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When solids become liquids: natural deep eutectic solvents for chemical process engineering

Periodic Reporting for period 3 - Des.solve (When solids become liquids: natural deep eutectic solvents for chemical process engineering)

Reporting period: 2019-04-01 to 2020-09-30

Sugars, aminoacids or organic acids are typically solid at room temperature. Nonetheless when combined at a particular molar fraction they present a high melting point depression, becoming liquids at room temperature. These are called Natural Deep Eutectic Solvents – NADES. NADES are envisaged to play a major role on different chemical engineering processes in the future, playing a significant role towards the development of greener and sustainable processes. Nonetheless, there is a significant lack of knowledge on fundamental and basic research on NADES, which is hindering their industrial applications. For this reason it is important to extend the knowledge on these systems, boosting their application development. NADES applications go beyond chemical or materials engineering and cover a wide range of fields from biocatalysis, extraction, electrochemistry, carbon dioxide capture or biomedical applications. Des.solve encompasses four major themes of research: 1 – Development of NADES and therapeutic deep eutectic solvents – THEDES; 2 – Characterization of the obtained mixtures and computer simulation of NADES/THEDES properties; 3 – Phase behaviour of binary/ternary systems NADES/THEDES + carbon dioxide and thermodynamic modelling; 4 – Application development. Starting from the development of novel NADES/THEDES which, by different characterization techniques, will be deeply studied and characterized, the essential raw-materials will be produced for the subsequent research activities. The envisaged action involves modelling and molecular simulations. Des.solve will be deeply engaged in application development, particularly in extraction, biocatalysis and pharmaceutical/biomedical applications. The knowledge that will be created in this action is expected not only to have a major impact in the scientific community, but also in society, economy and industry.
Development of NADES and THEDES
Production and characterization of NADES and THEDES
The production of NADES and THEDES will be prepared either by mixing the two components until a clear viscous liquid is obtained or after the evaporation of water solutions of each component in a rotary evaporator. NADES were prepared from different combinations of raw materials which include amino-acids, organic acids, sugars and choline derivatives. The choice of the combination is not completely random, as in the past couple of years a lot of new systems have been described and it is of general knowledge that a hydrogen bond donor and a hydrogen bond acceptor molecule are required. In this action, some of the molecules that were studied include menthol, lauric acid, stearic acid, myristic acid, ascorbic acid, citric acid, tartaric acid, galic acid, l-proline, sucrose, glucose, mannose, and xylose. Regarding the formulation of therapeutic mixtures, anti-inflammatory agents such as ibuprofen, flurbiprofen, naproxen, acetylsalicylic acid, anti-oxidants such as mandelic acid or antibacterials such as fatty acids were tested.
The different systems prepared and fully characterized are presented bellow in each one of the categories for which it was studied. Additionally, the characterization of the systems was carried out according to the expected application of each system. The characterization of NADES/THEDES involves the application of several different techniques, such as the water content determination; density and viscosity assessment; polarity; thermal behavior and cytotoxicity evaluation.

Modelling thermophysical data
Deep eutectic solvents (DESs) mostly comply with the principles of green chemistry. Therefore, they are being widely investigated due to their great potential for a variety of applications in the industries. However, it is necessary to have accurate (thermo)-physical property information on any solvent before it can pave its way into the industries. Among such properties, density as a function of temperature is one of the most important. In this study, a simple, accurate and global correlation is proposed to estimate the densities of a large number of DESs of different nature. The proposed model is a function of the critical temperature, critical volume and acentric factor of the DES, as well as the temperature of the system. In this way, the model is capable of accurately predicting the density of DESs without the need for any reference-temperature density data, in contrast to most of the literature models that do require a reference data point. The average relative error for 149 DESs of different natures was estimated to be 3.12% by the proposed model, indicating its accuracy compared to previous models.

Phase behaviour and theoretical modelling of binary and ternary mixtures of NADES/THEDES and supercritical carbon dioxide
Phase behavior – vapour liquid equilibrium
The implementation of this task has started late 2018 and therefore no major scientific results were obtained up to date. However, the team has started to determine the vapor-liquid equilibrium (VLE) of the binary mixtures THEDES + CO2 as this is crucial for their formulation (task 4.3). To be able to design formulation processes, such as particles from gas saturated solutions (PGSS) it is important to determine the VLE of the pseudo-binary system DES + CO2. THEDES systems based on anti-tuberculosis drugs ethambutol and L-arginine were studied. These will allow the understanding of the impact of its thermodynamic state in the design of innovative systems, from the appropriate choice of starting materials, conditions and technique, to the required final product properties or application outcome. In this work, the VLE was studied by determining pressure bubble points of different THEDES:CO2 ratios, in isothermic conditions, using variable-volume view cell methodology. Results suggest that it is possible to formulate drug delivery systems using the THEDES herein described by PGSS process, without compromising the integrity of the system

Application development
Extraction of natural bioactive compounds and lignocellulosic biomass processing
The Mediterranean diet is now recognized as healthy and with high nutritional value. Therefore, it is envisaged that the same value can be mined from the by-products and residues from the Mediterranean food industry to apply as dietetic supplements or cosmetics. NADES will be evaluated as extraction agents for polyphenols (gallic acid, resveratrol, chlorogenic acid, etc.) from grape pomace, apple pomace, spent coffee grounds or olive oil residues; carotenoids (lutein, beta-carotene, lycopene, etc.) from pumpkin residues, pepper residues and tomato residues; and flavonoids from broccoli residues; among others. The extracted material will be chemically characterized by HPLC and mass spectroscopy (MS) and evaluated for the antioxidant potential using common antioxidant tests (e.g. Folin-Ciocalteu, ABTS, DPPH, HORAC, ORAC and TBARS).
Selection of NADES as solvents for anti-oxidants extraction
Recently, the use of DES as an extraction media of bioactive compounds has surfaced as a greener alternative to common organic solvents. In order to determine suitable systems for the extraction of phenolic compounds from agricultural by-products a study of the state of the art was conducted. A comprehensive review was carried out to the systems reported in the literature and the data was grouped so that a rationalization between the systems studied, extractables and outcomes of the process could be drawn. From this study we can conclude that systems using choline chloride and either an organic acid or an alcohol have been mostly used, respectively in the extraction of flavonoids and phenolic acids. These systems show great promise in the extraction of phenolic compounds, namely flavonoids and phenolic acids. Furthermore, the functional group of the hydrogen bond donor affects the selectivity of the DES with organic acids and alcohols respectively extracting flavonoids and phenolic acids. In this part of the work we highlight the efficient yields reported with the functional groups present in the DES studied, and relate it with the available physico-chemical properties of the DES.
Extraction of polyphenols from Yerba-Mate
Superfoods have gained such a reputation due to their vast benefits that their consumption started to be almost mandatory for a healthy lifestyle. The antioxidant, anti-inflammatory, anti-obesity and cardioprotective effects typically associated to Mate, revolutionized the Mate market, which is now being commercialized worldwide. Mate is mainly composed by xantines, triterpenic saponins and phenolics, the major one responsible for all the good benefits, which accounts for 7-10% on dry basis. Recently, Mate tea started to be associated to some cancers, leading to questioning the real benefits of the tea. However, it was proven that the actual problem behind it was the temperature at which tea was drunk. Consequently, to have all the Mate potential, and overcome this disadvantage, there is the need to develop new ways to consume Mate. Phenolics are often extracted from agro-industrial products and incorporated in food products and nutraceutical supplements. One possible idea that might help reducing the heat problem, passes through the possibility of extracting these beneficial compounds and producing a shot of Mate phenolics that can be drunk, at more acceptable temperatures. In order to produce those, phenolics and other bioactive compounds from mate must be extracted with biocompatible and non-toxic solvents. A fractionated extraction using different classes of natural deep eutectic systems (NADES) was carried out and the optimization of the extraction conditions was performed, particularly in which concerns solid/liquid ratio, temperature and the use of ultrasounds.
The NADES used were menthol:lauric acid (2:1), lactic acid:glucose:water (5:1:3) and lactic acid:glycine:water (3:1:3). The systems were characterized by determination of polarity, using Nile red as solvatochromic probe and the water content was determined by Karl Fischer titration and Moisture analyzer. The viscosity and density of the systems as a function of temperature was also assessed. The results demonstrate that a sequential extraction of the mate leaves using first the system menthol:lauric acid and in a second step the lactic acid-based NADES leads to the production of two distinct extracts, the first rich in chlorophylls and beta-carotenes and the second rich in polyphenols, such as chlorogenic acid and, caffeine, among others. Finally, our studies demonstrate that the incorporation of the extract in the DES is beneficial for the stabilization of the antioxidants over time, maintaining their potential for up to 90 days.
Lignocellulosic biomass processing
Processing of lignin, cellulose and hemicellulose, from different biomass sources, such as forest and agricultural wastes will be evaluated. Particular interest will be devoted to the development of NADES that can solubilize or produce pure cellulose fractions of reduced crystallinity. In relation to the proposed, four natural deep eutectic solvents (NADES) systems were prepared at specific molar ratios, La:Bet (2:1) (lactic acid/betaine), La:Hist (9:1) (lactic acid/histidine), Ma:Bet:H2O (1:2:3) (malic acid/betaine/water) and Ma:Bet:Pro:H2O (1:1:1:2) (malic acid/betaine/proline/water). Their physical and thermodynamic properties were studied, namely viscosity, electrical conductivity, and heat capacity. The viscosity and electrical conductivity were determined as a function of temperature and the correlation for the temperature dependence was obtained and discussed based on Arrhenius theory. The heat capacity for all eutectic systems was measured by differential scanning calorimetry (DSC) over a temperature range of 293.15−363.15 K. The ability of these NADES to reduce cellulose crystallinity was evaluated. Cellulose crystallinity after suspension in these NADES was studied by X-ray diffraction. Cellulose suspended in Ma/Bet/H2O (1:2:3) suffer the highest crystallinity reduction among the systems studied and was about of 20%.

In this task the use of NADES as biocatalysis media will be studied for the transesterification of sec-alcohols for pharmaceutical applications, e.g. (R,S)-1-Phenylethanol and (R,S)-Menthol. In a first approach the transesterification of rac-1-phenylethanol with different vinyl esters using novozym 435 will be the model reaction of choice in order to gain a better understanding on the effect of NADES. The ultimate goal is however to study other sec-alcohols such as menthol. After the reaction step, and although as different chemical entities, the two enantiomers still coexist in the same phase. NADES similarities with ILs envisaged the viability of NADES to be used as extraction media in biphasic systems, particularly coupled with another green technology such as scCO2.The selective biotransformation of one enantiomer is the first step of the overall process which final cost depends heavily on the separation steps that follow and which may account for as much as 70% of the total production costs. A radical innovation will be the use of NADES as both solvent and substrate, in a solvent free reaction media approach. Because NADES are composed of organic molecules it will be possible to prepare a NADES with the two substrates of interest, a sec-alcohol and a carboxylic acid. Adding a lipase to the media, the esterification of one of the enantiomers of the alcohol would occur forming an ester. This product, being solid at room temperature and not part of the eutectic mixture, precipitates and can hence be recovered.
Enzymatic resolution is an effective route for the separation of racemic (rac) mixtures, and is thus of great importance for the pharmaceutical, food and cosmetic industries. In this work, we report on the use of DES in the enzymatic resolution of menthol, one of the largest traded flavours and aromas worldwide. Solid rac-menthol was mixed with solid lauric acid at different molar ratios, yielding a liquid at room temperature or slightly above. In this case rac-menthol was esterified with lauric acid in the absence of added solvent. The two solid species were combined to form a liquid DES that promptly reacted to yield menthyl laurate upon addition of Candida rugosa lipase. Only recently are DES starting to be used simultaneously as solvent and source of substrates for an enzymatic reaction. Compared to a previous report, our work goes one step further by using the racemic alcohol as DES component instead of just one of the alcohol enantiomers, thereby addressing the separation of the two alcohol isomers as well. The use of DES combined with enzymes paves the way for efficient and more sustainable biocatalytic processes.
When working with eutectic systems, it is important to know if the molar ratio used is suitable for the intended applications. In this case, it was important that the DES, in their dual capacity as source of substrates and solvent, were liquid at the temperature of reaction and did not hamper the diffusion of reaction species. Therefore the phase behaviour of the DES used was studied, and viscosity measurements obtained. Esterification reactions of rac-menthol with lauric acid were then carried out in (1:1), (2:1) and (3:1) M:LA DES, and the influence of several experimental parameters was assessed, such as substrate molar ratio, temperature, water activity. The two DES components reacted promptly when Candida rugosa lipase was added, and the conversions of rac-menthol reached 26-54% after 6 hours of reaction. Enzyme activity was dependent on the molar ratio of the substrates, as well as on the water activity in the DES.
Because the physical state of the DES varies with temperature, heating and cooling cycles may be an effective way to separate reaction species, and thus achieve an effective separation of enantiomers. This approach also relies on the SLE diagram of the system.
This work reinforces the notion that knowledge of the thermophysical behaviour of DES is important for the design of processes using these solvents, as in the case of enzymatic resolution and enantiomer separation.
Development of pharmaceutical and/or biomedical products
The preparation of THEDES by itself is a major contribution beyond the state of the art. The ability to form a liquid formulation of an active pharmaceutical ingredient may enhance the bioavailability and drug pharmacokinetics. The THEDES prepared were herein studied per se or in combination with a delivery system.

THEDES with antibacterial properties
In order to limit bacterial infections during wound treatment, it is interesting to consider the concept of loading
medical devices with antibacterial agents. With this in mind, an innovative system with thermosensitive properties was produced: loading a commercially available gauze with a fatty acid eutectic blend based on lauric acid (LA) and myristic acid (MA). This eutectic blend presents a melting point near physiological temperature, which together with its antibacterial properties make an appealing alternative in biomedical applications. At room temperature, the properties and the efficacy of the eutectic blend loaded onto gauzes are preserved, whereas at physiological temperature the eutectic blend undergoes a phase change that facilitates its diffusion from the gauze.

THEDES as wound healing enhancers
The breakthroughs achieved in recent years by deep eutectic solvents boost the appearance of different subclasses of these green solvents, among which are therapeutic deep eutectic solvents (THEDES), which possesses intriguing possible applications in the biomedical field. Thereby, the purpose of this study was to unravel the biomedical potential of hydrophobic THEDES based in menthol and saturate
d fatty acids (e.g. lauric acid (LA), myristic acid (MA) and stearic acid (SA)). Our comprehensive strategy resulted in the thermophysical characterization of different formulations, which allows to identify the appropriate molar ratio, as well as the intermolecular interactions behind the successful formation of THEDES. The evaluation of their biological performance was also performed towards bacteria and HaCaT cells. Among the different formulations of THEDES, the one based on menthol and SA, establishes stronger hydrogen bonding interactions, being also the most promising formulation as did not elicit any relevant cytotoxicity, and potentiate wound healing while presenting antibacterial properties against Staphylococcus aureus and. Staphylococcus epidermis strains, some of which methicillin resistant. This work provides clues on the future use of THEDES based on menthol:SA as potential candidates for the preparation of wound dressings.

THEDES with anticancer properties
Deep eutectic solvents have been recently reported as an interesting alternative to improve the therapeutic efficacy of conventional drugs, hence called therapeutic deep eutectic solvents (THEDES). Herein, different THEDES based on limonene (LIM) were developed to unravel the anticancer potential of such systems. THEDES were prepared by gently mixing capric acid (CA), menthol or ibuprofen (IBU) with LIM. Successful eutectic mixtures were obtained for Menthol:LIM (1:1), CA:LIM (1:1), IBU:LIM (1:4) and IBU:LIM(1:8). LIM-based THEDES were evaluated in terms of their cytotoxicity and antiproliferative effects. The results indicate that all the THEDES present antiproliferative properties, but IBU:LIM (1:4) was the only formulation able to inhibit HT29 proliferation without comprising cell viability. Therefore, IBU:LIM (1:4) was the formulation selected for further assessment of anticancer properties. The results suggest that the mechanism of action of LIM:IBU (1:4) is different from isolated IBU and LIM. Thus, THEDES comprises the protective and anti-inflammatory properties of IBU allied to the anticancer properties of LIM.

THEDES with anti-tuberculosis properties
The treatment for tuberculosis infection usually involves a prolonged regimen of multiple antibacterial drugs, which might lead to various secondary effects. For preventing drug resistance and side-effects of anti-tuberculosis drugs, new methods for improving the bioavailability of APIs were investigated. The strategy proposed consists at the preparation of therapeutic deep eutectic solvents (THEDES), that incorporate L-arginine and ethambutol. The eutectic mixtures were prepared by mixing the components at a certain molar ratio, until a clear liquid solution was formed. The solubility and permeability of the drugs when they are in the THEDES form was evaluated at 37°C, in phosphate buffered saline (PBS). Solubility studies showed an increase of the solubility of ethambutol when incorporated in the eutectic system. The cytotoxicity was evaluated using a model cell line (Caco-2), comparing the cytotoxicity of the API and with the API incorporated in the eutectic system. We observed that the cell viability in the THEDES was affected by the presence of citric acid, and higher cytotoxicity values were observed. Nonetheless, these findings do not compromise the possibility to use these systems as new delivery systems for ethambutol and arginine.

Development of novel formulations based on THEDES
The design of carriers able to deliver and protect the API’s until they reach the specific site of action, is not novel, nonetheless the preparation of a delivery system for a liquid is still a challenge to pharmaceutical sciences. The encapsulation of the bioactive compounds is one strategy that will be investigated. The preparation of particles from a gas saturated solution, so-called PGSS will be used. In this technique the gas, i.e. supercritical carbon dioxide, is dissolved in the THEDES + carrier mixture and the solution is expanded through a nozzle. The high shear-stresses which are generated in the process of dispersion contribute to the precipitation of small particles with a narrow particle size distribution. In this task it is predicted the optimization of the operating parameters to render a high quality product.

THEDES with anti-tuberculosis properties
The use of THEDES in combination with supercritical fluid technology using CO2 to explore controlled drug delivery systems has been explored. However, there is scarce information in the literature regarding the fundamentals of DES and CO2 mixtures in terms of phase equilibrium is a limiting factor. In this study, the possibility to encapsulate L-arginine-based THEDES in a lipidic matrix was evaluated. The phase equilibrium of THEDES and CO2 was studied to select the sc-CO2 method for THEDES encapsulation, namely, Particles from Gas Saturated Solution (PGSS) or Rapid Expansion of Supercritical Solutions (RESS). To be able to design formulation processes, such as particles from gas saturated solutions (PGSS) or rapid expansion of supercritical solutions (RESS) it is important to determine the VLE of the pseudo-binary system DES + CO2.
Results suggest that it is possible to formulate drug delivery systems using the THEDES herein described by PGSS process, without compromising the integrity of the system. The influence of the THEDES water-content in PGSS processing was also studied. Herein, the encapsulation of THEDES through PGSS was accomplished for the first time, with an average encapsulation efficiency of 75%. The results suggest that it is possible to formulate drug delivery systems using the THEDES herein described by PGSS process, without the interference of the THEDES-water content. Furthermore, the cell viability was measured in L929 fibroblasts comparing the cytotoxicity of the THEDES and the particles with THEDES encapsulated, through MEM extract test and the results show that the systems prepared are non-cytotoxic.

THEDES with antibacterial properties
Another strategy would be, upon optimization of the operating conditions of the PGSS process, the particles will be dispersed against a target substrate, for example a commercially available patch, the development of anti-bacterial or anti-inflammatory transdermal drug delivery patches for wound management.
The loading of the eutectic blend, based on lauric acid (LA) and myristic acid (MA), onto gauzes was performed using two different supercritical fluid technologies, namely, particle from gas saturated solutions (PGSS) and a derived version of rapid expansion of supercritical solution (D-RESS). The PGSS led to a heterogeneous dispersion of the eutectic blend in the gauze, whereas the D-RESS process led to the formation of a homogeneous dispersion along the surface of the gauze.
Additionally, with D-RESS no phase separation of the eutectic blend occurred, and the cytotoxicity was greatly improved compared with PGSS without compromising the antibacterial properties of the fatty acid eutectic blend. Hence, the present study highlights the potential use of the flexible D-RESS process to load the fatty acid eutectic blend with antibacterial properties onto medical devices in a controllable way. Overall, the effects produced by the loaded gauzes suggest the enormous potential of the developed technology in health-related areas.
Despite the knowledge that has been generated within Des Solve there are still many questions that remain unanswered. Until the end of the project efforts will be made to respond to the challenges proposed in Des Solve. Particularly, to enlighten the propensity of different pairs of compounds to form an eutectic system, to perceive the role of water on the properties of these new entities and develop molecular simulations and computational models which explain NADES formation. A theoretical approach based on molecular simulations to understand the mechanisms of formation of NADES will be put in place. Molecular simulations based on classical models play an important role on the understanding of how condensed phase properties are linked to chemical structure and composition. Molecular simulations of NADES and THEDES will be performed using a Monte Carlo simulation approach. This methodology is based on atomic charges, which are not always known. These can be predicted through charge derivation methods, such as the Restrained Electrostatic Potential (RESP) method. Molecular simulations will allow a better understanding of NADES/THEDES structure and the prediction of some relevant properties such as transport properties. Simulations will be complemented with experimental data from the characterization of the systems performed earlier, particularly the spectroscopic analysis (FTIR and NMR) of the interactions of the molecules which compose the NADES.
Under a different topic, not yet fully explored within Des Solve is the study of the phase behaviour of NADES in the presence of supercritical fluids and thermodynamic modeling of the binary and/or ternary mixtures. The prediction of the phase behavior will be performed using the Cubic Plus Association (CPA) Equation of State (EoS) and optimizations will be done using Genetic Algorithm.The design of separation processes requires the knowledge of the phase equilibrium. However, data regarding phase equilibra for systems involving NADES are almost inexistent in literature. For that purpose, binary phase equilibrium studies for the system NADES/scCO2 will be performed to determine the solubility of scCO2 in different NADES and the solubility of NADES in scCO2. The latter is essential to evaluate the feasibility of a two phase extraction system where virtually no NADES is lost in the gas phase. In line with this, modelling plays here an important role towards the design and scale-up of the processes envisaged (separation/extraction and formulation). Experimental equilibrium data of the binary and ternary systems, will be hence required to validate the models. The CPA EoS is among the best EoSs to study the phase behavior of associating mixtures. CPA actually adds to the physical interactions of a simple cubic EoS, a term that takes into account the contribution of the association interactions between molecules. Therefore, it has the potential to handle much more complex mixtures, as compared to the simple engineering EoSs. Because DESs are polar systems with different hydrogen bonding sites on their structure, the CPA EoS is among the best choices for modelling such mixtures, which can reliably handle the necessary phase equilibrium calculations. The optimizations will be done using Genetic Algorithm (GA), which is among the powerful and well known optimization algorithms. In order to have more comprehensive investigation, the simple conventional engineering equations of state, such as the Peng-Robinson (PR) and the Soave-Redlich-Kwong (SRK), the standard equations of commercial software, will also be investigated and their results compared to those of the CPA. This will provide valuable data on the binary/ternary systems NADES/THEDES + supercritical fluids, being not only relevant to the characterization of the systems, but it allows the unraveling of new processing methodologies, based on green technologies.
Other technological breakthroughs are expected on the application development plan. New systems and enhanced biological properties will be studied and explored and it is expected that other patent proposals will be submitted through the course of the project.