The purpose of this study was to establish a new experimental approach to determine the maximum amount of camptothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultracentrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubility of CPT in the buffer was improved by approximately a factor of 10 (from ~2.7 to 15-50 µg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ~100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ~20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrinsic water solubility of the drug.

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Two innovative 20-S-camptothecin (CPT) formulations, previously found suitable to achieve therapeutically relevant CPT concentrations, were assessed for their in vitro cytotoxic potential as compared to an aqueous CPT solution, using the MTT assay. The formulations, cationic CPT-containing liposomes (CPT-Lip), hydroxypropyl-beta-cyclodextrin (HP-beta-CD) complexed CPT (CPT-CD) and a saturated aqueous CPT solution (CPT-Sol), were diluted in culture medium to appropriate CPT concentrations (4.7-300 ng/ml), and incubated with HT-29 and SW-480 human colon carcinoma cell lines. IC50 values were calculated after 48 and 72 h incubation for the HT-29 and SW-480 cell lines, respectively, and were found to be of the same magnitude for all formulations, with only a slight difference (CPT-Sol

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