Amphipathic Homopolymers for siRNA Delivery: Probing Impact ofBifunctional Polymer Composition on TransfectionChristian 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-Zurich,CH-8093, Switzerland∥Institute of Organic Chemistry, University of Zurich, Switzerland

*S Supporting Information

ABSTRACT: In this study, we systematically explore theinfluence of the lipophilic group on the siRNA transfectionproperties of the polycationic-based delivery vectors. For this,a novel and modular synthetic strategy was developed for thepreparation of polymers carrying a cationic site and a lipophilicgroup at each polymer repeat unit. These bifunctionalpolymers could form a complex with siRNA and deliver it tohuman colon carcinoma cells (HT-29-luc). In general,transfection capability increased with an increase in the chainlength of the lipophilic moiety. The best transfection agent, apolymer containing ammonium groups and pentyl side chains,exhibited lower toxicity and higher transfection efficiency than branched and linear polyethylenimines (PEI). Moreover, asopposed 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.

■ INTRODUCTION

Delivery of small interfering RNA (siRNA) to cells represents apromising approach in tackling genetic disorders and viralinfections through gene silencing at the post-transcriptionallevel.1 However, siRNAs are susceptible to enzymaticdegradation. Moreover, their negatively charged backboneand relatively high molecular weight result in poor cellularuptake. To circumvent these issues, a variety of viral andnonviral delivery vectors have been developed.2−12 Viral vectorsare efficient gene transfecting agents. However, safety concerns,related to immune responses and random integration of viralgenomes, may limit their widespread clinical use.13,14 Nonviralsystems include lipids, peptides, proteins, and syntheticpolymers. Among these, polymers are particularly attractivedue to their chemical versatility and generally low productioncost.15−31 In the past decade, a major effort has been focusedon the development of cationic polymers that can formelectrostatic complexes with negatively charged siRNA, protectit from the harsh extracellular environment, and facilitate itscellular uptake. However, once the complexes enter the cell, inmost cases by endocytosis, enzymatic degradation by lysosomalnucleases and extracellular clearance may follow.32 To avoidthis problem and to deliver siRNA to the silencing machinery inthe cytosol, the polymer must also possess endosomaldestabilizing properties.33 This is often achieved throughincorporation of buffering groups that are thought to allowendosomal escape via the so-called “proton sponge” effect.34−36

An alternative design principle is provided by nature in which

cell penetrating peptides possess an amphipathic structure andtranslocate themselves into the cells via direct penetration ormembrane perturbation. Even though the membrane penetrat-ing mechanism is still poorly understood, lipophilic moieties areknown to play an important role in their intracellulartranslocation properties.37 Some of these peptides, such asKALA, exhibit endosomolytic properties via hydrophobicinteraction between lipophilic amino acid residues andendosomal membrane lipids, eliciting membrane perturbationand endosomal escape.38 In this context, a system that carriespositive charges for complexation with siRNA and lipophilicmoieties for endosomal escape seems ideally suited for genedelivery purposes. Such amphipathic structures would presentan alternative to the current systems that operate upon the“proton sponge” effect. A pioneering study from Rozemautilizing random sequences of an alkyl (with variable chainlength) and an ammonium-based vinyl ether monomerpolymerized through a cationic polymerization has alreadydemonstrated efficient membrane-lytic abilities, indicating apromising future of this strategy in the design of gene deliveryvectors.39 Recent reports from Stayton,40,41 Hollfelder,42

Zintchenko,43 Wagner,44 Frechet,15 and Anderson45 furtherindicate that lipophilic groups play a role in determining thetransfection efficiency of a delivery system.37 With a different

Received: January 24, 2014Revised: April 6, 2014Published: April 22, 2014

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perspective, Sanders, Matile, and Tew have carried outimpressive work in establishing the efficacy of amphipathicstructures for translocation purposes across vesicle mem-branes.46−50 It is surprising therefore to note that a systematicstudy to probe the influence of the lipophilic group by usingmolecularly precise polymers as nucleic acid delivery vehiclesremains poorly explored. Toward this end, in the present study,we employed homopolymer sequences to examine this aspectin more detail. To achieve the proposed goal, we utilized thefunctional group compatibility of the ATRP process51−55 toprepare a reactive and general polymer scaffold from acommercially available and inexpensive monomer (Figure 1).The scaffold was then transformed into a library of amphipathichomopolymers differing in the nature of the cationic and thelipophilic moieties. The cationic charge was provided by anammonium or guanidinium group whereas the lipophilicmoieties 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 transfectioncapability of this new family of amphipathic polymers was thenestablished.

■ EXPERIMENTAL SECTIONMaterials. 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 bovineserum (FBS), penicillin−streptomycin solution, phosphate-bufferedsaline (PBS), and SYBR Gold were obtained from Invitrogen(Carlsbad, CA). Heparin sodium salt (molecular weight from 8000to 25000) was purchased from AppliChem (Darmstadt, Germany).Firefly luciferase specific siRNA (Luc-siRNA) and nonspecific controlsiRNA with three mismatches (mm-siRNA) were obtained fromBioneer (Daejeon, South Korea). The fluorescently labeled bcl-2targeting siRNA (DY547-siRNA) was provided by DharmaconResearch (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 GdTdT-3′; mm-siRNA sense, 5′-CGU ACG CGG AAU ACU UCG AdTdT-3′; mm-siRNA antisense, 5′-UCG AAG UAU UCC GCG UAC

GdTdT-3′; DY547-siRNA sense, 5′-(DY547) GCA UGC GGC CUCUGU UUG AUU-3′; DY547-siRNA antisense, 5′-UCA AAC AGAGGC CGC AUG CUU-3′. Branched polyethylenimine (B-PEI) andlinear polyethylenimine (L-PEI) with molecular weights of 25000 wereobtained from Sigma-Aldrich (St. Louis, Mo) and Polysciences, Inc.(Warrington, PA), respectively. Bright-Glo Luciferase Assay Systemand 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 obtainedfrom Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich(St. Louis, MO), respectively.

Methods. Gel Retardation Assay. siRNA/polymer complexeswere prepared by mixing between 15 pmol of siRNA and polymers atN/P ratios (molar ratios of atomic nitrogen in polymer to phosphorusin siRNA) of 0.6, 1.1, 2.2, and 4.5 in 15 μL of diluted PBS (0.5 mMKH2PO4, 77.5 mM NaCl, 1.5 mM Na2HPO4, pH 7.4). After a 15 minincubation at room temperature, the complex solutions with 3 μL ofloading dye were loaded onto 1% agarose gel and the electrophoresiswas performed in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH8.0) at 180 mV for 20 min. The unbound siRNAs were visualized withethidium bromide staining by using a UV transilluminator (GelDocTM XR, Bio-Rad, Hercules, CA).

Heparin Displacement Assay. siRNA/polymer complexes wereprepared by mixing between 0.8 pmol of siRNA and polymers at anN/P ratio of 4.5 in 5 μL of diluted PBS (0.5 mM KH2PO4, 77.5 mMNaCl, 1.5 mM Na2HPO4, pH 7.4). After a 15 min incubation, thecomplexes 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 mMNa2HPO4, pH 7.4)) and transferred in a 384-well plate. After a 15 minincubation, the complex/heparin mixture was reacted with 10 μL ofdiluted SYBR Gold dye solution (2-fold concentrate in TAE buffer)for 10 min. The fluorescence intensities from the intercalated dye intofree siRNA were measured with an excitation wavelength of 495 nmand an emission wavelength of 537 nm by using a fluorophotometer(Infinite 200 PRO, Tecan, Mannedorf, Switzerland). The percentageof displaced siRNA was calculated with the fluorescence intensity froma control siRNA without polymer as 100%.

Cell Culture. HT-29-luc cells were maintained in 10% FBScontaining RPMI-1640 medium supplemented with 100 units/mLpenicillin and 100 μg/mL streptomycin at 37 °C in a 5% CO2humidified atmosphere. The cells with passage number between 11and 16 were seeded in a 96-well plate and 24-well plate at a density of

Figure 1. Molecular design and synthesis of amphipathic homopolymers.

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3 × 104 cells/well and 2 × 105 cells/well, respectively. The cells werecultured 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 inserum free medium for 5 h. The medium was changed with 10% FBScontaining fresh medium and the cells were further incubated for 17 h.The CCK-8 solution containing water-soluble tetrazolium salt wasused to measure cell viability in accordance with the manufacturer’sinstructions. The absorbance at 450 nm in the cells without anypolymer 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-siRNAcomplexed with synthesized polymers at an N/P ratio of 4.5, B-PEI atan N/P ratio of 24, and L-PEI at an N/P ratio of 12 in serum-freeRPMI-1640 medium for 3 h. The control cells were incubated inserum free medium without complexes. The cells were washed twicewith cold PBS and detached from the plate by treating with 150 μL oftrypsin-EDTA solution (0.05%, w/v) for 3 min. After the addition of350 μL of 10% FBS containing RPMI-1640 medium, the cells werecollected by centrifugation (10 min, 300g) at 4 °C and washed twicewith cold PBS containing 2 mM of EDTA and 0.5% BSA. To quenchsignals from membrane-bound complexes, trypan blue solution wasadded to the cells to a final concentration of 0.2% (w/v) beforemeasurement. The relative fluorescence of the cells was analyzed witha minimum of 10000 events per sample by using a FACScanto flowcytometer (BE Biosciences, San Jose, CA). The mean fluorescenceintensity (MFI) folds were calculated by dividing with the MFI valueof 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 ofsiRNA complexed with synthesized polymers at N/P ratios of 4.5 and9 in serum-free RPMI-1640 medium for 5 h. As controls, B-PEI and L-PEI were complexed with siRNA at N/P ratios of 24 and 12,respectively, which values were determined previously to elicit the besttransfection efficiency without any toxic signs. The transfected cellswere replaced with fresh 10% FBS containing medium and furtherincubated for 40 h. To compare the target luciferase silencingefficiency in serum containing medium, 154 nM of Luc-siRNAs andmm-siRNAs were complexed with polymer C5A, B-PEI and L-PEI atthe conditions described above. The complexes were treated to thecells for 10 h in 25% FBS containing RPMI-1640 medium and thetransfection medium was replaced with 10% FBS containing fresh

medium. After 40 h further incubation, the cells were lysed forluciferase analysis. The cytotoxicity assay was performed with theCCK-8 reagent just after the transfection step. To measure the relativeluciferase expression, the culture medium was removed and 100 μL ofGlo Lysis Buffer were treated for 30 min. After removing the celldebris by centrifugation, the 50 μL of supernatant were mixed with 45μL of Bright-Glo reagent. The luminescence was measured by using aluminometer (Infinite 200 PRO, Tecan, Mannedorf, Switzerland) aftera 3 min incubation in the dark. The luciferase expression values werenormalized to the amount of total protein determined by BCA assayand the normalized luminescence from nontreated cells was used as acontrol of 100%.

Transfection in the Presence of Bafilomycin A1. For thecomparison of transfection efficiencies between polymer and B-PEIin the presence of a proton pump inhibitor, HT-29-luc cells werecotreated with 100 or 200 nM of bafilomycin A1 and siRNA/polymerC5A 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. Themedium was replaced with 10% FBS containing medium and the cellswere incubated for 40 h before the luciferase analysis.

Statistical Analysis. All data were passed with the Shapiro-Wilknormality test and the equal variance test before using parametricanalysis. The one-way ANOVA test with Tukey’s posthoc test wasperformed for the pairwise comparison between multiple groups. TheStudent’s t test was used for the comparison of buffering capacitybetween B-PEI and polymer C5A. The significant difference wasassigned at p-values < 0.05.

■ RESULTS AND DISCUSSION

Polymer Design and Synthesis. In previous studies,random copolymers composed of two different monomers, onecarrying the cationic group and the other carrying the lipophilicgroup, were used.39−41 The random copolymerization process,however, yields polymers with an ill-defined monomersequence that is subject to change from reaction to reactioneven if the total percentage of the two monomers remainsconstant. Therefore, it is difficult to assume that a directproperty comparison, especially in the biomedical field in whichpharmacokinetic behavior is sensitive to the molecular structureof the vector,56 can be made in a polymer family that isprepared through a random copolymerization process. For this

Scheme 1. Synthesis of Amphipathic Homopolymers

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reason, we chose to synthesize homopolymers where the twoactive residues, cationic and lipophilic moieties, have to beplaced at each repeat unit so that all repeat units of the polymerchain exhibit the same chemical structure (Figure 1). Thiswould allow for a direct comparison to be made betweendifferent members of the same vector family. This goal ofsynthesizing amphipathic homopolymers was achieved bydeveloping 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 andcommercially available glycidyl methacrylate monomer, 2(Scheme 1).57 The aromatic proton resonances of the initiatorallowed for determination of the molecular weight of thepolymer by end-group analysis using 1H NMR spectroscopy.The degree of polymerization of the polymers ranged from 33to 45, while the polydispersity index ranged from 1.1 to 1.3.Reaction of poly(glycidyl methacrylate), 3, with t-butoxycar-bonyl (t-boc) protected cysteamine gave rise to hydroxyl-functionalized polymer 4 through the thiol-epoxy couplingreaction.57−61 Esterification of the hydroxyl group with an acidchloride furnished alkyl or phenyl substituted polymers (see theSupporting Information for synthesis and characterizationdetails). A simple precipitation into a nonsolvent was sufficientfor purification of the polymers. The postpolymerizationreactions did not increase the polydispersity of the structuresas can be judged from symmetric and narrow elutionchromatograms of the polymers before and after thebifunctionalization process (Figure S1). Acidic conditionswere then used to remove the t-boc groups to access theammonium cation-based amphipathic homopolymer familyC3A, C4A, C5A, C6A, PheA, and EtPheA (Chart 1). PolymersC3A, C4A, C5A, and C6A carried propyl, butyl, pentyl, andhexyl side chains, respectively, whereas polymers PheA andEtPheA contained phenyl and phenyl-ethyl groups, respec-tively. In order to further change the chemical nature of thecation, the guanidinium group was introduced through coupling

of the ammonium group in polymers C4A, PheA, and EtPheAto amidinopyrazole hydrochloride. This procedure yieldedguanidinium-based amphipathic polymers C4G, PheG, andEtPheG (Chart 1). The control polymer C0A was accessedthrough protective group removal of polymer 4. The polymerswere purified by a simple precipitation into a nonsolvent. Forbiological studies, the polymer solutions were prepared bydissolving them in deionized water at a concentration of 10mg/mL. Polymers containing hexyl (polymer C6A) or ethyl-phenyl (polymers EtPheA and EtPheG) side chains wereexcluded 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, atargeting ligand or an imaging probe can be attached to thepolymer chain. Moreover, due to the controlled nature of thepolymerization process, block copolymers, for example, with apoly(ethylene glycol) (PEG) block,57 can also be prepared, ifnecessary. The synthetic strategy also allows control overmolecular weight and hence chain length of the polymers andresulted in low polydispersity materials exhibiting both of theactive residues on each polymer repeat unit. Furthermore, dueto the generality of the synthetic design, one scaffold, inprinciple, can give rise to a vector library differing in thechemical composition to allow the establishment of thestructure−property relationships.

Complexation with siRNA. Polymers containing cationicmoieties complex negatively charged siRNA via ionicinteractions causing retardation of siRNA under gel electro-phoresis. Polymers C4A and C5A bound all the siRNAs abovean N/P ratio of 4.5 and other polymers retarded completely themigration of siRNA above an N/P ratio of 2.3 (Figure 2). Thepolymers with longer aliphatic carbon chains showed lowerbinding capacity to siRNA. No significant difference could beobserved in the siRNA complexation efficiency of the polymerscarrying guanidinium groups when compared to polymerscarrying primary ammonium groups. Linear polyethylenimine

Chart 1. Chemical Structures of the Amphipathic Homopolymers

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(L-PEI) bound to siRNAs as efficiently as the synthesizedpolymers but branched polyethylenimine (B-PEI) exhibited thehighest binding capacity.Complex Stability Test with Heparin. Strong affinity

between the siRNA and polymer is important for a stablecomplex formation. However, after cellular internalization thesiRNA should be released from the complex and bind withtarget mRNA in the cytosol to induce the RISC (RNA-inducedsilencing complex) mediated gene silencing. To test complexstability, heparin, a polyanionic glycan, can be used as itcompetes with siRNA to bind with positively charged polymers,resulting in release of siRNA from the complex (Figure 3).

Polymer C5A, B-PEI, and L-PEI released siRNA from thecomplex 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. Eventhough the previous gel electrophoresis did not show a freesiRNA band after the complexation to L-PEI at an N/P ratio of

4.5, more than 30% of siRNA in L-PEI complex was stained bySYBR Gold dye because siRNA was not efficiently condensedby 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 wasevaluated in human colon carcinoma cells (HT-29-luc) atpolymer concentrations ranging from 3 to 48 μg/mL (Figure4). These results were compared with the cytotoxicity of B-PEI

and L-PEI. Considering that positive charge density is one ofthe major factors that causes cell toxicity, B-PEI and L-PEI withhighest charge density, were found to be more toxic than thesynthesized polymers at concentrations of 3, 6, and 12 μg/mL.At a concentration of 24 μg/mL, all polymers showed asignificant cytotoxicity, with polymers C0A, C3A, C4G, andPheG being more toxic than polymers C4A, C5A, and PheA.Although the lipophilic moieties of the polymers can interactwith the cell membrane and cause toxicity, carbon chain lengthand type (aliphatic or aromatic) did not show any correlationwith the cell toxicity data. For example, polymer C0A, withoutany lipophilic moiety, was more toxic than other polymerscarrying 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 thanammonium containing polymers.64 For example, polymerC4A, containing butyl carbon chains and primary ammoniumgroups, was less toxic than its guanidinium counterpart C4G.Polymer PheA, containing phenyl rings and ammonium groups,was also less toxic than the corresponding guanidinium polymerPheG. All transfection experiments were performed in anontoxic concentration range.

Comparison of Intracellular Uptake. Polymer complexeswith dye-labeled siRNA (DY547-siRNA) were incubated withHT-29-luc cells, and the mean fluorescence intensity of the cellswas measured by using flow cytometry to compare theintracellular uptake efficiency (Figure 5). The carbon chainlength did not correlate with the order of uptake efficiency aspolymer C0A without lipophilic group did not show anysignificant difference to deliver siRNA compared to the otherpolymers, except for polymer PheA. In contrast to previousstudies demonstrating that the guanidinium group enhancedthe intracellular uptake via efficient interaction with the cellmembrane, polymers C4G and PheG, containing guanidinium

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

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

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

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groups, did not show a significant superiority. Surprisingly,polymer PheA containing ammonium groups and phenyl ringsshowed much higher uptake efficiency than other polymers, B-PEI, and L-PEI (an approximately 4-fold average of otherpolymers). Despite this remarkable uptake efficiency, polymerPheA is not able to release siRNA easily, which can be apotential limitation for the siRNA-mediated gene silencing(Figure 3).siRNA Transfection Assays. To understand the direct

relationship between chemical composition of the polymerand delivery efficiency, we performed siRNA-mediated genesilencing assays in a luciferase reporter system. HT-29-luc cells,stably expressing luciferase, were transfected with luciferasespecific siRNA in serum free medium after complexation withpolymers at N/P ratios of 4.5 and 9. As demonstrated earlier bygel retardation assay, all of the polymers complexed siRNA atthese ratios. At an N/P ratio of 4.5, polymer C5A showed thebest knockdown efficiency of target luciferase (the remainingluciferase expression was 23.5% of control; Figure 6). Polymers

C3A (46.3%) and C4A (45.4%) were second best, followed bypolymers 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 transfectionefficiency followed by polymers C3A and C4A containingthree and four carbon atom long aliphatic side chains,respectively. In addition, the control polymer C0A, having nolipophilic side chain, lacked transfection capability, suggestingthat the lipophilic part is an essential structural unit in themolecular design of the present delivery vectors. Betweenpolymers PheA and PheG that carried a phenyl group as thelipophilic side chain, polymer PheA with an ammonium cationwas better than polymer PheG with a guanidinium cation. Thisdifference between ammonium and guanidinium was alsoobserved in polymers C4A and C4G, both carrying four carbonatom long carbon side chains. According to previous studies,the transfection efficiencies of guanidinium-based polymers andammonium-based polymers are highly structure-dependent.For example, polylysine-mediated DNA delivery is moreefficient than a polyarginine mediated one,65 while arginine-modified dendrimers show better transfection efficiency thanlysine counterparts.66 Combined with our cytotoxicity results,which showed that the guanidinium groups are more toxic thanthe ammonium groups, polymers containing ammoniumgroups seem to be a more suitable choice, in the present setof materials, for siRNA delivery applications. At an N/P ratio of9, polymers C4A and C5A showed the highest silencingefficiency of 35.9 and 33.7%, respectively (Figure S7). Based onthese results, we envision that polymers containing longerlipophilic carbon chains produce higher transfection efficiency.Although both parameters, complex stability and intracellular

uptake, are important factors in designing an efficient siRNAdelivery vector, they are not straightforwardly correlated to thetransfection efficiency.42,67 In this study, polymer C5A showedthe best gene silencing effect even though its binding affinityand uptake are relatively low in comparison with otherpolymers. It is worth noting that the translocation of siRNAinto the cytosol as a free form is a critical factor to elicit thedesired gene silencing. It is likely that pentyl carbon chains inpolymer C5A successfully interact with the endosomalmembrane to expose the complex to the cytosol and thatthese long lipophilic carbon chains prevent strong binding withsiRNA, resulting in efficient release of free siRNA from thecomplex after cellular internalization.68

The silencing effect of polymer C5A was further evaluated atdifferent N/P ratios and compared with other nucleic aciddelivery carriers such as B-PEI and L-PEI (Figure 7). For the B-PEI/siRNA and L-PEI/siRNA complexes, the N/P ratio waschosen to be 24 and 12, respectively. At these ratios, thecomplexes showed the most efficient silencing effect withoutinducing any toxicity. By increasing N/P ratios from 1.1 to 4.5(Figure 7), the silencing effect of polymer C5A increased toreach a luciferase expression of 25.0% of the control, which wasmore efficient than that of B-PEI/siRNA (60.1%). The lowinhibition efficiency of L-PEI/siRNA complex (72.5%) wascaused by a lack of stable binding with siRNA, which wasevident in the complex stability test (Figure 3). Next, anonspecific control siRNA with 3 mismatched bases wasdelivered with polymer C5A at an N/P ratio of 4.5, and nosignificant decrease in luciferase was observed. This confirmedthat 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/polymercomplexes at an N/P ratio of 4.5 (B-PEI and L-PEI at an N/P ratio of24 and 12, respectively) by using flow cytometry. MFI: meanfluorescence intensity. Values are represented as a mean ± SD (n = 3).

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

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free medium, most likely due to the interaction of polymer/siRNA complex with serum proteins resulting in aggregation ordissociation in the medium before intracellular uptake.Nonetheless, to elicit significant silencing effects in 25%serum containing medium, cells were transfected with twicethe amounts of siRNA (154 nM) for a prolonged incubationtime (10 h) after complexation with polymer C5A at an N/Pratio of 4.5, B-PEI at an N/P ratio of 24, and L-PEI at an N/Pratio of 12 (Figure S8). Polymer C5A significantly inhibited theluciferase expression to 65.1%; this efficiency was higher thanthe luciferase expression with B-PEI and L-PEI (77.9 and81.0%, respectively; Figure S9). The complexes did not causeany visible cell toxicities during experiments, but the highamount of L-PEI caused nonspecific luciferase inhibition withmismatch siRNA.PEI, often considered as an efficient polymeric vector for

siRNA delivery, possesses protonable amine groups with abroad range of buffering capacity. After the endocytosis ofsiRNA/PEI complexes, the protonation of amine groups atendosomal pH (pH 5−6) causes a “proton sponge” effect,resulting in the endosome rupture and endosomal escape ofentrapped complexes. The buffering capacity of polymer C5A(38.4%) was higher than that of B-PEI (22.8%; Figure S10). Incontrast to the total amine content in B-PEI mediated siRNAdelivery (N/P ratio = 24), the comparatively low amine amountin polymer C5A mediated siRNA delivery (N/P ratio = 4.5)may not be sufficient to encompass the threshold in which theendosomal membrane rupture can be attained by a highosmotic pressure within the endosome.69 To examine if thebuffering capacity of polymer C5A contributed to its efficientsiRNA delivery via the “proton sponge” effect, bafilomycin A1,a proton pump inhibitor, was coincubated with siRNA/polymerC5A and siRNA/B-PEI complexes in serum free medium.Polymer C5A showed a similar silencing effect in the presenceor the absence of bafilomycin A1, whereas transfectionefficiency of B-PEI significantly decreased in the presence ofbafilomycin A1 (Figure 8). This result suggests that polymersequipped with a lipophilic moiety along with a cationic group

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

■ CONCLUSIONSTo summarize, we described the synthesis of a general reactivescaffold through the ATRP process of a commercially availableand inexpensive monomer. This scaffold can be converted intoa desired amphipathic structure in three linear synthetic steps.The first step is the thiol-epoxy coupling chemistry that installsthe cationic group in a protected form and unravels a reactivehydroxyl unit. This hydroxyl unit is used for installation of alipophilic moiety via an esterification reaction. Finally, removalof the protective group gives rise to water-soluble polymerscarrying a positively charged-hydrophilic and a neutral-lip-ophilic side chain at each polymer repeat unit. The developedsynthetic strategy allowed good control over molecular weightand hence chain length of the polymers and resulted in lowpolydispersity materials exhibiting both of the active residueson each polymer repeat unit. These polymers could formelectrostatic complexes with siRNA and deliver it to humancolon carcinoma cells (HT-29-luc). In general, cell viability andtransfection efficiency were higher in ammonium-containingpolymers than guanidinium-carrying polymers. In in vitrotransfection studies with polymers containing ammoniumgroups and aliphatic carbon chains, the silencing efficiencyincreased with an increase in the length of the lipophilic moiety.Polymer C5A, carrying pentyl carbon chains and ammoniumgroups, showed the best gene silencing effect even though itsintracellular uptake efficiency was lower in comparison with theother polymers. In addition, this polymer exhibited lowercytotoxicity and higher transfection efficiency than B-PEI andL-PEI. Unlike PEI, however, the present system does not seemto rely upon the “proton sponge” effect for siRNA delivery. Thesuccess of polymer C5A mediated siRNA delivery may beattributed to the long aliphatic chains in the polymer structure,facilitating the endosomal escape by interaction with endosomalmembrane lipids as well as the release of free siRNA in thecytosol. This study, therefore, details a modular and generalsynthetic strategy that gives facile access to a library of

Figure 7. Sequence-specific and dose-dependent silencing of siRNAcomplexes 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 betweentwo groups.

Figure 8. Effect of bafilomycin A1 on the transfection efficiency ofsiRNA 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 < 0.001and **P < 0.05 between two bars.

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amphipathic homopolymers and underlines the importance ofthe amphipathic structure in the design of cationic siRNAdelivery vectors that do not rely upon the “proton sponge”effect. Classical strategies such as PEGylation and the additionof a targeting ligand will have to be envisaged to decreaseinteraction with plasma proteins and trigger cellular uptake in aphysiological context.

■ ASSOCIATED CONTENT*S Supporting InformationSynthesis and characterization details of polymers, additional invitro transfection, and polymer titration are presented. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors(J.-C.L.) *E-mail: jean-christophe.leroux@pharma.ethz.ch.(A.K.) *E-mail: anzar.khan@mat.ethz.ch.

Author Contributions†These authors contributed equally to this work (C.B. andS.H.L.).

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support from the Gebert Ruf Foundation (GRS-041/11) is gratefully acknowledged. A.K. thanks Prof. A. D. Schluter(ETH-Z) for support.

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