DOPC,DOPE和神经酰胺对鞘磷脂/胆固醇双层膜结构的影响

用LB技术和原子力显微镜(AFM)研究了1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)、1,2-二油酸甘油-3-磷脂酰乙醇胺(DOPE)和神经酰胺(Ceramide)对鞘磷脂(SM)/胆固醇(Chol)结构的影响. 实验结果表明, 在表面压力较低时, 每种混合脂双层膜都呈现均匀分布的脂双层结构. 随着表面压力的增加, 形态发生了明显的变化: (1) SM/Chol二元组分双层膜形成均一的液态有序相微区结构, 衬底覆盖率达到80%; (2) DOPC的加入促使SM/Chol双层膜出现相分离现象, SM/Chol形成的液态有序相 “岛状” 微区结构漂浮在液态无序相的DOPC上部, 约占总面积的30%; (3) DOPE与SM/Chol形成的双层膜明显不同于DOPC/SM/Chol, 呈现出液态无序相、液态有序相及凝胶相3相共存的结构; (4) Ceramide诱导了SM/Chol双层膜结构发生重排, 两层脂分子间发生翻转形成囊泡结构, 部分神经酰胺从液态有序相中分离形成小颗粒结构. 在较高膜压下, 各系统都呈现出具有特定形态的双层膜结构. 分子官能团的成键能力决定了双层膜形态结构.     

DOPC/DPPC单层膜中分子间的相互作用及形态特征

利用Langmuir-Blodgett(LB)技术制备了不同表面压力下的1,2-二油酸-甘油-3-磷脂酰胆碱(DOPC)/1,2-二棕榈酸甘油-3-磷脂酰胆碱(DPPC)(摩尔比为1:1)和DOPC/DPPC/Chol(摩尔比为2:2:1)单层膜, 对单层膜内分子间的相互作用进行了热力学分析, 并用荧光显微镜和原子力显微镜对其形态进行了观测.热力学分析表明, DOPC与DPPC分子在单层膜结构中相互作用为排斥力, 诱导单层膜出现相变; DOPC, DPPC与胆固醇(Chol)间的相互作用均为吸引力, 当表面压力(π)大于18 mN/m时, DPPC与胆固醇的作用力大于DOPC.荧光显微镜观测表明, DOPC/DPPC单层膜出现明显相分离现象, 富含DPPC微区成“花形”结构, 且随着表面压力的升高微区逐渐增大, “花瓣”增多; 当胆固醇加入到DOPC/DPPC体系时, 单层膜相态由液相与凝胶相共存转变为液态无序相与液态有序相共存结构, 富含DPPC的微区形状从“花形”转变成“圆形”.原子力显微镜对单层膜的表征验证了荧光显微镜的观测结果, 表明胆固醇加入到DOPC/DPPC体系中对单层膜排列具有明显的影响, 压力和溶液状态等是影响脂膜结构的重要因素.     

Langmuir-Blodgett膜超分子SM/DOPC/Chol三元脂系的原子力显微镜观察

用原子力显微镜研究了胆固醇(Chol)对鞘磷脂(SM)/1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)二元脂系统结构的影响和神经酰胺对SM/DOPC/Chol三元脂系统结构的影响. 实验发现, 在SM/DOPC二元脂系统中, 胆固醇和带饱和脂肪酸链的磷脂发生相互作用形成微区结构, 随着胆固醇含量的增加, 微区的面积逐渐增大, 形成了稳定的片层结构. 当把神经酰胺加入到等摩尔配比的SM/DOPC/Chol三元脂系统中时, 随着神经酰胺比例的增加, 先形成紧密的聚集态结构, 然后逐渐演变成具有特定微区的网状结构. 研究结果表明, 微区的形成主要是由分子不同的官能团之间的相互作用所决定, 这可能在细胞信号传导等生理活动中起到重要的作用.     

α-生育酚在模型生物膜中的分子动力学模拟

用分子动力学方法模拟了280, 310和350 K下α-生育酚在二豆蔻酰磷脂酰胆碱、二豆蔻酰磷脂酰乙醇胺、二硬脂酰磷脂酰胆碱和二硬脂酰磷脂酰乙醇胺双层膜中的性质, 包括了空间位置、氢键、取向和动力学性质, 取得了如下的结论. 第一, 生育酚头部的羟基一般位于脂双层亲疏水界面的下方, 升高温度将促进羟基向膜双层的中心移动, 在350 K时观察到了在上下两个单层间的翻转. 第二, 生育酚主要与磷脂的酯基形成氢键, 几乎不与磷脂酰乙醇胺的氨基形成氢键; 比较生育酚与磷脂酰胆碱和乙醇胺形成的氢键后发现, 后者更稳定. 第三, 生育酚的头部在膜中取向多变, 与膜的法线夹角不固定, 尾部的构象也很复杂. 第四, 在温度较低时, 生育酚的侧向扩散系数与磷脂的相当, 但在350 K时其扩散速度明显加快; 在垂直方向生育酚的扩散速度很慢.     

二亚油酰磷脂酰胆碱分析

通过HPLC/HPLC-MS研究了二亚油酰磷脂酰胆碱的测定方法,磷脂样品首先经过HPLC分离得到磷脂酰胆碱,该磷脂酰胆碱再进一步通过不同的HPLC系统分离二亚油酰磷脂酰胆碱,并通过标准样品和质谱进行二亚油酰磷脂酰胆碱的定性.定量方法通过标准样品外标法定量,由方法的回收率和精密度试验可知,该方法已用来测定一般磷脂样品和磷脂酰胆碱样品中的二亚油酰磷脂酰胆碱含量.     

微生物脂肽与磷脂的混合单分子膜性质

研究了一种微生物脂肽--表面活性素与二肉豆蔻酰磷脂酰胆碱(DMPC)在气,液界面形成的混合单分子膜性质.测定了混合单分子膜的表面压.分子面积(л-A)曲线,根据л-A曲线获得了不同表面压下混合单分子膜的过剩面积(Aex)和混合过剩自由能(△Gmex)与混合单分子膜中表面活性素摩尔分数的关系.Aex和△Gmex的计算结果均表明,表面活性素与DMPC在纯水亚相上形成的混合单分子膜中不相容,二者之间 的相互作用主要是排斥力.通过原子力显微镜观察了在表面压15mN/m下的混合单分子膜的LB膜,发现表面活性素与DMPC发生了微相分离,说明二者在混合膜中的烷基链取向不同,这可能是二者发生排斥作用的主要原因之一.此外,还研究了亚相pH对混合单分子膜相容性的影响,发现表面活性素与DMPC在混合单分子膜中的相容性在碱性环境下增强,这可能与二者极性头基之间的相互作用有关.     

改性苯乙烯-马来酸酐共聚物色谱固定相用于磷脂分离分析北大核心CSCD

磷脂是重要的信号分子,磷脂的代谢与多种疾病密切相关。因此,开展磷脂的分离分析研究至关重要。苯乙烯-马来酸酐共聚物(SMA)作为一种新型两亲性交替共聚物可以插入生物膜的磷脂双分子层中,形成以膜蛋白质为中心的脂质纳米盘,对膜蛋白质和磷脂具有良好的增溶作用。本文基于“点击”反应和自由基聚合反应,将对磷脂具有良好增溶性能的SMA接枝到硅胶表面,然后以蛋氨酸甲酯盐酸盐(MME·HCl)为开环试剂,通过亲核开环反应对SMA进行修饰,制备了新型的改性SMA修饰色谱固定相(Sil-SMA-MME)。结合高效液相色谱-紫外检测法,利用酰胺类和核苷/核酸碱基类以及苯酚类3类小分子物质对填充Sil-SMA-MME色谱柱的保留机制和分离性能进行了系统评价,Sil-SMA-MME色谱柱具有典型的亲水作用保留机制,其柱效最高可达90900 N/m,并显示了良好的分离选择性。进一步结合高效液相色谱-蒸发光散射检测法,考察了Sil-SMA-MME色谱柱对磷脂样品的分离性能。二棕榈酰磷脂酰丝氨酸钠(DPPS)、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰乙醇胺(DPPE)和4种磷脂酰胆碱(PC)类标准品溶血卵磷脂(LysoPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)、二硬脂酰磷脂酰胆碱(DSPC)、二棕榈酰磷脂酰胆碱(DPPC)均可实现基线分离,并且成功地实现了南极磷虾油和人血清磷脂提取物的分离分析。以上结果表明,所制备的Sil-SMA-MME色谱柱在磷脂类物质分离分析中具有良好的应用潜力。     

膜材性质及制备方法调控下的脂质体负载干扰素的研究

依据干扰素(IFN)分子、磷脂分子本身的理化性质和结构特点, 分别用三种制备方法, 以四种脂质体为膜材, 制备IFN脂质体, 考察了不同膜材、不同制备方法对脂质体粒径及包封率的影响. 结果表明, 以二肉豆蔻酰胆碱和二棕榈酰磷脂酰胆碱复合材料为主要膜材, 采用薄膜蒸发法制备的IFN脂质体有良好的稳定性, 60 d内其粒径可以保持在200~350 nm, 包封率可保持30%~40%.     

仿生信号转导体系的构建:囊泡表面磷脂信号分子诱发的乳酸脱氢酶的激活

以细胞肌醇磷脂信号转导途径为原型,在合成肽脂囊泡(人工细胞膜模型)上,利用天然磷脂为信号分子,成功地激活了处于囊泡表面的乳酸脱氢酶．为此,将1,2-二-十四烷基磷脂酰乙醇胺等天然磷脂嵌入合成肽脂N,N-二-十六烷基-N^a-6-三甲胺基己酰基-L-甘氨酰胺囊泡中,制备了稳定的混合双层膜囊泡,用透射电子显微照相、动态光散射及差示扫描量热等手段确认了混合囊泡的形态及粒径分布．以磷酸吡哆醛等维生素B6类化合物为信号分子激活剂,利用它们与天然磷脂形成复合体,进而与Cu^2 形成强的金属配合物的性质,实现了对处于囊泡表面、被Cu^2 抑制的乳酸脱氢酶的激活,构建了一个新的仿生信号转导体系．紫外一可见光谱实验证实了以上结果．此外,结果还表明囊泡表面的疏水作用和静电引力是促进天然磷脂一磷酸吡哆醛复合体形成的主要因素．囊泡表面的疏水微环境作为反应场是构建此仿生信号转导体系不可缺少的要素．     

盐浓度及磷脂分子结构对多组分带电膜弯曲刚性的影响

磷脂膜弯曲刚性模量很难直接测量, 本实验用循环冻融法制备尺寸大小与膜弯曲刚性相关的熵稳定单层囊泡. 粒度仪测量发现, 囊泡尺寸随盐浓度增加呈现先剧烈减小然后缓慢增加的分段变化规律. 但当组分中含有头部带电同时尾链带有不饱和键的二油酰磷脂酰甘油酯时, 囊泡尺寸却在较大的盐浓度范围内不出现回升. 囊泡膜的负Zeta-电势绝对值均表现为先急剧减小然后趋于平稳的变化规律, 数值大小只与带电组分的含量有关. 而对直接水合法制备的多层囊泡的统计发现, 囊泡尺寸随盐浓度增加急剧减小, 随后趋于稳定值, 均不随分子组合变化而回升. 结果表明在不同的盐浓度范围里, 主导磷脂膜弯曲刚性模量的因素不同. 低盐浓度的体系, 静电屏蔽效应为主导因素; 高盐浓度的体系, 膜双电层中反离子的分布起主导作用. 磷脂分子头部与尾部的不同结构组合会影响膜双电层, 使膜的弯曲刚性不同. 多层囊泡体系中, 高盐浓度下膜的热涨落掩盖了分子结构及双电层分布差异对膜弯曲刚性的影响.     

Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane compositions

Asymmetric cell division is common in biology and plays critical roles in differentiation and development. Unicellular organisms are often used as model systems for understanding the origins and consequences of asymmetry during cell division. Although basic as compared to mammalian cells, these are already quite complex. We report complete budding and asymmetric fission of very simple nonliving model cells to produce daughter vesicles that are chemically distinct in both interior and membrane compositions. Our model cells are based on giant lipid vesicles (GVs, 10-30 μm) encapsulating a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a crowded and compartmentalized cytoplasm mimic. Ternary lipid compositions were used to provide coexisting micrometer-scale liquid disordered (L(d)) and liquid ordered (L(o)) domains in the membranes. ATPS-containing vesicles formed buds when sucrose was added externally to provide increased osmotic pressure, such that they became not only morphologically asymmetric but also asymmetric in both their interior and their membrane compositions. Further increases in osmolality drove formation of two chemically distinct daughter vesicles, which were in some cases connected by a lipid nanotube (complete budding), and in others were not (fission). In all cases, separation occurred at the aqueous-aqueous phase boundary, such that one daughter vesicle contained the PEG-rich aqueous phase and the other contained the dextran-rich aqueous phase. PEGylated lipids localized in the L(o) domain resulted in this membrane domain preferentially coating the PEG-rich bud prior to division, and subsequently the PEG-rich daughter vesicle. Varying the mole ratio of lipids resulted in excess surface area of L(o) or L(d) membrane domains such that, upon division, this excess portion was inherited by one of the daughter vesicles. In some cases, a second "generation" of aqueous phase separation and budding could be induced in these daughter vesicles. Asymmetric fission of a simple self-assembled model cell, with production of daughter vesicles that harbored different protein concentrations and lipid compositions, is an example of the seemingly complex behavior possible for simple molecular assemblies. These compartmentalized and asymmetrically dividing ATPS-containing GVs could serve as a test bed for investigating possible roles for spatial and organizational cues in asymmetric cell division and inheritance.     

Positioning lipid membrane domains in giant vesicles by micro-organization of aqueous cytoplasm mimic

We report localization of lipid membrane microdomains to specific "poles" of asymmetric giant vesicles (GVs) in response to local internal composition. Interior aqueous microdomains were generated in a simple model cytoplasm composed of a poly(ethyleneglycol) (PEG)/dextran aqueous two-phase system (ATPS) encapsulated in the vesicles. The GV membrane composition used here was a modification of a DOPC/DPPC/cholesterol mixture known to form micrometer-scale liquid ordered and liquid disordered domains; we added lipids with PEG 2000 Da-modified headgroups. Osmotically induced budding of the ATPS-containing GVs led to structures where the PEG-rich and dextran-rich interior aqueous phases were in contact with different regions of the vesicle membrane. Liquid ordered (L o) membrane domains rich in PEG-terminated lipids preferentially coated the PEG-rich aqueous phase vesicle "body", while coexisting liquid disordered (L d) membrane domains coated the dextran-rich aqueous phase "bud". Membrane domain positioning resulted from interactions between lipid headgroups and the interior aqueous polymer solutions, e.g., PEGylated headgroups with PEG and dextran polymers. Heating resulted first in patchy membranes where L o and L d domains no longer showed any preference for coating the PEG-rich vs dextran-rich interior aqueous volumes, and eventually complete lipid mixing. Upon cooling lipid domains again coated their preferred interior aqueous microvolume. This work shows that nonspecific interactions between interior aqueous contents and the membrane that encapsulates them can drive local chemical heterogeneity, and offers a primitive experimental model for membrane and cytoplasmic polarity in biological cells.     

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.     

Fission of giant vesicles accompanied by hydrophobic chain growth through polymerization-induced self-assembly

In order to clarify the formation mechanisms of micrometer-sized spherical vesicles through the polymerization-induced self-assembly of amphiphilic poly(methacrylic acid)-block-poly(methyl methacrylate-random-methacrylic acid), PMAA-b-P(MMA-r-MAA), the nitroxide-mediated photocontrolled/living radical polymerization initiated by a PMAA end-capped with 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl was performed in an aqueous methanol solution. The polymerization proceeded in a living manner during the self-assembly. The vesicles produced during the early stage of the polymerization were not completely spherical and had dents and very small holes on their surface. As the hydrophobic P(MMA-r-MAA) block chains grew by the polymerization, the contorted vesicles were changed into half-sized elliptical vesicles accompanied by enlargement of the dents and holes. The vesicles were finally transformed into much smaller spherical vesicles by further growth of the hydrophobic chains. The mechanisms of the vesicles by fission involved the outside separation by the expansion of the dents and holes on the surface and the inside separation by budding.     

Dynamic processes in endocytic transformation of a raft-exhibiting giant liposome

The dynamic response of a raft-exhibiting giant liposome to external stimuli, such as the addition of Triton X-100 or osmotic stress, was studied. We observed that daughter vesicles are generated inside of the liposome through endocytic budding. It was found that the budding to generate daughter vesicles is classified into two different routes, simple budding through the invagination of a whole raft and budding from the boundary of a raft accompanied by waving motion. Smaller rafts show a preference for simple budding, whereas large rafts mainly adopt the other process. We discuss the mechanism of this difference in terms of the kinetic pathway of internalization by considering the line energy and bending energy of the membrane.     

Cationic lipid vesicles as coating precursors in capillary electrochromatography: separation of basic proteins and neutral steroids

1,2-Dioleyl-3-trymethylammoniumpropane (DOTAP) lipid vesicles were employed as coating precursors to obtain a semipermanent cationic lipid bilayer in silica capillary. The coating procedure was relatively fast and simple. Reliable results for the separation of four basic proteins (alpha-chymotrypsinogen A, ribonuclease A, cytochrome C, lysozyme) were obtained by using an acetate buffer under acidic conditions. The RSDs of the migration times were not higher than 0.5% run-to-run and about 1% day-to-day (3 days), while the RSDs of the peak areas were within 7% day-to-day (3 days). The day-to-day RSD of the EOF mobility of about 1%, confirmed that the DOTAP coating was stable for the separation of basic proteins, under acidic buffers. In addition to basic proteins the DOTAP coating was found suitable under acidic conditions for the repeatable separation of neutral steroids. The potential of DOTAP as a carrier in background electrolyte solution was studied.     

Budding dynamics of individual domains in multicomponent membranes simulated by N-varied dissipative particle dynamics

We study the budding dynamics of individual domains in flat, multicomponent membranes using dissipative particle dynamics (DPD) simulations with varied bead number N, in which addition and deletion of beads based on their density at the membrane boundary is introduced. The budding process of a tubular bud, accompanied by a dynamical transition reflected in the energy and morphology evolutions, is investigated. The simulations show that budding duration is shortened with increasing line tension and depends on the domain size quadratically. At low line tension, increasing bending modulus accelerates budding at first, but suppresses the process as it increases further. In addition, the controlling role of the surface tension in the budding process is also explored. Finally, we use the N-varied DPD to simulate the experimentally observed multicomponent tubular vesicles, and the three bud growth modes are confirmed.     

Budding dynamics of multicomponent tubular vesicles

Real-time budding dynamics of multicomponent, tubular lipid vesicles was investigated. By using a fluorescence microscope, three typical growth modes of the buds were observed, corresponding, respectively, to bud growth through coalescence between flat patches, a bud and a patch, as well as that of two buds. The spatial and temporal scales measured in the observation were used to estimate the bending rigidity of the membrane. In the late stage, the continuing coalescence between the buds resulted in large shape deformation of the vesicles, from tubular to spherical vesicles, and the number of the buds decayed with time as N approximately t-2/3. This scaling relation was observed for the first time in experiment and confirmed early theoretical predictions. Our observation showed a difference between the diffusivity of the buds on the lipid membrane and that of the embedded membrane proteins.     

Transmission electron microscopy study of solvent-induced phase morphologies of environmentally responsive mixed homopolymer brushes on silica particles

We report in this work a transmission electron microscopy study of phase morphologies of environmentally responsive mixed poly( t-butyl acrylate) (P tBA)/polystyrene (PS) brushes and mixed poly(acrylic acid) (PAA)/PS brushes on 180 nm silica particles after treatments with nonselective good solvents and selective solvents, respectively. Mixed P tBA/PS brushes were grown from Y-initiator-functionalized silica particles by sequential atom transfer radical polymerization and nitroxide-mediated radical polymerization. Mixed PAA/PS brushes were prepared from mixed P tBA/PS brushes by removal of the t-butyl groups. For mixed P tBA/PS brushes with P tBA M n of 24.2 kDa and PS M n of 23.0 kDa and the corresponding mixed PAA/PS brushes, random worm-like, nearly bicontinuous nanostructures were formed from lateral microphase separation when the particles were cast from nonselective good solvents (chloroform for mixed P tBA/PS brushes and N, N-dimethylformamide for mixed PAA/PS brushes). The feature sizes were on the order of polymer chain root-mean-square end-to-end distances ( approximately 10 nm). In contrast, mixed P tBA/PS brushes with lower molecular weights (P tBA M n = 10.4 kDa and PS M n = 11.9 kDa) did not strongly phase separate after being cast from chloroform. After the solvents in the particle dispersions were gradually changed to selective solvents ( n-octane for mixed P tBA/PS brushes and H 2O for mixed PAA/PS brushes), isolated microdomains with an average size of 14-19 nm were formed as one grafted polymer collapsed and associated to form isolated microdomains, which were shielded by another grafted polymer yielding surface-tethered micellar structures. These results confirmed the theoretical predictions of the formation of "rippled" nanostructures and surface micellar structures of mixed homopolymer brushes induced by nonselective and selective solvents, respectively.