Resveratrol-Mediated Gold-Nanoceria Synthesis as Green Nanomedicine for Phytotherapy of Hepatocellular Carcinoma

Background : In the present study, resveratrol was used to prepare complexes of cerium and nanoceria, also coated with gold (CeO 2 @Au core-shells) to improve the surface interactions in physiological conditions. Methods : The CeO 2 @Au core-shells were characterized using powder X-ray diffraction (PXRD), Fourier transforms infrared spectroscopy (FTIR), transmission electron microscope (TEM) analysis, dynamic light scattering (DLS) and ζ potential. Results : The experiment was led to the successful synthesis of nanosized CeO 2 @Au core-shells, although agglomeration of particles caused the distribution of the larger particles. The TEM analysis demonstrated the particles sizes ranged from 20 nm to 170 nm. Moreover, the PXRD analysis showed that both nanoceria and gold with the same crystal systems and space groups. To investigate the anticancer activity of the CeO 2 @Au core-shells, the cytotoxicity of the nanoparticles was investigated against liver cancerous cell lines (HepG2). Conclusions : The results indicated biosynthesized NCs have significant cellular toxicity properties against HepG2 and could be utilized in hepatocarcinoma therapy. Further in vivo investigations is proposed to be designed to assess anti-cancer and safety effects of fabricated nanocomposites.


Introduction
Phytoalexins are diverse plant secondary metabolites possessing supportive roles against biotic and abiotic stressors, whereas they have represented potent biological properties [1,2].Resveratrol (3,4 ′ ,5-trihydroxy-trans-stilbene) as a natural polyphenolic phytoalexin, can abundantly found in grapes, peanuts, and berries, while it has recently attracted numerous attentions due to its important biotherapy characteristics [3].Different investigations have confirmed that resveratrol richness in the stressed leaves [4,5].Asides from diverse beneficial bioactivity effects of resveratrol, various studies have shown significant cardiovascular protective effects in people who consume rich resveratrol herbal products [6][7][8].Resveratrol efficiently targets extracellular ROS when administered orally, consequently acts as a great antioxidant [9].Furthermore, this compound displays useful effects for the treatment of cancer, hypertensive, inflammatory, Alzheimer' and metabolic diseases [10][11][12][13].
The cerium oxide (nanoceria, NCs) have demonstrated anticancer activities, due to its interesting changes in oxidation states, while it can act similar to the antioxidant enzymes (catalase and superoxide dismutase mimetic activities) [14][15][16], subsequently, showing protective attribute as an active scavenger of reactive oxygen/nitrogen species (ROS/RNS) with unlimited cycles that involves changes in oxidation states of Ce 3+ /Ce 4+ [17,18].The strong anticancer function of the NCs can occur via correcting homeostasis in tumor microenvironment and angiogenesis, preventing myofibroblast formation, invasion and tumor growth without any changes in ROS levels, enhancing the immune system, and in some cases, some paradoxical reports explain the genotoxicity and oxidative stress from ROS formation as the determining factors to death of cancer cells [19][20][21][22][23][24].Reports suggest the bioactivity of the NCs are size-, medium-, pH-and time-dependent [25,26].
The combination of NCs with gold could enhance the anticancer effects of the NCs along with the biocompatibility of the particles [27].Due to the potential application of gold in different nanoplatforms for the treatment of cancer, as an active agent or a nanocarrier, it can be a good choice to form nanocomposites with NCs [28][29][30].The gold inertness and its nontoxic nature are of great use in the design of new active nanomaterials and nanocomposites for biomedical applications [31][32][33][34].The factors that can be crucial in determining the final anticancer properties can be the size, shape, and ratio of the components [35,36].Gold nanoparticles' toxicity is directly proportional to the size of nanoparticles.The larger particles are less toxic, and the smaller ones have substantial toxicity towards cancer cells [37][38][39][40].Therefore, the NC seeds' size influences the bioactivity of CeO 2 @Au core-shells.The combination of NCs and gold, and even with other metals would be an innovative approach for advancing new nanomaterials with stronger anti-cancer properties.
Herein, the complexation of a phytochemical resveratrol and Ce 3+ was occurred because of the oxophilic nature of lanthanide ions.For the first time, the resveratrol and Ce 3+ complex was used as a precursor to synthesize NCs by thermal treatment.The prepared NCs were coated with gold and the formation of CeO 2 @Au core-shells was fully investigated by using the conventional characterization methods.The anticancer properties of CeO 2 @Au coreshells and NCs were measured compared with each other to find the effectiveness of the gold core-shells against liver cancer cells.

Instruments and Materials
The following analyses were performed for characterization of the nanoparticles: powder X-ray diffraction (PXRD), Fourier transforms infrared spectroscopy (FTIR), transmission electron microscopy (TEM), Dynamic light scattering (DLS), and ζ potential.The materials were purchased from Sigma-Merck merged chemical groups except the stated items.

Synthesis of CeO 2 @Au Core-Shells
To prepare the nanoceria, 0.012 mol resveratrol and 0.004 mol cerium nitrate hexahydrate (Ce(NO 3 ) 3 .6H 2 O) were dissolved in 100 mL water separately.The resveratrol was sonicated then stirred vigorously.The cerium ion solution was then added dropwise to the first solution under vigorous stirring.After the addition of cerium ion solution, a milky-like appearance of the mixture confirmed the formation of the resveratrol-cerium complex.Then, the complex was separated using centrifugation and put in an oven at 500 °C overnight (8 h).The prepared nanoceria was used without further purifications.Afterwards, 0.5 g of the as-synthesized nanoceria was dispersed in 2.5 mL of a concentrated solution of Chloroauric acid (HAuCl 4 , 0.2 M).Then, it was centrifuged and washed one time with 10 mL of distilled water (DW) and dispersed again in 10 mL DW.Eventually, a solution of ascorbic acid (100 mL, 1 M) was prepared and dispersed nanoceria was added to this solution dropwise under vigorous stirring.The CeO 2 @Au core-shells were separated and washed several times with DW and applied for characterization and biological tests.

Cell Culture and Toxicity Effect of Biosynthesized NCs
Liver cancer cell line (HepG2) and human foreskin fibroblasts (HFF) were attained from Iran's Pasteur Institute.Cell developmental was performed in RPMI and DMEM media, supplemented with 100 µg/mL penicillin plus 10% FBS, and as a final point transferred to the incubator.In each well of a 96-well plate, cells were seeded, incubated 24 h, and after that the viability of cells was assessed in 24, 48, or 72 h to the biosynthesized NCs at the 0.16, 0.31, 0.63, 1.25, 2.50, 5.00 mg/mL concentrations using MTT assay.

Powder X-ray Diffraction (PXRD)
The PXRD confirmed the formation of gold coated nanoceria (CeO 2 @Au core-shells), where the peaks of CeO 2 and Au core-shells were compatible with the reference codes of 00-001-0800 and 00-001-1172, respectively (Fig. 1).The crystal systems, space groups and their numbers of both nanoceria and gold core-shells were cubic, Fm-3m and 225.311).The comparison of the calculated and experimental data indicated the successful synthesis of cerium oxide and the reduction of Au 3+ .According to the Scherrer equation, the crystallite (grain) size was calculated ≈24.2 nm.

Fourier-Transform Infrared Spectroscopy (FTIR)
The absorption bands of FTIR between ≈3200-3500 cm −1 were associated with the hydroxyl groups of the capped ascorbic acid and possibly on the surface of CeO 2 (Fig. 2) [41][42][43].The bands at 3100-2840 cm −1 can be related to the C-H stretching of ascorbic acid.Ascorbic acid usually shows at absorption signal at ≈1750-1760, specifically assigned to the carbonyl stretching of the γ-lactone ring in the ascorbic acid.The band absence after reduction of gold nanoparticle and bounding to the surface of nanoparticle could prove CeO 2 @Au core-shells coated by ascorbic acid [44].The bands corresponded to C-H bending of the methyl group and carbonate species after calcination was appeared at 1454 and 1385 cm −1 , respectively.The bands at 1132 and 1002 were also associated with the C-O stretching of the -OH group of the alcohol.The band observed at 500 cm −1 can also be assigned to the Ce-O vibration [45].

Transmission Electron Microscopy (TEM)
The TEM analysis was performed to determine the size of CeO 2 @Au core-shells in the solid phase.As shown in Fig. 3, the TEM images have revealed spherical, semispherical, and oval morphologies with particles sizes in the range of ≈20-170 nm.The images revealed that the appearance of larger sizes could possibly be due to the agglomeration of the several particles.

Dynamic Light Scattering (DLS) and ζ Potential
The hydrodynamic size and the partial surface charge of the particles were measured using the DLS and ζ potential analyzer (Fig. 4).The analyses revealed the z-average of 229 nm, where nearly 50 and 90% of the particles showed particle sizes under 282 and 468 nm in order, respectively.The particle size according to intensity and number of size dispersion was 262 and 115 nm, respectively, whereas the partial surface charge was -24 mV.The analyses demonstrated that the information obtained from solid-phase sizes and the hydrodynamic sizes were compatible and acceptable.The PXRD and FTIR also confirmed the synthesis of the nanoparticles.The TEM images further indicated an aura around the particles that could be attributed to the formation of a gold layer around the nanoceria particles.

Anticancer Properties of NC
Nanotechnology-mediated delivery can be employed as a beneficial device in increasing the bioavailability of resveratrol.Nanoscience is an emerging part of investigation that operates at the crossways of biology, physico- chemicals, engineering, pharmacology, and medicine [22,[46][47][48].Designing of NPs (nano particles) for an effective and controlled delivery of anticancer factors is being considered as one of the most important applications of nanoscience [24,49,50].Few experiments have attempted to study potency of resveratrol for the synthesis of nanoparticles.It has been shown that the solid lipid NPs loaded with resveratrol can cross the cell membrane, while it can be enhanced via increasing the exposure time of cells to resveratrol [51].Another study utilizing bovine serum albuminbound resveratrol NPs in primary ovarian cancer of mice showed that not only the NP-bound resveratrol was easier to work with because of the improved solubility, it had also a greater influence on prevent of tumor growth, compared to pure resveratrol [52].The anti-cancer effect of gold nanoparticles against breast, testicular, liver, and lung cancer cells has been proven in a concentration-dependent manner [30,[51][52][53][54][55].In another study, the produced gold nanoparticles provided a safe and great system for the delivery of gapmers in cancerous cells, which meaningfully down-regulated mutant p53 proteins and changed molecular markers related to cell growth and apoptosis [56].
The anticancer activity of the produced NC was remarkable, whereas it possessed lower toxic effect on normal cells.The results clearly exhibited the toxicity of NC against cancer cells in a concentration and time dependent manner (Fig. 5).Although resveratrol alone was effective against cancer cells, higher efficacy in toxicity against cancer cell lines was observed in combination with nanoparticles.

Conclusions
The nanoceria particles were successfully synthesized using resveratrol to form complexes as a precursor for further calcination procedure.The synthesized nanoceria was also coated with a gold layer to increase their anticancer efficiency.The prepared nanoceria were fully characterized and analyzed through conventional methods.The experiment has shown the particle size of ≈20-170 nm in the solid phase with the z-average hydrodynamic size of 229 nm.The present study indicated the successful synthesis of CeO 2 @Au core-shells.The anticancer results demonstrated that resveratrol-mediated synthesized NCs have significant cellular toxicity properties against HepG2 and could be utilized in hepatocarcinoma therapy.Further in vivo investigations will significantly help to the anticancer and safety effects of fabricated nanocomposites.

Fig. 3 .
Fig. 3.The TEM images of CeO2@Au core-shells.The particles sizes are in the range of 20-170 nm.

Fig. 5 .
Fig. 5. Cellular toxicity effect of biosynthesized NCs and resveratrol against HepG2 as a cancerous cell lines (B and D) and HFF as a normal cell lines (A and C).