Current Trends in Green Solvents: Biocompatible Ionic Liquids

Abstract

Biocompatible Ionic Liquids (Bio-ILs) are a new class of ILs that are task-specifically designed to derive from naturally occurring compounds and/or derivatives thereof, as well as molecules well known for their biocompatibility (e.g., active pharmaceutical ingredients or non-toxic bioactive compounds) in terms of sustainability and biocompatibility. Owing to their remarkable physicochemical properties that can be tailor made to comply with the requirements of each application, Bio-ILs have emerged as novel, efficient and green technology, appropriate for a vast variety of scientific fields. Herein, this review describes the state-of-the-art scientific research regarding the synthesis, characterization and applications of Bio-ILs reported in the literature for the period between 2020 and 2022.

1. Introduction

Ionic liquids (ILs) are normally defined as liquids composed entirely of ions, which, in most cases, are synthesized by the combination of a bulky organic asymmetric cation (e.g., tetraalkylammonium, tetraalkylphosphonium, imidazolium, cholinium, pyridinium) and an organic or inorganic anion [1,2,3]. They usually have melting points lower than 100 °C and, in many cases, they are liquids at room temperature—known as room-temperature ionic liquids (RTILs)—because of the reduced electrostatic interactions and crystallization ability, due to the counter-ions’ asymmetry and charge dispersion [1,2]. There are various forces that exist in ILs, which co-determine their properties. The formation of these solvents is generally accomplished through ionic and hydrogen bond interactions between the components, which depend on their type and mixing ratio [1]. The asymmetry and the alkyl chain length of the counterions are factors that affect the physicochemical properties of ILs [4]. Some of their characteristics that make them so special are the low or in some cases negligible vapor pressure, the wide temperature range of the liquid phase, the incombustibility and thermal and chemical stability, which depend on their structure [3].

Despite their unique physicochemical properties, the eco-friendliness and biocompatibility of ILs have been debated. The majority of ILs, such as imidazolium, phosphonium and pyridinium, show increased toxicity and low biodegradability [1,5]. However, in the last few years, Biocompatible Ionic Liquids (Bio-ILs) emerged as alternative greener media for the development of sustainable and eco-friendlier processes [6].

ILs synthesized using natural products (primary and/or secondary metabolites) and their synthetic analogues and/or active pharmaceutical ingredients (APIs) or other bioactive compounds are mentioned in the literature as Bio-ILs and consist of a new eco-friendly family of ILs [2,3,6]. Choline is a compound usually found in organisms and, thus, it is regarded as a biocompatible component for ILs. Organic acids, sugars, amino acids, terpenes and purines are substances of natural origin, often used for the synthesis of Bio-ILs [2]. The use of natural products for the preparation of ILs is in agreement with the principles of Green Chemistry and, thus, Bio-ILs have gained significant interest [3].

Bio-ILs are considered as green, biodegradable or biocompatible due to their components. However, in recent years, their toxicity (cyto- and eco-microbial toxicity [7,8,9]), biocompatibility [10] and biodegradability [7,11,12,13] have been studied more thoroughly.

ILs have been characterized as “designer solvents” [4] since their physicochemical properties can be task-specifically tuned by the cautious selection of the IL’s ions. This characteristic gives them an enormous advantage over other conventionally used solvents, since it is feasible, via structural alterations, to affect the physicochemical properties as well as the overall green character of the ILs. In the last few years, the scientific research turned to the design of biodegradable and biocompatible ILs able to be used in a plethora of applications. The increasing arising interest regarding Bio-ILs becomes obvious, not only because of the scientific research papers in the literature but also due to the publication of review articles [10,14,15,16], indicating the need of the scientific community for data regarding this area.

Indicatively, ILs have been used lately as solvents and/or catalysts in chemical reactions [17,18,19,20], in biocatalysis [21,22,23], in nanotechnology [24,25,26] and, more recently, they have been studied as innovative materials for uses in the biomedical field. Their solubilizing activity, high stability and biocompatibility make them suitable to be used as active pharmaceutical ingredients (APIs) and/or carriers (emulsions, microemulsions) in various innovative drug delivery systems, as antibiofilm, antimicrobial and anti-cancer agents and ionogels, etc. [1,4].

At this point, it should be mentioned that there is also another class of green solvents that could derive from biocompatible materials, such as choline, carbohydrates, amino acids, etc., namely, Natural Deep Eutectic Solvents (NADESs). ILs and Deep Eutectic Solvents (DESs) have a lot in common, especially regarding their physical properties and their applications. Thus, some authors consider DESs to be a subclass of ILs and, thus, in their works, they use the term of IL instead of DES or NADES. On the contrary, other authors strongly disagree with this statement, since DESs and ILs are, by their nature, independent groups [27]. ILs are composed of ions and are synthesized via a chemical reaction (such as a proton transfer in the case of protic ILs or, in other cases, several synthetic steps are involved). By definition, mainly ionic interactions are involved in the formation of ILs. On the other hand, NADESs are eutectic mixtures of at least two components and their formation is a result of an extended and complicated hydrogen bond array created among the chemical species that they are made of.

An interesting example of this controversy is choline and geranate, CAGE: [27,28,29]. CAGE is mentioned in some scientific papers as an IL, since it largely comprises ionic species and has a melting point below 100 °C; however, it also contains neutral geranic acid and is mentioned as a DES in other references. The classification of systems that involve both ions and neutral species is complex, due to definition limitations and, thus, these systems are also included in this review [30,31].

Herein, a literature review of the past three years (2020–2022) is presented, regarding the synthesis, characterization and application of biocompatible ionic liquids in different scientific fields and, more specifically, in catalysis, biomedicine, separation processes and lubricants. There are also a few references regarding the use of choline-based ILs in the nanotechnology field in the period between 2020 and 2022 [26,32,33,34,35]; however, since this field has been recently reviewed [36,37], we opted not to include it in the present review.

According to Figure 1, it is noteworthy that the focus in this area is mainly on the cholinium-based ILs (ChILs) (43% of the research data in the reviewed period). Amino-acid-based ILs (AAILs) are considered as emerging and promising green media (15% of the research data in the reviewed period), while the combination of choline and amino acids for the synthesis of bio-based ILs, namely Ch-AA-ILs, is gaining the scientific community’s interest (Figure 1).

2. Synthesis and Properties

A great asset of this class of promising green solvents is the usually simple and straight-forward synthesis. Two different synthetic routes for the development of bio-ILs have prevailed, namely the metathesis reaction and the neutralization reaction. Briefly, the metathesis reaction is usually performed between a halide anion (from the cationic constituent) and a metal cation (Scheme 1(1)). The mixture is stirred at room temperature, followed by the precipitation of the corresponding salt and the formation of the IL. The salt is filtered and the filtrate is evaporated in vacuo to obtain the IL. For the neutralization approach, the appropriate acid (diluted in the appropriate amount of an organic solvent, such as ethanol) is mixed with selected amines and the water produced as a by-product is removed under reduced pressure (Scheme 1(2)).

Characteristic examples of ILs that can be synthesized via both methodologies are cholinium-based ILs, starting with either choline chloride or choline hydroxide (Scheme 1) [14,38]. Some research teams report the modification of the anions or cations prior to their use in IL synthesis [5,39,40,41,42].

The development of novel ionic liquids that comprise naturally occurring compounds has gained the attention of the scientific community in the past few years and, therefore, the investigation of bio-IL structure has become a priority since it does not only affect their environmental impact, but also their properties and their potential utilization for various applications.

Sharma et al. [43] based their research on the synthesis and characterization of cholinium-based ILs. Eleven naturally occurring carboxylic acids available in a variety of plants were used as anions and the corresponding ILs formed were characterized via ¹H NMR (Nuclear Magnetic Resonance), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), optical rotation, conductivity and rheological investigations. The results of these studies revealed that the examined Ch-ILs possess thermal stability (decomposition temperatures > 230 °C), high conductivity and low zero shear viscosities (with the exception of choline coumarin-3-carboxylate IL), while the thermal properties and the optical rotation of the examined ILs were greatly affected by the carboxylic acid’s structure used each time.

Among ILs consisting of choline and carboxylic acids, choline and geranic acid (CAGE) has captured the interest of researchers due to its exceptional properties that make it suitable for biomedical applications, such as drug delivery systems. In an attempt to investigate the structure–properties relationship in depth, Takeda et al. [44] exploited small-angle X-ray scattering (SAXS), NMR spectroscopy, Dynamic Light Scattering (DLS) and Polarized Light Microscopy (PLM) for the analysis of CAGE and CAGE/water mixtures. The structure of CAGE with water was successfully elucidated, proving that the structure of the IL remains the same with the addition of 17% water, while this is not observed with the addition of 25% water, where the IL converted to the lamellar phase, nor with the addition of more than 67% water, where the IL changed to the micellar phase. These findings are of great importance and should be considered when CAGE is applied to transdermal administration where the water content is increased.

Small- and wide-angle X-ray scattering analysis was also performed on six different Ch-ILs synthesized by choline and amino acids and their solutions in water and n-alkanols by Miao et al. [45], in order to establish a better understanding of the nanostructure of bio-ILs. The results confirmed the long-range amphiphilic nanostructure of the Ch-ILs examined, which can be tailor made by the appropriate selection of the anion used. This nanostructure is not greatly affected by the presence of water or ethanol (up to 40 wt.%); however, longer-range nanostructures appear when these ILs are diluted to longer n-alkanols (C₄H₉OH to C₁₀H₂₁OH). In certain cases, IL/n-alkanol mixtures can enhance amphiphilicity and improve miscibility and viscosity, broadening the applicability of the hybrid solvents.

Dhattarwal et al. [46] approached the structural investigation of cholinium amino-acid-based ILs (Ch-AA-ILs) using atomistic simulations. The simulated total X-ray scattering of six Ch-AA-ILs revealed a strong hydrogen bond network between the hydroxyl group of the cholinium cation and the carboxyl group of the amino acid anions, while the side chain of the amino acids greatly affects the nanoscale heterogeneity of the solvents. More specifically, the bio-ILs, which are formed by amino acids with longer or bulkier side chains, such as phenylalanine or methionine ([Ch][Phe] and [Ch][Met], respectively) (Scheme 2), demonstrated higher heterogeneity due to the fact that side chains interact with each other, leading to the formation of side-chain clusters. This, however, is not observed in cases where the anion side chains strongly interact with the backbone, hindering the formation of clusters.

Computational analyses were performed by the research group of Daso [47], who aimed to better comprehend the interactions of ILs with biomolecules, which can be a determining factor for biological applications. Fourteen ILs, consisting of either choline or glycine betaine as cations and three amphiphilic molecules, differentiated regarding their polarity, aromaticity and hydrogen bond formation ability, were selected and studied via COSMO-RS (Conductor-like screening model for real solvents) and Molecular Dynamics simulations. COSMO-RS was used to obtain sigma profiles of the IL bio-organic molecule hybrid mixtures and to predict viscosities and mixing enthalpies, while molecular dynamics simulations were performed on the optimal hybrids to further investigate the interactions between the components of the mixture. The results revealed that greater interactions appeared using the ChILs, while the hydrophobic/hydrophilic character of the amphiphile bio-organic compounds plays a major role in the formation of intermolecular interactions with the ILs.

Following a different approach, Uddin et al. [48] successfully synthesized three novel lipid-based ILs, comprising a long-chain phosphonium cation (1,2-dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine—EDMPC) and the fatty acids stearic, oleic and linoleic acid as anions. The novel solvents were structurally elucidated via ¹H NMR, Fourier-Transform Infrared Spectroscopy (FTIR), Mass Spectrometry (MS) and Thin-Layer Chromatography (TLC), while their thermal properties were determined using DSC. The lack of a peak related to the free carboxylic acids in the NMR spectrum of the synthesized ILs confirms the interaction of the carboxylate anions and the ammonium group of the cation, while the FTIR analysis presented similar results. DSC analysis revealed a significant correlation between the degree of unsaturation of the anion and the melting point of the IL, as it is observed that the presence of unsaturated bonds decreases the melting point. In an effort to examine the potential of the novel media to be exploited as drug delivery systems, some preliminary tests were performed regarding their solubility and their biocompatibility via a skin irritation test using artificial human cells. The lipid-based ILs were found to be soluble in both polar and non-polar solvents and partially soluble in water, whereas they exhibited increased biocompatibility, rendering them as potential drug delivery systems.

The green character of AAILs is expected to be helpful for the solubilization and stabilization of several biomolecules in aqueous solutions and even physiological fluids and, therefore, their buffering capacity plays a key role for many biological, medical, pharmaceutical and biotechnological procedures. Patil et al. [49] studied the ionic and molecular interactions of two protic ionic liquids (PILs) in aqueous solutions, as they previously claimed that the diverse ions in ILs demonstrate different behavior in aqueous solutions, leading to a variety of interactions (hydrogen bonds, Coulomb and dispersion forces). Density measurements were conducted in order to be used for the prediction of the PILs’ volumetric properties. The examined ILs consisted of cations from glycine and L-alanine and nitrate as an anion (Scheme 3). The slight decrease in apparent molar volume at low concentrations for both ILs suggests the dominance of ion−solvent interaction over the association of the two ions (ion pairing). On the other hand, the apparent molar volume increases with the increase in temperature as a result of strong ionic hydration. Hydrophobic and ionic hydration are affected by temperature increment and both ion−solvent and hydrophobic association create a compensation (expansion) effect on water structural effects. In general, the system’s volume is affected by hydrophobicity, electrostriction of water, thermal expansion and cooperative H bonding of the AAILs used in the aqueous solution. The basicity of the N⁺ charge center, the carboxylic group, the side alkyl chain−water interactions and cooperative H bonding affect the hydration and water-structure-making effects, whereas the [NO₃]⁻ as an anion is a water-structure breaker (chaotrope). Furthermore, the studied AAILs become strongly hydrated due to the hydrophobicity and cooperative H bonding of the cation in the case of L–alanine nitrate, while for glycine nitrate, there seems to be a balancing effect due to charge and other effects. The mode of interactions at the N⁺ center and effect due to the non-polar methyl group probably can be differentiated on the basis of temperature variation in the expansivity parameter. It seems that both hydrogen bonding and solute–solute association effects are at work and the extent depends on the temperature.

The development of ILs, combined with Active Pharmaceutical Ingredients (APIs), aims to overcome some of the major problems concerning the pharmaceutical industry, regarding the physicochemical and biological properties of the APIs, such as polymorphism, low solubility, low bioavailability and cytotoxicity. The wide variety of possible cation/anion combinations, of which the ILs consist, provides an opportunity to modulate and improve the properties of APIs, as well as reducing the undesired side effects of used drugs. Therefore, many research teams have been investigating the synthesis, physicochemical behavior and pharmacological profile of API-ILs.

In 2020, the API-ILs lidocaine ibuprofenate ([Lid][Ibp]) and lidocaine salicylate ([Lid][Sal]) (Scheme 4) were synthesized by Panić et al. [50], in an attempt to combine the local anesthetic action of lidocaine with the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and salicylate, which exhibit low solubility and transdermal administration, as a means of treatment for analgesia in animals.

IR, NMR and MS analyses were conducted for the structural characterization of the API-ILs. More specifically, by comparing the wavenumbers ν (cm⁻¹) of the different carbonyl groups of salicylic acid, ibuprofen, lidocaine and carboxylate anions, as well as the amides of the API-IL structures, the formation and purity of the desired products were confirmed. TG (thermogravimetric) and DSC measurements revealed the greater thermal stability (higher decomposition temperatures) of API-ILs, compared to the initial compounds. The lower density and higher thermal expansion coefficient (a_p) of lidocaine ibuprofenate indicated the weaker ion interactions and faster movement of the cation away from the anion, respectively, in contrast to lidocaine salicylate, which is more stable. In addition, the high viscosity of lidocaine salicylate was a limiting factor that made the determination of Newtonian/non-Newtonian behavior of this API-IL impossible. However, lidocaine ibuprofenate exhibited Newtonian behavior. Using electrical conductivity (k) measurements and molar conductivity (λ_m) calculations, Walden plot was applied, in order to predict ionicity for API-ILs. Lidocaine salicylate displayed better ionicity and similar conductivity, compared to lidocaine ibuprofenate, although it is more viscous, probably due to poorer ion association for lidocaine salicylate. MEP (molecular electrostatic potential) surfaces revealed a larger number of non-covalent interactions in the lidocaine ibuprofenate structure, thus, stronger ion interactions, in accordance with the results from the Walden plot. Further, the reactivity and stability of the API-ILs were investigated via calculation of the HOMO and LUMO energy gap (ΔE_gap), which is negatively correlated with increased reactivity, with lidocaine salicylate exhibiting a lower ΔE_gap, implying it is less stable than lidocaine ibuprofenate. Through MD (molecular dynamic) simulations, it was concluded that smaller-sized salicylate anions allow for a denser packing between lidocaine cations, leading to higher viscosity and density values.

All the Bio-ILs mentioned above, along with their studied physicochemical characteristics, are summarized in

Table 1. Overview of recently investigated Bio-ILs.

IL	Synthesis	Physicocemical Properties	Ref.
[Ch][Abt]	Neutralization reaction	Tg * = −46.3 °C Tm = −20.5 °C Td = 314 °C η = 107 Pa·s (25 °C)	[43]
[Ch][Asc]		Tg = −70.1 °C Tm = 15.3 °C Td = 251 °C η = 1.18 Pa·s (25 °C)
[Ch][Caf]		Tg = − 35.72 °C Tm = 92.2 °C Td = 350 °C η = not measurable (25 °C)
[Ch][C3C]		Tg = −88.3 °C Tm = −28.0 °C Td = 261 °C η = 6290 Pa·s (25 °C)
[Ch][DHB]		Tg = −66.5 °C Tm = 40.7 °C Td = 279 °C η = not measurable (25 °C)
[Ch][Fer]		Tg = −96.6 °C Tm = 16.8 °C Td = 230 °C η = 10.10 Pa·s (25 °C)
[Ch][D-Gal]		Tg = −63.3 °C Tm = 17.4 °C Td = 245 °C η = 21.21 Pa·s (25 °C)
[Ch][GA3]		Tg = −97.7 °C Tm = −26.1 °C Td = 275 °C η = 1.56 Pa·s (25 °C)
[Ch][Glc]		Tg = −47.8 °C Tm = 13.1 °C Td = 240 °C η = 1.20 Pa·s (25 °C)
[Ch][Qui]		Tg = −76.3 °C Tm = 14.3 °C Td = 330 °C η = 0.23 Pa·s (25 °C)
[Ch][Sin]		Tg = −90.8 °C Tm = 13.6 °C Td = 210 °C η = 3.99 Pa·s (25 °C)
[Ch][Ger]	Salt metathesis reaction	η = 908 Pa·s (25 °C)	[44]
[Ch][Lac]	Neutralization reaction	-	[45]
[Ch][iBut]		-
[Ch][Asp]		-
[Ch][Asp2-]		-
[Ch][PHe]		-
[Ch][Lys]		-
[Ch][Phe]	Simulation	-	[46]
[Ch][Met]		-
[Ch][Gln]		-
[Ch][Glu]		-
[Ch][Gly]		-
[Ch][Cys]		-
[Ch][Bic]	Simulation	-	[47]
[Ch][Cit]		-
[Ch][Pho]		-
[Ch][Glc]		-
[Ch][Lev]		-
[Ch][Ser]		-
[Ch][Cl]		-
[Gbet][Bic]		-
[Gbet][Cit]		-
[Gbet][Pho]		-
[Gbet][Glc]		-
[Gbet][Lev]		-
[Gbet][Ser]		-
[Gbet][Cl]		-
[Ch][Bic]		-
[Ch][Cit]		-
[EDMPC][Lin]	Salt metathesis reaction	Tm = 20.8 °C	[48]
[EDMPC][Ole]		Tm = 24.3 °C
[EDMPC][Ste]		Tm = 54.9 °C
[Gly][NO3]	Neutralization reaction	d = 1023.902 kg/m3 (25 °C, ∼0.5 mol/kg H2O)	[49]
[Ala][NO3]		d = 1022.800 kg/m3 (25 °C, ∼0.5 mol/kg H2O)
[Lid][Ibu]	Neutralization reaction	$$\begin{gathered} {\mathrm{T}}_{\mathrm{g}}=-30.30 °\mathrm{C} \\ \mathsf{\eta }=5606.9 \mathrm{mPa}·\mathrm{s} (25 °\mathrm{C}) \\ \mathrm{d}=1.02145 \mathrm{g}/{\mathrm{cm}}^3 (25 °\mathrm{C}) \\ \mathrm{k}=2.51\times {10}^4 {\mathrm{mS/cm}}^1 (25 °\mathrm{C}) \end{gathered}$$	[50]
[Lid][Sal]		$$\begin{gathered} \mathrm{T}{\mathrm{g}}_{ }=-13.10 °\mathrm{C} \\ \mathrm{T}{\mathrm{m}}_{ }=189.99 °\mathrm{C} \\ \mathsf{\eta }=5836.9\times {10}^{-4}\mathrm{mPa}·\mathrm{s} (25 °\mathrm{C}) \\ \mathrm{d}=1.12741 \mathrm{g}/{\mathrm{cm}}^3(45 °\mathrm{C}) \\ \mathrm{k}=0.4\times {10}^4 {\mathrm{mS/cm}}^1 (45 °\mathrm{C}) \end{gathered}$$

* T_g: glass transition temperature, T_m: melting temperature, η: viscosity, d: density, k: conductivity.

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3. Applications of Bio-ILs

3.1. Catalysis

In the past few years, incorporation of the principles of green chemistry in organic synthesis has gained increasing interest, aiming at the elimination of toxic, volatile organic solvents and the introduction of greener and safer solvents. Bio-ILs have emerged as a solution to this problem and have found application in organic synthesis as alternative green media that can play a dual role, that of the solvent and the catalyst [17,18,19].

In 2020, Brzeczek-Szafran et al. [11] synthesized seven new bio-ILs based on glucose and different amino acids and structurally and physicochemically characterized them. The synthesized bio-ILs were examined regarding their biodegradability, revealing that all bio-ILs were readily biodegradable, decomposing within 5–6 days and, thus, revealing the green character of the solvents, while their ability to act as catalysts was examined. The novel materials were applied in a model reaction between benzaldehyde and malonitrile for the synthesis of α,β-unsaturated compounds, via the Knoevenagel condensation reaction, using water as a solvent/co-solvent (Scheme 5). The AAILs not only promoted the reaction achieving high conversion yields (67–94%) and reducing the reaction time to 15 min, but were also able to be recycled and reused up to three times without any significant decrease in the reaction yield.

Seven AAILs were also tested regarding their catalytic capacity by Sun et al. [51] for the synthesis of monoacylglycerol (MAG) via glycerolysis of soybean oil. After screening the different reaction mediums, [TMA][Arg] (tetramethylammonium arginine) was selected as the best performing catalyst, providing MAG (Scheme 6) in satisfactory yields (55%) in a short reaction time and the reaction parameters (soybean oil/glycerol ratio, temperature, [TMA][Arg] load) were optimized via a response surface design. The resulting model of MAG conversion indicated that the optimum conditions were 105 °C, 10% [TMA][Arg] load and soybean oil/glycerol ratio 1:2, reaching a maximum yield of 64.89 ± 1.26%, higher than the corresponding yield when using conventional catalysts.

In an attempt to develop a greener protocol for the cyclization of 2-aminobenzonitrile with CO₂ to yield quinazoline-2,4(1H,3H)-dione, Weng et al. [52] investigated the catalytic role of eight biocompatible ILs, synthesized via a neutralization method. The effect of both anion and cation of the corresponding IL was examined, revealing that the IL 1,1,3,3-tetramethylguanidinium laevulinate [HTMG][Lev] demonstrated the best results (81% yield), while the reaction’s conditions were optimized, including temperature, CO₂ pressure, catalyst amount and time (Scheme 7). Under the optimized reaction conditions (70 °C, CO₂ 1.0 MPa, 6 h) and towards a more sustainable approach, the recyclability and reusability of the IL were investigated. After five catalytic circles, the isolated yield of the product was retained as high, while the composition of the IL was controlled, proving its structural stability. The applicability of the [HTMG][Lev] medium as solvent and catalyst was investigated using different substituted 2-aminobenzonitriles, while the role of the IL in the reaction mechanism was investigated.

All the Bio-ILs used as solvents and catalysts, along with their studied physicochemical characteristics, are summarized in

Table 2. Overview of recently investigated Bio-ILs in catalysis.

IL	Synthesis	Physicocemical Properties	Application	Ref.
[Glu][Gly]	Neutralization reaction	Tg * = –19 °C Td * = 198 °C η = 10,196 mPa·s (70 °C)	Organocatalysts in the Knoevenagel condensation reaction	[11]
[Glu][Ser]		Tg = −18 °C Td = 198 °C η = 28,645 mPa·s (70 °C)
[Glu][Leu]		Tg = 4 °C Td = 208 °C η = 359,841 mPa·s (70 °C)
[Glu][Arg]		Tg = −15 °C Td = 107 °C η = 247,626 mPa·s (70 °C)
[Glu][His]		Tg = −9 °C Td = 207 °C η = 106,930 mPa·s (70 °C)
[Glu][Trp]		Tg = 0 °C Td = 211 °C η = 408,284 mPa·s (70 °C)
[Glu][Tyr]		Tg = 6 °C Td = 209 η = 1,476,023 mPa·s (70 °C)
[Ch][Arg]	Neutralization reaction	-	Catalysts for the synthesis of Soybean oil-based monoacylglycerol	[51]
[Ch][Lys]		-
[Ch][His]		-
[Ch][Trp]		-
[Ch][Glum]		-
[TMA][Arg]		-
[TBA][Arg]		-
[HTMG][Lae]	Neutralization reaction	-	Solvent-catalyst for CO2 conversion into quinazoline-2,4(1H,3H)-diones	[52]
[HDBU][Lae]		-
[HDBN][Lae]		-
[P4444][Lae]		-
[HTMG][Iso]		-
[HTMG][LA]		-
[HTMG][Imi]		-
[HTMG][Glut]		-

* T_g: glass transition temperature, T_d: decomposition temperature, η: viscosity.

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3.2. Biomedical Applications

The use of ionic liquids for biomedical applications started in the late 1990s and early 2000s, where they were used as enhancers of thermal stability for enzymes and model proteins, as antimicrobial agents, in controlled release systems and as formulation excipients for small molecules of low water solubility, etc. [53]. Since then, ILs’ use expanded into these fields of applications due to their physicochemical properties and characteristics that can be task-specifically designed. A few of them, regarding the period 2020–2022, are briefly discussed below. According to the presented data, it is evident that the main research interest regarding biomedical applications focuses on the use of Bio-ILs in order to improve the water solubility of bioactive compounds, the solubility of specific drugs via API-IL synthesis or to enhance the transdermal permeability of desirable compounds improving drug delivery.

3.2.1. Bio-ILs as Skin Permeability and Bioavailability Enhancers for Transdermal and Oral Drug Administration

In the drug delivery area, the next main challenge after the successful drug formulation is the effective delivery of the drug to the desirable target. The use of task-specifically designed ILs in this area is promising since they were found to enhance skin penetration [53]. The lipophilic nature of the stratum corneum is a limiting factor for the penetration of hydrophilic macromolecules (such as hyaluronic acid) into epidermis or dermis.

Wu et al. [1] studied the potential enhancement in transdermal permeability of hyaluronic acid (a polysaccharide of natural origin, which acts as a moisturizing agent and maintains skin elasticity) using a variety of choline-based ILs, using, as anions, a variety of biocompatible organic acids (malic, sorbic, malonic, succinic, lactic, geranic, citric, oleic acid). The interaction between hyaluronic acid and the ILs is probably through hydrogen bonding, due to the hydroxyl groups of the drug molecule. The in vitro skin penetration of each IL was examined using Franz diffusion cells and it was found that all the prepared ILs enhanced transdermal delivery of hyaluronic acid. The structure of the used organic acid is a crucial factor: carbonyl groups and the presence of a double bond seem to contribute to a higher permeability. In addition to this, the high viscosity of IL impedes penetration. Finally, choline-citric acid 3:1 ([Ch][Cit]) had the strongest impact on the hyaluronic acid skin permeability enhancement, followed by choline-malonic acid 2:1 ([Ch][Mal]) and choline-malic acid 3:2 ([Ch][Mala]), whereas the more viscous choline-succinic acid 2:1 ([Ch][Suc]) did not promote permeability.

The study of the impact of ILs’ water content on their structure and activity is also imperative, as it is known that the increase in water content leads to the gradual breakdown of the polar network formed. Through the appropriate conductivity studies, it was found that the presence of water in ILs actually improves the permeation ability when the water content is held below 70% since, due to the reduced viscosity, a better contact between skin surface and the IL is accomplished. The disintegration of [Ch][Cit] is only partial within this water content range and, therefore, its properties are not entirely lost. The optimal permeation ability for hyaluronic acid was observed for 60% water content in [Ch][Cit] in SC and epidermis and 80% in dermis. ILs can, therefore, be combined with water or other aqueous solutions in order to increase the dermal penetration of biologically active compounds.

Furthermore, in the same study, nude mice models were used for the assessment of ILs’ in vivo skin protection ability in terms of anti-dehydration and it is shown that [Ch][Mal] and [Ch][Cit] carrying hyaluronic acid demonstrate the best moisturizing and protecting impact, with the first having a more stable effect and the latter being more suitable for long-term medication. The skin maintains its water balance, according to the values of transdermal water loss determined and, thus, it stays hydrated and elastic after delivery. It was noted that [Ch][Cit], [Ch][Mal] and choline-malic acid 3:2 [Ch][Mala] do not cause irritation to skin (erythema, edema, inflammation) after one week of administration, so these three ILs display the appropriate effectiveness and biocompatibility to behave as carriers for hyaluronic acid through the skin.

Another Bio-IL that recently gained the interest of the scientific community regarding the biomedical application field is the one derived from choline and geranic acid that is well known as CAGE. CAGE has been used to increase the transdermal delivery of desirable compounds of high-molecular-weight drugs (e.g., insulin [54]) and also, most recently, of small molecules.

Regarding the use of CAGE as carriers for smaller molecules, Shi et al. [55] described the potential use of CAGE (choline:geranic acid ratio of 1:2) for oral administration of a hydrophobic drug, sorafenib (SRF), a potent multikinase inhibitor used for the treatment of advanced renal cell carcinoma and hepatocellular carcinoma. Further, the 2D NOESY spectrum of the SRF-CAGE system suggested that the interactions between the ingredients of the IL are strengthened when the drug is loaded and, thus, the complex is protected when diluted. CAGE was proven to solubilize SRF tosylate at concentrations higher than 500 mg/mL. The peak plasma concentrations of SRF in vivo were increased by 2.2-fold, declaring enhanced intestinal transport, and so did the elimination half-life of the drug (16.2 h) and the mean absorption time (32 h). The biodistribution of SRF was remarkably enhanced in terms of accumulation (4.4-fold in lungs and 6.2-fold in kidneys after 9 h), which is possibly attributed to the colloidal structure spontaneously formed by SRF-CAGE solution (micelles of 427 nm mean diameter). It is noted that the CAGE micellar structures present in blood eventually dissociate into choline and geranate, as shown by UPLC-MS studies.

The nature of CAGE generates some important challenges for the incorporation of CAGE in a final product available for wide use, such as scalability, stability for long periods of time and safety. Ko et al. [56] was the first to report on the clinical translation of CAGE for the treatment of rosacea, a chronic skin disease of an inflammatory nature. CAGE has been proven to penetrate deeply into the skin, probably because it plays an important role for lipid extraction from the stratum corneum, and it also acts as an antimicrobial agent against pathogens, such as Staphylococcus epidermidis. The formulation used was a gel containing 40% w/w CAGE (choline:geranic acid ratio of 1:2). The seven steps of translation from lab scale to a clinical product include: (a) scale-up, (b) characterization, (c) stability studies, (d) determination of mechanism of action, (e) dosing, (f) preclinical toxicological tests on minipigs and (g) human clinical studies on safety and efficiency. More specifically, the quality of the IL produced on a >3 kg scale is assessed in terms of water content, pH, conductivity and physical appearance. As for characterization, ¹H NMR, HPLC-Modulated DSC, TGA and FTIR were employed for the examination of physical and chemical properties of CAGE and the identification of its components. Further, the formulation studied displayed exceptional stability under forced degradation conditions, while it exhibited important solution, microbial, chemical and physical stability over a one-year period. Since the generation of rosacea has been connected to the activation of the epidermal protease KLK-5, CAGE was tested for its ability to inhibit this enzyme and, in fact, its IC₅₀ against KLK-5 was determined to be ~30 mM. In addition to this, CAGE was found to have a minimum inhibitory concentration (MIC) against P. acnes (a model Gram-positive bacterium) of approximately 57 μM–0.45 mM, indicating significant antibacterial activity, so the mechanism of its action might be associated with these two inhibitory roles of CAGE. In vitro permeability of the components of CAGE was examined, using human cadaver skin, and the results showed that choline and geranate retain some interaction during skin penetration, despite the fact that their lipophilicities are significantly different and so are their permeabilities. It was also proven in vitro that approximately 9.5% of the applied CAGE dose permeated into dermis within a period of 24 h. After these in vitro studies, dermal toxicity in minipigs was investigated for 91 days, for a daily gel dose of 1 g, and it was generally tolerable, without any long-term side effects. Finally, the formulation was tested clinically on human volunteers, for a dose of ~0.2 g of gel applied twice a day, and it was again tolerated. After two weeks of administration, the average number of papules and pustules was reduced by ~35%, while the best results were noted at the 12th week (mean reduction ~72%).

3.2.2. Improvement in Drug Solubility with the Presence of Bio-ILs

Bio-ILs have been also used as media able to increase the solubility of important biomolecules and drugs. In this context, Gaikwad et al. [4] used Ch-AA-ILs in combination with an anti-asthmatic drug, such as zafirlukast (ZFL), in order to enhance its solubility as well as in order for this system to act synergistically. Satisfactory results were obtained using the Ch-ILs shown in Scheme 8, especially when using the choline-L-proline IL, [Ch][Pro].

According to ¹H NMR studies, when ZFL is loaded into the IL, a strong hydrogen bond is formed between the IL and the drug, as choline donates the proton from its hydroxyl group. L-proline can form additional hydrogen bonds with the carbamate functional group of ZFL through its amine groups, causing stronger molecular interactions, which are capable of increasing the solubility of ZFL by 35- to 37-fold at a low IL concentration 1% w/v (ZFL solubility 14.95 μg/mL), as shown by saturation solubility studies. [Ch][Pro] IL has the strongest impact on the ZFL solubility among the studied ILs and it was found that it increases linearly with an increasing quantity of [Ch][Pro] IL in water. For this reason, the authors characterized [Ch][Pro] IL as a “functional excipient solvent” for the enhancement of solubility in ZFL commercial configurations. As for toxicity, it was indicated by the tube dilution method that all prepared Ch-AA-ILs show low toxicity in vitro, depending on the type of anions and cations of the IL. The [Ch][Ala] IL displayed lower antimicrobial activity than the other ILs against E. coli (Gram-negative rods) and S. aureus (Gram-positive coccus), probably due to the increment in molecular weight caused by the lengthening of side chains and the functional group of amino acid. It is a general agreement that higher lipophilicity, in terms of long alkyl chains, leads to better antimicrobial activity. Lighter and smaller anions, on the other hand, provide more biocompatible ILs.

Sintra et al. [57] investigated the use of three ChILs and, more specifically, the cholinium vanillate, cholinium gallate and cholinium salicylate as hydrotropes in order to improve the aqueous solubility of two model drugs. Ibuprofen and naproxen were the selected drugs that demonstrate low aqueous solubility and high intestinal permeability. In this study, it was proven by saturation studies that cholinium vanillate and cholinium gallate are capable of increasing the solubility of ibuprofen up to 500-fold, whereas cholinium salicylate increases the solubility of ibuprofen up to 6000-fold. All three ILs show solubility enhancements up to 600-fold in the case of naproxen. The dissolution mechanism seems to depend on a synergistic effect of the ionic components of each IL. More specifically, both ions in every IL are amphiphilic, so they both aggregate around the drug molecule through their non-polar parts, minimizing the local charge of the cluster formed.

The improvement in the aqueous solubility of bioactive compounds was also investigated by Carreira et al. [2]. Purines were selected to be studied since they are the most abundant natural N-heterocycles, with poor aqueous solubility. Specifically, in this study, four purines (theobromine, theophylline, xanthine and uric acid) were employed in combination with tetrabutylammonium (cation) to create bio-ILs. The solubility of the synthesized purine-based ILs was measured and the results indicated that their water solubility was improved in comparison with the corresponding purine forerunners, 53-fold in the case of tetrabutylammonium theophyllinate ([TBA][Theop]) and 870-fold for tetrabutylammonium theobrominate ([TBA][Theob]) (Scheme 9). [TBA][Theop] had the highest solubility (2.2 mol/L) due to the presence of the two methyl groups that prevent the formation of inter-base hydrogen bonds. Perhaps the most important observation about these ILs is that, despite the use of purines of natural origin as anions, the resulting products are not harmless and eco-friendly, as shown by ecotoxicity studies on microalgae Raphidocelis subcapitata. In fact, toxicity was mostly dependent on the tetrabutylammonium cation, which is highly hydrophobic and, thus, interacts with hydrophobic moieties occurring at the cell wall, possibly leading to its disruption. Therefore, the need for ecotoxicity studies to be conducted in any case is urgent.

[TBA][Theob] and [TBA][Theop] were also found to be able to form aqueous biphasic systems (ABSs) with sodium sulfate and tripotassium citrate salts. ABSs consist of two aqueous immiscible phases and are often useful for the solubility enhancement of biomolecules for the development of drug delivery systems. [TBA][Theop] was proven to be the one with the highest potential to form ABSs with important thermoresponsive behavior, because of its low affinity with water, which results in the easier salting-out of the IL from the aqueous medium and, thus, an ABS can be prepared.

Furthermore, [TBA][Theob] and [TBA][Theop] seem to facilitate the aqueous solubilization of ferulic acid (4-hydroxy-3-methoxycinnamic acid) in a pH-dependent manner and, especially in alkaline pH, the solubilization of ferulic acid becomes extremely easy.

In the frame of the research where Bio-ILs are used for bioactive compound solubility enhancement, luteolin (LUT) was also investigated by Shimul et al. [5]. LUT consists of a naturally occurring biologically active compound, which is poorly soluble in water and often used as a food preservative. Shimul et al. implemented a COSMO-RS study for the screening of various ILs. Thus, 180 ILs consisting of 1 of the 20 standard amino acid ethyl esters (cation) and 1 of 9 phenolic acids (anion) underwent screening, based on the activity coefficients (gamma) of LUT at infinite dilution in IL. The ILs finally selected according to the model were four proline ethyl ester phenolate ILs (PEEP-ILs), every one of which consisted of a biocompatible proline ethyl ester (ProEt) and a naturally available bioactive phenolic acid (trans-ferulic, vanillic, p-coumaric and 4-hydroxybenzoic acid) (Scheme 10). All the PEEP-ILs demonstrate high thermal stability and significantly higher aqueous solubility than those consisting of choline as a cation and the aforementioned phenolic acids. The synthesized PEEP-ILs (60% in water) solubilized LUT in the following order: [ProEt][Fer] > [ProEt][Cou] > [ProEt][Van] > [ProEt][Ben] and the predictions of COSMO-RS were confirmed by the experimental data. The reason behind the high solubility of LUT in [ProEt][Fer] is the presence of non-polar–non-polar interactions between the phytochemical and the IL, due to the methoxy group and the ethylenic bond. In general, the solubilization of LUT was accomplished through multiple hydrogen bonding between the hydroxyl groups of the free LUT and the carbonyl groups of the ILs, π–π interactions between the benzene ring of the ILs and LUT and cation–π interactions between the amino group of the cations and the π-electron of the benzene rings in LUT, according to ¹H NMR and COSMO-RS.

Furthermore, in the same study, biocompatibility investigations showed that the PEEP-ILs studied are not hazardous and display lower ecotoxicity (MIC > 1000 μg/mL) than traditional imidazolium-, pyridinium-, pyrrolidinium-, piperidinium- and morpholinium-based ILs, against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Furthermore, the organoleptic properties of LUT dissolved in 0.5% [ProEt][Fer] aqueous solution on red apple slices were exceptional, indicating its food preservation ability for up to 120 h.

Another challenge for pharmaceutical research is the development of drug formulations for poorly soluble APIs, which demonstrate low dissolution in physiological fluids and, thus, low bioavailability. The interest for the employment of ILs as green solvents (or cosolvents) to overpower this issue is growing, because they have been proven to achieve high solubility of the drug while retaining the desired form. This results from the strong molecular interactions between the hydrophobic drug and the IL [4]. The development of cholinium–amino-acid-based ionic liquids (Ch-AA-ILs), in order to enhance the solubility of active pharmaceutical ingredients (APIs), was recently reported in the literature.

Yuan et al. [58] developed Ch-AA-ILs to enhance the solubility of ferulic acid and puerarin, as well as to study their skin permeation ability. In this study, six ILs were firstly synthesized: cholinium glycinate [Ch][Gly], cholinium alaninate [Ch][Ala], cholinium serinate [Ch][Ser], cholinium isoleucinate [Ch][Ile], cholinium asparticate [Ch][Asp] and cholinium lysinate [Ch][Lys]. Regarding the solubility of the APIs, it was found to be greatly improved in the presence of Ch-AA-ILs. More specifically, by increasing the Ch-AA-IL concentration, it was observed that APIs’ solubility also increased, in a similar manner for all ILs, except for [Ch][Lys]. According to the conducted cytotoxicity studies, Ch-AA-ILs displayed lower IC₅₀ values than other commonly used ILs (i.e., imidazolium ones), making them less toxic, while the molecular weight and the hydrophilic/hydrophobic character of the ILs’ anion affected their cytotoxicity. For instance, due to the [Lys] anion with higher molecular weight and the longest hydrophobic chain, [Ch][Lys] exhibited a higher toxicity, compared to the other Ch-AA-ILs. Different API concentrations were also tested in terms of cytotoxicity, revealing that cell viability was generally unaffected. The permeation ability of the APIs through polyethersulfone membranes was greatly enhanced in the presence of Ch-AA-ILs (up to 60%), even at a low concentration (4 mg/mL), as it was shown from the enhancing ratios (ERs) calculated, a parameter used to compare the observed results in the presence and absence of Ch-AA-ILs, with the ferulic-acid-based ILs showing a higher ER than purearin based, most likely as a result of the higher molecular weight of the latter.

3.2.3. Bio-ILs Derived from Active Pharmaceutical Ingredients (API-ILs)

Except from the use of Ch-ILs and Ch-AA-ILs as enhancers of solubility of various compounds, APIs or drugs, there are also some research teams that used the desirable drug or API as one of the selected components for the synthesis of new ILs that are well known as API-ILs.

Lai et al. [41] developed API-ILs based on novel cationic lipoaminoacids (LAAs), in an attempt to overcome some major drawbacks for both LAAs and APIs, such as cationic lipids’ toxicity (due to cell and membrane interactions) and low solubility of the drug tolfenamic acid (Tol). More specifically, LAAs were designed in such a manner as to resemble the endogenous lipids and, thus, being digested in the gastrointestinal tract via lipolysis enzymes and esterases, leading to non-toxic fatty alcohols and amino acids. The LAA cations decyl alanine ester HCl (Dec Ala) and decyl phenylalanine ester HCl (Dec Phe) led to the ILs [Tol][DecAla] and [Tol][DecPhe], respectively, while the decyl amine (Dec) led to the formation of the Tolfenamate decyl amine (Tol Dec), with the latter being solid, with a melting temperature over 100 °C; thus, it was not considered as an IL. The solubility of Tol in lipid-based formulations was enhanced by 2.5-fold in the form of [Tol][DecAla], probably due to its methyl side-chain group, which interferes with the lattice formation and, as a result, the drug loading capacity was increased. On the contrary, [Tol][DecPhe] exhibited lower solubility than Tol, because of its large-scale side chain. Simulated intestinal fluid was used, in order to evaluate the digestibility of the LAA cations, specifically, the decyl phenylalanine ester LAA, by determining the concentration of (Dec Phe) and the breakdown product (Phe). It was reported that LAA’s breakdown rate was extremely fast (<5 min), without the enzyme being significantly obstructed by the bulky phenyl group, and the inversely related concentrations of (Dec Phe) and (Phe) were observed. Additionally, lipolysis studies (dispersion and digestion) of Tol ILs in two types of SEDDS (self-emulsifying drug delivery systems) revealed that in type IIIA formulation, in the case of [Tol][DecPhe], more than 90% of the drug was recovered in the aqueous phase after 60 min digestion, while in the case of [Tol][DecAla], the drug precipitated, instead of transferring to the aqueous phase, possibly due to the higher levels of [Tol][DecAla] loading. Conversely, in type IIIB formulation, [Tol][DecAla] exhibited the highest possible drug concentration in the aqueous phase, because of its higher solubility, leading to a very effective oral exposure of the drug, even at large loading levels.

Janus et al. [39] synthesized and studied Ibuprofen (Ibu)-based API-ILs by utilizing the amino acid valine and especially L-valine alkyl esters (ValOR), with various alkyl group lengths (ethyl, propyl, isopropyl, butyl, pentyl and hexyl), in order to increase the water solubility, bioavailability and skin permeation of this highly lipophilic drug. The six [ValOR][Ibu] ILs were fully structurally characterized using various spectroscopic analyses and elemental analysis while using the DSC method determined their melting temperatures (ranging from 67.4 to 79.8 °C) and, via TGA, their thermal stability and degradation temperature were determined. It was reported that [ValOR][Ibu] IL aqueous and buffer solubility was greatly improved, especially for esters with shorter alkyl chains, while all presented a positive logP (partition coefficient), although it was lower than ibuprofen’s, indicating the reduced hydrophobicity of the ILs. Skin penetration of the ILs was also investigated, using abdomen porcine skin, revealing that propyl ester (382.35 μg Ibu cm⁻²) and isopropyl ester (341.20 μg Ibu cm⁻²) derivatives exhibited higher mass accumulation of ibuprofen in the acceptor phase, compared to the parent API (302.84 μg Ibu cm⁻²), while the lowest mass accumulation was observed for the ethyl ester derivative (215.19 μg Ibu cm⁻²). Additionally, propyl, isopropyl, butyl and pentyl ester derivatives displayed a higher permeation rate through the skin, concluding that [ValOPr][Ibu] and [ValOiPr][Ibu] were the most suitable candidates for topical use (Scheme 11). All the ILs, except for [ValOBu][Ibu], appeared to have a lower mass accumulation of ibuprofen in the skin, in comparison with the free acid. In general, valine ester cations, being less lipophilic than ibuprofen, are expected to have a lower toxicity.

Bastos et al. [59] also studied Ibu-based API-ILs. Cholinium ibuprofenate ([Ch][Ibu]) API-IL, comprising a cholinium cation and an ibuprofenate anion, was selected. After the successful characterization of the ibuprofen-based IL through NMR spectroscopy, the water content was determined by Karl Fischer titration (<0.05 wt%) and the measurement of the glass transition temperature (T_g) and melting temperature (T_m), for both ibuprofen (T_g = −43.57 °C, T_m = 74.89 °C) and cholinium ibuprofenate (T_m = 70.89 °C), was conducted via DSC. The melting temperature of the API-IL appeared to be slightly reduced, compared to that of ibuprofen, although this reduction was insufficient to enable API-IL to be used in pharmaceutical applications within the body, with the T_m cut-off being 37 °C. On the other hand, the solubility of [Ch][Ibu] in water and buffer solutions simulating biological fluids at 25 °C was found to be up to 28,000-times higher than ibuprofen and up to 4.4-times higher than the salt form of the parent API, sodium ibuprofen, due to the anion–cation interactions, thus, enhancing the bioavailability and efficiency of ibuprofen.

Additionally, researchers studied the cytotoxicity of the API-IL using human colon (Caco-2) and hepatocellular (HepG2) carcinoma cell lines, concluding that no significant changes were observed, regarding the cytotoxicity profiles of ibuprofen, sodium ibuprofen and ibuprofen-based IL. The high biocompatibility (low gastrointestinal toxicity) of the Ch-API IL was assessed by estimating the EC₅₀ (effective concentration that reduces cell viability to 50%) in the Caco-2 cell line, with the values of the API-IL being at the mM level and slightly higher than Ibu. The low toxicity of the Ch-API ILs was also confirmed by demonstrating ~0% hemolytic activity up to 3 mM. Finally, the API-IL’s anti-inflammatory properties were indirectly evaluated, via protein albumin denaturation assay, as well as directly, through inhibition of cyclooxygenases (COX enzymes). According to prevention of protein denaturation studies, [Ch][Ibu] exhibited a similar performance, compared to ibuprofen and ibuprofen sodium salt, while the %COX inhibition (anti-inflammatory activity of ibuprofen) was upgraded by the API-IL.

Wu et al. [60] also developed three API-ILs with different ratios (2:1, 3:1, 4:1) of choline and tretinoin (Tr) (which is an extensively utilized drug in the treatment of several skin conditions), aiming to improve its low aqueous solubility and limited skin permeability. The successful synthesis of Tr-based ILs was confirmed by FT-IR, ¹H NMR, NOESY (nuclear overhauser effect spectroscopy) and DSC analyses. The lowest number of circled crosspeaks in NOESY spectrum, also translated as the highest permeability, was observed for 2[Ch][Tr], while the degree of crystallinity and thermal stability of the API-ILs was evaluated using the experimental values of their glass transition temperature (T_g) and degradation temperature (T_d). After the characterization of the Tr-based ILs, a series of photostability and solubility studies was conducted, revealing that the differences between the degradation percentages of Tr-based ILs and Tr when exposed to LED light were insignificant, while the water solubility of Tr in the form of API-IL was highly improved, with Tr precipitation appearing after 2[Ch][Tr] dilution to <14%. The impact of water content on skin permeability of 2[Ch][Tr] was also investigated, showing that only 20% 2[Ch][Tr] managed to reach the receptor cells, while it demonstrated the optimal delivery efficiency of Tr in dermis and epidermis. The coating of 2[Ch][Tr] in the oil phase of o/w emulsions, in order to study the in vitro release and permeation of Tr, as well as to protect it from any possible damages, led researchers to the conclusion that the two emulsions managed to enhance the skin permeability of the drug, maintaining the release of Tr at a low rate. However, 20% 2[Ch][Tr] proved to be superior to both emulsions, achieving up to a 3.19-times higher permeability-enhanced effect in the first four dermal layers.

In an attempt to address issues associated with some APIs, such as polymorphism, low bioavailability and low solubility, while maintaining their enhanced antimicrobial activity, Santos et al. [61] declared the formation of API organic salts and ILs (OSILs) derived from two fluoroquinolones (FQs), ciprofloxacin (Cip) and norfloxacin (Nor) as anions and choline as a cation. DSC measurements of API-OSILs revealed that the API-OSILs exhibited lower melting temperatures, compared to the parent fluoroquinolones (ciprofloxacin: T_m = 322.0 °C/[Ch][Cip]: T_m = 111.2 °C, norfloxacin: T_m = 217.0 °C/[Ch][Nor]: T_m = 94.5 °C); however, the melting temperature of [Ch][Cip] exceeded 100 °C, thus, making it an organic salt. Water solubility was also investigated, showing that API-OSILs were more soluble in water than the parent APIs.

Through antimicrobial activity studies against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria, it was reported that both the API-OSILs and the parent FQs inhibited the growth of all bacteria strains, more than 50%. Nevertheless, the IC₅₀ values of [Ch][Cip] against K. pneumoniae appeared to be quite low and also exhibited reduced activity against B. subtilis, compared to parent ciprofloxacin, while [Ch][Nor], which was more efficient against Gram-positive bacteria, displayed lower antimicrobial activity, compared to [Ch][Cip].

Another recent application of the API-ILs is their use in order to resolve the current problem of evolving bacterial resistance to antibiotics. Ferraz et al. [40] synthesized a variety of ionic liquids and organic salts (OSILs), containing anionic penicillin G [seco-Pen] and amoxicillin [seco-Amx] hydrolysate derivatives, including two with choline as a cation ([Ch][seco-Amx] and [Ch][seco-Pen]), and tested their antibacterial activity against sensitive and resistant Escherichia coli and Staphylococcus aureus strains. Choline-based API-OSILs were retrieved as yellow solids with melting points above ([Ch][seco-Amx]: 143–144 °C) and below ([Ch][seco-Pen]: 69–71 °C) 100 °C. When tested on the sensitive bacteria S. aureus and E. coli, both [Ch][seco-Amx] and [Ch][seco-Pen] not only appeared to have limited antibacterial activity, but also increased the minimum inhibitory concentrations (MICs) of the parent APIs ([Amx] from 0.05 mM to >5.0 and K[Pen] from 0.5 mM to >5.0). However, when tested on the resistant Gram-negative E. coli strains CTX M9 and CTX M2, as well as the methicillin-resistant S. aureus (MRSA) ATCC 43,300, choline-based API-OSILs improved the antibacterial activity of the parent APIs: for E. Coli, [Ch][seco-Amx] reduced the MIC of [Amx] about 10- to 100-times (from >5 to 0.5 mM for E. coli CTX M9, from >5 to 0.05 mM for E. coli CTX M2), while [Ch][seco-Pen] selectively reduced the MIC of K[Pen] only in the case of E. coli CTX M9 (from >5 to 1.0 mM). The two choline-based API-OSILs also displayed higher antibacterial activity, compared to the parent APIs, against MRSA (from >5 to 0.5 mM and from >5 to 1.0 mM).

The antioxidant activity of Bio-ILs has also been investigated. In an effort to synthesize bioactive and biocompatible ILs, Czerniak et al. [3] combined L-carnitine and natural antioxidant compounds, such as ascorbic acid and phenolic acid derivatives. L-carnitine is a salt occurring in a zwitterionic form, present in humans, that plays a protective role for cells against oxidative damage. The L-carnitine-based ILs formed were evaluated for their activity against various types of radicals and provided some useful conclusions about the way the anion of the IL affects the antioxidant activity. More specifically, the antioxidant capacity of the obtained ILs was assessed using the DPPH (1,1-diphenyl-2-picrylhydrazyl radical) and ABTS (2,2-azinobis(3- ethylbenzothiazoline-6-sulfonic acid radical) assay. As for DPPH results, the highest efficiency in the neutralization of DPPH radicals was observed for L-carnitine gallate ([Car][Gal]) (6.56μM), followed by L-carnitine gentisate ([Car][Gen]), while the least active IL was L-carnitine coumarate ([Car][Cou]). It is worth mentioning that the ILs with anions derived from cinnamic acid analogues (except for the caffeate anion) had EC₅₀ values greater than 20 μM, presenting lower antioxidant activity than the ILs with anions derived from benzoic acid analogues. The ILs containing caffeate and sinapate anions displayed the biggest increase in efficiency, approximately 45% and 30%, respectively, in comparison with the reference compounds (the antioxidant acids). On the other hand, all ILs demonstrated a higher ability to neutralize the ABTS radical than the DPPH radical and, similar to the results obtained from DPPH assay, ILs based on gallic and gentisic acid had the highest radical scavenging efficiency. Only the IL containing a coumarate anion had higher efficiency than the respective acid (37% more active), according to ABTS assay. In addition, the ion reduction ability of the bio-ILs was evaluated using the FRAP method, in which the antioxidant ability of the ILs to reduce ferric(III) ion to ferric(II) ions is measured, and the CUPRAC method, in which the ability of the ILs to reduce copper(II) ion to copper(I) ions is assessed. The FRAP method proved that the highest ability to reduce ferric (III) ions is displayed by the [Car][Gal] IL (approx. 2 μmol Trolox Equivalents), similar to the reference acid, whereas the combination of syringate and ascorbate anions with L-carnitine resulted in a significant reduction in their activity. The CUPRAC assay showed that ILs based on caffeate and gallate anions had the largest potential to reduce copper (II) ions. According to these studies, the IL with the best combined antioxidant activity was [Car][Gal] (Scheme 12), which can be attributed to the presence of three hydroxyl groups attached to the aromatic ring of the gallate anion, while, on the other hand, the least active ILs in all studies were those containing ascorbate and coumarate anions. Despite all the aforementioned encouraging results, all ILs employed showed weak ability to inhibit the enzyme xanthine oxidase (XO), which is responsible for generating reactive oxygen species (ROS) in the human body. This was attributed to the properties of the acids used.

API-ILs have also been used for biomedical applications, such as in the area of wound dressings and tissue regeneration. Panić et al. [62], in an effort to develop a novel material that can be used as an advanced drug delivery system, developed novel electrospun nanofibers of poly(ethylene oxide) (PEO) (a biocompatible polymer) loaded with API-ILs based on the local anesthetic drugs lidocaine and procaine, as well as salicylate. Before the formation of the nanofibers, the solubility assay of the synthesized ILs, lidocaine salicylate [Lid][Sal] and procaine salicylate [Prc][Sal], revealed a 4- to 10-fold higher solubility in water, compared to their initial compounds. The final form of the nanofibers (diameter and morphology) was affected by both the IL:PEO ratio and the molar mass of PEO, while the viscosity of the polymer–IL blend solutions decreased when the IL content increased. The IL content was found to be a contributing factor to the electrical conductivity when increased, due to the ionic nature of the ILs. FTIR analysis confirmed the loading of the synthesized ILs to the PEO nanofibers, which was achieved through hydrogen bonding, without inducing any chemical reactions between them. PEO:IL and pure PEO nanofibers were thermally assessed via DSC, indicating the slightly lower melting temperature of the PEO:ILs, possibly as a result of the formation of smaller crystallites in the presence of ILs. It was also shown that the decomposition temperature of the ILs combined with the nanofibers was lower, compared to pure ILs, on account of the more reactive surface of the nanofibers.

3.2.4. Bio-ILs Used in Various Biomedical Applications

In the literature, there are also some examples of Bio-ILs that are not derived from APIs, however, displaying bioactivity. Due to their antimicrobial activity, choline-based ILs have been investigated for their potential use as eco-friendly and inexpensive biofilm controllers. Pathogens generated by biofilms may contaminate food products and the films cause equipment biocorrosion, so it is urgent for their growth to be controlled. Pereira et al. [63] evaluated the antimicrobial activity of choline alaninate—[Ch][Ala] and choline glycinate—[Ch][Gly] against Bacillus cereus and Pseudomonas fluorescens and confirmed their activity against the planktonic cells, which is, however, lower than that of the compounds used as reference, benzalkonium chloride (BAC) and cetyltrimethylammonium chloride (CTAC). The reason for this is the highest alkyl chain hydrophobicity and length of the latter, which results in stronger interactions with the cell membrane. The antimicrobial activity of the ILs against both bacterial species seems to be dose dependent, increasing non-linearly with increasing concentrations, until a minimum bactericidal concentration is reached. As for the time dependence of the antimicrobial activity of ILs, in B. cereus, the colony-forming unit reduction was stabilized after 5 min for both ILs, whereas in P. fluorescens, the reduction was total after 10 min upon exposure to [Ch][Ala] and after 30 min for [Ch][Gly]. Both ILs caused a similar reduction in the P. fluorescens film biomass (~83%), while [Ch][Gly] led to the highest B. cereus biofilm removal (56%). The antimicrobial activity of these ILs was attributed mostly to the irreversible disruption of the bacterial multilayer surface and, according to the results, these choline-amino acid ILs could be used for biofilm control, perhaps in combination with BAC and CTAC for synergistic effects (74% removal from B. cereus biofilm, 86% from P. fluorescens).

In 2021, Demurtas et al. [64] synthesized six ChILs using different hydroxycinnamic acids (HCAs), in order to enhance their poor water solubility. More specifically, ferulic, sinapic, o-coumaric, m-coumaric, p-coumaric and caffeic acid anions, combined with cholinium cations, led to the formulation of the [Ch][HCA] ILs, which were subsequently characterized, regarding their water solubility and protonation equilibria of the initial HCAs. It was established that the highest solubility is achieved when the aromatic ring of the acids is substituted with only one hydroxyl group, while adding other functional groups decreases the molecule’s solubility. More importantly, the HCAs appeared to have lower solubility, by 2/3 orders of magnitude, compared to the [Ch][HCA] ILs. The melting and decomposition temperatures were calculated via TGA and DSC, with the melting temperature of the solid [Ch][Caff] being over 100 °C (141 °C). Additionally, through the DPPH assay, the antioxidant activity of the ILs was estimated, revealing their high radical scavenging activity, presenting lower EC₅₀ values than the HCAs. The cytotoxicity of the synthesized [Ch][HCA] ILs on murine melanoma B16–F10 and murine fibroblast 3T3 cells using the MTT assay was also investigated and found to be absent at the applied concentration, with the exception of [Ch][Caff] when used at the highest dose, indicating their high biocompatibility. DFT calculations contributed to the structure optimization of the species involved in studying the most common antioxidant mechanisms that include the HCAs and their ionic forms. Demurtas et al. concluded that [Ch][HCA] ILs consist of promising solvents to be applied in pharmaceutical formulations.

All the Bio-ILs used in biomedical applications, along with their studied physicochemical characteristics, are summarized in

Table 3. Overview of recently investigated Bio-ILs in biomedical applications.

IL	Synthesis	Physicochemical Properties *	Application	Ref.
[Ch][LSar]	Salt metathesis reaction	-	Antimicrobial activity	[65]
[Ch][Doc]		-
[Ch][Ger]	Salt metathesis reaction	-	Oral delivery of sorafenib	[55]
[Ch][Mali]	Salt metathesis reaction	-	Dermal delivery of hyaluronic acid (In vitro skin penetration through porcine skin, in vivo skin protection effect and skin irritation tests)	[1]
[Ch][Sorb]		-
[Ch][Mal]		Tg * = −77.6 °C
[Ch][Suc]		-
[Ch][Lac]		-
[Ch][Ger]		Tg = 39.5 °C
[Ch][Cit]		Tg = −60.5 °C
[Ch][Ole]		-
[TBA][Theob]	Neutralization reaction	Tm * = 377.8 K Td * = 457 K ΔHm = 50,920 J mol−1	Ecotoxicity against the microalgae Raphidocelis subcapitata, formation of aqueous biphasic systems, solubility enhancers	[2]
[TBA][Theop]		Tm = 370.6 K Td = 486 K ΔHm = 36,853 J mol−1
[TBA][Xan]		Tm = 487 K Td = 495 K
[TBA][Ur]		Td = 505 K
[Car][Asc]	protonation reaction of a zwitterionic form of L-carnitine with a proper antioxidant acid	Tg = 38.1 °C Tonset5 * = 175 °C Tonset50 = 226 °C	Antioxidant activity (DPPH, ABTS, FRAP, CUPRAC, chelation of ferrous (II), inhibition of xanthine oxidase)	[3]
[Car][Proc]		Tg = 30.8 °C Tonset5 = 187 °C Tonset50 = 213 °C
[Car][Gen]		Tg = 9.7 °C Tonset5 = 205 °C Tonset50 = 231 °C
[Car][Gal]		Tg = 43.0oC Tonset5 = 186oC Tonset50 = 235oC
[Car][Syr]		Tg = 45.7 °C Tonset5 = 221 °C Tonset50 = 250 °C
[Car][Cou]		Tg = 27.9 °C Tonset5 = 189 °C Tonset50 = 294 °C
[Car][Caf]		Tg = 31.9 °C Tonset5 = 182 °C Tonset50 = 302 °C
[Car][Fer]		Tg = 21.6 °C Tonset5 = 163 °C Tonset50 = 202 °C
[Car][Sin]		Tg = 47.3 °C Tonset5 = 193 °C Tonset50 = 249 °C
[Ch][Val]	Neutralization reaction	-	Solubility of ibuprofen and naproxen	[57]
[Ch][Gal]		-
[Ch][Sal]		-
[Ch][Ger]	Salt metathesis reaction	Tg = −68 °C Conductivity = ∼1.3 mS cm−1	Antimicrobiological activity, KLK5 inhibition, human cadaver skin permeation evaluation. Dermal toxicity in minipigs Cosmetic study (Clinical evaluation in human volunteers)	[56]
[Ch][Ala]	Not mentioned	-	Planktonic and biofilm growth control of Bacillus cereus and Pseudomonas fluorescens	[63]
[Ch][Gly]		-
[ProEt][Fer]	Neutralization reaction	Tg= 6.6 °C Tm = 52.7 °C Tonset = 113.6 °C	Solubility of luteonin in IL Food preservation test	[5]
[ProEt][Cou]		Tg = 1.5 °C Tm = 86.4 °C Tonset = 116.4 °C
[ProEt][Ben]		Tg = 4.2 °C Tm = 127 °C Tonset = 141.5 °C
[ProEt][Van]		Tg = 3.6 °C Tm = 110.0 °C Tonset = 138.4 °C
[Ch][Pro]	Salt metathesis reaction	Tg = −59 °C TODT * = 179 °C Td = 231 °C	Solubility enhancement of poorly water-soluble drug, Zafirlukast (ZFL)	[4]
[Ch][Ala]		Tg = −52 TODT = 145 °C Td = 206
[Ch][Met]		Tg = −63.29 °C TODT = 168.5 °C Td = 281 °C
[Ch][Ser]	Neutralization reaction	-	Solubility οf APIs, Cytotoxicity, in vitro permeation behavior	[58]
[Ch][Ile]		-
[Ch][Ala]		-
[Ch][Gly]		-
[Ch][Lys]		-
[Ch][Asp]		-
[Ch][Ibu]	Neutralization reaction	Tm = 70.89 °C	Solubility studies, cytotoxicity assays, Hemolytic activity, protein albumin denaturation assay, cyclooxygenases (COX-1 and COX-2) inhibition Assays	[59]
[Ch][Tr] (2:1)	Salt metathesis reaction	Tg = 79.8 °C Td = 117.4 °C	Photostability studies, solubility studies, preparation of o/w emulsions, in vitro drug release studies, skin permeation test	[60]
[Ch][seco-Amx]	Neutralization, salt metathesis reaction	Tm = 143–144 °C	Antimicrobial activity	[40]
[Ch][seco-Pen]		Tm = 69–71 °C
[ValOEt][Ibu]	Neutralization reaction	Tm = 77.9 °C	Solubility studies, determination of partition coefficient Skin electrical impedance Skin permeation studies, accumulation in the skin	[39]
[ValOPr][Ibu]		Tm = 79.81 °C
[ValOiPr][Ibu]		Tm = 78.01 °C
[ValOBu][Ibu]		Tm = 76.80 °C
[ValOAm][Ibu]		Tm = 73.81 °C
[ValOHex][Ibu		Tm = 67.35 °C
[Ch][Cip]	Salt metathesis reaction	Tm = 111.2 °C	Solubility, critical micelle concentration, cytotoxicity and antimicrobial activity studies	[61]
[Ch][Nor]		Tg= 54.8 °C Tm = 94.5 °C
[Ch][Fer]	Neutralization reaction	Td = 105 °C	Protonation equilibria and solubility, antioxidant activity, cytotoxicity, DFT calculations	[64]
[Ch][Sin]		Td = 103 °C
[Ch][Caf]		Tm = 141 °C
		Td = 148 °C
[Ch][o-Coum]		Td = 180 °C
[Ch][m-Coum]		Td = 132 °C
[Ch][p-Coum]		Td = 118 °C
[Lid][Sal]	Neutralization reaction	Tg = 3.7 °C	Development of electrospun nanofibers loaded with ILs	[62]
		Td = 170–250 °C
		n * = 5836 × 10−4 mPa s (25 °C)
		d * = 1.12741 g cm−3 (45 °C)
		k * = 0.4 × 104 mS cm−1 (40 °C)
[Prc][Sal]		Tg = −21.6 °C,
		Td = 200–270 °C
		d = 1.18324 g cm−3 (40 °C)
		η = 137.67 Pa s (50 °C)
		k = 3.51 μS cm−1 (55 °C)
[Tol][DecAla]	Salt metathesis reaction	Tg = −16 °C	Solubility studies, in vitro lipolysis studies, in vitro digestibility, pharmacokinetic studies	[41]
		Tm = 90–178 °C
[Tol][DecPhe]		Tg = −6 °C
		Tm = 88–174 °C

* T_g: glass transition temperature, T_m: melting temperature, T_d: degradation temperature, T_ODT: onset decomposition temperature, T_onset50: decomposition temperature of 50% sample, η: viscosity, d: density, k: conductivity.

.

3.3. Separation Processes

In the past few years, biocompatible ILs have been extensively studied as alternative green media to conventional toxic organic solvents used in the industry and have found many applications in widely used separation processes, such as gas–liquid absorption and liquid–liquid extraction, including CO₂ capture, delignification, denitrogenation, desulfurization and more.

Santiago et al. [6] simulated 50 Ch-ILs using COSMO-RS methodology and investigated the potential application of them in traditional separation processes. The anions selected for these simulations were amino acids, carboxylic acids or phosphate-based molecules and the effect of the different anions used each time, on the ability of the ILs formed to absorb hydrofluorocarbons (used as refrigerant gasses), greenhouse gasses (CO₂, H₂S, NH₃) and volatile organic compounds (VOCs) was examined. Henry’s law constant (K_H) was evaluated as an indication, where lower K_H values lead to better IL absorbent ability. Regarding the refrigerant gasses and the acid gasses (CO₂, H₂S), the size of the anion seems to play a crucial role in the gas absorption, obtaining favorable absorptions when small polar anions are used, meaning carboxylic acids with small alkyl chains, small amino acids and the phosphate-based anions were examined. Comparing the results with literature reports, bicarbonate-based anions present enhanced results in the case of the refrigerant gasses; however, in the case of acid gasses, the predicted K_H values of bicarbonate- and acetate-based ILs were similar with the IL benchmark (the IL reported as reference to be the best candidate in the literature).

Regarding NH₃ and VOCs, where acetone was used as a reference, different results were observed. Big anions, with lower polarity, favored the absorption, with [Ch][NTf₂] presenting the lowest K_H value in both cases. The K_H value of the [Ch][NTf₂] is comparable with the values that appear in the literature for acetone and lower than the K_H values reported for NH₃, suggesting its great performance and applicability.

In addition to gas absorption, researchers also investigated the implementation of ChILs on extractions of solutes from both aqueous and hydrocarbon streams. In the first case scenario, the octanol–water partition coefficient was determined with the COSMO-RS method for the 50 anions, in order to examine the hydrophobicity of the ILs; however, the LLE (liquid–liquid equilibrium) calculations revealed that the formation of two liquid phases with water did not appear in any case. As for the hydrocarbon streams, different mixtures were examined: toluene/heptane as hydrocarbon mixture reference of dearomatization; tiophene/heptane as desulfurization process; and pyrrole/heptane as denitrogenation process. The partition coefficients and selectivities of the Bio-ILs were evaluated for all the mixtures, revealing that these two factors have the exact opposite trend, i.e., ILs with high partition coefficients present low selectivity, while ILs with high selectivity present low partition coefficients. In general, acetate- and phosphate-based anions are the best candidates in terms of partition coefficients and amino-acid-based anions, and benzoate was the best in terms of selectivity, allowing one to select them on a need basis.

The increasing problem of air pollution due to industrial emissions has encouraged scientists to seek greener and more effective technologies to regulate this problem. In this context, Fahri et al. [42] studied the ability of ILs to absorb both hydrophobic and hydrophilic VOCs. Four novel Bio-ILs, obtained from choline chloride and fatty acids, were synthesized and characterized. The thermal properties and the polarity of the green media were measured, as well as the viscosity of the novel solvents, revealing that an elongated alkyl chain increases the viscosity. In terms of their effectiveness to act as VOC absorbents, toluene, dichloromethane and methyl ethyl ketone were chosen as reference and the partition coefficient constants at 30 ℃ were measured for each pair of VOC/ILs.

According to the results of this study, it can be concluded that in most cases, ILs acted as better or similar absorbents to water, with [Ch-C₈][Lev] (Scheme 13) presenting the best performance in all three cases. In an attempt to amplify the process efficiency and green profile, the absorption capacity and recyclability of the best IL were explored, leading to exceptional results. No saturation of the solvent was detected up to a VOC concentration of 3000 g/m³, three-times higher than the concentrations used at classical absorption processes (1000 g/m³), while the IL was successfully utilized in five absorption/desorption cycles without any significant loss in the process yield.

In an effort to find a more sustainable way to reduce the environmental footprint of industrial emissions, Latini et al. [66] in 2022, focused on the use of Ch-AA-ILs for CO₂ absorption. In this study, six Ch-AA-ILs were synthesized and characterized. Solutions of different concentrations of the bio-ILs in DMSO were tested for their ability to absorb CO₂, with IL [Ch][Gly] possessing the highest absorption capacity. The recyclability of all the ILs-DMSO solutions, except for [Ch][Gly] and [Ch][Ala] due to the precipitation of the amino acids during the first cycle, was examined, presenting a small loss in the absorption performance after ten cycles. Furthermore, the toxicity of the IL [Ch][Ser] was evaluated in vivo in zebrafish, proving that the examined IL did not affect the embryogenesis and, therefore, can be characterized as biocompatible.

In addition to gas absorption, Bio-ILs have also found application in the extraction of the food industry’s by-products, in order to obtain value-added compounds and minimize the environmental impact of the produced waste. ILs have been used in these processes either as the extraction media or as delignification factors. Kumar et al. [67] utilized ChILs, using different carboxylic acids as anions, for the selective coagulation of κ-carrageenan from Kappaphycus alvarezii extract.

Different concentrations of the six bio-ILs synthesized in this project were applied to both water and alkali extracts of Kappaphycus alvarezii; however, only [Ch][Capr] and [Ch][Lau] (Scheme 14) afforded the coagulation of the κ-carrageenan, with the best results (14.8 ± 0.5%) in terms of extraction yield being 6% w/v [Ch][Lau] used in the alkali extract. The isolated polysaccharide was characterized and its physicochemical and thermal properties were similar to the ones obtained when using the conventional method.

On the other hand, five research groups utilized ChILs as delignification factors for the valorization of food by-products. Lignocellulosic biomass fractionation and conversion have been thoroughly studied as a source of renewable energy and value-added products. The need for a more sustainable and energy-efficient method to exploit biomass leads to the implementation of green solvents in both biomass fractionation and lignin depolymerization.

In this concept, Liu et al. [68] investigated the applicability of both ChILs and choline-based deep eutectic solvents (DESs) for the pretreatment of corn stover and for the depolymerization of the lignin extracted. Aqueous solutions (10% w/v) of three ILs, [Ch][Ala], [Ch][Gly] and [Ch][Lys], and three DESs, choline chloride/glycerol 1:2, choline chloride/ethylene glycol 1:2 and choline chloride/maleic acid 2:1, were examined for their ability to remove lignin, revealing that ILs had a significant advantage over DESs, with [Ch][Gly] and [Ch][Lys] demonstrating the best results, approximately 50% lignin removal. After pretreatment, the diluted lignin in the aqueous [Ch][Gly] solution was subjected to depolymerization, via a bi-enzyme system, containing aryl alcohol oxidase and lignin peroxidase. Preliminary biocompatibility tests revealed that increased concentrations of ILs and DESs lead to lower activity of the enzymes, observing an up to 36% decrease, when using 10% concentration of the solvents. The efficacy of lignin depolymerization was assessed by GC-MS, showing that the molecular weight of lignin decreased after the pretreatment of the biomass (from 8058 to 734 g/mol), while it was further reduced after enzymatic depolymerization for 6 days (488 g/mol).

Husanu et al. [69] implemented two types of green solvents in the valorization of chestnut shell waste, deep eutectic solvents as extraction media and [Ch][Gly] ionic liquid for the delignification of the extraction residue. More specifically, following the extraction of the chestnut shell, the undissolved residue was treated with the bio-IL at 90 °C for 16 h, with a biomass/IL ratio of 1/20 (w/w), and the extraction mixture was diluted with NaOH 0.1 M, affording a solid fraction rich in cellulose and a liquid fraction, which, after acidification with HCl (until pH = 2), provided the lignin precipitate. The extraction yields of cellulose and lignin were found to be 54 and 8% of the chestnut shell waste residue, respectively, and cellulose and lignin were characterized via FTIR and TGA analyses, while the IL was successfully recovered in great purity and without any significant mass loss.

Asim et al. [70] investigated the application of Ch-AA-IL, namely [Ch][Lys], for the pretreatment of rice and wheat straw and compared it with [DMBA][H₂SO₄], which was used as a benchmark. The effect of time and temperature on the delignification and hemicellulose removal was assessed using three different temperatures: 80, 100 and 120 °C, for time durations from 1 to 8 h. The same trend was observed regardless of the biomass used each time. Both time and temperature favored the delignification process, with the optimum conditions being 120 °C for 8 h, providing 87% and 85% delignification for rice and wheat, respectively, higher than when using [DMBA][H₂SO₄] (77%). However, the pulp recovery was lower. The remaining pulp was then hydrolyzed using food-grade enzymes to obtain glucose and the reaction conditions were optimized. The results indicated that the delignification process plays a substantial role in the saccharification, since the pulp quality is improved and the sugar yields are increased.

In 2021, Pimienta et al. [71] developed a one-pot (OP) process of ethanol conversion, which included a protic IL (PIL) pretreatment step of agave bagasse (AG), using the biocompatible and low-cost IL 2-hydroxyethyl-ammonium acetate ([2-HEA][OAc]), followed by enzymatic saccharification and ethanol fermentation. The IL [2-HEA][OAc] selected did not require pH adjustment or any separation processes before the enzymatic saccharification and fermentation steps that follow biomass pretreatment, rendering it suitable for an OP process. More specifically, the optimization of the IL pretreatment process was conducted in a central composite design (CCD), where the experimental variables examined were temperature (120–150 °C), IL concentration (60–90 wt%) and time (1.6–4.9 h), while xylan and glucan conversion were chosen as a response. Glucan conversion was mainly affected by temperature, while both time and temperature had an impact on xylan conversion, with higher temperatures leading to higher glucan/xylan conversion. Taking into account the biorefinery economics (reduced usage of IL, lower viscosity, improved mass transfer), as well as the necessity of pretreatment reactor operation using a high biomass loading, the optimum conditions of AG pretreatment were: 160 °C, 60 wt% IL loading and 1.5 h using 30 wt% solid loading, reaching 72.9% and 36.1% glucan and xylan conversion, respectively. Additionally, the optimal conditions were applied in different AG substrates’ delignification processes, resulting in similar sugar conversions. The cellulose crystallinity was also examined as an important factor that affects the ability of enzymes to hydrolyse cellulose during the sequential saccharification step. Researchers observed a decrease in crystallinity index to 32% at the optimized conditions, compared to the 36% achieved when untreated AG was used, which was attributed to the removal of amorphous cell wall components. Through preliminary experiments on three different S. cerevisiae strains, the absence of any inhibitory effect on them by the PIL was also established. The implemented PIL–OP scheme resulted in the production of 132 kg ethanol per ton of untreated biomass, which is higher than the ethanol produced using untreated biomass (121 kg per ton of untreated biomass) and 1-ethyl-3-methyl-imidazolium acetate as an IL, as well as a >50% delignification of biomass at 10% solid loadings.

Liang et al. [72] focused on the development of an efficient methodology for the recovery and regeneration of [Ch][Ace] IL after biomass pretreatment. Sugar bagass and [Ch][Ace] were stirred in a ratio of 1/10 w/w for 16 h at 110 °C. Following the reaction, cellulose fraction was obtained as a precipitate upon addition of ethanol/water (1/1 v/v), containing 97.8% cellulose, 85.2% hemicellulose and 52.3% of lignin. Acetone in the remaining solution was recovered while a lignin fraction was obtained as a precipitate upon the addition of water. The resulting aqueous [Ch][Ac] mixture was subjected to ultrafiltration and bipolar membrane electrodialysis in order to recover the IL. The ultrasonication step was considered essential in order to remove diluted lignin and biomass lysates that would obstruct the electrodialysis, achieving 6.3%, 14.0% and 91.4% removal of neutral sugars, uronic acids and lignin, respectively, without any significant loss of cholinium and acetate ions. The recovery and regeneration of [Ch][Ace] via bipolar membrane electrodialysis was based on the divided recovery of cholinium anion as choline hydroxide and acetate cation as acetic acid, while two parameters of the electrodialysis were optimized, namely the concentration of fed solution and the current density. The regenerated IL was successfully reused five times, while an economic analysis of the process was performed, rendering the suggested methodology economically feasible for scale-up use.

In an attempt to decipher the dissolution mechanism of grassy and woody biomass, during its pretreatment with Ch-ILs, Mohan et al. [73] studied the interactions between lignin dimers as model compounds (Lignin 4-O-5, Lignin β-O-4 and Lignin 5-5) and the ions of five ILs comprising different carboxylate anions, [Ch][For], [Ch][Ace], [Ch][But], [Ch][Hex] and [Ch][Oct], using COSMO–RS and molecular dynamics simulations. Firstly, electrostatic interactions between the 4-O-5 lignin compound and the ILs dominated the van der Waals interactions. More specifically, it was observed that by increasing the alkyl chain length of the anion, the electrostatic interactions decreased, due to lower polarity, while the van der Waals interactions were favoured. On the contrary, lignin–cation electrostatic interactions were higher for longer-alkyl-chain anions. Through dissociation constant (pK_a) calculations, the low solubility of lignin was attributed to the incapability of the ILs to deprotonate the phenolic protons of the lignin. Additionally, radial distribution functions (RDFs) for lignin–anion and lignin–cation structural interactions, along with coordination number calculations, indicated that lignin solubility is controlled by the anion selection. Hydrogen bond (HB) measurements revealed that, although shorter-alkyl-chain-length anions created a slightly higher number of HBs with lignin, compared to the larger ones, the solubility of lignin was found to be higher in [Ch][Oct] and [Ch][Hex] ILs, due to the longer HB lifetimes between these two ILs and lignin. In comparison to [Ch][Lys], an IL that is considered to be very efficient in biomass delignification, dissolution mechanism of lignin in [Ch][Oct] was studied. Biomass pretreatment experiments were also conducted, in which [Ch][Lys] exhibited a 77% removal of lignin, compared to 52% of [Ch][Oct]. Further, the calculation of pK_a values of lysinate showed that ILs based on this anion were able to deprotonate the majority of the carboxylic and phenolic protons of lignin, leading to a more efficient delignification. Finally, the stronger non-bonded interaction energy between [Ch][Lys] and lignin β-O-4 dimer, compared to the other two dimers, was attributed to its more polar nature and higher reactivity, which was confirmed via spatial distribution function (SDF) evaluation and HB calculations.

Towards different approaches on the usage of bio-based ILs in the separation process, Hawatulaila and Abdullah examined two different problems encountered in the oil industry. Hawatulaila et al. [74] turned their focus to marine oil-spill remediation and the development of a novel, green and efficient oil dispersant. To achieve this goal, five ILs were synthesized and combined in order to form ten different formulations of dispersants, consisting of water and all the ILs in different mass ratios. The formulation IL dispersants were examined regarding their dispersant stability, surface tension, critical micellar concentration, interfacial tension between oil and water and emulsion stability index, indicating the best performing formulation (wt/wt%: [Bmim][LSar] 10, [BDP][DDBS] 10, [TBA][Cit] 15, [TBA][PP] 5, [TBA][EEG] 5, water 55). To further examine the effectiveness of the selected IL formulation, its applicability was examined using four types of crude oil (light, medium, heavy), achieving satisfactory to high dispersion effectiveness (70.75–94.71%), depending on the oil properties. In addition, different dispersant-to-oil ratios (1:100, 1:50, 1:25, 1:20 and 1:10) and different % of salinity (1.0, 3.0 and 5.0 wt%) were examined regarding the effectiveness of the dispersant, revealing that the formulation was highly effective between dispersant-to-oil ratios 1:10 to 1:25, while the saline concentration does not have a significant impact on the ability of the formulation to act as a dispersant. Furthermore, the toxicity of the suggested dispersant against zebrafish and grouper fish was assessed, disclosing an acute fish toxicity (LC₅₀) of 173.78 mg/L after 96 h, characterizing it as non-toxic and a promising alternative to the existing toxic dispersants.

Abdullah et al. [75] attempted to synthesize novel amphiphilic curcumin-based ILs, able to tackle the problem of demulsification of crude oil emulsions, in order to avoid storage, transportation and refining problems occurring in the presence of brine. Two Bio-ILs were synthesized via the reaction of curcumin with 1,3-propanesultone and bromoacetic acid, followed by the reaction of the obtained acids with 12-(2-hydroxyethyl)-15-(4-nonylphenoxy)-3,6,9-trioxa-12-azapentadecane-1,14-diol (HNTA), forming the corresponding ILs GCP-IL and GRB-IL. The novel ILs were characterized structurally via ¹H NMR and FTIR, while the surface activity and the ability of the ILs to demulsify water/oil emulsions was investigated. The results indicated that an increasing concentration of curcumin-based ILs decreases the surface tension up to the critical micellar concentration, with GCP-IL inducing a greater decrease than GRB-IL. The same trend was observed for interfacial tension, where higher concentrations of the ILs tested seemed to decrease the interfacial tension as well. Regarding the demulsification efficiency, several crude oil/water ratios (50/50, 70/30, 90/10) and different IL concentrations (250 ppm, 500 ppm and 1000 ppm) were tested, concluding that better efficiency can be achieved with high concentrations and low ratios of water. Interestingly, between the two ILs, GCP-IL demonstrated higher and faster demulsification, while it provided cleaner demulsified water than GRB-IL, opening the road for more research on the topic.

All the Bio-ILs used in separation processes, along with their studied physicochemical characteristics, are summarized in

Table 4. Overview of recently investigated Bio-ILs in separation processes.

IL	Synthesis	Physicochemical Properties *	Application	Ref.
[Ch][Gly]	Neutralization reaction	-	Valorization of Chestnut Shell Waste	[69]
[Bmim][LSar]	Salt metathesis and neutralization reactions	-	Ιonic liquid dispersant for the effective oil spill remediation	[74]
[BDP][DDBS]		-
[TBA][Cit]		-
[TBA][PP]		-
[TBA][EEG]		-
[Chol-C6][Lev]	Salt metathesis reaction	Tg * = − 79.80 °C ENR * = 52.92 kcal/mol	Absorption of toluene, dichloromethane and methyl ethyl ketone. Determination of vapor–liquid partition coefficients	[42]
[Chol-C8][Lev]		Tg = − 82.59 °C ENR = 53.88 kcal/mol
[Chol-C6][Lac]		Tg = − 73.07 °C ENR = 52.54 kcal/mol
[Chol-C8][Lac]		Tg = − 68.69 °C ENR = 52.99 kcal/mol
GCP-IL	Neutralization reaction	-	Demulsification of Heavy Crude Oil Emulsions	[75]
GRB-IL		-
[Ch][Fu]	Salt metathesis reaction	-	Selective coagulation of κ-carrageenan from Kappaphycus alvarezii extract	[67]
[Ch][Adi]		-
[Ch][Cap]		-
[Ch][Capl]		-
[Ch][Capr]		-
[Ch][Lau]		-
[Ch][Ala]	Neutralization reaction	-	Pretreatment of corn stover and lignin depolymerization by a bi-enzyme system	[68]
[Ch][Gly]
[Ch][Lys]
[Ch][Lys]	Neutralization reaction	-	Delignification Rice and Wheat Residues for production of food-grade glucose	[70]
[DMBA][HSO4]		-
[Ch][Gly]	Salt metathesis reaction	η * = 1230 cP (25 °C) d * = 1.156 g/cm3	CO2 capture	[66]
[Ch][Ala]		η = 720 cP (25 °C) d = 1.130 g/cm3
[Ch][Ser]		η = 12,500 cP (25 °C) d = 1.201 g/cm3
[Ch][Pro]		η = 9810 cP (25 °C) d = 1.138 g/cm3
[Ch][Phe]		η = 55,300 cP (25 °C) d = 1.143 g/cm3
[Ch][Sar]		η = 1058 cP (25 °C) d = 1.116 g/cm3
[2-HEA][OAc]	Simulation	-	Optimization of agave bagasse pretreatment in a one-pot ethanol production process	[71]
[C2C1Im][OAc]		-
[Ch][For]	Simulation	-	COSMO–RS and MD simulations of lignin dissolution in ILs	[73]
[Ch][Ace]		d = 1.10 g/cm3
[Ch][But]		d = 1.07 g/cm3
[Ch][Hex]		d = 1.02 g/cm3
[Ch][Oct]		-
[Ch][Lys]		d = 1.09 g/cm3
[Ch][Ace]	Purchased	-	Delignification of sugar baggasse and recovery and regenerartion of IL	[72]
Cholinium + 50 anions (amino/carboxylic acids)	Simulation	-	COSMO RS Simulation for Gas absorption, liquid-liquid extraction	[6]

* T_g: Glass transition temperature, E_NR: electron transition in Nile red, η: viscosity, d: density.

.

3.4. Lubricants

Due to the ability to choose the appropriate combination of ionic components for the design of ILs for a specific application and, provided that the IL is compatible with the targeted base oil, ILs have gained attraction for their use in lubricants. However, many of them are proven to be hazardous and corrosive, such as those based on imidazolium or pyridinium cations and halogen-based anions, so the need for the development of environmentally friendly ILs containing natural ingredients is imperative.

Sadanandan et al. [76] studied the tribological behavior of three novel ILs derived from 1,1,3,3-tetramethylguanidinium (TMG) cation and the anions from the amino acids L-histidine, L-glutamic acid and L-aspartic acid (Scheme 15), to investigate their potential as lubricant additives to polyethylene glycol 200 (PEG 200). FTIR, ¹H and ¹³C NMR were used for the characterization of the ILs and TGA for thermal stability, while viscosity, viscosity index and density were also measured. The IL-PEG 200 blends were examined as for their anti-wear, anti-friction and anti-corrosion activity and, according to the results, all ILs were remarkably efficient in improving the tribological performance of the base fluid. SEM, EDX spectroscopy and elemental mapping proved the thin compact film formation by all the blends on the metal surface analyzed, which prevented direct contact between the metals. The blend containing the aspartic-acid-based IL demonstrated the best behavior, regarding wear resistance, friction reduction and corrosion inhibition at 2 %wt. [TMG][Asp], which was the optimum concentration. This is mainly attributed to the presence of two carboxylic groups, which have strong affinity with metal surfaces.

Lubricants that are intended to be used in high-temperature applications are usually derived from petroleum-based oils, which reduces their sustainable character and, thus, they need to be replaced by bio-based lubricants (for instance, composed of vegetable oils). However, a challenge that must be dealt with for biolubricants is their thermal instability, which precludes them from use in high temperatures. Room-temperature ILs (RTILs) have been employed for this reason, as they are synthesized from bio-based feedstock and have the ability to remain stable under thermal oxidation conditions, so their large-scale development needs to become feasible for less toxic lubricant applications.

Reeves et al. [77] created eco-friendly lubricants and investigated their potential for use in high-temperature processes (around 100 °C). The cation was either the bio-based 1,3-diakylimidazolium or the non-toxic, anti-microbial and biodegradable trihexyl(tetradecyl)phosphonium [P_666,14]. The anions used were carboxylate, such as salicylate, saccharinate and benzoate, which are regarded as eco-friendly and of low toxicity. The tribological and thermal behavior of the resulting RTILs was studied in comparison with other bio-based and petroleum-based oils, through pin-on-disk testing and TGA analysis, respectively. It was observed that RTILs based on longer-alkyl-chain imidazolium cations, when combined with bis(trifluoromethylsulfonyl)amide anions [Tf₂N], displayed better tribological properties in terms of low coefficient of friction (COF) and wear volume, probably because of the formation of a thicker adsorbed monolayer film on the steel surface. Longer alkyl chains of the imidazolium cation also led to higher thermal stability. The most important influence on the behavior of imidazolium and phosphonium ILs had the aromatic carboxylic organic anions. In fact, the lowest COF and wear rate were observed by [P_666,14][Sac], because of most rings occurring. All RTILs prepared seemed to have better tribological and thermal performance than bio- and petroleum-based oils, but especially phosphonium-based ILs with carboxylate anions have the greatest potential, with lower friction, wear and degradation rates, even better than the imidazolium-based ILs. Remarkably, an increase in the anion rog size causes minimal friction and wear. All these results were confirmed by surface analysis through SEM. The noticeable tribological and thermal performance of RTILs is generally attributed to their inherent lamellar-like liquid-crystal structure, resulting from the long chains of polar anion–cation molecules and also from the electrostatic potential between cations and anions (lattice energy).

All the Bio-ILs used as lubricants, along with their studied physicochemical characteristics, are summarized in

Table 5. Overview of recently investigated Bio-ILs as lubricants.

IL	Synthesis	Physicochemical Properties	Application	Ref.
[TMG][His]	Neutralization reaction	Td *= 302 °C Kinematic viscosity = 256.07 mm2 s−1 (40 °C) Kinematic viscosity = 19.780 mm2 s−1 (100 °C) Viscosity index = 88 d * = 1.1654 g/cm3 (40 °C)	Νoncorrosive lubricant additives for tribological performance	[76]
[TMG][Glm]		Td = 338 °C Kinematic viscosity = 282.10 mm2 s−1 (40 °C) Kinematic viscosity = 21.74 mm2 s−1 (100 °C) Viscosity index = 93 d = 1.1796 g/cm3 (40 °C)
[TMG][Asp]		Td = 304 °C Kinematic viscosity = 325.26 mm2 s−1 (40 °C) Kinematic viscosity = 23.244 mm2 s−1 (100 °C) Viscosity index = 89 d = 1.2063 g/cm3 (40 °C)
[C3mim][Tf2N]	Salt metathesis reaction	-	Potential high-temperature lubricants in steel-steel tribo-contacts	[77]
[C5mim][Tf2N]		Td = 446 °C
[C6mim][Tf2N]		Td = 448 °C
[C8mim][Tf2N]		Td = 450 °C
[C10mim][Tf2N]		Td = 451 °C
[C8mim][Sal]		-
[C10mim][Sac]		-
[P666,14][Cl]		-
[P666,14][Tf2N]		Td = 408 °C
[P666,14][Benz]		Td = 379°C
[P666,14][Sal]		Td = 371 °C
[P666,14][Sac]		Td = 410 °C
[P666,14][Cyc]		-

* T_d: degradation temperature, d: density.

.

4. Conclusions

It is evident that, although ILs have been extensively studied since their first appearance, the urgent need for green solutions in chemical processes has paved the way for more exhaustive research towards the development of biocompatible and non-toxic ILs based on the use of natural products, such as choline, lipids, amino acids, etc. The advantageous and tunable physicochemical and pharmaceutical profiles of this new generation of ILs have resulted in their application in a vast variety of scientific fields, including organic catalysis, lubricants, separation processes and pharmaceutics, with the later one being the most investigated so far. It is worth mentioning that API-ILs appear to be the cutting-edge technology in biomedical processes, since more and more scientists have been exploring the potential of utilizing these green tools to tackle solubility and bioavailability problems in APIs, while enhancing their bioactivity. However, in spite of the fact that the literature data with reference to Bio-ILs have increased the past few years, more fundamental research is imperative in order to apprehend, in depth, their structure-property dependence and broaden their scope.