Fig 1.
Schematic diagram of an Ussing chamber (Authors’ diagram).
The apparatus consists of two baths separated by, for example, a sheet of intestinal mucosa, one bath being on the mucosal side and the other on the serosal side. Each bath is filled with Krebs-Ringer solution kept at 37°C, and is constantly agitated by bubbling 95% O2-5% CO2 gas. The test drug solution is dropped into the mucosal bath, and any drug permeating into the serosal bath is measured upon collection after a given time.
Fig 2.
SEM micrograph of AZ crystals (A) and AZ/HAP formulation (B). A and B are enlargements of photographs a and b, respectively.
Fig 3.
Solubility testing of AZ and AZ/HAP formulation in water (A) and DTS 2 (B). Samples were taken at 3, 10, 30, 120, and 360 min from the start of the test. The black circles and white circles indicate AZ and AZ/HAP formulation, respectively. Each value is the mean ± S.D. obtained after testing AZ 3 times and the AZ/HAP formulation 6 times.
Fig 4.
Levels of blood concentration of AZ after oral administration of untreated AZ and AZ/HAP formulation in rats.
Samples were taken at 0.5, 1.0, 3.0 and 6.0 hours from the start of the test. Four rats were used in the AZ group (black circles) and seven in the AZ/HAP formulation group (white circles). Each value is the mean ± S.D. obtained. Asterisks show a significant difference (p <0.05) between the AZ group and AZ/HAP group using Student’s t-test.
Table 1.
Cmax and AUC (0–6) values obtained from the blood concentration of AZ in each drug administration group.
Fig 5.
Elution patterns of gel filtration chromatography for AZ, nano-HAP and AZ/HAP formulation.
Graphs A and B show the elution patterns of AZ and nano-Hap obtained from AZ solution and from nano-HAP suspension treated with DTS 2 on the gel filtration column, respectively. C and D show the respective elution patterns of AZ and nano-HAP obtained from the analysis of AZ/HAP formulation treated with DTS 2. E and F show the respective elution patterns of AZ and nano-HAP obtained from the analysis of AZ/HAP formulation treated with intestinal fluid. The horizontal axis shows the amount of elution, and the vertical axis shows the elution rate (%) of each component.
Fig 6.
Permeability assay of AZ and the AZ/HAP formulation using a Caco-2 cell monolayer kit.
The vertical axis indicates Papp value (cm/s). The bar above each column indicates S.D. No significant difference between the AZ group and the AZ/HAP formulation group was observed.
Table 2.
Change in TEER Value (%).
Fig 7.
Permeability of AZ through NPP tissue using an Ussing chamber.
Graph A shows the amount of AZ at each sampling time in the serosal bath when the final concentration of AZ in the mucosal bath was 560 ppm. Graph B shows the results when the final concentration of AZ in the mucosal bath was 2,240 ppm. Black and white columns show results for the AZ and AZ/HAP formulation groups respectively. Each value is the mean ± S.D. obtained (n = 4). Asterisks indicate a statistically significant difference (p <0.05) between the two groups. Note that A and B have different vertical scales.
Fig 8.
Permeability of AZ through PP tissue using an Ussing chamber.
Graph A shows the amount of AZ in the serosal bath at each sampling time when the initial concentration of AZ in the mucosal bath was 560 ppm. Graph B shows the results when the final concentration of AZ in the mucosal bath was 2,240 ppm. Black and white columns show results for the AZ and AZ/HAP formulation groups respectively. Each value is the mean ± S.D. obtained (n = 4). Note that A and B have different vertical scales.
Fig 9.
Localization of OsteoSense 680EX and Calcein double-labeled nano-HAP particles in rat intestinal villi (A) and Peyer’s patches (B) 30 min after oral administration. Photos in the right column show the fluorescence observed at each wavelength for OsteoSense 680EX (top), Calcein (middle), and DAPI (bottom), respectively. The photo on the left (Merge) is a layered version of these three photos. Arrows indicate the location of the nano-HAP particles seen with fluorescence.