Purpose: Obatoclax is a small-molecule inhibitor of anti-apoptotic BCL-2 family members that is currently in Phase I clinical trials in patients with lung cancer, leukemia and lymphoma among others. Obatoclax is poorly water-soluble and requires Tween 80 for solubilization. The present study aims to develop a nanoformulation for the concurrent delivery of paclitaxel and obatoclax without using any organic solvents.
Methods: Paclitaxel/obatoclax-loaded mixed micelles were prepared by a solvent evaporation method. Polyethylene glycol-b-distearoylphosphatidylethanolamine (PEG-DSPE, 28 mg) and tocopheryl polyethylene glycol 1000 succinate (TPGS, 28.5 mg), and the varying amount of paclitaxel and obatoclax were dissolved in chloroform (1 ml) in a 25-ml round-bottom flask, which was rotor-evaporated to dryness at 37 ℃ to form a homogeneous thin drug-polymer film. The resulting thin film was further dried overnight under vacuum to remove any residual solvent. The film was hydrated with HEPES-buffered saline (HBS, 10 mM, pH 7.4, 1 ml) and then was centrifuged at 15,000 rpm to remove any undissolved polymers. The hydrodynamic diameter of the micelles was assessed by dynamic light scattering using a Nano Zetasizer. A reverse phase HPLC methodology was used to simultaneously quantify the concentration of paclitaxel and obatoclax. The HPLC system (Waters) has a Waters 2695 pump, a Phenomenex C18 column, and a Waters 2996 photodiode array detector. As an internal standard, α-naphthoflavone (2 μM) was used. The detection wavelengths for paclitaxel, obatoclax, and α-naphthoflavone were 230 nm, 312 nm, and 280 nm, respectively. The isocratic mobile phase comprised 30% (v/v) sodium phosphate buffer (25 mM, pH 3.0) and 70% (v/v) methanol, and the flow rate was 1.2 ml/min. The release kinetics of paclitaxel and obatoclax from the micellar nanocarriers was evaluated by a dialysis method. The anticancer activity of the drug-loaded nanoformulation was evaluated in human lung carcinoma A549 cells, as well as paclitaxel-resistant A549 cells.
Results: The drug-loaded mixed micelles were readily formed with minimal precipitation. The encapsulation efficiency for paclitaxel and obatoclax is 97% and 83%, respectively. The average hydrodynamic diameter of paclitaxel/obatoclax-loaded PEG-DSPE/TPGS mixed micelles was about 11 nm with a narrow distribution. In release study, the incorporation of paclitaxel and obatoclax into PEG-DSPE/TPGS mixed micelles resulted in significantly prolonged release half-lives for both drugs compared to those of the free drugs (Fig.1). The combination of paclitaxel with obatoclax significantly enhanced the cytotoxicity in A549 cells and paclitaxel-resistant (PTX-R) A549 cells (Fig.2).
Conclusion: Paclitaxel and obatoclax can be efficiently and stably loaded into PEG-DSPE/TPGS mixed micelles. The prolonged release of both drugs from the mixed micelles suggests that this nanocarrier system may retain these drugs for the extended period of time in the circulation. Further evaluation of paclitaxel/obatoclax loaded mixed micelles in animal models are warranted.