*2.3. In Vitro Study of Drug Loading and Release*

Tetracycline (TC) was selected as the model drug for the evaluation of drug loading and release. p-HApFs (10 mg) was added to a TC aqueous solution (5 mg/mL, 10 mL) and stirred for 24 h. After the loading procedure, the amount of TC adsorbed onto p-HApFs, which calcined at N2 atmosphere 800 ◦C, was calculated by determining the difference in the TC concentration before and after loading. The TC concentration was analyzed at a wavelength of 360 nm using a UV-Vis spectrophotometer (Ultrospec 1100 Pro, Amersham Biosciences, Piscataway, NJ, USA).

The drug contents and loading efficiency were calculated according to the following formulas:

Drug content (*w*/*w*) = weight of the TC in the p-HApFs/weight of the p-HApFs

Loading efficiency (%) = (weight of the TC in the p-HApFs/initial weight of the p-HApFs) × 100%

After loading, the p-HApFs were dried using a lyophilization process. TC-loaded p-HApFs (20 mg) were added to a phosphate-buffered saline (PBS) solution (2 mL) and agitated in a horizontally shaking bath at 37 ◦C. The release medium was withdrawn and replaced with fresh PBS at each measurement. The mechanism of drug release was analyzed by fitting the experimental data to equations describing different kinetic orders. Linear regression analyses were performed for zero-order [Mt/M0 = K0·t] and first-order [ln(M0 − Mt)=K1·t] kinetics, as well as the Higuchi [Mt/M0 = KH·t 1/2] model, where K is the kinetic constant and Mt/M0 is the fraction of TC released at time t. The best-fitted model was assessed on the basis of the correlation coefficient (r2). The drug release data were further analyzed with the Ritger-Peppas equation [Mt/M<sup>∞</sup> = kr·t n], where Mt is the amount of drug released at time t, M<sup>∞</sup> is the amount of drug released at time ∞, and kr and n represent the release rate constant and release exponent, respectively.
