Methotrexate was gifted by Emcure Pharmaceuticals Ltd., Pune. d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS), N,N′-dicyclohexylcarbodiimide (DCC) and 4-(Dimethylamino)pyridine (DMAP), 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Annexin V-FITC, and propidium iodide (PI) were purchased from Sigma Aldrich, Mumbai. Dimethylformamide (DMF), HPLC grade acetonitrile, methanol, and water were purchased from Molychem, Mumbai. All other reagents used were of analytical reagent grade and were used without further purification.
The MTX-sensitive breast cancer cell line (MCF-7) and MDR epithelial human triple-negative breast cancer cell line (MDA-MB-231) were procured from ATCC, USA. Stock cells were cultured in DMEM supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) in a humidified atmosphere of 5% CO2 at 37 °C until confluent.
Synthesis of MTX-TPGS conjugate (MTC)
The MTC was synthesized using the Steglich esterification reaction as per the previous report [14, 15]. The MTX and TPGS were reacted at 1:1 molar ratio in DMF containing DCC and DMAP at the same molar concentration. The prodrug synthesized was then confirmed using FTIR and NMR techniques.
Characterization of MTC
To determine the amount of MTX present in the conjugate, the 100 ppm and 50 ppm solutions of MTC in methanol were analyzed using a UV-visible spectrophotometer at 301 nm.
Aqueous saturation solubility
After shaking the saturated solutions of MTC for 24 h, the supernatants were analyzed using a UV-visible spectrophotometer at 301 nm.
Critical micelle concentration (CMC)
The CMC of MTC was determined using the iodine UV-visible spectrophotometric method as per the previous report . Briefly, MTX-TPGS conjugate solutions of varying concentrations were prepared in double-distilled water and mixed with the same volume of standard KI/I2 solution (25 μL) and equilibrated for 12 h in the dark at room temperature. Finally, the absorbance of all samples was recorded at 366 nm using UV-visible spectrophotometer [15, 16].
Preparation of MTC micelles
MTC micelles were prepared using the film dispersion method. Briefly, 2.0 mg of MTC conjugate was dissolved separately in beakers containing 5 mL of methanol. The solvent was then allowed to evaporate at room temperature. The resultant film at the bottom of the beaker was then dispersed by adding double-distilled water (10 mL). The resultant solution was then bath sonicated for 5 min and centrifuged at 5000 rpm to obtain the clear supernatant micellar solution.
Mean particle size and zeta potential analysis
The mean particle size and zeta potential of prepared MTC micelles were determined using the nanoparticle analyzer, Horiba SZ-100 (Horiba Scientific, Japan). The measurements were performed in triplicate at 25 °C.
Confirmation of self-assembling nature by TEM
The self-assembling nature of MTC was assessed by TEM (FEI Tecnai T-20ST). Briefly, the air-dried micellar solution on a copper grid was mixed with a negative staining solution (phosphotungstic acid solution, 2% w/v), air-dried, and observed under TEM.
In vitro hemolysis study
The hemolytic ability of MTX and MTC solutions (5 and 50 μg/mL in PBS, pH 7.4) was determined using human RBCs in comparison to negative control and positive control. The hemolytic effect was determined, after 1 h incubation at 37 °C, by measuring the supernatant absorbance at 420 nm [14, 15].
In vitro cytotoxicity
The cytotoxicity of MTX and MTC against human breast cancer cell lines (MCF-7 and MDA-MB-231) was determined using the MTT dye reduction assay. The 50,000 cells/well were seeded into each well, and after overnight incubation, cells were treated with different concentrations of MTX and MTC for 24 h and 48 h in 5% CO2 atmosphere at 37 °C. The cytotoxic effect and then the IC50 values were determined as per the previous protocol [14, 15].
Cell cycle analysis
1 × 106 cells (MDA-MB-231) were seeded and cultured for 24 h in a 6-well plate containing 2 mL of medium. Cells were then incubated with the drug solution (10 μM, 2 mL) prepared in complete medium for 48 h. Cells were then harvested and centrifuged at 2000 rpm for 5 min at room temperature, and the supernatant was discarded carefully retaining the cell pellet. The cell pellet was washed twice by re-suspending in 2 mL of 1× PBS. Cells were then fixed by re-suspending in 300 μL of sheath fluid followed by the addition of 1 mL of chilled 70% EtOH drop by drop with continuous gentle shaking, and another 1 mL of chilled 70% EtOH was added at once. The cells were then stored at 4 °C overnight and centrifuged at 2000 rpm for 5 min, and the pellet was washed twice with cold 1× PBS (2 mL). The cell pellet was then re-suspended in 450 μL of sheath fluid containing 0.05 mg/mL propidium iodide (PI) and 0.05 mg/mL RNase A and incubated for 15 min in the dark. The percentage of treated and untreated cell populations in various stages of the cell cycle was determined using FACS Caliber (BD Biosciences, San Jose, CA) .
1 × 106 cells (MDA-MB-231) per well were seeded into a 6-well plate. After 24 h, the floating (dead) cells were removed by replacing the old culture medium with the new medium of the same volume containing drug solution (10 μM, 2 mL). After 48 h of incubation, the culture medium along with the cells was transferred into 15 mL tubes. The cell suspension was then centrifuged; cells were washed twice with cold PBS and then re-suspended in 1 mL of 1× Binding Buffer at a concentration of ~ 1 × 106 cells/mL. Then, 500 μL of cell suspension was aliquoted and 10 μL of PI and 5 μL of Annexin V was added. The suspension was then incubated for 15 min at room temperature in the dark. Post incubation, the cells were analyzed by flow cytometer as soon as possible (within 1 h) .
In vitro release study
The in vitro release profile of MTX from MTC micelles was studied using the dialysis bag method and was compared with the plain MTX solution. Briefly, plain MTX solution prepared in phosphate buffer solution (PBS) of pH 7.4 and MTC micelles equivalent to 2 mg of MTX were placed into dialysis bags (MWCO = 12000 Da) and tightly sealed. The end-sealed dialysis bags were then submerged fully into 50 mL of release medium and PBS of pH 7.4 containing 1% (w/v) tween-80 and maintained at 37 ± 2 °C. The release medium was kept under stirring at 150 rpm during the study. At pre-determined time intervals (1, 2, 4, 8, 16, and 24 h), 1 mL of sample was withdrawn from the release medium and the same volume was replaced with the fresh medium. The withdrawn samples were centrifuged at 2000 rpm for 5 min, and the supernatant was bath sonicated for 5 min and then analyzed using a UV-visible spectrophotometer at 301 nm. Experiments were performed in triplicate and release profiles were expressed in terms of percentage cumulative release and plotted vs. time .
Data are presented as the mean ± standard deviation of three independent experiments. GraphPad Prism software version 8 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. The obtained results were analyzed using one-way ANOVA. p < 0.05 was considered to indicate a statistically significant difference.