Anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated fluorescent nanoparticles probe for breast cancer imaging

Spectrochimica Acta Part A 74 (2009) 410–414

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated fluorescent nanoparticles probe for breast cancer imaging Xu Hun a,b,∗ , Zhujun Zhang c a

College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China c Department of Chemistry, School of Chemistry and Materials Science, Shaanxi Normal University, Xi’an 710062, China b

a r t i c l e

i n f o

Article history: Received 10 March 2009 Accepted 10 June 2009 Keywords: Anti-epidermal growth factor receptor (anti-EGFR) antibody Fluorescent nanoparticles (FNs) Breast cancer cell Fluorescence microscopy imaging

a b s t r a c t Fluorescent nanoparticles (FNs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. In this study, anti-EGFR antibody conjugated fluorescent nanoparticles (FNs) (anti-EGFR antibody conjugated FNs) probe was used to detect breast cancer cells. FNs with excellent character such as non-toxicity and photostability were first synthesized with a simple, cost-effective and ˝ environmentally friendly modified Stober synthesis method, and then successfully modified with antiEGFR antibody. This kind of fluorescence probe based on the anti-EGFR antibody conjugated FNs has been used to detect breast cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-EGFR antibody conjugated FNs can effectively recognize breast cancer cells and exhibited good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of breast cancer cells and offer a new method in detecting EGFR. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Although lung cancer is now the leading cause of cancer death among women [1–3], breast cancer still constitutes the most commonly diagnosed malignancy in women after skin cancer. Early cancer diagnosis, in combination with the precise cancer therapies could eventually save millions of lives. Over the last 70 years, despite tremendous advances in our understanding of the molecular and cellular processes of cancer, there has been no change in the age-adjusted mortality due to cancer [4]. In order to further reduce the morbidity and mortality due to cancer, the diagnosis of cancer at the early stage is extremely challenging and has been an active research area these days. The integration of nanotechnology with biology and medicine is expected to produce major advances in molecular diagnostics, therapeutics, molecular biology, and bioengineering [5]. Bio-labeling with fluorescent dyes has many applications in biomedical science, especially in the field of cancer imaging and cancer targeting. Fluorescence microscopy is among the most widely used approaches for high resolution, noninvasive imaging

∗ Corresponding author at: College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Zhengzhou Road 53, Qingdao 266042, China. Tel.: +86 532 84022946; fax: +86 532 84022750. E-mail address: [email protected] (X. Hun). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.06.033

of live organisms and organic fluorophores are the most commonly used tags for fluorescence-based imaging. Despite their considerable advantages in live cell imaging, organic fluorophores suffer from the disadvantages like the inefficiency of labeling, lacking of photostability in addition to the problem of relatively low fluorescence intensity of dyes. In addition to small organic fluorophores, FNs represent a promising new generation fluorescent label, owing to its inherent advantages. As the most important and be extensively studied FNs in past few years is the dye-doped silica nanoparticles showing following advantages [6–14]. (1) High intensity of the fluorescent signal for thousands of fluorescent dye molecules encapsulated in the nanoparticle matrix, (2) excellent photostability due to exclusion of oxygen by shell of nanoparticles, (3) efficient conjugation with various biomolecules due to the nanoparticles surface easily to be modified, (4) easy manufacturing process, (5) size uniformity and tunebility and (6) especially, the nanoparticles with silica surface are water miscible and can be easily modified. Using this kind of FNs, the feasibility of developing optical imaging technique for the sensitive detection of cancer has been recently demonstrated [15–24]. And out of the many techniques to prepare nanoparticles, the inverse microemulsion polymerization is one most widely used method to prepare this kind of FNs in the issued articles. However, this technique requires large amounts of surfactants and organic solvents. Therefore, scaling-up these procedures proved to be difficult because it is pollutional to the environment, high costs and

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time-consuming. In this article, we use a simple, cost-effective ˝ and environmentally friendly modified Stober synthesis method for the preparation of the FNs, and then the FNs were successfully modified with anti-epidermal growth factor receptor (anti-EGFR) antibody. This kind of fluorescence probe based on the anti-EGFR antibody conjugated fluorescent nanoparticles (FNs) has been used to detect MCF-7 breast cancer cells (which has levels of EGFR expression 104 receptors/cell) [25], breast cancer cells with fluorescence microscopy imaging technology. 2. Materials and methods 2.1. Reagents Tris(2,2 -bipyridyl)dichlororuthenium(II) (Ru(bpy)3 2+ ) was purchased from Sigma (St. Louis, MO, USA). (3-aminopropyl)triethoxysilane (APS) was obtained from Acros (Belgium, USA). Tetraethyl orthosilicate (TEOS) and ethanol were obtained from Shanghai Chemical Plant (Shanghai, China). KH2 PO4 , Na2 HPO4 and NH3 ·H2 O (28–30 wt.%) were purchased from Xi’an Chemical Reagent Company (Xi’an, China). Anti-EGFR antibody was obtained from Beijing Boisynthesis Biotechnology Co. Ltd. (Beijing, China). Unless otherwise stated, all chemicals and reagents used in this study were of analytical grade quality. 2.2. Synthesis of fluorescent nanoparticles Preparation of fluorescent nanoparticles (FNs) was carried out according to method described by previous paper with some changes [26,27]. The procedure is briefly described in the following. At first, 12.5 mL ethanol, 750 ␮L ammonium hydroxide and amount of Ru(bpy)3 2+ solution were mixed by magnetic force stirring at room temperature (RT) for 5 min. Then, the resulting mixture was added drop by drop to a TEOS and APS mixtured solution in ethanol (2.5 mL) under continuing stirring. When the solution was added completely, the reaction mixture was allowed to stir for 2 h. And finally, the particles were recovered by centrifugation at 8000 rpm for 10 min. Then they were washed using centrifugation and ultrasonication with water several times to remove physically adsorbed Ru(bpy)3 2+ dye from the particle’s surface. The particles were air dried. The synthesized FNs were characterized by spectrofluorometry for fluorescence intensity, transmission electron microscope (TEM; Hitachi H700) for size and morphology. 2.3. Cell culture The MCF-7 breast cancer cell and MRC-5 normal cell were obtained from Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). MCF-7 breast cancer cell and MRC-5 normal cell were routinely maintained in MEM medium (HyClone Biochemical Product (Beijing) Co. Ltd., Beijing, China) containing 5% FBS (fetal serum bovine) at 37 ◦ C in a humidified 5% CO2 –95% air atmosphere. To performance cell imaging using anti-EGFR antibody conjugated FNs the cells were first plated on poly-l-lysine-coated six-well plastic dishes for 24 h for cancer cell and 48 h for normal cell before microscopy observation. And then anti-EGFR antibody conjugated FNs-dispersed culture media was added. Incubated for another a fixed time, the cells were washed several times with phosphate buffer solution (pH 7.4 PBS) to remove nonspecifically adsorbed anti-EGFR antibody conjugated FNs. The plastic dishes with cells were mounted over the microscope stage for fluorescence microscopy observation. During observation by microscopy, an incubator was mounted on the stage for the maintenance of proper temperature and humidity.

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2.4. Covalent immobilization of anti-EGFR antibody onto fluorescent nanoparticles surface The anti-EGFR antibody was directly immobilized onto the FNs with well-established glutaraldehyde method [28,29] The immobilization protocols were the following: (1) 2 mg of FNs was dispersed into the PBS buffer containing 5% glutaraldehyde for about 2 h; (2) the nanoparticles were separated by centrifugation and washed with PBS three times. After the nanoparticles re-dispersed in PBS, they were further incubated with anti-EGFR antibody for 12 h at 4 ◦ C with shaking; (3) the anti-EGFR antibody conjugated FNs were washed with PBS several times to remove excess antibody and kept at 4 ◦ C in PBS. 2.5. Fluorescence microscopy imaging Fluorescence microscopy imaging was performed in Olympus inverted microscope system (Olympus, Model IX70, Toyko, Japan) with a 100 W high pressure mercury lamp (Olympus, Model BH2RFL-T3, Tokyo, Japan) used as the light source. The six-well plastic dishes used mentioned above was mounted over the microscope stage. The excitation light which comes from high pressure mercury was introduced through the inverted microscope objective from underneath the chamber. The fluorescence image of the cell is collected by a microscope objective [30]. A CCD camera (Pixera, model PVC100C, Los Gatos, CA, USA) interfaced with a Pentium computer was employed for the acquisition of imaging of the cell. The software of 1.2 Million Pixel Digital Camera v2.5 was used to deal with the imaging. 3. Results and discussion 3.1. Fluorescent nanoparticles formation Silica nanoparticles can be formed by either an acid-catalyzed ˝ reaction or a base-catalyzed (Stober) reaction. The base-catalyzed reaction, using ammonia, ethanol, water, and TEOS, can be controlled to yield spherical silica particles with low size polydispersity ˝ [31]. In this paper, the FNs were prepared with a modified Stober ˝ synthesis method. The Stober synthesis method was also applied to prepare the dye-doped silica nanoparticles, but most of them need of conjunction of dye and organoalkoxysilanes coupling agent [32,33]. We found that the electroactive Ru(bpy)3 2+ can be embedded into silica nanoparticles, which maybe was attributed to strong electrostatic attractions between the positively charged ruthenium complex and the negatively charged silica. Unlike inverse microemulsion-based methods often used to prepare luminescent silica particles this technique completely avoids the use of potentially toxic organic solvents and surfactants and also is timesaving. Further conjugation of the particles to biomolecules is easier because there is no need to wash the particles off surfactant molecules, which often requires multiple washing steps when inverse microemulsion techniques are used to prepare nanoparticles. So this method would be used easily in the research field of biology and medicine. 3.2. Fluorescent nanoparticles–anti-EGFR antibody conjugation The EGFR is a glycoprotein that is found to be vastly expressed in a wide variety of human tumors, especially in epithelial cancer cells. In this study, cell imaging was performed based on interaction between EGFR on the surface of breast cancer cell and anti-EGFR antibody conjugated FNs [34,35]. After the hydrolysis reaction of TEOS and APS, some amino groups have been introduced to the nanoparticle’s surface. These amino groups made the modification and the bioconjugation of the nanoparticles easier. FNs–anti-EGFR

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Fig. 1. Schematic representation of the fabrication of a FCFNs (a) and its capturing of a cancer cell (b).

antibody conjugation were developed using the coupling strategy between anti-EGFR antibody and nanoparticles using glutaraldehyde as the coupling agent. Fig. 1 depicts the fabrication of the anti-EGFR antibody conjugated FNs and how it captures a cancer cell. With such a unique labeling method, thousands of Ru(bpy)3 2+ could be labeled with the anti-EGFR antibody, which led to the strong fluorescence signal and finally resulted in the increased sensitivity. 3.3. Characteristics of fluorescent nanoparticles–anti-EGFR antibody conjugation ˝ The modified Stober synthesis method yielded relatively uniform FNs. These nanoparticles were characterized using microscopic methods. Fig. 2 shows transmission electron microscopic photographs of (left) FNs and (right) anti-EGFR antibody conjugated FNs. It can be seen the FNs and the anti-EGFR antibody conjugated FNs were found to be of good dispersivity. And the particle sizes of nanoparticles were about 45 ± 5 nm. The TEM photograph indicated that the conjugation step did not result in aggregation. This property allowed the anti-EGFR antibody conjugated FNs to keep their efficient reactivity in solution. In addition, spectrofluorometric measurements were used to characterize the FNs and the anti-EGFR antibody conjugated FNs. The FNs emitted fluorescence light at 595 nm when excited at 435 nm in aqueous solution. However the emission maximum of the anti-EGFR antibody conjugated FNs shifted with 5 nm to the longer wavelength, indicating that the spectral characteristics of the antiEGFR antibody conjugated FNs were changed only insignificantly after conjugation with anti-EGFR antibody (Fig. 3). The comparison of the two spectra indicated that the optical properties of anti-EGFR antibody conjugated FNs remain unchanged after their conjugation to anti-EGFR antibody molecules. Furthermore, the fluorescence intensity of anti-EGFR antibody conjugated FNs solution was investigated. It was found that the fluorescence intensity of the anti-EGFR antibody conjugated FNs solution varied with the variation of the concentration of the Ru(bpy)3 2+ solution. The Ru(bpy)3 2+ concentration in the prepara-

Fig. 2. TEM image of (left) FNs and (right) after their conjugation to anti-EGFR antibody. Scale bars are 100 nm.

Fig. 3. Fluorescence emission spectra of FNs (A) and after their conjugation to antiEGF antibody (B) in aqueous solution.

tion solution was optimized to yield anti-EGFR antibody conjugated FNs with the fluorescence intensity. We observed that the fluorescence intensity was enhanced significantly when the Ru(bpy)3 2+ concentration was increased from 4, 8, 12, 16, 20, 25 to 30 mmol/L. However, at concentrations >25 mmol/L the fluorescence intensity became more flattened. So the FNs prepared with 25 mmol/L Ru(bpy)3 2+ was used for further investigation (Fig. 4). 3.4. Cell imaging When incubated in the presence of nanoparticles, the cells grow at a normal rate and the nanoparticles target the cells. Using fluorescence microscopy, the imaging of anti-EGFR antibody conjugated FNs from single cells is obtained shown in Fig. 5(a). It was found that the FNs with anti-EGFR antibody could capture MCF-7 cell about 30 min incubation. Control experiments showed that the

Fig. 4. Effect of concentration of Ru(bpy)3 2+ in the preparation solution on the fluorescence intensity.

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Fig. 5. Fluorescence microscopic images of MCF-7 cells (a) and MRC-5 cells (b) after incubation with anti-EGF antibody conjugated FNs and (c) MCF-7 cells with anti-EGF antibody-free FNs.

anti-EGFR antibody conjugated FNs were incubated with MRC-5 cells (a anti-EGFR-deficient normal human cell) for 30 min under the same conditions, no capture occurred (Fig. 5(b)). For anti-EGFR antibody-free FNs, there was no obvious interaction between the FNs and MCF-7 cells after 30 min incubation. Therefore, no fluorescence was observed on the cell surface (Fig. 5(c)). All of these results indicate that the anti-EGFR antibody conjugated FNs can capture the MCF-7 cells through the specific recognition interaction between anti-EGFR antibody conjugated FNs and EGFR. Photobleaching is known to be dependent on solvent interactions and is thought to occur as a bimolecular reaction between the dye and, for example, dissolved oxygen [36,37]. To investigate the photostability of the FNs and anti-EGFR antibody conjugated FNs the intensity of the fluorescence was monitored vs. time [24,38]. Measurements were performed in solution of FNs, antiEGFR antibody conjugated FNs or free dye. The solution under stirring was continuously illuminated by the Xenon lamp which was 150 W at its optimal excitation for 60 min using spectrofluorophotometer (Shimadzu RF-540), and the fluorescence intensity was acquired every 2 min over a 60 min period. Fig. 5 shows the photobleaching behavior of the FNs, anti-EGFR antibody conjugated FNs and free dye. It can be seen that FNs and anti-EGFR antibody conjugated FNs show significantly less photobleaching (9.8%, 7.1%) than the free dye (55.3%), even after continuous excitation for 60 min. The photostablity of anti-EGFR antibody conjugated FNs targeting MCF-7 cells was also investigated. In this experiment the specimens were continuously illuminated for about 50 min with light from a 100 W high mercury lamp coming from the inverted microscope objective of the Olympus inverted microscope system. The experimental results showed that the fluorescence intensity of the targeting cells have no obvious change during the entire 50 min illumination period. And this result comes with the experimental phenomenon of Bagwe et al. [39] suggesting that the silica matrix surrounding Ru(bpy)3 2+ molecules acted as a barrier to protect Ru(bpy)3 2+ from the surrounding environment. 4. Conclusions FNs are a good alternative to the use of organic dyes and quantum dots for biological imaging. Their resistance to photobleaching and brightness make them more attractive for use in imaging. Additionally, the ability to add targeting receptors makes them attractive for use in detecting biomarkers for cancer. Preliminary results in our study show demonstrated a simple, cost-effective and environmen˝ tally friendly modified Stober synthesis method for the preparation of the FNs and a simple and rapid method for detection of MCF-7 cell. Anti-EGFR antibody conjugated FNs have been successfully targeted breast cancer cells. This research work clearly demonstrates

the feasibility of developing FNs probe-based breast cancer detection system. Acknowledgement We gratefully acknowledge Chinese Natural Science Foundation (Project no. 30470886) and the Scientific and Technical Development Project of Qingdao (09-1-3-45-jch) for financial support. References [1] L.P. Stabile, A.L. Davis, C.T. Gubish, T.M. Hopkins, J.D. Luketich, N. Christie, Cancer Res. 62 (2002) 2141–2150. [2] L. Stabile, J. Siegfried, Curr. Oncol. Rep. 6 (2004) 259–267. [3] R.J. Pietras, D.C. Marquez, H.W. Chen, E. Tsai, O. Weinberg, M. Fishbein, Steroids 70 (2005) 372–381. [4] Atlanta, Am. Cancer Soc. (2005) 2–3. [5] X.H. Gao, W.C.W. Chan, S.M. Nie, J. Biomed. Opt. 7 (4) (2002) 532–537. [6] Y.-E. Lee-Koo, Y. Cao, R. Kopelman, S.M. Koo, M.G. Brasuel, M.A. Philbert, Anal. Chem. 76 (2004) 2498–2505. [7] W.H. Tang, H. Xu, R. Kopelman, M.A. Philbert, Photochem. Photobiol. 81 (2005) 242–249. [8] J.P. Sumner, N.M. Westerberg, A.K. Stoddard, C.A. Fierke, R. Kopelman, Sens. Actuator B 113 (2005) 760–767. [9] S.M. Buck, H. Xu, M. Brasuel, M.A. Philbert, R. Kopelman, Talanta 63 (2004) 41–59. [10] J.P. Sumner, J.W. Aylott, E. Monson, R. Kopelman, Analyst 127 (2002) 11–16. [11] S.M. Buck, Y-E.L. Koo, E. Park, H. Xu, M.A. Philbert, M.A. Brasuel, Curr. Opin. Chem. Biol. 8 (2004) 540–546. [12] E.J. Park, M. Brasuel, C. Behrend, M.A. Philbert, R. Kopelman, Anal. Chem. 75 (2003) 3784–3791. [13] J.P. Sumner, R. Kopelman, Analyst 130 (2005) 528–533. [14] X. Hun, Z.J. Zhang, Microchim. Acta 159 (2007) 255–262. [15] S. Santra, D. Dutta, G.A. Walter, B.M. Moudgil, Cancer Res. Treat. 4 (2005) 593–602. [16] S. Santra, B. Liesenfeld, D. Dutta, D. Chatel, C.D. Batich, W.H. Tan, B.M. Moudgil, R.A. Mericle, J. Nanosci. Nanotechnol. 5 (2005) 899–904. [17] X.X. He, J.H. Duan, K.M. Wang, W.H. Tan, X. Lin, C.M. He, J. Nanosci. Nanotechnol. 4 (2004) 585–589. [18] M. Qhobosheane, S. Santra, P. Zhang, W.H. Tan, Analyst 126 (2001) 1274–1278. [19] S. Santra, D.B.M. Dutta, Food Bioprod. Process. 83 (2005) 136–140. [20] J.W. Lee, J.Y. Lu, P.S. Low, P.L. Fuchs, Bioorgan. Med. Chem. 10 (7) (2002) 2397–2414. [21] S. Santra, H. Yang, D. Dutta, J.T. Stanley, P.H. Holloway, W.H. Tan, B.M. Moudgil, R.A. Mericle, Chem. Commun. 24 (2004) 2810–2811. [22] M.F. Kircher, U. Mahmood, R.S. King, R. Weissleder, L. Josephson, Cancer Res. 63 (2003) 8122–8125. [23] M.M. Hüber, A.B. Staubli, K. Kustedo, H.B.M. Gray, J. Shih, S.E. Fraser, R.E. Jacobs, T.J. Meade, Bioconjugate Chem. 9 (1998) 242–249. [24] S. Santra, K.M. Wang, R.W.H. Tapec Tan, J. Biomed. Opt. 6 (2001) 160–166. [25] C. Mamot, D.C. Drummond, U. Greiser, K. Hong, D.B. Kirpotin, J.D. Marks, J.W. Park, Cancer Res. 63 (2003) 3154–3161. [26] C.E. Moran, G.D. Hale, N.J. Halas, Langmuir 17 (2001) 8376–8379. [27] L.M. Rossi, L.F. Shi, F.H. Quina, Z. Rosenzweig, Langmuir 21 (10) (2005) 4277–4280. [28] M.V. Kiselev, A.K. Gladilin, N.S. Melik-Nubarov, P.G. Sveshnikov, P. Miethe, A.V. Levashov, Anal. Biochem. 269 (1999) 393–398. [29] I. Willner, E. Katz, Angew. Chem. Int. Ed. 39 (2000) 1180–1218. [30] K.P. McNamara, Z. Rosenzweig, Anal. Chem. 70 (22) (1998) 4853–4859. ˝ [31] W. Stober, A. Fink, E. Bohn, J. Colloid Interf. Sci. 26 (1968) 62–69.

414 [32] [33] [34] [35]

X. Hun, Z. Zhang / Spectrochimica Acta Part A 74 (2009) 410–414

X.J. Zhao, R. Tapec-Dytioco, W.H. Tan, J. Am. Chem. Soc. 125 (2003) 11474–11475. M.M. Collinson, Trends Anal. Chem. 21 (1) (2002) 31–39. H. Lee, M. Hu, R.M. Reilly, C. Allen, Mol. Pharm. 4 (5) (2007) 769–781. M. Hu, D. Scollard, C. Chan, P. Chen, K. Vallis, R.M. Reilly, Nucl. Med. Biol. 34 (8) (2007) 887–896. [36] L. Song, E.J. Hennink, I.T. Young, H.J. Tanke, Biophys. J. 68 (1995) 2588–2600.

[37] S.A. Soper, H.L. Nutter, R.A. Keller, L.M. Davis, E.B. Shera, Photochem. Photobiol. 57 (1993) 972–977. [38] W. Lian, S.A. Litherland, H. Badrane, W.H. Tan, D.H. Wu, H.V. Baker, Anal. Biochem. 334 (2004) 135–144. [39] R.P. Bagwe, C.Y. Yang, L.R. Hilliard, W.H. Tan, Langmuir 20 (2004) 8336–8342.