EP0211042A1 - Liposome composition and method - Google Patents

Abstract

A composition is formed of liposomes having functionally active proteins attached to their surface, in a concentration of between 100 and 600 mug proteins / mumole of lipid. Proteins are attached by amide bonds to spacers 3 to 18 atoms in length anchored to the liposomes. New methods of preparing this composition involve direct coupling of the protein with activated carboxyl or N-hydroxysuccinimide groups.

Description

LIPOSOME COMPOSITION AND METHOD

1. Field of the Invention

The present invention relates to lipid vesicles having surface-bound proteins, and to protein-coupling methods.

2. References

The following references are referred to herein by corresponding number:

1. Tyrell, D.A., Biochem Biophys Acta (1976) 457:259.

2. Huang, A., et al, Biochim Biophys Acta (1982) 716: 140. 3. Shen. D.. et al, Biochim Biophys Acta (1982)

689:31.

4. Dunnick, J.K., J Nucl Med (1975) 16:483.

5. Torchilin. V.P., et al, Biochem Biophys Res Commun (1979) 85:1114. 6. Torchilin. V.P., Biochem Biophys Res Commun (1978) 85:983.

7. Heath, T.D., et al, Biochim Biophys Acta (1981) 640 : 66 .

8. Martin, et al. Biochemistry (1981) 26:4229. 9. Kinsky. S.C., J Immunol Methods (1983) 65:295.

10. Kinsky, S.C., Biochim Biophys Acta (1984) 769:543.

11. Dancey. G.F., et al. J Immunol (1979) 255:8015.

12. Szoka, F.C., et al. Ann Rev Biophys Bioenq (1980) 9:467.

13. Szoka, F.C., et al, Proc Nat Acad Sci (USA) (1978) 75:4194. 14. Olson. F., et al. Biochim Biophys Acta (1979) 557:9.

15. Lowry. O.H., et al. J Biol Chem (1951) 193:265.

3. Background of the Invention

A variety of therapeutic and diagnostic uses of liposomes have been reported. In many applications, liposomes are prepared to include surface-bound molecules such as small haptens, enzymes, antibodies, and other protein and non-protein molecules capable of conferring selected enzymatic or surface-recognition features to the liposomes. The surface molecules may function, in therapeutic applications, to target drug-containing liposomes to specific tissue or organ receptors (reference 1). In diagnostic applications of liposomes, the surface-bound molecules typically are ligands capable of binding with high affinity to analyte-related anti-ligand molecules, which may be either free in the assay reaction medium or carried on a solid surface. Binding between ligand and anti-ligand molecules leads to one of a variety of surface-attachment, agglutination, reporter-modulation or liposome-lysis events which can be used in determining the presence and/or concentration of analyte in the reaction medium.

In some specialized uses, the surface-bound molecules may be lipid or lipid-like antigens, such as cardiolipin or glycolipid, which can be incorporated directly into the liposomes as part of the lipid components used in forming liposomes. In the more typical case, the molecules are relatively water-soluble ligands, generally proteins, which are preferably covalently attached to surface lipid components of the liposomes. In one general method for preparing liposomes with surface-attached proteins, the protein is first derivatized with a lipid, such as a fatty acid, then mixed with vesicle-forming lipids or preformed liposomes in the presence of a mild detergent, such as deoxycholate, under conditions which lead to gradual diffusion of the protein into the liposomes. The residual detergent is then removed, typically by dialysis. The general method is illustrated in references 2 and 3. which describe derivatizing immunoglobulin with a palmitic acid/N-hydroxysuccinimide reagent, in the presence of deoxycholate, and diffusion of the protein into liposomes. Coupling ratios of more than 200 μg protein/umole lipid were achieved with selected immunoglobulins.

One limitation of the above method is that many proteins, when derivatized with a lipid, become aggregated and resist diffusion into liposomes, even in the presence of detergent. These proteins, which include a variety of immunoglobulins, cannot be attached to liposomes efficiently by the technique. Also natural membrane lipids, such as phospholipids, typically cannot be used as the derivatizing lipid, because of. the increased tendency of the derivatized protein to aggregate. The difficulty in removing residual detergent after liposome formation is another limitation. Several methods for chemically coupling soluble proteins directly to preformed liposomes have been reported. Some of these methods involve coupling of proteins to unmodified surface lipids by water-soluble cross-linking agents such as 1-ethyl,3-(3-dimethγlaminopropyl)carbodiimide (EDCI) (reference 4), glutaraldehyde (reference 5), or suberimidate (reference 6). Cross-linking methods of this type have not been entirely satisfactory in that significant cross-linking of vesicles, or proteins or both may occur. The extent of specific ligand binding achievable is also generally quite low. One protein coupling technique which allows relatively high levels of pfotein binding to liposomes has been described by Heath, et al (reference 7). The method involves periodate oxidation of glycosphingolipids in the liposome outer membranes, to form reactive surface aldehyde groups. Proteins are then attached to the aldehyde groups through Schiff-base formation. followed by reduction with NaBH ₄ or NaBH₃CN. Under optimal conditions, up to about 20% of the protein may be coupled to the oxidized vesicles, and protein concentrations of between 100-200 μg protein per μmole lipid may be achieved. One limitation of the method is the requirement for glycosphingolipids in the liposomes. General oxidative damage to liposomes caused by periodate oxidation, and the need to remove periodate before protein coupling is carried out, are other limitations.

A second protein coupling method which has been shown to produce high coupling ratios is disclosed in U.S. patent number 4,429,008 to Martin, et al. The liposomes in this method are formed by incorporating a thiol-reactive lipid, such as (N-[4-(p-maleimidophenyl)butγryl] phosphatidylethanolamine (MPB-PE) or (N-[3-(pyridyl-2-dithiopropionyl] phosphatidylethanolamine) (PDP-PE). The thiol-reactive liposomes are reacted with a protein bearing a free sulfhydryl group, or with one which has been thiolated to produce such a group, under conditions Which lead to disulfide or thioether coupling to the thiol-reactive surface lipid. Coupling concentrations greater than about 200 ug protein (IgG)/μmole lipid have been obtained (reference 8). The method is limited to proteins which have an available free sulfhydryl group or which can be thiolated without loss of protein activity. Methods for attaching dinitrophenol phosphate-lysine (DNP-lysine), a small amine-containing hapten to liposomes, by coupling to an

N-hydroxysuccinimide (NHS) lipid derivative incorporated into liposomes, have been reported (references 9 and 10). It has been suggested (reference 9) that this approach might also be applicable to coupling proteins to liposomes. The particular NHS compounds employed in reference 9 were NHS-succinyl derivatives of fatty acid, cholesterol and phosphatidylethanolamine (PE). In each of these compounds the lipid polar group is spaced from the NHS group by a hydrocarbon chain two atoms in length. Studies performed in support of the present invention indicate very poor protein coupling to liposomes containing PE-succinyl-NHS. The reason for the difference between hapten and protein coupling to the liposomes may be due to the apparent ability of the hapten to penetrate the bilayer regions of liposomes (reference 11).

4. Summary of the Invention

The present invention includes a liposome composition comprising lipid bilayer vesicles containing in their outer bilayer regions, coupling groups which are each composed of a phosphatidylethanolamine (PE) lipid moiety and a spacer arm, 3-20 atoms in length, attached to the lipid moiety through an amide linkage. Functionally active proteins are attached to the vesicle surfaces, at a protein concentration of between about 100-600 μg protein/μmole lipid. through amide linkages to the free ends of the spacer chains.

One method for forming the liposome composition of the invention uses a novel lipid coupling reagent having (a) a PE lipid moiety for anchoring the reagent to liposome bilayer region, (b) a carbon-containing spacer arm of between 3-20 atoms in length, and (c) a chain-terminus carboxyl group. This reagent, which forms part of the invention, is referred to herein as a carboxylated-PE or PE-CO₂H coupling agent. The coupling method is applicable to a variety of proteins, particularly immunoglobulins.

A second method for forming a liposome composition of the type described uses a coupling reagent similar to the above PE-carboxyl reagent, except that the chain has a reactive N-hydroxysuccinimide (NHS) group at its free end rather than a carboxyl group. This reagent is referred to herein as an N-hydroxysuccinimide-PE or PE-NHS coupling reagent. Both coupling methods can be performed under conditions which lead to immunoglobulin coupling efficiencies of up to about 70%, and to a concentration of functionally active surface-bound protein of up to about 600 μg protein/μmole lipid. It is one general object of the invention to provide a novel liposome composition having a high surface concentration of functionally active proteins.

A more particular object of the invention is to provide novel coupling reagents and methods for forming such a composition.

Still another object is to provide, in such a methods, protein-coupling efficiencies of up to about 70%, and final protein surface concentrations of up to 600 μg protein/μmole lipid. These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention.

Brief Description of the Drawings

Figure 1 illustrates one scheme for synthesizing PE-CO₂H(III) and PE-NHS (IV) coupling reagents used in the invention;

Figure 2 illustrates another scheme for synthesizing the PE-NHS coupling reagent;

Figure 3 shows a reaction scheme for attaching a protein to the PE-NHS coupling reagent in Figures 1 and 2, with the reagent carried in a liposome; and

Figure 4 shows a reaction scheme for attaching a protein to the PE-CO₂H coupling reagent, with the reagent carried in a liposome.

Detailed Description of the Invention

1. Preparing the Coupling Reagents

A. PE-CO₂H Reagent

The PE-CO₂H coupling reagent of the invention has the general formula

where PE is phosphatidylcholine, X is oxygen (O) or sulfur ( S ) and CH₂ - - - CH₂ is an amide-linked carbon-containing chain having a chain length of between 3 and up to about 20 atoms.

Purified and partially purified PE preparations are commercially available, or may be prepared by known methods. and these may be purified and/or modified in acyl chain composition according to known techniques.

The CH₂ - - -CH₂ chain in the spacer arm moiety includes a carbon-containing linear chain having various degrees of saturation and/or heteroatom compositions. One preferred type of chain is a simple saturated acyl chain of the form (CH₂)_n, where n=3-20. The carbon atoms in the chain may also be partially unsaturated, including either ethylenic or ethylenic bonds, and/or may include such heteroatoms as carbon-linked O, S or nitrogen (N) atoms, forming ester, ether, thioester, thioether, amide or amine linkages within the chain. The chain atoms themselves may be substituted with carbon, hydrogen, O, S, or N atoms, or groups containing these atoms such as short chain acyl groups or the like. Examples I-IV describe carboxylated PE reagents having various-length saturated acyl chain spacer arms. Example V describes a carboxylated PE reagent having as a spacer arm, a disuccinoylethylenediamine (DES) moiety of the form

where 1, m, n = 2; more generally, l+m+n=1-10. Another general type of spacer arm includes a polypeptide chain composed of preferably between about 2-7 amino acids.

A preferred method of forming the coupling reagent of the invention is outlined in Figure 1. The method involves first providing a dicarboxylic anhydride of the form

where CH₂ - - - CH₂ has the chain characteristics noted above. For some selected chains, the anhydride may be obtained commercially, such as illustrated in Example II where succinic anhydride is used. Where the anhydride is not commercially available, it may be prepared, typically from the dicarboxylic acid of the anhydride. The dicarboxylic acid may be obtained commercially, as in Examples I, III, and IV, or prepared from more basic starting materials, as in Example V. With reference to Figure 1, 2 mole equivalents of the selected dicarboxylic acid (I) are reacted with 1 mole equivalent of a carbodiimide coupling reagent. Dicyclohexylcarbodiimide (DCCI) is suitable for use in organic solvents, such as methylene chloride, which are typically used. The reaction is carried out preferably under a non-oxidizing atmosphere, such as nitrogen, for a period of up to several days, at a temperature normally between about 10° and 25°C. Dicyclohexylurea, which is formed in the reaction, need not be removed from the anhydride reaction product before further coupling the anhydride to PE.

In the second step of the method, shown in the second line in Figure 1, 1 mole equivalent of the anhydride is reacted with about 1 mole equivalent of PE in the presence of about 3 mole equivalents of a tertiary amine, such as triethylamine. The reaction is typically prepared by adding a solution of PE and the tertiary amine in an organic solvent which is miscible with the solvent used in the anhydride-forming reaction. The reaction is carried out preferably under a non-oxidizing atmosphere for a period of up to several days, also at a temperature of between about 10° and 24°C. The resulting product is a tertiary amine salt of the PE-amide of dicarboxylic acid (not shown). The reaction mixture is then washed with an acidified aqueous solution to form the PE-amine carboxylic acid reagent which is shown at III in the figure. After removing the aqueous phase, the organic phase mixture may be dried, for example, over anhydrous sodium sulfate.

The carboxylated PE reagent is purified by conventional chromatographic techniques, such as silica gel column chromatography. using a selected solvent system to elute the reagent. The eluate fractions can be monitored conventionally by thin-layer chromatography (TLC). Examples I, II. III. and IV detail the preparation and purification of the PE-amides of glutaric acid, succinic acid. 1,12-dodecanedicarboxylic and 1,20-eicosanedicarboxylic acid, respectively.

Example V describes the preparation and purification of the PE amide of DES.

B. PE-NHS Reagent

The PE-NHS coupling reagent used in the present method has the general formula:

where PE-NH₂ , NHS, CH₂ - - - CH₂ are as defined above.

One method of forming the PE-NHS coupling method is illustrated in Figure 1. Here the PE-CO₂H coupling reagent identified at III in the figure is reacted with an N-hydroxysuccinimide (NHS) in the presence of a suitable activating agent, such as DCCI. Reaction conditions are similar to those used in forming the anhydride intermediate (II), and are detailed in Example VIII. The resulting PE-NHS coupling reagent, a PE-amide carboxylic acid NHS ester, is shown at IV in the figure. The NHS which is used in the reaction may be an N-hydroxysuccinimide, N-hydroxysulfosuccinimide, N-hydroxγquinolinimide, N-hydroxyphthalimide, or other suitable N-hydroxysuccinimide ring compound.

The coupling reagent may be purified, following removal of the reaction solvent, by silica gel chromatography, using a suitable solvent system, such as a step gradient of methanol in chloroform. The purified product may be stored as a dry residue under nitrogen at 4°C for up to several months.

A second general method for forming the PE-NHS coupling reagent is shown in Figure 2. Here the dicarboxylic acid (I) used in the first synthetic scheme is reacted directly with an NHS in the presence of DCCI to form the corresponding di-N-hydroxysuccinimide ester (V) seen in the upper line in the figure. Typical reaction conditions are similar to those used in forming compound IV in Figure 1. The material may be employed without further purification for reaction with PE. For some dicarboxylic acids, such as suberic acid (n=6), the di-N-hydroxysuccinimide derivative (compound V) is commercially available. This compound is used in Example IX in forming the cor responding coupl ing reagent (PE-suberoyl-NHS ).

To form the desired NHS coupling reagent, the di-N-hydroxysuccinimide compound from above is reacted with PE in a suitable nonaqueous solvent, such a methylene chloride, under reaction conditions like those described in Example IX. The PE-NHS coupling reagent, a PE-amide carboxylic acid NHS ester, may be purified by silica gel chromatography. and stored in dry form, as above. 2. Preparing the Liposome Composition

A. Liposome Preparation

The method of forming the liposome composition of the invention includes the steps of (1) preparing lipid vesicles, or liposomes having a surface array of activated PE-CO₂H or PE-NHS coupling reagent molecules, and (2) reacting the liposomes with a protein solution under suitable protein-coupling conditions. To prepare the liposomes, vesicle-forming lipids are mixed with a selected mole percent of the coupling reagent, then formed according to conventional liposome preparation methods. The vesicle-forming lipid typically includes between about 20-80% phospholipid, such as phosphatidylcholine (PC) and phosphatidylglycerol (PG) ; between about 20-60 mole percent cholesterol; and between about 1 and 20 mole percent of the coupling reagent, although a higher mole ratio of reagent may be employed. One preferred liposome composition, described in Examples VI and X, contains 50 mole percent cholesterol, 45 ιole percent PC, and 5 mole percent coupling reagent.

The liposomes may be prepared by a variety of known methods. In one general method, lipid components. including the coupling reagent, are dried to a thin film, typically in a round-bottom flask, slowly rehydrated by the addition of aqueous medium, then agitated to form a suspension of liposomes. The resulting multilamellar vesicles (MLVs) have heterogeneous sizes between about 0.05 and 10 microns.

To prepare liposomes which are largely oligolamellar in structure, the reverse-phase evaporation method described in reference 12 is preferred. Here a solution of vesicle-forming lipids in a suitable organic solvent is mixed with an aqueous medium, in a volume ratio which produces a water-in-oil emulsion when the mixture is emulsified. The reverse-phase gel which forms on removal of the organic solvent is shaken with added aqueous solution to form reverse-phase vesicles (REVs). The method is especially useful for preparing liposomes with encapsulated vesicles, since high encapsulation efficiencies (up to about 50% or more) are achieved. Examples VI and X below describe the preparation of REV-type liposomes having different selected reagent chain lengths. Properties of and other methods for preparing liposomes have been detailed in the literature, and the reader is referred particularly to above references 12 and 13, and references cited therein, for a comprehensive discussion of the topic.

The liposomes, once formed, may be sized, for example, by extrusion through a micropore membrane and/or washed by known techniques (reference 14). The liposomes used in many of the examples below were sized by successive extrusion through 0.4 and 0.2 micron polycarbonate membranes, as detailed in Example X. After final preparation, the liposomes may be suspended to a final concentration of between about 1-5 μmoles lipid/ml in a suitable buffer, such as one containing

0.1 to 0.2 M NaCl. The pH of the buffer is preferably adjusted to between 7-9 for the PE-CO₂H liposomes and

5-7 for the PE-NHS liposomes.

According to one aspect of the invention, the surface molecules, e.g., proteins, in the liposome composition are coupled to the lipid vesicles through amide linkages involving protein amine groups, allowing virtually any relatively soluble protein to be successfully coupled to the vesicles. That is, there is no requirement for special reactive groups, such as free sulfhydryl groups. One broad class of useful proteins are binding proteins, such as antibodies and antibody fragments which are capable of binding specifically and with high affinity to a ligand which forms the opposite member of a binding pair. Representative classes of antibodies include IgG, IgM, IgA, and IgE. and their Fab'. Fab. and F(ab) fragments. The coupling of IgG molecules. to lipid vesicles is detailed in Example VII and XI. Other binding proteins suitable for the present invention include protein A, hormone-receptor binding proteins; lectin, for binding carbohydrate-containing ligands; and avidin. which binds specifically to biotin and biotinylated molecules. A liposome composition having surface-bound avidin is described below.

Likewise , a variety of ligand proteins--that is, proteins whose surface or epitopic features are specif ically recognized by binding proteins--can be coupled efficiently and at high surface concentration to lipid vesicles by the method of the invention. Such ligands include antibodies and other serum proteins and polypeptide hormones. Experiments conducted in support of the present application, and presented below, illustrate compositions having surface-bound IgG ligand molecules (where the IgG serves as the target of a soluble binding protein), and bovine serum albumin (BSA) derivatized with dinitrophenylphosphate (DNP).

Enzymes are another class of proteins which may be efficiently coupled to lipid vesicles according to the method of the invention. Representative classes of enzymes include oxidoreductases, typified by luciferase, glucose oxidase, galatose oxidase, and catalase; hydrolyases, typified by various types of phosphatases; glycoside hydrolases, such as ß-galactosidase; peptidases; and lyases.

B. Coupling to PE-CO₂H Liposomes To couple amine-containing surface molecules to liposomes containing surface-bound PE-CO₂H, the reagent-containing liposomes are first activated with a water-soluble linking agent which is capable of reacting with a carboxyl or thiocarboxyl group, to form an activated complex which can react with amine groups to form a stable amide or thioamide linkage. Exemplary linking agents include carbodiimides, preferably water soluble carbodiimides such as 1-ethyl-3-(3-dimethylaιtιinopropyl)-carbodiimide (EDCI) and imidazoles, such as N-hydroxy imidazole. The agent is added to a final concentration of between about 1-2 mole equivalents per mole of coupling reagent in the suspension, and the activation reaction is carried out usually for about 1 hr at room temperature. The activated complex need not be separated from unreacted linking agent before further coupling to the surface molecules. The liposome suspension is mixed with an aqueous solution of the binding molecules, and the mixture is allowed to react under conditions which lead to amide-bond formation between the activated reagent and the molecules, as shown in the lower line of Figure 4. The coupling reaction medium is preferably adjusted to a pH of about 7-9, and to a salt concentration of between about 0.1 and 0.2 M. The reaction is carried out normally between about 4° and 10°C. until a maximum ratio of surface binding has occurred. Overnight reaction times are normal. The reaction may be quenched by the addition of a suitable amine-containing compound, such as an amino acid. The liposomes and surface-bound molecules are separated from the non-lipid-associated molecules in the reaction mixture by conventional separation techniques, such as molecular-sieve chromatography or centrifugation, for example, through a metrizamide density gradient. The liposomes may be further washed to remove non-specifically bound surface molecules. Example VII details methods of the coupling IgG molecules to each of the four liposome suspensions prepared in Examples I-IV. The final surface concentration of bound molecules can be adjusted by varying the initial ratio of added binding molecules to liposome lipid. Optimal surface concentrations of bound protein molecules are achieved at initial protein/lipid concentration ratios of about 0.5 to 2.0 mg protein/μmole lipid. In each of the coupling methods described in Example VII, about 0.75 mg IgG was reacted with about 1.5 μmole liposome lipid.

As will be seen from data presented in Example VII. the several reagents formed with saturated hydrocarbon spacer arm chains with lengths f rom 3-20 atoms all showed coupling efficiencies of at least about 30% and coupling ratios, for IgG, of greater than about 150 μg proteins/μmole liposome lipid. At an optimal CH₂ - - - CH₂ chain length of 12, the coupling efficiency was about 60% and the coupling ratio, about 300 μg IgG/μmole lipid. This result is surprising in view of the expected tendency of long-chain acyl groups, such as a 12-atom chain to partition in the surface bilayer. For example, studies by others on the immunogenicity of liposomes sensitized with dinitrophenylphosphate-derivatized PE containing different-length spacers show highest activity at chain lengths about 3 or 4 atoms and a sharp dropoff in immunogenic activity at chain lengths about 7-10 atoms. (reference 11).

At a chain length of 2 or less, i.e., for CH₂-CH₂ and CH₂ "chains." low efficiencies and coupling ratios are observed. At chain lengths greater than about 20 atoms, the reagent may be difficult to synthesize, due in part to a general lack of commercial availability of long-chain dicarboxylic acids. Further, chains having lengths of about 20 atoms show reduced ratios of specific to control (non-specific) protein coupling.

C. Coupling to PE-NHS Liposomes

Coupling of amine-containing surface molecules, such as proteins, occurs by direct reaction with PE-NHS liposomes. The reaction medium is preferably adjusted to between 5.5 and 6, and no higher than about pH 7.0, since the NHS groups in the coupling show increased hydrolysis at basic pH. The salt concentration of the reaction medium is normally between about 0.05 and

0.2M. The reaction is carried out at room temperature or below, and typically between about 4°C and 10°C. for several hours. The reaction may be quenched by the addition of a suitable amine-containing compound, such as an amino acid, or by exposing the composition to pH 8.0 or above for several hours. The protein-coupled liposomes are separated from unreacted protein as above. Optimal surface concentrations of bound protein molecules are achieved at initial protein/lipid concentration ratios of about 0.2 to 2.0 mg protein/μmole lipid. In each of the coupling reactions described in Example XI, about 0.65 mg IgG was reacted with about 1.0 μmole l iposome l ipid , producing protein concentrations between about 425 and 480 μg protein/μmole lipid.

As will be seen from data presented in Example XI, the several reagents formed with saturated hydrocarbon spacer arm chains with lengths from 3-12 atoms, and either succinimide or sulfosuccinimide NHS groups, all showed coupling efficiencies of between about 60% and 70%, and coupling ratios for mouse IgG of greater than about 400 μg proteins/μmole liposome lipid.

The coupling ratios and efficiencies achieved with the PE-NHS reagent are comparable to those achieved with the PE-CO H reagent, and generally much higher than those which have been achieved with prior art methods. Also similar to the PE-CO₂H reagent, long-chain acyl groups, such as a 12-carbon chain, gave good coupling characteristics. The major difference between the two reagents is that the PE-NHS reagent shows substantially no chain-length effect on coupling characteristics. That is, longer chains showed the same high coupling efficiencies and ratios as shorter (e.g., n=3) chain reagents.

3. Utility

A. PE-CO₂H Liposomes

Several liposome assays using liposome reagents prepared by the method of the invention were studied to demonstrate the use of the method in coupling surface-binding molecules to PE-CO₂H liposomes. The first assay was designed to show coupling of a relatively large protein antigen, bovine serum albumin (BSA), to large liposomes, and agglutination of the BSA-carrying liposomes by anti-BSA antibody. The liposomes were formed of phosphatidylcholine (PC) :cholesterol:PE amide of glutaric acid, in a ratio of 20:8:2, and prepared to include encapsulated erioglaucine (a water-soluble blue dye). The BSA was reacted with activated liposomes at a ratio of about 0.5 mg protein/μmole lipid. The protein coupling ratio was about 90 μg protein/μmole lipid. Large-liposome agglutination assays were performed according to the general method described in co-owned patent application for "Large-Liposome Agglutination Reagent and Method", Serial No. 517,826, filed July 27, 1984. In a typical assay, 50 nmole of liposomes showed strong agglutination with 14μg rabbit anti-DNP BSA (obtained from Miles Labs. Elkhart, IN). No non-specific agglutination was seen with human IgG or rabbit anti-human IgM.

A similar type of large-liposome agglutination assay was d.esigned to show coupling of a nuclear antigen material to PE-CO₂H liposomes and agglutination of the liposomes by anti-nuclear antibodies (ANA). Large liposomes prepared to include PC: cholesterol:PE amide of glutaric acid:PE-rhodamine (a lipid-soluble pink dye) at a ratio of 20:8:2:0.078 were prepared as in the just-cited co-owned patent application. Calf thymus antigen, a mixture of macromolecular antigens derived from thymus nuclei, including DNA, DNA-histone complex, histones. ribonucleoprotein, and RNA, were reacted with activated liposomes at a ratio of about 0.075 mg antigen protein per μmole liposomes. In large-liposome agglutination assays performed as above, liposomes were agglutinated by positive ANA serum, but not negative serum.

A third type of assay was based on liposome-enhanced agglutination, as described generally in the co-owned patent application for "Enhanced Agglutination Method and Kit", Serial No. 486,793, filed April 20, 1983. Liposomes composed of

PC:PG: cholesterol:PE amide of glutaric acid, at a ratio of 7:2:10:1 were prepared by a reverse evaporation procedure described in the just-cited application. Lens culinaris protein, a sugar-binding lectin protein, was reacted with the activated liposomes. at a ratio of about 0.8 mg protein per 1.6 μmole liposome lipid. The coupling ratio was about 260 μg protein per μmole lipid. The liposome reagent was reacted, in a latex agglutination assay test for rheumatoid factor (an IgM antibody) containing positive rheumatoid factor serum and a latex agglutination reagent composed of latex having surface-bound, heat-denatured IgG. In this assay, the normal agglutination of the latex by cross-linking with rheumatoid factor analyte is enhanced by the presence of liposome-bound lens culinaris lectin, which binds to IgM antibodies. The lens culinaris liposome reagent enhanced latex agglutination specifically in the presence of rheumatoid factor, as anticipated.

A final assay procedure involved ligand-specific binding of an anti-ligand reporter conjugate to liposomes having ligand molecules coupled to the liposome surfaces by the method of the invention. Large liposomes composed of PC: cholesterol :PE amide of glutaric acid, at a ratio of 20:8:4, were prepared by a reverse evaporation method, to contain encapsulated erioglaucine. The activated liposomes were reacted with mouse monoclonal antibody against hepatitis B surface antigen (HBsAg), at a ratio of about 0.3 mg antibody per μmole liposome lipid. In a first binding study the liposomes were incubated with fluorescein-labeled rabbit antibody against mouse IgG. After reacting the antibody and liposomes, the unbound antibody was removed by centrifugation and the liposomes were viewed under a fluorescence microscope. A readily detectable fluorescence intensity was observed. This study demonstrates that the antigenic sites of the anti-HBsAg antibody were not destroyed by the coupling reaction. In a second method, aliquots of the liposomes were incubated with increasing amounts of horseradish peroxidase-labeled HBsAg antigen. After removing unbound material by centrifugation, and resuspending the liposomes in a suitable assay buffer, the enzyme activity was measured. The peroxidase-labeled antigen could be detected at a sensitivity level of about 20 μg enzyme-labeled antigen. The second test indicates that the antigen-binding activity of the anti-HBsAg antibody was not destroyed in the coupling reaction.

B. PE-NHS Liposomes

A variety of liposome compositions containing PE-NHS were prepared to demonstrate (a) retention of protein activity after attachment to liposomes, and (b) utility, of the compositions in different diagnosticapplications. One composition was prepared by coupling rabbit antiserum against BSA-DNP to large liposomes prepared from PC (50 mole percent), PE-suberoyl-NHS coupling reagent (10 mole percent), cholesterol (40 mole percent), and a trace amount of ¹²⁵I-PE. The liposomes contained encapsulated erioglaucine. a water-soluble blue dye. The coupling reaction was carried out in 10 mM PO₄ , 0.15 NaCl, pH 6, at 4°C overnight, at protein and lipid concentrations of 133 μg/ml and 1.0 μmole/ml, respectively. The composition was freed of unbound protein and suspended to a final concentration of about 5 μmole/ml. The composition was tested for retention of both antigenic and antigen-binding activity in large liposome agglutination assays. In a first assay, the liposome suspension was incubated with either goat anti-rabbit IgG or goat anti-human IgG (control), under reaction conditions like those described in the above-cited patent application for Large-Liposome Agglutination Reagent and Method. Strong liposome agglutination was observed with the goat anti-rabbit antibody, but not in the control, showing that the coupled antibody retained its antigenicity. In a second assay, the liposome reagent was mixed with 10 mg/ml BSA under similar agglutination conditions, to confirm the ability of the coupled antibody to bind immunofipecifically to BSA. Again, an easily observable agglutination reaction was observed.

A liposome composition having surface-bound avidin was prepared according to the present coupling method and tested for its ability to bind biotinylated ß-galactosidase. REVs containing 10 mole percent

PE-suberoyl-NHS were prepared as described in Example IX (for the synthesis of PE-suberoyl-NHS) and coupled to avidin under conditions like those described in Example XI. The composition was mixed with biotinylated ß-galactosidase, and binding to the liposomes, as measured by ß-galactosidase activity associated with the liposomes, was compared with two types of control REVs. The first control was prepared by adding avidin to REVs after hydrolyzing the reactive surface NHS groups at pH 8.0; the second control included REVs alone, without added avidin. The enzyme activity associated with the avidin/liposome composition was about 35 times that of the first control (non-specifically bound avidin) and about 70 times that of the second control (no avidin present).

The surface concentration of avidin in the above composition was about 600 μg protein/μmole lipid, as determined by Lowry analysis (reference 15). Based on the measured activity of liposome-bound enzyme, it was calculated that the total amount of bound biotinylated ß-galactosidase was about 14 μg/μmole lipid. Thus, the coupling method of the invention provides a novel method for coupling enzymes to liposomes without loss of enzyme activity. The method involves (a) forming a liposome composition having a high surface concentration of enzyme-binding molecules (e.g., avidin) and (b) attaching the enzyme to the liposomes through the binding molecules.

It is noted that the efficiency of coupling avidin to PE-NHS liposomes was about 25%, as compared with the typically 60%-70% coupling efficiencies seen with immunoglobulins. These results illustrate the variability in efficiencies which can be expected in coupling different types of proteins to liposomes, and is presumably due to differences in accessibility of protein amine groups for reaction with liposomes. However, with all proteins which have been studied, including avidin, protein coupling ratios above 500 μg/μmole lipid have been achieved. This ratio is substantially higher than that reported for any other prior art method, including methods in which protein/lipid complexes are diffused into liposomes during or following liposome formation.

From the foregoing, it can be appreciated how various objects and features of the invention are met. The PE-CO₂H and PE-NHS coupling reagents described herein can be easily synthesized with one of a number of different spacer chains having selected lengths and atom compositions. Where the dicarboxylic acid precursor of the spacer arm in the reagent is available , the reagent can be formed by a simple two-step procedure which requires no intermediate separation or product-isolation step. The reagent can be included in liposomes up to 20 mole percent or more, without significantly affecting desired membrane properties, since PE is itself a common lipid in natural and artificial bilayer membranes. The coupling method detailed herein allows the coupling of soluble proteins, such as immunoglobulins, to liposomes, at efficiencies up to 70% and at coupling ratios of up to about 600 μg protein per μmole ml lipid. The coupling efficiency is comparable with the most efficient coupling reagents known in the prior art. and the coupling ratios are better than the best coupling methods heretofore available. The coupling method does not inactivate surface molecules being attached to the lipid surfaces, as evidenced by the retention of both antigenic and antigen-binding activity of a number of types of surface molecules coupled to liposomes. Further, the coupling reactions are not restricted to proteins having free sulfhydryl groups or the like, and are therefore applicable to a wide range of soluble proteins.

The following examples are intended to illustrate the method and reagent of the invention, but in no way to limit the scope thereof.

Example I

Preparing the PE Amide of Glutaric Acid Glutaric acid was obtained from Aldrich Chemical Co. (Milwaukee.WI); dicyclohexyl carbodiimide (DCDI) from Aldrich Chemical Co. (Milwaukee, WI); PE from Avanti Polar Lipids (Birmingham. AL); and triethylamine from Pierce Chemical (Rockford. IL). Silica gel (E.M. Kieselgel 60, 70-230 mesh) was obtained from Van Waters & Rogers (San Francisco. CA), and thin-layer chromatography (TLC) silica gel plates were obtained from J.T. Baker (Phillipsburg. NJ).

To form the anhydride of glutaric acid, 10.6 mg of glutaric acid (0.080 mmoles) and 8.7 mg DCDI (0.042 mmoles) were combined in 2 ml methylene chloride in a screw-cap tube. The tube was capped and the mixture stirred under nitrogen at 23°C for 48 hours with a magnetic stirring flea.

A solution of PE (0.038 mmoles) in 2 ml chloroform and 15 μl of triethylamine (0.108 mmoles) were added to the glutaric anhydride/DCDI solution. The reaction mixture was sealed under nitrogen atmosphere and allowed to react at 23°C for three days. Following this, the mixture was acidified by adding 5 ml chloroform and 4 ml 0.02 M phosphate/0.02 M citrate buffer. pH 5.5. with vigorous shaking. The aqueous phase was separated by low speed centrifugation and discarded. The organic phase was dried over anhydrous sodium sulfate.

The desired N-glutaryl PE was purified by silica gel column chromatography. The dried chloroform solution was introduced into a 1 cm x 20 cm silica gel (Kieselgel 60) column and fractions eluted by passing through the column 50 ml chloroform effluent solutions containing successively. 0%, 10%, 20%, 30%, and 50% methanol. The fractions eluted at each of the five different methanol concentrations were analyzed by TLC on silica gel plates developed with chloroform: methanol: water (65: 25: 4 v/v/v). The presence of N-glutaryl PE (PE amide of gluatic acid) was detected by I vapor absorption. N-glutaryl PE had an R_f value of about 0.3.

Most of the product reagent was found in the 30% methanol effluent. Evaporation of the effluent fractions containing only the product reagent yielded 26.8 mg of a colorless wax when dried to a constant weight under high vacuum. The calculated phosphorus in N-glutaryl PE is 3.60%. The actual phosphorus measured was 4.01%.

Example II Preparation of PE Amide of Succinic Acid Succinic anhydride was obtained from Aldrich Chemical Co. (Milwaukee, WI). A mixture containing 0.03 mmoles of PE, 4.4 mg succinic anhydride (0.04 mmoles) and 10 μl of triethylamine (0.072 mmoles) in 2 ml chloroform was prepared in a screw-capped tube. The mixture was stirred under nitrogen for 24 hours at 23°C.

The reaction mixture was washed and acidified, as in Example I, and the resulting washed choloroform solution was chromatographed on a silica gel column as above. The column material was eluted, successively, with 50 ml chloroform solutions containing 0%, 10%, 20%, 25%, and 30% methanol. The eluates were monitored by TLC as in Example I. The desired N-succinyl PE (PE amide of succinic acid) showed an R_f value of about 0.26.

The bulk of the desired reagent eluted in the last part of the 25% methanol in chloroform effluent, and in the first part of the 30% methanol effluent. Fractions containing only the desired product, as monitored by TLC, were dried under vacuum yielding a colorless wax whose weight was near the total theoretical yield of 32 mg.

Example III Preparation of PE Amide of

1, 12-Dodecanedicarboxylic Acid

1, 12-dodecanedicarboxylic acid was obtained from Aldrich Chemical Co. (Milwaukee. WI). The anhydride of the acid was formed by reaction with DCDI in 2 ml methylene chloride, as described in Example I.

To the resulting anhydride/DCDI solution were added 2 ml of chloroform solution of 0.03 mmole PE and 15 μl triethylamine. The mixture was stirred under a nitrogen atmosphere for 24 hours at 23°C, and washed and acidified as in Example I.

The washed chloroform solution was chromatographed on a silica gel column as in Example I, using 50 ml solution volumes of chloroform containing, successively, 0%, 10%, 20%, 25%, or 30% methanol. The effluent fractions were monitored by TLC as in Example

I. The desired PE amide of 1-12-dodecanedicarboxylic acid showed an R_f value of about 0.60. This R_f value is about the same as PE in the chloroform: methanol :H₂O (65: 25: 4 v/v/v) developing solvent, but can be distinguished from PE by the absence of a color reaction when exposed to a ninhydrin spraying reagent. The desired compound eluted primarily in the late 25% and 30% methanol effluent solution. The fractions containing only the desired compound were combined and dried under vacuum, giving a colorless wax at an approximate yield of 42% of theoretical yield. Example IV Preparing PE Amide of 1,20-Eicosanedicarboxylic Acid 1,20-eicosanedicarboxylic acid was obtained from Pfalz & Bauer (Stamford. CT) . The anhydride of

1.20-eicosanedicarboxylic acid was formed as in Example I, and reacted with a chloroform solution PE and triethylamine. also in accordance with this example, to form the PE amide of the eicosanedicarboxylic acid. The chloroform solution was washed and acidified as in the above examples, and chromatographed on a silica gel column using 50 ml chloroform effluent solutions containing successively 0%, 10%, 15%, 20%, 30%, 50% methanol to elute the desired PE amide carboxylic acid reagent. The eluate fractions were analyzed by silica gel TLC, on which the reagent showed an R_f value of about 0.51 with the solvent system described in the examples above.

The fractions containing only the PE amide reagent were combined and dried under vacuum, yielding a colorless wax at a yield of about 40% of the theoretical yield. The calculated phosphorus was 2.8%; the actual phosphorus measured was 3.1%.

Example V

Preparing the PE Amide of Disuccinoylethylenediamine Ethylenediamine was obtained from Aldrich Chemical (Milwaukee, WI); N-hydroxy-succinimide from Aldrich Chemical (Milwaukee, WI); and tetrahydrofuran from Aldrich Chemical (Milwaukee, WI).

Disuccinoylethylenediamine (DES) was formed by reacting 2 gm succinic anhydride (Example II) (0.02 moles) with 0.6 gm ethylenediamine (0.01 mole) and 2 gm triethylamine in 45 ml methylene chloride: tetrahydrofuran (1:1 v/v). The reaction mixture was stirred for one hour at room temperature.

Following removal of the solvent under reduced pressure, the residue was acidified to pH 3.0 with the dilute HCl, causing a colorless solid to aggregate. Crystallization from 25 ml hot water, followed by cooling to 4°C, yielded 1.18 gram of colorless crystals which melted at 205°C. The anhydride of DES was formed as in Example

I, by reacting DES (a dicarboxylic acid) with about 0.5 mole equivalents of DCDI in methylene chloride under a nitrogen atmosphere.

To form the PE amide of DES, about 63 mg of the anhydride of DES from above (about 0.125 mmoles dissolved in 0.5 ml methylene chloride), was added to 2.1 ml chloroform containing 42.2 mg PE (0.05 mmoles) and 15 μl of triethylamine. The reaction mixture was permitted to stand for 18 hr at room temperature. The reacted mixture was washed and chromatographed by silica gel chromatography, also as described above, to give a colorless oil identified as the desired product.

Example VI Preparing Liposomes Containing PE Amide Dicarboxylic Acids The PE-amide of 1,8-octanedicarboxylic was prepared according to the general procedures of Examples I-IV. Large, oligolamellar vesicle suspensions, each containing one of the four PE amide dicarboxylic acid reagents from Examples I-IV or the PE amide of 1,8-octanedicarboxγlic acid, were prepared by a reverse evaporation phase method described generally in reference 12. For each suspension, cholesterol (10 μmoles), PC (9 μmoles) and a selected PE amide reagent (1 μ mole) were dissolved in 1 ml of diethyl ether. To this was added 325 μl of 10 mM NaPO₄. 0.15 NaCl (pH 5.0). and the two phases were emulsified by sonication for one min at 25°C in a bath sonicator. Ether was removed under reduced pressure at room temperature. The resulting gel was agitated by vortexing in 10 mM phosphate buffer, pH 5.0. containing 0.15 M NaCl, to a final concentration of between about 1-2 μmole lipid/ml.

Example VII Coupling IgG to Carboxylated PE Liposome Suspensions 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI) was obtained from Pierce Chemical Co. (Rockford. IL). Mouse IgG was obtained from Cappel Labs (Malvern, PA). Each of the five liposomal suspensions (1.5 μmole) from Example VI was mixed with EDCI (4 mg) in

1.5 ml of 10 mM NaPO₄ , 0.15 M NaCl, pH 5.0. The reaction was carried out at room temperature for one hr.

The liposome/EDCI mixture (1.5 ml) was mixed with 75 μl of mouse IgG (10 mg/ml) and 75 μl of 1 M NaCl, and the coupling-reaction mixture adjusted to pH 8.0. Each reaction was carried out overnight at 4°C.

Unreacted protein was separated from liposome-conjugated protein by metrizamide density gradient centrifugation, according to a standard procedure. Control coupling reactions were performed by substituting buffer for ECDI.

The amount of protein bound to the liposomes was determined by the Lowry protein assay described in reference 15. The concentration of liposomal lipid was determined from I¹²⁵ radioactivity levels, based on a known amount of PE-I^{12 5} included in the liposome preparations. Based on the measured protein and lipid concentrations, the protein to lipid coupling ratios, expressed in micrograms protein/μmole, lipid were determined. The values obtained are shown in Table I below, for each of the four different liposomal suspensions which were examined.

The coupling efficiencies, also shown in Table

I, were calculated as the ratio of liposome-bound protein to total protein added to the reaction mixture.

As seen, all five PE dicarboxylic acid reagents showed high coupling efficiencies and relatively high levels of protein coupling. The optimal coupling ratio, relative to control values, occurred at n=12. Although the n=20 reagent gave high coupling values, control values were also substantially higher than for the four shorter-arm reagents. Example VIII Preparing PE-NHS Coupling Reagents: Method 1

Glutaric acid (n=3), adipic acid (n=4), suberic acid (n=6), and 1,12-dodecanedicarboxylic acid (n=12) were obtained from Aldrich Chemical Co. (Milwaukee,WI); dicyclohexyl carbodiimide (DCCI), from Aldrich Chemical Co. (Milwaukee, WI); N-hydroxysuccinimide, from Aldrich Chemical (Milwaukee, WI); PE, from Avanti Polar Lipids (Birmingham, AL); and triethylamine from Pierce Chemical (Rockford, IL). Silica gel (E.M. Kieselgel 60, 70-230 mesh) was obtained from Van Waters & Rogers (San Francisco. CA) , and thin-layer chromatography (TLC) silica gel plates were obtained from J.T. Baker (Phillipsburg. NJ). To form the anhydride of each dicarboxylic acid, about 0.080 mmoles of the acid and 8.7 mg DCCI (0.042 mmoles) were combined in 2 ml methylene chloride in a screw-cap tube. The tube was capped and the mixture stirred under nitrogen at 23°C for 48 hours with a magnetic stirring flea.

A solution of PE (0.038 mmoles) in 2 ml chloroform and 15 μl of triethylamine (0.108 mmoles) were added to the acid anhydride/DCCI solution. The reaction mixture was sealed under nitrogen atmosphere and allowed to react at 23°C for three days. Following this, the mixture was acidified by adding 5 ml chloroform and 4 ml 0.02 M phosphate/0.02 M citrate buffer, pH 5.5, with vigorous shaking. The aqueous phase was separated by low speed centrifugation and discarded. The organic phase was dried over anhydrous sodium sulfate.

The desired PE amide of the dicarboxylic acid was purified by silica gel column chromatography. The dried chloroform solution was introduced into a 1 cm x 20 cm silica gel (Kieselgel 60) column and fractions eluted by passing through the column 50 ml chloroform effluent solutions containing successively, 0%, 10%, 20%, 25%, 30%. and 50% methanol. The fractions all eluted between about 25%-30% methanol. The eluted material was alyzed by TLC on silica gel plates developed wit chloroform: methanol: water (65: 25: 4 v/v/v). The presence of PE amide product was detected by I₂ vapor absorption. The compounds showed R_f values ranging from about 0.3 for the n=3 compound to about 0.6 for the n=12 compound.

To form the PE-NHS coupling reagent, corresponding to each of the above chromatographed PE-carboxylic acid compounds, about 0.07 mmoles of the selected PE-acid was mixed with 0.1 mmoles of

N-hydroxysuccinimide or N-hydroxysulfosuccinimide, and 0.1 mmoles DCCI in 3 ml of methylene chloride. The reaction was carried out at room temperature for, three days. Following evaporation of the solvent, the reaction mixture was chromatographed by silica gel thin-layer chromatography and developed with chloroform, methanol, and water (65:25:4, v/v/v). The reagent was identified by parallel silica gel chromatography using phosphate spray to identify the product.

Example IX Preparation of PE-suberoyl-NHS: Method 2 Di-N-hydroxysuccinimide suberate was obtained from Piece Chemical Co. (Rockford, IL).

Egg PE (Avanti) (20μmole/ml, 1.0 ml) was dried under nitrogen in a "vacutainer" tube and to the dry residue was added di-N-hydroxysuccinimido suberate (60 μmole. 22.1 mg) followed by chloroform (1.0 ml) and triethylamine (28 μl, 20.2 mg). The mixture was tightly capped, vortexed. flushed with nitrogen, and stirred at ambient temperature for 2 hr. Thin-layer chromatography (TLC). using a chloroform:methanol:water (65:25:4 v/v/v) system, indicated the completion of reaction at this time. Two spots could be seen after phosphate spray. The presumed product was the major spot at 0.56 while an unknown compound could be detected as a very faint spot at Rf 0.36. The spot at Rf 0.56 also showed a red color with hydroxylamine-ferric chloride spray confirming the presence of intact succinimide moiety. As an additional test, a small amount of the crude product was incubated with excess aminoethane-thiol (generated from aminoethane thiol hydrochloride and triethylamine) and the product was monitored by TLC in the same solvent system and sprayed with thiol specific Ellman's reagent. The major phospholipid component gave a yellow color with the reagent (the mobility of the product was slow because of derivatization). The reaction mixture was dried and the residue was dissolved in minimal amount of chloroform and applied on a glass pla'te coated with silica gel (20 x 20 cm. 250 micron thick) and developed in chloroform:methanol:water 65:25:4 (v/v/v). Bands were visualized by phosphate spray. The major phospholipid band was excised, extracted with the above solvent (30 ml) for 2 hr at ambient temperature. After filtration and evaporation of the solvents, the dry residue was redissolved in chloroform (1 ml) and filtered through a fine-fritted funnel to remove any silica gel particles. Evaporation of the organic solvent gave pure PE derivative as a colorless syrup. Phosphorus determination indicated the recovery of 8.64 μmole or 43% of product. This material was homogeneous by TLC and could be stored as a dry residue under nitrogen in the refrigerator for at least 3.5 months.

Example X Preparing Liposomes Containing PE-NHS Coupling Reagent Large, oligolamellar vesicles (REVs), each containing one of the eight PE-NHS and PE-SO₃-NHS coupling reagents from Example VIII were prepared by a reverse evaporation phase method described generally in reference 7. For each suspension, cholesterol (10 μmoles), PC (9 μmoles), and a selected PE-NHS reagent (1 μmole) were dissolved in 1 ml of diethyl ether. To this was added 325 μl of 10 mM NaPO₄, 0.15 NaCl (pH 5.0), and the two phases were emulsified by sonication for one min at 25°C in a bath sonicator. Ether was removed under reduced pressure at room temperature. The resulting gel was agitated by vortexing in 10 mM phosphate buffer. pH 5.0, containing 0.15 M NaCl, to a final concentration of between about 1-2 μmole lipid/ml. The liposome suspensions were extruded successively through 0.4 and 0.2 micron polycarbonate membranes obtained from Bio Rad (Richmond, CA). The extruded suspensions had liposome sizes predominantly in the 0.2-0.4 micron range.

Example XI Coupling IgG to PE-NHS Liposomes Mouse IgG was obtained from Cappel Labs (Malvern. PA). Each of the eight liposomal suspensions (1.5 μmole) from Example X was mixed with mouse IgG at a final concentration of 650 μg protein/μmole lipid in 40 mM 2-[N-morpholino]ethanesulfonic acid-3- [N-morpholino]propanesulfonic acid (MES-MOPS) buffer, and the coupling-reaction mixture adjusted to pH 6.0. Each reaction was carried out overnight at 4°C. Unreacted protein was separated from liposome-conjugated protein by metrizamide density gradient centrifugation. according to a standard procedure. Control coupling reactions were performed by first inactivating the liposomes by incubation in 100 mM glycine buffer, pH 9.0, for 4 hours at 37°C. The liposome suspension was then adjusted to pH 6.0 and the protein coupling reaction carried out as above.

The amount of protein bound to the liposomes was determined by the Lowry protein assay described in reference 11. The concentration of liposomal lipid was determined from I¹²⁵ radioactivity levels, based on a known amount of PE-I¹²⁵ included in the liposome preparations. The protein to lipid coupling ratios, expressed in micrograms protein/μmole. are shown in Table I below for each of the eight different liposomal suspensions which were examined. The coupling efficiencies, calculated as the ratio of liposome-bound protein to total protein added to the reaction mixture, ranged between 65% and 73%.

While the invention has been described in preferred embodiments and illustrated with specific examples, it will be appreciated that various changes and modifications can be made without departing from the invention.

Claims

IT IS CLAIMED:

1. A liposome composition comprising lipid bilayer vesicles containing in their outer bilayer regions, coupling groups which are each composed of a phosphatidylethanolamine lipid moiety anchored in a vesicle bilayer, and a carbon-containing spacer chain, 3-20 atoms in length, attached to the lipid moiety through an amide linkage, and functionally active proteins attached to the vesicle surfaces, at a concentration of between about 100-600 μg protein/μmole lipid through amide linkages to the free ends of said spacer chains.

2. The composition of claim 1, wherein the chain has the form:

where are amide linkages and n=3-20.

3. The composition of claim 2, wherein n=3-12.

4. The composition of claim 1, wherein the proteins include antibodies or antibody fragments.

5. The composition of claim 4, wherein the proteins are present at a concentration greater than about 300 μg protein/μmole lipid.

6. The composition of claim 1, wherein the proteins include avidin.

7. The composition of claim 6, which further includes a biotinylated enzyme bound to said avidin.

8. A method of forming a liposome composition made up of lipid bilayer vesicles having functionally active, surface-bound proteins, at a surface concentration of between about 100-600 μg protein/μmole lipid. said method comprising: preparing lipid vesicles containing between about 1 and 20 mole percent of a coupling reagent, each molecule of which has a phosphatidyl ethanolamine moiety carried within the lipid bilayer region of the vesicles, and a linear, carbon-containing chain, between 3 to about 20 atoms in length, connected to the phosphatidyl ethanolamine moiety through an amide linkage, and terminating at its free end in an activated end group which can react with an amine to form an amide linkage, and reacting the lipid vesicles with such protein, at a protein concentration of at least about 200 μg protein/μmole lipid vesicle lipid.

9. The method of claim 8, wherein the activated end group is an N-hydroxysuccinimide group and said reacting is carried out at a pH between about 5-7.

10. The method of claim 9. wherein the N-hydroxysuccinimide group is selected from the group consisting of N-hydroxysuccinimide, and N-hydroxysulfosuccinimide.

11. The method of claim 8, wherein the activated end group is formed by reacting a carboxyl end group with a water-soluble carbodiimide. and said reacting is carried out at a pH between about 7-9.

12. The method of claim 8, wherein the protein is an antibody or antibody fragment, and said reacting is effective to produce a protein coupling efficiency of greater than about 50%.

13. The method of claim 8, wherein said reacting is effective, in the presence of at least about 600 μg protein/μmole lipid, to produce a concentration of surface-bound protein of at least about 300 μg protein/μmole lipid.

14. The method of claim 8. wherein the carbon-containing chain is a hydrocarbon chain 3-12 atoms in length.

15. The method of claim 8. wherein the protein is avidin, and the method further includes mixing the lipid composition with a biotinylated enzyme, to attach the enzyme to the composition through avidin/biotin linkages.