Verteporfin

To prepare a polymer solution of hydrophobic molecules (e.g., rhodamine B) for fiber coating, a mixture of poloxamer (1000 mg, pluronic F127, Sigma-Aldrich), PLGA (330 mg, poly(d,l-lactide-co-glycolide), molecular weight: 30–60k, Sigma-Aldrich) in tetrahydrofuran (THF, 5 mL) was agitated vigorously in a 8 mL reaction vial until PLGA solution was homogenous and clear. Saturated rhodamine B/acetone solution was added into the PLGA/THF solution until solution was homogeneous and pink. Similarly, verteporfin (Cayman Chemical, Ann Arbor, MI)/PLGA solution was prepared by adding 2.5 mg/mL of verteporfin/THF solution and 5 mL PLGA solution into an 8 mL reaction vial.
To coat the fiber layer-by-layer with the polymer solution, a polycarbonate optical fiber was dipped into PLA/THF (100 mg/mL) solution and removed slowly so as to create a smooth coating. Next, it was placed into an air-tight chamber connected to vacuum. The coated fiber was dried under vacuum for 30 min before it was dipped into the rhodamine B / PLGA solution. The fiber was dried under vacuum again for 60 min. This was repeated until there were 11 layers of alternating polymer coatings (additional 1 PLA layer outside of rhodamine B/ PLGA). verteporfin-coated fiber was prepared similarly. To extend the release of verteporfin, three additional layers of PLA coatings were coated onto the verteporfin fibers.

Human liver cancer cell lines Hep3B, PLC/PRF/5, HepG2, SNU-182, SNU-387, SNU-398, SNU-423, SNU-449, and SNU-475, HLE and HLF were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The Huh-7 human liver cancer cell line was obtained from the Japanese Cancer Research Resource Bank (JCRB, Ibaraki, Japan). These cell lines were cultured in RPMI-1640 medium or Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. All cells were confirmed to be free from pathogens and mycoplasma. Regorafenib was provided by Bayer AG (Leverkusen, Germany). Regorafenib was dissolved in the mixture of PEG400, propylene glycol and pluronic F68. ABT-737 and Verteporfin were purchased from Cayman Chemical (Ann Arbor, MI, USA). These compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions and diluted in cell growth medium for assays.

To establish an orthotopic colon tumor model, CMT93 cancer cells (1 × 10⁶ cells in 30 mL of Hanks’ balanced salt solution) were inoculated into the cecal wall (submucosal layer) of BALB/c mice using 28G needles under inhalation anesthesia of sevoflurane (Maruishi Pharmaceutical Co. Ltd., Osaka, Japan) according to the previously published method [39 (link),40 (link)]. The mice were observed for 4 weeks following inoculation. After 4 weeks, spontaneous liver metastasis was found.
Pantoprazole (PPZ, Sigma, 50 mg/kg) was administered every two days by oral gavage. With five mice in each group, small interfering RNA (siRNA) against GR (siGR, 500 pmol/kg body weight, twice a week, i.p, Santa Cruz Biotechnology (Santa Cruz, CA, USA)) and/or verteporfin (Ver, 50 mg/kg body weight, every two days by oral gavage, Cayman Chemical, Ann Arbor, MI, USA) were administered. A vehicle control group was run in parallel. Mice were fed with a standard CE-2 diet (CLEA Japan, Tokyo, Japan).