Process optimization for spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery

RNA interference (RNAi) is a highly conserved mechanism for the post-transcriptional regulation of gene expression in eukaryotic cells. Small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC) and direct RISC towards sequence-specific binding to its target mRNA, which is subsequently cleaved and degraded. This also applies to otherwise undruggable genes. On the other hand, a therapeutic gain-of-function approach based on the introduction of larger nucleic acids, e.g. plasmid DNA (pDNA), provides the opportunity for gene replacement. This targeted downregulation or the ectopic overexpression of specific genes offer promising avenues in the treatment of diseases like cancer, viral infections or in regenerative medicine [1-2]. Spray-drying of nucleic acid-based drugs is associated with many advantages including storage stability and more facile handling as well as the possibility of pulmonary application. However, it is crucial to define optimal nanoparticles, excipients and spray-drying conditions in order to preserve the activity of the siRNA or pDNA after the drying process [3-4]. The use of polymeric nanoparticles is an efficient approach for the protection and delivery of nucleic acids in cell culture in vitro as well as therapeutically in vivo. From the family of polymer nanoparticles, various structural types have been described with high efficiency for gene or oligonucleotide delivery. These include branched or linear polyethylenimines (PEI) as well as polypropylenimine (PPI) dendrimers. In this study, we established optimal spray-drying conditions for various PEI-based nanoparticles containing large pDNA or small siRNAs. These conditions were further applied to nanoparticles based on chemically modified polymers, including tyrosine-grafted linear or branched PEIs (10 kDa), as well as a fourth generation PPI or a very low molecular weight PEI (2 kDa) with tyrosine-modification and additional disulfide cross-linking. During process optimization, the choice of the excipient was identified as a critical parameter for preserving biological activity upon spray-drying. Poly(vinyl alcohol) (PVA), but not lactose or trehalose, was found to preserve or even enhance the transfection efficacy of nanoparticles upon spray-drying when compared to their freshly prepared counterparts. In addition, the mesh size had a major influence on the dry powder product. While particle activity was maintained with a mesh size of 7 µm (big mesh), the use of 5.5 µm mesh spacing (medium mesh) led to an almost complete loss of transfection efficiency. On the other hand, the inlet temperature was found to play an only minor role with regard to complex activity. Under the optimized conditions, the obtained dry-powder product contained microparticles in a desirable size range for pulmonary application (~ 3.3 – 8.5 µm) and retained its biological activity even after prolonged storage at room temperature [5]. We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.