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  • br Penetration efficiency in multicellular

    2020-08-18


    2.2.5.2. Penetration efficiency in GSK-3 tumor spheroids. The internalization of targeted and non-targeted dendrimer conjugates into the spheroids was studied using confocal microscopy. F-G4-PEG-Biotin and F-G4-PEG were added to the spheroids grown in 8-well glass chamber slides and incubated for 1 h and 4 h. After that, the cancer cell spheroids were gently washed with PBS and visualized by laser scanning confocal microscope (Leica DMi8, Leica Microsystems, Germany) at 10X magnification. Z-stack images were captured to see the penetration, from the surface where fluorescence is observed to the center of spheroid at every 10 μm thickness. Image J software was used to process the captured images.
    2.2.5.3. Flow cytometry analysis of spheroid uptake of dendrimer conjugates. Flow cytometry study was performed to quantitatively determine the uptake of targeted and non-targeted dendrimer conjugates in 3D cell spheroids. Five-day-old spheroids were employed in the study and incubated with F-G4-PEG and F-G4-PEG-Biotin for 1 h and 4 h. After the treatment period, 10 spheroids each of every treatment were pooled up to achieve required cell population for flow cytometry analysis. The spheroids were gently washed with sterile PBS and dissociated by adding Accutase cell detachment solution. Accutase solution is used to create single cell suspensions from a clump of cells. It contains proteolytic and collagenolytic enzymes. The obtained cell suspension after Accutase treatment was centrifuged and the single cell suspension was prepared by adding PBS. Further, the samples were run using flow cytometer and atleast 10,000 events were collected for every sample. The fluorescence intensity histograms were captured in the FITC channel for each sample. A bar diagram was plotted with geometric mean fluorescence exhibited by biotin modified and unmodified dendrimer conjugates.
    2.2.5.4. Growth inhibition of multicellular tumor spheroids. The inhibitory effect of PTX formulations on the growth of A549 cancer cell spheroids was studied to evaluate the potential of synthesized conjugates in delivering the drug to tumors. The cancer cell spheroids were incubated in complete media added with free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin at a PTX concentration of 25 μg/mL. After 24 h, the growth medium with formulations was carefully discarded and fresh growth medium was added. The magnitude of spheroidal growth inhibited by dendrimer drug conjugates was visualized using inverted microscope (Leica DMi8, Leica Microsystems, Germany). Brightfield images were captured at 10X magnification. The diameter of the spheroids at regular intervals was measured and represented as a line  International Journal of Pharmaceutics 557 (2019) 329–341
    graph.
    2.2.5.5. In vitro cytotoxic activity in multicellular tumor spheroids. To evaluate the cytotoxic activity of targeted and non-targeted dendrimer conjugates in multicellular tumor spheroids, Presto Blue assay was performed as per the manufacturer’s instructions. Presto Blue reagent is a cell permeable resazurin-based solution which turns to a red fluorescent complex upon reduction by viable cells. The fluorescence/ absorbance of the sample is measured and the cell viability is calculated. On the day of study, 3–5 day old spheroids were selected and treated with free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin at a PTX concentration of 25 μg/ml. For each treatment, 10 individual spheroids were used as a group. Following the incubation for 24 h, the medium in the wells was carefully discarded, washed with PBS and then disaggregated by adding 50 μl of Accutase solution. The cell suspension resulted by mild shaking was subjected to centrifugation to obtain cell pellet. The samples for analysis were prepared by adding 10 µl of Presto Blue reagent to the cell pellet dispersed in 90 μl of growth medium. After a brief incubation for 2 h at 37 °C, the absorbance of the samples was measured at 570 nm with a reference wavelength of 600 nm.
    2.2.5.6. Live/dead cell assay in multicellular tumor spheroids. The amount of live and dead population developed as a result of various PTX treatments to A549 cell spheroids was assessed by live/dead cell assay using Calcein Blue AM reagent. In brief, dendrimer conjugates and free PTX were added to individual spheroids at a concentration of PTX equivalent to 50 µg/ml and incubated for 24 h. Following the treatment period, spheroids were washed with PBS and stained with Calcein Blue AM reagent and Propidium iodide (PI). This assay works on the principle of diffusion of Calcein Blue AM ester passively into the cells and cleaves to calcein blue in presence of cellular esterases present in viable cells. The resultant calcein blue remains inside the cytoplasm and emits bright blue fluorescence. Further, PI dye stains the dead population. The fluorescence emitted was observed using a fluorescence microscope (Leica DMi8, Leica Microsystems, Germany) with blue and red filters. The images were processed using Image J software.