83
(Olivares et al. 2018). Their antimicrobial activity was determined in vitro by both
a limiting dilution method to obtain the minimum inhibitory concentration (MIC)
and a disk diffusion assay where the formulations were incorporated into cellulose
disks and placed on agar plates containing the microorganisms. The test bacteria
used were Escherichia coli and Pseudomonas aeruginosa. When freshly prepared
solutions were used, the minimum inhibitory concentration of both antibiotics was
identical in aqueous solution and in the solvents. However, when the formulations
were kept at 25 °C for 7 days before adding them to the bacteria, the minimum
inhibitory concentration of the water solution of imipenem increased at least 32-fold
and that of aqueous clavulanic acid doubled, whereas no changes were observed for
the deep eutectic solvents compared to a fresh preparation. These results are in
agreement with stability studies. The disk assay was used because it allowed the
deep eutectic solvent to be added to the bacterial cultures without dilution that could
perturb the solvent structure. The size of the ring of growth inhibition of Pseudomonas
aeruginosa was similar for antibiotics in aqueous solution and in the deep eutectic
solvents, showing that they remained fully bioavailable in the latter formulation.
This opens up possibilities for the use of these solvents in sustained release formulations without loss of antibiotic activity.
Formulation of Photosensitizers in Deep Eutectic Solvents
for Antimicrobial Activity
As described above, deep eutectic solvents have been used to improve the solubility
and stability of photosensitizing compounds, and the resulting formulations have
been used for antimicrobial photodynamic therapy. This is reported in a series of
articles and a patent application from the group of Tønnesen and Wikene (Tønnesen
and Wikene 2016). The first compound to be used was the small, naturally occurring
molecule curcumin (Wikene et al. 2015a). Curcumin in a deep eutectic solvent
composed of maleic acid and choline chloride in 1:3 molar ratio was phototoxic
toward Escherichia coli at a concentration of 1.25 μM, lower than any previously
reported result. At this concentration, there was no contribution of the toxicity of the
deep eutectic solvent itself.
Two porphyrin-based photosensitizers were formulated in a similar way. Mesotetra-(4-carboxyphenyl)-porphine (TCPP) is an anionic porphyrin that could be
solubilized in acidic deep eutectic solvents, citric acid:sucrose 1:1, DL-malic
acid:D-fructose:D-glucose 1:1:1, and choline chloride:xylitol 5:2, with improved
photostability compared with methanolic solution (Wikene et al. 2016). The
phototoxicity of these preparations were tested against both Gram-positive
(Enterococcus faecalis, Staphylococcus aureus) and Gram-negative (Escherichia
coli) bacterial species. Before testing the phototoxicity of meso-tetra-(4carboxyphenyl)-porphine, it was necessary to establish the photo- and dark toxicity
of the deep eutectic solvent and define the dilution at which the effects of the
medium were not significant. In these conditions, meso-tetra-(4-carboxyphenyl)porphine in citric acid:sucrose or malic acid:fructose:glucose was much more
2 Deep Eutectic Solvents for Innovative Pharmaceutical Formulations
(Olivares et al. 2018). Their antimicrobial activity was determined in vitro by both
a limiting dilution method to obtain the minimum inhibitory concentration (MIC)
and a disk diffusion assay where the formulations were incorporated into cellulose
disks and placed on agar plates containing the microorganisms. The test bacteria
used were Escherichia coli and Pseudomonas aeruginosa. When freshly prepared
solutions were used, the minimum inhibitory concentration of both antibiotics was
identical in aqueous solution and in the solvents. However, when the formulations
were kept at 25 °C for 7 days before adding them to the bacteria, the minimum
inhibitory concentration of the water solution of imipenem increased at least 32-fold
and that of aqueous clavulanic acid doubled, whereas no changes were observed for
the deep eutectic solvents compared to a fresh preparation. These results are in
agreement with stability studies. The disk assay was used because it allowed the
deep eutectic solvent to be added to the bacterial cultures without dilution that could
perturb the solvent structure. The size of the ring of growth inhibition of Pseudomonas
aeruginosa was similar for antibiotics in aqueous solution and in the deep eutectic
solvents, showing that they remained fully bioavailable in the latter formulation.
This opens up possibilities for the use of these solvents in sustained release formulations without loss of antibiotic activity.
Formulation of Photosensitizers in Deep Eutectic Solvents
for Antimicrobial Activity
As described above, deep eutectic solvents have been used to improve the solubility
and stability of photosensitizing compounds, and the resulting formulations have
been used for antimicrobial photodynamic therapy. This is reported in a series of
articles and a patent application from the group of Tønnesen and Wikene (Tønnesen
and Wikene 2016). The first compound to be used was the small, naturally occurring
molecule curcumin (Wikene et al. 2015a). Curcumin in a deep eutectic solvent
composed of maleic acid and choline chloride in 1:3 molar ratio was phototoxic
toward Escherichia coli at a concentration of 1.25 μM, lower than any previously
reported result. At this concentration, there was no contribution of the toxicity of the
deep eutectic solvent itself.
Two porphyrin-based photosensitizers were formulated in a similar way. Mesotetra-(4-carboxyphenyl)-porphine (TCPP) is an anionic porphyrin that could be
solubilized in acidic deep eutectic solvents, citric acid:sucrose 1:1, DL-malic
acid:D-fructose:D-glucose 1:1:1, and choline chloride:xylitol 5:2, with improved
photostability compared with methanolic solution (Wikene et al. 2016). The
phototoxicity of these preparations were tested against both Gram-positive
(Enterococcus faecalis, Staphylococcus aureus) and Gram-negative (Escherichia
coli) bacterial species. Before testing the phototoxicity of meso-tetra-(4carboxyphenyl)-porphine, it was necessary to establish the photo- and dark toxicity
of the deep eutectic solvent and define the dilution at which the effects of the
medium were not significant. In these conditions, meso-tetra-(4-carboxyphenyl)porphine in citric acid:sucrose or malic acid:fructose:glucose was much more
2 Deep Eutectic Solvents for Innovative Pharmaceutical Formulations