A typical organic laboratory on the 4th floor of the Carre building. Fumehoods are filled with set-ups consisting of glass flasks connected with tubes. A couple of students are working with fluid-filled bottles, containing invisible, dissolved compounds. Here, the magic happens on an invisible scale. ‘In this flask, we make our nanoparticles’, Paulusse says. He points to a large glass round-bottom flask, filled with a clear liquid. Using a pipette, MSc student Bram van der Veen adds droplets containing dissolved polymers, long chains of molecules, that will react inside the flask and turn magically into nanoparticles.
Using a complicated, multi-step process, Paulusse managed to create these nanoparticles, identical in size and shape. To guarantee that the process is reproducible and to make sure that the nanoparticles are formed in the right way, the process has many checks and balances. It starts with purifying the particle’s building blocks, and ends with identifying the nanoparticles formed using sophisticated machinery. For example, PhD candidate Jan Willem Paats operates the NMR spectrometer to verify the composition of the nanoparticles. ‘Eventually we managed to develop a very mild and easy procedure to make this nanoparticle’, Paulusse explains. ‘The synthesis reaction can take place in water, just in the open air and at room temperature and can easily be scaled up to produce large quantities.’
Smaller dosage
In 2014 organic chemist Paulusse and his team started working on designing and engineering nanoparticles. Initially, this was fundamental, curiosity-driven research, but Paulusse had a clear application in mind: the use of nanoparticles as drug carriers for therapeutic purposes. ‘We aimed to develop a tiny nanoparticle of just ten nanometers for application in brain therapeutics’, he explains. ‘Due to their small size, they are as small as proteins, and such nanoparticles may travel unimpeded through the body, and possibly also into the brain.’ By attaching a therapeutic drug to such a particle, it is possible to deliver the drug to the target location with high precision. As a result, the medication only reaches the place in the body where it is needed, resulting in using much smaller dosages than previously required. With these applications in mind, the scientists designed a simple, elongated chain-like molecule, a polymer, as a basis. In a next step, the team attached reactive groups at several distances along the polymer chain. Paulusse holds a white nylon cord, representing the polymer. Pink-colored beads are attached at regular distances; they visualize the reactive side groups, essential for the formation of the nanoparticle. ‘These groups react with each other and subsequently bind and get attached’, Paulusse explains. ‘That happens along the whole molecule chain, and results in the polymer getting shorter and more entwined, eventually forming a ball-like, tangled shape: a nanoparticle.’ The trick is that each polymer only forms links with itself; thereby the polymer chain determines the size of the nanoparticle. Its small size, between five and ten nanometers, allows the nanoparticle to travel through the body, entering tissues and even cells. Paulusse: ‘The polymer basis of the particle has enough space, where drugs can be chemically attached. It can therefore be used to carry medication to specific locations in the body.’
Original approach
While Paulusse was fine-tuning the synthesis of his nanoparticle, by coincidence, he met a research team working on malaria. These scientists had an original approach to fight this disease: instead of treating infected people, they choose to treat infected mosquitos, to reduce the spread of the disease. The choice to target the parasite inside the mosquito, and not in humans, gives more options to fight it. For example, nanoparticles cannot be applied in humans yet due to potential health risks, but in mosquitoes this poses less problems. ‘The team needed a carrier to bring an anti-malarial drug inside an infected mosquito, to kill the parasites’, Paulusse says. ‘I was convinced the nanoparticle we developed could play a key role in their efforts to bring malaria down.’ Very relevant research, since malaria is a major problem in many tropical countries. Every year about 200 million people are infected, of which half a million people die, mostly children below five years old. The parasite is transmitted from an infected mosquito to people, where they invade the red blood cells to reproduce. Paulusse reasoned that the parasite likely binds to red blood cells partially using electrostatic forces. The parasites therefore may have positive charges on their surface. Paulusse used this positive charge on the parasite to his benefit. By making the nanoparticles negatively charged, these easily stick to the parasite. The principle worked flawless: ‘Together with our collaborators in Greece, we fed mosquitoes with green fluorescent nanoparticles in a sugar solution’, Paulusse explains, ‘demonstrating that the mosquitoes don’t mind eating nanoparticles and that they end up in its stomach’. Collaborators in Barcelona exposed the malaria parasites to the nanoparticles, ‘the particle adhered perfectly to the parasite, and since there are only a handful of parasites inside a mosquito, it should be relatively straightforward to target every one of them, making this approach extremely promising.’ The next step was to couple the standard anti-malaria medication, malarone (atovaquone), to the particle to kill the parasite.
Anti-cancer treatment
Unfortunately, the team discovered that the malarone-nanoparticle complex wasn’t able to kill the parasite, a major set-back. Most likely, malarone is not the best drug for killing the parasite in this specific mosquito stage. So, the search for the right anti-malarial drug continues. In the meantime, one of Paulusse’s recently graduated students, Dr. Naomi Hamelmann, discovered that previous research had found that malarone could also inhibit some cancers. Precise drug delivery inside the cancer cell by the nanoparticle could possibly improve such anti-cancer treatments. The first experiments were very hopeful: when the scientists tested the nanoparticle, with malarone attached, on a cervical cancer cell line, they found that the drug was effectively carried inside the cancer cell, subsequently killing it. Without the carrier, the cancer cells were not affected by the drug.
Tiny, promising and versatile
Although these results are extremely promising for future treatment of some cancers, there still are some massive obstacles. Using nanoparticles in human medicine is still very tricky, since the short- and long-term health effects are not sufficiently known. So, it will take some time before it can be applied to cancer patients. Paulusse is currently evaluating how to apply nanoparticles for other purposes, for example, to improve properties of different materials. Paulusse: ‘The fabrication method we developed for these tiny, promising and versatile particles allows the production of large quantities, and we plan to produce kilograms of nanoparticles in the new startup company IntriS. I am very optimistic about the future use of these nanoparticles for different applications, from controlled drug delivery to improving the properties of everyday materials.’
