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.

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.

A new strategy for the reversible attachment of methoxypoly(ethylene glycol) (mPEG) to an amino-containing substrate is described. The strategy is based on formation of a benzyl carbamate linkage substituted with a disulfide in the para or ortho position. While being stable under nonreducing conditions, the dithiobenzyl (DTB) urethane linkage is susceptible to cleavage by mild thiolysis with cysteine resulting in release of the parent amino component of the conjugate in its original form. The method is exemplified by preparation of mPEG-DTB-alcohol, its activation and attachment to distearoylphosphatidylethanolamine (DSPE). The resulting lipopolymer incorporates into liposomes, which are capable of losing their polymer coating under conditions approximating those existing in vivo. Implications for drug delivery are briefly discussed.

Plasma-stable liposomes (100 nm) were prepared from dioleoylphosphatidylethanolamine (DOPE) and 3--6 tool% of a new disulfide-linked poly(ethylene glycol)-phospbolipid 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.

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.