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Targeted delivery of 5-fluorouracil to cholangiocarcinoma cells using folic acid as a targeting agent
Materials Science and Engineering C 60 (2016) 411–415
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Targeted delivery of 5-fluorouracil to cholangiocarcinoma cells using folic acid as a targeting agent Nipaporn Ngernyuang a, Wunchana Seubwai b,c, Sakda Daduang d, Patcharee Boonsiri e, Temduang Limpaiboon f,g, Jureerut Daduang f,⁎ a
Chulabhorn International College of Medicine, Thammasat University, Pathum Thani 12120, Thailand Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kean 40002, Thailand Comprehensive Cancer Research Group, Faculty of Medicine, Khon Kaen University, Khon Kean 40002, Thailand d Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand e Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand f Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand g Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand b c
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 13 November 2015 Accepted 23 November 2015 Available online 24 November 2015 Keywords: Cholangiocarcinoma Cancer Nanomedicine Folate receptor Targeted therapy
a b s t r a c t There are limits to the standard treatment for cholangiocarcinoma (CCA) including drug resistance and side effects. The objective of this study was to develop a new technique for carrying drugs by conjugation with gold nanoparticles and using folic acid as a targeting agent in order to increase drug sensitivity. Gold nanoparticles (AuNPs) were functionalized with 5-fluorouracil (5FU) and folic acid (FA) using polyethylene glycol (PEG) shell as a linker (AuNPs-PEG-5FU-FA). Its cytotoxicity was tested in CCA cell lines (M139 and M213) which express folic acid receptor (FA receptor). The results showed that AuNPs-PEG-5FU-FA increased the cytotoxic effects in the M139 and M213 cells by 4.76% and 7.95%, respectively compared to those treated with free 5FU + FA. It is found that the cytotoxicity of the AuNPs-PEG-5FU-FA correlates with FA receptor expression suggested the use of FA as a targeted therapy. The mechanism of cytotoxicity was mediated via mitochondrial apoptotic pathway as determined by apoptosis array. In conclusion, our findings shed some light on the use of gold nanoparticles for conjugation with potential compounds and FA as targeted therapy which contribute to the improvement of anti-cancer drug efficacy. In vivo study should be warranted for its effectiveness of stability, biosafety and side effect reduction. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cholangiocarcinoma (CCA) arises from the epithelial cells of the intrahepatic and extrahepatic bile ducts [1,2]. Surgical resection is currently the only potentially curative treatment for CCA. However, less than 25% of patients are successfully resected [3,4]. Adjuvant chemotherapy has been used in an attempt to improve disease control and quality of life in patients with unresectable, recurrent and metastastic CCA [3]. 5-Fluorouracil (5FU), a water-soluble fluorinated pyrimidine analog that interferes with thymidylate synthesis, has a broad spectrum of activity against solid tumors including CCA. 5FU-based chemotherapy may be efficient in treatment of advanced CCA with less cost, thus, is an appropriate use in a resource-limited country [5]. However, 5FU has limitations including a short biological half-life (10–20 min) due to the rapid absorption through blood capillaries into systemic circulation with plasma, hence high doses are required to achieve ⁎ Corresponding author. E-mail address: [email protected] (J. Daduang).
http://dx.doi.org/10.1016/j.msec.2015.11.062 0928-4931/© 2015 Elsevier B.V. All rights reserved.
therapeutic concentration. Unfortunately, the high doses of this drug are related with the increased risk of serious side effects such as bone marrow depression, gastrointestinal tract reaction, leucopenia and thrombocytopenia. In order to prolong the circulation time of 5FU, increase its efficiency and reduce 5FU-associated side effects, several studies have demonstrated the modification of 5FU delivery by using polymer conjugates or incorporation of 5FU into particulate carriers [6–8]. Recently, gold nanoparticles (AuNPs) have been widely used as chemotherapy delivery vehicles to several cancer cells because of their unusual properties that include relatively high stability, ultra small size, catalytic activity, low toxicity, enhanced Raman signal, surface plasmon resonance (SPR), and facile chemical tailorability [9]. Moreover, AuNPs structures can support multiple functionalities on their surface. These properties of AuNPs may be applied to targeted drug delivery in CCA which could lead to increased efficacy of traditional chemotherapeutics. In addition, folic acid (FA), or vitamin B9, plays a vital role in mammalian cell growth. Moreover, it has been studied extensively as a targeting ligand for imaging and several cancer therapies [10–14]. FA
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has a high binding affinity with the folic acid receptor (FA receptor) which is up-regulated on the surface of many cancer cells, and limited distribution is found in normal tissues [15]. Several studies have reported nanoparticles with FA for site-specific targeting and improvement of FA receptor-mediated uptake by cancer cells [16–18]. In this study, we presented the synthesis and characterization of AuNPs functionalized with 5FU chemotherapeutic drugs and FAspecific ligand as targeted therapy for improvement of CCA treatment efficacy. This strategy could represent a novel modality for effective CCA treatment. 2. Experimental section
2.4. Cell culture Human CCA cell lines, namely M139 and M213, were isolated from Thai CCA patients and established at the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Thailand. Both cell lines were cultured in a RPMI medium supplemented with 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin (Gibco, Life Technologies, NY) at 37 °C in a 5% CO2 humidified incubator. The presence of Mycoplasma contamination was periodically checked.
2.5. Folic acid receptor expression
All glassware used in the preparation and storage of AuNPs was cleaned with HCl:HNO3 (3:1), rinsed with water, and oven-dried. The AuNPs were synthesized via citrate reduction in chloroauric acid (HAuCl4). Briefly, 100 mL of a 0.01 mM HAuCl4 aqueous solution was refluxed and followed by the addition of 10 mL of 1% (w/v) trisodium citrate aqueous solution. The reaction was determined to reach completion when the solution color changed from clear to deep red/purple. To obtain AuNPs with 15 nm diameter and SPR at 520 nm, the AuNPs were then purified by centrifugation at 24,000 ×g for 10 min and resuspended in deionized water. Gold colloid particles were measured by transmission electron microscopy (TEM) imaging. The core nanoparticle diameters were determined using the ImageJ software.
Total RNA was extracted from CCA cell lines using the TRIzol reagent (Gibco, Grand Island, NY) according to the manufacturer's instructions. Then, the first strand of complementary DNA (cDNA) was prepared from 1 μg of total RNA with oligo d (T) primers using the Improm II™ Reverse Transcriptase System (Promega, Madison, WI). Reverse transcription PCR was performed to detect the expression of FA receptor and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; reference gene). The sequences of the primers were as follows: FA receptor forward: 5′-CAAGGTCAGCAACTACAGCCGAG-3′ and reverse: 5′-CATG GCTGCAGCATAGAACCTCG-3′ (product size 109 bp); human GAPDH forward: 5′-GTCAGTGGTGGACCTGACCT-3′ and reverse: 5′-AGGGGA GATTCAGTGTGGTG-3′ (product size 118 bp). Primer sequences of FA receptor and GAPDH were designed based on the cDNA sequences of Homo sapiens; GenBank Accession No. NM_001113535.1 and NG_007073, respectively.
2.2. Functionalization of AuNPs-PEG-backbones-5FU with FA (AuNPs-PEG5FU-FA)
2.6. Cell viability
2.1. Synthesis of gold nanoparticles (AuNPs)
Gold nanoconjugates (AuNPs-PEG) containing polyethylene glycol (PEG-6K) were prepared using single-step incubation by mixing 200 μL of AuNPs solution and 100 μL of aqueous PEG-6 K (1 mM) and shaking at 37 °C for 4 h. After PEG stabilization, the unbound PEG linker was removed by centrifugation at 24,000 × g for 10 min at room temperature. The supernatant was removed and the remaining pellet was resuspended in water. The AuNPs-PEG were functionalized with 5FU by adding 5FU to get final concentration at 10 μM and incubated at 37 °C for 24 h. Subsequently, the solution was centrifuged at 24,000 ×g for 10 min at room temperature; the pellets were collected and resuspended in deionized water. The amount of unbound 5FU in supernatant was determined by the high-performance liquid chromatography (HPLC). The AuNPs-PEG-5FU were functionalized with FA by adding FA to get a final concentration at 5 μg/mL and incubated at 37 °C for 24 h. Subsequently, the solution was centrifuged at 24,000 ×g for 10 min at room temperature, the pellets were collected and resuspended in deionized water. The amount of unbound FA in supernatant was determined by HPLC.
Cell proliferation was assessed by measuring the viable cells using sulforhodamine B (SRB) assay. The cells were seeded in 96-well plates at a density of 8 × 103 cells/well, then incubated at 37 °C with 5% CO2, and left for 24 h to allow formation of confluent monolayer. After 24 h, the media were replaced with different conditions of 5FU, 5FU + FA, AuNPs, AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA in which the final concentration of 5FU and FA in each condition was 9 μM and 2.5 μg/mL, respectively. Cells were incubated at 37 °C with 5% CO2 for 48 h, after which the SRB assay was performed according the previous report [19]. The SRB solution was transferred to a new 96-well plate to prevent the interference of AuNPs absorption.
2.3. Determination of unbound 5FU and FA after AuNPs conjugation by HPLC To determine the binding percentage of 5FU and FA, indirect assay HPLC was used to determine unbound 5FU and FA from the supernatant derived from AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA, respectively. Thirty microliter of the supernatant was injected into the HPLC column, Luna C18 size 15 cm × 3.0 mm (Phenomenex, California). Gradient elution was conducted at a flow rate of 1 mL/min using absolute methanol and 0.5% phosphoric acid. The absorbance at 270 nm (Waters, UV 2479) was detected and the peaks were analyzed by the Clarity program (Waters). The 10 μM of 5FU and 5 μg/mL of FA were used as standards. Mean peak areas of unbound 5FU and FA in the supernatant were calculated by comparison with the standard areas.
Fig. 1. Characterization of AuNPs by UV–VIS spectrum showing peak absorbance at a λmax of 520 nm. The upper-right panel shows the TEM image of AuNPs in H2O at magnification of 100,000×. The average diameter of AuNPs is 15.17 ± 1.69 nm.
N. Ngernyuang et al. / Materials Science and Engineering C 60 (2016) 411–415
Fig. 2. Absorption spectra of AuNPs after functionalization with 9 μM 5FU or 9 μM 5FU + 2.5 μg/mL FA by UV–VIS spectrophotometer scanning in a range of 350–750 nm.
2.7. Detection of AuNPs-PEG-5FU-FA uptake into CCA cell lines M213 cells were seeded in 6-well plates containing DMEM highglucose (DMEM-HG) media (Invitrogen, Carls-bad, CA) for 24 h. Next, AuNPs-PEG-5FU-FA was added into the cells and incubated at 37 °C with 5% CO2 for 48 h. The cells were stained with 5 mg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and analyzed under a fluorescence microscope. To confirm the localization of gold nanoconjugates, M213 cells were treated with AuNPs-PEG-5FU-FA for 12 h. The cells were trypsinized and embedded into resin-embedded cells with uranyl acetate staining as previously described [20] and performed by TEM.
2.8. Apoptosis array The M213 cell line was treated with free drug or AuNPs-PEG-5FU-FA for 48 h and cellular protein was prepared. The protein from each condition (200 mg) was hybridized onto an array membrane containing 35 pro- and apoptotic proteins (ARY009, R&D Systems Inc., MN) according to the manufacturer's instructions. Immunoreactive dots were visualized using an enhanced chemiluminescence detection system (R&D).
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Fig. 4. Endogenous expression of FA receptor mRNA in CCA cell lines. Cells were grown to reach 80% confluence under serum conditions and cultured for 24 h, after which the cells were collected. (A) mRNA was prepared from harvested cells and determined for FA receptor mRNA expression using RT-PCR, in which GAPDH was used as a loading control. (B) mRNA expression of FA receptor was normalized to GAPDH. NTC represents negative control.
2.9. Statistical analysis Statistical analysis was performed by a two-tailed student t-test and P b 0.05 was considered statistically significant.
3. Results and discussion 3.1. Synthesis and characterization of AuNPs AuNPs were obtained by a standard wet chemical method using sodium acetate (CH₃COONa) as a reducing agent as described previously [21]. AuNPs were highly soluble in water and stable under physiological conditions such as in a culture medium. The visible absorption spectra of wine-red colloidal AuNPs showed a well-defined absorption band with λmax = 520 nm (Fig. 1). This value was characteristic of SPR absorbance of spherical AuNPs [22]. The morphology and size distribution of more than 100 gold colloid particles were measured by TEM images. A representative TEM image of these particles is given in Fig. 1. The mean of spherical particle diameter was 15.17 ± 1.69 nm.
Fig. 3. HPLC chromatogram of 5FU (10 μM) and FA (5 μg/mL) standard and unbound 5FU and FA after conjugation.
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3.2. Synthesis and characterization of AuNPs-PEG-5FU and AuNPs-PEG5FU-FA The FA receptor is overexpressed on a wide variety of tumors [15], thus, the FA coupled AuNPs may be used to specifically target cancer cells. In this study, we have developed a targeted nanomedicine by attaching AuNPs to 5FU and FA molecules. In order to conjugate an AuNPs with 5FU and FA, we first synthesized appropriate AuNPs as discussed above. We selected PEG shell as a linker between the AuNPs, 5FU and FA. The UV–VIS spectra of AuNPs, AuNPs-PEG-5FU, and AuNPs-PEG5FU-FA showed a shift of absorption spectrum with λmax 520, 521 and 522 nm, respectively (Fig. 2), suggesting that both 5FU and FA were attached to the gold surface. However, a new absorption around 670 nm was observed in AuNPs-5FU-FA, which may be a sign of aggregation. To prove this event, TEM image should be confirmed. The binding percentage of AuNPs-PEG-5FU-FA was determined by indirect assay of unbound 5FU and FA using HPLC according to 5FU and FA standard retention time at 4 and 12 min, respectively (Fig. 3). The results showed that only 10% of 5FU was found in the supernatant suggesting that 90% of 5FU was conjugated to AuNPs. In the case of FA, 50% of unbound FA was found indicating 50% conjugation. 3.3. The expression of FA receptor in CCA cell lines The endogenous mRNA expression of FA receptor was found in M139 and M213 (Fig. 4). As expected, our finding agreed with the previous report [23], suggesting the potential of FA as targeting molecule. 3.4. The cytotoxicity of gold nanoconjugates in CCA cell lines The cytotoxicity effects of 5FU, 5FU + FA, AuNPs, AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA on CCA cells were measured using SRB assay. AuNPs showed less effect on both M139 and M213 with 4.62% and 2.98%, respectively, compared to control which agreed with the study of Conner and colleagues [24]. Several studies showed that the cytotoxicity of drug conjugated with AuNPs was higher than that of the free drug [25–27]. By contrast, our study showed that cytotoxicity of AuNPs-PEG-5FU on M213 was slightly increased by 1.26% (P = 0.640), while it was slightly decreased in M139 (Fig. 5). Our finding was in accordance with the study of Mohamed et al.
Fig. 6. Localization of AuNPs-PEG-5FU-FA in M213 cell line. (A) Untreated M213 cell line (control, left) and treated with AuNPs-PEG-5FU-FA (right), as visualized by a fluorescence microscope. (B) TEM image of M213 cells incubated with AuNPs-PEG-5FU-FA in media after 12 h.
in colon carcinoma cell lines by which the low concentration of AuNPs5FU (10 μM) had slightly lower effective cytotoxicity than the free 5FU [28]. In addition, cell cytotoxicity assays clearly demonstrated the differences between 5FU + FA and AuNPs-PEG-5FU-FA (Fig. 5). The results showed that AuNPs-PEG-5FU-FA could improve anti-cancer efficacy in CCA cell lines; the cytotoxic effects in the M139 and M213 cells treated with AuNPs-PEG-5FU-FA were 4.76% and 7.95%, respectively, higher than those treated with free 5FU + FA. Our study was in accordance with a previous report [29]. M213 expressed higher FA receptor than M139, suggesting higher cellular uptake of AuNPs-PEG-5FU-FA. In vivo studies have reported the toxicity of AuNPs in blood chemistry, inflammatory and pathological responses [30–32]. However, the biosafety of AuNPs was dependent on their size, composition, and surface characteristics [33,34]. Semmler-Behnke and colleagues demonstrated that a considerable percentage of AuNPs with the size of 18 nm could be removed from the blood and trapped predominantly in the liver and spleen [35]. Our small molecule of AuNPs-PEG-5FU-FA (approximately 15.17 ± 1.69 nm) may be of value for further in vivo applications.
Fig. 5. Percentage of viable cells in different conditions: control, AuNPs, 5FU, 5FU + FA, AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA, respectively. P b 0.05 is considered as a statistical difference.
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Fig. 7. Expression of apoptotic-related proteins in M213 cells after treatment with AuNPs-PEG-5FU-FA. P b 0.05 is considered as a statistical difference.
3.5. Cellular uptake of AuNPs-PEG-5FU-FA The cellular uptake efficiency of drug-conjugated AuNPs may affect the successful therapy. Thus verification of cell loading is very important. Our study showed the localization of AuNPs-PEG-5FU-FA in cytoplasm (Fig. 6). The nanoparticles were up taken into the cells via endocytosis and localized mainly in cytoplasm [36]. 3.6. AuNPs-PEG-5FU-FA mediates cytotoxicity via mitochondrial apoptotic pathway Previous study reported the effect of 5FU on tumor growth by increasing cytochrome C, caspase-3 and caspase-9 [37]. Our finding indicated that AuNPs-PEG-5FU-FA mediates cytotoxicity via mitochondrial apoptotic pathway by predominantly increasing cytochrome C level (P = 0.020, Fig. 7). 4. Conclusion In this report, we synthesized and functionalized AuNPs with 5FU and FA. The biological property of this nanomedicine was tested for its cytotoxic effect on CCA cell lines. The presence of FA on nanoparticles enhances cellular uptake of chemotherapeutic drug which activates the release of mitochondrial cytochrome C leading to apoptosis in cancer cells. The applications of these nanoparticles in vivo regarding stability, biosafety and targeting efficacy should be focused. Acknowledgments This work was supported by the National Research Council of Thailand, grant no. 2556-22 and the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, grant no. CMDL-2557. References [1] P.C. de Groen, G.J. Gores, N.F. LaRusso, L.L. Gunderson, D.M. Nagorney, N. Engl. J. Med. 341 (1999) 1368. [2] M. Aljiffry, M.J. Walsh, M. Molinari, World J. Gastroenterol. 15 (2009) 4240. [3] S. Thongprasert, Ann. Oncol. 16 (2005), ii93. [4] S.A. Khan, B.R. Davidson, R. Goldin, S.P. Pereira, W.M. Rosenberg, S.D. TaylorRobinson, A.V. Thillainayagam, H.C. Thomas, M.R. Thursz, H. Wasan, Gut 51 (2002) VI1.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Targeted delivery of 5-fluorouracil to cholangiocarcinoma cells using folic acid as a targeting agent Nipaporn Ngernyuang a, Wunchana Seubwai b,c, Sakda Daduang d, Patcharee Boonsiri e, Temduang Limpaiboon f,g, Jureerut Daduang f,⁎ a
Chulabhorn International College of Medicine, Thammasat University, Pathum Thani 12120, Thailand Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kean 40002, Thailand Comprehensive Cancer Research Group, Faculty of Medicine, Khon Kaen University, Khon Kean 40002, Thailand d Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand e Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand f Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand g Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand b c
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 13 November 2015 Accepted 23 November 2015 Available online 24 November 2015 Keywords: Cholangiocarcinoma Cancer Nanomedicine Folate receptor Targeted therapy
a b s t r a c t There are limits to the standard treatment for cholangiocarcinoma (CCA) including drug resistance and side effects. The objective of this study was to develop a new technique for carrying drugs by conjugation with gold nanoparticles and using folic acid as a targeting agent in order to increase drug sensitivity. Gold nanoparticles (AuNPs) were functionalized with 5-fluorouracil (5FU) and folic acid (FA) using polyethylene glycol (PEG) shell as a linker (AuNPs-PEG-5FU-FA). Its cytotoxicity was tested in CCA cell lines (M139 and M213) which express folic acid receptor (FA receptor). The results showed that AuNPs-PEG-5FU-FA increased the cytotoxic effects in the M139 and M213 cells by 4.76% and 7.95%, respectively compared to those treated with free 5FU + FA. It is found that the cytotoxicity of the AuNPs-PEG-5FU-FA correlates with FA receptor expression suggested the use of FA as a targeted therapy. The mechanism of cytotoxicity was mediated via mitochondrial apoptotic pathway as determined by apoptosis array. In conclusion, our findings shed some light on the use of gold nanoparticles for conjugation with potential compounds and FA as targeted therapy which contribute to the improvement of anti-cancer drug efficacy. In vivo study should be warranted for its effectiveness of stability, biosafety and side effect reduction. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cholangiocarcinoma (CCA) arises from the epithelial cells of the intrahepatic and extrahepatic bile ducts [1,2]. Surgical resection is currently the only potentially curative treatment for CCA. However, less than 25% of patients are successfully resected [3,4]. Adjuvant chemotherapy has been used in an attempt to improve disease control and quality of life in patients with unresectable, recurrent and metastastic CCA [3]. 5-Fluorouracil (5FU), a water-soluble fluorinated pyrimidine analog that interferes with thymidylate synthesis, has a broad spectrum of activity against solid tumors including CCA. 5FU-based chemotherapy may be efficient in treatment of advanced CCA with less cost, thus, is an appropriate use in a resource-limited country [5]. However, 5FU has limitations including a short biological half-life (10–20 min) due to the rapid absorption through blood capillaries into systemic circulation with plasma, hence high doses are required to achieve ⁎ Corresponding author. E-mail address: [email protected] (J. Daduang).
http://dx.doi.org/10.1016/j.msec.2015.11.062 0928-4931/© 2015 Elsevier B.V. All rights reserved.
therapeutic concentration. Unfortunately, the high doses of this drug are related with the increased risk of serious side effects such as bone marrow depression, gastrointestinal tract reaction, leucopenia and thrombocytopenia. In order to prolong the circulation time of 5FU, increase its efficiency and reduce 5FU-associated side effects, several studies have demonstrated the modification of 5FU delivery by using polymer conjugates or incorporation of 5FU into particulate carriers [6–8]. Recently, gold nanoparticles (AuNPs) have been widely used as chemotherapy delivery vehicles to several cancer cells because of their unusual properties that include relatively high stability, ultra small size, catalytic activity, low toxicity, enhanced Raman signal, surface plasmon resonance (SPR), and facile chemical tailorability [9]. Moreover, AuNPs structures can support multiple functionalities on their surface. These properties of AuNPs may be applied to targeted drug delivery in CCA which could lead to increased efficacy of traditional chemotherapeutics. In addition, folic acid (FA), or vitamin B9, plays a vital role in mammalian cell growth. Moreover, it has been studied extensively as a targeting ligand for imaging and several cancer therapies [10–14]. FA
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has a high binding affinity with the folic acid receptor (FA receptor) which is up-regulated on the surface of many cancer cells, and limited distribution is found in normal tissues [15]. Several studies have reported nanoparticles with FA for site-specific targeting and improvement of FA receptor-mediated uptake by cancer cells [16–18]. In this study, we presented the synthesis and characterization of AuNPs functionalized with 5FU chemotherapeutic drugs and FAspecific ligand as targeted therapy for improvement of CCA treatment efficacy. This strategy could represent a novel modality for effective CCA treatment. 2. Experimental section
2.4. Cell culture Human CCA cell lines, namely M139 and M213, were isolated from Thai CCA patients and established at the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Thailand. Both cell lines were cultured in a RPMI medium supplemented with 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin (Gibco, Life Technologies, NY) at 37 °C in a 5% CO2 humidified incubator. The presence of Mycoplasma contamination was periodically checked.
2.5. Folic acid receptor expression
All glassware used in the preparation and storage of AuNPs was cleaned with HCl:HNO3 (3:1), rinsed with water, and oven-dried. The AuNPs were synthesized via citrate reduction in chloroauric acid (HAuCl4). Briefly, 100 mL of a 0.01 mM HAuCl4 aqueous solution was refluxed and followed by the addition of 10 mL of 1% (w/v) trisodium citrate aqueous solution. The reaction was determined to reach completion when the solution color changed from clear to deep red/purple. To obtain AuNPs with 15 nm diameter and SPR at 520 nm, the AuNPs were then purified by centrifugation at 24,000 ×g for 10 min and resuspended in deionized water. Gold colloid particles were measured by transmission electron microscopy (TEM) imaging. The core nanoparticle diameters were determined using the ImageJ software.
Total RNA was extracted from CCA cell lines using the TRIzol reagent (Gibco, Grand Island, NY) according to the manufacturer's instructions. Then, the first strand of complementary DNA (cDNA) was prepared from 1 μg of total RNA with oligo d (T) primers using the Improm II™ Reverse Transcriptase System (Promega, Madison, WI). Reverse transcription PCR was performed to detect the expression of FA receptor and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; reference gene). The sequences of the primers were as follows: FA receptor forward: 5′-CAAGGTCAGCAACTACAGCCGAG-3′ and reverse: 5′-CATG GCTGCAGCATAGAACCTCG-3′ (product size 109 bp); human GAPDH forward: 5′-GTCAGTGGTGGACCTGACCT-3′ and reverse: 5′-AGGGGA GATTCAGTGTGGTG-3′ (product size 118 bp). Primer sequences of FA receptor and GAPDH were designed based on the cDNA sequences of Homo sapiens; GenBank Accession No. NM_001113535.1 and NG_007073, respectively.
2.2. Functionalization of AuNPs-PEG-backbones-5FU with FA (AuNPs-PEG5FU-FA)
2.6. Cell viability
2.1. Synthesis of gold nanoparticles (AuNPs)
Gold nanoconjugates (AuNPs-PEG) containing polyethylene glycol (PEG-6K) were prepared using single-step incubation by mixing 200 μL of AuNPs solution and 100 μL of aqueous PEG-6 K (1 mM) and shaking at 37 °C for 4 h. After PEG stabilization, the unbound PEG linker was removed by centrifugation at 24,000 × g for 10 min at room temperature. The supernatant was removed and the remaining pellet was resuspended in water. The AuNPs-PEG were functionalized with 5FU by adding 5FU to get final concentration at 10 μM and incubated at 37 °C for 24 h. Subsequently, the solution was centrifuged at 24,000 ×g for 10 min at room temperature; the pellets were collected and resuspended in deionized water. The amount of unbound 5FU in supernatant was determined by the high-performance liquid chromatography (HPLC). The AuNPs-PEG-5FU were functionalized with FA by adding FA to get a final concentration at 5 μg/mL and incubated at 37 °C for 24 h. Subsequently, the solution was centrifuged at 24,000 ×g for 10 min at room temperature, the pellets were collected and resuspended in deionized water. The amount of unbound FA in supernatant was determined by HPLC.
Cell proliferation was assessed by measuring the viable cells using sulforhodamine B (SRB) assay. The cells were seeded in 96-well plates at a density of 8 × 103 cells/well, then incubated at 37 °C with 5% CO2, and left for 24 h to allow formation of confluent monolayer. After 24 h, the media were replaced with different conditions of 5FU, 5FU + FA, AuNPs, AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA in which the final concentration of 5FU and FA in each condition was 9 μM and 2.5 μg/mL, respectively. Cells were incubated at 37 °C with 5% CO2 for 48 h, after which the SRB assay was performed according the previous report [19]. The SRB solution was transferred to a new 96-well plate to prevent the interference of AuNPs absorption.
2.3. Determination of unbound 5FU and FA after AuNPs conjugation by HPLC To determine the binding percentage of 5FU and FA, indirect assay HPLC was used to determine unbound 5FU and FA from the supernatant derived from AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA, respectively. Thirty microliter of the supernatant was injected into the HPLC column, Luna C18 size 15 cm × 3.0 mm (Phenomenex, California). Gradient elution was conducted at a flow rate of 1 mL/min using absolute methanol and 0.5% phosphoric acid. The absorbance at 270 nm (Waters, UV 2479) was detected and the peaks were analyzed by the Clarity program (Waters). The 10 μM of 5FU and 5 μg/mL of FA were used as standards. Mean peak areas of unbound 5FU and FA in the supernatant were calculated by comparison with the standard areas.
Fig. 1. Characterization of AuNPs by UV–VIS spectrum showing peak absorbance at a λmax of 520 nm. The upper-right panel shows the TEM image of AuNPs in H2O at magnification of 100,000×. The average diameter of AuNPs is 15.17 ± 1.69 nm.
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Fig. 2. Absorption spectra of AuNPs after functionalization with 9 μM 5FU or 9 μM 5FU + 2.5 μg/mL FA by UV–VIS spectrophotometer scanning in a range of 350–750 nm.
2.7. Detection of AuNPs-PEG-5FU-FA uptake into CCA cell lines M213 cells were seeded in 6-well plates containing DMEM highglucose (DMEM-HG) media (Invitrogen, Carls-bad, CA) for 24 h. Next, AuNPs-PEG-5FU-FA was added into the cells and incubated at 37 °C with 5% CO2 for 48 h. The cells were stained with 5 mg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and analyzed under a fluorescence microscope. To confirm the localization of gold nanoconjugates, M213 cells were treated with AuNPs-PEG-5FU-FA for 12 h. The cells were trypsinized and embedded into resin-embedded cells with uranyl acetate staining as previously described [20] and performed by TEM.
2.8. Apoptosis array The M213 cell line was treated with free drug or AuNPs-PEG-5FU-FA for 48 h and cellular protein was prepared. The protein from each condition (200 mg) was hybridized onto an array membrane containing 35 pro- and apoptotic proteins (ARY009, R&D Systems Inc., MN) according to the manufacturer's instructions. Immunoreactive dots were visualized using an enhanced chemiluminescence detection system (R&D).
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Fig. 4. Endogenous expression of FA receptor mRNA in CCA cell lines. Cells were grown to reach 80% confluence under serum conditions and cultured for 24 h, after which the cells were collected. (A) mRNA was prepared from harvested cells and determined for FA receptor mRNA expression using RT-PCR, in which GAPDH was used as a loading control. (B) mRNA expression of FA receptor was normalized to GAPDH. NTC represents negative control.
2.9. Statistical analysis Statistical analysis was performed by a two-tailed student t-test and P b 0.05 was considered statistically significant.
3. Results and discussion 3.1. Synthesis and characterization of AuNPs AuNPs were obtained by a standard wet chemical method using sodium acetate (CH₃COONa) as a reducing agent as described previously [21]. AuNPs were highly soluble in water and stable under physiological conditions such as in a culture medium. The visible absorption spectra of wine-red colloidal AuNPs showed a well-defined absorption band with λmax = 520 nm (Fig. 1). This value was characteristic of SPR absorbance of spherical AuNPs [22]. The morphology and size distribution of more than 100 gold colloid particles were measured by TEM images. A representative TEM image of these particles is given in Fig. 1. The mean of spherical particle diameter was 15.17 ± 1.69 nm.
Fig. 3. HPLC chromatogram of 5FU (10 μM) and FA (5 μg/mL) standard and unbound 5FU and FA after conjugation.
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3.2. Synthesis and characterization of AuNPs-PEG-5FU and AuNPs-PEG5FU-FA The FA receptor is overexpressed on a wide variety of tumors [15], thus, the FA coupled AuNPs may be used to specifically target cancer cells. In this study, we have developed a targeted nanomedicine by attaching AuNPs to 5FU and FA molecules. In order to conjugate an AuNPs with 5FU and FA, we first synthesized appropriate AuNPs as discussed above. We selected PEG shell as a linker between the AuNPs, 5FU and FA. The UV–VIS spectra of AuNPs, AuNPs-PEG-5FU, and AuNPs-PEG5FU-FA showed a shift of absorption spectrum with λmax 520, 521 and 522 nm, respectively (Fig. 2), suggesting that both 5FU and FA were attached to the gold surface. However, a new absorption around 670 nm was observed in AuNPs-5FU-FA, which may be a sign of aggregation. To prove this event, TEM image should be confirmed. The binding percentage of AuNPs-PEG-5FU-FA was determined by indirect assay of unbound 5FU and FA using HPLC according to 5FU and FA standard retention time at 4 and 12 min, respectively (Fig. 3). The results showed that only 10% of 5FU was found in the supernatant suggesting that 90% of 5FU was conjugated to AuNPs. In the case of FA, 50% of unbound FA was found indicating 50% conjugation. 3.3. The expression of FA receptor in CCA cell lines The endogenous mRNA expression of FA receptor was found in M139 and M213 (Fig. 4). As expected, our finding agreed with the previous report [23], suggesting the potential of FA as targeting molecule. 3.4. The cytotoxicity of gold nanoconjugates in CCA cell lines The cytotoxicity effects of 5FU, 5FU + FA, AuNPs, AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA on CCA cells were measured using SRB assay. AuNPs showed less effect on both M139 and M213 with 4.62% and 2.98%, respectively, compared to control which agreed with the study of Conner and colleagues [24]. Several studies showed that the cytotoxicity of drug conjugated with AuNPs was higher than that of the free drug [25–27]. By contrast, our study showed that cytotoxicity of AuNPs-PEG-5FU on M213 was slightly increased by 1.26% (P = 0.640), while it was slightly decreased in M139 (Fig. 5). Our finding was in accordance with the study of Mohamed et al.
Fig. 6. Localization of AuNPs-PEG-5FU-FA in M213 cell line. (A) Untreated M213 cell line (control, left) and treated with AuNPs-PEG-5FU-FA (right), as visualized by a fluorescence microscope. (B) TEM image of M213 cells incubated with AuNPs-PEG-5FU-FA in media after 12 h.
in colon carcinoma cell lines by which the low concentration of AuNPs5FU (10 μM) had slightly lower effective cytotoxicity than the free 5FU [28]. In addition, cell cytotoxicity assays clearly demonstrated the differences between 5FU + FA and AuNPs-PEG-5FU-FA (Fig. 5). The results showed that AuNPs-PEG-5FU-FA could improve anti-cancer efficacy in CCA cell lines; the cytotoxic effects in the M139 and M213 cells treated with AuNPs-PEG-5FU-FA were 4.76% and 7.95%, respectively, higher than those treated with free 5FU + FA. Our study was in accordance with a previous report [29]. M213 expressed higher FA receptor than M139, suggesting higher cellular uptake of AuNPs-PEG-5FU-FA. In vivo studies have reported the toxicity of AuNPs in blood chemistry, inflammatory and pathological responses [30–32]. However, the biosafety of AuNPs was dependent on their size, composition, and surface characteristics [33,34]. Semmler-Behnke and colleagues demonstrated that a considerable percentage of AuNPs with the size of 18 nm could be removed from the blood and trapped predominantly in the liver and spleen [35]. Our small molecule of AuNPs-PEG-5FU-FA (approximately 15.17 ± 1.69 nm) may be of value for further in vivo applications.
Fig. 5. Percentage of viable cells in different conditions: control, AuNPs, 5FU, 5FU + FA, AuNPs-PEG-5FU and AuNPs-PEG-5FU-FA, respectively. P b 0.05 is considered as a statistical difference.
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Fig. 7. Expression of apoptotic-related proteins in M213 cells after treatment with AuNPs-PEG-5FU-FA. P b 0.05 is considered as a statistical difference.
3.5. Cellular uptake of AuNPs-PEG-5FU-FA The cellular uptake efficiency of drug-conjugated AuNPs may affect the successful therapy. Thus verification of cell loading is very important. Our study showed the localization of AuNPs-PEG-5FU-FA in cytoplasm (Fig. 6). The nanoparticles were up taken into the cells via endocytosis and localized mainly in cytoplasm [36]. 3.6. AuNPs-PEG-5FU-FA mediates cytotoxicity via mitochondrial apoptotic pathway Previous study reported the effect of 5FU on tumor growth by increasing cytochrome C, caspase-3 and caspase-9 [37]. Our finding indicated that AuNPs-PEG-5FU-FA mediates cytotoxicity via mitochondrial apoptotic pathway by predominantly increasing cytochrome C level (P = 0.020, Fig. 7). 4. Conclusion In this report, we synthesized and functionalized AuNPs with 5FU and FA. The biological property of this nanomedicine was tested for its cytotoxic effect on CCA cell lines. The presence of FA on nanoparticles enhances cellular uptake of chemotherapeutic drug which activates the release of mitochondrial cytochrome C leading to apoptosis in cancer cells. The applications of these nanoparticles in vivo regarding stability, biosafety and targeting efficacy should be focused. Acknowledgments This work was supported by the National Research Council of Thailand, grant no. 2556-22 and the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, grant no. CMDL-2557. References [1] P.C. de Groen, G.J. Gores, N.F. LaRusso, L.L. Gunderson, D.M. Nagorney, N. Engl. J. Med. 341 (1999) 1368. [2] M. Aljiffry, M.J. Walsh, M. Molinari, World J. Gastroenterol. 15 (2009) 4240. [3] S. Thongprasert, Ann. Oncol. 16 (2005), ii93. [4] S.A. Khan, B.R. Davidson, R. Goldin, S.P. Pereira, W.M. Rosenberg, S.D. TaylorRobinson, A.V. Thillainayagam, H.C. Thomas, M.R. Thursz, H. Wasan, Gut 51 (2002) VI1.
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