Amphipathic Homopolymers for siRNA Delivery: Probing Impact of

Article pubs.acs.org/Biomac

Amphipathic Homopolymers for siRNA Delivery: Probing Impact of Bifunctional Polymer Composition on Transfection Christian Buerkli,†,‡ Soo Hyeon Lee,†,§ Elena Moroz,§ Mihaiela C. Stuparu,∥ Jean-Christophe Leroux,*,§ and Anzar Khan*,‡ ‡

Department of Materials and §Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH-Zürich, CH-8093, Switzerland ∥ Institute of Organic Chemistry, University of Zürich, Switzerland S Supporting Information *

ABSTRACT: In this study, we systematically explore the influence of the lipophilic group on the siRNA transfection properties of the polycationic-based delivery vectors. For this, a novel and modular synthetic strategy was developed for the preparation of polymers carrying a cationic site and a lipophilic group at each polymer repeat unit. These bifunctional polymers could form a complex with siRNA and deliver it to human colon carcinoma cells (HT-29-luc). In general, transfection capability increased with an increase in the chain length of the lipophilic moiety. The best transfection agent, a polymer containing ammonium groups and pentyl side chains, exhibited lower toxicity and higher transfection efficiency than branched and linear polyethylenimines (PEI). Moreover, as opposed to PEI, the transfection efficiency of polymer/siRNA complexes remained unchanged in the presence of bafilomycin A1, a proton pump inhibitor, suggesting that the present system did not rely on the “proton sponge” effect for siRNA delivery.

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INTRODUCTION Delivery of small interfering RNA (siRNA) to cells represents a promising approach in tackling genetic disorders and viral infections through gene silencing at the post-transcriptional level.1 However, siRNAs are susceptible to enzymatic degradation. Moreover, their negatively charged backbone and relatively high molecular weight result in poor cellular uptake. To circumvent these issues, a variety of viral and nonviral delivery vectors have been developed.2−12 Viral vectors are efficient gene transfecting agents. However, safety concerns, related to immune responses and random integration of viral genomes, may limit their widespread clinical use.13,14 Nonviral systems include lipids, peptides, proteins, and synthetic polymers. Among these, polymers are particularly attractive due to their chemical versatility and generally low production cost.15−31 In the past decade, a major effort has been focused on the development of cationic polymers that can form electrostatic complexes with negatively charged siRNA, protect it from the harsh extracellular environment, and facilitate its cellular uptake. However, once the complexes enter the cell, in most cases by endocytosis, enzymatic degradation by lysosomal nucleases and extracellular clearance may follow.32 To avoid this problem and to deliver siRNA to the silencing machinery in the cytosol, the polymer must also possess endosomal destabilizing properties.33 This is often achieved through incorporation of buffering groups that are thought to allow endosomal escape via the so-called “proton sponge” effect.34−36 An alternative design principle is provided by nature in which © 2014 American Chemical Society

cell penetrating peptides possess an amphipathic structure and translocate themselves into the cells via direct penetration or membrane perturbation. Even though the membrane penetrating mechanism is still poorly understood, lipophilic moieties are known to play an important role in their intracellular translocation properties.37 Some of these peptides, such as KALA, exhibit endosomolytic properties via hydrophobic interaction between lipophilic amino acid residues and endosomal membrane lipids, eliciting membrane perturbation and endosomal escape.38 In this context, a system that carries positive charges for complexation with siRNA and lipophilic moieties for endosomal escape seems ideally suited for gene delivery purposes. Such amphipathic structures would present an alternative to the current systems that operate upon the “proton sponge” effect. A pioneering study from Rozema utilizing random sequences of an alkyl (with variable chain length) and an ammonium-based vinyl ether monomer polymerized through a cationic polymerization has already demonstrated efficient membrane-lytic abilities, indicating a promising future of this strategy in the design of gene delivery vectors.39 Recent reports from Stayton,40,41 Hollfelder,42 Zintchenko,43 Wagner,44 Fréchet,15 and Anderson45 further indicate that lipophilic groups play a role in determining the transfection efficiency of a delivery system.37 With a different Received: January 24, 2014 Revised: April 6, 2014 Published: April 22, 2014 1707

dx.doi.org/10.1021/bm5001197 | Biomacromolecules 2014, 15, 1707−1715

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Figure 1. Molecular design and synthesis of amphipathic homopolymers. GdTdT-3′; DY547-siRNA sense, 5′-(DY547) GCA UGC GGC CUC UGU UUG AUU-3′; DY547-siRNA antisense, 5′-UCA AAC AGA GGC CGC AUG CUU-3′. Branched polyethylenimine (B-PEI) and linear polyethylenimine (L-PEI) with molecular weights of 25000 were obtained from Sigma-Aldrich (St. Louis, Mo) and Polysciences, Inc. (Warrington, PA), respectively. Bright-Glo Luciferase Assay System and Glo Lysis Buffer were purchased from Promega (Madison, WI). Micro BCA Protein Assay kit was purchased from Pierce (Rockford, IL). Cell counting kit-8 (CCK-8) and bafilomycin A1 were obtained from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich (St. Louis, MO), respectively. Methods. Gel Retardation Assay. siRNA/polymer complexes were prepared by mixing between 15 pmol of siRNA and polymers at N/P ratios (molar ratios of atomic nitrogen in polymer to phosphorus in siRNA) of 0.6, 1.1, 2.2, and 4.5 in 15 μL of diluted PBS (0.5 mM KH2PO4, 77.5 mM NaCl, 1.5 mM Na2HPO4, pH 7.4). After a 15 min incubation at room temperature, the complex solutions with 3 μL of loading dye were loaded onto 1% agarose gel and the electrophoresis was performed in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 180 mV for 20 min. The unbound siRNAs were visualized with ethidium bromide staining by using a UV transilluminator (Gel DocTM XR, Bio-Rad, Hercules, CA). Heparin Displacement Assay. siRNA/polymer complexes were prepared by mixing between 0.8 pmol of siRNA and polymers at an N/P ratio of 4.5 in 5 μL of diluted PBS (0.5 mM KH2PO4, 77.5 mM NaCl, 1.5 mM Na2HPO4, pH 7.4). After a 15 min incubation, the complexes were treated with 5 μL of heparin solution (0.003, 0.03, 0.3, and 3 units/μL in PBS (1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4, pH 7.4)) and transferred in a 384-well plate. After a 15 min incubation, the complex/heparin mixture was reacted with 10 μL of diluted SYBR Gold dye solution (2-fold concentrate in TAE buffer) for 10 min. The fluorescence intensities from the intercalated dye into free siRNA were measured with an excitation wavelength of 495 nm and an emission wavelength of 537 nm by using a fluorophotometer (Infinite 200 PRO, Tecan, Männedorf, Switzerland). The percentage of displaced siRNA was calculated with the fluorescence intensity from a control siRNA without polymer as 100%. Cell Culture. HT-29-luc cells were maintained in 10% FBS containing RPMI-1640 medium supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2 humidified atmosphere. The cells with passage number between 11 and 16 were seeded in a 96-well plate and 24-well plate at a density of

perspective, Sanders, Matile, and Tew have carried out impressive work in establishing the efficacy of amphipathic structures for translocation purposes across vesicle membranes.46−50 It is surprising therefore to note that a systematic study to probe the influence of the lipophilic group by using molecularly precise polymers as nucleic acid delivery vehicles remains poorly explored. Toward this end, in the present study, we employed homopolymer sequences to examine this aspect in more detail. To achieve the proposed goal, we utilized the functional group compatibility of the ATRP process51−55 to prepare a reactive and general polymer scaffold from a commercially available and inexpensive monomer (Figure 1). The scaffold was then transformed into a library of amphipathic homopolymers differing in the nature of the cationic and the lipophilic moieties. The cationic charge was provided by an ammonium or guanidinium group whereas the lipophilic moieties comprised an alkyl (3−6 carbons) or phenyl group. A comparative study of the binding to siRNA, complex stability, cytotoxicity, cellular uptake, and in vitro siRNA transfection capability of this new family of amphipathic polymers was then established.

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EXPERIMENTAL SECTION

Materials. Luciferase stably expressing HT-29 (HT-29-luc, human colon carcinoma cells) cells were purchased from Caliper Life Sciences (Hopkinton, MA). RPMI medium 1640 (RPMI-1640), fetal bovine serum (FBS), penicillin−streptomycin solution, phosphate-buffered saline (PBS), and SYBR Gold were obtained from Invitrogen (Carlsbad, CA). Heparin sodium salt (molecular weight from 8000 to 25000) was purchased from AppliChem (Darmstadt, Germany). Firefly luciferase specific siRNA (Luc-siRNA) and nonspecific control siRNA with three mismatches (mm-siRNA) were obtained from Bioneer (Daejeon, South Korea). The fluorescently labeled bcl-2 targeting siRNA (DY547-siRNA) was provided by Dharmacon Research (Lafayette, CO). The sequences of siRNAs are as follows: Luc-siRNA sense, 5′-CUU ACG CUG AGU ACU UCG AdTdT-3′; Luc-siRNA antisense, 5′-UCG AAG UAC UCA GCG UAA GdTdT3′; mm-siRNA sense, 5′-CGU ACG CGG AAU ACU UCG AdTdT3′; mm-siRNA antisense, 5′-UCG AAG UAU UCC GCG UAC 1708


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Scheme 1. Synthesis of Amphipathic Homopolymers

3 × 104 cells/well and 2 × 105 cells/well, respectively. The cells were cultured for 24 h before in vitro experiments. Cytotoxicity Assay. To compare the toxicities of polymers, the cells in a 96-well plate were washed once with PBS and treated with 3, 6, 12, 24, and 48 μg/mL of synthesized polymers, B-PEI, and L-PEI in serum free medium for 5 h. The medium was changed with 10% FBS containing fresh medium and the cells were further incubated for 17 h. The CCK-8 solution containing water-soluble tetrazolium salt was used to measure cell viability in accordance with the manufacturer’s instructions. The absorbance at 450 nm in the cells without any polymer treatment was used as a control of 100%. Flow Cytometry Analysis. The cells in a 24-well plate were washed once with PBS and transfected with 100 nM of DY547-siRNA complexed with synthesized polymers at an N/P ratio of 4.5, B-PEI at an N/P ratio of 24, and L-PEI at an N/P ratio of 12 in serum-free RPMI-1640 medium for 3 h. The control cells were incubated in serum free medium without complexes. The cells were washed twice with cold PBS and detached from the plate by treating with 150 μL of trypsin-EDTA solution (0.05%, w/v) for 3 min. After the addition of 350 μL of 10% FBS containing RPMI-1640 medium, the cells were collected by centrifugation (10 min, 300g) at 4 °C and washed twice with cold PBS containing 2 mM of EDTA and 0.5% BSA. To quench signals from membrane-bound complexes, trypan blue solution was added to the cells to a final concentration of 0.2% (w/v) before measurement. The relative fluorescence of the cells was analyzed with a minimum of 10000 events per sample by using a FACScanto flow cytometer (BE Biosciences, San Jose, CA). The mean fluorescence intensity (MFI) folds were calculated by dividing with the MFI value of the control cell. Target Luciferase Inhibition Assay. HT-29-luc cells in a 96-well plate were washed once with PBS and transfected with 77 nM of siRNA complexed with synthesized polymers at N/P ratios of 4.5 and 9 in serum-free RPMI-1640 medium for 5 h. As controls, B-PEI and LPEI were complexed with siRNA at N/P ratios of 24 and 12, respectively, which values were determined previously to elicit the best transfection efficiency without any toxic signs. The transfected cells were replaced with fresh 10% FBS containing medium and further incubated for 40 h. To compare the target luciferase silencing efficiency in serum containing medium, 154 nM of Luc-siRNAs and mm-siRNAs were complexed with polymer C5A, B-PEI and L-PEI at the conditions described above. The complexes were treated to the cells for 10 h in 25% FBS containing RPMI-1640 medium and the transfection medium was replaced with 10% FBS containing fresh

medium. After 40 h further incubation, the cells were lysed for luciferase analysis. The cytotoxicity assay was performed with the CCK-8 reagent just after the transfection step. To measure the relative luciferase expression, the culture medium was removed and 100 μL of Glo Lysis Buffer were treated for 30 min. After removing the cell debris by centrifugation, the 50 μL of supernatant were mixed with 45 μL of Bright-Glo reagent. The luminescence was measured by using a luminometer (Infinite 200 PRO, Tecan, Männedorf, Switzerland) after a 3 min incubation in the dark. The luciferase expression values were normalized to the amount of total protein determined by BCA assay and the normalized luminescence from nontreated cells was used as a control of 100%. Transfection in the Presence of Bafilomycin A1. For the comparison of transfection efficiencies between polymer and B-PEI in the presence of a proton pump inhibitor, HT-29-luc cells were cotreated with 100 or 200 nM of bafilomycin A1 and siRNA/polymer C5A and siRNA/B-PEI complexes at N/P ratios of 4.5 and 24, respectively, in a 96-well plate for 5 h in serum-free medium. The medium was replaced with 10% FBS containing medium and the cells were incubated for 40 h before the luciferase analysis. Statistical Analysis. All data were passed with the Shapiro-Wilk normality test and the equal variance test before using parametric analysis. The one-way ANOVA test with Tukey’s posthoc test was performed for the pairwise comparison between multiple groups. The Student’s t test was used for the comparison of buffering capacity between B-PEI and polymer C5A. The significant difference was assigned at p-values < 0.05.

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RESULTS AND DISCUSSION Polymer Design and Synthesis. In previous studies, random copolymers composed of two different monomers, one carrying the cationic group and the other carrying the lipophilic group, were used.39−41 The random copolymerization process, however, yields polymers with an ill-defined monomer sequence that is subject to change from reaction to reaction even if the total percentage of the two monomers remains constant. Therefore, it is difficult to assume that a direct property comparison, especially in the biomedical field in which pharmacokinetic behavior is sensitive to the molecular structure of the vector,56 can be made in a polymer family that is prepared through a random copolymerization process. For this 1709


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Chart 1. Chemical Structures of the Amphipathic Homopolymers

of the ammonium group in polymers C4A, PheA, and EtPheA to amidinopyrazole hydrochloride. This procedure yielded guanidinium-based amphipathic polymers C4G, PheG, and EtPheG (Chart 1). The control polymer C0A was accessed through protective group removal of polymer 4. The polymers were purified by a simple precipitation into a nonsolvent. For biological studies, the polymer solutions were prepared by dissolving them in deionized water at a concentration of 10 mg/mL. Polymers containing hexyl (polymer C6A) or ethylphenyl (polymers EtPheA and EtPheG) side chains were excluded from further studies due to their poor water solubility. The developed synthetic strategy allowed control over the chemical nature of the polymer chain ends. Hence, if required, a targeting ligand or an imaging probe can be attached to the polymer chain. Moreover, due to the controlled nature of the polymerization process, block copolymers, for example, with a poly(ethylene glycol) (PEG) block,57 can also be prepared, if necessary. The synthetic strategy also allows control over molecular weight and hence chain length of the polymers and resulted in low polydispersity materials exhibiting both of the active residues on each polymer repeat unit. Furthermore, due to the generality of the synthetic design, one scaffold, in principle, can give rise to a vector library differing in the chemical composition to allow the establishment of the structure−property relationships. Complexation with siRNA. Polymers containing cationic moieties complex negatively charged siRNA via ionic interactions causing retardation of siRNA under gel electrophoresis. Polymers C4A and C5A bound all the siRNAs above an N/P ratio of 4.5 and other polymers retarded completely the migration of siRNA above an N/P ratio of 2.3 (Figure 2). The polymers with longer aliphatic carbon chains showed lower binding capacity to siRNA. No significant difference could be observed in the siRNA complexation efficiency of the polymers carrying guanidinium groups when compared to polymers carrying primary ammonium groups. Linear polyethylenimine

reason, we chose to synthesize homopolymers where the two active residues, cationic and lipophilic moieties, have to be placed at each repeat unit so that all repeat units of the polymer chain exhibit the same chemical structure (Figure 1). This would allow for a direct comparison to be made between different members of the same vector family. This goal of synthesizing amphipathic homopolymers was achieved by developing a synthetic scheme that is described as follows. Synthesis of a reactive scaffold was carried out by ATRP (using 4,4′-dinonyl-2,2′-bipyridine ligand) of initiator 1 and commercially available glycidyl methacrylate monomer, 2 (Scheme 1).57 The aromatic proton resonances of the initiator allowed for determination of the molecular weight of the polymer by end-group analysis using 1H NMR spectroscopy. The degree of polymerization of the polymers ranged from 33 to 45, while the polydispersity index ranged from 1.1 to 1.3. Reaction of poly(glycidyl methacrylate), 3, with t-butoxycarbonyl (t-boc) protected cysteamine gave rise to hydroxylfunctionalized polymer 4 through the thiol-epoxy coupling reaction.57−61 Esterification of the hydroxyl group with an acid chloride furnished alkyl or phenyl substituted polymers (see the Supporting Information for synthesis and characterization details). A simple precipitation into a nonsolvent was sufficient for purification of the polymers. The postpolymerization reactions did not increase the polydispersity of the structures as can be judged from symmetric and narrow elution chromatograms of the polymers before and after the bifunctionalization process (Figure S1). Acidic conditions were then used to remove the t-boc groups to access the ammonium cation-based amphipathic homopolymer family C3A, C4A, C5A, C6A, PheA, and EtPheA (Chart 1). Polymers C3A, C4A, C5A, and C6A carried propyl, butyl, pentyl, and hexyl side chains, respectively, whereas polymers PheA and EtPheA contained phenyl and phenyl-ethyl groups, respectively. In order to further change the chemical nature of the cation, the guanidinium group was introduced through coupling 1710


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4.5, more than 30% of siRNA in L-PEI complex was stained by SYBR Gold dye because siRNA was not efficiently condensed by L-PEI. This result is consistent with previous reports, showing that L-PEI exhibits a low siRNA binding affinity, probably due to the structural rigidity of siRNA.62,63 Cytotoxicity Assays. The cytotoxicity of the polymers was evaluated in human colon carcinoma cells (HT-29-luc) at polymer concentrations ranging from 3 to 48 μg/mL (Figure 4). These results were compared with the cytotoxicity of B-PEI

Figure 2. Gel retardation assay for complexation efficiency of the amphipathic homopolymer family at various N/P ratios.

(L-PEI) bound to siRNAs as efficiently as the synthesized polymers but branched polyethylenimine (B-PEI) exhibited the highest binding capacity. Complex Stability Test with Heparin. Strong affinity between the siRNA and polymer is important for a stable complex formation. However, after cellular internalization the siRNA should be released from the complex and bind with target mRNA in the cytosol to induce the RISC (RNA-induced silencing complex) mediated gene silencing. To test complex stability, heparin, a polyanionic glycan, can be used as it competes with siRNA to bind with positively charged polymers, resulting in release of siRNA from the complex (Figure 3).

Figure 4. Cytotoxicity of the amphipathic homopolymer family, B-PEI, and L-PEI. Values are represented as a mean ± SD (n = 3).

and L-PEI. Considering that positive charge density is one of the major factors that causes cell toxicity, B-PEI and L-PEI with highest charge density, were found to be more toxic than the synthesized polymers at concentrations of 3, 6, and 12 μg/mL. At a concentration of 24 μg/mL, all polymers showed a significant cytotoxicity, with polymers C0A, C3A, C4G, and PheG being more toxic than polymers C4A, C5A, and PheA. Although the lipophilic moieties of the polymers can interact with the cell membrane and cause toxicity, carbon chain length and type (aliphatic or aromatic) did not show any correlation with the cell toxicity data. For example, polymer C0A, without any lipophilic moiety, was more toxic than other polymers carrying aliphatic carbon chains in their repeat unit structure. The nature of the cationic moieties in the polymers, however, showed a pronounced effect on cytotoxicity. In general, guanidinium containing polymers were more toxic than ammonium containing polymers.64 For example, polymer C4A, containing butyl carbon chains and primary ammonium groups, was less toxic than its guanidinium counterpart C4G. Polymer PheA, containing phenyl rings and ammonium groups, was also less toxic than the corresponding guanidinium polymer PheG. All transfection experiments were performed in a nontoxic concentration range. Comparison of Intracellular Uptake. Polymer complexes with dye-labeled siRNA (DY547-siRNA) were incubated with HT-29-luc cells, and the mean fluorescence intensity of the cells was measured by using flow cytometry to compare the intracellular uptake efficiency (Figure 5). The carbon chain length did not correlate with the order of uptake efficiency as polymer C0A without lipophilic group did not show any significant difference to deliver siRNA compared to the other polymers, except for polymer PheA. In contrast to previous studies demonstrating that the guanidinium group enhanced the intracellular uptake via efficient interaction with the cell membrane, polymers C4G and PheG, containing guanidinium

Figure 3. Heparin displacement assay to measure siRNA/polymers complex stability. Values are represented as a mean ± SD (n = 3).

Polymer C5A, B-PEI, and L-PEI released siRNA from the complex at a lower amount of heparin than polymers C0A, C3A, and C4A. Interestingly, polymers PheA, C4G, and PheG, which contain either guanidinium groups or phenyl rings, barely released siRNA even at high amounts of heparin. Even though the previous gel electrophoresis did not show a free siRNA band after the complexation to L-PEI at an N/P ratio of 1711


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C3A (46.3%) and C4A (45.4%) were second best, followed by polymers PheA (63.6%), C4G (80.6%), and PheG (91.0%). Polymer C5A with the longest aliphatic carbon side chain (pentyl carbon chain) showed the highest transfection efficiency followed by polymers C3A and C4A containing three and four carbon atom long aliphatic side chains, respectively. In addition, the control polymer C0A, having no lipophilic side chain, lacked transfection capability, suggesting that the lipophilic part is an essential structural unit in the molecular design of the present delivery vectors. Between polymers PheA and PheG that carried a phenyl group as the lipophilic side chain, polymer PheA with an ammonium cation was better than polymer PheG with a guanidinium cation. This difference between ammonium and guanidinium was also observed in polymers C4A and C4G, both carrying four carbon atom long carbon side chains. According to previous studies, the transfection efficiencies of guanidinium-based polymers and ammonium-based polymers are highly structure-dependent. For example, polylysine-mediated DNA delivery is more efficient than a polyarginine mediated one,65 while argininemodified dendrimers show better transfection efficiency than lysine counterparts.66 Combined with our cytotoxicity results, which showed that the guanidinium groups are more toxic than the ammonium groups, polymers containing ammonium groups seem to be a more suitable choice, in the present set of materials, for siRNA delivery applications. At an N/P ratio of 9, polymers C4A and C5A showed the highest silencing efficiency of 35.9 and 33.7%, respectively (Figure S7). Based on these results, we envision that polymers containing longer lipophilic carbon chains produce higher transfection efficiency. Although both parameters, complex stability and intracellular uptake, are important factors in designing an efficient siRNA delivery vector, they are not straightforwardly correlated to the transfection efficiency.42,67 In this study, polymer C5A showed the best gene silencing effect even though its binding affinity and uptake are relatively low in comparison with other polymers. It is worth noting that the translocation of siRNA into the cytosol as a free form is a critical factor to elicit the desired gene silencing. It is likely that pentyl carbon chains in polymer C5A successfully interact with the endosomal membrane to expose the complex to the cytosol and that these long lipophilic carbon chains prevent strong binding with siRNA, resulting in efficient release of free siRNA from the complex after cellular internalization.68 The silencing effect of polymer C5A was further evaluated at different N/P ratios and compared with other nucleic acid delivery carriers such as B-PEI and L-PEI (Figure 7). For the BPEI/siRNA and L-PEI/siRNA complexes, the N/P ratio was chosen to be 24 and 12, respectively. At these ratios, the complexes showed the most efficient silencing effect without inducing any toxicity. By increasing N/P ratios from 1.1 to 4.5 (Figure 7), the silencing effect of polymer C5A increased to reach a luciferase expression of 25.0% of the control, which was more efficient than that of B-PEI/siRNA (60.1%). The low inhibition efficiency of L-PEI/siRNA complex (72.5%) was caused by a lack of stable binding with siRNA, which was evident in the complex stability test (Figure 3). Next, a nonspecific control siRNA with 3 mismatched bases was delivered with polymer C5A at an N/P ratio of 4.5, and no significant decrease in luciferase was observed. This confirmed that luciferase silencing by polymer C5A was sequence-specific. In serum-containing medium, the polymer transfection efficiency decreased significantly in comparison to the serum-

Figure 5. Intracellular uptake efficiency of dye-labeled siRNA/polymer complexes at an N/P ratio of 4.5 (B-PEI and L-PEI at an N/P ratio of 24 and 12, respectively) by using flow cytometry. MFI: mean fluorescence intensity. Values are represented as a mean ± SD (n = 3).

groups, did not show a significant superiority. Surprisingly, polymer PheA containing ammonium groups and phenyl rings showed much higher uptake efficiency than other polymers, BPEI, and L-PEI (an approximately 4-fold average of other polymers). Despite this remarkable uptake efficiency, polymer PheA is not able to release siRNA easily, which can be a potential limitation for the siRNA-mediated gene silencing (Figure 3). siRNA Transfection Assays. To understand the direct relationship between chemical composition of the polymer and delivery efficiency, we performed siRNA-mediated gene silencing assays in a luciferase reporter system. HT-29-luc cells, stably expressing luciferase, were transfected with luciferase specific siRNA in serum free medium after complexation with polymers at N/P ratios of 4.5 and 9. As demonstrated earlier by gel retardation assay, all of the polymers complexed siRNA at these ratios. At an N/P ratio of 4.5, polymer C5A showed the best knockdown efficiency of target luciferase (the remaining luciferase expression was 23.5% of control; Figure 6). Polymers

Figure 6. Target luciferase silencing by siRNA complexed with various polymers at an N/P ratio of 4.5 in the absence of serum. RLU: relative light units. Values are represented as a mean ± SD (n = 3). *P < 0.05 between two groups. 1712


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can deliver siRNA without requiring the help of the “proton sponge” effect.

Figure 7. Sequence-specific and dose-dependent silencing of siRNA complexes with various polymers in the absence of serum: Luc-siRNA, luciferase targeting siRNA; mm-siRNA, control mismatch siRNA. Values are represented as a mean ± SD (n = 3). *P < 0.05 between two groups.

Figure 8. Effect of bafilomycin A1 on the transfection efficiency of siRNA complexed with polymer C5A (N/P = 4.5) and B-PEI (N/P = 24). Values are represented as a mean ± SD (n = 3−6). *P < 0.001 and **P < 0.05 between two bars.

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free medium, most likely due to the interaction of polymer/ siRNA complex with serum proteins resulting in aggregation or dissociation in the medium before intracellular uptake. Nonetheless, to elicit significant silencing effects in 25% serum containing medium, cells were transfected with twice the amounts of siRNA (154 nM) for a prolonged incubation time (10 h) after complexation with polymer C5A at an N/P ratio of 4.5, B-PEI at an N/P ratio of 24, and L-PEI at an N/P ratio of 12 (Figure S8). Polymer C5A significantly inhibited the luciferase expression to 65.1%; this efficiency was higher than the luciferase expression with B-PEI and L-PEI (77.9 and 81.0%, respectively; Figure S9). The complexes did not cause any visible cell toxicities during experiments, but the high amount of L-PEI caused nonspecific luciferase inhibition with mismatch siRNA. PEI, often considered as an efficient polymeric vector for siRNA delivery, possesses protonable amine groups with a broad range of buffering capacity. After the endocytosis of siRNA/PEI complexes, the protonation of amine groups at endosomal pH (pH 5−6) causes a “proton sponge” effect, resulting in the endosome rupture and endosomal escape of entrapped complexes. The buffering capacity of polymer C5A (38.4%) was higher than that of B-PEI (22.8%; Figure S10). In contrast to the total amine content in B-PEI mediated siRNA delivery (N/P ratio = 24), the comparatively low amine amount in polymer C5A mediated siRNA delivery (N/P ratio = 4.5) may not be sufficient to encompass the threshold in which the endosomal membrane rupture can be attained by a high osmotic pressure within the endosome.69 To examine if the buffering capacity of polymer C5A contributed to its efficient siRNA delivery via the “proton sponge” effect, bafilomycin A1, a proton pump inhibitor, was coincubated with siRNA/polymer C5A and siRNA/B-PEI complexes in serum free medium. Polymer C5A showed a similar silencing effect in the presence or the absence of bafilomycin A1, whereas transfection efficiency of B-PEI significantly decreased in the presence of bafilomycin A1 (Figure 8). This result suggests that polymers equipped with a lipophilic moiety along with a cationic group

CONCLUSIONS To summarize, we described the synthesis of a general reactive scaffold through the ATRP process of a commercially available and inexpensive monomer. This scaffold can be converted into a desired amphipathic structure in three linear synthetic steps. The first step is the thiol-epoxy coupling chemistry that installs the cationic group in a protected form and unravels a reactive hydroxyl unit. This hydroxyl unit is used for installation of a lipophilic moiety via an esterification reaction. Finally, removal of the protective group gives rise to water-soluble polymers carrying a positively charged-hydrophilic and a neutral-lipophilic side chain at each polymer repeat unit. The developed synthetic strategy allowed good control over molecular weight and hence chain length of the polymers and resulted in low polydispersity materials exhibiting both of the active residues on each polymer repeat unit. These polymers could form electrostatic complexes with siRNA and deliver it to human colon carcinoma cells (HT-29-luc). In general, cell viability and transfection efficiency were higher in ammonium-containing polymers than guanidinium-carrying polymers. In in vitro transfection studies with polymers containing ammonium groups and aliphatic carbon chains, the silencing efficiency increased with an increase in the length of the lipophilic moiety. Polymer C5A, carrying pentyl carbon chains and ammonium groups, showed the best gene silencing effect even though its intracellular uptake efficiency was lower in comparison with the other polymers. In addition, this polymer exhibited lower cytotoxicity and higher transfection efficiency than B-PEI and L-PEI. Unlike PEI, however, the present system does not seem to rely upon the “proton sponge” effect for siRNA delivery. The success of polymer C5A mediated siRNA delivery may be attributed to the long aliphatic chains in the polymer structure, facilitating the endosomal escape by interaction with endosomal membrane lipids as well as the release of free siRNA in the cytosol. This study, therefore, details a modular and general synthetic strategy that gives facile access to a library of 1713


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amphipathic homopolymers and underlines the importance of the amphipathic structure in the design of cationic siRNA delivery vectors that do not rely upon the “proton sponge” effect. Classical strategies such as PEGylation and the addition of a targeting ligand will have to be envisaged to decrease interaction with plasma proteins and trigger cellular uptake in a physiological context.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization details of polymers, additional in vitro transfection, and polymer titration are presented. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

(J.-C.L.) *E-mail: [email protected]. (A.K.) *E-mail: [email protected]. Author Contributions

† These authors contributed equally to this work (C.B. and S.H.L.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Gebert Rüf Foundation (GRS-041/ 11) is gratefully acknowledged. A.K. thanks Prof. A. D. Schlüter (ETH-Z) for support.

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