In Situ Single-Cell Bacterial Imaging Provides Mechanistic Insight into the Photodynamic Action of Photosensitizer-Loaded Hydrogels

Hydrogels, three-dimensional hydrophilic polymeric networks with high water retaining capacity, have gained prominence in wound management and drug delivery due to their tunability, softness, permeability, and biocompatibility. Electron-beam polymerized poly(ethylene glycol) diacrylate (PEGDA) hydrogels are particularly useful for phototherapies such as antimicrobial photodynamic therapy (aPDT) due to their excellent optical properties. This work takes advantage of the transparency of PEGDA hydrogels to investigate bacterial responses to aPDT at the single-cell level, in real-time and in situ. The photosensitizer methylene blue (MB) was loaded in PEGDA hydrogels by using two methods: reversible loading and irreversible immobilization within the 3D polymer network. MB release kinetics and singlet oxygen generation were studied, revealing the distinct behaviors of both hydrogels. Real-time imaging of Escherichia coli was conducted during aPDT in both hydrogel types, using the Min protein system to report changes in bacterial physiology. Min oscillation patterns provided mechanistic insights into bacterial photoinactivation, revealing a dependence on the hydrogel preparation method. This difference was attributed to the mobility of MB within the hydrogel, affecting its direct interaction with bacterial membranes. These findings shed light on the complex interplay between hydrogel properties and the bacterial response during aPDT, offering valuable insights for the development of antibacterial wound dressing materials. The study demonstrates the capability of real-time, single-cell fluorescence microscopy to unravel dynamic bacterial behaviors in the intricate environment of hydrogel surfaces during aPDT.