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(ethyle ne glycol) (PEG), We studied three 'classical' coupling methods in whi ch Ab was attached at the bilayer surface of sL, and two new coupling methods in which Ab was attached at the PEG terminus. Parameters exami ned included 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 method in which ma leimide-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 mal eimide-derivatized Ab was coupled to thiolated PEG, moving the couplin g reaction away from the liposome surface, very high coupling efficien cies were achieved, and these immunoliposomes achieved good specific b inding to their target cells. Oxidizing the Fc region of the Ab and co upling it to the PEG terminus through a hydrazone bond was a less effi cient coupling method, but had the advantage of retaining Ab orientati on. Efficient remote-loading of doxorubicin was found for immunoliposo mes in which Ab was attached at the PEG terminus.