Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system.

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1beta (IL-1beta). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

Dynorphin-phospholipid membrane interactions: role of phospholipid head-group and cholesterol.

The interaction of the kappa-opioid receptor-selective heptadecapeptide dynorphin A(1-17) (Tyr1-Gly-Gly-Phe-Leu5-Arg-Arg-Ile-Arg-Pro10-Lys-Leu-Lys-Trp-As p15-Asn-Glu) with phospholipid membranes has been investigated by monitoring the leakage of the internal aqueous contents of liposomes, the changes in the tryptophan emission spectrum, and the collisional quenching of tryptophan fluorescence by brominated lipids. The peptide induces more extensive leakage of contents from phosphatidylserine than from phosphatidylcholine vesicles, and experiences a blue shift of the Trp fluorescence emission maximum in the presence of phosphatidylserine vesicles. In the presence of phosphatidylcholine vesicles, however, the Trp fluorescence intensity is reduced without a blue shift. In phosphatidylserine membranes containing 10 mol% phosphatidylcholine, the intensity of the blue-shifted fluorescence is enhanced. This avid interaction of dynorphin A(1-17) with phosphatidylserine membranes is likely to be mediated by the positively charged Arg and Lys groups. It is proposed that, while the N-terminus of the peptide may be embedded in the bilayer in analogy with dynorphin (1-13), the C-terminal region of dynorphin A (1-17) bends back onto the bilayer/water interphase, and that the Trp14 residue is stabilized in a hydrophobic pocked near the interphase by the interaction of the neighboring charged amino acids with the phosphate, carboxyl and amino groups on phosphatidylserine.

Human immunodeficiency virus type 1 (HIV-1) fusion with model membranes: kinetic analysis and the role of lipid composition, pH and divalent cations.

The kinetics and extent of HIV-1 fusion with model membranes was studied. HIV-1 was labeled with octadecyl rhodamine B chloride, and fusion was monitored continuously as the dilution of the probe into target membranes. The results were analyzed by a mass action model which yielded good simulations and predictions for the kinetics and final extents of fluorescence increase. The model determined the percent of virions capable of fusing and rate constants of fusion, aggregation and dissociation. Ultrastructural analysis of the virus and reaction products by electron microscopy also provided evidence of HIV-1 fusion with membranes lacking CD4. HIV-1 fusion activity depends on the target membrane lipid composition according to the sequence: cardiolipin (CL) > > phosphatidylinositol > CL/dioleoylphosphatidylcholine (DOPC) (3:7), phosphatidic acid > phosphatidylserine (PS), PS/cholesterol (2:1) > PS/PC (1:1), PS/phosphatidylethanolamine (1:1) > DOPC, erythrocyte ghosts. Reduction of pH from 7.5 generally enhances the rate and extent of HIV-1 fusion. Physiologically relevant concentrations of calcium stimulate HIV-1 fusion with several liposome compositions and with erythrocyte ghost membranes. The fusion products of HIV-1 with liposomes consist of a single virus and several liposomes. The mass action analysis revealed that, compared to intact virions, the fusion products show a striking reduction in the fusion rate constant. Like influenza and Sendai viruses, HIV-1 fusion with membranes containing its own envelope glycoprotein(s) is strongly inhibited. Unlike these viruses, HIV-1 fusion is promoted by physiological levels of calcium. HIV-1 fusion with liposomes is qualitatively similar to simian immunodeficiency virus fusion.

Liposomes modulate human immunodeficiency virus infectivity.

We have investigated the effects of the fusion of liposomes with human immunodeficiency virus type 1 (HIV-1LVA) on the ability of the virus to infect CD4+ and CD4- cells. Fluorescence dequenching measurements indicated that HIV-1 fuses with liposomes composed of either cardiolipin (CL) or N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) but not appreciably with dioleoylphosphatidylcholine (DOPC) liposomes. Pre-incubation of HIV-1 with DOTMA liposomes enhanced virus production (measured by p24 gag antigen production in the culture medium and in situ) in CD4+ A3.01 and H9 cells in a concentration-dependent manner, but did not mediate the infection of the CD4- cell line, K562. Preincubation of HIV-1 with between 10 and 30 microM-DOTMA liposomes, and subsequent incubation with A3.01 cells, resulted in the production of about 30-fold greater levels of virus than controls. The presence of DOTMA liposomes during the incubation of A3.01 cells with HIV-1 enhanced the infectivity of the virus up to 90-fold compared to controls. Conversely, preincubation of HIV-1 with CL liposomes inhibited infection of A3.01 cells, dependent on the concentration of liposomes; DOPC liposomes did not alter the infectivity of the virus under any of the incubation conditions. Our results thus indicate that fusion of HIV-1 with liposomes alters the ability of the virus to infect its target cells.

Fusion of simian immunodeficiency virus with liposomes and erythrocyte ghost membranes: effects of lipid composition, pH and calcium.

Simian immunodeficiency virus from macaques (SIVmac) is closely related in its structure and biological activity to human immunodeficiency virus, and is the best animal model for the acquired immunodeficiency syndrome. We investigated the kinetics of membrane fusion between SIVmac and phospholipid vesicles and the effects of various parameters on this process. Purified SIVmac was labelled with octadecyl rhodamine B chloride, and fusion was continuously monitored as the dilution of the probe in target membranes. These studies show that SIVmac fusion is strongly dependent upon the liposome composition. Fusion with pure cardiolipin (CL) liposomes is significantly faster than with CL/dioleoylphosphatidylcholine (DOPC) (3:7), phosphatidylserine (PS) or disialoganglioside (GD1a)/DOPC (1.5:8.5) vesicles. SIVmac does not fuse appreciably with pure DOPC liposomes. Reduction of pH from 7.5 to 4.5 greatly enhances the rate of SIVmac fusion with CL, CL/DOPC and PS membranes, but does not affect fusion with DOPC or GD1a/DOPC membranes. Calcium stimulates viral fusion with CL liposomes, but not with CL/DOPC or DOPC liposomes. SIVmac fuses with human erythrocyte ghost membranes only slowly at reduced pH. Our results indicate that SIVmac can fuse with membranes lacking the known viral receptor, CD4. Although the mechanism of SIVmac fusion with model and biological membranes remains to be determined, the fusion activity of SIVmac shares similarities with other lipid-enveloped viruses such as Sendai and influenza viruses.

The kinetic mechanism of cation-catalyzed phosphatidylglycerol transbilayer migration implies close contact between vesicles as an intermediate state.

We have investigated variations in the rate of Mn2+-catalyzed phosphatidylglycerol transbilayer migration [Lentz, Madden, & Alford (1982) Biochemistry 21, 6799] with changes in phospholipid and cation concentration over more than a 100-fold range of both parameters. The slope of a double logarithmic plot of the rate of transbilayer lipid migration versus lipid concentration was 1.7, suggesting that lipid redistribution was dependent on vesicle aggregation or collision. A model involving transitory dimerization of vesicles was able to account for the concentration dependence of the transbilayer redistribution rate. The observed variation in rate with the logarithm of Mn2+ concentration was complex: linear above 0.4 microM (corresponding to roughly 2.5 Mn2+ per vesicle) but involving a steeper dependence on Mn2+ below 0.04 microM (roughly four vesicles per Mn2+). The rate of transbilayer redistribution increased substantially between 37 and 56 degrees C, yielding a nonlinear Arrhenius plot. There was no evidence of either fusion or lipid exchange between vesicles at the low concentrations of Mn2+ needed for transbilayer redistribution. The data are consistent with a model suggesting transitory "micro-domains" of a dehydrated, interbilayer complex as involved in the transition state and are inconsistent with a model involving an inverted micelle-type structure for the transition state.

Spontaneous fusion of phosphatidylcholine small unilamellar vesicles in the fluid phase.

Using a high-sensitivity differential scanning microcalorimeter capable of performing cooling scans, we have examined the phase behavior of small unilamellar vesicles (SUV) as a function of time of storage above their order-disorder phase transition. Vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were examined. Cooling scans on fresh (5-7-h postsonication) samples revealed broad, relatively simple heat capacity peaks (peak temperatures: 19.9 degrees C for DMPC, 37.8 degrees C for DPPC) free of high-temperature spikes or shoulders. Subsequent heating scans displayed a sharp peak characteristic of previously described fusion products formed below the phase transition. SUV samples stored for 1 or more days above their phase transition displayed a moderately broad, high-temperature shoulder (23.8 degrees C for DMPC and 40.2 degrees C for DPPC) in the cooling profile. For DMPC, the enthalpy associated with this peak increased in a first-order fashion with time. Hydrolysis products were not detected until 12-20 days of storage. Both the rate and extent of shoulder appearance increased with temperature (k = 0.0017 h-1, fraction of total enthalpy = 0.1 at 36 degrees C; k = 0.0037 h-1, fraction = 0.2 at 42 degrees C). Freeze-fracture electron micrographs confirmed that an intermediate-sized vesicle population (diameters 400-500 A) appeared in SUV samples stored above their phase transition. Also, the trapped volume of DMPC SUV increased from 0.26 microL/mumol after 17 h of storage to 0.54 microL/mumol after storage for 16 days at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles.

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase behavior of membranes reconstituted from dipentadecanoylphosphatidylcholine and the Mg2+-dependent, Ca2+-stimulated adenosinetriphosphatase of sarcoplasmic reticulum: evidence for a disrupted lipid domain surrounding protein.

A new method was used for reconstituting active sodium deoxycholate solubilized Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Removal of the detergent by dialysis at the pretransition temperature of the pure lipid (22 degrees C) favored the formation of sheet-like structures with a lipid and protein content close to that of the detergent-solubilized sample. Freeze-fracture electron micrographs revealed the Ca2+-ATPase to be organized in rows corresponding to the typical banded pattern seen in low-temperature freeze-fracture micrographs of pure lipid bilayers. Incubation of the sheetlike structures at a temperature (38 degrees C) above the pure lipid main phase transition (33.5 degrees C) caused closure of the sheets into vesicles displaying homogeneous intramembranous particle distributions, at least for membranes containing less than 150 lipids per Ca2+-ATPase. However, in membranes of higher lipid content, free lipid patches were seen both above and below the lipid phase transition. By use of high-sensitivity differential scanning calorimetry, three classes of excess heat capacity peaks were observed in the vesiculated samples. A broadened "free lipid" peak occurred for samples containing between 550 and 200 lipids per protein (Tm = 33.5 degrees C, as for the order-disorder transition in pure lipid vesicles). Between 200 and 150 lipids per Ca2+-ATPase, a broad shoulder became apparent in the range of 29-32 degrees C. Below 150 lipids per Ca2+-ATPase, a peak at 26-28 degrees C became increasingly prominent with lower lipid content. At a lipid to protein ratio of about 30, no peaks in heat capacity were observed. The temperature dependence of diphenylhexatriene fluorescence anisotropy revealed a similar pattern of membrane phase behavior, except that a phase transition was detected at 33.5 degrees C in all membranes studied. On the basis of these observations, we propose that the Ca2+-ATPase is surrounded by a "lipid annulus" of motionally inhibited lipid molecules that do not contribute to a calorimetrically detectable phase transition. Beyond the annulus, "secondary domains" of disrupted lipid packing account for the peak at 26-28 degrees C and the 29-32 degrees C shoulders. At high lipid to protein ratios, the secondary domains coexist with protein-free, lipid-bilayer patches, which account for the peak at 33.5 degrees C.

Phase behavior of mixed phosphatidylglycerol/phosphatidylcholine multilamellar and unilamellar vesicles.

The phase behavior of dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) mixtures has been studied in both small, unilamellar vesicles and large, multilamellar vesicles. We have used both the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and high-sensitivity differential scanning calorimetry to detect temperature-dependent changes in membrane structure. Electron microscopy has demonstrated different fracture face morphologies for large, multilamellar vesicles depending on sample composition and temperature. These data have been interpreted in terms of proposed phase diagrams for this lipid mixture. The shapes of the proposed phase diagrams have led us to conclude that DMPC and DC15PG mix freely in the plane of a lipid bilayer only at less than 50 mol % DC15PG. At higher DC15PG content, the data have been interpreted as reflecting substantial compositional inhomogeneities in the plane of the bilayer, if not phase immiscibility, even in the fluid phase. In addition, small vesicles containing greater than 50 mol % DC15PG were unstable in the ordered phase and spontaneously converted to larger vesicles. Finally, the anisotropy of DPH fluorescence was found to be invariant with DC15PG content at temperatures just above the liquidus phase line in small, unilamellar vesicles. This demonstrated that inclusion of negatively charged phosphatidylglycerol does not noticeably affect the order within the acyl chain region of the bilayer, relative to phosphatidylcholine.