Nivolumab br The results of MTT cell viability assay
The results of MTT cell viability assay on HER 2 positive breast cancer cell line (SK-BR3) are reported in the bar charts of Fig. 5. The results showed that cell viability was dependent on CCM concentration and in-cubation time which has decreased by increasing the drug dose and ex-posure time. Accordingly, within 24 h incubation time (Fig. 6A), no significant toxicity was observed for any of free CCM, HSA/CCM NP, and Apt-HSA/CCM NP formulations (p N 0.05). As shown in Fig. 5B, after 48 h, no significant difference was seen between the cytotoxic ef-fect of free CCM and non-targeted HSA/CCM NPs (p N 0.05). However, targeted nanoparticles (Apt-HSA/CCM NPs) show a significant cytotoxic effect at concentrations of 15 and 30 μM compared to free drug and non-targeted HSA/CCM NPs (p b 0.0001). By increasing the concentration of CCM, at 70 μM, the inhibitory effect of targeted nanoparticles was re-duced compared to free CCM (p b 0.05). However, at this concentration, the difference between HSA/CCM NPs and Apt-HSA/CCM NPs was still statistically significant (p b 0.001).
The highest toxicity was achieved after 72 h incubation (Fig. 5C). The results of MTT assay showed that the cell viability of SK-BR3 Nivolumab treated with 70 μM Apt-HSA/CCM NPs was reached 35%, while for free CCM and HSA/CCM NPs with the same dosage of drug, the percentage of cell viability was higher than 50% (60 and 69% respectively). The in-hibitory effect of targeted nanoparticles in comparison with free CCM and non-targeted nanoparticles was statistically significant at the levels of p b 0.0001 and p b 0.001, respectively.
In brief, the cell viability of b50% for free-CCM treated cells was not observed even after 72 h. While at this exposure time, for targeted
nanoparticles (Apt-HSA/CCM NPs), IC50 value was obtained about 34 μM.
Fig. 6 shows the cell viability of MCF-7 treated cells with free CCM, HSA/CCM NPs and Apt-HSA/CCM NPs. As it can be seen, Apt-HSA/CCM NPs did not reduce cell viability, but increased cell viability in some dos-age of CCM which was not significant. In MCF-7 cells which express low levels of HER2, anti-HER2 aptamer did not improve the interaction be-tween nanoparticles and MCF-7 cells, and failed to enhance the cellular uptake and cytotoxicity of nanoparticles.
These results confirm that Apt-HSA/CCM NP compared to non-targeted HSA/CCM NP has a stronger ability to induce tumor cell death in SK-BR3 breast cancer cells which overexpress HER2, but this formu-lation is significantly less cytotoxic to MCF7 breast cancer cells which express low level of HER2. The enhanced anti-tumor effect of Apt-HSA/CCM NP is due to: 1) encapsulation of CCM in NPs increase the water solubility and stability of CCM. Also, the size of these NPs in the range of 200–300 nm facilitate passive targeting of NPs through the en-hanced permeability and retention (EPR) effect in tumor site. 2) active targeting of HSA NPs with HER2 aptamer to tumor cells overexpressing HER2 receptor results in much more anticancer effect, whereas mini-mizes the toxicity of anticancer drug on non-target cells and reduces
side effects. 3) After internalization of Apt-HSA/CCM NP to tumor cells, as it was observed in release studies, the release of drug has increased in pH 5 and medium containing GSH 10 mM.
A new apt-targeted HSA NPs loaded with CCM for active targeting of HER2 overexpressing breast cancer cells was successfully designed and characterized. Small size, narrow size distribution, long term stability, sustained drug release behavior and higher cytotoxicity of apt-targeted CCM-loaded NPs in comparison with free drug and untargeted formulation demonstrate the significant capabilities of this new drug platform. The result of this study shows that toxicity of HSA/CCM NPs is close to free CCM, while in some cases the toxicity effect of free CCM is more than HSA/CCM NPs, but it was not significant. Despite the fact that encapsulation of CCM in HSA nanoparticle increases solu-bility and stability, there is difficulty in cell internalization. Apt-
Fig. 6. In vitro cytotoxicity results of free CCM, untargeted HSA/CCM NPs and targeted Apt-HSA/CCM NPs at different times of (A) 24 h (B) 48 h and (C) 72 h in MCF-7 cells. Data are plotted as mean ± SD of three experiments. No significant difference was seen in cell viability among cells treated with free CCM, HSA/CCM NPs and Apt-HSA/CCM NPs. Cell viability slightly increased in cells treated with Apt-HSA/CCM NPs at 70 μM after 72 h (*p b 0.05) compared to cells treated with curcumin.
targeted nanoparticles had more cytotoxic effect than non-targeted nanoparticles and free CCM in SK-BR3 (HER2+) cells (p b 0.05). How-ever, cytotoxicity differences were not observed in (HER2−) cells be-tween targeted and non-targeted nanoparticles (p N 0.05); suggesting that the conjugation of Apt in HSA NPs formulation significantly has im-proved cytotoxicity of CCM in HER2+ cells. Accordingly, it can be pro-posed that HER2-conjugated drug-loaded HSA NPs possess higher