Crystal formation holds clues to fighting disease
Researchers at the Adolphe Merkle Institute have been investigating the formation of crystals that ensure the survival of malaria parasites in the bloodstream. This interdisciplinary effort could facilitate the selection of new anti-malarial compounds and help stem the disease’s progress.
Malaria occurs after infection by certain types of mosquitoes, which pass the disease from human to human as a secondary effect of their feeding habits. It is caused by the Plasmodium sp. parasites, which de-grade and digest the hemoglobin found in blood. Yet, they cannot digest heme, an iron-containing molecule found in many living organisms that serves as an oxy-gen carrier. In fact, heme is actually toxic to the para-site, which has to neutralize the molecules’ effects to survive. To do this, the parasite bio-crystallizes heme into insoluble crystals known as hemozoin. This survival mechanism has been targeted by some anti-malarial drugs, particularly those based on chloroquine, which have been chosen for their capacity to inhibit hemozoin formation. Over the years, however, parasites in different parts of the world where malaria is endemic have become resistant to these treatments, making their use ineffective. This has also led to an increase in malaria-related mortality, especially in Africa.
To counter this, not only new compounds are need-ed, but also a better understanding of the mechanism and the kinetics of the hemozoin crystallization, as well as of how it is inhibited. Researchers at the Adolphe Merkle Institute chose to investigate the biocrystallization using so-called dynamic depolarized light scattering (DDLS). This approach was selected because it is considered to be a viable method for characterizing non-spherical optical anisotropic particles, and can thus be applied to hemozoin crystals.
“It basically involves shining a laser beam on a solution containing the sample, and detecting and analyzing the statistical properties of the light that is scattered when it strikes the crystals. It is especially useful for analyzing any particulate matter that is dispersed or suspended in a liquid matrix,” says Dr. Sandor Balog, one of the two researchers in charge of AMI’s instrument platform, and a scattering specialist. “With light scattering, we can infer the shape and stage of development of crystals, as well as the time it takes them to form.”
To test their approach, the researchers used a synhetic version of hemozoin whose properties are similar to the natural one produced by the malaria parasite. The tests were performed both in the presence and absence of a chloroquine-based anti-malarial drug.
The researchers found that while the drug did not pre-vent the initial formation of hemozoin nuclei, it did hinder the crystals from growing substantially, as heme was inhibited from attaching itself to its surface. For Balog, and colleagues from AMI’s Macromolecular Chemistry and Soft Matter Physics groups, dynamic light scattering, and DDLS in particular, could easily find its way to anti-malarial studies addressing biocrystallization.
“More generally, the technique is suitable for any system exhibiting the physical features of self-assembly – or for that matter its reverse, dissolution – completely independently of the parameters of the system, such as the chemical reactions,” adds Balog. “It could also be miniaturized and integrated into microfluidic platforms and lab-on-a-chip assays, where automation and parallelization for high throughput are desired.”
This research was carried out in parallel to the development of a malaria diagnostic tool by AMI’s Macromolecular Chemistry group. Their project uses hemozoin as a highly-sensitive indicator of the presence of the malaria parasite in asymptomatic carriers. It catalyzes a polymer in a solution, at temperatures close to those of the human body, changing the liquid from transparent to turbid in presence of the parasite.
Reference: Rifaie-Graham, O., Hua, X., Bruns, N., Balog, S. The Kinetics of ß-Hematin Crystallization Measured by Depolarized Light Scattering, Small, 2018, 14, 1802295