15 th International Symposium on MICROENCAPSULATION, Parma (Italy), September 18-21, 2005 MICROENCAPSULATION OF POORLY SOLUBLE ANTI-CANCER DRUGS INTO MICELLES MADE OF POLYETHYLENE GLYCOL-PHOSPHATIDYL ETHANOLAMINE (PEG-PE) CONJUGATES Li Mu, Tamer A. Elbayoumi, Aruna Karkala, Suna Erdogan, Rupa D. Dabholkar, Tatyana S. Levchenko, Dmitry A. Mongayt, Vladimir P. Torchilin Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA E-mail: v.torchilin@neu.edu Key words: POORLY SOLUBLE DRUGS, POLYMERIC MICELLES, PEG-PE, PACLITAXEL, CAMPTOTHECIN, PHOTODYNAMIC THERAPY INTRODUCTION Poor water solubility of many anticancer agents (such as paclitaxel, PCT; camptothecin, CPT; and certain porphyrins like meso-tetraphenylporphine, TPP, used in photodynamic therapy, PDT) hinders their application and complicates direct parenteral administration. Various formulation strategies based on the use of drug carrier systems have been suggested to overcome their poor solubility, low stability, and toxic side effects (1,2). Among such systems, polymeric micelles have drawn much attention owing to their easily controlled properties and good pharmacological characteristics (3). Micelles prepared from PEG-diacyllipids conjugates, such as PEG- PE, are of particular interest (4). Here, we describe the preparation, properties, and activity against cancer cells in vitro of PCT-, CPT-, and TPP- loaded PEG-PE micelles as well as mixed micelles made of PEG-PE and D-α-tocopheryl polyetheyene glycol 1000 succinate (TPGS). EXPERIMENTAL METHODS Drug-loaded micelles were prepared by dispersing dry film of the mixture of micelle-forming material (PEG-PE or/and TPGS) and drug in an aqueous buffer solution. The content of the drug incorporated into the micelles was assayed by HPLC for PCT and CPT and by fluorescence spectroscopy for TPP. The particle size analysis was done by the dynamic light scattering. The in vitro drug release from various micelles was investigated in an aqueous solution containing sodium salicylate in order to create pseudo-sink conditions. Murine LLC1 Lewis lung carcinoma and human HT-29 colorectal adenocarcinoma and MCF-7 breast cancer cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) and Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in 5% CO 2 . Standard in vitro cytotoxicity tests were with PCT- and CPT-loaded PEG-PE micelles. For phototoxicity studies, cells were grown in a 96-well microplate overnight and then incubated at 37°C with various concentrations of the free TPP and TPP in PEG- PE micelles in the darkness for 6 and 18h. After the incubation, the cells were washed to remove the non-cell- associated drug, and the plates were photo-irradiated for 30 min with the light source (LC-122) 630 nm at 12 mm spot. The cells were (as incubated for another 24 h, and the viability of irradiated and non-irradiated cells control) was evaluated using the MTT [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide] assay. RESULTS AND DISCUSSION Drug-loaded micelles of various compositions have been prepared (PEG-PE micelles with variable MW of PEG, 2,000 and 5,000 Da; and mixed micelles of PEG-PE and TPGS). The size of all micelles was within the 5-to-30 nm interval. The methods used allowed for the samples containing 15-to-200 µg of a drug per mL of the micelle suspension depending on the drug and micelle composition (CPT is the least solubilized, while TPP is the best solubilized drug; mixed micelles provide better solubilization as well as micelles made of PEG-PE with “longer” PEG block). Micelles are rather stable, since the storage of drug-loaded micelles at either room temperature or at 4°C for over a month did not cause any changes in micelle size. They firmly retain the incorporated drug – some initial burst drug release was observed (about 15% of incorporated drug – probably, the drug loosely associated with micelle corona) with no further drug loss over weeks. In vitro antiproliferative activity of various preparations (typical data for CPT- and TPP-loaded micelles are shown in Figs 1 through 4) indicates that the cytotoxicity of drug-loaded PEG-PE-based micelles was markedly higher compared to the free drug (empty micelles do not show any cytotoxicity). Thus, micellar preparations of CPT were cytotoxic against LLC1 and HT-29 cells even at CPT concentrations below 50 ng/ml (Figs 1 and 2). A representative cytotoxicity for 6 and 18 hrs incubation with TPP-loaded PEG-PE micelles before and after the light treatment are shown in Figs. 3 and 4 for LLC and MCF-7 cells. In both cases, there was a dramatic increase in the post-light-irradiation cytotoxicity (PDT conditions) 5