The Lipid Bilayer Provides a Site for Cortisone Crystallization at High Cortisone Concentrations

Abstract

Cortisone is an injected anti-inflammatory drug that can cause painful side effects known as "steroid flares" which are caused by cortisone crystallizing at the injection site. We used molecular dynamics simulations and X-ray diffraction to study the interaction of cortisone with model lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at drug concentrations from 0 mol% to 50 mol%. Cortisone was found to partition in the lipid bilayer and locate in the hydrophilic to hydrophobic interface of the membranes. Cortisone strongly affects the integrity of the membrane, as quantified by a decreased membrane thickness, increased area per lipid, and decreased lipid tail order parameters. At cortisone concentrations of more than 20 mol%, signals from crystallized cortisone were observed. These crystallites are embedded in the bilayers and orient with the membranes. While the cortisone molecules align parallel to the bilayers at low concentrations, they start to penetrate the hydrophobic core at higher concentrations. Trans-membrane crystallites start to nucleate when the membrane thickness has decreased such that cortisone molecules in the different leaflets can find partners from the opposite leaflet resulting in a non-zero density of cortisone molecules in the bilayer center. We suggest that the lipid bilayer provides a site for cortisone crystallization.

Figure 1

(a) A simulation snapshot of a POPC membrane containing 10 mol% cortisone; POPC is show as sticks, cortisone as solid spheres. Water is not shown for clarity. (b) Potential of mean force (PMF) for cortisone across along the membrane normal z. z = 0 corresponds to the membrane center. (c) Electron density profiles calculated from simulations. (d) electron density of cortisone. (e) Area per phospholipid molecule as a function of cortisone content. (f) Deuterium order parameter of the saturated POPC tail as calculated from the simulations.

Figure 2. Two-dimensional X-ray intensity maps of bilayers with increasing cortisone concentration.

(a) 0 mol%, (b) 2 mol%, (c) 5 mol%, (d) 20 mol%, (e) 35 mol% and (f) 50 mol% cortisone. All samples exhibit well-spaced Bragg peaks along , and a broad peak at , indicative of oriented fluid-phase membranes. At >20 mol%, additional peaks are observed in-plane indicating crystallization of cortisone.

Figure 3

(a) Out-of plane X-ray diffraction of oriented POPC membranes containing cortisone. (b) Top: scaled electron densities across the axis perpendicular to the bilayer for 0 mol% and 5 mol% cortisone. Bottom: the difference of these curves shows a positive peak at  Å, indicating the position of cortisone, and a negative peak at  Å due to the thinning of the membrane. The difference curve from the MD simulations at 5 mol% is included for comparison. The slight disagreement in peak position and width between experiment in simulation is likely due to bilayer undulations. An example of is shown as an inset. (c) The lamellar spacing, , and the head-head distance, , as a function of cortisone concentration as determined from diffraction experiments. (d) Scattering along for the oriented membrane samples. Additional peaks, which appear for cortisone concentrations >20 mol%, can be indexed by crystalline cortisone. (e) The integrated intensities for the crystalline cortisone peak observed at  Å⁻¹ as a function of azimuthal angle ϕ. (f) Unit cell of the observed cortisone crystallites, as determined from the in-plane Bragg peaks.

Figure 4

Simulation snapshots for POPC membranes at 0, 10, 40 and 50 mol% (a–d). The purple bar indicates a 40 Å ruler. The structural model of the cortisone crystals based on X-ray experiments is shown in (e).