Article pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Curcumin-Conjugated Gold Clusters for Bioimaging and Anticancer Applications Saravanan Govindaraju,† Arunkumar Rengaraj,‡ Roshini Arivazhagan,§ Yun-Suk Huh,*,‡ and Kyusik Yun*,† †
Department of Bionanotechnology, Gachon University, Gyeonggi-do, 13120, Republic of Korea Department of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon, 22212, Republic of Korea § Center for Genomics and Proteomics, Institute for Regenerative Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 406-840, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Curcumin-conjugated gold clusters (CUR-AuNCs) were synthesized using a “green” procedure and utilized as an anticancer and a bioimaging agent. Curcumin is a well-known anticancer agent, which forms a cluster when reacting with a gold precursor under mild alkali condition. A fluorescence spectroscopy analysis showed that the CUR-AuNCs emitted red fluorescence (650 nm) upon visible light (550) irradiation. Fourier transform infrared spectroscopy analysis confirmed the stretching and bending nature between the gold atoms and curcumin. Meanwhile, transmission electron microscopy analysis showed a cluster of approximately 1−3 nm with a uniform size. Time-resolved fluorescence analysis demonstrated that the red fluorescence was highly stable. Moreover, laser confocal imaging and atomic force microscopy analysis illustrated that a cluster was well distributed in the cell. This cluster exhibited less toxicity in the mortal cell line (COS-7) and high toxicity in the cervical cancer cell line (HeLa). The results demonstrated the conjugation of curcumin into the fluorescent gold cluster as a potential material for anticancer therapy and bioimaging applications.
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INTRODUCTION Cancer is the leading cause of death worldwide. The World Health Organization has estimated that approximately 84 million people die because of cancer.1 The most important reasons for such high mortality rates is the failure of early diagnosis for most types of cancers and the prognosis of cancer.2 Despite the availability of an enormous number of drugs for cancer treatment, complete cure is still not successful in clinical therapy because of the resistance developed by cancer cells to several common chemotherapeutic agents.3 Another major drawback is cancer metastasis, where cancer therapy becomes unresponsive.4−6 Chemotherapy also remains unsuccessful because of the nonselectivity of specific cells, where normal cells are also drastically affected. Therefore, a growing demand for targeted cancer therapy is observed in clinical trials.7,8 The current trends in drug delivery research focus on using biocompatible nanomaterials as drug carriers for targeted therapy. The advantages of using nanomaterials are its physical, chemical, and mechanical properties, which specifically favor drug targeting and release to cancer cells.9 These nanocarriers are biocompatible. Hence, they can be easily degraded by cells and are less toxic. One of such nanocarriers, which has recently drawn much attention, is gold nanoparticles.10 Gold clusters © XXXX American Chemical Society
have a unique physicochemical property, are easy to synthesize, and have higher stability, so they have been widely discussed in diagnosis and imaging.11 Clusters consist of a small number of atoms as compared to a nanoparticle and exhibit strong fluorescence under irradiation. As compared to nanoparticles, clusters are less toxic because of lower utilization of surfactant and its uniform size. Consequently, the green synthesis of these clusters using a biomolecule extracted from plants, animals, and microbes is widely being discussed. These biomolecule−cluster conjugates can be applied for imaging applications and therapy. Curcumin is a flavonoid (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-2,5-dione) well-known for being an antioxidant against cancer, microbial infection, diabetic wounds, anorexia, rheumatism, and neuroprotective properties, and an inhibitor of angiogenesis and hepatic disorders.12−15 Curcumin is extracted from the rhizome of turmeric and widely utilized in Asian medicine. Curcumin has the ability to inhibit generation of free radical species, which plays in important role in the concept of the H atom from the C12 methylene group.16−18 A few researchers have recently demonstrated the anticancer Received: November 8, 2017 Revised: December 9, 2017
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DOI: 10.1021/acs.bioconjchem.7b00683 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 1. Characterization of CUR-AuNCs: (a) UV−vis and PL emission spectrum. (b) Stability of CUR-AuNCs from 0 to 120 min and the inset image is the emission peak in different excitation.
Figure 2. (a) XRD spectrum of CUR-AuNCs and (b) FTIR characterization of curcumin and CUR-AuNCs. Red trace indicates the spectrum for curcumin and black trace indicates the spectrum for CUR-AuNCs.
was used herein to prepare quantum-sized nanoclusters during the growth and nucleation process.20 NaOH addition was performed to improve the reducing power of the BSA by increasing the levels of the pH value.21 Once the gold atoms reached the saturation level, they started to aggregate in the form of gold clusters through homogeneous nucleation. The −OH present in curcumin was also used to reduce HAuCl4 to Au0 and act as a capping agent. The possible mechanism behind the formation of the CUR-AuNCs by curcumin is described as (1) deprotonation − OH of curcumin into CUR3−, (2) reduction − Au3+ into Au0, (3) nucleation − Au0 atoms form Au clusters, (4) growth, (5) cleavage, and (6) maturation.18 During the synthesis reaction, we added BSA and curcumin together to the HAuCl4, so the material was synthesized together with the BSA and curcumin. Optical Property of the CUR-AuNCs. The UV−vis absorbance (UV) and photoluminescence (PL) properties of the synthesized CUR-AuNCs were analyzed. In UV spectroscopy, the small peaks at 420 nm indicated that curcumin perfectly conjugated to the AuNCs. Meanwhile, the small peak at 280 nm implied the combination of BSA with the Au ions.22 No absorption peak was observed in the UV−vis because of the small size of the AuNCs. The conduction electrons on the gold surface can interact with light and generate the SPR effect. At the same time, when the size of the particles is in the quantum range, it has some distinguishable properties because the space
property of curcumin (abstraction of the H atom from the C12 methylene group) by inhibiting the free radicals in cancer. The chromophore has a strong absorption maximum at 428 nm and a weaker absorption of light above 300 nm. The clinical trials have indicated that humans can tolerate a dose as high as 12 g per day without any toxic side effects.19 In this study, we used BSA and curcumin as a reducing and stabilizing agent in the formation of the gold cluster. This approach brings down the utilization of reducing agents and the toxic effect of surfactants in cell lines. To the best of our knowledge, this is the first report on curcumin-conjugated gold clusters for bioimaging and anticancer applications. As the synthesized clusters have shown significant results in both bioimaging and anticancer applications, this study is goes further to confirm its efficiency. In addition, this synthesis process represents an added advantage because the limitation of curcumin insolubility is overcome, thereby increasing its bioavailability, and its conjugation with AuNCs ensures its bioactivity. Finally, we confirm several antitumorigenic properties of our formulation in the HeLa cancer cell lines.
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RESULTS AND DISCUSSION
CUR-AuNCs Synthesis. The CUR-AuNCs synthesis developed by curcumin was allowed to react with the HAuCl4 and BSA solution. The color changes after the NaOH addition indicated the formation of gold clusters. BSA B
DOI: 10.1021/acs.bioconjchem.7b00683 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 3. Morphology characterization of CUR-AuNCs: (a) TEM of CUR-AuNCs; the inset image is SAED pattern. (b) HR-TEM; arrow indicates the collections of atoms to form a cluster. (c) Bio-AFM height image of CUR-AuNCs. (d) 3D image of CUR-AuNCs.
between the energy levels was inversely proportional to the radius of the particles.23 The peak at approximately 520 nm indicated the presence of a lower amount of gold nanoparticles. As shown in Figure 1a, the obtained CUR-AuNCs were well dispersed in the DI solution and presented a strong red fluorescence (inset) under UV light at 365 nm. The maximal excitation was observed at 450 nm. The emission peak was at ∼650 nm. A small peak at 550 nm indicated the presence of curcumin. Accordingly, the stability of the prepared CURAuNCs was investigated (Figure 1b). Moreover, pure curcumin and CUR-AuNCs PL intensity was checked to confirm the presence of curcumin after the CUR-AuNCs formation (Figure S1). The CUR-AuNCs were highly stable up to 120 min at room temperature and remained in the same PL intensity. This observation showed that this material was perfectly suitable for bioimaging, especially for in vivo studies. The inset image in Figure 1b shows the emission spectra of the CUR-AuNCs at different wavelengths from 400 to 540 nm. The strongest fluorescent was red, which remained the same with a shift at approximately 650 nm. There are no changes in the fluorescent property at different excitation wavelengths. X-ray Diffraction (XRD) and Fourier Transmission Infrared Spectroscopy. The synthesized CUR-AuNCs crystallinity and diffracted angles were evaluated using the XRD pattern (Figure 2a) from 5° to 90°. Different 2θ peaks were observed for the CUR-AuNCs at 45.5° and 65.6° corresponding to the (111) and (220) lattice planes, respectively. The peak at (111) had a stronger energy than the (220) peak. Figure 2b illustrates the FTIR spectrum of the CUR-AuNCs. An analysis was conducted to identify the chemical interaction and the functional group of the
synthesized materials and curcumin. The black trace denotes the CUR-AuNCs, while the red trace denotes pure curcumin. Two phenolic groups and one enolic group of hydroxyl groups were in-plane bending bands at 1383 cm−1, 1233 cm−1, and 962 cm−1, respectively. These bands were completely absent in the CUR-AuNCs, which indicated the interaction of HAuCl4 at these sites. The heptadiene chain of curcumin was observed in the CUR-AuNCs bands at 767 and 1425 cm−1 corresponding to the olenic in-plane bending vibrations. Similarly, bands at 2920 cm−1, 1461 cm−1, 1024 cm−1, and 989 cm−1 appeared because of the aliphatic C−H stretches, vibration of CH3, and aromatic −CC−C and −CC−H of curcumin, which confirmed that curcumin perfectly attached to the AuNCs. Morphology Analysis. The morphological characterizations were evaluated using TEM and atomic force microscopy (AFM). The TEM images (Figure 3a) indicated that the average sizes of the CUR-AuNCs were 1−3 nm, and all the particles were distributed well. Small atoms were combined together to form the AuNCs and were clearly visible in the HRTEM in the inset of Figure 3a shows the selected diffraction pattern of the CUR-AuNCs, which indicated that the prepared nanocrystals were well organized. In Figure 3b these results demonstrated the high magnification and the crystallinity of the CUR-AuNCs in fcc of Au. The lattice fringe spacing could correspond to the (111) reflection of fcc Au. The AFM images indicated the average size and height of the CUR-AuNCs (Figure 3c). The AFM height image in Figure 3c showed that the particles were well distributed, and the height of the CURAuNCs was approximately 1−6 nm because the nanomaterials overlapped with other nanoparticles. The AFM 3D image in Figure 3d also confirmed the uniform size and height of the cluster at approximately 6 nm. C
DOI: 10.1021/acs.bioconjchem.7b00683 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 4. MTT assay to evaluate the cytotoxicity of CUR-AuNCs on normal and cancer cell line. Different concentrations of the formulation ranging from 0 to 100 μg/mL were treated to normal and cancer cells and incubated for up to 24 h. MTT reagent was added to each well and incubated in the dark for 4 h. Following this, the OD was recorded. The percentage of viable cells in each group was calculated and plotted as a graph. (a) COS-7 − Mortal cell line and showed much less cytotoxicity. (b) HeLa − Immortal cell line showed gradual increase in cytotoxicity with increasing concentrations of CUR-AuNCs. Each value represents the mean ± SE (n = 5) *p < 0.05.
Figure 5. Laser confocal imaging of HeLa cells treated with image CUR-AuNCs: (a) FITC (500−600 nm), (b) curcumin-AuNCs (600−700 nm), and (c) merged. Scale bar = 50 μm.
Cytotoxicity of the Prepared CUR-AuNCs in the Normal and Cancer Cell Lines. Several reports have been released on the cytotoxic effects of curcumin in several types of cancers. 24−27 Several mechanisms have explained how curcumin enhances cytotoxicity within the cancer cells. The most common mechanism extensively reported for curcumin is the p53 mediated cell. One study also emphasized apoptosis regulation in prostate cancer cells via mitochondrial damage.28 Another study reported apoptosis induction through the regulation of ROS in Caki-1 renal carcinoma cells29 and in MCF-7 and MDA-MB-231 breast cancer cells. 30 The cytotoxicity of the CUR-AuNCs was evaluated by an MTT assay in the HeLa cervical cancer cell line and COS-7 normal kidney fibroblast cell line. The cells were treated with different concentrations of 0 μg, 10 μg, 25 μg, 50 μg, 75 μg, and 100 μg/ mL formulations. Subsequently, the cells were subjected to the MTT assay after 24 h incubation. The percentage of viable cells in the HeLa and COS-7 cell line after 24 h treatment was evaluated and plotted as a graph. The results from the MTT assay showed that the CUR-AuNCs induced a significant cytotoxicity to HeLa cells at concentrations of 50 μg, 75 μg, and 100 μg/mL. Moreover, the concentration of BSA also acts as the main role in cell viability. The high concentration of BSA can enhance the cell viability.31 Here, we were keeping the
amount of BSA constant through the entire reaction. Free curcumin shows less viability compared to the conjugate nanoparticles. This is due to the enhanced solubility of conjugate curcumin during the cellular uptake process, where the BSA, curcumin, and gold combined to form the CURAuNCs, giving better anticancer property to the cell lines.32 Moreover, we have included the drug release assay in Figure S2. Therefore, these results confirmed the apoptotic property of curcumin as previously reported. Meanwhile, the COS-7 cells were less affected at these formulation concentrations (Figure 4). Therefore, the CUR-AuNCs induced cytotoxicity only to cancer cells. Live−Dead Assay. Figure 5 shows confocal images of the HeLa cell lines treated with the CUR-AuNCs. The spatial localization of the CUR-AuNCs was confirmed by the appearance of red fluorescence in the cell, which was evidence for the use of the synthesized nanocluster for cancer cell bioimaging. Figure 6a presents a direct observation of the proportion of living and dead cells in the CUR-AuNCs-treated HeLa and COS-7 cells. The HeLa cells treated with 100 μg/mL of the formulation showed a gradual increase in the number of dead cells at 24 and 48 h treatment. Figure 6b shows a representation of the percentage of survival rate of cells treated with D
DOI: 10.1021/acs.bioconjchem.7b00683 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
and the wound area was reduced in size at 24 h (Figure 8a). Figure 8b explains the percentage of wound area at 0 to 24 h compared with the control and treated cells. These results concluded the inhibitory property of the CUR-AuNCs against HeLa cell migration.
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CONCLUSION
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EXPERIMENTAL SECTION
In summary, the CUR-AuNCs were successfully synthesized, and their anticancer effects were evaluated. The prepared nanocluster exhibited a strong red fluorescence with the particles size of 1−3 nm. Curcumin, being an autofluorescence drug, maintained its fluorescence property and emitted fluorescence within the CUR-AuNCs-treated HeLa cells, suggesting the advantages of using it for bioimaging. Following this, the CUR-AuNCs-treated HeLa cells showed significant cytotoxicity and morphological damages at concentrations of 50, 75, and 100 μg/mL after 24 h. The treatment with the CUR-AuNCs also increased the number of dead cells with an increasing time point. The HeLa cell migration was significantly inhibited as compared to the control upon treatment for 24 h. However, in spite of these properties, the CUR-AuNCs showed a very negligible toxicity of the COS-7 normal kidney cells. Together, our results suggested that this formulation induced apoptosis only in cancer cells. Moreover, the luminescence property exhibited by the formulation facilitated their tracking during chemotherapy. Based on our results, we strongly believe that this novel gold cluster formulation has enhanced properties compared to gold nanoparticles and can act as a potent drug for cancer therapy. Further in vivo studies are required to translate this approach into clinical application. Therefore, as a next step, we intend to continue the effective targeted therapy of this formulation against metastatic lung cancer xenograft models.
Figure 6. Live/dead assay of COS-7 and HeLa cell line treated with CUR-AuNCs at different time intervals. Cells were treated with CURAuNCs and incubated for 24 and 48 h. The number of live and dead cells in CUR-AuNCs treated Cos-7 and HeLa cells was evaluated using the live/dead assay kit. Images were captured using fluorescence laser scanning microscope at 0, 24, and 48 h. Cos-7 cells showed fewer or no dead cells at 24 and 48 h. HeLa cells, however, showed an increase in the number of dead cells in 24 and 48 h treated group. Scale bar = 100 μm.
nanoclusters. These results were similar to those in the previous reports on the effect of curcumin in different types of cancer cells.33−35 In conclusion, the results suggested that the CURAuNCs induced a significant cytotoxicity only to cancer cells while leaving normal cells less affected. This finding further confirmed the results from the MTT assay. Nanomorphological Changes by AFM. AFM was used to better understand how the CUR-AuNCs may alter the ultrastructural cell morphology of the HeLa cells. The deflection image illustrated that the CUR-AuNCs increased the surface roughness of the cells and pore formations. The treated cells showed uneven, damaged cell surfaces and cell breakage at 24 and 48 h, respectively (Figure 7). These results suggested that the antitumorigenic activity of the CUR-AuNCs abrogated the morphological structure of the HeLa cancer cells, confirming the previous reports on the morphological damage in cancer cells induced by curcumin.36−39 CUR-AuNCs Controlled the HeLa Cell Migration. Curcumin is a potent anticancer drug extensively studied for its antimigratory property in different types of cancer cells.40−43 We validated whether our formulation conferred similar antimigratory effects on cancer cells by evaluating the regulation of the HeLa cell migration at 0 and 24 h. Accordingly, the cells were treated with 100 μg/mL of the CUR-AuNCs. The images were captured to evaluate the recovery of the scratch area at 0 and 24 h. The results showed that the number of migrating cells at 0 and 24 h in the treatment groups were lower compared to the control untreated cells. The CUR-AuNCs drastically inhibited the HeLa cell migration. Moreover, the wound was not sealed for up to 24 h. However, the control untreated cells migrated,
Materials. Gold(III) chloride trihydrate (HAuCl4·3H2O), sodium hydroxide (NaOH), and curcumin were purchased from Sigma-Aldrich (USA). Bovine serum albumin (BSA) was purchased from Millipore LEE (South Korea). The transmission electron microscopy (TEM) sample grid (with the following specifications: ultrathin carbon Type-A, 400 mesh, copper, approximately 42 μm grid hole size) was purchased from Ted Pella, Inc. (USA). All chemicals were used without any further purification. Instrumentation. Definite optical observations of UV−vis spectra were taken for the synthesized CUR-AuNCs using a Varian Cary 50 ultraviolet spectrophotometer. The fluorescent property was recorded using a PTI UV illuminator at 360 nm. The FTIR spectra were recorded using a Bruker vortex highresolution 70 FTIR spectrometer equipped with BRUKER FTIR Vertex 70 with a micro plate extension HTS-XT and ATRunits from Billerica (MA, USA). Moreover, the morphological characteristic was studied using an FEI Titan 80−300 highresolution transmission electron microscope and a JPK NanoWizard II bioatomic force microscope. The stained samples were analyzed using a Nikon Eclipse TE 2000-U laser scanning fluorescence microscope. CUR-AuNC Synthesis. A stock solution of 2 mg curcumin was prepared by dissolving in 5 mL dimethyl sulfoxide (DMSO) solution. In a typical experiment, 5 mL of a 10 mM HAuCl4 was heated until boiling under magnetic stirring at 800 rpm. After this, 5 mL of the BSA solution was mixed to the HAuCl4 solution. Subsequently, 2 mL curcumin was added. E
DOI: 10.1021/acs.bioconjchem.7b00683 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 7. Bio-AFM height, error, and 3D images of CUR-AuNCs treated with HeLa cell in different interval of time from 0, 24, and 48 h.
After 30 min, 500 μL of the NaOH solution was added and maintained at 50 °C under vigorous stirring for 3−4 h. The color was changed from light yellow to brown color, indicating the formation of the CUR-AuNCs. The samples were freezedried for further experiment. Cellular Uptake. The cellular uptake and the fluorescence property of the CUR-AuNCs nanoparticles were also assessed. HeLa cells were briefly seeded on a coverslip in a 35 mm dish and allowed to adhere for 24 h. The cells were treated with the desired concentration of CUR-AuNCs nanoparticles for 24 h at 37 °C. The cells were then fixed with 100% methanol for 20 min. After this, the cells were washed thrice with 1× Dulbecco’s phosphate-buffered saline (DPBS). The cellular uptake and the fluorescence property of the synthesized CUR-AuNCs nanoparticles were observed using a confocal laser scanning microscope. In Vitro Cytotoxicity Assay. The COS-7 normal kidney fibroblast cell line and the HeLa cervical cancer cell lines were grown in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained at 37 °C with 5% CO2 in a humidified incubator. The cells were then washed with 1× DPBS and trypsinized with a trypsin−ethylenediaminetetraacetic acid solution (1 × ). The cells were used to evaluate the cytotoxicity of the prepared formulation. Accordingly, the cells were seeded on a 96-well plate in an RPMI-1640 medium with 10% FBS and 1% P/S at a cell density of 1 × 104 cells per well, and incubated overnight.
Different concentrations of the formulation (i.e., 0, 10, 25, 50, 75, and 100 μg/mL) were mixed with the RPMI-1640 media, and then added to the cells. After 24 h incubation, 20 μL of the MTT reagent was added to each well. The media were removed after 4 h incubation in 37 °C. Subsequently, 100 μL DMSO was added to each well. The OD was measured at 570 nm using a microplate reader. Live−Dead Cell Staining. Similarly, COS-7 and HeLa cells were seeded on a glass slide and incubated for 24 h. The cells were then treated with media containing 100 μg/mL concentrations of CUR-AuNCs and incubated further for 24 and 48 h. Subsequently, the cells were washed thrice with 1× DPBS. Live−dead staining was then performed per the manufacturer’s protocol. The number of live (green fluorescent) and dead (red fluorescent) cells in the CUR-AuNCstreated COS-7 and HeLa were observed at 0, 24, and 48 h using a confocal laser scanning microscope. Atomic Force Microscopy (AFM). The surface topography of the CUR-AuNCs (100 μg/mL) treated HeLa cells were analyzed using AFM. Accordingly, cells were seeded on glass slides for 24 h followed by treatment with desired concentrations of the formulation and further incubated for 24 and 48 h. The cells were washed three times with 1× DPBS, fixed with 0.5% glutaraldehyde in RPMI for 1 h, and again washed three times with 1× DPBS. The slides containing the cells were used for AFM analysis. F
DOI: 10.1021/acs.bioconjchem.7b00683 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (No.2017R1A2B4004700).
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(1) Danhier, F., Feron, O., and Preat, V. (2010) To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Controlled Release 148, 135−146. (2) Chen, K. H., Weng, M. S., and Lin, J. K. (2007) Tangeretin suppresses IL-1β-induced cyclooxygenase (COX)-2 expression through inhibition of p38 MAPK, JNK, and AKT activation in human lung carcinoma cells. Biochem. Pharmacol. 73, 215−227. (3) Dai, L., Liu, J., Luo, Z., Li, M., and Cai, K. (2016) Tumor therapy: targeted drug delivery systems. J. Mater. Chem. B 4, 6758−6772. (4) Zheng, G., Li, L., Li, M., Feng, X., Pu, X., Zhang, B., Yu, P., He, G., Zhang, Y., and Xia, H. (2015) Effects of different functional groups on metastatic behavior of SPC-A-1/human lung cancer cells in selfassembled monolayers. RSC Adv. 5, 41412−41419. (5) Koumoutsakos, P., Pivkin, I., and Milde, F. (2013) The fluid mechanics of cancer and its therapy. Annu. Rev. Fluid Mech. 45, 325. (6) Arivazhagan, L., and Pillai, S. S. (2014) Tangeretin, a citrus pentamethoxyflavone, exerts cytostatic effect via p53/p21 upregulation and suppresses metastasis in 7, 12-dimethylbenz (α) anthracene-induced rat mammary carcinoma. J. Nutr. Biochem. 25, 1140−1153. (7) Liang, C., Xu, L., Song, G., and Liu, Z. (2016) Emerging nanomedicine approaches fighting tumor metastasis: animal models, metastasis-targeted drug delivery, phototherapy, and immunotherapy. Chem. Soc. Rev. 45, 6250−6269. (8) Roa, W. H., Azarmi, S., Al-Hallak, M. K., Finlay, W. H., Magliocco, A. M., and Lobenberg, R. (2011) Inhalable nanoparticles, a non-invasive approach to treat lung cancer in a mouse model. J. Controlled Release 150, 49−55. (9) Cui, W., Li, J., and Decher, G. (2016) Self-Assembled Smart Nanocarriers for Targeted Drug Delivery. Adv. Mater. 28, 1302−1311. (10) Alhalili, Z., Figueroa, D., Johnston, M. R., Shapter, J., and Sanderson, B. (2016) Effect of Modification Protocols on the Effectiveness of Gold Nanoparticles as Drug Delivery Vehicles for Killing of Breast Cancer Cells. Aust. J. Chem. 69, 1402−1412. (11) Zhang, W., Lin, D., Wang, H., Li, J., Nienhaus, G. U., Su, Z., Wei, G., and Shang, L. (2017) Supramolecular self-assembly bioinspired synthesis of luminescent gold nanocluster-embedded peptide nanofibers for temperature sensing and cellular imaging. Bioconjugate Chem. 28, 2224−2229. (12) Goel, A., Kunnumakkara, A. B., and Aggarwal, B. B. (2008) Curcumin as “Curecumin”: from kitchen to clinic. Biochem. Pharmacol. 75, 787−809. (13) Aggarwal, B. B., and Harikumar, K. B. (2009) Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int. J. Biochem. Cell Biol. 41, 40−59. (14) Anand, P., Kunnumakkara, A. B., Newman, R. A., and Aggarwal, B. B. (2007) Bioavailability of curcumin: problems and promises. Mol. Pharmaceutics 4, 807−818. (15) Singh, D. K., Jagannathan, R., Khandelwal, P., Abraham, P. M., and Poddar, P. (2013) In situ synthesis and surface functionalization of gold nanoparticles with curcumin and their antioxidant properties: an experimental and density functional theory investigation. Nanoscale 5, 1882−1893. (16) Hatcher, H., Planalp, R., Cho, J., Torti, F., and Torti, S. (2008) Curcumin: from ancient medicine to current clinical trials. Cell. Mol. Life Sci. 65, 1631−1652. (17) Balasubramanyam, M., Koteswari, A. A., Kumar, R. S., Monickaraj, S. F., Maheswari, J. U., and Mohan, V. (2003)
Figure 8. CUR-AuNCs inhibited the migration of HeLa cells. Cells were assessed for migration with wound healing assay. Accordingly, cells were seeded and cultured until confluence was reached. A scratch was made and cells were treated with or without CUR-AuNCs nanoparticles. (a) The area of the wound was evaluated at 0 and 24 h in control and treated cells. (b) Graph representing the percentage of wound area at 0 and 24 h in control and treated cells. Each value represents the mean ± SE (n = 5) *p < 0.05.
Wound Healing Assay. HeLa cells were seeded (∼1 × 106 cells) in a 6-well plate until monolayers formed. The cell layers were scratched with a sterile micropipet tip (Sigma), washed thrice with 1× DPBS, and incubated in fresh serum-free RPMI1640 medium with or without CUR-AuNCs (100 μg/mL) for 24 h. The number of migrating cells was observed under an inverter microscope and photographed at 0 and 24 h after scratching. Statistical Analysis. All experiments were performed in replicates of three. The results were presented as means ± standard deviations (SDs). The statistical significance was evaluated using Student’s t test. P values
