FENRETINIDE-LOADED EXTRACELLULAR VESICLES: CHARACTERIZATION AND BIOLOGICAL EVALUATION IN NEUROBLASTOMA 2-D AND 3-D CELL CULTURES

Neuroblastoma (NB) is the most common pediatric extracranial solid tumour and the outcome for patients with high-risk neuroblastoma remains poor. For this reason, novel treatment strategies are urgently needed. Fenretinide (4-HPR) is a synthetic retinoid that has shown cytotoxic activity against various solid tumours, meanwhile exhibiting low systemic toxicity and good tolerability in clinical trials in NB patients. However, the main limitation of this molecule relies in its poor solubility and bioavailability, with a consequent limited therapeutic efficacy. As a result, new oral and parenteral formulations are required to improve clinical outcomes. To date several attempts have been made and 4-HPR has been successfully entrapped into liposomes or polymeric micelles [1]. Recently, we opted for an endogenous drug delivery system such as mesenchymal stromal cell-derived extracellular vesicles. Mesenchymal stromal cells (MSCs) have undergone in-depth studies for their therapeutic roles, which appear to be derived from their paracrine activity mostly mediated by extracellular vesicles (EVs). EVs are cell-derived submicronic membranous vesicles that are recognized to be key carriers of information in cell-to-cell communication, exert similar effects as their parental cells and have some of the highly desired attributes of a drug delivery system. Our aim was to propose a new strategy based on the use of MSC-derived EVs as carrier capable of transporting 4-HPR in NB cells, thus improving its bioavailability. The study started from MSC isolation from human umbilical cord, then the MSCs were expanded for 15 days and finally exposed to high doses of 4-HPR for different time to assess the best passive drug loading conditions. The resulting 4-HPR-EVs were collected, purified by ultracentrifugation and characterized for size, concentration and loading by Nanoparticle Tracking Analysis and Dynamic Light Scattering. The drug amount encapsulated into the vesicles was determined by HPLC and to estimate the analyte recovery the internal standard N-(4-ethoxyphenyl)-retinamide has been previously synthetized [2]. Loaded and empty EVs showed a concentration of 1.25x1011 and 1.53x1011 vesicles/mL and a mean diameter of 120 and 140 nm, respectively. The Zeta potential was comprised between -10 e -15 mV, while the drug concentration was of 6 μM. Biological studies were performed on a panel of human NB cell lines: IMR-32, HTLA-230, SK-N-AS, SH-SY5Y. Particularly, cells were treated with 1.7 μM 4-HPR-EVs for 48 and 72 h and a time dependent cytotoxic effect was observed with a significant inhibition of cell viability. Following this, the effects of the free and encapsulated form of 4-HPR on cell viability were compared. Therefore, the most resistant (SH-SY5Y), and the most sensitive (IMR-32) NB cell lines were treated with 1.7 µM 4-HPR-EVs or increasing doses of free 4-HPR (1, 2.5 and 5 µM). The difference was markedly evident in the most resistant cells, where the loaded endogenous carrier showed an improved efficacy over time in inhibiting cell viability compared to free 4-HPR. To further confirm the increased cytotoxic effect exerted by 4-HPR-EVs, we examined the ability to induce apoptosis by Annexin V-FITC Assay using Flow cytometry. 1.7 μM HPR-EVs were statistically more effective in inducing apoptosis than free 4-HPR, even at the highest free drug concentration tested (5 μM) in SH-SY5Y cells. Furthermore, incisive results were also attained in a 3-D cell culturing model that better mimics the formation of a tumor mass. 4-HPR-EVs inhibited the viability of IMR-32 and SH-SY5Y spheroids, obtained by seeding cells in ultra-low-attachment 96-well plates, in a statistically significant manner, allowing to appreciate the stronger efficacy of the drug through the EV treatment. These results demonstrate the anti-tumor effect of 4-HPR-loaded-MSC-derived EVs against NB cells, and underline their potential as a novel drug delivery system against NB, opening to deepened pre-clinical investigations. In this study, we demonstrated for the first time the passive loading capacity of MSCs for the production of 4-HPR-containing EVs. The development of this endogenous carrier for the administration of 4-HPR could overcome its poor bioavailability, which up to now has strongly weakened the clinical success of previous trials.