Carotenoids, and β-carotene in particular, are important natural antioxidants. Singlet oxygen, the lowest excited state of molecular oxygen, is an intermediate often involved in natural oxidation reactions. The fact that β-carotene efficiently quenches singlet oxygen in solution-phase systems is invariably invoked when explaining the biological antioxidative properties of β-carotene. We recently developed unique microscope-based time-resolved spectroscopic methods that allow us to directly examine singlet oxygen in mammalian cells. We now demonstrate that intracellular singlet oxygen, produced in a photosensitized process, is in fact not efficiently deactivated by β-carotene. This observation requires a re-evaluation of β-carotene's role as an antioxidant in mammalian systems and now underscores the importance of mechanisms by which β-carotene inhibits radical reactions.

Riboflavin (Rf) is an endogenous photosensitizer, which can participate in Type I and Type II processes. We have recently shown that the yield of the triplet excited states of Rf is enhanced in the presence of pectin-coated silver nanoparticles (Pec@AgNP) due to formation of a complex between Rf and Pec@AgNP (Rf-Pec@AgNP). Consequently, under aerobic conditions, the amounts of singlet molecular oxygen and superoxide radical anion generated are also larger in the presence of the nanoparticles. This result made us suspect that the nanoparticles could have a beneficial effect in Rf-based PDT. To prove this hypothesis, we here compared the photodamage in HeLa cells incubated with Rf in the presence and in the absence of Pec@AgNP applying several optical assays. We used fluorescence imaging of irradiated HeLa cells incubated with Annexin V and propidium iodide to evaluate the occurrence of apoptosis/necrosis, the reduction of the tetrazolium dye MTT to formazan and neutral red uptake to prove cell viability, as well as synchrotron infrared microscopy of single cells to evaluate possible structural changes of DNA and nuclear proteins. The enhanced photodamage observed in the presence of Pec@AgNP seems to indicate that Rf enters into the cells complexed with the nanoparticles.

Plasmonic metal nanoparticles (NPs) can be used as enhancers of the efficiency of standard photosensitizers (PSs) in photodynamic therapy (PDT). Protein corona, the adsorption layer that forms spontaneously around NPs once in contact with biological fluids, determines to a great extent the efficiency of PDT. In this work, we explore the possibility that pectin-coated Au NPs (Au@Pec NPs) could act as adjuvants in riboflavin (Rf)-based PDT by comparing the photodamage in HeLa cells cultured in the presence and in the absence of the NPs. Moreover, we investigate the impact that the preincubation of Rf and Au@Pec NPs (or Ag@Pec NPs) at two very different serum concentrations could have on cell’s photodamage. Because reactive oxygen species (ROS) precursors are the excited states of the PS, the effect of proteins on the photophysics of Rf and Rf/plasmonic NPs was studied by transient absorption experiments. The beneficial effect of Au@Pec NPs in Rf-based PDT on HeLa cells cultured under standard serum conditions was demonstrated for the first time. However, the preincubation of Rf and Au@Pec NPs (or Ag@Pec NPs) with serum has undesirable results regarding the enhancement of Rf-based PDT. In this sense, we also verified that more concentrated protein conditions result in lower amounts of the triplet excited state of Rf and thus an expected lower production of ROS, which are the key elements for PDT’s efficacy. These findings point out the relevance of serum concentration in the design of in vitro cell culture experiments carried out to determine the best way to combine and use potential sensitizers with plasmonic NPs to develop more effective PDTs.