Impact of PEG Chain Length on the Physical Properties and Bioactivity

Mar 23, 2017 - Grazieli Olinda Martins , Maicon Segalla Petrônio , Aline Margarete Furuyama Lima , André Miguel Martinez Junior , Vera Aparecida de ...
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Impact of PEG chain length on the physical properties and bioactivity of PEGylated chitosan/siRNA nanoparticles in vitro and in vivo Chuanxu Yang, Shan Gao, Frederik Dagnaes-Hansen, Maria Jakobsen, and Jørgen Kjems ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on March 25, 2017

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Impact of PEG chain length on the physical properties and bioactivity of PEGylated chitosan/siRNA nanoparticles in vitro and in vivo Chuanxu Yang1,2*, Shan Gao1,2,4, Frederik Dagnæs-Hansen3, Maria Jakobsen1,2, Jørgen Kjems1,2* 1. Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark 2. Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark 3. Department of Biomedicine, Aarhus University, Bartholin Building Building 1240, Wilhelm Meyers Alle 4,8000 Aarhus C, Denmark 4. Suzhou Ribo Life Science Co., Ltd, Beijing, China. Correspondence Chuanxu Yang Interdisciplinary Nanoscience Center (iNANO) Aarhus University Gustav Wieds Vej 14 DK-8000 Aarhus E-mail: [email protected] Jørgen Kjems Interdisciplinary Nanoscience Center (iNANO) Aarhus University Gustav Wieds Vej 14 DK-8000 Aarhus E-mail: [email protected]

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Abstract PEGylation of cationic polyplexes is a promising approach to enhance the stability and reduce unspecific interaction with biological components. Herein, we systematically investigate the impact of PEGylation on physical and biological properties of chitosan/siRNA polyplexes. A series of chitosan-PEG copolymers (CS-PEG2k, CS-PEG5k and CS-PEG10k) were synthesized with similar PEG mass content but with different molecular weight. PEGylation with higher molecular weight and less grafting degree resulted in smaller and more compacted nanoparticles with relatively higher surface charge. PEGylated

polyplexes

showed

distinct

mechanism

of

endocytosis,

which

was

macropinocytosis and caveolae-dependent and clathrin-independent. In vitro silencing efficiency in HeLa and H1299 cells was significantly improved by PEGylation and CSPEG5k/siRNA

achieved

the

highest

knockdown

efficiency.

Efficient

silence

of

ribonucleotide reductase subunit M2 (RRM2) in HeLa cells by CS-PEG5k/siRRM2 significantly induced cell cycle arrest and inhibited cell proliferation. In addition, PEGylation significantly inhibited macrophage phagocytosis and unspecific interaction with red blood cells (RBCs).

Significant extension of in vivo circulation was achieved only with high

molecular weight PEG modification (CS-PEG10k), whereas all CS/siRNA and CSPEG/siRNA nanoparticles showed similar pattern of biodistribution with major accumulation in liver and kidney. These results imply that PEGylation with higher molecular weight PEG and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both in vitro and in vivo. Keywords: Chitosan, PEGylation, siRNA delivery, Nanoparticles, Blood circulation, Biodistribution

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1. Introduction Small interfering RNA (siRNA), known to specifically silence target genes in a sequencedependent manner, provides a promising therapeutic approach for treatment of various diseases including cancer and inflammatory diseases.1-2 However, the difficulties of efficient siRNA delivery limit its wide therapeutic applications. The large size (~13.3kDa) and negative charge of the siRNA makes it unable to cross cellular membranes and the rapid enzymatic degradation challenges its integrity in the plasma and intracellular cytosol. Therefore, improved vectors are necessary for both siRNA protection and cellular internalization. Cationic polymers or liposomes are often introduced to condense siRNA into nanocomplexes and promote cell endocytosis. Chitosan, a cationic polysaccharide, has attracted wide attention as a promising vector for siRNA delivery due to its excellent biocompatibility, biodegradability and low immunogenic properties.3-5 The repeated unit containing a primary amine group favors the electrostatic interactions with anionic siRNA to form nanosized polyplexes. The compact nanoparticles protect siRNA from degradation by enzymes while the overall positive charge contributes to the cellular adhesion and internalization. Additionally, the “proton sponge’’ effect of the amine groups helps siRNA escape from the endosome, explaining the robust knockdown accomplished by chitosan/siRNA complexes both in vitro and in vivo.6-7 However, chitosan is only soluble in acidic aqueous solutions with a pH below its pKa value (~6.5), which may be incompatible with some biological systems and reduced transfection efficiency under physiological pH. In addition, intravenous injection of cationic polyplexes often leads to particle instability, protein absorption and aggregation with red blood cells, which limits their potential clinical applications.8-9

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To overcome the issues of cationic nanocomplexes, surface modification of polyethylene glycol (PEG), a process generally known as PEGylation, is an attractive method to improve their stability by shielding their excess charge. PEG is an amphiphilic, non-toxic, biodegradable and biocompatible polymer that is widely used for surface coating to prevent protein adhesion and activation of the immune system.10-11 PEGylation has been widely applied for biomacromolecules delivery as well as nanoparticle and liposome surface coating to enhance stability both in vitro and in vivo.12-13 It has been shown that PEG grafted on cationic polymers poly (ethylene imine) (PEI) and poly (amidoamine) dendrimers (PAMAM) greatly improved their stability, gene silencing effect and biocompatibility.14-16 However, detailed study with PEGylated chitosan for siRNA delivery has been less studied, especially its effect on gene silencing and in vivo pharmacokinetics. Regarding PEGylation of polyplexes, two major factors need to be considered including PEG chain length and graft density. In this study, we systematically investigate the influence of PEG chain length on the physicochemical and biological properties of chitosan/siRNA based polyplexes. A series of Chitosan-PEG copolymers were synthesized with similar PEG mass content but with different molecular weight, including 2 kDa, 5 kDa and 10 kDa (CS-PEG2k, CS-PEG5k, CS-PEG 10k). We found that PEG length greatly influences both the physiochemical and biological properties of chitosan/siRNA polyplex. PEGylation with higher molecular weight PEG and less grafting degree resulted in smaller nanoparticles with relatively high positive charge. PEGylated polyplexes were mainly taken up by cell through macropinocytosis and caveolea pathways. In vitro silencing effect was significantly improved PEGylation and CS-PEG5k/siRNA achieved the highest knockdown efficiency. In addition, longer in vivo circulation time was achieved only with high molecular weight PEG modification. Therefore, PEG with higher molecular weight and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both

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in vitro and in vivo. These data provide detailed information for optimizing chitosan/siRNA nanoparticles for in vivo application as well as fundamental insights for design of PEGylated polyplexes for siRNA delivery. 2. Materials and Methods 2.1 Materials Chitosan (molecular weight 150 kDa, 95% deacetylation) was provided from Heppe Medical Chitosan GmbH (Frankfurt, Germany). Methoxy PEG succinimidyl ester (mPEGNHS) with MW 2 kDa, 5 kDa and 10 kDa were purchased from Rapp Polymere (Tubingen, Germany).

siRNA

against

GFP

(siGFP)

with

the

sequence:

sense,

5’-

GACGUAAACGGCCACAAGUTC-3’, antisense, 5’-ACUUGUGGCCGUUUACGUCGC-3’; siRRM2:

sense,

5’-GAUUUAGCCAAGAAGUUCAGA-3’,

antisense,

5’-

UGAACUUCUUGGCUAAAUCGC-3’ and Cy3 or Cy5 labeled siGFP were purchased from Ribotask (Odense, Denmark). 2.2 Synthesis of Chitosan-PEG (CS-PEG) copolymers Commercial chitosan was purified by first dissolving in 1% acetic acid solution and precipitated by slowly adding 1 M NaOH solution dropwise under stirring. The precipitate was filtered and washed with water until neutral pH before lyophilization. To synthesize CS-PEG copolymers, chitosan were dissolved in acetate buffer (200mM pH 5.5) and filtered through 0.45 µm Minisart® Syringe Filters (Sartorius stedim biotech) to get a 0.5% (w/v) solution. A solution of mPEG-NHS with desired amount (Table S1) in DMSO was then added to the solution and the reaction was conducted for 24 h at room temperature. The product was dialyzed against double distillated water using a dialysis tube (MWCO of 50 kDa,) for 3 days, followed by lyophilization. PEGylated chitosan was characterized by Fourier transform infrared (FTIR) spectroscopy using a Perkin-Elmer

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1

H nuclear magnetic resonance (NMR) spectra

(400MHz, Bruker) using CD3COOD/D2O (1%, v/v) as solvent. 2.3 Preparation and Characterization of Nanoparticles CS/siRNA or CS-PEG/siRNA nanoparticles were prepared by simple complexation method as in our previous report.17 Briefly, chitosan and CS-PEG were dissolved in acetate buffer (200 mM, pH = 5.5) at final concentration of 2 mg/mL and filtered through 0.22 µm Minisart® Syringe Filters. Twenty microgram siRNA in RNAse-free water were used for particle formulation. Various amounts of chitosan were mixed with siRNA at different N/P ratios under constant magnetic stirring for 1 h at room temperature. The obtained nanoparticles were characterized or used for cell transfection. The encapsulation efficiency of siRNA was determined using RiboGreen reagent (Invitrogen) as in our previous report.18 Briefly, siRNA loaded polyplex solution was mixed with a diluted RiboGreen solution (1 : 200 in TE buffer). The fluorescence emission at 520 nm was measured when excited at 480 nm using a Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific). Free siRNA and buffer was included as reference for efficiency calculation. The size of the nanoparticles was determined by photon correlation spectroscopy (PCS) and zeta potential by Laser Doppler Velocimetry (LDV) at 25°C using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). siRNA complexation was further confirmed by 4% agarose gel electrophoresis at constant voltage of 60 V for 1 h in TAE buffer. 2.4 FRET tracking of polyplex assembly Polyplexes were prepared with a mixture of FRET pair-labeled siRNA (Cy3-siRNA and Cy5-siRNA, 1:1, molar ratio). CS or CS-PEG solution was mixed with the above siRNA solution at desired ratio to obtain N/P ratio from 1 to 30. After incubation for 30 min, the samples were excited at 520 nm and the fluorescence intensity was recoded from 540 nm 6 ACS Paragon Plus Environment

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to 760 nm using a Horiba Jobin Yvon Fluoromax-3 fluorimeter. FRET efficiency was calculated as a ratio of the fluorescent intensity as follows: FRET =

‫଻଺ܫ‬଴ ‫ܫ‬ହ଺଴ + ‫଻଺ܫ‬଴

I560, I670 fluorescent intensity at emission of 560 nm and 670 nm, respectively. 2.5 siRNA releasing by heparin displacement Polyplexes prepared by FRET pair-labeled siRNA (N/P = 30) were incubated with heparin (Sigma) at different concentrations from 2 U/mL to 100 U/mL for 1 h. The emission spectrum was recorded as described before. FRET efficiency was calculated and normalized to the value of untreated polyplexes. In addition, siRNA releasing profile was further evaluated by agarose gel electrophoresis as described before. 2.6 Cell culture H1299 (human lung cancer cells), HeLa (human cervical carcinoma cells) and RAW 264.7 (Murine macrophage cells) were maintained in RPMI media supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 37°C in 5% CO2 and 100% humidity. H1299 cells stably expressing GFP (H1299-GFP) were kindly provided by Dr. Anne Chauchereau (CNRS, Villejuif, France) and HeLa cells stably expressing GFP (HeLa-GFP) were obtained by transfection with pEGFP-C1 (Clontech Laboratories, USA). Both of them were cultured in growth medium supplemented with 500µg/ml Geneticin (antibiotic G418 sulfate, Thermo Fisher Scientific). 2.7 In vitro gene silencing HeLa-GFP and H1299-GFP cells were plated on 24-well plates (1 x 105 cells/well) in full media before transfection. The CS/siRNA or CS-PEG/siRNA nanoparticles were added at 50 nM final siRNA concentration. After 12 hours incubation, the media was exchanged with fresh growth media. After total 48 hours transfection, the cells were then detached by 7 ACS Paragon Plus Environment

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trypsin-EDTA solution trypsin-EDTA (0.05% trypsin, 0.02% EDTA, GIBCO), washed with PBS and resuspended in PBS containing 1% BSA. The GFP expression level of each sample was quantified by flow cytometry (Becton Dickenson FACSCalibur). A commercial transfection reagent TransIT-TKO (Mirus, Madison, WI, USA) was included as positive transfection control according to manufacturer's specification. 2.8 Cellular uptake Cells were seeded in 24-well plates, respectively (2 × 105 cells/well) in complete growth media one day before transfection. Then, the media were changed and nanoparticles freshly prepared by Cy3 labeled siRNA were added at 50 nM final siRNA concentration. After 4 hours incubation at 37°C, 5% CO2, the cells were harvested by trypsin-EDTA and resuspended in PBS containing 1% BSA after washing. Cy3 fluorescent intensity of the cells was measured by flow cytometry. The uptake efficiency was quantified using the mean fluorescent intensity. To study the mechanism of endocytosis of polyplexes, HeLa cells were pre-incubated with endocytosis inhibitors, including ethyl-isopropyl-amiloride (EIPA, 50 µM), chlorpromazine (CPZ, 20 µM), genistein (GEN, 50 µM) or cytochalasin D (CCD, 1 µM) for 1 h before transfection. To visualize the cellular uptake and intracellular distribution, cells were plated in 8-well culture chambers (SARSTEDT, Germany) at density of 2 × 104 cells/well. After transfection for 4 hours, the cells were fixed with 4% PFA for 15 min at room temperature and nuclei were stained with DAPI and cell membranes were stained with Wheat Germ Agglutinin, Alexa Fluor 488 conjugate (WGA-488, Molecular Probes) according to the manufacturer’s protocol. For intracellular tracking, cells were incubated with LysoTracker Green DND-26 (Molecular Probes) for 30 min before fixation. The slides were further mounted with ProLong Gold Antifade reagent (Molecular Probes) and stored at 4°C overnight. Images were taken by using LSM 710 (Zeiss, Germany). Images processing was performed with

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ZEN program (Zeiss) and the Mander’s colocalization coefficients were analyzed using Fiji/ImageJ software. 2.9 Cytotoxicity analysis Cytotoxicity was tested using MTS assay (CellTiter 96® Aqueous One Solution Reagent). H1299 and HeLa cells in full growth media were seeded in 96-well plate (1 × 104 cells/well). After the cells attached on the plate, the media was changed to serum free media and chitosan/siRNA nanoparticles and TransIT/siRNA complex were added then incubated for 4 hrs. After replacing the media and another 44 hrs incubation, 20 µl MTS reagent was added to each well and incubated for additional 3 hours before measuring the absorbance at 490 nm. 2.10 Cell cycle analysis Cell cycle was analyzed using a two-step cell cycle analysis kit (ChemoMetec, Allerød, Denmark) on NucleoCounter NC-3000 (Chemometec) according to the manufacturer’s instructions. In brief, HeLa cells after CS-PEG5k/siRRM2 treatment for 48 h were harvested by trypsin-EDTA and resuspended in lysis buffer supplemented with DAPI (10 µg/mL) for 5 min at 37°C. Equal volume of stabilization buffer was further added and 10 µL of the sample suspension was loaded into the sample chamber (NC-Slide 8, Chemometec) and analyzed by the NucleoCounter. 2.11 Nanoparticles interaction with macrophages In vitro macrophage phagocytosis of nanoparticles was evaluated similarly to the cellular uptake study. The uptake was quantified using the mean fluorescent intensity from flow cytometry analysis. To examine the immunogenicity of nanoparticles, RAW 264.7 macrophages were plated in 24-well tissue culture plate (1 x 105 cells/well) and cultured overnight. Nanoparticles were added at 50 nM siRNA final concentration and incubated overnight before medium change. After another 24 hours, total RNA was isolated by Trizol 9 ACS Paragon Plus Environment

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reagent (Invitrogen) and cDNA was synthesized with RevertAid RT Reverse Transcription Kit (Thermo Scientific). The inflammatory cytokines TNF-α and IL-1β expression levels were quantified by real-time PCR (qRT-PCR) performed by SYBR Green kit (Invitrogen) on a LightCycler 480 Real-Time PCR system (Roche). Primers used in RT-qPCR: GAPDH, forward, 5’-GACGGCCGCATCTTCTTGTG-3’, reverse, 5’-GCGCCCAATACGGCCAAATC3’;

TNF-α,

forward,

5’-AGGCTGCCCCGACTACGT-3’,

GACTTTCTCCTGGTATGAGATAGCAAA-3’; CAGGCTCCGAGATGAACAAC-5’

,

IL-1β,

reverse,

5’-

forward,3’-

reverse,3’-GGTGGAGAGCTTTCAGCTCATA-5’.

Lipopolysaccharide (LPS, from E. coli., Sigma) stimulated cells were included as positive control by introducing 100 ng/mL of LPS to culture medium at 4 hours before RNA isolation. 2.12 Hemocompatibility test Whole blood was collected from Balb/cA mice and red blood cells (RBCs) were isolated by centrifugation and further washed three times by PBS. RBCs were incubated with nanoparticles containing Cy5.5-siRNA (final siRNA concentration of 10 µg/mL) at 37°C for 1 hour. The supernatant was collected after centrifugation (500 g, 5 min) and the fluorescent intensity was measured (Ex = 670 nm, Em = 700 nm) by Varioskan™ Flash Multimode Reader. The remaining siRNA was quantified by normalization to initial nanoparticles solution. For hemolysis assay, after incubation with nanoparticles, RBCs were then centrifuged and the supernatant was collected and the absorbance at 540 nm was measured by Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific). PBS and RIPA lysis buffer were included as negative and positive control, respectively. 2.13 In vivo circulation and biodistribution Balb/cA female mice at 4 - 5 weeks of age (Janvier Lab, France) were injected intravenously through tail vein with nanoparticles containing Cy5.5 labeled siRNA (n = 4 for 10 ACS Paragon Plus Environment

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each group). Blood samples were collected at 5, 10, 20 and 60 minutes after injection using 20 µL heparinized pipettes (Vitrex Medical A/S, Herlev, DK). The plasma was isolated after centrifugation and imaged by Typhoon scanner (GE Healthcare). Cy5.5 fluorescent intensity was quantified using ImageJ software. Two hours after injection, the mice were anesthetized with 3.75% isoflurane and blood samples were collected from retro-orbital puncture before sacrificing the mice. The fluorescent intensity in plasma was measured using a Horiba Jobin Yvon Fluoromax-3 fluorimeter. To examine the biodistribution of nanoparticles, the mice were sacrificed at 2 hours postinjection and major organs including heart, lung, liver, spleen and kidney were collected and scanned using an IVIS200 imaging system (Xenogen, Caliper Life Sciences, Hopkinton, MA). The distribution was further analyzed using Living Image 4.3 quantification software (Caliper Life Sciences). The animal experiment was performed with permission from the Danish Animal Experiments Inspectorate. 2.14 Statistics Experiments were repeated a minimum of three times. One-way ANOVA with Tukey test (OriginPro 8.1, OriginLab) was used to determine statistical significance and P < 0.05 was considered as significant difference. 3. Results 3.1 Synthesis and characterization of CS-PEG copolymers CS-PEG copolymers were synthesized by conjugation of mPEG-NHS to the primary amine groups on chitosan. Three different PEGs with molecular weight of 2 kDa, 5 kDa and 10 kDa (PEG2k, PEG5k and PEG10k) were used to synthesize three different types of CSPEG (CS-PEG2k, CS-PEG5k and CS-PEG10k). To systematically investigate the PEG chain length on the properties of polyplex, we optimized the reaction to obtain CS-PEG copolymers with similar PEG/CS mass ratio (Table S1). The success of PEGylation was 11 ACS Paragon Plus Environment

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confirmed by both FTIR and 1H-NMR spectra (Figure 1). The FTIR spectra presented in Figure 1A shows PEGylated chitosan with characteristic peaks at 2870 cm-1 (C-H stretching) and 1100 cm-1 (C-O stretching), which is correspond to the characteristic peaks of PEG. In addition, the increasing of peak at 1660 cm-1 (amide band) compared to 1600 cm-1 (amine band) of CS-PEG compared to CS, indicating the covalent conjugation of PEG to the amine groups on CS. From

1

H-NMR spectra, the highly enhanced peak 3.4 to 4.0 ppm

corresponds to the repeated ethyl group in PEG (-CH2-CH2-O-) and the peak at 3.25 corresponds to the methyl group of PEG (-O-CH3). The degree of substitution (DS) was calculated by the ratio between the increased integrity at 3.1-4.0 ppm and the monosaccharide residue (CH-NH-, 2.9-3.1 ppm) and further adjusted with PEG molecular weight to obtain molar substitution degree (molar DS) and PEG mass contents (mass DS).19-20 The mass DS of obtained CS-PEG2k, CS-PEG5k and CS-PEG10k copolymers were determined as 30.9%, 29.1% and 26.65%, while their molar DS were 3.88%, 1.46% and 0.65%, respectively (Table S1). 3.2 Characterization of polyplexes The ability of the chitosan to efficiently condensate siRNA is prerequisite for successful encapsulation. The encapsulation efficiency was quantified using RiboGreen reagent which becomes fluorescent upon binding with free siRNA but not if siRNA is condensed into polyplex. We investigated the influence of both amine-to-phosphate (N/P) ratio and PEG chain-length on siRNA encapsulation efficiency. From Figure 2A, PEGylation of chitosan significantly impaired siRNA complexation at N/P ratio 1, while complete association of siRNA was achieved for all CS and CS-PEG after N/P ratio reached 5. The complexation was further confirmed by gel electrophoresis (Figure 2B) that fully loading of siRNA was achieved by CS and CS-PEG at N/P > 15.

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To access the impact of PEGylation on intraparticle organization of siRNA, we tracked the polyplex assembly by measuring the FRET efficiency between Cy3-siRNA and Cy5-siRNA. As expected, FRET was only observed upon encapsulation of both Cy3 and Cy5 labeled siRNA (Figure S1). In line with siRNA encapsulation measurement, at N/P ratio 1, significantly lower FRET efficiency was observed for CS-PEG and longer PEG chain hinder the complexation at higher degree (Figure 3). However, at higher N/P ratio after full complexation (N/P = 5, 15 and 30), saturation of FRET was observed for all CS/siRNA and CS-PEG/siRNA nanoparticles, indicating that full complexation was achieved. Interestingly, CS-PEG2k/siRNA showed the lowest FRET efficiency after completed encapsulation (N/P ≥ 5), whereas CS/siRNA obtained the highest FRET efficiency. Therefore, although no difference of encapsulation efficiency at high N/P ratio was observed after PEGylation, the siRNA intraparticle condensation differs with different PEG chain length. The hydrodynamic diameters of nanoparticles at N/P ratio 30 were determined by DLS. As shown in Figure 4, both CS and CS-PEG could be formulated with negatively charged siRNA into nanosized polyplex with narrow size distribution.

The unmodified chitosan

exhibited the smallest size of 126.6 ± 2.18 nm with polydispersity index (PDI) of 0.268 and PEGylation resulted in larger size of polyplex. Interestingly, CS-PEG2k/siRNA showed the largest size of 175.6 ± 2.76 nm (PDI = 0.245), followed by CS-PEG5k/siRNA with the size of 151.4 ± 6.87 nm (PDI = 0.272) and CS-PEG10k/siRNA of 134.9 ± 1.28 nm (PDI = 0.232). Similar trend of size change was also observed at N/P ratio 15 and 60 (Figure S2). The increased size of PEGylated nanoparticles might be due to the PEG chain embedded into nanoparticles or protruding on the particle surface. From zeta potential measurements, CS/siRNA polyplexes showed the highest zeta potential of +26.7 ± 0.42 mV and CSPEG2k/siRNA was the lowest (+19.2 ± 0.41 mV), while CS-PEG5k/siRNA and CSPEG10k/siRNA polyplex were +20.6 ± 2.05 mV and 21.9 ± 1.83 mV, respectively (Figure

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4C). Since siRNA is fully encapsulated (≥ 95%) and efficient gene silencing by chitosan was previously reported at N/P ≥ 30 by us and other groups17, 21, we focused on N/P = 30 formulation for the following experiments. 3.3 siRNA releasing by heparin displacement To trigger gene silencing, siRNA should be released from the polyplexes after entering the cells. To evaluate the releasing profile of siRNA, we performed the FRET measurement and gel electrophoresis after incubating the polyplexes with heparin. As shown in Figure 5B, polyplex disassembly is depended on heparin concentration. At high heparin concentration (50 U/mL and 100 U/mL), the FRET efficiency of both CS/siRNA and CS-PEG/siRNA polyplexes was significantly impaired. Interestingly, CS-PEG/siRNA polyplexes showed better reversibility comparing to CS/siRNA, which could be beneficial for siRNA releasing after endocytosis.

The siRNA releasing was also confirmed by gel retardation assay

(Figure 5C), which showed efficient siRNA releasing at high concentration of heparin (50 U/mL and 100 U/mL). 3.4 Cell uptake To investigate the influence of PEGylation on the bioactivity of nanoparticles, we tested both the cellular uptake and gene silencing efficiency in vitro. HeLa cells were incubated for 4 hours with different nanoparticles containing Cy3-siRNA at N/P ratio 30 and analyzed by flow cytometry (Figure 6 A, B). CS/siRNA treated cells showed the highest fluorescent intensity and all the PEGylated nanoparticles resulted in significantly (P < 0.001) reduction of cell uptake to approximately 50%. A slightly higher uptake of CS-PEG10k/siRNA nanoparticles was observed compared to CS-PEG2k/siRNA (P < 0.05). To visualize the intracellular uptake of nanoparticles, transfected cells were fixed and stained for confocal imaging. From Figure 6 C, CS/siRNA treated cells showed much stronger Cy3-siRNA signal compared to CS-PEG/siRNA treated ones. However, most of the Cy3-siRNA were 14 ACS Paragon Plus Environment

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localized on the membrane. In contrast, CS-PEG/siRNA showed a large portion of Cy3siRNA inside of cytoplasm instead of aggregation on the membrane, especially CSPEG5k/siRNA and CS-PEG10k/siRNA, which was further confirmed by quantitative analysis of intracellular Cy3 intensity (Figure S3). This might be due to the enhanced stability of nanoparticles after PEGylation.6, 22 As the physiochemical properties of nanoparticles are shown to influence the endocytotic pathways,23-25 we further investigated the mechanism of endocytosis of CS/siRNA and CSPEG/siRNA polyplexes. As shown in Figure 7A, pre-incubation of cells with endocytosis inhibitors, including EIPA (macropinocytosis inhibitor), CPZ (clathrin pathway inhibitor), GEN (caveolae pathway inhibitor) and CCD (actin-dependent macropinocytosis inhibitor) resulted

in

significantly

lower

uptake

of

CS/siRNA

polyplexes,

indicating

that

macropinocytosis, clathrin and caveolae pathways are all involved in cell internalization. In contrast, CS-PEG5k/siRNA polypolexes mainly entered cells through clathrin-independent pathways (Figure 7B) as CPZ treatment without affecting cell uptake. Interestingly, macropinocytosis seemed to play a more dominant role for CS-PEG5k/siRNA compared to CS/siRNA, as EIPA and CCD treatment reduced cellular uptake by about 90%. In addition, uptake of CS-PEG2k/siRNA and CS-PEG10k/siRNA polyplexes appeared to utilize similar endocytosis mechanism as CS-PEG5k/siRNA (Figure S4). Endosome escaping is a critical step for successful delivery of siRNAs to trigger RNAi effect.26 To visualize the subcellular localization of internalized siRNA, we stained the cells with Lysotracker and conducted confocal imaging (Figure S5). For both CS/siRNA and CSPEG5k/siRNA treated cells, the majority of siRNA inside of cells was out of lysosome (red color) and only a limited number of siRNA was co-localized with lysosome (yellow dots), which indicates efficient endosome escaping of siRNA. 3.5 Impact of PEGylation and PEG chain length on gene silencing

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We further evaluated the effect of PEGylation on gene silencing efficiency. As shown in Figure 8A, HeLa-GFP cells with knockdown shifted to low fluorescent intensity range (Gate B), which is used to quantify the silencing efficiency. Interestingly, CS-PEG5k/siGFP and CS-PEG10k/siGFP transfected cells significantly enhanced silencing efficiency compared to CS/siGFP at N/P ratio of 30 and 60, however, CS-PEG2k/siGFP showed improved knockdown at high N/P ratio of 60, while impaired knockdown efficiency at lower ratio of 30 (Figure 8B). CS-PEG5k/siGFP achieved the highest knockdown efficiency of 95 %, similar to the efficiency of commercial transfection reagent TransIT-TKO. The knockdown of GFP was also directly visualized by fluorescent imaging (Figure S6). We further tested the knockdown efficiency of these nanoparticles on H1299-GFP cells. As shown in Figure 8C, at N/P ratio 30, CS/siGFP transfection achieved 65.3% target silencing, while

CS-PEG2k/siGFP,

CS-PEG5k/siGFP

and

CS-PEG10k/siGFP

obtained

the

knockdown efficiency of 40.2%, 66.3% and 53.5%, respectively. At N/P ratio 60, CSPEG/siRNA significantly enhanced knockdown compared to CS/siRNA. More importantly, the influence of PEG length on siRNA bioactivity showed similar trend with HeLa-GFP cells and CS-PEG5k/siRNA achieved the highest silencing efficiency (~92%) among the three PEGylated polyplexes. To further exploit the impact of PEGylation on in vitro silencing, we synthesized PEG5k and PEG10k modified chitosan with higher grafting density similar to CS-PEG2k (CS-PEG5kH and CS-PEG10kH, respectively) and evaluated their transfection efficiency. From NMR anlysis (Figure S8), the molar DS of CS-PEG5kH and CS-PEG10kH were 3.57% and 3.32%, respectively. Both copolymers achieved efficient siRNA loading capability at N/P > 15 (Figure S9A), and the size of CS-PEG5kH/siRNA and CS-PEG10kH/siRNA were 179.7 ± 8.2 nm and 203.7 ± 10.8 nm, respectively (Figure S9B). Interestingly, increasing of both PEG5k and PEG10k grafting density resulted in lower knockdown efficiency (Figure S10,

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CS-PEG5k/siRNA vs. CS-PEG5kH/siRNA, **p < 0.01; CS-PEG10k/siRNA vs. CSPEG10kH/siRNA, ***p < 0.001). 3.6 Biocompatibility Cationic polymer based gene delivery system could cause toxicity from interaction with cell membrane or intracellular organelles.8,

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chitosan/siRNA polyplex toxicity, MTS assay was performed 48 hours after transfection. We observed a slight reduction of cell viability after CS/siRNA treatment, while the commercial transfection reagent TKO showed significant cytotoxicity towards both HeLa and H1299 cells (Figure 9 A, B). In contrast, CS-PEG/siRNA nanoparticles showed neglectable effect on cell proliferation. The improved cell viability could be due to the shielding of nanoparticle surface charge by PEGylation and reduced aggregation on cell surface as observed by confocal microscopy. Another issue with nanoparticles is their interaction with immune cells to trigger immune response.28-29 To investigate the influence of PEGylation on the potential immunogenicity of chitosan polyplexes, we evaluated the phagocytosis of the different polyplexes and inflammatory cytokine expression by murine macrophages (RAW 264.7 cells) after polyplex treatment. As shown in Figure 9C, PEGylation significantly suppressed macrophage uptake with the trend of less uptake with increase in PEG length. Furthermore, from qRT-PCR analysis (Figure 9D), none of CS/siRNA or CS-PEG/siRNA nanopoarticles showed significant induction of the inflammatory cytokines TNF-α and IL-1β expression, indicating the low immunogenicity of chitosan based delivery materials. 3.7 Inhibition of cancer cell proliferation through RRM2 Knockdown To assess the biomedical application of the CS-PEG/siRNA polyplexes, we further performed an in vitro anti-cancer study by silencing RRM2, a potential therapeutic target.3031

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by CS/siRRM2 (Figure 10A), while siNC containing polyplexes did not affect RRM2 expression. The proliferation of HeLa cells was inhibited by PEG5k/siRRM2 following compared to PEG5k/siNC (Figure 10B) and ~ 50% inhibition of cell growth was observed at 72 h. The morphological change of HeLa cells to a flat large shape and less cell density with RRM2 knockdown for 72 h was clearly observed by microscopy (Figure 10C). As RRM2 is the critical enzyme for DNA synthesis and replication, CS-PEG5k/siRRM2 treatment also induced cell cycle arrest in G2/M phase (from 2.2% to 10.2%) as well as increased apoptotic population in pre-phase (from 4.9% to 16.2%). These results indicate the therapeutic potential of CS-PEG/siRNA system. 3.8 Blood circulation and biodistribution We first assessed the effect of PEGylation on the nonspecific interaction between polyplex and red blood cells. After incubation of different polyplexes (N/P = 30) with blood for 1 h, only 5 % of CS/siRNA was retained in plasma, whereas PEGylated polyplexes showed significant higher retention (Figure 11A). More importantly, longer PEG chain resulted in higher retention (18.7%, 24.5% and 33.8% for CS-PEG2k/siRNA, CS-PEG5k/siRNA and CS-PEG10k/siRNA, respectively). We further evaluated the hemolytic activity of different nanoparticles. Both CS/siRNA and CS-PEG/siRNA showed good hemocompatibility without disrupting the integrity of RBC membrane (Figure 11B). To assess the effect of PEGylation and the influence of PEG length on nanoparticle circulation, CS/siRNA, CS-PEG2k/siRNA, CS-PEG5k/siRNA and CS-PEG10k/siRNA (N/P = 30) were intravenously injected via tail vein. Blood samples were collected at 5 min, 10 min, 20 min, 1 h and 2 h and fluorescence intensity in plasma was analyzed to quantify the retained siRNA amount in circulation. As shown in Figure 12, > 50% of injected siRNA was cleared from blood circulation after 5 min for all the different nanoparticles. At 10 min postinjection, a clear trend of increasing siRNA retained in circulation with increment of PEG

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length, and only CS-PEG10k/siRNA showed statistical significance (P < 0.05) compared to CS/siRNA. In addition, at the time points of 1 h and 2 h, PEG10k also significantly improved the siRNA in blood level (P < 0.01). In contrast, only at 2 h, CS-PEG5k/siRNA showed significant higher siRNA level compared to CS/siRNA (P < 0.01). However, no significant difference of siRNA circulation was observed between CS-PEG2k/siRNA and CS/siRNA at all time points. In short summary, extended circulation was only significant in CSPEG10k/siRNA injected group compared to CS/siRNA injected group. In addition, gel electrophoresis analysis showed that both CS/siRNA and CS-PEG/siRNA polyplexes were stable in serum (Figure S11). Furthermore, the biodistribution of different nanoparticles was investigated by ex vivo fluorescent imaging. All the four groups showed similar pattern of biodistribution in major organs (Figure 13A). The majority of siRNA signal was observed in liver and kidneys and slight signal was visualized in lung, while no significant fluorescence could be detected in heart and spleen. The deposition of siRNA in each organ was further quantified using IVIS Living Image 4.3 program (Figure 13B). There was a clear trend that CS-PEG10k/siRNA treated groups showed more fluorescence in lung, liver and kidney, especially, significantly stronger signal presented in kidney compared to CS/siRNA treated group (P < 0.01). 4. Discussion Chitosan, a nature derived cationic polysaccharide, is a promising siRNA delivery material due to its good biocompatibility and effective transfection capability.6, 17 However, the low solubility and high density of positive charge of chitosan hinder its systemic administration in vivo. PEGylation is an attractive strategy to improve stability and extend the bioactivity of macromolecules and nanoparticles.10 In the present study we systematically investigated the impact of PEGylation on the physical properties and bioactivity of chitosan/siRNA nanopoarticles in vitro and in vivo.

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Regarding PEGylation of polyplex, two major issues need to be considered including PEG chain length and PEG grafting density. We observed that at similar mass content of PEG, higher molecular weight PEG and less grafting ratio favors the nanoparticle assembly with smaller size, and condensed siRNA packaging as demonstrated by DLS. However, PEG2k modification resulted in the lowest zeta potential, which could be due to its higher grafting density leading to reducing the number of primary amines on chitosan. Similarly, Mao et al. has systematically investigated the effect of PEGylation on the properties of PEI/siRNA polyplex,14 that showed higher density of shorter PEG grafting also resulted in larger nanoparticles with lower surface charge. To uncover the effect of different PEGylation strategies on polyplex assembly, we designed a simple FRET assay to probe the intraparticle distribution of siRNA. Fluorescent labeling of carrier materials with fluorophores or quantum dots have been widely used for FRET tracking of gene delivery.32-33 However, this strategy is time-consuming and difficult because of controlling labeling efficiency, which is critical to compare different carrier materials in parallel. In contrast, we utilized a mixture of FRET-pair (Cy3 and Cy5) labeled siRNA at equal molar ratio and monitor the FRET efficiency after introducing CS or CSPEG. Our result revealed that at N/P = 1, longer PEG chain hinders the complexation with siRNA, however, at higher N/P ratio after full complexation (N/P ≥ 5), CS-PEG2k/siRNA showed the lowest FRET efficiency. Due to the fact that FRET efficiency is determined by the proximity between the probes, we could suspect that short PEG and high grafting density resulted in polyplexes with more PEG embedding and less condensed siRNA packaging. Interestingly, we also observed a slightly decline of FRET efficiency at higher N/P ratio, which is similar to a previous report by Alabi, et al, that claims this phenomenon is due to the redissolving or swelling of siRNA with cationic delivery materials.34 This FRET assay provided more details into the physical properties of nanoparticles at intraparticle

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level, which could not be observed by either dye exclusion assay or fluorescent quenching assay. How does PEGylation influence the transfection efficiency of chitosan polyplexes? PEGylation of nanoparticles in general reduce cellular uptake due to their neutral charge and by lowering interaction with cell membrane.14, 35 Here, we observed large amount of CS/siRNA polyplex on cell membrane as aggregates rather than in side of cytoplasm. In contrast, significantly higher amounts of CS-PEG5k/siRNA and CS-PEG10k/siRNA were localized inside cells, which could be the reason for their enhanced transfection efficiency. More interestingly, the silencing efficiency of CS-PEG/siRNA increased with N/P ratio increment, while the knockdown efficiency of CS/siRNA significantly declined in HeLa cells or unchanged H1299 cells from N/P ratio 30 to 60. This opposite effect of N/P ratio on silencing activity could be due to their different stability. To prove this, we performed a stability test by challenging the polyplexes at N/P ratio of 30 and 60 against heparin. As shown in Figure S7, the size and PDI of CS/siRNA polyplexes at N/P ratio 60 significantly increased after incubation with heparin. However, all the CS-PEG/siRNA polyplexes (both at N/P ratio 30 and 60) showed only a slight increase in size and PDI. Therefore, increasing N/P ratio from 30 to 60 destabilized CS/siRNA polyplexes, but did not alter the stability of PEGylated polyplexes. To be noted, large number of aggregates were also observed in culture medium after transfection with CS/siRNA (N/P = 60). The influence of PEG chain length on knockdown in H1299 cells showed similar trend as in HeLa cells. In both cell lines CS-PEG5k/siRNA showed the best knockdown, which we suspect is due to the good compromise between siRNA condensation and intracellular releasing. Similarly, Mao et al. also observed that among different PEGylated PEI/siRNA polyplexes, PEI-PEG5k/siRNA demonstrated the best knockdown in NIH/3T3 cells.14

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Interestingly, PEGylation caused a significant change in the endocytosis pathway of chitosan based polylexes. CS-PEG/siRNA polyplexes were mainly taken up by cells through macropinocytosis and caveolae rather than clathrin-mediated pathway. The selection of endocytosis pathways is known to influence the intracellular processing of nanoparticles and transfection efficiency. Joanna et al. reported that polylexes taken up by caveolea, could escape endosome/lysosome compartment and mediate efficient gene transfer.36 Recently, Mihael et al. also reported that micropinocytosis and caveolae, but not the clathrin-mediated pathway lead to efficient gene silencing by PEI/siRNA polyplexes in HeLa cells.37 Therefore, the changed endocytosis mechanism of PEG-CS/siRNA polyplexes might favor siRNA intracellular trafficking and endosome escaping. As shown in Figure S5, CS-PEG5k/siRNA treated cells indeed showed lower colocalization between siRNA and endosome compared to the cells treated with CS/siRNA. In addition, although all CS/siRNA and CS-PEG/siRNA polyplexes demonstrated good biocompatibility, including cell viability and low immunogenicity, PEGylation significantly improved cell viability and reduced the phagocytosis by RAW 264.7 macrophages. This could be due to the “bio-inert” properties of PEG that shield the positive charge, reduction of aggregation as well as blocking the macrophage recognition, which is in line with the PEGylated hydrogel nanoparticles reported by Perry, et al.38 Erythrocytes or RBCs, as large fraction of blood, was directly interacted with intravenously injected nanoparticles.39 From our hemocompatibility test, PEGylation significantly improved the retention of chitosan polyplex after being incubated directly with RBCs (Figure 11B) and longer PEG achieved higher stability against RBCs. Although PEGylated polyplexes has been investigated for plasmid DNA delivery in vivo in several reports,40-41 the impact of PEGylation on in vivo behavior of siRNA associated polyplexes has been less studied. Due to the relatively smaller size compared to DNA

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plasmid, siRNA encapsulated polyplexes have been shown to suffer from fast excretion due to rapid decomplexation and macrophage phagocytosis.9, 25, 30 Recently, PEGylation of micelles and cationic polycarbonate polyplexes with varied PEG length has been studied in vivo for siRNA delivery.42-43 Here, we observed significant extension of blood circulation of CS-PEG10k/siRNA and a slight extension by CS-PEG5k/siRNA. This could be due to the reduction of RBCs association and macrophage phagocytosis as observed in vitro. Additionally, nanoparticles disassembly at glomerular basement membrane in the kidneys, similarly as we also observed here by heparin displacement, also serves as a major barrier for long circulation.44 The current study is focused on the investigation of PEG length on polyplex performance. However, it would be interesting to further evaluate the blood circulation of CS-PEG5k and CS-PEG10k with different PEG substitution degree. In addition, Nelson, et al reported that introducing hydrophobic content into cationic polymer significantly enhanced circulation time, hence,

another strategy to further boost the

circulation of CS-PEG10k/siRNA polyplexes could be by incorporating hydrophobic components.9 Moreover, further conjugation of a targeting ligand on the distal end of longer PEG (PEG5k or PEG10k), could be a promising strategy to achieve selective siRNA delivery to specific tissues or cell types. 5. Conclusion In this study, we systematically investigate the impact of PEGylation on the physicochemical and biological properties of chitosan/siRNA based polyplexes. A series of Chitosan-PEG copolymers were synthesized with similar PEG mass content but with different molecular weight, including 2 kDa, 5 kDa and 10 kDa (CS-PEG2k, CS-PEG5k, CS-PEG10k). Higher molecular weight PEG and less grafting degree resulted in smaller nanoparticles with less positive charge. In vitro silencing effect was significantly improved by PEGylation and CS-PEG5k/siRNA achieved the highest knockdown. In addition,

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extended in vivo circulation time was achieved only with high molecular weight PEG modification (PEG10k). Therefore, PEG with higher molecular weight and less grafting density is a promising strategy to improve chitosan/siRNA nanoparticle performance both in vitro and in vivo. Acknowledgements This project was founded by “the Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration” (LUNA). We also acknowledge the postdoctoral fellowships from Lundbeck Foundation. Conflict of Interest The authors declare no conflict of interest. Supporting Information Chemical composition of CS-PEG copolymers. Fluorescent spectra of CS/Cy3-siRNA, CS/Cy5-siRNA and CS/FRET-siRNA. Size of polyplexes at N/P ratio of 15 and 60. Quantification of intracellular Cy3-siRNA. Endocytosis mechanism of CS-PEG2k/siRNA and CS-PEG10k/siRNA polyplexes. Intracellular distribution of polyplexes. Fluorescent imaging of GFP silencing in HeLa-GFP cells. Stability test of polyplexes at N/P ratio of 30 and 60. 1H-NMR spectra of CS-PEG5kH and CS-PEG10kH. Characterization of CSPEG5kH/siRNA and CS-PEG10kH/siRNA polyplexes. Comparison of GFP silencing efficiency by CS/siGFP and CS-PEG/siGFP. Stability of CS/siRNA and CS-PEG/siRNA in serum.

Figures

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CS

CS-PEG2k Self-assembly CS-PEG5k siRNA CS-PEG10k

Scheme 1. Illustration the design and self-assembly of PEGylated chitosan/siRNA nanoparticle with different length of PEG.

Figure 1. Characterization of CS-PEG co-polymers. (A) FTIR spectra and (B) 1H-NMR spectra analysis of CS, CS-PEG2k, CS-PEG5k, CS-PEG10k.

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Figure 2. siRNA encapsulation efficiency. (A) Quantification of siRNA encapsulation efficiency at different N/P ratio by dye exclusion assay. (B) siRNA complexation was confirmed by gel electrophoresis assay.

Figure 3. FRET tracking of polyplex assembly. (A) Fluorescence spectra (excitation at 520nm) of CS/siRNA and CS-PEG/siRNA complexes at N/P ratio 1 (red), 5 (blue), 15 (cyan) and 30 (purple). Complexes containing only Cy5-siRNA (black) as control. (B) 26 ACS Paragon Plus Environment

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Quantification of FRET efficiency from emission spectra. The quantitative result represent as mean ± SD (n = 3). (C) Illustration the FRET mechanism during polyplex formation.

Figure 4. Nanoparticle size and zeta potential. (A) Size histogram of CS/siRNA, CSPEG/siRNA nanoparticles (N/P = 30) measured by DLS. Quantification of (B) size and (C) zeta potential of nanoparticles. Results represent mean ± SD (n = 3).

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Figure 5. siRNA releasing by heparin displacement. (A) Illustration the disassembly of CS/siRNA or CS-PEG/siRNA polyplexes (N/P = 30) and impaired FRET efficiency by heparin displacement. (B) FRET Monitoring of polyplexes disassembly at different heparin concentration. (C) Gel electrophoresis analysis of siRNA releasing by heparin displacement at 2 U/mL, 50 U/mL and 100 U/mL, respectively. (Lane 1, free siRNA; a, CS/siRNA; b, CS-PEG2k/siRNA; c, CS-PEG5k/siRNA; d, CS-PEG10k/siRNA.)

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Figure 6. siRNA uptake in HeLa cells. (A) Confocal imaging of intracellular uptake of nanoparticles. HeLa cells were incubated with polyplexes (N/P = 30) at siRNA concentration of 50 nM for 4 hours. Cell nucleus stained with DAPI (blue), membrane stained with WGA-488 (green) and Cy3 labeled siRNA (red). Images were taken by 29 ACS Paragon Plus Environment

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Zeiss 63×/ 1.40 oil immersion objective. (B) Histogram of cellular uptake by flow cytometry. (C) Quantification of siRNA uptake in HeLa cells from mean fluorescent intensity. The quantitative result represent as mean ± SD (n = 3). Significance: *p < 0.05, **p < 0.01, ***p < 0.001, N.S., no significant difference.

Figure 7. Mechanism of endocytosis of polyplexes. HeLa cells were incubated with CS/Cy5-siRNA (A) or CS-PEG5k/Cy5-siRNA (B) after 1 h pre-treatment with endocytosis inhibitors: EIPA (macropinocytosis, 50 µM), CPZ (clathrin pathway, 20 µM), GEN (caveolae pathway, 50 µM) or CCD (macropinocytosis, 1 µM). Cellular uptake was quantified by flow cytometry. Results represent mean ± SD (n = 3). Significance: ***p < 0.001, N.S., no significant difference.

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Figure 8. Influence of PEGylation on gene silencing efficiency. (A) Flow cytometry histogram of GFP stably expressing HeLa cells transfected with polyplexes (N/P = 30) at a final siRNA concentration of 50 nM for 48 hours (nanoparticles incubated with cells for 12 hours). Cells in Gate B as GFP silenced population, cells in Gate C as non-silenced population. Quantification of GFP silencing efficiency in HeLa-GFP cells (B) and H1299GFP cells (C) after 48 hours transfection. Results represent mean ± SD (n = 3). Significance: *p < 0.05, **p < 0.01, ***p < 0.001, N.S. as not significant (compared to CS/siGFP group).

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Figure 9. Biocompatibility of polyplexes in vitro. Cell viability of polyplexes (N/P = 30) in (A) Hela cells and (B) H1299 cells by a standard MTS assay. Significance: *p < 0.05, **p < 0.01 vs. untreated cells. (C) Quantification of macrophage phagocytosis of polyplexes at siRNA concentration of 50 nM after incubation for 4 hours. (D) Inflammation cytokines including TNF-α and IL-1β mRNA level normalized to GAPDH quantified by qRT-PCR. Results represent mean ± SD (n = 3). Significance: ***p < 0.001 vs. CS/siRNA treatment group.

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Figure 10. Silencing of RRM2 inhibited HeLa cell proliferation. (A) RT-qPCR quantification of silencing efficiency of RRM2 in HeLa cells transfected by CS/siRRM2 and CS-PEG5k/siRRM2 polyplexes (N/P = 30). (B) Viability of HeLa cells after treatment of CS-PEG5k/siRRM2 and CS-PEG5k/siNC by MTS assay. Results represent mean ± SD (n=3). Significance: *p < 0.05, **p < 0.01, ***p < 0.001. (C) Morphological changes of cells after silencing of RRM2 for 72 h (from left to right: untreated, CS-PEG5k/siNC, CSPEG5k/siNC) (D) Cytometry analysis of cell cycle after silencing of RRM2 for 48 h.

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Figure 11. Interaction of polyplex with red blood cells. (A) Remaining siRNA after incubation with RBCs for 1 h; (B) Hemolysis analysis of nanoparticles. Lysis buffer and PBS serves as positive and negative control, respectively. Insertion: color image of the supernatents from hemolysis assay (from left to right: lysis buffer, PBS, CS/siRNA, CSPEG2k/siRNA, CS-PEG5k/siRNA, CS-PEG10k/siRNA). Results represent mean ± SD (n = 3). Significance: ***p < 0.001 vs. CS/siRNA group.

Figure 12. The impact of PEGylation on chitosan/siRNA nanoparticles blood circulation in vivo. (A) Fluorescent scan of plasma samples at 5 min, 10 min, 20 min and 1 h post i.v. injection of different nanoparticles containing Cy5.5-siRNA. (B) Quantification of remained siRNA in plasma by measuring Cy5.5 fluorescence intensity in plasma

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samples. Results represent mean ± SD (n = 4). Significance: *P < 0.05, **P < 0.01 vs. CS/siRNA group.

Figure 13. Biodistribution of nanoparticles. (A) Ex vivo fluorescent imaging of major organs, including heart, lung, liver, spleen and kidney 2 hours after i.v. injection of (b) CS/Cy5.5-siRNA, (c) CS-PEG2k/Cy5.5-siRNA (d) CS-PEG5k/Cy5.5-siRNA, (e) CSPEG10k/Cy5.5-siRNA and (a) untreated mouse served as negative control to subtract tissue autofluorescence. (B) Quantitative analysis of in vivo distribution of different nanoparticles using the IVIS Living Image 4.3 software package (Caliper Life Science). Each bar represents the mean ± SD (n = 4). Significance: **P < 0.01 vs. CS/siRNA group.

References 1. Kim, H. J.; Kim, A.; Miyata, K.; Kataoka, K. Recent Progress in Development of siRNA Delivery Vehicles for Cancer Therapy. Adv. Drug Deliv. Rev. 2016, 104, 61-77. 2. Draz, M. S.; Fang, B. A.; Zhang, P.; Hu, Z.; Gu, S.; Weng, K. C.; Gray, J. W.; Chen, F. F. Nanoparticle-Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral Infections. Theranostics 2014, 4, 872-892. 3. Yang, C.; Nilsson, L.; Cheema, M. U.; Wang, Y.; Frokiaer, J.; Gao, S.; Kjems, J.; Norregaard, R. Chitosan/siRNA Nanoparticles Targeting Cyclooxygenase Type 2 Attenuate Unilateral Ureteral Obstruction-Induced Kidney Injury in Mice. Theranostics 2015, 5, 110-123. 4. Howard, K. A.; Paludan, S. R.; Behlke, M. A.; Besenbacher, F.; Deleuran, B.; Kjems, J. Chitosan/siRNA Nanoparticle-Mediated TNF-alpha Knockdown in Peritoneal Macrophages for Anti-Inflammatory Treatment in a Murine Arthritis Model. Mol. Ther. 2009, 17, 162-168. 35 ACS Paragon Plus Environment

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20. Jeong, Y. I.; Kim, D. G.; Jang, M. K.; Nah, J. W. Preparation and Spectroscopic Characterization of Methoxy Poly(Ethylene Glycol)-Grafted Water-Soluble Chitosan. Carbohydr. Res. 2008, 343, 282-289. 21. Malmo, J.; Sorgard, H.; Varum, K. M.; Strand, S. P. siRNA Delivery with Chitosan Nanoparticles: Molecular Properties Favoring Efficient Gene Silencing. J. Controlled Release 2012, 158, 261-268. 22. Zhang, Y. Q.; Chen, J. J.; Zhang, Y. D.; Pan, Y. F.; Zhao, J. F.; Ren, L. F.; Liao, M. M.; Hu, Z. Y.; Kong, L.; Wang, J. W. A Novel PEGylation of Chitosan Nanoparticles for Gene Delivery. Biotechnol. Appl. Biochem. 2007, 46, 197-204. 23. Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J. Y.; Yan, H.; Fan, C. H. Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells. Angew. Chem., Int. Ed. 2014, 53, 7745-7750. 24. Pei, H.; Zuo, X. L.; Zhu, D.; Huang, Q.; Fan, C. H. Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 550-559. 25. Naeye, B.; Deschout, H.; Caveliers, V.; Descamps, B.; Braeckmans, K.; Vanhove, C.; Demeester, J.; Lahoutte, T.; De Smedt, S. C.; Raemdonck, K. In Vivo Disassembly of IV Administered siRNA Matrix Nanoparticles at the Renal Filtration Barrier. Biomaterials 2013, 34, 2350-2358. 26. Wittrup, A.; Lieberman, J. Knocking Down Disease: A Progress Report on siRNA Therapeutics. Nat. Rev. Genet. 2015, 16, 543-552. 27. Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. Toxicity of Cationic Lipids and Cationic Polymers in Gene Delivery. J. Controlled Release 2006, 114, 100-109. 28. Szeto, G. L.; Lavik, E. B. Materials Design at the Interface of Nanoparticles and Innate Immunity. J. Mater. Chem. B 2016, 4, 1610-1618. 29. Love, S. A.; Maurer-Jones, M. A.; Thompson, J. W.; Lin, Y. S.; Haynes, C. L. Assessing Nanoparticle Toxicity. Annu. Rev. Anal. Chem. 2012, 5, 181-205. 30. Rahman, M. A.; Amin, A. R. M. R.; Wang, X.; Zuckerman, J. E.; Choi, C. H. J.; Zhou, B. S.; Wang, D. S.; Nannapaneni, S.; Koenig, L.; Chen, Z. J.; Chen, Z.; Yen, Y.; Davis, M. E.; Shin, D. M. Systemic Delivery of siRNA Nanoparticles Targeting RRM2 Suppresses Head and Neck Tumor Growth. J. Controlled Release 2012, 159, 384-392. 31. Zuckerman, J. E.; Davis, M. E. Clinical Experiences with Systemically Administered siRNABased Therapeutics in Cancer. Nat. Rev. Drug Discovery 2015, 14, 843-856. 32. Chen, H. H.; Ho, Y. P.; Jiang, X.; Mao, H. Q.; Wang, T. H.; Leong, K. W. Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed from Quantum Dot-FRET. Mol. Ther. 2008, 16, 324-332. 33. Lee, H.; Kim, I. K.; Park, T. G. Intracellular Trafficking and Unpacking of siRNA/Quantum Dot-PEI Complexes Modified with and without Cell Penetrating Peptide: Confocal and Flow Cytometric FRET Analysis. Bioconjugate Chem. 2010, 21, 289-295. 34. Alabi, C. A.; Love, K. T.; Sahay, G.; Stutzman, T.; Young, W. T.; Langer, R.; Anderson, D. G. FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA Nanocomplexes. ACS nano 2012, 6, 6133-6141. 35. Pozzi, D.; Colapicchioni, V.; Caracciolo, G.; Piovesana, S.; Capriotti, A. L.; Palchetti, S.; De Grossi, S.; Riccioli, A.; Amenitsch, H.; Lagana, A. Effect of Polyethyleneglycol (PEG) Chain Length on the Bio-Nano-Interactions between PEGylated Lipid Nanoparticles and Biological Fluids: From Nanostructure to Uptake in Cancer Cells. Nanoscale 2014, 6, 2782-2792. 36. Rejman, J.; Bragonzi, A.; Conese, M. Role of Clathrin- and Caveolae-Mediated Endocytosis in Gene Transfer Mediated by Lipo- and Polyplexes. Mol. Ther. 2005, 12, 468-474.

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