Background and Objective
Several photosensitisers (PS) used in experimental bacterial phototoxicity studies are poorly water- soluble and tend to aggregate. Specific vehicles can improve and control properties such as aqueous solubility, aggregation and photoreactivity. Natural deep eutectic solvents (NADES) are eutectic mixtures of two or more naturally occurring metabolite components in a specific molar ratio. The resulting eutectics are transparent liquids with a supramolecular hydrogen-bond structure that may solubilize a wide range of hydrophilic and hydrophobic molecules including pharmaceuticals and biomolecules . The composition and water content determine properties such as viscosity and polarity. Previously we have compared aqueous solubility and aggregation of various lipophilic PS dissolved in a series of NADES. The PS monomers were maintained in a supersaturated state without any precipitation inhibitor other than the NADES network. Selected NADES-PS samples were included in the present study on microbial phototoxicity  .
Materials and Methods
A series of 35 NADES was initially prepared and characterised. The following NADES were selected for the current studies on Gram-positive and Gram-negative bacteria: citric acid/sucrose (1:1) (CS); malic acid/glucose (1:1) (MG); maleic acid/choline chloride (1:3) (MC3); malic acid/fructose/glucose (1:1:1) (MFG). The samples were diluted in water 1:100 – 1:400.
The PSs studied were synthetic curcumin, the neutral 5,10, 15, 20-tetrakis(4-hydroxyphenyl)porphine (THPP) and the corresponding anionic 4-carboxyphenyl porphine (TCPP).
The phototoxic effect was determined by counting the bacterial CFU/ml pre- (106-107) and post-exposure to NADES-solubilised PS under blue light irradiation using an incoherent light source (400-550 nm; λmax 450 nm). The light dose was in the range 10-30 J/cm2.
Results and Discussion
- Exposure of coli to curcumin (1 μM) dissolved in MC3 combined with light reduced CFU/ml > 6 log units. Substitution with curcumin-CS showed no effect.
- Nanomolar concentrations of THPP with and without CS or MG in the presence of light reduced CFU/ml > 6 log units of faecalis and S. epidermidis (THPP-CS/MFG). A similar reduction of E.coli required NADES-solubilised THPP and three times higher light dose. CS or MFG alone caused a reduction in CFU/ml of both Gram-positive and -negative bacteria except E.coli. This effect was augmented by irradiation. In general, the phototoxic effect on E. coli and K. pneumoniae was higher in the presence of THPP-NADES than the effect induced by NADES in combination with light.
- The phototoxic effect of TCPP (1 nM) on faecalis, S. aureus and E.coli increased in the presence of NADES-CS/MFG (2-4 log units reduction of CFU/ml depending on type of NADES).
Protonation of the porphyrin leading to decreased PS aggregation may have contributed to an increase in porphyrin-induced phototoxicity.
The phototoxic effect of selected porphyrins and curcumin can be enhanced in combination with certain NADES. The effect is dependent on bacterial strain and independent of Gram classification.
- 1. Choi, YH., et al., Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology? Plant Phys, 2011. 156(4): p. 1701-1705.
- Wikene, KO., Ph.D. Thesis, School of Pharmacy, University of Oslo, Oslo, Norway. ISSN 1501-7710.
K. Wikene1*, E. Bruzell2, H. Valen2, H.H. Tønnesen1
1 School of Pharmacy, University of Oslo, Oslo, Norway * present employer: GE Healthcare, Oslo, Norway
2 Nordic Institute of Dental Materials (NIOM), Oslo, Norway, firstname.lastname@example.org