Human melanoma cell lines and reagents
Three human malignant melanoma cell lines, MelJuSo, UACC903, and WM1205, were obtained from the University of Vermont. All cells were maintained in 50:50 Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) containing 5% fetal bovine serum (FBS) and supplemented with penicillin (50 units/mL), streptomycin (100 μg/mL), hydrocortisone (100 μg/mL), insulin (2.5 μg/mL), transferrin (2.5 μg/mL), and selenium (2.5 μg/mL), and incubated at 37°C in 5% CO2. Cells were passaged weekly at a 1:10 ratio in to new flasks. Docetaxel (DOC) was obtained from Sigma (St Louis, MO.)
Treatment of melanoma cells with docetaxel for assessment of cell growth
Cells were plated into 12 well plates at a density of 25,000 cells per well in 0.5 mL of medium described above and allowed to adhere for 2 – 3 hours before treatment. Varying doses of DOC (with equal total amounts of dimethyl sulfoxide (DMSO), the solvent control) were made up in fresh media and 0.5 mL was added to cells already plated to achieve the desired final concentrations of DOC. At 24, 48 and 72 h, cells were trypsinized, collected, and counted with a heamocytometer to determine average total cell numbers remaining at each time point for each dose.
Scanning Electron Microscopy (SEM) of APMS-TEG interacting with MelJuSo melanoma cells
Acid prepared mesoporous spheres (APMS) tagged with tetra ethylene glycol (TEG) (APMS-TEG complex) were synthesized by Dr. Christopher Landry at the University of Vermont as previously described . MelJuSo cells were grown to confluence on thermonox plastic coverslips (Nalge Nunc International, Naperville, IL) and were administered APMS-TEG particles (0.1 mg/mL). At 1, 4 and 24 hours, coverslips were fixed and prepared for SEM analysis as done previously . In summary, coverslips were washed 2× for 5 minutes with 0.1 M Millonig’s phosphate buffer (pH 7.2), then fixed in 1:1 H2O dilution of Karnovsky’s fixative (2.5% glutaraldehyde, 1% paraformaldehyde) at 4°C for 45 minutes. Samples were then washed with Millonig’s phosphate buffer (pH 7.2), and post-fixed in osmium tetroxide (OsO4) at 4°C for 30–45 minutes. Samples were then dehydrated in graded ethanols, from 35% to 100%. Samples were critical point dried using liquid CO2 as the transition fluid in a Samdri PVT-3B critical point dryer (Tousimis Research Corporation, Rockville, MD). Specimens were mounted on aluminum specimen stubs using conductive graphite paint and allowed to dry, and were sputter-coated for 4–5 min with gold and palladium in a Polaron sputter coater (Model 5100; Quorum Technologies, Guelph, ON, Canada). Cells and APMS-TEG particles were then imaged on a JSM 6060 scanning electron microscope (JEOL USA, Inc., Peabody, MA) .
Melanoma cell growth in response to docetaxel loaded APMS micro particles
APMS particles (2 mg) were incubated with increasing concentrations of docetaxel in 5 mL sterile distilled water for 24 – 72 h at 4°C on a rocking platform. APMS particles were then spun down to remove unloaded docetaxel still in solution, washed in water, spun down again and resuspended in 5 mL of fresh media. A 0.5 mL suspension of the loaded particles in media of each docetaxel concentration were then added to respective wells of a 12 well plate containing previously plated cells as described above. Controls used were DMSO alone, APMS (0.2 mg/mL) and 1 nM unloaded docetaxel. Concentrations of docetaxel indicated in these experiments represent the concentration of docetaxel in the loading solution and thus the maximal concentration of DOC that can be released if there is complete uptake and release from the APMS-TEG particles. Cells were collected at 24, 48, and 72 hours and total cell numbers were counted as described above.
Release kinetics of docetaxel loaded APMS-TEG micro-particles
APMS particles (2 mg) were incubated with increasing concentrations of docetaxel in 5 mL sterile distilled water for 48 hours at 4°C on a rocking platform. APMS-TEG particles were then spun down with supernatant collected to quantitate the amount of unloaded docetaxel still in solution (unloaded). The particles were washed in water, spun down again, with the supernatant collected to quantitate the amount of DOC lost with washing (wash). Particles were resuspended in 10 mL of fresh media. A 1 mL suspension of the loaded particles in media of each docetaxel concentration was then added to respective wells of a 12 well plate. The control used was APMS (0.2 mg/mL) with no docetaxel. Concentrations of docetaxel indicated in these experiments represent the concentration of docetaxel in the loading solution and thus the maximal concentration of DOC released if there is complete uptake and release from the APMS-TEG particles. Media samples were collected at 1, 2, 4, 8, 24, and 48 hours.
High performance liquid chromatography for detection of docetaxel
As per the protocol developed by Andersen, et al., a 3 μm Purospher STAR RP-18e (3×125 mm) column from EMD Millipore (Darmstadt, Germany) was obtained and maintained at 55°C . Mobile phase consisted of 20 mM dibasic potassium phosphate buffer (pH 3): acetonitrile (57.5:42.5 v/v) run at 0.8 mL/min. Docetaxel peaks were seen between 7 – 8 minutes using a UV detector set to 227 nm. Fresh buffer and standards were prepared daily.
Statistical analysis was performed using GraphPad Prism 5.03/6.00. All data was analyzed by one way ANOVA followed by the Newman-Keuls Multiple Comparison Test or a student’s t test where indicated. Data with p <0.05 were determined to be significant.