Rational Design of a siRNA Delivery System: ALOX5 and Cancer Stem Cells as Therapeutic Targets

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 polyethyleneimine (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.

New Advances in Biomedical Application of Polymeric Micelles

“…Additionally, some Pluronics ® can interact with cell membranes and affect important cellular functions, potentially contributing to the effects of therapeutic load. The attractive inherent antitumor properties of Pluronic ® polymers in combination with other functional polymers, variety of therapeutic molecules and cell targeting, and stimuli-responsive ligands greatly improved the antitumoral therapeutic effects of tested drugs and are widely exploited in combination with gene therapy, phothodynamic, or immunotherapy [120,121]. In spite of that, the extraordinary complexity of biological challenges in the delivery of micellar drug payload makes their therapeutic potential still not exploited to the fullest, and therapeutic load stability and off-target delivery still need to be addressed [122][123][124].…”

A Novel Tendon Injury Model, Induced by Collagenase Administration Combined with a Thermo-Responsive Hydrogel in Rats, Reproduces the Pathogenesis of Human Degenerative Tendinopathy

“…Using hydrogels to optimize the delivery of intra-tendinous injected collagenase could facilitate the development of novel and more accurate tendinopathy models to imitate human degenerative tendon pathology. Hydrogels have been widely used for tissue engineering applications [71][72][73][74], controlled release of therapeutic agents [75,76], cell delivery [77], and new treatment strategies in cancer [78,79].…”

Advancements in Polymeric Nanocarriers to Mediate Targeted Therapy against Triple-Negative Breast Cancer

“…This detection segregates non-CSCs from CSC subpopulations and helps in monitoring CSC biological activity before, during and after therapy. Recently, siRNA inhibition of arachidonate 5lipoxygenase (Alox5) has shown down-regulation its gene expression in vitro and also decrease the cell invasion and malignant transformation of breast CSCs [217].…”