Enhanced Antimicrobial and Anticancer Activity of Silver and Gold Nanoparticles Synthesised Using Sargassum incisifolium Aqueous Extracts

A detailed, methodical approach was used to synthesise silver and gold nanoparticles using two differently prepared aqueous extracts of the brown algae Sargassum incisifolium. The efficiency of the extracts in producing nanoparticles were compared to commercially available brown algal fucoidans, a major constituent of brown algal aqueous extracts. The nanoparticles were characterised using TEM, XRD and UV/Vis spectroscopy and zeta potential measurements. The rate of nanoparticle formation was assessed using UV/Vis spectroscopy and related to the size, shape and morphology of the nanoparticles as revealed by TEM. The antioxidant, reducing power and total polyphenolic contents of the aqueous extracts and fucoidans were determined, revealing that the aqueous extracts with the highest contents produced smaller, spherical, more monodisperse nanoparticles at a faster rate. The nanoparticles were assessed against two gram-negative bacteria, two gram-positive bacteria and one yeast strain. In contrast to the literature, the silver nanoparticles produced using the aqueous extracts were particularly toxic to Gram-negative bacteria, while the gold nanoparticles lacked activity. The cytotoxic activity of the nanoparticles was also evaluated against cancerous (HT-29, MCF-7) and non-cancerous (MCF-12a) cell lines. The silver nanoparticles displayed selectivity, since the MCF-12a cell line was found to be resistant to the nanoparticles, while the cancerous HT-29 cell line was found to be sensitive (10% viability). The gold nanoparticles displayed negligible toxicity.


UV-Vis Spectroscopy
UV-Vis spectra of the aqueous extracts and fucoidans in water ( Figure S2) revealed the pure fucoidan samples Fucus vesiculosus (Fv) and Undaria pinnatifida (Up) to be featureless, as polysaccaharides are optically transparent. The fucoidans isolated from Macrocystis pyrifera (Mp), while also colourless, displays a broad peak around 250 nm, as do the aqueous extracts (AC and AR), Figure S2, though, the absorption peaks for AC and AR are more pronounced due to the presence of polyphenols. It is clear that the extracts and fucoidans will not mask the observation of the SPR bands for the AgNPs or AuNPs which are expected at ~420 nm and 532 nm, respectively. Absorbance (a.u)

NMR Spectroscopy
Further spectroscopic comparison of these extracts and fucoidans was accomplished through 1 H NMR and multiplicity-edited HSQC 2D spectra as shown in Figure S3 (and Figure S4). HSQC spectra provide information on direct 1 H-13 C correlations since the large number of overlapping proton signals makes the analysis of the 1 H-NMR spectrum (along the x-axis) very difficult. However, using 2D experiments such as HSQC, the carbon signals (along the y-dimension) are more 'spread out', providing a significant amount of information. The purpose of this analysis was not to assign the structures of the fucoidans and aqueous extracts, but rather to provide a basis which allows for the identification of distinct structural features for each of the extracts or fucoidans. In addition, it is also possible to distinguish between methyl, methylene and methine signals at a glance. Figure S3 shows an overlay of the HSQC NMR spectra of the S. incisifolium aqueous (AC) extract (black), and that of the Fv fucoidan (red). Firstly, a methyl signal at ~δ 18 is clearly apparent, as are the sugar oxymethine carbons between δ 60 and 80. Finally, the anomeric carbons of the sugar moieties are visible around δ 100. Most importantly, an overlay of the two spectra (red and black contours) clearly shows that there are significant differences in carbon and proton signals for the two samples suggesting that the major monosaccharides present in these extracts are quite different. Interestingly, only low intensity signals are present at δC/δH 102/6.2 (characteristic of phlorotannins/polyphenols) suggesting that phlorotannins are present as minor constituents in the S. incisifolium crude extract.
Similar trends were observed when comparing the HSQC spectra of the S. incisifolium aqueous extracts and the other fucoidans ( Figure S4). Figure S6 shows an overlay of HSQC spectra for all three of the commercially available fucoidans, showing significant overlap of the signals and suggesting a very similar monosaccharide composition for these fucoidans.

IR Spectroscopy
FT-IR spectroscopy was also used for comparison of the AC and AR extracts and the fucoidan samples ( Figure S5). Key differences in the IR spectra of the fucoidan and the aqueous extract (AR and AC) samples is observed at approximately 800 cm −1 which is attributed to the C-O-C group of monosaccharides and is observed in all the spectra except for AC extract. The Mp and Fv fucoidans exhibited a stretch at ~1200 cm −1 which may be due to sulphated polysaccharides. All fucoidan samples and aqueous extracts exhibited an OH moiety at around 3200-3400 cm −1 , as expected.
FT-IR spectroscopy was used to identify the characteristic peaks associated with the metabolites present in aqueous extract of S. incisifolium as well as the pure fucoidans involved in capping the NPs. The FT-IR spectra for AgNP and AuNP are shown in Figures S6 and S7, respectively. Figure S6 shows the C-O-C moiety 4 is still present in all AgNP samples though it has shifted to lower values from ~830 cm −1 to approximately 810 cm −1 . Furthermore, the peaks observed at approximately 1025 cm −1 and 1040 cm −1 which are thought to be due to the C-OH of polysaccharides remained largely unchanged. The biggest changes observed were for those peaks observed at approximately 1300 cm −1 where peaks that were attributed to the sulfonated polysaccharides were originally present at ~1200 cm −1 and a significant decrease in the 1600 cm −1 stretch in the AgNPs synthesised. The IR spectra of all the samples all had a broad peak at 3200-3300 cm −1 indicating the presence of the OH moieties. The FT-IR spectra shown in Figure S9 the spectra obtained for AuNPs synthesised. The AC-AuNP and AR-AuNP samples both displayed a peak at 1040 cm −1 which is indicative of a C-OH moiety present in polysaccharides. Both AC-AuNP and AR-AuNP contained broad peaks at approximately 3200-3300 cm −1 which indicates the presence of a OH group. In addition, the AR-AuNP sample also displayed a peak at 2922 cm −1 , attributed to CH stretching which was not clearly observed with the AC-AuNPs.

Transmission Electron Microscopy and Energy Dispersive X-ray Spectroscopy
Elemental analysis of the nanoparticles was accomplished using Energy Dispersive X-ray Spectroscopy (EDX), which was subsequently acquired with the TEM images. Elemental Ag and Au were detected in all samples as expected as shown in Figures S13 and S15. However, traces of other elements which may be part of the sample, sample holder or due to impurities are also observed such as copper, nickel, chlorine and oxygen. Copper and nickel are accounted for since holey carbon coated copper/nickel grids were used in the acquisition of data, while oxygen and chlorine may be due to the starting materials or due to sea salt (NaCl or KCl) which may still be present in the seaweed extract. Oxygen is easily adsorbed onto sample surfaces, which would account for some of its presence. The EDX spectra ( Figure S13) reveal traces of other elements such as oxygen and chlorine which may be due to the starting materials or more likely (because of the manner of sample work up) due to any sea salt (NaCl or KCl) which may still be present in the seaweed extract. The EDX spectra ( Figure S15) reveal traces of other elements such as oxygen and chlorine which may be due to the starting materials or more likely (because of the manner of sample work up) due to any sea salt (NaCl or KCl) which may still be present in the seaweed extract. Inset: enlarged area of EDX spectrum showing Ag peaks. Figure S14. TEM image and NP size distribution obtained for the SC-AuNPs.

Gold Nanoparticles
The EDX spectra ( Figure S15) reveal traces of other elements such as oxygen and chlorine which may be due to the starting materials or more likely (because of the manner of sample work up) due to any sea salt (NaCl or KCl) which may still be present in the seaweed extract.   Table S2. Antimicrobial activity (well-diffusion assay) of synthesised nanoparticles against a panel of microorganisms.

Cytotoxicity Studies
Some issues were encountered with using the MTT dye in these assays. Metallic nanoparticles are known to interfere with MTT absorbance by depleting the free MTT, leading to false positive or negative results. The formazan dye also absorbs in the 500-600 nm range. NPs can cleave the MTT tetrazolium ring and increase light absorption which may influence the final data collected. In our study, it appeared that the nanoparticles themselves did interfere to some extent with the absorbance readings of the formazan dye. Care was thus taken to remove excess NPs before addition of the MTT dye to minimise these effects. To ensure that the AuNPs and AgNPs do not interfere with the absorbance readings, the OD was measured at 570 nm, where the effect of the AuNP SPR band is much lower.