Efficient Dual siRNA and Drug Delivery Using Engineered

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Efficient Dual siRNA and Drug Delivery Using Engineered Lipoproteoplexes Che Fu Liu,†,◆ Raymond Chen,†,◆ Joseph A. Frezzo,† Priya Katyal,† Lindsay K. Hill,†,‡,§ Liming Yin,† Nikita Srivastava,† Haresh T. More,† P. Douglas Renfrew,○ Richard Bonneau,#,∇,○ and Jin Kim Montclare*,†,∥,⊥ †

Department of Chemical and Biomolecular Engineering, NYU Tandon School of Engineering, Brooklyn, New York 11201, United States ‡ Department of Biomedical Engineering, State University of New York Downstate Medical Center, Brooklyn, New York 11203, United States § Department of Radiology, New York University School of Medicine, New York, New York 10016, United States ∥ Department of Chemistry, New York University, New York, New York 10003, United States ⊥ Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, New York 11203, United States # Center for Genomics and Systems Biology, New York University, New York, New York 10003, United States ∇ Courant Institute of Mathematical Sciences, Computer Science Department, New York University, New York, New York 10009, United States ○ Center for Computational Biology, Flatiron Institute, Simons Foundation, 162 Fifth Avenue, New York, New York 10010, United States S Supporting Information *

ABSTRACT: An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000. When the lipoproteoplex is loaded with doxorubicin, the improved doxorubicin loading (∼40 μg Dox/mg CSP) results in a substantial decrease in MCF-7 cell viability.

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vehicles.8 Adding further complexity, some cancers develop resistance at the genetic level to even the most effective smallmolecule drugs by inhibiting small-molecule drug retention in the cell.9,10 The synergistic effect of siRNA targeting those genes that promote drug resistance combined with small-molecule drug delivery has been an area of expanding research interest. Several examples of synthetically derived nanoparticles developed as vehicles for combined siRNA and drug delivery have been reported.11−13 In one such example, a polyethylenimine-coated graphene oxide (PEI-GO) vehicle has been employed to adsorb both doxorubicin and Bcl-2-targeted siRNA onto the GO moiety through noncovalent adherence via π−π stacking, while the PEI acts to expedite cell membrane penetration.11 This PEI-GO

INTRODUCTION Both nucleic acids and small-molecule drugs possess critical instructions for controlling biological processes with tangible implications in medicine.1,2 The suppression of active mRNA transcripts that invoke and propagate disease states, such as malignant cancers, can be effectively diminished through RNA interference technologies such as small interfering RNA (siRNA).3,4 Phenotype modification by siRNA can be extremely effective; however, there are limitations to siRNA that have hampered clinical adoption.4−6 Issues including cell membrane penetration, lysosomal entrapment, susceptibility to nucleases and serum instability have necessitated the use of protective vectors or chemical modification of siRNA for safe, potent delivery.3,4 Similarly, small-molecule drugs such as anthracyclines (e.g., doxorubicin, epirubicin) and taxanes (e.g., paclitaxel, docetaxel) are effective suppressants of malignant neoplasms.7,8 However, there are still off-target effects, some of which are fatal, that therefore require the use of small-molecule delivery © 2017 American Chemical Society

Received: February 9, 2017 Revised: July 4, 2017 Published: July 7, 2017 2688

DOI: 10.1021/acs.biomac.7b00203 Biomacromolecules 2017, 18, 2688−2698

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transfection ability (60% knockdown), enhanced doxorubicin loading (∼40 μg Dox/mg CSP), and potent delivery to MCF-7 cells.

vehicle demonstrates negligible cytotoxicity as an empty vector and outperforms PEI 25K in siRNA transfection.11 In another example, a triblock polymer, formed via the reversible addition− fragmentation chain transfer (RAFT) agent 4-cyanopentanoic acid dithionapthalenoate (CPDPA) with poly(ε-caprolactone) flanked on each side by a polymerized dimethylaminoethyl methacrylate (PDMAEMA), shows promise as a vehicle for VEGF siRNA and paclitaxel delivery.12 This triblock demonstrates effective cell penetration, VEGF knockdown, and greater drug efficacy than the drug alone. Another triblock copolymer composed of N-succinyl chitosan-poly-L-lysine-palmitic acid (NSCPLL-PA) has been designed to safely harbor P-glycoproteintargeted siRNA and doxorubicin at pH 7.4; however, upon a decrease in the lysosomal pH of 5.3, the micelle destabilizes and leads to rapid release of the siRNA and drug cargo.13 While there are numerous examples of synthetic dual siRNA and drug-delivery vehicles, protein- and peptide-based vehicles show promise due to safe biodegradation, intricate 3D chemical moieties and structures that are difficult to replicate on the synthetic scale, as well as ease of specific residue manipulation and modification via protein engineering techniques.14−27 Furthermore, proteins and peptides demonstrate compatibility in the construction of multifunctional hybrid materials.18,23,24,28−32 Our recent work attests to these attributes of proteinbased hybrid materials as we combined an engineered supercharged coiled-coil protein (CSP), imparted with extensive positive charges, and the commercially available transfection reagent FUGENE.18 This engineered protein−lipid hybrid, or lipoproteoplex, exhibited an 8-fold improvement in transfection efficiency compared with FUGENE alone, while demonstrating negligible cytotoxicity.18 Herein, we present a dual-delivery lipoproteoplex system composed of the supercharged protein CSP and Lipofectamine 2000 (L2000) for cellular delivery of siRNA targeted to GAPDH mRNA transcripts and the small-molecule chemotherapeutic doxorubicin (Dox) (Figure 1). The CSP protein, derived from the homopentameric coiled-coil domain of Cartilage Oligomeric Matrix Protein (COMPcc),33 bears arginine residues at eight solvent exposed sites for nucleic acid condensation and, when assembled as a pentamer, possesses a net positive charge of +53 mV.18,32 We hypothesize that because COMPcc self-assembles into a homopentamer bearing a hydrophobic pore, the supercharged CSP would also maintain the pore for encapsulation of a small-molecule drug (Figure S1a). Our studies indicate that the pore of the COMPcc and CSP proteins can indeed bind the chemotherapeutic, Dox (Figure 1, Figure S1), which is prone to efflux from drug-resistant cancer cell lines.33,34 When the doxorubicin-loaded CSP is combined with GAPDH siRNA and the transfection reagent L2000, there is retention of overall

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MATERIALS AND METHODS

General. Tryptic soy agar, tryptone, yeast extract, isopropyl β-D-1thiogalactopyranoside (IPTG), Tris-base, urea, 0.2 μm syringe filters, and Sephadex G-25 medium resin were obtained from VWR. Sodium chloride (NaCl), Tris-HCl, imidazole, HisPur Ni-NTA agarose, dimethyl sulfoxide (DMSO), 3.5 kDa MWCO SnakeSkin dialysis tubing, Coomassie Brilliant Blue R-250 dye, ethidium bromide, and Pierce Bicinchoninic Acid Assay Kit (BCA) were purchased from Thermo-Fisher Scientific. Antibiotics (ampicillin, chloramphenicol), recombinant human insulin, and agarose were obtained from SigmaAldrich. Precision Plus Protein Unstained Standard and 30% acrylamide/bis solution, 37.5:1 were obtained from Bio-Rad. Doxorubicin (Dox, free base form) was obtained from Medkoo Bioscience (Chapel Hill, NC). Gibco alpha minimal essential medium (α-MEM), minimal reduced serum medium (Opti-MEM), Dulbecco’s modified Eagle’s medium (DMEM), Gibco Trypsin, Gibco fetal bovine serum (FBS), penicillin (5000 U/mL), streptomycin (5000 U/mL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA, cyanine 5 (cy5)-conjugated siRNA, SYBR Green RT-PCR master mix, 18S primers and GAPDH primers, Vybrant 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) cell proliferation assay kit, and Lipofectamine 2000 (L2000) were obtained from Life Technologies. CCK-8 cell proliferation assay kit was obtained from Dojindo Molecular Technologies. MCF-7 (ATCC HTB2) cells and Eagle’s minimum essential medium (EMEM) were obtained from ATCC. The highcapacity cDNA reverse transcription kit was obtained from Applied Biosystems and the RNeasy Mini Kit and RNase-free DNase Set were obtained from Qiagen. The following oligonucleotide primers were obtained from Integrated DNA Technologies for quantitative real-time PCR: for GAPDH, forward 5′-TCACCACCATGGAGAAGGC-3′ and reverse 5′-GCTAAGCAGTTGGTGGTGCA-3′; for 18S, forward 5′-CGGCTACCAGATCCAAGGAA-3′ and reverse 5′-GCTGGAATTACCGCGGCTG-3′. Folded capillary zeta cell cuvettes and disposable polystyrene low-volume cuvettes were obtained from Malvern Instruments. Protein Expression and Purification. CSP/PQE30 and COMPcc/PQE30 vectors were transformed into AF-IQ E. coli17,33 and XL-1 blue E. coli cells, respectively. The transformed cells were grown for 12−16 h at 37 °C on tryptic soy agar plates containing ampicillin (200 μg/mL) and chloramphenicol (35 μg/mL) for CSP and ampicillin (200 μg/mL) for COMPcc. Starter cultures were inoculated with a single colony in a culture tube containing 4 mL of lysogeny broth (LB) containing ampicillin (200 μg/mL) and chloramphenicol (35 μg/mL) for CSP and 4 mL of LB containing ampicillin (200 μg/mL) for COMPcc. The cultures were incubated overnight at 37 °C, 350 rpm. The starter culture was used to inoculate 800 mL LB media with respective antibiotics for large-scale expression. The flasks were incubated for 6 h at 37 °C and 250 rpm. After obtaining optical density (OD600) of ∼0.8 to 1.0, the culture was induced with 200 μg/mL

Figure 1. Illustration of supercharged protein design based on coiled-coil and cationic lipids resulting in lipoproteoplexes for gene (siRNA), drug (doxorubicin), or co-delivery. The doxorubicin bound in the CSP represents the lowest energy conformation from docking studies. 2689

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Figure 2. siRNA binding to (a) CSP and (b) COMPcc with increasing wt/wt ratio evaluated through electrophoretic mobility shift assay as visualized on 2% agarose gels. In both gels: L, 100 base-pair ladder; 1, siRNA alone. (a) CSP binding to siRNA at wt/wt ratio: 2, 0.5:1; 3, 1:1; 4, 1.5:1; 5, 2:1; 6, 2.5:1; 7, 3:1; 8, 3.5:1; 9, 4:1. (b) COMPcc binding to siRNA at wt/wt ratio 2, 2:1, 3, 5:1; 4, 8:1; 5, 10:1; 6, 20:1; 7, 35:1. IPTG for protein expression and incubated for another 3 h under the same conditions. Cells were harvested by centrifugation at 5000 rpm and 4 °C for 15 min. Cell pellets were stored at −80 °C until purification. Expression of protein was confirmed by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Figure S2). The cell pellet was thawed and resuspended in 40 mL (for a 400 mL pellet) of lysis buffer (50 mM Tris-HCl, pH 8.0, 6 M Urea, 20 mM imidazole, and 0.5 M NaCl). The cells were then lysed by sonication using the Q500 ultrasonic probe sonicator (QSonica) with 5 s on and 5 s off pulse sequence for a total of 90 s at 40% amplitude/power. This was repeated twice to obtain nearly complete lysis. The insoluble cell debris was separated by centrifugation at 13 500 rpm for 90 min at 4 °C. The supernatant was then incubated with 2.5 mL of lysis buffer-equilibrated HisPur Ni-NTA agarose beads (Thermo Fisher Scientific, Somerset, NJ) for 2 h at 4 °C. The beads were then washed with 40 mL of wash buffer (50 mM Tris-HCl pH 8.0, 20 mM imidazole, 0.5 M NaCl), followed by an increasing imidazole concentration gradient for elution of pure protein. Pure CSP protein was obtained in fractions eluted by buffer containing 500 mM and 1 M imidazole (Figure S3), and pure COMPcc was obtained in fractions eluted by buffer containing 200 mM imidazole. The proteins were dialyzed against 15 L of 50 mM Tris-HCl buffer pH 8.0 with 0.5 M NaCl to remove urea and imidazole. Electrophoretic Mobility Shift Assay. Binding of siRNA was evaluated through electrophoretic mobility shift assay (EMSA) carried out at increasing wt/wt ratios of protein to siRNA. For CSP, 100 ng of siRNA was mixed with increasing wt/wt ratios of protein from 0.5:1 to 4:1 CSP·siRNA (Figure 2). Similarly, 100 ng of siRNA was mixed with increasing COMPcc wt/wt ratios from 2:1 to 35:1 COMPcc·siRNA. The mixtures were incubated for 30 min at room temperature. The samples were subjected to electrophoresis on 2% agarose gels. The agarose gels were stained with ethidium bromide and imaged under UV-light exposure (ImageQuant 350, GE Healthcare, USA). Small-Molecule Binding Studies. Binding of Dox with CSP was achieved by passive loading into the pore of the protein. In brief, 50 μM of CSP was mixed with 100 μM of Dox (0.4% DMSO v/v) in 50 mM Tris-HCl, 0.5 M NaCl pH 8.0 buffer at a total volume of 500 μL and incubated for 2 h at room temperature. After incubation, free Dox was separated by size exclusion chromatography using Sephadex G-25 Medium resin (GE Healthcare, USA). Ten fractions of 0.5 mL were collected and the Dox content of each fraction was subsequently measured spectrophotometrically at an absorbance of 485 nm.35 Fractions containing Dox were further assessed for protein concentration using a Pierce BCA assay kit (Thermo Fisher Scientific). Dox concentrations coeluted with CSP were calculated using respective standards curves. The doxorubicin encapsulation efficiency (%) was calculated as (amount of doxorubicin entrapped/amount of doxorubicin loaded) × 100.35 An alternative dialysis method of Dox binding was also conducted with the added hindrance of siRNA binding to investigate potential small-molecule displacement by the siRNA. Formulations of CSP·Dox and L2000·Dox were incubated for 2 h at room temperature, followed by the addition of siRNA with incubation for 30 min at room temperature. Excess unbound Dox was removed by room-temperature dialysis using 3.5 kDa MWCO SnakeSkin dialysis tubing (Thermo-Fisher Scientific)

in a 200:1 v/v 50 mM Tris-HCl, 0.5 M NaCl pH 8.0 buffer solution. The CSP, Dox, L2000, and siRNA concentrations mirrored those used in the transfection and confocal microscopy experiments. At the conclusion of dialysis, the Dox content within the dialysis tubing was measured at an absorbance of 485 nm and the concentration was determined using a standard curve. The results represent an average of two independent trials. Computational Modeling. To determine a potential binding interaction between the CSP helical bundle and Dox, we generated an ensemble of ligand conformations and carried out small-molecule docking simulations (Figure S1). Initial coordinates for Dox were obtained from PubChem (CID number 31703). Dox conformers were generated using the BCL::CONFORMERGENERATOR.36 A smallmolecule rotamer library compiled from structures in the Cambridge Structural Database was used in place of the default library based on structures in the Protein Databank. Dox was docked in to CSP using the Rosetta Macromolecular Modeling Suite37 and recent improvements to the ROSETTALIGAND docking protocol38 that incorporate simultaneous rotation and translation of the molecule in the first stage of the protocol and have been shown to have improved performance on nonglobular ligands like Dox. Ten initial starting conformations were created manually by placing the Dox molecule into 10 positions in the central channel of the CSP helical bundle. 1000 trajectories were run for each of the starting conformations. Backbone and side-chain dihedral angles of residue positions within 6 Å of the ligand could move during the simulations. Models were ranked based on the total energy of the conformation. Circular Dichroism Spectroscopy. To study the secondary structure of CSP and the effect of siRNA upon CSP conformation, wavelength scans were performed on a Jasco circular dichroism (CD) spectropolarimeter (J-815) over a range of 200−250 nm at 1 nm intervals and 20 nm/min using a 1 mm Quartz cuvette (Hellma Analytics). CD experiments were conducted at room temperature and replicated three times to confirm results. Concentrations of 10 μM CSP were employed in the presence and absence of siRNA, respectively. For complexation studies, mixtures of a static concentration of protein and decreasing wt/wt ratios of siRNA were incubated for 30 min at room temperature prior to wavelength scans. After subtracting the baseline of buffer alone for each wavelength scan, the obtained ellipticity (Θ) was converted to mean residue ellipticity (MRE) using the standard equation: ΘMRE = Θ/(10cpl), where c is the molar concentration of the protein, p is the path length of the measuring cuvette in centimeters, and l is the number of amino acids per protein.33 The CSP·siRNA results reflect the average of two independent trials. Dynamic Light Scattering and Zeta Potential Studies. The zeta potential (ZP) and particle size of samples were measured on a Zetasizer Nano ZS90 (Malvern Instruments) with a laser source of 630 nm. The instrument calculated the ZP by measuring the electrophoretic mobility and fitting the acquired data into a Smoluchowski equation.39 Sample preparation was accomplished by mixing 10 μM (∼130 ng/μL) solution of nonsense siRNA with CSP at 2:1 and 8:1 CSP·siRNA wt/wt ratio, respectively. In addition, the 8:1 ratio was selected to bind to the Doxbound CSP. After a 30 min incubation at room temperature, L2000 was added to yield a L2000·CSP·siRNA mixture at ratios of 5:2:1 and 5:8:1 2690

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Figure 3. Impact of (a) CSP and (b) COMPcc secondary structure upon siRNA binding as measured by CD spectroscopy using fixed concentrations of protein and decreasing concentrations of siRNA. (a) CSP alone (solid line), CSP·siRNA 2:1 (circle), CSP·siRNA 4:1 (diamond), and CSP·siRNA 8:1 (triangle). (b) COMPcc alone (solid line), COMPcc·siRNA 2:1 (circle), COMPcc·siRNA 4:1 (diamond), and COMPcc·siRNA 8:1 (triangle). as well as 5:8:3:1 for the L2000·CSP·Dox·siRNA complex. The mixture was incubated for an additional 15 min at room temperature, followed by size and ZP measurements. A 5 μL sample was aliquoted to observe morphology of the lipoproteoplexes via transmission electron microscopy (TEM). All samples were prepared in ultrapure water. Three trials were performed on the same sample, and each trial consisted of 20 runs. Transmission Electron Microscopy. Approximately, 5 μL samples (aliquoted above) were deposited on Formvar/carbon-coated 400 mesh copper grids for 1 min at room temperature. Excess liquid was removed with filter papers. Subsequently, the grids were rinsed with 5 μL of dH2O twice and 5 μL of 1% w/v uranyl acetate. The samples were then stained with 5 μL of 1% w/v uranyl acetate for 1 min at room temperature and blotted with filter paper. Images were obtained using CM12 transmission electron microscope (Phillips). Transfection Studies. Transfection studies were performed on human breast cancer MCF-7 cells. Transfection of GAPDH siRNA or nonsense siRNA was performed by seeding 40 000 cells/well in a 24-well cell culture plate in DMEM media with 10% FBS and 1% penicillin−streptamycin (pen−strep) antibiotic, incubated for 24 h at 37 °C, 5% (v/v) CO2. Cells were then washed with PBS and replenished with fresh culture medium sans supplements. Lipoproteoplexes were prepared at different wt/wt ratios of L2000·CSP·siRNA. A positive control of L2000 and siRNA (5:1 wt/wt ratio) was prepared according to the manufacturer’s protocol. The lipoproteoplexes were added to appropriate wells in triplicates. The cells were then incubated for 6 h at 37 °C at 5% (v/v) CO2. The media containing treatments was removed and the cells were replenished with fresh media, 10% FBS and 1% pen-strep. They were allowed to grow for another 42−48 h at 37 °C at

5% (v/v) CO2 and harvested for mRNA extraction. The results reflect the average of three independent trials and error bars represent the mean standard deviation. mRNA Extraction, cDNA Synthesis, and Quantitative RealTime PCR. The cells were harvested and mRNA was extracted by following the manufacturer’s protocol (using the Qiagen RNeasy Mini Kit). In addition, DNase treatment was conducted during the mRNA extraction according to the manufacturer’s protocol (using the Qiagen RNase-free DNase Set). The concentration and purity of extracted mRNA was determined by Nanodrop (Thermo Scientific). After determining the concentrations, 300−500 ng of mRNA, depending on the available concentration and purity, was subjected to immediate cDNA conversion using a high-capacity reverse transcription kit (Applied Biosystems) in a thermocycler (BioRad MyCycler Personal Thermo Cycler). The cDNA was stored at −20 °C prior to quantitative real-time PCR (qPCR). GAPDH mRNA expression was quantified via qPCR using the SYBR Green master mix (Life Technologies) with standard qPCR protocol provided by the manufacturer. Primers used were specific to GAPDH to investigate changes in GAPDH expression due to siRNA transfection and to the 18S housekeeping gene for normalization and calculation of relative GAPDH mRNA expression using the double delta Ct (ΔΔCt) method.40 Confocal Microscopy. Cells were prepared by seeding 80 000 cells per well (40 000 cells/mL) on glass coverslips in a six-well cell culture plate in DMEM media supplemented with 10% FBS and 1% pen-strep antibiotic, incubated for 24 h at 37 °C, 5% (v/v) CO2. Lipoproteoplexes were prepared at six different wt/wt ratios of L2000 in Opti-MEM media. Cells were washed with PBS, and the media was substituted with 2691

DOI: 10.1021/acs.biomac.7b00203 Biomacromolecules 2017, 18, 2688−2698

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Biomacromolecules Table 1. Summary of Zeta Potential and Size Measurements sample L2000 CSP CSP·siRNA (2:1) CSP·siRNA (8:1) L2000·siRNA (5:1) L2000·CSP·siRNA (5:2:1) L2000·CSP·siRNA (5:8:1) L2000·CSP·Dox·siRNA (5:8:3:1)

Table 2. Summary of Doxorubicin Loading

zeta potential (mV)

size (nm)

sample

encapsulation efficiency %

weighted loading (μg/mg vehicle)

+82.33 ± 1.40 +6.35 ± 0.53 −0.60 ± 0.61 +15.17 ± 1.11 +68.00 ± 2.82 +48.50 ± 3.90 +21.73 ± 1.46 +16.80 ± 0.32

40.38 ± 0.58 13.56 ± 0.53 3546.67 ± 431.41 115.27 ± 1.75 42.40 ± 0.98 425.07 ± 76.35 85.72 ± 1.52 205.30 ± 5.06

CSP L2000

39.4 ± 4.0 35.0 ± 5.6

39.5 ± 4.0 21.1 ± 6.7

was evaluated by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Cell Counting Kit-8 (CCK-8) assays. For MTT assay, 10 μL of the 5 mg/mL stock MTT in PBS was added to each well and incubated at 37 °C, 5% (v/v) CO2 for 4 h. After incubation all but 25 μL of the medium was removed from each well and 50 μL of dimethyl sulfoxide was added. The cells were incubated at 37 °C, 5% (v/v) CO2 for 10 min. The absorbance of the solution was observed using a microplate reader Synergy at 540 nm, and the cell viability was calculated as a percent of cellular activity of the cells without treatment. For CCK-8 assay, 10 μL of the stock CCK-8 was added to each well and incubated at 37 °C, 5% (v/v) CO2 for 4 h. After incubation, the absorbance of the plate was observed directly using a microplate reader Synergy at 450 nm, and the cell viability was calculated as a percent of cellular activity of the cells without treatment. Data represent the average of four replicates per sample per trial, and error bars represent the mean standard deviation.

Opti-MEM. The lipoproteoplexes were added to the appropriate wells and the cells were incubated for 4 h at 37 °C, 5% (v/v) CO2 in darkness. The cells were washed with PBS after 4 h of incubation, and the coverslips were placed on slides for viewing. The samples were viewed on an inverted confocal microscope Zeiss Axiovert 200 using a HAL100 camera, with 63× oil immersion lens, Exfo X-Cite 120 fluorescent lamp, PerkinElmer Ultraview ERS 3E lens controller, and Melles Griot Krypton/Argon Ion Laser with laser lines at 488, 568, and 647 nm, and images were captured using the PerkinElmer Ultraview ERS system.41,42 The cy5-labeled GAPDH siRNA was excited using the 647 nm laser line and viewed under the emission exclusion setting in the red/far red wavelength at 670 nm. The Dox was excited using the 488 nm laser line and viewed under the emission exclusion setting in the yellow wavelength at 560−590 nm. Flow Cytometry. Cells were plated onto six-well plates at a density of 1 × 106 cells per well in DMEM media supplemented with 10% FBS and 1% pen-strep antibiotic and incubated for 24 h at 37 °C, 5% (v/v) CO2. The cells were treated with different formulations having Dox at a final concentration of 6 μg/mL and siRNA at a final concentration of 5 μg/mL for 3 h at 37 °C, 5% (v/v) CO2 in darkness. Following incubation, the media was removed and cells were washed with PBS, trypsinized, and transferred to 2 mL tubes and centrifuged. The cell pellets were washed with PBS and resuspended in 0.5 mL of PBS. Flow cytometry was performed using BD FACS Aria (Becton Dickinson, USA), and a total of 10 000 events were analyzed for uptake studies with an excitation wavelength of 488 and 633 nm (with an Allophycocyanin (APC) filter). Cell Viability Studies. MCF-7 cells were seeded in a 96-well plate at a cell density of 5 × 103 cells/well in MEM supplemented with 10% FBS, 1% pen-strep, and 0.01 mg/mL recombinant human insulin and incubated at 37 °C, 5% (v/v) CO2 overnight. The cells were rinsed with PBS and the media was replaced. The cells were treated with the complexes in quadruplicates for 48 h and incubated at 37 °C with 5% CO2. After treatment, the cells were rinsed gently with PBS once and replaced with 100 μL of fresh medium. The cytotoxicity of the complexes

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RESULTS AND DISCUSSION siRNA Complexation and Impact on Protein Secondary Structure. The intention of this work is to develop a protein− lipid hybrid material, or lipoproteoplex, that can bind and deliver both siRNA and the small molecule, doxorubicin. The protein component of this system is a mutated coiled-coil protein, CSP, shown to effectively bind and condense siRNA at 1:1 wt/wt ratios consistent with previous evidence of supercharged proteins complexing with either plasmid or siRNA.18,43 Binding and condensation of siRNA to the solvent-exposed surfaces of proteins was evaluated using an electrophoretic mobility shift assay. In these assays, a static concentration of siRNA is exposed to increasing concentrations of protein, CSP, endowed with extensive positive charges on the surface of the self-assembled pentamer. CSP demonstrates the ability to partially hinder the mobility of siRNA through an agarose gel with full immobilization of siRNA occurring at a 2:1 CSP·siRNA wt/wt ratio (Figure 2a). Conversely, COMPcc fails to immobilize siRNA even when subjected to substantially higher wt/wt ratios (Figure 2b). The effect of siRNA binding on the alpha helical structure of COMPcc and CSP proteins was measured by circular dichroism

Figure 4. TEM images of (a) L2000, (b) lipoplex L2000·siRNA (5:1), (c) CSP, proteoplex, (d) CSP·siRNA (2:1), and (e) CSP·siRNA (8:1) and lipoproteoplex (f) L2000·CSP·siRNA (5:2:1) and (g) L2000·CSP·siRNA (5:8:1). 2692

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(CSP·siRNA), lipoplex (L2000·siRNA), and lipoproteoplex (L2000·CSP·siRNA and L2000·CSP·Dox·siRNA) (Table 1). CSP, alone, exhibited the formation of small particles (13.56 ± 0.53 nm) carrying positive charge (6.35 ± 0.89 mV) (Figure 4c, Table 1), but when combined with siRNA at a lower content, large aggregates were formed (3546.67 ± 431.41 nm) with a nearly neutral charge (−0.60 ± 0.61 mV) and low colloidal stability (Figures 4d, Table 1). With increased protein content, however, CSP·siRNA developed particles with diameter of 115.27 ± 1.75 nm and an overall positive charge (+15.17 ± 1.11 mV) (Figure 4e, Table 1), suggesting the role of CSP in condensing the siRNA and adjusting the size of the proteoplexes. When L2000 was introduced, the lipoproteoplexes containing both low (2:1) and high CSP (8:1) content (Figure 4f,g) exhibited similar morphology as L2000 and lipoplex (Figure 4a,b) with increased sizes of 425.07 ± 76.35 and 85.72 ± 1.52 nm, respectively (Table 1); this indicated that the proteoplexes were condensed and encapsulated in L2000. Although the lipoproteoplex, L2000·CSP·siRNA (5:2:1), measured on dynamic light scattering (DLS) was >400 nm (Table 1), it exhibited similar morphology and size (400 nm observed by DLS could be attributed to the aggregation of CSP·siRNA (2:1) (Figure 4d). Given the overall increase in positive charge, we expected the surface charge of the proteoplexes to increase with CSP content. Interestingly, when condensed in L2000, the L2000·CSP·siRNA (5:2:1) lipoproteoplex possessed a zeta potential value of +48.50 ± 3.90 mV, while the L2000·CSP· siRNA (5:8:1) lipoproteoplex was less positively charged with +21.73 ± 1.46 mV (Table 1). We hypothesized that the aggregation of CSP·siRNA (2:1) would be unfavorable for L2000 to encapsulate, leaving L2000 predominantly empty with higher positive charge of 82.33 ± 1.40 mV (Table 1) and thus causing the overall charge to be more positive than the CSP·siRNA (8:1). In addition, the aggregation could trap free siRNA molecules and prevent them from approaching the L2000·CSP·siRNA (5:2:1) lipoproteoplex. By contrast, the L2000·CSP·siRNA (5:8:1) displayed a less positive charge, which could be due to the association with free siRNA. Finally, the full lipoproteoplex L2000·CSP·Dox·siRNA demonstrated a size of 205.30 ± 5.06 nm (Table 1), slightly larger than the other lipoproteoplex above without Dox, suggesting that the encapsulation with L2000 might be better when CSP is bound to both Dox and siRNA. As expected, the overall charge of +16.80 ± 0.32 mV (Table 1) exhibited by L2000·CSP·Dox·siRNA (5:8:3:1) is similar to L2000·CSP·siRNA with Dox being a neutral molecule. Additional physical characterization methods such as cryo-TEM or atomic force microscopy could provide a deeper understanding of the assembly of the lipoproteoplexes at both 5:2:1 and 5:8:1 ratio. Doxorubicin Loading. CSP, although mutated extensively, still retains all native core hydrophobic residues necessary for coiled-coil assembly and small-molecule binding.18,33,44 Dox loading into CSP was carried out under passive binding conditions at a 2:1 Dox molar excess. The Dox encapsulation efficiency was measured to be 41.5 ± 1.1% following size exclusion chromatography (Table 2). The dialysis experiment yielded 39.4 ± 4.0% Dox encapsulation efficiency and 39.5 ± 4.0 μg Dox loaded per milligram of CSP (Table 2). L2000 was shown to have similar Dox encapsulation efficiencies post dialysis (35.0 ± 5.6%), but due to the differences in weight, there was a calculated 21.1 ± 6.7 μg Dox loading per milligram of L2000 (Table 2). Thus the presence of CSP improved the loading by 38.9% over L2000.

Figure 5. GAPDH mRNA expression in MCF-7 cells following transfection with proteoplex (CSP·siRNA), lipoplex (L2000·siRNA), and lipoproteoplex (L2000·CSP·siRNA) using GAPDH siRNA (a) or nonsense siRNA (b) measured via quantitative real-time PCR, normalized to 18S mRNA levels. Data represents the average of three independent trials with mean standard deviation, where ns denotes nonsignificant data and * denotes statistical significance of p < 0.01 via student’s t-test.

(CD). As expected, siRNA impacted CSP structure as the negatively charged siRNA complexed with the positively charged surface of CSP (Figure 3a). Computational modeling would appear to predict this binding as there is uniform positive charge across the CSP pentamer surface (Figure S1b). Upon the addition of siRNA, there was an increase in the 208 nm minima, which indicated binding between the protein and siRNA. Conversely, the addition of siRNA to COMPcc had a negligible impact on secondary structure, indicating little to no interactions between the two molecule populations (Figure 3b). Lipoproteoplex Particle Size and Surface Charge Characterization. Zeta potential and size measurements were undertaken of the different formulations of the proteoplex 2693

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Figure 6. Confocal microscopy of MCF-7 cells treated with (a,b) Dox·CSP·L2000, (c,d) Cy5-conjugated siRNA·CSP·L2000, and (e−g) Dox·Cy5conjugated siRNA·CSP·L2000. Samples were imaged under brightfield (a,c,e) or fluorescence in which the samples with Dox (b,d,f) were excited with a 488 nm laser line and viewed with the emission exclusion setting at 560−590 nm and samples with the Cy5-conjugated siRNA (d,g) were excited with a 647 nm laser line and viewed with the exclusion setting at 670 nm. Using the PerkinElmer Ultraview ERS microscope system software, Dox fluorescence appears red and Cy5-conjugated siRNA fluorescence appears blue.

siRNA Transfection. Transfection studies were performed using GAPDH-specific siRNA to yield GAPDH knockdown in efficiently transfected cells and nonsense siRNA as a negative control. GAPDH gene knockdown by GAPDH siRNA-bound L2000·CSP·siRNA lipoproteoplex was investigated at varying CSP·siRNA wt/wt ratios (2:1, 8:1, and 10:1) compared with the standard L2000·siRNA lipoplex prepared according to the manufacturer’s recommended protocol (Figure 5a). Control studies were performed using the same complexes but bound to nonsense siRNA (Figure 5b). The endogenous housekeeping gene 18S was used to normalize the total cDNA content for correct measurement of GAPDH mRNA expression. For both GAPDH siRNA and nonsense siRNA plots, GAPDH mRNA expression levels in the control non-transfected cells were set to a level of 1, to which all transfection results were normalized. Unpaired student’s t-test was conducted between complexes to assess for significant differences in the resulting mRNA expression levels. When bound to GAPDH siRNA, CSP·siRNA at an 8:1 wt/wt ratio failed to demonstrate any significant knockdown of GAPDH. The L2000·siRNA exhibited 0.47 ± 0.04 normalized GADPH mRNA levels and the L2000·CSP·siRNA

(5:2:1) lipoproteoplex demonstrated similar knockdown efficiency (Figure 5a). Notably, both L2000·CSP·siRNA (5:8:1) and L2000·CSP·siRNA (5:10:1) revealed improved knockdown efficiencies compared with the L2000·siRNA lipoplex by 16%, p < 0.01 (Figure 5a), suggesting a supportive role for CSP in aiding transfection. These groups revealed no significant difference in GAPDH mRNA expression in the presence of nonsense siRNA (Figure 5b). Cellular Uptake of siRNA and Doxorubicin. To assess the ability of MCF-7 cells to uptake both Dox and Cy5-conjugated siRNA, confocal microscopy and flow cytometry were performed. Cells treated with Dox (Figure 6a,b) or Cy5-conjugated siRNA (Figure 6c,d), in the presence of L2000·CSP, indicated more diffuse spots of Dox, while Cy5-siRNA appeared as more punctate spots, respectively. When Dox and siRNA were delivered in the presence of L2000·CSP, bright spots were observed that colocalize within the cells. Interestingly, CSP alone also possessed the ability to deliver Dox relative to L2000·Dox (Supplementary Figure S4). To verify the uptake of Dox and siRNA, cells treated with different formulations were further analyzed by flow cytometry. 2694

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Figure 7. Flow cytometry analysis of MCF-7 cells treated with different formulations of Dox and siRNA. Samples with Dox (a) were excited with a wavelength of 488 nm, while samples with Cy5-conjugated siRNA (b) were excited using 633 nm, followed by APC filter. Samples containing both Dox and siRNA were observed under similar channels as Dox and Cy5-siRNA. The insets describe the subpopulation containing both Dox·Cy5-conjugated siRNA. Inset within (a) depicts percent of Dox containing cells bearing Cy5-siRNA, while inset in (b) represents percent of Cy5-siRNA containing cells having Dox within them. Error bars represent the standard error between two readings. All samples had doxorubicin at a final concentration of 6 μg/mL and Cy5-siRNA at a final concentration of 5 μg/mL.

Figure 8. Relative cell viability of MCF-7 cells after 48 h treatment based on MTT assay, normalized to the negative control that consists only of MEM and 50 mM Tris-HCl, 0.5 M NaCl pH 8 buffer. Numbers in horizontal axis represent Dox concentration in micromolar. Error bars represent standard deviation of three independent trials and across four replicates per trial.

CSP alone showed increased uptake of Dox (Figure 7a), Cy5siRNA (Figure 7b), and both (Figure 7a,b) under different fluorescence channels. A better uptake of siRNA with CSP was observed under APC channel (Figure 7b). Consistent results were obtained for cells subpopulation containing both Dox and siRNA (Figure 7a,b, Figure S5), further indicating colocalization of Dox/siRNA. Both CSP- and CSP·L2000-based formulations

exhibited enhanced uptake relative to no vehicle or in the presence of L2000 after 3 h of treatment. Confocal microscopy imaging and flow cytometry analysis indicate colocalization of Dox and siRNA. Although CSP alone shows a better uptake profile, we surmise that it could result in concentrating Dox/Cy5-siRNA in endosomal compartments or remain intact as clusters within the cytosol.45 When CSP is 2695

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Figure 9. Relative cell viability of MCF-7 cells after 24, 48, and 72 h treatment with L2000 transfection reagent based on MTT assay, normalized to the negative control that consists of only MEM, Opti-MEM, and 50 mM Tris-HCl, 0.5 M NaCl pH 8 buffer. Numbers in horizontal axis represent Dox concentration in micromolar. Data are an average of three independent trials. Error bars represent standard deviation across four replicates.

study discussed above at 0.4 μM Dox per 20 000 cells. L2000· Dox reduced cell viability down to 70.9 ± 10.3% at 0.4 μM after 72 h relative to the negative control (Figure 9, Tables S2 and S3). L2000·CSP·Dox had a higher cell viability, with 78.6 ± 2.2% at 0.4 μM relative to the negative control (Figure 9, Tables S2 and S3). The entire lipoproteoplex L2000·CSP·Dox·siRNA displayed the most prominent effect on the cell viability, with 61.0 ± 3.8% at 0.4 μM relative to the negative control (Figure 9, Tables S2 and S3). These data affirmed that the delivery of both Dox and siRNA could improve cell death. It is interesting to note the effect of CSP alone on cell viability; at higher concentrations beyond 0.025 μM, CSP has a visibly cytotoxic effect on the cells (Figure 8). This is consistent with literature that observes cytotoxic effects of the uptake of small cationic nanoparticles in nonphagocytic cells at higher concentrations.47,48 Dox cytotoxicity is observed across all concentrations, presumably entering the cell via passive diffusion. In contrast, a larger particle such as Dox-bound CSP may possibly be endocytosed, resulting in greater reduction of cell viability in the CSP·Dox sample than the Dox alone in treatments below 0.4 μM (p < 0.0005 at 0.2, 0.1, 0.05, and 0.0125 μM, p < 0.005 at 0.025 μM), which has been seen in some studies with Dox-loaded micelles.46 Further cell viability studies show that the lipoproteoplex without the scrambled siRNA is comparable to the L2000·Dox in reducing cell viability (p > 0.2 at 0.4 μM) (Figure 9). This may implicate the L2000 as the major driving force for cellular uptake due to surface interactions with the cell membrane but may also point to a difference in particle size due to the inclusion of CSP. The lipoproteoplex is more efficient in reducing cell proliferation than all other samples at all concentrations (p < 0.005 against L2000·Dox at 0.2 μM, p < 0.0005 against L2000·Dox·CSP at 0.2 μM), demonstrating a 39.0 ± 3.8% decrease in cell viability below the negative control after 72 h at a Dox concentration of 0.4 μM (Figure 9). The cytotoxicity of the GAPDH siRNA as a major contributor has been ruled out by comparing the relative cell viabilities between the L2000·siRNA and L2000·siRNA·CSP treatments (Figure S4). This suggests a combinatorial cytotoxic effect arising from siRNA and Dox in the lipoproteoplex. It is expected that delivery of siRNA targeting a gene more vital to cell survival along with Dox will push cell viability down even further.13

complexed with L2000, it could allow Dox/Cy5-siRNA to enter the cells via multiple pathways, including heparin sulfate proteoglycan engulfment, phagocytosis, and membrane fusion interactions,46 leading to improved knockdown efficiencies observed in our studies. As previously discussed, Dox is an excellent chemotherapeutic agent; however, greater Dox dosages result in toxic side effects such as irreversible cardiotoxicity, which therefore necessitates the use of Dox delivery vehicles.45 Our approach allows for lower total concentrations of Dox dosage to circumvent ineffective mechanism of free Dox diffusion. The enhanced delivery of Dox can be seen with our lipoproteoplex over free Dox delivery alone. Because Dox is encapsulated within the hydrophobic pore of CSP (Figures 1, Figure S1), the protein component may provide an extra boundary for Dox release from the global lipoproteoplex, which could be useful for delayed release of chemotherapeutic agents. Cell Viability upon Delivery of Lipoproteoplex. Cell viability studies were conducted on MCF-7 cells treated with varying combinations of CSP and decreasing Dox concentrations for 48 h at 37 °C. The CSP concentrations were scaled to Dox concentrations according to the optimal CSP·Dox molar binding ratio of 3:4, determined via the Dox loading experiment with size exclusion chromatography described above (Table 2). The negative cell control consisted only of EMEM and 50 mM TrisHCl, 0.5 M NaCl pH 8 buffer. The CSP vehicle began to exhibit toxicity at 0.1 μM with a relative cell viability of 71.3 ± 3.5%, leaving CSP concentrations of 0.05 μM and below suitable for delivery (Figure 8, Table S1). By contrast, the lower range of CSP·Dox treatments yielded a significant loss in cell viability at 45.3 ± 2.4% for 0.05 μM, 52.9 ± 2.7% for 0.025 μM, and 55.1 ± 3.6% for 0.0125 μM, demonstrating the effective delivery of soluble Dox (Figure 8, Table S1). At the same concentrations of 0.05 μM and below, cells treated with Dox alone resulted in cell death but with less potency when compared with CSP·Dox treatments (Figure 8, Table S1). This indicated that CSP improved the delivery and functional potency of Dox. An alternate cell viability study was conducted using the same method with the samples L2000·Dox, L2000·CSP·Dox, and L2000·CSP·Dox·siRNA scaled to lower Dox concentrations of 0.4 μM (Figure 9, Tables S2 and S3). These concentrations more closely reflected the values used in the confocal microscopy 2696

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(6) Barros, S. A.; Hinkle, G. Discovery and Development Strategies for Small Interfering RNAs. In Drug Discovery Toxicology; John Wiley & Sons, Inc: 2016; pp 39−51. (7) Hoelder, S.; Clarke, P. A.; Workman, P. Discovery of small molecule cancer drugs: Successes, challenges and opportunities. Mol. Oncol. 2012, 6 (2), 155−176. (8) Crozier, J. A.; Swaika, A.; Moreno-Aspitia, A. Adjuvant chemotherapy in breast cancer: To use or not to use, the anthracyclines. World Journal of Clinical Oncology 2014, 5 (3), 529−538. (9) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P. G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer 2013, 13 (10), 714−726. (10) Persidis, A. Cancer multidrug resistance. Nat. Biotechnol. 1999, 17 (1), 94−95. (11) Zhang, L.; Lu, Z.; Zhao, Q.; Huang, J.; Shen, H.; Zhang, Z. Enhanced Chemotherapy Efficacy by Sequential Delivery of siRNA and Anticancer Drugs Using PEI-Grafted Graphene Oxide. Small 2011, 7 (4), 460−464. (12) Zhu, C.; Jung, S.; Luo, S.; Meng, F.; Zhu, X.; Park, T. G.; Zhong, Z. Co-delivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA−PCL−PDMAEMA triblock copolymers. Biomaterials 2010, 31 (8), 2408−2416. (13) Zhang, C.-g.; Zhu, W.-j.; Liu, Y.; Yuan, Z.-q.; Yang, S.-d.; Chen, W.-l.; Li, J.-z.; Zhou, X.-f.; Liu, C.; Zhang, X.-n. Novel polymer micelle mediated co-delivery of doxorubicin and P-glycoprotein siRNA for reversal of multidrug resistance and synergistic tumor therapy. Sci. Rep. 2016, 6, 23859. (14) Elzoghby, A. O.; Samy, W. M.; Elgindy, N. A. Protein-based nanocarriers as promising drug and gene delivery systems. J. Controlled Release 2012, 161 (1), 38−49. (15) Kratz, F.; Elsadek, B. Clinical impact of serum proteins on drug delivery. J. Controlled Release 2012, 161 (2), 429−445. (16) Hume, J.; Sun, J.; Jacquet, R.; Renfrew, P. D.; Martin, J. A.; Bonneau, R.; Gilchrist, M. L.; Montclare, J. K. Engineered Coiled-Coil Protein Microfibers. Biomacromolecules 2014, 15 (10), 3503−3510. (17) Yuvienco, C.; More, H. T.; Haghpanah, J. S.; Tu, R. S.; Montclare, J. K. Modulating Supramolecular Assemblies and Mechanical Properties of Engineered Protein Materials by Fluorinated Amino Acids. Biomacromolecules 2012, 13 (8), 2273−2278. (18) More, H. T.; Frezzo, J. A.; Dai, J.; Yamano, S.; Montclare, J. K. Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex. Biomaterials 2014, 35 (25), 7188−7193. (19) More, H. T.; Zhang, K. S.; Srivastava, N.; Frezzo, J. A.; Montclare, J. K. Influence of Fluorination on Protein-Engineered Coiled-Coil Fibers. Biomacromolecules 2015, 16 (4), 1210−1217. (20) Megeed, Z.; Haider, M.; Li, D.; O’Malley, B. W., Jr; Cappello, J.; Ghandehari, H. In vitro and in vivo evaluation of recombinant silkelastinlike hydrogels for cancer gene therapy. J. Controlled Release 2004, 94 (2−3), 433−445. (21) Hubbell, J. A.; Chilkoti, A. Nanomaterials for Drug Delivery. Science 2012, 337 (6092), 303−305. (22) Frezzo, J. A.; Montclare, J. K. Exploring the potential of engineered coiled-coil protein microfibers in drug delivery. Ther. Delivery 2015, 6 (6), 643−646. (23) Haghpanah, J. S.; Tu, R.; Da Silva, S.; Yan, D.; Mueller, S.; Weder, C.; Foster, E. J.; Sacui, I.; Gilman, J. W.; Montclare, J. K. Bionanocomposites: Differential Effects of Cellulose Nanocrystals on Protein Diblock Copolymers. Biomacromolecules 2013, 14 (12), 4360− 4367. (24) Yamano, S.; Dai, J.; Yuvienco, C.; Khapli, S.; Moursi, A. M.; Montclare, J. K. Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolaemediated endocytosis. J. Controlled Release 2011, 152 (2), 278−285. (25) Haghpanah, J. S.; Yuvienco, C.; Civay, D. E.; Barra, H.; Baker, P. J.; Khapli, S.; Voloshchuk, N.; Gunasekar, S. K.; Muthukumar, M.; Montclare, J. K. Artificial Protein Block Copolymers Blocks Comprising Two Distinct Self-Assembling Domains. ChemBioChem 2009, 10 (17), 2733−2735.

CONCLUSIONS We demonstrate here the development of a dual siRNA and small-molecule vehicle, termed lipoproteoplex, composed of a commercially available transfection reagent and an engineered supercharged protein that both condenses siRNA and encapsulates doxorubicin. The assembly of each component is not restricted by harsh chemical reaction conditions or covalent bonding. As the lipid component appears to drive cell entry, there is flexibility in introducing different lipid materials for enhanced transfection or cell-specific transfection.49,50 This could provide an avenue for targeting cancer cells for both gene and drug therapy.49,50 Further experiments will focus on developing novel lipid materials for enhanced therapeutic siRNA transfection and doxorubicin delivery in an in vivo drug-resistant cancer model.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00203. Doxorubicin docking in CSP, SDS-PAGE analysis of protein expression and purification, confocal microscopy, flow cytometry histograms, quantification of cell viability studies, and relative cell viability data. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jin Kim Montclare: 0000-0001-6857-3591 Author Contributions ◆

C.F.L. and R.C. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF DMR-1505214 (J.K.M.) and in part by the NSF MRSEC Program under Award Number DMR-1420073 and NYU CTSI grant from the National Center for Advancing Translational Sciences (NCATS), NIH (UL1TR000038). P.D.R. and R.B. are funded by the Flatiron Institute. We thank the Flatiron Scientific Computing Core for computational support.

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REFERENCES

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DOI: 10.1021/acs.biomac.7b00203 Biomacromolecules 2017, 18, 2688−2698