Delivery of siRNA targeting tumor metabolism using non-covalent PEGylated chitosan nanoparticles: Identification of an optimal combination of ligand structure, linker and grafting method

    Delivery of siRNA targeting tumor metabolism using non-covalent PEGylated chitosan nanoparticles: Identification of an optimal combination of ligand structure, linker and grafting method Cyril Corbet, H´elo¨ıse Ragelle, Vincent Pourcelle, K´evin Vanvarenberg, Jacqueline Marchand-Brynaert, V´eronique Pr´eat, Olivier Feron PII: DOI: Reference:

S0168-3659(15)30272-8 doi: 10.1016/j.jconrel.2015.12.020 COREL 8019

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

23 September 2015 3 December 2015 12 December 2015

Please cite this article as: Cyril Corbet, H´elo¨ıse Ragelle, Vincent Pourcelle, K´evin Vanvarenberg, Jacqueline Marchand-Brynaert, V´eronique Pr´eat, Olivier Feron, Delivery of siRNA targeting tumor metabolism using non-covalent PEGylated chitosan nanoparticles: Identification of an optimal combination of ligand structure, linker and grafting method, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.12.020

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ACCEPTED MANUSCRIPT Delivery of siRNA targeting tumor metabolism using non-covalent PEGylated chitosan nanoparticles: identification of an optimal combination of ligand

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structure, linker and grafting method.

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Cyril Corbet 1,¥, Héloïse Ragelle 2,¥, #, Vincent Pourcelle 3,¥, Kévin Vanvarenberg 2,

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Jacqueline Marchand-Brynaert 3, Véronique Préat 2 and Olivier Feron 1,§ 1

Université catholique de Louvain, Pole of Pharmacology and Therapeutics, Institut

de Recherche Expérimentale et Clinique (IREC), 53 Avenue Mounier B1.53.09, B-

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1200 Brussels, Belgium 2

Université catholique de Louvain, Advanced Drug Delivery and Biomaterials,

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Louvain Drug Research Institute (LDRI), 73 Avenue Mounier B1.73.12, B-1200 Brussels, Belgium 3

Université catholique de Louvain, Molecules, Solids and Reactivity, Institute of

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Condensed Matter and Nanosciences (IMCN), UCL, 1 Place Louis Pasteur, L4.01.03,

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B-1348 Louvain-la-Neuve, Belgium.

These authors have equally contributed to this work.

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OF and VP have equally supervised this work.

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Present address: David H. Koch Institute for Integrative Cancer Research,

Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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Correspondence to: O. Feron, Pole of Pharmacology and Therapeutics (FATH),

Institut de Recherche Expérimentale et Clinique (IREC), UCLouvain, 53 Avenue E. Mounier B1.53.09, B-1200 Brussels, Belgium. Phone: +32-2-7645264; Fax: +32-27645269; E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT PEGylated chitosan-based nanoparticles offer attractive platforms for siRNA

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cocktails delivery into tumors. Still, therapeutic efficacy requires to select a rational

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combination of siRNAs and an efficient tumor delivery after systemic administration.

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Here, we showed that non-covalent PEGylation of chitosan-based nanoparticles loaded with siRNA targeting two key transporters of energy fuels for cancer cells, namely the lactate transporter MCT1 and the glutamine transporter ASCT2, could

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lead to significant antitumor effects. As a ligand, we tested variations of the

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prototypical RGD peptidomimetic (RGDp). A higher siRNA delivery was obtained with naphthyridine-containing RGDp randomly conjugated on the PEG chain by clip

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photochemistry and the use of a lipophilic linker than when using traditional chain-

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end grafting and RGDp with a hydrophilic linker. The antiproliferative effects resulting from ASCT2 and MCT1 silencing were validated separately in vitro in conditions

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mimicking specific metabolic profiles of cancer cells and in vivo upon concomitant delivery. The combination of those siRNA and the selected components of targeted

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RGDp nanoparticles led to a dramatic tumor growth inhibition upon peri-tumoral but also systemic administration in mice. Altogether these data emphasize the convenience of using non-covalent PEGylated chitosan particles to produce sheddable stealth protection compatible with an efficient siRNA delivery in tumors.

Keywords: siRNA, chitosan, RGD peptidomimetic, tumor metabolism, lactate, glutamine

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ACCEPTED MANUSCRIPT INTRODUCTION Inhibition of tumor growth by exploiting RNA interference (RNAi)-mediated

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gene silencing is a promising strategy for cancer therapy [1, 2]. One of the key

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challenges for the therapeutic use of small interfering RNA (siRNA) is to overcome

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the rapid degradation by plasma nucleases and the poor intracellular uptake by the target cells [3-5]. Chitosan-based nanoparticles (NP) have been described to offer such platforms for targeted siRNA delivery into tumors [6-10]. Chitosan is a natural

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polymer that is non-toxic, non-immunogenic, biodegradable and biocompatible [11,

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12]. Because of its cationic nature, chitosan can complex siRNA into NP and can increase the efficacy of cell binding through electrostatic interactions [13]. To achieve

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a significant antitumoral effect, key remaining issues in the field of chitosan-based

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siRNA-loaded NP are actually to optimize the NP/payload components with a specific focus on the targeting ligands, the PEGylation and a rational cocktail of siRNA.

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Although numerous studies describe the functionalization of NP with targeting moieties, the evaluation of the ligand characteristics and its repartition on the NP

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surface are generally underappreciated [14, 15]. Arg-Gly-Asp (RGD) tripeptide motif and peptidomimetics (RGDp) are known to preferentially bind to α vβ3 integrin, which is overexpressed both on endothelial cells lining tumor blood vessels and on some tumor cell types. RGDp thus enable a preferential tumor targeting following systemic administration of NP decorated with these ligands [16]. While RGDp are more stable than the RGD peptide because of the absence of easily cleavable peptidic link in their structure [17], differences between RGDp structures for the capacity to deliver siRNA are poorly understood. There is also room for optimization in the nature of the linker attaching RGDp to the NP and when coupled to PEG, to the localization of the targeting moiety along the chain.

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ACCEPTED MANUSCRIPT PEGylation drawbacks are also rarely considered. PEGylated NP are often referred as stealth NP because of their capacity to partly escape the surveillance of the

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reticuloendothelial system. However, although it prolongs the circulation half-life of

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NP, PEGylation may seriously hinder the NP uptake by tumor cells once they

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extravasate. PEGylation also prolongs NP payload exposure to healthy tissues thereby causing possible side effects. The ideal PEG coating should therefore come off from the NP in a time window that facilitates the drug release and avoids systemic

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over-exposure [18]. Since a balance between PEGylation and de-PEGylation is

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needed to produce a NP formulation that is potent and safe, non-covalent PEGylation may represent an option to offer a more easily sheddable coating. Physical adsorption (i.e., via hydrogen bonding or electrostatic cohesive forces) may thus

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represent an attractive alternative to covalent PEGylation of NP. Finally, the therapeutic target(s) of the siRNA included in the NP must also be

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optimized. Although the use of cocktails of therapeutic siRNA loaded in the same NP represents an obvious advantage, the tumor adaptation to the effects of siRNA may

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lead to resistance phenomena like for any drug treatment. It may therefore be relevant to choose siRNA that target genes that code for proteins involved in mutually compensatory mechanisms. Tumor metabolism is one example of the potential of tumor plasticity that may lead to treatment resistance. For instance, glutamine and lactate represent energy fuels that directly participate to the production of biosynthetic intermediates (while glucose is more involved in ATP generation). Concomitant blockade of both lactate and glutamine uptake in tumor cells may thus represent one therapeutic strategy that exploits different siRNA to stimulate synergism and reduce escape pathways for the tumor.

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ACCEPTED MANUSCRIPT In the present study, the therapeutic efficacy of siRNA targeting the lactate (MCT1) and the glutamine (ASCT2) transporters was investigated both in vitro and in

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vivo by testing non-covalent PEGylated chitosan-based NP differentially

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functionalized with RGDp. The RGDp differed by (i) their basic motif (amino-pyridine

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versus naphthyridine structure), (ii) the nature of the linker to the PEG chain (hydrophilic versus lipophilic) and (iii) the repartition of the RGDp on the PEG chain (chain-end versus randomly distributed along the chain) [19, 20]. At the end, this

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work led us to prove that targeting tumor cell metabolism with a mixture of siRNA

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directed against critical fuel transporters constituted an attractive therapeutic strategy, the in vivo feasibility of which requiring however to fine-tune the delivery

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system.

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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Material

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RGD peptidomimetics were synthesized as previously published [20-22]. O-

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succinimidyl-4-(1-azi-2,2,2-trifluoroethyl)benzoate (NHS-TPD clip) was prepared as

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previously published [23]. α-Methoxy--hydroxyl-poly(ethylene glycol) 5-kDa (PEG) was purchased from INEOS OXIDE (Belgium). Gly-Arg-Gly-Asp-Ser (GRGDS named

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RGD) peptide was purchased from PolyPeptide (France).

Chitosan (CS) (Kitozyme, Herstal, Belgium) had a molecular weight of 90 kDa, based

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on intrinsic viscosity measurements, and a deacetylation degree of 81.3%. Branched poly(ethylene imine) (PEI, 25 kDa) and tripolyphosphate (TPP) were purchased from

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Sigma-Aldrich (Diegem, Belgium). The green fluorescent protein (EGFP) siRNA

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(target sequence: 5’-ACCCUGAAGUUCAUCUGCACC-3’) and the corresponding control (CTRL) siRNA (target sequence: 5’-GAGCUCAUCGUUGCACUAUTT-3’)

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were supplied by Eurogentec (Seraing, Belgium). The SLC1A5 or ASCT2 siRNAs (target sequences: 5’-CCGGUCCUGUACCGUCCUCAA-3’ and 5’-

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UCGCUCAUACUCUACCACCUA-3’), SLC16A1 or MCT1 siRNA (target sequence: 5’-AAGAGGCUGACUUUUCCAAAU-3’) and control siRNA (5’CAGGGUAUCGACGAUUACAAA-3’) were purchased from Eurofins MWG Operon (Ebersberg, Germany). siRNA labelled with Alexa647 and Cy5.5 were purchased from Qiagen (Venlo, the Netherlands) or Eurogentec, respectively. Preparation of chitosan and PEG-RGDp assembly. We chose here to work with non-covalent association of CS with the different PEGRGDp. For the PEG-RGDp conjugates, two modalities were considered leading either to the random distribution of RGDp along the PEG chains or to the chain-end coupling. The “clip photochemistry”, which is based on the photoreaction of 6

ACCEPTED MANUSCRIPT trifluoromethylphenyldiazirine clip (NHS-TDP clip) [23-25], was used to produce PEGRGDp conjugates where the targeting moieties are randomly dispersed along the

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polymer chain (and proven to be accessible for integrin interaction) [22-25]. To

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prepare PEGs conjugated with RGDp at their distal ends, a 5-kDa PEG chain

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terminated with a diethoxy acetal function was produced using a methodology previously reported [26]. The CS/PEG assemblies were characterized by Gel Permeation Chromatography (GPC), 19F qNMR and Diffusion Ordered SpectroscopY

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(DOSY) NMR. GPC was performed on a polycationic column with aqueous 0.1 M

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trifluoroacetic acid (pH = 1) as mobile phase. DOSY NMR is a pseudo 2D NMR experiment that gives a formal separation of compounds depending on their diffusion coefficient in the solvent. Detailed procedures for the RGDp grafting methods and

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GPC analysis are described in the Supplementary Information. Formulation of the nanoparticles

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Unless otherwise stated, the solutions were prepared using RNase-free water (Life Technologies, Gent, Belgium) and filtered through 0.22 µm filters (VWR, Heverlee,

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Belgium). Throughout all experiments, RNase-free materials and conditions were carefully applied. The nanoparticles were prepared by using the ionic gelation method [8, 9, 27]. In brief, the negatively charged components, i.e. the siRNA (50 or 100 nM), and TPP (1 mg/ml) were added to the positively charged components, i.e. PEG/CS (1 mg/ml in 0.2 M sodium acetate buffer, pH 5.7) and PEI (1 mg/ml in RNase-free water, pH 7.4) and vortexed for 30 s. The mixtures were incubated for 1h at room temperature. The ratio between PEG/CS and PEI was 5/1 (w/w) in all formulations. The concentration of siRNA in the formulations was kept constant and was 11.35 µg/ml. The amount of TPP was optimized in order to obtain monodisperse nanoparticles. The (CS/PEG):TPP ratio was 2.7:1 or 3.3:1 (w/w) depending on the 7

ACCEPTED MANUSCRIPT CS/PEG. The average size, the polydiversity index and the zeta potential of the nanoparticles were determined using a Nanosizer NanoZS (Malvern Instruments Ltd.,

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Malvern, UK) by photon correlation spectroscopy and electrophoretic mobility,

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respectively.

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Cell culture

The human non-small cell lung carcinoma cell line H1299 expressing the enhanced

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green fluorescent protein (H1299-EGFP) was used for the in vitro tests as reported previously [8, 28]. H1299-EGFP cells were grown in RPMI 1640 medium (Life

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Technologies) supplemented with 10% heat-inactivated FBS (Life Technologies). SiHa and HeLa cervix cancer cells were maintained in Dulbecco’s modified Eagle’s

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medium (DMEM; Sigma-Aldrich) supplemented with 10% heat-inactivated FBS and

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1% penicillin-streptomycin, at 37°C, in 5% CO2-humidified atmosphere. Cell viability was determined by using the Presto Blue cell viability reagent (Life Technologies),

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according to the manufacturer’s instructions. Cellular uptake

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Cellular uptake of nanoparticles was quantified by flow cytometry. H1299-EGFP cells were seeded in 12-well plates (1.5x105 cells/well), 24 h before experiment. Cells were incubated with nanoparticles for 1 h at a concentration of 100 nM Alexa 647labelled siRNA. Cells were rinsed with phosphate-buffered saline (PBS), trypsinized and diluted with medium. After centrifugation (250 x g, 5 min, 4°C), the cell pellet was resuspended in 200 µl PBS. The measurements were done on a FACS Calibur cytometer (BD Biosciences, Erembodegem, Belgium) in triplicates and the data were analyzed using FlowJo software. A number of 2.104 cells were analyzed in each measurement. Fluorescence microscopy 8

ACCEPTED MANUSCRIPT The H1299-EGFP cells were seeded in 12-well plates (105 cells/well) containing a cover slip. After 24 h, the cells were incubated with the nanoparticles loaded with

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Alexa647-labelled siRNA (1h at 100 nM), then fixed with fresh paraformaldehyde (2%

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(w/v) in PBS) and rinsed three times with PBS. In order to stain the nuclei, the cells

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were incubated with 4',6-diamidino-2-phenylindole (DAPI; 0.1 µg/ml) for 5 min and rinsed three times with PBS. The cells were incubated for 1 h with AlexaFluor488conjugated concanavalin A (5 µg/ml in PBS, Molecular Probes) to stain the cell

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membranes. After washing, the cover slips were placed on a slide using the

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Vectashield® mounting medium. The slides were imaged using a structured illumination AxioImager microscope equipped with an Apotome module (Zeiss,

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Germany, magnification 40x).

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Detection of integrin expression

One million cells were incubated with 10 µg of anti-αvβ3 antibody (mouse monoclonal

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BV3 against αvβ3 integrin, GeneTex, Bio-Connect, Huissen, The Netherlands) or with 10 µg of isotype control antibody (APC Mouse IgG1, κ Isotype Ctrl (FC) Antibody,

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BioLegend, Imtec Diagnostics, Antwerp, Belgium) at 4°C for 30 min. The cells were rinsed twice with PBS and incubated with 2 µg of a secondary antibody (Alexa647 goat–anti-mouse IgG1, 2 mg/mL, Molecular Probes) at 4°C for 30 min. As control, 106 cells were incubated with 2 µg of the secondary antibody. The measurements were done on a FACS Calibur cytometer (BD Biosciences) in triplicates and the data were analyzed using FlowJo software [8]. RNA interference experiments EGFP silencing on H1299-EGFP cells The H1299-EGFP cells were seeded in 12-well plates at a density of 105 cells per well 24 h prior to the experiment. The following day, the cells were transfected with 9

ACCEPTED MANUSCRIPT the nanoparticles loaded with siRNA EGFP or with siRNA CTRL for 4 h. Subsequently, the medium was replaced with fresh medium. After 24 or 48 h, cells

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were washed with PBS, trypsinized and resuspended in 300 µl PBS prior to flow

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cytometry analysis. The percentage of gene silencing was calculated by using the

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FL-1 median (median of the GFP fluorescence histogram). Cells transfected with NP loaded with the siRNA CTRL were used as negative control. ASCT2 and MCT1 silencing in SiHa cells

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SiHa tumor cells were seeded in standard DMEM medium at 30% confluence in 96-

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well tissue culture plates (cell viability assays) or in 6-well plates (protein extraction). The next day, cells were transfected with the NP loaded with 100 nM siRNA for 6 h. Lipofectamine™ RNAiMAX transfection reagent (Life Technologies) was also used,

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as a transfection control, with 50 nM siRNA, following the manufacturer’s instructions.

Western blot

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Cell viability and protein expression were assessed after 72 h.

Tumor cells or excised tumors were lysed in a buffer containing 50 mM Tris-HCl pH

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7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Roche Applied Science, Vilvoorde, Belgium). Lysates were cleared (6000 x g, 10 min, 4°C) and stored at -80°C until analysis. Protein extracts were then separated by SDS-PAGE under reducing conditions and transferred onto PVDF membranes (Immobilon, Millipore, Overijse, Belgium). The membranes were blocked with 5% skimmed milk in TBS-0.1% Tween 20 and subsequently immunoblotted with primary antibody for overnight incubation at 4°C. The membranes were probed with horseradish peroxidase-conjugated secondary antibody and reactive proteins were detected using Amersham ECL detection 10

ACCEPTED MANUSCRIPT reagents (GE Healthcare Life Sciences, Diegem, Belgium). Anti-MCT1 (1/1000; home-made), anti-ASCT2 (1/1000; ABN73, Millipore) and anti-actin (1/10000; A5441,

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In vivo delivery of siRNA-loaded RGD nanoparticles

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Sigma-Aldrich), used as an equiloading control, were diluted in 5% skimmed milk.

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Each procedure was approved by the local authorities according to national animal care regulations. Seven week-old female NMRI nude mice were purchased from

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Elevage Janvier (Le Genest-St-Isle, France) and randomly assigned to a treatment group (6 mice per group). For tumor growth inhibition experiments, SiHa tumor cells

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were injected subcutaneously in both flanks of anesthetized (ketamine/xylazine) mice (2.106 cells/flank). When tumors reached a mean diameter of 5 mm, non-targeted

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NP, RGD NP or RGDp NP loaded with siCTRL or a combination siASCT2/siMCT1

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were given twice weekly through either peritumoral or intravenous (tail vein) injection. Tumor volume was tracked with an electronic caliper and calculated as follows:

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(length x width² x π)/6 and expressed in mm³. Xenogen IVIS 50 imaging system (PerkinElmer, Zaventem, Belgium) was also used

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to observe the ex vivo distribution of Cy5.5-labelled siRNA in nude mice bearing SiHa tumors. When tumor size reached ~300 mm³, formulations of non-targeting NP and RGDp NP loaded with Cy5.5-siRNA (50 µg) were administered via the tail vein. At 1h post-injection, the mice were sacrificed, and the tissues were excised and imaged at the appropriate wavelength. Statistical analysis The experiments were performed in triplicate, unless otherwise stated. Values are given as means ± s.e.m. One-way or two-way ANOVA tests were used where appropriate. * p<0.05, ** p<0.01 or *** p<0.001 were considered to be statistically significant. 11

ACCEPTED MANUSCRIPT RESULTS GRAFTING OF RGDP ON PEG CHAINS AND ASSEMBLY WITH CHITOSAN

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Different types of RGDp grafted on the PEG chains were investigated; they are

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based on a tyrosine scaffold and differ by their linker arms and basic moieties, as summarized in Figure 1. These RGDp were designed from the structure of a cyclic RGD (Cilengitide®) immobilized in the binding pocket of v3 integrins [29] and their

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specificity was previously documented (in competitive studies) using isolated v3

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integrins [22] and v3-expressing cells [17, 21, 30]. The first method of RGDp grafting was the chain-end coupling that allowed the RGDp to be exclusively linked at the end of the PEG chains, with a grafting of the PEG chains ranging from 35 to 70

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mol% (RGDp E1, E2, E3 and E4). The second grafting method was the clip photochemistry method that resulted in the random distribution of RGDp along the

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PEG chains, with 4 to 11 mol% of grafted PEG chains (RGDp R1, R2 and R3) [17]. The complete characterization of the different PEG conjugates (RGDp per PEG chain

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and PEG per chitosan) is described in the Supplementary Table 1. The linker between the RGDp and the PEG was either an amino-tri-ethylene glycol-carboxylate (OEG) which is hydrophilic [22] or an aminocaproic acid (or caproyl) which is lipophilic [21] (Fig. 1). Finally, we used two different RGDp chemical structures differing by their basic motif, i.e. naphthyridine or amino-pyridine (Fig. 1). Gly-ArgGly-Asp-Ser (GRGDS named RGD here below) peptide was grafted on the PEG chains using the clip photochemistry method with 10 mol% of grafted PEG chains; ungrafted PEG was also used to generate non targeted NP (see below).

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Figure 1. PEG-RGDp conjugate. Schematic representation of (A) the general structure of RGDp peptidomimetics, (B) the linkers and (C) the structures and grafting methods to produce the PEG-RGDp conjugates; the colors refer to the type of linkers.

The non-covalent assembly of CS and PEG was obtained by mixing both polymers in an acetate buffer that was dialysed after several days of stirring and freeze-dried. After this treatment, CS and PEG form close associations as proven by the nonremoval of 5-kDa PEG from the blend during the dialysis with a wide molecular cutoff [31]. Several studies have already evidenced that PEG and CS are linked together by hydrogen bonding either in solid phase [32] or in solution [33]. It was also 13

ACCEPTED MANUSCRIPT demonstrated that this association works better in blends than in copolymers [34]. This strong physical association (i.e., 50-75% PEG linked to CS) was further

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PHYSICO-CHEMICAL CHARACTERISTICS OF THE NANOPARTICLES

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documented by GPC and DOSY NMR (see Suppl. Table S2 and Fig. S1-S4).

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The rationale for the selection of the excipients to formulate siRNA-loaded PEGylated chitosan NP was based on previous optimization [9, 10]: TPP was used as an ionic

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crosslinker agent for ionic gelation and PEI was added to the formulation as an endosomal disrupting agent. The physicochemical characteristics of the NP are

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summarized in Table I. The mean diameter was between 200 and 300 nm and the polydispersity index (PDI) was generally below 0.2, indicating that the nanoparticle

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populations were monodisperse. The RGDp E1 NP had however a particular profile

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with a mean diameter of 467 nm and a PDI = 0.26. All the nanoparticles presented a

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positive surface charge, but slight differences in the zeta potential values could be observed depending on the amount of TPP in the formulation (that was optimized to obtain monodisperse NP). Indeed, some of the NP (NP grafted with RGDp R1, R2,

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R3, E1 and E4) were formulated at a (CS/PEG):TPP ratio of 2.7:1 (w/w) (zeta potential around 15 mV) while others (non-targeted NP, NP grafted with RGD, RGDp E2 and RGDp E3) were formulated at a 3.3:1 (CS/PEG):TPP ratio (zeta potential around 27 mV).

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ACCEPTED MANUSCRIPT Table I. Physicochemical characteristics of NP exhibiting different RGDp-linkerPEG combinations. Values represent mean ± SD (n=3). Size (nm)

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Zeta potential (mV)

None

239 ± 22

0.15 ± 0.02

27.8 ± 0.3

RGD

269 ± 18

0.16 ± 0.02

26.5 ± 1.1

RGDp E1

467 ± 130

0.26 ± 0.09

RGDp E2

250 ± 20

0.18 ± 0.02

27.3 ± 0.7

RGDp E3

274 ± 23

0.21 ± 0.02

26.6 ± 1.9

RGDp E4

285 ± 37

0.16 ± 0.01

18.3 ± 2.8

RGDp R1

244 ± 59

0.19 ± 0.06

13.6 ± 2.1

RGDp R2

270 ± 25

0.14 ± 0.02

15.8 ± 1.0

RGDp R3

334 ± 65

0.18 ± 0.04

16.3 ± 1.4

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Targeting moiety

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14.7 ± 0.0

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TARGETED NP INDUCED EGFP SILENCING ON H1299 EGFP CELLS

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Non-targeted and αvβ3-targeted NP were first evaluated for their ability to silence the expression of an EGFP reporter protein constitutively expressed in H1299 lung carcinoma cells; two siRNA concentrations were considered, namely 50 and 100 nM.

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The EGFP silencing obtained with the non-targeted NP was below 25% irrespective of the applied doses of siRNA whereas the extent of silencing dose-dependently increased following exposure to the αvβ3-targeted nanoparticles. The αvβ3-targeted NP randomly grafted with the clip photochemistry method (RGDp R1-R3) were globally more efficient than their end-chain grafted counterparts (RGDp E1-E4) (Fig. 2). In particular, at a concentration of 100 nM siRNA (Fig. 2B), 55-70% EGFP silencing was obtained with the clip-grafted NP while only 20-40% was observed by using the chain-end grafted NP. Of note, the differences in the zeta potential between the formulations (Table 1) did not influence the gene silencing efficiency.

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Figure 2. Comparison of the different RGDp-linker-PEG combinations for EGFP siRNA delivery. Extent of EGFP silencing in H1299-EGFP cells 48 h after exposure to (A) 50 nM and (B) 100 nM EGFP siRNA loaded in non-targeted nanoparticles (NP) and αvβ3-targeted nanoparticles grafted using the clip method (R-series) or the chain-end coupling method (E-series).

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ACCEPTED MANUSCRIPT ENDOCYTOSIS OF TARGETED NP IS MEDIATED BY THE αVβ3 INTEGRINS To further investigate the process of αvβ3-driven siRNA delivery, we used siRNA

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conjugated to the fluorophore Alexa 647. Fluorescence microscopy revealed that siRNA-loaded targeted NP were efficiently internalized in EGFP-expressing H1299

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lung carcinoma cells as soon as after 1 hour incubation (see intracellular red dots in Fig. 3A and white bars in Fig. 3C). We then evaluated the role of the αvβ3 integrin

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expressed in H1299 cells in the extent of siRNA delivered; as a positive control, we used the cervix cancer cells SiHa known to express very large amounts of this

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integrin at the plasma membrane, as confirmed by flow cytometry (Fig. 3B). The cellular uptake of the αvβ3-targeted NP (RGD NP and RGDp R1 NP) and non-

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targeted NP was then assessed using fluorophore-conjugated siRNA in these two

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cell lines as well as in cervix cancer HeLa cells that do not express α vβ3 integrins [35,

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36]. The siRNA fluorescence signal confirmed that the uptake of targeted NP was increased in proportion to the αvβ3 expression level (vs. non-targeted NP that showed the same profile of siRNA internalization in the three cell lines) (Fig. 3C). Of note, the

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uptake of fluorescent siRNA resulting from the use of targeted NP in the α vβ3negative HeLa cells was similar to that obtained with non-targeted NP in any cells. Altogether, these data indicated that both αvβ3 receptor-dependent and –independent endocytosis of NP can occur: non-targeted NP only enter cells through the latter while endocytosis of RGD/RGDp-decorated NP results from the combination of both mechanisms [8].

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Figure 3. Effects of the RGD peptide and RGDp R1 peptidomimetic on the extent of siRNA delivery in αvβ3 integrin-expressing cells. (A) Cellular uptake of Alexa647-conjugated siRNA (red) upon 1h-exposure of H1299-EGFP cells to RGD NP or RGDp R1 NP. Nuclei and cells are counterstained with DAPI (blue) and concanavalin A (green), respectively. (B) Detection of αvβ3 integrins in H1299-EGFP and SiHa cells. Data are shown as fluorescence histograms after flow cytometry 18

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analysis of cells incubated with an anti-αvβ3 antibody or an isotype control FC antibody, followed by incubation with an Alexa 647 secondary antibody. As controls, cells were only exposed to the secondary antibody. (C) Cellular uptake of 100 nM Alexa 647-labelled siRNA following1h-exposure of αvβ3-expressing SiHa and H1299EGFP cells, and αvβ3 non-expressing HeLa cells, to the indicated loaded NP, namely non-targeted NP, RGD NP and RGDp R1 NP.

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THE EXTENT OF MCT1 & ASCT2 SILENCING DEPENDS ON THE NATURE OF THE TARGETED NP To determine the potential therapeutic efficacy of gene silencing, we chose to knock

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down the expression of MCT1 and ASCT2, two proteins that support the high energydemand metabolism of tumor cells by mediating lactate and glutamine transport,

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respectively [37-40]. SiHa cells were selected for these experiments because they express high levels of αvβ3 integrins (see Fig. 3B) and pilot studies had shown their

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propensity to consume glutamine and lactate both in vitro and in vivo.

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We first evaluated the efficiency of the silencing of MCT1 (Fig. 4A) and ASCT2 (Fig.

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4B) by using non-targeted NP and NP decorated either with the RGD peptide or the peptidomimetic RGDpR1. Quantitative analysis of immunoblots was used to estimate

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the extent of silencing (vs. control siRNA). We found that both MCT1 and ASCT2 were significantly silenced after transfection with either RGD or RGDpR1 NP (Fig. 4A and 4B). Interestingly, the non-targeted NP were unable to significantly silence any of the targets (Fig. 4A and 4B).

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Figure 4. Comparison of the different RGDp-linker-PEG combinations for the delivery of siRNA targeting tumor cell metabolism. Western blot analysis of (A) MCT1 and (B) ASCT2 expression in SiHa cells following corresponding siRNA (100 nM) delivery using the indicated targeted or non-targeted NP; control transfection was performed using lipofectamine reagent. Extent of the cytotoxic effects of MCT1 silencing in SiHa cells incubated in the presence of (C) glucose- and (D) lactatecontaining medium 72 hours after exposure to the indicated NP or lipofectaminebased transfection. Extent of the cytotoxic effects of ASCT2 silencing (E) in native SiHa cells and (F) in acidic pH-acclimated SiHa cells treated for 72 hours as above.

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We investigated whether the silencing of the lactate transporter MCT1 induced the

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expected cytotoxic effects. SiHa cells were incubated with the NP loaded with MCT1-

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targeting siRNA in culture medium containing either glucose or lactate, and the cell viability was measured 72 h after exposure to NP.

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In the presence of glucose, MCT1 silencing barely influenced cell growth regardless the type of NP (Fig. 4C). However, in the conditions mimicking the tumor environment

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where blood-borne fuels such as glucose are limiting or exhausted, and where lactate represents the main energy fuel for cancer cells [37-40], MCT1 silencing led to

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significant cytotoxic effects. We found indeed that when MCT1 siRNA were delivered

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using targeted NP, 20 to 65% cell death were observed depending on the targeting moieties (Fig. 4D). By contrast, the use of non-targeted NP did not lead to tumor cell

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death, in accordance with the lack of MCT1 silencing observed in Fig. 4A. The RGDp R1 NP and the RGD NP were the most efficient formulations, inducing up to 50-65%

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cell death whereas the RGDp R2 NP and the RGDp R3 NP showed limited cytotoxic effect (approximately 20% cell death). Regarding the chain-end grafted NP, no influence of the linker on the silencing efficiency could be observed: approximately 40% cell death was obtained with RGDp E1, E2, E3 and E4 (Fig. 4D). The in vitro cytotoxicity resulting from ASCT2 silencing was evaluated on SiHa cells grown in glutamine-containing medium. SiHa cells acclimated to pH 6.5 were compared to native SiHa cells, as an acidic environment better reflects the in vivo tumor microenvironment and was recently showed by us to be associated with an increased dependency towards glutamine [41].

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ACCEPTED MANUSCRIPT In the acidic pH-adapted cells (Fig. 4F), the NP induced an overall higher antitumor effect (vs. native SiHa cells; Fig. 4E), validating further the glutamine transporter

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ASCT2 as a critical therapeutic target, particularly in the tumor acidic environment. In

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these glutamine-addicted tumor cells, RGD and RGDp R1 NP were again the most

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efficient NP to deliver therapeutic concentrations of ASCT2 siRNA. IN VIVO ANTI-TUMOR EFFECTS OF MCT1 & ASCT2 SILENCING DIFFER ACCORDING TO THE

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NATURE OF THE TARGETED NP

We first evaluated the in vivo anticancer effects of the NP loaded with a combination

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of ASCT2 and MCT1 siRNA, using the peritumoral route. Of note, we used immunodeficient mice in order to exclude any possible contribution of the immune

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system in the observed results and we consistently compared the effects of NP

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loaded either with siRNA of interest or with irrelevant siRNA. Based on the in vitro

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experiments, we chose the RGD NP and the RGDp R1, E1 and E2 NP as the αvβ3targeted NP exhibiting the highest potential of siRNA delivery (see Fig. 4). The tumor growth was regularly tracked following peritumoral injection of NP loaded either with

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MCT1 and ASCT2 siRNA or control siRNA (Fig. 5A). We found that the RGDp R1 NP led to a profound inhibition of tumor growth whereas the RGD NP, the other RGDp NP (RGDp E1 and E2) and the non-targeted NP, did not show any significant effect (vs. control siRNA NP) (Fig. 5A); only a trend towards an inhibitory tumor growth was observed with RGDp E2 NP. Ex vivo analysis of the tumors confirmed the major growth inhibitory effects of MCT1/ASCT2 siRNA delivered through the RGDp R1 NP (Fig. 5B).

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Figure 5. Comparison of different RGDp-linker-PEG combinations for the in vivo intratumoral administration of siRNA targeting tumor cell metabolism. (A) Time courses of SiHa tumor growth in mice after peritumoral injection of non-targeted NP and RGD, RGDp R1, RGDp E1 or RGDp E2 NP loaded with a mixture of MCT1 and ASCT2 siRNA (or control siRNA). (B) Representative pictures of tumors excised at the end of the treatment with non-targeted and RGDp R1 NP.

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ACCEPTED MANUSCRIPT In a second set of experiments, we examined whether similar effects could be obtained following intra-venous (i.v.) administration of the NP. We first examined the

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in vivo biodistribution of Cy5.5 fluorophore-conjugated siRNA delivered with RGDp

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R1 NP vs non-targeted NP. Ex vivo imaging of tumor and several organs was

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performed 1 hour post-i.v. injection. Tumor accumulation of Cy5.5-conjugated siRNA was only detected in mice treated with the αvβ3-targeted NP (vs. non-targeted NP) (Fig. 6A). In addition, while the liver and the kidney were Cy5.5-positive irrespective

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of the type of NP, accumulation of Cy5.5-siRNA in the lungs was only observed with

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untargeted NP (Fig. 6A).

We then i.v. injected mice bearing SiHa tumors twice a week with either RGDp R1 or non-targeted NP loaded with the MCT1/ASCT2 siRNA combination. A net tumor

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growth delay was observed in mice treated with the RGDp R1 NP whereas the nontargeted NP failed to show any antitumor effects (Fig. 6B). Immunoblot experiments

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confirmed the silencing of both targets ASCT2 and MCT1 in mice injected with RGDp

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R1 NP but not when siRNA were delivered using non-targeted NP (Fig. 6C).

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Figure 6. Effects of the RGDp R1 peptidomimetic for the in vivo systemic administration of siRNA targeting tumor cell metabolism. (A) Typical ex vivo imaging of tumor and organs one hour after i.v. injection of Cy5.5-siRNA-loaded NP. (B) Time courses of SiHa tumor growth in mice after i.v. injection of non-targeted NP and RGDp R1 NP loaded with a combination of MCT1 and ASCT2 siRNA (or control siRNA). (C) Western blot analysis of MCT1 and ASCT2 expression in SiHa tumors collected at day 12 after siRNA delivery using the indicated non-targeted and RGDp R1 NP.

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ACCEPTED MANUSCRIPT DISCUSSION The first main finding of this study is that targeting tumor metabolism using

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siRNA represents an attractive therapeutic strategy to treat cancer. The recent

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discovery by us and others of the major roles of metabolic fuels such as lactate and

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glutamine [37-41] found here some direct therapeutic applications. We have indeed documented that the blockade of the uptake of either substrate can be detrimental for

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tumor cell proliferation and is an achievable goal in vivo. Importantly, this work also emphasizes the advantage to simultaneously target different metabolic pathways in

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tumors. The combination of siRNA directed against the lactate transporter MCT1 and the glutamine transporter ASCT2 within the same NP appears particularly suited to

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oppose tumor metabolism plasticity (i.e., the capacity of tumor cells to adapt

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themselves in order to use an alternate metabolic route when a given pathway is blocked). Also, tumor heterogeneity may account for the dependency of different

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tumor areas for distinct metabolic fuels at the same time. We previously documented that MCT1 inhibition targets oxidative tumor cells using lactate instead of glucose [39]

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whereas blocking ASCT2 has major detrimental effects in tumor cells growing in the most acidic tumor areas [41]. This offers an additional rationale to exploit cocktails of siRNA simultaneously targeting several metabolic paths to reach the largest proportion of cancer cells within a tumor. A second major finding of this study is that even if targeted PEGylated chitosan NP represent well-known platforms to deliver siRNA, the extent of the in vivo delivery and thus the therapeutic efficacy is subtly dependent on the chemical structure of the ligand moiety, the nature of the linker used for the grafting of the ligand onto the PEG or the method (location) of grafting. To gain insights on the best combination of these parameters, we used RGDp as a prototypical model of ligand 26

ACCEPTED MANUSCRIPT and αvβ3 integrin-expressing cells including EGFP-H1299 and SiHa cancer cells expressing MCT1 and ASCT2. Reducing the intracellular fluorescence signal of the

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reporter protein EGFP and silencing MCT1/ASCT2 protein expression were used as

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read-outs of the efficacy of the different NP in delivering corresponding siRNA. In all

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these experiments, the targeted NP were always more efficient than the non-targeted NP. In addition, using fluorophore-conjugated siRNA, we could document that the siRNA delivery was proportional to the expression of the RGD/RGDp target, namely

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the αvβ3 integrin. Although non-targeted NP led to a significant amount of siRNA

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delivery into cells (Fig. 3C), this poorly influenced the expression of targeted genes (Figs 4D and 4F) indicating that the endocytic route of non-targeted NP is not compatible with an efficient delivery of active siRNA [8].

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Among the targeted ligands, the RGDp (R1-3) randomly conjugated on the PEG chain by clip photochemistry showed the highest capacity to silence EGFP (see Fig.

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2B), reaching the same efficacy as the RGD peptide. Moreover, in the experiments evaluating the cytotoxic effects of MCT1- and ASCT2-targeting siRNA (Fig. 4D and

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4F), RGDp R1 was significantly more efficient than the two other representatives of the clip-grafted family (RGDp R2 and R3), suggesting that a lipophilic linker (caproyl) improved targeted NP delivery efficacy (vs. the hydrophilic OEG linker). This could be explained by a less likely interaction of the lipophilic linker with the hydrophilic core of the NP and thus a more efficient presentation of the ligands on the NP surface. These data also showed that for the same RGDp structure (containing a caproyl linker and a naphthyridine motif), the clip photochemistry method of grafting (RGDp R1 NP) was more efficient than the traditional chain-end coupling (RGDp E1 NP) despite the lower ligand density obtained with the clip grafting (11% vs. 52% PEG grafting ratio, respectively) (see Supplementary Table 1).

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ACCEPTED MANUSCRIPT These results indicate that the coupling location is more determinant than the amount of ligands on the PEG chain. The binding of the RGDp targeting moieties to

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plasmalemmal integrins depends mainly on the availability of free ligands at the

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surface of the NP and even abundant ligands can be masked in the final formulation

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if they have a favourable interaction with the NP core. A parallel can be drawn with a study reporting that for PLGA-PEG NP, folate was acting as a less efficient targeting ligand than RGD because most of the folate was buried in the PLGA NP core due to

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hydrophobic interactions between PLGA and folate molecules [15]. For the same

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reason, i.e. the non-covalent association of PEG and CS, targeting ligands at the distal end of the PEG chain are very likely to be located next to the chitosan core (as modelled in Suppl. Fig. 5). On the contrary, ligands grafted along the chain may

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stand aside the backbone and emerge from a loop thus being more available for interaction with their receptors (see Suppl. Fig. 5). This model is also coherent with

[42-44].

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other studies documenting that lower density of ligands can lead to better targeting

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The in vitro evaluation of the different combinations of RGDp, linker and grafting method led us to identify the high potential of RGDp R1 NP to deliver therapeutic siRNA to mice bearing SiHa tumors. A net inhibition of tumor growth was observed upon peritumoral injection (Fig. 5) but also importantly following i.v. administration (Fig. 6B) of RGDp R1 NP loaded with the mixture of MCT1 and ASCT2 siRNA. The lack of effects of non-targeted NP in both conditions strongly suggests that the RGDp moiety not only allows to reach the tumor upon systemic administration (through a preferred interaction with αvβ3-expressing endothelial cells lining tumor blood vessels) but also largely contributes to the internalization and intracellular delivery of the NP payload into αvβ3-expressing tumor cells. The tumor targeting was confirmed 28

ACCEPTED MANUSCRIPT by ex vivo imaging following i.v. administration of NP loaded with Cy5.5-conjugated siRNA (Fig. 6A). Indeed, RGDp R1 NP consistently led to an increase in fluorescence

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in the tumors whereas non-targeted NP failed to do so. Moreover, liver and kidney

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were similarly Cy5.5-positive but lung accumulation of fluorescent siRNA was only

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detected when non-targeted NP were used. The silencing potential of delivered siRNA was further proven by ASCT2 and MCT1 immunoblotting of lysates of excised tumors from treated mice (Fig. 6C). These in vivo results also indirectly confirm that

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the non-covalent PEGylation of chitosan NP is a convenient and realistic option to

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generate NP offering an optimal kinetics of efficient siRNA delivery. Finally, it should be stressed that even if the RGD peptide moiety led to similar in vitro results as the best RGD peptidomimetics in terms of siRNA delivery, the RGD

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peptide failed to show any significant antitumor effects when used in vivo (see Fig. 5A). Protease degradation occurring in vivo is very likely to account for this lack of

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activity and underscores the need to optimize peptidomimetics to increase the potential of decorated NP to find their cell targets upon i.v. administration.

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In conclusion, this work provides clear evidence of the therapeutic potential of inhibiting tumor metabolism with siRNA loaded in chitosan-based NP. This strategy takes advantage of (i) the use of siRNA targeting different pathways and thus concomitantly acting on different metabolic cell phenotypes present in tumors and (ii) the careful selection of RGD peptidomimetic characteristics (structure, linker and grafting) that considerably influence the extent of bioactive siRNA delivery.

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ACCEPTED MANUSCRIPT CONFLICT OF INTEREST

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The authors declare that they have no competing interests.

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ACKNOWLEDGEMENTS

This work was supported by grants from the Fonds national de la Recherche

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Scientifique (FRS-FNRS), the Belgian Foundation against cancer, the J. Maisin Foundation, the interuniversity attraction pole (IUAP) research program #UP7-03

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from the Belgian Science Policy Office (Belspo), an Action de Recherche Concertée (ARC 14/19), and the Walloon Region (BioWin project Targetum). The funding

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sources had no involvement in the study design; in the collection, analysis, and

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interpretation of data; in the writing of the report; nor in the decision to submit the

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paper for publication. We acknowledge Dr. Raphael Riva for the GPC, and Dr. Cécile

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Le Duff for the DOSY NMR measurements.

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Graphical Abstract

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