Analysis of ROS dynamics based on dissolved oxygen sensing in upconversion nanoparticle-based photodynamic therapy

Photodynamic therapy (PDT) is a promising cancer treatment approach that relies on the localized generation of reactive oxygen species (ROS) to eliminate cancer cells. In particular, the nanophotonic approach based on upconversion nanoparticles (UCNPs) offers a key advantage by enabling the use of near-infrared (NIR) light, which enhances light penetration into tissue and expands clinical applicability of PDT. Real-time monitoring of ROS generation and degradation during the PDT process offers distinct advantages over conventional endpoint assays for elucidating PDT mechanisms, optimizing photosensitizer (PS) formulations and refining treatment protocols. In this study, we not only distinguish and quantify the relative contribution of NaYF₄:Yb³⁺,Tm³⁺ UC nano-antennas, Rose Bengal (RB) PS, NIR activation laser, and culture medium in UCNP-based PDT for the first time via real-time ROS analysis using dissolved oxygen (DO) data which cannot be achieved by endpoint assays but also introduce new and insightful concepts such as medium activation time (FWHM), maximum PL lifetime change (Δτ_max), and time to reach the maximum PL lifetime change ₍τ_max). This is realized by implementation of a 3D-printed optofluidic dissolved oxygen (DO) sensor for indirect analysis of ROS dynamics which infer from changes in the sensor's photoluminescence (PL) lifetime (τ). Thus, performance and optimum concentrations of NaYF₄:Yb³⁺,Tm³⁺ UCNPs and RB PS are first determined via MTT assays using A375 melanoma cells, and subsequent in-vitro PDT tests using a 980 nm laser. Quantitative analyses show that, UCNPs, RB, and the cell culture medium contribute approximately 25 %, 26 %, and 4 % to the total Δτ respectively. The maximum performance occurs when all components are present and activated, resulting in the highest ROS level with the longest activation time. Interestingly, even laser excitation of the medium alone or UCNPs without PS results in partial ROS generation. These findings provide valuable insights for optimizing UCNP-based PDT drugs for cancer treatment.