Microinjection of ricin entrapped in unilamellar liposomes into a ricin-resistant mutant of baby hamster kidney cells.

Fluorescein-labelled Ricin was entrapped in unilamellar liposomes; 14 microgram of protein was entrapped by 1 mg of lipids. Liposomes added to cells in culture in low serum medium can deliver entrapped Ricin to a Ricin-resistant mutant of baby hamster kidney(BHK)cells. Ricin entrapped in unilamellar liposomes inhibits protein biosynthesis at a concentration of 1.75 microgram/ml in Ricin-resistant cells. Ricin dissolved in medium at 50 microgram/ml does not affect protein synthesis in these cells.     

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type     

Introduction of ribonucleic acids into cells by means of liposomes.

A method of ultramicroinjection of nucleic acids into cultured cells by means of liposomes is described. Messenger RNA, ribosomal RNA and transfer RNA were entrapped in large unilamellar liposomes and subsequently the liposomes were fused with cells. The uptake of RNA by the cells was stimulated 6--8 times by our method. Possible applications of microinjection of RNA by means of liposomes are discussed.     

Biotin and choline replace the growth requirement of Madin-Darby canine kidney cells for high-density lipoproteins

MDCK cells maintained on extracellular matrix (ECM)-coated dishes and exposed to Dulbecco's modified Eagle's medium (DME) supplemented with transferrin and either high-density lipoproteins (HDLs) or phosphatidyl choline (PC) liposomes have a growth rate and final cell density similar to those of cultures exposed to serum-supplemented DME. When MDCK cells are exposed to a medium consisting of a mixture (1:1) of DME and F12 medium (D/F), the addition of transferrin (10 μg/ml) alone supports cell growth and the presence of HDLs or PC liposomes is no longer required. MDCK cells exposed to D/F medium supplemented with transferrin can be passaged for more than 50 generations in total absence of serum. The F12 components that support growth in the absence of HDLs or PC liposomes are biotin (which is absent in DME) and choline (which is present in insufficient concentration in DME). Supplementation of DME with transferrin, biotin (3.6 ng/ml), and choline (10 μg/ml) allows optimal growth of MDCK cells and permits serial propagation through more than 50 generations. The growth requirement of MDCK cells for HDLs or PC liposomes can therefore be replaced by adequate concentrations of biotin and choline. The widely observed fact that a combination of DME/F12 medium is more effective than DME alone in supporting cell growth may be due in part to the lack of biotin and suboptimal choline concentration in DME.     

Structural properties of the proteoliposomes catalyzing electron transport from formate to fumarate

The electron-transport chain catalyzing fumarate reduction by formate has recently been reconstituted from the formate dehydrogenase complex and the fumarate reductase complex from Vibro succinogenes, in a liposomal preparation containing vitamin K-1 (Unden, G. and Kröger, A. (1982) Biochim. Biophys. Acta 682, 258–263). We have now investigated the structural properties of this preparation. The preparation was found to consist of a homogeneous population of unilamellar proteoliposomes with an average diameter of about 100 nm and an internal volume of 2–4 ml / g phospholipid. The buoyant density (1.07 g / ml) was consistent with the protein / phospholipid ratio (0.2 g / g) of the preparation. Leakage of glucose from the internal spaces of the proteoliposomes was negligibly slow. Proteoliposomes prepared with either of the enzyme complexes showed peripheral projections mainly on the outer surface, when examined by electron microscopy after negative staining. The size, orientation and surface density of the projections were consistent with those of the enzymes. Most of the substrate and dye-reactive sites (70–90%) of the enzymes in the proteoliposomes were accessible to external non-permeant substrates. The proteoliposomes catalyzing electron transport were formed by freeze-thawing a mixture of liposomes and protein-phospholipid complexes which did not perform electron transport from formate to fumarate. Nearly the entire amount of the enzymes supplied (0.2 g protein / g phospholipid) was incorporated into the liposomes by this procedure. The transformation of liposomes into proteoliposomes was accompanied by exchange of the internal solutes with the external medium.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.     

HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z_HER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as “Affisomes” were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80–100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41°C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16–20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4–12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were?～70% at a protein-lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm?～0°C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained?>?90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.     

Uptake of Liposomally Entrapped Adenosine-3′-5′-Cyclic Monophosphate in Mouse Brain

[3H] Adenosine-3',5'-cyclic monophosphate (cAMP) could be entrapped efficiently into small unilamellar vesicles when bound to cAMP-dependent protein kinase. The leakage of [3H]cAMP protein kinase complex from liposomes was reduced by more than 60% as compared to free [3H]cAMP. Hyperosmolar mannitol increased the delivery of liposomally entrapped [3H]cAMP protein kinase to the brain with maximum uptake occurring at 10 min after mannitol administration. Optimal delivery to the brain was observed when vesicles composed of total brain lipids or phosphatidylcholine:cholesterol:sulfatides (7:2:1) were used. A slower clearance of liposomally entrapped material from brain tissue was seen under hyperosmolar conditions.     

Infection of cells with Sindbis virus nucleocapsids entrapped into liposomes

The nucleocapsid of Sindbis virus, a natural non-infectious complex of the viral RNA and protein molecules can be encapsulated in large, unilamellar vesicles and delivered efficiently to cells in an infectious form. It is shown that high infectivity of the vesicle entrapped nucleocapsids is partly due to the viral envelope proteins which enhance entrapment and liposome cell interaction.We believe that the efficiency of liposome mediated gene transfer of eukaryotic cells can be increased significantly by the insertion of fusogenic viral envelope proteins into the lipid bilayer of liposomes.     

Characterization of liposomes prepared using a microemulsifier

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 μmol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 μm in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 μmol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4°C, and less than 10% Ara-C at 37°C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

Distribution of solid liposomes on the surface of an epithelial cell layer

A study was made of the adhesion of liposomes, composed of dipalmitoyl- or di-stearoylphosphatidycholine, on the surface of epithelial cells in culture. Sodium fluorescein was entrapped in liposomes for their visualization by fluorescence microscopy. It is found that sonicated unilamellar liposomes adhere predominantly along the sheet margins. Multilamellar liposomes and lipid-coated carmine particles adhere over the whole cellular surface. However, their adhesion along sheet margins was stronger, as evidenced by a brief trypsin treatment. A prolonged trypsin treatment removed all types of liposomes from the cell surface. After the cells were partly detached from each other, small liposomes readily adhered to the newly accessible cell margins. The existence of special lipid membrane-binding proteins on the cell surface is suggested.     

Effect of microwave radiation on the permeability of carbonic anhydrase loaded unilamellar liposomes

The influence of 2.45 GHz microwave exposure (6 mW/g) on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied. The enzyme carbonic anhydrase (CA) was entrapped into cationic unilamellar vesicles. Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (PNPA) across intact liposome bilayer. A twofold increase in the diffusion rate of PNPA through the lipid bilayer was observed after 120 min of microwave radiation compared to temperature control samples. The microwave effect was time dependent. The enzyme activity, as a function of increased diffusion of PNPA, rises over 120 min from 22.3% to 80%. The increase in stearylamine concentration reduces the enzyme activity from 80% to 65% at 120 min. No enzyme leakage was observed. © 1994 Wiley-Liss, Inc.     

Injection of DNA into liposomes by bacteriophage lambda

Small unilamellar vesicles (75-100 nm diameter) and large liposomes (greater than 1 micron in diameter) were prepared containing the lamB protein, an outer membrane protein of Escherichia coli and Shigella which serves as the receptor for bacteriophage lambda. Bacteriophage were observed to bind to these liposomes and vesicles by their tails and in most cases the heads of the bound bacteriophage appeared empty or partially empty of DNA. The lambda DNA was usually only partially ejected from the bacteriophage head when small unilamellar liposomes were used, presumably because the vesicles are too small to contain all the DNA. The partially ejected DNA was not susceptible to DNase unless the vesicle bilayer was first disrupted suggesting that DNA injection of phage DNA into the vesicle had occurred. After disruption of these vesicles on electron microscope grids, the bacteriophage are seen to have partially empty heads and a small mass of DNA associated with their tails. Using larger liposomes prepared by the fusion of lamB bearing vesicles with polyethylene glycol and n-hexyl bromide, the heads of most of the bound bacteriophage appeared to be completely empty of DNA. Disruption of these preparations on electron microscope grids revealed circular arrays of empty-headed bacteriophage surrounding DNA which had apparently been contained within the intact liposomes. These results indicate that high molecular weight DNA can be entrapped within liposomes with high efficiency by ejection from bacteriophage lambda. The possible use of these DNA-containing liposomes to facilitate gene transfer in eukaryotic cells is discussed.     

Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Incorporation of influenza virus M-protein into liposomes.

M-protein from influenza virus vaccine was purified by sodium dodecyl sulfate-gel chromatography and incorporated into liposomes by solubilization with octylglucoside and subsequent dialysis. Liposomes containing M-protein formed a distinct population with a density of 1.22 g/ml on sucrose-gradient centrifugation, regardless of the net charge on the liposomes. Treatment of the liposomes by freeze-fracture followed by electron microscopic examination showed multilamellar structures in those liposomes without M-protein; liposomes containing M-protein were mulberry-like structures which appeared unilamellar. These studies show incorporation of M-protein into the lipid bilayer.     

Diffusion and Catalysis Processes in Unilamellar Liposomes

Abstract

This research concerns the study of enzyme loaded unilamellar vesicles as bioreactors. The plant enzyme Ascorbic Acid Oxidase (AAO) was entrapped in unilamellar liposomes.

Kinetic study shows that the enzyme activity is largely criptic and that the enzyme in liposomes is partially protected against proteolysis. In order to describe the reactivity of such a system, a kinetic model was developed which accounts both for the enzyme location and for the substrate diffusion across the lipid membrane. For the building of the kinetic model we have used information derived from previous studies of freeze-fracture and label fracture microscopy, which have shown that the protein is located both inside the aqueous core and across the lipid membrane of the vesicles.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

Reversible depression of the reticuloendothelial system by liposomes

The effect of various doses of different types (reverse phase evaporation vesicles and small unilamellar vesicles) of intravenously injected liposomes on reticuloendothelial activity, as measured by the blood clearance rate of intravenously injected carbon, was investigated. Also the effect of pretreatment with reverse phase evaporation vesicles on blood clearance and tissue distribution of a second dose of similar vesicles was determined. For all concentrations used reverse phase evaporation vesicles caused reduction in reticuloendothelial activity at least up to 4 h after injection. 24 h after administration the rate of carbon clearance returned to the control level. On the contrary small unilamellar vesicles did not block reticuloendothelial activity. Pretreatment with reverse phase evaporation vesicles (250 μmol/kg) caused an increased blood level and a decreased hepatic uptake of a second dose of the vesicles, injected 1 h after the first dose. This seems to be due to a depression of reticuloendothelial activity and not to a depletion of opsonins. Pretreatment with small unilamellar vesicles (250 μmol/kg) had no significant influence on the tissue distribution of a second dose of vesicles. Our results clearly indicate that reverse phase evaporation vesicles cause a reversible depression of reticuloendothelial activity and this depression seems to be induced by a saturation of reticuloendothelial cells with liposomes.     

Protective effect of HDL on endothelial NO production: the role of DDAH/ADMA pathway

Accumulating studies have demonstrated that the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine (DDAH/ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of high density lipoprotein (HDL) on endothelial NO production was related to its effect on DDAH/ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were prior exposed to HDL (10, 50, or 100 μg/ml) for 1 h, and then incubated with oxidized low density lipoprotein (ox-LDL) (100 μg/ml) for 24 h. The cultured medium was collected for measuring the concentration of NO and ADMA. The cells were collected for measuring the mRNA and protein expression of DDAH-II as well as DDAH activity. HUVECs treated with ox-LDL (100 μg/ml) for 24 h significantly decreased the concentration of NO, the mRNA and protein expression of DDAH-II as well as DDAH activity and increased the level of ADMA. Pretreatment with HDL (10, 50, or 100 μg/ml) could counteract these changes induced by ox-LDL (100 μg/ml). HDL significantly increased the attenuated endothelial cell NO production induced by ox-LDL, which was attributed to its effect on DDAH/ADMA pathway.