Doxycycline hyclate (DOX, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 5 mL of 50 mM HEPES buffer (pH = 7.2, Fisher Scientific, Pittsburgh, PA, USA) at three distinct concentrations (w/v): 10% (0.5 g), 20% (1 g), and 30% (1.5 g) by stirring (Fisher Thermix, model 310T, Fisher Scientific). The loading process started by mixing a predetermined amount (1.25 g) of pre-sieved (<45 µm) Halloysite® nanotubes (HNT, Dragonite 1415JM, Applied Minerals Inc., New York, NY, USA) in 5 mL of each DOX solution and followed by sonication for 2 h [19 (link)]. In order to minimize any air between and within the nanotubes, the solutions were placed in a vacuum (25 in.Hg) chamber for 1 h. Next, the solutions were mixed for 1 h and vacuum was reapplied [14 (link)]. Finally, the HNT+DOX solutions were centrifuged (3000 rpm) for 10 min. The supernatants were collected and stored at −20°C for high-pressure liquid chromatography (HPLC) analysis. Meanwhile, the HNT+DOX pellets were dried at 37°C, weighed, ground, and sieved.