Fabricating a low-temperature synthesized graphene-cellulose acetate-sodium alginate scaffold for the generation of ovarian cancer spheriod and its drug assessment

3D cell culture can mimic tumor pathophysiology, which reflects cellular morphology and heterogeneity, strongly influencing gene expression, cell behavior, and intracellular signaling. It supports cell–cell and cell–matrix interaction, cell attachment, and proliferation, resulting in rapid and reliable drug screening models. We have generated an ovarian cancer spheroid in interconnected porous scaffolds. The scaffold is fabricated using low-temperature synthesized graphene, cellulose acetate, and sodium alginate. Graphene nanosheets enhance cell proliferation and aggregation, which aids in the formation of cancer spheroids. The spheroids are assessed after day 7 and 14 for the generation of reactive oxygen species (ROS), expression of the hypoxia inducing factor (HIF-1⍺) and vascular endothelial growth factor (VEGF). Production of ROS was observed due to the aggregated tumor mass, and enhanced production of HIF-1⍺ and VEGF results from a lack of oxygen and nutrition. Furthermore, the efficacy of anticancer drug doxorubicin at varying concentrations is assessed on ovarian cancer spheroids by studying the expression of caspase-3/7 at day 7 and 14. The current findings imply that the graphene-cellulose-alginate (GCA) scaffold generates a reliable ovarian cancer spheroid model to test the efficacy of the anticancer drug.

Fourier Transform Infrared spectroscopy.
The FTIR spectra show a reduction in bound (3450 cm -1 ) and unbound (3640 cm -1 ) -OH, C-O, and C=O with the heating step.The graphene ranges have a similar pattern with reduced noise compared with the graphite spectra.The noise reduction is because graphite, composed of multiple layers, has few degrees of freedom, whereas graphene possesses separate layers.As all three samples are forms of carbon, the signature peaks can be seen from all the spectra visibly seen in (Fig. S1b).However, there is very little difference between graphite and graphene when considering the type of bonds observed.

X-ray Diffraction Analysis
XRD spectra prove the benefit of heating to obtain graphene.The graphitic carbon shows a sharp peak at 26.35°.The reduced graphene oxide shows the peak at 26.54° which shifts to 25.52° after the final heating step.Peak broadening is observed in graphene due to the unordered stacking of sheets.This stacking of sheets is because of the high static interaction between the layers.The slight shift between graphite and graphene suggests the presence of some residual oxygen content in the produced graphene (Fig. S1c).
Electronic Supplementary Material (ESI) for Nanoscale Advances.This journal is © The Royal Society of Chemistry 2023

Energy dispersive X-ray Analysis
The heating step leads to an increase in the concentration of carbon in the obtained graphene, thereby decreasing oxygen content and other impurities.A monolayer of oxygen is formed under high vacuum conditions; thus, oxygen contamination cannot be avoided.Consequently, few oxygen molecules are present in the graphene and cannot be removed with the help of chemical techniques.The sample must be heated at high temperatures in an inert atmosphere to reduce the oxygen impurities.Some sodium is present in the selection and is constant across all regions.Its presence might be due to sodium nitrate and sodium borohydride.Depth profiling checks the purity of different graphene layers and avoids the substrate effect.At low acceleration voltage, the presence of silicon substrate is negligible.

Cell viability
A significant increment in cell viability from day 1 to day 7 on the scaffold containing 5 µg/mL graphene concentration was observed, which can be seen in Fig. S2.

Scanning Electron Microscopy
The generated ovarian cancer spheroids on GCA scaffolds are confirmed by scanning electron on days 7 and 14.On days 7 and 14, cells are washed with PBS and fixed with 0.2% glutaraldehyde solution (Sigma Aldrich USA) at 4°C for 12 h.The glutaraldehyde solution is discarded, and scaffolds are submerged in 10% ethanol for 15 min.Further, 10% ethanol is discarded, and the previous step is repeated with 30%, 50%, 70%, 90%, and 100% ethanol.Scaffolds containing cancer spheroids are dried in a critical point drier, coated with gold, and observed under a scanning electron microscope.
Increased tumor mass and aggregation are observed from days 7 to 14, as shown in Fig S3.

Figure
Figure S1 a) UV-Vis spectra, b) spectra, and c) XRD spectra of graphite, reduced graphene oxide, and graphene.

Fig
Fig S2.Graph representing the growth of NIH 3T3 cells on the GCA scaffold with varying concentrations of graphene (1, 5, and 10 μg/mL) on days 1, 3, and 7.

Fig
Fig S3 SEM images of ovarian cancer spheroids grown on GCA scaffold on a) day 7 and b) day 14.

Table S1 :
Variation in the elemental concentrations of graphite and the synthesized rGO and graphene.