Preparation and Antibacterial\nActivity Evaluation\nof Daphnetin-Loaded Poloxamers/Polyvinylpyrrolidone Thermosensitive\nHydrogels

Antibiotic misuse and bacterial resistance are pressing\nissues\nthreatening public health. Natural plant extracts with bactericidal\nproperties offer potential alternatives to reduce or replace antibiotic\nuse. This study aims to develop a thermosensitive hydrogel containing\ndaphnetin (DAP-TG) using poloxamers 407 (P407), polyvinylpyrrolidone\n(PVP), and poloxamers 188 (P188). We systematically evaluated the\ngel’s antibacterial activity against <i>Escherichia coli</i> (E. coli) and Staphylococcus aureus (S. aureus), as well as its antibacterial mechanisms.\nBy examining the gelation temperature and time, degradation time,\nand in vitro release performance of DAP-TG, we produced a sustained-release\nDAP-TG with a rapid phase transition at (31.6 ± 0.1) °C.\nIts structure was characterized through Fourier transform infrared\n(FTIR) spectroscopy and scanning electron microscopy (SEM). The results\nindicated that the DAP thermosensitive hydrogel was formed and presented\na 3D network spatial structure. The biocompatibility of DAP-TG was\nexplored through the hemolysis test and cytotoxicity test. The results\nindicated that DAP-TG possessed excellent biocompatibility. The antibacterial\nefficacy of DAP-TG against E. coli and S. aureus was assessed using minimum inhibitory concentration\n(MIC), minimum bactericidal concentration (MBC), growth curve, and\ninhibition zone tests. Results showed that DAP-TG exhibited excellent\nantibacterial activity against both E. coli and S. aureus, with MIC values of\n1.28 and 0.32 mg/mL. The antibacterial mechanism of DAP-TG was preliminarily\nexplored through the investigation of bacterial cell content leakage,\nAKP leakage, membrane permeability, SEM, ROS production, and biofilm\ninhibition activity. DAP-TG induced irreversible damage to the cell\nmembranes of E. coli and S. aureus, resulting in enhanced permeability, elevated\nROS levels, and inhibited biofilm formation. Our study indicates that\nDAP-TG exhibits effective sustained-release and antibacterial properties\nagainst E. coli and S. aureus in vitro, making it a promising candidate\nfor antibacterial applications in food and pharmaceutical products.