Rational modification of oligoarginine for highly efficient siRNA delivery: structure-activity relationship and mechanism of intracellular trafficking of siRNA Dafeng Chu PhD, Wen Xu MS, Ran Pan MS, Yong Ding MS, Weiping Sui PhD, Pu Chen PhD PII: DOI: Reference:

S1549-9634(14)00430-4 doi: 10.1016/j.nano.2014.08.007 NANO 989

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

12 June 2014 17 August 2014 21 August 2014

Please cite this article as: Chu Dafeng, Xu Wen, Pan Ran, Ding Yong, Sui Weiping, Chen Pu, Rational modification of oligoarginine for highly efficient siRNA delivery: structureactivity relationship and mechanism of intracellular trafficking of siRNA, Nanomedicine: Nanotechnology, Biology, and Medicine (2014), doi: 10.1016/j.nano.2014.08.007

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ACCEPTED MANUSCRIPT Rational modification of oligoarginine for highly efficient siRNA delivery: structure-activity relationship and mechanism of intracellular trafficking of siRNA Dafeng Chu, PhD1, Wen Xu, MS1, Ran Pan, MS, Yong Ding, MS, Weiping Sui, PhD, Pu Chen, PhD*

These authors contributed equally.

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Word count for abstract: 148 Complete manuscript word count: 4926 Number of references: 49 Number of figures/tables: 8 (6 figures and 2 tables)

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Corresponding author Pu Chen Waterloo Institute for Nanotechnology Department of Chemical Engineering University of Waterloo 200 University Avenue West Waterloo, Ontario, Canada N2L 3G1 Tel: (519) 888-4567 x35586 Fax: (519) 746-4979 Email: [email protected]

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Department of Chemical Engineering and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada

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The authors state no competing interests. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Foundation for Innovation (CFI) and the Canada Research Chairs (CRC) program.

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ACCEPTED MANUSCRIPT Title: Rational modification of oligoarginine for highly efficient siRNA delivery: structure-activity relationship and mechanism of intracellular trafficking of siRNA Authors: Dafeng Chu1, Wen Xu1, Ran Pan, Yong Ding, Weiping Sui, Pu Chen*

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Affiliations: Department of Chemical Engineering and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada These authors contributed equally.

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Corresponding author: Tel.: +1 519-888-4567 ext. 35586; Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract Recently, cell-penetrating peptides (CPPs) have received much attention for cellular delivery of

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therapeutic molecules. However, in the case of CPPs as carriers for siRNA delivery, their utility is

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often restricted by low cellular uptake and/or entrapment in endosomes. Here, in order to deliver siRNAs with high efficiency, oligoarginine, prominent member in CPPs, are rationally modified with

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oligohistidine and stearyl moieties (STR-) by fully taking into account the formation of nanoparticles,

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uptake and intracellular trafficking. We show that when the ratio of histidine/arginine in a peptide sequence is >1.5, pronounced gene silencing is induced. Following this rule, STR-HnR8 (n=16 and 20)

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are developed, which show a high knockdown efficiency rarely reported before. Finally, we find that endosomal escape of siRNA induced by stearylated and oligohistidylated oligoarginine is only from

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“proton-sponge” effect. Taken together, our results suggest a new strategy for the improvement and

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optimization of CPP-based siRNA delivery systems.

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Key words: siRNA delivery, peptide, nanoparticle, endosomal escape, gene silencing

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ACCEPTED MANUSCRIPT Background

RNA interference (RNAi)-mediated silencing offers one of the most attractive methods of gene

demonstrated to be potential targets for RNAi based therapy

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therapy for many diseases. Many types of diseases including viral infections and cancers have been 1–3

; however efficient delivery of short

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interfering RNAs (20~25 base pairs) (siRNAs), the molecules that mediate RNAi, is still a key challenge in realizing the full potential of RNAi therapeutics 4.

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Passive diffusion of “naked” siRNAs across biological membranes is impeded by the

hydrophilicity

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physicochemical properties of the siRNA, including their high molecular weight, negative charge and . Thus, different systems have been developed for siRNA delivery by taking

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advantage of viral or nonviral vectors. Viral vectors are highly efficient but they bear an inherent risk for the patient to encounter severe immunological responses or even develop cancer

9–11

. These

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problems in viral delivery have initiated an intense search for efficient non-viral delivery systems.

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Common non-viral siRNA delivery systems include cationic lipids and polymers. These vectors have various levels of efficiency and toxicity, but for most clinical indications remain unsatisfactory

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.

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Hence, the development of appropriate delivery systems is still an essential requirement to turn these molecules into medicine.

Recently, cell-penetrating peptides (CPPs), consisting of short cationic or amphipathic sequences of about 5-30 amino acids, have received much attention owing to their low toxicity, along with their ability to translocate through plasma membranes

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, which is a significant barrier for siRNA to

access the intended targets in the cytoplasm. In most cases, however, the CPP/siRNA complexes were either internalized by the cells in very low amounts, or the internalized CPP/siRNA complexes were trapped in endosomes, therefore not inducing high gene silencing

23–26

. In terms of cellular uptake,

stearyl modification of CPPs has significantly improved the internalization of siRNA by cells in comparison to the unmodified peptide 27. For the CPP/siRNA entrapped in the endosomes, it is highly 4

ACCEPTED MANUSCRIPT desirable to modify these complexes in order to allow for their escape. Two main mechanisms are currently proposed to explain the escape of biologicals induced by peptides. The first is the “proton sponge” effect

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, that relies on protonable groups in the molecules with pKa values close to the

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endosomal/lysosomal pH. This will lead to a higher influx of protons, along with chloride ions and water, resulting in inevitable endosomal swelling and rupture. This release mechanism of contents to

is pH-sensitive membrane disruption

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28,29

. The second mechanism

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the cytosol has been established for polyhistidine based delivery systems

. The hypothesis is that as endosomes acidify, endocytosed

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and a pentadecaarginine (R15)-PLGA-PEG polymer

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with

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this concept, a histidine-rich peptide

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pH-sensitive peptides shift from an inactive state to an active, membrane-disruptive state. In line with

pH-sensitive membrane disruption capability have been developed.

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In this study, by taking all this knowledge into account, we undertook a rational design of CPP derivatives aiming to provide highly efficient siRNA delivery. First, oligoarginine, one group of the

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most widely utilized CPPs for intracellular delivery of biologicals, was employed as a model carrier

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because it can bind to the cell surface with high affinity 34 and is additionally more effective at entering cells than oligolysine, oligoornithine and arginine-rich peptides 35,36. Second, in order to overcome the

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lack of silencing activity induced by oligoarginine

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, we introduced stearic acid and various

oligohistidine modifications. Afterwards, we explored in detail the relationship between structures and properties of the oligoarginine based molecules with their biological activities and discovered the mechanism of intracellular siRNA release. Methods Peptides and siRNA The R8, R15, STR-R8 and STR-H12R8 were synthesized by CanPeptide Inc. (Pointe-Claire, Canada). The H16R8, STR-H8R8, H8R15, STR-H8R15, STR-H16R8 and STR-H20R8 were synthesized by Pepscan (Lelystad, The Netherlands). Silencer TM GAPDH siRNA (Life Technologies, 5

ACCEPTED MANUSCRIPT Carlsbad, USA) was used to target the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The sense sequence was 5'-GGU CAU CCA UGA CAA CUU Utt-3' and antisense sequence was 5'AAA GUU GUC AUG GAU GAC Ctt-3'. The SilencerTM Cy3-labeled GAPDH siRNA (Life

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Technologies, Carlsbad, USA) was used in confocal laser scanning microscopy (CLSM) and Fluorescence Activated Cell Sorting (FACS). The scrambled siRNA (Life Technologies, Carlsbad,

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USA) was used as negative control in the experiments.

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Cell culture

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CHO-K1 (Chinese hamster ovary) cells were purchased from American Type Culture Collection (ATCC CCL-61). Cells were cultured in F-12K (Thermo scientific, Ottawa, Canada) supplemented

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with 10% fetal bovine serum (FBS, Sigma-Aldrich, Oakville, Canada). The Hela cells were obtained from American Type Culture Collection (ATCC CCL-2), and cultured in Eagle's Minimum Essential

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Medium (Thermo scientific, Ottawa, Canada) with 10% heat-inactivated FBS. All of the cells were

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incubated at 37C in a humidified atmosphere containing 5% CO2.

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Preparation of the complexes

The complexes formulated with siRNA were prepared based on a modified method

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. Both of the

powders of the modified or unmodified peptides and siRNA were dissolved in RNase free water, respectively. The complexes were formed by mixing the two solutions at various molar ratios of arginine residues in peptides to phosphate groups of siRNA (R/P ratio). Lipofactamine 2000 (Lipo) (Life Technologies, Carlsbad, USA) was also diluted in RNase free water and mixed with the siRNA solution to form complexes with the final concentration of 20 L Lipo/mL. The final siRNA concentration was 1M for all the samples containing complexes, which were incubated for 20 minutes at room temperature before characterization and siRNA release study. The samples were

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ACCEPTED MANUSCRIPT diluted further to 10% of the original concentration in Opti-MEM medium (Life Technologies, Carlsbad, USA) for the cell culture assays.

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Particle size and zeta potential

The hydrodynamic diameter of the complexes were measured by dynamic lighter scattering (DLS)

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on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with a 4 mW He-Ne laser operating at 633 nm. A quartz microcell (45 μL) with a 3 mm light path was used and the scattered

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light intensities were collected at an angle of 173°. Zeta potential measurements were also performed

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on the same machine using clear disposable zeta cells. The zeta potential values were obtained with the multimodal algorithm CONTIN, Dispersion Technology Software 5.0. Three measurements were

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Atomic force microscopy (AFM)

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performed to generate the intensity-based size and zeta potential plot reported herein.

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The nanostructure of the complexes was imaged on AFM. 10 L of the sample solution containing complexes of H16R8 or STR-H16R8/siRNA with an R/P ratio of 10:1 was put on a freshly cleaved

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mica surface. The sample was incubated for 10 min at room temperature to allow the complexes to adhere onto mica surface. Mica was then washed at least five times with RNase free water to remove unattached complexes. After air drying, the mica surface was analyzed by a Dimension Icon AFM (Bruker, Santa Barbara, USA) at room temperature using the tapping mode with ScanAsyst-Air tips (Bruker, Santa Barbara, USA). All AFM images were obtained at a resolution of 512 × 512 pixels on a scale of 2 μm × 2 μm.

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ACCEPTED MANUSCRIPT Transmission electron microscopy (TEM) 10 L of sample solution containing the complexes of STR-H16R8/siRNA at R/P 10 or STR-H16R8 only in the same concentration was applied to a 400 mesh Formvar coated copper grid (Canemco-

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Marivac, Canton de Gore, Canada) for 3-5 minutes. The sample was then washed using 5 successive wash steps and dried overnight. The complexes was stained with uranyl acetate and analyzed using a

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JEOL JEM 1200 EX TEMSCAN transmission electron microscope (JEOL, Peabody, USA) operating at an accelerating voltage of 80kV. The images were acquired with an AMT 4 megapixel digital

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camera (Advanced Microscopy Techniques, Woburn, USA).

siRNA binding efficiency (see details in Supplementary Material section)

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RiboGreen assay was used to determine the siRNA loading efficiency using the manufacturer's

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protocol (Life Technologies, Carlsbad, USA).

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Gene silencing

CHO-K1 cells (40,000/well) were plated in a 24-well cell culture plate in F-12K medium with 10%

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FBS. 24 h later, the medium was removed and washed with PBS, and then 300 L of the sample solution containing the complexes of scrambled or GAPDH siRNA (100 nM) formulated with peptides or Lipo was added to the cells. 3 h later, 300 μL of F-12K with 20% FBS was added. In case of chloroquine (CQ) and bafilomycin A1 (Baf A1), both of them (50 μM and 25 nM, respectively) was dissolved in 300 μL of F-12K with 20% FBS, respectively, which was added to the cells 3 h after the cells were treated with the complexes. For transfection in serum, 300 μL of F-12K with 20% FBS was mixed with 300 L of the complex solution and then the 600 L of the mixture was added to the cells. Afterwards, the cells were incubated for 48 hours at 37 °C in a 5% CO2 atmosphere. The cultures were then washed with PBS. Total RNA was extracted from the cells with TRIzol reagent (Life

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ACCEPTED MANUSCRIPT Technologies, Carlsbad, USA), then treated with chloroform (Sigma, Oakville, Canada) and 2propanol (Sigma-Aldrich, Oakville, Canada) as recommended by the manufacturer. RNA concentrations were measured by Nanodrop spectrophotometer ND-1000 (Thermo scientific, Ottawa,

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Canada). All RNAs were reverse transcribed with Bio-Rad iScript cDNA synthesis kit (Bio-Rad, Mississauga, Canada). The cDNA synthesis was primed with a unique blend of oligo (dT) and random

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primers. The following pairs of primers were used for PCR: 5-TTGCTGTTGAAGTCGCAGGAG-3, 5-TGTGTCCGT- CGTGGATCTGA-3 (Sigma, Oakville, Canada). Here, the housekeeping gene

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cyclophilin was chosen as an internal control to normalize the GAPDH gene. The normalization was

AGGGTTTCTCCACTTCGATCTTGC-3

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performed by the amplification of mouse/rat cyclophilin mRNA with the following primers: 5and

5’-AGATGGCACAGGAGGAAAGA-

GCAT-3

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(Sigma, Oakville, Canada). PCR reaction was performed with Brilliant II Fast SYBR Green QPCR Master Mix (Agilent Technologies, Wilmington, USA) on an Mx3005P™ Real-Time PCR System

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(Agilent Technologies, Wilmington, USA).

Fluorescence-activated cell sorting (FACS)

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Cellular uptake of Cy-3 labeled GAPDH siRNA (100 nM) was studied using BD FACSCalibur Flow Cytometry (BD Biosciences, Mississauga, Canada). CHO-K1 cells were transfected with the complexes (R/P 10) according to the protocol listed above. Nontreated cells and naked siRNA served as a negative control. After 3 h incubation, the culture medium was discarded and cells were washed with PBS, Trypsin-EDTA was then added to detach the cells from the plate; cells were suspended in 4% PFA solution and collected.

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ACCEPTED MANUSCRIPT Confocal laser scanning microscopy (CLSM) CHO-K1 cells (80,000/well) were plated in a Nunc Lab-Tek 4-well glass chamber slides (Thermo scientific, Ottawa, Canada) 24 h before transfection. CHO-K1 cells were also treated with the same

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protocol as described above. Treated cells were incubated at 37C for 3 h with Opti-MEM medium. For endosomal labeling, the cells were incubated with 50 nM Lysotracker Green (Life Technology,

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Carlsbad, USA) for 30 min before fixing. The wells were then washed with 15 U/mL heparin (Grade IA, ≥180 USP U/mg, Sigma-Aldrich, Oakville, Canada) for three times in one hour at 37C and fixed

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with 500 L/well of fresh 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 37C for

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30 min. The fixation agent was aspirated, and the cells were washed twice with PBS before they were covered with Fluoroshield with DAPI mounting medium (Sigma-Aldrich, Oakville, Canada). Carl

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Zeiss LSM 700 confocal microscope (Zeiss, Jena, Germany) was used to visualize the cells. The microscope was equipped with Plan-Apochromat 20x/0.8 NA objective lens. The images were

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analyzed with LSM Zen 2009 software.

Membrane integrity assay

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CHO-K1 cells (8,000/well) were plated on 96-well plates and incubated overnight in 100 L of F12K medium containing 10% FBS. The cell culture medium was removed and washed with PBS. The modified and unmodified oligoarginine were dissolved in Opti-MEM medium with pH 7.4, pH 6.3 and pH 5.5, respectively. After stand for 5 min, the solution was added to the cells. After the incubation at 37°C for 3 h, the plates were centrifuged. 50 L of aliquots in each well were collected for the lactate dehydrogenase (LDH) assay. The LDH activity in these samples was determined using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, USA) according to the manufacturer’s protocol, which determined the LDH activity from the amount of produced red formazan product by a colorimetric assay. The amount of formazan produced was assessed by measuring the absorbance at

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ACCEPTED MANUSCRIPT 490 nm with a FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany). Percentages of the LDH activity in each well were calculated from a ratio of the obtained value to the

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control well containing 10 L lysis buffer.

pH-dependent siRNA release

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The solutions containing the GAPDH siRNA complexes at R/P 10 were diluted to 10% of the original concentration in a serial dilution of heparin in PBS, pH 7.4 or 5.5, respectively. 15 min later,

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the released siRNA was determined by the RiboGreen method as described above in “siRNA loading

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efficiency”.

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Cytotoxicity assay

CHO cells were plated in to 96-well plates (8,000 cells/well) in F-12K medium with 10% FBS. 24 h

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later, the medium was removed and washed with PBS. The solutions containing peptides or Lipo were

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prepared in RNase free water and diluted by Opti-MEM medium to 10% of the original concentration (The concentrations were the same as those in gene silencing assay). After stand for 5 min, 60 L of

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the solution was added to the cells. 3 h later, 60 L F12-K medium with 20% FBS was added. For cytotoxicity in the presence of serum, 60 μL of F-12K with 20% FBS was mixed with 60 L of the sample solution and then 120 L of the mixture was added to the cells. After incubation for 24 and 48 hours at 37 °C in a 5% CO2 atmosphere, the cultures were washed with PBS. 100 L of Opti-MEM medium with CCK-8 reagent was then added to each well. Cell viability was assessed by measuring the absorbance at 570 nm with a FLUOstar OPTIMA microplate reader and expressed as the ratio of the cells treated with the carriers over the nontreated cells (negative control).

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ACCEPTED MANUSCRIPT Results

Particle size and zeta-potential

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We first examined the ability of the ten compounds (Table 1) to form nano-sized complexes with siRNA. For all the un-stearylated and stearylated peptides only, no diameters were detected. At various

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molar ratios of arginine residues in peptides to phosphate groups of siRNA (R/P ratio), un-stearylated peptides of R8, R15, H8R15 and H16R8 failed to condense the siRNA into nanoparticles ( 1 m and polydispersity index (PDI) > 0.5. Conversely, peptides with stearic acid modification showed the particle sizes of < 200 nm at various R/P ratios (Table 2).

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AFM and TEM imaging supported the DLS size measurements, showing the presence of distinct nanoparticles with an overall diameter of ca. 70 nm for STR-H16R8/siRNA complexes (Figure 1A and

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1B) along with no regular nanoparticles for H16R8/siRNA and STR-H16R8 only (Figure 1C and 1D).

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However, the particle sizes increased significantly under physiological buffering condition (Figure S1). The ζ-potentials of R8/siRNA complexes at R/P 2.5 and 5 were 15.2 and 19.2 mV, but increased to

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22.4 mV at R/P 10 (Figure 1E). We only measured the ζ-potential of R15/siRNA complexes at R/P 2.5 to be 19.8 mV, because the particle size can be detected only at this R/P ratio (Table 2). The ζpotentials of stearylated peptides/siRNA complexes at R/P 2.5 to 10 increased slightly and were all in the range of 20~30 mV (Figure 1E).

siRNA binding capacity To gain deeper insight into the interactions between these oligoarginine-based molecules and siRNA, siRNA loading capacities were determined by RiboGreen assay. This assay was based on the strong fluorescence of RiboGreen upon intercalation with free siRNA. In this assay, H16R8 and H8R15 were

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ACCEPTED MANUSCRIPT not included as they were difficult to form nanoparticles with siRNA at various R/P ratios. Generally, R8, R15, STR-R15 and STR-(H)nR8 (n=0, 8, 12, 16 or 20) showed significant fluorescence quenching above an R/P ratio of 2.5, which means these molecules could condense siRNA efficiently above this

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ratio (Figure S2). R8 exhibited less siRNA binding capacity than R15 and the corresponding stearylated R8. Interestingly, after the conjugation of oligohistidine to stearylated R8, fluorescence

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quenching was clearly increased with extended chain length of oligohistidine in the order of STRH20R8 > STR-H16R8 > STR-H12R8 > STR-H8R8 > STR-R8. Furthermore, the binding capacity of

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STR-H8R15 is between STR-H12R8 and STR-H16R8. Agarose gel shift assay was also applied to

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evaluate the interaction between siRNA and STR-H16R8, where almost no free siRNA was detected at

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R/P 1 (Figure S3), which is similar to the result from RiboGreen assay.

Gene silencing

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The gene silencing efficacy of siRNA delivered by the individual oligoarginine-based carriers was

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examined using a GAPDH mRNA knockdown assay. The mRNA levels in the cells after treatment were measured by real-time PCR. As shown in Figure 2A and 2B, the peptides/scrambled siRNA and

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R8, R15 and STR-R8/GAPDH siRNA complexes did not induce GAPDH mRNA knockdown at various R/P ratios. The STR-H8R8/GAPDH siRNA formulations showed 16% and 26% lower mRNA levels than that of nontreated cells at R/P 5 and 10, respectively. STR-H8R15/GAPDH siRNA formulations revealed decreased mRNA levels by 17% at R/P 10, lower than that of STR-H8R8. Furthermore, with an increased chain length of oligohistidine, STR-H12R8 induced gene silencing with the knockdown efficiency of 31% at R/P 5 and 59% at R/P 10. STR-H16R8 showed significant gene knockdown levels of 50% at R/P 5 and 88% at R/P 10. The latter value was similar to that of a benchmark, Lipofactamine 2000 (Lipo), which is the most commonly utilized and efficient transfection reagent to introduce siRNA into cells, inducing 84% gene silencing in this assay. Moreover, the knockdown efficiency was improved further to 92% by STR-H20R8. Hela cells were also employed 13

ACCEPTED MANUSCRIPT for the mRNA knockdown study, which showed a similarly higher silencing efficiency induced by STR-H16R8 than that of Lipo (Figure S4A). Finally, gene silencing efficiency of STRH16R8/GAPDH siRNA was investigated in the presence of serum. When the concentrations of the

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carriers and siRNA were doubled compared with those in serum-free assays, STR-H16R8 (R/P 10) and

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lipo induced 81% and 72% gene knockdown, respectively (Figure S5A)

Cellular uptake

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Cellular uptake of Cy-3 labeled siRNA formulated with the carriers was measured by FACS. As

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shown in Figure 3A-3B, in comparison to the other modified peptides R8 and R15 mediated a markedly lower internalization of siRNA, which would be one main reason for its ineffectiveness in

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gene silencing. In terms of stearylated peptides, STR-R8 had the highest mean fluorescence intensity (MFI) of 260, with STR-H8R8, STR-H8R15, STR-H12R8, STR-H16R8 and STR-H20R8 inducing

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MFI of 160, 195, 187, 220 and 211, respectively (Figure 3C). These intensities were all much greater

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than that of Lipo, inducing a MFI of 59. The percentages of cells that were positive for fluorescence (Figure S6) also showed the same trend as MFI. In the presence of 10% serum, even when the

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concentration of siRNA is 200 nM, MFI is only 89 (R/P 5) and 181 (R/P 10) for STR-H16R8 and 46 for Lipo (the dose of Lipo was doubled compared with that in serum-free assays).

CLSM Figure S7 and 4 showed that the cells treated with the Lysotracker Green revealed a punctuate pattern and the Cy3 labeled GAPDH siRNA with red fluorescence delivered by STR-H16R8 revealed a punctuate distribution pattern at 1, 3 and 6 h as well. The majority of Cy-3 fluorescent-labeled siRNA appeared to be trapped in endosomal/lysosomal vesicles as demonstrated by its extensive colocalization with the LysoTracker Green marker, visualized as yellow punctuate fluorescence. This suggested that the uptake of the complexes followed an endosytic pathway. Moreover, all the green 14

ACCEPTED MANUSCRIPT color was overlapped by red color and the complexes were internalized by more cells at 3 and 6 h (Figure 4) compared with those at 1 h (Figure S7). At 6 h, the punctuate distribution pattern of yellow fluorescence increase in size and decrease in amount due to the fusion of endosome/lysosome . A small amount of red punctuate fluorescence can be found

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compartments into larger vesicles

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distributing in the cytosol, which indicated the endosomal escape of siRNA.

Endosomal escape

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Chloroquine (CQ) is an endosomolytic agent well known to facilitate the release of entrapped 29,42

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biomolecules by causing the swelling and disruption of endocytic vesicles by osmotic effects

the case of STR-R8, which induced a high uptake of siRNA but hardly detectable gene silencing, a 13%

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decrease in mRNA levels was observed when it was simultaneously treated with CQ. The effect of CQ was more pronounced with STR-H8R8 and STR-H16R8, showing a significant increase in gene

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silencing from 8% to 49% and 45% to 79%, respectively (Figure 5A). Focusing on STR-H16R8, we

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used an inhibitor for vacuolar ATPase proton pump, bafilomycin A1 (Baf A1), to reduce the acidification of the endosomes

. Figure 5B showed that the addition of Baf A1 decreased the gene

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silencing dramatically.

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The pH-dependent membrane disruption was further investigated by a membrane integrity study using the widely used LDH leakage assay

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. In terms of all the stearylated peptides, there was no

significant pH-dependent promotion of LDH leakage at various concentrations (Figure 5C), although pH-sensitive membrane disruption was demonstrated with R8 and R15. This suggested that after stearylation or stearylation/oligohistidylation modifications, the pH-sensitive membrane disruption capabilities of the oligoarginine molecules were restrained completely. siRNA dissociation from the stearylated peptides was investigated in phosphate buffered saline (PBS) at pH 5.5 and 7.4, mimicking the acidic environment in the endosome/lysosome and the physiological conditions during cell culturing. Negatively charged heparin was used to represent the 15

ACCEPTED MANUSCRIPT endogenous polyanions

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. In terms of the stearylated and oligohistidylated oligoarginine, no

pronounced change of siRNA release was found when the medium was acidified (Figure S8A and S8B). In both of the PBS solutions, extended chain lengths of oligohistidine and oligoarginine

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segments reduced the siRNA release against the heparin competition, which showed a similar order as

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the siRNA binding capacity of these oligoarginine based molecules (Figure S2).

Cytotoxicity

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When these oligoarginine based molecules are used as siRNA delivery agents, a low cytotoxicity is

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critical. The final investigation therefore was evaluation of the cytotoxicity of the peptides. The viability of cells treated with modified and unmodified peptides was almost the same as that in

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nontreated cells. However, Lipo showed slight cytotoxicity with only 87% and 93% of the cells surviving after 24 and 48 h (Figure 6A and 6B). STR-H16R8 did not show any cytotoxicity in Hela

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cells (Figure S4B) and in CHO-K1 cells in the presence of serum as well (Figure S5B), when a high

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Discussion

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knockdown efficiency was obtained.

It has been reported that oligoarginine uptake increases with increasing charges, peaking between octaarginine (R8) and pentadecaarginine (R15)

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. For this reason, in our approach, ten compounds

were investigated including R8 and R15, along with their derivatives obtained by modification with oligohistidine and a stearyl moiety. The DLS results indicated that the peptides only were not selfassembled, but freely dissolved in water. This highlights that hydrophilicity as well as the positive charges of the peptides impeded the aggregation of hydrophobic stearyl moieties. Ideally, cationic carriers/siRNA complexes should be smaller than 200 nm to: (i) ensure optimal diffusion through tumor tissues by the enhanced permeation and retention effect (EPR) 47; and (ii) reduce the recognition 16

ACCEPTED MANUSCRIPT and removal by phagocytic cells in vivo. However, only the complexes of stearylated peptides/siRNA exhibited the particle sizes of < 200 nm, which indicated that the stearic acid moiety was able to assist peptides to condense siRNA into small complexes. AFM and TEM imaging, which was hardly found

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in previous reports, confirmed that the hydrophobic stearyl moiety could significantly improve the formation of the small complexes between siRNA and the peptides. The reason would be that the

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negative charge on siRNA molecules neutralized the positive charge of the peptides, leading to a reduced intermolecular repulsion from arginine residues, and consequently improving the hydrophobic

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interaction of stearyl groups.

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siRNA binding assay indicated that the stearyl moiety could help condense siRNA more efficiently and R15 condensed more siRNA than R8. Furthermore, the extended chain length of oligohistidine

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improved the condensing of siRNA significantly. We assumed that a portion of histidine (pKa of the side chain is 6.0) residues in the stearyl-oligohistidine-oligoarginine were positively charged in

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aqueous solutions and interact with siRNA as well, so that they can show a more pronounced binding

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effect. However, in the case of STR-H8R15 this assumption is not suitable, since the number ratio of histidine/arginine in the sequence was only 0.53, far lower than those of STR-H8R8 and STR-H12R8

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with the histidine/arginine ratio of 1 and 1.5, respectively. This phenomenon verified that the longer oligoarginine had greater capability to condense siRNA compared with the short one.

The gene

silencing assay implied that the increased chain length and stearylation modification cannot improve the knockdown efficiency of siRNA induced by oligoarginine. However, When the ratio of histidine/arginine in a stearylated peptide sequence is >1.5, pronounced gene silencing was demonstrated and a high knockdown efficiency was induced by STR-HnR8 (n>12). The positive correlation between the ratios of histidine/arginine and knockdown efficiency indicated that histidine residues played an important role in the cellular delivery of siRNA. The reason would be high cellular uptake or efficient endosomal escape by “proton sponge” effect or pH sensitive membrane disruption, or high siRNA release from the complexes, or good protection of siRNA by the carriers. Based on the 17

ACCEPTED MANUSCRIPT FACS observations, we can conclude that stearylated peptides with various chain lengths of oligoarginine and oligohistidine resulted in similar cellular uptakes of the complexes. Therefore, significant differences in the efficacy of the stearylated peptides/siRNA complexes would not be

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attributed to the internalization of the complexes, which were only slightly different, but rather to the varying degrees of siRNA release into the cytosol. The assay with CQ confirmed that not only the

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stearylated peptides/siRNA complexes were internalized via endocytosis, but also the involvement of oligohistidine in STR-R8 enhanced the endosomal escape of siRNA substantially. Moreover, the

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endosomal acidification was proven important to help the release of siRNA formulated in the

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complexes from endosomes. Naturally, the inhibition of the proton pumps on the endolysosomes should weaken both the “proton-sponge” effect

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and the pH-dependent endosomal membrane

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disruption 30. However, the stearylated peptides possessed similar membrane disruption capabilities at pH 5.5, 6.3 and 7.4.

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The release of siRNA from the internalized complexes is crucial for effective gene silencing,

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whereby a low siRNA dissociation would most likely reduce the gene silencing efficiency 49. On the contrary, a premature extracellular release of siRNA is also undesired. These results indicated that the

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acidic environment did not significantly change the release of siRNA from the complexes. Also, siRNA dissociation from the internalized complexes would not be an issue in terms of hindering the effective gene silencing. This is because STR-R8 and STR-H8R8, exhibiting higher release of siRNA did not induce effective gene silencing, while STR-HnR8 (n=12, 16 or 20), inducing a higher knockdown efficiency showed a lower siRNA release. On the other hand, less replacement of siRNA by heparin indicated that siRNA could be better protected in the complexes formed with the molecules possessing longer oligohistidine and oligoarginine chains, presumably preventing siRNA from the degradation in the endosome/lysosome more effectively. However, STR-H8R15, which protected siRNA slightly better than STR-H12R8 showing a significant knockdown efficiency, did not induce efficient gene silencing. Thus, better protection of siRNA in the complexes, although important would 18

ACCEPTED MANUSCRIPT not be a determining factor to the more efficient gene silencing. Based on the comprehensive investigations on the mechanism of endosomal escape, we therefore concluded that the “proton-sponge” effect induced by histidine residues played a predominant role in the escape of siRNA from endosomes

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to cytosol.

In summary, by fully taking into account the formation of nanoparticles, cellular uptake and

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intracellular trafficking of siRNA, we have rationally modified oligoarginine with stearic acid and various oligoaringine in order to deliver siRNA with high efficiency. The relationship between the

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structures/properties of the oligoarginine based molecules and their biological activities have been

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investigated in detail. It was found that STR-HnR8 (n>12) induced outstanding knockdown efficiency without any cytotoxicity. In addition, we found that endosomal escape of siRNA induced by the

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peptides was only from “proton-sponge” effect induced by the oligohistidine segments. Future studies will focus on the efficacy and biodistribution of this newly developed siRNA delivery system in

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animals.

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Appendix A. Supplementary data

Supplementary data related to this article can be found at:

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ACCEPTED MANUSCRIPT Figure legends

Figure 1. (A) AFM and (B) TEM imaging of STR-H16R8/siRNA complexes. (C) AFM image of

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H16R8/siRNA complexes. (D) TEM image of STR-H16R8 only (the concentration was in accordance with that at R/P 10). (E) ζ-potential of the peptides/siRNA complexes determined by DLS (n=3). The

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scale bar on the TEM pictures is 100 nm.

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Figure 2. Knockdown efficiencies of GAPDH mRNA in CHO-K1 cells induced by the modified and

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unmodified oligoarginine/(A) scrambled and (B) GAPDH siRNA complexes (n=4).

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Figure 3. (A-C) Cellular uptake of Cy3-siRNA complexes formulated with the modified and

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unmodified oligoarginine at 3 h after the treatment of CHO-K1 cells.

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Figure 4. Intracellular localization of siRNA in CHO-K1 cells at 3 and 6 h after the treatment with STR-H16R8/Cy3-labeled siRNA (red) complexes determined by the confocal microscope. Both length

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and width are 80 μm.

Figure 5. (A) Effect of an endosomolytic agent, CQ, on gene silencing of siRNA formulated with STRR8, STR-H8R8 and STR-H16R8 at R/P 10. (B) Effect of an acidification inhibitor of endosomes, Baf A1, on gene silencing efficiency of siRNA formulated with STR-H12R8, STR-H16R8 and STRH20R8 at R/P 10 (n=4). (C) Membrane disruption induced by the unmodified and modified oligoarginine in CHO-K1 cells at pH 7.4, 6.3 and 5.5 measured by comparing the amount of leaked lactate dehydrogenase (LDH) in the sample wells with the control wells (n=5).

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ACCEPTED MANUSCRIPT Figure 6. Cytotoxicity of oligoarginine and modified oligoargine in CHO-K1 cells after (A) 24 and (B)

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48 h incubation tested with CCK-8 reagent (n=5).

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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ACCEPTED MANUSCRIPT Table 1. Sequences of the unmodified and modified oligoarginine along with their molecular weight

Sequences

MW

Purity

R8

Acetyl-RRRRRRRR-NH2

1,308.6

98.8

STR-R8

Stearyl-RRRRRRRR-NH2

1.533.0

96.8

STR-H8R8

Stearyl-HHHHHHHHRRRRRRRR-NH2

2,630.2

94.0

R15

Acetyl-RRRRRRRRRRRRRRR-NH2

2,401.9

98.8

H8R15

Acetyl –HHHHHHHHRRRRRRRRRRRRRRR-NH2

3,499.1

99.4

STR-H8R15

Stearyl-HHHHHHHHRRRRRRRRRRRRRRR-NH2

3,723.6

92.4

STR-H12R8

Stearyl-HHHHHHHHHHHHRRRRRRRR-NH2

3,178.7

96.0

H16R8

Acetyl-HHHHHHHHHHHHHHHHRRRRRRRR-NH2

3,503.0

99.9

STR-H16R8

Stearyl-HHHHHHHHHHHHHHHHRRRRRRRR-NH2

3,727.4

98.3

STR-H20R8

Stearyl-HHHHHHHHHHHHHHHHHHHHRRRRRRRR-NH2

4276.0

98.7

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and purity.

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ACCEPTED MANUSCRIPT Table 2. Particle size of the unmodified and modified oligoarginine/siRNA complexes, determined by DLS (n=3). R/P* 2.5 Name

PDI

R/P 5

†

R/P 10 PDI

PDI (nm)

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(nm)

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(nm) 415±154

0.48±0.17

288±13

0.18±0.03

364±19

0.39±0.1

STR-R8

120±35

0.31±0.06

89±15

0.29±0.04

73±14

0.24±0.04

STR-H8R8

65±16

0.22±0.02

71±9

0.27±0.01

78±7

0.29±0.03

R15

257±27

0.35±0.09

> 1m

> 0.5

> 1m

> 0.5

H8R15

> 1m

> 0.5

> 1m

> 0.5

> 1m

> 0.5

STR-H8R15

64±8

0.31±0.05

78±5

0.29±0.05

77±8

0.28±0.04

STR-H12R8

88±2

0.31±0.01

95±3

0.28±0.03

89±2

0.27±0.01

H16R8

> 1m

> 0.5

> 1m

> 0.5

> 1m

> 0.5

STR-H16R8

79±11

0.22±0.03

88±12

0.3±0.03

75±9

0.31±0.03

STR-H20R8

89±13

95±7

0.32±0.04

92±7

0.29±0.04

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0.29±0.05

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R8

* R/P: Molar ratio of arginine residues in peptides versus phosphate groups of siRNA PDI: Polydispersity index

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ACCEPTED MANUSCRIPT Graphic Abstract The complexes of siRNA/stearyl-(histidine)n-(arginine)8 (n>12) induced outstanding knockdown

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efficiency without any cytotoxicity, where oligoarginine can bind to the cell surface with high affinity;

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stearyl modification has significantly improved the cellular uptake of siRNA; the endosomal escape of siRNA induced by the peptides was only from “proton-sponge” effect induced by the oligohistidine

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segments.

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