unloaded drug DOX or SC was determined using centrifugal
unloaded drug (DOX or SC) was determined using centrifugal filter tube (Amicon® filter, molecular weight cutoff 100 kDa, Millipore, UK). For this purpose, 1 ml of formulation was diluted with 1 ml of Dimethyl sulfoxide (1% vol/vol) to solve the probable unloaded SC and then centrifuged at 4000 rpm for 8 min to separate the free DOX or SC from the encapsulated drugs. After that, the amount of unloaded drugs were measured by ultraviolet visible spectrophotometer (Ultrascope 2000®,
pharmacia biotech, UK) at λmax 480 and 291 nm for DOX and SC, re-spectively. For this purpose, ultraviolet-visible calibration curve of SC and DOX were prepared and amount of unloaded DOX and SC calcu-lated using acquired BMS-936558 from spectrophotometer and equation of calibration curve. Finally the EE of DOX and SC were calculated using the following equations: EE (%) = Ct Caq × 100
Where, Ct was the total added drug concentration, Caq was the unloaded drug concentration in aqueous phase. To assess the physical stability, the size, PDI and EE of optimum formulation was determined after 8 weeks storage at 2–6 °C by mentioned methods.
2.4. Cell viability assay
To study the in-vitro cytotoxicity of nanoformulation, human lung carcinoma A549 cells (1 × 104 cells/well) were cultured in RPMI medium with 10% FBS, 100 U/mL penicillin, and 100 μg/ml strepto-mycin at 5% CO2. Then, the different concentrations of blank NLC-RGD, free SC, free SC + DOX, [SC + DOX]-co-loaded NLC and [SC + DOX]-co-loaded NLC-RGD were used for treatment of A549 cells. After 48 h of incubation, the treated media were removed and substituted with 200 μL of fresh media containing 50 μL of MTT solution (5 mg/ml). Then, the cells incubated for further 3 h at 37 °C. After this time, the media were removed and 180 μL of DMSO and 20 μL Sorenson buffer were used to dissolve the formazan crystals. The optical density (OD) values were recorded at 570 nm compared to control cells using a micro-plate reader (Elx808, Biotek, USA).
2.5. Cellular uptake study
To confirm the targetability of NLC-RGD, the cellular uptake of nanoformulation (with and without RGD) were studied by flow cyto-metry and fluorescence microscopy. Moreover, since DOX is a fluor-escent drug, so the permeability of NLC in the cells was explored by encapsulated DOX. For the flow cytometry, the A549 cells were seeded in six-well plates at 3 × 105 cells per well and then treated with free
DOX, DOX + SC, [DOX + SC]-coloaded NLC, and [DOX + SC]-co-loaded NLC-RGD for 3 h. Then, a minimum of 1 × 106 cells were col-lected and washed three times with PBS for analyzing the DOX fluor-escence intensity using a FACS caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) for each sample.
2.6. Apoptosis assay
To identify necrosis, early and late apoptosis, Fluorescein iso-thiocyanate (FITC) labelled annexin V assay was performed. A549 cells were cultured in six-well plates at a density of 5 × 105 cells/well and then treated with 1.26 μM DOX, 1.26 μM DOX + 15 μM SC and equivalent doses [DOX + SC]-coloaded NLC (with and without RGD) for 24 h. Then, the cells harvested, centrifuged and washed twice with cold phosphate-buffered saline (PBS). Next, the cells were re-suspended in 200 μL of binding buffer containing 5 μl FITC-labelled annexin V and incubated for 20 min at ambient temperature in dark room. Finally, the cells were examined using a FACS calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).
To quantify the apoptotic nuclei by DAPI staining, A549 cells (5 × 105 /well) were seeded onto the glass coverslips placed in 6-well plates and incubated for 24 h to reach the 80% confluence. Then, the cells were treated with DOX, DOX + SC, [DOX + SC]-coloaded NLC (with and without RGD). After 24 h, the cells were washed twice with cold PBS and fixed with 4% paraformaldehyde and permeabilized in 0.1% (w/v) Triton X-100. After 15 min, the cells were stained with DAPI for further 30 min. Finally, the morphology of nuclear structure alteration was evaluated by fluorescence microscope to figure out the apoptotic cells.
2.8. RNA extraction, reverse transcription and analysis of gene expression
To examine expression of ABCB1, ABCC1, ABCC2 and nuclear factor-E2-related factor 2 (Nrf2), A549 cells were treated with 25 μM SC and equivalent doses of SC loaded NLC-RGD for 48 h. Subsequently total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s procedure and was quantified by nanoDrop (ND-1000, NanoDrop technology, Australia). Complementary DNA (cDNA) synthesized by using First Strand cDNA synthesis Kit (thermo scientific, Schwerte, Germany) and Real-time PCR (RT-PCR) was per-formed by the real time rotary analyzer (Rotor-Gene 6000, Corbet Life Science, Australia) based on SYBR Green chemistry. Sequence of pri-mers are indicated in Table 1. Pfaffl method was used to analyze the data and the cycle threshold (CT) values were standardized regarding to housekeeping gene (GAPDH) expression.