Description
Antibiotic resistance is one of the major global challenges, as resistance to all antibiotics currently in clinical use has been reported, while only a limited number of new medications are being developed. In this context, antimicrobial photodynamic therapy (aPDT) has emerged as a promising alternative for the treatment of chronic wound infections. This approach relies on light‑activated photosensitizers to generate reactive oxygen species (ROS) that kill pathogens without promoting resistance. Among available photoactive molecules, methylene blue (MB) is particularly attractive because it has been used clinically for over a century, exhibits broad antimicrobial activity, and absorbs light strongly in the red spectral region (660-670 nm), which enables deep tissue penetration.
To localize MB exposure and promote faster wound healing, the photosensitizer is commonly immobilized within alginate hydrogel systems. Alginate hydrogels provide a biocompatible and transparent matrix that helps reduce off‑target effects. However, most existing solutions rely on bulk alginate gels or polydisperse beads produced by batch emulsification methods. These approaches result in wide-size distributions and poorly controlled MB loading, resulting in undefined and irreproducible dosing. Furthermore, despite the central role of ROS in aPDT, current studies do not quantify ROS output per‑bead, nor address how ROS generation changes over repeated light activation cycles. This limits the predictability, optimization, and long‑term applicability of alginate‑based aPDT systems.
To overcome these limitations, we developed a water-in-oil droplet microfluidic system. A polydimethylsiloxane (PDMS) on glass microfluidic chip with a cross-junction geometry was designed to generate highly monodisperse water‑in‑oil droplets. The system was optimized by adjusting aqueous and oil phase flow rates, alginate concentration, and Tween 80 surfactant content in mineral oil, as well as CaCl2 crosslinking conditions. The resulting microbeads were characterized by optical imaging to determine size, uniformity, and stability. After loading with MB, ROS generation was quantified on a per‑bead basis under controlled light activation. This platform allows direct assessment of photodynamic behavior across microbead populations.
Overall, this work establishes a reproducible and quantitative framework for controlled aPDT dosing, enabling direct correlation between microbead design and photodynamic output. In the future, this approach may support the development of localized and reusable antimicrobial treatments, including wound dressings, catheter or implant coatings, and flow‑based disinfection systems where predictable and controllable photodynamic activity is required.