Materials
Ifosfamide (≥98 %) was purchased from Sigma Aldrich (St. Louis, MO, USA).Poly(d,l-lactic-co-glycolic acid) (PLGA) (Mw: 10,000; lactic acid : glycolic acid = 50:50) was procured from Wako Pure Chemical (Tokyo, Japan). Dextran from Leuconostocspp was also obtained from Sigma-Aldrich (China). All other chemicals were reagent grade and used without further purifications.
Synthesis of PLGA-Dextran block copolymer
Approximately 3 g of PLGA-COOH was dissolved in anhydrous methylene chloride and to this organic solution, 70 mg of NHS (N-hydroxysuccinimide) and 140 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) was added. The organic mixture was stirred continuously for 12 h at room temperature. The 12 h time period is sufficient for the complete activation of carboxylic acid group in PLGA. The formed PLGA-NHS was precipitated by the addition of ice cold ether, washed with organic solvent mixture, and dried.
Aminated dextran was prepared as reported previously. Briefly, dextran and cyanoborohydride was mixed in a DMSO medium and to this mixture hexamethylene diamine was added and allowed the reaction for 24 h. The amine group terminated dextran was collected, dialyzed, and lyophilized. To prepare the block copolymer, 100 mg of PLGA-NHS and 125 mg of dextran was dissolved in DMSO and inert atmosphere was maintained throughout the reaction time. The formed PLGA-dextran was dialyzed using dialysis membrane (molecular weight cutoff, 10,000 g/mol) for 3 days. The resulting products was lyophilized and dried under vacuum conditions.
Preparation of Ifosfamide-loaded polymeric nanoparticles
IFS-loaded polymeric nanoparticles (NP) were prepared by precipitation method. In brief, 25 mg of PLGA-dextran (PLD) and 5 mg of IFS were dissolved in 5 ml of DMSO and to this mixture 20 ml of ultra-pure water were added. The mixture was magnetic stirred for 2 h and followed by dialysis against distilled water. The dialysis process was continued for 3–4 h and the resulting drug-loaded polymeric NP was collected and lyophilized.
Drug loading
The loading efficiency and loading capacity was determined as follows. In brief, 10 mg of lyophilized NP was dissolved in 5 ml of DMSO and sonicated for 15 min. The organic solution was centrifuged and the supernatant was used to calculate the amount of drug loaded. The drug loading was quantified using HPLC method. The HPLC system (Shimadzu, Kyoto, Japan) consisted of LC-10AT pump, a SPD-10A UV/Vis detector and a DGU-14A degasser model. The flow rate was maintained at 1 ml/min. The wavelength of detection was 254 nm. 50 mM of KH 2 PO 4 (pH 5.0) was used as a mobile phase.
Particle size and size distribution analysis
The average particle size and size distribution analysis was performed using a Zetasizer Nano-S90 (Malvern Instruments, Malvern, UK) and a 633 nm He-Ne laser beam at a fixed scattering angle of 90°. A dilute solution of NP was used to analyse the particle size. The experiments were performed in triplicates.
Transmission electron microscopy
The morphology of the PD/IFS was examined on a transmission electron microscope (JEOL JEM-200CX). Before the examinations, NP dispersion was diluted many times with ultra-pure water. The aqueous solution was dropped on the carbon coated copper grid and counter stained with 2 % phosphotungistic acid. The samples were dried using an infrared lamp and viewed under TEM.
Drug release study
The IFS release from the PD/IFS NP system was determined using a dialysis method. Briefly, 30 mg of PD/IFS lyophilized powder was dissolved in 1 ml of water and sealed in a dialysis tube. The dialysis tube was in turn placed in a 50 ml of Falcon tube containing 25 ml of release media. Selective release media including phosphate buffered saline (PBS, pH 7.4) and acetate buffered saline (ABS, pH 5.5) was used. The main reason behind the selection of different pH was to mimic the conditions of tumor microenvironment. The sampling was done at specific time points such as 1,2,4.6,8,10,12,24,48,72,96,120 h. At each sampling point, 1 ml of release sample was withdrawn and replaced with equal volume of fresh media. The released IFS content in the released medium was determined by HPLC as previously described.
Cell culture
MG63 and Saos-2 osteosarcoma cancer cells were grown in DMEM supplemented with 10 % FBS, 100 units/mL penicillin and 100 μg/mL of streptomycin. Cells were maintained at 37 °C with 5 % CO2 in a humidified incubator.
Cell viability assay
Cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) calorimetric assay. MG63 and Saos-2 osteosarcoma cancer cells were seeded in a 96-well plate (4000 cells/well) and allowed to grow for 48–72 h. Next day, media was removed and replaced with fresh media containing blank PLGA-dextran, free IFS, and PD/IFS NP in a concentration-dependent manner. The formulations were incubated for 24 h and cell viability was estimated using MTT solution. MTT reagent 20 μL in PBS was added into each well and the plate was incubated for 4 h at 37 °C. The culture medium in the wells was removed and 200 μL of dimethylsulfoxide (DMSO) was added into the wells. The optical density of the solution was measured at 570 nm with a microplate reader. The mean value and standard deviation for each treatment were determined and then converted values relative to the control. IC50 were calculated using GraphPad Prism software.
Morphological cell imaging
Cover slips were rinsed in 70 % ethanol for 10 min and washed with PBS. The cells were seeded into the cover slips and allowed to attach for 12 h. The formulations as mentioned above was added to each well and further incubated for 24 h. Then samples were washed with PBS, fixed with formalin (Sigma), and viewed under Nikon Eclipse 60i microscope system.
Caspase-3 activity
The activity of caspase-3 was measured by colorimetric assay kits (Sigma-Aldrich) as per the manufacturer’s protocols. MG63 and Saos-2 osteosarcoma cancer cells were seeded in a 6-well plate (1 × 106 cells/well) and allowed to attach for 24 h. Next day, media was removed and replaced with fresh media containing blank PLGA-dextran, free IFS, and PD/IFS NP in a concentration-dependent manner. The cells were incubated with respective formulations for 24 h. Cell pellets were collected and treated with lysis buffer and incubated for 10 min in ice bath. The lysate was collected, centrifuged and supernatant was collected and evaluated for caspase-3 activity.
Apoptosis analysis
FACS analysis is considered to be a specific and objective method for quantitative determination of apoptosis. MG-63 and Saos-2 cells were seeded at a density of 5 × 105 cells in a 6-well plate and incubated for 24 h. When the cells reached 80 % confluence, cells were treated with free IFS, and PD/IFS NP formulations (1 μg/ml) and further incubated for 24 h. Following day, cells were harvested, washed, and incubated with a mixture of 0.25 mg/mL Annexin-V FITC and 10 mg/mL PI. The mixture was kept for 15 min at 37 °C. Excess PI and AV-FITC fluorescence were then washed off and cells were measured by flow cytometry (FACS Calibur, BD Biosciences). A minimum of 10,000 events was counted per sample by flow cytometry.
Statistical analysis
Results in the present study are presented as means ± standard deviations. Statistical significance was evaluated by analysis of variance (ANOVA), followed by Tukey’s post-hoc test. *P-values of p < 0.05 was considered to be statistically significant.
