基因载体材料聚乙烯亚胺与磷脂酰胆碱脂质体的相互作用对膜结构的影响

采用动态光散射、荧光光谱、zeta电位测定和等温滴定量热技术分析了分子量分别为25000,10000和1800的聚乙烯亚胺(PEI)与二油酰磷脂酰胆碱(DOPC)脂质体的相互作用及其对脂质体膜内环境极性和膜通透性的影响.结果表明,PEI通过氨基与DOPC的磷脂基团和胆碱基团产生氢键或范德华作用,从而与脂质体结合形成复合物;低浓度PEI(0.075 mg/mL)导致DOPC脂质体的聚集和表面电位的增加,但未引起脂质体膜融合和表面电位反转;进一步增加PEI的浓度对脂质体表面电位的影响很小,而结合在表面的PEI分子链之间的排斥作用阻碍了脂质体聚集.PEI分子与DOPC脂质体的结合降低了脂质分子碳氢链的堆积密度和脂质体膜内环境的疏水性,从而增强了钙黄绿素和槲皮素在脂质体膜中的通透性.PEI与DOPC脂质体的相互作用具有明显的分子尺寸效应,增大PEI的分子量可以增强与脂质体的相互作用及对脂质体膜结构的影响.     

动、静态光散射在线跟踪DPPC/PA脂质体的聚集和融合过程

结合动、静激光光散射在线跟踪了经低温培养的二棕榈酰基磷脂酰胆碱/棕榈酸(摩尔比为1:2)脂质体在25-41℃温度范围内的聚集和融合过程.在升温过程中,脂质体的尺寸和相对分子质量明显增大,表明有聚集或融合发生.另外,尺寸分布出现角度依赖性,证明囊泡结构遭到破坏.而在接下来的降温过程中,粒径和相对分子质量继续增大,没有回到初始状态.根据光散射结果,我们认为脂质体富集脂肪酸分子的区域随温度升高会发生黏合而形成聚集,该聚集体形态类似反六方柱状相.融合发生的比例较小.降温过程中的变化表明囊泡是动力学稳定态,一旦聚集发生,会自发继续进行聚集或融合.     

拉链型脂肽的温控开关效应

亮氨酸拉链型脂肽是由两条肽链以螺旋结构依靠疏水作用并列结合形成的二聚体,当温度升至其相变温度时,其螺旋结构解旋继而变为无序链状结构。利用该类脂肽的温敏性能,本文设计、合成得到一组具有温敏性的拉链型脂肽,将其与磷脂混合制备温敏性脂质体。用圆二色谱测定磷脂双分子层上脂肽的二级结构,动态光散射测定脂肽-脂质体的粒径及电位;荧光偏振法测定脂质体膜的流动性;采用紫外分光光度计考察阿霉素(DOX)在37.0、45.0°C下的释放行为。结果表明,含有脂肽的脂质体具备较好的温敏性,胆固醇含量、脂质体膜的流动性,对脂肽的温控开关效应有一定的影响。脂肽-脂质体作为一种新型的温敏性药物载体展现了其较好的应用前景。     

稀土离子及其配合物与磷脂脂质体的相互作用

用核磁共振(NMR)方法研究了稀土离子及其配合物与二棕榈酰磷脂酰胆碱(DPPC)和鞘磷脂(SPM)脂质体的相互作用.磷脂极性头平行于膜平面.稀土离子与磷脂极性头P—O键键合,与经典模型不同,键合后极性基团仍平行于膜平面,而不是垂直于膜平面.稳定的稀土配合物对磷脂脂双层结构影响很小.将稀土离子引入磷脂脂质体和小分子配体的混合物中,稀土首先与小分子配体配位.     

脂质体荧光探针检测磷脂酶C的活性

建立了一种以荧光标记脂质体为探针检测磷脂酶 C (PLC) 活性的新方法.此荧光探针是由二棕榈酰磷脂酰胆碱(DPPC)和丽丝胺罗丹明B标记的荧光磷脂(Liss Rhod PE)通过自组装形成有序的荧光标记脂质体,探针脂质体中Liss Rhod PE由于相互之间距离靠近产生自猝灭效应,因而作为探针的脂质体并不表现出荧光性质.当在此探针溶液中加入目标物PLC,PLC可以水解切割标记在磷脂酰基二位上的荧光团罗丹明,使其从脂质体释放到溶液中,导致自猝灭效应的减弱,溶液荧光信号增强,以此实现对PLC活性的检测.使用此探针检测PLC活性,荧光强度的增加值与PLC浓度在5~300 U/L范围内呈良好的线性关系,检出限为2 U/L(S/N=3).此外,此探针还可用于PLC抑制剂的筛选.     

L-精氨酸盐酸盐/磷脂酰胆碱脂质体的制备及其对ATP的分子识别

用L-精氨酸盐酸盐和磷脂酰胆碱制备了L-广精氨酸盐酸盐／磷脂酰胆碱脂质体受体,研究了受体与腺苷三磷酸(ATP)在脂质体亲脂区通过分子间氢键进行的分子识别。透射电镜表征发现脂质体呈明显的双层膜圆球形,粒径100-200mm;并对氢键识别作用进行了UV-Vis光谱研究。     

稀土离子对肌醇磷脂囊泡的作用--结合、诱导聚集和促进水解

肌醇磷脂在细胞信号转导系统中起重要作用, 其水解反应是信号转导过程中的关键环节. 应用荧光淬灭滴定, 光散射以及高压液相技术, 研究了La3 和Tb3 对肌醇磷脂囊泡的作用, 包括结合常数的测定, 和对囊泡的诱导聚集作用以及促进肌醇磷脂水解的作用, 发现稀土离子与肌醇磷脂间具有较高的亲和力, 同时这种结合也诱导了肌醇磷脂囊泡的聚集和水解, 其程度与稀土离子和肌醇磷脂的结合常数有关.     

用NMR方法研究中药山莨菪碱与磷脂脂质体的相互作用

用核磁共振(NMR)方法研究了中药山莨菪碱(anisodamine)与结构不同的3种磷脂脂质体相互作用,二棕榈酰磷脂酰胆碱(DPPC)极性基团空阻较大,山莨菪碱三级胺端不能与P-O键作用,仍处于水相,苯环只能嵌入到甘油骨架C-2附近。二棕榈酰磷脂酸(DPPA)极性头空间位阻较小,山莨菪碱苯环可以直接插入到靠近脂酰链γ-次甲基的位置,而三级胺端与极性头发生静电作用,并且药物可以提高DPPA脂质体的流动性。山莨菪碱通过三级胺端与鞘磷脂(SPM)极性头静电作用较强,而苯环位于SPM脂双层亲水和疏水区界面。药物对3种磷脂双层结构影响很小。     

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%.     

双(苯并噁唑)茋的光异构化反应(Ⅱ)溶剂性质的影响

对双(苯并噁唑)茋化合物在不同极性溶剂中的光谱、光物理及光顺反异构化问题进行了详细研究.在测定其荧光寿命及荧光量子产率基础上计算出它们的辐射及非辐射衰变速度常数.发现化合物(1)在极性溶剂中有较高的荧光量子产率,而在非极性溶剂中则有较高的异构化能力.工作还测定了化合物在两种溶剂中反顺异构化过程的活化能,并对所得结果进行了初步讨论.     

Synthesis of fluorocarbon phospholipids and the formation of their liposomes

Two novel types of fluorocarbon phospholipids were prepared with choline and phosphate as the polar head group respectively. They form liposomes under dispersion either by sonication or injection. The size of the liposome with choline group is in the range of 32–37 nm based on the measurements of electron microscopy and of dynamic light scattering. However, the light scattering result showed that the size of the liposome containing phosphate group is larger and increases with the increasing pH value of the system. The fluorocarbon liposome is a much lucid dispersion. It is similar to the conventional hydrocarbon analogues in the ability to incorporate the water-soluble substrates. There is a transition point at 53 °C in the plot of fluorescene intensity of the fluorecent probe ANS incorporated into the fluorocarbon liposome versus temperature.     

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.     

Interactions of complementary PEGylated liposomes and characterization of the resulting aggregates

The interaction of complementary liposomes bearing both recognizable and protective ligands at their external surface has been investigated. Aggregation of hydrogenated phosphatidyl choline/cholesterol (2:1 molar ratio) based liposomes was mediated by the molecular recognition of the complementary phosphate and guanidinium groups incorporated in separate unilamellar liposomes. The phosphate group was incorporated in the bilayer employing dihexadecyl phosphate, while the guanidinium moiety was introduced in the membrane through the incorporation of various guanidinium lipids. For the latter, anchoring ability and primarily introduction of a spacer group between their lipophilic part and the guanidinium group was found to affect the ability for molecular recognition. Also, poly(ethylene glycol) (PEG) introduced in both types of liposomes at various concentrations and up to 15% with respect to cholesterol modifies the interaction effectiveness and morphology of the obtained aggregates. Interaction of these complementary liposomes leads to large precipitating aggregates or fused liposomes, as shown by phase contrast microscopy and dynamic light scattering. Specifically, fusion of liposomes takes place under a nonleaking process involving lipid mixing, as demonstrated by calcein entrapment and resonance energy transfer experiments. Calorimetric parameters also correlate with the processes of aggregation and fusion. The interactions of non-PEGylated liposomes involve exothermic processes of higher enthalpic content than those of the PEGylated counterparts.     

Studies on the encapsulation of diclofenac in small unilamellar liposomes of soya phosphatidylcholine

The encapsulation of acid (AD) and sodium diclofenac (SD) in small unilamellar liposomes (SUV) as well as the interactions of the drug with the bilayer was studied. SUV was prepared by sonication from multilamellar liposomes containing soya phosphatidylcholine and diclofenac at various proportions. The size distribution obtained from dynamic light scattering showed that the incorporation of SD decreases significantly the size of the liposomes suggesting that the drug interacts with the bilayer of the liposomes. This size decrease is related with the phase transition of liposomes to mixed micelar solution. The encapsulation of the hydrophilic dye indocyanine green in the aqueous compartment of liposomes showed that the rate of captured dye decreases with SD concentration suggesting the transition of liposomes to mixed micelles. The (31)P NMR analysis indicates that SD interacts with the phosphate of phosphatidylcholine head groups. A schematic model for interaction of SD with phosphatidylcholine of the liposomes in which the diclofenac anion interacts with the ammonium group of the phospholipid and the dichlorophenyl ring occupies a more internal site of bilayer near phosphate group was proposed.     

Microfluidic directed formation of liposomes of controlled size

A new method to tailor liposome size and size distribution in a microfluidic format is presented. Liposomes are spherical structures formed from lipid bilayers that are from tens of nanometers to several micrometers in diameter. Liposome size and size distribution are tailored for a particular application and are inherently important for in vivo applications such as drug delivery and transfection across nuclear membranes in gene therapy. Traditional laboratory methods for liposome preparation require postprocessing steps, such as sonication or membrane extrusion, to yield formulations of appropriate size. Here we describe a method to engineer liposomes of a particular size and size distribution by changing the flow conditions in a microfluidic channel, obviating the need for postprocessing. A stream of lipids dissolved in alcohol is hydrodynamically focused between two sheathed aqueous streams in a microfluidic channel. The laminar flow in the microchannel enables controlled diffusive mixing at the two liquid interfaces where the lipids self-assemble into vesicles. The liposomes formed by this self-assembly process are characterized using asymmetric flow field-flow fractionation combined with quasi-elastic light scattering and multiangle laser-light scattering. We observe that the vesicle size and size distribution are tunable over a mean diameter from 50 to 150 nm by adjusting the ratio of the alcohol-to-aqueous volumetric flow rate. We also observe that liposome formation depends more strongly on the focused alcohol stream width and its diffusive mixing with the aqueous stream than on the sheer forces at the solvent-buffer interface.     

Interaction of Dipalmitoyl Phosphatidylcholine with n‐Hexadecanol in Monolayer and Liposome

The monolayer collapse behavior of n‐hexadecanol/dipalmitoyl phosphatidylcholine (DPPC) was investigated in this study at the air/water interface at 37 °C. Surface pressure variations with time for the mixed monolayers of DPPC with 20 mol% and 50 mol% n‐hexadecanol at corresponding collapse points were recorded by a Langmuir trough system. In addition, the interaction of n‐hexadecanol with a pure DPPC monolayer was identified by fluorescence microscopy (FM). The results demonstrated distinct differences between these systems; according to our observation, the higher the ratio of n‐hexadecanol to DPPC, the more nucleation domains can be induced. The FM images demonstrated that pronounced domain formation was associated with a longer relaxation time of the collapsed DPPC and DPPC/n‐hexadecanol monolayers, and the presence of n‐hexadecanol appeared to enhance the relaxation processes. The liposome was prepared by the thin‐film hydration method. The average diameter of DPPC and DPPC/n‐hexadecanol liposomes was investigated by dynamic light scattering. It is shown that the diameter of DPPC liposome with n‐hexadecanol is smaller than pure DPPC liposome at the initial state. After 24 hours, DPPC/n‐hexadecanol liposome became larger than pure DPPC liposome and lasted for the next four days. The effects of a greater ratio of n‐hexadecanol did not play an important role in DPPC liposome formation based on our dynamic light scattering analysis. Our result demonstrated that n‐hexadecanol might affect the DPPC liposome stability. The increased ratio of n‐hexadecanol in DPPC liposomes could only a play a minor role in DPPC liposome fusion.     

Kinetics of the adhesion of DMPC liposomes on a mercury electrode. Effect of lamellarity, phase composition, size and curvature of liposomes, and presence of the pore forming peptide mastoparan X

Liposomes suspended in aqueous electrolyte solutions can adhere at mercury electrodes. The adhesion is a complex process that starts with the docking and opening and leads to a spreading, finally resulting in the formation of islands of adsorbed lecithin molecules. The adhesion process can be followed by chronoamperometry, and a detailed analysis of the macroscopic and microscopic kinetics can be performed yielding rate constants and activation parameters. By using giant unilamellar liposomes and multilamellar liposomes, the effect of lamellarity and liposome size could be elucidated for liposomes in the liquid crystalline, gel, and superlattice phase states. Below the phase transition temperature, the time constant of opening of the liposomes (i.e., the irreversible binding of the lecithin molecules on the preliminary contact interface liposome|mercury and the therewith associated disintegration of the liposome membrane on that spot) is shown to be strongly size dependent. The activation energy, however, of that process is size independent with the exception of very small liposomes. That size dependence of time constants is a result of the size dependence of the initial contact area. The time constant and the activation energies of the spreading step exhibit a strong size dependence, which could be shown to be due to the size dependence of rate and activation energy of pore formation. Pore formation is necessary to release the solution included in the liposomes. This understanding was corroborated by addition of the pore inducing peptide Mastoparan X to the liposome suspension. The obtained results show that electrochemical studies of liposome adhesion on mercury electrodes can be used as a biomimetic tool to understand the effect of membrane properties on vesicle fusion.     

Liposome-templated supramolecular assembly of responsive alginate nanogels

Nanosized gel particles (nanogels) are of interest for a variety of applications, including drug delivery and single-molecule encapsulation. Here, we employ the cores of nanoscale liposomes as reaction vessels to template the assembly of calcium alginate nanogels. For our experiments, a liposome formulation with a high bilayer melting temperature (Tm) is selected, and sodium alginate is encapsulated in the liposomal core. The liposomes are then placed in an aqueous buffer containing calcium chloride, and the temperature is raised up to Tm. This allows permeation of Ca2+ ions through the bilayer and into the core, whereupon these ions gel the encapsulated alginate. Subsequently, the lipid bilayer covering the gelled core is removed by the addition of a detergent. The resulting alginate nanogels have a size distribution consistent with that of the template liposomes (ca. 120-200 nm), as confirmed by transmission electron microscopy and light scattering. Nanogels of different average sizes can be synthesized by varying the template dimensions, and the gel size can be further tuned after synthesis by the addition of monovalent salt to the solution.     

Liposome solubilization induced by surfactant molecules in a microchip

The dynamics of liposome solubilization was monitored by dynamic light scattering and optical microscopy. A newly designed Y-shape microchannel connected to a room was incorporated into a microchip and the reaction processes of the liposome suspension and surfactant solution were observed in the room after mixing the two fluids and stopping the flow. By using this microchip, we succeeded in real-time monitoring of liposome solubilization and the following dynamic processes of solubilization were proposed: 1) Deformed liposomes become spherical. 2) The liposome size increases until the surfactant/liposome ratio in the liposome membrane reaches a threshold value. 3) Mixed micelles of surfactants and phospholipids are released and the liposomes collapse.