Characterization of Protoporphyrin IX Species in Vitro Using Fluorescence Spectroscopy and Polar Plot Analysis

Protoporphyrin IX (PPIX) is a photodynamic therapy (PDT) agent for the treatment of various types of cancer. The effectiveness of PDT is believed to be associated with aggregation of PPIX in cells. However, the aggregation equilibrium of PPIX in the cellular environment and in solution is still poorly understood. This is attributed by the lack of a method that allows for controllable generation of PPIX aggregates and robust analysis technique for measuring their photophysical properties. In this study, the dynamics of PPIX aggregation were investigated under high pressure and different solvent conditions using time-resolved fluorescence spectroscopy. The data were analyzed on a polar plot, a model-free analysis method that has become increasingly popular for fluorescence lifetime studies. We discovered that increasing hydrostatic pressure enhanced the formation of J-type aggregates based on measured absorbance, spectra, and lifetime features. Formation of large aggregates, which have a subnanosecond lifetime in the excited state, was observed under the increasing concentration of divalent cations as well as under a solvent of around neutral pH. PPIX monomerizes from the aggregate as pH becomes more basic, not dimerization as proposed by previous studies. Here, we demonstrate that the combination of time-resolved measurement and polar plot analysis is very robust for monitoring the presence of different types of PPIX aggregates formed in various chemical environments.