In this study, the effects of two loading strategies, namely in situ and ex situ loading, on the release behavior, antimicrobial activity, and cellular compatibility of bacterial cellulose (BC)-based nanocomposites containing silver (Ag), copper oxide (CuO), and zinc oxide (ZnO) antimicrobial agents were investigated. Although numerous studies have reported the incorporation of different antimicrobial agents into BC matrices, a direct and systematic comparison of in situ and ex situ loading strategies for several common antimicrobial agents on the same BC substrate under identical evaluation conditions remains limited. Therefore, the novelty of the present work lies in examining the loading strategy as an independent variable across three systems containing Ag, CuO, and ZnO, in order to clarify its role in active-agent accessibility and the resulting biological outcomes.

Following synthesis, the samples were characterized using structural and chemical analysis methods. Their release behavior in phosphate-buffered saline (PBS), antimicrobial activity against Escherichia coli and Staphylococcus aureus, and cellular response were then evaluated. The results showed that after 72 h, the ex situ-loaded samples exhibited higher cumulative release of the active agents than their corresponding in situ-loaded counterparts. Specifically, the release percentages of Ag, Cu, and Zn in the ex situ samples were 38.0 ± 6.00%, 41.0 ± 6.00%, and 36.0 ± 5.50%, respectively, whereas the corresponding values for the in situ samples were 24.0 ± 3.51%, 29.0 ± 4.51%, and 21.0 ± 3.51%. In addition, both antimicrobial performance and cellular compatibility depended on the type of antimicrobial agent and the loading strategy employed.

Overall, the findings indicate that the loading strategy can significantly influence the release profile and, consequently, the final biological response of BC nanocomposites by altering the accessibility of the active agent. However, given the in vitro nature of this study, the limited number of formulations examined, and the sensitivity of the results to fabrication and washing conditions, these findings should be regarded as preliminary evidence for subsequent screening and optimization rather than as a basis for direct preclinical or clinical generalization.



Keywords: bacterial cellulose; antimicrobial nanocomposite; in situ loading; ex situ loading; controlled release; antimicrobial activity; cytocompatibility