Rafael D, Andrade F, Montero S, Gener P, Seras-Franzoso J, Martínez F, González P, Florindo H, Arango D, Sayós J, Abasolo I, Videira M, and Schwartz Jr. S. , Prec. Nanomed. 2018 July;1(2):86-105 DOI:10.29016/180629.1
https://precisionnanomedicine.com/article/12
Abstract
The search for an ideal gene delivery system is a long and laborious process in which several factors from the first idea to final formulation, including main challenges, peaks and troughs, should be deeply taken into consideration to ensure adequate biological safety and in vivo efficacy endpoints. Arachidonate 5-lipoxygenase (ALOX5), a crucial player related with cancer development and in particular with cancer stem cells malignancy. In this work we describe the process behind the development of a small interfering RNA (siRNA) delivery system to inhibit ALOX5 in cancer stem cells (CSC), as a model target gene. We started by screening chitosan polyplexes, among different types of chitosan in different complexation conditions. Due to the low silencing efficacy obtained, chitosan polyplexes were combined with Pluronic®-based polymeric micelles with recognized advantages regarding gene transfection. We tested different types of polymeric particles to improve the biological efficacy of chitosan polyplexes. Nevertheless, limited transfection efficiency was still detected. The well-established polyethylenimine (PEI) cationic polymer was used in substitution of chitosan, in combination with polymeric micelles, originating PEI-siRNA-Pluronic® systems. The presence of Pluronic® F127 in the formulation showed to be of utmost importance, because not only the silencing activity of the polyplexes was improved, but also PEI-associated toxicity was clearly reduced. This, allowed to increase the amount of PEI inside the system and its overall efficacy. Indeed, different types of PEI, N/P ratios and preparation methods were tested until an optimal formulation composed by PEI 10k branched-based polyplexes at an N/P ratio of 50 combined with micelles of Pluronic® F127 was selected. This combined micelle presented adequate technological properties, safety profile and biological efficacy, resulting in high ALOX5 gene silencing and strong reduction of invasion and transformation capabilities of a stem cell subpopulation isolated from MDA-MB-231 triple negative breast cancer cells.
