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Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96-99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12-48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60-1.74 for the 22-kDa CA and 2.37-2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

The appearance of protein bound to the surface of intact and microfluidized liposomes and its possible influence on their morphology was examined by freeze-fracture electron microscopy, cryo electron microscopy and small angle X-ray scattering (SAXS) techniques. Results obtained by the two microscopy techniques were in agreement with one another in terms of vesicle size and localization of protein (tetanus toxoid or immunoglobulin G) on the surface of vesicles. Surface-bound protein was observed as particles (10-12 nm diameter) by freeze-fracture electron microscopy and was confirmed by immunogold cryo microscopy. SAXS was shown to be a suitable means to further characterize liposomes with, or without bound protein., (Copyright 1998 Elsevier Science B.V.)

Erwinia carotovora L-asparaginase was coupled covalently to colominic acid, a low molecular mass polysialic acid, by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (50:1, 100:1 and 250:1), polysialylated constructs contained 4.2-8.1 molecules of colominic acid per molecule of enzyme. Such constructs retained most (82-86%) of the initial asparaginase activity and also maintained the Km values of the native enzyme towards the substrate asparagine. On exposure to (mouse) blood plasma at 37 degrees C, polysialylated asparaginase constructs exhibited resistance to proteolysis with 65-83% of the initial enzyme activity still present after 6 h. In contrast, most of the native enzyme was inactivated under the same conditions. In vivo experiments with intravenously injected mice revealed a significant increase in the half-life of the polysialylated asparaginase over that observed with the native enzyme. Such an increase was greatest (250%, about 38 h) for the construct with the highest degree of polysialylation. Results suggest that polysialylation of asparaginase and other proteins may provide an alternative means to improve their effective use in therapeutics.

Inclusion complexes of 14C-labelled hydroxypropyl-beta-cyclodextrin (HP beta-CD) with tritiated dehydroepiandrosterone (DHEA), retinol (RET) and dexamethasone (DEX) were entrapped in the aqueous phase of liposomes composed of distearoyl phosphatidylcholine. Incubation of liposome-entrapped complexes in the presence of rat blood plasma at 37 degrees C resulted in complex dissociation and drug leakage in the media at a rate which was minimal for DEX/HP beta-CD (11%), modest for RET/HP beta-CD (23%) and considerable for DHEA/HP beta-CD (56%; 60 min). In contrast, the HP beta-CD moiety of the complexes was retained by liposomes quantitatively. Free complexes injected intravenously into rats were cleared rapidly from the circulation with most (up to 94%) of the HP beta-CD moiety recovered in 24 urine together with lesser and variable amounts of drug (up to 46% of the dose) in a pattern (DHEA < RET < DEX) that was consistent with the extent of drug association with HP beta-CD in the presence of plasma. A significant proportion of the drugs (up to 25% of the dose)(but very little HP beta-CD) was removed by the liver where drugs were catabolised rapidly, probably following complex dissociation and transport to the tissue via plasma proteins. Injection of entrapped complexes revealed that liposomes alter their pharmacokinetics with only 6-13% of HP beta-CD and a moderate proportion of drugs (up to 26% of the dose) recovered in the urine. Much of the HP beta-CD moiety of the liposomal complexes was recovered in the liver (up to 83%) and spleen (up to 13% of the dose) by 30 min after injection, together with a variable proportion of drugs in a pattern (DHEA < RET < DEX) which reflected that of complex dissociation in vitro. Longer term experiments where animals injected with liposome-entrapped complexes were killed at time intervals (up to 24 days), revealed that HP beta-CD is eliminated from the tissues, albeit at a very slow rate. Moreover, the metabolism of individual drugs in the tissues following vesicle disintegration, appeared to depend on the rate of complex dissociation with DEX, for instance, reaching values of 4-6% (liver) only after 14 days. It is concluded that administration of drug/cyclodextrin inclusion complexes via liposomes could serve as a means to control the action of a wide range of therapeutic agents.

Tetanus toxoid and immunoglobulin G (IgG), model proteins for vaccines and targeting ligands respectively, were covalently coupled to preformed dehydration-rehydration vesicles (DRV) to produce vesicles with surface-bound proteins (DRV-protein) or to preformed small unilamellar vesicles (SUV) which were used to generate DRV with bound protein [(SUV-protein)DRV]. Of the amount of protein employed for coupling (1 mg), 13.8-45.1% was recovered with the liposomes, depending on the type of preparations and the proteins used. Microfluidization of similar DRV-protein or (SUV-protein)DRV for up to 10 cycles led to the formation of smaller vesicles (98-136 mm diameter) which, however, had modestly reduced (estimated as 8.8-21.7%) bound proteins, again depending on the type of preparation and protein used. Treatment of DRV-protein and (SUV-protein) DRV with proteinase revealed that 32.9-45.6% of the total bound protein was exposed on the liposomal surface. With microfluidized liposomes, the proportion of surface-exposed protein increased to 63.1-76.2%. Incubation of intact and microfluidized DRV-IgG and (SUV-IgG) DRV with a protein A-Sepharose 4B CL gel confirmed the presence of IgG on the liposomal surface (47.1-68.4 and 80.5-82.1% of total bound protein respectively). These studies were supplemented with freeze-fracture electron microscopy of (SUV-toxoid)DRV which demonstrated the presence of protein particles (up to 3; 12-14 nm diameter) on the surface of both intact and microfluidized individual liposomes.

Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide. The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride. The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction. At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions. Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis. Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation. Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin. The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.

Multilamellar liposomes composed of equimolar egg phosphatidylcholine and cholesterol and containing carboxyfluorescein or colloidal gold were injected subcutaneously into the footpad of the hind-leg of rats. The draining popliteal lymph nodes of animals killed at time intervals after injection were then dissected and sections examined by fluorescence microscopy (carboxyfluorescein), light microscopy using an immunogold silver kit to enhance gold particles or by transmission electron microscopy. Morphological observations confirmed that subcutaneously injected liposomes accumulate in large numbers in the draining lymph node. The majority of liposomes arrived at the subcapsular sinuses, probably via afferent lymphatic vessels, as such, i.e., in a non-cell bound form. Subsequently, liposomes were dispersed throughout the lymph node either by permeation as free vesicles along the sinuses or by cells involved in vesicle uptake. The majority of such cells were free macrophages, littoral cells and reticular cells (fixed macrophages). Once within cells, liposomes were seen digested by the lysosomal apparatus with varying loss of their lamellar structure, leaving free gold particles within the lysosomes.

Haemoglobin (Hb) was isolated from human erythrocytes under conditions which maintained NADH-cytochrome-b5 reductase activity and suppressed oxidation of Hb during storage at 4 degrees C (methaemoglobin values < 3% after 29 days). Hb was entrapped into liposomes composed of hydrogenated egg phosphatidylcholine and equimolar cholesterol according to the dehydration/rehydration procedure of Kirby and Gregoriadis ((1984) Biotechnology 2, 979). However, encapsulation of Hb in its intact form was poor (< 5%) as a result of its oxidation and denaturation during freeze-drying. The addition of cryoprotectants and the use of both, higher initial concentrations of Hb and very small void vesicles resulted in Hb-rich dehydration/rehydration vesicles (phospholipid/Hb molar ratio of about 200:1) of the preferred size of 110 nm (mean). Highly homogeneous and small void vesicles as starting material were prepared using the one-step method of Brandl et al. ((1990) Drug Dev. Ind. Pharm. 16, 2167). The cryoprotectants were chosen with respect to their sufficient protection of Hb without affecting its loading into vesicles during freeze-drying and rehydration. 51Cr-labelling of Hb was used for the in vivo monitoring of the fate of Hb-containing vesicles rather than 125I-labelling since the latter induced strong interactions of Hb with liposomes. Upon intravenous administration into rats, liposomal 51Cr-Hb showed greater blood levels and prolonged circulation times in the blood compared to free Hb. The present approach provides high yield entrapment of labile molecules into vesicles of small size known to exhibit long circulation time.

Bovine serum albumin (BSA), employed as a model ligand, was covalently linked (about 16% of the amount used) to small unilamellar vesicles (SUV) composed of phospholipid, cholesterol and N-(p-aminophenyl)stearylamide (APSA) (molar ratios 1:1:0.05). SUV with bound BSA were then used to generate dehydration-rehydration vesicles (DRV) in the presence of tetanus toxoid and/or carboxyfluorescein (CF). Nearly all of the SUV-bound BSA (about 15% of the original amount) was recovered in the multilamellar DRV formed, with a considerable proportion (42-62%) of the ligand becoming available on the outer bilayers. This apparent spatial reorientation of BSA within DRV also caused the entrapped toxoid to shift to some extent to the liposomal surface. There was no significant difference in the z average mean size between DRV with and without coupled BSA (543 and 555 nm diameter, respectively). Percent number diameter distribution data revealed that 71.2 (BSA-free) and 76.4% (BSA-containing DRV) of the vesicles had diameters of about 300-440 and 330-420 nm, respectively. However, in terms of percent mass diameter distribution, 69.5% (BSA-free) and 65.2% (BSA-containing DRV) of the mass was in vesicles with corresponding ranges of diameter of 1381-2975 and 1086-2840 nm. Vesicle size heterogeneity in both preparations was confirmed by freeze-fracture electron microscopy which also indicated that structures with or without bound BSA, were mostly vesicular of the multilamellar type. Judging from CF latency values, ligand-bearing DRV were stable on incubation with blood plasma at 37 degrees C for 24 h. Stability was, however, reduced significantly when the amount of ligand bound was excessive. The present approach allows for the coupling of ligands to and the entrapment of antigens and other labile solutes in liposomes independently, thus avoiding potential damage of such solutes by the coupling reagents.

Well-defined liposome systems have previously established the influence of size, surface charge lipid composition and surface ligands, on in vivo fate and behaviour of model compounds entrapped in liposomes. In the present study, preformed liposomes which quantitatively retain aqueous markers were covalenty coupled via dipalmitoylphosphatidyl-ethanolamine, to the hydrophilic polymer, monomethoxypoly(ethylene glycol) (MPEG 5000). Such liposomes retain the coating in the presence of plasma, and appear to adsorb plasma components more slowly than liposomes without the polymer, shown using an aqueous two-phase partitioning technique. MPEG-coupled liposomes were cleared from the blood circulation up to 30% more slowly than liposomes without MPEG after intravenous administration to mice, despite the unmodified liposomes being of a composition and size shown previously to favour achievement of maximum half-life. It is suggested that the polymer acts as a surface barrier to plasma factors which otherwise bind to liposomes in the blood and accelerate vesicle removal.

A number of SH-containing proteins or protein derivatives were coupled to small unilamellar liposomes. These were composed of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol (1:1, phospholipid/cholesterol molar ratio) and activated (DPPE moiety) with the heterobifunctional reagents N-hydroxysuccinimide ester of iodoacetic acid (hydroxysuccinimide iodoacetate), N-succinimidyl-4-(2-bromoacetylamino)benzoate (SBAB) or N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP). DPPE was activated with the reagents before or after its incorporation into liposomes. Protein coupling values varied widely depending on the reagent and the protein used, but were highest in the case of SPDP-activated liposomes and SPDP-modified immunoglobulin G (IgG). Monoclonal anti-Thy1 125I-IgG1-bearing liposomes (SPDP- or SBAB-activated) containing quenched carboxyfluorescein were incubated under a variety of conditions with mouse AKR-A cells expressing the cross-reactive Thy 1.1 antigen. The following observations were made; (a) binding of intact liposomes to the cells at 4 degrees C reached plateau values after about 1 h with at least 70% of the liposomes used being capable of associating with the target cells; (b) binding of liposomes to AKR-A cells was much more pronounced than when using another cell line (EL4-Tc); (c) binding to AKR-A cells could be effected with as little as 1.3 molecules (average) of IgG1 per vesicle; (d) binding was inhibited only modestly by the presence of 50% mouse plasma; (e) stability of IgG1-bearing liposomes in terms of entrapped solute and IgG1 retention in the presence of plasma at 37 degrees C was maintained quantitatively for at least 5.5 h, and by 24 h, 54% of the IgG1 was still associated with the liposomes. AKR mice were injected intravenously with 99mTc-labelled AKR-A cells and 2.5 min later with anti-Thy1 125I-IgG1-bearing liposomes containing quenched carboxyfluorescein and 111In-Ca-DTPA or with similar liposomes devoid of IgG1. In parallel experiments, AKR mice received either of the liposome preparations without previous injection of cells. On the basis of patterns of quenched carboxyfluorescein, 111In and 125I-clearance from the circulation, of 99mTc levels in the blood and of values of 111In in the liver and spleen, it appeared that IgG1-bearing liposomes were capable of binding to their target cells in the vasculature. Such binding accelerated the clearance of interacting moieties (i.e., AKR-A cells and liposomes). The present results suggest that targeting of liposomes to circulating in vivo is feasible.

The effect of high density lipoproteins (HDL) on the stability and clearance of injected liposomes was investigated under conditions of abnormal lipoprotein metabolism in vivo. Small unilamellar liposomes composed of phosphatidylcholine and containing quenched carboxyfluorescein were injected intravenously or intraperitoneally into normal mice or mice previously made lipoprotein deficient with 4-aminopyrazolo[3,4d]pyrimidine (4-APP). As evidenced from quenched carboxyfluorescein values in the blood, levels of stable liposomes in the circulation were increased and clearance rates reduced considerably in lipoprotein-deficient animals indicating increased bilayer integrity. This was confirmed by the demonstration that transfer of liposomal phosphatidylcholine to HDL, occurring in the presence of normal mouse plasma, was virtually abolished in the presence of plasma from lipoprotein deficient mice. The role of other lipoprotein species in destabilizing liposomes was also investigated. Plasma from lipoprotein-deficient mice was supplemented with increasing amounts of HDL, LDL + IDL or VLDL (to cover the physiological range of lipoprotein concentrations in mouse blood) prior to the addition of phosphatidylcholine liposomes, and incubated at 37 degrees C. It was shown that among the lipoprotein species studied only HDL was detrimental to liposomal stability under the conditions employed. Our results indicate that use of liposomal drugs in the treatment of patients must take into account HDL fluctuations in their blood as these could after liposomal membrane permeability to the drugs and thus upset therapeutic efficiency.

A procedure is described for the covalent coupling of p-aminophenyl-alpha-D-mannopyranoside, in the presence of carbodiimide, to a derivative of phosphatidylethanolamine (N-glutarylphosphatidylethanolamine) incorporated into the bilayers of multilamellar liposomes prepared by the dehydration-rehydration method. It appears that much of the phospholipid derivative exposed on the surface of the outer liposomal bilayer interacts with the aminosugar. The procedure is simple, does not destabilize liposomes and could be applied to other receptor-specific aminosugars.

Mouse monoclonal IgG1 specific for hepatitis B surface antigen and ovine polyclonal antibody raised against digoxin were covalently coupled by a diazotisation method to small unilamellar vesicles (SUV) composed of equimolar phospholipid and cholesterol supplemented with 6 mol% aminophenylstearylamine (APSA). Up to 33% of the antibody used was associated with vesicles, depending on the phospholipid and the antibody type used. Antibody-coated SUV were mixed with carboxyfluorescein (CF) or beta-galactosidase to generate multilamellar dehydration-rehydration vesicles (DRV) containing CF or active enzyme. In contrast, coupling of antibodies directly to beta-galactosidase-containing DRV resulted in total inactivation of the enzyme. About 85% of the SUV-bound antibody was recovered in DRV and of this, 78-82% was exposed on the liposomal surface, possibly because of reorientation of the APSA-antibody complex during DRV formation. Antibody-coated DRV remained stable in the presence of plasma at 37 degrees C and also under storage at 4 degrees C. Further, antibody coupled to such liposomes was capable of efficient interaction with the respective antigen. The present method allows the attachment of antibodies to the liposomal surface independently of entrapment of solutes, the activity of which is thus preserved, and could be adapted to alternative coupling procedures or ligands.

The previously established direct relationship between long half-life of uncharged small unilamellar liposomes in the circulation of injected animals and reduced permeability to entrapped solutes in the presence of blood plasma was investigated further. It was found that vesicle size and surface charge override state of membrane permeability in determining rates of vesicle clearance. Thus, half-lives of liposomes that were practically impermeable in plasma were much shorter for larger vesicles or vesicles with a negative charge on their surface. 111In-labelled bleomycin-containing small unilamellar liposomes with long half-lives accumulated in the liver after injection to a much lesser extent (e.g., 26%) than similar liposomes (55% of the dose) exhibiting a shorter half-life. Much of the injected long-lived liposomes (about 35%) were recovered in the carcass of the animals. Scanning of the carcasses revealed quantitative accumulation of radioactivity in the bones, presumably the phagocytic cells of the bone marrow. Long-lived liposomes appear suitable for drug delivery to, or imaging of the bone marrow.

The fate of small (30-60 nm) and large (about 400 nm diameter) liposomes composed of phosphatidylcholine or sphingomyelin and equimolar cholesterol and containing quenched carboxyfluorescein and 111In-labelled bleomycin after footpad injection of rats was investigated. Judging from the values of latent carboxyfluorescein in the plasma, phosphatidylcholine and sphingomyelin small liposomes entered the blood circulation intact, presumably via the lymphatics to reach 3 h after injection a peak of 16.9 and 23.1% of the dose per total blood, respectively. Of the 111In radioactivity given in phosphatidylcholine liposomes, about 32% was recovered in the liver. Hepatic uptake of 111In for sphingomyelin liposomes was lower (about 9%) reflecting their slower rate of clearance. No latent carboxyfluorescein could be detected in the blood after injection with phosphatidylcholine or sphingomyelin large liposomes and levels of 111In in the liver (and spleen) were very low. A proportionally much greater amount of liposomal 111In was intercepted by the popliteal and to a lesser extent, the lumbar lymph nodes. Such uptake was significantly higher (e.g. 463%) for small than for large (e.g. 195% per g popliteal nodes) liposomes. After foot pad injection of large liposomes into Walker 256 tumour-bearing rats, localization of 111In in the popliteal nodes was similar to that seen with control animals. However, 111In localization in the lumbar nodes was augmented more than 5-fold. These results suggest that large liposomes as used in this study, characterized by efficient drug entrapment, inability to reach the liver and spleen and improved localization in the lymph nodes of tumour-bearing animals may be preferable to vesicles of small size for the local treatment of lymphatic metastases.