• Bio- ILs
• stabilization DNA-based vaccines

In recent years, the rapid development of biopharmaceutical products, accelerated by the COVID-19 pandemic, has led to a significant interest in optimizing their manufacturing processes. The complex production process of mRNA vaccines, associated with the limitations of viral vectors and inactivated vaccines, opens promising perspectives towards developing plasmid DNA (pDNA)-based vaccines. In fact, pDNA-based vaccines are very simple to create, have a relatively low manufacturing cost, and induce both humoral and cellular immune responses. However, from the manufacturing stages until administration, one of the main challenges still unsolved is ensuring pDNA stability, which is a crucial feature influencing its transcriptional efficiency and efficacy. For this reason, it is essential to preserve the structural integrity from thermal and enzymatic degradation to ensure its therapeutic potential. In this context, bio-based ionic liquids (Bio-ILs), which are characterized by high biodegradability and low cytotoxicity, have proven to be promising candidates for pDNA stabilization while ensuring a more sustainable synthesis process. For this purpose, in this work new Bio-ILs were synthesised and their effect on pDNA stability was evaluated. Two sets of L-carnitine and betaine bromide esters have been synthesized via O-alkylation, yielding monocationic and dicationic ILs with different alkyl chain lengths. The third set of ILs was obtained by neutralization reaction between cholinium hydroxide and cinnamic acid derivatives. A model pDNA encoding for p53 protein was amplified by Escherichia coli fermentation, followed by recovery and purification using a commercial kit based on silica spin-columns. Electrophoretic analysis was applied to confirm the integrity of pDNA samples, while their concentration and purity were determined by UV-Vis spectrophotometry. Short-term stability tests were conducted in the presence of DNases, highlighting the potential stabilizing effect of all synthesized Bio-ILs. For mono and dicationic ILs, better performances were observed for the more hydrophilic ILs, emphasizing the influence of the length of the alkyl chain. In addition, the data showed that dicationic ILs offer a superior stabilizing effect on pDNA compared to their monocationic counterparts. Specifically, dicationic L-carnitine dibromide at all concentrations tested (10 mM, 50 mM, and 100 mM) revealed to be the most effective bio-IL in preserving pDNA stability against DNase-mediated degradation. Contrarily, the assays designed to evaluate the ability of ILs to protect pDNA from thermal degradation were not conclusive, requiring additional work to shed light on their potential in this regard. Overall, these results underscore the promising potential of bio-based ILs to protect pDNA from enzymatic degradation, paving the way for future applications for the stabilization of pDNA-based vaccines.