HER2-Specific Affibody -Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anti-Cancer Agents

摘要

Thermosensitive liposomes are attractive vehicles for delivery and release of drugs to tumors. To improve targeting efficacy for breast cancer treatment, an 8.3 kDa HER2-specific Affibody molecule (ZHER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nano particles denoted “Affisomes” were investigated. Thermo-sensitive small unilamellar vesicles (SUV) liposomes of (80–100 nm) diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41 °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 (tributylphosphine) for 16–20 hours under 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 ~70% at a protein/lipid ratio of 20 μg/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. Thermo-sensitivity of Affisomes was determined by monitoring release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as non-targeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg-phosphatidyl choline (Tm ~0 °C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained >90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in PBS supplemented with 10% heat-inactivated serum (FBS) did not result in destabilization of liposomes. Therefore, Affisomes present promising and novel drug delivery candidates for breast cancer targeting.