# Bacterial Cellulose-Polyvinyl Alcohol Based Complex Composites for Controlled Drug Release
## 2 Materials and Methods
### Composites Characterization  
The microstructure and the morphology of the materials was evaluated using a FEI Quanta Inspect F electron microscope (FEI Company, Hillsboro, OR, USA), operated at 20 kV. A thin and continuous layer of gold was deposited on the samples' surface before SEM investigation. The behavior of obtained systems in an aqueous environment was studied. Two parameters were determined: swelling degree, as a measure of moisture uptake capacity, and water solubility, as a parameter indicating matrix degradation and biodegradability. To determine the _swelling degree (SD)_, membranes were dried to a constant weight. Then, each sample was prepared by cutting it into 2 \(\times\) 2 cm square shapes and immersed in distilled water at room temperature (25 \({}^{\circ}\)C) for 2 h. The experiments were performed in triplicates. Based on the authors' best knowledge and a survey of the literature, 2 h were considered sufficient for equilibrium achievement [27; 28; 29]. With a very short time needed for the procedure, the membrane dissolution into the distilled water was neglected. Furthermore, the amount of TC-HCl contained in the active films and released in aqueous media was considered insignificant compared to the amount of absorbed water. The weight of each swollen sample was measured after gently blotting the wet surface with tissue paper to remove excess water. Swelling degree (SD) was obtained by measuring the initial weight, \(m_{i}\) (g), and the weight of the sample in a swollen state, \(m_{s}\) (g) using Equation (1):  
\[\mathrm{SD}=\left(m_{s}-m_{i}\right)/m_{i} \tag{1}\]  
_Water solubility (WS)_ was also determined in triplicate, in accordance with the method proposed by [PERSON] et al., 2010, but slightly modified [30]. Samples of membranes (square shapes of 2 \(\times\) 2 cm) were dried to a constant weight, and then immersed in glass beakers \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample ID** & **Structure** & **Composition** & \
\begin{tabular}{c} **Blending Ratio** \\ **(_w/w_) * \\ \end{tabular} & **Thickness (mm)** &
\begin{tabular}{c} **Drug Loading mg/g** \\ **d.m.** \\ \end{tabular} \\ \hline M0 & monolayer (control) & PVA, NpBC & 7,23 & 0.479 \(\pm\) 0.395 & NA \\ M1 & monolayer & PVA, NpBC, TC-HCl & 7,23,0.1 & 0.481 \(\pm\) 0.121 & 10.60 \\ M2 & monolayer & PVA, NpBC, TC-HCl & 7,23,0.5 & 0.523 \(\pm\) 0.158 & 50.86 \\ M1p & 3-layer & PVA/M1/PVA & 7,7/23,0.1/7 & 0.796 \(\pm\) 0.142 & 7.73 \\ M2p & 3-layer & PVA/M2/PVA & 7,7/2,0.5/7 & 0.785 \(\pm\) 0.155 & 35.71 \\ M1p\(\ell\) & 3-layer & PVA, NfBC/M1/PVA, NfBC & 7,15/7,2,3,0.1/7,15 & 0.793 \(\pm\) 0.151 & 7.24 \\ M2p\(\ell\) & 3-layer & PVA, NfBC/M2/PVA, NfBC & 7,15/7,2,3,0.5/7,15 & 0.817 \(\pm\) 0.148 & 35.20 \\ \hline \hline \end{tabular}  
* Weight in g of each constituent per 100 g casting solution.  
\end{table} 
Table 1: Structure, composition, and thickness of composite membranes. containing 50 mL distilled water. The beakers were periodically shaken and kept at room temperature (25 \({}^{\circ}\)C) for a longer time (72 h). Undissolved pieces of membranes were removed from the water and further dried to a constant weight. The water solubility of films was calculated according to Equation (2), where \(m_{i}\) is the initial weight of the sample, and \(m_{f}\) is the weight of the dried un-dissolved sample, both measured in grams:  
\[WS=\left(m_{i}-m_{f}\right)/m_{i} \tag{2}\]  
_Water vapor permeability (WVP)_ of each composite (prepared as monolayer or multilayer) was determined in triplicate. We followed a well-known procedure, earlier described by [PERSON] et al. [31]. The composites were cut into disks with a diameter of 3 cm. Each disk was carefully sealed on a cup containing silica gel (0% RH) and placed in a desicator equipped with a sensor for temperature and relative humidity. The desiccator contained distilled water as moisture source. The experimental conditions were 37 \({}^{\circ}\)C and 95% RH, maintained constant for the entire experiment.