(W1230-03-15) Development of Aerosolizable mRNA Incorporated Chitosan Nanoparticles for Efficient Lung Delivery

Purpose: Although gene therapy holds significant promise in the treatment of lung diseases, effectively delivering therapeutic genes via inhalation to the specific cells in need remains a major obstacle. The incorporation of mRNA into Chitosan nanoparticles and their drying using spray freeze-drying and inhalation via a Dry Powder Inhaler (DPI) could potentially protect the mRNA from degradation, enhance the delivery of therapeutic genes to the lungs, improve cellular uptake, and enable targeted delivery to lung cells [1]. Chitosan nanoparticles have shown great promise as a delivery system for nucleic acids due to their biocompatibility, biodegradability, and ability to protect the payload from enzymatic degradation. Furthermore, their small size makes them suitable for efficient cellular uptake. By incorporating mRNA into Chitosan nanoparticles and drying using spray freeze-drying, it may be possible to achieve a targeted and sustained delivery of therapeutic genes to the lungs [2].

Methods: The ionotropic gelation method involves the interaction between positively charged amino groups on Chitosan (CS) and negatively charged Sodium tripolyphosphate (TPP) ions. This interaction forms stable nanoparticles ranging in size from 50 to 800 nm. The nanoparticle size depends on many factors like CS's molecular weight, concentration of CS and TPP, pH of the solution, and mixing time and speed. In this study, 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 5 using 0.1 M NaOH. TPP stock solution of 0.1% was prepared and pH was adjusted to 5 using 0.1M HCl. Both CS and TPP solutions were then filtered through a 0.22 μm filter. The CS-TPP complexation was carried out at 25°C under magnetic agitation (750 rpm) for 30 minutes, followed by sonication (235W) for 40 minutes. The dispersion was left undisturbed for 16 hours, then centrifuged at 11800 x g for 15 minutes. The resulting nanoparticles were characterized for their particle size, zeta potential, and polydispersity index using a Zetasizer (Nanobrook 90 Plus PALS, Brookhaven). Formation of the nanoparticles was further confirmed by using Scanning electron microscopy (SEM) and FTIR spectral studies (Schimadzu IRSpirit). Luciferase mRNA was extracted from A549/Luciferase cell line using the Qiagen TurboCapture 96 mRNA kit as per the protocol and further purified by ethanol precipitation. Purity and Quantity of extracted mRNA were analyzed by measuring for absorbance at 260nm and 280nm wavelengths using NanoDrop® ND-1000 UV-Vis Spectrophotometer. Luciferase assay was performed using Promega Luciferase assay kit to confirm expression of Luciferase from A549 cells using UV Plate reader (Varioskan™ LUX multimode microplate reader).

Results: When CS and TPP were mixed at mass ratios of 5:1 (0.038%, 0.054%, 0.069%, 0.085%, and 0.1%), nanoparticles with a size range of 150-200 nm were obtained. Increasing the TPP concentration in the nanoparticles resulted in larger particle sizes ranging from 200 to 250 nm (Figure 1). A comparison of the infrared spectra of pure CS and CS-TPP nanoparticles revealed noticeable shifts in band frequencies. The C-O stretching band at 1647 cm−1 in pure CS shifted to 1738 cm−1 in CS-TPP nanoparticles, and the N-H stretching band at 1588 cm−1 in pure CS shifted to 1643 cm−1 in CS-TPP nanoparticles. These shifts indicate an interaction between CS and TPP, likely involving chemical bonding. (Figure 2A). The SEM images of the CS-TPP nanoparticles are shown in Figure 2B indicating their uniformity in size and elongated shape with a smooth surface. The extracted mRNA quantified for its purity using UV-Spectrophotometry confirmed that eluate 1 from the TurboCapture plate has pure mRNA with a A260/A280 of 1.9 – 2.1 (Figure 3A). As the luciferase assay progressed, the luminescence values initially increased to 347,500 light units at 1 minute, but then began to decrease steadily over subsequent time intervals (Figure 3B).

Conclusion: In conclusion the results indicated that increasing the CS concentration led to an increase in nanoparticle size, which could be due to the increased amount of polymer chains available for crosslinking with TPP, leading to the formation of larger nanoparticles. Whereas increasing the TPP concentration decreased the size due to increased cross-linking with CS, resulting in more compact and smaller nanoparticles. The shift in band frequencies observed in the infrared spectra of the nanoparticles indicated interaction and cross-linking between CS and TPP through chemical bonding. The results obtained from UV Spectrophotometry provide compelling evidence supporting the successful extraction and quantification of mRNA confirming a quantity of 2ng/µl. The confirmation of extracted mRNA as pure mRNA with an A260/A280 ratio of 1.9 - 2.1 validates the integrity and quality of the mRNA sample used in subsequent experiments. The findings of luciferase assay indicate that the luciferase activity follows a pattern of diminishing luminescence over a period of 10 minutes suggesting a successful luciferase expression in the cells.

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. Particle size of Chitosan-TPP Nanoparticles

Figure 2. A) FTIR Spectra of Chitosan-TPP nanoparticles B) SEM images of Chitosan-TPP Nanoparticles

Figure 3. A) mRNA Absorbance spectrum of extracted mRNA and B) Luminescence exhibited by cell supernatant of the A549 cells