Hybrid nanocomposite curcumin-capped gold nanoparticle-reduced graphene oxide: Anti-oxidant potency and selective cancer cytotoxicity

Abstract

Nanotechnology-based antioxidants and therapeutic agents are believed to be the next generation tools to face the ever-increasing cancer mortality rates. Graphene stands as a preferred nano-therapeutic template, due to the advanced properties and cellular interaction mechanisms. Nevertheless, majority of graphene-based composites suffer from hindered development as efficient cancer therapeutics. Recent nano-toxicology reviews and recommendations emphasize on the preliminary synthetic stages as a crucial element in driving successful applications results. In this study, we present an integrated, green, one-pot hybridization of target-suited raw materials into curcumin-capped gold nanoparticle-conjugated reduced graphene oxide (CAG) nanocomposite, as a prominent anti-oxidant and anti-cancer agent. Distinct from previous studies, the beneficial attributes of curcumin are employed to their fullest extent, such that they perform dual roles of being a natural reducing agent and possessing antioxidant anti-cancer functional moiety. The proposed novel green synthesis approach secured an enhanced structure with dispersed homogenous AuNPs (15.62 ± 4.04 nm) anchored on reduced graphene oxide (rGO) sheets, as evidenced by transmission electron microscopy, surpassing other traditional chemical reductants. On the other hand, safe, non-toxic CAG elevates biological activity and supports biocompatibility. Free radical DPPH inhibition assay revealed CAG antioxidant potential with IC50 (324.1 ± 1.8%) value reduced by half compared to that of traditional citrate-rGO-AuNP nanocomposite (612.1 ± 10.1%), which confirms the amplified multi-potent antioxidant activity. Human colon cancer cell lines (HT-29 and SW-948) showed concentration- and time-dependent cytotoxicity for CAG, as determined by optical microscopy images and WST-8 assay, with relatively low IC50 values (~100 μg/ml), while preserving biocompatibility towards normal human colon (CCD-841) and liver cells (WRL-68), with high selectivity indices (≥ 2.0) at all tested time points. Collectively, our results demonstrate effective green synthesis of CAG nanocomposite, free of additional stabilizing agents, and its bioactivity as an antioxidant and selective anti-colon cancer agent.

Fig 1. CAG nanocomposite synthesis illustration.

Fig 2. The proposed reduction sites action of CR.

The O^- moieties formed at alkaline pH are assumed to reduce Au⁺ ions to AuNPs [63].

Fig 3. TEM images of CAG vs. rGO-AuNP nanocomposites.

(A) CAG composite at 0.5 μm scale, (B) CAG at 100 nm scale with an inset of size distribution histogram. (C) rGO-AuNPs prepared using sodium citrate at 0.5 μm scale, (D) rGO-AuNPs at 100 nm scale.

Fig 4. Raman spectra of synthesized nanocomposite CAG, GO and graphite.

Fig 5. FTIR and XRD analysis.

(A) FT-IR spectra of GO, CAG, and CR. (B) XRD analysis of GO and CAG.

Fig 6. UV-Vis absorption spectra.

Panel of GO, citrate-AuNPs, CAG, and CR.

Fig 7. TGA curve trends.

TGA curves for CAG in comparison to graphite, CR, and GO.

Fig 8. Dynamic light scattering.

Particle size distribution of CAG nanocomposite dispersion in RPMI-1640 medium.

Fig 9. DPPH antioxidant assay.

CAG antioxidant activity compared to raw material GO, and citrate synthesized rGO-AuNPs nanocomposite. Results expressed as mean from triplicate analysis ± SEM. ^{a, b} and ^c indicates a significant increase in DPPH inhibition of respective treatments (CAG, rGO-AuNPs, and GO, respectively) compared to no treatment control (p<0.05). * Significant difference between treatment groups at respective concentration (p<0.05). Statistical analysis was performed using factorial ANOVA tests, SPSS software.

Fig 10. Optical phase-contrast microscopy images.

Untreated control cells (up; HT-29, bottom; SW-948) compared to CAG nanocomposite treatment at low and high concentrations.

Fig 11. In vitro viability results after CAG treatment.

Percentage viability of colon cancer cells (HT-29 and SW948 cell lines), normal colon cells (CCD841), and normal liver cells (WRL-68), upon exposure to CAG nanocomposite at different concentrations (62.5–1000 μg/mL), measured at three time points using WST-8 assay. Tests were performed and results were means from triplicate analysis. * Significant decrease (p<0.05) in viability percentage compared to untreated control, as analyzed by factorial ANOVA test, SPSS software.

Fig 12. Schematic illustration of the proposed CAG interaction and mechanism of action on cell.

(a) Antioxidant activity and free radical inhibition, (b) CAG-cell interaction and proposed subsequent mechanism.