Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID-BA or NSAID-BA-PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA-IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA-IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA-IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA-IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.

Figure 1

Chemical structures of (A) cholic acid (CA), (B) ibuprofen (IBU), and (C) dodecylphosphocholine (DPC). Key atoms discussed in the text are labeled. Color code: carbon (cyan), oxygen (red) and nitrogen (blue). Hydrogen atoms are not shown.

Figure 2

(A) Ensemble averaged N_W and N_N (main plot) and polydispersity index (inset). (B) Weighted probability distribution of the total number of CA and IBU molecules belonging to clusters of size CSn where CSn is defined as the number of CA-IBU mixed micelles of size n (n=2, 3, 4…) or the number of CA and IBU monomers (n=1). For this and subsequent figures the first 30ns of the trajectories was excluded.

Figure 3

The average number of IBU (black square) and CA (red circle) molecules belonging to a particular cluster size in simulation S_A (A), S_B (B), and S_C (C). The gray dotted lines represent the most populated micelle sizes shown in Figure 2B.

Figure 4

Bivariate distribution of I₁/I₂ and I₂/I₃ ratios for the predominant micelles from simulation S_A: CS19 (A), CS18 (B), CS15 (C), and CS10 (D); simulation S_B: CS15 (E); and simulation S_C: CS8 (F). Red indicates maximum probability. The principal moments of inertia were calculated from the last 10ns of each trajectory.

Figure 5

A proposed mechanism for the aggregation of CA and IBU to form mixed-micelles. CA is shown in green and IBU in blue. Representative snapshots from the simulations are shown for illustration. The information conveyed in this figure is qualitative because limitations in the simulation time scales and the complexity of the process did not allow us to quantify the relative role of each mechanism.

Figure 6

(A) A 3D representation of the molecular packing in a typical CA:IBU micelle. CA is shown in licorice green with C18 & C19 in yellow spheres. IBU is in blue space-filling model. The oxygen atoms in both CA and IBU are shown in red. (B) Average distances between the center of mass and the indicated atoms of micelle CS19 from simulation S_A.

Figure 7

NMR and DLS characterization of IBU/CA interaction. (A) Chemical shifts in a 25mM sample of CA (green), IBU (red) and IBU-CA (blue). (B) Portion of a 400 ms ROESY spectrum for a CA-IBU mixture. (C) DLS-derived micelle sizes at 25mM for CA (black), IBU (green, which lies on the x-axis) and IBU-CA (red). Micelle size distribution for a 50mM deoxyCA (gray dashed line) is shown for reference. IBU alone did not form micelles at 25mM (green line), and at 5mM had no effect on micelle formation by CA (not shown). Shown in the inset is the average micelle size against IBU/CA ratio, where the sigmoidal curve indicates increase in micelle size with increasing IBU level until a plateau is reached when IBU is ~3-times more than CA. Refer to Figure 1 for the numbering in (A) and (B).

Figure 8

(A) Weighted probability distributions of the total numbers of CA and IBU molecules belonging to clusters of size CSn from simulation S_E. Two clusters, CS2 and CS4 adsorbed on the DPC micelle, are shown in a space filling model. CA and IBU molecules that are not part of a cluster are shown in licorice. The predominant cluster CS10 found in the aqueous medium is also shown. Very similar results were obtained from S_D (data not shown). (B) Distribution of the distances between CA-C18 and IBU-C11 for all CA and IBU molecules in CS10 (gray) and for those within 6Å of the DPC micelle (black). The distribution is calculated for the last 10ns of the simulation. IBU is shown in blue, CA in green and DPC in magenta.

Figure 9

(A) The distribution of selected atoms as a function of distance from the center of mass of the DPC micelle. (B) Orientations of different regions of IBU as a function of distance from the center of mass of the mixed micelle (see text for detail).