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 beta-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 beta-hematin crystals. The latter have been compared with beta-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, beta-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.