ASSESSMENT OF FENRETINIDE IN MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR VESICLES

Fenretinide (N-(4-hydroxyphenyl)-retinamide, 4-HPR) is a synthetic retinoid with fewer adverse effects than natural retinoids, whose efficacy against ovarian, prostate, small cell lung, brain, neuroectodermal-derived tumors has been widely demonstrated in several in vitro and preclinical studies. Unfortunately, clinical responses both in adult and pediatric patients are often partial, revealing a limited activity of 4-HPR against existing disease [1]. The underlying causes of this slight therapeutic efficacy consist in its poor water solubility, low bioavailability (estimated at 16%) and high first-pass hepatic effect. All trials have employed oral gelatin capsules containing 4-HPR suspended in corn oil and polysorbate 80. The low plasma drug concentrations achievable and/or the poor patient compliance in taking the required number of capsules strongly limit the effectiveness of this strategy. As a result, new oral and intravenous formulations are required to enhance the bioavailability of 4-HPR. To this end, we herein propose a new 4-HPR formulation based on the use of mesenchymal stem cellsderived extracellular vesicles (EVs) as endogenous carriers for delivering 4-HPR. EVs are nonimmunogenic, biocompatible nanocarriers able to interact with multiple cell types within the immediate vicinity and remote. After isolation from human umbilical cord, the mesenchymal stem cells were treated with high doses of 4- HPR to achieve a passive drug loading. The resulting 4-HPR-EVs were collected, purified by ultracentrifugation and characterized for size, concentration and morphology by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA), using a Nano Sight 300 instrument. The vesicles showed a mean diameter of about 130 nm with spherical shape (Figure 1). The drug amount encapsulated into the vesicles was determined by HPLC. Briefly, prior 4-HPR quantification an extraction procedure was optimized and, to estimate the analyte recovery an internal standard was employed. Since for this purpose, N-(4-ethoxyphenyl)- retinamide (4-EPR) has been reported, we developed a new operator-friendly one-step procedure to synthetize highly pure 4-EPR in quantitative yield. After exposure of mesenchymal stem cells to 25 μM 4-HPR for 48 h, we obtained 1.8 x1011 EVs/mL corresponding the a final drug concentration of 12 μM. Studies are currently being conducted to determine the best drug loading conditions and the reproducibility of the protocol.