Design and in vitro study of etoposide loaded lipid nanomedicines for neuroblastoma treatment

Introduction: Neuroblastoma is the most frequent pediatric extracranial solid tumor being responsible for 8-10% of all childhood malignancies. Patient outcome is still a major issue due to neuroblastoma’s heterogeneity and complex tumor biology.1,2 Nanotechnology has been widely studied in cancer treatment with the aim of improving the therapeutic index of chemotherapeutic drugs.3 Among them, etoposide is a podophyllotoxin derivative given to neuroblastoma patients that often presents acute and late toxicities.4 In this study we have designed and characterized etoposide-loaded lipid nanoparticles (ETP-LN) with the aim of improving therapeutics in neuroblastoma management. Methods: ETP-LN were prepared by the hot homogenization followed by ultrasonication method which does not involve the use of any organic solvent.5 Nanoparticles physiochemical properties were characterized (size, homogeneity, surface charge) by dynamic light scattering and drug loading was evaluated by UV-vis and UHPLC-UV methods. Cell viability studies (MTS assay) were performed in multiple human neuroblastoma cell lines: SK-N-BE (2), SK-N-BE(2)C and NGP-A (MYCN-amplified cells) and SH-SY5Y, CHLA-90, NBL-S and SK-N-SH (MCYN-non-amplified cells). Results: Optimized ETP-LN showed a mean particle diameter of 105 ± 3.29 nm with a PDI value of 0.19±0.01, indicating homogenous particle distribution. The surface charge presented a negative zeta potential value of -19.9 ± 4.24 mV. UHPLC-UV showed better reproducibility than the UV-vis method. Nevertheless, both techniques indicated a similar drug loading (4.26 ± 0.18 µg/mg and 4.58 ± 0.60 µg/mg, respectively), corresponding to an encapsulation efficiency of 86.8 + 4.16 %. Preliminary in vitro cell-based cytotoxicity assay revealed that ETP-LN displayed similar IC50 values in the studied cell lines when compared to the free drug, indicating that the encapsulation process did not affect etoposide’s antitumor efficacy. Conclusions: ETP-LN were successfully developed and characterized. Further experiments in vitro and in vivo will be performed to evaluate the therapeutic potential of ETP-LN in neuroblastoma. References: Pastor, E. R. & Mousa, S. A. Current management of neuroblastoma and future direction. Crit. Rev. Oncol. Hematol. 138, 38–43 (2019). Park, J. R., Eggert, A. & Caron, H. Neuroblastoma: Biology, Prognosis, and Treatment. Hematology/Oncology Clinics of North America 24, 65–86 (2010). Rodriguez-Nogales, C., Gonzaiez-Ferna, Y., Aldaz, A., Couvreur, P. & Blanco-Prieto, M. J. Nanomedicines for Pediatric Cancers. (2018). Relling, M. V. et al. Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia. Leukemia 12, 346–352 (1998). Gonzalez-Fernandez, Y., Brown, H. K., Patino-Garcia, A., Heymann, D. & Blanco-Prieto, M. J. Oral administration of edelfosine encapsulated lipid nanoparticles causes regression of lung metastases in pre-clinical models of osteosarcoma. Cancer Lett. 430, 193–200 (2018).