Light-Activated Dyes Explained by a Foremost Expert

Belinda Heyne is one of the world's foremost experts in photochemistry and photobiology. She is a leading expert and a key opinion leader on photosensitizers (also known as Light-Activated Dyes or LADs) and their creation of singlet oxygen production. After earning her Ph.D. in Science from the University of Liege in Belgium in 2004, she completed a post-doctoral fellowship at the University of Ottawa in Canada under the supervision of Prof. J.C. Scaiano. She began her career at the University of Calgary in 2007 as an Assistant Professor in the Chemistry Department, where she is a Professor since July 2020. Her lab, The Heyne Lab for Photosciences at the University of Calgary, is one of a few labs in the world with the capability to measure directly singlet oxygen. Belinda was a crucial contributor in the WHO-led consortium DeMaND study and conducts ongoing research as part of Singletto Labs.

In this piece, we talk with Belinda about her career in fundamental research on photosensitizers.

Q. What made you interested in studying photochemistry?

Well, I joke about this, but it’s mostly true. I was fascinated with Star Wars and the light sabers growing up. That is what first peaked my interest and curiosity into light and how it worked. After studying chemistry in Belgium, I moved to Canada to complete my post-doc in 2004. There, at the University of Ottawa, I met Tito (Dr. J.C. Scaiano). He is internationally considered the “Godfather” of Photochemistry and he was a true mentor to me. We make the joke that people who have joined his group are part of the Scaiano Mafia, thus he was the Godfather. I have learned everything from him, and I thank him for where I am now. When I arrived in Canada unable to speak English and questioning if I had made the right move, he welcomed me as an employee, friend, and daughter. He was my number one supporter and I credit him for all my successes. Neither of us had a clue that our work in photochemistry would lead to such important pandemic-related breakthroughs; but, I am proud that it has.

Q. As a leading expert in photosensitizers, can you explain how they work?

A. Photosensitizers basically take out the energy from light. The light can come from the sun, a lightbulb in your home, or even a flashlight on your cell phone. The photosensitizers take the energy and transfer it to oxygen. This transfer of energy converts oxygen to a more excited state of oxygen. This energized, or excited, state of oxygen is called singlet oxygen. So, a photosensitizer's job is to take up the energy and give it to oxygen because oxygen is bad at taking the energy on its own. A photosensitizer can also be called a Light-Activated Dye (LAD). Most LAD are just colored molecules with a unique ability to take up the energy of light and give that energy to oxygen. Not every single colored molecule can do this; it requires something special. Molecules are made of atoms, and atoms contain electrons. It comes down to the electronic distribution and the type of electronic configuration reached upon light activation. That sounds complicated – maybe it's easier to imagine these LADs as the convertors you use in plugs when traveling. Not all converters will work in all countries, but the right converter (or the right photosensitizer in our case) will convert the energy.

Q. How many photosensitizers exist? Do you have a favorite one?

A. While I can't give an exact answer, I can say in the hundreds certainly. I mean, they are all around us. Photosensitizers also exist within the human body, which you are likely not aware of. Some of your DNA base pair are photosensitizers and can produce singlet oxygen.

There are many, many photosensitizers. Some we can see with the human eye, and some we cannot; but, because they have a UV spectrum, they can still absorb light and convert energy to singlet oxygen.  

My personal favorite is Rose Bengal. It's often used medically as a stain to detect damage in the eye, and it has many other great uses and abilities. We use it quite a bit in my lab. Plus, it's pink, which is lovely.

Q. Talk to us about the photosensitizer Methylene Blue and why it was chosen for the DeMaND study and other follow-up research.

A. Sure, Methylene Blue is one of the many photosensitizers with which I work and am familiar. We decided to use Methylene Blue because it's used quite often in the healthcare system. It's used in surgeries. It's safe to use for children; so, obviously safe for adults, as well. It also has a nice color that matches really well with the light we have from the sun and inside the home.

Q. How do the varying photosensitizers differ from each other?

A. There are three main differences: wavelength response, solubility, and size. Certain colored dyes respond to different wavelengths of light. Some are soluble in water, some only in other solvents like alcohol, and some are only soluble in lipids. And, they vary by size (how big they are) and how well they can organize together. Those are the differences but the similarity, rule #1 for being a photosensitizer (and for photochemistry in general), is they must have the ability to absorb light. They have a spectrum feature that is going to respond to light. The 2nd rule is they must have access to a state, which is the equivalent of the converter plug I mentioned before. That state doesn't exist for every single molecule. Molecules that do have this state, called triplet state, can take the energy of light and convert it so that it can be transferred to oxygen.

Q. How do you detect the presence of singlet oxygen created by these photosensitizers?

A. You have two methodologies for such: Direct and Indirect. We use both in my lab because they provide different information depending on the theories being explored.

Singlet oxygen is an amazing molecule with so much energy. Everything that gets energy is going to lose the energy eventually. One way for singlet oxygen to lose its energy is to react with other molecules. This is what we want it to do – react with a virus or bacterium. But, if it doesn't react with other molecules, it will lose its energy to the environment or by emitting light and then will become regular oxygen again. That's the beauty of it: it either reacts with bacteria and viruses around it or reverts back to the oxygen we need. When singlet oxygen loses its energy via light emission, we technically cannot see it with our eyes because it's in the near-infrared. In addition, it is a very rare process. In water, only 1 in 100,000 singlet oxygen molecule emits light. It is thus like finding a needle in a haystack. Still, we can measure it with a very sensitive instrument. This is a very tricky and complicated process, but it's the most precise, direct way and considered the holy grail in measuring singlet oxygen if done correctly.

The indirect method is way more conventional and easier to accomplish but requires careful attention to controls. Using a color indicator sensor that selectively reacts with singlet oxygen, you can follow the sensor's spectral pattern and see when singlet oxygen is consumed by the sensor, allowing you to quantify how much was produced.  

Q. What happens when singlet oxygen interacts with a virus like the coronavirus?

A. When singlet oxygen comes into contact with coronavirus, it can react with the virus in many ways: with the amino acids in the spike proteins, in the membrane's lipids, and/or with the RNA. So, basically, it can react with everything. It's not selective. It has a preference, though, for certain amino acids in the spike proteins: these are like the coronavirus's favorite food. But the whole virus is basically like food for the singlet oxygen.

Q. How much singlet oxygen does a typical photosensitizer make?

A. What you are asking about is called the singlet oxygen quantum yield: the number of singlet oxygen molecules produced per number of photons being absorbed. For Methylene Blue, the quantum yield is 0.5, so 50% of the absorbed light will be converted into singlet oxygen. So, if you have one molecule of Methylene Blue absorbing a hundred photons throughout its lifetime, it will generate 50 singlet oxygen molecules. So the amount of singlet oxygen created is determined by the number of Methylene Blue molecules (or other LADs) you have and the number of photons you have from the light source.

For the DeMaND study, light-activated Methylene Blue produced roughly 10 to the 15th singlet oxygen molecules (that's a quadrillion) every single second. Virus particles studied in the DeMaND study went up to 10 to the 6th (that’s one million) on the high end. So you can see the difference … we are talking about a quadrillion singlet oxygen molecules per second fighting a million virus particles. Or simplified, this would mean one billion singlet oxygen molecules to every one virus. That's a huge order of magnitude. I like to picture a car going through a car wash, but instead of getting water poured down, billions of little pebbles are dumped – think of all the damage that would be done. This is like what happens to the virus. It gets bombarded and destroyed by the billions of pathogen-destroying singlet oxygen molecules as it passes through the air.

Q. What is the lifetime of singlet oxygen? How far can it travel?

A. Singlet oxygen is very short-lived. Oxygen in this excited state needs to transfer the energy quickly. The distance it can travel depends on the environment. In aqueous solutions, singlet oxygen gives its energy to the water molecules around it. It's like it's at a party with friends, giving off energy and excitation to everyone around. So singlet oxygen is not going to travel very far because it will give its energy to the solvent. However, in other solvents, where there is less ability to transfer excitation, it can travel much further. So it would be like me telling you a joke in French. You might not get the same excitation as when I'm telling the joke to my mom in French. My mom gets the excitation; you may laugh eventually but maybe not for the joke. Excitation takes some time to release … if the molecules are not interacting with other molecules around them, they can travel much further. Typically in water, singlet oxygen has a lifetime of 3-4 microseconds. In the air, it can travel about 90 milliseconds, which is about 90,000 microseconds. Distance-wise, this means roughly several nanometers in water and several millimeters or centimeters in the air.

Q.  Methylene Blue is often used in medicine and was originally used as a textile dye back in the 1860s. It's listed on the World Health Organization's (WHO) list of essential medicines. It's known and has been extensively studied. Talk to us about its safety, as well as the safety of singlet oxygen.

A. Correct, Methylene Blue as a dye has probably been worn by many people who didn't even know they were wearing it. So, people have been exposed to singlet oxygen creation from such and other dyes for a long time. It is also used in many medical studies and clinical uses around the world at much higher concentration levels than we are using. In fact, clinical applications in Germany are using 1000xs the concentration of Methylene Blue in their nasal spray and mouth gargle solution for COVID-19 treatment. Further, the standard for Methylene Blue inhalation safety is no more than 700 parts per million. In our studies, we don't see any more than 6 parts per million. But, just like anything, too much of something can have negative effects, even water. For this reason, we conservatively dose the light-activated dye solutions.

Regarding our skin, the great thing is we have a layer of dead cells as our top layer. So that, combined with the very short lifetime of singlet oxygen (3-4 microseconds in water), means the singlet oxygen doesn't have much time to penetrate the skin (it being made up of upwards of 60% water). Singlet oxygen likes to react with what it has right in front of it. As far as inhalation, the singlet oxygen produced from a mask's surface, for example, reverts back to regular oxygen so quickly, it really doesn't have much time to reach one's mouth or nose for inhalation. The beauty of singlet oxygen is how quickly it loses its energy and returns to the regular oxygen we need to breathe.

Q. What does it mean for you to have been a part of the WHO-led DeMaND study and to see your life's work in photosensitizers having such an impact on the future of public health?

A. It's pretty incredible. I have been doing fundamental research on photosensitizers my whole career. I would never have guessed that one day we would be applying photosensitizers to masks to decontaminate and protect against a pandemic-level virus. This is why it's so important to do fundamental research, and unfortunately, this kind of research is getting cut worldwide for financial reasons. But, life is unexpected, and it's serendipitous that I have been studying for decades the very thing that will offer us serious protection in this pandemic and beyond. Very few people were excited about photosensitizers before … now, the World Health Organization is creating studies around such, and we at Singletto are developing truly remarkable solutions for the near future.

Q. Finally, for people who haven't yet heard of Singletto, can you describe the following in one word:

-Singletto Culture: Collaborative.

-Singletto People: Passionate. The team is so enthusiastic you feel energized (like singlet oxygen) after every meeting.

-Singletto Technology: Beautiful.

-Singletto Innovation: Disruptive.

-Singletto Mission: Protect. Singletto is a bit like the Justice League protecting humankind from an invisible threat.