Conjugation of monoclonal antibodies to super paramagnetic iron oxide nanoparticles for detection of her2/neu antigen on breast cancer cell lines.
Molecular probes for biomolecular recognition are of great importance in the fields of chemistry, biology, medical sciences and in biotechnology as well. These probes have been used in studies of biological functions and in ultrasensitive detection of biological factors responsible for many diseases (1). On the other front, the developments of nontoxic and biocompatible magnetic particles have been disclosed for biological applications since mid-1980s (2).
Recently, magnetic particles have attracted growing interest as high performance biomaterial which is used for transport and separation of cells or cell parts (2, 3), MRI (4), hyperthermia (5) and drug delivery (6). Biological samples such as blood, serum, cell suspensions and cell lysates are allowed to be exposed to specific ligand-coupled particles, and the captured molecules or cells are then rapidly separated using magnetic fields (3,7,8).
Magnetic particles conjugated with anti tumor monoclonal antibodies provide a new approach to identify tumor cells.
Antibodies labeled with magnetic nanoparticles give magnetic signals on exposure to a magnetic field. Iron oxide particles are usually coated with different organic shells including dextran, albumin or polyethylene glycol. Coated nanoparticles can be manufactured with a variety of functional groups (such as amino, aldehyde, hydroxyl, sulfate and carboxyl groups) on their surfaces. Considering these properties, we used super paramagnetic iron oxide (nanomag-D-SPIO 20nm) with COOH group on the surface, for conjugation to a humanized anti her2/neu monoclonal antibody (Herceptin) as a cancer targeting antibody.
Materials and Methods
Nanomag-D-SPIO 20 nm nanoparticles (surface COOH) and MACS separator with MS columns were purchased from Micromod (Miltenyi Biotech GmbH, Germany). The breast carcinoma cell lines SKBR-3 and T47D were obtained from Pasteur Institute of Iran. Other reagents and chemicals were obtained from Merck and Sigma.
Conjugation of anti her2 antibody (Herceptin) with nanoparticles by EDC method
N-ethyl-N-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC, 26mM) and 10 mM N-hydroxy succinimide (NHS) were dissolved in 0.1 M 2-(N-morpholino) ethane-sulfonic acid (MES) buffer (pH=8.3). The mixture was added to 1 ml of 5 mg/ml nanomag-D-SPIO 20 nm nanoparticles, and shaken at room temperature for 2 hours.
[FIGURE 1 OMITTED]
The particles were washed twice with phosphate buffered saline (PBS) pH=7.4 and then 0.5-1 mg/ml of Herceptin was added to the activated particles. The mixture was shaken for 3 hours and the reaction was quenched by the addition of glycine for 30 minutes (9, 10).
The unconjugated antibodies were separated from conjugated antibodies by MACS column. The amount of immobilized antibody was estimated based on the Bradford method.
Spectrophotometric measurement of Iron
Iron concentration of conjugated samples was obtained by potassium thiocyanate method (12,13). In brief, samples were diluted with 300 [micro]l 6N HCl containing %1 H2O2; under this condition, the iron in the samples is dissolved and oxidized to ferric state. The samples were then added to a 5% solution of potassium thiocyanate where the Fe III formed a red complex with the thiocyanate which could be measured by absorbance at 480 nm.
The her2/neu expressing cell lines SKBR3 and T47D were grown in RPMI 1640 medium with 10% (v/v) fetal calf serum and %1 penicillin/streptomycin. Cells were incubated at 37[degrees]C containing 5% Co2.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
To verify the expression of her2 proteins on the cells, the SKBR3 and T47D cells were incubated with anti her2/neu (Herceptin) at 10 [micro]g/ml concentration for 1 hour at 37[degrees]C. After being washed in PBS, FITC-labeled anti human IgG (diluted 1/20, Avicenna Research Institute, Tehran, Iran) was added and incubated for 1 hour at room temperature. Cells were then observed directly on a fluorescence microscope (Olympus, Japan).
In vitro cell labeling
SKBR3 and T47D cells were counted and adjusted to a suspension of 4x[10.sup.5] cells/ml; 100 [micro]l of each cell suspension were cytospined on microscope slides (Shandon cytospin 4, Thermo, Germany). The cells were incubated with 100 [micro]l magnetic nanoparticles (with or without antibody; 5 [micro]g Ab and 0.2 mg iron) for 1 hour at 37[degrees]C. Then cells were washed extensively with PBS to remove unbound particles.
The nanoparticles that bounded on the cell surface were detected by iron staining with Prussian blue staining method (14, 15).
We used 20nm nanoparticles (a magnetic core covered with dextran) with carboxyl group for conjugation to Herceptin as a cancer targeting antibody. The final products of conjugation were suspensions without precipitate and the amount of immobilized antibody was 20-36 [micro]g Ab/mg nanoparticles (Figure 1).
The amount of iron conjugated was determined by potassium thiocyanate method using FeCl3-6H2O as a reference (Figure 2).
The SKBR3 and T47D cell lines showed high levels of her2/neu expression, after incubation with Herceptin-FITC (Figure 3), but not with the isotype control antibody (data not shown).
These results confirmed the presence of antigen on the cell surface. Specific binding of the conjugates to the cells can be determined by using electron microscopy, MRI imaging and iron staining (13,18,19).
In this study after incubation of the cells with conjugated nanoparticles, the blue stain induced by iron staining showed the accumulation of conjugated nanoparticles on the cell surface (Figure 4).
[FIGURE 4 OMITTED]
Polymer-coated magnetic particles with particle sizes 5-500 nm have been employed in medicine or biotechnology for many years (16). In this study the 20 nm nanoparticles were coupled via their surface carboxyl group to the amino groups on the Herceptin antibody using the EDC method (10, 17).
After conjugation, the amount of immobilized antibody was approximately 20 [micro]g/mg magnetite. However by increasing concentration of antibody during the process, the efficiency of the conjugation did not improve. In other studies the efficiency of conjugation has been reported as 5-20 [micro]g Ab/mg particles (9,18). Conjugated nanoparticles bound specifically to the her2/neu antigen.
Iron staining, confirmed the presence of nanoparticles on the cell surface. In the present study we showed specific binding of Herceptin-nanomagnetic particle conjugates to her2/neu over expressing cells, suggesting a future application of Herceptin-magnetite for MR imaging of breast cancer.
This work was supported by a grant from the Nanotechnology Committee of Iran's Ministry of Health and Medical Education.
Received: 21 Feb 2009
Accepted: 21 March 2009
(1.) Santra S, Zhang P, Wang K, Tapec R, Tan W. Conjugation of biomolecules with luminophoredoped silica nanoparticles for photostable biomarkers. Anal Chem 2001;73(20):4988-4993.
(2.) Gruettner C, Teller J, Westphal F, Ivkov R. Magnetic nanoparticle compositions and methods related thereto. Patent application publication 2005. Pub No: US 2005 /0271745 A1 Pub Date: Dec 8, 2005.
(3.) Nagasaki Y, Kobayashi H, Katsuyama Y, Jomura T, Sakura T. Enhanced immunoresponse of antibody mixed-PEG coimmobilized surface construction of high performance immunomagnetic ELISA system. J Colloid Interface Sci 2007;309(2):524-530.
(4.) Baio G, Fabbi M, d.Totero D, Ferrini S, Cilli M, Derchi LE, Neumaier CE. Magnetic resonance imaging at 1.5T with immunospecific contrast agent in vitro and in vivo in a xeno-transplant model. Mag Reson Mater Phy 2006;19:313-320.
(5.) Itoa A, Kugaa Y, Hondaa H, Kikkawab H, Horiuchib A, Watanabeb Y, et al. Magnetite nanoparticle-loaded anti-her2 immunoliposomes for combination of antibody therapy with hyperthermia. Cancer Lett 2004;212(2):167-175.
(6.) Kawasaki ES, Player A. Nanotechnology, nanomedicine, and the development of new, effective therapies for cancer. Nanomedicine 2005;1(2): 101-109.
(7.) Lawson EL, Clifton JG, Huang F, Li X, Hixson DC, Josic D. Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes. Electrophoresis 2006;27(13):2747-2758.
(8.) Lee JH, Huh YM, Jun YW, Seo JW, Jang JT, Song HT, et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nat Med 2007;13(1):95-99.
(9.) Gruttnera C, Mullera K, Tellera J, Westphala F, Foremanb A, Ivkovb R. Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy. J Magn Mater 2007;311:181-186.
(10.) Denardo SJ, Denardo G, Miers L, Natarajan A, Foreman A, Gruettner C, et al. Development of tumor targeting bio probes (111In-Chimeric L6 Monoclonal Antibody nanoparticles) for alternating magnetic field cancer therapy. Clin Cancer Res 2005;11(19 Pt2):7087s-7092s.
(11.) Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.
(12.) Owen CS, Sykes NL. Magnetic labeling and cell sorting. J Immunol Methods 1984;73(1):41-48.
(13.) Funovicsa MA, Kapellerb B, Hoellerc C, Sud HS, Kunstfeldc R, Puiga S, et al. MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents. Magn Reson Imaging 2004;22(6):843-850.
(14.) Bancroft JD, Stevens A. Theory and Practice of Histological Techniques, 4th ed. New York: Churchill Livingstone Inc;1996.
(15.) Luna LG. Manual of Histological Staining Methods of the AFIP. 3rd ed. New York: McGraw Hill; 1968.
(16.) Wagner K, Kautz A, Ro [??] der M, Schwalbe M, Pachmann K, Clement JH, et al. Synthesis of oligonucleotide-functionalized magnetic nanoparticles and study on their in vitro cell uptake. Appl Organometal Chem 2004;18(10):514-519.
(17.) Prestvik WS, Berge A, Mork PC, Stenstad PM, Ugelstad J. Preparation and application of monosized particles in selective cell separation. In: Hafeli U, Schutt W, Teller J, Zborowski M (eds). Scientific and clinical applications of magnetic carriers. New York: Springer;1997,11-35.
(18.) Hilger I, Trost R, Reichenbach JR, et al. MR imaging of her2/neu protein using magnetic nanoparticles. Nanotechnology 2007;18(13):135103-135111.
(19.) Zhang C, Jugold M, Woenne EC, Lammers T, Morgenstern B, et al. Specific targeting of tumor angiogenesis by RGD-conjugated ultra-small superparamagnetic Iron oxide particles using a clinical 1.5-T magnetic resonance scanner. Cancer Res 2007;67(4):1555-1562.
Fereshteh Shamsipour , Amir Hassan Zarnani , Roya Ghods , Mahmood Chamankhah , Flora Forouzesh , Sedigheh Vafaei , Ali Ahmad Bayat , Mohammad Mehdi Akhondi , Mohammad Ali Oghabian , and Mahmood Jeddi-Tehrani  *
[1.] Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
[2.] Department of Immunology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
[3.] Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
[4.] Research Centre for Science and Technology in Medicine, Tehran University of Medical Sciences, Tehran, Iran
* Corresponding author: Mahmood Jeddi-Tehrani, Ph.D., Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, P.O. Box: 19615-1177
Tel: +98 21 22432020
Fax: +98 21 22432021
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|Title Annotation:||Original Article|
|Author:||Shamsipour, Fereshteh; Zarnani, Amir Hassan; Ghods, Roya; Chamankhah, Mahmood; Forouzesh, Flora; Vaf|
|Publication:||Avicenna Journal of Medical Biotechnology (AJMB)|
|Date:||Apr 1, 2009|
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