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  • br Fig Percent cell viability

    2021-03-03


    Fig. 2. Percent cell viability of B16F10 mouse melanoma CAY10683 after 48 h of treatment with pure EGCG, EGCG-loaded SLN (EGCG-SLN) and bombesin-conjugated EGCG-SLN (EB-SLN).  Chemistry and Physics of Lipids xxx (xxxx) xxx–xxx
    Table 2
    IC50 values (μg/mL) for epigallocatechin gallate (EGCG), EGCG-loaded solid lipid nanoparticles (EGCG-SLN) and bombesin conjugated SLN (EB-SLN) against MDA-MB-231 human breast cancer cells and B16F10 mouse melanoma cells after 48 h of treatment.
    Formulation IC50 values (μg/mL)
    4.3. Phase contrast microscopy studies
    Phase contrast microscopy was used to observe changes in the morphology of MDA-MB-231 cells after incubation with different EGCG formulations. Fig. 3 shows MDA-MB-231 cells treated with blank SLNs, EGCG, EGCG-SLN and EB-SLN at 24 h and 48 h post treatment with the respective formulations. Control cells were used as negative controls in the experiment. When cells are under stress and undergoing apoptosis, a few distinct morphological changes can be observed. MDA-MB-231 cells are characteristically elongated and tapered. During cell death, the cell becomes spherical and apoptotic bodies can be seen with the help of a nuclear staining dye (Elmore, 2007).
    Blank SLNs do not show any observable cytotoxicity, as is evident by the elongated healthy cells at 24 and 48 h (Fig. 3). The EGCG treated cells showed apoptotic cells, demonstrated by the rounding of the cells when compared to the control cells at both time points. Cells incubated with EGCG-SLN showed more spherical cells compared to EGCG-in-cubated cells which suggests greater toxicity of EGCG-SLN. This could be attributed to the increased stability of the drug within the SLNs as well as the sustained release due to encapsulation (Radhakrishnan et al., 2016). EB-SLN treated cells showed more apoptotic cells, with the number of dead or dying cells increasing at 48 h compared to 24 h. This could again be attributed to the sustained release of the drug within SLNs and more importantly greater uptake due to targeting of bombesin to GRPR receptors (Kulhari et al., 2014). This enhanced uptake would automatically lead to increased concentrations of the drug within the cells and hence greater cytotoxicity.
    4.4. Apoptosis studies
    Apoptosis is a process of programmed cell death that occurs after a cell is no longer needed in the body. In cancer cells, the factors affecting the extrinsic and intrinsic pathways of apoptosis are downregulated, causing the cells to continue to proliferate uncontrollably. EGCG has shown the ability to induce apoptosis in cancer cells by triggering both the extrinsic and intrinsic apoptotic pathways (Wang and Bachrach, 2002).
    To study apoptosis caused by the different formulations, MDA-MB-231 cells were incubated with blank-SLN, pure EGCG, EGCG-SLN and EB-SLN for 48 h followed by staining with Hoechst 33,342. Nuclear condensation is an important process during apoptosis, forming the principle of the nucleus-staining apoptosis assay. Hoechst 33,342 is a cell-permeable, blue fluorescence dye that emits blue fluorescence at 495 nm on binding with DNA (Allen et al., 2001). Fluorescence images in Fig. 4 clearly showed marked differences in nuclear staining in each of the treated cells. Hoechst 33,342 stains all nuclei, hence blue fluor-escence is visible throughout the samples. However, the intensity of fluorescence is more in cells undergoing apoptosis, owing to nuclear condensation. Control cells and cells treated with CAY10683 blank-SLN show no intense fluorescence. However, cells treated with EGCG show several intense fluorescence specks. These specks increase in the EGCG-SLN; however, the highest intensity is visible in the EB-SLN treated cells. It can be explained by an increased uptake of these nanoparticles in
    R. Radhakrishnan, et al. Chemistry and Physics of Lipids xxx (xxxx) xxx–xxx
    Fig. 3. Phase contrast microscopy images of MDA-MB-231 human breast cancer cells after 24 h (I) and 48 h (II) of treatment with A) control (without treatment), B) pure epigallocatechin gallate (EGCG), C) blank solid lipid nanoparticles (blank SLN), D) EGCG-loaded SLN (EGCG-SLN) and E) bombesin conjugated SLN (EB-SLN) equivalent to 20 μg/mL EGCG.
    R. Radhakrishnan, et al. Chemistry and Physics of Lipids xxx (xxxx) xxx–xxx
    Fig. 4. Nuclear fragmentation studies:
    Fluorescent microscopic images of MDA-MB-
    231 human breast cancer cells after 48 h of
    treatment with blank solid lipid nanoparticles (blank SLN), pure epigallocatechin gallate (EGCG), EGCG-loaded SLN (EGCG-SLN) and bombesin conjugated SLN (EB-SLN) equivalent to 20 μg/mL EGCG followed by staining with Hoechst 33,342.