Thermosensitive
liposomes are attractive vehicles for the delivery and release of drugs to
tumors. To improvethe targeting efficacy for
breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z(HER2:342)-Cys) was conjugated to the surface of
liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as "Affisomes" were investigated. Thermosensitive small unilamellar vesicle (SUV)
liposomes of (80-100 nm) a diameter consisting of
dipalmitoyl phosphatidylcholine (DPPC, Tm 41 degrees C) as the matrix
lipid and a
maleimide-conjugated pegylated
phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication.
Fluorescent probes were incorporated into
liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these
liposomes via its C-terminal
cysteine by incubation in the presence of a
reducing agent (e.g.,
tributylphosphine) for 16-20 hours under an
argon atmosphere.
Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4-12%
Bis/Tris gel under reducing conditions. Affibody conjugation yields were approximately 70% at a
protein-
lipid ratio of 20 microg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive
liposomes did not have any significant effect on the hydrodynamic size distribution of the
liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped
calcein (a water-soluble
fluorescent probe, lambdaex/em 490/515 nm) as a function of temperature.
Calcein was released from Affisomes (thermosensitive
liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive
liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90-100%) at 41 degrees C. In contrast,
liposomes prepared from Egg
phosphatidyl choline (Egg PC, Tm approximately 0 degrees C) under similar conditions released only 5-10%
calcein at 41 degrees C. Affisomes, when stored at room temperature, retained > 90% entrapped
calcein up to 7 days. Moreover, incubation of
liposomes in
phosphate-buffered saline, supplemented with 10% heat-inactivated
serum (fetal bovine serum) did not result in a destabilization of
liposomes. Therefore, Affisomes present promising, novel
drug-delivery candidates for
breast cancer targeting.