Evaluation of the Effects of Storage Conditions on Spray-Dried siRNA-LNPs Before and after Subsequent Drying

In an ideal world, pharmaceutical drugs would have infinite shelf life, no susceptibility to degradation, chemical reactions or loss of efficacy. In reality, these processes occur, however, making it desirable to extend a drugs’ shelf life. Nucleic acid-based drugs are most commonly stored as aqueous suspension where they are vulnerable to microbial growth and degradation processes. Drying procedures, such as lyophilization and spray drying, help to reduce the products’ residual moisture while increasing the products’ shelf life and stability. The present study was designed to evaluate 90 days of storage of spray-dried siRNA-lipid nanoparticles (LNPs) at 4 °C and 25 °C. An updated Onpattro® composition modified with a positively charged helper lipid was used as the LNP carrier system.

In an attempt to further reduce the residual moisture of our previously reported formulations, all LNP samples were subjected to a secondary drying step in the spray drying tower for 20 min. The measurement of physicochemical properties of spray-dried and subsequently dried LNPs resulted in sizes of 180 nm, PDI values of 0.1-0.15 and zeta potentials of +3 mV. Spray drying resulted in residual moisture levels of 3.6 – 4% and was reduced by subsequent drying to 2.8 – 3.1%. Aerodynamic properties after storage showed discrepancies depending on the storage conditions. MMADs remained at 2.8 µm when stored at 4 °C, whereas an increase to 5 µm at 25 °C was observed. Subsequent drying led to sizes of 3.6-3.8 µm, independent of the storage conditions.

Spray-dried LNPs maintained bioactivity resulting in > 95% protein downregulation and confirming the lack of cytotoxic effects in a lung adenocarcinoma cell line. Furthermore, the spray-dried and subsequently dried LNPs stored for 3 months at 4 °C and 25 °C achieved up to 50% gene silencing of the house-keeping gene GAPDH after deposition on the mucus layer of Calu-3 cells. This study confirms the stability of spray-dried and subsequently dried LNPs over at least 90 days at 4 °C and 25 °C emphasizing the potential of dry powder inhalation of RNA-loaded LNPs as a therapy option for pulmonary diseases.

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Materials

Dicer substrate double-stranded siRNA targeting green fluorescent protein (DsiRNA EGFP, 25/27) (siGFP), dicer substrate double-stranded siRNA targeting the house-keeping gene GAPDH (DsiRNA GAPDH) (siGAPDH) and scrambled, non-specific control (siNC) were purchased from IDT (Integrated DNA Technologies, Inc., Leuven, Belgium). [21-24] Tris-EDTA buffer solution 100x (T9285), RPMI1640 medium (R8758), EMEM medium, fetal bovine serum (FBS) (F9665), penicillin-streptomycin (P/S) (P4333), G418 disulfate salt solution (G8168), Dulbecco’s phosphate buffered saline (D-PBS) (D8537), methylthiazolyldiphenyl-tetrazoliumbromid (MTT), Triton X-100 were obtained from SigmaAldrich, a subsidiary of Merck KGaA (Darmstadt, Germany). 1,2-Dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoglycerol, sodium salt (DSPG) and 1,2-dioleoyl-3- trimethylammonium propane (DOTAP) were bought from Avanti Polar Lipids, Alabaster, USA. The ionizable cationic lipid used is a sulfur-containing analog of DLin-MC3-DMA (pKa 6.3-6.6). [21, 25] InhaLac®230, lactose monohydrate for dry powder inhalers, was purchased from Meggle Group (Wasserburg, Germany). Quant-itTM RiboGreen DNA reagent, black 96-well plates (10307451), power SYBRTM green PCR master mix and Aquastar® water standard oven 1% were bought from Thermo Fisher Scientific (Schwerte, Germany). Pumpsil® tubings were obtained from Watson-Marlow GmbH (Rommerskirchen, Germany) and had an inner diameter and a thickness of 1.6 mm. White 96-well PCR plate and 0.2 mL PCR tubes were purchased from Biozym Scientific GmbH (Hessisch Oldendorf, Germany). PneumaCult ALI differentiation medium, hydrocortisone and heparin were bought from Stemcell Technologies (Vancouver, Canada). Transwell® polyester membrane cell culture inserts were purchased from Corning (New York, USA)

Zimmermann, Christoph M. and Luciani, Paola and Merkel, Olivia M., Evaluation of the Effects of Storage Conditions on Spray-Dried siRNA-LNPs Before and after Subsequent Drying. Available at SSRN: https://ssrn.com/abstract=4548382 or http://dx.doi.org/10.2139/ssrn.4548382

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