Synthesis and characterization of CP-GNPs
Synthesis of 20 nm spherical GNPs was carried out as previously described by Enustun and Turkevich (18). Briefly, HAuCl4 solution )414 µl of 50% w/v( was added to double distilled water) 200 ml(, followed by heating in an oil bath until boiling. Then 4.04 ml of 10% solution of sodium citrate tribasic dihydrate (Sigma-Aldrich, Rehovot, Israel) were added, followed by 10 min stirring. After cooling to room temperature GNPs were coated with a layer of polyethylene glycol (9.47 mg SH-PEGCOOH; MW ≈ 1 kDa; Sigma-Aldrich). The mixture was stirred for 24 hr at room temperature. Glucosamine conjugation was performed by adding 52.5 µL 12 mg/mL D-(1)-glucosamine hydrochloride (Sigma-Aldrich) followed by excess EDC (1-ethyl- 3-(3-(dimethylamino)propyl) carbodiimide HCl, Thermo Scientific) and NHS (N-hydroxysulfosuccinimide sodium salt, ChemImpex International). 2 mg of Cl2-Pt-(NH3)2 (Sigma-Aldrich) (1 mg/mL) was added to the mixture, and stirred overnight at room temperature. GNPs were then centrifuged to a final Au concentration of 30 mg mL− 1 as measured by atomic absorption spectroscopy (17).
Size, shape and uniformity of the GNPs were measured using transmission electron microscopy (TEM) (JEM-1400, JEOL). Samples were prepared by drop-casting 5µL of the GNP solution onto standard carbon-coated film on a cooper grid. Samples were left to dry in a vacuum desiccator. The GNPs were further characterized using ultraviolet-visible spectroscopy (UV-1650 PC; Shimadzu Corporation, Kyoto Japan) at the different coating stages. Conjugation of CP to GNPs was verified using zeta-potential (ZetaSizer 3000HS; Malvern Instruments, Malvern, UK).
In Vitro Cell Survival Analysis
MB49 mouse bladder cancer cell line (Merck, SCC148) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries, Beth Haemek, Israel) supplemented with 10% heat-inactivated Fetal Bovine Serum (Biological Industries, Beth Haemek, Israel) and maintained in a 37°C and 5% CO2 incubator. This widely-used murine bladder cancer cell line shares several pivotal tumor characteristics with human bladder cancer, such as cell surface markers, sensitivity to apoptosis, and immunological profile (19). To assess cell survival, MB49 cells (104) were seeded in 14 mm dishes and grown in 1 mL DMEM with 5% fetal calf serum, 0.5% penicillin and 0.5% glutamine. Cells were incubated at 37ͦ C with either CP-GNP (7.3 µM CP, 78.4 µg Au), CP (7.3 µM), free GNP (78.4 µg Au), or left untreated (n = 3 samples per group). After 48 hrs, medium was removed and cells were washed twice with PBS. Live cells were quantified by cell counter. Percentage of live cells in each group was calculated with respect to t0.
Animal Model And In Vivo Experiments
The animal study was conducted in compliance with protocols approved by the Animal Care and Use Committees of Bar Ilan University, Ramat Gan, Israel, and performed in accordance with the National Institutes of Health guidelines and regulations.
For tumor induction, MB49 cells (100 µl; 106) were injected subcutaneously into the back flank area of C57BL6 male mice aged 8 weeks (n = 7/group for the different treatments, detailed below). Treatments were administered intraperitoneally (IP) once tumors reached a diameter of 5–8 mm (day 0).
Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis
To determine tumor accumulation of CP-GNP, tumor-bearing mice were treated IP with a single injection of free CP or CP-GNP (13 mg/kg or 20 mg/kg). The tumor was excised (on day 4 or 14) and melted in 1 mL aqua-regia acid (a mixture of nitric acid and hydrochloric acid in a volume ratio of (1:3), and then evaporated and diluted to a total volume of 10 mL. After filtration of the samples, Pt concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent Technologies) according to absorbance values and with correlation to calibration curves.
CP-GNP dosing regimens in MB49 tumor-bearing mice
For assessment of anti-tumor efficacy and safety of different dosing regimens, MB49 tumor-bearing mice received non-fractionated free CP or CP-GNP (single 20 mg/kg dose, IP), and were then monitored over two weeks for tumor volume, weight, and survival. Another group of MB49 tumor-bearing mice received fractionated dosing of either free CP or CP-GNP (10 mg/kg on day 2, 7 mg/kg on day 6, and 3 mg/kg on day 12 and day 16; IP injection), and were monitored over 20 days for tumor volume, weight, and survival. Untreated tumor-bearing mice served as control. Tumor growth measurements were conducted in a blinded fashion using a caliper and calculated according to standard formula (length × width2 × 0.5).
Histological analysis
At the conclusion of the study (day 20 after first treatment), mice (n = 5–7 from each group) were euthanized and kidney was isolated and dissected in 4 µm–thick sections. Sections were then stained with hematoxylin and eosin (H&E) to assess necrosis, or with Masson's trichrome stain to assess fibrosis, and assessed under light microscope (×20 magnification). Blind pathological analysis and scoring was conducted for all sections by Patho-Logica Ltd.
Statistical Analysis
Comparison of tumor growth rate and of data between two groups were performed using paired two-tailed Student’s t-test. p-values below 0.05 were considered significant. Survival curves were calculated by Kaplan–Meier method. Chi-squared test (χ²) was used for survival analysis.