Structural Effects and Translocation of Doxorubicin in a DPPC/Chol Bilayer: The Role of Cholesterol  Tyrone J. Yacoub, Allam S. Reddy, Igal Szleifer  Biophysical Journal  Volume 101, Issue 2, Pages 378-385 (July 2011) DOI: 10.1016/j.bpj.2011.06.015 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Chemical representation of neutral doxorubicin. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 Potential of mean force, ΔG, as a function of the distance between the centers of mass of DOX and the bilayer (ZDOX–BIL), for 0% (black line, bottom), 15% (blue line, middle), and 30% (red line, top) Chol systems. From left to right, the drug begins in the bulk water, penetrates into the center of the bilayer, and exits into the water on the opposite side. Calculations were performed on one side of the membrane, and assumed to be identical on the opposite side. The error is a propagation of mean force errors. Error bars omitted on the left side for clarity. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 Snapshots (A–C) show 0%, 15%, and 30% Chol systems with ZDOX–BIL = −1.9, −2.5, and −2.6 nm, respectively, corresponding to the most likely position of DOX based on the free energy minima in Fig. 2, and (D–F) show the corresponding systems with the drug in the center. Representations include water (blue), DPPC nitrogen (purple), DPPC phosphorous (green), and Chol oxygen (red). DOX is shown in a van der Waals representation, with oxygen (red), hydrogen (white), nitrogen (blue), and carbon (teal). Note the various degrees of water penetration, and the horizontal Chol molecule bound to the drug in snapshot (E). DOX may act as a facilitator of cholesterol flip-flop. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Snapshot of DOX in 30% Chol, with ZDOX–BIL = −0.1 nm. (A) DOX causes membrane curvature on both sides of the membrane by attracting DPPC headgroups from both leaflets. (B) Shows (A) with everything removed except the drug and water, showing water translocation. Snapshots are rendered as in Fig. 3, with DOX reduced in thickness to show its structure and interaction with water. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 5 Number density profiles in 30% Chol bilayers with DOX (A), see Fig. 4, and without DOX (B). Colors correspond to Figs. 3 and 4. Representations include water (blue), DPPC nitrogen (purple), DPPC phosphorous (green), Chol oxygen (red), and DOX (black). Note the penetration of water and a general broadening of the head-group atom densities in the presence of DOX. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 6 (A) Average DPPC order parameters, <SZ>, as a function of DOX-bilayer distance, ZDOX–BIL. To elucidate structural effects of DOX, the bilayer is split into four regions, as denoted in the legend of (A) and the corresponding colored regions of snapshot (B). Open data points account for DPPC with phosphorous within a 1.75 nm shell of the drug in the xy plane, while solid points are for chains beyond the shell. The simulation shown in (B) thus contributes the four data points at ZDOX–BIL = −0.9 nm in (A). Lines are regressions as a guide for the eye. Note strong coupling between leaflets, even with DOX far from the center. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 7 Area per lipid as a function of the distance between the center of mass of DOX from the center of the bilayer, ZDOX–BIL. Dashed lines correspond to the area per lipid calculated in DOX-free systems. Biophysical Journal 2011 101, 378-385DOI: (10.1016/j.bpj.2011.06.015) Copyright © 2011 Biophysical Society Terms and Conditions