(416e) Preparation and Characterization of Pegylated, Acid-Labile Dendrimer-Doxorubicin Conjugates and Their Aerosol Formulation in Propellant-Based Inhalers

Lung cancer is the leading cause of cancer death among both men and women in the United States.  Adenocarcinoma accounts for more than 40% of all lung cancer cases.  Doxorubicin (DOX) is an FDA-approved chemotherapeutic that has been widely used as primary anticancer drug in the treatment of a variety of cancers including lung cancers.  However, rapid elimination, uncontrolled release, and life-threatening cardiotoxicity, has hindered the applicability of DOX and other potent anti-cancer agents.  The potential efficacy of DOX in the treatment of lung adenocarcinoma is also limited by low accumulation in the lung tissue, which is usually administered through systemic route.  The development of polymeric carriers for the modulation of transport, targeting and controlled release of potent anti-cancer agents and their aerosol formulation for local lung delivery is of great potential relevance in the treatment of lung adenocarcinoma.

In this work, a series of PEGylated poly(amidoamine) dendrimer nanocarriers with acid-labile DOX conjugates were synthesized and characterized.  We employed a two-step PEGylation strategy to increase the payload of the hydrophobic DOX.  We investigated the impact of pH (neutral and acidic pH), PEGylation density (low, medium and high) and number of DOX conjugates (low and medium) on the release of DOX from the dendrimer nanocarrier, the kinetics of carrier uptake, intracellular release kinetics of DOX from the nanocarrier, and toxicity in an alveolar adenocarcinoma cell line (A549). PEGylation retards the release of DOX in acidic medium, and also intracellularly, as determined by co-localization studies with confocal microscopy.  We also observed that the kinetics of cellular entry of the nanocarrier with DOX increased significantly compared to free DOX, as determined by flow cytometry.  At the highest PEGylation density, the rate of internalization of the nanocarrier containing DOX was even higher than that of free DOX.  PEGylation density also affects cytotoxicity as seen by an increase in IC₅₀ for DOX-conjugated dendrimer compared to free DOX, due to the controlled release of the therapeutic. PEGylated dendrimer-DOX conjugates were directly formulated in the propellant-based metered-dose inhalers (pMDIs) with a trace amount of ethanol (ca. 0.2%), forming a solution aerosol formulation.  The stability and aerosol characteristics increased significantly as PEG density increased and DOX payload decreased.  The formulation of highly PEGylated dendrimer-DOX conjugates resulted in aerosols with high deep lung deposition (up to 80%).  These results are rationalized based on the phase behavior of the polymer in propellant HFA.