Abstract
Recently, the potential of messenger RNA (mRNA) as a promising therapeutic approach is beingunwrapped in the pulmonary medicine stream more than anywhere else. The unique function of
directing the cells in protein synthesis “in vivo” has raised the mRNA as an effective alternative for
a non-viral, inhalation delivery system. By leveraging the extensive surface area and vascularization
of the lungs, inhalation delivery presents a non-invasive method to achieve localized treatment
effects directly within the respiratory tract. The use of nanoparticles for mRNA delivery to the lungs
opens a promising way for treating a wide variety of pulmonary disorders, including genetic and
infectious diseases. Chitosan-tripolyphosphate (CS-TPP) nanoparticles present promising
opportunities due to their biocompatibility and ability to encapsulate negatively charged nucleic
acids. This research focuses on the intricacies of formulating CS-TPP nanoparticles that can
encapsulate mRNA and be effectively aerosolized for targeted lung delivery, aiming to achieve
localized therapeutic effects with high transfection efficiency.
The research developed an in-house, cost-effective mRNA extraction method followed by its
quantification and even purification if required. The mRNA-incorporated CS-TPP nanoparticles
were produced through the Ionotropic gelation method using two different formulation approaches,
with one approach containing Incremental concentrations of chitosan and the other in which
chitosan and mRNA are mixed as per their weight ratios. The nanoparticles were then characterized
for their particle size, zeta potential, polydispersity index, and encapsulation efficiencies. The blank
and mRNA-incorporated nanoparticles had an average particle size of 150nm and 212nm with 1.5
and 16.38 mv of zeta potential, followed by a poly dispersity index of 0.1 and 0.3. The results
indicated that the first formulation approach with increasing concentrations of chitosan produced
better encapsulation efficiencies and was used for further studies.
The aerodynamic characterization of these formulations was assessed using a vibrating mesh
nebulizer, and particle size separation was performed using a Next-generation impactor (NGI). The
aerodynamic characterization revealed that formulations had an optimal aerodynamic diameter
between 1-5μm. Other properties like fine particle fraction and geometric standard deviation could
be improved by optimizing the nebulization parameters further. The transfection efficiency of
formulations pre and post-nebulization was studied on A549 (lung adenocarcinoma) and BEAS-2b
(lung epithelial) cell lines. Cellular uptake and protein expression were observed using Confocal
imaging on the BEAS-2b cell line. Immunogenicity studies on transfected cells revealed a dosedependent
release of Inflammatory cytokines concerning the amount of Chitosan used in the
formulations.
In conclusion, the study developed and characterized mRNA-incorporated chitosan-tri
polyphosphate nanoparticles, demonstrating their potential as a stable delivery vehicle for the
inhalation delivery of mRNA.