Controlled binding of liposomes to cultured cells by means of lectins

Lectin-mediated binding of liposomes to Hela cells was analyzed as a function of different parameters. We show that the amount of lectin covalently bound to liposomes can be accurately controlled. We chose to work with 500 - to 1 000 molecules of WGA bound per liposome of 1 micron diameter. These liposomes bound very efficiently to Hela cells as demonstrated by fluorescent microscopy, and fluorescent cell-sorting. We show that the number of liposomes bound is proportional to the input, over a wide range of concentrations. The liposomes bound very tightly to cells and could not be removed by trypsin or N-acetylglucosamine, which competes with WGA binding.     

Antibodies to phospholipids and liposomes: binding of antibodies to cells

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.     

The binding of thyrotropin to liposomes containing gangliosides.

The relationship between uptake of Ca⁺⁺ and incorporation of sn-[¹⁴C]-glycerol-3-phosphate into phosphatidate, diglyceride, and triglyceride was evaluated in microsomes isolated from livers of normal fed male rats. Uptake of Ca⁺⁺ was dependent on concentration of Ca⁺⁺ (0.1 – 2.5 mM), and accompanied by a decrease in the rate of glycerolipid synthesis. The quantity of Ca⁺⁺ ion taken up at 20 μM CaCl₂ in the presence of ATP was equivalent to that observed with 2.5 mM CaCl₂ in the absence of ATP. The ATP dependent uptake of Ca⁺⁺, like the passive uptake at higher concentrations of Ca⁺⁺, was correlated with inhibition of incorporation of sn-glycerol-3-phosphate into phosphatidate. Accumulation of Ca⁺⁺ in hepatic microsomes, therefore, appears to result in a calcium-dependent decrease in biosynthesis of phosphatidate and other glycerolipids.     

Increased binding and killing of neuraminidase-galactose oxidase-treated tumor cells by normal macrophages.

The binding of Line 10 hepatoma cells to normal syngeneic guinea pig macrophages is increased when the tumor cells are treated with neuraminidase and galactose oxidase (NAGO) before they are added to the macrophage monolayers. The effect is abolished by exposure of the NAGO-treated tumor cells to sodium borohydride. Line 1 hepatoma cells treated with NAGO or with sodium periodate are killed to a greater extent than untreated tumor cells. This effect can also be reversed by sodium borohydride. Further, periodate-treated macrophages become cytotoxic for unmodified tumor cells. These results demonstrate that increased tumor cell killing occurs when artificial contacts (presumably via Schiff bases) are established between normal macrophages and tumor cells. They are consistent with the hypothesis that close cell to cell contact is necessary for macrophage-mediated cytotoxicity.     

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G_M1 ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G_M1 ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G_M1-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.28 μg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 μg/ml applied CTB could be observed under the fluorescence microscope.     

Trypsin purification by affinity binding to small unilamellar liposomes

A novel protein purification process using affinity-ligand-modified liposomes and membrane ultrafiltration is described. The feasibility of the process was tested using trypsin as the model protein and p-aminobenzamidine (PAB) as the affinity ligand for trypsin. The affinity liposomes were prepared by covalently attaching PAB to the surface of small unilamellar liposomes via the hydrophilic spacer arm diglycolic acid. The liposomes were comprised of dimyristoyl phosphatidyl choline, cholesterol, and dimyristoyl phosphatidyl ethanolamine to which the diglycolic acid was attached. The equilibrium binding constant between trypsin and immobilized PAB was shown to be dependent on the PAB density of the liposome surface. Bound trypsin was eluted from the liposomes by the trypsin inhibitor benzamidine. Trypsin was purified from a trypsin/chymotrypsin mixture and from one of its naturally occurring sources, porcine pancreatic extract. A recovery yield from the crude mixture of 68% was obtained with a trypsin purity of 98%. The affinity-modified liposomes were stable in the complex mixture and retained their trypsin binding capacity after multiple adsorption/elution cycles over a 30-day period.     

Increased binding of tumor cells by macrophages activated in vitro with lymphocyte mediators.

Following incubation in vitro with lymphocyte mediators, activated macrophages become capable of binding more tumor cells than nonactivated macrophages. Increased binding occurs rapidly (within 1 hr), does not require the presence of serum in the medium, and is inhibited by treatment with trypsin. The increased binding by activated macrophages is quantitatively selective for tumor cells. Incubation with lymphocyte mediators of cell types other than macrophages does not increase the binding of tumor cells to such monolayers. These results indicate that the binding of tumor cells by activated macrophages results from the stimulation of a specific macrophage function during the process of macrophage activation.     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

Iron binding to alpha-tocopherol-containing phospholipid liposomes

Tocopherols (vitamin E) located in the hydrophobic domains of biological membranes act as chain breaking antioxidants preventing the propagation of free radical reactions of lipid peroxidation. The naturally occurring form, d-alpha tocopherol is an exquisite molecule in that it is intercalated in the membrane in such a way that the hydrophobic tail anchors the molecule positioning the chromanol ring containing the hydroxyl group, which is the essence of its antioxidant function, at the polar hydrocarbon interface of phospholipid membranes. The interaction of this group with water soluble substances is not very well understood. In the present study, an investigation was made of the interaction of ascorbate and ferrous ions (Fe+2) initiators of lipid peroxidation with alpha tocopherol. The results show that tocopherol increases membrane associated iron. The formation of a tocopherol iron complex in the presence of phospholipid liposomes and ascorbate in its reduced form is indicated. These results suggest a new way in which tocopherols act to inhibit lipid peroxidation.     

Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells

Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.     

Microelectrophoresis studies of the binding of glycosaminoglycans to phosphatidylcholine liposomes.

The binding of the glycosaminoglycans (GAG) chondroitin sulfate and heparin and the homologous molecule dextran sulfate to multilamellar dimyristoyl phosphatidylcholine (DMPC), dilaureyl phosphatidylcholine (DLPC) and egg lecithin liposomes was investigated by microelectrophoresis measurements. Drastic changes of the zeta potential of the liposomes to negative values indicate the binding of the highly anionic macromolecules. Binding depends strongly on Ca2+ and NaCl concentrations in the medium and does not occur in the absence of Ca2+. The adsorption is saturated at concentrations of about 0.1 mg/ml chondroitin sulfate and heparin and 0.01 mg/ml dextran sulfate. In the gel state of the phospholipid bilayer more GAG can associate with the surface compared to the fluid state.     

The fluorescence study of the binding of zinc phthalocyanine to breast cancer cells solubilized in synthetic liposomes.

Steady state fluorescence spectroscopy is used to study the binding characteristics of zinc phthalocyanine (ZnPc) to MCF-7 human breast cancer cells solubilized in a liposomal vesicle prepared with L-alpha-phosphatidic acid, dipalmitoyl. The observed apparent binding constant, K', of 1.14 x 10(7) together with the free energy of binding, deltaG, of -40.38 kJ/mole suggests a very strong affinity and spontaneous binding between the breast cancer cells and ZnPc. The wavelength of excitation of ZnPc in the liposomal vesicle (611 nm) is favorable to cytotoxic reactive singlet oxygen (1O2*) production necessary for photooxygenation reaction with the cancerous cells and is within the energy threshold that has good penetration to normal tissues without undue skin necrosis.     

Characteristics of binding of zwitterionic liposomes to water-soluble proteins

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to ³¹P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).