Cryptotanshinone-Induced Permeabilization of Model Phospholipid Membranes: A Biophysical Study

The Danshen terpenoid cryptotanshinone (CPT) is gaining enormous interest in light of its various outstanding biological activities. Among those, CPT has been shown to interact with cell membranes and, for instance, to have antibacterial activity. Several works have shown that CPT alone, or in combination with other drugs, can effectively act as an antibiotic against various infectious bacteria. Some authors have related the mechanism underlying this action to CPT-membrane interaction. This work shows that CPT readily partitions into phosphatidylcholine membranes, but there is a limiting capacity of accommodation of ca. 1 mol CPT to 3 mol phospholipid. The addition of CPT to unilamellar liposomes composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) causes membrane permeabilization, as shown by fluorescent probe leakage. This process has been kinetically studied, as well as its modulation by incorporation of phosphatidylethanolamine or phosphatidylglycerol, as a model for pathogenic cell membranes. The thermotropic behavior of 1,2-dimyristoylphosphatidylcholine (DMPC) model membranes is weakly affected by CPT, but the terpenoid causes significant dehydration of the polar region of the bilayer and weak disordering of the acyl chain palisade, as observed in Fourier-transform infrared spectroscopy (FTIR) results. Small-angle X-ray scattering (SAXS) shows that CPT increases DMPC bilayer thickness, which could be due to localization near the phospholipid/water interface. Molecular dynamics (MD) simulations show that the lateral diffusion coefficient of the phospholipid increases with the presence of CPT. CPT extends from the polar head region to the center of the bilayer, being centered between the carbonyl groups and the unsaturated region of the POPC, where there is greater overlap. Interestingly, the free energy profiles of a water molecule crossing the lipid membrane show that the POPC membrane becomes more permeable in the presence of CPT. In summary, our results show that CPT perturbs the physicochemical properties of the phospholipid membrane and compromises its barrier function, which could be of relevance to explain part of its antimicrobial or anticancer activities.