Fetal bovine serum (fbs)

The cells used in this study are human vascular-derived cells (HVSC) harvested from the vena saphena magna from the Catharina Hospital Eindhoven. The tissues are considered surgical rest material, whereby the patient has been informed on potential use of rest material for scientific research purposes42 (link). Verbal informed consent was obtained from patients and tissues were handed over anonymously, without any patient-specific information except for gender. Procedures for secondary use of patient material were followed as described in the Dutch code of conduct for responsible use of patient material. According to the Dutch medical scientific research with human subjects act (WMO), secondary use of patient material does not need review by a Medical Ethics Examination Committee. HVSC have previously been characterized as myofibroblasts43 (link). The HVSC were cultured in advanced Dulbecco Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% Fetal Bovine Serum (Greiner Bio-one), 1% penicillin/streptomycin (Lonza), 1% GlutaMax (Invitrogen). Only cells at passage 7 were used in this study. Myofibroblasts have been shown to be sensitive and respond to mechanical cues44 (link)45 (link). This makes this cell type interesting for our study.

HT-29, a human colon cancer cell-line, was obtained from the ATCC. The cells were cultured at 37°C in 5% CO₂ humidified atmosphere in McCoy's SA medium (Invitrogen, 26600-023) with Fungizone (Bristol Myers Squibb B.V, 3440 AM Woerden), 10% fetal bovine serum (Greiner Bio One, 758093), and penicillin-streptomycin (Invitrogen, 15070-063). The HT29 cells were authenticated by STR DNA profiling (ATCC, 25/02/14).
An MTT assay as described by De Smet et al. was performed to evaluate Oxaliplatin cytotoxicity in vitro [33 (link)]. Briefly, cells were seeded in 96-well plates at a concentration of 8 × 10⁴ cells/ml. After 24 h, 20 μL medium is replaced by 20 μL of drug solution. Oxaliplatin concentrations of 100 μg/mL, 50 μg/mL, 20 μg/mL, 10 μg/mL, 5 μg/mL, and 1 μg/mL were assessed. Eight wells per concentration were used and all experiments were repeated (3x). Next, the plates were incubated for 60 min at 37°C and 10% CO₂. Then, the oxaliplatin solution was replaced by 200 μL fresh medium. Afterwards, the cells were incubated for 24 h at 37°C and 10% CO₂. Finally, the cytotoxicity of the formulations was evaluated via MTT assay and measured by assessing the optical density using an ELISA-plate reader (Paradigm Detection Platform, Beckman Coulter, Suarleé, Belgium).

The following chemicals were used as received: Crystal Violet (CV, Anedra Tigre, Argentina); Fetal Bovine Serum (Greiner Bio-One, Frickenhausen, Germany); Sabouraud Dextrose Broth (SDB, Difco, Detroit, MI); Sabouraud dextrose agar (Difco, Detroit, MI), Phosphate Buffer Solution (PBS); dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany); Calcofluor-White (Sigma-Aldrich Co, St Louis, MO, USA); MeOH (HPLC grade, Merck, Germany); Nitro Blue Tetrazolium (NBT, Sigma-Aldrich Co, St Louis, MO, USA); Methionine (Sigma-Aldrich); Riboflavin (Sigma-Aldrich Co, St Louis, MO, USA), Tiron (Sigma-Aldrich); Sodium azide (Sigma-Aldrich).
Deuterium oxide (99.9%) was purchased from Solvents Documentation Synthesis (Peypin, France). Deuterated PBS (D-PBS) was prepared by dissolving PBS powder in deuterium oxide.
1 and 2 were purified from benzene extracts of H. pustulata using a methodology described previously [10 (link),11 (link)]. They were unequivocally identified by their spectroscopic/spectrometric data (¹H NMR, ¹³C NMR, IR, UV–Vis, MS) [10 (link)]. The purity was 93.6% ± 0.1% for 1 and 93.8 ± 0.1% for 2, as established by HPLC analysis [13 (link)] (Fig 2 in S1 File).

CT26.WT murine colon carcinoma cells (American Type Culture Collection (ATCC; CRL-2638)) were cultured as a monolayer at 37°C and 5% CO₂ in RPMI-1640 medium (Invitrogen, Breda, The Netherlands), supplemented with 10% fetal bovine serum (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) and 50 U/ml penicillin/streptomycin (Lonza Bioscience, Basel, Switzerland). Early passages (5–10) of the original ATCC batch were used for inoculation.
10–12 week-old Balb/c mice (Charles River, Maastricht, The Netherlands) were inoculated with 2×10⁶ CT26.WT cells subcutaneously in the right hind limb. Approximately 10 days after inoculation, tumors became palpable in all animals.

To complete the evaluation of complexes C1, C3, and C4, in vitro biological behavior was assessed. Two different cell lines derived from prostatic adenocarcinoma were used, LNCaP, which express the AR, and PC3 cells, which do not express it.
The adherent cell line LNCaP (ATCC^® CRL-1740™, American Type Culture Collection, Manassas, VA, USA) was cultured in a conditioned RPMI 1640 medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (Capricorn Scientific), 100 U/mL of penicillin (Sigma-Aldrich, St. Louis, MI, USA), and 100 μg/mL of streptomycin (Sigma-Aldrich) in T75 flasks (Greiner Bio-one, Frickenhausen, Germany) at 37 °C and 5% CO₂. The studies were performed with monolayer cultures with less than 25 passages.
The adherent cell line PC3 (ATCC^® CRL-1435™, American Type Culture Collection, Manassas, VA, USA) was cultured in a DMEM medium (A1316, 9050 PanReac AppliChem, ITW Reagents, Darmstadt, Germany) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin in T75 flasks (Greiner Bio-one, Kremsmünster, Austria) at 37 °C and 5% CO₂. The studies were performed with monolayer cultures with less than 20 passages.
Cellular uptake, efflux, and binding studies for each complex were performed.