Stabilization of 5-aminolevulinic acid by cucurbit[7]uril complex with enhanced fluorescence detection and photodynamic therapy of breast cancer in vitro and in a CAM model.

The FDA-approved prodrug 5-aminolevulinic acid (ALA) is efficiently metabolized by cancer cells into protoporphyrin IX (PpIX), a potent photosensitizer widely used in photodynamic therapy (PDT) and fluorescence-guided tumor surgery. However, the poor membrane permeability of ALA led to the development of more lipophilic derivatives, such as the methyl (ALAm) and hexyl (ALAh) esters. Despite improved cellular uptake, ALA and its esters are highly unstable at physiological pH, resulting in premature decomposition, reduced PpIX accumulation, and limited therapeutic efficacy. In this study, the encapsulation of ALA, ALAm, and ALAh within cucurbit[7]uril (CB[7]) was evaluated as a strategy to enhance their stability and performance. Complex formation was confirmed by isothermal titration calorimetry, 1H NMR spectroscopy, and density functional theory (DFT) calculations. Decomposition kinetics at physiological pH showed that encapsulation significantly increased the stability of the ester derivatives. In vitro studies using 2D cell cultures demonstrated that the ALA@CB[7] complex enhanced intracellular conversion to PpIX, leading to increased phototoxicity, attributed to the improved stability of encapsulated ALA. In contrast, encapsulation of ALAh produced no significant change in phototoxicity, while encapsulated ALAm resulted in decreased phototoxicity in MCF-7 breast cancer cells. Experiments in 3D cell cultures revealed increased PpIX fluorescence and more homogeneous distribution for encapsulated ALA and ALAh, whereas ALAm showed no improvement. Finally, in vivo studies using the chorioallantoic membrane (CAM) model confirmed that encapsulated ALA and ALAh were more efficiently metabolized into PpIX, resulting in higher photosensitizer accumulation. Overall, these findings highlight CB[7] as a promising protective nanocapsule to improve the delivery, stability, and photodynamic efficacy of ALA-based prodrugs.