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  • br Determination of Caspase Activity br Fluorometric method

    2020-08-18


    2.5. Determination of Caspase-3 Activity
    Fluorometric method was used to evaluate the Caspase-3 activity. 12-well plates were seeded with cells and incubated for 24 h. Later the cells were treated with SeNPs, followed by exposure to X-ray and in-cubation for 24 h. Lysis buffer was used for cell lysis after harvesting and then incubated for 1 h in ice. Caspase-3 substrates (Ac-DEVD-AMC) were used to determine the Caspase activity by measuring the fluor-escence intensity at an excitation wavelength of 380 nm and 460 nm as an emission wavelength.  Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 123–127
    Fig. 1. UV–visible spectra change in color with respect to time.
    2.6. Live/Dead Analysis
    A 6-well plate was seeded with A549 cells at a density of 3 × 105 cells/well and was incubated for 24 h. New media with SeNPs with the presence and absence of X-ray was used to replace the older media and incubated for 24 h. Then, the cells were stained using Calcein AM as a live staining agent and ethidium homodimer-1 as a dead cell staining agent. The stained cells were further observed using the fluorescence microscope.
    3. Result and Discussion
    Fig. 1 displayed a time dependent color change in the sample, which was incubated at 30 °C for 48 h. A gradual color change of the solution with time from initial pale yellow to red color was observed. However, no further color change was identified after 48 h of incubation time. The excitation of surface plasmon vibrations of the selenium nano-particles has instigated the final red color to the sample. As expected, no change in color of the sample was noticed in the control experi-ments, which supported the formation of biogenic SeNPs was only be-cause of the bacteria and its protein. While in the control experiment, the high temperature and pressure of autoclave have degraded its proteins and killed the bacterial cells. An absorbance peak was ob-served at 330 nm in the UV–visible spectrometric analysis (Fig. 1B) corresponding to the protein. Further, a decrease in the intensity of Glycoursodeoxycholic acid peak was monitored with the increase in time, which re-presented the protein consumption during the reduction of SeO32− to Se0.
    (210) and (211) reflections of the pure hexagonal phase of selenium crystals with lattice parameters a = 4.366 Å and c = 4.9536 Å (JCPDS 06–0362). Alternatively, the sharp Bragg reflection attributed to the presence of selenium nanorods which is in agreement with that of the SAED findings. Scherrer's equation was used to calculate the average crystalline size of the selenium nanoparticles. D = k / cos (1)
    where, λ = X-ray wavelength, Kα = 1.54 Å, θ = Bragg angle, β = full width half maximum in radians, k = unknown shape factor. The cal-culated average crystalline size of synthesized selenium nanoparticles was found to be 88.89 nm that was in accordance with the TEM ana-lysis. The TEM images have displayed that the synthesized selenium
    Fig. 2. XRD pattern of synthesized selenium nanoparticles.
    nanoparticles could be enveloped as shown in Fig. 3.Certainly, the molecular structure of polysaccharides has reported reactive amino, hydroxyl or carboxyl groups which play a key role in the formation, stabilization and growth of selenium nanoparticles [13]. However, it was evident that the gum-arabic mediated SeNPs exhibited spherical morphology [14] while presented elliptical and rod-like morphology in
    0.1% chitosan-solution [15]. Henceforth, these diverse morphologies suggested that the polysaccharides had variable impact in the formation and elongation of SeNPs. The average diameter of the enveloped sele-nium nanoparticles was evidenced to be 60 nm. The SAED pattern of SeNPs has disclosed hexagonal ring structure with diffraction ring pattern. The EDS spectrum (Fig. 4) of the biosynthesized SeNPs has pre-sented an intense peak at 1.5 keV signifying the entire composition of Se in the developed SeNPs. In addition, few weak signals of Cu, P, Cl, Si and Na were also recorded possibly related with the glass underlay. The C, P, and O signals were associated with either capped or closely pre-sent biomolecules like enzymes, proteins, or bacterial bio-material. XRD studies helped to measure the phase of the synthesized SeNPs.
    MTT assay was performed to evaluate the radio-sensitizing effect of selenium nanoparticles under the X-ray influence. As displayed in Fig. 5a, A549 cells have presented reduced cell viability in a con-centration dependent manner with the treatment of SeNPs. The results display that cell viability of A549 cells were approximately ∼70%, ∼45% and ∼25% for the exposure of 20 μg/ml, 60 μg/ml, and 100 μg/ ml, respectively. Especially, the combination of SeNPs + X-ray has provoked a momentous cell destructing effect, thus presented effi-ciently increased anticancer activity in lung cancer cells. Also, the cell viability of A549 cells was recorded as∼45%, ∼18% and ∼5% for the exposure of 20 μg/ml, 60 μg/ml, and 100 μg/ml, respectively. This manifested that the synergistic effect of the SeNPs + X-ray might have enacted a crucial role in increasing the cell killing effect via cell