Figure 1 shows schematic representation for synthesis of MUC1 aptamer-conjugated hyperbranched bis-MPA polyester dendrimer. All the intermediate and final H40 polymer products were recorded on Bruker Avance III HD NMR 500 MHz spectrometer using DMSO as a solvent (Figures 2A–D). Tetraethylenepentamine (TEPA) signature peaks could be observed at 1.5 ppm (amine groups), confirming the formation of H40-TEPA (Figure 2B). The dendrimers were further conjugated with heterobifunctional polyethylene glycol (PEG) to increase biocompatibility and solubility and protect siRNAs. The peak of PEG was observed at 3.5 ppm in the NMR spectra (Figure 2C). To target cancer cells, the nanoparticles were modified with aptamer depicting NMR peaks from 1.8 to 2.10 ppm (Figure 2D).

The hydrodynamic size of the dendrimers was measured by dynamic light scattering. The non-targeted and targeted dendrimer had sizes of 46.23 ± 2.14 and 68.42 ± 3.78 nm, respectively (Supplementary Figure S1). Zeta potential analysis showed a surface charge of 9.67 ± 2.33 and 3.62 ± 1.24 mV for non-targeted and targeted dendrimers, respectively (Supplementary Figure S1). An increase in size and reduction in the zeta potential of targeted dendrimers compared to non-targeted dendrimers was due to the conjugation of MUC1 aptamer. siRNA-loaded targeted and non-targted dendrimers were also characterized for size and surface charge. The hydrodynamic size of the siRNA-loaded targeted and non-targted dendrimers was found to be 76.41 ± 2.27 and 58.77 ± 3.38 nm, respectively (Supplementary Figure S2). Zeta potential analysis of siRNA-loaded dendrimers showed a surface charge of −5.09 ± 1.44 and −2.32 ± 1.65 mV for targeted and non-targeted dendrimers, respectively (Supplementary Figure S2).

Nucleic acid molecules are effectively loaded onto cationic dendrimers, and the negative charge of siRNA can be neutralized, inhibiting the mobility of siRNA (Tambe et al., 2017). A gel retardation experiment was carried out to measure the dendrimers and siRNA interaction. The negatively charged siRNA molecules were complexed to targeted dendrimers, which reduced siRNA mobility in agarose gel electrophoresis. Different nitrogen-to-phosphate (N/P) ratios were used to investigate the potential of targeted dendrimers for the loading of siRNA in a stable manner. The agarose gel electrophoresis experiment revealed a gradual reduction in the movement of siRNA as the concentration of dendrimers increased. The complete complexation of the siRNA with targeted dendrimers and non-targeted dendrimers occurred at 25:1 and 20:1 N/P ratio, respectively (Supplementary Figure S3). The higher dendrimers to siRNA ratio for targeted dendrimers was due to the presence of aptamer.

To establish the toxicity level, NIH-3T3 cells were subjected to treatment with targeted dendrimers at five different concentrations, followed by an MTT experiment. The targeted dendrimers showed no toxicity at concentrations as high as 150 μg/mL (Supplementary Figure S4). When considerable concentrations of dendrimers are required, they must have a low cytotoxicity.

The cellular uptake of the aptamer-conjugated targeted dendrimer was assessed against non-targeted dendrimer using fluorescence-based confocal microscopy. MCF-7 cells showed significantly higher red fluorescence in the group treated with targeted dendrimers in comparison with non-targeted dendrimers (Figure 3). Here, the higher uptake of targeted dendrimer was due to the interaction of MUC1 aptamer present over dendrimer and receptor present over MCF-7 cells. To demonstrate the specificity of MUC1 aptamer towards MCF-7 cells, the uptake of the aptamer-conjugated targeted dendrimer and non-targeted dendrimer was also assessed on MUC1 negative MDA-MB-453 cells by fluorescence-based confocal microscopy. Results of the study showed that there was very little uptake in the MDA-MB-453 cell line for both targeted and non-targeted dendrimers (Supplementary Figure S5). Further, there was no difference between targeted and non-targeted dendrimer in MDA-MB-453 cells. Here, non differentiated uptake of both the dendrimers was due to absence of MUC1 (Yamada et al., 2008). This study demonstrates the specificity of MUC1 aptamer-conjugated dendrimers for MCF-7 cells.

Assessing the hemolytic potential of dendrimers is widely considered advantageous in establishing their suitability for medicinal purposes. To achieve this objective, a hemolysis experiment was conducted utilizing lipofectamine-2000, targeted dendrimers, and non-targeted dendrimers. Both dendrimers showed a substantially lower hemolysis activity towards red blood cells (RBCs) compared to commercially available transfecting reagent lipofectamine-2000 (Supplementary Figure S6). This study suggests that the produced dendrimers are suitable for intravenous injection.

The silencing efficacy was evaluated by transfecting MCF-7 cells with various siRNAs (Survivin and negative control siRNA) that were bound to targeted dendrimers, non-targeted dendrimers, and lipofectamine-2000 (Figure 4A). The use of targeted dendrimers for delivering siRNA into cells effectively suppressed the expression of target-specific genes. The qRT-PCR study showed that the use of targeted dendrimers for delivering siRNA led to a more significant decrease in survivin mRNA levels compared to the delivery using non-targeted dendrimers and lipofectamine-2000. The introduction of survivin siRNA-loaded targeted dendrimers into MCF-7 cells resulted in a higher silencing effect of ∼2.5-fold compared to the non-targeted dendrimers (∼1.3-fold) and lipofectamine-2000 (∼2-fold) (Figure 4A). The conjugation of TEPA and MUC1 aptamer greatly enhanced the silencing capability of the targeted dendrimers. The presence of the positively charged component TEPA has enhanced the efficacy of loading siRNA onto the dendrimers. Additionally, the MUC1 aptamer targets MCF-7 cells, hence increasing their ability to take up the dendrimers efficiently. Prior studies have demonstrated similar results where cationic moiety-modified nanocarriers were used for the siRNA delivery to suppress gene expression. Kumar et al. (2023) reported successful inhibition of the BCl-xL and BCL-2 genes in DOX-resistant MDA-MB-231 triple-negative breast cancer cells through the delivery of targeted siRNA-loaded cationic moiety-modified nanocarriers. The AS1411 aptamer binds explicitly to nucleolin, enhancing the silencing capacity of the nanocarrier modified with a cationic moiety (Kumar et al., 2023).

The activation of caspases was confirmed by observance of the green color inside the cells (Figures 4B–D). The cells treated with non-targeted dendrimer loaded with survivin siRNA showed lower caspase activation (lower green fluorescence) (Figure 4C). However, cells treated with targeted dendrimer loaded with survivin siRNA showed higher caspase activation (higher green fluorescence) (Figure 4D). The higher caspase activation in the targeted dendrimer group was due to efficient uptake of the dendrimer followed by higher survivin gene silencing. This study indicated that the synthesized targeted dendrimer loaded with survivin siRNA effectively activated caspases due to the downregulation of survivin inside breast cancer cells and follow apoptosis-mediated cell death.

Annexin V/PI dual staining has been used for quantitative determination of apoptosis. The increased presence of the phosphatidyl serine (PS) on the surface of plasma membrane is the key feature of apoptosis, which can be visualized by tagging with fluorescently labeled Annexin V molecules. Simultaneously, cells stained with propidium iodide (PI) enable visualization of the dead cells. The combination of both staining procedures ensures distinction among live, early apoptotic, late apoptotic and necrotic cells. Annexin V/PI dual staining study in MCF-7 cells showed that the targeted dendrimers loaded with survivin siRNA had higher cell death as compared to non-targeted dendrimers (Supplementary Figures S7, S8). At 12 h, targeted dendrimers, non-targeted dendrimers and Lipofectamine 2000 loaded with survivin siRNA showed 15.5% ± 2.64%, 6.9% ± 1.37% and 10.0% ± 1.19% apoptosis, respectively (Supplementary Figure S7). While at 48 h, significant higher cell killing was observed in cells treated with targeted dendrimers (86.2% ± 2.72% apoptosis) loaded with survivin siRNA as compared to non-targeted dendrimers (49.3% ± 3.49% apoptosis) and Lipofectamine 2000 (72.4% ± 2.29% apoptosis) (Supplementary Figure S8). Flow cytometry analysis confirmed apoptosis mediated cell death in MCF-7 cells due to silencing of survivin.