Pre-Clinical Evaluation of rHDL Encapsulated Retinoids for the Treatment of Neuroblastoma

Abstract

Despite major advances in pediatric cancer research, there has been only modest progress in the survival of children with high risk neuroblastoma (NB) (HRNB). The long term survival rates of HRNB in the United States are still only 30-50%. Due to resistance that often develops during therapy, development of new effective strategies is essential to improve the survival and overcome the tendency of HRNB patients to relapse subsequent to initial treatment. Current chemotherapy regimens also have a serious limitation due to off target toxicity. In the present work, we evaluated the potential application of reconstituted high density lipoprotein (rHDL) containing fenretinide (FR) nanoparticles as a novel approach to current NB therapeutics. The characterization and stability studies of rHDL-FR nanoparticles showed small size (<40 nm) and high encapsulation efficiency. The cytotoxicity studies of free FR vs. rHDL/FR toward the NB cell lines SK-N-SH and SMS-KCNR showed 2.8- and 2-fold lower IC50 values for the rHDL encapsulated FR vs. free FR. More importantly, the IC50 value for retinal pigment epithelial cells (ARPE-19), a recipient of off target toxicity during FR therapy, was over 40 times higher for the rHDL/FR as compared to that of free FR. The overall improvement in in vitro selective therapeutic efficiency was thus about 100-fold upon encapsulation of the drug into the rHDL nanoparticles. These studies support the potential value of this novel drug delivery platform for treating pediatric cancers in general, and NB in particular.

Keywords: all-trans-retinoic acid; drug delivery; fenretinide; nanoparticles; neuroblastoma; rHDL.

Figure 1

Incorporation efficiency of ATRA into rHDL nanoparticles as a function of initial ATRA concentration.

Figure 2

Chemical composition of rHDL/ATRA and rHDL/FR particles.

Figure 3

Drug entrapment efficiency of rHDL/ATRA and rHDL/FR nanoparticles.

Figure 4

Size distribution of rHDL/ATRA (A) and of rHDL/FR (B) nanoparticles with DLS.

Figure 5

Morphology of rHDL/ATRA (A) and of rHDL/FR (B) nanoparticles with Atomic Force Microscopy.

Figure 6

Differential cytotoxicity of free fenretinide vs. the rHDL encapsulated drug against non-malignant, ARPE-19 cells (A) and two neuroblastoma cell lines SMS-KCNR (B) and SK-N-SH (C).

Figure 7

Inhibition of uptake of ATRA and FR by in SMS-KCNR (neuroblastoma) cells from rHDL nanoparticles by human HDL.

Figure 8

Inhibition of ATRA uptake by SMS-KCNR (neuroblastoma) cells via the SR-B1 inhibitor BLT.

Figure 9

Size distribution of rHDL/FR nanoparticles determined by dynamic light scattering after storage under different conditions. (A) Storage at 4°C for 1 month, (B) Storage at −20°C for 1 month, (C) Post-Lyophilization.