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.
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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).
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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.
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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
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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.
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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.
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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).
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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.
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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 .
Keywo ds: cyclooligosaccha ides ·mac ocycles ·molecula
nanopa icles ·non- i al gene deli e y ·sel -assembling ·
ehalose
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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
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s uc u e (sphe ical mul ilamella o 1and 7, co e-shell o 6and cylind ical
o 10) as also depic ed
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