(T0930-05-27) Improving Lipid Nanoparticles Stability for mRNA Lung Delivery Through Nebulization

Purpose: After Covid-19 vaccination success, RNA-based therapies gained traction as therapeutics for pulmonary diseases, leveraging inhalation as a route of administration. However, RNA is vulnerable to degradation by RNases and alveolar macrophage response [1]. Degradation can be prevented through messenger RNA (mRNA) encapsulation in lipid nanoparticles (LNPs), usually produced through rapid mixing processes, such as microfluidics [2,3]. Nebulization is a preferred delivery strategy for pre-clinical and early stages due to its simplified formulation and process development, low material requirements, and low cost, with vibrating mesh nebulizers being the best choice for sensitive molecules [4]. The present study aimed to develop a stable formulation for lung delivery through the nebulization of transfer RNA (tRNA) and Green Fluorescent Protein-mRNA (GFP-mRNA) encapsulated in lipid nanoparticles.

Methods: tRNA-LNPs or GFP-mRNA-LNPs were produced by microfluidics, using previously optimized process parameters. Then, the different LNPs were dialyzed in different formulation buffer solutions shown in Figure 1 using a slide-a-lyzer mini dialysis device. After that, all formulations were nebulized with an Innospire Go vibrating mesh nebulizer. Particle size distribution and polydispersity index (PdI) were analyzed via dynamic light scattering (DLS). Encapsulation efficiency (EE) was assessed through the RiboGreen assay. RNA integrity was evaluated by HPLC and electrophoresis. Aerodynamic performance was measured with a NGI coupled to a Breathing Simulator. The quality attributes considered for RNA-LNPs included: particle size (≤ 200 nm), PdI (≤ 0.3), EE (≥ 80 %), and maintenance of mRNA integrity. Moreover, osmolality was targeted to achieve ~300 mOsm.

Results: The average results for all tRNA-LNPs batches produced (represented as blue bars in Figure 1) were a particle size of 51.18 ± 0.54 nm, a PdI of 0.20 ± 0.01, and a tRNA EE of 98 %, displaying the reproducibility of microfluidics as a production technique for RNA-LNPs. After dialysis, particle size increased while PdI decreased (see Figure 1, trials 1 – 13). This phenomenon has been reported in the literature, however, a clear understanding of the mechanism behind it is still lacking [5]. After nebulization, tRNA-LNPs particle size and PdI increased beyond the intended limits in all formulations. This increase was a consequence of the mechanical stress induced on tRNA-LNPs while passing through the nebulizer, leading to LNP destabilization. Nevertheless, two formulations – from trial 5 (PBS + Tween 80) and trial 8 (PBS + Poloxamer 188) – can be highlighted, since they led to lower levels of LNP destabilization, i.e., a lower increase in particle size and PdI. After an optimization in LNP/Buffer dilution of both solutions to a ratio of 1:10, Trial 15 formulation (PBS + Tween 80) showed better stabilization potential. This formulation was used in the following activities. After formulation screening with tRNA-LNPs, mRNA-LNPs were produced by microfluidics achieving a particle size of 97.00 ± 1.20 nm, a PdI of 0.20 ± 0.01, and encapsulation efficiency of 85 % as shown in Figure 2. Then, mRNA-LNPs were dialyzed using the lead formulation from trial 15. As shown in Figure 2, the encapsulation efficiency of mRNA-LNPs after nebulization falls below the established threshold (EE ≥ 80 %). Improvements can be made to enhance the process since the microfluidics parameters employed were optimized for a smaller molecule, tRNA.  The mechanical stress induced on mRNA-LNPs during nebulization led to a slight increase in particle size and PdI. However, these values successfully remained within the defined limits. mRNA integrity was maintained throughout the product life cycle as demonstrated by the absence of mRNA degradation in Table 1 and Figure 2. In this study, the mRNA-LNPs formulation in PBS with Tween 80 exhibited favorable aerodynamic performance. This formulation achieved quality attributes within the acceptable range: MMAD was found to be 4.6 µm, with an FPF of 51 % and an emitted dose of 92 %. These results are aligned with the reference values used in this nebulizer for a reference solution [7]. The data suggests that the mRNA-LNPs formulation has the capability to effectively deliver the LNPs into the lungs without degradation, ensuring efficient drug delivery to the targeted site

Conclusion: Formulation screening with tRNA-LNPs and different buffer solutions was performed to preserve colloidal stability after nebulization. The lead formulation from this first stage was applied to mRNA- LNPs.  Throughout the product's lifecycle, the colloidal stability of LNPs and mRNA integrity was maintained. Additionally, mRNA-LNPs formulation exhibited good aerodynamic performance, achieving an MMAD within 4.6 µm, FPF of 51 %, and an emitted dose of 92 %. This work represents a major breakthrough in the field of nebulization for nucleic acids, by presenting a formulation with the ability to withstand mechanical stress induced by a vibrating mesh nebulizer, while achieving acceptable aerodynamic performance for lung delivery.

References: [1]. W. Liang, H. W. Pan, D. Vllasaliu, and J. K. W. Lam: Pulmonary delivery of biological drugs, Pharmaceutics, vol. 12, no. 11. MDPI AG, pp. 1–28, Nov. 01, 2020. doi: 10.3390/pharmaceutics12111025.
[2]. X. Hou, T. Zaks, R. Langer, and Y. Dong: Lipid nanoparticles for mRNA delivery, Nature Reviews Materials, vol. 6, no. 12. Nature Research, pp. 1078–1094, Dec. 01, 2021. doi: 10.1038/s41578-021-00358-0.
[3]. M. J. W. Evers, J. A. Kulkarni, R. van der Meel, P. R. Cullis, P. Vader, and R. M. Schiffelers: State-of-the-Art Design and Rapid-Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery, Small Methods, vol. 2, no. 9. John Wiley and Sons Inc, Sep. 11, 2018. doi: 10.1002/SMTD.201700375.
[4]. J. S. Lass, A. Sant, and M. Knoch: New advances in aerosolized drug delivery: Vibrating membrane nebulizer technology, Expert Opin Drug Deliv, vol. 3, no. 5, pp. 693–702, Sep. 2006, doi: 10.1517/17425247.3.5.693
[5]. T. Terada et al., “Characterization of Lipid Nanoparticles Containing Ionizable Cationic Lipids Using Design-of-Experiments Approach,” Langmuir, vol. 37, no. 3, pp. 1120–1128, Jan. 2021, doi: 10.1021/acs. Langmuir.0c03039.
[6]. Philips InnoSpire Go. (n.d.). Retrieved July 13, 2023, from https://www.usa.philips.com/healthcare/product/HC1126593/innospire-go-portable-virtually-silent-mesh-nebulizer


Figure 1. Particle size and PdI results of tRNA-LNPs after microfluidics, after dialysis, and after nebulization with a description of each trial composition in the table. Only the formulation of PBS with Tween 80 at a dilution ratio of 1:10 (Trial 15) maintained a particle size and PDI inside the acceptable limits after nebulization (particle size ≤ 200 nm and PdI ≤ 0.300).


Figure 2. Particle size, PdI results of mRNA-LNPs for the best-performing formulation containing PBS with Tween 80 after microfluidics, after dialysis, and after nebulization (A) and Encapsulation efficiency (B) shows that the formulation was able to maintain colloidal stability in all steps. C) Gel electrophoresis results show that mRNA does not degrade upon nebulization.


Table 1: IP-RP-HPLC results after microfluidics and after nebulization compared with the standard mRNA sample. There is no significant degradation throughout the lifecycle.