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Hypericin is one of the most efficient photosensitizers used in photodynamic tumor therapy (PDT). The reported treatments of this drug reach from antidepressive, antineoplastic, antitumor and antiviral activity. We show that hypericin can be optically detected down to a single molecule at ambient conditions. Hypericin can even be observed inside of a cancer cell, which implies that this drug can be directly used for advanced microscopy techniques (PALM, spt-PALM, or FLIM). Its photostability is large enough to obtain single molecule fluorescence, surface enhanced Raman spectra (SERS), fluorescence lifetime, antibunching, and blinking dynamics. Sudden spectral changes can be associated with a reorientation of the molecule on the particle surface. These properties of hypericin are very sensitive to the local environment. Comparison of DFT calculations with SERS spectra show that both the neutral and deprotonated form of hypericin can be observed on the single molecule and ensemble level.
Glioblastoma WHO IV belongs to a group of brain tumors that are still incurable. A promising treatment approach applies photodynamic therapy (PDT) with hypericin as a photosensitizer. To generate a comprehensive understanding of the photosensitizer-tumor interactions, the first part of our study is focused on investigating the distribution and penetration behavior of hypericin in glioma cell spheroids by fluorescence microscopy. In the second part, fluorescence lifetime imaging microscopy (FLIM) was used to correlate fluorescence lifetime (FLT) changes of hypericin to environmental effects inside the spheroids. In this context, 3D tumor spheroids are an excellent model system since they consider 3D cell–cell interactions and the extracellular matrix is similar to tumors in vivo. Our analytical approach considers hypericin as probe molecule for FLIM and as photosensitizer for PDT at the same time, making it possible to directly draw conclusions of the state and location of the drug in a biological system. The knowledge of both state and location of hypericin makes a fundamental understanding of the impact of hypericin PDT in brain tumors possible. Following different incubation conditions, the hypericin distribution in peripheral and central cryosections of the spheroids were analyzed. Both fluorescence microscopy and FLIM revealed a hypericin gradient towards the spheroid core for short incubation periods or small concentrations. On the other hand, a homogeneous hypericin distribution is observed for long incubation times and high concentrations. Especially, the observed FLT change is crucial for the PDT efficiency, since the triplet yield, and hence the O2 activation, is directly proportional to the FLT. Based on the FLT increase inside spheroids, an incubation time 30 min is required to achieve most suitable conditions for an effective PDT.
Malignant primary brain tumors are a group of highly aggressive and often infiltrating tumors that lack adequate therapeutic treatments to achieve long time survival. Complete tumor removal is one precondition to reach this goal. A promising approach to optimize resection margins and eliminate remaining infiltrative so-called guerilla cells is photodynamic therapy (PDT) using organic photosensitizers that can pass the disrupted blood–brain-barrier and selectively accumulate in tumor tissue. Hypericin fulfills these conditions and additionally offers outstanding photophysical properties, making it an excellent choice as a photosensitizing molecule for PDT. However, the actual hypericin-induced PDT cell death mechanism is still under debate. In this work, hypericin-induced PDT was investigated by employing the three distinct fluorescent probes hypericin, resorufin and propidium iodide (PI) in fluorescence-lifetime imaging microscopy (FLIM). This approach enables visualizing the PDT-induced photodamaging and dying of single, living glioma cells, as an in vitro tumor model for glioblastoma. Hypericin PDT and FLIM image acquisition were simultaneously induced by 405 nm laser irradiation and sequences of FLIM images and fluorescence spectra were recorded to analyze the PDT progression. The reproducibly observed cellular changes provide insight into the mechanism of cell death during PDT and suggest that apoptosis is the initial mechanism followed by necrosis after continued irradiation. These new insights into the mechanism of hypericin PDT of single glioma cells may help to adjust irradiation doses and improve the implementation as a therapy for primary brain tumors.
Characterization of brain tumours requires neuropathological expertise and is generally performed by histological evaluation and molecular analysis. One emerging technique to assist pathologists in future tumour diagnostics is multimodal optical spectroscopy. In the current clinical routine, tissue preprocessing with formalin is widely established and suitable for spectroscopic investigations since degradation processes impede the measurement of native tissue. However, formalin fixation results in alterations of the tissue chemistry and morphology for example by protein cross-linking. As optical spectroscopy is sensitive to these variations, we evaluate the effects of formalin fixation on multimodal brain tumour data in this proof-of-concept study. Nonfixed and formalin-fixed cross sections of different common human brain tumours were subjected to analysis of chemical variations using ultraviolet and Fourier-transform infrared microspectroscopy. Morphological changes were assessed by elastic light scattering microspectroscopy in the visible wavelength range. Data were analysed with multivariate data analysis and compared with histopathology. Tissue type classifications deduced by optical spectroscopy are highly comparable and independent from the preparation and the fixation protocol. However, formalin fixation leads to slightly better classification models due to improved stability of the tissue. As a consequence, spectroscopic methods represent an appropriate additional contrast for chemical and morphological information in neuropathological diagnosis and should be investigated to a greater extent. Furthermore, they can be included in the clinical workflow even after formalin fixation.
