Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation Su Jung Hwang MS, Sang Hui Jun MS, Yohan Park PhD, Song-Hyun Cha MS, Minho Yoon PhD, Seonho Cho PhD, Hyo-Jong Lee PhD, Youmie Park PhD PII: DOI: Reference:
S1549-9634(15)00113-6 doi: 10.1016/j.nano.2015.05.002 NANO 1129
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
5 December 2014 30 March 2015 2 May 2015
Please cite this article as: Hwang Su Jung, Jun Sang Hui, Park Yohan, Cha Song-Hyun, Yoon Minho, Cho Seonho, Lee Hyo-Jong, Park Youmie, Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation, Nanomedicine: Nanotechnology, Biology, and Medicine (2015), doi: 10.1016/j.nano.2015.05.002
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Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation Su Jung Hwang, MSa,† Sang Hui Jun, MSa,† Yohan Park, PhDa,b,c,† Song-Hyun Cha, MSd
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Minho Yoon, PhDd Seonho Cho, PhDd Hyo-Jong Lee, PhDa,b,c,* and Youmie Park, PhDa,b,c,d,* a
College of Pharmacy, Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621-749, Republic of Korea
b
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u-Healthcare & Anti-aging Reearch Center (u-HARC), Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621-749, Republic of Korea c
Biohealth Products Research Center (BPRC), Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621749, Republic of Korea d
National Creative Research Initiatives (NCRI) Center for Isogeometric Optimal Design, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea
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Correspondence to: Youmie Park, professor, Ph. D, email: [email protected];
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phone: +82-55-320-3884; fax: +82-55-320-3940; Hyo-Jong Lee, professor, Ph. D, email: [email protected]; phone: +82-55-320-3458; fax: +82-55-320-3940.
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Author Contributions
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†These authors contributed equally to this work. Financial support: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Government, the Ministry of Education (2012R1A1A2042224) and the Ministry of Science, ICT & Future Planning (2013R1A1A1059709 and 2010-18282).
Word count: 151 for the abstract, 5294 for a complete manuscript Number of references: 30 Number of figures/tables: 8/1 1
ACCEPTED MANUSCRIPT Abstract
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Here we developed a novel green synthesis method for gold nanoparticles (CGA-
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AuNPs) using chlorogenic acid (CGA) as reductants without the use of other chemicals and validated the anti-inflammatory efficacy of CGA-AuNPs in vitro and in
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vivo. The resulting CGA-AuNPs appeared predominantly spherical in shape with an average diameter of 22.25 ± 4.78 nm. The crystalline nature of the CGA-AuNPs was
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confirmed by high-resolution X-ray diffraction and by selected-area electron diffraction analyses. High-resolution liquid chromatography/electrospray ionization mass spectrometry revealed that the caffeic acid moiety of CGA forms the quinone structure through a two-electron oxidation causing the reduction of Au3+ to Au0.
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When compared to CGA, CGA-AuNPs exhibited enhanced anti-inflammatory effects
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on NF-κB-mediated inflammatory network, as well as cell adhesion. Taken together, green synthesis of CGA-AuNPs using bioactive reductants and mechanistic studies
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based on mass spectrometry may open up new directions in nanomedicine and
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CGA-AuNPs can be an anti-inflammatory nanomedicine for future applications.
KEYWORDS
chlorogenic acid; gold nanoparticles; green synthesis; inflammation; NF-κB
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ACCEPTED MANUSCRIPT Background
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With recent advancements in nanotechnology, nanoscale materials have attracted
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considerable attention because they exhibit unique properties compared to their bulk counterparts. Among metal nanoparticles, gold nanoparticles (AuNPs) have
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emerged as an important tool for diverse applications1-3. The functionalization or bioconjugation of biologically active molecules onto the surface of AuNPs has been
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widely used to engineer biocompatible AuNPs for the diagnosis and treatment of diseases4,5. The most common synthetic method employs a chemical route utilizing chemical reductants to convert Au ions to AuNPs6,7.
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As an alternative to chemical reductants, green reductants have attracted much attention because of their potential to help realize current sustainability initiatives.
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Green reductants include diverse types of biological entities8-10. The green synthesis
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of AuNPs offers many advantages, including increased biocompatibility, convenient scale-up, and straightforward reaction procedures. Furthermore, the combination of
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AuNPs and green reductants may possibly result in synergistic biological activities. Among naturally occurring bioactive compounds, chlorogenic acid (CGA) is a polyphenol compound in plants including green coffee beans. CGA exhibits various functions, such as anti-oxidant, anti-diabetic and anti-tumorigenic effects11,12. Previously, we also reported that CGA inhibits endotoxin-induced inflammation in Raw 264.7 macrophages and mouse retinal inflammation models through the downregulation of the NF-κB pathway, which activates the transcription of numerous target genes, such as pro-inflammatory cytokine and adhesion molecules13. Furthermore, AuNPs (diameter, d = 10 ~ 15 nm) synthesized using sodium citrate 3
ACCEPTED MANUSCRIPT inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by blocking the activation of NF-κB and signal transducer and activator of
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transcription1 (STAT1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells14,
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and a single dose of AuNPs (diameter, d = 21 nm) reduces tumor necrosis factors (TNF)-α and interleukin 6 (IL-6) mRNA levels with no measurable organ or cell
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toxicity in mice15. These findings prompted us to study the potential of CGA as a
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green reductant for producing AuNPs, the characteristics exhibited by the resulting CGA-AuNPs, and the possibility of CGA-AuNPs exhibiting synergistic activity against inflammation.
In the present study, CGA was used as a reductant for the green synthesis of
the
CGA-AuNPs,
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CGA-AuNPs. Spectroscopic and microscopic techniques were used to characterize including
UV-Visible
spectrophotometry,
high-resolution
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transmission electron microscopy (HR-TEM), atomic force microscopy (AFM), field
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emission scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and inductively coupled plasma mass
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spectrometry (ICP-MS). Furthermore, the reduction mechanism of Au3+ to AuNPs was elucidated by characterizing the oxidized form of caffeic acid in CGA using highresolution liquid chromatography electrospray ionization mass spectrometry (HR-LCESI-MS). To verify the activity of CGA-AuNPs, we investigated the anti-inflammatory effects of CGA-AuNPs in LPS-inflamed murine Raw 264.7 macrophage cells and mouse retinal inflammation models. Our findings suggest that CGA-AuNPs may be potential candidates for use in the treatment of many inflammatory diseases, warranting further clinical studies to test whether they can effectively inhibit inflammation. 4
ACCEPTED MANUSCRIPT Methods Reagents and cells
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Raw 264.7 cells, widely used murine macrophages, were obtained from the Korean
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Cell Line Bank (KCLB) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and
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penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco BRL). Lipopolysaccharide
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(LPS), CGA, Griess reagent, caffeic acid, (-)-quinic acid, and hydrochloroauric acid trihydrate (HAuCl4·3H2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.
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Instrument measurements
UV-Visible spectra were recorded on a Shimadzu UV-1800 or UV-2600 (Shimadzu
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Corporation, Kyoto, Japan). HR-TEM images were obtained on a JEM-3010 model
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(JEOL Ltd., Tokyo, Japan) operating at 300 kV. The samples were freeze-dried in an FD5505 freeze dryer (Il Shin Bio, Seoul, Korea) for FT-IR and XRD analyses. The
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freeze-dried CGA-AuNP samples were subjected to FT-IR analysis in a Varian 640 IR in attenuated total reflectance (ATR) mode (Agilent Technologies, Santa Clara, CA, USA). A Bruker D8 Discover high-resolution X-ray diffractometer using CuKα radiation (λ = 0.1541 nm) was used for XRD analysis. AFM images were obtained on a Dimension® Icon® instrument (Bruker Nano, Inc., Santa Barbara, CA, USA) in tapping mode. FE-SEM images were acquired on a JSM-7100F SEM operated at an accelerating voltage of 15 kV (JEOL, Tokyo, Japan). HR-LC-ESI-MS analyses were performed on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap instrument (Thermo Scientific, USA) with an ESI interface in the negative-ion mode. LC 5
ACCEPTED MANUSCRIPT separation was conducted on a Dionex Ultimate 3000 RSLCnano HPLC system with an INNO 10 column (ODS, 5-μm particle size, 2.0 mm in i.d. × 100 mm in length,
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Young Jin Biochrom, Republic of Korea). The injection volume and the flow rate
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were 10 μL/min and 150 μL/min, respectively. The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B).
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Gradient elution was performed as follows: 0~15 min (5~25% B), 15~17 min (25% B),
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17~18 min (25~5% B), and 18~20 min (5% B). For HR-LC-ESI-MS analyses, the sample solution was subjected to ultracentrifugation at 21,130 g for 1 h at 18°C (5424R centrifuge, Eppendorf AG, Hamburg, Germany), and the supernatant was taken for HR-LC-MS analysis. ICP-MS samples were analyzed using an ELAN 6100
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(Perkin-Elmer SCIEX, Waltham, MA, USA).
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Green synthesis of CGA-AuNPs
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For the green synthesis of the CGA-AuNPs, a CGA solution (0.25 mM in deionized water, 800 µL) was placed into a glass vial with a cap and heated to boil on a hot
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plate (set at 220°C) with stirring for 2 min. To this solution, a solution of hydrochloroauric acid trihydrate (1.0 mM in deionized water, 200 µL) was added dropwise and stirred on the hot plate for an additional 1 min. Then, the reaction mixture was further incubated in an 85°C oven for 12 h. Caffeic acid and (-)-quinic acid were used as reductants to synthesize the AuNPs through the same procedure described above for the preparation of CGA-AuNPs. For the preparation of the CAAuNPs (or (-)-quinic acid-AuNPs), a caffeic acid (or (-)-quinic acid) solution (0.25 mM in deionized water, 800 µL) was placed into a glass vial with a cap and heated to boil on a hot plate (set at 220°C) with stirring for 2 min. To this solution, a solution of 6
ACCEPTED MANUSCRIPT hydrochloroauric acid trihydrate (1.0 mM in deionized water, 200 µL) was added dropwise and stirred on the hot plate for an additional 1 min. Then, the reaction
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mixture was further incubated in an 85°C oven for 12 h. For the measurement of
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anti-inflammatory activities, the CGA-AuNPs were concentrated 25-fold under an N2
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atmosphere.
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Nitric oxide (NO) assay
A NO assay was performed as described previously13.
Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR
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RT-PCR and real-time PCR were performed using the same primers described previously13. Real-time PCR was conducted on a Rotor-Gene Q real-time PCR
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cycler (Qiagen) using the SYBR Green RT-PCR kit (Qiagen) per the manufacturer's
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instructions.
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Western blot analysis
Western blotting was performed as described previously13 with antibodies against iNOS, β-actin, NF-κB, I-κB and GAPDH (Santa Cruz Biotechnology).
Cell viability assay A cell viability assay was performed using CellTiter 96 ® Aqueous One Solution Reagent (Promega, Madison, WI) and trypan blue exclusion test as described previously13.
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ACCEPTED MANUSCRIPT Cell adhesion assays A cell adhesion assay was performed as described previously13. Attached cells were
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stained with crystal violet and washed twice. After lysis with 0.2% NP-40, the
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absorbance of the lysates was analyzed by ELISA at 590 nm.
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Induction of endotoxin shock
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An endotoxin-induced inflammation model was prepared as described previously13. C57BL/6 mice (purchased from Hyochang Bio., Republic of Korea) were injected three times intraperitoneally (i.p.) with a dose of 6.6 μg of LPS (330 μg/kg of body weight) and/or intravitreally with a dose of 0.1 mg of CGA-AuNPs (5 mg/kg body
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weight) over 3 days. The animals were sacrificed 24 h after LPS administration. The handling and care of the animals were approved by the Institutional Animal Care and
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Use Committee (IACUC) of Inje University in Gimhae, Republic of Korea (Inje-2012-
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20). All experiments were conducted with effort made to minimize the number of
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animals used and the suffering caused by the procedures used in the present study.
Immunofluorescent staining Immunofluorescent staining was performed as described previously13. Antibodies used for immunostaining were Ninj1 (1:500, a generous gift from Dr. K.W. Kim) and iNOS (1:500, BD Biosciences). Nuclei were stained using 4'-6-diamidino-2phenylindole (DAPI, Invitrogen). Images were obtained with an Axiovert M200 microscope (Zeiss).
Gelatin zymography 8
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Cellular uptake of AuNPs
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MMP-9 using 10-fold concentrated conditioned media as described previously13.
The amount of gold per cell was determined by ICP-MS and the number of AuNPs
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per cell was calculated as described previously16.
Data analysis and statistics
All data are presented as means ± S.D. and have been converted to relative percentages. Statistical comparisons between groups were performed using
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Student’s t-test. P < 0.05 was considered statistically significant.
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Results
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UV-Visible, FT-IR Spectra and HR-XRD Analysis of CGA-AuNPs
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CGA (1) is composed of two natural compounds, caffeic acid (2) and (-)-quinic acid (3), linked through an ester bond (Fig. 1A). When CGA was used as a reductant, the CGA-AuNPs turned pink in color and exhibited a characteristic surface plasmon resonance band with maximum absorbance at 545 nm (Fig. 1B, line ii), whereas no color change was observed in the solution without the reductant (Fig. 1B, line i). The FT-IR spectrum of CGA standard was described in our previous report17. As shown in Figure 1C, the synthetic process dramatically altered the FT-IR spectra. The shift in the band at 3421 cm-1 in CGA to 3213 cm-1 in the CGA-AuNPs indicated that an – OH functional group was likely involved in the synthesis of the AuNPs (Fig. 1C). The 9
ACCEPTED MANUSCRIPT intense diffraction peaks from the CGA-AuNPs represented the face-centered cubic structure of an atomic gold crystal, confirming the crystalline nature of the CGA-
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AuNPs (Fig. 1D). The peaks from the CGA-AuNPs appeared at 38.2°, 44.7°, 64.7°,
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and 77.3°, corresponding to the characteristic diffractions of the (111), (200), (220), and (311) planes, respectively. The standard intensity ratios of the (200)/(111) and
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(220)/(111) peaks are reported to be 0.52 and 0.33, respectively18; however, our
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experimental values were 0.42 and 0.26, lower than the standard values. These results demonstrated that the (111) lattice plane was the main crystal orientation of the CGA-AuNPs. Considering the (111) peak, the average particle size was estimated using the Debye-Scherrer equation (D = 0.89·λ / W·Cosθ), where λ is the
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wavelength of the X-ray source (0.1541 nm), W is the full width at half-maximum of the (111) peak in radians, θ is the diffraction angle, and D is the particle size. The
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size of the AuNPs was estimated by this equation to be 18.48 nm.
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HR-TEM, AFM and FE-SEM Images of CGA-AuNPs The obtained CGA-AuNPs were visualized by HR-TEM (Fig. 2) and appeared predominantly spherical in shape (Fig. 2A-D). The images revealed that the AuNPs were well dispersed without any agglomeration, suggesting that CGA acted as both a reductant and a stabilizing agent. HR-TEM images supported the stabilizing effect of CGA in capping the AuNPs (Fig. 2A-D). The thin organic layers were observed in HR-TEM images which clearly confirmed the capping of AuNPs with CGA (red arrows in Fig. 2C-D). We previously observed similar capping of AuNPs when catechins were used as a reductant to synthesize the AuNPs19. The average 10
ACCEPTED MANUSCRIPT diameter was determined to be 22.25 ± 4.78 nm based on the measurement of 111 discrete AuNPs in the HR-TEM images (Fig. 2E). This result agrees with the
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diameter (18.48 nm) estimated using the Debye-Scherrer equation. As shown in
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Figure 2F, the bright circular spots in the selected-area electron diffraction (SAED) pattern also reflect the crystalline nature of the AuNPs. The 2- and 3-dimensional
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morphologies of the CGA-AuNPs were further visualized by AFM and FE-SEM (Fig.
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3), revealing primarily spherical shapes. In the 3-dimensional height image (Fig. 3A), the bright contrast indicates AuNPs with large heights. Section analysis was performed, the result of which are depicted in Figure 3E. Two AuNPs (lines a-b and c-d) in Figure 3D had heights of 24.62 nm and 19.48 nm. Moreover, the CGA-
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AuNPs displayed no aggregation or agglomeration in the FE-SEM image (Fig. 3F), suggesting that CGA acted as a stabilizing agent. In addition, the FE-SEM image
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further confirmed that the shape of the CGA-AuNPs was primarily spherical, in
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accord with the HR-TEM and AFM images.
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Mechanistic Study by HR-LC-ESI-MS for the Formation of CGA-AuNPs During the reduction of Au3+ in the synthesis of the AuNPs, CGA acted as a reductant, suggesting that CGA was converted to its oxidized form. The purification and characterization of the surface-bound, oxidized form following the synthesis of AuNPs have been attempted by other researchers using various spectroscopic techniques20; however, they have been unable to generate any meaningful results. This inability may be a result of the very low concentration of the oxidized form and its surface-bound state. We hypothesized that the majority of the oxidized CGA would be bound to the surface of the AuNPs; however, there was a possibility that 11
ACCEPTED MANUSCRIPT some oxidized CGA would remain in the supernatant after the ultracentrifugation of the CGA-AuNPs. Therefore, we collected the supernatant and analyzed it using FT13
C-NMR and HR-LC-ESI-MS. Unfortunately, these attempts were unable to
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identify the oxidized form of CGA from the spectral data.
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IR,
CGA (1) can be cleaved into two natural compounds, caffeic acid (2) and (-)-quinic
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acid (3), by the hydrolysis of an ester bond (Fig. 1A). To determine which moiety in 1
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is essential to the synthesis of AuNPs, the synthesis was also performed using either 2 or 3 alone as the reductant. Interestingly, the AuNPs were successfully synthesized using caffeic acid (2) (Fig. 1B, line iii) but not using (-)-quinic acid (3) (Fig. 1B, line iv). When caffeic acid (2) was used, the color of the solution turned
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purple upon synthesis of the AuNPs, whereas no color change was observed when using (-)-quinic acid (3). These results clearly indicated that the (-)-quinic acid (3)
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moiety in CGA (1) did not have a direct effect on the synthesis of the AuNPs. To
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confirm the presence of the oxidized product 6 (Fig. 1E), the supernatant of the AuNPs synthesized using compound 2 as a reductant was characterized using HR-
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LC-ESI-MS. Peaks eluted at 1.06 min and the interpretation of each peak are summarized in Table 1. The peaks were identified at m/z 177.0185 [the oxidized product 6 - H]- and m/z 377.0862 [caffeic acid (2) + caffeic acid (2) + H2O - H](Supplementary Fig. S1). Remarkably, the observed peak at m/z 177.0185 was identified as the oxidized product 6 , in which two hydrogens were lost from the caffeic acid moiety (2). Although the intensity of this peak was very low, the error in the observed peak was 1.1 ppm relative to the theoretical mass, further confirming the generation of oxidized product 6.
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ACCEPTED MANUSCRIPT CGA-AuNPs Suppresses iNOS Protein Levels in Stimulated Macrophages
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Next, we investigated the effects of CGA-AuNPs on NO production and iNOS
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expression and compared to that of CGA. CGA-AuNPs significantly inhibited LPSinduced NO production in a dose-dependent manner (Fig. 4A and Supplementary
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Fig. S2). Additionally, the inhibitory effect of CGA-AuNPs on NO production was stronger than that of CGA at the same concentration. To examine whether the
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inhibitory effect of CGA-AuNPs on NO production resulted from toxicity, we performed cellular uptake, toxicity test and organ toxicity test. The data show that CGA-AuNPs were endocytosed about 61.5% (Supplementary Fig. S3) and had no effect on cell viability up to a concentration of 20 μM in Raw 264.7 cells and
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zebrafish larva (Fig. 4B, Supplementary Fig. S4 and S5). These results suggest
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that CGA-AuNPs exhibit an inhibitory effect on NO production without cell toxicity. Next, we examined the effects of CGA-AuNPs on LPS-induced iNOS gene
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expression in Raw 264.7 cells. LPS-induced iNOS mRNA (Supplementary Fig. S6,
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top, and S7) and protein (Supplementary Fig. S6, bottom) expression were also significantly attenuated in a dose-dependent manner by CGA-AuNPs. Moreover, immunofluorescence staining indicated that LPS-induced iNOS upregulation was abolished by treatment with CGA-AuNPs (Fig. 4C). These results confirm that CGAAuNPs have an inhibitory effect on iNOS induction in Raw 264.7 cells.
CGA-AuNPs Inhibit Expression of Pro-inflammatory Cytokines via NF-κB We also examined the effects of CGA-AuNPs on the expression of proinflammatory cytokines, such as IL-1β and TNF-α, as well as other inflammation13
ACCEPTED MANUSCRIPT related genes, such as COX-2 and IL-6. The LPS-induced expression of proinflammatory cytokines and other inflammation-related genes was significantly
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inhibited by CGA-AuNPs (Fig. 5). Taken together, these results suggest that CGA-
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AuNPs might reduce the expression of pro-inflammatory cytokines and chemokines. Because the activation of NF-κB by LPS can induce the expression of pro-
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inflammatory mediators5, and both CGA and AuNPs are known to suppress the
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nuclear translocation of NF-κB8, we verified the effect of CGA-AuNPs on the NF-κB signaling pathway under LPS-induced inflammation. As expected, the marked decrease in IκB proteins elicited by LPS was notably impaired in the presence of CGA-AuNPs (Fig. 5B, top). Moreover, significant nuclear accumulation of NF-κB
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was observed in cells treated with LPS, and CGA-AuNP treatment significantly attenuated the observed nuclear translocation (Fig. 5B, bottom). Additionally, the
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inhibitory effect of CGA-AuNPs on nuclear accumulation of NF-κB was stronger than
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that of CGA at the same concentration. Taken together, these results indicate that the inhibition of the NF-κB signaling pathway by CGA-AuNPs may be the mechanism
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responsible for the suppression of NO and pro-inflammatory cytokines in LPSstimulated murine macrophages.
CGA-AuNPs Suppress Expression of Ninjurin1 and the Adhesion of Macrophages Adhesion molecules are essential for a recognition system between leukocytes and other cells or cellular matrix proteins during inflammation 21. Ninjurin 1 (Ninj1), an adhesion molecule, is known to increase movement to the site of inflammation and the
activity
of
leukocytes
in
inflammatory 14
responses
and
developmental
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induced expression of Ninj1 mRNA and protein was also significantly attenuated in a
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dose-dependent manner by CGA-AuNPs (Fig. 6A and Supplementary Fig. S8). Moreover, immunofluorescence staining of Ninj1 indicated that CGA-AuNPs
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abolished Ninj1 upregulation mediated by LPS treatment in vitro (Supplementary
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Fig. S9). To investigate the effect of CGA-AuNPs on the activation of macrophages in vivo, we used an LPS-inflamed mouse model and examined the entry of activating Ninj1-expressing macrophages into the retina. In untreated mice, Ninj1-expressing macrophages were not observed around the retinal vessels (Fig. 6B). When the
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mice were treated with LPS, the number of Ninj1-expressing macrophages increased, which were blocked by CGA-AuNP treatment (Fig. 6B). Next, we
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examined the effect of CGA-AuNPs on the cell-to-matrix adhesion of Raw 264.7
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cells. Under LPS-induced inflammatory conditions, the adhesion of Raw 264.7 cells on gelatin, collagen type I, laminin, and fibronectin matrices increased compared to
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that observed for the control (Fig. 7). However, CGA-AuNPs abolished the LPSinduced adhesion activity of Raw 264.7 cells in a dose-dependent manner (Fig. 7). Thus, these results showed that CGA-AuNPs downregulated Ninj1 expression and had an inhibitory effect on the adhesion activity of Raw 264.7 cells.
CGA-AuNPs Inhibit Activation of MMPs in Stimulated Macrophages During leukocytes homing, leukocytes are recruited to the site of inflammation by the degradation of extracellular matrix (ECM), in which transendothelial migration is attributed to matrix metalloproteinases (MMPs). To determine the effect of CGA15
ACCEPTED MANUSCRIPT AuNPs on the activity of MMP-2 and MMP-9, which are major MMPs in macrophages, Raw 264.7 cells were cultured in the presence of different
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concentrations of CGA-AuNPs. In addition, the gelatinase activity of the conditioned
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media was measured by zymography. Under LPS-induced inflammatory conditions, the enzymatic activities of MMP-2 and MMP-9 were increased compared to the
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activity of the control, and CGA-AuNP treatment caused significant inhibition of the
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activity of MMP-2 and MMP-9 (Fig. 8). This inhibitory mechanism of CGA-AuNPs on MMP-2 and MMP-9 production may in part be attributed to the direct inhibition of the activation of NF-κB and the subsequent binding of NF-κB to the MMP-2 and MMP-9
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promoter.
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Discussion
CGA has been reported in various functions such as anti-oxidant, anti-aging, and
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anti-diabetic11,12. For instance, it slows the release of glucose into the blood, helping
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counter the accumulation of body fat caused by excess blood glucose and reduce the risk of diabetes. Recently, the anti-inflammatory effect of CGA have been reported in LPS-inflamed murine macrophage cells, mouse retinal inflammation model, carbon tetrachloride (CCl4)-induced liver fibrosis model, and LPS-inflamed keratinocytes13,24,25. For this reason, CGA has received much attention and those taking a natural product or preparation containing CGA are on the increase. Therefore, it is worthwhile to make CGA more effective forms in aspects of absorption, stability, or efficacy with no detectable toxicity to cells and vital organs (heart and vessel). Recently, there has been increased research interest in the use 16
ACCEPTED MANUSCRIPT of nanoscale Au in medical applications. The green synthesis of AuNPs using naturally occurring bioactive compounds as reductants offers many advantages over
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traditional chemical methods. These advantages include increased biocompatibility,
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reduced use of toxic chemicals and possible synergistic activities from the combination of nanomaterials with bioactive compounds. In the present report, we
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synthesized CGA-AuNPs using CGA as a green reductant. The synthesis of CGA-
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AuNPs was facile and the resulting NPs were obtained in aqueous dispersions. The formation of CGA-AuNPs was certainly detectable by eyes due to the appearance of pink color and NPs were completely characterized the particle size, shape and crystal nature by spectroscopic and microscopic techniques. The distinct differences
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of FT-IR spectra between CGA and CGA-AuNPs suggested that –OH functional groups of CGA were most likely involved in the reduction of HAuCl 4. This
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observation was well correlated with the mechanistic study where two –OH groups of
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caffeic acid (2) moiety in CGA (1) were oxidized during the synthesis. In addition, any noticeable aggregation or agglomeration of CGA-AuNPs was not observed with
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the 25-fold concentration under N2 atmosphere, suggesting that CGA-AuNPs possessed an excellent colloidal stability. Maintaining a good colloidal stability of NPs is considered to be a crucial factor for desired biological activities. In case of the silver nanoparticles (AgNPs), there is a correlation between the colloidal stability and enhanced antibacterial activities26. For examination of redox mechanism between HAuCl4 and CGA (1), we tried to figure out oxidation product of 1 by using IR, NMR, and MS, but all attempts did not give any meaningful results. Indeed, many previous mechanistic studies in the synthesis of metal NPs drew the same failures because oxidation products of 17
ACCEPTED MANUSCRIPT organic reductants existed as a small amount and are attached to inorganic metal NPs20. To solve the problem, we designed a strategic approach, structurally
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decomposing original reductant 1 into caffeic acid (2) and (-)-quinic acid (3) (Fig. 1A).
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By the strategy, we expected two things: 1) at least one of 2 and 3 has a reducing potential; 2) structurally simplified reductant could make it easy to detect its oxidation
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form. As a result of adding each commercially available 2 or 3 to aqueous HAuCl4, it
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was found that only 2 has a reducing ability to synthesize AuNPs (Fig. 1E). To confirm the exact structure, oxidation product of reductant 2 was characterized by using HR-LC-ESI-MS (Fig. S1). This result solidly supported that oxidation product of 2 is compound 6 in Fig. 1E. Also, based on the results with reductant 2, we
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suggested the oxidation mechanism of CGA (1) in Fig. 1E. Particularly, confirming oxidation form by MS is a valuable trial because oxidation products in the synthesis
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of metal NPs have been generally observed using IR and NMR, not MS 24,27,28. To
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our knowledge, this result is the first success in detection with MS. We believe that our strategy‒structurally decomposing original reductant, detecting oxdation form of
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simplified reductant with MS, and applying oxidation form to original reductant‒is a consecutive protocol to solve the mechanistic problem in the research field of metal NPs. Next, we compared anti-inflammatory efficacy between CGA and CGA-AuNPs in mouse Raw 264.7 macrophages and inflamed retinal models. Interestingly, CGAAuNPs showed the better anti-inflammatory effects than CGA in most aspects without toxicities. For example, the inhibitory effect of 5 μM CGA-AuNPs on NF-kB translocation and cell adhesion was stronger than that of 20 μM CGA. However, further studies are warranted to understand the mechanisms by which CGA 18
ACCEPTED MANUSCRIPT increases the anti-inflammatory effect of AuNPs and to clarify whether combination of AuNPs and CGA (as bioactive reductants) produces additive or synergistic effects
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or not.
The use of NPs for inflammation treatment is currently under intensive investigation
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and chemical, optical, and electromagnetic properties of NPs may be strongly influenced by their size and shape29. Ji Su Ma et al. synthesized AuNPs (diameter, d
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= 10 ~ 15 nm) using sodium citrate and showed that AuNPs block NO production and iNOS expression through the inhibition of NF-κB and STAT1 in LPS-stimulated RAW 264.7 cells14. Chen Hui et al. reported that spherical AuNPs of 21.3 ± 0.7 nm significantly reduces TNF-α and IL-6 mRNA levels with no measurable organ or cell
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toxicity in male C57BL/6 mice15. The authors utilized the spherical AuNPs prepared
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by the standard citrate-reduction route to evaluate the anti-inflammatory efficacy of AuNPs15. It is noteworthy that the preparation of CGA-AuNPs presented in the
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current report is unique from previous reports. We combined two anti-inflammatory
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agents, AuNPs and CGA, by a green synthetic method, which is an eco-friendly technology, to increase or reinforce anti-inflammatory activities of AuNPs. However, AgNPs of 24 nm exhibit cytotoxic properties to macrophages and trigger an inflammatory response, although they showed beneficial effect such as antibacterial activity30. From our studies and the studies described above, therefore, it is necessary and interesting to study further whether functionalization or the combination of NPs with green reductants, possessing desired biological activity, can decrease harmful effect and increase beneficial effect of NPs simultaneously or not.
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ACCEPTED MANUSCRIPT In the present report, CGA was successfully used to reduce Au 3+ to AuNPs. As the first success in mechanistic study by HR-LC-ESI-MS, it was confirmed that the
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caffeic acid moiety of CGA (1) was essential to reduce Au3+. CGA-AuNPs inhibited
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pro-inflammatory cytokines and other inflammation-related genes (including MMPs and Ninj1) through the inhibition of NF-κB nuclear translocation. Compared to that of
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CGA, the anti-inflammatory effect of CGA-AuNPs was much stronger, indicating that
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functionalization or the combination of AuNPs with green reductants, which are known to have therapeutic or preventive properties, can provide a new strategy for the development of novel anti-inflammatory agents. These results clearly warrant further clinical studies to ascertain the in vivo efficacy of CGA-AuNPs for
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inflammatory or infectious diseases treatment.
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ACCEPTED MANUSCRIPT Figures legends
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Figure 1 Spectroscopic analyses of CGA-AuNPs. (A) Structural characteristics of
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CGA (1). (B) UV-Visible spectra of the AuNPs synthesized with reducing agents 1–3 with i) no reductant, ii) CGA (1), iii) caffeic acid (2), and iv) (-)-quinic acid (3). The
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detailed reaction conditions for the synthesis of AuNPs are described in the experimental section. (C) FT-IR spectrum and (D) HR-XRD analysis. (E) Two-
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electron oxidation of gallic acid,19 caffeic acid, and CGA upon the synthesis of AuNPs
Figure 2 HR-TEM images of the CGA-AuNPs. The scale bar represents (A) 100 nm,
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(B) 50 nm, (C) 20 nm, and (D) 5 nm. (E) size histogram and f) SAED pattern. The organic thin layer was indicated by red arrows which represents the capping of
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AuNPs by CGA.
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Figure 3 AFM and FE-SEM images of the CGA-AuNPs. (A) 3-D height (2.5 µm × 2.5 µm), (B) 3-D amplitude error (2.5 µm × 2.5 µm), (C) 3-D phase (1.0 µm × 1.0 µm),
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(D) 2-D height (1.0 µm × 1.0 µm), (E) section analysis of two NPs (lines a-b and c-d) in image (D), and (F) FE-SEM image of the CGA-AuNPs. The magnification of (F) 45,000×. The scale bar represents 100 nm in (F). Figure 4 Effects of CGA-AuNPs on iNOS expression and cytotoxicity in LPSstimulated Raw 264.7 cells. (A) NO assay results obtained using the Griess reagent; **P
S1549-9634(15)00113-6 doi: 10.1016/j.nano.2015.05.002 NANO 1129
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
5 December 2014 30 March 2015 2 May 2015
Please cite this article as: Hwang Su Jung, Jun Sang Hui, Park Yohan, Cha Song-Hyun, Yoon Minho, Cho Seonho, Lee Hyo-Jong, Park Youmie, Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation, Nanomedicine: Nanotechnology, Biology, and Medicine (2015), doi: 10.1016/j.nano.2015.05.002
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Green synthesis of gold nanoparticles using chlorogenic acid and their enhanced performance for inflammation Su Jung Hwang, MSa,† Sang Hui Jun, MSa,† Yohan Park, PhDa,b,c,† Song-Hyun Cha, MSd
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Minho Yoon, PhDd Seonho Cho, PhDd Hyo-Jong Lee, PhDa,b,c,* and Youmie Park, PhDa,b,c,d,* a
College of Pharmacy, Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621-749, Republic of Korea
b
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u-Healthcare & Anti-aging Reearch Center (u-HARC), Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621-749, Republic of Korea c
Biohealth Products Research Center (BPRC), Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621749, Republic of Korea d
National Creative Research Initiatives (NCRI) Center for Isogeometric Optimal Design, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea
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Correspondence to: Youmie Park, professor, Ph. D, email: [email protected];
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phone: +82-55-320-3884; fax: +82-55-320-3940; Hyo-Jong Lee, professor, Ph. D, email: [email protected]; phone: +82-55-320-3458; fax: +82-55-320-3940.
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Author Contributions
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†These authors contributed equally to this work. Financial support: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Government, the Ministry of Education (2012R1A1A2042224) and the Ministry of Science, ICT & Future Planning (2013R1A1A1059709 and 2010-18282).
Word count: 151 for the abstract, 5294 for a complete manuscript Number of references: 30 Number of figures/tables: 8/1 1
ACCEPTED MANUSCRIPT Abstract
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Here we developed a novel green synthesis method for gold nanoparticles (CGA-
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AuNPs) using chlorogenic acid (CGA) as reductants without the use of other chemicals and validated the anti-inflammatory efficacy of CGA-AuNPs in vitro and in
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vivo. The resulting CGA-AuNPs appeared predominantly spherical in shape with an average diameter of 22.25 ± 4.78 nm. The crystalline nature of the CGA-AuNPs was
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confirmed by high-resolution X-ray diffraction and by selected-area electron diffraction analyses. High-resolution liquid chromatography/electrospray ionization mass spectrometry revealed that the caffeic acid moiety of CGA forms the quinone structure through a two-electron oxidation causing the reduction of Au3+ to Au0.
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When compared to CGA, CGA-AuNPs exhibited enhanced anti-inflammatory effects
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on NF-κB-mediated inflammatory network, as well as cell adhesion. Taken together, green synthesis of CGA-AuNPs using bioactive reductants and mechanistic studies
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based on mass spectrometry may open up new directions in nanomedicine and
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CGA-AuNPs can be an anti-inflammatory nanomedicine for future applications.
KEYWORDS
chlorogenic acid; gold nanoparticles; green synthesis; inflammation; NF-κB
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ACCEPTED MANUSCRIPT Background
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With recent advancements in nanotechnology, nanoscale materials have attracted
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considerable attention because they exhibit unique properties compared to their bulk counterparts. Among metal nanoparticles, gold nanoparticles (AuNPs) have
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emerged as an important tool for diverse applications1-3. The functionalization or bioconjugation of biologically active molecules onto the surface of AuNPs has been
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widely used to engineer biocompatible AuNPs for the diagnosis and treatment of diseases4,5. The most common synthetic method employs a chemical route utilizing chemical reductants to convert Au ions to AuNPs6,7.
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As an alternative to chemical reductants, green reductants have attracted much attention because of their potential to help realize current sustainability initiatives.
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Green reductants include diverse types of biological entities8-10. The green synthesis
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of AuNPs offers many advantages, including increased biocompatibility, convenient scale-up, and straightforward reaction procedures. Furthermore, the combination of
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AuNPs and green reductants may possibly result in synergistic biological activities. Among naturally occurring bioactive compounds, chlorogenic acid (CGA) is a polyphenol compound in plants including green coffee beans. CGA exhibits various functions, such as anti-oxidant, anti-diabetic and anti-tumorigenic effects11,12. Previously, we also reported that CGA inhibits endotoxin-induced inflammation in Raw 264.7 macrophages and mouse retinal inflammation models through the downregulation of the NF-κB pathway, which activates the transcription of numerous target genes, such as pro-inflammatory cytokine and adhesion molecules13. Furthermore, AuNPs (diameter, d = 10 ~ 15 nm) synthesized using sodium citrate 3
ACCEPTED MANUSCRIPT inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by blocking the activation of NF-κB and signal transducer and activator of
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transcription1 (STAT1) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells14,
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and a single dose of AuNPs (diameter, d = 21 nm) reduces tumor necrosis factors (TNF)-α and interleukin 6 (IL-6) mRNA levels with no measurable organ or cell
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toxicity in mice15. These findings prompted us to study the potential of CGA as a
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green reductant for producing AuNPs, the characteristics exhibited by the resulting CGA-AuNPs, and the possibility of CGA-AuNPs exhibiting synergistic activity against inflammation.
In the present study, CGA was used as a reductant for the green synthesis of
the
CGA-AuNPs,
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CGA-AuNPs. Spectroscopic and microscopic techniques were used to characterize including
UV-Visible
spectrophotometry,
high-resolution
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transmission electron microscopy (HR-TEM), atomic force microscopy (AFM), field
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emission scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and inductively coupled plasma mass
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spectrometry (ICP-MS). Furthermore, the reduction mechanism of Au3+ to AuNPs was elucidated by characterizing the oxidized form of caffeic acid in CGA using highresolution liquid chromatography electrospray ionization mass spectrometry (HR-LCESI-MS). To verify the activity of CGA-AuNPs, we investigated the anti-inflammatory effects of CGA-AuNPs in LPS-inflamed murine Raw 264.7 macrophage cells and mouse retinal inflammation models. Our findings suggest that CGA-AuNPs may be potential candidates for use in the treatment of many inflammatory diseases, warranting further clinical studies to test whether they can effectively inhibit inflammation. 4
ACCEPTED MANUSCRIPT Methods Reagents and cells
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Raw 264.7 cells, widely used murine macrophages, were obtained from the Korean
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Cell Line Bank (KCLB) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and
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penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco BRL). Lipopolysaccharide
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(LPS), CGA, Griess reagent, caffeic acid, (-)-quinic acid, and hydrochloroauric acid trihydrate (HAuCl4·3H2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.
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Instrument measurements
UV-Visible spectra were recorded on a Shimadzu UV-1800 or UV-2600 (Shimadzu
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Corporation, Kyoto, Japan). HR-TEM images were obtained on a JEM-3010 model
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(JEOL Ltd., Tokyo, Japan) operating at 300 kV. The samples were freeze-dried in an FD5505 freeze dryer (Il Shin Bio, Seoul, Korea) for FT-IR and XRD analyses. The
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freeze-dried CGA-AuNP samples were subjected to FT-IR analysis in a Varian 640 IR in attenuated total reflectance (ATR) mode (Agilent Technologies, Santa Clara, CA, USA). A Bruker D8 Discover high-resolution X-ray diffractometer using CuKα radiation (λ = 0.1541 nm) was used for XRD analysis. AFM images were obtained on a Dimension® Icon® instrument (Bruker Nano, Inc., Santa Barbara, CA, USA) in tapping mode. FE-SEM images were acquired on a JSM-7100F SEM operated at an accelerating voltage of 15 kV (JEOL, Tokyo, Japan). HR-LC-ESI-MS analyses were performed on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap instrument (Thermo Scientific, USA) with an ESI interface in the negative-ion mode. LC 5
ACCEPTED MANUSCRIPT separation was conducted on a Dionex Ultimate 3000 RSLCnano HPLC system with an INNO 10 column (ODS, 5-μm particle size, 2.0 mm in i.d. × 100 mm in length,
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Young Jin Biochrom, Republic of Korea). The injection volume and the flow rate
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were 10 μL/min and 150 μL/min, respectively. The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B).
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Gradient elution was performed as follows: 0~15 min (5~25% B), 15~17 min (25% B),
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17~18 min (25~5% B), and 18~20 min (5% B). For HR-LC-ESI-MS analyses, the sample solution was subjected to ultracentrifugation at 21,130 g for 1 h at 18°C (5424R centrifuge, Eppendorf AG, Hamburg, Germany), and the supernatant was taken for HR-LC-MS analysis. ICP-MS samples were analyzed using an ELAN 6100
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(Perkin-Elmer SCIEX, Waltham, MA, USA).
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Green synthesis of CGA-AuNPs
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For the green synthesis of the CGA-AuNPs, a CGA solution (0.25 mM in deionized water, 800 µL) was placed into a glass vial with a cap and heated to boil on a hot
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plate (set at 220°C) with stirring for 2 min. To this solution, a solution of hydrochloroauric acid trihydrate (1.0 mM in deionized water, 200 µL) was added dropwise and stirred on the hot plate for an additional 1 min. Then, the reaction mixture was further incubated in an 85°C oven for 12 h. Caffeic acid and (-)-quinic acid were used as reductants to synthesize the AuNPs through the same procedure described above for the preparation of CGA-AuNPs. For the preparation of the CAAuNPs (or (-)-quinic acid-AuNPs), a caffeic acid (or (-)-quinic acid) solution (0.25 mM in deionized water, 800 µL) was placed into a glass vial with a cap and heated to boil on a hot plate (set at 220°C) with stirring for 2 min. To this solution, a solution of 6
ACCEPTED MANUSCRIPT hydrochloroauric acid trihydrate (1.0 mM in deionized water, 200 µL) was added dropwise and stirred on the hot plate for an additional 1 min. Then, the reaction
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mixture was further incubated in an 85°C oven for 12 h. For the measurement of
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anti-inflammatory activities, the CGA-AuNPs were concentrated 25-fold under an N2
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atmosphere.
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Nitric oxide (NO) assay
A NO assay was performed as described previously13.
Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR
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RT-PCR and real-time PCR were performed using the same primers described previously13. Real-time PCR was conducted on a Rotor-Gene Q real-time PCR
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cycler (Qiagen) using the SYBR Green RT-PCR kit (Qiagen) per the manufacturer's
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instructions.
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Western blot analysis
Western blotting was performed as described previously13 with antibodies against iNOS, β-actin, NF-κB, I-κB and GAPDH (Santa Cruz Biotechnology).
Cell viability assay A cell viability assay was performed using CellTiter 96 ® Aqueous One Solution Reagent (Promega, Madison, WI) and trypan blue exclusion test as described previously13.
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ACCEPTED MANUSCRIPT Cell adhesion assays A cell adhesion assay was performed as described previously13. Attached cells were
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stained with crystal violet and washed twice. After lysis with 0.2% NP-40, the
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absorbance of the lysates was analyzed by ELISA at 590 nm.
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Induction of endotoxin shock
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An endotoxin-induced inflammation model was prepared as described previously13. C57BL/6 mice (purchased from Hyochang Bio., Republic of Korea) were injected three times intraperitoneally (i.p.) with a dose of 6.6 μg of LPS (330 μg/kg of body weight) and/or intravitreally with a dose of 0.1 mg of CGA-AuNPs (5 mg/kg body
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weight) over 3 days. The animals were sacrificed 24 h after LPS administration. The handling and care of the animals were approved by the Institutional Animal Care and
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Use Committee (IACUC) of Inje University in Gimhae, Republic of Korea (Inje-2012-
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20). All experiments were conducted with effort made to minimize the number of
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animals used and the suffering caused by the procedures used in the present study.
Immunofluorescent staining Immunofluorescent staining was performed as described previously13. Antibodies used for immunostaining were Ninj1 (1:500, a generous gift from Dr. K.W. Kim) and iNOS (1:500, BD Biosciences). Nuclei were stained using 4'-6-diamidino-2phenylindole (DAPI, Invitrogen). Images were obtained with an Axiovert M200 microscope (Zeiss).
Gelatin zymography 8
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Cellular uptake of AuNPs
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MMP-9 using 10-fold concentrated conditioned media as described previously13.
The amount of gold per cell was determined by ICP-MS and the number of AuNPs
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per cell was calculated as described previously16.
Data analysis and statistics
All data are presented as means ± S.D. and have been converted to relative percentages. Statistical comparisons between groups were performed using
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Student’s t-test. P < 0.05 was considered statistically significant.
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Results
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UV-Visible, FT-IR Spectra and HR-XRD Analysis of CGA-AuNPs
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CGA (1) is composed of two natural compounds, caffeic acid (2) and (-)-quinic acid (3), linked through an ester bond (Fig. 1A). When CGA was used as a reductant, the CGA-AuNPs turned pink in color and exhibited a characteristic surface plasmon resonance band with maximum absorbance at 545 nm (Fig. 1B, line ii), whereas no color change was observed in the solution without the reductant (Fig. 1B, line i). The FT-IR spectrum of CGA standard was described in our previous report17. As shown in Figure 1C, the synthetic process dramatically altered the FT-IR spectra. The shift in the band at 3421 cm-1 in CGA to 3213 cm-1 in the CGA-AuNPs indicated that an – OH functional group was likely involved in the synthesis of the AuNPs (Fig. 1C). The 9
ACCEPTED MANUSCRIPT intense diffraction peaks from the CGA-AuNPs represented the face-centered cubic structure of an atomic gold crystal, confirming the crystalline nature of the CGA-
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AuNPs (Fig. 1D). The peaks from the CGA-AuNPs appeared at 38.2°, 44.7°, 64.7°,
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and 77.3°, corresponding to the characteristic diffractions of the (111), (200), (220), and (311) planes, respectively. The standard intensity ratios of the (200)/(111) and
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(220)/(111) peaks are reported to be 0.52 and 0.33, respectively18; however, our
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experimental values were 0.42 and 0.26, lower than the standard values. These results demonstrated that the (111) lattice plane was the main crystal orientation of the CGA-AuNPs. Considering the (111) peak, the average particle size was estimated using the Debye-Scherrer equation (D = 0.89·λ / W·Cosθ), where λ is the
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wavelength of the X-ray source (0.1541 nm), W is the full width at half-maximum of the (111) peak in radians, θ is the diffraction angle, and D is the particle size. The
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size of the AuNPs was estimated by this equation to be 18.48 nm.
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HR-TEM, AFM and FE-SEM Images of CGA-AuNPs The obtained CGA-AuNPs were visualized by HR-TEM (Fig. 2) and appeared predominantly spherical in shape (Fig. 2A-D). The images revealed that the AuNPs were well dispersed without any agglomeration, suggesting that CGA acted as both a reductant and a stabilizing agent. HR-TEM images supported the stabilizing effect of CGA in capping the AuNPs (Fig. 2A-D). The thin organic layers were observed in HR-TEM images which clearly confirmed the capping of AuNPs with CGA (red arrows in Fig. 2C-D). We previously observed similar capping of AuNPs when catechins were used as a reductant to synthesize the AuNPs19. The average 10
ACCEPTED MANUSCRIPT diameter was determined to be 22.25 ± 4.78 nm based on the measurement of 111 discrete AuNPs in the HR-TEM images (Fig. 2E). This result agrees with the
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diameter (18.48 nm) estimated using the Debye-Scherrer equation. As shown in
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Figure 2F, the bright circular spots in the selected-area electron diffraction (SAED) pattern also reflect the crystalline nature of the AuNPs. The 2- and 3-dimensional
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morphologies of the CGA-AuNPs were further visualized by AFM and FE-SEM (Fig.
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3), revealing primarily spherical shapes. In the 3-dimensional height image (Fig. 3A), the bright contrast indicates AuNPs with large heights. Section analysis was performed, the result of which are depicted in Figure 3E. Two AuNPs (lines a-b and c-d) in Figure 3D had heights of 24.62 nm and 19.48 nm. Moreover, the CGA-
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AuNPs displayed no aggregation or agglomeration in the FE-SEM image (Fig. 3F), suggesting that CGA acted as a stabilizing agent. In addition, the FE-SEM image
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further confirmed that the shape of the CGA-AuNPs was primarily spherical, in
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accord with the HR-TEM and AFM images.
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Mechanistic Study by HR-LC-ESI-MS for the Formation of CGA-AuNPs During the reduction of Au3+ in the synthesis of the AuNPs, CGA acted as a reductant, suggesting that CGA was converted to its oxidized form. The purification and characterization of the surface-bound, oxidized form following the synthesis of AuNPs have been attempted by other researchers using various spectroscopic techniques20; however, they have been unable to generate any meaningful results. This inability may be a result of the very low concentration of the oxidized form and its surface-bound state. We hypothesized that the majority of the oxidized CGA would be bound to the surface of the AuNPs; however, there was a possibility that 11
ACCEPTED MANUSCRIPT some oxidized CGA would remain in the supernatant after the ultracentrifugation of the CGA-AuNPs. Therefore, we collected the supernatant and analyzed it using FT13
C-NMR and HR-LC-ESI-MS. Unfortunately, these attempts were unable to
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identify the oxidized form of CGA from the spectral data.
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IR,
CGA (1) can be cleaved into two natural compounds, caffeic acid (2) and (-)-quinic
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acid (3), by the hydrolysis of an ester bond (Fig. 1A). To determine which moiety in 1
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is essential to the synthesis of AuNPs, the synthesis was also performed using either 2 or 3 alone as the reductant. Interestingly, the AuNPs were successfully synthesized using caffeic acid (2) (Fig. 1B, line iii) but not using (-)-quinic acid (3) (Fig. 1B, line iv). When caffeic acid (2) was used, the color of the solution turned
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purple upon synthesis of the AuNPs, whereas no color change was observed when using (-)-quinic acid (3). These results clearly indicated that the (-)-quinic acid (3)
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moiety in CGA (1) did not have a direct effect on the synthesis of the AuNPs. To
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confirm the presence of the oxidized product 6 (Fig. 1E), the supernatant of the AuNPs synthesized using compound 2 as a reductant was characterized using HR-
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LC-ESI-MS. Peaks eluted at 1.06 min and the interpretation of each peak are summarized in Table 1. The peaks were identified at m/z 177.0185 [the oxidized product 6 - H]- and m/z 377.0862 [caffeic acid (2) + caffeic acid (2) + H2O - H](Supplementary Fig. S1). Remarkably, the observed peak at m/z 177.0185 was identified as the oxidized product 6 , in which two hydrogens were lost from the caffeic acid moiety (2). Although the intensity of this peak was very low, the error in the observed peak was 1.1 ppm relative to the theoretical mass, further confirming the generation of oxidized product 6.
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ACCEPTED MANUSCRIPT CGA-AuNPs Suppresses iNOS Protein Levels in Stimulated Macrophages
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Next, we investigated the effects of CGA-AuNPs on NO production and iNOS
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expression and compared to that of CGA. CGA-AuNPs significantly inhibited LPSinduced NO production in a dose-dependent manner (Fig. 4A and Supplementary
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Fig. S2). Additionally, the inhibitory effect of CGA-AuNPs on NO production was stronger than that of CGA at the same concentration. To examine whether the
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inhibitory effect of CGA-AuNPs on NO production resulted from toxicity, we performed cellular uptake, toxicity test and organ toxicity test. The data show that CGA-AuNPs were endocytosed about 61.5% (Supplementary Fig. S3) and had no effect on cell viability up to a concentration of 20 μM in Raw 264.7 cells and
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zebrafish larva (Fig. 4B, Supplementary Fig. S4 and S5). These results suggest
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that CGA-AuNPs exhibit an inhibitory effect on NO production without cell toxicity. Next, we examined the effects of CGA-AuNPs on LPS-induced iNOS gene
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expression in Raw 264.7 cells. LPS-induced iNOS mRNA (Supplementary Fig. S6,
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top, and S7) and protein (Supplementary Fig. S6, bottom) expression were also significantly attenuated in a dose-dependent manner by CGA-AuNPs. Moreover, immunofluorescence staining indicated that LPS-induced iNOS upregulation was abolished by treatment with CGA-AuNPs (Fig. 4C). These results confirm that CGAAuNPs have an inhibitory effect on iNOS induction in Raw 264.7 cells.
CGA-AuNPs Inhibit Expression of Pro-inflammatory Cytokines via NF-κB We also examined the effects of CGA-AuNPs on the expression of proinflammatory cytokines, such as IL-1β and TNF-α, as well as other inflammation13
ACCEPTED MANUSCRIPT related genes, such as COX-2 and IL-6. The LPS-induced expression of proinflammatory cytokines and other inflammation-related genes was significantly
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inhibited by CGA-AuNPs (Fig. 5). Taken together, these results suggest that CGA-
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AuNPs might reduce the expression of pro-inflammatory cytokines and chemokines. Because the activation of NF-κB by LPS can induce the expression of pro-
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inflammatory mediators5, and both CGA and AuNPs are known to suppress the
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nuclear translocation of NF-κB8, we verified the effect of CGA-AuNPs on the NF-κB signaling pathway under LPS-induced inflammation. As expected, the marked decrease in IκB proteins elicited by LPS was notably impaired in the presence of CGA-AuNPs (Fig. 5B, top). Moreover, significant nuclear accumulation of NF-κB
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was observed in cells treated with LPS, and CGA-AuNP treatment significantly attenuated the observed nuclear translocation (Fig. 5B, bottom). Additionally, the
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inhibitory effect of CGA-AuNPs on nuclear accumulation of NF-κB was stronger than
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that of CGA at the same concentration. Taken together, these results indicate that the inhibition of the NF-κB signaling pathway by CGA-AuNPs may be the mechanism
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responsible for the suppression of NO and pro-inflammatory cytokines in LPSstimulated murine macrophages.
CGA-AuNPs Suppress Expression of Ninjurin1 and the Adhesion of Macrophages Adhesion molecules are essential for a recognition system between leukocytes and other cells or cellular matrix proteins during inflammation 21. Ninjurin 1 (Ninj1), an adhesion molecule, is known to increase movement to the site of inflammation and the
activity
of
leukocytes
in
inflammatory 14
responses
and
developmental
ACCEPTED MANUSCRIPT processes22,23. Thus, we further investigated the effect of CGA-AuNPs on the in vitro and in vivo expression of Ninj1 and the adhesive activity of macrophages. LPS-
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induced expression of Ninj1 mRNA and protein was also significantly attenuated in a
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dose-dependent manner by CGA-AuNPs (Fig. 6A and Supplementary Fig. S8). Moreover, immunofluorescence staining of Ninj1 indicated that CGA-AuNPs
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abolished Ninj1 upregulation mediated by LPS treatment in vitro (Supplementary
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Fig. S9). To investigate the effect of CGA-AuNPs on the activation of macrophages in vivo, we used an LPS-inflamed mouse model and examined the entry of activating Ninj1-expressing macrophages into the retina. In untreated mice, Ninj1-expressing macrophages were not observed around the retinal vessels (Fig. 6B). When the
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mice were treated with LPS, the number of Ninj1-expressing macrophages increased, which were blocked by CGA-AuNP treatment (Fig. 6B). Next, we
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examined the effect of CGA-AuNPs on the cell-to-matrix adhesion of Raw 264.7
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cells. Under LPS-induced inflammatory conditions, the adhesion of Raw 264.7 cells on gelatin, collagen type I, laminin, and fibronectin matrices increased compared to
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that observed for the control (Fig. 7). However, CGA-AuNPs abolished the LPSinduced adhesion activity of Raw 264.7 cells in a dose-dependent manner (Fig. 7). Thus, these results showed that CGA-AuNPs downregulated Ninj1 expression and had an inhibitory effect on the adhesion activity of Raw 264.7 cells.
CGA-AuNPs Inhibit Activation of MMPs in Stimulated Macrophages During leukocytes homing, leukocytes are recruited to the site of inflammation by the degradation of extracellular matrix (ECM), in which transendothelial migration is attributed to matrix metalloproteinases (MMPs). To determine the effect of CGA15
ACCEPTED MANUSCRIPT AuNPs on the activity of MMP-2 and MMP-9, which are major MMPs in macrophages, Raw 264.7 cells were cultured in the presence of different
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concentrations of CGA-AuNPs. In addition, the gelatinase activity of the conditioned
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media was measured by zymography. Under LPS-induced inflammatory conditions, the enzymatic activities of MMP-2 and MMP-9 were increased compared to the
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activity of the control, and CGA-AuNP treatment caused significant inhibition of the
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activity of MMP-2 and MMP-9 (Fig. 8). This inhibitory mechanism of CGA-AuNPs on MMP-2 and MMP-9 production may in part be attributed to the direct inhibition of the activation of NF-κB and the subsequent binding of NF-κB to the MMP-2 and MMP-9
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promoter.
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Discussion
CGA has been reported in various functions such as anti-oxidant, anti-aging, and
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anti-diabetic11,12. For instance, it slows the release of glucose into the blood, helping
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counter the accumulation of body fat caused by excess blood glucose and reduce the risk of diabetes. Recently, the anti-inflammatory effect of CGA have been reported in LPS-inflamed murine macrophage cells, mouse retinal inflammation model, carbon tetrachloride (CCl4)-induced liver fibrosis model, and LPS-inflamed keratinocytes13,24,25. For this reason, CGA has received much attention and those taking a natural product or preparation containing CGA are on the increase. Therefore, it is worthwhile to make CGA more effective forms in aspects of absorption, stability, or efficacy with no detectable toxicity to cells and vital organs (heart and vessel). Recently, there has been increased research interest in the use 16
ACCEPTED MANUSCRIPT of nanoscale Au in medical applications. The green synthesis of AuNPs using naturally occurring bioactive compounds as reductants offers many advantages over
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traditional chemical methods. These advantages include increased biocompatibility,
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reduced use of toxic chemicals and possible synergistic activities from the combination of nanomaterials with bioactive compounds. In the present report, we
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synthesized CGA-AuNPs using CGA as a green reductant. The synthesis of CGA-
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AuNPs was facile and the resulting NPs were obtained in aqueous dispersions. The formation of CGA-AuNPs was certainly detectable by eyes due to the appearance of pink color and NPs were completely characterized the particle size, shape and crystal nature by spectroscopic and microscopic techniques. The distinct differences
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of FT-IR spectra between CGA and CGA-AuNPs suggested that –OH functional groups of CGA were most likely involved in the reduction of HAuCl 4. This
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observation was well correlated with the mechanistic study where two –OH groups of
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caffeic acid (2) moiety in CGA (1) were oxidized during the synthesis. In addition, any noticeable aggregation or agglomeration of CGA-AuNPs was not observed with
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the 25-fold concentration under N2 atmosphere, suggesting that CGA-AuNPs possessed an excellent colloidal stability. Maintaining a good colloidal stability of NPs is considered to be a crucial factor for desired biological activities. In case of the silver nanoparticles (AgNPs), there is a correlation between the colloidal stability and enhanced antibacterial activities26. For examination of redox mechanism between HAuCl4 and CGA (1), we tried to figure out oxidation product of 1 by using IR, NMR, and MS, but all attempts did not give any meaningful results. Indeed, many previous mechanistic studies in the synthesis of metal NPs drew the same failures because oxidation products of 17
ACCEPTED MANUSCRIPT organic reductants existed as a small amount and are attached to inorganic metal NPs20. To solve the problem, we designed a strategic approach, structurally
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decomposing original reductant 1 into caffeic acid (2) and (-)-quinic acid (3) (Fig. 1A).
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By the strategy, we expected two things: 1) at least one of 2 and 3 has a reducing potential; 2) structurally simplified reductant could make it easy to detect its oxidation
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form. As a result of adding each commercially available 2 or 3 to aqueous HAuCl4, it
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was found that only 2 has a reducing ability to synthesize AuNPs (Fig. 1E). To confirm the exact structure, oxidation product of reductant 2 was characterized by using HR-LC-ESI-MS (Fig. S1). This result solidly supported that oxidation product of 2 is compound 6 in Fig. 1E. Also, based on the results with reductant 2, we
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suggested the oxidation mechanism of CGA (1) in Fig. 1E. Particularly, confirming oxidation form by MS is a valuable trial because oxidation products in the synthesis
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of metal NPs have been generally observed using IR and NMR, not MS 24,27,28. To
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our knowledge, this result is the first success in detection with MS. We believe that our strategy‒structurally decomposing original reductant, detecting oxdation form of
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simplified reductant with MS, and applying oxidation form to original reductant‒is a consecutive protocol to solve the mechanistic problem in the research field of metal NPs. Next, we compared anti-inflammatory efficacy between CGA and CGA-AuNPs in mouse Raw 264.7 macrophages and inflamed retinal models. Interestingly, CGAAuNPs showed the better anti-inflammatory effects than CGA in most aspects without toxicities. For example, the inhibitory effect of 5 μM CGA-AuNPs on NF-kB translocation and cell adhesion was stronger than that of 20 μM CGA. However, further studies are warranted to understand the mechanisms by which CGA 18
ACCEPTED MANUSCRIPT increases the anti-inflammatory effect of AuNPs and to clarify whether combination of AuNPs and CGA (as bioactive reductants) produces additive or synergistic effects
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or not.
The use of NPs for inflammation treatment is currently under intensive investigation
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and chemical, optical, and electromagnetic properties of NPs may be strongly influenced by their size and shape29. Ji Su Ma et al. synthesized AuNPs (diameter, d
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= 10 ~ 15 nm) using sodium citrate and showed that AuNPs block NO production and iNOS expression through the inhibition of NF-κB and STAT1 in LPS-stimulated RAW 264.7 cells14. Chen Hui et al. reported that spherical AuNPs of 21.3 ± 0.7 nm significantly reduces TNF-α and IL-6 mRNA levels with no measurable organ or cell
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toxicity in male C57BL/6 mice15. The authors utilized the spherical AuNPs prepared
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by the standard citrate-reduction route to evaluate the anti-inflammatory efficacy of AuNPs15. It is noteworthy that the preparation of CGA-AuNPs presented in the
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current report is unique from previous reports. We combined two anti-inflammatory
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agents, AuNPs and CGA, by a green synthetic method, which is an eco-friendly technology, to increase or reinforce anti-inflammatory activities of AuNPs. However, AgNPs of 24 nm exhibit cytotoxic properties to macrophages and trigger an inflammatory response, although they showed beneficial effect such as antibacterial activity30. From our studies and the studies described above, therefore, it is necessary and interesting to study further whether functionalization or the combination of NPs with green reductants, possessing desired biological activity, can decrease harmful effect and increase beneficial effect of NPs simultaneously or not.
19
ACCEPTED MANUSCRIPT In the present report, CGA was successfully used to reduce Au 3+ to AuNPs. As the first success in mechanistic study by HR-LC-ESI-MS, it was confirmed that the
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caffeic acid moiety of CGA (1) was essential to reduce Au3+. CGA-AuNPs inhibited
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pro-inflammatory cytokines and other inflammation-related genes (including MMPs and Ninj1) through the inhibition of NF-κB nuclear translocation. Compared to that of
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CGA, the anti-inflammatory effect of CGA-AuNPs was much stronger, indicating that
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functionalization or the combination of AuNPs with green reductants, which are known to have therapeutic or preventive properties, can provide a new strategy for the development of novel anti-inflammatory agents. These results clearly warrant further clinical studies to ascertain the in vivo efficacy of CGA-AuNPs for
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inflammatory or infectious diseases treatment.
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ACCEPTED MANUSCRIPT Figures legends
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Figure 1 Spectroscopic analyses of CGA-AuNPs. (A) Structural characteristics of
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CGA (1). (B) UV-Visible spectra of the AuNPs synthesized with reducing agents 1–3 with i) no reductant, ii) CGA (1), iii) caffeic acid (2), and iv) (-)-quinic acid (3). The
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detailed reaction conditions for the synthesis of AuNPs are described in the experimental section. (C) FT-IR spectrum and (D) HR-XRD analysis. (E) Two-
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electron oxidation of gallic acid,19 caffeic acid, and CGA upon the synthesis of AuNPs
Figure 2 HR-TEM images of the CGA-AuNPs. The scale bar represents (A) 100 nm,
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(B) 50 nm, (C) 20 nm, and (D) 5 nm. (E) size histogram and f) SAED pattern. The organic thin layer was indicated by red arrows which represents the capping of
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AuNPs by CGA.
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Figure 3 AFM and FE-SEM images of the CGA-AuNPs. (A) 3-D height (2.5 µm × 2.5 µm), (B) 3-D amplitude error (2.5 µm × 2.5 µm), (C) 3-D phase (1.0 µm × 1.0 µm),
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(D) 2-D height (1.0 µm × 1.0 µm), (E) section analysis of two NPs (lines a-b and c-d) in image (D), and (F) FE-SEM image of the CGA-AuNPs. The magnification of (F) 45,000×. The scale bar represents 100 nm in (F). Figure 4 Effects of CGA-AuNPs on iNOS expression and cytotoxicity in LPSstimulated Raw 264.7 cells. (A) NO assay results obtained using the Griess reagent; **P
