牛胰多肽(BPP)与脂质体的相互作用——对脂质体通透性的影响

本文报导用测量脂质体内含物羧基荧光素(CF)释放的荧光方法,研究BPP对脂质体通透性的影响,结果表明去氧胆盐(DOC)促进CF的漏出,增加脂质体的通透性,BPP抑制CF的释放,降低通透性,保护膜结构.从而进一步闸明BPP具有细胞保护作用的机理.     

膜融合剂对脂质体及血影膜膜流动性的影响

本文用荧光探针ANS,DPH与A研究了几种膜融合剂对脂质体与血影膜流动性的影响.蔗糖使PS脂质体的脂双层流动性降低,探针越是在极性区流动性越小,说明蔗糖主要作用于脂双层的极性区;蔗糖也使血影膜流动性降低,此作用是可逆的.油酸甘油脂(GMO)使PS脂质体的流动性增加,且越是在疏水区内部,流动性增加得越大,说明GMO主要是作用于脂双层的非极性区:GMO也使血影膜流动性增加,此作用是不可逆的.二甲亚砜(DMSO)对血影膜的作用,两种不同荧光探针不一样,对DPH的作用出现双相让,低浓度与高浓度的作用结果分别与蔗糖和GMO的作用一致.     

血影膜及脂质体的融合及其与膜流动性的关系

本文采用Tb/DPA荧光方法研究了血影膜及磷脂酰丝氨酸脂质体在不同Ca~(2 )浓度诱导下的融合动力学过程,并通过改变温度研究了血影膜、PS脂质体的融合与膜流动性的关系.血影膜、PS脂质体的最大融合程度在一定范围内随膜流动性增加而增大,超出此范围则随膜流动增加而减小.对此结果进行了讨论.     

竹红菌甲素对红细胞膜和几种磷脂脂质体膜的流动性的光敏损伤

本文以荧光探针为手段,通过测量膜偏振度的变化,探讨了竹红菌甲素光敏作用对红细胞膜和几种磷脂脂质体膜的流动性的损伤。结果表明,甲素光敏作用使不同种类的磷脂(DPPC,DPPC/DPPE,红细胞膜磷脂)脂质体的流动性增加,其对光敏作用的敏感程度为红细胞膜磷脂脂质体显著小于DPPC/DPPE脂质体及DPPC脂质体。对红细胞膜来说,甲素光敏作用使其流动性呈现先降低而后增加的现象。去除膜上的spectrin以及用胰蛋白酶处理可使这种流动性变化的幅度受到抑制。据此,我们认为,膜磷脂,膜蛋白对甲素光敏作用中膜流动性的变化有着不同的影响,膜蛋白,特别是spectrin,是其中极重要的因素。     

脂质体通透性与脂多型性关系的研究

本文以TPE和TPE/DOPE(1:1.mol:mol)制成包裹荧光分子calcein的脂质体,通过测量荧光强度随扫描温度的变化,探讨了脂质体通透性与脂多型性之间的关系.结果表明,在不发生双层相(L)变成六角形Ⅱ相(H)相转变时,脂质体悬液的荧光强度不增加;当发生该转变时,脂质体悬液的荧光强度开始增加;完成该相转变后,脂质体悬液的荧光强度仍继续增加.据此,我们认为:脂质体的通透性与脂的多型性密切相关,当发生L→HⅡ相转变时,脂质体的通透性增加.由于荧光强度的变化对相变非常敏感,我们建议用测量脂质体荧光强度随温度的变化来监测脂质体稀悬液中脂的多型性.     

脂质体通透性与脂多型性关系的研究

本文以TPE和TPE/DOPE(1:1.mol:mol)制成包裹荧光分子calcein的脂质体,通过测量荧光强度随扫描温度的变化,探讨了脂质体通透性与脂多型性之间的关系.结果表明,在不发生双层相(L)变成六角形Ⅱ相(H)相转变时,脂质体悬液的荧光强度不增加;当发生该转变时,脂质体悬液的荧光强度开始增加;完成该相转变后,脂质体悬液的荧光强度仍继续增加.据此,我们认为:脂质体的通透性与脂的多型性密切相关,当发生L→HⅡ相转变时,脂质体的通透性增加.由于荧光强度的变化对相变非常敏感,我们建议用测量脂质体荧光强度随温度的变化来监测脂质体稀悬液中脂的多型性.     

艾氏腹水癌细胞膜质子跨膜外转运与脂质体融合:Ca2+及脂质体膜脂成份的作用

研究了Ca^2 及脂质体膜脂成分对艾氏腹水癌细胞质膜质子跨膜转运驱动的脂质体融合中的作用。结果 表明Ca^2 促进质子跨膜转运驱动的质子跨膜转运驱动艾氏腹水癌细胞与脂质体间的融合,膜融合程度与膜表面电荷密度的相关曲线显示,在下述条件膜融合与膜表面电荷密度呈正相关：（1）介质Ca^2 浓度小于6mmol/L,脂质体磷脂组成为PE：PC：CL＝6：2：2;（2）介质Ca^2 浓度为6mmol/L,脂质体鳞脂组成为PE：PC：CL＝6：2：2;（3）无Ca^2 介质,脂质体磷脂组成为PE：CL＝8：2;（4）介质Ca^2 浓度10mmol/L,脂质体磷脂组成为PE：CL＝8：2。脂质体PE／PC含量对膜融合的影响表明,当PE含量减少PC含量增加时,膜融合程度不断下降,提示影响膜融合的另一因素可能是生物膜结构形成“柄”融合中介体的能力。     

阳离子脂质体运载基因的跨膜机制

阳离子脂质体等非病毒载体以其制备简单、低毒性、低免疫原性、可生物降解等优点,成为近年来基因转运中的常用载体。理解阳离子脂质体运载基因的机制对阳离子脂质体的研究具有重要意义。从跨膜机制和信号调控的角度,介绍了脂质体/DNA复合体以特定构象避免细胞外基质中核酸酶的降解,跨越细胞膜进入细胞的过程;阐明了DNA在信号调控的作用下,逃离溶酶体并安全释放的机制;讨论了基因穿过核被膜进入到细胞核的方式,为进一步阐明阳离子脂质体运载基因的分子机制奠定基础。     

微丝骨架通过调控线粒体膜透性影响拟南芥耐盐性

植物微丝参与了许多重要的细胞生理活动,与植物耐盐性有密切的联系。在微丝解聚剂Latrunculin B（LatB）存在的情况下,拟南芥会表现出盐胁迫敏感。本研究结果表明盐胁迫下LatB可增加拟南芥线粒体膜通透性转换孔（mitochondrial permeability transition pore,MPTP）开放度,导致线粒体膜电势下降和细胞色素C的释放。而加入MPTP抑制剂环孢素A（CsA）后,膜电势下降程度降低,细胞色素C释放减少,解聚微丝造成的盐敏感表型得到一定程度恢复。     

二价汞、铅、镉氯化物对脑磷脂平板双分子层膜电特性的影响

本文报道了重金属毒物对脑磷脂双分子层膜电性质的影响.二价汞、铅、镉氯化物的存在,使膜电阻下降,膜的通透性增加.并且随着浓度的增加,膜平均寿命下降,击穿电压减小,说明膜机械脆性增加,膜容易破裂.而其它二价的Mg~(2 )、Ca~(2 )、Cu~(2 )、Mn~(2 )氯化物并无此作用.可以认为重金属毒物对生命的毒害作用中包括了对膜脂双层的直接影响.     

Effect of microwave radiation on permeability of liposomes. Evidence against non-thermal leakage

The effect of 2.45 GHz microwave radiation on the permeability of unilamellar phosphatidylcholine liposomes has been studied. Leakage of 5(6)-carboxyfluorescein from the liposomes was measured using spectrofluorimetry after exposure to either microwaves or thermal heating for 5–20 min intervals. The exposure temperature, 37.6 ± 0.5°C, was well above the phase transition temperature of the lipid membrane. The microwave exposure did not result in any non-thermal increase in permeability above that produced by thermal heating. This study refutes the results reported by Saalman et al. [1] in which an increased liposome permeability due to microwave exposure was reported. The refined analysis in the present study shows that this increased liposome permeability was not a non-thermal microwave effect.     

Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.     

钙库调控的钙通道Orai1体外研究方法的建立

目的:建立钙通道Orai1的体外研究方法。方法:利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果:成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论:利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。     

Cholesterol-independent membrane disruption caused by triterpenoid saponins

The membrane-disrupting activity of 15 triterpenoid saponins, obtained from Chinese plants of the genus Aralia, was investigated using phosphatidylcholine liposomes with and without cholesterol. The permeability of the membrane was examined by monitoring the induced fluorescent dye release from the liposome. On the basis of the obtained results, the structure-activity relationship among glucuronides of oleanolic acid was discussed. This takes into account particularly the variation in the carboxyl function. Namely, the saponins could induce a permeability change on liposomal membrane without cholesterol when they are glycosylated at both C-3 and C-28 of the oleanolic acid. There also exists a great similarity in the time-course curves for dye-release within such saponins, reflecting their similar action with the lipid bilayer membrane. The saponins glycosylated only at C-3 could also exhibit the same activity with somewhat different action profiles when the glucuronic acid is esterified, while those with the free glucuronic acid required cholesterol in the liposomes to induce permeability change thereof.     

精胺对脂质体及细胞膜融合的作用

本文通过共振能量转移法与三氯化铽荧光法探讨了精胺及其与Ca~(2 )对脂质体及人红细胞膜融合的诱导作用.结果表明,精胺能诱导PS脂质体的凝聚,但不能诱导其融合.精胺能诱导血影膜的融合.精胺与Ca~(2 )一起使用.对脂质体及血影膜的融合都分别有协同增效作用.     

Mycobacteria glycolipids as potential pathogenicity effectors: alteration of model and natural membranes

Four mycobacterial wall glycolipids were tested for their effects on phospholipidic liposome organization and passive permeability and on oxidative phosphorylation of isolated mitochondria. From fluorescence polarization of diphenylhexatriene performed on liposomes it was concluded that the two trehalose derivatives (dimycoloyltrehalose and polyphthienoyltrehalose) rigidified the fluid state of liposomes, the triglycosyl phenolphthiocerol slightly fluidized the gel state, while the peptidoglycolipid ("apolar" mycoside C) just shifted the phase transition temperature upward. Dimycoloyltrehalose was without effect on liposome passive permeability, as estimated from dicarboxyfluorescein leak rates, and polyphthienoyltrehalose and triglycosyl phenolphthiocerol slightly decreased leaks, while mycoside C dramatically increased leaks. Activity of these lipids on mitochondrial oxidative phosphorylation was examined. The two trehalose derivatives have been tested previously: both had the same type of inhibitory activity, dimycoloyltrehalose being the most active. Triglycosyl phenolphthiocerol was inactive. Mycoside C was very active, with effects resembling those of classical uncouplers: this suggested that its activity on mitochondria was related to its effect on permeability. All these membrane alterations were called nonspecific because it is likely that they result from nonspecific lipid-lipid interactions, and not from recognition between specific molecular structures. Such nonspecific interactions could be at the origin of some of the effects of mycobacteria glycolipids on cells of the immune system observed in the last few years.     

Shear stress induced lipid order and permeability changes of giant unilamellar vesicles

BackgroundThe permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability.MethodsWe determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan.ResultsWe observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes.Major conclusionsThe observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition.General significanceOur study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed.     

Interaction of wasp venom mastoparan with biomembranes

Mastoparan-induced changes in the K+ permeability of rat peritoneal mast cells, human erythrocytes, Staphylococcus aureus and Escherichia coli were examined. Mastoparan did not efficiently increase the K+ permeability of cells except for S. aureus. The release of membrane phospholipids was also observed from S. aureus cells in the concentration range of the permeability enhancement. Mastoparan stimulated histamine release from mast cells, independently of a small efflux of K+. Mastoparan became markedly effective to E. coli cells whose outer membrane structure was chemically disrupted beforehand, showing that the peptide can enhance the permeability of the cytoplasmic membranes of both Gram-positive and -negative bacteria. In experiments using liposomes, mastoparan increased the permeability of the liposomes composed of egg phosphatidylethanolamine and egg phosphatidylglycerol, which are the lipid constituents of the cytoplasmic membrane of E. coli cells, while it showed a weak activity to the liposomes composed of egg phosphatidylcholine and cholesterol. The latter result related closely to the fact that this peptide acted weakly on erythrocytes and mast cells in which acidic lipids constitute a minor portion. Mastoparan decreased the phase transition temperature of dipalmitoylphosphatidylglycerol liposomes, but it did not affect that of dipalmitoylphosphatidylcholine liposomes. These results indicate that mastoparan penetrated into membranes mainly containing acidic phospholipids and disrupted the membrane structure to increase the permeability. The action of the wasp venom mastoparan was compared with that of a bee venom melittin.     

Liposomes disposition in vivo V. Liposome stability in plasma and implications for drug carrier function

The kinetics of [¹⁴C]sucrose release from multilamellar liposomes of fixed diameter (approx. 0.23 μm) incubated in human plasma (serum and blood) were quantified. Composition was various ratios of phosphatidylcholine, phosphatidic acid and cholesterol with α-tocopherol included as antioxidant. Considerable intra-individual variability was noted for liposome stability in blood and its derived fluids, yet reproducible results were obtained for pooled samples. The destabilizing effects of plasma decreased with increasing lipid concentrations. Results of fitting a kinetic model to the data showed that four of five model parameters were linearly related to liposome cholesterol content. Liposomes depleted plasma of its destabilizing factors, and when pre-incubated with plasma were partially stabilized to the effects of a subsequent plasma addition. Plasma caused a rapid rise in liposome membrane permeability which then declined non-linearly, presumably because of a rearrangement of membrane lipids and adsorbed proteins to form their most stable configuration. the therapeutic availability of drugs administered encapsulated in liposomes, which can be governed by the kinetics of their in vivo extracellular release, may be directly proportional to - and predictable from - the time-course and extent of release in plasma. The kinetic model was used in conjuction with simple pharmacokinetic assumptions to show that the effectiveness of a liposome drug carrier cannot be predicted based simply on its plasma stability; more stable liposomes may not be more effective drug carriers. Interestingly, plasma-induced solute release from liposomes serendipitously mimics an important facet of ideal carrier behavior.