Journal of Controlled Release 196 (2014) 28–36

Contents lists available at ScienceDirect

Journal of Controlled Release

j ourna l homepage: www.e lsev ie r .com/ locate / jconre l

Efficient intracellular delivery and multiple-target gene silencingtriggered by tripodal RNA based nanoparticles: A promising approach inliver-specific RNAi delivery

S. Sajeesh a,1, Tae Yeon Lee a,1, Joon Ki Kim a, Da Seul Son a, Sun Woo Hong a, Soohyun Kim b, Wan Soo Yun b,Soyoun Kim c, Chanil Chang d, Chiang Li e, Dong-ki Lee a,⁎a Global Research Laboratory for RNAi Medicine, Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Koreab Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Koreac Department of Medical Biotechnology, Dongguk University, Seoul 100-715, Koread BMT Inc., Seoul, 153-777, Koreae Skip Ackerman Center for Molecular Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

⁎ Corresponding author.E-mail addresses: dklee@skku.edu, dklee0318@gmail.c

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jconrel.2014.09.0160168-3659/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 March 2014Accepted 16 September 2014Available online 20 September 2014

Keywords:RNA interferenceTripodal-interfering RNA (tiRNA)NanoparticlesLiver targetingPolyethyleneimine

RNA interference (RNAi) triggering oligonucleotides in unconventional structural format can offer advantagesover conventional small interfering RNA (siRNA), enhanced cellular delivery and improved target gene silencing.With this concept, we present a well-defined tripodal-interfering RNA (tiRNA) structure that can induce simul-taneous silencing of multiple target genes with improved potency. The tiRNA structure, formed by the comple-mentary association of three single-stranded RNA units, was optimized for improved gene silencing efficacy.When combinedwith cationic polymers such as linear polyethyleneimine (PEI), tiRNA assembled to forma stablenano-structured complex through electrostatic interactions and induced stronger RNAi response over conven-tional siRNA-PEI complex. In combination with a liver-targeting delivery system, tripodal nucleic acid structuredemonstrated enhanced fluorescent accumulation in mouse liver compared to standard duplex nucleic acidformat. Tripodal RNA structure complexed with galactose-modified PEI could generate effective RNAi-mediatedgene silencing effect on experimental mice models. Our studies demonstrate that optimized tiRNA structuralformat with appropriate polymeric carriers have immense potential to become an RNAi-based platform suitablefor multi-target gene silencing.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Small interfering RNA (siRNA) structure with 19–21 base pairs hasbeen regarded as a robust tool in post-transcriptional gene silencing[1,2]. However, the classical siRNA structures failed to provide a properclinical mandate, largely due to their incompatibility with the deliverysystems [3,4]. The adaptability of RNAi machinery to accommodate un-conventional RNA triggers sustained the development of novel structur-al variants, devised to overcome the limitations posed by conventionalshort RNA duplex format [4–7]. Our efforts in this direction have beenfocused largely on expanding the structural repertoire of classicalsiRNA structures in a rather hypothesis-driven manner, with the aimof improving their potency, reducing non-specific effects or even to en-hance their association with delivery systems [8–11].

Evenwith the recent success from the lipid-based delivery platformsin the pre-clinical stages, polymer-based RNAi delivery technology

om (D. Lee).

seems to be more appealing from a safety and commercial point-of-view [12,13]. However, rigid structural format and poor charge densityof conventional siRNAs often limits its interactionwith the polymeric de-livery systems [14]. Development of unconventional RNAi-triggeringstructureswith higher charge density and improvedmolecular flexibilitywere undertaken to enhance the affinity between cationic polymers andRNA structures [4]. Chemical conjugation and complementary hybridi-zation strategies were employed to generate novel RNAi triggers withimproved structural features [4,15,16]. Nevertheless, irrational designof long RNA structures may jeopardize the specific RNAi effects and alogical approach should be devised for the development of second-gen-eration RNAi triggers. Long multimerized double-stranded RNA (liRNA),prepared via complementary base paring method, formed stable nano-complex upon association with the cationic polymers and induced po-tent gene silencing activity in a sequence-specific manner [17].

We have previously developed a branched, non-linear tripodal-interfering RNA (tiRNA) structure capable of simultaneously inducingmultiple-gene silencing [18,19]. In contrast to the generally acceptednotion,we observed that tiRNA structure triggered efficient gene silenc-ing activity via Dicer-independent pathway. Interestingly, the tiRNA

29S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

structure induced more potent gene silencing activity over the classicalsiRNA structure when complexed with cationic polymers [18].

In this study, we developed a liver-specific RNAi platform by inte-grating the structural advantages of tripodal RNAwith a targeted de-livery approach. Linear highmolecularweight polyethyleneimine (PEI),functionalizedwith galactose units, was used as the liver-targeting unit.Specific tiRNA sequences were assembled with the modified polymerand detailed in vitro investigations were performed to evaluate theirgene silencing efficacy. In vivo biodistribution studies were conductedto assess the liver-targeting ability of these polyplexes and liver-specificgene knock-down activity was verified with mouse models.

2. Materials and methods

2.1. Materials

Commercial low molecular weight (22 kDa) linear-PEI (jetPEI)was obtained from PolyPlus-transfection (Illkirch, France) andlinear poly(ethyleneimine) (MW 25 kDa) was purchased fromPolysciences, Inc. (Warrington, PA). 3-(2-Pyridyldithio)propionic acidN-hydroxysuccinimide ester (SPDP), 2-iminothiolane, 4-aminophenylβ-D-glucopyranoside (GAL), polyinosinic–polycytidylic acid sodiumsalt [Poly(I:C)] were purchased from Sigma-Aldrich (St. Louis, MO).

Chemically synthesized RNA oligos were obtained from BMT Inc.(Seoul, South Korea) and fluorescent labeled DNA/RNA oligos wereobtained from Bioneer (Daejeon, South Korea) and ST Pharma (Seoul,South Korea). The sequence details and the annealing pattern ofsiRNAs/tiRNA used in these experiments are shown in supplementarydata (Figures s1 and s4).

2.2. Preparation and evaluation of siRNA/tiRNA-PEI complex

2.2.1. siRNA/tiRNA complex formationTo prepare polyplexes, stock siRNA/tiRNA solutions (10 μM) were

mixed with appropriate amounts of PEI solution (1 mg/ml) and com-plex was diluted with 150 mM NaCl solution. Complex was vortexedfor 20 s and incubated for 20min at room temperature. Following initialoptimization experiments using surrogate DNA structure (supplemen-tary data; Figure s3A–C), we chose tiRNA structure composed of 32 ntRNA units for further experiments.

2.2.2. Particle size and zeta potential measurements of polyplexesZeta potential values and average particle size of siRNA/tiRNA-PEI

complex were analyzed by using Zeta-Sizer (Nano-ZS 90, MalvernInstrument) with a He–Ne Laser beam (633 nm, fixed scattering angleof 90°) at 25 ° C. Polyplex solutions were prepared by mixing siRNA/tiRNA-PEI solution and samples were diluted to a final volume of 1 mLbefore measurement. Zeta-potential values and average size of thesiRNA/tiRNA-PEI complex were presented as the average value of 3runs.

2.2.3. Atomic force microscope (AFM) measurementsThe polyplexes were examined by AFM to evaluate their size and

surface morphology. The siRNA/tiRNA-PEI complex was transferred toclean silicon surface and air dried. AFM images of RNA-PEI complexwere then obtained with NX AFM (Park Systems) in tapping mode.

2.2.4. Agarose gel electrophoretic analysis on the complex stabilitysiRNA/tiRNA binding ability of PEI was examined by agarose gel

electrophoresis. Agarose gel (2% w/v) containing ethidium bromidewas prepared in TBE (Tris-Borate-EDTA) buffer and the polyplexeswere prepared in different N/P ratios as mentioned previously. Thesamples were mixed with 6× RNA loading dye and electrophoresed at100 V for 10 min; and further examined by UV illuminator (Gel Docu-mentation Systems, Biovision).

2.2.5. Anionic (heparin) decomplexation assayFor heparin decomplexation assay, siRNA/tiRNA complexedwith PEI

at different N/P ratios were incubated with various amounts of heparin(heparin–RNAweight ratio 0, 1, 2, 3 and 5) for 15 min at room temper-ature. Thereafter, the samples were loaded onto 2% agarose gel andsubjected to electrophoresis.

2.2.6. Serum stability testFor serum stability assay, siRNA/tiRNA complexedwith PEI at an N/P

ratio of 10 was incubated with FBS at 10% final concentration. Sampleswere then incubated at 37 °C and aliquots were collected at differenttime points. Samples were then decomplexed with heparin incubationand then loaded on a 10% PAGE gel. RNA fractions were visualized bystaining with ethidium bromide.

2.3. Cellular uptake experiments with siRNA/tiRNA-PEI complex

HeLa cells (human cervix epithelial adenocarcinoma) were seededat a density of 1 × 105 cells/well in a 6-well plate with DMEM culturemedium to reach 60%–70% confluency. Culturemediumwas exchangedfor fresh serum-free DMEM and Cy3 labeled siRNA/tiRNA-PEI complexat 100 nM RNA (N/P 5 and 10) concentration was added and incubatedfor 3 h at 37 °C. Thereafter, the medium was aspirated off and the cellswere trypsinized, centrifuged and re-suspended in serum-freeDMEM containing 10 μg/mL of Hoechst 33342 dye. After incubationfor 15 min at 37 °C, the cell suspension was centrifuged and re-suspended in serum-free DMEM. Samples were immediatelyanalyzed on Nucleocounter NC-3000 Chemometec) with a user-adaptable protocol [20].

The cellular uptake of Cy3-labeled siRNA/tiRNA-PEI complex wasfurther examined by using fluorescent microscopy. Cells seeded in con-focal imaging dishes at a density of 5 × 104 cells/dish were treated withpolyplexes at 100 nM RNA concentration (N/P 5) for 3 h in serum-freeDMEM. After removing the complex from the confocal dish, cells werewashed with PBS solution containing trypan (0.02%) to quench extra-cellular fluorescence. The intracellular localization of Cy3-RNA wasthen analyzed using fluorescence microscope (Olympus IX-81) and im-ages were acquired with a Metaview imaging software.

2.4. Transfection experiments using siRNA/tiRNA-PEI complex

For transfection experiments, HeLa were seeded in 12-well platesand cultured for 24 h. tiRNA structure designed to silence threeknown cancer targets Lamin A/C, DBP (vitamin D-binding protein)and TIG3 (Tazarotene-induced gene 3) simultaneously were used at100 nMconcentration and corresponding siRNAs, with the same targetswere used as a simple mixture at the same concentration. Cells weretransfectedwith siRNAmixture or tiRNA using jetPEI as the transfectionreagent (N/P 5). After transfection, total mRNAs were extracted usingthe Isol-RNA Lysis Reagent kit (5 Prime) and used as templates forcDNA synthesis using ImProm-II™ Reverse Transcription System(Promega). Target gene expression levelswere analyzed by quantitativereverse transcription real-time polymerase chain reaction (qRT-PCR)using StepOne real-time PCR system (Applied Biosystems).

2.5. Preparation and evaluation of galactose-modified PEI for liver-specificRNAi delivery

2.5.1. Preparation of galactose-modified PEIPEI was modified with SPDP-based cross-linker to include thiol-

modified galactose units [21,22]. PEI (500 mg) was dissolved in PBSbuffer (supplemented with 2 mM EDTA) in a glass vial and heated inan oil bath set at 65 °C for 10minwithmagnetic stirring. After completedissolution of the polymer, 200 μl (1 N HCl) was added and stirred for30 min at room temperature. SPDP (5 mg) dissolved in 100 μl anhy-drous DMSO was slowly injected into the PEI solution and activated

30 S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

PEI solution was incubated for 2 h at room temperature. The productwas then applied to Sephadex column (PD-10, GE Healthcare)eluted with 0.01 M PBS buffer supplemented with 2 mM EDTA.4-Aminophenyl β-D-galactopyranoside (GAL) was thiolated by reactionwith 2-iminothiolane in phosphate buffer 8.0 supplementedwith 2mMEDTA. Activated PEI was then mixed with the thiol-modified galactoseand allowed to react for 12 h at room temperature. PEI-GAL was thenpurified by centrifugal dialysis method (MWCO 3000) to remove un-bound galactose. The galactose concentration in PEI-GAL was deter-mined by sulfuric acid micro-method [23], and we used 4% galactosemodified PEI for these studies.

2.5.2. Characterization of siRNA/tiRNA complex with PEI-GALsiRNA/tiRNA complex with PEI-GAL was prepared in an exactly

manner as described with PEI complex. siRNA/tiRNA-PEI-GAL complexwas further characterized for evaluating their particle size, surfacemor-phology and RNA binding efficacy.

2.5.3. Uptake experiments using PEI-GALThe cellular uptake of Cy3-labeled siRNA/tiRNA was further exam-

ined upon complexation with either PEI or PEI-GAL (N/P 5) by usingfluorescent microscopy on Hep3B (human hepatocyte carcinoma) andHepa 1–6 (mouse hepatoma cell line) cell lines. The effect of tiRNA/PEI-GAL on control cells (HeLa cells)was also verified. Cellswere seededin confocal imaging dishes at a density of 3 × 104 cells per dish and up-take studies were performed as described previously.

2.5.4. Knock-down of liver-specific genes in vitroFor transfection experiments with mouse-specific apolipoprotein B

(ApoB), Hepa 1–6 cells were plated at a density of 1.5 × 105 cells/wellin a 12-well plate and incubated for 24 h. Cells were transfected withApoB-specific siRNAs (3 different sequences targeting mouse ApoB) ortiRNA (encoded with the same ApoB sequences in tripodal format)using PEI/PEI-GAL at a dose of 30 nM, 50 nM and 100 nM at N/P 5.Mutant ApoB siRNAs/tiRNA sequences were used as controls in theseexperiments and cells were harvested 24 h post-transfection, subjectedto qRT-PCR quantification as described before.

2.6. In vivo evaluation of siRNA/tiRNA-PEI complex on mice models

2.6.1. In vivo biodistribution experimentsAll animal experiments in this study were reviewed and approved

by the Institutional Animal Care and Use Committee (IACUC) ofSungkyunkwan University School of Medicine (SUSM). To evaluatethe biodistribution and liver targeting, an in vivo near-infrared fluores-cence (NIRF) imaging method was adopted. C57BL/6 mice were usedas in vivo models in this study. First, we used 20 bp dsDNA (siDNAs)and 32 nt tripodal DNA (tiDNA) labeled with Cy 5.5 as surrogates forsiRNA/tiRNA. PEI or PEI-GAL was mixed with 60 μg siDNAs (combina-tion of 3 complementary siDNAs) or tiDNA-32 at N/P 5 and complexwas prepared in 5% glucose solution. Polyplexes were injected intrave-nously and major organs were excised 3 h post-injection. Images werethen obtained using an In-Vivo Imaging System FOBI (Neoscience,Korea). Following initial experiments, additional biodistribution exper-iments were performed using Cy5.5-labeled tiRNA complexedwith PEI-GAL, to compare the distribution pattern between tripodal DNA andRNA structures.

2.6.2. Liver targeting studiesThe in vivohepatocyte binding affinity of siRNA/tiRNA-PEI-GAL com-

plex was quantified by NC-3000 method. PEI-GAL complexed siRNA/tiRNA-Cy5.5 samples were administered intravenously at a dose of60 μg/mouse. After 3 h of injection, themicewere sacrificed, liver tissuewas excised and hepatocytes were isolated using liver digest medium(GIBCO). The cell suspension was passed through 70 μm cell strainer,centrifuged and washed twice with hepatocyte wash medium (GIBCO).

Isolated cells were stained with 20 μg/mL of Hoechst 33342 dye andwere analyzed immediately on NC-3000.

2.6.3. Immune stimulation testFor in vivo immune stimulation test, mice were intravenously

injectedwith a single dose siRNAs-Apo B/tiRNA-ApoB-PEI-GAL complexat 6 mg/kgmouseweight (N/P 5). Poly I:C, a potent immunostimulatorydsRNA analogue, and dsRNA (48 bp) possessing similar number ofnucleotides as the tripodal RNA (32 nt) structure were included as con-trols. Blood samples were collected from the mice 6 h after injection,and cytokine (IFN-γ, TNF-α, IL-6) levels were measured with commer-cially available enzyme-linked immunosorbent assay kits (eBioscience,USA) according to the manufacturer's instructions.

2.6.4. Knock-down of liver-specific gene in vivo using siRNA/tiRNAPEI-GAL complex

For in vivo gene silencing experiments, mice were intravenouslyinjectedwith a single dose siRNAs-Apo B/tiRNA-ApoB-PEI-GAL complexat 6 mg/kg mouse-weight (N/P 5) in 5% glucose solution. Mutant ApoBsiRNAs/tiRNA sequences at the same concentrations were used as con-trols in these experiments. After 48 h of injection, liver samples werecollected and total mRNA from the tissue samples was extracted. ApoBmRNA level was assayed using qRT-PCR method by GAPDH normaliza-tion. Serum low-density lipoprotein (LDL) cholesterol level wasmea-sured after ApoB treatment using a commercial LDL cholesteroldetection kit according to the manufacturer' s instructions (HDL andLDL/VLDL Cholesterol Quantification Kit, Abcam, UK).

2.7. Statistical analysis

The statistical significance of differences between the mean valueswas determined by using Student's t test and a p value of 0.05 or lesswas considered significant.

3. Results

3.1. Optimization of tripodal core structure using model DNA structures

To optimize the tiRNA structure having optimal complexing abilitywith cationic polymers, model tripodal core structure was developedwith DNA units. By annealing three individual DNA fragmentswith sim-ilar length, a non-linear tripodal structure could be obtained. As thecomplexation process between nucleic acids and the cationic polymersis largely driven by charge–charge interactions, DNA model structurewould be a reasonable surrogate for these optimization studies. Nativegel electrophoresis confirmed that the tiDNA constructed with single-stranded DNA (ssDNA) fragments shorter than 26 nt were unstableand failed to form the expected tripodal structure (supplementarydata; Figure s3A). In contrast, tiDNA construct with 26, 32 and 40 ntDNA units formed a stable tripodal construct, as they appeared as asingle band in the gel. Cellular uptake studies on HeLa cells usingfluorescence-tagged DNA-PEI complex demonstrated improved uptakefor tiDNA constructs derived from 32 or 40 nt DNA units (supplementarydata; Figure s3B and s3C). These results possibly suggest that stable tripo-dal nucleic acid structure can be obtained from 32 nt units, and thesestructures can effectively associate with cationic polymers for enhancedintracellular delivery. Based on these observations, tripodal structurecomposed of 32 nt RNA sequence was developed for further studies(supplementary data; Figure s1 and s4).

3.2. Characterization of siRNA/tiRNA complex with PEI

Morphological and physical characteristics of siRNA/tiRNA-PEI com-plexes were analyzed by AFM and DLS experiments. As shown in Fig. 1,the DLS studies revealed that the tiRNAmolecules were effectively con-densed by PEI at both N/P 5 and 10. The Z average value of tiRNA-PEI

31S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

complexes was 193 ± 22 nm at N/P 5 and 154 ± 15 nm at N/P 10. Onthe other hand, siRNA-PEI complexes had larger particle size, andobserved values were 460 ± 30 nm and 394 ± 42 nm at N/P 5 and10, respectively. At N/P ratio of 5, zeta potential values for both siRNA/tiRNA-PEI complex were positive. Values were in the range of 22.8 mVand 30.7 mV for siRNA-PEI and tiRNA-PEI complexes, respectively, atN/P 5. At higher N/P of 10, zeta potential values increased to 24.6 mVfor siRNA-PEI complex and tiRNA-PEI had a value of approximately31.3 mV.

AFM studieswere performedmainly to examine the surfacemorpho-logical properties of siRNA/tiRNA-PEI complexes (Fig. 2). As expected,siRNA-PEI complex had larger size and irregular surface morphology. Incontrast, tiRNA-PEI complex had rather uniform size and spherical mor-phology, and the average particle size was in the range of 100–200 nm.The variation in size and morphology between siRNA/tiRNA complexescan be attributed to the difference in their charge density and RNAchain length. tiRNA with increased chain length have much highercharge density than monomeric siRNA, and thus readily forms morecompact complex structures when complexed with the cationic agents.On the other hand, conventional double-stranded siRNA have a ratherrigid structural format and they tend to form loose and/or unstable com-plexes with cationic carriers [4,17].

3.3. Stability assessment of siRNA/tiRNA polyplexes

siRNA/tiRNA complex formation pattern with PEI was examined byagarose gel electrophoresis at different N/P ratios (supplementary data;Figure s5A). siRNA forms a rather weak complex with PEI at lower N/Pratios and complex stability observed at N/P 10 and above. WithtiRNA structure, the migration of RNA on the gel was retarded at N/Pratio 5 and above, indicating an improved stability for this complex.The relative stability of these complexes was examined by heparindecomplexation assay at N/P ratios of 5, 10 and 10 (supplementarydata; Figure s5B). Here, the siRNA stability with PEI carrier was alsoextremely poor at N/P 5 and 10, RNA easily migrated from the gelupon incubation with anionic molecules. The stability of the tiRNA-PEIcomplexwas relatively better, especially atN/P 10 and 15. This probablyindicates that the tiRNA form stable complexes with cationic polymersthan with simple siRNA. The higher stability of tripodal complexes canbe attributed to the formation of stable polyplexes by the increasedcharge density and improved backbone flexibility.

Fig. 1. Size and zeta potential of siRNA/tiRNA-PEI complex. Size distribution and zetapotential of siRNA/tiRNA with PEI at N/P 5 and 10. Error bars are standard deviations(±SD) of 3 measurements performed on the same sample. Values on primary axis repre-sent the hydrodynamic size (nm) of siRNA/tiRNA-PEI complex and values on secondaryaxis represent zeta potential values of the same complex.

siRNA/tiRNA stability against serum nucleases was further verified,in both native and PEI complexed conditions (supplementary data;Figure s5C). siRNA in its native state was degraded after 24 h of incuba-tion in FBS, whereas complexed siRNA remained protected even after48 h of incubation. tiRNA, on the other hand, was quite susceptible tonuclease degradation in its uncomplexed state. tiRNA destabilizationwas initiated within an hour of incubation and degradation was almostcomplete within 12 h of contact. PEI complexation improved tiRNAstability against serum nucleases and degradation was observed onlyafter 6 h of incubation.

3.4. Cellular uptake studies

Nucleocounter-based technique was performed to evaluate thecellular uptake behavior of Cy3-labeled siRNA/tiRNAupon PEI complex-ation. In this technique, the intra-cellular fluorescence intensity on agated cell population was quantified and the uptake efficacy of siRNA/tiRNA-PEI complex was evaluated at N/P 5 and 10 (Fig. 3). Within thesame N/P ratios (5 and 10) and RNA concentration (100 nM), the cellu-lar uptake of the tiRNA-PEI was significantly higher than that of nativesiRNA-PEI complex. Amore than 3-fold increase in uptakewas observedwith tiRNA-PEI over siRNA-PEI complex at both N/P ratios. To furthersubstantiate this, a fluorescent microscopic investigation was per-formed on these complexes. As quantified by Nucleocounter experi-ments, tiRNA complex displayed enhanced cellular uptake in HeLacells with PEI at N/P 5 (Fig. 4). siRNA-PEI complex displayed quiteweak fluorescent signals, signifying poor ability of PEI in deliveringconventional siRNA structure. These quantitative and qualitative uptakeexperiments seem to be a clear indication for the enhanced uptakepattern of tiRNA-PEI complexes.

3.5. In vitro gene knock efficacy of siRNA/tiRNA-PEI complex on HeLa cells

A salient feature of the tiRNA format is its ability to induce multipletarget silencingwith improved efficacy over conventional siRNA format.The multiple gene silencing ability of tiRNA structures was establishedonHeLa cells using Lamin/DBP/TIG3 (L/D/T) cancer targets. tiRNA struc-ture encoding these targets were used along with their siRNA counter-parts at the same conditions and L/D/T mRNA levels were normalizedwith the GAPDH housekeeping gene levels (Fig. 5). At 100 nM concen-tration and N/P 5, we observed 82.7 ± 2.4% and 79.9 ± 0.7% knock-down in Lamin and DBP mRNA levels, respectively, with tiRNA-jetPEItransfection. On the other hand, corresponding siRNAs induced nearly62.9 ± 5.9% and 68.9 ± 2.3% knock-down for Lamin and DBP targets,respectively, at the same RNA concentration. TIG3 had almost 90%knock-down with both siRNA and tiRNA transfection in these experi-ments. Consistent with our previous reports [18], this experiment clearlyvalidated our hypothesis that tiRNA format can successfully implementmultiple target knock-down ability with improved efficacy over simplecombination of siRNAs.

In the same study, we compared the efficacy of our newly designedtiRNA-32 structure against our reported tiRNA-38 structure [18]. Inter-estingly, both structures had similar RNAi efficacy, demonstrating theshorter version of tiRNA composed of 32 nt units could trigger efficientgene silencing, similar to the original tiRNA structure with 38 nt units.Because tiRNA-32 is smaller and easier to synthesize, we decided touse this tiRNA for the rest of the studies. We also attempted to evaluatethe activity of partial tiRNA structure, prepared by the annealing of two32 nt RNA strands. These partial structures failed to impose any appre-ciable level of gene silencing in HeLa cells (supplementary data;Figure s6), implying the significance of maintaining proper tripodalgeometry in inducing robust gene silencing efficacy.

Fig. 2. AFM images showing surface morphology of siRNA/tiRNA-PEI complex. (A) siRNA-PEI complex; (B) tiRNA-PEI.

32 S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

3.6. Liver specificity of galactose-modified PEI in vitro

In order to achieve a targeted delivery onto the liver, PEI (25 kDa)modified with galactose units were utilized. Galactose-modified poly-mers are recognized by the asialoglycoproteins (ASGP) receptors inliver and may facilitate a receptor-mediated uptake. First, the PEI wasreacted with SPDP linker and resultant PEI-PDP was coupled withthiol activated GAL (aminophenyl β-D-galactopyranoside). Physico-chemical characterization studies were performed to confirm thesiRNA/tiRNA binding efficacy of PEI-GAL. Consistent with the PEIcomplexation experiments, GAL-modified PEI formed stable nano-complex with tiRNA structures and retained their RNA binding efficacy

Fig. 3. NC-3000 based quantification of internalized siRNA/tiRNA-PEI complex. HeLa cellswere transfected with either Cy3 siRNA-PEI or Cy3 tiRNA-PEI complex at 100 nM concen-tration (N/P 5 and 10). The fluorescence intensitywas quantified byNucleocountermethodbased on intracellular Cy3 signals. Inset, histogram plot of uptake quantification studiesusing siRNA/tiRNA-PEI complex.

(supplementary data; Figure s7A–C). From these studies, it was obviousthat the GALmodification through SPDP linker has not significantly ham-pered the nucleic acid binding capability of native PEI.

GALmodification had a profound effect in improving cellular uptakeof tiRNA complexes in liver cells. From the microscopic experiments, itwas obvious that GAL modified PEI had better uptake in Hep3B cells(Fig. 6) and Hepa 1–6 cells (supplementary data; Figure s8A) comparedto unmodified PEI, when complexed with Cy3 labeled tiRNA. On theother hand, the uptake of tiRNAwith PEI and PEI-GALwere quite similaron HeLa cells (supplementary data; Figure s8B). In contrast, siRNAcandidates had a marginal difference in their cellular uptake behaviorbetween PEI and PEI-GAL complexes in Hep3B cells (Fig. 6). These

Fig. 4. Microscopic studies on cellular uptake of siRNA/tiRNA-PEI complex. Fluorescentmicroscopic images demonstrating cellular internalization of Cy3-labeled siRNA/tiRNA-PEI complex (100 nM, N/P 5) after 3 h of incubation in serum-free conditions. Imageswere acquired at 20× magnification in fluorescence mode-Cy3 channel (left panel) andDIC mode (middle panel), further overlapped to visualize the internalized polyplexes(right panel). A, control (untreated cells); B, siRNA-PEI complex; C, tiRNA-PEI complex.

Fig. 5. Gene silencing activity of siRNA/tiRNA-PEI complex in HeLa cells. Cells weretransfected with either siRNA mixture (Lamin, DBP, and TIG3) each at 100 nM or withtiRNA-32/tiRNA-38 at 100 nM with the same encoded sequences at N/P 5 using jetPEI(see supplementary data for sequence details on siRNA/tiRNA). L/D/T and GAPDH expres-sion levelswere analyzed byqRT-PCR24hpost-transfection andmRNA levelswerenormal-ized with GAPDH levels. All data in the graph represent the mean ± SD of 3 independentexperiments.

Fig. 6.Microscopic studies on cellular uptake of siRNA/tiRNA with galactose modified PEIin Hep3B liver cells. Fluorescent microscopic images demonstrating cellular internaliza-tion of Cy3-labeled siRNA/tiRNA (100 nM, N/P 5) with PEI or PEI-GAL after 3 h of incuba-tion in serum-free conditions. Imageswere acquired at 20×magnification in fluorescencemode-Cy3 channel (left panel) and DIC mode (middle panel), further overlapped to visu-alize the internalized polyplexes (right panel). A, control (untreated cells); B, siRNA-PEIcomplex; C, siRNA-PEI-GAL complex; D, tiRNA-PEI complex; E, tiRNA-PEI-GAL complex.

33S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

studies have confirmed that the addition of GAL on the PEI backbonemay provide receptor-mediated uptake in liver cells, while retainingthe inherent nucleic acid transferring ability of PEI units.

Next, we conducted gene knock-down experiments with siRNAs/tiRNA targeting mouse ApoB on Hepa 1–6 cells, to substantiate thetranslation of enhanced delivery efficacy to an actual RNAi effect. Inthis study, instead of multiple targets, we have encoded three differentmouse ApoB targeting sequences in the tiRNA format. As a control, cor-responding ApoB siRNA sequences in conventional duplex format wereused as physical mixture, with PEI and PEI-GAL carriers (Fig. 7). WithPEI-GAL carrier, tiRNA could induce stronger RNAi response in livercells than corresponding siRNAmixture, consistentwith the cellular up-take studies. Dose-dependent ApoB gene silencing was observed at 30,50 and 100 nM concentrations with both siRNAs/tiRNA complexes,and tiRNA could implement stronger response at all these concentra-tions. Nearly 34.6 ± 3.6%, 51.3 ± 5.4% and 70.6 ± 5.1% knock-downwas attained at 30, 50 and 100 nM RNA concentrations, respectively,with tiRNA-PEI-GAL transfection on Hepa 1–6 cells. With siRNAs-PEI-GAL, the ApoB knock-down level was 1.94 ± 2.9, 28.2 ± 12.2 and47.9 ± 9.3% at 30, 50 and 100 nM conditions, respectively. Even withunmodified PEI, tiRNA structure could induce a stronger RNAi responsethan with siRNA candidates at similar transfection conditions. Controlsequences (siRNAs-Mut/tiRNA-Mut) failed to impart any effect inthese experiments. These studies clearly suggest that the tiRNA struc-ture can implement robust RNAi activity in a sequence-dependentman-ner over the conventional siRNA system. Moreover, with the addition ofGAL as a targeting ligand, the PEI-based delivery system can be used asan effective liver-specific RNAi platform.

3.7. Biodistribution experiments

In vivo biodistribution experiments were performed to study theliver-targeting ability of GAL-modified PEI. After intravenous injectionof the complexes, the organs of interest were collected for imagingand fluorescence intensity level was quantified for different samples.As shown in Fig. 8, the group of mice that received tiDNA-PEI-GAL com-plex exhibited maximum fluorescence intensity in this study, and theirintensity was quite higher than animals injected with siDNAs-PEI-GAL

Fig. 7. Gene silencing activity of siRNAs/tiRNA in Hepa 1–6 mouse cells transfected usingLPEI/LPEI-GAL. Each siRNA (3 siRNAs targeting mouse ApoB) or tiRNA-32 encoding thesame siRNA sequence was transfected at 30, 50 and 100 nM concentrations at N/P 5.siRNAs-mut and tiRNA-mutwere used as the control in this experiments (see supplemen-tary data for sequence details on siRNAs/tiRNA). GAPDH expression levels were analyzedby qRT-PCR 24 h post-transfection and the ApoB mRNA levels were normalized with theGAPDH mRNA level. All data in the graph represent the mean ± SD of 3 independentexperiments (**P b 0.05, *P b 0.01).

Fig. 8. In vivo biodistribution studies. In vivo biodistribution pattern of surrogate nucleic acid structures with PEI/PEI-GAL complex. Cy5.5 labeled siDNAs (similar to siRNAs) or tiDNA-32(similar to tiRNA) complexed with polymers were injected into the tail vein of mice at a dose of 60 μg DNA (N/P 5). The mice were sacrificed after 3 h and major organs were excised forimaging studies using In-Vivo Imaging System (A). The fluorescence intensity in major organs (lung, liver, kidney, and spleen) was detected, and quantified for each sample and repre-sented as histogram plot (B). All data in the graph represent the mean ± SD of 3 independent experiments.

34 S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

complex. Interestingly, the animals that received siDNAs/tiDNA fromunmodified PEI had much lower fluorescence intensity and intensitylevels were comparable between siDNAs and tiDNA complexes. As ex-pected, the fluorescence accumulation in other organs were marginal,except in the kidney. This study supports our hypothesis that the tripo-dal structure indeed is effective in enhancing the intra-cellular deliveryof nucleic acids over conventional siRNA format even under in vivo con-ditions. GAL modification was effective in delivering oligonucleotides,specifically to the liver, and thus the efficacy of tiDNA structures oversiDNAs could clearly be acknowledged. On the other hand, unmodifiedPEI failed to provide any advantage for tiRNA structure and furtherin vivo efficacy tests were not designed with this delivery approach.

Furthermore, to verify the biodistribution pattern of surrogate DNAwith the functional RNA structure, additional experiments were per-formed with tiRNA-Cy 5.5 complexed with PEI-GAL. From this study, itwas obvious that both surrogate tiDNAand tiRNA also had similar distri-bution patterns and maximum fluorescent accumulation was obtainedin the liver region (supplementary data; Figure s9) with both of theseformulations. In addition, we verified the ability of PEI-GAL carriers todeliver siRNA/tiRNA to hepatocyte cells. NC-3000 experiments basedon Cy 5.5 signals clearly suggest improved fluorescence accumulationin hepatocytes for tiRNA-PEI-GAL complexes (supplementary data;Figure s10) compared to their siRNA counterparts.

3.8. In vivo cytokine response test

The administration of long double-stranded RNA can provoke stronginnate immune response and this may jeopardize the specific RNAi-mediated effects [24]. To verify the effect of siRNA/tiRNA on innate im-mune response, cytokine (IFN-γ, TNF-α and IL-6) expression in serumwas verified in vivo. In this study, no pronounced difference in cytokinelevels was observed for siRNA-treated mice groups with either PEI-GALcarrier (supplementary data; Figure s11). However, the tiRNA anddsRNA (48 bp) treatment induced slightly higher levels of inflammatorycytokine than the siRNA-treated groups. On the other hand, the positivecontrol, poly I:C could provoke strong cytokine response in this studywith PEI-GAL carriers [25].

3.9. In vivo gene silencing efficacy

In order to assess the target gene silencing ability of tiRNA/PEI-GALcomplex in vivo, the level of endogenous ApoB mRNA in the liver wasevaluated. Animals were treated with single-dose intravenous injec-tions of ApoB-targeting siRNAs/tiRNA with PEI-GAL (6 mg/kg) and

ApoB level was estimated 48 h after injection via qRT-PCR. As shownin Fig. 9A, ApoB mRNA level in mouse liver was reduced to 63.5 ±7.3% with a single dose of tiRNA/PEI-GAL complex treatment, and33.1 ± 18.9% reduction was attained with siRNAs/PEI-GAL complex.No significant silencing effect was observed in mouse groups that re-ceived either siRNAs-Mut or tiRNA-Mut with the same delivery system,and this confirmed that the ApoB knock-down in vivo was sequence-specific. Consistent with the in vitro experiments, these studies indis-putably confirmed the efficacy of the tiRNA format over siRNA underin vivo conditions.

ApoB is an essential component in the formation of LDL and theyplay a major role in the metabolism of dietary and endogenous choles-terol [26]. Therefore, we assessed the LDL level in serum 48 h after treat-mentwith ApoB siRNAs/tiRNA. In this study, comparedwith PBS-treatedcontrol mice groups, serum cholesterol levels were reduced for animalswith ApoB siRNAs/tiRNA treatment (Fig. 9B).

4. Discussion

By annealing three ssRNA units, tiRNA targeting three differentgenes were generated for this study. The tiRNA structure was designedsuch that the 5′ region of each antisense strandwas oriented toward theoutside, and this arrangement allows the structure to retain their multi-gene knock-down ability, unlike conventional duplex RNA format (sup-plementary data; Figure s2). The affinity between cationic polymers andRNA backbonewas greatly improvedwith the structural design and thiswas quite apparent in the characterization experiments. Importantly,the multiple target encoded tiRNA could impart a stronger RNAiresponse over the simple mixture of siRNAs.

Hepatocytes, the key parenchymal cells in the liver, have been tradi-tional target cells for drug delivery given their central role in several in-fectious and metabolic disorders [27]. The asialoglycoprotein receptor(ASGP-R) has been widely used as the target receptor for drug deliveryto these hepatocyte cells and galactose/lactosemoieties coupled to poly-mers/liposomes can facilitate ASGP targeting [28,29]. Polyethyleneiminemodifiedwith galactose units seems to be a potential candidate for liver-targeted genedelivery systems [30]; PEI backbone possesses nucleic acidbinding domain and their intrinsic endosomal-disrupting activity(“proton sponge” effect) helps in the delivery of nucleic acid cargoes inthe endosomal compartment. However, the biocompatibility of largemolecular weight branched versions of polyethyleneimine still remainsa major concern and their clinical usage have also been hampered bytheir deleterious effects [31]. Linear PEI remains the standard transfec-tion reagent for plasmid DNA; however, their efficacy towards siRNA

Fig. 9.A, gene silencing activity of siRNAs/tiRNA onmousemodels delivered using PEI-GAL in vivo on C57BL/6mice. Each siRNA (3 siRNAs targetingmouse Apo B) or tiRNA (32-mer)wereinjected via tail vein at 6 mg/kg at N/P 5 (single dose). siRNAs-mut and tiRNA-mut were used as control samples. Liver tissues were harvested 48 h post-injection and ApoB mRNA wasquantified by GAPDHnormalization. All data in the graph represent themean± SD of 3 independent experiments (**P b 0.05). B, serumHDL/LDL level after treatmentwith ApoB siRNAs/tiRNA. Mice treated with siRNAs/tiRNA-PEI-GAL samples at 6 mg/kg (N/P 5) and serum HDL/LDL was analyzed by Cholesterol Assay kit, 48 h post-injection.

35S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

candidates seems quite low. Nevertheless, linear PEI is the most pre-ferred choice for RNAi delivery, as they are less toxic than their branchedcounterparts. Lack of primary amino groups in linear PEI backbonemakes them less amenable to functional modifications. In this study,we used SPDP-based conjugation reaction for functionalization of PEIwith galactose, and this was utilized as a liver-homing device. The strat-egy here was to develop a biocompatible liver-specific platform fortiRNA delivery, by retaining the nucleic acid delivering capability of na-tive PEI. Both PEI and PEI-GAL had similar physicochemical properties(size/zeta potential and surface morphology), although PEI-GAL formedslightly larger complexes than the native PEI-RNA complexes. However,tiRNA-PEI-GAL complexes were more effective in inducing target geneknock-down under in vivo conditions, as they could perfectly combinenucleic acid transferring ability with liver-targeting efficacy.

Improved cellular uptake and superior RNAi effect was observedwith tiRNA-PEI-GAL complexes on in vitro liver cell models. As we ex-pected, GAL-modified PEI units could impart stronger RNAi responsewith tiRNA candidates. However, more clear and conclusive evidenceswere obtained with our in vivo studies. The biodistribution patternseems to suggest that the tripodal nucleic acid structure with PEI-GALcarrier was more readily localized into the liver than other polyplexesused in this study. With a targeted approach, we could deliver higheramounts of nucleic acids specifically into the liver region and this allowedus to discriminate the efficacy between the conventional duplex and tri-podal structures.With unmodified PEI, possibly due to their non-specificbiodistribution pattern,wewere unable tomake any clear distinction be-tween these structures.

The in vivo RNAi effect demonstrated by endogenous ApoB knock-down was a clear manifestation of tiRNA efficacy over conventionalsiRNAs. Even with the same combination of siRNAs and the carrier, wecould clearly demonstrate the stronger RNAi effect with tiRNA formatin mouse models. Thus, by integrating the structural features of tiRNAstructure with targeted delivery approach, an effective RNAi-basedtherapeutic platform could be devised for liver treatment.

5. Conclusion

siRNA structural variant, constructed in a well-defined tripodalformat, expanded the structural diversity repertoire suitable for multi-target gene silencing. When combined with cationic PEI deliverysystems, the tiRNA structure induced enhanced cellular uptake andimproved gene silencing potency over classical siRNA-PEI system. Line-ar PEI functionalized with galactose units facilitated effective gene

silencing effect on both in vitro and in vivomodels. Our studies suggestthat unconventional tiRNA structure, in combination with appropriatecationic polymers, is capable of executing multi-target gene silencingwith enhanced potency and could be utilized as a structural platformto develop efficient liver-targeted RNAi therapeutics.

Acknowledgment

This studywas supported by a Global Research Laboratory grant fromthe KoreanMinistry of Education, Science, and Technology (2008-00582)to D.K.L. S.S. was supported by a grant from the National Research Foun-dation of Korea funded by the Korean Government (Ministry of Educa-tion, Science and Technology) (NRF-2013R1A1A2064660). S.K. wassupported by Korea Ministry of Environment as "EI project" (ERL E211-41003-0007-0).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2014.09.016.

References

[1] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature411 (2001) 494–498.

[2] S.M. Elbashir, W. Lendeckel, T. Tuschl, RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev. 15 (2001) 188–200.

[3] Y.K. Oh, T.G. Park, siRNA delivery systems for cancer treatment, Adv. Drug Deliv. Rev.61 (2009) 850–862.

[4] S.H. Lee, B.H. Chung, T.G. Park, Y.S. Nam, H. Mok, Small-interfering RNA (siRNA)-based functionalmicro- and nanostructures for efficient and selective gene silencing,Acc. Chem. Res. 45 (2012) 1014–1025.

[5] D.H. Kim, M.A. Behlke, S.D. Rose, M.S. Chang, S. Choi, J.J. Rossi, Synthetic dsRNA dicersubstrates enhance RNAi potency and efficacy, Nat. Biotechnol. 23 (2005) 222–226.

[6] W. Salomon, K. Bulock, J. Lapierre, P. Pavco, T. Woolf, J. Kamens, Modified dsRNAsthat are not processed by dicer maintain potency and are incorporated into theRISC, Nucleic Acids Res. 38 (2010) 3771–3779.

[7] C.I. Chang, S.W. Hong, S. Kim, D.K. Lee, Structure-activity relationship study ofsiRNAs with structural variations, Biochem. Biophys. Res. Commun. 359 (2007)997–1003.

[8] C.I. Chang, H.A. Kim, P. Dua, S. Kim, C.J. Li, D.K. Lee, Structural diversity repertoire ofgene silencing small interfering RNAs, Nucleic Acid Ther. 21 (2011) 125–131.

[9] C.I. Chang, H.S. Kang, C. Ban, S. Kim, D.K. Lee, Dual-target gene silencing by usinglong, synthetic siRNA duplexes without triggering antiviral responses, Mol. Cells27 (2009) 689–695.

[10] C.I. Chang, J.W. Yoo, S.W. Hong, S.E. Lee, H.S. Kang, X. Sun, H.A. Rogoff, C. Ban, S. Kim,C.J. Li, D.K. Lee, Asymmetric shorter-duplex siRNA structures trigger efficient genesilencing with reduced nonspecific effects, Mol. Ther. 17 (2009) 725–732.

36 S. Sajeesh et al. / Journal of Controlled Release 196 (2014) 28–36

[11] C.I. Chang, T.Y. Lee, P. Dua, S. Kim, C.J. Li, D.K. Lee, Long dsRNA-mediated RNA inter-ference and immunostimulation: long interfering RNA (liRNA) as a potent anti-cancer therapeutics, Nucleic Acid Ther. 21 (2011) 149–155.

[12] E. Wagner, Polymers for siRNA delivery: inspired by viruses to be targeted, dynamic,and precise, Acc. Chem. Res. 45 (2012) 1005–1013.

[13] K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances insiRNA delivery, Nat. Rev. Drug Discov. 8 (2009) 129–138.

[14] H. Mok, S.H. Lee, J.W. Park, T.G. Park, Multimeric small interfering ribonucleic acidfor highly efficient sequence-specific gene silencing, Nat. Mater. 9 (2010) 272–278.

[15] S.Y. Lee, M.S. Huh, S.K. Lee, S.J. Lee, H.J. Chung, J.H. Park, Y.K. Oh, K.W. Choi, K.M. Kim,I.C. Kwon, Stability and cellular uptake of polymerized siRNA (poly-siRNA)/polyethylenimine (PEI) complexes for efficient gene silencing, J. Control. Release141 (2010) 339–346.

[16] J.H. Jeong, H. Mok, Y.K. Oh, T.G. Park, siRNA conjugate delivery systems, Bioconjug.Chem. 20 (2009) 5–14.

[17] S. Sajeesh, T.Y. Lee, S.H.Woo, P. Dua, J.Y. Choe, K. Aeyeon, Y.W. Soo, C. Song, S.H. Park, S.Lee, C. Li, D.K. Lee, Long dsRNAmediated RNA interference and immunostimulation: atargeted delivery approach using polyethyleneimine based nano-carriers, Mol. Pharm.11 (2014) 872–884.

[18] C.I. Chang, T.Y. Lee, S. Kim, X. Sun, S.W. Hong, J.W. Yoo, P. Dua, H.S. Kang, S. Kim, D.K.Lee, Enhanced intracellular delivery andmulti-target gene silencing triggered by tri-podal RNA structures, J. Gene Med. 14 (2012) 138–146.

[19] C.I. Chang, T.Y. Lee, J.W. Yoo, D. Shin, M. Kim, S. Kim, D.K. Lee, Branched, tripartite-interfering RNAs silence multiple target genes with long guide strands, NucleicAcid Ther. 22 (2012) 30–39.

[20] P. Dua, H.S. Kang, S.M.Hong,M.S. Tsao, S. Kim,D.K. Lee, AlkalinephosphataseALPPL-2 isa novel pancreatic carcinoma-associated protein, Cancer Res. 73 (2013) 1934–1945.

[21] M. Kursa, G.F. Walker, V. Roessler, M. Ogris, W. Roedl, R. Kircheis, E. Wagner, Novelshielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes forsystemic tumor-targeted gene transfer, Bioconjug. Chem. 14 (2003) 222–231.

[22] D. Schaffert, M. Kiss, W. Rödl, A. Shir, A. Levitzki, M. Ogris, E. Wagner, Poly (I:C)-mediated tumor growth suppression in EGF-receptor over expressing tumorsusing EGF-polyethylene glycol-linear polyethylenimine as carrier, Pharm. Res.28 (2011) 731–741.

[23] M. Monsigny, C. Petit, A.C. Roche, Colorimetric determination of neutral sugars by aresorcinol sulfuric acid micro-method, Anal. Biochem. 175 (1998) 525–530.

[24] L. Alexopoulou, A.C. Holt, R. Medzhitov, R.A. Flavell, Recognition of double-strandedRNA and activation of NF-kß by toll-like receptor, Nature 413 (2001) 732–738.

[25] M. Matsumoto, T. Seya, TLR3: interferon induction by double-stranded RNA includingpoly(I:C), Adv. Drug Deliv. Rev. 60 (2008) 805–812.

[26] J.R. Burnett, P.H. Barrett, Apolipoprotein b metabolism: tracer kinetics, models, andmetabolic studies, Crit. Rev. Clin. Lab. Sci. 39 (2002) 89–137.

[27] L. Li, H. Wang, Z.Y. Ong, K. Xu, P.L.R. Ee, S. Zheng, J.L. Hedricke, Y.Y. Yang, Polymer-and lipid based nanoparticle therapeutics for the treatment of liver diseases, NanoToday 5 (2010) 296–312.

[28] C.H. Wu, G.Y. Wu, Receptor-mediated delivery of foreign genes to hepatocytes, Adv.Drug Deliv. Rev. 29 (1998) 243–248.

[29] M. Hashida, S. Takemura, M. Nishikawa, Y. Takakura, Targeted delivery of plasmidDNA complexed with galactosylated poly (L-lysine), J. Control. Release 53 (1998)301–310.

[30] M.A. Zanta, O. Boussif, A. Adib, J.P. Behr, In vitro gene delivery to hepatocytes withgalactosylated polyethylenimine, Bioconjug. Chem. 8 (1997) 839–844.

[31] U. Lungwitz, M. Breunig, T. Blunk, A. Göpferich, Polyethylenimine- based non-viralgene delivery systems, Eur. J. Pharm. Biopharm. 60 (2005) 247–266.