Robust peptide/RNA complexes prepared with microfluidic mixing for pulmonary delivery by nebulisation

Small interfering RNA (siRNA) and messenger RNA (mRNA) have drawn considerable attention in recent years due to their ability to modulate the expression of specific disease-related proteins. However, it is difficult to find safe, robust, and effective RNA delivery systems suitable for pulmonary delivery to treat lung diseases. In this study, two cationic peptides, namely LAH4-L1 and PEG KL4, were employed as non-viral vectors for siRNA and mRNA delivery. Four formulations (i.e. LAH4-L1/siRNA; PEG KL4/siRNA; LAH4-L1/mRNA and PEG KL4/mRNA) were investigated. Microfluidic mixing method was utilised to fabricate RNA complexes in a controllable and reproducible manner. Upon optimisation of the microfluidic mixing protocol, a vibrating mesh nebuliser was employed to aerosolise the RNA complexes, and their transfection efficiency was evaluated on A549 and BEAS-2B cells. Following nebulisation, inhalable mist was generated for all RNA formulations with mass median aerodynamic diameter below 5 μm. Although the hydrodynamic particle sizes of the RNA complexes were significantly reduced to around 100 nm after nebulisation regardless of the original size of the complexes prior to nebulisation, the RNA binding efficiency and the in vitro RNA transfection ability of all the peptide formulations were successfully preserved with no significant differences compared to the same system before nebulisation. The current result indicates that both LAH4-L1 and PEG KL4 hold significant potential for future clinical application for pulmonary siRNA and mRNA delivery through nebulisation.