MUC1 aptamer-tethered H40-TEPA-PEG nanoconjugates for targeted siRNA-delivery and gene silencing in breast cancer cells

With a prevalence of 12.5% of all new cancer cases annually, breast cancer stands as the most common form of cancer worldwide. The current therapies utilized for breast cancer are constrained and ineffective in addressing the condition. siRNA-based gene silencing is a promising method for treating breast cancer. We have developed an aptamer-conjugated dendritic multilayered nanoconjugate to treat breast cancer. Initially, we transformed the hydroxyl groups of the hyperbranched bis-MPA polyester dendrimer into carboxylic groups. Subsequently, we linked these carboxylic groups to tetraethylenepentamine to form a positively charged dendrimer. In addition, the mucin-1 (MUC1) aptamer was attached to the dendrimer using a heterobifunctional polyethylene glycol. Characterizing dendrimers involved 1H NMR and dynamic light scattering techniques at every production stage. A gel retardation experiment was conducted to evaluate the successful binding of siRNA with targeted and non-targeted dendrimers. The targeted dendrimers exhibited no harmful effects on the NIH-3T3 fibroblast cells and RBCs, indicating their biocompatible characteristics. Confocal microscopy demonstrated significant higher uptake of targeted dendrimers than non-targeted dendrimers in MCF-7 breast cancer cells. The real-time PCR results demonstrated that the targeted dendrimers exhibited the most pronounced inhibition of the target gene expression compared to the non-targeted dendrimers and lipofectamine-2000. The caspase activation study confirmed the functional effect of survivin silencing by dendrimer, which led to the induction of apoptosis in breast cancer cells. The findings indicated that Mucin-1 targeted hyperbranched bis-MPA polyester dendrimer carrying siRNA could successfully suppress the expression of the target gene in breast cancer cells.


Purification of H40-OH
For the purification of dendrimer, 200 mg of H40-OH was dissolved in 2 mL of acetone under continuous stirring in an inert condition and incubated overnight.After this, the resultant solution was added to chilled diethyl ether dropwise and incubated for 4 hrs at -20°C.The obtained precipitate was recovered by centrifugation at 1200 RPM for 30 mins.The pellet was dried under vacuum till complete removal of solvent (Tu et al., 2013).

Synthesis of carboxylic group modified H40 dendrimer (H40-COOH)
H40-OH was converted to H40-COOH using succinic anhydride and TEA (Figure 1).In detail, 110 mg of H40-OH was reacted with 140 mg of succinic anhydride in the presence of 0.03 mL of TEA.The reaction was performed in anhydrous THF (2 mL) for 24 hrs under continuous stirring at room temperature.The obtained solution was washed with THF and diethyl ether four times (Chen et al., 2012).
1.4 Synthesis of TEPA-modified H40 dendrimer (H40-TEPA) Tetraethylenepentamine was conjugated over the -COOH modified dendrimer using DCC and NHS coupling reaction (Figure 1).For this, 130 mg of H40-COOH was dissolved in 5 mL of dimethyl sulfoxide (DMSO), and then 15 mg of DCC and 8 mg of NHS were added to the above solution and stirred for 12 hrs at room temperature under an inert atmosphere.Further, 30 mg of tetraethylenepentamine was added to the reaction and continued the reaction for 24 hrs.Upon completion of the reaction, the resultant solution was filtered to remove the by-product, i.e., dicyclohexylurea (DCU).Subsequently, the obtained product was dialyzed against DMSO for 24 hrs and against distilled water for 24 hrs using a 2 kDa molecular weight cut-off dialysis membrane to remove impurities (Chen et al., 2019).
For the synthesis of the targeted dendrimers, five molecules of PEG and MUC1 aptamer were conjugated over each dendrimer.For this, H40-TEPA was dissolved in DMSO and allowed to react with NHS-PEG-maleimide for 24 hrs in an inert atmosphere under continuous stirring.
Further, MUC1 aptamer treated with TCEP in water was added to the above reaction and allowed to continue the reaction for 24 hrs.After completion of the reaction, the above solution is dialyzed against water and lyophilized.Similarly, in the case of non-targeted dendrimer synthesis, NHS-PEG-OCH 3 was reacted with H40-TEPA (Li et al., 2012).

Characterization of dendrimer
1 H NMR was carried out to confirm synthesis of H40-COOH, H40-TEPA, H40-TEPA-PEG and H40-TEPA-PEG-MUC1.The 1 H NMR spectra of the samples were obtained by using a Bruker Avance III HD NMR 500 MHz spectrometer, and DMSO (d6) was used as the solvent.The size and zeta potential of both H40-TEPA-PEG-MUC1 and H40-TEPA-PEG-OCH 3 dendrimers were determined by dynamic light scattering (Malvern Zetasizer ZSP, Malvern, Germany).

Gel retardation assay
This assay was performed to assess the binding efficiency of targeted dendrimers with siRNA using agarose gel electrophoresis.The process of complexation between siRNA and dendrimers was carried out at different N/P ratios (N/P 5:1, 10:1, 15:1, 20:1, 25:1, and 50:1).Complexes were developed by incubating dendrimers with siRNA at room temperature for 1 hr and evaluated by performing gel electrophoresis using a 3% agarose gel at 65 mV.Free siRNA was employed as a negative control (Tambe et al., 2017).

Cell viability assay
The MTT assay was used to determine the possible cytotoxicity of the synthesized dendrimers.
For this, NIH-3T3 cells were seeded at a density of 1x10 4 cells per well in a 96-well plate and incubated at 37°C with 5% CO 2 .After 24 hrs, the medium was removed, and the cells were treated with targeted NPs at different concentrations (25, 50, 75, 100, and 150 μg/mL) for 48 hrs in incomplete medium.Then, 10 μL of MTT reagent (5 mg/mL) was added to each well and incubated for 4 hrs in the CO 2 incubator.Formed MTT formazan crystals in the wells were dissolved in 200 μL of DMSO.The absorbance was measured at 570 nm using the BioTek Synergy plate reader (Yu et al., 2014).

Cellular uptake visualization using confocal microscopy
In a 6-well plate, 1 × 10 5 MCF-7 or MDA-MB-453 cells were seeded per well and incubated at 37℃ for 24 hrs.The cells were then incubated with fluorescently tagged dendrimer (100µg/mL) for 2 hrs in serum-free media.The treatment was ended by removing the media and washing the cells with PBS.The cells were then fixed, permeabilized, stained, and imaged using a confocal microscope (Leica Microsystems, Model TCS-SP8, Germany) (Liu et al., 2015).

Hemolysis Study
The purchased EDTA stabilized sheep blood was diluted (1:1) in PBS and centrifuged at 3000 rpm for 5 min.The red blood cells (RBCs) in the pellet were dispersed in PBS, and this step was repeated three times.Further, obtained RBCs were diluted in a glucose solution (5% w/v).RBCs were treated with appropriately diluted targeted dendrimers, non-targeted dendrimers, and lipofectamine 2000.Triton-X 100 was used as a positive control, and PBS as a negative control.
The mixture was incubated at 37˚C for 2 hrs and then centrifuged to settle the cells.The absorbance of the supernatant was measured at 540 nm.The % hemolysis was calculated, and the graph was plotted (Vankoten et al., 2016).

Gene silencing
The gene silencing study was performed to check the silencing ability of synthesized dendrimers in MCF-7 breast cancer cells.For this study, MCF-7 cells were seeded on a 24-well plate at 10 5 cells/well density.Treatment was initiated once cells were attached to the substratum.The survivin gene silencing efficacy was investigated by administering a 100 nM siRNA for a duration of 24 hrs.Survivin siRNA complexed with targeted dendrimers (N/P ratio 25:1), nontargeted dendrimers (N/P ratio 20:1), and lipofectamine-2000 was added to cells in Opti-MEM media.Total RNA was extracted from the cell samples and then complementary DNA (cDNA) was prepared.Quantitative real-time PCR (qRT-PCR) (Light Cycler 480, Roche, Germany) was then used to investigate survivin gene expression levels in different treatment groups.The sequence of primers is given in table 1.To calculate the ΔCt value of the target gene for every group, the β-actin, and GAPDH gene was used as an internal control.The following formula was used to find the relative expression of the survivin gene: Relative quantification (RQ) = 2 -ΔΔCt For the gene-silencing analysis, the transfecting agent, lipofectamine 2000 (ThermoFisher Scientific, 11668019), was used as the positive control, whereas the negative control siRNAs (Merck, SIC001-10NMOL) complexed with targeted nanocarriers, non-targeted nanocarriers, and lipofectamine-2000 was used as a negative control (Kumar et al., 2023).

Caspase assay:
Caspase-3/7 Green Detection Reagent (Molecular probes, Invitrogen, USA) was used to confirm caspase-3 and caspase-7 activation in MCF-7 cells.In detail, MCF-7 cells (1x10 5 ) were treated with survivin siRNA loaded with non-targeted dendrimer and survivin siRNA loaded with targeted dendrimer for 24 hrs in an incomplete medium.Then, caspase-3/7 Green Detection Reagent was added to the treated cells and incubated for 1 hr.Then the cells were visualized under a fluroscence microscope (Nikon Eclipse TS 100, Japan) (Killinger et al., 2021).
1.13 Flow cyometry by Annexin V/PI staining 1x10 5 MCF-7 cells were seeded and grown overnight in 24 well plates at 37°C in CO 2 incubator.
The cells were then treated with non-targeted dendrimer, targeted dendrimer and Lipofectamine 2000 complex with the Survivin siRNA.After 12 and 48 hrs, cells were harvested and centrifuged (1000 RPM, 10 mins) to remove media residues.Cells were resuspended in 100 µL Annexin binding buffer.Thereafter, cells were stained with Alexa Fluor 488 Annexin V and propidium iodide (FITC annexin V/dead cell apoptosis kit, molecular probe) for 15 min at room temperature.The volume of the suspension was made up to 500 µL with annexin binding buffer.

Statistical analysis
Each experiment was performed in triplicate, data presented as means ± standard deviation (SD).
Statistical analysis was carried out by using GraphPad Prism software.The Statistical significance was checked by two-way analysis of variance (ANOVA) with Bonferroni posttests.P value <0.05 was found significant.