蜂毒肽浓度和磷脂组分对巨囊泡泄露的影响

蜂毒肽作为一种广谱抗菌肽已经得到广泛认知,用蜂毒肽构建载药体系攻击癌细胞研究正在兴起.基于仿生物膜模型探索其破坏机理,可以避免潜在活性细胞过程的影响.在此,我们选用细胞尺寸的单层巨囊泡膜模型,可在光学显微镜下直接观察和操作,获得仿正常细胞膜和仿癌细胞膜在不同蜂毒肽浓度刺激下的响应.研究得出,低浓度蜂毒肽诱导囊泡泄露实验表明中性磷脂囊泡以孔模式为主泄露,负电性磷脂囊泡以爆裂模式为主泄露;高浓度蜂毒肽诱导囊泡泄露实验表明负电性磷脂相较于中性磷脂可延迟蜂毒肽作用效果;蜂毒肽色氨酸残基荧光光谱表明囊泡膜表面蜂毒肽吸附量以及泄露模式依赖于磷脂组分.此外,推断了蜂毒肽对不同组分磷脂膜的破坏作用模型.研究为蜂毒肽在肿瘤细胞的作用机制及其衍生物的优化设计提供参考.     

蜂毒肽与多组分生物膜作用：电性与相态的影响

蜂毒肽非特异性地靶向杀伤细菌具有重要的生物医学应用前景. 利用荧光光谱与荧光显微考察了蜂毒肽与单组分、多组分磷脂膜的作用机制. 发现对于不同电性与相态的磷脂膜, 肽-膜作用呈现为稳定桶板型孔、非稳U型孔及变薄裂解等多种机制, 具有显著不同的内含物泄露效率. 多组分磷脂囊泡的泄露实验表明, 泄露由肽亲和性较强的磷脂组分决定. 相较于凝胶相磷脂, 蜂毒肽与液相磷脂的亲和性强, 凝胶-液相混合囊泡与纯液相磷脂囊泡的泄露性质相近; 相较于双电性磷脂, 蜂毒肽与负电性磷脂的亲和性强, 双电-负电混合囊泡与纯负电磷脂囊泡的泄露性质相近. 研究深化了多肽与多组分生物膜作用机制的理解.     

多聚赖氨酸诱导的负电性磷脂巨囊泡形变

利用荧光显微技术表征了多聚赖氨酸诱导的负电性磷脂巨囊泡的动力学响应行为.研究发现,多聚赖氨酸可吸附至二油酰磷脂酰胆碱和二油酰磷脂酸混合磷脂巨囊泡的表面,诱导其发生粘连、出"绳"及破裂现象.分析认为,在低盐环境中,膜形变由多聚赖氨酸吸附于二油酰磷脂酸富集区引起的膜两叶应力不对称,以及静电相互作用等因素产生.研究结果对基于聚合物-巨囊泡体系的药物输运控释、细胞形变、微控反应和基因治疗等方面的研究提供有价值的支持.     

磷脂跨膜交换机制的研究

膜间磷脂交换是一项重要的生理活动, 其对药物运输及膜功能研究有重要意义. 本文用石英晶体微天平及耗散系数测试仪研究囊泡与囊泡、囊泡与支撑膜间磷脂交换行为, 荧光光谱仪用来测量膜表面电性与膜组分对磷脂交换的影响. 实验结果表明: 磷脂跨膜交换速率与交换时间成反比, 膜表面异电性磷脂的增加会加速膜内相互作用和磷脂跨膜交换速率, 以及改变膜表面组分会对囊泡与支撑膜间的磷脂交换产生影响. 本文研究有助于加深理解磷脂跨膜交换机制, 并对药学研究提供参考.     

磷脂跨膜交换机制的研究

膜间磷脂交换是一项重要的生理活动,其对药物运输及膜功能研究有重要意义.本文用石英晶体微天平及耗散系数测试仪研究囊泡与囊泡、囊泡与支撑膜间磷脂交换行为,荧光光谱仪用来测量膜表面电性与膜组分对磷脂交换的影响.实验结果表明:磷脂跨膜交换速率与交换时间成反比,膜表面异电性磷脂的增加会加速膜内相互作用和磷脂跨膜交换速率,以及改变膜表面组分会对囊泡与支撑膜间的磷脂交换产生影响.本文研究有助于加深理解磷脂跨膜交换机制,并对药学研究提供参考.     

磷脂在膜结构间的交换:温度和离子强度的影响

磷脂跨膜交换对生物膜功能与药学研究有重要意义.石英电子微天平及耗散系数测量仪被用于研究囊泡与支撑膜间磷脂的交换行为.研究表明:首先,在磷脂跨膜输运过程中,热力学环境和离子强度对支撑膜表面吸附囊泡的形变程度影响较小,囊泡与支撑膜的总接触面积直接取决于囊泡的吸附数量;其次,交换过程中膜结构间最大总接触面积随着温度的升高和离子强度的降低而增大,温度和离子引起的囊泡吸附速率和跨膜交换速率的变化在其中发挥着关键调节作用.本研究有助于加深对磷脂在生理条件下跨膜输运过程的理解,并为基于脂质体的药物载运体系研究提供参考.     

磷脂囊泡在胺端基和羧端基修饰金表面的沉积

磷脂囊泡与阴, 阳离子性表面作用的深入理解对磷脂的生物纳米科技应用具有重要意义. 通过石英电子微天平及耗散系数测量仪, 研究了不同离子性磷脂囊泡在胺端基和羧端基修饰Au衬底的沉积行为. 实验观察到四条囊泡沉积路径: (ⅰ)吸附至临界值后破裂成膜; (ⅱ)吸附即破裂成膜; (ⅲ)吸附不破裂; (ⅳ)不吸附. 沉积路径由囊泡与衬底的双电层相互作用决定, 离子可发挥调节作用.     

磷脂囊泡在胺端基和羧端基修饰金表面的沉积

磷脂囊泡与阴,阳离子性表面作用的深入理解对磷脂的生物纳米科技应用具有重要意义.通过石英电子微天平及耗散系数测量仪,研究了不同离子性磷脂囊泡在胺端基和羧端基修饰Au衬底的沉积行为.实验观察到四条囊泡沉积路径:(ⅰ)吸附至临界值后破裂成膜;(ⅱ)吸附即破裂成膜;(ⅲ)吸附不破裂;(ⅳ)不吸附.沉积路径由囊泡与衬底的双电层相互作用决定,离子可发挥调节作用.     

磷脂微滴囊泡化的介观模拟

发展了一种非显示溶剂的粗粒化三粒子磷脂模型,该模型明确反映磷脂分子的双尾结构．模型分别采用变形的MIE作用势和Harmonic作用势描述分子间非成键和分子内成键相互作用,粗粒化力场参数通过拟合DPPC双分子层的结构和力学性质获得．该粗粒化模型成功重现了磷脂分子从随机初始态到双分子层和从盘状结构到囊泡的形成过程．应用该模型系统研究了球形和柱形磷脂微滴囊泡化的过程,结果表明此模型能有效地模拟介观尺度下复杂磷脂囊泡的形成及演化．     

组分混合磷脂球胶束化动力学

两亲性磷脂分子能够形成各种不同形态的胶束,其结构形成不仅依赖于磷脂分子结构和组成,还依赖于两亲性分子的自组装路径. 本工作采用粗粒化分子动力学方法模拟研究了二棕榈酰磷脂酰胆碱(DPPC)与十六烷基磷酸胆碱(HPC)混合磷脂球胶束化行为. 通过调节DPPC/HPC的组分比例和磷脂球尺寸,观察到多种不同胶束结构形成,例如：球形和非球形(扁平或长椭球)囊泡、盘形胶束、单环或双环胶束和蠕虫状胶束. 研究发现,由于原位胶束化作用,采用磷脂球作为初始态有利于形成囊泡和环形拓扑结构胶束. 模拟结果表明,结合初始态结构设定同时调节磷脂分子组成是一种有效调控磷脂胶束结构的方法.     

Isothermal volume variations in lipid vesicle suspensions. A new evidence of intervesicle fusion kinetics

Summary In the present study of fusion between lipid vesicles performed by thermomechanical analysis, isothermal volume variation has been shown to be a reliable tool to follow these kinetics without introducing perturbing probes. In fact, the fusion process is accompanied by bilayer strain release which causes an overall volume decrease of the fused vesicles. Volumetric variations induced by side processes, such as adhesion or ion binding onto the vesicle surface, were accounted for in our measurements. Moreover, by the same technique we followed segregation effects of the membrane lipid components in mixed vesicles. The systems examined were neutral and anionic phospholipids containing vesicles. The role of temperature, vesicle size, lipid composition as well as the influence of different cations were also investigated.     

On the changes in the microwave dielectric spectrum of aqueous phospholipid bilayer solutions at the ordered-fluid phase transition

The microwave part of the dielectric spectrum (ν ? 1 GHz) is considered of aqueous phospholipid solutions in the limit of high water content. A continuum model is presented which allows to calculate in the water relaxation region the frequency-dependent complex permittivity of solutions in which the bilayers form globular single-walled vesicles as well as multilamellar liposomes. The model is not only capable of explaining the strikingly small values of the extrapolated static permittivity and of the main dielectric relaxation time which became evident in many measurements on colloidal aqueous solutions of phospholipids. It also allows the positive and negative step-like changes in the dielectric properties of solutions, which have been found at the main (order-disorder) phase transition temperature of the bilayers, to be explained by dimensional changes as resulting from vesicle growth and fusion.     

In vitro ultrasound-mediated leakage from phospholipid vesicles

Interest in using ultrasound energy in wound management and intracellular drug delivery has been growing rapidly. Development and treatment optimization of such non-diagnostic applications requires a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the changes in membrane permeation (leakage mimicking drug release) in vitro during exposure to ultrasound applied in two frequency ranges: “conventional” (1 MHz and 1.6 MHz) therapeutic ultrasound range and low (20 kHz) frequency range. Phospholipids vesicles were used as controllable biological membrane models. The membrane properties were modified by changes in vesicle dimensions and incorporation of poly(ethylene glycol) i.e. PEGylated lipids. Egg phosphatidylcholine vesicles with 5 mol% PEG were prepared with sizes ranging from 100 nm to 1 μm. Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. Custom-built transducers operating at frequencies of 1.6 MHz (focused) and 1.0 MHz (unfocused) were used, the I_spta of which were 46.9 W/cm² and 3.0 W/cm², respectively. A commercial 20 kHz, point-source, continuous wave transducer with an I_spta of 0.13 W/cm² was also used for comparative purposes. Whereas complete leakage was obtained for all vesicle sizes at 20 kHz, no leakage was observed for vesicles smaller than 100 nm in diameter at 1.6 or 1.0 MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300 nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100 nm and 1 μm. This observation suggests that physico-chemical membrane properties play a crucial role in ultrasound mediated membrane permeation and that low frequency (tens of kilohertz) ultrasound exposure is more effective in introducing permeability change than the “conventional” (1 MHz) therapeutic one. The experimental data also indicate that the leakage level is controlled by the exposure time. The results of this work might be helpful to optimize acoustic field and membrane parameters for gene or drug delivery. The outcome of this work might also be useful in wound management.     

Accurate determination of elastic parameters for multicomponent membranes

Heterogeneities in the cell membrane due to coexisting lipid phases have been conjectured to play a major functional role in cell signaling and membrane trafficking. Thereby the material properties of multiphase systems, such as the line tension and the bending moduli, are crucially involved in the kinetics and the asymptotic behavior of phase separation. In this Letter we present a combined analytical and experimental approach to determine the properties of phase-separated vesicle systems. First we develop an analytical model for the vesicle shape of weakly budded biphasic vesicles. Subsequently experimental data on vesicle shape and membrane fluctuations are taken and compared to the model. The parameters obtained set limits for the size and stability of nanodomains in the plasma membrane of living cells.     

Negative tension induced by lipid uptake

Membrane fusion is an important process in cell biology. While the molecular mechanisms of fusion are actively studied at a very local scale, the consequences of fusion at a larger scale on the shape and stability of the membrane are still not explored. In this Letter, the evolution of the membrane tension during the fusion of positive small unilamellar vesicles with a negative giant unilamellar vesicle has been experimentally investigated and compared to an existing theoretical model. The tension has been deduced using videomicroscopy from the measurement of the fluctuation spectrum and of the time correlation function of the fluctuations. We show that fusion induces a strong decrease in the effective tension of the membrane which eventually reaches negative values. Under these conditions, we show that localized instabilities appear on the vesicle. The membrane finally collapses, forming dense lipid structures.     

Model system of self-reproducing vesicles

Development of self-reproducing vesicle systems is the first step for autopoietic cycles. We established a model self-reproducing vesicle system without the membrane molecule synthesis route. The model vesicle composed of cylinder- and inverse-cone-shaped lipids formed inclusion vesicles inside the mother vesicle, and the inclusion vesicles were then expelled by a temperature cycling. By changing the vesicle composition, the mother vesicles showed a budding-type self-reproduction pathway. A key concept of this system is the coupling of the main-chain transition and the shape of lipids.     

Spectroscopic and calorimetric studies of phospholipid–polyamine molecular interactions

The effect of polyamines (putrescine, spermine and spermidine) on the physical properties of liposomes of phospholipids with different amount of charge on the bilayer was studied. The measured effect depends both on the charge of the lipid and on the charge of the polyamine, which under physiological conditions can be considered as a polycation. No interaction was observed with a neutral phospholipid for all the polyamines considered. With charged phospholipids, polyamines modify the surface charge and, as a consequence, the inner structure of the core of the bilayer also changes. The polyamine with the highest positive charge and the phospholipid with the highest content of negative groups showed the strongest interaction.     

Quadrupolar ordering of phospholipid molecules in narrow necks of phospholipid vesicles

Shapes of phospholipid vesicles that involve narrow neck(s) were studied theoretically. It is taken into account that phospholipid molecules are intrinsically anisotropic with respect to the membrane normal and that they exhibit quadrupolar orientational ordering according to the difference between the local principal membrane curvatures. Direct interactions between oriented molecules were considered within a linear approximation of the energy coupling with the deviatoric field. The equilibrium shapes of axisymmetric closed vesicles were studied by minimization of the free energy of the phospholipid bilayer membrane under relevant geometrical constraints. The variational problem was stated by a system of Euler-Lagrange differential equations that revealed a singularity in the derivative of the meridian curvature at points where the effect of the orientational ordering exactly counterbalances the effect of the isotropic bending. The system of Euler-Lagrange differential equations was solved numerically to yield consistently related equilibrium orientational distribution of the phospholipid molecules and vesicle shape. According to our estimation of the model constants the formation of the neck is promoted if direct interactions between the oriented molecules are taken into account. It was shown that the energy of the equilibrium shapes is considerably affected by the quadrupolar ordering of phospholipid molecules.     

Hexagonal arrangement of phospholipids in bilayer membranes

The phospholipid membrane plays a key role in myriad biological processes and phenomena, and the arrangement structure of membrane determines its function. However, the molecular arrangement structure of phospholipids in cell membranes is difficult to detect experimentally. On the basis of molecular dynamic simulations both in a non-destructive way and at native environment, we observed and confirmed that the phospholipids self-assemble to a hexagonal arrangement structure under physiological conditions. The underlying mechanism was revealed to be that there are hexagonal arrangement regions with a lower free energy around each lipid molecule. The findings potentially advance the understanding of biological functions of phospholipid bilayers.