Table 2 CTT2-SL liposomes were made by pipetting the above-mentioned lipid mixture except the CTT2-PEG-lipid, to a round bottomed flask, dried under nitrogen and lyophilized for 2h to remove trace amounts of chloroform. Doxorubicin liposomes were prepared by using standard pH gradient technique [1]. To synthesize CTT2-PEG-3400-DSPE Caelyx/doxil-liposomes, CTT2-PEG-DSPE (1mg) was suspended in 400μl of buffer (100mM histidine, 55mM sucrose, pH 6.5), and 100μl of this CTT2-PEG-DSPE micelle suspension was added to 1ml Doxil/Caelyx solution or internally prepared similar to doxil-liposomes (Ortho Biotech).

In vivo murine studies were performed after incubating the mixture Inhibitors,research,lifescience,medical for 30min at 60°C. The incorporation efficiency, the percentage of total activity contained

in the liposome fractions, was measured by using radioisotope-labeled peptide and gel filtration to separate the unreacted micelle from the liposome; optimal reaction conditions Inhibitors,research,lifescience,medical were found to be 60°C at 30min (nearly 100% efficient). The doxorubicin leakage from the liposomes after the incorporation experiments was determined by comparing the amount of free doxorubicin versus liposome-bound doxorubicin before and after the experiment. The leakage was found to be minimal (the leakage before the incorporation Inhibitors,research,lifescience,medical was in product information Average 4.5% and after Inhibitors,research,lifescience,medical the reaction in average 4.2%). 2.4. Radiolabeling of Peptides Radiolabeling of peptides and all liposomal formulations with iodine-125 (125I) was performed using the IODOGEN (Pierce, Rockford, IL). The CTT2-PEG3400-DSPE peptide was labeled with 125I using iodogen as a catalyst. 5MBq of Na125I (Amersham, Buckinghamshire, England) in 0.5ml PBS was added to a tube containing Inhibitors,research,lifescience,medical 10μg dried iodogen and 100μg CTT2-PEG3400-DSPE peptide construct. The mixture was incubated for 20min at room temperature. The 125I-bound

particle fractions were purified by elution from PD-10 columns. The activity of the peptide was determined in a gamma counter (Cobra II, Packard Instruments). 2.5. Animal Models and Tumor Inoculation The mice were cared for according to the instructions of the animal facility, and the experiments were approved GSK-3 by an ethical committee of Helsinki University, Finland. Male athymic nu/nu mice (6–8weeks old, Harland) were provided with water and maintained on regular diets ad libitum. Subcutaneous human serous ovarian carcinoma (OV-90) xenograft models were generated by coinjecting equal volumes of cells (~5×106/100μl phosphate buffered saline, PBS) and matrigel subcutaneously into the hindlegs of nude mice. Average tumor volumes of 65mm3–200mm3were used for all studies. 2.6. In Vivo Biodistribution and Pharmacokinetics Following single i.v.