Effect of using amino acids in the freeze‑drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection

Abstract

Recently, small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) have become a crucial research tool for studying gene function. Easy and reliable siRNA transfection with a large set of siRNAs is required for the successful screening of gene function. Reverse (Rev)-transfection with freeze-dried siRNA lipoplexes is validated for siRNA transfection with a large set of siRNAs in a multi-well plate. In our previous study, it was shown that Rev-transfection with siRNA lipoplexes freeze-dried in disaccharides or trisaccharides resulted in long-term stability with a high level of gene-knockdown activity. In the present study, the effects of amino acids used as cryoprotectants in the freeze-drying of siRNA lipoplexes on gene knockdown via Rev-transfection were assessed. A total of 15 types of amino acids were used to prepare freeze-dried siRNA lipoplexes, and it was found that the freeze-drying of siRNA lipoplexes with amino acid concentrations >100 mM strongly suppressed targeted gene expression regardless of the amino acid type; however, some amino acids strongly upregulated or downregulated gene expression in the cells transfected with negative control siRNA. Amongst the amino acids tested, the presence of asparagine showed specific gene-knockdown activity, forming large cakes after freeze-drying and retaining a favorable siRNA lipoplex size after rehydration. These findings provide valuable information regarding amino acids as cryoprotectants for Rev-transfection using freeze-dried siRNA lipoplexes for the efficient delivery of siRNA into cells.

Recent progress in drying technologies for improving the stability and delivery efficiency of biopharmaceuticals

“…Despite its shortfalls such as high energy and time consumption (from days to weeks to complete) (Vass et al 2019 ), induced stresses in freezing and thawing stages, and lack of control over the size of the resultant particles (O’Sullivan et al 2022a , b ), FD is widely applied to produce more than half of the present biopharmaceuticals in today's market (Vass et al 2019 ). Freeze dried products include monoclonal antibodies (mAbs) for either inhalation or injection purpose (Hickey et al 2022 ), high protein concentration products for targeted delivery systems (Butreddy et al 2021 ), vaccines [DNA (Chen et al 2021a , b ), mRNA (Cohen 2022 ), siRNA- and mRNA (Zhao et al 2020 ) based therapeutics (Rehman et al 2021 ; Tang et al 2021 )], and room-temperature stable mAb solutions (Zhang et al 2021a , b ). Conventional excipient, with low glass transition (Tg) and low collapse temperature (Tc) such as sucrose can lead to a long and expensive lyophilization cycle, which could be substituted with new excipients, e.g., dextran (Haeuser et al 2020 ).…”