(T0930-05-29) Pulmonary Delivery of mRNA via Engineered Chitosan Nanoparticles: Development and Application

Purpose: Recently, the potential of messenger RNA (mRNA) as a promising therapeutic approach is being unwrapped 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. Using 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 [1]. 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.

Methods: The ionotropic gelation method prepared luciferase mRNA incorporated CS and TPP ions. This generally involves interacting with positively charged amine groups of CS and negatively charged mRNA and TPP ions. This interaction forms stable nanoparticles ranging in size from 50 to 500 nm. For this project, low molecular weight CS (50-190 kDa) was dissolved in 4.6 mM HCl at concentrations from 0.038% to 0.1%, with pH adjusted to 7 using 0.1 M NaOH. Two different formulation approaches were used to prepare nanoparticles, one with Increasing concentrations of CS and the other with CS and mRNA mixed according to their (w/w) ratios. The CS-mRNA complexation was carried out at 25°C under magnetic agitation (750 rpm) for 10 minutes, then mixed with TPP for 30 minutes. The formulations were incubated at 4°C for 16 hours to stabilize before analysis. The resulting nanoparticles were then characterized for their particle size and zeta potential, using Zetasizer (Nanobrook 90 Plus PALS, Brookhaven) and encapsulation efficiency using Quant-iT™ RNA Assay Kit. Aerodynamic studies were conducted on FITC-labeled CS-TPP mRNA nanoparticles by aerosolizing them using a vibrating mesh nebulizer (VMN) and passing them through a next-generation impactor. The CS-TPP mRNA nanoparticles were then nebulized using a VMN, followed by evaluations of transfection efficiency on the A549 and BEAS-2b cell line before and after Nebulization [2]. Using confocal microscopy, cellular uptake and protein expression were monitored.

Results: The particle size data indicates that as the concentration of CS stock solution increases, the size of both blank and mRNA-loaded CS-TPP nanoparticles also increases, with mRNA-loaded nanoparticles consistently more significant across all concentrations. When CS and mRNA were mixed at w/w ratios, the data revealed an inconsistent trend concerning particle sizes, where some w/w ratios had a bigger blank nanoparticle size than mRNA nanoparticles (Figure 1A and 1B). A similar trend was also observed in encapsulation efficiency, where increasing concentrations of chitosan produced better encapsulation efficiency ranging from 60% to 90% and continued for subsequent studies. For all concentrations of CS-TPP NPs, there is a noticeable decrease in transfection efficiency in both cell lines post-nebulization compared to pre-nebulization (Figure 2A and 2B). Aerodynamic studies revealed that CS-TPP mRNA nanoparticles had an optimum Mass Median aerodynamic diameter (MMAD) ranging from 1-5m but a sub-optimal Fine Particle Fraction (FPF) of ~50% and a high Geometric standard deviation (GSD) of 3.7-4.2 (Table 1). Confocal imaging on cellular uptake of FITC-labeled CS-TPP mRNA NPs showed significant internalization of nanoparticles. Successful mRNA translation was also confirmed by the strong GFP signal from protein expression of GFP mRNA incorporated CS-TPP nanoparticles.

Conclusion: In conclusion, the studies highlight the viability of aerosolizable Chitosan-mRNA nanoparticles for efficient lung-targeted gene delivery. The formulation parameters, including chitosan concentration and w/w ratios, were found to critically influence the characteristics of nanoparticles, affecting their size and encapsulation efficiency. The data indicated that although the formulations had an ideal MMAD, other parameters like FPF and GSD should be improved by optimizing the nebulization parameters or using different nebulizers like air-jet nebulizers or ultrasonic nebulizers. Invitro transfection efficiency studies revealed a specific decrease in transfection post-nebulization, but they still exhibited a functional capacity for gene expression compared to the untreated group. Cellular uptake and subsequent protein expression were visualized using confocal imaging, confirming the translation of mRNA into proteins. All these findings underscore the potential of Chitosan-mRNA nanoparticles to enhance non-invasive pulmonary gene delivery, marking a significant step forward in treating respiratory diseases.

References: 1. Lallana Ozores, Enrique & Rios de la Rosa, Julio & Tirella, Annalisa & Pelliccia, Maria & Gennari, Arianna & Stratford, Ian & Puri, Sanyogitta & Ashford, Marianne & Tirelli, Nicola. (2017). Chitosan/Hyaluronic Acid Nanoparticles: Rational Design Revisited for RNA Delivery. Molecular Pharmaceutics. 14. 10.1021/acs.molpharmaceut.7b00320.
2. Qiu, Y., Man, R. C. H., Liao, Q., Kung, K. L. K., Chow, M. Y. T., & Lam, J. K. W. (2019). Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide. Journal of controlled release: official journal of the Controlled Release Society, 314, 102–115. https://doi.org/10.1016/j.jconrel.2019.10.026

Acknowledgements: The authors thank Dr. Satish Agrawal, Resident Assistant Professor, Department of Pharmacy Sciences, School of Pharmacy and Health Professions, for his assistance in SEM imaging.

Funding: The authors gratefully thank the Department of Pharmacy Sciences, Creighton University, for the financial support.
Conflict of interest statement: We confirm that there are no conflicts of interest associated with this work, and there has been no significant financial support for this work that could have influenced its outcome.


Figure 1. A) Particle size of CS-TPP Blank and CS-TPP mRNA Nanoparticles by Increasing concentrations of Chitosan; B) Particle size of CS-TPP Blank and mRNA NPs mixed by Chitosan and mRNA (w/w) ratios


Table 1. Aerodynamic characteristics of CS-TPP mRNA nanoparticles


Figure 2. A) Transfection efficiency of CS-TPP mRNA NPs in Lung cancer cell line; B) Transfection efficiency of CS-TPP mRNA NPs in Lung epithelial cell line