Specific interaction of myeloma tumor cells with hapten-bearing liposomes containing methotrexate and carboxyfluorescein.

Hapten-bearing liposomes containing methotrexate and the fluorescent solute carboxyfluorescein were incubated with murine myeloma tumor cells expressing surface immunoglobulin with affinity for the hapten. Liposomes bearing the dinitrophenyl hapten became bound to MOPC 315 myeloma tumor cells, and liposomes bearing the phosphorylcholine hapten became bound in much larger amounts to TEPC 15 cells. In each case, fluorescence microscopy showed a patchy surface pattern, indicating intact liposomes at the cell surface. Few liposomes were bound to cells in the presence of excess soluble hapten or to cells lacking the relevant surface immunoglobulin. Cell-associated liposomes were quantitated by use of the fluorescence-activated cell sorter, and the pharmacological effect of the methotrexate was assessed from measurement of incorporation of radiolabeled deoxyuridine into the cells. Little inhibition of deoxyuridine incorporation was observed, because contents of the bound liposomes did not enter the cytoplasmic compartments of the cells.

Small liposomes are better than large liposomes for specific drug delivery in vitro.

We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 A, 2000 A or 4000 A, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2Kk monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 A vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes.

Weak acid-induced release of liposome-encapsulated carboxyfluorescein.

Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes' aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form: 9 X 10(-9) cm X s-1), acetic acid (greater than 1 X 10(-7) cm X s-1) and glycine (cation: 6 X 10(-9) cm X s-1). These results are consistent with low effective proton permeability of liposomes (less than 5 X 10(-12) cm X s-1) and with the permeability coefficient of HCl (3 X 10(-3) cm X s-1) reported by Nozaki and Tanford ( (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324-4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.

Immunopurification and insertion into liposomes of native and mutant H-2Kb: quantification by solid phase radioimmunoassay.

To study the interaction between T cells and isolated H-2Kb, we developed protocols for the immunopurification of the molecule from monoclonal anti-H-2Kb immunoadsorbent columns and for its insertion in lipid vesicles. Patterns of reactivity of two anti-H-2Kb monoclonal antibodies (mAb) (20-8-4 and Y3) on H-2 recombinant and H-2Kb mutant mice indicated that mAb Y3 reacted with all six mutant forms of H-2Kb tested. Binding competition studies indicated that Y3 and 20-8-4 recognized distinct epitopes of H-2Kb. A solid phase radioimmunoassay was established using these two mAb to monitor H-2Kb activity in detergent containing cell lysates, after immunopurification, and after insertion into liposomes. About 70% of H-2Kb activity could be eluted from anti-H-2Kb-immunoadsorbents in the presence of 3 M NH4SCN and octyl glucoside (pH 7.4). A procedure of liposome formation combining gel dilution and dialysis yielded liposomes bearing H-2Kb molecules which could inhibit antibody plus complement cytolysis and could stimulate in vivo primed T cells to generate cytotoxic T lymphocytes in vitro. The present protocol can be extended to immunopurify and obtain H-2Kbm1 bearing liposomes.

Immune clearance of liposomes inhibited by an anti-Fc receptor antibody in vivo.

In a study designed to evaluate the potential for in vivo manipulation of the circulation and tissue distribution of injected liposomes, mice were passively injected with antidinitrophenyl (anti-DNP) monoclonal antibodies of the IgG2a or IgG2b subclasses or were immunized with the nitrophenyl hapten bound to a protein carrier. They were then injected i.v. with 125I- and carboxyfluorescein-labeled, DNP-bearing liposomes. Circulation time of the DNP-bearing liposomes was markedly reduced in actively and passively immune mice, with increased deposition of liposomes in the liver. The increased clearance of liposomes could be abrogated by injection of a monoclonal antibody directed against the murine IgG Fc receptor (2.4G2). The results suggest that clearance of ligand-bearing reagent in the face of an immune response may be modified by specific immunologic manipulation in vivo.

Elimination or rescue of cells in culture by specifically targeted liposomes containing methotrexate or formyl-tetrahydrofolate.

Methotrexate (Mtx) inhibits cellular proliferation by blocking formyl tetrahydrofolate (THF) production. Mtx- or THF-containing liposomes were covalently coupled to Staphylococcus aureus protein A. These liposomes bound specifically in vitro to cells pre-incubated with relevant monoclonal antibodies. When liposomes contained Mtx, targeted cells died. When liposomes contained THF, targeted cells survived in the presence of quantities of Mtx in the medium sufficient to eliminate non-targeted cells. These negative or positive selection liposomes allow the recovery of either a small proportion of cells not bearing a given antigen, or a small proportion of cells bearing a given antigen, from heterogeneous cell populations.

A calmodulin antagonist increases the apparent rate of endocytosis of liposomes bound to MHC molecules via monoclonal antibodies.

We have investigated the molecular mechanisms required for endocytosis of MHC-encoded proteins by a cell line, TRH 42, that expresses endogenous murine and introduced human class I molecules. As probes we have used protein A-bearing liposomes which bind to cell surface determinants via monoclonal antibodies. The technique of fluorescence quenching release was used with liposome encapsulated quenched carboxyfluorescein as the marker for endocytosis. We demonstrate that the calmodulin antagonist trifluoperazine (TFP) enhances the apparent rate of endocytosis of liposomes bound to MHC class I molecules. Drugs that interfere with energy metabolism, microfilament organization, or phospholipase A2 activity all block endocytosis both in the presence and absence of TFP. The requirement of extracellular Ca2+ for endocytosis was found to be partial. The implications for the structural and enzymatic requirements of endocytosis of MHC class I molecules are discussed.

Interferon sensitive and insensitive MHC variants of a murine thymoma differentially resistant to methotrexate-containing antibody-directed liposomes and immunotoxin.

We evaluated the ability of methotrexate-containing liposomes or a ricin alpha-chain immunotoxin, both associated with monoclonal antibodies specific for the major histocompatibility complex-encoded class I molecule H-2Kk, to kill cells of the murine k haplotype thymoma RDM4. Cells were incubated with liposomes or immunotoxin in the presence or absence of interferon-gamma, which is known to augment the expression of the target class I molecules. The great majority of cells were killed by either of these reagents. Two types of mutant cells were obtained: type 1 cells, selected by methotrexate-containing liposomes, failed to express sufficient target H-2k molecules to be killed by liposomes in the absence of interferon-gamma. In the presence of interferon-gamma, these cells increased expression of all H-2 class I molecules and could be killed by targeted liposomes. Type 2 cells were immunoselected from cloned type 1 cells by liposomes in the presence of interferon. These cells failed to respond to interferon with expression of the H-2Kk molecule, but continued to augment H-2Dk expression in response to interferon. A third variant (type 3) selected from the wild type population by an H-2Kk specific immunotoxin in the absence of interferon phenotypically resembled type 1 cells. Type 1 but not type 2 cells respond to interferon by augmented synthesis of H-2Kk specific mRNA. The results suggest that for interferon-sensitive cell surface molecules of tumor cells, use of interferon improves the efficacy of targeted chemotherapy, but does not prevent development of mutants lacking the target molecule.

Monoclonal antibody covalently coupled to liposomes: specific targeting to cells.

We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar liposomes. Coupling of an IgG2 alpha monoclonal anti-beta 2-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter:580 A). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human beta 2-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigens specificity confirm that the technique can be generally applied.

Antiviral activity in L1210 cells of liposome-encapsulated (2'-5')oligo(adenylate) analogues.

Evidence is available for a role of a (2'-5')(A)n-activated endoribonuclease (RNase L) in the antiviral activity of interferon for several RNA viruses. (2'-5')(A)n and their analogues might thus provide an interesting alternative to exogenous interferons or their inducers in antiviral chemotherapy. In addition, the evaluation of the activity of (2'-5)(A)n as mediators of interferon's biological activities or as cell growth regulators requires biochemical studies using agonists or antagonists of the system. Non-disruptive techniques for the introduction of (2'-5')(A)n and their analogues into cell lines or tissues are required for these studies since these highly charged compounds are cell impermeable. (2'-5')(A)n oligomers and analogues of increased stability towards phosphodiesterases were derived by chemical modification of their 2' end and encapsulated in protein-A-bearing liposomes. The specific delivery of liposome contents into L1210 mouse leukemic cells was achieved with the help of monoclonal antibodies directed against the appropriate class I major histocompatibility complex-encoded proteins expressed by these cells. This intracellular delivery led to transient inhibition of protein synthesis and an antiviral activity, both compatible with activation of RNase L. This activity was enhanced for the analogues designed to resist degradation, with respect to the natural product.

Differential endocytosis of T and B lymphocyte surface molecules evaluated with antibody-bearing fluorescent liposomes containing methotrexate.

Antibody-bearing fluorescent liposomes containing methotrexate became bound to cells expressing determinants recognized by the antibody. The number of bound liposomes could be evaluated by fluorometry, and the internalization of liposomes was evaluated by the methotrexate-mediated inhibition of radio-labeled deoxyuridine incorporation. The effect of methotrexate transferred from the liposomes into the cells was a function not of the number of liposomes bound but of the nature of the cells and of the target molecules. Liposomes bearing antibodies with specificity for the H-2K or Mr 94,000 and 180,000 molecules were much more effective at drug delivery into T than B cells, even though these determinants were expressed by both cell types. B cells were more sensitive to the effect of methotrexate in anti-H-2 I-A and I-E liposomes than in anti-H-2K liposomes. Inhibition of the methotrexate effect by NH4Cl suggested that methotrexate entered the cell by endocytosis of the liposomes. The results are consistent with differential internalization of H-2K, I-A, I-E, and Mr 94,000 and 180,000 cell surface molecules by mitogen-stimulated T and B cells.

Drug transfer into lymphoblasts mediated by liposomes bound to distinct sites on H-2 encoded I-A, I-E, and K molecules.

Protein A from S. aureus was covalently coupled to small sonicated liposomes containing methotrexate (MTX) and carboxyfluorescein (CF). These liposomes were incubated with mitogen-stimulated CBA T or B cells preincubated with one of a panel of murine IgG2 monoclonal antibodies directed at distinct sites on H-2Kk, I-Ak, or I-Ek molecules. Liposomes became bound to the cells as a consequence of the interaction of protein A on the liposome and antibody on the cell. The number of bound liposomes was measured by fluorescence, and the endocytosis of liposomes was evaluated by the MTX-mediated reduction in radiolabeled deoxyuridine incorporation. T cells were sensitive to MTX in liposomes bound to all anti-H-2Kk antibodies tested, but B cells were essentially insensitive, even though they expressed more H-2Kk, bound more liposomes directed at this molecule, and were equally sensitive to free MTX. By contrast, B cells were sensitive to liposome-encapsulated MTX when liposomes were bound to any antibody directed at I-Ak. The situation was more complex with respect to I-Ek. Antibodies directed at one part of the molecule were quite effective at drug delivery, while antibodies directed at other, spatially separate domains were much less effective, even though the number of liposomes bound to these various determinants was the same. The results suggest differential regulation of the internalization of H-2K by T and B cells, and are compatible with the existence of functionally distinct I-E molecules.

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.

Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A.

Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human beta 2-microglobulin [antibody B1.1G6 (IgG2a, kappa) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

Endocytosis of HLA and H-2 molecules on transformed murine cells measured by fluorescence dequenching of liposome-encapsulated carboxyfluorescein.

We have studied internalization of cell surface proteins encoded by genes of the human major histocompatibility complex (HLA) transferred into murine L cells, in comparison with mouse (H-2) histocompatibility determinants. This internalization was measured by the use of monoclonal antibodies directed at these determinants, to which were bound methotrexate- and carboxyfluorescein-containing liposomes covalently coupled to protein A. In addition to the effect of methotrexate on the cells, the technique of fluorescence self-quenching release was used to measure the kinetics of internalization for single cells using a flow cytofluorometer. The native and foreign gene products were internalized in an apparently identical manner. These techniques can be applied to cells expressing genes with altered or deleted segments thus providing a basis for the analysis of the effect of sequence modifications on the internalization of the encoded molecule.

Receptor-mediated endocytosis of antibody-opsonized liposomes by tumor cells.

Specific receptor-mediated delivery of the contents of small, sonicated liposomes was studied with three murine tumor cell types: an IgG Fc receptor-negative nonphagocytic line (EL4); an Fc receptor-positive phagocytic line (P388D1); and an Fc receptor-positive nonphagocytic line (P388). The liposomes (formed from phosphatidylcholines, cholesterol, and dinitrophenyl-substituted phosphatidylethanolamine) contained carboxyfluorescein as a fluorescent marker and methotrexate as a pharmacologic agent. Binding and internalization of the liposomes were observed by fluorescence microscopy and measured by flow microfluorometry. The hapten-derivatized lipid was used as a binding point on the liposome for the antibody-combining site of the immunoglobulin. In the presence of IgG anti-dinitrophenyl, but not F(ab')2 or IgA anti-dinitrophenyl, liposomes bound to the Fc receptor-bearing cells. The liposomes underwent endocytosis by the P388D1 cells and, to a lesser extent, by the P388 cells. As measured by depression of [3H]deoxyuridine incorporation, methotrexate in IgG-opsonized liposomes had a much greater pharmacologic effect on the P388D1 cells than did the same amount in unopsonized liposomes or in free solution. This observation indicates that an appropriately chosen drug, incorporated in liposomes, can exert its effect on a cytoplasmic target after endocytosis. P388 cells showed a moderate effect of the drug in liposomes. Neither P388 nor P388D1 cells bound or ingested unopsonized liposomes, and the Fc receptor-negative EL4 line neither bound nor ingested opsonized liposomes. The data demonstrate specific interaction of opsonized liposomes with the cells' IgG Fc receptor.

Endocytosis of liposomes bound to cell surface proteins measured by flow cytofluorometry.

A new technique for the quantification of cellular receptor-mediated endocytosis has been developed based on the analysis by flow cytometry of ligand-bearing liposomes containing the fluorochrome carboxyfluorescein. Carboxyfluorescein encapsulated at high concentrations in protein A-bearing liposomes is self-quenched. Binding and internalization of such liposomes by cells via antibodies directed towards membrane surface determinants results in the release of the liposome-encapsulated carboxyfluorescein into the cytoplasm causing an increase in cell-associated fluorescence. This increase can be quantified on a flow cytofluorometer.