Review Challenges Inert Solvent Assumptions for Deep Eutectic Systems

A comprehensive review published in Discover Chemistry argues that natural deep eutectic solvents, or NADES, should not be classified as inert pharmaceutical excipients. Instead, the authors propose an “Active System Hypothesis,” suggesting that NADES are complex, supramolecular systems that actively regulate local physicochemical environments. This conceptual shift is driven by growing experimental and computational evidence indicating that NADES exhibit non-ideal behavior governed by cooperative hydrogen bonding and dynamic microheterogeneity, rather than the linear, additive properties assumed by traditional pharmaceutical frameworks.

The central challenge in contemporary drug delivery is translating physicochemical properties into predictable therapeutic performance. While therapeutic deep eutectic systems, or THEDES, have been shown to enhance the apparent solubility and permeability of drugs like ibuprofen, these effects are primarily governed by system-level intermolecular interactions. Specific examples include menthol-based systems containing ibuprofen, benzoic acid, and phenylacetic acid. Research by Aroso et al. demonstrated that various choline chloride and menthol-based systems form liquid THEDES at room temperature, exhibiting enhanced dissolution compared to the active pharmaceutical ingredients alone. However, the review highlights that these improvements are often misinterpreted as simple solubility increases, obscuring the underlying mechanistic complexity of the interactions.

A critical finding of the review is that apparent solubility enhancement in NADES often arises from kinetically stabilized supersaturation rather than a true shift in thermodynamic equilibrium. Time-dependent crystallization studies demonstrate delayed nucleation and prolonged metastable states, which challenge equilibrium-based solubility models. The authors explain that transient solute-NADES interaction networks inhibit nucleation and crystal growth, maintaining metastable supersaturation.

The role of water in NADES systems is another area where conventional assumptions fail. The review indicates that small compositional variations, particularly in water content, produce disproportionate changes in physicochemical behavior. Increasing water content induces a stepwise transition from a hydrogen-bonded supramolecular network to an aqueous-like system, reflecting the loss of DES structural integrity. This non-linear influence on viscosity and solvation thermodynamics undermines reproducibility and predictability. For instance, altering hydrogen bond donor ratios by less than 10% can induce more than five-fold changes in solubility and viscosity, highlighting the extreme sensitivity of these systems to microstructural reorganization.

Biological effects associated with NADES are frequently confounded by the intrinsic activity of their components. Many hydrogen bond donors and acceptors, including organic acids and amino acids, possess independent biological effects. The review notes that IC50 values for cytotoxicity can vary from approximately 15 mM to over 400 mM depending on the hydrogen bond donor selection. This variability indicates that supramolecular organization fundamentally alters biological interaction profiles, rendering component-based safety extrapolation unreliable. Without clear differentiation between controlled modulation and nonspecific toxicity, the biological relevance of NADES remains difficult to establish.

The review also addresses the concept of the “caging effect,” a mechanistic concept often used to describe solute confinement within stable hydrogen-bond networks. However, structural and spectroscopic evidence does not support the existence of discrete or static cages. Neutron scattering, NMR, and molecular dynamics studies show that these systems display spatial heterogeneity with short-lived interaction motifs that continuously reorganize at the molecular scale. Stabilization in these systems is therefore not a consequence of spatial confinement but of collective, fluctuating interactions that modulate local environments. This dynamic structuring challenges the static models often applied to describe solute behavior in NADES.

Hydrophobic natural deep eutectic solvents, or HNADES, represent a distinct group with limited miscibility with water and greater affinity for non-polar compounds. These systems are particularly interesting for the extraction and solubilization of lipophilic compounds, including bioactive molecules with poor aqueous solubility. HNADES may interact with biological membranes due to their affinity for lipid-rich environments, potentially enhancing skin permeability. However, such interactions are not necessarily beneficial, as hydrophobic DES-containing microemulsions have been shown to alter the lipid organization of the stratum corneum. This distinction is important when evaluating HNADES for pharmaceutical use, as increased permeability alone does not establish a safe or controlled delivery mechanism.

From a regulatory perspective, NADES occupy a grey area where neither excipient-based nor drug-based evaluation strategies are fully applicable. Current regulatory models are designed for single, chemically defined excipients with fixed composition and predictable behavior. In contrast, NADES are inherently multi-component, tunable, and dynamically interacting systems. This discrepancy becomes particularly pronounced with THEDES, where the distinction between excipient and active substance becomes ambiguous. The absence of standardized and universally applicable classification criteria that account for compositional variability and emergent behavior contributes to a regulatory gap that may limit clinical translation.

The authors propose that the Active System Hypothesis provides a structured framework for defining NADES as conditional functional systems. This framework establishes criteria for evaluating when NADES exhibit system-level effects and when observed outcomes are attributable to individual components. It shifts the field from descriptive interpretation toward mechanism-based classification, enabling the development of predictive models that integrate physicochemical structure with biological response. Validating this framework will require integrated experimental designs that decouple component activity, quantify non-additive interactions, and establish direct links between NADES structure and biological function.

Future research must move beyond reporting outcomes and directly probe structure-dynamics-function relationships under physiologically relevant conditions. A critical unresolved question is whether NADES persist as supramolecular assemblies in biological environments or rapidly dissociate into individual components. The review suggests that future work should employ isotopically labeled NADES components combined with in situ NMR spectroscopy and mass spectrometry imaging to track co-localization and co-diffusion across biological membranes. Additionally, quantitative interaction mapping is needed to distinguish between true thermodynamic solubilization and kinetic stabilization, using techniques such as temperature-dependent solubility measurements and time-resolved crystallization kinetics.

The advancement of NADES in pharmaceutical science requires a shift from component-centric assessment to a system-oriented regulatory paradigm. This approach must explicitly account for interaction-driven behavior and context-dependent variability. Without this transition, the classification of NADES as safe, functional, or active pharmaceutical systems will remain unsupported by the level of evidence required for regulatory acceptance. The review concludes that NADES should be viewed as context-dependent supramolecular systems, with their pharmaceutical value determined by their composition, structure, and biological environment, rather than as simple inert solvents.

The review identifies a significant gap in current experimental design: the inability to distinguish between intrinsic component activity and emergent system-level behavior. When embedded in a hydrogen-bonded network, these components alter their chemical environment, yet current studies rarely isolate individual contributions from system-level interactions. This ambiguity is particularly problematic for therapeutic deep eutectic systems, where the active pharmaceutical ingredient participates directly in eutectic formation while contributing to therapeutic function. Without orthogonal controls capable of separating cooperative interactions from intrinsic bioactivity, the classification of NADES as either inert excipients or active biological systems remains conceptually unresolved. The authors emphasize that observed effects are frequently attributed to enhanced solubility or stabilization rather than direct interaction between the NADES matrix and biological signaling machinery, such as the Nrf2 or PI3K/Akt pathways, which remain mechanistically unresolved in current literature.

Computational approaches, particularly molecular dynamics simulations, are highlighted as essential for resolving the mechanistic complexity of these systems. These methods enable atomistic-level characterization of solute-solvent interactions, hydrogen-bond dynamics, and spatial organization, providing descriptors that are not accessible through classical thermodynamic or empirical models. However, the translation of these insights into scalable, predictive models for formulation design remains an unresolved challenge. Current studies largely focus on isolated systems or specific compositions, lacking generalized models capable of translating atomistic interaction patterns into scalable, formulation-relevant predictions. The review notes that the absence of reliable quantitative structure-property relationship models for NADES explains why results often fail to translate across compositions, as macroscopic properties arise from collective behavior rather than pairwise molecular contributions.

From a safety perspective, the assumption that NADES are inherently safe due to their composition from naturally derived metabolites is not supported by systematic toxicological data. Multiple studies demonstrate pronounced, concentration-dependent cytotoxicity, membrane perturbation, and oxidative stress responses that cannot be reliably predicted from the properties of individual components alone. The compositional dependence of cytotoxicity is evident in varying IC50 values across different hydrogen bond donors, which limits pharmaceutical translation due to the absence of universal descriptors for batch-to-batch consistency. Furthermore, there is a lack of system-level pharmacokinetic characterization; NADES are often presumed to dissociate into metabolizable constituents upon administration, but there is little direct experimental evidence describing their absorption, distribution, persistence, or clearance as supramolecular systems. This assumption of rapid dissociation may overlook transient or localized system-level effects that influence bioavailability and tissue interaction.

The review outlines a translational roadmap requiring a shift from descriptive studies toward experimentally testable, mechanism-resolving strategies. This involves combining temperature-dependent solubility measurements with van’t Hoff analysis and time-resolved crystallization kinetics to explicitly separate equilibrium and non-equilibrium contributions. Additionally, membrane biophysics must be integrated with cellular assays using techniques like Langmuir monolayer studies and cryo-electron microscopy to quantify changes in lipid packing and domain organization upon NADES exposure.

Regulatory advancement depends on the establishment of standardized, system-level evaluation protocols. Current toxicity studies are highly variable and lack comparability due to inconsistencies in water content, concentration, and exposure duration. Future studies must adopt harmonized testing frameworks, including concentration-dependent cytotoxicity, oxidative stress markers, and membrane integrity assays under standardized conditions. Comparative studies across multiple NADES compositions are needed to identify structure-toxicity relationships. Regulatory classification will require clear evidence demonstrating whether NADES behave as independent systems or as mixtures of individual components under physiological conditions. Progress in this field requires integration of multi-scale experimental and computational approaches, linking molecular interaction, microstructure, transport, and biological response within unified experimental frameworks to enable direct mechanistic correlations rather than post hoc interpretations.

Subject of Research: Re-evaluation of natural deep eutectic solvents as active supramolecular systems in pharmaceutical applications

Article Title: Natural deep eutectic solvents as active systems rather than inert solvents

Article References: Kattel, A., Bhat, S., Sharma, A., & Chutia, D. (2026). Natural deep eutectic solvents as active systems rather than inert solvents. Discover Chemistry, 3(1), Article 605. https://doi.org/10.1007/s44371-026-01065-x

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01065-x

Keywords: NADES, pharmaceutical excipients, supramolecular systems, drug delivery, solubility enhancement, hydrogen bonding, regulatory frameworks, therapeutic deep eutectic systems, Natural, deep, eutectic, solvents