Head‐to‐Head Comparison of Selected Extra‐ and Intracellular CO‐Releasing Molecules on Their CO‐Releasing and Anti‐Inflammatory Properties

Abstract Over the past decade, a variety of carbon monoxide releasing molecules (CORMs) have been developed and tested. Some CORMs spontaneously release CO once in solution, while others require a trigger mechanism to release the bound CO from its molecular complex. The modulation of biological systems by CORMs depends largely on the spatiotemporal release of CO, which likely differs among the different types of CORMs. In spontaneously releasing CORMs, CO is released extracellularly and crosses the cell membrane to interact with intracellular targets. Other CORMs can directly release CO intracellularly, which may be a more efficient method to modulate biological systems. In the present study, we compared the efficacy of extracellular and intracellular CO‐releasing CORMs that either release CO spontaneously or require an enzymatic trigger. The efficacy of such CORMs to modulate HO‐1 and VCAM‐1 expression in TNF‐α‐stimulated human umbilical vein endothelial cells (HUVEC) was evaluated.


General Information
Unless otherwise specified, all reactions were carried out under inert conditions. Glassware was heat-dried under vacuum and flushed with argon (Linde® Argon 4.6 (99.996 %, <1 ppm water, <1 ppm oxygen). Solids were added under argon counter flow. Liquids were added with syringes through gas-tight septa. Before use, syringes were flushed with argon thrice.
Reagents were purchased from commercial suppliers (Sigma-Aldrich, Lancaster, Alfa Aesar, Carbolution, ABCR or Acros) and used without further purification.
The mixture was diluted with EtOAc (45 mL    and phosphate buffer (0.1 M, pH = 7.4, 1.0 mL). Subsequently, the vials were closed with gas-tight silicon/PTFE septa crimp caps (BGB Analytics, cat. No. 20030500). Then, a defined gas volume was substituted by CO or CO2 (0.00 mL, 0.05 mL, 0.10 mL, 0.25 mL, 0.50 mL, 1.00 mL, 1.50 mL, 2.00 mL). After equilibrating the vials for 10 min at 37 °C the composition of the gas phase was determined by headspace GC. These measurements were repeated three times and a calibration curve was generated.

In situ quantification of the CO release properties
The respective complex (36 μmol) was dissolved in DMSO (0.2 mL) and phosphate buffer (0.1 M, pH = 7.4, 1.0 mL) then PLE (15 mg) was added. The mixture was stirred at 37 °C and the amount of released CO assessed through headspace GC using the previously recorded calibration curve.
As a control, samples without addition of esterase were analyzed in the same manner. replicates were used.
Cell lysates were centrifuged for 10 minutes at 12000 G at 4 °C to remove undissolved cell debris.