Long dsRNA-Mediated RNA Interference and Immunostimulation: A

Article pubs.acs.org/molecularpharmaceutics

Long dsRNA-Mediated RNA Interference and Immunostimulation: A Targeted Delivery Approach Using Polyethyleneimine Based NanoCarriers S. Sajeesh,†,# Tae Yeon Lee,†,# Sun Woo Hong,† Pooja Dua,† Jeong Yong Choe,† Aeyeon Kang,‡ Wan Soo Yun,‡ Changsik Song,‡ Sung Ha Park,§ Soyoun Kim,∥ Chiang Li,⊥ and Dong-ki Lee*,† †

Global Research Laboratory for RNAi Medicine, Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Republic of Korea ‡ Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Republic of Korea § Department of Physics, Sungkyunkwan University, Suwon 440-746, Republic of Korea ∥ Department of Medical Biotechnology, Dongguk University, Seoul 100-715, Republic of Korea ⊥ Department of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, United States S Supporting Information *

ABSTRACT: RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy. KEYWORDS: siRNA delivery, nanotechnology, targeted delivery, immunostimulation, RNA interference, glioblastoma

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INTRODUCTION Small interfering RNA (siRNA) has been recognized as an efficient tool for post-transcriptional gene silencing.1,2 However, safe and efficient delivery of therapeutic siRNAs remains a major hurdle toward its clinical application.3 Polymers are regarded as a safe option for nucleic acid delivery; however, their efficacy toward siRNA delivery remains quite low.4,5 In contrast to plasmid DNA structure, siRNA has extremely low charge density and high stiffness, which limit its complexforming ability with cationic polymers.6−8 Controlled immunostimulation via targeted drug delivery is an interesting approach in anticancer treatment.9,10 The antiproliferative and immunostimulatory activity of long dsRNAs, such as polyinosinic/polycytidylic acid [poly(I:C)], was successfully implemented to treat tumor models. Intratumoral delivery of the epidermal growth factor (EGF) receptor-targeted PEI-poly (I:C) complex induced complete © 2014 American Chemical Society

regression of pre-established intracranial tumors on nude mice, with no obvious adversity.11 While RNAi therapeutics primarily focus on executing specific target gene silencing without inducing any innate immune response, bifunctional immunostimulatory RNAi molecules capable of provoking both immune response and target gene silencing could present an interesting option for anticancer or antiviral treatment.12,13 In a previous study, we presented a nicked long dsRNA structure, named long interfering dsRNA (liRNA), as a novel trigger for the immunostimulatory RNAi approach.14 The liRNA is composed of a 19 bp siRNA unit and is multimerized by base paring between long overhangs. liRNA is capable of Received: Revised: Accepted: Published: 872

September 9, 2013 December 17, 2013 February 12, 2014 February 12, 2014 dx.doi.org/10.1021/mp400541z | Mol. Pharmaceutics 2014, 11, 872−884

Molecular Pharmaceutics

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in these experiments are shown in the Supporting Information (Figures S1 and S2). In vitro Dicer cleavage assay was performed on siRNA/liRNA samples using Turbo Dicer siRNA Generation Kit (Genlantis, San Diego, CA) according to manufacturer’s protocol. Cleaved products were then analyzed on 15% nondenaturing PAGE gel, stained with EtBr, and visualized by UV transillumination. Synthesis of Cross-Linked PEI Polymers. bPEI-SS/bPEIIMI was obtained by coupling of DTPA/IDCA with bPEI (1.8 kDa) using an EDC/NHS activation reaction, as reported elsewhere.23 In brief, DTPA/IDCA (2 mmol) was activated by the addition of EDC (5 mmol) and NHS (2 mmol) in a reaction flask containing 25 mL of MES buffer (pH 6.5) for 1 h. bPEI was dissolved in 5 mL of distilled water and slowly added to the activated cross-linker solution, and the reaction was continued for 48 h at room temperature. Next, the polymer solution was purified by exhaustive dialysis (3500 MWCO, Pierce Biotech. Rockford IL) with deionized water for 2−3 days. Finally, samples were dialyzed against RNAase-free water for 24 h, and the polymer solution was filtered with a 0.2 μm syringe filter. Polymer samples were either freeze-dried or used as solution after assessing their dry weight. Characterization of Polymers. For the primary amine group quantification, bPEI and modified bPEI samples were diluted to 0.5 mg/mL and then mixed with 200 μL of 0.1% TNBS reagent. Sodium bicarbonate solution (200 μL, 4%) was added to the polymer solution, and samples were incubated for 2 h at 37 °C. The solution was neutralized with the addition of 200 μL of 2 N HCl, and the absorbance at 405 nm was then measured using a microplate reader (Thermo, MULTISKAN EX). Polyplex Preparation. To prepare polyplexes, both the RNA solutions (10 μM) and polymer stock solution (1 mg/ mL) were first diluted with 150 mM NaCl solution. The polymer-RNA solutions were then mixed in appropriate N/P ratios, vortexed, and incubated for 20 min at room temperature. JetPEI-RNA complex was prepared by following the manufacturer’s instructions. Particle Size and Zeta Potential. Zeta-potential values and average size of siRNA/liRNA-PEI polyplexes were examined by using Zeta-Sizer (Nano-ZS 90, Malvern Instrument) with a He−Ne Laser beam (633 nm, fixed scattering angle of 90°) at 25 °C. Polyplex solutions were prepared by mixing RNA-PEI solution in 150 mM NaCl, vortexing, and incubating for 20 min. Samples were diluted to a final volume of 1 mL with 150 mM NaCl solution before measurement. Measured zeta-potential values and average sizes were presented as the average values of 3 runs. AFM Measurements. The polyplexes were prepared as described above, and an aliquot of the sample was then deposited onto the cleaved mica substrate for AFM analysis. AFM images of the RNA-PEI complex were then obtained by using a Multimode Nanoscope (Veeco Inc.) in liquid tapping mode. Agarose Gel Electrophoresis. The siRNA/liRNA condensing ability of cationic PEI was examined by agarose gel electrophoresis. Agarose gel (2% w/v) containing ethidium bromide was prepared in a Tris-borate-EDTA (TBE) buffer. Polyplexes were prepared in different N/P ratios as mentioned previously. Thereafter, the samples were mixed with 6× loading dye and electrophoresed at 100 V for 10 min. RNA bands in the gels were visualized by a UV illuminator (Gel Documentation Systems, Biovision).

executing specific target gene silencing with a combined effect to trigger dsRNA mediated PKR activation and IFN induction. Site-specific delivery of these potent therapeutic agents will therefore warranty a superior antitumor activity on the target organs, with minimal interaction on the normal healthy tissues. Thus, we believe an approach combining the key elements of targeted drug delivery, in conjugation with the bifunctional immunostimulatory-RNAi effectors, should prove promising in the successful implementation of liRNA therapy. PEI is the most studied material for nucleic acid delivery due to its favorable biophysical properties, including the ability to bind negatively charged nucleic acids to form nanoscale complexes and high buffering capacity for facilitated endosomal release.15,16 High molecular weight PEI (25 kDa branched PEI and 22 kDa linear PEI) has been the gold standard for transfection experiments; however, they exhibit high levels of toxicity both in vitro and in vivo. While low molecular weight PEIs such as 1.8 kDa branched PEI display a favorable toxicity profile, they exhibit much less transfection efficacy than their high molecular weight versions.17,18 The strategy of linking small molecular weight bPEI either by a cleavable disulfide linker or by noncleavable linkers was effective in reducing the toxicity.19 The bioreducible versions are mostly reduced by intracellular reductants such as glutathione and thus reduces the toxicity of the polymer and facilitates the release of nucleic acids.20 Noncleavable cross-linked versions of bPEI are equally effective in delivering nucleic acids; however, their toxicity remains slightly higher than that of bioreducible versions.21,22 In this study, cationic polymeric carriers were synthesized by cross-linking low-molecular weight bPEI either by disulfide or imidazole linkers. The multimerized structure of liRNA was expected to form a stable nanocomplex with these cross-linked cationic carriers. Cross-linked PEI was complexed with siRNA/ liRNA candidates and was examined by AFM and DLS analysis. Cellular uptake experiments were performed to compare the uptake efficacy of the siRNA/liRNA-PEI complex on U87MG cells. Gene silencing efficacy of the polyplexes was evaluated using Survivin as the target gene. Finally, disulfide modified bPEI was further modified with transferrin (Tf), and their uptake ability in mouse brain endothelial cell models was evaluated in vitro.

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MATERIALS AND METHODS Commercial low molecular weight (22 kDa) linear-PEI (jetPEI) was purchased from PolyPlus-transfection (Illkirch, France). Branched poly(ethylene imine) with a molecular weight (MW) of 1.8 kDa (bPEI) and linear poly(ethylene imine) with a MW of 25 kDa (LPEI) were purchased from Polysciences, Inc. (Warrington, PA). Polyinosinic−polycytidylic acid sodium salt [Poly(I:C)], branched poly(ethyleneimine) with a MW of 25 kDa (bPEI 25), N-hydroxysuccinimide (NHS), 4,5-imidazoledicarboxylic acid (IDA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 3′-dithiobispropanoic acid (DTPA), thiazolyl blue tetrazolium bromide (MTT), 2-(N-morpholino)ethanesulfonic acid (MES), diethyl pyrocarbonate (DEPC), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) were purchased from Sigma-Aldrich (St. Louis, MO). Solutions were made RNase-free by treating with 0.1% DEPC prior to autoclaving. Chemically synthesized RNAs were purchased from BMT Inc. (Seoul, South Korea), and cyanine 3 (Cy3) tagged RNAs were obtained from Bioneer (Daejeon, South Korea). The sequences and the annealing pattern of siRNA and liRNA used 873

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analyzed, and the mean fluorescence intensity of cells with Cy3 signals was quantified. The cellular uptake of Cy3 labeled polyplexes was further examined using fluorescent microscopy. The cells were seeded in confocal imaging dishes (SPL, South Korea) at a density of 5 × 104 cells per dish. The polyplexes at 100 nM RNA concentration and N/P = 5 were prepared as mentioned above and were exposed to the U87MG cells in serum free DMEM medium. After 3 h of incubation at 37 °C, the polyplexes were removed, and the cells were washed with DPBS. Further, cells were treated with 200 μL of trypan blue solution (0.02%) to quench extracellular fluorescence. The localization of Cy3-RNA within the cells was observed using a fluorescence microscope (Olympus IX-81), and images were acquired with Metaview imaging software at 20× magnification. Transfection Experiments Using the siRNA/liRNA-PEI Complex. For transfection experiments, U87MG cells were seeded with a cell density of 3 × 104 in 12 well plates and cultured for 24 h. siRNA/liRNA polyplexes were prepared in 150 mM NaCl, and 100 μL of the prepared transfection complexes were added to the cells. Lipid mediated transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Transfected cells were incubated for 12 h in 10% serum supplemented DMEM culture medium, and total RNAs were extracted from cell lysates using the Isol-RNA Lysis Reagent kit (5 Prime). Isolated mRNAs were used as templates for cDNA synthesis, which was performed using ImProm-II Reverse Transcription System (Promega) according to the manufacturer’s protocol. Target gene expression levels were analyzed by quantitative reverse transcription real time polymerase chain reaction (qRT-PCR) using a StepOne real-time PCR system (Applied Biosystems) according to the manufacturer’s protocol. The details of all the primer sequences used in this study are given in the Supporting Information (Figure S6). The immunostimulatory activity of the liRNA-PEI complex was assessed by the mRNA level evaluation of Interferoninduced protein with tetratricopeptide (IFIT 1), 2′-5′oligoadenylate synthetase 2 (OAS 2), tumor necrosis factors (TNF-α), interleukin 6 (IL 6), and interferon β (IFN-β) in U87MG cells using qRT-PCR. In this experiment, cells were treated with 100 nM liRNA at N/P 10 using different PEI samples, and poly(I:C) at 1.3 μg/mL (complexed with jetPEI) was used as the positive control. Western Blot Analysis. For Western blot analysis, the cells were seeded in a 6-well plate and transfected with the siRNA/ liRNA-PEI complex at 30 nM concentration (N/P 5 and 10) for 24 h in 10% serum supplemented DMEM culture medium. Cell proteins were extracted, and total protein was quantified by the BCA protein assay kit (Thermo, USA); 20 μg of extracted protein was fractionated by SDS−PAGE and transferred to a PVDF membrane. Proteins were detected using primary antibody (Cell Signaling Technology) against GAPDH (1:10,000 dilution, mouse mAb) and Survivin (1:1000 dilution, rabbit mAb). After incubation with peroxidase-coupled secondary antibodies, blots were developed with the ECL system (Neuronex) according to the manufacturer’s protocol and exposed to X-ray films (Kodak). Cell Growth Inhibition Assay after siRNA/liRNA-PEI Treatment. For assessing the cell growth pattern after siRNA/ liRNA-PEI transfection, the cell viability assay was measured by direct counting. U87MG cells were seeded for 24 h before transfection with a cell density of 3 × 104 in 12-well plates.

Anionic (Heparin) Decomplexation Assay. For the heparin decomplexation assay, siRNA/liRNAs were first complexed with various polymer carriers at room temperature for 20 min. Then, various amounts of heparin (heparin/RNA weight ratio 0, 1, 2, 3, and 5) were added, and the mixtures were further incubated for 15 min at room temperature. The samples were loaded onto a 2% agarose gel and subjected to electrophoresis as described previously. Acid−Base Titration. The buffer capability of PEI polymers was determined by acid−base titration assays over a pH range from 10.0 to 4.0. Briefly, the polymer (5 mg) was dissolved in 20 mL of 150 mM NaCl solution. The solution was brought to a starting pH of 10.0 with 0.1 M NaOH and was then titrated by the sequential addition of 20 μL aliquots of 0.01 N HCl to a pH of 4. The pH profile for each polymer was obtained during this acid titration. Cell-Culture Studies. HeLa cells (human cervix epithelial adenocarcinoma), U87MG cells (human glioblastoma-astrocytoma), and bEnd.3 cells (immortalized mouse brain endothelial cell line) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin, and 100 μg/mL streptomycin. The cells were then maintained at 37 °C in a 5% CO2 humidified atmosphere. Cytotoxicity Assessment of Polymers. The cytotoxicity assessment of polymer carriers was carried out on HeLa cells by using an MTT assay. The cells were seeded in a 96-well plate at an initial density of 10,000 cells/well and were then cultured for 24 h in DMEM medium containing 10% FBS. The cells were then treated with different polymers at varying concentrations, and the polymer treated cells were incubated in a humidified environment with 5% CO2 at 37 °C for 4 h. After replacing the culture medium with a fresh medium, the cells were further maintained for another 20 h. After the specified time, MTT reagent (5 mg/mL) was added to each well, and the cells were incubated for another 4 h. The medium in each well was replaced with 50 μL of DMSO to dissolve the formazan crystals. The plate was gently agitated for 15 min, and the absorbance (O.D.) at 570 nm was recorded with a microplate reader. The cell viability was calculated by using the following equation: cell viability (%) = [O.D. (test)/O.D. (control)]× 100

where O.D. (test) and O.D. (control) are the absorbance values of the cells cultured with and without polymers, respectively. Cellular Uptake Experiments. U87MG cells were seeded at a density of 1 × 105 cells/well in a 6-well plate in a DMEM culture medium and grown to reach 60%−70% confluence. Before transfection, the medium of each well was exchanged for fresh serum-free DMEM medium. Cy3 labeled siRNA/liRNAPEI complex at 100 nM RNA (N/P = 5) concentration was prepared in 150 mM NaCl and incubated for 20 min at room temperature. The cells were treated with polyplex solutions for 3 h at 37 °C, the medium was aspirated off from the wells and the cells were washed with DPBS. The cells were further trypsinized, centrifuged, and resuspended in 100 μL of serum free DMEM containing 10 μg/mL of Hoechst 33342 dye. The cells were incubated for 15 min at 37 °C and were collected by centrifugation. The cell pellet was resuspended in 100 μL of serum free DMEM medium and immediately analyzed on Nucleocounter NC-3000 (Chemometec) using a user adaptable protocol as reported previously.24 Five thousand cells were 874



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Figure 1. Size and zeta potential of the siRNA/liRNA-PEI complex. (A) Size distribution and zeta potential of liRNA with PEI carriers at N/P 5 and 10. (B) Size distribution and zeta potential of siRNA with PEI carriers at N/P 5 and 10. Error bars are standard deviations (±SD) of 3 measurements performed on the same sample. Average polydispersity index (PDI) values obtained during size measurements is provided in the figure (values above the bar diagram). Gray bars: hydrodynamic size (nm) of the siRNA/liRNA-PEI complex at N/P 5. Open bars: size of the siRNA/liRNA-PEI complex at N/P 10. Values on secondary Y axis: (-○-) zeta potential (ZP) values of the siRNA/liRNA-PEI complex at N/P 10 and (-●-) ZP values of the siRNAliRNA-PEI complex at N/P 5.

study, are excellent candidates for nucleic acid delivery as they perfectly combine the favorable toxicity profile of low molecular weight PEI with the higher transfection efficiency of high molecular weight versions. Molecular weight estimation using gel permeation chromatography (GPC) showed a 2.98 fold increase in the MW of bPEI with disulfide addition and nearly a 2.2-fold increase with imidazole linking. The TNBS assay showed reduction in primary amino groups in bPEI (nearly 22% and 16% reduction for bPEI-SS and bPEI-IMI, respectively, in comparison with that of unmodified bPEI), indicating the formation of cross-linked bPEI networks. liRNA Forms a Nanocomplex with Cationic Polymers. The hydrodynamic diameter of siRNA/liRNA-PEI complexes was determined for N/P ratios of 5 and 10 through laser light scattering (Figure 1). The average size of the liRNA complexes was evaluated with different PEI derivatives at N/P values of 5 and 10. Compared to the siRNA complex, liRNA formed a stable nanocomplex with cationic polymers. The Z average of the liRNA-LPEI complexes was 250 ± 20 nm and 210 ± 15 nm at N/P = 5 and 10, respectively. The bPEI based liRNA complex had a larger size (Z average of 310 ± 10 nm) compared to that of the LPEI complex at N/P 5, but its size was drastically reduced at an N/P of 10 (Z average of 150 ± 15 nm). A similar trend was obtained with modified bPEI, as the Z average for both bPEI-SS and bPEI-IMI complexes were in the range of 160−180 nm at N/P 10. In the case of the siRNA-PEI complex, the average size at an N/P ratio of 5 was somewhat higher than that of the liRNA-PEI complex. The siRNA-LPEI complex was quite unstable at the lower N/P ratio, and the particle size was in the range of 550 nm, whereas siRNA-bPEI showed a size above 1 μm. At N/P 10, the siRNA-PEI complexes were condensed and showed reduced size distribution with both LPEI and bPEI. At N/P ratios of 5 and 10, the zeta potential values for both siRNA/ liRNA-PEI complexes were positive (Figure 1). The siRNA/ liRNA complex with both LPEI and bPEI had zeta potential in the range of 25 ± 2 mV at N/P 5, and the values were in the range of 30 ± 2 mV at N/P 10.

Polyplexes at three different siRNA/liRNA concentrations (10 nM, 30 nM, and 50 nM) were complexed with bPEI-SS (N/P = 5) in 150 mM NaCl. Mutant sequences of siRNA and liRNA at the same concentrations were used as the control. Immediately before transfection, the medium was changed to a serumcontaining culture medium, and 100 μL of the prepared transfection complexes was added to the cells. The cells were grown for 12 h, and cell growth inhibition was evaluated at a specified time after the transfection, using trypan blue viable cell counting. Uptake of the Transferrin Modified bPEI-SS-liRNA Complex on b.End3 Cell Monolayers. Tf was first thiolated by reaction with 2-iminothiolane in phosphate buffer at pH 8.0 supplemented with 2 mM EDTA, as described previously.25 The product was then applied to a Sephadex column (PD-10, GE Healthcare) and eluted with a 0.01 M PBS buffer. The protein fractions were collected, and thiol groups were quantified by Ellman’s test.26 Next, the bPEI-SS (2 mg/mL) in PBS solution (supplemented with 5 mM EDTA) was activated with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) (Sigma) for 2 h, and the unreacted SPDP was then removed by using a PD-10 column. Activated bPEI-SS was then mixed with the thiol modified Tf at an optimized weight ratio and allowed to react for 12 h. bEnd.3 cells were seeded onto a confocal dish plate at a density of 8 × 104 cells/dish and cultured for 24−48 h to obtain cell monolayers. The cells were treated with 100 nM liRNA complexed with either bPEI-SS or Tf-bPEI-SS at N/P = 5, and uptake experiments were preformed as mentioned previously. Similarly, quantification of cellular uptake on bEnd.3 cell monolayers in 6-well plates was performed using the NC-3000 method at 100 nM liRNA (N/P 5 and 10) using either bPEI-SS or Tf-bPEI-SS.

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RESULTS Synthesis and Characterization of Cross-Linked bPEI for liRNA Delivery. Cross-linked low molecular weight bPEI [bPEI (1.8)], cross-linked via EDC and NHS chemistry in this 875



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Figure 2. AFM images showing the surface morphology of the liRNA-PEI complex. (A) liRNA-LPEI complex. (B) liRNA-bPEI-SS complex. (C) siRNA-LPEI complex.

Figure 3. Gel retardation assay of the siRNA/liRNA complex with PEI at different N/P ratios. (A) siRNA-PEI complex or (B) liRNA-PEI complex at different N/P ratios (line 1, N/P 1.25; line 2, N/P 2.5; line 3, N/P 5; line 4, N/P 10; and line 5, N/P 15). RNA bands without PEI complexation (control) or that were dissociated from the polyplexes were separated by agarose electrophoresis and visualized by ethidium bromide staining.

The morphological and physical characteristics of liRNA complexes prepared with either LPEI or bPEI-SS were analyzed by atomic force microscopy (AFM), using the siRNA-LPEI complex as the control, at N/P 10. As shown in Figure 2, the liRNA molecules were effectively condensed by LPEI or bPEISS and formed well-dispersed, homogeneous nanoparticles with the particle size of approximately 100 nm. The liRNA-LPEI complex was spindle-shaped, whereas the liRNA-bPEI-SS was spherical in nature with smaller dimensions. However, the naked siRNA-LPEI complex had a higher particle size with irregular surface morphology. The difference in size and morphology between these two polyelectrolyte complexes can be attributed to the clear differences in their charge density and chain length. In contrast to plasmid DNA or long dsRNA, the double stranded siRNA with 21 bp has a smaller number of charges and forms a shorter, stiffer structure. Thus, when siRNAs are complexed with cationic condensing polymers, loose and unstable complexes are formed.27 liRNA exhibits an increased chain length as well as more negative charges than monomeric siRNA and therefore readily forms more compact complex structures when condensed with these cationic agents. liRNA-PEI Complex Is More Stable than the Naked siRNA-PEI Complex. siRNA/liRNA complex formation with the PEI polymer was examined by agarose gel electrophoresis at different N/P ratios. As shown in Figure 3, siRNA forms a weak complex with LPEI at the lower N/P ratios. Compared with

LPEI, both cross-linked bPEIs formed a more stable complex with naked siRNA at the same N/P ratio. However, liRNA formed a more stable complex than siRNA with all PEIs tested, especially with bPEI (1.8) and cross-linked bPEI. The migrations of liRNA-PEI on the gel were completely retarded at an N/P ratio of 5 and above, indicating that the cationic polymers form stable complexes with the RNA and thus retard their electrophoretic mobility. Cross-linked bPEI formed a very stable complex with liRNA, and mobility retardation was observed even at N/P 2.5. However, unmodified polymers [bPEI (25), bPEI (1.8) and LPEI] displayed slightly lower liRNA complexation ability compared to that of the crosslinked bPEI in this study. Next, the stability of these complexes was examined by a heparin decomplexation assay at different N/P ratios (Figure 4). Similar to the previous results, the siRNA complexation with cationic PEI was unstable at N/P 5 and 10. However, the liRNA-PEI complex was not readily dissociated with the addition of increasing concentration of anionic heparin. This result yet again confirms that liRNA forms more compact nanostructures with cationic polymers than with siRNA. Cross-Linking Reduces the Buffering Capacity of Branched PEI. An acid−base titration was used to examine whether the cross-linking of bPEI affected the buffering efficacy of polymers. Figure S3 (Supporting Information) shows the buffering capacity of different PEIs in the pH range of 10−4. 876



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Figure 4. Anionic (heparin) decomplexation assay of the siRNA/liRNA-PEI complex. The stability of the siRNA/liRNA-PEI complex was challenged with the addition of varying concentrations of heparin (0, 1, 2, 3, and 5 correspond to heparin/RNA weight ratios) at three different N/P ratios (N/P ratios denoted as 5, 10, and 15 are shown in the figure). (A) siRNA-LPEI complex, (B) siRNA-bPEI (1.8) complex, (C) siRNA-bPEI-SS complex, (D) siRNA-bPEI-IMI complex, (E) liRNA-LPEI complex, (F) liRNA-bPEI (1.8) complex, (G) liRNA-bPEI-SS complex, and (H) liRNA bPEI-IMI complex.

We found that the amount of acid required to reduce the pH from 10 to 4 decreased with the modification, as bPEI (25) and bPEI (1.8) showed the highest buffering capacity. Imidazolemodified bPEI has a slightly enhanced buffering action compared to that of its disulfide modified version, indicating the presence of imidazole groups in its polymer backbone. Cross-Linked bPEI Exerts Lower Toxicity than Standard Linear PEI. The cytotoxicity of modified/unmodified bPEI and LPEI was evaluated using the MTT assay on HeLa cells. As shown in Figure S4 (Supporting Information), cross-linked bPEI exerted lesser toxicity on HeLa cells than LPEI or bPEI (25) in this study. However, both cross-linked polymers were slightly more toxic than bPEI (1.8). liRNA-PEI Complex Demonstrate Higher Cellular Uptake Efficacy. To further assess the capacity of the various PEI derivatives for siRNA/liRNA complexation and subsequent cellular internalization, Cy3 labeled RNA was used in polyplex formation, and uptake was quantified by a nucleocounter based technique. The number of cells that showed red fluorescence due to the internalization of polyplexes was quantified for a gated cell population, and the ability of different PEIs to

enhance the uptake of siRNA/liRNA was then compared (Figure 5). Within the same N/P ratio and RNA concentration, the cellular uptake of the liRNA-polymer complex was significantly higher than the native siRNA-polymer complex. Particularly, the uptake of cross-linked bPEI-liRNA complexes was much higher than that of other polyplexes tested in this investigation. However, both LPEI and its commercial version (jetPEI) showed similar cellular uptake efficacy in these experiments (data not shown). To further confirm the data obtained with a nucleocounter, a fluorescent microscopic investigation was performed (Figure 6). As quantified by nucleocounter experiments, both bPEI-SS and bPEI-IMI showed improved cellular uptake in U87MG cells when complexed with liRNA than with siRNA. However, the uptake of liRNA was rather low when complexed with either LPEI or bPEI (1.8) under similar conditions. However, they still showed better internalization than the corresponding complexes made with siRNA. Time-course uptake experiments also suggest that the disulfide cross-linked bPEI was more effective in delivering the liRNA candidate in the U87MG cells (Figure S5, Supporting Information). For all PEIs tested, the siRNA-PEI 877



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Figure 5. NC-3000 based quantification of internalized siRNA/liRNA-PEI complex. U87MG cells were transfected with either Cy3 liRNA-PEI or Cy3 siRNA-PEI complex at 100 nM concentration (N/P 5). The fluorescence intensity of each sample was analyzed by neucleocounter method based on intracellular Cy3 signals. (A) Uptake quantification of the liRNA-PEI complex. (B) Uptake quantification of the siRNA-PEI complex. (C) Fluorescent intensity quantification data.

complex displayed quite weak signals, suggesting the poor ability of PEI in siRNA delivery. liRNA-PEI Complexes Induce Strong RNAi Activity in U87MG Cells. For gene knock-down experiments, we transfected siRNA/liRNA targeting Survivin into U87MG cells using different PEI polymers. Survivin mRNA levels were normalized with the house keeping gene glyceraldehyde 3phosphate dehydrogenase (GAPDH) in our studies. The siRNA (siSurvivin) used in this study induced no appreciable gene silencing activity with PEI mediated transfection. However, the same siRNA showed strong RNAi activity when transfected using lipid based reagent Lipofectamine 2000 (Figure S7, Supporting Information). This compromised gene silencing is probably due to the weak complex formation between siRNA and the polymer transfection reagents. To test whether the gene silencing activity of liSurvivin was sequence specific, siSurvivin and liSurvivin with mutation in the seed sequence (Figure S1, Supporting Information) were used as controls. When transfected with jetPEI at N/P 5 (30 nM), neither siSurvivin nor the mutated siSurvivin/liSurvivin (Figure S8, Supporting Information) showed target gene silencing activity. Only liSurvivin could induce potent gene silencing

activity in these experiments. At higher N/P values of 10, siSurvivin triggered mild silencing activity by reducing the Survivin mRNA level to 60%, whereas liSurvivin induced more than 95% target knock-down. The control samples (mutated siRNA/liRNA sequences) were still ineffective without any significant knock-down activity. This study confirms that the liRNA activity is sequence specific and seed sequence dependent, like any conventional siRNA. From these studies, we confirm that liRNA is a potent and robust gene silencing tool and that its activity can be optimized by the use of an appropriate cationic carrier system. Next, we studied the silencing activity of siRNA/liRNA with different polymeric carriers (Figure 7). At 30 nM concentration, the liRNA-PEI complex induced potent gene knockdown efficacy; more than 80% knock-down in Survivin mRNA level was attained by using either jetPEI or modified bPEI samples with no reduction in GAPDH level (Figure S8, Supporting Information). LPEI was not quite as effective as jetPEI in these experiments, even though their cellular uptake efficacy was quite similar. siRNAs were totally ineffective in these experiments and were not considered for the experiments with lower concentrations. Next, we attempted to study the 878



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Figure 6. Microscopic studies on the cellular uptake of the siRNA/liRNA-PEI complex. Fluorescent microscopic images demonstrating cellular internalization of the Cy3 labeled siRNA/liRNA-PEI complex (100 nM, N/P 5) after 3 h of incubation in serum free conditions. Images were acquired at 20× magnification in fluorescence mode-Cy3 channel (left panel) and DIC mode (middle panel), further overlapped to visualize the internalized polyplexes (right panel). (A) Control (untreated cells), (B) siRNA-LPEI complex, (C) siRNA-bPEI (1.8) complex, (D) siRNA-bPEI-SS complex, (E) siRNA-bPEI-IMI complex, (F) liRNA-LPEI complex, (G) liRNA-bPEI (1.8) complex, (H) liRNA-bPEI-SS complex, and (I) liRNA bPEI-IMI complex. Time dependent uptake experiments were performed with liRNA-LPEI/bPEI-SS samples with 1 and 2 h of incubation (data provided in Supporting Information, Figure S5).

complexes used failed to induce any knock down effect. At 20 nM concentration, both disulfide or imidazole linked bPEI showed nearly 70% knock-down in their target gene expression, whereas jetPEI and unmodified bPEI showed about 50% knock-down efficacy. Finally, the immunostimulatory effect of liRNA was investigated by evaluating the expression levels of IFIT1, OAS2, TNF-α, IL-6, and IFN-β after treatment with 100 nM liRNA concentration at N/P 10 (Figure S11, Supporting Information). Similar to our previous investigations, siRNA failed to induce any appreciable levels of cytokine response and was not investigated in the experiments in this study.28 Overall, the liRNA-PEI complex induced low levels of IFIT1 and OAS2, and the inflammatory cytokines were largely undetected in these experiments. The positive control poly(I:C)-PEI complex induced slightly higher expression levels for IFIT1 and OAS2; however, the GAPDH level and amount of total mRNA were reduced after poly(I:C) treatment. We found some reduction in the GAPDH level for bPEI and bPEI-IMI samples when complexed with liRNA at N/P 10, whereas both jetPEI and bPEI-SS were found to be quite suitable. Nevertheless, these experiments validate the hypothesis that the multimerized RNA structures, such as liRNA, are less toxic than the conventional double stranded immunostimulatory RNA structures and can be safely used for specific target knock-down when combined with an appropriate polymer carrier. Western blot analysis of Survivin levels in transfected U87MG cells showed decreased protein levels for liSurvivin treated samples in agreement with the results of qRT-PCR experiments (Figure 9). At N/P 5, only liSurvivin complexed

Figure 7. Gene silencing activity of siSurvivin/liSurvivin in U87MG cells transfected using different PEI samples. Each siRNA (30 nM) or liRNA (30 nM) was transfected at N/P 5 using either jetPEI, LPEI, bPEI (1.8), bPEI-SS, or bPEI-IMI. Survivin and GAPDH expression levels were analyzed by qRT-PCR 12 h post-transfection, and the Survivin mRNA levels were normalized with the GAPDH mRNA level. (Individual Survivin and GAPDH mRNA levels of each samples are shown in the Supporting Information, Figure S9.) All data in the graph represent the mean ± SD of 3 independent experiments.

gene-knock down effect of the liRNA-PEI complex at 10 and 20 nM concentrations (Figure 8). At 10 nM concentration, the gene silencing activity was almost negligible for most of the liRNA-PEI complexes. The liRNA-bPEI-IMI complex showed nearly 35% reduction in Survivin level, whereas other 879



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Figure 8. Gene silencing activity of the liSurvivin-PEI complex at lower RNA concentration. liSurvivin was transfected at N/P 5 using either jetPEI, LPEI, bPEI (1.8), bPEI-SS, or bPEI-IMI at 10 and 20 nM concentration. Survivin and GAPDH expression levels were analyzed by qRT-PCR 12 h post-transfection, and the Survivin mRNA levels were normalized with the GAPDH mRNA level. (Individual Survivin and GAPDH mRNA levels of each sample are shown in the Supporting Information, Figure S10.) All data in the graph represent the mean ± SD of 3 independent experiments.

Figure 9. Western blot analysis of Survivin expression after siRNA/liRNA-PEI transfection. U87MG cells were transfected with either jetPEI or bPEI-SS at N/P 5 and 10 for 24 h. NT: nontreated samples. GAPDH serves as an internal loading and protein expression control.

concurred with our expectation, as the specific Survivin mRNA knock-down can reduce the cell proliferation and induce cell cycle arrest. Transferrin Improves the Uptake of the bPEI-SS-liRNA Complex in bEnd.3 Cells. As an immortalized mouse brain endothelial cell line, bEnd.3 cells exhibit endothelial and blood−brain barrier (BBB) characteristics, and are typically chosen as a simple BBB model to study the brain delivery properties of nanoparticles in vitro.29 In this study, fluorescence imaging revealed that the bEnd.3 cellular uptake of liRNA complexed Tf-PEI-SS was higher than that of the liRNA-bPEISS complex (Figure 11). Further quantitative uptake studies using the nucleocounter method also proved that the uptake of the Tf modified nanocomplex was higher at N/P values of 5 and 10 for a 2 h incubation period (Figure S12, Supporting Information). Several studies have demonstrated that Tf increases the brain delivery of nanocarriers via receptor mediated transcytosis; however, the exact cellular internalization pathway of Tf modified nanocarriers remains unclear. Nevertheless, our studies clearly suggest that the uptake of bPEI condensed liRNA across BBB can be improved via Tf modification.

with jetPEI or modified bPEI (bPEI-SS/bPEI-IMI) showed reduction in the Survivin protein level. As expected, mutated siRNA/liRNAs sequences failed to impart any silencing effect on the Survivin protein expression levels, further confirming the specificity of the liRNA mediated gene silencing. liRNA-PEI Complex Induces Specific Inhibition in Cancer Cell Growth. In the next step, a cell viability assay was performed at transfection conditions by directly counting the cells. Here, the polyplexes at 3 different siRNA/liRNA concentrations (10 nM, 30 nM, and 50 nM) were evaluated with bPEI-SS as the transfection agent at N/P 5. As shown in Figure 10, the siSurvivin and mutant sequence used in this study failed to impose any significant change in the cell growth pattern, with respect to the untreated cells, even up to 50 nM concentration. This can be correlated with the poor RNAi effect induced by siSurvivin with PEI transfection. Likewise, at 10 nM concentration, liRNA-PEI complexes exerted marginal growth inhibitory effect. However, at 30 nM and 50 nM concentrations, a dose-dependent inhibition was observed for liRNAPEI treated cells. Nearly 50% and 70% reduction was registered at 30 and 50 nM, respectively, with liSurvivin-PEI treatment. Importantly, the liSurvivin mutant sequence failed to impart any appreciable reduction in cell count, and maximum 20% reduction was registered at 50 nM concentration. This 880



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efficacy when used with the cationic polymeric delivery systems such as PEI and poly L-lysine, etc. Our efforts have mainly focused on expanding the structural diversity repertoire of RNAi triggers, aimed at reducing the nonspecific effects triggered by classical siRNAs,31 the modulation of innate immune stimulation,28 enhanced cellular delivery, and, in some cases, triggering simultaneous multitarget gene silencing.32,33 Earlier, it was assumed that specific gene silencing cannot be achieved with dsRNAs longer than 30 bp.34 However, in contrast to popular belief, larger dsRNA structures have also been shown to implement target gene silencing.35,36 Validating these observations, our group has also demonstrated that dsRNAs as long as 38 bp can trigger specific gene silencing via the RNAi pathway.28 Even the unconventional dsRNA structure used in the present experiment triggered a potent RNAi mediated gene silencing activity in a sequence specific manner.16In this study, it was observed that the long dsRNA (liRNA) structure was cleaved by Dicer treatment to the smaller 20−23 nucleotide units (Figure S2B, Supporting Information). Thus, it can be assumed that the Dicer intervention helps in the breakdown of this dsRNA structure and that this may further facilitate the formation of the RNA induced silencing complex (RISC). Classical long dsRNAs are known to implement strong and uncontrolled induction of innate immune response. liRNAs, however, operate mainly via the RNAi effect and can induce low to mid levels of inflammatory response. Combining the structural advantages and potent gene silencing efficacy, this moderate level of immunostimulation may translate into a more potent anticancer effect in vivo. We believe that, along with the specific tumor-directed delivery system, the liRNA structure has immense potential to be developed as an anticancer therapeutic strategy. In our present investigation, we present a model of tumor directed delivery approach for liRNA based anticancer therapy. The rational design of polymeric systems seems to be the key to the successful implementation of any RNAi therapeutic approach. The crucial factor that determines the efficacy of siRNA mediated transfection is the vector unpacking and endosomal escaping of the genetic material.37 PEI polymers are known to trigger the endosomal release for RNAi molecules by a proton-sponge mechanism to facilitate RNAi mediated target gene silencing. Structural constraints arising from polymer backbone modification can result in lowering of the endosomal destruction property. Reinforcement/enhancement of endosomal rupturing activity of cationic polymers was achieved by specific modification with membrane lytic agents38 or by the attachment of imidazole/histidine units.39 In this study, we also attempted to enhance the endosomal escaping property of modified bPEI with imidazole linkers. However, the effect of imidazole linking was not quite appreciable in the acid−base titration experiments. Modification reduced the overall buffering effect of bPEI, and marginal enhancement could be observed in the pH range of 5−7 by imidazole incorporation. It remains unclear whether this could be actually translated into an endosomal disturbing property for RNAi molecules in vivo. Nevertheless, imidazole linked bPEI was used as a noncleavable version of cross-linked bPEI in our experiments. Long RNA structures are known to form stable nanocomplex structures with cationic polymeric reagents.6,27,40,41 liRNA used in our investigation formed a stable nanocomplex structure with PEI polymers, especially with cross-linked bPEI. Both AFM and DLS studies validated the hypothesis that the liRNA-PEI

Figure 10. Effect of Survivin siRNA/liRNA treatment on cell proliferation. Cell viability after treatment with three different siRNA/liRNA concentrations (10, 30, and 50 nM) at N/P 5 using bPEI-SS. Cell counting was performed after trypan blue treatment, and cell viability was assessed 48 h post-transfection. Mutant siRNA/liRNA complexed with bPEI-SS was used as the control. (-○-, siSurvivin-PEI; -●-, siSurvivin-Mut-PEI; -□-, liSurvivin-PEI; -■-, liSurvivin-Mut-PEI). All data in the graph represent the mean ± SD of 3 independent experiments.

Figure 11. Uptake of Tf liRNA-PEI complex in b.End3 cell monolayers. Fluorescent microscopic images demonstrating cellular internalization of the Cy3 labeled liRNA-PEI complex (100 nM, N/P 5) in b.End3 cell monlolayers after 2 h of incubation in serum free conditions. Images were acquired at 20× magnification in fluorescence mode (left panel) and DIC mode (middle panel), and are further overlapped to visualize the internalized polyplexes (right panel). (A) Untreated cells, (B) liRNA-bPEI-SS, and (C) liRNA-Tf-bPEI-SS.

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DISCUSSION The main limiting factor for polymer mediated siRNA delivery has been the structural incompetency of small RNA units to generate stable polyplexes with cationic polymers.5,27,30 As a result, the siRNA-polymer complex displays poor transfection 881



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sequences had minimal impact on cell proliferation. Survivin, a key member in the inhibitor of apoptosis family protein, is known to play a lead role in tumor survival and is a key regulator of mitosis.42,43 Survivin is now regarded as an attractive target for anticancer gene therapy because of its powerful apoptotic inhibition and cell division regulation.44 Here, we demonstrated that liRNA based Survivin knock-down on U87MG cells resulted in significantly reduced cell growth. As the liRNA molecules operate via both RNAi and the immunostimulatory effect, the contribution of each individual factor on cell survival is still unclear. As the liSurvivin mutant failed to impose a stronger inhibitory effect on cell growth, it seems the RNAi effect induced by specific Survivin knock-down is the key factor in cell growth inhibition. This study correlated very well with the overall gene silencing efficacy induced by the liRNA-PEI complex over the siRNA-PEI complex. Another major advantage of using bPEI based polymers is their ease of chemical modification to make them a target oriented delivery device. Branched PEI molecules, in contrast to linear PEI, contain free primary amino groups, making them quite susceptible for chemical modifications. As a proof of concept, Tf was used as a ligand to promote the uptake of the nanocomplex across model BBB. Membrane Tf receptor mediated endocytosis has been identified as an efficient cellular uptake pathway for drug delivery. The Tf receptor is overexpressed in many tumors, and even in the blood−brain barrier, a Tf-conjugated nanocomplex may enhance the delivery of liRNA to the brain.45,46 The mechanism of the enhanced efficacy of the nanocomplex with conjugated Tf appears to be due to a higher cellular drug uptake as evident by the microscopic and nucleocounter methods. Thus, a strong RNAi mediator capable of triggering both RNA interference and controlled immunostimulation was optimized for anticancer therapeutics. When combined with the cationic polymer PEI, liRNA provided robust RNAi mediated gene knock-down activity in the glioblastoma cell line without inducing any deleterious effect. Moreover, via the targeted delivery approach, these potent RNAi agents can be transported across the BBB to facilitate gene knock-down in glioblastoma cells. The new targeted and bioreducible carrier complexed with liRNA molecules may be a promising efficient RNAi delivery system for potential cancer gene therapy.

complex had a much lower size than that of the siRNA-PEI complex. Moreover, their complex formation ability and their stability were also better than those of the native siRNA-PEI complex. Disassembly of the RNA/polymer complex is the key rate-limiting step in siRNA delivery and is closely related to overall gene silencing efficacy. The premature disassembly of the polyplexes in the presence of anionic polymers such as proteoglycans and serum proteins can strongly influence the in vivo gene silencing activity.37 The liRNA-PEI complex, in this regard, seems to have an advantage over the siRNA structures and thus may provide an enhanced efficacy under in vivo conditions. However, the major issue is whether the enhanced stability and the superior physical characteristics of the liRNA-PEI complex could actually be translated into a gene knock-down effect. Our results based on cellular uptake and gene knockdown experiments certainly validate this. Both qualitative and quantitative cellular uptake experiments using Cy3 labeled siRNA/liRNA clearly demonstrated that the uptake was significantly higher for the liRNA-PEI complex in U87MG cells. It is quite well accepted that the stable RNA-polymer nanocomplexes are more readily internalized into the cells. Thus, the enhanced intracellular uptake of liRNA complexes could be attributed to their compact size and stability, compared to that of the conventional siRNA-PEI complexes. Further, with the qRT-PCR experiments, we observed that liSurvivin induced a strong sequence specific RNAi mediated gene knock down effect at a concentration of 30 nM (N/P-5). The liRNA mediated knock-down of the target protein (Survivin) was validated both at the mRNA and protein levels, in sequence specific manner. The effect was visible even at lower concentrations (20 nM) without any change in the GAPDH level, whereas the siRNA-PEI complex failed to induce any gene silencing effect at these conditions. liRNA was most effective when complexed with cross-linked bPEI and linear PEI (jetPEI). However, cross-linked bPEI with a disulfide linker seems to be the appropriate carrier for liRNA delivery. Imidazole linked bPEI was as equally effective as disulfide linked bPEI; however, it triggered some nonspecific effects when applied at higher N/P ratios and also induced variations at the house-keeping gene levels. Cross-linked bPEI had a slightly improved efficacy compared to the jetPEI commercial transfection reagent at lower concentrations, whereas 25 kDa LPEI and low molecular weight bPEI were not very effective in our experiments. The added advantage of cross-linked bPEI is its lower toxicity and the possibility of further chemical modifications to introduce active targeting motifs. Most of the studies reported using multi-RNA-polymer complexes either were carried out at higher RNA concentrations or used a higher polymer to RNA ratio to facilitate the effective condensation of the polyplexes.5,30,41 This, we believe could potentially induce a strong cytotoxic effect and even lead to nonspecific effects. liRNA at a concentration of less than 30 nM and N/P 5 was quite effective in reducing target gene expression, with no reduction in the house-keeping gene levels. This efficient gene silencing activity can be regarded as a result of the effective synergism between the multimerized long dsRNA structure and the cationic polymers. Cell growth inhibition studies clearly demonstrated that liSurvivin can mediate sequence specific growth inhibition when combined with cationic PEI. At 30 nM concentration, the liSurvivin-PEI complex induced cell growth inhibition, whereas siRNA (siSurvivin) or the seed mutated siSurvivin/liSurvivin

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ASSOCIATED CONTENT

S Supporting Information *

Schematic illustration of siRNA/liRNA structure; annealing test for siSurvivin/siSurvivin-mut and liSurvivin/liSurvivin-mut on 10% native PAGE gel; in vitro Dicer cleavage assay; acid/base titration experiments; cytotoxic study; time dependent liRNA uptake experiments; primer sequence used in this study; gene silencing activity of siSurvivin/liSurvivin in U87MG cells using lipid based transfection; specific gene silencing activity of siRNA/liRNA targeting Survivin mRNAs in U87MG cells; Individual GAPDH and Survivin mRNA level after siRNA/ liRNA transfection using different PEI carriers at N/P 5 (30nM); individual GAPDH and Survivin mRNA level after liRNA transfection using different polymeric carriers at 10 and 20 nM concentration (N/P 5); immunostimulatory effect of liRNA-PEI transfection; and Nucleocounter-3000 based quantification of the internalized liRNA-PEI complex in bEnd.3 cell monolayers. This material is available free of charge via the Internet at http://pubs.acs.org. 882



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

S.S. and T.Y.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a Global Research Laboratory grant (2008-00582) from the Korean Ministry of Education, Science, and Technology to D.-k.L.

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