Cationic alkyl chain length and nanoaggregate form of ionic liquids dominate biocompatibility and toxicity.

While ionic liquids (ILs) have diverse applications, their potential in biomedical applications remains largely untapped due to gaps in systematic understanding of the spectrum of IL biosafety (biocompatibility/toxicity). Here, we establish an IL library and identify an in vitro reduction in biocompatibility (increased toxicity) with increased ILs' cationic alkyl chain length. Particularly, we present compelling evidence for IL nanoaggregates in aqueous environment, thereby elucidating the mechanisms involved in cell interactions. ILs with short cationic alkyl chains (scILs) are restricted in intracellular vesicles, whereas ILs with long cationic alkyl chains (lcILs) accumulate to the mitochondria for inducing mitophagy and apoptosis. The occurrence of dysfunctional behaviour in lcILs is also observed in vivo, with a positive correlation between the lcIL signal in tissues and mitophagy/apoptotic levels. Irrespective of the administration routes (oral/intramuscular/intravenous), scILs exhibit ~30-80 times greater tolerance than lcILs. The feasibility of scIL nanoaggregates as carriers for insoluble drugs is thus validated, and an enhanced bioavailability over the commercial tablet is acquired. The findings obtained by integrating computational analysis with diverse cell/animal evaluations (from multiple cell lines, cell spheroids, patient-derived organoids to male murine and canine models) offer unique insights into the behaviour, mechanisms, and biomedical application scenarios of IL nanoaggregates.