**2.3 Assessment of** *in vitro* **bioactivity test**

The bioactivity of the obtained samples was examined using *in vitro* test by immerging the samples in SBF solution at body temperature, 37°C at pH 7.40 following Kokubo method [31]. The *in vitro* bioactivity was performed by soaking grainy 3DOM-MBGCs in the SBF solution at a temperature of 37.0°C for 1, 2, 3 and 7 days with daily refreshing of the SBF solution. The ratio of the glass powders weight to SBF volume was 1.5 mg/mL. After soaking, samples were removed from the SBF solution and washed with deionized water several times and air-dried at room temperature. The changes of the bioactive glass surfaces were examined by SEM and FTIR techniques.

### **2.4** *In vitro* **study of drug release**

### *2.4.1 Determination of drug concentration*

Gentamicin concentration was analyzed by measuring the UV/Vis absorbance of gentamicin-*o*-phthaldialdehyde complex at 333 nm [14]. The *o*-phthaldialdehyde reagent was prepared according to Huang et al., 2017 [17]. 1 mL of gentamicin solution, 1 mL of isopropanol and 1 mL of *o*-phthaldialdehyde reagent were reacted for 45 min at room temperature to prepare the sample to examine in UV/visible

*Three-Dimensionally Ordered Macroporous-Mesoporous Bioactive Glass Ceramics for Drug… DOI: http://dx.doi.org/10.5772/intechopen.95290*

spectroscopy [32]. Before determination, a calibration curve (R2 = 0.99) was made for each set of measurements and determined by taking absorbance vs. drug concentration between 1 to 150 ppm as parameters.

### *2.4.2 Encapsulation of gentamicin in the bioactive glass ceramics*

Encapsulation of gentamicin into the MBGCs (45S5 and S53P4) were carried out in PBS pH 7.4 at room temperature for 24 h. 200 mg of each MBGCs was immersed in 10 mL of PBS containing gentamicin with a concentration of 10 mg/mL and stirred for 24 h. After that, the drug loaded MBGCs were filtered and then the drug loading efficiency and drug loading content were determined. Drug loading efficiency was measured by depletion method, by determining the difference in gentamicin concentration in the loading medium before and after loading [14]. The drug-loading experiments were carried out in triplicate, and the statistical computations were performed with the IBM SPSS Statistics version 25. Drug loading efficiency and drug loading content are two important parameters for drug delivery study with MBGCs. Drug loading content represents the mass ratio of drugs in drug loaded MBGCs and drug loading efficiency reflects the utilization of drugs in feed during drug loading [33]. The drug loading efficiency and drug loading content are expressed according to the following Equations [33].

$$\text{Drug loading efficiency} \left( wt. \text{9\%} \right) = \frac{\text{Mass of the drug in MBSCs}}{\text{Mass of the drug in feed}} \times 100 \quad \text{(1)}$$

$$\text{Drug loading content} \left( wt.\% \right) = \frac{\text{Mass of the drug in MBGCs}}{\text{Initial mass of MBGCs}} \times 100 \quad (2)$$

### *2.4.3 In vitro drug release*

50 mg of drug loaded MBGCs were placed into 10 mL of PBS and subsequently agitated in a horizontal shaking incubator at 37°C. 2 mL of release medium was withdrawn at predetermined time intervals and replaced with fresh release medium (2 mL) at each measurement. The triplicate samples of each drug loaded MBGCs (45S5 and S53P4) were used to determine the drug release profile.

### *2.4.4 Kinetic analysis drug release profile*

To study the drug release kinetic from 45S5 and S53P4 MBGCs, the *in vitro* drug release data of gentamicin was fitted in Peppas-Korsmeyer kinetic model mentioned below;

$$\frac{M\_{\rm r}}{M\_{\rm o}} = Kt^{\rm n} \tag{3}$$

Where K is the Peppas-Korsmeyer constant, *Mt M*<sup>∞</sup> is the fractional solute release at time t, and n is the exponent indicative of the release mechanism. An exponential value in the range of 0.45 or less and 0.89 or above indicate respectively Fickian

diffusion and case II transport (typical zero-order release). Values between 0.45 and 0.89 indicate non-Fickian or anomalous release by both diffusion and erosion release [34].
