Research paper Short- and long-term stability study of lyophilized solid lipid nanoparticles for gene therapy A. del Pozo-Rodríguez, M.A. Solinís, A.R. Gascón, J.L. Pedraz * Pharmacy and Pharmaceutical Technology Laboratory, University of the Basque Country (UPV-EHU), Vitoria-Gasteiz, Spain article info Article history: Received 11 July 2008 Accepted in revised form 30 September 2008 Available online 7 October 2008 Keywords: Gene therapy Non-viral vectors Solid lipid nanoparticles Lyophilization Stability study abstract 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 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH. After lyoph- ilization 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 °C/60%RH and 6 months at 30 °C/65%RH. This stability was lost when harder conditions were employed (40 °C/75%RH). LyoSLNs maintained or increased the transfection efficacy (from 19% to approximately 40% EGFP positive cells) over time only at 25 °C/60%RH and 30 °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. Ó 2008 Elsevier B.V. All rights reserved. 1. Introduction The development of gene therapy has boosted the use of a new group of pharmaceutical agents for the treatment of human dis- eases [1–3]: gene delivery systems. Non-viral vectors are being extensively studied because of their greater security as compared to viral vectors. Non-viral vectors can be composed by polymers, lipids, peptides or mixtures of them. The typically employed lipidic systems are cationic liposomes, but it has been proved that cationic solid lipid nanoparticles (SLNs) can also provide good transfection levels [4–6]. Although the most well-known problem of non-viral systems is their low transfection efficiency, the poor physical sta- bility of these systems in aqueous suspensions is also a barrier to their development as medicaments [7]. Lipoplexes tend to form aggregates which decrease their transfection capacity. In order to avoid this limitation, clinical trials using lipoplexes have tradition- ally used systems freshly prepared at the bedside prior to injection [8,9]. However, the quality and the particle size of these extempo- raneous preparations are hardly ever controlled. Most studies in gene therapy are focused on developing more efficient non-viral vectors, ignoring their stability, even though physically and chem- ically stable vectors are necessary to achieve large easily shipped and stored batches. Different authors [9–12] have demonstrated that frozen DNA formulations maintain transfection rates, but they require strict storage and shipping temperatures, which imply a substantial in- crease in costs. This fact has generated an increasing interest in developing dehydrated formulations, which can be stored and shipped at room temperatures. Lyophilization is one of the most employed techniques to generate dried pharmaceuticals in general, and DNA-based formulations in particular [9,12–17]. Lyophilization subjects formulations to two important stresses, freezing and drying [7], which can damage biomolecules unless appropriate stabilizers, such as sugars, are used. Different sugars have been employed to stabilize lyophilized SLNs and non-viral vectors composed by cationic lipids and DNA [13,18–21]: mono- saccharides (glucose), disaccharides (trehalose, sucrose), oligosac- charides (inuline) or polysaccharides (hydroxyethyl starch, high molecular weight dextrans). Among these, trehalose is one of the most commonly employed sugars providing positive results. Although there are some studies about the long-term stability of SLNs for poorly water-soluble pharmaceutical drugs [22–24], we have not found any work about the long-term stability of lyophilized cationic SLNs for gene therapy, and only a few studies 0939-6411/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2008.09.015 * Corresponding author. Pharmacy and Pharmaceutical Technology Laboratory, Pharmacy Faculty, University of the Basque Country (UPV-EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain. Tel.: +34 945013091; fax: +34 945013040. E-mail addresses: ana.delpozo@ehu.es (A. del Pozo-Rodríguez), knppemuj@vc. ehu.es (J.L. Pedraz). European Journal of Pharmaceutics and Biopharmaceutics 71 (2009) 181–189 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb