Introduction

Molecular probes for biomolecular recog-nition 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 pajmb_articles have been disclosed for biological applications since mid-1980s (2).
Recently, magnetic pajmb_articles have attrac-ted 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). Bio-logical samples such as blood, serum, cell suspensions and cell lysates are allowed to be exposed to specific ligand-coupled pajmb_articles, and the captured molecules or cells are then rapidly separated using magnetic fields (3,7,8).
Magnetic pajmb_articles conjugated with anti tumor monoclonal antibodies provide a new approach to identify tumor cells.
Antibodies labeled with magnetic nano-pajmb_articles give magnetic signals on exposure to a magnetic field. Iron oxide pajmb_articles are usually coated with different organic shells including dextran, albumin or polyethylene glycol. Coated nanopajmb_articles can be manu-factured with a variety of functional groups (such as amino, aldehyde, hydroxyl, sulfate and carboxyl groups) on their surfaces. Con-sidering these properties, we used super para-magnetic 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 nanopajmb_articles (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 nanopajmb_articles by EDC method N-ethyl-N-(3-dimethyl aminopropyl) car-bodiimide 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 nanopajmb_articles, and shaken at room temperature for 2 hours.
The pajmb_articles 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 pajmb_articles. The mixture was sha-ken for 3 hours and the reaction was quenched by the addition of glycine for 30 minutes (9, 10).
The unconjugated antibodies were separ-ated 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 µl 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.
Cell culture
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°C containing 5% Co2.
Immunofluorescence staining
To verify the expression of her 2 proteins on the cells, the SKBR3 and T47D cells were incubated with anti her2/neu (Herceptin) at 10 µg/ml concentration for 1 hour at 37°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 fluo-rescence microscope (Olympus, Japan).
In vitro cell labeling
SKBR3 and T47D cells were counted and adjusted to a suspension of 4×105cells/ml; 100 µl of each cell suspension were cyto-spined on microscope slides (Shandon cyto-spin 4, Thermo, Germany). The cells were incubated with 100 µl magnetic nanopajmb_articles (with or without antibody; 5 µg Ab and 0.2 mg iron) for 1 hour at 37°C. Then cells were washed extensively with PBS to remove unbound pajmb_articles. The nanopajmb_articles that bounded on the cell surface were detected by iron staining with Prussian blue staining method (14, 15).

Result

We used 20nm nanopajmb_articles (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 µg Ab/mg nanopajmb_articles (Figure 1).
The amount of iron conjugated was deter-mined 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 deter-mined by using electron microscopy, MRI imaging and iron staining (13,18,19).
In this study after incubation of the cells with conjugated nanopajmb_articles, the blue stain induced by iron staining showed the accu-mulation of conjugated nanopajmb_articles on the cell surface (Figure 4).

Discussion

Polymer-coated magnetic pajmb_articles with particle sizes 5-500 nm have been employed in medicine or biotechnology for many years (16). In this study the 20 nm nanopajmb_articles 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 immo-bilized antibody was approximately 20 µg/mg magnetite. However by increasing concentra-tion 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 µg Ab/mg pajmb_articles (9,18). Conjugated nanopajmb_articles bound specific-ally to the her2/neu antigen.
Iron staining, confirmed the presence of nanopajmb_articles 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.

Acknowledgement

This work was supported by a grant from the Nanotechnology Committee of Iran’s Ministry of Health and Medical Education.