A novel capillary and method for melanin radical determination in preclinical studies of potential melanoma drugs using the Danio rerio embryo model

Radicals, melanoma, and zebrafish

Dr. Katerina Makarova from the Faculty of Pharmacy at the Medical University of Warsaw, together with her team, has developed an innovative method for studying melanin free radicals - one of the key drivers behind melanoma development. Her research utilizes an animal model based on the transparent embryos of a small freshwater fish: the zebrafish (Danio rerio). This technique significantly reduces the time, complexity, and cost of preclinical research into new melanoma drugs. "Thanks to a grant awarded by the Foundation for Polish Science under the Proof of Concept scheme - funded by the European Funds for a Modern Economy 2021–2027 (FENG) - we aim to conduct a market analysis of melanoma diagnostics and therapeutics in Poland, Europe, and the US, define our target customer groups and adapt our method accordingly, perform pilot studies on several melanoma cell lines, and prepare a commercialization plan," says Dr. Katerina Makarova.

Melanin is one of the most widespread natural pigments in nature. Responsible for the color of our skin, hair, and eyes, it plays a vital role in shielding the body from harmful solar radiation while participating in thermoregulation and toxin absorption. However, melanin also has a dark side: melanin radicals are a major contributing factor to melanoma development. With global melanoma incidence on the rise, there is an urgent need for effective new diagnostic and therapeutic methods. Recently, zebrafish (Danio rerio) embryos have emerged as a primary animal model for investigating melanoma progression mechanisms and potential anti-melanoma therapeutics. Why zebrafish? They are small, easy to breed, feature a short development cycle, and yield large clutches of offspring. Remarkably, over two-thirds of human genes have a direct zebrafish counterpart. Furthermore, zebrafish embryos develop externally in water and are completely transparent, allowing researchers to observe biological changes with ease.

"Electron paramagnetic resonance (EPR) is the only known technique capable of detecting and identifying specific types of melanin radicals. When applying this method to study radicals in zebrafish embryos, obtaining sufficient measurement sensitivity requires higher microwave power levels than those used in rat or mouse models. However, the stronger radiation causes samples placed in standard capillaries to overheat rapidly - by up to several degrees Celsius. Our breakthrough was the creation of a novel, non-heating capillary that enables EPR-based melanin radical analysis directly in Danio rerio embryos. It is a non-invasive method, allowing embryos inside our capillary to be monitored over several days, as the uniquely engineered shape protects them from overheating during measurements," explains Dr. Katerina Makarova.

This technology can be applied to measure biological and physical parameters in the zebrafish model system, including melanin radical levels. It enables precise investigation into disease development mechanisms (primarily melanoma) and the efficacy of anticancer drugs—with potential future applications extending to Alzheimer's and Parkinson's disease therapeutics. Ultimately, the method holds promise for personalized medicine in skin cancer patients by enabling rapid, simultaneous multi-drug testing on the model to select the most effective treatment combination.

Dr. Katerina Makarova originally hails from Belarus. She graduated with honors from the Faculty of Radiophysics at the Belarusian State University in Minsk, completing part of her master's thesis experimental work during a 3-month stay at the Biophysics Laboratory at Wageningen University in the Netherlands. From 2006 to 2008, she studied and worked at the Institute of Physics of the Polish Academy of Sciences, during which she also visited Ghent University in Belgium and the University of Tartu in Estonia. Between 2006 and 2011, she pursued her doctoral studies as a research fellow at Wageningen University's Biophysics Laboratory. Since 2009, she has been affiliated with the Department of Physical Chemistry (currently the Department of Organic and Physical Chemistry) at the Medical University of Warsaw. For the project titled "Capillary for in vivo determination of melanin radical using the EPR method on a zebrafish embryo model," she and Dr. Katarzyna Zawada were awarded a gold medal at the 15th INTARG International Trade Fair in 2022. The project was also honored with the Special Award at Euroinvent 2022 in Romania.