Bioconjugate Chem. 2009, 20, 2055–2061
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Hydrophobically Modified Oligoethylenimines as Highly Efficient Transfection Agents for siRNA Delivery Alexander Philipp,‡ Xiaobin Zhao,§ Peter Tarcha,§ Ernst Wagner,‡ and Arkadi Zintchenko*,†,‡ Center for Drug Research, Department of Pharmacy, Pharmaceutical Biology-Biotechnology, and Center for NanoScience (CeNS), Ludwig-Maximilians-University, Butenandstr. 5-13, D-81377 Munich, Germany, and Department of Advanced Drug Delivery, Abbott Laboratories, D-R43D, AP31-2, 200 Abbott Park Road, Abbott Park, North Chicago, Illinois 60064. Received April 7, 2009; Revised Manuscript Received August 19, 2009
RNA interference is a promising therapeutic strategy for treatment of diseases, in particular, cancer. Despite a huge number of targets identified for different cancer types, there are no effective delivery strategies available so far. Polymeric delivery vehicles are often based on large macromolecules. Such approaches often lead to accumulation of toxicity and narrow therapeutic windows. In the current paper, an alternative approach is presented. Low molecular weight oligoethylenimine (OEI) 800 Da was hydrophobically modified through the Michael addition of different alkyl acrylates. An optimal structure containing ten hexyl acrylate residues per one OEI chain (OEIHA-10) was found to be a promising candidate for siRNA delivery. Hydrophobic modification stabilized the siRNA polyplex structure, increased the colloidal stability of the nanoparticles, and provided lytic properties to OEI required for crossing cellular membranes in the delivery process. In addition, the acrylate ester bond enables fast degradation of OEI-HA-10 into far less toxic components. Further improvement of biological properties of the OEI-HA-10 polyplexes by different formulation strategies was demonstrated. In particular, a remarkable increase of biocompatibility without loss of efficiency could be achieved by coformulation of OEI-HA-10 with lauryl acrylate modified OEI-LA-5.
INTRODUCTION 1
Small interfering RNA (siRNA) is a promising tool for both basic and applied biology. The ability to knock down essentially any gene of interest has led to an increased interest in identifying important therapeutic genes and develop siRNA-based treatment. This is particularly interesting for cancer, where a large number of disease-related genes have been understood (1, 2). However, the successful application of siRNA-based therapy still represents a great challenge. The in vivo application of siRNAs, in reality, poses many hurdles, especially in humans. One of the main hurdles is the optimization of the delivery strategy, especially the carrier systems. Generally, the carrier must be effectively bound to siRNA, ensuring high stability against dissociation of nucleic acid in physiological fluids. It must provide effective transport of siRNA to the cytosol of the target cells and show low toxicity. Accumulation of the carrier in the body leads normally to accumulation of the toxicity (3), which narrows the therapeutic window and limits the dosing frequency of the therapeutics. Therefore, relatively fast biodegradation of the carrier is a desired property for the application of transfection vectors in vivo. Recently, several strategies for the development of polymeric carriers were introduced, which are mainly based on optimization of the conventional vectors for DNA delivery. Different polyethylenimines (PEIs), which are known to be powerful * Correspondence to Arkadi Zintchenko, tel +31 53 489 3272, fax +31 53 489 2155, e-mail
[email protected]. † Current address: University of Twente, Faculty of Science and Technology, Institute for BioMedical Technology (BMTI), Zuidhorst, NL-7500 AE Enschede, The Netherlands. ‡ Ludwig-Maximilians-University. § Abbott Laboratories. 1 Abbreviations: OEI 800, oligoethylenimine 800 Da; EA, ethyl acrylate; BA, butyl acrylate; HA, hexyl acrylate; LA, lauryl acrylate; EtBr, ethidium bromide; siRNA, short interfering RNA.
agents for DNA delivery, are far less effective for siRNA. Different approaches were utilized for the enhancement of their efficiency. In the case of linear PEI, the activity can be enhanced with “sticky” siRNA (ssiRNA) applied for polyplex formation (4). Acetylation of the amines of branched PEI or introduction of negatively charged carboxylic groups into structure remarkably increased the knockdown efficiency of the carrier (5). Large PEI macromolecules (>5 kDa) are generally nondegradable and cannot be cleared from the body. Thus, they are characterized by only limited potential for therapeutic applications. Low molecular weight PEIs are far less toxic, but also rather inefficient and require high excess of oligoamine in the formulation (6, 7). The polyplex stability of such formulations, especially in vivo, is a big concern. Covalent cross-linking of oligoamines by biodegradable linkers provides to the formulation the stability and efficiency of polyamines, and can be degraded in the body to excretable degradation products. Polyamidoamine approaches for cross-linking of oligoamines often lead to rather effective carriers (8, 9). However, slow degradation of amide bonds generally limits the therapeutic window of the formulation. Introduction of secondary break points into bifunctional diacrylate structures (i.e., disulfide) can provide much higher degradation rates (10). Degradable synthetic polypeptides (i.e., PLL) are rather inefficient in both DNA and siRNA delivery. Conjugation with endosomolytic agents enhanced efficiency of the carrier (11). “Dynamic polyconjugates” introduced by Rozema et al. (12) are based on biocompatible poly(vinyl alcohol) backbones modified by amine and hydrophobic alkyl groups. Such structures showed a pronounced lytic activity (13), which could be reversibly switched off by modification of amines with maleic anhydride-based reagent (14). These “dynamic polyconjugates” were rather nontoxic in vivo and showed good efficiency for delivery of siRNA to liver hepatocytes (12). Other hydrophobic pH-responsive polymer-based carriers were described by Convertine et al. (15). Copolymers
10.1021/bc9001536 CCC: $40.75 2009 American Chemical Society Published on Web 10/19/2009
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containing cationic binding block and pH-responsive hydrophobic block based on propylacrylic acid and butyl methacylate showed remarkable knockdown of the target genes with relatively low toxicity. In the current paper, an alternative approach is presented. Instead of cross-linking, OEI 800 was hydrophobically modified by Michael addition of alkyl acrylates. As expected, such structures formed polyplexes with siRNA and demonstrated increased stability against dissociation. Structures with enhanced endosomolytic properties (in particular, hexyl acrylate modified OEI) were efficient in siRNA delivery. Furthermore, coformulation with different agents could significantly improve the parameters of toxicity and efficiency of the carrier. The optimized formulation could represent a promising carrier for delivery to cancer cells.
EXPERIMENTAL PROCEDURES Materials. Ethyl, butyl, hexyl, and lauryl acrylate (EA, BA, HA, or LA respectively), were obtained from Sigma-Aldrich (Munich, Germany). The plasmid pEGFPLuc (Clontech Laboratories, Heidelberg, Germany) containing a CMV promoter driven fusion of the genes encoding for enhanced green fluorescent protein and luciferase was used for generation of stably transfected cells. Oligoethylenimine with an average molecular weight of 800 Da and all other chemicals were purchased by Sigma-Aldrich (Munich, Germany). Lipofectamine 2000 (LF2000), cell culture media, antibiotics, and fetal calf serum (FCS) were purchased from Invitrogen (Karlsruhe, Germany). Ready to use siRNA duplexes were purchased from Dharmacon (Lafayette, CO), namely, luciferase-siRNA: GL3 luciferase duplex: 5′-CUUACGCUGAGUACUUCGAdTdT-3′ (sense); control-siRNA: nontargeting control duplex: 5′-AUGUAUUGGCCUGUAUUAGUU-3′ (sense). Modification of OEI by Ethyl, Butyl, and Hexyl Acrylate (EA, BA, and HA). PEI (1 g) was dissolved in chloroform. The solution was heated to 40 °C. The desired amount of acrylate was added to the solution under stirring. The reaction proceeded at 40 °C. After 4 h, the chloroform was removed by rotor evaporator. The waxy product was dissolved in 15 mL water and neutralized by HCl to pH 4-5 and freeze-dried. The gravimetric yields were generally between 80% and 140%. Some yields above 100% were due to additional mass of chloride as a counterion in the lyophilized sample. The modification degree was analyzed by 1H NMR in D2O on a Jeol JNMR-GX400 (400 MHz) or a Jeol JNMR-GX500 (500 MHz) spectrometer from the ratio between the peaks of OEI (δ 2.4-3.3 ppm) and methyl of acrylates (δ ) 0.8 for HA, δ ) 0.9 for BA, and δ ) 1.3 for EA). The modification degree was expressed as the number of modifications per OEI molecule. According to FTIR spectra recorded on a Jasco FT/IR-410 spectrometer (Jasco Labor and Datentechnik GmbH, Germany), a pronounced ester peak (1730 cm-1) was detected. No significant amide band (1652 cm-1) was found. Modification of OEI by Lauryl Acrylate (LA). One gram of OEI was dissolved in chloroform and heated to 40 °C. After this, the calculated amount of lauryl acrylate (LA) was added dropwise to the OEI solution. The ratios of OEI to LA were 2.5, 5, and 7.5. The mixture was incubated for 4 h. The resulting solution was evaporated on a rotovap to 1/3 of the initial volume. An excess of diethyl ether was added resulting in a clear solution. After this, an excess of concentrated HCl (37% vol) was added, which resulted in precipitation of the product. The precipitate was filtered, washed with diethyl ether, frozen in liquid nitrogen, and dried under vacuum. The composition of the products was determined by NMR as a ratio between methyl protons of LA (0.8 ppm) and a broad peak of OEI (2.4-3.3 ppm). The peak at 4.1 ppm represents
Philipp et al.
the CH2-O proton of the ester bond. It was found to be stable over time and show good proportionality to the weights of all other peaks of LA (2:3 if compared to methyl). Preparation of Liposomes. Lipid mixtures were prepared as a film by chloroform evaporation method. Dioleoyl phosphatidylethanolamine (DOPE, Sigma-Aldrich, Germany), dioleoylphosphatidylcholine (DOPC, Sigma-Aldrich, Germany), and dipalmitoylphosphatidylcholine (DPPC, Sigma-Aldrich, Germany) were used. For the preparation of liposomes, about 10 mg of lipid (DOPE, DOPC, DPPC or mixtures) was dissolved in 0.1 mL of ethanol under mild heating (50 °C). The solution containing 0.5, 1.0, 2.0, or 5.0 mg of lipid was added to Eppendorf tubes and flash-mixed with 1 mL of OEI-HA-10 solution (1 mg/mL in water pH ∼3). The turbid suspension of nanoparticles was formed in all cases. Mixing of lipid (1 mg in 100 µL ethanol) with 1 mL of HBG buffer (without OEI-HA-10) resulted in precipitation of the lipid. The final suspensions were sonicated for 1-2 min resulting in nearly clear solutions in the case of DOPE/OEI-HA10 (weight ratios 0.5, 1, and 2) and slightly opalescent solution in the case of DOPE/OEI-HA10 with a weight ratio of 5. The size of the nanoparticles was analyzed using photon correlation (Zetasizer Nano ZS, Malvern Instruments, Herrenberg, Germany). All nanoparticles were around 80-110 nm in diameter. Preparation of Polymer Mixtures. For the preparation of OEI-HA-10/OEI mixtures, the amounts of OEI-HA-10 and OEI stock solutions were calculated according to the desired ratio in a final mixture (2/1, 1/1, or 1/2 w/w). The resulting solutions (1 mg/mL total OEI) were used as a stock for preparation of the polyplexes. Polyplex Formation. In all studies, the composition of polyplexes was characterized by the w/w ratio of the polymer to nucleic acid in the mixture. The weight of modified OEIs represents the weight of lyophilized product, which was previously neutralized and, therefore, includes the mass of counterions (chloride). Formulations for siRNA delivery were prepared in HBG (20 µL) as follows: First, different concentrations of the nucleic acid and oligoamine formulations were diluted at various polymer/nucleic acid ratios in separate tubes in HBG (HEPES buffered glucose solution; 20 mM HEPES, 5% glucose, pH 7). Then, the HBG solution of oligoamine was added to the HBG solution of the nucleic acid, mixed and incubated for 30-40 min at room temperature to form stable complexes. In the case of LF2000, formulations with siRNA were prepared according to the manufacturer’s standard protocol (Invitrogen). Agarose Gel Retardation Assay. Polyplexes were prepared as indicated in the corresponding experimental settings containing 500 ng siRNA in 10 µL. Then complexes were mixed with loading buffer (6 mL glycerine, 1.2 mL 0.5 M EDTA, 2.8 mL H2O, 0.02 g xylenecyanole) and placed into a 2.5% agarose gel in TBE buffer (trizma base 10.8 g, boric acid 5.5 g, disodium EDTA 0.75 g, and 1 L water) containing ethidium bromide (EtBr). Electrophoresis was performed at 80 V for 40 min and evaluated under UV-light. Ethidium Bromide Exclusion Assay. siRNA condensation ability was evaluated in HBG (20 mM HEPES, 5% glucose, pH 7) using an ethidium bromide exclusion assay. Aliquots of the respective polymer were added stepwise to a siRNA solution (10 µg/mL) in HBG containing 400 ng/mL EtBr, and the decrease of fluorescence was measured in a Cary Eclipse fluorescence spectrophotometer (Varian Deutschland GmbH, Darmstadt, Germany). EtBr/siRNA fluorescence (λex 510 nm and λem 590 nm) was set to 100% prior to addition of polycation, and results are given as relative fluorescence intensities to plain siRNA. Values are given as mean of triplicates ( SD.
Hydrophobically Modified Oligoethylenimines
Particle Size and Zeta-Potential Measurement. Particle size and zeta-potential of polyplexes were determined by laser light scattering using a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany). For measurement of zeta potentials, the polyplexes (prepared in HBG) were diluted with 1 mM NaCl to a final siRNA concentration of 10 µg/mL (total volume 1 mL). Values are given as mean of triplicates ( SD. Degradation of Hydrophobically Modified Polyamines. Degradation of conjugates was investigated in an aqueous solution (1 mL, D2O) at pH 7 at 37 °C. Resulting products were characterized by 1H NMR spectroscopy. Degradation results in formation of alcohol with a peak at δ 3.6 different from those of ester at δ 4.1 (Supporting Information Figure 1). The percentage of degraded ester bond was expressed as the ratio δ 3.6/(δ 3.6 + δ 4.1). Cell Culture. All cultured cells were grown at 37 °C in 5% CO2 humidified atmosphere. For siRNA delivery experiments, murine neuroblastoma cells Neuro2A/EGFPLuc, human hepatoma cells HUH7/EGFPLuc stably transfected with the EGFPLuc gene, or human lung carcinoma cells H1299/Luc stably transfected with the luciferase gene (kindly provided by Abbott Laboratories, Chicago) were used. Neuro2A/EGFPLuc cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, 1 g/L glucose), HUH7/EGFPLuc were grown in DMEM/Ham’s F-12 medium, and H1299/Luc cells were grown in RPMI 1640 medium (4.5 g/L glucose), whereby all media were supplemented with 10% FCS, 4 mM stable glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. All cells were seeded 24 h prior to transfection using 5000 cells per well in 96-well plates for analysis of protein expression. For the generation of stably transfected cells, the same protocol was used as previously reported (5). Cytotoxicity of Polymers. Neuro2A/EGFPLuc cells (5000/ well) were seeded into a 96-well plate in 100 µL medium. After 24 h, medium was replaced with 100 µL/well of serial dilutions of polymer stock solutions in serum containing (10% FCS) growth medium. After incubation at 37 °C for 48 h without medium change, the cell viability was analyzed using a MTTassay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) (Sigma-Aldrich, Germany) was dissolved in phosphate buffered saline at 5 mg/mL, and 10 µL aliquots were added to each well reaching a final concentration of 0.5 mg MTT/mL. After incubation for 1 h, unreacted dye with medium was removed. The purple formazan product was dissolved in 100 µL/well dimethyl sulfoxide and quantified by a plate reader (Tecan Spectrafluor Plus, Gro¨dig, Austria) at 590 nm with background correction at 630 nm. The relative metabolic activity (%) related to untreated control cells was calculated by [A] test/ [A] control × 100. Values are given as mean of triplicates ( SD. Erythrocyte Lysis Assay. Freshly collected citrate buffered murine blood was washed several times with PBS, and erythrocytes were resuspended in HBG/10% FCS at a concentration of 4% (v/v). 75 µL of a polymer solution was mixed with 75 µL erythrocyte suspension in a 96-well plate (NUNC, V-bottom, Denmark). After incubation for 45 min at 37 °C under constant shaking, blood cells were removed by centrifugation and 80 µL of the supernatant was transferred to a new 96-well plate. Hemoglobin absorption was determined at 405 nm using a microplate reader (Tecan Spectrafluor Plus, Gro¨dig, Austria). HBG/10% FCS and 1% Triton X-100 solution in PBS were used as negative and positive controls, respectively. Hemolysis is defined as percentage (ODpolymer - ODbuffer) × 100/(ODTritonX100 - ODbuffer). Values are given as mean of triplicates ( SD. In Vitro Gene Silencing with siRNA. In vitro gene silencing experiments were performed in stably transfected Neuro2A/ EGFPLuc, HUH7/EGFPLuc, and H1299/Luc cells using Luc-
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Figure 1. Structure of hydrophobically modified OEIs, which mediate steric stabilization of the polyplexes and cause cell membrane destabilization.
siRNA. siCONTROL was used as an unspecific control siRNA. In all experiments, siRNA delivery was performed in 96-well plates with 5000 cells per well in triplicates. Cells were seeded 24 h prior to transfection, and then, medium was replaced with 80 µL fresh growth medium containing 10% FCS or 50% FCS where indicated. Transfection complexes for siRNA delivery (20 µL in HBG) at different w/w ratios were added to each well and incubated at 37 °C without medium change for 48 h. In case of the 50% FCS experiments and H1299/Luc cells, a medium change was performed after 24 h with fresh serum containing (10% FCS) growth medium. Cells were washed with phosphate-buffered saline (PBS) and treated with 50 µL cell lysis buffer (25 mM Tris, pH 7.8, 2 mM EDTA, 2 mM DTT, 10% glycerol, 1% Triton X-100) 48 h following siRNA transfection. Luciferase activity in cell lysate (20 µL) was measured using a luciferase assay kit (100 µL luciferase assay buffer, Promega, Mannheim, Germany) on a luminometer for 10 s (Lumat LB9507 instrument, Berthold, Bad Wildbad, Germany). The relative light units (RLU) were related to untreated control cells. Statistical Analysis. Results are presented as mean ( s.d., and statistical significance of differences was evaluated by variance analysis (one-way ANOVA): p values smaller than 0.05 were considered to be significant, * p < 0.05.
RESULTS Synthesis of the Polymers. All polymer products were synthesized by Michael addition of acrylate to the amines of OEI at 40 °C for 4 h (Figure 1). Higher temperature or longer reaction times are known to cause the aminolysis of the pendant ester bond resulting in cross-linking of the OEI units. Isolation was performed either by evaporation of residual acrylate at high vacuum (
