Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonucleotide A. Arnedo, J.M. Irache, M. Merodio, M.S. Espuelas Milla ´n * Centro Gale ´nico, Departamento de Farmacia y Tecnologı ´a Farmace ´utica, Facultad de Farmacia, Universidad de Navarra, Irunlarrea 1, Ap. 177, 31080 Pamplona, Spain Received 11 April 2003; accepted 8 October 2003 Abstract The goal of this study was to evaluate the potential of albumin nanoparticles as a delivery system for antisense oligonucleotides. Nanoparticles were prepared by a coacervation process and cross-linkage with glutaraldehyde. Phosphodiester (PO) and phosphorotioate (PS) oligonucleotides were either adsorbed on the surface of nanoparticles (PO-NPA and PS-NPA) or incorporated in the nanoparticle matrix (PO-NPB and PS-NPB). When PO-loaded nanoparticles were incubated with phosphodiesterase, only NPB was able to keep the oligonucleotide hybridization capability for at least 60 min. The antiviral activity was evaluated in MRC-5 fibroblasts infected with human cytomegalovirus at a MOI of 0.0035. Both PO nanoparticle formulations significantly increased the antiviral activity of free PO ( P < 0.001) and NPB showed slightly higher efficacies than NPA ( P < 0.05). On the other hand, PS exhibited significant higher activity than free PO ( P < 0.001), however, no significant differences were found between PS-nanoparticle and PO-nanoparticle formulations. These findings were well correlated with the intracellular distribution observed for fluorescent oligonucleotide-loaded albumin nanoparticles. Even these carriers delayed and decreased the uptake of PO by MRC-5 cells, they finally induced a diffused cytoplasmic distribution and major nuclear accumulation. In summary, albumin nanoparticles partially protected a PO against enzymatic degradation and improved their presence in the nucleus and thus, increased its efficiency. D 2003 Elsevier B.V. All rights reserved. Keywords: Albumin nanoparticles; Oligonucleotide; Cellular uptake; Enzymatic stability; Antiviral 1. Introduction Antisense oligonucleotides are promising agents that may be widely used in the regulation of inappro- priate expression of genes in pathological situations by specific inhibition of the expression of their mRNA targets [1]. However, the widespread use of these molecules in clinic has been limited by two major obstacles, their enzymatic instability [2] and poor and inadequate intracellular delivery [3]. Oligonucleotide seems to be internalized through an endocytic mech- anism [4]. This sequestration of oligonucleotides into endosomal compartments means their degradation, impairing the interaction with their target mRNA and hence decrease their activity [5]. 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.10.009 * Corresponding author. Tel.: +34-948-42-5600; fax: +34-948- 42-5649. E-mail address: sespuelas@unav.es (M.S. Espuelas Milla ´n). www.elsevier.com/locate/jconrel Journal of Controlled Release 94 (2004) 217 – 227 GENE DELIVERY