Trifaceted Mickey Mouse Amphiphiles for Programmable Self-Assembly, DNA Complexation and Organ-Selective Gene Delivery

T i ace ed Mickey Mouse Amphiphiles o P og ammable Sel -Assembly, DNA Complexa ion and O gan-Selec i e Gene Deli e y Ana I. Ca bajo-Go dillo+,[a] Manuel González-Cues a+,[b] José L. Jiménez Blanco,[b] Juan M. Beni o,[a] Ma ía L. San ana-A mas,[c] Thais Ca mona,[d] Ch is ophe Di Gio gio,[e] Céd ic P zybylski,[ ] Ca men O iz Melle ,*[b] Conchi a T os de Ila duya,*[c] F ancisco Mendicu i,*[d] and José M. Ga cía Fe nández*[a] Abs ac : Ins illing seg ega ed ca ionic and lipophilic domains wi h an angula disposi ion in a ehalose-based i ace ed mac ocyclic sca old allows enginee ing pa chy molecula nanopa icles le e aging di ec ional in e ac ions ha emula e hose con olling sel -assembling p ocesses in i al capsids. The esul ing ilobula amphiphilic de i a i es, ea u ing a Mickey Mouse a chi ec u e, can elec os a ically in e ac wi h plasmid DNA (pDNA) and u he engage in hyd ophobic con ac s o p omo e condensa ion in o ans ec ious nano- complexes. No ably, he opology and in e nal s uc u e o he cyclooligosaccha ide/pDNA co-assemblies can be molded by ine- uning he alency and cha ac e is ics o he ca ionic and lipophilic pa ches, which s ongly impac s he ans- ec ion e icacy in i o and in i o. Ou s anding o gan selec i i ies can hen be p og ammed wi h no need o inco po a ing a bio ecognizable mo i in he o mula ion. The esul s p o ide a e sa ile s a egy o he cons uc ion o ully syn he ic and pe ec ly monodispe se non i al gene deli e y sys ems uniquely sui ed o op imiza ion schemes by making cyclooligosaccha ide pa chiness he ocus. In oduc ion Su ace aniso opy has p o en o be i al in biological sys ems. An example is he well- egula ed s uc u al con ol seen in i us capsids, which s ems om he o e all shape o he olded p o ein, he a angemen o hyd ophobic a eas on he p o ein su ace and he dis ibu ion o cha ged esidues, al oge he se ing up he sp ead o disease by sel -assembly o i al pa icles in i o.[1] In capsids, he in e aces esponsible o he p o ein-p o ein in e ac ions mus no only be in con ac , bu also ha e he app op ia e ela i e o ien a ion. I has long been he aim o chemis s o ins ill a i icial sys ems wi h simila ly di ec ional in e ac ions in o de o sel -assemble ad anced ma e ials capable o pe o ming sophis ica ed asks wi h limi ed human in e en ion.[2] The basic no ion is ha he e ogeneous a angemen s o unc ional elemen s on pa icle o mac o- molecule su aces, esul ing in he o ma ion o localized clus e s o “pa ches”, can bes ow he sys em wi h di e en [a] D . A. I. Ca bajo-Go dillo,+D . J. M. Beni o, P o . J. M. Ga cía Fe nández Ins i u e o Chemical Resea ch, IIQ CSIC-Uni . Se illa C/ Amé ico Vespucio 49, 41092 Se illa (Spain) E-mail: [email p o ec ed] [b] M. González-Cues a,+D . J. L. Jiménez Blanco, P o . C. O iz Melle Depa men o O ganic Chemis y, Facul y o Chemis y Uni e si y o Se illa C/ P o Ga cía González 1, 41012, Se illa (Spain) E-mail: [email p o ec ed] [c] M. L. San ana-A mas, P o . C. T os de Ila duya Depa men o Pha maceu ical Technology and Chemis y School o Pha macy and Nu i ion Uni e si y o Na a a 31080 Pamplona (Spain) E-mail: [email p o ec ed] [d] D . T. Ca mona, P o . F. Mendicu i Depa men o Analy ical Chemis y, Physical Chemis y and Chemical Enginee ing Ins i u o de In es igación Química “And és M. del Rio” (IQAR) Uni e si y o Alcalá Campus Uni e si a io C a. Mad id-Ba celona Km 33.600, 28871, Alcalá de Hena es (Spain) E-mail: [email p o ec ed] [e] D . C. Di Gio gio Ins i u de Chimie Nice, UMR 7272 Uni e si é Cô e d’Azu 28, A enue de Val ose, 06108 Nice (F ance) [ ] D . C. P zybylski CNRS, Ins i u Pa isien de Chimie Moléculai e, IPCM So bonne Uni e si é, Pa is (F ance) [+]These au ho s con ibu ed equally o his wo k. Suppo ing in o ma ion o his a icle is a ailable on he WWW unde h ps://doi.o g/10.1002/chem.202100832 © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley- VCH GmbH. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and e- p oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9429Chem. Eu . J. 2021,27, 9429–9438 © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9429/9438] 1 ea u es compa ed o hose wi h uni o mly dis ibu ed g oups, ansla ing in o unique capabili ies o sel -assemble in o 2D and 3D s uc u es o di e en opologies.[3] The ab ica ion o pa chy nanocons uc s ha a e highly monodispe se in pa ch numbe ( alency), size and posi ion ep esen s a main de y o he abo e channels.[4] Mos epo s ocus on Janus bodies wi h wo dissimila hemisphe es.[5] Meanwhile, he de elopmen o e icien s a egies o he elabo a ion o obus nanos uc u ed objec s wi h highe ace alency, displaying well-de ined s a ic pa e ns o seg ega ed domains, emains a daun ing endea o .[6] Molecula nanome ic en i ies (molecula nanopa icles, MNPs) exhibi ing de ined symme y and pe sis en shape and olume such as ulle enes, polyhed al oligome ic silsequioxanes (POSS), me al-o ganic amewo ks (MOFs), calixa enes, o cyclodex ins (CDs), in combina ion wi h p ecision syn hesis me hodologies, o e unique oppo uni ies owa ds his end.[7] The syn hesis o gene deli e y sys ems ( ec o s) based on monodispe se cyclodex in (especially β-cyclodex in; βCD) de i a i es is a pa adigma ic example: bo h he sel -assembling p ope ies and he abili ies o o m nanocomplexes wi h nucleic acids (CDplexes) a e igh ly dependen on he aniso opic disposi ion o clus e ized ca i- onizable and lipophilic g oups a opposi e ims o he mac o- cyclic co e in a Janus-like a chi ec u e (Figu e 1A).[8] Inc easing he numbe o ca ionic o lipophilic pa ches is expec ed o b oaden he oppo uni ies o egula e di ec ional ecogni ion phenomena in sea ch o a i icial i uses. Howe e , nei he cyclodex ins no any o he commonly used MNP pla o ms allows b eaking he wo- ace Janus bounda y.[9] Mac ocyclic sca olds based on he disaccha ide α,α’- ehalose (cyclo ehalans, CTs) ypi y a singula addi ion o he MNP coho .[10] CT ep esen a i es inco po a ing om wo o i e α,α’- ehalose b icks (CT2 o CT5) a e on eco d.[11] Di e - en ly om CDs, which a e in insically dissymme ic Janus molecules, CTs po ay as many aces as he numbe o cons i u i e α,α’- ehalose uni s, all o which bea six seconda y hyd oxyls and a e chemically iden ical in he canonical CT ep esen a i es. Homogeneous unc ionaliza ion o he CT2 (cyclo e asaccha ide) and CT3 (cyclohexasaccha ide) membe s was pu a wo k o access highly symme ical polyca ionic s a - shape polyme s (Figu e 1B) ha showed high gene deli e y e iciencies.[12] In e es ingly, CTs can po en ially be cons uc ed om di e - en ly unc ionalized building blocks, hen esul ing in aniso- opically- ace ed a chi ec u es. This no ion was i s ealized o he simples CT2 co e: pe ec Janus MNPs ea u ing ca ionic (C) and lipophilic (L) hal es we e elabo a ed ha o med mul i- lamella ans ec ious nanocomplexes (CTplexes) wi h plasmid DNA (pDNA) (Figu e 2A).[13] We hypo hesized ha inc easing he ace alency would enable new oppo uni ies o encode in o ma ion o p og ammable assembly. CTplex ine s uc u e and opology could hen be p ecas in o de o op imize gene ec o pe o mance and selec i i y. As a p oo o concep , he e we epo he syn hesis o pa chy MNPs combining C and L lobes in 1:2 (C1L2) o 2:1 (C2L1) ela ionships wi h a clea -cu angula disposi ion (Figu e 2B). By analogy wi h he e minol- ogy coined o colloidal pa icles wi h he same ype o pa chiness, we call his new p o o ypes Mickey Mouse molec- ula nanopa icles (MM-MNPs).[14] The syn hesis, cha ac e iza- ion, sup amolecula sel -assembling, pDNA nanocomplexa ion and in i o and in i o ans ec ion capabili ies o compounds 1–10 (Scheme 1) a e discussed. Figu e 1. A) S uc u e o β-cyclodex in (βCD) and schema ic ep esen a ion o a Janus- ype polyca ionic amphiphilic de i a i e. B) Gene al s uc u e o cyclo ehalans (CTs) and schema ic ep esen a ion o polyca ionic CT- cen e ed s a -shape polyme s. Figu e 2. A) P e ious wo k: Janus- ype CT2 de i a i es (le ) and ep esen a- i e TEM mic og aph o he nanocomplexes o med upon co-assembly wi h pDNA; a schema ic ep esen a ion o he laye ed ul a hin s uc u e is shown in he inse ( igh ).[13] B) This wo k: s uc u e o he new CT3-based Mickey Mouse- ype molecula ec o s wi h une en dis ibu ion o ca ionic (C) and lipophilic domains (L). Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9430Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9430/9438] 1 Resul s and Discussion Design c i e ia and syn hesis One can an icipa e ha going om he Janus- ype CT2 pa e n (Figu e 2A) o he C1L2 ilobula CT3 o ganiza ion (Figu e 2B, le ) would oughly mul iply by wo he olume o he hyd o- phobic a ea in he co esponding amphiphiles. Going o C2L1 MM-MNPs (Figu e 2B, igh ) will ins ead inc ease he e ec i e a ea o he ca ionic domain. Such di e ences a e expec ed o esul in signi ican ly dispa a e sel -assembling p ope ies, as well as pDNA complexing and ans ec ion capabili ies, which migh be u he adjus ed by ailo ing he ca ionic and lip- ophilic appendages.[15] In o de o es his no ion, we se led o de elop e icacious and e sa ile me hodologies, compa ible wi h he acile elabo a ion o bo h he ca ionic and lipid domains, o access bo h ypes o Mickey Mouse pa chy mac o- cycles o unable gene deli e y. The key s ep in ou syn he ic scheme was he in e molecu- la mac ocycliza ion eac ion be ween a C2-symme ic linea e asaccha ide a med wi h iso hiocyana e g oups a he dis al p ima y posi ions (s uc u e II o V) and a 6,6’-diamino-6-6’- dideoxy-α,α’- ehalose pa ne (s uc u e III o VI; Scheme 1).[16] The o ma ion o he CT3 co e is hen a sel - empla ed p ocess d i en by he igid conca e/con ex geome y o he ehalose building blocks.[17] The e asaccha ide p ecu so was ob ained om he homologous disaccha ide diiso hiocyana e (s uc u e I o IV)[16] by con olled sel -condensa ion in py idine-wa e , a eac ion ha p o ides he dime ic hiou ea wi h no in ol e- men o a ansien amine, hus p e en ing O!N acyl mig a ion side eac ions.[18] In he case o C1L2CT3 de i a i es, he dime ic diiso hiocyana e (s uc u e II) bea s wel e lipophilic ails Scheme 1. Syn hesis o he C1L2(le ) and C2L1( igh ) Mickey Mouse cyclo ehalans 1–6and 7–10. Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9431Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9431/9438] 1 ancho ed a he seconda y oxygen a oms h ough ei he es e (C6o C14) o e he unc ionali ies (C6), whe eas he diamine (s uc u e III) was equipped wi h six O-linked N- e -bu oxyca - bonyl (Boc)-p o ec ed cys eaminylp opyl heads. The la e we e ins alled e y e icien ly h ough mul iple hiol-ene click eac ion[19] be ween a hexa-O-allyl α,α’- ehalose de i a i e and Boc-p o ec ed cys eamine.[17] Coupling o building blocks II and III, ollowed by acid-p omo ed hyd olysis o he ca bama e g oups in he mac ocyclic adduc s, a o ded he a ge i- ace ed ca ionic amphiphiles 1–3 in 44–62% yield (Scheme 1; see he Suppo ing In o ma ion o de ails). No ewo hy, ch oma og aphic pu i ica ion could be e icien ly accomplished on he Boc-ended adduc s. The subsequen dep o ec ion s ep was a quan i a i e p ocess as con i med by he disappea ance o he e -bu yl p o on signal in he co esponding 1H NMR spec a. The inal compounds we e isola ed as pe hyd ochlo ide sal s upon wo- old lyophilisa ion om 0.1 M HCl solu ions. The eac ion o iso hiocyana es wi h amines o p oduce hiou eas is highly e icien wi hou need o ca alys and insensi i e o oxygen and mois u e, mee ing all he c i e ia o click chemis y, and is pa icula ly well-sui ed o mul iconjuga- ion schemes.[20] We ha e u he used i o he pos - modi ica ion o he ca ionic heads in 1–3 by addi ion o he iso hiocyana e-a med b anching elemen 11[21] and subsequen ca bama e hyd olysis. The newly gene a ed hiou ea unc ion- ali ies in he esul ing MM-MNPs 4–6 a e hen expec ed o con ibu e o nucleic acid binding h ough coope a i e hyd o- gen bonding in e ac ions.[22] In addi ion o wel e p ima y amines, he adduc s u he inco po a e six e ia y amine cen e s pe ca ionic pa ch in he b anching poin s, which will habili a e he so-called p o on sponge mechanism o endo- somal escape by impa ing bu e ing capabili ies.[23] An analo- gous eac ion sequence implying he cys eaminyl-equipped e asaccha ide diiso hiocyana e (s uc u e IV) and hexa-O- alkyla ed (C6o C14) disaccha ide diamines (s uc u e V) p o ided he e e se C2L1MM-MNPs 7and 8, which upon homologa ion by eac ion wi h 11 deli e ed he dend onized adduc s 9and 10 (Scheme 1; see he Suppo ing In o ma ion o de ails). The s uc u e and homogenei y o all MM-MNPs, as he co esponding pe hyd ochlo ide sal s, we e gauged by 1H and 13C NMR spec oscopy (Figu es S1-S28, Suppo ing In o ma ion), mass spec ome y (MS) and elemen al analysis. I is wo h no ing ha he new ec o s ha e a single C2axis o symme y, meaning ha he wo α-glucopy anoside cons i uen s o he α,α’- ehalose moie ies in he win lipid o ca ionic pa ches o C1L2(1-6) o C2L1(7-10) CT3 de i a i es a e no magne ically equi alen . This ansla es in o h ee di e en se o signals o he ca bohyd a e subuni s in he NMR spec a. Elec osp ay ioniza ion (ESI)-MS con i med he expec ed molecula masses o he Mickey Mouse amphiphiles ha keep he cys eaminyl g oups unde i a ized (1–3and 7,8; Figu es S29–S31, S35 and S36, Suppo ing In o ma ion). The co esponding pseudomolec- ula ions o he highe dend oidal homologues (4–6and 9,10) we e no clea ly obse ed unde he same condi ions because hei nume ous hiou ea and amine g oups p e en ed hem om ionizing. Fo hose cases we implemen ed an al e na i e s a egy, consis ing in he p e o ma ion o nonco alen com- plexes wi h a single-s anded 12-me DNA (5-AAGCCCGCCCAA- 3; DNAi).[24] Since he compounds a e concei ed o co-assembly wi h DNA, we ad anced ha an e icien associa ion would ake place, pa ly masking hei s ong polyca ionic cha ac e . We we e deligh ed o see ha he esul ing MM–MNP/DNAi complexes could be subjec ed o ma ix-assis ed lase deso p- ion ioniza ion ime-o - ligh (MALDI-TOF) MS, p o iding signals ha ma ched e y well he heo e ical masses (Figu es S32–S34, S37 and S38, Suppo ing In o ma ion). Sel -assembling and pDNA co-assembling p ope ies Dynamic ligh sca e ing (DLS) and mixed mode measu emen phase analysis ligh sca e ing (M3-PALS) o he hexanoyla ed o n-hexyla ed C1L2MM-MNPs 1,4and 3,6, espec i ely, p o ided poo quali y da a indica i e o insu icien pa icle coun s a he concen a ions used o pDNA complexa ion, which e lec s hei inabili y o o m s able assemblies. T ans- mission elec on mic oscopy (TEM, Figu e 3) e ealed he p esence o low densi ies o small agg ega es (<10 nm diame e ), p obably a ising om ansien clus e s o a ew molecules ha a e cap u ed upon d ying he sample on he TEM g id. Such clus e s become mo e s able o he hexacys ea- minyl-dodecamy is oyla ed de i a i e 2, as obse ed bo h by DLS (a e age hyd odynamic diame e , Dh, 15 nm; ζ-po en ial 38 mV) and TEM, likely due o i s dec eased wa e solubili y. Di e en ly, he dend onized homologue 5sel -assembled in o gian esicles (Dh320 nm; ζ-po en ial 41 mV). In he C2L1 cyclohexasaccha ide se ies, compounds 7and 10 did no agg ega e, whe eas 8a o ded sphe ical and cyclind ical micelles (Dh12 nm; ζ-po en ial 35 mV) and 9was ound by TEM o o m esicles o 30–150 nm (Figu e 3). The possibili y o bias he p e e ence o in eg a e high- o low-cu a u e sel -assembled cons uc ions by molding he MM–MNP ec o a chi ec u e was an icipa ed o u he impac he opology and s abili y o hei co-assemblies wi h nucleic acids. To es his p edic ion, he TEM images o he sup amolecula complexes p epa ed wi h pDNA (luci e ase- encoding pCMV-LucVR1216) and he MM-MNPs a p o onable ni ogen/phospho us (N/P) a io 20 in 4-(2-hyd oxye hyl)-1- pipe azinee hanesul onic acid (HEPES) bu e (pH 7.4, 10 mM) we e eco ded. The mic og aphs ob ained om 2/pDNA o mula ions showed i egula mo ula-like agg ega es o a ia- ble shape and size (150–300 nm), likely esul ing om elec o- s a ically-d i en diso de ed co-agg ega ion be ween clus e s o 2and pDNA, whe eas in he case o 5/pDNA CTplexes globula (100–150 nm) and ilamen ous objec s (>200 nm) coexis ed. The i s can a ise om he di ec in e ac ion o he plasmid wi h sel -assembled esicles o he ec o . Filamen ous and wo m-like opologies on hei side a e ypically obse ed o mac omolecula polyca ions wi h a ma ked p e e ence o linea o e cu ed a angemen s upon pDNA empla ion.[25] These ypes o complexes ha e been shown p e iously o be inadequa e o gene deli e y applica ions due o ine icien p o ec ion o he nucleic acid ca go[26] and we e no u he Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9432Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9432/9438] 1 pu sued. The o he C1L2MM-MNPs 1,3,4and 6a o ded quasi- sphe ical CTplexes o 30–50 nm diame e upon co-assembling wi h pDNA. The 1/pDNA and 3/pDNA nanocomplexes showed a sinusoidal, lamella ul a hin s uc u e cha ac e is ic o al e - na ed DNA segmen s (da k; high elec on densi y) and amphiphile bilaye s (ligh ; low elec on densi y).[27] This co- o ganiza ion mode is usually encoun e ed in complexes o DNA and polyca ionic amphiphilic cyclodex ins, being gene ally associa ed o high ans ec ion e icacy.[28] In he case o 4/ pDNA and 6/pDNA CTplexes such snake-like a angemen is missing. Ins ead, he pa icles appea as b igh whi e objec s in he TEM mic og aphs, s ongly sugges ing a co e-shell o gan- iza ion e oca i e o en eloped i uses:[29] a e an ini ial condensa ion phase p omo ed by elec os a ic in e ac ions, he nega i ely cha ged “da k” co e hus o med is enci cled by an ex e nal “ligh ” shell o he su ac an (Figu e 3; see also Figu e S39, Suppo ing In o ma ion). Analogous nanos uc u es ha e been p e iously assembled by sophis ica ed mul i o mula- ion s a egies[30] and, mo e ecen ly, om α,α’- ehalose-based Figu e 3. Rep esen a i e TEM images eco ded on he C1L2(1,2,4and 5) o C2L1- ype (7–10) Mickey Mouse molecula nanopa icles as well as on he co esponding CTplexes o mula ed wi h pDNA (luci e ase-encoding epo e gene pCMV-Luc VR1216) a N/P 20 (see also Figu e S39, Suppo ing In o ma ion, o selec ed high magni ica ion images). Schema ic ep esen a ions o he p oposed a angemen s o he molecula nanopa icle cons i uen s in he sphe ical mul ilamella (1/pDNA and 7/pDNA), mo ula (2/pDNA and 8/pDNA), co e-shell (4/pDNA) ilamen ous (5/pDNA; inse ), globula (9/pDNA) o cylind ical supe s uc u es (10/pDNA) a e also depic ed. The co esponding TEM mic og aphs o he C1L2MM–MNP ec o s 3and 6and o he espec i e 3/pDNA and 6/pDNA o mula ions a e e y simila o hose ob ained om 1and 4and a e no shown. Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9433Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9433/9438] 1 Siamese- win amphiphiles,[17] and ound o be app op ia e o nucleic acid ca go deli e y o a ge cells and speci ic o gans. In he C2L1MM–MNP se ies, compound 7(n-hexyla ed) likewise o med sphe ical agg ega es wi h a mul ilamella ul a hin s uc u e upon co-assembly wi h pDNA. The TEM images o he CTplexes o mula ed wi h 8(n- e adecyla ed) showed mo ula-like pa icles, sugges ing ha nanocomplex o ma ion in ol ed sel - assembled micelles and no he indi idual ec o molecules. The in luence o he hyd ophobic/hyd ophilic balance in de e mining he p e e ence o a lamella o a mo ula-like a angemen has been p e iously no iced, he la e a chi ec u e being mos ly de imen al o ans ec ion.[17,28a] Compound 9( he dend onized analogue o 7) o med ins ead globula pa icles pDNA, likely in ol ing he co-agg ega ion o sel -assembled esicle o he ec o wi h pDNA. This scena io is simila o ha obse ed o 2/ pDNA and 5/pDNA CTplexes, bu pa icles a e now smalle (�50 and 100 nm, espec i ely) and, a p io i, be e sui ed o ans- ec ion s udies. Finally, compound 10 ( he dend onized analogue o 8) was unique wi hin he whole se o MM-MNPs syn hesized in p o iding small cylind ical nanopa icles upon co-assembling wi h pDNA (Figu e 3; see also Figu e S39, Suppo ing In o ma ion). DLS o colloidal MM–MNP/pDNA nanocomplex solu ions a N/ P a ios 5, 10, and 20 in HEPES e idenced he o ma ion o unimodal popula ions o pa icles o mos o mula ions, wi h hyd odynamic diame e s comp ised be ween 65 and 150 nm and polydispe si y index alues <0.3. The excep ions we e he C1L2 CT3 my is oyla ed de i a i es 2(a all s udied N/P alues) and 5 (a N/P 5 and 10). I was ound ha he size o he CTplexes dec eased mono onically as he p opo ion o he ca ionic ec o inc eased, whe eas he ζ-po en ial ollowed he opposi e end, eaching posi i e alues in he ange 23–36 mV a N/P 20. O no e, compounds 4and 6o igina ed nega i ely cha ged nanopa icles upon co-assembling wi h pDNA a N/P 5, suppo ing he p oposed wo-phase (nuclea ion/coa ing) mechanism leading o co e-shell nanopa icles (Table 1). The abo e MM–MNP/pDNA o mula ions we e nex analyzed by elec opho esis mobili y shi assay (EMSA) in 0.8% aga ose gel, wi h s aining by he in e cala ing agen e hidium b omide, o assessing DNA complex o ma ion and p o ec ion as well as DNA in eg i y (Figu e 4; no e ha he lanes o in e es , coming om di e en EMSA expe imen s conduc ed unde iden ical condi ions, a e shown. The co esponding gel pic u es om which hese lanes we e aken, wi h he indi idual e e ences, a e collec ed in Figu e S40, Suppo ing In o ma ion). Excep o 2and 5(da a no shown), ull pDNA complexa ion and p o ec ion we e achie ed in all cases, as in e ed om he capaci y o he compounds o a es mig a ion o pDNA in he gel and he eco e y o he essen ially unal e ed pDNA a e DNase I/sodium dodecyl sul a e (SDS) ea men . These esul s, oge he wi h he a he small size and homogenei y o he CTplexes, make hem p omising candida es o he de elopmen o sys emic applica ions in i o by limi ing size- es ic ed di usion and epi helial pe mea ion and abso p ion. Table 1. Hyd odynamic diame e (Dh) and ζ-po en ial o he nanocomplexes o mula ed wi h he Mickey Mouse molecula nanopa icles 1–10 and pDNA (luci e ase-encoding pCMV-LucVR1216) a p o onable ni ogen/phospho us (N/P) a ios 5, 10, and 20 in 4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid (HEPES) bu e (pH 7.4, 10 mM).[a] N/P 5 10 20 Dh(nm) ζ(mV) Dh(nm) ζ(mV) Dh(nm) ζ(mV) 1145�2 16�2 147�2 25�1 102�3 31�2 2>500 – >500 – >500 – 3148�25 6�4 108�11 18�8 86�1 36�14 4230�325�1 177�3 16�2 123�4 33�2 5>500 – >500 – 157�6 35�2 6108�20 9�5 74�2 25�9 66�2 27�12 7108�6 21�1 93�1 23�2 82�1 29�2 8104�11 28�1 88�2 33�1 86�2 33�3 9138�11 28�17 106�28 23�6 93�4 28�8 10 151�21 9�5 80�10 21�9 65�1 23�2 [a] Excep o o mula ions based in compound 2(all N/P alues) and 5(N/P 5 and 10), polydispe si y index alues we e <0.3 in all cases. Figu e 4. EMSA gels o CTplexes o mula ed wi h compounds 1,3,4and 6– 10 a N/P 10, be o e (uppe panel) and a e ea men wi h DNAse I and subsequen dissocia ion o he complexes wi h SDS (lowe panel). Naked pDNA and e hidium b omide we e used as he con ol and s aining eagen , espec i ely. No ably, pDNA complexa ion esul s in a es ed mig a ion o he anode, whe eas deg ada ion leads o he disappea ance o any isible band due o dis up ion o he double helix, which p e en s in e cala ion o he luo escen p obe. Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9434Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9434/9438] 1 Compu a ional assessmen o CTplex assembly The abo e DLS and TEM da a illus a e he po en ial o shi ing be ween he C1L2and he C2L1Mickey Mouse CT3 p o o ypes o ine- une he opology o he ec o /pDNA co-agg ega es. The excep ion is he pai 1(o 3) e sus 7, combining unb anched cys eaminylp opyl ca ionic a ms and C6lipophilic ails: quasi- sphe ical mul ilamella nanocomplexes wi h simila ζ-po en ial, secu ing pDNA p o ec ion, we e obse ed in bo h se ies, in spi e o he expec ed subs an ial change in he hyd ophilic/hyd ophobic balance. P elimina y ci cula dich oism s udies we e consis en wi h nei he 1no 7sel -associa ing in HEPES a he concen- a ions used o CTplex o mula ion and bo h being able o in e mingle wi h DNA as indi idual molecules (Figu es. S46–S48, Suppo ing In o ma ion). To ge a deepe insigh on he in e ac ions a play, he s abili y o an ipa allel cyclohexasaccha ide dime s acing hei hyd ophobic domains, bo h in he bulk and in he con ined space be ween DNA agmen s, was in es iga ed by molecula mechanics (MM) and molecula dynamics (MD) simu- la ions conduc ed in explici wa e . Such dime s can be conside ed as he basic elemen s o ec o bilaye s.[31] Ini ially, he minima binding ene gy (MBE) s uc u es o 1o 7 pe hyd ochlo ide dime s we e op imized (MM), hen de oid om he chlo ine a oms (ne cha ge o each pa chy CT3 de i a i e +6 and +12 esu o 1and 7, espec i ely) and used as he s a ing con o ma ions o he compu a ion in wa e o (MM–MNP)2/(DNA)2 complexes (Figu es S41–S44, Suppo ing In o ma ion). Subsequen MD simula ions showed ha he cha ged cyclohexasaccha ide dime s eadily dissocia ed in he absence o he lanking DNA agmen s. Recip ocally, he DNA chains all apa in he absence o he in e posed ca ionic ec o s. Howe e , he sup amolecula (MM–MNP)2/(DNA)2complexes emained s able du ing 1 ns MD simula ions in he p esence o wa e . Some di e ences a ose, howe e , be ween he complex buil om he C1L2de i a i e 1 and he C2L1coun e pa 7: whe eas in he i s case he dis ance be ween he cons i u i e ec o monome s emained s able h oughou he whole MD ajec o y (Figu e 5A and B), in he second such dis ance became signi ican ly la ge (Figu e 5C and D). Concei ably, he p esence o a single lipophilic pa ch in 7 esul s in weake an de Waals in e ac ions be ween he ec o molecules as compa ed wi h 1, which is compensa ed because he ca ionic pa ches in 7can b idge he DNA segmen s in he complex. Such di e ences a e expec ed o lead o a iances in ex e nal/in e nal cha ge as well as in he deg adabili y o he co esponding CTplexes, which a e ac o s ha g ea ly in luence he in i o opism.[32] Bo h 1and 7we e hus e ained o he compa a i e assessmen o hei ans ec ion capabili ies in i o and in i o. Toxici y and in i o cell ans ec ion CTplexes o mula ed om he CT3-sca olded Mickey Mouse molecula nanopa icles 1,3,4,6–10 and he luci e ase-encoding epo e gene pCMV-Luc VR1216 a N/P 5, 10, and 20 we e nex assayed o hei ans ec ion capabili ies in i o in A ican g een monkey epi helial kidney COS-7, human hepa ocellula ca cinoma HepG2, human ce ical ca cinoma HeLa, mu ine emb yonic hepa ocy e BNL-CL2, and mu ine mac ophage RAW 264.7 cells (Figu e 6). The la e linage is known o be no ably mo e ecalci an o ans ec ion.[33] Polyplexes gene a ed omb anched polye hylenimine (bPEI, MW=25 kDa; N/P 10), he gold s anda d ca ionic polyme o non i al gene deli e y,[34] and polyplexes o mula ed om Lipo ec amine 3000® (LP, a comme cial ca ionic lipid o mula ion) we e also included in ou sc eening as con ols o compa a i e pu poses. All expe imen s we e conduc ed in he p esence o 10% se um, he op imal condi ions o he con ols. Pa chy CT3 de i a i es gi ing ise o sphe ical pa icles wi h a well-de ined lamella o ganiza ion, namely 1and 3in he C1L2 se ies and 7in he C2L1se ies, ou pe o med bPEI a all N/P alues and i alled Lipo ec amine 3000® pe o mance in all cell lines. Nanocomplexes wi h an analogous opology ob ained om polyca ionic amphiphilic cyclodex ins we e p e iously ound o en e he cell p e e en ially h ough a ca eolae-media ed endo- cy ic pa hway ha a o ed hei apid accumula ion a he icini y o he nucleus and led o high ans ec ion le els.[35] The e iden analogies sugges ha a simila mechanism migh ope a e he e. Excep ing in COS-7 cells (Figu e 6A), compounds 4and 6, a o ding co e-shell nanopa icles upon co-assembly wi h pDNA, also o e passed bPEI polyplexes in hese expe imen s. Howe e , he ans ec ion e iciency mono onically dec eased wi h he N/P alue, sugges ing ha o ma ion o he ex e nal compac shell esul s in e y s able CTplexes om which elease o he pDNA ca go in acellula ly akes place a a lowe a e. The si ua ion is Figu e 5. A) MD his o ies o he dis ances DNA(1)-1(1) (black), DNA(2)-1(2) ( ed), 1(1)-1(2) (blue) and DNA(1)-DNA(2) (g een). B) S uc u e o he (1- dime )(DNA)2nanocomplex a e aged h oughou he 1 ns MD ajec o y. C) MD his o ies o he dis ances DNA(1)-7(1) (black), DNA(2)-7(2) ( ed), 7(1)-7(2) (blue) and DNA(1)-DNA(2) (g een). D) S uc u e o he (7-dime )(DNA)2 nanocomplex a e aged h oughou he 1 ns MD ajec o y (DNA agmen s in g een; he indi idual ec o monome cons i uen s 1o 7a e colo ed in ligh and deep blue). Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9435Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9435/9438] 1 analogous o ha encoun e ed o CTplexes ob ained om dend onized CT3-cen ed s a polyme s.[12] The i egula mo ula and globula shaped CTplexes o mula ed om 8and 9showed compa a i ely poo e ans ec ion e iciencies. Indeed, nanocom- plexes ob ained om pDNA and molecula ec o s o ming s able micelles ypically ha e a highe endency o agg ega e and poo e ans ec ion capabili ies.[17,28a,36] Rema kably, he cylind ical 10/ pDNA CTplexes displayed e y high selec i i y owa ds he mac o- phage cell line RAW 264.7, eaching exp essions o he encoded luci e ase p o ein o e one o de o magni ude highe han bPEI polyplexes, abou 3- old as compa ed wi h LP lipoplexes (Fig- u e 6E). This is consis en wi h epo s showing ha mac ophages in e nalize ellipsoidal pa icles much as e han sphe ical pa icles.[37] Al oge he , he esul s demons a e ha he size, na u e and ela i e dis ibu ion o he ca ionic and lipophilic pa ches in he cyclohexasaccha ide co e exe s a no ewo hy in luence on he cell selec i i y o he esul ing MM–MNP based nanocomplexes, highligh ing he in e es o s a egies compa ible wi h mac omolecula ailo ing o speci ic applica ions. Mos no ably, se e al o he new ec o s pe o med be e han bPEI and Lipo ec amine 3000® o all he cell lines, e en a he lowes N/P 5 a io, while showing no oxici y in he whole ange o N/P a ios and cell linages sc eened (Figu e S45, Suppo ing In o ma- ion). In i o ans ec ion The shape and size o nanopa icles a e known o conside ably a ec hei biodis ibu ion and op ion o endocy ic pa hways and ha e been ound o be main de e minan s ega ding he in i o opism o molecula ec o -based pDNA nanocomplexes.[38] The possibili y o p og am di e en opologies a he nanoscale by ailo ing he ec o a chi ec u e a he molecula le el, enabled by he Mickey Mouse CT3 p o o ype, o e s exci ing p ospec s in his sense. To explo e his concep , CTplexes o mula ed om pDNA (pCMV-Luc VR1216) and he C1L2 ype MM-MNPs 1and 6o he C2L1membe s 7and 10 a N/P 5 and 10 we e sys emically injec ed in o mice, and hei ac i i y in he li e , hea , lungs, and spleen was compa ed wi h PBS and he naked DNA as nega i e con ols. All animals we e s udied in acco dance wi h guidelines es ablished by Di ec i e 86/609/EEC and wi h he app o al o he Commi ee on Animal Resea ch a he Uni e si y o Na a a (acc edi a ion numbe CEEA 017-19). The esul s, based on he luci e ase epo e gene exp ession, indica ed ha 24 h a e he in a enous adminis a ion o N/P 5 1/pDNA CTplexes, which o med sphe ical mul ilamella pa icles, simila ans ec ion le els we e eached a all he analyzed o gans. A N/P 10 ans ec ion occu ed p e e en ially a he spleen, bu i was s ill signi ican a he hea , li e and lung. Sha ply di e en ly, he co e-shell 6/pDNA nano- complexes exhibi ed a ma ked opism o he lung ha was sligh ly highe a N/P 5 han a N/P 10. Compound 7, which simila ly o 1a o ded quasi-sphe ical mul ilamella CTplexes, media ed ins ead ans ec ion in he li e wi h high selec i i y, especially a N/P 10. This is asc ibable o di e ences in he in e nal s uc u e as sugges ed by he compu a ional s udies abo e discussed. In u he suppo o his hypo hesis, in i o esul s ob ained o N/P 10 CTplexes o compa able shape and size o mula ed wi h compound 3po ayed an o gan ans ec ion p o ile closely ma ching ha o 1(less han 10% di e ence in no malized luci e ase exp ession alues in hea , li e , lung and spleen; da a no shown), wi h which 3sha es he same C2L1 a chi ec u e. Finally, he cyclind ical CTplexes o mula ed om 10 almos exclusi ely ans ec ed he spleen a ei he N/P a io, wi h negligible luminescence de ec ed in o he o gans (Figu e 7). Figu e 6. Luci e ase exp ession in (A) COS-7, (B) HepG2, (C) HeLa, (D) BNL- CL2, and (E) RAW 264.7 cells p omo ed by CTplexes o mula ed wi h he MM-MNPs 1,3,4o 6–10 and he luci e ase-encoding epo e gene pCMV- Luc VR1216 a N/P 5, 10, and 20 in he p esence o e al bo ine se um (FBS, 10%). Da a ob ained wi h Lipo ec amine 3000® polyplexes and bPEI polyplexes (N/P 10) unde iden ical condi ions a e included o compa a i e pu poses. The da a ep esen he mean �s anda d de ia ion (SD) alues o h ee wells and a e ep esen a i e o h ee independen de e mina ions. Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9436Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9436/9438] 1 The ac ha he MM–MNP ec o s allow simul aneous ans- ec ion o mul iple in e nal o gans o selec i e deli e y o he nucleic acid ca go o a speci ic o gan is ema kable. E en mo e no able is ha he o gan des ina ion can be swi ched be ween lung, li e o spleen using he same CT3 pla o m by app op ia ely designing he di e en pa ches on he CT3 co e, wi h no need o a a ge ing ligand. Di e en ial mRNA deli e y o he li e o he spleen has been p e iously achie ed wi h lipid nanopa icles (LNPs) by adjus ing he N/P a io, hus he cha ge o he lipoplexes.[39] Ve y ecen ly, Siegwa and co-wo ke s epo ed ha a a ie y o LNPs can be enginee ed o exclusi ely deli e mRNA o ex ahepa ic issues ia addi ion o supplemen al molecules ha allow uning he in e nal cha ge o he lipoplexes, so-called selec i e o gan a ge ing (SORT) molecules.[40] Lung-, spleen- and li e - a ge ed SORT LNPs we e designed in his manne . In ou case, uning he opological landscape o he nanocomplexes le achie e simila o gan-selec i e ans ec ion p o iles, wi h he no able di e ence ha he MM-MNPs he e epo ed a e single molecule, pe ec ly monodispe sed species e ec i e in mono o - mula ion. Conclusions The ensemble o da a p o ides conclusi e e idence on he s ong po en ial o judiciously ins alling di e en a angemen s o unc- ional elemen s on o a cyclo ehalan co e o e icien ly access pe ec ly monodispe se pa chy molecula nanopa icles wi h ailo ed s uc u es and p ope ies beyond he Janus a che ype. The Mickey Mouse ma e ials he e epo ed we e concei ed o condense pDNA in o nanocomplexes h ough concu en elec o- s a ic and hyd ophobic in e ac ions. The da a spo ligh he ad an age o mac omolecula di e si y-o ien ed s a egies com- pa ible wi h s ic con ol o e he s uc u al pa ame e s, including he na u e and alency o he pa ches, o modula e he sel - and co-assembling beha io s. The ema kable cell selec i i y and, especially, o gan selec i i y di e ences obse ed wi hin he se ies o MM-MNPs p epa ed emphasize he compelling e ec o bo h he ec o a chi ec u e and he nanocomplex opology on he biological ac i i y. Taken oge he , hese esul s in o m a e sa ile p o o ype o he cons uc ion o ully syn he ic aniso opic non i al gene deli e y sys ems uniquely sui ed o op imiza ion schemes. Expe imen al Sec ion Expe imen al de ails a e gi en in he Suppo ing In o ma ion. Acknowledgemen s We acknowledge he Minis e io de Ciencia, Inno ación y Uni e sidades, he Minis e io de Ciencia e Inno ación and he Agencia Es a al de In es igación (p ojec s RTI2018-097609-B-C21, RTI2018-097609-B-C22 and (PID2019-105858RB-I00), he Uni e si- dad de Alcalá (CCG19/CC-033) and he Eu opean Regional De elopmen Funds (FEDER-UE). A.I.C.-G. and M.G.-C. a e FPI ellows. The CITIUS (Uni e si y o Se ille), he Cen e Commun de Mic oscopie Appliquée (S. Pagno a, Uni e si é Cô e d’Azu ) and he Mass Spec ome y Co e Facili y (G. Clodic, So bonne Uni e si é) a e also acknowledged o echnical suppo . Con lic o In e es The au ho s decla e no con lic o in e es . 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Gang, ACS Nano 2020,14, 6823–6833. [5] a) H. Su, C.-A. Hu d P ice, L. Jing, Q. Tian, J. Liu, K. Qian, Ma e . Today Bio 2019,4, 100033; b) G. Ag awal, R. Ag awal, ACS Appl. Nano Ma e . 2019, Figu e 7. Gene exp ession conduc ed in i o a e in a enous adminis a- ion o 60 μg o pCMV-Luc VR1216 o mula ed wi h he MM-MNPs 1,6,7 and 10 a (A) N/P 5 o (B) N/P 10. Ba s ep esen he mean�SD (n=8 animals). Schema ic ep esen a ions o he nanocomplex shape and in e nal s uc u e (sphe ical mul ilamella o 1and 7, co e-shell o 6and cylind ical o 10) as also depic ed Chemis y—A Eu opean Jou nal Full Pape doi.o g/10.1002/chem.202100832 9437Chem. Eu . J. 2021,27, 9429–9438 www.chemeu j.o g © 2021 The Au ho s. Chemis y - A Eu opean Jou nal published by Wiley-VCH GmbH Wiley VCH F ei ag, 18.06.2021 2136 / 204573 [S. 9437/9438] 1