Structural Features of Interacting Complementary Liposomes Promoting Formation of Multicompartment Structures

The structural features of complementary liposomes and factors favoring formation of multicompartment systems are investigated. Specifically, liposomal formulations consisting of PEGylated unilamellar liposomes with guanidinium moieties located at the distal end of polyethylene glycol (PEG) chains interact with complementary multilamellar liposomes bearing phosphate moieties. Furthermore, the number of PEG chains attached to the unilamellar interface of the liposomes is enhanced by incorporating PEGylated cholesterol in their bilayer. While molecular recognition of the liposomes is the driving force for initiating multicompartmentalization, it is the enhanced PEGylation at the liposomal interface that synergistically promotes fusion resulting in large and well‐formed multicompartment systems. A mechanism is proposed according to which initial adhesion of the liposomes, followed by reorganization of their membrane lipids, leads to giant bilayer aggregates incorporating large liposomes.     

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.     

Characterisation of electroosmotic flow in capillary electrochromatography columns

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.     

Immobilized liposome chromatography to study drug-membrane interactions. Correlation with drug absorption in humans

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.     

Complementary liposomes based on phosphatidylcholine: interaction effectiveness vs protective coating

A prospective targeted drug delivery system was prepared by the introduction of complementary and protective moieties at the external surfaces of liposomes. Thus recognition between hydrogenated phosphatidylcholine-cholesterol-based liposomes was achieved by the interaction of the complementary phosphate and guanidinium groups incorporated in separate liposomes while polyethylene glycol chains (PEG) protected both liposomes from environmental factors. In general, protective coating of liposomes in the range of 5% molar incorporation exerted an inhibitory effect on their recognition but it also permitted effective interaction between complementary liposomes.     

Interaction of functional dendrimers with multilamellar liposomes: design of a model system for studying drug delivery

Multilamellar liposomes consisting of phosphatidylcholine-cholesterol-dihexadecyl phosphate (19:9.5:1 molar ratio) and dispersed in aqueous or phosphate buffer solutions were interacted with poly(propylene imine) dendrimers which were partially functionalized with guanidinium groups. The remaining toxic external primary amino groups of the dendrimers were reacted with propylene oxide, affording the corresponding hydroxylated derivatives. Microscopic, zeta-potential, and dynamic light scattering techniques have shown that liposomal-dendrimeric molecular recognition occurs due to the interaction between the complementary phosphate and guanidinium groups. Calcein liposomal entrapment experiments demonstrate a limited leakage, i.e., less than 13%, following liposomes interaction with the modified dendrimers. Calorimetric studies indicate that the enthalpy of the interaction is dependent on the number of guanidinium groups present at the dendrimeric surface and the medium. The process is reversible, and redispersion of the aggregates occurs by adding concentrated phosphate buffer. Two corticosteroid drugs, i.e., betamethasone dipropionate and betamethasone valerate, were encapsulated into the functionalized dendrimers. Drug transport from guanidinylated dendrimers to multilamellar liposomes ranges from 40% to 85%, and it is also dependent on the medium and the degree of dendrimer guanidinylation.     

Neutral liposome-mediated delivery process of fluorescein-modified oligonucleotides in cultured human keratinocytes

We propose a model of the intracellular delivery process in which fluorescein-labeled natural oligonucleotides (F-DNA) are transferred into the nuclei of cultured human keratinocytes. By encapsulation in neutral multilamellar lecithin liposomes, the F-DNA appeared to be protected against intracellular interactions with cellular materials and nuclease attacks in the cytoplasm during the process. The intracellular behavior of F-DNA and fluorescent phospholipid-labeled liposomes was observed by means of fluorescence analysis. Results showed that: F-DNA encapsulated in neutral multilamellar liposomes reached the cellular nuclei more efficiently than either free F-DNA, or F-DNA in unilamellar liposomes; the liposomal membranes appeared to be left in the cytoplasm. The reaction of F-DNA with complementary DNA was suggested by a rapid quenching of the fluorescence in the nucleus. In addition, the fluorescence decrease was evidently suppressed in the cytoplasm, indicating a protective effect of the neutral multilamellar liposomes against the interaction of F-DNA with cytoplasmic materials. The application of these findings to ‘photo’-antisense studies has been discussed, where suppression of a gene expression is attempted by using oligonucleotide-attached fluorescein with the aid of a photo-induced covalent binding property.     

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.     

Effect of a PEG lipid (DSPE-PEG2000) and freeze-thawing process on phospholipid vesicle size and lamellarity

Liposomes containing distearoylphosphatidylethanolamine with covalently linked polyethylene glycol of molecular weight 2,000 (DSPE-PEG2000) covering a range of 0–30 mol% were prepared by a mechanical dispersion or detergent-removal method. The effects of DSPE-PEG2000 on particle sizes and lamellarity of liposomes were investigated. The average diameters of vesicles prepared from both methods decreased when the concentration of DSPE-PEG2000 was increased. The decrease in vesicle size with increase in DSPE-PEG2000 was ascribed to the steric hindrance of strongly hydrated PEG. The significant decrease in the sizes of DSPE-PEG2000-containing EggPC vesicles prepared by the detergent-removal method could be explained by the postvesiculation size growth in the process of micelle–vesicle transition. For DMPC vesicles prepared by the detergent-removal method, electron micrographs showed that inclusion of DSPE-PEG2000 promoted vesicle formation. Based on the results of investigation of calcein entrapment efficiency, we concluded that the lamellarity of liposomes is reduced as PEG lipid concentration is increased. Fragmentation of multilamellar vesicles into smaller unilamellar vesicles occurred more readily when the liposome suspension was subjected to repetitive freeze-thawing. After five cycles of freezing and thawing, vesicles containing more than 0.5 mol% DSPE-PEG2000 were fragmented into unilamellar vesicles with diameters smaller than 300 nm.     

Novel asparagine-derived lipid enhances distearoylphosphatidylcholine bilayer resistance to acidic conditions

A novel asparagine-derived lipid analogue (ALA(11,17)) bearing a tetrahydropyrimidinone headgroup and two fatty chains (11 and 17 indicate the lengths of linear alkyl groups) was synthesized in high yield and purity. The thin film hydration of formulations containing 5 mol % or greater ALA(11,17) in distearoylphosphatidylcholine (DSPC) generated multilamellar vesicles (MLVs) that remained unaggregated according to optical microscopy, while those formed from DSPC only were highly clustered. The MLVs were processed into unilamellar liposomes via extrusion and were characterized by dynamic light scattering (DLS), zeta potential, turbidity, and scanning electron microscopy (SEM) analysis. Results show that the presence of ALA(11,17) in DSPC liposomes significantly alters the morphology, colloidal stability, and retention of encapsulated materials in both acidic and neutral conditions. The ability of ALA(11,17)-hybrid liposomes to encapsulate and retain inclusions under neutral and acidic conditions (pH < 2) was demonstrated by calcein dequenching experiments. DLS and SEM confirmed that ALA(11,17)/DSPC liposomes remained intact under these conditions. The bilayer integrity observed under neutral and acidic conditions and the likely biocompatibility of these fatty amino acid analogues suggest that ALA(11,17) is a promising additive for modulating phosphatidylcholine lipid bilayer properties.     

UV light induced photodegradation of liposome encapsulated fluoroquinolones: An MS study

Fluoroquinolone antibacterial agents are among the drugs most commonly causing phototoxic side effects. The phototoxicity may be originated in formation of reactive oxygen species upon ultraviolet exposure. Researches aiming the liposomal encapsulation of fluoroquinolones, expecting an increase in their therapeutic index, enhance the importance of studies on physicochemical properties and photostability of liposomal preparations. We studied the photodegradation of ciprofloxacin, ofloxacin and lomefloxacin by mass spectrometry upon various doses of UV irradiation. Lomefloxacin, the most phototoxic fluoroquinolone among them, was encapsulated into small unilamellar and multilamellar liposomes. Impact of vesicle structure and lipid composition – the presence of unsaturated fatty acid containing dioleoyl-phosphatidylcholine in dipalmitoyl-phosphatidylcholine liposomes – on the lomefloxacin photolysis was investigated; the structure of the main photoproducts was identified by mass spectrometry. It was found that the presence and type of lipids influence the ways of photodegradation process.     

Niosomes from alpha,omega-trioxyethylene-bis(sodium 2-dodecyloxy-propylenesulfonate): preparation and characterization

The synthesis and characterisation of new surfactants with peculiar physical-chemical properties are amongst the most promising and expanding issues in pharmacological colloid science. The most used vesicular carriers are liposomes prepared from a wide variety of natural and synthetic phospholipids, but several ionic and non-ionic amphiphiles have been used to form multilamellar and/or unilamellar vesicles. In the present study the synthesis of alpha,omega-trioxyethylene-bis(sodium 2-dodecyloxy-propylenesulfonate), an anionic Gemini surfactant, and its ability to form niosomes are elucidated. The compound forms vesicles with and without added cholesterol. The vesicular systems were characterized by size, shape and drug entrapment efficiency. The compounds to be incorporated are beta-carotene and ferulic acid, as antioxidants, acetyl salicylic acid, as FANS, and the antineoplastic 5-flurouracil, widely used in dermatological disorders. The results of this study show that alpha,omega-trioxyethylene-bis(sodium 2-dodecyloxy-propylenesulfonate) can be used for the preparation of niosomes entrapping lypophilic, amphiphilic or hydrophilic substances. These niosomes may be promising candidates as percutaneous carriers for the aforementioned drugs.     

Covalently anchored lipid structures on amine-enriched polystyrene

The study of the adhesion of lipid vesicles on surfaces is of increasing interest in the field of medical implants and tissue engineering (protein-resistant surfaces), drug delivery, biosensors, and biochips. In this work, lipid coverage was developed from PEG-coated vesicles (with sizes from 100 to 300 nm) by covalently binding poly(ethylene glycol)-alpha-disteroylphosphatidylethanolamine-omega-benzotriazole carbonate (DSPE-PEG-BTC) molecules onto the surface amine groups by carbamate chemistry. Lipid surface density and the surface structure of multilamellar (MLVs) and extruded unilamellar (LUVs) vesicles deposited on three types of polystyrene (PS) well-plates were probed by fluorescence and atomic force microscopy (AFM) imaging. A significant difference in the vesicle surface coverage of PS substrates was observed with a substantial increase in lipid multilayers on the amine-enriched PS surface using both unilamellar and multilamellar vesicles.     

Liposome formation of selectively-polymerized diene-containing phospholipids and their postpolymerization

Small and large unilamellar liposomes composed of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) are prepared by sonication and extrusion, respectively. They are polymerized with water-insoluble radical initiator, azobis(isobutyronitrile) (AIBN) which can selectively polymerize diene groups in 1-acyl chains of the lipids. Polymerized liposomes are freeze-dried to obtain the polymerized liposome powder. There are two methods to redisperse lyophilized liposomes into water. The extrusion is an effective method to disperse them because the energy at extrusion is necessary only for redispersion, whereas the excess energy at sonication gives damage on liposome structure. There is no difference in stability between polymerized liposomes before and after redispersion with extrusion. DODPC polymers, obtained from free radical-initiated polymerization with AIBN, are linear and have polymerizable diene groups in 2-acyl chains. The liposome powder is therefore soluble in organic solvents. Reconstruction of polymerized liposomes is performed with lipid polymers having low or high molecular weight. The lipid polymers having high molecular weight provide stable large unilamellar liposomes by ethanol injection, but unstable small unilamellar liposomes are formed by sonication. The liposomes reconstructed from lipid polymers having low molecular weight are unstable regardless of their size. After reconstruction of liposomes selectively polymerized by AIBN, diene groups in 2-acyl chains are polymerized by water-soluble radical initiator or UV-irradiation to yield highly crosslinked structure. Their stability is improved remarkably by this postpolymerization.     

Multilamellar LipoCEST Agents Obtained from Osmotic Shrinkage of Paramagnetically Loaded Giant Unilamellar Vescicles (GUVs)

Moving from nano‐ to micro‐systems may not just be a matter of scale, but it might imply changes in the properties of the systems that can open new routes for the development of efficient MRI contrast agents. This is the case reported in the present paper, where giant liposomes (giant unilamellar vesicles, GUVs) loaded with Ln^III complexes have been studied as chemical exchange saturation transfer (CEST) MRI contrast agents. The comparison between nanosized liposomes (small unilamellar vesicles, SUVs) and GUVs sharing the same formulation led to differences that could not be accounted for only in terms of the increase in size (from 100–150 nm to 1–2 μm). Upon osmotic shrinkage, GUVs yielded a saturation‐transfer effect three order of magnitude higher than SUVs consistent with the increase in vesicles volume. Confocal microscopy showed that the shrinkage of GUVs resulted in multilamellar particles whereas SUVs are known to yield asymmetrical, discoidal shape.     

Interaction of liposomes with cultured cells: possible involvement of lipid peroxidation in cell growth inhibition

Systematic analyses of the interaction between liposomes and cells were examined. Liposomes were found to affect the growth of mouse NIH 3T3 cells depending upon their size, net charge, and cholesterol content. Among the charged compounds, stearylamine was the most inhibitory and showed complete inhibition of cell growth at 100 microM. The cholesterol-rich and small unilamellar vesicles were more suppressive compared to the cholesterol-poor and multilamellar ones, respectively. The binding assay of liposomes to the cells showed a positive correlation between liposome binding and the extent of growth inhibition. Suppression of liposome uptake by inhibitors of the cytoskeletal system and energy metabolism were suggestive of an endocytotic mechanism for the cellular uptake of liposomes. The growth inhibitory effect seemed secondary to the intracellular uptake of liposomes, and peroxidation of incorporated lipids would lead to cellular damage. Therefore, it is highly recommended that potential growth inhibitory effects associated with the particular composition and other properties of liposomes should be carefully assessed in any human studies, especially for long-term use.     

Niosomes from 80s to present: The state of the art

Efficient and safe drug delivery has always been a challenge in medicine. The use of nanotechnology, such as the development of nanocarriers for drug delivery, has received great attention owing to the potential that nanocarriers can theoretically act as “magic bullets” and selectively target affected organs and cells while sparing normal tissues. During the last decades the formulation of surfactant vesicles, as a tool to improve drug delivery, brought an ever increasing interest among the scientists working in the area of drug delivery systems. Niosomes are self assembled vesicular nanocarriers obtained by hydration of synthetic surfactants and appropriate amounts of cholesterol or other amphiphilic molecules. Just like liposomes, niosomes can be unilamellar or multilamellar, are suitable as carriers of both hydrophilic and lipophilic drugs and are able to deliver drugs to the target site. Furthermore, niosomal vesicles, that are usually non-toxic, require less production costs and are stable over a longer period of time in different conditions, so overcoming some drawbacks of liposomes.     

A calorimetric evaluation of the interaction of amphiphilic prodrugs of idebenone with a biomembrane model

Lipoamino acids (LAA) are useful promoieties to modify physicochemical properties of drugs, namely lipophilicity and amphiphilicity. The resulting membrane-like character of drug-LAA conjugates can increase the absorption profile of drugs through cell membranes and biological barriers. To show the role of amphiphilicity with respect to lipophilicity in the interaction of drugs with biomembranes, in the present study we evaluated the mode of such an interaction of lipophilic conjugates of LAA with the antioxidant drug idebenone (IDE). DSC analysis and transfer kinetic studies were carried out using dimyristoylphosphatidylcholine (DMPC) multilamellar liposomes (MLVs) as a model. For comparison, two esters of IDE with alkanoic acids were synthesized and included in the analysis. The experimental results indicate that based on their different structure, IDE-LAA conjugates interacted at different levels with respect to pure IDE with DMPC bilayers. In particular, a progressive penetration inside the vesicles was observed upon incubation of IDE-LAA compounds with empty liposomes. The enhanced amphiphilicity of the drug due to the LAA moieties caused more complex interactions with DMPC bilayers, compared to those registered with the native drug or IDE alkanoate esters.     

Polyadenylic acid binding on cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine

In this work unilamellar liposomes doped with a novel non-ionic 5′-Uridine-head nucleolipid, Lauroyl Uridine (LU), were prepared and characterized for their ability to interact with the polynucleotide polyadenylic acid (poly-A). Vesicles, were made up of the cationic lipid DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), the zwitterionic lipid DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine), and the novel amphiphile Lauroyl Uridine. The influence of the non-ionic nucleolipid on essential liposomes properties, such as the structure and net charge was first investigated by a comparative analysis performed on the different lipoplex preparations by means of ζ-potential and size measurements. Both structure and net charge of liposomes were shown to be not modified by the presence of the non-ionic nucleolipid.The role of the synthetic lipid inserted as anchor in the liposome bilayer in the condensation process between vesicles and the polynucleotide poly-A was then analyzed by UV–vis, Circular Dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The data presented comparative UV–vis analyses that evidenced the occurrence of staking interactions in the poly-A only in LU containing lipoplexes. CD and NMR studies indicated the presence of H-bonding interaction between Lauroyl Uridine containing vesicles and the polynucleotide poly-A. The results presented in this work support a role for Lauroyl Uridine in A-U molecular recognition, thus, suggesting that cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine could represent a model system to study molecular interactions among single stranded polynucleotides and lipid anchor bearing the complementary bases.     

Interactions of anionic surfactants with methemoglobin

The liquid crystal morphologies of symmetrical diacy phosphatidylcholine liposomes examined in this research study were found to be dependent on saturated hydrocarbon chain length. Both powder X-ray diffraction and synchrotron mid-IR spectromicroscopy indicate that phosphatidylcholines with short hydrocarbon tails (i.e. ten and twelve carbons) are more likely to form unilamellar liposomes while those with long hydrocarbon tails (i.e. eighteen and twenty carbons) are more likely to form multilamellar liposomes. Hydrocarbon chain lengths of fourteen and sixteen represent a transitional zone between these two liquid crystal morphologies. The FTIR spectra where a shoulder develops on the peak at wavenumber 1750 cm(-1) particularly highlights the change in the packing of adjacent molecules in the transitional zone.