HER2/neu Oncogene Silencing in a Breast Cancer Cell Model Using Cationic Lipid-Based Delivery Systems

The overexpression of the human epidermal growth factor 2 (HER2/neu) oncogene is predictive of adverse breast cancer prognosis. Silencing the HER2/neu overexpression using siRNA may be an effective treatment strategy. Major requirements for siRNA-based therapy are safe, stable, and efficient delivery systems to channel siRNA into target cells. This study assessed the efficacy of cationic lipid-based systems for the delivery of siRNA. Cationic liposomes were formulated with equimolar ratios of the respective cholesteryl cytofectins, 3β-N-(N′, N′-dimethylaminopropyl)-carbamoyl cholesterol (Chol-T) or N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), with the neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE), with and without a polyethylene glycol stabilizer. All cationic liposomes efficiently bound, compacted, and protected the therapeutic siRNA against nuclease degradation. Liposomes and siRNA lipoplexes were spherical, <200 nm in size, with moderate particle size distributions (PDI < 0.4). The siRNA lipoplexes exhibited minimal dose-dependent cytotoxicity and effective HER2/neu siRNA transfection in the HER2/neu overexpressing SKBR-3 cells. The non-PEGylated Chol-T-siRNA lipoplexes induced the highest HER2/neu silencing at the mRNA (10000-fold decrease) and protein levels (>111.6-fold decrease), surpassing that of commercially available Lipofectamine 3000 (4.1-fold reduction in mRNA expression). These cationic liposomes are suitable carriers of HER2/neu siRNA for gene silencing in breast cancer.


Introduction
Millions of people succumb to cancer annually, breast cancer (BC) being the most prevalent and a major cause of cancer death among women. In 2020, 2.3 million women were diagnosed with BC, with 685,000 deaths worldwide [1]. Breast cancer mortality is a greater challenge in developing countries with poor access to medical treatment. Radiation, surgery, and chemotherapy have been the conventional treatment options. Still, despite advances in the formulation of novel chemotherapeutics, their use has been limited due to systemic toxicities, multidrug resistance, and lack of tumor cell specificity [2].
Overexpression of the HER2/neu oncogene has been found in approximately 30% of all invasive BCs and correlates with more unfettered and destructive tumor growth and greater resistance to cancer chemotherapy [3][4][5]. In HER2/neu amplified breast carcinomas, expression levels are significantly higher, varying from 500,000 to more than 2 million receptors per tumor cell, compared to 25,000 to 185,000 receptors per cell in non-amplified tumors [6,7]. The exploitation of the overexpression of the HER2/neu oncogene can be a potential therapeutic target for BC. Despite recent advances in treatment strategies, most, if not all, conventional treatments, such as chemotherapy, are limited by a lack of specificity for tumor cells and the cell cycle dependence of many chemotherapeutic agents. This has spurred efforts to develop unique anticancer agents with improved molecular target specificity.
Silencing the HER/neu oncogene expression using small interfering RNA (siRNA) may effectively treat patients with HER2/neu overexpressing BC. However, the delivery of therapeutic siRNA to intracellular targets for the induction of sequence-specific mRNA degradation creates an encumbrance that limits the success of siRNA therapeutics. These limitations include the polyanionic nature, hydrophilic character, and relatively high molecular weight, which hampers the direct association of the siRNA to the cell membrane, which creates difficulty in the cellular uptake of these molecules by passive diffusion [8,9]. Furthermore, siRNA molecules in the physiological milieu are prone to degradation by serum nucleases, non-targeted biodistribution, and activation of an immune response, limiting its' systemic applications [5]. Hence, stable, non-toxic nanocarriers are needed to transport therapeutic siRNA efficiently.
Liposomes have been the most popular of the many lipid-based systems available. Liposomes are vesicles made up of natural membrane constituents and containing an aqueous core. They are biocompatible and biodegradable in vivo, and their by-products are non-toxic [10][11][12], which increased their popularity as efficient siRNA delivery vehicles. Cationic liposomes, due to their positive charge, readily form lipoplexes with the negatively charged siRNA, ensuring favorable intracellular siRNA separation with high transfection and little or no adverse physiological effects [13,14]. The cationic lipids used include a hydrophilic positively charged head group, a lipid hydrophobic tail to anchor the head group to the liposomal membrane bilayer, and a linker to connect the hydrophilic and hydrophobic regions [15,16]. This has led to many novel and interesting cationic lipids being developed over the years [14,[17][18][19], including liposomes containing polysaccharides such as chitin and pectin [20]. Of note is the lipid-based siRNA drug partisan (Onpattro), which was approved in 2018 for the treatment of polyneuropathies caused by hereditary transthyretin amyloidosis. This has opened avenues for other siRNA-based nanotherapeutics [21].
When systemically applied, cationic liposomes can activate complement adsorption with plasma proteins [22], resulting in the formation of a protein corona which is recognized and eliminated from circulation by the reticuloendothelial system (RES) or by renal excretion [23]. Hence, liposomes are surface modified by incorporating biocompatible, hydrophilic moieties such as polyethylene glycol (PEG), which provides steric stabilization and 'masks' the liposome charge density, preventing opsonization. Hence, the liposome circulation time is increased, and the passive tumor targeting of the liposome is improved [23,24]. However, it was noted that multiple administrations of PEGylated liposomes could lead to increased clearance from the blood, reducing the therapeutic efficiency [25].
This study evaluated the ability of cationic lipid-based carrier systems to deliver targetspecific siRNA to breast cancer cells. Cationic cytofectins, 3β-N-(N , N -dimethylaminopropyl)-carbamoyl cholesterol (Chol-T) [26] and N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09) [27] that have been reported previously were used in the liposomal formulation together with DOPE. These liposomes were modified by adding PEG2000 (0-5 mol%) to afford stealth liposomes. The efficacy with which the liposomesvehiculated siRNA induced silencing of the HER2/neu oncogene overexpression in vitro was assessed at both the mRNA and protein levels using qRT-PCR and western blotting, respectively.

Liposome Preparation
Cationic liposomes and PEGylated cationic liposomes were prepared using the thin film evaporation method [28]. The two cationic cholesteryl cytofectins (CCC), Chol-T and MS09, were previously synthesized in the laboratory and reported [26,27]. The composition and molar ratio of the liposomal formulations used in the present study are outlined in Table 1. PEGylated liposomes were formulated with 2 or 5 mol% of PEG. The lipid mixture was vortexed and rotor-evaporated (Büchi RE121 Rotavapor, Büchi, Switzerland) to a thin film, which was hydrated overnight at 4 • C in 500 µL sterile HEPES buffered saline (HBS, 20 mM HEPES, 150 mM NaCl; pH 7.5). The sample was then vortexed and sonicated for 5 min at 21 • C and stored at 4 • C.

Liposome: siRNA Complex Preparation
Lipoplexes were freshly prepared by adding the siRNA to varying amounts of the cationic liposome suspensions to obtain different (w/w) ratios. The reaction mixtures were briefly vortexed and incubated at room temperature for 30 min to allow for lipoplex formation. The morphology of the cationic liposomes and lipoplexes (at optimal binding ratios) was examined by cryo-TEM. Liposomes or lipoplexes (2 µL) were deposited onto a formvarcoated copper grid (Ted Pella Inc., Redding, CA, USA) and contrasted 1:1 (v/v) with 4% saturated acidic uranyl acetate. The grids were plunged into liquid nitrogen at −180 • C using an injector system (Leica Microsystems EM CPC, Buffalo Grove, IL, USA), and the samples were examined using a JEOL JEM-1010 electron microscope (Jeol, Tokyo, Japan) operating at an accelerating voltage of 100 kV. The images were captured using the associated Soft Imaging System (SIS) MegaView III, bearing a side-mounted 3-megapixel digital camera.

Size, Zeta Potential, and Polydispersity Index
The hydrodynamic size, zeta potential, and polydispersity index (PDI) of the liposomes and lipoplexes were determined by dynamic light scattering (DLS) on a Malvern Nano-ZS ZetaSizer (Malvern Instruments, Worcestershire, UK), equipped with a 5 mW He-Ne laser beam (633 nm, fixed backscattering detection optics positioned at 173 • ) at 25 • C. All measurements were done in triplicate. All data were analyzed using the ZetaSizer software version 6.30 (Malvern Instruments, Worcestershire, UK).

Band-Shift Assay
This assay was used to determine the optimum binding of the siRNA to the cationic liposomes and prepared as previously described [29]. The siRNA: cationic liposomes (w/w) preparations are presented in Table 2, with siRNA kept constant at 0.32 µg. Lipoplexes were loaded into the wells of a 2% agarose gel and electrophoresed at 50 V for 30 min. Gels were visualized, and images were captured on a Vacutec Syngene G: Box BioImaging System (Syngene, Cambridge, UK) using GeneSnap Imaging Software version 7.05 (Syngene, Cambridge, UK). Naked siRNA served as a positive control.

Serum Nuclease Protection Assay
The siRNA lipoplexes at sub-optimum, optimum, and supra-optimum weight ratios obtained for the band shift assay were used (Table 3). After incubation, the lipoplexes were treated with fetal bovine serum (FBS) to a final concentration of 10% and incubated at 37 • C for 4 h. The reaction was terminated with 10 mM EDTA, and lipoplexes disassociated using 0.5% SDS. After incubation for 20 min at 55 • C, the samples were subjected to agarose gel electrophoresis as in Section 2.5. Control samples included untreated siRNA and FBS-treated siRNA.

Cell Viability Studies
The cytotoxicity of the lipoplexes was assessed in the HEK293, MCF-7, and SKBR-3 cells using the MTT assay. Cells were seeded at a density of 2.0 × 10 4 cells per well into 48-well cell plates containing 0.25 mL of complete medium (EMEM supplemented with 10% (v/v) FBS and 1% antibiotics) and incubated at 37 • C for 24 h. The siRNA lipoplexes were prepared as in Section 2.3 and Table 3. After incubation, the medium was replaced, and the lipoplexes were added to the cells. The cells were incubated at 37 • C for 48 h, after which the medium was removed and replaced with 25 µL of MTT (5 mg · mL −1 in PBS) in 0.25 mL medium. The cells were incubated for an additional 4 h at 37 • C, after which the MTT-containing medium was aspirated, and DMSO (0.2 mL) was added to dissolve the formazan crystals. Untreated cells were used as the positive control (100% cell viability), together with a Lipofectamine ® 3000 control at its optimal concentration as per manufacturer's protocol. All assays were conducted in triplicate. Absorbance was measured at 570 nm using a Vacutec, Mindray MR-96A microplate reader, with DMSO as a blank. The cell viability (%) was then calculated using Equation (1) Four different sequences of 19 nucleotides (ON-TARGETplus SMARTpool) were used as potential siRNAs targeting the HER2/neu gene. A non-targeting sequence siRNA was used as a non-specific siRNA control. The SKBR-3 cells were seeded into 6-well plates at a density of 1 × 10 5 cells per well and incubated at 37 • C for 24 h. The medium was then replaced with 1.5 mL fresh complete medium, followed by adding 10 µL of the siRNA lipoplexes (Table 3), with siRNA maintained at 0.64 µg. Controls included untreated cells and Lipofectamine ® 3000 (positive transfection control), where lipoplexes were prepared according to the manufacturer's instructions (5 µL Lipofectamine ® 3000 reagent and 2.5 µL siRNA (0.64 µg) in 250 µL EMEM for 5 min at room temperature). All assays were done in triplicate. Post 48 h and 72 h transfection, the cells were harvested for assessing HER2/neu gene silencing using qRT-PCR and western blotting, respectively.

RNA Extraction and qRT-PCR
Total cellular RNA from the SKBR-3 cells was extracted using TRIzol ® Reagent following the manufacturer's protocol. The RNA pellet was resuspended in 30 µL RNase-free water. The total RNA was converted into cDNA by reverse transcriptase PCR using the high-capacity cDNA Reverse Transcription (RT) Kit with RNase inhibitor, following the manufacturer's protocol. Gene expression was quantified by qRT-PCR using the TaqMan ® gene expression assays, which are FAM TM dye-labeled and possess a minor-groove binding (MGB) probe. The primers and probe used were the genes of interest HER2/neu (Assay ID Hs01001580_m1) and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID Hs03929097_g1). Singleplex PCR reactions were conducted as triplicates for all samples. Each reaction mixture (20 µL) contained 10 µL TaqMan ® gene expression master mix (AmpliTaq Gold ® DNA polymerase, deoxyribonucleotide triphosphates (dNTPs) with deoxyuridine triphosphate (dUTP), UP (Ultra Pure), Uracil-DNA glycosylase (UDG), ROX TM passive reference, as well as buffer components optimized for specificity, sensitivity, and precision), 1 µL 20× TaqMan ® gene expression assay mix, and 9 µL sample cDNA. The qRT-PCR was performed under the following conditions: 95 • C for 10 min (hold), followed by 40 cycles of 95 • C for 15 s (denature) and 60 • C for 1 min (anneal/extend) on a BioRad CFX 96 TM Real-Time System, C1000 Touch TM Thermal Cycler using CFX Manager Software version 3.0. (Hercules, CA, USA) Relative expression values of HER2/neu mRNA normalized to the level of GAPDH mRNA were determined using the 2 −∆∆Ct method [30].
where s represents the sample, c the calibrator (untreated cells), GOI the gene of interest HER2/neu, and norm the normalizer gene GAPDH.

Protein Extraction and Western Blotting
The extraction of total cellular proteins was performed 72 h after transfection, using the ReadyPrepTM Protein Extraction Kit, according to the manufacturer's specifications (Bio-Rad, Hercules, CA, USA), and quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Approximately 20 µg of protein were treated with an equal volume of 2× Laemmli sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 125 mM Tris-HCl, pH 6.8) and heated at 95 • C for 5 min. Before loading, Mini-PROTEAN ® TGXTM (10%) long shelf life precast gel cassettes (Bio-Rad Laboratories, Richmond, CA, USA) were placed in a Bio-Rad Mini-PROTEAN ® Tetra System, with the upper tank containing chilled 1× Tris/glycine/SDS running buffer (25 mM Tris, 190 mM glycine, and 0.1% (w/v) SDS, pH 8.3, Bio-Rad Laboratories, Richmond, CA, USA)., and the lower tank filled with chilled 1× Tris/glycine/SDS running buffer. The protein samples were loaded into the wells, and electrophoresis was conducted at room temperature for 30 min at 200 V cm −1 . A molecular weight marker (3 µL) (Precision Plus ProteinTM dual extra standards) was loaded into the first well. Following electrophoresis, protein transfer (blotting) was conducted using the Bio-Rad Trans-Blot ® TurboTM transfer system and RTA transfer kits following the manufacturer's protocol. The transfer was performed at 2.5 A, 25 V for 10 min to promote high Mw transfer (>150 kDa). The blot and gel were then placed in deionized water. Before antibody incubation, unoccupied sites on the blot were saturated in a solution of 3% blotting-grade blocker (non-fat dry milk) in Trisbuffered saline (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were incubated overnight at 4 • C in TBST containing either Neu, a mouse monoclonal antibody raised against a synthetic peptide corresponding to amino acids 1242-1255 of human Neu (1:5000) for HER2/neu protein detection, or β-Actin (1:200) used as an internal control for protein loading. The following day, the primary antibody was removed, and the membranes were washed in 20 mL TBST with agitation for 5 min (5×). The membranes were then incubated at room temperature in goat anti-mouse IgG-HRP secondary antibody (1:2000 in TBST). After 1 h, the secondary antibody was decanted, and the membranes were washed in 20 mL TBST with agitation for 5 min (5×). The membranes were developed using a ClarityTM Western ECL substrate kit following the manufacturer's instructions. The blots were incubated for 5 min at room temperature and then visualized using a Bio-Rad digital imager ChemiDocTM MP system. Band intensity was determined using Image Lab Software version 5.2.2 (BioRad, Hercules, CA, USA).

Statistical Analysis
Data are presented as means ± SD (n = 3). Statistical Analysis among mean values was performed using one-way ANOVA followed by the Tukey-Kramer multiple comparisons test between formulations. All statistics were performed using a 95% confidence interval and were considered significant when the p-value was less than 0.05 (p < 0.05).

Liposome and Lipoplex Characterization
Cryo-TEM enabled the direct examination of the ultrastructure of the colloidal carriers in their frozen-hydrated state. Figure 1A-F presents the TEM micrographs of the Chol-T and MS09 lipoplexes with siRNA. The images revealed a heterogeneous population of typically spherical or ellipsoidal structures with a bilayered membrane surrounding the internal aqueous core. The lipoplexes appeared well dispersed with little aggregation. The blots were incubated for 5 min at room temperature and then visualized using a Bio-Rad digital imager ChemiDocTM MP system. Band intensity was determined using Image Lab Software version 5.2.2 (BioRad, Hercules, CA, USA).

Statistical Analysis
Data are presented as means ± SD (n = 3). Statistical Analysis among mean values was performed using one-way ANOVA followed by the Tukey-Kramer multiple comparisons test between formulations. All statistics were performed using a 95% confidence interval and were considered significant when the p-value was less than 0.05 (p < 0.05).

Liposome and Lipoplex Characterization
Cryo-TEM enabled the direct examination of the ultrastructure of the colloidal carriers in their frozen-hydrated state. Figure 1A-F presents the TEM micrographs of the Chol-T and MS09 lipoplexes with siRNA. The images revealed a heterogeneous population of typically spherical or ellipsoidal structures with a bilayered membrane surrounding the internal aqueous core. The lipoplexes appeared well dispersed with little aggregation. DLS measurements revealed nanometer (nm) sized particles which were accompanied by moderate to narrow polydispersity indices (PDI) and good zeta (ζ) potentials, as summarized in Table 4. For Chol-T, sizes ranged from 71.64 nm to 121.07 nm, while the lipoplexes ranged from 149.61 nm to 187.97 nm. The MS09 liposomes and lipoplexes were slightly smaller (65.47 nm to 113.02 nm and 103.24 nm to 169.13 nm, respectively). The MS09-based lipoplexes also exhibited better zeta potentials (16.08 mV to 53.21 mV) than the Chol-T-based lipoplexes. For both liposomes, a size reduction upon functionalization was noted. Furthermore, functionalization with PEG reduced the zeta potential, especially in the Chol-T-based liposomes and lipoplexes. DLS measurements revealed nanometer (nm) sized particles which were accompanied by moderate to narrow polydispersity indices (PDI) and good zeta (ζ) potentials, as summarized in Table 4. For Chol-T, sizes ranged from 71.64 nm to 121.07 nm, while the lipoplexes ranged from 149.61 nm to 187.97 nm. The MS09 liposomes and lipoplexes were slightly smaller (65.47 nm to 113.02 nm and 103.24 nm to 169.13 nm, respectively). The MS09-based lipoplexes also exhibited better zeta potentials (16.08 mV to 53.21 mV) than the Chol-T-based lipoplexes. For both liposomes, a size reduction upon functionalization was noted. Furthermore, functionalization with PEG reduced the zeta potential, especially in the Chol-T-based liposomes and lipoplexes.

Liposome Binding and Protection Efficiencies
The agarose gel migration patterns illustrated that all liposomal formulations effectively bound the siRNA, as seen in Figure 2. The complete or endpoint binding ratios of the siRNA by the cationic liposomes are represented in Table 5. Ratios below the optimum exhibited bands that fluoresced brightly and then gradually lightened until the band completely disappeared, signifying that the negatively charged siRNA was neutralized entirely at those respective N/P charge ratios.  Table 4. Size, zeta (ζ) potential, and polydispersity indices (PDI) of the cationic liposomes and complexes with siRNA at their optimum binding ratios (w/w).

Liposome Binding and Protection Efficiencies
The agarose gel migration patterns illustrated that all liposomal formulations e tively bound the siRNA, as seen in Figure 2. The complete or endpoint binding rati the siRNA by the cationic liposomes are represented in Table 5. Ratios below the optim exhibited bands that fluoresced brightly and then gradually lightened until the band pletely disappeared, signifying that the negatively charged siRNA was neutralized tirely at those respective N/P charge ratios.    The nuclease protection assay showed the ability of the cationic liposomes to protect their siRNA cargo from degradation (Figure 3). The positive controls (lanes 1) and negative controls (lanes 2) assessed the degree of serum nuclease digestion of the lipoplexes. Both non-PEGylated lipoplexes offered better protection to their siRNA cargo than their PEGylated counterparts ( Figure 3A). The nuclease protection assay showed the ability of the cationic liposomes to protect their siRNA cargo from degradation (Figure 3). The positive controls (lanes 1) and negative controls (lanes 2) assessed the degree of serum nuclease digestion of the lipoplexes. Both non-PEGylated lipoplexes offered better protection to their siRNA cargo than their PEGylated counterparts ( Figure 3A).

Cell Viability Assay
The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the viability of cells exposed to the transfection complexes ( Figure 4). All cells maintained a viability of over 70%, except for the SKBR-3 cells exposed to MS09 5% PEG2000 lipoplexes which showed a 50% decrease in cell viability (p < 0.001). Figure  4A,B shows that for the HEK293 and MCF-7 cells, a dose-dependent cytotoxic effect with an increase in charge ratios was observed. The MS09-based lipoplexes were slightly less toxic than the Chol-T -based lipoplexes in the HEK293 cells ( Figure 4A). The MS09: siRNA lipoplexes at N/P ratios of 4.9 and 5.4 were non-toxic to the HEK293 cells and produced improved cell viability (>100%). In the SKBR-3 cells, a dose-independent decrease in cell viability was observed for the MS09-based lipoplexes, and in some instances, MS09 was slightly more toxic than Chol-T. This was seen for the MS09 lipoplex containing 5% PEG, where cell viability was slightly above 50%. Nevertheless, all other lipoplexes produced cell viability above 70%.

Cell Viability Assay
The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the viability of cells exposed to the transfection complexes ( Figure 4). All cells maintained a viability of over 70%, except for the SKBR-3 cells exposed to MS09 5% PEG2000 lipoplexes which showed a 50% decrease in cell viability (p < 0.001). Figure 4A,B shows that for the HEK293 and MCF-7 cells, a dose-dependent cytotoxic effect with an increase in charge ratios was observed. The MS09-based lipoplexes were slightly less toxic than the Chol-T -based lipoplexes in the HEK293 cells ( Figure 4A). The MS09: siRNA lipoplexes at N/P ratios of 4.9 and 5.4 were non-toxic to the HEK293 cells and produced improved cell viability (>100%). In the SKBR-3 cells, a dose-independent decrease in cell viability was observed for the MS09-based lipoplexes, and in some instances, MS09 was slightly more toxic than Chol-T. This was seen for the MS09 lipoplex containing 5% PEG, where cell viability was slightly above 50%. Nevertheless, all other lipoplexes produced cell viability above 70%.

Real-Time Quantitative PCR (qRT-PCR)
According to the qRT-PCR results depicted in Figure 5, the calibrator revealed high HER2/neu gene levels. The NT-siRNA showed no knockdown, whereas the uncomplexed HER2/neu target siRNA resulted in a slight decrease of the HER2/neu gene at the mRNA level. The gene expression levels of the housekeeping normalizer gene GAPDH did not differ significantly among the control and test samples. Each liposomal formulation could efficiently deliver the siRNA against the HER2/neu oncogene in the SKBR-3 BC cells in the presence of serum, as indicated by the downregulation of HER2/neu at the mRNA level (p < 0.001). A comparison of the non-PEGylated Chol-T and MS09 lipoplexes at the different charge ratios revealed that Chol-T:siRNA lipoplexes induced the highest HER2/neu silencing effect at all concentrations (p < 0.001), exceeding that of Lipofectamine ® -3000 (4.1fold decrease). Pharmaceutics 2023, 15, x FOR PEER REVIEW 10 of 18  differ significantly among the control and test samples. Each liposomal formulation could efficiently deliver the siRNA against the HER2/neu oncogene in the SKBR-3 BC cells in the presence of serum, as indicated by the downregulation of HER2/neu at the mRNA level (p < 0.001). A comparison of the non-PEGylated Chol-T and MS09 lipoplexes at the different charge ratios revealed that Chol-T:siRNA lipoplexes induced the highest HER2/neu silencing effect at all concentrations (p < 0.001), exceeding that of Lipofectamine ® -3000 (4.1-fold decrease).

HER2/neu Protein Expression
The HER2/neu protein expression levels were analyzed using western blotting. Results were analyzed based on densitometric values, and β-actin control was used to determine the normalization or equivalency of lane loading. High levels of HER2/neu protein expression (ratios) in negative control treatments relative to β-actin ( Figure 6A) were observed. In contrast, dramatic decreases in HER2/neu protein expression levels were observed for all the siRNA delivery systems employed in this study. Figure 5. Analysis of HER2/neu gene expression in SKBR-3 cells by qRT-PCR. The vertical axis represents the relative quantification of HER2/neu normalized against GAPDH mRNA level using the comparative quantification algorithm 2-∆∆Ct [30]. The mean ± SD of independent experiments (n = 3). *** p < 0.001 was considered statistically significant.

HER2/neu Protein Expression
The HER2/neu protein expression levels were analyzed using western blotting. Results were analyzed based on densitometric values, and β-actin control was used to determine the normalization or equivalency of lane loading. High levels of HER2/neu protein expression (ratios) in negative control treatments relative to β-actin ( Figure 6A) were observed. In contrast, dramatic decreases in HER2/neu protein expression levels were observed for all the siRNA delivery systems employed in this study.

Discussion
Both cationic liposomes were previously synthesized and characterized, and their complexes with DNA were described as clusters of spherical liposomes [26,27], as observed in the current study. However, some variations were noted, which could be due to the amorphous nature or flexibility of the liposomal membranes. The surface modifications of the liposomal vesicles with PEG chains are not clear under TEM due to the low contrast of PEG [31].
The size of the liposome influences its cellular interaction and uptake, with sizes of 50-250 nm proposed to bypass elimination and clearance by the RES [32,33]. Our results indicate that the liposomes were relatively stable and homogeneous and are within the size range generally considered ideal for both cellular uptake and systematic circulation [34,35]. Lipoplex formation resulted in a size increase (up to 187.97 nm), possibly due to the binding of the siRNA onto the liposome surface compared to its' entrapment within the liposomes. The decrease in size and PDI of the Chol-T and MS09 liposomes upon PEGylation may be attributed to the strong inter-bilayer repulsion that can overcome the attractive van der Waals forces, thus providing a steric barrier at the surface of the Graphs represent the HER2/neu/β-actin normalization ratios. Data are presented as means ± SD (n = 3). * p < 0.1, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 are considered statistically significant vs. Lipofectamine ® 3000.

Discussion
Both cationic liposomes were previously synthesized and characterized, and their complexes with DNA were described as clusters of spherical liposomes [26,27], as observed in the current study. However, some variations were noted, which could be due to the amorphous nature or flexibility of the liposomal membranes. The surface modifications of the liposomal vesicles with PEG chains are not clear under TEM due to the low contrast of PEG [31].
The size of the liposome influences its cellular interaction and uptake, with sizes of 50-250 nm proposed to bypass elimination and clearance by the RES [32,33]. Our results indicate that the liposomes were relatively stable and homogeneous and are within the size range generally considered ideal for both cellular uptake and systematic circulation [34,35]. Lipoplex formation resulted in a size increase (up to 187.97 nm), possibly due to the binding of the siRNA onto the liposome surface compared to its' entrapment within the liposomes. The decrease in size and PDI of the Chol-T and MS09 liposomes upon PEGylation may be attributed to the strong inter-bilayer repulsion that can overcome the attractive van der Waals forces, thus providing a steric barrier at the surface of the liposomes. PEG chains are reported to provide steric stabilization, prevent vesicle aggregation and encourage the formation of homogeneous, smaller liposomes with good colloidal stability and biocompatibility [36,37]. The zeta potential indicates the colloidal stability with values < −25 mV or > +25 mV considered stable. The zeta potentials of the liposomes and lipoplexes decreased upon the addition of PEG due to the masking of the positive charges. However, liposomes containing 2% PEG remained highly positive (>20 mV), indicating that the amount, length, and density of the PEG chains can affect the degree of stability offered [38]. It is important that the amount of positive charge present be sufficient to allow for siRNA binding and endosomal escape without affecting its disassociation from the lipoplex [39]. The successful transfection noted confirms the favorable properties of these liposomes. The MS09 5% PEG had higher PDI values, suggesting a slightly polydisperse liposomal population. It was proposed that for liposomes, a PDI of 0.3 is acceptable and indicates a homogenous population of vesicles [40].
More of the PEGylated cationic liposomes were required to completely bind the siRNA, possibly due to the partial masking of the positive charges by PEG. Although the 5% MS09 liposomes could retard the siRNA, no clear endpoint was established. Overall, the liposomes showed good protection of the siRNA in the presence of FBS, with the 5% PEG liposomes offering the least protection. Turetskiy and Coworkers (2017) observed sandwichlike structures for the siRNA lipoplexes and proposed that this improved their ability to protect the nucleic acid from degradation [41]. PEGylated liposomes were proposed to form looser complexes with siRNA, with PEG chains preventing the electrostatic interaction between the siRNA and the cationic lipid [42]. This could lead to the siRNA being exposed on the liposome's surface and easily attacked by nucleases present in serum. There was also some correlation with the zeta potentials obtained, indicating that liposomal instability can affect the ability of the carrier to protect its cargo from nucleases. A similar serum nuclease digestion profile was reported for DOTAP-based cationic liposomes complexed with a TNF-α-siRNA [43].
The low cytotoxicity observed for these lipoplexes could be attributed to monovalent cationic lipids, which are less toxic than multivalent cationic lipids [44]. Furthermore, it was suggested that monovalent cationic liposomes possess good gene knockout due to their favorable cellular uptake and endosomal escape due to the ion-pair mechanism [25]. The DOPE and cholesterol in the liposomal formulations contributed to the lipoplexes biocompatibility and stability, with cholesterol, a natural biomembrane component known to regulate the fluidity of the lipid bilayer [15,45]. These results are similar to previous reports where an increase in the length of the spacer segment resulted in reduced cytotoxicity in vitro [46]. Moreover, lipid-containing carbamate bonds have lower cytotoxicity, are stable under neutral conditions, and are prone to hydrolysis at low pH (in the endosome) [47][48][49]. The increased cytotoxicity exhibited by the PEGylated lipoplexes was proposed to be due to a higher cationic lipid concentration [50] and the possibility that these lipids generate reactive oxygen species (ROS). Since cancer cells exhibit greater ROS stress than normal cells [51,52], the SKBR-3 cell viability may have affected ROS production.
PEG-based Chol-T lipoplexes produced a reduced HER2/neu mRNA expression, which is in accordance with a previous report where PEGylation of DC-Chol/DOPE siRNA lipoplexes caused a decrease in size and zeta potential, and no HER2 silencing in SKBR-3 cells [50]. However, PEG-based MS09 lipoplexes significantly increased the HER2/neu gene silencing (p < 0.001) despite a size and zeta potential reduction. This may be related to their slightly lower cell viability. The HER2/neu protein expression results corroborated the qRT-PCR findings, suggesting that cationic liposomes bearing shorter aliphatic chains offer better transfection efficiency. It was proposed that increased transfection efficiency with shorter hydrocarbon chains is due to increased bilayer fluidity, leading to enhanced inter-membrane transfer and mixing of the lipid membrane [53]. Although the MS09 lipoplexes did not elicit HER2/neu gene silencing at the same level as the Chol-T lipoplexes, they were still effective. These MS09 liposomes have previously shown significant c-MYC silencing in MCF-7 and HT-29 cells, surpassing that of Lipofectamine ® -3000 [54]. Cationic liposomes have also been successfully used to deliver siRNA (siPlk1) to breast cancer cells in vitro [55].
Despite advances in HER2-targeted monoclonal antibody therapy [56], including the use of Trastuzumab or Herceptin [57], the need for chemotherapy is still crucial for breast cancer patients. In addition, the approval of the siRNA drug, Onpattro and its effectiveness in improving nerve function has highlighted the immense potential of RNA interference for the treatment of other disorders. Hence, nanoparticle-mediated gene silencing may be a viable option. Overall, findings from this study encourage future studies and optimizations of these cationic liposomes for their use in gene silencing in cancer.

Conclusions
This study highlights the immense potential of both PEGylated and non-PEGylated Chol-T and MS09 cationic liposomes in HER2/neu siRNA oncogene silencing in SKBR-3 cells. The findings support the hypothesis that these non-viral cationic liposome systems can facilitate future-therapeutic siRNA gene delivery. However, with the caveat that PEGylated cationic liposomes be used with caution since their effects varied with each of the cytofectins used in this study. Post-inserting PEG on preformed siRNA lipoplexes is conceivably a viable option to alleviate some of the adverse effects of using PEG. This strategy demonstrates better siRNA binding and the potential for masking siRNAs and protecting them from nuclease digestion. Among the cationic liposomes utilized in this study, non-PEGylated Chol-T lipoplexes induced the highest gene silencing at all tested N/P ratios, as indicated by the significant decrease in gene expression (10,000-fold decrease, p < 0.001). They exceeded the silencing effect obtained by Lipofectamine ® -3000 (4.1-fold reduction). Chol-T lipoplexes were well dispersed, colloidally stable, possessed the lowest N/P charge ratio, and exhibited minimal cytotoxicity in the SKBR-3 cells, which may have been attributed to their superior transfection. Given their biocompatibility and excellent transfection efficiency compared to the commercial transfection agent, these cationic liposomes offer a promising and attractive alternative for siRNA gene therapy in vivo. In addition, a combination therapy using siRNA and an anticancer drug can be employed as a further study.