Hereafter, the sections will showcase the study of breast cancer cell-lines with our novel dye administered. Since the present study involves establishing the binding properties of nDC with Estrogen receptors we chose MCF-7 (target): Estrogen receptors (ER) positive and MDA MB 231 (control): Triple Negative (ER, PR and HER negative) cell lines for our studies. Both are human breast tissue cell lines from mammary gland with adenocarcinoma. The main difference between the two cell lines is that MCF-7 (target) expressed estrogen receptor, whereas MDA MB 231 (control) did not express the same. The motive of choosing the above cell lines was to prove that novel dye had a specific binding affinity with ER-positive cell lines (MCF-7). And also, to show that there was no specific binding with ER-negative cell lines (MDA MB 231).
5.1 Cytotoxicity Studies
The cytotoxicity studies aimed to determine the viability of the cells post nDC administration. As discussed earlier, breast cancer cell lines chosen were MCF-7 and MDA-MB-231. A 30 mM stocks of nDC were made in DMSO. On day 1, a 96-well plate for cell seeding density of 10,000 cells/well was prepared with 100 µL of complete medium (Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum). An incubation condition in 5% CO2 was set at 37°C for 24 hours. On day 2, the nDC ester was weighed, and a 30 mM of working stock was made in DMSO with 1% FBS. Test sample concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µM of nDC were prepared and the cells were treated with the same. Fig. 4 shows the viability of cell for the various concentrations over 24 hours. Based on the above figure, it is evident that dose-dependent cell cytotoxicity was not observed for our novel dye in MCF-7 and MDA-MB-231 cells in the tested range of 10-100 µM up to 24-48 hours of treatment. On average, 90% of cells were viable after 24-48 hours of dye treatment. Hence, the novel dye proved negative in toxicity which enabled us to proceed with further studies.
5.2 Specific Binding Studies
In this section, we discuss some of our significant findings for endocytosis of the novel dye conjugate in the cell lines and its specific binding. DMEM/F12 medium was used to culture MDA MB 231 and MCF 7 with 10% fetal bovine serum in absence of phenol red. Each of 96-well culture plate were added with 50 µl cell suspension for a final 1x103 cells/well concentration which was incubated in carbon dioxide chamber at 37° C for 24 hours. Each well was added with 24.5mg/2ml of DMSO initial concentration and nDC, which were further incubated for 2 hours. Different concentration of DMSO (1:10, 1:50, 1:100, 1:500 and 1: 1000) were used to treat cell thereafter, and were incubated at about 25°C for an hour. Thereafter, cells were given three washes of phosphate buffered saline and were mounted with DPX (Distrene Plasticyser Xylene). These were then imaged using the Olympus Confocal microscope. We carried out in total of three tests with cell lines:
Test 1: Confocal imaging of the cell lines post nDC administration.
Test 2: Confocal imaging of the cell lines administered with nDC post Diethylstilbestrol (DES) and Tamoxifen Treatment.
Test 3: Confocal imaging of MCF-7 stained with ICG
Both the cell lines were stained with novel dye conjugate. Fig. 5(a) shows the confocal images of MCF-7 cell lines stained with nDC. Fig. 5(b) is a DIC image of 5(a). It can be observed that the fluorescent intensity at the nucleus is higher in comparison to the background. Localization of conjugated dye in the nucleus of MCF-7 (ER-positive) cell lines is evident in Fig. 5(a). The cell proliferation is controlled by estrogen. The functional group associated with the dye is instrumental in percolating the dye into the nucleus of MCF-7 cell lines and hence providing a unique distinction between the cells and background. Fig. 5(c) shows the confocal images of MDA MB 231 cell lines stained with nDC. These cell lines lack the estrogen activity in the nucleus therefore, it can be observed from the figure evidently that the fluorescent intensity at the nucleus is lower in comparison to the background. The fluorescence signal largely co-localized in the cytoplasm of the cells. Hence, the control, MDA-MB-231 showed staining only at the levels of the plasma membrane. Clearly, nDC endocytosis failed due to absence of estrogen receptors. From the above test, it is clear that nDC has a special binding ability with cancerous cell exhibiting estrogen receptors. Further to prove our claim, we carried out a test wherein the cell lines were treated with antagonist drugs as explained in the next section.
Diethylstilbestrol (DES) and Tamoxifen are commonly used antagonist drugs which blocks the estrogen receptors from binding with estrogens. MCF 7 cells were treated with various concentrations of DES and Tamoxifen separately for five days. Fig. 5(e) is confocal image of MCF-7 cell lines treated with DES then stained with nDC. Fig. 5(d) is confocal images of MCF-7 cell lines treated with Tamoxifen then stained with nDC. From both the images, it is evident that fluorescence signal is high at the cytoplasm and low in the nucleus. It clearly demonstrates that the nDC is blocked from entering the nucleus due to blocking of ER activity by DES and Tamoxifen treatment respectively. Hence, from test 1 and 2, it can be concluded that the entry of nDC was facilitated due to the presence of active estrogen receptors making our novel dye a target specific.
Indocyanine Green (ICG) is the commercially available nonspecific fluorescent dye. Fig. 5(f) shows cytoplasm level staining of MCF-7 cell lines by ICG as expected. The fluorescence signal is as low as nil at the center of the nucleus with high intensity in the cytoplasm. It shows an apparent distinction on non-specific binding when compared to specific binding of nDC. Thus, it proves that the nDC is superior in term of specificity of target enhancement.