br Experimental Section br All the chemicals
All the chemicals used here were purchased from Sigma-Aldrich with highest purity grade and used as supplied. The solvents like tet-rahydrofuran, ethanol etc. were supplied by S D Fine-Chem Limited with HPLC Grade. Deionized water (18 MU cm, Millipore Milli-Q water) was used to prepare the solutions where necessary. Cationic amphi-philic polymer CPF was synthesized by polymerizing 2,7-dibromo-9,9′-bis(6″- bromohexyl)fluorene with 1,4-phenyldiboronic Diphenylterazine (DTZ) by the Suzuki cross-coupling polymerization and afterward quarternization using triethylamine (TEA) according to our previous report [31,32,44]. The molecular weight (Mw) of polymer the precursor poly(9,9′-bis(6″-bromohexyl)fluorene-co-alt-1,4-phenylene), was determined to be ∼8000 Da with a polydispersity index (PDI) of 1.75 by gel permeation chromatography (GPC) using tetrahydrofuran (THF) as the eluent and Sensors & Actuators: B. Chemical 291 (2019) 337–344
polystyrene as a standard.
2.2. Preparation of solution for spectroscopic study
The CPF polymer is sparingly soluble in water and to increase the concentration of these polymers, a THF–water mixture was used for spectroscopic studies. 1 mg cationic polyfluorene CPF was dissolved in 100 mL solvent (10% THF in water) at pH 7. Folic acid solution (2 mM) is prepared in aqueous buﬀer solution at pH 9. The pH of the CPF-FA assembly was adjusted by adding dilute acid or base and measuring the resultant pH by using a bench-top probe pH-meter (IQ Scientific Instruments, Model-IQ240) which was calibrated with buﬀer of pH = 7.00 and pH = 4.00 prior to each measurement.
2.3. Spectroscopic measurements
The UV–vis spectra of the compounds were studied using a JASCO/ V-650 (190–900 nm) UV–vis spectrophotometer using a quartz cuvette with 1 cm path length. Fluorescence studies were done in a JASCO/FP-6300 (190–900 nm) fluorescence spectrometer at an excitation wave-length of 362 nm. The circular dichroism (CD) study of the solutions were performed in a spectrophotometer (JASCO, model J-1500) in a 1 cm quartz cuvette.
2.4. Morphological characterization
For evaluation of the morphology of CPF and CPF-FA assembly at diﬀerent pH, field emission scanning electron microscope (FESEM) was performed by drop casting the compounds on glass cover slip followed by drying in air at room temperature. The sample coated glass cover slips were finally dried in oven at 60 °C. It was coated with gold for 30 s prior to observation through a FESEM instrument by FEI, Apreo S with a 20 kV operating voltage.
2.5. Cell imaging study
Cell imaging studies were carried out using B16 F10 cells (cancer cell lines with over expression of folate receptors) as well as Human Corneal Epithelial (HCE) cells (normal cell lines). Briefly, B16 F10 cells were seeded in 12 well plate at a density of 50,000 cells/well in 500 μL of DMEM medium and incubated at 37 °C for 24 h. After 24 h, 6 out of 12 wells were saturated with folic acid solution (5 mg/mL in PBS) and the other 6 wells were kept as control (without folic acid treatment), and the cells were incubated for 4 h. Then, the media was replaced with freshly prepared CPF solution (1000 μL; 50 μg/mL in DMEM media) from all the wells and incubated for 4 h. The treatments were carried out in duplicate. Later the polymer solution was pipetted out and the wells were thoroughly rinsed with PBS to wash out the dead cells as well as left out polymer solution if any. The fluorescence of CPF inside the cells was observed under the fluorescence microscope (Leica Microsystems LAS version 4.8.0 (Switzerland) Ltd.) at 40x and the images were captured. The same procedure was repeated using Human Corneal Epithelial (HCE) cells, in DMEM-F12 medium.