Changes in surface charge density of lecithin liposomes by lipid peroxidation. A fluorescence study with 8-anilino-1-naphthalenesulfonate.

Treatment of lecithin liposomes with 100 microM ascorbic acid and 10 microM ferrous ion resulted in the formation of fluorescent products exhibiting an emission maximum at 430 nm and a decrease in the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) bound to the liposomes without change in the emission maximum. The degree of ascorbic acid/Fe(2+)-induced decrease in the ANS fluorescence was dependent on the extent of fluorescent product formation. The results of kinetic studies on ANS-binding to the liposomes showed that treatment of the liposomes with ascorbic acid/Fe2+ causes an increase of the apparent dissociation constant (Kd) of ANS-liposome complex. This indicates that lipid peroxidation of the liposomes by treatment with ascorbic acid/Fe2+ decreases the binding affinity of ANS to the liposomes. In addition, it was also found that there is a good correlation between degrees of the Kd value and the formation of fluorescent products. The fluorescence properties, i.e. emission maximum and response of the fluorescence intensity for borohydride reduction, of the products formed by lipid peroxidation of the liposomes were similar to those derived from modification of the liposomes with monofunctional aldehydes such as acetaldehyde and heptaldehyde. From these results, it is suggested that the decrease of ANS-binding affinity to the liposomes by treatment with ascorbic acid/Fe2+ may be due to changes in the surface charge density of the liposomes relating to the formation of fluorescent products.     

Fluorescence lifetimes study of alpha-tocopherol and biological prenylquinols in organic solvents and model membranes

We have found that for biological prenyllipids, such as plastoquinol-9, alpha-tocopherol quinol, and alpha-tocopherol, the shortest fluorescence lifetimes were found in aprotic solvents (hexane, ethyl acetate) whereas the longest lifetimes were those of ubiquinonol-10 in these solvents. For all the investigated prenyllipids, fluorescence lifetime in alcohols increased along with an increase in solvent viscosity. In a concentrated hexane solution, the lifetimes of prenylquinols considerably decreased. This contrasts with methanol solutions, which is probably due to the self-association of these compounds in aprotic solvents. We have also found a correlation of the Stokes shift of prenyllipids fluorescence with the orientation polarizability of the solvents. Based on data obtained in organic solvents, measurements of the fluorescence lifetimes of prenyllipids in liposomes allowed an estimation of the relative distance of their fluorescent rings from the liposome membrane surface, and was found to be the shortest for alpha-tocopherol quinol in egg yolk phosphatidylcholine liposomes, and increased in the following order: alpha-tocopherol in dipalmitoyl phosphatidylcholine liposomes < alpha-tocopherol < plastoquinol-9 < ubiquinol-10 in egg-yolk phosphatidylcholine liposomes.     

Carbazole as an excited state proton transfer fluorescent probe for lipid bilayers in alkaline medium

In our effort to look for novel excited state proton transfer (ESPT) fluorescent probes in alkaline pH range, we have examined carbazole as a possible candidate because of its high extinction coefficient, high quantum yield and a larger difference in ionization constant between the ground and excited state (pKa - pKa*). The photodissociation of carbazole was studied in liposome membrane by steady state fluorescence measurements at alkaline pH ranges. The neutral form and the anionic form of carbazole emit at 362 and 417 nm, respectively. This large shift in emission makes it convenient to monitor the physical properties of liposomes. The neutral form fluorescence intensity of carbazole is sensitive to phase changes in the membrane and also shows a maximum at phase transition temperature. This variation of intensity can be explained in terms of redistribution of probe between the surface and interior of the liposomes. Cholesterol induced phase changes of liposomes were also sensed by the ESPT of carbazole.     

Calcium ion fluorescence detection using liposomes containing Alexa-labeled calmodulin

This paper describes the preparation and characterization of calcium ion sensitive fluorescent liposomes and their application for the determination of calcium ions in aqueous samples. Calmodulin (CaM), a calcium ion-binding protein labeled with the fluorophore Alexa-488 is embedded in the membrane of unilamellar liposomes. Upon calcium ion binding, calmodulin undergoes a conformational change that exposes its hydrophobic core and affects the fluorescence intensity of the attached fluorophore. Characterization studies of Alexa-CaM-containing liposomes reveal that embedding calmodulin molecules in the bilayer membrane of liposomes extends the lifetime of the calcium ion binding activity of calmodulin by about fourfold compared to the lifetime of its calcium-binding activity in free solution. Moreover, the calcium ion response of Alexa-CaM-containing liposomes is about threefold higher than the calcium ion response of Alexa-CaM in solution. The improvement in the calcium ion detection properties is attributed to the interaction between calmodulin, a membranal protein, and the hydrophobic phospholipids of the liposomes. The analytical properties of the calcium ion sensitive fluorescent liposomes are discussed.     

Migration of a cationic polymer between lipid vesicles

The adsorption of a synthetic polycation, poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), on the surface of bilayer lipid vesicles (liposomes) and the migration of adsorbed macromolecules between the liposomes are studied. Liposomes of three types are used, including (1) traditional two-component liposomes composed of neutral phosphatidylcholine (PC) and anionic cardiolipin (CL); (2) three-component liposomes consisting of PC, CL, and cationic dicetyldimethylammonium bromide (DCMAB); and (3) anionic PC/CL liposomes with a nonionic surfactant, poly(ethylene oxide)-cetyl alcohol ether (Briij 58), incorporated into their bilayers. The adsorption of PEVP on the surface of PC/CL liposomes is accompanied by their aggregation. Using the fluorescence method, it is shown that the units (segments) of the polycation undergo partial redistribution between the liposomes inside the aggregates formed from PC/CL liposomes (with and without a fluorescent label) and PEVP. On the contrary, three-component PC/CL/DCMAB and PC/CL/Briij liposomes are not aggregated, even with the complete neutralization of their charges by adsorbed PEVP. In both cases, the migration of PEVP molecules between individual (nonaggregated) liposomes is observed. Possible reasons for the aggregative stability of the three-component PC/CL/DCMAB and PC/CL/Briij liposomes and the mechanism of interliposome migration of PEVP in such systems are discussed.     

Fluorescence study of the fluidity and cooperativity of the phase transitions of zwitterionic and anionic liposomes confined in sol-gel glasses

The current work makes use of different fluorescent reporter molecules and fluorescent spectroscopic techniques to characterize the thermotropic, physical, and dynamical properties of large unilamellar liposomes formed from either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] (DMPG) encapsulated in sol-gel matrixes. In particular, cooperativity of the phase transition is analyzed from steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), the interfacial properties are studied by measuring the spectral shift of Laurdan, and the structural organization (heterogeneity) of the lipid bilayer is determined from the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamical properties in the bulk hydrophobic core is obtained from time-resolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH). The spectroscopic study reveals that upon encapsulation, the basic thermodynamic properties as well as the fluidity of the lipid bilayer practically remain intact for DMPG liposomes but not for DMPC liposomes, whose lipid bilayer exhibits large gel-fluid heterogeneity. On the basis of these experimental results, electrostatic interactions between phospholipid polar heads and the porous surface of the host matrix seem to play a capital role for the preservation of the structural integrity of encapsulated bilayer.     

Intracellular distribution of fluorescent probes delivered by vesicles of different lipidic composition

In order to study mechanisms involved in liposome–cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle–cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker.     

三种非甾体类抗炎药与脂质体的相互作用

以卵磷脂脂质体为生物膜模型, 分别采用二阶导数吸收光谱法和以1-苯胺基-8-萘磺酸铵(ANS)为探针的荧光光谱法, 研究了三种丙二酸类非甾体抗炎药吲哚美辛、舒林酸和托美丁与脂质体的相互作用. 药物的二阶导数吸收谱成沟槽型,在脂质体中沟槽变浅, 但波长基本上不移动, 表明药物结合在磷脂双层的表面而没有进入到脂双层内部. 在荧光光谱中, 抗炎药可以结合到脂质体上, 游离出ANS, 猝灭ANS 在脂质体中的强烈荧光, 表观猝灭常数K_SV^app＞10^12 L·mol^(-1); 药物先行结合到脂质体上后, 会降低ANS结合到脂质体上的机率. 同样表明药物可以结合到脂质体表面磷脂分子的头部, 结合能力的强弱与吸收谱得到的结果一致, 舒林酸最强, 吲哚美辛其次, 托美丁的结合能力较弱.     

纳米脂质体包裹荧光试剂进入单细胞的研究

本研究首次使用直径约100 nm的小脂质体包裹荧光染料, 通过细胞的内吞作用或融合过程, 转移不透膜荧光物质进入细胞内, 标记细胞内组分.     

Signal enhancement of a micro-arrayed polydiacetylene (PDA) biosensor using gold nanoparticles

Polydiacetylene (PDA) liposomes possess unique properties that allow liposomes to change color and emit fluorescence in response to stimuli such as temperature, antibody-antigen interaction, pH, mechanical stress, and organic solvent. They have been studied extensively as signal transducers in biosensor applications. Here, we describe an antibody-based biosensor using PDA liposomes for detection of human immunoglobulin E (hIgE). Target hIgE chemically bound to hIgE monoclonal antibodies immobilized on PDA liposomes and the fluorescent signals were slightly increased depending on the target protein concentration. As the primary response, the hIgE could be detected to below 10 ng mL(-1). However, fluorescent signals were dramatically increased depending on the target protein concentration when gold nanoparticle-conjugated polyclonal antibody probes were added on the PDA liposomes after the primary immune reaction. A PDA liposome biosensor could detect the hIgE as low as 0.1 ng mL(-1) and the sensitivity was increased up to one hundred times higher than the primary response. As a result, we confirmed that gold nanoparticle-conjugated polyclonal antibody probes efficiently enhanced the fluorescent signal of the PDA liposome biosensor chip. This strategy can be useful to detect proteins of ultra-low concentration.     

Interactions of hemoglobin with lecithin liposomes

In this paper, the interaction of hemoglobin (Hb) with lecithin liposomes is studied by UV-vis spectroscopy, fluorescent spectroscopy, and transmission electron microscopy. The adsorption of Hb on liposomes is likely to be related to the hydrophobic interaction between Hb and liposomes, which brings about the increase of the microenvironmental polarity (I ₁/I ₃) and the decrease of the fluorescence polarization (P) of lecithin liposomes. These results are considered to be that the adsorption of Hb on liposomes makes the spaces between the lecithin molecules increase, and a temporary gap is consequently formed in the liposomal bilayer membranes. The leakage of aqueous-space marker from the liposomes is increased with the addition of Hb.     

脂质体荧光衰减技术预测10种中药单体的被动吸收

制备了DPH荧光脂质体,选用10种药理活性较好的中药单体,测定了这些中药单体进入荧光脂质体后引起的荧光衰减百分率,评估中药单体的细胞膜通透性。采用大鼠在体肠单向灌流实验测定了中药单体的小肠有效透皮系数(Peff),与中药单体引起的脂质体荧光衰减数据对比,两者相关性良好,从而证明荧光脂质体模型可以应用来预测中药单体的被动吸收。用DPH标记红细胞膜,测定了以上10种中药单体对荧光标记的红细胞膜的荧光衰减百分率,发现荧光脂质体与红细胞膜相关性良好,荧光脂质体模型的确可以一定程度上替代制备繁琐的红细胞膜,用来研究中药单体在真实细胞膜中的通透性。综合而言,荧光脂质体模型简单易行、重现性好、所需样品量少,适合于中药早期研究的高通量筛选。     

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.     

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.     

Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop

Characterization of the potential-induced changes of a lipid-coated Hg-0.1 M KCl interface through electrochemical techniques and newly developed in situ fluorescence microscopy is described. Fluorescence of a fluorophore-containing dioleoyl phosphatidylcholine (DOPC) layer deposited from the gas-solution interface was observed to be dependent upon the potential of the Hg surface. The largest changes occurred for potentials where the lipid layer was desorbed: the lipid moved away from the electrode surface, reducing the efficiency of metal-mediated quenching of the excited state resulting in an increase in fluorescence. Electric potential-induced changes in the morphology of the adsorbed or desorbed DOPC lipid monolayer were observed optically for the first time using this technique. The observed potential-dependent fluorescence was compared to previous studies on an octadecanol-coated Au(111) electrode. Fluorescence microscopy was also used to characterize the fusion of DOPC liposomes with a previously adsorbed DOPC layer. Large changes in fluorescence were observed for the DOPC layer after fusion with liposomes. The fusion was accomplished via potential-created defects in the adsorbed DOPC monolayer through which the liposomes interact. The integration of the liposomes into the adsorbed monolayer results in a hybrid layer in which some lipid exists further from the electrode surface, resulting in a large increase in fluorescence. Possibilities for the creation of a biomimetic adsorbed hybrid lipid layer on Hg are also discussed.     

Complexation of anionic liposomes with spherical polycationic brushes

Spherical polycationic brushes, consisting of polystyrene particles with linear cationic macromolecules grafted onto their surfaces, were electrostatically complexed with small unilamellar anionic liposomes. Complexation was monitored using a multimethod approach that included laser electrophoresis, dynamic light scattering, fluorescence, cryogenic transmission electron microscopy, and conductivity. Liposomes adsorb onto the outer edges of the brushes rather than penetrate into their dense polycationic layer. The integrity of the liposomes remains unaltered when the liposomes reside on the polycationic brushes. The resulting complexes (roughly 40 liposomes per brush) do not dissociate into their components upon exposure to physiological solutions. The system is potentially useful in that liposomes are gathered into well-defined clusters with a high encapsulating potential. Multicomponent constructs can be easily prepared if polycationic brushes are allowed to bind to a mixture of liposomes that encapsulate different guests. This work provides an example of "systems chemistry" whereby as many as eight components, each with its own particular location and function (i.e., polystyrene core, polycationic graft, egg lecithin, cardiolipin, two fluorescent dyes, water, and buffer), collectively self-assemble.     

An investigation of the interaction of cobra venom cytotoxins with liposomes by the fluorescent-probe method

The interaction of cytotoxins V_c1, V_c5, and V_c6 of the venom of the Central Asian cobra with liposomes having a negative surface charge prepared from a mixture of phosphatidylcholine and palmitic acid (1:1, molar) has been investigated with the aid of a pyrene fluorescent probe. It has been shown that on interacting with liposomes a cytotoxin increases the microviscosity of the hydrophobic region of membranes. This effect depends on the phase state of the lipids. Observations on the kinetics of the transfer of energy between pyrene probes and diphenylhexatriene have shown that on the addition of a cytotoxin to samples of liposomes with a negative surface charge an aggregation of the liposomes takes place without a disturbance of their integrity.     

Individual electrophoretic mobilities of liposomes and acidic organelles displaying pH gradients across their membranes

This report focuses on measuring the individual electrophoretic mobilities of liposomes with different pH gradients across their membrane using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The results from the individual analysis of liposomes show that, using surface electrostatic theories and the electrokinetic theory as the first approximation, zeta potential contributes more significantly to the electrophoretic mobility of liposomes than liposomal size. For liposomes with an outer pH 7.4 (pH(o) 7.4) and a net negative outer surface charge, the most negative electrophoretic mobilities occur when the inner pH (pH(i)) is 6.8; at higher or lower pH(i), the electrophoretic mobilities are less negative. The theories mentioned above cannot explain these pH-induced electrophoretic mobility shifts. The capacity theory, predicting an induced electrical charge on the surface of liposomes, can only explain the results at pH(i) > 6.8. In this report, we hypothesize that there is a flip-flop process of phospholipids, which refers to the exchange of phospholipids between the outer and inner layers of the membrane. This flip-flop is caused by the pH gradient and membrane instability and results in the observed electrophoretic mobility changes when pH(i) is <6.8. Furthermore, it is found that the mobilities of acidic organelles are consistent with the predictions of liposome models we used here.     

Synthesis and fluorescence properties of new fluorescent, polymerizable, metal-chelating lipids

Liposomes incorporating fluorescent, metal-chelating lipids find applications in molecular recognition of peptides, 2D protein recrystallization, protein targeting, and biological sensing. It would be advantageous to combine the usefulness of polymerizable, metal-chelating lipids and fluorescent lipids. Herein, we report the synthesis and fluorescence properties of several fluorescent, polymerizable, metal-chelating lipids. They have been successfully incorporated into liposomes and then polymerized. These lipids can be used as membrane probes to study the polymerizable liposomes in the unpolymerized state and to investigate lipid redistribution during polymerization. In addition, if a luminescent metal ion (e.g., Eu(3+), Tb(3+), etc.) is used to complex the headgroup, the lipids can probe the membrane interior and exterior simultaneously.     

Carbohydrate-Derived Metal-Chelator-Triggered Lipids for Liposomal Drug Delivery

Liposomes are versatile three-dimensional, biomaterial-based frameworks that can spatially enclose a variety of organic and inorganic biomaterials for advanced targeted-delivery applications. Implementation of external-stimuli-controlled release of their cargo will significantly augment their wide application for liposomal drug delivery. This paper presents the synthesis of a carbohydrate-derived lipid, capable of changing its conformation depending on the presence of Zn²⁺: an active state in the presence of Zn²⁺ ions and back to an inactive state in the absence of Zn²⁺ or when exposed to Na₂EDTA, a metal chelator with high affinity for Zn²⁺ ions. This is the first report of a lipid triggered by the presence of a metal chelator. Total internal reflection fluorescence microscopy and a single-liposome study showed that it indeed was possible for the lipid to be incorporated into the bilayer of stable liposomes that remained leakage-free for the fluorescent cargo of the liposomes. On addition of EDTA to the liposomes, their fluorescent cargo could be released as a result of the membrane-incorporated lipids undergoing a conformational change.