Gold Nanoparticles Biosynthesized and Functionalized Using a

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Functional Nanostructured Materials (including low-D carbon)

Gold nanoparticles biosynthesized and functionalized using a hydroxylated tetraterpenoid trigger gene expression changes and apoptosis in cancer cells Bing Tian, Jiulong Li, Renjiang Pang, Shang Dai, Tao Li, Yulan Weng, Ye Jin, and Yuejin Hua ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09206 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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ACS Applied Materials & Interfaces

Gold nanoparticles biosynthesized and functionalized using a hydroxylated tetraterpenoid trigger gene expression changes and apoptosis in cancer cells

Bing Tian# *, Jiulong Li#, Renjiang Pang, Shang Dai, Tao Li, Yulan Weng, Ye Jin, Yuejin Hua*

Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China

#

These authors contributed equally to this work and are co-first authors.

*

Corresponding author: Bing Tian ([email protected]); Yuejin Hua ([email protected]),

Institute of Nuclear-Agricultural Sciences, Zhejiang University, No.268, Kaixuan Road, 310029 Hangzhou, China, Tel/Fax: +86-571-86971215 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Understanding the synthetic mechanisms and cell-nanoparticle interactions of biosynthesized and functionalized gold nanoparticles (AuNPs) using natural products is of great importance for developing their applications in nanomedicine. In this study, we detailed the biotransformation mechanism of Au(III) into AuNPs using a hydroxylated tetraterpenoid deinoxanthin (DX) from the extremophile Deinococcus radiodurans. During the process, Au(III) was rapidly reduced to Au(I) and subsequently reduced to Au(0) by deprotonation of the hydroxyl head groups of the tetraterpenoid. The oxidized form, deprotonated 2ketodeinoxanthin (DX3), served as a surface-capping agent to stabilize the AuNPs. The functionalized DX-AuNPs demonstrated stronger inhibitory activity against cancer cells compared with sodium citrate-AuNPs and were nontoxic to normal cells. DX-AuNPs accumulated in the cytoplasm, organelles and nuclei and induced reactive oxygen species generation, DNA damage and apoptosis within MCF-7 cancer cells. In the cells treated with DX-AuNPs, 374 genes, including RRAGC gene, were upregulated; 135 genes, including the genes encoding FOXM1 and NR4A1, were downregulated. These genes are mostly involved in metabolism, cell growth, DNA damage, oxidative stress, autophagy and apoptosis. The anticancer activity of the DX-AuNPs was attributed to the alteration of gene expression and induction of apoptosis. Our results provide significant insight into the synthesis mechanism of AuNPs functionalized with natural tetraterpenoids, which possess enhanced anticancer potential.

KEYWORDS: gold nanoparticles, tetraterpenoid, biosynthesis, functionalization, cellnanoparticle interactions, anticancer, gene expression

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INTRODUCTION Gold nanoparticles (AuNPs) have attracted considerable attention for their potential applications in various fields and their novel properties.1 Functional gold-based nanomaterials can improve the efficacy of cancer diagnosis and therapy that remains a great challenge around the world. 2 In the ever-expanding field of nanomaterial research, AuNP biosynthesis has been proposed as an alternative to chemical and physical syntheses attributed to its milder process and decreased use of energy and toxic chemicals.3-5 Nanoparticles produced and capped with appropriate biomolecules display enhanced biocompatibility and functionalization in living bodies.6,7 Natural products that have antioxidant, antimicrobial and/or anti-carcinogenic properties (e.g., curcumin, chlorogenic acid and tannins) have been used to reduce metal precursors to form the corresponding nanoparticles.5,8,9 Tetraterpenoids, also called carotenoids, are derivatives of tetraterpenes from plants and bacteria and have been used for natural cancer chemoprevention. Treatment with tetraterpenoids at either high oxygen tension or high concentration can induce a pro-oxidant and cytotoxic effect on cancer cells.10 However, the low bioavailability of tetraterpenoids, which results from their poor solubility in aqueous solution, has hindered their applications.8 Recently, selected tetraterpenoids were used to synthesize colloidal AuNPs in dimethyl sulfoxide (DMSO) or methanol;11,12 however, the exact functional groups and oxidized products formed during the reduction of Au(III) by tetraterpenoids remain unclear. It is critical to explore the synthetic mechanism and function of tetraterpenoid-functionalized nanoparticles in aqueous solution and elucidate the cell-NP interactions for bioapplications. Deinoxanthin (2,1′-dihydroxy-3′,4′-didehydro-1′,2′-dihydro-β,ψ-caroten-4-one, DX) is a tetraterpenoid

extracted

from

the

nonpathogenic

extreme

bacterium

Deinococcus

radiodurans.13 Bacterial metabolites have gained attention in AuNP synthesis because bacteria can be cultured economically and their metabolites can be prepared without seasonal 3 ACS Paragon Plus Environment


and geographic effects.14,15 DX was identified as a strong antioxidant in D. radiodurans for defense against ionizing radiation and oxidants.16,17 DX, which contains multiple hydroxyl groups, is more polar and reactive than carotenes (β-carotene and lycopene) and xanthophylls (lutein and zeaxanthin).18-20 Recent studies demonstrated that DX was a potent chemopreventive agent and applicable in dietary supplementation.21,22 Herein, we report the facile method for the synthesis of AuNPs via incubation of hydroxylated tetraterpenoids with Au(III) in aqueous solution. We then unravel the underlying synthetic mechanism of DX-capped AuNPs (DX-AuNPs) using Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), high-performance Xray photoelectron spectroscopy (XPS) and liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). In addition, we evaluate the interactions between DXAuNPs and cancer cells based on physiological assays and gene expression profiles.

EXPERIMENTAL SECTION Materials. Deinococcus radiodurans (ATCC13939) was cultured aerobically at 32°C in tryptone glucose yeast medium on an orbital shaker at 220 rpm. Chloroauric acid (HAuCl4▪3H2O), astaxanthin (purity ≥97%), lutein (purity ≥97.0%), β-carotene (purity ≥95%) and Fluorometric Intracellular ROS Kit were purchased from Sigma-Aldrich Co. (St. Louis, MO,

USA).

MTS

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium, inner salt) was purchased from Promega Co. (Madison, WI, USA). Commercial AuNPs synthesized by sodium citrate (SC-AuNPs) with sizes of 30 nm were purchased from Shanghai So-Fe Biomedical Co. (Shanghai, China). Ultrapure Millipore water (18.25 MΩ) was used in all washing procedures and solution preparations. The Au(III) solutions used in this study were acquired through dissolving HAuCl4▪3H2O in ultrapure water. 4 ACS Paragon Plus Environment

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Preparation of DX. Five liters of the D. radiodurans culture (OD600nm=1.0) were harvested by centrifugation (8,000×g, 10 min). As described previously,13,19 cell pellets were extracted with acetone and methanol (7:2, v⁄v) in the dark until the pellets became colorless, and DX was purified using column chromatography and semipreparative high-performance liquid chromatography (HPLC) and identified by HPLC and mass spectrometry (MS). DX, with a purity >90%, was identified using its retention time, absorption spectrum features and MS analysis compared with references.19 The collected DX was evaporated under N2 and dissolved in ethanol for AuNP synthesis.

Synthesis of AuNPs using tetraterpenoids. A final concentration of 1 mM Au(III) solution was incubated with 15–45 µg/mL tetraterpenoids at pH 7.0 and 25°C. AuNP formation was monitored by the changes in color and absorption spectra of the suspension sampled at regular intervals using UV/Vis absorption spectroscopy (SpectraMax M5; Molecular Devices, San Jose, CA, USA). Controls, including the reaction mixtures containing the solvent plus Au(III) or tetraterpenoids alone, were measured simultaneously for comparison.

Characterization of DX-AuNPs. All suspensions containing DX-AuNPs were filtered using a 0.22 µm syringe filter. The suspensions were dialyzed repeatedly using a cellulose tube (Molecular Weight cutoff 14,000 Da) against 1 L of ultrapure water and ethanol for 48 h at 25°C to remove unreacted molecules and ions, if necessary. The suspension was subsequently lyophilized. The morphology, distribution, and size of the prepared DX-AuNPs were identified by transmission electron microscopy (TEM; Hitachi Model H-7650; Hitachi, Tokyo, Japan), scanning electron microscopy (SEM; Hitachi Model SU8010) and high5 ACS Paragon Plus Environment


resolution TEM (HR-TEM; Tecnai G2 F20 S-TWIN; FEI Co., Hillsboro, Oregon, USA) as described previously.23,24 The elemental analysis of samples was performed using Energydispersive X-ray spectroscopy (EDS). An X-ray diffractometer (X'Pert PRO; PANalytical Ltd., Almelo, Netherlands) with Cu Kα1 via radiation at a wavelength (λ) =1.540 Å was used to measure the XRD patterns of the prepared DX-AuNPs. A laser Doppler anemometer (Zetasizer Nano ZS; Malvern Instruments, Malvern, UK) was used to measure the particle sizes and zeta potentials of AuNPs at a wavelength of 632.8 nm using a He-Ne laser beam at 25°C. A 150 mV electric field was used to monitor the electrophoretic velocity of DX-AuNPs.

Detection of functional groups, surface-bound forms of DX and Au transient speciation during DX-AuNP formation. For the FTIR analysis of lyophilized DX-AuNPs, a pressed pellet composed of KBr and DX-AuNPs crushed in a mortar at a ratio of 100:1 was covered with a clip and straightway analyzed in the 400-4,000 cm−1 region using a Nicolet5700 FTIR spectrometer (Thermo Fisher Scientific Co., Waltham, MA, USA). For the FTIR analysis of the solution samples, the solutions containing tetraterpenoids in the presence or absence of Au(III) after an initial reaction time of 1 min were analyzed by attenuated total reflection (ATR)-FTIR with the appropriate calibration.25 Samples for NMR analysis were prepared by dissolving DX (20 mg) or DX (20 mg) plus chloroauric acid (40 mg) in solutions of DMSO-d6 and D2O (1:1, v/v). The NMR spectra of DX, DX plus Au(III), and the synthesized DX-AuNPs were acquired with a standard 5 mm probe on a 400 MHz Bruker 400 spectrometer (Bruker Biospin Corp., Billerica, MA, USA). Chemical shifts were calibrated to the residual solvent peaks. DX-AuNPs prepared by lyophilization were mounted onto a stainless steel holder, and a high-performance X-ray photoelectron spectrometer (Escalab 250Xi; Thermo Fisher Scientific Co.) was used for XPS analysis with monochromatic Al Kα radiation at an energy 6 ACS Paragon Plus Environment

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of 1486.6 eV. The C1s peak at 284.8 eV was regarded as the charge reference to determine the core-level binding energies.26 Oxidized forms of DX and products formed during the synthesis of DX-AuNPs were characterized using an Agilent 6460 LC-MS instrument equipped with a triple quadrupole mass spectrometer (Agilent, Santa Rosa, CA, USA).6 The filtered samples containing DX, intermediates, or DX-AuNPs were separated by HPLC using a Zorbax SB C8 column (3.5 µm, 150 mm × 2.1 mm). The 9-min gradient elution consisted of 0.1% formic acid solution (v/v) and acetonitrile (with a gradient proportion of 15% during the first 0.5 min, 15%–45% from 0.5–5.0 min, 45%–60% from 5.0–6.0 min, 60%–15% from 6.0–6.5 min, and 15%–30% from 6.5–9.0 min for acetonitrile). The flow rate and temperature were 0.4 mL/min and 35°C, respectively. MS analysis performed with an ESI interface in negative-ion mode at a capillary voltage of 3000 V was performed with a gas temperature of 325°C and a gas flow rate of 5 L/min. All experiments were performed independently in triplicate, and representative data are presented.

Cell cultures and evaluation of the anticancer activity of DX-AuNPs. The human breast adenocarcinoma cell line MCF-7, human kidney adenocarcinoma cell line ACHN and normal rat kidney cell line NRK were purchased commercially from CTCC BIOSCIENCE, Inc. (Wuxi, China) and cultured in regular growth medium composed of highglucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin under a humidified environment of 95% O2, 5% CO2, and 37°C. Cells seeded onto 96-well plates (5×103 cells/well) and maintained in an incubator overnight were used in an antiproliferative assay. The cell culture medium was replaced with



100 µL fresh complete medium containing the indicated concentrations of AuNPs including DX-AuNPs and SC-AuNPs (0, 2.5, 5, 25, and 50 µg/mL in medium). Subsequently, all cells were incubated identically for 24 h. Cell viability was determined using CellTiter 96® AQueous One Solution Reagent. The absorbance of the cell suspension was read at 490 nm. The proliferation rate of untreated cells, which were used as a control, was set to 100%. Reactive oxygen species (ROS) production in cells was analyzed by fluorometric assays using the Fluorometric Intracellular ROS Kit (Red Fluorescence) following the manufacturer's instructions. ROS react with the cell-permeable fluorescent probe from the ROS assay kit, resulting in a red fluorescent product proportional to the ROS amount. For relative quantification of intracellular ROS production using a fluorescence spectrophotometer (SpectraMax M5),27,28 untreated MCF-7 cells and MCF-7 cells treated with 50 µg/mL AuNPs were incubated with the fluorescent probe at 37°C for 30 min in the dark. The cells were then washed twice with phosphate-buffered saline. The level of ROS in the cells was monitored by measuring the fluorescence intensity at λex=520/λem=605 nm. The fluorescence intensity of untreated cells was used as a control. For qualitative observation using a fluorescence microscope,29 the cells were treated by the same method as mentioned above, and the ROSinduced cellular fluorescence images were obtained using a Zeiss AXIO Observer A1 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a fluorescent light source and filter. DNA damage and apoptosis in MCF-7 cells were detected using a terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay with an In situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Briefly, the prepared cell samples treated with fixation solution including 4% paraformaldehyde were permeabilized with 0.1% Triton X-100. The TUNEL reaction mixture (50 µL) was added to the sample, and the slides were incubated in a humidified atmosphere for 60 min at 37°C in the dark. The samples were visualized using a fluorescence microscope 8 ACS Paragon Plus Environment

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(Zeiss AXIO Observer A1). The internalization of the DX-AuNPs in cells was observed by TEM, and the content of DX-AuNPs was analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES; Optima 8000DV; PerkinElmer Inc., Waltham, MA, USA).

Gene expression analysis. RNA from untreated MCF-7 cells and MCF-7 cells treated with 50 µg/mL AuNPs was extracted and purified. The Illumina Solexa high-throughput sequencing platform was used for transcriptomic sequencing. Data were processed using the R statistical programming language. We used fragments per kilobase of transcript per million mapped reads (FPKM) as the gene expression level to screen genes with significant expression changes.30 Two independent cell treatments were performed and served as biological replicates. The expression levels of some selected genes were also demonstrated by quantitative real-time PCR (qRT-PCR) analysis. Pathway and Gene Ontology (GO) enrichment

analyses

were

demonstrated

using

the

clusterProfiler

R

package

(http://www.bioconductor.org/biocLite.R). Protein-protein interactions were built using STRING database (https://string-db.org) and Cytoscape 3.6.1 (The Cytoscape Consortium, New York, USA).

RESULTS AND DISCUSSION Synthesis of AuNPs by tetraterpenoids. We purified the DX from D. radiodurans (Figure S1) and dissolved it in ethanol. Following incubation with 1 mM Au(III) (water: ethanol, 9: 1, v⁄ v), the red color of DX was increasingly changed to a purple color together with the observed absorption peak at 535 nm (Figure 1A–B), which corresponded to excitation of surface plasmon vibrations of AuNPs,26 demonstrating that DX-AuNPs were formed. Moreover, two hydroxylated tetraterpenoids (astaxanthin and lutein) reacted with 9 ACS Paragon Plus Environment


Au(III) and formed the corresponding AuNPs, while nonhydroxylated carotene (β-carotene) did not synthesize AuNPs (Figure S2A–C). We believe that only the hydroxylated tetraterpenoids mediated the synthesis of AuNPs. The reaction system consisting of DX and Au(III) promptly changed to colorless within approximately 20 s, indicating that an intermediate complex in the reaction system was quickly formed by electrostatic interaction in the process;31-33 after approximately 2 h, the color subsequently shifted to light purple with the formation of DX-AuNP, followed by the appearance of an unchanged purple color at incubation over 6 h (Figure 1A), implying the formation of relatively stable DX-AuNPs in aqueous solution,34 which were consistent with their varied absorbance profile (Figure 1B). The stabilized DX-AuNPs exhibited no significant changes in color or characteristic absorption with a slight metal release of 3.02% ± 0.28% in aqueous solution within three months. Because DX contains multiple hydroxyl group substitutions and exhibits stronger in vitro antioxidant activity than lutein and β-carotene, it was used for subsequent experiments in this study.


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Figure 1. Characterization of DX-AuNPs. Changes in color (A) and absorbance spectra (B) of the solution containing 1 mM Au(III) and 30 µg/mL DX. Controls including solvent plus Au(III) (lane 1) or DX alone (lane 2) were measured for comparison. Lanes 3-5 show the solution at incubation times of 20 s, 2 h and 6 h. (C) TEM and (D) HR-TEM images of DXAuNPs. Arrows indicate the surfaces of AuNPs capped by DX, and the rectangle indicates the assembled Au atom-array inside. (E) SEM image of DX-AuNPs. (F) SEM-EDS analysis of DX-AuNPs. Scale bars in the images indicate the corresponding lengths.

Characterization of DX-AuNPs. The morphology, distribution and size of the DXAuNPs were identified using electron microscopy. Monodisperse DX-AuNPs with spherical 11 ACS Paragon Plus Environment


or polygonal morphology were observed (Figure 1C–E). The spherical AuNPs consisted of Au atomic arrays capped with a layer of DX (Figure 1D and Figure S3), demonstrating a self-assembled nanoparticle structure.35 The average AuNP size was calculated using ImageJ to be 29.83 ± 4.18 nm. EDS, which focused on a selected area in the SEM image (Figure 1E), revealed the presence of characteristic peaks of Au along with trace peaks of C and O (Figure 1F). The strong Au peak near 2.12 keV is characteristic of AuNPs.36 The C and O peaks likely arose from the X-ray emissions of the hydrocarbon, carbonyl or hydroxyl groups of DX, indicating that DX acted as a stabilizing or capping agent on the AuNPs. The crystallite nature of the purified DX-AuNPs was determined by X-ray diffraction (XRD). The (111) lattice plane of the face-centered cubic Au structure, with a 2θ of 38.16°, was the main crystal orientation of the DX-AuNPs (Figure S4A). The nanoparticle size distribution (PSD) and zeta potential of the DX-AuNPs were measured using dynamic light scattering (DLS). The DX-AuNPs had a hydrodynamic particle diameter of 42.44 ± 0.59 nm with a polydispersity index (PDI) of 0.136±0.017, indicating that the nanoparticles were nearly monodisperse (Figure S4B). The differences between the particle sizes measured by DLS and TEM were ascribed to the formation of surface capping on the nanoparticles. The zeta potential value of −24.95 ± 0.38 mV at pH 7 indicated the presence of repulsive forces between the nanoparticles, which may have resulted from the deprotonation of –OH groups on the AuNPs. These repulsive forces explain the relative stability of the DX-AuNPs.

Synthetic mechanism of DX-AuNPs. The chemical groups of DX involved in the synthesis of AuNPs were analyzed by FTIR at different incubation times. The strong band at 3,320 cm−1 in the spectrum of pure DX was attributed to the stretching vibration of –OH groups (Figure 2A–B) due to molecular association and reactive hydrogen.37 In the spectra of DX incubated with Au(III) for 1 min (Figure 2A) and the synthesized DX-AuNPs obtained at


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6 h (Figure 2B), the –OH peaks shifted to 3,263 and 3,340 cm−1, respectively. For DX that initially reacted with Au(III), the bands at 1,408, 1,379, 1,128 and 1,109 cm−1, which correspond to the in-plane deformation vibration of –OH from C–OH and CH–OH along with C–O stretching vibration in the C–OH and CH–OH,38-40 shifted slightly to 1,403, 1,371, 1,122 and 1,105 cm−1 with changes in strength or shape, respectively. However, these bands were notably weak in the spectra of the DX-AuNPs. Additionally, the small band at 1,758 cm−1 (C=O stretching vibration for DX) shifted to 1,797 cm−1 along with a distinct peak shown in the nanoparticles, suggesting that additional keto groups were formed upon the synthesis of DX-AuNPs. These results indicate that the –OH groups present in DX are involved in the reduction of Au ions and the surface capping of the AuNPs. The contribution of the CH–OH hydroxyl groups of tetraterpenoids to the initial reaction with Au(III) was also detected by incubation of lutein with Au(III) (Figure S5). Based on these results, we speculated that the CH–OH and C–OH groups of DX participated in the reduction of Au(III) by hydrogen donation and the –OH groups were progressively oxidized by Au(III) and finally transformed into C=O or C–O groups. This result is supported by previous reports that the –OH groups from curcumin or β-CD (β-cyclodextrin) are involved in the synthesis of AuNPs.8,41,42 Moreover, the intense bands at 1,658 cm−1 (C=C stretching vibration) showed little change or shift in the synthetic process (Figure 2A–B), indicating that the C=C system in tetraterpenoids was not responsible for the synthesis of AuNPs. Therefore, the hydroxyl head groups, but not the hydrophobic conjugate double bond groups, of tetraterpenoids contributed to the efficient synthesis of AuNPs. To verify the roles of the –OH groups of DX in DX-AuNP formation, samples from the reaction were prepared and characterized using NMR.43 Changes in chemical shifts as a consequence of the chemical environment of the system during the synthetic process were monitored in the range from 1.70 to 3.70 ppm. The varied signals observed in the 1H NMR spectra were ascribed to the chemical groups that reacted with Au(III) in the DMSO-d6 and 13 ACS Paragon Plus Environment


D2O solution (1: 1, v/v) (Figure 2C). The peaks at approximately 2.56 ppm were attributed to the DMSO-d6 solvent.44,45 The calculated shifts at 2.08 and 1.89 ppm were assigned to H2C(3) linked to the carbonyl group of DX.13,44 The signal of HC(2) was detected at near 3.36 ppm with multiplicity.13 The signals of both H2C(3) and HC(2) decreased within the reaction process and disappeared upon the formation of DX-AuNPs. In addition, the shift at 2.99 ppm corresponding to 1H of O=C-CH2-C=O emerged following the reaction and increasingly enhanced, suggesting that CH-OH on the ring end of the DX was gradually oxidized into C=O during the synthesis of DX-AuNPs, consistent with the FTIR results.

Figure 2. FTIR and NMR analyses of DX-AuNPs. (A–B) FTIR analysis of DX incubated with Au(III) at the initial reaction time of 1 min (A) and synthesized DX-AuNPs obtained at 6 h (B). Pure DX without Au(III) treatment was used as a control. Dotted lines and arrows indicate the changes of the corresponding bands. (C) 1H NMR analyses of DX, reaction mixture of DX with Au(III) at approximately 1 h (DX + Au(III)), and synthesized AuNPs at 6 h (DX-AuNPs). Changes in chemical shifts as a consequence of the chemical environment of the solution were monitored.


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The speciation of Au during the transformation of Au(III) into DX-AuNPs and the functional groups on the nanoparticles were studied using XPS. The peaks of Au4f, Cl2p, C1s and O1s were detected in the spectra of the DX-AuNPs (Figure 3A). The fitted Au4f spectra were composed of three sets of doublet peaks representing Au(III), Au(I), and Au(0) (Figure 3B). The 4f spectrum corresponding to 4f7/2 and 4f5/2 at 83.9 and 87.6 eV, respectively, was assigned to Au(0). The shift of approximately 1.0 eV in the binding energy for the Au4f corelevel spectrum suggested that the DX-AuNPs were bound with capping agents through Au–O bonds, as reported previously.8,46,47 The asymmetric peaks at 84.6 and 88.3 eV were ascribed to Au(I), corresponding to the intermediate chemical state of Au during the reduction of Au(III). The peaks at 86.5 and 90.4 eV were attributed to the residual Au(III) in the system.48 The core-level C1s spectrum was resolved into three components related to the surface capping of AuNPs (Figure 3C). The carbon bound with carbon (C–C or –C=C–) or hydrogen (C–H) had a binding energy at 284.6 eV. The binding energies located at 286.2 and 288.1 eV were attributed to carbon bound with oxygen (C–O and C=O). The O1s core levels presented at approximately 532.3 and 532.7 eV were assigned to the O–C and O=C groups, respectively (Figure 3D). The core-level spectra of C1s and O1s provided further evidence that the hydroxyl groups of DX (C–OH or CH–OH) could supply reduction and capping sites for DXAuNP formation through hydrogen donation and oxidation into C–O or C=O groups, consistent with the FTIR and NMR results. These results demonstrated that Au(III) were first reduced to Au(I) and subsequently reduced to Au(0), and stable DX-AuNP formation was achieved by capping with C–O or C=O from oxidized hydroxyl groups of DX.



Figure 3. XPS spectra of the prepared DX-AuNPs (A) and the core-level spectra of Au4f (B), C1s (C), and O1s (D).

In the reduction process of Au(III) into AuNPs, DX acted as a reducing and capping agent and could be converted into its oxidized forms. To confirm the presence of the oxidized forms and surface-bound states of DX on the AuNPs, the prepared samples were characterized by LC-ESI-MS. The identified dominant peaks are shown in Figure 4. The peak with a molecular ion at m/z 581.7 [DX – H]− was identified as DX, with a molecular mass of 582.13 The peaks at m/z 580.1 [DX1 – H]−, m/z 579.2 [DX2 – H]− and m/z 578.2 [DX3 – H]− corresponded to three oxidized forms of DX (Figure 4A). Among these, the dominant peak at m/z 580.1 was ascribed to DX1, with one hydrogen donated from the hydroxyl group at HC(2) of the ring end during the reduction of Au(III) by DX. The peak at m/z 579.2 corresponded to DX2, which was generated from the donation of two hydrogens from the hydroxyl group at 16 ACS Paragon Plus Environment

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HC(2) of the ring and the 1′-hydroxyl group at the chain end of DX. The peak at m/z 578.2 was assigned to the final oxidized product DX3 (deprotonated 2-ketodeinoxanthin), with three donated hydrogens (two from the HC(2)-hydroxyl group with the formation of C=O and one from the 1′-hydroxyl group). A product with a molecular ion of m/z 1366 [DX3 + 4Au – H]− (Figure 4B), matching the formula Au4C40H51O3, was identified and tentatively assigned as a DX-capped AuNP. This result indicates that a primary AuNP was composed of four Au(0) atoms capped by one DX3 through an Au–O bond. With increasing reaction time, the assembled AuNPs formed via the coalescence of DX-AuNP (Figure 1D), indicating that a relatively stable nanoparticle form was achieved.49-52 Moreover, LC-MS analysis of the forms of DX during the reduction process within 0.5 h identified an intermediate product (m/z 776.6, [DX2 + Au(I) – H]−), corresponding to a complex of DX2 (m/z 579.5) and Au(I) (Figure S6A). In further reduction of Au(I) to Au(0), DX was ultimately oxidized to DX3 (Figure S6B). The standard oxidation potential of alcoholic hydroxyl in the ketone/aldehyde system (EθROH/RCO/RCHO=1.80 V) is higher than the reduction potential of gold (EθAu(III)/Au(0)=1.50 V and Eθ(Au(I)/Au(0)=1.65 V).48 Therefore, the hydroxyl groups of DX have the capacity to reduce Au(III) to Au(I) and Au(0). Our current study suggested that the CH–OH of DX acted as the major reducing group for Au ions and was gradually oxidized into C=O. Subsequently, the DX3 was bound to the surface of AuNPs through the Au–O bond. Li et al. demonstrated that the length of the hydrophobic alkyl groups of the thioether lipids was a key parameter for their functionalized nanomaterials than the nature of the cationic head groups.53 Our findings revealed that it is the deprotonation of the hydroxyl head groups of the tetraterpenoid, but not their hydrophobic conjugate double bond groups, that contributes to the efficient synthesis of AuNPs.



Figure 4. LC-MS analyses of samples during the synthesis of DX-AuNPs. (A) Assignments of oxidized forms of DX at a retention time of 9.961 min and m/z of molecular ions from the reaction system containing 1 mM Au(III) and abundant DX (0.45 mg/mL). (B) Assignment of the DX-capped AuNP at a retention time of 12.836 min and m/z 1366 of molecular ions [DX3 + 4Au(0) – H]− matching the formula Au4C40H51O3. All components were detected at 480 nm by LC and identified by MS.

A schematic of the proposed synthetic process of DX-AuNPs is shown in Figure 5. First, DX interacted expeditiously with Au(III), which was reduced to Au(I) by the donation of hydrogens from the hydroxyl groups in the ring and chain end of DX, and the intermediate complex [DX2 + Au(I)] was formed. Subsequently, Au(I) was further reduced to Au(0) by the 18 ACS Paragon Plus Environment

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intermediates of DX (deprotonated DXs), leading to the formation of DX3 (deprotonated 2ketodeinoxanthin) and DX-AuNP, as suggested by the detection of AuNP nuclei capped by DX3 [DX3 + 4Au(0)]. Finally, relatively stable DX-AuNPs consisting of Au atomic arrays capped with a layer of DX were formed, as demonstrated in Figure 1D.

Figure 5. Proposed schematic mechanism of DX-AuNP synthesis using DX. Numbers 1-4 in the upper panel indicate the steps of DX-AuNP formation with changes in the DX and Au species: 1) DX is incubated with Au(III); 2) Au(III) is reduced to Au(I) by DX, leading to the formation of the complex [DX2 + Au(I)]; 3) Au(I) is further reduced to Au(0), leading to the formation of AuNP nuclei capped by DX3 [DX3 + 4Au(0)]; 4) Stable DX-AuNPs are formed from the assembled Au atom-array and capped with DX3. The structures of DX and its oxidized forms are indicated in the lower panel. 19 ACS Paragon Plus Environment


Anticancer activity of DX-AuNPs. AuNPs can accumulate in tumor tissues and be used in cancer theranostics.54 The inhibitory activity of DX-AuNPs against human cancer cells was investigated using the MTS assay. As illustrated in Figure 6A, the DX-AuNPs inhibited the cell viability of the human breast adenocarcinoma cell line MCF-7 in a dosedependent manner (2.5, 5, 25, and 50 µg/mL), with a half-maximal inhibitory concentration (IC50) of approximately 50 µg/mL. In contrast, the commercial AuNPs synthesized by sodium citrate (SC-AuNPs) had no obvious inhibitory effect on the cancer cells.51 The different cell viabilities of the kidney adenocarcinoma cell line ACHN treated with these nanoparticles also confirmed the higher anticancer activity of the DX-AuNPs compared to SC-AuNPs. However, the DX-AuNPs had no significant cytotoxicity on the normal cell line, NRK (Figure S7). The enhanced inhibitory activity of the DX-AuNPs against these cancer cells was primarily attributed to the effect of the surface capping of DX on AuNPs. Compared to the DX,21 the IC50 of the DX-AuNPs was lower, indicating that the anticancer activity of DX-AuNPs was stronger than DX. DX-AuNPs exhibit great potential as an effective agent against cancer cells.


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Figure 6. Anticancer activity of AuNPs. (A) Cell viability of MCF-7 cells treated with DXAuNPs or SC-AuNPs. The cell viability of untreated cells, which were used as a control, was set to 100%. Asterisks indicate significance (P

Gold Nanoparticles Biosynthesized and Functionalized Using a

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