Abstract.We have initiated studies to enhance targeted delivery of an anticancer agent, curcumin, for prostate cancer treatment by incorporating this agent into the liposomes (nanodelivery vehicles primarily composed of phospholipids) coated with prostate membrane specific antigen specific antibodies. We prepared curcumin-loaded liposomes of various lipid compositions by sonication at an average size of 100-150 nm. Un-entrapped curcumin was removed by size exclusion chromatography. Data show that curcumin preferentially partitioned into liposomes prepared from dimyristoyl phosphatidyl choline (DMPC) and cholesterol among the various compositions tested. The anti-proliferative activity of liposomal curcumin was studied using two human prostate cancer cell lines (LNCaP and C4-2B) by a tetrazolium dyebased (MTT) assay. Treatment of cells with liposomal curcumin (5-10 μM) for 24-48 h at 37˚C resulted in at least 70-80% inhibition of cellular proliferation without affecting their viability. On the other hand, free curcumin exhibited similar inhibition only at 10-fold higher doses (>50 μM). We also observed that LNCaP cells were relatively more sensitive to liposomal curcumin mediated block of cellular proliferation than C4-2B cells. We are currently developing liposome formulations with targeting ability to further improve the efficacy of curcumin in vivo.

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 (Z HER2:342 -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.