Dimethyl sulfoxide (DMSO) is a ubiquitous nonaqueous solvent in life sciences, classified as nontoxic by the FDA and ICH. Its use is widespread due to its ability to serve as a vehicle for hydrophobic compounds and as a cryoprotectant for cell storage. However, DMSO's inherent toxicity and its disruptive effects on cellular behavior present limitations. DMSO's cell permeability allows it to bind to various proteins, directly interfering with their functions, and indirectly altering cellular behavior through epigenetic mechanisms, even potentially causing apoptosis. This necessitates the search for a superior alternative. This paper introduces a zwitterionic liquid (ZIL) as a potential replacement. ZIL, a zwitterion-type ionic liquid with histidine-like modules, remains liquid at ambient temperature, unlike most zwitterions. Previous research demonstrated ZIL's ability to dissolve cellulose and its minimal toxicity to *E. coli*. This study expands on these findings, investigating ZIL's biocompatibility with animal cells and tissues and its potential as a cryoprotectant and drug vehicle. The research aims to establish ZIL as a multifunctional, biocompatible solvent offering significant advantages over DMSO.

The literature extensively documents DMSO's wide-ranging applications in various life science domains. Studies have highlighted its effectiveness as a cryoprotectant (Fayomi et al., 2019; Morris et al., 2019), a vehicle for hydrophobic drugs (Jacob & de la Torre, 2015), and its role in various biological processes (Golan et al., 2018; Tuncer et al., 2018). However, concerns regarding DMSO's toxicity and its effects on cell behavior have been raised (Verheijen et al., 2019; Thaler et al., 2012; Kang et al., 2017). The detrimental effects of DMSO on the activity of platinum-based anticancer drugs have also been noted (Hall et al., 2014; Kerrison & Sadler, 1977). Previous work by the authors introduced ZIL and demonstrated its unique properties (Kuroda et al., 2017; Satria et al., 2018), laying the groundwork for this investigation into its biocompatibility and potential as a DMSO replacement. The mechanism of action of cryoprotectants, including the role of polyampholytes and the importance of membrane stabilization, is reviewed, paving the way for the discussion of ZIL's cryoprotective effects. (Stubbs et al., 2019; De Santis & Coticchio, 2011; Huang et al., 2017; Leslie et al., 1995; Bailey et al., 2019; Rajan et al., 2016).

The study employed a multifaceted approach encompassing cell viability assays, molecular dynamics (MD) simulations, cell cycle analysis, immunoblotting, iPS cell culture and analysis, zebrafish embryo toxicity tests, drug solubility assays, and cryopreservation experiments. Human and mouse fibroblast cell lines (hNF-1, hNF-2, mNF), MDA-MB-231 breast cancer cells, WM266.4 melanoma cells, PC9 lung cancer cells, MDCK cells, and mouse primary astrocytes were used to assess ZIL's toxicity compared to DMSO at various concentrations. Cell viability was determined using CellTiter 96 Aqueous One Solution and neutral red assays. MD simulations using Amber18 and AmberTools19 software modeled ZIL's interaction with a DOPE-DOPG lipid bilayer to determine its cell permeability. Cell cycle analysis was conducted using EdU incorporation and DAPI staining, and immunoblotting was used to assess Rb phosphorylation. Human iPS cells were cultured and analyzed to assess ZIL's impact on stem cell differentiation. Zebrafish embryos were used to evaluate the toxicity of ZIL and DMSO during embryogenesis. The solubility of 12 hydrophobic compounds in ZIL and ZIL aqueous solutions was tested. The anticancer effects of cisplatin dissolved in ZIL aqueous solutions were compared to those dissolved in DMSO aqueous solutions using MDA-MB-231 cells and CellTiter 96 Aqueous One Solution. Cryopreservation experiments used hNF-2 cells and compared the viability after freezing with different media containing DMSO or ZIL. Differential scanning calorimetry (DSC) was used to analyze the phase behavior of the freezing media. Statistical analyses included one-way ANOVA with Dunnett's test and two-tailed unpaired Student's t-tests.

ZIL demonstrated significantly lower toxicity to human fibroblasts (hNF-1 and hNF-2) and mouse fibroblasts (mNF) than DMSO at high concentrations (10%). MD simulations showed that ZIL did not penetrate the cell membrane, unlike DMSO. At low concentrations (1-2%), DMSO suppressed cell cycle progression and dephosphorylated Rb, while ZIL had minimal effects. ZIL exhibited better compatibility with human iPS cells, maintaining the expression of undifferentiated markers unlike DMSO. In zebrafish embryos, DMSO caused high mortality and malformations, whereas ZIL had no adverse effects. ZIL effectively dissolved various hydrophobic compounds, including those insoluble in water and DMSO. Importantly, ZIL served as a solvent for cisplatin stock solutions without affecting its anticancer activity, in contrast to DMSO, which inactivated the drug. ZIL functioned as a cryoprotectant, preserving cell viability after freezing at −85°C, comparable to commercial freezing media containing DMSO. Interestingly, even a simple 5% (w/v) ZIL aqueous solution effectively cryopreserved hNF-2 cells, unlike a 5% (v/v) DMSO aqueous solution. DSC analysis showed that ZIL aqueous solutions formed a glass-like unfrozen phase, suggesting that the cryoprotective mechanism involves concentration of ZIL near the cell membrane, supported by MD simulations.

This study demonstrates that ZIL is a promising alternative to DMSO, possessing significantly improved biocompatibility and functionality. The non-cell-permeable nature of ZIL, confirmed by MD simulations and intracellular concentration measurements, mitigates the adverse effects associated with DMSO's intracellular actions. The ability of ZIL to dissolve hydrophobic compounds, including platinum-based anticancer drugs without affecting their activity, addresses a critical limitation of DMSO. Furthermore, its cryoprotective properties, possibly mediated by membrane interaction and glass-transition formation, broaden its potential applications. The results demonstrate ZIL's superior biocompatibility in various cell lines and even in developing zebrafish embryos. The findings offer compelling evidence for ZIL's potential to replace DMSO in many areas of life science research, particularly where DMSO's toxicity presents a limitation.

ZIL emerges as a superior alternative to DMSO, offering enhanced biocompatibility, functionality as a drug vehicle, and cryoprotective properties. Its ability to maintain the efficacy of platinum-based anticancer drugs is noteworthy, along with its promising applications in stem cell research and cryopreservation. Future research could focus on optimizing ZIL-based freezing media, exploring its efficacy in other cell types and organisms, and investigating its potential in diverse applications across various life science fields. The development of ZIL holds significant promise for advancing life science research and applications.

While the study demonstrates the considerable advantages of ZIL over DMSO, several limitations should be acknowledged. The sample sizes in some experiments were relatively small. Further investigations are needed to fully elucidate ZIL's mechanism of cryoprotection. The long-term effects of ZIL on various cell types and organisms require further investigation. The study predominantly focused on fibroblast cell lines; broader evaluation across different cell types is necessary to confirm ZIL's general applicability. Finally, while the cost-effectiveness of ZIL was noted, a detailed economic comparison with widespread use would be beneficial.