Physicochemical and Biological Evaluation of siRNA Polyplexes Based on PEGylated Poly(amido amine)s
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Use of RNA interference as novel therapeutic strategy is hampered by inefficient delivery of its mediator, siRNA, to target cells. Cationic polymers have been thoroughly investigated for this purpose but often display unfavorable characteristics for systemic administration, such as interactions with serum and/or toxicity.
We report the synthesis of a new PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone. Various amounts of PEGylated polymers were mixed with their unPEGylated counterparts prior to polyplex formation to alter PEG content in the final complex.
PEGylation effectively decreased polyplex surface charge, salt- or serum-induced aggregation and interaction with erythrocytes. Increasing amount of PEG in formulation also reduced its stability against heparin displacement, cellular uptake and subsequent silencing efficiency. Yet, for polyplexes with high PEG content, significant gene silencing efficacy was found, which was combined with almost no toxicity.
PEGylated poly(amido amine)s are promising carriers for systemic siRNA delivery in vivo.
KEY WORDSbiodegradable delivery PEGylation poly(amido amine)s siRNA
RNA interference (RNAi) using small interfering RNAs (siRNAs) has emerged as a powerful tool for knockdown of genes and holds great potential as a novel therapeutic strategy for a broad range of diseases (1,2). The primary limitation to clinical success, however, is the lack of efficient delivery systems that can deliver siRNA to the target cell population. siRNAs do not readily pass cellular membranes due to their negative charge and are rapidly degraded by serum nucleases.
Cationic polymers have widely been investigated for siRNA delivery due to their great flexibility, ease of manufacturing and modification possibilities (3). Typical examples include poly(ethyleneimine) (pEI) (4), poly(L-lysine) (pLL) (5), chitosan (6) and poly(2-(dimethyl-amino)ethylmethacrylate) (pDMAEMA) (7). Although significant progress has been made in improving these polymers for siRNA delivery, either low efficiency and/or high cytotoxicity remain to hinder their usefulness. Recently, we have described a new class of biodegradable polymers based on poly(amido amine)s with disulfide linkages in the backbone (SS-PAA) that were specifically tailored for delivery of siRNA (8,9). These copolymers were composed of N,N′-cystaminebisacrylamide (CBA), 4-amino-1-butanol (ABOL) and ethylene diamine (EDA) (p(CBA-ABOL/EDA)) and were able to complex siRNA into positively charged polyplexes that were efficiently taken up by cells and induced target gene silencing. Moreover, this was combined with low cellular toxicity, which encouraged us to perform further functional studies.
For cancer therapy, in order to reach distant tumors or metastases, systemic administration of siRNA polyplexes is inevitable. Upon intravenous injection however, positively charged polyplexes might potentially interact aspecifically with serum proteins or erythrocytes and other blood cells, leading to formation of aggregates, which causes rapid clearance by the reticulo-endothelial systems (RES) and sometimes significant toxicity (10). The biocompatibility of polyplexes can be enhanced by conjugation of poly(ethylene glycol) (PEG) to the cationic polymer (PEGylation). Complexation of oligonucleotides with PEG-containing copolymers leads to the formation of particles with a core-shell structure, in which the cationic polymer packs the oligonucleotide within the particle core and the hydrophilic, non-charged PEG chains form a shell layer around it (11). In general, PEGylation of polyplexes results in a lower surface charge, reduced interaction with blood components, prolonged blood circulation and lower cytotoxicity (12,13).On the other hand, steric shielding of the polyplex particles also leads to reduced cellular association and uptake, diminished endosomal escape properties, and inefficient siRNA release (14,15). These contrasting effects associated with the use of PEG in oligonucleotide delivery are also referred to as the ‘PEG dilemma’ (16), and raise the need for strategies to fine-tune the PEG content in polyplexes to optimize their physicochemical and biological properties. Recently, Brumbach et al. demonstrated the feasibility of altering and optimizing PEG content in polyplexes by formulating mixtures of a polycation and its corresponding PEGylated counterpart before complex formation, avoiding the need to synthesize multiple copolymers with varying degrees of PEGylation to identify optimal carrier candidates (17).
In this study we synthesized a new PEGylated polymer, p(CBA-ABOL/EDA/PEG), based on the successful p(CBA-ABOL/EDA) polymer and used mixtures of the PEGylated and unPEGylated polymer in order to vary the PEG content in the final polyplex. Polyplexes with different PEG content were compared to corresponding unPEGylated complexes regarding physicochemical characteristics, stability, cellular uptake, gene silencing activity and in vitro biocompatibility.
MATERIALS AND METHODS
All chemicals, 4-amino-1-butanol (ABOL, Merck), ethylene diamine (EDA, Sigma-Aldrich), N,N′-cystaminebisacrylamide (CBA, Polysciences) and α-amino-ω-hydroxy poly(ethylene glycol) (PEG-NH2, 3000 g/mol, Iris Biotech GmbH) were purchased in the highest purity and used without further purification.
siRNAs were chemically synthesized and supplied by Eurogentec (Maastricht, The Netherlands). Sequence of siRNA against luciferase (siLuc) was 5′-GAU-UAU-GUC-CGG-UUA-UGU-AUU-3′ (sense) and 5′-UAC-AUA-ACC-GGA-CAU-AAU-CUU-3′ (antisense). For cellular uptake studies, Alexa-488-modified siRNA was used (dye was attached to the 5′-end of the sense strand). Universal negative control siRNA (siNS) was purchased from Eurogentec.
Polymer Synthesis and Characterization
Synthesis of p(CBA-ABOL/EDA) was performed as previously described (9). p(CBA-ABOL/EDA/PEG) was synthesized by Michael addition polymerization of ABOL, EDA and PEG-NH2 with N,N′-cystaminebisacrylamide (CBA). Therefore, 424 mg (1.63 mmol) CBA, 98 mg (1.10 mmol) ABOL and 500 mg (0.17 mmol) PEG-NH2 were dissolved in methanol/water (4:1 v/v) and were allowed to react at 45°C in the dark in a nitrogen atmosphere. The reaction mixture became homogeneous within 1 h. After 6 days of prepolymerization, 22 mg (0.37 mmol) EDA was added and the reaction was proceeded for another 2 days. Then the polymerization was terminated by addition of a 10% molar excess EDA, to consume remaining toxic acrylamide endgroups. After termination, the reaction mixture was diluted with hydrochloric acid (1 M) and water, purified by ultrafiltration (MWCO 5000, pH 5) and recovered as its HCl salt by lyophilization. 1H NMR (D2O) δ (ppm) = 1.58 (m, 2H, CH 2 CH2NR); 1.79 (m, 2H, CH 2 CH2OH); 2.69 (t, 4H, NHCH 2 CH 2 NH); 2.77 (t, 2H, CH 2CONH); 2.83 (t, 4H, CH 2 SSCH 2 ); 3.22 (t, 2H, CH2 CH 2 NR); 3.32 (t, 2H, COCH2 CH 2 NH); 3.44 (t, 4H, CH 2 NRCH 2 ); 3.51 (t, 4H, NHCH 2 CH2SSCH2 CH 2 NH); 3.60 (t, 2H, CH 2 OH); 3.65–3.90 (m, 272H, OCH 2 CH 2 O). Polymers were characterized by 1H NMR (D2O), recorded on a Varian Innova spectrometer (300 MHz). Molecular weights were determined by GPC relative to PEG standards, using a GPCmax with an acetate buffer pH 4.5 containing 30% (v/v) methanol as eluent.
To prepare polyplexes at different polymer/siRNA (w/w) ratios, appropriate amounts of polymer and siRNA were each diluted in glucose-containing Hepes buffer (HBG: 20 mM Hepes, pH 7.4, 5 wt.% glucose). Next, polymer solution was added to siRNA solution (4:1, v/v), followed by 5 s vortexing and 30 min incubation at room temperature. To obtain polyplexes with different PEG content, expressed as the weight percentage of PEG to total polymer, polymer solutions of p(CBA-ABOL/EDA) and p(CBA-ABOL/EDA/PEG) were mixed prior to polyplex formation.
For gel retardation assays, polyplexes were prepared in 50 μl HBG at a final siRNA concentration of 2.5 μM and incubated for 1 h at 37°C in the presence or absence of 5 mM glutathione. After addition of loading dye (Fermentas, St. Leon-Rot, Germany), samples were run on a 4% agarose gel containing 0.5 μg/ml ethidium bromide at 90 V for 10 min. Hydrodynamic diameters and ζ-potentials were measured as previously described (8).
Complex Aggregation in Salt
Polyplexes were prepared in 400 μl HBG at a final siRNA concentration of 500 nM. Then, 2 ml PBS was added and hydrodynamic diameters were measured at indicated time points as described above.
Complex Aggregation in Serum
Polyplexes containing were prepared in 50 μl HBG at a final siRNA concentration of 15 μM. 180 μl fetal bovine serum (FBS) was added to 20 μl polyplex solution and samples were incubated for 10 min at 37°C. Aggregation in terms of turbidity increase of each sample was quantified by absorbance detection at 595 nm.
Stability of Polyplexes Against Heparin
Polyplexes were prepared in 50 μl HBG at a final siRNA concentration of 2.5 μM and incubated for 30 min with heparin solutions of different concentrations, expressed as heparin/siRNA (w/w) ratio. After addition of loading dye, samples were run on a 4% agarose gel containing 0.5 μg/ml ethidium bromide at 90 V for 10 min.
Erythrocyte Aggregation and Hemolysis
Erythrocytes were obtained from 200 μl whole murine blood by multiple centrifugation rounds (1000 g, 10 min, 4°C) followed by washing the pellet in PBS until the supernatant was clear. The final pellet was resuspended in 4 ml PBS. Subsequently, 160 μl erythrocyte suspension was added to 40 μl polyplex solution having a siRNA concentration of 4 μM and samples were incubated for 1 h at 37°C. Triton X-100 (1%) in PBS (100% lysis) and HBG (0% lysis) were used as controls. After a final centrifugation step, 150 μl of the supernatant was analyzed for hemoglobin release by absorbance detection at 550 nm. The pellet was resuspended in 50 μl PBS for microscopic evaluation using a Nikon TE2000-U microscope.
H1299 (human lung cancer) cells stably expressing firefly luciferase were cultivated at 37°C and 5% CO2 in RPMI 1640 (PAA laboratories GmbH, Pasching, Austria) supplemented with 10% (v/v) FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B.
Quantification of siRNA uptake was performed as previously described, with minor modifications (18). H1299 cells were seeded in 6-well plates at a density of 1.6 × 105 cells per well, 24 h before transfection. Cells were treated with 400 μl of different polyplexes containing Alexa-488 labeled siRNA at a final siRNA concentration of 125 nM in FBS-containing medium. After 4 h, cells were washed twice with PBS to remove non-internalized polyplexes. 300 μl lysis buffer (2% SDS, 1% Triton X-100 in PBS) was added to lyse the cells and dissociate polyplexes. Cells were lysed for 1 h on ice, after which the lysates were centrifuged (14000 g, 15 min, 4°C) to remove cell debris. 200 μl supernatant was transferred to a black 96-well plate to measure fluorescence using a Fluostar OPTIMA microplate-based multi-detection reader (Isogen Life Science, Maarssen, The Netherlands). The mean fluorescence intensity was normalized to the amount of protein present in the sample, determined using the MicroBCA™ protein assay (Pierce, Rockford, USA).
For microscopy, cells were seeded in 12-well plates on coverslips, 24 h before transfection. Cells were treated with 20 μl of different polyplexes containing Alexa-488 labeled siRNA at a final siRNA concentration of 125 nM. After 4 h, cells were washed with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. After fixation, slides were washed, counterstained with DAPI and mounted using Fluorsave (Calbiochem, San Diego, CA, USA). Cells were imaged using a Nikon TE2000-U fluorescent microscope (Nikon Benelux, Brussels, Belgium).
In Vitro Gene Silencing
H1299 cells were seeded in 96-well plates at a density of 8000 cells per well, 24 h before transfection. Cells were treated with 20 μl of different polyplexes at a final siRNA concentration of 125 nM in FBS-containing medium. After 4 h, medium was replaced, and cells were incubated for another 48 h. Then, cells were washed and lysed in 100 μl reporter gene lysis buffer (Promega, Leiden, The Netherlands). After a freeze/thaw cycle, 50 μl lysate was mixed with 50 μl luciferase assay reagent (Promega) and luciferase activity was determined by measuring the luminescence for 10 s at room temperature using a Fluostar OPTIMA microplate-based multi-detection reader equipped with a microinjector (Isogen Life Science). Luciferase activity of untreated cells was defined as 100% expression.
Results were analyzed using Student’s t-tests to assess statistical significances. For multiple comparisons, ANOVA with a Bonferroni post-test was used.
RESULTS AND DISCUSSION
Feed compositiona ABOL/EDA/PEG
Obtained compositionb ABOL/EDA/PEG
Wt % PEGc
Mw PAA (kg/mol)d
75 / 25 / 0
80 / 20 / 0
67 / 23 / 10
59 / 31 / 10
In order to prepare polyplexes with various amounts of PEG in the formulations, mixtures containing different ratios of p(CBA-ABOL/EDA) and p(CBA-ABOL/EDA/PEG) were taken and mixed with siRNA. As it has previously been shown that PEG conjugation to polymeric vectors can interfere with polyplex formation and siRNA complexation, we first investigated the physicochemical properties of polyplexes with different PEG contents.
The disulfide bridges in the poly(CBA-ABOL/EDA) polymers are expected to be cleaved in the cytosol because of its high redox potential as compared to the extracellular environment. To investigate whether the accessibility of these disulfide bonds for reducing agents like glutathione is restricted by PEGylation of the polyplexes, polyplexes were incubated for 1 h at 37°C in the presence of 5 mM glutathione and subjected to electrophoresis. For all formulations, this incubation resulted in total release of siRNA from the polyplexes, indicating that the PEG chains do not interfere with polymer reduction (Fig. 2a–d, right). Based on these results, further studies were performed with polyplexes prepared at polymer/siRNA w/w ratio 24.
Erythrocyte Aggregation and Hemolysis
Cellular Uptake and Gene Silencing
In this study, the synthesis of a novel PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone was described for the delivery of siRNA. Polyplexes with various PEG contents were prepared by mixing the PEGylated polymer with its unPEGylated counterpart prior to polyplex formation. PEGylation decreased polyplex surface charge, increased their stability against salt and serum and decreased polyplex interactions with erythrocytes. Controversially, PEGylated polyplexes showed decreased resistance against heparin displacement. Cellular uptake was lower for polyplexes with increasing PEG content, which resulted in reduced gene silencing efficiency, but also reduced toxicity. Polyplexes with PEG contents of 30 and 45 wt.% showed significant silencing efficiency in the absence of toxicity, which makes them promising carriers for delivery of siRNA in vivo. The addition of targeting ligands on the PEG chain ends is expected to further improve their cellular uptake and silencing potential.
ACKNOWLEDGMENTS & DISCLOSURES
The authors gratefully acknowledge M.J. van Steenbergen for his assistance with GPC measurements. This project is financially supported by the Technology Foundation STW of The Netherlands Organization for Scientific Research NWO grant UFA7468.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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