Understanding excipient interactions unlocks untapped potential of RNA-lipid nanoparticles in dry powder formulations for local pulmonary delivery

Abstract

Small interfering RNA (siRNA)-loaded lipid nanoparticles (LNPs) are a promising modality for gene silencing therapies. Pulmonary delivery offers an attractive, non-invasive route to target respiratory diseases. However, the development of stable dry powder formulations suitable for inhalation remains a key challenge.

In this study, we investigated the impact of spray drying on the physicochemical integrity and biological performance of siRNA-LNPs. Four LNP formulations differing in PEG-lipid helper lipid content were subjected to spray drying in the presence of a lactose matrix. The impact of formulation parameters on physicochemical integrity, colloidal stability, structural preservation, and biological behaviour was systematically evaluated before and after spray drying and predicted by molecular dynamic simulations.

Choosing this holistic approach demonstrates that LNP composition critically influences suitability for spray drying and provides key insights for the development of stable pulmonary siRNA therapies.

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Introduction

The coming of age of RNA-based vaccines during the pandemic is estimated to have saved >14 M lives [1], and novel RNA-based drugs are being developed for indications ranging from Alzheimer’s disease to cancers. A major challenge in developing these therapies is the need for targeted delivery, as off-target effects in non-target organs pose substantial risks. Local administration directly to target tissues can mitigate many toxicological issues associated with systemic delivery. While RNA therapeutics are theoretically well-suited for treating pulmonary disorders via localized lung administration, this route presents significant challenges in practice. To address these difficulties, dry powder formulations of RNA-loaded lipid nanoparticles (RNA-LNPs) have been proposed. These formulations can be delivered to the lungs using well-established dry powder inhalers, which offer a rapid, non-invasive method, and are associated with favorable storage stability.

However, conversion of LNPs into dry powder formulations remains technically challenging, and the underlying physical mechanisms are still incompletely understood. Spray drying (SD) and lyophilization (freeze-drying) are the two primary methods employed to generate dry RNA-LNP powders, with each introducing distinct stress factors that must be carefully controlled [2]. SD, in particular, imposes shear stress during pumping and during atomization of the liquid feed, and thermal stress during droplet drying. While the high inlet temperature of the drying gas may initially seem problematic, the evaporative cooling effect ensures that the internal droplet temperature remains relatively low during atomization and the early drying phase. However, as particles travel through the drying chamber and residual moisture decreases, the exposure to elevated outlet temperatures increases, which can potentially lead to lipid phase transitions, membrane fusion, or RNA degradation.

Empirical evidence underscores these concerns. Several studies have reported structural changes in RNA-LNPs following SD, most notably an increase in particle size upon rehydration. Zimmermann et al. reported SD siRNA-LNPs maintained in vitro efficacy after drying at an inlet temperature of 100 °C [3]. Nevertheless, dynamic light scattering (DLS) measurements revealed a significant increase in the hydrodynamic diameter of the LNPs after reconstitution, suggesting structural rearrangement. Similarly, Friis et al. formulated SD mRNA-LNPs for intratracheal delivery. Post-drying characterization revealed an increased proportion of particles exceeding 200 nm in diameter, as well as the formation of electron-dense lining around the LNP observed via cryo-TEM [4]. Interestingly, Gordon et al. found that spray-freeze drying, a technique which does not induce thermal stress, also led to size increases of LNPs after redispersion, pinpointing that drying-induced reorganization is not solely thermally driven [5].

To protect lipid membranes during drying, the incorporation of sugars, which are known to provide lyo- and cryoprotective effects on biomembranes, is a widely adopted strategy. Two principal models have been proposed to explain their protective mechanisms: the preferential exclusion model and the preferential interaction model. The preferential exclusion model describes that the sugars are excluded from the immediate vicinity of the membrane and thereby preserve a hydration shell that stabilizes the lipid bilayer [[6], [7], [8], [9], [10], [11]]. The preferential direct interaction model, in contrast, encompasses three, not mutually exclusive, hypotheses: (i) water replacement, in which sugars substitute for water molecules through hydrogen bonding with lipid headgroups; (ii) entrapment, where sugars immobilize residual water, reducing ice crystallization; and (iii) vitrification, whereby the formation of a glassy sugar matrix physically stabilizes membrane structures during drying or freezing [[12], [13], [14], [15], [16], [17], [18], [19]].

In addition to the technological challenges of drying RNA-LNPs, several biological barriers must be overcome for efficient pulmonary delivery. The LNPs must traverse the mucus layer in the upper airways or the lung lining fluid in the alveoli, with factors like mucus clearance and turnover time being crucial. Upon reaching the target tissue, the LNPs must cross the extracellular matrix for cellular uptake. For LNPs, cellular internalization is significantly influenced by the proteins adsorbed onto their surface, forming a protein corona after administration. This phenomenon has been utilized in early LNP formulations to promote an apolipoprotein (ApoE)-rich corona, enhancing uptake by hepatocytes through the low-density lipoprotein receptor (LDL-R). In pulmonary delivery, LNPs are exposed to different proteins, and the effects of these interactions on LNP behaviour in the lungs remain mostly unclear.

Here, we dissect the mechanisms underlying the performance of dry powder-formulated LNPs for pulmonary RNA delivery. We systematically characterize SD-induced structural changes using an orthogonal set of advanced methodologies, including molecular dynamics simulations, cryo-TEM, and membrane fluidity measurements. We show that the concentration of PEG-lipid and the choice of helper lipid critically modulate interactions between excipients and the LNP membrane, promoting hydrogen bonding and partial excipient incorporation that drive morphological alterations. Even after reconstitution, membranes remain in a less fluid state, with PEG and helper lipid content dictating the extent of this effect. These structural changes influence LNP diffusion through mucus, cellular uptake, and RNA silencing efficiency. Using in situ diffusion assays, fluorescence correlation spectroscopy, and air–liquid interface (ALI) cell models, we further demonstrate formulation-specific differences in LNP transport across lung barriers and intracellular delivery. Finally, we characterize the composition of the protein corona that forms on LNPs upon contact with human bronchoalveolar lavage fluid, offering what is, to our knowledge, the first reported analysis of its kind.

Materials

((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,23,31-tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA) was obtained from MedChemExpress (Monmouth Junction, USA) and Corden Pharma (Plankstadt, Germany). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 3β-hydroxy-5-cholesten (cholesterol), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG 2000), as well as absolute ethanol, highly purified water (HPW), 6-dodecanyl-2-dimethylaminonaphthalene (Laurdan), TNS reagent (6-(p-toluidino-2-naphthalenesulfonic acid sodium salt)), and dimethyl sulfoxide (DMSO), sodium phosphate, sodium borate, sodium citrate, sodium hydroxide, and EDTA (2 mM), foetal bovine serum (FBS), penicillin-streptomycin (1 %), G418 (0.4 %), RPMI 1640 media, and EMEM media, sterile egg yolk emulsion, mucin (bovine submaxillary gland), Atto 647 N DOPE, calf thymus DNA (Type I), DTPA solution (1 mg/mL), sodium chloride, sucrose and potassium chloride were purchased from Sigma-Aldrich (Taufkirchen, Germany). eGFP siRNA and siNC were obtained from Integrated DNA Technologies (IDT, Leuven, Belgium) and Sigma-Aldrich (Taufkirchen, Germany). RiboGreen® RNA quantification reagent and TE buffer, phosphate buffered saline (PBS), LysoTracker™ Red DND-99, Alexa Fluor 647, and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific (Darmstadt, Germany). Lactose monohydrate (Inhalac® 230) was purchased from Meggle (Wasserburg, Germany).

The human non-small lung carcinoma cell line H1299 stably expressing enhanced green fluorescent protein (H1299-eGFP) was purchased from ATCC (VA, USA), and Calu-3 cells were sourced from LGC Standards (Wesel, Germany). PneumaCult™ ALI medium was sourced from STEMcell Technologies (Vancouver, Canada). Rhodamine-labelled lipid 18:1 Liss Rhod PE was purchased from Avanti Research (Birmingham, AL, USA). Plastics and consumables, including Vivaspin 500 columns (10,000 MWCO PES), Vivaspin 6 columns (30,000 MWCO PES), 384-well plates, 96-well black flat-bottom plates, 24-well plates, Transwell plates (24-well, 6.5 mm, PET, 8 μm pores), and Corning Transwell inserts (6.5 mm, 0.4 μm), were purchased from VWR (Darmstadt, Germany) and Greiner (Frickenhausen, Germany). 3.5 kDA, Pur-A-Lyzer TM Mega Dialysis kit was purchased from Sigma-Aldrich (Taufkirchen, Germany). 8-well μ-slides were obtained from ibidi GmbH (Martinsried, Germany). 0.22 μm Acrodisc® syringe filters were obtained from Pall (Dreieich, Germany) and disposable DLS cuvettes from Brand GmbH & Co. KG (Wertheim, Germany).

Nora Martini, Leonie Deßloch, Taras Sych, Otto Berninghausen, J. Merl-Pham, Sjoerd Dijkstra, Simone P. Carneiro, Marion Frankenberger, Roland Beckmann, Jürgen Behr, Gabriele Matschiner, Christine Schuberth-Wagner, A. Önder Yildirim, David C. Jürgens, Erdinc Sezgin, Olivia M. Merkel, Benjamin Winkeljann, Understanding excipient interactions unlocks untapped potential of RNA-lipid nanoparticles in dry powder formulations for local pulmonary delivery, Journal of Controlled Release, 2025, 114539, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2025.114539.

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