In the nanotransporter to the site of action

Novel vehicles increase the efficiency of antibiotics against chronic infections

Scientists from the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) Germany have developed a strategy to deliver antibacterial agents in a way that makes them many times more effective. Their findings promise hope in the fight against hospital-acquired infections and antibiotic resistance. The study appears in the international issue of the journal Angewandte Chemie. HIPS is a site of the Braunschweig Helmholtz Centre for Infection Research (HZI) in cooperation with Saarland University.

At present, the world is looking spellbound at pharmaceutical research to relieve us of the burden of the new SARS-CoV-2 coronavirus. In addition to the search for a vaccine, the continued development of other medicines is critical right now and well beyond the crisis. For example, many of those who are particularly severely ill with COVID-19 need to be treated with antibiotics because chronic pre-existing disease or superinfection with bacteria stresses the lungs and dramatically worsens the course of the disease. The treatment of these patients is made more difficult by the ever emerging resistance of germs to antibiotics. A typical colonizer of the lungs is Pseudomonas aeruginosa. The bacterium is known to cause hospital infections and make life difficult for people with cystic fibrosis. It forms a protective layer of complex, long-chain molecules in infected tissues. This biofilm forms a barrier against antibiotics, much to the patients' distress.

Prof. Claus-Michael Lehr has been researching biological barriers for decades. "The intestinal wall, the skin, the air-blood barrier in the lungs or, indeed, biofilms: they all get in the way of the active ingredient," says the head of the "Drug Transport" department at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS). "To reach the site of action, drugs need a suitable vehicle that is designed for the specific active ingredient and the respective barrier."

In collaboration with the French pharmacist Patrick Couvreur in Paris, Lehr's doctoral student Duy-Khiet Ho produced special nanoparticles that would transport antibiotics into the biofilm formed by pseudomonads. He synthesized a novel substance for this purpose. The molecules of this substance and the nanoparticles formed from it are amphiphilic, meaning they dissolve in both water and fat.

Under the microscope, the scientists were able to observe how the particles entered and even penetrated a cultivated layer of bacterial biofilm. However, when they packaged the antibiotic tobramycin, commonly used to fight Pseudomonas aeruginosa infections, into the nanoparticles, they saw no increased effect against the germs in the biofilm. The reason for this is explained by Dr. Brigitta Loretz, who developed the method together with Lehr: "In the biofilm, the bacteria shut down their metabolism. The antibiotic, which actually blocks protein production, therefore can't have much effect." If you throw a stick into a gearbox that is at a standstill anyway, you won't achieve a braking effect with it either. That's why the researchers took advantage of another point of attack by the germs: quorum sensing. This is the mechanism that bacteria use to determine whether they have reached a certain population density. Only then does it make sense for them to form a biofilm. Among other things, quorum sensing regulates the production of the polymers used to build up the biofilm. Prof. Rolf Hartmann and Dr. Martin Empting are researching novel substances in the neighboring "Drug Design" department at HIPS that can flip an important switch in this mechanism. Lehr used one of these substances: He loaded his nanoparticles with the quorum sensing inhibitor QSI(1) in addition to the antibiotic tobramycin. When he treated his biofilm cultures with the double-loaded particles, a fraction of the antibiotic concentration was enough to kill the bacteria and dissolve the biofilm. "We turn off quorum sensing right in the biofilm," Loretz says. "The bacteria ramp up their metabolism and the antibiotic takes effect."

Medically, it is of immense importance to be able to target infections with the help of low concentrations of antibiotics. "This is how we prevent bacteria from developing resistance to the active ingredients," Loretz explains.

"We have produced a first generation of anti-infective nanocarriers," says Lehr, who is now further developing the novel nanoparticles. In their grown biofilm cultures, he and his colleagues can not only observe the particles. They can also measure exactly how much of the active substances has penetrated the biofilm. "This is a distinct advantage over tests in experimental animals," he says. His strategy is to investigate as many parameters of a new active substance as possible "in the test tube." To do this, the passionate tinkerer develops complex models, preferably from human cells, that reflect the conditions in the body in as much detail as possible. "It's like a modular system," he says. "In vitro, for example, I can culture different cells in different combinations to understand the role of each cell type." Lehr and his colleagues recently published the first protocol for a three-dimensional cell culture model of respiratory infections, in which he grows mucosal and defense cells together with biofilm-forming bacteria. In such a model, various inflammatory parameters can be monitored and antibiotics tested simultaneously. Animal testing is needed only for confirmation after extensive in vitro studies, he says. His goal is to test new active substances in humans as quickly as possible. "After all, we don't want to cure mice," he says.