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  • Dynamic Amphiphile Libraries To Screen for the Fragrant Deliveryof siRNA into HeLa Cells and Human Primary FibroblastsCharlotte Gehin,, Javier Montenegro,,, Eun-Kyoung Bang, Ana Cajaraville,, Shota Takayama,,

    Hisaaki Hirose,,# Shiroh Futaki, Stefan Matile,*, and Howard Riezman*,

    School of Chemistry and Biochemistry, National Centre of Competence in Research (NCCR) Chemical Biology, University ofGeneva, Geneva, SwitzerlandInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

    *S Supporting Information

    ABSTRACT: Dynamic amphiphiles are amphiphiles withdynamic covalent bridges between their hydrophilic headsand their hydrophobic tails. Their usefulness to activateion transporters, for odorant release, and for dierentialsensing of odorants and perfumes, has been demonstratedrecently. Here, we report that the same fragrant dynamicamphiphiles are ideal to screen for new siRNA transfectionagents. The advantages of this approach include rapid accessto fairly large libraries of complex structures, and possibletransformation en route to assist uptake and minimizetoxicity. We report single-component systems that exceedthe best commercially available multicomponent cocktailswith regard to both eciency and velocity of EGFPknockdown in HeLa cells. In human primary broblasts,siRNA-mediated enzyme knockdown nearly doubled from>30% for Lipofectamine to >60% for our best hit. Theidentied structures were predictable neither from literaturenor from results in uorogenic vesicles and thus support theimportance of conceptually innovative screening approaches.

    RNA interference (RNAi) has emerged as a powerful methodto inhibit the biosynthesis of specic proteins with theircomplementary small interfering RNA (siRNA).1 Because of thegreat potential for applications in biology and medicine, thequestion of how to deliver siRNA to the cytosol has attractedsignicant scientic attention.1 Extensive experience with genetransfection could be adapted partially to yield a high number ofnonviral siRNA transporters, including cationic amphiphiles, peptides,polymers, dendrimers, nucleobase-lipid hybrids, and more complexarchitectures.1,2 Triggerable amphiphiles3a that transform in responseto the pH drop in the endosome include Thompsons beautifulvinyl ethers,3b acetals,3c activated esters,3df or very elegant ortho-esters3g and phosphotriesters.3h Pioneering examples for genetransfection with acid-labile hydrazone bridges have suggestedthat degradation en route into cells could be advantageous tominimize cytotoxicity and maximize oligonucleotide release.4a

    However, other studies have suggested that hydrazone-bridgedamphiphiles4 are inferior to disulde-bridged amphiphiles.4b Theunique power of cytosolic disulde reduction to minimizetoxicity and release the substrate is also attracting much attentionwith cell-penetrating poly(disulde)s.5

    Despite these impressive eorts and the enormous potentialfor applications in biology and medicine, problems concerning

    their delivery into cells remain, particularly with regard to in vivoapplications. They include the frequent need of multicomponentcocktails in practice, variable eciencies with dierent cell linesdepending on the specic properties of their cellular barriers,and, most importantly, the scarcity of general structureactivityguidelines for rational design. This situation calls for screeningapproaches.1d Dynamic amphiphiles appeared most attractive forrapid access to fairly large libraries. They are amphiphiles withdynamic covalent bonds between their hydrophilic heads andtheir hydrophobic tails. Dynamic covalent bonds are interestingtools because they can combine the advantages of strong covalentbonds and weak non-covalent bonds, depending on con-ditions.611 Dynamic amphiphiles have been introduced recentlyas activators of ion transporters,7 for slow release of odorants,8

    dynamic micelles, vesicles and gels,9 and as biosensors7 or dif-ferential sensors10 that function in lipid bilayers. Here, we showthat the same odorant libraries used in articial noses 10 can beused to screen for the fragrant delivery of siRNA. Facile accessto expanded libraries with hydrazone, oxime and disuldebridges11 is used to screen up to 900 amphiphiles, either in modelvesicles,10,11 cells, or both. The screening results for GFP knock-down in HeLa cells disclosed in the following include hits withfairly complex, totally unpredictable structures that can competewith multicomponent systems on the market. They operate assingle-component systems, with ability to deliver siRNA intohard-to-transfect human primary broblasts, high reproducibilityand low toxicity.Our formal library of 900 dynamic amphiphiles was con-

    structed from 18 heads and 50 tails (Figures 1 and 2). The headscontain one to two ammoniums (A) or guanidinium (G) cationsplus one to six reactive groups to form hydrazone (H) or oxime(O) bridges with aldehyde or ketone containing tails (T1T50).For doubly bridged amphiphiles, preformed disulde bridges (S)were placed in the head part before amphiphile formation.The synthesis of all peptide dendrons used as scaolds in the

    head groups has been reported.10,11Most tails were commerciallyavailable, some had to be prepared following or adapting straight-forward procedures (Scheme S1).12 Dynamic amphiphiles wereprepared by incubation of heads (e.g., G2H4) and tails (e.g.,T20) in DMSO, usually for 1 h at 60 C (Figure 1). The for-mation of hydrazone-, oxime-, or disulde-bridged amphiphiles

    Received: April 26, 2013



    XXXX American Chemical Society A dx.doi.org/10.1021/ja404153m | J. Am. Chem. Soc. XXXX, XXX, XXXXXX

  • (e.g., G2H4T20) was conrmed by ESI-MS as describedpreviously.10,11 Some amphiphiles were eliminated early onbecause of poor physical properties. The characterization of mostamphiphiles as activators of DNA as cation transporters in uo-rogenic vesicles has been reported.10,11 Several underperformingamphiphiles were also eliminated at this level. The remainingfocused library of 160 amphiphiles was tested for cellular uptake.For robotic library screening, a GFP silencing assay was

    performed in HeLa GPI-EGFP cells (genetically engineeredHeLa cells that stably express GPI-anchored green uorescentproteins at their plasma membrane). A liquid-handler (BiomeckFX) automatically mixed amphiphiles in serum-containingmedium,with a custom siRNA sequence that when properly delivered, hasthe ability to knockdown EGFP expression (siEGFP). The HeLaGPI-EGFP cells were incubated with this mixture for 72 h andEGFP expression was quantied with a uorimeter. All experimentswere carried out at constant concentrations of siRNA (33.8 nM)and DMSO (0.25% (v/v)) and increasing concentrations ofdynamic amphiphiles (Figure 2). No pre-incubation step of siRNAand dynamic amphiphiles in low serummedia (i.e., Opti-MEM)wasrequired before transfection.For comparison, parallel experiments were performed with

    scrambled siRNA as a negative control. G2H4T20 was used asthe reference in positive control experiments due to its similartransfection eciency compared with Lipofectamine RNAiMax(Figure S1). EGFP knockdown eciency was calculated as thepercentage of uorescence decrease observed in cells transfectedwith siEGFP compared to transfection with scrambled siRNA.To facilitate high-throughput screening, cell viability was rst

    evaluated as the percentage of uorescence decrease in samplestransfected with complexes made of scrambled siRNA andamphiphiles compared to untreated cells in medium supple-mented with 0.25% (v/v) DMSO (see SI for experimentaldetails).12 Only amphiphiles with cell viability up to 70% wereretained for further validation and optimization. In furtherexperiments, nontoxicity of selected amphiphiles was conrmedusing a commercially available kit (cytotoxicity detection kit, Roche)that detects cell death by measuring activity of lactate dehydrogenase(LDH) released from cells whose plasma membrane was damaged.The results from robotic high-throughput screening of our

    library conrmed that heads and tails alone (33.8 M nalconcentration) were neither toxic nor able to transfect siRNA(Figure 2g, ). The best results were generally obtained forhydrazones with one or two guanidinium heads linked to three orfour aliphatic tails, either long unsaturated (T19, T20, T21, T22,and T24) or shorter saturated alkyl chains (T12, Figures 2, S2, andS3). The structures of most of the hits were predictable neitherfrom literature nor from activities in vesicles.10,11 G2H4T31 andA2H4T31 have been previously described to be very active to

    transport DNA across uorogenic vesicles10 but this propertywas weaker in the HeLa GPI-EGFP assay (Figure 2). Whereasthe oleyl tails inG2H4T20 are well precedented,1 lauryl tails havebeen implied as less suitable for cellular uptake.11,13 Althoughparticularly promising in vesicles,10,11 fragrant amphi-philes with tails from jasmine and cyclamen T44 and T45 did notperformwell.We also noticed thatminor structural changes down tosingle carbon homologues could cause large dierences in activity.This overall poor predictability conrmed the importance of facileaccess to large amphiphile libraries as well as methods developmentfor high-throughput robotic screening of siRNA delivery.The enhanced detergent activity observed in vesicles for the

    lengthened oxime and disulde amphiphiles11 was translated intoan increase of the toxicity in cells (Figures 2, S2, andS3).Replacementof the guanidinium head by an ammoniumdecreased the transfectioneciency of our cationic amphiphiles (Figures 2, S2, and S3).Screening a panel of increasing concentrations for each amphiphilesalso allowed overcoming the issue of false negative results due tocytotoxicity. Indeed, some amphiphiles (e.g.,G2H4T24) were evenmore active when lowering their concentration (Figures 2 and S2)