*3.6. Internalization and Endosome Escape of M-MTX/ FAM-siRNA in FR-Overexpressing HeLa Cell*

The M-MTX/FAM-siRNA complexes could be specifically taken up by HeLa cells. However, the process of internalization, cytoplasmic delivery, subcellular localization, and drug release of FAM-siRNA still remained unclear. Thus, we applied another fluorescent reagent, Lyso Tracker™ Red DND-99, to label the endosomes and to investigate whether FAM-siRNA could escape from the endosomes. As shown in Figure 3, M-MTX/ FAM-siRNA (green signal) started to be internalized to the cells, and the endosomes (red signal) were also found near the cell membrane at the first hour. At the second hour, the overlap of green and red signals gradually increased and the overlapping orange signal started to transport into the cytoplasm. At the fourth hour, the fluorescence signal was completely located in the cytoplasm and a rich green signal was observed to clearly separate from the red signal, indicating that FAM-siRNA were successfully released into the cytosol. The results demonstrated the endocytic process of M-MTX/FAM-siRNA, which finally achieved drug release in the HeLa cells. The proton sponge effect of the cationic polymer of bPEI may play the most important role in achieving endosome escape and drug release [34]. Although some of the amines of bPEI had been reacted with LA and MTX, M-MTX still had a proton sponge effect and achieved siRNA endosomal escape.

**Figure 3.** The internalization and endosome escape of M-MTX/siRNA in folate receptor (FR)-overexpressing HeLa cells: The internalization and endosome (red) escape of M-MTX loaded with FAM-siRNA (green) in the Hela cells was visualized by the confocal laser scanning microscopy (CLSM) at the first, second, and fourth hours. The red bar in the images was 20 μm.

#### *3.7. In Vitro Biological Activities of M-MTX/Survivin-siRNA*

The synergy of MTX and survivin-siRNA was first studied in HeLa cells by an MTT assay. M-MTX/survivin-siRNA showed enhanced cell cytotoxicity (\*\*\* *p* < 0.001) compared to MTX and siRNA alone in vitro (Figure 4A). Simultaneously, the cytotoxic efficiency of the M-MTX/survivin-siRNA-treated group was reversed by free FA(1 mM) while no significant cytotoxicity difference was found between the M/siRNA-treated group preincubated with free FA (1 mM) and the M/siRNA-treated group without FA (1 mM) addition (Figure 4A). The western blot results were also consistent with the cytotoxicity experiments (Figure 4B). M-MTX/survivin-siRNA had the lowest protein expression and could be reversed by the addition of FA (1 mM). MTX was conjugated to bPEI-LA through an amide bond which had previously been demonstrated to be a stable chemical bond and was difficult to degrade. Thus, we assumed that the inhibition of cell viability and protein expression was achieved by MTX-bPEI-LA. To verify this, we investigated the inhibitory activity of MTX and MTX-bPEI-LA on DHFR. As shown in Figure 4C, MTX-bPEI-LA with a larger structure also had a good inhibitory efficiency against DHFR, although slightly lower than MTX, and exhibited a concentration-dependent inhibitory effect. The large structural steric hindrance of MTX-bPEI-LA may result in the reduced binding ability to DHFR. Interestingly, M-MTX had a higher protein expression inhibitory effect in the western blot assay after 48 h compared with free MTX although MTX-bPEI-LA was shown to have a lower inhibitory activity against DHFR (Figure 4B). Survivin expression is closely related to the efficient delivery of survivin siRNA and MTX to the targeted site. This could be explained by a much higher uptake of M-MTX, which had a positive charge and was able to interact with the negatively charged cell membranes. Free MTX, M-MTX, M-MTX/siRNA negative control, and M-MTX/siRNA complexes were incubated with cells only for 4 h and replaced with fresh medium in the western blot assay. M-MTX had a significant decrease in survivin expression due to more M-MTX uptake compared to MTX and the M-MTX/siRNA negative control. The greatest cytotoxicity and protein inhibition caused by M-MTX/siRNA may depend primarily on the enhanced tumor cell uptake of MTX conjugates, while M-siRNA without MTX conjugates with a much smaller amount of cellular uptake showed

lower cytotoxicity and more survivin expression. Thus, we proposed that M-MTX had enhanced the anticancer therapy compared to the free MTX and could serve as both a targeting agent and a chemotherapeutic agent for tumor therapy [31,32,41,42].

**Figure 4.** The cytotoxicity, protein expression, and dihydrofolate reductase (DHFR) inhibition activity of M-MTX/siRNA. (**A**) The cell viability was obtained by an MTT assay after incubation with MTX, M-MTX, M-MTX/siRNA negative control, M-MTX/siRNA, M, and M/siRNA for 48 h. \* *p* < 0.1, \*\*\* *p* < 0.001. (**B**) Survivin expression was further measured by a western blot assay. The competitive inhibition was also investigated by preincubated with FA (1 mM) in advance. (**C**) The inhibition activity of MTX-bPEI-LA to DHFR was performed to evaluate the difference of inhibition activity of the conjugated MTX compared to free MTX.
