Intracellular PK/PD Relationships of Free and Liposomal Doxorubicin: Quantitative Analyses and PK/PD Modeling.

Nanomedicines are widely studied for intracellular delivery of cancer drugs. However, the relationship between intracellular drug concentrations and drug responses are poorly understood. In this study, cellular and nuclear concentrations of doxorubicin were quantified with LC/MS after cell exposure with free and liposomal doxorubicin (pH-sensitive and pegylated liposomes). Cellular uptake of pegylated liposomes was low (∼3-fold extracellular concentrations) compared with doxorubicin in free form and pH-sensitive liposomes (up to 280-fold extracellular concentrations) in rat glioma (BT4C) and renal clear cell carcinoma (Caki-2) cells. However, after the cell exposure with pegylated liposomes, intracellular doxorubicin was distributed into the nuclear compartment in both cell types. Despite high drug concentrations in the nuclei, Caki-2 cells showed strong resistance toward doxorubicin. A model was successfully built to describe PK/PD relationship between drug concentrations in nucleus and cytotoxic responses in BT4C cells. This model is the first step to link target site concentration of doxorubicin into its effect and can be a useful part of more comprehensive future in vivo PK/PD models.

Human placental cell and tissue uptake of doxorubicin and its liposomal formulations.

The anticancer drug doxorubicin and its liposomal formulations are in clinical use, doxorubicin also during pregnancy. However, little is known about how doxorubicin and its liposomal formulations are taken up by placental cells and whether they can cross human placenta. We therefore investigated quantitative cellular uptake and toxicity of doxorubicin and its two liposomal formulations, pH-sensitive liposomal doxorubicin (L-DOX) and commercially available pegylated liposomal doxorubicin (PL-DOX), in human placental choriocarcinoma (BeWo) cells. PL-DOX showed significantly lower cellular uptake and toxicity compared with doxorubicin and L-DOX. In preliminary studies with human placental perfusion, PL-DOX did not cross the placenta at all in 4h, whereas doxorubicin and L-DOX crossed the placenta at low levels (max 12% of the dose). Furthermore, PL-DOX did not accumulate in placental tissue while doxorubicin did (up to 70% of the dose). Surface pegylation probably explains the low placental cell and tissue uptake of PL-DOX. Formulation of doxorubicin thus seems to enable a decrease of fetal exposure.

Targeted delivery via avidin fusion protein: intracellular fate of biotinylated doxorubicin derivative and cellular uptake kinetics and biodistribution of biotinylated liposomes.

In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.