HG-9-91-01 br The in vitro evaluation of the synthesized
The in vitro evaluation of the synthesized conjugates was performed in human lung cancer cells, A549 which were procured from National Center for Cell Sciences (NCCS, Pune, India). Cells were grown in Dulbecco’s minimum essential medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin solution (Himedia Labs, Mumbai). During the period of study, cell cultures were maintained in an in-cubator operated at 37 °C with 5% CO2.
22.214.171.124. Cellular uptake by confocal microscopy. Cellular internalization International Journal of Pharmaceutics 557 (2019) 329–341
of multifunctional dendrimer conjugates was studied using confocal microscopy. Briefly, A549 HG-9-91-01 were harvested from culture flasks at 80% confluence and counted. The cells were seeded at a cell density of 50,000 cells/well onto round coverslips placed in 12-well plates. On the day of study, cells were treated with conjugates F-G4-PEG and F-G4-PEG-Biotin (20 µg/ml) and incubated at 37 °C for 1 h and 4 h. Also, to assess the mechanism of uptake of biotin tagged dendrimer conjugates, the cells were pre-incubated with excess free biotin (300 µM for 30 min) to saturate the biotin receptors. Further, the wells containing medium with free biotin was removed and F-G4-PEG-Biotin was added to the cells. Following incubation, cells were washed three times with sterile PBS, stained with DAPI (1 µg/ml) for 5 min, washed and fixed with 4% para-formaldehyde for 15 min. Using a mounting medium (Fluoromount-G), the coverslips containing cells were placed on the microscopic slides. At 40 X magnification, cells were visualized under confocal microscope (Leica DMi8, Leica Microsystems, Germany), Photographs of cells were taken in FITC and DAPI fluorescence filters. The obtained data were processed using Image J software.
126.96.36.199. Cellular uptake by flow cytometry. To quantitatively determine the cellular association of dendrimer conjugates, flow cytometry experiments were performed in A549 cells. The 6-well microplates were seeded with A549 cells at a cell population of 0.8 × 106 cells/well and allowed to attach overnight. Similar to the confocal microscopy study, biotin receptors were saturated by pre-incubating the cells with excess of free biotin (300 µM for 30 min) which receive F-G4-PEG-Biotin treatment to observe if the cellular uptake was mediated via biotin receptors. Cells were treated for 1 h and 4 h with F-G4-PEG and F-G4-PEG-Biotin at a concentration of 20 µg/ml. After the incubation period, cells were washed with sterile fresh PBS and then trypsinized. Cells were centrifuged and the obtained cell pellet was re-dispersed in PBS before analyzing the samples by flow cytometer (Amnis, EMD Millipore, USA). Control cells did not receive any treatment. At least 10,000 events were collected for each sample. The data was captured as geometric mean fluorescence and was calculated using the IDEAS software V6.0.
188.8.131.52. Cell viability study. The cytotoxic activity of free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin was studied by MTT colorimetric assay. A549 cells were seeded in 96-well plates at a population of 10,000 cells/well and allowed to attach overnight. On the day of the study, cells were treated with free PTX and PTX conjugates (0–50 µg/ ml) and incubated for 24 h and 48 h at 37 °C. Following incubation, formulations were removed and 50 µl of MTT reagent (5 mg/ml solution) was added to each well. The plates were further incubated for another 4 h, and MTT reagent was discarded. The purple colored formazan crystals formed in the wells were dissolved by adding DMSO (150 µl) to each well. The absorbance of the wells was measured at 570 nm using a microplate reader (Spectramax™, Molecular Devices, USA) with a reference wavelength of 630 nm. Control cells did not receive any treatment containing PTX. The cytotoxicity caused due to various treatments was calculated as percentage cell viability and is represented as a bar graph against the concentration of PTX.
2.2.5. Evaluation of biotin tagged PAMAM dendrimer conjugates in multicellular tumor spheroids
184.108.40.206. Formation of A549 spheroids. Multicellular spheroids are 3D structures which mimic the in vivo tumors. In the current study, A549
S.V.K. Rompicharla et al.
tumor spheroids were prepared by liquid overlay method (Perche and Torchilin, 2012; Sarisozen et al., 2014). In brief, 1.5% (w/v) agar solution was prepared in serum free DMEM medium and autoclaved. A volume of 50 µl of the agar solution was added to each well of a 96-well plate at the bottom to prevent cell adhesion. Care should be taken so as to not let the agar solution solidify before adding to wells. For confocal microscopy study, 8-chambered glass slides were used and to each well 150 µl of agar solution were dispersed on the inner bottom.
From the culture flasks, A549 cells were detached using trypsin and cell pellet was collected by centrifugation. To each well, cells were seeded at a density of 8000 cells. Culture plates were centrifuged at room temperature for 15 min. After centrifugation, plates are left in the incubator. The spheroid formation was constantly supervised using an inverted microscope (Leica DMi8, Leica Microsystems, Germany). For further studies, 3–5 days old spheroids were used.