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Nano-assembly of Pamitoyl-bioconjugated Co-Enzyme-A for Combinatorial Chemo-Biologics in Transcriptional Therapy Santosh K. Misra, Taylor L Kampert, and Dipanjan Pan Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00117 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Nano-Assembly of Pamitoyl-Bioconjugated Co-Enzyme-A for Combinatorial Chemo-Biologics in Transcriptional Therapy Santosh K. Misra, Taylor L. Kampert and Dipanjan Pan* Department of Bioengineering; Beckman Institute of Advanced Science and Technology, Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Illinois, USA; Mills Breast Cancer Institute, Carle Foundation Hospital, 502 N. Busey, Urbana, Illinois, USA, 61801. *Corresponding author: [email protected]

ABSTRACT: Pathogenesis, the biological mechanism that leads to the diseased state, of many cancers is driven by the interruptions to role of Myc oncoprotein, a regulator protein that codes for a transcription factor. One of the most significant biological interruptions to Myc protein is noted as its dimerization with Max protein, another important factor of family of transcription factors. Binding of this heterodimer to E-Boxes, enhancer box as DNA response element found in some eukaryotes that acts as a protein-binding site and has been found to regulate gene expression, are interrupted to regulate cancer pathogenesis. The systemic effectiveness of potent small molecule inhibitors of Myc-Max dimerization has been limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. The potential of gene therapy for targeting Myc can be fully realized by successful synthesis of a smart cargo. We developed a ‘nuclein’ type nanoparticle “siNozyme” (45±5 nm) from nano-assembly of pamitoyl-bioconjugated acetyl co-enzyme-A for stable incorporation of chemotherapeutics and biologics to achieve remarkable growth inhibition of human melanoma. Results indicated that targeting transcriptional gene cMyc with siRNA with co-delivery of a topoisomerase inhibitor, amonafide caused ~90% growth inhibition and 95% protein inhibition.

Introduction A well-documented transcription factor c-Myc gene is involved and overexpressed in a large percentage of human cancer.1 The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation.2 Myc target genes are involved in protein synthesis, cell cycle, survival, cell adhesion and metabolism.3 c-Myc activity is dependent upon heterodimerization with its obligatory counterpart Max to control target gene transcription.1,2 Downregulation of c-Myc has been found to cause arrest of cell growth, accumulate in the G0/G1 phase of the cell cycle, and rapidly undergo apoptosis.47 Transcription factor's relative position downstream as integrators of multiple signaling cascades makes them an attractive therapeutic target.8-11 Approaches for inhibition of Myc function included antisense strategies, RNA interference, and disruption of Myc-Max dimerization using small molecules inhibitors.12-14 As transformation by Myc is dependent upon dimerization with the basic-helix-loop-helix leucine zipper domain (bHLHZIP) protein Max, pathogenesis of cancer can be easily regulated by significant biological interruptions to Myc proteins. Interruption to these heterodimer and further binding to to E-Boxes, enhancer box as DNA response element found in some eukaryotes that acts as a protein-binding site and has been found to regulate gene expression, can play a

role as therapeutic interventions. However, Myc remains a challenging target due to the difficulty of inhibiting protein– protein or protein–DNA interactions15 with small molecules and only recently, several small molecule inhibitors of the Myc-Max interaction were reported.16-21 However, these compounds, although effective in their Myc-Max disruption, shown to undergo rapid metabolism, poor bioavailability, or inability of the drug to reach inhibitory concentrations in tumors.22 So, the pursuit for an effective strategy to target Myc is still ongoing to overcome these hurdles. The systemic utility of potent small molecule inhibitors of Myc-Max dimerization was limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. Interestingly, topoisomerase II inhibition is known to alter DNA conformation most dramatically at and near promoters, at sites likely to experience strong unwinding stresses.23 Therefore, by using topoisomerase inhibitors changes could be induced in transcriptional activities including c-Myc. We hypothesized that a combination of downregulating c-Myc siRNA and a small molecule topoisomerase II inhibitor could be effective strategy for impeding c-Myc-Max dimerization and for inducing cell growth inhibition in melanoma.24-26 Furthermore, we anticipate that our design of a novel nano-assembly from a unique precursor will result in a stable incorporation of chemotherapeutics and biologics to achieve remarkable growth inhibition.

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The unprecedented potential of gene therapy cannot be fully realized until the successful discovery of an efficacious, controllable and safer delivery vector.27-31 The majority of the vectors used thus far, are cationic in nature, which impart toxicity and complement activation issues in vivo, plaguing a meaningful systemic application.32-36 Things get even more complex when two drugs, e.g. chemotherapeutics and biologics are attempted to deliver using a single nano system. One

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Results and Discussion Cargo-based delivery of oligo/polynucleotides49-51 and drugs5254 originated due to numerous physiological barriers55-57 encountered by these agent58-60 which reduces their overall activity in biological systems. Nanocarriers from molecular assemblies are one of the most widely explored categories in gene and drug delivery research but their translation is bottle-

Figure 1. (A) Molecular structures of palmitoyl coenzyme A and amonafide used in preparation of siNozyme-Am nanoparticles and (B, C) mode of their probable interactions with head-group functionalities of palmitoyl coenzyme A. can turn to “Mother Nature” to gain inspiration in developing a ‘nuclein’ type nanoparticle essentially comprised of organic functionalities abundant in the nucleus of a cell, consisting chiefly of proteins, phosphoric acids, and nucleic acids.37-41 Biomimetics have given rise to new technologies inspired by biological solutions at macro and nanoscales. Use of molecules with biological abundance can be very useful for carrying the desired cargo i.e. drug, gene and antisense sequences in intracellular space. Palmitoyl CoA, an important metabolic product of palmitic acid, with required amphiphilic nature and biological presence, could be important molecule to generate efficient carrier system. As already revealed, importance of palmitoyl CoA in biological system comes from metabolic degradation of palmitic acid, which gets modified in the liver to enter hepatic mitochondria in the process of β-oxidation.42,43 Although this molecule has so important niche in biological system and pre-requisites of amphiphilic nature, it has never been utilized as carrier system. With this background, our design utilized a self-assembled nanoparticle (Pcozyme) from amphiphilic palmitoyl-grafted acetyl co enzyme A (PCA). Acetyl-CoA is known for its critical function in cellular metabolism, biochemical reactions and for contributing to the cell’s energy supply.44-48 To this end, we have synthesized and biologically evaluated a ‘nuclein’ type particles self-assembled from palmitoyl-CoA. Through a series of chemical and biological studies we demonstrate that these nanoparticles can be used successfully to co-deliver chemo-biologics (Figure 1).

necked due to side-effects.61-63 Majority of these agents are cationic in nature and consequently becomes immunoresponsive during in vivo application.64,65 An alternative strategy which does not rely on cationic charges for oligo/polynucleotides binding could be able to avoid such side effects. Many a times, therapy using oligo/polynucleotides cease to succeed due to lack of follow-up control on protein/enzyme machinery post gene therapy.66,67 An additional incorporation of drug molecule in nano-assemblies with oligo/polynucleotides can impose improved therapeutic regime on required subject.68-70 Here, we used a nanoscale assembly of pamitoyl-bioconjugated co-enzyme-A for combinatorial chemo-biologics in transcriptional therapy. To improve the cellular acceptability, this two-compartmental agent used CoA while palmitoyl conjugation inculcated amphiphilicity to the agent to facilitate its self-assembly. A co-incorporation synthetic pathway was followed to load siRNA and amonafide (Am) in siNozyme-Am nanoparticles. Methanol solution of palmitoyl co A (1 mg/200 µL of MeOH) was mixed with Am and or siRNA (buffer) and evaporated immediately under reduced vacuum with continuous rotation. Produced membrane was hydrated in phosphate buffer (pH 7.4) for 4h at 4 °C and vortexed mild with intermittent heat thaw cycle of 4-37 °C. A low bath sonication was performed for 30 sec before storing the sample at 4 °C and given mild vortexing before using any experiment. Contrary to commonly pursued approaches, this process uses incorporation of siRNA and a mixture of siRNAamonafide during the preparation of


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siNozyme and siNozyme-Am, respectively. An induced molecular arrangement of palmitoyl CoA head group moieties with intermittent siRNA and amonafide presumably be fortifying the involvement of nitrogen bases of oligonucleotide and pi-sextet of aromatic rings from amonafide during these preparations.

Figure 2. Physico-chemical studies showing (A) change in hydrodynamic diameter of siNozyme and Pcozyme in buffer (pH 7.4) along time of incubation; (B) zeta potential of siNozyme assemblies and their comparison with Pcozyme and siNozyme-Am while siNozyme-I and siNozyme-II are made with 25 and 50 nM of siRNA; (C) representative TEM image showing anhydrous state morphology of siNozyme-Am and (D) UV-Vis absorption patterns of Pcozyme and siNozyme showing no distinguishability in patterns. siRNA loading and release studies revealing (E) gel retardation pattern of siRNA

in siNozyme obtained from Lane 1: siRNA alone (100 ng), Lane 2: siNozyme compassing 100 ng c-myc siRNA in assembly of plamitoyl coA and post-incubated with RNase I (0.1 U), and siNozyme incubated with SDS Lane 3: 0.25 mM, Lane 4: 0.5 mM and Lane 5: 1 mM for 30 min compared to Lane 6: Pcozyme alone. (F) DNA interaction properties of various components of siNozyme-Am nanoparticles showed no degradation or retardation for Lane: plasmid DNA, Lane 2: amonafide, Lane 3: Pcozyme-Am, Lane 4: siNozyme-Am, Lane 5: siNozyme, Lane 6: Pcozyme and Lane 7: plasmid DNA. Small angle x-ray diffraction studies showing (G) single ring 2D pattern and (H) q value graph. This ring pattern shows a typical single shot scattering and grainy structures on the concentric ring. It is characteristic of a well-developed speckle pattern, also indicative of the highly coherent nature of the source. The q value graph correlates d-spacing values of lamellar structures of siNozymes. As synthesized nanoparticles were characterized for hydrodynamic diameter and found to be 45±5 nm for siRNA loaded PCAs (siNozymes), slightly bigger than PCA particles of 30±2 nm (Pcozymes). Stability of these nanoparticles were followed for 72h and found to be satisfactory (Figure 2A). Zeta potential studies performed on various formulations showed negative potential of Pcozymes (-10 mV) changed to more negative potential of -20 and -30 mV in case of siNozyme and siNozyme-Am nanoparticles (Figure 2B). This signifies that the introduction of siRNA and additional amonafide allowed exposure of phospho-di-ester backbone. Anhydrous morphology of siNozyme-Am was found to be ~25 nm spherical nanoparticles (negative staining with uranyl acetate) (Figure 2C). UVVis absorption pattern of Pcozyme and siNozyme did not differ much with common absorption bands at 285 and 410 nm (Figure 2D). Physico-chemical properties of these agents were in accordance with their co-assembly generation patterns. The comparative hydrodynamic diameter of Pcozyme, siNozyme and siNozyme-Am were found to follow a trend (30 ± 2; 45 ± 5 and 50 ± 5, respectively) where incorporation of siRNA in Pcozyme assembly increased the size of the assembly and further addition of amonafide resulted in increased size to some extent (Figure S1A). It is likely that the extended orientation of siRNA increased the size of the assembly and added amonafide sand-witched between layers to increase the dspacing (Figure S1B) as well as the overall size. Similar patterns were observed by the size distribution in anhydrous state but with overall reduction in size of all the assemblies. Preparation of samples for anhydrous state size determination using TEM was done with removal of all the solvent molecules (water) from the assembly which causes the loss of layer of hydration. This loss of layer of hydration leads to reduction of assembly size under TEM investigations (Figure S1C, Figure S1C, D). Introduction of siRNA in Pcozyme did not change the UV absorption pattern due to presence of similar absorbing moieties (nitrogen bases) (Figure S1D) but addition of amonafide appeared as an extra absorptions band around 350 nm (Figure S1B).



Stability and triggered release pattern of siRNA from siNozyme was verified by using gel electrophoresis. Here siRNA retardation studies were relied on disappearance of siRNA band running out of wells rather than band intensity in wells. Similarly, DNA interaction and cleaving studies were relied on expected new DNA bands running out of the wells. It was found that siNozyme could encapsulate all the used siRNA with no free siRNA migrating out of well even after being incubated with RNase I (Lane 2, Figure 2E). An addition of anionic surfactant SDS (Lane 3: 0.25 mM, Lane 4: 0.5 mM and Lane 5: 1 mM) for 30 min showed an increase in released siRNA amount from 25-85%, respectively (Figure 2E). The probability of nanoparticles interacting with genomic

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actions between palmitic chains of two adjacent molecules of palmitoyl CoA possibly co-existing in these assemblies. These indicate the over-powering associative interactions between these components compared to repulsive one and expected to be the reason behind the justification of carrier-cargo chargecharge mitigations and preparation of such assemblies. Success of oligonucleotide delivery using synthetic carriers depends on its interactions with various physiological factors including high serum content in systemic circulation and low pH in tumor micro-environment. Mimicking these conditions and correlating their stabilities can reflect on their potential for successful in vivo use. We performed stability studies of the developed agents, i.e. Pcozyme, siNozyme and siNozyme-Am

Figure 3. Cell internalization study and after effect on C32 melanoma cells. (A)Bright field image of untreated C32 cells and cells under FITC channel incubated with (B) Pcozyme; (C) free FITC-siRNA and (D) FITC-siNozyme. Yellow arrow show cells with high fluorescence due to internalized FITC-siRNA. Scale bar represent a length of 90 µm. Flow analysis on C32 cells (E) untreated or treated with (F) Pcozyme; (G) free FITC-siRNA and (H) FITC-siNozyme. Population more than 102 level of fluorescence represent cell population with internalized FITC-siRNA while yellow population represent cell population with highly enriched FITC-siRNA. DNA could be verified by incubation of various components of siNozyme-Am. It was found that none of the formulations could cleave or retard a model duplex plasmid DNA (Lane 1, 7) after 30 min of incubation at RT including Lane 2: amonafide, Lane 3: Pcozyme-Am, Lane 4: siNozyme-Am, Lane 5: siNozyme, Lane 6: Pcozyme (Figure 2F). Composition of siNozymes with inter-lamellar arrangement of siRNA loaded in PCA assembly could be verified with small angle xray diffraction arrangements. The broadened single ring is a consequence of a weakly correlated lamellar arrangement (Figure 2G) with an inter-layer d-spacing of ~3.14 nm (Figure 2H). Small angle X-ray scattering (SAXS) investigations on siNozyme-Am revealed a higher d spacing of ~5 nm, which matches with hypothesized pattern of Amonafide incorporation in siNozyme assembly, leading to an increased d-spacing (Figure S2). The occurrence of more than one plausible interaction among these participating molecules could be hypothesized. Involvement of the π− π interaction among head group moieties of palmitoyl-CoA with intermittent siRNA and amonafide aromatic rings, repulsive forces between phosphate groups in palmitoyl CoA and siRNA and associative hydrophobic inter-

samples over a period of 168h at 37 °C. Results revealed that all the nanoparticles were significantly stable for 48h in 10% fetal bovine serum concentration, while much prolonged time points indicated destabilizations (Figure S3). Change in their hydrodynamic diameter was noted for all the siNozyme-Am and the controls. siNozyme-Am was found to be more prone to change in their size at low pH, compared to siNozyme and Pcozyme alone even at lower time points. This signifies the likely role of acidic environment in dissociation of these assemblies in presence of siRNA sequences, plausible due to the presence of pH responsive backbones and change in hydration patterns. These characterized NPs were used for antisense-small molecule combinatorial therapy in human melanoma cells C32. Beyond better loading and release of siRNA, a better performance of anti-sense therapy also depends on higher cellular uptake of siRNA loaded nanoparticles. Internalization of siNozymes in cells was quantified using flow assisted cell sorting and followed by fluorescence imaging (Figure 3). Cells (bright field Figure 3A) treated with Pcozyme (Figure 3B) and siNozymes with siRNA tagged with FITC and incubated with cells at a siRNA concentration of 50 nM for 1h before visual-


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izing under fluorescence microscope. A significantly higher population of cells were found enriched with internalized FITC tagged siNozymes (Figure 3D) where free FITC-siRNA treatment did not show any significant cell population with FITC fluorescence (Figure 3C) compared to cells alone in FITC channel. Cells with FITC-siRNA enrichment were found to be at a significantly high level of around 30% (Figure 3H) compared to almost none with free FITC-siRNA treatment (Figure 3G) as very similar to untreated cells (Figure 3E) or with Pcozyme (Figure 3F). At higher magnification for representative siNozyme-Am formulation incubation internalized FITC-siRNA in intracellular space could easily be seen (Figure S4) An improved internalization of siRNA in siNozyme should be able to improve the anti-sense mediated cell growth regression in C32 cells and assistive improvement in growth regression with co-incorporated amonafide. Cell growth regression study was performed using formulations including siRNA alone, Pcozyme, siNozyme, siNozyme-Am, Am alone and siRNA complexed with a commercially available delivery agent as siPlex. Initially growth regression was performed by MTT assay on C32 cells treated with Pcozyme (Figure S5B), free FITC-siRNA (Figure S5C) and FITC-siNozyme (Figure S5D) in an increasing order of produced formazan crystals directly proportional to the mitochondrial respiration/reduction of MTT added to the growing cells and in turn viable cell population. Decreased level of formazan crystals produced in siNozyme treated cell population indicated the high growth regression ability probably via down-regulation of cMyc gene through anti-sense mechanism. After visual inspection of improved siRNA delivery via siNozyme preparation, a model study for anti-sense therapy was performed in 2D culture of melanoma cells. Study was further extended to a combinatorial therapy protocol with siRNA formulation co-encapsulated with amonafide in PCA particles. After being treated with various formulations, MTT assay was performed on cells after 24, 48 or 72h of time point. Nanoparticles were prepared with or without 50 nM of siRNA and/or incorporation of 25 nM of amonafide. Irrespective of the time on treatment, siNozymes were best with ~40, 50 and 60% of cell growth regression in 24, 48 and 72h, respectively compared to ~25, 30 and 40% in case of siPlex treatment using commercial siRNA delivery agent (sc29528; with 25 nM of loaded siRNA (Figure S3)). At same concentration and time point free siRNA was not able to show any effect on cell viability. Scrambled siRNA (ScsiRNA) was used as negative control at same concentration and time period for the treatment with same mw of oligo-nucleotide sequence as c-Myc siRNA with no anti-sense property. At none of the

time points ScsiRNA with or without being loaded to ScsiNozymes could influence the cell viability to any appreciable extent, supporting the role of anti-sense c-Myc sequence present in siRNA parting its role in cell growth regression. The safety profile of Pcozyme at same treatment concentration was found to be considerably safe across all the used time points. Figure 4. Effect of combinatorial therapy on C32 cells using Anti-sense siRNA and drug loading to single delivery vehicle. Cell viability was calculated from MTT assay post treatment for different time points of (A) 24; (B) 48 and (C) 72h. Protein expression analysis on treated C32 cells. (D) Comparative quantification of protein expression respect to background protein amount calculated from (E) western blots from SDS page gel for cMyc protein expression in cell population treated with 1: untreated; 2: +Pcozyme; 3: siNozyme-I (25 nM siRNA); 4: siNozyme-II (50 nM siRNA); 5: siNozyme-Am-I (25 nM siRNA); 6: siNozyme-Am-II (50 nM siRNA) and 7: siPlex (50 nM siRNA). Biostatistical analysis has been performed using ONE WAY ANOVA with Bonferroni post test where ***, represent p value of < 0.001 and ns as non-significant.

Optimized siRNA and amonafide loaded formulations were used to evaluate maximum cell growth regression which can be achieved with change in amount of siRNA loaded and codelivery of amonafide. MTT experiments were performed on C32 cells after 24, 48 and 72h of incubations with various formulations. It was found that siNozyme-Am was best formulation for C32 growth regression with decrease in viable cell population of around 85-90% across all the used time lines of treatment (Figure 4), while amonafide alone at same concentration of treatment could only allow 75% of the cells with no specific effect from increasing time of incubation. Studies with siNozyme carrying 50 nM of siRNA could reduce the growing cell population to ~20% at 24h time point but with increasing time of incubation a slight increase in cell growth was reported as ~25 and 30% at 48 (Figure 4B) and 72h (Figure 4C). Presumably cell division after 24h time point allows more sense mRNA sequences to transcript in cells without being blocked by anti-sense siRNA sequence, which in turn slowly nullified the downregulation effect on c-Myc. Results were compared with positive control of siPlex, composed of commercial siRNA carrier with c-Myc siRNA (50 nM) and found to be at least three-fold less effective compared to siNozyme-Am in cell growth regression (Table S2). As expected Pcozyme did not show any significant change in cell viability across any of the used incubation times (Figure S6). Biostatical analysis represents the cell growth inhibition efficiency of siNozyme, Amonafide and siNozyme-Am as highly significant with p value of < 0.001 for siNozyme and siNozyme-Am samples compared to siPlex efficiency presented as ‘***’ while non-significant for amonafide treatment represented as ‘ns’. A reduced effect was seen for a lower c-Myc siRNA (25 nM) loading (Figure S7).



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or both during cell treatment process, Annexin-v staining assay was performed on C32 cells treated with siPlex, amonafide, siNozyme and siNozyme+amonafide. It was found that maximum cell population (~22%) underwent apoptosis compared to cells treated with siPlex (~12%), amonafide (~10%) or siNozyme (~10%), alone (Figure 5). It was also noted that co-assembly of siNozyme+Amonafide could undergo apoptosis at least two-fold more cell population compared to siNozyme and amonafide alone. This further supports the combinatorial effect of these individual treatments.

Conclusions

Figure 5. Apoptosis assay with AnnexinV-FITC positive cells. Representative dot plot of (A) untreated cells and (B) cells treated with siNozyme+amonafide. Quadrants represent LL = Live cells; LR = early apoptotic cells; UL = necrotic cells and UR = late apoptotic cells. (C) Representative histogram showing higher green fluorescence from siNozyme+amonafide treated early apoptosis positive cells. (D) Comparative apoptotic cell population from treatments of siPlex, amonafide, siNozyme and siNozyme+amonafide. Biostatistical analysis has been performed using ONE WAY ANOVA with Bonferroni post-test where * represents p value of < 0.05 for changes compared to cells alone.

Protein expression analysis on treated C32 cells was performed by western blotting (Figure 4D, E) to reveal increasingly high c-Myc protein inhibition for treatments of siNozyme-I (25 nM siRNA), siNozyme-II (50 nM siRNA), siNozyme-Am-I (25 nM siRNA), siNozyme-Am-II (50 nM siRNA) (Figure 4D) as calculated from protein bands of cMyc compared to β-actin (Figure 4E). c-Myc protein in cells treated with Pcozyme were unaffected and siPlex prepared from commercial siRNA carrier agent were considerably significant in c-Myc protein inhibition to a level of ~70% while siNozyme-Am-II (50 nM siRNA) could inhibit it to a level of ~95%. It also revealed that combinatorial use of c-Myc siRNA and amonafide in form of siNozyme-Am-II (50 nM siRNA) with ~95% protein inhibition was significantly better than delivery of c-Myc siRNA alone in form of siNozyme-II (50 nM siRNA) with only 60% protein inhibition. siNozymes worked synergistically with amonafide for growth regression and was found to give CI (50), CI (75), CI (90) and CI (97) as 0.4, 0.47, 0.55 and 0.66, respectively (Table S2). A 75% growth inhibition even at smaller time point of ~24h post transfection and found to be significantly better than one of the commercial siRNA delivery agent used here. The co-loaded siNozyme particles with amonafide and siRNA were able to reduce the cell growth by ~90% even at 24h time point with non-significant improvement in cell growth even at higher time points of 48 and 72h. The fundamental achievement of this platform was to avoid coulombic interactions between siRNA and carrier assembly, use of a biomolecule Co-A derivative for making the nanoassembly and co-incorporation of small molecule drug of inducing a combined effect. Because of its size, siNozyme-Am assembly is expected to follow enhanced permeability and retention (EPR) path to enrich tumour in vivo. This unique combinatorial strategy through a cellular-friendly ‘nuclein’ type nano-assembly may provide a way ahead to the anti-sense therapeutics and solve major issues preventing combinatorial delivery of chemotherapeutics and biologics.

Experimental section Palmitoyl coenzyme A (870716P) was purchased from Avanti Lipids. siRNA (sc-29226), FITC-siRNA, scrambled siRNA (ScsiRNA) and siRNA delivery agent (sc-29528) were purchased from Santa Cruz Biotechnology. Sodium Dodecyl Sulfate and Methanol was purchased from Fisher Scientific (PA, USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide) and dimethyl sulfoxide (DMSO) was purchased from Sigma Aldrich. Amonafide (ab142983) was purchased from abcam. Preparation of Pcozyme, siNozyme and siNozyme-Am nanoparticles Palmitoyl coenzyme A (1 mg/200 µL of MeOH) was mixed with c-myc siRNA (25 µL of 10 µM stock) with or without amonafide (50 µM) resulting in 0.2 and 0.3 wt% of c-myc siRNA and Amonafide, respectively. Mixture was evaporated immediately under reduced vacuum with continuous rotation. Produced membrane was hydrated in phosphate buffer (pH


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7.4) for 4h at 4 °C and vortexed mild with intermittent heat thaw cycle of 4-37 °C. A low bath sonication was performed for 30 sec before storing the sample at 4 °C and given mild vortexing before using any experiment. Dynamic Light Scattering The hydrodynamic size distribution of the nanoparticles was determined through dynamic light scattering measurements on Malvern Zetasizer ZS90 instrument (Malvern Instruments Ltd, United Kingdom) at fixed angle of 90o. A 10 µL of particle suspension was mixed with 990 µL of nanopure water to run the samples in DLS machine. A photomultiplier aperture of 400nm was used and the incident laser power was so adjusted to obtain a photon counting rate between 200 and 300 kcps. Measurements for which the measured and calculated baselines of the intensity autocorrelation function were within 0.1% range were used for diameter values. All measurements were carried out in triplet of thirteen consecutive measurements. Stability of siNozyme Samples Stability of different samples in probable physiological condition of low pH of 4.5 and high serum concentration represented as 10% FBS solution were performed to show their plausible fate in vivo. Samples (Sample 1: Pcozyme; Sample 2: siNozyme and Sample 3: siNozyme-Am) were incubated at 37 C for 168h. Experiments were performed in triplicate and % changes in hydrodynamic diameter of different samples were compared at different time points. The statistical analysis was performed using TWO Way ANOVA where *, **, **** represent p values of

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