二棕榈酰磷脂酰胆碱/豆固醇脂质体液态有序相的结构性质:同步辐射X光衍射研究

用同步辐射小角和宽角X光衍射实验技术研究了由二棕榈酰磷脂酰胆碱(DPPC)和豆固醇所形成的脂质体的液态有序相的结构性质. 结果表明液态有序相的小角X光衍射d值(d-spacing)随着固醇温度和浓度的变化仅有微小的改变. 与凝胶相及液晶相的宽角X光衍射d值相比, 液态有序相的宽角X光衍射d值有更宽的变化范围, 在30到52 °C的温度范围内, 液态有序相的宽角X光衍射d值从0.422 nm变化到0.460 nm. 电子云密度计算表明液态有序相的脂双层厚度和水层厚度都要大于与之平衡共存的液晶相的脂双层厚度和水层厚度. 电子云密度计算结果还表明液态有序相的脂双层厚度随温度升高而降低. 本研究结果对于从定量的角度认识 生物膜的相态及深入认识生物膜中的有序结构具有重要意义.     

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.     

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.     

β-谷甾醇、豆甾醇与DPPC相互作用的单分子膜研究

植物甾醇对生物膜具有重要的调节作用,甾醇尾部的饱和程度对生物膜性质的影响一直是个热点问题.通过LB技术和原子力显微镜分别研究了尾部是单键的β-谷甾醇(β-sito)和尾部是双键的豆甾醇(stig)与二棕榈酰磷脂酰胆碱(DPPC)的相互作用,深入分析了β-谷甾醇、豆甾醇对DPPC单分子膜液态扩张-液态聚集(LE-LC)相变过程的影响.实验结果表明:β-谷甾醇、豆甾醇添加到DPPC单分子膜中,膜的压缩性以及分子的排列都发生了较大变化.当植物甾醇含量Xsterols=0.8时,β-谷甾醇、豆甾醇与DPPC相互作用具有相同的规律,过量分子面积和过量吉布斯自由能均为负值,说明分子间的吸引力比排斥力更强;在低浓度下,Xsterols=0.2,0.4时,两种系统的过量分子面积和过量吉布斯自由能呈现出明显的区别,尾部是单键的β-谷甾醇和DPPC混合单分子膜的排斥力更强烈,而尾部是双键的豆甾醇和DPPC混合单分子膜的吸引力更强烈,说明尾部是双键的豆甾醇比尾部是单键的β-谷甾醇更容易和DPPC发生凝聚,AFM数据进一步证实了这些结果.     

LB膜中的缺陷畴区及其诱导形成环形草酸钙图形

利用存在缺陷的LB膜来模拟受损伤的肾上皮细胞膜诱导肾结石矿物草酸钙(CaOxa)晶体生长. 经2.5 mmol·L-1的草酸钾溶液对二棕榈酰磷脂酰胆碱(DPPC)的LB膜进行处理后, 可强化LB膜中液态扩张相(LE)和液态凝聚相(LC)之间的结构差异, 原子力显微镜(AFM)证实了圈状缺陷的存在. 以LB膜的圈状缺陷为模板, 诱导了一水草酸钙(COM)晶体环状图案. 相比之下, 未经草酸钾处理的LB膜只诱导生成零散的六边形COM晶体.     

巨型囊泡的侧向相分离与出芽

用电形成法制备含三组分二油酰磷脂酰胆碱(DOPC)/二棕榈酰磷脂酰胆碱(DPPC)/胆固醇(Chol)的巨型囊泡, 以TR-DPPE为荧光染色剂, 在荧光显微镜下直接观察膜的侧向相分离与微区相凸起出芽的耦合. 发现囊泡膜内的相分离具有诱导期, 相分离速度很快, 形成的微相区在整个囊泡球面上均衡分布. 各微相区的出芽不是同时进行, 为逐个随机发生. 每次出芽的时间小于0.5 s. 分相与出芽的耦合使球面上的不同微区之间不会相互融合成更大的微区.     

衍生于咔唑的双氰基二苯代乙烯型双光子荧光脂筏探针

制备了一个衍生于咔唑的双氰基二苯代乙烯型双光子荧光脂筏探针——(E)-2-甲基-5-{2-[9-正辛基(3-咔唑基)]乙烯基}对苯二甲腈(DLR), 并对其结构进行了表征. 结果表明, DLR属于推-拉电子结构(供体-桥-受体, D-π-A), 其最大发射波长随介质极性递增, 而其荧光强度却随极性递减. DLR在二棕榈酰磷脂酰胆碱 (DPPC)中的发射强度是在二油酰磷脂酰胆碱(DOPC)中的20倍, 其对DPPC, 模拟脂筏[n(DOPC)∶n(鞘磷脂)∶n(胆固醇)=1∶1∶1]和DOPC的荧光强度比为20∶12.8∶1, 在DPPC中的荧光寿命是在DOPC中的2.2倍以上, 表明DLR能很好地区分DPPC与DOPC. DLR在DPPC和DOPC中的双光子发射截面(Φδ)分别为1350和67 GM, 表明DLR能够很好地识别脂筏, 成像脂筏在细胞与组织中的分布动态.     

两亲性分子聚集体的相变及其协同性

两亲性分子聚集体是一类重要的软物质,它们有着丰富而复杂的相行为.本文主要从两个方面综述了作者所在的研究组在两亲性分子聚集体相变研究方面的工作进展.(1)磷脂相关体系相变热力学:归纳了多种小分子(二甲基亚砜、甘油、海藻糖、尿素等)对于磷脂体系相行为的调控,比较并讨论了固醇类分子和葡萄糖神经酰胺分子诱导磷脂分子形成液态有序相的能力,还介绍了计算机模拟磷脂相行为的工作进展.(2)两亲性分子聚集体相变的协同性:先介绍了相变协同性(即分子头部、尾部、界面等基团在相变过程中的一致性)问题的提出,然后通过双十八烷基二甲基溴化铵分子和硬脂酰溶血卵磷脂两个体系的研究实例,说明两亲性分子聚集体相变过程中存在着头尾不一致的现象.对这个问题的研究,将为我们打开挑战相态转变的一系列重大问题(如相变动力学、相态多型性、相态稳定性以及相变可逆性等)的新窗口.     

血清中非胆固醇类固醇检测方法研究进展

人体血清中除含有胆固醇外,还含有微量的角鲨烯和非胆固醇类固醇.近年的研究发现:血清中的角鲨烯和胆固醇前体固醇可以反映机体的内源性胆固醇合成效率,而植物固醇可反映机体的外源性胆固醇吸收效率~([1]).胆固醇前体固醇和植物固醇是一类以环戊烷全氢菲为骨架的醇类化合物.在某些家族性血脂代谢异常的患者血清中,非胆固醇类固醇的含量会明显偏高~([2]).因此,分析固醇类物质含量与血脂代谢的关系,就显得尤为重要.     

乌苏酸/DPPC 混合单层膜相互作用的研究

乌苏酸(UA)为五环三萜羧基酸类化合物, 是一种中药有效成分. 它进入细胞的过程与膜脂分子有密切关系. 选用生物膜系统中的膜脂分子二棕榈酰基磷脂酰胆碱(DPPC)为代表, 通过LB(Langmuir-Blodgett)膜技术获得乌苏酸与DPPC 混合单层膜的表面压力/平均分子面积(π-A)曲线. 分析了混合单层膜的平均和过量分子面积、弹性系数等热力学参量, 并用原子力显微镜进行了研究. 比较了乌苏酸/DPPC 与胆固醇/DPPC 混合单层膜的异同. 实验结果表明: 乌苏酸能促使DPPC 的凝聚; 乌苏酸/DPPC 两组分的物质的量比与混合单层膜的膜压对单层膜的压缩性、稳定性和热力学特性有影响, 对单层膜中不同组分间的混合性以及分子间的相互作用具有重要的影响.     

Liquid Ordered Phase of Binary Mixtures Containing Dipalmitoylphosphatidylcholine and Sterols

The effect of cholesterol, desmosterol, stigmasterol, sitosterol, ergosterol, and androsterol on the phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) was studied to understand the role of the side chain in the formation of ordered phases of the type observed in membrane rafts. Thermotropic changes in the structure of mixed dispersions and transition enthalpies were examined by synchrotron X-ray diffraction (XRD) and differential scanning calorimetry (DSC). The observations indicated that cholesterol was more efficient than phytosterols (stigmasterol and sitosterol) or ergosterol in its interaction with DPPC to form the liquid ordered phase (L_o). The L_o induced by cholesterol or desmosterol was stable over a wide temperature range, whereas, the liquid ordered phase containing phytosterols or ergosterol was profoundly dependent on temperature, which should be distinguished as L_oβ and L_oα, representing the phases below and above the main transition temperature. The characteristics in forming ordered structures of cholesterol and other sterols imply that the evolution may have selected cholesterol as the most efficient sterol for animals to form rafts in their cell membranes.     

Phase diagram of androsterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

The effect of androsterol, whose structure resembles that of cholesterol but without the alkyl side chain, on the phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine has been studied to understand the role of the side chain played in the formation of ordered phases of the type observed in membrane rafts. Thermotropic changes in the structure of mixed dispersions and transition enthalpies have been examined by synchrotron X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. From these results a partial phase diagram of the binary system has been constructed. The three-phase line is determined to be 34.5 degrees C, which is 3-5 degrees C lower than that observed in binary mixtures of cholesterol, ergosterol, or stigmasterol with dipalmitoylphosphatidylcholine. The proportions of androsterol in mixtures representing the "left end point" and "right end point" of the three-phase line are 11.1 and 30.9 mol %, respectively. These proportions are greater than that seen in phase diagrams of other sterols codispersed with dipalmitoylphosphatidylcholine. We conclude that androsterol is less effective in promoting the formation of an ordered phase, and furthermore, this ordered phase is less compact than the normal lamellar liquid-ordered phase.     

Phase Transitions and Structural Changes in DPPC Liposomes Induced by a 1-Carba-Alpha-Tocopherol Analogue

Steady-state emission spectroscopy of 1-anilino-8- naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescence anisotropy, and DSC methods were used to characterize the interactions of the newly synthesized 1-carba-alpha-tocopherol (CT) with a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane. The DSC results showed significant perturbations in the DPPC structure for CT concentrations as low as 2 mol%. The main phase transition peak was broadened and shifted to lower temperatures in a concentration-dependent manner, and pretransition was abolished. Increasing CT concentrations induced the formation of new phases in the DPPC structure, leading to melting at lower temperatures and, finally, disruption of the ordered DPPC structure. Hydration and structural changes of the DPPC liposomes using ANS and DPH fluorescent probes, which are selectively located at different places in the bilayer, were studied. With the increased concentration of CT molecules in the DPPC liposomes, structural changes with the simultaneous formation of different phases of such mixture were observed. Temperature studies of such mixtures revealed a decrease in the temperature of the main phase transition and fluidization at decreasing temperatures related to increasing hydration in the bilayer. Contour plots obtained from concentration–temperature data with fluorescent probes allowed for identification of different phases, such as gel, ordered liquid, disordered liquid, and liquid crystalline phases. The CT molecule with a modified chromanol ring embedded in the bilayer led to H-bonding interactions, expelling water molecules from the interphase, thus introducing disorder and structural changes to the highly ordered gel phase.     

Orientation of tie-lines in the phase diagram of DOPC/DPPC/cholesterol model biomembranes

We report the direction of tie-lines of coexisting phases in a ternary diagram of DOPC/DPPC/cholesterol lipid bilayers, which has been a system of interest in the discussion of biological rafts. For coexisting Ld and Lo phases, we find that the orientation angle α of the tie-lines increases as the cholesterol concentration increases and it also increases as temperature increases from T = 15 °C to T = 30 °C. Results at lower cholesterol concentrations support the existence of a different two-phase coexistence region of Ld and So phases and the existence of a three-phase region separating the two two-phase regions. Our method uses the X-ray lamellar D-spacings observed in oriented bilayers as a function of varying hydration. Although this method does not obtain the ends of the tie-lines, it gives precise values (±1°) of their angles α in the ternary phase diagram.     

Calorimetric and spin-label ESR studies of PEG:2000-DPPE containing DPPC/lyso-PPC mixtures

The thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) multibilayers containing up to 10 mol% of lyso-palmitoylphosphatidylcholine (lyso-PPC) with and without low content of poly(ethylene glycol:2000)-grafted dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) has been studied by high sensitivity differential scanning calorimetry (DSC) and electron spin resonance (ESR) using the spin probe di-tert-butyl-nitroxide (DTBN). The three lipids, dispersed in buffer at appropriate concentrations, form thermosensitive liposomes used as site-specific drug-delivery systems. Without polymer–lipids, the DPPC main transition temperature is downshifted of 1.2–1.3 °C at the highest lyso-PPC content. The molar enthalpy and the cooperative unit of the DPPC main transition first decrease rapidly, then more slowly and finally slightly increase with lyso-PPC content. Moreover, in the mixed dispersions, the membrane fluidity increases at any temperature. The addition up to 5 mol% of PEG:2000-DPPE to DPPC/10 mol% lyso-PPC mixtures does not affect neither the thermotropic phase behavior nor the transition cooperativity and the fluidity of the dispersions.     

Interaction of Natural and Modified β-Cyclodextrins with a Biological Membrane Model of Dipalmitoylphosphatidylcholine

Lipid vesicles made up of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were used as a biological membrane model to investigate the interaction between natural and modified β-cyclodextrins and these membrane bilayers. Differential scanning calorimetry was used to study the thermotropic behavior of the DPPC vesicles and any change caused by the presence of cyclodextrins. The presence of dimethyl-β-cyclodextrin (DM-β-CyD) triggered a reduction in the enthalpy values related to the main transition peak from gel state to liquid crystal phase of DPPC aqueous dispersions, as a function of the DM-β-CyD molar fraction: the larger the amount of DM-β-CyD, the greater the reduction in ΔHvalues. This effect was probably due to the ability of DM-β-CyD to extract and to complex the DPPC molecules forming the phospholipid vesicles. The presence of β-cyclodextrin (β-CyD) or hydroxypropyl-β-cyclodextrin (HP-β-CyD) caused no particular alteration in the thermotropic parameters of DPPC vesicles, whereas trimethyl-β-cyclodextrin (TM-β-CyD) at molar fractions higher than 0.12 caused broadening of the transition peak due to a possible interaction with the hydrophobic part of the bilayers. Experiments on DPPC–cholesterol (10 mol%) vesicles showed the capability of β-CyD and TM-β-CyD to extract cholesterol from the ordered bilayer structures, triggering an alteration in the lipid constituents of the membranes. HP-β-CyD caused no variation in the thermotropic parameters of the DPPC–cholesterol (10 mol%) vesicles. The findings show that HP-β-CyD seems the most suitable molecular drug carrier forin vivoadministration.     

A DSC study of paeonol-encapsulated liposomes, comparison the effect of cholesterol and stigmasterol on the thermotropic phase behavior of liposomes

Thermotropic phase behaviors of paeonol-encapsulated liposomes containing stigmasterol or cholesterol have been investigated by differential scanning calorimetry. We compared the thermotropic phase behavior of pure dipalmitoylphosphatidylcholine (DPPC) liposomes, sterol/DPPC liposomes, and paeonol/sterol/DPPC liposomes increasing the ratio of paeonol to sterol from 0 to 1, by analyzing the calorimetric parameters of main phase transition of liposomes including phase transition temperature (onset temperature and peak temperature) and phase transition cooperativity. The results showed that paeonol could incorporate into the hydrophobic region of DPPC, thus, decrease phase transition temperature of DPPC. Though stigmasterol interacts with DPPC less favorably than cholesterol, thermotropic phase behavior of paeonol/cholesterol/DPPC liposomes and that of paeonol/stigmasterol/DPPC liposomes are very similar. A phase separation occurred when the molar ratio of paeonol to sterol reached 1:1 in paeonol-encapsulated liposomes, where a paeonol-rich domain coexisted with a sterol-rich domain. The packing order of acyl chains of DPPC in sterol-rich domain is a little higher than that in paeonol-rich domain.     

Interaction of 3β-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.     

Interaction of propyl paraben with dipalmitoyl phosphatidylcholine bilayer: a differential scanning calorimetry and nuclear magnetic resonance study

The influence of the preservative, propyl paraben (PPB) on the biophysical properties of dipalmitoyl phosphatidyl choline (DPPC) vesicles, both in multilamellar vesicle (MLV) and unilamellar vesicle (ULV) forms, has been studied using DSC and (¹H and ³¹P) NMR. The mechanism by which PPB interacts with DPPC bilayers was found to be independent of the morphological organization of the lipid bilayer. Incorporation of PPB in DPPC vesicles causes a significant depression in the transition temperature and enthalpy of both the pre-transition (PT) and the gel to liquid crystalline transition. The presence of the PPB also reduces the co-operativity of these transitions. However, at high PPB concentration the PT disappears. DSC and NMR findings indicate that: (i) PPB is bound strongly to the lipid bilayer leading to increased headgroup fluidity due to reduced headgroup–headgroup interaction and (ii) the PPB molecules are intercalated between the DPPC polar headgroups with its alkyl chain penetrate into the co-operative region. MLV incorporated with high PPB concentration shows additional transitions whose intensity increases with increasing PPB concentration. This phase segregation observed could probably be due to co-existence of PPB-rich and PPB-poor phospholipid domains within the bilayers. The effect of inclusion of cholesterol in the PPB-free and PPB-doped DPPC dispersion was also studied. Equilibration studies suggest that PPB molecules are very strongly bound and remain intercalated between the polar headgroup for prolonged time.