Acid-Responsive Linear Polyethylenimine for Efficient, Specific, and

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Bioconjugate Chem. 2009, 20, 488–499

Acid-Responsive Linear Polyethylenimine for Efficient, Specific, and Biocompatible siRNA Delivery Min Suk Shim†,‡ and Young Jik Kwon*,‡,§,| Department of Macromolecular Science and Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, and Department of Chemical Engineering and Materials Science, Department of Pharmaceutical Sciences, and Department of Biomedical Engineering, University of California, Irvine, California 92697. Received October 13, 2008; Revised Manuscript Received December 23, 2008

Efficient intracellular processes including cytosolic release and unpackaging of siRNA from the carrier in the cytoplasm are efficiency-determining steps in achieving successful gene silencing. In this study, acid-degradable ketalized linear polyethylenimine (KL-PEI) was synthesized for efficient, intracellular target-specific, and biocompatible siRNA delivery. The siRNA/KL-PEI polyplexes resulted in much higher RNA interference efficiency than unmodified L-PEI via selective cytoplasmic localization of the polyplexes and efficient disassembly of siRNA from the polyplexes, which were promoted upon acid-hydrolysis of amino ketal linkages. Confocal laser scanning microscopy demonstrated that siRNA was efficiently disassembled from the siRNA/KL-PEI polyplexes that were selectively localized in the cytoplasm. On the contrary, siRNA and unmodified linear PEI were colocalized in both the cytoplasm and the nucleus, and limited unpackaging of siRNA from the polyplexes was observed. In addition, ketalization further reduced the cytotoxicity of linear PEI but did not alter its serum-independent gene delivery efficiency. Therefore, KL-PEI is a promising nonviral vector for efficient and biocompatible siRNA delivery.

INTRODUCTION Silencing gene expression in a sequence-specific manner by double stranded small interfering RNA (siRNA) has attracted great attention as a potential therapeutic approach for treating diseases arising from genetic defects (1-3). However, the use of siRNA is restricted by its inherent low stability against degradation and poor cellular uptake into cells in vitro or in vivo (4, 5). Therefore, the development of effective carrier systems, which can protect and deliver siRNA to the cytoplasm, where siRNA encounters target mRNA, is a major key to exploit the successful therapeutic and research use of siRNA in mammalian cells (6, 7). Among the gene carriers that have been investigated, nonviral vectors have recently attracted more and more attention in comparison to viral vectors, due to their advantages, including the ease of synthesis, delivery of large quantities of genes, and low immune response (8). Recently, cationic polymer-based nonviral systems for siRNA delivery have been popularly developed since the cationic polymers form stable polyplexes via electrostatic interaction and protect siRNA from enzymatic digestion (9-13). Common challenges in using the cationic polymer for siRNA delivery often involve high toxicity and limited siRNA delivery efficiency (14). One of the major bottleneck steps in nonviral gene transfer is the escape of nucleic acid-containing carriers from the endosomal compartment, where acidic pH and nucleases ef* Corresponding author. 916 Engineering Tower, Irvine, CA 926972575. Tel: +949 824 8714. Fax: +949 824 2545. E-mail: [email protected]. † Case Western Reserve University. ‡ Department of Chemical Engineering and Materials Science, University of California, Irvine. § Department of Pharmaceutical Sciences, University of California, Irvine. | Department of Biomedical Engineering, University of California, Irvine.

ficiently digest nucleic acids, and the release of nucleic acids in their desired intracellular targets (e.g., cytoplasm for siRNA) (15-18). Therefore, the facilitated release of siRNAcontaining polyplexes from the digestive endosome and efficient polyplex unpackaging in the cytoplasm are indispensible design goals of developing efficient carriers for gene silencing. In recent years, polyethylenimine (PEI)-based carriers have been highlighted as efficient nonviral nucleic acid delivery carriers (4, 15, 19-22). Among a variety of selections in terms of molecular weights (0.8-800 kDa) and topological isomers (branched or linear structures), linear high molecular weight PEI (L-HMW PEI) (e.g, 22 kDa) and branched high molecular weight PEI (B-HMW PEI) (e.g., 25 kDa) have demonstrated sufficient efficiency. Although both polymers have shown high gene delivery efficiency in vitro (15, 23), L-HMW PEI has shown much higher in vivo gene expression with less cytotoxicity in comparison to branched PEI (B-PEI) (22-26). However, linear PEI shows limited efficiency in siRNA delivery, although the reason for such a discrepancy between DNA and siRNA delivery efficiencies is unclear (20, 27). For this reason, finding a strategy to improve siRNA delivery efficiency using linear PEI is of great interest. In this study, 25 kDa linear PEI was modified to accomplish enhanced siRNA delivery with low cytotoxicity. It was hypothesized that the incorporation of acid-degradable amino ketal branches to secondary amines of linear PEI would facilitate endosomal escape, by broadened buffering capacity of primary, secondary, and tertiary amines, and the subsequent release of siRNA to the cytoplasm, thus resulting in efficient RNA interference. Under the acidic conditions in the endosome, hydrolyzed ketal side branches were expected to induce endosomal destabilization by increased osmotic forces and swollen polyplexes. It was shown that ketalized linear PEI resulted in high RNA interference (RNAi) efficiency, even in the presence of serum, through exclusive cytoplasmic localization of the siRNA-containing polyplexes and efficient unpackaging of

10.1021/bc800436v CCC: $40.75  2009 American Chemical Society Published on Web 02/06/2009

Ketalized Linear PEI for siRNA Delivery

siRNA from the hydrolyzed polyplexes. This study implies that ketalized linear PEI is a promising siRNA delivery carrier offering substantially improved RNAi efficiency and biocompatibility.

Bioconjugate Chem., Vol. 20, No. 3, 2009 489 Scheme 1. Synthesis of Ketalized Linear PEI

MATERIALS AND METHODS Materials. Linear polyethylenimine (25 kDa, 2.5 kDa) was purchased from Polyscience, Inc. (Warrington, PA). Branched polyethylenimine (25 kDa) and 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazoliumbromide (MTT) were purchased from Sigma Aldrich (Milwaukee, WI). 2-Aminoethanol, pyridinium ptoluenesulfonate, ethyl trifluoroacetate, 2-methoxypropene, molecular sieves, and 1,4-diaminobutane dihydrochloride were supplied from Acros (Morris Plains, NJ). Acryloyl chloride was supplied from Alfa Aesar (Ward Hill, MA). Ethidium bromide was purchased from Fisher Scientific (Pittsburgh, PA). Enhanced green fluorescent protein (eGFP)-encoding plasmid DNA was a generous gift from Dr. Pamela Davis (Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH). Alexa Fluor 488 carboxylic acid succinimidyl ester dye was purchased from Molecular Probes (Eugene, OR), and DRAQ5 nuclear dye was purchased from BioStatus (Leicestershire, UK). Silencer GFP siRNA and Silencer Cy3-labeled negative control siRNA were purchased from Ambion (Austin, TX). NIH 3T3 cells (ATCC, Rockville, MD) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (MediaTech, Herndon, VA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), unless otherwise noted. Polymer Characterization. Electrospray mass spectra of various monomers were obtained on a Micromass LCT mass spectrometer (Micromass Ltd., Manchester, UK). 1H NMR spectrum of various ketalized linear PEI was obtained by a Bruker Avance 500 MHz NMR spectrometer (Bruker Biospin Corporation, Billerica, MA). Chemical shifts for 1H NMR spectra were reported relative to (2,2-dimethyl-2-silapentane5-sulfonate sodium salt) (DSS, δ ) 0.015 ppm) when D2O was used as solvent. The molecular weights and molecular weight distributions of various ketalized linear PEI were determined by a gel permeation chromatography (GPC) system equipped with Agilent 1100 series (Agilent Technologies, Santa Clara, CA). The eluent was water, and a flow rate of 1.0 mL/min was used. The sample was diluted to 0.5 mg/mL in DI water and filtered through 0.45 µm PVDF syringe filters, and 100 µL of sample was injected into the GPC system for analysis. The molecular weights were calibrated against poly(ethylene oxide) standards. Synthesis of Ketalized Linear Polyethylenimine (KLPEI). Acid-degradable ketalized linear polyethylenimine in which acid-degradable ketal linkages were grafted was synthesized via Michael addition conjugation of acrylated ketal monomers (Scheme 1). Synthesis of Compound 2 [N-(2-(2-(2-Aminoethoxy)propan-2-yloxy)ethyl)-2,2,2-trifluoroacetamide]. [1,1′-(2,2′-(Propane-2,2-diylbis(oxy))bis(ethane-2,1-diyl))diurea] (compound 1) was prepared as previously reported (28). Compound 1 (2.2 g, 13.58 mmol, 1 equiv) was dissolved in 10 mL of methanol with 2.06 g of triethylamine (20.37 mmol, 1.5 equiv). Ethyl trifluoroacetate (1.93 g, 13.58 mmol, 1 equiv) was dissolved in 10 mL of methanol, and the resulting mixture was slowly added dropwise to the solution of compound 1 on ice. The reaction mixture was stirred for 6 h on ice, and then the mixture was extracted with 3 × 30 mL of dichloromethane. The organic layers were combined and evaporated. The product was purified by silica gel chromatography using a gradient from hexane to 60/40 hexane/tetrahydrofuran to obtain the product as a white solid (1.57 g, 6.11 mmol, 45% yield). ESI-MS [M + H]+ calcd 259.1; found, 259.2. 1H NMR (500 MHz, CDCl3): δ 1.37 (s,

6H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 1.51 (br, 2H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 2.88 (t, 2H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 3.52 - 3.56 (m, 4H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 3.70 (t, 2H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 9.50 (br, 1H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NH2). Synthesis of Compound 3 [N-(2-(2-(2-(2,2,2-Trifluoroacetamido)ethoxy)propan-2-yloxy)ethyl)acrylamide. Compound 2 (1.40 g, 5.42 mmol, 1 equiv) was dissolved in 10 mL of dioxane with 2.19 g of triethylamine (21.68 mmol, 4 equiv), and the mixture solution was kept in an ice bath. Acryloyl chloride solution (0.98 g, 10.84 mmol, 2 equiv) was prepared in 10 mL of dioxane and slowly added to the solution of compound 2. The reaction mixture was stirred for 10 min, and the product was extracted using 3 × 100 mL of ethyl acetate and then purified by silica gel chromatography using a gradient from hexane to 1/1 hexane/ethyl acetate to obtain the product as a yellow oil (1.17 g, 3.75 mmol, 69% yield). ESI-MS [M + H]+ calcd 313.1; found, 313.2. 1H NMR (500 MHz, CDCl3): δ 1.36 (s, 6H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH2); δ 3.48 - 3.53 (m, 8H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH2); δ 5.67 (dd, 1H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH’H”); δ 6.01 (br, 1H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH2); δ 6.14 (dd, 1H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH2); δ 6.33 (dd, 1H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH’H”); δ 7.05 (br, 1H, CF3CONHCH2CH2OC(CH3)2OCH2CH2NHCOCHCH2). Synthesis of Compound 4. Linear low molecular weight PEI (L-LMW PEI, 2.5 kDa) and linear high molecular weight PEI (L-HMW PEI, 25 kDa) (0.1 g, 2.33 mmol of secondary amine groups, 1 equiv) were dissolved in 2 mL of methanol followed by evaporating the methanol under high vacuum for 30 min. The resulting solution was dissolved in 10 mL of dimethylformamide (DMF) with 0.73 g of compound 3 (2.33 mmol, 1 equiv), and 0.23 g of triethylamine (2.33 mmol, 1 equiv), which served as a deprotonating agent as well as a preventer of hydrolysis of ketals, was added. The resulting mixture was stirred for 120 h at 45 °C, and the polymer was precipitated into anhydrous diethyl ether and dried under vacuum. The precipitated polymer was dissolved in 10 mL of 1:9 MeOH/6 M NaOH solution and stirred for 12 h to deprotect trifluoro-

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acetate groups. After deprotection, the polymer was dissolved in DI water and purified by dialysis against DI water at 4 °C (Mw cutoff of dialysis membrane ) 500 for L-LMW PEI and Mw Cutoff ) 6000 for L-HMW PEI). After 24 h of dialysis, each polymer was lyophilized overnight to obtain the product as a powder with a hint of yellow color [60 mg of ketalized linear low molecular weight PEI (KL-LMW PEI) (26% yield) and 72 mg of ketalized linear high molecular weight PEI (KLHMW PEI) (36% yield)]. 1H NMR (500 MHz, D2O): δ 1.41 (s, -NCH2CH2CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 2.7 - 3.0 (m, -CH2-CH2-NH-, -NCH2CH2CONHCH2CH2OC(CH3)2OCH2CH2NH2, and - NCH2CH2CONHCH2CH2OC(CH3)2OCH2CH2NH2); δ 3.3 - 3.7 (m, -NCH2CH2CONHCH2CH2OC(CH3)2OCH2CH2NH2, -NCH2CH2CONHCH2CH2OC(CH3)2OCH2CH2NH2, and - NCH2CH2CONHCH2CH2OC(CH3)2OCH2CH2NH2). Hydrolysis Kinetics of KL-PEI. The hydrolysis rates of KLPEI at different molecular weights and pH values were investigated by incubating KL-PEI in (10 mg) pH 5.0 acetate, 6.0 acetate, and pH 7.4 Tris-HCl buffers in D2O at 37 °C (0.6 mL) for various periods of time. Disappearance of the ketal linkage peak (1.41 ppm) was quantified by using 1H NMR spectroscopy in comparison with the constant buffer salt peak. Half-lives of the various PEI at different pH values were obtained by using the Arrhenius equation. Preparation and Characterization of siRNA/PEI Polyplexes. Two micrograms of siRNA with various amounts of polymers were mixed in 100 µL of DI water at various N/P ratios, followed by incubation for 30 min at room temperature. Then the resulting polyplex solution was diluted with additional 800 µL of DI water. Mean particle diameter (Z-average) and zeta potential of various siRNA/PEI polyplexes were measured at 25 °C by using ZEN3600 Zetasizer (Malvern Instruments, Worcestershire, UK)y. The viscosity (0.887 mPa/s) and the refractive index (1.333) of water at 25 °C were used to analyze the data. The mean values of each sample were yielded from the data obtained from at least 10 measurements. Each sample was analyzed in triplicate. Ethidium Bromide Exclusion Assay. The ability of KLPEI to condense siRNA was evaluated by a standard ethidium bromide exclusion assay. After one microgram of ethidium bromide and 1 µg of siRNA in 40 µL of DI water were incubated for 15 min at room temperature, desired amounts of the polymer solution in 60 µL of DI water were added in order to achieve various N/P ratios. After 30 min of incubation, fluorescence intensity was measured using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) with an excitation wavelength (λex) of 320 nm and an emission wavelength (λem) of 600 nm. Normalized fluorescence was determined as (F - F0)/(FsiRNA - F0), where F, F0, and FsiRNA represent the fluorescence of the solution containing ethidium bromide and the polyplexes, the solution of ethidium bromide without siRNA and polymers, and siRNA/ethidium bromide solution, respectively. Reduced fluorescence intensity was used as a quantitative indicator of siRNA condensation in polyplexes. Agarose Gel Electrophoresis Assay. The binding of siRNA with PEI was determined by 1.2% agarose gel electrophoresis. Desired amounts of ketalized and unketalized L-PEI dissolved in phosphate buffered saline (PBS) were mixed with 1 µg of siRNA in DI water (17 µL in total) to yield various N/P ratios, followed by incubation for 15 min. Three µL of a gel loading dye was added to each polyplex solution, and 20 µL of the resulting polyplex solution was loaded on a gel containing 1 µg/mL of ethidium bromide. To verify the dissociation of the siRNA from the polymers upon hydrolysis, 2 µg of siRNA and desired amounts of polymer were mixed in 8 µL of DI water to obtain various N/P ratios. Then the mixture was incubated for 20 min at R.T. followed by the addition of 26 µL of pH 5.0

Shim and Kwon

acetate buffer. The polyplexes were further incubated for 4 h at 37 °C. A 17 µL aliquot of the sample containing 1 µg of siRNA was mixed with 3 µL of gel loading dye, and 20 µL of the mixture solution was loaded on the agarose gel. The samples were electrophoresed in Tris-acetate-EDTA (TAE) buffer at 60 V for 15 min. The siRNA bands were then visualized under a UV transilluminator at a wavelength of 365 nm. Transmission Electron Microscopy. Size and morphological changes of various polyplexes upon hydrolysis were analyzed by transmission electron microscopy (TEM). Briefly, siRNA/ KL-PEI polyplexes were prepared by mixing 1 µg of siRNA with various PEI in 20 µL of DI water to obtain the N/P ratio of 100. A 2 µL aliquot of the sample solution was dropped on a carbon-coated copper TEM grid (Electron Microscopy Sciences, Hatfield, PA) and air-dried for 10 min at R.T. Grids were then stained with uranyl acetate and dried. To prepare hydrolyzed particles, 20 µL of siRNA/KL-PEI polyplex solution was mixed with 40 µL of pH 5.0 acetate buffer and further incubated for 4 h at 37 °C. Then a 6 µL aliquot of the resulting polyplex solution was deposited on a grid and dried. TEM images were acquired using Philips CM20 (Philips Electronic Instruments, Mahwah, NJ) operated at 200 kV. Fluorescence Labeling of Polyplexes and Quantification. Desired amounts of various polymers were mixed with 4 µg of siRNA in DI water (400 µL total) to obtain various N/P ratios. The polyplexes were incubated for 30 min at room temperature and then fluorescently labeled with Alexa Fluor 488 dye (Molecular Probes, Inc., Eugene, OR) by following the manufacturer’s instructions. Labeled siRNA/L-PEI polyplexes were purified from unreacted dyes by a PD-10 size exclusion column (GE Healthcare, Piscataway, NJ) using DMEM as eluent. To quantify Alexa Fluor 488 conjugation to siRNA/KLPEI polyplexes, the fluorescence intensity of the Alexa Fluor 488-conjugated polyplexes was compared with the one of Alexa Fluor 488 added for conjugation. Briefly, siRNA/KL-PEI polyplexes were prepared by mixing 71.3 µg of KL-LMW PEI with 4 µg of siRNA in DI water (500 µL total) to achieve the N/P ratio of 100. After 30 min of incubation, KL-LMW PEI (0.11 mmol of primary amines linked to ketal branches, 1 equiv) complexing siRNA reacted with Alexa Fluor 488 carboxylic acid succinimidyl ester dye (1 equiv), and the resulting mixture was incubated for 2 h at R.T. Then unreacted Alexa Fluor 488 dyes were removed using a PD-10 size exclusion column. Fluorescence of the Alexa Fluor 488 labeled-polyplexes was measured using a microplate reader with an excitation wavelength (λex) of 488 nm and an emission wavelength (λem) of 520 nm. In addition, a fluorescence standard curve was constructed using various concentrations of Alexa Fluor 488 dye stock solution in the range of 0.01 mM to 10 mM. In order to quantitatively estimate the amount of free Alexa Fluor 488 released from the ketalized linear PEI upon hydrolysis, Alexa Fluor 488-labeled polyplexes were incubated in pH 5.0 acetate buffer for 4 h at 37 °C. Released free Alexa Fluor 488 dye was removed by eluting the polyplexes through a PD-10 size exclusion column, and the reduced fluorescence intensity of the eluted solution was compared with the one of the unhydrolyzed ployplexes-containing solution. Confocal Laser Scanning Microscopy. Confocal laser scanning microscopy was performed to observe the intracellular localization of various siRNA/PEI polyplexes. NIH 3T3 cells were inoculated at a density of 2.0 × 104 cells/well in a Falcon 8-well cultureslide (BD Biosciences, Franklin Lakes, NJ), 15 h prior to the incubation with the polyplexes. Five hundred twenty microliters of polyplex solution containing 0.6 µg of siRNA in DMEM, prepared as described above, was added to each well. After 6 h of incubation, the cells were washed with PBS four times and fixed with 2% p-formaldehyde solution for 30 min


at 4 °C. Then, the nuclei of the cells were counter-stained by treating 1 µM nuclear dye DRAQ5 solution in PBS (BioStatus, Leicestershire) for 20 min, and the cells were washed with PBS. Fluorescence images were acquired using an Olympus IX2 inverted microscope equipped with a Fluoview 1000 confocal scanning microscopy setup (FV10-ASW, Olympus America, Melville, NY) and a 40X/1.3 NA oil immersion planapochromat objective lens. Alexa Fluor 488 was excited with a 488 nm multiple argon laser light, and emission was collected using a SDM 560 dichroic mirror and a 505-605 nm band-pass filter. Images of DRAQ5-stained nuclei were acquired using a 635 nm helium-neon laser excitation light, a 635 nm dichroic mirror, and a 655-755nm band-pass barrier filter. Digital image recording and analysis were carried out using FV10-ASW 1.6 viewer (Olympus America). The cells were scanned in three dimensions as a z-stack of two-dimensional images (1024 × 1024 pixels). An image cutting horizontally through approximately the middle of the cellular height was selected out of a z-stack of images to differentiate the fluorescence from the polyplexes located in the perinuclear and intranuclear areas. To observe siRNA and polymer in NIH 3T3 cells, polyplexes were prepared with Cy3-labeled siRNA, as described earlier. Then linear PEI complexing Cy3-labeled siRNA were labeled with Alexa Fluor 488 dye. Unreacted dyes were removed using PD-10 size exclusion columns. Desired amounts of polyplex solutions containing 0.6 µg of siRNA in DMEM were added to each well containing NIH 3T3 cells inoculated at a density of 2.0 × 104 cells. After 4 h of incubation, the cells were washed several times with PBS and fixed with 2% p-formaldehyde solution for 30 min at 4 °C. Then, the fixed cells were counterstained by DRAQ5 and washed with PBS. Fluorescence images of Alexa Fluor 488 were acquired using a 488 nm excitation light from a multiple argon laser, a SDM560 dichroic mirror, and a 505-540 nm band-pass barrier filter. A 559 nm helium-neon laser, a SMD640 dichroic mirror, and a 575-620 nm band-pass barrier filter were used to obtain the images of Cy3-labeled siRNA. Images of DRAQ5-stained nuclei were acquired using a 635 nm helium-neon laser, a 635 nm dichroic mirror, and a 655-755 nm band-pass barrier filter. Each excitation light was scanned separately for the individual excitation of the dyes to eliminate cross-talk. RNA Interference. eGFP-expressing NIH 3T3 cells were obtained by retroviral transduction and subsequent G418 selection (29). Cells were seeded in a 24-well plate at a density of 2 × 104 cells/well. siRNA-containing polyplexes were prepared by mixing various amounts of polymer stock solutions in PBS with anti-eGFP siRNA or nonspecific control siRNA solutions (75 pmol) in DI water to obtain various N/P ratios in the final volume of 75 µL, followed by 30 min of incubation at R.T. After 24 h, the culture medium in each well was replaced with 300 µL of FBS-free DMEM or 10% FBS-containing DMEM. Then 75 µL of polyplex solutions was added to the cells to obtain a final siRNA concentration of 200 nM. After 4 h of incubation, the medium in the well was replaced with 0.5 mL of fresh DMEM containing 10% FBS, and the cells were further cultured for 72 h. After the cells were harvested by trypsinization and washed with PBS, their eGFP fluorescence was analyzed by using a Guava EasyCyte Plus flow cytometer (Guava Technologies, Hayward, CA). The silencing of eGFP gene expression was quantified by mean fluorescence intensities of the cells incubated with various siRNA-containing polyplexes in comparison with the cells incubated without polyplexes. Cytotoxicity Assay. eGFP-expressing NIH 3T3 were inoculated at a density of 4 × 103 cells/well in a 96-well plate and incubated for 24 h. The culture medium was replaced with the mixtures of 60 µL of FBS-free DMEM and 15 µL of anti-eGFP siRNA or nonspecific control siRNA (15 pmol) polyplex

Bioconjugate Chem., Vol. 20, No. 3, 2009 491

Figure 1. 1H NMR spectrum of ketalized linear low molecular weight PEI (KL-LMW PEI). The peaks appearing at 1.1 ppm and at 2.4 ppm (denoted by P) indicate the -CH3 and -CH2- protons of the residual propionyl moiety in commercial linear PEI, respectively.

solutions in a final siRNA concentration of 200 nM in a well. After 4 h of incubation, the medium was replaced with 100 µL of fresh DMEM supplemented 10% FBS. After the cells were further incubated for 24 h at 37 °C, the medium was replaced with the mixture of 10 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT) in PBS (10 mg/mL) and 90 µL of growth medium. After 3 h of incubation, the MTT solution was discarded from each well, and the mixtures of 200 µL of DMSO and 20 µL of glycine buffer (0.1 M glycine, 0.1 M NaCl) were added to dissolve the blue formazan crystals formed by live cells. The relative viability of the cells treated with various polyplexes was determined by UV absorbance of formazan products at 561 nm. Statistics. Triplicate data were analyzed using one-way analysis of variance (ANOVA) on the significance level of p < 0.01 and presented as mean ( standard deviation.

RESULTS AND DISCUSSION Design and Synthesis of Ketalized Linear PEI. As intracellular targets for DNA transfection and RNA interference are different (30, 31), the design of nonviral carrier for siRNA delivery should be distinguished from the one for DNA delivery in order to obtain desired therapeutic effects. It is hypothesized that efficient cytoplasmic release of free siRNA is required to maximize the therapeutic efficacy and avoid undesirable effects. Therefore, in this study, acid-degradable ketalized linear PEI (KL-PEI) was specifically designed and synthesized to achieve efficient cytoplasmic localization of polyplexes and unpackaging of the polyplexes through hydrolysis of ketal branches grafted to the polymer backbone. Acrylamide groups at the end of amino ketal monomers were conjugated to secondary amines of linear PEI via a Michael addition reaction. A sharp singlet peak at 1.41 ppm (i.e., methyl protons of ketal linkage) in the 1H NMR spectrum of ketalized linear low molecular weight (KL-LMW) PEI indicates successful conjugation of acid-degradable branches (Figure 1). It was found that ketal hydrolyzed at pH 7.4 at 4 °C with a half-life of approximately 200 h. The hydrolysis test using 1 H NMR showed that approximately 8.0% of ketal linkages hydrolyzed during the dialysis for the purification of ketalized linear PEI. To quantify the degree of hydrolysis during the dialysis, integral ratios of methyl protons (1.41 pm) of ketal linkage peak to methyl protons (1.1 ppm) of the residual propionyl moiety of linear PEI in 1H NMR spectra were measured before and after dialysis, respectively. Ketalization ratios relative to available secondary amines were calculated


Shim and Kwon

Table 1. Characterization and Hydrolysis Kinetics of Various Ketalized Linear PEI half-lives (h) polymer

degree of ketalization (mol %)

Mn (kDa)

Mw (kDa)

PDI

pH 5.0

pH 6.0

pH 7.4

L-HMW PEI L-LMW PEI KL-HMW PEI KL-LMW PEI

0 0 24 22

23.8 2.2 47.2 5.0

45.5 3.4 85.8 8.2

1.91 1.55 1.82 1.65

N/A N/A 2.3 1.9

N/A N/A 6.0 4.7

N/A N/A 21.6 20.3

to be approximately 22% for ketalized linear low molecular weight PEI (KL-LMW PEI) and 24% for ketalized linear high molecular weight PEI (KL-HMW PEI), by integral ratios of the peak at 1.41 ppm to the peak at 2.7-3.0 ppm. The polydispersity of the ketalized PEI was not increased during the synthesis (Table 1). It was also found that the buffering capacity of linear PEI was not significantly changed by ketalization (Supporting Information, Figure S1). Acid-Degradability of KL-PEI. Hydrolysis rates of KL-PEI at endosomal and physiological pH values were investigated by incubating the polymer in acetate and Tris-HCl buffers in D2O at 37 °C for various periods of time. The disappearance of the ketal linkage peak at 1.41 ppm relative to the constant buffer salt peak at different incubation time points was quantified by 1H NMR spectroscopy. As shown in Table 1, the half-lives of KL-LMW PEI at pH 5.0, 6.0, and 7.4 were calculated to be 1.9, 4.7, and 20.3 h, respectively, while the half-lives of KLHMW PEI at each different pH were 2.3, 6.0, and 21.6 h, respectively (Table 1). The results show that the KL-PEI hydrolyzed about 10 times faster at an endosomal pH of 5.0 than at a physiological pH of 7.4, regardless of molecular weights. It is desirable for nucleic acids to be released from the carrier by an intracellular stimulus (e.g., low pH) (32), and KLPEI was found to be capable of intracellular release of siRNA via effective hydrolysis in the acidic endosome, which facilitates endosomal membrane destabilization and unpackaging of siRNA/ KL-PEI polyplexes. Acid hydrolysis of ketal linkages in a test tube depends on the concentration of amines, which can scavenge protons and change pH, but roles of the polymer concentration dependency of ketal hydrolysis in a cell have not been confirmed. In this study, ketal hydrolysis was tested at the concentration of 16.7 mg/mL in a test tube, which was substantially higher than the highest polymer concentration of incubation (i.e., 66 µg/mL at the N/P ratio of 140), although it is very difficult to assess what exactly would be the concentration of the polymer (or amines) in the endosome/lysosome. In addition, the proton concentration in the endosome/lysosome changes by active influx via proton pumps. Complexation of siRNA with KL-PEI. Condensation of nucleic acids by a gene carrier plays a key role in determining extracellular stability, endocytosis of polyplexes, and eventual gene expression (33, 34). The effects of ketalization on siRNA condensation in various PEI polyplexes were confirmed by size reduction. As shown in Figure 2A, the size of siRNA/B-PEI (25 kDa) polyplexes at the N/P ratio of 5 and above was relatively constant in the range of 110 to 160 nm. Unmodified L-LMW and L-HMW PEI formed siRNA-containing polyplexes in similar sizes of 130 to 220 nm in diameter at the N/P ratio of 10 to 120. It was shown that KL-PEI effectively condensed siRNA into nanosized particles at high N/P ratios, allowing them to enter the cell through nonreceptor-mediated endocytosis. The size of siRNA/KL-LMW PEI polyplexes was in the range of 120 to 190 nm at the N/P ratio of 10 to 120, which was close to the size of siRNA/L-LMW PEI and siRNA/L-HMW PEI polyplexes. However, KL-HMW PEI formed polyplexes with diameters of 170 to 400 nm at the observed N/P ratios, indicating lower complexation efficiency of siRNA by KL-HMW PEI than by the other types of PEI.

Ethidium bromide exclusion analysis was also conducted to quantitatively evaluate the abilities of polymers to condense siRNA in the polyplexes. As shown in Figure 2B, siRNA/BPEI, siRNA/L-LMW PEI, and siRNA/L-HMW PEI showed

Figure 2. (A) Particle size of various siRNA/PEI polyplexes at various N/P ratios, determined by DLS analysis. (B) siRNA condensation efficiency of various siRNA/KL-PEI polyplexes at various N/P ratios, measured by ethidium bromide exclusion assay. (C) Surface charge density of various siRNA/PEI polyplexes at various N/P ratios.



Figure 3. Agarose gel electrophoresis of various siRNA/linear PEI polyplexes at different N/P ratios. (A) siRNA/L-LMW PEI polyplexes, (B) siRNA/L-HMW PEI polyplexes, (C) siRNA/KL-LMW PEI polyplexes before hydrolysis, (D) siRNA/KL-LMW PEI polyplexes after hydrolysis, (E) siRNA/KL-HMW PEI polyplexes before hydrolysis, and (F) siRNA/KL-HMW PEI polyplexes after hydrolysis. Numbers indicate N/P ratios. (0 N/P ratio: naked siRNA only.)

efficient fluorescence quenching, indicating their abilities to efficiently condense siRNA. In contrast, both KL-LMW PEI and KL-HMW PEI showed less efficient siRNA condensation ability compared to unmodified L-PEI. Particularly, siRNA/KLHMW PEI polyplexes exhibited relatively low condensation efficiency, even at high N/P ratios. The reason for the low condensation efficiency of siRNA by both KL-LMW PEI and KL-HMW PEI can be explained by their spatially hampered interactions with siRNA resulting from bulky ketal arms, especially when they were conjugated to high molecular weight linear PEI (28). Generally, it was observed that the siRNA condensation efficiency of various polyplexes determined by the ethidium bromide exclusion assay corresponded to size reduction of the polyplexes (Figure 2A and B). The measured zeta potentials of various siRNA/PEI polyplexes were shown in Figure 2C. All of the polyplexes had positive surface charges at the observed range of N/P ratios, suggesting that negatively charged siRNA was successfully condensed into the polymers. All KL-PEI and unmodified L-PEI polyplexes prepared at the N/P ratio of 60 and above exhibited relatively constant positive zeta potentials of +16 to +22 mV. The polyplexes formed by both KL-LMW PEI and KL-HMW PEI did not show a noticeable difference in surface charges compared to those of the polyplexes formed by unmodified L-LMW PEI and L-HMW PEI (p > 0.3). Polyplexes prepared by siRNA and B-PEI showed the highest cationic surface charge among the various polyplexes, obviously due to the highest cationic density of the B-PEI (35). Efficient Dissociation of siRNA from Hydrolyzed KL-PEI Polyplexes. The formation of various siRNA/PEI polyplexes and the dissociation of the siRNA from the polyplexes upon hydrolysis were demonstrated by agarose gel electrophoresis assay. As shown in Figure 3A and B, siRNA was partially retained by unmodified L-PEI at the N/P ratio up to 20 and completely retarded at the N/P ratios of 40 and above. However, for siRNA/KL-PEI polyplexes, the siRNA was completely retarded even at the N/P ratio of 10, indicating that KL-PEI could efficiently interact with negatively charged siRNA at lower N/P ratios than unmodified L-PEI (Figure 3C and E). This result is expected because of improved interactions of siRNA with amine-bearing branches, which can more closely access to siRNA than the linear cationic backbone of PEI. By the incorporation of ketal branches to linear PEI, secondary amines of linear PEI were altered to tertiary amines, and additional primary amines were generated from the ketal branches. Therefore, the newly added primary amines will make

Figure 4. TEM micrographs of (A) siRNA/L-LMW PEI polyplexes (N/P ) 40), (B) siRNA/L-HMW PEI PEI polyplexes (N/P ) 40), (C) siRNA/KL-LMW PEI polyplexes (N/P ) 100) before hydrolysis, (D) siRNA/KL-LMW PEI polyplexes (N/P ) 100) after hydrolysis, (E) siRNA/KL-HMW PEI polyplexes (N/P ) 100) before hydrolysis, and (F) siRNA/KL-HMW PEI polyplexes (N/P ) 100) after hydrolysis. Scale bar ) 100 nm.

linear PEI more protonatable and can interact more closely with siRNA than unmodified linear PEI. Various siRNA/KL-PEI polyplexes were treated with pH 5.0 acidic buffer and electrophoresed to demonstrate the disassembly of siRNA from polyplexes upon hydrolysis of ketal branches, which simulates the release of siRNA in the acidic endosome. It was clearly confirmed that the hydrolysis of ketal branches in KL-PEI at mild acidic pH 5.0 facilitated siRNA release from the polyplexes even at high N/P ratios (e.g., 100 and 120) as shown in Figure 3D and F. No detectable degradation of naked siRNA incubated at pH 5.0 for 4 h at 37 °C was observed (data not shown). The results obtained from the gel electrophoresis assay indicate that ketalization of L-PEI did not alter siRNA condensation but facilitated siRNA release from the polyplexes upon hydrolysis in mild acidic environments. Morphological Changes of siRNA/KL-PEI Polyplexes upon Hydrolysis. Transmission electron microscopy images were acquired to observe the changes of sizes and the morphologies of various siRNA/PEI polyplexes before and after hydrolysis (Figure 4). All of the unmodified L-PEI and KLPEI formed spherical and compact siRNA-containing polyplexes with a diameter of 40-100 nm (Figure 4A, B, C, and E). In addition, no significant size difference among the polyplexes formed by L-LMW PEI, L-HMW PEI, and KL-LMW PEI was observed (p > 0.8). However, the size of siRNA/KL-HMW PEI polyplexes formed at the N/P ratio of 100 was slightly larger that of siRNA/unmodified L-PEI polyplexes (Figure 4E). The larger size of siRNA/KL-HMW PEI polyplexes compared to those of other polyplexes was consistent with the results obtained from DLS analysis (Figure 2A). Again, taken together with the results from ethidium bromide exclusion assay, these results imply that the siRNA complexation ability of KL-HMW PEI was lower than the ones of unmodified L-PEI and KL-LMW PEI, possibly because of steric hindrance from ketal branches. Importantly, the TEM images showed swollen and disrupted siRNA/KL-PEI polyplexes after hydrolysis (Figure 4D and F), indicating that hydrolysis of ketal branches of KL-PEI at the endosomal pH of 5.0 may be able to destabilize the polyplexes and therefore facilitate the disassembly of siRNA from the destabilized polyplexes.


Shim and Kwon

Figure 5. Laser scanning confocal micrographs illustrating the intracellular localization of various siRNA/L-PEI polyplexes in NIH 3T3 cells. (A) siRNA/L-LMW PEI polyplexes (N/P ) 30), (B) siRNA/LHMW PEI polyplexes (N/P ) 30), (C) siRNA/KL-LMW PEI polyplexes (N/P ) 100), and (D) siRNA/KL-HMW PEI polyplexes (N/P ) 100). The upper panel depicts merged images of Alexa Fluor 488stained polyplexes (green) and DRAQ5-counter-stained nuclei of the cells (blue). The lower panel depicts overlaid images of the upper panel and differential interference contrast (DIC) images of the cells. Images cutting horizontally through approximately the middle of the cell were selected out of a z-stack of images to differentiate the polyplexes in the perinuclear area from ones in the nucleus. Scale bar ) 25 µm.

Intracellular Localization of Polyplexes. The intracellular localization of siRNA is an important factor for successful RNA interference. To investigate the intracellular localization of various siRNA/PEI polyplexes after cellular uptake, NIH 3T3 cells were incubated with various siRNA/PEI polyplexes and observed by confocal laser scanning microscopy. Various ketalized and unmodified L-PEI were used to complex unlabeled siRNA or Cy3-labeled siRNA, and the resulting polyplexes were further labeled with Alexa Fluor 488 dye. It was quantified that 42% of primary amines of KL-PEI were conjugated with Alexa Fluor 488. Since 1 equivalent Alexa Fluor 488 was used to react with the 1 equivalent primary amines of the entire KL-PEI complexing siRNA, and Alexa Fluor 488 was conjugated after siRNA complexation, it is presumed that most primary amines on the surface of the polyplexes were conjugated with Alexa Fluor 488 (i.e., actual conjugation ratio of Alex Fluor 488 to primary amines on the surface of the polyplexes was significantly higher than 1). Since Alexa Fluor 488 conjugation to ketalized linear PEI with abundant primary amines and unmodified linear PEI with mainly secondary amines in the backbone is not identical, internalizations of fluorescently labeled and unlabeled polyplexes cannot be directly compared. Therefore, relative numbers of the polyplexes in the cytoplasm to the ones in the nucleus were considered to explore intracellular localizations. Localization of the various polyplexes was observed in the middle focal plane, which traverses both the cytoplasm and the nucleus of a cell (Figure 5). As shown in Figure 5A and B, a large number of the polyplexes prepared by using unmodified L-PEI were found inside cells regardless of their molecular weights, and these polyplexes were localized not only in the cytoplasm but also in the nucleus. This observation was in agreement with a previous study reporting that L-PEI delivers nucleic acids to the nucleus (36, 37). On the contrary, siRNA/ KL-LMW PEI and siRNA/KL-HMW PEI polyplexes (and possibly free Alexa Fluor dye released by acid hydrolysis) were exclusively localized in the cytoplasm where siRNA mediates its function (Figure 5C and D), demonstrating that ketalization of L-PEI greatly redirects the intracellular distributions of the polyplexes to the cytoplasm. Hydrolysis of siRNA/KL-PEI polyplexes is hypothesized to make the polyplexes become larger and destabilized as the hydrolyzed KL-PEI has neutrally charged hydroxyl side chains, which hinder tight complexation with the siRNA. Therefore, cytoplasmic localization of the

Figure 6. Laser scanning confocal micrographs illustrating the intracellular colocalization of Cy3 labeled-siRNA (red) and Alexa fluor 488 (green)-labeled linear PEI in NIH 3T3 cells. (A) siRNA/L-LMW PEI polyplexes (N/P ) 30), (B) siRNA/L-HMW PEI polyplexes (N/P ) 30), (C) siRNA/KL-LMW PEI polyplexes (N/P ) 100), and (D) siRNA/ KL-HMW PEI polyplexes (N/P ) 100). The left upper panel depicts images of Alexa Fluor 488-labeled linear PEI (i), and the right upper panel depicts images of Cy3-labeled siRNA (ii). The left lower panel depicts merged images of Alexa Fluor 488-stained polyplexes and Cy3labeled siRNA with nucleus staining (blue) (iii). The right lower panel depicts overlaid images of image (iii) and DIC image of the cells. Scale bar ) 10 µm.

siRNA/KL-PEI polyplexes might be ascribed to their increased size and loose siRNA complexation in the cytoplasm, which prevent transport through nuclear pores. Localization of Alexa fluor 488-labeled linear PEI (green, i) and Cy3-labeled siRNA (red, ii) was investigated, and the colocalized polymer and siRNA were represented as yellow dots (iii) in Figure 6. When the siRNA/unmodified linear PEI polyplexes were incubated with cells, siRNA and unmodified linear PEI were clearly colocalized both in the cytoplasm and the nucleus as shown in Figure 6A and B. On the contrary, when siRNA/KL-PEI polyplexes were incubated with the cells, both siRNA and the KL-PEI were mainly colocalized in the cytoplasm instead of entering the nucleus as shown in Figure 6C and D. More importantly, there were significantly more red spots, which represent stably labeled siRNA alone, in the cell incubated with siRNA/KL-PEI polyplexes than the ones incubated with siRNA/unmodified L-PEI polyplexes [Figure 6C (iii) and D (iii)] after 4 h of incubation in the cells. In comparison, siRNA/unmodified linear PEI polyplexes resulted in exclusive colocalization of siRNA and unmodified PEI [(Figure 6A (iii) and B (iii)]. Quantitative estimation of the amount of free Alexa Fluor 488 dye released from the ketalized linear PEI upon hydrolysis showed that 70% of the conjugated Alexa Fluor 488 dye was released during hydrolysis. This indicates that some yellow spots in the cells incubated with siRNA/KL-PEI polyplexes probably represent colocalized siRNA and free Alexa Fluor 488. Again, attention should be given to the fact that there were more red spots (i.e., free siRNA alone) in the cells incubated with KL-PEI polyplexes than the ones incubated with unketalized L-PEI polyplexes. This finding clearly demonstrates efficient intracellular disassembly of siRNA from ketalized linear



Figure 7. eGFP gene silencing efficiency of various siRNA/PEI polyplexes at various N/P ratios. eGFP knockdown in NIH 3T3 cells by various (A) siRNA/PEI polyplexes in the absence of serum, (B) siRNA/PEI polyplexes in the presence of serum, and (C) siRNA/PEI polyplexes synthesized with control (nonspecific) siRNA in the presence of serum. Specific eGFP silencing efficiency (D) was determined by subtracting nonspecific gene silencing efficiency of siRNA/PEI polyplexes synthesized with control siRNA from the gene silencing efficiency of siRNA/PEI polyplexes synthesized with anti-eGFP siRNA in the presence of serum. Notice that there was an insufficient number of live cells to analyze for gene silencing after being incubated with siRNA/B-PEI at N/P ratio of 40 and above due to severe cytotoxicity.

PEI polyplexes in the cytoplasm and efficient subsequent processes toward RNA interference. Efficient Gene Silencing with Low Cytotoxicity by siRNA/KL-PEI Polyplexes. RNAi efficiency of various siRNA/ PEI polyplexes at various N/P ratios was evaluated by quantifying the reduced expression of eGFP in NIH 3T3 cells. As shown in Figure 7A, to some extent, KL-PEI-mediated RNAi efficiency tended to increase with N/P ratios. This might be due to increased endosomal escape caused by higher numbers of amines (17). siRNA/B-PEI polyplexes showed the highest gene silencing efficiency among the various polyplexes in the absence of serum as shown in Figure 7A. Both unmodified L-HMW PEI and L-LMW PEI showed minimal RNAi efficiency at the entire N/P ratios in contrast to their superior DNA transfection efficiency as previously reported (22-26). In contrast, ketalization of L-PEI remarkably enhanced the RNAi efficiency (p , 0.001), and the highest gene silencing efficiencies of KLHMW PEI and KL-LMW PEI were observed at N/P ratios of 100 and 120, respectively. A confocal laser scanning microscopy study showed that siRNA and unmodified L-PEI were exclusively colocalized in both the cytoplasm and the nucleus, although a large number of siRNA/unmodified L-PEI polyplexes were localized in the cytoplasm (Figure 6A and B). This finding indicates limited dissociation of siRNA from the polyplexes in the cytoplasm possibly due to highly stable siRNA complexation by L-PEI, resulting in limited RNAi activity (Figures 2B and 7A). However, acid-degradable KL-PEI was able to facilitate efficient polyplex unpackaging in the cytoplasm and subsequent siRNA release (Figure 6C and D), suggesting that the improve-

ment of RNAi efficiency mediated by KL-PEI compared to that of unmodified L-PEI might be ascribed to more efficient endosomal escape and unpackaging of siRNA/KL-PEI polyplexes in the cytoplasm as well as its targeted cytosolic delivery of siRNA. As shown in Figure 7A, both KL-LMW PEI and KL-HMW PEI resulted in still slightly lower gene silencing efficiencies than B-PEI in the absence of serum. However, the gene silencing efficiency of siRNA/B-PEI polyplexes dramatically decreased in the presence of serum (p < 0.005), which might be attributed to an undesirable interaction of the polyplexes with negatively charged serum proteins (38). For example, the gene silencing by siRNA/B-PEI polyplexes reduced from 85.7% in the absence of serum to 65.3% in the presence of 10% serum at the N/P ratio of 20. Regardless of molecular weights, unmodified linear PEI did not show significant decrease in RNAi efficiency in the presence of serum (p > 0.3), as previously reported (39). Similarly, high gene silencing efficiencies of siRNA/KL-HMW PEI and siRNA/KLLMW PEI polyplexes prepared at the N/P ratio of 80 to 140 remained without significant changes (Figure 7A and B). This result was also clearly presented by confocal laser scanning micrographs (Figure 8) as well as a flow cytometry histogram (Supporting Information, Figure S2). The effects of various serum concentrations on gene silencing efficiency by ketalized PEI were also investigated (Supporting Information, Figure S3). Results showed that KL-PEI, regardless of molecular weights, was able to silence eGFP expression more efficiently than unketalized branched and linear PEI in the presence of up to 50% serum as shown in Figure S3B (Supporting Information)


Shim and Kwon

Figure 8. Confocal micrographs of eGFP-expressing NIH 3T3 cells (A) untreated used as a control and incubated with (B) siRNA/L-LMW PEI polyplexes (N/P ) 60), (C) siRNA/KL-LMW PEI polyplexes (N/P ) 120), and (D) siRNA/B-PEI polyplexes (N/P ) 10), in the presence of 10% serum. The upper panel depicts fluorescent images of eGFP-expressing NIH 3T3 cells. The lower panel depicts differential interference contrast (DIC) images of the cells. Scale bar ) 20 µm.

Figure 9. Viability of NIH 3T3 cells incubated with siRNA/PEI polyplexes at different N/P ratios (A) in the absence of serum and (B) in the presence of serum (10%). NIH 3T3 cells were incubated with polyplexes in serum-free or serum-containing medium for 4 h followed by 24 h of incubation with fresh medium supplemented with 10% FBS.

(p < 0.003), although KL-PEI showed slightly less efficient siRNA condensation capability than unketalized branched and linear PEI (Figure 2B). In order to exclude nonspecifically reduced gene expression by the polyplexes in gene silencing quantification, the eGFP expressing NIH 3T3 cells were also incubated with the polyplexes prepared by nonsequence-specific (control) siRNA and PEI. For the polyplexes prepared by using unmodified and ketalized linear PEI, nonspecific eGFP knockdown was found to be very minimal (e.g., less than 8% of gene silencing) at low N/P ratios (e.g., N/P e 60), although nonspecific effects slightly increased with N/P ratios (Figure 7C). However, siRNA/ B-PEI polyplexes showed significant nonspecific gene silencing in a N/P ratio-dependent way (p , 0.001) (Figure 7C). In other words, specific gene silencing by siRNA/PEI polyplexes must be considered after subtracting nonspecific effects (Figure 7D). Specific eGFP silencing by siRNA/KL-LMW PEI and siRNA/ KL-HMW PEI polyplexes in the presence of serum was much higher than that of siRNA/B-PEI polyplexes. The maximum specific eGFP silencing by siRNA/KL-LMW PEI polyplexes at the N/P ratio of 100 was around 63%, which was about 2-fold higher than specific eGFP silencing efficiency by siRNA/BPEI polyplexes at the N/P ratio of 10. Cytotoxicity of siRNA/KL-PEI polyplexes was slightly lower than that of siRNA/unketalized L-PEI polyplexes (p > 0.02) but significantly lower than that of siRNA/B-PEI polyplexes as shown in Figure 9 (p , 0.001). Cells incubated with the siRNA/KL-PEI polyplexes remained more than 70% viable at

the N/P ratios up to 80 whether they were incubated with serum or not. On the contrary, siRNA/B-PEI polyplexes showed significant cytotoxicity even at the N/P ratio of 20, regardless of the presence of serum (p , 0.001). This high cytotoxicity seems to contribute to nonspecific gene silencing by siRNA/ B-PEI polyplexes (Figure 7C). Unmodified linear PEI showed relatively low cytotoxicity compared to B-PEI as previously reported (27, 36). It is well-known that high cytotoxicity of B-PEI is correlated with its high charge density (40). Since zeta potentials of siRNA/L-PEI and siRNA/KL-PEI polyplexes were a lot lower than that of B-PEI (p , 0.001), this low cationic charge density of the polyplexes probably contributed to high cell viability even at high N/P ratios. It seems that low cytotoxicity of KL-PEI is not only a consequence of the inherently low cytotoxicity of L-PEI but also the reduced interactions of hydrolyzed KL-PEI, which has neutral hydroxyl overhangs, with endogenous genes as previously discussed (28, 41). The reason for similar characteristics of KL-PEI to ones of unmodified L-PEI with regard to serum-independent gene transfer and low cytotoxicity might be due to a low degree of ketalization (i.e., 22 and 24%). Although linear PEI was transformed to a branched form to a certain extent by ketalization, its advantageous properties (e.g., low cytotoxicity and serum-independence) seem to be preserved. One of the practical challenges for in vivo gene delivery mediated by cationic nonviral vectors is inhibition of gene delivery efficiency by serum and high cytotoxicity (42). Although branched PEI, one of the efficient cationic nonviral gene delivery carriers, has been



Figure 10. Effects of ketalization of L-PEI on eGFP gene silencing efficiency and cytotoxicity. eGFP knockdown in NIH 3T3 cells mediated by various (A) siRNA/PEI polyplexes in the presence of serum and (B) siRNA/PEI polyplexes synthesized with control (nonspecific) siRNA in the presence of serum, and (C) specific eGFP silencing efficiency of various siRNA/PEI polyplexes in the presence of serum. (D) Cytotoxicity of acid-degradable KL-LMW PEI and nondegradable modified L-LMW PEI (ND-L-LMW PEI) at various N/P ratios. eGFP expressing NIH 3T3 cells were incubated with various siRNA-containing polyplexes in the 10% FBS supplemented medium. After 4 h of incubation, the medium was replaced with fresh DMEM containing 10% FBS, and the cells were further cultured for 72 h.

popularly used for years due to its high transfection efficiency in vitro (15), its in vivo application has been limited because of its serum-dependent gene transfection efficiency and high cytotoxicity (25, 36, 38, 43). In contrast to branched PEI, KLPEI showed serum-independent high RNAi efficiency and low cytotoxicity. Moreover, KL-PEI showed higher specific eGFP silencing efficiency than B-PEI (Figure 7D). Our previous study showed that the transfection efficiency of ketalized low molecular weight branched PEI was also influenced by the presence of serum to some extent such as unmodified 25 kDa branched PEI, although ketalized low molecular weight PEI showed enhanced transfection efficiency and significantly reduced cytotoxicity (28). Therefore, serum-independent and nontoxic siRNA delivery by KL-PEI in this study implies that the critical drawbacks of PEI-mediated gene delivery for clinical trials can be ameliorated. Enhanced Gene Silencing by Acid-Degradable Ketal Branches. Contribution of the acid-degradability of KL-PEI to the improved RNA interference was evaluated by comparing RNAi efficiency of modified L-LMW PEI with nondegradable amine-bearing branches. This nondegradable counterpart was prepared by conjugating L-LMW PEI with nondegradable N-(4(2,2,2-trifluoroacetamido)butyl)acrylamide followed by deprotecting the trifluoroacetate group. The synthetic scheme, detailed synthesis procedures, and characterization are described in Supporting Information (Figure S4). As shown in Figure 10A, the eGFP knockdown by siRNA/KL-LMW PEI polyplexes in NIH 3T3 cells was much higher than the one by nondegradable

L-LMW PEI (ND-L-LMW PEI) at all observed N/P ratios (p < 0.004). Interestingly, siRNA/ND-L-LMW PEI polyplexes showed a similar level of RNAi efficiency in comparison to that of siRNA/unmodified L-LMW PEI polyplexes. This result clearly demonstrates that the acid degradability of KL-LMW PEI remarkably contributed to the efficient RNAi by siRNA/ KL-LMW PEI polyplexes. Moreover, siRNA/ND-L-LMW PEI polyplexes showed some nonspecific gene silencing at high N/P ratios (e.g., 80-140) (nonspecific gene silencing degree: g 10%), although it was not significantly different from the one by siRNA/KL-LMW PEI polyplexes (e.g., less than 10% of nonspecific gene silencing) (p > 0.08) (Figure 10B). The cytotoxicity result shown in Figure 10D indicates no significant difference in cell viability between degradable and nondegradable polyplexes (p > 0.34). These results imply that ketalization of L-PEI indeed increased gene silencing, while keeping the minimal nonspecific RNA interference and cytotoxicity of L-PEI.

CONCLUSIONS In this study, pH-sensitive ketalized linear PEI was synthesized for siRNA delivery. The conjugation of acid-degradable amino ketal branches resulted in enhanced cytoplasmic release of free siRNA, which could be a crucial part of the reason for the achieved efficient RNA interference. The ketalization preserved the advantages of linear PEI including low cytotoxicity, serum-independent gene delivery, and sequence-specific


gene silencing. It appeared that KL-PEI could be a promising candidate for a nonviral siRNA delivery carrier in vivo.

ACKNOWLEDGMENT We thank the staff at the Optical Biology Core (OBC) facility in UC Irvine for assistance with the confocal laser scanning microscopy and Ms. Shirley Wong (UC Irvine) for proofreading the manuscript. The authors are also grateful to Dr. Jian-Guo Zheng for his assistance of TEM imaging and analysis at the Materials Characterization Center in UC Irvine. Supporting Information Available: Buffering capacity of KL-PEI, flow cytometry histogram of eGFP-exprssing cells incubated with various polyplexes, effects of various serum concentrations on RNA interference, and synthesis of nondegradable modified linear polyethylenimine (ND-L-PEI). This material is available free of charge via the Internet at http:// pubs.acs.org.

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