Improved delivery of Mcl-1 and survivin siRNA combination in breast cancer cells with additive siRNA complexes

This study aimed at investigating the influence of commercial transfection reagents (Prime-Fect, Leu-Fect A, and Leu-Fect C) complexed with different siRNAs (CDC20, HSP90, Mcl-1 and Survivin) in MDA-MB-436 breast cancer cells and the impact of incorporating an anionic additive, Trans-Booster, into siRNA formulations for improving in vitro gene silencing and delivery efficiency. Gene silencing was quantitatively analyzed by real-time RT-PCR while cell proliferation and siRNA uptake were evaluated by the MTT assay and flow cytometry, respectively. Amongst the investigated siRNAs and transfection reagents, Mcl-1/Prime-Fect complexes showed the highest inhibition of cell viability and the most effective siRNA delivery. The effect of various formulations on transfection efficiency showed that the additive with 1:1 ratio with siRNA was optimal achieving the lowest cell viability compared to untreated cells and negative control siRNA treatment (p < 0.05). Furthermore, the combination of Mcl-1 and survivin siRNA suppressed the growth of MDA-MB-436 cells more effectively than treatment with the single siRNAs and resulted in cell viability as low as ~ 20% (vs. non-treated cells). This aligned well with the induction of apoptosis as analyzed by flow cytometry, which revealed higher apoptotic cells with the combination treatment group. We conclude that commercial transfection reagents formulated with Mcl-1/Survivin siRNA combination could serve as a potent anti-proliferation agent in the treatment of breast cancers.


Introduction
Breast cancer ranks first in cancer incidence among women with more than two million new breast cancer cases in 2020 with an increasing trend [1]. In majority of the cases, development of breast cancer involves estrogen, progesterone and epidermal growth factor receptors, while another sizable fraction of 10-15% [2] involves triple negative cases. Either surgery or surgery in combination with radiotherapy is performed in local therapy of breast cancers and, if necessary, this is followed by systematic treatment with pharmacological agents. Optimal treatment strategy for each breast cancer patient is based on tumor subtype, stage of cancer and patient preferences [3]. Trends in development of novel cancer therapeutics have shifted from conventional to more specific targeted methods. Conventional targeted treatment strategies focus on the use of monoclonal antibodies [4] to seek out target cells that display the corresponding receptor proteins on their surface, for delivering small molecular inhibitors. While this strategy leverages the presence of cell surface proteins to achieve targeted therapy, it possesses little capacity to target intracellular proteins [4]-a limitation of the conventional delivery method [5].
Targeting oncogenes in malignant cells by inhibiting oncoprotein synthesis is a relatively new approach for molecular targeting. Small interfering RNA (siRNA) is a promising intervention in this regard since it can overcome the limitation of small molecular pharmacological agents by targeting previously undruggable cancer drivers at will. siRNA implements its gene silencing activity by incorporating itself into the RISC (RNA-induced silencing complex), which then binds to the target messenger RNA (mRNA) in order to degrade it, resulting in attenuated protein synthesis [6] . The relatively larger microRNA (miRNA) can also downregulate the expression of desired genes, but siRNA displays more targeting specificity. However, the anionic nature of siRNA makes it impermeable to cell membranes, and hence it requires a carrier for cell entry. Several carriers such as liposomal nanoparticles, cationic lipids, and inorganic and organic polymers have been explored for this purpose. Cationic carriers are often utilized to deliver siRNA as they can form nanoscale complexes with siRNA via electrostatic interaction, prevent enzymatic degradation of siRNA and aid in cellular uptake [7]. Neutral and anionic carriers usually require addition of other components to forge a more stable interaction with siRNA and undertake effective delivery [8].
Polyethyleneimine (PEI) is a cationic polymer widely used in non-viral gene delivery. PEI can condense the anionic nucleic acid and has high cellular uptake efficiency resulting in inhibition of gene expression both in vivo and in vitro [9,10]. In addition to its high stability and low cost, PEI is able to form siRNA polyplexes at the nano-scale range, which is highly suitable for cell uptake [10]. PEI displays low toxicity when used at low molecular weights (MW < 5 kDa), but it is not effective in its native state. The facile chemistry of PEI allows for chemical manipulation to enhance its stability and transfection efficiency while retaining its ability to form complexes with nucleic acids. Numerous strategies have been explored over the years for this purpose. Inclusion of hydrophilic polymers such as polyethyleneglycol (PEG), polyhydroxypropylmethacrylamide (pHPMA) and polyvinylpyrrolidine into PEI complexes, resulted in higher transfection efficiency [11]. The tertiary pDNA/PEI complex coated with poly(γ-glutamic acid) (γ-PGA) can convert its cationic surface charge to anionic charge without changing the particle size and lowering the cytotoxicity of cationic PEI [11]. A low MW PEI (4.7 kDa) when modified with C16 and C18 chains modified by ethyleneoxide (PEG) units (Cx-EO) for stability [12], during the self-assembly process, allowed enhanced interaction between PEI and plasma membrane, resulting in better cellular uptake [8]. In contrast, the complexation of anionic siRNA with anionic polymers such as hyaluronic acid (HA) can prevent excretion via the glomerular capillary wall owing to repulsive force formation between the anionic species [13]. However, inclusion of anionic γ-PGA into chitosan/siRNA complexes has shown to facilitate intracellular release of siRNA, and consequently improve gene silencing efficacy [14].  investigated the effect of incorporating different polyanions into complex assembly on siRNA delivery efficiency [15], and demonstrated that hyaluronic acid-formulated additive polyplexes lead to the highest transfection and silencing activity with target siRNA in breast cancer cells [15].
Mcl-1, a member of the anti-apoptotic BCL-2 family, is necessary for cell survival and is responsible for resistance to chemotherapy induced apoptosis via the mitochondrial apoptotic pathway. Mcl-1 is highly expressed in various cancer cells, making it an attractive target for tumor treatment [16]. An elevated level of Mcl-1 expression is observed in breast cancer cases owing to its short half-life [17]. In MDA-MB-435 cells, Mcl-1 silencing was effectively achieved with siRNA complexed with cholesteryl-substituted poly(ethyleneoxide)-block-poly(e-caprolactone-graftedspermine) (PEOb-P(CL-g-SP)) micelles [18] To further establish the feasibility of using Mcl-1 as a therapeutic target in breast cancer, this study explored several commercial delivery agents for siRNA delivery and gene silencing efficiency. Transfection reagents were derived from the low MW PEI (1.2 kDa) after optimal balance of hydrophobic (lipidic) groups were chemically conjugated to the PEI [20]. Several formulations of the commercial reagents were explored for the feasibility of retarding the growth of breast cancer cells using specific siRNAs.

Cell culture
MDA-MB-436 breast cancer cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin under a humidified atmosphere with 5% CO 2 at 37 °C. When cells were ~ 80% confluent, the spent medium was removed followed by rinsing the monolayer once with sterile 1X HBSS (pH 7.4). Cell monolayers were incubated at 37 °C for 3 min with 1 mL of 0.25% trypsin/EDTA to promote cell dissociation. The suspended cells were centrifuged at 600 rpm for 5 min and resuspended in 10 mL of fresh medium after removing the supernatant. The cells were sub-cultured at a split ratio of 1:9 for subsequent passage and remaining cells were seeded into 96-well plates for transfection experiments.

siRNA complex preparation
The Trans-Booster additive was added to siRNA solution that was dissolved in RPMI at a 1:1 siRNA:additive ratio (w/w) with a final siRNA concentration of 40 or 80 nM in the tissue culture medium. The 1:1 siRNA:additive ratio (w/w) was chosen since this 1:1 ratio gave the highest cellular uptake in a previous study [23]. The desired transfection reagents were then added at a 1:5 or 1:10 (w/w) transfection reagent:siRNA ratio and the mixture was then incubated for 30 min at room temperature to allow for optimal complexation [15]. Table 1 summarizes preparation of typical complexes.

Characterization of siRNA complexes
The ζ-potential of siRNA complexes was measured by LITE-SIZER 500 (Anton Paar, Austria). siMcl-1 and siNC were formulated into various complexes using siRNA:Prime-Fect:additive ratios of 1:10:0, 1:10:1, 1:5:0, and 1:5:1 (w/w/w) and yielding a final concentration of 80 nM siRNA in 20 µL RPMI. The complexes were then incubated for 30 min at room temperature. Prior to the size and ζ-potential measurements, the prepared complexes were further diluted with RPMI to a final volume of 1 mL before each measurement. The instrument was set at a 175° back scatter and 25 °C.
Analysis of the morphology of the polyplexes was performed using a 200kv JEOL 2100 Transmission Electron Microscope (TEM). Samples were prepared over a carboncoated grid immediately after a glow discharge treatment under vacuum by adding a droplet of the polyplex suspension for 5 min and blotting it out using filter paper. After that, the samples were negatively stained using a 2% uranyl acetate solution for 30 s.

siRNA binding by SYBR green dye exclusion assay
The SYBR Green II stain was used to test the ability of the transfection reagents to bind siRNA. In black 96-well plates, 200 µL of SYBR Green II (1X) was added to each well, followed by 4 µL of 0.025 µg/µL siNC. Different volumes of 0.005 µg/µL of the transfection reagent (diluted from 1 mg/ mL stock solution) were added to wells to achieve transfection reagent:siRNA ratios ranging from 1:1 to 70:1 (w/w). The plate was incubated for 30 min in the dark at room temperature and the fluorescence of the samples was then measured with a multiwell plate reader (Thermo Ascent; λ ex 485 nm, λ em 527 nm) to determine the amount of free siRNA. The percentage of bound siRNA vs. transfection reagent:siRNA ratio (w/w) was plotted to compare the relative siRNA binding efficiency of different transfection reagents.

Cell viability assay
MTT Assay was used to assess the effect of siMcl-1 treatment in MDA-MB-436 cells. The treatment was carried out in complete medium (DMEM/10% FBS/Pen-Strep) in triplicates in 96-well plates. 6 × 10 3 cells in a volume of 150 µL were seeded in wells and incubated for 24 h at 37 °C in 5% CO 2 atmosphere. The siRNA complexes were prepared as described above and 15 μL of siRNA polyplexes were added to the wells to give a final siRNA concentration of 40 to 80 nM. The cells were typically incubated for 3 or 6 days after polyplex treatment. At the desired timepoint, MTT solution (5 mg/mL) was added to the cells so as to attain a final concentration of 1 mg/mL in each well. The plates were incubated for an additional 2 h at 37 °C. The residual MTT medium was removed, and the formazan crystals formed were dissolved with DMSO. The absorbance (450 nm) was measured using a microplate reader (Molecular Devices, Spectramax 250) with pure DMSO serving as a blank solution. Percent cell viability was calculated with respect to the absorbance of non-treated cells. Analysis of Variance (oneway ANOVA) was applied to determine statistically significant differences among the study groups, with a confidence interval of 95% (p-value < 0.05) (GraphPad, version 8.0, San Diego, CA). Tukey's post hoc test was also performed for comparing all possible pairs of means in all groups.

Analysis of apoptosis
MDA-MB-436 cells were seeded (8 × 10 4 cells/well) in 24-well plates for 24 h before being treated with 40 nM and 80 nM siNC, siMcl-1, siSur and siMcl-1/siSur complexes for 72 h. The cells were washed once with HBSS, then collected and transferred to 1.5 mL centrifuge tubes after Accutase® digestion, washed (× 2) with 1X binding buffer, and centrifuged for 5 min at 900 rpm. Cells were then resuspended in 300 µL of binding buffer and transferred to 5 mL polystyrene round-bottom tubes. Each sample was incubated with 2.5 µL of FITC-Annexin V and 2.5 µL of Propidium Iodine (Apoptosis kit from BD Biosciences) for 15 min before fluorescence measurement by flow cytometry.

Real-time RT-qPCR for gene silencing
MDA-MB-436 cells (3.2 × 10 5 cells/well) were seeded in triplicates in 12 well plates and incubated for 24 h at 37 °C in 5% CO 2 . The cells were treated with siRNA complexes in complete media for 3 days. After washing (× 2) cells with HBSS, 0.5 mL TRIzol reagent was added to isolate total RNA. The lysates were incubated at room temperature for 5 min, followed by addition of 100 µL chloroform, and centrifuged for 15 min at 8900 rpm at 4 °C. The upper phase was transferred to a new tube and mixed with 250 µL of isopropanol. The mixture was centrifuged for 15 min at 8900 rpm at 4 °C [24]. The total RNA pellet was stored in 1 mL of 75% ethanol overnight at -20 °C and then centrifuged at 7500 rcf for 5 min. The pellet was then airdried for 30 min, resuspended in nuclease-free water, and then placed in a water bath at 60 °C for 10 min. One µg of extracted RNA was converted into cDNA by using the SensiFAST™ cDNA Synthesis kit as per the instructions of the manufacturer (Meridian Bioscience, OH, USA). For real time RT-qPCR analysis, human Beta-actin was used as an endogenous housekeeping gene. All RT-qPCR reactions were performed by the reaction mixtures incubated at 95 °C for 10 min followed by 40 amplification cycles at 95 °C for 15 s, 65 °C for 1 min using StepOnePlus (Applied Biosystems, CA, USA) real time thermal cycler. The fold change was calculated by the ΔΔC t method. Significant differences among study groups were analyzed using one-way ANOVA (GraphPad, v8.0; San Diego, USA), followed by Tukey's multiple comparison tests with significance level set at p < 0.05. Microarray analysis was used to further inspect human apoptotic gene expression (RT2 Profiler™ PCR Array Human Apoptosis; PAHS-012ZC) in treated cells. RNA extraction from treated cells and conversion of extracted RNA to cDNA was carried out as mentioned above. The RT2 Profiler™ PCR ArrayHuman Apoptosis plate was loaded with the SensiFAST™ SYBR Hi-ROX mastermix and cDNA template (5 ng/µL). Using a StepOnePlus (Applied Biosystems, CA, USA) thermal cycler, real time RT-qPCR was performed as outlined above. The ΔΔC t method was used to calculate the fold change.

Cellular uptake of siRNA polyplexes
To quantify siRNA uptake, MDA-MB-436 cells were transfected with 30 nM FAM-labeled siRNA at siRNA:reagent:additive ratios of 1:10:0 and 1:10:1 (w/w/w). After 24 h of transfection, cells were trypsinized and fixed with 3.7% formaldehyde. The mean fluorescence in cells and FAM-positive cell population were quantified using BD Accuri C6 Plus flow cytometer (BD Biosciences, Franklin Lakes, NJ). In addition, the mean fluorescence of the recovered cell population and the percentage of cells showing FAM-fluorescence were determined after gating a representative portion of nontreated cells for autofluorescence (typically set at 1% of total population) [25].
A fluorescence microscopy analysis of MDA-MB-436 cells transfected with FAM-labeled siRNA complexes was performed after 24 h and 72 h of cell uptake. Cells were washed (× 2) with HBSS, fixed with 3.7% formaldehyde, and stained with DAPI (Brunschwig Chemie, Amsterdam, the Netherlands) to visualize the nuclear border using the fluorescent microscope model FSX100 Bio-imaging navigator (Olympus America, Center Valley, PA). 15 NCG male mice, aged 6-8 weeks, were purchased from Charles River Labs (Quebec City, Canada) and used in this study. The animal study was carried out in compliance with the Canadian Council on Animal Care guidelines, with approval from the University of Alberta's Animal Care and Use Committee (AB, Canada). The MDA-MB-436 cell line was injected subcutaneously (2 × 10 6 cells in 50 µL of PBS and 50 µL of Matrigel) to generate tumor xenografts, and the mice were treated after the tumors reached a size of ≥ 50 mm 3 . Mice were treated via intravenous (tail-vein) injection of siNC and 1:1 combination of siMcl-1 and siSur polyplexes. The siRNA dose was 20 μg, formulated with 20 μg of Trans-Booster additive and 200 μg of Prime-Fect (giving a siRNA:Trans-Booster:Prime-Fect ratio (w/w) of 1:1:10) in 100 µL of RPMI media. The siRNA polyplexes were administered every third day and mice were observed daily. Tumor size was measured using a calliper on days 0, 3, 6 and 9 and the mean/standard error of relative tumor volume was determined in each group using the formula: 0.5LW 2 (the tumor's long diameter is L, and its short diameter is W.)

Mcl-1 and survivin silencing in vivo
The level of gene knockdown in the tumor was measured using real-time RT-qPCR. RNA from the tumors was extracted using the TRizol method and converted to cDNA using the SensiFAST™ cDNA Synthesis kit. The real time RT-qPCR reactions were performed as outlined above. The ΔΔC t technique was used to calculate the fold change.

Effect of siMcl-1 treatment on cell viability
The siRNA binding affinity of transfection reagents is summarized in Fig. 1A. All reagents displayed a similar binding pattern with siRNA, with Leu-Fect A displaying a more complete binding at higher reagent:siRNA ratios. The effect of transfection reagent:siRNA ratio and Trans-Booster additive on the complex size was evaluated (Fig. 1B). Increasing the reagent:siRNA ratio resulted in smaller complexes with Prime-Fect (225 vs. 306 nm), but did not alter the size of polyplexes for other transfection reagents used in complexation process. The typical size distribution is displayed in Fig. 1C. The presence of the additive resulted in a slight increase in size of Leu-Fect A and Leu-Fect C complexes (Fig. 1A). With increasing transfection reagent:siRNA ratio, the zeta-potential increased, but it dropped significantly when the additive was introduced in Prime-Fect, Leu-Fect A and Leu-Fect C polyplexes (Fig. 1D).
The TEM images of siRNA complexes with Prime-Fect are shown in Fig. 2, The complexes were prepared at Prime-Fect:siRNA ratios of 5 and 10, with and without the additive. In the absence of the additive, a more heterogeneous complex structures were evident, where smaller, presumably single particles were present as well as apparently aggregated particles. The agglomerates were constituted in some cases by more than 10 particles of variable sizes in a close-packed conformation, measuring up to 1 µm (upper right portion of Fig. 2A). It is likely that the aggomorated particles were a result of the TEM processing and especially the drying process. In the presence of the additive (1:5:1 and 1:10:1 samples), more uniform particle sizes and shapes were evident where individual spherical particles were evident without any signs of agglomorated particles. There was, however, some variation in particle sizes even with the additive.
The siRNA mediated inhibition of cell growth was then investigated in MDA-MB-436 cells by employing three different siRNAs (siCDC20, siHSP90, and siMcl-1), which were promising targets in past studies from the authors' group. The transfection reagent Prime-Fect was used for siRNA delivery as well as different siRNA:transfection reagent ratios (1:5 and 1:7.5) and siRNA concentrations (40, 60 and 80 nM). Among the three siRNAs, only Mcl-1 was effective in this cell model with ~ 35% inhibition (compared to non-treated group) in cell viability at the highest siRNA concentration (80 nM) (Fig. 3A). No growth inhibition was observed in the siCDC20 and siHSP90 treated groups. With the Trans-Booster additive ( Fig. 3B and C), the growth inhibition with siMcl-1 became more pronounced, giving significant differences between the siNC and siMcl-1 at 1:10:1 siRNA:transfection reagent:additive formulation. Different ratios of formulation were explored to identify the optimal ratio, but 1:10:1 appeared to be optimal in inhibiting cell viability with siMcl-1 (Fig. 1S), so that ratio was used for the rest of this study. The efficiency of commonly used transfection reagent Lipofectamine 2000 (Fig. 3C) was then examined for siMcl-1 delivery. Lipofectamine generally showed higher toxicity than the complexes formed with Prime-Fect, despite the lower transfection reagent ratio used (1:2, ratio recommended by the manufacturer), and clear inhibition of cell growth with the specific siRNA was only evident at a single siRNA concentration (60 nM). When the cell seeding density was increased from 1,500 to 6,000 cells/well (Fig. 3D), the non-specific toxicity of siNC was decreased from ~ 55% to ~ 15% inhibition without altering the response to siMcl-1. With prolonged treatment (6 days), maximum cell death was observed after transfection was carried out at the formulation ratio of 1:10:1 with 80 nM of siMcl-1 (Fig. 3E). Cell viability was ~ 35% on day 6 vs. ~ 50% on day 3 after siRNA transfection. With complexes at 1:5:1 and 1:10:1 ratios (siRNA/transfection reagent/additive), the Prime-Fect transfection reagent showed lower toxicity than other transfection reagents after 3 and 6 days of treatment.

Combination of Mcl-1 and survivin siRNAs
Treatment with a combination of siMcl-1 and siSur was then explored to determine if it was more effective than treatment with a single siRNA (Fig. 4). The combination siRNA treatment resulted in a strong MDA-MB-436 cell growth inhibition (~ 80% decrease in cell viability), which was significantly lower than single siRNA treatments. The siMcl-1/siSur combination was evaluated in an additional breast cancer cell model, MDA-MB-231 cells. The results showed a similar trend, although the response was relatively lower than the MDA-MB-436 cells. To investigate the effect of siRNA treatments on target mRNA levels, MDA-MB-436 cells were treated with siNC and siMcl-1 s at 60 and 80 nM siRNA and target transcript levels were quantified after 3 days of treatment (Fig. 4C). The siMcl-1 treated cells displayed significant Mcl-1 gene silencing (60% and 70% silencing at 60 and 80 nM respectively), as compared to the siNC treated cells, which did not show any silencing effect.
Although silencing of target mRNAs was clearly observed in the combination treatment, a trend of overexpression of non-target gene (survivin in the case of siMcl-1 treatment alone, and vice versa) was noted. This is not surprising given the equivalent roles of these two anti-apoptotic proteins and the possibility of compensating for reduced availability of one functional protein with a similar protein. A microarray of human anti-apoptotic genes was utilized to screen for other expression patterns after the siMcl-1/siSur treatment in combination ( Fig. 4E; see Fig. 2S for raw data). In the microarray assay, both Mcl-1 and survivin mRNA levels were found to be suppressed to ~ 40% and ~ 17%, respectively. Additionally, other genes including BCL2L10, CASP14, CD40, CIDEA, LTA, and PYCARD, were downregulated by Fig. 1 A) The binding of different polymers to scrambled siRNA by SYBR Green II dye exclusion assay (n = 3). B) Size in nm (mean ± SD) of Prime-Fect, Leu-Fect A and Leu-Fect C polyplexes (n = 2). C) Typi-cal size distribution obtained with 1:10:1 Prime-Fect polyplexes. D) ζ-potential (mean ± SD) of the three types of polyplexes at different formulations (n = 2) more than 80%. The mRNA levels of BIRC3, HRK, and BCL2A1 genes were observed to be upregulated by 9.9-, 5.3-, and 5.3-fold, respectively, possibly indicating their involvement to enhance cell survival when Mcl-1/survivin are suppressed.

Pro-apoptotic effect of siRNA treatment
The Annexin-V/PI apoptosis assay was used to quantify the effect of single and combination siRNA treatments on inducing apoptosis. The percentage of total apoptotic population in the Mcl-1 and survivin (Fig. 5C) siRNA treated groups was greater than the siNC treated group, which was evident in both early and late apoptotic populations (Fig. 5A-B). The siRNA combination treated group harboured ~ 25% late apoptotic population and ~ 30% total apoptotic population, which were significantly greater than the single siRNA treated groups (Fig. 5C).

Cellular uptake of siRNA complexes
The MDA-MB-436 cells were transfected with FAMlabeled siRNA and uptake was assessed after 1 and 3 days by measuring siRNA positive cell population and mean fluorescence intensity per cell (Fig. 6). The FAM-positive population was ≥ 80% on day 1 for all formulations with the polymer, but FAM-positive cells dramatically decreased in groups transfected with siRNA:transfection reagent ratio (1:5) on day 3 (Fig. 6Ai). Presumably, the higher amount of polymer with the 1:10 complex treated groups could better protect the siRNA from degradation. Based on mean FAM fluorescence intensity/cell, the additive polyplexes showed a higher uptake at both siRNA:polymer ratios (Fig. 6Aii). Despite the decrease in siRNA levels on day 3, better cellular uptake and FAM-positive population for the 1:10:1 formulation was evident compared to the lower ratio (1:5:1) formulation.
The uptake of siRNA complexes was confirmed using fluorescence cell imaging. From Fig. 6B, a higher uptake in the additive complex group was evident as compared to additive-free polyplexes for both the 1:5:1 and 1:10:1 formulations, in line with the flow cytometry data in Fig. 6A. At day 3 (Fig. 6Bii), the siRNA uptake was less than day 1, but the uptake demonstrated by the additive polyplexes was still higher than the additive-free groups.

Animal study
Tumor volumes were measured on days 3, 6 and 9 after RPMI (no treatment), siNC and siMcl-1/siSur combination injections every third day. Although tumor growth was observed up to tumor sizes of 50 mm 3 , there was no exponential growth observed in this model. Upon indicated treatments, reduction in tumor volume was not observed since the cell line (MDA-MB-436) utilized for generating tumor xenografts might not have been compatible with the specific strain of mice used. However, both Mcl-1 and survivin gene knockdown was observed (Fig. 7A and B) in the tumors treated with the siRNA combination compared to those treated with siNC and no treatment groups. Approximately 38% Mcl-1 silencing and 31% survivin silencing was seen in combination siRNA treated tumors compared to non-treated tumors.

Discussion
Nucleic acid-based approaches can form the foundation of future therapeutic agents with a greater specificity for inhibiting cancer cell proliferation than the traditional targeted therapies [26]. The siRNA therapy can be used to target specific cells if the aberrant mRNA sequence is known; however, an effective siRNA carrier must be developed to improve extracellular siRNA stability and siRNA uptake in target cells [27]. The transfection reagents evaluated here are based on lipid modification of low MW PEIs to improve The results show that supplementing the complexes with the anionic Trans-Booster can reduce the size of complexes assembled with Prime-Fect, the most effective delivery agent among those explored in this study. It is likely that the increased anionic charge of the complexes minimized the interactions among the complexes to yield fewer aggregates and smaller complexes. The reduced surface charge, as indicated by the zeta-potential, can be expected to hamper the interaction between cationic complexes and cell membranes [29]. But contrary to this expectation, the Trans-Booster incorporated complexes exhibited higher cellular uptake as shown in Fig. 6 The more negatively charged complexes may also be beneficial in lowering the cytotoxicity of complexes [30], as well as protecting the siRNA from enzymatic degradation. Moreover, the additive complexes may also improve the availability of siRNA intracellularly due to due to better siRNA release [14] from complexes, which is consistent with the results of Prabhakar et al. mesoporous silica nanoparticles modified with PEI and anionic dithiothreitol charges with a lower zeta-potential had a higher DNA release than nanoparticles with a higher zeta-potential [31]. The morphology of the polyplexes was studied with TEM. At siRNA:PRIME-Fect ratio of 1:5, particles were found either as single spheres or agglomerates, which explains the increased size obtained by the DLS. Increasing the siRNA/PRIME-Fect ratio from 5 to 10 generated less agglomeration and smaller particle sizes. The presence of the additive resulted in a well-dispersed sample with no presence of agglomerates, probably due to the shifting of zeta potential to more negative values. Larger particles in this sample showed a darker region at their center (Fig. 2E). Figure 2F shows a scheme of changes in agglomeration as a consequence of polyplex composition. In short, the increase in polycation concentration reduces the agglomerate size by reducing the number of particles in agglomerates. For both polycation compositions, the presence of additive avoided the formation of agglomerates.
To identify an effective target, siRNAs against the targets CDC20, HSP70 and Mcl-1, known mediators that are highly expressed [30,32,33] and implicated in breast cancer malignancy, were screened. These three targets were selected since they are involved in different pathways for controlling cell growth. The transcription factor CDC20 activates the Anaphase-Promoting Complex (APC) during mitosis, leading to chromatid separation [34]. HSP90 is a protein that stabilizes the cells against heat stress and helps with folding of a number of proteins required for tumor growth [35]. Mcl-1, a member of the BCL-2 family gene, blocks cytochrome C secretion from mitochondria to prevent induction of cell apoptosis [36]. Mcl-1 was found to be the most potent among the investigated targets and was hence chosen as a candidate for inhibition of malignant cell proliferation.
The data in Fig. 3B and C show that higher inhibition of cell growth can be achieved with higher transfection reagent:siRNA ratio. This was due to better intracellular delivery of the siRNA cargo at higher ratio, as seen in the flow cytometry-based uptake results. The optimal ratio was 1:10 and higher ratios led to undesired toxicity on the cells (data not shown). The efficiency of siRNA delivery with the selected transfection reagents was also compared with Lipofectamine, a commonly used commercial gene carrier. Lipofectamine was observed to be equivalent to Prime-Fect in efficacy, but displayed more toxicity even when it was used in low amounts. Wang et. al. had also reported a high toxicity of Lipofectamine in Huh-7 liver cancer cells, SHSY5Y neuroblastoma cells and MCF-7 breast cancer cells [37]. These studies were conducted with the optimal cell seeding density in multiwell plates where too low a cell density resulted in higher toxicity than desired, while too high a cell density hampered Mcl-1 gene silencing. Beyond the transfection reagent:siRNA ratio, another critical variable was the inclusion of Trans-Booster in the complexes. Such additive complexes led to robust inhibition of cell growth (as much as 60% vs. non-treated cells with siMcl-1) while the conventional additive-free complexes showed minimal inhibition of cell growth (< 15% inhibition). This was evident when using complexes with different ratios (1:5 and 1:10) as well as concentration of siRNA. The enhanced ability of additive complexes in reducing cell viability may be a direct consequence of higher cellular uptake seen with them compared to additive-free complexes. The cell killing activity of additive complexes improved with increase in duration siSur was also examined in our study since it is an antiapoptotic gene that is widely expressed in malignant cells [38], and has been shown to be a viable target in the management of breast cancer [39]. The combination of siMcl-1 and siSur showed an additive effect in inhibiting cell viability in both MDA-MB-436 and MDA-MB-231 cells. At a lower siRNA concentration of 40 nM in MDA-MB-231 cells, a synergistic activity was observed where the ineffective siMcl-1 improved the cell killing activity when delivered in combination with siSur (Fig. 4). The additive effect of Mcl-1 and siSur was also confirmed by analysis of apoptotic cell population after treatment (Fig. 5). The effect of siMcl-1 and its combination with siSur has not been previously reported with MDA-MB-436 cells. The synergistic effects of siRNA and chemotherapy drugs are routinely reported in the literature [38][39][40]. In the microarray PCR assay, other genes such as AKT1, an anti-autophagy gene, were down-regulated since the down-regulation of survivin and Mcl-1 can induce autophagy in cells. Norouzi et. al. for example, used doxorubicin in combination with siSur in PEI-modified silk fibroin nanoparticles in 4T1 cells and discovered a synergistic effect between the small molecular drug and siRNA [40]. The siMcl-1 and siSur combination was recently investigated by Pengnam et al. [41]. but they showed no effective response (i.e., cell killing) with siMcl-1 and siSur in MDA-MB-231 cells using a delivery system of cationic niosomes (PCN-B). The positive results reported in this study were presumably due to better siRNA transfection efficiency achieved by the specific transfection reagents employed.
An in vivo study was carried out in NCG mice with MDA-MB-436 xenografted tumors to assess siRNA polyplex activity. The NCG mice were able to establish human MDA-MB-436 tumors, but the xenografts in our hands did not grow as expected (i.e., exponentially) as in the other conventional xenograft models (Fig. 3S). After systemic (IV) administration of siRNA combination targeting Mcl-1 and survivin genes, reduced levels of Mcl-1 and survivin mRNA were noted, although the difference did not reach statistically significant levels (p ~ 0.78, p ~ 0.34 for Mcl-1 and survivin vs. Negative control, respectively). Our preliminary result suggests that NCG mice were unsuitable as an immunocompromised host for preclinical studies of MDA-MB-436 tumors. Future studies will be designed to employ a more established mouse strain for breast cancer grafting, such as the nude mouse or NOD/SCID mouse that were previously used to this end [42,43]. siSur and siMcl-1 individually (not in combination) were previously used in various xenograft models (4T1, SKBr-3, MCF7, MDA-MB-468, and MDA-MB-435) in mice successfully, leading to significant tumor reduction [44][45][46][47]. The effect(s) of anti-apoptotic protein silencing should be better revealed in an animal model with exponentially growing tumors.

Conclusions
The incorporation of Trans-Booster additive resulted in improved cellular uptake of siRNA, effective self-assembly of complexes and enhanced inhibition of breast cancer cell growth with the use of commercial transfection reagent Prime-Fect. Using optimal formulations, the siRNA treatment achieved ~ 65% decrease in cell viability compared to the no treatment group. The combination of siMcl-1 and siSur augmented the effect of the single Mcl-1 or siSur delivery alone, where the growth inhibition with the combination siRNA treatment reached ~ 80% after three days of treatment. This study provides evidence for the feasibility of exploring different target siRNA combinations, relevant in breast cancer malignancy, both in vitro and in vivo, with an optimized siRNA delivery system that is commercially available.

Declarations
Consent for publication All the authors agree to the publication of this manuscript.