Hyaluronic acid-functionalized liposomes for CD44-targeted anticancer therapy: In Vitro induced cytotoxicity in HeLa...

This study investigates liposomes as a CD-44-targeted delivery system with bound porphyrinic photosensitizer TMPyP using high molecular weight sodium hyaluronate (HA) for cancer cell targeting to enhance photodynamic therapy outcomes. The effects of TMPyP in its free and liposomal forms are assessed on HeLa and NIH3T3 cells, focusing on intracellular accumulation, oxidative stress, metabolic disruption, and DNA damage. DNA damage, evaluated through γ-H2AX immunofluorescence, indicates double-stranded breaks (DSBs). Free TMPyP causes DSBs in NIH3T3 cells, while liposomal TMPyP promotes a repair mechanism, shown by reduced γ-H2AX. These results are supported by cytotoxicity assays, ROS production, mitochondrial membrane potential depolarization, cytochrome C release and caspase-8 activity, indicating a possible extrinsic apoptotic pathway. The cell death analysis shows that, unlike liposomal TMPyP, free TMPyP damages NIH3T3 cells. Colocalization analysis revealed higher free TMPyP accumulation in the mitochondria of NIH3T3 cells, whereas liposomal TMPyP predominantly accumulates in the mitochondria of HeLa cells. The FLIM analysis reveals shifts in mitochondrial and nuclear NAD(P)H and FAD lifetimes, supporting the nonspecific effects of free TMPyP and highlighting the advantages of its liposomal form. These findings suggest that liposomes composed of cardiolipin, DPPC, and cholesterol with bound HA can selectively deliver TMPyP to HeLa cells for effective PDT.