Anti- c-myc cholesterol based lipoplexes as onco-nanotherapeutic agents in vitro

Background: Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNA-based, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated siRNA delivery systems. Methods: This study has focused on cationic liposomes prepared with equimolar quantities of the cytofectin, N,N-dimethylaminopropylamido-succinylcholesteryl-formylhydrazide (MS09), and cholesterol (Chol) for the development of a simple, but effective anti- c-myc onco-nanotherapeutic agent. Liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) in place of Chol as the co-lipid were included for comparative purposes. Results: Liposomes successfully bound siRNA forming lipoplexes of less than 150 nm in size, which assumed bilamellar aggregrates. The liposome formulations were well tolerated in the human breast adenocarcinoma (MCF-7) and colon carcinoma (HT-29) cells, which overexpress c-myc. Lipoplexes directed against the c-myc transcript mediated a dramatic reduction in c-myc mRNA and protein levels. Moreover, oncogene knockdown and anti-cancer effects were superior to that of Lipofectamine™ 3000. Conclusion: This anti- c-myc MS09:Chol lipoplex exemplifies a simple anticancer agent with enhanced c-myc gene silencing potential in vitro


Introduction
Cancer is one of the leading causes of death world-wide. Deaths due to cancer have been reported to outnumber those due to acquired immune deficiency syndrome (AIDS), malaria and tuberculosis combined 1 . Cancer treatment currently involves surgery, chemotherapy and/or radiation depending on the type and stage of the disease. Despite advances in understanding tumorigenesis and disease progression, these treatments are limited by harsh side-effects, the possibility of recurrences, and are heavily dependent on early detection and diagnosis for success 2 . With the global cancer burden projected to increase to 21.7 million new cases and 13 million deaths by the year 2030, it is clear that more effective treatment strategies are required 1 .
The altered activity of the c-myc proto-oncogene has been identified as an important element in the initiation and maintenance of the cancerous state of a cell. The c-myc gene encodes a nuclear phosphoprotein that is widely recognized for its role as a transcription factor. The c-Myc protein is believed to participate in the regulation of 10-15% of all genes 3 . These include genes involved in cell-cycle progression, metabolism, cell growth, differentiation, adhesion, and apoptosis 4-10 . Hence, advances in the design of appropriate c-myc-silencing systems may prove useful in treating a broad range of cancers 11 .
In theory, effective c-myc silencing may be achieved using endogenous cellular machinery, provided that the designed small interfering RNA (siRNA) molecule is successfully introduced. Several factors militate against the success of naked siRNA molecules in vivo. Naked siRNA molecules are highly susceptible to serum nucleases and are rapidly cleared by the kidneys 11,12 , while the size (~14 kDa) and negative charge of the siRNA prevents its passage across biological membranes 13 . Therefore, an appropriate carrier is required to protect the siRNA from damage and elimination, as well as to disguise its negative charge. This system must have low toxicity, afford stability in serum, avoid recognition by the immune system, avoid renal clearance, and successfully deliver its contents to the RNAi machinery in the diseased cells.
Since nucleic acids can electrostatically associate with positively charged agents, a variety of cationic molecules as potential carrier vehicles have been investigated, cationic lipids receiving much attention, both in laboratory-scale experiments and clinical trials [14][15][16] . Cationic liposomes formed by the self-assembly of cationic and neutral or helper lipids, are the earliest cationic lipid-based delivery systems 17,18 . These form lipoplexes, when associated with siRNA. Their favorable characteristics such as safety, biocompatibility, and amenability to modification, have sustained their interest in cationic liposomal-siRNA delivery 19 . Although several novel liposomal-siRNA systems have shown promise, none have resulted in a commercially available treatment 16 . Major barriers to their application include poor stability in the bloodstream and early recognition by the immune system, as these positively charged lipoplexes associate with anionic serum proteins such as albumin and lipoproteins. This results in opsonization by serum components, destabilization of the lipoplex, and damage to the nucleic acid cargo before it reaches the diseased cells. Furthermore, lipoplexes often aggregate forming larger particles that accumulate in the lung and are rapidly cleared by the reticuloendothelial system, reducing dosage and circulation time [20][21][22] . Increase of the mechanical strength of the liposome bilayer to render it more resistant to the destabilizing action of serum proteins can be achieved by the incorporation of rigid, membrane-stabilizing lipids such as cholesterol (Chol) 23,24 .
Synthesis of MS09 and liposome formulation MS09 was prepared from cholesterylformylhydrazide hemisuccinate (MS08) via an active ester intermediate according to the method previously published 23 . Briefly, the N-hydroxysuccinimide ester of cholesterylformylhydrazidehemisuccinate (0.083 mmol) and dimethylpropylamine (0.35 mmol) were dissolved in a H 2 O:pyridine:DMF (13:7:10, v/v/v) mixture. The product (MS09) was monitored and purified by thin layer chromatography. For liposome formulation, the lipids were combined in quantities as in Table 1 and concentrated to a thin film in vacuo (Büchi Rotavapor R rotary evaporator). The film was rehydrated (4°C, 48h) in sterile HEPES buffered saline (HBS, 20 mM HEPES, 150 mM NaCl, pH 7.5; 0.5 ml), vortexed and, then sonicated in a Transsonic (T460/H) bath-type sonicator (Singen, Germany). Liposome preparations were stored at 4°C and sonicated prior to use.

Liposomes and lipoplex characterization
The morphology of liposomes and siRNA lipoplexes were evaluated using cryogenic transmission electron microscopy (cryo-TEM), using a JEOL JEM.1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Images were captured using an Olympus MegaView III digital camera in conjunction with SIS iTEM Universal Imaging Platform software (Shinjuku, Japan). Particle size and zeta (ζ) potential were determined using the Nanoparticle tracking analysis (NTA, NanoSight NS500, Malvern Instruments, Worcestershire, UK) fitted with a ZetaSight™ module and NTA 3.0 software.

Gel retardation assay
Lipoplexes were prepared ( w / w ) with varying amounts of the liposomes and a fixed quantity of siGENOME non-targeting siRNA (0.3 μg), as per Table 2. Lipoplexes (10 μl in HBS) were mixed with gel loading buffer (40% sucrose, 0.25% xylene cyanol, 0.25% bromophenol blue; 2.5 μl) and loaded onto 2% agarose gels. Electrophoresis was carried out for 30 min in a Mini-Sub ® Cell GT electrophoresis cell (Bio Rad, Richmond, CA) containing tris-phosphate-EDTA (TPE) running buffer (36mM Tris-HCl, 30mM NaH 2 PO 4 , 10mM EDTA, pH 7.5) at 50 V. Gels were stained with ethidium bromide (0.1 μg/ml in water) for 30 min, and images were viewed and captured using a Vacutec Syngene G:Box gel documentation system, fitted with GeneSnap software, version 7.05.02 (Syngene, Cambridge, UK). Densitometric analysis was performed with the associated GeneTools software. The fluorescence intensities of unbound siRNA were expressed as a percentage against that of a naked siRNA control. The amount of liposomeassociated siRNA at each MS09:siRNA ( w / w ) ratio was determined as follows: % bound siRNA = 100 -% free siRNA  Nuclease protection assays Lipoplexes each containing 0.3 μg non-targeting siRNA were assembled at ( w / w ) ratios of 12:1-32:1. These were incubated with 10% FBS at 37°C for 4h. A control, 0.3 μg naked siRNA was treated similarly. Nuclease activity was terminated using EDTA (10mM), and complexes destabilized with 0.5% ( w / v ) SDS (55°C, 25 min). This was followed by electrophoresis as described previously.
Cell viability assays MCF-7 and HT-29 cells were seeded at densities of 4.0 × 10 4 cells/well in 48 well plates, and maintained at 37°C for 24h, followed by treatment with lipoplexes containing non-targeting siRNA (14 nM) at ratios used in the nuclease protection assay. Controls with naked siRNA (14 nM) were included. Lipofectamine™ 3000 (LF3K), prepared according to the manufacturer's protocol was used as a positive control.

Gene expression assays
In all ensuing experiments, MS09:Chol and MS09:DOPE lipoplexes (MS09:siRNA w / w = 16:1) contained 12 nM siRNA. MCF-7 and HT-29 cells were seeded in 6-well plates at densities of 3.0 ×10 5 cells/well and incubated as previously described. Cells were transfected with lipoplexes as previously described. Lipoplexes were assembled with 12 nM of either non-targeting siRNA or ON TARGETplus SMARTpool Human myc-siRNA (anti-c-myc-siRNA). Transfections were carried out in triplicate, followed by cell harvesting for total RNA and protein.

RNA extraction and RT-qPCR
Total cellular RNA was extracted using TRIzol ® Reagent, and cDNA synthesis was carried out using the gDNA Clear cDNA Synthesis Kit, as per manufacturer's instructions. For a single reaction, 0.8 μg of the RNA sample was used. Reaction mixtures were prepared on ice and the reactions were carried out in a C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories (PTY) Ltd., Richmond, USA). The reaction was performed as follows: DNA digestion (25 °C, 5 min), DNase inactivation (75 °C, 5 min), and samples were maintained at 4 °C for 10 min. The RT supermix (4 μl) was added and cDNA synthesis was carried out as follows: priming (25 °C, 5 min), reverse transcription (46 °C, 20 min), RT inactivation (95 °C, 1 min) and samples were held at 4 °C for 10 min. Two cDNA synthesis reactions per RNA isolate were performed in parallel i.e. one reaction containing the RT supermix and a no RT control in which the no-RT supermix was added instead. cDNA samples were diluted to a final concentration of 25 ng/μl in nuclease-free water and stored at 4 °C, for no more than a week. The product of each cDNA synthesis reaction was subjected to RT-qPCR. A single reaction mixture (20 μl) contained SsoAdvanced™ Universal SYBR ® Green Supermix (10 μl); primers (1 μl) specific for either the target gene, c-MYC (PrimePCR™ SYBR ® Green Assay: MYC, Human) or reference gene, β-actin (PrimePCR™ SYBR ® Green Assay: ACTB, Human); cDNA sample (25 ng, 1μl) and nuclease-free water (8 μl). Reaction mixtures in which DNA templates (either PrimePCR™ Template for SYBR ® Green Assay: MYC, Human; or PrimePCR™ Template for SYBR ® Green Assay: ACTB, Human; 1 μl) were substituted for cDNA served as positive controls. Reaction mixtures where nuclease-free water (1 μl) was substituted for either primers or cDNA were included as negative controls. All reactions were performed in triplicate.
Reaction mixtures were prepared, on ice, in Hard-Shell ® 96 well plates, sealed, briefly centrifuged and then loaded in a C1000 Touch™ Thermal Cycler (CFX 96 Touch™ Real-Time PCR Detection System, Bio-Rad Laboratories (PTY) Ltd., Richmond, USA). Data was analyzed with CFX Manager™ Software version 3.0, and c-myc expression was normalized to β-actin using the ΔΔCq comparative quantification algorithm.
Total protein expression and ELISA MCF-7 and HT-29 cells were seeded in 6-well plates at densities of 3.0 ×10 5 cells/well and incubated as previously described. Cells were transfected with lipoplexes as previously described. The medium was removed, and cells washed twice with ice-cold PBS (1 ml/well). Cold (4°C) RIPA buffer (100 μl/well) was then added and cells placed on ice for 5 min with gentle shaking to dislodge cells. Cell suspensions were centrifuged (14 000 × g, 4°C, 15 min) and lysates/protein extracts were immobilized onto the wells of a 96-well plate with 50 mM carbonate-bicarbonate coating buffer (pH 9.6, at 4°C), overnight. Three replicates per isolate were performed. Each well received 10 μg protein in 100 μl coating buffer. The coating buffer was removed, and wells were rinsed twice with TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween 20 (TBST, 100 μl/well). Wells were then treated with 5% non-fat dry milk in TBST (100 μl) with gentle agitation to saturate unoccupied attachment sites. The blocking agent was removed, and wells rinsed twice with TBST (100 μl/well). Either c-myc (1:2000, in TBST) or β actin (1:10 000, in TBST) primary antibodies were added (100 μl/well) and incubated at room temperature for 1h. Primary antibodies were removed and wells washed with TBST (4x, 100 μl/well) for 5 min each, with agitation. The secondary antibody (1:2000, in TBST) was then added and incubated at room temperature for 1h. Wells were drained and washed with TBST, as previously. TMB (100 μl/well) was applied (room temperature, 30 min), and the reaction was terminated by the addition of 2M H 2 SO 4 (100 μl/well). Absorbance was measured at 450 against a TMB (100 μl)/2M H 2 SO 4 (100 μl) blank. Wells containing BSA (10 μg) served as negative controls. Antibody-free and substrate-free controls were included. Expression of c-myc was normalized to β actin and presented relative to untreated cells.

Statistical analysis
Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey's Multiple Comparison Test to compare between groups using GraphPad Prism version 5.04 (GraphPad Software Inc., USA). P values less than 0.05 were considered significant.

Results
Liposome and lipoplex characterization TEM presented MS09:DOPE and MS09:Chol formulations as round to irregular shaped unilamellar vesicles, respectively (Figure 1a,b). The liposome-siRNA complexes (Figure 1c,d) assumed structures that were different from the vesicles, emphasizing the heterogeneity and assembly of the liposome-siRNA complexes.
NTA results (Table 3) show that MS09:DOPE and MS09:Chol liposome sizes were below 150 nm in size and may be classified as small unilamellar vesicles. The substitution of DOPE with Chol seems to have no significant effect on size or zeta potential, which remained high. It was observed that Chol is not likely to influence the electrical surface potential of liposomes because it does not bear an ionizable group 26 . Lipoplexes were less than 150 nm in size (Table 3), which is important for passive targeting of tumour cells via the enhanced permeability and retention effect. Zeta potential measurements are based on the interaction of the particle with ions in the medium in which it is dispersed. In this study, liposomes were dispersed in HEPES buffer, which can influence the zeta potential of a bilayer depending on the orientation of the molecule's ionizable groups relative to the membrane 27,28 in the electrical double layer. Hence, the negative zeta potential values do not necessarily imply that the liposomes would be unable to associate with siRNA molecules. Lipoplexes similarly displayed negative zeta potentials (Table 3). Although it is accepted that the net positive charge of lipoplexes allows for binding to anionic membrane-associated proteoglycans to initiate cellular uptake 29 , it is also possible for siRNA lipoplexes with negative zeta potential to enter cells and successfully facilitate gene silencing.

Gel retardation assay
The gel retardation assay or band shift assay is widely documented as the first step in assessing the siRNA-binding ability of cationic carriers 30-32 . This assay is based on the premise that the migration of siRNA is retarded in an electric field when bound to a carrier due to the formation of electroneutral complexes that are unable to permeate the gel matrix. MS09: DOPE was capable of fully preventing the migration of siRNA (Figure 2a), whereas Figure 2b shows that although there was a decrease in unbound siRNA with increasing liposome content for the MS09:Chol formulation, unbound siRNA was evident at all MS09:siRNA ( w / w ) ratios. Densitometric analysis (Figure 3) confirmed that the binding of siRNA by these liposome formulations increased until a point, whereupon free siRNA in the gel persisted despite addition of liposome. This MS09:siRNA ( w / w ) ratio was taken as the end-point/optimum binding ratio of these liposomes.

Nuclease protection study
Attempts at lipid-mediated siRNA delivery are often frustrated by adverse interactions with serum. Figure 4 shows that, while naked siRNA was entirely degraded under experimental conditions (lanes 2), intact siRNA bands, less intense than the untreated control, were clearly visible in all instances. This shows that the liposomal formulations partially protected siRNA at the respective MS09:siRNA (w/w) ratios. Densitometric analysis of gels ( Figure 5) provided further insight into the siRNA-protecting capabilities of the liposomes. Maximum intact siRNA of 73% afforded by MS09:Chol    liposomes at the MS09:siRNA ( w / w ) ratio of 24:1 was significantly less than that of MS09:DOPE. 25% of siRNA was likely to be surface associated, as it was so loosely bound that it was coaxed off during electrophoresis. Such siRNA is readily detached from the liposomal bilayer upon exposure to serum nucleases 33 .

Cell viability studies
It was important that any growth inhibitory effects in the cancer cells be attributed solely to the delivered siRNA, and not due to any intrinsic harmful effect of the liposomal carrier. This assay is based on the principle that enzymes of the mitochondria of living cells reduce soluble MTT, a tetrazolium salt, to formazan crystals 34 . The intensity of the resultant purple solution correlates with the extent of MTT reduction, and the number of viable cells 35 . No significant reduction in viability was detected upon treatment of all cells with naked siRNA at concentrations contained in lipoplexes ( Figure 6). Cells retained viability of at least 88% with exposure to LF3K-siRNA complexes. In general, cell survival after exposure to the liposomal formulations were greater than 85% with no severe cytotoxicity evident. Gene silencing mediated by MS09:DOPE and MS09:Chol lipoplexes Given that the initial RNAi effect is exerted at the mRNA level, the effect of transfection with anti-c-myc lipoplexes on c-myc transcripts in cancer cells was studied using RT-qPCR. Figure 7 shows a decrease in c-myc-mRNA in instances where a transfecting agent was used to deliver anti-c-myc-siRNA sequences. Quantification of cellular c-myc protein by ELISA (Figure 8) showed that a decrease in c-myc mRNA levels was, in all cases, accompanied by a concomitant reduction in protein expression. Complexes assembled with non-targeting siRNA were without effect. This confirmed that the observed reduction in c-myc-mRNA contributed to the RNAi effect of the delivered anti-c-myc-siRNA. The fact that naked anti-c-myc-siRNA did not influence c-myc expression in any way, highlights the need for a delivery vehicle.
MS09:Chol and MS09:DOPE lipoplexes produced significant gene silencing compared to LF3K in both cell lines. In the MCF-7 cell line, MS09 lipoplexes achieved 8-and 3.5-fold greater knockdown of c-myc than LF3K at the mRNA and protein levels, respectively. In HT-29 cells, the decrease in c-myc-mRNA and protein was 5-and 2.8-fold more for MS09 lipoplexes. The superior performance of MS09 lipoplexes is highlighted by the fact that these contained at half the final siRNA concentration as LF3K.

Apoptosis assay
A desirable feature is for cancer treatment to induce cancer cell death without causing harm to surrounding healthy tissue. Hence, several anticancer approaches exploit the natural mechanisms of cell death such as apoptosis, that normally eliminates damaged and/or harmful cells in a regulated fashion 36 . The AO/EtBr method is based on the principle that AO enters cells with intact plasma membranes and binds to DNA to emit green fluorescence, while EtBr enters cells with defective membrane integrity and fluoresces red-orange when bound to DNA. Differentiation between normal, early apoptotic, late apoptotic and necrotic cells was made based on observations of nuclear morphology (Figure 9 and Figure 10). Live cells were characterized by a bright green nucleus in the center of the cell. The nuclei of early apoptotic cells, with undamaged membranes, also stained green, but appeared to be fragmented or condensed. In contrast, the nuclei of late apoptotic cells, with compromised membrane integrity, stained orange with evidence of fragmentation or condensation. Finally, necrotic cells were characterized by an intact bright orange nucleus 37 .
The major mechanism of cell death observed with these anti-c-myc lipoplexes was apoptosis, which is similar to other studies demonstrating that inhibition of c-myc in cancer cells leads to apoptosis 38-40 . Importantly, necrosis, a non-specific form of cell death that is associated with an inflammatory response 41 , was negligible in all instances, accounting for less than 3% of total cells per sample. Hence, MS09:Chol and MS09:DOPE-mediated anti-c-myc-siRNA delivery is capable of destroying cancer cells without damaging healthy tissue. The application of anti-c-myc-siRNA on its own did not result in any anticancer activity. Hence we can conclude that the anticancer effects can be ascribed to the RNAi activity of liposome bound anti-c-myc-siRNA.

Discussion
MS09, a cationic lipid comprising three structural domains viz. a hydrophobic cholesteryl anchor and a polar dimethylammonium head group, separated by a 15Å spacer arm, was originally co-formulated with DOPE for the delivery of DNA 23 and siRNA 42 into mammalian cells. DOPE is a commonly used helper lipid in cationic liposome formulations 43,44 , but may not be suitable for intravenous administration. Early studies TEM revealed that the liposomes were unilamellar, spherical vesicles that were capable of forming lipoplexes with the siRNA. Both the liposomes and lipoplexes were less than 150 nm in size, favoring targeting as nanoparticles of a suitable size will not pass through the tight junctions of normal blood vessels but can access tumor cells by passing through their more permeable vasculature and are retained because of reduced lymphatic drainage 50 . These properties are valuable as determinants of lipoplex performance because they impact on the circulation time of lipoplexes in the body, accumulation at target sites, interaction with cells, the efficacy of cellular uptake and, gene silencing activity 51 . Others have reported ligand-modification of anti-c-myc lipid-based nanoparticles 40,52 . While these may render delivery to tumour cells more effective and possibly alleviate short-term effects of c-myc inhibition in normal cells, it may not be an entirely necessary feature. This is with reference to findings that presented the feasibility of systemic c-myc inhibition with a non-discriminate c-myc inhibitor 53 and, that systemic administration of anti-c-myc siRNA in non-targeted liposomes did not inhibit the growth of cells with low c-Myc levels 54 . Therefore, this study has focused on passive targeting of lipoplexes through optimising physical features to exploit the enhanced permeability and retention effect for effective intratumoural delivery of siRNA. In fact, preliminary experiments (not herein reported) show that the lipoplexes were not taken up by a non-transformed cell line. Moreover, a passive targeting strategy may pave the way for less elaborate anti-c-myc siRNA lipid nanoparticles that can be more easily and economically produced 55 .
The negative zeta potential of the liposomes and lipoplexes did not affect their cellular uptake. It was reported that siRNA lipoplexes with negative zeta potentials were internalized by breast cancer cells via endocytosis and, that cellular uptake was dependent upon the activity of microtubules and actin 56 . Negatively charged lipid-based siRNA nanocomplexes may be useful as they can avoid aggregation through interaction with erythrocytes and anionic proteins in biological fluids and have also been associated with lower toxicities than complexes carrying a net positive charge 57,58 . MS09:DOPE and MS09: Chol liposomes showed good siRNA binding, but replacing DOPE with Chol at the same molar ratio in MS09 formulations weakened the siRNA interaction with MS09:Chol. It is possible that Chol may have induced arrangement of cytofectin molecules during vesicle formation such that a greater number of cationic centers were positioned inwards rather than on the surface of the bilayer. A further explanation may arise from the fact that Chol is a more rigid lipid than DOPE and results in liposomal bilayers that are less malleable. Therefore, during the process of lipoplex formation, the Chol-containing liposomes may not change conformation as easily as their DOPE-containing counterparts to completely encompass siRNA molecules. An early study with DOTAP/Chol liposomes, also demonstrated that Chol widened the interlamellar spaces of the resultant lipoplexes 59 .
Both liposomes were capable of protecting the siRNA against nuclease degradation and were well tolerated by the MCF-7 and HT-29 cell lines. Cytotoxicity testing was performed under the same conditions as those employed in gene silencing experiments, except that lipoplexes were assembled with non-targeting siRNA so as to rule out the possibility of cell death due to silencing of any functional genes. In general, cell survival after exposure to the MS09:Chol formulation was better than its DOPE-containing counterpart. This could be attributed to Chol as an endogenous lipid, and that DOPE is pH-sensitive while Chol is not. This property of DOPE, which is regarded as important for transfection, has also been associated with toxicity, because it causes destabilization of lysosomes and release of debris within the cell 60 .
Gene silencing experiments revealed that both MS09 liposomal formulations exhibited enhanced c-myc gene silencing in the MCF-7 and HT-29 cell lines, superior to that of LF3K. It was observed that c-myc inhibition at both levels of expression by all transfecting agents was more pronounced in the MCF-7 cells than in the HT-29 cells (P<0.05). This is underscored by the fact that HT-29 cells are considerably more difficult to transfect than other human cell lines 61,62 . This was also evident for the LF3K reagent with a markedly lower oncogene knockdown (1.4-fold reduction). In HT-29 cells cellular uptake of MS09:Chol and MS09:DOPE lipoplexes was comparable (P>0.05). The similar reduction in c-myc-mRNA and protein implied that the MS09 lipoplexes facilitated RISC-engagement of intact anti-c-myc-siRNA molecules with near-equal efficiency. However, in the MCF-7 cells, the MS09:Chol lipoplex, at 12 nM siRNA, facilitated effec-tivesiRNA delivery compared to DOPE-containing lipoplexes  A significant decrease in c-myc expression was correlated with anticancer effects following apoptosis analysis, which included inhibition of cancer cell migration, loss of cell viability and elimination of cancer cells through apoptosis, with the exception of anti-c-myc-LF3K in HT-29 cells. Here c-myc inhibition was too low to induce significant apoptosis and reduce cancer cell numbers. Overall, both anti-c-myc lipoplexes produced better anticancer activity than LF3K, in a given cell line. Furthermore, the comparable gene silencing activity mediated by MS09:Chol and MS09:DOPE lipoplexes was coupled with anticancer effects of near-equal potency.
Although c-myc inhibition with MS09 lipoplexes was more pronounced (3.8-and 2.8-fold differences at the mRNA and protein levels, respectively) in MCF-7 cells than in HT-29 cells, their associated impact on cell migration, growth and apoptosis induction were similar. This could be due to the level of c-myc inhibition required to elicit anticancer activity differs among cell lines, or that more potent oncogene knockdown does not necessarily correspond with enhanced anticancer activity.
Overall, our study has shown that effective c-myc gene silencing was achieved with both MS09 lipoplexes. Gene silencing experiments with both liposomal formulations showed superior gene silencing compared to Lipofectamine™ 3000 in vitro.
It is worth mentioning that preliminary cellular uptake experiments (not reported in this study) under physiological serum concentration (50% v / v ) have highlighted the serum-resistance of the MS09:Chol lipoplexes. Taken together with the results reported, the new formulation is likely to be a good candidate for in vivo evaluation, so as to provide a clearer indication of its clinical applicability.

Conclusion
The findings from this study show the potential of MS09-based siRNA cholesterol-rich lipoplexes for the effective silencing of the c-myc oncogene in the MCF-7 and HT-29 cells. This co-formulation of MS09 and Chol for siRNA delivery and its application in c-myc gene silencing was not previously explored but was shown to be an effective c-myc silencing agent with results comparable to its DOPE-containing counterpart. On a positive note, the fact that both MS09 lipoplexes have performed better in a recalcitrant cell line than the standard transfection reagent, confirms their applicability as oncogene silencing agents in difficult-to-transfect cancer cells, adding credence to their potential as broad-range anti-c-myc agents.
Overall, these novel MS09:Chol liposomes are endowed with physicochemical properties that may render them more suitable for in vivo siRNA delivery than their MS09:DOPE counterparts, and that their anti-c-myc-siRNA lipoplexes should be developed further as a posttranscriptional intervention treatment modality for mono-and polygenic human diseases.

Open Peer Review
prevention, diagnosis, and treatment of cancer, hence anti-c-myc cholesterol can be an example.
Therefore, I want to use this medium to congratulate the authors and suggesting that the authors should carry out this experiment in vivo in future (if possible) so as to re-validate the in vitro results.
Is the work clearly and accurately presented and does it cite the current literature? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly not even come close to approximating in vivo conditions. Moreover, preliminary results obtained with cellular uptake experiments of MS09:Chol lipoplexes in MCF-7 and HT-29 cells in 50 % v/v serum (physiological serum concentration) are encouraging. TEM image of the liposome is not clear and even in a small field of view the heterogeneity is not at all close to the state of the art 1 . They should provide TEM images of the large field, size distribution (with the sampling number), etc.

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The liposome after complexing with siRNA (lipoplex) looks like an aggregate structure rather than a single structure, this must be rectified.

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This has been corrected in the abstract.
In the Abstract author specify the liposome as bilamellar but in the Results section that stated/appears to be unilamellar. Also, the size said in the abstract is less than 200 nm which far away from the result. The author should be very objective in the abstract itself.

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The liposomes were unilamellar. However, the complexes of liposomes and siRNA (lipoplexes) to which reference is made in the abstract were bilamellar The comparison of the liposome is done with Lipofectamine™ 3000, which is not the commercial benchmark for the siRNA transfection. They should provide efficacy compare to the Lipofectamine™ RNAiMAX in all the cases.

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Most of the figure captions are inadequate, they must be more informative.