Synthetic Hemozoin (β-Hematin) Crystals Nucleate at the Surface of Neutral Lipid Droplets that Control Their Sizes

Emulsions of monopalmitoylglycerol (MPG) and of a neutral lipid blend (NLB), consisting of MPG, monostearoylglycerol, dipalmitoylglycerol, dioleoylglycerol, and dilineoylglycerol (4:2:1:1:1), the composition associated with hemozoin from the malaria parasite Plasmodium falciparum, have been used to mediate the formation of β-hematin microcrystals. Transmission electron microscopy (TEM), electron diffraction, and electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) have been used to characterize both the lipid emulsion and β-hematin crystals. The latter have been compared with β-hematin formed at a pentanol/aqueous interface and with hemozoin both within P. falciparum parasites and extracted from the parasites. When lipid and ferriprotoporphyrin IX solutions in 1:9 v/v acetone/methanol were thoroughly premixed either using an extruder or an ultrasound bath, β-hematin crystals were found formed in intimate association with the lipid droplets. These crystals resembled hemozoin crystals, with prominent {100} faces. Lattice fringes in TEM indicated that these faces made contact with the lipid surface. The average length of these crystals was 0.62 times the average diameter of NLB droplets, and their size distributions were statistically equivalent after 10 min incubation, suggesting that the lipid droplets also controlled the sizes of the crystals. This most closely resembles hemozoin formation in the helminth worm Schistosoma mansoni, while in P. falciparum, crystal formation appears to be associated with the much more gently curved digestive vacuole membrane, which apparently leads to formation of much larger hemozoin crystals, similar to those formed at the flat pentanol–water interface

Insights into the Role of Heme in the Mechanism of Action of Antimalarials

By using cell fractionation and measurement of Fe­(III)­heme-pyridine, the antimalarial chloroquine (CQ) has been shown to cause a dose-dependent decrease in hemozoin and concomitant increase in toxic free heme in cultured <i>Plasmodium falciparum</i> that is directly correlated with parasite survival. Transmission electron microscopy techniques have further shown that heme is redistributed from the parasite digestive vacuole to the cytoplasm and that CQ disrupts hemozoin crystal growth, resulting in mosaic boundaries in the crystals formed in the parasite. Extension of the cell fractionation study to other drugs has shown that artesunate, amodiaquine, lumefantrine, mefloquine, and quinine, all clinically important antimalarials, also inhibit hemozoin formation in the parasite cell, while the antifolate pyrimethamine and its combination with sulfadoxine do not. This study finally provides direct evidence in support of the hemozoin inhibition hypothesis for the mechanism of action of CQ and shows that other quinoline and related antimalarials inhibit cellular hemozoin formation