PCL (number average molecular weight 70,000–90,000) was purchased from Sigma-Aldrich (St Louis, MO, USA). Sorafenib was purchased from LC Laboratories (Woburn, MA, USA). All solvents, ie, dichloromethane, tetrahydrofuran, and chloroform, purchased from Honeywell (Morristown, NJ, USA) were of extra-pure grade, while other solvents used were of high-performance liquid chromatography grade.
A sorafenib-coated stent was prepared with bio-spray-coating equipment (EBS ES-Biocoater; Nano NC, Seoul, South Korea). The electrospray consisted of a high-voltage power supply, syringe pump, X-Y robotic system, and Drum-roll collector. One hundred milligrams of PCL was dissolved in 10 mL of dichloromethane. To this solution, sorafenib dissolved in dimethyl sulfoxide (DMSO; 200 mg/mL) was added, and a final concentration of sorafenib was adjusted to 2%–10% (w/w) versus PCL. Ten milliliters of this solution (100 mg PCL and 10 mg sorafenib) was introduced into a syringe and then sprayed onto a rolling collector (diameter 1 cm, length 10 cm, rolling speed 800 rpm, voltage 10 kV) at a speed of 100 mL/minute. Next, the sorafenib-coated stent was dried for 3 hours and then carefully isolated from the collector and weighed.
The morphology of the polymer surface was examined using a field-emission scanning electron microscope (S-4800; Hitachi, Tokyo, Japan) at 25 kV.
In vitro release studies
The sorafenib-coated stent prepared by the method described above was placed in 15 mL conical tubes with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS). This tube was placed in an orbital shaker at 100 rpm at 37°C. At specific time intervals, the whole media was taken to measure the released drug and was replaced with fresh media every day. Drug concentration was determined by high-performance liquid chromatography analysis. Furthermore, sorafenib-released media was used to test whether sorafenib maintained its biological activity during the stent-coating process and drug-release experiment. At 1, 5, 10, 15, 20, and 30 days of the drug-release experiment, the collected media were used to test anticancer activity.
The unloaded PCL film or sorafenib-loaded PCL film (10% [w/w] sorafenib) was immersed in 100 mL of phosphate-buffered saline (PBS; 0.01 M, pH 7.4), artificial bile solution (15 mM sodium phosphate buffer, pH 7.4, 62 mM NaCl, 2.5% glucose, 6.6 mM sodium taurocholate, 0.6 mM phosphatidylcholine), and 0.01 N NaOH (pH 12). The degradation test was carried out at 100 rpm at 37°C. The media was changed every week for 3 months. At predetermined time intervals, the films were rinsed with distilled water and dried to analyze weight loss of the polymer surface:
where Wi is the initial weight of the film and Wd is the weight of the film after the degradation time interval.
HuCC-T1 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan). To measure growth inhibition of cancer cells, 3 × 103 cells were seeded in 96-well plates and incubated overnight in an incubator with 5% CO2 at 37°C. Following incubation, sorafenib or sorafenib-released media (sorafenib-released media from polymer described above) were added to this plate. Control was 0.1% v/v DMSO. The cells were incubated for an additional 32 hours. Subsequently, 25 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; 3 mg/mL) was added to each well. After 4 hours, 100 μL of sodium dodecyl sulfate (SDS)-hydrochloric acid (HCl) solution (SDS 10% w/v, 0.01 M HCl) was added to each well, and after 12 hours absorbance was measured at 570 nm (Infinite M200 Pro microplate reader; Tecan, Männedorf, Switzerland). Viable cells were expressed as a percentage of control. Results were calculated as the means ± standard deviation of three different experiments.
Gelatin zymography was performed as described previously. A total of 5 × 105 HuCC-T1 cells seeded in six-well culture plates were cultured with serum-free media. The cells were treated with sorafenib or sorafenib-released media and then incubated for an additional 32 hours. The conditioned media were then collected and centrifuged to remove cell debris. Concentrated proteins (50 mg) were mixed with nonreducing sample buffer (0.5 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue) at a 1:1 ratio and electrophoresed on 8% SDS-polyacrylamide gels (SDS-PAGE) containing 2 mg/mL gelatin (Bio Basic, Markham, ON, Canada) under nonreducing conditions. After electrophoresis, the gel was washed three times for 30 minutes at room temperature in a 2.5% (v/v) Triton X-100 solution to remove SDS and then incubated in zymogram development solution (50 mM Tris-HCl pH 7.5, 5 mM CaCl2, 200 mM NaCl) for 24 hours at 37°C. The gel was stained with Coomassie Brillant Blue R-250 (0.2% Coomassie Brillant Blue R-250, 20% methanol and 10% acetic acid in H2O), then destained (20% methanol and 10% acetic acid in H2O).
Flow cytometry analysis
Fluorescein isothiocyanate-annexin V and propidium iodide (PI) were used to identify apoptosis and necrosis of HuCC-T1 cells. Cells were treated with various concentrations of sorafenib or sorafenib-released media for 24 hours. Following treatment, the cells were collected and washed with PBS. The collected pellets were resuspended with binding buffer (10 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) containing fluorescein isothiocyanate-annexin V (1 μg/mL) and further incubated for 30 minutes. Ten minutes prior to the termination of incubation, PI (10 μg/mL) was added to stain necrotic cells under dark conditions. The cells were immediately analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA).
Matrigel invasion assay
Invasion assays were performed using a transwell chamber.19
A polyethylene terephthalate (PETE) membrane (pore size 8 μm; BD Biosciences) was coated with Matrigel (BD Biosciences) diluted in serum-free RPMI 1640 medium (RPMI:Matrigel = 4:1) at 4°C. A total of 2 × 104
HuCC-T1 cells in 100 μL of serum-free media were seeded in the upper compartment of transwells and allowed to invade the PETE membrane in the lower chamber for 2 days. The lower chamber was filled with RPMI medium supplemented with 10% FBS with various concentrations of sorafenib and sorafenib-released media. After that, noninvaded cells on the upper surface of the membrane were removed, and the invaded cells on the lower surface of the membrane were stained with the Hemacolor rapid staining kit (Merck KGaA, Darmstadt, Germany). The invaded cells were observed with an optical microscope (Micros, St Veit/Glan, Austria). The number of cells in four randomly selected microscopic fields per membrane was counted.
An angiogenesis assay was performed as reported by Okabe et al.20
When HuCC-T1 cells filled the dishes to 70%–80% confluence, the medium was replaced with serum-free RPMI 1640. The cells were then treated with various concentrations of sorafenib or sorafenib-released media for 32 hours. After that, the media were centrifuged at 1000 rpm for 5 minutes, and the collected supernatant was used as conditioned media. The protein content of the conditioned media was determined by a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and aliquots were stored at −80°C until use. Human umbilical vein endothelial cells (HUVECs; 1 × 104
cells/well) were suspended in a mixture of conditioned media/EGM-2 medium (100 μL/100 μL) with 0.5% FBS and seeded on 50 μL of Matrigel in 96-well plates. These plates were incubated for 12 hours, and then the morphology of the cells in each well was examined. The total capillary tube length and branching points were examined in three random view-fields per well, and average values were calculated.
A wound-healing assay of HuCC-T1 cells was performed using a wound-healing assay kit containing Ibidi (Planegg, Germany) culture inserts. Aliquots containing 5 × 105 cells in RPMI 1640 media were seeded on six-well plates, and the cells were exposed to sorafenib or sorafenib-released media at 37°C and 5% CO2 for 24 hours. The cells were washed twice with PBS and harvested. Next, 5 × 104 cells in serum-free media were seeded into culture inserts following incubation for 24 hours. The zone of wound healing and migrated cells was observed using light microscopy.
Effect of sorafenib-coated stent on the growth of HuCC-T1 cells
The films cast in glass plates with a diameter of 3 cm were sterilized under ultraviolet radiation. A total of 5 × 105 HuCC-T1 cells were seeded on the films and cultured for 32 hours. The nuclei of cancer cells were stained with eosin using the Hemacolor rapid staining kit according to the manufacturer’s protocol.
In vivo animal tumor xenograft study
HuCC-T1 cells (1 × 107 cells) in a total volume of 100 μL were subcutaneously injected into the back of male nude mice (5 weeks old and 20–25 g in weight, Orient, Seongnam, South Korea). When the tumor diameter reached about 6 mm, a sorafenib-eluting PCL film was surgically implanted under the tumor. Treatment dose was adjusted to 200 μg of sorafenib (10 mg/kg). A total of 30 mice were divided into three groups, as follows: (1) nonimplanted, (2) empty PCL film-implanted, and (3) sorafenib-loaded film-implanted. Body weight and tumor volume were measured twice weekly, starting on the first day of treatment. Two perpendicular diameters of the tumor were measured, and tumor volume was calculated using the formula V = (a × [b]2)/2, with a dUDT being the largest and b being the smallest diameter.
Animal study was carried out according to the guidelines of the Animal Treatment and Research Council of Pusan National University.
Tumors were removed 30 days after film implantation, fixed in 4% formamide, paraffin-embedded, and sliced for hematoxylin and eosin staining or for terminal deoxynucleotidyl transferase dUDT nick-end-labeling (TUNEL) assay.
Immunohistochemical staining of paraffin sections of the tumors was done with matrix metalloproteinase (MMP)-9 antibody at a dilution of 1:100, with caspase-3 antibody at a dilution of 1:200, with cleaved caspase-3 antibody (cell-signaling technology) at a dilution of 1:1600, and with B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter, Bcl-x, signal transducer and activator of transcription (STAT)-5, pan-janus kinase/stress-activated protein kinase 1, ERK1, and Fas/CD95/APO-1 (BD Biosciences) at a dilution of 1:100. Staining was done using an Envision kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol.