Stability of asymmetric phospholipid membranes.

Bilayers (black films) composed of phosphatidylserine are unstable under conditions of asymmetric distribution of calcium or hydrogen ions with respect to the membrane. Addition of calcium ions to the solution (100 millimolar sodium chloride, pH 7.0) on one side only, produces lowering of the direct-current resistance and results in breaking of the membrane. However, with calcium ions on both sides the membranes are stable and show very high electrical resistance.

Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times.

We have previously reported on liposome formulations with reduced uptake by the reticuloendothelial system, prolonged circulation time, and enhanced accumulation in transplanted tumors. One of these formulations, consisting of hydrogenated phosphatidylinositol (HPI), hydrogenated phosphatidylcholine (HPC), and cholesterol (Chol) (HPI-HPC-Chol), and a control formulation, consisting of phosphatidylglycerol (PG), phosphatidylcholine (PC), and Chol (PG-PC-Chol), were loaded with doxorubicin (DXR) and injected intravenously into BALB/c mice for pharmacokinetic studies. Although both formulations were similar in vesicle size, fraction of negatively charged lipid, and drug-to-lipid ratio, there were striking pharmacokinetic differences. DXR was cleared much faster in PG-containing liposomes than in HPI-containing liposomes. Liposome-associated drug was detectable in plasma up to 5 hours after injection in the case of PG-PC-Chol and as late as 72 hours after injection in the case of HPI-HPC-Chol. In agreement with the plasma clearance curves, peak drug concentrations in the liver were observed at 1/2, 5, and 24 hours after injection for free DXR, DXR in PG-PC-Chol, and DXR in HPI-HPC-Chol, respectively. Both types of liposomes reduced considerably the amount of drug accumulating in the heart compared with that accumulating after injection of free DXR.

Cytotoxicity of antibody-directed liposomes that recognize two receptors on K562 cells.

Encapsulation of methotrexate-gamma-aspartate in antibody-conjugated liposomes increased its toxicity for K562 cells, a human leukemia cell line that expresses Fc receptors and human glycophorin A. The liposomes were conjugated with either nonspecific mouse IgG, which interacts with an Fc receptor, or with monoclonal anti-human glycophorin, which interacts simultaneously with an Fc receptor and human glycophorin in the cell membrane. The drug in antibody-directed liposomes was up to 20 times more effective than the free drug, and it was 55 times more effective than the drug in liposomes bearing no surface ligand. The efficacy of drug delivery with liposomes directed only to an Fc receptor was reduced ninefold in the presence of soluble human IgG. Efficacy of drug delivery with liposomes directed both to the Fc receptor and to glycophorin A was not reduced by human IgG or soluble antiglycophorin A, but it was reduced twofold in the presence of both soluble ligands. These results were qualitatively consistent with previous studies on the binding of targeted liposomes to K562 cells.

Specific enhancement of drug delivery to AKR lymphoma by antibody-targeted small unilamellar vesicles.

Antibody targeting of drug-containing liposomes to specific cell populations provides the opportunity to improve cancer chemotherapy. We report here the efficacy of targeted liposomes containing methotrexate-gamma-aspartate against two murine T-lymphomas, AKR/J SL2 and R1.1. Both large and small unilamellar vesicles conjugated to anti-Thy-1.1 antibody associated with AKR lymphoma cells in 10-fold greater amounts than nonconjugated liposomes or liposomes conjugated to a nonspecific antibody. Cell association was inhibited by two different anti-Thy-1.1 monoclonal antibodies, but not by nonspecific antibody. Vesicle size is the critical factor determining drug delivery of targeted liposomes to both AKR and R1.1 T-lymphoma cells. Although targeted large unilamellar vesicles (mean diameter, 0.45 micron) specifically bind to lymphoma cells, they probably are not internalized, because they fail to enhance the efficacy of the drug for growth inhibition of either AKR or R1.1 cells. In contrast, drug encapsulated in targeted small unilamellar vesicles (mean diameter, 0.053 micron) is up to 22 times more effective than free drug against AKR cells, and is 40 times more effective against R1.1 cells. We have also demonstrated the efficacy of small compared to large unilamellar vesicles using two different target antigens, Thy-1.1 for AKR cells and H-2Kk for R1.1 cells. These experiments establish a system which can be used to test the antitumor efficacy of targeted liposomes against AKR/J SL2 lymphoma implanted in AKR/Cu mice.

Inhibition of tumor cell growth in vitro and in vivo by 1-beta-D-arabinofuranosylcytosine entrapped within phospholipid vesicles.

Phospholipid vesicles have been used as a carrier vehicle to enhance the cytotoxic activity of 1-beta-D-arabinofuranosyl-cytosine (ara-C) and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate against several tumor cell lines. The activity of both compounds in free solution or entrapped within phospholipid vesicles was compared against L1210 cells, Ehrlich ascites cells, and SV40-transformed 3T3 cells in vitro. In addition, the activity of vesicle-entrapped ara-C against L1210 cells was also studied in vivo. The results obtained in vitro with ara-C indicated no difference in the concentration needed to inhibit growth of cells by 50% between free ara-C and vesicle-entrapped ara-C. In contrast, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate entrapped in phospholipid vesicles was a more potent inhibitor of L1210 in culture (ID50, 2 X 10(-8) M) compared to the relatively inactive free 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (id50 greater than 10(-7) M). Experiments carried out with L1210 cells in mice showed that, after a single i.p. dose (10 mg/kg) of vesicle-entrapped ara-C, the average survival times of mice inoculated with 10(5) L1210 cells were increased by over 90%. In control experiments, free ara-C or vesicles plus free ara-C (10 mg/kg) did not prolong survival of mice.

Surface properties of phorbol esters and their interaction with lipid monolayers and bilayers.

The potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) is surface active and was found to occupy a limiting area of 62 sq A/molecule in monolayers at the air-water interface. The interfacial tension of aqueous TPA solutions is decreased by increasing the bulk-phase TPA concentrations up to 2 x 10(-6) M,beyond which no further decreases were observed. This concentration is in agreement with the apparent solubility limit previously obtained. The apparent aqueous solubility limit of the more hydrophobic phorbol-didecanoate is 5 x 10(-8) M. Interaction of TPA with egg phosphatidylcholine monolayers at the air-water interface was shown by an increase in the surface pressure of the monolayer from 22 dynes/cm, initial film pressure, to 34 dynes/cm 90 min after introduction of TPA into the aqueous subphase. It was shown by gel filtration chromatography that a similar phorbol derivative, tritiated phorbol-didecanoate, binds to phospholipid vesicles. Differential scanning calorimetry also indicated that the addition of either TPA or an inactive stereoisomer, 4-alpha-phorbol-didecanoate, to phospholipid bilayers results in a marked reduction of the enthalphy of the minor transition of dipalmitoylphosphatidylcholine liposomes. Several fluorescence polarization probes for membrane fluidity indicate that TPA does not affect this membrane parameter. Further, the presence of TPA induces no measurable change in the cation permeability of phospholipid vesicles, the conductance of planar bilayer membranes, or the electrophoretic mobility of negatively charged liposomes. The lack of a specific effect with bilayers alone, combined with the documented physiological effects at low TPA concentrations, point to the possibility of a specific membrane component as the receptor for TPA at the plasma membrane.

Antibody-directed targeting of liposomes to human cell lines: role of binding and internalization on growth inhibition.

Small unilamellar liposomes containing methotrexate or methotrexate-gamma-aspartate were conjugated to Staphylococcus aureus protein A and were thus able to bind cell-specific immunoglobulins for targeting to malignant human B- and T-cell lines. We were able to demonstrate enhanced protein A liposome uptake and growth inhibition by targeting with an anti-major histocompatibility complex class II antibody recognizing two different B-cell lines. The enhanced growth inhibition was specific for the targeting antibody and amounted to a 2- to 3-fold lowering of the concentration of drug required to inhibit cell growth by 50% as compared to nontargeted liposomes or liposomes targeted with an antibody not recognizing a cell surface antigen. A strong association between enhanced growth inhibition and liposome internalization as assessed by fluorescent-activated cell sorter analysis of carboxyfluorescein containing protein A liposomes was seen. By contrast, specific enhancement of growth inhibition was not seen with several anti-idiotype antibodies or antibodies to T-cell differentiation antigens. Liposome internalization did not occur with these antibodies. Failure of growth inhibition and PA liposome internalization could not be explained by differences in cell binding of the antibody PA liposomes or the degree of protein A binding of the targeting antibody. Although the ability of the targeting antibody to bind to the cell and to protein A are important, these factors alone are not sufficient to guarantee internalization and growth inhibition. Variations in rates of internalization of various cell surface antigen-antibody complexes may account for different protein A liposome mediated cytotoxicities.

Effect of liposome composition and other factors on the targeting of liposomes to experimental tumors: biodistribution and imaging studies.

We have examined the distribution of radiolabeled liposomes in tumor-bearing mice after i.v. injection. Two mouse tumors (B16 melanoma, J6456 lymphoma) and a human tumor (LS174T colon carcinoma) inoculated i.m., s.c., or in the hind footpad were used in these studies. When various liposome compositions with a mean vesicle diameter of approximately 100 nm were compared using a radiolabel of gallium-67-deferoxamine, optimal tumor localization was obtained with liposomes containing a phosphatidylcholine of high phase-transition temperature and a small molar fraction of monosialoganglioside or hydrogenated phosphatidylinositol (HPI). At 24 h after injection, average values of tumor uptake higher than 10% of the injected dose per g and liver-to-tumor ratios close to 1 were reproducibly obtained. Increasing the molar fraction of HPI from 9% to 41% of the total phospholipid resulted in enhancement of liver uptake and decrease of tumor uptake. Methodological aspects that influence vesicle size appear to affect significantly liposome localization in the tumor. However, varying the phospholipid dose within a 10-fold range caused only minor changes in the percent of injected dose recovered in the tumor. A high uptake by tumors was also observed using other radiolabels [[3H]inulin and indium-111-labeled bleomycin (111In-Bleo)] in monosialoganglioside- and HPI-containing liposomes. In the case of 111In-Bleo, encapsulation in liposomes resulted in approximately 20- to 40-fold increase in tumor accumulation of the radiolabel at 24 h after injection. The marked localization of liposomes in the mouse footpad inoculated with tumor as opposed to the contralateral mock-injected footpad was also documented by imaging experiments with gallium-67-deferoxamine and 111In-Bleo-labeled liposomes. These results support the contention that some glycolipid-containing liposomes previously shown to have long circulating half-lives accumulate significantly in a variety of tumors and are promising tools for the delivery of anti-tumor agents.

Use of lipid vesicles as carriers to introduce actinomycin D into resistant tumor cells.

Unilamellar lipid vesicles have been used as a carrier vehicle to enhance the uptake of actinomycin D into an actinomycin D-resistant Chinese hamster tumor cell line (DC-3F/ADX). The DC-3F/ADX cell line is resistant to actinomycin D as a result of its decreased capacity to transport actinomycin D across the plasma membrane and is able to grow in the presence of concentrations of actinomycin D that are cytotoxic for the sensitive parent cell line (DC-3F). Incubation of resistant DC-3F/ADX cells with actinomycin D-containing vesicles produced a 5-fold increase in intracellular drug concentration over that achieved by exposure to identical concentrations of the drug added to the culture medium. Vesicle-mediated uptake of actinomycin D into resistant cells produced inhibition of cellular RNA synthesis and cell growth at actinomycin D concentrations that had no inhibitory effect when added as free drug in the culture medium. Dose-response measurements established that direct introduction of actinomycin D into resistant cells by means of lipid vesicles resulted in a 200-fold reduction in the concentration of actinomycin D required to inhibit cellular RNA synthesis and a 120-fold reduction in the dose of actinomycin D required to produce a 50% inhibition of cell growth. These results lend strong support to the hypothesis that cellular resistant to actinomycin D is due to a lower capacity to take up actinomycin D as a result of a reduction in the permeability of the cellular plasma membrane to this drug. The potential value of lipid vesicles for introducing other classes of drugs into cultured cells and their possible use in chemotherapy are also discussed.

Liposome-based therapy of human ovarian cancer: parameters determining potency of negatively charged and antibody-targeted liposomes.

Liposomes containing cytotoxic agents may be highly efficacious for intracavitary therapy of malignancies such as ovarian carcinoma, which resides principally in the peritoneal cavity. We have examined in vitro the cytotoxicity of a variety of liposome-drug formulations against OVCAR-3, a human ovarian cancer cell line. Two drugs tested, methotrexate-gamma-aspartate and 5-fluoroorotate, show increased cytotoxicity on various cultured cell lines following encapsulation in liposomes and can be considered liposome-dependent agents. With the optimal lipid composition used in this study, the maximal increase in potency on OVCAR-3 is 2.6-fold for methotrexate-gamma-aspartate and 5.2-fold for 5-fluoroorotate. Studies on liposome-cell association suggest a low capacity of OVCAR-3 to bind and internalize phospholipid vesicles, which limits the in vitro potency of liposomes for these cells. OC-125, a monoclonal antibody recognizing an antigen common to a number of human ovarian cancers (CA-125), has been coupled covalently to the liposome surface. Liposomes bearing OC-125 and containing methotrexate-gamma-aspartate show an 8-fold increase in potency against OVCAR-3 cells in a 96-h growth inhibition assay. Briefer exposure of tumor cells to treatment accentuates the advantage of targeted liposomes. The cytostatic effect of 1 h exposure to OC-125 liposomes is 100-fold greater than the equivalent exposure to free drug and equal to the maximal cytostatic effect achieved with free drug for 96 h. Attachment of OC-125 antibody also confers upon liposomes the capacity to recognize OVCAR-3 cells growing as an ascites tumor in nude mice. After i.p. injection, control liposomes bind tumor cells in relatively low numbers, while fluorescent OC-125 liposomes can be observed bound specifically to tumor cell masses for periods of days.

Role of ligand in antibody-directed endocytosis of liposomes by human T-leukemia cells.

The rate of uptake and intracellular processing of ligand-directed drug carriers may depend heavily on the endocytic pathway of the target antigen. We examined the role of the target antigen and type of antibody-liposome linkage in determining endocytosis of liposomes by three human T-cell leukemias, Jurkat, CEM, and Molt-4. Liposome-cell binding and internalization over time were studied using two independent assays for intracellular delivery of liposome contents: a new fluorescence assay using a pH-sensitive fluorescent dye; and a growth inhibition assay for delivery of cytotoxic drug, methotrexate-gamma-aspartate. Liposomes targeted against the transferrin receptor showed greater surface binding, internalization, and growth inhibition than liposomes targeted against the T-cell surface antigens, CD2, CD3, or CD5. Furthermore, liposomes made by conjugating the targeting antibody directly to the liposome surface were more efficiently internalized and retained than were liposomes linked to antibody-coated cells via Protein A. Selection of the type of antibody-liposome conjugate as well as the appropriate surface receptor to facilitate endocytosis is essential in antibody-directed drug treatment of cancer.

Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.

Using light and electron microscopy, we investigated the in vivo distribution of liposomes sterically stabilized by specific lipids which prolong their circulation in blood. Tissue distribution of sterically stabilized liposomes composed of distearoyl phosphatidylcholine:cholesterol:monosialoganglioside GM1 (10:5:1)-encapsulated 67Ga-Desferal indicates that more than 30% of liposomes still remain in the blood at 24 h after tail vein injection. Moreover, such liposomes accumulated in tumors (C-26 colon carcinoma cells implanted s.c.), reaching almost the same level of uptake as liver (approximately 20% injected dose/g tissue). The microscopic localization of liposomes labeled with encapsulated colloidal gold or rhodamine-labeled dextran coincided well with the tissue distribution. To evaluate circulation parameters, two sizes of gold-containing egg phosphatidylcholine:cholesterol:distearoyl phosphatidylethanolamine (derivatized at its amino position with a 1900 molecular weight segment of polyethylene glycol) (10:5:0.8) liposomes were injected. The plasma was examined by electron microscopy of negative-stained preparations at 0.5, 4, and 24 h after liposome injection. It was found that the ratio of small (less than 100 nm diameter) to large (greater than 100 nm) liposomes increased with time, indicating a much faster clearance of the larger liposomes. To detect the localization of liposomes in various tissues, appropriate samples were fixed 24 h after the injection of gold-containing liposomes (between 80 and 100 nm in diameter) composed of egg phosphatidylcholine:cholesterol:monosialoganglioside GM1 (10:5:1) or egg phosphatidylcholine:cholesterol:derivatized distearoyl phosphatidylethanolamine. The tissues examined for this study included normal liver, bone marrow, and implanted neoplasms. Silver-enhanced colloidal gold was found predominantly within Kupffer cells in the normal liver and within macrophages in the bone marrow. Rarely were any silver-enhanced gold particles detected in hepatocytes. In all preparations, electron microscopy revealed the presence of gold in endosomes and lysosomes of fixed sinusoidal lining macrophages in the liver and bone marrow. Peripheral to the implanted tumors, silver enhancement revealed gold in small blood vessels and focally beyond the vessel boundaries in extracellular spaces around tumor cells. Gold particles were not observed within the tumor cell cytoplasm. At the tumor border, nonenhanced gold was occasionally seen by electron microscopy in cells of the mononuclear phagocyte system. We obtained the same localization pattern as with silver enhancement by using an alternative aqueous content marker, rhodamine B isothiocyanate-dextran. We conclude that liposomes of specific composition, which have the ability to remain in circulation with a half-life of 12-24 h, are also able to transverse the endothelium of small blood vessels, including those in tumors, and extravasate into extracellular spaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics and therapeutics of sterically stabilized liposomes in mice bearing C-26 colon carcinoma.

Three different liposome types were compared for blood clearance and tissue uptake in mice bearing C-26 colon carcinoma growing either s.c. or in liver. Therapeutic experiments were performed with the liposome preparation showing the highest tumor uptake. Liposomes were composed of solid-phase phosphatidylcholine, either distearoyl phosphatidylcholine or hydrogenated soy phosphatidylcholine, and cholesterol at a 2:1 molar ratio. These liposomes were compared with similar but sterically stabilized liposomes (SL) which, in addition, contained either GM1 ganglioside or phosphatidylethanolamine derivatized with poly(ethylene glycol). Pharmacokinetic analysis of drug disposition was based on the areas under the curve for liposome-entrapped 67Ga uptake per gram of tissue up to 96 h following i.v. injection. The highest tissue area under the curve values with both liposome types were obtained in spleen, liver, and tumor. However, the sterically stabilized liposomes gave an area under the curve value 2-3-fold higher in the s.c. tumor and about 2-fold lower in liver and spleen. The therapeutic efficacy of doxorubicin (DOX) and epirubicin (EPI) encapsulated in poly(ethylene glycol)-derivatized phosphatidylethanolamine-containing liposomes was compared with that of free drug at two doses, 6 and 9 (or 10) mg/kg animal weight. Liposomes containing drug were injected either as a single dose, at different times following tumor implantation, or as three weekly doses starting 10 days after implantation. When injected as a single dose, liposome-encapsulated DOX had the maximal effect on tumor growth when injected 6 to 9 days after tumor implantation. When injected as three weekly doses, with treatment starting with a delay of 10 days, tumors which had grown to a size of approximately 0.05-0.1 cm3 regressed in groups of animals treated with either liposome-encapsulated drug (SL-DOX or SL-EPI) but continued to grow unabated in untreated mice and in mice receiving either of the free drugs. Survival of tumor-bearing animals treated with either SL-EPI or SL-DOX was significantly prolonged. Animals receiving saline, EPI, or DOX survived a mean of 50, 62, and 49 days, respectively, following tumor implantation. Eight of nine and nine of 10 animals receiving 6 and 9 mg/kg SL-EPI, respectively, survived to 120 days. Ten of 10 animals in both groups receiving 6 and 9 mg/kg SL-DOX survived to 120 days. None of the surviving animals in the SL-EPI and SL-DOX group showed any histological evidence of tumor at the conclusion of the experiment (120 days).(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody-directed liposomes: comparison of various ligands for association, endocytosis, and drug delivery.

We have developed and compared the cytotoxicity of methotrexate-gamma-aspartate encapsulated in several liposome formulations which bind mouse monoclonal antibody in order to define conditions for screening cell lines and antibodies for liposomal efficacy. Liposomes conjugated to Staphylococcus aureus Protein A were more potent than liposomes conjugated to either rabbit or affinity-purified goat anti-mouse immunoglobulin (Ig) when incubated with AKR/J SL2 cells sensitized with specific antibody. The antibody-directed Protein A liposomes were also 10-fold more potent than liposomes conjugated directly to specific antibody against the AKR/J SL2. We examined the effect of antibody specificity, concentration, and isotype on liposome-mediated drug delivery to AKR/J SL2 cells. The growth-inhibitory effect of the drug in the antibody-directed Protein A liposomes varied with the target antigen on the cell. The potency of the liposomes with a given antibody was proportional to their relative binding and endocytosis by the cells, and to the reactivity of the particular antibody with the cell as demonstrated by indirect immunofluorescence. The Protein A liposomes maintained maximal potency down to antibody concentrations as low as 1 microgram/ml with the anti-Thy 1.1-sensitized AKR/J SL2 cells, thus demonstrating the possible use of these liposomes for hybridoma screening. Use of isotype-switched variants of the anti-Thy 1.1 antibody with the AKR/J SL2 cells showed the superior efficacy of the IgG2a, IgG2b, and IgG3 isotypes to the IgG1 with the Protein A liposomes. The large differential potency of the free drug and the drug encapsulated in antibody-directed Protein A liposomes was maintained even at short incubation times, thus providing a system which may be useful for eradication of tumor cells from bone marrow in vitro.

Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft.

Microvascular permeability and interstitial penetration of sterically stabilized liposomes in both normal s.c. tissue and human colon adenocarcinoma LS174T xenograft were quantified by using the dorsal skin-fold chamber implanted in severe combined immunodeficient mice and intravital fluorescence microscopy. Significant extravascular accumulation was the dominant feature of liposome distribution in tumors, whereas only minimal intramural accumulation in postcapillary and collecting venules was observed in normal s.c. tissue. The extravasated liposomes in tumors distributed heterogeneously and formed perivascular clusters that did not move significantly and could be observed for up to 1 week. The effective permeability of tumor vessels to liposomes (2.0 +/- 1.6 x 10(-8) cm/s; n = 23) was six times smaller than that to bovine serum albumin (1.2 +/- 0.5 x 10(-7) cm/s; n = 6). These results provide new insights into the mechanisms of transendothelial pathways of liposomes and improvements in liposome-mediated drug delivery.

Fluctuations in red cell flux in tumor microvessels can lead to transient hypoxia and reoxygenation in tumor parenchyma.

Hypoxia occurs in two forms in tumors. Chronic or diffusion-limited hypoxia is relatively well characterized. In contrast, intermittent or perfusion-limited hypoxia is not well characterized, and it is not known how common it is in tumors. The purpose of this study was to determine whether spontaneous fluctuations in tumor microvessel flow rate can modify vessel oxygen tension (pO2) sufficiently to cause intermittent hypoxia (IH; tissue pO2 < 3 mmHg) in the tumor parenchyma supplied by such vessels. Microvessel red cell flux (RCF) and perivascular pO2 were measured simultaneously and continuously in dorsal flap window chambers of Fischer-344 rats with implanted R3230Ac tumors. In all vessels, RCF was unstable, with apex/nadir ratios ranging from 1.5 to 10. RCF and pO2 were temporally coordinated, and there were linear relationships between the two parameters. Vascular pO2 was less sensitive to changes in RCF in well-vascularized tumor regions compared with poorly vascularized regions. Simulations of oxygen transport in a well-vascularized region of a tumor demonstrated that two-fold variations in RCF can produce IH in 30% of the tissue in that region. In poorly vascularized regions, such fluctuations would lead to an even greater percentage of tissue involved in transient hypoxia. These results suggest that IH is a relatively common phenomenon. It could affect binding of hypoxic cytotoxins to tumor cells, in addition to being an important source of treatment resistance. Intermittent hypoxia also could contribute to tumor progression by providing repeated exposure of tumor cells to hypoxia-reoxygenation injury.

Liposomes and hyperthermia in mice: increased tumor uptake and therapeutic efficacy of doxorubicin in sterically stabilized liposomes.

We have shown that sterically stabilized (Stealth) liposomes (SL), can accumulate in the extracellular space within tumors, and may improve pharmacokinetics and therapeutic efficacy of encapsulated doxorubicin (SL-DOX). When SL-DOX were incubated in vitro at different temperatures with 50% bovine serum, approximately 20% of the encapsulated DOX was released at 42 degrees C within 1 min, compared with less than 1% DOX released at 37 degrees C. In vivo, mice were implanted s.c. with C-26 colon carcinoma in both flanks to produce matched tumors 6-10 mm in diameter. Topical hyperthermia treatment consisting of 42 degrees C minimum tumor temperature for 30 min was applied with a microwave device to the tumor on one side only at 1 h after i.v. injection of SL-DOX or free DOX. Tumor DOX concentration in the group which was given injections of SL-DOX and sacrificed 2 h after drug injection was 1.5-fold higher compared with the nonheated tumor in mice given injections of SL-DOX. At 24 h after injection the thermal enhancement ratio for DOX accumulation in tumor remained at 1.5. In addition, there was a 15-fold higher concentration of DOX in tumor from the group given injections of SL-DOX compared to mice given injections of free doxorubicin. To assess therapeutic efficacy, we treated mice with hyperthermia for 15 min either at 1, or at 24 h or at both time points after injection of SL-DOX. We have found that the life span of the group of mice treated with SL-DOX and two 15-min hyperthermia treatments increased 51% compared with control groups receiving the same dosage of SL-DOX but without hyperthermia, and 59% compared to those receiving two hyperthermia treatments but with free DOX. A single hyperthermia treatment either at 1 or 24 h was less effective in increasing life span compared with two treatments, but all groups treated with SL-DOX and single hyperthermia were still superior to the control groups, showing a 27-38% increase in life span.

Control of membrane fusion by phospholipid head groups. I. Phosphatidate/phosphatidylinositol specificity.

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca2+ or Mg2+ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90 degrees light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca2+ and Mg2+ at threshold concentration ranges of 0.03-0.1 mM (Ca2+) and 0.07-0.15 mM (Mg2+) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca2+ or Mg2+ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5-10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.

The role of surface charge and hydrophilic groups on liposome clearance in vivo.

The effect of negative surface charge and hydrophilic groups on liposome clearance from blood was investigated in mice using liposome-entrapped 67gallium-deferoxamine as a label. The presence of negatively-charged lipids may retard or accelerate liposome clearance. Physicochemical features contributing to optimal retardation of liposome clearance include a hydrophilic carbohydrate moiety and a sterically hindered negatively-charged group. The relevance of the negative charge steric effect is suggested by the finding that phosphatidylinositol phosphate (PIP) and trisialoganglioside (GT1) are less effective than phosphatidylinositol (PI) and monosialoganglioside (GM1), respectively, in retarding liposome clearance. The need for negative charge in addition to the carbohydrate group for optimal effect on retardation of clearance is indicated by the observation that asialoganglioside (AGM1) is less effective than GM1 in this respect. The negative charge effect is observed with liposome bilayers having both low and high temperature phase-transitions. Increasing the molar fraction of negatively-charged lipid (hydrogenated PI derived from soya) from 23 to 41% resulted in a dramatic acceleration of liposome clearance. The clearance-accelerating effect of the high negative charge was specifically directed to the liver with selective reduction of spleen uptake. Increasing liposome size also had an accelerating effect on clearance but in this case it was accompanied by a non-specific concomitant increase of both liver and spleen uptake.

Conjugation of apolipoprotein B with liposomes and targeting to cells in culture.

Mixed phospholipid/cholesterol (2:1 molar ratio) liposomes were conjugated with native and acetylated apolipoprotein B (apoB), the protein part of low density lipoprotein (LDL). The objective was to increase the specificity of the cellular uptake of liposomes by utilization of the LDL and scavenger receptor pathways. The method of choice for the conjugation of liposomes with apoB proved to be the detergent solubilization and removal procedure. Two detergents were tested;sodium cholate (NaC) and octyl glucoside (OG). The integrity of the resulting complexes was demonstrated by Sepharose CL-4B gel chromatography and Metrizamide gradient centrifugation. The conjugates showed a good physical stability and the leakiness was only marginally larger than for unconjugated liposomes. The interaction of apoB- and acetyl apoB-liposome conjugates with CV-1 and J774 cells, respectively, was monitored by an encapsulated pH-sensitive fluorophore, pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)). This dye provides means of detecting binding and endocytosis of conjugates in living cells. The internalization was a fast process and about 10-times faster for the OG-conjugates than for the corresponding unconjugated liposomes. The conjugates showed a clear concentration-dependent association of dye with cells, while this was less prominent with liposomes. The uptake was nearly an order of magnitude faster with CV-1 cells than with J774 cells. Acidification of intracellular conjugates proceeded fast during the first 30 min of incubation and reached a minimum value of approx. pH 6 after 3 h. The specificity of binding of apoB-liposome conjugates to CV-1 cells was demonstrated by displacement experiments with native LDL. The results indicate that apoB-liposome conjugates may be used as a delivery vehicle for bioactive subtsances to cells.