To direct stem cell fate, a delicate control of gene expression through small interference RNA (siRNA) is emerging as a new and safe promising strategy. In this way, the expression of proteins hindering neuronal commitment may be transiently inhibited thus driving differentiation. Mesenchymal stem cells (MSC), which secrete tissue repair factors, possess immunomodulatory properties and may differentiate towards the neuronal lineage, are a promising cell source for cell therapy studies in the central nervous system. To better drive their neuronal commitment the repressor Element-1 silencing transcription (REST) factor, may be inhibited by siRNA technology. The design of novel nanoparticles (NP) capable of safely delivering nucleic acids is crucial in order to successfully develop this strategy. In this study we developed and characterized two different siRNA NP. On one hand, sorbitan monooleate (Span(®)80) based NP incorporating the cationic components poly-l-arginine or cationized pullulan, thus allowing the association of siRNA were designed. These NP presented a small size (205 nm) and a negative surface charge (-38 mV). On the other hand, lipid nanocapsules (LNC) associating polymers with lipids and allowing encapsulation of siRNA complexed with lipoplexes were also developed. Their size was of 82 nm with a positive surface charge of +7 mV. Both NP could be frozen with appropriate cryoprotectors. Cytotoxicity and transfection efficiency at different siRNA doses were monitored by evaluating REST expression. An inhibition of around 60% of REST expression was observed with both NP when associating 250 ng/mL of siRNA-REST, as recommended for commercial reagents. Span NP were less toxic for human MSCs than LNCs, but although both NP showed a similar inhibition of REST over time and the induction of neuronal commitment, LNC-siREST induced a higher expression of neuronal markers. Therefore, two different tailored siRNA NP offering great potential for human stem cell differentiation have been developed, encouraging the pursuit of further in vitro and in vivo in studies.