OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis

For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.     

Stability of desmopressin loaded in liposomes

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of alpha-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against alpha-chymotrypsin digestion.     

Stability of Desmopressin Loaded in Liposomes

Abstract

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of α-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against α-chymotrypsin digestion.     

Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.     

Temperature sensitization of liposomes using copolymers of N-isopropylacrylamide.

To obtain liposomes which release the contents in response to ambient temperature, liposomes modified with copolymers of N-isopropylacrylamide with varying lower critical solution temperatures have been designed. Poly(N-isopropylacrylamide-co-acrylamide)s with various compositions were synthesized by free-radical copolymerization. The lower critical solution temperature of the polymer increased with increasing acrylamide content in the polymer. Poly(N-isopropylacrylamide-co-acrylamide-co-N, N-didodecylacrylamide)s were also prepared via the same method as the thermosensitive polymers having anchor groups to the liposome membrane. Calcein-loaded dioleoylphosphatidylethanolamine/egg yolk phosphatidylcholine (6:4, w/w) liposomes were coated with these polymers by incubating the liposomes with aqueous solutions of the polymers. The liposomes hardly released the contents below the lower critical solution temperature of the polymer, but the release was greatly enhanced above that temperature. The liposomes were also made from a mixture of the same lipids and the polymer. The liposome revealed a more drastic release property than the liposomes prepared by the incubation with the polymer solution, because the polymer chains were bound on both surfaces of the membrane. The close relationship between lower critical solution temperatures of the polymers and temperature regions where enhancement of the release from the polymer-fixed liposomes demonstrates that the release was triggered by alteration of the polymers from a hydrophilic state to a hydrophobic state occurring at their lower critical solution temperatures.     

STABILITY OF MARINE LIPID BASED-LIPOSOMES UNDER ACID CONDITIONS. INFLUENCE OF XANTHAN GUM

Liposomes are potential candidates in Human nutrition to supplement diets in essential lipo- and hydro-soluble nutriments. In this context, marine lipid based-vesicles, rich in polyunsaturated fatty acids, and containing vitamin B1 (thiamine) were prepared. Two methods were envisaged for vitamin trapping, passive encapsulation and active loading through the use of a transmembrane pH gradient. These methods were compared in terms of thiamine trapping efficiency. Influence of xanthan addition was also studied. Although, the active loading yielded to higher encapsulation ratios than the passive method, encapsulation efficiency was markedly increased, in both cases, as soon as the hydrocolloid was added after the lipid hydration phase. Liposome stability was assayed by microscopy observation and membrane permeability to thiamine in a medium that mimic that faced by the structures administrated by the oral route, i.e., an acid pH medium, as in the Human stomach. Low pH value led to an instantaneous vesicle aggregation. Thiamine was totally released from liposomes prepared without xanthan after 24 h storage in a neutral medium. This time was shortened to 1 h at pH 1.5. However, thiamine retention was significantly improved when liposomes were prepared in presence of the hydrocolloid, whatever the encapsulation method used. On the whole, these results were interpreted in terms of xanthan coating around the liposome surface that would result from lipid-hydrocolloid interactions occurring during the centrifugation steps of liposome preparation.     

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.     

Interaction of arginine oligomer with model membrane

Short oligomers of arginine (R8) have been shown to cross readily a variety of biological barriers. A hypothesis was put forward that inverted micelles form in biological membranes in the presence of arginine oligomer peptides, facilitating their transfer through the membranes. In order to define the role of peptide-lipid interaction in this mechanism, we prepared liposomes as the model membrane to study the ability of R8 inducing calcein release from liposomes, the fusion of liposomes, R8 binding to liposomes and membrane disturbing activity of the bound R8. The results show that R8 binding to liposome membrane depends on lipid compositions, negative surface charge density and interior water phase pH values of liposomes. R8 has no activity to induce the leakage of calcein from liposomes or improve liposome fusion. R8 does not permeabilize through the membrane spontaneously. These peptides delivering drugs through membranes may depend on receptors and energy.     

Biophysical studies on the liposome-albumin system

The potential use of liposomes as a delivery system is still limited by the poor understanding of their interaction mechanisms with biological media. In the present work, interaction between bovine albumin (BA) and liposomes was studied using phase transition and dielectric measurements as well as solubilization process using non-ionic detergent octylglucoside (OG). After liposomes were incubated with diluted and concentrated BA, phase transition, characterizing the liposome membrane exhibited a shift towards higher temperatures, together with initiation of multiple phase transitions. The relaxation time of liposome membrane molecules also increased in a concentration-dependent manner. The solubilization profiles of incubated samples also showed remarkable changes, especially in beginning of solubilization stages. Moreover, amount of detergent needed to completely solubilize membrane was also increased. It was concluded that BA significantly altered the physical state of liposome membrane, which may be attributed to BA interaction with liposomes surface and/or by its incorporation within the bilayer membrane.     

Characterization of surface-immobilized layers of intact liposomes

Surface-immobilized liposome layers are of interest for various potential applications such as localized drug delivery, but their characterization is challenging. We have employed an AFM method and fluorescent dye release to analyze anchored liposomes. In addition, we studied whether the liposomes are surface-bound solely via specific interaction (NeutrAvidin/biotin) or whether physisorptive binding also plays a role. Liposomes containing PEG-biotin lipids were affinity bound to NeutrAvidin molecules which had been immobilized onto solid supports via three different hydrogel interlayers. After liposome docking, approaching the surface with a colloid probe mounted onto an AFM cantilever showed considerable compression behavior, consistent with expectation based on intact, deformable liposomes but not lipid bilayers, thus showing that disruption of liposomes did not occur upon immobilization onto these support surfaces. Plastic deformation suggestive of liposome disruption on compression was not observed. The kinetics of fluorescent dye release also demonstrated that intact liposomes had been successfully immobilized onto all three supports. Blocking surface-immobilized NeutrAvidin molecules with excess biotin in solution before exposure to liposomes showed that the docking of liposomes was dependent largely but not exclusively on biotin-NeutrAvidin affinity binding, with evidence for some nonspecific physisorption, as the extent of liposome binding onto blocked NeutrAvidin surfaces was appreciably lower than for unblocked surfaces but not zero. Finally, consecutive addition of further NeutrAvidin and liposome layers enabled fabrication of multilayers, and this was clearly seen in AFM compressibility and fluorescent dye release measurements.     

A radiolabel and electron microscopy study of the interaction of liposomes with synaptosomal membranes

The quantitative and qualitative interaction of liposomes with synaptosomes isolated from rat brain was examined using radiolabeled phospholipids and electron microscopy. Liposomes were prepared by sonication and detergent dialysis. Binding (adsorption) of radiolabeled phospholipid to synaptosomes was saturable when liposomes were in the liquid-crystalline state, were electrically neutral (egg-phosphatidylcholine), or carried increasing fractions (10:2 and 10:4 molar ratio) of negatively charged phosphatidic acid. Analysis using the Langmuir isotherm equation indicated a biphasic adsorption behavior. Adsorption increased with increasing temperature (4 degrees C and 37 degrees C). Binding was nonsaturable when liposomes were positively charged with stearylamine or composed of dimyristoylphosphatidylcholine and phosphatidylinositol (10:2 molar ratio). Due to the latter composition's solid state at 4 degrees C, temperature dependency was inverse. Electron micrographs revealed disc-shaped areas of adsorption that were free of integral membrane particles which appeared to form a condensed layer surrounding the areas of liposome adsorption. Following interaction with stearylamine-containing liposomes the vesicular structure of synaptosomes appeared largely destroyed. It is concluded that both liposome surface charge and membrane fluidity determine the extent of interaction with biological membranes.     

Interaction of a hydrophobic protein with liposomes evidence for particles seen in freeze fracture as being proteins

Freeze fractures of liposome membranes showed a smooth fracture surface while liposomes into which a hydrophobic protein from human myelin had been incorporated showed a particulate fracture surface. The evidence suggests that at least some of the analogous particles present on fracture surfaces of biological membranes could be proteins, embedded in a phospholipid bilayer.     

Liposome-mediated gene delivery into plant cells

Liposomes may offer several advantages as vectors for gene delivery into plant cells: (1) enhanced delivery of encapsulated DNA by membrane fusion, (2) protection of nucleic acids from nuclease activity, (3) targeting to specific cells, (4) delivery into a variety of cell types besides protoplasts by entry through plasmodesmata, (5) delivery of intact small organelles. Realization of these advantages calls for the construction of efficient liposomes, for appropriate fusion conditions and for an understanding of the nature of liposome-cell interactions. Various characteristics and techniques of the liposome-cell system are described (mode of delivery, liposome types and composition, and means of promoting delivery of liposome contents). Data of liposome-mediated delivery of various macromolecules into plant cells, with special reference to protoplasts, calli and pollen are reviewed. This includes data obtained by the use of fluorescent probes, radioactive-labelled DNA, viral nucleic acids and expression of plasmid-DNA. Structure and characteristics of plant surfaces and plasmodesmata are discussed with respect to DNA entry. It is suggested that liposome-mediated gene delivery into plant cells, and not only protoplasts, will be advantageous in certain specific tissues and situations.     

Structure of planar membrane formed from liposomes

Lipid vesicles with incorporated ion channels from polyene antibiotic amphotericin B were used to investigate structures of planar membranes formed by Shindler's techniques. A planar membrane assembled on the aperture in a lavsan film from two layers generated at the air-aqueous liposome suspension interface is not a simple bilayer but a bimolecular membrane containing numerous partly fused liposomes. A complete fusion of liposomal membranes with the planar bilayer is an unlikely event during membrane formation. A planar bimolecular lipid membrane without incorporated liposomes can be made by a method consisting of three stages: formation of a lipid layer on the air-water interface of a suspension containing liposomes, transfer of this layer along the surface of the solution into a chamber containing a solution without liposomes where a lipid monomolecular layer forms gradually (within about 20 min) at the air-water interface, assembling of the planar bilayer membrane from this monolayer. The knowledge of the planar membrane structure may be useful in experiments on incorporation of membrane proteins into a planar lipid bilayer.     

A new approach to the study of lipid-protein interactions in the structure of biological membranes.

The lytic action of a number of N-acyl amino acids on lecithin liposomes was examined. The agents' affinity for lecithin liposome membrane was measured and the results obtained were treated to estimate the interactions of the amino acid residues with the lecithin polar head group at the surface of the liposome membrane. The data were considered in relation to the study of the surfactant effects on the erythrocyte volume. The ability of the suggested approach to obtain information on protein-lipid interactions inaccessible by other techniques is briefly commented on.     

47 Incorporation of alternative nucleic acid chemistries into G-quadruplex structure

Nucleic acids that form G-quadruplex (G4) structure have found applications in a host of research and technology regimes. Numerous G4 based aptamer drugs have been identified with pharmacological activity against cancer, HIV, prions, and blood coagulation (1). In the field of nanotechnology, G4 based sensors and nano-machines have also received much attention. The ability to synthesize nucleic acid ex-vivo allows for the site-specific incorporation of non-natural chemistries into nucleic acids that can be used to tune their physical and pharmacological properties. We summarize the results of a series of studies investigating the effective incorporation of alternative nucleic acid chemistries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, and Locked nucleic acid (LNA) modifications to the ribose sugar. We report primarily on the effect of these modifications on G-quadruplex folding topology, thermal stability, and structure. The substitution of LNA-guanosine into the core guanine tetrads disrupts structure in specific structural environments. On the other hand, 2′-F- and 2′-F-ANA guanosine can generally be incorporated without disrupting the structure when substituted into guanine bases in certain structural conformations. We find that 2′-F-ANA-guanosine and 2′-F-guanosine are powerful tools for controling the conformation of G4 structures (2). Functionalization at the C8 of the guanine base stabilizes in a manner dependent on the glycosidic conformation of the base, with different modification chemistries stabilizing to varying extents (3). The results of these studies provide useful insight on how to effectively incorporate some useful chemical tools from the growing toolbox of modified nucleic acid chemistries into G-quadruplex nucleic acid.     

脂质体递药系统的靶向修饰

作为药物递送载体，脂质体（LPs）由于免疫原性低、稳定性好、毒性低和成本低而被认为是有前途的纳米药物递送系统。然而，LPs的靶向递送效果并不理想，往往会对正常的机体细胞造成伤害，因此，如何优化LPs药物，使其具有靶向性仍然是当前研究的重点。本文结合近年来国内外相关研究进展，重点介绍了多肽、抗体、糖类、配体，以及核酸适配体等靶向修饰物对LPs功能的影响，并归纳总结了各种靶向修饰目前存在的优势与挑战，以期对LPs给药系统的进一步研究提供科学参考及新药研发提供理论依据。     

Membrane potential generation by two reconstituted mitochondrial systems: Liposomes inlayed with cytochrome oxidase or with ATPase

Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB⁻), a penetrating anion probe, was used.

The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB⁻ uptake by liposomes. The PCB⁻ response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.

Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F₁and F_c, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB⁻ uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.

The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).     

Permeability of amino acids into liposomes.

1. A simple and rapid assay for the measurement of permeability of amino acids into liposome membrane was carried out by using the liposomes trapping D-amino acid oxidase (D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) inside the membrane. 2. Permeability of amino acids into liposomes depended on the lipid composition of the membrane. Permeability of amino acids into phosphatidylcholine-cholesterol liposomes depended critically on temperature. 3. Permeability also depended on the structure of amino acids. The order of permeability was norvaline greater than isoleucine greater than leucine greater than phenylalanine greater than tryptophan greater than methionine greater than tyrosine, valine greater than threonine greater than serine greater than alanine greater than glycine.