Eutectogels as Promising Materials in Biocatalysis

The entrapment of deep eutectic solvents (DES) and eutectic systems into porous scaffolds renders a new class of soft and nonvolatile materials called eutectogels that have recently stepped into the spotlight in different areas ranging from electronics to drug delivery. Recent progress in the use of DES in biocatalysis, where they have been demonstrated to improve substrate supply, conversion, and enzyme stability, has opened an unparalleled opportunity to exploit the merits of eutectogels for immobilizing biological catalysts. The resulting functional materials could outperform traditional hydrogels and ionic liquid gels, offering fresh perspectives to broaden the application scope of many enzymes. In this perspective, we go into the potential of eutectogels as innovative scaffolds that support biocatalytic reactions and discuss different applications where these systems could show plain benefits compared to traditional materials. Future directions for this newly developed technology are highlighted.

materials could outperform traditional hydrogels and ionic liquid gels, offering fresh perspectives to broaden the application scope of many enzymes.In this perspective, we go into the potential of eutectogels as innovative scaffolds that support biocatalytic reactions and discuss different applications where these systems could show plain benefits compared to traditional materials.Future directions for this newly developed technology are highlighted.

Context
Eutectic systems were early described by F. Guthrie in 1884 as mixtures of two or more components showing a minimum temperature of liquefaction at a particular proportion (eutectic point). [1]Nowadays, it is known that all mixtures of pure compounds that are entirely or partly immiscible in the solid phase present a eutectic point. [2]Still, in 2003, Abbot et al. discovered an abnormally large melting point depression at the eutectic composition of the choline chloride (ChCl, melting point, T m � 302 °C): urea (U, T m � 133 °C) mixture (1 : 2 mol fraction), resulting in a liquid a room temperature ("reline", T m � 12 °C). [3]This landmark work introduced the term deep eutectic solvents (DES) and postulated the hydrogen bonding between the mixture components, typically called hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA), as the root cause of their unexpected liquid behavior. [4]Nevertheless, a recent study using betaine as HBA has demonstrated that hydrogen bonding in a system is neither a necessary nor sufficient condition for its components to form a DES, and instead, it depends on the strength of the interactions between them compared to those in their pure state. [5]here is still some uncertainty in the realm regarding the differences between traditional eutectic solvents and DES, and establishing a working definition for these neoteric systems is urgently necessary.Some researchers have defined DES as eutectic solvents whose components present enthalpic-driven negative deviations from thermodynamic ideality, as this behavior is observed in the archetypical reline that shows asymmetrical deviations in the urea liquidus curve. [6]Therefore, marked negative deviations from thermodynamic ideality could create low-temperature melting mixtures, providing unusual liquid environments for new biochemistries.Figure 1 displays solid-liquid equilibrium phase diagrams for hypothetical binary mixtures illustrating the difference between eutectic solvents and DES.In general terms and for a binary mixture of components A and B, negative deviations occur when the interactions between them are stronger than the interactions with themselves (AÀ B > AÀ A, BÀ B).
Indeed, DES technology has been actively explored for biocatalysis since Kazlauskas et al. proved enzymatic reactions in these non-conventional solvents in 2008. [7]DES has been Figure 1.Schematic illustration of solid-liquid equilibrium phase diagrams for hypothetical mixtures of A and B components displaying thermodynamic ideal behavior (dashed black line, example of a eutectic solvent) or negative deviations from the thermodynamic ideality (dashed red line, instance of a DES).Adapted with permission from reference [6].demonstrated to enhance substrate solubility and stability of different enzymes, including hydrolases, oxidoreductases, peroxidases, transferases, and lyases, spanning from single to multistep transformations involving organocatalyst/enzyme and metal/enzyme combinations. [8]14][15][16][17] In this context, we see an unparalleled opportunity for pushing this field toward enzyme immobilization into DEShosting soft materials, called "eutectogels", benefiting from the well-known advantages of solid supports such as higher enzyme stability, broader biocatalyst applicability, recycling, and continuous operation possibilities. [18]This perspective presents a picture of biocatalysis in DES, emphasizing eutectogels as innovative support materials to open up exciting horizons for future applications (Scheme 1).Their potential in the biomedical space, biomineralization processes, biosensors, and others are discussed.By placing eutectogels into this context, we hope the biocatalysis research community gains insight into how these materials could influence scientific progress, which may inspire the future design of novel systems.

Biocatalysis in DES
Water is the predominant solvent in biocatalysis, yet its extensive polarity poses a significant limitation in biocatalytic processes.This arises from the incongruence between the high polarity of water and the hydrophobic characteristics of numerous target reactants, resulting in a diminished solubility of substrates within the aqueous reaction milieu.Therefore, non-conventional reaction media, including organic solvents, supercritical fluids, and ionic liquids (IL), have been considered as media in biocatalysis. [19]However, the utilization of these solvents is often limited by their toxicity and high cost.In this context, DES have emerged as a greener and cost-effective alternative, sharing many traits with IL, such as non-flammability and low vapor pressure.Hence, there is a growing interest in employing DES in biocatalysis, considering their ability to enhance substrate solubility and stabilize enzymes.As described by Holtmann and coworkers, various enzymes, including living microbial cells, have proven successful in effecting biotransformations within DES environments. [19]This section is dedicated to exploring the utilization of DES in pertinent biocatalytic reactions.Some of the reactions studied in DES are tabulated in Table 1 and Figure 2. The examination has extensively delved into single reactions catalyzed by hydrolases in DES and DES-aqueous mixtures, specifically lipases.The appeal of hydrolase-catalyzed reactions in organic synthesis lies in their user-friendly nature and the absence of specific cofactor requirements for optimal functioning.This has sparked significant interest in advancing such reactions, encompassing a diverse array of enzymes, including but not limited to lipases, esterases, amidases, nitrilases, proteases, and epoxide hydrolases (EHs).
hemoglobin and immunoglobulin G experienced thermal stabilization in the presence of DES. [45,46]In this context, Kim et al. verified the enhanced enzymatic activity of lipase when DES were employed as cosolvents. [49]Furthermore, a comparative analysis demonstrated the superiority of ChCl-based DES over ChAc-based counterparts in boosting the activity of horseradish peroxidase. [50]ChCl : Gly and ChCl : EG eutectic solvents were employed to maintain the relative activity of cellulase at levels exceeding 80 %, even following a 48-hour incubation at 50 °C. [47]The observed preservation of enzymatic activity was notably more pronounced in ChCl : EG compared to ChCl : Gly.In DES, water molecules are mostly confined, preventing a complete hydration layer, in contrast to osmolytes' preferential exclusion/hydration mechanism.Additionally, structural fluctuations of proteins are suppressed in DES, implying slow conformational dynamics.This suggests a potential kinetic stabilization mechanism for protein stabilization in DES attributed to the presence of a rigid solvation shell in the absence of water, as revealed by molecular dynamics simulations, although experimental validation remains challenging. [42]anchez-Fernandez et al. demonstrated the efficacy of ChCl : U in storing and preserving lysozyme.The protein exhibited stability over 40 days at room temperature, retaining its native conformation and activity upon rehydration. [60]In another study, a re-entrant behavior was unveiled in the amino acid environment and secondary structure of proteins as bovine serum albumin, lysozyme, and immunoglobulin G in ChCl : U and ChCl : Gly, depending on solvent hydration. [61]A domeshaped transition occurred at very low (< 10 wt% H 2 O) and high (> 40 wt% H 2 O) DES hydration, with varying protein structures, unfolding, and oligomerization observed under different hydration levels, highlighting the complex interplay between DES composition, hydration, and protein behavior. [61]hile the extensive application of DES has been welldemonstrated in biocatalysis, they encounter various challenges.These include issues such as recyclability, the homogeneous nature leading to difficulties in separating biocatalysts after reactions, and the vulnerability of enzymes to denaturation in relatively severe conditions, all of which hampers their large-scale industrial use.Consequently, a more sustainable approach to utilizing eutectic solvents in biocatalysis is imperative.This approach should focus on protecting enzymes for use in harsh conditions, ensuring that the resulting biocatalytic system is robust, and facilitating ease of manufacturing.

Enzyme Immobilization in Eutectogels
Utilizing an appropriate supporting medium for immobilization presents an appealing approach to enhance the stability and reusability of these biocatalysts with ease of separation.Besides, this strategy allows the confinement of the enzymes with cofactors or chemical catalysts in given volumes, offering distinctive possibilities for developing synergistic combinations.The traditional approach for protein encapsulation involves covalently attaching it to a heterogeneous supporting material.
Alternatively, a non-covalent method, known as entrapment, confines the enzyme within a material, typically a porous matrix.This process creates an engineered environment that shields the protein from poisoning and denaturation.In entrapment, the enzyme in a solution becomes enveloped by the matrix and is physically immobilized during a polymerization event, forming a gel.
Entrapment alters the immediate surroundings of the protein by constraining the enzyme within the solvent held in the matrix.One such immobilization method has already been demonstrated recently by Imam et al.where lipase was entrapped in IL supramolecular gels. [62]The porous matrix functions as a selective barrier, creating a distinction between the enzyme and the surrounding solution.This method contrasts surface immobilization, where exposure to external environments remains unaffected.This idea could be extended by using eutectic solvents instead of IL and creating eutectogels of different chemical natures that can be molded in various shapes depending on the application.
The term eutectogels was coined by Joos et al. in 2018 to describe the embedment of a Li + -conducting DES within a silica matrix, [63] although now it is broadly used to define any gel in which the swelling agent is a traditional eutectic solvent or a DES.[69][70] There is still much to be done in this emerging topic, principally expanding the application of eutectogels to other areas.
Indeed, immobilizing enzymes within eutectogels is an entirely unexplored concept, unlike other soft materials such as hydrogels, which have been widely investigated for this goal. [71]ybrid protein/eutectogels could come with unique synergistic properties where, for example, the therapeutic activity of THEDES can be combined with enzymatic reactions that are valuable in the biomedical space, at the same time that the eutectic medium boosts the biocatalyst performance.
One interesting case may be the immobilization of catalase in eutectogels containing eutectic solvents based on either fatty acids with antimicrobial activity or polyphenols with antiinflammatory properties, which could be highly attractive as therapeutic patches for diabetic chronic wound treatment.In these functional materials, catalase would provide oxygen to the wound microenvironment, as it plays a crucial role in the healing process by promoting cell proliferation and protecting them from hypoxia-related damage.[74] This enzyme could also be interesting for designing electroactive eutectogel dressings, as electrical stimulation can promote cell migration and proliferation, accelerating wound closure.
Glucose oxidase (GOx) is another biomedically relevant enzyme that has recently gained significant interest in starvation therapy for cancer treatment. [75]This therapy is based on the catalytic depletion of intracellular glucose, cutting off the nutrition source of cancer cells and consequently inhibiting their proliferation. [76,77]One exciting option to explore is combining GOx with eutectic solvents based on anti-cancer drugs that would improve the tumor permeability, solubility, and diffusion rate as they remain in a permanent liquid state.Thus, it becomes obvious that the beauty of eutectic technology relies on the possibility of liquefying a substance with a target property that otherwise would be solid at the desired application temperature.Then, injectable supramolecular eutectogels stabilizing GOx in chemotherapeutic DES would be a highly innovative system for intratumoral synergistic cancer treatment.
The shape and size of eutectogels containing biocatalysts can be adjusted by using molds of various geometries and carefully controlling the gelation process.The gel precursor is poured into these molds, allowing it to take on the desired form as it solidifies.Temperature gradients can also be applied to introduce gel properties or shape variations.Advanced 3D printing techniques, like extrusion-based or stereolithographybased methods, enable precise manipulation of eutectogel shapes, facilitating the creation of intricate structures with exceptional accuracy.Enhancing the stability of eutectogels involves adjusting the concentration of gelling agents.This fortifies the gel against deformation and collapse, ensuring good mechanical stability for recycling the biocatalyst and its operation in reactors.
Another good entrapment strategy is forming polymerizable DES (PDES) in which the HBD or HBA is a monomer for free radical polymerization.[80] It is conceivable to suggest that the reactive component of DES undergoes a process of evolution or decomposition, leaving behind the inert component embedded within the resultant product of the transformation.Consequently, free radical polymerization can manifest through various combinations: (a) involving a polymerizable HBD and an inert HBA, (b) commprising a polymerizable HBA and an inert HBD, and (c) incorrporating a polymerizable external monomer within an inert DES.Depending on the selected DES matrix and its targeted application, initiators or photoinitiators, alongside crosslinkers, may be introduced in conjunction with enzymes to instigate the polymerization process. [66,81,82][85][86][87][88][89][90] Therefore, this approach could also be explored for enzyme encapsulation, forming nanocapsules/nanogels by adding cross-linkers and tuning the chemical structure of the DEM.
This opens up an entirely new avenue in biomedical science for sustainably developing nanocarriers.We elaborate on this notion by proposing the concept of creating eutectogels wherein the DES serves a dual role, functioning concurrently as both the reaction medium and the substrate reservoir.Very recently, a similar strategy has been employed in which eutectogels were synthesized by free radical polymerization of AAm in the presence of an α-helical peptide cross-linker (PC) in ChCl : U or ChCl : EG eutectic solvents. [91]AAm, PC, and a photoinitiator were dissolved in DES and then exposed to UV light resulting in eutectogel formation.Recently, a supramolecular eutectogel was synthesized through in situ ring-opening polymerization of thioacetic acid within a PDES, forming eutectogels capable of encapsulating liquid metal. [92]These gels exhibit underwater self-healing and self-adhesive properties.
In this context, the entrapment of inorganic constituents, including metal nanoparticles/clusters, MXenes, perovskites, and metal-organic frameworks (MOFs), alongside enzymes, establishes an innovative avenue for applications in biocatalysis, sensing, wearable or implantable devices, flexible materials for soft robotics, and artificial skins (Figure 3). [93,94]Integrating this novel fabrication approach with the inherent stability of DES holds promise for engineering intelligent structures characterized by a harmonious blend of lightweight and robust attributes, particularly conducive to biomimetic soft robotics.The meticulous choice of DES, informed by a thorough understanding of their structural nuances and impact on biomacromolecules and inorganic components, represents a paradigm shift in biohybrid soft materials, opening tremendous perspectives.
This approach holds potential advantages for protein engineering, particularly for enzymes dependent on cofactors, as the presence of responsive metals nearby will benefit activity.Furthermore, it will be efficient for one-pot chemoenzymatic reactions that necessitate the collaboration of both artificial and natural catalysts.

Final Remarks
Eutectic technology has started to shine in many areas, and the current library of these liquid mixtures is expanding fast, incorporating drugs, metal alloys, polymers, and industrially relevant organic molecules, which bring new opportunities for chemists and material scientists.In this encouraging context, we envision a promising future for a new generation of hybrid soft materials combining eutectogels with enzymes that could be exploited in various applications.Perhaps we will witness the rise of a new realm, the "eutectozymes".Materials design from the macro to the nanoscale (still unreached for eutectogels) and the exciting idea of new liquid environments where enzymes can boost their performance unveil unlimited opportunities for technological innovations.We invite the scientific communities working in biocatalysis and eutectogels to join efforts to advance in this coming field and hope this perspective serves as an inspiration for creating new exciting materials with unseen functions and propelling technological breakthroughs.

Figure 2 .
Scheme 1. Schematic representation of the concept of enzyme immobilization within eutectogels.This technology could also be used to support chemoenzymatic reactions due to the exceptional properties given by the scaffold: three-dimensionality, excellent solvent environment, and the possibility of confinement.Created with BioRender.com. Figure 2. Summary of different biocatalytic reactions that could be carried out in DES-supporting eutectogels.Created with BioRender.com.

Figure 3 .
Figure 3. Representation of the photopolymerization process of a DEM in the presence of an enzyme to produce a hybrid PDES supporting network.Created with BioRender.com.

Table 1 .
Summary of biocatalytic reactions performed in DES.