Periodic Reporting for period 5 - Des.solve (When solids become liquids: natural deep eutectic solvents for chemical process engineering)
Reporting period: 2022-01-01 to 2022-10-31
Sugars, aminoacids and organic acids are typically solid at room temperature, but, when combined at a particular molar fraction, present a high melting point depression, becoming liquids. These are called Natural Deep Eutectic Solvents (NADES). NADES are envisaged to play a major role on different chemical engineering processes towards the development of greener and sustainable processes. Nonetheless, there is a significant lack of knowledge on their fundamental and basic research, which is hindering their industrial applications. Des.solve encompasses 4 major themes of research to extend the knowledge on these systems: 1 – Development of NADES and THEDES; 2 – Characterization of the obtained mixtures and computer simulation of NADES/THEDES properties; 3 – Phase behaviour of binary/ternary systems NADES/THEDES + CO2 and thermodynamic modelling; 4 – Application development. Des.solve will be deeply engaged in application development, particularly in extraction, biocatalysis and pharmaceutical/biomedical applications, and it is expected that the created knowledge have a major impact in the scientific community, society, economy and industry.
The work performed in the scope of the project involves the development of NADES and THEDES (production and characterization) for several application developments.
Modelling thermophysical data
A simple, accurate and global correlation was proposed to estimate the densities of many different DES. The proposed 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.
Phase behaviour and vapour liquid equilibrium
To design formulation processes, such as particles from gas saturated solutions (PGSS), the vapor-liquid equilibrium (VLE) of the binary mixtures THEDES + CO2 was determined. THEDES systems based on anti-tuberculosis drugs ethambutol and L-arginine were studied. Results suggest that it is possible to formulate drug delivery systems using the THEDES by PGSS process, without compromising the integrity of the system.
Extraction of bioactive compounds
NADES were evaluated as extraction agents for polyphenols from grape pomace, apple pomace or olive oil residues; carotenoids from pumpkin residues, pepper residues and tomato residues; and flavonoids from broccoli residues; among others. The extracted material was characterized by HPLC and mass spectroscopy and evaluated for the antioxidant potential using common antioxidant tests.
To obtain phenolics and other bioactive compounds from mate, a fractionated extraction using different NADES was carried out. The results demonstrated that a sequential extraction, using first the system menthol:lauric acid and then the lactic acid-based NADES, led to 2 distinct extracts, the first rich in chlorophylls and beta-carotenes and the second rich in polyphenols. The incorporation of the extract in the DES is beneficial for antioxidants stabilization up to 90 days.
Processing of lignin, cellulose and hemicellulose, from different biomass sources was evaluated and the ability of NADES to reduce cellulose crystallinity was evaluated. Cellulose suspended in Ma/Bet/H2O (1:2:3) suffered the highest crystallinity reduction among the studied systems.
Biocatalysis
The use of NADES as biocatalysis media was studied for the transesterification of sec-alcohols for pharmaceutical applications, envisaging its viability as extraction media in biphasic systems, particularly coupled with scCO2. Esterification reactions of rac-menthol with lauric acid were carried out, and the 2 DES components reacted promptly when Candida rugosa lipase was added. The use of DES combined with enzymes paves the way for efficient and more sustainable biocatalytic processes.
Pharmaceutical and/or biomedical applications
The ability of THEDES to form a liquid formulation of an active pharmaceutical ingredient (API) may enhance the bioavailability and drug pharmacokinetics.
To limit bacterial infections during wound treatment, it is interesting to consider the loading of medical devices with antibacterial agents. A commercially available gauze was loaded with a fatty acid eutectic blend based on lauric acid (LA) and myristic acid (MA), using 2 supercritical fluid technologies: PGSS and a derived version of rapid expansion of supercritical solution (D-RESS). PGSS led to a heterogeneous dispersion of the eutectic blend in the gauze, whereas the D-RESS led to a homogeneous dispersion. 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.
The biomedical potential of hydrophobic THEDES based in menthol and saturated fatty acids (e.g. lauric acid (LA), myristic acid (MA) and stearic acid (SA)) was studied towards bacteria and HaCaT cells. Among the different formulations of THEDES, the one based on menthol and SA was the most promising formulation. It potentiated wound healing, while presenting antibacterial properties against Staphylococcus aureus and. Staphylococcus epidermis strains, some of which methicillin resistant. So, THEDES based on menthol:SA can be used as potential candidates for wound dressings.
Different THEDES based on limonene (LIM) were developed to unravel the anticancer potential of such systems. All the THEDES presented antiproliferative properties, but IBU:LIM (1:4) was the only formulation able to inhibit HT29 cells proliferation without compromising cell viability. 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 ibuprofen allied to the anticancer properties of LIM.
The use of THEDES that incorporate L-arginine and ethambutol was investigated. It was showed an increase of the solubility of ethambutol when incorporated in the eutectic system. Also, the use of THEDES with CO2 supercritical fluid technology for controlled drug delivery systems was shown to be possible.
Modelling thermophysical data
A simple, accurate and global correlation was proposed to estimate the densities of many different DES. The proposed 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.
Phase behaviour and vapour liquid equilibrium
To design formulation processes, such as particles from gas saturated solutions (PGSS), the vapor-liquid equilibrium (VLE) of the binary mixtures THEDES + CO2 was determined. THEDES systems based on anti-tuberculosis drugs ethambutol and L-arginine were studied. Results suggest that it is possible to formulate drug delivery systems using the THEDES by PGSS process, without compromising the integrity of the system.
Extraction of bioactive compounds
NADES were evaluated as extraction agents for polyphenols from grape pomace, apple pomace or olive oil residues; carotenoids from pumpkin residues, pepper residues and tomato residues; and flavonoids from broccoli residues; among others. The extracted material was characterized by HPLC and mass spectroscopy and evaluated for the antioxidant potential using common antioxidant tests.
To obtain phenolics and other bioactive compounds from mate, a fractionated extraction using different NADES was carried out. The results demonstrated that a sequential extraction, using first the system menthol:lauric acid and then the lactic acid-based NADES, led to 2 distinct extracts, the first rich in chlorophylls and beta-carotenes and the second rich in polyphenols. The incorporation of the extract in the DES is beneficial for antioxidants stabilization up to 90 days.
Processing of lignin, cellulose and hemicellulose, from different biomass sources was evaluated and the ability of NADES to reduce cellulose crystallinity was evaluated. Cellulose suspended in Ma/Bet/H2O (1:2:3) suffered the highest crystallinity reduction among the studied systems.
Biocatalysis
The use of NADES as biocatalysis media was studied for the transesterification of sec-alcohols for pharmaceutical applications, envisaging its viability as extraction media in biphasic systems, particularly coupled with scCO2. Esterification reactions of rac-menthol with lauric acid were carried out, and the 2 DES components reacted promptly when Candida rugosa lipase was added. The use of DES combined with enzymes paves the way for efficient and more sustainable biocatalytic processes.
Pharmaceutical and/or biomedical applications
The ability of THEDES to form a liquid formulation of an active pharmaceutical ingredient (API) may enhance the bioavailability and drug pharmacokinetics.
To limit bacterial infections during wound treatment, it is interesting to consider the loading of medical devices with antibacterial agents. A commercially available gauze was loaded with a fatty acid eutectic blend based on lauric acid (LA) and myristic acid (MA), using 2 supercritical fluid technologies: PGSS and a derived version of rapid expansion of supercritical solution (D-RESS). PGSS led to a heterogeneous dispersion of the eutectic blend in the gauze, whereas the D-RESS led to a homogeneous dispersion. 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.
The biomedical potential of hydrophobic THEDES based in menthol and saturated fatty acids (e.g. lauric acid (LA), myristic acid (MA) and stearic acid (SA)) was studied towards bacteria and HaCaT cells. Among the different formulations of THEDES, the one based on menthol and SA was the most promising formulation. It potentiated wound healing, while presenting antibacterial properties against Staphylococcus aureus and. Staphylococcus epidermis strains, some of which methicillin resistant. So, THEDES based on menthol:SA can be used as potential candidates for wound dressings.
Different THEDES based on limonene (LIM) were developed to unravel the anticancer potential of such systems. All the THEDES presented antiproliferative properties, but IBU:LIM (1:4) was the only formulation able to inhibit HT29 cells proliferation without compromising cell viability. 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 ibuprofen allied to the anticancer properties of LIM.
The use of THEDES that incorporate L-arginine and ethambutol was investigated. It was showed an increase of the solubility of ethambutol when incorporated in the eutectic system. Also, the use of THEDES with CO2 supercritical fluid technology for controlled drug delivery systems was shown to be possible.
Despite the generated knowledge until now, there are still some challenges, such as to enlighten the propensity of different pairs of compounds to form a eutectic system, to understand the role of water, and to develop molecular simulations and computational models to explain NADES formation. Additionally, it is necessary to increase the knowledge about the several NADES applications, mainly in the pharmaceutical and/or biomedical field. The performed work in this project already showed important achievements in terms of the characterization of the systems, namely through a new developed model for thermophysical properties, and valuable data on the binary/ternary systems NADES/THEDES + supercritical fluids, through vapour liquid equilibrium studies. In terms of applications, the results showed progress beyond the state of the art in the extraction of bioactive compounds and biocatalysis applications, as well as in the development of pharmaceutical and/or biomedical products. Until the end of the project, we expect to continue exploring the applications of these systems, through new approaches and/or optimizing the solutions in study. 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.