Short- and long-term stability study of lyophilized solid lipid nanoparticles for gene therapy.

Most studies in gene therapy are focused on developing more efficient non-viral vectors, ignoring their stability, even though physically and chemically stable vectors are necessary to achieve large easily shipped and stored batches. In the present work, the effect of lyophilization on the morphological characteristics and transfection capacity of solid lipid nanoparticles (LyoSLN) and SLN-DNA vectors (Lyo(SLN-DNA)) has been evaluated. The lyophilized preparations were stored under three different sets of temperature and humidity ICH conditions: 25 degrees C/60%RH, 30 degrees C/65%RH and 40 degrees C/75%RH. After lyophilization we found an increase in particle size which did not imply a reduction of "in vitro" transfection capacity. Stability studies of formulations lyophilized with trehalose showed that SLNs were physically stable during 9 months at 25 degrees C/60%RH and 6 months at 30 degrees C/65%RH. This stability was lost when harder conditions were employed (40 degrees C/75%RH). LyoSLNs maintained or increased the transfection efficacy (from 19% to approximately 40% EGFP positive cells) over time only at 25 degrees C/60%RH and 30 degrees C/65%RH. Lyo(SLN-DNA) resulted in almost no transfection under all conditions. LyoSLNs showed less DNA condensation capacity, whereas in Lyo(SLN-DNA) the plasmid became strongly bound, hampering the transfection. Furthermore, the storage of lyophilized lipoplexes stabilized with the disaccharide trehalose did not affect cell viability.