(512g) TPP-Modified Dendrimer-siRNA Complexes and Their Aerosol Formulation for siRNA Delivery to the Lungs

The regulation of genes utilizing the RNA interference (RNAi) mechanism via the delivery of synthetic siRNA as a therapeutic molecule has great potential in the treatment for a variety of prevalent diseases that affect the lungs including lung cancer, asthma, and cystic fibrosis.  However, there are many challenges in delivering siRNA to the lungs including the poor bioavailability of free siRNA and difficulty in formulating biomacromoleucles in portable inhalers.  The use of nonviral vectors such as cationic dendrimers as siRNA nanocarriers has shown promise to improve siRNA bioavailability, but has had limited due to low effective transfection efficiency.  However, the modification of dendrimers with various targeting ligands such as triphenylphosphonium (TPP), a mitochondrial targeting agent, has improved targeting, biocompatibility, and transfection efficiency of dendrimers. The delocalized positive charge and hydrophobic nature of TPP enhances cellular uptake and efficient membrane fusion as well as efficient escape from endolysosomes to target mitochondria.  The presence of TPP on dendrimer surfaces as helps modulate the interaction between nucleic acid (siRNA) and dendrimer, which may allow for the bioavailability of free siRNA once delivered into cells and thus increase its transfection ability.  Furthermore, TPP can passively target cancerous cells due to the increased mitochondrial membrane potential within these cells, which may benefit cancer treatment strategies.

In this work we investigate the ability of a triphenylphosphonium (TPP) modified generation 4 poly(amdoamine) (PAMAM) dendrimers (G4NH2-TPP) as a nanocarrier of siRNA, and the aerosol formulation of the complexes in pressurized-metered dose inhalers (pMDIs) and  dry powder inhalers (DPIs). We will discuss the synthesis and characterization of the dendrimers, and the preparation and characterization of the complexes of siRNA and G4NH2-TPP (dendriplexes).  The complexation efficiency via gel electrophoresis and in vitro gene knockdown ability in stably eGFP expressing lung alveolar epithelial (A549) cells were assessed as a function of the number density of TPP conjugated to G4NH2, as well as the N/P ratio of the dendriplexes.  G4NH2-TPP dendriplexes were prepared with TPP densities of 0, 4, 8, and 12 per dendrimer and increasing N/P ratios of 5, 10, 20, and 30.  An increase in TPP density and N/P ratio led to an increase in vitro gene knockdown with G4NH2-12TPP dendriplex at N/P ratio 30 showing the highest in vitro gene knockdown of all groups.  To assess the potential of G4NH2-TPP dendriplexes for pulmonary use, we also develop micron particle technologies for both pMDI and DPIs, and determine their aerosol characteristics utilizing an Andersen Cascade Impactor (ACI).  The proposed particle engineering strategy is shown to be effective in producing aerosol properties of suitable for deep lung deposition and respirable fractions up to 66%, as well as showing no impact on the in vitro gene knockdown efficiency of the siRNA present in those formulations.  This work demonstrates the potential benefits of utilizing TPP-conjugated dendrimers in the formation of dendriplexes and in the aerosol formulation of siRNA for the pulmonary delivery to the lungs using portable inhalers.