br Scanning electron microscopy SEM br The morphology
3.1.6. Scanning electron microscopy (SEM)
The morphology and size distribution of MNPs were investigated by SEM analysis. SEM images showed a uniform pattern in both MNP-Si and MNPs, with a similar size distribution (Fig. 6).
3.2. Herceptin conjugation to MNP-Si
As shown in scheme 1A, to coat the surface of MNP-Si with
Fig. 10. Fluorescence microscopy images of 100 stained SK-BR-3 cells in whole blood after magnetic separation. a,c) Without, and b,d) with fluorescent lighting. c,d) With local magnification.
Herceptin, antibodies need to be initially activated with EDC/NHS be-cause MNP-Si has an amine group (reverse direction). The MNP-Si were added to the active antibodies, and an unstable intermediate product was formed which reacted with the primary amine group, and then an amide bond was formed between the two materials. Studies have shown that antibodies have two types of amines: primary amines will settle at the site of antibody binding to its antigen, and a large number of sec-ondary amines will be located in other sites. EDC/NHS-activated Protease Inhibitor Cocktail reacts with primary amines . Consequently, if the MNPs contain an acidic group and are initially activated by EDC/NHS, they will react with the primary amine in a specific site (paratope), thereby reducing the antibody’s ability to conjugate to the antigen. In the reverse di-rection this problem was resolved, because the specific areas or para-topes did not react. The MNPs in this study did not contain an acidic group, so the reverse reaction approach was used, with interesting re-sults as explained below.
Since Herceptin is a human IgG , an anti-human IgG1 antibody conjugated to a fluorescent dye was used to verify the conjugation of Herceptin to MNPs, and changes in fluorescence intensity observed for MNP-Si with and without Herceptin were considered as a criterion for eﬃcient coating. In many previous studies, complicated and time-consuming tests such as gel electrophoresis, the Bradford test, western blotting and SDS-PAGE have been used to investigate the binding of a protein or antibody to a nanoparticle [7,59]. But here we applied a flow cytometric-based method which is not only an accurate and reliable method, but also an easier and faster one compared to other ap-proaches.
As shown in Fig. 7, after conjugation of the antibody to MNP-Si, mean fluorescence intensity (MFI) of MNP-Si was dramatically in-creased [2 in MNP-Si without Herceptin (Fig. 7c) vs. 185 in MNP-Si with Herceptin (Fig. 7d)], indicating eﬀective coating of MNP-Si with Herceptin in the appropriate conformation. As a result, the use of MFI is a method that can be considered to verify the conjugation of antibodies or ligands to MNPs. As shown here, after conjugation with Herceptin, based on forward and side scatter parameters, size, and size distribu-tions of MNPs did not change significantly, and these results were
concordant with SEM findings.
3.3. SK-BR-3 cell isolation using Herceptin-conjugated MNP-Si
Based on recent studies, 15–25% of women with BC are HER2-po-sitive, which means HER2 is highly expressed on their cells. We chose SK-BR-3 cells as a model of HER2+ BC, with 1–2.6 million HER2 re-ceptors on their surface compared to normal cells, which have only 20,000 HER2 receptors . This diﬀerence in HER2 expression levels on the cell surface helps to determine the eﬃciency of HER2-coated MNP-Si in the separation of positive and negative cells. Accordingly, PBMCs were mixed with diﬀerent numbers of SK-BR-3 cells and then subjected to separation by Ab/MNP-Si as shown in Scheme 1B. To distinguish SK-BR-3 cells from PBMCs, anti-human antibodies against Ep CAM (tumor marker) and CD45 (panleukocyte marker) were used. 7-AAD vital dye was also used to distinguish live cells from dead ones. As shown in Fig. 8, Herceptin-coated MNP-Si was eﬃciently able to bind to cancer cells and separate them, which confirmed the eﬀective conjugation of Herceptin antibody to MNP-Si. As summarized in Table 1, the percentage of SK-BR-3 cells at a concentration of 4 × 105 increased from 11% before separation to 68.8% after separation (Fig. 8c and d). The estimated yield was 84%, as the number of PBMCs showed a sharp decrease from 30% to 8% which confirmed the ability of Ab/ MNP-Si to bind to SK-BR-3 cells and eﬃciently separate and enrich them.
In addition, to investigate the eﬀect of the number of cells on Ab/ MNP-Si binding to and separation of SK-BR-3 cells, 10,000 and 1 × 105 cells were added to the same number of PBMCs. As shown in Fig. 8, despite a significant reduction in the number of isolated cells compared to 4 × 105 cells, separated SK-BR-3 increased from 4% to 19% in a reaction containing 1 × 105 cells (Fig. 8e and f) and from 0.2% to 11% in a reaction containing 10,000 cells (Fig. 8g and h). Ac-cordingly, the estimated yield eﬃciency was 77% and 98% respec-tively, which demonstrated the high performance of Ab/MNP-Si even in media containing very small numbers of cells.