Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most The use of phospholipid vesicles (liposomes) in biology and medicine, a promising new area of research (1), will depend to a large degree on technological improvements in the formation of vesicles of various sizes and properties. As more attempts are made to modify cellular physiology by introducing regulatory molecules into the cell or to improve chemotherapy in the whole animal, the need for a vesicle preparation that entraps a large percentage of the aqueous phase has become apparent.The original liposome preparations of Bangham et al. (2), consisting of multilamellar vesicles (MLV), have been admirably suited in defining many membrane properties (3, 4) and were the basis for the development of the sonicated unilamellar vesicles (SUV) (5). However, both preparations show a relatively low volume of entrapped aqueous space per mole of lipid and restricted ability to encapsulate large macromolecules. This is because in MLV most of the lipid is participating in the internal lamellae, and the close apposition of the adjacent concentric bilayers restricts the internal water space. In SUV, which are single-compartment vesicles, the ratio of surface area to encapsulated volume is so large that only a small aqueous volume per mole of lipid can be attained. Attempts to circumvent these shortcomings (6-8) have been only partially successful. The ethanol injection method produces vesicles of about the same size as SUV with the same shortcomings. The ether infusion technique produces large unilamellar vesicles with high captured volumes per mole of lipid, but the efficiency of encapsulation is relatively low. Other useful techniques for preparing large volume vesicles either use specialized conditions (9) or are restricted to a single phospholipid (10). Methods based upon solvent evaporation have been attempted in the past but have resulted in the formation of multilamellar vesicles (6,11,12). A method designed to form asymmetric vesicles by centrifugation of a suspension of dense aqueous inverted micelles through an organic solvent/water interface has been reported (13). The report indicated that the internal volume was small and the vesicles themselves relatively unstable (13). Techniques based upon the removal of detergents-yield vesicles slightly larger than SUV (14). These are suitable for membrane reconstitution experiments but, like the SUV, fail to encapsulate the aqueous phase efficiently. Recently a method that combines detergent dialysis and solvent evaporation has been described (15). This technique leaves an equal weight of detergent per phospholipid in the resu...

The results obtained in this study esablis that liposome formulations incorporating a synthetic polyethylene glycol-derivatied phospholipid have a pronounced effect on liposome tissue dt n and can produce a large increase in the pharmaologial eflkacy of encapsulated antitumor drugs. This effect is i ly greater than that observed previously with conventionl lposomes and is ith a more than 5-fold

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The rapid clearance of circulating liposomes from the bloodstream, coupled with their high uptake by liver and spleen, has thus far been an obstacle to any attempts at targeting to tumors. We have assessed the impact of liposome composition on their clearance from the circulation in normal and tumor-bearing mice and on their uptake by tumors and various normal tissues. By selective changes in lipid composition, while maintaining a mean particle diameter of '100 nm, we have achieved up to a 60-fold increase in the fraction of recovered dose present in blood 24 hr after i.v. i 'ection. Concomitantly, there was a decrease by a factor of 4 of the recovered dose localizing in the liver and spleen, the major organs of the reticuloendothelial system. Parallel experiments in tumor-bearing mice demonstrated a 25-fold increase of the liposome concentration in the tumor when formulations with long and short blood residence time were compared. The most favorable results were obtained with liposomes containing a small molar fraction of a negatively charged glycolipid, such as monosialoganglioside or phosphatidylinositol, and a solidphase neutral phospholipid as the bulk component. The biodistribution of such formulations is of considerable therapeutic potential in cancer for increasing the concentration of cytotoxic agents in tumors while minimizLing the likelihood of toxicity to the reticuloendothelial system. Liposome encapsulation has been proven useful for reducing the toxicity of certain drugs (1-6) as well as for enhancing the efficacy of macrophage-activating factors (7,8) and drugs directed against parasites residing within the reticuloendothelial system (RES) (9)(10)(11)(12). The propensity of the RES to remove liposomes from the circulation has thus far limited the prospect oftargeting liposomes to tissues other than liver, spleen, and lung (13-15).Designing liposomes with prolonged circulation time would require a reduction of the rate of their clearance by the RES and of the leakage of liposome contents in the bloodstream. Our strategy to achieve these goals was based on the following prior observations. (i) The inclusion of cholesterol (Chol) and solid-phase phospholipids such as sphingomyelin and distearoyl phosphatidylcholine has been shown to increase liposome stability in plasma as determined by the degree of retention of various liposome-encapsulated markers in vitro (16-18). (ii) The same solid-phase phospholipids (sphingomyelin or distearoyl phosphatidylcholine) in the form of small sonicated liposomes are cleared more slowly after intravenous injection than those made with fluid phospholipids such as egg yolk phosphatidylcholine (PtdCho) (19)(20)(21)(22). (iii) The inclusion of certain gangliosides, which confer to the liposome surface a negative charge and increased hydrophilicity, synergizes with Chol to enhance liposome stability in plasma (23) and prolongs liposome half-life in blood with a concomitant decrease in liver and spleen uptake (24). In addition, inclusion of phosphatidylinositol (P...

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Differential scanning calorimetry (DSC) and fluorescence polarization of embedded probe molecules were used to detect phase behavior of various phospholipids. The techniques were directly compared for detecting the transition of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) dispersed in aqueous salt solutions. Excellent agreement occurred in the case of phosphatidylcholine; however, in the case of phosphatidic acid, at pH 6.5, transitions detected by fluorescence polarization using the disc-like perylene molecule occurred about 10 degrees lower than those detected by DSC. Discrepancy between fluorescence and DSC methods is eliminated by using a rod-like molecule, diphenylhexatriene (DPH). Both techniques show that doubly ionizing the phosphate group reduces the Tc by about 9 degrees. Direct pH titration of fluidity can be accomplished and this effect is most dramatic when membranes are in their transition temperature range (ca. 50 degrees). Phosphatidic acid transitions occur at higher temperatures, and have appreciably lower transition enthalpies and entropies than phosphatidylcholine. These effect could not be explained simply on the basis of double layer electrostatics and several other factors were discussed in an attempt to rationalize the results. Addition of monovalent cations (0.01-0.5 M) is shown to increase the Tc of dipalmitoylphosphatidylglycerol by less than 3 degrees. However, addition of (1 x 10-3 M) Ca2+ abolishes the phase transition of both phosphatidyglycerol and phosphatidylserine in the range 0-70 degrees. Preliminary X-ray evidence indicates the phosphatidylserine-Ca2+ bilayers are in a crystalline state at 24 degrees. In contrast, 5 x 10-3 M Mg2+ only broadens the transition and increases the Tc indicating a considerable difference between the effects of Ca2+ and Mg2+. Neutralization of PS increases the Tc from 6 degrees (at pH 7.4) to 20-26 degrees (at pH 2.5-3.0) but does not abolish the transition, suggesting the Ca2+ effect involves more than charge neutralization. Addition of Ca2+ to mixed phosphatidylserine-phosphatidylcholine dispersions, induces a phase separation of the dipalmitoyl- (and also distearoyl-) phosphatidylcholine as seen by the appearance of a new endothermic peak at 41 degrees (58 degrees). Similarly, in mixed (dipalmitoyl) phosphatidic acid-phosphatidylcholine (2:1) dispersions, Ca2+ again can separate the phosphatidylcholine component.

The method of fluorescence recovery after photobleaching has been used to measure the temperature dependence of the lateral diffusion coefficients (D) of two fluorescent lipid analogues in phospholipid multibilayers of various compositions. The probes employed were 3,3-dioctadecyloxocarbocyanine (diO-C18(3) and N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE). In fluid egg phosphatidylcholine multibilayers at 25 degrees C, D was about 4 X 10(-8) cm2/s for NBD-PE and 1.5 X 10(-7) cm2/s for diO-C18(3) and was moderately temperature dependent (2-fold change over 10 degrees C). Equimolar cholesterol reduced D for NBD-PE in these multibilayers by a factor of 2. A greater than 100-fold decrease in D was detected in dimyristoylphosphatidylcholine multibilayers at approximately 23 degree C, which coincides with the gel-to-liquid-crystalline transition temperature, Tm (D 5 X 10(-8) cm2/s at T greater than Tm to D less than 5 X 10(-10) cm2/s at T less than Tm). Equimolar cholesterol abolished this transition behavior, raising D below Tm and decreasing D above Tm. These results confirm and extend previous studies of lateral diffusion employing magnetic resonance and other optical techniques and give additional confidence in the fluorescence methods.

Liposomes (70-100 nm) of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, and poly(ethylene glycol) (PEG)-modified phosphatidylethanolamine (PEG-DSPE) were conjugated to Fab' fragments of a humanized recombinant MAb against the extracellular domain of HER2/neu to create sterically stabilized immunoliposomes (anti-HER2 SL) as a drug carrier targeting HER2-overexpressing cancers. Conjugation employed maleimide-terminated membrane-anchored spacers of two kinds: a short spacer, providing attachment of Fab' close to the liposome bilayer, or a long spacer, with Fab' attachment at the distal terminus of the PEG chain. Confocal microscopy and spectrofluorometry of HER2-overexpressing breast cancer cells incubated with fluorescently labeled anti-HER2 SL prepared with either spacer showed binding of liposomes (8000-23000 vesicles/cell) followed by endocytosis (rate constant ke = 0.012-0.033 min-1) via the coated-pit pathway, evidenced by intracellular acidification and colocalization with transferrin. Uptake of anti-HER2 immunoliposomes by breast cancer cells with low HER2 expression, or after preincubation of cells with free anti-HER2 Fab', was less than 0.2% and 4.3%, respectively, of the uptake by HER2-overexpressing cells. Increasing PEG-DSPE content (up to 5.7 mol %) in anti-HER2-SL prepared with the short spacer decreased liposome-cell binding affinity 60-100-fold, while ke decreased only 2-fold; however, when Fab' fragments were conjugated via a PEG spacer, both binding affinity and ke were unaffected by PEG-DSPE content. Cell binding and internalization of anti-HER2 immunoliposomes increased at higher surface density of conjugated Fab' fragments, reaching plateaus at approximately 40 Fab'/liposome for binding and approximately 10-15 Fab'/liposome for internalization. Uptake of anti-HER2 immunoliposomes correlated with the cell surface density of HER2 and significantly (p < 0.005) correlated with the antiproliferative effect of the targeting antibody but not with the total level of cellular HER2 expression. The results obtained were used to optimize in vivo preclinical studies of anti-HER2 SL loaded with antineoplastic drugs.