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  • 1134-47-0 br The cytotoxicity exhibited by the


    The cytotoxicity exhibited by the dendrimer-drug conjugates com-pared to free drug in A549 1134-47-0 was studied by MTT assay. For the experiment, cells were treated with free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin at a concentration range of 0–50 µg/ml and placed the well plates in incubator for 24 and 48 h. From the MTT assay, it was observed that there was an overall decrease in the cell viability with increase in treatment time and concentration of the drug given to the cells (Fig. 5). Of all the treatments, G4-PTX-PEG-Biotin was found to be more active in killing the cells compared to other treatments. After 24 h, cells treated with G4-PTX-PEG-Biotin exhibited 32.7 ± 5.14% cell viability against 51.62 ± 3.9% for G4-PTX-PEG and 62.3 ± 3% for free PTX, respectively. Furthermore, the cell viability declined to 14.5 ± 3.44%, 29.8 ± 3.2%, and 43.2 ± 2.8% for G4-PTX-PEG-Biotin, G4-PTX-PEG, and free PTX, respectively after 48 h. Dendrimer-drug conjugates were more active at all the time points compared to free PTX. Moreover, G4-PTX-PEG-Biotin had shown superior cell killing which can be ascribed to its capability of internalizing actively into the cells via the biotin receptors which in turn delivers greater concentra-tions of PTX to cancer cells than non-targeted dendrimer or free drug. The uptake of the dendrimer conjugates by receptor mediated/ad-sorptive endocytosis could avoid the drug efflux by P-glycoprotein. A reason for lesser cytotoxicity noticed in free PTX treated cells could be due to the drug being pushed out of the cell by P-glycoprotein trans-porter system.
    3.3. Evaluation of biotin tagged PAMAM dendrimer conjugates in multicellular tumor spheroids
    3.3.1. Penetration efficiency in multicellular tumor spheroids Multicellular tumor spheroidal models simulate the in vivo tumors in
    terms of tumor structure and microenvironment. 3D spheroids retain cancer cells in a natural morphology in presence of extracellular matrix, pH and oxygen gradients resembling actual tumors. Also, spheroid models mimic drug resistance in solid tumors to a greater extent com-pared to 2D monolayer cell system (van den Brand et al., 2017; Yang et al., 2017). These features make 3D spheroidal cancer cell model an attractive alternative to understand the movement of molecules into the cancer tissue.
    From the Z-stack images captured using confocal microscopy, it was observed that the fluorescence was deeper in spheroids treated with biotin tagged dendrimers in comparison to the non-targeted conjugate. Further, the conjugates moved into the core of the spheroids as evi-denced by the bright green fluorescence at depths with increase in the time of incubation from 1 h to 4 h (Fig. 6). Biotin receptor mediated
    Fig. 4. Cellular uptake of fluorescently tagged G4 conjugates in A549 cells after 1 h and 4 h incubation as assessed by flow cytometer and represented as histograms and bar graph of geo mean fluorescence (Mean ± SD, n = 3).
    S.V.K. Rompicharla et al.
    uptake of F-G4-PEG-Biotin resulted in superior diffusion of dendrimer conjugates into the 3D tumor spheroids compared to non-targeted dendrimers. This observation ascertains the advantage of active tar-geting to tumors by biotinylation of the delivery systems.
    3.3.2. Uptake of dendrimer conjugates in multicellular spheroids by flow cytometry
    The internalization of biotin targeted and non-targeted dendrimers in the 3D multicellular cancer spheroids was quantified by flow cyto-metry. The fluorescence intensity was observed to be higher with F-G4-PEG-Biotin conjugate treatment compared to F-G4-PEG. The biotin tagged dendrimer conjugate exhibited a 1.63 fold and 1.7-fold higher geo mean fluorescence value compared to non-targeted dendrimer conjugate following 1 h and 4 h of incubation, respectively.
    The flow cytometry data supports the confocal microscopy results, where time-dependent intensification of the green fluorescence due to accumulation in the spheroids was seen. It can be affirmed that the biotin conjugation on the surface remarkably improved the uptake of the dendrimer conjugates. Data collected from flow cytometer was re-presented in Fig. 7 as fluorescence intensity histogram and a bar graph.
    3.3.3. Growth inhibition of multicellular tumor spheroids
    Growth inhibition caused due to exposure of multicellular cancer spheroids to free PTX, G4-PTX-PEG, and G4-PTX-PEG-Biotin was eval-uated by measuring the diameter of the tumor spheroids over a period of time. The spheroids were examined for 6 days and the microscopic images were captured on Day 0, Day 3, and Day 6 using brightfield microscope. The diameter of the spheroids was noted down to analyze their growth. As depicted in Fig. 8A, the diameter of the spheroids was significantly less on treatment with PAMAM dendrimer-drug conjugates compared to free drug. Moreover, the diameter of control spheroids increased drastically up to 1000 µm diameter during the study. In the treatment groups, the average diameter of spheroids at the end of 6 days was measured to be 531.3 ± 13.5 µm, 647 ± 32.6 µm,