Table 1.
Viability of H9c2 cardiac myoblast cells treated with sodium selenite and SGG nanoparticles.
Figure 1.
Particle size distribution of nanoparticle.
Nanoparticles were prepared by nanoprecipitation method. The prepared nanoparticles were subjected to particle size analysis. (a) GG nanoparticle had an average particle size varies from 41 to 132 nm range and (b) SGG nanoparticle prepared by nanoprecipitation method shows an increase in particle size from 69 to 173 nm.
Figure 2.
TEM images of (a) GG showing spherical morphology with a size of ∼40 nm with few larger particles (b) SGG nanoparticles were also spherical in shape, upon incorporation of selenium the particle size has been increased to the range of 50–100 nm (c) EDX spectrum of SGG show the presence of Se peaks, thereby confirming the presence of Se in SGG.
Figure 3.
XRD images of (a) GG (b) sodium selenite and (c) SGG nanoparticle.
GG nanoparticle appears amorphous in nature in their morphology as per XRD data (Fig. 3a). While sodium selenite appears crystalline nature in the XRD pattern (Fig. b) The presence of major peaks of selenium in SGG confirm the incorporation of selenium in GG nanoparticle (Fig. 3c).
Figure 4.
Morphological examination of cells with nanoparticles.
Images of H9c2 cells from different experimental groups under phase-contrast microscope (10x). (a) Control cells; (b, c, d) Cells treated with 5, 25 and 50 nM Se respectively; (e, f, g) Cells treated with 5, 25 and 50 nM SGG, respectively. H9c2 cells showed no significant alteration in morphology upon treatment with Se and SGG of any concentrations reported in the study. Scale bar corresponds to 100 µm.
Figure 5.
A. Uptake of SGG by H9c2 cardiac myoblasts after 1(a) Control; (b) MGG; (c, d, e) Cells treated with 5, 25 and 50 nM of SGG.
The autofluorescent property of guar gum was utilized for the uptake study. The cells were counter stained with AO that binds with DNA of live cells to distinguish nucleus from cytoplasm. The green colour represents nucleus and blue colour is the autofluorescence emitted by GG. Figure 5b appears like blue cloud due to the autofluorescence of MGG in the medium. The presence of blue fluorescence (Fig. 5c, d, e) in the cytoplasm of cells clearly reveals the uptake of SGG by the cells in a dose dependent manner. Scale bar corresponds to 100 µm. B. Uptake of SGG by H9c2 cardiac myoblasts after 6 h incubation (a) Control; (b) MGG; (c, d, e) Cells treated with 5, 25 and 50 nM SGG. The intensity of fluorescence is more in these figures compared to 5A (c, d, e) revealing the duration dependent uptake of SGG by cells. Red arrow points cytoplasm and yellow arrow point nucleus respectively. Scale bar corresponds to 100 µm. C. Uptake of SGG by H9c2 cardiac myoblasts after 24 h incubation (a) Control; (b) MGG; (c, d, e) Cells treated with 5, 25 and 50 nM SGG. Fluorescent intensity of the cells in this group was high compared to the other 2 groups (Figs. 5B c, d, e) confirming the duration dependent uptake of SGG by cells. Scale bar corresponds to 100 µm.
Figure 6.
In vitro cellular uptake of Se from sodium selenite and SGG.
Se uptake study using diaminonaphthalene method shows that uptake of Se by cell is higher for SGG (red) than sodium selenite (blue). 25 nM of both Se and SGG showed maximum uptake of Se by cells.
Figure 7.
Alteration in DNA integrity with Se and SGG nanoparticle.
Photomicrographs of H9c2 cells with AO/EtBr staining to check induction of apoptosis with Se and SGG (20×); (a) Control cells stained with AO; (b) Control cells stained with EtBr; (c) superimposed image of a and b (d) H9c2 cells treated with 5 nM Se stained with AO; (e)H9c2 cells treated with 5 nM Se stained with EtBr; (f) Superimposed images of d and e; (g) H9c2 cells treated with 25 nM Se stained with AO; (h) H9c2 cells treated with 25 nM Se stained with EtBr; (i) Superimposed images of g and h; (j) H9c2 cells treated with 50 nM Se stained with AO; (k) H9c2 cells treated with 50 nM Se stained with EtBr; (l) Superimposed images of j and k respectively; (m) H9c2 cells treated with 5 nM SGG stained with AO; (n) H9c2 cells treated with 5 nM SGG stained with EtBr; (o) Superimposed images of m and n; (p) H9c2 cells treated with 25 nM SGG stained with AO; (q) H9c2 cells treated with 25 nM SGG stained with EtBr; (r) Superimposed images of p and q (s) H9c2 cells treated with 50 nM SGG stained with AO; (t) H9c2 cells treated with 50 nM SGG stained with EtBr; (u) Superimposed images of s and t. The green color represents viable cells stained with AO. This reveals clearly that there was no induction of apoptosis with Se or SGG. Scale bar corresponds to 100 µm.
Figure 8.
A. DNA damage protection by SGG.
Modulating effect of SGG against Fentons reaction induced plasmid conformational change. Figure 8A shows gel view of plasmid DNA clevage assay, OC and SC indicate the open circular and supercoiled plasmid forms respectively. (a) Control DNA pUC18 plasmid alone, (b) the plasmid DNA pUC18 was incubated with Fentons reagent, (c) with ellagic acid 50 nM, with different concentrations of Se (d) 5 nM (e) 25 nM (f) 50 nM and different concentration of SGG (g) 5 nM (h) 25 nM (i) 50 nM. SGG nanoparticle effectively protects the DNA from plasmid breakage in a dose dependent manner. B. The effect of nanoparticle on DNA. This gel view shows plasmid conformation with various doses of Se and SGG revealing clearly the non toxicity of the particle on plasmid. (a) Control DNA pUC18 plasmid alone, (b, c) with ellagic acid 5 nM and 50 nM, with different concentrations of Se (d) 5 nM (e) 25 nM (f) 50 nM and different concentration of SGG (g) 5 nM (h) 25 nM (i) 50 nM.
Figure 9.
A. Evaluation of ROS with various doses of Se and SGG after 1
Fluorescent microscopic images of H9c2 cells stained with DCFDA (20×). (a) Control cells; (b, c, d) Cells treated with 5, 25 and 50 nM Se, respectively; (e, f, g) Cells treated with 5, 25 and 50 nM of SGG, respectively; (h) The fluorometric analysis supported the microscopic data. It is clear from the fluorescent images that treatment of cells with Se and SGG induce ROS generation i.e., there is a linear increase in fluorescence intensity in dose dependent way (Fig. 9a to g). This indicates ROS generation with Se and SGG in a dose dependent manner. Scale bar corresponds to 100 µm. B. Evaluation of ROS with various doses of Se and SGG after 6 h. Fluorescent microscopic images of H9c2 cells stained with DCFDA (20×). (a) Control cells; (b, c, d) Cells treated with 5, 25 and 50 nM Se, respectively; (e, f, g) cells treated with 5, 25 and 50 nM of SGG, respectively; (h) The fluorometric analysis data. It is interesting to note that the magnitude of ROS generation had been decreased in 6 h of incubation compared to 1 h. Scale bar corresponds to 100 µm. C. Evaluation of ROS with various doses of Se and SGG after 24 h. Fluorescent microscopic images of H9c2 cells stained with DCFDA (20×). (a) Control cells; (b, c, d) cells treated with 5, 25 and 50 nM Se, respectively; (e, f, g) cells treated with 5, 25 and 50 nM of SGG, respectively; (h) The decrease in ROS production with Se and SGG is the sign of non toxicity of particle to cells for longer duration (24 h). The initial outburst of ROS (Fig. 9A) is most probably due to the self adaptation mechanism of cell to the entry of foreign particle in the form of Se and SGG nanoparticles. Scale bar corresponds to 100 µm.
Figure 10.
A. Mitochondrial transmembrane potential with Se and SGG after 1
Images of H9c2 cells stained with JC1 for mitochondrial study (20×). (a) Control cells; (b) Positive control valinomycin; (c, d, e) Cells treated with 5, 25 and 50 nM Se, respectively; (f, g, h) Cells treated with 5, 25 and 50 nM SGG, respectively; (i) Relative fluorescence intensity. There was no shift from red to green fluorescence with any groups indicating no change in mitochondrial transmemberane potential. Scale bar corresponds to 100 µm. B. Mitochondrial transmembrane potential with Se and SGG after 6 h. Images of H9c2 cells stained with JC1 for mitochondrial study (20×). (a) Control cells; (b, c, d) cells treated with 5, 25 and 50 nM Se, respectively; (e, f, g) Cells treated with 5, 25 and 50 nM SGG, respectively; (h) Relative fluorescence intensity. Here also there is no change of mitochondrial transmembrane potential with any dose of Se and SGG. Scale bar corresponds to 100 µm. C. Mitochondrial transmembrane potential with Se and SGG after 24 h. Images of H9c2 cells stained with JC1 for mitochondrial study (20×). (a) Control cells; (b, c, d) Cells treated with 5, 25 and 50 nM Se, respectively; (e, f, g) Cells treated with 5, 25 and 50 nM SGG, respectively; (h) Relative fluorescence intensity. Long duration of exposure of Se and SGG caused some alterations in transmembrane potential of mitochondria at higher dose (50 nM). Scale bar corresponds to 100 µm.
Figure 11.
Effect of Se and SGG on cytoskeleton of H9c2 cells.
Fluorescent microscopic images of H9c2 cells stained with phalloidin (20×). (a) Control cells; (b, c, d) cells treated with 5, 25 and 50 nM Se, respectively; (e, f, g) cells treated with 5, 25 and 50 nM SGG, respectively. The green colour indicates the cytoskeleton F-actin stained with phalloidin. The blue colour indicates the nucleus stained with DAPI. There were no significant alterations in cytoskeleton with any groups. This reveals clearly that SGG does not cause alteration to cytoskeleton. Scale bar corresponds to 100 µm.