-responsive properties facilitates lowering temperature-triggered drug release during therapeutic hypothermia.     

Polymer encapsulation within giant lipid vesicles

We report encapsulation of polymers and small molecules within individual giant lipid vesicles (GVs; 3-80 microm), as determined by confocal fluorescence microscopy. Polymer-bound or free dyes were encapsulated within GVs by including these molecules in the aqueous solution during vesicle formation via gentle hydration. Encapsulation efficiencies of individual GVs (EE(ind)) were determined from the fluorescence intensity ratio inside vs outside the vesicle. EE(ind) varied considerably from vesicle to vesicle, with interior solute concentrations for GVs within the same batch ranging from much less than to slightly more than the initial concentration. The majority of GVs had high internal concentrations of polymer or small-molecule encapsulants equal to or slightly greater than the external concentration. EE(ind) decreased for high molecular weight polymers (e.g., dextran 500 000), but was relatively insensitive to the GV diameter, membrane composition, or incubation temperature in our experiments. Knowledge of EE(ind) is important for quantitative evaluation of reactions occurring within GVs (e.g., enzymatic processes) and for optimizing encapsulation conditions.     

Protein nanotubes comprised of an alternate layer-by-layer assembly using a polycation as an electrostatic glue

We present the synthesis and structure of various protein nanotubes comprised of an alternate layer-by-layer (LbL) assembly using a polycation as an electrostatic glue. The nanotubes were fabricated by sequential LbL depositions of positively charged polycations and negatively charged proteins into a porous polycarbonate (PC) membrane, followed by release of the cylindrical core by quick dissolution of the template with CH(2)Cl(2). This procedure provides a variety of protein nanotubes without interlayer cross-linking. The three-cycle depositions of poly-L-arginine (PLA) and human serum albumin (HSA, M(w)=66.5 kDa) into the porous PC template (pore diameter, D(p)=400 nm) yielded well-defined (PLA/HSA)(3) nanotubes with an outer diameter of 419+/-29 nm and a wall thickness of 46+/-8 nm, revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. The outer diameter of the tubules can be controlled by the pore size of the template (200-800 nm), whereas the wall thickness is always constant, independent of the D(p) value. The (PEI/HSA)(3) (PEI: polyethylenimine) nanotubes showed a slightly thin wall of 39+/-5 nm. CD spectra of the multilayered (PEI/HSA)(n) film on a flat quartz plate suggested that the secondary structure of HSA between the polycations was almost the same as that in aqueous solution. The three-cycle LbL depositions of PLA and ferritin (M(w)=460 kDa) or myoglobin (Mb, M(w)=1.7 kDa) into the porous PC membrane also gave cylindrical hollow structures. The wall thickness of the (PLA/ferritin)(3) and (PLA/Mb)(3) nanotubes were 55+/-5 nm and 31+/-4 nm; it depends on the globular size of the protein (ferritin>HSA>Mb). The individual ferritin molecule was clearly seen in the tubular walls by SEM and TEM measurements.