PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo

Accepted Manuscript PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo

Gudrun Aldrian, Anaïs Vaissière, Karidia Konate, Quentin Seisel, Eric Vivès, Frédéric Fernandez, Véronique Viguier, Coralie Genevois, Franck Couillaud, Héléne Démèné, Dina Aggad, Aurélie Covinhes, Stéphanie Barrère-Lemaire, Sébastien Deshayes, Prisca Boisguerin PII: DOI: Reference:

S0168-3659(17)30532-1 doi: 10.1016/j.jconrel.2017.04.012 COREL 8757

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

7 February 2017 9 April 2017 10 April 2017

Please cite this article as: Gudrun Aldrian, Anaïs Vaissière, Karidia Konate, Quentin Seisel, Eric Vivès, Frédéric Fernandez, Véronique Viguier, Coralie Genevois, Franck Couillaud, Héléne Démèné, Dina Aggad, Aurélie Covinhes, Stéphanie Barrère-Lemaire, Sébastien Deshayes, Prisca Boisguerin , PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Corel(2017), doi: 10.1016/j.jconrel.2017.04.012

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ACCEPTED MANUSCRIPT PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo. Gudrun Aldrian2*, Anaïs Vaissière1*, Karidia Konate1*, Quentin Seisel 1, Eric Vivès1, Frédéric Fernandez3 , Véronique Viguier3, Coralie Genevois4 , Franck Couillaud4, Héléne Démèné 5, Dina Aggad6 , Aurélie Covinhes7, Stéphanie Barrère-Lemaire 7, Sébastien Deshayes1, Prisca Boisguerin1#

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Affiliations:

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1 - Centre de Recherche de Biologie cellulaire de Montpellier, CNRS UMR 5237, 1919 Route de Mende,

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34293 Montpellier Cedex 5, France

2 - Sys2Diag, UMR 9005-CNRS/ALCEDIAG, 1682 Rue de la Valsière, 34184 Montpellier CEDEX 4, France 3 - Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier, France

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4 - EA 7435 IMOTION (Imagerie moléculaire et thérapies innovantes en oncologie), Université de Bordeaux, 146 rue Leo Saignat, 33076 Bordeaux, France

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5 - Centre de Biochimie Structurale, CNRS UMR 5048-INSERM U1054-UM, 29, rue de Navacelles, 34090 Montpellier, France

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6 - Institut des Biomolécules Max Mousseron, CNRS UMR 5247, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 05, France

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7 - IGF, CNRS, INSERM, Universités de Montpellier, 141 rue de la Cardonille, 34094 Montpellier, France.

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* Authors contributed equally to the work.

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# Corresponding author: [email protected]

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Keywords: PEGylation, retro-inverso, siRNA delivery, cell penetrating peptides, nanoparticle, gene knock-

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ACCEPTED MANUSCRIPT ABSTRACT Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by selfassembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene

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glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties.

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A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in

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zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤ 20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of

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PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labelled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after

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administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation

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constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of

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RNAi-based cancer therapeutics.

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functional moieties to develop safe and biocompatible delivery systems for the clinical application of

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ACCEPTED MANUSCRIPT 1. Introduction

Since several years, short interfering RNAs (siRNAs) have been widely considered as a successful tool with a great potential for therapy, as they are highly specific for the potential targeting of any single or group of genes [1,2]. siRNAs are able to catalytically silence mRNA translation by interacting with their complementary mRNA sequence which induces, in many cases, mRNA degradation by the RISC

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machinery.

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However, upon entering the bloodstream, siRNAs are vulnerable to degradation by endogenous

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nucleases, and renal excretion due to their small size and highly anionic character. Moreover, these negative charges also prevent binding to and crossing through the cell membrane, which severely restricts their ability to reach their active site within the cells. Various molecular siRNA analogues have

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been designed mainly to resist enzymatic hydrolysis [3,4]. However, delivery of siRNAs in their native form has several advantages over using chemically modified analogues because the induction of RNA

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interference and the straightforward use of easy-to-synthesize and cheap siRNA molecules. Therefore,

oligonucleotides (ON) delivery systems.

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the bottleneck associated with this therapy still remains the development of effective native

As potent drug delivery systems (DDSs), non-viral vectors based on many different classes of

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compounds, including various cationic lipids, polymers, peptides, or carbohydrate analogs have been developed and their use is steadily increasing [5,6]. Among these, small delivery peptides, termed cell-

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penetrating peptides (CPPs), have gained considerable interest [7–9]. CPPs are usually up to 30 amino acids long and are characterized by their remarkable ability to convey different cargo molecules into cells

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and tissues. The first CPP sequences were derived from natural proteins such as penetratin [10] and Tat [11]. They can be divided into two main classes: amphipathic CPPs, containing both a hydrophobic and a

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hydrophilic domain (e.g. MPG) [12] and polycationic CPPs containing a high number of positively charged residues (e.g. poly-Arginine) [13,14]. There are two main strategies to vectorize ONs using CPPs: by directly conjugating ONs to the CPPs (covalent conjugation approach) or by forming non-covalent complexes between the CPPs and ONs (nanoparticle-based approach). The formation of non-covalent nanoparticles (NPs) has been particularly efficient in the CPP-mediated delivery of oligonucleotides carrying multiple negative charges and therefore able to form electrostatic complexes with cationic peptides. Moreover such stable electrostatic complexes should provide greater ON-protection against nucleases. In addition, complex formation is easy to perform since no chemical modification is required

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ACCEPTED MANUSCRIPT to allow NP assembly. Therefore formation of electrostatic complexes remain the most popular application for CPP-mediated siRNA delivery, and some significant progresses have been made both in vitro and in vivo by several groups using either natural or synthetic CPPs such as PepFect and NickFect [15,16], MPG and CADY [17,18], hCT-variants (human calcitonin) [19], C6M1 [20], or CADY-K and RICK developed in our group [21,22]. We recently designed the RICK CPP, as the retro-inverso form of the CADY-K peptide [22]. RICK

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conserves the main biophysical features of its L-parental homologue, keeps the ability to associate with

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siRNA in stable peptide-based nanoparticles (PBNs) and to internalize cells via direct translocation. The

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main advantage of RICK-based NPs lays in resistance to enzymatic degradation and increased protection of the siRNA. RICK-based NPs are able to rapidly internalize siRNAs into different cell lines (U87, B16 and Neuro-2a) and to induce specific inhibition of gene expression at a low dose (20 nM siRNA) without any

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significant cytotoxicity. Compared to other CPP-based siRNA carriers [23–26], we showed that this knockdown effect is remarkable since other systems need higher siRNA concentration to obtain the same

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knock-down efficiency. These new PBNs present a promising strategy for future in vivo applications, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins).

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The major limitation of using these NPs is their short life span in the blood circulation. However, thanks to a plethora of peptide chemistry processes, they can be further engineered with functional

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moieties to improve for example their blood circulation [4]. Along this line, PEGylation is extensively used [27,28] to improve their physical stability in vivo, while preventing both recognition by the

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mononuclear phagocytic system (MPS) in the liver and spleen and interactions with blood components [29–31]. A successful example of PEGylated NPs is given by Genexol-PM [methoxy-PEG-poly(D,L-

[32,33].

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lactide)Taxol], the first polymeric micellar NP succeeding phase II clinical trials in the United States

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In the present work, we report the PEGylation of our newly develope d RICK-based NPs [22]. All the physicochemical properties of NPs such as size, shape, surface charge, surface chemistry (PEG moieties), which affect the cellular activity and the biodistribution of the NPs, have been evaluated in details. We notably showed the NPs incorporating PEGylated peptide display improved pharmacokinetics with enhanced initial blood circulation and reduced off-target organ accumulation. The capacity to yield NPs with this set of traits is imperative for the successful use for systemic siRNA administration.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Materials. Peptides: RICK (NH2-kwllrwlsrllrwlarwlg-CONH2) and Cysteine-RICK (NH2-Ckwllrwlsrllrwlarwlg-CONH2) were purchased from LifeTein. PEGylation and Atto633-labeling of the RICK peptide were performed as described in the Supporting Information or elsewhere [21], respectively. siRNA: Unlabeled or Cy5-labeled siRNA were obtained from Eurogentec and siRNA-Cy3b from BioSynthesis. The different sequences are for anti-firefly luciferase (siFLuc) : 5’-CUU-ACG-CUG-AGU-ACU-

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UCG-AdTdT-3’ (sense strand) and a scrambled version of the anti-luciferase (siSCR): 5’-CAU-CAU-CCC-

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UGC-CUC-UAC-UdTdT-3’ (sense strand) used as control. The sequence for the siRNA anti -cyclin

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dependent kinase 4 (siCDK4) is 5′-CAG-AUC-UCG-GUG-AAC-GAU-GdTdT-3′ (anti-sense strand) and the scrambled version: 5′-AAC-CAC-UCA-ACU-UUU-UCC-CAA-dTdT-3′ (anti-sense strand) based on [34]. Large

Unilamellar

Vesicles

(LUVs)

were

prepared

as

previously

reported

[35]

using

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dioleylphosphatidylcholine (DOPC) and 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) phospholipids, cholesterol (Chol) and sphingomyelin (SM) (purchased from Avanti Polar Lipids).

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2.2. Circular dichroism (CD) measurements. CD spectra were recorded with an optical path of 1 mm on a Jasco 810 (Japan) dichrograph in quartz suprasil cells (Hellma). For each condition, same concentrations

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of peptide (40 µM) were used. Spectra of 3 accumulations were recorded between 190 and 260 nm (0.5 nm data pitch, 1 nm bandwidth) using the standard sensitivity.

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2.3. NMR measurements: Incorporation of RICK and PEG-RICK peptides in sodium dodecyl sulfate (SDS) micelles was done following the procedure of Killian et al . [36]. This procedure yielded in our hands to

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the most homogeneous samples, as estimated by inspection of the NMR proton linewidths. Briefly, the peptide was dissolved in deuterated Trifluoroethanol (d3-TFE, Eurisotop), and then mixed to an equal

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amount of water containing 17 times the final desired concentration of deuterated SDS (Sigma-Aldrich). Water was then added to yield a 16:1 water:TFE ratio. The solution was lyophilized overnight and the

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dried products were rehydrated by adding deionized water. pH was set to 5.3 by adding small aliquots of 5 M HCl. The final peptide and SDS concentrations were 200 µM and 100 mM respectively. 2D Total Correlated SpectroscopY (TOCSY) (mixing time, 60 ms) and NOESY (mixing time, 200 and 500 ms) experiments [37], were recorded on a 700 MHz Bruker Avance spectrometer equipped with a cryogenic H/C/D/N probe with a Z-axis gradient at 323K. Proton chemical shifts were referenced relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid resonances. All NMR Spectra were processed and analyzed using Gifa [38] and NMRView softwares [39]. 2.4. Nanoparticle formulation. Stock solutions of peptide were prepared at 1 mg/ml in ultrapure water. After sonication (10 min) and centrifugation (5 min, 13,500 rpm), the supernatant of the peptide solution

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ACCEPTED MANUSCRIPT was filtered (0.22 mm). Finally, peptide concentration was determined by absorption spectrometry using a NanoDrop 1000 (Thermo Scientific). For the nanoparticle formulation, the peptides were first placed in solvent at the desired concentration then siRNA was added to the peptide solution at room temperature at the indicated molar ratio (R). Then the solution was mixed by 5 up and downs and le ft for 30 min at room temperature for complex formation. As an example, a molar ratio of 20 (R 20) corresponds to 20fold higher molar concentration of peptide compared to the siRNA.

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2.5. Agarose gel shift assay. CPP:siRNA complexes were formulated at different molar ratios in 5%

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glucose and analyzed by agarose gel (1% w/v) electrophoresis stained with GelRed (Interchim) for UV

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detection as descripted previously [21].

2.6. Dynamic light scattering (DLS). CPP:siRNA NP formation was evaluated with a NanoZS (Malvern) in terms of mean size (Z-average) of the particle distribution and of homogeneity (PdI). All results were

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repeated three times with three runs by measurement at 25°C.

2.7. Cell culture conditions. The efficiencies of siRNA-mediated gene silencing were investigated in U87

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cell lines (U87 MG, human glioblastoma) stably transfected with firefly and NanoLuc luciferases (FLucNLuc) encoding plasmid (details of cell line generation are given in the supplementary material). Cells

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were grown in a complete medium: DMEM (+ GlutaMAX™ supplement, Life Technologies), with 100 units/ml penicillin (Life Technologies), 100 mg/ml streptomycin (Life Technologies), 10% fetal bovine

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serum (FBS) (PAA), non-essential amino acids NEAA 1X (LifeTechnologies) and selection antibiotics hygromycine B (Invitrogen) (50 µg/ml). Cells were maintained in a humidified incubator with 5% CO 2 at

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37 °C.

2.8. Transfection experiments. For flow cytometry measurements, 100,000 cells were seeded 24 h

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before experiment into 24-well plates. Cells were incubated with 175 µl of fresh pre-warmed serum-free DMEM and 75 µl of NP solution were added. After 1 h of incubation, cells were washed twice with D-PBS

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and incubated with 100 µl 0.05% Trypsin (Gibco) for 10 min (37°C, 5% CO 2). By adding 400 µl PBS containing 5% FBS, the cells were transferred in Eppendorf tubes and centrifuged (1,200 g, 10 min, 4°C). Cell pellets were resuspended in 500 µl D-PBS containing 5% FBS and analyzed by flow cytometry (Fortessa and FACSDiva software from Becton Dickinson). Living cells were selected by the addition of DAPI (1 µg/ml, Sigma-Aldrich). For Luciferase and LDH assays, 5,000 cells were seeded 24 h before experiment into 96-well plates as descripted elsewhere [21,22]. In brief, cells were incubated with 70 µl of fresh pre-warmed serum-free DMEM and 30 µl of NP solution were added. After 1h30 of incubation, 100 μl DMEM supplemented with 20% FBS were added to the transfection solution, and cells were then incubated for another 36 h. The

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ACCEPTED MANUSCRIPT experimental procedure was designed to test CPP:siRNA NPs at a peptide:siRNA molar ratio of R = 20, containing siRNA concentrations of 5, 10 and 20 nM in the final volume of 200 µl. For Western blot assay, 75,000 cells were seeded 24 h before experiment into 24-well plates. Cells were incubated with 175 µl of fresh pre-warmed serum-free DMEM and 75 µl of NP solution were added. After 1h30 of incubation, 250 μl DMEM supplemented with 20% FBS were added to the transfection solution, and cells were then incubated for another 24 h. The experimental procedure was designed to

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test CPP:siRNA NPs at a peptide:siRNA molar ratio of R = 20, containing siRNA concentrations of 5, 10

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and 20 nM in a final volume of 500 µl.

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For microscopy experiments, 400,000 cells were seeded 24 hours before imaging into a Fluoro Dish (World Precision Instruments). Before microscopy imaging, cells were washed twice with D-PBS and covered with 1600 µl of complete medium. 400 µl of NPs (Atto633-RICK:siRNA-Cy3b; R = 20), formulated

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as previously described [22] in 5% glucose water, were directly added on the cells at the very beginning of imaging.

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2.9. Measurement of cell cytotoxicity. 50 µl of supernatant were taken to estimate the cytotoxicity induced by the NPs with the Cytotoxicity Detection KitPlus (LDH, Roche Diagnostics) following the

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manufacturer instructions.

2.10. Luciferase reporter gene silencing assay. The evaluation of siRNA delivery and biological activity

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using the different vectors were carried out by measuring the remaining FLuc and NLuc activity in cell lysates. Briefly, the medium covering the cells was carefully removed after 48 h and replaced by 50 µl of

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0,5X Passive Lysis Buffer (PLB; Promega). After 30 min of shaking at 4°C, plates containing the cells were centrifuged (10 min, 1,800 rpm, 4°C) and 5 µl of each cell lysate supernatant were finally transferred into

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a white 96-well plate. FLuc and NLuc activities were quantified using the Dual Luciferase Assay Reagents (Promega) on a plate-reading luminometer (POLARstar Omega, BMG Labtech). Results are expressed as

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percentage of relative light units (RLU) in non-treated cells (%FLuc and %NLuc), then normalized on %NLuc to obtain the Relative Luc Activity (%FLuc/%NLuc). 2.11. Western blot evaluation. Transfected cells were lysed and proteins were separated by 4 - 20% Mini-PROTEAN® TGXTM Precast Gel (Bio-Rad) and transferred onto Trans-Blot® Turbo™ Mini PVDF Transfer membrane (Bio-Rad). As antibodies (all from Cell Signaling), anti-CDK4 rabbit mAb D9G3E, antiVinculin rabbit mAb E1E9V, anti-Vinculin rabbit mAb E1E9V and anti-rabbit IgG HRP were used. More details are given in the Supporting Information. 2.12. Liposome leakage assay. Large unilamellar vesicles (LUV) reflecting the plasma membrane were prepared as described in detail in the supplementary material. Leakage was measured as an increase in

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ACCEPTED MANUSCRIPT fluorescence intensity upon addition of either the CPP alone or NP (CPP final concentration of 500 nM) to 2 ml of LUVs (100 μM) in buffer (20 mM HEPES, 145 mM NaCl, pH 7.4). 100% fluorescence was achieved by solubilizing the membranes with 0.1% (v/v) Triton X-100 resulting in the completely unquenched probe. 2.13. Cell imaging by confocal microscopy: For microscopy experiments, the U87 cells or GUVs were placed into a glass bottom cell culture dish (Greiner bio-one) and incubated with NPs at various amounts

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of PEGylation (20%, 50% and 100% of PEG-RICK) (400 nM RICK/PEG-RICK:20 nM siRNA-Cy3b). Confocal

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images of GUVs were immediately obtained with an inverted LSM780 multi -photon microscope (Zeiss).

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The confocal images were projected and treated with the software ImageJ.

2.14. Spinning disk confocal microscopy: To study the entrance of the NPs inside living cells, we used an inverted microscope (Nikon Ti Eclipse) coupled to a spinning disk (ANDOR) system as described

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elsewhere [22]. Along the 2 h of measurements, cells were maintained at 37°C by a cage incubator (Okolab). Control experiments were performed to ensure that no emission bleed through was observed

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between the different channels. Images were recorded every 2 min for 120 min with an EM-CCD camera (iXon Ultra) and projected with the software Andor IQ3. The image treatment was performed with the

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ImageJ software.

2.15. Zebra fish experiments: The use of casper zebrafish for experimental purposes was conducted in

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accordance with the European guidelines. Zebrafish were maintained at 28°C and embryos were collected from natural crosses of adult fishes. Collected embryos were maintained in Egg water (60

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µg/ml "Instant Ocean" Sea Salts in distilled water) at 28°C. Embryos were staged according to hours postfertilization (hpf) at 28°C and morphological criteria [21]. siRNA-Cy3 loaded NPs were injected into each

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egg at one-cell stage (2 nl containing 3 µM siRNA (R = 20) per embryo). Confocal images of zebrafish were obtained 24 h post-fertilization with an inverted LSM780 multi-photon microscope (Zeiss). The

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confocal images were projected and treated with the software ImageJ. 2.16. Mice experiments: C57BL/6 mice (male, 8 weeks, Charles River) are handled in the animal facility according to the guidelines set forth by the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication 8th Edition, 2011). Mice were anaesthetized and an intracardiac injection was performed with RICK:siRNA-Cy5, 20% and 100% PEGRICK:siRNA-Cy5 as well as with siRNA-Cy5 alone (2.5 µg siRNA in 20 µl). Retro-orbital blood samples (~40 μL each) were taken at 10, 30, 60 and 180 min time points following injection using heparinized glass capillaries. Thereafter, blood was transferred to tubes containing some µl of heparin and stored at -20°C until analysis. 24 hours after siRNAs injection, mice were sedated again with the same anesthetic mixture

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ACCEPTED MANUSCRIPT and sacrificed by heart harvesting. Then organs (lungs, liver, spleen, kidneys, and brain) were harvested, rinsed in cold PBS and stored at -80°C. To determine the siRNA blood circulation, 20 μl of blood were transferred to a black 96-well plate and the sample was diluted 1.5 times in MilliQ water. To evaluate the siRNA organ accumulation, tissues were placed for some minutes in liquid nitrogen, homogenized using a Freezer Mill 6770 homogenizer (Fisher Scientifics) and lysed using 1X Passive Lysis Buffer (PLB, Promega) (250 µl for spleen and heart,

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500 µl for lung, kidney and brain, 1000 µl for liver).

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siRNA-Cy5 fluorescence was measured on a PolarStar fluorimeter (BMG) at λex = 650 nm and λem =

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670 nm. For the siRNA blood concentration, acquired values were normalized to siRNA concentration calculated from calibration curves and for siRNA organ accumulation, acquired values were normalized

More details are given in the Supporting Information.

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3. Results

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to protein content (Pierce) to get FU/mg. 3 technical replicates were used for data analysis.

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3.1. Characterization of PEGylated RICK nanoparticles.

Many studies, including ours [21,22], have highlighted the necessity of a conformational change during

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CPP interaction with cargos, proteoglycans of the extracellular matrix or phospholipids [40,41]. For that reason, we first analyzed the structural behavior of the PEGylated-RICK CPP (PEG-RICK) alone and in the

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presence of siRNA or lipids. CD profile of PEG-RICK alone had a maximum at 203 nm and a weak shoulder at 220 nm (Figure 1A), suggesting a mainly random coil conformation with few helical contributions. In

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the presence of siRNA, PEG-RICK adopted a left-handed α-helical structure (minimum at 191 nm, maxima at 210 nm and 220 nm) which was not impacted by addition of zwitterionic large unilamellar vesicles

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(LUVs) made of DOPC/SM/Chol (2:2:1). Curiously, contributions of close tryptophan/tryptophan interaction observed through the band at 228 nm for the RICK CPP alone was not observed for the PEG-RICK CPP. The missing tryptophan/tryptophan interaction was not due to a cross-reaction of the PEG moiety, because CD measurements performed with a mixture of RICK and PEG (at molar ratio) did not influence the 228 nm band (Figure 1B). To gain more insight in the tryptophan/tryptophan interaction, we performed NMR measurements with both peptides (200 µM RICK and PEG-RICK) in the presence of SDS micelles. Incorporation of peptides in SDS micelles by contrast provoked the appearance of well -defined peaks in the range of 10

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ACCEPTED MANUSCRIPT ppm to 6 ppm, corresponding to the frequency resonance range of amide and aromatic protons. Assignment of proton resonances was done using standard procedures. Despite the use of a procedure aimed at minimizing structural heterogeneity in the mixed peptide-SDS micelles, for both peptides, some residues exhibited two very close amide protons resonances, especially the segments centered around the tryptophan residues (w2, w6, w13, w17). Tryptophan aromatic protons exhibited also double resonances. w2 of PEG-RICK exhibited even three resonances for its amide proton and for some of its

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aromatic protons. For all residues, the two amide resonances were differing by around 0.05 ppm,

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showing minimal chemical environment differences. No resonance doubling was observed for aliphatic

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proton resonances and the same patterns of NOEs were found for all residues, whatever the resonances considered. Hence, we concluded that the two forms were folded into the same overall structures, and

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that the doubling of resonances might originate from a small reorientation of tryptophan side chains.

Figure 1: Characterization of PEGylated RICK nanoparticles. (A) Conformational changes of PEG-RICK (100%) in the presence of siRNA (R = 20) and in the presence of siRNA + liposomes (R = 20 / r = 10) measured by CD. (B) Evaluation of the influence of PEG moiety on RICK structure. CD spectrum of RICK (40 µM) was compared to CD spectra of RICK mixed with different

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ACCEPTED MANUSCRIPT concentration of PEG as well as to PEG-RICK (40 µM). (C): Summary of sequential and medium range NOEs found for native and PEG-RICK. Intensity of NOEs represented by the thickness of the corresponding bar. (D) Expansion of the 1H NMR spectrum of RICK and PEG-RICK peptides (323 K, in presence of 100 mM SDS) showing the differences in the H1 proton resonances of w2 and w6 for native RICK (black line) and PEG-RICK (grey line). Each H1 proton exhibit two close resonances (see text), and

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even more for w2 of PEG-RICK.

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For both RICK and PEG-RICK, the NOESY spectra exhibited a continuous pattern of NH i/NHi+1 NOEs,

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suggesting the presence of helices. The inspection of the sequential and medium range NOEs confirmed this folding for both peptides (Figure 1C), with the helix covering the 2-19 region and the 4-19 region for the native and PEGylated peptide, respectively. In addition, analysis of NOESY maps revealed the

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presence of NOEs between w2 and w6 protons for the native peptide (w6H1-w2 H1, H2 and w2 2Hw6 H1, 6H2), compatible with its folding into a helix extending to w2, that could lead to a partial

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stacking of both w2 and w6 aromatic rings. The PEGylation of the RICK peptide had a destabilizing effect on this helix, which begins only at residue 4 for PEG-RICK according to the sequential and medium NOE

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pattern. The NOEs between w2 and w6 could not be found on the NOESY maps of PEG-RICK. The loss of interaction between w2 and w6 in the PEGylated peptide could explain the difference of the proton

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chemical shifts observed for these residues (Figure 1D), since the aromatic protons of w2 and w6

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exhibited substantially less shielded resonances for PEG-RICK than for native RICK.

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3.2. Nanoparticle assembly characterization depending on the PEG percentage. Knowing that percentage of PEGylation has to be adjusted depending on the used NPs [30,42], we were interested in modifying the functionalization rate of comple xes and therefore we tested different

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proportions of PEG-RICK replacing the parent peptide, thereby yielding a CPP:siRNA complex with the desired functionalization percentage. We prepared a series of complexes, composed of 3 different components RICK, RICK-PEG and siRNA to obtain NPs with different percentage of PEG substituents per RICK peptide (5% to 100%). To assess complexation of siRNA, we looked on the CPP:siRNA complex formation at different PEG percentage by agarose shift assay (Figure 2). This assay was used to follow the complexation state in a molar ratio-dependent manner because siRNA migration into the agarose gel will be prevented by CPP interactions. Although a slight difference could be noticed with the different PEG-percentages at a molar ratio R = 10, all RICK/PEG-RICK ratios were clearly able to complex siRNA in a similar manner with optimal 11

ACCEPTED MANUSCRIPT complexation at peptide:siRNA molar ratio of R = 20 (Figure 2). This ratio, used for all experiments mentioned below, corresponds to a charge ratio of CR = 3. In conclusion, agarose gel assay clearly demonstrated that the PEGylation rate did not affect the siRNA encapsulation. To determine the PEGylated RICK:siRNA complex size and surface charge dependent on the PEG-RICK percentage, DLS measurements were carried out. Because light diffused by bigger particles is one million fold stronger than those from small ones, we decided to evaluate the particle size distribution on the

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diffused light intensity (%) as well as on number (%) (Table 1). Indeed, size distribution based on number

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(%) obtained using algorithms could give us a more accurate information about size distribution as

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previously shown for RICK:siRNA [22]. Size distribution by intensity showed that all NPs have a mean size in the range of 67 – 99 nm. However, it seemed that complexation of siRNA with 10% or 20% PEG-RICK moieties was more compacted with smaller sizes (~68 nm) compared to RICK :siRNA or 100% PEG-

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RICK:siRNA. As expected, size distribution by number suggested the presence of smaller nanoparticles in addition to those found by the intensity-based size distribution. These smaller particles had a diameter

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of ~24 nm for RICK:siRNA and the PEGylated versions (Table 1). For all conditions the polydispersity index (PdI) was around 0.3, which is in the expected range for PBNs [21,22].

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Changes in the surface charge (zeta potential, ZP) occurred from 43 mV to 19 mV with the increased percentage of PEGylation confirming the shielding property of the PEG moiety. Furthermore, all values

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concerning mean sizes, PdIs and ZPs did not change when the NPs were stored 7 days at 4°C, as previously shown for 3 days [22]. Furthermore, 100% PEG-RICK:siRNA revealed a stable NP (size

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distribution by intensity (%): ~100 nm or by number (%): ~30 nm) even if the NPs were formulated in PBS (0.5X) or in 154 mM NaCl (physiological salt concentration). This was not the case for RICK:siRNA or all of

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the other percentages of PEGylated NPs, highlighting the importance of PEG moiety grafting. In sum, between the native and the PEG-RICK peptide-based NPs, no significant differences were

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observed in mean size. This was further confirmed for 100% PEG-RICK:siRNA by environmental scanning electron microscopy (ESEM) measurements and transmission electron microscopy (TEM) revealing a mean size of ~250 nm in dry conditions (Figure S1A) and the presence of small (~30 nm) and bigger (~120 nm) nanoparticles in solution (Figure S1B). Only changes in the surface charge (0% PEG-RICK = 40 mV to 100% PEG-RICK = 19 mV) revealed the implementation of the PEG-RICK molecules in the NPs with the corresponding shielding effect of the PEG-moieties. The global positive surface charge demonstrated that in theory all formulated NPs could internalize the cell via direct translocation as shown for the parental peptide CADY [43] in contrast to the PepFect family (negative surface charge) which internalized via class A scavenger receptors (SCARA)[44].

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Table 1: Characterization of siRNA-loaded nanoparticles by DLS. Immediately measured Nanoparticles

Mean size (nm)

Measured after 7 days

PdI

ZP (mV)

Mean size (nm)

PdI

ZP (mV)

30 ± 7

0.37 ± 0.06

53 ± 9

80 ± 6

18 ± 6

0.34 ± 0.01

47 ± 1

39 ± 4

70 ± 6

27 ± 2

0.28 ± 0.04

40 ± 1

37 ± 1

72 ± 1

24 ± 8

0.27 ± 0.02

37 ± 1

0.30 ± 0.08

28 ± 2

71 ± 12

23 ± 10

0.28 ± 0.04

29 ± 1

25 ± 3

0.27 ± 0.03

22 ± 2

77 ± 16

23 ± 2

0.34 ± 0.01

24 ± 1

23 ± 5

0.35 ± 0.04

19 ± 2

17 ± 1

0.45 ± 0.07

23 ± 1

Nb (%)

0% PEG-RICK

86 ± 17

22 ± 4

0.26 ± 0.02

40 ± 3

91 ± 16

5% PEG-RICK

71 ± 2

27 ± 1

0.27 ± 0.03

43 ± 1

10% PEG-RICK

67 ± 2

20% PEG-RICK

69 ± 1

24 ± 4

0.27 ± 0.05

20 ± 3

0.29 ± 0.05

50% PEG-RICK

73 ± 10

28 ± 9

75% PEG-RICK

82 ± 11

100% PEG-RICK

99 ± 22

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I (%)

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Nb (%)

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I (%)

72 ± 14

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Footnote: Percentage of PEG-RICK is indicated. All CPP:siRNA complexes were formed at R = 20 using a siRNA concentration of 500 nM in an aqueous solution of 5% glucose for mean size acquisition and in an

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aqueous solution of 5% glucose supplemented with 5 mM NaCl for Zeta potential (ZP) assessment. Mean sizes were calculated using the intensity [I (%)] and the number [Nb (%)] distributions. All values

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represent the mean  SD of n ≥ 2 independent measurements.

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Differences in the NP formation between RICK:siRNA and the PEGylated versions were clearly shown in a leakage assay using LUVs with a complex lipid composition (DOPC/SM/Chol (2:2:1)) reflecting the

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plasma membrane. In the absence of peptides (or NPs), no leakage was observed based on the low permeability of the phospholipid vesicle membrane to the fluorophore/quencher mix (base line during

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the first 100 ms, Figure 2B and 2C). Addition of RICK or PEG-RICK (peptide alone) on the LUVs induced a significant increase in fluorescence revealing an important leakage of 60  10% and 55  6% (p = ns),

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respectively, after 15 min incubation compared to Triton (positive control of each run) (Figure 2B). In the range between 100 s and 300 s, the leakage rate seemed to be slower for PEG-RICK compared to RICK, even if end-point results turned out to be not significant. The occurrence of leakage confirmed the ability of RICK and PEG-RICK peptides to cross plasma membrane by direct cell membrane translocation as published for the parent peptide CADY [43]. Finally, as expected, the PEG moiety alone did not have an influence on the LUV leakage.

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Figure 2: Evaluation of the PEGylation rate of RICK-based NPs compared to the native version. (A) Pre-formed native and PEGylated RICK:siRNA complexes were analyzed by electrophoresis on

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agarose gel (1% wt/vol) stained with GelRed. Data represent: mean ± SD, with n = 2. (B) Comparison of the leakage properties of RICK, PEG-RICK and PEG moieties on LUVs. (C) Comparison of the leakage

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properties of RICK:siRNA, 20%, 50% and 100% PEG-RICK:siRNA on LUVs. For all measurements CPPs were used at a final concentration of 500 nM with R = 20 for the NPs. LUVs were composed of the following

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lipids: DOPC/SM/Chol (2:2:1). Peptides/NPs were injected at 100 s and the Triton (positive control) at 1,000 s for each assay (= 100% leakage). (n ≥ 2 for each condition). ns = non-significant and * for p < 0.05 after one-way ANOVA with Tukey’s post-test.

The leakage of the RICK:siRNA NP was two-fold weaker (28  3%) than the one observed for the RICK peptide alone. This was in agreement with the fact that within the RICK:siRNA complex one part of the peptide interacted with the siRNA and the other part with the lipids. NPs formed by 20% PEG-RICK:siRNA showed a slightly higher leakage which was not significantly different compared to RICK:siRNA (37  11%,

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3.3. Cellular internalization of the PEGylated nanoparticles.

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To determine the cellular uptake of the NPs, U87 human glioblastoma cells were incubated for 1 h with

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siRNA-Cy5 loaded PBNs with different PEGylation percentages. Subsequent analysis by flow cytometry of

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the living cells revealed a week residual median fluorescence of the siRNA-Cy5 alone (750  30 a.u.) corresponding to the background (600  40 a.u.) (Figure 3A, see also Figure S2 for histogram visualization). Using NPs made of RICK alone, or 10%, 20% and 50% PEG-RICK, we obtained a significant

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increase of the fluorescence signal (~3.500  350 a.u.) demonstrating the efficient internalization of siRNA-Cy5. Using NPs composed of 75% or 100% PEG-RICK, we revealed a reduction in median

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fluorescence intensity (20% and 60%, respectively). This correlated with a reduced leakage property of 100% PEG-RICK:siRNA as observed in Figure 2B.

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Next, confocal microscopy measurements were performed to define the subcellular localization of 20%, 50% and 100% PEG-RICK NPs in living U87 cells (Figure 3B). A dotted cytoplasmic pattern of the

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internalized siRNA-Cy5 could be observed for PEG-RICK NPs. However, the fluorescence intensity lowered with the increased percentage of PEGylation as shown in the graphical representation of

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intracellular siRNA-Cy5 fluorescence quantification according to PEG ratio (Figure S3). Here again, the NPs bearing 20% PEGylation had an apparent close internalization level as the non-PEGylated one.

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Finally, we compared quantitatively the internalization of RICK:siRNA-Cy3b and 20% PEG-RICK:siRNACy3b by spinning disk confocal microscopy. Over 2 h, images were acquired every 2 min and the

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fluorescence intensity inside one cell was measured with ImageJ software. Fluorescent values were then normalized compared to the maximum value of fluorescence for each cell and plotted on a kinetic graph (Figure 3C). Based on the graphical representation, we clearly confirmed that the siRNA internalization occurred in the first 10 min (up to 40%). Between the 12th and the 20th minute of the internalization, we observed a reduction in the siRNA-Cy3b signal and thereafter again a constant increase in fluorescence signal till the end of the measurement. We hypothesized that the decrease in fluorescence intensity could be due to the membrane repair response (MRR) activation by the direct membrane transduction of the NPs as reported by Palm-Apergi et al. [45]. Finally, addition experiments with longer incubation (>24

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shown).

Figure 3: Internalization of PEGylated RICK nanoparticles in living U87 glioma cells.

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(A) FACS analysis of siRNA-Cy5 loaded RICK-based NPs without or with different percentages of PEGylation. Cells were incubated with the NPs (siRNA-Cy5 = 20 nM, R = 20) for 1 h at 37°C. After a

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trypsinization step to remove membrane-bound NPs, the median intracellular fluorescence was measured by FACS. (B) Example of confocal microscopy images showing the internalization of 20%, 50%

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and 100% PEG-RICK:siRNA-Cy5 internalization in living U87 cell line. The white bar represent 40 µm. (C) Comparison of RICK:siRNA-Cy3b and 20% PEG-RICK-siRNA-Cy3b internalization kinetic by spinning disk

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(siRNA-Cy3b = 20 nM, R = 20) with image recorded every 2 min for 120 min. Thereafter, the percentage of mean grey values were plotted against the time (mean  SD; 2 - 5 individual counted cells from 3 - 5 independent experiments).

3.4. Knock-down efficiency of PEGylated nanoparticles. The knock-down efficiency of the different NPs was performed on U87 cell line stably transfected for constitutive expression of FLuc/NLuc reporter genes (U87-FRT-CMV/Fluc-CMV/iRFP-IRES-NLuc). Peptide:siRNA NPs have been formulated in 5% glucose, R = 20, with a siRNA targeting FLuc (siFLuc) using three concentrations (5 nM, 10 nM and 20 nM). To evaluate the influence of the PEGylation on the FLuc

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ACCEPTED MANUSCRIPT knock-down efficiency, different percentages of PEGylated NPs (10%, 20%, 50%, 75% and 100% PEG) were formulated and used on U87 cells (Figure 4A). In addition, a scrambled siRNA version (siSCR) was used as negative control. FLuc activities were normalized to the NLuc levels and to non-treated cells and then plotted as Relative Luc Activity (%). As shown in Figure 4A, the knock-down efficiency of 10% and 20% PEG-RICK NPs were not significantly different to the RICK:siRNA complex. However, a decrease of the FLuc expression when using NPs increasing PEG percentages was observed, until reaching a

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luciferase expression at the level of the non-treated cells with 100% PEG-RICK:siRNA. Combined with our

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confocal microscopy results (Figure 3C), the reduction of biological activity clearly correlated with the

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lower internalization of the 100% PEG-RICK NPs. We also tested the efficiency of siRNA transfection with a commercial transfection reagent (RNAiMAX), but the cytotoxic effect of their formulation resulted in false-positive knock-down effects in the luciferase assay (data not shown).

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In parallel to the luciferase assay, we quantified on the same samples LDH release as an indication of cytotoxicity. For all conditions, the LDH values were close to those measured for untreated ce lls (0%) and

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not higher than 10% (except for the negative control Triton X-100). These results clearly demonstrate that we can obtain with 20 nM siRNA more than 80% knock-down efficiency with NP formulated with

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RICK or 5% to 20% PEG-RICK without any cytotoxicity.

Gene silencing was also undertaken on the endogenous protein cyclin-dependent kinase 4 (CDK4),

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known to be overexpressed in glioblastoma [46]. After incubation of the cells with the different NPs, the resulting protein expression (blot signal intensities) was first normalized to the loading control vinculin

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and thereafter to the non-treated condition (N.T. cells) (Figure 4B and 4C). First of all, no effect using RICK:siSCR (first lane) was seen. As expected, we clearly observed a specific ~90% knock-down of CDK4

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for 10% and 20% PEG-RICK:siCDK4 NPs as observed with RICK:siCDK4 (p = ns for the three conditions). Again, the reduction of CDK4 expression level was correlated with the increase of PEG-content (50%,

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75% and 100% PEG-RICK NPs) as observed in the luciferase assay (Figure 4A). In the case of 50% PEG-RICK:siRNA, the knock down efficiency was reduced to ca. 50% in both cases (FLuc and CDK4) even if the internalization property seemed to be unaffected (same transfection yield as RICK:siRNA in Figure 3A). At this point, we could only hypothesize that siRNA releasing within the cell could be affected by the PEGylation ratio.

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Figure 4: Knock down activity of NPs in U87 glioma cells. (A) Relative Luc activity (%FLuc/%NLuc) and relative toxicity (LDH quantification) after transfection with RICK-based complexes made of different percentages of PEGylation in U87-FLuc-NLuc cells (siRNA as shown in the graph, R = 20 in 5% glucose). (B) Example of the Western blot analysis of CDK4 gene silencing using RICK-based complexes made of

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ACCEPTED MANUSCRIPT different percentages of PEGylation (20 nM siRNA, R = 20 in 5% glucose) in absence of serum. (C) Graphical representation of signal intensities obtained by Western blot analysis as exemplified in (B). (D) Example of the Western blot analysis of CDK4 gene silencing using RICK-based complexes made of different percentages of PEGylation (20 nM siRNA, R = 20 in 5% glucose) in the presence of 10% serum. (E) Graphical representation of signal intensities obtained by Western blot analysis as exemplified in (A). All graphs represent the mean ± SD of 2-3 independent experiments in triplicates. ns = non-significant

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after one-way ANOVA with Tukey post-test. Abbreviations: siSCR = scrambled siRNA, N.T. = non-treated

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cells, siFLuc = siRNA anti-firefly luciferase, siCDK4 = siRNA anti-cyclin dependent kinase 4 and Vin. =

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Vinculin.

3.5. Influence of serum on RICK and PEG-RICK nanoparticle stability.

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One requirement for gene delivery vectors is to protect efficiently their cargo from degradative enzymes such as nucleases (or proteases in the case of peptides). Therefore, we extracted siRNAs from serum-

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treated NPs and evaluated the siRNA stability. In details, we incubated siRNA-Cy3 loaded RICK NPs without PEG, or with 20% and 100% PEG-RICK in the presence of serum. After a 24 h incubation at 37°C,

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siRNAs were extracted and quantified by Cy3 detection after acrylamide gel migration. As expected, we observed a 300-fold increase in the stability of siRNA-Cy3 in the RICK:siRNA construct over the naked

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siRNA, thus confirming the protective effect of the retro-inverso RICK peptide (Figure S4). Similar siRNA protective effect was observed for 20% PEG-RICK NPs (200-fold increase). Surprisingly, fully PEGylated

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NPs appeared more sensitive to serum destabilization since only a 70-fold signal increase was observed. Similar results were obtained by gel shift experiments, when increasing heparin concentrations (up to 10

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equivalents of negative charges related to the positive charges of the RICK peptide) were added to RICK, 20% and 100% PEG-RICK NPs complexed to siRNA (data not shown).

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To further evaluate the effect of serum on the NP biological activity, CDK4 knock-down experiments descripted above were repeated in the presence of 10% serum during the NP incubation of the cells (Figure 4D and 4E). Compared to the CDK4 knock-down efficiency in the absence of serum, we clearly revealed a lower reduction of CDK4 expression (67%, 55% and 44% for RICK:siRNA, 10% and 20% PEGRICK:siRNA, respectively) in the presence of serum. However, the knock-down efficiencies of RICK, 10% or 20% PEG-RICK NPs were not significantly different (p = ns for the three conditions). Higher content of PEG (50%, 75% and 100%) on NPs did not stabilize the complexes despite the more neutral surface charge (see Table 1) expected to prevent unspecific interactions with serum proteins.

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3.6. In vivo evaluation of the non-PEGylated and PEGylated nanoparticles. Next, we tested the behavior of PEGylated complexes in in vivo conditions (zebrafish and mouse model). For this purpose, we compared fluorescence-labeled RICK:siRNA-Cy3 only with 20% PEG-RICK:siRNA-Cy3

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(conditions showing a knock-down efficiency in serum). Furthermore, we include d 100% PEG-RICK as a

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negative control (no internalization, no knock-down). NPs were injected in the cell of zebrafish eggs at 1-

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to 2-cells stage. 24 h post-fertilization, fluorescence signals of the siRNA-Cy3 were followed by confocal microscopy (Figure 5A). Independently of the PEGylation rate, most of the siRNA signals were observed in the egg yolk. Highest siRNA signals were revealed in the body of zebrafish larvae after internalization

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via the RICK-based NPs. Looking on the conditions using PEGylated PBNs, it seemed that the siRNA signal was reduced for 20% PEG-RICK and more or less completely lost for 100% PEG-RICK. Figure 5B

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summarized the distribution of the fluorescence intensities estimated in all measured zebrafish larvae ( ≥ 50): we observed a high fluorescence intensity (+++ population: for 9 of the 15 counted larvae = 60%) for

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RICK:siRNA-Cy3, an intermediate (+ population: 12 of the 21 counted larvae = 57%) for 20% PEGRICK:siRNA-Cy3 and no fluorescence (- population: 7 of the 12 counted larvae = 58%) with 100% PEG-

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RICK:siRNA-Cy3.

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Figure 5: Evaluation of the NPs in vivo. (A). Distribution of RICK:siRNA-Cy3, 20% and 100% PEG-

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RICK:siRNA-Cy3 in zebrafish 24 h post fertilization (siRNA = 20 nM, NP with R = 20). Images were represented in false colors with siRNA-Cy3 in green. Arrows indicate the fluorescence in the zebrafish

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body. (B) Determination of the fluorescence intensity in the body of the zebra larvae: +++ = high fluorescence, + = medium fluorescence, - = less or without fluorescence (>50 counted zebrafish from 3 independent experiments). (C) Circulation kinetics of siRNA alone, RICK:siRNA, 20% and 100 % PEG-

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RICK:siRNA (with Cy5-labelled siRNA). (D) Organ biodistribution of intravenously administered siRNA alone, RICK:siRNA, 20% and 100 % PEG-RICK:siRNA assessed 24 h post NP injection (with Cy5-labelled siRNA). Statistical significance (for C and D) determined by two-way ANOVA with Tukey post-test with ns = non-significant, * for p < 0.05 ** for p < 0.01 and **** for p < 0.0001 (n = 3 per condition).

To assess the pharmacokinetic effect of PEGylation, siRNA-Cy5 alone or complexed to RICK, 20% or 100% PEG-RICK were administrated to mice via an intracardiac injection. At several time points post injection, siRNA-Cy5 levels were measured in collected blood samples. As can be seen in Figure 5C, naked siRNA was fully cleared at 180 min post-injection, while the RICK:siRNA and 20% PEG-RICK:siRNA

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ACCEPTED MANUSCRIPT persisted to 24% and 34%, respectively. Moreover, 100% PEGylation decreased the rate of elimination showing a 1.8 times (p = *) and a 3.5 times higher (p = **) siRNA blood concentration compared with complexes without PEG (p interaction < 0.003, 2-way ANOVA with Tukey post-test). This suggested that NP functionalization with PEG moieties inhibited their contact with bl ood serum elements allowing them to circulate longer in the blood circulation. We then sought to determine whether the different primary half-life of the PEGylated NPs might be

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linked with differential capture in various organs. To quantify the effect of P EG incorporation on organ

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accumulation, we performed a biodistribution study in mice. At 24 h after intravenous administration of

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the PEGylated PNs, we measured significant reductions in off-target hepatic (1.6-fold for 20% PEG-RICK and 1.8-fold for 100% PEG-RICK), and renal (5.0-fold and 5.3-fold, respectively) accumulation of siRNA cargo compared to non-PEGylated NPs (Figure 5D). Meanwhile, siRNA accumulation remained

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unchanged in the others organs such as spleen, heart, lung and brain. Overall, 100% PEG-RICK NPs maintained higher blood concentrations during the first 3 hours after administration, however 20% PEG-

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RICK seemed to be sufficient to greatly decrease distribution into off-target organs and increase the

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presence of siRNA inside body.

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4. Discussion

As a therapeutic approach, gene silencing using siRNA provides a solution to the major drawbacks of

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traditional pharmaceutical drugs: ‘non-druggable’ targets. All protein targets can be inhibited by siRNA, which can be rapidly and rationally screened, designed and synthesized. However, the clinical

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advancement of this strategy has been difficult to reach, in particular because of the low nuclease resistance, the rapid elimination and the poor cellular internalization of naked siRNA [47]. Such

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limitations emphasize the need for an efficient and safe drug delivery system to modulate the siRNA pharmacokinetics and biodistribution. In this context, peptide-based NPs fulfilled some of the requisites necessary for an intravenous administration of siRNA such as, high internalization efficiency, protection against nucleases and size around 100 nm. Nevertheless, those features are not efficient enough to promote an effective systemic siRNA delivery to sites other than the liver or the kidneys, highlighting the importance of strategies typically using poly(ethylene glycol) (PEG) chains of molecular weight 2,000 or 5,000 g/mol [27,28].

4.1. Important structural features relevant for the formulation of PEGylated NPs.

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ACCEPTED MANUSCRIPT To face the limitations of in vivo administrations and the risks of a too fast clearance, most DDSs have evolved with additional motifs, such as targeting sequences and PEGylation. In this context, PEGylation was proposed as an alternative approach to increase life time in the circulating system and to improve bioavailability of NPs in vivo [28]. In particular, recent works reported the successful association of PEG to PBN [30,48]. Based on these observations and on our previous work with the retro-inverso peptide RICK, we investigated new PBN formulations including different ratios of RICK and its PEGylated form. As

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RICK was able to keep the main structural properties of it L-parent peptide [22], PEG-RICK was also

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studied for its conformational state, ability to interact with siRNA and self-assembling into PBN. RICK is a

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secondary amphipathic peptide and this specific innate feature makes its able to undergo conformational versatility favoring interactions and self-assembly with siRNA [17]. The insertion of a PEG motif in N-terminus of RICK could hence modify, improve or abolish most of these skills.

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Compared to RICK, PEG-RICK was mainly unfolded with few helical contributions and without any significant w/w interaction band at 228 nm (Figure 1A). As PEG did not induced any modifications in RICK

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CD profile (Figure 1B), the absence of the band at 228 nm suggested that the conjugation of PEG to the N-terminus of RICK induced a reorganization of w/w interactions. In the presence of siRNA (R = 20) and

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with lipids (R = 20/r = 10), PEG-RICK clearly adopted a left-handed α-helix (Figure 1A), suggesting a structural behavior similar to RICK [22]. This structure was confirmed by NMR analyses that also pointed

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out a α-helical conformation for both RICK and PEG-RICK peptides in SDS micelles (Figure 1C and 1D). However, the PEG induced a slight decrease of helicity in the RICK N-terminal region, characterized by

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the loss of w2 and w6 interactions. This last observation could also explain the disappearance of the

[22].

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band at 228 nm in the CD spectrum of the PEGylated peptide as compared to the CD spectrum of RICK

Structural similarities between RICK and PEG-RICK in the presence of siRNA suggested equivalent

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interactions with siRNA. In this context the ability of RICK and PEG-RICK to form mixed NPs was analyzed and NPs with different PEGylation rates were formulated. Indeed, as the PEG motif could interfere in the NP formulation, the maintenance of PBN formation should be confirmed. In this context, analysis of several NP formulations with various PEGylation rates revealed a similar complexation of siRNA with optimal CPP:siNA molar ratio of R = 20 (Figure 2A). All analyzed NPs showed mean size of 67 - 99 nm measured by intensity distribution and of 20 – 27 nm measured by number distribution (Table 1). In addition, although no significant variation of NP size was observed, a significant decrease of surface charge was noticed for increasing amounts of PEGylated peptide (40 mV for RICK:siRNA to 10 mV for 100% PEG-RICK:siRNA) (Table 1). This latter observation, which was the only significant difference in the

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reported previously for similar PEGylated peptides [30,48]. Moreover this indicated that grafting of any

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other ligand (e.g. targeting motifs) at the N-terminus of RICK should keep the ability of RICK to fold into

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the secondary amphipathic helix through interactions with siRNA in order to self-assemble into NPs with similar charges and sizes. In the particular case of PEGylation, colloidal characterization of NPs only

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revealed a decrease of surface charge - a decrease expected to improve stability and in vivo half-life [49].

4.2. What are the relevant features for siRNA-loaded PEGylated NPs activity in cellulo.

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The “PEG dilemma” regarding the use of PEG in drug delivery systems described the balance of the NP shielding with PEG which improved generally the stability in vivo but reduced the cellular internalization

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[50]. Therefore, it was crucial to adjust and to monitor the percentage of PEGylation required to improve the stability of the NPs without reducing their drug delivery property. Different NPs with PEGylation

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rates ranging from 0% to 100% were prepared by mixing siRNA with variable proportions of RICK and PEG-RICK. First of all, we evaluated the percentage of PEGylation which provided knock-down efficiency

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equivalent to the one observed for native RICK NPs. Therefore, a subset of 21 different NPs combining different PEG incorporations and 3 different siRNA concentrations were screened (Figure 4A). We clearly

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revealed that 100% PEG composition in the NPs completely abolish luciferase firefly knock-down efficiency in transfected human U87 glioma cells. 50% and 75% PEG substitution provided an

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intermediate effect, whereas 5% to 25% PEGylation rates induced a knock-down similar to those obtained with the native RICK NPs. This finding correlated with the internalization profile (Figure 3A and B) as well as the leakage properties (Figure 2C) of PEGylated NPs. Higher PEG percent composition of the NPs induced a decrease of the zeta potential (Table 1), thus probably altering the interaction with proteoglycans or negatively charged membrane lipids and subsequently reduced siRNA-dependent gene silencing [43,51]. These electrostatic interactions are the first step occurring during the direct membrane translocation procedure of the NPs. Furthermore, an increase of the local NPs concentration at the plasma membrane can induce transient pore formation which activates the MRR. This repair process occurring within few seconds could be the reason for fluorescence decrease observe d between the 12th

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ACCEPTED MANUSCRIPT and the 20th minute (Figure 3C). Indications for the direct translocation are given by the fact that RICKbased NPs have the same biophysical properties (shape and surface charge) as the parental peptides CADY-K [21] or CADY [51] which were able to directly translocate themselves as well as their cargoes through the plasma membrane. As previously reported, no co-localization with endocytosis markers such as cholera toxin subunit B (CtB), transferrin, lysotracker, caveolin or Rab5 were observed [43]. Furthermore, leakage assays performed in this study also demonstrated that RICK as well as 20% PEG-

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RICK NPs could destabilize model membranes with a lipid composition reflecting the plasma membrane

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(Figure 2B and C).

Figure 6: Summary of the results obtained with the siRNA-loaded NPs at different PEGylation ratio in

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vitro and in cellulo.

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We observed a similar knock-down profile following a PEG-dependent manner for the inhibition of the endogenous protein CDK4 in U87 cells (Figure 4B) as obtained for the luciferase gene inhibition. A

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summary of the obtained results are given in the schematic representation in Figure 6. Discrepancies between good internalization and low knock-down efficiency of 50% PEG-RICK:siRNA could be due to low siRNA release within this cell affected by the PEGylation ratio. Indeed, Miteva et al. have shown the lack of cellular knock-down activity due to higher intracellular siRNA unpacking as visualized by confocal microscopy and quantitative FRET and studies [52]. Finally, compared to RICK:siRNA NPs, formulations with PEGylated peptides seem to be slightly less stable according to siRNA protection ability (in vitro incubation in serum) (Figure S3) and transfection efficacy in the presence of serum (Figure 4D and 4E). Although these results suggest weaker complex stability in degradative environment, it does not exclude the possibility that slightly decreased stability in

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4.3. Fine-tuning of the PEGylation rate is important for the in vivo application of the NPs. PEGylation enhances the therapeutic efficacy of the drugs in vivo by bringing in several advantageous

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modifications over the non-PEGylated products such as increasing serum half-life and decreasing hepato-

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renal elimination of the conjugate. Few factors such as protection from absorption by reticuloendothelial

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cells, cleavage by proteolytic enzymes and reduced immunogenicity upon the formation of a protective hydrophilic shield are the key benefits of PEG molecule grafting thus providing an improved pharmacokinetic (PK) profile of our PBNs.

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To evaluate the PK profile, we compared the native NPs with 20% and 100% PEG-RICK NPs in zebrafish and in mice (Figure 5). Confocal imaging of zebra embryos (24 h post-fertilization) which were

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injected at the one-cell stage egg with the three NPs, revealed a similar siRNA distribution in the egg yolk (Figure 5A). However, we could clearly detect a high siRNA related fluorescence signal in the larva body

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in 60% of the RICK:siRNA treated zebrafishs, an intermediate one in 57% of the 20% PEG-RICK:siRNA condition, and no fluorescence in 58% of the zebrafishs injected with 100% PEG-RICK:siRNA (Figure 5B).

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At this stage, the zebrafish model should be carefully considered because we observed here the biodistribution through cell division and not through the blood circulation.

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Therefore, we decided to compare the intracardiac injected native RICK:siRNA NPs with the 20% and 100% PEG-RICK NPs to mice by collecting blood samples (10 min, 30 min, 60 min and 180 min) and

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organs (24 h) to quantify the fluorescently labeled siRNA (Figure 5C). As expected, encapsulation of the siRNA results in a longer blood circulation (2-fold increase with RICK at 30 min) and 100% PEG-RICK NPs

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circulated in the blood (~3 nM) over the whole 3 h period. More importantly, the addition of only 20% PEGylation reduced the accumulation in organs implicated in drug elimination such as the liver and the kidneys in the same way as 100% PEG-RICK (p = ns between both in Figure 5D). This finding was in agreement with recent data showing that 20% PEGylated NickFect55/pDNA particles exhibited the highest in vivo activity with a reduced lung accumulation [48]. In our case, lung accumulation was not observed for all applied conditions. Taking together, increase in blood circulation and reduction in organ accumulation were also observed for PEGylated PepFect [30] NPs or other systems such as liposomes [53–55].

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5. Conclusions With the rapid progression of biotechnology, non-viral gene delivery strategies will attract more attention and achieve long-term development in cancer gene therapy. In this context, peptide-based NPs are innovative drug delivery systems with multiple applications [7,56,57]. Here, we showed that PEGylation did not alter the NP formation (only decrease in zeta potential) compared to the native form.

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At low PEG percentage (≤ 20%) NPs retained their capacity of cellular internalization and activity. More

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importantly no accumulation in the eliminating organs such as the liver and the kidneys were observed.

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Overall, this approach constitutes a modular DDS that allows a wide range of formulations and which may be extended to other CPPs or to other types of functional moieties due to the simple formulation and functionalization of CPP:siRNA complexes, even if fine-tuning of the composition is essential to

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develop safe and biocompatible delivery systems for the clinical application of RNAi -based cancer

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therapeutics.

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Acknowledgments: We thank Sylvain De Rossi (Montpellier RIO imaging microscopy platform) and Nicolas Cubedo (Zebrafish platform) for technical advices. This work was supported by the Fondation de

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la Recherche Médicale (DBS 20140930769), the Labex TRAIL TARGLIN (ANR-10-LABX-57) and the Centre National de la Recherche Scientifique. This work used (HD) the platform of the IBISA GIS of Montpellier

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(support from the “Agence nationale de la recherche” of France ANR-10-INSB-05-2 and ANR-10-INSB-05-

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0 “French Infrastructure for Integrated Structural Biology—FRISBI“).

Supporting Information available: Additional information concerning the materials and methods

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sections; Validation of the cellular model for the activity evaluation of the nanoparticles; Figure S1 – S3.

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