Direct measurement of a tethered ligand-receptor interaction potential.

Many biological recognition interactions involve ligands and receptors that are tethered rather than rigidly bound on a cell surface. A surface forces apparatus was used to directly measure the force-distance interaction between a polymer-tethered ligand and its receptor. At separations near the fully extended tether length, the ligands rapidly lock onto their binding sites, pulling the ligand and receptor together. The measured interaction potential and its dynamics can be modeled with standard theories of polymer and colloidal interactions.

Impact of polymer tether length on multiple ligand-receptor bond formation.

The promoters of cell adhesion are ligands, which are often attached to flexible tethers that bind to surface receptors on adjacent cells. Using a combination of Monte Carlo simulations, diffusion reaction theory, and direct experiments (surface force measurements) of the biotin-streptavidin system, we have quantified polymer chain dynamics and the kinetics and spatial range of tethered ligand-receptor binding. The results show that the efficiency of strong binding does not depend solely on the molecular architecture or binding energy of the receptor-ligand pair, nor on the equilibrium configuration of the polymer tether, but rather on its "rare" extended conformations.

Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells.

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.

Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes.

Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (approximately 18 IgG per 100 nm liposome) exhibited long circulation times (MRT = 8.5 h, Cl = 0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRT < or = 0.7 h, Cl > or = 7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab' fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.

Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures.

Several coupling methods for binding antibodies (Ab) to liposomes have previously been developed. We were interested in examining if some of these methods would be suitable for attaching Ab to long-circulating formulations of liposomes (SL), sterically stabilized with poly(ethylene glycol) (PEG). We studied three 'classical' coupling methods in which Ab was attached at the bilayer surface of SL, and two new coupling methods in which Ab was attached at the PEG terminus. Parameters examined including binding efficiency, antibody surface density, the ability of the immunoliposomes to remote-load the anticancer drug doxorubicin, and the specific binding of the resulting immunoliposomes to target cells. The non-covalent biotin-avidin coupling method resulted in low Ab densities at the cell surface, as did a coupling in method in which maleimide-derivatized Ab was attached to the liposome surface through a thiolated phospholipid incorporated into the liposomes. The low levels of Ab achieved in these method was likely due to interference by PEG with the access of the Ab to the liposome surface. However, when a maleimide-derivatized Ab was coupled to thiolated PEG, moving the coupling reaction away from the liposome surface, very high coupling efficiencies were achieved, and these immunoliposomes achieved good specific binding to their target cells. Oxidizing the Fc region of the Ab and coupling it to the PEG terminus through a hydrazone bond was a less efficient coupling method, but had the advantage of retaining Ab orientation. Efficient remote-loading of doxorubicin was found for immunoliposomes in which Ab was attached at the PEG terminus.

A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells.

The development of long-circulating formulations of liposomes (S-liposomes), sterically stabilized with lipid derivatives of poly(ethylene glycol) (PEG), has increased the likelihood that these liposomes, coupled to targeting ligands such as antibodies, could be used as drug carriers to deliver therapeutic drugs to specific target cell populations in vivo. We have developed a new methodology for attaching monoclonal antibodies to the terminus of PEG on S-liposomes. A new end-group functionalized PEG-lipid derivative pyridylthiopropionoylamino-PEG- distearoylphosphatidylethanolamine (PDP-PEG-DSPE) was synthesized for this purpose. Incorporation of PDP-PEG-DSPE into S-liposomes followed by mild thiolysis of the PDP groups resulted in formation of reactive thiol groups at the periphery of the lipid vesicles. Efficient attachment of maleimide-derivatized antibodies took place under mild conditions even when the content of the functionalized PEG-lipid in S-liposomes was below 1% of total lipid. The resulting S-immunoliposomes showed efficient drug remote loading, slow drug release rates and increased survival times in circulation compared to liposomes lacking PEG. When antibodies recognizing several different tumor-associated antigens were coupled to the PEG terminus of S-liposomes a significant increase in the in vitro binding of liposomes to the target cells was observed. The binding of S-immunoliposomes containing entrapped doxorubicin to their target cell population resulted in increased cytotoxicity compared to liposomes lacking the targeting antibody.

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.

Nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump.

Folic acid, attached to polyethyleneglycol-derivatized, distearoyl-phosphatidylethanolamine, was used to target in vitro liposomes to folate receptor (FR)-overexpressing tumor cells. Confocal fluorescence microscopic observations demonstrated binding and subsequent internalization of rhodamine-labeled liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiFR cells), with inhibition by free folic acid. Additional experiments tracking doxorubicin (DOX) fluorescence with DOX-loaded, folate-targeted liposomes (FTLs) indicate that liposomal DOX is rapidly internalized, released in the cytoplasmic compartment, and, shortly thereafter, detected in the nucleus, the entire process lasting 1-2 h. FR-mediated cell uptake of targeted liposomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-HiFR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contrast to uptake of free DOX, based on verapamil-blockade experiments with quantitation of cell-associated DOX and flow cytometry analysis. Delivery of DOX by FTLs to M109R-HiFR cells increased continuously with time of exposure, reaching higher drug concentrations in whole cells and nuclei compared with exposure to free DOX. The in vitro cytotoxic activity obtained with DOX-loaded FTLs was 10-fold greater than that of the nontargeted liposome formulation, but was not improved over that of free DOX despite the higher cellular drug levels obtained with the targeted liposomes in M109R-HiFR cells. However, if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week time span, FTL DOX was significantly more tumor inhibitory than free DOX. It is suggested that the biological activity of liposomal DOX released inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an in vivo environment for full expression.

Cardioprotection by liposome-conjugated sialyl Lewisx-oligosaccharide in myocardial ischaemia and reperfusion injury.

OBJECTIVES: Selectins are important adhesion molecules which utilize a carbohydrate ligand such as sialyl Lewisx (SLex). Our objective was to study the effects of a liposome-conjugated SLex (Lipo-SLex) in myocardial ischaemia (MI) and reperfusion (R) injury in order to further clarify the actions of this carbohydrate. METHODS: We studied the efficacy of Lipo-SLex in a feline model of MI (90 min) and R (270 min) injury in vivo. Lipo-SLex (400 micrograms SLex/kg, iv) was administered intravenously 10 min prior to R. We also utilized an in vitro system of neutrophil adherence to thrombin-stimulated coronary endothelium to validate the efficacy of Lipo-SLex. RESULTS: Lipo-SLex significantly attenuated myocardial necrosis (8.6 +/- 1.2 vs. 29.5 +/- 3.1% of area-at-risk, P < 0.01) and plasma creatine kinase activities (P < 0.01) compared to vehicle (liposome alone). Moreover, endothelium-dependent relaxation to acetylcholine and A23187 in ischaemic-reperfused coronary rings obtained from cats treated with Lipo-SLex was significantly preserved compared to cats given liposomes without SLex (P < 0.01). After reperfusion, ex vivo PMN adherence to ischaemic-reperfused coronary endothelium was significantly increased in vehicle-treated cats, however, this was significantly attenuated in Lipo-SLex-treated cats (82 +/- 7 vs. 28 +/- 3 PMNs/mm2, P < 0.01). Myeloperoxidase activity in the ischaemic myocardium, a marker of PMN accumulation, was also significantly attenuated in Lipo-SLex-treated cats compared to liposomes without SLex (P < 0.01). CONCLUSIONS: Liposome-conjugated SLex-oligosaccharide attenuates myocardial necrosis and preserves coronary endothelial function following MI/R in vivo. The mechanism appears to be mediated by inhibition of the initial PMN-endothelial interaction and eventual accumulation into the ischaemic cardiac tissue. The liposome-SLex complex may be an efficient drug formulation for acute inflammatory diseases.

Long circulating, cationic liposomes containing amino-PEG-phosphatidylethanolamine.

Ligand attachment to polyethylene glycol (PEG) grafted, long circulating liposomes at the polymer terminus is of interest for targeting but the effect of positively charged groups is unknown. Amino-polyethylene glycol-phosphatidylethanolamine (AminoPEG-PE), prepared in four steps from alpha-amino-omega-hydroxy-PEG, was tested for influence on liposome interactions in vivo: blood circulation and biodistribution. Despite surface amines on each liposome conferring cationic behavior, in vivo properties are comparable to those obtained with methoxy-PEG-PE. The consequences are profound for targeting and possibly systemic delivery of cationic lipidic-polynucleotide complexes.

Liposomes with detachable polymer coating: destabilization and fusion of dioleoylphosphatidylethanolamine vesicles triggered by cleavage of surface-grafted poly(ethylene glycol).

Plasma-stable liposomes (100 nm) were prepared from dioleoylphosphatidylethanolamine (DOPE) and 3-6 mol% of a new disulfide-linked poly(ethylene glycol)-phospholipid conjugate (mPEG-DTP-DSPE). In contrast to similar preparations containing non-cleavable PEG-phospholipid conjugate, thiolytic cleavage of the grafted polymer chains facilitated rapid and complete release of the liposome contents. Furthermore, the detachment of PEG from DOPE liposomes resulted in liposomal fusion. Finally, while formulation of pH-sensitive DOPE/cholesterol hemisuccinate liposomes with mPEG-DTP-DSPE abolished the pH sensitivity, cleavage of the PEG chains completely restored this property. These are the first examples of new useful properties of liposomes grafted with cleavable polymer.

Improved imaging of infections by avidin-induced clearance of 99mTc-biotin-PEG liposomes.

UNLABELLED: This article describes the preparation and optimization of biotin-polyethyleneglycol (PEG) liposomes and their application in experimental infection models to improve the scintigraphic imaging of infection and inflammation. METHODS: Biotin was coupled to PEG-distearoylphosphatidylethanolamine (DSPE) and subsequently incorporated in the PEG liposomes. Biotinylated liposomes were radiolabeled with 99mTc-hydrazinonicotinamide. In vitro binding studies were performed to find the optimal biotin concentration in the liposomes. In rats the biodistribution of the 99mTc-biotin-PEG liposomes was compared with the biodistribution of normal (nonbiotinylated) 99mTc-PEG liposomes. Furthermore, in vivo studies in rats were performed to study both the effect of the biotin content and the optimal avidin dose for efficient clearance of the liposomes. Liposomes containing 0.5 or 1.0 mol% biotin-PEG-DSPE were compared in rats with a Staphylococcus aureus infection in the left calf muscle. Avidin was injected 4 h after injection of the liposomes. RESULTS: Biotinylation of the liposomes did not affect their in vivo behavior. All biotin-PEG liposome formulations tested showed good in vitro avidin binding with 50% inhibitory concentrations ranging from 36 to 8 micromol/L. With avidin doses higher than 100 microg, both preparations rapidly cleared from the circulation. As a result, abscess-to-blood ratios increased 5-fold. To illustrate the potential of the avidin-induced clearance of radiolabeled PEG liposomes, we also studied the 99mTc-biotin-PEG liposomes in rabbits with a subcutaneous S. aureus abscess. The infection was visualized only after injection of 100 microg avidin. CONCLUSION: This study shows that biotin-coated 99mTc-PEG liposomes in combination with the injection of avidin can lead to improved imaging of infection or inflammation localized especially in regions with high blood-pool activity.

Evaluation of blood clearance rates and biodistribution of poly(2-oxazoline)-grafted liposomes.

Two amphipatic polymers of the poly(2-oxazoline) family, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ), were synthesized with the carboxylic group positioned at either the initiation or termination ends of the polymer chains. Distearoylphosphatidylethanolamine was covalently linked to the carboxyl groups of the polymers, resulting in conjugates which incorporate readily into liposomes. Systematic evaluation of plasma clearance kinetics and biodistribution of liposomes containing hydrogenated soy phosphatidylcholine, cholesterol, and 5 mol % the polymer-lipid conjugates in mice revealed the following. Both polymers, PMOZ and PEOZ, exhibited long plasma lifetimes and low hepatosplenic uptake. PMOZ was more effective at decreasing blood clearance rates than PEOZ. The best results, which were quantitatively comparable to the results obtained with the optimized preparations of methoxypolyethylene glycol(PEG)-2000-grafted liposomes, were obtained with formulations containing PMOZ of molecular weight 3260.

Synthesis of an end-group functionalized polyethylene glycol-lipid conjugate for preparation of polymer-grafted liposomes.

Synthesis of a distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) conjugate, bearing a hydrazide group at the unattached end of the polymer chain, was achieved using a new heterobifunctional polymeric reagent. The heterobifunctional PEG derivative carrying on one end a reactive succinimidyl carbonate (SC) group and at the other terminal a tert-butyloxycarbonyl (Boc) protected hydrazide group was prepared by an efficient four step process from readily available PEG-2000. The SC-end group of the polymer reacted readily with the amino group of DSPE forming a stable urethane attachment between lipid and PEG. Acidolytic removal of the Boc group yielded a hydrazide-PEG-lipid conjugate suitable for preparation of polymer-grafted liposomes. Taking advantage of the well-documented chemical versatility of hydrazide groups, various biologically relevant ligands can be linked to this type of functionalized liposomes.

Enzymatic activity of chymotrypsin and its poly(ethylene glycol) conjugates toward low and high molecular weight substrates.

Native chymotrypsin and its polyethylene glycol (PEG) conjugates, obtained using the succinimidyl carbonate of methoxy-PEG (SC-PEG) as the amino group modifying reagent, were tested for their activity toward several low and high molecular weight substrates. Tripeptide and tetrapeptide p-nitroanilides either as N alpha-benzyloxycarbonyl derivatives or attached via their N-terminals to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers or to PEG, as well as the proteins albumin and azoalbumin, were used as substrates. Extensively modified chymotrypsin bearing on average of 14 PEG chains per chymotrypsin molecule (PEG14CHT) and a moderately modified one containing 10 PEG chains (PEG10CHT) both degraded the low molecular weight p-nitroanilides at rates comparable to, and in the case of PEG10CHT, greater than, the rates exhibited by the native enzyme. Synthetic high molecular weight substrates and azoalbumin were also degraded by the PEG-modified enzymes. However, rates of such enzymatic reactions were lower for the PEG-enzymes than for the native one. Native albumin, as compared to azoalbumin, resisted degradation by both PEGCHTs yet was readily digested by the native chymotrypsin. The results obtained indicate that substrate-size-dependent specificity of PEG-modified enzymes cannot be explained solely by steric hindrance considerations. The decreased activity of PEG-enzymes toward protein substrates is consistent with the well-documented ability of PEG to exclude proteins from its surroundings and with the influence of protein unfolding on the susceptibility to degradation.

New amphipatic polymer-lipid conjugates forming long-circulating reticuloendothelial system-evading liposomes.

Lipid-conjugates of two amphipatic polymers, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ) (degree of polymerization approximately 50) were synthesized by linking glutarate esters of the polymers to distearoylphosphatidylethanolamine (DSPE) or alternatively by termination of the polymerization process with DSPE. Surface-modified liposomes (90 +/- 5 nm) prepared from either conjugate (5 mol % of total lipid) were injected into rats and followed by blood level and tissue distribution measurements. Both polymers PEOZ and PMOZ were found to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as polyethylene glycol (PEG), the best known material for this purpose. This is the first demonstration of protection from rapid recognition and clearance conveyed by alternative polymers, which is equal to the effect of PEG.

A convenient general method for synthesis of N alpha- or N omega-dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification.

N alpha-Dithiasuccinoyl (Dts) amino acids needed for solid-phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side-chain protected amino acid derivatives are first reacted with a polymeric xanthate, following which the free alpha-carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride. Upon aqueous work-up, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer-bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N-(iso-propyldithio)carbonyl derivative of proline is also presented.

Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins.

Methoxypolyethylene glycol of molecular weight 5000 was converted to a reactive succinimidyl carbonate form (SC-PEG). The usefulness of this new polymeric reagent for the covalent attachment of polyethylene glycol to proteins was evaluated. SC-PEG was found to be sufficiently reactive to produce extensively modified proteins under mild conditions within 30 min, showing the highest reactivity around pH 9.3. The commonly used succinimidyl succinate derivative of methoxypolyethylene glycol (SS-PEG) served as a reference standard to which the new reagent was compared. The stability of the polymer-protein linkages, studied on a series of PEG-modified bovine serum albumins, provided the single most important difference between the two activated polymers. Urethane-linked PEG-proteins obtained through the use of SC-PEG showed considerably higher chemical stability than SS-PEG-derived conjugates. The measured rate constants of aminolysis (using N alpha-acetyllysine) and hydrolysis showed that SC-PEG is slightly less reactive yet more selective of the two reagents. Hydrolysis of the active groups on SC-PEG was on average twofold slower than that on SS-PEG. The differences in the rates of aminolysis were even smaller than those in hydrolysis. PEG-trypsin conjugates produced by both activated polymers showed similar properties: they had no proteolytic activity, well-preserved esterolytic activity, and enhanced activity toward p-nitroanilide substrates. Michaelis-Menten constants of the modified enzymes were determined using N alpha-benzyloxycarbonyl-L-arginine p-nitroanilide. These measurements indicated that the attachment of PEG to trypsin caused an increase in both the rate of turnover of the substrate and its affinity toward the modified enzymes. Through a series of experiments involving the appropriate polymeric and low-molecular-weight model compounds, it was demonstrated that these increases in amidolytic activity were unrelated to tyrosyl residues acylation by either one of the activated polymers.

Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains.

Novel conjugates tailor-made for inclusion in liposomal formulations, containing distearoylphosphatidylethanolamine (DSPE) as a lipid anchor, heterobifunctional polyethylene glycol (PEG) with a molecular weight of 2000 as a linking moiety, and a biological cell adhesive ligand [YIGSR peptide or Sialyl Lewis(X) oligosaccharide (SLX)], were synthesized. They were characterized by NMR, chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Inclusion of either of the ligand-PEG-lipid conjugates (2 mol %) in a lecithin/cholesterol/ methoxy-PEG2000-DSPE (55:40:3 mole ratio) lipid mixture followed by preparation of unilamellar vesicles (100 nm) resulted in positioning of 55% of the YIGSR and 63% of the SLX ligands on the periphery of the outer surface-grafted polymeric "brush", as determined by a combination of specific enzymatic alterations of each ligand and HPLC. Similar densities of ligand-bearing PEG chains were incorporated into liposomes by simply incubating (37 degrees C, 5 h) either one of the ligand-PEG-lipid conjugates with preformed lipid vesicles. This conjugate insertion process was aggregation free. Using enzymatic derivatization-HPLC, it was demonstrated that all the ligands incorporated into lipid membranes by this new approach were positioned exclusively on the outer leaflet of the liposomal bilayers. Since liposomes of this type are intended for in vivo use as long-circulating, ligand-presenting platforms, the insertion approach is preferable because of the more efficient utilization of ligand-PEG-lipid conjugates.

Targeting of stealth liposomes to erbB-2 (Her/2) receptor: in vitro and in vivo studies.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.