br To evaluate whether the inhibition
To evaluate whether the inhibition of cell proliferation by [email protected] NPs was due to the induction of apoptosis, cell apoptosis was evaluated by calcein-AM/PI double staining (Fig. 3D). More apoptotic cells were observed upon treatment with [email protected] NPs than with free DOX or OA treatment, confirming apoptosis to be important in the anti-cancer effects of the NPs.
In order to investigate the effect of [email protected] NPs on tu-moral cell apoptosis in depth, it was necessary to perform a molecular level evaluation. RT-qPCR was used to detect the mRNA expression levels of several important apoptosis-related genes (Bcl-2, Bax, Caspase-3, and Caspase-9) in MDA-MB-231 cells. Bcl-2 is a vital proto-oncogene and the first confirmed gene able to participate in preventing apoptosis of cells and prolonging cell survival . As shown in Fig. S4A, com-pared with the PBS control group all drug treatments significantly (P < 0.05) inhibited the expression of Bcl-2 mRNA in breast cancer cells and promoted the process of cancer cell apoptosis. Furthermore, the downregulation effect of the [email protected] NPs was greater than that of other formulations (P < 0.01). The Bax (an inhibitor of Bcl-2), Caspase-3 and Caspase-9 genes (members of the cysteine-aspartic Filipin III protease family) are activated in apoptotic cells both by extrinsic and intrinsic pathways, and were found to be upregulated in MDA-MB-
231 cells after incubation with DOX, OA, FA-CS-g-OA or [email protected] DOX NPs (Fig. S4B, C, D). Cells treated with [email protected] NPs exhibited the highest mRNA expression levels for these three pro-apoptosis genes. It is hence clear that [email protected] NPs can in-duce apoptosis in tumor cells.
3.4. In vitro cellular uptake
In vitro cellular uptake experiments were performed using human breast carcinoma MDA-MB-231 cells (known to significantly over-express the FA receptor), with HUVEC cells (no overexpression of FA receptor) utilized as controls. Cells were incubated with [email protected] DOX NPs or free DOX for 2 h, and then examined by CLSM. As revealed in Fig. 4A, the uptake of [email protected] NPs by MDA-MB-231 cells was noticeably higher than those cultured with free DOX. For HUVEC cells, minimal fluorescence was observed regardless of whether they were treated with [email protected] NPs or free DOX. Moreover, the red fluorescence which can be seen in the cytoplasm in MDA-MB-231 cells is greater and more closely situated around the nuclei (blue) than that in the control HUVEC cells. The main reason for this is that far fewer FA receptors are expressed on HUVEC cells than on MDA-MB-231 cells, resulting in much reduced receptor-mediated endocytosis in the former case.
The specific targeting ability of the FA receptor by [email protected] DOX NPs was further verified by a competitive inhibition assay, in which MDA-MB-231 cells were pretreated with free FA (to saturate the FA receptors on the cell surface) before being cultured with [email protected] NPs or DOX. A sharply decreased uptake of the NPs is ob-served (Fig. 4A). In addition, no obvious differences in DOX fluores-cence are observed between MDA-MB-231 cells incubated with free DOX and those incubated with the NPs. The FA ligands on the NPs therefore play an important role in increasing cellular uptake via FA-receptor-mediated pathways. To provide further evidence, flow cytometry was employed to ex-plore the cellular uptake of the [email protected] NPs. Free DOX and [email protected] NPs were incubated with HUVEC and MDA-MB-231 cells for 4 h and then the cellular uptake of DOX quantitatively in-vestigated. The results (Fig. 4B) are consistent with those from fluor-escence microscopy. No significant difference in fluorescence intensity
Fig. 3. Cell viability of (A) HUVEC and (B) MDA-MB-231 cells after incubation with DOX, OA, FA-CS-g-OA and [email protected] NPs for 24 h. (C) IC50 values against MDA-MB-231 cells for different formulations. n = 15; results are shown as mean ± S.D. *P < 0.05, **P < 0.01 compared to free DOX. (D) Fluorescence images of calcein-AM (green)/PI (red) double stained cells treated with the different formulations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)