(456h) Uptake, Co-Localization, Cytotoxicity and Transepithelial Transport of Acid-Labile Doxorubicin-Dendrimer Conjugates in an in Vitro Model of the Lung Adenocarcinoma Epithelium

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.  However, rapid elimination, uncontrolled release, systemic distribution, and life-threatening cardiotoxicity, has hindered the applicability of DOX and other potent anti-cancer agents.  The development of polymeric carriers for the modulation of transport, targeting and controlled release of potent anti-cancer agents and their formulation for local lung delivery is of great relevance in the treatment of lung adenocarcinoma.

In this work, a series of PEGylated poly(amidoamine) dendrimer nanocarriers with acid-labile and acid-nonlabile 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, toxicity in an alveolar adenocarcinoma cell line (A549) and effect of PEGylation degree on transport of dendrimer nanocarriers across lung epithelial cell line (calu-3).  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.  Mucus layer secreted by airway epithelial cells (calu-3) retained most of dendrimer carriers on apical side. However, high PEGylation degree significantly helped dendrimer nancarriers escape mucus trapping compared to bare dendrimer, aiding their transport to the basolateral side.  Reduced transepithelial electrical resistance (TEER) and limited cellular internalization demonstrated paracellular transport plays a vital role in transporting dendrimer nanocarriers across airway epithelium.  Dendrimer-DOX conjugates were formulated in the portable metered-dose inhalers using a new particle engineering strategy, and their aerosol characteristics were observed to be conducive to deep lung deposition.

Key words: Polyamidoamine dendrimer (PAMAM), doxorubicin, lung cancer, pressurized metered-dose inhalers (pMDI), controlled release.