International Journal of Molecular Sciences

Review The Role of Ionic Liquids in the Pharmaceutical Field: An Overview of Relevant Applications

Sónia N. Pedro , Carmen S. R. Freire, Armando J. D. Silvestre and Mara G. Freire * Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, 3810-193 Aveiro, Portugal; [email protected] (S.N.P.); [email protected] (C.S.R.F.); [email protected] (A.J.D.S.) * Correspondence: [email protected]



Received: 25 September 2020; Accepted: 2 November 2020; Published: 5 November 2020


Abstract: Solubility, bioavailability, permeation, polymorphism, and stability concerns associated to solid-state pharmaceuticals demand for effective solutions. To overcome some of these drawbacks, ionic liquids (ILs) have been investigated as solvents, reagents, and anti-solvents in the synthesis and crystallization of active pharmaceutical ingredients (APIs), as solvents, co-solvents and emulsifiers in drug formulations, as pharmaceuticals (API-ILs) aiming liquid therapeutics, and in the development and/or improvement of drug-delivery-based systems. The present review focuses on the use of ILs in the pharmaceutical field, covering their multiple applications from pharmaceutical synthesis to drug delivery. The most relevant research conducted up to date is presented and discussed, together with a critical analysis of the most significant IL-based strategies in order to improve the performance of therapeutics and drug delivery systems.

Keywords: active pharmaceutical ingredients; drug delivery systems; formulations; ionic liquids; solubility; permeability

1. Introduction Pharmaceuticals play a major role in medical care, boosting life quality and expectancy, especially when considering chronic diseases [1]. The global prescription of medicines is forecast to grow up to nearly $1.2 trillion by 2022 [2]. Although active pharmaceutical ingredients (APIs) can be commercialized in several dosage forms, crystalline forms have been the preferred option [3,4]. However, 40 to 70% of the drugs under development present low water-solubility, which may compromise the bioavailability and therapeutic efficacy and, thus, fail in the later stages of development [5,6]. The irregular gastrointestinal absorption of solid forms, along with the low therapeutic efficiency and possible toxicity and side-effects of polymorphs, are major concerns to overcome [7]. For instance, large differences in bioavailability among different polymorphs require different drug dosages [8]. On the other hand, the therapeutic dosage of a certain API can correspond to a toxic or potential lethal dose if the wrong polymorph is administered. Polymorphism issues result in significant economic losses in sales and in R&D to enable novel formulations back into the market [9,10]. Beyond the well-known downsides of polymorphism, the APIs’ solubility in aqueous solution, dissolution, and bioavailability are also dependent on particle size and properties [11]. Attempting to improve the drugs solubility in water as well as their bioavailability, several strategies have been investigated, especially when the oral route is envisaged [5,6]. Nevertheless, most of these strategies still use large quantities of organic solvents in the manufacturing process of these formulations, particularly to induce the crystallization of a given polymorphic form and particle size, having associated health and environmental concerns [12]. Furthermore, solvent molecules can be incorporated into the crystal structure of the API during the crystallization process [13]. Therefore, when considering the use of

Int. J. Mol. Sci. 2020, 21, 8298; doi:10.3390/ijms21218298 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 8298 2 of 50 organic solvents, they must be removed from the API or their levels must be controlled in order to ensure human consumption safety [12]. Despite the existence of extensive literature describing novel and “greener” solvents to this purpose, there is still some reluctance by the pharmaceutical industry to accept and implement these alternatives [14–16]. In the above context, liquid forms of APIs are appealing solutions to avoid both polymorphism and improve low-water solubility constraints, while allowing to reduce organic solvents use. The pharmaceutical industry has relied on eutectic mixtures for this purpose, shortly exploring other optionsInt. J. Mol. for Sci. commercialization 2020, 21, x FOR PEER REVIEW [17 ,18]. In addition to these, ionic liquids (ILs)2 discloseof 51 high potential in the pharmaceutical field, which is mainly due to their high versatility in terms of chemical must be controlled in order to ensure human consumption safety [12]. Despite the existence of structure designextensive towards literature a targetdescribing application. novel and “greener” ILs are molten solvents salts to this that purpose, are composed there is still of asome large organic cation andreluctance an organic by the/inorganic pharmaceutical anion. industry The largeto accept dimensions and implement of theirthese alternatives ions lead [14–16]. to charge dispersion, which makesIn di theffi cultabove the context, formation liquid forms of of a APIs regular are appealing crystalline solutions structure to avoid both [19, polymorphism20]. ILs display and a set of unique features,improve fromlow-water which solubility is possible constraints, to highlight, while allowing if properly to reduce designed, organic solvents their highuse. The thermal and pharmaceutical industry has relied on eutectic mixtures for this purpose, shortly exploring other options chemical stabilityfor commercialization and a strong [17,18]. solvation In addition ability to these, for ionic a wide liquids variety (ILs) disclose of compounds high potential [21 in]. the The proper selection ofpharmaceutical cation-anion field, combinations which is mainly indue ILs to their enables high versatility the use in of terms drugs of chemical as ion components,structure design allowing for the conversiontowards a target of solid application. active ILs pharmaceutical are molten salts that ingredients are composed into of liquida large organic forms cation (API-ILs). and an Thus, this strategy solvesorganic/inorganic the problem anion. ofThe polymorphism large dimensions of and their provides ions lead improvedto charge dispersion, bioavailability, which makes and ideally difficult the formation of a regular crystalline structure [19,20]. ILs display a set of unique features, from boosts therapeutic properties [3,22]. which is possible to highlight, if properly designed, their high thermal and chemical stability and a strong Becausesolvation of the ability unique for a wide properties variety of compounds of ILs, their [21]. application The proper selection in the of pharmaceuticalcation-anion combinations field has been extended farin ILs beyond enables the the developmentuse of drugs as of ion novel components, liquid formsallowing (API-ILs), for the conversion being investigatedof solid active as well in other stagespharmaceutical of drug development ingredients into and liquid delivery. forms The(API-ILs). number Thus, ofthis publications strategy solves related the problem to the of application of ILs in thepolymorphism pharmaceutical and provides field hasimproved grown bioavailability, exponentially and ideally in the boos pastts therapeutic 20 years, properties as illustrated [3,22]. in Figure1. Because of the unique properties of ILs, their application in the pharmaceutical field has been ILs have beenextended applied far beyond in the the development development of novel purification liquid forms platforms (API-ILs), for being pharmaceuticals investigated as well (an in application out of the scopeother stages of this of drug review), development for which and delivery. some recent The number review of manuscriptspublications related exist to [ the23– application25]. Other relevant reviews andof ILs book in the chapterspharmaceutical recognizing field has grown the exponentially advances of in ILsthe past in 20 di ffyears,erent as illustrated areas of in pharmaceuticals Figure development,1. ILs spanning have been fromapplied their in the formulation, development biologicalof purification activity, platforms and for application pharmaceuticals on drug (an delivery application out of the scope of this review), for which some recent review manuscripts exist [23–25]. are also availableOther relevant [22, 26reviews–34]. and However, book chapters most recogniz of theseing focusthe advances on a specificof ILs in applicationdifferent areas of of ILs in the pharmaceuticalpharmaceuticals field. On development, the other hand,spanning this from review their formulation, compiles andbiological discusses activity, the and most application relevant works and overallon applicationsdrug delivery are of also ILs available in the [22,26–34]. pharmaceutical However, most field, of namelythese focus on on thea specific role application of ILs as solvents, reagents, andof ILs/or in catalyststhe pharmaceutical in the APIs’ field. On synthesis, the other hand, in the this APIs review crystallization, compiles and discusses as solvents, the most co-solvents, relevant works and overall applications of ILs in the pharmaceutical field, namely on the role of ILs and emulsifiersas solvents, to improve reagents, and/or drugs catalysts solubility, in the as APIs’ a way synthesis, of producing in the APIs liquidcrystallization, forms as of solvents, APIs, and in the developmentco-solvents, of drug and delivery emulsifiers systems. to improve Special drugs attentionsolubility, as is a drawn way of producing to the most liquid important forms of APIs, achievements reported soand far in onthe thedevelopment use of ILs of drug in the delivery described systems. applications Special attention and is drawn the resulting to the most benefits important in terms of pharmaceuticalachievements formulations reported so and far on pharmacological the use of ILs in the activity. described applications and the resulting benefits in terms of pharmaceutical formulations and pharmacological activity.

Figure 1. FigureNumber 1. Number of publications of publications per per year year in in aa twentytwenty years years perspective perspective related related to ILs and to active ILs and active pharmaceuticalpharmaceutical ingredients ingredients (APIs) (APIs) (number (number ofof articles,articles, reviews reviews and andbook bookchapters chapters according according to a to a ScienceDirect database search using as keywords “ionic liquids”, “active pharmaceutical ScienceDirectingredients”, database and search “drug delivery”) using as (left keywords). Overview “ionic of theliquids”, ILs’ applications “active in the pharmaceutical pharmaceutical field ingredients”, and “drugreported delivery”) hitherto (left (right). Overview). of the ILs’ applications in the pharmaceutical field reported hitherto (right).

Int. J. Mol. Sci. 2020, 21, 8298 3 of 50 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 51 2. ILs in the Synthesis of Pharmaceutical Compounds 2. ILs in the Synthesis of Pharmaceutical Compounds The increase in environmental awareness led to the proposal of the so-called Environmental The increase in environmental awareness led to the proposal of the so-called Environmental factor (E-factor), which assesses the environmental impact of manufacturing processes, being defined factor (E-factor), which assesses the environmental impact of manufacturing processes, being defined by the ratio of the mass of waste per mass of product [35]. The pharmaceutical E-factor is one of by the ratio of the mass of waste per mass of product [35]. The pharmaceutical E-factor is one of the the highest in the industry context (25–100) [35]. The waste production that is generated by the highest in the industry context (25–100) [35]. The waste production that is generated by the pharmaceutical industry is mainly attributed to solvent losses. In order to reduce these losses and pharmaceutical industry is mainly attributed to solvent losses. In order to reduce these losses and minimize the environmental impact, it is essential to consider alternative solvents, i.e., to develop minimize the environmental impact, it is essential to consider alternative solvents, i.e., to develop more sustainable processes. To this purpose, ILs have been studied as (i) solvents; (ii) catalysts; (iii) more sustainable processes. To this purpose, ILs have been studied as (i) solvents; (ii) catalysts; (iii) reagents; and, (iv) enantioselectivity enhancers in the synthesis of different APIs [36–38]. Reactions in reagents; and, (iv) enantioselectivity enhancers in the synthesis of different APIs [36–38]. Reactions these solvents may be faster and involve fewer steps than those that were carried out in conventional in these solvents may be faster and involve fewer steps than those that were carried out in organic solvents, and additionally be easier to implement [39]. However, an initial assessment of conventional organic solvents, and additionally be easier to implement [39]. However, an initial conditions must be performed, since the kinetic of reactions that were carried out in ILs differ from assessment of conditions must be performed, since the kinetic of reactions that were carried out in those performed in conventional organic solvents [40]. The following described examples intend to ILs differ from those performed in conventional organic solvents [40]. The following described illustrate the multifunction role displayed by ILs in APIs’ synthesis, in which some have been even examples intend to illustrate the multifunction role displayed by ILs in APIs’ synthesis, in which combined. Figure2 provides an illustrative summary of the applications of ILs in the synthesis of APIs some have been even combined. Figure 2 provides an illustrative summary of the applications of ILs and their precursors, giving one example of each application discussed in this section, with the goal of in the synthesis of APIs and their precursors, giving one example of each application discussed in replacing the use of volatile organic solvents. this section, with the goal of replacing the use of volatile organic solvents.

Reaction media Catalyst Reagent Enzyme stability and enantioselectivity enhancer Anti-inflammatory Antiviral APIs precursors Antifungal Anti-inflammatory Pravadoline Trifluridine 2,3-dihydroquinazolin-4(1H)-one core Clioquinol Ibuprofen OH

F OH F

F N O

O N O H

[C4C1im][PF6] [(C1OC2)C1im][MsO] [Nbmd][OH] [C4C1py][DCI] [C4C1im][PF6] 90-94% yield 91% yield 98% yield 93% yield 3-fold increase

Figure 2.2.Multiple Multiple roles roles of ILsof ILs in the in synthesisthe synthesis of diff oferent different APIs and APIs their and respective their respective efficiency (adequateefficiency references(adequate arereferences given along are given the current along the section). current section).

Given the high high ILs’ ILs’ applicability applicability in in different different chem chemicalical processes, processes, they they have have been been applied applied in the in theproduction production of pharmaceutical of pharmaceutical precursors, precursors, such such as aslactam lactam [41], [41 ],pyrazolone pyrazolone [42], [42], thiazole thiazole [43], [43], imidazoleimidazole [44[44],], and and thiazolidine thiazolidine [43 ,[43,45]45] cores, cores, which which are APIs’ are precursorsAPIs’ precursors with vast with biological vast biological activities. Dueactivities. to their Due charged to their nature,charged ILs nature, can provide ILs can provid fast microwavee fast microwave heating, heating, resulting resulting in faster in andfaster more and emoreffective effective reactions. reactions. For example, For example, this approach this appr has beenoach successfullyhas been successfully applied to the applied direct lactamizationto the direct oflactamization lactones in aof one-pot lactones reaction in a one-pot with high reaction yields with (>80%), high obtained yields (>80%), in short obtained reaction in times short ( 35reaction min.) ≤ (Figuretimes (≤335)[ min.)41]. The (Figure combination 3) [41]. The of combination ILs with ultrasound of ILs with irradiation ultrasound can irradiation be also useful can be to also accelerate useful organicto accelerate reactions, organic allowing reactions, for allowing synthetizing for synthe APIs’ precursorstizing APIs’ at precursors room temperature at room temperature with high yields with (95%)high yields [46]. (95%) [46].

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 51

FigureFigure 3. 3.Fast Fast and and acid-free acid-free one-pot one-pot IL-basedIL-based microwave methodology methodology for for direct direct synthesis synthesis of oflactams lactams from lactones and primary amines proposed in [41]. fromFigure lactones 3. Fast and and primary acid-free amines one-pot proposed IL-based in microwave [41]. methodology for direct synthesis of lactams from lactones and primary amines proposed in [41]. TheThe versatile versatile nature nature ofof ILsILs allowsallows forfor reducing the the volume volume of of solvents solvents and and the the use use of ofmetal metal catalystscatalystsThe in in APIs versatile APIs synthesis synthesis nature [ 47[47].of]. ILs DespiteDespite allows thethe for varietyvariety reducing of cations cationsthe volume and and anions,of anions, solvents imidazolium-based imidazolium-based and the use of saltsmetal salts havehavecatalysts been been the thein mostAPIs most synthesis studiedstudied as[47]. solvents solvents Despite for for the the the variety sy synthesisnthesis of cationsof APIs of APIs and and andanions, their their precursors imidazolium-based precursors [45,48–51]. [45,48 salts In–51 ]. 2000, Seldon and coworkers reported the first high yield (90–94%) IL-based route to produce a non- In 2000,have Seldonbeen the and most coworkers studied as reportedsolvents for the the first synthesis high yield of APIs (90–94%) and their IL-based precursors route [45,48–51]. to produce In steroidal anti-inflammatory (NSAID) drug, pravadoline, using an imidazolium-based IL as solvent, a non-steroidal2000, Seldon anti-inflammatoryand coworkers reported (NSAID) the first drug, high pravadoline, yield (90–94%) using IL-based an imidazolium-based route to produce a non- IL as namely 1-butyl-3-methylimidazolium hexafluorophosphate ([C4C1im][PF6]) (Figure 4) [51]. solvent,steroidal namely anti-inflammatory 1-butyl-3-methylimidazolium (NSAID) drug, pravadolin hexafluorophosphatee, using an imidazolium-based ([C C im][PF ]) (FigureIL as solvent,4)[ 51 ]. Conventionally, the reaction to produce pravadoline is carried out in volatile4 1 organic6 solvents, such Conventionally,namely 1-butyl-3-methylimidazolium the reaction to produce pravadoline hexafluorophosphate is carried out in([C volatile4C1im][PF organic6]) (Figure solvents, 4) such[51]. as as dimethylformamide (DMF), while using sodium hydride as a base that additionally presents dimethylformamideConventionally, the (DMF), reaction while to produce using sodium pravadoline hydride is carried as a base out thatin volatile additionally organic presents solvents, health such healthas dimethylformamide and environmental (DMF), concerns while [52,53]. using The sodium proposed hydride reaction as a baseusing that the additionally IL as solvent presents and andpotassium environmental hydroxide concerns as base [ 52allowed,53]. The for proposedimproving reactionthe conventional using the reaction IL as solvent yield (70–91%) and potassium up to hydroxidehealth asand base environmental allowed for improvingconcerns [52,53]. the conventional The proposed reaction reaction yield using (70–91%) the IL up as to solvent 95%, simply and 95%,potassium simply hydroxideby heating asthe base IL at allowed 150 °C for improving2 min. With the this conventional strategy, it isreaction possible yield to easily (70–91%) separate up to bythe heating API product, the IL at recycle 150 ◦C and for 2reuse min. the With solvent, this strategy, and the itonly is possible chemical to waste easily generated separate thein the API process product, 95%, simply by heating the IL at 150 °C for 2 min. With this strategy, it is possible to easily separate recycle and reuse the solvent, and the only chemical waste generated in the process is an aqueous is thean aqueousAPI product, solution recycle of potassium and reuse chloride.the solvent, and the only chemical waste generated in the process solution of potassium chloride. is an aqueous solution of potassium chloride.

Figure 4. Pravadoline synthesis in an IL media proposed in [51].

FigureFigure 4. Pravadoline4. Pravadoline synthesis synthesis in in an an IL IL media media proposed proposed inin [[51].51].

Int. J. Mol. Sci. 2020, 21, 8298 5 of 50

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 51 Int. J. Mol.A variety Sci. 2020 of, 21 pharmaceutical, x FOR PEER REVIEW agents (e.g., antibiotics, antifungals, alkaloids, or cardiac glycosides)5 of 51 have an heterocyclic structure to mimic the structure and, thus, the biological action of natural A variety of pharmaceutical agents (e.g., antibiotics, antifungals, alkaloids, or cardiac glycosides) compounds [54]. Reactions that were carried out in IL solvent media have high regioselectivity have an heterocyclic structure to mimic the structure and, thus, the biological action of natural compounds and, for this reason, have been successfully applied in the synthesis of different heterocyclic [54]. Reactions that were carried out in IL solvent media have high regioselectivity and, for this reason, APIs [55,56]. Imidazolium-based ILs have been used as solvents in the synthesis of antiviral have been successfully applied in the synthesis of different heterocyclic APIs [55,56]. Imidazolium-based drugs as brivudine, stavudine and trifluridine [56]. Figure5 provides a summary on the ILs have been used as solvents in the synthesis of antiviral drugs as brivudine, stavudine and trifluridine synthesis time and yield of nucleoside-based antiviral drugs in IL media. Trifluridine, for [56]. Figure 5 provides a summary on the synthesis time and yield of nucleoside-based antiviral drugs in example, was produced as a single product in IL media. Among others, the best results were IL media. Trifluridine, for example, was produced as a single product in IL media. Among others, the best obtained with 1-methoxyethyl-3-methylimidazolium methanesulfonate ([(C OC )C im][MsO]), using results were obtained with 1-methoxyethyl-3-methylimidazolium1 2 1methanesulfonate 4-dimethylaminopyridine (DMAP) as catalyst and acetic anhydride as acylating agent. Trifluridine ([(C1OC2)C1im][MsO]), using 4-dimethylaminopyridine (DMAP) as catalyst and acetic anhydride as was obtained with 91% yield in 20–25 min. without the need of extra-purification steps. The IL was acylating agent. Trifluridine was obtained with 91% yield in 20–25 min. without the need of extra- recycled and reused up to four times. purification steps. The IL was recycled and reused up to four times.

1. Brivudine 2. Stavudine 3. Trifluridine

IL media Time(min) 60 10 20 [(C1OC2)C1im][MsO]) [(C1OC2)C1im][MsO]) Yield(%) 70 89 91

Figure 5. Synthesis of nucleoside-bas nucleoside-baseded antiviral drugs in IL media proposed in [[56].56].

Antifungal and antiprotozoal drugs, such as iodoquinol and clioquinol, respectively, have been prepared by aa simplesimple andand eefficientfficient iodinationiodination method,method, whilewhile taking advantageadvantage of the ILs’ILs’ multiplemultiple roles in synthesis, as summarized in Figure 6 6[ [57].57]. The The IL IL 1-butyl-3-methylpyridinium 1-butyl-3-methylpyridinium dichloroiodate dichloroiodate ([C 4C1 py][DCI]) was used both as solvent and iodinating agent in the absence of any oxidant, catalyst, ([C44C1py][DCI]) was used both as solvent and iodinating agent in the absence of any oxidant, catalyst, or base. It was possible to regenerate the IL for up to fivefive runs byby additionaddition ofof IClICl (1.2(1.2 eq.),eq.), withwith >>90%90% yield, without losinglosing itsits iodinatingiodinating activity.activity.

Recovered IL IL OH N I N + + 80˚C

Cl Clioquinol

Recycled ICl /1.2 e.q.

Figure 6. ILIL application application as as solvent solvent and and iodinating iodinating agent agent in the the synthesis synthesis of clioqu clioquinolinol proposed in [[57].57].

Naproxen was initially initially synthesized synthesized and and commercialized commercialized by by Syntex Syntex while while using using b-naphthol b-naphthol as asprecursor precursor for forits itssynthesis synthesis [58]. [58 However,]. However, this this process process uses uses several several undesirable undesirable reagents, reagents, such such as asnitroaromatic nitroaromatic compounds, compounds, ammoni ammoniumum sulfide sulfide sodium sodium hydride, hydride, and and methyl methyl iodide. iodide. In order In order to toovercome overcome these these drawbacks, drawbacks, new new procedures procedures were were considered, considered, increasing increasing the yields the yields from fromless than less 50% to 90%, but the formation of undesired side products and use of metal catalysts in these processes remained [59]. Recently, 1-butyl-3-methylimidazolium tetrafluoroborate ([C4C1im][BF4]) was applied as a reaction medium in the electrosynthesis of naproxen through the electrocarboxylation of 2-(1-

Int. J. Mol. Sci. 2020, 21, 8298 6 of 50 than 50% to 90%, but the formation of undesired side products and use of metal catalysts in these processes remained [59]. Recently, 1-butyl-3-methylimidazolium tetrafluoroborate ([C4C1im][BF4]) Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 51 was applied as a reaction medium in the electrosynthesis of naproxen through the electrocarboxylation of 2-(1-chloroethyl)-6-methoxynaphthalene using CO2 [60], as summarized in Figure7. This process chloroethyl)-6-methoxynaphthalene using CO2 [60], as summarized in Figure 7. This process allowed allowed for achieving high yields (89%) and conversion rates (90%), with 65% of atom economy when for achieving high yields (89%) and conversion rates (90%), with 65% of atom economy when considering the recovery of the solvent. Despite the new adapted synthesis routes mentioned above considering the recovery of the solvent. Despite the new adapted synthesis routes mentioned above also allowing for obtaining similar high yields, the process in IL media uses cheaper and more available also allowing for obtaining similar high yields, the process in IL media uses cheaper and more catalysts (electrons) and CO2 instead of CO, a well-known pollutant, contributing for the development available catalysts (electrons) and CO2 instead of CO, a well-known pollutant, contributing for the of “greener” routes in the synthesis of APIs. development of “greener” routes in the synthesis of APIs.

FigureFigure 7.7. SchematicSchematic representationrepresentation ofof naproxen’snaproxen’s electrosynthesiselectrosynthesis underunder CO2CO2 atmosphereatmosphere proposedproposed inin [[60].60].

BiologicallyBiologically active compounds,compounds, withwith antidiuretic,antidiuretic, anti-inflammatory,anti-inflammatory, andand antihypertensiveantihypertensive activities,activities, usually possess possess 2,3-dihydroquinazolin-4(1H)-one 2,3-dihydroquinazolin-4(1H)-one (DHQ) (DHQ) cores. cores. Due Due to their to their importance importance for forthe production the production of such of compounds, such compounds, the synthesi thes synthesisof substituted of substitutedDHQ derivatives DHQ has derivatives attracted hassignificant attracted attention, significant and different attention, synthetic and di stratefferentgies synthetic have been strategies developed. have In alternative been developed. to the Inconventional alternative harsh to reaction the conventional conditions, gemini harsh basic reaction ILs (such conditions, as (4,4′-(butane-1,4-diyl)bis(4-dodecyl- gemini basic ILs (such as (4,4morpholin-4-ium)hydroxide0-(butane-1,4-diyl)bis(4-dodecyl-morpholin-4-ium)hydroxide ([Nbmd][OH])), have been proposed ([Nbmd][OH])), as catalysts and have reaction been media proposed for astheir catalysts synthesis and [61]. reaction In addition media forto the their high synthesis reported [61 yields]. In addition (98%), it to was the shown high reported the possibility yields (98%),to reuse it wasthe ILs shown up to the five possibility times without to reuse significant the ILs up losses to five in times catalytic without activity. significant losses in catalytic activity. SeveralSeveral industrialindustrial processes processes apply apply enzyme-catalyzed enzyme-catalyzed reactions reactions in organic in mediaorganic for media the production for the ofproduction APIs. An of exampleAPIs. An ofexample the advantageous of the advantageous use of ILsuse inof ILs biocatalysis in biocatalysis was demonstratedwas demonstrated in the in transesterificationthe transesterification of ribavirin, of ribavirin, a nucleoside a nucleoside antiviral antiviral drug, drug, using usingC. C. antarctica antarcticalipase lipase [ 55[55].]. TheThe useuse ofof thethe ILIL [C[C44C11im][BF44]] as as adjuvant adjuvant allowed allowed to to improve improve the re regioselectivity,gioselectivity, the reaction yieldyield andand reactionreaction raterate aboutabout 3.53.5 times.times. The enantioselectivity and enzyme activity inin IL media was comparable oror eveneven improvedimproved when compared to the results that were obtained in traditional organic solvents. Furthermore,Furthermore, lipase catalyzed transterificationtransterification reactions can be 25 times more eeffectiveffective in ILIL mediamedia thanthan inin conventionalconventional solventssolvents [[62].62]. StereoselectivityStereoselectivity andand enantioselectivity enantioselectivity are are major ma concernsjor concerns regarding regarding the bioavailability the bioavailability and safety and ofsafety pharmaceutics. of pharmaceutics. In this context,In this ILscontext, have beenILs ha studiedve been in studied order to in improve order theto improve kinetic resolution the kinetic of APIsresolution and API of APIs precursors. and API Enantiopure precursors. chiral Enantiopure alcohols chiral have beenalcohols shown have to been be versatile shown chiralto be buildingversatile blockschiral building for the synthesis blocks for of chiralthe synthesis pharmaceuticals of chiral [63pharmaceuticals]. For instance, [63]. (S)-3-chloro-1-phenyl-1-propanol For instance, (S)-3-chloro-1- ((S)-CPPO)phenyl-1-propanol is a useful ((S)-CPPO) chiral building is a useful block chiral for the building synthesis block of anti-depressantfor the synthesis drugs of anti-depressant [64]. Aiming todrugs produce [64]. Aiming (S)-CPPO to withproduce high (S)-CPPO yields and with selectivity, high yields a variety and selectivity, of ILs were a variety tested of as ILs media, were wheretested [Cas 4media,C1im][NTf where2] [C was4C1 ultimatelyim][NTf2] was selected ultimately for increasing selected for the increasing solubility the of solubility the (S)-CPPO’s of the (S)-CPPO’s precursor, 3-chloro-1-phenyl-1-propanoneprecursor, 3-chloro-1-phenyl-1-propanone (3-CPP), in (3-CPP), a IL/water in mixturea IL/water [65 mixture]. The use [65]. of The the IL,use allowed of the IL, to dramaticallyallowed to dramatically increase the increase concentration the concentration of 3-CPP and of 3-CPP the yield and of the the yield target ofcompound, the target compound, where the where the yeast reductase YOL151W was able to convert the 3-CPP enantioselectively into (S)-CPPO exclusively, with an enantiomeric excess of >99%. The selected examples provide evidences where the use of ILs leads to comparable or superior reaction conditions and yields, and they may also simplify the separation and purification steps of some target products. The possibility to recycle and reuse ILs, without compromising the synthesis yield and lack of toxic by-products production, as shown in some examples, further reinforces the

Int. J. Mol. Sci. 2020, 21, 8298 7 of 50 yeast reductase YOL151W was able to convert the 3-CPP enantioselectively into (S)-CPPO exclusively, with an enantiomeric excess of >99%. The selected examples provide evidences where the use of ILs leads to comparable or superior reaction conditions and yields, and they may also simplify the separation and purification steps of some target products. The possibility to recycle and reuse ILs, without compromising the synthesis yield and lack of toxic by-products production, as shown in some examples, further reinforces the advantageous properties of ILs from the environmental and pharmaceutical perspectives. However, as happens with organic solvents, the amount of residual IL in the final product must be limited in order to guarantee the safety of the drug in the final dosage form. Following this, it is essential for a more careful monitorization of these contaminants in the final product and to study their impact in the drug’s therapeutic performance and toxicity.

3. ILs as Solvents, Co-Solvents or Emulsifiers for APIs Solubilization The therapeutic efficacy of APIs is mostly defined by their solubility/bioavailability, since higher solubilities in aqueous solutions may allow for better achieving the desired concentration of a drug in systemic circulation [66]. An API is considered highly soluble when its highest dose strength is soluble in 250 mL or less of aqueous medium over a pH range 1–6.8 at human body’s temperature [67]. Nevertheless, many APIs from different pharmacological classes do not fulfill these requirements and they are only sparingly soluble in water. Although water is the preferable medium when considering human consumption, occasionally the use of non-aqueous solvents is considered since their presence may positively influence the API absorption [68]. Organic solvents like ethanol, methanol or dimethyl sulfoxide (DMSO) are generally used as solvents or co-solvents in pharmaceutical formulations to improve solubility [30]. To address this solubility challenge, ILs have been investigated as alternative neat solvents [69]. The good solvation ability of ILs also allowed for increasing the aqueous solubility of APIs by cosolvency, hydrotropy and micellization phenomena, as summarized in Table1. ILs represent a novel class of hydrotropes with superior performance to enhance the solubility of poorly water-soluble compounds in aqueous solution, driven by the formation of API–IL aggregates [21]. Cosolvency, unlike the hydrotropic mechanism, is not based on the formation of aggregates, but on the solvation of the solute by a mixed solvent (water + IL), acting by disrupting the water self-association and by reducing the interfacial tension between the API and the solvent medium [70]. The use of surface-active agents, on the other hand, acts by taking advantage of their amphiphilic nature and by incorporating hydrophobic APIs into the micelles core [71]. It has been demonstrated that the selection of IL anion/cation combinations has a significant impact on the solubilization mechanism of poorly water-soluble APIs and solvation ability, as shown by solubility enhancements for antifungal, analgesic, and nonsteroidal anti-inflammatory drugs that are listed in Table1. In particular, it has been demonstrated that the solubility of drugs like amphotericin B, albendazole, itraconazole, paclitaxel, or etodolac, which are very low-water soluble, can be enhanced by several orders of magnitude (from 700–5.6 106-fold) by adding ILs. × Int. J. Mol. Sci. 2020, 21, 8298 8 of 50 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 51 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 51 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 51 Table 1. Solubility of different APIs in selected ILs and comparison with their water solubility. Table 1. Solubility of different APIs in selected ILs and comparison with their water solubility. Table 1. Solubility of different APIs in selected ILs and comparison with their water solubility. Int. J. Mol. Sci. 2020, 21, x FOR PEERAPI REVIEWTable 1. Solubility of Structuredifferent APIs in selected Water ILs and Solubility comparison with their water IL solubility. Solubility8 of 51 Reference API Structure Water Solubility IL Solubility Reference a API Structure Water Solubility 666(14)IL 2 [P Solubility][NTfa ] Reference 0.0524 *[72] API Structure Water Solubility [P666(14)IL][NTf 2] 666(14)Solubility0.0524 2* Reference[72] a a 4-Hydroxycoumarin - [P2 666(14)1 ][NTf3 [C2]3 C im][CF0.0524O a *S][72] 0.1107 * 4-HydroxycoumarinTable 1. Solubility of different APIs in selected ILs and- comparison [Cwith[P2C666(14) 1theirim][CF][NTf water3O2]3 S]solubility.2 1 0.05240.1107 3 a 3* [72] 4-Hydroxycoumarin - [C2C1im][CF3O3S] 0.1107 a * a [C4C1im][CF3O[C3S]4 C1im][CF0.09073O a 3*S] [73] 0.0907 *[73] 4-HydroxycoumarinAPI Structure Water Solubility- [CIL24 C1im][CF3OSolubility3S] 0.11070.0907Reference * [73] 4 1 3 3 a [C C im][CF O S] 0.0907 a * [73] [P666(14)[C][NTf4C1im][CF2] 3O30.0524S] a * 0.0907[72] * [73] 5-Fluorouracil 12.21 b * b [C4C1im]Br 31.19 b * [74] b 5-Fluorouracil5-Fluorouracil4-Hydroxycoumarin 12.21- 12.21* [C*2C1im][CF[C3O4C3S]1im]Br 0.1107[C 4a *C 1im]Br31.19 * [74] 31.19 * [74] 5-Fluorouracil 12.21 b * [C4C1im]Br 31.19 b * [74] b [C4C1im][CF3O3S] 0.0907 a * [73]b Int. J. Mol. Sci. 5-Fluorouracil2020, 21, x FOR PEER REVIEW 12.21 * [C4C1im]Br 31.198 of 51* [74] c c 98.8 c [C4C1im][BF4] >132 c [69] c b c [C Cb im][BFc ] >132 [69] 5-Fluorouracil 12.2198.8 * [C4C1im]Br[C48C 1im][BF4]31.19 4 * 1 >132126[74] c4 [69] Table 1. Solubility of different APIs in selected ILs and comparison98.8 cwith their water solubility.[C48C 1im][BF4] >132126 c [69] c [C C im][BFc ] 126 [C84C1im][BF46] 8 1 126 c 4 Acetaminophen API Structure Water Solubility IL Solubility[C 84CReference1im][PF 46] 52 c 98.8 c c [Cb4C1im][BF[C C4]im][BF ] >132 c 126[69] c Acetaminophen a [C C im][PFc ] 52 Acetaminophen [P98.8666(14)][NTf19.162] 0.0524[C * 48C1im][PF[72] 6] 4 1 52 c 6 [C8C1im][PF6] c 10 c Acetaminophen [C8C1im][BF[Ca 4C41]im][PF 6] 126 5210 4-Hydroxycoumarin - [C2C1im][CF3O3S] 0.1107 * c c b [C86C1im][PF6] [C8C1im][PF10 6b] 10 19.16[C4C1im][CF b 3O3S] 0.0907 [Ca * 6C1im][PF[73] 6] c 13.21c b [75] Acetaminophen 19.16 [C4C1im][PF[C8C61]im][PF 6] 52 13.2110 [75] b 19.16 b [C6C1im][PF6] [C C im][PF13.21 ab ] [75] 13.21 [75] b [C8C1[Cim][PF2C1im][CF6] 3O3S]10 6 c 1 0.1711 6ab * 5-Fluorouracil 12.21 b * 19.16[C4 C1 im]Br 31.19[C [Cb2 *C 61Cim][CF1im][PF[74] 3O6]3 S] 0.171113.21 * [75] a 19.16 b [C6C[C1im][PF2C1im][CF6] 3O3S]13.21 b 0.1711[75] a * a [C4C1im][CF3O3S] 0.1088 aa * 98.8 c [C4C1im][BF4] >132[C c2 C1im][CF[69] 3O[C3S]2 C1im][CF0.17113O*3 S] 0.1711 * [C2C1im][CF4 13O3S] 3 30.1711a * aa [C8C1im][BF4] 126[C c C4 im][CF1 O2S] 0.1088 a * a Acetylcysteine - [C4 4C1 1im][NTf3 32] 0.0866 a * [73] [Cc C im][CF O[CS]4 C1 aim][CF 0.10883O3* S] 0.1088 * AcetylcysteineAcetaminophen [C- 4C1im][PF6] [C4C1im][CF52 [C 4C3O1im][NTf3S] 20.1088] * 0.0866 a a* [73] [Cc 6C1im][NTf2] 0.0635 a a a AcetylcysteineAcetylcysteine [C- 8C1im][PF-6] 10 [C46C1im][NTf 2][C C a im][NTf0.08660.0635 * ] [73] 0.0866 * [73] Acetylcysteine - [C4C1im][NTf2] 0.08664 1* [73] a2 19.16 b [C6C1im][PF6] 13.21[C b 6C1im][NTf[75] 2] 0.0635 a [C10C1im][NTf2] a 0.0102 a a * a 6 1 [Ca 106 C112im][NTf22] 0.0102 * [C2C1im][CF3O3S] [C C0.1711im][NTf[C* C im][NTf] ]0.0635[C 6C1 im][NTf0.0635 2 ] 0.0635 [C10C1im][NTf2] 0.0102 ca * [C4C1im][CF3O3S] 0.1088 [Ca * 4C1im][BF4] a 1.49 c a a [C10C1im][NTf[C[C104CC11im][NTf2im][BF] 4]0.01022[C ] C* im][NTf0.01021.49 * ] 0.0102 * Acetylcysteine - [C4C1im][NTf2] 0.0866 a * [73] 10 1 c 2 [C4C1im][BF4] c 1.49 c [C4C1im][BF[C a 6C41]im][BF 4] 1.49 2.97 cc [C6C1im][NTf2] 0.0635[C 46C1im][BF4] 1.492.97 a c c [C10C1im][NTf2] 0.0102 * 6 1 4 [C Cc im][BF ] 1.49 [C6C1im][BF[C C4]im][BF ] 2.974 1 2.97 c c4 c 86 1 44 c [C4C1im][BF4] 1.49[C[C 8C1im]im][BF [BF4]] 2.977.2 c c [C cC im][BF c ] 2.97 [C6C1im][BF4] [C8C2.971im][C [BF8C14im]] [BF4] 7.26 1 7.2 4 [C[C84C11im]im] [BF[PF46]] 7.229 cc c [C8C1im] [BF4] 7.2 c[C 4C1im] [PF6] [C cC im]29 [BF ] 7.2 c c[C4C1im] [PF6] 298 1 c 4 Albendazole 0.0020c 4 1 6 [69] [69] AlbendazoleAlbendazoleAlbendazole 0.00200.0020 [C 4C10.0020im] [PF6] 29 c[C C im] [PF ] 29[69] c [69] c c c [C6C1im] [PF6] 53 cc Albendazole Albendazole 0.0020 0.0020 [C6C1im][C [PF46C16im]][69] [PF6] [C534 cC 1im]2953 [PF 6][69] 29 [C6C1im]c [PF6] 53 c Albendazole 0.0020 [C6C1im] [PF6] 53 c [69] c [C6C1im] [PF6] [C6C1im]53 [PF c 6] 53 [C8C1im] [PF6] >75 c c [C8C1im][C [PF8C16im]] [PF6] [C>758 Cc 1im]>75 [PF c 6] >75 [C8C1im] [PF6] >75 8 1 6 c [C2C1im][CH3COO] 85 b[C C im] [PF ] >75 [C8C1im] [PF6] >75 c b [C4NH3][CH3COO] 30 b [C2C1im][CH3COO] 85 [C2C1im][CH3COO] 85 b [C6NH3][CH3COO] [C30 2b C1im][CH3COO] 85 b b [C2C1im][CH3COO][C4NH b 3][CH853 COO] 30 [C8NH3][CH3COO][C 4NH320][CH b 3COO] 30 [C2C1im][CH3COO] 85 b [C4NH3][Oleate] [C<5 b4 NH3][CH3COO] 30 b b [C6NH[C[C3][CH24CNH1im][CH3COO]3][CH 33COO][C6NH30 b 3][CH85303 COO] 30 [C6NH3][Oleate] <5 b4 3 3 bb Amphotericin B 4 b [C6NH3][CH[76] 3COO] b 30 bb b Amphotericin B 2.0 × 10−4 b 2.0 10[C−8NH[C3][CH46NH3COO]3][CH 3COO][C8NH20 3][CH303COO] 20 [76] 6 3 3 bb × [C4NH[C3][Oleate]8NH3][CH 3COO]<5 b 3020 b b [C68NH3][CH3COO][C NH ][Oleate]3020 b 4 3 b <5 [C6NH[C3][Oleate]8NH3][CH 3COO]<5 b 20 b 8 3 [C b 4NH3][Oleate] <5 bb b [C NH ][Oleate] [C<5[C8NH4NH3][CH3][Oleate]3COO] 20<5[76] −4 b [C6NH3][Oleate] b <5 Amphotericin B 2.0 × 10 [C46NH3][Oleate] <5 b [C46NH3][Oleate] <5 b b [C8NH3][Oleate] b [76] <5 −4 b [C6NH3][Oleate] <5 [76] Amphotericin B 2.0 × 10−4 b [C6NH3][Oleate] <5 b [76] Amphotericin B 2.0 × 10−4 b [76] Amphotericin B 2.0 × 10−4 b [C8NH3][Oleate] <5 b

b [C8NH3][Oleate] <5 b [C8NH3][Oleate] <5 b [C8NH3][Oleate] <5 b

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 51 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 51

[C4C1im] [BF4] 18.9 c [69] Int. J. Mol. Sci. 2020, 21, 8298 [C4C1im] [BF4] 18.9 c [69] 9 of 50 HO [C8C1im] [BF4] >59 c HO [C8C1im] [BF4] >59 c [C4C1im] [PF6] 11.9 c [C4C1im] [PF6] 11.9 c H 8 1 6 c H H c [C C im] [PF ] 35 c Danazol 0.00030 c Table 1. Cont.[C8C1im] [PF6] 35 Danazol H 0.00030 d H [C6C6OCOpy][N(CN)2 >90 d [77] Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW H [C6C6OCOpy][N(CN)2 >90 [77] 9 of 51 API Structure Water Solubility IL Solubility Reference d [C6C6OCOpy][NTf2] 25 d N [C6C6OCOpy][NTf2] c 25 N O [C4C1im] [BF4] 18.9 [69] c O [C4C1im] [BF4] 18.9 [69] HO c Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW [C8C[C1im]4C 1[BFim]4] [NTf2] >599 of 0.03751 a * c [C4C1im] [NTf2][C 8C1im]0.037 [BF a4 *] >59 4 1 6 c [C c C im] [PF ] 11.9 c [C4C1im] [BF4] 18.9 [C10[69]C 1im] [NTf2[C] C im]0.072 [PF a ] 11.9 HOH 4 1 6a [C8C1im] [BF4] c>59 c 8 [C1 10C 1im]6 [NTf2] c 0.072 Danazol H c0.00030 [C C im] [PF ] 35 Danazol 0.00030 c c [C4C1im] [PF6] 11.9 [C C im] [PF ] 35 H 8 1 d 6 H [C8C1im] [PF6] [C635C c6 OCOpy][N(CN) 2 >90 [77] Danazol H 0.00030 c 666(14) a d [P ]Cl 0.085 a d H [C6C6OCOpy][N(CN)2 >90 [77][P 666(14)]Cl[C 6C6OCOpy][N(CN)0.085 2 >90 [77] d [C6C6OCOpy][NTf2] [C256C d 6OCOpy][NTf 2] 25 d N N O [C6C6OCOpy][NTf2] 25 O Erythromycin [C4C1im]- [NTf2] 0.037 a * [78] Erythromycin - a [78] a [C10C1im] [NTf2] 0.072[C a4 C1im] [NTf2] 0.037 * [N4,1,1,1][NTf2][C 4C1im]0.053 [NTf a2 ] 0.037 * 4,1,1,1 2 a a [N ][NTf ] a 0.053 a [P666(14)]Cl 0.085[C 10 C1im] [NTf2] [C100.072C1im] [NTf2] 0.072 Erythromycin - a [78] Erythromycin - [78] [P666(14)a ]Cl 0.085 a [P666(14)]Cl 0.085 [N4,1,1,1][NTf2] 0.053 [N ][NTf ] 0.053 a 4,1,1,1 2a [Pyrr4,1][NTf2] 0.017 a a [Pyrr4,1][NTf2] [Pyrr4,1][NTf0.017 2 ] 0.017 Erythromycin [Pyrr- 4,1][NTf2] 0.017 a [78] [N4,1,1,1][NTf2] 0.053 a

Etodolac Insoluble [C4C1im] [PF6] 374.33 b * [79] b b EtodolacEtodolac InsolubleInsoluble [C4C1im] [PF6] [C C im]374.33 [PF ]* [79] 374.33 * [79] Etodolac Insoluble [C4C1im] [PF6] 4 1 374.33 6b * [79] [C6C6OCOpy][N(CN)2] >125 d [Pyrr4,1][NTf2] 0.017 a

d Fenofibrate - [C6C6OCOpy][N(CN)[77] 2] >125 d d d [C6C6OCOpy][NTf2] >130[C 6C6OCOpy][N(CN)[C6C6OCOpy][N(CN)2] >125 2] >125 d Etodolac Insoluble [C4C1im] [PF6] [C6C374.336OCOpy][NTf b * [79]2] >130

Fenofibrate - [77] Fenofibrate - d [77] Fenofibrate - [C6C6OCOpy][N(CN)[C6C6OCOpy][NTf2] 2] >125 >130 d [77] [C6C6OCOpy][NTf2] >130 d

Fenofibrate - [77] [C6C6OCOpy][NTf2] >130 d

Int. J. Mol. Sci. 2020, 21, 8298 10 of 50 Int.Int. J.J. Mol.Mol. Sci.Sci. 2020,, 21,, xx FORFOR PEERPEER REVIEWREVIEW 10 of 51

Table 1. Cont.

Int. J. Mol. Sci. 2020, 21, APIx FOR PEER REVIEW Structure Water Solubility IL10 of 51 Solubility Reference

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 51 Glibenclamide 2.4 × 10−−66 bb * [Ch][Try] 9.89 bb * [80]

GlibenclamideGlibenclamide 2.4 × 10−6 b * 2.4 [Ch][Try]10 6 b * 9.89 b * [80] [Ch][Try] 9.89 b * [80] × −

Glibenclamide 2.4 × 10−6 b * [Ch][Try] 9.89 b * [80]

[C4C1im] [PF6] 6.95 b [75] b bb b Ibuprofen 0.124 [C6C1im] [PF6] [C[C44C26.3811im]im] [PF[PF66]] [C 4C1im]6.956.95 [PF 6 ] [75][75] 6.95 [75] a b [P666(14)][NTf2] [C66C0.052811im] [PF66] [72] 26.38 b b IbuprofenIbuprofenIbuprofen 0.124 0.124 [C C im] [PF ] [C6C1im]26.38 [PF 6 ] 26.38 [DDA][NO3] 0.0452 a * [81] a [P ][NTf aa] 0.0528 [72] [P666(14)666(14)][NTf a 22] 666(14)0.0528 2 [72] [C2][NTf2] [P 0.0235][NTf ] [82] 0.0528 [72] [C4C1im] [PF6] 6.95 b [75] Isoniazid - [C4C1im][NTf2] 0.004 c b a a [C6C1im] [PF6] [DDA][NO26.38 33] [DDA][NO 0.0452 a] * [81] 0.0452 *[81] Ibuprofen 0.124 [C6C1im][NTf2] [DDA][NO0.003 c ] 0.04523 * [81] a [P666(14)][NTf2] 0.0528 c [72] a a [P666(14)][NTf2] [C[C0.065122][NTf][NTf 22]] [72][C 2][NTf0.02350.02352] a [82][82] 0.0235 [82] b [C2C[DDA][NO1im][CH3COO]3] 0.0452<5 a * [76][81] c c - 44 11 c IsoniazidIsoniazidIsoniazid -- [C[C C im][NTf2]im][NTf2]b [C 4C1im][NTf2]0.0040.004 0.004 [C4NH[C32][CH][NTf3COO]2] 0.0235<5 a [82] c c NN 6 1 b c Isoniazid - [C[C6NH4C13im][NTf2]][CH3COO] [C[C6C10.004im][NTf2]im][NTf2]<5 c [C 6C1im][NTf2]0.0030.003 0.003 O b c N [C[C8NH6C13im][NTf2]][CH3COO] 0.003<5 cc c 1.0 × 10−6 b [P[P666(14)666(14)][NTf][NTf22]] [P ][NTf0.06510.0651 ] [72][72] 0.0651 [72] b 666(14) 2 [C[P4NH666(14)3][Oleate]][NTf2] 0.0651<5 c [72] bb [C22C11im][CH b 33COO] <5 [76] [C[C2C6NH1im][CH3][Oleate]3COO][C C im][CH<5 b COO][76] <5 [76] b b [C2C1im][CH3COO] <5 [76] [C[C4NH8NH3][CH3][Oleate]3COO] <5 b bb N [C[C44NH33][CH][CH33COO] <5 d b NN [C6C6OCOpy][N(CN)3 3 2] 40 b [77] NNNN [C NH ][CH COO] <5 [C4NH 3][CH3 COO]b <5 [C[C66NH33][CH][CH33COO] <5 b N O b N [C8NH3][CH3COO] <5 b 1.0 × 10−6 b Itraconazole OO b [C6NH3][CH3 COO] bb <5 NN [C4NH3][Oleate][C[C 88NH33][CH][CH<5 33COO] <5 1.0 × 10−−66 bb b 1.0 × 10 [C6NH3][Oleate] <5 b [C NH ][CH COO]b <5 [C[C44NH33][Oleate]][Oleate]8 3 <5<53 b 8 3 b O [C NH ][Oleate]6 b <5 b N 1.0 10− [C NH ][Oleate] bb <5 [C6C6OCOpy][N(CN)[C[C2] 66NH4033][Oleate]][Oleate] d [77] 4 3 <5<5 × d N - [C6C6OCOpy][NTf2] 8 b Itraconazole O N b [C[C88NH33][Oleate]][Oleate][C 6NH3][Oleate]<5<5 b <5 N Itraconazole N O NN dd b Cl [C[C66C66OCOpy][N(CN)[C822NH]] 3][Oleate]4040 [77][77] <5 d NN [C C OCOpy][N(CN) ] 40 [77] O 6 6 2 Itraconazole Cl Itraconazole d d N - [C6C6OCOpy][NTf-2] 8 [C6C 6OCOpy][NTf2] 8 Paclitaxel O <4.0 × 10−6 b [Ch][Gly] 22.34 b [83] N N O

Cl

OO

Cl dd NN -- [C[C66C66OCOpy][NTf22]] 88 −6 b b Paclitaxel OO <4.0 × 10 [Ch][Gly] 22.34 [83]

NN O NN O ClCl

ClCl Paclitaxel <4.0 × 10−−66 bb [Ch][Gly][Ch][Gly] 22.3422.34 bb [83][83]

Int. J. Mol. Sci. 2020, 21, 8298 11 of 50

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW Table 1. Cont. 11 of 51 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 51 Int. J. Mol. Sci. 2020, 21, x FOR PEERAPI REVIEW Structure Water Solubility IL Solubility11 of 51 Reference [Ch][Ala] 18.52 b O [Ch][Ala] 18.52 b b b b O [Ch][Ala][Ch][Pro] 18.52[Ch][Gly] 16.16 22.34 O [Ch][Pro] 16.16 b [Ch][Pro][Ch][Phe] 16.16 b 14.15 b b HN [Ch][Ala] b 18.52 HN [Ch][Phe][Ch][Phe] 14.15 b 14.15 b HN [Ch][Ile] [Ch][Pro]9.39 16.16 b b b [Ch][Ile][Ch][Ile] 9.39 9.39 b OH [Ch][Ser] 7.32 b [83] O [Ch][Phe] b b 14.15 O OHOH [Ch][Ser][Ch][Ser] 7.32 7.32 O 6 b b Paclitaxel O O <4.0 10− [Ch][Ile] 9.39 O × OO b O [Ch][Ser] 7.32 OO [Ch][Leu] 6.61 b O O O OH b b OH OO [Ch][Leu][Ch][Leu] 6.61 6.61 OH b HOHO O [Ch][Leu] 6.61 HO H OO H O O H O OO O H O H O H O [C2C1im] [NTf2] 0.0048 a [84] a [C2C1im] [NTf2[C] C im]0.0048 [NTf a ][84] 0.0048 [84] 2 1 a2 [C4C1[Cim]2C [NTf1im]2 ][NTf 2] 0.0054 a*0.0048 [84] a 4 1 2 a O [C C im] [NTf[C] 4C1im]0.0054 [NTf2* ] 0.0054 * [C6C1im]4 [NTf1 2] 2 0.0050 a * a O [C C im] [NTf ] 0.0054 a * a N [C6C1im] [NTf2[C] C im]0.0050 [NTf* ] 0.0050 * O [C8C1im] [NTf2] 60.00521 a a 2 NH2 [C6C1im] [NTf2] 0.0050 * Pyrazinecarboxamide N - a a [C8C1im] [NTf2] a0.0052 N NH2 [C10C1im] [NTf2] [C80.0046C1im] [NTf2] 0.0052 PyrazinecarboxamidePyrazinecarboxamide - - [C8C1im] [NTf2] 0.0052 a N O NH2 a a Pyrazinecarboxamide H - [C10C1im] [NTf2] a0.0046 [C10C1im][CF3O3S] [C100.0116C1im] [NTf a2] 0.0046 N O [C10C1im] [NTf2] 0.0046 a a a NH O [C[C2][NTf10C1im][CF2] 3[CO3S]10 0.0165C1im][CF 0.01163O[81]3 S] 0.0116 H [C10C1im][CF3O3S] 0.0116 a [P666(14)][NTf[C2][NTf2] 2] 0.0125[C ][NTf a 0.0165] a[72] [81] 0.0165 a [81] [C2][NTf2] 2 0.01652 a [81] [P666(14)][NTf2] [P ][NTf0.0125 a] [72] 0.0125 a [72] [P666(14)][NTf2] 666(14)0.01252 a [72] Thymoquinone - [P666(14)][NTf2] 0.1105 a [72]

Thymoquinone - [P666(14)][NTf2] 0.1105 a [72] a a ThymoquinoneThymoquinone - -[P666(14)][NTf2] [P666(14)][NTf0.11052] [72] 0.1105 [72] a: molar fraction; b: mg mL−1; c: mmol L−1; d: mg g−1. * Solubility determined at body’s temperature (34–38 °C). If not specified, the reported solubilities were assessed

at a temperature range −from1 21 to 30 −°C.1 Relevant−1 solubilities determined at >38 °C are further addressed in this chapter. a: molar fraction; b: mg mL−1; c: mmol L1 −1; d: mg g−11. * Solubility1 determined at body’s temperature (34–38 °C). If not specified, the reported solubilities were assessed a: molar fraction;a: molar b: mg fraction; mL ; b: c: mgmmol mL− L; c:; d: mmol mg g L− .; * d: Solubility mg g− . * determined Solubility determined at body’s at temperature body’s temperature (34–38 (34–38 °C). If◦C). not If specified, not specified, the the reported reported solubilities solubilities werewere assessed assessed at a temperature at a temperature range from 21 to 30 °C. Relevant solubilities determined at >38 °C are further addressed in this chapter. at a temperaturerange range from 21from to 30 21◦ C.to Relevant30 °C. Relevant solubilities solubilities determined determined at >38 ◦C are at further >38 °C addressed are further inthis addressed chapter. in this chapter.

Int. J. Mol. Sci. 2020, 21, 8298 12 of 50

The application of pure ILs for APIs solubilization was first reported in 2008 by Jaitely et al. [85], who investigated the ILs [C4 8C1im][PF6] on the solubilization of potassium penicillin V,dexamethasone − dehydroepiandrosterone, and progesterone. Although these ILs are immiscible in water, it is possible to use these formulations to enhance the release of some solutes into an aqueous medium. The studied ILs were submitted to a current flow (over the range 1–5 mA) allowing to increase the release rate of the APIs from the IL to the aqueous medium. The release of these APIs slowly increased with the increase of the alkyl chain length of the IL cation (up to three-fold). The partition of APIs in the studied ILs/water systems was further correlated with the APIs octanol-water partition coefficient. To infer the safety of these solvents for pharmaceutical applications, their cytotoxicity was evaluated in Caco-2 cell lines, which suggests that, with the exception of 1-octyl-3-methylimidazolium hexafluorophosphate ([C8C1im][PF6]), these compounds are non-toxic (90% cell viability) at the conditions and concentrations studied, and might be considered as excipients in pharmaceutical formulations. The same trend was observed by Mizuuchi et al. [69], who studied similar ILs in order to solubilize albendazole, danazol, acetaminophen, and caffeine. The increase in the hydrophobicity of + the imidazolium cation ([C4 8C1im] ) resulted in a higher solvation ability for hydrophobic drugs. − However, this trend resulted in a decrease in the solubility of hydrophilic drugs. The authors demonstrated that it is possible to increase albendazole’s solubility more than 37,000-fold while using [C8C1im][PF6] as solvent. In a different study by Forte et al. [82], the variation of the anion in + 1-decyl-3-methylimidazolium-based ILs and the alkyl chain length of the cation ([C2-10C1im] ) were studied to infer their effect on isoniazid’s (antibiotic) solubility. The results showed that the presence + of an acidic proton at the 2-position of [C2C1im] increases the ILs ability to hydrogen-bond with isoniazid, leading to higher solubility values. Among the ILs studied, 1-decyl-3-methylimidazolium trifluoromethanesulfonate ([C10C1im][CF3O3S]) was found to be the best solvent for isoniazid (at T > 38 ◦C). Furthermore, the increase of the alkyl chain length at the imidazolium cation decreases the acidity of the proton at the 2-position, thus increasing the API’s solubility in the IL. Overall, the trends that were obtained in the described studies demonstrate that the influence of the cation’s alkyl chain length differs according to the IL and the APIs nature, and accordingly with the molecular-level mechanisms involved. These differences make difficult the establishment of heuristic rules and development of predictive models that could be used in a widespread manner. Although interactions between the IL anion and the API have been reported as relevant, mainly via hydrogen-bonding, the influence of the IL anion in which the IL hydrogen-bond basicity can be strongly tuned is less studied and still not completely understood. Although promising results have been reported in terms of APIs solubility, comparisons between the results obtained in IL media with the ones that were obtained with other common solvents, such as water or ethanol, are shortly explored along with the bioavailability profiles. These assays should be considered in order to support the advantageous use of IL-based solvents alternatives. In addition to the study of pure ILs as solvents for APIs, the application of ILs as co-solvents has been investigated. For instance, cholinium-based ILs have been successfully applied as co-solvents in paclitaxel formulations for chemotherapeutic treatment [83]. Paclitaxel is a low-water soluble 1 API (<4 µg mL− ) that is used in the treatment of different types of cancer; however, its parenteral formulation requires the use of ethoxylated castor oil (CrEL) and ethanol as solubilizing agents. The use of these agents can be associated with major hypersensitivity reactions and side effects. By using cholinium-amino-acid-based ILs as co-solvents, it was possible to remarkably enhance the APIs solubility in aqueous media (>5500-fold) and decrease the formulation toxicity and hypersensitivity, while maintaining the antitumor therapeutic action of the API. In a different study, also using amino-acid-based ILs, and in particular cholinium tryptophan, it increased the glibenclamide’s (an antidiabetic drug) solubility in aqueous solutions with 6.5 wt% of IL from 400 to 2000-fold [80]. The establishment of hydrogen bonds and π–π interactions between the API and the IL anion were described as playing a major role in the obtained solubility improvements in both studies. Int. J. Mol. Sci. 2020, 21, 8298 13 of 50

In a more fundamental study, tetrabutylammonium-, phosphonium-, imidazolium-, pyridinium-, piperidinium- and pyrrolidinium, and cholinium-based ILs were investigated in aqueous solution regarding their ability to act as hydrotropes and improve the solubility of ibuprofen [86]. It was found that the IL cation and anion synergistically contribute to the hydrotropic mechanism of solubilization. Among the cations that were investigated in a chloride-based IL series, imidazolium- and phosphonium-based ILs lead to a higher increase in the drug solubility. In order to evaluate the influence of the IL anion on ibuprofen’s solubility, [C4C1im]- and sodium-based hydrotropes were investigated, where a higher hydrotropic activity was disclosed with 1-butyl-3-methylimidazolium thiocyanate ([C4C1im][SCN]) and 1-butyl-3-methylimidazolium dicyanamide ([C4C1im][N(CN)2]). These ILs increase the aqueous solubility of ibuprofen by 60- and 120-fold, respectively, in comparison to the API in pure water. The formation of IL-drug aggregates was proved based on dynamic light scattering and molecular dynamics simulations. In particular, ILs have been reported as powerful catanionic hydrotropes [23,86]. The use of surfactants above the critical micelle concentration (CMC) can be also an appealing alternative to increase API’s solubility. By varying the cation type and alkyl chain length and the nature and size of the counterion it is possible to change the ILs’ hydrophilic-hydrophobic balance. ILs with surfactant behavior are usually referred to as surface-active ionic liquids (SAILs), displaying high potential to increase the solubility of pharmaceutical agents in aqueous media. Sanan et al. [87] studied 1-dodecyl-3-methylimidazolium chloride ([C12C1im]Cl)-ibuprofen mixtures in aqueous solution, ranging from monomeric to micellar regions. Aggregate assemblies in aqueous media (mixed micelles) were observed, depending on the mixture composition. The formation of these complexes was mainly attributed to the establishment of hydrophobic, electrostatic and hydrogen-bonding interactions. Faria et al. [88] investigated surface-active ILs, both cationic and anionic, as well as composed of different cations and anions, for the solubilization of the nutraceutic ursolic acid in aqueous media. For this, different ILs constituted by long alkyl side chains with known surface-active characteristics ([C8-18C1im]X with X = Cl and [C8H17SO4]) and tributyltetradecylphosphonium chloride ([P444(14)]Cl) were considered. The use of the best SAIL aqueous solutions allows for solubility enhancements of ursolic acid in 8 orders of magnitude when compared to pure water. More recently, aqueous solutions of [C12C1im]Cl were used to increase the solubility of the nutraceutical oleanolic acid [89]. An increase 1 in the IL concentration up to 1000 mM improved the solubility of oleanolic acid to 21.10 mg mL− , indicating that aqueous solutions of SAILs leads to a remarkable increase (up to 106-fold) on the solubility of the target compound in water. Although significant results have been disclosed on the use of ILs as solvents, co-solvents, or surfactants to improve the APIs solubility, most studies reported so far focus on imidazolium-based ILs. This trend is probably associated with the fact that these ILs are commercially available, and well studied and characterized in literature. Although few other IL combinations were investigated, the results reported hitherto on drug solubility enhancements promote the evolution of ILs further from their solvent applications to the study of novel drug delivery approaches, where stability, absorption, and bioavailability can be improved. Furthermore, special care must be taken for the mutual administration of ILs and APIs, since these can also induce multixenobiotic/multidrug cell resistance and/or reduce the effectiveness of therapies, an issue that is usually not addressed in the reported studies. More biocompatible combinations are expected to be studied and more complete studies are still required to boost IL research and enable their use in pharmaceutical formulations.

4. ILs in APIs Crystallization The properties of crystalline APIs, such as their solubility, structural stability, and dissolution rates, are mainly dependent on the respective polymorphs [8,90]. Thus, designing the correct polymorph represents a critical requirement in drug development and production as it impacts the APIs bioavailability and shelf life. Crystallization from organic or aqueous-based reaction media is often a key step in pharmaceuticals isolation and purification [91]. APIs polymorphs can be originated Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 51

Several studies have reported the application of ILs as adjuvants in APIs crystallization [94–96], allowing for not only the design of new polymorphic forms, but also to manipulate the crystal form and habit to present enhanced properties, and ultimately, to separate and isolate specific polymorphic forms that are not achievable with conventional solvents (Figure 8). IL-based crystallization techniques, which include solvent-antisolvent [97], cooling crystallization [98], or drowning-out [92] techniques, were proposed in order to promote the correct habit and polymorphic forms of several drugs. The use of ILs has shown the possibility to design new APIs’ polymorphs with enhanced thermal stability [92]. The IL 1-allyl-3-ethylimidazolium tetrafluoroborate ([(CH2CH=C2)C2im][BF4]) has been applied to design polymorphs of adefovir dipivoxil, through drowning-out crystallization. This process can be considered to be one of the most important techniques to be applied when the separation of solutes from multicomponent solutions is envisaged [99]. This method relies on the supersaturation of the solution by adding specific substances, drowning-out agents to the initial solution in order to reduce the solubility of the solute. The use of the IL in the crystallization process allowed for obtaining the form-II of the API, and upon an increase of the crystallization temperature two new polymorphic structures and hydrated crystals form. A significant increase in the thermostability in aqueous solutions was verified when using the IL. By the application of the IL [(CH2CH=C2)C2m][BF4] as solvent and 1-butyl-2,3- dimethylimidazolium tetrafluoroborate ([(C4C1C1m][BF4]) as antisolvent, a new form of adefovir dipivoxil crystal was obtained at a crystallization temperature below 50 °C [97]. This new polymorphicInt. J. Mol. Sci. 2020 form, 21, was 8298 achieved due to unique intermolecular interactions between API molecules14 of 50 that were promoted by the IL, resulting in different molecular packing during crystallization. However, when considering the use of the ILs [(CH2CH=C2)C2m][BF4], 1,3-diallylimidazolium from the variety of intermolecular interactions between the molecules, which are dependent on the tetrafluoroborate ([(CH2CH=C2)2im][BF4]) and 1-ethyl-3-methylimidazolium ethylsulfate crystallization conditions (solvent, temperature, additives, and supersaturation) [92]. It is possible ([C2C1im][EtSO4]), the conventional form-I polymorph was obtained. The possibility to use high to fine-tune the polymorphic form by adjusting the crystallization conditions, introducing guest crystallization temperatures enabled the formation of a stable polymorph, with enhanced solubility molecules or promoting a preferred crystal nucleation while using additives [93]. Several studies and thermal stability over 100 °C (two-fold increase in the decomposition temperature). Using a have reported the application of ILs as adjuvants in APIs crystallization [94–96], allowing for not only similar approach, rifampicin nanosized particles were prepared to improve the solubility and the design of new polymorphic forms, but also to manipulate the crystal form and habit to present dissolution kinetics of poorly-water soluble APIs [100]. To this purpose, the use of the IL 1-ethyl-3- enhanced properties, and ultimately, to separate and isolate specific polymorphic forms that are not methylimidazolium methylphosphonate ([C2C1im][CH3OHPO2]) as alternative solvent and achievable with conventional solvents (Figure8). IL-based crystallization techniques, which include phosphate buffer as an antisolvent was considered, allowing for obtaining API crystals with <1 μm. solvent-antisolvent [97], cooling crystallization [98], or drowning-out [92] techniques, were proposed Reducing the particle size down to the submicron range allowed not only a faster dissolution ability, in order to promote the correct habit and polymorphic forms of several drugs. but also to increase the APIs’ solubility by 30%.

Figure 8. Applications ofof ILsILs inin APIs’APIs’ crystallization crystallization processes processes (adequate (adequate references references are are given given along along the thecurrent current section). section).

The use of ILs has shown the possibility to design new APIs’ polymorphs with enhanced thermal stability [92]. The IL 1-allyl-3-ethylimidazolium tetrafluoroborate ([(CH2CH=C2)C2im][BF4]) has been applied to design polymorphs of adefovir dipivoxil, through drowning-out crystallization. This process can be considered to be one of the most important techniques to be applied when the separation of solutes from multicomponent solutions is envisaged [99]. This method relies on the supersaturation of the solution by adding specific substances, drowning-out agents to the initial solution in order to reduce the solubility of the solute. The use of the IL in the crystallization process allowed for obtaining the form-II of the API, and upon an increase of the crystallization temperature two new polymorphic structures and hydrated crystals form. A significant increase in the thermostability in aqueous solutions was verified when using the IL. By the application of the IL [(CH2CH=C2)C2m][BF4] as solvent and 1-butyl-2,3 -dimethylimidazolium tetrafluoroborate ([(C4C1C1m][BF4]) as antisolvent, a new form of adefovir dipivoxil crystal was obtained at a crystallization temperature below 50 ◦C[97]. This new polymorphic form was achieved due to unique intermolecular interactions between API molecules that were promoted by the IL, resulting in different molecular packing during crystallization. However, when considering the use of the ILs [(CH2CH=C2)C2m][BF4], 1,3-diallylimidazolium tetrafluoroborate ([(CH2CH=C2)2im][BF4]) and 1-ethyl-3-methylimidazolium ethylsulfate ([C2C1im][EtSO4]), the conventional form-I polymorph was obtained. The possibility to use high crystallization temperatures enabled the formation of a stable polymorph, with enhanced solubility and thermal stability over 100 ◦C (two-fold increase in the decomposition temperature). Using a similar approach, rifampicin Int. J. Mol. Sci. 2020, 21, 8298 15 of 50 nanosized particles were prepared to improve the solubility and dissolution kinetics of poorly-water soluble APIs [100]. To this purpose, the use of the IL 1-ethyl-3-methylimidazolium methylphosphonate ([C2C1im][CH3OHPO2]) as alternative solvent and phosphate buffer as an antisolvent was considered, allowing for obtaining API crystals with <1 µm. Reducing the particle size down to the submicron range allowed not only a faster dissolution ability, but also to increase the APIs’ solubility by 30%. Imidazolium-based ILs have been studied to control the crystallization of gabapentin, a neuroleptic drug used to treat epilepsy [101]. This API presents three polymorphic forms (forms II, III, and IV) and a hydrated form (form I) [102]. The commercialized polymorph is the form II due to its highest thermodynamic stability [103]. However, forms III and IV are commonly obtained through crystallization in ethanol at room or high temperature, where the form IV cannot be completely isolated [102]. Distinct IL cation/anion combinations were studied in order to access their ability in directing the crystallization process towards the isolation and stabilization of less stable polymorphs [101]. Using 1-hexyl-3-methylimidazolium tetrafluoroborate ([C6C1im][BF4]), the API form IV was isolated, which is a highly unstable polymorph. ILs have been investigated as well for the isolation of specific polymorphic forms of paracetamol [94]. Imidazolium-based ILs were studied regarding their impact on the API’s solubility, mainly by tailoring their hydrogen-bond ability with the API. Because the solubility of the API was shown to be governed by the basicity of the IL anion, 1-ethyl-3-methylimidazolium acetate ([C2C1im][CH3COO]) was then selected for the crystallization process. The strong interactions between the IL and the studied antisolvents (ethanol, acetic acid, and 1,1,1,3,3,3-hexafluoroisopropanol) allowed for engineering the crystallization in the form of polymorph I (the most stable) and manipulating the system’s interactions to obtain crystallization yields greater than 88% at room temperature. Attempting to control the crystallization of paracetamol, Smith et al. [104] studied the ILs [C4C1im][PF6] and 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6C1im][PF6]). The selected ILs, the respective concentration, and the method of crystal growth considered (cooling crystallization) have shown impact on the crystal habit and size. When [C6C1im][PF6] was used at the lowest 1 concentration (16 mg mL− ), tetragonal bypyramids were formed; by increasing the concentration 1 of the IL, it was possible to move from plate particles to more tubular structures (30–69 mg mL− ) as a consequence of growth post spontaneous nucleation. At higher concentrations, 69 mg mL 1, ≈ − particles with 23 to 206 µm were obtained. To avoid the use of an antisolvent, Weber et al. [98] proposed the cooling crystallization in IL media, with 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C2C1im][NTf2]), also acting as a purification methodology for twelve APIs with anti-inflammatory, antifungal and antipyretic properties. From those, ten of the APIs proved to be highly soluble in the IL at their melting temperature. Among the studied systems, similar or higher purities with improved yields to those that were obtained with antisolvent crystallization were achieved, enabling the possibility to use this method for APIs purification. Despite the promising results reported so far and the growing interest on the use of ILs to tailor the APIs polymorphs, there is still a gap in a systematic and comprehensive research on this topic, particularly to better understand the IL cation and anion effects. The use of computational methods can be an advantageous alternative to understanding the IL-API interactions driving the formation of specific polymorphic forms and habits, as it is already performed for other conventional solvents [105]. Furthermore, the development of effective separation methods and research on techniques to avoid the IL contamination in the final product are highly demanding.

5. ILs with Biological Activity Because of the myriad of anion-cation combinations in ILs, these solvents may display specific biological activities [106]. Although the application of ILs in this area is emerging, promising results were already disclosed. Figure9 provides an overview on the biological activities of ILs, namely Int. J. Mol. Sci. 2020, 21, 8298 16 of 50 antioxidant, anti-tumoral, and antimicrobial activity, covering some of the main types of cation-based ILsInt. studied J. Mol. Sci. so 2020 far., 21, x FOR PEER REVIEW 17 of 51

FigureFigure 9. 9.Biological Biological activities activities reported reported for for ILs ILs and and the the respective respective studied studied cations cations (adequate (adequate references references areare given given along along the the current current section). section).

Imidazolium-Imidazolium- and and pyridinium-based pyridinium-based ILs ILs have have been been largely largely investigated investigated in in what what regards regards their their antibacterialantibacterial activity activity [107 [107–110].–110]. Although Although some some exceptions exceptions may may appear appear and and have have been been reported, reported, in in general,general, an an increase increase in in the the alkyl alkyl chain chain length length of of the the IL IL cation cation (1-alkyl-3-methylimidazolium (1-alkyl-3-methylimidazolium based based ILs ILs ++ + + ([C([C4-84-8CC1im]1im])) and and 1-alkyl-N-methylpyrrolidinium 1-alkyl-N-methylpyrrolidinium based based ILs ILs ([C ([C4-84-8CC1pyr]1pyr])))) leads leads to to higher higher antibacterial antibacterial activity.activity. Furthermore, Furthermore, ILs ILs cations cations with with longer longer aliphatic aliphatic chains chains and and more more alkyl alkyl group group substituents substituents on on thethe cation cation ring ring exhibit exhibit higher higher antibacterial antibacterial activity activity against against Gram-positive Gram-positive bacteria bacteria (e.g.,: (e.g:Staphylococcus Staphylococcus aureusaureus, Bacillus, Bacillus subtilis subtilis) and) and Gram-negative Gram-negative bacteria bacteria (e.g.,; (e.g.,;Escherichia Escherichia coli coli, Pseudomonas, Pseudomonas fluorescens fluorescens) and) and SaccharomycesSaccharomyces cerevisiae cerevisiae[110 [110].]. From From these, these,B. subtilis B. subtilisdemonstrated demonstrated the higher the higher susceptibility susceptibility to the tested to the ILs.tested Later ILs. studies Later studies with ILs with with ILs longer with longer alkyl chain alkyl lengthschain lengths (1-alkyl-3-methylimidazolium (1-alkyl-3-methylimidazolium based based ILs + + + + ([CILs8-14 ([CC1im]8-14C1)im] and) 1-alkyl-N-methylpyrrolidiniumand 1-alkyl-N-methylpyrrolidinium based ILs based [C8-14 ILsC1pyr] [C8-14))C provided1pyr] )) provided further insights further oninsights the antimicrobial on the antimicrobial activity of activity ILs [111 of]. Overall,ILs [111]. the Overall, effect ofthe the effect tested of the ILs tested against ILs Gram-positive against Gram- microorganismspositive microorganisms was similar was or similar even higher or even than higher that than displayed that displayed by a common by a common antimicrobial antimicrobial agent, cetyltrimethylammoniumagent, cetyltrimethylammonium chloride. chlo Basedride. on Based the exposed, on the theexposed, ILs antibacterial the ILs antibacterial efficiency canefficiency be tuned can bybe varying tuned bothby varying the alkyl both chain the length alkyl andchain modifying length and the modifying head group the at thehead cation. group The at increasethe cation. in theThe susceptibilityincrease in the of thesesusceptibility pathogens of canthese be pathogens assigned to can the be ability assigned of ILs to to the interact ability or of disturb ILs to biological interact or membranes,disturb biological which membranes, leads to cell deathwhich [leads112]. Into allcell of death these [112]. cases, In varying all of these the anioncases, varying identity the did anion not revealidentity a significant did not reveal effect a on significant the ILs’ antimicrobial effect on the activity.ILs’ antimicrobial activity. DoriaDoria et et al. al. [113 [113]] synthesized synthesized a a series series of ofN N-cinnamylimidazolium-cinnamylimidazolium salts salts with with di differentfferent alkyl alkyl chain chain lenghtslenghts (1, (1, 6, 6, 8, 8, and and 10 10 carbons), carbons), and and evaluated evaluated their their antibacterial antibacterial activity activity against against skin skin and and soft soft tissue tissue infections.infections. These These ILs ILs were were synthesized synthesized while while employing employing microwave microwave radiation radiation under under solvent-free solvent-free conditions,conditions, attempting attempting to to minimize minimize the the environmental environmental concerns concerns that that were were related related to to conventional conventional IL IL synthesis.synthesis. Based Based on on antimicrobial antimicrobial activityactivity studies,studies, itit waswas possiblepossible to verify that as as the the alkyl alkyl chain chain is isincreased, increased, the the antibacterial activityactivity increasedincreased withwith aa dose-dependent dose-dependent e ffeffect.ect. Molecular Molecular dynamics dynamics simulationsimulation studies studies allowed allowed for explainingfor explaining this antimicrobialthis antimicrobial behavior, behavior, as the result as the of theresult ILs’ of insertion the ILs’ ininsertion the lipidic in double the lipidic layer, double facilitating layer, the facilitating subsequent the di subsequentffusion to the diffusion intracellular to the space. intracellular While higher space. aliphaticWhile higher chain lenght aliphatic ILs extendchain tolenght the interior ILs extend of the membrane,to the interior the lackof the of hydrophobicity membrane, the of lowerlack of alkylhydrophobicity chain lenght of ILs lower reduces alkyl this chain phenomenon lenght ILs [reduces113]. this phenomenon [113]. More recently, predictive models for the antimicrobial and antifungal activities of ILs started to be developed, aiming at a rational design of ILs to be included in pharmaceutical applications. Cho et al. [114] reported six quantitative structure-activity relationship (QSAR) models, which were developed while using linear free energy relationship (LFER) descriptors calculated by density functional theory and a conductor screening model, to predict the minimal inhibitory concentration (MIC) and minimal biocidal concentration (MBC) of ILs against E. coli and S. aureus. Later, QSAR and

Int. J. Mol. Sci. 2020, 21, 8298 17 of 50

More recently, predictive models for the antimicrobial and antifungal activities of ILs started to be developed, aiming at a rational design of ILs to be included in pharmaceutical applications. Cho et al. [114] reported six quantitative structure-activity relationship (QSAR) models, which were developed while using linear free energy relationship (LFER) descriptors calculated by density functional theory and a conductor screening model, to predict the minimal inhibitory concentration (MIC) and minimal biocidal concentration (MBC) of ILs against E. coli and S. aureus. Later, QSAR and molecular docking were applied in order to address the antibacterial activity of 131 imidazolium-based ILs against S. aureus ATCC 25923 and its clinical isolate [115]. The developed models presented robustness, predictive power and reliability, with 80–82% of accuracy. The obtained results reveal that ILs with C12 alkyl chains or with two identical C8 and C9 alkyl chains seem to have higher activity against the pathogen, and they can be foreseen to design novel strategies against this microorganism. Attempting to investigate IL candidates as antibacterial agents, Zheng et al. [116] studied a series of ILs by molecular dynamics. The cations of ILs were found to insert into the lipid bilayer spontaneously, regardless of the cation types. Furthermore, imidazolium-based ILs with different alkyl chain lengths not always keep the preferential orientation, presenting the alkyl side chain of the cations close to the tail groups of the bilayer and the imidazolium ring close to the head groups of the lipid bilayer. This spontaneous insertion and reorientation inside the lipidic bilayers might be the cause of disorder and disruption of membranes and, thus, influence antibacterial activity [116]. In addition to antibacterial properties, ILs were disclosed as presenting antiviral activity. A systematic analysis investigated the effects of defined structural elements of 55 ILs (by changing the cation core, anion, and the length of the cation alkyl side chains) on virus activity, namely on the human norovirus surrogate phage MS2 and phage P100, representing non-enveloped DNA viruses [117]. Imidazolium-based ILs ([C1-10C1im]Cl) did not show particular effectiveness against the phages, except for the IL with a higher alkyl chain cation that exhibited a reduction in the phages number. The antiviral activity shown to be IL concentration-dependent. Because the phages used in the previous study are nonenveloped, the observed inactivation by long alkyl chain ILs could be mainly attributed to protein denaturation (as the capsid of the phages consists of proteins), rather than membrane disturbance typically caused by surfactant-like behavior. However, the effect of the IL anion on the antiviral activity remains unclear. Despite their antibacterial and antiviral properties, some ILs can present antifungal activity, even 1 at low concentrations (0.28 µg mL− ). Bergamo et al. [118] reported an in vitro antifungal activity of 1-hexadecyl-3-methylimidazolium chloride ([C16C1im]Cl) against multidrug-resistant Candida tropicalis isolates, whereas other authors [107] have shown the potential of [C6-14C1m]Cl ILs to control planktonic bacteria and biofilm formation. ILs with tetraalkylammonium and pyridinium cations were combined with anions that were derived from artificial sweeteners (saccharinate and acesulfamate) attempting to pair the biological activity inherent in the cation with the anion’s biological function [119]. These ILs were tested for their antifungal activity against C. albicans. However, these ILs present equal or decreased antifungal activity towards the microorganism than the starting compounds. Previous findings have shown that the IL effect on fungal metabolism is more intricated than the attribution of their activity to the ILs’ toxicity [120]. In this regard, Suchodolski et al. [121] synthesized novel menthol-based ammonium ILs ([C10-12-Am-Men]Cl) attempting to understand the mechanisms underlying their antifungal activity on C. albicans. As it happens for the previous mentioned works, the antimicrobial activity increased with the increase of the alkyl chain length of the cation, being the most effective ILs the ones that present more than 11 carbon atoms. When used at 50 µM, these ILs cause partial decomposition of the cell wall and promote the detachment of fungal cells. These novel ILs can be considered as disinfectants due to their antifungal activity and low hemolytic activity. Antioxidant properties have gained interest in the pharmacological context due to the possibility to reduce the free radicals’ concentration at skin, and thereby preventing and repairing damages caused by oxidative stress. Some phenolic compounds present high antioxidant and anti-inflammatory activities; however, their limited aqueous solubility represents a disadvantage towards their incorporation in Int. J. Mol. Sci. 2020, 21, 8298 18 of 50 water-rich pharmaceutical formulations and skin care products. To overcome these drawbacks, phenolic acids have been considered as IL anion sources to increase their solubility and antioxidant activity. However, it should be remarked that due to their action and when considered for therapeutic purposes, these may be also considered as new active principle ingredients in the form of ILs (API-ILs), which are discussed below. Overall, ILs have proven to possess high antioxidant activities, as summarized in Table2. Sintra et al. [ 122] synthetized five cholinium-based ILs using gallate, caffeate, vanillate, syringate, and ellagate anions. The resulting ILs demonstrated a solubility in water 3 orders of ≈ magnitude higher than the corresponding phenolic acids. These ILs presented not only similar, but even higher antioxidant activities, as well as comparable cytotoxicity and lower ecotoxicity profiles than their acidic precursors, being the most promising results obtained with the IL dicholinium ellagate. In another study, hydroxyl functionalized ammonium dicationic ILs containing natural derived ions and ether linkage between cationic head groups were synthesized and described as a novel form of antioxidants, where protocatechuic acid (also known as 3,4-dihydroxybenzoic acid), a natural compound with not only antioxidant properties but also chelation ability, was considered as anion [123]. All of the in vitro studies indicated that the antioxidant activity of dicationic ILs was significantly higher than that of free acids or commonly used antioxidants. More recently, attempting to understand the effect of the cation structure towards the antioxidant activity, Ahmad et al. [124] evaluated five ferulate-based ILs with ammonium cations comprising different alkyl chain lengths. The prediction of their antioxidant activity based on the σ-potential of the COSMO-RS model was in agreement with the experimental DPPH free radical scavenging results, revealing that tertiary alkanolamine-based ILs have higher antioxidant activities than secondary alkanolamine-based ILs. All of the synthesized ILs showed higher antioxidant activities than the ferulic acid precursor, even at low concentrations (up to 12.93 µM). In a different approach, novel ILs derived from natural sources were designed to enhance other biological properties in aqueous solutions, such as analogues of glycine-betaine (AGB-ILs) [125]. AGB-ILs, namely triethyl [2 -ethoxy-2-oxoethyl]ammonium bromide ([(C2)3NC2]Br), have recently been studied regarding their potential to enhance anti-inflammatory and antioxidant activities. These ILs allowed for increasing the antioxidant/anti-inflammatory activities of nutraceutical extracts, being possible to use them in nutraceutical formulations.

Table 2. Antioxidant activity of ILs and comparison with reference compounds.

DPPH Free DPPH Free Reference IL Radical Radical Reference Compound Scavenging (µM) Scavenging (µM) 2-(methylamino)ethanol ferulate 17.40 2-(propylamino)ethanol ferulate 16.61 2-(butylamino)ethanol ferulate 16.34 Ferulic acid 21.40 [124] 3-dimethylamino-1-propanol ferulate 12.93 3-diethylamino-1-propanol ferulate 14.09 Bis(ammonium) protocatechuate 5.06–5.98 Protocatechuic acid 15.83 [123] Cholinium caffeate 2.55 Caffeic acid 1.99 [122] Cholinium syringate 2.44 Syringic acid 2.04 [122] Cholinium vanillate 16.03 Vanillic acid 80.46 [122] Dicholinium ellagate 1.22 Ellagic acid 0.79 [122]

ILs have been largely tested in different cell lines in order to assess their antitumoral activity. From these, cancer cells lines have provided relevant results towards the understanding of the ILs potential as anticancer agents [126]. Phosphonium-, tetralkylammonium-, and, later, imidazolium-based ILs have been tested in vitro in 60 human tumor cell lines [126,127]. Phosphonium-based ILs were found to have higher anti-tumor activity than ammonium-based ones. Imidazolium-based ILs showed particularly high activity against leukemia cell lines, whereas an increase in the alkyl chain length leads to significant Int. J. Mol. Sci. 2020, 21, 8298 19 of 50 improvements in antitumor activity. Thus, multiple possibilities of IL cation-anion combinations can rule the resulting biological activity and associated cytotoxicity, playing a major role in therapeutic applications. The apoptotic mechanism that was caused by alkyl-methylimidazolium-based ILs was later unveiled using rat pheochromocytoma (PC12) cells [128]. As the analysis of the biological activity of ILs is strongly dependent on the organism considered, it is important to consider a broader study for each IL in order to obtain a complete profile of its activity. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 20 of 51 The proper cation/anion combination will allow for the synthesis of ILs with specific pharmacodynamic properties. TheseAs the analysis ILs with of the biological biological properties activity of IL cans is strongly be applied dependent not only on the as organism novel active considered, compounds, but ideally,it is andimportant by taking to consider advantage a broader of their study solubilization for each IL in ability, order to be obtain designed a complete to enhance profile the of its solubility of a givenactivity. API andThe supplementproper cation/anion and/or combination potentiate theirwill allow therapeutic for the action.synthesis Furthermore, of ILs with specific a closer look is requiredpharmacodynamic towards the understandingproperties. These IL ofs ILs’with distinctbiological mechanisms properties can of be membrane applied not only binding, as novel insertion, active compounds, but ideally, and by taking advantage of their solubilization ability, be designed to and disruption. enhance the solubility of a given API and supplement and/or potentiate their therapeutic action. Furthermore, a closer look is required towards the understanding of ILs’ distinct mechanisms of 6. API-ILs as Liquid Forms of APIs membrane binding, insertion, and disruption. The design of novel liquid forms of APIs where they are at least one the constituting ions in 6. API-ILs as Liquid Forms of APIs ILs (API-ILs) is an appealing strategy to overcome the solubility, bioavailability, and polymorphism drawbacks.The Their design charged of novel and liquid liquid forms state of allow APIs where to overcome they are theat least melting one the enthalpy constituting barrier ions andin ILs improve (API-ILs) is an appealing strategy to overcome the solubility, bioavailability, and polymorphism solubility/bioavailability. The large variety of IL cation–anion combinations allows for obtaining new drawbacks. Their charged and liquid state allow to overcome the melting enthalpy barrier and drugs inimprove the form solubility/bioavailability. of API-ILs with specific The large physicochemical variety of IL cation–anion and biological combinations properties, allows and foreven to possessobtaining dual pharmacological new drugs in the form action. of API-ILs These with ILs specific can be physicochemical also obtained and using biological oligomeric properties, ions or by applyingand the even prodrug to possess strategy dual pharmacological to one of the ionsaction. of These an API-IL. ILs can Figurebe also obtained10 summarizes using oligomeric the options of design forions API-ILs or by applying found the in theprodrug literature. strategy During to one of this the section, ions of an several API-IL. alike Figure summarizing 10 summarizes images the will be providedoptions with of design the information for API-ILs found collected in the from literature. the worksDuring tothis be section, discussed. several alike summarizing images will be provided with the information collected from the works to be discussed.

Figure 10.FigureSchematic 10. Schematic examples examples of API-ILs of API-ILs pharmaceutical pharmaceutic formulationsal formulations (adequate(adequate references are are given given along the current section). along the current section). API-ILs were first reported by Rogers and coworkers [129] in 2007, with the synthesis of ranitidine docusate ([Ran][Doc]), liquid at room temperature. Ranitidine, which is a histamine H2-

Int. J. Mol. Sci. 2020, 21, 8298 20 of 50

API-ILs were first reported by Rogers and coworkers [129] in 2007, with the synthesis of ranitidine Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 21 of 51 docusate ([Ran][Doc]), liquid at room temperature. Ranitidine, which is a histamine H2-receptor antagonist, is well known for its polymorphic conversion that affects its pharmaceutical action. receptor antagonist, is well known for its polymorphic conversion that affects its pharmaceutical The authors demonstrated its conversion into an IL form by incorporating the docusate anion, which, action. The authors demonstrated its conversion into an IL form by incorporating the docusate anion, in addition to overcoming the polymorphism concern, also improves the API absorption. After this which, in addition to overcoming the polymorphism concern, also improves the API absorption. pioneering work, an increasing number of reports on API-ILs emerged over the years, where the After this pioneering work, an increasing number of reports on API-ILs emerged over the years, structural and chemical properties of the ions and a wise selection of counterions led to the formation of where the structural and chemical properties of the ions and a wise selection of counterions led to the IL forms of the pristine drugs, with single or dual therapeutic action [130–134]. However, it should be formation of IL forms of the pristine drugs, with single or dual therapeutic action [130–134]. taken into account that, upon dissolution, the combination of APIs with simple and inert counterions However, it should be taken into account that, upon dissolution, the combination of APIs with simple or other active agents will dissociate in the body fluids, and the cationic and anionic components will and inert counterions or other active agents will dissociate in the body fluids, and the cationic and follow their independent pharmacokinetic and metabolic pathways [135]. anionic components will follow their independent pharmacokinetic and metabolic pathways [135]. API-ILs of different pharmacological classes have been reported, including the following APIs: API-ILs of different pharmacological classes have been reported, including the following APIs: lidocaine [106,136], sulfacetamide [129,137], ibuprofen [137,138], indomethacin [139], procaine [136], lidocaine [106,136], sulfacetamide [129,137], ibuprofen [137,138], indomethacin [139], procaine [136], aspirin [131,136], salicylic acid [136], piperacillin [137], penicillin G [137], and docusate [138]. Most of aspirin [131,136], salicylic acid [136], piperacillin [137], penicillin G [137], and docusate [138]. Most of the reported works comprise the API as the anion combined with an “inert” cation, as summarized in the reported works comprise the API as the anion combined with an “inert” cation, as summarized Figure 11, such as cholinium or phosphonium cations. These cations are mostly explored since they in Figure 11, such as cholinium or phosphonium cations. These cations are mostly explored since they are widely characterized in the literature and, especially, in the case of cholinium, this is a safe and are widely characterized in the literature and, especially, in the case of cholinium, this is a safe and low-costlow-cost cationcation source.source. However, thethe possibilitypossibility toto predictpredict ifif thethe selectedselected ionsions willwill resultresult inin anan API-IL andand theirtheir resultingresulting propertiesproperties isis stillstill aa challengechallenge toto bebe tackled.tackled.

FigureFigure 11.11. Selected examples of cations and anions used in API-ILs preparation (adequate(adequate referencesreferences areare givengiven alongalong thethe currentcurrent section).section).

InIn general,general, API-ILs API-ILs exhibit exhibit improved improved solubility solubility in waterin water and and bioavailability bioavailability when when compared compared with thewith original the original and non-charged and non-charged APIs. Figure APIs. 12 depictsFigure examples12 depicts of examples promising solubilityof promising enhancements solubility thatenhancements are achieved that by this are approach, achieved which by this can approach be advantageous, which alternatives can be advantageous for different pharmacologicalalternatives for classes.different pharmacological As an example, classes. betulinic As acid,an example, a low-water betulinic soluble acid, a natural low-water product soluble with natural anti-cancer, product anti-inflammatory,with anti-cancer, anti-inflammatory, and anti-HIV properties, and anti-HIV has been properties, converted has into been an converted API-IL using into choliniuman API-IL using cholinium as counterion [140]. The cholinium-based derivative has a significantly higher solubility in water than betulinic acid (by 100-fold) and its half maximal inhibitory concentration (IC50) was considerably improved (from 60 to 22 μg mL−1), meaning that a higher ability of liberating its latent biological activity for inhibition of HIV-1 protease. The API-IL approach has been also

Int. J. Mol. Sci. 2020, 21, 8298 21 of 50 as counterion [140]. The cholinium-based derivative has a significantly higher solubility in water than betulinic acid (by 100-fold) and its half maximal inhibitory concentration (IC50) was considerably 1 improved (from 60 to 22 µg mL− ), meaning that a higher ability of liberating its latent biological activityInt. J. for Mol. inhibition Sci. 2020, 21, x of FOR HIV-1 PEER protease.REVIEW The API-IL approach has been also applied to increase 22 of 51 the solubility of nalidixic acid by its conversion into cholinium nalixidixate ([Ch][Nal]) and of niflumic acid by itsapplied conversion to increase into cholinium the solubility niflumate of nalidixic ([Ch][Nif]), acid by where its conversion an increase into of 3300-foldcholinium andnalixidixate 53,0000-fold in solubility([Ch][Nal]) in aqueous and of niflumic media, respectively,acid by its conversi was observedon into cholinium [141]. Furthermore, niflumate ([Ch][Nif]), the in vitro wherestudy an on two increase of 3300-fold and 53,0000-fold in solubility in aqueous media, respectively, was observed human cell lines, Caco-2 colon carcinoma cells and HepG2 hepatocellular carcinoma cells, revealed that [141]. Furthermore, the in vitro study on two human cell lines, Caco-2 colon carcinoma cells and the cytotoxicity of these APIs is preserved upon their conversion into ILs. Other poorly water-soluble HepG2 hepatocellular carcinoma cells, revealed that the cytotoxicity of these APIs is preserved upon APIs,their such conversion as diclofenac, into ILs.ibuprofen, Other poorly ketoprofen, water-soluble naproxen, APIs, such as sulfadiazine, diclofenac, ibuprofen, sulfamethoxazole, ketoprofen, and tolbutamide,naproxen, were sulfadiazine, converted intosulfamethoxazole, tetrabutylphosphonium-based and tolbutamide, ILs, andwere their converted solubility into in water as comparedtetrabutylphosphonium-based to the free acids and ILs, sodium and their salts solubili [142].ty Tetrabutylphosphonium-basedin water as compared to the free acids ILs improveand the solubilitysodium salts of [142]. the corresponding Tetrabutylphosphonium-based API in aqueous ILs media, improve where the solubility significantly of the corresponding higher maximum concentrationsAPI in aqueous were media, reached where with significantly ibuprofen ( high80-fold),er maximum ketoprofen concentratio ( 60-fold),ns were naproxen reached ( with70-fold), ≥ ≥ ≥ sulfadiazineibuprofen (130-fold), (≥80-fold), and ketoprofen sulfamethoxazole (≥60-fold), ( 50naproxen fold). In ( another≥70-fold), study, sulfadiazine the conversion (130-fold), of ibuprofenand sulfamethoxazole (≥50 fold). In another study,≥ the conversion of ibuprofen into the IL 1-ethanol-3- into the IL 1-ethanol-3-methylimidazolium ibuprofenate ([C2OHC1im][Ibu]) allowed for increasing methylimidazolium ibuprofenate ([C2OHC1im][Ibu]) allowed for increasing the API’s solubility in the API’s solubility in water by 155,000-fold in comparison to the original API [143]. water by 155,000-fold in comparison to the original API [143].

FigureFigure 12. Example 12. Example of solubilityof solubility enhancements enhancements provided by by API-ILs API-ILs in inaqueous aqueous media media in comparison in comparison −1 with the parent APIs and their dissolution in water (mg mL1 ) (adequate references are given along with the parent APIs and their dissolution in water (mg mL− ) (adequate references are given along the the current section). current section). Ferraz et al. [144] reported the conversion of ampicillin into ampicillin salts/ILs, including API- FerrazILs liquid et al. [below144] reported body’s thetemperature, conversion ofnamely ampicillin trihexyltetradecylphosphonium into ampicillin salts/ILs, including ampicillin API-ILs liquid([P below666(14)][Amp]) body’s and temperature, 1-hydroxy-ethyl-3-methylimidazolium namely trihexyltetradecylphosphonium ampicillin ([C2OHC ampicillin1im][Amp]), ([P666(14) as well][Amp]) and 1-hydroxy-ethyl-3-methylimidazoliumas a water-soluble alternative with low ampicillinmelting temperature ([C2OHC 1(Tim][Amp]),m = 58 °C), ascholinium well as aampicillin water-soluble alternative([Ch][Amp]). with lowLater, melting the water temperature solubility (T(atm room= 58 and◦C), body’s cholinium temperature) ampicillin and ([Ch][Amp]). the respective Later, the waterhydrophilic/lipophilic solubility (at roombalance and of these body’s API-ILs temperature) was evaluated and the[145]. respective The solubility hydrophilic for [Ch][Amp]/lipophilic balanceand of [C these2OHC API-ILs1im][Amp] was was evaluated found to [ 145be ].comparable The solubility to the for water [Ch][Amp] solubility and of [C the2OHC respective1im][Amp] was foundampicillin to be sodium comparable salts and to it theincreased water by solubility nine-fold of at thebody’s respective temperature ampicillin in comparison sodium with salts the and it increasedpure bysalt, nine-fold thus standing at body’s as a temperaturecompetitive altern in comparisonative to the with marketed the pure drug. salt, For thus both standing ILs, an as a enhancement in the octanol–water partition coefficient by 11- and 7-fold was relatively verified to the competitive alternative to the marketed drug. For both ILs, an enhancement in the octanol–water starting ampicillin. The authors also evaluated the antibacterial activity of the prepared ampicillin- partitionbased coe ILsffi cient[146]. by The 11- studied and 7-fold ILs wasshowed relatively increase verifiedd growth to theinhibition starting for ampicillin. some Gram-negative The authors also evaluatedresistant the bacteria, antibacterial achieving activity MIC of values the prepared 10–1000 higher ampicillin-based than the ampicillin ILs [146 salt,]. Theincluding studied the ILs action showed increasedagainst growth resistant inhibition bacterial forstrains. some More Gram-negative recently, the resistantanti-tumoral bacteria, activity achieving of these ampicillin-based- MIC values 10–1000 higherILs than was theassessed ampicillin in human salt, cancer including cell lines the in action order againstto evaluate resistant the possibility bacterial to strains.have multiple More and recently, enhanced biological activity in the liquid form. To this purpose, the anti-tumoral activity was

Int. J. Mol. Sci. 2020, 21, 8298 22 of 50 the anti-tumoral activity of these ampicillin-based-ILs was assessed in human cancer cell lines in order to evaluate the possibility to have multiple and enhanced biological activity in the liquid form. To this purpose, the anti-tumoral activity was assessed in T47D (breast), PC3 (prostate), HepG2 (liver), MG63 (osteosarcoma), and RKO (colon) cell lines [147]. [C2OHC1im][Amp] shown to be the most relevant ampicillin-based IL, with a higher antiproliferative activity and lower cytotoxicity being associated towards healthy cells. Following the previous reports with ampicillin [144–147], primaquine-based ILs were synthesized aiming to treat malaria infection [148]. Primaquine blocks disease transmission due to its gametocytocidal action; however, its action is overshadowed by toxicity issues and by the drug’s poor activity against blood-stage parasites. Aiming to tackle these drawbacks, the API was combined with different cinnamates as counterions and screened against blood-stage chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum parasites. The novel API-ILs showed an increased in vitro blood-stage activity when compared to the pure API. Later, it was discovered that primaquine-derived ILs may contribute to increasing the API’s permeation into malaria-infected erythrocytes, behavior that considerably diverges from that of the parent drug [149]. This trend was explained based on more pronounced electrostatic interactions of the charged anti-malarial drugs with the polar head groups of the phospholipids in the erythrocyte membranes, thus potentiating treatment efficacy [149]. The possibility of replacing the “inert” counterion of an API-IL by a second API may lead to dual therapeutic function ILs. However, the physicochemical and pharmaceutical properties of both APIs in the API-IL form, i.e., the resulting melting temperature, solubility, bioavailability, and stability will most likely be different for the two APIs in comparison to the precursors, and their adequate characterization is mandatory. Both lidocaine and etodolac, in the form of lidocainium etodolac ([Lid][Eto]), can exhibit superior water solubility than both APIs alone, with an increase of >90-fold for etodolac and two-fold for lidocaine [150]. Rogers and co-workers reported the enhancement of aqueous solubility, thermal stability and topical analgesic effect of lidocainium docusate ([Lid][Doc]) in comparison with the parent API (lidocaine hydrochloride)[129]. Lidocaine is known for its anesthetic effect, where docusate was selected due to its action as dispersing agent in formulations. Studies of anti-nociception in mice suggested that [Lid][Doc] produces a longer duration of antinociceptive effect than the original APIs. The longer pain relief afforded by this API-IL is provided by the synergistic effect of both APIs that impact pharmacokinetic, resulting in a different mechanism of action with higher therapeutic efficacy. The bioavailability of [Lid][Doc] was later addressed in vivo through its transdermal application on Sprague–Dawley rats [151]. The concentration of lidocaine in blood plasma was evaluated over time after the topical application of creams containing 5.0 wt% of [Lid][Doc]. However, the total systemic lidocaine absorption was almost undetectable even after 240 min. of the transdermal application. Despite the fact that many APIs display higher solubility in water and higher therapeutic efficacy when solubilized in ILs or when converted into ionic liquid salts, addressing the in vivo bioavailability of the developed formulations is a crucial requirement. The conversion of APIs into liquid forms as API-ILs may not always require a stochiometric approach. Oligomeric ion formation can be a possible alternative [152]. The preparation of oligomeric API-ILs, which are hydrogen-bonded moieties that include both the ions and the neutral non-ionized material, may contribute to the expansion of liquid drug formulations by simply changing the stoichiometry and/or complexity of the ions, i.e., by introducing the free acid/base of the conjugate base/acid within the salt formulation, thus lowering the API’s melting point [22]. This concept was first introduced in 2010, by Bica and coworkers [153], with tetrabutylphosphonium salicylates, ([PBu4][Sal]nHm 1). The combination of tetrabutylphosphonium hydroxide and salicylic acid beyond − one equivalent originated several liquid compositions in a range of [PBu4][Sal]1.3–3H0.7–2, where the proton transfer is stronger. This behavior can be explained by the formation of hydrogen-bonded dimer complexes between salicylic acid and the salicylate anion, which can reach the composition of [PBu4][Sal]3H2 (Figure 13). MacFarlane et al. [154] presented a library of nine compounds and also four oligomeric API-ILs were synthesized and prepared. Benzoic, salicylic, and gentisic acids were Int. J. Mol. Sci. 2020, 21, 8298 23 of 50 chosen for this study, as these are frequently found in pharmaceutical formulations. These oligomers Int.may J. Mol. modulate Sci. 2020 membrane, 21, x FOR PEER transport REVIEW properties in vivo, enabling a higher permeability of APIs in24 of this 51 form than the starting materials. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 24 of 51 Oligomeric anion - Oligomeric anion - + +

Tetrabutylphosphonium salicylate Salicylic acid [PBu4][Sal]1.3-3H0.7-2

Tetrabutylphosphonium salicylate Salicylic acid [PBu4][Sal]1.3-3H0.7-2

Figure 13. Schematic representation of the formation of oligomeric ions based on salicylate/salicylic acidFigure proposedFigure 13. Schematic 13. Schematicin [153]. representation representation of of the the formationformation of of o oligomericligomeric ions ions based based on onsalicylate/salicylic salicylate/salicylic acid proposedacid proposed in [ 153in [153].]. Another way of enhancing the APIs therapeutic action and their delivery is by the prodrug AnotherAnother way way of enhancingof enhancing the the APIs APIs therapeutictherapeutic action action and and their their delivery delivery is by is the by prodrug the prodrug approach.approach. The Theprodrug prodrug concept concept has has be beenen refined refined over thethe years, years, and, and, nowadays, nowadays, is defined is defined by IUPAC by IUPAC approach. The prodrug concept has been refined over the years, and, nowadays, is defined by IUPAC as anyas compoundany compound that that undergoes undergoes biotransformation biotransformation beforebefore exhibiting exhibiting pharmacological pharmacological effect effect [155]. [155]. as any compound that undergoes biotransformation before exhibiting pharmacological effect [155]. A prodrugA prodrug only only has has activity activity after after enzymatic enzymatic and/orand/or chemicalchemical transformation transformation in vivoin vivo in order in order to to Arelease prodrugrelease the activethe only active hasparent parent activity drug drug into after into the enzymaticthe therapeuti therapeuti andc target/or chemical [156]. [156]. The The transformation prodrug prodrug strategy strategy inmay vivo may enablein enable orderthe the to optimizationreleaseoptimization the active of severalof parent several drug drug drug prop into properties, theerties, therapeutic i.e.,i.e., achieveachieve target higherhigher [156 stab stab].ility, Theility, improved prodrug improved strategysolubility solubility mayand/or and/or enable increasedthe optimizationincreased permeability. permeability. of several Yet, Yet, drug solid solid properties, prodrugs prodrugs can i.e.,can suffersuffer achieve fromfrom higher the the same same stability, problems problems improved as any as any solid solubility solid API andAPI and and/or notablyincreasednotably polymorphism permeability. polymorphism Yet,[157]. [157]. solid Therefore, Therefore, prodrugs thethe can combcomb sufferinationination from of theof API-IL sameAPI-IL advantages problems advantagesas with any with a solid prodrug a prodrug API and strategy can be an appealing alternative to enhancing the APIs efficacy. The functionalization of APIs strategynotably polymorphismcan be an appealing [157]. alternative Therefore, theto enhancing combination the of APIs API-IL efficacy. advantages The functionalization with a prodrug strategyof APIs with easily biochemically cleavable (e.g., by hydrolysis) ionic functional moieties, which can be withcan be easily an appealing biochemically alternative cleavable to enhancing (e.g., by thehydr APIsolysis) effi cacy.ionic Thefunctional functionalization moieties, which of APIs can with be easilycombined biochemically with selected cleavable counterions, (e.g., by is hydrolysis) the basis of ionicprodrug functional API-IL development. moieties, which Similar can to beother combined IL combinedstrategies, with it selected is possible counterions, to design the is prodrug the basis API- ofIL prodrug for a specific API-IL therapeutic development. purpose Similar by the proper to other IL with selected counterions, is the basis of prodrug API-IL development. Similar to other IL strategies, it strategies,selection it is of possible the counterion, to design which the may prodrug also include API-IL a for second a specific API (dual-function therapeutic purposeprodrug) byor eventhe proper a is possible to design the prodrug API-IL for a specific therapeutic purpose by the proper selection of selectionpermeation of the counterion,enhancer [22,158]. which A reportedmay also example include of a this second approach API encompasses (dual-function liquid prodrug) paracetamol- or even a permeationthe counterion,based drugs enhancer whichcombined [22,158]. may with also imidazolium,A includereported a example second pyrrolidi API ofnium, this (dual-function pyridinium, approach encompasses and prodrug) phosphonium or liquid even paired aparacetamol- permeation with basedenhancerdocusate drugs [22 combined, 158(Figure]. A 14) reported with [158]. imidazolium, The example resulting of this pyrrolidiprodrugs approach nium,present encompasses pyridinium, lower water liquid andsolubilities ph paracetamol-basedosphonium than the pairedneutral drugs with docusatecombinedparacetamol (Figure with imidazolium, and14) [158].slower Therelease pyrrolidinium, resulting profiles prodrugs in aqueous pyridinium, present media. andlower These phosphonium water differences solubilities in paired the thanparacetamol with the docusate neutral (Figureparacetamolderivative 14)[158 and properties]. Theslower resulting may release be prodrugs advantageous profiles present in aqueousfor lowerthe development media. water solubilities These of controlled differences than release the neutralin systems.the paracetamol paracetamol The promising results that were obtained with paracetamol may boost the study of this strategy for other derivativeand slower properties release profiles may inbe aqueous advantageous media. Thesefor the di developmentfferences in the of paracetamol controlled release derivative systems. properties The APIs. promisingmay be advantageous results that forwere the obtained development with paraceta of controlledmol may release boost systems. the study The of promisingthis strategy results for other that wereAPIs. obtained with paracetamol may boost the study of this strategy for other APIs.

Cl + Cl O Cl Acetaminophen Cl (Acetam) + O

Acetaminophen (Acetam) O Acetam anion exchange35 withAcetam silver docusate Acetam Acetam P(Bu)3 O Cl Yield 79% Yield 97% Yield 83% Yield 35% O Acetam anion exchange35 withAcetam silver docusate Acetam Acetam P(Bu)3 Anion exchange with silver docusate O Cl Yield 79% Yield 97%Prodrug docusate ILs Yield 83% Yield 35%

Anion exchange with silver docusate Figure 14. Synthesis of cationic acetaminophenProdrug prodrug docusate ILs paired ILs with the docusate anion proposed in [158].

Despite being shortly explored, prodrug API-ILs can be designed to target specific tissues. FigureFollowing 14. SynthesisSynthesisthis notion, of of cationica cationic promising acetaminophen acetaminophen protecting-group prodrug -free IL ILs ssynthesis paired with for the the docusate modification anion of proposed tertiary- in [[158].158].

Despite being shortly explored, prodrug API-ILs can be designed to target specific tissues. Following this notion, a promising protecting-group-free synthesis for the modification of tertiary-

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and heteroaryl-amineDespite being shortly containing explored, complex prodrug small API-ILs molecules can with be designedquaternary-ammonium to target specific linkers tissues. has Followingrecently been this proposed notion, a as promising a chemoselecti protecting-group-freeve approach for targeted synthesis delivery for the [159]. modification of tertiary- and heteroaryl-amineThe diversity in the containing API-IL toolbox complex stands small as molecules a unique with possibility quaternary-ammonium to provide new biologically linkers has recentlyactive combinations been proposed that as must a chemoselective be properly investigated. approach for The targeted largest delivery fraction [of159 published]. works focus on enhancingThe diversity the APIs in the solubility; API-IL toolbox however, stands the assessment as a unique of possibility in vitro and to provideespecially new in vivo biologically studies activeof API-ILs combinations are still less that reported. must be properlyFurthermore, investigated. the lack The of pharmacokinetic largest fraction of and published pharmacodynamic works focus onstudies enhancing with new the APIsAPI-ILs solubility; hinders however, the understanding the assessment of the of changesin vitro andon the especially therapeuticin vivo action,studies the ofmetabolic API-ILs pathways are still less that reported. are involved Furthermore, in their uptake the lack and of pharmacokineticthe alterations in and toxicity pharmacodynamic in comparison studieswith the with precursor new API-ILs API. This hinders gap themight understanding be partially attributed of the changes to the on lack the of therapeutic guidelines action, from the metabolicpharmaceutical pathways entities that for are API-ILs, involved which in their makes uptake difficult and thetheir alterations formulation in toxicity and correct in comparison testing as withnew drugs. the precursor Establishing API. these This gaplines mightwill allow be partially for overcoming attributed these to drawbacks the lack of and guidelines also considering from the pharmaceuticalother challenges, entities such for as API-ILs,the scale-up, which makespurification, difficult stability, their formulation and delivery and correct of liquid testing forms as new of drugs.therapeutics. Establishing these lines will allow for overcoming these drawbacks and also considering other challenges, such as the scale-up, purification, stability, and delivery of liquid forms of therapeutics. 7. IL-Based Drug Delivery Systems 7. IL-Based Drug Delivery Systems In the field of drug delivery, ILs have been applied as novel pharmaceutical forms (API-ILs) and the respectiveIn the field drug of drug delivery delivery, appraised, ILs have and been as appliedsolvents asor novel as polymerizable pharmaceutical monomers forms (API-ILs) for the anddevelopment the respective of polymer drug delivery drug delivery appraised, systems and as[75, solvents79,80,160–166]. or as polymerizable ILs are excellent monomers solvents for for the a developmentwide range of of biopolymers, polymer drug such delivery as proteins systems [167,168], [75,79,80 DNA,160– [169,170],166]. ILs areand excellent polysaccharides solvents [171– for a wide173], rangebeing ofused biopolymers, in their processing such as proteins into films [167 and,168 micro], DNA and [169 nanoparticles,170], and polysaccharides with potential [ 171for– drug173], beingdelivery. used Additionally, in their processing ILs have into shown films and promising micro and ability nanoparticles to functionalize with potential ionogels, for drugopening delivery. new Additionally,routes for designing ILs have advanced shown materials, promising including ability to th functionalizeeir use as drug ionogels, release systems opening [174,175]. new routes Figure for designing15 depicts advancedthe versatility materials, of ILs including in the development their use as drugof novel release drug systems delivery [174 systems.,175]. Figure Adding 15 depicts up to theexisting versatility reviews of ILson inthis the area development [30,161,176], of our novel discussion drug delivery focuses systems. on the Adding advances up made to existing for each reviews type onof thisadministration area [30,161 ,route.176], our The discussion selection focuses of the onadministration the advances maderoute foris eachmostly type dependent of administration on the route.physicochemical The selection properties of the administration of the API route (e.g., is melting mostly dependent temperature, on the molecular physicochemical weight, properties polarity, ofsolubility, the API (e.g.,etc.), meltingits pharmacokinetic temperature, profile molecular and weight,ultimately polarity, the intended solubility, target etc.), site its pharmacokineticof action [6,177]. profileThis selection and ultimately is also dependent the intended on other target factors, site of su actionch as [invasiveness,6,177]. This selection where non-invasive is also dependent routes onof otheradministration factors, such are aspreferable. invasiveness, where non-invasive routes of administration are preferable.

Figure 15.15. Schematic representationrepresentation on on the the use use of of ILs ILs in in drug drug delivery delivery systems systems (adequate (adequate references references are givenare given along along the currentthe current section). section).

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7.1. Intravenous Drug Delivery 7.1. Intravenous Drug Delivery Intravenous drug administration is a preferable choice when aiming to bypass biological Intravenous drug administration is a preferable choice when aiming to bypass biological absorption absorption barriers [178]. The selection of this route offers several advantages, since it provides the barriers [178]. The selection of this route offers several advantages, since it provides the most complete most complete drug bioavailability with a minimal delay. Foreseeing intravenous drug delivery drug bioavailability with a minimal delay. Foreseeing intravenous drug delivery applications, ILs have applications, ILs have recently been explored for the development of polymer nanocomplexes. In this recently been explored for the development of polymer nanocomplexes. In this field, the conjugation field, the conjugation of SAILs with chitosan was studied [179]. The SAILs 1-butyl-3- of SAILs with chitosan was studied [179]. The SAILs 1-butyl-3-methylimidazolium octylsulfate methylimidazolium octylsulfate ([C4C1im][C8OSO3]) and 3-methyl-1-octylimidazolium chloride ([C4C1im][C8OSO3]) and 3-methyl-1-octylimidazolium chloride ([C8C1im]Cl) induced the formation ([C8C1im]Cl) induced the formation of chitosan nanoparticles at low concentrations. While the of chitosan nanoparticles at low concentrations. While the negative charge of the counter ion Cl- of negative charge of the counter ion Cl- of [C8C1im]Cl could promote the IL-chitosan agglomeration [C8C1im]Cl could promote the IL-chitosan agglomeration (particles size of 450 nm), [C4C1im][C8OSO3] (particles size of 450 nm), [C4C1im][C8OSO3] allowed for the formation of particles with smaller size allowed for the formation of particles with smaller size due to stronger electrostatic interactions due to stronger electrostatic interactions between the positively charged chitosan chain and [C8OSO3]- between the positively charged chitosan chain and [C OSO ]- ions (particles with 300 nm). Thus, work ions (particles with 300 nm). Thus, work focused on8 the 3preparation and characterization of these focused on the preparation and characterization of these particles; however, further studies regarding particles; however, further studies regarding the encapsulation and release profile of a selected drug the encapsulation and release profile of a selected drug from these particles, as well as the knowledge from these particles, as well as the knowledge of their cytotoxicity profile, are required issues to better of their cytotoxicity profile, are required issues to better appraise their potential in drug delivery. More appraise their potential in drug delivery. More recently, the IL 1-butyl-3-methylimidazolium acetate, recently, the IL 1-butyl-3-methylimidazolium acetate, ([C4C1im][CH3COO]), was applied as solvent ([C4C1im][CH3COO]), was applied as solvent media for the synthesis of an amphiphilic derivative of media for the synthesis of an amphiphilic derivative of a chitosan oligosaccharide grafted with linoleic a chitosan oligosaccharide grafted with linoleic acid-(LCOS), followed by self-assembly in aqueous acid-(LCOS), followed by self-assembly in aqueous media [180]. The use of the IL allowed for a higher media [180]. The use of the IL allowed for a higher degree of grafting when compared to the values degree of grafting when compared to the values obtained with a conventional procedure using DMSO. obtained with a conventional procedure using DMSO. It was then possible to obtain ibuprofen- It was then possible to obtain ibuprofen-loaded LCOS micelles in aqueous solution with an average loaded LCOS micelles in aqueous solution with an average size lower than 200 nm, suitable for size lower than 200 nm, suitable for intravenous administration. Despite the possibility to recover and intravenous administration. Despite the possibility to recover and reuse the IL from the reaction reuse the IL from the reaction media, the cytotoxicity of the loaded LCOS micelles was not assessed. media, the cytotoxicity of the loaded LCOS micelles was not assessed. In a different approach, polydopamine nanoparticles that were loaded with doxorubicin and the IL In a different approach, polydopamine nanoparticles that were loaded with doxorubicin and the [C4C1im][PF6] were recently developed for cancer treatment (Figure 16)[181]. The IL was employed as a IL [C4C1im][PF6] were recently developed for cancer treatment (Figure 16) [181]. The IL was employed microwave sensitizer to prepare these novel nanoplatforms for combined chemotherapy and microwave as a microwave sensitizer to prepare these novel nanoplatforms for combined chemotherapy and thermal therapy by intravenous administration. The antitumor efficacy of doxorubicin-loaded microwave thermal therapy by intravenous administration. The antitumor efficacy of doxorubicin- IL-polydopamine nanoparticles was demonstrated in in vitro and in vivo experiments in the treatment loaded IL-polydopamine nanoparticles was demonstrated in in vitro and in vivo experiments in the of tumors in mice, after intravenous injection via tail vein. The referred nanoparticles exhibited high treatment of tumors in mice, after intravenous injection via tail vein. The referred nanoparticles inhibition effect when combined with the microwave thermal irradiation, acting in the tumor ablation exhibited high inhibition effect when combined with the microwave thermal irradiation, acting in the without inducing significant tissue toxicity. tumor ablation without inducing significant tissue toxicity.

Figure 16.16. Schematic illustration of the preparation of IL-polydopamine (PDA) nanoparticles loaded withwith doxorrubucindoxorrubucin forfor combinedcombined chemotherapychemotherapy andand microwavemicrowave thermalthermal therapytherapy proposedproposed inin [[181].181].

Micelles self-assembled from amphiphilic block copolymers were proposed for the administration of prednisone, a glucocorticoid that was mostly used to suppress the immune system

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Micelles self-assembled from amphiphilic block copolymers were proposed for the administration of prednisone, a glucocorticoid that was mostly used to suppress the immune system [182]. To this end, the IL 1-allyl-3-methylimidazolium chloride ([(CH2CH=C2)C1im]Cl) was used as solvent in order to prepare cellulose grafted with polylactic acid by ring opening graft polymerization of l-lactide. Colloidal solutions comprising micelles of cellulose-g-PLLA were prepared in aqueous media by a membrane-dialysis method. The resulting micelles exhibited spheric morphology within a size range of 30–80 nm, and allowed for sustained drug release. The IL removal and the cytotoxicity evaluation in 3T3 mouse fibroblasts cell line showed low toxicity towards cells, reinforcing their potential applicability as drug carriers. ILs can be used to form IL-in-oil (IL/O) [183], IL-in-water (IL/W) [79,184], oil-in-IL (O/IL), and water-in-IL (W/IL) [185] emulsions. The possibility to manipulate and design the IL structure allows for the use of IL-based vesicles and micelles as novel carriers of low-water soluble APIs. Accordingly, hydrophobic nontoxic ILs were used to prepare novel IL/W nanoemulsions for intravenous administration of amphotericin B [186]. Amphotericin B is an antifungal agent that, due to its 1 low-water solubility (<1.0 µg mL− ) and self-aggregation in aqueous media, presents undesired side-effects, thus being its intravenous drug delivery a challenge. In a preliminary study, high 1 contents (>5.0 mg mL− ) of the API were solubilized in a new hydrophobic dicholinium-based IL with the bis(trifluoromethanesulfonyl)imide ([NTf2]−) anion. The mixture of this hydrophobic IL with a hydrophilic cholinium-based IL resulted in the solubilization of the drug, preventing the concentration-dependent aggregation with controlled release of the API. Despite the maintenance of the antifungal activity of the API, and the low toxicity towards embryo-larval zebrafish models, further studies are required in order consider this formulation adequate for intravenous administration.

7.2. Oral Drug Delivery The challenges that are faced in oral drug delivery are primarily related with the API’s poor bioavailability, i.e., related with the API’s dissolution, permeability, and solubility. An example of using ILs in the development of oral drug delivery systems comprises their application as monomers in the synthesis of positively charged polymers loaded with naproxen, in the form of an anionic API [187]. The drug delivery systems were prepared by free radical polymerization while using two IL monomers, 1-(4-vinylbenzyl)-3-methyl imidazolium hexafluorophosphate and 1-(4-vinylbenzyl)-4-(dimethylamino)-pyridinium hexafluorophosphate, and methyl styrene. The resulting positively charged polymers were loaded with naproxen and provided a controlled release of the API, avoiding the delivery in acidic and neutral media (pH 2–6.5). Given their pH-dependent behavior, these systems can be envisaged to target the intestine delivery. Similarly, ILs with a pH-sensitive character have been used in order to modify positively charged silica nanoparticles for oral delivery of methotrexate, a chemotherapeutic drug [188]. Imidazolium-based ILs were grafted to silica nanoparticles that were also grafted with polymethacrylic acid to form stimuli-responsive nanoparticles. These systems allowed for high drug encapsulation efficiencies (76%) due to the strong electrostatic interactions established between the nanoparticles and the API. Recently, cholinium geranate ([Ch][geranate2(H)]) has been used for insulin solubilization as a novel formulation for the administration of this API [189]. The IL-based insulin formulation was coated with Eudragit L-100 and orally administrated, exhibiting a promising, in vivo, pharmacokinetic and pharmacodynamic outcome. The respective oral bioavailability of the IL-insulin formulation was found to be 51% relative to subcutaneous injection of insulin. When orally administrated, this formulation significantly enhanced the paracellular transport of insulin, protecting it from enzymatic degradation, which resulted in a sustained decrease in blood glucose down to 45% in 2.5 h. This delivery system was also investigated for its cytotoxicity and stability, showing no relevant toxicity and a constant stability over two months at room temperature and for at least four months under refrigeration. Despite tolerability tests still being required to establish a direct comparison between this system and the injection administration of insulin, the possibility to develop alternative and less invasive routes of administration must Int. J. Mol. Sci. 2020, 21, 8298 27 of 50 be highlighted as an advantage of the use of ILs for delivery purposes. Additionally, this type of formulations preserves the API structure, avoiding immunological reactions or loss of the API in a Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 28 of 51 multistep system development. In a different approach, API-ILs have been incorporated in carrier materials for the development of In a different approach, API-ILs have been incorporated in carrier materials for the development IL-based oral drug delivery systems. For instance, tetrabutylphosphonium ibuprofenate ([P ][Ibu]) of IL-based oral drug delivery systems. For instance, tetrabutylphosphonium ibuprofenate4444 and lidocainium ibuprofenate ([Lid][Ibu]) were successfully immobilized into mesoporous silica ([P4444][Ibu]) and lidocainium ibuprofenate ([Lid][Ibu]) were successfully immobilized into particles for a fast and complete API release when placed into an aqueous environment, where mesoporous silica particles for a fast and complete API release when placed into an aqueous approximately within 5 min. all IL was released from the solid support [190]. Zhang et al. [191] proposed environment, where approximately within 5 min. all IL was released from the solid support [190]. an all-in-one concept for the application of API-ILs as drug delivery systems. The pharmaceutically Zhang et al. [191] proposed an all-in-one concept for the application of API-ILs as drug delivery active IL itself works as both the carrier and the active drug. The API-IL self-assemble into vesicles systems. The pharmaceutically active IL itself works as both the carrier and the active drug. The API- in aqueous solution due to the combination of an anionic surfactant (sodium dodecylsulfate) with a IL self-assemble into vesicles in aqueous solution due to the combination of an anionic surfactant cationic drug with anti-depressive properties (amitriptyline hydrochloride) (Figure 17). Furthermore, (sodium dodecylsulfate) with a cationic drug with anti-depressive properties (amitriptyline the referred IL-based vesicles have high drug loading contents, an advantage over conventional drug hydrochloride) (Figure 17). Furthermore, the referred IL-based vesicles have high drug loading delivery systems. Also, it was possible to control the release profile of the API, moving from a total contents, an advantage over conventional drug delivery systems. Also, it was possible to control the release of 74% and 82% to 28% and 32% in just 2 h, using the correspondent API-IL, at pH 7.4 and release profile of the API, moving from a total release of 74% and 82% to 28% and 32% in just 2 h, pH 1.2, respectively. using the correspondent API-IL, at pH 7.4 and pH 1.2, respectively.

Figure 17. IllustrationIllustration of the the API-IL API-IL self-assembling into vesicles in aqueous media proposed in [[191].191].

Itraconazole and cinnarizine were converted into lipophilic ILs (cinnarizine decylsulfate, Itraconazole and cinnarizine were converted into lipophilic ILs (cinnarizine decylsulfate, itraconazole dioctyl sulfosuccinate) aiming to facilitate their incorporation into lipid-based itraconazole dioctyl sulfosuccinate) aiming to facilitate their incorporation into lipid-based formulations [192]. The resulting API ILs were completely miscible or highly soluble in lipid-based formulations [192]. The resulting API−ILs were completely miscible or highly soluble in lipid-based self-emulsifying drug delivery systems (SEDDs). These systems, composed of long or medium chain self-emulsifying drug delivery systems (SEDDs). These systems, composed of long or medium chain glycerides, surfactants, such as Kolliphor-EL, and cosolvents, like ethanol, were easily incorporated glycerides, surfactants, such as Kolliphor-EL, and cosolvents, like ethanol, were easily incorporated into lipid-based formulations for in vivo oral drug delivery. The pharmacokinetic evaluation upon the into lipid-based formulations for in vivo oral drug delivery. The pharmacokinetic evaluation upon administration of SEDDs revealed higher drug plasma exposure for the API-IL formulations (2-fold for the administration of SEDDs revealed higher drug plasma exposure for the API-IL formulations (2- cinnarizine and 20-fold for itraconazole) in comparison with the SEDDs with the respective parent fold for cinnarizine and 20-fold for itraconazole) in comparison with the SEDDs with the respective APIs. The use of API-ILs for the development of these formulations enabled obtaining liquid SEDDs, parent APIs. The use of API-ILs for the development of these formulations enabled obtaining liquid increasing the oral exposure to the API. The increase in the drug absorption is enabled by the increase SEDDs, increasing the oral exposure to the API. The increase in the drug absorption is enabled by the in the APIs’ solubility and by promoting the gastrointestinal lipid absorption pathways. The design of increase in the APIs’ solubility and by promoting the gastrointestinal lipid absorption pathways. The the IL structure was also employed for optimization of the solubility of danazol, an API used in the design of the IL structure was also employed for optimization of the solubility of danazol, an API treatment of endometriosis and fibrocystic breast disease, and itraconazole, an antifungal drug [77]. used in the treatment of endometriosis and fibrocystic breast disease, and itraconazole, an antifungal Like many other low-water soluble APIs, itraconazole and danazol present low-water and lipidic drug [77]. Like many other low-water soluble APIs, itraconazole and danazol present low-water and solubilities. Such conditions hinder the selection of formulation excipients that can enhance these lipidic solubilities. Such conditions hinder the selection of formulation excipients that can enhance drugs’ bioavailability. The solubility of danazol and itraconazole was increased 20-fold and >500-fold these drugs’ bioavailability. The solubility of danazol and itraconazole was increased 20-fold and using 1-hexyl-3-hexyloxycarbonylpyridinium dicyanamide ([C6C6OCOpy][N(CN)2]), when compared >500-fold using 1-hexyl-3-hexyloxycarbonylpyridinium dicyanamide ([C6C6OCOpy][N(CN)2]), when to its solubility in soybean oil (a common lipid excipient). These solubility enhancements surpass compared to its solubility in soybean oil (a common lipid excipient). These solubility enhancements those that were provided by using co-solvents like PEG and ethanol. The oral administration of the surpass those that were provided by using co-solvents like PEG and ethanol. The oral administration danazol-containing self-emulsifying IL formulation rises up to 4.3-fold the API’s bioavailability when of the danazol-containing self-emulsifying IL formulation rises up to 4.3-fold the API’s bioavailability compared to the respective crystalline drug. Not only the absorption of the API was improved, but also when compared to the respective crystalline drug. Not only the absorption of the API was improved, but also its release sustained, as the formulation prolonged the plasma exposure to the API when compared with the respective lipid formulation.

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7.3. Topical andand TransdermalTransdermal DrugDrug DeliveryDelivery When considering the the topical topical application application of of APIs, APIs, the the drug drug delivery delivery system system must must target target one one or ormore more different different skin skin layers layers and andunderlying underlying tissues, tissues, or skin or associated skin associated structures structures (sebaceous (sebaceous or sweat or sweatglands, glands, etc.) [193]. etc.) [193Transdermal]. Transdermal drug drug delivery, delivery, in particular, in particular, aims aims to to reach reach systemic systemic circulation, representing anan alternative alternative to to parenteral parenteral and and oral oral routes, routes, while while avoiding avoiding pre-systemic pre-systemic metabolism metabolism [194]. Despite[194]. Despite being being justrecently just recently explored, explored, ILs ILs have have been been studied studied as as promising promising pharmaceutical pharmaceutical agentsagents or formulationformulation components in order to tackletackle thethe challengeschallenges inin topicaltopical andand transdermaltransdermal deliverydelivery systems, presenting a widewide rangerange ofof applications,applications, asas illustratedillustrated inin FigureFigure 1818[ [195].195]. ILs havehave beenbeen investigatedinvestigated inin microemulsions, microemulsions, nanoparticles, nanoparticles, (bio)polymer-based (bio)polymer-based drug deliverydrug delivery systems systems (e.g., patches (e.g., andpatches membranes), and membranes), and as permeation and as permeation enhancers enhancer aiming tos aiming deliver to poorly deliver water-soluble poorly water-soluble drugs at topical drugs andat topical transdermal and transdermal level, as summarized level, as summarized in Table3. in Table 3.

Figure 18.18. ILIL applicationsapplications inin thethe developmentdevelopment ofof drugdrug deliverydelivery systemssystems forfor topicaltopical andand transdermaltransdermal administration (adequate(adequate referencesreferences areare givengiven alongalong thethe currentcurrent section).section).

Microemulsions have have been been proposed proposed in inorder order to improve to improve transdermal transdermal delivery, delivery, especially especially when whencomprising comprising ILs. A ILs. single A singleIL can IL replace can replace different different components components in a ingiven a given drug drug formulation, formulation, by bysubstituting substituting the the oil, oil, water water or orsurf surfactantactant phase phase in in microemulsions, microemulsions, ac actingting as as the the permeation permeation enhancer andand/or/or beingbeing thethe APIAPI itself,itself, asas aforementioned.aforementioned. Furthermore, the fine-tuningfine-tuning of SAILs allows for manipulating the structure and dynamics of theirtheir micellarmicellar aggregates,aggregates, making these ILs promisingpromising vehiclesvehicles forfor APIs delivery due to their ability to enhance the solubility and specially the permeability of the drug acrossacross biologicalbiological membranes.membranes.

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Table 3. IL application in topical and transdermal strategies.

Strategy IL IL Role API Reference Dopamine hydrochloride Micellar system [C C im]Br Surfactant [196] 14 1 Acetylcholine chloride

Micellar system [C12C1im]Cl Surfactant Ibuprofen [87] [C C im]Cl Lidocaine Micellar system 12 1 Surfactant [197] [C14C1im]Cl hydrochloride [C C im]Cl Reichardt’s dye Microemulsion 6 1 Aqueous/Oil phase [29] [C4C1im][PF6] (drug model)

Microemulsion [C4C1im][PF6] Oil phase Etodolac [79]

Microemulsion [C1C1im][(CH3O)2PO2] Aqueous phase Acyclovir [198] [Ch][formate] Non-aqueous [Ch][lactate] Microemulsion phase; Surfactant in Acyclovir [199] [Ch][propionate] oil phase [Ch][oleate]

Microemulsion [C1C1im][(CH3O)2PO2] Aqueous phase Methotrexate [200] [C OHC ]Cl Aqueous phase; Microemulsion 2 1 Dencichine [201] [C1C1im][C12SO3] Surfactant phase Bacterial [Ch][Caf] Caffeic acid nanocellulose API [202] [Ch][Gal] Gallic acid membranes Bacterial [Ch][Ibu] Ibuprofen nanocellulose [Ch][Nap] API Naproxen [203] membranes [Ch][Ket] Ketoprofen

Bacterial [Ch][B3] Niacin nanocellulose [Ch][B5] API Pantothenic acid [204] membranes [Ch][B6] Pyridoxine Lidocaine Patch [Lid][Eto] Dual API [205] Etodolac [Lid][Nap] Naproxen Polyvinvylidene [Lid][Ibu] Dual API Ibuprofen [206] fluoride membrane [Lid][Dicl] Diclofenac [Ch][Phe] PLGA [Ch][Glu] API solubilization Rutin [207] nanoparticles

[C im]Cl Permeation 8 Membrane [C C im]Cl Testosterone [208] enhancers 1 8 disruption [C1C1C8im]Cl Permeation [ProOEt][Ibu] API Ibuprofen [209] enhancers

[mPEG3N444][Sal] Permeation [HN ][Sal] 444 API Salicylic acid [210] enhancers [Ch][Sal] [C1Pyrr][Sal] Permeation [Ch][geranate (H)] API Geranic acid [211] enhancers 2

The design of IL-excipient with tunable lipophilicity/hydrophilicity character is advantageous, especially when used as a solubility-enhancing agent for complex amphiphilic APIs, like amphotericin Int. J. Mol. Sci. 2020, 21, 8298 30 of 50

B and itraconazole, which are antifungal drugs [76]. Mahajan et al. [196] studied the performance of the SAIL 1-methyl-3-tetradecylimidazolium bromide ([C14C1im]Br) as a drug carrier, and then compared it to a conventional cationic surfactant tetradecyltrimethylammonium bromide (TTAB). [C14C1im]Br revealed not only superior surface activity, but also acts as better drug carrier of APIs, namely dopamine hydrochloride and acetylcholine chloride, than the traditional TTAB. Further, Sanan et al. [87] established the effect of the aggregate morphology and dilution on the micellar transition of [C12C1im]Cl and ibuprofen mixtures. The transition is driven by the release of the API from the mixed micelles due to the solubility disparity between both components. Similar SAILs, [C12C1im]Cl and 1-methyl-3-tetradecylimidazolium chloride ([C14C1im]Cl) have shown capability to form aggregates with lidocaine hydrochloride, improving the drug’s dissolution into aqueous media [197]. Furthermore, the reported thermal stability of nonaqueous IL microemulsions (up to 150 ◦C) was shown to be compatible with sterilization processes, an advantage over conventional formulations [212]. All of the described works envisioned the application of the studied emulsions in transdermal and topical drug delivery; however, permeation studies on skin models were not conducted for evaluating the ability of these systems to improve dermal delivery. The influence of imidazolium-based ILs on the properties and stability of oil-in-water (W/O) and water-in-oil (O/W) emulsions was investigated by Dobler et al. [29] using a fluorescent probe, namely Reichardt’s dye, as a drug model. A hydrophilic IL, 1-hexyl-3-methylimidazolium chloride ([C6C1im]Cl), and a hydrophobic IL, [C4C1im][PF6], were incorporated as water and oil phase components, respectively, resulting in stable formulations. Skin permeation across pig’s ear skin was studied in vitro on Franz glass diffusion cells. A permeation enhancement was observed when using these formulations due to the disruption of the lipidic bilayer packing by the ILs used. These ILs present antimicrobial activity and preservative efficacy within these formulations, being possible to fulfil the requirements as novel preservatives. Similar ILs where studied for an IL/W microemulsion designed for etodolac’s topical delivery [79], which is used in the treatment of inflammation and pain that are associated with rheumatoid arthritis and osteoarthritis [213]. The prepared microemulsion was based on [C4C1im][PF6], Tween 80 as surfactant, and ethanol as co-surfactant. The IL/W-based formulation was able to efficiently enhance the solubility and ex-vivo permeability of the API for its transdermal delivery [79]. Skin penetration was evaluated while using the fluorescent dyes sodium fluorescein (a hydrophilic fluorescence marker) and Nile red (lipophilic), which in the presence of ILs revealed a more efficient penetration into the deeper skin layers. Likewise, in-vivo pharmacodynamic evaluation showed improved anti-arthritic and anti-inflammatory activities in comparison to O/W microemulsions and marketed etodolac’s formulations, exhibiting the high capability of these formulationsin order to enhance the API’s performance. The first IL-based microemulsion reported for transdermal delivery aimed to improve membrane transport of a sparingly soluble API, namely the antiviral drug acyclovir [198]. In this work, a blend of two nontoxic surfactants, Tween-80 and Span-20, was used in combination with imidazolium-based ILs to form stabilized IL droplets. In the referred microemulsion, the external phase (oil phase) is constituted by isopropyl myristate (Figure 19). Among the investigated ILs, dimethylimidazolium dimethylphosphate ([C1C1im][(CH3O)2PO2]) presented superior ability to dissolve the selected API and form more stable droplets in the formulation. This improvement was justified by the hydrogen bonding interactions between the polar groups of acyclovir and the IL anions. The in vitro study across Yucatan micropig porcine skin (performed on Franz diffusion cells), allowed for verifying an increase in acyclovir’s skin permeability of several orders of magnitude, as well as the API’s transdermal permeation when using the IL/O system as drug carrier. Int. J. Mol. Sci. 2020, 21, 8298 31 of 50 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 32 of 51

FigureFigure 19.19. IL-in-oilIL-in-oil microemulsionsmicroemulsions basedbased onon [C[C1C1C11im][(MeO)im][(MeO)22PO2]] as as drug carrierscarriers ofof acycloviracyclovir proposedproposed inin [[198].198].

Cholinium-basedCholinium-based ILs ILs comprising comprising anions anions that that were were derived derived from from carboxylic carboxylic acids acids were were also also used used in ILin/ OIL/O microemulsions microemulsions to increase to increase the transdermal the transdermal delivery delivery of acyclovir, of acyclovir, as non-toxic as non-toxic and non-irritating and non- alternativesirritating alternatives [199]. Hydrophilic [199]. Hydrophilic ILs (cholinium ILs (cholinium formate, cholinium formate, lactatecholinium and choliniumlactate and propionate) cholinium werepropionate) used as were the non-aqueous used as the non- polaraqueous phase and polar a surface-active phase and a surface-active IL (cholinium oleate)IL (cholinium as the surfactant oleate) as inthe combination surfactant within combination a co-surfactant, with Span a co-surfactant, 20, in a continuous Span 20, oil phase.in a continuous An enhancement oil phase. in skin An permeationenhancement due in toskin the permeation modification due and to the disruption modification of the and regular disruption arrangement of the regular of the arrangement corneocytes ofof thethe stratumcorneocytes corneum of thewas stratum observed, corneum and was related observed, to the ionicand related character to the of theionic IL. character Cytotoxicity of the tests IL. revealedCytotoxicity a high tests cell revealed survival a high rate (cell>90%) survival in comparison rate (>90%) with in comparison Dulbecco’s with phosphate-bu Dulbecco’sff eredphosphate- saline solution,buffered highlightingsaline solution, the highlighting potential of the these potential formulations of these as formulations low toxic drug as low carriers. toxic Similarly,drug carriers. the ILSimilarly, [C1C1im][(CH the IL 3[CO)1C2PO1im][(CH2] was3 usedO)2PO in2] anwas IL used/O microemulsion in an IL/O microemulsion in order to improve in order the to transdermalimprove the deliverytransdermal of the delivery sparingly of solublethe sparingly chemotherapeutic soluble chem methotrexateotherapeutic [200 methotrexate]. In this work, [200]. drug In permeation this work, acrossdrug permeation a similar skin across model a tosimilar the previous skin model enounced to the was previous evaluated, enounced revealing was a significantevaluated, transdermal revealing a permeationsignificant transdermal of the API in permeation comparison of to the the API application in comparison of other to typicalthe application O/W and of O other/W formulations. typical O/W IL-basedand O/W microemulsions formulations. IL-based were also microemulsions developed for enhancing were also the developed skin permeation for enhancing of dencichine, the skin a haemostaticpermeation agentof dencichine, [201]. An a initial haemos screeningtatic agent with [201]. fourteen An initial imidazolium-based screening with ILs fourteen was performed, imidazolium- from whichbased 1-(2-hydroxyethyl)-3-methylimidazoliumILs was performed, from which chloride1-(2-hydroxyethyl)-3-methylimidazolium ([C2OHC1im]Cl) and dimethylimidazolium chloride dodecanesulfate([C2OHC1im]Cl) ([Cand1C dimethylimidazolium1im][C12SO3]) were selected dodecanesulfate and incorporated ([C1C1im][C into the12SO aqueous3]) were and selected surfactant and phases,incorporated respectively, into the with aqueous an enhancement and surfactant on skin phas permeationes, respectively, of approximately with an enhancement 10-fold. However, on skin despitepermeation that theof approximatelyin vivo pharmacodynamic 10-fold. However, activity wasdespite found that to the be inin goodvivo correlationpharmacodynamic with the inactivity vitro permeability,was found to hemostatic be in good activity correlation studies with revealed the in no vitro statistic permeability, difference betweenhemostatic these activity formulations studies andrevealed dencichine no statistic aqueous difference solution. between these formulations and dencichine aqueous solution. TheThe applicationapplication of of API-ILs API-ILs represents represents an an advantage advantage in thein the design design of topicalof topical delivery delivery systems systems due due not onlynot only to the to possibility the possibility of providing of providing new biologically new biologically active combinations active combinations and enhancing and theenhancing therapeutic the action,therapeutic but also action, due to but the possibilityalso due toto enhancethe possib the transdermalility to enhance delivery the of pharmaceuticals.transdermal delivery The use of ofpharmaceuticals. API-ILs might allowThe use significant of API-ILs developments might allow insignificant delivery developments systems, since in these delivery can be systems, designed since to self-aggregate,these can be designed trapping theto self-aggregate, drug in a micelle, trapping or to displaythe drug tunable in a micelle, hydrophilic-lipophilic or to display balancetunable tohydrophilic-lipophilic potentiate the drugs balance permeation. to potentiate In addition the drugs to the permeation. incorporation In addition of ILs into to the microemulsions incorporation asof transdermalILs into microemulsions drug delivery as systems, transdermal the combination drug delivery of API-ILs systems, with the polymers combination and biopolymersof API-ILs with has beenpolymers recently and investigated. biopolymers Morais has been et al.recently [202] synthesized investigated. cholinium-based Morais et al. [202] ILs synthesized paired with anionscholinium- that arebased derived ILs paired from phenolicwith anions acids, that namely are derived gallic, from caffeic, ph andenolic ellagic acids, acids. namely These gallic, ILs werecaffeic, incorporated and ellagic intoacids. bacterial These ILs nanocellulose were incorporated membranes into bacteria (BC). Thel nanocellulose developed drug membranes delivery (BC). systems The showed developed superior drug antioxidantdelivery systems activity showed to the starting superior materials, antioxidant and withactivity controlled to the starting diffusion materials, of the active and compounds with controlled from thediffusion wet membranes. of the active The compounds obtained resultsfrom the demonstrated wet membranes. that theThe dissolution obtained results profiles demonstrated were essentially that governedthe dissolution by the profiles solubility were of the essentially ILs rather governed by their interactionsby the solubility with the of BCthe nanofibrils. ILs rather Forby boththeir BC-[Ch][Caf]interactions with and BC-[Ch][Gal]the BC nanofibrils. wet membranes, For both approximatelyBC-[Ch][Caf] and 70% BC-[Ch][Gal] dissolution of wet the ILmembranes, content in approximately 70% dissolution of the IL content in membranes was reached after 6 h. Regarding the cytotoxicity of these delivery systems, it was shown that they do not cause any decrease in cell

Int. J. Mol. Sci. 2020, 21, 8298 32 of 50

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 33 of 51 membranes was reached after 6 h. Regarding the cytotoxicity of these delivery systems, it was shown thatviability they at do the not concentrations cause any decrease investigated. in cell viability Additionally, at the the concentrations exposure of investigated.cells to BC-ILs Additionally, membranes thesignificantly exposure decreases of cells to the BC-ILs LPS-induced membranes NO significantlyproduction, indicating decreases thea relevant LPS-induced anti-inflammatory NO production, and indicatingantioxidant a potential relevant anti-inflammatory(Figure 20). The permeation and antioxidant flux of potentialboth API-ILs (Figure from 20 the). TheBC membranes permeation fluxwas ofassessed both API-ILs in vitro fromin human the BC epidermal membranes skin, was at body’s assessed temperature,in vitro in humanon Franz epidermal diffusion skin, cells. at The body’s skin temperature,permeation assay on Franz showed diff usionthe possibility cells. The to skin obtain permeation a slow and assay sustained showed release the possibility profile while to obtain using a slowthese anddrug sustained delivery releasesystems profile over 5 while h of administration. using these drug delivery systems over 5 h of administration.

Figure 20. IllustrationIllustration of of BC-IL BC-IL membranes’ membranes’ with with antioxidant antioxidant and anti-inflammatory anti-inflammatory action for dermal application proposed in [[202].202].

Chantereau et al. [[203]203] later incorporated NSAID-basedNSAID-based ILs also into BC membranes envisaging their use in transdermal drug delivery systems. Th Theseese [Ch][NSAID] ILs allowed for increasing up to 100-fold the solubility of the respective NSAID precursors (ibuprofen, naproxen and ketoprofen) in aqueous media. The impregnation of of BC BC membranes with with these these ILs ILs also also increased, by by 18 to 26-fold, the rehydration ability of thethe membranes,membranes, allowing their potential application on the absorption of exudates. GivingGiving thethe obtained obtained results, results, the the developed developed systems systems are promisingare promisin alternativesg alternatives for the for design the ofdesign transdermal of transdermal patches forpatches anti-inflammatory for anti-inflammato drugsry delivery. drugs Moredelivery. recently, More the recently, same researchers the same investigatedresearchers investigated the possibility the possibi to developlity to membranes develop membranes that were that loaded were with loaded vitamin with vitamin B-based B-based ILs for dermalILs for applicationsdermal applications [204]. Three [204]. ILs, Three namely ILs, cholinium namely nicotinatecholinium ([Ch][B3]), nicotinate cholinium ([Ch][B3]), pantothenate cholinium ([Ch][B5]),pantothenate and ([Ch][B5]), cholinium and pyridoxylate cholinium ([Ch][B6]), pyridoxylate were also([Ch][B6]), synthesized were andalso incorporated synthesized in and BC, resultingincorporated in more in BC, thermally resulting stable in more forms thermally for the vitamins stable forms without for toxicity the vitamins associated without for skin toxicity cells. Theassociated solubility for ofskin these cells. ILs The in aqueoussolubility media of these was IL highers in aqueous than their media vitamin was precursors,higher than with their solubility vitamin enhancementsprecursors, with up solubility to 30.6-fold. enhancements The increase up on to the 30.6-fold. re-hydration The increase ability ofon BC-IL the re-hydration membranes, ability allowed of forBC-IL obtaining membranes, a complete allowed and for fasterobtaining release a complete profile ofand ILs faster in aqueous release profile media of than ILs in the aqueous release media of the precursorthan the release vitamins. of the These precursor ILs also vitamins. displayed These a plasticizing ILs also edisplayedffect on the a plastici BC membranes,zing effect favoring on the theBC applicationmembranes, of favoring these systems the application on irregular of th skinese regions.systems on irregular skin regions. The IL [Lid][Eto] has been incorporated into a topi topicalcal delivery system [205]. [205]. The The patch patch (Etoreat), (Etoreat), studied by ILIL PharmaPharma Inc.Inc. (MEDRx, (MEDRx, Kagawa, Kagawa, Japan), Japan), contains contains the the only only API-IL that has reached clinical trials. This API-IL based patch for alleviating pain caused by inflammation inflammation was tested for the treatment of ankle sprains and low back pain, due to its enhancedenhanced skinskin absorption.absorption. However, due to the lack of of statistic statistic significant significant results results between between Etor Etoreateat and and placebo placebo administration, administration, its its further further stage stage of ofdevelopment development was was suspended. suspended. In Inaddition addition to tothis this example, example, other other dual-active dual-active API-ILs API-ILs have been considered for improving transdermal delivery of analgesic and anti-inflammatoryanti-inflammatory APIs in wound healing [206]. Lidocainium naproxen ([Lid][Nap]), [Lid][Ibu], and lidocainium diclofenac ([Lid][Dicl]) were synthesized, which resulted in liquid mixtures at room temperature. These API-

Int. J. Mol. Sci. 2020, 21, 8298 33 of 50 healing [206]. Lidocainium naproxen ([Lid][Nap]), [Lid][Ibu], and lidocainium diclofenac ([Lid][Dicl]) were synthesized, which resulted in liquid mixtures at room temperature. These API-ILs displayed significantly higher solubility in aqueous media than the parent APIs, except for [Lid][Dicl]. These ILs were then incorporated into a bilayer wound dressing, which was composed of a hydrophobic polyvinylidene fluoride membrane that acts as a drug reservoir and a biocompatible hyaluronic acid layer. The assessment of anti-inflammatory activity revealed similar therapeutic efficacy when compared with the original APIs, through the inhibition of LPS-induced production of nitric oxide and prostaglandin E2 by macrophages. These systems enable higher permeation of both APIs in API-IL form than the parent APIs without compromising the fibroblasts proliferation. Furthermore, the hyaluronic acid that was used in these systems played a protective effect on the cytotoxicity since it minimized the potential antiproliferative effects attributable to the APIs, allowing the simultaneous delivery of anti-inflammatory and analgesic drugs to the injured area without compromising skin regeneration. ILs were also studied as solubility enhancers in conjugation with polymers as novel nanoparticle hybrid systems for the delivery of poorly water-soluble drugs [207]. Two amino-acid-based ILs, namely cholinium phenylalanine ([Ch][Phe]) and cholinium L-glutamine ([Ch][Glu]), were used in blends with poly-(lactic-co-glycolic acid) (PLGA) that was loaded with rutin, which shows antidiabetic, antihypertensive, and antilipidemic activities. These systems allowed for a drug loading capacity higher than 50% for both ILs, and up to 76% for [Ch][Phe] while using only 0.2% (v/v) of IL. The systems provided a sustained release of rutin, with 85% released after 72 h, without toxicity to associated skin cells. Zhang et al. [208] used testosterone as a model drug to investigate the transdermal delivery enhancement provided by twenty imidazolium-based ILs. The conducted study revealed an interdependence between the API permeation enhancement and the structure and composition of the IL. ILs with longer alkyl side chains (N-octylimidazolium chloride ([C8im]Cl)), 1-octyl-3-methyl imidazolium chloride ([C1C8im]Cl) and 1-octyl-2,3-dimethyl imidazolium chloride ([C1C1C8im]Cl)) led to higher transdermal delivery enhancements. Additionally, the number of alkyl groups at the cation, as well as the anionic constitution, was demonstrated to have an impact on the drug penetration through skin. However, this trend follows the cytotoxicity profile and disruptive character of ILs with longer alkyl chain lengths, in which the most cytotoxic compromise in a larger extent the structural integrity of biological membranes. The enhancement of API permeation was attributed to the change in skin permeability, rather than the change in drug concentration. Evaluations by ATR-FTIR and atomic force microscopy of skin membrane indicated that the ILs can disrupt the regular and compact arrangements of the corneocytes, altering the skin structure to a more permeable state. Although this mechanism was not observed for all ILs, further information must be gathered in order to properly understand the process of the interaction between these ILs and skin cells to better design IL-based permeation enhancers. This subject has been addressed by Jing and coworkers [214], which confirmed that amphiphilic ILs could disrupt the lipid bilayer by IL insertion, endcapping the hydrophobic edge of the lipid bilayer, and eventually disintegrating the membrane (Figure 21). This destabilization is directly related with the IL concentration, the length of the IL cation alkyl chain, and anion hydrophobicity, which are also correlated with the IL cytotoxicity. Hydrophilic and hydrophobic ILs seem to act on the APIs transportation through the stratum corneum by different mechanisms [215]. While hydrophilic ILs, particularly imidazolium-based ones, fluidize the cell membrane in order to create pathways for the diffusion of molecules (paracellular transport), hydrophobic ILs, on the contrary, modify the APIs partitioning by providing channels through biological membranes (transcellular transport) [211,216]. Int. J. Mol. Sci. 2020, 21, 8298 34 of 50 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 35 of 51

Figure 21. SchematicSchematic representation representation of IL effect effect and insertioninsertion into the lipid bilayer proposed inin [[214].214].

DiDifferentfferent ILsILs thatthat were were based based on on carboxylic carboxylic acids acid [217s [217],], aliphatic aliphatic amines, amines, amino amino acids acids [209 ,[209,218]218] and polyethyleneand polyethylene glycol glycol derivatives derivatives [210] have [210] been have also studiedbeen also as alternativestudied as permeation alternative enhancers. permeation An aminoenhancers. acid esterAn amino (proline acid ethylester) ester (proline was used ethylester) in combination was used with in ibuprofen combination as a novelwith API-ILibuprofen (proline as a ethylesternovel API-IL ibuprofenate (proline ethylester ([ProOEt][Ibu])) ibuprofenate to improve ([ProOEt][Ibu])) transdermal to improve delivery transdermal [209]. An approximatelydelivery [209]. 10-foldAn approximately enhancement 10-fold in the enhancement cumulative amount in the cumula of APItive was amount achieved of at API 96 hwas when achieved compared at 96 with h when the controlcompared sample with whilethe control using sample [ProOEt][Ibu]. while using [ProOEt][Ibu]. ILs generated by a neutralizationneutralization reaction between aliphatic carboxylic acids (octanoic acid or isostearic acid) and aliphatic amines (diisopropan (diisopropanolamineolamine or triisopropanolamine) were proposed in order to study the mechanism of permeability enhancementenhancement of model hydrophilic and hydrophobic APIs [[217].217]. The model formulation containing th theseese ILs exhibited a more pronouncedpronounced permeationpermeation enhancement under acidic excess conditions than underunder neutralneutral environments.environments. Despite that these formulations displayed superior controlled release for the hydrophilic model API, thethe mechanismmechanism that was was responsible responsible for for this this behavior behavior was was not not further further explored. explored. Later, Later, the difference the difference between between API- API-ILsILs transport transport across across membranes membranes and the and respective the respective commercial commercial sodium sodium salts was salts studied was for studied salicylic for salicylicacid [210]. acid The [210 membrane]. The membrane transport transport ability and ability rate and of ratethe ILs of thetriethylene ILs triethylene glycol glycolmonomethyl monomethyl ether ethertributylammonium tributylammonium salicylate salicylate ([mPEG ([mPEG3N4443N][Sal]),444][Sal]), tributylammonium tributylammonium salicylate salicylate ([HN([HN444444][Sal]),][Sal]), cholinium salicylate ([Ch][Sal]), and 1-methylpyrrolidiniumsalicylate1-methylpyrrolidiniumsalicylate ([C 1Pyrr][Sal]),Pyrr][Sal]), as as well as the respective sodium salt (Na[Sal]) and neutral form (HSal), was evaluated while using a Franz Franz diffusion diffusion cell system. The cation influence influence in the efficiency efficiency of the membrane transport was also highlighted, as [mPEG3N444444][Sal]][Sal] showed showed a a transport transport enhancement enhancement by by ~2.5 ~2.5-fold-fold in in comparison comparison to to PEG-free PEG-free cations. cations. Zakrewsky et al. [211] [211] investigated the application of ILs in in a a multiple multiple context context for for drug drug delivery, delivery, where the the IL IL [Ch][geranate [Ch][geranate2(H)]2(H)] was was used used not not only only to improve to improve transdermal transdermal delivery, delivery, but also but to also tackle to tackleskin biofilm-protected skin biofilm-protected microbial microbial infections. infections. This IL This allowed IL allowed to increase to increase by 16-fold by 16-fold the delivery the delivery of a model antibiotic, cefadroxil, into deeper skin layers when compared to its aqueous solution. The potential clinical efficacy of the IL formulation was accessed in vivo based on its antimicrobial activity

Int. J. Mol. Sci. 2020, 21, 8298 35 of 50 of a model antibiotic, cefadroxil, into deeper skin layers when compared to its aqueous solution. The potential clinical efficacy of the IL formulation was accessed in vivo based on its antimicrobial activity against biofilm-infected wounds. [Ch][geranate2(H)] enabled reducing Pseudomonas aeruginosa and Salmonella enterica bacterial viability by >95% after 2 h. The IL ability to disrupt the bacterial biofilm allowed for delivering the antibiotic with increased efficacy, improving the pathogens susceptibility to the antibiotic. For topical and transdermal, as it happens for intravenous and oral delivery systems, it is important to understand and better evaluate the activity, in vivo behavior, and safety to achieve more effective and less toxic options with a desired drug activity. Although an increase in in vivo results on the administration of IL-based formulations has been observed, the available information is still scarce. The evaluation of pharmacokinetic and pharmacodynamic parameters as well as the therapeutic efficacy must be encouraged to be pursued to provide missing insights on this strategy. In the future, other IL-(bio)polymer combinations are expected and the understanding of their mechanistic levels should be encouraged to be unveiled. The understanding of these therapeutic options and the increase in the research of ILs in the nanoparticle field will allow for developing targeted-specific drug delivery systems that will reduce drug side-effects and fluctuation in circulating drug levels, optimizing the treatment efficacy. For this, ILs use in drug delivery should be further explored from the use of imidazolium-based ILs to options with low toxicity and known cytotoxicity profiles. Additionally, it is expected an increase in the studies comprising IL-(bio)polymer-based systems with stimulus-responsiveness, and investigation on their ability to protect drugs from degradation while providing controlled drug release.

8. Conclusions and Future Perspectives Organic volatile solvents have been a main choice in the pharmaceutical industry, particularly in the reaction and purification steps, but they still raise concerns on the contamination of the final product and on the related environmental and health impacts. To overcome some of these drawbacks, ILs have been investigated as solvents, reagents, or catalysts in the synthesis of APIs and applied in the crystallization process of drugs. Still, additional studies should focus on the development of more sustainable strategies to remove ILs after the APIs synthesis. A careful monitorization of these contaminants in the final product and study of their impact in the drug’s performance and toxicity must be taking into consideration. Furthermore, the use of solvents and co-solvents, like ethanol, methanol or DMSO, hydrotropes, and surface-active agents, to improve the API’s solubility in pharmaceutical formulations has been challenged by applying ILs to this purpose. ILs have been shown to allow the aqueous solubility improvement of APIs from distinct pharmacological classes in several orders of magnitude (to be best of our knowledge and up to date, up to 5.6 106-fold), standing as competitive × alternatives to organic solvents. However, these formulations require a more comprehensive study in what concerns the stability, absorption and bioavailability of APIs. Furthermore, more recent studies employing aqueous solutions of ILs instead of pure ILs can be the key for their use and acceptance by the pharmaceutical field. Because the APIs’ solubility in aqueous solution and bioavailability can be limited by polymorphism, controlling this process is essential for obtaining a stable and high-quality drug product. The study of ILs in the crystallization process of APIs has enabled the possibility to design new polymorphs, with higher thermal stability, to select the crystal form and habit, and even isolate and purify the correct API polymorph through crystallization. To expand the research on this topic, it is still mandatory to comprehensively understand the IL-API interactions that drive the formation of specific polymorphic forms and habits. The use of computational tools can be helpful in designing ILs in such a way. Furthermore, as it happens with the APIs synthesis, the research on effective separation methods and the limitation of the IL contamination in the final product are highly demanding issues. ILs can be designed to present biological activities due to the broad number of anion-cation combinations. In this sense, ILs have been successfully studied regarding their antimicrobial, Int. J. Mol. Sci. 2020, 21, 8298 36 of 50 antioxidant, and anti-tumoral activities. So far, IL activities have been mainly studied in vitro and have focused on imidazolium-based ILs. To expand this field of research, it is necessary to unveil the mechanisms of interaction between the IL and biological membranes and, consequently, establish a correlation with their biological activities, and in which computational tools may also play a crucial role. The possibility to manipulate the cation-anion combinations also allowed for obtaining new drugs with desired chemical and biological properties, while avoiding polymorphism concerns. API-ILs have provided double action in therapeutic formulations for topical and transdermal delivery, namely by providing the API facilitating its permeation through biological membranes. In this field, API-ILs stand as novel liquid forms that can be designed with a specific or dual pharmacological action, obtained by incorporating APIs as IL ions, using oligomeric ions or by applying a prodrug strategy. However, in this field, only few works conducted bioavailability assays, however allowing to demonstrate the increase in the API’s bioavailability and the therapeutic efficacy of these novel drugs. Because API-ILs can present contrastive, enhanced or even dual effect when compared to the initial precursors, in vivo pharmacokinetic and pharmacodynamic tests are mandatory in understanding the pathways that are involved in their absorption, metabolism, and routes of elimination. These assays are required to enable the acceptance of these new drugs by the pharmaceutical industry, favoring the establishment of guidelines for their development and research. Until their implementation, additional obstacles are expected to be faced. The pharmaceutical industry is majorly prepared in order to produce solid APIs; thus, the scale-up implementation might be a lengthy process. Years of routinely working with solid forms of APIs might make the development of standardized procedures for the liquid drugs’ purification difficult. The flexibility of ILs allowed for the development of tailored (bio)polymer drug delivery systems as well, both due to their polymerizable character and polymer solvation ability. Because ILs can enhance the APIs solubility in aqueous media, they have successfully allowed the incorporation and delivery of several low-water soluble drugs, enabling the consideration of new administration routes. However, the lack of more complete studies on this topic that can assist the conscious development of more effective drug delivery options still confines their use and commercialization. Advances in this area should comprise integrated studies where the IL can be designed with a specific biological activity and/or therapeutic action. These designed ILs can simultaneously have a specific role in the development of the drug delivery systems. In this line, stimuli-responsive drug delivery systems, promoted by the IL and/or by the polymer, also are of particular relevance. Overall, ILs have potential to overcome solubility, bioavailability, permeation, polymorphism, and stability concerns that are associated to solid-state pharmaceuticals. Furthermore, ILs are promising alternatives to volatile organic solvents when applied as solvents, reagents, and anti-solvents in the synthesis and crystallization of active pharmaceutical ingredients (APIs). More recent studies demonstrated their potential to improve the performance of drug-delivery-based systems. The results and advances herein revised support the multiple roles of ILs in the pharmaceutical field, encouraging new ways of taking advantage of their unique properties.

Author Contributions: S.N.P., Writing and Original Draft Preparation; M.G.F., Conceptualization and Review-Editing and Supervision; C.S.R.F. and A.J.D.S., Review-Editing and Supervision. All authors have read and agreed to the published version of the manuscript. Funding: This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. This work was financially supported by the project IonCytDevice (POCI-01-0145-FEDER-031106, PTCD/BTA-BTA/31106/2017) funded by FEDER, through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI), and by national funds (OE), through FCT/MCTE). S. N. Pedro acknowledges the PhD grant SFRH/BD/132584/2017. Conflicts of Interest: The authors declare no conflict of interests. Int. J. Mol. Sci. 2020, 21, 8298 37 of 50

Abbreviations

General 3-CPP 3-chloro-1-phenyl-1-propanone AGB-ILs Ionic liquids analogues of glycine-betaine APIs Active pharmaceutical ingredients API-ILs Ionic liquid comprising active pharmaceutical ingredients BC Bacterial cellulose Cellulose-g-PLLA Cellulose-graft-poly (L-lactide) CrEL Ethoxylated castor oil DHQ 2,3-Dihydroquinazolin-4(1H)-one DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethyl sulfoxide DPPH 2,2-diphenyl-1-picrylhydrazyl FTIR–ATR Fourier transform infrared spectroscopy—attenuated total reflectance HSal Salicylic acid (neutral form) IC50 Half maximal inhibitory concentration IL Ionic liquid IL/O IL-in-oil IL/w IL-in-water LCOS Linoleic acid-grafted chitosan oligosaccharide LFER Linear free energy relationship MIC Minimal inhibitory concentration MBC Minimal biocidal concentration NSAID Non-steroidal anti-inflammatory [Na][Sal] Sodium salicylate O/IL Oil-in-IL PC12 Pheochromocytoma cells PLGA Poly (lactic-co-glycolic acid) QSAR Quantitative structure-activity relationship SAIL Surface-active ionic liquid (S)-CPPO (S)-3-chloro-1-phenyl-1-propanol SEDDs Self-emulsifying drug delivery systems TTAB Tetradecyltrimethylammonium bromide W/IL Water-in-IL Ionic Liquids Ammonium [C4C1im][BF4] 1-butyl-3-methylimidazolium tetrafluoroborate [(C4C1C1m][BF4] 1-butyl-2,3dimethylimidazolium tetrafluoroborate [C6C1im][BF4] 1-hexyl-3-methylimidazolium tetrafluoroborate [C8C1im][BF4] 1-octyl-3-methylimidazolium tetrafluoroborate [(CH2CH=C2)C2m][BF4] 1-allyl-3-ethylimidazolium tetrafluoroborate [C4C1im]Br 1-butyl-3-methylimidazolium bromide [C14C1im]Br 1-methyl-3-tetradecylimidazolium bromide [C8im]Cl N-octylimidazolium chloride [C1C8im]Cl 1-dodecyl-3-methylimidazolium chloride [C1C1C8im]Cl 1-methyl-3-tetradecylimidazolium chloride [C6C1im]Cl 1-hexyl-3-methylimidazolium chloride [C8C1im]Cl 3-methyl-1-octylimidazolium chloride [C12C1im]Cl 1-hexadecyl-3-methylimidazolium chloride [C14C1im]Cl 1-octyl-3-methyl imidazolium chloride [(CH2CH=C2)]Cl 1-allyl-3-methylimidazolium chloride [C2C1im][CH3COO] 1-ethyl-3-methylimidazolium acetate Int. J. Mol. Sci. 2020, 21, 8298 38 of 50

[C2C1im][CH3OHPO2] 1-ethyl-3-methylimidazolium methylphosphonate [C1C1im][(CH3O)2PO2] Dimethylimidazolium dimethylphosphate [C4C1im][C8OSO3] 1-butyl-3-methylimidazolium octylsulfate [C1C1im][C12SO3] Dimethylimidazolium dodecanesulfate [C2C1im][EtSO4] 1-ethyl-3-methylimidazolium ethylsulfate [C4C1im][N(CN)2] 1-butyl-3-methylimidazolium dicyanamide N-ethyl-2-hydroxy-N,N-dimethylethanammonium [C ][NTf ] 2 2 bis(trifluoromethylsulfonyl)amide) [C2C1im][NTf2] 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C4C1im][NTf2] 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C6C1im][NTf2] 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C10C1im][NTf2] 1-decyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C2C1im][CF3O3S] 1-ethyl-3-methylimidazolium trifluoromethanesulfonate [C4C1im][CF3O3S] 1-butyl-3-methylimidazolium trifluoromethanesulfonate [C10C1im][CF3O3S] 1-decyl-3-methylimidazolium trifluoromethanesulfonate [C4C1im][PF6] 1-butyl-3-methylimidazolium hexafluorophosphate [C6C1im][PF6] 1-hexyl-3-methylimidazolium hexafluorophosphate [C8C1im][PF6] 1-octyl-3-methylimidazolium hexafluorophosphate [C4C1im][SCN]) 1-butyl-3-methylimidazolium thiocyanate [(C2)3NC4]Br Triethyl[2-ethoxy-2-oxoethyl]ammonium bromide [C4NH3][CH3COO] N-butylammonium acetate [C6NH3][CH3COO] N-hexylammonium acetate [C8NH3][CH3COO] N-octylammonium acetate [C4NH3][oleate] N-butylammonium oleate [C6NH3][oleate] N-hexylammonium oleate [C8NH3][oleate] N-octylammonium oleate [(C1OC2)C1im][MsO] 1-methoxyethyl-3-methylimidazolium methanesulfonate [C2OHC1im]Cl 1-(2-hydroxyethyl)-3-methylimidazolium chloride [DDA][NO3] Didecyldimethylammonium nitrate [N4,1,1,1][NTf2] N-trimethyl-N-butylammonium bis(trifluoromethanesulfonyl)imide Cholinium [Ch][Ala] Cholinium alaninate [Ch][Ile] Cholinium isoleucine [Ch][geranate2(H)] Cholinium geranate [Ch][Glu] Cholinium L-glutaminate [Ch][Gly] Cholinium glycinate [Ch][Leu] Cholinium leucinate [Ch][Phe] Cholinium phenylalanine [Ch][Pro] Cholinium prolinate [Ch][Se] Cholinium serinate [Ch][Try] Cholinium tryptophan Morpholinium [Nbmd][OH] 4,40-(butane-1,4-diyl)bis(4-dodecyl-morpholin-4-ium)hydroxide Phosphonium [P444(14)]Cl Tributyltetradecylphosphonium chloride [P6,6,6,14]Cl Trihexyltetradecylphosphonium chloride [P6,6,6,14][NTf2] Trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide Pyridinium [C4C1py][DCI] 1-butyl-3-methylpyridinium dichloroiodate [C6C6OCOpy][N(CN)2] 1-hexyl-3-hexyloxycarbonylpyridinium dicyanamide [C6C6OCOpy][NTf2] 1-hexyl-3-hexyloxycarbonylpyridinium bis(trifluoromethylsulfonyl)imide Int. J. Mol. Sci. 2020, 21, 8298 39 of 50

Pyrrolidinium [Pyrr4,1][NTf2] Butylmethylpyrrolidinium bis(trifluorosulfonyl)amide API-ILs [C4C1im][Ibu] 1-butyl-3-methylimidazolium ibuprofenate [(C10)2C1C1im][Doc] Didecyidimethylammonium docusate [(C10)2C1C1im][[Ibu] Didecyldimethylammonium ibuprofenate [(C10)2C1C1im][Pen G] Didecyldimethylammonium penicillin G [(C10)2C1C1im][Sal] Didecyldimethylammonium salicylate [Ch][Amp] Cholinium ampicilate [Ch][BA] Cholinium betulinate [Ch][B3] Cholinium nicotinate [Ch][B5] Cholinium pantothenate [Ch][B6] Cholinium pyridoxylate [Ch][Caf] Cholinium caffeate [Ch][Gal] Cholinium gallate [Ch][Ibu] Cholinium ibuprofenate [Ch][Ket] Cholonium ketoprofen [Ch][Nal] Cholinium nalixidixate [Ch][Nap] Cholinium naproxen [Ch][Nif] Cholinium niflumate [Ch][Sal] Cholinium salicylate [Ch]2[Ell] Dicholinium ellagate [C2OHC1im][Amp] 1-hydroxy-ethyl-3-methylimidazolium ampicilate [C2OHC1im][Ibu] 1-ethanol-3-methylimidazolium ibuprofenate [C1Pyrr][Sal] 1-methylpyrrolidinium salicylate [HN444][Sal] Tributylammonium salicylate [Lid][Asp] Lidocainium acetylsalicylate [Lid][Dicl] Lidocainium diclofenac [Lid][Doc] Lidocainium docusate [Lid][Eto] Lidocainium etodolac [Lid][Ibu] Lidocainium ibuprofenate [Lid][Nap] Lidocainium naproxenum [Ran][Doc] Ranitidine docusate [mPEG3N444][Sal] Triethylene glycol monomethyl ether tributylammonium salicylate [P4444][Ibu] Tetrabutylphosphonium ibuprofenate [P666(14)][Amp] Trihexyl-tetradecyl phosphonium ampicilate [PBu4][Sal]nHm-1 Tetrabutylphosphonium salicylates [ProOEt][Ibu] Ethylester ibuprofenate

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