Acid-Degradable Cationic Poly(ketal amidoamine) for Enhanced RNA

Dec 16, 2012 - Efficient delivery of small interfering RNA (siRNA) is one of major challenges in the successful applications of siRNA in clinic. In th...
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Acid-Degradable Cationic Poly(ketal amidoamine) for Enhanced RNA Interference In Vitro and In Vivo Hyungsuk Lim,† Joungyoun Noh,‡ Yerang Kim,† Hyungmin Kim,† Jihye Kim,† Gilson Khang,‡ and Dongwon Lee*,†,‡ †

Department of BIN Fusion Technology and ‡Polymer Fusion Research Center, Department of Polymer·Nano Science and Technology, Chonbuk National University, Jeonju, 561-756, Korea ABSTRACT: Efficient delivery of small interfering RNA (siRNA) is one of major challenges in the successful applications of siRNA in clinic. In the present study, we report a new acid-degradable poly(ketal amidoamine) (PKAA) as a siRNA carrier, which has high delivery efficiency and low cytotoxicity. PKAA was designed to have acid-cleavable ketal linkages in the backbone of cationic biodegradable poly(amidoamine). PKAA efficiently self-assembled with siRNA to form nanocomplexes with a diameter of ∼200 nm and slightly positive charges, which are stable under physiological conditions, but rapidly release siRNA at acidic pH. PKAA exhibited sufficient buffering capability and endosomolytic activity due mainly to the presence of secondary amine groups in its backbone and rapid degradation in acidic endosomes, leading to the enhanced release of siRNA to cytoplasm. Cell culture studies demonstrated that PKAA is capable of delivering anti-TNF (tumor necrosis factor)-α siRNA to lipopolysaccharide (LPS)-stimulated macrophages and significantly inhibits the expression of TNFα. A mouse model of acetaminophen (APAP)-induced acute liver failure was used to evaluate in vivo siRNA delivery efficacy of PKAA. PKAA/anti-TNF-α siRNA nanocomplexes significantly reduced the ALT (alanine transaminase) and the hepatic cellular damages in APAP-intoxicated mice. We anticipate that acid-degradable PKAA has great potential as siRNA carriers based on its excellent biocompatibility, pH sensitivity, potential endosomolytic activity, and high delivery efficiency. cells.15,16 Besides their essential characteristics, cationic polymers have abilities to facilitate endosomal escape of siRNA via “proton sponge effects”.17−19 Upon cellular uptake, siRNA nanocomplexes are confined in endosomes which have acidic pH (5.5−6.5). Endosomes are then fused with lysosomes, which are intracellular degrading machinery with pH 4.5−5.5, leading to rapid destruction of siRNA and no release into cytosol.20 Therefore, cationic polymers with endosomolytic activity have tremendous advantages as siRNA carriers. However, it should be noted that dissociation of nanocomplexes for the release of intact siRNA into the cytosol is required to achieve the maximum gene-silencing activity of the siRNA.2,16 Cationic polymers such as polyethyleneimine (PEI) and poly(L-lysine) have demonstrated excellent transfection efficiency and endosomolytic activity, but their applications as siRNA carriers are limited by their cytotoxicity, nondegradability, and slow release of siRNA to cytoplasm.1,21,22 There is, therefore, great need for the development of cationic biodegradable polymers, which are able to meet the fundamental requirements for siRNA delivery, such as biocompatibility, high delivery efficacy, fast release of siRNA to cytoplasm, and stimulus-responsiveness.23,24

1. INTRODUCTION RNA interference is a process whereby double stranded RNA mediates the endogenous cleavage of a target mRNA by incorporating into the RNA-induced silencing complex (RISC).1,2 The small double-stranded RNA is 20−25 nucleotides in length and is celled siRNA. siRNA-mediated gene silencing mechanism in a sequence specific manner has shown great potential as an alternative therapeutic strategy for treating various gene-related diseases whose conventional treatments are limited.3 However, the successful applications of siRNA in mammalian cells have been limited by its inherent instability and poor permeability across biological membranes.2 Therefore, therapies integrating RNA interference rely on the efficient and safe delivery of siRNA to target cells and enormous efforts have been directed toward the development of effective siRNA carriers.4−6 So far, the delivery systems for siRNA include cationic polymers, biodegradable polymer particles, lipids, lipid-like materials, iron oxide nanoparticles, and gold nanoparticles.3,7−11 Efficient siRNA delivery has also been achieved through chemical modification and conjugation to small molecules or polymers.12−14 To date, cationic polymers have been a leading class of materials for siRNA delivery because of their ability to form stable nanocomplexes with siRNA by electrostatic interactions. Cationic polymers are also capable of protecting siRNA from enzymatic degradation and facilitating entry into target © 2012

Received: October 30, 2012 Revised: December 12, 2012 Published: December 16, 2012 240

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2.2. Synthesis of 2,2′-(Propane-2,2-diylbis(oxy))diethanamine. N-(2-Hydroxyethyl) phthalimide (5.0 g, 26 mmol) was dissolved in benzene (80 mL) to which 2,2′-dimethoxypropane (1.4 g, 13 mmol) and pTSA (55 mg, 0.32 mmol) were added. The reaction was allowed at 90 °C for 12 h using a short path distillation head. The reaction was terminated by the addition of triethylamine (1.0 mL). The product, 2,2′-(propane-2,2-diylbis(oxy))bis(ethane-2,1diyl) bis(isoindoline-1,3-dione), was purified by column chromatography eluting with 1:1 hexanes/ethyl acetate and obtained as white solid after vacuum drying (yield: 30%). Its chemical structure was identified with a 400 MHz 1H NMR spectrometer with CDCl3; δ 7.7− 7.9 (−ArH), 3.6−3.8 (−NCH2CH2O), 1.4 (−CCH3CH3−). 2,2′-(Propane-2,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(isoindoline1,3-dione) was added to 15 mL of 6 M NaOH and the mixture was refluxed overnight at 120 °C. The product, 2,2′-(propane-2,2diylbis(oxy))diethanamine was extracted using dichloromethane and the organic layer was evaporated to obtain amber-colored oil (yield 30%). The chemical structure of 2,2′-(propane-2,2-diylbis(oxy))diethanamine was identified with a 400 MHz 1H NMR spectrometer with CDCl3. 2.3. Synthesis of PKAA. PKAA was synthesized from a Michael addition polymerization between 2,2′-(propane-2,2-diylbis(oxy))diethanamine and N,N′-methylenebisacrylamide. In brief, 2,2′(propane-2,2-diylbis(oxy))diethanamine (2.0 g, 1.23 mmol) and N,N′-methylenebisacrylamide (1.9 g, 1.23 mmol) were dissolved in 7 mL of dry methyl alcohol. Polymerization reaction was performed at 70 °C for 48 h under mechanical stirring. The resulting product was completely dried using a rotary evaporator, followed by vacuum drying overnight. The chemical structure of polymers was identified with a 400 MHz 1H NMR spectrometer with D2O. 2.4. Acid−Base Titration. The ability of PKAA to protonate was determined by acid−base titration. Briefly, 100 mg of PKAA or PEI was dissolved in 10 mL of 150 mM NaCl solution. The sample solution was first titrated with a 0.5 N NaOH solution to a pH of 9. HCl (0.5 N) was gradually added to the solution and the pH value was monitored. 2.5. Preparation and Characterization of PKAA/siRNA Nanocomplexes. PKAA/siRNA nanocomplexes were prepared by simple mixing of PKAA and siRNA solutions in phosphate buffer solution (pH 7.4). The size and size distribution of PKAA/siRNA nanocomplexes were characterized by dynamic light scattering and transmission electron microscope. The zeta potential of PKAA/siRNA nanocomplexes was measured using an ELS-6000 (Otsuka Inc., Japan) at 25 °C. The integrity of siRNA was examined using the agarose gel retardation assay. The ability of nanocomplexes to release siRNA after a challenge with the competing polyanionic heparin was also determined as a measure of nanocomplex stability. Nanocomplexes were prepared with various composition ratios to ensure complete binding of siRNA by the polymers, and then incubated with 2 IU of heparin at 37 °C for 3 h. The solution was run on agarose gel to determine the integrity of siRNA. 2.6. Cytotoxicity Assay. The cytotoxicity of PKAA was evaluated using a 3-(4,5-dimethylathiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Both HEK293 cells and NIH3T3 cells were seeded at a density of 5.0 × 104 cells/well in a 24-well plate and incubated for 24 h to reach ∼90% confluency. Cells were treated with various amounts of PKAA or PEI 25k for 24 h. Each well was given 100 μL of MTT solution and was incubated for 4 h. Dimethyl sulfoxide (1000 μL) was added to cells to dissolve the resulting formazan crystal. After 10 min of incubation, the absorbance at 570 nm was measured using a microplate reader (Synergy MX, BioTek Instruments Inc. Winoosk, VT). The cell viability was obtained by comparing the absorbance of PKAA-treated cells to that of untreated cells. 2.7. Confocal Laser Scanning Microscopy. HEK293 cells were seeded on a 35 mm coverglass bottom dish (MatTek Corp, Ashland, MA) and incubated overnight in 1 mL of DMEM (Dulbecco’s Modified Eagle Medium, Invitrogen, Carlsbad, CA) containing 10% FBS (fetal bovine serum). After the medium was replaced with fresh medium, cells were treated with calcein (Sigma-Aldrich, St. Louis,

A rational design approach for biodegradable polymers as effective siRNA carriers involves exploitation of stimuli in cell compartments or diseased sites. Acidic pH in endosomes, lysosomes, and the tumor site has been widely exploited as a stimulus to trigger hydrolytic degradation and conformational change of biopolymers.19 Acid-degradable polymers have acidlabile linkages in their backbone, including hydrazone, acetal, and ketal linkages.25 Among them, ketal has advantages as an acid-cleavable linkage due to its high acid sensitivity and biocompatible and neutral degradation products such as alcohol and acetone.11,26,27 In this work, we developed acid-cleavable ketal containing cationic poly(amidoamine) (PKAA) as siRNA carriers. Poly(amidoamine) (PAA) was chosen as a platform of biodegradable polymer because of its water solubility, cationic nature, excellent biocompatibility, ease of synthesis, and synthetic flexibility.15 We reasoned that acid-degradable cationic PKAA forms nanocomplexes with siRNA and undergoes rapid acid-catalyzed hydrolysis in the acidic environment of endosomal compartments, leading to the rapid endosomal escape and intact siRNA release to cytosol, which are critical to achieve maximal gene silencing effects of siRNA (Scheme 1). Scheme 1. Schematic Diagram Illustrating the Enhanced RNA Interference by a New Family of Cationic AcidDegradable Polymer, PKAA

PKAA was synthesized by Michael type-addition polymerization of bis(acrylamide) and ketal containing diamine. We investigated the physicochemical properties, biocompatibility and siRNA delivery efficacy of acid-degradable cationic PKAA in LPS-stimulated cells. The delivery of siRNA to macrophages in vivo was also evaluated using a mouse model of APAP(acetaminophen)-induced acute liver failure. Based on the results of the experiments, we anticipate that aciddegradable cationic PKAA has great potential as siRNA delivery systems.

2. MATERIAL AND METHODS 2.1. Materials. N-(2-Hydroxyethyl) phthalimide and 2,2′-dimetoxypropane, N,N′-methylenebisacrylamide, p-toluenesulfonic acid (pTSA), and PEI 25k were purchased from Sigma-Aldrich (St. Louis, MO). pTSA was recrystallized from methanol and chloroform. Benzene was purified by distillation over calcium hydride. MTT reagents for cell viability were obtained from AMRESCO (Solon, OH). Double-stranded tumor necrosis factor-α (TNF-α) siRNA (mouse; sense strand: 5′-GAC AAC CAA CUA GUG GUG CUU-3′) was synthesized by Bioneer (Daejeon, Korea). 241

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Scheme 2. Synthesis and Degradation of PKAA: (a) Synthesis of 2,2′-(Propane-2,2-diylbis(oxy))diethanamine; (b) Polymerization and Acid-Catalyzed Degradation of PKAA

Figure 1. 1H NMR spectra of 2,2′-(propane-2,2-diylbis(oxy))diethanamine (a) and PKAA (b). MO) and LysoTracker Red (Molecular Probes, Eugene, OR) for 1 h at 37 °C. Cells were washed with fresh medium and 50 μL of medium containing PKAA (2 μg/μL) was added to each dish, followed by incubation for 4 h. PKAA containing media were removed and cells were washed three times with fresh media and then fluorescence images of cells were made using a confocal laser scanning microscope (LSM 510 Meta, Carl Zeiss, Inc. Germany). 2.8. Suppression of TNF-α Expression by PKAA/siRNA Nanocomplexes In Vitro. RAW 264.7 macrophages (2 × 105 cells/well, 24-well plate containing 1 mL of culture medium) were incubated with 1.3 μg of free TNF-α siRNA or various formulations of PKAA/TNF-α siRNA, PAA/TNF-α siRNA and PEI 25k/TNF-α siRNA nanocomplexes for 24 h. The cells were washed with fresh medium and then stimulated with LPS (100 ng/mL) for 4 h to induce TNF-α expression. The TNF-α level in cells treated was measured with an ELISA kit as advised by the manufacturer (eBioScience, San Diego, CA). 2.9. Mouse Model of APAP-Induced Acute Liver Failure. ICR mice (∼20 g) were purchased from Orient Bio (Seoul, Korea) and divided into five groups. Mice were fasted for 12 h prior to the experiments and acute liver failure was induced by the intraperitoneal injection of 400 μL of APAP (25 mg/mL). After 1 h, mice were injected with 0.7 μg of free TNF-α siRNA, PKAA/TNF-α siRNA nanocomplexes (10:1), or PAA/TNF-α siRNA nanocomplexes (10:1) through the tail vein, giving siRNA concentrations of 35 μg/kg. Mice were sacrificed 24 h after APAP intoxication and whole blood and livers were collected. The activity of ALT in serum was determined using an ALT enzymatic assay kit (Asan Pharma, Seoul, Korea). The liver tissues were fixed with 4% formalin (Sigma-Aldrich, St. Louis, MO) and embedded into paraffin. Histological sections were made and stained with hematoxylin and eosin (H&E). All the animal

experiments were carried out according to the guidelines of the institution animal ethical committee.

3. RESULTS AND DISCUSSION 3.1. Design and Synthesis of Acid-Degradable PKAA. We hypothesized that rapid acid-catalyzed hydrolysis of cationic polymers in endosomes would facilitate the endosomal escape of siRNA nanocomplexes via ‘proton sponge effects’ and release of intact siRNA to the cytoplasmic compartment, thereby enhancing the RNA interference (Scheme 1). To address this hypothesis, rapidly acid-cleavable ketal linkages were incorporated into the backbone of cationic linear poly(amidoamine) (PAA). Poly(amidoamine)s, dendritic or linear, contain aminoand amido groups regularly arranged along the backbone chain and have demonstrated potential for intracytoplasmic and endosomolytic vectors for delivery of drugs, proteins and nucleic acids.2,15,19,28 Poly(amidoamine)s are known to be biodegradable, but they degrade into oligomeric products in aqueous media within days or weeks, depending on their structure.28 Zhao et al. reported that PAA underwent weight loss of 4% after 27 days in phosphate buffer solution (pH 7.4).29 In addition, clinical use of nondegradable or slowly degrading polymer fragments is reported to cause long-term vacuolization.15 We therefore developed PKAA with pHdependency and enhanced degradability in which rapidly acid-cleavable ketal linkages are incorporated along the backbone of polymers. 242

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immersion in neutral D2O. These observations suggest that PKAA rapidly degrades in acidic endosomal compartments and produce acetone and diol. We also determined the hydrolysis half-life of PKAA to be ∼1.5 h at pH 5.5 by comparing the ratio of methyl protons of ketal linkages and methyl protons of acetone. Cationic polymers, such as PEI, which contains a large number of secondary and tertiary amine groups along its backbone, have buffering effects in acidic endosomal compartments. Buffering effects are known to induce an increase of osmotic pressure in endosomes, resulting in the disruption of endosomal membrane to facilitate the release of siRNA nanocomplexes into cytosol. We hypothesized that secondary amines in the backbone of PKAA provide buffering capacity for “proton sponge effects”, which facilitate the endosomal escapes of PKAA/siRNA nanocomplexes. To confirm the buffering capacity of PKAA, acid−base titration was performed (Figure 3), and a comparison was made with NaCl solution and PEI

PKAA was synthesized from the Michael-type addition polymerization of N,N′-methylenebisacrylamide with 2,2′(propane-2,2-diylbis(oxy))diethanamine. We first synthesized 2,2′-(propane-2,2-diylbis(oxy))diethanamine in two steps (Scheme 2a). N-(2-Hydroxyethyl)phthalimide was reacted with 2,2-dimethoxypropane in the presence of a catalytic amount of pTSA in dry benzene using azeotropic distillation to drive the reaction forward. The intermediate was obtained as white solid with a yield of ∼30%. 2,2′-(Propane-2,2-diylbis(oxy))diethanamine was obtained as amber color liquid after deprotection of the intermediates with 6 M NaOH and column chromatography. The chemical structure was confirmed by 1H NMR (Figure 1a). The peaks at ∼3.5 ppm correspond to methylene protons next to ketal linkages and methyl protons of ketal linkages appeared at ∼1.2 ppm. PKAA was synthesized by Michael-type addition polymerization, in which polymerization can be accomplished in a single step and purification steps are unnecessary.30 PKAA was synthesized from a reaction of 2,2′-(propane-2,2-diylbis(oxy))diethanamine with commercially available N,N′-methylenebisacrylamide in methanol at 70 °C. PKAA was obtained as an amber-colored highly viscous polymer after vacuum drying. The molecular weight was determined to be ∼8000 Da by gel permeation chromatography. The chemical structure was confirmed by 1H NMR. As shown in Figure 1b, two multiplet peaks at ∼2.5 and ∼2.7 ppm correspond to methylene protons adjacent to amine groups, demonstrating polymerization of diamine with bisacrylamide. 3.2. Characteristics of PKAA. PKAA was designed to degrade rapidly in acidic environments. To confirm the acidtriggered hydrolysis of PKAA, we performed 1H NMR spectroscopy using a CD3COOD/D2O solution (pH 5.5). Figure 2 shows the 1H NMR spectrum of PKAA after 4 h of hydrolysis in CD3COOD/D2O solution. Methyl protons and methylene protons of ketal linkages were initially observed at 1.2 ppm and ∼3.4 ppm in D2O, but dramatically decreased after hydrolysis. In addition, methyl protons of acetone and hydroxyl protons of degradation products appeared at ∼1.8 and ∼3.7 ppm. In contrast, no change was observed after 24 h of

Figure 3. Acid-based titration of PKAA.

25k at the same concentration. The initial pH of NaCl solutions rapidly decreased by the addition of 0.5 N of HCl solution, suggesting little buffering capacity. However, PKAA (10 mg/ mL) showed a slow and gradual reduction in the pH value by the addition of HCl because of the protonation of amine groups along the polymer backbone, comparable buffering capacity to the same concentration of PEI 25k. The sufficient buffering capacity of the PKAA may play an important role in the endosomal escape of PKAA/siRNA nanocomplexes and enhance the cytosolic delivery of siRNA. 3.3. Characterization of PKAA/siRNA Nanocomplexes. PKAA, a new family of acid-degradable cationic poly(amidoamine)s, has the propensity to combine with negatively charged siRNA to form nanocomplexes through electrostatic interactions in an aqueous solution. PKAA/siRNA nanocomplexes were prepared by simple mixing of PKAA solution and siRNA solution with different weight ratios. Figure 4 shows that PKAA/siRNA nanocomplexes at a 5:1 weight ratio are spherical, with a mean diameter of ∼237 nm. The size and morphology of PKAA/siRNA nanocomplexes were not significantly influenced by the weight ratio. Additionally, the PKAA/siRNA nanocomplexes had a zeta potential of ∼+3 mV at pH 7.4. We hypothesized that PKAA/siRNA nanocomplexes are stable at neutral conditions and rapidly release siRNA in acidic environments due to acid-cleavable ketal linkages. To confirm

Figure 2. 1H NMR spectrum of PKAA after 4 h of hydrolysis in CD3COOD/D2O solution. 243

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Figure 4. Characterization of PKAA/siRNA nanocomplexes. A representative transmission electron micrograph (a) and dynamic light scattering (b) of PKAA/siRNA nanocomplexes with a weight ratio of 5:1.

Figure 5. Agarose gel electrophoresis of PKAA/siRNA nanocomplexes at various weight ratios.

for 24 h. Figure 6 shows the viability of cells treated with various amounts of PKAA, with a comparison with PEI 25k.

the ability of PKAA to form stable nanocomplexes and pHdependent siRNA release profiles, we performed agarose gel electrophoresis of PKAA/siRNA nanocomplexes with various weight ratios, after 4 h of incubation at pH 5.4 and 7.4. Figure 5 illustrates the migration of PKAA/siRNA nanocomplexes on agarose gel. After incubation at pH 7.4, siRNA migration was dependent on the weight ratio. At weight ratios (10:1 and 15:1), migration of siRNA was completely retarded. However, after 4 h of incubation at pH 5.4, siRNA was released and completely migrated at all weight ratios. These results demonstrate that PKAA forms stable nanocomplexes with siRNA, but rapidly degrades at acidic pH to release siRNA. We also found that PKAA/siRNA nanocomplexes at a 10:1 weight ratio were stable for ∼12 h, after which siRNA release was initiated. The physical stability of the PKAA/siRNA nanocomplexes was also investigated in the presence of heparin which is known to interfere the electrostatic interactions between cationic polymers and siRNA, facilitating the dissociation of nanocomplexes and siRNA release.31 In the presence of 2 IU of heparin for 3 h, more migration was observed at weight ratios less than 5:1. However, a little fraction of siRNA was migrated at a weight ratio of 10:1, suggesting that PKAA can condense siRNA into nanocomplexes, which have excellent stability at this weight ratio. 3.4. Cytotoxicity and Intracellular Trafficking of PKAA. Cytotoxicity is one of the critical concerns involving the development of drug carriers. In vitro cytotoxicity of PKAA was evaluated using the MTT assay. Two different cells (NIH3T3 and HEK293 cells) were treated with various amounts of PKAA

Figure 6. Cytotoxicity of NIH3T3 and HEK293 cells incubated with various amounts of PEI and PKAA for 24 h. The values are the mean ± SD (n = 4).

PEI at a dose of 50 μg/mL caused more than ∼40% of reduction in cell viability. However, the viability of cells was not influenced by PKAA up to 1000 μg/mL, demonstrating the excellent biocompatibility of PKAA as a biomaterial. Cationic polymers have an ability to disrupt acidic endosomes via buffering effects and osmotic pressure buildup associated with chloride accumulation, which is called “proton sponge effects”.32 When siRNA nanocomplexes are internalized into the cells, they are enclosed in endosomes. To realize 244

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3.5. Suppression of TNF-α by PKAA/siRNA Nanocomplexes In Vitro. The abilities of PKAA to form stable nanocomplexes with siRNA and to induce endosomal escapes of siRNA motivated us to explore its potential as siRNA carriers using a mouse model of APAP-induced liver failure. Anti-TNFα siRNA nanocomplexes were prepared with PKAA, PAA, or PEI 25k at various weight ratios and given to LPS-stimulated macrophages. TNF-α is a pro-inflammatory cytokine involved in systemic inflammation and is produced by activated macrophages. We therefore measured the level of TNF-α expression from LPS-stimulated macrophages to evaluate their siRNA delivery efficiency. Figure 8 shows RNA interference mediated by PKAA/siRNA nanocomplexes, in a comparison with PAA and PEI 25k. Free

maximum gene silencing effects, siRNA nanocomplexes should escape the endosome and release intact siRNA to cytosol. We hypothesized that PKAA, after cellular uptake, undergoes rapid acid-catalyzed hydrolytic degradation and increases osmotic pressure, resulting in endosomal rupture and endosomal escape. To confirm our hypothesis, we studied intracellular trafficking of PKAA using membrane impermeable calcein and LysoTracker Red as a pH-sensitive fluorescence marker of endolysosomal compartments. Cells pretreated with both calcein and LysoTracker Red were treated with PKAA and then observed under a confocal laser scanning microscope. For comparison purposes, we also synthesized a linear poly(amidoamine) (PAA), which has chemical structure similar to PKAA, but has no ketal linkage in its backbone. Cells treated with PAA show colocalization of calcein and LysoTracker Red in endolysosomal compartments in the periphery of cells, evidenced by the yellow fluorescence in a merged image (Figure 7).

Figure 8. Enhanced delivery of anti-TNF-α siRNA by PKAA in LPSstimulated cells. The amount of siRNA was 1.3 μg. **P < 0.01 relative to PAA groups at the same weight ratio (n = 4, ± S.D).

siRNA exhibited negligible RNA interference, but PEI 25k enhanced RNA interference significantly. PAA/siRNA nanocomplexes also showed the ability to transfect and silence TNFα production, in a weight ratio-dependent manner. However, PKAA/siRNA nanocomplexes demonstrated even higher level of transfection and RNA interference than PAA/siRNA nanocomplexes. Both PAA and PKAA contain secondary amine groups along the backbone and have abilities to disrupt endosomes via “proton sponge effects”, leading to effective delivery of siRNA to cytosol. However, at the same weight ratio of nanocomplexes, PKAA induced significantly higher gene silencing effects of siRNA than PAA. As afore addressed, the enhanced RNA interference mediated by PKAA can be explained by the combined effects of ‘proton sponge effects’ and colloid osmotic mechanism of PKAA which undergoes rapid acid-catalyzed hydrolytic degradation in acidic endosomes.34,35 3.6. Mouse Model of APAP-Induced Acute Liver Failure. The ability of PKAA to enhance the delivery of siRNA in vivo was investigated in mice suffering from APAPinduced acute liver failure. APAP is a dose-dependent hepatotoxicant and APAP-induced toxicity has been the most frequent cause of acute liver failure in the United States.35 Therefore, a mouse model of APAP-induced acute liver failure is clinically relevant and is one of the most popular experiments in vivo to test the efficacy of various therapeutic compounds.36 In addition, Kupffer cells (liver macrophage) secret TNF-α which plays a major role in the development of several inflammatory diseases such as acute liver failure.37 We therefore investigated if PKAA could enhance the delivery of anti-TNF-α

Figure 7. Representative confocal fluorescence micrographs of cells treated with PKAA or PAA for 4 h in the presence of calcein and LysoTracker Red.

On the other hand, treatment with PKAA resulted in a wide distribution of calcein and no red fluorescence in the whole area of cytosol. No colocalization of calcein and LysoTracker Red in the endosomes indicates that PKAA rapidly induces endosome rupture and release of calcein and LysoTracker Red into cytosol. LysoTracker Red had no fluorescence because it loses its fluorescence under neutral conditions.20 The rapid and efficient endosomal escape by PKAA may be attributed to the combined effects of “proton sponge effects” and colloid osmotic mechanism of PKAA which contains a number of secondary amines and rapid acid-cleavable ketal linkages in its backbone.33−35 We reasoned that PKAA rapidly degrades in acidic endosomes to release hundreds of free small molecules, leading to the further increase in colloid osmotic pressure. The increased osmotic pressure is expected to induce the further swelling of endosomes and accelerate disruption of endosomes. The cytosolic delivery of siRNA is one of major challenges in the delivery of siRNA, in particular to macrophages, which rapidly degrade phagocytosed materials in their phagosomes (endosomes). Therefore, the PKAA with endosomolytic activities and rapidly acid-cleavable linkages has promising potential as a siRNA carrier. 245

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We next performed the histological studies to further confirm the enhanced therapeutic efficacy of PKAA/TNF-α siRNA nanocomplexes in APAP-induced liver injury. Figure 10 shows that APAP-intoxication caused extensive liver damages and disruption of tissue architecture, evidenced by destruction of hepatocytes and leukocyte infiltration. Free siRNA showed no effects on the histopathological alterations. However, PKAA/ siRNA nanocomplexes remarkably reduced the liver tissue damage and histopathological alterations. The results demonstrate that PKAA/anti-TNF-α siRNA nanocomplexes showed effective protection of hepatocytes from APAP-induced liver damage. It can be reasoned that the enhanced RNA interference of anti-TNF-α siRNA with PKAA is attributed to many factors, including effective protection of siRNA from serum nucleases, passive targeting to liver, endosomal escape, and efficient dissociation of siRNA from the nanocomplexes. Particulate drug delivery systems have the natural propensity to localize to the mononuclear phagocyte systems, in particular, liver macrophages. Previously, it was reported that a large population of liver macrophages in contact with blood get activated and enter the injured liver tissues once liver is damaged by toxins.35,38 We therefore postulate that PKAA/siRNA nanocomplexes are easily taken up by macrophages in liver during APAP-intoxication, leading to passive liver targeting. Then, as expected from Figure 7, PKAA facilitated endosomal escape of siRNA and released siRNA payloads into the cytoplasmic compartments, leading to the maximal gene silencing activity.

siRNA, targeted against TNF-α and alleviate APAP-induced acute liver failure. Mice were treated with various siRNA formulations 1 h after APAP injection. A group of mice was treated with saline and served as a control. Mice were sacrificed 24 h after APAPtreatment and the activity of ALT (alanine transaminase) was determined to evaluate the liver injury (Figure 9). The level of

Figure 9. Activity of ALT in serum of APAP-intoxicated mice after PKAA/anti-TNF-α siRNA nanocomplexes. The values are the mean ± SD (n = 4) *P < 0.05 relative to PAA groups at the same weight ratio.

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ALT is correlated with the severity of liver injury and therefore ALT has been widely used as a surrogate clinical marker for liver injury.35 APAP-intoxication for 24 h remarkably increased the ALT activity, suggesting the severe damage to liver tissues. Free siRNA at a dose of 0.7 μg showed no effects on the ALT level due mainly to degradation by serum nucleases. The same dose of siRNA complexed with PAA at a weight ratio of 10:1 reduced the activity of ALT to some extent, not significantly. In contrast, the same dose of PKAA/siRNA nanocomplexes induced significant reduction in the level of ALT.

CONCLUSIONS We developed a new acid-degradable cationic polymer, PKAA as siRNA carriers. PKAA was designed to have acid-cleavable ketal linkages along the backbone and synthesized from a Michael type-addition polymerization of ketal containing diamines and methylenbisacrylamide. PKAA exhibited excellent buffering capability and biocompatibility. PKAA condensed with siRNA to form stable nanocomplexes through electrostatic

Figure 10. Representative micrographs of liver tissues stained with H&E. 246

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Biomacromolecules

Article

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interactions in an aqueous solution. The nanocomplexes were stable at neutral conditions, but released siRNA rapidly under acidic conditions. PKAA/TNF-α siRNA nanocomplexes could effectively transfect and silence the TNF-α production in LPSstimulated macrophages. In addition, PKAA/TNF-α siRNA nanocomplexes significantly reduced the hepatic cellular damages from APAP-induced acute liver failure. Given its excellent biocompatibility, endosomolytic activity, and pHsensitivity, PKAA has great potential as siRNA carriers.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 82-63-270-2344. Fax: 82-63-270-2341. E-mail: dlee@ jbnu.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the World Class University program (R31-20029) funded by the Ministry of Education, Science and Technology, Korea.

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REFERENCES

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dx.doi.org/10.1021/bm301669e | Biomacromolecules 2013, 14, 240−247