Udo Bakowsky

Targetability of novel immunoliposomes prepared by a new antibody conjugation technique

In order to develop long-circulating immunoliposomes (IL), which combine sterical stabilization with a superior targetability, we have introduced a new methodology for attaching monoclonal antibodies directly onto the distal ends of liposome-grafted polyethylene glycol (PEG) chains. Therefore, we have synthesized a new PEG-PE derivative, which had been endgroup-functionalized with cyanuric chloride. Antibodies can simply be coupled to this membrane anchor in mild basic conditions (pH 8.8) without the need for previous antibody derivatizations. The coupling results have been determined with consideration to various liposome parameters and have been compared to several established antibody coupling procedures, where antibodies had been linked directly to the liposome surface in the presence of PEG (conventional IL). To investigate the targetability of the resulting new IL, anti E-selectin mAb have been coupled and the degree of binding selectin-containing cells has been analyzed. The terminal coupled antibodies show a 1.8-fold higher degree of in vitro cell binding compared to conventional IL, which has been attributed to the antibody position being more easy accessible at the PEG termini. Furthermore, we have illustrated the liposome surface topology and the coupled antibodies by atomic force microscopy, which for such fluid IL has been used first. These images have finely corresponded to the cell binding results, and have been discussed in terms of antibody position and flexibility at the liposome surface.

The role of glycolipids in mediating cell adhesion: a flow chamber study

Selectins constitute a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium by recognizing various carbohydrate ligands in response to inflammation. To test the hypothesis that multivalent binding of selectins to their ligands is the molecular basis for achieving sufficient binding forces, we have performed this flow chamber study. Selectin-containing Chinese hamster ovarial cells (CHO-E) bind and roll along a support-fixed phospholipid membrane containing a defined concentration of a synthetic Sialyl Lewisx (sLex) glycolipid ligand. Ligands are either homogeneously distributed, or arranged in defined lateral clusters, as illustrated here for the first time. The lateral glycolipid clusters which appear as recognition motifs are essential for mediating cell rolling. Furthermore, the transition from firm cell adhesion to cell rolling depends on the site density of ligands. Rolling velocity shows little dependence on shear forces within a broad range. As we found out that cells do not roll along the model membranes with homogeneous ligand distribution, our results therefore support the hypothesis of multivalent binding events. Since these investigations suggest that lipid-anchored sLex, functionally embedded in a lipid matrix, can mediate cell rolling, this study demonstrates the relationship between dynamic glycolipid binding to selectins with the hypothesis of multivalency of binding for the first time.

Investigation of the orientation of purple membrane sheets in Langmuir-Blodgett films by a quartz crystal microbalance.

By means of the quartz crystal microbalance (QCM), a convenient method was developed to determine the degree of orientation of purple membrane (PM) sheets on the air/water interface. Langmuir‐Blodgett films from both wild‐type and SH‐mutant PM (bR D36C) were vertically deposited on the surface of goldsputtered AT‐cut quartz crystals. The shift of resonance frequency of the QCM during a special washing protocol allowed us to differentiate between physically adsorbed PM fragments and any PM attached to the gold surface via chemical bonds. By washing with organic solvents, complete desorption of the wild‐type PM was achieved, whereas for the SH‐mutant, 60% of the PM fragments could not be detached from the surface. These PM sheets should be oriented with the cytoplasmic side facing the water subphase so that their SH‐groups can chemically bind to the gold surface of the quartz plate.