Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery

Accepted Manuscript Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery

Alessio Malfanti, Anna Scomparin, Sabina Pozzi, Hadas Gibori, Adva Krivitsky, Ronit Satchi-Fainaro, Francesca Mastrotto, Paolo Caliceti, Stefano Salmaso PII: DOI: Reference:

S0168-3659(19)30469-9 https://doi.org/10.1016/j.jconrel.2019.08.005 COREL 9892

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

18 April 2019 4 August 2019 6 August 2019

Please cite this article as: A. Malfanti, A. Scomparin, S. Pozzi, et al., Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery, Journal of Controlled Release, https://doi.org/10.1016/ j.jconrel.2019.08.005

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ACCEPTED MANUSCRIPT

Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery Alessio Malfanti1 , Anna Scomparin2,3 , Sabina Pozzi2 , Hadas Gibori2 , Adva Krivitsky2 , Ronit Satchi-Fainaro2 *, Francesca Mastrotto1 , Paolo Caliceti1 *, Stefano Salmaso1 1

Departement of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F.

2

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Marzolo, 5, 35131 Padova – Italy. Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University

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69978 Tel Aviv – Israel. 3

Department of Drug Science and Technology, University of Turin, Via P. Giuria 9, 10125 Turin –

*Correspondence should

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Italy.

be addressed

to

Paolo

Caliceti: Department of Department of

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Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova – Italy; Tel: +39 049 8275695; e-mail: [email protected]; Ronit Satchi-Fainaro:

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Department of Physiology and Pharmacology, Sackler, Faculty of Medicine, Tel Aviv University,

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Room 607, Tel Aviv 69978 – Israel; e-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract Novel bioconjugates (Agm6 -M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped “head” (Agm6 -M) to a polymeric “tail” (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm6 -M-PEG-FA and Agm6 -M-PEG-OCH3 , respectively). Gel electrophoresis showed that the

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bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies

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performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take

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was obtained with the 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size <80 nm with

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narrow polydispersity and rod-shaped morphology. The polyplexes were stable for several hours in plasma while ON was released in the presence of heparin concentration 16-times higher than the

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physiological one. The polyplexes displayed negligible cytotoxicity, hemolysis and low proinflammatory TNF-α release. Studies performed with FR-overexpressing HeLa and MDA-MB-231

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cells using siRac1 revealed that the folated polyplexes caused significantly higher gene silencing

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(86.1±9.6%) and inhibition of cell migration (40%) than the non-folated polyplexes obtained with Agm6 -M-PEG-OCH3 only. Although cytofluorimetric analyses showed similar cell uptake for both folated and non-folated polyplexes, confocal, TEM and competition studies showed that the folated

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polyplexes were taken-up by lysosome escaping caveolin-mediated pathway with final polyplex localization within cytosol, while non-folated polyplexes were preferentially taken-up via clathrinmediated pathway to localize in the lysosomes. Finally, preliminary in vivo studies carried out in mice revealed that the folated polyplexes dispose in the tumor mass.

Keywords Polyplexes, oligonucleotide delivery, siRNA, polycationic polymers, supramolecular carriers, folate receptor, active targeting, endocytotic pathways.

ACCEPTED MANUSCRIPT Introduction Therapeutic oligonucleotides (ONs) such as sequence-specific oligonucleotide (SSO), small interfering RNA (siRNA) and microRNA (miRNA), offer unique therapeutic opportunities for the treatment of a variety of diseases by regulating the gene expression in cells. Nevertheless, the clinical translation of ONs suffers from their rapid elimination by degradation and kidney

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ultrafiltration, low membrane permeability, lack of target selectivity, and activation of innate

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immune system, which result in poor bioavailability, high toxicity and low gene knockdown [1-4].

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In an attempt to overcome the hurdles limiting the therapeutic exploitation of ONs, a variety of molecular and nanoscale delivery strategies have been explored including chemical manipulations

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(i.e. bioconjugation) [5, 6] and physical combinations within natural carries such as viral vectors, exosomes [7] or synthetic vectors [8-11].

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Cationic vectors represent a broad class of materials, namely polymers, lipids, peptides and other supramolecular structures, which have been successfully exploited for ONs delivery [12, 13]. These

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carriers easily form stable polyplexes by simple coulombic association with ONs without chemical

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manipulations or complicate processes [14, 15]. Furthermore, polycationic carriers can be properly designed to yield ON protection from degrading enzymes and the immune system [16], prolonged polyplex circulation in the bloodstream and extravasation-dependent accumulation into tissues with

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leaky vasculature such as tumours [17]. Polycationic carriers have also been harnessed with targeting agents [18-22] to enhance cell selectivity and uptake through endocytic mechanisms (i.e. clathrin, dynamin and caveolae-mediated transport and macropinocytosis) [23, 24]. For example, transferrin targeted cyclodextrin based nanocarrier is the first siRNA selective vector approved for phase I clinical trial in human cancer treatment [25-27]. However, even though polycationic targeted and non-targeted ON carriers yielded promising in vitro and in vivo cell transfection, little clinical progresses have been achieved, mainly due to their intrinsic toxicity, poor polyplex in vivo stability and insufficient target bioavailability [1, 28-30].

ACCEPTED MANUSCRIPT For such reasons, these delivery systems mostly remained at the preclinical investigational phase, with an unmet need for more effective ON carriers with enhanced delivery profiles. The results reported in the literature clearly show that the composition and architecture of the polycationic

carriers

dictate

the

biopharmaceutical

features,

stability,

biocompatibility,

pharmacokinetic and biodistribution profiles and cell trafficking of polyplexes, and in turn, their

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therapeutic performance [31, 32].

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In order to develop new polycationic architectures with enhanced ON delivery properties, we

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designed a supramolecular ON carrier that, according to evidences reported in the literature, combines structural key elements for therapeutic applications. Unlike other supramolecular ON

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carriers described in the literature, the bioconjugates presented here possess a non-linear polycationic star-like shaped “head” where guanidyl groups were conjugated to a small

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oligosaccharide, which was further functionalized with a PEG “tail”. In order to bestow selective cell bio-recognition, the carrier was upgraded with folic acid (FA) as targeting agent model since it

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possesses high affinity for the folic acid receptor (α isoform of the FR, Kd ~ 0.1 nM) overexpressed by many tumor cells [33-35]. Furthermore, β-isoform of the folate receptor can promote the

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targeting of macrophages in tumors and inflamed tissues

[36]. Accordingly, these systems can be

exploited to treat either tumors or inflammatory diseases, such as arthritis.

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A reproducible chemical protocol was set-up to produce bioconjugates with defined chemical structures. The physicochemical, biopharmaceutical and biological properties of polyplexes were characterized and the cell trafficking profiles were investigated in order to elucidate the targeting mechanism.

Furthermore,

biodistribution.

preliminary

in

vivo

studies

were

performed

to

evaluate

their

ACCEPTED MANUSCRIPT Materials and Methods Materials Linear 5 kDa α-methoxy-ω-amino-poly(ethylene glycol) (mPEG5kDa-NH2 ) and linear 6 kDa bis

(Marktredwitz,

glycol)

Germany).

(NH2 -PEG6kDa-NH2 )

were obtained

Cyanine-3-N-hydroxysuccinimde

from Iris Biotech GmbH

(Cy3-NHS)

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amino-poly(ethylene

was

supplied

by

Lumiprobe GmbH (Hannover, Germany). Folic acid (FA), maltotriose, N-hydroxysuccinimide [1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride]

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(NHS),

(EDC),

morpholino-

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ethan-sulphonic acid buffer (MES), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] were purchased from Sigma Aldrich (St. Louis, MO, USA) or Merck (White House

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Station, NJ, US). [3 H]-folic acid ([3 H]FA) sodium salt was purchased by American Radiolabeled

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Chemicals, Inc. (St. Louis, MO, USA). Rac1 siRNA (siRac1), Cy5-labeled siRac1 (siRac1-Cy5) were kindly supplied by QBI Enterprise (Ness Ziona, Israel). Lipofectamine®2000 was obtained

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from Life Technologies, (Grand Island, NY, USA). psiCHECK™ reporter assay was purchased from Promega (Madison, Wisconsin, USA). ProLong Gold antifade with DAPI mounting medium

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was purchased from Invitrogen (Eugene, OR, USA). Organic solvents were purchased from Carlo Erba (Milan, Italy), VWR International (Lutherworth, UK) and Sigma-Aldrich (St. Louis, MO,

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USA). All the other reagents and salts were obtained from Fluka Analytical or Sigma-Aldrich. The cell lines were purchased from the ATCC (Manassas, VA, USA), unless otherwise indicated. Forty mM Tris, 20 mM acetic acid, 1 mM EDTA (TAE) electrophoresis buffer and all the tissue culture reagents were purchased from Biological Industries Ltd. (Beit Haemek, Israel).

ACCEPTED MANUSCRIPT Synthesis of bioconjugates: Agm6 -M-PEG-OCH3 , Agm6 -M-PEG-FA and Agm6 -M-PEG-Cy3 The glycol)

synthesis 5

kDa

of

(agmatinyl)6 -maltotriosyl-N-acetyl-amino-hexanoate-α-methoxy

(Agm6 -M-PEG-OCH3 ),

poly(ethylene

(agmatinyl)6 -maltotriosyl-N-acetyl-amino-hexanoic

acid

(Agm6 -M-COOH) was carried out according to a modified procedure reported in the literature [37, 38]. Briefly, the anomeric carbon of maltotriose (M) was derivatized with amino-terminating

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hexanoic acid and the resulting Schiff base was stabilized by acetylation using acetic anhydride to

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obtain M-COOH. The hydroxyl groups of M-COOH were derivatized with acryloyl-agmatine by

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ATRP reaction to obtain the polycationic hexa-guanidyl derivative Agm6 -M-COOH which was further derivatized with NH2 -PEG-OCH3 .

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The synthesis of (agmatinyl)6 -maltotriosyl-N-acetyl-amino-hexanoate-poly(ethylene glycol) 6 kDafolate (Agm6 -M-PEG-FA) was carried out by adding 291.9 mg (1.88 mmol) of 1-ethyl-3-(3-

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dimethylaminopropyl)carbodiimide (EDC) and 216.3 mg (1.88 mmol) of N-hydroxysuccinimide (NHS) to 5 mL of a 100 mg/mL (37.6 mM) solution of Agm6 -M-COOH in 1:2 v/v DMSO/100 mM

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morpholino-ethan-sulphonic acid buffer (MES), pH 4.7. The solution was maintained under stirring for 30 min and then added of 3 mL of a 314 mg/mL (52.3 mM) poly-(ethylene glycol)6kDa-folate

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(NH2 -PEG-FA) [39] solution in 1:2 v/v DMSO/100 mM MES, pH 4.7. The mixture was maintained under stirring for 72 h at room temperature and then dialyzed against deionized water for 48 h using

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a 3.5-5.0 kDa MWCO dialysis membrane. The solution was lyophilized to obtain a dry yellow powder (780 mg, 0.092 mmol) with 55.2% molar yield. The PEG-FA conjugation degree was calculated by normalizing the guanidine content determined by the Sakaguchi assay [40] for the PEG content determined by the iodine assay [41] and was 96%. The

synthesis

of

(agmatinyl)6 -maltotriosyl-N-acetyl-amino-hexanoate-poly(ethylene

glycol)6kDa-

Cyanine3 (Agm6 -M-PEG-Cy3) is described in the Supporting Information (SI), which resulted in 93% conjugation yield

ACCEPTED MANUSCRIPT Polyplex preparation Aliquots of ON stock solutions, siRac1 (22 pb, ~13 kDa) or siCtrl (22 pb, ~13 kDa) in 10 mM phosphate buffer, 0.15 M NaCl (PBS), were added to Agm6 -M-PEG-OCH3 , Agm6 -M-PEG-FA and Agm6 -M-PEG-Cy3 solutions prepared in the same buffer to yield polyplexes with fixed compositions, concentrations, and nitrogen/phosphate ratios (N/P). The detailed description of

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polyplex preparation is reported in the SI.

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Electrophoretic mobility shift assay (EMSA)

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Polyplexes with different composition (0:100, 50:50, 75:25 and 100:0 w/w Agm6 -M-PEGOCH3 /Agm6 -M-PEG-FA ratio and 0.5, 1, 2, 3, 4 and 5 N/P molar ratio) were prepared by mixing

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95 µL of 9, 18, 35, 53, 71, 88 μM bioconjugate concentration (Agm6 -M-PEG-OCH3 +Agm6 -MPEG-FA) in 500 µL of PBS, pH 7.4, with 5 μL of 53 μM siRac1 in the same buffer. Fifteen μL of

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these solutions were added to 5 μL of loading buffer and loaded into 2% agarose gel supplemented with 10 g/mL ethidium bromide in TAE buffer. The electrophoresis analysis was performed using

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TAE as running buffer at 100 V for 15 min. The migrated siRNA was visualized under UV light.

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Dynamic Light Scattering and Zeta potential analysis 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplex solutions in PBS, pH 7.4, (500 µg/mL polymer equivalent concentration) were analysed by dynamic light scattering (DLS) at 25°C using a Malvern Zetasizer NanoZS (Malvern Instruments Ltd., UK) supported by Zetasizer Software (version 6.12) to estimate the mean size, polydispersity index (PDI) and zeta potential.

ACCEPTED MANUSCRIPT Transmission Electron Microscopy 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplex solutions in PBS, pH 7.4, (100 µg/mL polymer equivalent concentration) were analysed by transmission electron microscopy (TEM). The samples were deposited on a small copper grid (400 mesh), covered by “holey film” carbon layer and analyzed by negative staining mode using 1% w/v uranyl acetate

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solution in milliQ water as contrast agent.

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Heparin-siRNA competition assay

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Twenty μL of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplex solution (2.5 μM siRac1 equivalent concentration) were maintained at 37°C for 6 hours. After

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incubation, 5 μL of polyplex solutions were added to 10 μL of RNase-free water or heparin solutions in RNase-free water to yield final heparin concentrations in the range of 0-15 IU/mL. The

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samples were incubated overnight at 37°C and mixed with loading buffer. The samples were finally

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analyzed by gel electrophoresis according to the procedure reported above.

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Polyplex stability in plasma

Samples were prepared by adding 5 μL of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEGFA/siRac1 3 and 5 N/P polyplex solution (2.5 μM siRac1-equivalent concentration) to 15 μL of whole mouse plasma. The samples were incubated for 0, 0.5, 1, 3, 5, 8 and 24 h at 37°C. At scheduled times, 10 μL of samples were added of 5 µL of ultrapure water or 90 IU/mL heparin in water and incubated at for 15 min at 37°C. The samples were added of 5 μL of loading buffer and loaded on 2% agarose gel supplemented with 5 µL of a 0.7 mg/mL ethidium bromide solution in TAE buffer. The gel electrophoresis analysis was performed as reported above.

ACCEPTED MANUSCRIPT Hemolysis assay Mouse blood (2 mL) was centrifuged at 1100 rpm for 5 min at 4°C and the supernatant was removed. The sedimented red blood cells (RBCs) were washed 3 times with 40 mL of 0.15 M NaCl in ultra-pure water and centrifuged. The pellet containing RBCs was dispersed in PBS at a final concentration of 2% w/w. Samples were prepared by adding 75:25 w/w Agm6 -M-PEG-

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OCH3 :Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplexes to the cell suspension to yield 0.0001-1.0

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mg/mL range of polymer concentrations and incubated at 37°C for 1 h. Control samples were

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prepared by adding serial dilutions of sodium dodecyl sulfate (SDS) or 70 kDa dextran (0.0001-1.0 mg/mL) to the RBC suspension. The samples were centrifuged at 1100 rpm for 5 min and 100 μL

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of the supernatants were plated into a 96-well plate and spectrophotometrically analyzed at 550 nm using SpectraMax® M5e plate reader (Molecular Devices LLC., Sunnyvale, CA, USA). The

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hemolysis degree was calculated according to the absorbance readings normalized to that obtained

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for RBC treatment with 1% v/v of Triton X-100 solution (100% hemolysis).

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TNF-α cytokine releases from human peripheral blood mononuclear cells Leukocyte enriched whole blood (50 mL) from human healthy donors (Sheba Medical Center Blood

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Bank, Ramat Gan, Israel) was diluted to 215 mL with PBS supplemented with 2 mM EDTA. The solution was gently overlaid onto 15 mL lymphoprep™ (Ficoll) (Axis-Shield, Dundee UK). The suspensions were centrifuged at 20 °C, 400 g for 40 min. The upper layer was removed and the opaque-light human peripheral blood mononuclear cells (hPBMCs) rings were collected and suspended in 50 mL of PBS supplemented with 2 mM EDTA. The suspension was centrifuged at 200 g for 15 min at 20°C. The supernatant was completely removed and hPBMCs were dispersed in RPMI 1640 growth medium supplemented with 10% FBS, 2 mM L-Glutamine, 100 µg/mL Streptomycin, 100 IU/mL Penicillin, 12.5 IU/mL Nystatin to yield 3.0×106 cells/mL.

ACCEPTED MANUSCRIPT hPBMCs (1.5×106 cells/well) were seeded in 12 well plates and treated with 75:25 w/w Agm6 -MPEG-OCH3 :Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplexes (125, 250 and 500 nM siRac1equivalent doses) or 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA (0.08 mg/mL polymer concentration) polymer mixture 3 and 5 N/P or siRac1. 4 μg/mL of lipopolysaccharides (LPS) and PBS, were used as positive control and negative control, respectively. After 12 h, the cells were

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centrifuged at 800 g for 7 min and TNF-α cytokine secreted in the supernatant from hPBMCs was

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estimated by using the DuoSet ELISA kit (DY210, R&D Systems, Minnesota, USA) according to

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the protocol’s manufacturer.

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Cell lines

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Human cervical adenocarcinoma HeLa cells and human mammary adenocarcinoma MDA-MB-231 cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with

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10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin, and 12.5 IU/mL Nystatin. Cells were cultured in folate-free RPMI (FF-RPMI), in

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order to avoid competition with FA in the medium, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL Penicillin, 100 µg/mL Streptomycin, and 12.5 IU/mL

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Nystatin. The cells were incubated at 37°C at 5% CO 2 .

Dual Luciferase reporter assay HeLa cells (1×106 cells/well) were cultured overnight in a 10 cm Petri dish. The cells were then transfected with 4 μg Rac1-psiCHECK™-2-based plasmids reporter assay (Promega Madison, Wisconsin, US) using 4 μL of Lipofectamine® (Life Technologies, Grand Island, NY) as described by Polyak et al. [42]. Following 5 h incubation, 2.5×103 transfected HeLa cells/well were seeded in 96-wells plate and incubated overnight. Then, the medium was removed and the cells were treated with 100:0, 75:25, 50:50 and 0:100 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 and Agm6 -

ACCEPTED MANUSCRIPT M-PEG-OCH3 :Agm6 -M-PEG-FA/siCtrl 3 and 5 N/P polyplexes (125, 250 and 500 nM siRac1 and siCtrl equivalent) in medium supplemented with 10% FBS. siRac1 and siCtrl (50 nM) complexed with Lipofectamine® and siRac1 and siCtrl (125, 250 and 500 nM) were used as controls. Lipofectamine® was used as described in the manufacturing protocol. After 72 h incubation, the cells were lysed using a lysis buffer (Promega, Wisconsin, US). Renilla luciferase and Firefly

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luciferase levels were evaluated using Dual-Luciferase® Assay kit (Promega, Wisconsin, US)

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according to the manufacturer procedure. The results were normalized to untreated cells (controls).

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MDA-MB-231 cells (1×106 cells/well) in FF-RPMI medium supplemented with 10% FBS were transfected with 4 μg Rac1-psiCHECK™-2-based plasmids and treated as reported with siRac1,

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siCtrl, Agm6 -M-PEG-OCH3 /siRac1 or 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1, 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siCtrl 3 N/P or Agm6 -M-PEG-OCH3 /siCtrl

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polyplex solutions (125, 250, 500 nM siRNA concentration).

Cell viability studies

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HeLa and MDA-MB-231 cells were seeded in 96 well plate (2.5×10 3 cells/well) in DMEM supplemented with 10% FBS and FF-RPMI supplemented with 10% FBS, respectively. After 24 h

w/w

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incubation, the medium was removed and the cells were treated with 100:0, 75:25, 50:50 and 0:100 Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1

and

Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-

FA/siCtrl 3 and 5 N/P polyplexes (125, 250 and 500 nM siRac1 and siCtrl equivalent) in medium supplemented with 10% FBS. siRac1 and siCtrl (50 nM) complexed with Lipofectamine® were used as positive controls. siRac1 and siCtrl (250 and 500 nM) were used as negative controls. After 72 h incubation with the polyplexes, the cell viability was assessed using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolinium bromide (MTT) assay [43]. The results were normalized to untreated cells (controls).

ACCEPTED MANUSCRIPT Cell migration assay MDA-MB-231 cells (7.0×104 cells/well) were seeded in a 96-well plate and incubated for 24 h FFRPMI medium supplemented with 10% FBS. A scratch was made in each well using the IncuCyte® WoundMaker (Essen BioScience, Inc.,Michigan, USA). The cells were then treated with 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1, 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-

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FA/siCtrl, Agm6 -M-PEG-OCH3 /siRac1 and Agm6 -M-PEG-OCH3 /siCtrl, 3 N/P polyplexes (250 nM

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siRNA equivalent) in medium supplemented with 10% FBS. Cells treated with siRac1 or siCtrl or

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PBS only were used as controls. The plates were placed in the IncuCyte® ZOOM incubator and images were taken at scheduled times. After 12 h of incubation with the polyplexes, the Relative

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Wound Density was calculated using IncuCyte®Software and the images were analyzed using

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ImageJ version 1.51g software.

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Confocal microscopy studies

MDA-MB-231 cells (8×104 cell/well) in FF-RPMI medium were seeded in 24 well plates with 12

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mm cover slips and incubated for 24 h. The cells were treated for 24 h with 99:1 w/w Agm6 -MPEG-OCH3 :Agm6 -M-PEG-Cy3/siRac1-Cy5 and 74:25:1 w/w/w Agm6 -M-PEG-OCH3 :Agm6 -M-

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PEG-FA:Agm6 -M-PEG-Cy3/siRac1-Cy5 3 N/P polyplexes (125 nM siRNA equivalent) in medium containing 10% FBS. Then, the cells were washed, and fixed with 4% v/v paraformaldehyde, for 20 min in an ice bath. Cover glasses were mounted on microscope slides using the ProLong™ Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA) and analyzed using a Leica SP8 confocal imaging system (60x Magnification). Lasers with emission wavelengths at 405, 568 and 666 nm were used to detect DAPI, Agm6 -M-PEG-Cy3 and siRac1-Cy5, respectively. The images were processed with ImageJ version 1.51g software.

ACCEPTED MANUSCRIPT Cell uptake of polyplexes on MDA-MB-231 MDA-MB-231 cells (1.5×106 cells/well) in FF-RPMI supplemented with 10% FBS were seeded in a 6 well plate and incubated for 24 h. The cells were then treated with 99:1 w/w Agm6 -M-PEGOCH3 :Agm6 -M-PEG-Cy3/siRac1 and 74:25:1 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA:Agm6 M-PEG-Cy3/siRac1 3 N/P polyplexes (125 nM siRac1-equivalent). After 0.08, 0.5, 1, 3, 6 and 24 h

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incubation with the polyplexes in full medium supplemented with 10% FBS, the cells were washed,

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harvested and centrifuged at 1100 rpm for 5 minutes at 4°C. The pellets were re-suspended in PBS,

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imaging flow cytometer (Amnis Corp., Seattle, WA).

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pH 7.4, supplemented with 2% FBS and the samples were analyzed by ImageStream multispectral

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Folate competition study

MDA-MB-231 cells (1×106 cells/well) in 500 μL of FF-RPMI were seeded in 24 well plate and

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treated with folic acid (FA), 95:5, 90:10, 75:25, 50:50, 0:100 w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siRac1 3 N/P polyplex solutions (2 μM of FA-equivalent dose). Similarly, cells were with

Agm6 -M-PEG-OCH3 /siRac1

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treated

3

N/P

polyplex

solution

(1.0

mg/mL

carrier

concentration). After 3 h incubation, 10 μL of [3 H]FA were added to each well and the cells were

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incubated for further 3 h. Then, the medium was removed, and the cells were washed, harvested and centrifuged at 1200 rpm for 5 min. The pellets were washed 3 times with 300 μL of PBS, pH 7.4, and lysed overnight by with 0.5 N NaOH. The samples were then neutralized with 0.5 N HCl. After 30 min, 600 μL of the suspension was added to 3 mL of scintillation fluid. The amount of bound FA was

determined

by radioactivity emission (Tri-Carb® 2100TR liquid

Waltham, MA, USA).

scintillation counter,

ACCEPTED MANUSCRIPT Immunofluorescence analysis MDA-MB-231 cells (8.0×104 cell/well) in FF-RPMI medium were seeded in a 24 well plate on 12 mm cover slips. After 24 h incubation, the cells were treated with Agm6 -M-PEG-OCH3 /siRac1-Cy5 and 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1-Cy5 3 N/P polyplex solutions (200 nM siRac1-Cy5 equivalent). After 3 h incubation, the cells were washed 3 times with 250 μL of

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PBS, pH 7.4, and fixed with 500 μL of 4% v/v paraformaldehyde for 20 min in ice bath. Membrane

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permeabilization was performed by incubating cells with 0.25% v/v Triton X-100 solution in PBS

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for 10 min.

The permeabilized cells were blocked for 30 min with normal mouse and normal rabbit (Santa

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Cruz, Heidelberg, Germany) antibodies diluted in PBS supplemented with 2% BSA.

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Staining of early endosomes and lysosomes was performed with mouse anti-EEA1 (Early endosomes Antigen 1, BD, New Jersey, US) and rabbit anti-LAMP1 (Lysosomal-associated

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membrane protein 1, Cell Signaling Technology, Massachusetts, US) antibodies diluted 1:100 in PBS supplemented with 2% BSA for 1 h at RT. The cells were then washed 3 times with 250 μL of

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PBS, pH 7.4, supplemented with 50% v/v FBS and 3 times with 250 μL of PBS, pH 7.4 . The cells were then incubated for 30 min goat anti-mouse-rhodamine and goat anti-rabbit-AlexaFluor 488

2% BSA.

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secondary antibodies (Santa Cruz, Heidelberg, Germany) diluted 1:100 in PBS supplemented with

Staining of caveolae and clathrin was performed using the mouse anti-Caveolin-1 and the rabbit anti-Clathrin heavy chain antibodies diluted 1:100 in PBS supplemented with 2% BSA (Abcam, Cambridge, UK) for 1 h. The cells were then washed and incubated for 30 min with the goat anti mouse-rhodamine and goat anti rabbit-AlexaFluor 488 (Santa Cruz, Heidelberg, Germany) diluted 1:100 in PBS supplemented with 2% BSA. The glass slides were washed 3 times with PBS, pH 7.4, mounted on microscope slides using the ProLong™ Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA) and analyzed using a

ACCEPTED MANUSCRIPT Leica SP8 confocal imaging system (60x Magnification). Lasers with emission wavelengths at 405, 525, 583 and 666 nm were used to detect DAPI, AlexaFluor 488, Rhodamine and siRac1-Cy5, respectively. Representative images of each dye were captured, and the images were processed with

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ImageJ version 1.51g software. Cells treated with secondary antibodies only were used as control.

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Live cell imaging for intracellular polyplex colocalization with lysosomes

PEG-OCH3 :Agm6 -M-PEG-FA/siRac1-Cy5

at

N/P

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MDA-MB-231 cells were treated with Agm6 -M-PEG-OCH3 /siRac1-Cy5 and 75:25 w/w Agm6 -Mratio

of

3

and

125

nM

siRNA

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concentration. After 2.5 h, Lyostracker Green (Invitrogen) and Hoechst 33342 (Invitrogen) at a concentration of 2.5 μg/ml and 100 nM, respectively, were added and the cells were incubated for

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additional 30 minutes. Then, cells were washed twice with PBS and imaged using Leica SP8

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confocal imaging systems (X63 Magnification) (Leica Microsystems, Wetzlar Germany).

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TEM analysis of MDA-MB-231 cells in presence of polyplexes MDA-MB-231 cells (3×105 cells/well) were seeded in 12 well plates and grow for 24 h. The cells

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were then treated with Agm6 -M-PEG-OCH3 /siRac1 and 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siRac1 3 N/P polyplex solutions (200 nM siRac1 equivalent). After 3 h, the medium was removed, and the cells were washed three times with PBS. The cells were fixed with 0.1 M sodium cacodylate buffer supplemented with 2.5% w/v glutaraldehyde for 1 h at 4°C. Then, the fixed cells were treated using 1% w/v osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h, dehydrated using ethanol and embedded in fresh EPON resin. Ultrathin sections of the samples were cut and observed with a Tecnai G2 Transmission Electron Microscope (FEI, Oregon, USA). The size of vesicles was evaluated using ImageJ version 1.51g software.

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Animals and Ethics Statement All animal procedures were performed in compliance with Tel Aviv University guidelines approved

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by the Institutional Animal Care and Use Committee (IACUC).

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Tumor cell inoculation

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Athymic 7-week-old nu/nu nude mice (Envigo Ltd., Rehovot, Israel) were anesthetized using ketamine (100 mg/kg) and xylazine (12 mg/kg) and intra-mammary inoculated with MDA-MB-231

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tumor cells (2.0×106 cells in 100 μL). The mice body weight and tumor growth were monitored twice a week using a caliper. After 21 days, mice developing circa 100 mm3 tumor were included in

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the in vivo experiments.

Maximum tolerated dose (MTD)

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MDA-MB-231 tumor bearing nu/nu nude mice were daily treated for 3 days by intravenous (i.v.) injection of 100 μL Agm6 -M-PEG-OCH3 :siRac1 or 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-

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PEG-FA/siRac1 3 N/P polyplex solutions (1, 2, 4 and 6 mg/kg siRac1 equivalent doses). Mice were monitored and weighed for 1 month.

Tumor accumulation of siRac1-polyplexes Tumor bearing nu/nu nude mice were daily treated for 3 days by i.v. injection of 100 µL of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1, 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siCtrl 3 N/P polyplexes (4 mg/kg siRNA equivalent dose). On day 4, the animals were

ACCEPTED MANUSCRIPT euthanized, and the tumors were collected, weighed and homogenized. The RNA was isolated using EZ-RNA II total RNA isolation kit (Biological industries) following the manufacturer’s protocol. The isolated RNA was then converted to cDNA using EZ-first strand cDNA synthesis kit for RTPCR (Biological industries). Finally, target genes expression levels were assessed by SYBR green real time PCR (Fast

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SYBRTM green Master Mix, Applied Biosystems; StepOne plus, Life Technologies).

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Biodistribution of folated polyplexes and histological studies

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Tumor-bearing nu/nu nude mice were injected intravenously with 100 μL of 60:25:15 w/w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA:Agm6 -M-PEG-Cy3/siRac1 3 N/P polyplex solutions (4

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mg/kg equivalent siRNA dose). After 0.5, 3 and 6 h, the mice were euthanized, and tumor, spleen, liver, lungs, heart and kidneys were collected and analyzed by CRI Maestro T M fluorescence imaging

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system. Multispectral image-cubes were used through 500–720 nm spectral range in 10 nm steps using excitation (554 nm) and emission (568 nm) filter set. The organ auto-fluorescence and

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background signals were eliminated by spectral analysis and linear un-mixing algorithm. A quantitative analysis of the fluorescence intensity was carried out using the Maestro software and

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the intensity measurements were normalized according to the tumor/organ size. The tumors were fixed in 4% paraformaldehyde for 3 h, maintained in 0.5 M D-sucrose solution for 1 h and moved to 1 M D-sucrose solution for the overnight. The tumors were embedded in OCT (Scigen Inc., Gardena, CA, USA) on dry ice and stored at -80°C. Immunohistochemistry of intramammary tumor was performed using 5 μm thick OCT frozen tissue sections. Staining was performed using the BOND RX autostainer (Leica Biosystems, Wetzlar, Germany). Frozen tumor sections were stained for blood vessels using rat anti-mouse CD31 antibodies (BD biosciences, Caesarea,

Israel)

diluted

1:25,

and

goat

anti-rat-Alexa-488

secondary

antibodies

for

immunofluorescence (Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:300 with PBS.

ACCEPTED MANUSCRIPT The tissues were fixed and mounted on microscope slide, and the nuclei were counterstained with ProLong™ Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). Fluorescence images were captured using a fluorescence microscope (Evos FL Auto, Life Technologies) at 10x and 40x magnification. Lasers with emission wavelengths at 405, 525 and 555 nm were used to

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detect DAPI, Alexa-488 and -Cy3 labelled polyplexes. Untreated tumor was used as control.

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Statistical Analysis

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All the experiments were performed in triplicate, unless otherwise stated. Values are given as means

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were considered to be statistically significant.

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± SD. Two-way ANOVA tests were used where appropriate. * p <0.05, ** p <0.01 or *** p <0.001

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Results and Discussion

Synthesis and characterization of ON carriers

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The novel supramolecular bioconjugates developed for ON delivery were designed according to the evidences reported in the literature showing that polycationic cyclic architectures containing 6

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guanidyl groups efficiently associate with ONs [44], and di-block macromolecules terminating with a few guanidyl groups are less toxic and display higher transfection activity than linear macromolecules with cations distributed along a polymeric backbone [45]. PEG was introduced in the bioconjugate structure to enhance the solubility of the bioconjugates and bestow stealth properties to the carrier, a requisite for intravenous administration of colloidal drug delivery systems [46]. Furthermore, PEG can be easily end-functionalized with a variety of targeting agents endowing systems with bioselectivity. Accordingly, we synthesized di-block bioconjugates formed by: 1. an oligo-cationic star-like shaped “head” (Agm6 -M-COOH) containing 6 guanidyl moieties (agmatines, Agm) attached to the

ACCEPTED MANUSCRIPT hydroxyl groups of a small oligosaccharide (maltotriose, M); 2. a polymeric “tail” (PEG) conjugated to the anomeric carbon of maltotriose through a short alkyl chain; 3. a terminal-PEG function, methoxyl group (OCH3 ), folic acid (FA) or Cyanine 3, to produce non-targeted, targeted and fluorescent bioconjugates, respectively. The structures of the polycationic bioconjugates are

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described in Scheme 1.

Scheme 1. Chemical structure of star-like polymers: A. (agmatinyl) -maltotriosyl-N-acetyl-amino-hexanoate-αmethoxy poly(ethylene glycol) poly(ethylene

glycol)

poly(ethylene glycol)

6kDa

6kDa

5kDa

-folate

6

(Agm6 -M-PEG-OCH3 ); B. (agmatinyl) -maltotriosyl-N-acetyl-amino-hexanoate6

(Agm6 -M-PEG-FA);

C.

(agmatinyl) -maltotriosyl-N-acetyl-amino-hexanoate6

-Cyanine3 (Agm6 -M-PEG-Cy3).

According to the peculiar reactivity of the anomeric carbon of sugars, maltotriose was first derivatized with aminohexanoic acid to obtain M-COOH, which was stabilized by reaction with

ACCEPTED MANUSCRIPT acetic anhydride. This carboxyl function was used as anchoring group to conjugate the NH2 -PEG-X moiety, which could not be straightforward attached to the anomeric carbon. The hydroxyl groups of M-COOH were derivatized with of 2-bromo-isobutyryl-bromide and acryloyl-agmatine by ATRP reaction to introduce guanidyl groups into the oligosaccharide. The star-like oligo-guanidyl “head” (Agm6 -M-COOH) was functionalized with NH2 -PEG-OCH3 to

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yield the non-targeted Agm6 -M-PEG-OCH3 described in Scheme 1A.

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The folated bioconjugate Agm6 -M-PEG-FA described in Scheme 1B was obtained by NH2 -PEG-FA

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conjugation to Agm6 -M-COOH (Scheme ISI) to target folate receptor (FR) overexpressing tumor cells [33, 47, 48].

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The fluorescent bioconjugate Agm6 -M-PEG-Cy3 shown in Figure 1C was synthesized to track polyplexes in biological studies. This bioconjugate was obtained by Agm6 -M-COOH reaction with

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high excess of NH2 -PEG-NH2 to avoid crosslinking and the resulting Agm6 -M-PEG-NH2 was reacted with Cyanine-3-NHS (Cy3-NHS).

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In order to enhance the exposure of the targeting agent on the surface of polyplexes formed by Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA mixtures, the bioconjugates were obtained using PEGs

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with different chain length [49]. Agm6 -M-PEG-OCH3 was obtained by using 5 kDa PEG while Agm6 -M-PEG-FA were obtained with 6 kDa PEG, that should favor the recognition of FR on the

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cell surface.

Polyplex composition selection The optimization of the polyplex composition, namely Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA and N/P ratios, was achieved by ON complexation, gene silencing and cytotoxicity studies performed with polyplexes with different compositions (0:100, 50:50, 75:25 and 100:0 w/w Agm6 -M-PEGOCH3 :Agm6 -M-PEG-FA and 0-5 N/P). siRac1 was used as model ON because this siRNA silences the expression of Rac1, a protein member of GTPase of Rho family overexpressed in many cancer cells that promotes cell migration, adhesion, invasion and finally metastasis [50]. Importantly,

ACCEPTED MANUSCRIPT siRac1 possesses only cytostatic activity, which facilitates the evaluation of gene silencing of the polyplexes with respect to their cytotoxicity. The gel electrophoresis mobility shift assay (EMSA) showed that all the tested carrier compositions displayed identical association profiles, with complete association at 3 N/P ratio, indicating that the negative charge of FA in the Agm6 -M-PEG-FA structure does not interfere with the association of

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the polyanionic siRNA. As an example, Figure 1 reports the gel electrophoresis profile of siRac1

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associated with 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA at increasing N/P ratio.

Figure 1: A. Agarose gel electrophoresis profiles obtained with 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEGFA/siRac1 at increasing N/P ratio (0-5). B. Rac1 silencing (siRac1/siCtrl silencing %) performed on HeLa cells with different Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 compositions C. MTT assay of polyplexes with different Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 compositions. 3 N/P ratio (■); 5 N/P ratio (■). Lipofectamine®/siRac1 (L, □) and unformulated siRac1( ) were used as control positive and negative control, respectively. 0% FA (100:0 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1); 25% FA (75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1), 50% FA (50:50 w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siRac1) and 100% FA (0:100 w/w Agm6 -M-PEG-OCH3 :Ag m6 -M-PEG-FA/siRac1)

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The gene knockdown and cell toxicity studies of the polyplexes were carried out using Rac1psiCHECK T M reporter vector transfected into HeLa cells according to the method described by Polyak et al. [42]. In a preliminary screening (Figure 1SI), this cell line was shown to overexpress the FR. Rac1-psiCHECK®-2 exploits cells transfected with two gene reporters expressing

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fluorescent proteins: Renilla luciferase and Firefly luciferase. The gene reporter of Renilla

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luciferase is fused with siRac1. Therefore, the Rac1 silencing silences the Renilla luciferase expression while the expression of Firefly luciferase is not affected by siRac1, and for such a reason

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is used for normalization of the luminescence signal. Non-formulated siRac1 and scrambled siRNA

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(siCtrl), which does not affect the Rac1 silencing, were used as siRNA reference. Commercial Lipofectamine® (L) was used as carrier reference.

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The gene silencing of the siRac1 formulations was calculated with respect to that obtained with the siCtrl counterparts. The results summarized in Figure 1B (full screening data in Figure 2SI) show polyplexes compositions containing siRac1,

regardless the Agm6 -M-PEG-

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that all folated

OCH3 :Agm6 -M-PEG-FA and N/P ratios, yielded dose dependent gene silencing. Maximal silencing

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was obtained with 250 nM siRac1 while no silencing improvement was obtained with higher doses of siRac1 (Figure 2SI). Furthermore, the gene silencing obtained with the folated polyplexes was

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significantly higher compared to non-folated polyplexes, polyplexes containing siCtrl (Figure 2SI) or non-formulated siRac1. At 250 nM siRac1 dose, 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siRac1 3 N/P yielded about 80% silencing compared to equivalent siRNA doses of 75:25 w/w

Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siCtrl

3

N/P

and

non-formulated

siRac1.

Interestingly, the polyplex with lower content of folated bioconjugate (75:25 w/w Agm6 -M-PEGOCH3 :Agm6 -M-PEG-FA) showed the highest activity indicating that gene silencing did not require high folate density in the ON carrier. This result is in agreement with evidences reported in the literature demonstrating that high folate density on a nanocarrier surface may yield FA residues dimerization, which starves the nanocarriers from cell biorecognition and uptake [51-54].

ACCEPTED MANUSCRIPT The results reported in Figure 1C show that the 3 N/P polyplexes possessed negligible cytotoxicity, while the 5 N/P polyplexes displayed higher cytotoxicity compared to the 3 N/P polyplexes. As expected, the positive control (Lipofectamine®/siRac1 polyplexes) was highly active at a low ON dose (50 nM) but it displayed high cytotoxicity. According to EMSA data showing that the siRNA association was not affected by the carrier

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composition and the evidence that high density of FA did not improve significantly the gene

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silencing, the carrier containing 25% of folated conjugate (75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -

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M-PEG-FA) was selected for further biological studies. The 3 and 5 N/P ratios were also selected because they represent formulations corresponding to the N/P association threshold and formulation

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above the N/P association threshold, respectively.

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Biophysical and biopharmaceutical properties of the polyplexes The DLS profiles reported in Figure 2A and 2C show that siRac1 associated with 75:25 w/w Agm6 -

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M-PEG-OCH3 :Agm6 -M-PEG-FA to form nanosized polyplexes with narrow distribution size. The 3 and 5 N/P polyplexes had 73.16±9.11 (PDI 0.312) and 66.05±7.98 nm (PDI 0.268) “sphere

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equivalent hydrodynamic diameters”, respectively, which is a requisite for prolonged circulation in the bloodstream and passive accumulation in solid tumours by EPR effect [55-57]. Interestingly,

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despite the excess of guanidyl groups with respect to the phosphate groups, the 3 and 5 N/P polyplexes displayed only slight positive -potentials: +3.0 mV and +5.3, respectively. This result seems to indicate that PEG shields the positive charges of the polyplex confirming the hypothesis that the oligo-cationic “head” is in the core of the polyplexes while the polymer “tail” is exposed forming a surface corona. Interestingly, the transmission electron microscopy (TEM) analyses showed that the polyplexes possessed unusual rod-shape morphology compared to the spherical structures of polyplexes described in the literature. It is worth to note that also Lipofectamine® based polyplexes used as reference in the following biological studies form spherical structures. The nano-rods obtained at 3

ACCEPTED MANUSCRIPT and 5 N/P ratios with the novel bioconjugates possessed different aspect ratio, 3.3±0.9 and 5.6±1.2, respectively suggesting that the structure of the polyplexes depends on the ON/polymer ratio. These results are in agreement with those obtained with non-folated polyplexes composed of Agm6 -MPEG-OCH3 . Previous in silico studies showed in fact that the rod-shaped structures result from the decrease of the coulombic repulsion between the charged constituents of a large super-complex of

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many dsDNA assemblies. Calorimetric studies showed that the ON association with the polymer

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involves different enthalpic and entropic phenomena depending on the ON/polymer ratio [37].

Figure 2. DLS size distribution of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/ON 3 and 5 N/P polyplexes

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(Figure 2A and 2C, respectively). TEM images of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA(ON 3 and 5 N/P polyplexes (Figure 2B and 2D, respectively).

The gel electrophoresis images reported in Figure 3A and 3B show that the 3 and 5 N/P polyplexes in buffer were stable in the presence of physiological concentrations of heparin (first raw), an anionic polymer that can compete with the ON for the association with the carrier. Polyplex dissociation was obtained only above 2.5 IU/mL heparin, which is 16-times higher concentration than the physiological value (0.15 IU/mL) [58].

ACCEPTED MANUSCRIPT The images reported in Figure 3C and 3D show that the 3 and 5 N/P polyplexes are stable in plasma at least for 8 h and 24 h, respectively. The higher stability of the 5 N/P polyplex is in agreement with a shift of the association equilibrium towards the associated species in the presence of an excess of complexing bioconjugate. The polyplex incubation with plasma followed by siRNA displacement with heparin yielded band of siRNA that correspond to the double stranded ON, as it

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has the same migration shift of the native free duplex (Figure 3SI). Accordingly, both polyplexes

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are expected to be stable in the bloodstream during the biodistribution process, which takes place in

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a few hours, and in the extracellular matrix (ECM) of the tumor tissue before being taken-up by the

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cells [59].

Figure 3. Heparin displacement assay with 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplexes, (Figure 3A and 3B, respectively). Plasma stability of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEGFA/siRac1 3 and 5 N/P polyplexes (Figure 3C and 3D, respectively).

ACCEPTED MANUSCRIPT Biocompatibility of polyplexes The 75:25 w/w Agm6 -M-PEG-OCH3 /Agm6 -M-PEG-FA/siRac1 3 and 5 N/P polyplexes displayed low hemolysis and negligible pro-inflammatory activity due to the immunoresponse, which are usually the main drawbacks for exploitation of these therapeutic systems [60-62]. The results reported in Figure 4A show that, similarly to dextran (negative control), the polyplexes

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incubated with mouse red blood cells (RBC) elicited negligible hemolysis, even at high

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concentrations, while SDS (positive control) induced high hemolysis. Figure 4B shows that

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undetectable TNF-α (early indicator of inflammatory process) release was observed by human peripheral blood monocyte cells (hPBMCs) incubation with 125 and 250 nM siRac1 dose

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equivalent polyplexes. Even at very high polyplex doses (500 nM siRac1-equivalent) the TNF-α release was remarkably lower than that observed with the positive control LPS. Very high doses of

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polyplexes showed also that the 3 N/P ratio polyplexes were slightly less immunostimulating than the 5 N/P ones, while the carrier alone was more immunostimulating than the corresponding

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polyplexes (Figure 4SI).

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Figure 4. A. Hemolysis test of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 at 3 (green line) and 5 (blue

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line) N/P polyplexes tested at increasing concentrations. Dextran (orange line) and sodium dodecyl sulfate (SDS) (brown line) were used as negative and positive control, respectively. B. TNF-α cytokine secretion from human

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peripheral blood mononuclear cells after 12 h incubation with 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEGFA/siRac1 polyplexes at 3 ( ) and 5 ( ) N/P. 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA polymer mixtures were tested at 3 ( ) and 5 ( ) N/P polymer equivalent dose, respectively. The cells were treated with 125 or 250 nM of

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siRNA equivalent doses. siRac1 alone was tested at 250 nM ( ). Lipopolysaccharides were used as positive control ( ). Untreated cells were used as reference.

The slightly higher hemolytic and immunostimulating effect of 5 N/P polyplexes compared to the 3 N/P ones is probably due to the presence of non-complexed bioconjugate, which has been found to be less biocompatible when it is not complexed with ON. This is also in agreement with the literature reporting that free polycationic macromolecules used for ON delivery may be responsible of poor biocompatibility of drug delivery systems [63].

ACCEPTED MANUSCRIPT Notably, the biocompatibility study showed that the polyplexes induced limited hemolysis and TNF-α release even at higher concentrations than those expected to be administered in vivo [64]. According to these results and the evidences reported above indicating that the 3 N/P polyplexes are less cytotoxic than the 5 N/P polyplexes, 3 N/P ratio was selected for the biological investigations

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Bioactivity of polyplexes.

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Cell transfection and trafficking of 75:25 w/w Agm6 -M-PEG-OCH3 /Agm6 -M-PEG-FA/siRac1 3 N/P polyplexes were investigated using HeLa and MDA-MB-231cell lines transfected with Rac1-

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psiCHECK T M reporter vector (Figure 1SI). MDA-MB-231 cells were used in addition to HeLa cells

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because they represent a suitable model to evaluate the efficacy of siRac1 in breast cancer progression. Furthermore, these cells express higher number of FR and display lower phagocytic

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activity than HeLa cells allowing for better evaluation of the silencing effect of polyplexes on cell migration and their cell uptake.

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The results reported in Figure 5A and 5B show that the 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siRac1 polyplexes induced higher gene silencing than the Agm6 -M-PEG-OCH3 /siRac1

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polyplexes while in the case of both folated and non-folated formulations containing scrambled siRNA (siCtrl) gene silencing was very low. To note that a non-specific silencing was also observed

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in the case of Lipofectamine®, suggesting that this effect could be attributable to off-target effect of siCtrl. The folated polyplexes containing siRac1 showed similar dose-dependent behaviour in the two cell lines: at siRac1 dose of 125 nM, the folated and non-folated polyplexes yielded about 45% and 5% gene silencing, respectively, while at 250 nM the gene silencing was 77.6% and 46.8%, respectively. In the case of HeLa cells, Lipofectamine®/siRac1 polyplexes elicited high gene silencing at very low siRac1 dose (97.5±1.2 % at 50 nM), which is in agreement with the high transfection properties of this carrier.

Surprisingly,

in the case of MDA-MB-231 cells

Lipofectamine®/siRac1 did not yield gene silencing. This unexpected results are in agreement with

ACCEPTED MANUSCRIPT data reported in the literature and could be ascribable to the lower endocytic activity of MDA-MB231 compared to HeLa resulting in lower transfection [65, 66]. The results reported in Figure 5C and 5D confirm the low polyplex cytotoxicity (>85% cell viability) observed in the preliminary studies and the high cytotoxicity of Lipofectamine® (70.6% cell viability) even at the low amount used in the assay.

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The cell migration images of MDA-MB-231 scratched cell cultures reported in Figure 5E show the

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cell mobility inhibition induced by siRac1. The data reported in Figure 5F show that after 12 h

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incubation, the folated and the non-folated polyplexes inhibited the cell migration by about 40% and 10%, respectively, while the non-formulated siRac1 did not inhibit the cell migration. The

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results obtained with the non-folated polyplexes were similar to those obtained with folated and

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non-folated polyplexes containing siCtrl (Figure 5SI).

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Figure 5. Silencing activity and cell viability study on HeLa (A and C) and MDA-MB-231 (B and D) cells. 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 ( ) and Agm6 -M-PEG-OCH3 /siRac1 ( ) polyplexes were incubated with cells for 72 h and the final gene knockdown was measured. Lipofectamine ®/siRac1 ( ) was used as commercial standard. 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siCtrl ( ), Agm6 -M-PEG-OCH3 /siCtrl ( ) polyplexes and Lipofectamine®/siCtrl ( ). Representative images of wound healing closure of MDA -MB-231 cells treated with various polyplexes (E) relative wound density quantification after 12 h cells treatment (F). 75:25 w/w Agm6 -M-PEGOCH3 :Agm6 -M-PEG-FA/siRac1 ( ) and Agm6 -M-PEG-OCH3 /siRac1 ( ) polyplexes were incubated with cells and the cell migration was measured. siRac1 alone ( ) was used as control. 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEGFA)/siCtrl ( ), Agm6 -M-PEG-OCH3 /siCtrl ( ) polyplexes. Cells treated with PBS, pH 7.4 were used as reference ( ).

ACCEPTED MANUSCRIPT Folated and non-folated polyplexes display similar cellular uptake but different intracellular trafficking. The mechanism of cell uptake and trafficking were studied using MDA-MB-231 cells incubated with folated and non-folated polyplexes double labelled with fluorescent Agm6 -M-PEG-Cy3 and siRac1-Cy5.

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Surprisingly, the confocal images reported in Figure 6A and 6B show that folated and non-folated

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polyplexes yield fluorescent spots in the cytosol indicating that both of them were remarkably

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taken-up by the cells. The Agm6 -M-PEG-Cy3 and siRac1-Cy5 signals overlapped indicating that the polyplexes were taken-up by cells without dissociation, which is in agreement with the high

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stability of the polyplexes previously documented. Furthermore, the quantitative data obtained by Imagestream flow cytometer analysis of MDA-MB-231 cells incubated with Cy3-labeled folated

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and non-folated polyplexes reported in Figure 6C show a progressive increase of Cy3 associated fluorescence indicating that the polyplexes were progressively taken-up by cells over the 24 h

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incubation.

The similar cell uptake profiles of folated and non-folated polyplexes were in contrast with the

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different gene silencing observed with the two carriers suggesting that the highest bioactivity obtained with the folated polyplexes could not be simply ascribed to their high cell internalization

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but, likely, to different cell uptake mechanisms. Therefore, in order to elucidate the uptake mechanism and trafficking, FR competition studies were undertaken by MDA-MB-231 cell coincubation with tritiated folic acid ([3 H]FA) and polyplexes containing increasing concentrations of Agm6 -M-PEG-FA. Parallel studies were carried out using non-radioactive FA and siRNA-free Agm6 -M-PEG-FA. Interestingly, the results of competition studies performed using MDA-MB-231 cells summarized in Figure 6D show that neither the folated bioconjugate alone nor the folated polyplexes competed with the [3 H]FA cell uptake. However, the higher activity obtained with the folated polyplexes was in fair agreement with the evidences reported in the literature demonstrating that nanocarriers

ACCEPTED MANUSCRIPT bearing folic acid yield effective transfection despite the low folated polyplexes/FR binding capacity [54, 67-69]. This evidence together with the results reported here seems to indicate that the folated

carrier uptake occurs through an “alternative” FA/FR-mediated

cell internalization

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mechanism.

Figure 6. Confocal analysis of MDA-MB-231 cells incubated with 74:25:1 w/w/w Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA:Agm6 -M-PEG-Cy3/siRac1 polyplexes

(A) and 99:1 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-Cy3/siRac1

polyplexes (B). Single XY plane imaging of Agm6 -M-PEG-Cy3 (red), siRac1-Cy5 (green) and DAPI (blue) nuclei staining showed accumulation of the polyplexes in the cytoplasm after 24 h of incubation. Merged images showed a significant co-localization of the polymer and siRNA. Scale bar is 25 μm.

C. MDA-MB-231 intracellular uptake

quantification at different time points for non-folated ( ) and folated polyplexes ( ) by Imagestream analysis of 99:1 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-Cy3/siRac1 non-folated polyplexes and 74:25:1 w/w/w Agm6 -M-PEGOCH3 :Agm6 -M-PEG-FA:Agm6 -M-PEG-Cy3/siRac1 folated polyplexes. D. [3 H]FA/folated polyplex competition for folic acid receptor displayed on MDA-MB-231 cell surface. FA and polyplexes containing 0%, 5%, 10%, 25%, 50%,

ACCEPTED MANUSCRIPT 100% w/w of Agm6 -M-PEG-FA were incubated for 3 h with MDA-MB-231 cells and [3 H]FA. Cells incubated with [3 H]FA/FA and [3 H]FA only were used as control.

Intracellular trafficking of Agm6 -M-PEG-OCH3 /siRac1-Cy5

and

75:25

w/w Agm6 -M-PEG-

OCH3 :Agm6 -M-PEG-FA/siRac1-Cy5 3 N/P polyplexes in MDA-MB-231 cells was further

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investigated by immunofluorescence analysis after 3 and 24 h incubation. The confocal images reported in Figure 7A1 and 7A2 show that after 3 h incubation, siRac1

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formulated with non-folated polyplexes accumulate into the early endosomes while the images

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reported in Figure 7C1 and 7C2 show that after 24 h incubation siRac1 is predominantly localized

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into the lysosomes. Oppositely, Figure 7B1 and 7B2 shows that after 3 h incubation, siRac1 formulated as folated polyplexes was only partially associated with endosomes and limited traces

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were found into the lysosomes. The images reported in Figure 7D1 and 7D2 show that after 24 h incubation, siRac1 was mainly localized into the cytosol matrix with negligible siRac1 localization

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into lysosomes or endosomes suggesting that folated polyplexes possess endosomal-escape capacity.

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These results are in agreement with the reports by Duarte et al. [69, 70] and Reddy et al. [52] who demonstrated that the folated polyplexes undergo endosomal escape, intracellular dissociation and

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efficient gene silencing. As reported in literature, the bypass of the endo-lysosomal pathway, could explain the silencing efficacy of the folated polyplexes that can escape endosomes and dissociate into the cytosol [71]. These results were confirmed by confocal imaging of living cells showing that the folated polyplexes colocalized to a lower extent with LisoTracker than the non-folated ones (Figure 7SI)

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ACCEPTED MANUSCRIPT Figure 7. Co-localization studies of folated and non-folated polyplexes in MDA-MB-231 cells with endosomes (A1, B1 and C1) and lysosomes (A2, B2 and C2). A1, A2: Agm6 -M-PEG-OCH3 /siRac1-Cy5 polyplexes after 3 h incubation. C1 and C2: Agm6 -M-PEG-OCH3 /siRac1-Cy5 polyplexes after 24 h incubation. B1, B2: Agm6 -M-PEG-OCH3 :Agm6 -MPEG-FA/siRac1-Cy5 polyplexes after 3 h incubation. D1 and D2: 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEGFA/siRac1-Cy5 polyplexes after 24 h incubation. Cells were stained for early endosomes (red) and lysosomes (cyan). Polyplexes were labelled using siRac1-Cy5

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(green). Nuclei were stained with DAPI (blue). The scale bar is 5 μm.

This hypothesis was confirmed by cell uptake studies performed by the confocal and TEM analysis

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of MDA-MB-231 cell incubated with Agm6 -M-PEG-OCH3 /siRac1-Cy5 and 75:25 w/w Agm6 -M-

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PEG-OCH3 :Agm6 -M-PEG-FA/siRac1-Cy5 3 N/P polyplexes. In the confocal studies, caveolae were labelled with mouse anti-caveolin-1 antibodies (anti-Cav-1) and rhodamine-labeled goat anti-

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mouse secondary antibodies, and clathrin was labelled with rabbit anti-clathrin heavy chain antibodies (anti-CHC) and Alexa Fluor® 488 labeled goat anti-rabbit secondary antibodies.

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The images reported in Figure 8A1 and 8A2 show that siRac1-Cy5 formulated with non-folated polyplexes mainly associated within clathrins. Conversely, Figure 8A3 and 8A4 shows that siRac1-

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Cy5 formulated with folated polyplexes remarkably associated with caveolae. The TEM images reported in Figure 8B1 and 8B2 show that the non-folated polyplexes are associated with 80-100

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nm pit-coated invaginations and vesicles, which are typical of clathrin vesicles, while folated polyplexes are associated with surface connected large invaginations known as caveosomes that originate small caveolae vesicles [72, 73]. Unfortunately, it was not possible to appreciate the presence of the polyplexes since they are soft-matter and are not visible with this technique. However, the TEM images show differences on the cell membrane morphology (Figure 6SI). In light of the results obtained by confocal analysis, we speculated that these differences are ascribable to the polyplexes composition. Therefore, these results support that the non-folated polyplexes are cell internalized through a clathrin-dependent endocytosis to be transported to early endosomes and finally accumulated into lysosomes. This behavior can be explained with the findings reported in

ACCEPTED MANUSCRIPT the literature, which demonstrate that particles with aspect ratio around 4 are typically taken-up by cells by clathrin pathways [74-76]. Nevertheless, the folated polyplexes are preferentially internalized by a caveolin-dependent mechanism with final disposition within the cytosol indicating that the cell uptake of the targeted polyplexes is not dictated by the morphology but by specific cell surface interaction and suggesting that folic acid prevents the internalization of the rod-shaped

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polyplexes through the clathrin-dependent mechanism. This is in agreement with the results

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obtained by Gabrielson et al. [77] who demonstrated that folated polyethylenimine/pDNA

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polyplexes undergo caveolin-mediated cell uptake resulting in efficient gene transfection and silencing [71]. It was also demonstrated that early endosomal vesicles generated by the caveolin-

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which may result in the ON degradation [78].

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mediated uptake may have a different intracellular fate with respect to clathrin-mediated transport,

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ACCEPTED MANUSCRIPT

Figure 8 A. Intracellular trafficking study of Agm6 -M-PEG-OCH3 /siRac1-Cy5 (Figure A1, A2) and 75:25 w/w Agm6 M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1-Cy5 (Figure A3, A4) 3 N/P polyplexes in MDA-MB-231 cells. Colocalization of siRac1-Cy5 (green) with caveolin-1 (red) and clathrin heavy chain (cyan) was observed after 3 h of incubation. Nuclei were stained with DAPI (blue). The scale bar is 5 μm. B. TEM images obtained after 3 h incubation of folated and non -folated polyplexes with MDA-MB-231. B1 clathrin vesicles obtained by non-folated polyplexes; B2. caveolae vesicles obtained with folated polyplexes. Untreated cells in Figure 8SI were used as control. Scale bar is 500 nm.

ACCEPTED MANUSCRIPT In vivo studies. Preliminary in vivo studies were carried out to evaluate the in vivo toxicity and biodistribution of polyplexes. Toxicity studies were carried out by intravenous administration of 1 to 6 mg/kg siRac1 equivalent doses of 75:25 w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA/siRac1 3 N/P polyplexes to

administration without signs of pain or weight loss (Figure 9SI).

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Nu/Nu female mice. All animals were in good health (Table 1SI) over the seven days from

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Biodistribution studies were carried by intravenous administration of Agm6 -M-PEG-Cy3-labeled

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folated 3 N/P polyplexes and unformulated siRac1 to mice bearing orthotopic intra-mammary MDA-MB-231 human adenocarcinoma tumors. The polyplexes biodistribution was evaluated by

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siRac1 tumor accumulation, organ fluorescence imaging and fluorescence tissue microscopy. The polyplex accumulation in the tumor after 3 h

was about 2.53 times higher than in the liver

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(6.36 scaled counts s-1 cm-2 and 2.51 scaled counts s-1 cm-2 , respectively), while no significant accumulation was observed in the spleen, kidneys, lungs and heart (Figure 10SI).

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Figure 9A shows the siRac1 levels measured by RT-PCR. After 4 days from administration, the siRac1 levels obtained with the folated formulation resulted ~500-fold higher than those expressed

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after administration of folated polyplexes with siCtrl, respectively. The histology images reported in Figure 9B show that the polyplexes progressively accumulate in

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the tumor resulting in increased fluorescent spots. Histological images of tissue from untreated animals are reported in Supporting Information (Figure 11SI). Finally, the in vivo studies showed a significant time-dependent accumulation of the polyplexes into the tumor tissues, which is ascribable to the colloidal size and stability of the polyplexes. The delayed accumulation in the liver and the negligible disposition in the mononuclear phagocyte system (MPS) organs, namely lungs, spleen and kidneys, responsible for the clearance of opsonized particles (Figure 5SI), confirmed the PEG shielding from protein adsorption and erythrocytes aggregation [79, 80].

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Figure 9 A. Tumor accumulation of 75:25 w/w Agm6 -M-PEG-OCH3 :A1gm6 -M-PEG-FA/siRac1 3 N/P polyplexes following systemic administration into orthotopic MDA -MB-231 tumor bearing mice. PBS and 75:25 w/w Agm6 -MPEG-OCH3 :Agm6 -M-PEG-FA/siCtrl polyplexes were used as control. B. Histological analysis of tumor tissue after 30 min, 3 and 6 h administration of 74:25:1 w/w/w Agm6 -M-PEG-OCH3 :Agm6 -M-PEG-FA:Agm6 -M-PEG-Cy3/siRac1 3 N/P polyplexes. Tumor tissues were stained with DAPI (blue) and CD31-FITC (green). Polyplexes were stained with Cyanine 3 (red). Scale bar is 100 μm.

ACCEPTED MANUSCRIPT Conclusions The novel bioconjugates for ON delivery described here have been found to form nanorods with high ON association properties, remarkable silencing profile and negligible toxicity either in vitro or in vivo [81]. The nanorod shaped polyplexes are unspecifically taken-up by cells and localize mainly in lysosomes, which results in limited bioactivity. On the contrary, the polyplexes bearing a

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targeting agent have been found to skip the lysosome disposition. These polyplexes were found to

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undergo cell up-take through a caveolin pathway that yield the cytosol delivery of the ON.

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This study suggests the possible crosstalk between the surface properties for biorecognition and the morphology of colloidal carriers which, in combination, impact the intracellular and in vivo fate of

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these system resulting in unique biopharmaceutical performances.

However, further investigations are required to better understand the mechanisms behind the

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intracellular uptake and tumor accumulation of the polyplexes.

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Conflict of interest

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The authors declare no conflict of interest.

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Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript

ACCEPTED MANUSCRIPT Acknowledgements This research has been partially supported from EU Horizon 2020 research and Innovation program under the MCS grant agreement number 722717 (OCUTHER). The Satchi-Fainaro laboratory's research leading to these results received partial funding from the European Research Council

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(ERC) under the European Union's Seventh Framework Programme / ERC Consolidator Grant Agreement n. [617445]- PolyDorm. We would like to thank the Sackler Cellular & Molecular

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Imaging Center (SCMIC) for the imaging service, and special thanks to Dr. Alexandra Lichtenstein,

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Ph.D., for her kind and professional assistance.

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Supporting Information

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The Supporting Information is available free of charge on the Elsevier Publications website.

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Data Availability

References

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on reasonable request.

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All data generated and analyzed during current study are available from the corresponding author

[1] S. Akhtar, I.F. Benter, Nonviral delivery of synthetic siRNAs in vivo, The Journal of clinical investigation, 117 (2007) 3623-3632. [2] R.L. Juliano, The delivery of therapeutic oligonucleotides, Nucleic acids research, 44 (2016) 6518-6548. [3] A. Judge, I. Maclachlan, Overcoming the innate immune response to small interfering RNA, Human gene therapy, 19 (2008) 111-124. [4] M. Edbrooke, N. Clarke, RNAi Therapeutics: Addressing Targets? Eur, Pharm. Rev, 4 (2008) 11-17. [5] G.F. Deleavey, M.J. Damha, Designing chemically modified oligonucleotides for targeted gene silencing, Chemistry & biology, 19 (2012) 937-954.

ACCEPTED MANUSCRIPT [6] N. Shokrzadeh, A.-M. Winkler, M. Dirin, J. Winkler, Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents, Bioorganic & medicinal chemistry letters, 24 (2014) 5758-5761. [7] L. Kumar, S. Verma, B. Vaidya, V. Gupta, Exosomes: natural carriers for siRNA delivery, Current pharmaceutical design, 21 (2015) 4556-4565. [8] P. Zhang, K. An, X. Duan, H. Xu, F. Li, F. Xu, Recent advances in siRNA delivery for cancer therapy using smart nanocarriers, Drug Discov Today, 23 (2018) 900-911.

PT

[9] Y. Xin, M. Huang, W.W. Guo, Q. Huang, L. zhen Zhang, G. Jiang, Nano-based delivery of RNAi in cancer therapy, Molecular cancer, 16 (2017) 134. [10] K. Gupta, A. Puri, B.A. Shapiro, Functionalized non-viral cationic vectors for effective siRNA induced cancer therapy, DNA and RNA Nanotechnology, 4 (2017) 1-20.

RI

[11] D. Bumcrot, M. Manoharan, V. Koteliansky, D.W. Sah, RNAi therapeutics: a potential new class of pharmaceutical drugs, Nature chemical biology, 2 (2006) 711-719.

SC

[12] W. Gelbart, R. Bruinsma, P. Pincus, V. Parsegian, Phys. Today, 53, No., 9 (2000) 38.

NU

[13] V.S. Trubetskoy, L.J. Hanson, P.M. Slattum, J.E. Hagstrom, V.G. Budker, J.A. Wolff, Selfassembly of DNA—polymer complexes using template polymerization, Nucleic acids research, 26 (1998) 4178-4185.

MA

[14] S. May, D. Harries, A. Ben-Shaul, The phase behavior of cationic lipid–DNA complexes, Biophysical Journal, 78 (2000) 1681-1697. [15] J.O. Rädler, I. Koltover, T. Salditt, C.R. Safinya, Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes, Science, 275 (1997) 810-814.

ED

[16] P.V.G.M. Rathnayake, B.G.C.M. Gunathunge, P.N. Wimalasiri, D.N. Karunaratne, R.J.K.U. Ranatunga, Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study, Journal of Biophysics, 2017 (2017) 12.

EP T

[17] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer research, 46 (1986) 6387-6392.

AC C

[18] M. Ogris, G. Walker, T. Blessing, R. Kircheis, M. Wolschek, E. Wagner, Tumor-targeted gene therapy: strategies for the preparation of ligand–polyethylene glycol–polyethylenimine/DNA complexes, Journal of Controlled Release, 91 (2003) 173-181. [19] M. Hashida, M. Nishikawa, F. Yamashita, Y. Takakura, Cell-specific delivery of genes with glycosylated carriers, Advanced drug delivery reviews, 52 (2001) 187-196. [20] C.H. Wu, J.M. Wilson, G. Wu, Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo, Journal of Biological Chemistry, 264 (1989) 16985-16987. [21] D.V. Schaffer, D.A. Lauffenburger, Targeted synthetic gene delivery vectors, Curr. Opin. Mol. Ther, 2 (2000) 155. [22] Y. Liu, K. Li, B. Liu, S.-S. Feng, A strategy for precision engineering of nanoparticles of biodegradable copolymers for quantitative control of targeted drug delivery, Biomaterials, 31 (2010) 9145-9155. [23] D. Vercauteren, J. Rejman, T.F. Martens, J. Demeester, S.C. De Smedt, K. Braeckmans, On the cellular processing of non-viral nanomedicines for nucleic acid delivery: mechanisms and methods, Journal of controlled release, 161 (2012) 566-581.

ACCEPTED MANUSCRIPT [24] G. Sahay, D.Y. Alakhova, A.V. Kabanov, Endocytosis of nanomedicines, Journal of controlled release, 145 (2010) 182-195. [25] C. Chakraborty, A.R. Sharma, G. Sharma, C.G.P. Doss, S.-S. Lee, Therapeutic miRNA and siRNA: moving from bench to clinic as next generation medicine, Molecular Therapy-Nucleic Acids, 8 (2017) 132-143. [26] N.C. Bellocq, S.H. Pun, G.S. Jensen, M.E. Davis, Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery, Bioconjugate chemistry, 14 (2003) 11221132.

PT

[27] M.E. Davis, The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic, Molecular pharmaceutics, 6 (2009) 659-668.

RI

[28] S. Nazir, T. Hussain, A. Ayub, U. Rashid, A.J. MacRobert, Nanomaterials in combating cancer: therapeutic applications and developments, Nanomedicine: Nanotechnology, Biology and Medicine, 10 (2014) 19-34.

SC

[29] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer therapy, Advanced drug delivery reviews, 56 (2004) 1649-1659.

NU

[30] S.M. Moghimi, P. Symonds, J.C. Murray, A.C. Hunter, G. Debska, A. Szewczyk, A two-stage poly (ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy, Molecular Therapy, 11 (2005) 990-995.

MA

[31] D.J. Gary, H. Lee, R. Sharma, J.-S. Lee, Y. Kim, Z.Y. Cui, D. Jia, V.D. Bowman, P.R. Chipman, L. Wan, Influence of nano-carrier architecture on in vitro siRNA delivery performance and in vivo biodistribution: polyplexes vs micelleplexes, ACS nano, 5 (2011) 3493-3505.

ED

[32] C.E. Nelson, J.R. Kintzing, A. Hanna, J.M. Shannon, M.K. Gupta, C.L. Duvall, Balancing cationic and hydrophobic content of PEGylated siRNA polyplexes enhances endosome escape, stability, blood circulation time, and bioactivity in vivo, ACS nano, 7 (2013) 8870-8880.

EP T

[33] A.R. Hilgenbrink, P.S. Low, Folate receptor‐ mediated drug targeting: From therapeutics to diagnostics, Journal of pharmaceutical sciences, 94 (2005) 2135-2146. [34] P.S. Low, W.A. Henne, D.D. Doorneweerd, Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases, Accounts of chemical research, 41 (2007) 120-129.

AC C

[35] D.M. Kranz, T.A. Patrick, K.E. Brigle, M.J. Spinella, E.J. Roy, Conjugates of folate and antiT-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis, Proceedings of the National Academy of Sciences, 92 (1995) 9057-9061. [36] A. Puig-Kröger, E. Sierra-Filardi, A. Domínguez-Soto, R. Samaniego, M.T. Corcuera, F. Gómez-Aguado, M. Ratnam, P. Sánchez-Mateos, A.L. Corbí, Folate receptor β is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages, Cancer research, 69 (2009) 9395-9403. [37] A. Malfanti, F. Mastrotto, Y. Han, P. Král, A. Balasso, A. Scomparin, S. Pozzi, R. SatchiFainaro, S. Salmaso, P. Caliceti, Novel Oligo-Guanidyl-PEG Carrier Forming Rod-Shaped Polyplexes, Molecular Pharmaceutics, 16 (2019) 1678-1693. [38] S. Bersani, S. Salmaso, F. Mastrotto, E. Ravazzolo, A. Semenzato, P. Caliceti, Star-like oligoarginyl-maltotriosyl derivatives as novel cell-penetrating enhancers for the intracellular delivery of colloidal therapeutic systems, Bioconjugate chemistry, 23 (2012) 1415-1425. [39] C. Brazzale, R. Canaparo, L. Racca, F. Foglietta, G. Durando, R. Fantozzi, P. Caliceti, S. Salmaso, L. Serpe, Enhanced selective sonosensitizing efficacy of ultrasound-based anticancer treatment by targeted gold nanoparticles, Nanomedicine, 12 (2016) 3053-3070.

ACCEPTED MANUSCRIPT [40] S. Sakaguchi, A new color reaction of protein and arginine, J. Biochem, 5 (1925) 25-31. [41] G. Sims, T. Snape, A method for the estimation of polyethylene glycol in plasma protein fractions, Analytical biochemistry, 107 (1980) 60-63. [42] D. Polyak, A. Krivitsky, A. Scomparin, S. Eliyahu, H. Kalinski, S. Avkin-Nachum, R. SatchiFainaro, Systemic delivery of siRNA by aminated poly (α) glutamate for the treatment of solid tumors, Journal of controlled release: official journal of the Controlled Release Society, 257 (2017) 132-143.

PT

[43] A.H. Cory, T.C. Owen, J.A. Barltrop, J.G. Cory, Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture, Cancer communications, 3 (1991) 207-212.

RI

[44] M. Ahmed, Peptides, polypeptides and peptide–polymer hybrids as nucleic acid carriers, Biomaterials science, 5 (2017) 2188-2211.

SC

[45] H. Wei, D.S. Chu, J. Zhao, J.A. Pahang, S.H. Pun, Synthesis and evaluation of cyclic cationic polymers for nucleic acid delivery, ACS macro letters, 2 (2013) 1047-1050. [46] Y. Ikeda, Y. Nagasaki, Impacts of PEGylation on the gene and oligonucleotide delivery system, Journal of Applied Polymer Science, 131 (2014).

NU

[47] E. Gallon, T. Matini, L. Sasso, G. Mantovani, A. Armiñan de Benito, J. Sanchis, P. Caliceti, C. Alexander, M.J. Vicent, S. Salmaso, Triblock copolymer nanovesicles for pH-responsive targeted delivery and controlled release of siRNA to cancer cells, Biomacromolecules, 16 (2015) 1924-1937.

MA

[48] N. Parker, M.J. Turk, E. Westrick, J.D. Lewis, P.S. Low, C.P. Leamon, Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay, Analytical biochemistry, 338 (2005) 284-293.

EP T

ED

[49] T. Shiokawa, Y. Hattori, K. Kawano, Y. Ohguchi, H. Kawakami, K. Toma, Y. Maitani, Effect of polyethylene glycol linker chain length of folate-linked microemulsions loading aclacinomycin A on targeting ability and antitumor effect in vitro and in vivo, Clinical cancer research, 11 (2005) 2018-2025. [50] H.K. Bid, R.D. Roberts, P.K. Manchanda, P.J. Houghton, RAC1: an emerging therapeutic option for targeting cancer angiogenesis and metastasis, Molecular cancer therapeutics, 12 (2013) 1925-1934.

AC C

[51] R.J. Lee, P.S. Low, Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis, Journal of Biological Chemistry, 269 (1994) 3198-3204. [52] J. Reddy, C. Abburi, H. Hofland, S. Howard, I. Vlahov, P. Wils, C. Leamon, Folate-targeted, cationic liposome-mediated gene transfer into disseminated peritoneal tumors, Gene therapy, 9 (2002) 1542. [53] F. Ciuchi, G. Di Nicola, H. Franz, G. Gottarelli, P. Mariani, M.G. Ponzi Bossi, G.P. Spada, Self-recognition and self-assembly of folic acid salts: columnar liquid crystalline polymorphism and the column growth process, Journal of the American Chemical Society, 116 (1994) 7064-7071. [54] S.K. Jones, A. Sarkar, D.P. Feldmann, P. Hoffmann, O.M. Merkel, Revisiting the value of competition assays in folate receptor-mediated drug delivery, Biomaterials, 138 (2017) 35-45. [55] R.A. Petros, J.M. DeSimone, Strategies in the design of nanoparticles for therapeutic applications, Nature reviews Drug discovery, 9 (2010) 615-627. [56] H. Lee, A.K. Lytton-Jean, Y. Chen, K.T. Love, A.I. Park, E.D. Karagiannis, A. Sehgal, W. Querbes, C.S. Zurenko, M. Jayaraman, Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery, Nature nanotechnology, 7 (2012) 389-393.

ACCEPTED MANUSCRIPT [57] K. Itaka, K. Yamauchi, A. Harada, K. Nakamura, H. Kawaguchi, K. Kataoka, Polyion complex micelles from plasmid DNA and poly (ethylene glycol)–poly (l-lysine) block copolymer as serumtolerable polyplex system: physicochemical properties of micelles relevant to gene transfection efficiency, Biomaterials, 24 (2003) 4495-4506. [58] H. Engelberg, A. Dudley, Plasma heparin levels in normal man, Circulation, 23 (1961) 578581. [59] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Advanced drug delivery reviews, 65 (2013) 71-79.

PT

[60] M. Robbins, A. Judge, I. MacLachlan, siRNA and innate immunity, Oligonucleotides, 19 (2009) 89-102.

RI

[61] J.T. Marques, B.R. Williams, Activation of the mammalian immune system by siRNAs, Nature biotechnology, 23 (2005) 1399.

SC

[62] X. Chen, Y. Liu, L. Wang, Y. Liu, W. Zhang, B. Fan, X. Ma, Q. Yuan, G. Ma, Z. Su, Enhanced humoral and cell-mediated immune responses generated by cationic polymer-coated PLA microspheres with adsorbed HBsAg, Molecular pharmaceutics, 11 (2014) 1772-1784.

NU

[63] D.J. Gary, J. Min, Y. Kim, K. Park, Y.Y. Won, The effect of N/P ratio on the in vitro and in vivo interaction properties of PEGylated poly [2‐ (dimethylamino) ethyl methacrylate]‐ based siRNA complexes, Macromolecular bioscience, 13 (2013) 1059-1071.

MA

[64] L. Dai, L. Wang, L. Deng, J. Liu, J. Lei, D. Li, J. He, Novel multiarm polyethylene glycoldihydroartemisinin conjugates enhancing therapeutic efficacy in non-small-cell lung cancer, Sci Rep, 4 (2014) 5871.

ED

[65] T. Wang, L. Larcher, L. Ma, R. Veedu, Systematic screening of commonly used commercial transfection reagents towards efficient transfection of single-stranded oligonucleotides, Molecules, 23 (2018) 2564.

EP T

[66] M. KOBAYASHI, T. HOSHINO, Phagocytic activity of HeLa cells after thymidine treatment, Archivum histologicum japonicum, 46 (1983) 479-489. [67] W. Guo, R.J. Lee, Receptor-targeted gene delivery viafolate-conjugated polyethylenimine, The AAPS Journal, 1 (1999) 20-26.

AC C

[68] S. Yang, X. Yang, Y. Liu, B. Zheng, L. Meng, R.J. Lee, J. Xie, L. Teng, Non-covalent complexes of folic acid and oleic acid conjugated polyethylenimine: An efficient vehicle for antisense oligonucleotide delivery, Colloids and Surfaces B: Biointerfaces, 135 (2015) 274-282. [69] S. Duarte, H. Faneca, M.C.P. de Lima, Folate-associated lipoplexes mediate efficient gene delivery and potent antitumoral activity in vitro and in vivo, International journal of pharmaceutics, 423 (2012) 365-377. [70] S. Duarte, H. Faneca, M.C.P. de Lima, Non-covalent association of folate to lipoplexes: a promising strategy to improve gene delivery in the presence of serum, Journal of controlled release, 149 (2011) 264-272. [71] J. Rejman, M. Conese, D. Hoekstra, Gene transfer by means of lipo-and polyplexes: role of clathrin and caveolae-mediated endocytosis, Journal of liposome research, 16 (2006) 237-247. [72] Z. Wang, C. Tiruppathi, R.D. Minshall, A.B. Malik, Size and dynamics of caveolae studied using nanoparticles in living endothelial cells, ACS nano, 3 (2009) 4110-4116.

ACCEPTED MANUSCRIPT [73] S.E. Miller, S. Mathiasen, N.A. Bright, F. Pierre, B.T. Kelly, N. Kladt, A. Schauss, C.J. Merrifield, D. Stamou, S. Höning, CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature, Developmental cell, 33 (2015) 163-175. [74] X. Liu, F. Wu, Y. Tian, M. Wu, Q. Zhou, S. Jiang, Z. Niu, Size dependent cellular uptake of rod-like bionanoparticles with different aspect ratios, Scientific reports, 6 (2016) 24567. [75] S. Shukla, F.J. Eber, A.S. Nagarajan, N.A. DiFranco, N. Schmidt, A.M. Wen, S. Eiben, R.M. Twyman, C. Wege, N.F. Steinmetz, The impact of aspect ratio on the biodistribution and tumor homing of rigid soft‐ matter nanorods, Advanced healthcare materials, 4 (2015) 874-882.

PT

[76] Y. He, K. Park, Effects of the microparticle shape on cellular uptake, Molecular pharmaceutics, 13 (2016) 2164-2171.

RI

[77] N.P. Gabrielson, D.W. Pack, Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells, Journal of Controlled Release, 136 (2009) 54-61.

SC

[78] A.L. Kiss, E. Botos, Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation?, Journal of cellular and molecular medicine, 13 (2009) 1228-1237.

NU

[79] D.B. Fenske, I. MacLachlan, P.R. Cullis, Long-circulating vectors for the systemic delivery of genes, Current opinion in molecular therapeutics, 3 (2001) 153-158.

MA

[80] P.R. Dash, M.L. Read, L.B. Barrett, M.A. Wolfert, L.W. Seymour, Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery, Gene Ther, 6 (1999) 643-650.

AC C

EP T

ED

[81] S. Yamano, J. Dai, A.M. Moursi, Comparison of transfection efficiency of nonviral gene transfer reagents, Molecular biotechnology, 46 (2010) 287-300.