br untreated cells displayed negligible apoptotic as well as
untreated cells displayed negligible apoptotic as well as necrotic fea-tures. The apoptotic extent was higher in Ru-Fu complex (33%) treated cells than the cells treated with rutin (25%), fucoidan (21%) and their mixtures (27%). Ru-Fu complex treated cells showed low population of necrotic cells (31%), while the control group did not significantly show either the apoptotic or necrotic population (p ≤ 0.01). Anti-pro-liferative activity of fucoidan in various cancer cell lines has been va-lidated by studies by using flow cytometry analysis [61–63].
3.9. Eﬀect on Galactose 1-phosphate distribution
To validate the synergistic eﬀects of prepared Ru-Fu complex, the treated and PI stained HeLa cells were assessed for the distribution of cells in G0/G1, S, and G2/M phases of cell cycle using flow cytometry (Fig. 9). The cell cycle arrest in G0/G1 was increased in Ru + Fu mix-tures and Ru-Fu complex treated cells (p ≤0.01) but the arrest of G2/M phase was decreased against the treatment by individual compounds (Fig. 9). However, the extent of S phase (%) in Ru + Fu and Ru-Fu treated cells was significantly higher than the control cells (p ≤0.05) (Fig. 9). This increase could be due to an increased population of cells in G0/G1 phase. This observation was in consistent with apoptosis induc-tion, as we showed that the Ru-Fu complex arrested the growth of HeLa
cells in G0/G1 phase (Fig. 9). From these observations, it can be sub-stantiated that Ru-Fu complex exhibits cell cycle arrest at crucial points such as G0/G1 and S phases indicating its potential to reduce cell pro-liferation, and the complex can serve as potential chemo-preventive anticancer agent. Flavonoids could promote cell cycle arrest in distinct phases of cancer cells [77,78]. Rutin was reported to execute cell cycle arrest in G1 checkpoint that controlled the progress of the cells into S phase and prevented the replication of DNA .
The potential of fucoidan in inducing cell cycle disturbances in cancer cells has been documented in several studies especially in G0/G1 phase [20,80]. Studies with rutin and Rutin–zinc(II) complex proposed that the complex enhanced the expression of VEGF, Cyclin D1, Caspase-3 and Caspase-8 proteins in EAC cells of tumor bearing mice which are linked to cell cycle progress . It was reported that the fucoidan extracted from Undaria pinnatifida promoted cell cycle arrest of G0/G1 phase in prostate cancer cell line PC-3 as confirmed by flow cytometry analysis .
3.10. Enhanced biocompatibility of Ru-Fu complex in human RBCs
Assessment of hemolytic properties of drug like materials is re-garded as an important test to study their interactions with blood
Fig. 10. Eﬀect of Ru-Fu on RBC membrane integrity. (a). RBC suspensions are exposed to Ru-Fu at various concentrations (2.5, 5, 10, 25, 50 and 100 μg/ml) for 3 h followed by centrifugation. The presence of red hemoglobin in the supernatant indicates damaged RBCs. (+) and (−) symbols indicate positive (RBCs suspended in deionized water) and negative (RBCs suspended in phosphate-buﬀered saline, PBS) controls, respectively. (b). Hemolysis profile of RBCs incubated with complex at diﬀerent concentrations. Error bars denote mean ± SD of three independent experiments performed in triplicates. (c). Microscopic images (magnification of 200X) of human RBC treated with Ru-Fu at diﬀerent concentrations. PBS is used as a negative control and deionized water as a positive control.
components and the activity is determined by the release of hae-moglobin upon treatment of RBCs with substances. In this study, the extent of hemolysis of the prepared complex at all the tested con-centrations was found to be < 5% and the extent was only 0.95% for Ru-Fu treatment at 100 μg/ml concentration (Fig. 10b). Since hemolysis extent by biomaterials up to 5% is considered permissible, the values for our study complex Ru-Fu were much lower than 5% indicating its hemocompatibility for drug delivery applications. Fig. 10a shows the photographs of RBCs subjected to hemolytic test using the Ru-Fu complex. When deionised water (hypotonic solvent; positive control) was added to RBCs, hemolysis occurred leading to release of hae-moglobin indicated by red colour of the suspension. RBCs incubated with PBS were used as negative controls and showed no hemolysis. The supernatant from cells of Ru-Fu complex treatment at diﬀerent con-centrations is achromatic, which is comparable to the RBCs incubated with PBS. Thus, Ru-Fu at the tested concentration exhibited no sig-nificant hemolysis. The microscopic images of RBCs indicated that the Ru-Fu complex at various concentrations did not display either hemo-lysis or morphological changes when compared to controls, implying the biocompatibility of the complex (Fig. 10c).