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/).