Impact of Crystalline and Amorphous Matrices on Successful Spray Drying of siRNA Polyplexes for Inhalation of Nano‐in‐Microparticles

Abstract

To develop stable and inhalable dry powder formulations with long shelf life, polyplexes consisting of siRNA and a polyethylenimine (PEI)‐based block copolymer in presence of mannitol or trehalose are spray dried. The effect of inlet (T‐In) and outlet (T‐Out) temperature on the recovery of siRNA and adsorption effects within the tubing material are investigated. Choosing a low abrasion silicon tubing prevented siRNA loss due to adsorption. Mannitol and trehalose formulations preserved siRNA integrity regardless of excipient concentration and temperature at T‐Out below the siRNA melting temperature. Trehalose formulations allowed full siRNA recovery whereas mannitol formulations resulted in spray drying induced losses of ≈20% siRNA and of 50–60% polymer. Mannitol formulations showed optimal aerodynamic characteristics as confirmed by next generation impaction analysis based upon siRNA content. All spray dried formulations resulted in green fluorescent protein (GFP) silencing comparable or better than freshly prepared polyplexes. To test if the observed results could be transferred, formulations of siRNA and transferrin‐PEI conjugates are spray dried, characterized, and used to transfect primary human T cells ex vivo. Results confirmed successful silencing of the transcription factor GATA3 in primary CD4+ T cells with spray dried formulations as a potential treatment for severe asthma.

Inhibition of SARS-CoV-2 replication in the lung with siRNA/VIPER polyplexes

“…Considering the pulmonary administration route, the encapsulation of siRNA in a polymer-based system appears preferable. While lipid-based systems might be affected by stability problems once nebulized for inhalation [ 17 ], polymer-based formulations can be easily nebulized or processed to produce a dry powder for inhalation [ 18 ]. On this basis, we decided to formulate siRNA with VIPER (virus-inspired polymer for endosomal release), a block copolymer that showed high efficiency for both pDNA [ 19 ] and siRNA [ 20 ] delivery.…”

Can pulmonary RNA delivery improve our pandemic preparedness?

“…4 ) include atomizers and soft mist inhalers (SMIs) for liquid formulations, pressurized metered dose inhalers (pMDIs) which are most commonly used for small molecule inhalation, and dry powder inhalers (DPIs). Development of pMDI [ 47 ] and DPI [ 48 ] formulations of nucleic acid nanoparticles requires far greater development effort, however, compared to liquid formulations. Therefore, liquid formulations are generally developed, particularly for in vitro assessment.…”

“…My group has also taken on the challenge of developing dry powder formulations from RNA loaded nanosuspensions for DPI development by spray-drying [ 48 , 53 ]. The challenge in this endeavor is in fact not only the spray-drying process itself but also the question if the dry powder redisperses into nanoparticles with unchanged parameters in comparison to the freshly prepared formulation after spray-drying.…”

“…In an inhalable solid dosage form, chemical instabilities and microbial contaminations are significantly limited, and physical instabilities such as sedimentation, aggregation, coalescence or creaming are avoided [ 54 ]. Development of DPIs requires process engineering and optimization [ 48 , 53 ]. But in the light of the required ultra-low temperature storage requirements of Comirnaty®, for example, the development of a spray-drying platform technology for a range of different RNA nanoformulations seems very attractive for improved storage and transport conditions, particularly in developing countries.…”

“…This size does not refer to the nanoparticles within a nebulized suspension, however, but rather to the droplet size produced by the nebulizer. Other parameters that are important in the development of RNA aerosol medicines are the influence of heat, interfaces and shear stress on the macromolecule during nebulization [ 63 ], as well as RNA integrity and recovery in order to optimize engineering processes, surface materials, and to determine therapeutic doses [ 48 ].…”