Nano-Formulated Hydroxyurea Enhances Drug Delivery in K562 Cells

The vasculopathy of sickle cell disease (SCD), characterized by a chronic hemolytic anemia, endothelial dysfunction and recurrent vaso-occlusive events, underlies most of the clinical complications. Hydroxyurea (HU), the only FDA approved medication to treat patients with SCD, acts via the induction of fetal hemoglobin (HbF) production. Recent studies suggest that HU increases endothelial nitric oxide (NO) production and reduces adhesion molecule expression as another potential mechanism for the observed reduction in vaso-occlusive events and improved survival in SCD. Despite its benefits, HU is currently under-utilized and we hypothesize that improved targeted drug delivery may increase its usage. The use of nano-emulsions, a class of stable emulsions formed by a monolayer of phospholipids and/or biodegradable material, is a mechanism by which to enhance targeted cellular delivery of therapeutics. We hypothesized that a nano-formulation of HU would enhance drug delivery to the bone marrow and this may be a direct way of impacting HbF expression in SCD. Nano-formulated HU (NHU) was developed using a phospholipid approach and this compound was found to be biologically stable for > 6 months.

Chronic myelogenous leukemia bone marrow (K562) cells were treated with a NHU or HU suspension in concentrations ranging from 25µM to 250µM over a course of 4, 5 and 6 days. Cells were harvested for RNA isolation and qPCR was performed using primers for HbF and basal cell adhesion molecule (BCAM, a cellular adhesion molecule on red and white blood cells. On day 5, when HbF expression in K562 cells peaks, there was a five-fold increase in HbF expression treated with lower concentrations of NHU compared to untreated cells and a two-fold increase in expression compared with HU treated cells. With increasing concentrations of NHU and HU, HbF expression became similar. This suggests that the use of NHU can provide increased HbF expression at a lower concentration with likely less toxicity. Treatment of K562 cells with NHU and HU resulted in decreased BCAM expression on day 6 of treatment. Treatment with NHU produced a four-fold decrease in BCAM expression compared to the same concentration of a HU suspension which supports the concept that NHU can impact gene expression at lower doses than unformulated HU. Nanoformulated HU produces a molecule which is highly stable, and able to achieve higher intracellular concentrations with increased efficacy in K562 cells. This suggests a potential novel therapeutic for patients with sickle cell disease.