(M0930-09-55) Spray-Dried Inhalable RNA-Lipid Nanoparticles for Pulmonary Delivery

Purpose: The pulmonary delivery of RNA-based therapeutics through inhalation has numerous potential applications, including vaccination, protein replacement therapy, cancer immunotherapy, and gene editing [1]. This non-invasive, patient-friendly method minimizes systemic exposure and off-target effects. Additionally, the lung's large surface area can be exploited to induce effective systemic as well as mucosal immune responses, resulting in localized effects at sites of infection [2]. Lipid nanoparticles (LNPs) have been increasingly utilized as RNA delivery vehicles to protect RNA from chemical (RNase enzymes, hydrolysis) and physical degradation (pH, temperature) and facilitate efficient cytoplasmic delivery [3]. However, a major hurdle is that the RNA-LNP-based products necessitate ultracold chain storage conditions for the entire product shelf life (e.g., −90 to −60 °C for 12–18 months for Comirnaty^®) [4], which are not only costly but also challenging to implement in clinical settings. In this work, we have successfully demonstrated the formulation and processing of an inhalable dry powder of polyadenylic acid [poly(A), as a model for RNA]-LNPs suitable for lung delivery.

Methods: Poly(A) (molecular weight 700–3500 kDa, chain length 2,100–10,000 nucleotides) was initially utilized as a surrogate for RNA and was formulated into poly(A)-LNPs using the nanoprecipitation technique described prior [4]. An organic phase was prepared by dissolving an ionizable lipid, phospholipid, cholesterol, and PEGylated lipid in ethanol at molar ratios of 40:5:38.5:0.5, respectively. An aqueous solution of poly(A) was concurrently prepared in citrate buffer (pH 3, 25 mM). The organic phase was manually pipetted into the aqueous phase at a 1:3 v/v ratio. Consequently, poly(A)-LNPs were subjected to overnight dialysis in RNase-free water. Feedstocks were prepared by incorporating excipient 1 and excipient 2 solutions into the poly(A)-LNPs at a predetermined ratio prior to spray drying (Fig. 1). The hydrodynamic particle size, polydispersity index (PDI), and encapsulation efficiency (%EE) of poly(A)-LNPs were assessed pre- and post-spray drying through dynamic laser scattering (DLS) and the Quant-iT Ribogreen RNA assay, respectively. Additionally, aerosol properties of the spray-dried dry powder were assessed using the Next Generation Impactor (NGI) at a pressure drop of 4 kPa with the RS00 device. Furthermore, comprehensive powder characterizations were performed utilizing scanning electron microscopy (SEM), X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and laser diffraction techniques.

Results: The hydrodynamic particle size, PDI, and %EE of poly(A)-LNPs following dialysis were consistently measured within the ranges of 100-130 nm, 0.18-0.23, and 88-93%, respectively, utilizing the nanoprecipitation technique. Initially, the spray-drying process parameters, including inlet temperature (T_in) and solid content, were investigated to determine the optimal processing conditions and to identify excipients that can effectively stabilize poly(A)-LNPs against shear and thermal stress during the spray-drying procedure. Spray drying at elevated T_in >85°C, coupled with a solid content of 5% or less, showed increased particle size and polydispersity index (PDI >0.35), potentially due to the aggregation of nanoparticles. However, feedstocks prepared by combining poly(A)-LNPs with excipients 1 and 2 (known as pre-SD, Fig. 1B) did not show significant changes in particle size, PDI, and %EE. More importantly, the reconstituted dry powder obtained post-spray drying (F1-F4) formulations showed similar or no significant changes compared to pre-spray drying. Despite a 4-fold increase in poly(A) content, the particle size remained below 150 nm, PDI was less than 0.3, and encapsulation efficiency was >84% (Fig. 1B, 1C, and 1D). Additional excipients were also investigated; however, they did not lead to the stabilization of poly(A)-LNPs. The F1 dry powder formulation demonstrated desirable aerosol properties, with an average fine particle fraction (FPF-recovered) >50% and a mass median aerodynamic diameter (MMAD) of 4.8 ± 0.76 μm. The F1 dry powder formulation exhibited a spherical morphology with a smooth surface (Fig. 2B), low moisture content (~1%) and >68% yield (Fig. 2A). Furthermore, the F1 dry powder formulation exhibited amorphous characteristics following spray drying.

Conclusion: Using poly(A), as a model for RNA, we demonstrated that dry powder formulations of poly(A)-LNPs can be prepared using spray-drying, addressing thermal and shear stress challenges in nucleic acid stability. The dry powders had low moisture content, higher Tg, spherical morphology, and adequate aerosol properties for direct delivery into the lungs, demonstrating the potential for efficient aerosolization and effective pulmonary delivery.

References: 1. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6:1078–94.
2. He S, Gui J, Xiong K, Chen M, Gao H, Fu Y. A roadmap to pulmonary delivery strategies for the treatment of infectious lung diseases. Journal of Nanobiotechnology 2022 20:1. 2022;20:1–22.
3. Huang X, Kong N, Zhang X, Cao Y, Langer R, Tao W. The landscape of mRNA nanomedicine. Nature Medicine 2022 28:11. 2022;28:2273–87.
4. AboulFotouh K, Southard B, Dao HM, Xu H, Moon C, Williams RO, et al. Effect of lipid composition on RNA-Lipid nanoparticle properties and their sensitivity to thin-film freezing and drying. Int J Pharm. 2024;650:123688.

Figure 1. A) poly(A)-LNPs formulation composition with increased poly(A) loading; B) hydrodynamic particle size and polydispersity index (PDI); C) encapsulation efficiency (%EE) of poly(A)-LNPs and F1-F4 formulations pre- and post-spray drying; and D) representative image from DLS showing minimal change in hydrodynamic particle size of F4 formulation pre- and post-spray drying.

Figure 2. A) Spray-dried dry powder characteristics and aerosol performance properties of F1 formulation; and B) representative SEM image of F1 formulation.