Interactions of immunoliposomes with target cells

We have covalently attached a monoclonal antibody (11-4.1) against the murine major histocompatibility antigen, H-2Kk, on the surface of liposomes. The interaction of these antibody-coated liposomes (immunoliposomes) with target cells, RDM-4 lymphoma (H-2Kk), was investigated. About 90% of the immunoliposomes taken up by target cells at 4 degrees C could be removed by a mild protease treatment of the cells, whereas only 30% of the uptake at 37 degrees C was labile to the same treatment. Furthermore, the uptake of immunoliposomes at 37 degrees C was inhibitable by cytochalasin B or by a combination of 2-deoxyglucose and NaN3. These results suggest that immunoliposome binding to the target cell surface is the primary uptake event at 4 degrees C and that the surface-bound liposomes are rapidly internalized by the cells at 37 degrees C, probably via an endocytic pathway. Studies with fluorescence microscopy of target cells treated with immunoliposomes containing carboxyfluorescein also supported this conclusion. If endocytosis is the mechanism by which immunoliposomes gain entry into target cells, the efficacy of a cytotoxic drug encapsulated would depend on the resistance of the drug to lysosomal inactivation and its ability to escape from the lysosomal system. Consistent with this notion, we observed that methotrexate encapsulated in liposomes bearing 11-4.1 antibody specifically inhibited deoxy[6-3H]uridine incorporation into DNA in target RDM-4 cells but not in P3-X63-Ag8 myeloma cells (H-2Kd) at the same doses. The observed cytotoxic effect of encapsulated methotrexate could be reversed by the treatment of cells with a lysosomotropic amine, chloroquine, which has been shown to increase the intralysosomal pH of mammalian cells. On the other hand, cytosine-beta-D-arabinofuranoside encapsulated in immunoliposomes showed no target-specific killing, probably because the drug is readily inactivated in the lysosomal system. These results are discussed in terms of the drug carrier potential of immunoliposomes.     

Incorporation of acylated antibody into planar lipid multilayers: characterization and cell binding

Multiple (up to 14) layers of lipid were deposited onto an alkylated glass surface by dialysis of egg phosphatidylcholine (PC) and deoxycholate mixed micelles in the presence of alkylated glass coverslips. The amount of lipid associated with the coverslips was measured by using radioactive PC. It was found that the number of PC molecules in the multilayer increased with increasing initial lipid concentration in the dialysis mixture. Inclusion of cholesterol resulted in a significant increase in the amount of total lipid deposited in the multilayer. However, the PC/cholesterol ratio was up to 2-fold higher in the multilayers than in the liposomes present in the same dialysis bag. In addition, mouse monoclonal anti-H2Kk antibody which had previously been derivatized with palmitic acid could be readily incorporated into the lipid multilayer during dialysis. Measurements of lateral mobility with the fluorescence recovery after photobleaching technique on fluorescently labeled lipid or antibody in the multilayer showed that the lipid molecules diffused rapidly while the antibodies were essentially immobile. Lymphoma cells such as RDM4 cells expressing surface H2Kk glycoproteins could rapidly bind to the antibody-containing multilayers. The binding was blocked by free antibody or by goat anti-mouse immunoglobulin G, indicating the immunospecificity of the binding. Cell binding to the multilayer also exhibited a threshold dependence on the antibody density of the multilayer. A lower threshold was found for cells expressing a higher surface density of H2Kk. This system may be useful for model studies of cellular recognition.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and characterization of heat-sensitive immunoliposomes

Immunoliposomes able to bind specifically to target cells and to release their encapsulated contents upon brief heating were prepared. Monoclonal anti-H2Kk was covalently derivatized with palmitic acid by the method of Huang, A. et al. (Huang, A., Tsao, Y.S., Kennel, S.J. and Huang, L. (1982) Biochim. Biophys. Acta 716, 140-150). The palmitoyl antibody was injected at a controlled rate into a suspension of fused unilamellar dipalmitoylphosphatidylcholine liposomes maintained at a constant temperature. The final protein-to-lipid ratio of the resultant liposomes with incorporated antibody (immunoliposomes) was dependent upon the rate of antibody injection and the lipid concentration. Injection of palmitoyl antibody into a liposome suspension containing 50 mM carboxyfluorescein at 41 degrees C resulted in simultaneous antibody incorporation and entrapment of dye. Immunoliposomes were able to release the entrapped carboxyfluorescein upon heating. The release of dye at temperatures between the pre- and main-transition temperatures of DPPC was abolished by the addition of calf serum (5%). Furthermore, the presence of serum resulted in an increase in the temperature of the maximal release rate and also in the rate of release at that temperature. Retention of antigen-binding capacity was demonstrated by the ability of the immunoliposomes to bind specifically to the target cells. Rapid release of entrapped carboxyfluorescein from immunoliposomes bound to target cells at 4 degrees C was achieved upon brief exposure (less than 3 min) at 41 degrees C. These heat-sensitive immunoliposomes may be useful in enhancing drug delivery to target cells.     

Targeting of drug loaded immunoliposomes to herpes simplex virus infected corneal cells: an effective means of inhibiting virus replication in vitro

The goal of our studies was to develop liposomes containing antiviral drugs and targeted with antiviral antibody (immunoliposomes) that would be effective at inhibiting replication of herpes simplex virus (HSV) in vitro. To achieve this, a monoclonal antibody to glycoprotein D of HSV was derivatized with palmitic acid and was incorporated into the lamellae of dehydration-rehydration vesicles. The gD containing immunoliposomes were shown to bind specifically to HSV-infected rabbit corneal cells in vitro, whereas control immunoliposomes prepared with a monoclonal antibody of the same class as the anti-gD failed to preferentially bind to virus-infected cells. The gD immunoliposome binding was inhibitable by pretreatment with rabbit anti-HSV serum but not by aggregated normal serum. Thus liposome binding was judged to represent an antigen-antibody reaction not binding to Fc receptors expressed by cells infected with HSV. Immunoliposomes loaded with iododeoxyuridine (IUDR) leaked drug rapidly at 37 degrees C, whereas acyclovir (ACV)-loaded liposomes still contained 48% of drug after 24 hr at 37 degrees C. The ACV-liposomes retained 44% of drug after 14 days at 4 degrees C. The ability of immunoliposomes to inhibit virus replication was compared with that of untargeted and empty liposomes by means of virus yield assays in vitro, Immunoliposomes loaded with either IUDR or ACV inhibited virus replication, although ACV-containing immunoliposomes were the most efficacious. The implications of our in vitro results for the development of immunoliposomes suitable for the treatment of ocular herpes infection are briefly discussed.     

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.     

Antibody-directed liposomes Determination of affinity constants for soluble and liposome-bound antifluorescein

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.     

Preparation and characterization of immunoliposomes for targeting of antiviral agents

Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission spectra of palmitoylated IgG have exhibited a shift in emission maximum from 330 to 370 nm when it was incorporated into the liposomes. At least 50% of the incorporated antibody molecules were found to be oriented towards the outside in the liposomes. The average size of the liposome was found to be 300 A, and on an average, 15 antibody molecules were shown to be present in a liposome. When adriamycin encapsulated in immunoliposomes was incubated in a medium containing serum for 72 h, about 75% of the drug was retained in liposomes. In vivo localization studies, revealed an enhanced delivery of drug encapsulated in immunoliposomes to the target tissue, as compared to free drug or drug encapsulated in free liposomes. These data suggest a possible use of the drugs encapsulated in immunoliposomes to deliver the drugs in target areas, thereby reducing side effects caused by antiviral agents.     

Stable target-sensitive immunoliposomes.

Interaction of immunoliposomes composed of dioleoylphosphatidylethanolamine (DOPE) (80%), dioleoylphosphatidic acid (DOPA) (20%), and a small amount of specific antibody with Herpes Simplex virus (HSV) were studied by detecting the immune-dependent lysis of liposomes. DOPA was used as the principal stabilizer of the immunoliposomes. Antibodies conjugated with N-glutarylphosphatidylethanolamine or oxidized GM1 served as the target-specific ligands of immunoliposomes. These immunoliposomes (d = 160-180 nm) were stable for at least one month when stored at 4 degrees C. However, they undergo a rapid aggregation and lysis reaction in the presence of a membrane-bound target such as intact HSV virions. We have also employed epitope peptide-containing liposomes (target liposomes) to mimic the virus and showed that the immunoliposomes could be aggregated and lysed by the target liposomes in an antigen-dependent manner. Immunoliposome lysis could be accelerated by increasing the incubation temperature to 60-70 degrees C. No immunoliposome lysis was observed if the target liposomes were absent, indicating the prolonged stability of the immunoliposomes. Liposome lysis was always accompanied by liposome aggregation. However, the aggregation-induced liposome destabilization is unique to the HII phase-forming lipids such as DOPE. DOPC-containing immunoliposomes did not lyse despite the fact that massive liposome aggregation had taken place.     

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.     

Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells

In the present study the cellular uptake of targeted immunoliposomes by interleukin-1 activated human endothelial cells has been analysed by several spectroscopical and microscopical fluorescence techniques. Previous in vitro experiments demonstrated that the targeting of immunoliposomes to vascular selectins is a potential way for a selective drug delivery at inflammatory sites. In attempts to further adapt the targeting experiments to physiological conditions, we demonstrate that E-Selectin-directed immunoliposomes are able to bind their target cells under the simulated shear force conditions of capillary blood flow cumulatively for up to 18 h. In order to consequently follow the fate of liposomes after target binding, we analysed the route and degree of liposome internalization of the cells concentrating on cell activation state or various liposomal parameters, e.g., sterical stabilization, type of antibody or antibody coupling strategy. The use of NBD-labelled liposomes and subsequent fluorescence quenching outside the cells with dithionite show that circa 25% of the targeted immunoliposomes were internalized. According to inhibition experiments with agents that interfered with the endocytotic pathway, we found out that the major mechanism of liposome entry is endocytic. The entry involves, at least in part, receptor-mediated endocytosis via E-Selectin, a liposome accumulation in the endosomes and their acidification was proved by pyranine spectroscopic results. Furthermore, microscopical investigations demonstrate that also a fusion of liposomes with the cell membrane occurs followed by a release of entrapped calcein into the cytoplasm. These observations gain insight into the behaviour of E-Selectin-targeted immunoliposomes and indicate that these immunoliposomes have great potential to be used as drug carriers for intracellular drug delivery at inflammatory sites.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. II: Biotinylated liposomes and antibiotin antibody

The aggregation of biotinylated phospholipid vesicles (liposomes) cross-linked by antibiotin IgG was studied experimentally and theoretically. The liposomes were either low density liposomes that contained 0.4 mol% biotinylated phospholipid ( approximately 100 exposed biotin molecules per liposome), or high density liposomes that contained 2.7 mol% biotinylated phospholipid ( approximately 1000 exposed biotin molecules per liposome). The solution turbidity and mean particle size measured by quasi-elastic light scattering (QLS) were monitored throughout the aggregation. Three different lots of antibiotin antibodies, each with different association constants and binding heterogeneities, were used. The antibody binding characteristics affected the aggregation rates. The aggregation kinetics were analyzed using a model based on the Smoluchowski theory of aggregation, fractal concepts of aggregate microstructure, and Rayleigh and Mie light scattering theory. The experimental conditions of liposome concentration, protein concentration, and ligand density under which aggregation occurred correlated well with calculated sticking probabilities based on isotherms describing the adsorption of antibiotin antibody to the liposomes. These results are compared with prior observations made when avidin was used as the cross-linking protein. (c) 1996 John Wiley & Sons, Inc.     

Interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. II. Levels of expression of H-2K, H-2D, and H-2L in different mouse strains

The numbers of MHC class I molecules expressed by spleen cells from various mouse strains were determined by using MHC-specific monoclonal antibodies and a radioactive binding assay. Although small differences were found to exist in some cases, our general conclusion is that different mice of the same strain, congenic mice of different haplotypes, and syngeneic mice of varying background all express similar numbers of class I antigens. B10.A mice (8 to 10 wk old), for example, express 5.3 X 10(4) Kk molecules/cell, 5.4 X 10(4) Dd molecules/cell, and 2.2 X 10(4) Ld molecules/cell. Some of the differences observed in class I antigen expression included: 1) the level of Kk expression increased to a small but significant extent with age in B10.A mice; 2) female B10.A mice expressed slightly higher amounts of Kk than male mice; and 3) B10.A(2R) and B10.A(4R) recombinant strains expressed elevated levels of K-end antigens and slightly decreased levels of D-end antigens when compared with the unrecombinant B10.A strain. In several strains, F1 mice express approximately 50% as many copies of each parental antigen as do the homozygous parents. B10 mice, which are negative for the L antigen, nevertheless express the same total number of D-end molecules as do B10.A mice. The data suggest that the levels of expression of MHC class I molecules are controlled by at least two factors: gene dosage and another factor(s) that gives rise to the small variations in class I antigen expression seen with age, sex, and strain, and to the low expression of Ld relative to Dd and Kk.     

Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.     

Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies

In an attempt to delineate spatial relationships between various allodeterminants of cell surface MHC antigens, competitive binding studies were performed using 4 different monoclonal antibodies, each of which reacted with the H-2Kk antigen but with different serologic specificities. Competition was studied by examining the effect of unlabeled antibodies on the binding of each 125I-labeled antibody to spleen cells of the H-2a haplotype. Mutual inhibition was observed between 2 of the antibodies, and a 3rd antibody of lower affinity was inhibited by the first 2 antibodies but did not itself inhibit the binding of these antibodies. The 4th antibody did not block the binding of the other 3 labeled antibodies, and binding of this 4th labeled antibody was only partially inhibitable by the other 3 antibodies. These results indicate the presence of at least 2 spatially distinct allodeterminants on H-2Kk molecules expressed on the cell surface.     

Purification of the H-2Kk molecule of the murine major histocompatibility complex.

The intact H-2Kk antigen has been detergent-solubilized and purified using an immunoabsorbent column prepared from the 11-4.1 monoclonal antibody described by Oi et al. (Oi, V. T., Jones, P. P., Goding, J. Current Topics in Microbiology and Immunology (Melchers, F., Potter, M., and Warner, N. L., eds) Vol. 81, pp. 115-129, Springer-Verlag, New York). The mild conditions used for elution from the column, 0.5% deoxycholate in 10 mM Tris buffer, pH 8, with 0.14 M NaCl, result in recovery of 70 to 100% of the allogeneic serological activity. A murine lymphoma, RDM-4, was found to express high levels of H2-Kk; approximately 2 X 10(6) molecules/cell. Milligram quantities of H-2Kk can be purified readily using these cells.     

Immunomagnetic Particle Induced Lysis of Antibody-Conjugated Liposomes

Abstract

We have prepared liposomes of dioleoylphosphatidylethanolamine (DOPE) which have been stabilized by addition of 9-12 mol% N-biotinyl- phosphatidylethanolamine. Liposomes composed of DOPE/N-biotinyl-PE are quite stable and non-leaky although they exhibit strong temperature-dependent leakage following incorporation of palmitylated murine monoclonal antibodies as a targeting ligand. Addition of magnetic chromium dioxide particles coated with anti-mouse antibody to these immunoliposomes lead to their aggregation and the release of entrapped calcein. The lytic event was biphasic with an initial rapid release of 20% dye within 5 min. followed by a slower rate which reached nearly 40% release after 80 min. The rapid release phase was dependent upon the concentration of the liposomes and that of the multivalent particles. Lysis was immunospecific since no release was observed upon addition of nonspecific immunomagnetic particles to the immunoliposomes or if no antibody was incorporated into the liposome. Lysis could also be blocked by the addition of free murine antibody to the solution. The ability of these liposomes to release their contents in response to binding a multivalent antigen validates their potential for therapeutic or diagnostic applications.     

An improved method for covalent attachment of antibody to liposomes

A rapid and simple method is described for the incorporation of monoclonal antibody coupled with palmitic acid into liposomes prepared by the reverse-phase evaporation method (Szoka, F. and papahadjopoulos, D. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4194–4198). Palmitoyl antibody in 0.15% deoxycholate is added to a liposome suspension after the majority of the organic solvent has been removed by evaporation. Efficient incorporation (over 80%) of palmitoyl antibody occurred without leakage of the encapsulated drug. Native, unmodified antibody did not incorporate under identical conditions. About 50% of the incorporated antibodies could be readily digested by protease, while most of an internal protein marker was not, suggesting that about half of the antibodies were exposed on the outer surfaces of liposomes. Target-specific binding of antibody-liposomes has also been demonstrated in vitro with the RDM-4 lymphoma cells. This method offers a rapid and highly efficient attachment of functional antibody molecules to liposomes with high capture efficiency of drugs, and therefore should be useful in target-specific delivery of drugs mediated by liposomes.