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

Area of Science:

• Biotechnology
• Nanomedicine
• Respiratory Medicine

Background:

• Small interfering RNA (siRNA) and messenger RNA (mRNA) offer therapeutic potential for lung diseases by modulating protein expression.
• Effective and safe pulmonary delivery systems for RNA therapeutics remain a significant challenge.

Purpose of the Study:

• To evaluate cationic peptides LAH4-L1 and PEG12KL4 as non-viral vectors for pulmonary delivery of siRNA and mRNA.
• To assess the feasibility of using a microfluidic mixing and nebulization approach for RNA complex preparation and delivery.

Main Methods:

• Fabrication of four RNA formulations (LAH4-L1/siRNA, PEG12KL4/siRNA, LAH4-L1/mRNA, PEG12KL4/mRNA) using microfluidic mixing.
• Optimization of microfluidic mixing and subsequent aerosolization of RNA complexes using a vibrating mesh nebulizer.
• Evaluation of transfection efficiency in A549 and BEAS-2B lung cell lines post-nebulization.

Main Results:

• Nebulization produced inhalable mist (MMAD < 5 μm) for all RNA formulations.
• Hydrodynamic particle size was reduced to ~100 nm post-nebulization, irrespective of initial size.
• RNA binding efficiency and in vitro transfection ability were preserved after nebulization for both peptide formulations.

Conclusions:

• LAH4-L1 and PEG12KL4 peptides are effective non-viral vectors for pulmonary siRNA and mRNA delivery.
• Microfluidic mixing and nebulization are viable methods for preparing and delivering these RNA therapeutics.
• These findings support the potential clinical application of these peptide-based systems for treating lung diseases.