No abstract

Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.

No abstract

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.

No abstract

Liposomes (70-100 nm) of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, and poly(ethylene glycol) (PEG)-modified phosphatidylethanolamine (PEG-DSPE) were conjugated to Fab' fragments of a humanized recombinant MAb against the extracellular domain of HER2/neu to create sterically stabilized immunoliposomes (anti-HER2 SL) as a drug carrier targeting HER2-overexpressing cancers. Conjugation employed maleimide-terminated membrane-anchored spacers of two kinds: a short spacer, providing attachment of Fab' close to the liposome bilayer, or a long spacer, with Fab' attachment at the distal terminus of the PEG chain. Confocal microscopy and spectrofluorometry of HER2-overexpressing breast cancer cells incubated with fluorescently labeled anti-HER2 SL prepared with either spacer showed binding of liposomes (8000-23000 vesicles/cell) followed by endocytosis (rate constant ke = 0.012-0.033 min-1) via the coated-pit pathway, evidenced by intracellular acidification and colocalization with transferrin. Uptake of anti-HER2 immunoliposomes by breast cancer cells with low HER2 expression, or after preincubation of cells with free anti-HER2 Fab', was less than 0.2% and 4.3%, respectively, of the uptake by HER2-overexpressing cells. Increasing PEG-DSPE content (up to 5.7 mol %) in anti-HER2-SL prepared with the short spacer decreased liposome-cell binding affinity 60-100-fold, while ke decreased only 2-fold; however, when Fab' fragments were conjugated via a PEG spacer, both binding affinity and ke were unaffected by PEG-DSPE content. Cell binding and internalization of anti-HER2 immunoliposomes increased at higher surface density of conjugated Fab' fragments, reaching plateaus at approximately 40 Fab'/liposome for binding and approximately 10-15 Fab'/liposome for internalization. Uptake of anti-HER2 immunoliposomes correlated with the cell surface density of HER2 and significantly (p < 0.005) correlated with the antiproliferative effect of the targeting antibody but not with the total level of cellular HER2 expression. The results obtained were used to optimize in vivo preclinical studies of anti-HER2 SL loaded with antineoplastic drugs.

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.

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.