Development of inhaled siRNA formulation for pulmonary delivery

Abstract

Pulmonary delivery of therapeutic siRNA is a novel strategy for the treatment of many respiratory diseases. Local targeted gene silencing following pulmonary delivery of naked siRNA has been demonstrated in animals and clinical trials, potentially alleviating the health burden associated with respiratory diseases. These studies focused on evaluating the biological responses of siRNAs, without paying attention to the siRNA formulation. Although liquid-based formulations of siRNA were frequently used in animal studies, dry powder formulation is a superior pharmaceutical form of siRNA for clinical use due to the enhanced stability and the patient-friendly inhaler design. Nevertheless, an inhalable powder formulation of naked siRNA has not been reported. To develop an inhalable naked siRNA powder formulation, the siRNA must retain its structural integrity upon drying to preserve its bioactivity, and the powder must be aerodynamically favourable for inhalation. In this study, spray drying technology was used to prepare siRNA powder formulations, using mannitol as the bulking agent. The effect of bovine serum albumin (BSA) and L-leucine as particle dispersion enhancer was investigated. Naked siRNA powders were prepared with preserved siRNA structure as revealed by gel electrophoresis. The fine particle fractions (FPF, fraction of particles with aerodynamic diameter < 5 μm) of the formulations were measured using the Next Generation Impactor. Using BSA as the dispersion enhancer, the FPF of the best performing formulation, which contained 0.75% w/w siRNA and 10% BSA as the excipient, was 62.1%. Regarding L-leucine, the FPF of the best performing formulation, which contained 2% siRNA and 50% L-leucine as the excipient, was 44.4%. These formulations exhibited good aerosolization performance, considered that the FPF of commercial dry powder inhaler products ranged from 20% to 40%. Analysis of particle surface composition suggested that both dispersion enhancers were enriched on the particle surfaces, potentially altering interparticle interactions, thereby promoting powder dispersibility. Particles also exhibited rougher surfaces with dispersion enhancers in the formulation as observed under electron microscope. Since naked siRNA cannot transfect cells in vitro, evaluation of the biological activities of naked siRNA powders required a robust and easily accessible animal model. Intranasal administration of lipopolysaccharide (LPS) markedly upregulated MCP (Monocyte chemoattractant protein)-1 expression in the lungs of mice. Intratracheal instillation of naked siRNA targeting MCP-1 two hours prior to LPS stimulation inhibited MCP-1 expression in broncho-alveolar lavage fluid by 45% six hours after LPS administration, making it a plausible model to evaluate siRNA powders. However, in a disease model exploring the therapeutic potential of siRNA powders, daily intratracheal instillation of siRNA targeting nucleocapsid viral protein did not reduce body weight loss or improve survivability in H1N1 infected mice. Inhalable powder formulations of naked siRNA were prepared for the first time. The powder formulation contained 2% w/w siRNA, which was the highest concentration ever reported. It is anticipated that the same formulation can be adopted to deliver siRNAs of different sequences. Future investigations will focus on increasing the siRNA concentration in the formulation to reduce powder required to achieve therapeutic dose, and optimizing the animal model for evaluating siRNA powder formulations.