Water solubilization of Hypericin
Since HYP must be water-soluble for in vivo application, it was complexed to Polyvinylpyrrolidone 25 (PVP) by hydrogen bonds without losing its characteristic absorption spectrum and photosensitizing properties . The water-soluble Hypericin- Polyvinylpyrrolidone 25 complex (HYP-PVP25) was prepared according to the protocol of Kubinet al. (2008): HYP (10 mg, HWI pharma Service GmbH, Rülzheim, Germany) was dissolved in EtOH (2.5 ml) and sonicated for 2 min. PVP25 (1 g, Sigma Aldrich, St. Louis, USA) was dissolved in water (8 ml) and added to the HYP. The solution was stirred for 5 min at 70 °C and for 10 min after adding water (5 ml). The solvent was cautiously removed under reduced pressure resulting in a solid red mass. HYP-PVP25 complex was dissolved in sterile water (Aqua ad iniectablia, B. Braun Melsungen AG, Melsungen, Germany) to form a final HYP-PVP25 working solution of 100 µg/ 200 ml, which was stored at 4 °C under light protected conditions.

HIPEC and Hypericin-based PDD and PDT

Treatment was performed 21 days after xenotransplantation (RH-30). An overview about the experimental setup is shown in Figure 1A. HYP-PVP25 was injected i.p. over the left lower abdomen in all mice, four hours prior to PDD/PDT. The mice were kept in the dark preventing premature HYP-based cytotoxicity. Whereas the first group received no HIPEC, the remaining mice underwent i.p. lavage over 60 minutes with cisplatin (30 or 60 mg/m2) heated up to 37 or 42 °C three hours after the HYP-PVP25 injection (four groups). All groups (each group n=16) with the corresponding treatment, dosages and temperatures are shown in Table 1. The HIPEC treatment was performed according to our established murine HIPEC model . Four hours after the injection of HYP-PVP25 or at the end of HIPEC, a median laparotomy was performed. Tumor dissemination was documented visually using the peritoneal carcinomatosis index (PCI) according to the principle of Jacquet and Sugarbaker and adapted for the animal model (Figure 1C). Visual examination of the tumor spread was repeated under HYP-PVP25-based fluorescence guidance (PDD) with blue light and recorded as PDD-PCI. Subsequently HYP-PVP25-based photodynamic therapy (PDT) was performed. Therefore, one tumor node was resected immediately after PDD (Non-PDT) followed by PDT of a representative tumor bulk under white light for ten minutes. The light source for PDT was firmly positioned three centimeters above the tumor. HYP fluorescence detection and photodynamic therapy were performed using KARL STORZ D-light C System, a 3 mm 0° laparoscope with a 300 Watt short-arc lamp and filter options for white light (400 – 700 nm) and fluorescence excitation (390 – 420 nm). Additionally, a long pass filter (> 450 nm) was integrated in the eyepiece of the laparoscope blocking the reflected blue excitation light without blocking the red fluorescence of HYP. Tumors with and without PDD and its corresponding regions were photographedvia laparoscope camera.

Immunohistochemistry and Ki-67 proliferation marker detection

Tissues (tumor, liver, spleen, peritoneum) were harvested after laparotomy for the histological work-up as described previously : Tissues were fixed in 3.7% formalin, paraffin-embedded and cut into 3-5 μm thin sections. The sections were primary labeled with anti-Ki-67 (#M7240, Dako Cytoformation, Glostrup, Denmark, dilution 1:100, mouse-monoclonal) antibody and secondary labeled with a polymer−horseradish peroxidase (HRP) antibody (Dako Envision+ Kit; Dako, Glostrup, Denmark) with AEC (3-amino-9-ethylcarbazol). Sections were counterstained with Mayer’s hemalum solution (Merck KGaA, Darmstadt, Germany) and digitalized as whole slide image (WSI) using the PreciPoint M8 scanning microscope and ViewPoint Light microscopy software (PreciPoint, Freising, Germany). The Ki-67 proliferation index was analyzed and recorded by the software Cognition Master Professional Suite (Ki67 Quantifier, VMscope GmbH, Berlin, Germany).

Analysis of tumor affine Hypericin uptake

HYP uptake was measured by red fluorescence. For mounting and nuclear counterstaining of HYP penetrated tissue sections a VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI (VECTOR Laboratories, Burlingame, USA) was applied to the formalin-fixed, paraffin-embedded and 3 µm thin cut tissue sections. Fluorescence microscopic images were captured on a wide field microscope (Leica DM5500, Leica, Wetzlar, Germany).

TUNEL assay

For investigations on induced apoptosis TUNEL assays were performed on 3 µm thin tissue sections as reported previously using the terminal desoxyribosyl-transferasemediated dUTP nick-end labeling test (In Situ Cell Death Detection Kit, Fluorescein, Roche Diagnostics GmbH, USA) . Control tissues were only treated with the labelling solution without enzyme as negative control and with 0.3 mg/ml DNase I (Roche Diagnostics GmbH) as positive control. Tissue sections were mounted and nuclear counterstained with VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI (VECTOR Laboratories, Burlingame, USA). Fluorescence microscopic images were captured on a wide field microscope (Leica DM5500, Leica, Wetzlar, Germany).

Statistical analysis

All numerical data sets were expressed as means ± standard deviations (SD). Statistical significance was determined by the unpaired student’s t -test or one-way ANOVA (P values: ***P < 0.001; **P < 0.01; *P < 0.05) using the software Microsoft Excel 2017 and Graphpad Prism Version 8 (http://www.graphpad.com/scientific-software/prism/).