Compound of sucrose and citric acid as natural deep eutectic solvents kill bacteria

Photodynamic therapy has been used in the treatment of certain types of cancer for a number of years. The treatment is a result of a combination of light activated materials (photosensitizers) and light itself kills cancer cells or precursor state of cancer.

Kristine Opsvik Wikene has used her PhD to study how phototherapy can be used against bacteria in infected surface wounds. The results are so startling that the University's own innovation initiative, Inven2, has applied for a patent together with Opsvik Wikene and her main tutor, Professor Hanne Hjorth Tønnesen.

"In principle, the scientists have discovered a new type of solvents applied to the light activated materials used in bactericidal phototherapy, but we are looking into several possibilities. There is a substantial need for this type of treatment in the market," says Technology Strategy Manager Jan Solberg at Inven2- a university innovation company.

You might as well learn a new scientific term straight away: Eutectic solvents. In fact, the patent application is about destroying bacteria using eutectic solvents of natural substances. The solvents are named NADES, short for "Natural deep eutectic solvents." A eutectic solvent is a solvent that have a melting point below that of the original solvent substances.

"The original idea was to make NADES mixtures to dissolve the photosensitizers, a group of substances that have a bactericidal effect when illuminated by light with the "right" wavelength.

But then we discovered that the NADES by themselves, without photosensitizers, had a bactericidal effect," Hjorth Tønnesen explains.

The bactericidal effects are there already when the solvents are used by themselves, but the effect is enhanced when the solvents are exposed to light at a certain wavelength. And if we additionally add the photosensitizers then the effect is even stronger. We have shown that in some circumstances only a tiny quantity is necessary, says Opsvik Wikene.

It is quite likely that photodynamic therapy kills bacteria resistant to antibiotics just as easily as other bacteria.

This makes the patent application even more interesting because antibiotic resistant bacteria is a rapidly increasing problem within modern health care.

"There have been attempts at creating bacteria that is resistant to photodynamic therapy, but so far the bacteria seem defenseless. In comparison it took only a couple of years after penicillin was in use before penicillin resistant bacteria was a fact," says Opsvik Wikene.

It looks like Kristine Opsvik Wikene and Hanne Hjorth Tønnesen have improved antimicrobial photodynamic therapy considerably.

"The results indicate that our special compounds are very well suited for treatment of body surface infections. For example, a growing problem is diabetics developing foot wounds and we believe that our compounds can stop harmful infections emerging in these types of wounds. The compounds should also be effective against acne, infected burns or in the treatment of ulcers in the oral cavity," says Hjorth Tønnesen.

The two scientist emphasize that it will take some time before you can buy the new antimicrobial substances in your local pharmacy.

"The bactericidal effect is so far just demonstrated in the lab. We have to do experiments involving animals followed by clinical studies on humans before this can become a commercial product. But we wholeheartedly believe we have discovered something very exciting," says Opsvik Wikene.

"One test tube contains sucrose, the same as the sugar you keep in your home cupboard. Sucrose is a solid substance at room temperature. The second test tube contains a type of sugar called glucose, also a solid substance -- and the third tube contains a compound made up of the two sugars," she explains.

As if by magic, the remarkable thing is that the third tube containing the compound is a liquid substance. The liquid compound contains exactly the same amount of molecules of sucrose and glucose; it's the compound itself that creates the distinct structure that makes it liquid.

Opsvik Wikene has also discovered NADES compounds proportioned differently. A common treat in all the compounds is that they contain substances present in several types of cells; substances such as sugars, amino acids, organic acids as citric acid and so on.

The bactericidal effect of the NADES compounds is probably the result of the creation of free radicals, short lived and reactive chemical compounds, when the compounds are in contact with the bacteria in the vicinity of light.

"We think these free radicals attack all types of molecules in a bacteria cell -- both the outer membrane and the DNA inside the cell. The bacteria are left defenseless because it doesn't manage to develop resistance against such a substantial attack: It's almost like running over the bacteria with a bulldozer," Hjorth Tønnesen proclaims.

The free radicals will in principle also attack healthy cells close to the bacteria that is being killed, but luckily photosensitizers dissolved in NADES is more quickly transmitted to bacteria than healthy body cells.

"This means we have a time frame: If we apply a NADES with photosensitizer and illuminate the infested area immediately, we can kill the bacteria before the surrounding area is affected," according to Hjorth Tønnesen.

The NADES solvents have a special ability; they can dissolve both water-soluble substances and non-water-soluble substances. This gives the NADES the possibility to use a wide range of photosensitizers.

"If this treatment proves as promising as it looks we can easily have a huge commercial success on our hands, says Jan Solberg at Inven2. He has assisted Opsvik Wikene and Hjorth Tønnesen with the patent application and thinks it contains a number of interesting things.

"The most immediate usage is to apply these methods on infested leg wounds and other superficial type of wounds. It is common that older people, especially smokers and diabetics can develop wounds on their legs because of limited blood circulation. These wounds are often difficult to treat, painful and easily infected. But with this new method we imagine home care nurses can apply NADES solvent on these kinds of wounds and subsequently activate the bactericidal substances with light of the "right wavelength," says Solberg.

The leg wounds will not disappear just because the bacteria dies, but it will be very advantageous to get rid of all the infections.

Solberg points out that bactericidal photodynamic therapy has been known for a long time, but that it has become an ever more interesting field of research after antibiotic resistance has become an increasing problem.

"The substances used when killing bacteria with this method are often large molecules that are difficult to dissolve. However, the NADES compounds have characteristics that makes it easier to dissolve these substances and that opens a lot of possibilities. The patent application is especially interesting also because the solvents in themselves seem to have a bactericidal effect, both with and without the use of light," says Solberg.

Inven2 is now hopeful that a pharmaceutical company will buy the rights described in the patent or that some investors could get interested and start up a new company.

"It is usually necessary with substantial clinical testing before a new pharmaceutical can be approved, but in this case at least a few of the NADES compounds are made up of substances that have a documented effect. Kristine Opsvik Wikene has for example described a NADES that simply consist of sucrose and citric acid, both substances you will find in your common cup of tea. We hope that an approval the treatment of wounds will be easier to obtain with these common substances," Solberg says.

However, Inven2 wish to facilitate in such a way that the scientists can get the resources to for example study which bacteria is most common in leg wounds, and how effectively NADES destroys them.

"Henceforth, we look to apply to the Norwegian Research Council for funds from the Biotek2021 initiative. That will pave the way for a more substantial funding for this interesting project," explains Solberg.

The two scientists have so far tested the effect of NADES on among others these bacteria: Escherichia coli, common intestinal bacteria, Enterococcus faecalis that often cause infections inside the oral cavity, Staphylococcus aureus that can cause a long line of infections, Staphylococcus epidermidis that can create a biofilm on catheter and other implants, Pseudomonas aeruginosa that is common in infected wounds, and Klebsiella pneumoniae that often cause pneumonia and hospital infections.

The tests have been conducted at Nordic Institute of Dental Materials (NIOM) as part of a larger collaboration project. NADES compounds also destroys the fungus Candida albicans that can cause mouth and genital infections, especially in women.

FACTS ON NADES Eutectic solvents based on synthetic and often toxic substances have previously been used as solvents in Organic synthesis. In 2011, Dutch scientists discovered that substances present in many types of cells, often in large quantities, have at certain proportions the ability to create eutectic solvents. This discovery forms the background for this new research at UiO. But what is NADES really? Opsvik Wikene presents three small test tubes to explain what it is all about.