Indomethacin forms elusive nanoparticles with calcium in situ causing enhanced permeation across a biomimetic barrier

Indomethacin is a poorly soluble weak acid and a widely used model drug in enabling formulations. When using microdialysis for sampling of indomethacin from a buffer containing calcium, we observed the formation of nanoparticles of a poorly water-soluble indomethacin calcium salt. The nanoparticles were not detected during solubility experiments where filtration had been used to separate the solid phase because the nanoparticles were unusually small in size, less than 2 nm in diameter as determined by DLS. The nanoparticles only formed in the presence of tris-maleate, a common buffer for in vitro lipolysis. Molecular dynamics simulations indicated that indomethacin strongly interacted with tris and maleate causing aggregation on a molecular level. Counterintuitively, the nanoparticles significantly enhanced permeation of indomethacin across a biomimetic barrier (Permeapad®), probably due to charge neutralization. This agrees with literature on in vivo enhanced bioavailability and demonstrates the biomimetic properties of the barrier. By microdialysis sampling, drug release from enabling formulations during conditions mimicking gastrointestinal processing (lipolysis) can be assessed. This sampling technique excludes drug associated with colloidal structures. Thus, a combination of conventional and microdialysis sampling can reveal the interplay between free drug and drug associated with colloidal structures in a time-resolved manner. Using only conventional filtration or bench top centrifugation for separation, those elusive nanoparticles would have remained undiscovered.