Mesoporous silica microparticles gated with a bulky azo derivative for the controlled release of dyes/drugs in colon

Mesoporous silica microparticles were prepared, loaded with the dye safranin O (M-Saf) or with the drug budesonide (M-Bud) and capped by the grafting of a bulky azo derivative. Cargo release from M-Saf at different pH values (mimicking those found in the gastrointestinal tract) in the absence or presence of sodium dithionite (a reducing agent mimicking azoreductase enzyme present in the colon) was tested. Negligible safranin O release was observed at pH 6.8 and 4.5, whereas a moderate delivery at pH 1.2 was noted and attributed to the hydrolysis of the urea bond that linked the azo derivative onto the external surface of the inorganic scaffold. Moreover, a marked release was observed when sodium dithionite was present and was ascribed to the rupture of the azo bond in the molecular gate. Budesonide release from M-Bud in the presence of sodium dithionite was also assessed by ultraviolet-visible spectroscopy and high performance liquid chromatography measurements. In addition, preliminary in vivo experiments with M-Saf carried out in mice indicated that the chemical integrity of the microparticles remained unaltered in the stomach and the small intestine, and safranin O seemed to be released in the colon.


HPLC conditions and procedures
The calibration method of HPLC was prepared as a previously reported. [1] The mobile phase consisted of methanol: water (80:20 v/v). The flow rate was 1 mL/min. The wavelength of detection was 244 nm (λmax for BUD). The injection volume was 20 µL.

Preparation of stock and standard solutions.
Methanol: water (50:50 v/v) was used as a solvent for preparation of both stock as well as standard solutions. The stock solution of budesonide was prepared by dissolving 10.0 mg of drug in 100 mL solvent, creating a 100 µg/mL solution of budesonide. This solution was diluted with solvent a needed to prepare different standard solutions (100, 50, 25, 10, 5 and 1 µg/mL). Standard solutions each in three replicates were injected into the system. The method of linear regression was used for data evaluation. Peak area ratios of standard budesonide were plotted against theoretical concentrations of standards solutions Figure S12. Linearity was expressed as a correlation coefficient. Drug release studies.
In a typical experiment, 5 mg of M-Bud was suspended in water (10 mL), sodium dithionite (3 mg) was added and aliquots were separated after a 5 minutes and 4 hours, centrifuged. A known maximum drug release was then injected to HPLC systems. The chromatogram show a single peak at t R = 4.20 min. and no indication of degradation of budesonide at tR = 2.0 min. The peak area of budesonide (tR = 4.20 min) was concentration. Drug release at 5 minutes was 24 µg/mg material and at 4 hours was 31.2 µg/mg material.

In vitro dye release studies from M-Saf at different pH values.
Dye delivery from M-Saf was followed by UV-vis spectroscopy monitoring the safranin O band centered at 520 nm (See Figure S14). These studies were carried out using three different buffers, to simulate the GIT, (hydrochloric acid pH 1.2, sodium acetate pH 4.5 and sodium phosphate pH 6.8) that were prepared according to the Ph Eur. In a typical experiment, M-Saf (5 mg) was suspended in water (17 mL) at selected pH (6.8, 4.5 and 1.2) in the absence or in the presence of sodium dithionite (3 mg). Aliquots were taken at scheduled times, filtered off in order to eliminate the suspended microparticles and the absorbance of the safranin O released from M-Saf was determined. Then the filtered solution and the microparticles were returned to the initial suspension until the next measurement.  Figure S17. UV-Vis spectra of M-Saf in buffer sodium phosphate pH 6.8.  M-Saf buffer 6.8

With Na2S2O4
Time  In vivo release studies.
The studies reported here adhere to the Principles of Laboratory Animal Care and were approved by the institutional ethics committee of the Conselleria de Agricultura, Medio Ambiente, Cambio Climático y Desarrollo Rural (Generalitat Valenciana), according to 2016/VSC/PEA/00158.
In a typical experiment, 10 mg of M-Saf was suspended in water (200 µL). The mixture was administrated to the mouse by oral gavage by using a cannula provided with a rounded tip. The mouse, with free access to water and food, was observed during 4 hours showing a normal behaviour. The mouse was euthanized with an overdose of anesthesia (dolethal®) and then the abdomen was opened and the digestive system was removed. In one of then, the digestive system was dissected to locate the microparticles through it. The M-Saf was observed to pass freely without causing any ulcer or absorption in the other tissues. After this time, an appreciable amount of closed material was present in the stomach. In addition, the microparticles located along the small intestine remained also closed. The material isolated in each section, was evaluated using sodium dithionite and the safranin O delivered was followed by fluorescence spectrometry (Figure 5 and S19). In addition, another mouse was studied during 24 h after oral administration of M-Saf. After 24 h the mouse was euthanized in similar conditions and the digestive system was dissected to locate the microparticles through it. After this time, no appreciable amounts of closed material were present in the stomach, intestine or colon, and the corresponding release studies using Na2S2O4 did not show the presence of safranin O in the extracted solutions. In addition, the stool was also evaluated. The stool was homogenized and suspended in water (5 mL) during 24 h at room temperature. The mixture was centrifuged and the supernatant was separated. The solid was suspended again in water (5 mL) and safranin O release was evaluated using sodium dithionite. After this time, safranin O was not observed ( Figure S20).