The approval of mRNA and self-amplifying mRNA lipid-nanoparticle vaccines against COVID-19 has demonstrated the effectiveness and safety of these platforms and carriers. However, their low thermostability requires (ultra)cold storage conditions that hamper their distribution and accessibility, especially in low-income countries. Therefore, we investigated the ability and potential benefits of our linear polyethylenimine-random-polypropylenimine copolymer (L-PEI-ran-PPI) carrier in maintaining stable, functional saRNA-polyplexes after freezing and freeze-drying. First, a freeze-thaw study was performed at -80 and - 20 °C, which showed the excellent stability of our saRNA-polyplexes in aqueous solutions with or without a cryoprotectant (CP; glucose, sucrose or trehalose) for at least one month. Moreover, an additional freeze-thaw cycle did not compromise the transfection efficiency for the tested formulations. This suggests an inherent 'cryopreservative' property of our L-PEI-ran-PPIpolymer in the form of saRNA-polyplexes. Only the aqueous saRNA-L-PEI-ran-PPIformulations without cryoprotectant showed acceptable performance after one week of storage at 4 °C. Based on these results, the ease of use and the beneficial cost-effectiveness, glucose-containing formulations were selected for freeze-drying, with the formulation without CP as reference. Stability of saRNA-L-PEI-ran-PPIpolyplexes without CP could be enhanced upon 24 h of lyophilisation, with successful retention of activity during storage at 23 °C for one day, and at least 1 month at 4 °C. Intriguingly, when glucose was used as CP the same freeze-drying process enabled successful storage at 23 °C for at least 1 month, without relevant changes in transfection efficiency, size and percentage of encapsulation comparable to the fresh formulations. However, when tested in vivo, this formulation never showed a relevant bioluminescence. In contrast, lyophilised saRNA-polyplexes with glucose, stored at 4 °C for 1 month, were successful in demonstrating comparable bioluminescence to the fesh formulation across this in vivo study in mice. x y x y x y x y
