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.     

Tripolyphosphate-sensitive egg phosphatidylcholine liposomes incorporating hydrophobically modified poly(ethylene imine)

Liposomes, which release their contents in answer to tripolyphosphate (TPP, a penta-anion), were prepared by immobilizing hydrophobically modified poly(ethylene imine) (HmPEI) on the surface of egg phosphatidylcholine (egg PC) liposome. HmPEI was prepared by covalently attaching decanoyl chloride to PEI through a condensation reaction. According to the ¹H NMR spectrum, the number of decanoyl chloride per one molecule of PEI was about 21, and HmPEI was air/water interface-active. HmPEI could readily complex with TPP in HEPES buffer (30 mM, pH 7.0), confirmed by Fourier transformed infrared spectrophotometer spectroscopy. The complexation increased with increasing the concentration of HmPEI and TPP, investigated through the measurement of optical density and light scattering intensity. Liposomes incorporating HmPEI were prepared by a film hydration and sonication method. The liposomes were multi-lamellar vesicles, observed on transmission electron microscope. Liposomes free of HmPEI did not release calcien when they were mixed with TPP. Liposomes whose egg PC/HmPEI was relatively low (e.g., 20:1 and 20:2) released calcein but not extensively (less than 10%) when mixed with TPP. Liposomes whose egg PC/HmPEI was relatively high (e.g., 20:4 and 20:20) released calcein extensively. For example, when the liposomes with lager amount of HmPEI were mixed with TPP so that HmPEI/TPP weight ratio was 8:1, the release degree in 60 sec was more than 70%. HmPEI can complex with TPP through electrostatic interaction and the complexation was thought to cause perturbation in the liposomal membranes and trigger the release.     

Reconstitution of neutral amino acid transport from partially purified membrane components from Ehrlich ascites tumor cells

Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X-100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine-binding activity has been detected in the Triton X-100 solubilized membrane fraction. The leucine-binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.     

Development of Phosphatidylethanolamine Liposomes That Efficiently Retain Encapsulated Vinorelbine Bitartrate

A hydrophilic and temperature-induced degradation drug, vinorelbine bitartrate (VB)-loaded phosphatidylethanolamin sterically stabilized liposomes (PSLs) were prepared by the thin film hydration method. Liposomes were made of phosphatidylethanolamine: cholesteryl: oleic acid (PE: CHOL: OA, 6:4:3 mass/mass). The mean particle size of the PSLs ranged from 600 to 650 nm. The transmission electron microscope (TEM) images displayed that the shape of the PSLs was multilamellar vesicles with smooth surface. The highest entrapment efficiency (EE) and drug loading capacity (DL) could reach up to 81.2 and 16.6%, respectively. The studies of drug release showed that the drug release could last for much more than 48 hours. The PSLs was evaluated by comparing the rate of release of encapsulated VB in different phosphate buffer solution (PBS).     

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.     

Sterically Stabilized Vesicles

After significant developments in liquid crystal and polymer research, scientists became interested in lyotropic systems containing polymers. These studies investigated, for instance, phase behavior and stability characteristics of suspensions of colloidal particles containing water-soluble or surface-adsorbed polymers or block copolymers. The most frequently studied were micelles, latex prticles, and lipid vesicles (liposomes). Liposomes aggregate and fuse in the presence of hydrophilic polymers but their properties were difficult to explain when block copolymers were adsorbed or surfactants with larger polymeric polar heads were inserted into the liposome membrane, because such systems are inherently ill defined. Liposomes containing diacyl surfactants with covalently linked, longer polymer chains display many new properties with very important consequences for both basic and applied research. They stimulate fundamental studies on phase behavior and polymer conformation, scaling laws, colloidal and surface properties, and cell function: applications deal predominantly with liposomes as drug delivery systems. While in basic research theory is currently more advanced than experiment, in medical applications theoretical understanding lags behind experimental achievements. It was discovered only relatively late that liposomes with an appropriate polymer coating are significantly more stable in a biological milieu, a necessary condition for their utility as drug carriers. In particular in medical applications, this practice has rejuvenated the field of anticancer therapy and targeted drug deliviery. All these advances were made possible by an effective and synergistic overlap of many different disciplines.     

Observation of inhomogeneity in the lipid composition of individual nanoscale liposomes

Liposomes, or vesicles, have been studied extensively both as models of biological membranes and as drug delivery vehicles. Typically it is assumed that all liposomes within the same preparation are identical. Here by employing pairs of fluorescently labeled lipids we demonstrated an up to 10-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm. Since the physicochemical properties of liposomes are directly linked to their composition, a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble.     

Active uptake of drugs into photosensitive liposomes and rapid release on UV photolysis

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-L-alpha-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4'-n-butylphenyl)azo-4'-(gamma-phenylbutyroyl))-glycero-3- phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30 degrees C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photo-induced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.     

Ultra magnetic liposomes for MR imaging, targeting, and hyperthermia

Magnetic liposomes offer opportunities as theranostic systems. The prerequisite for efficient imaging, tissue targeting or hyperthermia is high magnetic load of these vesicles. Here we describe the preparation of Ultra Magnetic Liposomes (UMLs), which may encapsulate iron oxide nanoparticles in a volume fraction of up to 30%. This remarkable magnetic charge provides UMLs with high magnetic mobilities, MRI relaxivities, and heating capacities for magnetic hyperthermia. Moreover, these UMLs are rapidly and efficiently internalized by cultured tumor cells and, when they are administered to mice, they can be vectorized to tumors by an external magnet.     

Effect of the phase state of the lipid bilayer on the structure and characteristics of the polycation-(anionic liposome) complex

Interaction of the cationic polymer poly-N-ethyl-4-vinylpyridinium bromide with bilayer vesicles (liposomes) composed of zwitterionic dipalmitoylphosphatidylcholine and anionic cardiolipin (the molar fraction of the negatively charged cardiolipin groups is 0.2) is studied. The composition and characteristics of the polycation-liposome complex are shown to be controlled by the phase state of the lipid membrane. Liposomes whose membranes exist in their LC state (“liquid” liposomes) keep their integrity in the complex with polycation. The adsorbed polycation can be completely removed from the liposomal membrane by the addition excess amounts of a competing polyanion. The adsorption of polycation on the surface of liposomes whose membranes exist the gel state (“solid” liposomes) leads to the formation of defects in the membrane, and the polycation’s adsorption with such liposomes becomes irreversible. The defects that form are also preserved when solid liposomes on whose surface the polycation is sorbed are transformed into the liquid state. Moreover, the reversible contact between polycation and liquid liposomes becomes irreversible once the liposomal membranes bound to the polycation transform into the solid state.     

Gelatin Coating for the Improvement of Stability and Cell Uptake of Hydrophobic Drug-Containing Liposomes

Purpose: Most therapeutic agents have limitations owing to low selectivity and poor solubility, resulting in post-treatment side effects. Therefore, there is a need to improve solubility and develop new formulations to deliver therapeutic agents specifically to the target site. Gelatin is a natural protein that is composed of several amino acids. Previous studies revealed that gelatin contains arginyl-glycyl-aspartic acid (RGD) sequences that become ligands for the integrin receptors expressed on cancer cells. Thus, in this study, we aimed to increase the efficiency of drug delivery into cancer cells by coating drug-encapsulating liposomes with gelatin (gelatin-coated liposomes, GCLs). Methods: Liposomes were coated with gelatin using electrostatic interaction and covalent bonding. GCLs were compared with PEGylated liposomes in terms of their size, zeta potential, encapsulation efficiency, stability, dissolution profile, and cell uptake. Results: Small-sized and physically stable GCLs were prepared, and they showed high drug-encapsulation efficiency. An in vitro dissolution study showed sustained release depending on the degree of gelatin coating. Cell uptake studies showed that GCLs were superior to PEGylated liposomes in terms of cancer cell-targeting ability. Conclusions: GCLs can be a novel and promising carrier system for targeted anticancer agent delivery. GCLs, which exhibited various characteristics depending on the coating degree, could be utilized in various ways in future studies.     

Impact of chitosan coating of anionic liposomes on clearance rate, mucosal and systemic immune responses following nasal administration in rabbits

Liposomes have been identified as effective immunological adjuvants and have potential for the intranasal and oral delivery of protein antigen. Anionic MLV liposomes were prepared by dehydration–rehydration method. For coating, liposomes were incubated in chitosan solution. Efficiency of coating was confirmed by the evaluation of FITC-labelled chitosan-coated liposomes using a fluorescent microscope. Liposomes morphology and size were studied by optical microscope and size analyzer. Mucoadhesion potential of liposomes was evaluated in human nose by gamma-scintigraphy using ^99mTc-labelled liposomes. Rabbits (4 animals per group) were nasally immunized in weeks 0, 2 and 4 by liposomes encapsulated with 40 Lf TT. Bleedings and lavage collections were taken place in weeks 3 and 6, and IgG and sIgA titers were measured by ELISA method.Liposomes had a mean diameter of 2.38 μm. Loading of TT was 58.7 ± 12.4%. The mucoadhesion (clearance rate from nose) of both coated and non-coated liposomes was similar (P > 0.05). Among the immunized animals, the highest nasal lavage sIgA titers were seen in non-coated liposomes followed by coated ones. The serum IgG titers (2nd bleeding) in animals immunized by both kinds of liposome were similar (P > 0.05), and were lower than the TT solution group (P < 0.05). Immunization by i.m. injection of TT solution resulted in the lowest sIgA and highest IgG titers (P < 0.05) compared with liposomal groups.The results were indicative of good potential of negatively charged liposomes in the induction of mucosal immunity. Coating of liposomes by chitosan, failed to increase both the residence time of liposomes in nasal cavity and systemic responses. Conversely, coated liposomes could not induce the mucosal responses as efficiently as non-coated liposomes. It seems that the coating of liposomes affected their interaction potential with nasal associated lymphoid tissue cells.     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

LIPOSOME MICROENCAPSULATIONS WITHOUT USING ANY ORGANIC SOLVENT

Liposomes are microscopic vesicles composed of phospholipid bilayers surrounding discrete aqueous compartment. Eventhough phospholipid bilayers are formed spontaneously in water, their conventional preparation methods involve either organic solvents or detergent molecules. Organic solvents may create a serious safety problem during liposome manufacturing especially on a large scale and can cause denaturation of proteins and affect the membrane properties. Liposomes are very promising carriers for protein drugs which have aroused great interests in the field of pharmaceutics in these recent years.

The objective of this study was to develop a new dispersion process to prepare liposomes without the usage of organic solvents and detergents. The approach involved the usage of a microfludizer for the extrusion and homogenization of aqueous dispersion of phospholipids, and a modified evaporator in order to increase lipid thin film surface area. The properties of the liposomes prepared by the novel method were superior to the liposomes prepared by conventional methods.     

Pharmacokinetics and Tissue Distribution of Vinorelbine Bitartrate after Intraveous Administration of Liposomal and Injectable Formulations

A hydrophilic and temperature-induced degradation drug, vinorelbine bitartrate (VB)-loaded phosphatidylethanolamin liposomes (PSLs), was prepared by the thin-film hydration method. Liposomes were made of phosphatidylethanolamine: cholesteryl: oleic acid (PE: CHOL: OA, 3:3:1 mass/mass). The mean particle size of the PSLs ranged from 293.06 nm. The transmission electron microscope (TEM) images displayed that the shape of the PSLs was multilamellar vesicles with smooth surface. The highest entrapment efficiency (EE) and drug loading capacity (DL) could reach up to 68.5% and 6.23%, respectively. The PSLs was evaluated by comparing the rate of release of encapsulated VB in different phosphate buffer solution (PBS), and the result showed that the rate of drug release in acid medium was faster than in pH 7.4. Pharmacokinetic characteristics in vivo and the tissue distribution in mice were investigated, which provided experimental and theoretical basis for utilizing liposomes in malignant tumor chemotherapy.     

Catabolism of hemoglobin-haptoglobin complex in microsome subfractions.

After internalization of hemoglobin-haptoglobin complex (Hb-Hp) via receptor-mediated endocytosis (RME) into liver parenchymal cells, organelles containing the complex distribute in the microsome fraction (Ms). Prior to the catabolism, Hb-Hp dissociates symmetrically into two 82,000-dalton (82 kDa) subunits. In the present investigation, the first event of Hb-Hp metabolism in Ms were further examined after [3H-heme, 14C-glogin]Hb-Ho or [125I-Hb]Hp injection to rats. Shortly after the internalization of Hb-Hp, this complex in Ms was intact. At 60 min after injection, radioactive materials of Ms extracted by freezing and thawing (F&T) with yield of 15% were composed of Hb-Hp, 82 kilodaltons (kDa) subunits and Hb metabolites with a ratio of 1:6:13. The heme metabolites were identified as [3H]bilirubin by high performance liquid chromatography (HPLC). The ratio of Hb-Hp/82 kDa subunits/Hb metabolites in microsome residue of the F&T was 40:8:1. The radioactivity in Ms at 60 min localized microsomes subfraction except Golgi light fraction. In electron microscope radioautography of microsome subfraction using [125I]Hb-Hp, silver grains were observed over or within morphologically heterogenous vesicles, e.g. vesicles containing very low density lipoprotein (VLDL) particles with appendage like multi-vesicular body (MVB) or compartment of uncoupling of receptor and ligand (CURL) in Goligi light and intermediate fractions. These studies suggest that Hb-Hp internalized by RME is dissociated symmetrically into two 82 kDa subunits in organelles of Ms, and that organelles with MVB or CURL-like structures are associated with Hb-Hp metabolism.     

Cationic liposomes modified with non-ionic surfactants as effective non-viral carrier for gene transfer

A defined change in formulation components affects the physical and chemical characteristics of cationic liposomes (CLs) carriers in many ways. Therefore, a great degree of control can be exercised over the structure by modifying the CLs with various materials, leading to new innovations for carrier improvement. In the present study, surface modifications of cationic liposomes with non-ionic surfactants—sorbitan monoesters serials (Span 85, 80, 40 and 20) were carried out for developing a new gene transfer carrier. Span modified cationic liposomes (Sp-CLs) were prepared by reverse phase evaporation method (RPV) and self-assemble complexes of antisense oligonucleotides/surfactant modifying cationic liposomes were prepared by auto-coacervation through electrostatic effect. Characterization of Sp-CLs and the self-assembled complex was performed by electron microscope, particle size, zeta potential, turbidity and agarose electrophoresis. Furthermore, in vitro cellular uptake experiment showed that Span plays a role in enhancing the cellular uptake of encapsulated oligonucleotides mediated by Sp-CLs by the endocytosis-dependent route. CLs modified with Span 40 significantly facilitated the cellular uptake by COS-7 cells and HeLa cells; also showed some positive effect on gene expression. That suggests it is a potential non-viral carrier for efficient gene transfer.     

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.

Deformable liposomes containing alkylcarbonates of γ-cyclodextrins for dermal applications

Liposomes made with hydrogenated soya lecithin (HPC) mixed with dodecylcarbonate γ-cyclodextrin (C12CD) at 20:1, 10:1 and 5:1 w/w ratios were prepared by the solvent evaporation method. C12CD had emulsifying properties and the possibility of producing deformable liposomes, as topical delivery system of progesterone (PG), was evaluated. Liposome size, deformability and drug entrapment were determined and the interaction between C12CD and HPC was investigated using differential scanning calorimetry (DSC). The size and the amount of PG loaded in the liposomes depended on the lipid:C12CD ratio: the smallest liposomes were obtained using 20:1 ratio and the maximum drug entrapment at 5:1 ratio. DSC analysis suggested that C12CD interacted with liposomes disrupting and fluidizing the lipid bilayer. PG transepidermal permeation through intact pig skin and PG skin uptake from deformable liposomes were assessed and compared to the values obtained from aqueous suspension and conventional liposomes. The PG permeations were negligible for all systems, while skin uptake increased for liposomes containing C12CD. This was attributed to the deformability and to the increase in the drug entrapment efficiency of these liposomes. The use of C12CD in liposome formulations can improve PG topical therapy.     

Mannan-coated liposome delivery of gadolinium-diethylenetriaminepentaacetic acid, a contrast agent for use in magnetic resonance imaging.

Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), a paramagnetic contrast agent for use in magnetic resonance imaging (MRI) was bound to stearylamine and incorporated into the liposomal membranes (Gd-DTPA liposomes). In addition, the Gd-DTPA liposomes were coated with mannan (cholesterol-aminoethylcarbamylmethyl mannan), a polysaccharide, to obtain the mannan-coated liposomes. An in vitro MRI study showed that the Gd-DTPA liposomes produced a greater intensity of contrast than did the Gd-DTPA solution with a reduced T1 relaxation time. Intravenous injection of the Gd-DTPA liposomes containing 153Gd or liposomes containing 153Gd or 14C-DTPA to mice showed an accumulation of Gd-DTPA primarily in the liver and lung. When the mannan-coated liposomes were administered, an increased uptake of Gd-DTPA by these tissues was demonstrated. The mannan-coated liposomes may enhance contrast of the liver in MRI at a lower dose of Gd-DTPA.