2.1. Antibacterial Properties of OA-DESs
As many organic acids exhibit antibacterial properties, it seems rational to try to improve their efficiency through their formulation as DESs, given that the DESs physical properties (high viscosity and low vapor pressure) may prolong their interaction with tissues. This should allow OA-DESs, for example, to exhibit a synergistic effect on biofilms and the possibility of controlled API release if needed.
A prime example of the antimicrobial properties of OA-DESs was reported in 2016 by Zakrewsky et al. [
31], who investigated a mixture of geranic acid and choline bicarbonate (CAGE) in a 2:1 molar ratio. CAGE has been tested on 47 pathogens, including bacteria, fungi, and viruses (
Figure 6). It displayed excellent antimicrobial properties while being benign to human cells and tissues. Geranic acid does not mix with water. However, after heating together with water-soluble choline bicarbonate, it could form a stable mixture. CAGE has been shown to have extremely low MBC of around 0.2–0.5% for
M. tuberculosis, Methicillin-resistant
S. aureus (MRSA),
P. acnes,
C. albicans and
A. niger. In addition, it was also very effective against Herpes Simplex Virus (HSV) Type-1 and 2 (
Table 1). Based on dynamic light scattering (DLS) analysis, it was suggested that the possible mechanism of bacterial inhibition occurs through their inactivation rather than destruction by OA-DES.
CAGE has been tested with benzalkonium chloride (commonly found in disinfectants), povidone-iodine and chlorhexidine acetate (presurgical sanitizer) for its cytotoxicity against keratinocytes. CAGE maintained almost 100% cell viability up to 10 mM, while referral substances killed practically 100% of the cells at concentrations as low as 0.01 mM. This result highlights that this OA-DES is an excellent candidate for a new generation of topical partially hydrophobic disinfectants [
31].
Regarding limited hydrophilicity, a fascinating group of organic acids is saturated fatty acids. They share some of the properties of simple carboxylic acids. Still, the presence of saturated carbon chains makes them partially dissolve in lipids, which might be a key to the penetration of the biological membranes. Saturated fatty acids can be used to synthesise DESs; however, during microbiological tests, the factor of diffusion of DESs in the medium must be considered. DESs are liquids, and due to diffusion-related phenomena when compared to solid substances like many antibiotics and disinfectants, they should be investigated by using disk-diffusion assays and assessing their MIC/MBC. In addition, in the case of fatty acids, which do not dissolve in water, a small amount of DMSO has to be used to optimize the measurements and improve their solubility while limiting the cytotoxicity towards healthy (human) cells [
47].
Combining fatty acids with APIs is possible, which results in THEDES. As an example, menthol (M), which is non-soluble in water (0.4 mg/L), is capable of forming stable DESs with stearic (SteA), lauric (LauA) and myristic (MyrA) acids [
48]. Further investigation revealed that a mixture of M:SteA exhibited the lowest cytotoxicity towards HaCaT cells. Stearic acid showed no MIC/MBC due to a complete lack of solubility, while MIC and MBC values of menthol were between 4–8 and 8–16 mM, respectively (depending on the strain investigated (
S. aureus, MRSA, MRSE)). After solubilization in the form of DESs, the MIC decreased to around 3.52–6.52 mM and MBC to 6.52–13.03 mM. Interestingly, DESs of M: SteA have been proven to boost the wound healing process by increasing the recovery of wound area by around 20% compared to control.
The same fatty acids, mixed as binary OA-DESs with capric acid (CapA), have been tested for bacterial eradication and biofilm removal potential [
47]. While none of the OA-DESs inhibited the growth of Gram-negative
E. coli and
P. aeruginosa, they were effective against Gram-positive
S. aureus (MRSA), MRSE and yeast strains of
C. albicans. Such behavior is not surprising as fatty acids’ antibacterial effect might be hindered by the presence of lipopolysaccharides [
52,
53]. Based on the initial results, a mixture of capric acid and lauric acid (CapA:LauA) has been chosen to study the effectiveness of biofilm removal of
E. coli, MRSA and
C. albicans. Interestingly, CapA:LauA was able to eliminate around 80–90% of all the biofilms within 20 min., with more than 50% of the biofilm removal in the first 5 min (
Figure 7). These results indicate that fatty-acid-based OA-DESs might comprise two effects: antibacterial properties (observed by the presence of inhibition halos and MICs/MBCs) and biofilm removal potential. The second effect might result from the dissolution of biofilm components in DESs. Such behavior is justified as a biofilm matrix comprising lipids, extracellular DNA, EPS, and extracellular vesicles [
54,
55].
When considering fatty acid-based OA-DESs, one realizes how broad the definition of DESs has become. As a result, mixtures such as those mentioned above can no longer be classified using the classical quadruple division proposed by Abbott et al. This also highlights the importance of thorough thermal and microscopic (POM) investigation of novel mixtures to prove that eutectic point exists.
Fatty acids seem to be very effective against Gram-positive bacteria. However, most dermal infections are caused by the formation of biofilms composed of Gram-negative and Gram-positive bacteria. Therefore, to increase the effectiveness of OA-DESs, alternative approaches have been studied. Following the idea of CAGE, where much smaller molecules are used, formulations that comprised menthol mixed with phenylacetic (PhaA), acetylsalicylic (AcA) or benzoic (BenzA) acid have been studied as well [
30]. These substances have been proven effective antimicrobial agents [
56,
57,
58,
59,
60,
61]; thus, their DESs formulations have been assessed against
E. coli,
S. aureus and
B. subtilis. In their approach, Aroso et al. [
30] applied the above-mentioned acids as APIs, whose effects have been boosted by menthol. All the formulations have been effective against all the tested strains with MIC as slow as 1 mg/mL for
B. subtilis and as high as 4 mg/mL for Gram-negative
E. coli.
In the case of DESDs, ternary systems comprising organic acids have also been tested. One of the examples is the OA-DES of betaine (B), malic acid (MalA) and glucose (G) or proline (P) in a 1:1:1 ratio [
49]. Betaine acts as an HBA, while malic acid and glucose or proline are HBDs. This mixture seems to be an excellent formulation due to the use of small metabolites expected to exhibit low cytotoxicity and good biocompatibility. The formulations were effective against
E. coli,
P. mirabilis,
S. Typhimurium,
P. aeruginosa and
S. aureus. In the same study, the authors demonstrated that OA-DESs that comprised citric acid (together with glucose or fructose and glycerol) were even more effective towards the same strains of bacteria. It also seems that using small metabolites as PSs affected the EC
50 values towards HeLa, HEK293T, and MCF-7 cells with values over 2 mg/mL. The joint effect of high antibacterial potential with low cytotoxicity makes such combinations even more promising materials, which may be looked into in the near future.
Based on the examples above, we can state that OA-DESs own great potential as active antimicrobial agents. Not only do they manage to kill Gram-positive effectively, but also Gram-negative bacteria. The latter is particularly interesting due to another double-lipid membrane and periplasmic space, which inhibit most substances from penetrating their cytoplasm. Also, as evidenced for fatty acid-based OA-DESs, particular specificity may be reached based on the presence of the Gram layer. OA-DESs have proven to be also effective against MRSA, MRSE and HSV—pathogens being hard to neutralize. This makes OA-DESs a promising alternative to small-molecule antibiotics. Even though not every reported OA-DES is soluble in water (i.e., ones that comprised menthol)—they can also be applied topically for the potential removal of biofilms, which should enhance the process of wound healing. Overall, OA-DESs are a group of materials which will be developed further in this direction, even though more testing for their cytotoxicity towards healthy human cells is required. They act as antibiotics while also having protein extraction potential, which is rarely tested ex vivo.
2.2. Drug Delivery Using OA-DESs
Another valuable aspect of medicine is drug delivery. However, drug delivery often encounters the problem of low bioavailability, which could be due to several factors:
route of administration
permeability
solubility
stability
There are different ways of tackling these challenges. In general, one can either change the route of administration (if possible), chemically modify the drug to enhance its solubility, and/or encapsulate it to improve its solubility and stability. Different strategies have been proposed over the recent years, but the general target is the same, i.e., achieve the lowest possible final drug concentration, which still ensures its effectiveness. That is why technologies targeted at improving drug permeability while increasing solubility and maintaining stability are crucial to resolving this hurdle. Here, OA-DESs might be a handy tool to ensure all these aspects. As OA-DESs function as microenvironments, they provide an elastic hydrogen bond lattice able to stabilize most of the molecules. Furthermore, depending on the composition of OA-DESs, one can alter the hydrophilicity of the solvent, enabling more significant dissolution of drugs that poorly dissolve in water. The high viscosity of OA-DESss also limits the contact of the API with oxygen, which prevents it from atmospheric oxidation.
The first ever work on the use of DESs in the delivery of API was reported in 1998 by Stott et al. The authors proposed OA-DESs, composed of ibuprofen and terpenes:
ld-menthol,
l-menthol, thymol, and 1,8-cineole [
28]. By measuring the flux of ibuprofen, they found that all the investigated terpenes enhanced the flux of ibuprofen, with thymol-driven enhancement by 11 times. This work sparked interest in transdermal delivery enhancement by DESs—mainly, that organic acids seemed to be excellent HBDs.
Following that, transdermal delivery of ibuprofen using OA-DES was reported in 2015 by Park and Prausnitz [
62]. Their study demonstrated the effectiveness of ibuprofen/lidocaine DES in vivo as an anesthetic. In addition, they found out that the solubilization of lidocaine with ibuprofen affected its permeability and increased the absorption of lidocaine in rat models. Another example of the application of ibuprofen as PS of OA-DES was described in 2019 by Pereira et al. [
63]. They have investigated four formulations of limonene with menthol, capric acid or ibuprofen. All the OA-DESs were effective antiproliferative agents, even though only OA-DES of ibuprofen and limonene (in 1:4 ratio) inhibited HT29 proliferation without comprising cell viability, inferring that this formulation is a potential anti-cancer drug and similar formulations may be developed soon, either as a direct use as a drug or as a medium for synthesis of other drugs [
64].
In the case of drug delivery, non-steroidal anti-inflammatory drugs (NSAIDs) are also of particular interest. Such drugs as ibuprofen, acetaminophen or naproxen have poor solubility in water, and thus their effect may be inhibited when administered in aqueous solutions. Here, OA-DESs may be a viable means, as they can improve the solubility of different substances. Formulations proposed by Lu et al. [
65] seem to be promising. Mixtures of different HBAs/HBDs proved very effective when solubilizing NSAIDs. Among the 18 tested formulations, 10 of them comprised organic acids. For HBAs, choline chloride, choline bitartrate (ChBT), betaine, tetrapropylammonium bromide (TPAB) and ethylammonium chloride (EACl) were investigated. At the same time, malonic (MaloA), oxalic acid, levulinic acid (LevuA), lactic acid (LacA), glutaric acid (GluA), and glycolic acid (GlycA) have been tested. Stable OA-DESs have been assessed as solvents for five NSAIDs: aspirin, naproxen, ibuprofen, ketoprofen and acetaminophen (
Table 2). The results were impressive—thanks to the use of OA-DESs, the solubility of aspirin and acetaminophen was increased 5 to 20 times. As for the poorly soluble drugs (ibuprofen/naproxen/ketoprofen), OA-DESs improved their solubility by 4000 times. As the type of HBD seems to play a significant role in the solubility of API, HBA will also affect this. A comparison of OA-DESs comprising levulinic acid revealed that switching ChCl for other HBA improved the solubility of all the drugs, especially those poorly dissolving in water.
It is worth noting here that OA-DESs have also been applied as effective antibiotic carriers. In their original work, Zakrewsky et al. [
31] used CAGE to treat ear infections in vivo on a rat model. When compared with saline, CAGE itself was much more effective than 1% solution of clindamycin. However, combining the two reduced rat ear thickness by ca. 70% after four days, compared to the eight days required for pure CAGE. This indicates that CAGE successfully boosted the antibacterial effect of the antibiotic by improving its transdermal delivery.
However, the inner dissolution of substances is also an important criterion when it comes to the synthesis of DESs. Based on the work of Aroso et al. [
30], the dissolution efficiency of three APIs—PhaA, AcA and BenzA increased when combined with menthol to form a THEDES (
Table 3). PhaA, AcA and BenzA have been previously investigated for their antimicrobial properties and proved to be effective agents against fungi and bacteria [
66,
67,
68]. Interestingly, MIC and MBC values of the tested DESs were higher than those of pure PSs. This indicates that, even though the dissolution of the APIs has increased, their effect has been slightly hindered. In contrast, similar studies by Duarte et al. [
29] showed that OA-DES formulations of ibuprofen, benzoic acid and phenylacetic acid with menthol had much better permeability and diffusion coefficient when compared to neat parent substances (
Table 3).
High permeability and a high diffusion coefficient resulted in much lower diffusion times through polyethersulfone membranes, making this type of OA-DESs a very promising delivery system. As the influence of the structure of HBA/HBD was not so often discussed, the authors used NMR to assess how the structure of organic acid affects diffusion and found that the size of the API molecule does not influence its diffusion [
29].
OA-DESs and DESs are good solvents for the dissolution of small molecules and for much bigger, high-molecular-weight biomolecules. Here, one of the first studies conducted in 2013 by Choi et al. [
69] demonstrated that DNA (from male salmon), albumin, and amylase displayed good solubility in certain NADESs, with DNA having 46% higher solubility in OA-DESs consisting of malic acid and proline when compared to water. Following this work, Dai et al. [
70] assessed more than 70 NADESs for solubilizing small biomacromolecules. In the final group of 5 tested NADESs, OA-DES of lactic acid, glucose, and water seemed particularly effective in dissolving gluten (88 times more effective) and DNA (34 times more effective). Moreover, the same OA-DESs enabled the dissolution of starch up to a concentration of 7.5 mM.
Proteins themselves are interesting components in the aspect of their delivery. However, their structure-dependent function relies on many weak interactions, namely hydrogen bonds, hydrophobic interactions, π-π stacking, and electrostatic interactions (including van der Waals forces) [
71,
72]. This makes their function susceptible to environmental changes, often limiting their availability and solubility. Here, OA-DESs, characterized by highly developed hydrogen bond lattice and elasticity of molecules with high coordination potential, might be interesting. Choi et al. [
69] first raised awareness of the role of NADESs, naturally found in living organisms, on the stability of proteins. This hypothesis was reevaluated in 2013 by Esquembre et al. [
73], in 2015 by Su and Klibanov [
74], and in 2017 by Sanchez-Fernandes et al. [
75] using model proteins and demonstrated that DESs are well-structured and activity preserving solvents.
Parallel to the studies mentioned above, applications of choline-based OA-DESs in the dissolution and transport of certain proteins have been conducted (
Table 4). In 2018, Tanner et al. [
33] delivered transdermal insulin using CAGE and its variants. Formulations of 1:1, 1:2, 1:4 and 2:1 of CAGE have been investigated for their conductivity and viscosity, thermal stability as well as by NMR to characterize the internal interactions of solvents. In their research, the authors first studied the interactions of CAGE formulations on the
stratum corneum (outermost layer of skin) to characterize the possible transport mechanism. As revealed by FT-IR spectroscopy, due to its partial hydrophobicity, CAGE acts as a solvent for lipids, disrupting lipid bilayers. Moreover, the extraction potential strictly depends on the amount of geranic acid in CAGE, with a 1:4 ratio being as effective as the neat geranic acid itself. Interestingly, though the 1:2 and 1:4 mixtures were the most effective in transporting insulin (as verified by confocal microscopy using diffusion Franz cell), the effectiveness of other ratios was comparable to PBS.
Furthermore, OA-DESs have also been used to transport biomolecules such as monoclonal antibodies. These specific antibodies are currently used in the therapy of (among others) cancer [
77,
78,
79] and arthritis [
80,
81]. They are usually administered via subcutaneous or intravenous injection, but OA-DESs might open a possibility for their delivery via the gastrointestinal tract. In their recent work, Angsantikul et al. [
76] studied the effectiveness of a mixture of choline chloride and glycolic acid (“CGLY”, in 2:1, 1:1 and 1:2 molar ratios) in the transport of TNFα antibody, focusing on the stability of the antibody in OA-DESs, in vitro transport and in vivo uptake. Interestingly, CGLY (2:1 and 1:1 ratios) had a negligible effect on the stability of the antibodies, which have retained their high binding activity even at 20%
v/
v of saline. At the same time, the 1:2 mixture inhibited the binding almost wholly. CGLY 2:1 and 1:1 did not affect the functionality of TNFα when stored at 4 °C; however, the 2:1 ratio inhibited the binding ability of the antibody when stored at room temperature. Cell viability on the Caco-2 cell line resulted in ratio-dependent IC
50—the smaller the fraction of choline bicarbonate, the lower the IC
50. The ratio of 2:1 turned out to be the best candidate and was tested further. The final results have underlined that CGLY 2:1 was an excellent transporter of the antibody, exerting no adverse effect on the rat model. Interestingly, thanks to its micro-extraction potential, this OA-DES decreased the viscosity of mucus, which enhanced the antibody uptake.
An interesting formulation of OA-DES was also proposed by Haraźna et al. [
82]. They have investigated bacteria-derived hydroxy fatty acids HFA as HBDs, while three ammonium salts, namely choline chloride, tetrabutylammoniummethyl chloride (TBMACl), and 1-ethyl-3-methylimidazolium chloride (EMlmCl) served as HBAs. The formulated OA-DESs have been compared with similar DESs where, instead of a mixture of HFAs acids, a mixture of aliphatic nonanoic and heptanoic acids (FA) was used (
Table 5). Obtained data suggested an HBA-dependent solubility of lignin in the investigated OA-DESs. EMlmCl seemed to be the best HBA in the investigated formulations, as the solubility of lignin increased 100 times (up to 30 mg/mL) compared to neat PS. On the other hand, the efficiency of TBMACl was much lower, and for HFA-based OA-DESs, it was only 2–3 times higher than that of PS and 7 and 15 times higher when FA was used. Surprisingly, ChCl was the least effective as an HBA, and lignin dissolution was only observed for one of the four investigated OA-DESs.
Looking at all the examples above, we can clearly state that OA-DESs significantly impact the dissolution of drugs. Not only do they improve the solubility of small molecular drugs, but they can also act as APIs themselves. Owing to their physicochemical properties, they seem to be an excellent environment for the dissolution of high molecular weight molecules such as proteins and peptides—a challenge we still face today. It must be pointed out that even though using OA-DESs increases the solubility of certain APIs, reaching therapeutic concentrations is still a challenge. Here again, we must look at the main factors limiting drug bioavailability. If by implementing OA-DESs, we can increase the solubility and stability of the API for certain routes of administration. More drugs will reach the target. If more drugs will be delivered using OA-DESs compared to other administration strategies, then adaptation of OA-DESs is beneficial and a frog leap in this field. Hopefully, in the following years, we will see a clear development in this topic and, finally, a successful application in vivo.